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Use of MRI to study excitotoxic spinal cord injury

University of Florida Institutional Repository

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USE OF MRI TO STUDY EXCITOTOXIC SPINAL CORD INJURY By SARA ANN BERENS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by SARA ANN BERENS

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This document is dedicated to my fianc, Ja son, and my family for their continued love and encouragement.

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iv ACKNOWLEDGMENTS First, I would like to thank my committee members, Drs. Robert Yezierski, Tom Mareci, Steve Blackband, and Doug Anderson, for their guidance and encouragement. Each has contributed to my development as a scientist in many different ways. I would like to thank my mentor, Dr. Yezierski, for t eaching me all there is to know about spinal cord injury and pain, as well as performi ng surgeries; Dr. Mareci, for shaping my knowledge of MR imaging, as well as show ing me how to operate several of the magnet systems; Dr. Blackband, for providing me a f oundation on the principles of MR Imaging; and Dr. Anderson for providing his expertise on spinal cord injury. I appreciate their encouragement to write as often as I did, through publications and grants; it made the final writing process less strenuous. I am indebted to all of the current and former lab members of the Yezierski and Mareci labs that I have had the opportunity to work with over the years. In particular, I would like to thank Dr. Chen-Guang Yu for pe rforming some of the surgeries; Jacquie Rodgers, for moral support and lending a helpin g hand in the care of my animals; my lab mate, Chris King, for moral support, helping w ith surgeries and animal care when I could not be there, for sharing an understanding of similar frustrations, and for being a friend; and Victoria Henry, for her continued mo ral support and guidance, problem solving skills, all of our many lunch walks, and, mo st importantly, for being my work mom. I appreciate all those who have contributed to my success in imaging, especially Danny Colvin and Christy Amwake, for build ing the surface coils; Evren zarslan for

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v helping me collect and analyze my in vitro MR data; Lari McEdwards, Suril Patel, Matthew Feldman, and Aaron Richardson for providing assistance in data analysis; Ty Black for his expertise and problem solving on the analysis software; and Nelly Voland, Chris Taylor, Min Sig Hwang, Hector Sepul veda, Lan Hoang Minh, Allan Elfstrm, Matthew Feldman, and Rashid Jawdat for pr oviding me the opportunity to take a lunch break during my many long hours of imaging. I would also like to give credit to th e Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) facility in the McKni ght Brain Institute of the University of Florida and the staff. I thank Raquel Torres and Xeve Silver for their help in placing tail vein catheters and general te chnical support; Barbara Beck a nd Kelly Jenkins, for their expertise in coil setup, designing the volume coil, and helping me solve signal and tuning problems; Gary Blaskowski, for technical support and making sure the instrument ran smoothly; and Jim Rocca and Dan Plant for checking up on me and making sure everything was running properly. I am also grateful for th e significant financial support provided by the McKnight Brain Institute. I would also like to thank Dr. Joseph Riley for his expertise and help with complicated statistics. On a more personal note, I would also like to thank my family for their continuous love, support, and encouragement throughout th e years, and for shaping me to become the person I am today. The countless phone calls and emails helped combat stress, allowing me to keep my sanity. My fianc, Jason, deserves special recognition, for taking the brunt of my frustra tion, letting me vent when th ings got tough, enduring all of

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vi my tribulations first hand, for caring and l oving me, and for supporting me in all I set out to do, never allowing me to give up.

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vii TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv TABLE OF CONTENTS..................................................................................................vii LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xi ii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW....................................................1 Spinal Cord Injury........................................................................................................1 Overview...............................................................................................................1 Primary and Secondary Injury Mechanisms..........................................................2 Excitotoxic Model of Spinal Cord Injury..............................................................3 In Vivo Magnetic Resonance Imaging..........................................................................4 Overview...............................................................................................................4 Dynamic Contrast-Enhanced Magnetic Resonance Imaging, Blood-Spinal Cord-Barrier.......................................................................................................6 In Vitro Magnetic Resonance Imaging.........................................................................7 Neuroprotective Strategies for Treat ment of Spinal Cord Injury.................................9 Overview...............................................................................................................9 Neuroprotective Effects of Agmatine..................................................................10 2 METHODOLOGICAL DEVELOPMENT................................................................13 Overview.....................................................................................................................13 Anesthetic/Experimental Drugs..................................................................................13 Magnetic Resonance Imaging.....................................................................................15 Catheter Placement..............................................................................................15 4.7T Coil Development.......................................................................................15 11.1T Coil Development.....................................................................................16 Coil Setup............................................................................................................18 11.1T Revised Imaging Protocol.........................................................................19

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viii 4.7T versus 11.1T Images...................................................................................19 Magnetic Resonance Im aging Obstacles....................................................................20 Heating Effects of the 11.1T Imaging Protocol...................................................20 Signal-to-Noise Ratio..........................................................................................23 3 EVALUATION OF THE PATHOLOGICAL CHARACTERISTICS OF SPINAL CORD INJURY WITH MAGNETI C RESONANCE IMAGING.............................25 Introduction.................................................................................................................25 Materials and Methods...............................................................................................26 Animal Preparation..............................................................................................26 Intraspinal Injection Procedure............................................................................27 In Vivo MR Imaging............................................................................................28 In Vivo High Resolution MR Imaging Data Acquisition....................................28 In Vivo DCE-MR Imaging Data Acquisition......................................................30 In Vitro MR Microscopy.....................................................................................31 Histological Procedures.......................................................................................31 Data Analysis.......................................................................................................32 Results........................................................................................................................ .33 In Vivo High Resolution MR Imaging.................................................................33 In Vivo DCE-MR Imaging...................................................................................37 In Vitro MR Microscopy.....................................................................................38 Histologic Findings.............................................................................................40 Discussion...................................................................................................................41 4 USE OF MAGNETIC RESONAN CE IMAGING TO STUDY THE NEUROPROTECTIVE EFFECTS OF AGMATINE FOLLOWING SPINAL CORD INJURY IN THE RAT...................................................................................47 Introduction.................................................................................................................47 Materials and Methods...............................................................................................48 Animals, Surgery, and In Vivo MR Imaging.......................................................48 Neuroprotection...................................................................................................48 In Vivo High-Resolution Magnetic Resonan ce Imaging Data Acquisition.........48 In Vivo DCE-MR Imaging Data Acquisition......................................................50 In Vitro Three-Dimensional MR Microscopy.....................................................51 Data Analysis.......................................................................................................51 Volume Analysis.................................................................................................52 Results........................................................................................................................ .53 In Vivo High Resolution Magnetic Resonance Imaging.....................................53 In Vivo Dynamic Contrast-Enhanced Ma gnetic Resonance Imaging.................60 Volume Analysis.................................................................................................62 Discussion...................................................................................................................68

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ix 5 DIFFUSION WEIGHTED MR IMAGI NG OF THE EXCISED QUISQUALIC ACID LESIONED RAT SPINAL CORD..................................................................72 Introduction.................................................................................................................72 Materials and Methods...............................................................................................73 Animals, Surgery, and Neuroprotection..............................................................73 In Vitro Three Dimensional MR Micr oscopy and Diffusion Weighted Imaging............................................................................................................73 Data Analysis.......................................................................................................74 Results........................................................................................................................ .75 Discussion...................................................................................................................85 6 CONCLUSION...........................................................................................................90 Summary of Findings.................................................................................................90 Future Directions........................................................................................................91 LIST OF REFERENCES...................................................................................................93 BIOGRAPHICAL SKETCH...........................................................................................107

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x LIST OF TABLES Table page 4-1 Summary of Significant Differences in Longitudinal Profiles of Edema, Cavitation, and Blood Vessel Leak age, and BSCB Disruption...............................67 4-2 Summary of Significant Differences in Volumes of Edema, Blood Vessel Leakage, and Cavitation...........................................................................................67

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xi LIST OF FIGURES Figure page 2-1 Comparison of MR images collected on the 4.7T magnet versus the 11.1T magnet. ...................................................................................................................20 2-2 Temperature measurements collected on the 4.7T and 11.1T magnets using a tissue equivalent phantom simulating rat abdomen. ..............................................22 2-3 Comparison of images collected with high versus low SNR on the 11.1T magnet. ...................................................................................................................24 3-1 Representative pathologic findings in the same animal (S05) at the same location observed in vivo (T2-weighted, TR/effective TE = 2000 ms/62.5 ms; NA = 6), in vitro (TR/TE = 2500 ms/20 ms; NA = 8), a nd in histologic sections. .34 3-2 Sagittal images for a ll QUIS-injured animals..........................................................35 3-3 Transverse images for all QUIS-injured animals. ..................................................36 3-4 Total injury lengths determined by using in vivo in vitro and histologic methods for each QUIS-injured animal. ................................................................37 3-5 Summary of in vivo in vitro and histologic data collected for a representative QL2 animal (S06). ..................................................................................................38 3-6 Summary of in vivo in vitro and histologic data collected for a representative QL1 animal (X03). .................................................................................................39 4-1 Surgery and MR im aging timeline. ........................................................................50 4-2 Representative T2-weighted (TR/e ffective TE = 2000 ms/62.5 ms; NA = 12) MR images for each injury/treatment group for each day. ....................................54 4-3 Longitudinal profile of th e presence of edema for all injury/treatment groups as determined from in vivo T2-weighted transverse MR images. ..............................55 4-4 Longitudinal profile of the presence of cavitation fo r all injury/treatment groups as determined from in vivo T2-weighted transverse MR images. ..........................56

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xii 4-5 Longitudinal profile of the pres ence of blood vessel leakage for all injury/treatment groups as determined from in vivo T2-weighted transverse MR images. ....................................................................................................................57 4-6 Longitudinal profile of th e total pathology inju ry length (the presence of edema, blood vessel leakage, and cavit ation combined) for all injury/treatment groups as determined from in vivo T2-weighted transverse MR images. ..............................58 4-7 Summary of DCE-MRI BSCB disruption. Peak enhancement is represented as a percentage. ..............................................................................................................61 4-8 Volume of edema for all injury/t reatment groups as determined from in vivo T2weighted transverse MR images. ...........................................................................62 4-9 Volume of blood vessel leakage for a ll injury/treatment gr oups as determined from in vivo T2-weighted transverse MR images. .................................................63 4-10 Volume of cavitation for all injury /treatment groups as determined from in vivo T2-weighted transverse MR images. ......................................................................64 4-11 Volume of total injury (volume of edema, blood vessel leakage, and cavitation combined) for all injury/treatment groups as determined from in vivo T2weighted transverse MR images (days 1, 3, 7, 15, and 25) and in vitro transverse 3D MRM images (day 30).......................................................................................66 5-1 S0 (b = 100 s/mm2) transverse MR image from a QUIS lesioned animal showing regions of interest drawn for anal ysis of the white matter. ....................................76 5-2 S0 (b = 100 s/mm2) transverse MR image from a QUIS lesioned animal showing regions of interest drawn for anal ysis of the gray matter. ......................................76 5-3 Average diffusivity and fractional anisot ropy values for Control white matter. ....78 5-4 Average diffusivity and fractional anisotropy values for Saline-SCI white matter. .....................................................................................................................7 9 5-5 Average diffusivity and fractional anis otropy values for QUIS-SCI white matter. ............................................................................................................................... ...80 5-6 Average diffusivity and fractional anisot ropy values for Control gray matter........81 5-7 Average diffusivity and fractional anisot ropy values for Saline-SCI gray matter. ............................................................................................................................... ...82 5-8 Average diffusivity and fractional anisot ropy values for QUIS-SCI gray matter. .83 5-9 Probability maps calculated from diffu sion weighted datasets collected from excised normal and QUIS injured spinal cords........................................................84

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xiii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy USE OF MRI TO STUDY EXCITOTOXIC SPINAL CORD INJURY By Sara Ann Berens August 2006 Chair: Robert P. Yezierski Major Department: Neuroscience Spinal cord injury (SCI) is an important cause of morbidity and mortality in modern society. Understandi ng the mechanisms responsible for acute and chronic spinal cord injury pathology as we ll as secondary consequences and the development of effective strategies for therap eutic intervention is of great clinical importance. Although most of what is known about the pathological changes associated w ith experimental SCI has come from histological studies, the ability to non-invasively char acterize spinal cord pathology provides a way to study the temporal profile of pathologi cal changes in the injured spinal cord using the same animal at multiple time points. A technique with the capability of achieving this objective is high-resolution magnetic resonance imaging (MRI), which is becoming a preferred method for the visualization of SCI. Threedimensional magnetic resonance microscopy and diffusion weighted imaging (DWI) are additional techniques that can be used to study SCI. Collectively, these MRI methods were used to evaluate the progression of excitotoxic injury, including changes in pathology, blood-spinal cord-barrier integrity, and white and gray matter fiber tracts, as

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xiv well as evaluate the neuropr otective effects of a pharm acological agent with known neuroprotective properties on the excitotoxic lesion. It was found that high-resolution MRI can be used to study experimental SCI quantitatively and qualitatively with high field magnets using customized coils and optimized methods. This method had the sensi tivity to detect significant differences in pathological characteristics between two different strategies of injury, as well as effectively evaluate the progression of pat hological changes longitudinally in the same animal. Magnetic resonance imaging had the capability to monitor the efficacy of a pharmacological agent with known neuroprot ective properties to protect the injured spinal cord. Additionally, st ructure and orientation of white and gray matter fiber tracts were evaluated with sophisticated diffusi on weighted MR imaging methods. Diffusion weighted MR imaging had the sensitivity to detect fiber patterns in a normal cord and reveal changes in fiber orientation in quis qualic acid lesioned spinal cords. With continued improvements and application of these techniques, it will be possible to determine additional insights into the mechanism responsible for the progression of secondary pathologic change s following spinal injury.

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1 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Spinal Cord Injury Overview Spinal cord injury (SCI) is an important cause of morbidity and mortality in modern society (Dumont et al., 2001; Feh lings et al., 2001; Sekhon and Fehlings, 2001; Carlson and Gorden, 2002). An estimated 600, 000 Americans are living with functional deficits due to SCI (Carlson and Gorden 2002; Hulsebosch, 2002), with about 11,000 new cases each year, costing an estimated $9.7 billion annually. At present, the recommended treatment for acute SCI is methylprednisolone, a drug that has been documented to affect several important components of spinal injury including inflammation and free-radical formation (Br acken, 2001; Xu et al., 2001; Rabchevsky et al., 2002). In spite of intense research directed to repair the injure d spinal cord, current long-term treatment is limited to rehabil itative physiotherapy (Wells et al., 2003). Because the success of these strategies is hi ghly variable, many patients with SCI remain physically impaired as a result of their inju ry (Dumont et al., 2001; Fehlings et al., 2001; Sekhon and Fehlings, 2001; Carlson and Gorden 2002; Wells et al., 2003). Therefore, understanding the mechanisms responsible fo r acute and chronic spinal cord injury pathology as well as secondary consequences an d the development of effective strategies for therapeutic intervention is of great clinical importance.

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2 Primary and Secondary Injury Mechanisms The pathophysiology of SCI involves both primary and secondary injury mechanisms. Primary injury is a direct result of mechanical trauma leading to hemorrhage, membrane disruption, and vascular damage. This initial insult initiates a progressive wave of secondary injury, whic h activates a cascade of pathophysiological mechanisms that exacerbate the injury. This spread of damage is thought to be due to activation of biochemical events leading to ce llular death (apoptosis and necrosis). There are several mechanisms that contribute to th is wave of destruction including breakdown of the blood-spinal-cord barrier (BSCB), ischemia, forma tion of free radicals and nitric oxide, invasion of neutrophils, activation of astrocytes and microglia, apoptosis of oligodendrocytes, Wallerian degenerati on, and oxygen radical-mediated lipid peroxidation (Anderson et al., 1985; Hall et al., 1992; Anderson and Hall, 1993; Hausmann, 2003). Following ischemic or traumatic SCI, there is a transient increase in extracellular excitatory amino acids (EAA), which results in glutamate receptor-mediated excitotoxic events (Mills et al., 2001). Activation of glutamate receptors initiates numerous intracellular signaling pathways, causing l ong-lasting pathological and ultimately functional deficits following SCI. Additionally, there is accumulation of intracellular sodium, producing edema and a concomitant el evation in intracellu lar calcium (Young et al., 1982; Stokes et al., 1983). Influx of cal cium is also associated with glutamate receptor activation and the incr eased release of EAAs leadin g to activation of calciumactivated proteases (e.g., calpains), formation of free radicals, stimulation of nitric oxide synthase (NOS), activation of proteases a nd endonucleases, mitoc hondrial damage, and

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3 induction of apoptosis (Lu et al., 2000; Carl son and Gorden, 2002; Germano et al., 2002; Hausmann, 2003; Kwon et al, 2004; Park et al., 2004; Profyris et al., 2004). Following spinal cord injury, damage is not restricted to the spinal cord. Studies have shown that damage occurs to ascendi ng pathways, leading to deafferentation of supraspinal structures (Yeziersk i, 2002). With deafferentati on, it is likely that cortical and subcortical regions could undergo significan t reorganization, resulting in widespread supraspinal functional changes following spinal injury (Morrow et al., 2000). Recently, Lee et al. (2004) evaluated the role of apopt osis in the secondary injury of the brain following acute spinal injury. They found ev idence for cell death in the sensorimotor cortex caused by injury. This would indicate that apoptosis can occu r remotely in regions distant from the lesion epicen ter. Similarly, evidence of apoptosis was found in the pyramidal cell layer of the motor cortex afte r spinal transection injury (Hains et al., 2003). Realizing that injury is not limited to the spinal cord but can have remote effects on the brain, is important, because in designi ng therapeutic treatments, one needs to take into consideration all available targets in order to maximize potential sites of intervention. Excitotoxic Model of Spinal Cord Injury In recent years, studies have shown that there exists a striking similarity between clinical and experimental SCI pathophysiology. In order for an animal model to have clinical relevancy, it needs to imitate the pathological features of human SCI, as well as be reliable, consistent, and reproducible. The ex citotoxic model of spin al cord injury is a reduced injury model that has been shown to result in pathological changes similar to those associated with traumatic and ischemic SCI (Yezierski et al., 1993; Yezierski et al., 1998) and parallels the pathol ogical sequela associated wi th human SCI (Kakulas, 1987;

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4 Bunge et al., 1993; Bunge et al., 1997). It is produced by the intraspinal injection of the AMPA/metabotropic receptor agonist, quisqua lic acid (QUIS). Previous studies have shown that the excitotoxic injury model re sults in pathophysiologi cal changes, pain behaviors, NMDA receptor phosphorylation and up-regulated gene expression for NMDA receptors, opioid peptides (PPE and PPD ), substance P receptor, inflammatory genes (iNOS and COX-2), apoptotic genes (F AS-L and TNF-related apoptosis-inducing ligand, TRAIL), and cytokine genes (IL-1 and TNF) (Yezierski and Park, 1993; Yezierski et al., 1993; Liu et al., 1997a; Brewer and Yezier ski, 1998; Yezierski et al., 1998; Brewer et al., 1999; Sc hwartz et al., 1999a; Fairbanks et al., 2000; Morrow et al., 2000; Abraham et al., 2001; Gorman et al., 2001; Plunkett et al., 2001; Yezierski, 2001; Pattany et al., 2002; Siddall et al., 2002; Y ezierski 2002; Yezierski and Burchiel, 2002; Caudle et al., 2003; Yu et al., 2003; Yu and Yezierski, 2005). The similarities between QUIS induced pathology and clinical pathology of SCI support the us e of the excitotoxic model in the study of pathophysiology and secondary injury mechanisms following spinal injury. Another advant age of the excitotoxic injury model is that it does not produce the mortality, morbidity, or challe nges with animal husbandry that result from ischemic or contusion models of injury. In Vivo Magnetic Resonance Imaging Overview Although most of what is known about the pathological changes associated with experimental SCI (i.e., edema, neuronal loss, hemorrhage, and cavitation) has come from histological studies, the abil ity to non-invasively charac terize spinal cord pathology provides a way to study the temporal profile of pathological changes in the injured spinal cord using the same animal at multiple time points. A technique with the capability of

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5 achieving this objective is high-resolution magnetic resonance imaging (MRI), which is becoming a preferred method for the visualizatio n of SCI (Falconer et al., 1994; Ohta et al., 1999; Bilgen et al., 2000). Clinically, MRI has been a useful tool for the study of secondary complications associated with SCI. For example, MRI can play an important role in the study of posttraumatic syringomyelia. The excitotoxic injury model has been shown to produce similar pathological changes to those observed in this condition (Yezierski et al., 1993; Schwartz et al., 1999a). Considering the lack of understanding of the pathological mechanism responsible for cavity formation in the injured cord (Madsen et al., 1994), it may be possible to gain insight in to the mechanism of cavitation using in vivo MR imaging at different time-points following excitotoxic SCI. In the past, clinicians have relied on patie nt descriptions of behavioral changes in determining clinical outcome (Williams, 1990, Oakes, 1996). However, recently MRI has been used to monitor changes associated with intraspinal transplantation, graftmediated functional recovery and changes in syrinx morphology af ter transplantation (Wirth et al., 2002), and to differentiate betw een cyst, scar, or tumor formation with the help of MR contrast agent administration (S chwartz et al., 1999a). With the importance and many uses for MRI, continued research is needed to enhance this technique and expand its uses to provide a better understa nding of the mechanisms responsible for the pathological changes associated with SCI. For example, an important question related to SCI is the temporal profile of pathological changes in the acute and chronic stages postinjury. Using in vivo MRI, it is possible to monitor these ch anges in the same animal.

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6 Dynamic Contrast-Enhanced Magnetic Resonance Imaging, Blood-Spinal CordBarrier In recent years, the integrity of the BS CB has been studied using MRI. Normally, the BSCB maintains a highly regulated homeostatic environment in the spinal cord as a result of tight junctions between neighbor ing capillary endothe lial cells surrounded by pericytes and astroglial foot processes (Purves et al., 2001) The BSCB functions to limit the penetration of a wide range of hydrophili c (water-soluble) molecules, proteins, and cellular elements into the spinal cord. This barrier is disrupted in spinal injury, making it permeable to toxic substances via vesicular tr ansport from intravascular to interstitial spaces (Popovich et al., 1996). Dynamic contrast-enhanced MRI is a method for studying this disruption. Breakdown of the ba rrier is detected with the use of an exogenous, water-soluble, paramagnetic contrast agent, gadolinium-d iethylene-triaminepentaacetic, bis-methylamine (Gd). Norma lly, Gd circulates throughout body tissues except in the central nervous system (CNS ) because of the highly regulated bloodnervous-tissue barrier. However, when the BSCB is disrupted, Gd can leak into the interstitial tissue; i.e., it no longer remains intravascular (Bilgen et al., 2001). These BSCB breaches can be observed with great sensitivity as an enhancement on T1weighted MR images. Long-term analysis can also reveal how long the BSCB remains permeable. Current studies have shown that the BS CB plays a vital role in activating and regulating secondary events following SCI, sp ecifically, the post-traumatic inflammatory response and edema formation (Hausmann, 2003); however, the exact mechanism of SCI-induced inflammatory response is not fully understood. Following injury, there is an ischemic environment at the injury epicente r as well as in adjacent areas of the white

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7 matter (Tator and Koyanagi, 1997). Edema deve lops and spreads, paralleling necrotic tissue at the epicenter (Tator and Fehlings, 1991). Changes fu rther associated with this disruption of microvasculature networks in clude hemorrhage, vasospasm, thrombosis, and impaired autoregulation, which in tu rn leads to further breakdown of the BSCB (Tator, 1991; Tator and Fehlings, 1991). Disr uption of the BSCB results in inflammatory invasion of neutrophils, macrophages, and T-cells (Bareyre and Schwab, 2003), which further contributes to secondary injury mechanisms (Lu et al., 2000; Hausmann, 2003; Kwon et al., 2004; Profyris et al., 2004). U nderstanding the time course of permeability of the BSCB and the subsequent pathologi cal consequences would be of significant benefit to understanding the secondary mechanisms associated with spinal cord injury. In Vitro Magnetic Resonance Imaging Three-dimensional magnetic resonan ce microscopy (3D MRM) and diffusion weighted imaging (DWI) are additional techniqu es that can be used to study SCI. These techniques allow for in vitro measurements, where motion artifacts are eliminated (Lemaire et al., 1990). This allows the signal -to-noise ratio (SNR) to be optimized. In addition, prolonged scan times and higher field strengths are not limiting factors (Guilfoyle et al., 2003). Both techniques allow for greater se nsitivity in accessing macroscopic changes in the injured cord, and with DWI, the integrity of the white and gray matter can be evaluated, which is limited or not capable with conventional in vivo methods of MRI. Diffusion imaging is also sensitive in evaluating spinal cord axonal integrity, degree of injury, and extent of ne uroprotection following injury (Ford et al., 1994a; Nevo et al., 2001), and can be used to detect cyst formation in gray matter following excitotoxic spinal cord injury, before conventional MRI is able to detect cavities (Schwartz et al., 1999b).

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8 Diffusion weighted MR imaging is deve loping into an important tool to characterize the organization of white matter in the brain and spinal cord (Beaulieu, 2002; Guilfoyle et al., 2003). Diffusion imaging is ba sed on intrinsic propert ies within gray and white matter, and relies on the phenomenon that water diffusion is highly anisotropic in tissues of the central nervous system. The overall tissue microstructure influences the mobility of diffusing water molecules, as th e tissue components (i.e., myelin sheath and axon cellular membranes) act as barriers and create indivi dual compartments. Diffusion characteristics are used to identify struct ural subtypes, where anisotropy is directly related to the geometry of th e fibers (Beaulieu, 2002). The direction of fibers may be inferred by the direction of greatest wate r diffusion, and by seeking out adjacent anisotropic voxels, a fiber can be mapped (Mel hem et al., 2002). Following spinal injury, there is a decrease in anisotr opy; i.e., there are less geom etric restrictions to water translational motion resulting in decreased pr eferential water diffusion in a particular direction. Having the capability to map white matter fibers allows for the determination of the effects of injury on selected sensory and motor fiber tracts in the white matter, which thus far is not apparent wi th other imaging techniques. Following spinal cord injury, apoptosis of oligodendrocytes occurs, which leads to chronic demyelination, ultimately causing anterograde neurodegeneration of ascending and descending white matter tracts (Li et al., 1996; Crowe et al., 1997; Liu et al., 1997b; Yong et al., 1998; Springer et al., 1999; Hausmann, 2003). This process of disintegration of the axon from its myelin sheath and breakdown of axons is known as Wallerian degeneration. Oligodendrocytes appear to be the predominant cellular target of glutamate in white matter tissue. Recent studies have shown oligodendrocytes to be particularly

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9 sensitive to excitotoxic insu lt (Yoshioka et al., 1996; Matu te et al., 1997; McDonald et al., 1998). Diffusion weighted imaging has the capability of detecting changes associated with this progressive condition (Becerra et al., 1995; Werring et al., 2000; Thomalla et al., 2004). Neuroprotective Strategies for Treatment of Spinal Cord Injury Overview An important consideration in the study of SCI is the fact that in the treatment of injury there is believed to be an optimal time for intervention. Secondary injury processes, including a number of cellular and biochemical cascades, are potential targets for neuroprotection (Amar and Levy, 1999; Du mont et al., 2001; Sekhon and Fehlings, 2001; Carlson and Gorden, 2002; Hausmann et al., 2002; Hulsebosch, 2002). Various neuroprotective strategies targ eting secondary injury have be en examined experimentally, including inhibitors of lipid peroxidation, nitric oxid e, calpain, apoptosis, caspase, glutamate receptor antagonist s, anti-inflammatory agen ts, and other compounds of uncertain mechanisms (Amar and Levy, 1999; Blight and Zimber, 2001; Hall, 2001). Strategies have been demonstrated to improve outcome after brain and spinal injury in pre-clinical studies, but are la rgely unsuccessful in clinical trials (Albers et al., 1995; Gaviria et al., 2000; Martinez -Vila and Sieira, 2001; Fade n, 2002; Hong and Liu, 2003; Wells et al., 2003). Reasons for the failure may be that previous research strategies 1) do not address the therapeutic window and effective dose requi red for treatment optimization; and 2) only target a single injury mechanism (Yam et al., 2000; Tekkok and Goldberg, 2001; Akins et al., 2002; Faden, 2002; Wells et al., 2003). Spinal cord injury is a complex condition in which multiple cell death (apoptosis and necrosis) and cell signaling pathways are in delicate balan ce with one another (Amar and Levy, 1999;

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10 Dumont et al., 2001; Carlson and Gorden, 2002) It is likely that a single drug with multiple targets or a combination drug treatment strategy that affects multiple components of the secondary injury cascade will exert superior ne uroprotective efficacy (Carlson and Gorden, 2002; Faden, 2002; Wells et al., 2003). Ther efore, understanding the temporal profile of different secondary in jury processes, as well as the impact of agents with known neuroprotectiv e effects, is critical in order to design a reasonable strategy of intervention. Neuroprotective Effects of Agmatine Previous studies have been carried out to examine the effects of putative neuroprotective agents on the spread of da mage following excitotoxic injury (Yezierski, 2002; Yu et al., 2003). One drug that has been shown to have therapeutic effects is the neurotransmitter/neuromodulator, agmatine. Agmatine [(NH2(CH2)4NH2C(NH=)HN] is a butyl chain with a guanidino group at one e nd and an amino group at the other, and is synthesized by the decarboxylati on of arginine. It meets th e criteria for an endogenous neurotransmitter/neuromodulator in that a) it is synthesized in the central nervous system (Li et al., 1994; Li et al., 1995; Fairbanks et al., 2000); b) it is stored in axon terminals in association with storage vesicles (Reis et al., 1998); c) it can be released by depolarization (Reis and Regunathan, 1998); d) it is inactivated by reuptake and/or degraded enzymatically by agmatinase (Sastre et al., 1996; Sastre et al., 1997); and e) it can bind to (Anis et al., 1990) and modulat e (Yang and Reis, 1999) the actions of the NMDA subclass of glutamate receptors. Because of the polar character of agmatine, questions have come about as to the extent to which it crosses the blood-central nervous system-barrier. Evidence has indicated that agmatine administered periphe rally does readily cross the barrier in a

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11 concentration-dependant manner and has dire ct effects on the central nervous system (Piletz et al., 2003). In addition, there ha ve been numerous studies (for review, see Nguyen et al., 2003) conducted in which system ically administered agmatine has affected phenomena mediated by central processing. However, there is no data suggesting a mechanism for such transport into nervous tissue, although an agmatinergic transporter has been suggested (Nguyen et al., 2003). Pharmacologically, treatment with exogenous agmatine is non-toxic (Gilad et al., 1996; Fairbanks et al., 2000), blocks NM DA receptors (Olmos et al., 1999; Yang and Reis, 1999), inhibits all isoforms of nitric oxide synthase (A uguet et al., 1995; Galea et al., 1996), reduces glutamate excitotoxicity (F airbanks et al., 2000; Zhu et al., 2003), and exerts significant neuroprotectiv e effects in models of ischem ic brain injury (Gilad et al., 1996; Gilad et al., 2000), and traumatic and ex citotoxic spinal injury (Fairbanks et al., 2000; Yu et al., 2000). Recently, it was show n that systemic treatment with agmatine significantly reduces neuronal loss (Fairbanks et al., 2000; Yu et al., 2000; Yu et al., 2003), improves locomotor function (Yu et al., 2000), and affects multiple signaling pathways following SCI. For example, molecular changes brought about by agmatine include inhibiting NF-kB activity, up-regulat ing AKT activity, and down-regulating painand injury-related gene expres sion following excitotoxic and traumatic spinal cord injury. These data suggest that agmatine may serve as a multifaceted agent that targets many of the secondary processes involve d in the mediation of cell death and the progression of secondary injury in SCI. The multiple site s of action of this endogenous molecule make agmatine a promising therapeutic agent for trea tment of spinal cord injury and secondary

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12 complications, as well as a useful pharm acological agent to study the impact of neuroprotective interventions with known therapeutic targets.

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13 CHAPTER 2 METHODOLOGICAL DEVELOPMENT Overview During the course of experiments descri bed in Chapters 3-5, new methodologies were learned and developed. With any new pr otocol, challenges arise that need to be addressed before the method is successful. Be low is a description of these learned and newly developed procedures, as well as associ ated complications and solutions to address each issue. Anesthetic/Experimental Drugs With each experiment, there is a signifi cant amount of preparation involved, from animal care, anesthetics, treatment, experime ntation, to a successful recovery. With anesthetics, it is crucial to administer proper doses, as well as prepare correct concentrations of the anesthetic mixture. It is equally important that each component of the cocktail be high in quality and freshly mi xed. When any one of these components is overlooked, problems can surface. An example of a problem that occurred with anesthetics was encountered during post-surgery survival with the last group of anim al surgeries. At first, the anesthetic used in Chapter 3 [mixture of ketamine (100 mg /mL, 3.0 mL), acepromazine (10 mg/mL, 1.0 mL), and xylazine (20 mg/mL, 3.0 mL; mixture at 0.65 mL/kg, subcutaneously)] was believed to be the cause of th e problem. It was believed that during times of infrequent surgeries, the cocktail mixture could have deteriorated, so fresh mixtures were made. However, animals were still not surviving, regardless of post-operative care. After

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14 talking with Animal Care Services and othe r labs using similar anesthetics on rats, a different cocktail recipe was developed [mix ture of ketamine (100 mg/mL, 0.5 mL) and xylazine (100 mg/mL, 0.1 mL)] in hopes that rats would have a more successful recovery following surgery, and that the anesthetic woul d act more effectively, i.e., animals would remain anesthetized for the duration of surg ery, with no supplements required. Tests were done with two doses of this new recipe (mixture at 0.75 mL/kg and 1.0 mL/kg) and through two different routes of administration (i ntraperitoneally versus subcutaneously). Based on results, it was decided that 1.0 m l/kg administered intraperitoneally was the most effective dose and route of administrati on. Intraspinal injection surgeries went on as scheduled, using the new cocktail recipe; howev er, animal survival was still an issue. Next, we tested the experimental drug, agmatine, to determine if it was the cause of failed recovery. At first, it was suspected th at the higher experimental dose (100 mg/kg) was too high, as there have been previous reports that higher doses of agmatine caused hypothermia (Yezierski, unpublished observation s). So, the dose was decreased to 80 mg/kg, however, this did not remedy the proble m. To further test the agmatine, a fresh supply was made; one rat received a dose of th e old supply and one ra t received a dose of the fresh supply. The rat that received the old agmatine supply, died several hours later, while the one that received th e fresh supply survived. Ther efore, on the next scheduled surgeries, rats received the new anesthetic cocktail and a dose of the fresh Agmatine supply at the lower dose (80 mg/kg). However, after a third attempt, animals were still not surviving; they seemed to die due to re spiratory distress. Once again, Animal Care Services was contacted and made aware of the problem in hopes that they might shed light on this ongoing problem. At this time, we discovered that the higher concentration

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15 of xylazine (100 mg/mL vs 20 mg/mL) was know n to cause pulmonary edema in rodents. Therefore, it was thought that the cocktail had never been the problem in the first place, and that the agmatine had been the main cause of animal survival problems. So, we switched back to the original cocktail recipe and used the fresh supply of agmatine (80 mg/kg), and finally the last group of animal surgeries were performed successfully, with no further survival issues. During the time the above problems were being addressed, a total of 16 animals were lost following preliminary MRIs and subsequent intraspinal injection of QUIS. Magnetic Resonance Imaging Catheter Placement A tail vein catheter was needed to administer contrast agent used in the dynamic contrast-enhanced MR imaging. Placing a tail vein catheter requires patience and a great deal of practice to perfect. Once the animal was anesthe tized with a mixture of oxygen and isofluorane, as described in Chapter 3, th e catheter was filled with a heparin/sterile saline solution (also described in Chapter 3), and inserted into the tail vein. Correct placement was confirmed with blood flow into the catheter, at whic h point, 2-3 cc of the heparin/sterile saline solution was injected to prevent further clotti ng. The catheter was then taped in place. 4.7T Coil Development Initial experiments were carried out on the 4.7T magnet system (please see Chapter 3). A highly-sensitive quadrature MR surface coil was constructed for this system, by Danny Colvin, specifically for imaging the ra t spinal cord. The coil consisted of two rectangular coil elements (3 x 3 cm) placed ne xt to each other on the inside of a halfcylinder cradle 4 cm in diameter. Each co il was constructed using copper tape, 5 mm in

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16 width, with a 3.3 pF ceramic-chip capaci tor (American Technical Ceramics (ATC), model 3R3C) inserted in series with the copper tape in the middle of each arc at the ends of each rectangle to provide distributed capacitance around the coil. The rectangular coils were overlapped along the inner edges to cancel their mutual inductance. The overlapping coils formed an arc of 120 degr ees with an overall arc length of 5.5 cm. Each coil was individually tuned and matche d with a combination of fixed series capacitors of 1 pF (ATC, model 1R0C) and va riable series and parallel capacitors (Johanson Manufacturing Corp, Boonton, NJ, m odel 5641) arranged on a custom circuit board. The tune and match circuit was in terfaced to the transmitter-receiver with an input-voltage balancing network on the same circuit board and a 50 ohm coaxial cable containing a parallel-resonance trap on the coax ial shield to eliminate surface currents. The trap was created by forming a loop in th e cable and attaching a 4.7 pF ceramic-chip capacitor (ATC, model 4R7C) to the shield of the cable across the loop. 11.1T Coil Development Following the first study, as described in Chapter 3, it was decided to move all MR imaging to the 11.1T magnet system for the remainder of the in vivo experiments (see Chapter 4). This would allow for greater signal-to-noise, resulting in a much higher resolution image, showing greater detail of in jury pathology. Two MR coils were used in the measurements of MR images. A circul arly polarized, transmit-only birdcage coil was used for excitation of resonance a nd was provide by the Advanced Magnetic Resonance Imaging and Spectroscopy Facility of the McKnight Brain Institute (see Coil Setup for description). Then, the resonance signal was measured with a smaller receiveonly, circular-polarized coil placed very near the spine. This coil was constructed by Christy Amwake. The receive-only coil was passively decoupled from the transmit coil

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17 during excitation and the transmit-only coil was actively decoupled from the receive-only coil during signal measurement. The receive-only circularly polari zed MR surface coil was constructed specifically for imaging the rat spinal cord at 470 MHz. The coil consisted of two rectangular inductive elements (3 x 3 cm) placed next to each other (with a common side) on the outside of a half-cylinder cradle 4.7 cm in outside-diameter and 77 cm long. Each rectangular section was constructed using c opper tape (5 mm in width), with the outer inductor-corner copper tape angl ed at 45 degrees to the side s. Four 4.7 pF non-magnetic ceramic-chip capacitors (American Technica l Ceramics (ATC), model ATC100B-4R7FWN-1000XC) were inserted in series with the copper tape around the outside of these inductors. These capacitors were inserted in the middle of each arc (at the ends of each rectangular section, opposite th e input circuit), and in the middle of the outer side of each rectangular section, in order to provide distributed capacitance around the coil. The common copper-tape element, joining the two i nductors, was constructed with a variable non-magnetic capacitor (1-16 pF, Voltronics, Denville, NJ) and inserted in series in the middle of the element. This variable capac itor was used to cancel the mutual inductance between the two inductive sections. The coil formed by the two rectangular sections had an arc of 170 degrees with an overall arc length of 8 cm. Each inductor was passively decoupled from the MR transmitter coil duri ng excitation of the signal. This was accomplished by adding a hand-wound inducto r and double-headed non-magnetic diodes (M/A-COM, Lowell, MA, model MA45471) in para llel with 4.7 pF capacitor located in the middle of each inductor arc. The hand-wound inductor was a 2 mm inner diameter solenoid of 2.5 turns made from 20 AWG ma gnet wire. Each MR coil inductor was

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18 individually tuned and matched with two va riable series and on e parallel non-magnetic capacitor (1-16 pF, Voltronics, Denville, NJ) ar ranged on a custom circuit board. Each tune and match circuit was interfaced to th e receiver with a 50 ohm coaxial cable containing a parallel-resonance trap on the coax ial shield to eliminate surface currents. The trap was created by forming a loop in th e co-axial cable, approximately 2 cm in diameter, and attaching two parallel 4.3 pF non-magnetic ceramic-chip capacitor (ATC, model ATC100B-4R3B-WN-1000XC) to the shield of the cable acro ss the loop. This configuration of coil outputs pr ovided two points along one side of the coil arc, separated by approximately 90 degree, for interface to the receiver to provide detection of the circular polarized signal. This was accomp lished by combining the two detected signals, within a 90-degree splitter-combiner (Ana ren Corp, East Syracuse, NY, model 10013-3), to provide a single input to the receiver. Coil Setup Separate transmit and receive coils were used to increase the SNR and achieve greater depth penetration. A volume coil used for transmission allowed for the development of 90 degree tip angles over a large region of the sample, eliminating the fast changing tip angle variation with depths that occurs with surface coil excitation. On the other hand, the surface co il provides greater sensitivity than the volume coil because of its smaller size. In setting up the coils, it is importa nt to avoid mutual coupling between the transmit-only volume coil and the receive-only surface coil or signal loss and mismatch will occur. Specifically, it is critical that the surface coil be decoupled during transmission and the birdcage be decoupled during reception. The transmit-only birdcage on the 11.1T is an eight leg, shielded, low-pass birdcage of 8.8 cm diameter and length.

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19 It is turned off during reception by switching high impedance circuits into every leg of the coil. This effectively blocks current fr om flowing in the coil. The high impedance circuit is a series combination of an inducto r and diode placed in parallel with a capacitor on the leg. The inductor and capacitor have equal reactance and creates high impedance when the diode is forward biased (parallel re sonance). Conversely, during transmission, the diode is reverse biased removing the hi gh impedance circuit from the leg and the birdcage is fully functional. The diode bi asing is controlled by a pulsed signal from the spectrometer. 11.1T Revised Imaging Protocol After switching to the 11.1T magnet, it was evident that the increase in field strength resulted in an increased sensitivity to breathing artifacts. For this reason, the imaging protocol originally used on the 4.7T magnet had to be modified accordingly for use on the 11.1T magnet system. The modifica tions made for the 11.1T imaging protocol are described in detail in Chapter 4. Most notably, the read direction was changed to head-foot (sagittal MR images) or left-righ t orientation (transverse MR images) to eliminate aliasing, and a slice saturation pulse was added to the sequence of high resolution images to eliminate breathing artifacts. 4.7T versus 11.1T Images One of the most notable advantages in moving from the 4.7T magnet to the 11.1T magnet was the increase in SNR (see Fig. 2-1) and the lack of aliasing due to changes made in the protocol used for the 11.1T. The images collected on the 11.1T magnet had a much higher resolution and showed greater de tail of injury pathology after excitotoxic spinal cord injury. In addition, there was an increase in gray-white matter contrast in T1weighted transverse images collected on the 11.1T magnet.

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20 A B C D Figure 2-1. Comparison of MR images collected on the 4.7T magnet versus the 11.1T magnet. T1-weighted sagittal (A and C) and transverse (B and D) MR images collected in vivo pre-injury (Day 0) on the 4.7T magnet (A, B) or the 11.1T magnet (C, D). Magnetic Resonance Imaging Obstacles Heating Effects of the 11.1T Imaging Protocol After moving to the 11.1T and optimizi ng the imaging protocol, there was a problem in animal survival during the MRIs. At first, it was thought that the animals were becoming too deeply anesthetized and an anesthetic scavenger line was needed in the magnet to remove excess anesthesia. In addition, the cradle apparatus was lowered to

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21 ensure the animals had ample room to breath. A deltaphase isothermal pad (Braintree Scientific, Inc., Braintree, MA) was al so placed on the animal during the MRI acquisition. In thinking that the proble m had been solved, experiments proceeded, however, the issue of animal survival pers isted, and experiments were postponed until the problem could be solved. At this point, it was thought that temperature might be an issue, as animals were coming out of the magnet following imaging sessions much warmer than expected. Temperature measurem ents were performed on both the 4.7T and 11.1T magnets using a tissue equivalent phantom simulating a rat abdomen (64.1% water, 32.0% sucrose, 1.6% salt, 2.3% TX -151; Beck et al., 2004), and compared. The data revealed that the new sequences that we re devised for the 11.1T magnet (described in Chapter 4), which were identical to the 4.7T (described in Chapter 3) except they included slice saturation pulses to eliminate breathing artifacts (the 11.1T is much more sensitive to breathing), were causing extensiv e heating of the coils and ultimately over heating the animals by several degrees (see Figs 2-2A, #5; 2-2B, # 1-6). The transverse images versus the sagittal images were causi ng a significant rise in temperature, which was due to the increase in duty cycle, not magnet frequency. A temperature and respiration monitoring system was then acqui red for the 11.1T, which allowed for close monitoring of respiration and temperature, as well as a f eedback loop for consistent temperature control. Transverse images in the dynamic contrast-e nhanced MRI protocol were also eliminated from the sequence. The final imaging protocol for the 11.1T magnet included the first five pulse sequen ces shown in Figure 2-2B (described in Chapter 4). During this time, a total of 8 animals were lost due to overheating.

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22 21 22 23 24 25 26 0:001:122:243:364:486:00 Time (hr:min)Temperature (C) 7 12 11 10 9 3 8 6 5 2 4Imaging Protocol with Slice Saturation Pulse 1 T1_trans 2 T1_sag 3 T2_trans 4 T2_sag 5 DCE trans/sag interleaved 6 DCE sag only 7 Cool Down Imaging Protocol, No Slice Saturation Pulse 8 T1_trans 9 T1_sag 10 T2_trans 11 T2_sag 12 DCE trans/sag interleavedA 21 22 23 24 25 26 0:001:122:243:364:486:00 Time (hr:min)Temperature (C) 7 8 6 5 4 3 2 1 Imaging Protocol with Slice Saturation Pulse 1 T1_trans 2 T1_sag 3 T2_trans 4 T2_sag 5 DCE sag only 6 DCE trans/sag interleaved 7 Cool Down 8 DCE trans/sag interleaved, Gradients turned offB Figure 2-2. Temperature measurements collec ted on the 4.7T and 11.1T magnets using a tissue equivalent phantom simulating ra t abdomen. The measurements were taken using (A) the protocol original ly devised on the 4.7T (no slice saturation); and (B) the new protocol devised for the 11.1T (including slice saturation). The new protocol (included slice saturation) resulted in a heating of the phantom on both magnet systems, which did not occur using the old protocol (no slice saturati on), thus confirming that the heating was a result of the increase in duty cycle, and not the increase in frequency.

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23 Signal-to-Noise Ratio The setup used for all in vivo imaging protocols is quite extensive and includes many components where problems can arise, including two coils, cables, and an abundance of hardware and software. One of the biggest issues, in addition to animal survival, was SNR problems. On numerous occasions, an imaging session would begin with problem solving due to low or lack of signal. When this occurred, the coils were inspected, all cables were checked to make sure they were not loose or faulty, correct cable connections were confirmed, and the d ecoupling box was tested to make sure it was functioning. Throughout the course of this project, several cables had gone bad, a fuse was replaced in the decoupling box, diodes were replaced in the volume coil, and capacitors were re-soldered on the surface co il. Occasionally, the problem was more severe and involved magnet hardware, such as the RF amplifiers or gradient power supplies, in which case new parts had to be ordered and as a result, the magnet became inoperable. As a result of poor signal, due to any combination of the above problems, the images had a low SNR when compared to imag es collected on days of optimal signal (see Fig. 2-3).

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24 A B Figure 2-3. Comparison of images collected with high versus low SNR on the 11.1T magnet. (A) T1-weighted (TR/TE = 1000 ms/15 ms, NA = 4) transverse MR image with high SNR. (B) T1-weighted (TR/TE = 1000 ms/15 ms, NA = 4) transverse MR image with low SNR.

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25 CHAPTER 3 EVALUATION OF THE PATHOLOGICAL C HARACTERISTICS OF SPINAL CORD INJURY WITH MAGNETIC RESONANCE IMAGING* Introduction The intraspinal injection of the alpha -amino-3-hydroxl-5-methyl-4-isoxazolepropionate/metabotrophic receptor agonist, qu isqualic acid, produces an excitotoxic spinal cord injury SCI, which has been us ed to study the molecular, pathophysiologic, and behavioral characteristics of SCI (Yezi erski, 2002). These injections result in pathologic changes similar to those associated with ischemic and traumatic SCI (Yezierski et al., 1993; Yezier ski et al., 1998). Although most of what is known about the pathologic changes associated with e xperimental SCI (ie, edema, neuronal loss, hemorrhage, and cavitation) has come fr om histologic studies, noninvasive highresolution MR imaging is becoming a prefer red method for the visualization of SCI (Falconer et al, 1994; Ohta et al., 1999; Bilgen et al., 2000). In recent years, MR imaging has become a valuable diagnostic tool in studies examining the pathologic changes following SCI (Flanders et al., 1990; Yamash ita et al., 1990; Yamashita et al., 1991; Ohshio et al., 1993; Flanders et al., 1999; Se lden et al., 1999; Rattli ff and Voorhies, 2000; Ishida and Tominaga, 2002). One well-documen ted change associated with SCI is the breakdown of the bloodspinal co rd barrier. The pa thologic characteri stics associated with BSCB disruption following SCI can be measured quantitatively with dynamic Reprinted with permission from Berens SA, Colvin DC, Yu CG, Yezierski RP, Mareci TH. Evaluation of the Pathological Characteristics of Spinal Cord Injury with MR Imaging. AJNR Am J Neuroradiol 2005; 26(7):1612-1622. by American Society of Neuroradiology

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26 contrast-enhanced MR imaging, by using exogenous paramagnetic contrast agents (Runge et al., 1997; Bilgen et al., 2001; Bilgen and Narayana, 2001; Bilgen et al, 2002). Fine details of SCI pathology can also be de termined with intact excised cords by using MR microscopy. With this te chnique, motion artifacts are eliminated, allowing the signal intensityto-noise ratio to be optimized with prolonged scan times and higher field strengths (Guilfoyle et al., 2003). Thus far, the pathologic characteristics determined with in vivo MR imaging have not been compared with in vitro MR imaging and histologic examination by using the excitotoxic injury model. We hypothesize that in vivo MR imaging has the sensitivity to detect significant differences in pathol ogic characteristics between two different strategies of injury, which can be correlated with in vitro MR imaging and histology. A preliminary description of this study has been reported (Berens et al., 2003). Materials and Methods All experiments were carried out in accord ance with National Institutes of Health guidelines for the care and use of animal s and approved by the University of Florida Institutional Animal Care and Use Committee. In vivo and in vitro MR methods, along with histologic techniques, were used, because these were the most direct measures of pathologic changes following injury. A comp arison of findings with different methods was carried out. Animal Preparation The technique of intraspinal injection was similar to that described elsewhere (Yezierski et al., 1998). In brief, Spra gue-Dawley male rats weighing 250 g were anesthetized with a mixture of ketamine (3 .0 mL), acepromazine (1.0 mL), and xylazine (3.0 mL; mixture at 0.65 mL/kg, subcutaneous ly). Supplemental doses of anesthetic were

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27 given, if necessary, when rats responded to a noxious pinch applied to the glabrous skin of an extended hind paw. Animals were placed in a stereotaxic frame and the vertebral column immobilized with a vertebral clam p. One injection window for intraspinal injections was made by laminectomy between spinal segments T12L2. Once the cord was exposed, the dura was incised longit udinally and reflec ted bilaterally. A small hole for unilateral injections was made in the pia mater. After injection, muscles were closed in layers and the sk in was closed with wound clips. The day following the final in vivo image acquisition, animals were anesthetized with sodium pentobarbital and euthanized by transcardial perfusion with normal saline followed by 10% formalin. Spinal cords were re moved and stored in 10% formalin. Intraspinal Injection Procedure Before injection, QUIS, 125 mmol/L, was mixed in sterile saline, corrected to physiologic pH, and was then injected unilatera lly at one level of the cord (T12L2). To compare injury pathology between lesions at different depths, two types of injection strategies were used: injections at a depth of 500 m ( QUIS lesion 1 [QL1], two animals) and at a depth of 1000 m (QUIS lesion 2 [QL2], five animals) below the surface (Yezierski, 2001). Injections were made be tween the dorsal root entry zone and the dorsal vein on the right or left side, dependi ng on vascularity on the surface of the cord. The total volume of QUIS injected was 1.2 L in three tracks (0 .4 L/track; distance between tracks 0.5 mm in the rostrocaudal di rection). A Hamilton microliter syringe with a glass micropipette ex tension (tip diameter, 5 m) was used for intraspinal injections. The syringe was positioned in a microinjector unit (Kopf 5000; Kopf, Tujunga, CA) attached to a micromanipulator A sham injury group was prepared by injecting saline in the same manner as described above.

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28 In Vivo MR Imaging On the basis of previous correlations between the extent of pathology and the temporal onset of injury-i nduced behavioral changes, in vivo MR imaging was performed 17 days (mean, 22 days) after injury (Gorma n et al., 2001; Yu et al., 2003). Animals were initially anesthetized by inhalation of a mixture of 5% isofluorane with oxygen at a flow rate of 2 L/min and maintained via a nose cone with a mixtur e of 2% isofluorane with oxygen at a flow rate of 1 L/min. To determine whether there was disruption of the BSCB following injury, DCE-MR imaging was pe rformed with the in travenous injection of gadolinium-diethylene-tr iamine-pentaacetic, bis-methylamine ([Gd] Omniscan, Nycomed, Oslo). For intravenous delivery of Gd, the tail vein was cannulated with a 24G, .75-inch intravenous catheter (Terumo Surflo, Webster Veterinary Supply, Inc., Sterling, MA). Before catheterization, the catheter was filled with heparin solution (mixture of 6 mL of saline and 0.2 mL of 1000 U/mL) to prevent clotting. Once the catheter was in place, a syringe filled w ith the appropriate vo lume (0.6 mL/kg body weight) of Gd (0.3 mmol/kg) was attached to the catheter and stabilized with surgical tape. A pulse oximeter was used on the tail to monitor heart rate and blood oxygenation saturation. Core body temperature was mon itored during experiments by using a rectal probe and was kept between 35C and 37C. Re spiration rate was also monitored during data acquisition. In Vivo High Resolution MR Imaging Data Acquisition All in vivo NMR measurements were performed on a 4.7T, 33-cm-bore Oxford magnet (Oxford Instruments, Oxford, UK) at 200 MHz by using a Bruker BIOSPEC Avance DBX console (Bruker NMR Instrument s, Billeria, MA) and 100 mT/m actively shielded gradients. A highly sensitive quadrature MR surface coil was constructed

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29 specifically for imaging the ra t spinal cord. The coil co nsisted of two rectangular overlapping coil elements (3 x 3 cm), cons tructed by using copper tape, placed on the inside of a half-cylinder cradle 4 cm in diameter. All data acquisition was performed with Bruker ParaVision software. General image processing and analysis of the time series DCE data were performed by using custom software written in the Interactive Data Language (Research Systems, Boulder, CO). Rats were placed supine in a custom-built half cylinder cradle/coil apparatus. The 200-MHz quadrature MR surface coil was tuned with a Hewlett Packard HP8752C network analyzer (Hewlett Packard, Englew ood, CO). The position of the animal was confirmed with pilot images to locate the injury site. Pilot images were T1 weighted (TR/TE = 500 ms/5 ms; NA = 1), with a field of view (FOV) of 6 cm in each direction, a 1 mm section, and a 128 x 128 matrix. Fat suppression was done by using a CHESS-type sequence (Haase et al., 1985). Once the positi on was confirmed, a se t of fat-suppressed, high-resolution sagittal and tr ansverse images was collected. Sagittal T1-weighted (TR/ TE = 1000 ms/10.5 ms; NA = 4) spin-echo images were acquired over five sections, each 1 mm thick without a gap between sections. A TR of 1000 ms was chosen to provide optimum signal intensity strength with T1-weighting for anatomic definition. Additional acquisition parameters were a FOV of 4 cm x 2 cm, read out in the head-to-foot direction, and a matrix of 256 x 128. The scan time with these parameters was 8.67 minutes. Transverse T1-weighted (TR/TE = 1000 ms/10. 5 ms; NA = 4) spin-echo images were acquired over 14 sections, each 1 mm thick without a gap between sections. Other acquisition parameters were a FOV of 2 cm x 2 cm, read out in the anteroposterior direction, and a matrix of 128 x 128. The scan time was 8.67 minutes.

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30 Sagittal T2-weighted (TR/effective TE = 2000 ms/62.5 ms; NA = 6) fast spin-echo images with phase-encode segmentation were acquired over five sections, each 1 mm thick without a gap between sections. Add itional acquisition parame ters were a FOV of 4 cm x 2 cm, read out in the head-to-foot di rection, phase-encode se gmentation factor of 4, and a matrix of 256 x 128. The scan time with these parameters was 6.5 minutes. Transverse T2-weighted (TR/effective TE = 2000 ms/62.5 ms, NA = 6) fast spin-echo images with phase-encode segmentation were acquired over 14 sections, each 1 mm thick without a gap between sections. Other acqui sition parameters were a FOV of 2 cm x 2 cm, read out in the anteroposterior direction, phase-encode segmentation factor of 4, and a matrix of 128 x 128. The scan time w ith these parameters was 6.5 minutes. In Vivo DCE-MR Imaging Data Acquisition After four sets of high-resolution imag es were collected, a series of dynamic contrast-enhanced images of the rat spinal cord was acquired. Tr ansverse and sagittal images were acquired before and after the contrast agent was injected. Transverse images were acquired with a FOV of 2 cm x 2 cm and a matrix of 64 x 64, over eight sections, and sagittal images with a FOV of 4 cm x 2 cm and a matrix of 128 x 64, over three sections, both 1 mm thick without a gap between sections. Th ree sets (transverse and sagittal interleaved) of T1-weighted pr e-contrast multi-section images (TR/TE = 330 ms/9.4 ms; NA = 8) were collected. After ac quisition of the pre-cont rast images, Gd was injected in approximately 5 seconds with continuous pressure, to prevent backflow, through the implanted catheter while the anim al was in the magnet. Immediately after injection, 810 sets (6 minutes for each set) of T1-weighted post-contrast multi-section images (TR/TE = 330 ms/9.4 ms; NA = 8) were collected, with tran sverse and sagittal data-set acquisitions interleaved. We collected these images over a 60-minute time

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31 frame, which was chosen on the basis of previous experience, with a peak enhancement for the cord at 3 minutes. The receiver gain was adjusted to one-third the value used for the high-resolution images because Gd provide s approximately three times the contrast enhancement. In Vitro MR Microscopy All in vitro NMR measurements were perfor med on a 14.1T, 51-mm-bore Oxford magnet, at 600 MHz, by using a Bruker BIOSPEC Avance DBX console. All data acquisition was performed with Bruker ParaVi sion software. Before imaging, spinal cords were placed in phosphate-buffered saline (PBS) overnight. The cords were placed in 5-mm tubes and imaged in PBS by using a standard 5-mm MR coil. Three sets of high-resolution multislice two-dimensional images oriented in three orthogonal directions were collected (TR/TE = 2500 ms/20 ms; NA = 8) The transverse images were acquired over 60 sections (200 m thick without a gap between sections; FOV = 5 mm x 5 mm; matrix = 256 x 256), the sagittal images we re acquired over 20 sect ions (200 m thick without a gap between sections; FOV = 20 mm x 5 mm; matrix = 1024 x 256), and the coronal images were acquired over 20 sect ions (200 m thick without a gap between sections; FOV = 20 mm x 5 mm; matrix = 1024 x 256). Immediately after the final imaging session, spinal cords were placed in fixative. General image processing and analysis was performed by using custom so ftware written in the Interactive Data Language (IDL, from Research Systems, Boulder, CO). Histological Procedures Once imaging was completed, spinal cords were placed in 10% sucrose overnight. Serial sections (75 m) were cut on a freezing microtome, collected in PBS, and mounted on gelatin-coated slides. Sections were stai ned with cresyl violet and cover-slipped. To

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32 confirm that the hypointense signals from MR images correlated with hemorrhage, sections sampled from the injury site we re stained with the Prussian-blue reaction (Bancroft and Cook, 1984). Data Analysis To compare results obtained with the three different methods used, total longitudinal injury lengths were determined. Statistical analysis of these data included a repeated-measures ANOVA test to determine differences in distances determined by the three methods (SPSS, Chicago, IL). This was followed by a paired comparison to interpret the significance of the ANOVA. To determine the total longitudinal injury length, the total number of transverse s ections from each method, with evidence of excitotoxic (ie, cavity formation, hemorrhag e, and neuronal loss) damage (Yezierski et al., 1998; Gorman et al., 2001; Plunkett et al ., 2001), were counted and multiplied by the section thickness appropriate for the method used. Transverse images were used for injury length calculation instead of sagittal images, because the boundaries of the injury (eg, neuronal loss in the dorsa l horna classical characterist ic of excitotoxic SCI) were best observed in transverse images. For the calculation of injury length by using in vivo MR imaging, T2-weighted transverse images were counted and multiplied by 1 mm; for in vitro MR imaging, transverse images were counted and multiplied by 200 m; and for histology, sections were c ounted and multiplied by 75 m (Gorman et al., 2001). Histologic determination of injury length wa s done via microscopic examination with 4x and 10x objectives. In addition to injury lengt h, pathologic characteristics (ie, cavity formation, central canal expansion, hemorrhag e, and neuronal loss) of the injury were compared between the two types of injec tion strategies used (ie, QL1 vs QL2).

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33 Injury lengths were also compared between QL1 and QL2. Statistical analysis of these data included a one-way between subjects ANOVA test to determine differences in injury severity between the four injury/treat ment groups (SPSS, Chicago, IL). This was followed by a Tukey HSD post hoc test to interpret the significance of the ANOVA. For DCE-MR imaging processing, regions of interest (region of interest) were selected in the injured and normal cord, muscle, and bone. A 1 mm x 2 mm elliptical region of interest was drawn in regions of injury and normal cord because the enhanced region was typically elliptical. A 1 mm circle region of interest was drawn for muscle and bone and was analyzed to determine the success of the Gd injection. Ten percent peak enhancement was used as the cutoff value because regions of interest of normal cord showed 10% peak enhancement because of the presence of contrast agent in the cord vasculature. Results In Vivo High Resolution MR Imaging Figure 3-1 shows a comparison of the pat hologic findings from the same animal (S05) at the same location for all three methods used in this study. The appearance of images was compared with normal controls. When an image region relative to a control region appeared darker, we classified it as hypo intense. When an image region relative to a control region appeared bright er, we classified it as hyperintense. No signal intensity changes were observed in the normal cord on either T1-weighted or T2-weighted images (data not shown). The only significant inform ation obtained from T1-weighted images of QUIS-injured animals was the injection tracks, which were seen as weak hypointense (ie, dark) signals.

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34 Figures 3-2A and 3-3A show the sa gittal and transverse T2-weighted in vivo images, respectively, for each injured animal. Using in vivo transverse images, it was possible to determine the anatomic level of the sections by examining the size and shape of the cord as well as gray and white matter. Injection tracts for QUIS injections were visible in both T2-weighted (Figs 3-1 and 33) and T1-weighted transverse images (not shown) in all animals. Hypointense signals can be seen within the cord on T2-weighted images of all QUIS-injured anim als; however only T2-weighted in vivo images of QL2 animals (S01, S02, and S04S06) showed hyperintense (ie, bright) signals. Figure 3-1. Representative pa thologic findings in the same animal (S05) at the same location observed in vivo (T2-weighted, TR/effective TE = 2000 ms/62.5 ms; NA = 6), in vitro (TR/TE = 2500 ms/20 ms; NA = 8), and in histologic sections. Many of the same pathologi c details are visible with all three methods albeit with different levels of resolution. In vivo and in vitro images are displayed with image si ze = 0.92 cm x 0.77 cm ( in vivo ) and 0.50 cm x 0.50 cm ( in vitro ). Letters label the most distinctive anatomic and pathologic characteristics observed in the three imag es as follows: A indicates expanded central canal; B, injection track; C, dorsa l root ganglion; D, dorsal root; E, hemorrhage at injury site; F, white matter; G, gray matter; H, ventral root; I, cavity; J, anterior spinal vessel. In vivo images show cavitation as hyperintense signals due to T2 weighting, whereas, on in vitro images, cavities appear isointense in thes e proton attenuation-weighted images. In vivo images showed consistent differen ces between QL1 and QL2 animals (Figs 3-4 to 3-6). It is important to note that hyperintense signals, indi cating expansion of the

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35 central canal and cavitation, were not seen in images from QL1 animals (Fig 3-3A, X01 and X03; Fig 3-6). Although the only differen ce in QUIS injection parameters was depth of injection, there were dramatic differences in the extent of pathologic damage (e.g., laterality, cavitation, and expansion of the central canal). There were also significant differences (p < 0.001, one-way between subjects ANOVA, F = 34.134, p < 0.001) in injury lengths between QL1 (X01 and X03) and QL2 animals (S01, S02, and S04S06; Fig 3-4). Figure 3-2. Sagittal images for all QUIS-injured animals. (A) In vivo (T2-weighted, TR/effective TE = 2000 ms/62.5 ms; NA = 6); and (B) in vitro (TR/TE = 2500 ms/20 ms; NA = 8). Images are oriented with rostral at the top and dorsal on the right. Arrows represent the location of the epicenter of the injury. The epicenter was defined as the regi on of maximal pathologic damage. Displayed image size = 0.77 cm x 2.15 cm ( in vivo ) and 0.40 cm x 1.50 cm ( in vitro ). Animals designated S01, S02, S04, S05, and S06 received QL2, whereas animals designated X01 and X03 received QL1. The vertebral column and laminectomy site were easily observed in both in vivo and in vitro sagittal images.

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36 Figure 3-3. Transverse images for all QUIS-injured animals. (A) In vivo (T2-weighted, TR/effective TE = 2000 ms/62.5 ms; NA = 6); and, (B) in vitro (TR/TE = 2500 ms/20 ms; NA = 8). Images are sampled from the epicenter of the injury (arrows in Fig 3-2) and show comp arable pathologic changes between methods. See Fig 3-1 for labeling of anatomic and pathologic findings. Images are oriented with dorsal at the top. L, left side of cord; R, right side of cord, for all images. Displayed image size = 0.92 cm x 0.77 cm ( in vivo ) and 0.50 cm x 0.50 cm ( in vitro ). A, Hypointense signals correlated with hemorrhage (see Histologic Findings) and hyperintense signals correlated with the presence of fluid filled cavities. For example, notice the hyperintense expanded central canal in the cord of animals S01, S02, and S04S06 and the hyperintense cavity in the gray matter on th e right side of th e cord in animal S02. B, Hypointense regions were obse rved bilaterally in the gray matter in all QL2 animals (S01, S02, and S04S06), whereas, in QL1 animals, evidence of hemorrhage was observed only ipsilate ral to the side of QUIS injection (X01 and X03). In addition, hypointense areas were observed in the dorsal columns in all the QUIS-injured animals except S01. Central canal expansion was clearly defined in four of the five QL2 animals (S01 and S04S06), and injection tracks were visible in all injured animals. Cavity formation was also seen in all QL2 animals bilaterally.

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37 Figure 3-4. Total injury lengths determined by using in vivo in vitro and histologic methods for each QUIS-injured animal. Total injury lengths in millimeters reflect the presence of excitotoxic tissu e damage. No significant differences are observed between in vitro and histology (p > 0.05). Significant differences are observed between in vivo and in vitro /histology (p > 0.05), but these differences are within the range of section thickness difference between in vivo and in vitro images. A single asterisk denotes the QL2 animals, and double asterisks denote the QL1 animals. Significant differences ( p < 0.001, +p < 0.01) in injury lengths were f ound between QL1 and QL2 animals for all three methods. In Vivo DCE-MR Imaging To determine whether there was BSCB disr uption at the 17 day interval (mean = 22 days) post-injury, DCE-MR imaging wa s performed by using Gd as a contrast agent. Disruption of the BSCB was indicate d by a significant Gd enhancement (>10%) within the cord at the site of injury. No significant Gd enhancement was observed in the normal and sham groups and no significant Gd enhancement was observed within the cord at the site of injury in any of the QUIS-injured animals. Gadolinium enhancement, 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 S01*S02*S04*S05*S06*X01**X03**Total Injury Length (mm) In Vivo In Vitro Histology + +

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38 however, was observed in surrounding muscle of all animals, approximately 300% peak signal intensity enhancement, which indicates that the procedure was performed correctly. Figure 3-5. Summary of in vivo in vitro and histologic data collect ed for a representative QL2 animal (S06). Repres entative sections (1) are sampled from the same rostrocaudal location in the spinal cord with each method used. Image sizes are the same as in Figs 3-2 and 3-3. S ee Fig 3-1, for labeling of anatomic and pathologic findings. Although sagittal im ages show the rostral-caudal extent of the injury, transverse sections prov ide greater detail of the pathologic changes associated with this injury model. In Vitro MR Microscopy Images obtained in vitro had greater resolution and more clearly defined the pathologic characteristics (i.e., neuronal loss, hemorrhage, and cavitation) of the injury compared with the in vivo MR images. No abnormal signals were observed in the normal control animal. For the saline-injected animal, however, a hypointense region was

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39 observed at the site of injection, which correlated with hemorrhage (see Histologic Findings). Figures 3-2B and 3-3B illu strate the sagittal and transverse in vitro images, respectively, at the epicenter for each QUI S-injured animal. In both sagittal and transverse images, white matter and gray matter were clearly distinguishable and the cord level was easily determined. In all QUIS-injured animals, hypointense regions were observed at the site of injury, correlating with hemorrhage. Figure 3-6. Summary of in vivo in vitro and histologic data collect ed for a representative QL1 animal (X03). Representative se ctions (1) are sampled with each method from the same rostrocaudal location in the spinal cord. Image sizes are the same as in Figs 3-2 and 3-3. See Fig 3-1, for labeling of anatomic and pathologic findings. Although sagittal im ages show the rostral-caudal extent of the injury, transverse sections prov ide greater detail of the pathologic changes associated with this injury model. Total injury lengths measured with in vitro MR images were significantly greater than lengths determined with in vivo MR images, because of differences in section

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40 thickness (Fig 3-4); repeated-measures ANOVA (F = 40.476; p = 0.001). Pair-wise comparisons indicated that in vivo MR imaging gave significantly shorter injury lengths than in vitro MR imaging (p = 0.001) and histolo gy (p = 0.001), but were within the range of difference in secti on thickness (length difference standard deviation = 0.50 mm) for each method used (section thickness in vitro of 0.2 mm and in vivo of 1.0 mm). Additionally, there were significant differen ces (p < 0.001, one-way between subjects ANOVA, F = 34.134, p < 0.001) in injury lengths between QL1 (X01 and X03) and QL2 animals (S01, S02, and S04S06; Fig 3-4). Histologic Findings Histologic results confirmed the pat hologic characteristics observed with in vivo and in vitro MR imaging. Injection tracts were vi sible in all QUIS-injured animals, as well as saline-injected animals. Neuronal loss in the dorsal horn ipsilateral to the injury was observed in all QUIS-injured animals. Neuronal loss in the dorsal horn contralateral to the injection site was observed in all QL 2 animals but not in QL1 animals. Central canal expansion was observed in four of five QL2 animals and cavity formation was observed only in QL2 animals, being present both ipsilateral and contralateral to the side of QUIS injection. Sections from three of three animals (S01, S06, and X03) stained positive for the Prussian Blue reaction, conf irming the presence of hemorrhage at the same location of hypointense signal intensity as observed in the in vivo and in vitro images. Because of histologic artifact s, complete injury lengths could not be determined for all cords. In animals with minimal histol ogic damage (all but X03), however, the total injury lengths determined from histologic ev aluation showed no significant differences to those determined with in vitro images (Fig 3-4; p = 0.05). However, here were

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41 significant differences (p < 0.01, one-way between subjects ANOVA, F = 34.134, p < 0.001) in injury lengths between QL1 (X01 and X03) and QL2 animals (S01, S02, and S04S06; Fig 3-4). Figures 3-5 and 3-6 show a summary of da ta collected from a representative QL2 (S06) and QL1 (X03) animal, respectively. Sufficient detail is visible in the in vivo images to determine the type and extent of pathologic damage. There was narrowing of the dorsal horn with sparing of the superficial laminae bilaterally in all QL2 animals. This is in contrast to QL1 animals, in which sparing was observed only unilaterally. A significant observation in this study was that following the more superficial injury (laminae IIII, QL1); damage occurred only ipsi lateral to the side of QUIS injections and included only evidence of hemorrhage. In c ontrast, lesions deeper in the gray matter (QL2) produced damage both ipsi lateral and contralateral to the injection site, showing evidence of both hemorrhage and cavitation. In addition, evidence for differences between QL1 and QL2 injuries were seen in th e rostrocaudal and ventral extent of injury (e.g., less extensive in QL1 animals compared with QL2 animals [Fig 3-6]). Discussion Clinically, MR imaging has been a us eful tool for the study of secondary complications associated with SCI. For example, MR imaging can play an important role in the study of post-traumatic syringomyelia. The excitotoxic inju ry model has been shown to produce pathologic changes simila r to those observed in this condition (Yezierski et al., 1993; Schwartz et al., 1999a). Considering the lack of understanding of the pathologic mechanism responsible for cavit y formation in the inju red cord (Madsen et al., 1994), it may be possible to gain insight into the mechanism of cavitation by using in vivo MR imaging at different time points fo llowing excitotoxic SCI. In the past,

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42 clinicians have relied on patient descripti ons of behavioral changes in determining clinical outcome (Williams, 1990; Oakes, 1996) ; however, MR imaging recently has been used to monitor changes associated with in traspinal transplantation, graft-mediated functional recovery, and changes in syrinx mo rphology after transplantation (Wirth et al., 2002) and to differentiate between cyst and sc ar or tumor formation with the help of MR contrast agent administration (Schwartz et al., 1999a). With the importance and many uses for MR imaging, continued research is needed to enhance this technique and expand its uses to provide a better understanding of the mechanisms responsible for pathologic changes associated with SCI. For example, an important question related to SCI is the temporal profile of pathologic changes in the acute and chronic stages post-injury. Using in vivo MR imaging, it should be possible to mo nitor these changes in the same animal. In the present study the feasibil ity of using a complement of in vivo and in vitro MR imaging, along with histologic methods to study experimental SCI was examined. The results have shown that MR images provide details of pathologic changes, which correlated with histologic fi ndings, and were easily detected both in vivo and in vitro These changes include cavities, central canal expansion, hemorrhage, and tissue loss. These characteristics of injury are similar to those previously reported in both experimental (Weirich et al., 1990; Wirth et al., 1992; Falconer et al ., 1994; Wirth et al., 1995; Ohta et al., 1999; Schwar tz et al., 1999a; Bilgen et al ., 2000; Metz et al., 2000) and clinical studies (Flanders et al., 1990; Weirich et al., 1990; Yamashita et al., 1990; Yamashita et al., 1991; Ohshio et al., 1993; Flanders et al., 1999; Selden et al., 1999; Rattliff and Voorhies, 2000; Is hida and Tominaga; 2002). The present study is one of the

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43 first that has attempted to compare all three methods (see also a recent study in the mouse [Bonny et al., 2004]). An important finding in this study was the sensitivity of in vivo MR imaging to detect differences in pathology between two di fferent strategies of injury. Despite the small differences in injury depths betw een QL1 and QL2, there were significant differences in the overall pathology detected in the in vivo MR images. Following the shallower injury (i.e., QL1), damage was obs erved only unilaterally in the gray matter and did not show any cavitation or central can al expansion. In contrast, following the deeper injury (i.e., QL2), damage was more severe, and showed cavitation, hemorrhage, and neuronal loss bilaterally. These differences could be due to the connectivity between regions of the cord at different depths in the gray matter. For example, propriospinal connections involving lamin ae IVVII are far more extensive within and between segments than those involving more superfic ial laminae (Yezierski et al., 1980; Menetrey et al., 1985). Although speculative, it is possi ble that, once damaged, a signal is relayed to other parts of the gray ma tter that induces further damage. The deeper lesion (QL2) resulted in damage of the contralateral side of the cord similar in pathologic characteristics to the side th at received the QUIS injecti on. This suggests a similar mechanism of injury (i.e., excitotoxicity). From this, one might conclude that propriospinal connections exert a trophic infl uence on other parts of the gray matter and once damaged, leads to an injury induced release of excitatory amino acids and subsequent pathologic changes. This obser vation may be important from the standpoint of providing insight into the mechanism re sponsible for the progression of pathologic changes following spinal injury.

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44 There was a corresponding loss of gray matter illustrated in in vitro MR images and histology. These changes could be quantifie d with sufficient information about volumes of normal white and gray matter. Volume quantification of white and gray matter loss is important; however, it was beyond the scope of this study and was considered in future studies (Chapter 4). All three methods used in this study were directly compared, because they all showed similar pathologic changes. For example, in vitro MR images were the most precise in determining injury length, rather than histologic secti ons due to histologic artifacts. Although significant differences were detected between tota l lengths of injury, these differences were within the thickness of sections of the methods used. For this reason, the present data support the conc lusion that in vivo MR imaging is a useful technique to study the evoluti on of pathologic changes followi ng SCI. This feature may be especially important when correlated with the profile of changes related to the disruption of the BSCB. The observation of changes in the BSCB is important in characterizing the excitotoxic model of SCI, because BSCB disr uption can contribute to events leading to detrimental pathologic processes followi ng SCI (Rapoport, 1976; Bradbury, 1979). At the time points used in this study, 17 da ys post-injury, our measurement of the BSCB indicated that the barrier was intact; howev er, previous studies indicate an opening at earlier time points following cont usion injury (Runge et al., 1997; Bilgen et al., 2001). Therefore, longitudinal studies will be necessary to detect the temporal characteristics of how the BSCB is affected following excitotoxi c SCI. Preliminary data from a subsequent

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45 study suggest that there are changes over time in the integrity of the BSCB following excitotoxic injury (S. A. Berens, P. Yezierski, T. H. Mareci; Chapter 4). There are several advantages and disadva ntages associated with each of the methods used in the present study. In vivo MR imaging can be used to non-invasively monitor the progression of pathologic changes over time in the same animal. This feature provides the opportunity to study the neuroprot ective and neurorestorative properties of therapeutic interventions (e.g., transplants, drugs, etc). In vivo MR imaging methods, however, lack the hi gher resolution that in vitro MR imaging and histologic methods provide. Despite the higher resolution, in vitro MR imaging and histologic methods have the disadvantage that they cannot be used at different time points in the same animal, and there is a risk of losing tissu e in histologic analysis because of processing artifacts. Another advantage of in vivo MR imaging is the ability to analyze the injured cord in transverse and sagittal planes of section at di fferent time points in the same animal. This capability allows one to easily determine the overall extent of injury as well as gross pathologic changes, two importa nt characteristics used in the evaluation of therapeutic interventions. With continue d technological improvements in in vivo MR imaging, this technique will continue to be an importan t analytical tool in the study of SCI. In some regions, in vivo MR images, collected on a 4.7T magnet, appear different from in vitro MR images collected on a 14.1T magnet. The in vivo MR images were collected by using T2-weighted fa st spin-echo sequences, whereas in vitro MR images were collected by using proton dens ityweighted spin-echo sequences. In vivo MR images show cavitation as hyperintense signals due to T2-weighting, which is

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46 comparable to what is used in human MR imaging. This is in contrast with in vitro MR images, because cavities appear isointense due to proton attenuation weighting. A limited number of studies have used in vivo MR imaging in experimental animal models, with an even smaller number using in vitro MR imaging techniques. In the present study, we have used customized ha rdware (coils) and high magnetic fields to obtain high-quality in vivo and in vitro images in the rat. Continued improvement of these MR components should lead to an increased utility of these methods. In our study, in vivo MR images show high resolution and great er pathologic deta il than reported in previous MR imaging studies at this magne tic field strength or lower (Hackney et al., 1986; Runge et al., 1997; Frai dakis et al., 1998; Ohta et al ., 1999; Iannotti et al., 2002; Brodbelt et al., 2003). As expect ed, higher quality MR images can be obtained at higher magnetic fields (Bonny et al., 2004; Franconi et al., 2000) or with chronically implanted MR coils (Bilgen et al., 2000; Bilgen et al., 2001; Wirth et al., 1993; Ford et al., 1994b; Fenyes and Narayana, 1998; Narayana et al ., 1999; Silver et al., 2001). Although we have been involved in the development of implanted coil technology (Wirth et al., 1993; Silver et al., 2001), we did not use implanted coils in this study because we wanted to eliminate the possible conf ounding effects of coil implan tation and also eliminate additional damage to the cord. By comparing the results of in vitro and histologic methods, details seen in vivo can be confirmed, thus streng thening the rationale for using in vivo MR imaging as an important t ool in the study of SCI.

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47 CHAPTER 4 USE OF MAGNETIC RESONAN CE IMAGING TO STUDY THE NEUROPROTECTIVE EFFECTS OF AGMA TINE FOLLOWING SPINAL CORD INJURY IN THE RAT Introduction The excitotoxic events following a quisqualic acid lesion play an important role in the pathologic sequelae of spin al injury (Regan and Choi, 1994). The use of antagonists of glutamate receptor subtypes has provided evidence for the involvement of excitotoxic cascades in the pathological outcomes of QUIS induced spinal injury (Fairbanks et al., 2000). Studies involving the QUIS model ha ve shown, for example, pathophysiological changes, up-regulation in gene expression fo r NMDA receptors and inducible nitric oxide synthase (Plunkett et al., 2001). Agmatine (decarboxylated arginine) is an endogenous neurotransmitter/neuromodulator that has been shown to have both NMDA receptor antagonist (Yang and Reis, 1999) and NOS i nhibitor properties (Auguet et al., 1995; Galea et al., 1996). The multifaceted capacity of this agent makes it a promising therapeutic agent in targeting secondary injury. In the present research, the effect s of agmatine, an agent with known pharmacological actions, was evaluated in relation to blood-sp inal cord-barrier disruption, edema and cavity formation, and bl ood vessel leakage. It is believed that limiting BSCB disruption reduces the entry of inflammatory mediators into the spinal cord. The BSCB may therefore represent an impo rtant target of therapeutic intervention. Applying the use of MRI to monitor the above pathological characteristics longitudinally following administration of a known neuroprotect ive agent will provid e insight into the

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48 site of action and mechanism of a drug w ith known pharmacological actions as well as further validate the use of MRI in the study of spinal cord injury. A preliminary description of this study has been reported (Berens et al., 2004). Materials and Methods Animals, Surgery, and In Vivo MR Imaging Sprague-Dawley male rats weighing 250-300g were subjected to excitotoxic spinal cord injury as described in Chapter 3. Rats received an in vivo MRI prior to injury to serve as their own controls, with animal prep aration as described in Chapter 3. Following MRI, animals were assigned to one of four injury/treatment groups (see Neuroprotection below) and then were inject ed with QUIS or saline (sham injury) at a depth of 500 m. Neuroprotection Agmatine was purchased from Sigma (St. Louis, MO), and dissolved in 0.9% saline. Animals were separated into 4 injury /treatment groups as follows: (1) Saline-SCI (sham injury, N = 5); (2) QUIS-SCI plus vehi cle (0.9% saline, N = 6); (3) QUIS-SCI plus Agmatine I (25 mg/kg, N = 7); and (4) QUISSCI plus Agmatine II (80 mg/kg, N = 7). Rats were given treatment intraperitoneally 30 minutes post-QUIS injury and once a day for 14 days (refer to Fig. 4-1 for a summary of the surgery and MR imaging timeline). In Vivo High-Resolution Magnetic Reso nance Imaging Data Acquisition In vivo MRI and DCE-MRI were performed on days 1, 3, 7, 15, and 25 post-injury (see Fig. 4-1). All in vivo NMR measurements were performed on an 11.1 T, 40 cm clear bore Oxford magnet at 470 MHz by usi ng a Bruker BIOSPEC Avance DBX console (Bruker NMR Instruments, Billeria, MA) and 200 mT/m actively shielded gradients. A highly sensitive quadrature MR surface coil was constructed specifically for imaging the

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49 rat spinal cord (please refer to Chapter 2 for coil specifications). Pilot images were T1weighted (TR/TE = 1000ms/15 ms, NA = 1), with a FOV of 8 cm in each direction, a 1 mm slice, and a 128 x 128 matrix. Fat s uppression was done using a CHESS-type sequence (Haase et al., 1985). To compensa te for breathing artifacts and eliminate aliasing, the read direction wa s changed to head-foot (sagittal MR images) or left-right orientation (transverse MR images), and a slice saturation pulse was added to the sequence of high-resolution images. The slice saturation pulse had a mao4-180 (modified sinc) pulse shape and was a single 30 mm thick slice in the coronal direction. Once the position was confirmed, a set of fat suppressed, sl ice saturated, high-resolution sagittal and transverse images was collect ed. Sagittal T1-weighted (TR/TE = 1000 ms/15 ms, NA = 4) spin echo images were acquired over 5 slices, each 1 mm thick without a gap between slices. Additional acquisition parameters were a FOV of 2 cm x 2 cm, read out in the head-foot direction, and a matrix of 128 x 128. The scan time with these parameters was 8.58 min. Transverse T1-weighted (TR/TE = 1000 ms/15 ms, NA = 4) spin echo images were acquired over 14 slic es, each 1 mm thick without a gap between slices. Other acquisition parameters were a F OV of 2 cm x 2 cm, read out in the left-right direction, and a matrix of 128 x 128. The scan time was 8.58 min. Sagittal T2-weighted (TR/effective TE = 2000 ms/62.5 ms, NA = 12) fast spin echo images with phase-encode segmentation were acquired over 5 slices, each 1 mm thick without a gap between slices. Additional acqui sition parameters were a FOV of 2 cm x 2 cm, read out in the head-foot direction, pha se-encode segmentation factor of 4, and a matrix of 128 x 128. The scan time with these parameters was 13.35 min. Transverse T2-weighted (TR/effective TE = 2000 ms/62.5 ms NA = 12) fast spin echo images with

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50 phase-encode segmentation were acquired over 14 slices, each 1 mm thick without a gap between slices. Other acquisition parameters we re a FOV of 2 cm x 2 cm, read out in the anterior-posterior direction, phase-encode segmentation factor of 4, and a matrix of 128 x 128. The scan time with these parameters was 13.35 min. Figure 4-1. Surgery and MR imaging timeline. Each animal was subjected to a pre-injury MRI, and then assigned to one of four injury/treatment groups. Animals then received an in vivo MRI on days 1, 3, 7, 15, and 25 post-injury, and were sacrificed on day 30 post-injury. Cords were excised and undergo in vitro three dimensional MR microscopy. In Vivo DCE-MR Imaging Data Acquisition After 4 sets of high-resoluti on images were collected, a series of dynamic contrastenhanced MR images was acquired as described in Chapter 3. Only sagittal images were acquired before and after the contrast agent was injected due to extreme heating effects caused by the transverse DCE-MR image acqui sition (as described in Chapter 2). Fat suppressed, slice saturated sa gittal images were acquired with a FOV = 2 cm x 2 cm, matrix = 96 x 96, over 3 slices, both with 1 mm thick, without a gap between slices. Three sagittal T1-weighted pre-contrast mu lti-slice images (with a 3 minute gap between each image) (TR/TE = 330 ms/9.4 ms, NA = 6) were collected. After acquisition of the Day 0 pre-injury In vivo MRI 11.1T Day 30 post-injury Euthanization (A) Saline-SCI (N = 5) (B) QUIS-SCI + Vehicle (N = 6) (C) QUIS-SCI + Agmatine I (N = 7) (D) QUIS-SCI + Agmatine II (N = 7) Day 1 post-injury In vivo MRI 11.1T Day 3 post-injury In vivo MRI 11.1T Day 7 post-injury In vivo MRI 11.1T Day 15 post-injury In vivo MRI 11.1T Day 25 post-injury In vivo MRI 11.1T Day 30 post-injury In vitro 3D-MRM 14.1T

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51 pre-contrast images, Gd was injected over a period of approximately 5 seconds with continuous pressure, to prevent backflow, through the implanted catheter while the animal was in the magnet. Immediately after injection, nine sagittal T1-weighted postcontrast multi-slice images (3 minutes between each image) (TR/TE = 330 ms/ 9.4 ms, NA = 6) were collected. These images were collected over a 60 minute timeframe, which was chosen based on previous experience with a peak enhancement for the cord at 3 min. The receiver gain was adjusted to one-third the value used for the high-resolution images because Gd provides approximately 3 times the contrast enhancement. In Vitro Three-Dimensional MR Microscopy All excised cords were subjected to in vitro three-dimensional MR microscopy, with cord preparation as described in Chap ter 3. Images were acquired with a threedimensional gradient echo pulse sequence using a TR = 150 ms, TE = 10 ms with NA = 2. The image FOV was 2 cm x 0.5 cm x 0.5 cm in a matrix of 512 x 128 x 128 in a total data acquisition time of 1.5 hrs. Therefore, MR images were acquired with an isotropic resolution of 40 microns. A 3D Fourier tran sformation was applied to the acquired data matrix to produce the 3D image. General im age processing and analysis was performed using custom software written in the Interactive Data Language (IDL, from Research Systems, Boulder, CO). Data Analysis To compare results obtained between th e four injury/treatment groups, the amount of edema, blood vessel leakage, cavitation, and total longitudinal injury lengths were determined and compared. To determine the amount of edema, the total number of MR transverse image slices (please refer to Chapte r 3 for justification in using transverse slice orientation), with evidence of edema (diffu se hyperintense signal on T2-weighted MR

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52 images, evident in early time points) were counted and multiplied by the section thickness appropriate for the method used. For in vivo MR imaging, T2-weighted transverse images were counted and multiplied by 1 mm; for in vitro MR imaging, transverse images were counted and multiplied by 39 m. This same method was used to determine blood vessel leakage (hypointense signal on T2-weighted images), cavitation (focal hyperintense signal, evident in later time points), and total longitudinal injury length (summation of the individual pathologi cal changes). Results for each group for each day were averaged together and compared Statistical analysis of these data included a one-way between subjects ANOVA te st to determine differences in injury severity between the four in jury/treatment groups (SPSS, Chicago, IL). This was followed by a Tukey HSD post hoc test to interpret the significance of the ANOVA. Power analysis (power = 0.80) was done fo r results showing non-significant trends, including: (A) blood vessel leakage and (B) total longitudinal injury length. Dynamic contrast-enhanced MR imaging pr ocessing was performed as described in Chapter 3. Peak enhancement was determined for each animal each day. Results were averaged for each injury/treatment group for each day and compared. Statistical analysis of these data included a one-way between s ubjects ANOVA test to determine differences in injury severity between th e four injury/treatment groups (SPSS, Chicago, IL). This was followed by a Tukey HSD post hoc test to interpret the significance of the ANOVA. Volume Analysis For further quantification of injury characteristics, volumes were calculated for edema, blood vessel leakage, cavitation, a nd total injury for each of the four injury/treatment groups. Volume analysis was done using the OsiriX-Dicom Viewer ( http://homepage.mac.com/rossetantoine/osirix ). To calculate the volume of edema for a

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53 specific cord, a data set was imported into OsiriX, and an ROI was drawn around all areas showing edema for each slice. Once th e ROIs were drawn, they were summed together (to obtain an area) and multiplied by the slice thickness appropriate for each method used (to obtain a volume). For in vivo MR imaging, areas were multiplied by 1 mm; for in vitro MR imaging, areas were multiplied by 39 m. Results were averaged for each group for each day and compared. Stat istical analysis of these data included a one-way between subjects ANOVA test to de termine differences in injury severity between the four injury/treat ment groups (SPSS, Chicago, IL). This was followed by a Tukey HSD post hoc test to interpret the significance of the ANOVA. Power analysis (power = 0.80) was done for results show ing non-significant trends (volume of blood vessel leakage). Results In Vivo High Resolution Magnetic Resonance Imaging Figure 4-2 shows a representative collecti on of T2-weighted MR images for the four injury/treatment groups for each day. Please refer to Chapter 3, Figure 3-1 for a description of the pathologi cal findings seen on an in vivo T2-weighted MR image. All post-injury MR images for an animal were compared to their respective day 0 MR images, thus each animal served as its own c ontrol. T2-weighted images gave the best representation of injury, which showed hyperi ntense (i.e., bright) and hypointense (i.e., dark) signals, compared to a day 0 T2-weighted MR image, representing edema or cavitation and blood vessel leak age, respectively. The only information obtained from T1-weighted images was the in jection track, which was seen as a hypointense signal as compared to a day 0 T1-weighted MR image.

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54 Figure 4-2. Representative T2-weighted (T R/effective TE = 2000 ms/62.5 ms; NA = 12) MR images for each injury/treatment group for each day. In vivo MR images are displayed with image size = 1.0 cm x 1.0 cm. MR images are sampled from the epicenter of the inju ry. Images are oriented with dorsal at the top. L, left side of cord; R, right side of cord, for all images. The presence of edema was distinguished from the presence of cavitation, which both show as hyperintense signals on T2-w eighted MR images, by the observation that edema is only observed in the earlier time poi nts (completely subsides between days 7 and 15, Fig. 4-3) and is seen as a more di ffuse, widespread hyperintense signal, whereas the presence of cavitation is only observed as early as day 15 (see Fig. 4-4) and is seen as a more intense and focal hyperintense signal. For example, in Figure 4-2, refer to the QUIS-SCI plus Vehicle MR images. Notice the difference in th e hyperintense signal Saline-SCI QUIS-SCI + Agmatine II QUIS-SCI + Agmatine I QUIS-SCI + Vehicle Day 0 Day 1 Day 3Day 7Day 15 Day 25 L R

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55 seen on days 1, 3, and 7 versus days 15 and 25. The hyperintense signal is more widespread throughout the injured cord on the in jection side on days 1, 3, and 7, whereas, the hyperintense signal seen on days 15 and 25 is more intense and focused mainly in the injection track. To quantify the pathological changes (e dema, cavitation, and blood vessel leakage) following either saline or QUIS injury, the number of transverse MR slices that contained edema (Fig. 4-3), cavitation (Fig. 4-4), or bl ood vessel leakage (Fig. 4-5) were counted and multiplied by the slice thickness for each da y to show a longitudinal profile of each Figure 4-3. Longitudinal profile of the pres ence of edema for all in jury/treatment groups as determined from in vivo T2-weighted transverse MR images. ( ) p < 0.02, QUIS-SCI + Agmatine I versus Saline-SCI. Error bars represent one standard deviation. Data is represented as the number of transverse slices in millimeters showing edema. Edema is present on day 1 post-injury and subsides completely by day 15 fo r all injury/treatment groups. 0 2 4 6 8 10 01371525Time (Days)# Transverse Slices (mm) Saline-SCI QUIS-SCI + Vehicle QUIS-SCI + Agmatine I QUIS-SCI + Agmatine II

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56 pathological change. Additionally, the numbe r of transverse slices that showed any combination of the above-mentioned pathological changes was summed and multiplied by the slice thickness to give a total injury length (Fig. 4-6). In Figure 4-3, edema is observed immediat ely following injury (day 1 post-injury) in all injury/treatment groups, persists until day 7, and completely subsides by day 15. For the QUIS-SCI plus Agmatine I group, ther e was a significant increase in the number of transverse slices containing edema on da ys 3 (p = 0.018, one-way between subjects ANOVA, F = 3.459, p = 0.024) and 7 (p = 0.007, one-way between subjects ANOVA, F = 4.390, p = 0.008) as compared to the Saline-SCI group. There were no significant 0 2 4 6 8 10 01371525Time (Days)# Transverse Slices (mm) Saline-SCI QUIS-SCI + Vehicle QUIS-SCI + Agmatine I QUIS-SCI + Agmatine II Figure 4-4. Longitudinal profile of the pres ence of cavitation for all injury/treatment groups as determined from in vivo T2-weighted transverse MR images. Error bars represent one standard deviation. Data is represented as the number of transverse slices in millimeters showing cavitation. Cavitation is present beginning on day 15 post-injury and c ontinues on day 25 for all QUIS-SCI groups. No cavitation was observed in the Saline-SCI group.

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57 differences in the presence of edema for the QUIS-SCI plus Vehicle and QUIS-SCI plus Agmatine II groups as compared to the Salin e-SCI group for any of the time-points. In Figure 4-4, cavitation is observed be ginning at day 15 for only the QUIS-SCI injury groups, regardless of treatment, and persists on day 25. No cavitation was observed for the Saline-SCI group. There we re no significant differences in the number transverse slices containing cavitation fo r any of the QUIS-SCI injury groups as compared to the Saline-SCI group. Figure 4-5. Longitudinal profile of the presen ce of blood vessel leakage for all injury/treatment groups as determined from in vivo T2-weighted transverse MR images. ( ) p < 0.01, QUIS-SCI + Agmatine I versus Saline-SCI; ( ) p < 0.05, QUIS-SCI + Vehicle versus Salin e-SCI. Error bars represent one standard deviation. Data is represented as the number of transverse slices in millimeters showing blood vessel leakage. Blood vessel leakage is observed on day 1 post-injury and continues to in crease until day 7, at which point it seems to stabilize for all injury/treatment groups. 0 2 4 6 8 10 01371525Time (Days)# Transverse Slices (mm) Saline-SCI QUIS-SCI + Vehicle QUIS-SCI + Agmatine I QUIS-SCI + A g matine II

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58 In Figure 4-5, blood vessel leakage is observed throughout all experimental timepoints for all injury/treatment groups, increasi ng from day 1 to day 7, at which point, the amount of blood vessel leakage appears to re main constant. There was a significant increase, versus Saline-SCI, in the number of transverse slices containing blood vessel leakage on days 7 (one-way between subjects ANOVA, F = 4.661, p = 0.006) for the QUIS-SCI plus Vehicle (p = 0.012) and QUISSCI plus Agmatine I (p = 0.007) groups; 15 (one-way between subjects ANOVA, F = 5.037, p = 0.004) for the QUIS-SCI plus Figure 4-6. Longitudinal profile of the total pathology inju ry length (the presence of edema, blood vessel leakage, and cavitation combined) fo r all injury/treatment groups as determined from in vivo T2-weighted transverse MR images. ( ) p < 0.05, QUIS-SCI + Agmatine I versus Saline-SCI; ( ) p < 0.02, QUIS-SCI + Vehicle versus Saline-SCI. Error bars represent one standard deviation. Data is represented as the number of transverse slices in millimeters showing any combination of edema, blood vesse l leakage, and cavitation. The presence of edema is the largest contributor to the total injury length in the earlier time-points (days 1, 3, and 7) whereas, blood vessel leakage is the main contributor in the later time-points (days 15 and 25). 0 2 4 6 8 10 01371525Time (Days)Total Injury Length (mm) Saline-SCI QUIS-SCI + Vehicle QUIS-SCI + Agmatine I QUIS-SCI + A g matine II

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59 Vehicle (p = 0.007) and QUIS-SCI plus Agma tine I (p = 0.006) groups; and 25 (one-way between subjects ANOVA, F = 4.390, p = 0.008) for the QUIS-SCI plus Vehicle (p = 0.042) and QUIS-SCI plus Agmatine I (p = 0. 007) groups. There were no significant differences in the presence of blood vessel leakage for the QUIS-SCI plus Agmatine II group as compared to the Saline-SCI group for any of the time-points. Although no significant differences were detected betw een QUIS-SCI plus Agmatine II and QUIS-SCI plus Vehicle groups, power analysis revealed that 8 animals for day 7, 8 animals for day 15, and 35 animals for day 25 would be require d to obtain a significan ce at p = 0.05 at a power = 0.80. The longitudinal profile of total injury length measured with in vivo transverse MR images is represented in Figure 4-6. The pres ence of edema is the primary contributor to the total injury length in the earlier post-i njury time-points (days 1, 3, and 7), whereas, blood vessel leakage is the foremost contributor in the later post-inj ury time-points (days 15 and 25). For the QUIS-SCI plus Vehicle gr oup, there was a significant increase in the total injury length on days 15 (p = 0.007, one-way between subjects ANOVA, F = 5.037, p = 0.004), and 25 (p = 0.006, one-way between subjects ANOVA, F = 4.577, p = 0.007) as compared to the Saline-SCI group. Fo r the QUIS-SCI plus Agmatine I group, there was a significant increase in the total injury length on days 1 (p = 0.049, one-way between subjects ANOVA, F = 2.672, p = 0.058), 3 (p = 0.009, one-way between subjects ANOVA, F = 4.087, p = 0.013), 7 (p = 0.029, one-way between subjects ANOVA, F = 3.430, p = 0.024), 15 (p = 0.006, one-way between subjects ANOVA, F = 5.037, p = 0.004), and 25 (p = 0.019, one-way between subjects ANOVA, F = 4.577, p = 0.007), as compared to the Saline-SCI group. There were no significant differences in

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60 total injury length for the QUIS plus Agma tine II group as compared to the Saline-SCI group for any of the time-points. Although no significant differences were detected between QUIS-SCI plus Agmatine II animal s and QUIS-SCI plus Vehicle, power analysis revealed that 31 animals for day 7, 13 animals for day 15, and 9 animals for day 25 would be required to obtain a significance at p = 0.05 at a power of 0.80. Refer to Table 4-1 for a summary of significance. In Vivo Dynamic Contrast-Enhanced Magnetic Resonance Imaging To determine whether there was BSCB disrupti on at the day 0 and days 1, 3, 7, 15, and 25 post-injury time-points, DCE-MR imagi ng was performed using Gd as a contrast agent. Disruption of the BSCB was indicate d by a significant Gd enhancement (>10%) within the cord at the site of injury. Fi gure 4-7 provides a peak enhancement profile for all four injury/treatment groups for all timepoints. No significant Gd enhancement was observed in any of the four injury/treatment groups at the day 0 (pre-injury) time-point. Gadolinium enhancement, however, was observe d in surrounding muscle of all animals, approximately 300% peak signal intensity enhancement, which indicates that the procedure was performed correctly. For the Saline-SCI group (sham injury), th ere was a significant increase in peak enhancement (one-way between subjects ANOVA, F = 13.966, p < 0.001) on days 1 (p = 0.001) and 3 (p < 0.001) as compared to da y 0. For the QUIS-SCI plus Vehicle group, there was a significant increase in peak enhancement (one-way between subjects ANOVA, F = 52.422, p < 0.001) on days 1 (p < 0.001), 3 (p < 0.001), and 7 (p = 0.02) as compared to day 0. For the QUIS-SCI pl us Agmatine I group, there was a significant increase in peak enhancement (one-way between subjects ANOVA, F = 59.234, p < 0.001) on days 1 (p < 0.001), 3 (p < 0.001), and 7 (p = 0.012) as compared to day 0. For

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61 the QUIS-SCI plus Agmatine II group, ther e was a significant increase in peak enhancement (one-way between subjects ANOVA, F = 31.807, p < 0.001) on days 1 (p < 0.001), 3 (p < 0.001) and 7 (p = 0.005) as compared to day 0. When comparing between groups on individual days, there we re no significant differences. Figure 4-7. Summary of DCE-MRI BSCB disrup tion. Peak enhancement is represented as a percentage. There were significan t differences in peak enhancement for all injury/treatment groups on days 1 and 3 as compared to day 0. There were significant differences in peak enhancement for the QUIS-SCI injury groups, but not the saline-SCI group on day 7 as co mpared to day 0. No significant disruption was observed on days 15 and 25 as compared to day 0. Additionally, no significant differe nces were observed between the injury/treatment groups for any of the days. (*) p < 0.001, ( ) p < 0.01. Error bars represent one standard deviation. 0 20 40 60 80 100 120 140 160 Day 0Day 1Day 3Day 7Day 15Day 25Peak Enhancement (%) Saline-SCI QUIS-SCI + Vehicle QUIS-SCI + Agmatine I QUIS-SCI + Agmatine II * * * *

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62 Volume Analysis To further quantify pathological change s (edema, cavitation, and blood vessel leakage) following either saline or QUIS inju ry, regions of interest were drawn, using transverse MR images, around areas showi ng edema (Fig. 4-8), blood vessel leakage (Fig. 4-9), or cavitation (Fig. 4-10), and we re summed and multiplied by the appropriate slice thickness for each animal and time-point to obtain a volume for each of the above mentioned pathological changes. Additionally a region of interest was drawn around the entire injured area (edema, bl ood vessel leakage, and cavitati on combined) for each slice, Figure 4-8. Volume of edema for all inju ry/treatment groups as determined from in vivo T2-weighted transverse MR images. (*) p 0.05. Error bars represent one standard deviation. Data is represented as the volume in cubic millimeters. 0 5 10 15 20 25 30 35 Day 1Day 3Day 7Day 15Day 25Volume (mm3) Saline-SCI QUIS-SCI + Vehicle QUIS-SCI + Agmatine I QUIS-SCI + Agmatine II *

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63 summed and multiplied by the appropriate slice thickness for each animal and time-point to give an overall volume of injury (Fig. 4-11). The volume of edema peaks at day 3 and starts diminishing day 7 for all injury/treatment groups except the QUIS-SC I plus Agmatine I group. This profile closely matches that shown in Figure 4-3. There was a significant increase in volume of edema on day 1 (one-way between subjects ANOVA, F = 3.286, p = 0.03) between the QUIS-SCI plus Agmatine II and Saline-SCI groups (p = 0.014). Additionally, the QUISSCI plus Vehicle group showed a significantl y higher volume of edema (p = 0.05, oneway between subjects ANOVA, F = 3.053, p = 0.037) as compared to the Saline-SCI 0 1 2 3 4 5 6 7 8 9 Day 1Day 3Day 7Day 15Day 25Volume (mm3) Saline-SCI QUIS-SCI +Vehicle QUIS-SCI + Agmatine I QUIS-SCI + Agmatine II Figure 4-9. Volume of blood vessel leakage fo r all injury/trea tment groups as determined from in vivo T2-weighted transverse MR imag es. Error bars represent one standard deviation. Data is represented as the volume in cubic millimeters.

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64 group. No significant differences were observed on day 7 for any of the QUIS-SCI groups as compared to the Saline-SCI group. Figure 4-9 illustrates the volume of blood vessel leakage profile, and shows an increase in volume through day 7 and a slight decrease in volume on days 15 and 25. The increase seen is consistent with that seen in Figure 4-5; however, volume analysis becomes a more sensitive outcome measure for days 15 and 25, as Figure 4-5 shows a constant level of blood vessel leakage (due to the method of quantification), whereas the volume shows a slight decrease. No signi ficant differences in volume of blood vessel 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Day 1Day 3Day 7Day 15Day 25Volume (mm3) Saline-SCI QUIS-SCI + Vehicle QUIS-SCI + Agmatine I QUIS-SCI + A g matine II Figure 4-10. Volume of cavita tion for all injury/treatment groups as determined from in vivo T2-weighted transverse MR images. Error bars represent one standard deviation. Data is represented as the volume in cubic millimeters.

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65 leakage were detected between any of the QUIS-SCI groups versus the Saline-SCI group. Although no significant differences were de tected between QUIS-SCI plus Agmatine II animals and QUIS-SCI plus Vehicle, power anal ysis revealed that 9 animals for day 7, 252 animals for day 15, and 19 animals fo r day 25 would be required to obtain a significance at p = 0.05 at a power of 0.80. The volume of cavitation following injury is presented in Figure 4-10. The volume profile is also consistent with the longitudina l profile of the presen ce of cavitation (Fig. 4-4). Cavitation was observed in all QUIS-SCI groups, but was not present in the SalineSCI group. Similar to the longitudinal profile data, there are no significant differences in the volume of cavitation following a QUIS le sion as compared to a saline lesion. Finally, the total injury volume, which c onsisted of edema, blood vessel leakage, and cavitation is shown is shown in Figure 4-11. As seen with the individual volumes of pathological changes, the total injury volume profile is similar to the total longitudinal injury length profile (Fig. 4-6). The edema volume is highly represented in the earlier time-points post-injury, whereas the volume of blood vessel leakage is more highly represented in the later time-points. The onl y significant differences in total injury volume was seen on day 1 (one-way betw een subjects ANOVA, F = 3.801, p = 0.017) between the QUIS-SCI plus Agmatine II group and the Saline-SCI group (p = 0.007). This is consistent with the significance seen in Figure 4-8. Refer to Table 4-2 for a summary of significance.

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66 Figure 4-11. Volume of total injury (vol ume of edema, blood vessel leakage, and cavitation combined) for all injury/tr eatment groups as determined from in vivo T2-weighted transverse MR images (days 1, 3, 7, 15, and 25) and in vitro transverse 3D MRM images (day 30). (*) p = 0.007. Error bars represent one standard deviation. Data is represented as the volume in cubic millimeters. 0 5 10 15 20 25 30 35 40 Day 1Day 3Day 7Day 15Day 25Day 30Volume (mm3) Saline-SCI QUIS-SCI + Vehicle QUIS-SCI + Agmatine I QUIS-SCI + Agmatine II( In Vitro )

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67Table 4-1. Summary of Significan t Differences in Longitudinal Pr ofiles of Edema, Cavitation, an d Blood Vessel Leakage, and BSCB Disruption. Injury/Treatment Group versus Saline-SCI Edema (longitudinal profile) (A) Figure 4-3 Cavitation (longitudinal profile) (B) Figure 4-4 Blood Vessel Leakage (longitudinal profile) (C) Figure 4-5 Total Injury Length (A+B+C) Figure 4-6 BSCB Disruption Figure 4-7 QUIS + Vehicle p < 0.05 (days 7, 15, 25) p < 0.02 (days 15, 25) QUIS + Agmatine I p < 0.02 (days 3, 7) p < 0.01 (days 7, 15, 25) p < 0.05 (days 1, 3, 7, 15, 25) QUIS + Agmatine II Table 4-2. Summary of Significant Di fferences in Volumes of Edema, Bl ood Vessel Leakage, and Cavitation. Injury/Treatment Group versus Saline-SCI Edema (Volume) (A) Figure 4-8 Blood Vessel Leakage (Volume) (B) Figure 4-9 Cavitation (Volume) (C) Figure 4-10 Total Injury Volume (A+B+C) Figure 4-11 QUIS + Vehicle p < 0.05 (day 3) QUIS + Agmatine I QUIS + Agmatine II p < 0.05 (day 1) p < 0.01 (day 1)

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68 Discussion The goal of the present study was to use magnetic resonance imaging to evaluate the neuroprotective effects of an agen t, with known pharmacologic actions, on the excitotoxic spinal cord injury model at different time-points in the same animal. Major findings include: (1) high resolution, in vivo MRI can be used to quantitatively and qualitatively study the dynamic changes in sp inal cord injury pa thology and blood-spinal cord-barrier integrity over time in the same animal; and (2) administration of 80 mg/kg agmatine (Agmatine II) post-injury resulted in neuroprotection against blood vessel leakage and edema following QUIS-SCI when compared to the Saline-SCI group. The results have shown that MR images provide details of pathologic changes, easily detected both in vivo and in vitro These changes include cavities, blood vessel leakage, cavitation, and tissue loss. Injury wa s observed unilaterally in some animals and bilaterally in others. This difference is ma inly due to inconsistency in the injection technique. Despite every effo rt to control the depth of injection, variance was found among injury depths, most likely accounting fo r differences in injury severity. As discussed previously (refer to Chapter 3, D iscussion), injuries following a deeper injection resulted in a more extensive injury both rostrocaudally and ventrally, showing bilateral blood vessel leakage and cavitation, whereas with a shallower lesion, damage was less severe resulting only in ip silateral blood vessel leakage. Differences in injury severity may also account for the significant increase in edema following the administration of 25 mg /kg agmatine (Agmatine I). There were variances in injection depth in each group; the QUIS-SCI plus Agmatine I group showing the highest variability in injury severity. More animals would need to be analyzed to reduce the variability and rule out the action of the drug as the cause. Additionally,

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69 although there were no significant differen ces observed between any of the four injury/treatment groups, a neuroprotectiv e trend was observed following 80 mg/kg agmatine (Agmatine II) seen in the volume of edema on days 3 and 7; blood vessel leakage on days 3, 7, 15, and 25; and total injury volume on days 3, 7, 15, and 25. Increasing the number of animals would also reduce variability and provide significance. An equally important finding in this st udy was the sensitivity and capability of in vivo MR imaging to study the neuroprotective effects of agmatine in a progressive model of spinal injury over time in the same animal. It is important in research to affectively minimize resources and cost. With the capability of using an animal as its own control in a time-course study, the need for an excessive number of animals to obtain multiple data time-points is eliminated, as shown in the curr ent study. The data is thus more accurately comparable, as it allows you to compare preand post-injury data for a single animal. Secondary injury events, such as neural degeneration and cavitation in spinal injury, both a result of glutamate induced excitotoxicity, have been well documented (Faden and Simon, 1988; Nag and Riopelle, 1990; Hao et al., 1991; Yezierski et al., 1993; Wrathall et al, 1994). These studies indi cate that elevations in glutamate signify the initiation of cellular cascades that in fluence excitability and survivability of surrounding neurons. Limiting such events through the use of neuroprotective agents would have a profound impact on secondary pr ocesses involved in th e mediation of cell death and the progression of s econdary injury in SCI. Agmatine, a cationic amine formed by the decarboxylation of argini ne, is such an agent that has been shown to affectively block NMDA receptor activity (Yang and Reis, 1999) and reduce the production of nitric oxide by inhibiting NOS (Auguet et al., 1995; Galea et al., 1996).

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70 The present results showed that during a survival period of 30 days, 80 mg/kg agmatine (Agmatine II) provided neuroprotecti on most strongly against edema and blood vessel leakage. The vehicle and 25 mg/kg agmatine (Agmatine I) treatment groups did not show neuroprotection, as expected. Although a higher volume of edema was observed in the QUIS-SCI plus Agmatine II gr oup compared to the Saline-SCI group, it was not as widespread, which is evident in the longitudinal profile of edema, meaning that the edema was more concentrated around th e epicenter of the injury. The same is true for the total volume of injury; while the QUIS-SCI plus Agmatine II group had a higher volume of injury compared to the Saline-SCI group, the injury was not as widespread along the rostral-cauda l axis of the cord. This ra ises the question of whether volume or rostral-caudal extent is more im portant. Previous studies have shown that length is a more determining factor in rela tion to behavioral outcome. For example, studies have shown a relationship between th e onset of excessive grooming behavior and the longitudinal extent of ex citotoxic damage produced by a QUIS lesion (Yezierski et al., 1998; Gorman et al., 2001; Plunkett et al., 2001; Yu et al., 2003). Yu et al. (2003) suggested that by limiting the spread of QUIS induced excitotoxic damage via neuroprotection, the expression of injury-induced pain behaviors may lessen. This is one indication that the longitudinal profile versus volume results hold a stronger indication of neuroprotection. Observing changes in the dynamics of th e BSCB is important in understanding the destructive pathologic pro cesses resulting from an exci totoxic lesion (Rapoport, 1976; Bradbury, 1979). Understanding the time cour se of permeability of the BSCB and the subsequent pathological consequences is crucial in understanding the secondary

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71 mechanisms associated with spinal cord inju ry. Previous studies by Runge et al. (1997) and Bilgen et al. (2001) involving the cont usion injury have shown cord-barrier disruption during the acute phase of injury. Previously, it was shown that in later timepoints (i.e.,17-24 days post-injury), the ba rrier was intact following a QUIS lesion (Chapter 3, Berens et al., 2005). At the tim e-points used in this study, a dynamic profile of BSCB disruption was achieved. The BSCB showed disruption immediately (day 1) following injury, and persisted until day 7 pos t-injury, with a maximum disruption on day 3 post-injury. Repair of the barrier was ev ident, because on day 7, even though there was a significant increase in peak enhancement, there was an overall decrease in value as compared to earlier time-points (days 1 and 3) No significant diffe rences were observed between any of the injury/treatment groups compared to the Saline-SCI group indicating that agmatine may not play a significant ro le in attenuating cord-barrier disruption.

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72 CHAPTER 5 DIFFUSION WEIGHTED MR IMAGING OF THE EXCISED QUISQUALIC ACID LESIONED RAT SPINAL CORD Introduction Diffusion weighted MR imaging of water tr anslational diffusion has been utilized to provide information beyond the anatomic and spatial resolu tion of conventional magnetic resonance imaging. It has a greater sensitivity, compared to conventional MR techniques, in evaluating the integrity of injure d white matter (Ford et al., 1994a), and assessing the degree of neur oprotection, axonal sprouting, and regeneration following therapy (Nevo et al., 2001; Schwartz et al., 2003; Schwartz et al., 2005). White and gray matter vary in their diffusi on characteristics due to their structural makeup. When considering diffusion prope rties, it is important to consider microstructure properties, such as cell size and density, cell orientation, membrane permeability, and intracellular and extracellular viscosity of the tissue. In addition, cell membranes and myelin can also restrict wa ter diffusion along the axis perpendicular to white matter tracts (Beaulieu, 2002; Schwartz and Hackney, 2003). Water diffusion in gray matter is fairly unrestricted and its direc tionality is more random than white matter. However in white matter, diffusion is along bundles of parallel, myelinated axons, is restricted (Moseley et al., 1990). Thus, norma l spinal cord is considered anisotropic, since there are more barriers to diffusion in th e plane transverse to the long axis of the cord (Schwartz and Hackney, 2003).

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73 In the present study, DWI was used to detect changes in fiber structure in white and gray matter following excitotoxi c spinal injury. It is hypothesized that DWI can be employed to evaluate the long term effects of excitotoxic injury on fiber tracts, and that it has the sensitivity to detect differences in gray matter fiber structure between control (non-injured) and QUIS lesioned cords. Materials and Methods Animals, Surgery, and Neuroprotection Sprague-Dawley male rats weighing 250-300g were subjected to excitotoxic spinal cord injury as described in Chapter 4. Rats were randomly assigned to one of three injury groups: (1) Control (no injury N = 3); (2) Saline-SCI (s ham injury, N = 5); and (3) QUIS-SCI (N = 8). Animals were sacrificed on day 30 post-injury, and cords prepared as described in Chapter 3. Different groups were used than those described in Chapter 4 in order to verify the technique. Differences needed to be observed between the three groups listed above before using this me thodology in the assessment of neuroprotective treatment (see Chapter 6, Future Directions). In Vitro Three Dimensional MR Microscopy and Diffusion Weighted Imaging All in vitro NMR measurements were perfor med on a 14.1T, 51-mm-bore Oxford magnet, at 600 MHz, using a Bruker BIOSPEC Avance DBX console. All excised cords were subjected to in vitro three-dimensional MR microsc opy as described in Chapter 4, with cord preparation described in Chapte r 3. Following 3D MRM acquisition, diffusion weighted MR images were acquired using a spin-echo, high angular resolution diffusion imaging (HARDI, Tuch et al., 2002) ac quisition sequence (TR/TE = 1400 ms/25 ms) with gradients applied in 46 di rections with a gradient stre ngth of 664 mT/m (b-value of 1250 s/mm2). A low b-value image was also obt ained with a gradient strength of 175

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74 mT/m (b-value of 100 s/mm2) applied in 6 directions. Additional parameters were echo crusher duration = 0.12ms, value = 17.5 ms, value = 1.5 ms, bandwidth = 32 kHz, FOV = 4.2 x 4.2 x 12.0 mm3, matrix = 70 x 70 with 40 slices, and resolution = 60 x 60 x 300 m3. Scan times were 9.5 hours for each cord. General image processing and analysis was performed using custom software written in the Interactive Data Language (IDL, from Research Systems, Boulder, CO). Data Analysis Diffusion weighted MR data were fit to a rank-2 tensor model as the initial method of analysis. To determine the fractional an isotropy and average diffusivity of the white and gray matter for each animal of each inju ry group, regions of interest (ROI) were drawn using the S0 MR image in the following regions of white matter (Fig. 5-1): A, dorsal column, right side; B, dorsal column, left side; C, lateral, right side; D, lateral, left side; E, ventral, right side; and F, ventral, left side; and in the following regions of gray matter (Fig. 5-2): A, dorsal horn, non-injured side; B, lateral, right side; C, lateral, left side; D, ventral, right side; and E, ventral, left side. Regions of interest were not drawn in the dorsal horn on the injured side, because a value of zero would result; an outcome due to the hypointensity of the signal. For each outcome measurement (i.e., fractional anisotropy and average diffusivity), values were averaged for each ROI and injury group. Saline and QUIS injury groups were then compared to the control group. To further characterize the QUIS lesions, a diffusion orientation transform (DOT) method (Ozarslan, et al., 2006) was applied to the analysis of excised rat spinal cord, diffusion-weighted image data acquired with the HARDI acquisition scheme. The DOT has recently been developed to resolve the ar chitecture of complex structured tissue. The DOT transforms the HARDI measured diffusi vity profiles into displacement probability

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75 profiles, representing the probabili ty of diffusion in a particular direction. Rather than fit the measured diffusion signal to a rank-2 m odel of diffusion, the DOT method assumes the signal attenuation (as a f unction of diffusion weighting) is represented by a monoexponential decay. Then this signal attenua tion is used to directly calculate the displacement probability, as a function of position, using a q-space Fourier transformation (Callaghan, 1991). Therefore, the peaks in th ese displacement probability profiles correspond to distinct fiber orientat ions. Using the DOT, the QUIS lesion can be further characterized to delineate and descri be complex fiber structures, specifically in the gray matter. Results First, to determine the effects of saline and QUIS lesions on white matter as compared to control (no injury ), average diffusivity (AD) and fractional anisotropy (FA) values were calculated for the six white ma tter regions described above for each injury group (see Data Analysis, Fig. 5-1). Data for each group was combined and averaged by matching and centering injured areas for each cord, because values along the rostrocaudal axis in a normal cord did not vary (i.e., it was not necessary to match the slices anatomically). Fractional anisot ropy values of one indicate anisotropic (unidirectional) diffusion; zero indicates isotropic (non-prefer ential) diffusion. Figures 53 through 5-5 show the AD and FA values of white matter for the Control, Saline-SCI, and QUIS-SCI groups, respectively. The AD values in control cords were very consistent along the rostral-caudal axis (Fig 5-3A). However, there was a marginal increase in AD from rostral to caudal in bot h Salineand QUIS-SCI groups (Figs. 5-4A and 5-5A, respectively). Fractional anisotropy decreased slightly from rostral to caudal for all three groups but was equivalent between all six regions of interest in white matter

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76 (Figs. 5-3B, 5-4B, and 5-5B). There were no significant differences in AD or FA values between any of the six white matter regions of interest for any of the three groups. There were no significant differences in AD or FA between any of the injury groups as compared to control. Figure 5-1. S0 (b = 100 s/mm2) transverse MR image from a QUIS lesioned animal showing regions of interest drawn for an alysis of the white matter. A, dorsal column white matter, right side; B, dorsal column white matter, left side; C, lateral white matter, right side; D, lateral gray matter, left side; E, ventral gray matter, right side; F, ventral gray matter, left side. Figure 5-2. S0 (b = 100 s/mm2) transverse MR image from a QUIS lesioned animal showing regions of interest drawn for an alysis of the gray matter. A, dorsal horn gray matter, non-injured side; B, late ral gray matter, righ t side; C, lateral gray matter, left side; D, ventral gray ma tter, right side; E, ventral gray matter, left side. A B D C E F A B D C E

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77 Second, to determine the effects of the saline and QUIS lesions on gray matter as compared to control (no injury), AD and FA values were also calculated for the five gray matter regions described above for each inju ry group (see Data Analysis, Fig. 5-2). Figures 5-6 through 5-8 show the AD and FA values of gray matter for the Control, Saline-SCI, and QUIS-SCI groups, respectively. The AD values for gray matter for all three groups were very consistent along the ro stral-caudal axis (Figs. 5-6A, 5-7A, 5-8A). Fractional anisotropy remained constant from rostral to caudal for the Saline-SCI group but had a negligible increase in the Control and QUIS-SCI gr oups (Figs. 5-6B and 5-8B, respectively). There were no significant diffe rences in AD or FA values between any of the five gray matter regions of interest for any of the three groups. Surprisingly, there were no significant differences in AD or FA between any of the injury groups as compared to control. Since, no differences were detected with th e initial series of analysis, DOT analysis was used to determine if this more sensitiv e method could detect changes in white and gray matter fibers in control and QUIS-SCI co rds (Fig. 5-9). In the Control group (Fig. 5-9A, C), white matter fiber orientation was obs erved to be out of the plane of section (i.e., along the long axis of the cord), as e xpected. Gray matter fibers showed a much more complex orientation and were observed to be in the plane section, also as expected. Fibers situated in the ventral regions of the gray matter appear to be more complex than those in dorsal regions of the dorsal horn. Mo st interestingly, however, are fibers in the substantia gelatinosa; they appear to ali gn along the long axis of the cord. One would expect fibers to be within the transverse pl ane versus along the long axis, as fibers enter the substantia gelatinosa from the dorsal roots and traverse into the dorsal horn.

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78 0.00E+00 5.00E-05 1.00E-04 1.50E-04 2.00E-04 2.50E-04 3.00E-04 3.50E-04 4.00E-04 135791113151719212325PositionMean AD Value Dorsal Column White Matter-Right Dorsal Column White Matter-Left Lateral White Matter-Right Lateral White Matter-Left Ventral White Matter-Right Ventral White Matter-LeftA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 135791113151719212325PositionMean FA Value Dorsal Column White Matter-Right Dorsal Column White Matter-Left Lateral White Matter-Right Lateral White Matter-Left Lateral White Matter-Right Ventral White Matter-LeftB Figure 5-3. Average diffusivity and fractional anisotropy values for Control white matter. Position is rostral (left side of graph) to caudal (ri ght side of graph). (A) Average diffusivity along the rostral-caudal axis of white matter is consistent. No significant differences were detected between any of the control white matter regions for either of the measurements. (B) There was a slight decrease in FA from rostral to caudal for all white matter regions.

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79 0.00E+00 5.00E-05 1.00E-04 1.50E-04 2.00E-04 2.50E-04 3.00E-04 3.50E-04 4.00E-04 13579111315171921232527PositionMean AD Value Dorsal Column White Matter-Right Dorsal Column White Matter-Left Lateral White Matter-Right Lateral White Matter-Left Ventral White Matter-Right Ventral White Matter-LeftA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 13579111315171921232527PositionMean FA Value Dorsal Column White Matter-Right Dorsal Column White Matter-Left Lateral White Matter-Right Lateral White Matter-Left Ventral White Matter-Right Ventral White Matter-LeftB Figure 5-4. Average diffusivity and fracti onal anisotropy values for Saline-SCI white matter. Position is rostral (left side of graph) to caudal (right side of graph). (A) Average diffusivity along the rost ral-caudal axis of white matter is consistent, with a slight increase along the rostral caudal axis. No significant differences were detected between any of the control white matter regions for either of the measurements. (B) Ther e was a slight decrease in FA from rostral to caudal for all white matter regions.

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80 0.00E+00 5.00E-05 1.00E-04 1.50E-04 2.00E-04 2.50E-04 3.00E-04 3.50E-04 4.00E-04 135791113151719212325PositionMean AD Value Dorsal Column White Matter-Right Dorsal Column White Matter-Left Lateral White Matter-Right Lateral White Matter-Left Ventral White Matter-Right Ventral White Matter-LeftA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 135791113151719212325PositionMean FA Value Dorsal Column White Matter-Right Dorsal Column White Matter-Left Lateral White Matter-Right Lateral White Matter-Left Lateral White Matter-Right Ventral White Matter-LeftB Figure 5-5. Average diffusivity and fracti onal anisotropy values for QUIS-SCI white matter. Position is rostral (left side of graph) to caudal (right side of graph). (A) Average diffusivity along the rost ral-caudal axis of white matter is consistent, slightly increasing from rostral to caudal. No significant differences were detected between any of the control white matter regions for either of the measurements. (B) Ther e was a slight decrease in FA from rostral to caudal for all white matter regions.

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81 0.00E+00 1.00E-04 2.00E-04 3.00E-04 4.00E-04 5.00E-04 6.00E-04 13579111315171921232527PositionMean AD Value Dorsal Horn Gray Matter-Non-injured Lateral Gray Matter-Right Lateral Gray Matter-Left Ventral Gray Matter-Right Ventral Gra y Matter-LeftA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 13579111315171921232527PositionMean FA Value Dorsal Horn Gray Matter-Non-Injured Lateral Gray Matter-Right Lateral Gray Matter-Left Ventral Gray Matter-Right Ventral Gra y Matter-LeftB Figure 5-6. Average diffusivity and fractional anisotropy values for Control gray matter. Position is rostral (left side of graph) to caudal (ri ght side of graph). (A) Average diffusivity along the rostral-cauda l axis of gray matter is consistent from rostral to caudal. No significant differences were detected between any of the control white matter regions fo r either of the measurements. (B) Fractional anisotropy values also remain consistent from rostral to caudal for all gray matter regions.

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82 0.00E+00 1.00E-04 2.00E-04 3.00E-04 4.00E-04 5.00E-04 6.00E-04 13579111315171921232527PositionMean AD Value Dorsal Horn Gray Matter-Non-Injured Lateral Gray Matter-Right Lateral Gray Matter-Left Ventral Gray Matter-Right Ventral Gra y Matter-LeftA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 13579111315171921232527PositionMean FA Value Dorsal Horn Gray Matter-Non-Injured Lateral Gray Matter-Right Lateral Gray Matter-Left Ventral Gray Matter-Right Ventral Gra y Matter-LeftB Figure 5-7. Average diffusivity and fracti onal anisotropy values for Saline-SCI gray matter. Position is rostral (left side of graph) to caudal (right side of graph). (A) Average diffusivity along the rost ral-caudal axis of gray matter is consistent from rostral to caudal. No significant differences were detected between any of the control white matter regions for either of the measurements. (B) Fractional anisotropy values also remain consistent from rostral to caudal for all gray matter regions.

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83 0.00E+00 1.00E-04 2.00E-04 3.00E-04 4.00E-04 5.00E-04 6.00E-04 135791113151719212325PositionMean AD Value Dorsal Horn Gray Matter-Non-Injured Lateral Gray Matter-Right Lateral Gray Matter-Left Ventral Gray Matter-Right Ventral Gray Matter-LeftA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 135791113151719212325PositionMean FA Value Dorsal Horn Gray Matter-Non-Injured Lateral Gray Matter-Right Lateral Gray Matter-Left Ventral Gray Matter-Right Ventral Gray Matter-LeftB Figure 5-8. Average diffusivity and fracti onal anisotropy values for QUIS-SCI gray matter. Position is rostral (left side of graph) to caudal (right side of graph). (A) Average diffusivity along the rost ral-caudal axis of gray matter is consistent from rostral to caudal. No significant differences were detected between any of the control white matter regions for either of the measurements. (B) Fractional anisotropy values also remain consistent from rostral to caudal for all gray matter regions.

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84 C D Figure 5-9. Probability maps calculated from diffusion weighted datasets collected from excised normal and QUIS injured spinal cords. (C) and (D) are enlargements taken from (A) normal; and (B)QUIS injure d spinal cords, respectively. The orientation surfaces are color-coded such th at portions in or out of the plane of section are blue and green, respectivel y. Notice the loss of complexity of fiber structure in the dorsal horn, specif ically along the medial regions (green circle) in the QUIS injured cord.

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85 While comparing the effects of a QUIS lesi on (Fig. 5-9 B, D) versus Control (Fig. 5-9A, C), a loss of complexity in gray matte r fiber structure is obs erved in the QUIS-SCI group. There appears to be a change in fiber orientation or a lining up of fibers along the medial dorsal horn gray matter (Fig. 5-9D, green circle) in the injured cord compared to a control cord. No changes were observed in fiber structure or orie ntation in the white matter or ventral regions of the gray matter in the QUIS injured cords. Discussion The goal of the present study was to us e diffusion weighted MR imaging to evaluate changes in white and gray matter fibers in the spinal cord following excitotoxic injury. Major findings include: (1) DWI can be used to quantitativ ely and qualitatively study excitotoxic spinal cord injury; and (2) DWI has the sensitivity to detect changes in gray matter fiber structure between contro l (non-injured) and QUIS lesioned cords. Differences were observed in FA and AD va lues between white and gray matter as expected. Their diffusion characteristics differ because of differences in their structural makeup. Gray matter consists of cell bodies and is reasona bly unrestricted, with more random directionality in water diffusion (Mos eley et al., 1990) than white matter. In contrast, white matter consists of long parallel bundles of myelinated axons, where water diffusion is mainly restricted to the long or ro strocaudal axis of the cord (Moseley et al., 1990). Thus, FA values in gray matter were lower that those in white matter and conversely, AD values higher in gray matter than white matter, because gray matter is considered more isotropic (unrestricted diffusi on in all directions), whereas, diffusion in white matter is considered anisotropic (re stricted diffusion in one direction). With DOT, fiber structure and orientation in the white matter was fairly uniform. Contrastingly, fiber orientation in the gr ay matter varied among dorsal and ventral

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86 regions. Fibers situated in the ventral regi ons of the gray matter appear to be more complex than those in the dorsa l regions of the dorsal horn. This is because propriospinal connections involving lamin ae IVVII are far more extensive within and between segments than those involving more superfic ial laminae (Yezierski et al., 1980; Menetrey et al., 1985). Since DOT was able to define fiber orientation and had the sensitivity to detect changes following injury, it may be possible to apply this technique to gain further insight into the differences seen with the tw o different injury stra tegies described in Chapter 3 and ultimately more details about the timing of events and the resolution of spatial location of pathologic changes following spinal injury could finally be discovered. When comparing FA and AD values in differe nt regions of the cord, in Control and QUIS-SCI groups, results were three-fold. Firs t, there were no differences in FA or AD values in any of the regions of white (6 ROIs) or gray matter (5 ROIs), separately, meaning all white matter in a particular section had similar values; the same being true for gray matter. However, values varied s lightly along the rostrocau dal axis of the cord because of the change in structural makeup. Secondly, results predictably revealed no significant differences in FA or AD values in the white matter, because the QUIS lesion is a gray matter lesion. Thirdly, results surp risingly showed that th ere were no significant differences between Control and QUIS-SCI groups in the gray matter. A loss in FA and an increase in AD were expected in the QUI S-SCI group around the in jury site in the initial analysis versus Control. Explanations might be that the method of analysis did not have the sensitivity to detect changes in FA or AD values for this particular injury model (i.e., changes may have been too subtle).

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87 With this in mind, DOT analysis was then used to evaluate fiber structure in both white and gray matter. DOT, an accurate and fast technique for fiber orientation mapping (zarslan et al., 2005), is a relatively new method for DWI analysis with far greater sensitivity in detecting changes in fibers follo wing injury. With this method, it was then possible to identify fiber structure change s in the QUIS lesioned cord. QUIS-SCI animals showed a loss in complexity of fiber structure in the dorsal horn, specifically along the medial regions in the gray matter comp ared to Control. This may be attributed to a loss in fibers, a re-growth of fibers in a different orientation, or perhaps a reorganization of fibers in the post-injury state. The change s observed are likely a loss in fibers versus a re-growth or re-organization because QUIS lesions result in excitotoxic cell death of the gray matter. Future histolog ical analysis will be critical in evaluating and validating these changes, in order to resolve these differences. Experimentally, diffusion weighted MRI ha s given researchers a new tool to study disease and injury. Diffusion weighted MR imaging in ex vivo studies of spinal cord injury have been shown to be more sensitiv e in detecting changes in areas surrounding a contusion injury than conventi onal MRI (Ford et al., 1994a). It is also more sensitive than conventional MR techni ques in early detection of cavity formation following excitotoxic spinal cord injury (Schwartz et al., 1999b). Addi tionally, Nevo et al. (2001) used DWI to study neuroprotection following a contusion injury in rats and found the method successful in evaluating neuroprotective effects and that the results corresponded with behavioral outcome. Diffusion weight ed MRI has also proven to be a successful method in assessing the effects of brain derive d neurotrophic factor secreting fibroblasts following spinal injury (Schwartz et al., 2003). This technique shows considerable

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88 possibilities and has higher se nsitivity in revealing pathol ogy that would otherwise not show on conventional MR images. Diffusion weighted imaging has also become a valuable tool in the clinical setting. Diffusion weighted MR images have been us ed to show diffusion changes within the spinal cord, which can sometimes be missed with conventional MRI, showing no signal abnormality, as demonstrated in patients with canal stenosis (Clark and Werring, 2002). Diffusion weighted MRI has also become an established method for the evaluation and early diagnosis of acute cereb ral ischemia (van Gelderen et al., 1994; Schlaug et al., 1997; Lvblad et al., 1998; Loher et al., 2003). With Wallerian dege neration, th ere is a change in signal intensity as seen with conventional MRI; howe ver, they cannot be differentiated from edema (Becerra et al ., 1995; Schwartz et al., 2003). Previous diffusion studies using DWI have also shown th at water diffusion may be altered in white matter tracts following Wallerian degenera tion (Segawa et al., 1993; Beaulieu et al., 1996; Pierpaoli et al., 1996; Makris et al., 1997; Pierpaoli et al., 1998; Castillo and Mukherji, 1999). Ries et al ., (2000) used diffusion weight ed imaging to investigate patients suffering from narrowing of the cervic al cord and concluded that DWI had the sensitivity to detect changes in diffusion characteristics in the cord. They were able to localize structural fiber damage in the spin al cord where T1and T2-weighted images were unable to detect any lesion. DWI is cl early a useful tool in the investigation of disease, if one puts aside the expense and ma gnitude of space require d in the laboratory. There are several advantages and disadvantag es associated with diffusion weighted MR imaging as compared with conventional MRI and histological methods in tracking neural pathways. It has the capability to provide more detailed information about tissue

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89 structure and integrity than conventional MR methods (Schwartz et al., 2003). Diffusion weighted MRI also has the potential to dete ct ongoing changes in cerebral and spinal connections, specifically those in neural deve lopment and the acute and chronic study of diseases that affect white and gray matter (Rye, 1999). It is exceedingly sensitive in resolving white matter myelination (Sakum a et al., 1991; Wimberger et al., 1995) and determining the involvement of pathways by pa thological processes (M akris et al., 1997) and evaluation of acute ischemic stroke (Koroshetz and Gonzal ez, 1997; Fisher and Albers, 1999). Additionally, it has the capab ility to enhance detection and provide insights into the diagnosis and treatment of disorders such as multiple sclerosis, encephalomyelitis, and the leukodystophies all of which involve dysfunctional myelinated fibers (Rye, 1999). Unfortunate ly, however, diffusion weighted imaging cannot resolve individual components of larg e axon bundles (i.e., affe rent and efferent fibers) and there are fiber-tracking difficulties when two pathways are adjacent and diverge near their terminations (Rye, 1999; Basser et al., 2000). Histological methods are advantageous over DWI and remain the gold standard for the visu alization of fiber pathways (i.e., Nauta method, electron micr oscopy) and it is convenient to use (Rye, 1999). However, the use of histological tracer molecules to label pathways has considerable shortcomings because active transp ort requires viable surgical specimens or post-mortem tissue and inadequate transport prevents visualization of most pathways (Rye, 1999).

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90 CHAPTER 6 CONCLUSION Summary of Findings High-resolution MRI can be used to st udy experimental SCI quantitatively and qualitatively with high field magnets using customized coils and optimized methods. In vivo MRI was sensitive enough to detect si gnificant differences in pathological characteristics between two different strategi es of injury (QL1 versus QL2). Although significant differences were found am ong injury lengths calculated from in vivo and in vitro images, they were within the range of difference in the slice thickness for each method used. These results indicate that in vivo MRI may underestimate total injury length by no more than one sl ice thickness due to volume aver aging in the end slices. High-resolution, in vivo MRI methods can also be used to effectively evaluate the progression of pathological changes longitudinally in the same animal. Additionally, in vivo MRI has the capability to monitor the e fficacy of a pharmacological agent with known neuroprotective properties to protect the injured spinal cord. Results revealed a specific temporal profile of pathological changes (edema, cavitation, and blood vessel leakage) as well as a specific pattern of change in the integrity of the blood-spinal cordbarrier following excitotoxic spinal cord injury. The use of 80 mg/kg agmatine following spinal cord injury has a significant effect on pathological outcomes, specifically blood vessel leakage and edema. Structure and orientation of white and gray matter fiber tracts can be effectively evaluated with sophisticated diffusion weighted MR imaging methods. Diffusion

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91 weighted MRI had the sensitivity to detect fiber patterns in a normal cord, showing orientation differences between white and gr ay matter, as well as differences within regions of gray matter. When comparing to Control, DWI also had the capability to detect changes in fibers in a QUIS lesioned spinal cord. Future studies will need to be carried out to understand and validate these changes in fiber orientation. Future Directions Numerous experiment ideas arose from the experiments described in Chapters 3-5, as expected. Below is a description of several future studies that would nicely complement the aforementioned experiments. In order to further understand changes in pathology following the excitotoxic lesion, magnetic resonance spectroscopy (MRS ) could be used to gain noninvasive insight into the biochemical changes respons ible for the dynamic changes in pathology following spinal cord injury. Currently, it is known that vasoactive substances (e.g., reactive oxygen molecules, bradykinins, hist amines, and nitric oxide) are released by trauma-activated endothelial and glial cells, which facilitate the crossing of plasmaderived molecules into the cord, further ex acerbating the injury (Hausmann, 2003). Using MRS, it may be possible to discover additiona l events that take place leading up to and consequences of BSCB disruption. Future studies could also use MRI techniques for the study of angiogenesis as well as examine the use of different contrast agents (smart contrast agents) for the noninvasive study of gene expression following SCI (Bell and Taylor-Robinson, 2000). These agents have the capability to be switched on in response to physiological changes within a cell. This could lead to a further understanding of events following cha nges in the integrity of the BSCB. In vivo highresolution MRI could also be us ed clinically and experimental ly to observe neural tissue

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92 transplantation (Reier et al ., 1992a; Reier et al., 1992b; Thom pson et al., 2001; Wirth et al., 2001) following spinal cord injury at multiple time-points. Future studies could examine the effects of other neuroprotec tive agents (e.g., LPhenylalanine derivatives; Kagiyama et al., 2004) using similar out come measures as those described in previous chapters and compare the efficacy of these agents to agmatine. An obvious future direction will be to correlate the neuroprotective effects with functional outcomes. Additionally, agmatine could be analyzed further, using other outcome measures at multiple time-points (e.g., Yu and Yezierski, 2004). Diffusion weighted MR imaging is becomi ng an undeniably valuable tool in understanding changes in spinal cord fibe r tracts following spinal injury. Chapter 5 involved comparing normal and QUIS lesioned cords. Histology, which serves a gold standard for the visualization of fiber pathways, can be done to further comprehend and verify the changes observed. Additionally, once these changes are confirmed, DWI could then be used to further u nderstand the neuroprotective effects of agmatine, as well as other treatment regimens. With continue d improvements to these techniques, more details about the timing of events and the re solution of spatial location of pathologic changes following spinal injury could finally be discovered.

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106 Yezierski RP, Liu S, Ruenes GL, Kajander KL, Brewer KL. Excitotoxic spinal cord injury: Behavior and morpholog ical characteristics of a central pain model. Pain 1998; 75:141-155. Yezierski RP, Park SH. The mechanosensiti vity of spinal sensory neurons following intraspinal injections of quisquali c acid. Neurosci Lett 1993; 157:115-119. Yezierski RP, Santana M, Park SH, Mads en PW. Neuronal degeneration and spinal cavitation following intraspina l injections of quisqualic acid. J Neurotrauma 1993; 10:445-456. Yong C, Arnold PM, Zoubine MN, Citron BA, Watanabe I, Berman NE, Festof BW. Apoptosis in cellular compartments of rat sp inal cord after severe contusion injury. J Neurotrauma 1998; 15:459-472. Yoshioka A, Bacskai B, Pleasure D. Pathophys iology of oligodendrogli al excitotoxicity. J Neurosci Res 1996; 46:427-437. Young W, Yen V, Blight A. Extracellular calcium ionic activity in experimental contusion injury. Brain Res 1982; 253:105-113. Yu CG, Marcillo AE, Fairbanks CA, W ilcox GL, Yezierski RP. Agmatine improves locomotor function and reduces tissue da mage following spinal cord injury. NeuroReport 2000; 11:3203-3207. Yu CG, Fairbanks CA, Wilcox GL, Yezierski RP. Effects of agmatine, interleukin-10, and cyclosporine on spontaneous pain behavi or after excitotoxic spinal cord injury in rats. J Pain 2003; 4: 129-140 Yu CG, Yezierski RP. Activation of the ER K1/2 signaling cascade by excitotoxic spinal cord injury. Mol Brain Res 2005; 138:244-255. Yu, CG, Yezierski RP. Agmatine targets multiple signaling pathways to produce neuroprotective effects following spinal co rd injury. Society for Neuroscience. 2004; San Diego, CA. Zhu MY, Piletz JE, Halavis A, Regunathan S. Effect of agmatin e against cell death induced by NMDA and glutamate in neur ons and PC12 cells. Cell Mol Neurobiol 2003; 23:865-872.

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107 BIOGRAPHICAL SKETCH Sara Ann Berens was born in Hays, Kansas, in 1979, but grew up in Jacksonville, Florida. During her years at Mandarin Hi gh School, Sara was a member of the National Honor Society. Sara also played clarinet and alto clarinet in the band throughout her high school years, during which the band part icipated and placed highly in numerous marching and concert competitions. After gra duating with honors in 1998, Sara enrolled at the University of Florida, becoming a me mber of the National Society of Collegiate Scholars. Sara had worked all through her undergraduate years, gaining research experience in hopes of attending medical sc hool. She gained valuable laboratory experience as a laboratory stockroom assistant for the organic chemistry lab. Simultaneously, she also began working for Dr Robert Yezierski, as an undergraduate laboratory student assistant. However, a nother professor, Dr. Wolfgang Streit, whom Sara collaborated with, had enlightened he r about the graduate opportunities in the University of Florida College of Medicines Interdisciplinary Program (IDP) in Biomedical Sciences, and she decided that this route was more suited to her interests. Sara graduated with a Bachelor of Scien ce in chemistry in 2002, and entered the IDP program that Fall, where she joined the laborat ory of Dr. Yezierski once again. It was his interest for collaboration with Dr. Thomas H. Mareci that set Sara up for her dissertation work. She played the role as bridge be tween two collaborators who might not have worked together due to the volume of time and work involved, thereby interconnecting two unrelated fields of expertise. Her dissertation work has focused on the use of

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108 magnetic resonance imaging in the study of the dynamics of excitotoxic spinal cord injury. Upon graduation, Sara hopes to cont inue her research in a neuroscience related field in the pharmaceutical, medical, or biomedical industry.


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Permanent Link: http://ufdc.ufl.edu/UFE0015380/00001

Material Information

Title: Use of MRI to study excitotoxic spinal cord injury
Physical Description: xiv, 108 p.
Language: English
Creator: Berens, Sara Ann ( Dissertant )
Yezierski, Robert P. ( Thesis advisor )
Mareci, Tom ( Reviewer )
Blackband, Steve ( Reviewer )
Anderson, Doug ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2006

Subjects

Subjects / Keywords: Neuroscience (IDP) thesis, Ph.D   ( local )
Dissertations, Academic -- UF -- Neuroscience (IDP)   ( local )

Notes

Abstract: Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy USE OF MRI TO STUDY EXCITOTOXIC SPINAL CORD INJURY By Sara Ann Berens August 2006 Chair: Robert P. Yezierski Major Department: Neuroscience Spinal cord injury (SCI) is an important cause of morbidity and mortality in modern society. Understanding the mechanisms responsible for acute and chronic spinal cord injury pathology as well as secondary consequences and the development of effective strategies for therapeutic intervention is of great clinical importance. Although most of what is known about the pathological changes associated with experimental SCI has come from histological studies, the ability to non-invasively characterize spinal cord pathology provides a way to study the temporal profile of pathological changes in the injured spinal cord using the same animal at multiple time points. A technique with the capability of achieving this objective is high-resolution magnetic resonance imaging (MRI), which is becoming a preferred method for the visualization of SCI. Three-dimensional magnetic resonance microscopy and diffusion weighted imaging (DWI) are additional techniques that can be used to study SCI. Collectively, these MRI methods were used to evaluate the progression of excitotoxic injury, including changes in pathology, blood-spinal cord-barrier integrity, and white and gray matter fiber tracts, as well as evaluate the neuroprotective effects of a pharmacological agent with known neuroprotective properties on the excitotoxic lesion. It was found that high-resolution MRI can be used to study experimental SCI quantitatively and qualitatively with high field magnets using customized coils and optimized methods. This method had the sensitivity to detect significant differences in pathological characteristics between two different strategies of injury, as well as effectively evaluate the progression of pathological changes longitudinally in the same animal. Magnetic resonance imaging had the capability to monitor the efficacy of a pharmacological agent with known neuroprotective properties to protect the injured spinal cord. Additionally, structure and orientation of white and gray matter fiber tracts were evaluated with sophisticated diffusion weighted MR imaging methods. Diffusion weighted MR imaging had the sensitivity to detect fiber patterns in a normal cord and reveal changes in fiber orientation in quisqualic acid lesioned spinal cords. With continued improvements and application of these techniques, it will be possible to determine additional insights into the mechanism responsible for the progression of secondary pathologic changes following spinal injury.
Subject: agmatine, barrier, berens, blood, cavitation, cord, diffusion, edema, excitotoxicity, hemorrhage, imaging, injury, magnetic, neuroprotection, neuroscience, quisqualic, rat, resonance, secondary, spinal, weighted
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 122 pages.
General Note: Includes vita.
Thesis: Thesis (Ph.D.)--University of Florida, 2006.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0015380:00001

Permanent Link: http://ufdc.ufl.edu/UFE0015380/00001

Material Information

Title: Use of MRI to study excitotoxic spinal cord injury
Physical Description: xiv, 108 p.
Language: English
Creator: Berens, Sara Ann ( Dissertant )
Yezierski, Robert P. ( Thesis advisor )
Mareci, Tom ( Reviewer )
Blackband, Steve ( Reviewer )
Anderson, Doug ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2006

Subjects

Subjects / Keywords: Neuroscience (IDP) thesis, Ph.D   ( local )
Dissertations, Academic -- UF -- Neuroscience (IDP)   ( local )

Notes

Abstract: Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy USE OF MRI TO STUDY EXCITOTOXIC SPINAL CORD INJURY By Sara Ann Berens August 2006 Chair: Robert P. Yezierski Major Department: Neuroscience Spinal cord injury (SCI) is an important cause of morbidity and mortality in modern society. Understanding the mechanisms responsible for acute and chronic spinal cord injury pathology as well as secondary consequences and the development of effective strategies for therapeutic intervention is of great clinical importance. Although most of what is known about the pathological changes associated with experimental SCI has come from histological studies, the ability to non-invasively characterize spinal cord pathology provides a way to study the temporal profile of pathological changes in the injured spinal cord using the same animal at multiple time points. A technique with the capability of achieving this objective is high-resolution magnetic resonance imaging (MRI), which is becoming a preferred method for the visualization of SCI. Three-dimensional magnetic resonance microscopy and diffusion weighted imaging (DWI) are additional techniques that can be used to study SCI. Collectively, these MRI methods were used to evaluate the progression of excitotoxic injury, including changes in pathology, blood-spinal cord-barrier integrity, and white and gray matter fiber tracts, as well as evaluate the neuroprotective effects of a pharmacological agent with known neuroprotective properties on the excitotoxic lesion. It was found that high-resolution MRI can be used to study experimental SCI quantitatively and qualitatively with high field magnets using customized coils and optimized methods. This method had the sensitivity to detect significant differences in pathological characteristics between two different strategies of injury, as well as effectively evaluate the progression of pathological changes longitudinally in the same animal. Magnetic resonance imaging had the capability to monitor the efficacy of a pharmacological agent with known neuroprotective properties to protect the injured spinal cord. Additionally, structure and orientation of white and gray matter fiber tracts were evaluated with sophisticated diffusion weighted MR imaging methods. Diffusion weighted MR imaging had the sensitivity to detect fiber patterns in a normal cord and reveal changes in fiber orientation in quisqualic acid lesioned spinal cords. With continued improvements and application of these techniques, it will be possible to determine additional insights into the mechanism responsible for the progression of secondary pathologic changes following spinal injury.
Subject: agmatine, barrier, berens, blood, cavitation, cord, diffusion, edema, excitotoxicity, hemorrhage, imaging, injury, magnetic, neuroprotection, neuroscience, quisqualic, rat, resonance, secondary, spinal, weighted
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 122 pages.
General Note: Includes vita.
Thesis: Thesis (Ph.D.)--University of Florida, 2006.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0015380:00001


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Full Text












USE OF MRI TO STUDY EXCITOTOXIC SPINAL CORD INJURY


By

SARA ANN BERENS













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

SARA ANN BERENS






















This document is dedicated to my fiance, Jason, and my family for their continued love
and encouragement.















ACKNOWLEDGMENTS

First, I would like to thank my committee members, Drs. Robert Yezierski, Tom

Mareci, Steve Blackband, and Doug Anderson, for their guidance and encouragement.

Each has contributed to my development as a scientist in many different ways. I would

like to thank my mentor, Dr. Yezierski, for teaching me all there is to know about spinal

cord injury and pain, as well as performing surgeries; Dr. Mareci, for shaping my

knowledge of MR imaging, as well as showing me how to operate several of the magnet

systems; Dr. Blackband, for providing me a foundation on the principles of MR Imaging;

and Dr. Anderson for providing his expertise on spinal cord injury. I appreciate their

encouragement to write as often as I did, through publications and grants; it made the

final writing process less strenuous.

I am indebted to all of the current and former lab members of the Yezierski and

Mareci labs that I have had the opportunity to work with over the years. In particular, I

would like to thank Dr. Chen-Guang Yu for performing some of the surgeries; Jacquie

Rodgers, for moral support and lending a helping hand in the care of my animals; my lab

mate, Chris King, for moral support, helping with surgeries and animal care when I could

not be there, for sharing an understanding of similar frustrations, and for being a friend;

and Victoria Henry, for her continued moral support and guidance, problem solving

skills, all of our many lunch walks, and, most importantly, for being my "work mom."

I appreciate all those who have contributed to my success in imaging, especially

Danny Colvin and Christy Amwake, for building the surface coils; Evren Ozarslan for









helping me collect and analyze my in vitro MR data; Lari McEdwards, Suril Patel,

Matthew Feldman, and Aaron Richardson for providing assistance in data analysis; Ty

Black for his expertise and problem solving on the analysis software; and Nelly Voland,

Chris Taylor, Min Sig Hwang, Hector Sepulveda, Lan Hoang Minh, Allan Elfstrom,

Matthew Feldman, and Rashid Jawdat for providing me the opportunity to take a lunch

break during my many long hours of imaging.

I would also like to give credit to the Advanced Magnetic Resonance Imaging and

Spectroscopy (AMRIS) facility in the McKnight Brain Institute of the University of

Florida and the staff. I thank Raquel Torres and Xeve Silver for their help in placing tail

vein catheters and general technical support; Barbara Beck and Kelly Jenkins, for their

expertise in coil setup, designing the volume coil, and helping me solve signal and tuning

problems; Gary Blaskowski, for technical support and making sure the instrument ran

smoothly; and Jim Rocca and Dan Plant for checking up on me and making sure

everything was running properly. I am also grateful for the significant financial support

provided by the McKnight Brain Institute.

I would also like to thank Dr. Joseph Riley for his expertise and help with

complicated statistics.

On a more personal note, I would also like to thank my family for their continuous

love, support, and encouragement throughout the years, and for shaping me to become

the person I am today. The countless phone calls and emails helped combat stress,

allowing me to keep my sanity. My fiance, Jason, deserves special recognition, for

taking the brunt of my frustration, letting me vent when things got tough, enduring all of









my tribulations first hand, for caring and loving me, and for supporting me in all I set out

to do, never allowing me to give up.
















TABLE OF CONTENTS


page

A C K N O W L E D G M E N T S ................................................................................................. iv

TABLE OF CONTENTS .......................................................................................................... vii

LIST OF TABLES ............... ................. ........... ........................ .... .x

LIST OF FIGU RE S ......... ......................... ... .......... ........... xi

A B S T R A C T ..................................................................................... x iii

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW....................................................1

Spinal C ord Injury ....................................................... 1
Overview ....................................... .................
Prim ary and Secondary Injury M echanism s............................... .....................2
Excitotoxic M odel of Spinal Cord Injury............... .............................................3
In Vivo M agnetic R esonance Im aging................................... .................................... 4
O v e rv iew ..................... .. ..... .... ... ..... ........ .. ...... .....................4
Dynamic Contrast-Enhanced Magnetic Resonance Imaging, Blood-Spinal
C ord-B carrier .................................................................... .. ............. .6
In Vitro M agnetic Resonance Im aging ................ ................ ................................... 7
Neuroprotective Strategies for Treatment of Spinal Cord Injury ..............................9
O v erview ............................................................ ................. 9
Neuroprotective Effects of Agmatine.......................... ................................10

2 METHODOLOGICAL DEVELOPMENT ..................................... ...............13

Overview ............... ............................................................ 13
Anesthetic/Experimental Drugs ......................... ... ....................................... 13
M agnetic R esonance Im aging.................................................... ..................... .... 15
C atheter P lacem ent ............................ .................................. ....... .. ..... 15
4.7T Coil D evelopm ent ......... ................... ................. ..... ............... 15
11.1T Coil D evelopm ent ............................ .. ... ......... ..... ...............16
C o il S etu p .............. ............. .................. .............. .......................18
11.1T Revised Im aging Protocol .............. ............. ............. ............. .. 19









4.7T versus 11. T Im ages .............. .... .............. ................................... 19
Magnetic Resonance Imaging Obstacles ........................ ...........................................20
Heating Effects of the 11.1T Imaging Protocol................................................20
Signal-to-N oise R atio ........................ .. ................................ .... ............ 23

3 EVALUATION OF THE PATHOLOGICAL CHARACTERISTICS OF SPINAL
CORD INJURY WITH MAGNETIC RESONANCE IMAGING.............................25

In tro d u ctio n ...................................... ................................................ 2 5
M materials and M methods ....................................................................... ..................26
A nim al P reparation ........ .............................................................. .... .... .... 26
Intraspinal Injection Procedure................................... ............................... ....... 27
In Vivo M R Im aging ................................................... ............ ..... .................. 28
In Vivo High Resolution MR Imaging Data Acquisition ....................................28
In Vivo DCE-MR Imaging Data Acquisition ............................................... 30
In Vitro M R M icroscopy .............................................................................. 31
H istological Procedures......................................................... ................ ..... 31
D ata A naly sis .................................................... 32
R e su lts ............................ .. .. ......... .. .....................................3 3
In Vivo High Resolution M R Imaging...................................... ............... 33
In Vivo D CE-M R Im aging........................................................ ............... 37
In Vitro M R M icroscopy .............................................................................. 38
H istologic F findings ....................... .. .......................... ...... ........... 40
Discussion ..................................................41

4 USE OF MAGNETIC RESONANCE IMAGING TO STUDY THE
NEUROPROTECTIVE EFFECTS OF AGMATINE FOLLOWING SPINAL
C O R D IN JU R Y IN TH E R A T ......................................................... .....................47

In tro d u ctio n ...................................... ................................................ 4 7
M materials an d M eth od s .................................................................... .....................4 8
Anim als, Surgery, and In Vivo M R Imaging .............................. .....................48
N europrotection ............ ..... ...... .... ...... ... .. ........... .. ............. .. 48
In Vivo High-Resolution Magnetic Resonance Imaging Data Acquisition.........48
In Vivo DCE-MR Imaging Data Acquisition ............................................... 50
In Vitro Three-Dimensional MR Microscopy .............. ............ ............... 51
D ata A n aly sis ................................................ ................ .. 5 1
V olum e A naly sis ...................................................... .. ........ .... ..... ...... 52
Results ......................................... ...................... ..................53
In Vivo High Resolution Magnetic Resonance Imaging ...................................53
In Vivo Dynamic Contrast-Enhanced Magnetic Resonance Imaging ...............60
Volum e Analysis .................................. .. .. .. ........ ...............62
D iscu ssion ............... ..................................... ........................... 68









5 DIFFUSION WEIGHTED MR IMAGING OF THE EXCISED QUISQUALIC
ACID LESIONED RAT SPINAL CORD ............ ..................... ... ..............72

Introduction .............. ....... ............. ..................................... 72
M materials and M methods .................................................................... ............... 73
Anim als, Surgery, and N europrotection..................................... .......................73
In Vitro Three Dimensional MR Microscopy and Diffusion Weighted
Im ag in g .........................................................................7 3
D ata A n a ly sis ................................................................................................. 7 4
R e su lts .................................. ...................... .............................. 7 5
D iscu ssio n .................85...........................................

6 C O N C L U SIO N ......... ...................................................................... .......... ......90

Sum m ary of Findings ...................................... .. ...... ...............90
F utu re D direction s ................................................................ 9 1

L IST O F R E FE R E N C E S ......................................................................... ...................93

BIOGRAPHICAL SKETCH ................................................... ........................ 107
















LIST OF TABLES


Table pge

4-1 Summary of Significant Differences in Longitudinal Profiles of Edema,
Cavitation, and Blood Vessel Leakage, and BSCB Disruption. ...........................67

4-2 Summary of Significant Differences in Volumes of Edema, Blood Vessel
Leakage, and Cavitation. ............................................... ............................... 67
















LIST OF FIGURES


Figure page

2-1 Comparison of MR images collected on the 4.7T magnet versus the 11.1T
m agnet. ...................................................................................20

2-2 Temperature measurements collected on the 4.7T and 11.1T magnets using a
tissue equivalent phantom simulating rat abdomen. ............................................22

2-3 Comparison of images collected with high versus low SNR on the 11.1T
m agnet. ...................................................................................24

3-1 Representative pathologic findings in the same animal (S05) at the same
location observed in vivo (T2-weighted, TR/effective TE = 2000 ms/62.5 ms;
NA = 6), in vitro (TR/TE = 2500 ms/20 ms; NA = 8), and in histologic sections. .34

3-2 Sagittal images for all QUIS-injured animals. ................................. ............... 35

3-3 Transverse images for all QUIS-injured animals. ............................................. 36

3-4 Total injury lengths determined by using in vivo, in vitro, and histologic
methods for each QUIS-injured animal. ........... .......................... ...............37

3-5 Summary of in vivo, in vitro, and histologic data collected for a representative
Q L 2 anim al (S06). ........................ ...... ................... ........... ...... .... .. 38

3-6 Summary of in vivo, in vitro, and histologic data collected for a representative
Q L 1 anim al (X 03). ......................................... ................... .......... 39

4-1 Surgery and M R im aging tim eline ....................................................................... 50

4-2 Representative T2-weighted (TR/effective TE = 2000 ms/62.5 ms; NA = 12)
MR images for each injury/treatment group for each day. ............................. 54

4-3 Longitudinal profile of the presence of edema for all injury/treatment groups as
determined from in vivo T2-weighted transverse MR images. ...........................55

4-4 Longitudinal profile of the presence of cavitation for all injury/treatment groups
as determined from in vivo T2-weighted transverse MR images. .......................... 56









4-5 Longitudinal profile of the presence of blood vessel leakage for all
injury/treatment groups as determined from in vivo T2-weighted transverse MR
im ages. ............................................................................. 57

4-6 Longitudinal profile of the total pathology injury length (the presence of edema,
blood vessel leakage, and cavitation combined) for all injury/treatment groups as
determined from in vivo T2-weighted transverse MR images. ...........................58

4-7 Summary ofDCE-MRI BSCB disruption. Peak enhancement is represented as a
percentage. ......................................... ............................. 61

4-8 Volume of edema for all injury/treatment groups as determined from in vivo T2-
weighted transverse M R images. ......................................................................62

4-9 Volume of blood vessel leakage for all injury/treatment groups as determined
from in vivo T2-weighted transverse MR images. ..........................................63

4-10 Volume of cavitation for all injury/treatment groups as determined from in vivo
T2-weighted transverse M R images. ........................................... ............... 64

4-11 Volume of total injury (volume of edema, blood vessel leakage, and cavitation
combined) for all injury/treatment groups as determined from in vivo T2-
weighted transverse MR images (days 1, 3, 7, 15, and 25) and in vitro transverse
3D M R M im ages (day 30). ........................................................... .....................66

5-1 So (b = 100 s/mm2) transverse MR image from a QUIS lesioned animal showing
regions of interest drawn for analysis of the white matter. ...................................76

5-2 So (b = 100 s/mm2) transverse MR image from a QUIS lesioned animal showing
regions of interest drawn for analysis of the gray matter. .....................................76

5-3 Average diffusivity and fractional anisotropy values for Control white matter. ....78

5-4 Average diffusivity and fractional anisotropy values for Saline-SCI white
m matter. ........................................................... ................ 7 9

5-5 Average diffusivity and fractional anisotropy values for QUIS-SCI white matter.
........................................................................................................ . 8 0

5-6 Average diffusivity and fractional anisotropy values for Control gray matter. ......81

5-7 Average diffusivity and fractional anisotropy values for Saline-SCI gray matter.
........................................................................................................ . 8 2

5-8 Average diffusivity and fractional anisotropy values for QUIS-SCI gray matter. .83

5-9 Probability maps calculated from diffusion weighted datasets collected from
excised normal and QUIS injured spinal cords........................................... ........... 84















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

USE OF MRI TO STUDY EXCITOTOXIC SPINAL CORD INJURY

By

Sara Ann Berens

August 2006

Chair: Robert P. Yezierski
Major Department: Neuroscience

Spinal cord injury (SCI) is an important cause of morbidity and mortality in

modern society. Understanding the mechanisms responsible for acute and chronic spinal

cord injury pathology as well as secondary consequences and the development of

effective strategies for therapeutic intervention is of great clinical importance. Although

most of what is known about the pathological changes associated with experimental SCI

has come from histological studies, the ability to non-invasively characterize spinal cord

pathology provides a way to study the temporal profile of pathological changes in the

injured spinal cord using the same animal at multiple time points. A technique with the

capability of achieving this objective is high-resolution magnetic resonance imaging

(MRI), which is becoming a preferred method for the visualization of SCI. Three-

dimensional magnetic resonance microscopy and diffusion weighted imaging (DWI) are

additional techniques that can be used to study SCI. Collectively, these MRI methods

were used to evaluate the progression of excitotoxic injury, including changes in

pathology, blood-spinal cord-barrier integrity, and white and gray matter fiber tracts, as









well as evaluate the neuroprotective effects of a pharmacological agent with known

neuroprotective properties on the excitotoxic lesion.

It was found that high-resolution MRI can be used to study experimental SCI

quantitatively and qualitatively with high field magnets using customized coils and

optimized methods. This method had the sensitivity to detect significant differences in

pathological characteristics between two different strategies of injury, as well as

effectively evaluate the progression of pathological changes longitudinally in the same

animal. Magnetic resonance imaging had the capability to monitor the efficacy of a

pharmacological agent with known neuroprotective properties to protect the injured

spinal cord. Additionally, structure and orientation of white and gray matter fiber tracts

were evaluated with sophisticated diffusion weighted MR imaging methods. Diffusion

weighted MR imaging had the sensitivity to detect fiber patterns in a normal cord and

reveal changes in fiber orientation in quisqualic acid lesioned spinal cords. With

continued improvements and application of these techniques, it will be possible to

determine additional insights into the mechanism responsible for the progression of

secondary pathologic changes following spinal injury.














CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW

Spinal Cord Injury

Overview

Spinal cord injury (SCI) is an important cause of morbidity and mortality in

modern society (Dumont et al., 2001; Fehlings et al., 2001; Sekhon and Fehlings, 2001;

Carlson and Gorden, 2002). An estimated 600,000 Americans are living with functional

deficits due to SCI (Carlson and Gorden, 2002; Hulsebosch, 2002), with about 11,000

new cases each year, costing an estimated $9.7 billion annually. At present, the

recommended treatment for acute SCI is methylprednisolone, a drug that has been

documented to affect several important components of spinal injury including

inflammation and free-radical formation (Bracken, 2001; Xu et al., 2001; Rabchevsky et

al., 2002). In spite of intense research directed to repair the injured spinal cord, current

long-term treatment is limited to rehabilitative physiotherapy (Wells et al., 2003).

Because the success of these strategies is highly variable, many patients with SCI remain

physically impaired as a result of their injury (Dumont et al., 2001; Fehlings et al., 2001;

Sekhon and Fehlings, 2001; Carlson and Gorden, 2002; Wells et al., 2003). Therefore,

understanding the mechanisms responsible for acute and chronic spinal cord injury

pathology as well as secondary consequences and the development of effective strategies

for therapeutic intervention is of great clinical importance.









Primary and Secondary Injury Mechanisms

The pathophysiology of SCI involves both primary and secondary injury

mechanisms. Primary injury is a direct result of mechanical trauma leading to

hemorrhage, membrane disruption, and vascular damage. This initial insult initiates a

progressive wave of secondary injury, which activates a cascade of pathophysiological

mechanisms that exacerbate the injury. This spread of damage is thought to be due to

activation of biochemical events leading to cellular death (apoptosis and necrosis). There

are several mechanisms that contribute to this wave of destruction including breakdown

of the blood-spinal-cord barrier (BSCB), ischemia, formation of free radicals and nitric

oxide, invasion of neutrophils, activation of astrocytes and microglia, apoptosis of

oligodendrocytes, Wallerian degeneration, and oxygen radical-mediated lipid

peroxidation (Anderson et al., 1985; Hall et al., 1992; Anderson and Hall, 1993;

Hausmann, 2003).

Following ischemic or traumatic SCI, there is a transient increase in extracellular

excitatory amino acids (EAA), which results in glutamate receptor-mediated excitotoxic

events (Mills et al., 2001). Activation of glutamate receptors initiates numerous

intracellular signaling pathways, causing long-lasting pathological and ultimately

functional deficits following SCI. Additionally, there is accumulation of intracellular

sodium, producing edema and a concomitant elevation in intracellular calcium (Young et

al., 1982; Stokes et al., 1983). Influx of calcium is also associated with glutamate

receptor activation and the increased release of EAAs leading to activation of calcium-

activated proteases (e.g., calpains), formation of free radicals, stimulation of nitric oxide

synthase (NOS), activation of proteases and endonucleases, mitochondrial damage, and









induction of apoptosis (Lu et al., 2000; Carlson and Gorden, 2002; Germano et al., 2002;

Hausmann, 2003; Kwon et al, 2004; Park et al., 2004; Profyris et al., 2004).

Following spinal cord injury, damage is not restricted to the spinal cord. Studies

have shown that damage occurs to ascending pathways, leading to deafferentation of

supraspinal structures (Yezierski, 2002). With deafferentation, it is likely that cortical

and subcortical regions could undergo significant reorganization, resulting in widespread

supraspinal functional changes following spinal injury (Morrow et al., 2000). Recently,

Lee et al. (2004) evaluated the role of apoptosis in the secondary injury of the brain

following acute spinal injury. They found evidence for cell death in the sensorimotor

cortex caused by injury. This would indicate that apoptosis can occur remotely in regions

distant from the lesion epicenter. Similarly, evidence of apoptosis was found in the

pyramidal cell layer of the motor cortex after spinal transaction injury (Hains et al.,

2003). Realizing that injury is not limited to the spinal cord, but can have remote effects

on the brain, is important, because in designing therapeutic treatments, one needs to take

into consideration all available targets in order to maximize potential sites of

intervention.

Excitotoxic Model of Spinal Cord Injury

In recent years, studies have shown that there exists a striking similarity between

clinical and experimental SCI pathophysiology. In order for an animal model to have

clinical relevancy, it needs to imitate the pathological features of human SCI, as well as

be reliable, consistent, and reproducible. The excitotoxic model of spinal cord injury is a

reduced injury model that has been shown to result in pathological changes similar to

those associated with traumatic and ischemic SCI (Yezierski et al., 1993; Yezierski et al.,

1998) and parallels the pathological sequela associated with human SCI (Kakulas, 1987;









Bunge et al., 1993; Bunge et al., 1997). It is produced by the intraspinal injection of the

AMPA/metabotropic receptor agonist, quisqualic acid (QUIS). Previous studies have

shown that the excitotoxic injury model results in pathophysiological changes, pain

behaviors, NMDA receptor phosphorylation and up-regulated gene expression for

NMDA receptors, opioid peptides (PPE and PPD), substance P receptor, inflammatory

genes (iNOS and COX-2), apoptotic genes (FAS-L and TNF-related apoptosis-inducing

ligand, TRAIL), and cytokine genes (IL-1 3 and TNF-a) (Yezierski and Park, 1993;

Yezierski et al., 1993; Liu et al., 1997a; Brewer and Yezierski, 1998; Yezierski et al.,

1998; Brewer et al., 1999; Schwartz et al., 1999a; Fairbanks et al., 2000; Morrow et al.,

2000; Abraham et al., 2001; Gorman et al., 2001; Plunkett et al., 2001; Yezierski, 2001;

Pattany et al., 2002; Siddall et al., 2002; Yezierski 2002; Yezierski and Burchiel, 2002;

Caudle et al., 2003; Yu et al., 2003; Yu and Yezierski, 2005). The similarities between

QUIS induced pathology and clinical pathology of SCI support the use of the excitotoxic

model in the study of pathophysiology and secondary injury mechanisms following

spinal injury. Another advantage of the excitotoxic injury model is that it does not

produce the mortality, morbidity, or challenges with animal husbandry that result from

ischemic or contusion models of injury.

In Vivo Magnetic Resonance Imaging

Overview

Although most of what is known about the pathological changes associated with

experimental SCI (i.e., edema, neuronal loss, hemorrhage, and cavitation) has come from

histological studies, the ability to non-invasively characterize spinal cord pathology

provides a way to study the temporal profile of pathological changes in the injured spinal

cord using the same animal at multiple time points. A technique with the capability of









achieving this objective is high-resolution magnetic resonance imaging (MRI), which is

becoming a preferred method for the visualization of SCI (Falconer et al., 1994; Ohta et

al., 1999; Bilgen et al., 2000).

Clinically, MRI has been a useful tool for the study of secondary complications

associated with SCI. For example, MRI can play an important role in the study of post-

traumatic syringomyelia. The excitotoxic injury model has been shown to produce

similar pathological changes to those observed in this condition (Yezierski et al., 1993;

Schwartz et al., 1999a). Considering the lack of understanding of the pathological

mechanism responsible for cavity formation in the injured cord (Madsen et al., 1994), it

may be possible to gain insight into the mechanism of cavitation using in vivo MR

imaging at different time-points following excitotoxic SCI.

In the past, clinicians have relied on patient descriptions of behavioral changes in

determining clinical outcome (Williams, 1990, Oakes, 1996). However, recently MRI

has been used to monitor changes associated with intraspinal transplantation, graft-

mediated functional recovery and changes in syrinx morphology after transplantation

(Wirth et al., 2002), and to differentiate between cyst, scar, or tumor formation with the

help of MR contrast agent administration (Schwartz et al., 1999a). With the importance

and many uses for MRI, continued research is needed to enhance this technique and

expand its uses to provide a better understanding of the mechanisms responsible for the

pathological changes associated with SCI. For example, an important question related to

SCI is the temporal profile of pathological changes in the acute and chronic stages post-

injury. Using in vivo MRI, it is possible to monitor these changes in the same animal.









Dynamic Contrast-Enhanced Magnetic Resonance Imaging, Blood-Spinal Cord-
Barrier

In recent years, the integrity of the BSCB has been studied using MRI. Normally,

the BSCB maintains a highly regulated homeostatic environment in the spinal cord as a

result of tight junctions between neighboring capillary endothelial cells surrounded by

pericytes and astroglial foot processes (Purves et al., 2001). The BSCB functions to limit

the penetration of a wide range of hydrophilic (water-soluble) molecules, proteins, and

cellular elements into the spinal cord. This barrier is disrupted in spinal injury, making it

permeable to toxic substances via vesicular transport from intravascular to interstitial

spaces (Popovich et al., 1996). Dynamic contrast-enhanced MRI is a method for

studying this disruption. Breakdown of the barrier is detected with the use of an

exogenous, water-soluble, paramagnetic contrast agent, gadolinium-diethylene-triamine-

pentaacetic, bis-methylamine (Gd). Normally, Gd circulates throughout body tissues

except in the central nervous system (CNS) because of the highly regulated blood-

nervous-tissue barrier. However, when the BSCB is disrupted, Gd can leak into the

interstitial tissue; i.e., it no longer remains intravascular (Bilgen et al., 2001). These

BSCB breaches can be observed with great sensitivity as an enhancement on Tl-

weighted MR images. Long-term analysis can also reveal how long the BSCB remains

permeable.

Current studies have shown that the BSCB plays a vital role in activating and

regulating secondary events following SCI, specifically, the post-traumatic inflammatory

response and edema formation (Hausmann, 2003); however, the exact mechanism of

SCI-induced inflammatory response is not fully understood. Following injury, there is an

ischemic environment at the injury epicenter as well as in adjacent areas of the white









matter (Tator and Koyanagi, 1997). Edema develops and spreads, paralleling necrotic

tissue at the epicenter (Tator and Fehlings, 1991). Changes further associated with this

disruption of microvasculature networks include hemorrhage, vasospasm, thrombosis,

and impaired autoregulation, which in turn leads to further breakdown of the BSCB

(Tator, 1991; Tator and Fehlings, 1991). Disruption of the BSCB results in inflammatory

invasion of neutrophils, macrophages, and T-cells (Bareyre and Schwab, 2003), which

further contributes to secondary injury mechanisms (Lu et al., 2000; Hausmann, 2003;

Kwon et al., 2004; Profyris et al., 2004). Understanding the time course of permeability

of the BSCB and the subsequent pathological consequences would be of significant

benefit to understanding the secondary mechanisms associated with spinal cord injury.

In Vitro Magnetic Resonance Imaging

Three-dimensional magnetic resonance microscopy (3D MRM) and diffusion

weighted imaging (DWI) are additional techniques that can be used to study SCI. These

techniques allow for in vitro measurements, where motion artifacts are eliminated

(Lemaire et al., 1990). This allows the signal-to-noise ratio (SNR) to be optimized. In

addition, prolonged scan times and higher field strengths are not limiting factors

(Guilfoyle et al., 2003). Both techniques allow for greater sensitivity in accessing

macroscopic changes in the injured cord, and with DWI, the integrity of the white and

gray matter can be evaluated, which is limited or not capable with conventional in vivo

methods of MRI. Diffusion imaging is also sensitive in evaluating spinal cord axonal

integrity, degree of injury, and extent of neuroprotection following injury (Ford et al.,

1994a; Nevo et al., 2001), and can be used to detect cyst formation in gray matter

following excitotoxic spinal cord injury, before conventional MRI is able to detect

cavities (Schwartz et al., 1999b).









Diffusion weighted MR imaging is developing into an important tool to

characterize the organization of white matter in the brain and spinal cord (Beaulieu, 2002;

Guilfoyle et al., 2003). Diffusion imaging is based on intrinsic properties within gray and

white matter, and relies on the phenomenon that water diffusion is highly anisotropic in

tissues of the central nervous system. The overall tissue microstructure influences the

mobility of diffusing water molecules, as the tissue components (i.e., myelin sheath and

axon cellular membranes) act as barriers and create individual compartments. Diffusion

characteristics are used to identify structural subtypes, where anisotropy is directly

related to the geometry of the fibers (Beaulieu, 2002). The direction of fibers may be

inferred by the direction of greatest water diffusion, and by seeking out adjacent

anisotropic voxels, a fiber can be mapped (Melhem et al., 2002). Following spinal injury,

there is a decrease in anisotropy; i.e., there are less geometric restrictions to water

translational motion resulting in decreased preferential water diffusion in a particular

direction. Having the capability to map white matter fibers allows for the determination

of the effects of injury on selected sensory and motor fiber tracts in the white matter,

which thus far is not apparent with other imaging techniques.

Following spinal cord injury, apoptosis of oligodendrocytes occurs, which leads

to chronic demyelination, ultimately causing anterograde neurodegeneration of ascending

and descending white matter tracts (Li et al., 1996; Crowe et al., 1997; Liu et al., 1997b;

Yong et al., 1998; Springer et al., 1999; Hausmann, 2003). This process of disintegration

of the axon from its myelin sheath and breakdown of axons is known as Wallerian

degeneration. Oligodendrocytes appear to be the predominant cellular target of glutamate

in white matter tissue. Recent studies have shown oligodendrocytes to be particularly









sensitive to excitotoxic insult (Yoshioka et al., 1996; Matute et al., 1997; McDonald et

al., 1998). Diffusion weighted imaging has the capability of detecting changes associated

with this progressive condition (Becerra et al., 1995; Werring et al., 2000; Thomalla et

al., 2004).

Neuroprotective Strategies for Treatment of Spinal Cord Injury

Overview

An important consideration in the study of SCI is the fact that in the treatment of

injury there is believed to be an optimal time for intervention. Secondary injury

processes, including a number of cellular and biochemical cascades, are potential targets

for neuroprotection (Amar and Levy, 1999; Dumont et al., 2001; Sekhon and Fehlings,

2001; Carlson and Gorden, 2002; Hausmann et al., 2002; Hulsebosch, 2002). Various

neuroprotective strategies targeting secondary injury have been examined experimentally,

including inhibitors of lipid peroxidation, nitric oxide, calpain, apoptosis, caspase,

glutamate receptor antagonists, anti-inflammatory agents, and other compounds of

uncertain mechanisms (Amar and Levy, 1999; Blight and Zimber, 2001; Hall, 2001).

Strategies have been demonstrated to improve outcome after brain and spinal injury in

pre-clinical studies, but are largely unsuccessful in clinical trials (Albers et al., 1995;

Gaviria et al., 2000; Martinez-Vila and Sieira, 2001; Faden, 2002; Hong and Liu, 2003;

Wells et al., 2003). Reasons for the failure may be that previous research strategies 1) do

not address the therapeutic window and effective dose required for treatment

optimization; and 2) only target a single injury mechanism (Yam et al., 2000; Tekkok and

Goldberg, 2001; Akins et al., 2002; Faden, 2002; Wells et al., 2003). Spinal cord injury is

a complex condition in which multiple cell death (apoptosis and necrosis) and cell

signaling pathways are in delicate balance with one another (Amar and Levy, 1999;









Dumont et al., 2001; Carlson and Gorden, 2002). It is likely that a single drug with

multiple targets or a combination drug treatment strategy that affects multiple

components of the secondary injury cascade will exert superior neuroprotective efficacy

(Carlson and Gorden, 2002; Faden, 2002; Wells et al., 2003). Therefore, understanding

the temporal profile of different secondary injury processes, as well as the impact of

agents with known neuroprotective effects, is critical in order to design a reasonable

strategy of intervention.

Neuroprotective Effects of Agmatine

Previous studies have been carried out to examine the effects of putative

neuroprotective agents on the spread of damage following excitotoxic injury (Yezierski,

2002; Yu et al., 2003). One drug that has been shown to have therapeutic effects is the

neurotransmitter/neuromodulator, agmatine. Agmatine [(NH2(CH2)4NH2C(NH=)HN] is

a butyl chain with a guanidino group at one end and an amino group at the other, and is

synthesized by the decarboxylation of arginine. It meets the criteria for an endogenous

neurotransmitter/neuromodulator in that a) it is synthesized in the central nervous system

(Li et al., 1994; Li et al., 1995; Fairbanks et al., 2000); b) it is stored in axon terminals in

association with storage vesicles (Reis et al., 1998); c) it can be released by

depolarization (Reis and Regunathan, 1998); d) it is inactivated by reuptake and/or

degraded enzymatically by agmatinase (Sastre et al., 1996; Sastre et al., 1997); and e) it

can bind to (Anis et al., 1990) and modulate (Yang and Reis, 1999) the actions of the

NMDA subclass of glutamate receptors.

Because of the polar character of agmatine, questions have come about as to the

extent to which it crosses the blood-central nervous system-barrier. Evidence has

indicated that agmatine administered peripherally does readily cross the barrier in a









concentration-dependant manner and has direct effects on the central nervous system

(Piletz et al., 2003). In addition, there have been numerous studies (for review, see

Nguyen et al., 2003) conducted in which systemically administered agmatine has affected

phenomena mediated by central processing. However, there is no data suggesting a

mechanism for such transport into nervous tissue, although an agmatinergic transporter

has been suggested (Nguyen et al., 2003).

Pharmacologically, treatment with exogenous agmatine is non-toxic (Gilad et al.,

1996; Fairbanks et al., 2000), blocks NMDA receptors (Olmos et al., 1999; Yang and

Reis, 1999), inhibits all isoforms of nitric oxide synthase (Auguet et al., 1995; Galea et

al., 1996), reduces glutamate excitotoxicity (Fairbanks et al., 2000; Zhu et al., 2003), and

exerts significant neuroprotective effects in models of ischemic brain injury (Gilad et al.,

1996; Gilad et al., 2000), and traumatic and excitotoxic spinal injury (Fairbanks et al.,

2000; Yu et al., 2000). Recently, it was shown that systemic treatment with agmatine

significantly reduces neuronal loss (Fairbanks et al., 2000; Yu et al., 2000; Yu et al.,

2003), improves locomotor function (Yu et al., 2000), and affects multiple signaling

pathways following SCI. For example, molecular changes brought about by agmatine

include inhibiting NF-kB activity, up-regulating AKT activity, and down-regulating pain-

and injury-related gene expression following excitotoxic and traumatic spinal cord injury.

These data suggest that agmatine may serve as a multifaceted agent that targets many of

the secondary processes involved in the mediation of cell death and the progression of

secondary injury in SCI. The multiple sites of action of this endogenous molecule make

agmatine a promising therapeutic agent for treatment of spinal cord injury and secondary






12


complications, as well as a useful pharmacological agent to study the impact of

neuroprotective interventions with known therapeutic targets.














CHAPTER 2
METHODOLOGICAL DEVELOPMENT

Overview

During the course of experiments described in Chapters 3-5, new methodologies

were learned and developed. With any new protocol, challenges arise that need to be

addressed before the method is successful. Below is a description of these learned and

newly developed procedures, as well as associated complications and solutions to address

each issue.

Anesthetic/Experimental Drugs

With each experiment, there is a significant amount of preparation involved, from

animal care, anesthetics, treatment, experimentation, to a successful recovery. With

anesthetics, it is crucial to administer proper doses, as well as prepare correct

concentrations of the anesthetic mixture. It is equally important that each component of

the cocktail be high in quality and freshly mixed. When any one of these components is

overlooked, problems can surface.

An example of a problem that occurred with anesthetics was encountered during

post-surgery survival with the last group of animal surgeries. At first, the anesthetic used

in Chapter 3 [mixture of ketamine (100 mg/mL, 3.0 mL), acepromazine (10 mg/mL, 1.0

mL), and xylazine (20 mg/mL, 3.0 mL; mixture at 0.65 mL/kg, subcutaneously)] was

believed to be the cause of the problem. It was believed that during times of infrequent

surgeries, the cocktail mixture could have deteriorated, so fresh mixtures were made.

However, animals were still not surviving, regardless of post-operative care. After









talking with Animal Care Services and other labs using similar anesthetics on rats, a

different cocktail recipe was developed [mixture of ketamine (100 mg/mL, 0.5 mL) and

xylazine (100 mg/mL, 0.1 mL)] in hopes that rats would have a more successful recovery

following surgery, and that the anesthetic would act more effectively, i.e., animals would

remain anesthetized for the duration of surgery, with no supplements required. Tests

were done with two doses of this new recipe (mixture at 0.75 mL/kg and 1.0 mL/kg) and

through two different routes of administration (intraperitoneally versus subcutaneously).

Based on results, it was decided that 1.0 ml/kg administered intraperitoneally was the

most effective dose and route of administration. Intraspinal injection surgeries went on

as scheduled, using the new cocktail recipe; however, animal survival was still an issue.

Next, we tested the experimental drug, agmatine, to determine if it was the cause of

failed recovery. At first, it was suspected that the higher experimental dose (100 mg/kg)

was too high, as there have been previous reports that higher doses of agmatine caused

hypothermia (Yezierski, unpublished observations). So, the dose was decreased to 80

mg/kg, however, this did not remedy the problem. To further test the agmatine, a fresh

supply was made; one rat received a dose of the old supply and one rat received a dose of

the fresh supply. The rat that received the old agmatine supply, died several hours later,

while the one that received the fresh supply survived. Therefore, on the next scheduled

surgeries, rats received the new anesthetic cocktail and a dose of the fresh Agmatine

supply at the lower dose (80 mg/kg). However, after a third attempt, animals were still

not surviving; they seemed to die due to respiratory distress. Once again, Animal Care

Services was contacted and made aware of the problem in hopes that they might shed

light on this ongoing problem. At this time, we discovered that the higher concentration









of xylazine (100 mg/mL vs 20 mg/mL) was known to cause pulmonary edema in rodents.

Therefore, it was thought that the cocktail had never been the problem in the first place,

and that the agmatine had been the main cause of animal survival problems. So, we

switched back to the original cocktail recipe and used the fresh supply of agmatine (80

mg/kg), and finally the last group of animal surgeries were performed successfully, with

no further survival issues. During the time the above problems were being addressed, a

total of 16 animals were lost following preliminary MRI's and subsequent intraspinal

injection of QUIS.

Magnetic Resonance Imaging

Catheter Placement

A tail vein catheter was needed to administer contrast agent used in the dynamic

contrast-enhanced MR imaging. Placing a tail vein catheter requires patience and a great

deal of practice to perfect. Once the animal was anesthetized with a mixture of oxygen

and isofluorane, as described in Chapter 3, the catheter was filled with a heparin/sterile

saline solution (also described in Chapter 3), and inserted into the tail vein. Correct

placement was confirmed with blood flow into the catheter, at which point, 2-3 cc of the

heparin/sterile saline solution was injected to prevent further clotting. The catheter was

then taped in place.

4.7T Coil Development

Initial experiments were carried out on the 4.7T magnet system (please see Chapter

3). A highly-sensitive quadrature MR surface coil was constructed for this system, by

Danny Colvin, specifically for imaging the rat spinal cord. The coil consisted of two

rectangular coil elements (3 x 3 cm) placed next to each other on the inside of a half-

cylinder cradle 4 cm in diameter. Each coil was constructed using copper tape, 5 mm in









width, with a 3.3 pF ceramic-chip capacitor (American Technical Ceramics (ATC),

model 3R3C) inserted in series with the copper tape in the middle of each arc at the ends

of each rectangle to provide distributed capacitance around the coil. The rectangular

coils were overlapped along the inner edges to cancel their mutual inductance. The

overlapping coils formed an arc of 120 degrees with an overall arc length of 5.5 cm.

Each coil was individually tuned and matched with a combination of fixed series

capacitors of 1 pF (ATC, model 1ROC) and variable series and parallel capacitors

(Johanson Manufacturing Corp, Boonton, NJ, model 5641) arranged on a custom circuit

board. The tune and match circuit was interfaced to the transmitter-receiver with an

input-voltage balancing network on the same circuit board and a 50 ohm coaxial cable

containing a parallel-resonance trap on the coaxial shield to eliminate surface currents.

The trap was created by forming a loop in the cable and attaching a 4.7 pF ceramic-chip

capacitor (ATC, model 4R7C) to the shield of the cable across the loop.

11.1T Coil Development

Following the first study, as described in Chapter 3, it was decided to move all MR

imaging to the 11.1T magnet system for the remainder of the in vivo experiments (see

Chapter 4). This would allow for greater signal-to-noise, resulting in a much higher

resolution image, showing greater detail of injury pathology. Two MR coils were used in

the measurements of MR images. A circularly polarized, transmit-only "birdcage" coil

was used for excitation of resonance and was provide by the Advanced Magnetic

Resonance Imaging and Spectroscopy Facility of the McKnight Brain Institute (see "Coil

Setup" for description). Then, the resonance signal was measured with a smaller receive-

only, circular-polarized coil placed very near the spine. This coil was constructed by

Christy Amwake. The receive-only coil was passively decoupled from the transmit coil









during excitation and the transmit-only coil was actively decoupled from the receive-only

coil during signal measurement.

The receive-only circularly polarized MR surface coil was constructed

specifically for imaging the rat spinal cord at 470 MHz. The coil consisted of two

rectangular inductive elements (3 x 3 cm) placed next to each other (with a common side)

on the outside of a half-cylinder cradle 4.7 cm in outside-diameter and 77 cm long. Each

rectangular section was constructed using copper tape (5 mm in width), with the outer

inductor-corer copper tape angled at 45 degrees to the sides. Four 4.7 pF non-magnetic

ceramic-chip capacitors (American Technical Ceramics (ATC), model ATC100B-4R7F-

WN-1000XC) were inserted in series with the copper tape around the outside of these

inductors. These capacitors were inserted in the middle of each arc (at the ends of each

rectangular section, opposite the input circuit), and in the middle of the outer side of each

rectangular section, in order to provide distributed capacitance around the coil. The

common copper-tape element, joining the two inductors, was constructed with a variable

non-magnetic capacitor (1-16 pF, Voltronics, Denville, NJ) and inserted in series in the

middle of the element. This variable capacitor was used to cancel the mutual inductance

between the two inductive sections. The coil formed by the two rectangular sections had

an arc of 170 degrees with an overall arc length of 8 cm. Each inductor was passively

decoupled from the MR transmitter coil during excitation of the signal. This was

accomplished by adding a hand-wound inductor and double-headed non-magnetic diodes

(M/A-COM, Lowell, MA, model MA45471) in parallel with 4.7 pF capacitor located in

the middle of each inductor arc. The hand-wound inductor was a 2 mm inner diameter

solenoid of 2.5 turns made from 20 AWG magnet wire. Each MR coil inductor was









individually tuned and matched with two variable series and one parallel non-magnetic

capacitor (1-16 pF, Voltronics, Denville, NJ) arranged on a custom circuit board. Each

tune and match circuit was interfaced to the receiver with a 50 ohm coaxial cable

containing a parallel-resonance trap on the coaxial shield to eliminate surface currents.

The trap was created by forming a loop in the co-axial cable, approximately 2 cm in

diameter, and attaching two parallel 4.3 pF non-magnetic ceramic-chip capacitor (ATC,

model ATC100B-4R3B-WN-1000XC) to the shield of the cable across the loop. This

configuration of coil outputs provided two points along one side of the coil arc, separated

by approximately 90 degree, for interface to the receiver to provide detection of the

circular polarized signal. This was accomplished by combining the two detected signals,

within a 90-degree splitter-combiner (Anaren Corp, East Syracuse, NY, model 10013-3),

to provide a single input to the receiver.

Coil Setup

Separate transmit and receive coils were used to increase the SNR and achieve

greater depth penetration. A volume coil used for transmission allowed for the

development of 90 degree tip angles over a large region of the sample, eliminating the

fast changing tip angle variation with depths that occurs with surface coil excitation. On

the other hand, the surface coil provides greater sensitivity than the volume coil because

of its smaller size.

In setting up the coils, it is important to avoid mutual coupling between the

transmit-only volume coil and the receive-only surface coil or signal loss and mismatch

will occur. Specifically, it is critical that the surface coil be decoupled during

transmission and the birdcage be decoupled during reception. The transmit-only birdcage

on the 11.1T is an eight leg, shielded, low-pass birdcage of 8.8 cm diameter and length.









It is turned off during reception by switching high impedance circuits into every leg of

the coil. This effectively blocks current from flowing in the coil. The high impedance

circuit is a series combination of an inductor and diode placed in parallel with a capacitor

on the leg. The inductor and capacitor have equal reactance and creates high impedance

when the diode is forward biased (parallel resonance). Conversely, during transmission,

the diode is reverse biased removing the high impedance circuit from the leg and the

birdcage is fully functional. The diode biasing is controlled by a pulsed signal from the

spectrometer.

11.1T Revised Imaging Protocol

After switching to the 11.1T magnet, it was evident that the increase in field

strength resulted in an increased sensitivity to breathing artifacts. For this reason, the

imaging protocol originally used on the 4.7T magnet had to be modified accordingly for

use on the 11.1T magnet system. The modifications made for the 11.1T imaging protocol

are described in detail in Chapter 4. Most notably, the read direction was changed to

head-foot sagittall MR images) or left-right orientation (transverse MR images) to

eliminate aliasing, and a slice saturation pulse was added to the sequence of high

resolution images to eliminate breathing artifacts.

4.7T versus 11.1T Images

One of the most notable advantages in moving from the 4.7T magnet to the 11.1T

magnet was the increase in SNR (see Fig. 2-1), and the lack of aliasing due to changes

made in the protocol used for the 11.1T. The images collected on the 11.1T magnet had a

much higher resolution and showed greater detail of injury pathology after excitotoxic

spinal cord injury. In addition, there was an increase in gray-white matter contrast in Tl-

weighted transverse images collected on the 11.1T magnet.
























A B















C D

Figure 2-1. Comparison of MR images collected on the 4.7T magnet versus the 11.1T
magnet. T1-weighted sagittal (A and C) and transverse (B and D) MR images
collected in vivo pre-injury (Day 0) on the 4.7T magnet (A, B) or the 11.1T
magnet (C, D).

Magnetic Resonance Imaging Obstacles

Heating Effects of the 11.1T Imaging Protocol

After moving to the 11.1T and optimizing the imaging protocol, there was a

problem in animal survival during the MRI's. At first, it was thought that the animals

were becoming too deeply anesthetized and an anesthetic scavenger line was needed in

the magnet to remove excess anesthesia. In addition, the cradle apparatus was lowered to









ensure the animals had ample room to breath. A deltaphase isothermal pad (Braintree

Scientific, Inc., Braintree, MA) was also placed on the animal during the MRI

acquisition. In thinking that the problem had been solved, experiments proceeded,

however, the issue of animal survival persisted, and experiments were postponed until the

problem could be solved. At this point, it was thought that temperature might be an

issue, as animals were coming out of the magnet following imaging sessions much

warmer than expected. Temperature measurements were performed on both the 4.7T and

11.1T magnets using a tissue equivalent phantom simulating a rat abdomen (64.1%

water, 32.0% sucrose, 1.6% salt, 2.3% TX-151; Beck et al., 2004), and compared. The

data revealed that the new sequences that were devised for the 11.1T magnet (described

in Chapter 4), which were identical to the 4.7T (described in Chapter 3) except they

included slice saturation pulses to eliminate breathing artifacts (the 11.1T is much more

sensitive to breathing), were causing extensive heating of the coils and ultimately over

heating the animals by several degrees (see Figs. 2-2A, #5; 2-2B, # 1-6). The transverse

images versus the sagittal images were causing a significant rise in temperature, which

was due to the increase in duty cycle, not magnet frequency. A temperature and

respiration monitoring system was then acquired for the 11.1T, which allowed for close

monitoring of respiration and temperature, as well as a feedback loop for consistent

temperature control. Transverse images in the dynamic contrast-enhanced MRI protocol

were also eliminated from the sequence. The final imaging protocol for the 11.1T

magnet included the first five pulse sequences shown in Figure 2-2B (described in

Chapter 4). During this time, a total of 8 animals were lost due to overheating.














26 Imaging Protocol with
Slice Saturation Pulse
1 T1 trans
2 T1_sag
25 3 T2_trans
4 T2_sag
5 DCE trans/sag
interleaved
24 6 DCE sag only
7 Cool Down


a-
E 23
1-



22




21


4
*

*


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***


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6
*
*
*
*
*
*r *

'* 7
*
*
*

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Imaging Protocol, No
Slice Saturation Pulse
8 T1 trans
9 T _sag
10 T2 trans
11 T2_sag
12 DCE trans/sag
interleaved


8- 910 12 *

11


2:24 3:36

Time (hr:min)


7
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*4 *



2 -*


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Imaging Protocol with Slice
Saturation Pulse
1 T1 trans
2 T1_sag
3 T2 trans
4 T2_sag
5 DCE sag only
6 DCE trans/sag interleaved
7 Cool Down
8 DCE trans/sag interleaved,
Gradients turned off


21
0:00


Time (hr:min)


Figure 2-2. Temperature measurements collected on the 4.7T and 11.1T magnets using a

tissue equivalent phantom simulating rat abdomen. The measurements were

taken using (A) the protocol originally devised on the 4.7T (no slice

saturation); and (B) the new protocol devised for the 11.1T (including slice

saturation). The new protocol (included slice saturation) resulted in a heating

of the phantom on both magnet systems, which did not occur using the old

protocol (no slice saturation), thus confirming that the heating was a result of

the increase in duty cycle, and not the increase in frequency.


0
0
0
3


25




24


a2

E 23
1-









Signal-to-Noise Ratio

The setup used for all in vivo imaging protocols is quite extensive and includes

many components where problems can arise, including two coils, cables, and an

abundance of hardware and software. One of the biggest issues, in addition to animal

survival, was SNR problems. On numerous occasions, an imaging session would begin

with problem solving due to low or lack of signal. When this occurred, the coils were

inspected, all cables were checked to make sure they were not loose or faulty, correct

cable connections were confirmed, and the decoupling box was tested to make sure it was

functioning. Throughout the course of this project, several cables had gone bad, a fuse

was replaced in the decoupling box, diodes were replaced in the volume coil, and

capacitors were re-soldered on the surface coil. Occasionally, the problem was more

severe and involved magnet hardware, such as the RF amplifiers or gradient power

supplies, in which case new parts had to be ordered and as a result, the magnet became

inoperable. As a result of poor signal, due to any combination of the above problems, the

images had a low SNR when compared to images collected on days of optimal signal (see

Fig. 2-3).























A B

Figure 2-3. Comparison of images collected with high versus low SNR on the 11.1T
magnet. (A) T1-weighted (TR/TE = 1000 ms/15 ms, NA = 4) transverse MR
image with high SNR. (B) T1-weighted (TR/TE = 1000 ms/15 ms, NA = 4)
transverse MR image with low SNR.















CHAPTER 3
EVALUATION OF THE PATHOLOGICAL CHARACTERISTICS OF SPINAL CORD
INJURY WITH MAGNETIC RESONANCE IMAGING*

Introduction

The intraspinal injection of the alpha-amino-3-hydroxl-5-methyl-4-isoxazole-

propionate/metabotrophic receptor agonist, quisqualic acid, produces an excitotoxic

spinal cord injury SCI, which has been used to study the molecular, pathophysiologic,

and behavioral characteristics of SCI (Yezierski, 2002). These injections result in

pathologic changes similar to those associated with ischemic and traumatic SCI

(Yezierski et al., 1993; Yezierski et al., 1998). Although most of what is known about

the pathologic changes associated with experimental SCI (ie, edema, neuronal loss,

hemorrhage, and cavitation) has come from histologic studies, noninvasive high-

resolution MR imaging is becoming a preferred method for the visualization of SCI

(Falconer et al, 1994; Ohta et al., 1999; Bilgen et al., 2000). In recent years, MR imaging

has become a valuable diagnostic tool in studies examining the pathologic changes

following SCI (Flanders et al., 1990; Yamashita et al., 1990; Yamashita et al., 1991;

Ohshio et al., 1993; Flanders et al., 1999; Selden et al., 1999; Rattliff and Voorhies, 2000;

Ishida and Tominaga, 2002). One well-documented change associated with SCI is the

breakdown of the blood-spinal cord barrier. The pathologic characteristics associated

with BSCB disruption following SCI can be measured quantitatively with dynamic


* Reprinted with permission from Berens SA, Colvin DC, Yu CG, Yezierski RP, Mareci TH. Evaluation of
the Pathological Characteristics of Spinal Cord Injury with MR Imaging. AJNR Am J Neuroradiol 2005;
26(7):1612-1622. 0 by American Society of Neuroradiology









contrast-enhanced MR imaging, by using exogenous paramagnetic contrast agents

(Runge et al., 1997; Bilgen et al., 2001; Bilgen and Narayana, 2001; Bilgen et al, 2002).

Fine details of SCI pathology can also be determined with intact excised cords by using

MR microscopy. With this technique, motion artifacts are eliminated, allowing the signal

intensity-to-noise ratio to be optimized with prolonged scan times and higher field

strengths (Guilfoyle et al., 2003).

Thus far, the pathologic characteristics determined with in vivo MR imaging have

not been compared with in vitro MR imaging and histologic examination by using the

excitotoxic injury model. We hypothesize that in vivo MR imaging has the sensitivity to

detect significant differences in pathologic characteristics between two different

strategies of injury, which can be correlated with in vitro MR imaging and histology. A

preliminary description of this study has been reported (Berens et al., 2003).

Materials and Methods

All experiments were carried out in accordance with National Institutes of Health

guidelines for the care and use of animals and approved by the University of Florida

Institutional Animal Care and Use Committee. In vivo and in vitro MR methods, along

with histologic techniques, were used, because these were the most direct measures of

pathologic changes following injury. A comparison of findings with different methods

was carried out.

Animal Preparation

The technique of intraspinal injection was similar to that described elsewhere

(Yezierski et al., 1998). In brief, Sprague-Dawley male rats weighing 250-300 g were

anesthetized with a mixture of ketamine (3.0 mL), acepromazine (1.0 mL), and xylazine

(3.0 mL; mixture at 0.65 mL/kg, subcutaneously). Supplemental doses of anesthetic were









given, if necessary, when rats responded to a noxious pinch applied to the glabrous skin

of an extended hind paw. Animals were placed in a stereotaxic frame and the vertebral

column immobilized with a vertebral clamp. One injection window for intraspinal

injections was made by laminectomy between spinal segments T12-L2. Once the cord

was exposed, the dura was incised longitudinally and reflected bilaterally.

A small hole for unilateral injections was made in the pia mater. After injection,

muscles were closed in layers and the skin was closed with wound clips. The day

following the final in vivo image acquisition, animals were anesthetized with sodium

pentobarbital and euthanized by transcardial perfusion with normal saline followed by

10% formalin. Spinal cords were removed and stored in 10% formalin.

Intraspinal Injection Procedure

Before injection, QUIS, 125 mmol/L, was mixed in sterile saline, corrected to

physiologic pH, and was then injected unilaterally at one level of the cord (T12-L2). To

compare injury pathology between lesions at different depths, two types of injection

strategies were used: injections at a depth of 500 [im (QUIS lesion 1 [QL1], two animals)

and at a depth of 1000 [m (QUIS lesion 2 [QL2], five animals) below the surface

(Yezierski, 2001). Injections were made between the dorsal root entry zone and the

dorsal vein on the right or left side, depending on vascularity on the surface of the cord.

The total volume of QUIS injected was 1.2 [iL in three tracks (0.4 [iL/track; distance

between tracks 0.5 mm in the rostrocaudal direction). A Hamilton microliter syringe

with a glass micropipette extension (tip diameter, 5-10 [im) was used for intraspinal

injections. The syringe was positioned in a microinjector unit (Kopf 5000; Kopf,

Tujunga, CA) attached to a micromanipulator. A sham injury group was prepared by

injecting saline in the same manner as described above.









In Vivo MR Imaging

On the basis of previous correlations between the extent of pathology and the

temporal onset of injury-induced behavioral changes, in vivo MR imaging was performed

17-24 days (mean, 22 days) after injury (Gorman et al., 2001; Yu et al., 2003). Animals

were initially anesthetized by inhalation of a mixture of 5% isofluorane with oxygen at a

flow rate of 2 L/min and maintained via a nose cone with a mixture of 2% isofluorane

with oxygen at a flow rate of 1 L/min. To determine whether there was disruption of the

BSCB following injury, DCE-MR imaging was performed with the intravenous injection

of gadolinium-diethylene-triamine-pentaacetic, bis-methylamine ([Gd] Omniscan,

Nycomed, Oslo). For intravenous delivery of Gd, the tail vein was cannulated with a 24-

G, .75-inch intravenous catheter (Terumo Surflo, Webster Veterinary Supply, Inc.,

Sterling, MA). Before catheterization, the catheter was filled with heparin solution

(mixture of 6 mL of saline and 0.2 mL of 1000 U/mL) to prevent clotting. Once the

catheter was in place, a syringe filled with the appropriate volume (0.6 mL/kg body

weight) of Gd (0.3 mmol/kg) was attached to the catheter and stabilized with surgical

tape. A pulse oximeter was used on the tail to monitor heart rate and blood oxygenation

saturation. Core body temperature was monitored during experiments by using a rectal

probe and was kept between 350C and 370C. Respiration rate was also monitored during

data acquisition.

In Vivo High Resolution MR Imaging Data Acquisition

All in vivo NMR measurements were performed on a 4.7T, 33-cm-bore Oxford

magnet (Oxford Instruments, Oxford, UK) at 200 MHz by using a Bruker BIOSPEC

Avance DBX console (Bruker NMR Instruments, Billeria, MA) and 100 mT/m actively

shielded gradients. A highly sensitive quadrature MR surface coil was constructed









specifically for imaging the rat spinal cord. The coil consisted of two rectangular

overlapping coil elements (3 x 3 cm), constructed by using copper tape, placed on the

inside of a half-cylinder cradle 4 cm in diameter. All data acquisition was performed

with Bruker ParaVision software. General image processing and analysis of the time

series DCE data were performed by using custom software written in the Interactive Data

Language (Research Systems, Boulder, CO).

Rats were placed supine in a custom-built half cylinder cradle/coil apparatus. The

200-MHz quadrature MR surface coil was tuned with a Hewlett Packard HP8752C

network analyzer (Hewlett Packard, Englewood, CO). The position of the animal was

confirmed with pilot images to locate the injury site. Pilot images were T1 weighted

(TR/TE = 500 ms/5 ms; NA = 1), with a field of view (FOV) of 6 cm in each direction, a

1 mm section, and a 128 x 128 matrix. Fat suppression was done by using a CHESS-type

sequence (Haase et al., 1985). Once the position was confirmed, a set of fat-suppressed,

high-resolution sagittal and transverse images was collected. Sagittal T1-weighted (TR/

TE = 1000 ms/10.5 ms; NA = 4) spin-echo images were acquired over five sections, each

1 mm thick without a gap between sections. A TR of 1000 ms was chosen to provide

optimum signal intensity strength with T1-weighting for anatomic definition. Additional

acquisition parameters were a FOV of 4 cm x 2 cm, read out in the head-to-foot direction,

and a matrix of 256 x 128. The scan time with these parameters was 8.67 minutes.

Transverse T1-weighted (TR/TE = 1000 ms/10.5 ms; NA = 4) spin-echo images were

acquired over 14 sections, each 1 mm thick without a gap between sections. Other

acquisition parameters were a FOV of 2 cm x 2 cm, read out in the anteroposterior

direction, and a matrix of 128 x 128. The scan time was 8.67 minutes.









Sagittal T2-weighted (TR/effective TE = 2000 ms/62.5 ms; NA = 6) fast spin-echo

images with phase-encode segmentation were acquired over five sections, each 1 mm

thick without a gap between sections. Additional acquisition parameters were a FOV of

4 cm x 2 cm, read out in the head-to-foot direction, phase-encode segmentation factor of

4, and a matrix of 256 x 128. The scan time with these parameters was 6.5 minutes.

Transverse T2-weighted (TR/effective TE = 2000 ms/62.5 ms, NA = 6) fast spin-echo

images with phase-encode segmentation were acquired over 14 sections, each 1 mm thick

without a gap between sections. Other acquisition parameters were a FOV of 2 cm x 2

cm, read out in the anteroposterior direction, phase-encode segmentation factor of 4, and

a matrix of 128 x 128. The scan time with these parameters was 6.5 minutes.

In Vivo DCE-MR Imaging Data Acquisition

After four sets of high-resolution images were collected, a series of dynamic

contrast-enhanced images of the rat spinal cord was acquired. Transverse and sagittal

images were acquired before and after the contrast agent was injected. Transverse

images were acquired with a FOV of 2 cm x 2 cm and a matrix of 64 x 64, over eight

sections, and sagittal images with a FOV of 4 cm x 2 cm and a matrix of 128 x 64, over

three sections, both 1 mm thick without a gap between sections. Three sets (transverse

and sagittal interleaved) of T1-weighted pre-contrast multi-section images (TR/TE = 330

ms/9.4 ms; NA = 8) were collected. After acquisition of the pre-contrast images, Gd was

injected in approximately 5 seconds with continuous pressure, to prevent backflow,

through the implanted catheter while the animal was in the magnet. Immediately after

injection, 8-10 sets (6 minutes for each set) of T1-weighted post-contrast multi-section

images (TR/TE = 330 ms/9.4 ms; NA = 8) were collected, with transverse and sagittal

data-set acquisitions interleaved. We collected these images over a 60-minute time









frame, which was chosen on the basis of previous experience, with a peak enhancement

for the cord at 3 minutes. The receiver gain was adjusted to one-third the value used for

the high-resolution images because Gd provides approximately three times the contrast

enhancement.

In Vitro MR Microscopy

All in vitro NMR measurements were performed on a 14. IT, 51-mm-bore Oxford

magnet, at 600 MHz, by using a Bruker BIOSPEC Avance DBX console. All data

acquisition was performed with Bruker ParaVision software. Before imaging, spinal

cords were placed in phosphate-buffered saline (PBS) overnight. The cords were placed

in 5-mm tubes and imaged in PBS by using a standard 5-mm MR coil. Three sets of

high-resolution multislice two-dimensional images oriented in three orthogonal directions

were collected (TR/TE = 2500 ms/20 ms; NA = 8). The transverse images were acquired

over 60 sections (200 [m thick without a gap between sections; FOV = 5 mm x 5 mm;

matrix = 256 x 256), the sagittal images were acquired over 20 sections (200 [im thick

without a gap between sections; FOV = 20 mm x 5 mm; matrix = 1024 x 256), and the

coronal images were acquired over 20 sections (200 [m thick without a gap between

sections; FOV = 20 mm x 5 mm; matrix = 1024 x 256). Immediately after the final

imaging session, spinal cords were placed in fixative. General image processing and

analysis was performed by using custom software written in the Interactive Data

Language (IDL, from Research Systems, Boulder, CO).

Histological Procedures

Once imaging was completed, spinal cords were placed in 10% sucrose overnight.

Serial sections (75 itm) were cut on a freezing microtome, collected in PBS, and mounted

on gelatin-coated slides. Sections were stained with cresyl violet and cover-slipped. To









confirm that the hypointense signals from MR images correlated with hemorrhage,

sections sampled from the injury site were stained with the Prussian-blue reaction

(Bancroft and Cook, 1984).

Data Analysis

To compare results obtained with the three different methods used, total

longitudinal injury lengths were determined. Statistical analysis of these data included a

repeated-measures ANOVA test to determine differences in distances determined by the

three methods (SPSS, Chicago, IL). This was followed by a paired comparison to

interpret the significance of the ANOVA. To determine the total longitudinal injury

length, the total number of transverse sections from each method, with evidence of

excitotoxic (ie, cavity formation, hemorrhage, and neuronal loss) damage (Yezierski et

al., 1998; Gorman et al., 2001; Plunkett et al., 2001), were counted and multiplied by the

section thickness appropriate for the method used. Transverse images were used for

injury length calculation instead of sagittal images, because the boundaries of the injury

(eg, neuronal loss in the dorsal horn-a classical characteristic of excitotoxic SCI) were

best observed in transverse images. For the calculation of injury length by using in vivo

MR imaging, T2-weighted transverse images were counted and multiplied by 1 mm; for

in vitro MR imaging, transverse images were counted and multiplied by 200 [im; and for

histology, sections were counted and multiplied by 75 tm (Gorman et al., 2001).

Histologic determination of injury length was done via microscopic examination with 4x

and 10x objectives. In addition to injury length, pathologic characteristics (ie, cavity

formation, central canal expansion, hemorrhage, and neuronal loss) of the injury were

compared between the two types of injection strategies used (ie, QL1 vs QL2).









Injury lengths were also compared between QL1 and QL2. Statistical analysis of

these data included a one-way between subjects ANOVA test to determine differences in

injury severity between the four injury/treatment groups (SPSS, Chicago, IL). This was

followed by a Tukey HSD post hoc test to interpret the significance of the ANOVA.

For DCE-MR imaging processing, regions of interest (region of interest) were

selected in the injured and normal cord, muscle, and bone. A 1 mm x 2 mm elliptical

region of interest was drawn in regions of injury and normal cord because the enhanced

region was typically elliptical. A 1 mm circle region of interest was drawn for muscle

and bone and was analyzed to determine the success of the Gd injection. Ten percent

peak enhancement was used as the cutoff value because regions of interest of normal cord

showed 10% peak enhancement because of the presence of contrast agent in the cord

vasculature.

Results

In Vivo High Resolution MR Imaging

Figure 3-1 shows a comparison of the pathologic findings from the same animal

(S05) at the same location for all three methods used in this study. The appearance of

images was compared with normal controls. When an image region relative to a control

region appeared darker, we classified it as hypointense. When an image region relative to

a control region appeared brighter, we classified it as hyperintense. No signal intensity

changes were observed in the normal cord on either T1-weighted or T2-weighted images

(data not shown). The only significant information obtained from T1-weighted images of

QUIS-injured animals was the injection tracks, which were seen as weak hypointense (ie,

dark) signals.









Figures 3-2A and 3-3A show the sagittal and transverse T2-weighted in vivo

images, respectively, for each injured animal. Using in vivo transverse images, it was

possible to determine the anatomic level of the sections by examining the size and shape

of the cord as well as gray and white matter. Injection tracts for QUIS injections were

visible in both T2-weighted (Figs 3-1 and 3-3) and T1-weighted transverse images (not

shown) in all animals. Hypointense signals can be seen within the cord on T2-weighted

images of all QUIS-injured animals; however only T2-weighted in vivo images of QL2

animals (S01, S02, and S04-S06) showed hyperintense (ie, bright) signals.

In Vivo In Vitro Histology



B .* ..-- B

-A



C J J G J I

Figure 3-1. Representative pathologic findings in the same animal (505) at the same
location observed in vivo (T2-weighted, TR/effective TE = 2000 ms/62.5 ms;
NA = 6), in vitro (TR/TE = 2500 ms/20 ms; NA = 8), and in histologic
sections. Many of the same pathologic details are visible with all three
methods albeit with different levels of resolution. In vivo and in vitro images
are displayed with image size = 0.92 cm x 0.77 cm (in vivo) and 0.50 cm x
0.50 cm (in vitro). Letters label the most distinctive anatomic and pathologic
characteristics observed in the three images as follows: A indicates expanded
central canal; B, injection track; C, dorsal root ganglion; D, dorsal root; E,
hemorrhage at injury site; F, white matter; G, gray matter; H, ventral root; I,
cavity; J, anterior spinal vessel. In vivo images show cavitation as
hyperintense signals due to T2 weighting, whereas, on in vitro images,
cavities appear isointense in these proton attenuation-weighted images.


In vivo images showed consistent differences between QL1 and QL2 animals (Figs

3-4 to 3-6). It is important to note that hyperintense signals, indicating expansion of the








central canal and cavitation, were not seen in images from QL1 animals (Fig 3-3A, X01

and X03; Fig 3-6). Although the only difference in QUIS injection parameters was depth

of injection, there were dramatic differences in the extent of pathologic damage (e.g.,

laterality, cavitation, and expansion of the central canal). There were also significant

differences (p < 0.001, one-way between subjects ANOVA, F = 34.134, p < 0.001) in

injury lengths between QL1 (X01 and X03) and QL2 animals (S01, S02, and S04-S06;

Fig 3-4).


A Sol IS2 S04


lei


W* S06 XO1


*rs ~o a


Figure 3-2. Sagittal images for all QUIS-injured animals. (A) In vivo (T2-weighted,
TR/effective TE = 2000 ms/62.5 ms; NA = 6); and (B) in vitro (TR/TE = 2500
ms/20 ms; NA = 8). Images are oriented with rostral at the top and dorsal on
the right. Arrows represent the location of the epicenter of the injury. The
epicenter was defined as the region of maximal pathologic damage.
Displayed image size = 0.77 cm x 2.15 cm (in vivo) and 0.40 cm x 1.50 cm (in
vitro). Animals designated S01, S02, S04, S05, and S06 received QL2,
whereas animals designated X01 and X03 received QL1. The vertebral
column and laminectomy site were easily observed in both in vivo and in vitro
sagittal images.


X03




4





























SoU S02 S04 05






B S06 XG1 X03

Figure 3-3. Transverse images for all QUIS-injured animals. (A) In vivo (T2-weighted,
TR/effective TE = 2000 ms/62.5 ms; NA = 6); and, (B) in vitro (TR/TE =
2500 ms/20 ms; NA = 8). Images are sampled from the epicenter of the injury
(arrows in Fig 3-2) and show comparable pathologic changes between
methods. See Fig 3-1 for labeling of anatomic and pathologic findings.
Images are oriented with dorsal at the top. L, left side of cord; R, right side of
cord, for all images. Displayed image size = 0.92 cm x 0.77 cm (in vivo) and
0.50 cm x 0.50 cm (in vitro). A, Hypointense signals correlated with
hemorrhage (see "Histologic Findings") and hyperintense signals correlated
with the presence of fluid filled cavities. For example, notice the hyperintense
expanded central canal in the cord of animals S01, S02, and S04-S06 and the
hyperintense cavity in the gray matter on the right side of the cord in animal
S02. B, Hypointense regions were observed bilaterally in the gray matter in
all QL2 animals (S01, S02, and S04-S06), whereas, in QL1 animals, evidence
of hemorrhage was observed only ipsilateral to the side of QUIS injection
(X01 and X03). In addition, hypointense areas were observed in the dorsal
columns in all the QUIS-injured animals except S01. Central canal expansion
was clearly defined in four of the five QL2 animals (S01 and S04-S06), and
injection tracks were visible in all injured animals. Cavity formation was also
seen in all QL2 animals bilaterally.


5U3












11.0
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0


* In Vivo
* In Vitro
0 Histology


S01* S02* S04* S05* S06* X01** X03**



Figure 3-4. Total injury lengths determined by using in vivo, in vitro, and histologic
methods for each QUIS-injured animal. Total injury lengths in millimeters
reflect the presence of excitotoxic tissue damage. No significant differences
are observed between in vitro and histology (p > 0.05). Significant
differences are observed between in vivo and in vitro/histology (p > 0.05), but
these differences are within the range of section thickness difference between
in vivo and in vitro images. A single asterisk denotes the QL2 animals, and
double asterisks denote the QL1 animals. Significant differences (Ap < 0.001,
+p < 0.01) in injury lengths were found between QL1 and QL2 animals for all
three methods.

In Vivo DCE-MR Imaging

To determine whether there was BSCB disruption at the 17-24 day interval (mean

= 22 days) post-injury, DCE-MR imaging was performed by using Gd as a contrast

agent. Disruption of the BSCB was indicated by a significant Gd enhancement (>10%)

within the cord at the site of injury. No significant Gd enhancement was observed in the

normal and sham groups and no significant Gd enhancement was observed within the

cord at the site of injury in any of the QUIS-injured animals. Gadolinium enhancement,








however, was observed in surrounding muscle of all animals, approximately 300% peak
signal intensity enhancement, which indicates that the procedure was performed
correctly.
QL2 animal (S06)
Rostral


viVo hI vitro


2 2
4 n~~r


H4h!





. Histolog


In viva

Figure 3-5. Summary of in vivo, in vitro, and histologic data collected for a representative
QL2 animal (S06). Representative sections (1-5) are sampled from the same
rostrocaudal location in the spinal cord with each method used. Image sizes
are the same as in Figs 3-2 and 3-3. See Fig 3-1, for labeling of anatomic and
pathologic findings. Although sagittal images show the rostral-caudal extent
of the injury, transverse sections provide greater detail of the pathologic
changes associated with this injury model.
In Vitro MR Microscopy
Images obtained in vitro had greater resolution and more clearly defined the
pathologic characteristics (i.e., neuronal loss, hemorrhage, and cavitation) of the injury
compared with the in vivo MR images. No abnormal signals were observed in the normal
control animal. For the saline-injected animal, however, a hypointense region was









observed at the site of injection, which correlated with hemorrhage (see "Histologic

Findings"). Figures 3-2B and 3-3B illustrate the sagittal and transverse in vitro images,

respectively, at the epicenter for each QUIS-injured animal. In both sagittal and

transverse images, white matter and gray matter were clearly distinguishable and the cord

level was easily determined. In all QUIS-injured animals, hypointense regions were

observed at the site of injury, correlating with hemorrhage.

QL I animal (X03)
Rostral












2iudl ln vog s
In rvio In vitrqo




Caudal In vrHo Hisbtoloy


In vivo

Figure 3-6. Summary of in vivo, in vitro, and histologic data collected for a representative
QL1 animal (X03). Representative sections (1-5) are sampled with each
method from the same rostrocaudal location in the spinal cord. Image sizes
are the same as in Figs 3-2 and 3-3. See Fig 3-1, for labeling of anatomic and
pathologic findings. Although sagittal images show the rostral-caudal extent
of the injury, transverse sections provide greater detail of the pathologic
changes associated with this injury model.

Total injury lengths measured with in vitro MR images were significantly greater

than lengths determined with in vivo MR images, because of differences in section









thickness (Fig 3-4); repeated-measures ANOVA (F = 40.476; p = 0.001). Pair-wise

comparisons indicated that in vivo MR imaging gave significantly shorter injury lengths

than in vitro MR imaging (p = 0.001) and histology (p = 0.001), but were within the

range of difference in section thickness (length difference standard deviation = 0.50 mm)

for each method used (section thickness in vitro of 0.2 mm and in vivo of 1.0 mm).

Additionally, there were significant differences (p < 0.001, one-way between subjects

ANOVA, F = 34.134, p < 0.001) in injury lengths between QL1 (X01 and X03) and QL2

animals (S01, S02, and S04-S06; Fig 3-4).

Histologic Findings

Histologic results confirmed the pathologic characteristics observed with in vivo

and in vitro MR imaging. Injection tracts were visible in all QUIS-injured animals, as

well as saline-injected animals. Neuronal loss in the dorsal horn ipsilateral to the injury

was observed in all QUIS-injured animals. Neuronal loss in the dorsal horn contralateral

to the injection site was observed in all QL2 animals but not in QL1 animals. Central

canal expansion was observed in four of five QL2 animals and cavity formation was

observed only in QL2 animals, being present both ipsilateral and contralateral to the side

of QUIS injection. Sections from three of three animals (S01, S06, and X03) stained

positive for the Prussian Blue reaction, confirming the presence of hemorrhage at the

same location of hypointense signal intensity as observed in the in vivo and in vitro

images.

Because of histologic artifacts, complete injury lengths could not be determined for

all cords. In animals with minimal histologic damage (all but X03), however, the total

injury lengths determined from histologic evaluation showed no significant differences to

those determined with in vitro images (Fig 3-4; p = 0.05). However, here were









significant differences (p < 0.01, one-way between subjects ANOVA, F = 34.134, p <

0.001) in injury lengths between QL1 (X01 and X03) and QL2 animals (S01, S02, and

S04-S06; Fig 3-4).

Figures 3-5 and 3-6 show a summary of data collected from a representative QL2

(S06) and QL1 (X03) animal, respectively. Sufficient detail is visible in the in vivo

images to determine the type and extent of pathologic damage. There was narrowing of

the dorsal horn with sparing of the superficial laminae bilaterally in all QL2 animals.

This is in contrast to QL1 animals, in which sparing was observed only unilaterally. A

significant observation in this study was that following the more superficial injury

(laminae I-III, QL1); damage occurred only ipsilateral to the side of QUIS injections and

included only evidence of hemorrhage. In contrast, lesions deeper in the gray matter

(QL2) produced damage both ipsilateral and contralateral to the injection site, showing

evidence of both hemorrhage and cavitation. In addition, evidence for differences

between QL1 and QL2 injuries were seen in the rostrocaudal and ventral extent of injury

(e.g., less extensive in QL1 animals compared with QL2 animals [Fig 3-6]).

Discussion

Clinically, MR imaging has been a useful tool for the study of secondary

complications associated with SCI. For example, MR imaging can play an important role

in the study of post-traumatic syringomyelia. The excitotoxic injury model has been

shown to produce pathologic changes similar to those observed in this condition

(Yezierski et al., 1993; Schwartz et al., 1999a). Considering the lack of understanding of

the pathologic mechanism responsible for cavity formation in the injured cord (Madsen et

al., 1994), it may be possible to gain insight into the mechanism of cavitation by using in

vivo MR imaging at different time points following excitotoxic SCI. In the past,









clinicians have relied on patient descriptions of behavioral changes in determining

clinical outcome (Williams, 1990; Oakes, 1996); however, MR imaging recently has been

used to monitor changes associated with intraspinal transplantation, graft-mediated

functional recovery, and changes in syrinx morphology after transplantation (Wirth et al.,

2002) and to differentiate between cyst and scar or tumor formation with the help of MR

contrast agent administration (Schwartz et al., 1999a). With the importance and many

uses for MR imaging, continued research is needed to enhance this technique and expand

its uses to provide a better understanding of the mechanisms responsible for pathologic

changes associated with SCI. For example, an important question related to SCI is the

temporal profile of pathologic changes in the acute and chronic stages post-injury. Using

in vivo MR imaging, it should be possible to monitor these changes in the same animal.

In the present study the feasibility of using a complement of in vivo and in vitro MR

imaging, along with histologic methods to study experimental SCI was examined.

The results have shown that MR images provide details of pathologic changes,

which correlated with histologic findings, and were easily detected both in vivo and in

vitro. These changes include cavities, central canal expansion, hemorrhage, and tissue

loss. These characteristics of injury are similar to those previously reported in both

experimental (Weirich et al., 1990; Wirth et al., 1992; Falconer et al., 1994; Wirth et al.,

1995; Ohta et al., 1999; Schwartz et al., 1999a; Bilgen et al., 2000; Metz et al., 2000) and

clinical studies (Flanders et al., 1990; Weirich et al., 1990; Yamashita et al., 1990;

Yamashita et al., 1991; Ohshio et al., 1993; Flanders et al., 1999; Selden et al., 1999;

Rattliff and Voorhies, 2000; Ishida and Tominaga; 2002). The present study is one of the









first that has attempted to compare all three methods (see also a recent study in the mouse

[Bonny et al., 2004]).

An important finding in this study was the sensitivity of in vivo MR imaging to

detect differences in pathology between two different strategies of injury. Despite the

small differences in injury depths between QL1 and QL2, there were significant

differences in the overall pathology detected in the in vivo MR images. Following the

shallower injury (i.e., QL1), damage was observed only unilaterally in the gray matter

and did not show any cavitation or central canal expansion. In contrast, following the

deeper injury (i.e., QL2), damage was more severe, and showed cavitation, hemorrhage,

and neuronal loss bilaterally. These differences could be due to the connectivity between

regions of the cord at different depths in the gray matter. For example, propriospinal

connections involving laminae IV-VII are far more extensive within and between

segments than those involving more superficial laminae (Yezierski et al., 1980; Menetrey

et al., 1985). Although speculative, it is possible that, once damaged, a signal is relayed

to other parts of the gray matter that induces further damage. The deeper lesion (QL2)

resulted in damage of the contralateral side of the cord similar in pathologic

characteristics to the side that received the QUIS injection. This suggests a similar

mechanism of injury (i.e., excitotoxicity). From this, one might conclude that

propriospinal connections exert a trophic influence on other parts of the gray matter and

once damaged, leads to an injury induced release of excitatory amino acids and

subsequent pathologic changes. This observation may be important from the standpoint

of providing insight into the mechanism responsible for the progression of pathologic

changes following spinal injury.









There was a corresponding loss of gray matter illustrated in in vitro MR images and

histology. These changes could be quantified with sufficient information about volumes

of normal white and gray matter. Volume quantification of white and gray matter loss is

important; however, it was beyond the scope of this study and was considered in future

studies (Chapter 4).

All three methods used in this study were directly compared, because they all

showed similar pathologic changes. For example, in vitro MR images were the most

precise in determining injury length, rather than histologic sections due to histologic

artifacts. Although significant differences were detected between total lengths of injury,

these differences were within the thickness of sections of the methods used. For this

reason, the present data support the conclusion that in vivo MR imaging is a useful

technique to study the evolution of pathologic changes following SCI. This feature may

be especially important when correlated with the profile of changes related to the

disruption of the BSCB.

The observation of changes in the BSCB is important in characterizing the

excitotoxic model of SCI, because BSCB disruption can contribute to events leading to

detrimental pathologic processes following SCI (Rapoport, 1976; Bradbury, 1979). At

the time points used in this study, 17-24 days post-injury, our measurement of the BSCB

indicated that the barrier was intact; however, previous studies indicate an opening at

earlier time points following contusion injury (Runge et al., 1997; Bilgen et al., 2001).

Therefore, longitudinal studies will be necessary to detect the temporal characteristics of

how the BSCB is affected following excitotoxic SCI. Preliminary data from a subsequent









study suggest that there are changes over time in the integrity of the BSCB following

excitotoxic injury (S. A. Berens, P. Yezierski, T. H. Mareci; Chapter 4).

There are several advantages and disadvantages associated with each of the

methods used in the present study. In vivo MR imaging can be used to non-invasively

monitor the progression of pathologic changes over time in the same animal. This feature

provides the opportunity to study the neuroprotective and neurorestorative properties of

therapeutic interventions (e.g., transplants, drugs, etc). In vivo MR imaging methods,

however, lack the higher resolution that in vitro MR imaging and histologic methods

provide. Despite the higher resolution, in vitro MR imaging and histologic methods have

the disadvantage that they cannot be used at different time points in the same animal, and

there is a risk of losing tissue in histologic analysis because of processing artifacts.

Another advantage of in vivo MR imaging is the ability to analyze the injured cord in

transverse and sagittal planes of section at different time points in the same animal. This

capability allows one to easily determine the overall extent of injury as well as gross

pathologic changes, two important characteristics used in the evaluation of therapeutic

interventions. With continued technological improvements in in vivo MR imaging, this

technique will continue to be an important analytical tool in the study of SCI.

In some regions, in vivo MR images, collected on a 4.7T magnet, appear different

from in vitro MR images collected on a 14.1T magnet. The in vivo MR images were

collected by using T2-weighted fast spin-echo sequences, whereas in vitro MR images

were collected by using proton density-weighted spin-echo sequences. In vivo MR

images show cavitation as hyperintense signals due to T2-weighting, which is









comparable to what is used in human MR imaging. This is in contrast with in vitro MR

images, because cavities appear isointense due to proton attenuation weighting.

A limited number of studies have used in vivo MR imaging in experimental animal

models, with an even smaller number using in vitro MR imaging techniques. In the

present study, we have used customized hardware (coils) and high magnetic fields to

obtain high-quality in vivo and in vitro images in the rat. Continued improvement of

these MR components should lead to an increased utility of these methods. In our study,

in vivo MR images show high resolution and greater pathologic detail than reported in

previous MR imaging studies at this magnetic field strength or lower (Hackney et al.,

1986; Runge et al., 1997; Fraidakis et al., 1998; Ohta et al., 1999; lannotti et al., 2002;

Brodbelt et al., 2003). As expected, higher quality MR images can be obtained at higher

magnetic fields (Bonny et al., 2004; Franconi et al., 2000) or with chronically implanted

MR coils (Bilgen et al., 2000; Bilgen et al., 2001; Wirth et al., 1993; Ford et al., 1994b;

Fenyes and Narayana, 1998; Narayana et al., 1999; Silver et al., 2001). Although we

have been involved in the development of implanted coil technology (Wirth et al., 1993;

Silver et al., 2001), we did not use implanted coils in this study because we wanted to

eliminate the possible confounding effects of coil implantation and also eliminate

additional damage to the cord. By comparing the results of in vitro and histologic

methods, details seen in vivo can be confirmed, thus strengthening the rationale for using

in vivo MR imaging as an important tool in the study of SCI.














CHAPTER 4
USE OF MAGNETIC RESONANCE IMAGING TO STUDY THE
NEUROPROTECTIVE EFFECTS OF AGMATINE FOLLOWING SPINAL CORD
INJURY IN THE RAT

Introduction

The excitotoxic events following a quisqualic acid lesion play an important role in

the pathologic sequelae of spinal injury (Regan and Choi, 1994). The use of antagonists

of glutamate receptor subtypes has provided evidence for the involvement of excitotoxic

cascades in the pathological outcomes of QUIS induced spinal injury (Fairbanks et al.,

2000). Studies involving the QUIS model have shown, for example, pathophysiological

changes, up-regulation in gene expression for NMDA receptors and inducible nitric oxide

synthase (Plunkett et al., 2001). Agmatine (decarboxylated arginine) is an endogenous

neurotransmitter/neuromodulator that has been shown to have both NMDA receptor

antagonist (Yang and Reis, 1999) and NOS inhibitor properties (Auguet et al., 1995;

Galea et al., 1996). The multifaceted capacity of this agent makes it a promising

therapeutic agent in targeting secondary injury.

In the present research, the effects of agmatine, an agent with known

pharmacological actions, was evaluated in relation to blood-spinal cord-barrier

disruption, edema and cavity formation, and blood vessel leakage. It is believed that

limiting BSCB disruption reduces the entry of inflammatory mediators into the spinal

cord. The BSCB may therefore represent an important target of therapeutic intervention.

Applying the use of MRI to monitor the above pathological characteristics longitudinally

following administration of a known neuroprotective agent will provide insight into the









site of action and mechanism of a drug with known pharmacological actions as well as

further validate the use of MRI in the study of spinal cord injury. A preliminary

description of this study has been reported (Berens et al., 2004).

Materials and Methods

Animals, Surgery, and In Vivo MR Imaging

Sprague-Dawley male rats weighing 250-300g were subjected to excitotoxic spinal

cord injury as described in Chapter 3. Rats received an in vivo MRI prior to injury to

serve as their own controls, with animal preparation as described in Chapter 3. Following

MRI, animals were assigned to one of four injury/treatment groups (see

"Neuroprotection" below) and then were injected with QUIS or saline (sham injury) at a

depth of 500 im.

Neuroprotection

Agmatine was purchased from Sigma (St. Louis, MO), and dissolved in 0.9%

saline. Animals were separated into 4 injury/treatment groups as follows: (1) Saline-SCI

(sham injury, N = 5); (2) QUIS-SCI plus vehicle (0.9% saline, N = 6); (3) QUIS-SCI plus

Agmatine I (25 mg/kg, N = 7); and (4) QUIS-SCI plus Agmatine II (80 mg/kg, N = 7).

Rats were given treatment intraperitoneally 30 minutes post-QUIS injury and once a day

for 14 days (refer to Fig. 4-1 for a summary of the surgery and MR imaging timeline).

In Vivo High-Resolution Magnetic Resonance Imaging Data Acquisition

In vivo MRI and DCE-MRI were performed on days 1, 3, 7, 15, and 25 post-injury

(see Fig. 4-1). All in vivo NMR measurements were performed on an 11.1 T, 40 cm clear

bore Oxford magnet at 470 MHz by using a Bruker BIOSPEC Avance DBX console

(Bruker NMR Instruments, Billeria, MA) and 200 mT/m actively shielded gradients. A

highly sensitive quadrature MR surface coil was constructed specifically for imaging the









rat spinal cord (please refer to Chapter 2 for coil specifications). Pilot images were Tl-

weighted (TR/TE = 1000ms/15 ms, NA = 1), with a FOV of 8 cm in each direction, a 1

mm slice, and a 128 x 128 matrix. Fat suppression was done using a CHESS-type

sequence (Haase et al., 1985). To compensate for breathing artifacts and eliminate

aliasing, the read direction was changed to head-foot sagittall MR images) or left-right

orientation (transverse MR images), and a slice saturation pulse was added to the

sequence of high-resolution images. The slice saturation pulse had a mao4-180

(modified since) pulse shape and was a single 30 mm thick slice in the coronal direction.

Once the position was confirmed, a set of fat suppressed, slice saturated, high-resolution

sagittal and transverse images was collected. Sagittal T1-weighted (TR/TE = 1000 ms/15

ms, NA = 4) spin echo images were acquired over 5 slices, each 1 mm thick without a

gap between slices. Additional acquisition parameters were a FOV of 2 cm x 2 cm, read

out in the head-foot direction, and a matrix of 128 x 128. The scan time with these

parameters was 8.58 min. Transverse T1-weighted (TR/TE = 1000 ms/15 ms, NA = 4)

spin echo images were acquired over 14 slices, each 1 mm thick without a gap between

slices. Other acquisition parameters were a FOV of 2 cm x 2 cm, read out in the left-right

direction, and a matrix of 128 x 128. The scan time was 8.58 min.

Sagittal T2-weighted (TR/effective TE = 2000 ms/62.5 ms, NA = 12) fast spin echo

images with phase-encode segmentation were acquired over 5 slices, each 1 mm thick

without a gap between slices. Additional acquisition parameters were a FOV of 2 cm x 2

cm, read out in the head-foot direction, phase-encode segmentation factor of 4, and a

matrix of 128 x 128. The scan time with these parameters was 13.35 min. Transverse

T2-weighted (TR/effective TE = 2000 ms/62.5 ms, NA = 12) fast spin echo images with









phase-encode segmentation were acquired over 14 slices, each 1 mm thick without a gap

between slices. Other acquisition parameters were a FOV of 2 cm x 2 cm, read out in the

anterior-posterior direction, phase-encode segmentation factor of 4, and a matrix of 128 x

128. The scan time with these parameters was 13.35 min.

Day 1 post-injury Day 7 post-injury Day 25 post-injury
In vivo MRI 11. T In vivo MRI 11. T In vivo MRI 11.IT
Day 0 pre-injury Day 3 post-injury Day 15 post-injury Day 30 post-injury
In vivo MRI 11.1T In vivo MRI 11.T In vivo MRI 11.T In vitro 3D-MRM 14.1T







(A) Saline-SCI (N = 5) Day 30 post-injury
(B) QUIS-SCI + Vehicle (N= 6) Euthanization
(C) QUIS-SCI + Agmatine I (N = 7)
(D) QUIS-SCI + Agmatine II (N = 7)

Figure 4-1. Surgery and MR imaging timeline. Each animal was subjected to a pre-injury
MRI, and then assigned to one of four injury/treatment groups. Animals then
received an in vivo MRI on days 1, 3, 7, 15, and 25 post-injury, and were
sacrificed on day 30 post-injury. Cords were excised and undergo in vitro
three dimensional MR microscopy.

In Vivo DCE-MR Imaging Data Acquisition

After 4 sets of high-resolution images were collected, a series of dynamic contrast-

enhanced MR images was acquired as described in Chapter 3. Only sagittal images were

acquired before and after the contrast agent was injected due to extreme heating effects

caused by the transverse DCE-MR image acquisition (as described in Chapter 2). Fat

suppressed, slice saturated sagittal images were acquired with a FOV = 2 cm x 2 cm,

matrix = 96 x 96, over 3 slices, both with 1 mm thick, without a gap between slices.

Three sagittal T1-weighted pre-contrast multi-slice images (with a 3 minute gap between

each image) (TR/TE = 330 ms/9.4 ms, NA = 6) were collected. After acquisition of the









pre-contrast images, Gd was injected over a period of approximately 5 seconds with

continuous pressure, to prevent backflow, through the implanted catheter while the

animal was in the magnet. Immediately after injection, nine sagittal T1-weighted post-

contrast multi-slice images (3 minutes between each image) (TR/TE = 330 ms/ 9.4 ms,

NA = 6) were collected. These images were collected over a 60 minute timeframe, which

was chosen based on previous experience with a peak enhancement for the cord at 3 min.

The receiver gain was adjusted to one-third the value used for the high-resolution images

because Gd provides approximately 3 times the contrast enhancement.

In Vitro Three-Dimensional MR Microscopy

All excised cords were subjected to in vitro three-dimensional MR microscopy,

with cord preparation as described in Chapter 3. Images were acquired with a three-

dimensional gradient echo pulse sequence using a TR = 150 ms, TE = 10 ms with NA =

2. The image FOV was 2 cm x 0.5 cm x 0.5 cm in a matrix of 512 x 128 x 128 in a total

data acquisition time of 1.5 hrs. Therefore, MR images were acquired with an isotropic

resolution of 40 microns. A 3D Fourier transformation was applied to the acquired data

matrix to produce the 3D image. General image processing and analysis was performed

using custom software written in the Interactive Data Language (IDL, from Research

Systems, Boulder, CO).

Data Analysis

To compare results obtained between the four injury/treatment groups, the amount

of edema, blood vessel leakage, cavitation, and total longitudinal injury lengths were

determined and compared. To determine the amount of edema, the total number of MR

transverse image slices (please refer to Chapter 3 for justification in using transverse slice

orientation), with evidence of edema (diffuse hyperintense signal on T2-weighted MR









images, evident in early time points) were counted and multiplied by the section

thickness appropriate for the method used. For in vivo MR imaging, T2-weighted

transverse images were counted and multiplied by 1 mm; for in vitro MR imaging,

transverse images were counted and multiplied by 39 rm. This same method was used to

determine blood vessel leakage (hypointense signal on T2-weighted images), cavitation

(focal hyperintense signal, evident in later time points), and total longitudinal injury

length (summation of the individual pathological changes). Results for each group for

each day were averaged together and compared. Statistical analysis of these data

included a one-way between subjects ANOVA test to determine differences in injury

severity between the four injury/treatment groups (SPSS, Chicago, IL). This was

followed by a Tukey HSD post hoc test to interpret the significance of the ANOVA.

Power analysis (power = 0.80) was done for results showing non-significant trends,

including: (A) blood vessel leakage and (B) total longitudinal injury length.

Dynamic contrast-enhanced MR imaging processing was performed as described in

Chapter 3. Peak enhancement was determined for each animal each day. Results were

averaged for each injury/treatment group for each day and compared. Statistical analysis

of these data included a one-way between subjects ANOVA test to determine differences

in injury severity between the four injury/treatment groups (SPSS, Chicago, IL). This

was followed by a Tukey HSD post hoc test to interpret the significance of the ANOVA.

Volume Analysis

For further quantification of injury characteristics, volumes were calculated for

edema, blood vessel leakage, cavitation, and total injury for each of the four

injury/treatment groups. Volume analysis was done using the OsiriX-Dicom Viewer

(http://homepage.mac.com/rossetantoine/osirix). To calculate the volume of edema for a









specific cord, a data set was imported into OsiriX, and an ROI was drawn around all

areas showing edema for each slice. Once the ROI's were drawn, they were summed

together (to obtain an area) and multiplied by the slice thickness appropriate for each

method used (to obtain a volume). For in vivo MR imaging, areas were multiplied by 1

mm; for in vitro MR imaging, areas were multiplied by 39 im. Results were averaged

for each group for each day and compared. Statistical analysis of these data included a

one-way between subjects ANOVA test to determine differences in injury severity

between the four injury/treatment groups (SPSS, Chicago, IL). This was followed by a

Tukey HSD post hoc test to interpret the significance of the ANOVA. Power analysis

(power = 0.80) was done for results showing non-significant trends (volume of blood

vessel leakage).

Results

In Vivo High Resolution Magnetic Resonance Imaging

Figure 4-2 shows a representative collection of T2-weighted MR images for the

four injury/treatment groups for each day. Please refer to Chapter 3, Figure 3-1 for a

description of the pathological findings seen on an in vivo T2-weighted MR image. All

post-injury MR images for an animal were compared to their respective day 0 MR

images, thus each animal served as its own control. T2-weighted images gave the best

representation of injury, which showed hyperintense (i.e., bright) and hypointense (i.e.,

dark) signals, compared to a day 0 T2-weighted MR image, representing edema or

cavitation and blood vessel leakage, respectively. The only information obtained from

Tl-weighted images was the injection track, which was seen as a hypointense signal as

compared to a day 0 T1-weighted MR image.







































Figure 4-2. Representative T2-weighted (TR/effective TE = 2000 ms/62.5 ms; NA = 12)
MR images for each injury/treatment group for each day. In vivo MR images
are displayed with image size = 1.0 cm x 1.0 cm. MR images are sampled
from the epicenter of the injury. Images are oriented with dorsal at the top. L,
left side of cord; R, right side of cord, for all images.

The presence of edema was distinguished from the presence of cavitation, which

both show as hyperintense signals on T2-weighted MR images, by the observation that

edema is only observed in the earlier time points (completely subsides between days 7

and 15, Fig. 4-3) and is seen as a more diffuse, widespread hyperintense signal, whereas

the presence of cavitation is only observed as early as day 15 (see Fig. 4-4) and is seen as

a more intense and focal hyperintense signal. For example, in Figure 4-2, refer to the

QUIS-SCI plus Vehicle MR images. Notice the difference in the hyperintense signal









seen on days 1, 3, and 7 versus days 15 and 25. The hyperintense signal is more

widespread throughout the injured cord on the injection side on days 1, 3, and 7, whereas,

the hyperintense signal seen on days 15 and 25 is more intense and focused mainly in the

injection track.

To quantify the pathological changes (edema, cavitation, and blood vessel leakage)

following either saline or QUIS injury, the number of transverse MR slices that contained

edema (Fig. 4-3), cavitation (Fig. 4-4), or blood vessel leakage (Fig. 4-5) were counted

and multiplied by the slice thickness for each day to show a longitudinal profile of each


- Saline-SCI
- QUIS-SCI + Vehicle
- QUIS-SCI + Agmatine I
-- QUIS-SCI + Agmatine II


0 1 3 7 15 25
Time (Days)


Figure 4-3. Longitudinal profile of the presence of edema for all injury/treatment groups
as determined from in vivo T2-weighted transverse MR images. (m) p < 0.02,
QUIS-SCI + Agmatine I versus Saline-SCI. Error bars represent one standard
deviation. Data is represented as the number of transverse slices in
millimeters showing edema. Edema is present on day 1 post-injury and
subsides completely by day 15 for all injury/treatment groups.










pathological change. Additionally, the number of transverse slices that showed any

combination of the above-mentioned pathological changes was summed and multiplied

by the slice thickness to give a total injury length (Fig. 4-6).

In Figure 4-3, edema is observed immediately following injury (day 1 post-injury)

in all injury/treatment groups, persists until day 7, and completely subsides by day 15.

For the QUIS-SCI plus Agmatine I group, there was a significant increase in the number

of transverse slices containing edema on days 3 (p = 0.018, one-way between subjects

ANOVA, F = 3.459, p = 0.024) and 7 (p = 0.007, one-way between subjects ANOVA, F

= 4.390, p = 0.008) as compared to the Saline-SCI group. There were no significant

Saline-SCI
10 -- QUIS-SCI + Vehicle
SQUIS-SCI + Agmatine I
QUIS-SCI + Agmatine II
8


w 6


4







0 1 3 7 15 25
Time (Days)

Figure 4-4. Longitudinal profile of the presence of cavitation for all injury/treatment
groups as determined from in vivo T2-weighted transverse MR images. Error
bars represent one standard deviation. Data is represented as the number of
transverse slices in millimeters showing cavitation. Cavitation is present
beginning on day 15 post-injury and continues on day 25 for all QUIS-SCI
groups. No cavitation was observed in the Saline-SCI group.






57


differences in the presence of edema for the QUIS-SCI plus Vehicle and QUIS-SCI plus

Agmatine II groups as compared to the Saline-SCI group for any of the time-points.

In Figure 4-4, cavitation is observed beginning at day 15 for only the QUIS-SCI

injury groups, regardless of treatment, and persists on day 25. No cavitation was

observed for the Saline-SCI group. There were no significant differences in the number

transverse slices containing cavitation for any of the QUIS-SCI injury groups as

compared to the Saline-SCI group.


Saline-SCI
- QUIS-SCI + Vehicle
QUIS-SCI + Agmatine I
SQUIS-SCI + Agmatine II


0 1 3 7 15 25


Time (Days)


Figure 4-5. Longitudinal profile of the presence of blood vessel leakage for all
injury/treatment groups as determined from in vivo T2-weighted transverse
MR images. (m) p < 0.01, QUIS-SCI + Agmatine I versus Saline-SCI; (A) p
< 0.05, QUIS-SCI + Vehicle versus Saline-SCI. Error bars represent one
standard deviation. Data is represented as the number of transverse slices in
millimeters showing blood vessel leakage. Blood vessel leakage is observed
on day 1 post-injury and continues to increase until day 7, at which point it
seems to stabilize for all injury/treatment groups.






58

In Figure 4-5, blood vessel leakage is observed throughout all experimental time-

points for all injury/treatment groups, increasing from day 1 to day 7, at which point, the

amount of blood vessel leakage appears to remain constant. There was a significant

increase, versus Saline-SCI, in the number of transverse slices containing blood vessel

leakage on days 7 (one-way between subjects ANOVA, F = 4.661, p = 0.006) for the

QUIS-SCI plus Vehicle (p = 0.012) and QUIS-SCI plus Agmatine I (p = 0.007) groups;

15 (one-way between subjects ANOVA, F = 5.037, p = 0.004) for the QUIS-SCI plus


U


7U


,_-n


- Saline-SCI
- QUIS-SCI + Vehicle
-QUIS-SCI + Agmatine I
.. TT T n ,' T


QUIS


-SUI + Agmatine II


A
~zczIj~
U


Time (Days)

Figure 4-6. Longitudinal profile of the total pathology injury length (the presence of
edema, blood vessel leakage, and cavitation combined) for all injury/treatment
groups as determined from in vivo T2-weighted transverse MR images. (m) p
< 0.05, QUIS-SCI + Agmatine I versus Saline-SCI; (A) p < 0.02, QUIS-SCI
+ Vehicle versus Saline-SCI. Error bars represent one standard deviation.
Data is represented as the number of transverse slices in millimeters showing
any combination of edema, blood vessel leakage, and cavitation. The
presence of edema is the largest contributor to the total injury length in the
earlier time-points (days 1, 3, and 7), whereas, blood vessel leakage is the
main contributor in the later time-points (days 15 and 25).









Vehicle (p = 0.007) and QUIS-SCI plus Agmatine I (p = 0.006) groups; and 25 (one-way

between subjects ANOVA, F = 4.390, p = 0.008) for the QUIS-SCI plus Vehicle (p =

0.042) and QUIS-SCI plus Agmatine I (p = 0.007) groups. There were no significant

differences in the presence of blood vessel leakage for the QUIS-SCI plus Agmatine II

group as compared to the Saline-SCI group for any of the time-points. Although no

significant differences were detected between QUIS-SCI plus Agmatine II and QUIS-SCI

plus Vehicle groups, power analysis revealed that 8 animals for day 7, 8 animals for day

15, and 35 animals for day 25 would be required to obtain a significance at p = 0.05 at a

power = 0.80.

The longitudinal profile of total injury length measured with in vivo transverse MR

images is represented in Figure 4-6. The presence of edema is the primary contributor to

the total injury length in the earlier post-injury time-points (days 1, 3, and 7), whereas,

blood vessel leakage is the foremost contributor in the later post-injury time-points (days

15 and 25). For the QUIS-SCI plus Vehicle group, there was a significant increase in the

total injury length on days 15 (p = 0.007, one-way between subjects ANOVA, F = 5.037,

p = 0.004), and 25 (p = 0.006, one-way between subjects ANOVA, F = 4.577, p = 0.007)

as compared to the Saline-SCI group. For the QUIS-SCI plus Agmatine I group, there

was a significant increase in the total injury length on days 1 (p = 0.049, one-way

between subjects ANOVA, F = 2.672, p = 0.058), 3 (p = 0.009, one-way between

subjects ANOVA, F = 4.087, p = 0.013), 7 (p = 0.029, one-way between subjects

ANOVA, F = 3.430, p = 0.024), 15 (p = 0.006, one-way between subjects ANOVA, F =

5.037, p = 0.004), and 25 (p = 0.019, one-way between subjects ANOVA, F = 4.577, p =

0.007), as compared to the Saline-SCI group. There were no significant differences in









total injury length for the QUIS plus Agmatine II group as compared to the Saline-SCI

group for any of the time-points. Although no significant differences were detected

between QUIS-SCI plus Agmatine II animals and QUIS-SCI plus Vehicle, power

analysis revealed that 31 animals for day 7, 13 animals for day 15, and 9 animals for day

25 would be required to obtain a significance at p = 0.05 at a power of 0.80. Refer to

Table 4-1 for a summary of significance.

In Vivo Dynamic Contrast-Enhanced Magnetic Resonance Imaging

To determine whether there was BSCB disruption at the day 0 and days 1, 3, 7, 15,

and 25 post-injury time-points, DCE-MR imaging was performed using Gd as a contrast

agent. Disruption of the BSCB was indicated by a significant Gd enhancement (>10%)

within the cord at the site of injury. Figure 4-7 provides a peak enhancement profile for

all four injury/treatment groups for all time-points. No significant Gd enhancement was

observed in any of the four injury/treatment groups at the day 0 (pre-injury) time-point.

Gadolinium enhancement, however, was observed in surrounding muscle of all animals,

approximately 300% peak signal intensity enhancement, which indicates that the

procedure was performed correctly.

For the Saline-SCI group (sham injury), there was a significant increase in peak

enhancement (one-way between subjects ANOVA, F = 13.966, p < 0.001) on days 1 (p =

0.001) and 3 (p < 0.001) as compared to day 0. For the QUIS-SCI plus Vehicle group,

there was a significant increase in peak enhancement (one-way between subjects

ANOVA, F = 52.422, p < 0.001) on days 1 (p < 0.001), 3 (p < 0.001), and 7 (p = 0.02) as

compared to day 0. For the QUIS-SCI plus Agmatine I group, there was a significant

increase in peak enhancement (one-way between subjects ANOVA, F = 59.234, p <

0.001) on days 1 (p < 0.001), 3 (p < 0.001), and 7 (p = 0.012) as compared to day 0. For






61


the QUIS-SCI plus Agmatine II group, there was a significant increase in peak

enhancement (one-way between subjects ANOVA, F = 31.807, p < 0.001) on days 1 (p <

0.001), 3 (p < 0.001) and 7 (p = 0.005) as compared to day 0. When comparing between

groups on individual days, there were no significant differences.


*



* 1
..Je


100

80

60


* Saline-SCI
* QUIS-SCI + Vehicle
M QUIS-SCI + Agmatine I
* QUIS-SCI + Agmatine II


Day 0


Day 1


Day 3


Day 7


Day 15 Day 25


Figure 4-7. Summary of DCE-MRI BSCB disruption. Peak enhancement is represented
as a percentage. There were significant differences in peak enhancement for
all injury/treatment groups on days 1 and 3 as compared to day 0. There were
significant differences in peak enhancement for the QUIS-SCI injury groups,
but not the saline-SCI group on day 7 as compared to day 0. No significant
disruption was observed on days 15 and 25 as compared to day 0.
Additionally, no significant differences were observed between the
injury/treatment groups for any of the days. (*) p < 0.001, (A) p < 0.01. Error
bars represent one standard deviation.






62


Volume Analysis

To further quantify pathological changes (edema, cavitation, and blood vessel

leakage) following either saline or QUIS injury, regions of interest were drawn, using

transverse MR images, around areas showing edema (Fig. 4-8), blood vessel leakage

(Fig. 4-9), or cavitation (Fig. 4-10), and were summed and multiplied by the appropriate

slice thickness for each animal and time-point to obtain a volume for each of the above

mentioned pathological changes. Additionally, a region of interest was drawn around the

entire injured area (edema, blood vessel leakage, and cavitation combined) for each slice,





35 -
Saline-SCI

30 QUIS-SCI + Vehicle
QUIS-SCI + Agmatine I
25 QUIS-SCI + Agmatine II


20-


15 -


10 -


5


0
Day 1 Day 3 Day 7 Day 15 Day 25

Figure 4-8. Volume of edema for all injury/treatment groups as determined from in vivo
T2-weighted transverse MR images. (*) p < 0.05. Error bars represent one
standard deviation. Data is represented as the volume in cubic millimeters.









summed and multiplied by the appropriate slice thickness for each animal and time-point

to give an overall volume of injury (Fig. 4-11).

The volume of edema peaks at day 3 and starts diminishing day 7 for all

injury/treatment groups except the QUIS-SCI plus Agmatine I group. This profile

closely matches that shown in Figure 4-3. There was a significant increase in volume of

edema on day 1 (one-way between subjects ANOVA, F = 3.286, p = 0.03) between the

QUIS-SCI plus Agmatine II and Saline-SCI groups (p = 0.014). Additionally, the QUIS-

SCI plus Vehicle group showed a significantly higher volume of edema (p = 0.05, one-

way between subjects ANOVA, F = 3.053, p = 0.037) as compared to the Saline-SCI



9 Saline-SCI

8 QUIS-SCI +Vehicle
QUIS-SCI + Agmatine I
7 QUIS-SCI + Agmatine II

,6

5



3

2

1-

0
Day 1 Day 3 Day 7 Day 15 Day 25


Figure 4-9. Volume of blood vessel leakage for all injury/treatment groups as determined
from in vivo T2-weighted transverse MR images. Error bars represent one
standard deviation. Data is represented as the volume in cubic millimeters.









group. No significant differences were observed on day 7 for any of the QUIS-SCI

groups as compared to the Saline-SCI group.

Figure 4-9 illustrates the volume of blood vessel leakage profile, and shows an

increase in volume through day 7 and a slight decrease in volume on days 15 and 25.

The increase seen is consistent with that seen in Figure 4-5; however, volume analysis

becomes a more sensitive outcome measure for days 15 and 25, as Figure 4-5 shows a

constant level of blood vessel leakage (due to the method of quantification), whereas the

volume shows a slight decrease. No significant differences in volume of blood vessel



5
Saline-SCI
4.5 0 QUIS-SCI + Vehicle

4 QUIS-SCI + Agmatine I
QUIS-SCI + Agmatine II
3.5

S 3

a 2.5

S 2

1.5

1-

0.5 T

0
Day 1 Day 3 Day 7 Day 15 Day 25


Figure 4-10. Volume of cavitation for all injury/treatment groups as determined from in
vivo T2-weighted transverse MR images. Error bars represent one standard
deviation. Data is represented as the volume in cubic millimeters.









leakage were detected between any of the QUIS-SCI groups versus the Saline-SCI group.

Although no significant differences were detected between QUIS-SCI plus Agmatine II

animals and QUIS-SCI plus Vehicle, power analysis revealed that 9 animals for day 7,

252 animals for day 15, and 19 animals for day 25 would be required to obtain a

significance at p = 0.05 at a power of 0.80.

The volume of cavitation following injury is presented in Figure 4-10. The volume

profile is also consistent with the longitudinal profile of the presence of cavitation (Fig.

4-4). Cavitation was observed in all QUIS-SCI groups, but was not present in the Saline-

SCI group. Similar to the longitudinal profile data, there are no significant differences in

the volume of cavitation following a QUIS lesion as compared to a saline lesion.

Finally, the total injury volume, which consisted of edema, blood vessel leakage,

and cavitation is shown is shown in Figure 4-11. As seen with the individual volumes of

pathological changes, the total injury volume profile is similar to the total longitudinal

injury length profile (Fig. 4-6). The edema volume is highly represented in the earlier

time-points post-injury, whereas the volume of blood vessel leakage is more highly

represented in the later time-points. The only significant differences in total injury

volume was seen on day 1 (one-way between subjects ANOVA, F = 3.801, p = 0.017)

between the QUIS-SCI plus Agmatine II group and the Saline-SCI group (p = 0.007).

This is consistent with the significance seen in Figure 4-8. Refer to Table 4-2 for a

summary of significance.










* Saline-SCI
* QUIS-SCI + Vehicle
* QUIS-SCI + Agmatine I
* QUIS-SCI + Agmatine II


T*


TI


TT IIL


Day 3


Day7


Day 15


Day 25


Day 30
(In Vitro)


Figure 4-11. Volume of total injury (volume of edema, blood vessel leakage, and
cavitation combined) for all injury/treatment groups as determined from in
vivo T2-weighted transverse MR images (days 1, 3, 7, 15, and 25) and in vitro
transverse 3D MRM images (day 30). (*) p = 0.007. Error bars represent one
standard deviation. Data is represented as the volume in cubic millimeters.


25

20

15


Dayl












Table 4-1. Summary of Significant Differences in Longitudinal Profiles of Edema, Cavitation, and Blood Vessel Leakage, and BSCB


Disruption.
Injury/Treatment
Group versus
Saline-SCI


QUIS + Vehicle

QUIS + Agmatine I


QUIS + Agmatine II


Edema
(longitudinal
profile)

(A) Figure 4-3


Cavitation
(longitudinal
profile)

(B) Figure 4-4


T, p < 0.02
(days 3, 7)


Blood Vessel
Leakage
(longitudinal
profile)
(C) Figure 4-5
T, p < 0.05
(days 7, 15, 25)
p, p < 0.01
(days 7, 15, 25)


Table 4-2. Summary of Significant Differences in Volumes of Edema, Blood Vessel
Injury/Treatment Edema Blood Vessel Cavitation
Group versus (Volume) Leakage (Volume)
Saline-SCI (Volume)


QUIS + Vehicle


QUIS + Agmatine I

QUIS + Agmatine II


(A) Figure 4-8
f, p < 0.05
(day 3)


(B) Figure 4-9


(C) Figure 4-1(


T, p < 0.05
(day 1)


Total Injury Length
(A+B+C)


Figure 4-6
T, p < 0.02
(days 15, 25)
T, p < 0.05
(days 1, 3, 7, 15,
25)


Leakage, and Cavitation.
Total Injury
Volume
(A+B+C)
0 Figure 4-11


BSCB Disruption



Figure 4-7


t, p < 0.01
(day 1)









Discussion

The goal of the present study was to use magnetic resonance imaging to evaluate

the neuroprotective effects of an agent, with known pharmacologic actions, on the

excitotoxic spinal cord injury model at different time-points in the same animal. Major

findings include: (1) high resolution, in vivo MRI can be used to quantitatively and

qualitatively study the dynamic changes in spinal cord injury pathology and blood-spinal

cord-barrier integrity over time in the same animal; and (2) administration of 80 mg/kg

agmatine (Agmatine II) post-injury resulted in neuroprotection against blood vessel

leakage and edema following QUIS-SCI when compared to the Saline-SCI group.

The results have shown that MR images provide details of pathologic changes,

easily detected both in vivo and in vitro. These changes include cavities, blood vessel

leakage, cavitation, and tissue loss. Injury was observed unilaterally in some animals and

bilaterally in others. This difference is mainly due to inconsistency in the injection

technique. Despite every effort to control the depth of injection, variance was found

among injury depths, most likely accounting for differences in injury severity. As

discussed previously (refer to Chapter 3, "Discussion"), injuries following a deeper

injection resulted in a more extensive injury both rostrocaudally and ventrally, showing

bilateral blood vessel leakage and cavitation, whereas with a shallower lesion, damage

was less severe resulting only in ipsilateral blood vessel leakage.

Differences in injury severity may also account for the significant increase in

edema following the administration of 25 mg/kg agmatine (Agmatine I). There were

variances in injection depth in each group; the QUIS-SCI plus Agmatine I group showing

the highest variability in injury severity. More animals would need to be analyzed to

reduce the variability and rule out the action of the drug as the cause. Additionally,









although there were no significant differences observed between any of the four

injury/treatment groups, a neuroprotective trend was observed following 80 mg/kg

agmatine (Agmatine II) seen in the volume of edema on days 3 and 7; blood vessel

leakage on days 3, 7, 15, and 25; and total injury volume on days 3, 7, 15, and 25.

Increasing the number of animals would also reduce variability and provide significance.

An equally important finding in this study was the sensitivity and capability of in

vivo MR imaging to study the neuroprotective effects of agmatine in a progressive model

of spinal injury over time in the same animal. It is important in research to effectively

minimize resources and cost. With the capability of using an animal as its own control in

a time-course study, the need for an excessive number of animals to obtain multiple data

time-points is eliminated, as shown in the current study. The data is thus more accurately

comparable, as it allows you to compare pre- and post-injury data for a single animal.

Secondary injury events, such as neural degeneration and cavitation in spinal

injury, both a result of glutamate induced excitotoxicity, have been well documented

(Faden and Simon, 1988; Nag and Riopelle, 1990; Hao et al., 1991; Yezierski et al.,

1993; Wrathall et al, 1994). These studies indicate that elevations in glutamate signify

the initiation of cellular cascades that influence excitability and survivability of

surrounding neurons. Limiting such events through the use of neuroprotective agents

would have a profound impact on secondary processes involved in the mediation of cell

death and the progression of secondary injury in SCI. Agmatine, a cationic amine formed

by the decarboxylation of arginine, is such an agent that has been shown to effectively

block NMDA receptor activity (Yang and Reis, 1999) and reduce the production of nitric

oxide by inhibiting NOS (Auguet et al., 1995; Galea et al., 1996).









The present results showed that during a survival period of 30 days, 80 mg/kg

agmatine (Agmatine II) provided neuroprotection most strongly against edema and blood

vessel leakage. The vehicle and 25 mg/kg agmatine (Agmatine I) treatment groups did

not show neuroprotection, as expected. Although a higher volume of edema was

observed in the QUIS-SCI plus Agmatine II group compared to the Saline-SCI group, it

was not as widespread, which is evident in the longitudinal profile of edema, meaning

that the edema was more concentrated around the epicenter of the injury. The same is

true for the total volume of injury; while the QUIS-SCI plus Agmatine II group had a

higher volume of injury compared to the Saline-SCI group, the injury was not as

widespread along the rostral-caudal axis of the cord. This raises the question of whether

volume or rostral-caudal extent is more important. Previous studies have shown that

length is a more determining factor in relation to behavioral outcome. For example,

studies have shown a relationship between the onset of excessive grooming behavior and

the longitudinal extent of excitotoxic damage produced by a QUIS lesion (Yezierski et

al., 1998; Gorman et al., 2001; Plunkett et al., 2001; Yu et al., 2003). Yu et al. (2003)

suggested that by limiting the spread of QUIS induced excitotoxic damage via

neuroprotection, the expression of injury-induced pain behaviors may lessen. This is one

indication that the longitudinal profile versus volume results hold a stronger indication of

neuroprotection.

Observing changes in the dynamics of the BSCB is important in understanding the

destructive pathologic processes resulting from an excitotoxic lesion (Rapoport, 1976;

Bradbury, 1979). Understanding the time course of permeability of the BSCB and the

subsequent pathological consequences is crucial in understanding the secondary









mechanisms associated with spinal cord injury. Previous studies by Runge et al. (1997)

and Bilgen et al. (2001) involving the contusion injury have shown cord-barrier

disruption during the acute phase of injury. Previously, it was shown that in later time-

points (i.e.,17-24 days post-injury), the barrier was intact following a QUIS lesion

(Chapter 3, Berens et al., 2005). At the time-points used in this study, a dynamic profile

of BSCB disruption was achieved. The BSCB showed disruption immediately (day 1)

following injury, and persisted until day 7 post-injury, with a maximum disruption on day

3 post-injury. Repair of the barrier was evident, because on day 7, even though there was

a significant increase in peak enhancement, there was an overall decrease in value as

compared to earlier time-points (days 1 and 3). No significant differences were observed

between any of the injury/treatment groups compared to the Saline-SCI group indicating

that agmatine may not play a significant role in attenuating cord-barrier disruption.














CHAPTER 5
DIFFUSION WEIGHTED MR IMAGING OF THE EXCISED QUISQUALIC ACID
LESIONED RAT SPINAL CORD

Introduction

Diffusion weighted MR imaging of water translational diffusion has been utilized

to provide information beyond the anatomic and spatial resolution of conventional

magnetic resonance imaging. It has a greater sensitivity, compared to conventional MR

techniques, in evaluating the integrity of injured white matter (Ford et al., 1994a), and

assessing the degree of neuroprotection, axonal sprouting, and regeneration following

therapy (Nevo et al., 2001; Schwartz et al., 2003; Schwartz et al., 2005).

White and gray matter vary in their diffusion characteristics due to their structural

makeup. When considering diffusion properties, it is important to consider

microstructure properties, such as cell size and density, cell orientation, membrane

permeability, and intracellular and extracellular viscosity of the tissue. In addition, cell

membranes and myelin can also restrict water diffusion along the axis perpendicular to

white matter tracts (Beaulieu, 2002; Schwartz and Hackney, 2003). Water diffusion in

gray matter is fairly unrestricted and its directionality is more random than white matter.

However in white matter, diffusion is along bundles of parallel, myelinated axons, is

restricted (Moseley et al., 1990). Thus, normal spinal cord is considered anisotropic,

since there are more barriers to diffusion in the plane transverse to the long axis of the

cord (Schwartz and Hackney, 2003).









In the present study, DWI was used to detect changes in fiber structure in white and

gray matter following excitotoxic spinal injury. It is hypothesized that DWI can be

employed to evaluate the long term effects of excitotoxic injury on fiber tracts, and that it

has the sensitivity to detect differences in gray matter fiber structure between control

(non-injured) and QUIS lesioned cords.

Materials and Methods

Animals, Surgery, and Neuroprotection

Sprague-Dawley male rats weighing 250-300g were subjected to excitotoxic spinal

cord injury as described in Chapter 4. Rats were randomly assigned to one of three injury

groups: (1) Control (no injury, N = 3); (2) Saline-SCI (sham injury, N = 5); and (3)

QUIS-SCI (N = 8). Animals were sacrificed on day 30 post-injury, and cords prepared as

described in Chapter 3. Different groups were used than those described in Chapter 4 in

order to verify the technique. Differences needed to be observed between the three

groups listed above before using this methodology in the assessment of neuroprotective

treatment (see Chapter 6, "Future Directions").

In Vitro Three Dimensional MR Microscopy and Diffusion Weighted Imaging

All in vitro NMR measurements were performed on a 14. IT, 51-mm-bore Oxford

magnet, at 600 MHz, using a Bruker BIOSPEC Avance DBX console. All excised cords

were subjected to in vitro three-dimensional MR microscopy as described in Chapter 4,

with cord preparation described in Chapter 3. Following 3D MRM acquisition, diffusion

weighted MR images were acquired using a spin-echo, high angular resolution diffusion

imaging (HARDI, Tuch et al., 2002) acquisition sequence (TR/TE = 1400 ms/25 ms)

with gradients applied in 46 directions with a gradient strength of 664 mT/m (b-value of

1250 s/mm2). A low b-value image was also obtained with a gradient strength of 175









mT/m (b-value of 100 s/mm2) applied in 6 directions. Additional parameters were echo

crusher duration = 0.12ms, A value = 17.5 ms, 6 value = 1.5 ms, bandwidth = 32 kHz,

FOV = 4.2 x 4.2 x 12.0 mm3, matrix = 70 x 70 with 40 slices, and resolution = 60 x 60 x

300 im3. Scan times were 9.5 hours for each cord. General image processing and

analysis was performed using custom software written in the Interactive Data Language

(IDL, from Research Systems, Boulder, CO).

Data Analysis

Diffusion weighted MR data were fit to a rank-2 tensor model as the initial method

of analysis. To determine the fractional anisotropy and average diffusivity of the white

and gray matter for each animal of each injury group, regions of interest (ROI) were

drawn using the So MR image in the following regions of white matter (Fig. 5-1): A,

dorsal column, right side; B, dorsal column, left side; C, lateral, right side; D, lateral, left

side; E, ventral, right side; and F, ventral, left side; and in the following regions of gray

matter (Fig. 5-2): A, dorsal horn, non-injured side; B, lateral, right side; C, lateral, left

side; D, ventral, right side; and E, ventral, left side. Regions of interest were not drawn in

the dorsal horn on the injured side, because a value of zero would result; an outcome due

to the hypointensity of the signal. For each outcome measurement (i.e., fractional

anisotropy and average diffusivity), values were averaged for each ROI and injury group.

Saline and QUIS injury groups were then compared to the control group.

To further characterize the QUIS lesions, a diffusion orientation transform (DOT)

method (Ozarslan, et al., 2006) was applied to the analysis of excised rat spinal cord,

diffusion-weighted image data acquired with the HARDI acquisition scheme. The DOT

has recently been developed to resolve the architecture of complex structured tissue. The

DOT transforms the HARDI measured diffusivity profiles into displacement probability









profiles, representing the probability of diffusion in a particular direction. Rather than fit

the measured diffusion signal to a rank-2 model of diffusion, the DOT method assumes

the signal attenuation (as a function of diffusion weighting) is represented by a mono-

exponential decay. Then this signal attenuation is used to directly calculate the

displacement probability, as a function of position, using a q-space Fourier

transformation (Callaghan, 1991). Therefore, the peaks in these displacement probability

profiles correspond to distinct fiber orientations. Using the DOT, the QUIS lesion can be

further characterized to delineate and describe complex fiber structures, specifically in

the gray matter.

Results

First, to determine the effects of saline and QUIS lesions on white matter as

compared to control (no injury), average diffusivity (AD) and fractional anisotropy (FA)

values were calculated for the six white matter regions described above for each injury

group (see "Data Analysis", Fig. 5-1). Data for each group was combined and averaged

by matching and centering injured areas for each cord, because values along the

rostrocaudal axis in a normal cord did not vary (i.e., it was not necessary to match the

slices anatomically). Fractional anisotropy values of one indicate anisotropic

(unidirectional) diffusion; zero indicates isotropic (non-preferential) diffusion. Figures 5-

3 through 5-5 show the AD and FA values of white matter for the Control, Saline-SCI,

and QUIS-SCI groups, respectively. The AD values in control cords were very

consistent along the rostral-caudal axis (Fig. 5-3A). However, there was a marginal

increase in AD from rostral to caudal in both Saline- and QUIS-SCI groups (Figs. 5-4A

and 5-5A, respectively). Fractional anisotropy decreased slightly from rostral to caudal

for all three groups but was equivalent between all six regions of interest in white matter






76


(Figs. 5-3B, 5-4B, and 5-5B). There were no significant differences in AD or FA values

between any of the six white matter regions of interest for any of the three groups. There

were no significant differences in AD or FA between any of the injury groups as

compared to control.


-C


-E


Figure 5-1. So (b = 100 s/mm2) transverse MR image from a QUIS lesioned animal
showing regions of interest drawn for analysis of the white matter. A, dorsal
column white matter, right side; B, dorsal column white matter, left side; C,
lateral white matter, right side; D, lateral gray matter, left side; E, ventral gray
matter, right side; F, ventral gray matter, left side.


-A

-B


C

E .


Figure 5-2. So (b = 100 s/mm2) transverse MR image from a QUIS lesioned animal
showing regions of interest drawn for analysis of the gray matter. A, dorsal
horn gray matter, non-injured side; B, lateral gray matter, right side; C, lateral
gray matter, left side; D, ventral gray matter, right side; E, ventral gray matter,
left side.









Second, to determine the effects of the saline and QUIS lesions on gray matter as

compared to control (no injury), AD and FA values were also calculated for the five gray

matter regions described above for each injury group (see "Data Analysis", Fig. 5-2).

Figures 5-6 through 5-8 show the AD and FA values of gray matter for the Control,

Saline-SCI, and QUIS-SCI groups, respectively. The AD values for gray matter for all

three groups were very consistent along the rostral-caudal axis (Figs. 5-6A, 5-7A, 5-8A).

Fractional anisotropy remained constant from rostral to caudal for the Saline-SCI group

but had a negligible increase in the Control and QUIS-SCI groups (Figs. 5-6B and 5-8B,

respectively). There were no significant differences in AD or FA values between any of

the five gray matter regions of interest for any of the three groups. Surprisingly, there

were no significant differences in AD or FA between any of the injury groups as

compared to control.

Since, no differences were detected with the initial series of analysis, DOT analysis

was used to determine if this more sensitive method could detect changes in white and

gray matter fibers in control and QUIS-SCI cords (Fig. 5-9). In the Control group (Fig.

5-9A, C), white matter fiber orientation was observed to be out of the plane of section

(i.e., along the long axis of the cord), as expected. Gray matter fibers showed a much

more complex orientation and were observed to be in the plane section, also as expected.

Fibers situated in the ventral regions of the gray matter appear to be more complex than

those in dorsal regions of the dorsal horn. Most interestingly, however, are fibers in the

substantial gelatinosa; they appear to align along the long axis of the cord. One would

expect fibers to be within the transverse plane versus along the long axis, as fibers enter

the substantial gelatinosa from the dorsal roots and traverse into the dorsal horn.











--Dorsal Column White Matter-Right
4.00E-04 -Dorsal Column White Matter-Left
Lateral White Matter-Right
3.50E-04
Lateral White Matter-Left
3.00E-04 --Ventral White Matter-Right
-- Ventral White Matter-Left


S2.50E-04

S2.00E-04

S1.50E-04

1.00E-04


5.00E-05

0.00E+00


1 3 5 7 9 11 13 15 17 19 21 23 25

Position


0.9

0.8

0.7

0.6

C 0.5
S.5 -*- Dorsal Column White Matter-Right

g 0.4 Dorsal Column White Matter-Left
0.3 Lateral White Matter-Right
Lateral White Matter-Left
0.2 Lateral White Matter-Right
n, --- Ventral White Matter-Left


1 3 5 7 9 11 13 15 17

Position


19 21 23 25


Figure 5-3. Average diffusivity and fractional anisotropy values for Control white matter.
Position is rostral (left side of graph) to caudal (right side of graph). (A)
Average diffusivity along the rostral-caudal axis of white matter is consistent.
No significant differences were detected between any of the control white
matter regions for either of the measurements. (B) There was a slight
decrease in FA from rostral to caudal for all white matter regions.


aj











-- Dorsal Column White Matter-Right
4.00E-04 -- Dorsal Column White Matter-Left
-- Lateral White Matter-Right
Lateral White Matter-Left
3.50E-04 --- Ventral White Matter-Right
-- Ventral White Matter-Left
3.00E-04

S2.50E-04

0 2.00E-04

1.50E-04

1.00E-04

5.00E-05

0.00E+00
1 3 5 7 9 11 13 15 17 19 21 23 25 27

Position
A
1-


0.9

0.8

0.7

S0.6

C 0.5

S0.4 -*- Dorsal Column White Matter-Right
a *- Dorsal Column White Matter-Left
0.3 Lateral White Matter-Right
Lateral White Matter-Left
0.2 --Ventral White Matter-Right
*-Ventral White Matter-Left
0.1


1 3 5 7 9 11 13 15 17 19 21 23 25 27

Position
B


Figure 5-4. Average diffusivity and fractional anisotropy values for Saline-SCI white
matter. Position is rostral (left side of graph) to caudal (right side of graph).
(A) Average diffusivity along the rostral-caudal axis of white matter is
consistent, with a slight increase along the rostral caudal axis. No significant
differences were detected between any of the control white matter regions for
either of the measurements. (B) There was a slight decrease in FA from
rostral to caudal for all white matter regions.











--Dorsal Column White Matter-Right
-- Dorsal Column White Matter-Left
-- Lateral White Matter-Right
Lateral White Matter-Left
--Ventral White Matter-Right
-- Ventral White Matter-Left


1 3 5 7 9 11 13 15
Position


17 19 21 23 25


- Dorsal Column White Matter-Right
-* Dorsal Column White Matter-Left
- Lateral White Matter-Right
Lateral White Matter-Left
-- Lateral White Matter-Right
- Ventral White Matter-Left


1 3 5 7 9 11 13 15
Position


17 19 21 23 25


Figure 5-5. Average diffusivity and fractional anisotropy values for QUIS-SCI white
matter. Position is rostral (left side of graph) to caudal (right side of graph).
(A) Average diffusivity along the rostral-caudal axis of white matter is
consistent, slightly increasing from rostral to caudal. No significant
differences were detected between any of the control white matter regions for
either of the measurements. (B) There was a slight decrease in FA from
rostral to caudal for all white matter regions.


4.00E-04

3.50E-04

3.00E-04

S2.50E-04

2.00E-04

2 1.50E-04

1.00E-04

5.00E-05

0.00E+00


S0.6










Dorsal Horn Gray Matter-Non-injured
--Lateral Gray Matter-Right
- Lateral Gray Matter-Left
-- Ventral Gray Matter-Right
-- Ventral Gray Matter-Left


1 3 5 7 9 11 13 15 17 19 21 23 25 27
Position


Dorsal Horn Gray Matter-Non-Injured
Lateral Gray Matter-Right
Lateral Gray Matter-Left
--Ventral Gray Matter-Right
-- Ventral Gray Matter-Left













1 3 5 7 9 11 13 15 17 19 21 23 25 27
Position


Figure 5-6. Average diffusivity and fractional anisotropy values for Control gray matter.
Position is rostral (left side of graph) to caudal (right side of graph). (A)
Average diffusivity along the rostral-caudal axis of gray matter is consistent
from rostral to caudal. No significant differences were detected between any
of the control white matter regions for either of the measurements. (B)
Fractional anisotropy values also remain consistent from rostral to caudal for
all gray matter regions.


6.00E-04


5.00E-04


4.00E-04


S3.00E-04
2.E-04
g 2.00E-04


1.00E-04


0.00E+00


0.7
S0.6
< 0.5

0.4
0


~~pL~L~t~~











Dorsal Horn Gray Matter-Non-Injured
-- Lateral Gray Matter-Right
- Lateral Gray Matter-Left
-- Ventral Gray Matter-Right
- Ventral Gray Matter-Left


.'. -~ -.---.- I -


1.00E-04


0.00E+00


1 3 5 7 9 11 13 15 17 19 21 23 25 27

Position


Dorsal Horn Gray Matter-Non-Injured
1- -- Lateral Gray Matter-Right

.9 Lateral Gray Matter-Left
-- Ventral Gray Matter-Right
.8 -- Ventral Gray Matter-Left


0.6

< 0.5

0.4

0.3

0.2

0.1


1 3 5 7 9 11 13 15 17 19 21 23 25 27

Position


Figure 5-7. Average diffusivity and fractional anisotropy values for Saline-SCI gray
matter. Position is rostral (left side of graph) to caudal (right side of graph).
(A) Average diffusivity along the rostral-caudal axis of gray matter is
consistent from rostral to caudal. No significant differences were detected
between any of the control white matter regions for either of the
measurements. (B) Fractional anisotropy values also remain consistent from
rostral to caudal for all gray matter regions.


6.00E-04


5.00E-04


4.00E-04


S3.00E-04

2.E-04
g 2.00E-04












6.00E-04


5.00E-04


Dorsal Horn Gray Matter-Non-Injured
- Lateral Gray Matter-Right
-- Lateral Gray Matter-Left
-- Ventral Gray Matter-Right
-*- Ventral Gray Matter-Left


4.00E-04

S3.00E-04


2.00E-04


1.00E-04


0.00E+00


1 3 5 7 9 11 13 15 17 19 21 23 25
Position


Dorsal Horn Gray Matter-Non-Injured
-*- Lateral Gray Matter-Right
Lateral Gray Matter-Left
-- Ventral Gray Matter-Right
-Ventral Gray Matter-Left


I 0.6
C 0.5
= 0.4
S0.3


1 3 5 7 9 11 13 15 17 19 21 23 25
Position


Figure 5-8. Average diffusivity and fractional anisotropy values for QUIS-SCI gray
matter. Position is rostral (left side of graph) to caudal (right side of graph).
(A) Average diffusivity along the rostral-caudal axis of gray matter is
consistent from rostral to caudal. No significant differences were detected
between any of the control white matter regions for either of the
measurements. (B) Fractional anisotropy values also remain consistent from
rostral to caudal for all gray matter regions.


F~p* ~bf* ~ _h












































C 0 D

Figure 5-9. Probability maps calculated from diffusion weighted datasets collected from
excised normal and QUIS injured spinal cords. (C) and (D) are enlargements
taken from (A) normal; and (B)QUIS injured spinal cords, respectively. The
orientation surfaces are color-coded such that portions in or out of the plane of
section are blue and green, respectively. Notice the loss of complexity of
fiber structure in the dorsal horn, specifically along the medial regions (green
circle) in the QUIS injured cord.









While comparing the effects of a QUIS lesion (Fig. 5-9 B, D) versus Control (Fig.

5-9A, C), a loss of complexity in gray matter fiber structure is observed in the QUIS-SCI

group. There appears to be a change in fiber orientation or a lining up of fibers along the

medial dorsal horn gray matter (Fig. 5-9D, green circle) in the injured cord compared to a

control cord. No changes were observed in fiber structure or orientation in the white

matter or ventral regions of the gray matter in the QUIS injured cords.

Discussion

The goal of the present study was to use diffusion weighted MR imaging to

evaluate changes in white and gray matter fibers in the spinal cord following excitotoxic

injury. Major findings include: (1) DWI can be used to quantitatively and qualitatively

study excitotoxic spinal cord injury; and (2) DWI has the sensitivity to detect changes in

gray matter fiber structure between control (non-injured) and QUIS lesioned cords.

Differences were observed in FA and AD values between white and gray matter as

expected. Their diffusion characteristics differ because of differences in their structural

makeup. Gray matter consists of cell bodies and is reasonably unrestricted, with more

random directionality in water diffusion (Moseley et al., 1990) than white matter. In

contrast, white matter consists of long parallel bundles of myelinated axons, where water

diffusion is mainly restricted to the long or rostrocaudal axis of the cord (Moseley et al.,

1990). Thus, FA values in gray matter were lower that those in white matter and

conversely, AD values higher in gray matter than white matter, because gray matter is

considered more isotropic (unrestricted diffusion in all directions), whereas, diffusion in

white matter is considered anisotropic (restricted diffusion in one direction).

With DOT, fiber structure and orientation in the white matter was fairly uniform.

Contrastingly, fiber orientation in the gray matter varied among dorsal and ventral









regions. Fibers situated in the ventral regions of the gray matter appear to be more

complex than those in the dorsal regions of the dorsal horn. This is because propriospinal

connections involving laminae IV-VII are far more extensive within and between

segments than those involving more superficial laminae (Yezierski et al., 1980; Menetrey

et al., 1985). Since DOT was able to define fiber orientation and had the sensitivity to

detect changes following injury, it may be possible to apply this technique to gain further

insight into the differences seen with the two different injury strategies described in

Chapter 3 and ultimately more details about the timing of events and the resolution of

spatial location of pathologic changes following spinal injury could finally be discovered.

When comparing FA and AD values in different regions of the cord, in Control and

QUIS-SCI groups, results were three-fold. First, there were no differences in FA or AD

values in any of the regions of white (6 ROI's) or gray matter (5 ROI's), separately,

meaning all white matter in a particular section had similar values; the same being true

for gray matter. However, values varied slightly along the rostrocaudal axis of the cord

because of the change in structural makeup. Secondly, results predictably revealed no

significant differences in FA or AD values in the white matter, because the QUIS lesion

is a gray matter lesion. Thirdly, results surprisingly showed that there were no significant

differences between Control and QUIS-SCI groups in the gray matter. A loss in FA and

an increase in AD were expected in the QUIS-SCI group around the injury site in the

initial analysis versus Control. Explanations might be that the method of analysis did not

have the sensitivity to detect changes in FA or AD values for this particular injury model

(i.e., changes may have been too subtle).