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1 INFLUENCE OF NEUROARCHITECTURE ON INFUSATE DISTRIBUTION: APPLICATIONS FOR A NOVEL EPILEPSY THERAPY By SVETLANA KANTOROVICH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMEN T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Svetlana Kantorovich
3 To my parents, James and Adele ; sister, Yelana ; and brother, Vladimir
4 ACKNOWLEDGMENTS I have many people to thank for wh o and wh ere I am today. First and foremost, I would like to thank the chair and members of my committee who have shaped the scientist I have become. My advisor Dr. Paul Carney, generously provided me with scientific freedom to pursue my interests and conc omitantly bestowed incredible opportunities for translational research. I would also like to express my gratitude to Dr s Malisa Sarntinoranont, William Ogle, Jake Streit and Michael King for serving on my committee and being exceptional mentors Special thanks to Dr. King for being a consistent source of reliable information, patience, and advice, and to Dr. Thomas Mareci for meticulous analysis and critique of my experiments and publications. I am very thankful for all the wonderf ul members of my laborat ory, past and present Thank you Rabia for teaching me everything I know in the world, especially the serious things. Thank you Mansi for laying down smooth tracks to follow and staying for the next generation. Thank you Stephen for being my recording guru and a source of great wisdom. Thank you eMalick for your intellectual curiosity, without which we would have never become collaborators and good friends. Thank you Garrett for becoming a MRI genius so that our experiments were completed without a hitch. I am grateful I had the opportunity to work with and learn from you. Thank you Phil and Aaron for your friendship and support, and many thanks to Frank, Shiva, Eric, Matt, Dave and Junli for providing an exciting and encouraging lab environment Finally, I wish to thank my family and friends. I have been blessed with the best parents, James and Adele Kantorovich, who have never wavered in their support or confidence in my abilities. Without the sacrifices they have made, I would never be where I am today. I to thank my brother, Vladimir, a role model whose footsteps I
5 have been following, and my twin sister, Yelana, a n interactive designer who is the reason my p resentation slides always look professionally amazing. Last but not least, t hank you Jeanne tte for being the best friend I could ask for, and Wade for sticking with me from day one. I could never have done it without you.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 BACKGROUND ................................ ................................ ................................ ...... 16 Motivatio n ................................ ................................ ................................ ............... 16 Epilepsy ................................ ................................ ................................ .................. 17 Classification of the Epilepsies ................................ ................................ ......... 17 Temporal Lobe Epilepsy ................................ ................................ ................... 18 Limbic system circuitry ................................ ................................ ............... 19 Etiology and pathophysiology of TLE ................................ ......................... 21 Current Treatments for Epilepsy ................................ ................................ ....... 23 Animal Models of Epilepsy ................................ ................................ ............... 25 Comparative Neuroanatomy of the Rat and Human Hippocampal Formation .. 26 Drug Delivery ................................ ................................ ................................ .......... 28 Disruption of the BBB ................................ ................................ ....................... 29 Passage Through the BBB ................................ ................................ ............... 30 Drug design ................................ ................................ ................................ 30 Mediated transport ................................ ................................ ..................... 3 1 Passage Behind the BBB ................................ ................................ ................. 33 Extracellular space ................................ ................................ ..................... 33 Intracerebroventricular/intrathecal delivery ................................ ................ 36 Intracerebral delivery ................................ ................................ ................. 37 2 CONVECTION ENHANCED DELIVERY IN THE NORMAL RAT HIPPOCAMPUS ................................ ................................ ................................ ..... 45 Introduction ................................ ................................ ................................ ............. 45 Methods ................................ ................................ ................................ .................. 48 Animal Preparation and Surgical Procedures ................................ ................... 48 MR Imaging and Image Segmentation ................................ ............................. 50 Histology ................................ ................................ ................................ ........... 51 Microscopy ................................ ................................ ................................ ....... 51 Results ................................ ................................ ................................ .................... 52 Infusion Site ................................ ................................ ................................ ...... 52
7 Gd albumin Distribution in the Septal Hippocampus ................................ ........ 52 Gd albumin Distribution in the Temporal Hippocampus ................................ ... 53 Backflow ................................ ................................ ................................ ........... 54 Image Segmentation ................................ ................................ ........................ 54 Histological Analysis ................................ ................................ ......................... 55 Discussion ................................ ................................ ................................ .............. 56 Distrib ution Profile and Shape ................................ ................................ .......... 56 Analysis of Shape and Volume ................................ ................................ ......... 60 Conclusions ................................ ................................ ................................ ............ 63 3 INFLUENCE OF LIMBIC SYSTEM INJURY ON INFUSATE DISTRIBUTIONS IN THE RODENT HIPPOCAMPUS ................................ ................................ ......... 68 Introduction ................................ ................................ ................................ ............. 68 Methods ................................ ................................ ................................ .................. 70 Animals ................................ ................................ ................................ ............. 70 Surgical Procedures ................................ ................................ ......................... 70 Induction of Self Sustaining Limb ic Status Epilepticus (SE) by Hippocampal Electrical Stimulation ................................ ................................ ..................... 71 MR Imaging ................................ ................................ ................................ ...... 72 Infusion of Gd albumin ................................ ................................ ..................... 73 Immunohistochemistry ................................ ................................ ...................... 74 Image Segmentation and Statistical Analysis ................................ ................... 75 Results ................................ ................................ ................................ .................... 76 SE Induced Injury ................................ ................................ ............................. 76 Volumes of Distribution of Gd albumin and Changes in Hippocampal Volume ................................ ................................ ................................ .......... 77 Characteristics of Gd albumin Distribution ................................ ....................... 78 Histology 2 4 Hours Post SE ................................ ................................ ............. 79 Histology 60 Days Post SE ................................ ................................ .............. 80 Discussion ................................ ................................ ................................ .............. 81 Acute SE Induced injury ................................ ................................ ................... 82 Injury and Final Infusate Dis tribution Volume ................................ ................... 83 Injury and Pattern of Infusate Distribution ................................ ......................... 85 Other Factors Affecting Final Infusate Distribution ................................ ........... 86 Conclus ions ................................ ................................ ................................ ............ 88 4 CONVECTION ENHANCED DELIVERY OF THERAPEUTIC AGENT CARRIERS ................................ ................................ ................................ ............. 99 Introduction ................................ ................................ ................................ ............. 99 Gene Therapy for Epilepsy ................................ ................................ ............. 100 Stem Cell Therapy for Temporal Lobe Epilepsy ................................ ............. 102 Proof of Principle Studies for Viral Vector and Stem Cell CED Delivery ........ 103 Methods ................................ ................................ ................................ ................ 104 Animals ................................ ................................ ................................ ........... 104 Vector Construction ................................ ................................ ........................ 104
8 Transduction and Isolation of NSCs ................................ ............................... 105 Surgical Procedures and CED Infusions for NSC Experiments ...................... 105 Surgical Procedures and CED Infusions for Viral Vector Experiments ........... 106 Perfusion and Immunochemistry ................................ ................................ .... 106 Quantification of NSC Transplant Dimensions ................................ ............... 107 Image Segmentation and 3D Reconstruction of NSC Engraftments .............. 108 Results ................................ ................................ ................................ .................. 108 Distribution of Viral Vectors in the Hippocampus ................................ ............ 108 Distribution of Transplanted NSCs in the Hippocampus ................................ 110 Distribution of Transplanted NSCs in the Thalamus and Striatum .................. 111 Discussion ................................ ................................ ................................ ............ 111 Viral Vector Distribution ................................ ................................ .................. 111 NSC Distribution ................................ ................................ ............................. 113 Conclusions ................................ ................................ ................................ .......... 114 5 CONCLUSIONS AND FUTURE WORK ................................ ............................... 121 Conclusions ................................ ................................ ................................ .......... 121 Identifying Approaches to Prevent Epilepsy or Its Progression ...................... 121 Developing and Optimizing New Strategies for Targeted Therapies .............. 122 Developing Animal Models for the Progression of Epilepsy ........................... 124 Future Work ................................ ................................ ................................ .......... 125 LIST OF REFERENCES ................................ ................................ ............................. 127 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 150
9 LIST OF TABLES Table page 3 1 Index of injury classification 24 hours post SE epilepticus.. ............................... 91 4 1 Features of gene expression vectors ................................ ................................ 116 4 2 Results from viral vector infusions ................................ ................................ .... 116
10 LIST OF FIGURES Figure page 1 1 The epilepsies are classified based on etiology, semiology, origin of seizures, and epilepsy syndromes.. ................................ ................................ ................... 42 1 2 Schematic of the various routes of molecular t ransport across the BBB. ........... 43 1 3 Final CED distribution with and without backflow of an MR contrast agent tagged with a fluorescent tracer in the rodent septal hippocampus. ................... 44 1 4 Lentivirus and neural stem cells targeted to the septal hippocampus via CED .. 44 2 1 Damage induced by the infusion cannula in the septal hippocampus.. .............. 64 2 2 Sagittal images of a single rat demonstrating the apparent disconnect between the septal and temporal hippocampus. ................................ ................ 64 2 3 High resolution T1 weighted MR images of septal hippocampus infusions. ....... 65 2 4 High resolution T1 weighted MR images of temporal hippocampus infusions. ... 65 2 5 Histological images following infusate CED into the septal hippocampus showing Evans blue dye spreading throughout the septal hippocampus. .......... 66 2 6 Histological images following infusate CED into the temporal hippocampus showing Evans blue dye spreading thoughout the temporal hippocampus. ....... 67 3 1 Experimental protocol flow chart ................................ ................................ ......... 89 3 2 T2 weighted coronal images of 19 different rodent brains acquired post induction of SE reveal injury within regions of the limbic circuitry. ...................... 90 3 3 Increasing classificat ions of injury were correlated with volumes of distribution in 24 hour animals ................................ ................................ ............ 91 3 4 High resolution T1 weighted images of Gd albumin infusions into the septal hippocampus of 19 different rodent brains post SE. ................................ ........... 92 3 5 Characterization of hippocampal damage 24 hours post SE. ............................. 93 3 6 Characterization of parahippocampal damage 24 hours post SE. ...................... 94 3 7 Damage to the ventral subiculum was seen in 6/17 rats 24 hours post SE. ....... 95 3 8 Characterization of thalamic inju ry 24 hours post SE. ................................ ........ 96
11 3 9 T2 weighted MR image with corresponding histology of a spontaneously seizing animal 60 days post SE. ................................ ................................ ......... 97 3 10 T2 weighted MR image and corresponding histology of another spontaneously seizing animal 60 days post SE. ................................ ................. 98 4 2 Infusions of viral vectors into the left rat septal hippocampus. .......................... 117 4 3 Infusions of four AAV serotypes exhibit specific distribution patterns throughout the hippocampal septo temporal axis. ................................ ............ 118 4 4 Short term en graftments of NSCs expressing GFP demonstrate the hippocampus specifically features anisotropic transport. ................................ .. 119 4 5 Geometric analyses of NSC infusions. ................................ ............................. 120
12 LIST O F ABBREVIATIONS AAV Adeno associated virus AED Anti epileptic drug AP Anterior posterior anatomical direction BBB Blood brain barrier CA Cornu ammonis subfield of the hippocampus CC Corpus callosum CD 68 Cluster of Differentiation 68 CED Convection enhance d delivery CNS Central nervous system CSF Cerebrospinal fluid DAB 3,3' diaminobenzidine DG Dentate gyrus DV Dorsal ventral anatomical direction ECS Extracellular space EEG Electroencephalography FJC Flouro Jade C GABA aminobutyric acid Gd albumin human s erum albumin labeled with gadolinium chelated by diethylene triamine pentaacetic acid, macromolecular magnetic resonance imaging contrast agent Gd DTPA gadolinium chelated by diethylene triamine pentacetic acid, low molecular weight magnetic resonance imag ing contrast agent GFP Green fluorescent protein GFAP Glial fibrillary acidic protein HF Hippocampal fissure
13 ICV Intracerebroventricular ILAE International League Against Epilepsy IP Intraperitoneal mTLE mesial temporal lobe epilepsy ML Medial lateral anat omical direction MR Magnetic Resonance MRI Magnetic Resonance Imaging PEEK P olyaryletheretherketone SE Status epilepticus SNR Signal to noise ratio SSLSE Self sustained limbic status epilepticus T Tesla T1 Longitudinal relaxation time constant T2 Transvers e relaxation time constant TLE Temporal lobe epilepsy VI Velum interpositum
14 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 Philo sophy INFLUENCE OF NEUROARCHITECTURE ON INFUSATE DISTRIBUTION: APPLICATIONS FOR A NOVEL EPILEPSY THERAPY By Svetlana Kantorovich May 2012 Chair: Paul R Carney Major: Medical Sciences Neuroscience Temporal Lobe Epilepsy (TLE) is the most common partial onset epilepsy and often refractory to antiepileptic drugs. Despite pharmacological advances in epilepsy treatment seizures cannot be controlled in many patients because current drugs do not target causes of the disorder Convection enhanced deliv ery (CED) a local drug delivery technique has the potential to deliver novel therapeutic s while avoiding systemic toxicity and blood brain barrier limitations. However, the efficacy of CED depends on appropriate tissue targeting which requires a deeper understanding of the effect of neuroarchitecture on interstitial transport. To investigate the influence of microanatomy on CED, the volume and pattern of infusate distribution was examined after varying the site of infusion, integrity of structure, and co mpound infused. Gd albumin, a magnetic resonance (MR) contrast agent, was infused in to two sites in normal rat hippocampi. Infusions were repeated in animals injured by an episode of status epilepticus (SE) a prolonged seizure known to cause neuronal dama ge and edema in the hippocampus and associated structures Infusions in injured animals were implemented 24 hours post SE, as prophylactic treatment has the potential to reduce structural damage, diminish associated cognitive impairment, or prevent
15 epilept ogenesis Infusions were also implemented at 60 days post SE, during chronic TLE High resolution T1 and T2 relaxation weighted MR images were acquired at 11.1 Tesla in vivo to visualize Gd albumin distribution and morphological changes, respectively. His tological analysis was performed to validate infusions and characterize injury at higher resolution. Finally, information gained from infusion studies with Gd albumin was used to guide proof of principle studies with neural stem cells and viral vectors. In fusions in normal hippocampi spread along preferential paths parallel to fiber directions and within fissures, with limited penetration across densely packed cell layers. Infusions in injured hippocampi followed similar patter n s, but resulted in significan tly larger distribution volumes that correlated with increased injury severity Therapeutic carrier syste ms exhibited comparable spatial characteristics to Gd albumin infusion s but were also influenced by cell interactions These studies show anisotropic hippocampal architecture plays a leading role in the distribution of infusate by CED This information can be applied to improve targeting guidelines, incorporated into injury specific computation models, and considered in treatment strategies involving no vel therapeutic agents
16 CHAPTER 1 B ACKGROUND Motivation Epilepsy is a chronic neurological disorder that affects about 1% of people in the United States, with approximately 50 million people affected worldwide ( Browne and Holmes, 2001 ) There are over 40 types of epilepsies, diverse in etiology but similarly characterized by the recurrence of spontaneous seizures. Seizures are transient episodes of abnormal, excessive, or synchronous neuronal activi ty in a focal area or throughout the brain ( Sander, 1993 ) Unfortunately, d espite b oldest recognized conditions, treatments for epilepsy lag behind developments of other neurological disorders. This is especially true for temporal lobe epilepsy (TLE), in which one third of patients are resistant to available antie pileptic drugs (AEDs). As temporal lobe epilepsy is the most common form of epilepsy in the adult population this leaves hundreds of thousands of patients with uncontrolled seizures. Therefore, t he objective of the research presented in this dissertation wa s to develop and optimize a novel therapeutic strategy for the prevention and treatment of focal epilepsy using convention enhanced delivery (CED). CED is a local drug delivery technique that uses positive pressure to deliver infusate directly into paren chymal interstitial space. This mechanism of delivery is applicable to a wide variety of potential treatments because it does not require passage through the blood brain barrier (BBB). The introduction of inhibitory neuropeptides, viral agents, or stem cel l therapy is impossible using currently available delivery options. Using CED, t hese agents can be delivered in a targeted manner over clinically relevant volume s
17 CED has the potential to overcome many obstacles in drug delive ry but detailed knowledge a bout the influence of biophysical properties of the brain on delivery into complex regions is lacking In addition to the extensive work being done to understand the influence of infusion parameters and cannula design, a thorough understanding of interstit ial flow in both normal and injured areas is necessary to provide direction for safe and effective delivery. This body of work addresses these concerns through the characterization of direct intracranial de livery in normal animals (C hapter 2) and in an ani mal model of epilepsy ( C hapter 3). Chapter 4 discusses applications of this strategy in specific ther apeutic modalities. Finally, Chapter 5 summarizes findings and proposes future conduits for the progression of this work. Epilepsy Epilepsy is one of the w recognized neurological disorder s The term is reserved for ( Commission on Epidemiology and Prognosis of the International L eague Against Epilepsy, 1993 ) manifestations of the disorder across geographical, racial, or social boundaries. This section will introduce the various syndromes, describe the etiology, p athophysiology, common treatments, and discuss animal models used to study th e disorder. Classification of the Epilepsies The epilepsies are classified based on etiology, semiology, origin of seizures, and epilepsy syndromes. Two systems of classification of epilepsies are in use today. The first developed by the International League Against Epilepsy (ILAE) i s based on seizure semiology ( Commission on Classification and Terminology of the International
18 League Against Epilepsy, 1981 ) This scheme divides seizures into three broad types based on EEG obs ervations : 1) partial (focal) seizures that involve one area of the brain, 2) generalized seizures that involve the whole brain, and 3) unclassifiable seizures which may be generalized and partial, continuous, multifocal, or generalized to only one hemisp here. These broad types are further subdivided by the appearance of additional clinical observations such as specific motor signs or l oss of consciousness This classification scheme is detailed in Figure 1 1 A The ILEA 1981 system is easy to implement, bu information about the severity, cause, or prognosis of the disease. Th us, another system of classification based on epilepsy syndromes was developed by the ILAE eight years later ( Commission on Classification and Terminology of the International League Against Epilepsy, 1989 ) Similar to the first system, the epilepsies a re divided broadly into four groups: 1) localization related seizures that in volve distinct parts of the brain 2) generalized seizures that involve both sides of the brain, 3) undetermined seizures that may be localized or generalized, and 4) special syndromes. These groups are then further divided by etiology, i.e. whether the ca use is unknown (idiopathic), identifiable (symptomatic) or hidden (cryptogenic). This classification is shown in Figure 1 1B The ILEA 1989 system is helpful for diagnostic purposes and is used as a complement to the 1981 system. For example, TLE is classi fied as a symptomatic localization related syndrome. People suffering from TLE may experience simple partial seizures, complex partial seizures, or secondarily gen eralized tonic clonic seizures. Temporal Lobe Epilepsy The focus of this dissertation is on T LE, t he most common form of epilepsy in adults ( Engel, 2001a ) and among the most frequent types of intractable epilepsy
19 ( Engel, 2001b ) TLE refers to spontaneous recurrent seizures originating in the limbic system. There are two main types of TLE: 1) mesial TLE (mTLE), in which seizures begin in the hippocampus, parahippocampal gyrus, or amygdala, and 2) lateral TLE, in which seizures arise from the neocortex. mTLE, the more common form, is poorly controlled with pharmacological intervention and hence most likely to benefit from alternative therapeutic strategies. Limbic system circuitr y Given that TLE affects the limbic system, it is necessary to introduce the limbic system circuitry ( Amaral and Lavenex, 2007 ) that underlies pathological features of the disorder. Limbic areas commonly affected in TLE include the hippocampal formation, parahippocampal gyrus, thalamus, and septum. The hippocampal formation consists of the d entate gyrus, Cornu Ammonis (CA) fields CA1, CA2, and CA3, subiculum, presubiculum, parasubiculum, and entorhinal cortex. These regions are cytoarchitectonically distinct, but linked by largely unidirectional neuronal pathways to form functional circuits. The dentate gyrus (DG) is a trilaminate region that receives direct input from the entorhinal cortex via the perforant path. It has a relatively cell free molecular layer, a granule cell layer, and a polymorphic cell layer (hilus) containing mossy cells an d interneurons. The molecular and granule cell layers form a sideways U shape in which the blade superior to the CA3 cell layer is referred to as the suprapyramidal blade and the blade inferior to the CA3 cell layer is referred to as the infrapyramidal bla de. Granule cells, the principal excitatory cells of the DG synapse bilaterally on mossy cells in the polymorphic layer and ipsilaterally on CA3 pyramidal cells.
20 The CA1, CA2, and CA3 regions are known specifically as the hippocampus proper. Inputs and o utputs to the hippocampal proper are organized within distinct layers or strata The pyramidal cell layer contains principle cells. Stratum oriens contains basal dendrites, interneurons, and some CA3 axons. Stratum radiatum contains apical dendrites, som e CA3 axons, and several classes of interneurons. Stratum lacunosum moleculare contains entorhinal cortex fibers, afferents from subcortical regions, and interneurons. This layer abuts the hippocampal fissure, a cell free region continuous with ventricular space that is lined by pia mater and filled with cerebrospinal fluid ( CSF ) and blood vessels ( Humphrey, 1967 ) Stratum lucidum is a thin acellular layer in the CA3 containing m ossy fibers. The alveus is a fiber containing layer composed of axons from the pyramidal cells in the subiculum and hippocampus. It eventually merges with the fimbria, which goes on to become the fornix. Information from mossy fibers in the DG advances to the CA3, and is then carried from the CA3 to the apical and basal dendrites of the CA1 via Shaffer collaterals. The CA3 also has a massive associational network which includes projections to the CA3 and CA2, and commissural connections to the contralateral CA3, CA2, and CA1. The CA1 has very limited associational projections and only weak commissural projections. The CA1 projects axons to the deep layers of the entorhinal cortex and to the deep portion of the subicular molecular layer. The subiculum is a main output of the hippocampus; it sends projections to the deep layers of entorhinal cortex, perirhinal cortex, amygdaloid complex, endopiriform nucleus, diencephalon (nucleus reuniens, paraventricular nucleus, paratenial nucleus), neocortex (prelimbic co rtex, infralimbic cortex, retrosplenial cortex, orbitofrontal cortex), basal forebrain (septal nucleus, nucleus accumbens), mammillary nuclei, and brain stem. It does not give rise to
21 commissural connections, but has a substantial association projection th at extends temporally from the cells of origin. The subiculum also projects to the presubiculum and parasubiculum, which distributes processed information to a series of cortical and subcortical structures. There many extrinsic connections with the hippoc ampus and other limbic regions. Most external input comes from the entorhinal cortex and is known as the perforant path. Neurons of layer II of the entorhinal cortex project axons to the DG and CA3 while l ayer III of the entorhinal cortex project s to the CA1 and the subiculum CA1 also receives input from the amygdaloid complex, perirhinal and postrhinal cortices, nucleus reunions of the thalamus, and the septum. The CA3 receives connections from the amygdaloid complex as well, and has reciprocal bilateral connections with the lateral septum. Etiology and p athophysiology of TLE The precise cause of TLE is unknown in most cases, but it is typically seen after an initial precipitating injury such as status epilepticus (SE), brain injury, tumors, meningitis, e ncephalitis, and febrile seizures during childhood ( French et al., 1993 ; Mathern et al., 1996 ; Pitkanen and Sutula, 2002 ; Lewis, 2005 ) The hallmark pathology of TLE is hippocampal sclerosis ( Blumcke et al., 2002 ; de Lanerolle and Lee, 2005 ) although cases witho ut those changes exist as well. Histological evaluation of biopsy specimens from chronic e pilepsy reveal hippocampal atrophy and scarring with varying degrees of regional neuronal loss and gliosis Classic hippocampal sclerosis consists of selective loss of pyramidal cells in the CA1 and hilus ( de Lanerolle et al., 2003 ) but n eurodegener ation has also been described in the CA2 and CA3 as well ( Bruton, 1988 ) Recurrent connectivity within the dentate gyrus has
22 been proposed as a major epileptogenic mechanism ( Mathern et al., 1995a ) due to several observations in hippocampal sclerosis. Loss of interneuronal subtypes ( Mathern et al., 1995b ) a nd excitatory mossy cells ( Blumcke et al., 2000 ) h av e been noted with a bnormal hippocampal neurogenesis, dispersion of the dentate granule cell layer, and mossy fiber sprouting ( Thom et al., 2005 ) Cytological alterations, including enlargement of neurofilaments, abnormal dendritic nodular swellings, ramification of end folial neurons, hav e also been reporte d though these features may represent adaptive rather than prima ry abnormalities ( Blumcke et al., 1999 ) In addition to the hippocampus and dentate gyrus pathological changes have commonly b een reported in the human amygdala ( Hudson et al., 1993 ) entorhinal cortex ( Du et al., 1993 ) and thalamus ( Margerison and Corsellis, 1966 ; Bruton, 1988 ) It remains unc ertain whether neuropathological findings represent a substrate for TLE development or are a consequence o f repeated seizures. In other words, the century ( Gowers, 1881 ) Animal models have been used to address this issue and have shown that recurrent seizures can produce hippocampal damage (for review, see ( Ben Ari et al., 200 8 ) ) Clinical evidence is inconclusive ( Hauser and Lee, 2002 ) because there are epilepsy syndromes that are progressive, and there are syndromes that remit regardless of the number of seizures experienced. How ever in human TLE, the remission of seizures after the surgical removal of a damaged focus ( Wiebe et al., 2001 ) suggests this damage is only one feature of a diseas e process that include s other predispositions.
23 Current Treatments for Epilepsy The most common treatment of epilepsy is pharmacotherapy. There are a number of anti epileptic drugs (AEDs) available for the management of epileptic seizures that are delivered through the use of pills or intravenous injections These may function by decreasing the excitation of neur ons by blocking sodium or calcium channels, or by enhancing their inhibition with the potentiation of inhibitory neurotransmitters, like aminobuty ric acid ( GABA ) The most commonly used older AEDs for TLE are ph en ytoin, carbamazepine, primidone, valproate, and phenobarbital. Newer drugs, such as gabapentin, topiramate, lamotrigine, levetiracetam, pregabalin, tiagabine lacosamide, and zonisamide hav e also been incorporated into the clinic Unfortunately, these medications are only effective for about two thirds of patients ( Brodie and Dichter, 1996 ) and often come with a number of side effects ( Baker et al., 1997 ) Some intractable epilepsy cases are remediable with surgical resection of the epileptic focus Not all individuals are candidates for surgery however, and among those who are, nearly 20% will refuse to accept the risks of a major surgical procedure ( Berg et al., 2003 ) Moreover, although epilepsy surgery is often considered the only causal treatment of epilepsy, in most patients anti epileptic drug treatment must be continued after surgery to achieve seizure control ( Loscher and Schmidt, 2002 ) In fact, the probability of achieving a one year remission from surgery is only about 57%. This suggests that in many patients undergoing epilepsy surgery, the focal tissue contributing to intractability is removed, rather than the complex epileptogenic network underlying the epileptic process.
24 The ketogenic diet is a less common treatment that was advocated after 19 21, when it was noted that ketosis and acidosis induced by a high fat/low carbohydrate diet had anticonvulsant effects ( Geyelin, 1921 ) This diet consists of very large amounts of fat ( minimum 1g per kg per day of protein) with a typical fat to carbohydrate ration of 4:1 or 3:1. Whil e those who respond to the diet show dramatic improvement, its use is primarily for generalized epilepsies associated with diffuse brain abnormalities L ess success is seen in patients with complex partial seizures or epileptiform discharges in the tempora l region ( Beniczky et al., 2010 ) Ketone bodies appear throughout the brain, so this treatment is not considered targeted and is difficult to maintain. Furthermore, there are some potential concerns regarding its effects on growth in children ( Rubenstein, 2008 ) and the effect of an almost all fat diet on cardiac status P atients may also have the option of receiving electrical stimulation as a potential treatment for intractable epilepsy. The most common form of stimulation treatment is the vagus nerve s timulator, although there is increasing int erest in deep brain stimulation and direct regional stimulation of epileptic regions ( Theodore and Fisher, 2004 ) The stimulation of the vagus nerve causes an increase in inhibition and a decrease in excitability, therefore raising the threshold for seizure ( Vonck et al., 2001 ) The mechanism of action is unclear, but it does not require craniotomy, and efficacy is comparable to adjunctive antiepileptic drugs ( DeGiorgio et al., 2000 ; Sirven et al., 2000 ) Similarly, stimulation of the anterior thalamic nucleus has been shown to be important in generalized seizures ( Mirski and Ferrendelli, 1986 ) Mir ski et al. showed they could inhibit the anterior thalamic nucleus with high frequency (100 Hz) stimulation. Timed electrical stimulation in response to measured preictal brain dynamical changes
25 has also shown to prevent seizures ( Nair S, 2005 ) Despite these encouraging results, difficulties and risks persist. The vagus nerve stimulator could result in complications such as left vocal cord paralysis, lower facial weakness, sternocleidomastoid spasm, and transient bradycardia or asystole ( Charous et al., 2001 ) The above mentioned limited therapeutic options leave a large number of patients untreated. Despite pharmacological, surgical, and electrical adva nces in the treatment of epilepsy, seizures cannot be controlled in many patients because current therapies target the symptoms of the disease (seizures) once it is already fully developed It is important to note that t here are current ly no protocols for preventative treatments, although m ounting evidence suggests prophylactic treatment after an initial insult may result in a positive modifying effect on the development of epilepsy (for review see [4, 5]) Early treatment has the potential to reduce struct ural damage, diminish associated behavioral and cognitive impairment, or prevent epileptogenesis. This subject is addressed in more detail in Chapter 3. Animal Models of Epilepsy Many different animal models of epilepsy have been described for various purp oses. In general, there are genetic animal models and models in which seizures are induced in normal animals. Induced seizures may be created with electrical or chemical application, and can result in the development of spontaneous (chronic) or on demand ( acute) seizures. Both electrical and chemical a cute seizure models, such as the maximal electroshock seizure (MES) and pentylenetetrazole (PTZ) models ( White et al., 1995 ) and are commonly used for AED discovery because they are easy to use and time efficient. MES and PTZ models induce seizures in healthy rodents. Alternatively, e lectrical kindling is an on demand seizure model in which the repeated application of
26 electrical stimuli induces permanent susceptibility to seizures. Kindling models are generally use d to characterize the anticonvulsant potential of a compound screened with the initial screening tests ( Loscher, 2011 ) Chronic, or post SE, animal models are typically used to evaluate e pilepsy prevent ion or disease modification because the epileptic condition results as a consequen ce of injur y followed by a latent period. Chemical SE models, such as kainate ( Hellier et al., 1998 ) and pilocarpine ( Cavalheiro, 1995 ) are routinely used, but have high mortality rates and considerable inter ani mal variability. T he self sustaining limbic SE model ( Nitsch and Klatzo, 1983 ; van Vliet et al., 2007 ) on the other hand, is often considered to have the greatest parallels with human TLE ( Schmidt and Loscher, 2009 ) This is an electrically induced SE model that replicates essential characteristics of epilepsy as it occurs in humans ( Lothman et al., 1990 ) including comparable electrophysiological correlates, pathological changes in the limbic system, and histological changes in the hippocampus ( Falconer, 1974 ; Babb, 1987 ; Bertram et al., 1990 ; Goodman, 1998 ; Marchi et al., 2010 ) This animal model is used in the studies described within this work because it most closely approximates structural changes. Importantly, a fraction of these animals also exhibit pharmaco resistance to current AEDs ( Loscher, 1997 ) Although more labor intensive, appropriate models of refractory epilepsy should also be incorporated into the development of novel epilepsy therapies Comparative N euroanatomy of the R at and H uman Hippocampal F ormation The description of the limbic system circuitry in this chapter has dealt primarily with the rat hippocampal formation because much of the neuroanatomical information available has been gained from studies of the rat, and the work described in this disserta tion involves the rat hippocampus. There are, however, a number of differences
27 in morphologic variations from species to species ( Amaral and Lavenex, 2007 ) For example, the CA1 and entorhinal cortex are disproportionately larger in primates than rodents. The volume of the dentate gyrus and hippocampus is 100 times larger in humans than in r ats (3300 mm 3 versus 32 mm 3 ). There are 15 times more dentate granule cells in humans compared to rats, and the human CA1 has 35 times more pyramidal cells than the rat CA1. Additionally, a lthough a common topography in the entorhinal hippocampal projectio n seems to be present in rat and human, there is also more extensive interchange of information between the hippocampal formation and the neocortex. The full extent of the differences between these species cannot yet be accurately gauged; there very well m ay be substantial variation in the cellular morphology, connectivity, and chemical neuroanatomy across species. Fortunately, the characteristic architecture of the hippocampal formation presents little variation with phylogenetic development. Both the rat and human hippocampus have a basic morphology of an elongated, curved, and rod like structure. Densely packed cell layers are obvious in rats and humans, with progressive lamination from dentate gyrus to entorhinal cortex. Many models for temporal lobe epi lepsy have been advanced based on cell degeneration and fiber sprouting in the rat hippocampus that have been verified in humans as well. The hippocampal formation is also in a similar location, close to olfactory structures in all mammals. Due to the sim ilarities in dense cell layers, hippocampal fissures, and hippocampal lamination, the rat hippocampus still presents a good model for spread of infusate within the human hippocampus, especially if these feat ures are capable of significantly influencing dis tribution.
28 Drug Delivery AEDs prescribed today must enter the brain by crossing over from the blood. Theoretically, the transvacular route, composed of 100 billion capillaries separated by only 40 m 2 ( Pardridge, 2003b ) has the potential to distribute drugs throughout the brain. In reality, this method is limited by the proclivity for systemic toxicity and notably the blood brain barrier (BBB). The BBB is a specialized inte rface between circulating blood and the brain responsible for maintaining CNS homeostasis and limiting entry of substances that can alter neuronal function ( Brad bury, 1985 ; Goldstein and Betz, 1986 ) It consists of tight junctions between endothelial cells that are supported and reinforc e d with the glia limitans The tight junctions result in a very high re sistance between cells that limits para cellular transport (Figure 1 2A ) while e nzymes present inside the endothel ial cells degrade most solutes travelling trans cellularly (Figure 1 2B ) With the exception of small (<1000 Daltons ) hydrophobic molecules t hat can freely diffuse through the membrane, the BBB is extremely efficient at restricting passage of substances from the bloodstream T reatment responsiveness of pharmacoresistant partial (focal) epilepsy is dependent on clinically relevant drug concentra tion s at the focus. Hence, t his neuroprotective role of the BBB presents a major challenge for the delivery of medication especially non lipophilic therapeutic agents that have been shown to be effective in experimental systems The challenge to circumven t the BBB for drug delivery has been met with methods developed to disrupt the BBB, pass through the BBB, or deliver substances behind the BBB. These drug delivery strategies are described in the rest of this section
29 Disruption of the BBB Systemic adminis tration of drugs with concomitant BBB disruption has been a strategy pursued to increase parenchymal drug concentrations. These include infusion of solvents (dimethyl sulfoxide or ethanol) and metals, X irradiation, induction of pathological conditions, or administration of antineoplastic agents. These techniques are usually toxic and not clinically useful. Intracarotid injection of an inert hypertonic solution such as mannitol or arabinose has been employed to increase the permeability of the BBB temporari ly Osmotic dehydration of the endothelial cells enlarges pore size between tight junctions to allow drugs to enter the brain ( Rapoport, 2000 ) and it is short lasting and spontaneously revers ible ( Siegal et al., 2000 ) This method has been used to deliver chemotherapeutic drugs for treating brain tumors ( Doolittle et al., 2002 ; Haluska and Anthony, 2004 ) Other factors have also been transported into the CNS with the use of mannitol: manganese for ne uroimaging ( Fa et al., 2011 ) neurotrophic factors for experimental stroke treatment ( Yasuhara et al., 2010 ) and a rabies virus glycoprotein labeled nanocarrier ( Hwang et al., 2011 ) D espite favorable results obtained in some patients with brain tumors, this method is probably not the right treatment for epi lepsy One study found seizures occur r ed 7% of the time during hyperosmotic BBB opening in cancer patients who were previously sei z ure free ( Haluska and Anthony, 2004 ) Another study documented seizures began directly following BBB opening in 25% of the procedures delivering mannitol with chemotherapy ( Marchi et al., 2007 ) The increa se of seizure risk is most likely because t his procedure indiscriminately opens the BBB to any circulating toxins or endogenous serum components that can disturb the homeostasis of extracellular ions ( Friedman, 2011 )
30 Therefore, there is considerable risk of infection or passage of un wanted molecules/proteins in addition to the targeted drug. Passage T hrough the BBB Drug design Another strategy for drug delivery is to sneak drugs into the brain through the BBB. BBB penetration is favored by low molecular weight, lack of ionization at p hysiological pH, an d lipophilicity ( Pardridge, 1988 ) Small lipophilic molecules can diffuse passively across t he BBB (Figure 1 2B ). C reating hydrophobic analogues of small hydrophilic drugs is a strategy employed to transport compounds into the brain However, this strategy has been met with disappointment. The price of increasing lipophilicity for better permeabi lity is decreased plasma solubility, leading to increased binding to plasma proteins and lower concentrations of drug available ( Misra et al., 2003 ) A potential solution to this problem i s to create prodrug s, pharmacologically inactive compound s that are converted to the ir active form with a chemical modification once inside the BBB Esterification or amidation of hydroxyl amino or carboxylic acid containing drugs are added to enhance lipid solubility then hydrolysis of the modifying group will release s the active compound once in the CNS ( Huttunen et al., 2011 ) Prodrugs have been created for valpr oi c acid ( Trojnar et al ., 2004 ; Peura et al., 2011 ) phenytoin ( Fechner et al., 2008 ; Rautio et al., 2008 ) and gabapentin ( Cundy et al., 2004 ; Rautio et al., 2008 ) Although a cleve r solution, t his strategy, too, suffers from important limitations. Increased lipophilicity increases uptake into other tissues, which may exacerbate toxicity at non target sites. Additionally, increased lipophilicity enhances efflux processes, resulting i n poor tissue retention and short biological action. Finally, hydrolysis of the prodrug may lead to unwanted metabolites that contribute to
31 the toxicity of the compound. ( Bodor and Kaminski, 1987 ; Lambert, 2000 ) Recently, prodrugs have been developed to include multi step activation and other moieties to enhance target specificity and prevent unwanted metaboli c conversions ( Huttunen et al., 2011 ) With improvement, t he pro drug approach may prove to be a feasible way to transport drugs into the CNS. Mediated transport There are several transport systems involved in the movement of macromolecules across the BBB that may be exploited for drug delivery. For example, t he b rain requires essential small and large hydrophilic molecules for normal functioning and survival that cannot passively diffuse across the BBB. These substances are recognized by specific transporters on endothelial cells and transported into the brain (Fi gure 1 2 ). Carrier mediated transport pathways (Figure 1 2C ) consist of membrane transporter proteins expressed at the luminal and basolateral endothelial membrane that allow certain small, hydrophilic circulating nutrients or peptides to pass through the endothelial cell These pathways include: Hexose transport system for glucose and mannose Neu t ral amino acid transport system Acidic amino acid transport system for glutamate and aspartate Basic amino acid transport system for arginine and lysine b amino acid transport system for b alanine and taurine Monocarbox i lic acid transport system for lactate and short chain fatty acids Choline transport system for choline and thiamine Amine transport system for mepyramine Nucleoside transport system for purine base s such as adenine and guanine Peptide transport system for small peptides such as enk e phalins Drugs can be modified to increase their affinity for a specific carrier system to increase their BBB penetration through an endogenous approach T he drug Levodop a, an exogenous precursor of dopamine is transported this way. Levadopa has a high affinity
32 for the large neutral amino acids system and is decarboxyl a te d to dopamine once inside the BBB. Another mediated transport system, receptor mediated transcytosis (Figure 1 2 D), allows the transfer of other essenti al molecules, such as hormones and lipoproteins, into the brain A protein or antibody that is transported this way under normal conditions may be conj ugated to the drug of interest to facilitate its tran sport through the BBB. Various enzymes, growth factors and neurotrophic factors have been delivered to the brain by targeting the transferrin receptor ( Zhang and Pardridge, 2005 ) a transporter for an essential protein needed for iron delivery to cells, the huma n insulin receptor ( Coloma et al., 2000 ; Pardridge, 2003a ) and the low density lipoprotein receptor rela ted proteins ( Spencer and Verma, 2007 ; Demeule et al., 2008 ; Karkan et al., 2008 ) Alternatively, drugs can also be encapsulated by a delivery system recognized by specific receptor involved in membrane transport Polymer nanoparticles and liposomes have been the delivery system most studied, but dendrimer s, micelles, carbon nanotubes, emulsions, solid lipid nanoparticles, and nanostructured lipid carriers have are also being developed ( Hughes, 2005 ) These carriers can be targeted to a specific receptor or delivered to the b rain via adsorptive mediated transcytosis (Fig 1 2 E), the vesicular endocytosis of charged substances ( Agyare et al., 2008 ) It is important to note that the kin etics, structural binding requirements, and in vivo activity post modification must be considered when using BBB transporter proteins. Certain drugs do not retain their pharmaceutical function following transformations, and dissociation from receptors may be challenging if there is high binding affinity ( Gabathuler, 2010 ) F urthermore, an essential p oint to note is that peripheral organs
33 express these proteins as well; therefore, systemic toxicity remains a concern for all of these strategies. Passage B ehind the BBB As described so far in this section, there have been many advances in drug design that show potential in treating CNS diseases. However, the risk factors associated with the successes to date have prompted researchers to pursu e another class of strategies that do not rely on the car diovascular system. Drug manipulation is not necessary because the following methods are geared toward circumventing the BBB altogether. The result is higher concentrations within the CNS without the risk of systemic side effects. However, any type of intr acerebral drug delivery involves navigation through the extracellular space (ECS), which makes up 15 25% of the CNS tissue volume ( Sykova, 1997a ) The ECS can slow or facilitate the movement of various substances in the CNS, and is thus an important factor in drug dist ribution. The next section describe s the ECS in more detail and is followed by a description of several approaches for drug delivery directly into the CNS. Extracellular s pace The ECS is a system of interconnected channels that is occupied by interstitial fluid and extracellular matrix components. Extracellular matrix components include glycosaminoglycans (e.g. hyalurona te), glycoproteins, and proteoglycans, which provide structural support, regulate intercellular communication, and bind proteins. Various extracellular matrix adhesion molecules have also been described, such as fibronectin, tanescin, and laminin ( Thomas and Steindler, 1995 ) Interstitial fluid is essentially the same as CSF, but local i onic changes occur as a result of neuronal activity. Interstitial fluid also contains metabolites (glucose, O2, CO2, HCO3 ), free radical scavengers,
34 amino acids, catecholamines, neurotransmitters, DNA, RNA, peptides, lipids, hormones, growth factors, cyto kines, chemokines, and various enzymes. The membranes, macromolecules, and charged particles in the ECS, together with neuronal and glial cell processes, all affect the size and shape of ECS channels. Irregular geometry of these channels can slow or facili tate the movement of various substances in the CNS, including the transport of exogenous drugs. The macroscopic properties of the ECS are described by volume fraction and tortuosity. The volume fraction is the quotient of the volume of the ECS and the tota l tissue volume. The volume fraction in a normal isotropic region of the brain is estimated to be 0.2, i.e., the ECS makes up approximately 20% of brain tissue ( Van Harreveld, 1972 ; Fenstermacher and Kaye, 1988 ) Tortuosity is another ECS parameter that quantifies how much the diffusion of molecules is hindered in tissue compared to free medium. T ortuosity in homogenous and is otropic brain regions is about 1.6, but larger molecules (with relative molecular mass above 10kDa) generally exhibit larger tortuosity ( Nicholson and Sykova, 1998 ) Hetero geneity in tortuosity is often tested by measuring diffusion along three orthogonal axes. A difference in tortuosity along different axes indicates anisotropy. Within the hippocampus, both volume fraction and tortuosity have shown considerable regional va riation implying the ECS is not a fixed volume, but varies among the hippocampal subfields. These parameters exhibit lower values in CA1 as compared to CA3 and dentate gyrus, specifically in stratum pyramidale as compared to other strata ( McBain et al., 1990 ; Perez Pinzon et al., 1995 ) Within stratum radiatum, isotropic tortuosity is present along medi o lateral and antero posterior directions, but is
35 increased along the dorso ventral axis depending on the distance from stratum pyramidale ( Mazel et al., 1998 ; Hrabetova, 2005 ) Local differences in ECS parameters may be a result of varying distances between cellular membranes, diffusion barriers in the extracellular matrix, or differential sizes of cell bod ies or processes in each region ( McBain et al., 1990 ) For instance, the pyramidal cell layer is more tightly packed in CA1 than CA3; CA3 cells (300 700 m 2 ) are l arger than those in CA1 (~193 m2 ); and the massive associational network which is so apparent in CA3 is largely missing in CA1 ( Amaral and Lavenex, 2007 ) T he decrease in CA1 volume fraction may be due to overall homogenously smaller intercellular channels or it may reflect h eterogenous channels that are on average smaller. The latter is su pported by measurement s taken of gaps between cells, which range from 38 64 nm in width, with uneven distentions in some regions as compared to others ( Brightma n, 1965 ; Van Harreveld et al., 1965 ; Bondareff and Pysh, 1968 ; Cragg, 1979 ) However, histological fixation in older morphological studies may have resulted in artificial narrowing in some regions ( Hrabetova and Nich olson, 2007 ) While the shape and size of cells and their processes create obstacles that diffusing molecules have to circumnavigate, it is not clear how these and other elements in the brain increase or decrease ECS parameters. C hanges in volume fractio n and extracellular tortuosity are often independent. Osmotic or ischemic insults result in reduction of the volume fract ion and a rise in tortuosity, presumably due to cellular swelling ( Sykova et al., 1994 ; Perez Pinzon et al., 1995 ; Hrabetova and Nicholson, 2000 ) I nflammatory and demyelinating diseases result in an increase in volume fraction and a decrease in tortuosity, perhaps due to an increase in blood brain barrier
36 permeability ( Simonova et al., 1996 ) However, during astrogliosis, a persistant increase in tortuosity without a decrease in volume fraction has been found ( Sykova et al., 1999a ) The converse has also been observed. S eizure act ivity which induces large increases in extracellular potassium ( Dietzel et al., 1980 ; Dietzel et al., 1989 ) results in subsequent water movement i nto surrounding cells and thus a reduction in extracellular volume fraction, but not necessarily a change in tortuosity ( Mc Bain et al., 1990 ) There remain many unknowns regarding what elements change ECS parameter and how these parameters a ffect spread and clearance mechanisms of the brain. Moreover, although pathological insults have been associated with changes in ECS and extracellular matrix, the ability of ECS to bind, slow, or facilitate the migration of substances rema ins an open question. However, t he heterogeneity of ECS architecture does affect i ntercellular communication, nutrient and metabolite trafficking ( McBain et al., 1990 ; Sykova, 1997b ; Nicholson and Sykova, 1998 ; Sykova and Nicholson, 2008 ) and p otentially as is described in the rest of Chapter 1, drug delivery in the brain. Intra cerebro ventricular/intrathecal delivery In intracerebroventricular (ICV) administration, drug is introduced directly into the cerebrospinal fluid ( CSF ) Theoretically, w hen drugs are infused directly into the CSF, not only is systemic toxicity reduced, but the drugs have longer half lives due to decreased protein binding and enzymatic activi ty relative to drugs in plasma. High local concentrations of drug have been admini stered into the CSF with the Ommaya reservoir, a plastic reservoir implanted subcutaneously in the scalp and connected to the ventricles within the brain via an outlet catheter ( Ommaya, 1963 ) Pumps have similarly been used to elevate drug concentrations in the CSF ( Bakhshi and North, 1995 ) In 2006, continuous ICV infusio ns of valproic acid were compared to bolus ICV
37 injections and intraperitoneal (IP) injections in a rat kindling model of epilepsy ( Serralta et al., 2006 ) The ICV bolus injection resulted in the highest concentrations of VPA in the brain, but all three methods were able to control generalized and focal seizures. The ICV infusion was the only one to achieve anticonvulsant effects with minimal toxicity. In 2007, Oommen et al. researched the effectiveness on a osmotic pump delivering gabapentin on flurothyl induced seizures in rats and found delivery delayed onset of generalized tonic clonic seizures ( Oommen et al., 2007 ) Despite these favorable results, ICV delivery is still plagued by diffusion. S olutes within CSF must diffuse into brain parenchyma, a slow process in which concentration decreases logarithmically with e ach millimeter of brain tissue ( Blasberg et al., 1975 ) The ECS of the brain is extremely tortuous, so drug diffusing from ventricles into this space is very s low and inversely proportional to the molecular weight of the drug ( Pardridge, 1997 ) Although s ome molecules have been effectively distributed through intracerebroventricular (ICV) injection ( Barcia and Gallego, 2009 ) in general, drug distribution from CSF into parenchyma is log orders slower than CSF clearance (and clearance of drugs infused into CSF) from the brain ( Pardridge, 2005 2007 ) ICV injection has been useful in cases where high drug concentrations in the CSF or immediately adjacen t parenchyma are de sired ( Harbaugh et al., 1988 ) Int racerebral delivery The most dire ct way of administering drug to a specific area in the brain is to deliver it directly to the brain in terstitium. Like ICV injections, th is method can yield high concentrations and minimal systemic exposure without the limitation the BBB poses to size or c hemical properties of therapeutic agents Additionally, direct targeting of the
38 seizure focus is more desirable due to the drop of drug concentration from the distance of the implant or injection site ( Saltzman and Olbricht, 2002 ) Implantable p olymers. Implantation of biocompatible sustained release polymers is a strategy employed to bypass the BBB. A drug can be loaded into these polymers and diffuse into the surrounding tissue at a controllable rate. For non biodegradable polymers such as ethylene viny l acetate copolymer (EVAc), the rate of drug diffusion into surrounding tissue is dependent on the loaded agent. For biodegradable polymers, such as the polylactic co glycolicacid (PLGA) family, the release of drug is dependent on the diffusivity of the ma cromolecule and the degradation rate of the polymer ( Luo et al., 1999 ) This method has been shown to release drugs successfully, but it still relies on diffusive transport, which yields a concentration profile drop off and is dependent on molecular size. Convection enhanced delivery. Convect ion enhanced delivery (CED) is an approach developed to overcome the obstacles in diffusion dependent central nervous system (CNS) delivery methods. CED was first described by Bobo et al. (1994 ) a s a local drug delivery technique that uses a small hydrostatic positive pressur e gradient to deliver infusate directly into parenchymal interstitial space Because CED relies on bulk flow, it can overcome slow diffusivity and result in a more widespread distribution volume as compared with diffusion based approaches When transport i s dominated by diffusion, a large concentration gradient develops from the site of infusion to the margins of the distribution volume as drug molecules move passively from regions of high to low concentration In contrast, CED provides a uniform concentrat ion throughout the region with a sharp drop off in concentration at the borders ( Rogawski, 2009 )
39 Dispersion of agents is powered by gradients of pressure in addition to gradients of concentration. As a result, convection driven flow overcomes slow diffusivity and can result in a larger volume of distribution as compared with other diffusion based drug delivery methods (for review, see ( Raghavan et al., 2006 ) ) Furthermore, CED is generally independent of molecular size ( Bobo et al., 1994 ; Sampson et al., 2011 ) as long as macromolecules are within the ECS width of CNS tissues (38 64 nm, ( Thorne and Nicholson, 2006 ) ) Previous studies have demonstrated intracranial CED into either white or gray matter can be used to distribute small or large molecular weight molecules in a ho mogenous, ta rgeted, and safe manner ( Bobo et al., 1994 ; Chen et al., 1999 ) Underlying the success of CED as a therapeutic drug delivery system is a good understanding of the principles governing delivery and flow of macromolecules within the CNS. There are several known factors affecting the final distribution of infusate in the brain. Firstly, infusion pump param eters, such as flow rate and duration of infusion, will affect the inflow of the solution. Because infusate is delivered into the ECS transport is dominated by convection and controlled through the flow rate, duration, and pressure of the infusion ( Raghavan et al., 2006 ) Secondly, cer tain infusate properties have a significant impact on distribution. These properties include viscosity ( Jagannathan et al., 2008 ) surface propertie s, ( Chen et al., 2005 ) particle charge ( Saito et al., 2006 ) and particle coating ( MacKay et al., 200 5 ) B ackflow of the infusate along the infusion cannula can also affect distribution. Backflow may be a result of tissue disruption along the catheter track, allowing the infusate to flow into low resistance spaces ( Guarnieri et al., 2005 ) or the pressure from the infusion may push the tissues away from the catheter, defined as intrinsic backflow ( Morrison et al., 1999 )
40 B ackflow is an important issue to take into account because it can result in the spread of infusate into unintende d regions and diminish the dose needed within the target tissues (Figure 1 3) This problem is addressed by using thinner catheters ( Lonser et al., 2002 ) step design catheters ( Krauze et al., 2005 ) and gui delines for catheter placement in humans ( Raghavan et al., 2006 ) and animals ( Kim et al., 2009 ; Astary et al., 2010 ) These guidelines will be further addressed in Chapter 2. CED for epilepsy. CED has already been proposed as a novel therapeutic therapy to treat epilepsy ( Rogawski, 2009 ) and continues to show promise. AEDs currently prescribed and developed are limited to orally active or BBB permeable agents. CED is applicable to a wide variety of potential anti epileptic drugs since it does not re quire passage through the BBB. CED can be used deliver anticonvulsant or neuroprotective peptides and other high molecular weight molecules that have already shown promis e as epilepsy treatments experimentally ( McLaughlin et al., 2000 ; Haberman et al., 2003 ; Richichi et al., 2004 ) An example of CED delivery of novel therapeutic agents can be seen in Figure 1 4. While CED for epilepsy remains experimental, several studies have already shown the feasibility of underta king it for epilepsy treatment ( Stein et al., 2000 ; Heiss et al., 2005 ; Gasior et al., 2007 ) CED has also been used clinically in malignant glioma ( Voges et al., 2003 ) growth factor ( Gill et al., 2003 ) and gene therapy vector ( Marks et al., 2008 ) delivery studies. W ith the high dr ug concentrations that can be achieved at specific sites, CED has a wide range of applications in the field and treatment of epilepsy. However, the efficacy of the procedure at this stage remains poorly determined partly due to the heterogeneity of drug di stribution and the effect of edema
41 on interstitial transport. More detailed knowledge about the anatomical and biophysical features of the brain is necessary for optimization of delivery into complex or injured brain regions This topic is the focus of Cha pter 2 and Chapter 3
42 Figure 1 1. The epilepsies are classified based on etiology, semiology, origin of seizures, and epilepsy syndromes. Two systems of classification of epilepsies are in use today. A) The International League Against Epilepsy (ILAE) d eveloped the first system based on seizure semiology in 1981. B) ILAE developed the second system based on epilepsy syndromes in 1989.
43 Figure 1 2 Schematic of the various routes of molecular transport across the BBB. These pathways can be exploited to transport biochemically altered drugs from the systemic circulation into the brain. A) Small water soluble agents travel through tight junctions connecting endothelial cells. B) Small, lipophilic molecules diffuse through endothelial cell membranes. C) C ar rier mediated transport systems are available for specific molecules such as amino acids, peptides, nucelosides, glucose, etc. D) Receptors are expressed for the transport of transferrin, insulin, lipoproteins, and other molecules that are actively transpo rted across the BBB. E) Adsorptive transcytosis involves nonspecific binding of charged ligands, such as albumin, to membrane surface charges followed by endocytosis.
44 Figure 1 3. Final CED distribution with and without backflow of an MR contrast agent ta gged with a fluorescent tracer in the rodent septal hippocampus. A,C) During successful CED, the tracer (A, red fluorescence) and contrast agent (C) travel along the contours of the hippocampal laminar structure. B,D) When backflow occurs, much of the infu sate travels away from the targeted site into white matter tracks, or other low resistance pathways EB = Evans blue dye; DAPI = 4',6 diamidino 2 phenylindole nuclear counterstain ; DG = dentate granule cells; CA3 = Cornu Ammonis hippocampal subfield 3; CA1 = Cornu Ammonis hippocampal subfield 1; CC = corpus callosum. Figure 1 4 Lentivirus and neural stem cells targeted to the septal hippocampus via CED. A) Lentiviral transduction of CAMKII ChR2/YFP in CA1 pyramidal cells and dentate granule cells. B) E ngraftment of neural stem cells expressing GFP in the CA1 subfield of the septal hippocampus. Counterstained with DAPI, 4',6 diamidino 2 phenylindole nuclear counterstain
45 CHAPTER 2 CONVECTION ENHANCED DELIVERY IN THE NORMAL RAT HIPPO CAMPUS Introduction Cu rrent CED research focuses on evaluating the efficacy of drug carriers, optimizing infusion parameters and infusion hardware (e.g. flow rate and duration, cannula design), and understanding the influence of the underlying tissue structure on the final dist ribution of the infused agent in the CNS ( Raghavan et al., 2006 ; Sampson et al., 2007b ; Jagannathan et al., 2008 ; Song and Lonser, 2008 ) With sufficient understanding of the controlling influences, CED might be used to target local delivery of the rapeutics into complex regions of the brain with heterogeneous and intricate neuroanatomy. One such structure is the hippocampus, which is comprised of densely packed layers o f neurons (gray matter ), and their axonal projections (white matter) in a tightly rolled, banana shaped structure. In addition, the hippocampus includes perivascular spaces and pial surfaces that line the ventricular compartments continuous with hippocampal fissures. The hippocampus is vulnerable to damage as a result of trauma ( Tate and Bigler, 2000 ) and is the central component of rare conditions such as limbic encephalitis ( Corsellis et al., 1968 ) and dementia with isolated hippocampal sclerosis ( Dickson et al., 1994 ) Hippocampal involvement is critical to the manifestation of TLE ( Bertram, 2009 ) and has been recognized in co morbidities of epilepsy such as schizophrenia ( Maier et al., 1995 ; Nelson et al., 1998 ) and Parts of this chapter are reprint ed with permission from Elsevier from Astary GW, Kantorovich S, Carney PR, Mareci TH, Sarntinoranont M (2010) Regional convection enhanced delivery of gadolinium labeled albumin in the rat hippocampus in vivo. J Neurosci Methods 187: 129 137 This work was made possible by the collaboration with Dr. Garrett Astary. Dr. Astary helped with the infusions and distribution analysis, and performed the MR imaging and image segmentation described in this chapter.
46 ( deToledo Morrell et al., 2007 ; Ohm, 2007 ; Duyckaerts et al., 2009 ) If the CED distribution of a therapeutic agent within the hippocampus can be predicted, this may enabl e the application of CED to the treatment of TLE and other hippocampal disorders. However, accurate prediction of distribution profiles in the hippocampus requires an understanding of how the underlying tissue architecture influences transport of a deliver ed agent. With the use of contrast agents, magnetic resonance (MR) imaging provides a means of non invasively monitoring distribution profiles of agents delivered by CED and can provide insight into the influence of anatomy on tracer distributions. Typical ly, gadolinium based contrast agents are used, which contain a paramagnetic center that interacts with surrounding water to reduce the longitudinal and transverse relaxation times ( Lauffer et al., 1987 ) In a relaxation weighted image, the reduced longitudinal re laxation times (T1) result in a higher signal in regions of tissue exposed to the contrast agent, provided transverse relaxation times (T2) are not substantially reduced. MR can also be used to monitor CED by observing an increase in water signal seen in T 2 weighted images ( Heiss et al., 2005 ) Gadolinium based contrast agents have been co infused with therapeutic agents to track their distribution in real time. C o infusion of glucocerebrosidase and diethylene triamene penta acetic acid chelated gadolinium (Gd DTPA) into the region of the right facial and abducens nuclei was used to treat a patient ibution of the agent as well as observe the agent cross a pial surface to enter the third ventricle ( Song and Lonser, 2008 ) Other studies have also been performe d to investigate the effect of pial surfaces on final distribution volumes of small molecular weight (Gd DTPA) and large
47 molecular weight (Gd DTPA bound albumin (Gd albumin)) tracers infused into the primate brainstem ( Jagannathan et al., 2008 ) As a free ion, gadolinium is highly toxic but is regarded as safe when administered as a chelated compound. Gd DTPA has been used in animal and human CNS studies, wi thout showing signs of toxicity ( Song and Lonser, 2008 ; Ding et al., 2009 ) ; however, it has been shown to have an adverse affect on patients with pre existing kidney disease ( Abujudeh et al., 2009 ) Liposomal drug carriers containing Gd have also been synthesized to prov ide a more direct method of monitoring the distribution of these therapeutic agent carriers ( Krauze et al., 2008 ; Fiandaca et al., 2009 ) MR has been used to monitor the distribution of these delivery vehicles as well as evaluate the performance of backflow resistant cannulae ( Fiandaca et al., 2009 ) MR has also been used to evaluate the effect of infusate viscosity on final distribution volumes in rat brain striatal tumors ( Mardor et al., 2009 ) However, no previous studies have implemented high resolution MR to investigate CED for delivering an agent into a structure as complex as the hippocampus. In addition to MR, histology has also been used to evaluate tracer distri bution of agents infused into the CNS. Light microscopy was used to detect the presence of Evans blue dye infused into the striatum of a mouse brain via an implantable microfluidic device designed for chronic CED ( Foley et al., 2009 ) Fluorescence microscopy has been used to observe the distribution of polyethylene glycol coated liposomal doxorubicin infused into the rat brain parenchyma with an intracranial tumor ( Kikuchi et al., 2008 ) Not only can histology provide higher resolution visualization of distribution at the cellular level, but histological staining protoco ls can verify particular structural details that may influence distribution.
48 This chapter describes the effect of tissue structure on infusate distributions after CED and limited diffusion in the hippocampus of normal animals Final distribution patterns o f the MR contrast agent, Gd albumin, labeled with Evans blue dye, infused into the left side septal and right side temporal hippocampus of a rat were evaluated with two currently available imaging modalities: (1) in vivo imaging of contrast agent distribut ion using high resolution MR imaging and (2) fluorescence microscopy of the distribution of Evans blue in histological slices. Gd albumin is an ideal model for macromolecular flow through the interstitial space due to low reactivity, convection dominated t ransport, and ease of labeling with contrast agent. MR provided a means of non invasively monitoring distribution profiles of contrast agents delivered by CED in vivo, while optical microscopy yielded higher resolution of finer structural detail. Black gol d staining was used to label myelinated white matter structures, and Cresyl violet staining was used to visualize cell bodies. The results of this study demonstrate the influence of infusion site and normal hippocampal structure on CED delivery Methods An imal Preparation and Surgical Procedures Experiments were performed on 2.5 month old male Sprague Dawley rats (n = 7) using protocols and procedures approved by the University of Florida Institutional Animal Care and Use Committee. Anesthesia was initiated with xylazine (10 mg/kg, SQ) and isoflurane (4%) in 1 L/min oxygen, then animals were placed in a stereotaxic Kopf apparatus, and inhalation anesthesia (1.5% in 1.5 L/min oxygen) was delivered via a nose mask. The skull was exposed by a mid sagittal incis ion that began between the eyes and extended caudally to the level of the ears to expose bregma and lambdoidal sutures. One hole was drilled into the skull above the left side septal hippocampus and
49 a second hole was drilled above the right side temporal h ippocampus. Then 5.0 L of Gd DTPA albumin (10 mg/m L in PBS solution; MW ~ 87 kDa,; ~35 Gd DTPA molecules per albumin molecule; R. Brasch Laboratory, University of California, San Francisco, CA), tagged with Evans Blue dye was infused into the septal denta te gyrus of the hippocampus [ 3.7mm AP, 2.2 mm ML 3.4 mm DV] and another 5 L into the temporal CA1 subregion of the hippocampus [ 5.0 mm AP 4.9 mm ML 5.0 mm DV] at a rate of 0.3 L /min. Over concerns that the Gd albumin may be aggregating, high performa nce liquid chromatography (HPLC) was used to evaluate the macromolecular constituents of the infusate solution. HPLC resulted in a single elutant peak suggesting the Gd albumin was not aggregating and the covalent bonds attaching the Gd DTPA molecules to a lbumin were intact. L gas tight syringe (Hamilton, Reno, NV) driven by a syringe pump (Cole Parmer, Vernon Hills, IL) connected to polyaryletheretherketone (PEEK) tubing (ID = 0.381 mm, OD = 0.794 mm, length ~ 0.5 m Upchurch Scientific, Oak Harbor, WA). The PEEK tubing was coupled to a silica cannula (ID = 50 m, OD = 147 m, Polymicro Technologies, Phoenix, AZ) via a microfluidic connector. Immediately following the infusion surgery (~30 min), animals were transpor ted to the 11.1 Tesla (T) magnet for MR imaging. At the end of the experiment, animals under inhalation anesthesia (1.5% in 1.5 L/min oxygen) were given xylazine (10 mg/kg, SQ) and ketamine (80 mg/kg, IP). Upon ensuring deep anesthesia, the chest activity was opened to expose the heart, and a needle connected to an infusion pump was inserted into the left ventricle. 200 300 m L of 0.9% saline solution was circulated by the heart, followed by 200 300 m L of 4%
50 formaldehyde solution. The brain was then extract ed from the skull following decapitation and stored in 4% formaldehyde solution overnight. MR Imaging and Image Segmentation MR experiments were per formed using a Bruker Avance imaging console (Bruker NMR Instruments, Billeria, MA) connected to a Magnex Sc ientific 11.1 T horizontal bore magnet system (Varian, Inc., Magnex Scientific Products, Walnut Creek California). A custom made 130 degree arc, 3.5 cm rectangular linear field surface coil constructed on a 4 cm diameter half cylinder was used for linear t ransmission and detection of MR signal. Two sets of high resolution T1 weighted images, with slices oriented in the coronal and sagittal directions, were acquired using a spin echo sequence with a 2 cm 2 cm field of view in a matrix of 160 160, recover y time of 1000 ms, echo time of 10 ms and 20 slices Coronally oriented and sagittally oriented data were acquired with 8 averages and 6 averages respectively. Final distribution volumes of Gd albumin were calculated by performing semi automatic image segmentation on the high resolution T1 weighted coronal images using the ITK SNAP open source medical image segmentation tool ( ( Yushkevic h et al., 2006 ) ; http://www.itksnap.org/). Septal and temporal hippocampus infusion volumes were segmented separately with the following specific threshold criteria. Voxels were included in the infusion volume if their signal intensity was at least 6 sta ndard deviations higher than the signal intensity in the corresponding region contralateral to the site of infusion. Final distribution volumes in the septal and temporal hippocampus were calculated by counting the number of voxels included in each segment ed region and multiplying by the volume of a single voxel.
51 Histology Black Gold was used to stain myelin in mounted sections. Black Gold II powder (Histo Chem Inc., Jefferson, AR) was resuspended in saline solution (0.9% NaCl) to a final concentration of 0 .3%. The solution was heated to 60C, and rehydrated tissue sections were incubated for 12 18 minutes, until desired intensity was achieved. The sections were then rinsed in double distilled water for 2 minutes, followed by sodium thiosulfate solution (1%) for 3 minutes. Finally, sections were rinsed three times with double distilled water for 5 minutes per rinse. Cresyl violet staining was performed to stain cell bodies in mounted sections. Slides were incubated in Cresyl violet solution for 2 3 minutes u ntil desired intensity was achieved. Slides were then dehydrated using a series of gradated alcohols (75%, 95%, 100%) for 5 minutes each. The dehydrated sections were then cleared in xylene for 2 minutes and cover slipped with mounting media. Microscopy Fo llowing mounting and staining, slides were examined on an Olympus BH 2 brightfield and epifluorescence microscope (Olympus America Inc., Center Valley, PA) with a Hitachi KP D581 color digital video camera (Hitachi Medical Systems America, Inc., Twinsburg, OH) interfaced with an Integral Technologies frame grabber (Pelco, Clovis, Ca) in a desktop computer. Motorized stage and focus (Prior Scientific, Rockland, MA), and image acquisition were controlled through ImagePro Plus (Media Cybernetics, Silver Spring s, MD). Anatomical structures were mapped to coronal sections of the Paxinos and Watson rat brain atlas ( Paxinos, 1998 )
52 Results Infusion S ite Infusions (n=14) were targeted into the dentate gyrus of the left side septal hippocampus (n=7) and into the right side CA1 of the temporal hippocampus (n=7). Actual infusion sites were confir med with MR and histology. Damage due to insertion of the cannula was minimal with some bleeding at the site of the cannula tip (Figure 2 1 A) and at the interface between the corpus callosum and a lveus of the hippocampus (Figure 2 1 B), as visualized with h istology. Contrast agent infused into the septal hippocampus was observed to have only limited penetration into the ipsil ateral temporal hippocampus (Figure 2 2A C). Similarly, contrast agent infused into the temporal hippocampus showed severely limited pe netration into the ipsilateral septal hippocampus (Fig ure 2 2B, D) with small amounts observed in the fimbria. Infusion sites were clearly identifiable in all subjects in the septal hippocampus and 6 of the 7 subjects in the temporal hippocampus. In the sep tal hippocampus infusions, 6 of 7 infusion sites were located in stratum radiatum of the CA1 subfield of the hippocampus One infusion site was located in the polymorphic layer of the dentate gyrus. In 4 of 7 temporal hippocampus infusion subjects, the inf usion site was located in stratum radiatum of the CA1 subfield of the hippocampus. In 2 of the subjects, the infusion site was determined to be in stratum oriens of the CA1 subfield of the hippocampus. Gd albumin Distribution in the Septal H ippocampus The profile of the contrast agent distribution into the septal hippocampus was easily distinguishable from surrounding tissue. Exposed regions displayed a hyperintense signal with respect to surrounding regions in T1 weighted images (Fig ure
53 2 3). MR images sho wed that the contrast agent entered the CA1, CA3 and dentate gyrus subfields of the hippocampus in all anima ls (Figure 2 3) and suggest that contrast agent penetrated poorly into the dense dentate gyrus granule cell lay er and CA1 pyramidal cell layer. T hes e regions remain hypointense with respect to the surrounding subfields and are clearly distinguishable in co ronal images (arrowheads in Figure 2 3). The contrast agent was seen to cross the midline of the brain in 3 of the 7 subjects. In two of these subj ects, the contrast agent crossed the midline of the brain by entering the corpus callosum and traveling medially to the side of the brain contralateral to the infusion site. In one subject, the contrast agent also entered the septal hippocampal commissure and was visible in a small portion of the CA1 subfield of the contralateral hippocampus. Contrast agent penetration into the fimbria subfield of the hippocampus was not seen in any of the subjects suggesting the densely packed cell layer CA1 and CA3 subfie lds served as a barrier to transport into this region (Figure 2 3). Gd albumin D istribution in the Temporal H ippocampus The contrast agent penetrated the CA1 and CA2 subfields of the temporal hippocampus in all subjects. In 5 of 7 subjects, contrast agen t was seen in the dentate gyrus, CA1, CA2, and CA3 subfields of the hippocampus (Fig ure 2 4B,C,E F). However in two of the subjects, penetration of the contrast agent into CA1 and CA2 was limited and primarily located in the alveus of the hippocampus (Figu re 2 4G H), most likely due to the lateral location of the infusion site (see above nfusion S ite ). The contrast agent did not appear to enter the granule cell layer and hippocampal fissure, since these regions were hypointense relative t o the neighboring dentate gyrus. Contrast agent was also observed at the interface between the corpus callosum and the cortex in these subjects.
54 Backflow Severe backflow, resulting in a significant amount of contrast agent entering the cortex, was seen in 3 of the 7 septal infusions (Figure 2 3C,E,F). Mild backflow resulted in minor exposure of the cortex to contrast agent in 2 of the 7 subjects (Fig ure 2 3D H). In 3 of the 7 subjects, backflow allowed the contrast agent to enter the corpus callosum and tra vel in both the medial and lateral directions along this white matter fibrous structure (Fig ure 2 3B,C F). Severe backflow in temporal infusions resulted in significant amounts of the contrast agent entering the cortex in 3 of the 7 subjects (Fig ure 2 4B, C E). Minor backflow was observ ed in 3 of the 7 subjects (Figure 2 4D,F G) and no back flow was seen in 1 subject (Figure 2 4H). In cases of minor backflow, contrast agent did not enter the cortex and remained in the hippocampus, usually penetrating the alv eus of the hippocampus. Image Segmentation The three dimensional contrast agent distributions were visualized with a semi automated segmentation of the contrast agent enhanced regions. Because MR imaging was conducted approximately 30 minutes after CED, t he observed distribution profiles include the effects of CED as well as post CED diffusion. Distribution volumes, including the effects of CED and diffusion, were calculated from the segmentations for each data set and included back flow volumes. For the s eptal hippocampus infusion, the mean L For the temporal hippocampus infusion, the mean and standard deviation of the calculated L The tempora l hippocampus distribution volume was greater than the septal
55 = 0.99). The contribution of diffusion after the end of CED to the measured distribution volumes was estimated using an analytical solu tion of one dimensional diffusion from a sphere. The radius of the sphere was determined such that the volume of the sphere would be equal to distribution volumes of the contrast agent in the septal and temporal hippocampus. The diffusion coefficient of al bumin in rat cortical slices, D = 1.63 10 7 cm 2 /s ( Tao and Nicholson, 1996 ) was used in this estimation. Based on these results we estimate that diffusion after the end of CED may increase distribution volumes up to 40%. This diffusional spread is equivalent to the contrast agent traveling 2 3 voxels (0.250 0.375 mm) during the time delay between CED and MR imaging (for co mparison, the average anterior posterior spread of the tracer was measured to be 5.4 mm for septal infusions and 4.75 mm for temporal infusions) Histological A nalysis Evans blue fluorescence confirmed the distributions seen in MR imaging. Dense cell layer s that appeared hypointense in MR images likewise did not fluoresce in histological images (arrowheads, Fig ure 2 5 and 2 6), indicating little or no penetration of the infusate. However, infusate was seen to distribute around the dense cell layers then pen etrate the dentate gyrus and CA1 CA3 subregions in all septal infusions and 5 of 7 temporal infusions, which is consistent with MR results. Preferential distribution was dependent upon location of the cannula tip. When the cannula tip was located in the in terface between the CA1 and hippocampal fissure (asterisks, Fig ures 2 5 and 2 6 ), fluorescence was greatest in the molecular layer of the dentate gyrus and CA1 immediately adjacent to the hippocampal fissure. In one subject (Fig ure 2 3D), the cannula tip w as in the polymorphic layer of the dentate gyrus, which resulted in a larger volume of contrast agent accumulated internal to the dentate gyrus granule cell layer.
56 In 2 of 7 temporal infusion subjects, Evans blue was observed to be predominately distribute d within the alveus of the hippocampus (closed arrows, Fig ure 2 6 C) and corpus callosum (open arrows, Fig ure 2 6 C), as was seen i n MR (Figure 2 4G H). These distributions were observed in sections displaying cannula tracts and those with no visible tissue damage. Due to a more lateral infusion site in these two subjects, infusate traveled along the axis of the white matter fiber tract and was limited mediolaterally by the pyramidal cell layer and the cortex adjacent to the fiber tracts (Fig ure 2 6 C). One t emporal infusion showed Evans blue in the perivascular space (Fig ure 2 6 A). Discussion This study compared the distribution profiles of Gd albumin in the septal and temporal hippocampus after CED and limited diffusion in normal animals Distribution of the contrast agent was visualized with high resolution MRI; shape and volume analysis was performed with segmentation; and validation was completed with histology, which also provided finer resolution to further elucidate the role of tissue structures on fina l distribution patterns. Images from histology and fluorescence microscopy were compared to MR images acquired in vivo to confirm the distribution of the infusate in hippocampal subregions. R esults demonstrate that the distribution profile and shape of the infusions are dependent upon infusion site and underlying neuroanatomical and cytoarchitectonic structure. Distribution Profile and S hape The infusion site was a critical factor influencing distribution of the con trast agent. The temporal infusions distri buted throughout the posterior dorsoventral hippocampus, while the septal infusion distributed throughout the anterior end to the septal pole of the
57 hippocampus. In addition, infusion into the septal target site resulted in a smaller distribution area comp ared to the temporal site and an apparent disconnect was noted between septal and temporal hippocampal infusion sites. Infusion site variability within the septal and temporal hippocampus also influenced the distribution profile of the infused agent. For t he septal hippocampus infusions, variability (~1 mm) in the cannula placement within the medial lateral/anterior posterior plane had negligible effects on tracer distribution, as observed in both MR and fluorescence microscopy (Fig ure 2 3). Although occurr ing in only one animal, variability in the depth of the cannula tip seems to have the most impact on final tracer distribution (Figure 2 3 D ), which was most apparent in fluorescence imaging (data not shown). In this subject, the most intense fluorescence s ignal was seen interior to the granule cell layer of the dentate gyrus. In contrast, the most intense fluorescence signal was seen around the hippocampal fissure and CA1 subregion of the hippocampus for all other subjects. In the temporal hippocampus infus ions, variability (~ 1 mm) in the depth of cannula penetration had little impact on final distributions; however, variability in the location of the cannula tip in the medial lateral direction had a significant impact. This is seen in two subjects where in fusions lateral to the targeted infusion site resulted in tracer distributing entirely within the alveus of the hippocampus and the corpus callosum (Fig ure 2 4G H). Since the infusion sites were stereotaxically targeted using an atlas developed from a fixe d rat brain, deviations between the fixed rat brain and in vivo rat brain, anatomical variability between rats, and experimental error may contribute to variability in the infusion site.
58 The dorsoventral disconnect may have several explanations. Since infu sions were only conducted at one volume, it is possible that the volume used was not sufficiently large enough to distribute throughout the entire hippocampus. Alternatively, anatomical a larger volume of the infusate. The fissure is a cell free region continuous with ventricular space that is lined by pia mater and filled with CSF and blood vessels ( Humphrey, 1 967 ) It could act as a mass sink for the contrast agent, especially since Gd albumin is able to cross pial boundaries ( Jagannathan et al., 2008 ) Indeed hyperintense regions were observed in the MR imaging within and surrounding the hippocampal fissure (Fig ures 2 3 and 2 4), and this finding was confirmed with fluorescence imaging (Fig ures 2 5 and 2 6). Although this finding may be explained by targeting a preferential distribution into the fissure is also seen in images where the cannula tip is not positioned in the fissure (Figure 2 6 ), indicating the contrast agent may be following the path of least resistance and collecting within the hippocampal fis sure. A third explanation for the dorsoventral disconnect may arise from the effect of differential axonal projections to, from, and between septal and temporal hippocampi. For example, different densities of projections have been found to the septal and t emporal hippocampus from the entorhinal cortex ( Krettek and Price, 1977 ; Dolorfo and Amaral, 1998 ) amygd ala ( Krettek and Pric e, 1977 ) ventral tegmental area, and locus coerul e us ( Haring and Davis, 1985 ; Verney et al., 1985 ) Hil ar ( Fricke and Cowan, 1978 ) and CA3 projections ( Ishizuka et al., 1990 ; Li et al., 1994 ) are also coded toward specific areas of the hippocampus. These axonal structural differences likely underlie
59 functional differences between the septal and temporal hippocampus ( Moser et al., 1993 ; Jung et al., 1994 ; Esclassan et al., 2009 ) and may affect CED distribution. This study demonstrat es that neuroanatomical structure influence s CED distribution of contrast agent at the molecular level. Although contrast agent entered all subfields of the hippocampus in each subject, limited penetration was observed in the granule cell layer in the dentate and pyramidal cell layer in the CA3 and CA1. These cell layers consist of densely packed excitatory cells that appeared as hypointense regions in the MR images (arrowheads, Fig ure s 2 3 and 2 4), and displayed weak or no response to fluoresce nce imaging (arrowheads, Fig ure s 2 5 and 2 6 ). Hydraulic conductivity describes the ease with which a fluid can move through a porous medium. In the case of densely packed cell layers, the hydraulic conductivity would be low and permeation of the infused a gent into these regions would be limited. Furthermore, the pyramidal cell layer in the CA3 of the septal hippocampus appeared to prevent infusate from entering the fimbria (Figure 2 3), while the CA1 and CA2 pyramidal cells layers served as a boundary in t he temporal hippocampus (Figure 2 4). It is likely that the contrast agent traveled around these structural boundaries, along the trisynaptic circuit ( Andersen et al., 1969 ) of the hippocampus. The trisynaptic circuit is comprised of axonal fibers connecting several subregions of the hippocampus. Preferential directions of water diffusion have been found to correspond to the average aligned fiber directions within a voxel ( Basser and Jones, 2002 ) Hence, t he hydraulic conductivity along th e direction of these fibers would be lower than that perpendicular to the fiber direction leading to a preferential distribution along the trisynaptic circuit. However, further
60 studies with in vivo dynamic contrast enhanced MRI (DCE MRI) are necessary to c onfirm this hypothesis. It should be noted that other factors may influence the distribution patterns of agents delivered by CED into the brain parenchyma. For example, choice in cannula design and flow rate can impact the severity of backflow while the to tal infusion volume will ultimately influence the distribution volume and exposure of structures to the agent. In this study, flow rate and infu respectively, for all subjects. Thus, we cannot comment on how th ese factors would influence distributions in the hippocampus based on our results. However, it is surmised that increasing the flow rate would contribute to backflow. Backflow would also be dependent on cannula design with generally smaller diameter cannul a resulting in less backflow (Morrison et al., 1999). A step design cannula has also been proposed that L /min ( Krauze et al., 2005 ) Although this study employed the use of a small diameter cannula and low flow rate, several cases of severe backflow were observed. This backflow could be due to tissue entering the cannula tip during insertion and obstruct ing flow. This tissue blockage would cause the pressure in the infusion system to rise until the blockage is cleared and then a volume of infusate would be injected into the tissue at a high flow rate. Further investigations evaluating the effects of flow rate, infusion volume and cannula design on hippocampal distribution volumes are warranted. Analysis of Shape and V olume Shapes segmented from the MR images matched well the shapes of the septal and temporal hippocampus, suggesting that the infusate distr ibuted throughout each region. Certain anomalies, such as severe backflow or contrast agent entering the
61 corpus callosum, were also easily identified in the 3D segmentations. The 3D segmentations also allowed quantitative comparisons between the septal and temporal hippocampus infusion volumes. Assuming a brain tissue porosity of 0.2 ( Mazel et al., 1998 ; Sykova a nd Nicholson, 2008 ) L The septal hippocampus distribution volume calculated in this study was similar to this value; however, the volume distribution calculated in the temporal hippocampus was significantly higher than the distri bution volume calculated in the septal hippocampus. This suggests that the temporal hippocampus may have a lower porosity than the septal hippocampus, or factors other than porosity may be influencing final distribution volumes. One potential factor is the proximity of the septal hippocampus infusions to the hippocampal fissure. Because the hippocampal fissure penetrates a larger portion of the septal hippocampus than the temporal hippocampus, a larger region of the septal hippocampus is in proximity to thi s mass sink. Another potential explanation for the observed difference in distribution volumes is the more compact shape of the septal hippocampus. Although it would be expected that the contrast agent would distribute throughout the septal hippocampus and then enter the temporal hippocampus, the dense pyramidal cell layer may serve as a barrier to this transport and may confine the distribution of the contrast agent to the septal hippocampus. The observed distribution profiles include the effects of CED an d diffusion during the time delay between the final infusion and MR imaging. We estimate that the effect of diffusion may increase the measured distribution volumes by up to 40% which is equivalent to the contrast agent traveling 2 3 MR imaging voxels by d iffusion during the time delay. It is important to recognize this post infusion transport; however, the analysis of influence of hippocampal
62 tissue architecture on CED distributions and method of image segmentation for determining final distribution volume s are still valid since both convective and diffusive extracellular transport are influenced by tissue boundaries and preferential transport routes. To avoid observer bias, the segmentation of contrast agent distribution within the infused structures was c onducted using a semi automatic routine employing the selection of a lower limit threshold that was set high to assure accurate segmentation. All voxels above the threshold within the infused regions of the brain were included in the segmented volumes. Thi s lower limit threshold was not based on a percentage of the maximum signal observed in the MR images. The absolute value of the signal in the presence of the contrast agent depends on the contrast agent relaxivity and the baseline T1 values within that pa rticular tissue ( Caravan et al., 1999 ; Burtea et al., 2008 ) Thus, establishing a threshold based solely on a percentage of the maximum observed signal is not adequate for the quantitative determination of contrast agent distribution. To establish the threshold value, the average signal was measured in the contralateral, unexposed structure. The threshold value was then set to six times the standard deviation above this average signal By setting the lower limit threshold to 6 times the standard deviation above the average signal observed in contralateral structures, the threshold excludes over 99% of voxels tha t have a measured signal greater than the baseline value due to solely a fluctuation in noise. A similar method has been employed to establish a lower signal enhancement limit when calculating the concentration profile of a contrast agent infused into an a garose gel ( Chen et al., 2008 ) Since the segmented volume is sensitive to the thresholding criteria, lowering the
63 criteria would result in larger calculated infusion volumes; however, the difference between the septal and temporal hippocampus distribution profiles would probably not substantially change. C on clusions This is the first study to observe CED delivery of MR detectable agents into the hippocampus. Injury was limited to damages induced directly by the cannula. The o bserved infusate distribution did not cover the entire hippocampus, but rather distributed according to known neuroanatomic features of the hippocampus with a detailed dependence on the infusion site. It is important to note that these results describe dis tributions in normally developed hippocampi. It is reasonable to expect variability of infusate distribution in CNS injury that results in structural pathology. Understanding extracellular transport in complex and/or edematous regions is paramount for targ eted delivery of therapeutics. When structural rearrangements in injured hippocampi render other treatment options ineffective, targeted and predictable delivery of therapeutics via CED might provide a method for delivery. Moreover, use of MR imaging to ob serve distributions of therapeutic agents co infused with contrast agents may allow targeted treatment in cases of variability in individual brain anatomy. The following chapter (Chapter 3) will describe studies examining infusate distribution within the i njured hippocampus.
64 Figure 2 1. Damage induced by the infusion cannula in the septal hippocampus. A) Blood at the tip of the cannula. B) Blood within the alveus/corpus callosum boundary. Figure 2 2 Sagittal images of a single rat demonstrating the a pparent disconnect between the septal hippocampus (top row) and temporal hippocampus (bottom row) A) The disconnect is seen when the contrast agent is infused into the septal hippocampus (A and C) and temporal hippocampus (B and D)
65 Figure 2 3. High re solution T1 weighted MR images of septal hippocampus infusions. A) Schematic of key structures in the septal hippocampus adapted from ( Paxinos, 1998 ) B H) MR image coronal slice of infusion site for septal hippocampus infusions in 7 rats. Filled arrow heads, dentate gyrus granule cell layer; unfilled arrow heads, CA1 pyramidal cell layer; asterisk, hippocampal fissure. Figure 2 4. High resolution T1 weighted MR images of temporal hippocampus infusions. A) Schematic of key structures in the temporal hippocampus adapted fr om ( Paxinos, 1998 ) B H) MR image coronal slice of temporal hippocampus infusions into 7 rats. Filled arrow heads, dentate gyrus granule cel l layer; unfilled arrow heads, CA1 pyramidal cell layer; asterisk, hippocampal fissure, filled arrow, alveus; unfilled arrow, corpus callosum
66 Figure 2 5. Histological images following infusate CED into the septal hippocampus showing Evans blue dye sprea ding thro ughout the septal hippocampus. A, C) Fluorescence images of 2 subjects showing limited penetration in the septal hippocampal dense granule cell layer (filled arrowhead) and pyramidal cell layer (unfilled arrowhead). Preferential distribution can b e seen in the hippocampal fissure (asteris ks) and alveus (filled arrow). B) Black gold stain ed image in close proximity to ( A) confirming alveus and dense cell layer approximations. D) Cresyl violet staining of a section in close proximity to (C) confirmin g dense cell layers.
67 Figure 2 6. Histological images following infusate CED into the temporal hippocampus showing Evans blue dye spreading thoughout the temporal hippocampus. Arrowheads denote granule cell layer of the dentate gyrus and py ramidal cell la yer of the CA1. A) A fluorescence image of Evans blue seen preferentially in the temporal hippocampal fissure (asterisks), alveus (filled arrow) and corpus callosum (unfilled arrow). B) Cresyl violet stained image of a sect ion in close proximity to (A). C) Fluorescent image of Evans blue seen preferentially in the alveus and corpus callosum Chevron shows Evans b lue in the perivascular space. D) Cresyl violet stained image of a section in close proximity to (C).
68 CHAPTER 3 INFLUENCE OF LIMBIC SYSTEM INJURY ON INFUSATE DISTRIBU TIONS IN THE RODENT HIPPOCAMPUS I ntroduction The work shown in Chapter 2 established the local heterogeneity of neuro structural characteristics as a governing feature of CED. The dependence of i nterstitial flow on normal hippocampal stru cture suggests pathological changes within the hippocampus would introduce variability in the distributions, especially if myelin integrity or dense cell layers we re affected. It is well documented that that SE ( Meierkord et al., 1997 ; Kim et al., 2001 ; Fabene et al., 2003 ) and temporal lobe epilepsy ( Babb, 1987 ; Kuzniecky et al., 1987 ; Bruton, 1988 ; Bernasconi et al., 2004 ; Parekh et al., 2010 ) result in structural changes within the limbic system, yet previous studies investigating the influences o f p athology on hippocampal distributions are lacking. Therefore, i n an effort to optimize targeted CED delivery into injured regions, this chapter describes the influence of microstructural changes in the hippocampus on the distribution of infusate in a pre c linical SE m odel of chronic limbic epilepsy. Limbic system injury was ranked using MR imaging and then corroborated at a cellular level using immunohistochemical and neurodegeneration markers. MR imaging was used to monitor and measure the distribution of Gd albumin and then distributions were compared to the results from control animal infusions described in Chapter 2 I nfusions were performed at acute and chronic points of TLE to illustrate the effect of different pathologies on infusate distributions in the hippocamp us. Infusions Excerpts from this chapter have been submitted for publication to Neurotherapeutics. This work was completed with the help of Dr. Garrett Astary. Dr. Astary helped with the infusions, and performed the MR imaging and image segmentation described in this chapter.
69 performed 24 hours after an episode of self sustaining limbic SE translate to a window for prophylactic treatment. Many treatment studies address only chronic time points in TLE, even though structural changes occur progressivel y from shortly after SE up to the appearance of spontaneous seizures ( Parekh et al., 2010 ) Clinically about 30% of epilepsies can be linked to an identifiable injury to the brain that triggers the development of the disorder ( Manford et al., 1992 ; Hauser, 1997 ) Treatment after initial insults have the potential to reduce structural damage, diminish associated behavioral and cognitive impairment, or prevent epileptogenesis (for review see ( Pitkanen, 2002a ; Sutula, 2002 ) ). W hile there is mounting experimental evidence that preventative therapy can be beneficial in positive disease modification ( Pitk anen, 2002b ; Brandt et al., 2003 ; Nicoletti et al., 2008 ; Zafar et al., 2012 ) clinical evidence is controversial ( Willmore, 2005 ) The f ailure of clinical trials ha s been attributed to the use of inappropriate treatment strategies ( Pitkanen, 2002a ) and pharmacokinetic issues, i.e. the lack of therapeutic levels in the brain after systemic administration ( Brandt et al., 2003 ) The latter has been specifically raised in cases of inconsistent or absent monitoring of therapeutic levels in studies ( Liu and Bhardwaj, 2007 ) This work addresses such concerns through employing CED, which, unlike systemic administration ensure s the application of a clinically relevant dosage and allows for provision of novel treatment strategies. Infusions were also performed at 60 days po st SE, once ani mals were spontaneously seizing. These infusions translate to the treatment of chronic TLE, and are specifically relevant for one third of patients with epilepsy who are resistant to current AEDs. CED
70 at this time point can be used to maximi ze the effectiveness of therapeutic agents by increasing drug concentration at the focus. Both 24 hours and 60 day time points represent important treatment windows for epilepsy, but exhibit distinct pathologies. The findings from this study add injury sp ecific evaluations to the collection of p rinciples influencing delivery of macromolecules within the CNS. Information from this study can be applied to improve targeting guidelines for CED, incorporated into computational CED transport models ( Sarntinoranont et al., 2006 ; Kim et al., 2010 ) and considered in the planning of preventative delivery strategie s of novel therapeutic agents Methods Animals Male Sprague Dawley rats (Harlan Labs, Indianapolis, IN) weighing 225 250 g on arrival were allowed one week to acclimate to the 12 h light/dark cycle and given food and water ad libitum. All procedures were a pproved by the University of Florida Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Experimental Animals. A flow chart depicting the order of experiments can be seen in Figure 3 1. Surgical Procedures Anesthesia was initiated with xylazine (10 mg/kg, SQ) and 4% isoflurane in 1 L/min oxygen. Once animals were securely placed in a Kopf stereotaxic apparatus, anesthesia was maintained at 1.5% isoflurane in 0.4 L/min oxygen. The lan dmarks bregma and lambda were exposed by a mid polyamide coated tungsten microwire electrodes (Plastics One, Roanoke VA) were implanted for the induction of self sustaining SE. Two electrodes were implanted into
71 t he right temporal hippocampus [ 5.3 mm AP, 4.9 mm ML, 5.0 mm DV] for stimulating and recording, and one was implanted as a reference electrode into the corpus callosum [ 3.3 mm AP, 4.9 mm ML, 2.4 mm DV]. A cannula guide was also secured to the skull region abo ve the left septal hippocampus [ 3.7 mm AP, 2.2 mm ML] for the future infusion of contrast agent. Infusion was planned contralateral to electrodes to avoid the confound of electrode implantation on infusate distribution. Four nylon anchoring screws were pla ced in the skull to allow for maximum support of the headset, which was permanently secured with Cranioplast cement (Plastics One, Roanoke VA). All animals were given one week to recover from the implantation surgery before the stimulation procedure began. Induction of Self Sustaining Limbic Status Epilepticus (SE) by H ippocampal Electrical Stimulation Approximating the evolution of structural changes that result in human epilepsy took precedence for the animal model chosen in this study because changes in cellular structure will undoubtedly affect migration in the interstitium. The self sustaining SE model replicates essential characteristics of epilepsy as it occurs in humans (See Chapter 1), including the presence of a latent period. To create the model, o ne week post electrode implantation, animals (n=2 8 ) were electrically stimulated to induce self sustaining SE, a prolonged seizure lasting 30 90 minutes, as described in ( Lothman et al., 1989 ) Stimulus trains (50 Hz of 1ms biphasic square wave pulses) were delivered for 10s on and 2s off for a total of 78 16 minutes. A modified Racine scale ( Racine, 1972 ; Borowicz et al., 2003 ) was used to grade the behavioral seizures as follows: grade 0 for no seizure response; grade 1 for immobility, eye closure, ear twitching, twitching of vibrissae, sniffing, facial clonus; grade 2 for head nodding associated with
72 more severe facial clonus; grade 3 for clonus of one forelimb; grade 3.5 for bilateral forelimb clonus without rearing; grade 4 for bilateral forelimb clonus with rearing; grade 5 for rearing and losing balance. Animals (n=1 9 ) that experienced Racine grade 4 or 5 seizures during the hippocampal stimulation and experienced electrographic seizure activity for at least 2 hour s post stimulation were included in the study. Animals were continuously recorded with time locked video EEG until 24 hours or 60 days post SE T hese inclusion criteria were used to create a comparable injury across animals that would likely lead to the de velopment of spontaneous seizures ( Lothman et al., 1989 ; Bertram and Cornett, 1994 ; Bertram, 1997 ; Sanchez et al., 2006 ; Parekh et al., 2010 ) MR Imaging Twenty four hours or 60 days post induction of SE, high resolution T1 and T2 weighted MR imaging was performed to generate a reference for contrast enhancement images (T1) and to visualize morphological changes (T2). Immediately following infusion of Gd albumin (see next section), high resolution T1 weighted imaging was repeated to visualize distribution profiles of the contrast agent. MR measurements were performed using a Bruker Avance imaging console (Bruker NMR Instruments, Billeria, MA) or Agilent Direct Drive imaging console (Agilent Technologi es, Santa Clara, CA, USA) connected to a Magnex Scientific 11.1 T horizontal bore magnet system(Varian, Inc., Magnex Scientific Products, Walnut Creek, CA). A custom made 130 degree arc, 3.5cm rectangular linear field surface coil constructed on a 4cm diam eter half cylinder was used for linear transmission and detection of MR signal. High resolution T1 weighted images, with slices oriented in the coronal direction, were acquired using a spin echo sequence with a 2.5cm2.5 cm field of view in a matrix of 20 0200, recovery time of 1000 ms, echo time of 10ms, 8 averages 30 slices 500 L slice thickness T2
73 weighted data were acquired using a fast spin echo sequence with 30 slices oriented in the coronal direction and a 2.5cm2.5cm field of view in a matrix o f 208208, recovery time of 3500ms, RARE factor of 8 and effective echo time of 45 ms. T2 weighted images (30 coronal slices per brain) were examined and used to classify injury in animals. The high water content of edematous tissue results in prolonged T2 relaxation times and manifests as a hyperintense signal in T2 weighted images. Hyperintense signal was defined as injury in these images and then validated post mortem (see Immunohistochemistry section). T2 weighted images were scored for injury as follow s: 1 = unilateral piriform cortex and/or amygdala damage, 2 = score of 1 plus injury in the septal nuclei, 3 = a score of 2 plus injury in the middle thalamic nuclei, 4 = a score of 3 plus damage in the lateral thalamic nuclei, 5 = a score of 4 plus damage in the ventral subiculum, 6 = bilateral piriform cortex/amygdala damage, septal injury, and damage in the middle and lateral thalamic nucle i. An early indication of the development of spontaneous limbic seizures in this animal model is the presence of ede ma in the parahippocampal gyrus ( Parekh et al., 2010 ) Because the purpose of this study was to examine injury before or after epileptogenisis, animals imaged at 24 hours post SE that did not exhibi t parahippocampal gyrus were excluded from subsequent analyses. Infusion of Gd albumin Twenty four hours (n=17) or 60 days (n=2) post induction of SE, animals were infused with 5.0 L of Gd albumin (10 mg/mL in PBS solution; MW 87 kDa, 35 Gd DTPA molecules per albumin molecule; R. Brasch Laboratory, University of California, San Francisco, CA), tagged with Evans Blue dye (1 mg dye/50 mg Gd albumin) into the septal dentate gyrus of the hippocampus [ 3.7 AP, 2.2 ML 3.4 DV]. The infusion was
74 performed throu gh the previously implanted cannu la guide using the same equipment and infusion parameters as described in Chapter 2 Results from experimental animals infused in this study were compared to results from control animals infused under s imilar conditions (se e Chapter 2) The only difference between infusions performed in control animals and those performed in injured animals was a ~30 minute time delay between infusion and MR imaging in control animals. The contribution of diffusion after the end of CED in th ese animals was estimated in Chapter 2. Immunohistochemistry Following the last MR measurement, animals were transcardially perfused with 200 mL saline solution followed by 300 mL of 10% buffered formalin phosphate. Brains were extracted and stored in the formalin solution overnight at 4C, then equilibrated in 30% sucrose solution for 72 hours. Brains were then sectioned coronally using a cryostat set at 50 m. Every fifth section in succession was collected for staining with either Fluoro Jade C (FJC), Bl ack Gold II, glial fibrillary acidic protein (GFAP), macrophage acti vation (CD 68), or Perl stain. GFAP, CD 68, and Perl stained sections were counterstained with cresyl violet for visualization of cell bodies. FJC staining ( Schmued et al., 2005 ) was used to visualize degenerating neurons with a modification by Lee et al ( Lee et al., 2011 ) Mounted tissue sections were first immersed in 1% sodium hydroxide in 80% ethanol for 5 minutes. They were then rinsed for 20 minutes in 70% ethanol, followed by 2 minutes in distilled water, and then incubated in 0.02% potassium permanganate solution for 3 minutes. Slides were t hen rinsed for 2 minutes in distilled water and transferred for 15 minutes into a 0.0002% solution of Fluoro Jade C (Histo Chem Inc., Jefferson, AR) dissolved in a 0.1% acetic acid vehicle.Sections were stained for myelin using Black Gold II ( Schmued et al.,
75 2008 ) Mounted slides were incubated in a 0.3% Black Gold II solution (Histo Chem Inc., Jefferson, AR) at 60C for 12 15 minutes, rinsed, then transferred to a 1% sodium thiosulfate solution for three minutes. For assessment of micro and astrogliosis, free floating sections we re incubated in 10mM citrate buffer, pH 9.0, for 25 minutes at 80C for antigen retrieval. After a brief wash, they were stained for microglial activation and astrocytosis overnight using primary monoclonal antibodies against CD 68 (AbD Serotec; Raleigh, N C) or GFAP (G A 5, Sigma Chemicals Co.; St. Louis, MO), respectively, at a concentration of 1:400. Sections were washed and incubated overnight in 1:10,000 biotinylated anti mouse immunoglobulin G, reacted with a 1:1,000 Extravidin peroxidase solution for 2 hours, then visualized with 0.05% 3,3' diaminobenzidine (DAB) in 0.0012% hydrogen peroxide in PBS. Image Segmentation and Statistical Analysis Final distribution volumes of Gd albumin were analyzed by performing semi automatic image segmentation on the T 1 weighted coronal images using routines written in MATLAB (The Ma thWorks Inc., Natick, MA, USA ) with the following specific threshold criteria. Voxels were included in the infusion volume if their signal intensity was higher than at least 6 standard dev iations of the noise in the corresponding region contralateral to the site of infusion (control regions containing no Gd albumin). The segmentation output of the MATLAB routine was refined using the ITK SNAP open source medical image segmentation tool ( Yushkevich et al., 2006 ) Dynamic and final distribution volumes in the septal and temporal hippocampus were calculated by counting the number of voxels inclu ded in each segmented region and multiplying by the volume of a single voxel.
76 Total hippocampal volumes were calculated by manually segmenting the T2 weighted pre infusion images in ITK SNAP. The borders of the hippocampus (e.g. corpus callosum, thalamus) were determined by white matter/gray matter contrast in the T2 weighted images, anatomical landmarks such as the velum interpositum and by referring to a rat brain atlas ( Paxinos, 1998 ) Distribution volumes of 24 hour animals were compared to injury ratings using This non parametric test is used for ordi nal data and is The Kendall tau correlation coefficient estimate of the corresponding population param e ter and has more accurate p values in small sample sizes ( Gibbons, 1993 ) a measure of the strength of a relationship and does not have a meaningful operational interpretation ( Bland, 1995 ) The significance of differences in volume of distributions between control animals, 60 day animals, and injury classifications of 24 day animals was calculated by analysis of variance (ANOVA). Post hoc testing for individual classification differences was done with Newman Keuls test. All tests were two tailed; a p <0.05 was considered signifi cant. Results SE Induced Injury Animals (n=1 9 ) were electrically stimulated in the temporal hippocampus to experience one episode of SE. T2 weighted images ( Figure 3 2 ) were acquired 24 hours (n=17) or 60 days (n=2) post induction of SE to reveal edema wit hin regions of the limbic circuitry. An early indication of the development of spontaneous limbic seizures in this animal model is the presence of edema in the parahippocampal gyrus
77 ( Parekh et al., 2010 ) One animal did not exhibit edema in the parahippocampal gyrus and was not included in the subsequent injury analyses. Of the remaining 24 hour group, 3 of 16 animals exhibited edema in the bilateral parahippocam pal gyrus ( Figure 3 2P R ) and 1 3 of 16 animals exhibited unilateral edema ( Figure 3 2C O). Out of the 1 3 animals that exhibited unilateral edema, injury was ipsilateral to the stimulating electrode in 3 animals ( Figure 3 2 D,I,L ), and contralateral in 10 an imals ( Figure 3 2 C,E H,J K,N O ). Table 3 1 presents the T2 injury index classification of each animal at 24 hours post SE Of the 2 animals that were imaged 60 days post SE (Figure 3 2S T) only one revealed contralateral parahippocampus edema (Figure 3 2T ) and enlarged ventricles in MR imaging Neither of the animals exhibited edema in any other limbic structures Volumes of Distribution of Gd albumin and Changes in Hippocampal Volume Increasing classifications of injury were correlated with volumes of dis tribution of Gd albumin in 24 hour animals ( tau = 0.51 p = 0.0 06 ), which averaged 21.2 3.6 L for animals classified as Class 1 2, 26.8 5.4 L for animals classified as 3 4, and 33.2 6.0 L for animals classified 5 6 ( Figure 3 3). Distributions in ani mals with severe injury (Class 5 and above) were significantly increased as compared to previously measured control animals in Chapter 2 (p=0.01 8 ; average 23.4 L 1.8 L ) and as compared to 60 day TLE animals (p=0.00 2 average 18.5 2.1 L ) This incre ase may be underestimated, as distribution volume of control animals includes ~30 extra minutes of diffusion that occurred in the time delay between infusion and MR imaging. The contribution of diffusion was estimated in Chapter 2 to increase control distr ibution volumes up to 40% (2 3 voxels, 0.250 0.375 mm). Therefore, the relative increase of distribution volume s in injured animals may actually be larger No significant differences
78 were found between animals presenting with ipsilateral versus contralater al parahippocampal injury. Total hippocampal volumes of 24 hour animals pre infusion were measured and compared to volumes of distribution post infusion. These volumes were not significantly different between injury classes (Class 1 2:18.4 1.0, Class 3 4 :18.6 1.6, Class 5 6:19.2 1.7), indicating that in this study, the larger infusion volumes seen in injured animals cannot be solely explained by differences in hippocampal size. Characteristics of Gd albumin Distribution While volumes of distribution correlated with magnitude of injury ( Figure 3 3), the pattern of infusate spread was consistent between animals exhibiting various levels of injury and at different time points ( Figure 3 4). All infusions showed clear demarcation within the hippocampus, wi th very minimal backflow only along the cannula tract in 9/1 9 ( 47 %) animals. Six animals ( 32 %) showed limited backflow within the corpus callosum overlying the hippocampal infusion site, and 4 animals (21%) exhibited no backflow at all. As described in con trol animals (see Chapter 2) spread of the contrast agent in experimental animals was seen in the dentate gyrus, CA3, CA2, CA1, and subiculum of the hippocampus. Additionally, both the dentate granule cell layer and pyramidal cell layers of CA3 CA1 and su biculum were clearly distinguishable from the surrounding hyperintense subfields, indicating poor contrast agent penetration. This was especially salient in the CA3 subregion in 8 animals ( 42 %), where infusate coverage tapered at stratum pyramidale, not qu ite reaching stratum oriens ( Figure 3 4 B,F,G,K,L,R T) In 4 animals (2 1 %), infusate filled all layers of the CA3 subregion and also penetrated the fimbria ( Figure 3 4 I J,M,P ). As in controls, infusate did not extend to the contralateral hippocampus or any other subcortical structures in any animals; however, enhancement
79 was observed as hyperintensity in T1 images within extraventricular regions surrounding the septal and temporal hippocampus. Twelve animals (6 3 %) exhibited leakage into the velum interpositu m of the septal hippocampus, and 1 4 animals (7 4 %) exhibited leakage into the midbrain cisterns of the temporal hippocampus, suggesting a portion of the contrast agent distribution was not accounted for within the hippocampal distr ibution volumes. Furthermo re, 12 animals (6 3 %) also exhibited leakage into the lateral ventricle ipsilateral to the infusion site. Histology 24 H ours P ost SE MR visualized injury was validated and characterized using histological assessments of CNS injury. For animals infused 24 ho urs post SE injury was visualized in several limbic regions. Within the hippocampus proper ( Figure 3 5), neurodegeneration was observed via FJC staining in the CA3/CA2 subfield of all rats and in the hilus of rats with more severe injury (10/17 animals, Class 3 6). Positive FJC staining was not seen in the hippocampus of animals in injury Class 0 1 (compare Figure 3 5D,F ). A minority (5/17) of animals in more severely injured classes (Class 4 6) also exhibited positive FJC staini ng in hippocampal subfield CA1 Activated microglia were detected by expression of CD68 ( Figure 3 5 G I arrows) in the CA3 of 10/17 rats encompassing injury C lasses 2 6. Only a minority of animals exhibited activated microglia in the CA1 and hilus (5 rats in Classes 2 6 and 4/17 ra ts in Class 4 6, respectively). CD68 positive staining was not detected in animals of Class 0 1 (compare Figure 3 5 G,I ). GFAP staining was used to visualize the enlarged soma and processes typical of reactive astrocytes ( Figure 3 5 J L arrows). Hypertrophi c astrocytes were seen in the hilus ( Figure 3 5 K arrows) of the majority of rats (14/17 animals, Class 2 6) and
80 in the CA3 of 5 rats (Class 2 6). Activated astrocytes were only seen in the CA1 of 1 rat in Class 6, the most severe injury class. All hyperi ntense regions identified in T2 weighted imaging were also immunostained for CNS injury markers. Hyperintensity observed in the p arahippocampal gyrus ( Figure 3 6) during in vivo T2 weighted imaging corresponded to fluid filled cavities ( Figure 3 6, asteris ks), myelin degradation ( Figure 3 6 A C ), neuronal degeneration ( Figure 3 6 D F ), macrophage activation ( Figure 3 6 G I ), and astrogliosis ( Figure 3 6 J L ) 24 hours post SE Injury in the ventral subiculum ( Figure 3 7), which resulted in a classification of Cl ass 5 or above, resulted in considerable degeneration of myelin (compare Figure 3 7 A,B ) and neurons, as measured by FJC staining (compare Figure 3 7 C,D ) and cell layer integrity in cresyl violet staining (compare Figure 3 7 E,F and G,H ). Thalamic injury ( Fi gure 3 8) did not consist of notable myelin degradation ( Figure 3 8 A ), but did encompass neuronal degeneration ( Figure 3 8B arrows) and microglia activation ( Figure 3 8 C arrows). Activated astrocytes were seen in the medial habenular nucleus ( Figure 3 8 D arrows). No obvious difference was observed in the hippocampi of rats that exhibited ipsilateral versus contralateral edema in the parahippocampal gyrus. Animals in higher classes of injury (4 and above) exhibited increased activation of FJC, CD68, and GFAP in the CA1 subfield, and increased activation of CD68 in the hilus. All animals exhibited injury near the electrode stimulation site at the temporal hippocampus. Histology 60 D ays P ost SE Histological analysis of 60 day animals revealed much less inju ry than acutely injured animals In one animal (Figure 3 9), no positive staining of neurodegeneration, microglia activation, or myelin degradation was seen in the hippocampus (red boxes),
81 thalamus (green boxes) or parahippocampus (blue boxes). Astrocytosi s was visible in the hippocampus, but not in other regions. The other animal (Figure 3 10) exhibited slight microglia activation and substantial astrocyte activation in the hippocampus (red boxes) and thalamus (green boxes), but no neurodegeneration. Cavit ation, astrocytosis, microgliosis, neurodegeneration, and myelin degradation were all seen in the parahippocampus of this animal (blue boxes). Discussion This study investigated the effect of injury at different time points on CED contrast agent distributi on profiles and volumes in the rat septal hippocampus. Distribution of Gd albumin was visualized with high resolution MR imaging, then volume analysis was performed with segmentation. Injury was rated based on edema visualized with T2 weighted MR images, a nd characterized with staining against neuronal degeneration, myelin degradation, astrocytosis, and macrophage act ivation The main finding in this study is that the volume of infusate distribution is increased in animals with more severe acute injury rel ative to mildly injured animals aged matched controls and chronically epileptic animals In normal hippocampi, CED performed under similar infusion conditions resulted in distribution volumes roughly 5 times greater than the volume infused (see Chapter 2 ) This spread corresponds to an estimated brain tissue porosity of 0.2 ( Mazel et al., 1998 ; Sykova and Nicho lson, 2008 ) which assumes isotropic porous medium and no CSF leaks. The volumes of distribution in mildly injured animals at 24 hours (Class 1 4) in this study are similar to those measured in control animals (Class 0). However, volumes are significantl y larger in animals that presented with a more severe CNS injury 24 hours post SE, even with the presence of CSF leakage. This effect was not present after 60 days; implying injury may
82 confer measurable effects on distribution volumes via changes in the ex tracellular volume fraction. Acute SE I nduced injury Following brain injury, a cascade of pathological events evolves over minutes, days, and weeks. Severity of CNS injury was classified in T2 weighted MR images and identified with several histological st ains. Although stimulation was always applied to the right hippocampus, injury was observed on both sides of the brain. At 24 hours post SE, d egeneration of neurons and myelin, in addition to activation of microglia and astrocytes was detected in the hippo campus, parahippocampal gyrus, thalamus, and septal nuclei. Injury in these areas is consistent with other histological reports at acute time points ranging from 8 to 48 hours. Damage has been reported in the piriform and amygdalar cortices after administr ation of pilocarpine ( Wall et al., 2000 ; Nairismagi et al., 2006 ) kainate ( Nakasu et al., 1995 ; Brandt et al., 2003 ) angular bundle stimulation ( Gorter et al., 2003 ) and rostral forebrain stimulation ( Handforth and Treiman, 1994 ) Damage has been reported in the CA1 and CA3 after administration of pilocarpine ( Nicoletti et al., 2008 ) kainate ( Brandt et al., 2003 ; Hsu et al., 2007 ) and rostral forebrain stimulation ( Handforth and Treim an, 1994 ) Injury in t he entorhinal cortex is commonly reported after angular bundle stimulation ( Gorter et al., 2003 ) SSLSE, kainate, and pilocarpine models ( Du et al., 1995 ) Midline thalamic nuclei were affected after rostral forebrain ( Handforth and Treiman, 1994 ; Brandt et al., 2003 ) and angular bundle stimulation ( Gorter et al., 2003 ) In addition, as in this study, the work by Brandt et al. ( Brandt et al., 2003 ) and Gorter et al. ( Gorter et al., 2003 ) report more extensive damage associated with lateral thalamic nuclei.
83 Injury and Final Infusate Distribution Volume SE is a major risk factor for the developm ent of chronic epilepsy in both humans ( Sloviter, 1999 ) and animal models ( Lothman et al., 1990 ; Cavalheiro, 1995 ; Hellier et al., 1998 ) Both acute and chronic time points present opportuni ties for treatment. The pathological states observed in this and other studies at acute time p oints include morphological changes, such as neuronal death, glial cell loss or proliferation, glial swelling, production of damaging metabolites, inflammation, edema, demyelination, and loss of ionic, pH, and amino acid homeostasis. They may also be accom panied by substantial changes in ECS ionic composition ( Sykova, 1983 ) and various changes in ECS diffusion parameters ( Sykova, 1983 ; Sykova et al., 1994 ; Sykova, 1997a ; Sykova et al., 1998 ; Sykova and Nicholson, 2008 ) suggesting reduction of the ECS In fact, ECS volume shrinkage is likely a compensation mechanism for acute neuronal and glial swelling ( Sykova, 1997a ) The presence of cellular debris and inflammatory markers paired with ECS changes increases the tortuosity, or hindrance to diffusion, within the ECS Geometrically, tortuosity is quantified as degree of curvature and complexity of the curve. Biologically, tortuosity is quantified as the ratio of the diffusivity in free space to that in the brain. Tortuosity in both applications changes with edema and neuropil remodeling. This has been documented in several models of brain injury, including co rtical stab wounds ( Vorisek et al., 2002 ) neural tissue grafts ( Sykova et al., 1999b ) and hypoxia ( Sykova et al., 1994 ) where the ECS volume fraction in rat cortex was specifically measured to decrease by 80% and was accompanied by an increase in tortuosity. Hypertrophic astrocytes, which have been observed here and in many other studies, were postulated to be the cause. Hence, an acute insult resulting in cellular debris and/or the swelling of cells and fine glial processes would increase intercellular
84 tortuosity, affect the size of the intercellular channels, and change diffusional characteristics. Accordingly, the consequence of smaller intercellular channels would be a larger volume of Gd albumin distribution in rats that experienced a greater degree of injury. On the other hand, the microstructural injury present at chronic time points (weeks years) has different characteristics and perhaps opposite effects on infusate distribution. A longitudinal study ( Parekh et al., 2010 ) has shed light on the progressive changes occurring during epileptogenesis in the SE animal model At acute phases, MR measurements (decreased average diffusivity (AD) and T2) in the hippocampi suggested the presenc e of cytotoxic edema and ongoing neurodegeneration that was confirmed with histology. At the onset of spontaneous seizures, MR measurements (increase in AD and T2) are indicative of vasogenic edema, and observed histologically as ongoing degeneration, and myelin degradation. Increased FA in the dentate gyrus thalamic damage was also seen that corresponded to microgliosis. Like the SE animal model, human mesial temporal lobe e pilepsy is also commonly associated with hippocampal pathology with varying degrees of regional neuronal loss and gliosis. Additionally, pathological changes in the human amygdala ( Hudson et al., 1993 ) entorhi nal cortex ( Du et al., 1993 ) and thalamus ( Margerison and Corsellis, 1966 ; Bruton, 1988 ) have often been reported. Histological evaluation of biopsy specimens from chronic epilepsy reveal a majority of TLE patients have the hippocampal atrophy lerosis, in addition to other abnormalities, such
85 as seizure associated postnatal neurogenesis and mossy fiber sprouting ( Thom et al., 2005 ) Sclerotic hippocampi in chronic epilepsy have even been associated with increased ECS ( Wieshmann et al., 1999 ) a development that may explain the change in distribution volumes compared to acutely injured hippocamp i. As opposed to acute damage, infusate spread in chronic TLE hippocampi was found to be decreased, perhaps due to enlarged interstitial spaces. While this study only observed two animals at chronic time points, histological analysis revealed less microgli a activation and no neurodegeneration in the hippocampus. The more injured animal also exhibited enlarged ventricles, which may result in more drainage of the infusate and contribute to the decrease in infusate distribution volume. Injury and Pattern of In fusate Distribution The data in this study suggest acute SE induced injury is significantly correlated with infusate distribution volumes. Interestingly, patterns of distribution within the septal hippocampus were consistent with those in control animals. Largely underestimated, the biophysical properties of the local tissue architecture at the infusion site are a critical consideration for planning the coverage of therapeutic agents in both normal and injured areas of the brain. Even in normal rat brains, measured tortuosity in vivo was found to be higher in regions including a dense cell layer, such as stratum pyramidale, as compared to stratum radiatum, which contains mostly fibers ( Sykova et al., 1998 ) Isotropic, dense gray matter regions will affect CED distribution differently than directional w hite matter regions. Anisotropic regions such as axonal bundles often exhibit increased infusate transport along fiber directions ( Vorisek and Sykova, 1997 ) while laminar structures such as the hippocampus may cause more widespread
86 dispersion. Additionally, ventricles and perivascular spaces can act as mass sinks in which infusate may pool or be directed towards subarachnoid spaces ( Neuwelt, 2004 ; Astary et al., 2010 ) Gd albumin access to fluid fille d spaces was observed in this study, where a majority of animals exhibited Gd albumin leakage into the lateral ventricle, the velum interpositum, and the midbrain cisterns. Other Factors Affecting Final Infusate Distribution In this study, the severity of injury was significantly correlated with increases in measured distribution volumes. It is important to note that the volume differences between controls and injured animals may be underestimated because the observed distribution profiles in control animal s include the effects of CED and diffusion during a ~30 minute time delay between the final infusion and MR imaging. The analysis of influence of hippocampal tissue architecture on CED distributions and method of image segmentation for determining final di stribution volumes are still valid since both convective and diffusive extracellular transport are influenced by tissue boundaries and preferential transport routes. There are also several other factors (discussed in more detail in the subsection Chapter 1) that may add to the remaining variability in the final distribution of infusate (for review, see ( Krauze et al., 2006 ) ). Firstly, infusion pump parameters, such as flow rate and duration of infusion, will influence interstitial transport ( Bobo et al., 1994 ) However, these factors were kept con stant across animals, and thus were deemed to have a negligible effect on inter subject variability. Secondly, distribution will vary based on properties of the infusate itself, including size, shape, viscosity, solubility, binding characteristics, concent ration, and rate of efflux from the brain. Under certain infusion conditions, highly viscous solutes display greatly increased volumes of
87 distribution ( Mardor et al., 2009 ) and water soluble, non transported compounds display slower efflux rates ( Groothuis et al., 2007 ) Since the same compound was used for all exper iments in this study, the physico chemical properties of Gd albumin do not explain the inter subject variability. However, it should be noted that these properties pose a significant influence on the rates and routes of delivery and efflux of various solut es. A third key factor that introduces variability in final distribution is backflow, which results in the spread of infusion into unintended regions. This issue can be addressed with preventive cannula designs ( Morrison et al., 1999 ; Guarnieri et al., 2005 ; Ivanchenko and Ivanchenko, 2011 ) but re mains an important va riable in drug delivery studies In this study, roughly half of the infusions exhibited very minimal backflow only along the cannula tract. About a quarter of the infusions resulted in no backflow, and another quarter had backflow alon g the overlying corpus callosum When compared to infusions with no backflow, the distribution volumes of infusions exhibiting backflow were not significantly affected (p=0.18). Finally, infusion site is a key factor in the final distribution of infusate d ue to the influence of normal local structural differences in CNS anatomy (see Chapter 2). Infusions in this study were targete d to the same stereotaxic coordinates in each experiment due to inter animal variability. The infusion site variability of the majority of animals in this study was very minimal; however, two Class 4 animals had infusion sites that were too lateral in the hippocampus. The resulting infusion coverage in these animals failed to cover the medial aspect of the CA1 subregion or the dorsal subiculum, resulting in artificially l ow distribution volumes that fell within the average of Class 1 2
88 animals. This type of infusion site variability can be avoided with careful MR guided cannula insertion, but it underlines the effect of local tissue variability on the distribution of infus ate. Conclu s ions This is the first study to observe CED delivery into the hippocampus of a pre clinical rodent model of chronic epilepsy. This type of delivery bypasses systemic circulation and opens the possibility of therapy to a wide variety of p otential compound s, since it does not require passage through the BBB or consideration of systemic safety. Several studies have described CED in normal hippocampi in different species, but understanding the influence of structural changes on extracellular transport in injured regions is critical for planning drug delivery studies. Historically, CNS injury has been associated with changes in the ECS swelling of cellular elements, and overall increased tortuosity within the interstitial space. This was obser ved here as neuronal degeneration, myelin degradation, and macro and microglia activation as an acute response to SE. Such changes can affect diffusion parameters and in this study, were found to be correlated with the final volume distribution of Gd albu min. Recovery of acute changes at chronic time points resulted in significantly reduced infusion volumes as well. These results illustrate the importance of biophysical influences on CED and should be incorporated in the planning of future studies tracking therapeutic agents Chapter 4 will describe two examples of CED of therapeutic carriers.
89 Figure 3 1. Experimental protocol flow chart
90 Figu re 3 2 T2 weighted coronal images of 1 9 different rodent brains acquired post induction of SE reveal injury wi thin regions of the limbic circuitry. Hyperintense re gions signify areas of injury. A ) Schematic of affec ted structures adapted from ( Paxinos, 1998 ) B R) MR images of injury induced in 17 different animals 24 hours post SE Structures most affected were the CA3 hippocampal subfield, ventral subiculum, piriform cortex (P), entorhinal cortex (ENT), amygdalar nuclei (AMYG), middle thalamic nuclei (MT), and laterodorsal/laterop osterior thalamic nuclei (LT). S T ) MR images of injury induced in two animals 60 days post SE. Electrode implantation is on the left side of each image
91 Table 3 1 Index of injury classification 24 hours post SE epilepticus Injured regions were identified using T2 weighted coronal images obtained in vivo prior to CED infusions Injury class Injury description Number of animals Percentage of animals Class1 Unila teral edema in the piriform cortex and amygdalar nuclei 2 12.5% Class 2 Class 1 plus edema in septal nuclei 1 6.25% Class 3 Class 2 plus edema in the middle thalamic nuclei 2 12.5% Class 4 Class 3 plus edema in the laterodorsal/lateroposterior thalamic nuclei 5 31.25% Class 5 Class 4 plus edema in the ventral subiculum 3 18.75% Class 6 Class 5 plus bilateral edema in the piriform cortex and amygdalar nuclei 3 18.75% Figure 3 3 Increasing c lassifications of injury were correlated with volumes of distribution in 24 hour animals ( tau = 0. 51 p = 0.0 06 ), which averaged 21.2 3.6 L for animals classified as Class 1 2, 26.8 5.4 L for animals classified as 3 4, and 33.2 6.0 L for animals classified 5 6. Distributions in animals Class 5 and above w ere significantly increased as compared to previously measured control animals (Class 0), which averag ed 23.4 1.8 L (p = 0.01 8 see Chapter 2) ) and to animals infused 60 days post SE (average 18.48 2.1 L p=0.00 2 ). **
92 Figure 3 4 High resolution T1 weig hted images of Gd albumin infusions into the septal hippocampus of 1 9 different rodent brains post SE A ) Schematic of key structures in the septal hippocampus adapted from ( Paxinos, 1998 ) B R ) MR images of contrast agent distributions in the septal hippocampus 24 hours post SE S T) Contrast agent distributions in the hippocampus o f 2 animals 60 days post SE Hyperintense regions are voxels containing Gd albumin. Distribution patterns contour along hippocampal circuitry with minimal backflow or exposure to extra hippocampal regions. CC = Corpus callusom; CA1 = CA1 pyramidal cell lay er; hf=hippocampal fissure; DGC = Dentate granule cell layer; CA3 = CA3 pyramidal cell layer
93 Figure 3 5 Characterization of hippocampal damage 24 hours post SE Representative stained sections of affected hippocampi (left column:low magnification, midd le column:high magnification) are compared to unaffected hippocampi (right column) for A C ) myelin degradation, D F ) neuronal degeneration, G I ) macrophage activation, and J L ) astrocytosis. Higher magnification of boxed areas show ongoin g neurodegeneratio n within CA3 ( E arrows) that corresponds with macrophage activation ( H arrows). Cell loss is corroborated through loss of cresyl violet staining in CA2 (closed arrowheads, compare G I and J L ) and CA3 (open arrowheads, compare G,I and J L ). Astrocytosis was seen predominantly in the hilus ( K arrows), while myelin degradation was not appreciably different within the hippocampus (compare A C ). BG = Black gold II; FJC/DAPI = Flouro jade C with 4',6 diamidino 2 phenylindole nuclear counterstain; GFAP = Glial fibrillary acidic protein; CD68 = Cluster of Differentiation 68; CV = Cresyl violet. Scale bar is 50m for b,e,h,k; 200m for all other
94 Figure 3 6 Characterization of parahippocampal damage 24 hours post SE Hyperintense regions observed in the parah ippocampal gyrus during in vivo T2 weighted imaging corresponded to A C ) myelin degradation, D F ) neuronal degeneration, G I ) macrophage activation, J L ) astrocytosis, and A,G,J ) cavitation. Asterisks denote cavitation. Compare injured (left column:low mag nification, middle column:high magnification) to uninjured (right column). BG = Black gold II; FJC/DAPI = Flouro jade C with 4',6 diamidino 2 phenylindole nuclear counterstain; GFAP = Glial fibrillary acidic protein; CD68 = Cluster of Differentiation 68; C V = Cresyl violet. Scale bar is 50m for b,e,h,k; 200m for all other images
95 Figure 3 7 Damage to the ventral subiculum was seen in 6/17 rats 24 hours post SE Affected (left column) is compared to unaffected hippocampi (right column). Injury in the v entral subi culum resulted in considerable A B ) myelin degradation, asterisks and C D ) ongoing neuronal degeneration of the pyram idal cell layer, white arrows. E H ) Cresyl violet staining reveals significant cell layer degradation, asterisks. E F ) activate d microglia, black arrows, were also seen in the ventral subiculum while G H ) total activated astrocytes were minimal, black arrows. BG = Black gold II; FJC/DAPI = Flouro jade C with 4',6 diamidino 2 phenylindole nuclear counterstain; GFAP = Glial fibrill ary acidic protein, CD68 = Cluster of Differentiation 68; CV = Cresyl violet. Scale bar is 50m
96 Figure 3 8 Characterization of t halamic injury 24 hours post SE A ) myelin staining did not s how notable fiber degradation. B ) ong oing neuronal degeneratio n and C ) macrophage activation within middle thalamic nuclei. D ) Activated astrocytes, arrows, were not seen in middle thalamic nuclei, but were present in middle habenular nuclei. BG = Black gold II; FJC/DAPI = Flouro jade C with 4',6 diamidino 2 phenylin dole nuclear counterstain; GFAP = Glial fibrillary acidic protein, CD68 = Cluster of Differentiation 68; CV = Cresyl violet. Scale bar is 50m
97 Fig ure 3 9. T2 weighted MR image with corresponding histology of a spontaneously seizing animal 60 days post SE Each row displays histological staining for GFAP, CD68, FJC, and BG (from left to right, see Figures 3 5 to 3 8 for abbreviations). Colored boxes correspond to colored regions outlined on the MR image.
9 8 Figure 3 10. T2 weighted MR image and correspond ing histology of another spontaneously seizing animal 60 days post SE Each row displays histological staining for GFAP, CD68, FJC, and BG (from left to right, see Figures 3 5 to 3 8 for abbreviations). Colored boxes correspond to colored regions outlined on the MR image.
99 CHAPTER 4 CONVECTION ENHANCED DELIVERY OF TH ERAPEUTIC AGENT CARR IERS I ntroduction Chapters 2 and 3 describe CED distributions of Gd albumin in normal and injured hippocampi These studies emphasize the importance of local tissue structure on infusate distribution specifically the presence of dense cell layers, fissures, white matter bundles, and edema However, they likely reveal distributions because albumin is a non binding, low reactivity protein tracer. As mentione d in t he in CED distribution is also introduced by physico chemical properties (size, shape, viscosity, solubility, binding, concentration, rate of efflux, etc.) of the infusate. T he in itial Gd albumin studies described in Chapters 1 and 2 can be used as a guide for predicting the distribution of therapeutic agents with more complex properties such as viral vectors or neural stem cells (NSCs). As described further in th is chapter, the use of viral vectors and NSCs to deliver therapeutic agents are novel strategies that have shown promise experimentally hese systems also exhibit interesting mi gratory properties of which CED studies are lacking. F urther work analyzing the effect of other variables such as cellular interaction and chemotaxis, will allow for more accurate prediction of Excerpts from this chapter are reprinted fr o m Njie eG, Kantorovich S, Ast ary GW, Green C, Zheng T, Semple Rowland SL, Steindler DA, Sarntinoranont M, Streit WJ, Borchelt DR (2012) A Preclinical Assessment of Neural Stem Cells as Delivery Vehicles for Anti Am yloid Therapeutics. PLoS ONE 7 : e34097 doi:10.1371/journal.pone.0034097 This work was completed with the help of Drs. eMalick Njie and Garrett Astary. Dr. Njie provided the NSCs used in this study, helped with surgical procedures, and performed the quantification of transplant dimensions. Dr Astary loaded the infusion pump for the surgeries and performed the image segmentation.
100 distribution Therefore, a s a follow up to initial CED invest igations, pr oof of principle studies using viral and NSC drug carriers were undertaken to elucidate whether these agents share the spatial and temporal properties of small molecule infusate distributions. Gene Therapy for Epilepsy Gene therapy is often p roposed as a solution for correcting or supplementing defective genes responsible for disease development. The generation of viral vectors, which can be engineered to induce specific and stable gene transfer offers an attractive strategy for epilepsy trea tment Thus far, therapeutic strategies have focused on modulating the signaling of neurotransmitters and neuroactive p e ptides that may have anticonvulsant or neuroprotective effects. Raol et al. (2006 ) showed the incidence of developing chronic seizures was significantly lower in pilocarpine treated animals that overexpressed the GABA A receptor subunit Due to reports of reduced numbers of GABAergic neuropeptides in experimental epilepsy ( Gant et al., 2009 ) gene therapy has also been designed to resto re galanin ( McCown, 2006 ; Kanter Schlifke et al., 2007 ; Loscher et al., 2008 ) neuropeptide Y ( McCown, 2006 ; Noe et al., 2007 ; Noe et al., 2008 ) and somatostatin ( Zafar et al., 2012 ) Neuroprotective effects have also been demonstrated with the overexpression of the glucose transporter 1 and the apoptosis inhibitor Bcl 2 ( McLaughlin et al., 2000 ) Experimental evidence s hows gene therapy is a promising utility for epilepsy therapy with several caveats. V ector systems differ in important aspects and the choice of system should be based on a combination of safety and effectiveness of the vector in a particular strategy. Th e adeno associated virus (AAV) vector system has several advantages over other systems such as lentiviruses or retroviruses. Lentiviruses, a
101 subclass of retroviruses 80 100nm in diameter, exhibit insertional mutagenesis. Overall they seem to be less mutage nic than parent retroviruses ( Modlich and Baum, 2009 ) because they generally integrate into gene regions less likely to disturb regulation and expression of h ost genes. Retroviruses integrate in promoter regions and CpG islands that may influence the acitivity of the host promoter or give rise to new full length transcripts ( Wu et al., 2003 ) AAV, on the other hand, exhibits low imm unogenicity similar to lentivirus, but does not integrate into the genome. AAV is a small (20 nm) replication defective, nonpathogenic human parvovirus that requires co infection with a helper virus to replicate ( Mandel et al., 2006 ) It is possible to concentrate and purify AAV vectors to very high titers, resulting in widespread and sta ble transduction with low toxicity. Eleven strains of AAV have been identified, and much effort has been put into modifying the tropism of the vector through capsid manipulation and its specificity through promoter design. AAV serotypes are distinct from e ach other in their capsid regions, allowing binding and entry into different cell types. Tissue tropisms of AAV vectors likely arise due to the cumulative effects of viral binding to multiple cell surface receptors, cellular uptake, intracellular processin g, nuclear delivery of vector genomes, uncoating, and second strand DNA conversion ( Wu et al., 2006 ) AAV2 shows moderate efficiency in the CNS, but AAV1 and 5 have been shown to exhibit higher tranduction frequencies ( Davidson et al., 2000 ; Burger et al., 2004 ) AAV8 and AAV 9 ( Cearley and Wolfe, 2006 ; Klein et al., 2006 ; Klein et al., 2007 ) are also capable of achiev ing high levels of neuronal expression but AAV4 appears to transduce ependyma and astrocytes in th e subventricular zone ( Davidson et al., 2000 ) The differential c ellular interaction s of AAV serotype s introduce another variable to CED
102 distributions. In this study, distributions of AAV1, 5, 8, and 9 were chosen to evaluate after CED due to their increased efficiency and ne uronal tropism. Stem Cell Therapy for Temporal Lobe Epilepsy The need for alternative therapeutic approaches for the evolution of TLE has also resulted in intervention strategies involving the grafting of neural stem cells. Due to its focal nature, TLE is one of the neurological disorders that could benefit from transplantation of cells. This strategy has the potential to curb epileptogenesis, ease chronic seizures, and benefit learning and memory impairments ( Shetty and Hattiangady, 2007 ) Although this field is still in its infancy there have been sundry approaches to repla ce lost neurons or glia, produce more GABA ergic cells, or deliver neuroprotective fact ors via stem cells. Replacement of degenerated neurons in the injured hippocampus via grafting of fetal hippocampal cells has shown promise for restraining epileptogenic changes and controlling seizures ( Shetty et al., 2005 ; Rao et al., 2007 ) Grafting of cells engineered to produce GABA and fetal GABA ergic cells into the epileptic foci have been shown to t ransi ently reduce seizures in a variety of animal models ( Gernert et al., 2002 ; Thompson, 2005 ; Castillo et al., 2006 ) Transplantation of GABA producing cells in the dentate gyrus was associated with increased GABA levels, enhanced local electrical seizure threshold, and delayed onset of behavioral seizures in the kindling model of epilepsy ( Thompson, 2005 ) In the kainic acid model of TLE in rats, grafts containing fetal neural precursors from CA3 were effective in increasing GABAergic function ( Shetty and Turner, 2000 ) and grafts of striatal precursor cells decreased the frequency of spontaneous seizures ( Hattiangady et al., 2008 ) Limited repair of the adult
103 hippocampus is also possible with grafts of neural stem cells with more restricted neural or glial fates ( Shetty et al., 2008 ) In general, stem cell based approaches focus on developing strategies to activate NSCs to produce new neurons, facilitate differentiation into GABA ergic neurons, and to suppress established seizures. However, it would also be interesting to develop methods for testing whether NSCs could prevent chronic epilepsy. Chu et al. (2004 ) transplanted human NSCs intravenously after a pilocarpine injection and found a positive result in the suppression of the formation of spontaneous seizures. Embryonic stem (ES) cell derived neuronal precursors engineered to release adenosine protected against developing generalized seizures in a kindling model of TLE ( Li et al., 2007 ) and suppression of spontaneous seizures in mice ( Li et al., 2008 ) To be successful, grafts of stem cells must not only survive, but also migrate correctly to the appropriate sites to establish appropriate synaptic connections. Spatial dynamics of implanted cells have been described after grafting ( Hoehn et al., 2002 ) but are solely attributed to pronounced migration to wa rds chemotactic signals. This chapter describes the pre migrational mobility of NSCs. Proof of Principle Studies for Viral Vector and Stem Cell CED Delivery Although much progress has been made in developing viral vector and NSC based therapies for epileps y, translation of either of these techniques require s control, or at the very least, understanding of their interstitial mobility so risks of aberrant migration could be weighed against potential benefits. O ne of the major hurdles in development today is b eing able to control dispersion of these carriers The effect of the initial distribution of both AAV and NSCs has often been overlooked because these other factors (migration, cellular uptake) are assumed to play a larger role in the final
104 distribution. H owever, enhancing initial distribution with CED can affect final outcome. This has been shown through increase of viral spread and transduction efficiency in the striatum ( Bankiewicz et al., 2000 ) CED delivery of AAV has not been studied in the hippocampus; and transduction efficiency varies markedly from one region to another ( McCown et al., 1996 ) No CED studies of NSC delivery exist; in fact, NSC distribution outside of migration has not been studied. Therefore, this chapter presen ts preliminary data to 1) illustrate the utility of CED in delivering NSCs and viral vectors in the hippocampus, 2) reveal the effect of initial CED distribution on NSC spread, and 3) describe the effect of vector tropism and size on hippocampal CED distri bution. Methods Animals NSC e xperiments were performed on 8 14 month C67/ B6 non transgenic mice. Both host mice and NSC donor mice were congenic on the C57BL/6J background, eliminating p roblems with graft rejection. AAV experiments were performed on male S prague Dawley rats weighing 225 250 g. Vector Construction Standard cloning techniques were used to construct recombinant AAV based plasmids. GFP was subcloned in an expression cassette that had the cytomegalovirus immediate early enhancer and the chicken beta actin promoter (construct pTRUF11, UF vector core) This was subcloned at the UF vector core into the rAAV backbone flanked by rAAV2 inverted terminal repeats and pseudotyped with serotype 1, 5, 8, and 9 capsid proteins (titers of 1.68 x 10 13 vg/mL, 9 .02 x 10 12 vg/mL, 6.47 x 10 12 vg/mL, 6.56 x 10 12 vg/mL, respectively).
105 Lentivirus plasmids containing CaMKII Channelrhodopsin 2/GFP were obtained from Karl Deisseroth (Stanford University) and packaged by Dr. Phil Barish. Transduction and I solation of NSC s GFP expressing NSCs were provided by eMalick Njie. The NSCs were isolated by removing the subependymal zone of neonatal B6 mice, processing with trypsin/ethylenediaminetetraacetic acid, and dissociating into a single cell suspension. Cells were maintaine d in culture flasks and then transduced with self inactivating Lentiviruses containing plankton copepod green fluorescent protein ( courtesy of Dr. Sue Semple Rowland) Cells were then enriched with fluorescence activated cell sorting (FACS). Prior to tran splantation, monolayers were detached from flasks washed, and diluted to 5 x 10 4 cells/ L Concentration was determined with two reference cell counts on a hemacytometer. Surgical Procedures and CED Infusions for NSC Experiments Mice were deeply anestheti zed with 1 5% isoflurane and then securely mounted with ear bars and a nose bar to a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). The anesthesia mixture was delivered through an inlet within the nose bar enclosure for the duration of the surgery. The top of the head was shaved and sterilized with alternating swipes of betadine antiseptic and 70% ethanol. A sterile scalpel was used to make a small incision into the skin above the skull. The skin was reflected in order to expose Bregma and a burr ho le s were drilled to allow cells to be injected bilaterally to the thalamus (n=7; [ 2.0mm AP, +/ 1.5mm ML, 4mm DV ] ) striatum (n=3; [ +1.18mm AP, +/ 1.5mm ML, 4mm DV ] ) and three depths within the hippocamp us [ 2.00mm AP, +/ 2.0mm ML, 1.5mm DV, 2.1 mm DV, and 2.8mm DV ] Hippocampal DV coordinates correspond to the superior aspect (n=5) dorsoventral center (n=5) and
106 inferior aspect (n=7) of the sep tal hippocampus, respectively. Approximately 5 x 10 5 cells (9.8 L) were infused into each region. Pe r experiment, 4 6 confluent T75 flasks were harvested a nd concentrated at 5 x 10 4 cells/L in order to reliably have enough volume for transplantation and for void volume in the infusion system. In general, the infusion parameters used here are established protocols in other studies ( Park et al., 2006 ; Yamasaki et al., 2007 ; Tang et al., 2008 ) T he cell numbers and volumes used here are similar to intracranial surgeries by Park et al. (2006 ) in which mouse brains wer e infused with 3 x 10 5 NSCs in 8 L. All infusions were delivered via CED at 1 L/min using the same infusion system mentioned in Chapters 2 and 3 Following injection, the needle was left in place for 3 minutes to minimize backflow before being slowly wit hdrawn. Surgical Procedures and CED Infusions for Viral Vector Experiments Surgical procedures to expose bregma and lambda on the skull were performed as described in Chapters 2 and 3. Once exposed, burr holes were drilled over the l eft hippocampus for th e injection of viral vectors (n=4; at [ 3.80 mm AP, 2.50 mm ML, 3.10 DV] ) Injections were performed at 0.3 L/min with a 10 mL Hamilton syringe (33 gauge, point style 4) attached to an infusion pump. The needle was kept in place for 5 additional minutes after cessation of injection to allow for distribution of the vector before retraction. Volumes of injection were adjusted per serotype (2.0 5.19 L) to equalize the number of vector genomes (vg) delivered to be 3.36 x 10 10 vg Perfusion and Immunochemis try Tissue harvesting was performed within 15 minutes of NSC injection to visualize immediate pre migrational distribution. To harvest tissues, mice were deeply anesthetized and euthanized by isoflurane overdose followed by immediate
107 exsanguination and per fusion with cold 1x PBS. Whole brains were quickly dissected out and submerged in cold 4% paraformaldehyde fixative overnight. Fixed brains were then cryoprotected in 30% sucrose and section ed at 20 m inte rvals with a cryostat. Sections were stored in ant i freeze media at 20C until staining with 4',6 diamidino 2 phenylindole (DAPI, Invitrogen, Carlsbad, CA). Immunostained cells and tissue sections were photographed with an Olympus DP71 camera mounted on an Olympus BX60 microscope. Tissues from viral vect or injected animals were harvested one month post infusion. Perfusion, brain extraction, and tissue sectioning was performed as d escribed in Chapter 3. No staining was performed on these sections. Quantification of NSC Transplant Dimensions NSC transplant dimensions were quantified using a method adapted from M azel et al. (1998 ) An index of anisotropy, desi gnated A, was determined by dividing the distance of engraftment (x) along the transverse (i.e. mediolateral) axis by the distance of engraftment (y) along the vertical (i.e. dorsoventral) axis With this m ethod, spherical engraftments would result in A values close to 1, while anisotropical engraftments would result in larger or smaller values of A For each infusion, one to two 20 m sections containing engraftments were analyzed with AxioVision LE softwar e (Zeiss, Jena, Germany). Images of samples with equal pixel/micrometer value were used to determine the distance of x and y. An unpaired, two test using Microsoft Excel was used to compare A from different sites of infusion. A p value o f <0.05 was conside red statistically significant.
108 Image Segmentation and 3D Reconstruction of NSC Engraftments A semi automatic image segmentation routine was implemented in MATLAB (The MathWorks, Inc., Natick, M A USA) that distinguished regions of GFP ex pression from control regions of the brain by means of a threshold unique to each histological section. RGB images of the histological sections were imported into MATLAB ; however, only the green channel of the images was used for image segmentation. The a verage background signal was determined by averaging the signal intensity in GFP free regions of interest for each section. Similarly, the standard deviation of noise was determined by evaluating the noise behavior in tissue free regions in images. Thresho lds were calculated by adding six times the standard deviation of noise to the control signal intensity for each section. The image segmentations were refined in ITK SNAP ( Yushkevich et al., 2006 ) an open source medical image segmentation tool. Refinements included removing regions of auto fluorescence (false positives) from the image segmentation as well as segmentation of annotations such as scale bars. Th ree dimensional reconstructions were generated by first aligning the histological sections prior to image segmentation and finally generating a mesh from the 3D image segmentation using ITK Results Distribution of Vir al Vectors in the Hippocampus Four neuronal serotypes of AAV GFP and a lentivirus vector were infused into the dorsal hippocampus (Figure 4 2 4 3 ) A summary of these results can be found in Table 4 1. AAV1 GFP (Figure 4 2A 4 3 ) transduced cells in the d entate granule cell layer, hilus, CA1, CA2, and medial CA3 throughout approximately 2800 m of the hippocampal septo temporal axis A portion of cells in Layer VI of the overlying cortex
109 were also transduced and projections in the lateral posterior thalami c nucleus were visible throughout ~800 m in the anterior posterior direction Contralateral projections of transduced cells were visible in all subregions of the hippocampus proper. AAV5 GFP (Figure 4 2B 4 3 ) transduced cells were visible mostly in the CA2, with less transduction in the CA3 and CA1 through approximately 2400 m of the septo temporal axis H ilar cells were also transduce d but almost no granule cells expressed GFP. Interestingly, contralateral projections of tran s duced cells were visible in all subregions of the hippocampus proper and in the inner molecular layer of the dentate gyrus, the target of hilar mossy cells. AAV8 GFP (Figure 4 2C 4 3 ) transduced cells in the CA3 CA3, and CA1, with minimal transduction in the hilus and dentate gr anule cells Transduction was visible through approximatel 2400 m along the septo temporal axis. Commissural projections were vis ualized in the contralateral CA1 and CA2. AAV9 GFP (Figure 4 2D 4 3 ) transduced cells mainly in the CA1, CA2, and Layer V1 o f the overlying cortex. CA3 cells expressed GFP in the septal hippocampus, but GFP in CA3 was not visible ~4.5 mm behind bregma through the temporal hippocampus Projections from transduced c ells could be visualized in the contralateral CA1, CA2, and in la teral posterior thalamic nuclei for ~1000 m in the anterior posterior direction AAV9 GFP expression was noticeably the most widespread and exhibited the highest intensity of GFP expression. The lentiv ector (Figure 4 2E) transduced pyramidal cells in the CA1 and granule cells of the dentate gyrus While AAV transduction occurred throughout the septo
110 temporal axis, GFP expression from lentivirus was only visualized along 600 m of the septo temporal axis. D istribution of T ransplanted NSCs in the Hippocampus NSCs were bilaterally infused into the hippocampus and host mice sacrificed within fifteen minutes of surgery to visualize pre migrational distribution via CED Across various depths of infusion into the hippocampus, well defined ellipsoid distributions o f GFP fluorescence we re consistently observed in the transverse plane ( Figure 4 4 A B, E F). GFP fluorescence was found distributed largely in three structures: the corpus callosum, the velum interpositum and the hippocampal fissure. Specifically, NSCs that were infused into the superior aspect of the septal hippocampus yielded GFP fluorescence distributions throughout the corpus callosum ( Figure 4 4 A; n=5). C ells that initially colonized white matter tracts moved laterally within this structure but did not migrate into adjacent gray matter ( Figure 4 4 A ). NSCs targeted to the inferior aspect of the septal hippocampus produced GFP fluorescence distributed mediolaterally along the velum interpositum ( Figure 4 4 B; n=7). The velum interpositum is a soft tissue pa rtition between the diencephalon and the telencephalon rather than a cavity or space ( Tubbs et al., 2008 ) Functionally, the velum interpositum forms the roof of the 3rd ventricle and connects the choroid plexus of this ventricle to that of the lateral ventricle. The distribution of GFP fluorescence in this region extended hundreds of micrometers from the 3rd ventricle to above the dor sal lateral geniculate nucleus. NSCs targeted to the dorso ventral center commonly distributed in the hippocampal fissure, corpus callosum, and velum inter positum (Figure 4 4 E). In some instances, GFP fluorescence was not only at the position the cells were targeted, but also in areas hu ndreds of micrometers away (Figure 4 4 E F).
111 Distribution of Transplanted NSCs in the Thalamus and Striatum NSCs were also i nfused into the thalamus and striatum to determine whether engraftment patterns in the hippocampus are unique. NSCs that were infused into the thalamus ( Figure 4 4 C; n=7) and striatum ( Figure 4 4 D; n=3) produced spherical distributions of GFP fluorescence that were indicative of isotropic transport. 3D reconstruction of serial sections of a representative corp us callosum infusion illustrates the sheet like spread of GFP fluorescence across the wide, flat bundle of corpus colossal fibers ( Figure 4 5 A). In co ntrast, thalamic infusion reconstruction shows a globular spread of GFP fluorescence ( Figure 4 5 B). These patterns of distribution were consistent with the cells having been distributed along structure specific pathways at the time of injection ( Figure 4 5 C). Discussion Viral Vector Distribution Comparisons of five viral vector infusions into the septal rat hippocampus revealed primarily neuronal expression patterns that differed across serotype and vector I ntensity of GFP expression was higher with AAV 1 and 9 than AAV 5 or 8. AAV9 resulted in the widespread expression in CA1 and CA2, but AAV1 and 8 resulted in better expression in CA3. AAV 1 and 9 resulted in GFP fluorescence in the thalamus, though AAV9 showed the most widespread expression Viral genomes were equalized across all AAV serotypes by varying infusion volume, which cannot be discounted as an influence on distribution. However, the largest volume infused was of AAV 8, which resulted in the second to worst transduction efficiency. Alternatively, the second to least volume infused was of AAV1, which resulted in the next best transduction efficiency. Another study in which viral genomes are equal across serotypes is needed to
112 determine the effect of volume on AAV distribution. Despite this limitatio n, these results are similar to that of other comparative studies. Previous comparisons have shown that transgene expression was greater with AAV1 and AAV8 than AAV5, but no difference between AAV 1 or 8 ( Klein et al., 2006 ) Another study demonstrated that AAV1 and AAV5 are of simila r efficiency ( Burger et al., 2004 ) A study examining transgene expression after systemic injection in mice revealed that AAV9 > AAV1, 8 > AAV5 ( Zincarelli et al., 2008 ) It is important to note results pertaining to serotype tropism should be interpreted cautiously due to inter study variations in vector titers, doses, and promoters. T he preliminary stud ies described in this chapter are distributions from one animal per vector and do not represent quantifiable comparisons They do, however, provide a practical illustration of the targeting strategies presented by viral vector choice. On one hand, AAV infu sions result in widespread and efficient gene expression that can be modulated by serotype. The disadvantage of AAV is the small (4 5 kb) cloning capacity of the vector. This constrains the choice of genetic regulatory elements, which allow for modulation of gene expression. Alternatively l entiviruses do not have signi ficant limitations on gene size With a cloning capacity of up to 8kb, l entiv ectors have the ability to indu ce long term stable expression and low immunogenicity ( McGrew et al., 2004 ) However, lentivectors can range from 80 100 nm in size, while AAV particles are approximately 25 nm. In contrast to AAV vectors, the lentivirus infusion resulted in a very focal pattern of expression of <1 mm which may be due to larger particle sizes relative to AAV
113 As mentioned in Chapters 2 and 3, infusion site presents a considerable influence on distribution. Infusion site could be visualized through a higher de nsity and intensity of GFP expression. Infusion with AAV5 was approximately 1 mm lateral as compared to the other infusions a potential explanation for the difference in transduction for this animal. The widespread expression from AAV9 in CA1 may be due t o a more dorsal infusion site as compared to the other serotypes. As described in Chapter 1, p referential distribution in the septal hippocampus i s dependent upon location of the cannula tip along the dorsoventral axis Further studies with more animals ar e needed to distinguish this influence from serotype cellular interactions. NSC Distribution To characterize NSC distribution patterns, transplants were analyzed immediately after i nfusion into areas of diverse anisotropic properties. The main finding of t his study revealed areas of low fluid resistance (namely corpus callosum, hippocampal fissure, velum interpositum) were a prevailing influence on the distribution of NSCs. Anisotropic distribution patterns were most prominent in the hippocampus, but absent in other regions such as the thalamus and striatum. Interestingly, the e ctopic engraftment pattern of NS C s is long term, as demonstrated by the fact that NSC s engrafted in the corpus callosum under identical conditions do not redistribute into gray matter after a month ( Njie et al., 2012 ) In fact, NSCs deposited into various hippocampal and cortical sites paradoxically distribute mainly in the corpus callosum and to a lesser extent, the hippocampal fissure ( Olstorn et al., 2007 ; Blurton Jones et al., 2009 ; Radojevic and Kapfhammer, 2009 ) Similar distribution characteristics have been reported in epilepsy models as well. Hattiangady et al. (2008 ) grafted striatal precursor cells 4 days post kainate induced SE and analyzed 9 12 months later. The grafts were targeted to CA3 of
114 the septal hippocampus, but were later found to be in the ve lum interpositum as well as the targeted region. Engraftment patterns in short and long term studies may be an artifa ct of injection parameters, such as the rate, volume of cells, or infusion site. This is unlikely, due to the range of parameters used in various studies. Furthermore, infusion site was varied in this study to include three different depths within the hipp ocampus. All three depths resulted in NSC spread to areas of least resistance. Alternative explanations for the ectopic engraftments in the long term may be due to cell viability. It is possible that NSCs distributed throughout the hippocampus, but only th ose within low resistance spaces remained viable at the time of observation. Regardless, t hese results raise fundamental questions regarding how engrafted NSCs are ultimately distributed after transplantation. Cell migration is clearly one determinant of g raft distribution. Indeed subventricular zone NSCs transplanted into the lateral ventricle follow migrational cues in the rostral migrational stream ( Zheng et al. 2006 ) In this study, NSC infusions showed neuroarchitecture may play an underappreciated role in distribution of engraftments. H ost mice harvested only minutes after surgery had remarkably similar distributions of NSCs in paths of least resistance to reports of long term distributions T his pattern of distribution is similar to anisotropic distributions observed in the work described in Chapters 2, 3, and previously with other small molecule infusions ( Vorisek and Sykova, 1997 ; Mazel et al., 1998 ; Astary et al., 2010 ) although to an exaggerated extent du e to the greater size constraints for NSCs Conclu s ions Gene and stem cell transfer have the capability to induce expression of neuroprotective compounds, anticonvulsant agents, or supply supportive replacement
115 cells for temporal lobe epilepsy. A co mbination therapy of gene and stem cell therapy may even be potential strategy in which GABA ergic cell transplants supplement viral vector delivery of anti convulsants. Unfortunately, there are still many hurdles for stem cell treatments for intractable e pilepsy. Cells must first be capable of exhibiting enduring survival and maintaining neurotransmitter release on a long term basis in the epileptic brain ( Zaman et al., 2000 ) Second, fetal precursor cells have not been shown generate specific types of neurons or glial cells needed to replace damaged cell types. Embryonic stem cells can be used to generate neural or glial precursors, but risk of tumor formation is high because they are pluripotent and mitotically active. As s tem cell therapies grow in popularity, these challenges are among the studies being undertaken. Chapter 4 describes proof of concept applications for CED in epilepsy using carrier vehicles comm only proposed as novel therapeutic strategies. CED delivery of these carriers resulted in distributions along paths of least resistance and exhibited a dependence on infusion site, akin to infusions of Gd albumin. Distributions of NSCs particularl y, were influenced by hippocampal specific anatomical constraints that resulted in previously uncharacterized anisotropic transport These results reiterate the significance of biostructural factors in targeting strategies of delivery approaches in addition to th e influence of serotype and chemotaxis
116 Table 4 1. Features of gene expression vectors Viruses Packaging capacity Inflammatory Response Advantage Disadvantage Retrovirus 8 kb Low Large cloning capacity; stable transgene expression Does not infect non divi ding cells. Insertional mutagenesis Lentivirus 8 10 kb Low Infects diving and non diving cells with 30% efficiency, large cloning capacity Insertional mutagenesis Adenovirus 8 kb High Infects all cell types with 100% efficiency ; does not integra te with host High inflammatory response AAV 4.7 kb Very Low Infects all cell types ; non pathogenic does not integrate with host Small packaging capacity Table 4 2. Results of hippocampal viral vector infusions Viral vector Volume Infused Length o f A/P Transduction Length of M/L Transduction Hippocampal subregions tranduced AAV1 3.73 L 2800 m 2739 m CA1 CA3, some DGC and hilar AAV5 2.00 L 2400 m 1557 m CA1 CA3, some hilar AAV8 5.19 L 2400 m 3789 m CA1 CA3, some hilar AAV9 5 .09 L 3200 m 4036 m CA1 CA3 Lentivirus 3.6 L 600 m 900 m CA1, DGC
117 Figure 4 2. Infusions of viral vectors into the left rat septal hippocampus. A) AAV1 GFP, B) AAV5 GFP, C) AAV8 GFP, D) AAV9 GFP E) Lentivirus CaMKII ChR2/GFP Right side shows contralateral projections of tran s duc ed cells.
118 Figure 4 3. Infusions of four AAV serotypes exhibit specific distribution pat terns throughout the hippocampal septo temporal axis Each column shows the GFP expression per AAV serotype at specific coro nal sections along the septo temporal plane. Each row depicts a coronal section posterior to the first row by the distance indicated on the y axis
119 Figure 4 4 Short term engraftments of NSCs expressing GFP demonstrate the hippocampus specifically featur es anisotropic transport. NSCs infused into the septal hippocampus distributed anisotropically along A) the corpus callosum ( CC ) when deposited in the superior aspect of the septal hippocampus and B) the velum interpositum (VI) when deposited in the infer ior aspect of the hippocampus. In contrast, engraftments into C) the thalamus (THAL) and D) the striatum resulted in isotropic distributions that did not spread preferentially along the transverse plane. E) When injected into the dorsoventral center of the hippocampus, 38.5% of infusions resulted in target such as the hippocampal fissure (HF), the CC, and the VI. F) Backflow in thalamic infusions resulted in anisotropic transport along the VI.
120 Figure 4 5 Geometric analyses of NSC infusions. A B) 3 dimensional reconstructions of serial histological sections shows engraftments form A) a sheet like spread in the corpus callosum, and B) a globular spread in the thalamus. C) Anisotropy (A) was quan tified by dividing the spread along the transverse plane (x) by the spread along the vertical plane (y). Compared to the thalamus, A is 7.3 times larger in the corpus callosum, 3.7 times larger in the hippocampal fissure, and 6.8 times la rger in the velum interpositum (**, p<0.01, comparison to the thalamus).
121 CHAPTER 5 CONCLUSIONS AND FUTU RE WORK Conclusions TLE is a devastating disease that affects people of all ages, races, and socioeconomic backgrounds. Even with an advanced understanding of underlying mechanisms the current treatment approach has not changed in decade s. Systemic administration of AEDs is the main approach to treating s eizures, even though one third of patients are resistant to curren t pharmacotherapies. Furthermore, treatment occurs on ly at chronic time points; there are currently no drugs available that prevent the development of epilepsy after an initial insult, such as SE Thus, the National Institute of Health has listed several important benchmarks for epilepsy research including 1) identifying approaches to prevent epilepsy or its progression, 2) developing and optimizing new strategies for targeted therapies and 3 ) developing animal models for the progression of epilepsy The work presented herein addresses these NIH benchmarks through the development of CED for targeted, prophylactic treatment of TLE Identifying Approaches to Prevent Epilepsy or Its Progression CED provides a targeting strategy applicable at any stage of epilepsy progression that is amenable to delivery of var ious therapeutic agents -far more than systemic administration is capable of transmitting to the brain. As discussed in Chapter 1, CED results in widespread distributions that are capable of reaching clinically relevant volumes without the risk of systemic side effects. In this work, CED was applied to the infusion of NSCs and several viral vectors into the rat hippocampus as a proof of principle approach to delivering therapeutic compounds at a local level (Chapter 4) We
122 found NSCs distributed according t o previously uncharacterized anisotropic conduits (corpus callosum, hippocampal fissure, velum interpositum), which had a significant impact on the final residence of the transplanted cells. Moreover, t he distribution of NSCs shared spatio temporal similar ities with small molecule distributions (Chapter 2) validating the use of surrogate markers to enable real time monitoring and prediction of the distributed agents Because NSCs are many orders of magnitude larger than most tracers, we suspect size was a major factor in this transport pattern This was also observed when viral vectors were test ed. Lentivirus (80 100 nm) expression in the hippocampus was markedly reduced as compared AAV (25 nm) expression Within CED distributions of different AAV serotypes AAV9 was found to have the most widespread transduction efficiency, followed by AAV1 and 8, and lastly, AAV 5. The variability in transduction distribution was likely due to both capsid interaction and infusion site. N o adverse effects were observed from the infusion of NSCs or viral vectors in animal behavior as assessed by daily handling, or cellular structure as analyzed through DAPI staining CED of these therapeutic carriers is a possible approach for the direct application of anti epileptic drugs, viral mediated gene transfer of inhibitory pe ptides, or cell based therapies A combination therapy of gene and stem cell therapy may be another potential strategy in which GABA ergic cell transplants supplement viral vector delivery of anti convulsants. Developing and Optimizing New Strategies for Targeted Therapies Apart from molecular and enzymatic targeting strategies, physical targeting strategies are commonly overlooked and underappreciated in drug delivery Unfortunately, the failure of several CED clinical trials ( Sampson et al., 2007a ; Sampson et al., 2010 ) has prompted researchers to look more clos ely at the influence of
123 neurostructure on infusate distribution. Properties of CED have been well established in homogenous gray and white matter, but only one study ( Heiss et al., 2005 ) has examined CED in the hippocampus, a structure commonly affected in TLE. Therefore, to develop and optimize CED targeting for epilepsy treatment, infusions of Gd albumin were performed in normal (Chapter 2) and injured (Chapter 3) rat hippocampi at two time points We found hippocampal distributions to be influenced by the site and size of the target structure, density of cellular elements, presence and orientation of pia l surfaces, and potentially, orientation of local axonal pr ojections. We also found that distribution volumes varied with the extent of brain injury with diffuse limbic system edema increasing the distribution volumes up to twice the volume measured in normal animals. Differences in distributions of injured ani ma ls are likely due to decrease s in the size of extracellular channels, potentially resulting from ongoing neuronal degeneration and accumulation of neuroinflammatory markers in the interstitium. These changes, in addition to swelling of cellular elements an d glia, affect diffusion parameters within the hippocampus and influence the spread of infusate. This is supported by the fact that the volume of infusate distributions 60 days post SE returned to control ranges, accordant with the abatement of these marke rs The effect of varying the infusion site and integrity of structure on distribution volume supports the hypothesis that anisotropic hippocampal neuroarchitecture plays a prominent role in the distribution of infusate These results not only demonstrate a strategy for targeting and tracking therapies to the hippocampus, but they also provi de guidance for the planning of infusion therapy in heterogenous tissue.
124 Developing Animal Models for the Progression of Epilepsy The idea of preventative treatment for TLE was initiated in an article by Dr. Murray Falconer ( Falconer, 1974 ) Dr. Falconer, and man y others since, believe s the latent period may offer a therapeutic window for the prevention of epileptogenesis. Experimental evidence suggests there is a cascade of morphologic and biological changes after an initial insult that are potential targets for the administration of neuroprotective or anti ictal substances. SE model s are ideal for studying the progression of epilepsy because they exhibit a latent period similar to the human condition Preventative studies are currently performed on kindling, pilo carpine, or kainate models. However, these models are poor approximations of structural rearrangements that occur in humans. Kindling models produce little to no damage, while chemical models result in sizable injury with a high mortality rate. Moreover, c hemical models act on sp ecific receptors, which present a confound to pharmacological research and drug design. In the electrical SE model presented here, we investigated the neuropathological changes occurring within 24 hours of SE (Chapter 3). This mode l is often considered to have the greatest parallels to human TLE, and thus was developed as a testing paradigm for CED infusions. At 24 hours post SE d iffuse limbic injury was observed in the hippocampus (specifically CA3 and CA1), amygdala, piriform cor tex, entorhinal cortex, middle thalamic and lateral thalamic nuclei, and lateral septum. Additionally, previous work ( Parekh et al., 2010 ) has determined the pr esence of parahippocamp al edema in this model is predictive of the development of spontaneous seizures. These early structural changes present a target for prophylactic treatment that resolve by the time spontaneous seizures begin, when animals exhibit var ying amounts of neuronal
125 loss and gliosis, similar to human hippocampal injury. We found the extent of injury was a significant variable for infusate distribution volumes. This finding underscores the importance of using appropriate models for drug deliver y. Future Work This dissertation discusses CED for the infusion of therapeutic agents for epilepsy treatment. Future work will focus on three extensions of these studies Firstly, a larger CED stud y will be undertaken at chronic time points. Since there ar e millions of people in the world suffering from chronic epilepsy today, i t is important to quantify distributions using more animals at this treatment time point Preliminary data from two animals suggests distribution volumes at 60 days post SE are not s ignificantly different from controls implying that control Gd albumin distributions can reliably predict infusion patterns in chronic brain injury We suspected ongoing neurodegeneration, cell loss, and Wallerian degeneration w ould create pooling within t he enlarged ECS and decrease infusate volumes in the brain Preliminary studies do not seem to support this hypothesis, but more animals are needed to make that conclusion. Second ly one important dimension not measured in the studies described is the dist ribution of concentration. Quantifying v olume distributions is particularly relevant for infusions of toxic or lytic agents that should be controlled carefully. It is also important to know which structures are being targeted. However, the increase or decr ease of distribution volumes in the brain implies differences in concentration across animals. Acquiring concentration measurements with MR are a future goal of these studies. Concentration can currently be determined with autoradiography studies post mort em, but q uantifying concentration maps using MR in a ddition to volumetric analyses will allow for true co registration of infusate concentration and spatial spread in vivo
126 Finally a third, and natural extension of these st udies is to use CED to deliver therapeutic agent s Work is currently being done to deliver light activated protein channels into the hippocampus to evoke cell type specific activity with light. These proteins can be virally transduced into any cell type with gene therapy, and then used to modulate cell firing. This approach can be used to explore the causal function of individual neuron types in epileptic circuitry and characterize the underlying cellular dysfunction in the hippocampus. Future studies also include using CED to deliver n anoparticles encapsulating or conjugating potential drugs. Depending on the ir molecular weight and stability nanoparticles can release drugs over h ours and days to several months ( Yasukawa et al., 2005 ) In general, nanoparticles are considered optimal for drug delivery to the brain as their mean size allows traveling through physical restrictions presented by the brain interstitial space. Nanoparticle encapsu lation of the compound MRZ 2/576 resulted in an increased duratio n of its antiepileptic activity ( Friese et al., 2000 ) E fforts have also been made to prepare an d optimize nano sized carrier systems for phenytoin ( Thakur and Gupta, 2006 ) carbamazepine ( Douroumis and Fahr, 2007 ) clonaze pam ( Jeong et al., 1998 ; Ryu et al., 2000 ) d iazepam ( Abdelbary and Fahmy, 2009 ) and valproic acid ( Darius et al., 2000 ) Although experimental progress has been made in development of nano carriers for AED release, pharmacokinetic data on the use of nanoparticles to deliver these dru gs are very limited Future work will focus on using CED to deliver and study in vivo pharmacodynamics.
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150 BIOGRAPHICAL SKETCH Svetlana Kantorovich was born in Vinnitsa, U kraine where she lived with her parents, twin sister, and older brother before moving to St. Louis, MO at the age of four. After completing high school in St. Louis, she attended Washington University in St. Louis to pursue her undergraduate degree. She gr aduated with honors in 2007 with a Bachelor of Arts in Biology and a minor in psychology. In the fall of 2007, Svetlana began graduate school in the Interdisciplinary Program in Biomedical Sciences (IDP) at the University of Florida with a concentration in Neuroscience. She joined the laboratory of Dr. Paul R. Carney in 2008 where she began characterizing the effect of normal and pathological tissue structure on infusate distribution profiles following convection enhanced delivery for epilepsy Her work cul minated in a Ph.D. from the University of Florida in the spring of 2012. Following graduation, Svetlana plans to widen her kn owledge and skill sets through the continuation of training in the field of biomedical sciences.