Citation
The Protective Effects of Plasma Gelsolin and Alpha 1 -Antitrypsin on Ischemic Stroke Outcome in Rats

Material Information

Title:
The Protective Effects of Plasma Gelsolin and Alpha 1 -Antitrypsin on Ischemic Stroke Outcome in Rats
Creator:
Moldthan, Huong Le
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmaceutical Sciences
Pharmaceutics
Committee Chair:
Song, Sihong
Committee Members:
Hughes, Jeffrey
Hochhaus, Guenther
King, Michael A
Batich, Christopher D
Graduation Date:
5/5/2012

Subjects

Subjects / Keywords:
Actins ( jstor )
Animal models ( jstor )
Blood flow ( jstor )
Brain damage ( jstor )
Brain ischemia ( jstor )
Inflammation ( jstor )
Ischemia ( jstor )
Plasmas ( jstor )
Rats ( jstor )
Strokes ( jstor )
1-antitrypsin
alpha
anti-cell
anti-inflammation
death
endothelin-1-induced
gelsolin
ischemic
mcao
pathways
plasma
stroke
Genre:
Unknown ( sobekcm )

Notes

General Note:
Plasma gelsolin and alpha 1-antitrypsin, used in this study, are human serum proteins with multiple functions, including anti-cell death pathways and anti-inflammatory effects that can be utilized to develop ischemic stroke therapy. The effects of the two proteins were tested on middle cerebral artery occlusion (MCAO) induced by the potent vasoconstrictor peptide endothelin-1 (ET-1). In this model, ET-1 is injected adjacent to left middle cerebral artery (MCA) in rats. To test the effects of the candidate therapeutic agents in the MCAO model, rats were post-treated by a discrete injection of pGSN or AAT at the same site of ET-1 injection or by an intravenous injection of AAT at the same time of ET-1 injection. Artificial cerebrospinal fluid (aCSF) and sterile saline were utilized as negative controls. The MCAO was verified using a laser Doppler perfusion imaging system in separate groups of animals. Cylinder and vibrissae tests were performed before and 72 hours after MCAO to assess motor/sensorimotor functional deficits. Infarct volumes were determined via infarct area calculation 72 hours after MCAO using magnetic resonance imaging (MRI) and 2,3,5-Triphenyltetrazolium chloride (TTC) assay. Results from these experiments showed: 1) pGSN or AAT (both local and systemic deliveries) treatment did not interrupt ET-1 induced MCAO as a disease model; 2) pGSN or AAT treatments significantly mitigated ET-1 induced functional deficits assessed by both cylinder and vibrissae tests; 3) pGSN local delivery, AAT local delivery or AAT systemic delivery markedly reduced brain injury (infarct volumes) by 49%, 83%, and 63%, respectively, compared to controls. Taken together, these findings show that plasma gelsolin and alpha 1-antitrypsin are potential novel therapeutic drugs for the protection against neurodegeneration following ischemic stroke.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright by Huong Thu Le. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
5/31/2014

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1 THE PROTECTIVE EFFEC TS OF PLASMA GELSOLIN AND ALPHA 1 ANTITRYPSIN ON ISCHEMIC STROKE OUTCOME IN RATS By HUONG LE MOLDTHAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Huong Le Moldthan

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3 To my beloved grandmother, parents and husband, Matthew Michael Moldthan

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4 ACKNOWLEDGMENTS Obtaining my doctorate has been the most challenging goal of my life so far. Yet, starting is the best decision that I will never regret. During the journey to this dissertation, both figurative as well as literal, I am deeply indebted to the many people who have provided me inspiration and supported my studies. My greatest gratitude goes to Dr. Jeffrey Hughes, my former mentor who gave me an opportunity to do research in his lab and for his excellent supervision, as well as his great sense of humor. I would like to gratefully thank Dr. Sihong Song for continuing to guide me in a new project when Dr. Hughes left. I also would li ke to express my thanks to Dr. Michael King and other committee members, Dr. Christopher Batich and Dr. Guenther Hochhaus for their detailed comments and great suggestions. Without their guidance, I would not have had such a great experience in completing this challenging project. I would like to express my special thanks to Dr. Aaron Hirko, for his constant help, patience and invaluable advice. A sincere thank you goes to Jeffrey Thinschmidt, who was always willing to help me with handling animals perform ing laser Doppler imaging experiments provi di ng great comment s on dissertation as well as my talks and contributing to a very happy lab environment. I also would like to thank Matthew Isaacson for analyzing the data. I truly acknowledge the Department of Pharmaceutics for all of their administrative work. I also would like to thank the Department of P harmacology and Therapeutics, the McKnight Brain Institute and Department of Veterans Affairs where I performed most of the animal work and whose facilities I used. I would like to send my many thanks to my professors, teachers, supervisors, lab mates and friends at Utrecht University in the Netherlands and to those whom I was fortunate enough to meet during my studies Coincidently, I met Dr. Wouter Driessen when I came to the University

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5 of Florida. He was a former graduate student at Utrecht University. Even as he was busy finishing up his doctorate, he was willing to teach, work, and gi ve great advice for my project. I would like to express my sincere thanks for his invaluable support intelligence and enthusiasm. I am truly grateful for wonderful old friends in Vietnam. I greatly appreciate your kindness and friendship via the internet or personally, w hen I went home. Although I am not able to name each of you here, I will never forget any of you! Lastly, I would like to express my deepest thanks for my family members. Most important among them are my parents, who love me unconditionally, support me by all means, and trust me implicitly. They are willing to do anything I need or even what they think is good for me. I would like to express my eternal gratitude to my siblings. My sister, Ha Thu Le, is not only my best friend but also my soul mate. She shares my lifes point of view and constantly inspires me to reach my goals, especially during my time in the U.S., which is made difficult by the nearly ten thousand miles separating me from my home town and my family. My two brothers, Hoc Van Le and Hien Van Le always wish me the best of luck and deeply sympathize with me simply because I am living o n the other side of the world. I would not have easily accomplished this important goal in my life without the constant support, l ove inspiration and patient proof reading, of my husband, Matthew Michael Moldthan I would like to thank my parents in law, Maxine and Rollan Ross who are constant encourage me to finish this dissertation. I would like to thank my grandmother for her most loving assistance, as well as insistence on my attending graduate school. She would cook, take care of my apartment and do anything I needed in order for me to continue my efforts to attend graduate school. She is in her 90s now but she is still healthy and vibrant. I love her dearly and it pains me that my studies have taken me so far away from her.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF FIGURES .........................................................................................................................9 LIST OF ABBREVIATI ONS ........................................................................................................10 ABSTRACT ...................................................................................................................................14 CHAPTER 1 INTRODUCTION ..................................................................................................................16 History of Stroke .....................................................................................................................16 Ischemic Stroke ......................................................................................................................17 Overview of Stroke ..........................................................................................................17 Focal Ischemic Stroke .....................................................................................................18 Animal Mod els for Focal Ischemic Stroke ......................................................................20 Intraluminal MCAO model ......................................................................................20 Embolus models .......................................................................................................20 Endothelin1induc ed MCAO model .......................................................................21 Pathophysiology of Focal Ischemic Stroke .....................................................................21 Mechanisms of cellular cell death following cerebral ischemia ..............................22 Sources of free radical generation contribute to exacerbate brain injury. ................25 Inflammatory response to cerebral ischemia ............................................................27 Blood brain barrier and focal ischemic stroke ..........................................................31 Research Approach .................................................................................................................36 The Current Options for Ischemic Stroke Therapy are Limited ......................................36 Therapeutic Strategies with pGSN and AAT ..................................................................39 Plasma GSN and ischemic stroke .............................................................................40 Alpha 1antitrypsin and ischemic stroke ..................................................................44 Hypothesis .......................................................................................................................47 2 METHODOLOGY .................................................................................................................48 Rats Used as an Animal Model for Focal Ischemic Stroke ....................................................48 Endothelin1Induced Middle Cerebral Artery Occlusion Model ..........................................51 Materials .................................................................................................................................52 Experimental Procedures ........................................................................................................53 Animal Model and Treatments ........................................................................................53 Laser Doppler Perfusion Imaging ...................................................................................54 Behavioral Tests ..............................................................................................................56 Cylinder test .............................................................................................................56 Vibrissae test ............................................................................................................57 Histopathology ................................................................................................................57

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7 Magnetic Resonance Imaging .........................................................................................58 Image Analysis ................................................................................................................59 Statistical Analysis ..........................................................................................................59 3 THE PROTEC TIVE EFFECTS OF PLASMA GELSOLIN ON ISCHEMIC STROKE OUTCOME IN RATS ............................................................................................................61 Introduction .............................................................................................................................61 Results .....................................................................................................................................63 Treatment of pGSN did not Interrupt ET 1 Induced Artery Contraction ........................63 Treatment of pGSN Significantly Reduced ET 1 Induced Behavioral Deficits .............64 Cylinder test .............................................................................................................64 Vibrissae test ............................................................................................................64 Treatment of pGSN Reduced MCAO Induced Brain Damage .......................................65 Discussion ...............................................................................................................................65 Conclusion ..............................................................................................................................67 4 ALPHA 1 ANTITRYPSIN MITIGATED ISCHEMIC STROKE DAMAGE IN RATS ......72 Introduction .............................................................................................................................72 Results .....................................................................................................................................74 Human AAT Did not Affect the ET 1 Induced Ischemia ...............................................74 Local Delivery of hAAT Mitigates ET 1 Induced S troke Outcome ...............................75 Systemic Delivery of hAAT Mitigated ET 1Induced S troke Outcome .........................77 Discussion ...............................................................................................................................79 Conclusions .............................................................................................................................81 5 GENERAL DISCUSSION, CONCLUSIONS AND FUTURE WORK ................................87 General Discussion .................................................................................................................87 Conclusions ...........................................................................................................................102 Future Work ..........................................................................................................................104 LIST OF REFERENCES .............................................................................................................106 BIOGRAPHICAL SKETCH .......................................................................................................143

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8 LIST OF TABLES Table page 21 Treatment schedule for laser Doppler perfusion imaging .................................................60 22 Treatment schedule of for aCSF, ET 1 alone, ET 1+pGSN, and ET 1+hAAT (i.c.) groups .................................................................................................................................60 23 Treatment schedule for ET 1+saline and ET 1+hAAT (i.v.) groups ................................60

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9 LIST OF FIGURES Figure page 31 Perfusion measurements of pGSN study ...........................................................................68 32 Cylinder test of pGSN study ..............................................................................................69 33 Vibrissae test of pGSN study .............................................................................................70 34 Infarction area labeled for mitochondrial activity of pGSN study ....................................71 41 Perfusion measurements of AAT study .............................................................................82 42 Cylinder test of ET 1 alone vs. ET 1 and intracerebral hAAT delivery ............................83 43 Vibrissae test of ET 1 alone vs. ET 1 and intracerebral hAAT delivery ...........................84 44 Infarction area labeled for mitochondrial activity of AAT study ......................................85 45 Cylinder test of ET 1 alone vs. ET 1 and intravenous hAAT delivery .............................85 46 Vibrissae test of ET 1 alone vs. ET 1 and intravenous hAAT delivery ............................86 47 Infarction area calculated from MRI images using Image J v5.0 ......................................86

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10 LIST OF ABBREVIATIONS AA Arachidonic acid AAT Alpha 1antitrypsin aCSF Artificial cerebrospinal fluid AGP 1 acid glycoprotein AIF Apoptosis inducing factor AMPA amino 3hydroxy 5methyl 4 propionate AMRIS Advanced magnetic resonance imaging and spectroscopy ANOVA Analysis of Variance Apaf 1 Apoptotic protease activating factor 1 APC Activated protein C APP Acute phase protein ATP Adenosine triphosphate BBB Blood brain barrier cAMP Cyclic adenosine monophosphate CBF Cerebral blood flow CDC Centers for Disease Control and Prevention cGSN Cytosolic gelsolin CINC Cytokine induced neutrophil chemoatractant CNS Central nervous system COPD Chronic obstructive pulmonary disease COX Cyclooxygenase CRP C reactive protein

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11 CVA Cerebral vascular (or Cerebrovascular) accident DAG Diacylglycerol ECASS European cooperative acute stroke study ECM Extracellular matrix ESRD End stage renal disease ET1 Endothelin1 FAF Familial amyloidosis of Finnish type FDA Food and Drug Administration FIB Fibrinogen GFAP Glial fibrillary acidic protein GSN Gelsolin hAAT Human alpha 1antitrypsin HNE Human neutrophil elastase ICAM 1 Intracellular cell adhesion molecule 1 IL Interleukin iNOS Ind ucible nitric oxide synthase IFN Interferon JAM Junction adhesion molecule JNK JunN terminal kinase LDL Low density lipoprotein LPA Lysophosphatidic acid LPS Lipopolysaccharide LRP Low density lipoproteinreceptor related protein

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12 LTP Lipoteichoic acid MC Mast cells MCA Middle cerebral artery MCAO Middle cerebral artery occlusion MCP 1 Monocyte chemoatractant protein 1 MHC Major histocompatibility complex MMPs Matrix metalloproteinases MPO Myeloperoxidase NADPH Nicotinamide adenine dinucleotide phosphate NF Nuclear factor NMDA N methyl aspartate NO Nitric oxide NVU Neurovascular unit PAF Platelet activating factor PAR 1 Proteaseactivated receptor 1 PARP 1 Poly(adenosine diphosphate ribose) polymerase 1 PBF Phosphate buffered formaldehyde PBS Phosphate buf fered saline pGSN Plasma gelsolin PI(4,5)P2 Phosphatidylinositol 4,5bisphosphate PMN Polymorphonuclear neutrophil PTGS Prostaglandinendoperoxide synthase RAP Receptor activated protein

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13 rhu pGSN Recombinant human plasma gelsolin RNS Reactive nitrogen species ROS Reactive oxygen species rtPA Recombinant tissue plasminogen activator s.e.m. Standard error of the mean SAINT (I, II) Stroke acute ischemic NXYtreatment trial (I, II) STAIR Stroke treatment academic industry roundtable TIA Transient ischemic attack TJ Tight junction tMCAO Transient middle cerebral artery occlusion TNF Tumor necrosis factor TNF Tumor necrosis factor alpha TTC 2,3,5triphenyltetrazolium chloride TWEAK Tumor necrosis factor like weak inducer of apoptosis uPA Urokinase plasminogen activator VCAM 1 Vascular cell adhesion molecule1 VEGF Vascular endothelial growth factor VSM Vascular smooth muscle VSMC Vascular smooth muscle cell XO Xanthine oxidase XOR Xanthine oxidoreductase

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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 Philosophy THE PROTECTIVE EFFECTS OF PLASMA GELSOLIN AND ALPHA 1 ANTITRYPSIN ON ISCHEMIC STROKE OUTCOME IN RATS By Huong Le Moldthan May 2012 Chair: Sihong Song Major: Pharmaceutical Sciences Plasma gelsolin and alp ha 1antitrypsin, used in this study, are human serum proteins with multiple functions including anti cell death pathways and anti inflammatory effects that can be utilized to develop ischemic stroke therapy. The effects of the two proteins were tested on middle cerebral artery occlusion (MCAO) induced by the potent vasoconstrictor peptide endothelin1 (ET 1) In this model, ET1 is injected adjacent to left middle cerebral artery (MCA) in rats To test the effects of the candidate therapeutic agents in the MCAO model, rats were post treated by a discrete injection of pGSN or AAT at the same site of ET 1 injection or by an intravenous injection of AAT at the same time of ET 1 injection Artificial c erebrospinal fluid ( a CSF) and sterile saline were utilized as negative controls. The MCAO was verified using a laser Doppler perfusion imaging system in separate group s of animals Cylinder and vibrissae tests were performed before and 72 hours after MCAO to ass ess motor/sensorimotor functional deficits. Infarct volumes were determined via infarct area calculation 72 hours after MCAO using magnetic resonance imaging (MRI) and 2,3,5Triphenyl tetrazolium chloride (TTC) assay. Results from these experiments showed: 1) pGSN or AAT (both local and systemic deliveries) treatment did not interrupt ET 1 induced

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15 MCAO as a disease model; 2) pGSN or AAT treatments significantly mitigated ET 1 induced fu nctional deficits assessed by both cylinder and vibrissae tests; 3) pGSN local delivery, AAT local delivery or AAT systemic delivery markedly reduced brain injury (infarct volumes) by 49%, 83%, and 63%, respectively, compared to controls. Taken together, t hese findings show that plasma g elsolin and alpha 1 antitrypsin are potential novel therap eutic drugs for the protection against neurodegeneration following ischemic stroke.

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16 CHAPTER 1 INTRODUCTION Th is chapter start s with a brief history of stroke followed by an extensive review of stroke animal models, pathophysiology of ischemic stroke, and current ischemic stroke therapies, with a focus on focal ischemic stroke, the main topic of this dissertation. Furthermore, an overview of plasma gelsolin and alpha 1antitrypsin as therapeutic strategies and the central hypothesis behind using these therapeutic proteins in stroke are discussed. History of Stroke The fi rst mention in medical literature of what is called stroke today, was during Hippocrates time. It was called apoplexy, which means struck down by violence in Greek. This was due to the fact that a person developed sudden paralysis and a change in well being. Physicians had little knowledge of the anatomy and function of the brain, the cause of stroke, or how to treat it ( 1) The word stroke first appears in recorded English i n 1599 ( 2) In the mid 17th century, Johann Jacob Wepfer did post mortem examinations of patients that died with apoplexy and discovered tha t they had bleeding in the brain ( 3) Based on these findings he was the first to hypothesi ze that death was caused by this bleeding. He also discovered that a blockage of the major arteries that supply blood to the brain could have similar effects. In the 20th century, the term cerebral vascular accident, or CVA, ente red the medical terminology following the development of angiography techniques in the early 1900s ( 4) and apoplexy was divided into categories based on the cause of the blood vessel problem. Stroke is also known as "brain attack" to represent the fact that it is caused by a lack of blood supply to the brain which is similar to a heart attack caused by a lack of blood supply to the heart. The term brain attack also implies that it requires immediate intervention s

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17 Ischemic Stroke Overview of S troke Stroke is defined as the rapid ly developed clinical signs of focal or global disturbance of cerebral function, with symptoms lasting more than 24 hours or leading to death, with no apparent cause other than of vascular origin ( 5) (page 114). Although this definition does not include transient ischemic attack (TIA), which is discussed later in this section, it covers most cases of strokes. Stroke occurs so quickly, it is also known as a brain attack or a cerebrovascular accident as it is primarily caused within the vasculature. It result s from the interruption of the blood supply to the brain, often because a blood vessel burst s or becomes blocked by a clot. This causes insufficient oxygen and nutrients suppl ied to the brain, ultimately leading to brain damage Broadly, strokes can be classified into two major categories: hemorrhagic and ischemic. A hemorrhagic stroke is the bleeding into or around the brain, which causes the compression of the brain tissues, reduc ing blood supply to those tissue s which subsequently leads to ischemia This can occur in several ways. One common cause is a bleeding ane urysm, a we a k or thin part of an artery wall which can break under arter ial pressure and spill blood into surrounding brain tissues Another cause of h emorrhagic stroke is when an artery break s open due to plaque deposited artery walls. Also, a n individual with arteriovenous malformation also has a risk of having a hemorrhagic stroke (Stroke: Hope through research, National Institute of Neurological Disorders and Stroke, access ed March 15th, 2012) Arteries ruptured within the brain result in hemorrhagic stroke called intracerebral hemorrhage. Arteries ruptured outside the brain result in hemorrhagic stroke called subarachnoid hemorrhage in which blood bleeds under the meninges or outer membranes of the brain into the thin fluid filled space below the arachnoid layer and above the pia mater layer surrounding the

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18 brain (Stroke: Hope through research, National Institute of Neurological Disorders and Stroke, access ed March 15th, 2012) An ischemic stroke results from the blockage of blood flow to a certain area of the brain lead ing to ischemia and eventually caus ing brain tissue death Ischemic strokes can be further divided into two subtypes global and focal Global strokes are caused by the decrease in blood supply to the entire brain ( 6) which is primarily involved in cardiac arrest F ocal strokes are often caused by the occlusion of a major cerebral artery, such as the middle cerebral artery ( 7) Transient ischemic attack also called a mini stroke, is defined as a brief episode of neurological dysfunction caused by focal brain or retinal ischemia, with clinical symptoms typically lasting less than one hour, and without evidence of acute infarction ( 8) (page 1715). This definition illustrates the import ance of paying attention to TIA, giving a quick evaluation and treatment to avoid cerebral ischemia. Since the symptoms of TIA initially are like a stroke but then resolves with no noticeable effect TIA used to be consider ed a benign event. However, it recently has become a critical indicator of impending stroke ( 9) Ischemic strokes, in origin, are approximately 87% of all stroke s worldwide ( 10) with the remainder being hemorrhagic strokes Th is has led ischemic stroke to be a n extensively studied field in the last few decades Animal models have been used extensively to study ischemic stroke, par ticularly focal ischemic stroke, which is more common than global ischemic stroke G lobal stroke models are generally associated with the model s of circulation resultant from cardiac arrest rather than stroke ( 11, 12) .T his dissertation focus es on focal ischemic stroke. Focal Ischemic Stroke F ocal cerebral ischemia involves a sufficient enough reduction in regional cerebral blood flow (CBF ) in a specific vascular territory to alter cerebral function and usually occurs clinically as an ischemic stroke due to blood clots. Blood clots can result i n ischemia and ultimately

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19 infarction in several ways A clot formed in a part of the body other than the brain can travel through blood vessels and become lodged in a brain artery. This clot is called an embolus, and is often form ed in the heart. A blood c lot formed in an artery which stays attached to the arter ial wall until it develops large enough to block the artery is called a thrombus Cerebral ischemia can also be due to stenosis, a narrow ing of the artery result ing from the buildup of plaque along the arterial wall e.g. atherosclerosis which is the gradual deposition of cholesterol and other lipids in the innermost layer of arter ial walls (National Institutes of Neurological Disorders and Stroke, accessed March 6th, 2012). In a typical focal ischemic stroke animal model t he middle cerebral artery is occluded either permanently or only temporarily, allow ing subsequent reperfusion The latter was chosen for this study because p ermanent ischemia model results in a region of severe ischemic d amage which represents only a minority of human strokes ( 13) Transient MCAO model s produce both ischemia and reperfusion which mimics the majority of human strokes Transient ischemic stroke induces varying degrees of ischemic injury depending on the durat ion ( 14) location, and intensity of ischemia ( 15) Therefore, one can manipulate conditions suitable to the study objectives. It is also one of the most relevant models as it correlates with the conditions such as TIA, spontaneous thrombolysis, and treatment induced thrombolysis, e.g. tissue plasminogen activator the standard therapy for ischemic stroke patient s T his is also one of the models which best mimics the clinical situation in which patients undergo therapeutic recanalization of the cerebral blood vessel following stroke ( 16) Furthermore t ransient ischemic stroke models offer a higher survival rate in study animals in comparison to permanent ischemia, allowing for long term effe cts in drug studies

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20 Animal Models for Focal Ischemic Stroke Several t ransient ischemic stroke models have been developed over the years. Examples include an intraluminal MCAO, embolus models and endothelin 1induced MCAO model which are relatively extens ive ly used in the development for stroke therapies. These models can be performed on both mice and rats ( 17) Intraluminal MCAO model This model involves inserting a monofilament suture into the internal carotid artery to block blood flow to the MCA for a certain period of time depending on the design of the stud y then wi thdrawing the suture to allow reperfusion The detail ed techniques and procedures have been described in numerous studies ( 14, 1820) The typical induced infarct areas are in the lateral cau datoputamen and frontopari etal cortex ( 21) The in farct is re producible, reperfusion is easil y obtain ed when the suture is withdrawn, and animals can survive up to months, making the model beneficial for functional outcome evaluations in testing neuroprotective drugs ( 22) Although this model has been popular since in the 1980s, it has several shortcomings which can affect the lesion size. For instance, slight physical differences in suture s ( 23 ) insertion position of the suture ( 24 ) accidental prematur e reperfusion ( 25) and spontaneous hyper thermia due to long duration of surgery ( 26) can significantly al ter the infarct size and may obliterate the testing agent. In addition to this, the technique requires adequately trained and experience d personnel. Embolus models Thromboembolic and photochemical MCAO models are commonly used. In the thromboembolic model the blockage of the MCA is induced by injecting a blood clot directly i nto the common carotid artery or into the carotid artery via a retrograde catheter placed in an external carotid artery ( 27 ) Scientists have shown great interest in this model because of its utility and resemblance of human ischemic stroke. Also, it can be used to evaluate thrombotic

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21 therapies ( 28 ) However, the main disadvantage s of this model are inhomogeneous infarct size diffuse location of infarcts and risk of microembolization ( 27) P hotochemical MCAO is induced by systemically injecting a photoactive dye in combination with irradiation of several branches of the distal MCA ( 29) This model produces consistent infarct and is m ore reliable in the Sprague Dawley than in the Wistar rat strain ( 30) The disadvantage is that the photochemical reaction can cause microvascular injury vasogenic edema, and BBB disruption which does not allow penumbra formation ( 31) Therefore, this model may not be used for neuroprotective drugs. Endothelin1induced MCAO model The model is induced by the intracranial injection of endothelin1 ( E T 1) a potent natur al vasoconstrictor peptide, proxim al to the MCA using a stereotaxic system. After ET 1 application, CBF decreases and induces significant cerebral ischemic injury within the MCA vicinity ( 32, 33) Although the model has some limitations (discussed in Chapter 5), the consistency in location, size, seve rity, duration of the infarct, and the avoidance of surgery complication s provide distinct advantages over other models ( 17) This model may be useful for neuroprotective agent studi es and is utilized in this dissertation pro ject. P athophysiology of Focal Ischemic S troke Interruption or decrease ( approximately 50% compared to base line ) of blood supply to the brain initiates a complex cascade of neuronal events within minutes. Insufficiency of major energy substrates including oxygen and glucose, slows or stops adenosine triphosphate (ATP) synthesis in mitochondria. As energy failure occurs ion pumps situated in the cell membrane malfunction leading to massive influ x of sodium and calcium; and efflux of potassium which together cause depolarization. This is accompanied by an inflow of water, resulting in intracellular edema (swelling cells) Once cell membrane is depolarize d, neurons release

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22 neurotransmitters, including glutamate and aspartate. Uncontrolled release of g lutama te at ischemic site s activates N methyl amino 3hydroxy 5 methyl 4propionate (AMPA), and metabotropic glutamate receptors causing an increase in intracellular calcium Depolari zation also opens presynaptic voltage activated calcium channels, releasing even more glutamate. Calcium overload ultimately results in excitotoxicity which leads to cell death. This is known as necrosis, and it occurs rapidly (within 510 min) ( 34 ) The primary necrotic region resulting from severe ischemia is commonly called the core of the infarct or um bra of a stroke ( 16) In addition to primary cell death due to oxygen and glucose deprivation, the core of the infarct is surrounded by a penumbra of compromised tissues in which program med cell death or apoptosis and inflammation occur ( 16 ) In the penumbral region, the cells can repolarize at the expense of further energy consumption and depolarize again in response to high concentrations of glutamate and potassium ions. Such repetitive depolarizations /repolarizations result in increased release of neurotransmitters ( 35) This, coupled with the generation of free radicals release of other biologically active molec ules from damaged mitochondria and dead cells, plus infiltra tion of leukocytes can continuously exacerbate injury in the penumbra Following the hours to weeks it takes for the secondary pathology to develop and revolve; necrosis is replaced by other death mechanisms. Mechanisms of cellular cell death following cerebral ischemia Multiple c ellular death pathways including necrosis and three known types of genetically programmed cell death appear to be triggered by ischemia and are involved in mitochondrial malfunction These death pathway s probably can concomitantly be present in the same cell ( 36 ) following c erebral ischemia; and a cell may switch back and forth between different pathways ( 37)

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23 Necrotic cell death pathway As mentioned above, necrosis is characterized by the loss of cellular membrane integrity as well as the irreversible swelling of the cytoplasm and its organelles Typically, the necrotic cells spill their contents into surrounding tissues, resulting in an inflammatory response (discussed later in this section) Type I genetically programmed cell death (Type I apoptosis ) also known as c aspase mediated cell death can be initiated by the loss of mitochondrial membr ane integrity cytoplasmic cytochrome C release, and indirectly released cytokines It has been demonstrated that in cerebral ischemia, mitochondrial cytochrome C, an essential membrane protein of the mitochondrial respiratory chain is translocated from mitochondria to the cytosolic compartment ( 38) Upon releas e to the cytoplasm, cytochrome C binds apoptotic protease activating factor 1 (Apaf 1) and activates caspase9, resulting in the formation of cytosolic apoptosome s Caspase9 then activates caspase 3, a frequently activated death protease, leading t o random cleavage of DNA fragments via the activation of endonucleases ( 39) Caspase 3 and 9 have also been shown to play crucial roles in neuronal death following ischemia ( 40, 41) Moreover, in caspase11 knockout animal model s a reduction in apoptosis and a defect in caspase 3 activation have been demonstrated following cerebral ischemia, suggesting that caspa se 11 is a crucial initiator responsible for the activation of caspase 3 ( 42) Caspase11 is also responsible for caspase1 activation which has the ability to enhance apoptosis ( 43) Caspase1 is known to produce cytokines, such as IL 18 ( 44) ; thus cytokines in apoptosis can also be considered as effectors during brain ischemia. Type II genetically programmed cell death ( Autophagy) is a caspaseindependent c ell death and involves autophagy. Autophagy is a regulated process of degradation and recycling of cellular constituents, involved in bioenergetic management of starvation ( 45) It has recently

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24 been shown to be upregulated in many stress condition s, such as cardiac ischemia/reperfusion ( 46) It also has been shown that cerebral ischemia enhances autophagy and the autophagic death cells have been detected at different time points post ischemia in vitro and in vivo ( 47, 48) Autophagocytosis can occur when the NMDA receptor is activated excessively in neuronal cell culture The neuronal death in rat organotypic hippocampal slices exposed to NMDA associated with a utophagy and endocytosis, which completely prevented by c JunN terminal kinase (JNK) inhibitor, suggesting that autophagy as well as endocytosis is mediated by the JNK ( 49) It has been reported that apoptosis inducing factor (AIF) rapidly migrates from mitochondria to the nuclei of injured neurons following transient MCAO (tMCAO) ( 50) When AIF is released in to the cytosol it initiates a caspaseindependent cellular suicide involving chromatin condensation and cleavage of DNA into characteristic 50kb fragments, inducing cell death ( 39) Decrease in AIF expression leads to a reduction in infarct size and cell death in the ischemic penumbra. Also, inhibition of poly(adenosine diphosphate ribose) polymerase 1 (PARP 1), an enzyme responsible for AIF release from mitochondria, lessens AIF migration and reduces neuronal cell death by more than 80% ( 50 ) Taken together, activated PARP 1 promotes the release of AIF which is a mediator of caspase independent apoptosis. Type II genetically programmed cell death is an important mechanism for dealing with protein aggregation and nonfunctional organelles (e.g. lysosomes, and especially mitochondria ). However, when it becomes excessive it can result in cell death in response to starvation, osmotic stress, and TNF release ( 51) Type III genetically programmed cell death (Parthanatos ) t he last intrinsic cell death mechanism driven by generation of oxygen a nd nitrogen species ( ROS/RNS) is also known as parth a natos ( 52 ) Free radicals s peci fic ally from the mitochondrial membran e, cause DNA

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25 damage via oxidation, methylation, depurination, and deamination ( 53) DNA damage activates PARP 1, an important nuclear enzyme. Activated PARP 1 promotes the release of AIF from the membrane of mitochondria. These findings imply type III apoptosis is interrelated to type II apoptosis. S ources of free radical generation contribute to exacerbate brain injury Reperfusion injur y after transient ischemia is associated with overgeneration of free radicals, e.g. ROS/RNS resulting in oxidative and nitrosative stress. T hree primary sources of free radicals, mitochondrial dysfunction, two enzymes ( xanthine oxidase (XO) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase) and activated leukocytes are discussed below Mitochondria are a major cellular site of free radical generation in phys iological and pathological condition s It is estimated that 1 to 4% of all oxygen used by mitochondria is converted into ROS. This is caused by electron leak at the respiratory chain transporting electrons from reduced pyridine nucleotides to O2 molecule s ( 54) primarily resulting in O2 generation As the reducing state of the respiratory chain increases, the more electrons can leak f r om the chain, promoting the production of O2 radicals. This process depends on the concentration and tension of oxygen ( 55) Superoxide anions then rapidly dismutate to H2O2 either spontaneously or enzymatically via manganese sup eroxide dismutase. The rate of O2 and/or H2O2 production increases as mitochondrial Ca2+ increases ( 56) The rate of H2O2 production is a function of the oxidationreduction state of the electron carriers, decreased carriers result in increasing the H2O2 product ion. During ischemic events, the respiratory chain is in a reduced state due to little or no oxygen supplied and overload of Ca2+ in mitochondria. Th is may lead to production of O2 radicals. Paradoxically during reperfusion, the quick influx of oxygen t o ischemic tissue r esults in overgeneration of ROS. The mitochondrial burst of ROS

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26 production can escape cellular antioxidant defenses and cause damage to DNA, proteins, and membrane lipids. As discussed earlier an increase in Ca2+ concentration can activate nitric oxide synthase (NO S ), provoking the formation of 2 to produce ONOO (peroxynitrite) radical a potent oxidizing agent. Following cerebral ischemia, reperfu sion or reoxygenation can cause oxidative stress to damage th e brain tissues. X anthine oxidase and NADPH oxidase are other important sources of ROS formation in post ischemic tissues E nzyme xanthine oxidase (XO) also known as xanthine oxidoreductase (XOR) plays crucial role in purine nucleotide catabolism in humans During ischemia, intracellular Ca2+ levels rise activating a protease that converts xanthine dehydrogenase (XDH) into XO In the mean time, purines are catabolized, and XO substrates, hypoxanthine and xanthine accumulate. In addition, a denosine triphosphate ( ATP ) breakdown due to hypoxic conditions causes the accumulation of XO substrates. When oxygen is again available (on reperfusion), xanthine oxidase m ay produce more superoxide (O2 ), consequently, of OH ( 57, 58) Niconinamide adenine dinucleotide phosphat e (NADPH) oxidase, a membranebound enzyme complex, expressed in neutrophils ( 59) It has been documented that activated NADPH oxidase contributes to ROS generation ( 60, 61) The activation of NADPH is triggered by protein kinase C ( PKC) or by increase intracellular calcium concentration during cerebral ischemia ( 62) Activated leukocytes contribute to ROS/RNS generation Myeloperoxidase (MPO), mainly released by activated neutrophils and monocyte s ( subtypes of leukocytes), is characterized by powerful pro oxidative and proinflammatory properties ( 63) MPO catalyz es the conversion of hydroge n peroxide and chloride anion to hypochlorous acid (HOCl), a cytotoxic radical. Thus, l eukocyte infiltration promotes inflammation and secretion of MPO

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27 produces free radicals and may prolong the phase of free radical generation and increase oxidative damage. In summary formation of reactive oxygen and nitrogen species involves multiple injury mechanisms, such as mitochondrial inhibition, activation of xanthine oxidase and NADPH oxidase enzymes and infiltrating leukocytes. Excessive amount of free radical leads to oxidative and nitrosative stress w hich contributes to the pathology of ischemic stroke. O xida tive and nitrosative stress resulting from excessive amount of free radicals, debris from death cells induced by various pathways, and blood clots from re canalization of the occluded blood vessel in the brain all con tribute to immune response signaling The role of the m ajor inflammatory components (microglia, astrocytes, leukocytes, and mast cells) is detailed below. Inflammatory response to cerebral ischemia Microglia play a bifunctional role during post ischemic inflammation Microglial cells the resident macrophages of the brain, are very sensitive to delicate alterations in their neuronal micro environment. Microglia comprise the innate immunity in the brain and defend against infectio ns as well as toxic substances released from dying cells in the brain by scavenging and engulfing unwanted pathogens and cellular debris. In normal condition, microglia often present in a ramified state, the resting form, which composes of long branching p rocesses projecting out from the small cellular body When the sensors of the microglial membrane bind to pathogenderived molecules or other m icroglial activating agents, the cells transform into an activated stage associated with amberoid morphology and expression of inflammatory genes. Although the precise role and the exact time course of microglia in stroke are still debatable, t he resident microglia are the fir st inflammatory cells that rapidly respond to cerebral ischemia. Experimental data have been shown that microglia are activated within minutes of ischemia onset and r elease

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28 plethora of pro inflammatory mediators, including IL tumor necrosis factor alph a ( TNF ) and chemokines e.g. monocyte chemoat t ractant protein 1 ( MCP 1) all of which exacerbate tissue damage ( 6467 ) M icroglial proliferation typically reaches a maximum at 48 72 hours after focal cerebral ischemia and may continue for several weeks after the initial insult ( 68, 69) However, microglia can also produce cytoprotective substances, such as brainderived neurotrophic factor (BDNF), insulinlike growth factor I (IGF I), and several other growth factors ( 70 ) These data impl y that microglia activation during ischemia, as an inflammatory indicator; have both deleterious and beneficial effects depending on the balance between pro and anti inflammatory secreted molecules. Astrocytes secret e inflammatory factors Astrocytes, the primary glial cells in the CNS play a variety of physiological role s in CNS homeostasis They are also known as astroglia and express glial fibrillary acid ic protein (GFAP), a protein required for normal functions such as maintenance of BBB integrity. In ischemic stroke, GFAP expression is upregulated, leading to astrocyte structural and functional changes ( 71) Reactive oxygen species produced by transient oxygen glucose deprivation have been demonstrated to cause astrocyte mitochondrial membrane depolarization. ROS have also been shown to damage astrocytes through activating the PARP 1 cell death pathway ( 72) The expression of astrocyte inducible nitric oxide (iNO), an ROS, has been found within several hours and is maximal 2 to 3 days after ischemia ( 73, 74) Astrocytes have the capability to express major histocompatibility complex (MHC) antigens and co stimulatory molecules that are critical for antigen presentation and T cell activation in brain inflammation. Astrocytes also participate in promoting Th2 (antiinflammation) response and suppressing interleukin 12 (IL 12) ex pression ( 75) Following cerebral ischemia, activated astrocytes secrete many inflammatory factors such as interleukins (IL 1, IL 6 ), interferons (I FN -

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29 I FN MMPs, and tumor necrosis factor (TNF ( 75 77) that can contribute to delayed neuronal death. M oreover, tumor necrosis factor like weak inducer of apoptosis (TWEAK), a member of tumor necrosis factor superf amily, has been reported to be produced by neurons, endothelial cells, and astrocytes ( 78, 79) and can induce the generation of proinflammatory mediators including IL 8 and IL 6, by interaction w ith its Fn14 receptor found on astrocytes ( 80) Both TWEAK and Fn14 proteinlevels are elevated in the ischemic penumbra and injection of a soluble Fn14 decoy receptor in an animal model of MCAO, can decrease the infarct volume ( 79) These data indicate ischemia i nduced astrocyte activation can potentially elevate brain inflammation. Finally, n eurons cannot survive in the brain without close interactions with astrocytes. Therefore, in any region where astrocytes fail to survive, there may well be failure of neurona l survival. Leukocytes produce inflammatory mediators In the subacute phase (hours to days), infiltrating leukocytes release cytokines and chemokines, especially excess amount of ROS and induction of MMP (mainly MMP 9). Infiltration of leukocytes occurs through three stages: rolling on the surface of endothelial cells, adhesion to the endothelial wall, and migration through the endothelium or diapedesis. These processes are mediated by adhesion molecules, including selectins (P E and L selectins), immu noglobulin superfamily (vascular cell adhesion molecule 1 (VCAM 1), intracellular cell adhesion molecule (ICAM 1)), and integrins (CD11a c) ( 81) L eukocytes express cathepsins, a class of globular liposomal proteases that degrade extracellular matrix via MMPs. Leukocytes (lymphocytes) contain granzymes that degrade DNA, hence inducing caspase independent apoptosis cascade (type II ap optosis) Granzymes,

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30 cathepsins A and G are serine proteases, and they could be targets of serine protease inhibitors (serpins) to mitigate brain inflammation induced by leukocyte activation. Mast cells are responsible for BBB damage, brain edema, and hemo rrhage Mast cells (MCs) are present in the brain, particularly inside the BBB (>96%) ( 82) They contain many granules (i.e. histamine and heparin) which are released upon activation. It is important to note that M Cs can respon d to activation either via exocytosis of the content of secretory granules, commonly known as degranulation ( 83) leading to acute release of mediators into the extracellular environment or under a controlled fashion resulting in differential release of mediator s ( 84, 85) This permits stimulus specific release of individual mediators, which also may be di fferent among MC types ( 86) D ifferent MC triggers, such as IL 1, IL 33, and LP S can produce multiple mediators independent of exocytosis ( 83) Brain edema, a consequen ce of ischemia, can be affected by M C activation. In animal model s, it ha s been s hown that ischemic brain edema can be reduced by 40% by treatment with the MC stabilizing agent sodium cromoglycate ( 87) Moreover, histamine plays a role in brain edema in global ischemic stroke ( 88, 89) extrapolating that MCs may also be involved in edema of focal ischemia since histamine is a major MC mediator. This evidence indicate s that mast cells may contribute to aggravated edema in ischemic stroke. Mast cells play a crucial role in CNS inflammation, par ticularly by recruiting leukocytes to sites of vascular inflammation by inducing all of the primary mechanisms involved in leukocyte infiltration during acute inflammation via secretion of histamine, adhesion molecules, selectins, and platelet activating factor (PAF) ( 90 ) MCs activation in immature animals due to hypoxia ischemia also results in exacerbating brain damage. It has been demonstrated that a rapid increase in MC count and their activation results in neuronal loss during hypoxic ischemic brain damage of immature rats, and the damage is

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31 reduced more than 50% by controlling the number of MCs ( 91) It is possibly caused by the contribution of MC derived histamine ( 92) Importantly, inhibition of MCs with cromoglycate can decrease hemorrhage formation during cerebral ischemiareperfusion following post i schemic administration of recom binant plasminogen activator (r t PA ), a thrombolytic agent ( 93) The reduction in hemorrhage formation may play a critical role in improving neurological outcomes and reducing mortality Blood b rain b arrier and focal ischemic s troke In addition to the activation of inflammatory processes via stimulation of major components of the immune system, such as microglia, astrocytes, leukocytes, and mast cells isc hemic stroke ultimately leads to disruption of the BBB The BBB is a semipermeable barrier between the CNS and the systemic circulation. It can be considered as the bodyguard of the CNS serving to maintain the fragile homeostasis of the brain. In addition to endothelial cells, t he BBB is constitut ed of pericytes, astrocytes, neurons, and extracellular matrix (ECM), which have been commonly known as the neurovascular unit (NVU) ( 94) E ach component of the NVU operates in harmony t o re gulate BBB functions such as microvascular permeability, ion gradients, nutrient uptake, toxic waste removal, and cerebral hemodynamics. Indeed, an alteration of any of the individual constituents may cause BBB impairment. BBB disruption directly contributes to cerebral vasogenic edema, hemorrhagic transformation, and poor outcomes of the disease. Both distinct stages of ischemic stroke, ischemia and reperfusion, can devastate BBB permeability and tight junction s (TJ s ) regulation, leading to complex clinical prognosis of the disease. Ischemia results in loss/shortage of oxygen and nutrients in the c ore and to the surrounding tissues. The endothelium at the core sustains the greatest severity of insult and likelihood for dysregulation and disassembly. The increase in paracel lular permeability of BBB TJ s ha s been observed after several hours following ischemia ( 95) Within minutes of ischemic

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32 onset, the internal capillary diameter shrinks due to endothelial swelling. Ischemic tissues become acidic due to anaer obic respiration, which release lact ate ( 96) Lac t acidosis caused by the lactate (lactic acid), directly contributes to swelling of endothelial cells, neurons, and astrocytes. Moreover, induction of proteases (i.e. tissue plasminogen activator ( t PA), MMPs, cathepsins, and heparanases ) via activation of inflammatory molecules contributes to BBB degradation. Continuous biochemical events following ischemia are strongly linked with downstream endothelial response s As mentioned earlier, within the penumbra region, human brain expresses of sev eral proteins, including caspases 1, 3,8, 9, death receptors, Apaf 1, and p53, which are capable of activating apoptotic pathways. Pro inflammatory mediators (e.g. IL 1, TNF induced, followed by upregulation of chem okines (e.g. MCP 1), and cytok ine induced neutrophil chemoat t ractant (CINC) involved in the activation of endothelium ( 97) Moreover, i ncreased expression of intercellular adhesion molecules and cytokines initiate s endothelial inflammatory activation subsequently promot ing leukocyte infiltration into the brain. L eukocyte s (both circulating and infiltrating ) and activated microgl ia are recruited to the ischemic zone further accel erating inflammation and toxic free radical production. Increased leukocyte migration perturbs the molecular organization of the TJ complex, including breakdown of occludin, a membrane protein found a t high concentrations in BBB TJs, and zonula occluden1 (ZO 1), a cytoplasmic protein involved in BBB TJ formation and regulation ( 98) The entry of neutrophil s in the ischemic brain also causes d isorgani zation of the actin cytoskeleton leading to increased BBB TJ permeability ( 99 ) Reperfusion or compensation through collateral circulation is a process (either spontaneous or via re canalization of the blood occlusion) which can re establish blood supply and limit the

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33 brain damage. However, it can contribute to additional ph ysiological complication s and has the potential for hemorrhagic transformation. For instance, blood vessels weakened by ischemic stroke break turning to hemorrhagic stroke form. Increased BBB TJ opening or paracellular permeability may occur upon reperfus ion followin g three stages: hyperemia, phase1, and 2 (also known as Biphasic permeability) ( 94 ) The degree of permeability depend s on a number of factors, including duration of ischemia, degre e of reperfusion, and experimental model ( 94) Hyperemia occurs as the result of an acute elevation in regional CBF. It couples with the loss of cerebral auto regulation and therefore is passively dependent on perfusion. After hyperemia, hypope rfusion of ischemic region initiates resulting in lack ing of nutrient s necessary for tissue recovery. Hypoperfusion may also induce neutrophil adhesion, following inflammatory response in the most susceptible tissues, and further induce to the next stage of increased BBB paracellular permeability. Phase1 of biphasic permeability occurs within 38 hours of reperfusion onset. Alteration in permeability in this phase is primarily caused b y enzymatic ECM degradation, increased inflammation and oxidative stress ( 100, 101) A c ontinuous period of ischemia results in increase in edema, and consequently initiate s phase 2 of biphasic permeability ( 102) This final phase initiates 1896 hours after r eperfusion and is coupled with angiogenesis and increased vasogenic edema ( 103 105) Neutrophil (leukocyte) extravasation time span during reperfusion is also dependent on the severity and form of ischemic insult. It has been shown that the highest concentration of neutrophil infiltration occurs a t 6 and 48 h following a transient middle cerebral artery occlusion (tMCAO), and 12 and 72 h after a permanent MCAO in rats ( 106) The association between neutrophil infiltration and paracellular permeability responses of the BBB TJ indicates that the inflammatory mediator is related to BBB dysfunction.

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34 Blood brain barrier dysfunction caused by ischemic stroke appears to be associated with inflammatory response. Initial induction of proinflammatory cytokines and adhesion molecules provokes the activation and migration of leukocyte s (i.e. neutrophils) across endothelium. Once activated at parenchyma, leukocytes and microglia produce more inflammatory mediators, including cytokines and TNF I nterleukin6, and TNF of ischemic stroke patients ( 107) and in ischemic brains of animal s ( 108) C ytokines TNF and IL 6 have been demonstrated to disrupt BBB in vitro study and the activation of cyclooxygenase (COX) could be a key role i n this neuroinflammation ( 109) The BBB permeability is reduced by COX inhibitor s in rat model s (e.g. indomethacin) ( 110) Cyclooxygenase enzymes or prostaglandinendoperoxide synthases (PTGS) catalyze the reaction in metabolism of arachidonic acid (AA), which is released from phospholipid membrane breakdown. This implies that AA cascades in ischemia reperfusion increases BBB disruption. In addition, it has been documented that TNF upregulat ed by neurons and astrocytes via activation of MMPs in the ischemic re gion precedes BBB permeability ( 111) Cytokines act ing upon the vascular endotheli um can indirectly stimulate the production and release of MCP 1, a major component involved in leukocyte extravasation into parenchyma and CINC ( 97 ) a member of the inflammatory chemokine IL 8 family. Cytokines/chemokines also stimulate induction of endothelial adhesion molecu les such as ICAM 1, P and E selectin, and leukocytes ( 112) I nterleukinngs about delayed and localized neutrophil recruitment, and breakdown of the BBB. Administration of chronic IL 1 in rat striatum has shown the marked in crease in recruitment, vasodil ation, and increased BBB breakdown at the highest concentration of IL 1 ( d ay s 8 and 14 post injection) ; activation of microglia and astrocytes was identified on day 14 post injection ( 113) Other mediators, including i NOS free radicals, and MMPs induced

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35 by neutrophils and mononuclear phagocytes ( 100 ) can directly or indirectly signal to accelerate the BBB paracellular permeability. Regarding TJ protein, it has been demonstrated that junction adhesion molecule ( JAM ) is directly associated with inflammation. JAM 1 has been shown redistributed following TNF upregulation from the junctions to the endothelial surfaces in vitro ( 114, 115) There is abundant eviden ce showing that mast cells are involved in sequential events during cerebral ischemia via inducing many vas oactive mediators; it is rational to hypothesize that MCs contribute to BBB regulation. Indeed, in the rat model of focal cerebral ischemia a 50% increase in BBB breakdown following the triggering of MC degranulation has been reported. Interestingly, pharmacological regulation (i.e. cromoglycate) of MCs reduces BBB breakdown by 50% over the control ( 87, 116) In similar studies with additional administration of tPA there has been shown a reduction in tPA mediated hemorrhagic transformation ( 117) One of the strong possibilities is that MCs bioactive mediators perturb BBB regulation and the basal lamina integrity Some evidence suggests proteases, such as MMPs, plasminogen activators, and cathepsins, in the disruption of the matrix of the basal lamina ( 116) MMPs, a family of zinc containing endopeptidases, can degrade most of extracellular matrix components, i.e. collagens, gelatin, elastin, fibronectin, and vitronectin ( 118) Specifically, MMP 2 and MMP 9 have involvement in the degradation of basal lamina subsequent BBB breakdown and hemorrhagic transformation ( 119121) Collectively, the p athoph ysiology of ischemic stroke is extremely complex. It occurs in a series of biochemical cascades i nvolving multiple factors and components with over lapping and redundant features. Insightful understanding of the disease is necessary for more effective intervention and drug development.

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36 Research Approach The Current Options for Ischemic Stroke Therapy are Limited Tissue plasminogen activator (t PA ) is a serine protease which catalyz es the conversion of plasminogen into active plasmin. In plasma, plasmin primarily acts to dissolve fibrin filaments in blood clot s This subjects the blood clot to further proteolysis by ot her enzymes, thus dissolving the clot and restoring the blood flow i n the occluded blood vessel. The Food and D rug Administration (FDA) has approved t PA as a therapy for acute ischemic stroke patients since 1996 with limited application, e.g. within 3 h (European Cooperative Acute Stroke Study, ECASS III, extended to 4.5 h in 2008 ( 122) ) time to treatment window and with no apparent hemorrhage Genentech, Inc (San Francisco, CA, U.S.A .) has manufactured recombinant tPA (rt PA; generic name: Alteplase; b r and name: Activase) The drug is given intravenously with a dose of 0.9 mg/kg and ( 123) and in some cases may be given directly into an artery within a relative ly narrow time window L ess than 2% of patients qualify for this treatment ( 124) because most patients do not seek medical assistance quickly enough, since the proper diagnostic procedure is requires time and the drug itself can increase risks of cerebral hemorrhage and brain injury The major side effects of the drug possibly occur through MMPs (e.g. MMP 9), and other signaling pathways associated with protease activated receptor 1 (PAR 1), low density lipoproteinreceptor related protein (LDL receptor related protein or LRP), and NMDA receptor and are detail ed bel ow. Administratio n of t PA can lead to BBB disruption and cerebral hemorrhage via activation of MMP 9. L evels of MMP 9 are increased both in plasma and brain of acute ischemic stroke patien ts, and further elevated after t PA administration ( 125127) It has been well established that MMP 9 is involved in mediating BBB disruption as well as inducing neurotoxicity in thrombolytic therapy ( 128) In early stage s of th e ischemic cascade, elevated MMP 9 levels can

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37 lead to degradation of ECM and vascular basement membrane, thereby opening the BBB. Breaking down of BBB leads to leukocyte infiltration, brain edema, and hemorrhagic stroke transformation ( 129) Logically inhibition of MMPs can reduce hemorrhage and improve outcome in experimental emb olic stroke model treated with tPA ( 130) However, t PA MMP 9 activation could also have beneficial effects on brain tissue recovery after stroke during the later stage by promoting vascular remodeling, angiogen esis, neurogenesis, axonal regeneration ( 131 ) as well as synaptic plasticity ( 132 ) and glial remodeling by degradation antagonistic molecules within glial scar ( 133) This implies that combination therapies of MMP inhibitor and other tPA related pathways may limit the neurotoxic effects and extend the therapeutic time window of tPA in brain ischemia. Tissue plasminogen activator can cause neu rotoxicity and increased BBB permeability through PAR 1. It has been reported that plasminogen and tPA (endogenous and exogenous) can be present in the brain. Therapeutically administered tPA and plasminogen are able to cross the BBB ( 129, 134 ) while endogenous tPA is synthesized by neurons and glia, and is particularly abundant in the hippocampus and hypothalamus ( 135, 136) In the brain, tPA can convert plasminogen into plasmin, which provokes PAR 1. Protease activated receptors (PARs) are G proteincoupled receptors that are activated by proteolytic cleavage of their N terminus The upregulation of PAR 1 during experimental ischemia in hippocampal slice culture suggests that PAR 1 may play a role in pathological effects. Indeed, in PAR 1 knockdown mice, the infarct volume was reduced 3 .1 times after transient ischemic stroke ( 137) This provides evidence that the toxic effects of tPA may be mediated by PAR 1 activation. Tissue plasminogen activator can also exacerbate brain injury via the LRP pathway and oxidativ e stress L ow density lipoproteinreceptor related protein, LRP, is a multifunctional cell

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38 membrane receptor, expressed in live r and throughout the brain, particularly high in the cerebellum, cortex, hippocampus, and brain stem ( 138) During cerebral ischemia, tPA induces the shedding of the extracellular domain of LRP in neurons and in perivascular astrocytes ( 139) The interaction between LRP and urokinase plasminogen activator (uPA), a serine protease used as a thrombolytic agent, has been shown to regulate vascular contractili ty ( 140) and BBB permeability ( 141, 142) In tPA deficient mice, the cerebral infarct is reduced 50% vs. wild type animal after ischemi c stroke, suggesting that tPA can increase stroke induced injury, and that increase is independent of tPAs thrombolytic effects ( 143) Inhibition of tPA activity using neuroserpin, a potent endogenous tPA inhibitor, can reduc e infarct volume by 64% at 72 hr ( 144) Blood brain barrier permeability increa ses when injecting tPA into CSF of mice without ischemia, and this is mitigated by an LRP antagonist, a receptor associated protein (RAP), implying tPA induces neurotoxicity via LRP receptor In addition to crossing disrupted BBB, tPA may be able to cross intact BBB via LRP mediated transcytosis to enter the parenchyma under either ischemic or nonischemic conditions ( 145, 146) It has been shown that LRP sign aling has an important role in the tPA induced expression and activation of MMP 3 ( 147) and MMP 9 in tissue cell culture as well as animal models of stroke ( 148) In vitro, MMP 3 expression increases in mouse brain endothelial cell line s under ischemic stress and following treatment with tPA This increase was suppressed by the LRP inhibitor or nuclear factor (NF) activation ( 147) Recombinant tPA (rtPA) upregulates MMP 9 expression in cultured human brain ECs; this response is remarkably inhibited in the ECs treated with small interfering RNA (siRNA) to suppress LRP ( 148) Taken together tPA co ntributes to BBB damage via activation of MMP 3 and MMP 9, which is regulated by LRP and NF s Furthermore, the tPA LRP interaction has resulted in activation of NF

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39 following MCAO in rodent model ( 149) This might be an additional source of ROS, eventually leading to more BBB disruption in ischemic stroke. Tissue plasminogen activator interacts with NMDA receptor resulting in excitotoxicity effects. The NMDA receptor (named for the selective glutamate receptor agonist, N methylaspartate) consists of variety of NMDA subunits including NR1 subunit ( 150) Glutamate is the main excitatory neurotransmitter in the brain. In cerebral ischemia, increased glutamate levels may cause BBB opening and induce further brain damage ( 151 ) The modulation of the NMDAinduced neuronal death by tPA has demonstrated that tPA forms a stable complex with the NR1 subunit in vitro ( 152) therefore amplifying the influx of calcium during ische mic excitotoxicity B locking the tPA NR1 interaction reduces excitotoxic necrosis in mice ( 153, 154) Also recent finding s ha ve shown that the surface trafficking of tPA NMDA receptor increases by enzymatic activity of MMP 9 ( 155 ) whic h can promote BBB disruption, hemorrhaging, and further induce excitotoxicity Taken together, evidence has strongly supported that tPA can induce neurotoxicity, edema, and hemorrhagic transformation through a number of mechanisms, such as activating MMPs (especially MMP 9) and protease activated receptor 1, and interacting with LRP and NMDA receptor. Thus, to improve it, it is of utmost importance to overcome the undesired effects of tPA which may be possible by applying combination therapy with other neuroprotective agent(s). Therapeutic Strategies with pGSN and AAT As described above, t he pathological mechanisms of ischemic stroke are e xceptionally complex involving multiple processes, which can have both beneficial and detrimental effects. Limiting the permanent damage and promoting recovery mechanisms are critical to improve the outcome of stroke. P lasma gelsolin ( pGSN) and alpha 1an titrypsin (AAT) could contribute to limiting cell death, promoting recovery mechanisms, and ultimately improve stroke outcome.

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40 The remainder of this chapter focuses on properties of the two proteins for enhancing stroke therapy strategies. Plasma GSN and i schemic stroke Gelsolin (GSN ) is a ubiquitous protein ( 156, 157) and is also known as brevin or actindepolymerizing factor ( 158) It was first discovered and isolated from rabbit lung macrophages in 1979. It is a calcium dependent regulatory protein which regulates the reversible gel sol transformations which occur in cytoplasm ( 159) hence the name gel sol in. Originally described as an actin binding protein, GSN exists in both intracellular (cytoplasmic protein, cGSN) and extracellular (a secreted protein or plasma gelsolin, pGSN) forms, and a single gene on human chromosome 9 encodes both cytoplasmic and secreted variants ( 160 ) Alter native transcription initiation sites and selective RNA processing lead to distinct mRNA messages that produce unique protein products. Plasma GSN consists of a single 755 amino acid polypeptide chain (84 kDa) including a 25amino acid N terminal extension ( 160) that distinguishes it from cGSN (82 kDa). Most cells secrete pGSN, however smooth, skeletal and cardiac muscle cells produce larger amounts of pGSN ( 156) It has been discovered a second cytoplasmic isoform named gelsolin 3, 11 amino acid longer than cGSN, which is coded by the same gene as the other isoforms and expressed in cyto plasm of oligodendrocytes in white matter tracts of the rat CNS ( 161) Gelsolin 3 has been shown to involve in myelin formation a nd CNS development ( 161) Gelsolin is also expressed throughout the human CNS, particularly in CSF ( 101, 162) where it mostly comes from ne urons ( 162, 163) and neuronal growth cones ( 164) suggesting that GSN may have a certain role in the brain. Plasma GSN has a relatively long half life of 2.3 days ( 165 ) a nd it circulates in serum with concentrations from 190 to 300 mg/mL ( 2.3 to 3.6 M) ( 166) Plasma GSN plays a vital role in primary tissue injury via actin binding activity A ctin, the most abundant protein in mammalian cells has fundamental roles in cell integrity,

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41 structure, and mobility. M onomer ic globular actin (G actin ), 42 kDa, can noncovalently (and reversibly) polymerize into filamentous actin ( F actin) under physiological conditions ( 167) W hen G actin is released from damaged or dying cells its polymerization is thermodynamically liberated in the extracellular space leading to toxic F actin formation Rats receiv ing an i ntravenous injection of large quant ities of G actin die quickly with symptoms of pulmonary venous obstruction by actin filaments, pulmonary microthrombi, and endothelial injury ( 168 ) Human actin containing sera dir ectly induce toxic ity to endothelial cells ( 169) Long actin filaments increase the blood viscosity, interfere with microcirculation and cause the development of secondary tissue injury GSN, a part of an actin scavenger system, has three distinct actin biding sites ( 170172) and m ay prevent the se toxic effects Similar to cGSN, pGSN can actively bind ( association constants, Ka = 109/mol/L) to the barbed end of F actin capping it to prevent the addition of monomers ( 171) and bind to the sides of F actin and s ever actin filaments. Th ese interactions are regulated by Ca2+ ion (at micromolar levels), pH (<6.5), and polyphosphoinositides ( 173) Consequently, pGSN can depolymerize F actin and remove it from circulation, thus prevent ing actin exposure toxicity, an increase in blood viscosity and possible blood flow disturbance ( 168) Therefore, massive actin released from damaged tissues can result in pGSN depletion. Inde ed, there is abundant evidence demonstrating that depletion of pGSN is asso ciated with various clinical conditions T here has been consistent observation of lowered levels of pGSN in hepatic failure ( 174, 175) acute lung injury ( 176 177) malaria ( 178 ) cardiac injury ( 175) sepsis ( 179, 180) and major trauma ( 181) Moreover, p revious studies have implicated that pGSN in CSF is a marker for a number of neuropathologies. For example, gelsolin levels in Alzheimer patients CSF w ere sig nificantly reduced compared to healthy age matched control s ( 145 ) Another example is GSN c68, the 68kDa carboxyl terminal fragment of

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42 gelsolin identified as a source of the insoluble peptide in familial amyloidosis of the Finnish type (FAF), was found in the CSF of a subject diagnosed with FAF ( 146) It has been reported that CSF gelsolin concentration is varied in certain neurological conditions, i.e. under normal conditions CSF gelsolin concentration is approximately 5 g/mL ( 182) ; is 2.1 0.7 g/mL in patients with multiple sclerosis and is 1.95 2.9 g/mL in patients recovering from a subarachnoid hemorrhagic stroke ( 183) The vari ation of CSF gelsolin concentrations indicates that it may play an important role in managing inflammation and/or other biological processes in the CNS. A recent study has shown that pGSN levels in stroke patients are significantly reduced compared to controls, a nd it can be a biomarker for prognosis after ischemic stroke ( 184) Gelsolin has also been shown to be involved in actin reorganization in microglia, promoting neuronal regenerative processes following brain inflammation. These processes include the production of anti inflammatory cytokines, synaptic stripping, and recruit ment of neurons and astrocytes to the damages area ( 185) Moreover, it has been demonstrated that gelsolin mediated actin depolymerization results in reduced calcium influx through NMDA receptors and voltage dependent calcium channels, and serves a neuroprotective role following excitotoxic and ischemic insults ( 186, 187) Plasma GSN has anti inflammation propert ies by interacting with potent inflammatory mediators C atastrophic actin leaked from cellular injury may induce pro inflammatory cytokine production (e.g. TNF impair the microcirculation as well as damage multiple organs ( 168, 188, 189 ) Actin binds to pGSN causing local and systemic pGSN depletion that allow s localized mediators to be released and initiate systemic inflammatory response s Recombinant pGSN has been shown to be protective against acute inflammatory response associated with tissue injury ( 190) Besides binding to actin, pGSN also binds to a

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43 number of bioacti ve lipids including lysophosphatidic acid (LPA), lipoteichoic acid (LTA), and lipopolysaccharide (LPS) ( 87, 191193 ) Lysophosphati dic acid has multiple biological functions, such as a lipid mediator as well as a precursor in phospholipid biosynthesis Indeed, pGSN binds LPA with an affinity Kd = 6 nM ( 194 ) and LPA inhibits the F actin severing activity of pGSN ( 87) It has been reported that pGSN inhibits PAF m ediated P selectin expression by 77% ( 195) Plasma GSN also strikingly inhibited PAF induced superoxide anion production of human peripheral neutrophils ( p olymorphonuclear neutrophils, PMN) in a concentration dependent manner ( 195) Platelet activating factor (PAF, 1 O alkyl 2acetyl sn glycero 3phosphocholine), is a potent, pro inflammatory phospholipid with diverse physiological and pathological effects ( 126) PAF does not circulate in the blood under normal conditions, but it is upregulated in septic and trauma pati ents, and PAF concentration may increase further at the site of local inflammation ( 141) The ability to inhibit LPA and PAF bioactivity suggests that pGSN has anti inflammatory activity during acute inflammation. Plasma GSN is a substrate for MMPs ( 196) Matrix metalloprotein ases zinc dependent endopeptidases, are upregulated during the inflammatory response ( 197) and in stroke ( 198) It has been demonstrated that, in the fluids of a burn wound, there was an inverse correlation between pGSN and metalloproteinase levels; gelsolin proteolytic fragments of 49 kDa were also detected ( 197 ) probably because pGSN is cleaved by MMPs and gelsolin fragments also play a certain role in the process. Plasma GSN has anti apoptotic activity. It has been documented that changes in dynamics of actin cytoskeleton result in the release of ROS and subsequent programmed cell death ( 199) It is also well known that GSN is a cytoskeletal regulator and has roles in apoptosis.

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44 GS N is a substrate for caspase3, a key mediator of apoptosis and N terminal GSN fragments have been shown to promote morphological changes in numerous cell types ( 200) and it is implicated that the GSN fragment s are effectors of apoptosis However, the full length of GSN suppresses apoptosis by forming a complex with phosphatidylinositol 4,5bisphosphate (PI(4,5)P2) which inhibits caspase 3 and 9 activity ( 201 ) It has also been shown that GSN inhibits apoptosis by blocking the loss of mitochondrial membrane potential and preventing caspase 3 activation ( 202 203) In several models of neuronal cell death GSN has demonstrated protect ion against excitotoxicity induced apoptosis by altering the actin cytoskeleton in response to Ca2+ influx ( 203) GSN knockout neurons have augment ed cell death and are susceptible to glucose/oxygen deprivation. In support of the protective effect of GSN in vivo, GSNknockout mice have shown to be more vulnerable to brain ischemia ( 187) Therefore, increasing GSN expression may ameliorate neurodegeneration during ischemic stroke. Most studies indicate that the restoration and/or increase in the levels of pGSN in injured animals have beneficial effects. Further, Critical Biologics Corporation has investigated recombinant human pGSN (rhupGSN) as a replacement therapy for the treatment of hypogelsolinemia in high risk patients presenting with end stage renal disease (ESRD), cystic fibrosis patients, and other critical care complications. ( http://www.lehigh.edu/~inbios21/PDF/Fall2008/Palmer_11032008.pdf ) Alpha 1 antitrypsin and ischemic stroke Alpha 1antitrypsin (AAT or A1AT 1antitrypsin), also known as alpha 1 proteinase inhibitor ( A1PI) was first isolated in 1955 and the name antitrypsin came from its ability to inhibit pancreatic trypsin ( 204) 1 part of the name comes from the alpha band of globulins formed dur ing protein electrophoresis and is part of subregion 1 of that band. AAT, a serpin (serine prote nase inhibitor ), is a globular, single chain 394amino acid glycoprotein of 52

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45 kDa that is about 15% carbohydrate ( 205) AAT is produced primarily in hepatocytes and re leased into blood circulation through the liver ( 206) It is also produced by other tissues, such as Paneth cell s of the gastrointestinal tract, kidney, and lung ( 207, 208) In normal conditions, concentrations of AAT in serum are between 1.0 to 2.5 mg / m L ( 2050 M) ( 206) in CSF is approximately 7.6g/mL ( 209) and it is fairly stable with a half life of 4 5 days in humans ( 206) It can cross the plasma membrane o f several types of cells and exert distinct extracellular and intracellular functi on s. For mice, serum AAT peaks within 10 min., and the half l ife is similar to humans after intraperitoneal (i.p.) or subcutaneous (s.c.) injection ( 210) A AT has anti inflammatory propert ies A AT is a primar y serpin in the blood and is a major protector against proteolytic digestion by human neutrophil elastase (HNE) through inhibiting HNE at the associate rate constant (Ka) of about 107M1S1 ( 211) The result ant complex renders the proteinase i nactive and the complex is then removed from circulation ( 212) Serin e proteinases and their inhibitors play important roles in several inflammatory processes including blood coagulation, fibrinolysis, and complement activation. A AT is a primary acute protein with potent anti inflammatory activities, such as the efficient inhibition of neutrophil elastase and proteinase s as well as activity against cathepsin G thrombin, trypsin, and chymotrypsin ( 213) Many of these proteinases target receptor proteins related to proinflammatory cytokine expression and cell signaling ( 214 ) Human AAT (hAAT) appears to be an inhibitor of both extracellular matrix degrad ation and PMN influx in the lung s of the m ice exposed to the cigarette model ( 215) Similar matrix degradation and infiltration occurs in inflammation following stroke and AAT may be able to interfere with matrix degradation and leukocyte infiltration that promote s edema, hemorrhage, and neurotoxicity. AAT is one of many acute phase proteins (APP) (e.g. C 1 glycoprotein (AGP), and

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46 fibri nogen (FIB)) elevated in human blood in response to transient brain ischemia ( 216) The inflammatory reaction following stroke may exacerbate the clinical outcome of stroke patients and these patients require treatments for inflammation ( 216) It is not known whether these elevated AAT levels correlate with functional outcome, but AAT serves as a major an tiinflammatory serum protein. It inhibits the production of proinflammatory cytokines interleukin (IL) 6, IL 8 and TNF inflammatory cytokine IL 10 production through increasing cellular cyclic adenosine monophosphate (cAMP) l evels ( 217, 218) This subsequently inhibits the leukocyte recruitment in in flammation and it is independent from AAT protease inhibitor activity AAT mediated interruption of IL 8 binding to its receptors limits neutrophil infiltration in lungs ( 219 ) AAT has also been shown to decrease neutrophil influx and TNF ( 220) Therefore, AAT may similarly act to reduce the recruitments of inflammatory mediators to inflammatory sites in the brain, preventing the organ damage. AAT has anti apoptotic and cytoprotective effects In addition to inhibiting serine protea ses, AAT also inhibits cysteine prote i nase, e.g. caspase3 that plays a central role in apoptosis ( 221, 222) For instance, AAT has been demonstrated to restrict apoptosis by internalizing into alveolar cells (lung cell s ) and interact ing with caspase 3 ( 223) It has been shown that AAT c an prot ect pancreatic cells against apoptosis in vitro and in vivo via caspase 3 inactivation and is a potenti al therapeutic for diabetics ( 222, 224) in animal models of ischemia in the kidney ( 220) as well as in liver cells in a model of hepatic ischemia/reperfusion injury ( 225 ) It has r ecently been documented that caspase 8 and caspase 3/7 are involved in activation of microglia and subsequent inflammatory mediated neurotoxicity ( 226) This suggests that AAT anticaspase activity may be highly significant in limiting stroke pathology

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47 that results from intrinsic inflammatory processes. Furthermore cathepsin and calpain are hypothesized to play key roles in autophagic cell death ( 227) and AAT, a member of the serpin family, can inhibit calpain and cathepsin therefore reduce apoptosis. Cytoprotection is critical in protection of health y tissue s immediately following an initial ischemic insult, preserv ation of organ functions, and prevention of organ failure. Therefore AAT could be a good candidate for ischemic stroke. Taken together with its antiinflammatory, anti apoptotic, anti hemorrhagic, and potential antioxidant properties ( 228, 229) and by inhibiting enzymatic activity of granzymes, calpains, and caspases AAT appears as a promising therapeutic agent for ischemic stroke. AAT is approved by the FDA for use in treating disease s associated with insufficient AAT activity usually due to genetic vari ants. It has an excellent safety profile based on growing use in chronic obstructive pulmonary disease and genetic insufficiencies, with no apparent cytotoxicity ( 218, 230) Three commercial formulations available Prolastin, Zemaira, and Aralast ( 231) are purified extracts from human plasma and delivered intravenously. Hypothesis The central hypothesis of this dissertation is that outcomes following ischemic stroke will be improved by delivery of pGSN and hAAT. This will be tested in a rat model using both localized and systemic deliver y.

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48 CHAPTER 2 M ETHODOLOGY Ischemic stroke occurs most common ly in the vicinity of the middle cerebral artery (MCA) in humans. Data coming from experimental animal model s delineate important mechanisms and the development of novel therapeutic strategies because of the difficulty in obtaining information from humans in terms of expense time, and ethic s in certain circumst ances. An e ndothelin1induced middle cerebral artery occlusion model has been shown to produce well defined areas of infarct, is reprod ucible, a minimally invasive procedure, and has the ability to achieve reperfusion in rats. This method can be utilized with the management of a laser Doppler imaging system to ascertain perfusion/reperfusion in cerebral ischemia. The outcome parameters are defined by neurological status ( motor and sensorimotor functions) and ischemic brain lesion quantification (either in vivo with magnetic resonan ce imaging (MRI) or post mortem with brain tissue staining). Rats Used as an Animal Model for Focal Ischemic Stroke Studies in human stroke are extremely limited due to the difficulty in collecting post mortem tissues at different time points after stroke onset where neuronal death occurs Therefore, ischemic stroke res earch concentrates mainly on experimental ischemic stroke in animal models. There are many animal models to study mechanism s and neuroprotective strategies. Using the appropriate animal models is very important to predict the efficacy of drugs that may ben efit humans However, each animal model has its own advantages and disadvantages. Rats were selected as animal model for the specific objectives of this project because the following reasons Rat m odel s have been shown to approximate human conditions well, such as cerebral anatomy and physiology, ability of analyzing physiology and brain tissues, genetic homogeneity within strains, low cost, and public and institutional acceptability in term of ethical aspect

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49 compared to large animals ( 97, 232, 233) Lar ge animals (higher species) are often used in later studies once positive results are achi e ved in small animal models prior to clinical trials (recommendations of Stroke Therapy Academic Industry Roundtable, STAIR, published in 1999). S troke outcome measur ements include testing motor and sensorimotor functions corresponding to brain damage as well as evaluating infarct size The r at has a moderate size which permits easy monitoring of physiologic parameters and evaluati on of brain specimen s ( 234) Somatosensory deficits are often present in humans following stroke ( 235) ; this ha s commonly been reported in rats as well ( 236, 237) The moveme nts of a human required to reach for a target object are similar to rats ( 238, 239) Tests have been established which measure motor and sensorimotor deficits acquired following focal brain damage, including t he cylinder and vibrissae stimulated forelimb placing tests ( 240, 241) There are also several issues regarding the use of rat s in animal models for stroke disease in humans. The se include the infarct size, reperfusion, and functional recovery Stroke can occur when a small or large artery is occluded which is associated with small or large infarct volume which the arteries suppl y, respectively. Human strokes are often relatively small (range 28 80 mm3) ( 13) while animal models usually ind uce bigger infarct volumes ( the infarct size of in Sprague Dawley rat MCAO ranges from 60 400 mm3) ( 242244) Therefore, the findings from animal models may introduce a potential bias for large clinical trials including stroke patient s with less sever e and hence smaller infarct volumes particularly in the first ever stro ke group ( 245, 246) In addition, the ratio of the volume of gray matter to white matter in the cerebral hemisphere in h umans is smaller than that of rats and preclinical studies have extensively examined neuroprotective gray matter in rats ( 247) Neuroprotective agents which

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50 work on gray matter do not necessarily have the same effects on white matter, which makes up a large proportion in the human brain. Reperfusion occurr ing in stroke patients comes primarily from three sources. First, approximately 1518.8% of all strokes have early spontaneously recanalization as early as 6 8 hours from stroke onset ( 248, 249) Second, collateral blood flow via the circle of Willis and leptomeningeal vessels has been observed using a regional angiographic system ( 250, 251) Last, the thrombolytic agent, tPA (intravenous injection within 34.5 hours from stroke onset ) or the intravascular clot retrieval devic es, Ancrod ( 252) can promote reperfusion. Moreover, leptomeningeal vessels have been shown to provide blood flow in the penumbra and improve stroke outcome ( 253, 254 ) Recovery after stroke includes at least three processes: resolution of acute tissue damage, behavioral compensation, and neuroplasticity ( 255) Cellular reparation within the penumbra involves axonal sprouting to establish new patterns of cortical connections ( 255) newly generated neurons possibly to replace losses in the penumbra ( 256, 257) and reorganization of cortical maps ( 258) In fact, connection between animal models to these areas is not wellestablished as animal models often produce a wide spread damage to many d ifferent brain regions, such as rost r al and medial frontal cortex, lateral temporal cortex, and occipital cortex ( 259261) therefore it is difficult to study for the patte rns of cellular reparation following stroke. Functional recovery of stroke involves behavioral deficits. However, most experimental studies focus on reducing damage areas in rodent models As mentioned above human functional deficits after stroke occur with damage to specific circuits, including cortical maps The recovery of motor sensory ( 262) and language ( 263) involves a progressive reparation and recovery of the penumbra, and beh avioral compensation following stroke ( 264, 265)

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51 Endothelin1Induced Middle Cerebral Artery Occlusion Model Human ischemic stroke in origin is more prominent than hemorrhagic stroke and mainly caused by occlusion of MCA Therefore MCAO model s are the most widely used in stroke research, with the goal of evaluating of new treatments ( 266, 267) Rat models have been shown to produce well defined areas of infarct within the neocortex and caudate nucleus which are feasible for quantification ( 268) MCAO results in a reduction of CBF in both the cortex and striatum, however, the degree and the reduction of blood flow depend on the duration of MCAO, the site of occlusion along the MCA, and the amount of the collateral blood into MCA territory. MCAO models can be pe rmanent, or temporary to allow reperfusion, depending on the study objectives. Permanent ischemia models al low the study of cerebral ischemia without interference of reperfusion effects, temporal/transient ischemia models allow investigating reperfusion injury which often occurs in human (due to recanalization or thrombus disintegration) ( 269, 270) It is understandable that there is no ideal ischemic stroke model due to the diversity of human stroke itself. The best model selected for investigating therapeutic agents should s atisfy the following criteria: (A) simulating the physiological conditions in human stroke; (B) creating re producible lesions; (C) employing relatively simple technique s and minimal invasive ness ; (D) showing the capability of monitoring physiological parameters; (E) allowing outcome measurements, such as behavior evaluations and brain analysis; and (F) l ow cost ( 271) The dissertation studies employ MCAO induced by e ndothelin1 (ET 1) a potent vasoconstrictor peptide, in rats as a model to evaluate the effects of two proteins, pGSN and AAT. Microinjection of ET 1 adjacent to the rat MCA has shown a reproducible pattern of focal cerebral infarction ( 272) The technique is relatively easy to perform (i.e., intracerebral injection follows stereotaxic coordinates) and minimizes the surgery complications ( 33) The d ata that have been collected from the present project include behavior tests and brain damage

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52 measurements using histology and MRI. Further discussion about advantages and disadvantages of the model are detailed in Chapter 5. ET1, a small peptide belongs to an endothelin (ET) peptide family. The ET family includes three 21amino acid peptide s ET1, 2, and 3 ( 273) ET1 is the predominant isoform in vasculature and has the most potent endogenous vasoconstrictor agent (the order of potency is ET1 > ET 2 >> ET 3). ET 1 acts in both paracrine and autocrine fashion by interacting with ET receptors in vascular smooth muscle ( VSM) and endothelial cells, thereby modifying vascular function ( 274) There are three known ET receptors, ETA, ETB, and ETC. ETA and ETB receptors are widely expressed in VSM cells (VSMC s ) however, the ETA receptor appears to be the predominant subtype in medial VS M C s The mechanism of vasoconstriction of ET 1 is its bi nding to the specific cell surface ETA receptor in VSMC, which induces G proteindependent stimulation of phospholipase C, leading to the formation of inositol 1,4,5triphosphate (IP3) and diacylglycerol (DAG) by hydrolysis of phosphatidyl inositol. IP3 upregulates the intracellular concentration of calcium (Ca2+), which in turn results in vasoconstriction ( 275, 276) In healthy adults, ET 1 concentration s in basal plasma are 0.7 5 pg/mL ( 277, 278 ) Studies of the kinetics of ET 1 clearance in a threecompartment model showed an approximate terminal half life of 455 minutes. It has been shown that ET1 presents long lasting action in many in vitro vascular tissue preparations e.g. cerebral, cor onary, and mesenteric arteries ( 279283) Furthermore, ET 1 injection in a dose dependent manner, in vivo directly into the rat brain parenchyma with induces local CBF to ischemic levels ( 33, 284286) Materials Rat ET1, MW = 2492.0, purchased from American Peptide C ompany, Inc, (CA, U.S.A.) was dissolved in sterile phosphate buffered saline (PBS) to make a stock concentration of 80 M (80 pmol /l). The stock solution was stored at 20oC. ET1 was thaw ed centrifuged, and placed

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53 on ice until ready to inject. Human pGSN 1 mg/mL in sterile saline stock solution, was a generous gift from Critical Biologics Corporation (MA, U.S.A.) Artificial cereb rospinal fluid (aCSF) was obtained from Fisher Scientific, Inc. (PA, U.S.A.) Human AAT (alpha 1 protein inhibitor, Prolastin C) dissolved in sterile water, was from Grifols Inc. (NC, U.S.A.) Other materials, such as phosphate buffer saline (PBS), saline, formalin, and isoflurane were purchased from Fisher Scientific (NH, U.S.A.) Experimental Procedures Animal Model and Treatments All procedures were performed with prior approval from the University of Florida Institutional Animal Care and Use Committee ( IACUC ). M ale SpragueDawley rats, 78 weeks in age, (weight 220250 g ) were purchased from Charles River Laboratories International, Inc. (MA, U.S.A.) Pair housed r ats were maintained in controlled environments on a 12/12 hour light/dark cycle in plastic c ages, received ad libitum food and water. The rats were acclimated for three or more days before the start of any experiments during the light phase Animals with incomplete dat a set s due to death, and nonresponse to behavior test s are eliminated from the study data. A model of transient/reversible middle cerebral artery occlusion (MCAO) using ET 1 was performed as previously described ( 287) Briefly, animals were anesthetized ( induced with 5% isoflurane in 1 L/min oxygen and maintained at 1.52.0% and 0.5 L/min) placed prone in a stereotaxi c frame (Braintree Scientific, Inc., MA, U.S.A.) and secured in the flat skull position. Under aseptic conditions, t he scalp was then retracted and a midline incision was made in the skin from the point above bregma until the lambda point, using a stainles s steel sterile blade. A small hole (3 mm i.d.) was drilled in the cranium adjacent to the left MCA at 0.2 mm anterior, 5.2 mm lateral ( 33) and 1.0 mm from the skull bottom ( 3 L of methylene blue was injected in

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54 other animals with the same coordinates to verify the injection site then the brain was sectioned to expose the MCA and the injection site was visualized, data not shown). A 27 gauge needle was used to inject ET 1 or pGSN or hAAT at a rate of 1 L/min. After ET 1 injection, the needle was left in place for an additional 3 min before being slowly withdrawn. The injection system was flushed by distilled water and loaded with subsequent treatment agents. The detailed schedule for treatments is described in tables 21, 22 and 23. Body temperature was maintained between 35 C and 37C throughout surgery, using a circulating water blanket (YSI telethermometer and Gaymar T pump system) Respiratory rate and general skin color were observed as an indirect assessment of heart rate. The scalp was then closed with suture wound clips, or thread wound sutures for the group subjected to MRI later. The animals were kept at 37C until totally rec overed. After full recovery, a nimals were returned to their home cages conditions for outcome evaluation 72 h after surgery During the acute post operative period and anytime that there is any acute concern, any animal displays the signs of distress or a loss > 15% body weight, animals will be treated or e uthanized following the approved protocol. Laser Doppler Perfusion Imaging Laser Doppler imaging system (MOORLDI system at the Department of Pharmacology and Therapeutics, University of Florida) was used to measure the relative flux/perfusion of the brain which is strongly associated with the spatial and temporal characteristics of the changes in cerebral blood flow (CBF) response ( 288290 ) The principle of the technique is that the dynamic blood flow in the vasculature results in a Doppler frequency shift of the scattered laser light, which is photodetected and then processed to build a color coded ma p of blood flow. The technique allows for repeatedly assessing perfusion changes over a wide brain by scanning a low power laser beam across the skull ( 290)

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55 Imaging studies measuring sequential perfusion changes after ischemia often require the animals to be immobilized for long periods of time (at least 2.5 h) I sofluran e a volatile anesthetic, has been shown to reduce early neuronal death in animal models of focal cerebral i s chemia ( 291) Therefore animals used in the s e study exposed to isoflurane for approximately 3 h, were not used for testing the effects of the treatment age nts to avoid the influence of isoflurane, and were sacrificed following the last measurement (survival time was about 4 h) The s calp was retract ed from animals head and t wo holes (~3 mm i.d.) were made in each hemisphere. The treatment details were exactly the same as the treatments in the Animal Model and Treatments above, except that an aCSF group was not included, there were three animals per group, one animal died during imaging due to isoflurane sensitivity (overdosed) and was replaced The i maging was performed at four different time points: pre injection, 1020 min, 3035 min, and 5565 min after ET 1 injection The pseudocoloring of relative flux (number of red blood cells multiplied by speed) illustrates cool colors for relative low flux and warm colors for relative high flux The 16bit color scans were made with an arbitrarily assigned unit from 0 (lower limit) to 1,000 or more (upper limit). The scan speed was 10 ms/pixel and the total scan (scan area was about 1.8 cm x 2.3 cm) duration per a nimal was approximately 10 min. Actual blood flow is highly correlated to flux except at supranormal pressures ( 292 ) Indeed, percentage reductions of perfusion on the i njected side of the brain were calculated using the following equation 21. Flux ( % ) = Fi Fc 100 ( Equation 21) where Fi is the flux value of the ipsilateral side (injected side) and Fc is the flux value of the contralateral side (opposite or unaffected side).

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56 Behavioral T ests In brain models of focal ischemic stroke, the sensorimotor cortex or striatum in one hemisphe re is frequently damaged with severe injury compared to other area of the brain. Unilateral injury to the forelimb region of the rat brain motor and sensorimotor cortex causes immediate deficits in the somatosensory functions. The deficit in the use of the cont ralateral forelimb can be measured ( 293) The behavioral tests such as cylinder and vibrissae tests, allow assessing motor and sensorimotor asymmetries. These tests have been useful in studies of recovery of function following central nervous system (CNS) injury as well as for evaluation of pharmacological i nterventions ( 294299) Cylinder test Cylinde r test is also known as forelimb use for vertical lateral exploration test The test examines the levels of preference for using the nonimpaired fore limb for weight shifting movements during spontaneous vertical exploration of the walls of a cylinder after unilateral cerebral ischemia. The test encourages the rodent (rats in th is study) inside a specially designed cylinder (40 cm height, 20 cm i.d.) to use the walls for upright support and vertical exploration. The cylinder walls reveal forelimb asymmetries that have resulted from different forms of brain injury, including cort ical damage ( 300) and nigrostriatal neurodegeneration ( 240) A video camera with slow motion playback capability records (3 min each animal) the number of times the animal uses the ipsilateral (affected) or contra lateral (less affected) forelimb alone or uses both simultaneously for upright support. Intact animals typically use both limb s equally for upright support. But after damage to the motor system, animals show an asymmetric reliance on the less affected (ipsilateral) limb An experimenter blinded to treatment conditions calcul ates the

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57 percentage of two forelimb s used from the number of total attempts for upright support and vertical expl oration. Vibrissae test Vibrissae test is also c alled vibrissaeevoked fore limb placing (unskilled reaching for a stable surface) test The test studies the sensorimotor/proprioception deficits. To determine whether an animal has asymmetrical sensorimotor perception, the test is designed to hold the anima l by the torso with its forelimb s hanging freely, and then slowly move the animal laterally toward the edge of the table or countertop until the vibrissae of one side make contact with the edge. Intact anim als typically quickly place the ipsilater al forelimb on the edge of the surface when the ipsilateral vibrissae brush the table edge. In contrast animal s with damage to the motor system often respond slowly ( or do not respond) to vibrissae stimulation on t he ipsilateral side compared to contralateral side ( 240) A camera records ten iterations of vibrissae stimu lation f or each side and the corresponding responses. An experimenter blinded to treatment conditions calculates the time between vibrissae stimulation and "placing response" for each side us ing Window s Movie M aker 2007. Histopathology Among a number of histological methods, the 2,3,5triphenyltetrazolium chloride (TTC) staining assay is one of the most commonly use d to measure ischemic lesion s The TTC assay has been shown to be reliable and consistent compared to other traditional histologic markers, e.g hematox y lin and eosin (H&E) assay ( 301 ) thionin staining ( 302) and cresyl violet staining technique ( 303) TTC is a white crystalline powder and soluble in water. It is enzymatically reduced to red lipid soluble compound, 1,3,5triphenylformazan (TPF) i n living tissues due to the activity of various dehydrogenases (important enzymes in oxidation of organic compounds,

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58 abundant in the inner membrane of mitochondria ( 304) ) Thus, pale or white TTC tissues indicate dead tissues, since these enzymes have been denatured or degraded. Animals in model and treatment groups were allowed to survive for 3 days. The brains were quickly isolated, placed in cold PBS (0 4oC) for 30 min and then sectioned into 2mmthick coronal slices using a brain slice matrix (Leica Microsystem, IL, U.S.A.) The tissue sections were held in cold PBS for 3 min before they were incubated in TTC solution (0.05% TTC in PBS) for 30 min at 37oC. The sections were washed three times (one minute each) with PBS and fixed in 0.1 M phosphate buffered formaldehyde (PBF). Calibrated digital images of tissue sections were made at 600 dpi scanner resolution with 48bit color and saved as TIFF files. The infarct areas were quantified by visual thresholding of TTC labeled (normal) and unlabeled (infarct) tissue, and the measurement of each area was completed using Image J version 5.0 (NIH). Infarct volume was determined as the sum of the infarct areas of all sections of each brain. The ratio of average infarct volumes of treated rats to untreated rats was used as a dependent measure for evaluating pGSN or hAAT effects. Magnetic R esonance I maging A m a gnetic resonance imaging (MRI) scanner uses information about the movement of the water molecules to determine the infarct after ischemic stroke in living subjects. Measurement of infarct volume in ischemic str oke patients using MRI have shown that the tec hnique is an accurate predictor of clinical outcome in human s and it is a relevant measure to use to assess the outcome of therapy both in human and animal studies ( 305) Three days following ET 1 induced ischemic stroke, damage was visualized in live animals from the groups ET1 alone (N = 8) and ET 1+ hAAT (i.v.) (N = 14) maintained under 1 2% isoflurane anesthesia using Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) facility in the McKnight Brain Institute of the University of Florida. An 11T/470 MHz

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59 MRI Spectrometer (Magnex Instruments, U .K .) using a Bruker Avance Console and Paravision software (Bruker BioSpin MRI, Inc MA, U.S.A.) were used to generate a standard T2 weighted spinecho sequence (rapid acquisition with relaxation enhancement) through continuous 1mmthick s ections covering the entire brain region. Body temperature was monito red and maintained at 35 37C for the entire scan time with heated ventilation. Total acquisition time for MR imaging was about 35 minutes. Image analysis and infarct area quantification are done by Jim version 5.0 and Image J. An experimenter blinded to treatment conditions calculated the total infarct areas of animals. Image Analysis Infarct areas of brain slices from all experiments, including from TTC assay and MRI imaging were analyzed in a blinded manner using the NIH Image J version 5.0. Regions of interest (ROI) were created encompassing infarct areas of each sections (in mm2), a nd total infarct volume were the estimated using the sum of infarct area of all slices of each animals Statistical Analysis Unless specified, all data are expressed as arithmetic means s.e.m. Two way ANOVA (time and treatment) was followed by Bonferroni post hoc test to compare behavioral dependent variables between groups. Comparisons of infarct area were made by two tailed Students ttest; and pvalues less than 0.05 were considered to be significant.

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60 Table 2 1. Treatment schedule for laser Doppler perfusion imaging Group Treatment Number of animals Survival rate Date collected ET 1 alone 3 L of ET 1 (i.c.) 3 3/3 3 ET1+pGSN 3 L of ET 1 (i.c.) 3 3/3 3 3 L of pGSN (i.c.) ET1+hAAT(i.c.) 3 L of ET 1 (i.c.) 3 3/3 3 3 L of hAAT (i.c.) ET1+hAAT(i.v.) 3 L of ET 1 (i.c.) 3 3/3 3 1 mL of hAAT (i.v.) Table 2 2. Treatment schedule of for aCSF, ET 1 alone, ET 1+pGSN, and ET 1+hAAT (i.c.) groups Group Treatment Number of animals Survival rate Date collected ET 1 alone 3 L of ET 1 ( i.c.) 3 3/3 3 ET1+pGSN 3 L of ET 1 (i.c.) 3 3/3 3 3 L of pGSN (i.c.) ET1+hAAT(i.c.) 3 L of ET 1 (i.c.) 3 3/3 3 3 L of hAAT (i.c.) ET1+hAAT(i.v.) 3 L of ET 1 (i.c.) 3 3/3 3 1 mL of hAAT (i.v.) Note: Two animals in ET 1 alone group were excluded because of death and incomplete data sets. One animal in ET 1+pGSN group was excluded due to difficulty of defining the infarct area. Table 2 3. Treatment schedule for ET 1+saline and ET 1+hAAT (i.v.) groups Group Treatment Number of animal s Survival rate Date collected ET1+saline 3 L of ET 1 (i.c.) 8 8/8 8 1 mL of saline (i.v.) ET1+hAAT 3 L of ET 1 (i.c.) 12 12/12 12 1 mL of hAAT (i.v.)

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61 CHAPTER 3 THE PROTECTIVE EFFEC TS OF PLASMA GELSOLI N ON ISCHEMIC STROKE OUTCOME IN RATS1Introduction Stroke or brain attack occurs when the blood supply to the brain is interrupted, usually because a blood vessel is blocked by a clot or loses structural integrity permitting hemorrhage. The disease is not subject to a particular race or ethnic group ( 306) In 2009, 795 000 strokes occur red in the United States, i.e. a stroke occurs once every 40 seconds and a death occurs every 4 minutes ( 10) According to the Centers for Disease Control and Prevention (CDC), the total cost of stroke was $68.9 billion and the number is expected to rise. Of all strokes, 87% are ischemic ( 10) Currently, recombinant tissue plasminogen activator (rtPA) is the only FDA approved therapeutic agent for ischemic stroke. rt PA is effective only if intravenously administered within 3 to 4.5 h of stroke onset, and can have adverse neurotoxic effects even with proper use ( 122) The drug can only be used within a narrow time window after a stroke begins and only about two percent of stroke patients are able to access rtPA therapy. Therefore, development of new agents for stroke is essential. The mechanisms involved in stroke injury and repair are extremely complex, involving e xcitotoxicity and necrotic cell death occurring within minutes of stroke onset ( 307) As well, there is increasing evidence showing that genetically programmed cell death during post ischemic tissue inflammation (that can last days to weeks) has a detrimental effect ( 308, 309) Therefore, therapeutic strategies targeting that delay or dampening inflammatory responses could inhibit the progression of the tissue damage and improve the overall out come of stroke. 1 This part is mainly a manuscript that was published in Experimental & Translational Stroke Medicine, 2011, 313

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62 Gelsolin (GSN) is a ubiquitous ( 156, 157) actin filamentsevering, capping and actin nucleation protein of eukaryotes. Originally described as an actin binding protein, GSN exists in both intracellular (cytoplasmic protein, cGSN) and extracellular (a secreted protein or plasma gelsolin, pG SN) forms. pGSN, also known as brevin and actindepolymerizing factor, consists of a single 755 amino acid polypeptide chain (84 kDa) including a 25amino acid N terminal extension ( 160) that distinguishes it from cGSN (82 kDa). Most cells secrete pGSN, howeve r smooth, skeletal and cardiac muscle cells produce larger amounts of pGSN ( 156) The plasma concentration of pGSN is 200300 mg/L ( 159, 310 311) and isolated human and rabbit pGSN have a plasma half life of 2.3 days ( 165) Because pGSN derives from muscle tissue, it must pass through interstitial fluid of the extracellular matrix to loc alize in the blood. pGSN also exists in human cerebrospinal fluid (CSF) ( 162) Although certain functions for t he intracellular isoforms have been described, the function(s) of the plasma isoforms remain unclear. The high affinity of pGSN for filamentous actin (F actin) (Ka > 109/mol/L) ( 312 ) suggests that its physiological function is likely related t o its actin binding properties. pGSN may scavenge actin leaked from injured tissue and limit subsequent damage instigated by extracellular filamentous actin ( 166) Studies have shown that large amounts of F actin could potentially increase the blood viscosity and perturb blood flow through the microvasculature ( 168) It is also well established that pGSN levels decrease in blood in acute inflammation conditions that involve tissue damage ( 176, 313315) Consistent w ith the idea that pGSN is not only a biomarker for inflammation but also an important protective factor, repletion of pGSN in a mouse model of endotoxemic sepsis led to solubilization of circulating actin aggregates and significantly reduced mortality in mice ( 179 ) GSN knockout mice neurons are vulnerable to g lucose/oxygen deprivation, and pharmacological brain actin depolymerization restored

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63 resistance to ischemic stroke in knockout mice ( 187) The knockout mice results could not determine which endogenous form of gelsolin is responsible, or whether gelsolin in or near the infarct me diates neu roprotective effects. Gelsolin overexpressing transgenic mice demonstrate neuroprotection against experimental stroke ( 187) but it is not known whether these effects are mediated by pGSN or cGSN, or whether it i s GSN near the infarct that mediates the protection. To test the hypothesis that proximal administration of pGSN can antagonize stroke pathology, we induced transient middle cerebral artery occlusion (tMCAO) in male rats via intracranial injection of ET 1, a potent vasoconstrictor, and post treated with discrete brain injection of pGSN. Cylinder and vibrissae tests were used to examine sensorimotor function before and 72 h after MCAO to assess functional deficits. Whole brain laser Doppler perfusion imaging was performed through the skul l to verify MCAO effectiveness. I nfarct volumes were examined 72 h after MCAO using 2,3,5triphenyltetrazolium chloride (TTC) assay. Results Treatment of pGSN d id not I nterrupt ET 1 Induced Artery C ontraction In order to test the effect of pGSN and ET 1 on MCAO, cohorts (n = 3) of rats were injected with ET 1 to induce transient middle cerebral artery occlusion (MCAO). Approximately 5 10 min after ET 1 injection, pGSN or saline was injected at the same l ocation. The time p oints of scanning were based on ET 1 injection time. The relative perfusion unit (PU) or blood flow values of animals before injection were in the range of 8001,600 PU and the difference between two hemispheres of the brain was not st atistically significa nt. After 20 min following ET 1 injection, the flux values on the injection side dropped to the range of 300600 PU. The calculations were made using equation 21 described in the methods. Relative flow values showed a rapid decrease to ~ 50% of baseline i n all animals and flow remained maximally decreased for 20 30 min after ET 1 injection (Fig ure 3 1). ET 1 injection resulted in

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64 a drop in perfusion immediately after injection regardless of pGSN injection at the same location. Reperfusion was observed in both groups after ~ 60 min. These results indicate that pGSN injection did not interrupt the induction of artery contraction by ET 1. Treatment of pGSN S ig nificantly Reduced ET 1 Induced Behavioral D eficits To test the protective effect of pGSN, behavioral studies were conducted. Initially, five rats were randomly assigned into ET 1 injection alone or ET 1 plus pGSN group. In the ET 1 only group, one rat died and one rat was paralyzed after ET 1 injection and was euthanized immediately. In ET 1 plus pGSN tr eated group, one rat did not show any infarction by TTC staining and was excluded. Therefore, three animals in ET 1 group and four animals in ET 1 plus pGSN group were examined for behavioral deficits and brain damage. Cylinder test As shown in Figure 3 2, ET1 induced MCAO resulted in profound impairment of contralateral forelimb function three days following injection. ET1 treated animals showed significant reduction of symmetrical forelimb use during wall exploration (from approximately 80% to 20%), while artificial cerebrospinal fluid (aCSF) injection had no effect. In the pGSN treatment group, the percentage of symmetrical forelimb use was significantly higher than the control group (20% vs. 70%, pvalue < 0.01). The number of attempts to explore the wall was also lower than before surgery (data is not shown). These results indicate that pGSN treatment significantly prevented MCAO induced damage to the motor system. Vibrissae test To test the effect of pGSN on sensorimotor system, we also performed vibrissae testing. As expected, vibrissaestimulated placing of the ipsilateral forelimb was not affected 3 days after experimental ischemic stroke (Fig ure 33A ). Contralateral forelimb placing was significantly slowed (from 1 s to 18 s) in the control (ET 1 alone) group (Fig ure 3 3B). Intriguingly, pGSN

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65 treatment significantly reduced the time of contralateral forelimb placing compared to control group (18 s vs. 9 s, pvalue < 0.01). These resul ts indicated that pGSN treatment significantly prevented the loss of sensorimotor function induced by MCAO Treatment of pGSN Reduced MCAO Induced Brain D amage In the control rats, ET 1 produced large and reproducible unilateral infarcts that involved the rostro central dorsolateral cortex and basal ganglia, corresponding to the full extent of the MCA territory. The infarction volume in the pGSN treatment group was reduced by 49% compared with the control group (Fig ure 34). Sparing was observed in both cor tical and subcortical structures. Discussion The current study reports for the first time the use of pGSN as a protein drug to reduce injury after transient local ischemic stroke. We demonstrate that pGSN can improve sensorimotor recovery in conjunction wi th substantial reduction in infarct volume present three days after stroke. Transient middle cerebral artery occlusion (tMCAO) induced by ET 1, a potent vasoconstriction peptide, decreased relative cerebral blood flow in brain tissue served by the MCA by 5 0% in all groups. In previous studies, injection of ET 1 adjacent to MCA has been shown to reduce blood flow 3075% in the region supplied by the artery including cortex and basal ganglia, and to produce subsequent ischemic neuropathology in these regions of the brain ( 32, 286, 316) Our results demonstrate that after 10 20 min following ET 1 intracranial injection, the ipsilateral cerebral relative blood flow decreased approximately 50% in all groups of animals (Fig ure 3 1), indicating that all animals initially have comparable ischemic strokes and that pGSN did not reduce the magnitude of the original ischemic event, but limited the subsequent

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66 degeneration and associated loss of function. This suggests that pGSN does not interfere with ET1 action on its receptors, and corresponds to studies in which GSN knockout mice had larger infarct volume at 22 h even though reductions in CBF during MCAO were not altered ( 187) Although this study shows that pGSN in or near an infarct can reduce neuropathology and functional loss due to ischemic stroke, the mechanism by which pGSN mediate s protection are not yet clear. One likely mechanism involves actin depolymerization. Upon tissue injury due to glucose/oxygen deprivation (CBF significantly reduced), large amounts of actin can be released from damaged cells into the extracell ular space. Since the ionic conditions in the extracellular fluid favor actin polymerization, high amounts of F actin could be released to potentially increase the viscosity of blood and perturb blood flow through the microvasculature. The actin severing protein gelsolin has a secreted plasma isoform (called plasma gelsolin), which is constitutively active in the high extracellular calcium concentrations of plasma. Plasma gelsolin severs extracellular F actin to short filaments, and by capping barbed ends, prevents polymerization and favors monomer release. Therefore, pGSN acts as debris cleaner limiting inflammation and possibly decrease blood clogging [22] Another possible mechanism is through anti apoptotic activity of pGNS. In Jurka t cells, overexpression of gelsolin inhibits cytokine induced apoptosis ( 317) It has been reported that gelsolin can form complex with phosphatidylinositol 4,5bisphosphate and inhibit caspase 3 and 9 activities ( 201) In addition, pGSN may also play an important role in regulating inflammation Histone deacetylase inhibitor mediated neuroprotection against MCAO has been associated with GSN upregulation and reductions in filamentous actin, neither of which was shown to occur in GSNknockout mice in which the treatment was ineffective ( 318) Also, GSN

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67 can modulate the actin cytoskeleton regulation of numerous ion channels responsible for elevated cytotoxic intracellular calcium and glutamate excitotoxicity ( 186, 319, 320) Gelso lin is regulated by phosphatidylinositol 4,5bisphosphate (PIP2), and contains a lipid signaling binding domain. This domain has been shown to bind to a number of bioactive lipids including lysophosphatidic acid (LPA), lipoteichoic acid (LTA), and lipopoly saccharide (LPS) ( 87, 191193) LPA levels have been shown to be increased in patients suffering ischemic stroke ( 321) LPA signaling has also been shown to regulate a number of proinflammatory genes ( 322) Increasing gelsolin levels during stroke may serve to modulate the inflammatory response thereby offering protection against the inflammation related neurodegeneration following stroke. Further emphasis of the potential importance of GSN in stroke comes from recent reports that circulating pGSN levels are reduced in ischemic stroke suffers and is highly predictive for first year mor tality from ischemic stroke ( 184) Matrix metalloproteinases (MMPs), zinc containing endopeptidases that participate in both normal and pathological processes, are upregulated during inflammatory conditions ( 197) including stroke ( 198) Plasma gelsolin is cleaved in vitro by MMP 3, MMP 2, MMP 1, MMP 14 and MMP 9 ( 323) which may be the cause of the severe depletion of pGSN observed in patients who suffer ischemic stroke. Replacing lost pGSN may interrupt proinflammatory cascade and result in decreased brain damage. Conclusion The current study offers a proof of principle that delivery of pGSN following ischemic stroke results in neuroprotection and can reduce both sensory and motor deficits that arise following stroke. Future research aimed at characterizing improved delivery, dose response, temporal, safety, pharmacokinetic issues, and physiological mechanisms for further preclinical development of this promising strategy are called for.

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68 Figur e 3 1. Perfusion measurements of pGSN study (A) Color photographs of an animal brain recorded at different time points (pre injection, 1020 min, 3035 min, and 6065 min from ET 1 injection time) by a digital camera of laser Doppler system, corresponding closely with the blood flow image. Lookup table shows arbitrarily assigned perfusion unit (PU) from the lower limit 0 to upper limit 1,000 and above. The skulls demonstrate the holes and injection sites. (B) Percentage perfusion reduction as a function of time calculated using equation 21. All r ats were injected with ET 1 (240 pmol in 3 L PBS) proximal to the left middle cerebral artery. About 510 min after ET 1 injection, pGSN (35.71 pmol in 3 L saline) was intracranially injected ( i.c.) at the same site in a group and no injection in the other group. The dotted line represents the average levels in pGSN treated group (N = 3). The solid line represent levels in control group (N = 3). The differences at all time points were not statically significant.

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69 Figure 3 2. Cylinder test of pGSN study Rats were placed in a transparent cylinder for 3 min. Animal forelimb s used during exploration were scored. Each bar represents the average percentages s.e.m. of using both forelimb s in the indicated group. ET 1 group, N = 3, ET 1+pGSN, N = 4,*: pvalue<0.05; **, pvalue < 0.01, ***, pvalue < 0.001.

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70 Figure 3 3. Vibrissae test of pGSN study (A) Time in seconds to ipsilateral forelimb placement on the countertop. (B) Time in seconds to contralateral forelimb placement on the countertop, ET 1 group, N = 3, ET 1 + pGSN, N = 4, **: pvalue < 0.01, ***: pvalue < 0.001. Data are means s.e.m.

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71 Figure 3 4. Infarction area labeled for mitochondrial activity of pGSN study ( 2,3,5triphenyltetrazolium chloride, TTC). ET 1alone group N = 3, pGSN treatment group, N = 4. *, pvalue < 0.05. Four TTC staining slices from two animals, two slices from each animal, one animal from each group, at the area receiv ing the most blood from the MCA. Off white color areas show dead tissues while red areas indicate vital tissues. Data are means s.e.m.

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72 CHAPTER 4 ALPHA 1 ANTITRYPSIN MITIGATED ISCHEMIC STROKE DAMAGE IN RATS Introduction World wide, there are more than 50 million survivors of stroke and TIA ( 324) producing an immense burden on the healthcare infrastructure as well as national economies ( 124) At present, recombinant tissue plasminogen activator (rtPA) is the only FDA approved therapeutic agent for ischemic stroke and is effective only if administered intravenously within 3 to 4.5 h of ischemic stroke onset. The major functions of rtPA are dissolving blood clots and promoting reperfusion. Paradoxically, it can cause neurotoxicity through the N methyl aspartate (NMDA) receptors and disruption of the neurovascular matrix through matrix metalloproteinase (MMP) dysregulation ( 325) Also in the United States, less than 2% of stroke patients are able to access the tP A treatment due to a narrow time window after stroke begins and the diagnostic procedure ( 124) Therefore, more effective therapies are needed in order to improve the outcome of stroke. Ischemic stroke, the dominant type of all strokes, initiates a series of events, including cellular bioenergetic failure, excitotoxicity, oxidative stress, BBB disruption, microvascular injury, homeostatic activation, and inflammation ( 326) The inflammation starts within a few hours of stroke onset and characterizes the secondary or delayed response to ischemia ( 327) It involves activation of micro glia and astrocytes, as well as influx of hematogenous cells recruited by cytokines, adhesion molecules and chemokines. These proinflammatory mediators penetrate activated blood vessel walls and invade into the parenchyma ( 81, 210) Once activated, leukocytes and microglia produce more inflammatory cytokines and chemokines, nitric oxide (NO) via inducible nitric oxide synthase s (iNO S s ), reactive oxygen species (ROS) and matrix metalloproteinases (MMPs). Pro inflammatory mediators and toxic molecules contribute to inflammation response and further potentiate brain injury, leading to apoptosis and necrotic cell

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73 death of the potentially viable tissue. I ncreasing eviden ce show s that genetically programmed cell death during post ischemic tissue inflammation (which can last days to weeks) has a detrimental effect ( 308, 309) Therefore, therapeutic strategies which target that delay or mitigate inflammatory responses, could inhibit the progression of the tissue damage and improve the overall outcome of stroke. Human alpha 1antitrypsin (hAAT) is a serum proteinase inhibitor which has anti inflammatory and apoptotic, and cytoprotective properties. AAT is primarily synthesized in the liver ( 205) secreted into a serum level of 1.0 2.5 m g/ mL (20 50 M) CSF level of 7.6 g/mL at normal condition, ( 209, 230, 328, 329) and is relatively stable with a half life of 45 days in human ( 330, 331) but as an acute phase reactant, the hAAT serum level can rise two to four fold in response to tis sue injury ( 332, 333 ) AAT synthesis mediated by LPS can be raised to eight fold ( 334) and even 100 fold in A549 cells when stimulated with cytokines and transforming growth factor b ( 335) P revious studies have demonstrated that AAT inhibits the production of pro inflammatory cytokines inte rleukin (IL) 6, IL 8 and tumor necrosis factor alpha (TNF anti inflammatory cytokine IL 10 production by increasing cellular cyclic adenosine monophosphate (cAMP) levels ( 217, 218) AAT has shown anti apoptotic and anti inflammatory effects as early as 2 h and as late as 24 h followi ng ischemia/reperfusion in an animal model by inhibiting neutrophil superoxide production, TNF caspase 1 and 3 like activities ( 220, 336) AAT mediated interruption of IL 8 binding to its receptors limits neutrophil infiltration in the lungs ( 337) In addi tion to inhibiting serine prote ases, AAT also inhibits cysteine proteinase (e.g., caspase 3) activity. Coupled with the cells lung kidney and liver cells) against apoptosis ( 222)

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74 Important note that the total protein concentration in CSF is about 200 times lower than in the blood plasma ( 338) and 80% CSF protein is serum derived and 20% is produced intrathecally ( 339) therefore AAT levels increase in neurodegenerative diseases such as, Alzheimer disease or dementia ( 209) may come from peripheral source by crossing BBB. Moreover, recent studies have shown that neuroserpin, a serin protease inhibitor (related to AAT) expressed in neurons ( 340) ex erts a neuroprotective effects after ischemic stroke due to its ability to complex with serine proteases tPA, uPA, and plasmin which are similar to AAT. The negative correlation between the decreased neuroserpin serum levels and levels of molecular markers (e.g. TNF 1) of brain damage suggesting that neuroprotective properties of neuroserpin might be associated with the inhibition of excitotoxicity, inflammation, and BBB compromise following ischemic stroke ( 341) Collec tive evidence implicates that AAT may hold therapeutic potential in limiting stroke pathology that results from intrinsic inflammatory processes. In the present study, we tested the protective effects of AAT in ET 1induced transient middle cerebral artery occlusion (tMCAO) in rats Results Human AAT D id n ot A ffect the ET 1I nduced I schemia In order to test the effect s of hAAT on middle cerebral artery occlusion (MCAO) induced by ET 1, cohorts (nine rats were divided randomly into three equal groups) of rats were stereotaxically injected with ET 1adjacent to the left MCA of the brain. About 510 min after ET1 injection hAAT or saline was intracranially injected at the same site in two groups ET1+hAAT (i.c.) group or ET 1+saline group, and the third group was intravenously injected, ET1+hAAT (i.v.) group. The relative reduction flux in rat brain was measured using a l aser Doppler scanner. As shown in Figure 41A, a pproximately 20 min following the ET 1 injection,

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75 the flux statistic at the injection site dropped markedly from ~ 1,600 PU to less than 900 PU in all groups (i.e. ET1+saline group, ET1+hAAT (i.c.), and ET 1+hAAT (i.v.) groups). The calculations were made using equation 21, (METHODOLOGY) The average relative flux values in all groups were drastically reduced by 40 50% compared to the base line. The maximally reduced flux was maintained for up to 35 min, followed by the initiation of reperfusion (Figure 41B). N o significant difference among the three groups was detected, illustrating that hAAT both locally and systemic ally delivery did not alter the ischemic induction of ET 1. Local Delivery of hAAT Mitigat es ET1Induced Stroke Outcome In a separate experiment, five rats were intracranially injected with ET 1 and hAAT as previously described. Data from the control groups, receiving aCSF or ET 1 alone were retrieved from our previous study ( 287 ) as the set of experiments were performed during the same time period and using identical methodology Three days after ET 1 injection, behavioral tests and brain damage evaluation were performed. Cylinder test results showed that animal behavior was normal before treatment, i.e. percentages of both forelimbs used for postural support in aCSF, ET 1 alone, and ET 1+AAT (i.c.) groups were 80.07.0%, 79.205.16%, and 82.504.15%, respectively; and no difference among the three groups (Figure 42). T hree days after ET 1 injection the use of both forelimbs i n ET1 alone group was significantly reduced (22.66.3%), indicating profound impairment of contralateral forelimb function whereas no impairment was observed in the group which received the aCSF injection (76.674.08%). In the hAAT treatment group, ET 1+A AT (i.c.), the use of forelimb was significantly higher than that in ET 1 alone group ( 60.06.1%, pvalue < 0.05) These results clearly demonstrate that local administration of hAAT mitigated the deficit of motor function caused by ischemic stroke in rats

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76 Vibrissae tests were performed to test the effect of hAAT on sensorimotor function. Data in Figure 4 3A shows the ipsilateral forelimb placing response to vibrissae stimulation before and 72 h post MCAO measured in seconds. Before ET 1induced ischemic str oke, the response time periods in aCSF, ET 1 alone, and ET 1+hAAT (i.v.) group were 1.540.04s, 1.760.18s, and 1.070.21s, respectively. Three days after ischemic stroke they were 1.920.71s, 2.150.19s, and 1.340.37s, respectively; and there is no stati stically significant difference among the three groups. This indicates ipsilateral forelimb placing was not affected by the middle cerebral artery occlusion in rats after three days. As shown in Figure 43B, c ontralateral forelimb placing time in ET1+hAAT (i.c.) group was significantly shorter than that in ET 1 alone group (5.79 1.17s, 18.92 4.14s, respectively, and p value < 0.001), suggesting that hAAT treatment is able to limit the loss of sensorimotor function induced by MCAO in rat model. The contrala teral forelimb placing time period in aCSF group remained the same (1.100.08s) after surgery vs. before surgery, and significantly shorter compared to ET 1 alone model group. This implies that and ET1 injection induced profound sensorimotor deficits whic h were not affected by the surgery procedure. After the above behavioral tests, all animals were sacrificed and subjected to brain damage evaluation. Brains were sectioned into 2 mmthick sections and stained using TTC assay. Images of the brain sections w ere obtained from a scanner and the infarct areas were quantified using Image J v5.0. Total infarct volume of each animal was defin ed by the sum of the infarct area of all sections. Results are presented in Figure 4 4, the average of total infarct areas of the model group (receiving ET 1 alone) was significantly larger than that of the hAAT treatment group (198.9 13.3 mm2and 33.72.8 mm2, respectively, p value < 0.001). Typically, the infarct in the

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77 model group involves the rostrocentral dorsolateral cor tex, subcortex, and basal ganglia while in the treatment group the infarct area was limited to the cortex region. Syste mic Delivery of hAAT Mitigated ET 1Induced Stroke O utcome Although direct local injection of hAAT clearly prevented brain damage from ET1induced MCAO, the procedure is invasive and may not be practically translated into clinical application. In order to test the effect of another route of hAAT delivery on ischemic stroke, we performed experiments using separate groups of animals Since we found no effects from aCSF in the previous studies, only control and hAAT treatment groups were utilized Eight rats in the control group received ET 1 (i.c.) and 1 mL of sterile saline (i.v.) Fourteen rats in AAT treatment group were intracranially (i.c.) injected with ET 1 and intravenously (i.v.) injected with hAAT ( 40 mg /kg) immediately after ET 1 injection. The rationale for choosing this dose is based on the normal concentration of AAT in humans From o ur laboratory experience, at a dose of 50 mg/kg injected intraperitone ally in mice, hAAT can prevent streptozotocincell apoptosis ( 222) ; a dose of 25 mg/kg used in mouse model for autoimmune arthritis has shown hAAT is able to reduce inflammation ( 342) Another study used a dose of 60 mg/kg injected intraperitoneally in mouse model for myocardial ischemia/reperfusion, demonstrated the anti inflammatory and tissue protective propert ies of hAAT ( 343) Therefore, 40 mg/kg was selected for treatment in rats in this study as it was in the experimental range. Cylinder test results showed that animal behavior was normal before treatment, i.e. percentages of both forelimbs used for postural support against cylinder walls of ET 1+saline and ET 1+hAAT (i.v.) groups were 84.51.2% and 87.93.2%, respectively; and no statistical difference between the two groups (Figure 4 5) Three days after the injections, both groups revealed significant deficit compared to pre treatment. However, the hAAT treatment group

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78 showed significant deficit r eduction in using two forelimbs for cylinder exploration compared to the controls (72.66.2% and 35.45.9%, respectively, p value < 0.001) suggesting intravenous delivery of hAAT can ameliorate motor function deficit induced by ET 1induced cerebral ischemia. Vibrissae test results were presented in Figure 4 6. Relative response time represented time to place forelimb as a percentage of pre treatment tests, using equation 41. The results show the ipsilateral on the left and the contralateral on the right. As expected, the unaffected side limb (ipsilateral to the injection side) was not affected by the MCA occlusion, the relative response time of forelimbs of animals in ET 1+saline and ET 1+hAAT (i.v.) groups were approximately 100% (127.323.5% and 99.211.5% respectively). The relative response time of t he affected side forelimb (contralateral to the injection side) of both groups were significantly increased (Figure 4 6); however the hAAT treatment group showed significantly less delay in response to vibrissae stimulation compared to control ET 1+saline group ( 238.743.4% and 400.987.4% respectively ) This indicates intravenous delivery of hAAT mitigate s sensorimotor f unction deficit by ischemic stroke. Relative response time = Seconds to place ( after surgery ) Seconds to place ( before surgery) 100 ( Equation 41) Brain damage was evaluated following ischemic insult 72 h using MRI Infarct size calculation is based on the T2 relaxation maps of each image (1 mm thickness) using Image J v5.0. Total infarct volume was defined by the sum of the infarct area of all secti ons. R esults are shown in Figure 47, the infarct size of ET 1+saline and ET 1+hAAT (i.v.) were

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79 141.837.6 mm2 and 53.0 17.7 mm2, respectively, p value < 0.05, indicating that systemic hAAT administered significantly reduced by ~ 63 % Discussion Human alpha 1antitrypsin has been used for more than two decades as augmentation therapy for chronic obstructive pulmonary disease (COPD) that is related to AAT deficiency. Our laboratory has been investigating its functions and has shown its beneficial effects in s everal different experimental models of diseases including rheumatoid arthritis ( 344) diabetes ( 172, 222, 345) and bone loss ( 346) However, the effect of AAT on brain injury induced by stroke has never been reported. In the present study, we showed that hAAT therapy protect ed against brain tissue injury and improve d stroke outcome. Considering that hAAT treatment has been proven to be safe, these results imply a new clinical application of hAAT for the treatment of stroke, and perhaps other brain diseases associated with inflammation and programmed cell death Pathogenesis of stroke is complex and involves inflammation and cell death. Within seconds to minutes after loss of blood flow to a region of the brain, the ischemic cascade is rapidly initiated. Cerebral ischemic excitotoxic mechanism is activated in the territory which leads to apoptotic cell death due to depletion of cellular energy stores. These injured neurons and microglia in both the core and periphery of the lesion are producing pre inflammatory mediators, such as cytokines and reactive oxygen species (ROS), which activa te microglial cells. Therefore, anti inflammatory and cytoprotective approaches hold great potential for the treatment of stroke. The increasing body of literature shows that hAAT has anti inflammatory and cytoprotective properties ( 347 ) In the present study, we showed that local delivery of hAAT protected ET 1 induced brain injury and mitigated behavioral deficits. Although the mechanism of this protection remains to be further investigated, it is possible that hAAT inhibits ischemia and

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80 reperfusioninduced neuron cell death by inhibiting caspase 1 and 3 ( 343) Human AAT may also inhibit local expression of ischemia and reperfusioninduced inflammatory cytokines and activated astrocytes or microglia. Therefore, administration of hAAT to the site of ischemic stroke insult may mitigate cell death and inflammatory responses. It is intriguing that systemic delivery of hAAT significantly improves stroke outcome. Since AAT is a 52 kd glycoprotein and may not be able to pass through an intact BBB ( 348) the following possible mechanisms may contribute to its protective effects. First, ET 1 is a known mediator that increases BBB permeability ( 349) Also, stroke increases BBB permeability to macromolecules, e.g. Immunoglobulin M (IgM, 450 kd) ( 350) Second, ET 1 induced ischemia reperfusion can lead to dysfunction of endothe lial cells and temporary leaking of the BBB ( 351) Third, the injection of ET 1 mechanically, possibly leads to local damage of the BBB. In all cases, circulating hAAT may pass through the BBB, reaching the impaired area of the brain and displaying loc al protective functions. A fourth possibility is that hAAT may indirectly reduce brain tissue damage by inhibiting the infiltration and migration of lymphocytes to the penumbra of the ischemic region of the brain ( 219) Considering that ischemic stroke often leads to local BBB damage in human patients, systemic administration of hAAT may be clinically beneficial for patients with early onset of stroke. Although the beneficial effects of systemic adminis tration hAAT may be limited after the recovery of BBB, such treatment may protect the brain from the recurrence of stroke, which occurs commonly in humans. In addition, since BBB damage in stroke is primarily induced by the inflammatory response and result s in vasogenic edema and brain damage, hAAT, as an anti inflammatory protein, can also contribute to recovery of the BBB.

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81 In the present study, approximately 20 min after ET 1 injection relative cerebral blood flow rapidly decreased to 4050% of baseline i n all animals and remained maximally decreased for at least 2030 min. The result is consistent with the previous study that showed microinjection of ET 1 near MCA induced a blood flow reduction of 3075% in the region supplied by the artery ( 33, 316) There was no significant differenc e among the three groups in CBF reduction, indicating that hAAT had no direct influence on ET 1 induced MCAO. Therefore, our results indicate that the protective effects of hAAT are mediated not through blocking the activity of ET 1, but rather by blocking the subsequent biochemical/inflammatory responses and limiting damage. Conclusions Present study demonstrated that b oth local and systemic deliveries of hAAT result ed in neuroprotection and mitigation of behavioral deficits induced by ischemic stroke in rats. Results from this study indicate a promising new application of hAAT as a therapy for ischemic stroke.

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82 Figure 4 1. Perfusion measurements of AAT study (A). Color photographs of an animal brain recorded at different time points (pre injection, 1020 min, 3035 min, and 6065 min following ET1 injection) by a digital camera of a laser Doppler system, corresponding closely with the blood flow image. Lookup table shows arbitrarily assigned perfusion unit (PU) from the lower limit of 0 to an upper limit of 1,000 and above. The skulls illustrate the holes and injection sites. The top, middle, and bottom panels are images of ET 1+saline, ET1+ hAAT (i.c .) and ET 1+ hAAT (i.v.) groups respectively. (B). Percentage perfusion reduction as a function of t ime calculated using equation 21. All rats were injected with ET 1 (240 pmol in 3 L PBS) proximal to the left MCA. About 510 min after ET 1 injection, hAAT (1.35 nmol in 3 L saline for i.c.) or (192 nmol in 1 mL saline for i.v.) was administ er ed at the same site in the two groups. The dotted, closed diamond solid and closed triangle solid lines represent the average levels in ET 1 alone, hAAT ( i.c.) and hAAT (i.v.) groups, respectively. N = 3 for all groups. The differences at all time points of all groups were not stati sti cally significant. Data are means s.e.m.

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83 Figure 4 2. Cylinder test of ET 1 alone vs. ET 1 and intracerebral hAAT delivery. Rats were placed in a transparent cylinder for 3 min. Forelimb use during exploration by the rat s was scored. Each bar represents the average percentages s.e.m. of using both forelimb s in the indicated group. ET 1 group, N = 3, ET 1 + hAAT group, N = 5,*, **, ***: pvalue < 0.05; 0.01; 0.001, respectively.

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84 Figure 4 3. Vibrissae test of ET 1 alone vs. ET 1 and intracerebral hAAT delivery. (A). Time in seconds to ipsilateral forelimb placement on the countertop. (B). Time in seconds to contralateral forelimb placement on the countertop, ET 1 group, N = 3, ET 1 + hAAT, N = 5, ***: pvalue < 0.001. Data are means s.e.m.

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85 Figure 4 4. Infarction area labeled for mitochondrial activity of AAT study ( 2,3,5triphenyltetrazolium chloride, TTC). Two TTC staining slices from two animals representi ng each group, the left one for group receiving ET 1 alo ne, and the right one for groups receiving ET 1 and intracranial hAAT administration. Off white color areas show dea d tissues while red areas indicate vital tissues. ET 1alone group N = 3, ET 1 and hAAT treatment group, N = 5. ***: pvalue < 0.001. Data are means s.e.m. Figure 4 5. Cylinder test of ET 1 alone vs. ET 1 and intravenous hAAT delivery. Rats were placed in a transparent cylinder for 3 min. Forelimb use during exploration by the rats was scored. Each bar represents the average percentages s.e.m. of using both forelimb s in the indicated group. ET 1 alone group, N = 8, ET 1 + hAAT (i.v.), N = 14,*: pvalue < 0.05, ***: pvalue < 0.001.

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86 Figure 4 6. Vibrissae test of ET 1 alone vs. ET 1 and intravenous hAAT delivery. The percentage of preMCAO response time calculated using equation 41. Data are means s.e.m. Figure 4 7. Infarction area calculated from MRI images using Image J v5.0. *: pvalue < 0.05. Data are means s.e.m.

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87 CHAPTER 5 GE NERAL DISCUSSION CONCLUSIONS AND FUTURE WORK General Discussion Despite the fact that stroke or cerebrovascular accident (CVA) has been known for more than two thousand years and has been intensively researched in the last several decades it still is t he third leading cause of death (following isch emic heart disease and cancer); and the most frequent cause of permanent disability worldwide ( 352, 353) Stroke poses a massive health burden and becomes a huge financial burden for society In the United States alone, as many as 800 000 people suff er from the disease annually, i.e. stroke occurs every 40 seconds ( 10) Stroke is well associated with age and other ag ed related diseases, so the absolute number s of stroke patients are likely to rise with an aging population. Y et it has limited effective therapeutic options e.g. only one FDA approved drug for acute ischemic stroke exists however, it is only available to less than 5% of all patients ( 124, 354) because of strict time restraints on administration post insult Therefore, innovative and effective therapies are greatly needed. Because the events leading to neuronal damage are h eterogeneous and involve many factors, it is challenging to translate data from animal studies to clinical trials. However, in order to rationally decide on the use of novel treatment regimens it is important to review pre clinical and clinical results of therapeutic strategies undertaken to date. Ischemic cascade following an ischemic stroke first affects brain cells (mostly neurons) exposed to extreme reductions in blood flow (the ischemic core) which leads to the loss of their membrane potential, cellular structural integrity, and eventually irreversible damage. In surrounding regions (the ischemic penumbra), the reduction in blood flow is sufficient to compromise neuronal function but does not immediately cause neuronal death. Inhibition of such delayed effects offers a promising window for neuroprotective strategies. O ver the past ten years

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88 there have been more than 1000 experimental papers and over 400 clinical trials focusing on neuroprotective strategies while there were hardly any publications on this topic prior to the 1990s ( 355) Major triggers of ischemic neuronal death are overloaded intracellular calcium concentrations, accumulation of free radicals resulting in oxidative and nitr osative stress, various cell death signaling pathways, and inflammatory reactions. Below is a summary of some of the findings and promising approaches for ischemic stroke therapies based on recent insight of stroke mechanisms. Calcium antagonist s (Nimodipine ) Following cerebral ischemia raised cytosolic calcium concentration s affect many processes associated with neurotoxicity. That is, influx of calcium into the cells (e.g. through voltage gated calcium chan nel and NMDA channels) and increased cycling across ischemia damaged membranes lead to a sustained rise in cytoplasmic calcium, leading to cell depolarization and neurotransmitters being released from neurons; and calcium overload in mitochondria, thereby resulting in cellular deat h ( 356 ) Cytoplasmic calcium activates enzymes (particularly proteases, lipases, and endonucleases) causing membrane damage ( 357 ) Cellular membrane damage and calcium iNOS activation provide high concentration s of nitric oxide and fatty acid substrates for free radical generation which damages many important biomolecules, e.g. proteins and DNA ( 358) Glutamate release is stim ulated by calcium dependent exocytosis and has toxic effects on surrounding cells ( 359, 360) Based on this rationale, nearly 3400 ischemic stroke patients were enrolled in a clinical trial to test the therapeutic benefit of N imodipine, a well known calcium channel antagonist ( 355) The drug showed positive results in animal models of acute focal ischemic stroke then was started in ischemic stroke patients and showed beneficial effects (30 mg every six hours

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89 begun within 24 hours of the onset the s ymptoms) however, the effects were limited to men ( 361) Based on these positive resu lts, more clinical trials of the drug were conducted at multiple centers. Unfortunately, the results from these studies were inconsistent Three considerations for why Nimodipine failed follow. First, intracellular translocations of calcium, resultant from the derangement of mitochondrial sequestr ation during severe ischemia, might not be susceptible to calcium blockers. Second, Nimodipine is possibly ineffective enough to saturate the Ca2 + binding sites as another calcium antagonist may. Third, the postischemic infusion may need to be longer for a more sustained improvement in the delayed hypoperfusion, which may contribute to ischemic neuronal injury. Consequently, both pre clinical and clinical studies of this drug showed to be ineffective ( 355, 362365) However, intracellular calcium influx can be attributable to activation of glutamate receptor; inhibition of these receptors may bring other potential therapeutics to stroke therapy in the future. Glutamate antagonist s Under ischemic conditions, i ntracellular calcium influx is attributable to stimulation of glutamate receptors, thus inhibition of the se receptors offer more targets in stroke therapy. E xcessive amount s of glutamate, a major excitatory CNS neurotransmitter, are released and capable of inducing excitotoxicity ( 366 368) Glutamate and related excitatory amino acids activate several receptors, such as NMDA and AMPA, which are relevant to neuroprotection. There have been several compounds in pre clinical and clinical trials which have tested the neuroprotective efficacy of glutamate antagonists following cerebral ischemia. MK 801 ( Dizocilpine ), a noncompetitive NMDA antagonist, is the most extensively studied compound. MK 801 binds to NMDA receptors with high affinity and showed infarct

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90 reduction in several experimental models within 1 2 h of ischemia ( 369, 370) Dextromethorphan (a centrally acting dextrorotatory analog of codeine) and its derivatives, noncompetitive NMDAchann el blocker s also demonstrated neuroprotective effects in animal studies of focal ischemic stroke ( 371, 372) Nevertheless, both MK 801 and dextromethorphan were terminated in clinical trials due to side effects (the phencyclinedine like effects, such as hallucination and agitation) lack of efficacy ( 373, 374 ) Apt iganel ( Cerestat CNS1102) is also noncompetitive NMDA antagonist which decreases infarct volume and behavioral deficits up to 2 h after focal ischemia ( 375 ) Studies of Aptiganel s afety and tolerability in patients at a low dose ( 4.5mg intravenous bolus by infusion, nonweight adjusted doses) was shown to be a tolerable and to have neuroprotective effects in animal models, however, it causes hypertension and CNS excitation ( 376) The drug was suspended in phase III because of both a lack of efficacy and safety ( 377) CGS 19755 (S elfotel), a competitive NMDA antagonist, has shown ~ 50% infarct reduction in animal models delivered at 75 min following ischemic insult ( 378) However it was discontinued in phase III trials due to lack of efficacy and a trend toward higher mortality ( 379, 380) With regard to AMPA receptor antagonism, the AMPA anta gonist ZK200755, in phase II trials at multiple centers, transiently worsened the neurological condition in patients with acute ischemic stroke ( 381) The trial was terminated after 61 patients f or safety reason s and no further studies have been reported. Another AMPA antagonist YM872 (Z onampanel) has potential neuroprotective effects (reduces infarct volume 3040%) when administered within 2 h of permanent and transient stroke onset in rats ( 382, 383) YM872 also lessened stroke damage and augmented tPA efficacy

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91 in rat model of thromboembolic stroke ( 384) Unfortunately, no reports have been made after clinical trials began in 2001 in human ( 355) Antioxidants/radical scavengers Imbalance between oxidants and antioxidant s results in oxidative stress. Oxidative and nitrosative stress involves formation of oxygen and nitrogen species through various mechanisms, including mitochondrial inhibition, intracellular calcium overload, reperfusion injury, and inflammation ( 385) During ischemia and reperfusion, cytosolic levels of free radicals (ROS/RNS) accumulate in the brain tissues and accelerate neuronal damage through membrane lipid peroxidation, DNA damage, and protein dysfunction ( 386, 387) Hence, free radicals are considered to be an important therapeutic target for improving stroke outcome. Several compounds have shown neuroprotective effects and are undergoing clinical studies. For instance, Ebselen (2 phenyl 1,2benzisolennazol 3(2H) one), a selenium compound with glutathione peroxidase like activity, has reduced ischemic damage when administered prior to ischemia in mice ( 388) It has also shown brain injury reduction and neurological improvement in MCAO model in rats ( 389) A phase III study of 394 patients with maximum time from onset of 24 h in multiple centers are ongoing (Internet Stroke Center, last updated on 19 February 2009; accessed date 27 February, 2012). Another antioxidant candidate is Edaravone (MCI 186; 3methyl1phenyl 2pyrazolin 5one). Edaravone is an oxygen r adical scavenger and blocker of lipid peroxidation. A p hase II I trial of 252 ischemic stroke patients with a 72h window of treatment reported that E daravone improved outcome in patients at 3 months with no serious safety issue s ( 390) In April 2001, Edaravone was approved by the Japanese Ministry of Health, Laboratory and Welfare for

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92 treatment of acute stroke patient s, and a post marketing study has shown that the drug is welltolerated in all patients ( 391) In addition, the novel radical scavenger NXY 059 protected brain tissues in various animal ischemia models, e.g. reducing infarct volume by 77% in MCAO model in rats ( 392) produced a 51% brain damage reduction in permanent MCAO model s in a primate s ( marmosets ) ( 393) and redu ced hemorrhage when combined with tPA in a n embolus model in rabbits ( 394, 395) The drug passed the first clinical trial, Stroke AcuteIschemicNXYTreatment trial I (SAINT I) involving 1722 patients with acute ischemic stroke and showed significant improvement of primary stroke outcome ( 396) However, in a larger number of patient s (3306, SAINT II) NXY 059 appeared to be ineffective ( 397) and a depletion of potassium was reported as a side effect ( 398) Anti inflammatory therapy Inflammation has detrimental effects on brain tissues after stroke. It occurs within hours of the initial stroke and can last for several months. Suppression of inflammation using a variety of agents has shown reduction in brain damage and clinica l outcome improvement in animal models of stroke. One benefit may be extend ing the therapeutic window of tPA and decreasing the risk of hemorrhage Although anti inflammatory candidates have not yet proved their therapeutic effects in clinical studies it is important to understand the mechanism s of inflammation in stroke as it would offer potential opportunities for improvement of stroke outcome. Inflammatory response s following cerebral ischemia initiates with the up regulation of proinflammatory pathways in neurons, astrocytes, microglia, pericytes, mast cells, and endothelial cells due to ROS, activated proteases, and intracellular components from necrotic cells in the ischemic core. Chemokines and cytokines are secreted, microglia are activated, and

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93 adh esion molecules are up regulated in vascular endothelial cells Increased expression of adhesion molecules has been found in thromboembolic stroke, and in permanent and transient foc al ischemic stroke in rat ( 399) and baboon ( 400 ) models Adhesion molecules and chemokines mediate the recruitment of circulating leukocytes to the vessel wall. Leukocytes, especially neutrophi ls migrate and infiltrate into parenchyma within several hours Infiltrating leukocytes and activated microglia produce proinflammatory mediators which accelerate the inflammatory reactions, as well as various mediators, including cytokines, NO via inducible nitric synthase (iNOS) ROS, and MMPs. Cytokines can directly lead to cell death. MMPs and other inflammatory effectors (e.g. plasmin ogen activators and cat hepsin) d amage the endothelium and other components of BBB resulting in vasogenic edema, microvas cular ischemia, and increased susceptibility to hemorrhagic transformation of the infarct. Reperfusion (as discussed in detail earlier) can contribute to ROS generation and promote inflammation. Migrating leukocytes and other hematogenous inflammatory cell s can also form aggregates that perturb cerebrovascular microvessels, worsening microvascular perfusion or preventing effective reperfusion ( 401, 402) Moreover, other chronic inflammatory states can be deleterious and effector s of stroke outcome. It has been documented that approximately 30% of ischemic stroke patient s ha ve a previous infection, and 30% develop an infection while they are in the hospital ( 403) For inst ance, atherosclerosis is a chronic inflammatory response in blood vessel walls associated with poor outcome in experimental stroke It is me diated partly by activation of CD36 ( 404) which is a multiligand scavenger receptor invo lv ed in the pathogenesis of arthrosclerosis ( 405) Subacute physiological stress is unfavorable for stroke outcom e partially by initiating tolllike receptor 4 (TLR 4) mediated inflammation in brain ische mic mice ( 406) Moreover, c hronic

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94 hypertension, obesity, and diabetes which are associated with inflammation, exacerbate stroke pathology in animal models of cerebral ischemia ( 407409) Recently, modeling peripheral inflammation by injecting lipopolysaccharide (LPS) in r ats showed delayed infection 24 h after MCAO and increased the infarct size by 85%. This is associated with microglia/macrophage and infiltrating leukocyte up regulation and greater functional deficits ( 403) Several agents have been proven to ameliorate inflammation in animal models as well as stroke patients They focus mainly on leukocyte trafficking (e.g. anti adhesion molecule therapy) effector molecules (e.g. MMP inhibition ) and inflammatory mediators (e.g. albumin therapy) Leukocyte trafficking Anti selectin antibody administration before and after transient MCAO in rat model reduced brain damage and hemorrhage ( 410) Blockade of P selectin using a monoclonal antibody also decreased infarct volume and improved reperfusion in mice ( 411) A clinical trial of a murine monoclonal anti human intercellular adhesi on molecule (ICAM 1) antibody Enlimomab was not effective, and potential ly worsen ed stroke outcome ( 412) To examine several potential mechanisms for the negative outcome in clinical stroke trial of Elimomab, the murine antirat ICAM 1 antibody, 1A29, was injected in rats after focal brain ischemia. The 1A29treated group did not reduce infarct volume, rather generated rat anti mouse antibodies and exacerbated the infarct size. Thus, the possible reason why Elimomab failed in the clinical trial is that it activates the immune sy s tem in response to foreign protein ( 413) Effector molecules MMPs, the largest class of human proteases, comprise of more than 25 different secreted and cell surface bound molecules ( 414) It is well documented that dysregulation of MMP activity may cause the degradation of extracellular matrix and basal lamina proteins which promote brain injury following stroke ( 415) MMP activity during the

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95 delayed neuroinflammatory response may be beneficial for stroke recovery ( 128 ) The standard hypothesis postulates that some MMPs play a cent r al role in the pathology of ischemic stroke. Many MMPs are significantly increased in brain tissues and plasma following i schemic stroke both in humans and experimental animals ( 416, 417) MMP mediated brain injury occurs through a variety of mechanisms, including break down of components of the ECM or via activation of other bioactive compounds (e.g. chemokines and cytokines) In experimental animal studie s of stroke MMP levels are associated with BBB disruption, edema formation, and hemorrhagic transformation events ( 119) It was demonstrated that inhibition of MMP 9 or treatment with an MMP 9 neutralizing antibody ameliorate d brain injury after permanent MCAO in rats ( 418) MMP 9 knockout mice had significant smaller ischemic lesions compared to wild type mice after permanent focal ischemia, implicating the deleterious role of MMP 9 in the development of brain injury after focal ischemia ( 419) In addition, M MP 9 has been shown to mediate the hypoxia edema formation via vascular endothelial growth factor (VEGF) ( 420 ) MMPs indu ce hemorrhagic transform ation following ischemic stroke via degradation of basal lamina and subsequent BBB disruption. It was reported that MMP 2 levels increased in early stages after MCAO in a primate species ( 421) MMP 2 degrades a protein, c laudin5 which is one of the proteins in TJ s resulting in increased BBB degradation ( 422) In addition to MMP 2 and 9, MMP 3 and 13 have also recently been shown to be activated after ischemiareperfusion contributing to BBB dysfunction and worse ning stroke outcome in murine models ( 423427) Importantly, MMP 9 and 2 are also elevated in brain tissues and plasma in the ischemic human brain ( 428) To date, 12 pharmacological inhibitors of MMP s have been in experimental studies demonstrating reduction of infarct size BBB opening, brain edema, decreased in delayed

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96 neurovascular remodeling, rescuing neurons from apoptosis, and reduction of tPA induced hemorrhage ( 414) Although MMP inhibitors have not yet been approved for stroke therapy, they appear as promising potential therapeutic s A number of studies, both in animals and humans, illustrate the possibilities to improve stroke outcome using several types of MMP inhibitors, including anti inflammatory agents. Interestingly, they can be combined with the current acute stroke therapy, tPA with protagonist/supportive effects. Perhaps, successful targeting of MMPs requires optimizing inhibition of MMP s deleterious activity without interfering with the beneficial effects of brain reparation during stroke recovery Learning from failures o f the past, blocking calcium channels and receptors associated with calcium overload, antioxidants, and even some promising anti inflammatory agents ha ve failed or shown only mild efficacy to prove their neuroprotective effects. Because stroke is complex having heterogeneous mechanisms involving multiple factors, i t is unlikely that in any approach targeting a single mechanism may provide an effective treatment for stroke patients, not to mention the translation from animals to humans. Plasma g elsolin and alpha 1 antitrypsin as novel treatment regimens in stroke. We investigated two new potential therapeutic proteins, plasma gel solin and alpha 1antitrypsin, for their ability to improve stroke outcome in animal models. Because of the complexity of events leading to neuronal damage following stroke we hypothesized that it is unlikely that any single mechanism approach would provide an effective treatment for stroke patients. Both pGSN and AAT have multiple effects that should be beneficial to i schemic stroke outcome. They possess a myriad of properties which may interrupt or inhibit multiple cell death pathways to prevent secondary injury, and promote regenerative mechanisms Both are naturally

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97 present in humans, have excellent safety profiles, and one of them (AAT) is has been an FDA approved drug since 1987 with a dose of 60 mg/kg weekly intravenous infusion for augmentation therapy for AAT deficiency patients ( 230) Plasma GSN and hAAT have distinct mechanisms that enable them useful for stroke therapy. However, t here are overlapping mechanisms of action also including anti a poptosis and anti inflammation C ombination of pGSN and AAT may not provide additive effects due to completive mode of action and/or interaction between the two proteins and it requires elaborate study design to evaluate efficacy of the proteins at different stages of the disease. Within minute s, the lack of blood supply to the brain can cause cell s to die in the ischemic core of the infarct Plasma GSN can prevent actin released from dead cells from polymerization (F actin) which is toxic to the surrounding tissues in the penumbra. It has been well established that the penumbra can r egain its ability to survive by interrupting the process of genetically programmed cell death (apoptosis) ( 429) Both pGSN and AAT can contribute to delayed programmed cell death in the penumbra by suppressing apoptotic caspase activation; pGSN can inhibit type I apoptosis while AAT can inhibit all types. Plasma GSN and AAT both can reduce inflammatory response through different mechanisms. Plasma GSN suppresses inflammation by binding to several bioactive molecules including LPA which is unregulated during cerebral ischemia. Plasma GSN also interact s with MMPs which are effector molecules in inflammatory process. Alpha 1 antitrypsin by other mechanisms, suppresses inflammatory reactions through inhibition of neutrophil elastase and proteinases cathepsins, thrombin, and trypsin; inhibition of proinf lammatory cytokines and enhancement of anti inflammatory cytokine IL 10 production via increasing cAMP; anti neutrophilic inflammation by inhibition of calpain activity ( 430 )

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98 It is interesting that at the site of injury pGSN levels drop due to actin binding while AAT concentrations can raise up to 34 times in response to without inducing toxicity ( 431) It was reported that AAT levels were elevated in the blood of ischemic stroke patients, but there is no evidence of these increased AAT levels correlated with stroke outcome. Increased APP levels are correlated with inflammatory reactions, so AAT, an acute phase reactant, is in response to tissue injury and inhibits inflammation. B oth proteins a re com monly considered as markers of inflammation ( 216, 432) Imp ortantly pGSN and AAT cannot only be effective therapy for acute stroke therapy but may also be utilized for prevention therapy. Indeed, approximately 2535% of the 795,000 Americans who have stroke each year will have another stroke within their life time, there i s a 40% chance of having another stroke after the first stroke (a transient ischemic attack or mini stoke) within 5 years, and recurrent strokes often have a higher rate of mortality and disability due to damage to the brain already injured by the original one (Recurrent Stroke Prevention Resources, National Stroke Association, accessed 3 March 2012). It has been shown that as ma ny as 30% of stroke patients have pre inflammation, the antiinflammatory proteins can lower the high risk in individuals with systemi c inflammation ( 433) and those with clinical conditions such as cardiovascular disease, hypertension, high cholesterol levels, diabetes, obesity, heavy alcohol consumption, and stress. Having been approved as a longterm treatment therapy with a good safety profile AAT coupled with a lasting inhibition of inflammation, is likely beneficial for prolonged rehabilitation therapy. Plasma GSN and AAT have multiple properties that target various pathophysiological stroke pathwa ys including biochemical and cellular pathways of post ischemic cell death,

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99 thereby they provid e neuroprotection, or they indirectly reduce the progression of brain injury and improve the functional outcome. Althoug h the recombinant tissue plasminogen activator is current ly the standard treatment for ischemic stroke, it can only be effective within 3h (extended to 4.5 h in Europe) of symptom onset, and it can cause neurotoxicity, edema, and hemorrhagic transformation ( 433) The primary bene ficial action of tPA is restoring blood flow and promoting reperfusion, which is associated with the induction of inflammatory mechanisms. Either pGSN or AAT can possibly mi tigate the inflammation in the brain by limiting brain cell death or injury from pro infl ammatory responses, which may extend the utility of tPA as well as minimize its undesired effects and consequently improve stroke outcomes. Some limitations and streng th s of the study T his study inevitably has some limitations including ET 1 induced middle cerebral artery occlusion model, behavioral tests, comparison of cortical motor function in rats and humans, time course, dose response and the pharmacokine tic s of pGSN and AAT in cerebral spinal fluid and comparison of pGSN and AAT in rats and human. The study utilized ET 1, a potent vasoconstrictor peptide, to induce middle cerebral occlusion. It has been reported that the r eperfusion process is ET 1dose depe ndent ( 33) in our experiments utilizing laser Doppler perfusion imaging, reperfusion began as early as 55 min to as long as more than two hours following ET 1 application implicating the variab ility of the experiment. Another issue is that ET1 can induce astrocytosis and facilitates axonal sprouting that may interfere with the production and interpretation of neural repair experiments ( 13) ET1 injection to induce a vasospasm model does not induce blood clot which is typical in thrombotic

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100 and embolic strokes in humans Lastly, b rain ischemia cannot be performed in conscious rats, so it requires anesthesia (isoflurane in this study ) which may influence testing agent effects. On the other hand, ET 1induce d MCAO model has some important advantages. The model produces a transient middle cerebral artery occlusion which mimic s stroke in human and provide s high ly reproducible ischemic damage and significant motor deficits ( 434) Reperfusion occurs in a range of hours ( 435) which may be more representative of human stroke than the immediate reduction and reperfusion that occur s with intraluminal suture models if the ET1 dose is adjusted appropriately to avoid variabi lity Also, ET1induced MCAO model with intracranial injection of ET 1, dose dependent and minimal edema ( 436) makes it possible to target the infarct to specific regions, particularly subcortical areas which are more difficult to induce in other models, such as intraluminal suture or direct surgical MCAO which requires the use of a clip or silk suture to ligate an artery ( 437 ) The model can induce penumbra so it is sui table for studies of neuroprotective agents as well as examining behavioral alterations during postischemic stroke. Microi njection of ET 1 to MCA vicinity is a minimally invasive and simple technique; it provides a high animal survival rate ( 17) and in our study it was 96% (two dead out of 52 animals studied) which is important for longterm studies of neuroprotective drugs. The study purposely selected male, young ( 7 8 week old) Sprague Dawley rats for testing the neuroprotective effects of the two proteins Male rats were selected t o avoid variability due to the known effects estrogen has on ischemic outcome ( 438, 439) Similarly, animal age also a ffects ischemic outcome ; e.g. older rats have been shown to develop a larger infarct compared to young er animals ( 440, 441) The animal strains may also affect cerebral ischemia ( 30) Therefore, gender age, and strain of the animal should be taken into consideration.

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101 The study utilized only a single dose of both testing protei ns. Thus, dose/response experiments are necessary to provide important information for optimizing the maximal effective doses as well as predicting the side effects which may occur. Although not studied in this project, the availability of the proteins throughout the time course of ischemic stroke is beneficial for maximizing the effective treatment. Importantly, pharmacokinetics (PK) data on both pGSN and AAT in CSF and serum in ischemic conditions are absent in this study, optimized doses of these proteins cannot be determined until their serum levels are investigated. A s ingle dose of 3 g of pGSN (local injection), or 70 g of hAAT (local injection), or 10mg ( 40 mg/ kg) of hAAT (intravenous injection) has shown significant reduction of 49%, 83%, and 63% in infarct size respectively The dose/response experiments can be established based on these findings for instance 1/3 and 3X of the dose could be tested. Also, the infarct measurem ents wer e made 72 h post ischemia and found the effects of both proteins, therefore the time course can also be established to find if multiple doses are need to maximize the outcome. Two behavioral tests cylinder and vibrissae, were performed in this study. The cylinder test is to examine the animal forelimb use for postural support. The test encourages the animal, inside a specially designed cylinder, to use the walls for upright support and vertical ex ploration. The test results reveal only forelimb sensitivity, thus another test to demonstrate the best prediction of the degree of injury to a specific area of the brain linked to the test should be done to assess the hindlimb function. Another potential disadvantage of the cylinder test is that it may not be repeat ed too often as animals may lose interest in exploring a novel environment and will not perform at all. In fact, in our study, some rats did not move when the test was performed again after thre e days, indicating the test may be not sensitive enough ( 442) Besides, the cylinder test has several n otable advantages, including its simplicity and relative eas e to perform,

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102 it does not require a particular exper tise or training, it does not require pre training of the ani mals, the brief duration of the procedure (in this study, 3 min/animal) and the data are rapidly obtained ( 443) In addition, t he test provides a true measure of spontaneous forelimb use as the movements that rats exhibit in the home cage The absence of comparison between rodent cortical motor and human cortical motor can be a caveat drawback of this study. Vibrissae or vibrissae s timulated forelimb placing test is a method for revealing sensorimotor/proprioception. The animal is held by the torso with its forelimbs hanging freely, and moved slowly toward a table or countertop so that the vibrissae on the one side contact with the t able or counter top, the limb on the same side readily to move forward to gain weight support. The test requires training and highly experienced personnel ( 444 ) Animal struggle and abrupt movements may influence on results, so care must be taken ( 444 ) However, the test is evaluate an important role that vibri ssae play in rats sensory environment as rats are thought to use vibrissae (whiskers) as the primary tools for explore their natural environment Also, the test can be modified for studying the neural events in sensorimotor system that occurs across the m idline ( 445) the anatomical reorganization between two hemispheres following brain damage ( 299, 446) The neuroprotective effects of the proteins have been shown 72 h post ischemia, while the secon dary stroke injury can last weeks to months. It cannot conclude the efficacy of the testing proteins for long term outcome. Also, the pGSN and AAT used in this study come from human serum, the examination of the immune response as well as the functional differences between rat and human pGSN and AAT which were unavailable in this study. Conclusion s The major impetus in stroke research is that stroke may be preventable, and once developed it may be manageable by therapeutic interventions. The delayed ons et of neuronal

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103 death following cerebral ischemia occurs via a cascade of events that offers numerous opportunities to intervene with neuroprotective agents. Excitotoxicity, genetically programmed cell death, and inflammation are all involved in the delayed neuronal death Plasma gelsolin and alpha 1 antitrypsin possess multiple functions, including anti apoptotic and anti inflammatory effects, which may improve stroke outcome through multiple mechanisms of action, and which are different from several neurop rotective agents targeting individual pathways (e.g. only anti inflammation, antiapoptosis, or MMP inhibitors). The data demonstrate significant reduction in brain damage and improvement in motor and sensorimotor functions in animal model of middle cerebr al artery occlusion. A model of reversible focal ischemia induced by endothelin1 (ET 1) showed substantial reduction (~50% compared to base line) in cerebral perfusion, an estimation of cerebral ischemia, in all groups, including ET 1 alone, ET 1+pGSN, ET 1+hAAT (i. c .) and ET 1+hAAT (i.v.), using a laser Doppler perfusion imaging system. This suggested that all groups initially receiving MCAO showed no stati sti cal difference between treatments, and neither pGSN nor hAAT interfere d with the vasoconstrictive activity of ET 1. Reperfusion was attained after approximately 1 hour of ET 1 application. Intracranial delivery of a single dose, 3 g or 35 pmol, of pGSN significantly prevented both motor and sensorimotor deficits three days following ischemic insult. In the cylinder test, examining animal forelimb use for postural support, animals with pGSN treatment performed better than animals in ET 1 alone control group, 70% vs. 20% ( pvalue < 0.01 ) respectively. In the vibrissae test (vibrissae stimulated forelimb placing test), which tests the sensorimotor response, the pGSN treatment group showed less delay in response to vibrissae stimulation compared to the control group (9 s vs. 19 s, pvalue < 0.01), respectively. Plasma gelsolin also

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104 markedly reduced total infarct volume, cortical and subcortical regions, 49% compared to control group (p value < 0.05) I ntracranial and intravenous administration s of human AAT at dose s of 70 g or 1.35 nmol and 40 mg/kg (10 mg/animal), showed significant reduction in infarct size, 83% and 63%, respectively. As expected, the motor and sensorimotor functions were diminished, in cylinder test though, animals performed much better in hAAT (i.c.) treatment group than in the control (60 % vs. 23% pvalue < 0.05), and in vibrissae test hAA T (i.c.) treatment group showed less delay in response to vibrissae stimulation (6 s vs. 19s, pvalue < 0.001) The study of the effects of intravenously delivered hAAT was performed in two separate groups of animals ET1 alone (N = 8) and ET 1+hA AT (i.v.) (N = 12). Human AAT (i.v.) treated group also illustrated significant improvement of motor function ( 73% in hAAT treatment vs.35% in control group, pvalue < 0.001) and sensorimotor function ( ~ 50% decrease in delayed time in hAAT treatment group compared to ET 1 alone group, p value < 0.05) The dissertation studies have shown that two proteins, plasma gelsolin and alpha 1antitrypsin, perform multiple functions that may provide multiple beneficial effects for ischemic therapy These novel therapies may give a promise for ischemic stroke patients, including preventing stroke recurrence. Future Work These two endogenous proteins offer several avenues of research toward neuroprotection following cerebral ischemia Further research to validat e the modes of action of plasma gelsolin and alpha 1antitrypsin, including the inhibition of cell death pathways and anti inflammatory properties of the both proteins are essential for further pre clinical development. The dose/response and time course nee d to be determined in order to optimize the protective effects of the agents. Although pGSN and A AT are natural pro teins, the safety/toxicity and study of

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105 their long term effect s are important for the treatment of th is disease. Also, pharmacokinetic studies should be performed to determine levels of the proteins in both serum and cerebral spinal fluid during cerebral ischemia; this data is essential for dosing adjustment, multiple dose regimens as well as to know whether transient ischemia has any eff ects on the testing agents. Study ing the effects of pGSN and AAT in combination with tissue plasminogen activator could be useful to see whether the proteins can be beneficial for extending the effective time window or necessary dose for tPA thrombolysis. Inclusion of more behavioral tests that assess motor /sensorimotor function sensibility and cognitive function can strengthen the translational applications.

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143 BIOGRAPHICAL SKETCH Huong Thu Le ( in her native language) was born in Vinh Phuc Vietnam on October 1975. Huong grew up in a large family with other three siblings. At a very young age, Huong showed herself as a dynamic girl. She likes helping people, which showed in the fact that at the age of 14, she started running errands to help her family. She chose to attend the Hanoi College of Pharmacy among the several schools to which she received admission in 1993. She got her Pharmacist Degree in 1998, and worked for a Japanese pharmaceutical company immediately following graduation. After working seven years in Hanoi, Huong decided to trade Vietnam and the pharmaceutical indust ry for the Netherlands and academia, starting a master degree in Drug Innovation at Utrecht University. During this master program, she joined Dr. Jeffrey Hughes Lab at the University of Florida as an intern. She got her master of science in early August 2007 and returned to Florida where later that month she was admitted into their Ph.D. program in the Department of Pharmaceutics. She has enthusiastically continued to work under the supervision of Dr. Jeffrey Hughes, and completed the project she began wo rking on during her internship. She was assigned to work in Dr. Sihong Songs Lab on another project when Dr. Hughes left for industrial work. Despite life's many obstacles and hardships, she received her Ph.D. from the University of Florida in the spring 2012 eagerly sought work in her field following graduation. In the last year of her Ph.D. program she met her fianc, Matthew Michael Moldthan whom she married in December of 2011.