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Qualitative identification of human retinal pigment epithelial (hRPE) cells attached to microcarriers… Flores, Joseph 2004

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QUALITATIVE IDENTIFICATION OF HUMAN RETINAL PIGMENT EPITHELIAL (HRPE) CELLS ATTACHED TO MICRO CARRIERS: A POTENTIAL NEW CELL THERAPY FOR PARKINSON'S DISEASE  by  Joseph Flores FJ.Sc., McGill University, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Graduate Program in Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA December, 2004 © Joseph Flores, 2004  Abstract:  Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta, leading to striatal DA depletion. Cell therapies such as human fetal cell transplants, which replace depleted DA by transplanting DA-producing cells, have been studied as a form of treatment. However, unconvincing clinical results and ethical and logistical hurdles have deemed such therapies unsuccessful. Human retinal pigment epithelial (hRPE) cells have been recently proposed as a tissue transplant alternative for PD. HRPE cells produce L-DOPA as part of the eumelanin pathway, they are easily grown in culture and can be stored for extended periods of time. Furthermore, when attached to gelatin microcarriers (hRPE-GM), immunosuppression is not needed. Recent studies in non-human primates, as well as open label studies in PD patients, have shown complete, / sustained reversal of parkinsonian symptoms without complications. However, there is no data on hRPE cell survival in the host. In this thesis, methods were developed to morphologically characterize hRPE-GM in vitro and to assess its short-term and long-term survival when implanted into the brain. Human RPE cells were grown to confluence in culture, harvested and passively attached to 45-63pm diameter gelatin microcarriers. In vitro HRPE-GM was immunohistochemically (IHC) characterized by using a human specific (NuMA Ab2) and epithelial specific (EMMPRIN) antibody. Next, fourteen male Sprague Dawley 6-OHDA lesioned rats receiving a unilateral striatal hRPEGM implant (without immunosuppression) were processed for IHC and ultrastructural electron microscopy (EM) analyses at various time points post-implant. A histological analysis revealed NuMA- and EMMPRIN-positive cells attached to the microcarriers in vitro and in vivo at various times post-implant. E M confirmed these results: morphologically characterized hRPE cells were seen at 48 hrs and 5 months post-implant. These results support the long-term survival of hRPEGM in the rat without immunosuppression.  ii  Table of Contents: Abstract: Table of Contents: List of tables: List of Figures Abbreviations Acknowledgements  ii iii v vi viii x  Chapter 1: Introduction 1. Introduction to Research 1.1. Epidemiology of Parkinson's disease 1.2. Clinical Features and Diagnosis of PD 2. The Basal Ganglia and Pathophysiology of PD 2.1. Introduction to the Basal Ganglia 2.1.1. The Direct and Indirect Pathways 2.1.2. Pathophysiology of the Basal Ganglia 3. Animal Models of PD 3.1. Animal Models of PD - How Axe They Useful?  1 1 1 2 4 4 5 6 .9 9  Chapter 2: Treatments for Parkinson's Disease 1. Evaluation Methods of PD Severity and Treatment Efficacy 1.1. Clinical Rating Scales 1.2. Objective Assessment Methods: Brain Imaging 2. Current Treatments for PD 2.1. Drug Therapies 2.2. Surgical Treatments for PD 2.2.1. Lesion and Stimulation 2.2.2. Cell Therapies: Transplantation 2.2.2.1. Fetal Ventral Mesencephalic (Nigral) Tissue Transplantation 2.2.2.2. Porcine Cell Transplantation 2.2.2.3. Glial cell line-derived neurotrophic factor (GDNF) 2.2.2.4. Embryonic Stem Cell Transplantation  12 12 12 12 13 13 15 15 18 18 22 24 26  Chapter 3: Human Retinal Pigment Epithelial Cells: A Cell Alternative for the Treatment of Parkinson's Disease 1. Fundamental Properties of hRPE Cells 1.1 HRPE-Cell Functioning in the Retina 1.2. Important hRPE-Cell Properties in the Treatment of PD 2. HRPE Cells Attached to Gelatin Microcarriers 3.1. Rodent hRPE-GM Efficacy Study 3.2. Non-Human Primate hRPE-GM Efficacy Studies 3.3. HRPE-GM Clinical Studies 4. Rationale and Objectives  29 29 29 29 33 35 35 37 38  Chapter 4: Methods 1. HRPE-Cell Culturing Techniques and In vitro Preparation  40 40  iii  1.1. HRPE Cell Isolation 1.2. HRPE Cell Attachment to Gelatin Microcarriers 1.3. In vitro Studies: hRPE-GM Preparation and Embedding 2. In vivo hRPE-GM Implantation Studies 2. In vivo hRPE-GM Implantation Studies 2.1. Animals 2.2. 6-Hydroxydopamine (6-OHDA) Lesions 2.3. HRPE-GM Implantation 3. Post Mortem Processing and Histology 4. Evaluation of Lesion Severity: Autoradiography Phosphor Imaging 5. Immunohistochemistry (IHC) 6. Electron Microscopy (EM)  40 40 41 43 43 43 43 44 45 46 46 48  Chapter 5: Results 1. Evaluation of the 6-OHDA Lesion Characteristics and Choice of Further Model 1.1. Surgical Observations 1.2. Behavioral Analyses 1.3. Phosphor Imaging Autoradiography 2. HRPE-GM Characterization 2.1. Surgical Considerations 2.2. Qualitative Immunofluorescence 2.2.1. In vitro Characterization 2.2.2. In Vivo Characterization of Implanted hRPE-GM  49 49 49 49 50 51 51 54 54 55  Chapter 6: Discussion and Conclusions 1. Cell Culture 2. In vitro Development of Immunohistochemical Methods 3. In vivo Pilot Studies '. 3.1. Behavioral Evaluations 3.2. In vivo Immunohistochemistry 3.3 Electron Microscopy 4. Alternate Considerations 4.1. Immune and Inflammatory Reactions to hRPE-GM 4.2. Issues Between Xenotransplantation and hRPE-GM Implants: Is the Rat Model the Right Model? 5. Comparison Between hRPE-GM Implants and Other Transplantation Techniques 5.1. Cell Handling and Surgical Approaches 5.2. Efficacy: Mechanism of Action of hRPE Cells 6. Future Directions 7. Conclusions Reference List  73 73 74 75 75 76 77 79 79  iv  80 81 81 83 84 86 89  List of tables: Table 1: Different pathogenic models of PD  9  Table 2: Advantages and disadvantages of F V M transplantation for PD  19  Table 3: Advantages and disadvantages of Porcine F V M transplantation for PD....  22  Table 4: Comparison of advantages and disadvantages of GDNF infusion for PD  25  Table 5: Advantages and disadvantages of Embryonic Stem Cells for PD  26  Table 6: Physiologic functions of the RPE in the retina  32  Table 7: Comparison of the advantages and disadvantages of hRPE-cell implantation for PD  34  Table 8: Calculated dose and pre- and post-cell viability of different cells lines used for hRPEGM implants 53 Table 9: Primary antibody marker characterization for hRPE-GM studies  53  Table 10: Pattern of fluorescent staining at different time-points post implant  56  v  List of Figures Figure 1: Circuitry of the classical basal ganglia model  5  Figure 2: Alteration in the basal ganglia circuitry after PD  6  Figure 3: Schematic drawing of RPE in the eye  30  Figure 4: Schematic diagram of the metabolic pathways leading to the production of eumelanin and pheomelanin ; 31 Figure 5: Experimental timeline of 6-OHDA lesion, hRPE-GM implantation and immunohistochemical (IHC), electron microscopy (EM), and behavioral analyses..  42  Figure 6: Schematic diagram of hRPE implants at anterior sites 1, 2 (A) and posterior sites 3, 4 (B) in the striatum 44 Figure 7: Behavioral characterization of unilateral 6-OHDA lesions  50  Figure 8: In vitro proliferation of hRPE cells in T-25 flasks  52  Figure 9: In vitro characterization of hRPE-GM  57  Figure 10: In vitro characterization of NuMA (Ab2) and GFAP in hRPE-GM  58  Figure 11: EMMPRIN characterization of hRPE-GM in vitro  59  Figure 12: Co-labeling of NuMA (Ab2) and EMMPRIN in hRPE-GM in vitro  60  Figure 13: In vivo hRPE-GM demonstrating co-labeling of NuMA (Ab2) (green) and GFAP (red) positive cells at different time-points post-implant 61 Figure 14: NuMA (Ab2)-positive cells at 5 months post-implant  62  Figure 15: In vivo co-labeling of NuMA (Ab2) and GFAP within the implant tract  63  Figure 16: In vivo EMMPRIN fluorescence at different time-points post-implant  64  Figure 17: In vivo hRPE-GM demonstrating co-labeling of NuMA (Ab2)-EMMPRIN positive cells 65 Figure 18: EM micrograph of single hRPE-GM 48 hrs post-implant  68  Figure 19: Acute inflammatory response at 48 hrs post-implant  69  Figure 20: EM micrograph of hRPE-GM 5 months post-implant  70  vi  Figure 21: Collagen fibers in hRPE cells 5 months post-implant  71  Figure 22: Melanin pigment production 5 months post-implant  72  Figure 23: Phenotypic variability between different hRPE-cell culturing points  87  Figure 24: Behavioral improvements after hRPE-GM  88  vii  Abbreviations 6-OHDA F-DOPA AADC ADL AIR BBB BSA CNS COMT CyA DA DAT DBS DBH DIC DMEM DMSO DOPA ED EGF EM EMMPRJN ER ES EtOH FBS FCS FGF FVM GABA GDNF GFAP GLP GM GPi GPe HBSS H&E hRPE hRPE-GM HY ICV IHC IGF 18  6-hydroxydopamine [ F]fluorodopa Aromatic L-amino decarboxylase Activities of daily living scale Amphetamine/apomoiphine induced rotation Blood-brain barrier Bovine serum albumin Central nervous system Catechol-O-methyltransferase Cyclosporin-A Dopamine Dopamine transporter Deep brain stimulation Dopamine beta hydroxylase Differential interference contrast Dulbecco's Modified Eagle Medium Dimethyl sulphoxide 3,4-dihydroxyphenylalanine Embryonic day Epidermal growth factor Electron microscopy Extracellular matrix metalloproteinase inducer Endoplastic reticulum Embryonic stem Ethanol Fetal bovine serum Fetal calf serum Fibroblast growth factor Fetal ventral mesencephalic y-amino-butyric-acid Glial cell line-derived neurotrophic factor Glial fibrillary acidic protein Good laboratory procedure Gelatin microcarriers Internal portion of Globus Pallidus External portion of Globus Pallidus Hank's Balance Salt Solution Hematoxylin and eosin Human Retinal Pigment Epithelial Human Retinal Pigment Epithelial cells attached to gelatin microcarriers HoehnandYahr Intracerebroventricular Immunohistochemistry Insulin-like growth factor 18  viii  IP L-DOPA MHC MPTP MRI NGS NuMA (Ab2) PBS PD PDGF PEDF PERV PET PMN RBC ROI SNr SNc SPECT SPIO STN TBS TGFP TH UPDRS VA VEGF VL VMAT  .  Intraperitoneal Levodopa Major histocompatibility complex 1 -methyl-4-phenyl-1,2,5,6-tetrahydropyridine Magnetic resonance imaging Normal goat serum Nuclear mitotic apparatus protein Phosphate-buffered saline Parkinson's disease Platelet-derived growth factor Pigment epithelial-derived factor Porcine endogenous retrovirus Positron emission tomography Polymorphonuclear leukocytes Red blood cell Region of interest Substantia nigra pars reticulata Substantia nigra pars compacta Single photon emission tomography Superparamagnetic iron oxide Subthalamic nucleus Tris-buffered saline Transforming growth factor beta Tyrosine hydroxylase Unified Parkinson's disease Rating Scale Ventral-anterior Vascular endothelial growth factor Ventrolateral Vesicular monoamine transporter  IX  Acknowledgements This work is dedicated to my family: my mom and dad, my sister Heidi, my grandmother, and Emma. I wouldn't have been able to do any of this without all your love and support. Thank you for everything that you have done for me. I would also like to thank my supervisor, Dr. Doris Doudet, for all her support and confidence in me and my work. In addition, I would like to thank Dr. John O'Kusky and Dr. Mike Cornfeldt for all their support and time mentoring me. Your encouragements have made me become a better researcher. I would also like to acknowledge Titan Pharmaceuticals for their time and support training me, and for all the great opportunities they have given me during this project.  x  Chapter 1: Introduction 1. Introduction to Research  Human retinal pigment epithelial (hRPE) cells have been recently proposed as a tissue transplant alternative for Parkinson's disease (PD). Recent studies in rats and non-human primates, as well as open label studies in humans with PD, have shown that hRPE cells attached to gelatin microcarriers and implanted into the striatum resulted in sustained reversal of parkinsonian symptoms without side effects. However, to date, there is no data concerning hRPE-cell survival in the host. Therefore, the present experiments were designed to develop an in vitro and in vivo method of hRPE-cell identification for assessing short-term and long-term survival and function of hRPE cells when implanted into the host striatum. 1.1. Epidemiology of Parkinson's disease  Parkinson's disease is a progressive, debilitating disorder that affects millions of people worldwide. Although many PD symptoms were observed as early as the second century by the Greek physician Galen (Kolb and Whishaw, 1990), it wasn't until 1817 when the London physician James Parkinson recognized the disease entity. At that time, James Parkinson published his famous monograph "An essay on the shaking palsy"; an account of six patients suffering from resting tremor, bent irregular posture, and a progressive motor disability. This disorder, originally called "shaking palsy" or "paralysis agitans" (Feldman et al., 1997), was later named Parkinson's disease by the French neurologist Charcot. PD is the second most prevalent neurodegenerative disease: its incidence is estimated at 1% of the general population (Samii et al., 2004), and rises to greater than 2%-3% in the population over the age of 60. Early studies showed the mean age of PD onset in the late 50s: however, in recent years, PD onset has been as late as the mid-60s. There does not seem to be any ethnic predisposition to PD; however, men are more likely to develop the disorder than women (Samii et al., 2004). Most reported cases of PD are idiopathic ("of spontaneous origin"). However, cases of early-onset PD (where symptoms present themselves between ages 21-40), and juvenile-onset PD (where symptoms occur before the age of 20 years) have also been observed.  1  1.2. Clinical Features and Diagnosis of PD  Motor dysfunction in PD can be separated into two main groups: positive symptoms, characterized by movements that are rarely seen in healthy individuals; and negative symptoms, which are deficits in normal behavioral capacity (Feldman et al., 1997). The positive symptoms of PD are tremor, muscular rigidity, and involuntary movements. PD patients exhibit a resting tremor with a 3-5 Hz frequency, and is typically the first symptom (in approximately 70% of PD patients) to appear. It is prominent in the patient's distal extremities, is usually asymmetric, and worsens with stress, contralateral motor activity, and during exercise. Tremor of the hands is another common symptom. Classically called "pill-rolling" tremor, hand tremors resemble hand movements that occur when rolling a pill between the thumb and forefinger. The second symptom, muscular rigidity, is described as increased resistance in passive joint movement. It is characterized as having a cogwheel quality: the movement force is initially resisted, but eventually a short movement occurs. The force is resisted again, and the process is repeated, resulting in a series of repeated jerky movements that resemble the movement of a cogwheel. Involuntary movement (akathesia) is the third type of positive symptom, which is characterized by constant shifts in posture normally used to relieve tremor or joint stiffness (Kolb and Whishaw, 1990). Bradykinesia and postural instability are the two main negative symptoms. Bradykinesia is the poverty or slowness of movement, and is the most disabling symptom in early PD. Bradykinesia is manifested by difficulties in walking, absence in facial expression and eye blinking, and lack of speech. Patients also lose fine motor control, thereby having difficulties doing normal behavioral tasks such as buttoning up clothes or handwriting. Bradykinesia is often exacerbated by postural instability in late stage PD. PD patients have a difficult time initiating walking and maintaining a normal gait, leading them to shuffle their feet in short steps. In addition, instability leads to poor balance, making falling while walking more likely. In time, a PD patient's shuffling gait becomes slower, and they eventually lose their ability to turn. The definite diagnosis of PD requires a patient autopsy; therefore, thorough clinical evaluations are essential to diagnose an individual with PD. Patient history, physical examination, and  2  symptomatic improvement after anti-parkinsonian drugs are factors that determine the patient's diagnosis. Fortunately, clinical diagnosis has become increasingly rigorous; different rankings characterize PD onset as either clinically possible, clinically probable, or definite (Calne et al., 1992; Samii et al., 2004). These criteria are necessary to separate PD from similar disorders including Parkinson-plus syndrome or normal pressure hydrocephalus, less common disorders like pallidoponto-nigral degeneration or dopa-responsive dystonia, or deficits due to normal aging. Characterizing the different stages of PD has become particularly important, especially in the development of new treatments. One approach to classifying different clinical stages of PD was established in the 1960s by Hoehn and Yahr (1967). The Hoehn and Yahr (HY) scale is still used today to assess PD patients. It is composed of five different stages, each with its' own set of criteria (Hoehn and Yahr, 1967): Stage 1. Unilateral involvement only, with minimal or no functional impairment. Stage 2. Bilateral or midline involvement, with no impairment of balance. Stage 3. First sign of impaired righting reflexes. Functionally, the patient is restricted to his activities, but may have some work potential, depending upon the type of employment. Patients are physically capable of leading independent lives, and disability is mild to moderate. Stage 4. Fully developed, severely disabling disease; the patient is still able to walk and stand unassisted but is markedly incapacitated. Stage 5. Confinement to bed or wheelchair unless aided. The HY scale is one of the most commonly used scales to clinically assess PD (Goetz et al., 2004). Importantly, the HY scale is used to enroll patients in clinical trials for new antiparkinsonian treatments. For example, progression to HY stage 3 was the main reason for the initiation of L-DOPA treatment in the past (Goetz et al., 1987; 2004). New treatments are assessed based on their ability to delay disease progression; they can prolong the latency between successive stages by as much as 5 years. Therefore, during a clinical trial, a PD patient can be assessed based on the severity of clinical features and disability before and during the course of the clinical trial. This scale determines whether a treatment is most beneficial when started in the  3  early or late stages of the H Y and whether new treatments can delay the time course of progression of the disease.  Although motor dysfunction is the most prevalent symptom seen in PD, there are other types of psychiatric impairments that are present. Many PD patients exhibit varying degrees of cognitive dysfunction including impairments in memory, language, and other cognitive processes. Approximately 25% of PD patients develop an Alzheimer-type dementia with features including memory deficits, aphasia, and apraxia (Marsh, 2000). Another common finding in PD patients is depression (Marsh 2000; Leentjens, 2004). Depression is the most common neuropsychiatric disturbance in PD; there is an estimated 40% prevalence rate of depression among PD patients (Cummings, 1992; Marsh, 2000). Nevertheless, depression in PD doesn't seem to be identical to clinical depression; there are subtle increases in dysphoria and pessimism, irritability, and suicidal ideation (Cummings, 1992). These observations suggest that there may be a diseasespecific depressive syndrome in PD that is distinct from major depression.  2. The Basal Ganglia and Pathophysiology of PD 2.1. Introduction to the Basal Ganglia The regulation of movement is carried out by a complex set of feedback loops that involves the cerebral cortex, the thalamus, and the basal ganglia. The basal ganglia is an integral part of the motor circuit and the pathophysiology of many motor disorders. It is composed of four interconnected nuclei: the caudate and putamen (which together comprise the striatum), the internal and external portions of the globus pallidus (GPi and GPe, respectively), the substantia nigra (comprised of the pars reticulata, SNr, and the pars compacta, SNc), and the subthalamic nucleus (STN). In the past few decades, advances in the understanding of neuroanatomical, physiological, and neurochemical processes of the basal ganglia has helped put together a functional model that explains both normal and abnormal motor behavior.  4  Cerebral Cortex (Glutamate) (Glutamate) (GAB A, enkephalin)  (GAB A, substance P, dynorphin) Thalamus  1 f (GAB A)  STN  SNr (Glutamate)  (GABA) GPi  Figure 1: Circuitry of the classical basal ganglia model. T h e striatum innervates output n e u r o n s i n the internal g l o b u s p a l l i d u s ( G P i ) a n d substantia n i g r a pars reticulata ( S N r ) t h r o u g h the direct p a t h w a y , a n d t h r o u g h the external g l o b u s p a l l i d u s ( G P e ) a n d s u b t h a l a m i c n u c l e u s ( S T N ) v i a the indirect p a t h w a y . D o p a m i n e r g i c input from the substantia n i g r a pars c o m p a c t a ( S N c ) is thought to i n h i b i t n e u r o n a l input (through D 2 receptors) i n the indirect p a t h w a y , w h e r e a s it excites activity ( t h r o u g h D l receptors) i n the direct p a t h w a y . 2.1.1. The Direct and Indirect Pathways T h e b a s a l g a n g l i a m o d e l d e v e l o p e d m o r e than a d e c a d e ago established that v o l u n t a r y m o v e m e n t is m e d i a t e d t h r o u g h t w o interdependent, o p p o s i n g p a t h w a y s ( A l b i n et a l . , 1989; A l e x a n d e r a n d C r u t c h e r , 1990) ( F i g u r e 1). T h e direct p a t h w a y consists o f striatal n e u r o n s that m o n o s y n a p t i c a l l y s e n d G A B A e r g i c projections to the G P i a n d S N r . T h e s e n e u r o n s c a n be d i s t i n g u i s h e d b a s e d o n their d o p a m i n e ( D A ) receptor a n d neuropeptide expression; n e u r o n s that c o m p r i s e the direct p a t h w a y express the D i D A receptor a n d the neuropeptides substance P a n d d y n o r p h i n . In contrast, the i n d i r e c t p a t h w a y consists o f a separate p o p u l a t i o n o f striatal n e u r o n s that express the D D A receptor subtype a n d the neuropeptide e n k e p h a l i n . T h e s e n e u r o n s s e n d G A B A e r g i c 2  projections to the G P e . G P e n e u r o n s then s e n d G A B A e r g i c projections to the S T N , w h i c h m a k e  5  excitatory connections with the GPi and SNr cell population. It is the GPi/SNr complex, which projects to the ventrolateral (VL) and ventral-anterior (VA) portions of the thalamus, which is responsible for the coordination of movement.  Cerebral Cortex  i  (Glutamate)  Striatum (Caudate/ Putamen) 1  (GAB A, enkephalin)  (Glutamate)  • (GAB A, substance P, dynorphin)  SNc  Thalamus  STN  1 1  1  (Glutamate)  (GABA)  Figure 2: Alteration in the basal ganglia circuitry after PD. The thickness in the arrows reflects the degree of activation in the projection. In PD, the absence of D A leads to reduced inhibition in the direct pathway, and increased inhibition of the GPe in the indirect pathway leading to disinhibition of the STN. Overactivity in the STN leads to excessive excitation of SNr/GPi neurons, over-inhibiting thalamo-cortical neurons reducing the control of voluntary movement. 2.1.2. Pathophysiology of the Basal Ganglia The pathophysiological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the SNc, leading to the significant loss of D A content within the striatum (Obeso et al., 2 0 0 0 ; Obeso et al., 2 0 0 2 ) . Anatomically, the neuronal cell bodies in the SNc possess long axonal projections innervating the striatum, making up the nigrostriatal pathway. In general, the loss of striatal D A leads to a significant increase in neuronal activity in the GPi/SNr complex (Figure 2).  An increase in GPi/SNr activity leads to the disproportionate inhibition of the V L and V A thalamus, reducing their thalamocortical activity. ; _  '  In the direct pathway, striatal GABAergic neurons (which are normally stimulated by DAergic activity) can no longer inhibit the GPi. Therefore, the GPi exerts an inhibitory action on the thalamus. In contrast, the indirect pathway (which is normally inhibited by normal dopaminergic functioning) exerts an inhibitory effect on the GPe. This, leads to disinhibition of the STN, resulting in the over-activation of the GPi/SNr complex. The net result is the inhibition the V L and V A thalamus and subsequent cortical motor centers. There are notable characteristics of neuronal degeneration and the loss of DAergic activity in PD. DAergic systems do not all degenerate to the same degree: while the nigrostriatal DA system is the most severely degenerated, other DAergic systems such as the mesocortical and mesolimbic DA systems are less affected. Within the striatum, the greatest DA deficit is in the rostral portion of the striatum, particularly in the dorsolateral or post-commisural putamen (rather than in the caudate nucleus) (Agid et al., 1987). This observation parallels well with the clinical features of PD; given the role of the putamen in motor control, this explains why motor dysfunction is the most prominent symptom of PD. In PD patients, there is a strong correlation between the severity of symptoms and the decrease in striatal DA concentrations. In addition, the most severe symptoms are observed on the side that is contralateral to the most severely affected SN. The pathophysiology of PD has been attributed to a greater than 80% loss of striatal DA levels. Post-mortem histopathological studies of PD patients demonstrated an equally significant loss of DA-producing SNc neurons (Kopin, 1993). There is a considerable loss of SNc neurons with age; however, the extent of degeneration in PD patients is substantially larger. The premise that the first parkinsonian symptoms appear when at least 80% of the striatal DA is lost stems from two observations: 1) in one hemi-parkinsonism case, the side contralateral to the patient's most affected side had a greater than 80% loss of striatal DA content, whereas the side showing no PD symptomotology showed deficits less than 75% (Agid et al., 1987); 2) in a study of 39 patients, a  7  mean DA deficiency of at least 70% was needed before any PD-like symptoms occurred, and the patient's motor deficits increased in correlation to the increasing loss of DA. There is other evidence demonstrating the degeneration of the nigrostriatal pathway in PD. Tyrosine hydroxylase (TH) activity, the rate-limiting enzyme in DA production, and the enzyme aromatic L-amino decarboxylase (AADC) (involved in the conversion of dopa to DA), are both severely reduced in the SN and striatum (Agid et al., 1987). In addition, DA metabolites such as dihydroxyphenylacetic acid and homovanillic acid are also reduced. Metabolic and electrophysiological evidence have been used to describe the anatomical dysfunction in PD. In situ hybridization for cytochrome oxidase I, a neuronal marker for metabolic activity, showed increased metabolism in the STN and GPi/SNr complex in MPTP-treated monkeys (Vila et al., 1997). There was also increased mRNA expression for glutamic acid decarboxylase, an enzyme involved in the synthesis of GAB A, in the SNr (Vila et al., 1996). As predicted in the basal ganglia model, you would expect an increase in metabolic activity in the STN or GPi/SNr complex. Bergman and colleagues (1994) studied the electrophysiological properties of the STN and GPi in PD. MPTP-treated monkeys showed a significant increase in firing rate when compared to their neuronal discharge prior to MPTP treatment. Wichmann and colleagues (1994) showed similar findings: by pharmacologically manipulating the STN and GPi in parkinsonian monkeys, they were able to temporarily reduce neuronal activity in the STN or GPi, alleviating parkinsonian symptoms.  Taken together, these results provide useful markers for the development of pharmaceutical or surgical therapies for PD. Researchers have focused on many different pathophysiological areas of PD which include normalizing the direct and indirect pathways or replacing the degenerated DA system. However, many treatments have demonstrated only moderate, temporary behavioral improvement. Therefore, to this day, researchers are looking for alternate means of alleviating PD symptoms.  8  3. Animal Models of PD 3.1. Animal Models of PD - How Are Thev Useful?  Experimental animal models of disease serve two important functions. First, they offer the opportunity to study certain aspects of the disease, providing insight into the possible mechanisms involved in its pathophysiology and progression. Second, experimental animal models provide a way to test new therapeutic strategies. Developing new drug therapies often involves established experimental models. This is the case in Parkinson's research: the development of new drugs or transplant therapies are first carried out in non-human primate or rat models of PD (Table 1).  Species Used  Experimental Model  •  Intracerebral 6-OHDA injections  • •  •  Systemic, intracerebral MPTP treatment  Non-human primates, rat, mouse, guinea pig, cat Non-Human primates, mouse, dog, cat  Table 1: Different pathogenic models of PD  Clinical Features seen in animals  • Akinesia • Tremor • Bradykinesia • Rigidity • Postural abnormalities • Dyskinesia  (adapted from Cenci et al., 2002).  The pattern of degeneration in PD patients can be reproduced in non-human primates by the systemic administration of l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP). MPTP is converted into the active neurotoxin, MPP+, where it enters DA terminals, disrupts mitochondrial function, and leads to neuronal death. MPTP enables researchers to induce a parkinsonian syndrome in non-human primates that is similar to the one observed in human PD patients. Primates show an abnormal body posture, slowness and lack of spontaneous movements, rigidity, and tremor (Cenci et al., 2002). Conversely, these symptoms can be reversed with the introduction of antiparkinsonian medications, making the MPTP model a useful testing ground. Nevertheless, the non-human primate MPTP model has economical, logistical, and ethical constraints that make it a difficult model to incorporate into research on an extensive basis.  9  A much older advancement in modeling PD was the introduction in the 1960s' of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) (Ungerstedt, 1968). 6-OHDA is transported into aminergic (both DAergic and noradrenergic) cell bodies causing massive nerve terminal degeneration. The neurotoxicity of 6-OHDA is due to its potent inhibitory effect on mitochondrial respiratory enzymes. Blocking these enzymes leads to a metabolic deficit whereby neurons can no longer exert normal physiological functioning, leading to cell death (Heikkila et al., 1989). Relative selectivity for DA neurons can be achieved by injecting 6-OHDA directly into the SN or medial forebrain bundle targeting DAergic pathways. Also, a co-systemic injection of the noradrenergic transporter blocker desipramine can inhibit the uptake of 6-OHDA into noradrenergic neurons, thereby reducing noradrenergic cell death. Rats with bilateral 6-OHDA lesions have been shown to produce motor disturbances reminiscent of those seen in PD patients. Rats exhibit postural abnormalities and have reduced capacity to maintain balance after a destabilizing challenge (Cenci et al., 2002). Spontaneous movement is also greatly reduced, as demonstrated in tests for normal exploratory movement (Cepeda et al., 2004: unpublished results). Moreover, footprint analysis of gait shows that bilaterally lesioned animals take shorter steps compared to normal rats (Cepeda et al., 2004: unpublished results). However, bilateral 6-OHDA lesion models are not commonly used. Even a partial bilateral lesion in rats leads to devastating neurological deficits, often requiring intensive post-operative care to avoid high morbidity and mortality rates. The animal model that is undoubtedly the most widely used in preclinical PD research is the unilateral 6-OHDA lesion model. A unilateral 6-OHDA lesion produces a hemiparkinsonian state, whereby one side of the body (contralateral to the lesioned side) is severely affected while the opposite (ipsilateral) side remains intact, acting as an intrinsic control. Researchers have experimented with different dose concentrations and injection sites for 6-OHDA. The most effective area for 6-OHDA injection seems to be directly along the medial forebrain bundle (Deumens et al., 2002). This lesioning method closely parallels degeneration seen in idiopathic PD; there is an almost complete loss (>90%) of striatal DA levels and a post-synaptic DA receptor supersensitivity ipsilateral to the lesioned side.  10  Behaviorally, unilaterally lesioned animals show body posture asymmetry, deficits in spontaneous and skilled motor movements (Barneoud et al., 1995; Kirik et al., 1998) and sensorimotor difficulties (Cenci et al., 2002). Most importantly, unilateral 6-OHDA lesions cause prominent asymmetry in motor behavior that can be quantified. Following a unilateral lesion, rats will preferentially turn towards the side of the lesion (Ungerstedt, 1968). When challenged with different DAergic agonists, the imbalance in DA activity between the two striata will lead to active rotational behavior. A rat challenged with amphetamine (a pre-synaptic DA agonist which releases DA from the unlesioned nigrostriatal nerve terminals) will preferentially rotate towards (ipsilateral to) the side of the lesion. Conversely, if challenged with a post-synaptic DA agonist (such as apomorphine) which acts on the denervated, supersensitive side, the rat will rotate away from (contralateral to) the lesioned side. Researchers have relied heavily on the asymmetric characteristics associated with the unilateral lesion model. Ipsilateral rotation after an amphetamine challenge has been used to monitor the effects of different neural transplantation techniques and neuroprotective treatments. Conversely, apomorphine-induced contralateral rotation has been used to assess new anti-parkinsonian drugs (Schwarting and Huston, 1996). In this regard, the normalization (or reduction) of rotational behavior after treatment is suggestive of the restoration of symmetric DA levels. However, there are caveats with using drug-induced rotational behavior to develop new treatments. It is not known how new treatments interact with different drugs. There are potential sensitivity issues between the drug and the new treatment regimen. In addition, the procedure itself (such as surgical striatal implants) may induce false-positive effects due to the stimulation of internal mechanisms. As a result, recent studies have employed non drug-induced behavioral procedures that may have less intrinsic effects. In conclusion, the 6-OHDA rodent model of PD is a useful tool for studying new antiparkinsonian treatments. Functionally, the model has been well characterized; it can parallel idiopathic PD. In addition, the model is relatively easy to use with large sample sizes. When the limitations to the different behavioral measures are overcome, the 6-OHDA model will be an invaluable method of testing new treatment strategies for PD.  11  Chapter 2: Treatments for Parkinson's Disease 1. Evaluation Methods of PD Severity and Treatment Efficacy 1.1. Clinical Rating Scales There are various clinical methods of evaluating behavioral improvements after treatment. However, these clinical scales are subjective and based either on physician assessments or patient evaluations. The few most commonly used are described below.  As described before, the five-stage H Y scale is a good means of assessing a PD patient's behavioral dysfunction before and during the course of the clinical trial (Hoehn and Yahr, 1967). This scale determines whether a new treatment can delay the progression of the disease. Another commonly used evaluation method is the Unified Parkinson's Disease Rating Scale (UPDRS). The UPDRS is a comprehensive inventory of symptoms of PD, including mood and mentation, activities of daily living, motor performance, muscle rigidity, speech, and gait (Freed et al., 2001). The scores for the UPDRS range from 0 (normal) to 176 (worst possible). It contains both motor (UPDRS-M) and non-motor components. Changes in the UPDRS-M are often used as an outcome measure to evaluate efficacy of various treatments. Other behavioral measures that are commonly used are the Schwab and England scale, which measures performances in daily life activities, total activities of daily living (ADL) score, and subjective self-assessment scales where patients record daily improvement in a diary.  1.2. Objective Assessment Methods: Brain Imaging Positron emission tomography (PET) or single photon emission tomography (SPECT) is another evaluation method that is used to correlate behavioral assessments with in vivo DAergic functioning. [ F]fluorodeoxyglucose imaging, which measures glucose metabolism, was one of 18  the first applied imaging techniques to study PD in vivo (Reivich et al., 1979; Eidelberg et al., 1990). There are several more D A specific ligands that assess PD dysfunction. [ F]fluorodopa 18  ( F-DOPA) is an analog of levodopa which is taken up by striatal DAergic terminals and can be 18  converted into F - D A . Therefore, F - D O P A is traditionally used for direct in vivo imaging of 18  18  dopa decarboxylase activity and storage in D A terminals. [ C]-raclopride, a selective DAergic n  D2/3 receptor antagonist, can be used as a surrogate marker to look at changes in endogenous DA.  12  [' 'CJraclopride competes with D A for the D 2 receptor binding site; therefore, any decrease in [ C]-raclopride binding suggests an increase in D A release and vice versa (Laruelle, 2000). n  Other ligands have been developed for studying DAergic synthesis and storage. For example, ligands such as [' 'CJmethylphenidate bind with high affinity to the D A transporter (DAT) (Volkow et al., 1995) whereas [ C]dihydrotetrabenazine binds to the vesicular monoamine n  transporter (VMAT) (Frey et al., 1996). Taken together, PET imaging can be an invaluable method for assessing new treatments.  2. Current Treatments for PD 2.1. Drug Therapies The discovery of levodopa (L-DOPA) in the 1960s modernized the treatment for PD and is still the best available treatment. It has become the gold standard against which new treatments are compared. Since its introduction, L-DOPA therapy has provided significant symptomatic improvement in most PD patients. L-DOPA produces a stable antiparkinsonian effect in the early stages of PD but is associated with increasing side effects as time goes on.  L-DOPA is an analog of the D A precursor 3,4-dihydroxyphenylalanine (dopa). Dopa is decarboxylated by A A D C to form DA. Since L-DOPA is administered orally, it must pass through the lumen of the intestine into hepatic and systemic circulation. In addition, it must pass through an endothelial cell-lined blood-brain barrier (BBB) before entering the brain. D A is not used as a treatment as it cannot pass through the BBB. In addition, intestinal walls, liver, kidneys, and the brain endothelium contain large amounts of A A D C and the enzyme catechol-Omethyltransferase (COMT) which catabolizes L-DOPA into 3-OM-DOPA. Therefore, L-DOPA treatment is normally accompanied with an A A D C inhibitor, such as carbidopa or benserazide, which are found to affect mainly peripheral A A D C in the concentrations used (Kopin, 1993). A A D C inhibitors are beneficial because they increase the bioavailability of L-DOPA by limiting the decarboxylation of L-DOPA in the peripheral tissues and in the endothelial barrier.  L-DOPA is extremely effective in the first decade following PD onset. However, there are two main problems with L-DOPA treatment. The first is the potential toxicity of L-DOPA on D A neurons (Olanow et al., 2004). In vitro data has demonstrated that L-DOPA exerts toxic effects  13  on cultured DA neurons. L-DOPA is known to undergo auto-oxidation, which leads to the formation of oxidative stress radicals including quinones, superoxide radicals, and hydrogen peroxides (Graham et al., 1978; Olanow et al., 2004). Research has shown that fetal mesencephalic cell cultures exposed to high levels of L-DOPA result in reduced TH-positive neurons, signs of apoptotic cell death, neuronal cell shrinkage, and DNA fragmentation (Mytilineou et al., 1993). However, several in vivo studies have not confirmed these findings. Chronic administration of L-DOPA in 6-OHDA lesioned animals has led to mixed results, ranging from small reductions in DA neurons to significant recovery of TH-positive neurons (Blunt et al., 1993; Datla et al, 2001). Additionally, in the recently completed ELLDOP A clinical study (Fahn and Parkinson's Study Group, 2002), L-DOPA-treated patients demonstrated less motor deterioration compared to control patients; however, imaging analyses demonstrated a greater decline of striatal function. It remains unclear whether L-DOPA has any adverse effect on PD. Nevertheless, most clinicians prescribe L-DOPA in the early stages of PD as the symptomatic effect is unwarranted. Future clinical studies are needed to clarify whether LDOPA is toxic in PD. The second and most significant limitation to chronic L-DOPA use is the development of motor complications in late stage PD. After five to ten years of L-DOPA treatment, approximately 5080% of PD patients develop motor fluctuations despite continuous L-DOPA administration. Patients experience a "wearing o f f of L-DOP A-induced improvement before administration of the next dose. Later, the patient begins to show "on-off phenomena; namely, the patient shows sudden signs of tremor, freezing, or rigidity interspersed with phases of mobility in a pattern that cannot be predicted by the L-DOPA regimen. Advanced PD patients may show signs of extreme dyskinesias (abnormal involuntary movement) and psychoses. These drug-induced symptoms ultimately confound the progression of PD, and they can be so severe as to completely disable the PD patient. Evidence suggests that L-DOP A-induced motor complications are due to L-DOPA's short halflife as well as its potential to induce pulsatile stimulation of DA receptors (Olanow et al. 2004). Based on these hypotheses, several strategies have been explored to restore L-DOPA efficacy. The dosages and timing of L-DOPA administration have been manipulated, and methods for  14  continuous systemic L-DOPA administration have been explored. However, in practice, many of these methods have proven impractical and intolerable to PD patients. Recent therapies have aimed at extending L-DOPA's half-life and controlling its release, such as developing coating for L-DOPA pills that slow release (Sinemet-CR). Another approach involves the administration of peripheral C O M T inhibitors. Preliminary evidence has been positive, demonstrating reduced dyskinesia and improved motor improvement when co-administered with L-DOPA. However, this treatment regimen is not yet fully established in PD patients and may lead to side effects of its own.  2.2. Surgical Treatments for PD 2.2.1. Lesion and Stimulation Due to the limitations of L-DOPA treatment, alternative techniques have been investigated. Advances in understanding the pathophysiology of PD, together with refined surgical stereotactic techniques, have lead to the resurgence of surgical treatments for PD. Theory suggests that the STN and GPi are overactive in PD (Obeso et a l , 2000; Obeso et al., 2002); therefore, the premise behind surgical treatments for PD is that reducing either STN or GPi neuronal activity normalizes basal ganglia output.  1. Lesions. Pallidotomy, which refers to the destructive lesioning of the GPi, was originally popular in the 1950s and 1960s. However, pallidotomy procedures were replaced with the introduction of L-DOPA in the 1960s. Nevertheless, advances in the understanding of the anatomy of the basal ganglia, stereotactic surgical techniques, and brain imaging, led to the resurgence of pallidotomy in the 1990s (Laitinen et al., 1992; Zesiewicz and Hauser, 2001). In one study, posteroventral pallidotomies in 38 patients resulted in significant improvements in tremor, rigidity, bradykinesia, and dyskinesia (Laitinen et a l , 1992). Dogali et a l , (1995) reported similar results in 18 patients; in this study, UPDRS scores improved 65%. Another group performed blinded assessments on 14 patients (Lozano et a l , 1995). Six months after pallidotomy, total motor score in the "off state improved by 30% and the total akinesia score improved by 33%. In a long-term prospective study, 40 late-stage pallidotomy PD patients demonstrated sustained, moderate improvement in "off period motor behavior (including bradykinesia and rigidity) up to two years post-surgery (Lang et al., 1997). There was also  15  marked improvement in L-DOPA-induced dyskinesia. Ipsilateral dyskinesias were also improved, albeit to a lesser degree, at one year (but not at two years) post-surgery. Other studies have reported similar results (Baron et al., 1996; Uitti et al., 1997): pallidotomies improve tremor, bradykinesia, and contralateral drug-induced dyskinesias.  A review of pallidotomy patients has revealed certain complications associated with the procedure (Laitinen, 2000). Temporary side effects were observed, including drowsiness, confusion, and memory deficits. More importantly, a small subset of patients demonstrated permanent memory deficits and mental deterioration. Because of this, pallidotomies have been infrequently used in the past few years. Motor improvements in most patients are minimal, are only beneficial for younger patients (Baron et al., 1996), and may not be sufficient to outweigh the costly detrimental effects associated with the surgical technique. Pallidotomies necessitates a destructive brain lesion which can potentially lead to more severe neurological deficits, particularly in bilateral surgical procedures.  Thalamotomy is another potential surgical technique capable of reducing contralateral tremor and rigidity. Different studies reported significant reductions in contralateral tremor, with improvements ranging from 75%-86% of the patient population (Fox et al., 1991; Osenbach and Burchiel, 1998; Linhares and Tasker, 2000). Diederich and colleagues (1992) demonstrated that thalamotomies resulted in tremor reduction in patients followed up to ten years post-surgery. However, there were transient complications with the surgery, which included contralateral weakness, confusion, aphasia, dysarthria, ataxia, and dystonia (Hallett et al., 1999). In addition, thalamotomy was not effective in treating bradykinesia, gait, or speech (Tasker et al., 1983).  STN lesions have also been considered. Alvarez and colleagues demonstrated improved UPDRS scores in PD patients who received subthalamotomies (Alvarez et al., 2001). However, lesioning the STN can lead to the risk of hemiballismus, an involuntary movement manifested through jerking movements of the extremities. In conclusion, due to the complications associated with the surgical procedure, attempts have been made to develop better surgical interventions.  16  2. Stimulation. A recently-developed surgical technique is high frequency deep brain stimulation (DBS). DBS is a non-ablative surgical procedure: instead of lesioning, hyperactive areas (such as the GPi and STN) are modulated by implanting electrodes and applying a highfrequency electrical current that blocks neural activity in surrounding areas (Bjarkam et al., 2001; Hammerstad and Hogarth, 2001). However, the means by which DBS works is unknown. Possible mechanisms include a depolarization blockade or the jamming of abnormal neuronal firing patterns (Benazzouz and Hallett, 2000). Nevertheless, DBS stimulation emulates the effects of a destructive lesion.  GPi DBS has led to behavioral improvement in a limited number of studies (Siegfried and Lippitz, 1994; Ghika et al., 1998; Volkmann et a l , 1998). Volkmann and colleagues (1998) conducted an open-label study in nine patients who received bilateral GPi stimulation. Patient UPDRS scores significantly improved after 3 and 12 months. In a comparable study, six patients were followed up to 24 months (Ghika et al., 1998). Results were similar to those of the Volkmann et al., (1998) study: patients demonstrated improvements in UPDRS scores as well as dyskinesia. However, clinical improvement declined after 12 months. In addition, although GPi DBS was effective in treating dyskinesia, some reports suggest it has a minimal effect on bradykinesia (Krack et al., 1998; Bejjani et al., 1997)  STN DBS has generated more promising results (Limousin et al., 1998; Krack et a l , 1999; Houeto et a l , 2000). Limousin and colleagues (1998) evaluated 24 bilateral STN DBS patients for 12 months and found that UPDRS and motor scores improved by 60%. In another study, 23 patients were followed for 6 months (Houeto et al., 2000). These patients demonstrated significant improvements in UPDRS and A D L scores, reductions in H Y disability score, and decreased motor fluctuations. Taken together, STN DBS seems particularly effective in improving parkinsonian symptoms.  In 2001, a DBS research group conducted a double-blind study that looked at the effects of bilateral DBS surgery in the GPi or STN (The Deep-Brain Stimulation for Parkinson's Disease Study Group, 2001). Ninety-six patients were implanted in the STN and thirty-eight patients were implanted in the GPi; all were followed up to six months post surgery. Both STN and GPi  17  DBS resulted in increased "on" time (without dyskinesia) and decreased "off time. In addition, there was a 49% and 37% increase in motor improvement in the STN and GPi group, respectively. A home diary assessment showed significant motor improvements: patients reported prolonged periods of mobility without involuntary movements.  Despite these positive results, there are certain limitations inherent to the procedure (Hariz, 2002). Nearly all patients exhibited one or more adverse events relating to the surgery. During the course of the study, patients showed signs of mood changes, language deficits, weakness and paralysis. In addition, approximately 37% of DBS patients experienced device-related adverse effects (Watts et a l , 2003) including hemorrhage, infections, and seizures. In some cases, PD patients demonstrated more severe motor impairment and other PD symptoms. Based on these results, DBS is a potential alternative for the treatment of PD. However, the sophisticated nature of this technique and potential complications (due to the surgical procedure or hardware) warrant further investigation.  2.2.2. Cell Therapies: Transplantation Cell replacement therapies have been investigated in the hope of finding a long-term, continuous DAergic source (reviewed in Bjorklund and Lindvall, 2000; Borlongan and Sanberg, 2002). There are numerous reasons why cell replacement therapies are suitable alternatives for treating PD. The pathophysiology of PD has been well-defined anatomically and is surgically accessible. Also, since neuronal loss is type-specific, cell replacement therapy has an easier task of replacing only a single cell type to restore DAergic activity. For these reasons, several studies have been conducted to test the efficacy of different cell transplants. Many of these studies have resulted in significant behavioral improvements, as well as evidence of survival and reinnervation of DAergic neurons into the host striatum. Therefore, many clinical trials were conducted in the 1990s to determine the safety and efficacy of cell transplants in human PD patients.  2.2.2.1. Fetal Ventral Mesencephalic (Nigral) Tissue Transplantation Fetal ventral mesencephalic (FVM) cell transplants are the most widely-studied transplant procedure for PD (Olanow et al., 1996; Bjorklund and Lindvall, 2000; Bjorklund et al., 2003) (Table 2). Over 300 patients worldwide have undergone fetal cell tissue transplants  18  (Subramanian, 2001; Borlongan and Sanberg, 2002); however, long-term follow up has only been conducted on fewer than 30 patients (Olanow et al., 1997). Several studies have reported both significant clinical improvements and increased DAergic activity up to twenty-four months post-transplant (Olanow et al., 1996; Hallett et al., 1999; Subramanian, 2001). However, collectively interpreting these results is a difficult task: there were variations in the surgical technique, the transplant target selection, and the quality and quantity of the tissue used for the transplant across different clinical studies.  Advantages  Disadvantages  •  • •  • •  F V M transplantation forms synaptic connections with host to recreate nigrostriatal system Long-term behavioral improvement in some patients Increase in D A activity as measured by PET  • • • • •  Ethical concerns Unknown large amount of F V M tissue (from several donors) needed to treat single patient Limited storage time Immunosuppression may be necessary Improvements not immediate Late-onset dyskinesias and other motor dysfunction Unsuccessful double-blind placebo study  Table 2: Advantages and disadvantages of F V M transplantation for PD.  Several open-label studies have reported clinical improvements after F V M cell transplants (Freed et a l , 1992; Spencer et al., 1992; Freeman et al., 1995; Wenning et al., 1997). In one study, four H Y stage 4 patients were assessed after receiving a unilateral F V M cell transplant into the right caudate nucleus (Spencer et al., 1992). Each patient received cryopreserved tissue from one donor at 7-11 weeks gestational age. There was moderate bilateral motor improvement in all patients; three of four patients were able to lower their L-DOPA dose. However, despite showing motor improvement, the patients were still markedly affected. There were no differences in H Y scores in the 1 year assessment period. Freed and colleagues (1992) reported moderate behavioral improvements in seven patients after F V M transplants from single donor embryos (gestational age 7-8 weeks). These patients demonstrated a reduction in "off period dyskinesias, and an average 38% reduction in L-DOPA dose. Mean H Y score improved slightly (from 3.71 to 2.50). Similar results were documented in bilateral F V M cell transplants (Freeman  19  et a l , 1995; Hauser et al., 1999), and have been confirmed in two collaborative studies (Sawle et al., 1992; Lindvall et a l , 1994). Two H Y stage 3 patients underwent F V M cell transplantation (from 8-9 week-old fetuses) and were serially studied using PET. Post-operative scans revealed that both patients had increased F - D O P A uptake at 13 months post-transplant (Sawle et al., 18  1992), which was maintained up to three years post-transplant (Lindvall et al., 1994). Patients showed moderate behavioral improvement in the first year after transplant, but became more pronounced between one and three years after surgery.  The longest reported follow-up after a F V M cell transplant has been ten years (Piccini et al., 1999). In this single-patient study, the patient demonstrated sustained, moderate improvement. At the patient's ten-year follow-up, researchers observed "continuous marked benefit with no rigidity, minor hypokinesia, intermittent, mild resting tremor and no 'on-off fluctuations" (Piccini et al., 1999). PET imaging indicated a gradual increase in F - D O P A levels in the ten 18  years post-transplant.  The results of these studies should be viewed with caution. Many studies had small sample sizes, and their open-label design can potentially be influenced by clinician bias or a patient placebo response (Freeman et al., 1999). To date, two studies investigated F V M transplantation using a double-blind, sham-surgery-controlled clinical trial (Freed at al., 2001; Olanow et al., 2003). One study followed 40 patients who received either a unilateral F V M tissue transplant or sham surgery up to twelve months post-transplant (Freed et al., 2001). Patients assigned to the transplant group received F V M tissue from four embryos aborted at seven to eight weeks. The results demonstrated no differences in total UPDRS scores between the two treatment groups. Only the younger patient subgroup (aged sixty and under) reported any clinical recovery and demonstrated improvement in UPDRS scores when compared to the sham-surgery group. Similarly, the Schwab and England test showed significant improvement in only the younger patients. Interestingly, 84% (16 of the 19 transplant patients) showed a significant increase in 18  F - D O P A uptake compared to the sham-surgery group. A n additional double-blind, placebo-  controlled clinical trial was conducted in 2003 (Olanow et al., 2003). In this study, 34 advanced PD patients were assigned to one of three groups: 1) bilateral F V M tissue transplant using one fetal donor per side, 2) bilateral F V M tissue transplant using four donors per side, or 3) bilateral  20  sham surgery procedure, and were followed up to 24 months. Interestingly, there was no significant overall treatment effect. Both the sham surgery and one donor transplant group deteriorated behaviorally, and the four donor group improved only slightly. However, F V M transplantation resulted in a significant increase in F - D O P A uptake in both the one donor and 18  four donor groups.  These studies have brought up some important issues. Some transplant patients went on to develop some atypical behavioral features suggestive of a Parkinson-plus syndrome (Spencer et al., 1992; Wenning et al., 1997), and in one study, (Freed et al., 2001), 15% of the patients who received F V M transplants developed dyskinesias three years after the transplant (Freed et a l , 2001). In the Olanow study (Olanow et al., 2003), over 56% of the transplanted patients developed "off period dyskinesias during the course of the study; there were no complications in the placebo group. The dyskinesias were characterized as stereotypic movements that affected one or both lower extremities. For three patients, the dyskinesia was so severe that surgical intervention was necessary after the completion of the study (Olanow et al., 2003).  Another important issue is the required number F V M donors needed for transplantation. The precise number of D A neurons required to ensure any clinical improvement is unknown, and there are certain issues pertaining to the optimal number of grafts needed for transplantation. First, the ratio of DAergic neurons to total cell number in a F V M graft is variable between grafts and cannot be controlled for. In addition, the number of surviving D A neurons post-transplant is unknown. Many studies have suggested that a minimum of four to eight F V M donors is needed in order to restore normal physiological levels (Olanow et al., 1996). However, in one case, an autopsied transplant patient (who died of unrelated causes) demonstrated a survival of only 5% of the theoretical total of DAergic neurons from six fetal donors (Olanow et al., 1996).  The disappointing results from the double-blind placebo-controlled clinical trials suggest that there are many issues that need to be resolved. The large number of fetal donors needed for one patient, as well as the ethical considerations in obtaining fetal tissue, has limited the utility of this procedure for treating PD. In addition, the development of disabling late-onset dyskinesias has urged researchers to more closely assess issues relating to the quality and quantity of F V M tissue  21  to be transplanted. Although the efficacy, morbidity and mortality rates in the published reports appear to be good, many of the techniques require further modification.  2.2.2.2. Porcine Cell Transplantation Recognizing the limitations of human F V M tissue transplantation, researchers have investigated alternative xenogeneic DAergic cell sources. Studies have shown that porcine F V M tissue may be a suitable alternative for tissue transplantation in PD (Pakzaban et al., 1995; Fink et a l , 2000; Schumacher et al., 2000). The pig is considered to be the most desirable xenogeneic source for different whole-organ transplantations due to similarities between human and porcine major histocompatibility complex (MHC) antigens. Pigs have large Utter sizes and can provide an unlimited supply of donor tissue, eliminating donor availability issues. Pig donors can provide optimally-staged DAergic tissue that is physiologically similar to human DAergic tissue. Also, porcine xenotransplantation allows for the sterile harvesting and extensive screening for any bacterial or viral contamination (Table 3). Based on these characteristics, studies have been performed to test the efficacy of porcine F V M tissue in treating PD.  Advantages  Disadvantages  • •  •  •  No ethical concerns with availability Large litter sizes can produce potential unlimited access to F V M porcine tissue Can form synaptic connections with the host nigrostriatal system  • • • •  Lifelong immunosuppression/antigen masking is needed Risk of rejection due to discordance between humans and pigs Risk of porcine endogenous retrovirus (PERV) Only moderate behavioral improvements not immediate No increase in D A activity (through PET)  Table 3: Advantages and disadvantages of Porcine F V M transplantation for PD.  In preclinical studies, the striatal implantation porcine F V M tissue in 6-OHDA lesioned rats resulted in significant improvements in parkinsonian symptoms (Isacson et al., 1995; Galpern et al., 1996). Porcine F V M tissue (from embryonic day (ED) 27 porcine fetuses) was transplanted into lesioned, cyclosporine-A (CyA) treated or non-CyA treated rats (Galpern et al., 1996), and  22  functional recovery was assessed by amphetamine-induced rotation (AIR). The porcine F V M transplant group that was pre-treated with CyA resulted in significant AIR reduction up to 18 weeks post-transplant. Interestingly, there was only transient recovery in the non-CyA treated group. In situ hybridization (for a porcine repeat D N A probe) and T H immunohistochemistry demonstrated significantly larger survival rates in the CyA-treated rats compared to the non CyA-treated rats. Other studies have documented similar results (Isacson et al., 1995; Pakzaban et al., 1995). By immunosuppressing the host or masking the M H C antigens of porcine F V M tissue, porcine tissue implants survived well, and were able to proliferate and send axonal projections to the host striatum.  Based on these results, pilot experiments were conducted on twelve late-stage PD patients that underwent unilateral porcine F V M tissue transplantations (from E D 25-28 porcine fetuses) (Fink et a l , 2000; Schumacher et al., 2000). Patients were assessed using the UPDRS postoperatively at 3, 6, 9, and 12 months. Half of the patients received CyA treatment, whereas the other half was transplanted with anti-MHC I-treated porcine tissue. At 12 months post-transplant, there was moderate 19% improvement in UPDRS scores in 10 out of the 12 patients. Three patients showed greater than 30% improvement, two patients showed a milder improvement (19% and 25%), whereas the remaining five patients showed an 11% improvement. Improvements were first evident at 3 months post-transplant, and were similar between the C y A pre-treated and anti MHC-I treated group. However, these results should be taken with caution; the open-label design in these studies could warrant the same potential bias seen in the human F V M transplant studies (Freeman etal., 1999).  Interestingly, there was no correlation between DAergic functioning and clinical improvement in any patient, as PET did not reveal any significant changes in F - D O P A uptake. One patient 18  enrolled in the study (who died suddenly of a pulmonary embolism 7 months after transplant) underwent an autopsy to look for porcine tissue survival (Deacon et al., 1997). Immunohistochemistry identified three (of the four) transplants; however, only a small amount of porcine tissue survived. There was a mean 638 TH-positive cells at each transplant site compared to the 40,000 surviving fetal DAergic cells observed in human F V M transplant patients.  23  Low porcine F V M cell numbers were likely due to the transplant rejection. In preclinical studies, the absence of C y A immunosuppression resulted in poor transplant survival (Galpern et al., 1996) . Despite all patients receiving C y A immunosuppression, preliminary post-mortem analyses in one patient suggests that CyA immunosuppression may not be enough (Deacon et al., 1997) . This observation parallels a xenotransplant characterization study done in rodents; C y A immunosuppression was protective only during the first week after transplant and failed to provide long-term survival of porcine tissue (Larsson et al. 2001).  Another concern with using porcine tissue is the risk of contracting infections from a pig reservoir (zoonosis) such as porcine endogenous retrovirus (PERV) infection (Schumacher and Isacson, 1997). Although there have been no reported PERV transmission, in vitro studies have shown that PERV could infect human cells (Subramanian, 2001). A n assay was developed to test all patients who received porcine F V M tissue transplants. Fortunately, there has been no evidence of PERV transmission.  Taken together, preliminary results have shown that porcine fetal transplants may be a viable form of treatment for PD. However, the moderate clinical improvement and low cell survival warrant further improvement. In addition, the need for constant immunosuppression, as well as the theoretical risk of zoonotic infection, may reduce its feasibility as a future transplant procedure.  2.2.2.3. Glial cell line-derived neurotrophic factor (GDNF) Converging evidence has suggested that glial cell line-derived neurotrophic factor (GDNF) is able to reverse DAergic degeneration and alleviate parkinsonian symptoms (Bjorklund at al., 1997) (Table 4). GDNF is a member of the transforming growth factor-p superfamily, and was first purified from a rat glial cell line based on its ability to promote D A uptake in F V M cell cultures (Lin et a l , 1993). Many neurotrophic factors have the ability to promote D A neuron survival in vitro; however, GDNF has been the only trophic factor to demonstrate significant in vivo protection of D A neurons in PD. Different methods of applying GDNF have been studied, including implanting GDNF-modified fibroblasts (Duan et al., 2004), and lentiviral or adenoviral GDNF delivery (Bilang-Bleuel et al., 1997).  24  Advantages  Disadvantages  • • •  • •  • •  No ethical concerns Consistent neuroprotection for D A neurons Can be implanted through a number of different carriers No concerns with availability and storage No immunosuppression needed  • •  Antibody development against GDNF Side effects including nausea, anorexia and vomiting Hyponatremia and paresthesias in high dose GDNF patients Safety concerns  Table 4: Comparison of advantages and disadvantages of G D N F infusion for PD.  Initial studies demonstrated that continuous nigral injections of GDNF into 6-OHDA lesioned rats resulted in complete protection of SN neurons up to four weeks post-lesion (Sauer et al., 1995). SN neuronal preservation has also been shown after intrastriatal GDNF injections up to twenty weeks post-GDNF (Shults et al., 1996; Duan et al., 2004). GDNF neuroprotection has been studied in parkinsonian monkeys (Kordower et al., 2000; Grondin et al., 2002; Palfi et al., 2002). In these studies, the chronic intracerebroventricular (ICV) or striatal infusion of GDNF in MPTP-treated monkeys led to significant behavioral improvements (Grondin et al., 2002). This was accompanied by a >20% increase in TH-positive SN neurons, a >30% in SN neuronal cell size, and a 233% increase in striatal D A levels. Palfi and colleagues (2002) found similar results; using a lentiviral delivery technique, GDNF treatment led to an eightfold increase in D A neuron survival in MPTP-treated monkeys.  Based on these findings, several open-label studies were conducted to test the safety and efficacy of GDNF treatment in PD patients. In one pilot study, five patients were implanted with a GDNF-releasing minipump for two years (Hotten et al., 2004). Mean UPDRS scores improved 41%, and PET analysis showed a 60% increase in F - D O P A uptake at the transplant site. 18  Recently, multicenter double-blind studies were conducted (Nutt et al., 2001; Lang et al., 2004). In one study, 50 patients were given monthly ICV injections of GDNF or placebo for 8 months (Nutt et al., 2001). This study generated disappointing results. There were no changes in UPDRS score compared to the placebo group. In addition, GDNF patients presented numerous side effects including nausea, anorexia, and vomiting. Patients who received higher doses of GDNF presented signs of weight loss and symptomatic hyponatremia. Paresthesias, or abnormal  25  neuronal sensations, were also seen in GDNF-treated patients, and only resolved after discontinuation of GDNF treatment. Similar results were found in a separate study (Lang et a l , 2004). Six months after the procedure, there were no differences in UPDRS-M scores between the placebo and GDNF-treated group, and F - D O P A uptake was only moderately improved in 18  the GDNF-treated group. Side effects were also common: they included paresthesias, headache, and upper respiratory infection. Interestingly, four of the patients developed antibodies to G D N F during the study, and one patient developed antibodies shortly after switching from the placebo group to the GDNF group. As a result, G D N F treatments have been withdrawn from all clinical trials until these issues have been resolved.  2.2.2.4. Embryonic Stem Cell Transplantation Recently, several reports have suggested that stem cells may provide a source for D A replacement (Freed, 2002; Roybon et al., 2004). Human stem cells are undifferentiated cells that have a high proliferative capacity and can differentiate into a wide variety of cells. They can be derived from either fetal or adult tissue, they are relatively easy to maintain in culture, and a single donor can be used to potentially produce large quantities. PD has been the exemplar disease to show how stem cells can work: they have the potential to differentiate into D A neurons as a cell replacement therapy for PD (Table 5).  .  Advantages  Disadvantages  • •  • •  Can be derived from fetal or adult tissue Potential unlimited supply of D A neurons  • •  Early stages of development Unlimited growth can lead to tumor formation No known method for differentiating into D A neurons Poor survival in animal models of PD  Table 5: Advantages and disadvantages of Embryonic Stem Cells for PD.  Stem cell experiments have focused largely on mouse embryonic stem (ES) cells and their ability to adopt a DAergic phenotype (Roybon et a l , 2004). That is, ES cells that differentiate into D A neurons should be identical to native D A neurons in the SN. Therefore, ES cells should not only  26  express adequate T H levels, but should also express other DAergic markers such as V M A T , D A T , but lack the D A beta hydroxylase (DBH) enzyme for noradrenaline synthesis. Even under a variety of growing conditions, ES cells do not spontaneously proliferate into DAergic neurons in vitro. However, recent research using genetically engineered DA-related transcription factors has demonstrated successful differentiation of ES cells into D A neurons (Saucedo-Cardenas et al., 1998; Cazorla et al., 2000).  Nevertheless, in vitro conditions do not mimic the conditions in the brain, and it is important to know whether ES cells are capable of maintaining a DAergic phenotype and establishing neuronal connections with proper striatal targets in vivo. Unfortunately, most animal studies have produced negative results; ES cells transplanted into lesioned rats have resulted in either poor cell survival or over-proliferation leading to the formation of teratomas (Roybon et al., 2004). Only one animal study has successfully reported the differentiation of ES cells into DAergic neurons in the brain (Bjorklund et al., 2002). In this study, a low dose mouse ES cell transplant in lesioned rats resulted in significant improvements in rotational behavior. There was also histological evidence of TH-positive cells co-expressing D A T and A A D C at the transplant site.  The molecular mechanisms involved in the differentiation of ES cells into a DAergic phenotype are unknown. Despite the success of the Bjorklund et al. (2002) study, many other studies have produced poor results. ES-cell research, to date, faces a number of dilemmas: to reduce the risk of teratoma formation, cell differentiation should take place in vitro before they are transplanted into the brain. However, the ability for ES cells to differentiate into D A neurons in vitro is premature and warrants further investigation. Therefore, before ES cells can be used as a form of treatment, this and a number of other important issues need to be resolved.  Many different cell therapy procedures have been studied for PD; all of them have demonstrated the potential to alleviate parkinsonian symptoms. Nevertheless, despite showing behavioral improvements in pre-clinical rodent and non-human primate models of PD, clinical results have been somewhat disappointing. Issues such as the availability of accessible DAergic sources, teratoma formation, or the risk of infections and side effects must be resolved. In this regard,  27  there is the need for an easily available, stable, long-lasting DAergic source for the treatment of PD.  28  Chapter 3: Human Retinal Pigment Epithelial Cells: A Cell Alternative for the Treatment of Parkinson's Disease 1. Fundamental Properties of h R P E Cells 1.1 HRPE-Cell Functioning in the Retina Human retinal pigment epithelial (hRPE) cells are retinal support cells located in between the choroid capillary bed and the photoreceptor layer (Figure 3) (Marmor and Wolfensberger, 1998; Schraermeyer and Heimann, 1999). By virtue of their position, hRPE cells form tight-junctions comprising the blood-retina barrier. HRPE cells play an integral part in normal retinal functioning (Table 6): they are responsible for the net movement of ions from the retina to the choroid and are involved in the uptake, processing and transport of different retinoids (Schraermeyer and Heimann, 1999). HRPE cells are also active phagocytic cells. They phagocytize dead or detached components of rod or cone organelles, and have the regenerative capacity to renew or reassemble these organelles. In addition, hRPE cells give nutritive support to adjacent retinal structures. Apart from their versatility, hRPE cells are also extremely robust. Under normal circumstances, hRPE cells, once differentiated, do not renew themselves; they remain viable throughout their lifetime. That is, a hRPE cell must perform all of its functions during the lifetime of its host (which may last over one hundred years).  1.2. Important hRPE-Cell Properties in the Treatment of PD HRPE cells are a potential cell therapy source for PD because of their DAergic properties (Marmor and Wolfensberger, 1998). HRPE cells are melanin-producing cells; thus, they contain the necessary machinery to produce melanin. In the RPE, dopa is synthesized from tyrosine in a reaction catalyzed by tyrosinase. Tyrosinase is a TH-analog which is involved in a series of metabolic steps that leads to melanin production (Figure 4). Although dopa seems to be reoxidized quickly (to dopaquinone) in normal adult functioning, embryonic hRPE contains more stable dopa concentrations, and has been hypothesized to be the dopa source in retinal development (Kubrusly et al., 2003). Apart from TH-like activity, hRPE cells possess other molecular machinery involved in D A production and regulation (Marchionini et al., 1999). HRPE cells express the D A D 2 autoreceptor, and V M A T involved in D A vesicular transport. In addition, in vitro studies  29  I Ir-zx i  IMsnrm  Figure 3: Schematic drawing of R P E in the eye (adapted from Marmor and Wolfensberger, 1998).  30  EUMELAN1NS (black/brown pigments)  PHEOMELANINS (yellow/red pigments)  Figure 4: Schematic diagram of the metabolic pathways leading to the production of eumelanin and pheomelanin. Note the production of dopa (red arrow) as a cofactor in the early pathway via the enzyme tyrosinase (adapted from Marmor and Wolfensberger, 1998).  31  have shown that embryonic hRPE cells express A A D C and are capable of producing D A (Kubrusly et al., 2003). However, hRPE cells do not express D A T or the Di receptor subtype. Taken together, hRPE cells have the necessary machinery to produce dopa and have the potential to become a reliable source of substrate for D A neurons when implanted into the brain.  Pigment functions • • • •  Light adaptation and screening Detoxification and binding Lipofuscin accumulation Antigenic properties  Interphotoreceptor matrix and retinal adhesion • Specialized matrix ensheathment of rods and cones • Metabolic control of adhesion  Environment and metabolic control  Repair and reactivity  • • •  • • • •  Blood-retina barrier Nutrient/ion transport Enzyme, growth factor, pigment synthesis  Repair and regeneration Immunologic interactions Scarring and pigment migration Modulation of fibrovascular proliferation  Outer segment phagocytosis and aging • • • •  Phagocytosis of outer segment tips Digestion and recycling of membrane material Aging effects: lipofuscin Deposits and alterations in Bruch's membrane  Table 6: Physiologic functions of the R P E in the retina (adapted from Marmor and Wolfensberger, 1998). HRPE cells have also been shown to produce a number of trophic factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulinlike growth factor (IGF), vascular endothelial growth factor (VEGF), nitric oxide, fas ligand, and pigment epithelial-derived factor (PEDF) (Marmor and Wolfensberger, 1998). Trophic factor production has only been studied in vitro or in its native environment; its expression and release when implanted in vivo has not yet been established. Nevertheless, as discussed later, it can be hypothesized that the co-expression of different trophic factors in hRPE cells can lead to the greater possibility of survival and functioning.  32  Well established cell culturing techniques provide another advantage of hRPE-cell use. These techniques allow hRPE cells to be dissected from the donor eye and grown to confluence on a permeable substrate (Gallemore et al., 1995; Hernandez et a l , 1995). The use of cultured hRPE cells from expanding primary tissue has several advantages. First, hRPE cells from a single donor can be grown to produce the number of cells needed for surgery. Since primary human tissue is always in short supply, expanding primary hRPE cells can help treat a large number of patients eliminating donor availability issues. Second, hRPE cells can be maintained in culture for extended periods of time (Hu et al., 1994). In previous F V M cell transplantation procedures, F V M tissue that was stored for long periods of time in culture resulted in the reduction in both cell viability and survivability after transplantation (Olanow et al., 1996). In comparison, HRPE cells maintain good viability over time, even when stored in culture for an extended period. Also, culturing procedures allow sufficient time for hRPE cells to be extensively tested for any viral or bacterial diseases, reducing the risk of contamination. Finally, since hRPE cells are allogeneic, there is no risk of zoonosis infection.  Table 7 summarizes the advantages of using hRPE-cell implants as a cell therapy source for treating PD. The development of hRPE-specific cell culturing procedures has improved the availability, storage, and viability of cell transplants. In addition, since hRPE cells are allogeneic, immunosuppression may not be necessary. As described in the next sections, hRPE cells are currently being studied in open-label and double-blind clinical trials to determine their safety and effectiveness in PD patients.  2. H R P E Cells Attached to Gelatin Microcarriers A critical problem with the transplantation of neuronal or non-neuronal cells into the brain has been low cell survival rates. Human and porcine F V M transplant studies have resulted in low survivability and rejection, even in the presence of immunosuppression. This is attributed to the host immune response even though the central nervous system (CNS) is considered an immune privileged site. Therefore, current research has focused on finding ways of improving cell survival.  33  Advantages  Disadvantages  •  •  •  • • •  Ethical concerns are minimized because single donor can treat numerous patients Established and effective cell culturing techniques for hRPE cell storage and proliferation No time constraints on hRPE cell storage and surgical scheduling Tissue can be extensively tested prior to implantation No risk of porcine retrovirus (PERV)  Does not form or re-establish synaptic connections with the host striatum  Table 7: Comparison of the advantages and disadvantages of hRPE-cell implantation for PD. Gelatin microcarriers have recently been used in cell culturing procedures to enhance cell viability and survival. Correspondingly, when cells are passively attached to biocompatible microcarriers and transplanted into the brain, there is prolonged survival even in the absence of immunosuppression (Cherksey et al., 1996; Saporta et al., 1997; Borlongan et al., 1998). In one study, F V M cells attached to microcarriers survived remarkably well when transplanted into the rat striatum (Saporta et al., 1997). In contrast, there were no signs of surviving F V M cells alone. In a similar experiment, Borlongan et al., (1997) studied functional recovery of transplanted adrenal chromaffin cells attached to microcarriers in hemiparkinsonian rats. Chromaffin cells attached to microcarriers resulted in significant behavioral recovery compared to rats that received chromaffin cells alone. Taken together, microcarrier technology appears to be a potential solution to some of the cell survival problem.  Our studies employ this technology; namely, hRPE cells are attached to gelatin microcarriers (hRPE-GM) prior to surgery (refer to methods section). The gelatin microcarriers are 40-60 pm diameter crosslinked porcine microcarriers. Because hRPE cells are anchorage-dependent cells, hRPE cells undergo apoptosis in vivo and in vitro in the absence of a support matrix. Therefore, gelatin microcarriers provide a way of enhancing the survival of hRPE cells when implanted into the brain.  34  3. Preclinical Studies on hRPE-Cell Implant Efficacy 3.1. Rodent h R P E - G M Efficacy Study Using the unilateral 6-OHDA rodent model of PD, Subramanian and colleagues (2002) were the first to show that hRPE-GM produces significant behavioral improvements in parkinsonian rats. In this study, each rat was assigned to one of the four treatment groups: hRPE-cells attached to microcarriers (hRPE-GM), hRPE-cells alone, microcarriers alone, or tract alone. All four treatment groups demonstrated a reduction in rotation scores (at 4 and 8 weeks post-implant); however, there was a gradual return to baseline rotational levels. At 12 and 18 weeks postimplant, only the hRPE-GM treatment group showed significant reductions in rotational scores.  Striatal tissue from each group was processed and stained for cresyl violet and immunohistochemical markers for a host immune response. Post-mortem analyses revealed surviving cells that "resembled hRPE cells" in the hRPE-GM group. In addition, there was minimal immune activation: there were few GFAP positive and no M H C class I, M H C class II, or activated microglia staining. There was no indication of cell survival in the hRPE-cell only group. Nevertheless, the researchers did not use any hRPE-specific markers for identification, and according to current research (Flores et al., 2004), it is unfeasible to identify hRPE cells by simple, morphological characteristics using light microscopy. Despite these discrepancies, the Subramanian group (2002) clearly illustrated that hRPE-GM can produce significant parkinsonian improvement.  3.2. Non-Human Primate h R P E - G M Efficacy Studies There are primate studies that tested the efficacy and safety of hRPE cells (Subramanian et al., 1998, 1999). In one study, three MPTP-treated monkeys were implanted with 10,000 hRPEGMs per site at five different targets in the left striatum using a MRI-guided stereotactic technique, and then observed for eight months post-implant. All monkeys showed significant improvements in mUPDRS scores during the observation period (Subramanian et al., 1998; Subramanian, 2001). In a follow-up study, a blinded placebo-controlled preclinical trial was conducted on sixteen MPTP-treated monkeys that underwent a procedure similar to the one previously described. Each monkey was assigned to one of four different implant groups: hRPEG M (10,000 cells/target), hRPE-GM (50,000 cells/target), gelatin microcarriers alone, or needle  35  tract alone (Subramanian et al., 1999). Implantations were placed at five sites (two sites in the caudate nucleus, three sites in the putamen) in the striatum (Subramanian et al., 1999;Subramanian, 2001). A blinded behavioral assessment demonstrated a strong mean improvement (50-60%) in mUPDRS scores in the high dose (50,000 cells/target) hRPE-GM group, and was statistically significant compared to the sham (microcarrier only or needle tract only) groups. These scores were consistent throughout the 12 month observation period.  Supplementary studies have been conducted to delineate the possible mechanisms for hRPE-GM induced behavioral improvements (Doudet et al., 2004). In our laboratory, five bilateral MPTPtreated monkeys were striatally implanted with hRPE-GM, and a PET analysis was performed to determine any hRPE-GM induced increase in DAergic functioning. There was a significant increase in F - D O P A accumulation and concomitant decrease in C-raclopride binding at the I8  n  implant site over the first two months post-implant. In addition, although behavioral improvement was not the primary focus of this study, average clinical motor scores significantly improved. Monkeys showed improvements in bradykinesia, balance and coordination, as well as general positive changes in daily activities. Taken together, this study suggests that hRPE-GM induced increase in DAergic functioning is responsible for the amelioration of parkinsonian symptoms.  In all aforementioned studies, monkeys recovered quickly from surgery, implants were well tolerated, and post-mortem analyses demonstrated only minimal inflammatory responses even in the absence of immunosuppression. To further ascertain the safety of hRPE-GM implants, a separate Good Laboratory Procedure (GLP) study was conducted on 32 monkeys implanted with hRPE-GM or microcarriers alone (Chang et al., 1999). At two different time points (6 months and 16 months post-implant) the monkeys were evaluated for cell survival, clinical signs, changes in body weight or body organs, pathological or gross necroscopy findings, and histological changes in response to the implant. The study resulted in no treatment-related mortality or clinical abnormalities. There were no changes in body or body organ weights. Postmortem analyses at 6 and 16 months indicated no pathological abnormalities. There were signs of chronic inflammation and glial fibrosis at six months post-implant; however, these are common features related to the surgical procedure which resolved by 16 months.  36  In conclusion, hRPE-GM implantations have demonstrated behavioral improvements as well as positive changes in F - D O P A and C-raclopride binding. However, it is not known whether 18  n  hRPE-GM induced increase in DAergic activity is sustained. In addition, there has been no definitive evidence suggesting that hRPE cells survive long term. Therefore, it would be advantageous to establish a method of hRPE identification in the primate brain.  3.3. H R P E - G M Clinical Studies Based on the primate results, the Food and Drug Administration approved an open-label clinical trial to implant hRPE-GM into six H Y stage 3-4 PD patients (Watts et al., 2001a, 2001b, 2002, 2003; Bakay et al., 2004). Using a three-dimensional MRI-guided stereotactic surgical procedure, each patient received a total of 325,000 hRPE cells attached to microcarriers at five different striatal sites contralateral to the most affected limbs. Patients did not receive any immunosuppression prior to surgery.  All six implant patients were evaluated at 1, 3, 6, 12, 24 and 36 months post-implant. Every patient demonstrated improvements in UPDRS-M scores in the "off state at all time points postimplant, with an improvement range between 44%-61%. At 12 months post-implant, the mean improvement in total UPDRS was 43%, and the UPDRS-M score improved from a mean baseline of 52 to 26 (Watts et al., 2001a, 2001b, 2002). Improvements were also seen in secondary behavioral measures. There was an average 38% improvement in the PDQ-39 Quality of Life assessment scale, and a 10% (in "on" state) and 41% (in "off state) improvement in the Schwab and England A D L Scale. Some patients experienced bilateral behavioral improvements with most prominent improvements seen in limbs contralateral to the implant. Importantly, there were positive changes in the dyskinesia rating scale and no signs of "off state" dyskinesia in any of the six patients.  All six patients tolerated the surgical procedure well, experiencing no serious adverse effects. There were periodic evaluations of each patient's hematology, serum chemistry, urinalysis, neuropsychological functioning, and MRI scans post implant. With the exception of one patient who had a small asymptomatic intraoperative hemorrhage near the implant tract, no adverse  37  effects were observed. MRI scans at 3, 6, and 12 months demonstrated normal healing processes. To this day (approximately 4 years post-implant) all six patients are still showing marked behavioral improvement without any adverse effects. Last year, a double-blind, placebocontrolled clinical trial was launched for over thirty patients in clinical centers throughout the United States. Results are expected to be available within the next few years.  4. Rationale and Objectives There are inherent limitations in the previously described hRPE-GM studies. First, hRPE-GM implants have only been assessed based on induced behavioral improvements; that is, there in no indication as to the survival of hRPE-GM in vivo. The only correlation between behavioral improvement and hRPE-cell survival has been the increase in F - D O P A binding in hRPE-GM 18  implanted monkeys (Doudet et a l , 2004). However, this effect was transient and observed only up to two months post-implant. The Subramanian group (2002) attempted to characterize hRPEG M survival in rodents through T H immunohistochemistry; however, there were certain methodological issues (particularly the absorption of peroxidase by gelatin microcarriers) that the researchers could not overcome. Even if the Subramanian group (2002) was successful, T H is not a direct marker for hRPE cells and may not necessarily indicate the survival of hRPE cells, particularly in a partial 6-OHDA rodent model of PD.  Another important limitation is the lack of morphological characterization of hRPE-GM. It is well known that hRPE cells alter their phenotypic profile depending on their host microenvironment (Hara et al., 2000; Knoernschild et al., 2003). In addition, there is the potential that hRPE cells can change their morphological characteristics in response to the gelatin microcarriers they are attached to. Since the attachment of hRPE cells to gelatin microcarriers and the implantation of hRPE-GM into the brain are relatively new phenomena, there is no data regarding the morphology of hRPE-GM in this setting. Therefore, more research is needed to elucidate this problem.  The aim of this thesis was to develop the appropriate methodologies to answer some of these questions:  38  Our first step was to adapt proper methods of hRPE-cell culturing and attachment to microcarriers as well as the adequate implantation parameters for use in rodent studies. In doing this, our goal was to become consistent in our techniques to minimize variability in hRPE cell viability across future experiments. Our next step was to develop an in vitro method of hRPE-GM identification by using hRPE-cell specific markers for potential use in post-mortem immunohistochemical studies after hRPE-GM striatal implants for variable time periods. Our final goal was to determine the qualitative survival and morphological characteristics of hRPE-GM implanted into the brain. Our ultimate goal was to lay the ground work for future studies of the functional characteristics of hRPE implanted-cells and stereological evaluation of cell survival.  39  Chapter 4: Methods  1. HRPE-Cell Culturing Techniques and In vitro Preparation 1.1. H R P E Cell Isolation HRPE cells are harvested at Titan Pharmaceuticals Inc. (Somerville, NJ, USA) and shipped to our laboratory under maintained cryopreservation (for an in depth explanation, please refer to Castillo et al. 1995). Briefly, the sensory retina was removed, the RPE monolayer was exposed, and then carefully dissected and dissociated with trypsin. The cell suspension was then centrifuged and placed in a cell culture flask with Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal calf serum (FCS). HRPE cells were grown to confluence and doubled x2. They were harvested by trypsinization and frozen in cryovials containing cryopreservative transfer medium (7.5% DMSO, 20% FCS, D M E M ) .  Cryopreserved hRPE cells were immediately put into vapor phase liquid nitrogen storage upon delivery. Five to seven days prior to implant, cryopreserved hRPE cells were rapidly thawed in a 37°C water bath and resuspended in high glucose medium containing DMEM-10% fetal bovine serum (FBS). The cell suspension was centrifuged, and the remaining cell pellet was seeded to an unlaminated T-25 cell culture flask containing high glucose DMEM-10%FBS. HRPE cells were grown to 100% confluence (for approximately 5-7 days) in an incubator (at 37°C) prior to microcarrier attachment and implantation. The medium was changed every 48 hours and examined regularly for proper cell growth and health (as shown in figure 8).  1.2. H R P E Cell Attachment to Gelatin Microcarriers The day before the implantation, dry microcarriers (40-60 pm diameter) (proprietary, Theracell Inc.) were hydrated in a microcentrifuge tube with phosphate-buffered saline (PBS) for a minimum of 1.5 hrs and then autoclaved (121°C, 15 psi, 15 min) for sterility. Sterile microcarriers were then resuspended and washed in fresh PBS, and stored in culture medium until attachment time.  The 100% confluent hRPE cell line was removed from the incubator, washed with PBS, and harvested by trypsinization and mechanical agitation. The hRPE cell suspension was transferred  40  to a polypropylene-coated tube containing culture medium and centrifuged. After centrifugation, the hRPE cell pellet was resuspended with medium, and a small (10 pi) sample of hRPE cells was taken and assessed for cell viability and vial concentration using the trypan blue exclusion method. Once criteria for viable cells was reached (> 80% cell viability; > 1x10 cells/ml 6  solution), the cells were passively attached to the microcarrier suspension. Once combined, the hRPE-GM suspension was stored in medium and placed in a 37°C incubator for 15-18 hours.  For hRPE-GM immunohistochemical (IHC) analyses, after the 15-18 hour incubation period, the medium was removed and the cell suspension was washed with sterile Hank's Balance Salt Solution (HBSS). The cell suspension was fixed in sterile 10% formalin for 10 minutes, washed in HBSS, and stored in 70% ethanol (EtOH).  1.3. In vitro Studies: h R P E - G M Preparation and Embedding For in vitro analyses, the hRPE-GM suspension (immersed in sterile 70% EtOH) was sent out (Department of Pathology, UBC) and paraffin embedded. Prior to embedding, the hRPE-GM suspension was immersed in agar to maintain suspension integrity. Briefly, paraffin embedding was comprised of the following steps: suspension blocks were dehydrated with graded concentrations of EtOH, washed in xylene, then placed in paraffin using a Shandon Citadel 2000 automatic tissue processor. A vacuum (at 60°C) was employed during the paraffin steps to ensure proper infiltration. After the final automated step, the sample blocks were placed in a paraffinfilled holding well (Shandon Histocentre 2 embedding centre), molded, and allowed to cool down. Processing time was approximately 16 hours. Sections (4 pm) were made through the paraffin block, placed on superfrost plus charged slides (Fisher Scientific), and placed in a 37°C oven overnight. Every tenth section was taken and stained with hematoxylin and eosin (H&E) for hRPE-GM identification. Adjacent sections were IHC processed.  41  48 Hrs Post: IHC; EM 7 Days Post: IHC / /  5 Weeks Post: IHC Surgical Recovery  5 Months Post: IHC; EM  Le o" 3  r-o  PL NT  o'  2  >  Figure 5: Experimental timeline of 6-OHDA lesion, hRPE-GM implantation and immunohistochemical (IHC), electron microscopy (EM), and behavioral analyses. For bilaterally lesioned rats, there was a two week recovery period between each hemispheric 6-OHDA lesion surgery, followed by an 6-8 week surgical recovery period prior to h R P E - G M implant. IHC processing was performed at 48hrs, 7 days, 5 weeks, and 5 months post-implant. E M was performed at 48 hrs and 5 months post-implant.  2. In vivo h R P E - G M Implantation Studies 2.1. Animals Fourteen Sprague-Dawley Rats (University of British Columbia Animal Facility) weighing 300375 g were used for this experiment. All animals were housed either in pairs or in groups of three in plastic cages with ad libitum access to food and water. A 12-hour light-dark cycle was maintained (where the animals' dark (active) cycle was 12:00 noon to 12:00 midnight) to accommodate the investigators' daytime cycle and facilitate behavioral assessments. Rats that survived until later time points (5 months) were lightly food restricted to minimize weight gain. Animal housing, animal care and all experimental procedures were approved by the U B C Committee of Animal Care.  Figure 5 illustrates the experimental timeline. Each rat underwent 6-OHDA lesion surgery, and after an 8 week recovery period, each rat was unilaterally injected with a calculated hRPE-GM dose. Following implantation, each rat was assigned to one of four different processing time points.  2.2. 6-Hvdroxvdopamine (6-OHDA) Lesions Rats were anesthetized with isoflourane and randomly assigned to one of two 6-OHDA lesion groups. Prior to surgery, every rat received a 25 mg/kg intraperitoneal (IP) injection of desipramine to reduce non-specific monoamine degeneration. Approximately half of the experimental rats received a unilateral stereotaxic 6-OHDA injection (10 pg in 4 pi ascorbic acid in saline) into two sites along the nigrostriatal pathway: Site 1 coordinates lead to administration of 6-OHDA directly in the SNc: AP -2.8 mm, M L ± 1.8 mm (from bregma), D V -8.0 (from bregma); Site 2 coordinates correspond to a location along the medial forebrain bundle: AP - 4.7 mm, M L ± 1 . 5 mm (from midline), D V - 7.9 (from injection site). The other half of the experimental rats received bilateral 6-OHDA injections (8 pg in 4 pi ascorbic acid in saline) at the same two sites used in the unilateral injection protocol. The 6-OHDA solution was kept on ice and covered to prevent inactivation (due to light exposure). The solution was delivered at a rate of 1 pl/min with a 125 pi Hamilton syringe through a 26-gauge needle using a Hamilton mini-pump. Following injection, the needle was left in place for an additional four minutes to ensure complete diffusion of 6-OHDA. After needle withdrawal, the rat was checked for  43  hemostasis and sutured. Each rat was immediately placed in a heated incubator to compensate for heat loss during surgery, and then returned to its home cage for recovery.  2.3. H R P E - G M Implantation HRPE-GM implants were made 7-8 weeks post-lesion. The morning of implantation, the hRPEG M suspension was washed in sterile HBSS. Prior to use, HBSS was removed until a liquid meniscus formed just above the hRPE-GM suspension. A small (10 pi) sample was taken, treated with a dispase solution to break down the microcarriers, and assessed for cell viability and dose concentration using the trypan blue exclusion method.  Figure 6: Schematic diagram of hRPE implants at anterior sites 1, 2 (A) and posterior sites 3,4 (B) in the striatum. Under aseptic conditions and isofluorane anesthesia, rats were implanted at four different striatal sites using the following (flat-skull) coordinates: Sites 1, 2: AP 1.6, M L ±2.5, D V -4.0/-6.0; Sites 3, 4: AP -0.4, M L ± 3 . 5 , D V -4.0/-6.0 (Figure 6). Each site was injected with an equal volume (3 pi) of hRPE-GM for a total injected volume of 12 pi. The hRPE cell suspension was drawn into a sterile 20 pi Hamilton syringe (pre-coated with medium and preloaded with sterile HBSS) and injected manually in a pulsatile motion (to maximize hRPE-GM diffusion) through a 22-gauge micropolished beveled needle. Rats were first injected at sites one and two (starting with the most ventral position and moving dorsally), the solution then replaced with a new cell  44  suspension, and injected into sites three and four. After each set of injections, the injection needle was held in position for an additional four minutes before needle withdrawal. After hemostasis was achieved, the wound was sutured. The rat was placed in a heated incubator until awake, then returned to its home cage.  3. Post Mortem Processing and Histology After completing all behavioral testing (see Cepeda et al., 2004), rats from each representative group were separated into different post-mortem histological or autoradiographical analyses. IHC analyses were made at 48 hours, 1 week, 5 weeks, and 5 months post-implant, and electron microscopy (EM) was conducted at 48 hours and 5 months post-implant.  The extent of the lesions using our methods was assessed in a few rats (n=2) at each time point using phosphor imaging autoradiography. Rats that underwent phosphor imaging analyses were euthanized by decapitation. Briefly, rats were handled in one of their familiar behavioral rooms until they became docile. After calming down, rats were moved to the perfusion room and decapitated. The brain was immediately removed and rapidly frozen in an isopentane solution cooled by dry ice. Frozen brains were immersed in cryoprotectant medium and stored at -80° C. Minimal or no anesthesia was used to sedate the rat due to potential anesthetic interference with autoradiographic binding assays.  For IHC or E M analyses, rats were deeply anesthetized with an IP injection of a 2:1 ketamine HCkxylazine solution. For IHC, rats were euthanized by a sequential transcardial perfusion (through the ascending aorta) with cold saline (4°C) and 70% EtOH in distilled water. The brain was removed and immersed in additional 70% EtOH (at 4°C) for 24-48 hours. For E M analyses, rats were euthanized by a transcardial perfusion with a 4% paraformaldehyde plus 1% glutaraldehyde in 0.1 M PBS fixative for 60 min. After, the brain was removed and placed in additional 4% paraformaldehyde plus 1% glutaraldehyde (at 4°C) until use.  The frozen brains for phosphor imaging autoradiography were coronally sectioned (18-20 pm) through the striatum using a cryostat. Adjacent sections were taken and placed on separate slides to separate total binding from non-specific binding control. For IHC, the brains were sent out  45  (Department of Pathology, UBC) following the immerse fixation with 70%EtOH and paraffin embedded. Coronal sections (4 pm) were made through the implant site, placed on superfrost plus charged slides (Fisher Scientific), and placed in a 37°C oven overnight. Every tenth section was taken and stained with H & E for basic cell implant identification. Adjacent sections were processed for IHC.  4. Evaluation of Lesion Severity: Autoradiography Phosphor Imaging WIN 35 428, a marker for DAT, was used to evaluate lesion severity and loss of D A striatal innervation through phosphor imaging autoradiography. Striatal D A T binding (total binding= specific + non-specific) was assessed by incubating sections with 15 n M [ H] WIN 35,428. Non3  specific binding was assessed by incubating adjacent sections with a saturating dose (10 pM) of the D A uptake inhibitor nomifensine.  Briefly, the following steps were followed (Strome et al., 2002): thawed sections were preincubated in Tris buffered saline (TBS), then adjacent sections were incubated with either 15 n M [ H] WIN 35,428 or 10 p M nomifensine for forty minutes. At the end of the incubation period, 3  the slides were washed in TBS and allowed to dry under dessication for two days. Sections were then apposed to multisensitive phosphor screens in standard film cassettes for 72 hours. A standard curve was produced for each screen by apposing an activity scale plate with the sections. The phosphor screens were scanned using a Canberra Packard Cyclone storage phosphor system and analyzed using the system's Optiquant software. For analysis, small regions of interest (ROIs) were placed in the striatum of one total binding and one non-specific adjacent section. The data from all ROIs was averaged, and non-specific binding was subtracted from total binding to get a measure of specific striatal D A T binding.  5. Immunohistochemistry (IHC) Prior to IHC, sections were deparaffinized in 2-3 washes of xylene and hydrated in descending grades of EtOH. After deparaffinization, in vitro hRPE-GM sections underwent twenty minutes of microwave heat-induced antigen retrieval in lOmmol/L pH 6.0 citric acid buffer. Tissue sections that were fixed in 70% EtOH did not need antigen retrieval. After cooling, sections were treated with a blocking solution for one hour (at room temperature) containing 10% normal goat  46  serum (NGS), 2%-5% bovine serum albumin (BSA), 0.2% non-fat dry milk, and 0.3% Triton-X to minimize nonspecific binding of the primary antibody. Sections were incubated overnight at 4 ° C with the following primary antibodies diluted in blocking solution:  1) Anti-NuMA (Ab2) mouse monoclonal antibody (1:500): Nuclear Mitotic Apparatus Protein (Oncogene Research Products). N u M A is a 240 kDA nuclear matrix protein derived from the human cervical carcinoma cell line ME-180. According to the distributor, N u M A (Ab2) is reactive to human (but not to rat) nuclear matrix antigens. 2) Anti-EMMPRJN rabbit polyclonal antibody (1:500): Extracellular Matrix Metalloproteinase Inducer (Zymed Laboratories). EMMPRIN is a 58 kDA protein purified from the plasma membrane of human LX-1 lung carcinoma cell line. EMMPRIN is found on the outer surface of tumor cells, where it interacts with fibroblasts to stimulate the expression of metalloproteinase. Anti-EMMPRIN is epithelial specific: it is reactive to the cell membrane of normal epithelium. 3) Anti-GFAP rabbit polyclonal antibody (1:1000) (DAKO): Glial Fibrillary Acidic Protein. Anti-GFAP reacts against glial fibrillary acidic protein in astrocytes.  Negative control sections were incubated with no primary antibody. Sections treated with antiNuMA mouse monoclonal were washed with PBS and incubated with the fluorochromeconjugated secondary antibody Alexa Fluor 488 goat anti-mouse IgG (1:500, Molecular Probes). Similarly, EMMPRIN and GFAP-treated tissue were incubated with either Alexa Fluor 488 or Alexa Fluor 555 goat anti-rabbit IgG (1:500, Molecular Probes) fluorescent secondary antibodies. Secondary antibody incubation was for one hour at room temperature. Sections were coverslipped and sealed using an antifade mounting medium (Molecular Probes). Immunofluorescence was detected using an Olympus Optical Fluoview 500 confocal laser scanning microscope. Differential interference contrast (DIC) images of sections were taken with each scan and viewed using the Olympus Optical Fluoview software. All images were saved as TIFF files and edited with Adobe Photoshop.  47  6. Electron Microscopy (EM)  From whole brain tissue at 48 hours and 5 months post-implant, 4-6 blocks of tissue through the implant site were sectioned and sampled for EM. Each block measured approximately 5.0 x 5.0 x 10.0 mm and was cut with the long axis oriented in the dorsal-ventral direction. Individual tissue blocks were washed in 0.1 M PBS and post-fixed overnight in 1% buffered osmium tetroxide. The following day, blocks were washed in acetate buffer and stained with 2% aqueous uranyl acetate for one hour. Tissue blocks underwent dehydration through ascending grades of EtOH, equilibration in propylene oxide, and then embedded in Epon. Each block was trimmed down further, and serial ultrathin (70 nm) sections were taken and individually mounted on Formvarcoated slot grids and stained with lead citrate (refer to O'Kusky et al, 2000, 2003 for additional explanation of methods). Electron micrographs were photographed with a 3.25 x 4.00 inch Kodak 4489 plate film, and negatives were scanned on an Epson scanner using Epson Twain-Pro software. All images were taken as TIFF files and edited with Adobe Photoshop.  48  Chapter 5: Results All transplanted animals survived the implantation procedure and entire post-implant period. All animals recovered well; there were no signs of morbidity throughout the experiment, and all animals naturally continued to gain weight after surgery.  1. Evaluation of the 6 - O H D A Lesion Characteristics and Choice of Further Model 1.1. Surgical Observations All animals recovered well after unilateral 6-OHDA lesions. There were no signs of morbidity, and mortality rate was low (<10%). Two rats died during the lesion procedure due to anesthetic fluctuations. There was little postoperative care; all animals stopped gaining weight during the first two to three days post-surgery; however, they began to naturally gain weight afterwards. At two to three weeks post-lesion, each animal's unilateral 6-OHDA lesion behaviorally characterized using the cylinder test for forelimb asymmetry (Figure 7). All animals showed significant reductions in forelimb use contralateral the lesioned side. Analyses for T H activity on the SN will be made in the future.  Bilaterally lesioned animals demonstrated different recovery characteristics. There was increased morbidity and mortality rate was higher (20-25%) compared to the unilateral lesion group. The majority of rats that died did so within three to four days post-surgery. There was considerable post-operative care; each rat was given sub-cutaneous fluids two times a day, and the most severe rats were manually fed with a high protein liquid diet. All animals lost approximately 20% of their weight during the first two weeks post-surgery; however, this weight was regained. Rats that lost greater than 20% of their weight were euthanized.  1.2. Behavioral Analyses Behavioral characterization of the lesion was assessed only after the weight of the rat was equal to or exceeded their pre-surgery weight. The tests used were designed to  49  50 |  -125  f  ' Pre-Lesion  Post-Lesion  Figure 7: Behavioral characterization of unilateral 6 - O H D A lesions. Lesion strength of individual rats (n=8) assessed by limb-use asymmetry (cylinder) test (Schallert et al., 2000) Cylinder test measures changes in forelimb use (pre- and post-lesion) during vertical exploration. 0 values represent equal (right and left) forelimb use. Positive values represent forelimb contralateral to the lesioned side. Results show preferential use of ipsilateral forelimb after 6O H D A lesion (negative values). show limb asymmetry both in the upper limb ("cylinder" test) and lower limb ("ledged beam test"). As this aspect of the study was the responsibility of another graduate student and not the subject of this thesis, we will only report the final conclusion of the behavioral testing. All rats demonstrated behavioral deficits, particularly a lack of spontaneous movement.  Interestingly,  even in bilaterally lesioned animals, the left side (right hemisphere) was consistently more impaired compared to the right side.  1.3. Phosphor Imaging Autoradiography Phosphor-imaging autoradiography helped quantify the lesion strength of bilaterally 6-OHDA lesioned animals. At the 8pg dose, the two-site bilateral 6-OHDA method produced a terminal  50  bilateral 7 0 % - 9 8 % l e s i o n as m e a s u r e d b y [ H ] W I N 35, 428 b i n d i n g . T h e l e s i o n w a s a l m o s t 3  a l w a y s a s y m m e t r i c , irrespective o f w h i c h h e m i s p h e r e w a s lesioned first, w i t h the right striatum s h o w i n g greater D A loss.  2. HRPE-GM Characterization 2.1. Surgical Considerations A l l h R P E c e l l cultures d i s p l a y e d healthy signs i n the five i n c u b a t i o n days p r i o r to c e l l attachment. T h e i r characteristic e l l i p t i c a l - s h a p e d nucleus a n d e l o n g a t e d c e l l shape w a s retained, a n d they r e a c h e d 100% c o n f l u e n c e i n T - 2 5 flasks w i t h i n 4-6 days after s e e d i n g ( F i g u r e 8). C o n f l u e n t h R P E c e l l lines h a v e p r o d u c e d consistent results: c e l l n u m b e r s r a n g e d f r o m 2-2.5 m i l l i o n cells p e r flask, w i t h c e l l viabilities b e t w e e n 80%-92%. O n l y after cells w e r e 100% confluent ( a n d before they started to proliferate b e y o n d the m o n o l a y e r ) w e r e they t r y p s i n i z e d a n d p r e p a r e d for m i c r o c a r r i e r attachment.  H R P E - G M dose estimates p r i o r to i m p l a n t a t i o n h a v e resulted i n greater than 8 4 % v i a b i l i t y w i t h c a l c u l a t e d d o s e concentrations b e t w e e n 2 0 0 0 - 3 5 0 0 cells p e r p i o f h R P E - G M s o l u t i o n ( T a b l e 8). T h e h R P E - G M s l u r r y was k e p t o n s h a v e d ice t h r o u g h o u t surgery to m a k e the c e l l s o l u t i o n easily m a n a g e a b l e for l o a d i n g into a n d i n j e c t i n g f r o m syringes. O c c a s i o n a l l y , 2-3 h o u r s after the storage time start, the h R P E - G M s o l u t i o n b e c a m e slightly t h i c k a n d h a d to b e r e s u s p e n d e d i n H B S S for 10 minutes. O v e r l y d r y h R P E - G M suspensions are difficult to l o a d into the syringe, c a u s i n g fluctuations i n actual injected dose concentrations. F r o m past e x p e r i e n c e w i t h this p r o c e d u r e , I h a v e c o n c l u d e d that r e - s u s p e n s i o n does not s i g n i f i c a n t l y alter h R P E c e l l v i a b i l i t y . A t the e n d o f the s u r g i c a l d a y , c e l l assessments were m a d e to d e t e r m i n e changes i n c e l l v i a b i l i t y a n d concentration w i t h storage. A s expected, c e l l concentrations d e c r e a s e d s i g n i f i c a n t l y to a p p r o x i m a t e l y 6 0 0 - 1 2 0 0 cells per p i o f solution; h o w e v e r , the c e l l v i a b i l i t y d e c r e a s e d o n l y slightly to a m i n i m u m o f 80%. F i g u r e 9 illustrates a n H & E stain a n d D I C m i c r o g r a p h o f the m o r p h o l o g i c a l features o f in vitro h R P E - G M . A s s h o w n , G M s are v a r i a b l e - s i z e d , s p h e r i c a l s h a p e d b e a d structures that possess grooves. G M s s e e m to absorb the H & E stain, staining a p i n k (eosin) c o l o r  51  Day 1  *  30% confluent  60% confluent  Day 6  100% confluent  Figure 8: In vitro proliferation of h R P E cells in T-25 flasks. H R P E cells can easily be expanded in culture, reaching confluence in a T-25 flask within 5-7 days of seeding. When 100% confluent, h R P E cells form a stable monolayer which can subsequently be split and doubled producing large quantities of h R P E cells.  Cell Viability PreImplant  Cell Viability PostImplant  h R P E Cell-Line  Calculated number h R P E cells/ul cell suspension  XMCB**02#168  2508  91%  N/A  XMCB**02#169  2288  90%  N/A  XMCB**02 #166,167  2200  94%  82%  XMCB**02#161, 173  3232  86%  81%  XMCB**03#321  2500  85%  N/A  XMCB**03#322  2000  97%  88%  XMCB**02#323  2300  88%  80%  XMCB**03#217  2600  84%  84%  Table 8: Calculated dose and pre- and post-cell viability of different cell lines used for h R P E - G M implants. characteristic of cytoplasmic staining. The majority of hRPE cells attach to the outer edges of the microcarriers, exhibiting an elongated cell shape along the G M surface. HRPE cells are irregularly shaped and do not resemble the hRPE cells in culture. In addition, the H & E (Figure 9A) stain did not differentiate between nuclear and cytoplasmic areas. This may be related to the level and thickness of sectioning, as 4pm sections will only expose a portion of a typical 1520pm thick hRPE cell.  Marker  Dilution  h R P E - G M in vitro  hRPE-GM Implanted  Rat Brain Striatum  N u M A (Ab2)  1:500  +++  +++  -  EMMPRIN  1:500  +++  +++  -  1:1000  -  ++  +++  GFAP  Table 9: Primary antibody marker characterization for h R P E - G M studies.  53  2.2. Qualitative Immunofluorescence  2.2.1. In vitro Characterization All primary antibodies were previously studied for hRPE-cell specificity and non-specificity to rat tissue (Qualtek molecular labs; unpublished results). Table 9 illustrates these results. According to the product distributor, N u M A (Ab2) is specific to human but not to rat tissue. Current testing confirms these results. N u M A (Ab2) demonstrates good staining of hRPE cells, with minimal or no staining of microcarriers or rat tissue. Similarly, EMMPRIN also demonstrates good specificity to human epithelial structures in hRPE cells but not to rat tissue or microcarrier components.  H R P E - G M characterization by IHC is illustrated in Figures 11 and 12. N u M A (Ab2) (Figure 10) and EMMPRIN (Figure 11) staining demonstrated hRPE cells attached to the outer edges of microcarriers. In some cases, hRPE cells penetrated within the G M grooves. For a control, GFAP immunofluorescence was conducted on hRPE-GM in vitro and produced negative staining (Figure 10). A double fluorescence protocol using both N u M A (Ab2) and EMMPRIN demonstrated identical fluorescence characteristics (Figure 12); that is, both N u M A (Ab2) and EMMPRIN stained similar cell properties. This is surprising despite the different staining specificities of N u M A and EMMPRIN.  There are other issues that must be taken into consideration. As noticed in both N u M A and EMMPRIN images, few hRPE cells were not attached to any microcarriers. Hypothetically, HBSS washes prior to surgery should eliminate any unattached hRPE cells; apparently, this wasn't fully accomplished. Therefore, if implanted into the brain, these unattached cells should die. It is not known whether unattached cells are counted when calculating cell dose concentrations prior to surgery. Also, figure 11 illustrates a hRPE-cell "peeling" off the G M surface. This is likely a sectioning artifact: in some cases, the sectioning blade may pull the hRPE cell away from the G M , creating an illusion of de-attachment. Studies to resolve this artifact are ongoing.  54  2.2.2. In Vivo Characterization of Implanted h R P E - G M During sectioning, injection tracts were identified and assessed for placement. Most injection tracts were placed accurately within the striatum. In some instances, the posterior implant tract (site 3, 4) was slightly more posterior than the intended coordinate; however, the tract was still well within the striatum. Observation of the tract site demonstrated G M clumps within the implant tract. The G M clumps were within the center of the striatum. In one rat, GMs were found in the cortical areas and corpus callosum coinciding with the penetration site, and H R P E - G M was not found in one rat. These rats were not used for qualitative analyses  Figure 13 illustrates qualitative N u M A fluorescence at different time-points post-implant. At all times there were NuMA-positive cells surrounding the microcarriers. There was a non-uniform staining pattern and gradual decrease in N u M A fluorescence over time. At 48 hours postimplant, there was some undesirable non-specific N u M A fluorescence (as seen in our negative control sections) despite the maximization of blocking solution in the fluorescence protocol. Correspondingly, early time-point negative control sections demonstrated some non-specific staining. One possibility is autofluorescent red blood cell (RBC) accumulation due to the relative temporal proximity of processing time and surgery. Figure 14 is a high magnification DIC micrograph and accompanying N u M A fluorescence in a rat five months post-implant. There was a decrease in NuMA-positive cells compared to earlier time-points; however, cell positivity was more uniformly distributed and correlated well with the calculated hRPE cells/bead ratio detailed in Subramanian et al. (2002). A low magnification micrograph in figure 15 reveals the entire tract area. Although the outer edges of the implant tract demonstrated irregular staining, N u M A positivity within the implant tract was more uniform, resembling the staining feature seen in figure 14. In addition, the co-labeling of N u M A and GFAP demonstrated GFAP fluorescence on the outer edges of the implant site, but there was no GFAP reactivity within the implant site.  EMMPRIN IHC demonstrated similar results. Figure 16 illustrates EMMPRIN positive cells at different time-points. EMMPRIN demonstrated non-uniform staining in the early (48 hours postimplant) time-points; however, EMMPRIN positivity decreased and became more uniformly distributed around the microcarrier at later time-points. There was also non-specific binding at early (48 hours post-implant) time-points, suggestive of R B C accumulation due to surgery. In  55  certain areas, fluorescent staining resembled RBC morphology; however, these staining qualities were reduced through time. Figure 16B illustrates a high magnification DIC image of a single implanted hRPE-GM and the accompanying EMMPRIN fluorescence. Note similar EMMPRIN qualities to those seen in hRPE-GM in vitro; hRPE cells were stretched out along the microcarrier. A NuMA/EMMPRIN double fluorescence protocol was performed to determine the inctracellular localization characteristics of N u M A and EMMPRIN staining (Figure 17). Results determined that N u M A and EMMPRIN bind to similar cell features resulting in identical fluorescent characteristics. Table 10 summarizes the pattern of fluorescent staining for N u M A and EMMPRIN across time.  Marker  48 hrs PostImplant  7 days PostImplant  5 weeks PostImplant  5 months PostImplant  N u M A (Ab2)  ++++  +++  +++  ++  EMMPRIN  ++++  +++  +++  ++  ++/+++  ++/+++  ++/+++  GFAP  ++/+++*  Table 10: Pattern of fluorescent staining at different time-points post implant. GFAP patterns reflect GFAP staining in whole brain sections.* Rats at the 5 month time points showed no GFAP reactivity within the implant site.  56  cn  Figure 9: In vitro characterization of hRPE-GM. 100x H&E stained (A) and 60x DIC micrograph (B) of h R P E cells attached to gelatin microcarriers.  00  Figure 10: In vitro characterization of NuMA (Ab2) and GFAP in hRPE-GM. 60x DIC image of h R P E G M and accompanying fluorescence. Note N u M A (Ab2)-positive cells attached to variable-sized microcarriers. No GFAP-positive reactivity to h R P E cells.  ^1-  Figure 11: EMMPRIN characterization of hRPE-GM in vitro. 60x DIC image of hRPE-GM and accompanying EMMPRIN staining. EMMPRIN staining patterns are similar to N u M A (Ab2) staining patterns. Also note hRPE cells "peeling" off from microcarriers (arrows).  59  ^^ j > 1  fVT mm \M  A  1*^*^81^%  y  1  J 1  1  1  E M M P R I N  Figure 12: Co-labeling of N u M A (Ab2) and E M M P R I N in h R P E - G M in vitro. 60x micrograph showing N u M A (Ab2) and E M M P R I N staining identical h R P E - c e l l elements.  60  48 hrs  7 days  5 weeks  5 months  •  *y -  Awl  **  *  •V 4  %  '  1  1  Figure 13: /n wVo hRPE-GM demonstrating co-labeling of NuMA (Ab2) (green) and GFAP (red) positive cells at different time-points post-implant. 60x N u M A (Ab2) positivity suggests hRPE-cell survival up to 5 months post-implant. Micrographs were taken along the outer edges of implant tract in order to show NuMAG F A P double fluorescence within the same section.  Figure 14: N u M A (Ab2)-positive cells at 5 months post-implant. 60x(1.5x zoom) DIC micrograph and accompanying N u M A (Ab2) staining of a single hRPE-GM in vivo.  62  In vivo co-labeling o / N u M A (Ab2) and G F A P within the implant tract. (C) 20x micrograph of implant tract 5 months post-implant. (A) NuMA (Ab2) staining shows positive staining within the implant with some irregular staining on outer edges of the tract. (B) antiGFAP demonstrates astrocytic processes on outer edges of tract with no infiltration within the implant. (D) Composite image of (A) and (B) showing some overlap on edges of implant. Figure 15:  63  48 hrs  7 days  Figure 16: In vivo EMMPRIN fluorescence at different time-points post-implant. (A) 60x E M M P R I N positivity observed up to 5 months postimplant. (B) illustrates a single h R P E G M in a rat 5 months post-implant.  Figure 17: In vivo h R P E - G M demonstrating co-labeling of N u M A (Ab2)-EMMPRIN positive cells. (C) 60x DIC image of hRPE-GM 5 weeks post-implant. Implant demonstrates (A) N u M A (Ab2) positive and (B) EMMPRIN positive cells. (D) demonstrates N u M A (Ab2)EMMPRIN overlap.  65  3. Electron Microscopy The morphological features of hRPE-GM 48 hrs post-implant are shown in figure 18. HRPE morphology is different from that seen in cultured hRPE-GM. The nucleus was stretched out, more elongated and lobular, and had a thick nuclear membrane. In some cases (Figure 19), the nucleolus was dark and pronounced. The cytoplasm was dark, and contained visible processes. Within the cytoplasm, the endoplasmic reticulum was visible and dilated. There was evidence of an acute inflammatory response at 48 hours post-implant. Figure 19 illustrates the presence of polymorphonuclear leukocytes (PMNs) at the implant site surrounding the G M . In the left inset is a P M N phagocytizing debris adjacent to a morphologically characterized hRPE cells. The inflammatory response was evident in other sections (not shown); there was P M N accumulation throughout the entire implant site.  Figures 20-22 demonstrates morphologically characterized hRPE cells at 5 months post-implant. Note the difference in hRPE-cell morphology compared to 48 hour hRPE cells. The cytoplasm was uniformly dense but with fewer cytoplasmic processes. Numerous nuclei were observed; they were smaller in size and had thinner, ruffled-like nuclear membranes. It is unclear whether this cluster consisted of many hRPE cells with individual nuclei and clumped cytoplasm, or a single hRPE cell with a single fragmented nucleus. In contrast to 48 hours implants, there was minimal inflammatory response observed at five months. P M N accumulation was diminished; however, there were some cells morphologically similar to macrophages (Figure 20).  Apart from morphologically characterizing hRPE-GM at five months, further analyses led to the description of hRPE cell-specific protein elements that could be used in future identification studies. On the outer edges of hRPE cells are small ladder-like protrusions (Figure 21). These protrusions were identified as cross-linked collagen fibers. The collagen fibers made from hRPE cells are morphologically dissimilar from collagen-made microcarriers, and so far collagen fibers have not been identified in rats implanted with G M only. There was no evidence of collagen production within the brain; therefore, metabolically active collagen may be a future source of hRPE-cell identification. In addition, figure 22 illustrates dark granule clusters located within the cytoplasm of several hRPE cells. These granules are hypothesized to be melanin, and are also identifiable on semi-thin l-2pm sections (data not shown). The only significant source of  66  melanin within the brain is the SN; therefore, if implants are far enough away from the SN, melanin location may be another means of identifying hRPE cells. However, both collagen and melanin formation are only seen at five months. For this reason, these qualities may only be beneficial at later time-points post-implant.  67  Figure 18: E M micrograph of single h R P E - G M 48 hrs post-implant. Note characteristic feature of hRPE cells: elongated nucleus with relatively thick nuclear membrane, dense cytoplasm with visible processes and dilated ER. (GM) - Gelatin Microcarriers.  68  Figure 19: Acute inflammatory response at 48 hrs post-implant. The presence of polymorphonuclear leukocytes (PMNs) (arrows) adjacent to morphologically characterized h R P E cells (with nucleus fragment embedded in cytoplasm) suggests early inflammatory reaction. Note phagocytic activity of P M N adjacent to the h R P E cell (A). (B) is continuing image of (A).  Figure 20: E M micrograph of h R P E - G M 5 months post-implant. h R P E cells are morphologically different than hRPE cells observed at 48 hours. Cytoplasm is still  dense, but with fewer observable processes. Numerous smaller nuclei present with ruffled-like nuclear membrane. Minimal morphologically characterized lymphocyte activity was present (red arrows).  70  Figure 21: Collagen fibers produced from hRPE cells 5 months post-implant. (A) Collagen fibers emanate from h R P E cells (inset) that were not previously found at the 48 hr timepoint. (B) Cross-linked collagen demonstrate ladder-like appearance (arrow).  Figure 22: Melanin pigment production 5 months post-implant (inset). Pigment granules not present at 48 hrs. Pigment also identified on semi-thin 1-2 pm sections (not shown).  72  Chapter 6: Discussion and Conclusions  1. Cell Culture The first step in these studies was to learn about and set up hRPE-specific cell culturing techniques. To begin with, we established standards to produce hRPE cells for use in hRPE-GM studies. These standards were based on the in vitro morphological characteristics of hRPE cells, the cell culture selection process prior to attachment, and the cell viability and cell concentration counts pre- and post-attachment.  1. Morphological characteristics of hRPE cells in vitro. As illustrated in figure 23, hRPE cells displayed phenotypic variability from seeding time to microcarrier attachment. Once seeded, hRPE cells were grown until 100% confluent in T-25 flasks. HRPE-cells expressed a characteristic phenotype; there was some heterogeneity between cells in culture. HRPE cultures remained pigmented, cells had distinct, spherical-shaped nuclei with a dark nucleolus, and cytoplasmic extensions that made cell-cell connections with adjacent hRPE cells. At confluence, hRPE cells formed a stable monolayer; it was only when they began to show this "epithelioid" or "cobblestone" appearance that they were harvested and prepared for attachment.  2. HRPE cell culturing and the selection process. In primate and human studies, the number of passages that hRPE-cell lines went through was minimized. Namely, R P E - G M attachments were prepared immediately after the thaw process (see methods section). In this study, hRPE cells were grown in culture prior to attachment. This step provided a number of advantages. First, we were able to perform a selection process whereby only healthy cells were used. Approximately 24 hours after hRPE-cell seeding, the culture medium was exchanged. In theory, exchanging the medium should have removed hRPE cells that had not yet formed or attached to a support matrix. Since hRPE cells undergo apoptosis if not attached to a substrate, and because the flasks (that hRPE cells are seeded onto) are not substrate coated, eliminating unattached cells at 24 hours only left "healthier" cells that found a way to generate a substrate matrix.  3. Cell concentrations and viability. The second advantage to culturing hRPE cells prior to attachment was that larger numbers of "healthier" cells were produced. Approximately 30% to  73  40% of hRPE cells were lost after the 24-hour medium change, leaving roughly 500,000 to 600,000 cells. HRPE cells were regularly grown to confluence for 4 to 6 days, giving consistent hRPE cell counts of 1.5 to 2 million cells per flask. This produced plenty of cells needed for attachment; in some cases, enough hRPE cells were produced for two solution attachments. Employing the pre-attachment culturing and selection process was an especially important step in rodent studies. Since only a limited volume of hRPE-GM (20,000 to 32,000 hRPE cells in 12 pi total volume) was implanted, it was absolutely necessary to select the healthiest cells to ensure optimal survival. This process has less importance in non-human primate studies since primates are implanted with a calculated total of 300,000 to 500,000 hRPE cells in a 50 pl/tract (5 tracts per striatum) volume.  The selection process also led to consistent viability between experiments. All cell viabilities were in the acceptable range (greater than 80%) with dose concentrations within the normal range of 2000 to 3000 cells per pi of hRPE-GM solution. Post-surgery viability counts were taken to determine viability changes between the beginning and end of a routine surgical day (i.e. 4-6 hours). Using our storage methods during surgery, viability was consistent and never dropped below 80%. Determining these viability counts gave us confidence that the majority of implanted cells were healthy, living hRPE cells.  2. In vitro Development of Immunohistochemical Methods The primary objective of this thesis was to characterize potential antibodies that could be used for future, post-mortem hRPE-cell identification. N u M A (Ab2) and EMMPRIN, which (according to their product suppliers) are human and epithelial specific, respectively, were chosen strictly for their robust specificity and relatively easy interpretation of results. Characterization studies on N u M A (Ab2) and EMMPRIN in the past demonstrated their specificity (unpublished results: Qualtek Molecular Labs, Santa Barbara): positive staining was observed in hRPE monolayers and RPE/choroid tissue, but was negative in rat striatum. Our studies confirmed these results: positive N u M A (Ab2) and EMMPRIN staining was seen in hRPE-GM in vitro. In addition, there was no GFAP expression in hRPE-GM cultures, and there was minimal antibody absorbance from gelatin microcarriers.  74  During this study, some methodological issues appeared that raised questions: as seen in figure 12, despite different antigen specificities between N u M A (Ab2) (nuclear marker) and EMMPRIN (cytoplasmic marker), similar staining patterns were observed. That is, using double labeling IHC techniques for N u M A (Ab2) and EMMPRIN, we demonstrated positive staining for what appeared to be the same hRPE cell components. The reason for this is unknown. Since this was the first study to simultaneously look at N u M A (Ab2) and EMMRPIN staining in hRPE-GM, we hypothesized that the antigenicity of hRPE cells was due, in part, to the morphological properties of hRPE cells when attached to gelatin microcarriers. Previous IHC data on hRPE cells in vitro parallel our findings (unpublished results: Qualtek Molecular Labs, Santa Barbara). Nuclear DAPI staining in hRPE-GM in vitro did not demonstrate nuclear signaling that would be found in typical cell types. DAPI labeling demonstrated irregular staining with no distinct nuclear region. Similarly, despite finding N u M A labeling in hRPE-GM preparations in our studies (Figure 11), nuclear specificity was not clear. This was later confirmed in our future IHC and E M analyses: in vivo hRPE-GM showed great morphological variability including changes in nuclear and cytoplasmic architecture. Current studies are underway to ascertain these changes.  3. In vivo Pilot Studies 3.1. Behavioral Evaluations Although behavioral assessment was not the focus of this thesis, another graduate student in our laboratory tested the same rats for hRPE-GM induced behavioral improvements at certain times pre- and post-implant (see figure 5 for behavioral timeline). We will briefly mention these behavioral observations as they suggest hRPE -cell survival in the animals (Cepeda et a l , 2004).  The (5 week and 5 month post-implant) rats were behaviorally assessed using an uphill tapered ledged-beam walking task (Fleming et al., 2002). Briefly, the beam was composed of three sections (wide, medium, and narrow width) of increasing difficulty with a transparent ledge spanning its entire length. Rats with depleted D A (after lesion) manifested their motor impairment by using the ledge as support for their hind limb side contralateral to the denervated striatum. Therefore, the lesioned rat gradually made more footfaults (errors) with increasing section difficulty. All rats demonstrated improvement in the contralateral hindlimb post implant  75  (Figure 24). In some cases, rats with bilateral lesions also showed ipsilateral behavioral improvement. On the narrow (most difficult) beam section, unilaterally-lesioned rats with hRPEG M implants showed a 14% improvement at 4 weeks post-implant and a 55% improvement at 18-20 weeks post-implant. Bilaterally-lesioned rats with unilateral implants demonstrated a 30% improvement at 2-4 weeks, and a 31% improvement at 8-10 weeks. Rats that received microcarriers or tract alone did not improve. These results strongly suggest that hRPE-GM implants have long-term behavioral effects.  3.2. In vivo Immunohistochemistry This was the first study to show histological evidence of long-term hRPE-GM survival when implanted into parkinsonian rats. N u M A (Ab2) and EMMPRIN revealed positive staining at all time-points up to five months post-implant (Figures 13 and 16). Furthermore, high magnification images of N u M A (Ab2) labeling (Figure 14) demonstrated uniformly distributed cells that correlated with the hRPE cell/bead ratio detailed in Subramanian et al., (2002). This suggests that hRPE-GM can survive long-term in vivo in the rodent brain without immunosuppression.  One issue that arose was the steady decrease in hRPE-positive staining over time. Taking into account the fraction of positive staining at early time-points due to R B C autofluorescence, there was still an apparent decrease in hRPE-cell positivity between early (48 hours and 7 days) and late (5 months) time-points. However, this is not surprising taking into account that alternate transplantation procedures have also shown substantial decreases in cell concentrations post transplant. Rodent studies on F V M xenotransplants demonstrated that only a small fraction of F V M D A neurons survived after transplant (Brundin et al., 1988). Also, F V M cell survival was only apparent in immunosuppressed rats. In our study, long-term hRPE-cell survival was apparent (albeit less than that seen at earlier time-points) at later (5 months) time-points despite the rats receiving no immunosuppression. Although it was possible to immunosuppress rats with CyA, we would not have been able to study long-term functioning since maintaining rats on immunosuppressive drugs can be difficult to do long-term. Therefore, although there was a decrease in long-term hRPE cell labeling (suggestive of decreased survival), it was still remarkable to find surviving hRPE-cells in a non-immunosuppressed rat.  76  Another issue that arose related to the processing methods. Initially, brain tissue from another set of rats (not shown here) was sectioned with a freezing microtome, as we hoped to use adjacent tissue for autoradiographic evaluation of lesion severity with [ H] WIN 35 428. However, we 3  observed that in rats processed up to 7 days post-implant, the implant site containing the microcarriers was destroyed. That is, the microcarriers in the implant area were falling out during the sectioning process, leaving only an open cavity. Only few microcarriers were left attached to the outer edges of the tract. Therefore, all the brains used in this study were paraffin embedded. Paraffin significantly increased the preservation of the hRPE-GM implant.  Nevertheless, although paraffin improved our processing techniques, it led to a sectioning artifact that could potentially alter our results. As shown in figure 11, sectioning led to hRPE cells peeling off the microcarriers. This represents a significant problem for future stereological studies as this could change the quantification of surviving hRPE cells when we compare the calculated dose to the actual hRPE-cell dose in vivo. We are currently working on alternate processing methods to overcome this problem. In collaboration with another laboratory (Dr. O'Kusky, Department of Pathology), we are testing an acrylic embedding resin system (LR White) as a new form of processing. Not only may this system reduce any sectioning artifact, but it may also be used for simultaneous IHC and E M analyses.  3.3 Electron Microscopy Our IHC experiments demonstrated that the highly variable and irregular labeling characteristics of hRPE-GM made the reliance on IHC findings alone a potentially flawed process. Thus, we evaluated other methods to parallel our IHC techniques in hope of finding a technique for unequivocal hRPE cell identification. As a result, an E M analysis was performed on 2 rats to determine the phenotypic profile of hRPE-GM at 48 hours and 5 months post-implant (due to fixation problems, the 5 week post implant rat set aside for E M analysis could not be used). Our results confirmed our previous speculation that hRPE cells expressed irregular morphological characteristics when attached to the microcarriers. At 48 hours post-implant, the nucleus and cytoplasmic areas were present and distinct; the nuclear region was abnormally stretched out along the microcarrier (as shown in IHC labeling), contained a thick nuclear membrane, and seemed to cover greater than 75% of the cell volume. The cytoplasmic region, despite being  77  small, contained prominent cytoplasmic processes (especially a very developed ER, characteristic of highly secretory cells such as hRPE cells). These observations paralleled our N u M A (Ab2) IHC results and raised the possibility that the nuclear volume became large enough so that N u M A (Ab2) fluorescence appeared to label the entire cell volume.  The phenotypic profile at five months was drastically different to the hRPE phenotype 48 hours after implant. At five months, the cytoplasmic volume was larger, but contained fewer processes. In contrast, the nuclear region was smaller and occupied less volume: nuclear fragments appeared small and elongated, had a ruffled nuclear membrane, and were dispersed within the cytoplasmic area. It is not known whether this hRPE-cell cluster was composed of a number of hRPE cells with individual nuclei and integrated cytoplasm, or a single hRPE cell with a single fragmented nucleus dispersed within its cytoplasm. The elucidation of this question is important as it raises the possibility that at least some cell IHC positivity at five months post-implant was due to cellular debris rather than the survival of healthy hRPE cells. Nevertheless, some hRPE cells appeared to contain melanin, a sign of healthy hRPE cells (Figure 22). Also behavioral improvements observed in these rats at five months suggest that there is long-term survival and function in hRPE-GM implants.  Studies have demonstrated that hRPE cells alter their phenotypic properties depending on their host microenvironment (Hara et al., 2000; Knoernschild et al., 2003). Interestingly, our E M observations demonstrated that hRPE cells also express different phenotypes with respect to time after implant: despite the same microenvironment, different morphological characteristics were seen between the 48 hour and 5 month time-points. Nevertheless, both hRPE phenotypes that we demonstrated have been observed in other studies. The 48 hour hRPE phenotype was similar to the morphology of hRPE cells that move into the vitreous cavity following surgery for retinal detachment in a condition known as proliferative vitreoretinopathy (Machemer and Laqua, 1975; Smith et al., 1976). Interestingly, the vitreous is composed primarily of collagen, the same material that gelatin microcarriers are composed of. These cells maintain some of the same characteristic properties: they retained their elongated cell shape, thick nuclear membrane, and dark cytoplasmic processes. HRPE-cell morphology at 5 months post-implant was more difficult to elucidate; however, this phenotype has also been characterized in the past (Knoernschild et al.,  78  2003). HRPE-cells at 5 months acquired more of a fibroblast-like appearance (however, not one that is commonly seen in the brain). Cytoplasmic processes were less pronounced and few cytoplasmic pigment granules were present. HRPE cell nuclei were small and elongated, the nuclear membrane had a ruffled-like appearance and there were increased amounts of collagen fibers emanating from within the cell. In addition, most hRPE cells come into close contact with each other, which was also seen at the 5 month time-point. Taken together, if we presume that hRPE-cell morphology depends on the host microenvironment in which hRPE cells are transplanted into, then we can hypothesize that changes in the host environment may contribute to changes in hRPE phenotype over time. One change may be due to the variations in the immune and inflammatory response in the brain.  4. Alternate Considerations 4.1. Immune and Inflammatory Reactions to h R P E - G M Immune reaction and tissue rejection have always been main issues regarding the transplantation of various tissues. Since the identification of hRPE cells was the primary purpose of this study, only a minor investigation was performed on the host immune response. IHC demonstrated normal GFAP positive staining throughout the striatum, but there seemed to be minimal or no GFAP infiltration within the implant site. The E M ultrastructural analysis revealed the presence of PMNs in the early (48 hour) time-point, indicative of an acute inflammatory response. At the later (5 month) time-point, there were few signs of a chronic immune response: only sporadic macrophages were present at the implant site, and there were no signs of activated microglia.  A rodent study done by Subramanian and colleagues (2002) suggested that the host immune response was minimal after hRPE-GM implantation. In their study, IHC staining for rat activated microglia (OX-42), M H C class I (OX-18), M H C class II (OX-6), and astrocytes (GFAP) revealed little immune activity in non-immunosuppressed hRPE-GM implanted rats up to 18 weeks. Typically, in a non-immunosuppressed rat, the host immune response to a xenotransplant begins within 48 hours after the transplant and usually leads to the complete rejection of the xenotransplant within 30 days. Our results suggested that hRPE-GM xenotransplants survived with minimal chronic inflammatory and immune presence at 5 months.  79  Since the brain is considered an immune-privileged site (Barker and Billingham, 1977), it seems natural for a tissue implant to survive in vivo for a prolonged amount of time when compared to implants in other, non immune-privileged sites (Billingham and Boswell, 1953). However, under certain conditions, immune reactions in the brain can take place as effectively as those seen in non-immune-privileged sites. That is, the brain can generate an immune response that can quickly lead to implant rejection. Therefore, the immune privilege of the brain is not absolute; tissue survival depends on several factors, including the regulation of the host immune response and the type of tissue being implanted.  Interestingly, the fibroblast-like morphology demonstrated at 5 months post-implant was comparable to the morphology of hRPE cells implanted into non-immune-privileged sites in the retina (such as the subconjunctival space) (Knoernschild et al., 2003). In this case, hRPE cells implanted into non-immune-privileged site were smaller, more elongated, and contained fewer cytoplasmic processes than typical hRPE cells. In addition, an inflammatory response was observed only when hRPE cells were implanted into non-immune-privileged sites of the retina: there was minimal immune activity when implanted into immune-privileged areas. The question, therefore, is why hRPE cells developed a phenotype suggestive of an implant into a non-immune-privileged environment? Despite being an immune-privileged site, there are certain factors that could contribute to the brain inducing a severe immune reaction to the implant. One factor may stem from the surgical technique associated with transplantation. Neuronal implantation required inserting a cannula into the brain disrupting the BBB. Through time, B B B disruption can lead to the entry of immune and inflammatory factors that would not normally be seen in immune-privileged sites. This can cause an immune reaction and potential rejection of the implant. In this case, the difference in hRPE-cell phenotype between 48 hours and 5 months post-implant may represent the gradual B B B breakdown and infiltration of inflammatory elements that may otherwise not be present in immune-privileged tissue.  4.2. Issues Between Xenotransplantation and h R P E - G M Implants: Is the Rat Model the Right Model? There is speculation as to whether the rodent PD model is a feasible method for studying hRPEG M implantation. Allogeneic hRPE-GM studies in humans induce an immune response that is  80  dependent on the M H C immunological identity between the donor and host tissue. Concordant xenotransplants (between two closely related species such as human hRPE tissue implanted into a non-human primate host) depend on immune reactions against xenogeneic M H C antigens and xenogeneic peptides; however, the immune response generally resembles what would be observed in an allograft response. On the other hand, discordant xenotransplants (between two distantly related species), as is the case studied here, invoke a more complex immune response (Brevig et al., 2000). Not only does the immune response involve host-induced immune and inflammatory reactions, but also several innate immune responses including the complement system, natural killer cell activation, and xenoreactive natural antibody activation (Cascalho and Piatt, 2001). Moreover, innate immune reactions in discordant xenotransplants usually result in a hyperacute rejection of the implant.  The reduction in hRPE cell survival over time may be indicative of this process; therefore, it may not parallel the level of hRPE-cell survival that would normally be seen in human patients (or in non-human primates) receiving hRPE-GM implants. However, it is important (for logistic and economical reasons) that a hRPE-GM rat model be established. There are certain factors expressed by hRPE cells that can increase cell survivability in non-immunosuppressed rats. RPE cells express CD95 antigens and release transforming growth factor beta  (TGF0), both of which  may be responsible for local immunosuppression (Liversidge and Forrester, 1998; Wenkel and Streilein, 2000). In addition, placing hRPE cells on microcarriers can increase their survival and immune-privilege after implant (Bakay et al., 2004). Nevertheless, it is important that further research be done on the differences in immune response to hRPE-GM across different species.  5. Comparison Between h R P E - G M Implants and Other Transplantation Techniques 5.1. Cell Handling and Surgical Approaches F V M tissue transplantation has always been the gold standard for neural transplantation in PD. Many studies in the 1990s demonstrated the therapeutic potential of F V M tissue transplants (Hallett et al., 1999). However, double-blind placebo studies produced disappointing results. Only moderate behavioral improvement was observed and was demonstrated only in the younger patients. In addition, patients experienced disabling late-onset "on-off' phenomena (Olanow et  81  al., 2003). How then, does F V M transplantation compare to hRPE-GM implants, and how can hRPE-GM implants overcome some of these past problems?  The double-blind placebo study may have had a disappointing outcome because of its unusual technical features. After the dissection of F V M tissue, the tissue block was passed through a needle creating a tissue "noodle". The "noodle" was then placed in culture for variable amounts of time (for up to four weeks) prior to the transplantation procedure. It is speculated that this long storage time may be responsible for the reduced viability and survivability of D A neurons which, in turn, contributed to the lack of clinical efficacy (Freed et al., 2001). In earlier studies, cryopreservation was used to store fetal tissue prior to transplantation (Redmond et al., 1988; Olanow et al., 1996); however, cryopreservation storage of F V M tissue has been associated with significant decreases in cell viability (Collier et al., 1993; Olanow et al., 1996). In open label studies, tissue was stored for up to 72 hours, minimizing any potential viability loss. Nevertheless, it is clinically improbable to rigorously set maximum storage times in culture. Since F V M tissue is difficult to acquire, the needed level of patient and clinician cooperation makes this an impractical method. In contrast, HRPE cells are epithelial cells and, as such, can be grown and survive well in culture for extended periods of time (Hu et al., 1994). Since there is no time constraint or availability issue to deal with, hRPE donors can be extensively screened for bacterial, viral, microplasmic, or prion contamination, increasing the safety of the transplanted tissue. In addition, hRPE cells that are stored by cryopreservation (as the cells used in this study) maintain good viability for three years. Therefore, donor RPE cells can be stored indefinitely with minimal loss of cell viability.  Another advantage of hRPE-GM implants is that there is no specific time period for hRPE cell dissection. F V M tissue has a narrow margin (between 6.5 to 8 weeks) for dissection in order to maximize the number of DA-producing neurons (Olanow et al., 1996). Currently, all hRPE cells that have been used have been primary cell cultures from fetal or neonatal retinal tissue (no specific gestational date). Our collaborators have been studying the molecular properties of adult hRPE cells, and we have begun testing a commercial immortalized hRPE-cell line. In the future, we hope to broaden our range of acceptable hRPE cell lines for use in PD treatment.  82  A final technical aspect that should be re-considered is the surgical method for transplantation. In the double-blind F V M studies, a frontal transaxial surgical approach was used to place the transplants into the putamen. This approach allowed the surgeon to minimize the number of tracts in each patient; however, this also meant that the transplant trajectory traversed a different path than ones used in most transplantation procedures. In contrast, hRPE injection techniques have been extensively studied and improved, creating a standard procedure for hRPE-GM injection. H R P E - G M implantation requires a rapid, pulsatile injection (in a dorsal-ventral trajectory similar to what was used in open-label F V M studies). This is the method we used to implant hRPE-GM in rats, and it is the same technique used in the in vivo primate and human studies. Histological studies (Bakay et al., 2004) and residual analyses have confirmed that the pulsatile technique provides a high level of hRPE-GM delivery and good hRPE-cell survival.  5.2. Efficacy: Mechanism of Action of h R P E Cells Behavioral improvements in rats, monkeys, and humans implanted with hRPE-GM have been attributed to the ability of hRPE cells to synthesize dopa through a DA-like mechanism . A combined F-DOPA/raclopride PET study in hRPE-GM implanted monkeys in our laboratory 18  (Doudet et al. 2004) showed an 8% to 10% increase in F - D O P A uptake in the hRPE-GM 18  implanted putamen of bilaterally MPTP-treated monkeys. In addition, there was a concurrent decrease in raclopride binding, indicative of an increase in endogenous synaptic D A concentration. However, these observations could not be reproduced in animals at 6 months or later post implant (unpublished results: Doudet et al.). Thus, it is unlikely that the clinical efficacy of hRPE-GM is solely due to increased dopa concentrations. Furthermore, unilateral hRPE-GM implants led to bilateral behavioral improvements in both monkeys and patients (Doudet et al., 2004; Watts et al., 2001, 2002). Dopa is minimally present in extracellular tissue: dopa is rapidly metabolized as part of the eumelanin pathway, and hRPE cells have been shown to synthesize D A in only small amounts. Taken together, these observations cannot account for the robust behavioral improvement seen in all species (Subramanian, 2001) and question the hypothesis that hRPE cells behave strictly as a dopa "pump". Therefore, other mechanisms could potentially be involved in hRPE-cell functioning.  83  HRPE cells are retinal "support" cells and, intrinsic to their role, are able to produce various pro and anti-inflammatory factors, cytokines, and trophic factors. Therefore, one can speculate that hRPE cells, when implanted into the host striatum, alter their regular production and "adapt" their metabolism to the different striatal conditions. Interestingly, many of the trophic factors that hRPE cells express have been previously studied in the treatment of PD (Ishida et a l , 1997; Siegel and Chauhan, 2000; Arnhold et a l , 2004). RPE cells have recently been found to produce GDNF (Koeberle and Ball, 2002). Although GDNF implantation clinical trials have been stopped due to safety concerns, hRPE cells may express normal physiological amounts of GDNF which may be beneficial for the neuroprotection of remaining D A terminals. Basic FGF (bFGF) is another trophic source that could potentially contribute to hRPE-cell functioning. Although there has been no direct connection between bFGF and the amelioration of parkinsonian behavior (Zeng et al., 1996), researchers have shown that the preincubation and cotransplantation of bFGF with F V M tissue improves tissue survival in parkinsonian rats (Takayama et al., 1995; Clarkson et al., 2001). Thus, bFGF could indirectly promote the survival of hRPE cells, acting as an intrinsic self-protective mechanism.  HRPE cells have also been found to produce and contain large amounts of the trophic factor PEDF (Tombran-Tink et a l , 1991). Evidence has suggested that PEDF can exert a neuroprotective effect on different cell types. Within the retina, PEDF prevents photoreceptor degeneration (Cayouette et al., 1999) and protects retinal neurons against H202-induced cell death (Cao et al., 1999). PEDF has also been demonstrated to protect spinal motor neurons (Bilak et al., 2002), cerebellar granule neurons (Taniwaki et al., 1997), and hippocampal neurons (DeCoster et a l , 1999) against toxicity and apoptosis. To date, no studies have looked at the protective effect of PEDF on basal ganglia D A neurons; therefore, whether PEDF is involved in the neuroprotection of D A neurons warrants further investigation.  6. Future Directions To date, we have only performed qualitative analyses on hRPE-GM. Based on our IHC and E M results, we have confirmed the survival of hRPE-GM up to five months post-implant. However, we have not yet attempted to find a quantitative measure of hRPE-cell survival. Before we attempt to quantify our results, we need to address the following issues: 1) find alternate  84  antibodies with good specificity and minimal cross-reactivity; 2) establish definitive morphological criteria of hRPE cells when implanted into the brain; and 3) test new processing methods for optimal long-term hRPE-GM identification.  We are currently improving our techniques and characterization criteria for future hRPE-GM studies. We have started a rat study using BrDU pre-labeled hRPE-cells, and we are in the process of finding other pre-labeling fluorescent techniques for in vivo identification. In addition, we are looking at alternate antibodies for hRPE-GM IHC. Although N u M A (Ab2) demonstrated good hRPE-cell labeling in vitro and in vivo in the rat, the cross reactive nature of N u M A (Ab2) may prevent its use in future primate and human studies. Some examples of new target proteins are keratin, melanin, or collagen; many of which are either not found or found only in specific areas of primate or human brains.  There have been recent reports using superparamagnetic iron oxide (SPIO) nanoparticle cell labeling techniques as an in vivo method to determine cell survivability through magnetic resonance imaging (MRI). We are in the process of adapting this method to hRPE cells, with our initial experiments investigating in vitro, the efficacy and toxicity of labeling hRPE cells using ferumoxides, a suspension of dextran-coated SPIO nanoparticles used as a MRI contrast agent. Our first experiments will consist of hRPE cells transfected with the ferumoxide complex, and to compare the short-term cell viability or any changes in phenotype to unlabeled hRPE cells in vitro. Success will be followed by the evaluation of an in vitro sample of ferumoxide-transfected cells, and hopefully, the in vivo tracking of hRPE cells in the rat brain by MRI. SPIO labeling would allow 1) the in vivo follow up of the hRPE-GM implant and 2) a more accurate postmortem identification of hRPE cells. Ideally, as cell-SPIO labeling is becoming more and more widely used (and is in the process of receiving F D A approval), this method (after testing in primates) could be applied to human hRPE-GM studies.  Another potential avenue of interest would be the investigation of alternate mechanisms of action either by hRPE cells themselves or through the initiation of sustained molecular processes. This includes the in vivo production of different trophic factors or anti-inflammatory agents of the  85  host itself, or the activation of indigenous stem cells in the presence or absence of hRPE cells.  With these proposed techniques, our objective is to develop a quantitative method that determines the number of surviving hRPE cells at various times post-implant. These methods will determine the degree to which hRPE-cells die across time when implanted into the brain and (in collaboration with different members of the lab) will allow us to correlate behavioral improvements with hRPE cell survival and function.  7. Conclusions In conclusion, we present here the development of specific methods for production, attachment, implantation and post mortem identification of hRPE cells as a new potential therapy for PD. In addition, using a small sample of animals, we now present the first evidence of long-term survival and morphological characterization of hRPE-GM in parkinsonian rats. Using IHC and E M techniques, we have shown that hRPE-GM survive up to five months post-implant. In a collaborative study, the same rats demonstrated behavioral improvement as measured by a nondrug-induced beam walking behavioral test (Cepeda et al., 2004). Although hRPE-cell transplantations are currently being studied in a multi-center double-blind placebo clinical trial in the U.S., there are still many issues that need to be resolved.  HRPE-cell implants offer several advantages: they can be easily obtained and expanded in culture, and a single donor can treat a large number of patients. Cells can be stored in culture or cryopreserved for extended periods of time, giving the opportunity for extensive testing of hRPE cells prior to use. Also, hRPE-GM negates the need for immunosuppression. The greatest advantage to hRPE cells is their combination of key features that contribute to their therapeutic potential and increased survivability. HRPE are complex cells by themselves: they contain the machinery necessary to synthesize dopa and produce D A , and they produce numerous trophic factors that can potentially contribute to self-protection and to the neuroprotection of remaining D A neurons. Also, they express certain immune factors that may be involved in local immunosuppression. It is these features that make hRPE-GM implants a potential alternative for ' the treatment of PD.  86  Figure 23: Phenotypic variability between different hRPE-cell culturing time points. At hRPE-cell seeding (A), h R P E cells express characteristic in vitro morphology. They form a "cobblestone" appearance at confluence (B), and continue to morphologically change when attached to microcarriers (C).  Figure 24: Behavioral improvements after h R P E - G M . Tapered ledged beam-walking test on unilaterally 6-OHDA lesioned (A) and bilaterally 6-OHDA lesioned (B) rats receiving unilateral hRPE-GM implants. 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