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Parkinson's disease : etiology, prevention and treatment Tabatabaei, Ali Reza 1991

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PARKINSON'S DISEASE: ETIOLOGY, PREVENTION A N D T R E A T M E N T by Ali Reza Tabatabaei B.Sc, The University of British Columbia, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES ( Department of Pharmacology & Therapeutics ) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1991 © Ali Reza Tabatabaei, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbil Vancouver, Canada Department DE-6 (2/88) T A B A T A B A E I , A. ii Abstract: This thesis consists of three chapters dealing with different aspects of Parkinson's disease (PD). 3-Acetylpyridine (3-AP), a naturally occurring neurotoxin, was studied for its neurodegenerative properties on the mesostriatal dopaminergic system in rats as a possible environmental cause of idiopathic PD. Chronic administration of this compound to rats caused a moderate but insignificant reduction of striatal dopamine (determined by HPLC measurement of striatal dopamine) and a more substantial degeneration of cerebellar neurons and their neurotransmitters (determined by amino acid analysis of cerebellum). Prophylactic use of a high dose of nicotinamide prevented the reduction of dopamine in the striatum as well as the severe behavioural manifestations induced by 3-AP in rats. The cerebellar damage, however, was not affected. Different mechanisms of damage by 3-AP in these structures were presumed based on the protective effects of nicotinamide in the substantia nigra but not in the cerebellum. Possible protective properties of MK-801 (a noncompetitive N M D A antagonist) and nicotinamide against MPTP neurotoxicity were also examined in mice. MK-801 treatment provided a substantial protection against MPTP-induced reduction of striatal dopamine. Nicotinamide on the other hand provided no such protection. Finally, a new controversial approach to the treatment of parkinsonism was evaluated. Nervous tissue from 13-15 day-old fetuses was transplanted into MPTP-treated mice. The transplanted material was harvested from different areas of the fetal brain and was prepared by various procedures to examine the possible bases of any improvement in the host animal. After two studies, we did not find a biochemical improvement in transplanted mice treated with MPTP regardless of the nature of the transplanted materials. T A B L E O F C O N T E N T S TABATABAEI, A. iii Contents Page Abstract ii Table of contents iii List of figures v List of tables vi Dedication viii Acknowledgements ix 1. Introduction 1 2. Chapter 1: NEUROCHEMICAL EFFECTS OF 3-ACETYLPYRIDINE IN STRIATUM A N D CEREBELLUM 11 2.1. Introduction 11 2.2. Materials and Methods 14 i. Animals and Experimental Materials 14 ii. Biochemical Methods 15 iii. Data Analysis 17 2.3. Results 17 2.4. Discussion 20 3. Chapter 2: N M D A RECEPTOR BLOCKADE A N D PARKINSONISM 25 3.1. Introduction 25 3.2. Materials and Methods 29 i. Animals and Experimental Materials 29 ii. Biochemical Methods 30 iii. Data Analysis 31 3.3. Results 31 3.4. Discussion 34 TABATABAEI. A. iv TABLE OF CONTENTS Contents Page 4. Chapter 3: FETAL DOPAMINERGIC CELL TRANSPLANTATION IN MPTP-TREATED MICE 36 4.1. Introduction 36 4.2. Materials and Methods 41 I. Experiment A 41 I.i. Animals and MPTP Exposure 41 I.ii. Fetal tissue preparation 41 I.iii. Neurotransplantation 43 I. iv. Biochemical Methods 45 II. Experiment B 45 n.i. Animals and MPTP Exposure 45 II. ii. Fetal tissue preparation 45 Il.iii. Neurotransplantation 46 Il.iv. Data Analysis 48 4.3. Results 48 4.4. Discussion 51 5. Concluding Remarks 57 6. References 59 TABATABAEI, A. v LIST OF FIGURES Figure Page 1. Schematic drawing of the connections of the basal ganglia 3 2. Enzymatic conversion of cysteine to taurine 22 3. Plot of % MAO-B activity in the presence of increasing concentrations of MK-801 33 4. Schematic illustration depicting the techniques involved with the stereotaxic transplantation of fetal nervous tissue into the adult recipient (experiment "A") 44 5. Schematic illustration depicting the techniques involved with the stereotaxic transplantation of fetal nervous tissue into the adult recipient (experiment "B") 47 TABATABAEI, A. vi LIST OF TABLES Table Page I. Striatal Dopamine and Metabolites in Rats Following Chronic 3-AP Treatment, with (Group C) or without (Group B) Nicotinamide pretreatment 18 II. Content of Neurotransmitter Amino Acids in Cerebellum of Rats Chronically Treated with 3-AP, with (Group C) or without (Group B) Nicotinamide pretreatment 19 HI. Striatal Dopamine and Metabolites in Mice Treated with MPTP (Group B), MPTP+Nicotinamide (Group C), or MPTP+MK-801 (Group D) 32 IV. Striatal Dopamine and Metabolites in MPTP-Treated C57 BL/6 Mice Transplanted with Fetal Nervous Tissue 49 V. Left and Right Striatal Dopamine and Metabolites in MPTP-Treated C57 BL/6 Mice Transplanted with Fetal Nervous Tissue 50 T A B A T A B A E I , A. v i i In memory of Dr. Thomas L. Perry T A B A T A B A E I , A . vi i i DEDICATION This thesis is dedicated to my parents for their continuous support and belief in me-at all times. ACKNOWLEDGEMENTS TABATABAEI, A. ix I am indebeted to the late Dr. Thomas L. Perry for allowing me the opportunity to study under his guidance. I am very grateful to Mrs. Shirley Moller-Hansen for her friendship and continuous moral and technical support. I would also like to thank Dr. Voon Wee Yong for performing the histological studies on animals in trial one of the transplantation studies and Mr. Etienne Grima for performing the stereotaxic placement of the transplanted material. I am grateful to Dr. Richard A. Wall for guidance with HPLC techniques. Dr. David V. Godin deserves special mention for his constructive comments and criticism during the preparation of this thesis. I am appreciative of the helpful suggestions made by Dr. James McLarnon and Dr. Michael J.A.Walker. The funds for this work were provided by a grant to Dr. Thomas L. Perry from the Medical Research Council of Canada and by a grant to me from the British Columbia Medical Services Foundation. TABATABAEI, A. 1 1. Introduction: Parkinson's disease (PD) was first described by James Parkinson in 1817. It is a chronic progressive disease characterized by bradykinesia, rigidity, tremor at rest and loss of postural reflexes. Among Caucasians, a prevalence of 84 to 187 per 100,000 population has been estimated. The prevalence of PD among the black population as well as the Japanese and Chinese population is lower than among Caucasians; however, environmental factors may be affecting the prevalence ratio. The age of onset of PD is between 50 and 79 years . The incidence of this disease increases with age of the population, being the lowest at the age group below 30-40 years and highest in the range 70-79 years. The latter age group has a yearly incidence of 1 or 2 persons per 1000 (Alvord et al.. 1987). The major neuropathological characteristics of PD are the formation of Lewy bodies and progressive degeneration of dopaminergic neurons of the substantia nigra pars compacta and loss of their ascending axonal projections onto the striatum, one of the major connecting routes of the basal ganglia. Degeneration of the nigrostriatal pathway leads to decreased levels of dopamine in the striatum (Hornykiewicz et al., 1989). A 70-80% reduction of dopamine-containing cells in the substantia nigra pars compacta precedes parkinsonian symptoms (Koller, 1987). Examination of Parkinsonian postmortem brain indicates that another pigmented area of the brain affected in this disease is the locus ceruleus. The basal ganglia are a group of associated gray matter structures in the brain extending through the telencephalon, diencephalon, and midbrain. These structures, with their extensive interconnecting pathways, are believed to be involved in modulating and facilitating motor programs. •Lidsky et al. (1985) have proposed that the basal ganglia act to regulate the interconnection of sensory inputs and motor impulses and hence allow particular sensory stimuli to initiate motor responses while others are ignored. In the last decade, our understanding of the function and anatomy of basal ganglia has increasingly improved. In 1977 Mensah TABATABAEI. A. 2 (Mensah, 1977) showed that the striatum is not homogeneous and that islands of increased cell density exist. The existence of two striatal components distinguishable on the basis of staining for acetylcholinesterase (ACHE) was later demonstrated; these regions were subsequently referred to as the striosomes and the matrix. It was shown by these investigators that in developing fetuses striosomes are intensely colored by A C H E staining, whereas the surrounding area (the matrix) is lightly stained. However, in adults the differential staining pattern is reversed (Graybiel et ah, 1987). A description of the organization of the basal ganglia is necessary for the understanding of the neurological basis of movement disorders. A simplified map of the basal ganglia and the connective pathways are presented below. In most mammals striatum consists of the caudate and the putamen which are separated by the fibers of the internal capsule. Most afferents to the striatum come from the cerebral cortex, and these corticostriatal neurons probably contain glutamic acid as their neurotransmitter (Kim et al., 1977; McGeer et al., 1977). The striatum also receives input from the substantia nigra pars compacta (SNc). These dopamine-containing fibers of the SNc project onto the striosome where they exhibit a higher density than in the matrix. In return, the striatum also projects directly onto the SNc. The major output structures of the basal ganglia are the medial globus pallidus (MGP) and the substantia nigra pars reticulata (SNr). These two structures receive direct input from the matrix of the striatum with fibers containing y-aminobutyric acid (GABA) and substance P. There is also an indirect input to these nuclei from the striatum to the lateral globus pallidus (LGP) (these fibers contain GABA and enkephalin as neurotransmitters). LGP, in turn, projects onto the subthalamic nucleus (STN). The efferent fibers from this nucleus terminate in LGP as well as in the MGP and SNr. The neurotransmitter of fibers originating in STN has not as yet been identified; however, glutamic acid has been proposed as a possible candidate (Smith et al., 1988). STN also receives direct input from the cortex. TABATABAEI, A. 3 .. S4>-u<.>. \ .^S-twMW I £ v :25£ • :2E-I I THALAMUS ) f VENTRAL LATERAL, "VENTRAL"] i ^ANTERIORJ SUBSTANTIA NIGRA . [PARS COMPACTA] GLOBUS PALLIDUS [MEDIAL] SUBSTANTIA NIGRA [PARS RETICULATA] GLOBUS PALLIDUS | [LATERAL] j SUBTHALAMIC NUCLEUS Figure: 1 Schematic drawing of the connections of the basal ganglia. Connections are indicated by the arrows. Neurotransmitters are differentiated by shade: (~ \ - --) = GABA; ) = Dopamine; {mmm ) = Glutamic acid TABATABAEI, A. 4 The output from the basal ganglia (MGP and SNr) is mostly onto the ventral-anterior and ventral-lateral (VA/VL) thalamic nuclei; these projections are believed to be GABAergic in nature. These regions of the thalamus, in turn, project with an excitatory neurotransmitter onto the prefrontal cortex and supplementary motor areas . This simplified map of nuclei and connecting pathways of basal ganglia completes a circle originating in the cortex, through the basal nuclei and returning to the cortex (Fig. 1) (Albin et al., 1989; Young et al., 1988). A better understanding of the anatomical and biochemical nature of the basal ganglia, including the nonhomogeneity of the striatum and the existence of separate striatal projecting neurons to the LGP and MGP/SNr with only a few collaterals have led Young and Penney (Albin et al., 1989; Young et al., 1988) to propose a model of basal ganglia function and dysfunction. In this model any challenge to the integrity of these nuclei or their connecting pathways leads to a variety of movement disorders. Damage to the STN (in ballism) or degeneration of GABAergic/enkephalin-containing neurons of striatum projecting to the LGP (in Huntington's disease) leads to hyperkinetic movement disorders. PD is a prototypic hypokinetic movement disorder. In the model of Young and Penney degeneration of DA-containing cells of SNc leads to decreased activity of striatal neurons projecting on to the MGP and SNr, which in turn leads to inhibition of thalamic nuclei and hence prevents initiation and facilitation of voluntary movements. Although idiopathic Parkinson's disease was described over 170 years ago, its etiology remains a mystery. Various hypotheses have been formulated, including genetic factors, viral infection, aging and toxic compounds of endogenous and exogenous origin. A genetic basis of PD was one of the early hypotheses as to the etiology of this disease. A genetically transmitted disease has the same prevalence in monozygotic twins. Ward et al. (1983) collected information on 43 monozygotic twin pairs of TABATABAEI, A. 5 whom at least one had PD. In this study only one pair was demonstrated to be concordant. Another study of this type was performed in Finland (Martila et al., 1986). This study included 18 monozygotic twin pairs, of which none showed concordance for PD. These observations do not support the genetic nature of PD. Therefore, the influence of some environmental factors seems likely. However, it should be kept in mind that a combination of environmental factors and genetic predisposition could be the cause of idiopathic Parkinson's disease. Viral infections, including encephalitis lethargica, encephalitis and syphilis, have been reported to lead to parkinsonism. After an epidemic of encephalitis lethargica (occurring during 1915-1919) in Europe and the United States, 60% of the affected population developed parkinsonian symptoms. These cases, however, had an early (about 27 years old) age of onset (Koller, 1987). The neuropathological characteristics of these patients were similar to those of idiopathic PD. Examination of 12 patients with post-encephalitis parkinsonism has shown a 90% decrease in striatal dopamine and severe degeneration of nigral cells (Bernheimer et al.y 1973). These observations suggest that some forms of parkinsonism can be produced by viral infection. The incidence of PD has been reported to remain relatively constant between 1935 and 1979 (Rajput, 1984), despite the nearly complete disappearance of encephalitis lethargica in 1935. Furthermore, brains of patients affected by Parkinson's disease did not show signs of viral infection and no virus could be isolated from these brains (Calne et al., 1983). On the other hand, Nocardia (a common soil bacterium) has been recently proposed as a possible cause of PD (Beaman, 1990). An increased rate of physiological aging has also been considered as a cause of PD. It has been shown that the neurons of the substantia nigra diminish in number with increasing age as does the level of striatal dopamine (McGeer et al., 1977; Hornykiewicz, 1966). Once again, studies of monozygotic twin pairs have provided evidence against this hypothesis since the physiological aging process is strongly TABATABAEI. A. 6 determined by genetic factors and should proceed at the same rate in monozygotic twins (Calne and Langston, 1983). These investigators proposed that a combination of the normal age-dependent reduction of nigral cells and striatal dopamine with an environmental insult may be a cause of idiopathic PD. Since a 80-90% depletion of dopaminergic neurons precedes the development of parkinsonian symptoms, it seems possible that an environmental insult at an early age could reduce the number of cells in the substantia nigra and produce a subclinical parkinsonian state. The affected individual may develop PD later in life due to the normal age-related reduction in the remaining dopamine-containing neurons. This hypothesis is under investigation by Calne who is following individuals exposed to l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), but who are not presently affected by parkinsonian symptoms. These individuals have been shown by positron emission tomography to have sustained damage to nigrostriatal pathways, but the damage is not sufficient to produce overt parkinsonism. The long-term observation of these individuals may provide evidence to support or refute the above hypothesis (Calne et al., 1985). On the other hand, aging has been known to be accompanied by an increased level of monoamine oxidase-B (MAO-B) in humans, monkeys and rodents (Robinson et al., 1985; Somorajski et al., 1973). It has also been demonstrated that MPTP has a greater and more widespread effect on reducing dopamine concentration in aged as compared with young animals. These observations provide grounds for the general hypothesis of the etiology of PD that age may render individuals more susceptible to some toxin(s) which could be encountered throughout life (Date et al., 1990; Ricaurte et al., 1987). Endogenous neurotoxins have also been implicated as a possible cause of idiopathic PD. Dopamine itself has been linked to development of PD through its oxidative metabolism and concomitant generation of free radical species such as hydrogen peroxide (H2O2), superoxide (0"-2) and hydroxyl radicals (HO) (Graham, TABATABAEI, A. 7 1984; Marker et al., 1981). Formation of these endogenous reactive oxidants can lead to membrane damage and cell death. The decreased levels of glutathione (GSH) observed in the substantia nigra of parkinsonian patients as compared to normals supports the idea that the nigrostriatal system is vulnerable to oxidative damage (Perry et al., 1982). More recently, glutamic acid has been linked to various neurodegenerative disorders including olivopontocerebellar atrophy (Plaitakis et al., 1982), spinal cord injury (Faden and Simon, 1988), Alzheimer's disease (Maragos et al., 1987), and Huntington's disease (Perry and Hansen, 1990; Coyle and Schwarcz, 1976). The mechanism of the toxicity produced by glutamate and other excitatory amino acids may involve an excessive intracellular influx of C a 2 + . This increased cytosolic C a 2 + can subsequently lead to the production of superoxide radicals and hydroxyl radicals (Dykens et al., 1987). Exogenous toxins have attracted considerable attention in the study of the etiology of PD. The ability of manganese (Mn) to produce parkinsonism in exposed individuals has been known for many years (Mena et al„ 1967). A progressive parkinsonian state follows exposure to a high concentration of Mn or Mn-containing compounds. As expected, the basal ganglia demonstrate some degree of neurodegeneration, but, unlike PD, there is extensive involvement of pallidum and striatum. There is also an intention tremor rather than a resting tremor present in Mn-induced parkinsonism (Koller, 1987). Other compounds such as carbon monoxide (CO), carbon disulfide, cyanide and methanol have been reported to be linked with parkinsonian symptoms (Koller, 1987). Until the genetic transmission of PD was refuted by the report of Ward et al. (1983), the environmental toxin hypothesis was not extensively investigated. However, accidental discovery of MPTP, which was produced as a byproduct during synthesis of an illicit narcotic (l-methyl-4-phenyl-4-propionooxypiperidine) (MPPP) TABATABAEI, A. 8 stimulated a vast search for environmental toxins that might be the cause of idiopathic PD. A number of heroin addicts injected with MPTP-contaminated MPPP developed a severe parkinsonism which was distinguished by its sudden onset (Langston et al., 1983). The neuropathological changes elicited by the administration of MPTP to primates produce a severe deficit virtually indistinguishable from the symptoms of idiopathic PD (Langston and Irwin, 1989; Jenner and Marsden, 1986). The extensive and highly selective destruction of the neuronal constituents of the substantia nigra, gliosis and the presence of Lewy bodies (Forno, 1986) make the manifestations of MPTP intoxication nearly identical to the changes associated with PD. The selective dopaminergic neurotoxicity of MPTP has been associated with the specific uptake of l-methyl-4-phenylpyridinium ion (MPP+) by the neuronal dopamine reuptake system. It is presumed that MPTP itself is not the neurotoxin causing parkinsonian symptoms, but rather a metabolic byproduct (Langston et al„ 1984). The first step of MPTP conversion requires MAO-B and produces the intermediate compound, l-methyl-4-phenyl-2,3-dihydropyridine (MPDP+) (Chiba et al., 1984; Chiba et al., 1985). There is growing evidence indicating that MAO-B inhibitors such as deprenyl and pargyline can protect experimental animals from the effects of MPTP by blocking its conversion to MPDP + (Glover et al„ 1986; Markey et al., 1984). MPDP+ is subsequently converted to MPP+ which is assumed to be the active neurotoxin. These oxidation processes are believed to occur mainly in glial mitochondria (Castagnoli et al., 1985). This extraneuronally generated compound (MPP+) is selectively taken up and concentrated in dopaminergic neurons by the dopamine reuptake system. The fact that dopamine uptake blockers such as mazindol, amfolenic acid and cocaine can prevent the toxic effects of MPTP lends support to this suggestion (Sundstorm and Jonsson, 1985; Mayer et al„ 1985). MPP+ is subsequently concentrated in the matrix of striatal mitochondria (Ramsay and Singir, 1986). This carrier system is capable of TABATABAEI, A. 9 concentrating the neurotoxin within the inner mitochondrial membrane by about 100-fold. Once a critical concentration of MPP + has been achieved within the matrix, M P P + is believed to introduce a block at the site of ubiquinone resulting in inhibition of ATP synthesis, and the abolition of oxidative phosphorylation. In parkinsonian patients, a reduction of NADH-CoQ reductase has been observed which is restricted to the cell bodies of substantia nigra cells (Schapira et al., 1990). This finding supports the hypothesis that MPTP acts through inhibition of complex I of the electron transport chain. The search for a possible cause of PD and the need for development of new and effective therapeutic agents has stimulated research dealing with the development of the various methods of producing artificial parkinsonism. Carlsson et al. (1958) were the first to show that a parkinson-like motor-induced akinesia accompanied by an affective disorder, could be elicited in animals by injection of reserpine; however, symptoms generally disappear once reserpine is discontinued. Another means of producing a reversible parkinsonian state involves the use of alpha-methyltyrosine (a-MT). Other methods include: blocking post-synaptic dopamine receptors by neuroleptics, chemical damage or ablative surgery of critical pathways of the basal ganglia. Manganese-containing compounds have also been used to produce parkinsonism (Gianutsos and Murray, 1982). 6-Hydroxydopamine (6-OHDA) has been used extensively in causing parkinsonism in experimental animals. The specificity of this compound in damaging the catecholaminergic system has been associated with the high affinity of the reuptake system for 6-OHDA. A significant disadvantage in the use of 6-OHDA relates to its inability to cross the blood-brain-barrier. Therefore, central effects require intraventricular administration or direct injection into the substantia nigra. The discovery of MPTP, as mentioned earlier, has provided a reliable method of inducing parkinsonism. MPTP can be administered peripherally and through its selective destruction of the nigrostriatal dopaminergic system produces a better TABATABAEI, A. 10 model of PD than does 6-OHDA. At this time, although the precise mechanism of action of MPTP is not well understood, the reproducible, specific and extraordinary similarity of neuronal degeneration it produces to the idiopathic PD in humans has led to the extensive use of this compound for induction of a parkinsonian state in animals. The following presents three series of experiments considering different aspects of Parkinson's disease. In the first chapter the effects of the chronic administration of 3-acetylpyridine, a naturally occurring neurotoxin, to rats have been examined. The second chapter considers a possible method of prevention of MPTP neurotoxicity by blockade of the N M D A subtype of glutamate receptors and hence the possible involvement of glutamate in the etiology of PD. Finally, the new controversial approach to the treatment of parkinsonian patients involving fetal tissue transplantation into the brain of patients with PD has been evaluated in MPTP-treated mice. TABATABAEI, A. 11 2. CHAPTER 1 NEUROCHEMICAL EFFECTS OF 3-ACETYLPYRIDINE IN STRIATUM A N D CEREBELLUM 2.1. Introduction: Olivopontocerebellar atrophy (OPCA) was described by Dejerrine and Andre-Thomas in 1900. The term OPCA refers to a series of heterogeneous diseases which show neuronal loss of the ventral portion of pons, the inferior olives and the cerebellar cortex as a common pathology (Berciano, 1982). In an extensive review of 132 cases of OPCA reported in the literature, Berciano (1988) estimated an average age of onset of 28 years and 49 years for the familial and sporadic types of OPCA, respectively. Symptoms exhibited by OPCA patients generally include cerebellar ataxia involving gait, visual defects, fatigue of lower limbs, dementia and generalized spasticity. The major pathological manifestations include deterioration of the arcuate, pontine, inferior olivary, ponto-bulbar nuclei and the cerebellar cortex. Parkinsonian symptoms have been reported in 35-57% of OPCA patients. In 46-49% of cases an OPCA-associated lesion of the substantia nigra is also present (Berciano, 1988). Perry (1984) demonstrated the existence of four distinct classes of dominantly inherited O P C A based on biochemical variations. The amino acid content of autopsied brain of 16 OPCA patients was quantitatively characterized and variations in the levels of four putative neurotransmitters of cerebellum (glutamate, aspartate, taurine and GABA) were observed. These observations further indicated that OPCA encompasses a heterogeneous group of disorders with some common neuropathological features. In a recent biochemical and neuropathological study of ten living OPCA Cuban patients and seven autopsied Cuban patients, respectively, Orozco et al. (1989) reported general pathological changes the same as those described by Berciano (1982) (marked reduction of Purkinje cells, decreased number TABATABAEI, A. 12 of granule cells and degenerated olivary and pontocerebellar neurons). Although these investigators did not observe parkinsonian symptoms in 81 living patients examined, six of the seven autopsied patients studied presented an approximately 87% reduction in neurons of substantia nigra. They have also noted reductions of 60% and 48% in 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) respectively in the CSF of OPCA patients (Orozco et al., 1989). This substantial reduction of nigral cells indicates that neurodegeneration associated with OPCA is not restricted to the cerebellum and brain stem. The observation that some compounds such as 3-acetylpyridine (Schut, 1950), phenytoin (Mimaki et al„ 1980; Utterback et al., 1958) and alcohol (Torvik et al., 1983) are able to cause damage to cerebellar neurons in experimental animals and humans which is similar in part to the pathology of OPCA, suggests the possibility that the neurodegeneration in OPCA patients may occur as a result of specific neurotoxins which may not be completely destroyed and cleared due to an inherited mutation in genes coding for some degrading enzyme(s). Furthermore, OPCA-associated lesions include damage to the substantia nigra; an environmental toxin which can produce OPCA-associated parkinsonism or a structurally related compound with a higher degree of specificity for cells of substantia nigra might be a cause of idiopathic PD. 3-AP is an environmental neurotoxin which has been found in Burley tobacco cigarette smoke (Quin et al., 1961), white bread crust (Folkes and Grimshaw, 1981), filberts (Kinlin et al., 1972), coffee (Sasaki et al„ 1987), brandy (Ough and Almy, 1986), snuff (Lavoie et al., 1989) and beer (Narziss et al., 1988). Therefore, it is possible that humans could be exposed to low doses of this compound over a long span of time. This compound has been known to induce degeneration of the inferior olive and of the climbing fibers rising from this nucleus to the cerebellum. These are some of the main structures affected in OPCA. The neurotoxicity of 3-acetylpyridine has been known for many years. Desclin and Escubi (1974) presented TABATABAEI, A. 13 an overview of studies describing the cerebellar degeneration caused by this compound in mice, rats, rabbits and cats. These investigators also reported discrete lesions of the substantia nigra. Later, these findings of 3-AP neurotoxicity were confirmed by Balaban (1985), who reported that the main 3-AP-induced lesion sites include the inferior olive, nucleus ambiguus, hypoglossal nucleus, dorsal raphe nucleus, interpeduncular nucleus, dentate gyrus, and the hippocampus. In this study the degeneration of dopaminergic neurons of pars compacta and occasional damage of pars reticulata and ventral tegmental area were also observed. The caudate-putamen (CP) which receives ascending axons of substantia nigra was affected laterally and dorsally by the administration of large doses of the neurotoxin 3-AP. Deutch et al. (1989) have recently reported a modest degeneration of the nigrostriatal pathways in Sprague-Dawley rats 6 weeks after a single dose of 3-AP (80 mg/kg). Based on the above observations, chronic administration of sublethal doses of 3-AP may lead to substantial reduction of striatal dopaminergic function. The involvement of one or more environmental neurotoxins in the etiology of PD is strongly supported; however, at this time no other compound except MPTP is known to induce a parkinsonian state in humans and other primates. Some naturally occurring structural analogues of MPTP such as tetrahydroisoquinoline (TIQ), 2-phenylpyridine (2-PP) and 3-phenylpyridine (3-PP) have been examined for their neurotoxic properties. These compounds have not shown the ability to produce such neurodegeneration as MPTP does. Therefore, should 3-AP or a structurally related compound (a pyridine compound not related to MPTP) be shown to produce parkinsonism, the hypothesis that an environmental neurotoxin contributes to the etiology of idiopathic PD would be strongly supported. The following study was conducted to examine the neurotoxic properties of 3-AP on nigrostriatal neurons in rats. Furthermore, since the neurotoxic effects of 3-AP are presumed to be the result of formation of an antimetabolite, 3-acetylpyridine TABATABAEI, A. 14 adenine dinucleotide, it seemed worth-while to evaluate nicotinamide as a protective agent against the toxicity of 3-AP through competitive inhibition of the antimetabolite formation. 2.2. Materials and Methods: i. Animals and Experimental Materials Female Sprague-Dawley rats, weighing 177-188 g upon arrival in laboratory, were obtained from Charles River Canada Inc.. The animals were housed in polycarbonate cages, maintained on 12 hour light/dark cycle and had free access to food and water. 3-Acetylpyridine and nicotinamide were purchased from Sigma Corporation and were dissolved in 0.9% saline. Doses were calculated as the free base. The animals were divided into three groups. Group A animals (n=12) were used as controls and were injected s.e. with 0.9% NaCl. The volumes of injection were adjusted according to changes in the weight of the animals and corresponded to the volumes given to the experimental groups. Animals in group B (n=20) were injected s.e. with 3-AP (50 mg/kg). Group C animals (n=13) received s.e. injections of 3-AP (50 mg/kg) as in group B animals as well as s.e. injections of nicotinamide ( 100 mg/kg) immediately prior to the 3-AP injections. Each group received a total of seven injections of 3-AP at intervals ranging from 7 to 19 days over a two-month period. Due to the severe effects of 3-AP on the animals it was necessary to vary the elapsed time between injections in order to maintain a reasonable number of animals in each group. Group A and C rats displayed no neurological symptoms, behaved normally and continued to gain weight. Rats in group B, on the other hand, showed symptoms of hyperkinesis, muscular coordination disturbance, tremor and were increasingly ataxic with intervals of tonic spasm. These animals did not gain weight as did the animals of the other groups and began dying after the fourth injection. This dose of 3-AP (50 mg/kg) was chosen based on the observation of previous TABATABAEI, A. 15 experiments of Balaban (1985) in which a group of four rats receiving intraperitoneal injection of 3-AP (75-80 mg/kg) followed by vehicle (saline) injections died within 16 hours of the 3-AP administration. Perry et al. (1976) also reported a 20-50% death rate after intraperitoneal administration of 3-AP (65 mg/kg) to rats. Furthermore, the purpose of this study was to determine the effects of chronic exposure to this neurotoxin. The experiment was terminated 24 hours after the seventh injection due to the high rate of mortality (40%) of group B rats (final n=12). At this time, the surviving animals were sacrificed by cervical dislocation. The skull was exposed, cut along the midline and deflected to each side to expose the brain. The brain then was removed to a glass plate, dissected and frozen within an average of 80 seconds (60-155 seconds) of the animal's death. The cerebellum was frozen in liquid nitrogen for 20 seconds at which time it was packaged and maintained at -70 °C until analyzed chemically. The remainder of the brain was frozen on dry ice immediately after the original dissection. After thawing in -8 °C freezer to a proper consistency for dissection, striatum was dissected out on a cold plate, weighed and stored at -70 °C prior to biochemical analysis. The rapid removal and freezing of the brain after death is of utmost importance. It is known that while the contents of taurine, glutamic acid and glutamine do not change appreciably after death, glutathione (GSH) content is only stable for ten minutes and thereafter diminishes continuously for up to 48 hours (Perry et al., 1981). The level of G A B A in brain is also very unstable and increases rapidly immediately after death; therefore, the rapid removal and freezing of brain was necessary for accurate measurements of the GABA content of cerebellum (Perry et aL, 1981). ii. Biochemical Methods Contents of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured simultaneously in TABATABAEI, A. 16 striatal homogenates by high-performance liquid chromatography (HPLC) with electrochemical detection. Striata were homogenized in 0.8 ml of ice-cold medium (0.1 M H C I O 4 , 0.4 M NaSC>3) in a 1.5 ml Kontes tube fitted with a matching pestle. Homogenates were then incubated on ice for 30 minutes before centrifugation at 10,000 x g for 10 minutes at 4 °C. The supernatant then was filtered through a 0.45 urn filter (Millipore). 20 ul of the filtrate was then injected into the HPLC system. This system consisted of a reverse-phase chromatographic column (60 x 4.6 mm packed with ODS Hypersil, 3 [im, Shandon) and an LC-4A amperometric detector (Bioanalytical Systems Inc.) equipped with a glassy carbon electrode. External standards were used for quantification; standards were injected after every third or fourth sample for accurate measurement. A sodium phosphate buffer was used as the mobile phase (0.1 M N a 2 H P 0 4 , 60 mg/L Na 2 EDTA and 200 mg/L sodium octyl sulfate). The p H of this buffer was adjusted to 3.70 with 1 M H 3 P O 4 and methanol was added to a final concentration of 1% (Perry et al., 1985). The amino acid content of cerebellum was determined using the following procedure. The cerebellum was weighed, homogenized and deproteinized in 0.4 M H C I O 4 (5 x volume) in a Potter-Elvehjem homogenizer. The homogenate was then centrifuged at 8000 rpm for 10 minutes. The precipitate was resuspended in 0.4 M H C I O 4 (3 x volume) and rehomogenized. After recentrifugation, the p H of the supernatant was adjusted to 2.2.-2.5 by the addition of diluted potassium hydroxide (KOH) solution prior to a centrifugation at 8000 rpm for 10 minutes to remove precipitated KCIO4. The supernatant was stored at -70 °C until amino acid analysis was performed. A volume of supernatant containing 40 mg of cerebellum was placed on a previously regenerated column of Technicon Chromobeads (approximately 120 x 0.6 cm of 17 urn spherical particles). Norleucine (0.1 umole) was added to the samples as an internal standard. The elution buffer gradient consisted of lithium citrate buffers (containing Li3C6HsO, L iOH, Brij 35 solution and thiodiglycol) at p H values of 2.80, 3.80, 6.10 adjusted with HC1. For 6.5 hours the TABATABAEI, A. 17 column was maintained at 35 °C until glutamine was eluted and thereafter the temperature of the column was automatically increased to 70 °C. The eluted compounds were detected using ninhydrin. The area under each peak was integrated and the amount of each compound calculated accordingly (Perry et al., 1968). iii. Data Analysis Data were analyzed by means of unweighted means analysis of variance and subsequent Newmans Keuls post-hoc test. 2.3. Results: Table I displays the mean contents of dopamine and its metabolites (DOPAC and HVA) in the whole striatum from the three groups. Treatment of rats with chronic doses of 3-AP (50 mg/Kg) resulted in an approximately 11% reduction of striatal dopamine. This change, however, fell short of a statistically significant reduction. The striatal dopamine level of animals treated with 3-AP alone (group B) is, however, significantly lower than the animals pretreated with nicotinamide (group C). Animals in this group are not different from control animals in regard to the striatal dopamine level. A reduction of striatal dopamine level in rats was observed by Deutch et al. (1989) who report an approximately 25% reduction of striatal dopamine six weeks after a single injection of a large dose (80 mg/kg) of 3-AP to rats. In a recent paper, these investigators report the reduction of striatal dopamine to be limited to the dorsolateral section of the striatum. This reduction of dopamine level has been reported to be as high as 40% in rats treated with a single injection of 3-AP (80 mg/kg) (Deutsch et al., 1990). Table II lists the mean content of seven amino compounds found in the cerebellum of the control and treated rats. These compounds include taurine, aspartate, glutamate, glycine, y-aminobutyric acid (GABA), glutamine and total glutathione. Chronic s.e. administration of 3-AP led to a significant reduction (26%) in the taurine content of cerebellum. This reduction in cerebellar taurine levels was TABATABAEI, A. 18 TABLE I Striatal Dopamine and Metabolites in Rats Following Chronic 3-AP Treatment, with (Group C) or without (Group B) Nicotinamide Pretreatment. Normal Controls 3-AP 3-AP+Nicotinamide Compound Group A (n=12) Group B (n=12) Group C (n=13) Dopamine 10.07 ±0 .40 8.98 ±0 .40 10.85 ± 0.381 DOPAC 1.34 ±0.06 1.19 ±0.06 1.34 ±0.05 H V A 1.02 ±0 .05 0.81 ± 0 . 0 5 * 0.85 ± 0 . 0 5 * Values (mean ± SEM) expressed in p.g/g wet weight. * Significantly different from controls, P<0.05. t Significantly different from 3-AP group, P<0.05. TABATABAEI, A. 19 TABLE II Content of Neurotransmitter Amino Acids in Cerebellum of Rats Following Chronic 3-AP Treatment, with (Group C) or without (Group B) Nicotinamide Pretreatment. Compound Controls 3-AP 3-AP+Nicotinamide Group A (n=ll) Group B (n=10) Group C (n=10) Taurine 3.98±0.13 2.95±0.14 * 3.77±0.14 Aspartic acid Glutamic acid 2.19+0.04 10.14±0.24 1.90+0.04 * 8.91±0.25 * 1.81±0.04 * 9.16±0.25 * Glutamine 5.04+0.13 Total glutathione 0.9910.05 (as GSH) Glycine 1.26±0.08 G A B A 1.31±0.03 5.28±0.13 0.89±0.05 1.33±0.08 1.22±0.03 * 4.82±0.13 0.91±0.06 1.41±0.08 1.18±0.03 * Values ( meant SEM ) expressed in urnol/g wet weight. * Significantly different from the control group, P<0.05. TABATABAEI, A. 20 prevented by administration of high doses of nicotinamide immediately prior to 3-AP injection. The chronic treatment of rats with 3-AP also led to modest although still significant reductions (10-15%) in aspartate, glutamate and GABA. The reduction in the levels of these compounds was not prevented by nicotinamide injection. 2.4. Discussion: Chronic s.e. administration of 3-AP at a sublethal dose did not cause a significant reduction of striatal dopamine levels in Sprague-Dawley rats. This finding is not in agreement with the observation of Balaban (1985) of damage to the substantia nigra pars compacta and the observation of Deutch et al. (1989; 1990) of reduction in striatal dopamine levels following 3-AP treatment. Our finding of a slight but not significant reduction of striatal dopamine content in rats does not provide any evidence that 3-AP may be a neurotoxin causing the parkinsonism associated with OPCA. The lack of a significant reduction of striatal dopamine in this experiment might be due to a requirement of a threshold dose of 3-AP before a severe toxic effect is produced. Furthermore, high rate of death in animals treated with 3-AP alone might have eliminated the more susceptible animals. It is also possible that the dissection of the whole striatum rather than the dorsolateral striatum masked a more significant reduction in dopamine level. Despite the substantial (40%) reduction in dopamine in rat dorsolateral striatum as observed by Deutch et al. (1990) six weeks after a single high dose (80 mg/kg) treatment with 3-AP, this compound is unlikely to be the much-searched-for cause of idiopathic PD, since a 70-80% reduction of dopaminergic cells in the substantia nigra pars compacta precedes the manifestation of parkinsonian symptoms. In the present experiment, in spite of severe behavioural abnormalities, chronic administration of doses of this compound which were relatively high as compared to the trace amounts parkinsonian patients would have been exposed to in their diets, led to a relatively small striatal dopamine reduction which was not statistically significant. Despite TABATABAEI, A. 21 the absence of a significant reduction of striatal dopamine in rats treated with 3-AP alone, these animals exhibited severe behavioural abnormalities which were prevented by nicotinamide pretreatment. The prophylactic use of nicotinamide immediately prior to 3-AP injection prevented the reduction of striatal dopamine levels. It is known that the administration of 3-AP to rats leads to the synthesis of an antimetabolite, 3-acetylpyridine adenine dinucleotide (Herken, 1968). This false pyridine nucleotide can interfere with cellular metabolism and cause cellular damage and death. Furthermore, it is possible that the very large amount of nicotinamide injected into rats prior to 3-AP exposure in the present study favoured the formation of NAD+ over that of 3-AP adenine dinucleotide thereby preventing the toxic effects of 3-AP. Repeated administration of 3-AP also caused reduction in the cerebellar levels of taurine, aspartate, glutamate and G A B A , putative neurotransmitters of cerebellum. Degeneration of particular subtypes of neurons may be detected by a decrease in the local level of the neurotransmitters of the degenerating neurons. Therefore, the reduction of these neurotransmitters was interpreted as being indicative of neuronal damage caused by 3-AP as reported by other investigators. High doses of nicotinamide prevented the reduction of taurine observed in cerebellum of animals treated with 3-AP. The reduction in taurine was assumed to be the result of formation of 3-AP adenine dinucleotide, as explained above. Taurine is formed from cysteine by a three step process in which the first step involves cysteine dioxygenase (Fig. 2). This initial step also requires N A D + as a co-factor in rat brain (Misra, 1975). Hence, an antimetabolite formed due to high levels of 3-AP in certain neurons might prevent the formation of taurine. Animals treated with repeated injections of 3-AP were severely ataxic and tremulous. They also showed signs of muscular incoordination and hyperkinesis. These symptoms were not observed in animals receiving nicotinamide prior to 3-AP exposure. As mentioned earlier, the reduction of cerebellar taurine and the TABATABAEI, A. 22 Figure: 2 Enzymatic conversion of cysteine to taurine. C ^ Represents compounds, Represents enzymes. TABATABAEI, A. 23 slight (but insignificant) reduction of striatal dopamine were also prevented by nicotinamide pretreatment. The modest reductions of aspartate, glutamate and G A B A , however, were not prevented by nicotinamide. Striatal reduction of dopamine (11%) does not seem adequate for such severe behavioural symptoms. Although the behavioural symptoms are prevented, as is the reduction of striatal dopamine, it is unlikely that the severe behavioural symptoms observed in this experiment are due to the slight nigrostriatal damage. The reduction of taurine in cerebellum is also assumed not to be the cause of the severe ataxia and tremor since in biochemical assessment of autopsied brains of 16 OPCA patients taurine was found to be unaffected or increased. Therefore, it was concluded that the behavioural symptoms observed in this study were not due to the reduction of cerebellar taurine. It is possible that another area of the brain not studied in this experiment could be damaged by 3-AP treatment and cause the behavioural symptoms observed. The 3-AP-induced damage to such structure(s) is preventable by prophylactic use of nicotinamide. The mechanism of action of 3-AP is not well understood. It seems logical to assume that a mechanism of uptake with some degree of specificity is involved, since the neurotoxicity of 3-AP treatment has been observed in some structures of the brain and not in others (Balaban, 1985). Furthermore, it seems that the mechanisms of cellular damage in cerebellum and in substantia nigra are not the same since nicotinamide prevented the small reduction in striatum but not in the cerebellum. It is, therefore, possible that long-term exposure to low doses of this neurotoxin could produce cellular damage to the substantia nigra without the cerebellar damage associated with OPCA. Furthermore, humans might be much more sensitive to 3-AP than are rats. Long-term treatment of primates with low doses of 3-AP seems necessary to further support or abandon 3-AP as a possible cause of idiopathic PD. TABATABAEI, A. 24 The present study, therefore, does not provide any evidence that 3-AP may be a cause of OPCA-associated parkinsonism in predisposed individuals. The possibility of involvement of this compound in the etiology of PD remains unresolved until further experiments are undertaken. TABATABAEI, A. 25 3. CHAPTER 2 N M D A RECEPTOR BLOCKADE A N D PARKINSONISM 3.1. Introduction: During the last decade, excitatory amino acids (EAA) have been increasingly implicated in a variety of neurodegenerative disorders. The effects of E A A such as glutamate and aspartate or environmental excitotoxins have been linked with the pathogenesis of neurodegenerative disorders, such as olivopontocerebellar atrophy (OPCA) (Plaitakis et al„ 1982), spinal cord injury (Faden and Simon, 1988), Alzheimer's disease (Maragos et al., 1987), and Huntington's disease (Perry and Hansen, 1990; Coyle and Schwarcz, 1976). Endogenous E A A such as the neurotransmitters, glutamate and aspartate, and metabolites such as quinolinate, homocysteate and cysteine (Olney et al., 1990) have been proposed as possible excitotoxins leading to neuronal damage. The mechanism of such neurotoxicity is not clear; however, it seems activation of ion channels leads to an increased intracellular C a 2 + which is well correlated with the time and degree of cellular damage. The toxic consequences of increased intracellular C a 2 + might involve a variety of mechanisms including activation of a host of enzymes leading to cellular damage. Production of free radicals as a result of such enzyme activation might be one cause of cellular damage (Meldrum et al., 1990). An alternative mechanism of cellular damage may involve free radicals. The neurodegenerative property of kainate is well documented. The involvement of superoxide or secondarily produced hydroxyl radicals in the neurotoxicity of kainate was demonstrated by the prevention of such detrimental effects of kainate by addition of scavengers such as superoxide dismutase and catalase or mannitol in cell cultures. The neuronal damage was also prevented by allopurinol, a specific inhibitor of xanthine oxidase through reduction of superoxide radical generation (Dykens et al., 1987). Reactive oxidants such as hydrogen peroxide have also been TABATABAEI, A. 26 suggested as a possible cause of PD (Cohen, 1986). The oxidative stress induced by MPTP oxidation has not been ruled out as a possible mechanism of damage produced by this compound. More recently, the excitotoxic effects of L-glutamate, which is accepted as the putative neurotransmitter of afferent pathways of the basal ganglia, have also been associated with Parkinson's disease. Klockgether and Turski (1989) have suggested that compounds with antagonistic effects at N-methyl-D-aspartate (NMDA) receptors (a subtype of glutamate receptors) might be useful as antiparkinsonian drugs. These investigators proposed the subthalamic nucleus as the site of action of such inhibitors, since degenerating nigrostriatal dopaminergic pathways lead to reduced GABAergic activity of LGP and hence overactivity of the subthalamic nucleus due to an imbalance of excitatory cortical input and inhibitory effects of LGP. On the other hand, the site of action of N M D A antagonists has been suggested to involve the caudate-putamen and the neurotransmitter balance of dopamine-glutamate in the striatum (Schmidt et al., 1990). Although Klockgether and Turski support the concept that NMDA-block may have antiparkinsonism effects, they propose the striatal output neurons with N M D A receptors as the site of action. Despite the controversial site of action of such antagonists, the potential use of N M D A receptor-ion channel complex blocking agents such as MK-801 in treatment of PD has been considered. Carlsson and Carlsson (1989) demonstrated that systemic administration of MK-801 in monoamine-depleted mice (albino mice treated with 10 mg/kg reserpine and 250 mg/kg oc-MT), stimulates locomotor activity. Other investigators also have demonstrated the anticataleptic effects of MK-801 in rats rendered parkinsonian by haloperidol treatment (Schmidt and Bubser, 1989). Furthermore, memantine which is used as a therapeutic agent in treatment of parkinsonian patients is known to have NMDA-blocking characteristics. The therapeutic efficacy of this agent was mostly attributed to its dopaminergic properties; however, whole-cell patch-clamp studies of spinal neurons of mice have TABATABAEI, A. 27 demonstrated a noncompetitive blockade of N M D A receptors by memantine with a similar potency to MK-801 (Borman, 1986). Previous to these observations, Olney et al. (1987) tested a series of antiparkinsonian agents and showed that the compounds tested block N M D A toxicity in a noncompetitive manner. Hence, the central mechanism of action of such drugs known to antagonise peripheral acetylcholine receptors was questioned. The antiparkinsonian properties of MK-801 have also been studied in MPTP-treated monkeys. The cumulative intramuscular administration of 0.1-30 fig/kg of MK-801 did not show clear changes in parkinsonian symptoms of monkeys. A single 30 |ig/kg dose, on the other hand, exacerbated the symptoms. These investigators, however, did not provide any data nor indicate the methods of rating (Crossman et al., 1989). Later, it was demonstrated by Graham et al. (1990) that local administration of MK-801 in the medial globus pallidus leads to amelioration of parkinsonian symptoms in MPTP-treated monkeys. Not only has MK-801 been studied in regard to its therapeutic use as an antiparkinsonian agent, but also the close association of dopamine with E A A has been studied. A direct excitatory effect of N M D A on release of newly synthesized [3H]dopamine from rat striatal slices has been demonstrated (Krebs et al„ 1991). This might explain the reduced amelioration of parkinsonian symptoms by 3,4-dihydroxyphenylalanine (L-DOPA) after systemic MK-801 administration in monkeys as reported by Crossman et al. (1989). A direct interaction of dopamine and glutamate at a molecular level has been suggested by Girolt et al. (1990). They propose an opposite effect of dopamine and glutamate in striatum through regulation of protein phosphorylation. D- l receptor stimulation leads to an increased cAMP level with a resulting activation of cAMP-dependent protein kinase. This leads to the phosphorylated DARPP-32, a small phosphoprotein which is found in high concentration in striatonigral cells. Phosphorylated DARPP-32 is a potent inhibitor of phosphatase-1. This inhibition of dephosphorylation is TABATABAEI, A. 28 suggested to enhance other effects of DA. A calcium-activated protein (calcineurin) is responsible for dephosphorylation of DARPP-32 and reactivation of phosphatase-1 and hence reversal of the effects of DA. In rat striatal slices, N M D A leads to dephosphorylation of DARPP-32. This action of N M D A is inhibited by MK-801; therefore, N M D A blockade would allow a facilitated action of DA. The site of action of N M D A blockers as antiparkinsonian agents remains unresolved and multiple sites of action may be involved. Furthermore, the question that remains is: are excitatory amino acids the cause of the selective neurodegeneration of PD and, if so, can this degenerating effect of excitotoxins be prevented by N M D A receptor blockade? The toxicity of E A A in vitro or when administered locally in vivo has been demonstrated. However, the regional selectivity of cellular damage observed in PD cannot be explained by exci to toxicity of EAA. Olney et al. (1990) have examined the toxicity of L-DOPA and 6-OHDOPA through E A A receptors on a chick embryo retinal preparation. These investigators proposed a possible defective conversion of DOPA to DA and hence accumulation of L-DOPA. The leakage of L-DOPA or other toxic derivatives, as well as glutamate released from corticostriatal neurons, then, might cause damage to dopaminergic neurons. Other investigators have recently examined the neuroprotective effects of NMDA-blockade by MK-801 against the neurotoxicity of MPTP. In their report, Sonsalla et al. (1989) concluded that MK-801 does not prevent MPTP toxicity in mice sacrificed three days after the MPTP-treatment. This report, however, may overlook the possible short term protection of MK-801 which was last administered three hours after the first MPTP injection (four doses of MPTP (20 mg/kg) were administered intraperitoneally every 2 hours). The mechanism of action of MPTP in producing nigrostriatal damage is not clearly understood. The majority of investigations point to involvement of complex I of the mitochondrial transport chain requiring N A D + as a co-factor; however, the oxidative stress induced by MPTP, TABATABAEI, A. 29 which is also one of the suggested mechanisms responsible for neuronal damage caused by E A A (Dykens et al., 1987) has not been ruled out (Markey, 1986). It was, therefore, the aim of this study to examine the effects of prophylactic use of an N M D A blocking agent (MK-801) on the neurotoxicity of MPTP. We have also examined the protective capability of nicotinamide as a source of increased concentration of NAD+ to facilitate N A D H - C o Q reductase activity in viable mitochondria against MPTP neurotoxicity. The MAO-B activity of fresh brain homogenates was measured in vitro in the presence of various concentrations of MK-801 to examine a possible inhibitory effect of MK-801 on MAO-B activity. 3.2. Materials and Methods: i. Animals and Experimental Materials Female litter mate 70-77 day old C57 BL/6 mice were obtained from Charles River Canada Inc. and were maintained on 12 hour light/dark cycle. Food and water were provided ad libitum. Nicotinamide (Sigma) and MK-801 (Merck Sharp & Dohme Research LAB) were dissolved in 0.9% NaCl. Doses were calculated as the free base. Phenylethylamine hydrochloride p*-[ethyl-14C] was obtained from N E N Research Products. Crystalline MPTP (Aldrich) was converted to its hydrochloride salt and dissolved in 0.9% NaCl solution. The p H of the solution was adjusted to 5-6 with 5 N NaOH. The MPTP solution was then diluted with 0.9% saline to obtain a concentration of 2 mg/ml (calculated as the free base). Animals were randomly assigned to one of four groups. Group A animals (n=10) were spared treatment and were used as base-line controls. Group B animals (n=15) received a single injection of MPTP (40 mg/kg) s.e. The other two groups received the same dose of MPTP, but group C animals (n=15) were also injected with nicotinamide (100 mg/kg) s.e. immediately prior to the MPTP injection and every 12 hours thereafter for 48 hours (a total of 5 nicotinamide injections) to maintain a relatively high concentration of N A D H in the animals until MPTP was completely cleared. Group D animals (N=19), in addition to MPTP, received 9 injections of MK-TABATABAEI, A. 30 801. The first injection (immediately before the MPTP exposure) was given at a dose of 1 mg/kg s.e. The remaining injections were administered at a dose of 0.5 mg/kg s.e. every 6 hours for 48 hours. All animals except one in group D survived the treatment period. These mice were allowed to survive for a four-week period at which time they were sacrificed by cervical dislocation. The brains were removed rapidly as described in the previous experiment and were immediately frozen on dry ice; after thawing in a -8 °C freezer to a proper consistency for dissection, striata were dissected out, weighed and stored at -70 °C until the biochemical analyses were performed. All specimens were coded at this time and the subsequent biochemical analyses were performed blinded. ii. Biochemical Methods The striata were homogenized and deproteinized as described in the previous experiment. To compensate for the lower weight of striatum in mice, however, 0.4 ml of homogenisation buffer was used. Contents of DA and its metabolites were then measured in the striatal homogenates by HPLC with electrochemical detection as described previously. Monoamine oxidase-B activity was measured in fresh brain homogenates. Mice were sacrificed by cervical dislocation and the brains were removed immediately, weighed and homogenized in Potter-Elvehjem homogenization vessels in 100 x (w/v) of buffer (100 mM N a H 2 P 0 4 / p H 7.4 adjusted with NaOH, 0.1% (v/v) of Triton X-100) and were kept on ice at all times. The assay mixture consisted of 100-130 ul of phosphate buffer, 0-30 |il of MK-801 (at various concentrations), 50 ul of fresh brain homogenate and 20 |il phenylethylamine (3-[ethyl-14C] at a final concentration of 400 u.M and a specific activity of 12.5 mCi/mmol. The reaction mixture was incubated at 37 °C for 15 minutes with continuous shaking before the reaction was stopped by the addition of 100 |il of 2 M citric acid. The labeled phenylacetic acid then was extracted into 3 ml of toluene (Fisher Scientific TABATABAEI, A. 31 Company) (Glover et al.. 1982). The radioactivity of 2 ml of the organic layer was measured in a liquid scintillation counter (Beckman LS 9000). Preliminary experiments were performed to confirm and adjust the amount of substrate required to obtain V m a x . The linearity of the reaction with respect to amount of enzyme present was also confirmed, iii. Data Analysis Data were analyzed by means of unweighted means analysis of variance and subsequent Newman Keuls post-hoc test. 3.3 Results: The contents of D A and its metabolites in whole striatum of the four experimental groups are presented in Table III. A single injection of MPTP (40 mg/kg) led to a substantial reduction of DA, DOPAC and H V A in the striata of animals treated with MPTP alone (group B). MK-801/MPTP treated mice (group D) had significantly higher levels of striatal DA (52% of normal) in comparison with MPTP-treated animals (18% of normal). The nicotinamide/MPTP-treated group did not show any significant change in striatal levels of DA when compared with the MPTP exposed group. Animals receiving nicotinamide treatment recovered from the behavioural effects of MPTP (hypokinesis, lack of spontaneous movement and somnia which lasted approximately 24 hours for group B) more rapidly than animals receiving MPTP alone. Mice receiving MK-801, on the other hand, were severely ataxic and quite inactive for up to 4 hours after the last MK-801 injection. The IC50 for each set of data of the activity of freshly prepared MAO-B of C57 BL/6 mouse brain in the presence of increasing amounts of MK-801 was obtained. An average IC50 of 2.8 mM was determined for MK-801 (Fig. 3). TABATABAEI, A. 32 TABLE III Striatal Dopamine and Metabolites in Mice Treated with MPTP (Group B), MPTP+Nicotinamide (Group C), MPTP+MK-801 (Group D). Normal MPTP MPTP + MPTP + Control Nicot. MK-801 (A) (B) (C) (D) Compound (n=10) (n=15) (n=15) (n=18) Dopamine 14.39±0.55 * 2.66±0.45 2.98±0.45 7.38±0.41 § DOPAC 1.18±0.05 * 0.33±0.04 0.36±0.04 0.68±0.04 § H V A 1.54+0.08 * 0.66±0.07 0.81±0.07 1.10+0.06 § Values (mean ± SEM) expressed in (ig/g wet weight. § Significantly different from control, MPTP-treated, MPTP+Nicot. mice, P<0.05. * Significantly different from mice treated with MPTP, P<0.05. TABATABAEI. A. 33 100 T • ———. J. IVITY 80 NT rj < 60 -% OF MAO 40 20 n L—l i l i 1 • i I i i i m l I as, i i i i nil 1 A i • n 1 1 1e-05 1e-04 1e-03 1e-02 1e-01 1e+00 1e+01 1e+02 MK-801 FINAL C O N C E N T R A T I O N m M Figure: 3 Plot of % M A O - B activity in the presence of increasing concentrations of MK-801. The bars represent S.D. The dotted line represents the IC50. TABATABAEI, A. 34 3.4. Discussion: The possible neuroprotective effect of MK-801, a noncompetitive N M D A receptor antagonist, was examined against the specific neurodegenerative properties of MPTP. Nicotinamide was also tested for its possible protective properties against MPTP neurotoxicity. Treatment with MK-801 in the present study substantially lessened the MPTP-induced reduction of striatal DA. It is possible that MK-801 protects against MPTP neurotoxicity by inhibiting MAO-B, thereby preventing the conversion of MPTP to MPDP. In a previous experiment MK-801 ( 1-2 mg/kg) administration led to a significant reduction in striatal DOPAC and H V A content, perhaps as a consequence of inhibition of MAO-B (Perry et al., 1989). To investigate a possible direct effect of MK-801 on MAO-B activity we measured the activity of MAO-B in freshly prepared brain homogenates of mice in the presence of increasing concentrations of MK-801. An IC50 of 2.8 mM was obtained. A half life of 2.05 hours in brain of rats has been determined for MK-801 (Vezzani et al., 1989). Furthermore, Hucker et al. have demonstrated an approximately equal distribution of MK-801 throughout the body of rats (Hucker et al., 1983). Based on the rate of clearance and the approximate distribution of MK-801 in rodents the calculated maximum concentration of MK-801 in brain is 0.003 mM in our experiment. The IC50 obtained in the in vitro enzyme studies is three orders of magnitude grater than the higest possible concentrationof MK-801 estimated to be present in brain under the dose scheduale used in our study. Therefore this IC50 indicates a non-specific effect of MK-801 on MAO-B activity and does not provide any support for the above hypothesis. MK-801 may directly block the dopamine reuptake system which would prevent the uptake of MPP+ in MPTP treated mice; more experiments are required to examine this hypothesis. On the other hand, it is of interest to examine the possibility that this protection may be due to an indirect effect of N M D A receptor blockade on the mechanism of action of MPTP. A few months after completion of this experiment, a report by Turski and TABATABAEI, A. 35 colleagues (Turski et al., 1991) presented an experiment confirming our observations. In their experiment, MPP + was focally administered to the substantia nigra pars compacta. Competitive [(2-amino-7-phosphonoheptanoate)(AP7)] and noncompetitive antagonists [ ( 3 - ( ( ± ) - 2 - c a r b o x y p i p e r a z i n - 4 - y l ) - p r o p y l - l -phosphonate)(CPP) and MK-801] of N M D A receptors prevented the degenerating effects of MPP+. The quisqualate antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX) did not provide such protective effects. This study also did not examine the possibility of blockade of MPP+ uptake by MK-801. Whether MPTP itself is excitatory or simply potentiates the action of endogenous E A A to cause neuronal damage remains unresolved. The majority of investigations into the mechanism of action of MPTP point to the involvement of complex I of the mitochondrial transport chain requiring N A D + as co-factor and resulting in depletion of the ATP stores of the affected neurons. In parkinsonian patients, a specific reduction in the activity of N A D H -CoQ reductase, which is restricted to the cell bodies of the substantia nigra, has been observed (Schapira et al., 1990). This finding supports the hypothesis that the mechanism of action of MPTP involves inhibition of complex I. Therefore, increasing the concentration of N A D to facilitate NADH-CoQ reductase activity in unaffected mitochondria might be expected to provide some degree of cellular protection. Our findings, however, did not provide evidence to support this hypothesis. Although this experiment suggests some useful properties of MK-801 as a means of slowing or possibly preventing the progression of parkinsonian symptoms, the mechanism of such effects remains unresolved and the many side effects of MK-801 may limit the clinical usefulness of this compound for the management of patients with PD. TABATABAEI, A. 36 4. CHAPTER 3 FETAL DOPAMINERGIC CELL TRANSPLANTATION IN MPTP-TREATED MICE 4.1. Introduction: Over the last decade transplantation of neuronal tissue has been investigated as a possible tool for the replacement of particular neurotransmitters in brain where specific subclasses of neurons have degenerated. Experiments performed on 6-hydroxydopamine (6-OHDA)-treated rats in 1979 provided the first evidence of functional intracerebral grafts (Bjorklund and Stenevi, 1979; Perlow et al., 1979). In these early experiments, rats with unilateral 6-OHDA-induced lesions of the nigrostriatal pathway were shown to have reduced rotational behaviour in response to apomorphine and amphetamine after transplantation of ventral mesencephalic tissue of 13-15 day old fetuses. These early observations led to an enthusiasm of investigators for this technique. Various experiments have been performed in rats using two main procedures: 1) transplantation of minced fetal tissue into the ventricle or onto the caudate/putamen (Freed et al., 1980; Bjorklund and Stenevi, 1979; Perlow et al., 1979); or 2) transplantation of dissociated cells in suspension into the caudate/putamen (Bjorklund et al., 1983; Schmidt et al., 1981; Bjorklund et al., 1980). These transplants were only effective if the implants were placed within or directly on the surface of the striatum. Transplantation of fetal mesencephalic tissue in substantia nigra or along the nigrostriatal pathway has not been successful (Dunnet et al., 1983). However , in a more recent study, Yurek et al. (1990) have demonstrated that fiber outgrowth of grafted neurons can be stimulated by simultaneous grafting of dopaminergic neurons with their embryonic target cells. They have hypothesized that a reduction or lack of nerve growth factors in adult transplanted animals does not allow an extensive outgrowth of fibers, whereas co-transplantation of dopaminergic and embryonic target cells (striatal cells) provides favorable conditions for survival and growth of dopaminergic implants. Other TABATABAEI, A. 37 studies have assessed the effectiveness of such transplants in MPTP-induced parkinsonian non-human primates. Successful transplantation of fetal ventral mesencephalic tissue into the striatum of rhesus monkeys (Bakay et al., 1985), African green monkeys (Redmond et al., 1986) and marmosets (Fine et al., 1988) has been reported. Another source of dopaminergic cells for implants has been the adrenal medulla. Chromaffin cells of the adrenal medulla are known to produce dopamine, albeit in low levels (Snider and Carlsson, 1972). Studies involving transplants of adrenal medullary tissue to the lateral ventricle or to the dopamine-depleted striatum in rats and monkeys also have been performed (Stromberg et al., 1985; Morihisa et al., 1984; Freed et al., 1981). Although survival of such transplanted cells has been reported in rats, the chromaffin cells require nerve growth factors (NGF) for long-term survival and outgrowth of neuron-like processes (Stromberg et al., 1985). The effects of such treatment with adrenal medullary tissue in rats have not been long lasting or very effective. The current therapeutic strategies for PD include treatment with dopaminergic and anticholinergic drugs. Experimentally, the possibility of slowing or interrupting the progression of the disease with antioxidant therapy and monoamine oxidase-B inhibitors has been explored. Studies of adrenal-brain transplantation and fetal neurotransplantation in animals have shown potential in rats and, to a lesser degree, in monkeys. These results have led to a large number of clinical implantations of grafts of adrenal medullary or dopaminergic fetal tissue in parkinsonian patients. In most cases, the transplantation has been carried out in patients with end-stage PD when the response to levodopa therapy is lost. Adrenal medulla chromaffin and fetal nigral cells are capable of synthesis and storage of DA and hence are strong candidates as donor sources for such transplantation. Despite the modest and transient benefit observed in adrenal medullary tissue transplantation in experimental animals, autologous adrenal medulla was used as the donor source in humans to avoid immunological and ethical TABATABAEI, A. 38 complications. The first clinical transplantations of this sort were autologous adrenal medullary tissue transplanted using a stereotaxic apparatus in a 55 year-old man and a 46 year-old woman (Backlund et al„ 1985). This report did not provide any evidence of functional improvement. Shortly afterwards in 1987, Lindvall et al. (1987) reported a trial of adrenal medullary tissue transplants involving two parkinsonian patients. They reported a modest but not long-lasting improvement in motor function. At this time, Madrazo et al. (1987) reported transplantation of adrenal medulla tissue to the head of the caudate where transplanted tissue is in contact with the target tissue (caudate) and is continuously bathed by the CSF. In this procedure, simultaneous right adrenalectomy and right frontal craniotomy were performed. The right caudate was exposed through the right lateral ventricle, and a small bed was constructed on the head of the caudate to receive the prepared fragments of medullary adrenal gland. These investigators reported an impressive and continued clinical improvement shortly after the transplant (15 and 6 days). One patient was reported to have continued to improve up to ten months post-transplant. In the following years the number of clinical transplants increased dramatically going from single cases to 100 cases in 1988 (Lindvall, 1989). Many investigators utilized the technique of Madrazo for adrenal medulla transplants to the head of the caudate with the hope of reproducing the impressive results reported from Mexico. Goetz et al. (1989) reported on attempted adrenal medullary transplant using Madrazo's technique. In this study, 19 patients with severe PD were given implants of autologous adrenal medulla. Post-operatively, the mean "on" (periods of improved mobility) time was increased and the mean "off" (periods of marked akinesia) time was decreased; however, the dosage of antiparkinsonian medication could not be decreased and no major motor function improvement was observed. Other investigators also have attempted autologous and homologous transplants of adrenal medulla to the striatum of parkinsonian patients, who showed varying degrees of improvement. None of these studies, however, TABATABAEI, A. 39 indicated comparable success to that reported by Madrazo (Kelly et al., 1989; Zhang, 1989; Penn et al.. 1988). Fetal neurotransplantation has also been used in clinical trials in the hopes of alleviating parkinsonian symptoms. Some experiments involving transplantation of human fetal dopaminergic neurons into rats preceded the grafting of fetal substantia nigra cells in humans. These experiments provided crucial information for successful neuronal transplantation. The functional effects of transplantation were observed approximately 2-3 months post-operation which is the time required for the growth of implanted neurons and formation of synapses with striatal neurons of the host (Brundin et al., 1988; Clarke et al., 1988). The optimal age of the fetus as a good source of dopaminergic cells to be used in transplantation was determined to be eight to ten gestational weeks. These neurons were also shown to have growth of axons extending from the transplanted tissue into the striatum of the host animal. In 1988, Madrazo et al. (1988) reported the transplantation of fetal substantia nigra in one patient and fetal adrenal medulla in a second patient using their original technique of microsurgery and transplantation to the head of caudate. These investigators reported improvement eight weeks after surgery in both cases without any complications. Shortly afterwards, Hitchcock et al. (1988) reported fetal neurotransplantation in two parkinsonian patients. In this study fetal mesencephalic tissue was kept in a tissue medium and mechanically disaggregated into a thick cell suspension. This suspension was then implanted stereotaxically into the head of the right caudate. They indicated a functional improvement eight hours after the transplant in the absence of any antiparkinsonian medication. Three months after the operation, medication which had been reinstated 30 days after the operation, was reduced. Although they reported a subjective functional improvement, objective rating scales did not support such an improvement. These investigators regarded their results as positive and recommended further trials. TABATABAEI, A. 40 Lindvall et al. (1988) also reported on two cases of stereotaxic transplantation of human fetal dopaminergic neurons into the putamen contralateral to the side most affected by PD. These patients demonstrated no change in on-off periods; however, some degree of improvement in motor function six months after the transplant was reported. Positron emission tomography (PET) performed six months after the transplant did not show any evidence of survival of the transplanted neurons. These investigators concluded that fetal mesencephalic tissue can be transplanted stereotaxically into the brain of parkinsonian patients with no complications for the recipient. This procedure, however, did not provide a functional improvement of parkinsonian symptoms. An improved procedure was reported in the case of an immunosuppresed 49-year old man transplanted with fetal ventral mesencephalic tissue (aged 8 to 9 weeks). Lindval et al. followed the procedure of their original experiment with some minor changes; the size of cannula used for implantation was reduced, as was the time elapsed between tissue dissection and transplantation. In this experiment, the medium was also changed from saline to a balanced, pH stable salt solution. A general improvement in parkinsonian symptoms was found two months after the transplantation. They also provide a comparison of pre- and post-operation PET scans as evidence of graft survival (Lindvall et al., 1989). The procedure remains controversial, despite the many attempts at various modes of transplantation. The mechanism of improvement, if indeed present, is not clearly understood and has also been the center of controversy. The following experiments were designed to answer the following questions: 1) does neurotransplantation of fetal tissue induce partial biochemical and histological recovery of MPTP-induced reduction of striatal dopamine? 2) is this recovery mediated by non-dopaminergic (rostral portion, astrocytes and frontal pole cells) or dopaminergic (caudal portion and mesencephalic flexure) fetal cells? 3) is this recovery due to growth of fetal cells into the striatum of the host animal? And finally, 4) is this recovery (if present) due to one or more growth factor(s) (rostral TABATABAEI, A. 41 and caudal portion, astrocytes in experiment "A", frontal pole cells and mesencephalic flexure in experiment "B") released from fetal cells, inducing growth of the remaining dopaminergic neurons of the host animal? A second set of experiments was performed to correct possible problems in the original attempt. The experimental design was improved by the use of younger fetal tissue and decreased time between sacrifice of the embryos and transplantation. The number of dopaminergic cells in the grafts was also increased by the use of ventral mesencephalic flexure tissue rather than the caudal portion of the brain. The coordinates of the transplant were also corrected based on histological observations of the first experiment. In the later experiment, we also explored the bilateral effects of unilateral transplant to examine the possible involvement of prolonged release of nerve growth factors. 4.2. Materials and Methods: I. Experiment A Li. Animals and MPTP Exposure Young female littermate C57 BL/6 mice were obtained from Charles River Canada Inc. and were maintained on a 12 hour light/dark cycle. Animals were housed in polycarbonate cages and were given food and water ad libitum. Crystalline MPTP (Aldrich) was converted to its hydrochloride salt and dissolved in 0.9% NaCl solution. The p H of this solution was adjusted to 5-6 with 5 N NaOH. The concentration of MPTP solution then was adjusted with 0.9% saline to obtain a concentration of 2 mg/ml (calculated as free base). Ninety 50-57 day old mice were injected s.e. with the MPTP solution at a dose of 40 mg/kg and this was repeated one week later with an equal dose. Fifty six mice survived this regimen of MPTP exposure. Ten littermates were spared MPTP injection to be used as normal controls. I.ii. Fetal Tissue Preparation Female C57 BL/6 mice which were kept separated for two weeks from the male mice were "painted" with the pheromone (urine) of the male mice. These TABATABAEI, A. 42 animals were kept separated for an additional three days. Female mice then were transferred to the males' cages overnight and were removed the following morning after observation of the vaginal plug. This day was counted as day zero. Animals were caged and maintained separately. On the 15 t h day of gestation, the pregnant mice were sacrificed by cervical dislocation; the abdominal wall was washed with 70% ethanol and cut to expose the internal organs, the uterus containing the fetuses was removed and placed into a sterile phosphate buffered saline (PBS) bath on ice. The fetuses (crown to rump 15 mm) were then removed from the uterus and amniotic sac. To hold the cranium in place, curved, serrated eye-dressing forceps (Graefe forceps, Fine Science Tools Inc.) were placed around the cervical vertebra accessed dorsally. The cranium was cut along the midline using straight sharp-ended surgical scissors. A fine spatula was used to peel back the skin and thin skull bone. The brain was then removed using the spatula and was placed on a filter paper saturated with ice-cold PBS in a Petri dish on ice. A razor blade was lowered perpendicularly to bisect the brain approximately 1/5 of the distance from the rostral end in the rostral/caudal plane. This rostral portion was removed with a large fire-polished, siliconized pasteur pipette to a Petri dish. The caudal (remaining) portion of the brain was transferred to a separate Petri dish. These tissues were minced and then were dissociated by means of trypsin treatment (final concentration of 0.1% and 15 minute incubation at 37 °C). The tissues were then centrifuged at 1000 rpm for 6 minutes and the supernatant was removed. Cells were washed three times with 5 ml of PBS. Minced tissues were then mechanically dissociated using progressively smaller bore fire-polished siliconized pipettes. Al l of the above steps were performed by naked eye or under a magnifying glass. Cell suspensions were then counted in trypan blue using a hemocytometer and the volume was adjusted to provide 4 x 107 cells/ml. Cultured C57 BL/6 mouse astrocytes were obtained from Dr. V.W.Yong (Montreal Neurological Institute) and were maintained in an incubator at 37 °C in TABATABAEI, A. 43 an atmosphere of 5% CO2 until they were prepared and used in neurotransplantation. I.iii. Neurotransplantation 38 days after the second MPTP treatment, transplantation of 15' day fetal nervous tissue (prepared fresh) was performed stereotaxically under sodium pentobarbital anesthesia (48 mg/kg). Mice were placed in the stereotaxic apparatus, an incision was made in the midline of the head, the scalp was deflected and a dental drill, fitted with a 2 mm stainless steel dental burr, and fixed to the stereotaxic injection arm was used to penetrate the skull in the appropriate location. A 25 |il Hamilton syringe equipped with a 26 gauge, blunt, stainless steel needle loaded onto the stereotaxic arm was used to transplant the suspended cells. All animals received the appropriate injections into the left lateral ventricle at the following coordinates using bregma as a marker: 0.5 mm caudal, 1.0 mm lateral and 2.5 mm ventral to dura (Fig. 4). 5 ul of suspended cells were infused over a one minute period and the needle was left in place for an additional minute before it was slowly retracted to allow for the intracerebroventricular pressure to redistribute and to avoid back leakage of the suspended cells along the needle track. However, an estimated 2 \d of back flow was observed. MPTP-treated mice were randomly divided into four groups. Group A mice (n=14) received a 5 |il injection of PBS. Group B (n=13) and C (n=14) were injected with 5 u.1 of rostral cell suspension and caudal cell suspension, respectively. Group D mice (n=14) received a 5 u.1 injection of cultured astrocyte suspension in PBS (all cell suspensions were at a concentration of 4 x 107 cells/ml). After the transplant, the skin was sutured and animals were placed in heated cages to recover from anesthesia. All animals (except one, which died, presumably due to the anesthetic) recovered well and appeared normal by the following day. Animals were allowed to survive for 35 days at which time they were sacrificed by cervical dislocation and brains were removed as described previously. The brains of 22 mice (5 from each group and 2 from non-MPTP-treated controls) were placed on a glass TABATABAEI, A. 44 ENZYMATIC TISSUE DISSOCIATION MECHANICAL TISSUE DISSOCIATION STEREOTAXIC TRANSPLANTATION Figure: 4 Schematic illustration depicting the techniques involved with the stereotaxic transplantation of fetal nervous tissue into the adult recipient (experiment "A" TABATABAEI, A. 45 plate and were cut sagittally using a razor blade. Left hemispheres of these animals were fixed in 4% paraformaldehyde solution for histological studies. The right hemispheres and the remaining whole brains were immediately frozen on dry ice. The striata were then dissected out after warming the brain to about -15 °C by placing the deep frozen brains in a -8 °C freezer. The striata were then weighed and maintained at -70 °C until the biochemical analyses could be performed. All specimens were coded at this step and the subsequent biochemical measurements were performed blinded. I. iv. Biochemical Methods The striata were homogenized and deproteinized as previously described. Contents of dopamine and its metabolites (DOPAC and HVA) were measured simultaneously in striatal homogenates by HPLC and electrochemical detection as described in chapter one. II. Experiment B ILL Animals and MPTP Exposure Young female littermate C57 BL/6 mice were obtained from Charles River Canada Inc. and were maintained as in the previous experiment. Ninety mice, 63-70 days old, were injected s.e. with the hydrochloride salt of MPTP dissolved in 0.9% NaCl at a dose of 20 mg/kg followed at 24 hours by a 40 mg/kg dose. During the following week 13 mice died, while the rest began to regain weight to a normal level. Sixteen littermates were spared MPTP treatment to use as normal controls and for breeding purposes as a source of fetal tissue for transplantation. Il.ii. Fetal Tissue Preparation The procedure outlined in the previous experiment was followed to obtain pregnant mice with known date of gestation. After 13-14 days of gestation the pregnant mice were sacrificed by cervical dislocation, the abdominal wall was swabbed with 70% ethanol. The skin was lifted and cut along the midline, the uterus containing the fetuses was removed to a Petri dish containing Hanks TABATABAEI, A. 46 balanced salt solution (HBSS) at room temperature, in which the fetuses (crown to rump 10-12 mm) were removed from the uterus and amniotic sac. The fetuses were transferred to a fresh bath and the brains were removed from the cranium under a dissecting microscope by means of two number five Dumont micro-surgery forceps (Fine Science Tools Inc.). The fetuses were held in place with one pair of forceps and skin, thin bone and other tissue covering the brain was removed by gently lifting the tissue from the brain. An attempt was made to remove as much meninges as possible. The brains were then dissected under a dissecting microscope by means of Vannas spring scissors and a number five Dumont micro-surgery forceps (Fine Science Tools Inc.). Ventral mesencephalic tissue was dissected out and transferred to a new bath. This tissue was minced into pieces of approximately 0.5 mm 3 using the Vannas spring scissors . Frontal pole tissue was also dissected out. Dissection was performed within 50 minutes from sacrifice of the mother. Frontal pole tissue was dissociated in 0.1% trypsin in HBSS as explained in the previous experiment. A 93% cell viability was obtained after frontal pole tissue dissociation and the volume of the final suspension was adjusted to obtain 6.8 x 107 cells /ml. The pieces of ventral mesencephalic tissue were microscopically examined in trypan and a viability of 75% was estimatedblue after the transplantation procedures were completed. II. iii. Neurotransplantation 37 days after the MPTP treatment, transplantation of 13-14 day fetal nervous tissue was performed stereotaxically. Animals were prepared as in the previous experiment and the stereotaxic procedure was performed accordingly. In this experiment, however, a 25 |il Hamilton syringe with a 22 gauge, blunt, stainless steel needle was used to graft 3-5 small pieces (approximately 0.5 mm3) of fetal ventral mesencephalon tissue suspended in HBSS into the left lateral ventricle (n=28) at the following coordinates, using bregma as a marker: 0.5 mm caudal, 1 mm lateral and 1.7 mm ventral to the dura. Using the same method, 28 mice received grafts into the left putamen ( 2.25 mm lateral, 0.2 mm rostral and 3.0 mm ventral to the dura). TABATABAEI, A. 47 V 20 X J MECHANICAL TISSUE DISSOCIATION STEREOTAXIC T R A N S P L A N T A T I O N Figure: 5 Schematic illustration depicting the techniques involved with the stereotaxic transplantation of fetal nervous tissue into the adult recipient (experiment "B"). TABATABAEI, A. 48 This tissue was transplanted into the appropriate target rapidly in a 1-2 ul bolus; the needle was checked after each injection to ensure that the tissue was ejected. The remaining mice received dissociated frontal pole cells (n=10) or a vehicle injection (n=ll) into the left lateral ventricle. Three p.1 of the prepared cell suspension (6.8 xlO 7 cell/ml) or HBSS were injected over a one-minute period by means of a 25 |il Hamilton syringe equipped with a 26 gauge, blunt, stainless steel needle. The needle was left in place for one minute after which it was slowly retracted (Fig. 5). We were not able to use dissociated ventral mesencephalic cells due to the formation of clumps trapping the cells upon trypsin treatment. The formation of these gel-like suspensions was attributed to excess remaining meninges or the failure to use DNA-ase in the mixture. After the transplantation period, the skin was sutured and animals were placed in heated recovery cages. All animals recovered well and appeared normal by the following day. A total of 77 mice were transplanted and allowed to survive for 66 days, at which time the mice were sacrificed by cervical dislocation. Brains were removed and dissected as were the brains in the previous experiment. Left hemispheres of 18 mice were fixed in 10% buffered formalin solution for histological studies. All specimens were coded for biochemical and histological studies which were performed blinded. Il.iv. Data Analysis Data were analyzed by means of unweighted means analysis of variance and subsequent Newman Keuls post-hoc test. 4.3. Results: Table IV presents the level of dopamine and its metabolites in the whole striatum of MPTP-treated animals which received transplantation of dissociated rostral, caudal or astrocyte tissue into the left lateral ventricle (experiment A). The regimen of MPTP used in this experiment led to a substantial reduction of striatal dopamine. None of the transplanted groups of animals showed any improvement of striatal DA levels when compared to the animals treated only with MPTP. TABATABAEI, A. 49 TABLE IV (EXPERIMENT A) Striatal Dopamine and Metabolites in MPTP-treated C57 BL/6 Mice Transplanted with Fetal Nervous Tissue Normal MPTP-Treated  Controls Vehicle Astrocytes Caudal Rostral Compound (n=9) (n=14) (n=14) (n=14) (n=12) Dopamine 16.64+0.37 * 1.91±0.18 1.94±0.12 1.7910.16 1.86±0.12 DOPAC 1.22±0.05 * 0.23±0.01 0.25±0.02 0.23±0.01 0.26±0.04 H V A 1.83±0.07 * 0.62±0.04 0.59±0.03 0.55±0.02 0.66±0.04 Values (mean ± SEM) expressed in U-g/g wet weight. * Significantly different from vehicle-injected-MPTP-treated mice, P<0.05. TABATABAEI, A. 50 TABLE V (EXPERIMENT B) Left and Right Striatal Dopamine and Metabolites in MPTP-treated C57 BL/6 Mice Transplanted with Fetal Nervous Tissue Compound Normal Controls (n=ll) MPTP-Treated Vehicle in LLV (n=9) Right Left Frontal pole in LLV (n=8) Right Left Dopamine 16.25 ± 0.33* 2.65 ± 0.37 3.07 ± 0.37 3.39 ± 0.39 3.53 ± 0.39 DOPAC 1.12 ±0 .03* 0.30 ±0 .03 0.33 ±0 .03 0.35 ±0 .04 0.36 ±0 .04 H V A 1.56 ±0 .05* 0.73 ±0 .05 0.77 ±0 .05 0.80 ± 0.05 0.84 ±0.05 Compound MPTP-Treated Mesencephalic Flexure in LLV (n=22) Right Left Mesencephalic Flexure in LP (n=21) Right Left Dopamine 2.86 ± 0.23 3.06 ± 0.23 3.02 ±0.24 2.99 ±0.24 DOPAC 0.02 H V A 0.32 ± 0.02 0.79 ± 0.03 0.32 ± 0.02 0.79 ± 0.03 0.34 ± 0.02 0.39 ± 0.72 ± 0.03 0.71 ± 0.03 Values (mean ± SEM) expressed in |ig/g wet weight. * Significantly different from mice receiving vehicle injection, P<0.05. LLV, Left Lateral Ventricle. LP, LeftPutamen. TABATABAEI, A. 51 Histological studies supported the biochemical findings. Tyrosine hydroxylase immunoreactivity of striatum from transplanted animals was not greater than that of the striatum of animals receiving MPTP treatment alone. Furthermore, no tyrosine hydroxylase-immunoreactive cell bodies were observed in the striatum, indicating that no catecholaminergic grafts had survived the recovery period. It was also apparent that not all transplants were placed in the lateral ventricle. Only 32% of transplants in the brains examined histologically were placed in the lateral ventricle or at the tip of the dorsal striatum, presumably in contact with CSF. Table V lists the levels of DA and its metabolites in the left and right striatum of mice transplanted in the second experiment designed to correct for possible problems of the original design (experiment B). The lower dose of MPTP used in this experiment also caused a substantial reduction of DA level bilaterally. No significant improvement was observed in any of transplanted groups when compared with the non-transplanted, MPTP-treated mice. Histological studies indicate correct placement of the transplant tissue at the intended site. 4.4. Discussion: C57 BL/6 mice were treated with MPTP. These animals showed symptoms of somnia and hypokinesia for up to 24 hours after each s.e. injection of MPTP; thereafter, surviving animals behaved normally. These behavioural observations are in agreement, with the observations of Perry et al. (1985) who also reported a substantial reduction of DA and its metabolites in the striatum of MPTP-treated mice as was observed in the present study (89% reduction of striatal dopamine levels). Histological studies of the transplanted animals did not provide any evidence for survival of graft tissue transplanted into the left lateral ventricle. Tyrosine hydroxylase immunoreactivity did not show any evidence of sprouting of the remaining host cells or of the implanted fetal tissue. Furthermore, not all the implants were placed in the lateral ventricle (only 32% of brains studied histologically indicated deposition in the lateral ventricle or surface of the caudate TABATABAEI, A. 52 in contact with CSF). The unsuccessful results of the primary experiment might be due to the low number of dopaminergic cells in the grafts, the age of the fetal tissue, the relatively long elapsed time between the sacrifice of the fetus and transplant or the high degree of membrane damage caused by trypsin and mechanical dissociation. Based on these observations, the coordinates of transplantation in the second set of experiments were adjusted and ventral mesencephalic flexure tissue of younger fetuses replaced the caudal portion of the brain used in the original design. Trypsin dissociation was eliminated and dissection of fetal tissue and transplantation was performed expeditiously. In this experiment, the striatal dopamine of MPTP-treated mice was reduced to approximately 18% of untreated animals (103 days after MPTP treatment). MPTP-treated mice received injections of vehicle, dissociated frontal pole cells, ventral mesencephalic flexure minced tissue into the left ventricle and ventral mesencephalic flexure minced tissue into the left putamen in randomly assigned groups of mice. The levels of dopamine and its metabolites in left (transplanted) and right striatum were statistically indistinguishable. Not only was no significant difference between the two hemispheres of the brain found, but no significant improvement was observed in comparison with the vehicle-injected animals. The 66 day recovery period was chosen to allow for the sprouting of the grafted tissue or the remaining dopaminergic cells of the host. An approximately 2 month delay post-transplant was observed before any signs of improvement were evident in a clinical trial (Lindvall et al., 1989). Transplants of fetal human dopaminergic cells into the striatum of DA-depleted rats also confirmed a 2-3 month delay for functional effects to become apparent. This time also coincides with the time required for growth and synapse formation of grafted cells (Brundin et al., 1988; Clarke et al„ 1988). However, this extended delay of sacrifice after MPTP-treatment may mask a possible boosting effect of transplant on recovery of the surviving TABATABAEI, A. 53 dopaminergic cells of the host animals in this study. The striatal dopamine level of MPTP-treated mice is known to increase spontaneously from 10% of normal one week after MPTP insult to 40% by 4.5 months post-treatment (Perry et al , 1985). It is possible that transplantation may lead to an increased rate of recovery of the surviving cells of substantia nigra; therefore, a shorter period of recovery might show a statistically significant improvement of striatal dopamine in comparison with the vehicle-injected group as reported by Bohn et al. (1990). In their study, adrenal medullary tissue was transplanted in the head of the caudate of MPTP-treated mice. Although the dopamine content of striatum was examined after MPTP-treatment, the striatum was not studied biochemically after the transplant. The tyrosine hydroxylase immunoreactive fibers are reported to be denser up to 6 weeks post-transplant. This recovery is also reported in mice receiving sham grafts; however, no supporting data were provided. These investigators concluded that some neurotrophic factors released due to the transplant procedure induced recovery and sprouting of the dopaminergic host cells. The mechanism by which transplanted adrenal medullary or fetal dopaminergic tissue improves parkinsonian symptoms (if this indeed occurs) is not clearly understood. The improvement might be due to a diffuse release of DA by the grafted cells in the striatum or CSF. This hypothesis is the suspected mechanism of action of adrenal medullary grafts. The grafted neurons may be capable of sprouting and forming synapses in the dopamine-depleted striatum. On the other hand, some neurotrophic factors released from the grafted tissue or the host animal itself might induce the recovery and sprouting of the remaining dopaminergic cells of the host. Of course, the placebo effect of such treatment in humans with a high degree of expectation of recovery should not be ignored. The results of various experiments are inconsistent and difficult to compare. Generally, the recovery observed with adrenal medullary tissue transplants has been transient and modest, if present at all. The validity of the observations of Madrazo TABATABAEI, A. 54 et al. (1987) has now been challenged (Lewin, 1988) due to the modest improvement observed by other investigators (Kelly et al., 1989; Zhang, 1989; Penn et al.. 1988; Lindvall et al., 1987; Backlund et al., 1985). The sustained survival of these transplanted cells has not been supported by autopsy findings in patients receiving the adrenal medullary implants (Peterson et al., 1989). Survival of such grafts in monkeys has also not been impressive; a modest survival of grafted chromaffin cells transplanted into the striatum of parkinsonian monkeys has been reported (Findaca et al., 1988; Morihisa et al., 1984). Transplantation of human fetal adrenal medullary tissue into the striata of MPTP-treated monkeys has also not been successful (Young et al., 1989). Implant sprouting and synapse formation has been demonstrated by implants of human nerve tissue into the striatum of rats. This outgrowth process took at least 2-3 months and generally did not appear until 4 to 5 months after the transplant. The idea of recovery due to a rich axonal growth of ventral mesencephalic flexure tissue implanted into the striatum of parkinsonian patient is not supported by the rapid recovery reported after the transplant of such tissue in Mexico and England (Madrazo et al., 1988; Hitchcok et al., 1988). The first report of recovery after stereotaxic transplantation of fetal dopaminergic cells (except the very dramatic improvements of Madrazo's patients) was reported by Lindval et al. (1989). The validity of clinical improvement reported by these investigators has been challenged based on the effect of the high expectations of the patients (Freed, 1990). Freed (1990) also argues that the observed improvements are not more impressive than the observations of Goetz et al. (1989) of patients transplanted with adrenal medullary tissue; the time-course of improvement is similar in both studies. In their original study Bohn et al. (1987) reported an increased tyrosine hydroxylase-immunoreactivity at six weeks after sham grafts of Gelfoam in MPTP-treated mice. This observation indicates that improvement might be due to a transplant-activated release of neurotrophic factors from the brain, inducing the TABATABAEI. A. 55 growth and sprouting of the remaining viable dopaminergic cells of the parkinsonian animals. In a later study, this observation was supported by transplantation of adult mice adrenal medulla, dead cells of adrenal medulla (cells were killed by repeated cycles of freezing and thawing), postnatal (day 7) adrenal medulla and sham grafts of Gelfoam. In this study, only the grafts of postnatal (day 7) adrenal medulla survived up to one month after the transplant; however, the three transplanted groups showed significant recovery of striatal dopamine level (Bohn and Kanuicki, 1990). This observation also suggests that a host response to the grafted tissue might lead to a release of neurotrophic factors causing the observed improvement. Other experiments studying the effects of neurotrophic factors also provide some evidence for involvement of nerve growth factor(s) (NGF) in such improvement. Pezzoli et al. (1988) reported an equivalent recovery of unilaterally 6-OHDA-denervated rats by transplants of sciatic nerve or fat (tissues which do not produce catecholamines) plus nerve growth factor and by transplants of adrenal medulla and nerve growth factor. The exogenous and endogenous release of neurotrophic factors seems to be the cause of recovery in these experiments. Furthermore, Date et al. (1990) reported a regenerating effect of acidic fibroblast growth factors (aFGF) injected stereotaxically into the striatum unilaterally (two days after MPTP-treatment followed by two other injections repeated five days apart) in the nigrostriatal dopaminergic system of MPTP-treated mice, and demonstrated a biochemical and histological improvement of young (2 month-old) mice. Similar neurotrophic effects of basic fibroblast growth factors (bFGF) in the striatum of MPTP-treated young mice have also been reported (Otto and Unsicker, 1989). More recently, Shults et al. (1991) transplanted 15 parkinsonian patients with autologous adrenal medulla tissue by means of Madrazo's surgical technique. Five to nine months after the transplant these patients generally showed modest improvement as assessed by a subjective scoring of various motor functions. These TABATABAEI, A. 56 investigators also measured chromogranin A (Cg A) in ventricular cerebrospinal fluid (VCSF), which is a soluble protein in chromaffin granules and which is released with catecholamines (Takiyyuddin et al., 1990), one week, three months and 5-9 months after surgery, as an indicator of graft survival and continued release of DA. There was no significant change of Cg A at any time compared with levels in Cg A of VCSF obtained at the time of surgery. These samples of CSF were examined for the presence of bFGF but none was detected. It was concluded that the transplanted medullary tissue did not survive or the surviving cells did not continue to release catecholamine by exocytosis. The release of bFGF from the transplanted tissue or the induction of release from the host cells was also refuted by these observations. Despite some indications of modest improvement in parkinsonian symptoms by such surgical procedures, the validity of adrenal medullary tissue or fetal dopaminergic nervous tissue transplantation as a clinical intervention in Parkinson's disease is not well established. The mechanism of action of any improvement (which is modest in most reported cases) is not clear. Following our experiments involving stereotaxic transplantation of fetal nervous tissue into the striatum of MPTP-treated C57 BL/6 mice, we have found that under the given conditions and in C57 BL/6 mice neurotransplantation does not increase the striatal DA levels. Although our finding does not attest to the effectiveness of this procedure, we do not reject neurotransplantation as a possible treatment of PD. However, more animal studies and long-term observation of present clinical trials are required before the validity of neurotransplantation as a clinical treatment is confirmed or refuted. TABATABAEI, A. 57 5. Concluding Remarks: We have explored a possible environmental cause of idiopathic PD as well as methods of prevention and treatment. We examined 3-AP, a possible environmental pyridine compound, for its neurotoxicity on the mesostriatal dopaminergic system in rats. Chronic administration of this naturally occurring compound led to a moderate but insignificant reduction in striatal dopamine and a marked degeneration of cerebellar neurons. Prophylactic use of nicotinamide prevented the striatal reduction of DA but not the cerebellar damage, indicating a possible difference in the mechanism of action of 3-AP at each site. Although it is unlikely that the severe behavioural abnormalities observed in 3-AP treated animals, but not the nicotinamide pretreated animals, are due to the modest reduction of striatal DA, it is possible that due to the different mechanism of action a very long-term low dose exposure to 3-AP could lead to more substantial nigral damage without severe cerebellar damage in rodents and possibly in humans. It is also possible that 3-AP causes severe damage to other structures in the brain which were not examined in this study and that these structures are protected by high doses of nicotinamide. The recent finding of a close relationship between dopaminergic and glutamatergic neurons reinforces the proposed association of E A A with the pathology of PD. We have examined the effects of N M D A receptor blockade on MPTP neurotoxicity. We have also examined a possible neuroprotective effect of nicotinamide against MPTP-induced DA reduction. Our findings suggest that the mechanism of neurotoxicity of MPTP involves N M D A receptor-ion channel complex and this effect can be substantially reduced by prophylactic MK-801 injection. Therefore, N M D A receptor blockade can be used not only as means of symptomatic treatment as proposed by other investigators, but also as a means of halting the progression of the disease at an early stage. Nonetheless, the wide range TABATABAEI, A. 58 of untoward effects of MK-801 and the relatively small amount of available data at this time precludes the clinical use of this drug as an antiparkinsonian treatment. We have observed a complete behavioural protection by nicotinamide against 3-AP treatment; furthermore, MPTP-treated mice receiving nicotinamide exhibited less behavioural abnormalities compared with the mice receiving MPTP alone. However, nicotinamide treatment did not prevent the reduction in striatal DA levels of mice treated with MPTP and there was only a very small reduction of striatal dopamine in rats treated with 3-AP (unlikely to produce such severe behavioural abnormalities) which was prevented by prophylactic use of nicotinamide. This finding may indicate the involvement of other pathways outside the DA pathway of basal ganglia. The possibility of striatonigral pathways containing substance P has been proposed as a possible alternative damaged structure in idiopathic PD (Barker, 1986). Finally, we have examined a new controversial approach to the treatment of parkinsonian patients. Transplantation of fetal dopaminergic neurons has been used with a varying degree of improvement in animals and humans. After two consecutive studies, we did not find any evidence of biochemical improvement in transplanted mice regardless of the nature of the transplanted materials. 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