Unilateral Injection Of 6-OHDA Research Articles

Two praxiological methods in evaluation the substantia nigra compacta lesion size of rats

目的 观察黑质多巴胺能(DA)神经元毁损大鼠行为学和形态学变化特点,探讨两者之间的相关性,寻找一种能敏感反应DA毁损程度的行为学指标.方法利用6-羟基多巴胺(6-OHDA)单侧一点注射大鼠黑质致密区(SNc),特异毁损DA能神经元,采用开野实验和阿朴吗啡(APO)诱导的旋转实验观察术后不同时间点行为学变化;利用Nissl染色、HE染色、免疫组织化学方法,观察各时间点黑质形态学变化.结果术后1d约50%黑质DA神经元丢失,21d毁损比近90%;开野实验中旋转、探究、后肢站立和穿梭行为在术后1d即有显著改变,与毁损程度有良好的相关性(r分别为0.471、-0.719、-0.589、-0.594,P＜0.01),在3 d、5 d时有恢复倾向,修饰行为与毁损程度无相关性(r=-0.227,P＞0.05).术后7d大鼠出现APO诱导旋转行为,14d转速＞7圈/min,与毁损程度呈正相关(r=0.942,P＜0.01),没有改善迹象.结论开野实验可作为评价黑质毁损程度的敏感行为学指标。

The role of TNF in in vitro and in vivo models of neurodegeneration

Using our novel engineered dominant negative Tumor Necrosis Factor variants (DN-TNFs) in models of Parkinson’s Disease (PD) and Alzheimer’s Disease (AD), we are identifying TNF-dependent signaling mechanisms that elicit death of DA neurons as well as TNF-dependent microglial activities that exacerbate amyloid beta-associated neurotoxicity and death of cholinergic neurons. In mouse models of AD, peripheral inflammation (LPS) exacerbates amyloid and tau pathology in hippocampus; we investigated whether blocking soluble TNF signaling with the infusion of DN-TNFs could block the progressive neuropathology or ameliorate the LPS effects. Inhibition of TNF signaling in vitro with DN-TNFs attenuates LPS- induced microglia activation and loss of DA neurons; delayed TNF inhibition still rescues DA neurons and provides more robust rescue than anti-oxidant pretreatment. Experiments are underway to investigate which signaling pathways transduce the neuroinflammatory and neurotoxic effects of TNF. In vivo, unilateral intrastriatal injections of 6-OHDA or intranigral chronic low dose LPS infusion result in a 70–80% loss of ipsilateral nigral DA neurons. Co-administration of DN-TNFs on the lesion side reduces neuronal loss by half and attenuates ipsiversive circling behavior. These results suggest that targeting the TNF pathway may be a reasonable therapeutic approach to halt or slow the progressive loss of DA neurons in PD. Funding by MJ Fox Foundation, American Health Assistance Foundation, and The University of Texas Southwestern Medical Center at Dallas Alzheimer’s Disease Center; NIH,NIA P30AG12300.

6‐Hydroxydopamine‐induced alterations in blood–brain barrier permeability

Vascular inflammation is well known for its ability to compromise the function of the blood--brain barrier (BBB). Whether inflammation on the parenchymal side of the barrier, such as that associated with Parkinson's-like dopamine (DA) neuron lesions, similarly disrupts BBB function, is unknown. We assessed BBB integrity by examining the leakage of FITC-labeled albumin or horseradish peroxidase from the vasculature into parenchyma in animals exposed to the DA neurotoxin 6-hydroxydopamine (6OHDA). Unilateral injections of 6OHDA into the striatum or the medial forebrain bundle produced increased leakage in the ipsilateral substantia nigra and striatum 10 and 34 days following 6OHDA. Microglia were markedly activated and DA neurons were reduced by the lesions. The areas of BBB leakage were associated with increased expression of P-glycoprotein and beta 3-integrin expression suggesting, respectively, a compensatory response to inflammation and possible angiogenesis. Behavioural studies revealed that domperidone, a DA antagonist that normally does not cross the BBB, attenuated apomorphine-induced stereotypic behaviour in animals with 6OHDA lesions. This suggests that drugs which normally have no effect in brain can enter following Parkinson-like lesions. These data suggest that the events associated with DA neuron loss compromise BBB function.

Reactive oxidative and nitrogen species in the nigrostriatal system following striatal 6-hydroxydopamine lesion in rats

Oxidative stress is a major contributing factor in the pathogenesis of Parkinson's disease. We therefore investigated the effect of the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on hydroxyl-free radical and peroxynitrite formation in the intrastriatal 6-OHDA rat model of Parkinson's disease. The hydroxylation product of salicylate (2,3-dihydroxy-benzoic acid) as well as the hydroxylation and nitration products of d-phenylalanine (2- and 3-hydroxyl-phenylalanine, nitrotyrosine and nitrophenylalanine) were assessed in tissue samples of the striatum and, for the first time, the substantia nigra of adult rats at four different time points (25 min, 2 h, 4 h and 7 days) after unilateral stereotaxic intrastriatal injection of 6-OHDA. In the striatum, maxima of hydroxylating and nitrating markers were found at early time points after 6-OHDA lesion. These results suggest a direct interrelation between 6-OHDA-autoxidation and/or the increased dopamine turnover and hydroxyl-free radical and peroxynitrite formation. In the substantia nigra, i.e., at a distance from the injection site of the neurotoxin, an increase in hydroxyl-free radical formation was observed at 7 days after 6-OHDA lesion, with this modification possibly being independent of 6-OHDA autoxidation and rather representing a long-term effect of the toxin. Furthermore, we conclude that apart from the formation of reactive oxygen species, the production of reactive nitrogen species occurs in this experimental Parkinson's disease model. Finally, the similarity between the 6-OHDA model and Parkinson's disease supports the notion that reactive oxygen species as well as reactive nitrogen species may play an important role in the pathogenesis of this neurodegenerative disorder.

A proteomic approach in the study of an animal model of Parkinson's disease

Background The aetiology of Parkinson's disease (PD), an age-related disorder characterized by a progressive degeneration of dopaminergic neurons of the substantia nigra (SN) pars compacta, remains unclear. Current treatments, such as administration of L-DOPA, are only symptomatic and do not stop or delay the progressive loss of neurons. In fact, it has been suggested that the dopamine precursor L-DOPA, increases generation of reactive oxygen species (ROS) leading to further neuronal damage. A similar loss in nigrostriatal dopaminergic neurons is produced on intracerebral administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). In an animal model of PD, termed ‘the hemiparkinsonian rat’, unilateral injection of 6-OHDA into the nigrostriatal pathway results in extensive loss of dopaminergic cells in the ipsolateral SN. In an attempt to identify some of the proteins that are involved in dopaminergic neuronal death, we used the proteomic methods to analyze this animal model of PD. Methods Five hemiparkinsonian rats were obtained by intranigral stereotaxic injection of 6-OHDA.The right 6-OHDA-lesioned substantia nigra and striatum tissues along with the left, unlesioned controlateral tissues, were excised and homogenized, using urea-based buffer, to extract the tissues protein. The separation of the protein mixtures and the visualization of the protein patterns obtained were performed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein profiles of control and treated tissues were compare by the PDQuest 2D-gel analysis software (BIO-Rad laboratory). The protein spots showing differential expression were analysed by matrix assisted laser desorption/ionizing time of flight (MALDI-TOF) mass spectrometry. Results The brain protein extraction and solubilization protocol was validated obtaining a satisfactory protein profile. In comparison to the normal rats, hemiparkinsonian animals exhibited a different expression in α-enolase and β-actin in substantia nigra and striatum, respectively. Conclusion The proteomic study of 6-OHDA-induced lesions in the nigrostriatial pathway allowed us to identify two proteins, α-enolase and β-actin, showing increased levels in the 6-OHDA-lesioned brain tissues compared to control. Previous studies described the same proteins as oxidized and proteins in Alzheimer's disease (AD) brain. Our preliminary data could mirror those results pointing out a common mechanism of neurodegenerative diseases.

Effect of intrastriatal 6-OHDA lesion on dopaminergic innervation of the rat cortex and globus pallidus

The present study examined in the rat the effect of a partial lesion of the nigrostriatal dopaminergic pathway induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA), on the dopaminergic innervation of the cortex and the globus pallidus as revealed using tyrosine hydroxylase (TH) immunoreactivity. Twenty-eight days after unilateral injection of 6-OHDA into the dorsal part of the striatum, TH-positive fiber density was reduced by 41% in the dorsal and central part of the structure, and was accompanied by a retrograde loss of 33% of TH-positive neurons in the substantia nigra (SN), while the ventral tegmental area was completely spared. In the SN, TH-positive cell loss was most severe in the ventral part of the structure (−55%). In the same animals, a substantial loss of TH-positive fibers was evident in the dorsal part of the globus pallidus, and involved both thick fibers of passage and thin varicose terminal axonal branches. In the cortex, a loss of TH-positive fibers was prominent in the cingulate area, moderate in the motor area and less affected in the insular area, while the noradrenergic innervation revealed using dopamine-β-hydroxylase immunoreactivity was preserved in all of these cortical subregions. These results demonstrate that the intrastriatal 6-OHDA lesion model in rats produces a significant loss of dopaminergic axons in extrastriatal structures including the pallidum and cortex, which may contribute to functional sequelae in this animal model of Parkinson's disease.

Bilateral Increase of Perforated Synapses after Unilateral Dopamine Depletion

In recent years attention has been focused on perforated synapses considering their possible involvement in synaptic plasticity in the nervous system. It has been hypothesized that an increase in the number of synapses may represent a structural basis for the enduring expression of synaptic plasticity during some events that involve memory and learning; also it has been suggested that perforated synapses increase in number after some experimental situations. The aim of this study was to analyze whether the dopamine depletion produces changes in the synaptology of the corpus striatum of rats after the unilateral injection of 6-OHDA. The findings suggest that after the lesion, both contralateral and ipsilateral striata present a significant increment in the number of perforated synapses, suggesting brain plasticity that might be an intent to recuperate the contact surface lost after endogenous or exogenous aggressions.

Long-term restoration of nigrostriatal system function by implanting GDNF genetically modified fibroblasts in a rat model of Parkinson's disease.

The motor behavior and levels of dopamine and its metabolites in the striatum were studied in rats that received a unilateral injection of 6-OHDA and underwent grafting of rat-derived primary fibroblasts that had been genetically modified to express lacZ and human glial cell line-derived neurotrophic factor (GDNF). Rotation behavior tests were performed each week and striatal levels of DA and its metabolites were measured every 4 weeks after grafting of fibroblasts that expressed lacZ, with or without additional transfection of the GDNF transgene. Rats grafted with GDNF-producing fibroblasts showed a significant improvement in motor behavior as determined by the rotation test, with a less pronounced reduction in the levels of dopamine and its metabolites in the striatum as compared with those in the control animals or brain parts. In addition, there was a lower decrease in the number of TH immunoreactive neurons in the substantia nigra ipsilateral to the lesion in rats with GDNF-producing fibroblasts than in rats with lacZ-expressing fibroblasts. These results support the notion that intracerebral grafting of fibroblasts that express GDNF is a potentially useful therapeutic strategy for treating Parkinson's disease.

Neuroprotective effect of Ginkgo biloba L. extract in a rat model of Parkinson's disease.

The neuroprotective effects of a standardized extract of Ginkgo biloba L. (EGb 761) were investigated on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in the nigrostriatal dopaminergic system of the rat brain. Rats were given a week of pretreatment with daily administrations of EGb 761. Unilateral striatal injection of 6-OHDA was followed by treatment with EGb 761 for a week. Serial measurement of contralateral forepaw adjusting steps revealed a progressive deficit in motor activity. At 8 weeks after 6-OHDA lesion the number of contralateral forepaw adjusting steps was significantly higher in rats that were treated with high doses of EGb 761 (100 mg/kg daily) than in those treated with low doses (50 mg/kg) or with the vehicle. Dopamine neuron loss in the substantia nigra and a depletion in striatal dopamine corresponded with behavioural deficit. These data suggest that the neuroprotective effects of EGb 761 reduce the behavioural deficit in 6-OHDA lesions in rat and also indicates a possible role for the extract in the treatment of Parkinson's disease.

Voluntary running provides neuroprotection in rats after 6-hydroxydopamine injection into the medial forebrain bundle.

Neurotoxic drugs such as 6-hydroxydopamine (6-OHDA) have been used to mimic a Parkinsonian state in a rat model. The toxic effect of 6-OHDA has been shown to be reduced in rats that were forced to use the impaired limb immediately after unilateral 6-OHDA injection. The aim of this study was to determine whether dopamine neurons in the substantia nigra are spared in rats that exercise voluntary. Two groups of rats were placed in cages with attached running wheels 7 days prior to injection of 6-OHDA (10 microg/4 microL saline) into the medial forebrain bundle. The running wheels of the control group were immobilized for the duration of the study. After 6-OHDA injection, the rats were returned to their respective cages where they remained for a further period of 14 days. Wheel revolutions during free running were recorded daily in the experimental group. At the end of this period the rats were injected with apomorphine (0.5 mg/kg, s.c.) and the number and direction of rotations was recorded. Rats that exercised in the running wheels did not rotate contralaterally in response to apomorphine injection, suggesting that dopamine neurons had been spared sufficiently from the toxic effects of 6-OHDA injection to prevent upregulation of postsynaptic dopamine receptors in the striatum.

NMDA receptor antagonists ameliorate the stepping deficits produced by unilateral medial forebrain bundle injections of 6-OHDA in rats.

To test the hypothesis that excess glutamatergic transmission at NMDA receptors may contribute to the pathogenesis of Parkinson's disease (PD), we examined the effects of various NMDA receptor antagonists on a recently developed rat model of PD. Following unilateral injections of 12 microg 6-OHDA into the medial forebrain bundle of male Long Evans rats, stepping with both front paws was measured separately as the paws were dragged backwards and laterally. The effects of i.p. injections of varying doses of L-dopa, the non-competitive NMDA receptor antagonist dizocilpine [(+)-MK-801], the competitive NMDA receptor antagonist CPP, and combinations of L-dopa and NMDA receptor antagonists were then examined on stepping in three separate groups of rats. The lesioned rats stepped less often with their contralateral paw than with their ipsilateral paw, and the magnitude of this stepping deficit was positively correlated with the amount of DA depletion in the ipsilateral dorsal striatum. L-dopa (1-25 mg/kg) dose dependently enhanced stepping with the contralateral paw, and 0.15-0.3 mg/kg dizocilpine and 1.5-6.25 mg/kg CPP enhanced stepping with the contralateral paw as much as did 8 mg/kg L-dopa. The combinations of L-dopa and each of the NMDA receptor antagonists did not significantly improve stepping more than either drug alone. Moreover, none of the drugs completely eliminated the stepping deficits, and high doses began to impair stepping with the ipsilateral paw by inducing turning. These data indicate that deficits in contralateral stepping are a reliable and sensitive measure of akinesia in unilateral 6-OHDA-lesioned rats, and they support the hypothesis that excess glutamatergic transmission at NMDA receptors may play a role in the expression of PD symptomology.

Dopaminergic mechanisms underlying bladder hyperactivity in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway.

1. This study was undertaken to elucidate dopaminergic mechanisms underlying bladder hyperactivity in a rat model of Parkinson's disease (PD) induced by a unilateral 6-OHDA injection into the substantia nigra pars compacta. 2. In 6-OHDA-lesioned rats, voided volume per micturition (0.41+/-0.04 ml, mean+/-s.e.m.) measured during 24 h in a metabolic cage was significantly smaller than in sham-operated rats (0.67+/-0.07 ml). 3. Cystrometrograms (CMG) in conscious animals revealed significantly smaller bladder capacity (BC) (0.46+/-0.03 ml) in 6-OHDA-lesioned rats than in sham rats (0.72+/-0.06 ml). 4. SKF38393 (D1/D5 receptor agonist, i.v.) significantly increased BC in 6-OHDA rats without apparent effects in sham rats. SKF38393 applied intracerebroventricularly (i.c.v.) under urethane anesthesia also increased BC in 6-OHDA-lesioned rats and by a smaller increment in sham rats. 5. In contrast, quinpirole (D2/D3/D4 receptor agonist, i.v.) significantly reduced BC in sham and 6-OHDA-lesioned rats. Intrathecal injection of quinpirole similarly reduced BC in sham and 6-OHDA-lesioned rats. 6. PD128907 (D(3)-receptor agonist) did not have significant effects on BC in 6-OHDA-lesioned rats. 7. These results indicate that a rat model of PD exhibited bladder hyperactivity as observed in patients with PD, and that stimulation of D1/D5 dopamine receptors at a supraspinal site can suppress bladder hyperactivity in PD, whereas stimulation of D2/D4, but not D3, dopamine receptors had the opposite effect to reduce bladder capacity. Thus, D1/D5 dopamine receptor agonists might be effective in treating neurogenic bladder hyperactivity in PD.

Sensorimotor deficits in a unilateral intrastriatal 6-OHDA partial lesion model of Parkinson’s disease in marmoset monkeys

Animal studies investigating the efficacy of neurotrophic factors as treatments for Parkinson’s disease (PD) ideally require partial dopamine (DA) lesion models. The intrastriatal 6-hydroxydopamine (6-OHDA) lesion model may be suitable for this purpose. Although this model has been well characterized in rodents, it has not previously been used in monkeys. The goal of the present study was to characterize the behavioral effects of unilateral injections of 6-OHDA in the basal ganglia of common marmoset monkeys ( Callithrix jacchus). Cell counts from tyrosine hydroxylase immunochemistry 5 months postlesion revealed DA cell loss in the substantia nigra on the lesioned side to ∼46% of relative to the unlesioned side. 6-OHDA lesioned monkeys showed a variety of behavioral deficits. Apomorphine induced rotation and simple sensorimotor measures (head position bias and PD disability rating score) were most affected by the lesion. The largest deficits were seen at 1 or 2 weeks postsurgery but had recovered by week 10. 6-OHDA lesioned monkeys took longer to complete a more complex sensorimotor staircase task. At 3.5 months postlesion, 6-OHDA monkeys also showed deficits on an object retrieval task designed to measure sensorimotor planning and skilled hand use. α-Methyl- p-tyrosine, a tyrosine hydroxylase inhibitor, reinstated those deficits which had undergone recovery in the lesioned animals and also exacerbated the deficits on the staircase task. This model has potential in assessing treatments for PD aimed at curtailing disease progression such as continuous delivery of neurotrophic factors.

Glial Cell Line-Derived Neurotrophic Factor (GDNF) Gene Delivery Protects Dopaminergic Terminals from Degeneration

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for β-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [125I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 α-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.

Comparison of unilateral and bilateral intrastriatal 6-hydroxydopamine-induced axon terminal lesions: evidence for interhemispheric functional coupling of the two nigrostriatal pathways.

Partial lesions of the nigrostriatal dopamine system can be induced reliably by the intrastriatal injection of 6-hydroxydopamine (6-OHDA) and are considered to be analogous to the early stages of human Parkinson's disease. Previous studies have established a clear correlation between different doses and placements of the 6-OHDA toxin and the degree of neurodegenerative changes and behavioral impairments. In the present study, the influence of the interdependence between the two nigrostriatal systems in both hemispheres on the effects on sensorimotor behavioral performances after terminal 6-OHDA lesions was investigated. The behavioral effects were correlated to the extent of nigral dopamine neuron cell and striatal tyrosine-hydroxylase (TH)-positive fiber loss. Sprague-Dawley rats receiving unilateral intrastriatal 6-OHDA injections (4 x 5 microg) exhibited a 30-70% reduction in striatal TH-positive fiber density along an anterior-posterior gradient, an 80% loss of nigral dopamine neurons and a mild degree of behavioral impairments as revealed by amphetamine-induced rotational asymmetry, and a reduced performance in the stepping and postural balance tests. When the same amount of toxin was injected twice into both hemispheres (2 x 4 x 5 microg), additional behavioral deficits were observed, consisting of a significant, but temporary, weight loss, a stable reduction in general locomotor activity and explorational behavior, and a long-term deficit in skilled forelimb use. This is interesting in light of the morphological findings, in which uni- and bilaterally lesioned animals did not differ significantly in the extent of TH-immunoreactive fiber and dopamine neuron loss within the nigrostriatal system in each lesioned hemisphere. These results indicate that the interdependent regulation of the two nigrostriatal systems may provide some compensatory support for the function and behavioral performance of the lesioned side via the normal unlesioned side, which is lost in animals with bilateral lesions of the nigrostriatal system. Therefore, this model of uni- and bilateral partial lesions of the nigrostriatal system, as characterized in the present study, may foster further exploration of compensatory functional mechanisms active in the early stages of Parkinson's disease and promote development of novel neuroprotective and restorative strategies.

Time course of degenerative alterations in nigral dopaminergic neurons following a 6-hydroxydopamine lesion.

The neurotoxin 6-hydroxydopamine (6-OHDA) has been used extensively in animal models of Parkinson's disease. Typically, rodents develop severe unilateral movement deficiencies coupled with apomorphine-induced rotation behavior at least 1 week after an ipsilateral 6-OHDA lesion of the nigrostriatal dopamine (DA) system. The short-term morphological effects of 6-OHDA have not been determined in detail, however, and the exact process by which neurons die has not been elucidated. Thus, novel degenerative markers were used to determine the temporal pattern of acute phenotypic and degenerative alterations following a unilateral 6-OHDA injection into the medial forebrain bundle of adult rats. 6-Hydroxydopamine administration resulted in an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining as early as 6 hours postlesion. Staining for FluoroJade, a marker of neuronal degeneration, was evident at all time points examined but was maximal at 48 hours. Loss of tyrosine hydroxylase (TH) immunoreactivity began in axons at 6 hours, and progressed to cell bodies at later time points postlesion. Morphological examination of these neurons supported the conclusion of their death via apoptosis. Thus, whereas behavioral manifestations typically become evident 1 week or more following a 6-OHDA lesion, it is evident that nigral cell degeneration begins much earlier. This suggests multiple therapeutic possibilities, including the prevention of apoptosis, in affected neurons.

Early behavioral changes after nigro-striatal system damage can serve as predictors of striatal dopamine depletion

1. 1. Rats which had received a unilateral injection of 6-OHDA into the substantia nigra were assigned to four lesion groups according to the degree of DA depletion in the neostriatum. In these animals, behavioral changes in the open-field were investigated during the first post-operative week. Overall, this analysis showed that the animals could adequately be characterized by behavior on day 1 and day 7 after lesion. 2. 2. On the first day after lesion, the groups with the severest DA depletions ( > 80% and 55–80%) showed an ipsilateral asymmetry in turning. After one week, these groups showed a tendency to recover from this deficit; however, the group with the most strongest lesions ( > 80%) was still asymmetric. 3. 3. In scanning behavior, in contrast to turning, all the lesion groups displayed an initial ipsilateral asymmetry. On day 7 after lesion, only the group with DA depletions of > 80% still had an ipsilateral asymmetry. Locomotor activity and rearing were initially reduced after lesion, and showed a tendency to recover, especially in the group with the most severe DA depletions ( > 80%). There were no differences between groups neither on day 1 nor on day 7 by grooming, but this behavior increased in all the lesion groups with time. 4. 4. The correlational analyses yielded a positive relationship between the asymmetry in turning and neostriatal DA depletion. Locomotor activity and rearing on day 1 were both negatively correlated with DA depletion. The present results show that a number of behavioral parameters obtained in the open-field are affected by unilateral lesions of the nigro-striatal DA system. The degree of deficit, its time course and relation to lesion size differs among the various behavioral measures. Some of these early behavioral changes after unilateral nigrostriatal lesion are related to DA depletion and should therefore be useful to predict lesion size. 5. 5. Together, these data suggest that the study of such behavioral changes can provide an important tool, to investigate the compensatory mechanisms underlying striatal DA depletion and to understand preclinical states of the Parkinson's disease.

Behavioral and cellular protection of rat dopaminergic neurons by an adenoviral vector encoding glial cell line-derived neurotrophic factor.

Previously, we observed that an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF), injected near the rat substantia nigra (SN), protects SN dopaminergic (DA) neuronal soma from 6-hydroxydopamine (6-OHDA)-induced degeneration. In the present study, the effects of Ad GDNF injected into the striatum, the site of DA nerve terminals, were assessed in the same lesion model. So that effects on cell survival could be assessed without relying on DA phenotypic markers, fluorogold (FG) was infused bilaterally into striatae to retrogradely label DA neurons. Ad GDNF or control treatment (Ad mGDNF, encoding a deletion mutant GDNF, Ad lacZ, vehicle, or no injection) was injected unilaterally into the striatum near one FG site. Progressive degeneration of DA neurons was initiated 7 days later by unilateral injection of 6-OHDA at this FG site. At 42 days after 6-OHDA, Ad GDNF prevented the death of 40% of susceptible DA neurons that projected to the lesion site. Ad GDNF prevented the development of behavioral asymmetries which depend on striatal dopamine, including limb use asymmetries during spontaneous movements along vertical surfaces and amphetamine-induced rotation. Both behavioral asymmetries were exhibited by control-treated, lesioned rats. Interestingly, these behavioral protections occurred in the absence of an increase in the density of DA nerve fibers in the striatum of Ad GDNF-treated rats. ELISA measurements of transgene proteins showed that nanogram quantities of GDNF and lacZ transgene were present in the striatum for 7 weeks, and picogram quantities of GDNF in the SN due to retrograde transport of vector and/or transgene protein. These studies demonstrate that Ad GDNF can sustain increased levels of biosynthesized GDNF in the terminal region of DA neurons for at least 7 weeks and that this GDNF slows the degeneration of DA neurons and prevents the appearance of dopamine dependent motor asymmetries in a rat model of Parkinson's disease (PD). GDNF gene therapy targeted to the striatum, a more surgically accessible site than the SN, may be clinically applicable to humans with PD.

6-hydroxydopamine lesion of rat nigrostriatal dopaminergic neurons differentially affects nicotinic acetylcholine receptor subunit mRNA expression.

Nicotinic acetylcholine receptor (nAChR) subunit mRNA expression in the rat substantia nigra (SN) was assayed by semiquantitative RT-PCR following 6-hydroxydopamine (6-OHDA) lesion of nigrostriatal dopaminergic neurons. Six months after unilateral injection of 6-OHDA or saline into the SN, total RNA was isolated from ipsilateral and contralateral tissue samples. RT-PCR amplifications were performed with template titration using primers specific for sequences encoding 1. nAChR alpha 2-alpha 7 and beta 2-beta 4 subunits 2. Glutamic acid decarboxylase 3. Glyceraldehyde 3-phosphate dehydrogenase for normalization of template mass. PCR products specific for alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, beta 2, beta 3, and glutamic acid decarboxylase were detected in the reactions containing SN RNA. This is the first evidence that alpha 7 may be expressed in the SN. alpha 2 and beta 4 PCR products were not detected in SN reactions, although they were observed in hippocampus and thalamus control reactions. A comparison of ipsilateral and contralateral SN RT-PCR reaction products showed substantial decreases in alpha 5, alpha 6, and beta 3 product yields following 6-OHDA, but not sham treatment. Neither the SN of sham-lesioned rats nor the thalamus of 6-OHDA-lesioned rats yielded similar results, indicating that the effects observed in 6-OHDA-treated SN were not caused by local mechanical damage or a nonspecific response, respectively. Effects of 6-OHDA treatment on alpha 3, alpha 4, alpha 7, beta 2, or glutamic acid decarboxylase product yields from SN samples were small or undetectable. The results suggest that alpha 5, alpha 6, and beta 3 subunit-encoding mRNAs are expressed at substantially higher levels in dopaminergic than in nondopaminergic cell bodies in the SN.

Long-term protection of the rat nigrostriatal dopaminergic system by glial cell line-derived neurotrophic factor against 6-hydroxydopamine in vivo.

Glial cell-line-derived neurotrophic factor (GDNF) has been shown to enhance the survival of dopaminergic neurones both in vitro and in vivo, and to protect the rodent dopaminergic system from neurotoxic damage. However, most previous studies have only examined the short-term protective effects of GDNF. We have investigated the long-term effects of GDNF on a 6-hydroxydopamine (6-OHDA)-induced lesion of the rat medial forebrain bundle (MFB), which results in complete and irreversible destruction of the nigrostriatal pathway, and is a robust model of Parkinson's disease. GDNF was administered ipsilaterally above the substantia nigra and into the lateral ventricle immediately before a unilateral 6-OHDA injection into the MFB. The effects of GDNF were examined in vivo by behavioural testing and positron emission tomography (PET) at weekly intervals, for 12 weeks. GDNF prevented the development of amphetamine-induced rotations at all time-points. PET studies, using [11C]-RTI-121 as a tracer for the dopamine transporter, indicated that GDNF prevented 6-OHDA-induced reduction of dopamine reuptake sites in the ipsilateral striatum. Post-mortem neurochemical analysis at 13 weeks after surgery found that GDNF significantly inhibited 6-OHDA-induced loss of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the ipsilateral striatum. Immunocytochemistry showed that GDNF reduced 6-OHDA-induced loss of tyrosine hydroxylase-positive neurones in both the substantia nigra pars compacta and ventral tegmental area. We have shown that a single treatment with GDNF can confer long-term protective effects against a 6-OHDA lesion, which suggests that this factor may be useful for the treatment of Parkinson's disease.