Dopaminergic neuroprotection and regeneration by neurturin assessed by using behavioral,biochemical and histochemical measurements in a model of progressive Parkinson's disease

Trophic effects of neurturin, a member of the glial cell line-derived neurotrophic factor-family, have been demonstrated on mesencephalic dopaminergic neurons, suggesting its therapeutic potential for Parkinson's disease. This study was designed to test the neuroprotective and regenerative effects of an intrastriatal injection of neurturin based on behavioral, neurochemical and histochemical changes in a rat model of progressive Parkinson's disease. An extensive and progressive dopaminergic lesion was unilaterally made by intrastriatal convection-enhanced delivery of 6-hydroxydopamine (6-OHDA), in which 20 microg of 6-OHDA dissolved in 20 microl of vehicle was infused at a rate of 0.2 microl/min. For neuroprotection study, recombinant human neurturin (5 microg in 5 microl of vehicle) was stereotaxically injected into the unilateral striatum. The 6-OHDA lesion was made on the ipsilateral side 3 days after the neurturin treatment. Tyrosine hydroxylase (TH)-immunoreactive neurons of the substantia nigra were protected from progressive degeneration in the neurturin-treated animals compared with the vehicle-treated animals 2 and 8 weeks after the 6-OHDA lesion. Eight weeks after the 6-OHDA lesion, dopamine concentration significantly increased in the striatum of neurturin-treated animals with improvement of methamphetamine-induced rotation behavior. For neuroregeneration study, 5 microg of neurturin was injected into the striatum 12 weeks after the 6-OHDA lesion. Four weeks after neurturin or vehicle injection, there were no significant differences in the survival of nigral TH-immunoreactive neurons between the groups. However, TH-immunoreactive fibers were thicker and more abundant in the striatum of the neurturin-treated rats compared to those of the control group, suggesting neurturin-induced growth of the dopaminergic axons. Striatal dopamine levels also significantly increased in the neurturin-treated rats compared with those in the control group of rats, accompanied by the recovery of methamphetamine-induced rotation in the neurturin-treated rats. In conclusion, an intrastriatal injection of neurturin is a useful method to protect nigral dopaminergic neurons from extensive cell death in a model of progressive Parkinson's disease, as well as to promote the axonal regeneration and dopaminergic function.     

Delivery of a GDNF gene into the substantia nigra after a progressive 6-OHDA lesion maintains functional nigrostriatal connections

The effects of delivering GDNF via an adenoviral vector (AdGDNF) 1 week after lesioning dopaminergic neurons in the rat substantia nigra (SN) with 6-hydroxydopamine (6-OHDA) were examined. Rats were unilaterally lesioned by injection of 6-OHDA into the striatum, resulting in progressive degeneration of dopaminergic neurons in the SN. One week later, when substantial damage had already occurred, AdGDNF or a control vector harboring beta-galactosidase (AdLacZ) was injected into either the striatum or SN (3.2 x 10(7) PFU/microl in 2 microl). Rats were examined behaviorally with the amphetamine-induced rotation test and for forelimb use for weight-bearing movements. On day 30 postlesion, the extent of nigrostriatal tract degeneration was determined by injecting a retrograde tracer (FluoroGold) bilaterally into the lesioned striatum. Five days later, rats were sacrificed within 2 h of amphetamine injection to examine amphetamine-induced Fos expression in the striatum, a measure of dopaminergic-dependent function in target neurons. AdGDNF injection in the SN rescued dopaminergic neurons in the SN and increased the number of dopaminergic neurons that maintained a connection to the striatum, compared to rats injected with AdLacZ. Further support that these spared SN cells maintained functional connections to the striatum was evidenced by increased Fos expression in striatal target neurons and a decrease in amphetamine-induced rotation. In contrast to the effects observed in rats injected with AdGDNF in the SN, rats injected with AdGDNF in the striatum did not exhibit significant ameliorative effects. This study demonstrates that experimentally increasing levels of GDNF biosynthesis near the dopaminergic neuronal soma is effective in protecting the survival of these neurons and their function even when therapy is begun after 6-OHDA-induced degeneration has commenced. Thus, GDNF gene therapy may ameliorate the consequences of Parkinson's disease through rescuing compromised dopaminergic neurons.     

Subthalamic Nucleus Lesion in Rats Prevents Dopaminergic Nigral Neuron Degeneration After Striatal 6-OHDA Injection: Behavioural and Immunohistochemical Studies

Several studies have shown that antagonists of N -methyl-D-aspartate receptors provide protection of the dopaminergic nigrostriatal pathway in animal models of Parkinson's disease. Since the substantia nigra compacta receives a moderate glutamatergic innervation from the subthalamic nucleus, we tried to determine whether subthalamic nucleus lesion could prevent the toxicity of the selective dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Experiments were carried out on four groups of rats. Group 1 ( n = 10) received a unilateral injection of 6-hydroxydopamine in the striatum and group 2 ( n = 10) received kainic acid in the subthalamic nucleus. Group 3 ( n = 10) received an injection of kainic acid in the subthalamic nucleus and 1 week later an injection of 6-OHDA in the striatum. Group 4 ( n = 5) received the same treatment but kainic acid was replaced by saline. Apomorphine induced an ipsilateral rotation in rats of groups 2 and 3 and a contralateral rotation in rats of groups 1 and 4. The number of tyrosine hydroxylase-immunoreactive cells in the pars compacta of the substantia nigra was not significantly different between injected and non-injected sides in rats of groups 2 and 3, but was significantly decreased on the side ipsilateral to 6-OHDA striatal injection in rats of groups 1 and 4. These results show that subthalamic nucleus lesion provides neuroprotection of the dopaminergic nigrostriatal pathway against 6-OHDA toxicity and opens a new way for slowing or stopping the progression of Parkinson's disease.     

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.     

N-terminal tripeptide of IGF-1 (GPE) prevents the loss of TH positive neurons after 6-OHDA induced nigral lesion in rats

The effect of the N-terminal tripeptide of insulin-like growth factor (IGF)-1, glycine-proline-glutamate (GPE), as a neuroprotective agent for nigro-striatal dopaminergic neurons was examined in the present study using a rat model of Parkinson's disease. A unilateral nigro-striatal lesion was induced in rats by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB). GPE (3 microgram) or its vehicle was administered intracerebroventricularly (i.c.v.) 2 h after the 6-OHDA lesion. Tyrosine-hydroxylase (TH) immunohistochemistry in the substantia nigra compacta (SNc) and the striatum were examined 2 weeks after the lesion. Following 6-OHDA injection, the number of TH immunopositive neurons in the ipsilateral SNc was reduced. The density of TH immunostaining was also reduced in the ipsilateral SNc and the striatum. Treatment with a single dose of GPE (n=9) significantly prevented the loss of TH immunopositive neurons (p<0. 001) and restored the TH immunoreactivity in both the SNc and the striatum compared with the vehicle control group (n=9, p<0.001). The results suggest that GPE showed promise as a potential treatment for Parkinson's disease.     

Neuroprotective effect of rasagiline in a rodent model of Parkinson's disease

Sprague-Dawley rats received a unilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum and were treated daily for 6 weeks with increasing doses of monoamine oxidase type B inhibitor rasagiline [R(+)-N-propargyl-1-aminoindane] or saline (controls). Both doses of rasagiline markedly increased the survival of dopaminergic neurons in the lesioned substantia nigra, compared to controls (+97% and +119%, respectively). Treatment with the lower dose of rasagiline also abolished the motor stereotypies associated with nigrostriatal lesion. Our study supports the neuroprotective potential of chronic rasagiline administration in an experimental model of Parkinson's disease (PD).     

Unilateral axonal or terminal injection of 6-hydroxydopamine causes rapid-onset nigrostriatal degeneration and contralateral motor impairments in the rat

Unilateral injection of the catecholamine neurotoxin 6-hydroxydopamine into the axons or terminals of the nigrostriatal pathway is commonly used to model Parkinson's disease in experimental animals. Although the terminal lesion paradigm is considered to induce a more progressive lesion when compared to the axonal lesion, few studies have directly compared the early time-course for lesion development in these two models. Thus, this experiment sought to establish the temporal pattern of nigrostriatal degeneration and emergence of contralateral motor impairment in these models. Young adult male Lister Hooded rats were used. After baseline testing on a battery of spontaneous motor tests, standard stereotaxic techniques were used to inject 6-hydroxydopamine into the nigrostriatal axons or terminals at the level of the medial forebrain bundle or striatum respectively. From the day after lesion surgery, a subset of the rats was tested for motor performance, while another subset was used for immunohistochemical analysis. Quantitative tyrosine hydroxylase immunohistochemistry revealed that although both lesions caused a similar temporal pattern of immunopositive cell loss from the substantia nigra, the terminal lesion caused a more rapid loss of immunopositive terminals from the striatum. Despite these differences in striatal dopaminergic deafferentation, both lesion types caused a profound loss of contralateral motor function from the first day after lesion surgery. These findings illustrate the rapidity of the neuropathological and behavioural consequences of either axonal or terminal injection of 6-hydroxydopamine into the nigrostriatal pathway, and further highlight the need for a more progressive model of human Parkinson's disease.     

Evidence that L-dopa-induced rotational behavior is dependent on both striatal and nigral mechanisms

Parkinson's disease results from the death of the dopamine-containing neurons in the substantia nigra pars compacta (SNC). This is accompanied by a loss of dopamine in brain regions, such as the corpus striatum, which receives input from dopaminergic neurons in the substantia nigra (SN). Since the corpus striatum is the primary target for these dopaminergic neurons, it has long been thought that the corpus striatum is the principal region affected. It was, therefore, natural to assume that replenishing dopamine in the striatum might be an effective treatment for Parkinson's disease. In fact, the dopamine precursor L-dihydroxyphenylalanine (L-dopa), the current drug of choice for treatment of Parkinson's disease, is believed to exert its therapeutic effect by replenishing dopamine levels in the corpus striatum via enzymatic decarboxylation within the synaptic terminals of surviving nigrostriatal neurons (Hornykiewicz, 1974). However, dopamine is also synthesized, stored, and released from the dendrites of SNC neurons that arborize in the substantia nigra pars reticulata (SNR) (Cheramy et al., 1981). Using a classic animal model for Parkinson's disease (rats with a unilateral 6-hydroxydopamine lesion of the SN), we show that L-dopa is also converted to dopamine in significant amounts within the 6-OHDA-lesioned SN. Furthermore, in contrast to the situation in the striatum where dopamine levels are only elevated for a short time, dopamine levels in the SN remain elevated until the behavioral effects of L-dopa have subsided. This elevation of nigral dopamine levels produces rotation that can be blocked by injecting a selective D1 dopamine receptor antagonist (SCH 23390, 2 micrograms in 1 microliter) directly into the SN pars reticulata. Infusion of SCH 23390 into the ipsilateral striatum produced only a modest reduction in L-dopa-induced circling behavior. These results suggest that D1 dopamine receptors in the SN may be at least as important as D1 dopamine receptors in the striatum as a site for the effects of L-dopa. This may have important implications for the therapy of Parkinson's disease.     

Recombinant adeno-associated viral vector (rAAV) delivery of GDNF provides protection against 6-OHDA lesion in the common marmoset monkey (Callithrix jacchus)

Glial cell line-derived neurotrophic factor (GDNF) has shown potential as a treatment for Parkinson's disease. Recombinant adeno-associated viral vectors expressing the GDNF protein (rAAV-GDNF) have been used in rodent models of Parkinson's disease to promote functional regeneration after 6-OHDA lesions of the nigrostriatal system. The goal of the present study was to assess the anatomical and functional efficacy of rAAV-GDNF in the common marmoset monkey (Callithrix jacchus). rAAV-GDNF was injected into the striatum and substantia nigra 4 weeks prior to a unilateral 6-OHDA lesion of the nigrostriatal bundle. Forty percent of the dopamine cells in the lesioned substantia nigra of the rAAV-GDNF-treated monkeys survived, compared with 21% in the untreated monkeys. Fine dopaminergic fibres were observed microscopically in the injected striatum of some rAAV-GDNF-treated monkeys, suggesting that rAAV-GDNF treatment may have prevented, at least in part, the loss of dopaminergic innervation of the striatum. Protection of dopamine cells and striatal fibre innervation was associated with amelioration of the lesion-induced behavioural deficits. rAAV-GDNF-treated monkeys showed partial or complete protection not only in the amphetamine and apomorphine rotation but also in head position and the parkinsonian disability rating scale. Therefore, our study provides evidence for the behavioural and anatomical efficacy of GDNF delivered via an rAAV vector as a possible treatment for Parkinson's disease.     

Partial loss of dopaminergic neurons in the substantia nigra,ventrotegmental area and the retrorubral area -- model of the early beginning of Parkinson's symptomatology?

Summary. To test substances which might have protective effects on the dopaminergic system it is necessary to use models with a pathological symptomatology of the early beginning, i.e. models in which the chance exists to arrest the otherwise progressive pathological processes (see Heim et al., 2001). 6-hydroxydopamine (6-OHDA) injected unilaterally into the ventrolateral striatum of rats (6 μg dissolved in 2 μl 0.2% ascorbic acid) leads to specific stereotyped movements after subcutaneous injection of apomor-phine both 3 and 13 weeks after surgery. Ten weeks after surgery decreased spontaneous motor activity could be observed. Twelve weeks after 6-hydroxydopamine injection, the animals had difficulties in performing a spatial navigation task when the submerged escape platform was moved to another position. The switching of motor programs was less pronounced. The application of tyrosine-hydroxylase-staining showed a loss of ipsilateral neurones of the substantia nigra compacta as well as of dendrites in the pars reticulata, neurones in the ventral tegmental area and in the retrorubral area ipsilaterally as well as a loss of dopaminergic fibres both ipsilaterally and contralaterally in the striatum which should belong to the contralateral acting substantia nigra afferents. The loss of the neurones and the afferents was induced by the retrograde denervation following the 6-OHDA injection within the ventrolateral striatum. The question arises whether the model used here with the partially loss of dopaminergic neurons and fibres reflects some of pathological symptoms of Parkinson's disease in the early states. Received December 21, 2001; accepted February 25, 2002     

Striatal 6-hydroxydopamine induces apoptosis of nigral neurons in the adult rat

The massive dopaminergic neuronal loss that occurs in Parkinson's disease shows features of apoptosis. In the current study we have characterised the neuronal death in an animal model of Parkinson's disease. 6-Hydroxydopamine infused in the striatum of adult rats induced progressive loss of dopamine neurons, identified as tyrosine hydroxylase immunoreactive profiles, in the ipsilateral substantia nigra starting at day 5 post-lesion (32%). Silver staining revealed the presence of apoptotic profiles with neuronal morphology in the substantia nigra ipsilateral to the intrastriatal 6-hydroxydopamine injection. These apoptotic nuclei were first observed at day 6 post-lesion, peaked between days 7 and 10 and then abruptly declined. The apoptotic morphology of 6-hydroxydopamine-induced neuronal death was confirmed by electron microscopic studies. These data show that intrastriatal 6-hydroxydopamine-induced dopaminergic neuronal death in the adult rat is apoptotic and supports the use of this lesion protocol as an animal model of Parkinson's disease.     

Single or continuous injection of glial cell line-derived neurotrophic factor in the striatum induces recovery of the nigrostriatal dopaminergic system

Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor beta superfamily and acts as a neurotrophic factor for the nigrostriatal dopaminergic system. GDNF at a dose of 100 micrograms was injected stereotactically into the striatum of Sprague-Dawley rats that had been treated with intrastriatal 6-hydroxydopamine (6-OHDA) injection four weeks earlier. Immunocytochemical and behavioral analyses showed significant recovery of the nigrostriatal dopaminergic system after a single GDNF injection or continuous GDNF injection. Immunocytochemical and behavioral study showed that there was no significant difference between the results obtained from the two different injection methods. This result demonstrates the potential usefulness of GDNF for the treatment of Parkinson's disease.     

Single administration of GDNF into the striatum induced protection and repair of the nigrostriatal dopaminergic system in the intrastriatal 6-hydroxydopamine injection model of hemiparkinsonism

Purpose: Neurotrophic factor delivery into the brain is a promising approach in the treatment of Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent neurotrophic factors for dopaminergic neurons. Although multiple injections of GDNF into the brain are commonly performed in experimental studies, the present study investigates the efficacy of using a single injection of GDNF, which may be useful in elinically applying this treatment. Methods: Unilateral 6-hydroxydoparnine (6-OHDA) administration into the striatum was perforrned in Sprague-Dawley rats to create a partial lesion of the nigrostriatal DA system. These parkinsonian model rats received a single injection of human recombinant GDNF into the same portion of the striatum either 24 h before or 4 weeks after 6-OHDA treatrnent. Results: GDNF injected into the striatum before 6-OHDA administration potently protected the dopaminergic system, as shown by the numbers of mesencephalic dopaminergie neuron cell bodies and dopaminergic nerve terminal densities in the striatum. Dopaminergic neuron cell bodies and fiber densities were also significantly restored when GDNF was given after 6-OHDA administration, although the degree of restoration was lower than in the protective experiment. ODNF administration ameliorated apomorphine-induced rotational behavior in animals receiving it either before or after 6-OHDA treatment. However, the degree of improvement was less prominent when GDNF was iniected after 6-OHDA. Conclusion: Intracerebral GDNF adininistration exerts both protective and regenerative effects on the nigrostriatal dopaminergic system, a finding which may have implications for the development of new treatment strategies for Parkinson's disease.     

Simultaneous intrastriatal and intranigral grafting (double grafts) in the rat model of Parkinson's disease

Experimental and clinical studies of neural transplantation in Parkinson's disease have focused on the placement of fetal dopaminergic grafts not in their ontogenic site (substantia nigra) but in the main nigral target area (striatum). The reason for this is the apparent inability of intranigral nigral grafts to extend axons for long distances reinnervating the ipsilateral striatum. This review presents previous work by our laboratory [I. Mendez, M. Hong, Reconstruction of the striato-nigro-striatal circuitry by simultaneous double dopaminergic grafts: a tracer study using fluorogold and horseradish peroxidase, Brain Res. 778 (1997) 194–205; I. Mendez, D. Sadi, M. Hong., Reconstruction of the nigrostriatal pathway by simultaneous intrastriatal and intranigral dopaminergic transplants, J. Neurosci. 16 (1996) 7216–7227] using a new transplantation strategy aimed at restoring dopaminergic innervation of the nigra and striatum by simultaneous dopaminergic transplants placed in the substantia nigra and ipsilateral striatum (double grafts) in the 6-hydroxydopamine lesioned adult rat brain. These double grafts achieve not only greater striatal reinnervation than the standard intrastriatal grafts but also produce a faster and more complete behavioural recovery six weeks after transplantation. Injection of the retrograde tracer fluorogold into the striatum and nigra resulted in fluorescent labeled cells within the intranigral graft and the intrastriatal graft and surrounding striatum, respectively suggesting that these double grafts promote at least partial reconstruction of the nigrostriatal dopaminergic pathway. This double graft strategy may have potential implications in clinical neural transplantation for Parkinson's disease.     

The locus coeruleus and dopaminergic function in rat brain: implications to parkinsonism.

Clinical and experimental evidence suggests that degeneration of the locus coeruleus (LC) may be responsible for certain symptoms of Parkinson's disease (PD). We have, therefore, studied the effects of LC lesion on dopamine (DA) metabolism in the rat striatum. Unilateral depletion of norepinephrine (NE) was obtained by stereotaxic injection of 6-hydroxydopamine (6-OHDA) into the dorsal NE bundle (DNEB). Rats were sacrificed 1 or 3 weeks after lesioning. 6-OHDA induced approximately 50% depletion of NE in the ipsilateral hippocampus at 1 week postinjection, and over 75% depletion after 3 weeks. DNEB lesions had no effect on DA or DOPAC levels in the ipsi- or contralateral striatum at either time point. Lesions also failed to affect DA synthesis or utilization in either striatum. The metabolism of exogenous levodopa in the striatum was also unaffected. It is suggested that any possible effect of the LC on DA transmission in the striatum is not mediated by the DNEB.     

Single administration of GDNF into the striatum induced protection and repair of the nigrostriatal dopaminergic system in the intrastriatal 6-hydroxydopamine injection model of hemiparkinsonism

Purpose: Neurotrophic factor delivery into the brain is a promising approach in the treatment of Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent neurotrophic factors for dopaminergic neurons. Although multiple injections of GDNF into the brain are commonly performed in experimental studies, the present study investigates the efficacy of using a single injection of GDNF, which may be useful in elinically applying this treatment. Methods: Unilateral 6-hydroxydoparnine (6-OHDA) administration into the striatum was perforrned in Sprague-Dawley rats to create a partial lesion of the nigrostriatal DA system. These parkinsonian model rats received a single injection of human recombinant GDNF into the same portion of the striatum either 24 h before or 4 weeks after 6-OHDA treatrnent. Results: GDNF injected into the striatum before 6-OHDA administration potently protected the dopaminergic system, as shown by the numbers of mesencephalic dopaminergie neuron cell bodies and dopaminergic nerve terminal densities in the striatum. Dopaminergic neuron cell bodies and fiber densities were also significantly restored when GDNF was given after 6-OHDA administration, although the degree of restoration was lower than in the protective experiment. ODNF administration ameliorated apomorphine-induced rotational behavior in animals receiving it either before or after 6-OHDA treatment. However, the degree of improvement was less prominent when GDNF was iniected after 6-OHDA. Conclusion: Intracerebral GDNF adininistration exerts both protective and regenerative effects on the nigrostriatal dopaminergic system, a finding which may have implications for the development of new treatment strategies for Parkinson's disease.     

Neurorescue effects of VEGF on a rat model of Parkinson's disease

Vascular endothelial growth factor (VEGF) has been shown to display neuroprotective effects on dopaminergic (DA) neurons. Here, we investigated the neurorescue effects of VEGF on 6-hydroxydopamine (6-OHDA)-treated DA neurons in vitro and in vivo. Initially, we examined in vitro whether 1, 10, or 100 ng/ml of VEGF administration at 2 or 4 h after 6-OHDA treatment rescued DA neurons derived from E14 murine ventral mesencephalon. The earlier treatment of VEGF suppressed 6-OHDA-induced loss of DA neurons more than the delayed treatment. Next, we examined whether the continuous infusion of VEGF had neurorescue effects in a rat model of Parkinson's disease. We established a human VEGF secreting cell line (BHK-VEGF) and encapsulated the cells into hollow fibers. The encapsulated cells were unilaterally transplanted into the striatum of adult rats at 1 or 2 weeks after 6-OHDA lesions, and animals subsequently underwent behavioral and immunohistochemical evaluations. Compared to lesioned rats that received BHK-Control capsules, lesioned rats transplanted with BHK-VEGF capsules showed a significant reduction in the number of amphetamine-induced rotations, a significant preservation of TH-positive neurons in the substantia nigra pars compacta, and a remarkable glial proliferation in the striatum, with the earlier transplantation exerting much more benefits than the delayed transplantation. Parallel studies revealed that the observed in vitro and in vivo neurorescue effects were likely mediated by VEGF's angiogenic and glial proliferative effects, as well as its direct effects on the neurons. Our results suggest that VEGF is a highly potent neurorescue molecule for Parkinson's disease therapy.     

Specificity of dopaminergic neuronal degeneration induced by intracerebral injection of 6-hydroxydopamine in the nigrostriatal dopamine system.

The neurotoxic specificity of injections of 6-hydroxydopamine (6-OHDA) into areas containing either dopamine (DA) cell bodies (substantia nigra) or DA axon terminals (striatum) was studied. This selective effect was compared to the unspecific effects of copper sulfate (CuSO4) injection and electrocoagulation. One to two days after unilateral nigral injection of 2 mug of either 6-OHDA or CuSO4 into the nigra the volume of the unspecific lesions around the tip of the cannula was very similar. Only the 6-OHDA-induced lesions were associated with elective degeneration of the nigral DA neurons. Ten days after the administration of the same compounds the gliosis in the substantia nigra was much more extensive in CuSO4-than in 6-OHDA-treated rats; however, the reduction of DA concentrations in the ipsilateral striatum was only noticeable after 6-OHDA (-62%). A somewhat similar decrease of striatal DA levels (-52%) was observed after large electrocoagulation of the substantia nigra. Ten days after 6-OHDA (8mug) or electrolytic lesion of the striatum the Km for DA, serotonin and choline uptakes were similar in the striata of both sides, suggesting that the uptake process in the non-damaged neurons of the lesioned side was functionally normal. Following electrolytic lesion of the striatum, serotonin and choline Vmax values were decreased to about the same extent as the striatal reduction in weight and DA levels. When directly administered into the striatum 6-OHDA also produced a decline in DA concentration and Vmax but in contrast did not affect serotonin and choline uptake (Vmax), suggesting that the drug specifically destroyed dopaminergic neurons. The present data confirm that selective DA denervation can be achieved when appropriate amounts of the drug are injected into brain tissue in order to limit the unspecific lesion.     

Neuroprotective effect of citicoline in 6-hydroxydopamine-lesioned rats and in 6-hydroxydopamine-treated SH-SY5Y human neuroblastoma cells

Citicoline (CDP-choline or cytidine 5'-diphosphocholine) has been used as a therapeutic agent in combination with levodopa in the treatment of Parkinson's disease (PD). The present study examines the effects of citicoline by using validated in vivo and in vitro models. Citicoline reduces the cytotoxic effect of 6-hydroxydopamine (6-OHDA)-treated human dopaminergic SH-SY5Y neuroblastoma cells as measured cellular redox activity with 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) and increases the levels of reduced glutathione (GSH), a major antioxidant agent. Moreover, citicoline (500 mg/kg i.p.) administered for 7 days ameliorates functional behaviour by significantly reducing the number of apomorphine-induced contralateral rotations in 6-OHDA rats. Finally, citicoline significantly attenuates substantia nigra (SN) dopaminergic cell dropout and tyrosine hydroxylase immunoreactivity in the ipsilateral striatum in rats injected intrastriatally with 6-hydroxydopamine (6-OHDA).     

Potentiation of parkinsonian symptoms by depletion of locus coeruleus noradrenaline in 6-hydroxydopamine-induced partial degeneration of substantia nigra in rats

Parkinson's disease is characterized not only by a progressive loss of dopaminergic neurons in the substantia nigra but also by a degeneration of locus coeruleus noradrenergic neurons. The present study addresses the question of whether a partial neurodegeneration of dopaminergic neurons using 6-hydroxydopamine in rat, not sufficient to produce motor disturbances, is potentiated by prior selective denervation of locus coeruleus noradrenergic terminal fields using N-ethyl-2-bromobenzylamine. Two types of denervations, one causing dopamine deficiency alone and the other causing noradrenaline and dopamine deficiency, were performed. Noradrenaline, 5-hydroxytryptamine, 5-hydroxyindole acetic acid, dopamine and its metabolites were analysed in various brain regions. Behaviour was evaluated by catalepsy tests and activity box. N-ethyl-2-bromobenzylamine selectively depleted noradrenaline from neurons of locus coeruleus origin. Decreased dopamine content in the striatum, substantia nigra and pre-frontal cortex was observed after dopaminergic lesion with 6-hydroxydopamine (42.9%). Additional locus coeruleus noradrenaline depletion with N-ethyl-2-bromobenzylamine aggravated the dopamine depletion (61.2%). The lesion in the noradrenergic and dopaminergic neurodegenerated group was not sufficient to induce consistent catalepsy and akinesia. However, after a subthreshold dose of haloperidol (0.1 mg/kg), the expression of catalepsy and akinesia was strong in the dual-lesioned group and less in the 6-hydroxydopamine-lesioned group. These results indicate that denervation of locus coeruleus noradrenergic terminals with N-ethyl-2-bromobenzylamine potentiates the 6-hydroxydopamine-induced partial dopaminergic neurodegeneration and parkinsonian symptoms. Based on the present findings and existing reports, it can be concluded that noradrenergic neurons of locus coeruleus have neuromodulatory and neuroprotective properties on the dopaminergic neurons of basal ganglia and that noradrenergic degeneration may contribute to the aetiology and pathophysiology of Parkinson's disease.