Striatal membrane-bound and soluble catechol-O-methyl-transferase after selective neuronal lesions in the rat

Summary Activities of the two forms of catechol-O-methyltransferase (COMT), viz. the soluble (S-COMT) and the membrane-bound (MB-COMT), have been studied in the rat striatum to characterize their localization in relation to the nigrostriatal dopaminergic neurons. Selective unilateral nigrostriatal dopaminergic lesions were produced by an intranigral injection of 6-hydroxydopamine (6-OHDA; 8g/site). 6-OHDA caused an extensive lesion of the dopaminergic neurons as revealed by non-detectable concentrations of dopamine in the striata of the lesioned sites. In spite of that neither S-COMT nor MB-COMT activities were altered in comparison with the intact control striata. The intrastriatal injection of kainic acid significantly increased S-COMT activity but to some extent decreased MB-COMT activity. Kainic acid did not alter the striatal concentration of dopamine.These results suggest that both S-COMT and MB-COMT reside postsynaptically the nigrostriatal dopaminergic neurons. S-COMT seems to be found mainly in striatal glial cells, whereas striatal MB-COMT might be located both in postsynaptic neuronal and extraneuronal cells.     

Cerebrospinal dopamine metabolites in rats after intrastriatal administration of 6-hydroxydopamine or 1-methyl-4-phenylpyridinium ion

Dopamine (DA) and its main cerebral metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in striatum and cerebrospinal fluid (CSF) from cisterna magna in rats bilaterally lesioned by intrastriatal administration of 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium ion (MPP⁺). 6-OHDA caused a progressive lesion in striatum that is only moderately reflected in the decrease in dopamine metabolite concentration in CSF. MPP⁺ caused an acute but less selective lesion in the dopamine striatal system, as indicated by a significant reduction in striatal GABA content, followed by a slow recovery in dopamine striatal metabolism and content. The locomotor activity was dramatically reduced in both groups 48 hours after the treatment but remained significantly decreased after two months only in 6-OHDA lesioned animals. A positive correlation was found between HVA CSF concentration and striatal DA content in MPP⁺ lesioned rats, but not in 6-OHDA lesioned rats. It is concluded that the concentration of dopamine metabolites in CSF can be altered only after a severe striatal lesion: reduction of striatal dopamine content below 50% of normal values and involvement of neuronal or non-neuronal elements other than the dopaminergic system, similarly to the lesions caused by MPP⁺. These results may partly explain why CSF dopamine metabolites concentrations were significantly decreased both in advanced stages of parkinsonism and in other neurodegenerative disorders.     

Vitamin D3 attenuates 6-hydroxydopamine-induced neurotoxicity in rats

Previous reports have demonstrated that exogeneous administration of glial cell line-derived neurotrophic factor (GDNF) reduces ventral mesencephalic (VM) dopaminergic (DA) neuron damage induced by 6-hydroxydopamine (6-OHDA) lesioning in rats. Recent studies have shown that 1,25-dihydroxyvitamin D₃ (D3) enhances endogenous GDNF expression in vitro and in vivo. The purpose of present study was to investigate if administration of D3 in vivo and in vitro would protect against 6-OHDA-induced DA neuron injury. Adult male Sprague–Dawley rats were injected daily with D3 or with saline for 8 days and then lesioned unilaterally with 6-OHDA into the medial forebrain bundle. Locomotor activity was measured using automated activity chambers. We found that unilateral 6-OHDA lesioning reduced locomotor activity in saline-pretreated animals. Pretreatment with D3 for 8 days significantly restored locomotor activity in the lesioned animals. All animals were sacrificed for neurochemical analysis 6 weeks after lesioning. We found that 6-OHDA administration significantly reduced dopamine (DA), 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanilic acid (HVA) levels in the substantia nigra (SN) on the lesioned side in the saline-treated rats. D3 pretreatment protected against 6-OHDA-mediated depletion of DA and its metabolites in SN. Using primary cultures obtained from the VM of rat embryos, we found that 6-OHDA or H₂O₂ alone caused significant cell death. Pretreatment with D3 (10⁻¹⁰ M) protected VM neurons against 6-OHDA- or H₂O₂-induced cell death in vitro. Taken together, our data indicate that D3 pretreatment attenuates the hypokinesia and DA neuronal toxicity induced by 6-OHDA. Since both H₂O₂ and 6-OHDA may injure cells via free radical and reactive oxygen species, the neuroprotection seen here may operate via a reversal of such a toxic mechanism.     

A continuous striatal infusion of 6-hydroxydopamine produces a terminal axotomy and delayed behavioral effects

Rat models of Parkinson's disease typically employ a rapid nigral injection of 6-hydroxydopamine (6-OHDA) to produce a near-complete loss of nigrostriatal dopamine neurons, and thus model end stage disease. The present report describes the use of a continuous, low dose infusion of 6-OHDA into the striatum which produces a terminal axotomy of nigrostriatal dopamine neurons and protracted behavioral response. A solution of 6-OHDA in 0.4% ascorbate, delivered at 37°C from osmotic minipumps, was stable for 8 days as determined by its retained toxicity to a dopaminergic neuroblastoma cell line. The continuous infusion of 0.2 μg 6-OHDA per h did not affect the striatal uptake of [³H]GABA, [³H]choline, or [³H]glutamate but reduced [³H]dopamine uptake by 55% within 1.5 days after the start of the infusion. The striatal infusion of 6-OHDA produced a dose-dependent reduction of striatal dopamine and DOPAC levels but did not alter HVA, 5-HT, or 5-HIAA. An increase in amphetamine-induced ipsiversive rotations occurred within 1.5 days after the acute striatal injection of 20 μg or 30 μg of 6-OHDA but required 4 days to develop with the continuous 6-OHDA infusion. The topography of the lesion mapped by [³H]mazindol binding showed that, begining by 1.5 days, a diffuse depletion of terminals encompassed much of the striatum in the 30 μg acute injection group, whereas in the continuously infused rats, the lesion was apparent only by 4 days and was restricted to a smaller and more completely lesioned area. Unlike acutely lesioned animals, continuously infused rats revealed no obvious loss of dopamine neurons in the pars compacta by 5 weeks after 6-OHDA. The continuous striatal infusion of 6-OHDA can produce a topographically limited terminal axotomy of dopamine neurons and a protracted behavioral impairment.     

Sustained pharmacological inhibition of nitric oxide synthase does not affect the survival of intrastriatal rat fetal mesencephalic transplants

The objective of the present study was to investigate the potential role of the free radical nitric oxide (NO) in the development of fetal rat mesencephalic neurons grafted in a 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson's disease. First, using nitric oxide synthase (NOS)-immunocytochemistry and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, we investigated the presence of the neuronal isoform of NOS (nNOS) in intrastriatal mesencephalic grafts. During the course of the experiment (16 weeks) an increase in the staining intensity and the number of nNOS/NADPH-d positive cells within the grafts was observed, as well as a gradual maturation of dopaminergic neurons. In addition, within both the host striatal and grafted mesencephalic tissue, a NO-dependent accumulation of cyclic guanosine monophosphate (cGMP) was detected, indicating the presence of guanylate cyclase, i.e., the target-enzyme for NO. Secondly, to determine the impact of NO on the survival of grafted dopaminergic neurons, 6-OHDA lesioned rats received mesencephalic grafts and were subsequently treated with the competitive NOS-inhibitor N^ω-nitro-

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-arginine methylester (

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-NAME). After chronic treatment for 4 weeks, tyrosine hydroxylase immunocytochemistry revealed no apparent differences between the survival of grafted dopaminergic neurons in control- or

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-NAME treated animals, respectively. As the maturation of grafted dopaminergic neurons coincides with a gradual increase in the expression of nNOS within the graft and since dopaminergic cell numbers are not changed upon administration of

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-NAME, it is concluded that endogenously produced and potentially toxic NO does not affect the survival of grafted fetal dopaminergic neurons.     

Calretinin-containing axons and neurons are resistant to an intrastriatal 6-hydroxydopamine lesion

Relative preservation of dopaminergic axons in patches and a subcallosal layer was observed in the dorsal, lateral and caudal striatum 4 weeks after intrastriatal injection of 6-hydroxydopamine (6-OHDA), a neurotoxin selective for catecholaminergic neurons. Since calcium binding proteins are reported to provide neuroprotective influence in neurons, differences in the distribution of the calcium binding proteins might be related to the different vulnerabilities of dopaminergic neurons and axons to neurotoxins. To address this possibility, we characterized patches of relatively dense tyrosine hydroxylase-immunoreactive (TH-IR) axons in intrastriatal 6-OHDA lesioned rats, focusing on two calcium binding proteins, calbindin (CB) and calretinin (CR). The patches and subcallosal layer of preserved dopaminergic axons in the striatum of rats lesioned with 6-OHDA contained CR, a 31-kDa calcium-binding protein, but interestingly not CB. Dopaminergic neurons containing CR in the substantia nigra pars compacta (SNpc) were relatively spared compared to those that did not contain CR. Taken together, our data indicate that dopaminergic axons and neurons containing CR in the nigrostriatal pathway are more resistant to 6-OHDA lesion than those that do not contain CR.     

Enhanced survival of cultured dopamine neurons by treatment with soluble extracts from chemically deafferentiated striatum of adult rat brain

A soluble fraction was extracted from a chemically deafferentiated striatum of adult Wistar rats after unilateral lesioning of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) injection. The soluble extract from the lesioned side enhanced the survival of cultured mesencephalic dopamine (DA) neurons of 14-day-old rat embryos as evidenced by quantitative counting of tyrosine hydroxylase-like immunoreactive cells. The neurotrophic activity of this striatal extract for DA neurons was highest 14 days after 6-OHDA injection and became negligible in 28 days. The extract showed no promoting effects on cultured γ-aminobutyric acid (GABA)-containing mesencephalic neurons. These observations indicate that the striatum of adult rats may initiate de novo synthesis of trophic substance(s) for DA neurons but not for GABA neurons when subjected to nigral dopaminergic deafferentiation.     

Bromocriptine treatment in a murine parkinson's model: Ultrastructural evaluation after dopaminergic deafferentation

The objective of this article was to identify the effects of bromocriptine on the ultrastructure of the caudate nucleus in rats with a 6-hydroxidopamine (6-OHDA) unilateral lesion of the substantia nigra pars compacta. Eighteen Wistar male rats were stereotactically lesioned with 6-OHDA (n = 12), or sham lesioned (n = 6). Two days after rotational behavior was tested, and 2 days later, 6 rats were treated with 0.3 mg/Kg bromocriptine orally for a month and 6 rats were kept for the same time without treatment. The neuropile of the sham operated and bromocriptine-treated rats was well preserved contrary to the non-bromocriptine-treated rats. Also, it was found that there was a significant difference in the number of synaptic endings with edema in caudate of bromocriptine-treated rats compared with non-treated rats; however, the size of the synaptic endings were different to those found in the sham lesioned rats. Also, as in the sham lesioned group, the bromocriptines showed more synaptic contacts with dendritic spines contrasting to the non-treated group. The results suggest that bromocriptine possesses antioxidant properties because it decreased the ultrastructural alterations after 6-OHDA lesion.     

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.     

Vasoactive Intestinal Peptide Enhances Striatal Plasticity and Prevents Dopaminergic Cell Loss in Parkinsonian Rats

Destruction of the nigrostriatal dopaminergic pathway by the administration of 6-OHDA generates an animal model of Parkinson's disease. The main characteristic of this progressive neurological disorder is the loss of the dopaminergic neurons located in the substantia nigra pars compacta (SNc). Dopaminergic inputs from the SNc innervate the medium spiny neurons of the striatum and modulate the spontaneous activity of the primary output nuclei of the basal ganglia, globus pallidus interna, and substantia nigra pars reticulata. In our previous studies, we showed that systematically administered vasoactive intestinal peptide (VIP) is effective at reversing motor deficits, decreasing neuronal cell death, and repairing the myelin sheet in parkinsonian rats. In the current study, the effects of VIP on the dendritic morphology of the striatal neurons and the number of dopaminergic neurons in the SNc were examined in 6-OHDA-lesioned rats using Golgi-Cox staining and design-based stereological methods, respectively. Adult Sprague-Dawley rats were separated into sham-operated, bilaterally 6-OHDA lesioned and lesioned + i.p. VIP-injected (25?ng/kg) groups. VIP was first injected 1?h after the intrastriatal 6-OHDA microinjection (every 2?days for 15?days). The 6-OHDA significantly decreased the total number of dopaminergic neurons, branching, and spine density of the medium spiny neurons in the striatum. VIP significantly increased the number of neurons immunostained with tyrosine hydroxylase and the density of spines without altering the branching and the total length of dendrites. In conclusion, VIP might display synaptogenetic activity by enhancing the spine density in the striatum of the parkinsonian rats.     

Striatal glutamatergic function: Modifications following specific lesions

The effects of specific lesions of the striatum: (a) hemidecortication; (b) striatal injection of(±) ibotenate; and (c) 6-hydroxydopamine injections into the substantia nigra, were investigated on specific [³H]glutamate binding to striatal membranes. One month after decortication, there was a substantial reduction of calcium-dependent, stimulated glutamate release from striatal slices, indicating effective loss of glutamatergic fibres. Striatal glutamate binding increased by approximately 30% and this supersensitivity could be attributed solely to an increased receptor density. Ibotenate lesions which destroy target neurones for the glutamatergic fibres (sparing terminals), reduced glutamate binding in the striatum, as did nigral 6-OHDA lesions which delete striatal dopaminergic terminals. This finding supports the concept of there being glutamate receptors on pre-synaptic dopamine terminals in the striatum, involved in regulation of dopamine release. 6-OHDA lesions also result in a supersensitivity of the dopamine receptors localized on the cortico-striatal afferent terminals, as evidenced by the enhanced ability of dopamine to inhibit the K⁺-evoked, calcium-dependent release of endogenous striatal glutamate.     

Dopamine receptor activation promotes adult neurogenesis in an acute Parkinson model

Cell proliferation of neural progenitors in the subventricular zone (SVZ) of Parkinson disease (PD) patients and animal models is decreased. It was previously demonstrated that the neurotransmitter dopamine modulates cell proliferation in the embryonic brain. The aim of the present study was to analyze whether oral treatment with the dopamine receptor agonist pramipexole (PPX) modulates adult neurogenesis in the SVZ/olfactory bulb system in a dopaminergic lesion model. 6-Hydroxydopamine (6-OHDA) lesioned adult rats received either PPX (1.0 mg/kg) or PBS orally twice daily and bromodeoxyuridine (BrdU, a cell proliferation marker) for 10 days and were perfused immediately after treatment or 4 weeks after PPX withdrawal. Stereological analysis revealed a significant augmentation in SVZ proliferation by PPX. Consecutively, enhanced neuronal differentiation and more new neurons were present in the olfactory bulb 4 weeks after PPX withdrawal. In addition, dopaminergic neurogenesis was increased in the olfactory bulb after PPX treatment. Motor activity as assessed by using an open field paradigm was permanently increased even after long term PPX withdrawal. In addition, we demonstrate that D2 and D3 receptors are present on adult rat SVZ-derived neural progenitors in vitro, and PPX specifically increased mRNA levels of epidermal growth factor receptor (EGF-R) and paired box gene 6 (Pax6).Oral PPX treatment selectively increases adult neurogenesis in the SVZ–olfactory bulb system by increasing proliferation and cell survival of newly generated neurons. Analyzing the neurogenic fate decisions mediated by D2/D3 signaling pathways may lead to new avenues to induce neural repair in the adult brain.     

Effect of glial cell line-derived neurotrophic factor (GDNF) co-transplantation with fetal ventral mesencephalic cells (VMC) on functional restoration in 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson’s disease: neurobehavioral, neurochemical and immunohistochemical studies

Among trophic factors already known, glial cell line-derived neurotrophic factor (GDNF) and other members of its family have potent and specific action on dopaminergic neurons. In the present investigation an attempt has been made to validate the role of GDNF co-transplantation with fetal ventral mesencephalic cells (VMC) on functional viability and restoration using neurobehavioral, neurochemical and immunohistochemical parameters at 6 weeks post-transplantation in 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson's disease (PD). A significant restoration (P<0.01) in D-amphetamine induced rotations, spontaneous and apomorphine induced locomotor activity in rats co-transplanted with VMC and GDNF was observed as compared to VMC alone transplanted rats. Level of dopamine (DA), 3,4-dihydroxy-phenyl acetic acid (DOPAC) and dopamine D2 (DA-D2) receptors in the caudate putamen (CPu) were significantly (P<0.001) restored in co-transplanted group as compared to VMC transplanted or GDNF administered animals. The functional viability of transplanted VMC was confirmed by tyrosine hydroxylase (TH) expression and quantification of TH-positive cells by image analysis revealed a significant restoration in TH-IR fibers density as well as TH-IR neurons counts in co-transplanted animals over VMC transplanted animals. Results suggest that co-transplantation of VMC and GDNF may be a better approach towards functional restoration in 6-OHDA lesioned rat model of Parkinson's disease.     

Chronic L-DOPA administration is not toxic to the remaining dopaminergic nigrostriatal neurons, but instead may promote their functional recovery, in rats with partial 6-OHDA or FeCl(3) nigrostriatal lesions.

In this study, we have examined the effects of chronic L-3,4-dihydroxyphenylalanine (L-DOPA) administration on the remaining dopaminergic neurons in rats with 6-hydroxydopamine (6-OHDA) or buffered FeCl(3) partial lesions to the nigrostriatal tract. L-DOPA administration increased the turnover of dopamine in the striatum. L-DOPA administration for 1 week produced an increase in the level of striatal RTI-121 binding, a specific marker for dopamine uptake sites on the dopaminergic nerve terminals in the striatum. However, longer periods of L-DOPA treatment decreased the level of RTI-121 binding in the striatum. In the partial 6-OHDA lesion model, L-DOPA treatment had a time-dependent effect on the number of neurons demonstrating a dopaminergic phenotype i.e., neurons that are tyrosine hyrdoxylase (TH)-immunopositive, on the lesioned side of the brain. In the first few weeks of treatment, L-DOPA decreased the number of TH-positive neurons but with long-term treatment, i.e., 24 weeks, L-DOPA increased the number of neurons demonstrating a dopaminergic phenotype. Even in the buffered FeCl(3) infusion model, where the levels of iron were increased, L-DOPA treatment did not have any detrimental effects on the number of TH-positive neurons on the lesioned side of the brain. Consequently, chronic L-DOPA treatment does not have any detrimental effects to the remaining dopaminergic neurons in rats with partial lesions to the nigrostriatal tract; indeed in the 6-OHDA lesion model, long-term L-DOPA may increase the number of neurons, demonstrating a dopaminergic phenotype.     

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 beta-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 [(125)I]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 alpha-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.     

Dopaminergic neurotoxicity by 6-OHDA and MPP+: differential requirement for neuronal cyclooxygenase activity

Cyclooxygenase (COX), a key enzymatic mediator of inflammation, is present in microglia and surviving dopaminergic neurons in Parkinson's disease (PD), but its role and place in the chain of neurodegenerative events is unclear. Epidemiologic evidence showed that regular use of nonsteroidal antiinflammatory drugs (NSAIDs), specifically non-aspirin COX inhibitors like ibuprofen, lowers the risk for PD; however, the putative cause-and-effect relationship between COX activity in activated microglia and neuronal loss was challenged recently. We examined whether neuronal COX activity is involved directly in dopaminergic cell death after neurotoxic insult. Using low concentrations of 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridium ion (MPP+), neurotoxicants used to model selective dopaminergic cell loss in PD, and cultures of embryonic rat mesencephalic neurons essentially devoid of glia, we tested whether the nonselective COX inhibitor ibuprofen attenuated 6-OHDA and MPP+ neurotoxicity. At levels close to its IC50 for both COX isoforms, ibuprofen protected dopaminergic neurons against 6-OHDA but not MPP+ toxicity. Experiments with selective inhibitors of COX-1 (SC-560) and COX-2 (NS-398 and Cayman 10404), indicated that COX-2, but not COX-1, was involved in 6-OHDA toxicity. Accordingly, 6-OHDA, but not MPP+, increased prostaglandin (PG) levels twofold and this increase was blocked by ibuprofen. At concentrations well above its IC50 for COX, ibuprofen also prevented MPP+ toxicity, but had only limited efficacy against loss of structural complexity. Taken together, our data suggest that selective 6-OHDA toxicity to dopaminergic neurons is associated with neuronal COX-2, whereas MPP+ toxicity is COX independent. This difference may be important for understanding and manipulating mechanisms of dopaminergic cell death.     

Ginsenosides Rb1 and Rg1 effects on survival and neurite growth of MPP<Superscript>+</Superscript>-affected mesencephalic dopaminergic cells

Summary. Ginsenosides Rb1 and Rg1 are the main active ingredients of Panax ginseng C.A. Meyer (Araliaceae). They appear to exert protection against ischaemia and anoxic damage in animal models, suggesting an antioxidative and cytoprotective role. In our study, primary cultures from embryonic mouse mesencephalon are applied to examine the effects of these two ginsenosides on neuritic growth of dopaminergic cells and their survival affected by 1-methyl-4-phenylpyridinium-iodide (MPP⁺). Ginsenoside Rb1 (at 10µM) enhanced the survival of dopaminergic neurons by 19% compared to untreated control. MPP⁺ (at 1µM) significantly reduced the number of dopaminergic neurons and severely affected neuronal processes. Both ginsenosides counteracted these degenerations and significantly protected lengths and numbers of neurites of TH⁺ cells. Both compounds however could not prevent the cell loss caused by MPP⁺. Our study thus indicates partial neurotrophic and neuroprotective actions of ginsenosides Rb1 and Rg1 in dopaminergic cell culture.     

CX3CR1 Disruption Differentially Influences Dopaminergic Neuron Degeneration in Parkinsonian Mice Depending on the Neurotoxin and Route of Administration

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons accompanied by an inflammatory reaction. The neuron-derived chemokine fractalkine (CX3CL1) is an exclusive ligand for the receptor CX3CR1 expressed on microglia. The CX3CL1/CX3CR1 signaling is important for sustaining microglial activity. Using a recently developed PD model, in which the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin is delivered intranasally, we hypothesized that CX3CR1 could play a role in neurotoxicity and glial activation. For this, we used CX3CR1 knock-in mice and compared results with those obtained using the classical PD models through intraperitonal MPTP or intrastriatal 6-hydroxydopamine (6-OHDA). The striatum from all genotypes (CX3CR1^+/+, CX3CR1^+/GFP and CX3CR1-deficient mice) showed a significant dopaminergic depletion after intranasal MPTP inoculation. In contrast to that, we could not see differences in the number of dopaminergic neurons in the substantia nigra of CX3CR1-deficient animals. Similarly, after 6-OHDA infusion, the CX3CR1 deletion decreased the amphetamine-induced turning behavior observed in CX3CR1^+/GFP mice. After the 6-OHDA inoculation, a minor dopaminergic neuronal loss was observed in the substantia nigra from CX3CR1-deficient mice. Distinctly, a more extensive neuronal cell loss was observed in the substantia nigra after the intraperitoneal MPTP injection in CX3CR1 disrupted animals, corroborating previous results. Intranasal and intraperitoneal MPTP inoculation induced a similar microgliosis in CX3CR1-deficient mice but a dissimilar change in the astrocyte proliferation in the substantia nigra. Nigral astrocyte proliferation was observed only after intraperitoneal MPTP inoculation. In conclusion, intranasal MPTP and 6-OHDA lesion in CX3CR1-deficient mice yield no nigral dopaminergic neuron loss, linked to the absence of astroglial proliferation.     

Effect of the additional noradrenergic neurodegeneration to 6-OHDA-lesioned rats in levodopa-induced dyskinesias and in cognitive disturbances

Parkinson’s disease is a motor and cognitive disorder characterised by a progressive loss of the substantia nigra pars compacta (SNc) dopaminergic neurons as well as of the locus coeruleus (LC) noradrenergic neurons. It has been suggested that LC neurodegeneration might influence levodopa-induced motor disturbances and cognitive performance. We investigated the influence of dopaminergic and noradrenergic lesions on levodopa-induced dyskinesias and on working memory in rats. Two groups of animals were used: (1) rats with a dopaminergic lesion induced by a unilateral administration of the neurotoxin 6-hydroxydopamine (6-OHDA), and (2) rats with a combined lesion of the dopaminergic and noradrenergic systems induced by 6-OHDA and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), respectively. Dyskinesias were evaluated on days 1, 8, 15 and 22 of chronic levodopa treatment (6 mg/kg, twice at day, i.p.). Working memory was evaluated by a radial-arm maze (1) before lesions, (2) before levodopa administration and (3) after 22 days of levodopa treatment. Total, axial, limb and orofacial dyskinesias not differed significantly between both groups. Working memory tasks worsened in both lesioned groups reaching significance in terms of time of performance (P < 0.05). The number of repeated entries in the same arm (errors) was only significant in the double-lesioned group (P < 0.05). This behaviour was not different from the one observed after chronic levodopa treatment. These results suggest that levodopa-induced dyskinesias in the 6-OHDA-lesioned rats were not affected by the additional noradrenergic lesion, whereas this last condition was sufficient to worse the cognitive performance deficit produced by the dopaminergic lesion.     

重组人促红细胞生成素预处理对黑质多巴胺能神经元凋亡的影响

目的研究重组人促红细胞生成素(rhEPO)对离体帕金森病模型中黑质多巴胺神经元凋亡的影响。方法以6-羟基多巴胺(6-OHDA)为毁损剂建立大鼠离体帕金森病(PD)模型。用6u/mlrhEPO预处理黑质多巴胺神经元,然后用免疫组化方法观察黑质中酪氨酸羟化酶(TH)免疫反应阳性细胞数和半胱天冬酶-3(Caspase-3)免疫反应阳性细胞数的变化,TUNEL法观察黑质中多巴胺神经元的凋亡情况。结果与6-OHDA组(44.2±5.0)相比,rhEPO预处理组TH免疫反应阳性细胞(63.8±6.2,P<0.01)增多;与6-OHDA组(22.3±2.8)相比,rhEPO预处理组多巴胺神经元中Caspase-3表达减少,Caspase-3免疫反应阳性细胞染色较淡,数量减少(13.7±1.8,P<0.01);与6-OHDA组(20.3±3.1)相比,rhEPO预处理组TUNEL阳性细胞染色较淡,数量减少(10.7±1.5,P<0.01)。结论rhEPO预处理可以减轻6-OHDA对离体帕金森病模型中多巴胺神经元的损伤,其机制可能与rhEPO抑制黑质多巴胺神经元凋亡有关。