Positive Neurons In Substantia Nigra Research Articles

Identification of Clec7a as the therapeutic target of rTMS in alleviating Parkinson's disease: targeting neuroinflammation

Parkinson's disease (PD) is a neurodegenerative disease. Repetitive transcranial magnetic stimulation (rTMS) is a therapeutic tool in PD. High-throughput sequencing was performed to screen potential therapeutic targets in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. The candidate gene, Clec7a, was screened out and validated. Clec7a is a pattern recognition receptor involved in neuroinflammation. The higher expression of Clec7a was observed in the substantia nigra (SN) and striatum of PD rats with dopaminergic neurons damage and was mainly localized in the microglial. Adeno-associated virus (AAV)-mediated specific knockdown of Clec7a in microglial alleviated 6-OHDA induced motor deficits and nigrostriatal dopaminergic neuron damage of rats, as evidenced by the increase of tyrosine hydroxylase (TH) -positive neurons in SN, as well as dopaminergic nerve fibers in the striatum. Clec7a knockdown restrained the neuroinflammation by suppressing inflammatory factors (IFN-γ, TNF-α, IL-1β, IL-18, and IL-6) release in SN, which might result from enhanced Arg-1 expression (M2 polarization) and defective inducible nitric oxide synthase (iNOS) expression (M1 polarization). The same phenomena were also observed in the LPS inflammatory rat model of PD. In vitro, α-synuclein fibrils induced upregulation of Clec7a expression and microglia polarization to a pro-inflammatory state of BV2 cells, leading to increased release of cytokines. However, Clec7a knockdown reversed those changes and induced a shift to an anti-inflammatory phenotype in BV2 cells. In conclusion, our study suggested that Clec7a was involved in PD pathogenesis, and its inhibition might protect rats from PD by depressing neuroinflammation through microglial polarization.

Sodium butyrate ameliorates gut dysfunction and motor deficits in a mouse model of Parkinson's disease by regulating gut microbiota.

A growing body of evidence showed that gut microbiota dysbiosis might be associated with the pathogenesis of Parkinson's disease (PD). Microbiota-targeted interventions could play a protective role in PD by regulating the gut microbiota-gut-brain axis. Sodium butyrate (NaB) could improve gut microbiota dysbiosis in PD and other neuropsychiatric disorders. However, the potential mechanism associated with the complex interaction between NaB and gut microbiota-gut-brain communication in PD needs further investigation. C57BL/6 mice were subjected to a rotenone-induced PD model and were treated intragastrically with NaB for 4 weeks. The gut function and motor function were evaluated. The α-synuclein expression in colon and substantia nigra were detected by western blotting. Tyrosine hydroxylase (TH)-positive neurons in substantia nigra were measured by immunofluorescence. Moreover, gut microbiota composition was analyzed by 16S rRNA sequencing. Fecal short chain fatty acids (SCFAs) levels were determined by liquid chromatography tandem mass spectrometry (LC-MS). The levels of glucagon like peptide-1 (GLP-1) in tissues and serum were evaluated using enzyme-linked immunosorbent assay (ELISA). NaB ameliorated gut dysfunction and motor deficits in rotenone-induced mice. Meanwhile, NaB protected against rotenone-induced α-synuclein expression in colon and substantia nigra, and prevented the loss of TH-positive neurons. In addition, NaB could remodel gut microbiota composition, and regulate gut SCFAs metabolism, and restore GLP-1 levels in colon, serum, and substantia nigra in PD mice. NaB could ameliorate gut dysfunction and motor deficits in rotenone-induced PD mice, and the mechanism might be associated with the regulation of gut microbiota dysbiosis.

Hydrogen sulfide attenuates induction and prevents progress of the 6-hydroxydopamine-induced Parkinsonism in rat through activation of ATP-sensitive potassium channels and suppression of ER stress

PurposeStudies argue in favor of hydrogen sulfide (H2S) as the next potent therapeutic agent for neurodegenerative diseases. In present study, we investigated the effect of long term treatment with NaHS (as donor of H2S) on induction and progress of the 6-hydroxydopamine (6-OHDA) -induced Parkinsonism in rat. MethodsThe 6-OHDA was injected into medial forebrain bundle of right hemisphere by stereotaxic surgery. Behavioral tests and treatments were carried out to eight weeks after the toxin. Immunohistochemistry and western blotting were carried out to evaluate the survival of tyrosine hydroxylase (TH) -positive neurons in substantia nigra (SN) and also expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), the markers of endoplasmic reticulum (ER) stress, in striatum and SN. ResultsEight weeks assessment of the behavioral symptoms showed that NaHS especially at dose of 100 μmol/kg attenuates remarkably induction of the Parkinsonism and prevents its progress. NaHS also increased the survival of TH- positive neurons and suppressed 6-OHDA- induced overexpression of GRP78 and CHOP. Blockade of ATP-sensitive potassium (K-ATP) channels with glibenclamide (Glib) prevented markedly the effect of NaHS on both the induction phase and survival of TH- positive neurons. But Glib did not affect the preventing effect of NaHS on the progress phase and its suppressing effect on the overexpression of ER stress markers. ConclusionH2S attenuates induction of the 6-OHDA- induced Parkinsonism and also increases the survival of dopaminergic neurons through activation of K-ATP channels. H2S also prevents progress of the Parkinsonism probably through suppression of ER stress.

HDAC Inhibition by Valproic Acid Induces Neuroprotection and Improvement of PD-like Behaviors in LRRK2 R1441G Transgenic Mice.

Parkinson’s disease (PD) is one of the late-onset neurodegenerative movement disorder. Major pathological markers of PD include progressive loss of dopaminergic neurons, Lewy body formation, genetic mutations, and environmental factors. Epigenetic regulation of specific gene expression via impaired histone acetylation is associated with neuronal dysfunction in various neurodegenerative diseases. In this study, we hypothesized that histone deacetylase (HDAC) inhibitor, valproic acid (VPA), can improve motor function by enhancing cell survival in PD genetic model mice with LRRK2 R1441G mutation. To address this question, we administered VPA in LRRK2 R1441G transgenic mice to determine whether VPA affects 1) histone acetylation and HDAC expression, 2) dopaminergic neuron survival, 3) inflammatory responses, 4) motor or non-motor symptoms. As results, VPA administration increased histone acetylation level and the number of tyrosine hydroxylase (TH) positive neurons in substantia nigra of LRRK2 R1441G mice. VPA reduced iba-1 positive activated microglia and the mRNA levels of pro-inflammatory marker genes in LRRK2 R1441G mice. In addition, VPA induced the improvement of PD-like motor and non-motor behavior in LRRK2 R1441G mice. These data suggest that the inhibition of HDAC can be further studied as potential future therapeutics for PD.

Sophora Tomentosa Extract Prevents MPTP-Induced Parkinsonism in C57BL/6 Mice Via the Inhibition of GSK-3β Phosphorylation and Oxidative Stress.

Sophora species are used as dietary medicines in aging-associated symptoms. Sophora tomentosa L. (ST) is a native medicinal plant in Southeast Asia; however, there is no pharmacological literature about ST extract. The present study evaluates the antioxidant phytoconstituent contents and radical scavenging capacities of ST extract. The further investigation was to clarify the neuroprotective mechanism of ST extract against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism by assaying the activities of the dopaminergic system and antioxidant defenses, glycogen synthase kinase 3β (GSK3-β) phosphorylation, and α-synuclein levels in C57BL/6 mice. The results show that ST extract alleviated the motor deficits in MPTP-induced Parkinsonism with four behavioral tests, including a rearing locomotor, catalepsy test, balance beam walking test, and pole test. ST extract reversed the number of tyrosine hydroxylase (TH)-positive neurons in substantia nigra (SN) that had decreased by MPTP. ST extract also restored the decreased levels of dopamine and the expression of tyrosine hydroxylase (TH) in the striatum. Furthermore, ST extract restored the levels of glutathione (GSH) and the activities of antioxidant enzymes, and decreased the elevated levels of malondialdehyde (MDA) in mouse striatum. ST extract also decreased α-synuclein overexpression and GSK-3β phosphorylation in mouse striatum. In vitro, ST extract exerted higher 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging capacities through its higher phenolic contents, especially protocatechuic acid and epicatechin. These results suggest that ST extract has the potential to counteract MPTP-induced motor deficit. The neuroprotective mechanism of ST extract against MPTP-induced Parkinsonism might be related to decreasing GSK-3β phosphorylation and restoring the activities of striatal antioxidant defenses to restore the nigrostriatal dopaminergic function and decrease α-synuclein accumulation.

Neuroprotective effect of IDPU (1-(7-imino-3-propyl-2,3-dihydrothiazolo [4,5-d]pyrimidin-6(7H)-yl)urea) in 6-OHDA induced rodent model of hemiparkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative motor disorder characterized by the degeneration of dopaminergic nigrostriatal neurons. Levodopa (l-DOPA) is the most effective therapy for PD, however, PD progression continues with significant side effects in long term, thus necessitating the search for effective therapy that impedes PD progression. PD therapy through non-dopaminergic pathways offers treatment without the risk of extrapyramidal effects. In this regard, earlier, we had reported, a novel compound IDPU with potential adenosine A2A receptor antagonist effect in haloperidol (chronic treatment) induced Parkinson model. In the present study, we extended our investigation towards i) evaluation of IDPU in well-established 6-OHDA induced Parkinson rat model to establish its role in the therapy of PD ii) its function in alleviating the neuronal loss. We carried the IDPU administration (i.p.) in rats for two weeks after establishing 6-OHDA induced unilateral lesions. The behavioral activity, neurochemical alteration, oxidative stress marker and tyrosine hydroxylase positive neurons in substantia nigra were analyzed. The results showed that IDPU significantly reduced motor and non-motor deficits induced by 6-OHDA in the behavioral tasks such as apomorphine, rota rod and force swim test. Furthermore, the results of oxidative stress biomarkers revealed that IDPU successfully modulated oxidative stress associated biomarkers such as MDA, catalase, superoxide dismutase, nitric oxide and reduced glutathione level. Additionally, IDPU significantly elevated intracellular dopamine, decreased glutamate and calcium levels in brain as compared to 6-OHDA alone treated animals which is evocative of its neuroprotective behavior. Thus, the investigations clearly validated IDPU as a potent anti-parkinsonian agent which showed immense capability to protect neurodegeneration.

Nimodipine, an L-type calcium channel blocker attenuates mitochondrial dysfunctions to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice

Parkinson’s disease (PD), the most common progressive neurodegenerative movement disorder, results from loss of dopaminergic neurons of substantia nigra pars compacta. These neurons exhibit Cav1.3 channel-dependent pacemaking activity. Epidemiological studies suggest reduced risk for PD in population under long-term antihypertensive therapy with L-type calcium channel antagonists. These prompted us to investigate nimodipine, an L-type calcium channel blocker for neuroprotective effect in cellular and animal models of PD. Nimodipine (0.1–10 μM) significantly attenuated 1-methyl-4-phenyl pyridinium ion-induced loss in mitochondrial morphology, mitochondrial membrane potential and increases in intracellular calcium levels in SH-SY5Y neuroblastoma cell line as measured respectively employing Mitotracker green staining, TMRM, and Fura-2 fluorescence, but only a feeble neuroprotective effect was observed in MTT assay. Nimodipine dose-dependently reduced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian syndromes (akinesia and catalepsy) and loss in swimming ability in Balb/c mice. It attenuated MPTP-induced loss of dopaminergic tyrosine hydroxylase positive neurons in substantia nigra, improved mitochondrial oxygen consumption and inhibited reactive oxygen species production in the striatal mitochondria measured using dichlorodihydrofluorescein fluorescence, but failed to block striatal dopamine depletion. These results point to an involvement of L-type calcium channels in MPTP-induced dopaminergic neuronal death in experimental parkinsonism and more importantly provide evidences for nimodipine to improve mitochondrial integrity and function.

Neuroprotective effects of Aceglutamide on motor function in a rat model of cerebral ischemia and reperfusion.

To investigate the effect and underlying mechanism of Aceglutamide on motor dysfunction in rats after cerebral ischemia-reperfusion. Adult male Sprague-Dawley rats were subjected to 2 h transient middle cerebral artery occlusion (MCAO). Aceglutamide or vehicle was intraperitoneally given to rats at 24 h after reperfusion and lasted for 14 days. Subsequently functional recovery was assessed and number of tyrosine hydroxylase (TH)-positive neurons in substantia nigra (SN) was analyzed. Tumor necrosis factor receptor-associated factor 1(TRAF1), P-Akt and Bcl-2/Bax were determined in mesencephalic tissue by Western blot method. PC12 cells and primary cultured mesencephalic neurons were employed to further investigate the mechanism of Aceglutamide. Aceglutamide treatment improved behavioral functions, reduced the infarction volume, and elevated the number of TH-positive neurons in the SN. Moreover, Aceglutamide significantly attenuated neuronal apoptosis in the SN. Meanwhile Aceglutamide treatment significantly inhibited the expression of TRAF1 and up-regulated the expression of P-Akt and Bcl-2/Bax ratio both in vitro and in vivo. Aceglutamide ameliorated motor dysfunction and delayed neuronal death in the SN after ischemia, which involved the inhibition of pro-apoptotic factor TRAF1 and activation of Akt/Bcl-2 signaling pathway. These data provided experimental information for applying Aceglutamide to ischemic stroke treatment.

Efficacy of repetitive transcranial magnetic stimulation treatment with MPTP Parkinson's disease mice

Objective To observe the efficacy of the treatment of repetitive transcranial magnetic stimulation (rTMS) on the 1 ⁃ methyl ⁃ 4 ⁃ phenyl ⁃ 1, 2, 3, 6 ⁃ tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). Methods Fifty⁃six C57BL/6J male mice were divided into 4 groups: PD model group, sham magnetic stimulation group (s⁃rTMS group), rTMS group and control group. Mice were given intraperitoneal injection of MPTP (15 mg/kg, every 2 h, 4 times) to prepare mouse model of Parkinson's disease 24 h before accepting repetitive transcranial magnetic stimulation. The stimulation were controlled at 1.00 Hz, 1.00 T, and lasted for 25 s, being performed at fixed time for 14 d. At the 1, 3, 7 and 14 d after MPTP injection, Rotarod Test were performed to evaluate physical coordination ability and freedom of movement of mice. Immunohistochemical staining was used in the midbrain and striatum to count the number of tyrosine hydroxylase (TH) ⁃ positive cells, and high ⁃ performance liquid chromatography ⁃ electrochemical detection (HPLC ⁃ ECD) of striatal dopamine content was applyed. Results The rotary number of mice in rTMS group（85.89±3.74）were significantly higher than that in PD group (59.71 ± 8.33; P = 0.000). TH⁃positive neurons of substantia nigra in rTMS group (36.67 ± 3.82) were significantly more than PD group (31.67 ± 3.35; P = 0.000). Dopamine concentration in striatum of PD group (152.35 ± 1.64) was significantly lower than that in rTMS group (258.70 ± 1.06; P = 0.000). Conclusion The repetitive transcranial magnetic stimulation could improve the motor coordination of MPTP mice and protect injured TH ⁃ positive neurons in substantia nigra and TH ⁃ positive nerve fibers in striatum, as well as elevating dopamine level in striatum. DOI：10.3969/j.issn.1672-6731.2011.01.014

Nurr1 deficiency predisposes to lactacystin-induced dopaminergic neuron injury in vitro and in vivo

Parkinson's disease (PD) has been proposed to result from a combination of genetic susceptibility and environmental exposure. Dysfunction of the ubiquitin–proteasome system (UPS) has been implicated in neuron degeneration and in pathogenesis of PD. Nurr1, a member of nuclear receptor superfamily, is a potential susceptibility gene for PD. In this in vitro and in vivo study, we investigated whether Nurr1 deficiency may predispose to environmental proteasome inhibitors-induced neuron injury. We found that lactacystin, an irreversible proteasome inhibitor, caused greater injury to SH-SY5Y cells that Nurr1 expression has been suppressed by small interference RNA (siRNA). On the contrary, the Nurr1 overexpressed SH-SY5Y cells by Nurr1 expression vector transfection rescued the lactacystin-induced injury. In vivo, stereotactic microinjection with lactacystin into right median forebrain bundle (MFB) of mice caused significant inhibition of the proteasome activity in both Nurr1 knock out heterozygous ( Nurr1 +/−) mice and their littermate wild-type ( Nurr1 +/+) mice. At same time, we found that there was a severer loss of tyrosine hydroxylase (TH)-positive neurons in substantia nigra (SN) and greater reduction of striatal dopamine (DA) levels in Nurr1 +/− mice as compared with that in Nurr1 +/+ mice. Furthermore, lactacystin-induced increase of cleaved PARP, cleaved caspase3 and p53 and decrease of bcl-2 in SN was significantly enhanced in Nurr1 +/− mice. These findings suggest that reduction in Nurr1 expression increases susceptibility to DAergic neuron injury induced by UPS impairment.

Distribution of nitric oxide synthase positive neurons in the substantia nigra of rats with liver cirrhosis

Background Nitrogen monoxide plays an important role in the physiological activity and pathological process of striatum in substantia nigra, and the nitric oxide synthase in substantia nigra may have characteristic changes after liver cirrhosis. Objective To observe the distribution and forms of nitric oxide synthase (NOS) positive neurons and fibers in substantia nigra of rats with liver cirrhosis. Design A comparative observational experiment. Settings Beijing Friendship Hospital; Capital Medical University. Materials Twenty 4-month-old male Wistar rats (120–150 g) of clean grade, were maintained in a 12-hour light/dark cycle at a constant temperature with free access to standard diet and water. Cryostat microtome (LEICA, Germany); All the reagents were purchased from Sigma Company. Methods The experiment was carried out in the Department of Anatomy (key laboratory of Beijing city), Capital Medical University from July 2000 to March 2002. The rats were randomly divided into normal group ( n =10) and liver fibrosis group ( n =10). Rats in the liver fibrosis group were subcutaneously injected with 60% CCl 4 oil at a dose of 5 mL/kg for the first time, and 3 mL/kg for the next 14 times, twice a week, totally 15 times. Liver fibrosis of grades 5–6 was taken as successful models. Whereas rats in the normal group were not given any treatment. Four months after CCl 4 treatment, all the rats were anesthetized to remove brain, and frontal frozen serial sections were prepared. The expressions of nitric oxide synthase positive neurons in substantia nigra of rats were observed under inverted microscope. The number and gray scale of cell body of nitric oxide synthase positive neurons in substantia nigra were detected with NADPH-diaphorase staining. Main outcome measures ▪ Number and gray scale of cell body of nitric oxide synthase positive neurons in substantia nigra; ▪ Expressions of nitric oxide synthase positive neurons in substantia nigra. Results All the 20 rats were involved in the analysis of results. ▪ The nitric oxide synthase positive neurons in substantia nigra were obviously fewer in the liver cirrhosis group than in the normal group ( P < 0.01), and the gray scale of the positive cell body was higher in the liver cirrhosis group than in the normal group ( P < 0.05). ▪ Abundant nitric oxide synthase positive neurons were observed in substantia nigra of normal rats, the cell body of nitric oxide synthase positive neurons was clear and transparent, with short own cloudy processes. In substantia nigra of rats with liver cirrhosis, the body of nitric oxide synthase positive neurons were observed shrink obviously, less fibrin than normal. Conclusion Rats with liver cirrhosis may suffer from the physiological dysfunction of neurons due to lack of fibers. The nitric oxide synthase positive neurons in substantia nigra can shrink and reduce.

A stereological study of substantia nigra in young and old rhesus monkeys

The number of pigmented and non-pigmented neurons in the substantia nigra (SN) of 10 old and six young female Macaca mulatta monkeys and in three old alpha male monkeys were estimated using new stereological cell counting methods. No systematic right-left differences were noted, nor were old animals different from young ones with respect to SN volume (68.9 mm 3 vs. 62.8 mm 3) or absolute number of nerve cells (320,000 vs. 312,000). However, the total number of pigmented neurons was about eight times higher in old animals compared with young ones (166,000 vs. 21,400) while the total number of non-pigmented SN neurons was less than half in old animals compared with young ones (139,000 vs. 285,000). These differences create difficulties in generalizing experimental results from the rhesus animal model to man. It seems unlikely that a simple correlation can be made between pigmented and tyrosine hydroxylase (TH) positive neurons in SN in monkeys. Instead of estimating the total number of pigmented and non-pigmented cells, only SN neurons positive for TH using immunohistochemical techniques might be used as an indicator of the total number of dopaminergic neurons in SN in monkeys.