Cold water stress attenuates dopaminergic neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice.

In the present study, we tested the effect of cold water stress (CWS) on dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse model, and found that CWS pretreatment elicited less MPTP neurotoxicity. To understand the molecular mechanism underlying this phenomenon, we detected the expression of heat shock protein 70 (Hsp70) in the striatum of the experimental mice, and found that CWS pretreatment could significantly increase striatal Hsp70 in MPTP-treated mice. Furthermore, in parallel with the induction of Hsp70, the MPTP-induced increase of striatal α-synuclein was inhibited in the CWS + MPTP-treated mice. CWS pretreatment also significantly inhibited the reduction of anti-apoptotic molecule Bcl-2 expression in the striatum and enhanced Bcl-2 transcription in the substantia nigra of MPTP-treated mice. Taken together, these data indicated that Hsp70 might be an important intermediate for the neuroprotective effect of CWS against MPTP-induced dopaminergic toxicity.

[Protective effects of transplantation of hCNTF-transfected cell line on axotomy-induced degeneration of retinal ganglion cells].

OBJECTIVE: To investigate the protective effects of transplantation of cell line stably expressing and secreting human ciliary neurotrophic factor (hCNTF) on the degeneration of retinal ganglion cells (RGCs) after optic nerve transection. METHODS: Plasmid encoding hCNTF was transfected into human lung fibroblast (HLF) cell line, then the stably transfected clones were selected with methopterin. In adult SD rats, RGCs were labeled with retrograde axonal tracer fluorogold (FG) injected to their targets including dorsal lateral geniculate nuclei (dLGN) and superior colliculi (SC). Seven days later, the optic nerve was transected alone or in combination with transplantation of HLF cells. Five, 14, 17, 21 and 28 days after axotomy, the retinas were mounted and examined under fluorescence microscope to observe the RGCs. RESULTS: Compared to the controls, the density of RGC in axotomy group decreased by 67.44% and 82.73% on the 14th and 28th day, respectively. In the eyes with hCNTF-transfected HLF cells transplantation, RGC density was higher than that of the axotomy group on the 5th, 17th, 21st day after axotomy (P < 0.05). On the 5th day, the morphology of RGC in the hCNTF group remained the same as the controls, whereas the morphology of RGC in the axotomy alone group began to change. CONCLUSIONS: hCNTF administered at the time of optic nerve transection can protect RGC from degeneration, increasing the numbers of surviving RGCs and delaying the death of RGCs.

[Expression of transient receptor potential channel 4 in striatum and hippocampus of rats is increased after focal cerebral ischemia].

This paper was designed in middle cerebral artery occlusion (MCAO) model of rats, to explore the role of transient receptor potential channel 4 (TRPC4) as Ca(2+) selective channel by detecting the changes of the expression of TRPC4 in different parts of cerebral tissues under the condition of focal cerebral ischemia. The rats were sacrificed after MCAO surviving time 6 h, 12 h, 1 d, 3 d. As determined by Western blot, the expressions of TRPC4 in striatum and hippocampus of 12 h, 1 d, 3 d groups were significant higher than that in the control group (P<0.05). Immunohistochemical staining showed that the TRPC4 immunoreactive substances were present in the membrane of neurons. Compared with the control group, immunostaining positive cells increased in hippocampus and striatum of cerebral ischemia groups. The TRPC4 immunostaining positive cells increased significantly in 1d-group and 3d-group (P<0.05). It suggests that as a Ca(2+) selective channel, the variance of the expression of TRPC4 may play a role in acute and delayed neuronal injury in focal cerebral ischemia.

[The expression of nestin in ischemia-injured brain of adult rat].

Immunohistochemistry and double immunofluorescent labeling techniques combined with confocal laser scanning microscope analysis were used to investigate the characteristic spatial induction profile of nestin following a transient middle cerebral artery occlusion in adult rat brain. The results showed that nestin was induced in ischemic core at 1 day after reperfusion. In addition to ischemic core, the expression of nestin increased in peri-ischemic I, II and III regions at 3 days and 1 week, then it decreased and narrowed along the rim of ischemic core 2 weeks after reperfusion. Double immunofluorescent labeling showed that nestin positive cells were mostly co-stained with GFAP,a astrocyte marker, in peri-ischemic I region 3 days after reperfusion. At 2 weeks, however nestin cells showed a long process and the cells double stained with nestin and NSE,a neuonal specific marker,increased in the ischemic brain. The results suggest that cerebral ischemia induces nestin expression in damaged neurons which might favor the neuroprotection against ischemic damage.

miR-15b suppression of Bcl-2 contributes to cerebral ischemic injury and is reversed by sevoflurane preconditioning.

Ischemic neuroprotection afforded by sevoflurane preconditioning has been previously demonstrated, yet the underlying mechanism is poorly understood and likely affects a wide range of cellular activities. Several individual microRNAs have been implicated in both the pathogenesis of cerebral ischemia and cellular survival, and are capable of affecting a range of target mRNA. Conceivably, sevoflurane preconditioning may lead to alterations in ischemia-induced microRNA expression that may subsequently exert neuroprotective effects. We first examined the microRNA expression profile following transient cerebral ischemia in rats and the impact of sevoflurane preconditioning. Microarray analysis revealed that 3 microRNAs were up-regulated (>2.0 fold) and 9 were down-regulated (< 0.5 fold) following middle cerebral artery occlusion (MCAO) compared to sham controls. In particular, miR-15b was expressed at significantly high levels after MCAO. Preconditioning with sevoflurane significantly attenuated the upregulation of miR-15b at 72h after reperfusion. Bcl-2, an anti-apoptotic gene involved in the pathogenesis of cerebral ischemia, has been identified as a direct target of miR-15b. Consistent with the observed downregulation of miR-15b in sevoflurane-preconditioned brain, postischemic Bcl-2 expression was significantly increased by sevoflurane preconditioning. We identified the 3'-UTR of Bcl-2 as the target for miR-15b. Molecular inhibition of miR-15b was capable of mimicking the neuroprotective effect of sevoflurane preconditioning, suggesting that the suppression of miR-15b due to sevoflurane contributes to its ischemic neuroprotection. Thus, sevoflurane preconditioning may exert its anti-apoptotic effects by reducing the elevated expression of miR-15b following ischemic injury, allowing its target proteins, including Bcl-2, to be translated and expressed at the protein level.

Focal cerebral ischemia activates neurovascular restorative dynamics in mouse brain.

Cerebral ischemia triggers regeneration of neural stem/progenitor cells (NSCs/NPCs), which are associated with neovascularization and white matter repair in the brain. This study analyzed the dynamics of neurogenesis, neovascularization, and white matter injury/repair after middle cerebral artery occlusion (MCAO) and elucidated their temporal association. Mice were subjected to MCAO for 60 minutes and sacrificed up to 28 days after reperfusion. Neurogenesis and angiogenesis, as measured by double staining of 5-bromo-2-deoxyuridine (BrdU) with DCX or tomato lectin, respectively, were substantially activated soon after ischemia and persisted for 4 weeks. Despite the moderate recovery of functional vessels in infarct margin from 7 days post-ischemia, a significant decrease in vascular density remained over time. Clusters of immature neurons localized proximal to angiogenic blood vessels beginning 14 days after ischemia, suggesting interplay between neurogenesis and revascularization. Progenitors of oligodendrocytes (NG2+) constitutively presented in the normal brain and proliferated soon after ischemia. However, axon damage and the loss of white matter integrity after ischemic stroke were almost irreversible, as revealed by sustained decreases of myelin basic protein (MBP) and neurofilament-200 expression.

Sevoflurane preconditioning confers neuroprotection via anti-inflammatory effects.

Neuroprotection afforded by volatile anesthetic preconditioning (APC) has been demonstrated in both in vivo and in vitro experiments, yet the underlying mechanism is poorly understood. We therefore investigated whether suppression of p38 MAPK, NF-kappa B and the downstream pro-inflammatory signaling cascade contribute to sevofurane APC-induced neuroprotection. Male Sprague-Dawley rats were exposed for 30 min/day on 4 consecutive days to ambient air or to sevoflurane (1.2% or 2.4%). Then rats were subjected to filament occlusion of the middle cerebral artery (MCAO) for 60 min, and euthanized 3 days after MCAO for measuring infarct volume. APC with sevofurane markedly improved neurological performance of stroke rats, significantly decreased infarct volume, and robustly suppressed activation of NF-kappa B and p38 MAPK, and expression of inflammatory cytokines. Furthermore, APC with sevofurane showed a direct inflammation-suppressing effect in rat brain receiving intracerebroventricular infusion of a dose of LPS that doesn't cause overt brain damage. Thus, the data suggest that APC with sevoflurane confers neuroprotection against focal ischemic brain injury, at least in part, by the anti-inflammatory effects of sevoflurane.

Sevoflurane preconditioning protects blood-brain-barrier against brain ischemia.

Sevoflurane preconditioning has recently been demonstrated to protect ischemic brain in vivo and in vitro. However, mechanisms underlying this neuroprotection have not been delineated. We therefore assessed the hypothesis that sevoflurane pretreatment protected blood-brain-barrier (BBB) via suppression of cell adhesion molecules (CAMs) and matrix metalloproteinases (MMPs) after ischemia. Repeated sevoflurane preconditioning was administered 24 hours before transient middle cerebral artery occlusion (MCAO). Neurologic deficits and expression of CAMs, MMPs and occludin were examined up to 3 days after ischemia. Evans blue (EB) extravasation and electron microscopy was detected at 2 days after ischemia. The data showed that sevoflurane pretreatment markedly improved BBB integrity and neurological outcomes after ischemia, robustly suppressed ischemia-induced decreases of occludin and increases of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), MMP-2, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1). Sevoflurane pretreatment also suppressed the activation of astrocytes and microglias in ipsilateral cortex and corpus callosum. In conclusion, repeated sevoflurane preconditioning confered potent protection against brain ischemia, partly by improving BBB integrity.

Alteration of NRSF expression exacerbating 1-methyl-4-phenyl-pyridinium ion-induced cell death of SH-SY5Y cells.

Neuron-restrictive silencer factor (NRSF)/neuronal repressor element-1 silencing transcription factor (REST) and its neuron-specific truncated form REST4 participates in the pathological processes of nervous system diseases, such as global ischemia, epilepsy, Huntington disease and so on. In this paper, we investigated the changes of NRSF and REST4 in a cellular model of Parkinson's disease (PD). Our results showed that neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP(+)) treatment triggered the mRNA and protein expression of NRSF and REST4, and caused both NRSF and REST4 proteins relocalized between the nucleus and cytoplasm in human dopaminergic SH-SY5Y cells. Redistribution of NRSF and REST4 derepressed the expression of target genes at late time points. Furthermore, alteration of NRSF and REST4 expression by overexpression or RNAi techniques elicited deleterious effects on cell viability of SH-SY5Y treated with toxic MPP(+).

Bcl-2 enhances neurogenesis and inhibits apoptosis of newborn neurons in adult rat brain following a transient middle cerebral artery occlusion.

To determine whether Bcl-2 could influence adult neurogenesis and prevent apoptosis of newborn neurons, we injected Bcl-2 expressing plasmid into the lateral ventricle of rat brain immediately following a 30-min occlusion of the middle cerebral artery (MCAO). We found that Bcl-2 increased neural progenitor cells (BrdU+-DCX+) in the ipsilateral striatum, newborn immature neurons (BrdU+-Tuj-1+) and newborn mature neurons (BrdU+-MAP-2+) in the ipsilateral striatum and frontal cortex at 1 to 4 weeks following MCAO. Bcl-2 overexpression promoted development of newborn neurons into GABAergic and cholinergic neurons in the ipsilateral striatum. Moreover, Bcl-2 significantly decreased the apoptosis of newborn neurons, determined by double staining of Tuj-1 and activated caspase-3 (Tuj-1+-Casp+). These results indicate that overexpression of Bcl-2 in adult rat brain enhances neurogenesis and survival of newborn neurons. Increasing neurogenesis and preventing the death of newborn neuron may be a strategy to aid in the repair of adult brain after stroke.

Melatonin attenuates MPTP-induced dopaminergic neuronal injury associated with scavenging hydroxyl radical.

To clarify the relationship between melatonin's hydroxyl radical (*OH) scavenging ability and its protective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal injury, in the present study, the salicylate trapping method combined with high-performance liquid chromatography (HPLC)-electrochemical detection were used to measure the contents of dihydroxybenzoic acid (DHBA) and dopamine (DA) in brain tissues of C57BL/6 mice. Immunocytohistochemistry was used to detect tyrosine hydroxylase (TH)-like positive staining neurons. Results show that MPTP treatment induced an increase in the content of DHBA and decrease in the level of DA as well as the number of TH positive stained neurons in the mouse brain. However, melatonin dose-dependently inhibited the increase of DHBA levels in ventral midbrain tissues, the decrease of DA content and the loss of dopaminergic neurons. Moreover, the relationship between the changes of DHBA and DA levels in the brain of mice following MPTP and melatonin treatment showed a statistically significant negative correlation. Present results suggest that melatonin can ameliorate MPTP-induced dopaminergic neuronal lesions probably, at least partially, because of its inhibition of *OH generation.

Role of vascular endothelial growth factor in neuronal DNA damage and repair in rat brain following a transient cerebral ischemia.

The antisense knockdown technique and confocal laser scanning microscopic analysis were used to elucidate vascular endothelial growth factor (VEGF) induction and its effect on DNA damage and repair in rat brain following a transient middle cerebral artery occlusion. Immunohistochemical study and in situ hybridization showed that the expression of VEGF and its mRNA was enhanced in the ischemic core and penumbra of ischemic brain. Western blot analysis further illustrated that VEGF induction was time-dependently changed in these areas. Double-staining analysis indicated that VEGF-positive staining existed in the neuron, but not in the glia, and it colocalized with excision repair cross-complementing group 6 (ERCC6) mRNA, a DNA repair factor. VEGF antisense oligodeoxynucleotide infusion reduced VEGF induction and resulted in an enlargement of infarct volume of the brain caused by ischemia. Moreover, it also increased the number of DNA damaged cells and lessened the induction of ERCC6 mRNA in ischemic brains. These results suggest that the induction of endogenous VEGF in ischemic neurons plays a neuroprotective role probably associated with the expression of ERCC6 mRNA.