Inosine pretreatment of pregnant rats ameliorates maternal inflammation-mediated hypomyelination in pups via microglia polarization switch.

Periventricular leukomalacia (PVL) is a neurological condition observed in premature infants, characterized by hypomyelination and activation of microglia. Maternal inflammation-induced brain injury in offspring significantly contributes to the development of PVL. Currently, there are no clinical pharmaceutical interventions available for pregnant women to prevent maternal inflammation-mediated brain injury in their offspring. Inosine has been shown to modulate the immune response in diverse stressful circumstances, such as injury, ischemia, and inflammation. The aim of this investigation was to examine the potential prophylactic impact of inosine on offspring PVL induced by maternal inflammation. This was accomplished by administering a 1 mg/ml inosine solution (40 ml daily) to pregnant Sprague-Dawley (SD) rats for 16 consecutive days prior to their intraperitoneal injection of lipopolysaccharide (350 µg/kg, once a day, for two days). The results showed that maternal inosine pretreatment significantly reversed the reduction in MBP and CNPase (myelin-related markers), CC-1 and Olig2 (oligodendrocyte-related markers) in their PVL pups (P7), suggesting that inosine administration during pregnancy could improve hypomyelination and enhance the differentiation of oligodendrocyte precursor cells (OPCs) in their PVL pups. Furthermore, the protective mechanism of inosine against PVL is closely associated with the activation and polarization of microglia. This is evidenced by a notable reduction in the quantity of IBA 1-positive microglia, a decrease in the level of CD86 (a marker for M1 microglia), an increase in the level of Arg 1 (a marker for M2 microglia), as well as a decrease in the level of pro-inflammatory factors TNF-α, IL-1ß, and IL-6, and an increase in the level of anti-inflammatory factors IL-4 and IL-10 in the brain of PVL pups following maternal inosine pretreatment. Taken together, inosine pretreatment of pregnant rats can improve hypomyelination in their PVL offspring by triggering the M1/M2 switch of microglia.

Necrostatin-1s Suppresses RIPK1-driven Necroptosis and Inflammation in Periventricular Leukomalacia Neonatal Mice.

Periventricular leukomalacia (PVL), a predominant form of brain injury in preterm survivors, is characterized by hypomyelination and microgliosis and is also the major cause of long-term neurobehavioral abnormalities in premature infants. Receptor-interacting protein kinase 1 (RIPK1) plays a pivotal role in mediating cell death and inflammatory signaling cascade. However, very little is known about the potential effect of RIPK1 in PVL and the underlying mechanism. Herein, we found that the expression level of RIPK1 was drastically increased in the brain of PVL neonatal mice models, and treatment of PVL neonatal mice with Necrostatin-1s (Nec-1s), an inhibitor of RIPK1, greatly ameliorated cerebral ischemic injury, exhibiting an increase of body weights, reduction of cerebral infarct size, neuronal loss, and occurrence of necrosis-like cells, and significantly improved the long-term abnormal neurobehaviors of PVL. In addition, Nec-1s significantly suppressed hypomyelination and promoted the differentiation of oligodendrocyte precursor cells (OPCs), as demonstrated by the increased expression levels of MBP and Olig2, the decreased expression level of GPR17, a significant increase in the number of CC-1-positive cells, and suppression of myelin ultrastructure impairment. Moreover, the mechanism of neuroprotective effects of Nec-1s against PVL is closely associated with its suppression of the RIPK1-mediated necrosis signaling molecules, RIPK1, PIPK3, and MLKL. More importantly, inhibition of RIPK1 could reduce microglial inflammatory injury by triggering the M1 to M2 microglial phenotype, appreciably decreasing the levels of M1 marker CD86 and increasing the levels of M2 markers Arg1 or CD206 in PVL mice. Taken together, inhibition of RIPK1 markedly ameliorates the brain injury and long-term neurobehavioral abnormalities of PVL mice through the reduction of neural cell necroptosis and reversing neuroinflammation.

Activation of A2B adenosine receptor protects against demyelination in a mouse model of schizophrenia.

The purpose of the present study was to explore the effects of A2B adenosine receptor (A2BAR) on learning, memory and demyelination in a dizocilpine maleate (MK-801)-induced mouse model of schizophrenia (SCZ). BAY 60-6583, an agonist of A2BAR, or PSB 603, an antagonist of A2BAR, was used to treat SCZ in this model. The Morris Water Maze (MWM) was utilized to determine changes in cognitive function. Moreover, western blotting, immunohistochemistry and immunofluorescence were conducted to investigate the myelination and oligodendrocyte (OL) alterations at differentiation and maturation stages. The MWM results showed that learning and memory were impaired in SCZ mice, while subsequent treatment with BAY 60-6583 alleviated these impairments. In addition, western blot analysis revealed that myelin basic protein (MBP) and chondroitin sulphate proteoglycan 4 (NG2) expression levels were significantly decreased in MK-801-induced mice, while the expression of G protein-coupled receptor 17 (GPR17) was increased. Additionally, the number of anti-adenomatous polyposis coli clone CC-1/OL transcription factor 2 (CC-1+/Olig2+) cells was also decreased. Notably, BAY 60-6583 administration could reverse these changes, resulting in a significant increase in MBP and NG2 protein expression, and in the number of CC-1+/Olig2+ cells, while GPR17 protein expression levels were decreased. The present study indicated that the selective activation of A2BAR using BAY 60-6583 could improve the impaired learning and memory of SCZ mice, as well as protect the myelin sheath from degeneration by regulating the survival and maturation of OLs.

A2B adenosine receptor inhibition ameliorates hypoxic-ischemic injury in neonatal mice via PKC/Erk/Creb/HIF-1α signaling pathway.

Periventricular leukomalacia (PVL), the dominant cerebral white matter injury disease, is induced by hypoxia-ischemia and inflammation in premature infants. The activation of A2B adenosine receptor (A2BAR) is shown to involve into inflammation, ischemia, and other typical stress reactions, but its exact function in PVL has not been clarified. We gained initial insight from PVL mouse model (P9) by the induction of hypoxia-ischemia with right carotid ligation followed by exposure to hypoxia and intraperitoneal (i.p.) injection of Lipopolysaccharide (LPS). The results showed that treatment of PSB-603, an A2BAR selective antagonist, greatly ameliorated cerebral ischemic injury by increasing bodyweights, reducing infarct volume, brain injury,inflammation andcontributing to long-term learning memory functionalrecoveryof the PVL mice. Meanwhile, PSB-603 treatment suppressed neurons apoptosis as characterized byreducing of Caspase-3 level, inhibited microglia activation and attenuated hypomyelination through promoting MBP expression and oligodendrocytes differentiation. A2BAR inhibition also augmented PKC expression, the activity of PKC downstream signaling molecules were then explored. Erk expression and Creb phosphorylation exhibited upregulation in PSB-603 treatment group compared with the control group. Hypoxia Inducible Factor-1α (HIF-1α), a direct target of hypoxia, which is a key regulator of adenosine signaling by binding to the A2BAR promoter to induce expression of A2BAR, was shown to be decreased by PSB-603. Taken together, A2BAR inhibition can ameliorate hypoxic-ischemic injury in PVL mice maybe through PKC/Erk/Creb/HIF-1α signaling pathway.

The impact of quetiapine on the brain lipidome in a cuprizone-induced mouse model of schizophrenia.

The antipsychotic effect of Quetiapine (Que) has been extensively studied and growing evidence suggests that Que has a beneficial effect, improving cognitive functions and promoting myelin repair. However, the effects of Que on the brain lipidome and the association between Que-associated cognitive improvement and changes in lipids remain elusive. In the present study, we assessed the cognitive protective effects of Que treatment and used a mass spectrometry-based lipidomic approach to evaluated changes in lipid composition in the hippocampus, prefrontal cortex (PFC), and striatum in a mouse model of cuprizone (CPZ)-induced demyelination. CPZ induces cognitive impairment and remarkable lipid changes in the brain, specifically in lipid species of glycerophospholipids and sphingolipids. Moreover, the changes in lipid classes of the PFC were more extensive than those observed in the hippocampus and striatum. Notably, Que treatment ameliorated cuprizone-induced cognitive impairment and partly normalized CPZ-induced lipid changes. Taken together, our data suggest that Que may rescue cognitive behavioral changes from CPZ-induced demyelination through modulation of the brain lipidome, providing new insights into the pharmacological mechanism of Que for schizophrenia.

Dichloromethane extraction from Piper nigrum L. and P. longum L. to mitigate ischemic stroke by activating the AKT/mTOR signaling pathway to suppress autophagy.

Dichloromethane fraction (DF) of Piper nigrum L. and P. longum L. (PnL and PlL), has been found to exert a protective effect against ischemic stroke in rats. However, the regulatory mechanism exerted by PnL and PIL have not been fully elucidated. In this study, we found that DF greatly ameliorated cerebral ischemic injury in a rat model of permanent middle cerebral artery occlusion (pMCAO). The neurological score, behavioral assessment, brain infarct volume, phosphorylation of AKT (p-AKT), phosphorylation mTOR (p-mTOR), and Atg7 protein levels were determined. Additionally, we discovered that DF pretreatment reduced infarct volume, neurological score, and brain damage. Furthermore, DF therapy caused the downregulation of Atg7 and p-AKT expression, as well as the upregulation of p-mTOR expression. In conclusion, our findings indicated that DF treatment can reduce brain damage and inhibit apoptosis and autophagy by activating the Akt-mTOR signaling pathway in ischemic stroke.

Effect of Sox10 on remyelination of the hippocampus in cuprizone-induced demyelinated mice.

OBJECTIVE: The low number of oligodendrocytes (OLs) in the hippocampus of patients with schizophrenia suggests that hippocampal demyelination is changed in this condition. Sox10 is expressed throughout OL development. The effect of Sox10 on myelin regeneration is unknown. This study aimed to analyze changes in Sox10 expression in the hippocampus and its regulatory role in hippocampal myelin regeneration in a mouse model of demyelination. METHODS: Mice were fed 0.2% cuprizone (CPZ) for six weeks to establish the acute demyelinating model (CPZ mice). Behavioral changes of these mice were assessed via open field and tail suspension tests. The ultrastructure of the myelin sheaths in the hippocampus was observed by transmission electron microscopy. The expression levels of myelin sheath-related proteins and the transcription factor Sox10 were detected via immunohistochemistry and Western blots. Furthermore, Sox10-overexpressing adeno-associated virus was injected into the hippocampus after establishing the demyelinating model to investigate effects of Sox10 on remyelination. RESULTS: CPZ mice showed abnormal behavioral changes, a large number of pathological changes in the myelin sheaths, and significantly reduced protein expression of the myelin sheath markers myelin basic protein and proteolipid protein. This confirmed that the demyelinating model was successfully established. Meanwhile, the protein expression of the oligodendrocyte precursor cell marker neural/glial antigen 2 (NG2) increased, whereas Sox10 expression decreased. After Sox10 overexpression in the hippocampus, the abnormal behavior was improved, the ultrastructure of the myelin sheaths was restored, and the expression of myelin sheath protein was reversed. NG2 expression was upregulated. CONCLUSION: Overexpression of Sox10 promotes hippocampal remyelination after CPZ-induced acute demyelination.

NEP1-40 alleviates behavioral phenotypes and promote oligodendrocyte progenitor cell differentiation in the hippocampus of cuprizone-induced demyelination mouse model.

BACKGROUND: Studies have demonstrated that the failure of oligodendrocyte precursor cells (OPCs) differentiation as a major cause of remyelination failure in demyelinating disease. The reasons for this failure are not completely understood. We hypothesized that the present of myelin debris in CNS play an important role in poor OPCs differentiation in the mouse model of demyelinating disease. METHODS: Mice were fed by the food mixed with normal or 0.2 % cuprizone (CPZ) for 6 weeks. Then the learning and memory impairment were tested by Morris water maze test. The spontaneous alternation behavior and depression-like symptoms were assessed by tail suspension test and open filed test. The number of OPCs and oligodendrocytes were counted by immunofluorescence. After exposed to CPZ for 6 weeks, the mice were then receiving stereotactic injection of NEP1-40 into the CA3 of hippocampus. The behavioral, learning and memory changes were assessed by tail suspension test and open field test. The differentiation of OPCs were detected by immunofluorescence and western blot. RESULTS: The mice in CPZ group are more likely to show signs of depression and they showed impairment of long-term learning and memory function. The differentiation of OPCs were impaired in CPZ group. We found that mice treated with NEP1-40 showed less depression-like symptom in TST and higher locomotor activity in the OFT than the mice treated with PBS. CONCLUSIONS: Our study suggest that NEP1-40 can promote OPC differentiation and survival. Further study should focus on the effect of NEP1-40 on the differentiation and survival of OPCs in vitro.

N-methyl-D-aspartate receptor subunit 1 regulates neurogenesis in the hippocampal dentate gyrus of schizophrenia-like mice.

N-methyl-D-aspartate receptor hypofunction is the basis of pathophysiology in schizophrenia. Blocking the N-methyl-D-aspartate receptor impairs learning and memory abilities and induces pathological changes in the brain. Previous studies have paid little attention to the role of the N-methyl-D-aspartate receptor subunit 1 (NR1) in neurogenesis in the hippocampus of schizophrenia. A mouse model of schizophrenia was established by intraperitoneal injection of 0.6 mg/kg MK-801, once a day, for 14 days. In N-methyl-D-aspartate-treated mice, N-methyl-D-aspartate was administered by intracerebroventricular injection in schizophrenia mice on day 15. The number of NR1-, Ki67- or BrdU-immunoreactive cells in the dentate gyrus was measured by immunofluorescence staining. Our data showed the number of NR1-immunoreactive cells increased along with the decreasing numbers of BrdU- and Ki67-immunoreactive cells in the schizophrenia groups compared with the control group. N-methyl-D-aspartate could reverse the above changes. These results indicated that NR1 can regulate neurogenesis in the hippocampal dentate gyrus of schizophrenia mice, supporting NR1 as a promising therapeutic target in the treatment of schizophrenia. This study was approved by the Experimental Animal Ethics Committee of the Ningxia Medical University, China (approval No. 2014-014) on March 6, 2014.

Repetitive high-frequency transcranial magnetic stimulation reverses depressive-like behaviors and protein expression at hippocampal synapses in chronic unpredictable stress-treated rats by enhancing endocannabinoid signaling.

The anti-depressant effect of repetitive transcranial magnetic stimulation (rTMS), a clinically-useful treatment for depression, is associated with changes to the endocannabinoid system (ECS). However, it is currently unknown whether different frequencies of rTMS alter the ECS differently. To test this, rats exposed to chronic unpredictable stress (CUS) were treated with rTMS at two different frequencies (5 (high) or 1 Hz (low), 1.26 Tesla) for 7 consecutive days. Twenty-four hours after the final rTMS treatment, we evaluated depressive-like behaviors and the expression of several synaptic proteins and ECS-related proteins in the hippocampus. In addition, we knocked-down diacylglycerol lipase alpha (DAGLα) and cannabinoid type 1 receptor (CB1R), two important components of the ECS, and measured depressive-like behaviors and synaptic protein expression following rTMS. Furthermore, we measured the expression levels of several components of the ECS system in hippocampal-derived astrocytes and neurons exposed to repetitive magnetic stimulation (rMS) with different parameters (5 or 1 Hz, 0.84 or 1.26 Tesla). Interestingly, we found that only high-frequency rTMS ameliorated depressive-like behaviors and normalized the expression of hippocampal synaptic proteins in CUS-treated rats; this effect was eliminated by knockdown of DAGLα or CB1R. Moreover, we found that rMS at 5 Hz increased the expression of DAGLα and CB1R in hippocampal astrocytes and neurons. Collectively, our results suggest that high-frequency rTMS exerts its anti-depressant effect by up-regulating DAGLα and CB1R.

Early intervention with electroacupuncture prevents PTSD-like behaviors in rats through enhancing hippocampal endocannabinoid signaling.

Electroacupuncture (EA) is a clinically useful physiological therapy that has been recently adopted to treat several brain disorders. However, the potential role of early EA intervention in the prevention of posttraumatic stress disorder (PTSD) as well as its potential cellular and molecular mechanism has never been investigated previously. In the present study, we used an enhanced single prolonged stress (ESPS) model to access the effects of early EA intervention on the prevention of anxiety-like and fear learning behaviors, as well as the influence of the expression of post-synaptic density protein 95 (PSD95), synaptophysin (Syn), brain derived neurotrophic factor (BDNF), diacylglycerol lipase alpha (DAGLα) and cannabinoid type 1 receptor (CB1R) in the hippocampus with or without DAGLα or CB1R knockdown by a short hairpin RNA (shRNA) in the hippocampus. Moreover, the effects of electrical stimulation with different parameters on the expression of DAGLα and CB1R in the hippocampal astrocytes were also observed. The results showed that Early EA intervention improved hippocampal synaptic plasticity and ameliorated PTSD-like behaviors and also increased expression of BDNF, DAGLα and CB1R. However, either DAGLα or CB1R knockdown by a short hairpin RNA (shRNA) eliminated the neuroprotective effects of early EA intervention. Furthermore, electrical stimulation with 2/15 Hz 1 mA elevated the expression of DAGLα and CB1R. Altogether, our findings provide new insights regarding the possibility of using early EA intervention in the prevention of PTSD, and the protective effects of EA is involving the activation of DAGLα and CB1R.

Effect of NMDA on proliferation and apoptosis in hippocampal neural stem cells treated with MK-801.

The purpose of the present study was to investigate effects of N-methyl-D-aspartate (NMDA) on proliferation and apoptosis of hippocampal neural stem cells (NSCs) treated with dizocilpine (MK-801). Cultures of hippocampal NSCs were randomly divided into four groups consisting of an untreated control, cells treated with MK-801, NMDA and a combination of MK801 and NMDA (M+N). Proliferative and apoptotic responses for each of the experimental groups were determined by MTS and flow cytometry. The results revealed that MK-801 and NMDA exerted significant effects on hippocampal NSCs proliferation. Cell survival rates decreased in MK-801, NMDA and M+N treated groups compared with the control group. Cells survival rates in NMDA and M+N treated groups increased compared with the MK-801 treated group. MK-801 and NMDA were demonstrated to significantly affect apoptosis in hippocampal NSCs. Total and early stages of apoptosis in MK-801 and NMDA groups significantly increased compared with the control group. Total and early apoptosis of NSCs in the M+N group significantly decreased compared with MK-801 and NMDA groups. Late apoptosis of NSCs in MK-801 and NMDA groups significantly decreased compared with the control group. Late apoptosis of NSCs in the M+N group significantly increased compared with MK-801 and NMDA groups. The present study revealed that MK-801 inhibited proliferation and increased apoptosis in hippocampal NSCs. NMDA may reduce the neurotoxicity induced by MK-801, which may be associated with its activity towards NMDA receptors and may describe a novel therapeutic target for the treatment of schizophrenia.

Expression of NR1 and apoptosis levels in the hippocampal cells of mice treated with MK­801.

The aim of the present study was to investigate the characteristics of N­methyl­D­aspartate receptor R1 (NR1) expression and apoptosis in the nerve cells of the hippocampus in schizophrenia­like mice. C57BL/6 mice were randomly allocated to the following groups: i) Blank group; ii) MK­801 group; iii) MK­801+NMDA group, according to body weight. The NMDAR antagonist, MK­801 (0.6 mg/kg/d) was intraperitoneally injected daily for 14 days to induce a schizophrenia­like phenotype mouse model, and the effect of the NMDA injection via the lateral ventricle was observed. The results demonstrated that the number of NR1 positive cells in the MK­801 group increased in the CA1 and DG regions, indicating that NMDA may reverse this change. The level of damage decreased in the MK­801 treated group when compared with the blank group in the CA3 region. The protein expression of NR1 increased however, at the mRNA expression level, NR1 was lower in the MK­801 treated group when compared to the blank group; NMDA also reversed this change. In addition, early and total apoptosis detected in the hippocampal nerve cells was significantly increased in the MK­801 group when compared with the blank group, which was reversible following treatment with NMDA. These results indicated that NMDA may regulate the expression of NR1 and suppress apoptosis in hippocampal nerve cells in schizophrenia­like mice. Thus, NR1 may be a promising therapeutic target for the treatment of schizophrenia.

Directed differentiation of postnatal hippocampal neural stem cells generates nuclear receptor related­1 protein­ and tyrosine hydroxylase­expressing cells.

Parkinson's disease (PD) is a severe neurodegenerative disorder. Although the detailed underlying molecular mechanism remains to be elucidated, the major pathological feature of PD is the loss of dopaminergic (DA) neurons of the substantia nigra. The use of donor stem cells to replace DA neurons may be a key breakthrough in the treatment of PD. In the present study, the growth kinetics of hippocampal neural stem cells (Hip­NSCs) isolated from postnatal mice and cultured in vitro were observed, specifically the generation of cells expressing DA neuronal markers nuclear receptor related­1 protein (Nurr1) and tyrosine hydroxylase (TH). It was revealed that Hip­NSCs differentiated primarily into astrocytes when cultured in serum­containing medium. However, in low serum conditions, the number of ßIII tubulin­positive neurons increased markedly. The proportion of Nurr1­positive cells and TH­positive neurons, significantly increased with increasing duration of directed differentiation of Hip­NSCs (P=0.0187 and 0.0254, respectively). The results of the present study reveal that Hip­NSCs may be induced to differentiate in vitro into neurons expressing Nurr1 and TH, known to be critical regulators of DA neuronal fate. Additionally, their expression may be necessary to facilitate neuronal maturation in vitro. These data suggest that Hip­NSCs may serve as a source of DA neurons for cell therapy in patients diagnosed with PD.

Vasonatrin peptide, a novel protector of dopaminergic neurons against the injuries induced by n-methyl-4-phenylpyridiniums.

Vasonatrin peptide (VNP), a novel manmade natriuretic peptide, is known as a cardiovascular active substance. However, its neuroeffects are largely unknown. Here, cultured dopaminergic neurons from ventral mesencephalon of mouse were exposed to N-methyl-4-phenylpyridinium (MPP(+)), and the effects of VNP on the neurotoxicity of MPP(+) were investigated. As a result, MPP(+) caused injuries in the dopaminergic neurons. VNP significantly reduced the cytotoxicity of MPP(+) by increasing axon number and length of dopaminergic neurons, and by enhancing the cell viability. Also, the MPP(+)-induced depolymerization of ß-Tubulin III was attenuated by the treatment of VNP. In addition, VNP significantly increased the intracellular levels of cGMP. These effects of VNP were mimicked by 8-br-cGMP (a cell-permeable analog of cGMP), whereas inhibited by HS-142-1 (the antagonist of the particulate guanylyl cyclase-coupled natriuretic peptide receptors), or KT-5823 (a cGMP-dependent protein kinase inhibitor). Taken together, VNP attenuates the neurotoxicity of MPP(+) via guanylyl cyclase-coupled NPR/cGMP/PKG pathway, indicating that VNP might be a new effective reagent in the treatment of neuron degeneration of dopaminergic neurons in Parkinson's disease (PD).

Hyperphosphorylation of tau protein by calpain regulation in retina of Alzheimer's disease transgenic mouse.

Aim to investigate phosphorylated tau expression and its pathogenic mechanism in eye of Alzheimer's disease (AD) transgenic mice. Levels of tau, phosphorylated tau and other related factors (p35/p25, Cyclin-dependent kinase 5 (Cdk5), calpain) were observed by western blot. ß-Amyloid (Aß) plaques and neuron-fibrillary tangles (NFTs) in APP/PS1 double transgenic mice were detected by immuno-histochemistry. We found that hyper-expression of phosphorylated tau was detected in retina, and only a few or no expressed in optic nerve, cornea and lens of transgenic mice. Increased senile plaques (Aß) and NFTs were observed in transgenic mice accompanying with increased tau phosphorylation. The increased tau phosphorylation was associated with a significant increase in production of p35 and p25, and up-regulation of calpain. In conclusion, phosphorylated tau level was highly expressed in retina of AD transgenic mice. The pathogenic mechanism of AD was triggered by accelerating tau pathology via calpain-mediated tau hyper-phosphorylation in retina of an AD mice model.

Early Blockade of TLRs MyD88-Dependent Pathway May Reduce Secondary Spinal Cord Injury in the Rats.

To determine the role of toll-like receptors (TLRs) myeloid differentiation factor 88 (MyD88) dependent pathway in the spinal cord secondary injury, compression injury was made at T8 segment of the spinal cord in adult male Sprague-Dawley rats. Shown by RT-PCR, TLR4 mRNA in the spinal cord was quickly elevated after compression injury. Intramedullary injection of MyD88 inhibitory peptide (MIP) resulted in significant improvement in locomotor function recovery at various time points after surgery. Meanwhile, injury area, p38 phosphorylation, and proinflammation cytokines in the injured spinal cord were significantly reduced in MIP-treated animals, compared with control peptide (CP) group. These data suggest that TLRs MyD88-dependent pathway may play an important role in the development of secondary spinal cord injury, and inhibition of this pathway at early time after primary injury could effectively protect cells from inflammation and apoptosis and therefore improve the functional recovery.

The protective effects of inosine against chemical hypoxia on cultured rat oligodendrocytes.

Inosine is a purine nucleoside and is considered protective to neural cells including neurons and astrocytes against hypoxic injury. However, whether oligodendrocytes (OLs) could also be protected from hypoxia by inosine is not known. Here we investigated the effects of inosine on primarily cultured rat OLs injured by rotenone-mediated chemical hypoxia, and the mechanisms of the effects using ATP assay, MTT assay, PI-Hoechst staining, TUNEL, and immunocytochemistry. Results showed that rotenone exposure for 24 h caused cell death and impaired viability in both immature and mature OLs, while pretreatment of 10 mM inosine 30 min before rotenone administration significantly reduced cell death and improved the viability of OLs. The same concentration of inosine given 120 min after rotenone exposure also improved viability of injured mature OLs. Immunocytochemistry for nitrotyrosine and cellular ATP content examination indicated that inosine may protect OLs by providing ATP and scavenging peroxynitrite for cells. In addition, immature OLs were more susceptible to hypoxia than mature OLs; and at the similar degree of injury, inosine protected immature and mature OLs differently. Quantitative real-time PCR revealed that expression of adenosine receptors was different between these two stages of OLs. These data suggest that inosine protect OLs from hypoxic injury as an antioxidant and ATP provider, and the protective effects of inosine on OLs vary with cell differentiation, possibly due to the adenosine receptors expression profile. As OLs form myelin in the central nervous system, inosine could be used as a promising drug to treat demyelination-involved disorders.