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Prenatal alcohol exposure (PAE) could lead to developmental disorders of the central nervous system (CNS) and mental retardation. Toll-like receptor (TLR) 4 plays an important role in PAE-induced neurodevelopmental defects. However, how PAE affects TLR4 response in the brain remains controversial. Using a moderate PAE model by feeding pregnant rats with liquid ethanol diet, we investigated the TLR4-mediated response to intraventricular injection of lipopolysaccharide (LPS) in the hippocampus of PEA rats at postnatal day (PND) 30. The results showed that PAE significantly up-regulated the expression of Toll-Interleukin-1 Receptor (TIR)-domain-containing adaptor protein inducing interferon (IFN)-β (TRIF), TNF-α, and IL-1β in the rat hippocampus in the absence of LPS, indicated by western blot assay. LPS treatment dramatically up-regulated the expressions of TLR4 and its downstream molecules in the hippocampus of paired-food and control groups. But no such significant changes of those molecules were found in the hippocampus of PAE animals. Moreover, the LPS stimulation even down-regulated the levels of TLR4 and TRIF in the PAE group. These data suggest that the relatively moderate level of PAE may lead to a mild neuroinflammation and a suppression of TLR4-mediated response to LPS in the hippocampus of young rats. As innate immunity plays crucial roles in CNS development, moderate PAE-induced suppression of TLR4-mediated response may serve as a new candidate mechanism of CNS developmental defects.

Our previous studies revealed that etomidate, a non-barbiturate intravenous anesthetic agent, has protective effects on retinal ganglion cells within 7 days after optic nerve transection. Whether this process is related to anti-oxidative stress is not clear. To reveal its mechanism, we established the optic nerve transection injury model by transecting 1 mm behind the left eyeball of adult male Sprague-Dawley rats. The rats received an intraperitoneal injection of etomidate (4 mg/kg) once per day for 7 days. The results showed that etomidate significantly enhanced the number of retinal ganglion cells retrogradely labeled with Fluorogold at 7 days after optic nerve transection. Etomidate also significantly reduced the levels of nitric oxide and malonaldehyde in the retina and increased the level of glutathione at 12 hours after optic nerve transection. Thus, etomidate can protect retinal ganglion cells after optic nerve transection in adult rats by activating an anti-oxidative stress response. The study was approved by the Animal Ethics Committee at Air Force Medical University, China (approval No. 20180305) on March 5, 2018.

Purpose This study's aim was to investigate the beneficial effects of branched-chain amino acids (BCAAs) on the neuronal survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) transection. Method The experimental rats received daily BCAA injections through the caudal vein after left intra-orbital ON transection. Neuroprotection was evaluated by counting Fluorogold-labeled RGCs. The role of mammalian target of rapamycin (mTOR) pathway activation in promoting RGC survival was studied after rapamycin administration. Moreover, a peripheral nerve (PN) graft was transplanted onto the transected ON to study the effects of BCAAs on axon regeneration of injured RGCs. Results Our results showed that BCAAs alleviated the death of RGCs 7 and 14 days after ON transection, accompanied by an activation of mTOR pathway in RGCs. Blocking mTOR pathway with rapamycin eliminated such neuroprotective effects of BCAAs. Moreover, BCAAs also promoted axon regeneration of injured RGCs into a PN graft. Conclusion Our results suggest a neuroprotection of BCAAs through the activation of mTOR pathway. BCAAs also have a beneficial effect on axon regeneration of injured RGCs. Therefore, BCAAs could be considered for the clinical treatment of ON injury.

Cell therapy has been shown to be a key clinical therapeutic option for central nervous system diseases or damage. Standardization of clinical cell therapy procedures is an important task for professional associations devoted to cell therapy. The Chinese Branch of the International Association of Neurorestoratology (IANR) completed the first set of guidelines governing the clinical application of neurorestoration in 2011. The IANR and the Chinese Association of Neurorestoratology (CANR) collaborated to propose the current version “Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)”. The IANR council board members and CANR committee members approved this proposal on September 1, 2016, and recommend it to clinical practitioners of cellular therapy. These guidelines include items of cell type nomenclature, cell quality control, minimal suggested cell doses, patient-informed consent, indications for undergoing cell therapy, contraindications for undergoing cell therapy, documentation of procedure and therapy, safety evaluation, efficacy evaluation, policy of repeated treatments, do not charge patients for unproven therapies, basic principles of cell therapy, and publishing responsibility. Refereed/Peer-reviewed

Neuroprotection of lithium for axotomized retinal ganglion cells (RGCs) is attributed to upregulated intraretinal Bcl-2. As lithium also upregulates brain-derived neurotrophic factor (BDNF) which can rescue axotomized RGCs, it is hypothesized that lithium could protect RGCs through BDNF. This study investigated this hypothesis and a possible relationship between the dose and protection of lithium. All adult experimental rats received daily intraperitoneal injections of lithium chloride (LiCl) at 30, 60 or 85 mg/kg·bw until they were euthanized 2, 7 or 14 days after left intraorbital optic nerve (ON) transection. Our results revealed that RGC densities promoted and declined with increased dose of LiCl and the highest RGC densities were always in the 60 mg/kg·bw LiCl group at both 7 and 14 day points. Similar promotion and decline in the mRNA and protein levels of intraretinal BDNF were also found at the 14 day point, while such BDNF levels increased in the 30 mg/kg·bw LiCl group but peaked in the 60 and 85 mg/kg·bw LiCl groups at the 7 day point. These findings suggested that lithium can delay the death of axotomized RGCs in a dose-dependent manner within a certain period after ON injury and such beneficial effect is interrelated with an upregulated level of intraretinal BDNF.

We aimed to investigate whether peripheral low-dose lipopolysaccharide (LPS) induces the breakdown of the blood–brain barrier (BBB) and/or the activation of toll-like receptor 4 (TLR4) in the neonatal rat brain. Neonatal rats received intraperitoneal injections of low-dose LPS (0.3 mg/kg∙bw), and the BBB compromise was detected by Evans Blue extravasation and electron microscopy. Meanwhile, TLR4, adaptin myeloid differentiation factor 88 (MyD88), nuclear transcription factor kappa-B (NF-κB) p50 and tumor necrosis factor alpha (TNFα) in the neonatal rat brain were determined by quantitative real-time polymerase chain reaction (PCR) and Western Blot. Immunohistochemistry was used to determine the distribution and activation of microglia in the brain after LPS administration. It was demonstrated that Evans Blue extravasation was not observed in the brain parenchyma, and that tight junctions of cerebral endothelial cells remained intact after systemic injections of LPS in neonatal rats. Although intracerebroventricular injections of LPS activated microglia and up-regulated the expression of TLR4, MyD88, NF-κB p50 and TNFα in the neonatal rat brain, systemic LPS did not induce these responses. These findings indicate that while the neonatal rat brain responds to the direct intra-cerebral administration of LPS through robust TLR4 activation, systemic low-dose LPS does not induce the innate immune reaction or compromise the BBB in neonatal rats.

Recent studies demonstrated that mature atrocytes have the capacity for de-differentiating into neural stem/progenitor cells (NSPCs) in vitro and in vivo. However, it is still unknown what signals endow astroglial cells with a de-differentiation potential. Furthermore, the signaling molecules and underlying mechanism that confer astrocytes with the competence of NSPC phenotypes have not been completely elucidated. Here, we found that sonic hedgehog (Shh) production in astrocytes following mechanical injury was significantly elevated, and that incubation of astrocyes with the injured astrocyte conditioned medium (ACM) causes astrocytes to gradually lose their immunophenotypical profiles, and acquire NSPC characteristics, as demonstrated by down-regulation of typical astrocytic markers (GFAP and S100) and up-regulation of markers that are generally expressed in NSCs, (nestin, Sox2, and CD133). ACM treated astrocytes exhibit self-renewal capacity and multipotency similar to NSPCs. Concomitantly, in addition to Ptc, there was a significant up-regulation of the Shh downstream signal components Gli2 and Cyclin D1 which are involved in cell proliferation, dramatic changes in cell morphology, and the disruption of cell-cycle G1 arrest. Conversely, the depletion of Shh by administration of its neutralizing antibody (Shh n-Ab) effectively inhibited the de-differentiation process. Strikingly, Shh alone had little effect on astrocyte de-differentiation to NSPCs. These data above suggest that Shh is a key instructive molecule while other molecules secreted from insulted astrocytes may synergistically promote the de-differentiation event.

Purpose To compare neuroprotection and therapeutic time windows of two diazepam regimens on retinal ganglion cells (RGCs) after rat optic nerve transection (ONT). Methods Adult rats received initial intraperitoneal diazepam injections 30 minutes before left ONT, followed by daily diazepam (regimen-A) or every 8 hours for 3 days (regimen-B) until they were killed at day 7 or 14. Initial diazepam in regimen-A and regimen-B was delayed to 3, 6, 7, 9, 10, 12 and 6, 7, 8, 9, 10, 12 hours after ONT and these animals survived for 7 days. The effect of daily combinational uses of diazepam and bicuculline was assayed at 7 days. Results Regimen-A induced higher RGC densities than those in control and regimen-B groups at day 7, but lower density than regimen-B did at day 14. When initial diazepam was delayed beyond 6 or 8 hours after ONT with regimen-A and regimen-B, the promoting effects of diazepam on RGC densities disappeared. Bicuculline completely inhibited the protection of diazepam. Conclusions Prolonged neuroprotection on RGCs at day 14 and extended therapeutic time window for 8 hours can be achieved by regimen-B, while regimen-A induces a stronger neuroprotection at day 7. Diazepam neuroprotection is mediated through GABAA receptor.

To mimic multilevel nerve root compression and intervertebral foramina stenosis in human, we established a new animal model of the chronic compression of unilateral multiple lumbar DRGs (mCCD) in the rat. A higher occurrence of signs of spontaneous pain behaviors, such as wet-dog shaking and spontaneous hind paw shrinking behaviors, was firstly observed from day 1 onward. In the meantime, the unilateral mCCD rat exhibited significant bilateral hind paw mechanical and cold allodynia and hyperalgesia, as well as a thermal preference to 30°C plate between 30 and 35°C. The expression of activating transcription factor 3 (ATF3) was significantly increased in the ipsilateral and contralateral all-sized DRG neurons after the mCCD. And the expression of CGRP was significantly increased in the ipsilateral and contralateral large- and medium-sized DRG neurons. ATF3 and CGRP expressions correlated to evoked pain hypersensitivities such as mechanical and cold allodynia on postoperative day 1. The results suggested that bilateral neuropathy of primary sensory neurons might contribute to bilateral hypersensitivity in the mCCD rat.

In adult mammals, restoration of function after peripheral nerve injury is often poor and effective therapies are not available. Previously we have shown in mice that a peptide which functionally mimics the human natural killer cell (HNK)-1 trisaccharide epitope significantly improves the outcome of femoral nerve injury. Here we evaluated the translational potential of this treatment using primates. We applied a linear HNK-1 mimetic or a functionally inactive control peptide in silicone cuffs used to reconstruct the cut femoral nerves of adult cynomolgus monkeys (Macaca fascicularis). Functional recovery was evaluated using video-based gait analysis over a 160-day observation period. The final outcome was further assessed using force measurements, H-reflex recordings, nerve histology, and ELISA to assess immunoreactivity to HNK-1 in the treated monkeys. Gait deficits were significantly reduced in HNK-1 mimetic-treated compared with control peptide-treated animals between 60 and 160 days after injury. Better outcome at 160 days after surgery in treated versus control animals was also confirmed by improved quadriceps muscle force, enhanced H-reflex amplitude, decreased H-reflex latency, and larger diameters of regenerated axons. No adverse reactions to the mimetic, in particular immune responses resulting in antibodies against the HNK-1 mimetic or immune cell infiltration into the damaged nerve, were observed. These results indicate the potential of the HNK-1 mimetic as an efficient, feasible, and safe adjunct treatment for nerve injuries requiring surgical repair in clinical settings.

Intraorbital transection of the optic nerve (ON) always induces ultimate apoptosis of retinal ganglion cells (RGCs) and consequently irreversible defects of vision function. It was demonstrated that transplanted olfactory ensheathing cells (OECs) in partially injured spinal cord have a distant in vivo neuroprotective effect on descending cortical and brain stem neurons. However, this study gave no answers to the question whether OECs can protect the central sensitive neurons with a closer axonal injury because different neurons respond variously to similar axonal injury and the distance between the neuronal soma and axonal injury site has a definite effect on the severity of neuronal response and apoptosis. In the present study, we investigated the effect of transplanted OECs on RGCs after intraorbital ON transection in adult rats. Green fluorescent protein (GFP)-OECs were injected into the ocular stumps of transected ON and a significantly higher number of surviving RGCs was found together with a consistent marked increase in the mRNA and protein levels of BDNF in the ON stump and retina in the OEC-treated group at 7 days, but not 2 and 14 days, time point when compared to the control group. Our findings suggest that OEC transplantation induces the expression of BDNF in the ocular ON stump and retina and delays the death of axotomized RGCs at a certain survival period.

Spinal cord injury (SCI) in mammals not only damages the focal area, but also leads to wallerian degeneration (WD) of axons and myelin distal to the injury. In the present study, we investigated cellular responses within areas of WD of a sensory pathway, the fasciculus gracilis, after a T8-9 lateral spinal hemisection in the adult monkey Macaca fascicularis. Spinal cord segments rostral and caudal to the injury at two clinically-relevant time points, 1 week and 4 weeks post-SCI, representing subacute and chronic stages, respectively, were examined. We observed marked axon degeneration in the areas of WD at the subacute stage, and minimal axonal neurofilament staining at the chronic stage. At the ultrastructural level, however, many degenerating axonal profiles remained at the chronic stage. Myelin breakdown was a much-delayed process. A large number of residual myelin sheaths was observed at the chronic stage. In contrast to rodents, a substantial astrogliotic response was not found in the WD regions up to 4 weeks post-injury. Microglia activation was evident in the WD areas at the subacute stage, and was enhanced at the chronic stage. However, the lack of round reactive microglia/macrophages in these regions suggests that microglial activation was either delayed or incomplete. Thus it appears that many pathological characteristics of WD in monkeys are much delayed compared to those in rodents, but are similar to those in humans. Our results suggest that non-human primate SCI models are useful for evaluating repair strategies before they are translated to clinical trials of human SCI.

Protective effect of inosine against cell death after photochemically induced focal cerebral ischemia was investigated in adult rats. Intraperitoneal injection of inosine was started 24 h before operation and continued till the animals were sacrificed. The TUNEL method and Nissl staining were used to identify apoptosis and necrosis, respectively. TUNEL-positive cells were located in the transitional zone between the necrotic core and normal cortex. The mean maximum area of the necrotic cores and mean number of TUNEL-positive cells 24 h after operation were reduced significantly in the inosine-treated rats when compared with those in the saline-treated control animals. We suggest that inosine can significantly reduce both necrosis and apoptosis following cerebral ischemia induced by photothrombosis in adult rats.

Sphingosine-1-phosphate and its structural analog FTY720 (fingolimod) are important in the inhibition of osteoclast differentiation and bone resorption, however, it remains unknown whether they enhance osteogenic differentiation of the bone marrow mesenchymal stem cells (BM‑MSCs). The present study investigated the effect of FTY720 on the osteogenic differentiation of BM‑MSCs from the femurs of the ovariectomized (OVX) rats. Three different concentrations (1, 10 and 100 nM) of FTY720 were demonstrated to markedly upregulate mRNA expression levels of Runt‑related transcription factor 2 (Runx2) and Sp7 transcription factor (Sp7) at 2 weeks, and alkaline phosphatase (ALP) at 3 weeks. The osteocalcin (OCN) expression was similar at weeks 2 and 3. The protein expression levels of Runx2, Sp7, OCN and ALP induced by three different concentrations of FTY720 were higher than those in the control groups at 3 weeks in the OVX and sham groups. The findings of the current study suggested a beneficial effect of FTY720 on bone formation in OVX rats, and provided a potential therapeutic method of FTY720 to prevent alveolar bone resorption in patients with post‑menopausal osteoporosis.

Nogo-66 receptor was first identified in neurons. Recently, it was demonstrated in glial cells as well. Our previous study on the expression of Nogo-66 receptor in the cerebellum of the rat surprisingly found its location at the glial gap junctions. Here, we present our study on Nogo-66 receptor in the rat posterior pituitary, which is densely packed with pituicytes, a special type of astrocyte, and is known to be rich in gap junctions. We were able to demonstrate with immunohistochemistry and immuno-electron microscopy abundant Nogo-66 receptor immunoreactive gap junctions between pituicytes. This study, together with our prior one, strongly suggests that the Nogo-66 receptor may play a role in regulating the function of the gap junctions.

Assessment of the potential protective effects of inosine on an animal model of spinal cord injury. Our previous studies have demonstrated that inosine can directly protect neurons in vitro from zinc-induced injury and axotomized retinal ganglion cells of rats in vivo. This investigation was carried out to examine the possible protective effects of inosine on spinal cord secondary degeneration. Institute of Neurosciences, The Fourth Military Medical University, Xi'an, China. Compressive spinal cord injury (95-g load for 1 min) model was established in rats, and inosine was administrated beginning at different time points (2, 12, or 24 h) after spinal cord injury. Using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and hematoxylin and eosin staining, our study demonstrated that administration of inosine as late as 12 h after injury significantly reduced the total volume of spinal cord degenerative areas and the number of apoptotic cells 3 days following the trauma. Inosine can significantly reduce the spread of secondary degeneration and the cell death following spinal cord injury in adult rats. These findings may find a clinical application in the treatment of acute spinal cord injury.

The present study was initiated to investigate the role of extracellular signal-regulated kinases (ERK) 1/2 signaling pathway in the early response of spinal cord and associated dorsal root ganglion (DRG) to rhizotomy by using Western blotting and immunohistochemical techniques in a rat model of L3 and L4 dorsal root transection. The results showed that there were a considerable amount of total and phosphorylated ERK 1/2 protein in both spinal cord and DRG in normal animals killed under pentobarbital anesthesia. The total ERK 1/2 distributed in both glia and neurons, while phosphorylated ERK 1/2 dominantly existed in the latter in the gray matter of spinal cord, as demonstrated with double immunofluorescent staining. Twenty-four and forty-eight hours after axotomy, the phosphorylation level of ERK 1/2 in the operation side of dorsal spinal cord was much higher than that in the contralateral side, while the total ERK 1/2 level seemed unchanged. The increased expression of Fos protein was also seen in the dorsal spinal cord at lesion side twelve and twenty-four hours after axotomy. Double fluorescent staining proved that the phosphorylated ERK 1/2 positive cells in the ipsilateral dorsal spinal cord after axotomy predominantly were microglia and small portion was oligodendrocytes, whereas the Fos expression was mainly in neurons. In normal DRG, most neurons, especially the medium and small-sized ones, and the satellite cells contained total ERK 1/2-like immunoreactivity, whereas only a small portion of neurons and satellite cells contained phosphorylated ERK 1/2. After unilateral dorsal rhizotomy, there were no detectable changes for the phosphorylation of ERK 1/2 in either neurons or satellite cells in DRG.Collectively, the present results suggest that both ERK and Fos signal pathways involve the cellular activation in the spinal cord following dorsal rhizotomy, with ERK mainly in microglia and Fos in neurons. The increase of phosphorylation of ERK 1/2 in microglia of spinal cord after rhizotomy implicates that ERK signaling pathway involves intracellular activity of microglia responding to the experimental injury.

We explored the possibility of cytochrome oxidase (CO) involvement in spinal cord regeneration in adult rats. The spinal cord was hemitransected at T9. After one month's survival, the animals were deeply anesthetized and perfused. The spinal cord segments including the lesion site were removed and sectioned horizontally for CO histochemistry. Under light microscope, a substantial number of CO-reactive nerve fibers and boutons were identified in the lateral funiculus adjacent to the lesion site. Under electron microscope, moderately to highly CO-reactive mitochondria could be seen within nerve fibers and boutons. Synaptic contacts were identified among them. The increase in CO activity in nerve fibers and boutons may indicate their high-energy demand for synaptic and spontaneous activity following spinal cord hemisection.

Damage to the central nervous system (CNS) is always followed by an irreversible axon degeneration of injured neurons. The purine nucleoside inosine has been shown to induce neurons to regenerate axons in culture and in vivo. In the present study, we investigated the in vivo effects of inosine on the axon regeneration of axotomized retinal ganglion cells (RGCs) in adult rats, using the model of peripheral nerve (PN) grafting onto the ocular stump of the transected optic nerve. Animals were allowed to survive for 4 weeks after surgery with repeated intraperitoneal injections of inosine 1 day before PN grafting till they were killed. Treatment with inosine induced a significant increase (62%) in the number of FluroGold -labeled RGCs regrowing their axons into the PN graft, when compared with the control animals. The axon outgrowth-promoting effect of inosine in adult rodents may represent a potential clinical treatment for injured or degenerated CNS.

It has long been promulgated that microglial cells serve beneficial roles in the central nervous system (CNS). The beneficial role of microglial cells is considered to be linked with microglial activation and consequent up-regulation of various trophic factors. However, what triggers microglial activation and consequent elevated level of trophic factors, especially brain-derived neurotrophic factor (BDNF), following traumatic CNS injury has become a crucial but elusive issue. Furthermore, an effort still remains in understanding of the cellular and molecular mechanisms underlying the endogenous neuroprotection of activated microglial cells. In this study, we demonstrated that mechanically-injured astrocyte conditioned medium (ACM) could provoke beneficial activation of microglial cells and thus promote the transcription, synthesis and release of BDNF in cultured microglial cells. The microglia-derived BDNF can exerted a demonstrable biological role in promoting neurite outgrowth and intimate terminal contacts of dorsal root ganglion (DRG) neurons co-cultured with microglial cells. Moreover, ACM induced remarkable p38MAPK phosphorylation in cultured microglial cells that preceded the burst of BDNF. Activating p38-MAPK by anisomycin resulted in salutary effects similar to those seen with ACM, whereas specific inhibition of the p38MAPK by SB203580 abrogated all the positive effects of ACM, including BDNF promotion and subsequent neurite outgrowth of DRG neurite outgrowth of DRG neurons and their intimate terminal contacts with microglial cells. Together, our results indicated that the neuroprotection of the microglial source is mainly caused by micro-environmental soluble molecules released from injured astrocytes, and ACM-induced BDNF production and release from microglial cells may be mediated through p38-MAPK signaling pathway. Therefore, these findings may lay a foundation to further investigations on the microglial beneficial activation role in the repair of traumatic CNS injury and neurodegenerative diseases.

In addition to a fast activating and immediately inactivating inward sodium current, many types of excitable cells possess a noninactivating or slowly inactivating component: the persistent sodium current (I(NaP)). The I(NaP) is found in normal primary sensory neurons where it is mediated by tetrodotoxin-sensitive sodium channels. The dorsal root ganglion (DRG) is the gateway for ectopic impulses that originate in pathological pain signals from the periphery. However, the role of I(NaP) in DRG neurons remains unclear, particularly in neuropathic pain states. Using in vivo recordings from single medium- and large-diameter fibers isolated from the compressed DRG in Sprague-Dawley rats, we show that local application of riluzole, which blocks the I(NaP), also inhibits the spontaneous activity of A-type DRG neurons in a dose-dependent manner. Significantly, riluzole also abolished subthreshold membrane potential oscillations (SMPOs), although DRG neurons still responded to intracellular current injection with a single full-sized spike. In addition, the I(NaP) was enhanced in medium- and large-sized neurons of the compressed DRG, while bath-applied riluzole significantly inhibited the I(NaP) without affecting the transient sodium current (I(NaT)). Taken together, these results demonstrate for the first time that the I(NaP) blocker riluzole selectively inhibits I(NaP) and thereby blocks SMPOs and the ectopic spontaneous activity of injured A-type DRG neurons. This suggests that the I(NaP) of DRG neurons is a potential target for treating neuropathic pain at the peripheral level.

To investigate the intra-retinal expression of neuroglobin (Ngb) and death of retinal ganglion cells (RGCs) in acute retina ischemia rats.It was an experimental study. The acute retina ischemia model was established by specific hypothesised left retina artery of Sprague-Dawley rats. Forty rats were divided into four groups (0, 15, 30, 60 min) by the time of retina ischemia. Every group has 10 rats, in one group random 3 rats were detected by Western blotting; 4 rats were detected by ganglion cell counted by hematoxylin and eosin stain and immunohistochemistry fluorescence intensity analysis. The rest 3 rats were detected by Western blotting. The difference among different data were analyzed statistically by One-factor analysis of variance and LSD-t analysis.The intra-retinal expression of Ngb reached maximum after acute ischemia 15 minute (P = 0.000). then the expression began decreasing. After 30 minute acute ischemia, the expression of Ngb had approached normal (P = 0.728), while, the cell number of RGCs began lower than 0 min group (P = 0.011); after 60 minute acute ischemia, the expression of Ngb had been obviously lower than 0 min group (P = 0.001), the cell number of RGCs had been further lower than 0 min group (P = 0.000). The expression of Ngb in RGCs layer was highest in rat retina. The expression in inner plexiform layer and external plexiform layer were lower than the former. The expression of Ngb RGCs was mostly intracytoplasm. After 30 minute acute ischemia, the expression of Ngb were detected in mitochondrial outer compartment and mitochondrial cristae, but in cytoplasm of inner nuclear layer and outer nuclear layer the Ngb was not found.Ngb quickly steps-up when RGCs die in acute retina ischemia, and mainly expresses intracytoplasm of RGCs. It has tense relationships with nerve cells' survival in hypoxia.

Transplantation of olfactory ensheathing cells (OECs) becomes one of the promising strategies in restoring lost functions of injured central nervous system. Elevated level of expressed brain-derived neurotrophic factor (BDNF) was revealed in the previous studies to be related to the protective effects of OECs on injured cortical and brain stem neurons as well as retinal ganglion cells (RGCs), but no evidence has been obtained to demonstrate whether transplanted OECs protect injured central neurons directly by their secreted BDNF. In the present study, the effects of BDNF neutralization on the neuroprotection of adult OEC-conditioned medium (OEC-CM) on scratch-insulted RGCs were examined. The results showed that OEC-CM protected cultured RGCs from scratch insult, and neutralization of BDNF by BDNF neutralizing antibody attenuated such neuroprotection of the medium. It is thus concluded that neurotrophic factors including BDNF secreted by OECs can protect injured OECs in vitro and BDNF plays a major role in such a protection of OECs.

In order to develop a simplified method for long-term primary culture of highly-pure rat embryonic hippocampal neurons of low-density (10(3) cells/cm(2)), we optimized and modified conventional culturing methods. The modifications of our simplified method include: (1) combinational application of two growth substrates, tail collagen and poly-L-lysine, to coat plastic culture dishes and coverslips for a better neuronal attachment; (2) dissociation of hippocampal tissues with combinational use of two milder enzymes (collagenase and dispase) and trypsin of a lower concentration to minimize enzymatic damages to cultured neurons; (3) a cell pre-plating step to preliminarily eliminate the contaminating non-neuronal cells; (4) a modified culture medium as a critical step to promote highly pure neurons of low-density for a long term; and (5) appropriately reduced frequency and volume of refreshment of the culture medium. Using our modified method, the beta-tubulin III-immunostained and Hoechst 33342 counterstained neurons harvested a steady and healthy growth with a longer culture time of over 35 days, and a clear distinction between TAU-1- and MAP2-immunoreactive neurites was apparent at the early culturing period. In addition, the purity of neurons was over 95% at the different time points in comparison with the control culture using conventional serum-free method in which most neurons degenerated and died within 5 days. Thus, our modified method proved to be a simple, feasible as well as time- and resource-saving approach for a long-term survival of pure rat embryonic hippocampal neurons of low-density.

In recent years, a small number of cells that have stem cell properties were identified in human gliomas called brain tumor stem cells (BTSCs), which were thought to mainly contribute to the initiation and development of gliomas and could be identified by the surface marker CD133. However, recent studies indicated that the expression of CD133 might be regulated by environmental conditions such as hypoxia and that there might be CD133(-) BTSCs. Genetic mouse models demonstrated that some gliomas originated from transformed neural stem cells (NSCs). Therefore, we investigated the expression of CD15, a surface marker for NSCs, in tumor spheres derived from astrocytoma and ependymoma. CD15(+) cells isolated from these tumor spheres had properties of BTSCs including self-renewal, multidifferentiation, and the ability to recapitulate the phenocopy of primary tumors. CD15 exhibited stable expression in long-term cultured tumor spheres, which sustained BTSCs properties, whereas CD133 expression decreased significantly in late passages. Furthermore, CD15(+)CD133(-) cells isolated from early or late passages of tumor spheres showed similar characteristics of BTSCs. Examination of glioma samples by immunohistochemistry showed that CD15 was expressed in a subset of human brain tumors. Therefore, CD15 can be used as a marker of stem-like cells derived from brain tumors that might contain CD133(-) BTSCs.

Our previous study definitely demonstrated that the mature astrocytes could undergo a de-differentiation process and further transform into pluripotential neural stem cells (NSCs), which might well arise from the effect of diffusible factors released from scratch-insulted astrocytes. However, these neurospheres passaged from one neurosphere-derived from de-differentiated astrocytes possessed a completely distinct characteristic in the differentiation behavior, namely heterogeneity of differentiation. The heterogeneity in cell differentiation has become a crucial but elusive issue. In this study, we show that purified astrocytes could de-differentiate into intermediate precursor cells (IPCs) with addition of scratch-insulted astrocyte-conditioned medium (ACM) to the culture, which can express NG2 and A2B5, the IPCs markers. Apart from the number of NG2(+) and A2B5(+) cells, the percentage of proliferative cells as labeled with BrdU progressively increased with prolonged culture period ranging from 1 to 10 days. Meanwhile, the protein level of A2B5 in cells also increased significantly. These results revealed that not all astrocytes could de-differentiate fully into NSCs directly when induced by ACM, rather they generated intermediate or more restricted precursor cells that might undergo progressive de-differentiation to generate NSCs.

Thymosin-beta4 (Tbeta4) is a major actin monomer-binding peptide in mammalian tissues and plays a crucial role in the nervous system in synaptogenesis, neuronal survival and migration, axonal growth, and plastic changes of dendritic spines. However, it is unknown whether Tbeta4 is also involved in challenges with external stress such as ethanol-induced neurotoxicity. In the present study, we investigated the effects of Tbeta4 on ethanol-induced neurotoxicity in cultured cerebral cortical astrocytes and the underlying mechanisms. Primarily cultured astrocytes were treated with 1 microg/ml Tbeta4 2 h prior to administration of 100 mM ethanol for 0.5, 1, 3 and 6 days, respectively. The results showed that ethanol caused neurotoxicity in cultured astrocytes, as shown by declined cell viability, distinct astroglial apoptosis and increased intracellular peroxidation. Tbeta4 markedly promoted cell viability, ameliorated the injury of intracellular glial fibrillary acidic protein-immunopositive cytoskeletal structures, reduced the percentage of apoptotic astrocyte and cellular DNA fragmentation, suppressed caspase-3 activity and upregulated Bcl-2 expression, inhibited the accumulation of reactive oxygen species and production of malondialdehyde in ethanol-treated astrocytes in a time-dependent manner. These data indicated that Tbeta4 attenuates ethanol-induced neurotoxicity in cultured cortical astrocytes through inhibition of apoptosis signaling, and one of the mechanisms underlying the capacity of Tbeta4 to suppress apoptosis may in part be due to its effect of anti-peroxidation.

Background The incidence of spinal injury with spinal cord contusion is high in developed countries and is now growing in China. Furthermore, spinal cord injury happens mostly in young people who have a long life expectance. A large number of patients thus are wheelchair bound for the rest of their lives. Therefore, spinal cord injury has aroused great concern worldwide. Despite great efforts, recovery from spinal cord injury remains unsatisfactory. Based on the pathology of spinal cord contusion, an idea of early neurosurgical intervention has been formulated in this study. Methods A total of 30 patients with "complete" spinal cord injury or classified as American Spinal Injury Association (ASIA)-A were studied. Orthopedic treatment of the injured vertebra (e), internal fixation of the vertebral column, and bilateral laminectomy for epidural decompression were followed directly by neurosurgical management, including separation of the arachnoid adhesion to restore cerebrospinal fluid flow and debridement of the spinal cord necrotic tissue with concomitant intramedullary decompression. Rehabilitation started 17 days after the operation. The final outcome was evaluated after 3 months of rehabilitation. Pearson chi-square analysis was used for statistical analysis. Results All the patients recovered some ability to walk. The least recovered patients were able to walk with a wheeled weight support and help in stabilizing the weight bearing knee joint (12 cases, 40%). Thirteen patients (43%) were able to walk with a pair of crutches, a stick or without any support. The timing of the operation after injury was important. An optimal operation time window was identified at 4 - 14 days after injury. Conclusions Early neurosurgical intervention of spinal cord contusion followed by rehabilitation can significantly improve the locomotion of the patients. It is a new idea of a therapeutic approach for spinal cord contusion and has been proven to be very successful.

Pedestrian detection plays an important role in automated surveillance system. However, it is challenging to detect pedestrian robustly and accurately in a cluttered environment. In this paper, we propose a new cooperative pedestrian detection method using both colour and thermal image sequences, which is compared with the method using only colour image sequence and that using multi-modal fusion. Experiment results show that our cooperative detection mechanism could get more accurate pedestrian areas, a lower false alarm rate and a higher detection precision. Therefore, it has broad application prospects in the field of industry and military.

In this article, the selective visual attention mechanism and curve detection by Connect The Dots model is introduced to small and dim target detection in deep space background. The greyscale and movement continual significance are fully taken into account to get focus of attention integration map. A curve detection method which based on Connect The Dots model is designed to detect the target trajectory. Qualitative and quantitative results prove that the proposed algorithm has strong anti-noise performance and improve calculate efficiency of detection system effectively.

To study the role of cAMP in repair of hemisection of spinal cord in rats models.Rats models of spinal cord hemisection were made and cAMP were injected once in the motor cortex or continuously input in the lesion area or in the subarachnoid cistern for 3 d. NFs, GFAP, CSTs and spinal axons in the lesion areas were observed by immunohistochemistry and hind limb movements were evaluated in BBB scales.Many regenerated axons were presented in the lesion areas in cAMP groups though no continuous long regenerated axons traversed the lesion area when cAMP was input in the motor cortex or in the local lesion area. In control group, no regenerated axon were presented in the lesion areas. When cAMP was input in the subarachnoid cistern, only few-labelled CST axon survived and presented in the lesion area comparing no labelled CST axon presented in the lesion area. More NFs and less GFAP were distributed and extended in the lesion area in the cAMP groups. All the rats restored to normally walk 4-5 weeks after operations and no significance existed between cAMP groups and control groups comparing the BBB scales of hind limb movements.cAMP injected in the brain cortex or continuously input in the lesion area can induce the axonal regeneration.

Thoracic spinal cord transections were performed in adult rats. The animals were divided into two groups, with or without internal fixation of the involved vertebral column. Histologic and immunohistochemical studies were performed to compare the effect of internal fixation of the vertebral column.To find out the aspects and extent of beneficial effects of vertebral column fixation for spinal cord repair.Vertebral column fixation is a routine procedure in clinical spinal cord surgery. Paradoxically, most, if not all, animal spinal cord experiments seem to have ignored the importance of vertebral column fixation. During trunk movements, the vertebral column flexes to different directions, accompanied by bending of the spinal cord. Following spinal cord lesions, with frequent bending of the cord there will be repeated bleeding, inflammation, and other pathologic processes at the lesion site. Thus, the healing process will be hampered. The severity of the damages that will be brought about by bending of the cord is, to a certain degree, unpredictable. There will be rather big individual variations in injury and repair among the same type of experiments, rendering quantification and conclusion difficult.Adult Sprague-Dawley rats were used. The thoracic spinal cord was transected. Strong stainless steel wires were used for internal fixation of the vertebral column. The histology of the horizontal sections of the spinal cord segment, which included the lesion site, was examined at the 14th postoperative day. The volumes of the secondary degeneration and meningeal scar, the gap between the borders of the proximal and distal stumps of the transected spinal cord, the thickness of the meningeal scar, the astrocytic reaction, and the abundance of regenerating nerve fibers at the lesion site were compared between the vertebral column fixed and nonfixed groups. Whenever possible, the results were evaluated quantitatively.In all these aspects, the internally fixed group was consistently far better than the unfixed group. The quantitative analyses were as follows (fixed/unfixed): 1)volume of secondary degeneration: 1.07 +/- 0.20/1.81 +/- 0.43 mm3 (P0.01); 2) volume of meningeal scar: 2.38 +/- 0.55/4.34 +/- 1.40 mm3 (P0.05); 3) distance between cord stumps: 1.38 +/- 0.34/2.35 +/- 0.79 mm (P0.05); 4) the mean thinnest dimension of the meningeal scar: 0.90 +/- 0.43/1.98 +/- 0.85 mm (P0.05).Vertebral column fixation is a crucial procedure for spinal cord animal experiments.

A major issue in analysis of experimental results after spinal cord injury is spontaneous functional recovery induced by remaining nerve fibers. The authors investigated the relationship between the degree of locomotor recovery and the percentage and location of the fibers that spared spinal cord transection.The spinal cords of 12 adult rats were transected at T9 with a razor blade, which often resulted in sparing of nerve fibers in the ventral spinal cord. The incompletely-transected animals were used to study the degree of spontaneous recovery of hindlimb locomotion, evaluated with the BBB rating scale, in correlation to the extent and location of the remaining fibers.Incomplete transection was found in the ventral spinal cord in 42% of the animals. The degree of locomotor recovery was highly correlated with the percentage of the remaining fibers in the ventral and ventrolateral funiculi. In one of the rats, 4.82% of remaining fibers in unilateral ventrolateral funiculus were able to sustain a certain recovery of locomotion.Less than 5% of remaining ventrolateral white matter is sufficient for an unequivocal motor recovery after incomplete spinal cord injury. Therefore, for studies with spinal cord transection, the completeness of sectioning should be carefully checked before any conclusion can be reached. The fact that the degree of locomotor recovery is correlated with the percentage of remaining fibers in the ventrolateral spinal cord, exclusive of most of the descending motor tracts, may imply an essential role of propriospinal connections in the initiation of spontaneous locomotor recovery.

We successfully constructed a retrovirus expression vector pRev-TRE-NT3. The supernatant with highest retroviral titers was obtained by transfection of two retrovirus vector, pRev-TRE-NT3 and pRev-Tet-On, into an ecotropic Ecopack-293 cells. Primarily cultured rat olfactory ensheathing cells (OECs) were co-infected with retrovirus following pRev-TRE-NT-3 and pRev-Tet-On system of produced retrovirus. By adding different concentrations of Doxycline, genetically modified OECs were induced to secrete human NT-3. The secreted NT-3 in OEC culture supernatant was detected with western-blot, and its biological activity was confirmed by the growth bioassay of dorsal root ganglion (DRG) cells. The results are as follows: (1) 0.78 kb hNT-3 cDNA was harvested and successfully cloned into pRev-TRE vector by PCR and restriction enzyme digestion methods. The constructed plasmid was identified with electrophoresis, and the direction of hNT-3 cDNA inserting pRev-TRE vector was integrated correctly; (2) NT-3-modified OEC culture supernatant contained 28 kd NT-3 which can bind NT-3 antibody in western-blotting assay, and NT-3 expression was concentration-dependent on Doxycline. NT-3 was not detected in control groups; and (3) Numerous DRG cells migrated from DRG tissue mass in NT-3-modified OECs experimental groups, and these cells had long and thin processes with strong dioptre. These processes formed complicated networks. As for unmodified OEC control groups, a few DRG cells migrated from the tissue mass, and the processes of these cells were rather short. In blank groups, no cells migrated and grew. And their processes in control groups are significantly shorter than NT-3-modified OECs experimental groups. Our results indicate that Tet-On-regulated NT-3 expression in OECs was efficient, offering novel material of in vivo transplantation for the repair of CNS injury.

It has been reported that inosine has no direct neuroprotection against respiratory inhibitors due to the absence of purine nucleoside phosphorylase in neurons. Recent evidence, however, has shown that inosine has direct neurotrophic effects. Thus, lack of direct neuroprotection, as reported before, may not be a general conclusion, but is related to special types of injury. We used PC12 cells to explore direct neuroprotection of inosine against high concentration of zinc sulfate, an injury different from the previous one and found that inosine reduced the mortality of PC12 cells significantly in a dose dependent manner. The results indicate that inosine can directly protect neurons from zinc-induced injury, and such effect might be mediated via mechanism(s) other than purine nucleoside phosphorylase.