Effects of recombinant adenovirus-mediated hypoxia-inducible factorlalpha gene on proliferation and differentiation of endogenous neural stem cells in rats following intracerebral hemorrhage

Objective:To investigate the effects of adenovirus(Ad)-mediated hypoxia-inducible factor-1alpha(HIF-1α)gene on proliferation and differentiation of endogenous neural stem cells(NSCs)in rats following intracerebral hemorrhage(ICH)and the underlying mechanisms.Methods:A total of 120 specific pathogen-free,adult,male Sprague-Dawley rats were included in this study.After establishment of ICH models in rats,PBS,Ad,or Ad-HIF-1αwas administered via the ischemic ventricle.On the 1st,7th,14th,21st and 28th d after ICH,rat neurological deficits were scored,doublecortin(DCX)expression in the subventricular zone cells was detected by immunohistochemical staining,and 5-bromo-2’-deoxyuridine(Brdtl)-,BrdU/DCX-,and BrdU/glial fibrillary acidic prolein-posilive cells in the subventricular zone were counted using immumofluorescence method among PBS,Ad,and Ad-HIF-1αgroups.Results:On the 7th,14th,21st and 28th d after ICH,neurological deficit scores in the Ad-HIF-1αgroup were significantly lower than in the PBS and Ad groups(P【0.05).In the Ad-HIF-lαgroup,DCX expression was significantly increased on the 7th d,peaked on the 14th d,and then gradually decreased.In the Ad-HIF-1αgroup,BrdU-positive cells were significantly increased over time course,and significant difference in BrdU-positive cell counts was observed when compared with the PBS and Ad groups at each time point(P【0.01 or 0.05).On the 7th,14th,21st and 28th d after ICH,the number of DCX-,BrdU-,BrdU/DCX-,and BrdU/DCX-positive cells in the Ad-HIF-1αgroup was significantly greater than in the PBS and Ad groups(P【0.05).Conclusions:HIF-1αgene can promote the proliferation,migration and differentiation of endogenous neural stem cells after ICH,thereby contributing to neurofunctional recovery after ICH.

Basic fibroblast growth factor increases the numbe of endogenous neural stem cells and inhibits the expression of amino methyl isoxazole propionic acid receptors in amyotrophic lateral sclerosis mice

This study aimed to investigate the number of amino methyl isoxazole propionic acid （AMPA） receptors and production of endogenous neural stem cells in the SOD1 G93AG1H transgenic mouse model of amyotrophic lateral sclerosis, at postnatal day 60 following administration of basic fibroblast growth factor （FGF-2）. A radioligand binding assay and immunohistochemistry were used to estimate the number of AMPA receptors and endogenous neural stem cells respectively. Results showed that the number of AMPA receptors and endogenous neural stem cells in the brain stem and sensorimotor cortex were significantly increased, while motor function was significantly decreased at postnatal days 90 and 120. After administration of FGF-2 into mice, numbers of endogenous neural stem cells increased, while expression of AMPA receptors decreased, whilst motor functions were recovered. At postnatal day 120, the number of AMPA receptors was negatively correlated with the number of endogenous neural stem cells in model mice and FGF-2-treated mice. Our experimental findings indicate that FGF-2 can inhibit AMPA receptors and increase the number of endogenous neural stem cells, thus repairing neural injury in amyotrophic lateral sclerosis mice.

Effect of valproic acid on endogenous neural stem cell proliferation in a rat model of spinal cord injury

BACKGROUND： Valproic acid has been reported to decrease apoptosis, promote neuronal differentiation of brain-derived neural stem cells, and inhibit glial differentiation of brain-derived neural stem cells. OBJECTIVE： To investigate the effects of valproic acid on proliferation of endogenous neural stem cells in a rat model of spinal cord injury. DESIGN, TIME AND SETTING： A randomized, controlled, neuropathological study was performed at Key Laboratory of Trauma, Buming, and Combined Injury, Research Institute of Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA between November 2005 and February 2007. MATERIALS： A total of 45 adult, Wistar rats were randomly divided into sham surgery （n = 5）, injury （n = 20）, and valproic acid （n = 20） groups. Valproic acid was provided by Sigma, USA. METHODS： Injury was induced to the T10 segment in the injury and valproic acid groups using the metal weight-dropping method. The spinal cord was exposed without contusion in the sham surgery group. Rats in the valproic acid group were intraperitoneally injected with 150 mg/kg valproic acid every 12 hours （twice in total）.MAIN OUTCOME MEASURES： Nestin expression （5 mm from injured center） was detected using immunohistochemistry at 1,3 days, 1, 4, and 8 weeks post-injury. RESULTS： Low expression of nestin was observed in the cytoplasm, but rarely in the white matter of the spinal cord in the sham surgery group. In the injury group, nestin expression was observed in the ependyma and pia mater one day after injury, and expression reached a peak at 1 week （P 〈 0.05）. Expression was primarily observed in the ependymal cells, which expanded towards the white and gray matter of the spinal cord. Nestin expression rapidly decreased by 4 weeks post-injury, and had almost completely disappeared by 8 weeks. At 24 hours after spinal cord injury, there was no significant difference in nestin expression between the valproic acid and injury groups. At 1 week, there was a significant increase in the number of nestin-positive cells surrounding the central canal in valproic acid group compared with the injury group （P 〈 0.05）. Expression reached a peak by 4 weeks, and it was still present at 8 weeks. CONCLUSION： Valproic acid promoted endogenous neural stem cell proliferation following spinal cord injury in rats.

Effects of leukemia inhibitory factor on endogenous neural stem cell proliferation and glycoprotein-130 expression in a mouse model of cerebral infarction

Leukemia inhibitory factor （LIF） has been shown to promote proliferation of endogenous neural stem cells. In this study, we treated mice with cerebral infarction using LIF to investigate whether the LIF receptor subunit glycoprotein （gp）130 is involved in neuroprotection. After LIF treatment, the motor function of model mice was significantly improved. Immunofluorescence histochemistry showed increased numbers of endogenous neural stem cells surrounding the infarct foci. Western blot analysis revealed that gp130 expression was significantly decreased surrounding the infarcted foci. Results demonstrated that LIF promoted the proliferation of endogenous neural stem cells by inhibiting gp130 protein expression.

Effects of leukemia inhibitory factor and basic fibroblast growth factor on free radicals and endogenous stem cell proliferation in a mouse model of cerebral infarction

The present study established a mouse model of cerebral infarction by middle cerebral artery occlusion, and monitored the effect of 25 tJg/kg leukemia inhibitory factor and （or） basic fibroblast growth factor administration 2 hours after model establishment. Results showed that following administration, the number of endogenous neural stem cells in the infarct area significantly increased, malondialdehyde content in brain tissue homogenates significantly decreased, nitric oxide content, glutathione peroxidase and superoxide dismutase activity significantly elevated, and mouse motor function significantly improved as confirmed by the rotarod and bar grab tests. In particular, the effect of leukemia inhibitory factor in combination with basic fibroblast growth factor was the most significant. Results indicate that leukemia inhibitory factor and basic fibroblast growth factor can improve the microenvironment after cerebral infarction by altering free radical levels, improving the quantity of endogenous neural stem cells, and promoting neurological function of mice with cerebral infarction.

Extracellular vesicles released by transforming growth factor-beta 1-preconditional mesenchymal stem cells promote recovery in mice with spinal cord injury

Spinal cord injury(SCI)causes neuroinflammation,neuronal death,and severe axonal connections.Alleviating neuroinflammation,protecting residual cells and promoting neuronal regeneration via endogenous neural stem cells(eNSCs)represent potential strategies for SCI treatment.Extracellular vesicles(EVs)released by mesenchymal stem cells have emerged as pathological mediators and alternatives to cell-based therapies following SCI.In the present study,EVs isolated from untreated(control,C-EVs)and TGF-β1-treated(T-EVs)mesenchymal stem cells were injected into SCI mice to compare the therapeutic effects and explore the underlying mechanisms.Our study demonstrated for the first time that the application of T-EVs markedly enhanced the proliferation and antiapoptotic ability of NSCs in vitro.The infusion of T-EVs into SCI mice increased the shift from the M1 to M2 polarization of reactive microglia,alleviated neuroinflammation,and enhanced the neuroprotection of residual cells during the acute phase.Moreover,T-EVs increased the number of eNSCs around the epicenter.Consequently,T-EVs further promoted neurite outgrowth,increased axonal regrowth and remyelination,and facilitated locomotor recovery in the chronic stage.Furthermore,the use of T-EVs in Rictor/SCI mice(conditional knockout of Rictor in NSCs)showed that T-EVs failed to increase the activation of eNSCs and improve neurogenesis sufficiently,which suggested that T-EVs might induce the activation of eNSCs by targeting the mTORC2/Rictor pathway.Taken together,our findings indicate the prominent role of T-EVs in the treatment of SCI,and the therapeutic efficacy of T-EVs for SCI treatment might be optimized by enhancing the activation of eNSCs via the mTORC2/Rictor signaling pathway.

Identification of key genes involved in recovery from spinal cord injury in adult zebrafish

Zebrafish are an effective vertebrate model to study the mechanisms underlying recovery after spinal cord injury.The subacute phase after spinal cord injury is critical to the recovery of neurological function,which involves tissue bridging and axon regeneration.In this study,we found that zebrafish spontaneously recovered 44%of their swimming ability within the subacute phase(2 weeks)after spinal cord injury.During this period,we identified 7762 differentially expressed genes in spinal cord tissue:2950 were up-regulated and 4812 were down-regulated.These differentially expressed genes were primarily concentrated in the biological processes of the respiratory chain,axon regeneration,and cell-component morphogenesis.The genes were also mostly involved in the regulation of metabolic pathways,the cell cycle,and gene-regulation pathways.We verified the gene expression of two differentially expressed genes,clasp2 up-regulation and h1m down-regulation,in zebrafish spinal cord tissue in vitro.Pathway enrichment analysis revealed that up-regulated clasp2 functions similarly to microtubule-associated protein,which is responsible for axon extension regulated by microtubules.Down-regulated h1m controls endogenous stem cell differentiation after spinal cord injury.This study provides new candidate genes,clasp2 and h1m,as potential therapeutic intervention targets for spinal cord injury repair by neuroregeneration.All experimental procedures and protocols were approved by the Animal Ethics Committee of Tianjin Institute of Medical&Pharmaceutical Sciences(approval No.IMPS-EAEP-Q-2019-02)on September 24,2019.

Application of the sodium hyaluronate-CNTF scaffolds in repairing adult rat spinal cord injury and facilitating neural network formation

The present study aimed to explore the potential of the sodium hyaluronate-CNTF (ciliary neurotrophic factor) scaffold in activating endogenous neurogenesis and facilitating neural network re-formation after the adult rat spinal cord injury (SCI). After completely cutting and removing a 5-mm adult rat T8 segment, a sodium hyaluronate-CNTF scaffold was implanted into the lesion area. Dil tracing and immunofluorescence staining were used to observe the proliferation, differentiation and integration of neural stem cells (NSCs) after SCI. A planar multielectrode dish system (MED64) was used to test the electrophysiological characteristics of the regenerated neural network in the lesioned area. Electrophysiology and behavior evaluation were used to evaluate functional recovery of paraplegic rat hindlimbs. The Dil tracing and immunofluorescence results suggest that the sodium hyaluronate-CNTF scaffold could activate the NSCs originating from the spinal cord ependymal, and facilitate their migration to the lesion area and differentiation into mature neurons, which were capable of forming synaptic contact and receiving glutamatergic excitatory synaptic input. The MED64 results suggest that functional synapsis could be established among regenerated neurons as well as between regenerated neurons and the host tissue, which has been evidenced to be glutamatergic excitatory synapsis. The electrophysiology and behavior evaluation results indicate that the paraplegic rats’ sensory and motor functions were recovered in some degree. Collectively, this study may shed light on paraplegia treatment in clinics.