Proliferation And Differentiation Of Endogenous Neural Stem Cells After Spinal Cord Injury And Related Mechanism

Degeneration, necrosis of numerous neural cells in damaged lesionafter spinal cord injury (SCI) always leads to the patients with limbmovement and sensation dysfunction. How to regulate the regeneration ofneural cells in injured spinal cord after SCI to repair the tissue structure andlimb function has been the focus and difficulty of the neuroscientists formany years.The proposaling of endogenous neural stem cells (ENSCs) bring hopefor the regeneration of spinal cord injury. Experiments confirmed thatendogenous neural stem cells are found in the central canal of the spinal cordand its surroundings. After SCI, ENSCs showed varying degrees of stemcell-like reaction, such as proliferation, migration and differentiation.However, its distance of migration is quite short, proliferation isinsurfficient, differentiation also becomes to more GFAP positive astrocytesand no or small propotion of functional neurons which is the best choice ofcompletely renewable in SCI. Accordingly, exploring the molecularmechanism of ENSCs proliferation and differentiation would provide theoretical basises and experimental evidences for regulation of ENSCs infurther SCI treatment.In terms of central nervous system (CNS) development, many factorsparticipate in the regulation of neural stem cells (NSCs) in theirdifferentiation process to neurons and gliocytes. The proliferation andsubtype’s determination of neurons or gliocytes formation were largelycontrolled by Notch pathway and bHLH family. Activating bHLH familyfactors includ Mash, Math, Ngn, Olig, while the the inhibiting factorsalways refere to Hes1/3/5. The activating factors highly expressed, NSCswill differentiate into neurons while the inhibiting genes upregulated NSCswill maintain its properties of stem cells, and differentiate into gliacytes.However, the relationship between the expression levels of these factorsand ENSCs proliferation, differentiation after SCI is still rarely reported.Therefore, we firstly labeled to ENSCs by DIL fluorescentl dye usingthe measure of intracerebroventricular micro injection; then the proliferationand differentiation of ENSCs after SCI were obsereved by HE stianing andimmunofluorescent in mild SCI model of rats; finally, some of the bHLHfamily factors were analysised after SCI by some molecular biologymethods in order to explore its possible mechanisms.Main techniques and methods：1.10μl DAPI or DIL were stereotaxically injected to the lateralventricle of rats respectively. After the operation, neurological status of the rats’ wrer evaluated. The labeled ENSCs wrer observed at different timepoints of both fluorescent dyes.2. Allen’s impactor was used to prepare with a10g12.5mm forcesrats model of mild SCI. BBB score and evoked potential testing were used toevaluate the hind limb motor function and nerve conduction velocity. HEstaining of damaged spinal cord tissue and changes in the number ofependymal cells were recorded. Double immunofluorescence of ENSCs atdifferent times after SCI was examined and the GFAP or β-tubulinIIIpositive cells were counted.3. The expression of Notch1and some of bHLH family members likeHes1, Ngn2, Olig2was estimated by western-blot, RT-PCR andimmunofluorescence after SCI. The possible mechanism of ENSCs’proliferation and differentiation direction was discussed.Mian results：1. Ventricule ependymal cells were fine labeled by DAPI，also was thechoroid membrane. However, there was no spinal cord ependymal wereobserved labeled by DAPI at any time after injection. Ependymal of theventricles and spinal cord was labeled well24hours after the DIL injection.The labeling time of DAPI and DIL is pretty long after the injection，forthere were still no significant attenuation observed at day14th(P>0.05).2. Neurological severity scores (NSS) showed that theintracerebro-ventricular injection of DAPI or DIL is a relatively safe and harmless operation to rats.3. Mild Allen’s strike of SCI model was still able to cause rats withobvious paralysis of the hind limb. BBB score and electrophysiological ofMEP testing showed significant difference compared with the sham group(P<0.05). HE staining indicated bleeding, tissue dameging, cavitation andglial scar formation in injuried spinal cord. Recover in rats after SCI wasobserved, but the degree remians limited.4. Apparent proliferation of ependymal cells and short distancemigration was obsrved after SCI. Most proliferation of the ependymal cellsdifferentiated to the GFAP positive astrocytes, while the β-tubulinIII positivenerons were much fewer compared with the former (P <0.05).5. RT-PCR and Western-blot detected that activating genes Ngn2andOlig2transient increased (P <0.05) at1,3days after SCI but significantlyreduced and lasted to day14th (P <0.05); Notch1and the inhibiting gene andHes1were apearently raised after SCI, and continued until14days postinjuried (P <0.05).6. Double Immunofluorescent: Notch1and Hes1co-expressing cells,β-tubulinIII and Ngn2co-expressing cells co-expressing cells, GFAP andHes1co-expressing cells were observed around the central canal of theinjuried spinal cord.Conclusions：1. Fluorescent dye DIL was suitable for a long period of brain and spinal cord ENSCs’ labeling, while DAPI was suitable for ventriculeependymal region of endogenous neural stem cells’ labeling.2. Mild SCI in rats can induce the proliferation and differentiation ofENSCs, but the majority number of ENSCs differentiated into astrocytes notneurons. This is one of the important reasons why the ability of spontaneousrepairing of SCI patients is poor.3. Expressions of Nocth1bHLH transcription factors after SCI havestrong corelation with ENSCs’ proliferation and differentiation. Duringwhich, sustained high expression of the Notch1and inhibiting Hes1withlower expression of the activating Olig2and Ngn2may lead to ENSCs’proliferation and differentiation limitation after SCI.