| Objective Traditionally, people believe that the central nervous system(CNS) hardly has the capability to regeneration. In recent years, people found that persistent neurogenesis exists in some areas in CNS. Neural stem cells (NSC)generated from subventricular zone(SVZ) migrate to olfactory bulb(OB) along rostral migratory-stream(RMS)where they differentiate to granule cells, which is very important to olfactory study and memory, and bring hope to the therapy of cerebral ischemia. However, the ability to repair the damage to the CNS is still very limited after ischemia. There are some reasons for this phenomenon. Firstly, the ability of proliferation and migration of progenitor cells is very limited. Secondly, some inhibitors obstruct axonal regeneration, such as repulsive guidance molecule A (RGMa). Here, we exerted two kinds of intervention including OB lesion and stimulation to normal rats to investigate the effects of OB lesion and stimulation on proliferation, migration and differentiation on NSC in SVZ, then we focused on the results of electrical stimulation of OB on proliferation, migration, differentiation on NSC in SVZ and RGMa expression after focal cerebral ischemia/reperfusion injury in rats.Methods 1. Normal rats were exerted two kinds of intervention including OB lesion and electrical stimulation. OB lesion was induced by N-methyl-D-aspartic acid (NMDA) injection. Electrical stimulation was performed via a bipolar electrode implanted in the right OB of rats. Bromodeoxyuridine (Brdu) was injected intraperitoneally to label NSC. Immunohistochemistry staining was used to detect the proliferation and migration of NSC in SVZ. Immunofluorescence was used to investigate the differentiation of NSC in OB. 2. Rats were suffered from focal cerebral ischemia/reperfusin insults. Focal cerebral ischemia/reperfusin (I/R) was induced by intraluminal right middle cerebral artery occlusion (MCAO). 5-bromo-2-deoxyuridine (Brdu) was injected intraperitoneally to label NSC. Immunohistochemistry staining was used to detect the proliferation of NSC in SVZ. Fluorescent double staining for Brdu/Neuronal nuclei antigen (Neun)and Brdu/Glial fibrillary acidic protein (GFAP) was performed finally. 3. Rats were suffered from focal cerebral ischemia/reperfusin insults and exerted electrical stimulation on OB. The behavioral test was conducted by using the modified neurological severity score (mNSS).The changes in the expression of RGMa were analysed by reverse transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemistry, respectively. Neurofilament protein 200 (NF-200) immunohistochemical staining was used to assess axonal regeneration. Another group of rats were divided into sham-operated group, I/R group, sham-stimulated group and stimulation group. The infarct volume of brain was determined by triphenyltetrazolium chloride (TTC) staining.Results 1. In SVZ, Brdu-positive cells began to increase 1 day after OB lesion had been made (P<0.05), reached to maximum at week 1 (P<0.01) and still sustained a high level at week 4. Five weeks after the lesion was induced, the Brdu-positive cells significantly increased in RMS and OB (P<0.01). By using fluorescence double staining, we showed that most of Brdu-positive cells co-localized with neuronal nucl nuclei antigen (Neun), and a portion of Brdu-positive cells expressed glial fibrillary acidic protein (GFAP).In SVZ, Brdu-positive cells began to increase 1 day after stimulation(P<0.01), reach to maximum at week 1(P<0.01), then decrease to normal at week 3. Four weeks after injection of Brdu, there were more Brdu-positive cells in RMS and OB (P<0.01) in stimulation group than normal group. Fluorescence double staining showed that stimulation of OB had no effect on the differentiation of NSC into neurons or gliocytes. 2. In SVZ of rats, Brdu-positive cells began to increase 48h after ischemia in I/R group and stimulation group. There were more Brdu positive cells in stimulation group than I/R group at 48h (P<0.05) and 7d (P<0.01) after ischemia/reperfusion in rats. Four weeks after stimulation, the Brdu-positive cells significantly increased in cortex in stimulation group than I/R group (P<0.01). Fluorescence double staining showed that stimulation of OB had no effect on the differentiation of NSC into neurons or gliocytes (P>0.05). 3. The behavioral test showed that neurological function was better in stimulation group than in I/R group and sham-stimulated group. The expression of RGMa mRNA and protein were greatly elevated at both 48 hours and 7days after ischemia/reperfusion in I/R group and sham-stimulated group. The level of RGMa expression was lower in stimulation group than I/R group and sham-stimulated group. Immunohistochemistry staining showed that OB stimulation upregulated NF-200 expression in ischemic cortex of rats. OB stimulation also diminished infarct volume in rats with cerebral ischemia/reperfusion injury.Conclusions 1. OB lesion and stimulation improved proliferation of NSC in SVZ and made more NSC migrate to OB in rats. Those newborn cells in lesion group not only differentiated into neurons, but also gliocytes which might repaire the lesion. Electrical stimulation of OB has no effect on the differentiation of NSC into neurons or gliocytes. 2. Electrical stimulation of OB might enhance proliferation of NSC in SVZ and promote migration of NSC to ischemic cortex, but OB stimulation has no effect on differentiation of NSC. 3. Electrical stimulation of OB downregulated RGMa expression, reduced infarct volume, and promoted axonal regeneration and functional recovery after cerebral ischemia/reperfusion injury in rats.
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