| Background Although studies has shown that the involvement of N-methyl D-aspartate receptor(NMDAR)in neurogenesis of rat's subventricular zone.NMDAR plays an important role in the proliferation of neural stem cell(NSCs),differentiation,survival and migration.However,NMDAR subunits effects on neurogenesis is unclear and little is known about the express of NMDAR subunits in the SVZ of rats at early postnatal rats.At present,the role of NR1(NMDA receptor subunit)subunit in the mechanism of neurogenesis in the SVZ after cerebral ischemia-reperfusion is not clear.This study includes two parts.First PartObjectives The animals received either intraperitoneal injection of NMDAR agonist NMDA or NMDAR antagonist,and then we determined observation of the cell proliferation in SVZ of the neonatal Sprague-Dawley rats.We also study the effects of NMDAR agonist and NMDAR antagonist on neural stem cells proliferation in subventricular zone of the neonatal Sprague-Dawley rats.Immunohistochemical staining was used to assay the number of Brdu and Nestin-posifive cells in the SVZ.Methods The normal rats at early postnatal age were randomly divided into six groups,which were the normal group,NMDA group,MK-801 group,Ro25-6981group,NVP-AAM077 group as well as the saline control group.Each group was fed normally to the relevant time point i.e.,postnatal day 7(PND-7),PND-14,PND-21,and PND-28.Rats were intraperitoneally injected NMDA receptor agonist NMDA(2mg/kg/d),selective non-competitive NMDA receptor antagonist MK-801(10 mg/kg),NR2A antagonist NVP-AAM077(10 mg/kg)and NR2B antagonist Ro25-6981(40mg/kg)respectively at the age PND-3.At each time point,Brdu was injected intraperitoneally to label proliferation cell.Immunohistochemical staining was used to assay the number of Brdu posifive cells and Nestin-posifive cells in the SVZ.In the normal group,hematoxylin-eosin(HE)staining was used to assay total number of cells.Results 1)The result of HE staining showed that the number of positive cells in the SVZ decreased with age during the early postnatal period.2.The result of BrdU immunohistochemical staining showed that at the age P7 d,the number of BrdU positive cells in the SVZ of NMDA group,MK-801 group and Ro25-6981group decreased significantly compared with the saline control group(P<0.05);Compared to MK-801 group,the number of positive cells in the NVP-AAM077group and the saline control group increased significantly(P<0.05).At the age P14 d,compared to the saline control group,NMDA group,MK-801 group,Ro25-6981group the number of positive cells decreased significantly(P<0.05);while the number of positive cells in rats'SVZ of the saline control group,NMDA group and NVP-AAM077 group increased significantly compared with MK-801 group(P<0.05).At the age P21 d,Compared with the saline control group,the number of positive cells in the SVZ of NMDA group and Ro25-6981 group increased significantly(P<0.05).And compared to MK-801 group,the number of positive cells in the NMDA group increased significantly and in the NVP-AAM077 group reduced significantly(P<0.05).At the age P28 d,compared to the saline control group as well as MK-801 Group,the cell count of NMDA group increased significantly(P<0.05).2)The result of Nestin immunohistochemical staining showed that at the age P7 d,compared with the saline control group,the number of positive cells decreased significantly in the NMDA group,MK-801 group and Ro25-6981 group(P<0.05).Compared with MK-801 Group,the number of positive cells increased significantly in the saline control group,NMDA group and NVP-AAM077 group(P<0.05).At the age P14 d,compared with the saline control group,the number of positive cells in the NMDA group,MK-801 group and Ro25-6981 group decreased significantly(P<0.05).Compared with MK-801 Group,the number of positive cells in the saline control group,NMDA group and NVP-AAM077 group were increased significantly(P<0.05).At the age P21 d,compared to the saline control group,the number of positive cells in the NMDA group increased significantly.At the age P28d,compared to the saline control group,the number of positive cells in the NMDA group and Ro25-6981 group increased significantly(P<0.05).Conclusions There are large number of proliferating cells and neural stem cells in the SVZ of rats at early postnatal age.The number of positive cells in the SVZ showed the downtrend with age during the early postnatal period.Moderate activation of NMDA receptors could promote the cell proliferation in the SVZ.On the contrary,inhibition of NMDA receptors will make the cell proliferation decrease through inhibiting the subunit NR2B in the SVZ.Second PartObjectives This study sought to determine the distribution and subcellular localization of NR1 in NSCs of adult rat SVZ,using pre-embedding double labeled immunoelectron microscopy,in order to provide morphological evidence for the NR1 function in SVZ NSCs.Methods To developed a transient middle cerebral artery occlusion(MCAO)model in rats.Rats were randomly divided into 4 groups:Nomal group,MCAO group and Sham group.SVZ tissues were sliced into coronal sections with a vibrating slicer(Vibratoivie,Bannockburn,IL,USA)at 150?m thickness.Sections were immersed in an increasing sucrose gradient for 2 hours.After freezing and thawing,the sections were returned to the ice crystal protected fluid.Sections were rinsed with PBS three times,5 minutes each,and then incubated with 10%normal rabbit serum at room temperature for 3 hours.Immunohistochemical staining was used to assay nestin expression and Immunogold staining of NR1 expression.NR1/nestin immune double-labeled staining,the same sections underwent both nestin immunohisto-chemistry and NR1 immunogold labeling.Specimens were prepared for electron microscopy.SVZ immunoreactive tissues were cut into 1-mm~3 pieces under the stereomicroscope and fixed with 1%osmium tetroxide at room temperature for45minutes.Pieces were rinsed with deionized water for 1 hour,before gradient ethanol dehydration,resin replacement,saturation,embedding and polymerization.Ultrathin sections were cut at 70 nm and stained with uranyl acetate and lead citrate,then observed under transmission electron microscopy.Results 1)NR1-positive cells were observed by light microscopy on the membrane of various types of SVZ cells in each group.Compared to the normal group,the number of NR1-positive cells not increased significantly(P>0.05).The IOD values of NR1 positive cells were increased at 1 day after ischemia-reperfusion.After 7 days of cerebral ischemia-reperfusion,the number of NR1-positive cells were reached maximum in the MCAO group,and after 14 days of cerebral ischemia-reperfusion the number of positive cells were reduced,but it remained at 3days level.By using immune double-labeled staining,Nestin/NR1 positive cells were observed by light microscopy in the SVZ in each group.Compared with normal group,the IOD values of Nestin/NR1 positive cells were not significant change(P>0.05).The IOD values of Nestin/NR1 positive cells were increased at 1day after reperfusion.After 7 days of cerebral ischemia-reperfusion,the number of NR1-positive cells were reached maximum in the MCAO group,and after 14 days of cerebral ischemia reperfusion the number of positive cells were reduced,it remained at 3 days level.2)Under electron microscopy,the nestin immunoreactive product was a black,electron-dense flocculent substance which stained all cells.Nestin-positive cells were small,with irregular shape,high nuclear-cytoplasm ratio,one or two irregular nuclei,membrane retraction and abundant chromatin.Little cytoplasm,slight staining,low electron density,and a single cilium,directly or indirectly contacting the lateral ventricle were observed.Clusters of free ribosomes were occasionally observed,accompanying a small rough endoplasmic reticulum and Golgi complex.Electron microscopy revealed two different positive labeling signals in the NSCs of adult brain SVZ.One was the nano-gold-silver particles,and the other was nestin immunoreactivity products.These two products are easily distinguished.Nestin immunoreactivity products were flocculent and distributed throughout the NSC cytoplasm.NR1 immunogold-silver particles were black,electron-dense particles,which were located in the NSC membrane,cytoplasm,rough endoplasmic reticulum,and the Golgi complex.Compared with the normal group and sham group,after cerebral ischemia,the number of NR1-positive cells increased significantly in the neural stem cells residing in the adult rats subventricular zone.After cerebral ischemia 1 day,NR1 positive cells were increased,at 1 days after cerebral ischemia,the number of NR1-positive cells reached maximum,and then the number of neural stem cells reached maximum.Conclusions 1)Ischemic damage stimulated proliferation of the adult rat brain SVZ neural stem cell.2)Early stage of cerebral ischemia can induce the number of NR1 cells increased which in neural stem cells of SVZ,because the mode of NR1-positive cells increased in accordance with the neural stem cells increase induced by cerebral ischemia.So we speculate that NR1 may participate in the SVZ neural stem cell proliferation. |
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