| Part I CXCL12inhibits cortical neuron apoptosis after traumatic brain injury in rats by increasing the ratio of Bcl-2/BaxObjective:To investigate the effect of CXCL12on neuron apoptosis after traumatic brain injury (TBI) in rats and the mechanisms involved.Methods:1. Rat model of TBI was established by a fluid percussion device.2. The rats were randomly divided into4groups:(1) the sham group, the rats were performed craniotomy only without any injury or injection to the brain tissue,(2) the control group, normal saline were injected into the ipsilateral cortex after TBI,(3) the CXCL12group, CXCL12were injected into the ipsilateral cortex after TBI, and (4) the CXCL12+AMD3100group, CXCL12+AMD3100were injected into the ipsilateral cortex after TBI.3. At3days after TBI, we subjected brain tissues to in situ terminal deoxynucleotide transferase dUTP nick end labeling(TUNEL) assay and immunofluorescent staining with antibodies to NeuN and GFAP to detect apoptotic cells and their cell types. Immunofluorescent double-labeled staining were performed to assess the expression levels of CXCR4, active caspase-3, Bcl-2, and Bax in tissue sections.4. Western Blot assay was used to measure the protein expression levels of active caspase-3, Bcl-2, and Bax in different groups.5. Real time PCR was used to assess the mRNA levels in different groups.Results:1. At3days after TBI, apoptotic reaction around the injury site occurred primarily in neurons, which also expressed CXCR4. After treatment with CXCL12, the apoptosis index (AI) was significantly decreased compared with that of the control group. In the CXCL12+AMD3100group, the AI was significantly increased compared with that of the CXCL12group but still significantly lower than the control group.2. The protein and gene expressions of active caspase-3were significantly down-regulated in the CXCL12group than in the control group. CXCL12+AMD3100treatment significantly increased the expression level of active caspase-3compared with CXCL12treatment only, but it still significantly lower than that in the control group.3. The results showed significant up-regulation of Bcl-2and down-regulation of Bax in the CXCL12group compared with the control group. Using AMD3100together with CXCL12, the expression of Bcl-2was down-regulated and Bax was up-regulated compared with the CXCL12group. CXCL12significantly increased the Bcl-2/Bax ratio and AMD3100partially abolished this effect of CXCL12.Conclusions:Our results indicate that after TBI in rats CXCL12combing CXCR4receptors can inhibit the caspase-3pathway by up-regulating Bcl-2/Bax ratio, which protect neurons from apoptosis.Part ⅡCXCL12promotes proliferation and migration of neural precursor cells after traumatic brain injury in ratsObjective:To investigate the effect of the CXCL12on neural precursor cells proliferation and migration after traumatic brain injury (TBI) in rats and the mechanism involved.Methods:1. Rat model of TBI was established by a fluid percussion device.2. The rats were randomly divided into4groups:(1) the sham group, the rats were performed craniotomy only without any injury or injection to the brain tissue,(2) the control group, normal saline were injected into the ipsilateral cortex after TBI,(3) the CXCL12group, CXCL12were injected into the ipsilateralcortex after TBI, and (4) the CXCL12+AMD3100group, CXCL12+AMD3100were injected into the ipsilateral cortex after TBI.3. At7days after TBI, the brain tissues were subjected to immunofluorescent double-labeled staining with the antibodies of the BrdU/Nestin, BLBP/Nestin, BLBP/Vimentin, BLBP/SOX2, BLBP/CXCR4, DCX/BLBP, DCX/BrdU, DCX/GFAP, DCX/NeuN, MMP-2/DCX, MMP-2/GFAP, MMP-2/NeuN, CXCR4/DCX; immunofluorescent staining was used to detect the expression of MMP-2.4. Western Blot assay and Real time PCR were used to measure the protein and gene expression levels of Nestin, BLBP, Vimentin, and MMP-2in different groups.5. Neural stem cells (NSCs) were isolated and cultured from embryonic rat hippocampus. Immunofluorescent double-labeled staining of BrdU/Nestin and SOX2/CXCR4were used to detect their embryonic characteristics and self-proliferating ability. Immunofluorescent staining of GFAP, MAP-2, CNP were used to detect their capacities of multipotent differentiation.6. The cultured NSCs were divided to3groups:the control group were added with TBI rat extract, the CXCL12group were added with TBI rat extract and CXCL12, the CXCL12+AMD3100group were added with TBI rat extract and CXCL12+AMD3100. Immunofluorescent staining of DCX was used to detect the differentiation of NSCs to DCX positive cells.Results:1. Compared with the control group, CXCL12treatment significantly increased the positive cells double stained with BrdU/Nestin, BLBP/Nestin, and BLBP/Vimentin around the injured cortex and corpus callosum areas. CXCL12+AMD3100treatment significantly decreased the number of these cells compared with the CXCL12treatment and control group. The protein and gene expressions of Nestin, BLBP, and Vimentin had the same change trends as the results of immunofluorescent staining.2. The BLBP/Vimentin positive cells presented with the astrocyte pattern around the injured cortex area but with the RGCs pattern around the injured corpus callosum area, where most of these cells had long bipolar axon. The BLBP positive cells also expressed CXCR4and SOX2.3. Compared with the control group, the number CXCR4/DCX positive cells in the CXCL12treatment group were significantly increased around the injured corpus callosum areas. The area occupied by these cells also increased and changed from chain to radial distribution. Outside these areas, there were more dispersed CXCR4/DCX cells. CXCL12+AMD3100treatment significantly decreased the number and distribution area of the CXCR4/DCX positive cells compared with the control group. DCX positive cells could not form chain or radial distribution.4. There was a small amount of DCX/BLBP positive cells in the ipsilateral corpus callosum and most of the BLBP positive cells located very closely to the DCX positive cells. In the DCX/BrdU double staining, part of the DCX positive cells showed BrdU positive staining. In the DCX/GFAP double staining, DCX positive cells located immediately adjacent to the inner side of the GFAP positive cells and we did not observed any cells simultaneously stained with DCX and NeuN.5. Compared with the control group, the number of MMP-2positvie cells was significantly increased after treatment with CXCL12in the ipsilateral brain. CXCL12+AMD3100treatment significantly decreased the number of these cells compared with the CXCL12treatment and control group. The protein and genes expressions of MMP-2had the similar change trends as the results of immunofluorescent staining.MMP-2could be secreted by DCX, GFAP and NeuN positive cells.6. The cultured NSCs showed no significant difference in the differential capacity to DCX positive cells in three experimental groups.Conclusion:CXCL12promotes the proliferation of neural precursor cells by combing to its receptor, CXCR4. The proliferating neural precursor cells presents radial glial cell like cells, which also showed the morphologic characteristics long bipolar axons in the ipsilateral corpus callosum. CXCL12/CXCR4axis can improve the migration of the immature neurons (neuroblast) along the corpus callosum by stimulating the MMP-2secretion of different types of cells. The immature neurons can be differentiated from the radial glial cells, but CXCL12cannot change the differentiation of NSCs to immature neurons. The migration of the immature neurons in the corpus callosum area shows tangential and radial characteristics.Part III CXCL12promotes angiogenesis after traumatic brain injury in ratsObjective:To investigate the effect of the CXCL12on proliferation of blood vessel after traumatic brain injury (TBI) in rats and the mechanism involved.Methods:1. Rat model of TBI was established by a fluid percussion device.2. The rats were randomly divided into4groups:(1) the sham group, the rats were performed craniotomy only without any injury or injection to the brain tissue,(2) the control group, normal saline were injected into the the ipsilateral cortex after TBI,(3) the CXCL12group, CXCL12were injected into the the ipsilateral cortex after TBI, and (4) the CXCL12+AMD3100group, CXCL12+AMD3100were injected into the the ipsilateral cortex after TBI.3. At7days after TBI, immunofluorescent staining was used to detect the expression of CD31、vWF and VEGF. We calculated the microvessel density (MVD) with the staining results of CD31and vWF. We also calculate the optical density (OD) of VEGF.4. Western Blot assay and Real time PCR were used to measure the protein and gene expression levels of VEGF in different groups.Results:1. The MVD of CD31and vWF were significantly increased in the CXCL12treatment group compared with those in the control group. After treatment with CXCL12+AMD3100, the MVD of CD31and vWF were significantly decreased compared with the treatment of CXCL12as well as control group.2. The OD values of VEGF were significantly increased in the CXCL12treatment group compared with those in the control group. After treatment with CXCL12+AMD3100, the OD values of VEGF were significantly decreased compared with the treatment of CXCL12as well as control group.3. The expression levels of VEGF protein and gene were significantly increased in the CXCL12treatment group compared with those in the control group. After treatment with CXCL12+AMD3100, the expression levels of VEGF protein and gene were significantly decreased compared with the treatment of CXCL12as well as control group.Conclusion:CXCL12improves proliferation of microvessel in rat brain after TBI, which is related to the high expression of VEGF induced by the CXCL12/CXCR4axis. |
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