| PART 1 Recombinant Annexin A2 improves early neurobehavioral performance through blood-brain barrier protection following controlled cortical impactObjectiveThe aim of this study was to determine the role of Annexin A2 (A2) in neurobehavioral recovery, BBB permeability and edema formation following TBI.MethodsExperiment one:The temporal and cell-type specific expression of endogenous A2 protein after CCI.After establishing the CCI model on adult male mice, the time course of annexin A2 protein expression in injuried hemisphere before and after TBI was examined by western blot. Then the brain section from animals at 7 days after CCI was double-stained with anti-A2 and other cell type antibodies (CD31, GFAP, NeuN), respectively.Experiment two:Effect of rA2 on BBB permeability, edema and neuronal survival in hippocampus following CCIAfter eatablishing the injury model on cultured endothelial cells, the TEER and FITC-dextran leakage assay was measured to describe the endothelial permeability. Following CCI model on mice, BBB permeability in vivo was quantitated with Evans blue extravacation in the hemispheres. The water content in wet-dry protocol was applied to elucidate edema formation in the hemispheres. In addition, hippocampal neurons was quantified following CCI by immunofluorescence, and cellular viability of neurons under hypoxia treated with endothelial-conditioned medium was measured between rA2 and control groups.Experiment three:Effect of rA2 on early neurobehavior performance and lesion size following CCINSS test and wire-gripping test was performed to describe the neurological behavior in the early phase following CCI between rA2 and control groups. The effect of rA2 on lesion size was detected in HE staining of CCI brain section.Experiment four:Mechanism of rA2 protection on BBB integrity following CCIThe gelatin zymography was applied to analyse MMP-9 activity in the injuried hemispheres following CCI. Then the content of VEGF, Occludin, Claudin-5 and ZO-1 protein in the injuried hemispheres was quantified by western blot. Following injury model in vitro, the effect of rA2 on the stress fiber formation within cultured endothelial cells was determined.ResultsExperiment one:Following CCI model, endogenous A2 expression increased since day 3 (P<0.05) and peaked on day 7 (P<0.05). Since then the protein level decreased gradually and returned to baseline on day 21. Compared with sham group, the expression of A2 in the endothelial cells increased on day 7 following CCI. The A2 expression can be detected in both astrocytes and neurons before and after injury.Experiment two:With different administrative dose (0.75mg/kg, 1mg/kg,1.5mg/kg) and timing (2h,4h,6h), the evans blue extravacation in the injuried hemisphere was attenuated by rA2 treatment with significance (P<0.05). However, no significant effect on edema formation following CCI was found in rA2 group (P>0.05). At 7 days after CCI, the rA2 group exhibited significantly improved neuronal survival in both CA1 and CA3 areas (P<0.05). In vitro the neuronal viability at 24 hours after hypoxia increased significantly following rA2 treatment (P<0.05)Experiment three:The NSS test result indicated that the motor function was improved by rA2 administration in 7 days following significantly (P<0.05). In the wire-gripping test, it showed no difference in the performance between two groups (P>0.05). The lesion size at 7 days after CCI was negatively affected by rA2 treatment (P>0.05)Experiment four:At 7 days following CCI, no significant effect was found neither in the MMP-9 activity by rA2 treatment (P>0.05), nor the VEGF, Occludin and Claudin-5 expression in the injuried hemisphere (P>0.05). In the contrast, rA2 significantly increase ZO-1 expression (P<0.05). Furthermore, the fluorescent intensity for F-actin stress fiber formation in the cultured endothelial cells following injury was significantly ameliorated by rA2 treatment (P<0.05)ConclusionWe thus propose exogenous rA2 as a critical regulator of BBB permeability following TBI, via correcting ZO-1 expression and stress fiber formation, hence improves clinical outcome.PART 2 Recombinant Annexin A2 improves long-term functional recovery through promoting angiogenesis following controlled cortical impactObjectiveThe aim of this section is to evaluate the biological effect of recombinant A2 on the long-term functional recovery following TBI. Then the underlying mechanism would be explored.MethodsExperiment one:The effect of rA2 on long-term functional recovery and lesion size following TBI.NSS test, Rotor-rod test and Morris water maze test were performed to compare the neurological behavior following CCI between rA2 and control groups. The effect of rA2 on lesion size in the late phase was detected in HE staining of CCI brain section.Experiment two:Effect of rA2 on angiogenesis of CCI animals and its underlying mechanism.The effect of rA2 on endothelial viability, tube formation and migration was measured in vitro. The brain section following CCI were double-stained with CD31 and Brdu for enthelial proliferation. The functional vessel density was quantified in the positive area of intravenously-injected Lectin. The cortical microvessel was isolated from CCI brain hemispheres for in situ plasmin activity detection in the vessel.Experiment three:Effect of rA2 on neurogenesis of CCI animals and its underlying mechanism.The brain section following CCI were double-stained with Nestin and Brdu for neural proliferation. The newly-born neurons on CCI brain section were defined as NeuN/Brdu double-positive cells. The brain section following CCI were double-stained with PSA-NCAM/Brdu for neuroblasts migration. The level of BDNF and VEGF in the injuried hemisphere was quantified by western blot. Furthermore, the mRNA level of BDNF and VEGF was detected from cortical microvessel of injuried hemisphere.ResultsExperiment one:The NSS test and Rotor-Rod test indicated that the motor function was improved by rA2 administration in 28 days following significantly (P<0.05). In the water maze test, it showed no difference in the learning ability between groups (P>0.05). However, rA2 significantly elevated the spatial memory of CCI animals (P<0.05). The lesion size at 28 days after CCI was negatively affected by rA2 treatment (P>0.05)Experiment two:At 48 hours after rA2 exposure, the endothelial viability, tube formation and migration capability was significantly elevated (P>0.05). Durning the first 7 days following CCI, increased endothelial proliferation in the both hippocampus and lesioned area was detected in the rA2 group(P<0.05). Consistently, the functional vessel density was elevated by rA2 treatment (P<0.05). In contrast, no difference was observed in the hippocampus area (P>0.05). The plasmin activity in the microvessel was higher in the rA2 group compared with the control group.Experiment three:No significant difference was found in the neural stem cell proliferation in the SVZ and SGZ area by rA2 treatment at 7 days following CCI(P>0.05). Nevertheless, the newly-born neuron number in the peri-lesion and dentate gyrus (DG) area was elevated by rA2 with significance in the 28 days (P<0.05). Meanwhile, the rA2 treatment leaded to increased neuroblasts migration to the injuried area (P<0.05). rA2 significantly increases pro-angiogenic and trophic factor protein (BDNF/VEGF) expression in peri-lesion brain tissues at 7 days after CCI (P<0.05). Moreover, higher level of mRNA of above factors was detected in the isolated microvessel by rA2 treatment (P<0.05)ConclusionAfter TBI, rA2 can promote angiogenesis via increased plasmin activity in the microvessels, coupled with increased pro-angiogenic and trophic factor protein (BDNF/VEGF) expression, which can attract the mobile neuroblasts migrating to lesion area along the vessel. After arriving the destination (peri-lesion area), naive neuroblasts can develop into mature neurons, integrated into existed "neurotransmission network". |
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