| Background and Objective:Recently years, more and more extremely weather and climate events frequently happen all over the world. For example, the extremely cold and snowy weather event happened in southern China in the beginning of the year 2008. The extremely cold and snowy weather event not only can result traffic accidents and building collapse, increasing the incidence rate of trauma, but also often can get trauma patients exposed to low temperature a period of time following injury because of slippery road and traffic jams. Traumatic brain injury (TBI) is a leading cause of death and disability wordwide thar threatens our life and health seriously. However, it is still unclear that what changes will occur about the pathophisiology of TBI following low temperature exposure. Hence, it is necessary to carry out pathophisiological studies of TBI following low temperature exposure actively in order to mastering its pathogenesis regularity and preventive and therapeutic strategis better.A large number of neural cells necrosis leads to permanent loss of neurological function after TBI. So far, there are no effective treatments that could facilitate the repair of the loss of neurological functional. More and more studies have demonstrated that TBI can induce endogenous neural stem cells (NSCs) proliferation and differentiation in the subgranular zone (SGZ) of hippocampal dentate gyrus (DG) and subventricular zone (SVZ) and reapair the loss of neurological function. Therefore, improving the proliferation and differentiation ability of NSCs after injury will be a new therapeutic stratege of TBI. MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as important post-transcriptional repressors and have been implicated in many biological processes. However, which and how miRNAs are involved in trauma-induced proliferation and differentiation of NSCs are still largely unknown; whether low temperature exposure can affect miRNAs expression after TBI is still unclear yet.In our study, mice were suffered closed head injury (CHI), and then were exposed at 4℃for 4 hours. We detected hippocampal miRNA profiles after TBI by miRNA microarray, screened abnormally expressed miRNAs that are involved in NSCs proliferation and differentiation according to references and bioinformatics, and then identified their expression by real-time PCR. Subsequently, we investigated expression levels of 1-2 of these miRNAs and their targets and endogenous proliferation of NSCs in the hippocampus after TBI following low temperature exposure to explore the regulation role of miRNAs on neural stem cells proliferation after traumatic brain injury following low temperature exposure.Methods:1. Closed head injury mice model and experiment groupsMice were suffered closed head injury using a modified weight-drop device. At 24 hours after injury, brain tissues were isolated and stained by HE staining to assess whether CHI model was successfully established. Animals were randomly divided into normal temperature exposure TBI group (NTBI) and low temperature exposure TBI group (LTBI). Each group was divided into sham group,4 hours,1 day and 3 days group post-TBI. NTBI groups were kept at～25/℃. LTBI groups were kept at～4℃for 4 hours, and then were kept at～25℃. Sham group was not suffered CHI as control. Animals were killed at 4 hours,1 day and 3 days after injury. Ipsilateral hippocampus tissues were extracted for examining.2. Expression changes and validation of hippocampal miRNA profiles after TBImiRNA microarry was used to determine hippocampal miRNA profiles at 4 hours after TBI. Abnomally expressive miRNAs that regulate NSCs proliferation and differentiation after TBI were screened according to references and bioinformatics. And then, their expression levels were validated using real-time PCR.3. Expression changes of miR-34a, Notch1 and proliferation of endogenous NSCs in hippocampus after TBI3.1 real-time PCR was used to examine miR-34a expression changes in the hippocampus at different time points after TBI3.2 real-time PCR was used to detect Notch1 mRNA expression changes in the hippocampus at different time points after TBI; Notchl immunofluorescence was used to identify Notch1 protein expression changes in the DG at 3 days post TBI. 3.2 Immunofluorescence was used to identify NSCs proliferation in DG at 3 days after TBI by Nestin staning.Results:1. Assessment of CHI mice model:Pathology observation showed that seriously cerebral contusion, highly cellular edema, a large number of neurons necrosis and astrocyte proliferation after TBI, demonstrating CHI mice model was successfully established.2. Expression changes of hippocampal miRNA profiles after TBI2.1 miRNAs microarray results:(1) Compared to sham group,24 miRNAs were more than 2-fold up-regulated,7 miRNAs were more than 2-fold down-regulated in NTBI group; 20 miRNAs were more than 2-fold up-regulated,3 miRNAs were more than 2-fold down-regulated in LTBI group, reaveling that TBI can induce lots of miRNAs expression changed in the hippocampus.(2) Compared to control group, the expression levels of miR-200b in NTBI group and LTBI group were up-regulated (2.45±0.21) fold and (1.1±0.15) fold, respectively; the expression levels of miR-34a were down-regulated (2.37±0.28) and (1.65±0.16) fold, respectively, suggesting TBI can change miR-200b and miR-34a expression in the hippocampus.2.2 Validation of miR-200b and miR-34a expression levels by real-time PCR:(1) Compared to control group, the expression levels of miR-200b were were up-regulated (2.95±0.17) fold and (1.22±0.11) fold, respectively; the expression levels of miR-34a were down-regulated (2.08±0.09) and (1.47±0.03) fold, respectively. Real-time PCR essentially showed, the same pattern of expression changes of the two miRNAs as observed with the microarray analysis.(2) The expression levels of miR-200b in NTBI group were apparently up-regulated (P<0.05), whereas in LTBI group showed no significantly change (P>0.05) compared to sham group; the expression levels of miR-34a in NTBI group and LTBI group were both apparently down-regulated compared to sham gourp (P<0.05). These results suggested that TBI can change the expression levels of miR-200b and miR-34a. (3) The up-regulation level of miR-200b and the down-regulation level of miR-34a in LTBI group were both less compared to NTBI group, suggesting low temperature exposure can affect the two miRNAs expression after TBI.3. Expression changes of miR-34a and Notchl and proliferation of NSCs in the hippocampus after TBI3.1 Expression changes of miR-34a:real-time PCR revealed that, (1) in NTBI groups, miR-34a expression levels were significantly down-regulated at 4 hours,1 day and 3 days after injury compared to sham group (P<0.05); (2) in LTBI groups, miR-34a expression levels were significantly down-regulated at 4 hours,1 day and 3 days after injury compared to sham group, too (P<0.05); (3) the down-regulation levels of miR-34a at 4 hours,1 day and 3 days post injury were less compared to NTBI relative time points groups (P<0.05), suggesting TBI can dow-regulated miR-34a expression,whereas low temperature can inhibit its down-regulation.3.2 Expression changes of Notchl signaling(1) Expression changes of Notchl mRNA:real-time PCR revealed that, (1) in NTBI groups, Notchl mRNA expression levels were significantly up-regulated at 4 hours,1 day and 3 days after injury compared to sham group (P<0.05); (2) in LTBI group, Notchl mRNA expression levels show no statistical significance at 4 hours,1 day after injury compared to sham group (P>0.05), but up-regulated significantly at 3 days (P<0.05); (3) the up-regulation levels of Notchl mRNA at 4 hours,1 day and 3 days post injury were less compared to NTBI relative time point groups (P<0.05), suggesting TBI can induce Notchl mRNA up-regulation, whereas low temperature can inhibit its up-regulation.(2) Expression changes of Notchl protein:immunofluorescence show that, (1) the number of Notch1+cells in the ipsilateral hippocampal DG of NTBI 3 days group was significantly increased compared to NTBI sham group (18.2±3.56 vs 0.4±0.55, P<0.01); (2) the number of Notchl+ cells in the ipsilateral hippocampal DG of LTBI 3 days group was also significantly increased compared to LTBI sham group (10±2.55 vs 0.6±0.55, P<0.01); (3) the number of Notchl+ cells in the ipsilateral hippocampal DG of LTBI 3 days group was less compared to NTBI 3 days group (P<0.01), suggesting TBI can induce Notch1 protein up-regulation, whereas low temperature can inhibit its up-regulation.3.3 Endogenous NSCs proliferation:Immunofluorescence show that, (1) the number of Nestin+ cells in the ipsilateral hippocampal DG of NTBI 3 days group was significantly enhanced compared to NTBI sham group (21.8±5.07 vs 0.8±0.45, P<0.01); (2) the number of Nestin+ cells in the ipsilateral hippocampal DG of LTBI 3 days group was also significantly enhanced compared to LTBI sham group (16.0±1.59 vs 0.4±0.55, P<0.01); (3) the number of Nestin+ cells in the ipsilateral hippocampal DG of LTBI 3 days group was less compared to NTBI 3 days group (P<0.05), suggesting TBI can induce NSCs proliferation in DG, whereas low temperature can inhibit its proliferation.Conclussions:1. Lots of miRNAs (such as miR-200b and miR-34a) expression were changed in the hippocampus after TBI, whereas low temperature can affect their expression post injury.2. TBI can significantly decrease miR-34a expression, increase its target Notch1 signaling expression and induce NSCs proliferation in the hippocampus, suggesting miR-34a/Notch1 pathway might be involved in trauma-induced NSCs proliferation.3. Low temperature exposure might affect NSCs proliferation by inhibiting the role of miR-34a/Notch1 pathway in the hippocampus after TBI, suggesting low temperature exposure may affect TBI prognosis. |
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