| Objective: To investigate the relationship between mild hypothermia treatment and gene transcription and protein expression of receptor-interacting protein kinase-1(RIPK-1) and RIPK-mediated necroptosis signal pathways following by traumatic brain injury(TBI) in rats.Methods: In experiment one(animal experiments), Male Wistar rats were randomly divided into four groups: normothermic uninjuried group(Sham group), Sham+HT group, normothermic treatment TBI group, mild hypothermic treatment TBI group(TBI+HT group). Neurological deficit was evaluated using a modified neurological severity score(m NSS)24h、48h after CCI. Analysis for Activation of Changes in the RIPK-1 Pathways with using real-time PCR and Western blotting after Traumatic Brain Injury. Pretreatment with hypothermia therapy inhibited signal makers of RIPK-1 Pathways expression around the injured cerebral cortex site post-TBI in immunohistochemistry and immunofluorescence. Effects of Controlled Cortical Impact(CCI) injury and hypothermia intervention on cerebral blood flow. Ultrastructural characteristics of mitochondria present in injured cerebral cortex.In experiment two(cell experiments), cultured SH-SY5 Y human neuroblastoma cells, gradient against human neuroblastoma SH-SY5 Y cells validated CIC-II instrument damage performance, conventional culture trypsin digestion of SH-SY5 Y cells, the cells were divided into four groups as animal experiments.Results:TBI injury with hypothermia therapy improved behavioral outcomes following TBITBI induced motor and neurologic deficits in rats, as determined by m NSS scores in each group. We observed histologic changes of the brain tissue around the injured cortex after 48 h. The brain tissue around the injured cortex at 48 h post-injury.In TBI + HT group the hemorrhage and hyperemia were alleviated. Neuron swelling was not also obvious in the molecular layer of the hippocampus. There were no differences between the sham and sham + HT groups in the test. TBIinjury in the rats’ right hemispheric cortex resulted in functional deficits in the evaluated rats using the m NSS scoring system after 24 h and 48 h. The TBI group showed severe neurological deficits, and m NSS scores were significantly higher on sham and sham + HT group(*p<0.05, **p<0.01, ## p<0.01). Nevertheless, the interference of hypothermia therapy in TBI rats exhibited a significant reduction in neurological deficits to compare with TBI group.(## p<0.01).Analysis for Activation of Changes in the RIPK-1 Pathways after Traumatic Brain InjuryThe cortex brain tissue around the injured showed neurons necrosis, the RIPK-1 pathways may be attributable to this pathophysiologic process. We next sought to determine the changes in RIPK-1 pathways after TBI using the same set of brain samples with using real-time PCR and Western blotting. There were three upstream signal membrane proteins, TNF-α, Fas L, TRAIL, triggering the RIPK-1 pathways, Caspase-3, Caspase-8, RIPK-1, RIPK-3, locating in cytoplasm, and PARP-1, and FADD in nerve cells. Real-time PCR was used to determine the relative target products level in each samples using the recorded standard curve method. Western blots were used to measure the immunoreactivity of a post-TBI marker protein as described above, in cortex brain tissues obtained 48 h after TBI. We performed densitometric measurements of both upper and lower bands either together for the total level or separately for the individual level. As shown in Figure 2C, real-time PCR and WB analysis showed that RIPK-1 pathways playing an important role in TBI injury m RNA expression and protein expression was significantly increased in compared with sham and sham + HT group(*p<0.05, **p<0.01), whereas, TBI rats with the interference of hypothermia therapy indicated a significant decreasing in neurological deficits to compare with TBI group.(## p<0.01). β-actin was used to normalize the content of these bio-makers in the different lanes.Pretreatment with hypothermia therapy inhibited Signal Makers of RIPK-1 Pathways expression around the injured cerebral cortex site post-TBI in Immunohistochemistry and ImmunofluorescenceTo research the regional and cellular distribution of Signal Makers of RIPK-1 Pathways after TBI, we used light microscope to image sham, sham + HT control, aswell as the TBI and TBI + HT groups parietal cortical regions at 48 h after TBI. Sections were observed by microscope and probed with relative antibodies of RIPK-1 signaling pathways. Both TNF-α and Fas L were localized on the plasmalemma of dendrites, axons. Caspase-3, Caspase-8, RIPK-1, RIPK-3, and FADD were localized to cytoplasm and PARP-1 was localized in neurons nucleus. The relative antibodies of RIPK-1 signaling pathways were unchanged or increased slightly, whereas, relative to sham and sham + HT control, relative antibodies of RIPK-1 signaling pathways were markedly increased in immunoreactivity in the parietal ipsilateral cortical area showing an increased number of positive cells that indicated RIPK-1 signaling pathways activated in the TBI injury intervention. There were no differences in number of positive cells between the sham and sham + HT groups. To identify the vital role of hypothermia treatment that is responsible for RIPK-1 signaling pathways involving neurogenesis and neurons apoptosis and necrosis after TBI, we utilized the hypothermia to treat TBI rats. As shown in our study, the number of positive cells in TBI + HT group in rats were obviously decreased showing statistical significance in compared with TBI injury rats at different levels. Tendency detected by relative antibodies in different localizations in RIPK-1 signaling pathways was similar. We also observed Immunofluorescence of PARP-1 and TRAIL in RIPK-1 signaling pathways in neurons. In the cerebral cortex of sham and sham + HT control rats, few or no PARP-1 and TRAIL positive neurons were observed. Furthermore, at 48 h post-TBI, the number of PARP-1 and TRAIL double-labeled cells seen in the group in the traumatized cortex exhibited robust PARP-1 and TRAIL immunoreactivity. When compared to TBI-injured with hypothermia-treated, TBI + HT rats showed a markedly decreased PARP-1 and TRAIL immunoreactivity in the majority of evaluated. It clearly demonstrates that hypothermia post-TBI suppressed RIPK-1 signaling pathways transduction, reducing the expression of PARP-1 and TRAIL. In addition, there was no significant difference between the number of immunopositive cells 48 h after injury.Effects of Controlled Cortical Impact(CCI) Injury and Hypothermia Intervention on Cerebral Blood FlowCerebral blood flow did not change significantly from baseline in rats thatunderwent sham injury and hypothermia Intervention. There were no significant differences between sham and sham + HT group with respect to CBF levels, however, the CBF in the ipsilateral hemisphere of sham + HT animals( p > 0.05) was higher than only sham animals. Moreover, hypothermia also significantly increased r CBF in the contralateral hemisphere 48 h post-TBI(124.3 ± 4.5 %, p < 0.01) compared with the TBI group. Forty eight hours after TBI, the CBF values level in the rats was decreased broadly across the ipsilateral hemisphere, here, the CBF values was much lower compared with all other groups. Scanned images showing that there were no significant differences among the experimental groups in post-TBI the ipsilateral hemispheres, and no major differences among the average baseline CBF values images(data not shown). During the TBI period following hypothermia for 6 h, rats experienced similar increased in CBF values.Ultrastructural Characteristics of Mitochondria Present in Injured Cerebral Cortex.Mitochondria characterized by a moderately dense matrix and a normal cristate architecture. Some mitochondria exhibited a tightly condensed matrix and widely expanded intracristate space, indicative of a ‘‘condensed conformation.’’ End-stage degenerating mitochondria are typified by spherical fission products, some showing high amplitude swelling and swollen collapsing mitochondria. Some of end-stage degenerating mitochondria are enveloped by residual membranes of autophagic vacuoles(mitophagy). Furthermore, there were also some characteristic performance appearing nuclear chromatin condensation and margination and the deformation of nuclear.Conclusions: In conclusion, we may reach three conclusions: a. Our studies from our laboratories have demonstrated that the expression of RIPK1 increased dramatically following by TBI, but degraded obviously after mild hypothermia treatment which may act as neuroprotection by inhibiting the expression of RIPK1 and delayed the pathological process of necroptosis. b. RIPK1-RIPK3 necroptosis-inducing protein complex motivated by upstream signal molecule being a central and critical factor in RIPK-mediated necroptosis signal pathways after TBI. c. The role of FADD in RIPK-mediated necroptosis signal pathways after TBI exhibits positive effects onRIPK1-induced programmed necrosis. |
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