Studies On The Protection And Mechanism Of Cold Shock Proteins Of Subarachnoid Hemorrhage By Therapeutic Hypothermia In Early And Secondary Brain Injury

Background:Subarachnoid hemorrhage(SAH)is a high mortality and disability disease,which is caused by cerebral vascular lesion rupture and blood invasion into the subarachnoid cavity,defined by primary or secondary damage.The treatment of vasospasm,clinically,during the last decade has made great progress,but still left the serious problem of fully recovering functions after SAH damage especially direct central nervous system(CNS)dysfunction after injury.These injuries include brain inflammatory reactions induced by secondary brain damage,which will directly affect the quality of life and mortality of patients with SAH.In particular,direct brain injury(early brain injury)of the whole brain within 72 hours after the occurrence of SAH has direct toxic effects on the CNS including early brain edema,oxidative stress,apoptosis of neurons and the development of cerebral infarction.Therefore,in this study,the expression of CIRP(cold inducible RNA binding protein,CIRP)(and C23 proteins)during early brain injury and factors related to secondary cerebral injury(RNA binding motif protein3,RBM3)were measured and analyzed,and we also investigated the regulation of such response factors and potential mechanisms to reduce neuronal damage.Method:Primary neuronal cells from C57BL/6 mice were obtained and a stable cell line of primary neuronal cell cultured,which were then established as SAH cell and experimental rat SAH models by prechiasmatic cistern respectively.The distribution characteristics of CIRP and RBM3 neurons in the brain were determined by observing the immunofluorescence staining of cerebral temporal cortex neurons in the rat SAH model.The mRNA relative and protein expression levels of neurons at different time points after SAH were detected by real-time PCR and western blotting.Then,the CIRP inhibitor C23 protein was used to block the actions of CIRP in rat brain,and the damage and recovery of neurological function in rats identified by immunohistochemistry,Nissl staining,TUNEL apoptosis and rat rotating rod experiments.Changes in apoptosis-related proteins,neuronal apoptosis and motor functions after SAH were observed to investigate the effects of hypothermia on CIRP and RBM3 factors and neuroprotective effects in neurons after SAH.Results:Male rat SAH models by prechiasmatic cistern methodology were successfully established.High mRNA and protein expression of CIRP was detected in the temporal cortex of rats 12 hours and 1 day after modeling.mRNA and protein expression of RBM3 were high at 3 and 7 days after modeling.Low expression of bcl-2,related to apoptosis,and high expression of Bax,caspase-3,caspase-9 and cytochrome c were also detected.In addition,SAH rats exposed to hypothermia were able to recover to the level of the control sham group after 7 days,and the low temperature itself had the effect of increasing the apoptosis rate of neurons and also increasing brain tissue damage of rats in the normal sham group.However,the low temperature treatment of the SAH group inhibited apoptosis of neurons and damage to brain tissue.Using fluorescent staining,we confirmed that CIRP and RBM3 were mainly expressed in neurons in rat coronal sections,while astrocytes only contributed a small component of the expression.Therefore,rat neurons were the main cells that expressed CIRP and low temperature treatment of SAH in rat brain significantly decreased the cell apoptosis rate.Immunohistochemical DAB staining revealed that the expression of CIRP in SAH rat brain by low temperature was obviously reduced.Nissl staining and electron microscopic examination showed that exposure of SAH rats to a low temperature led to a lower rate of neuron apoptosis and a restoration of mitochondrial functions,and also enhanced the activity of SAH rats in turn bar functions.At the same time,we found that the CIRP antagonist C23 protein had an inhibitory effect on CIRP binding to cell surface receptors,which effectively reduced SAH-induced apoptosis of neurons,reduced the inflammatory response and restored motor functions in rats.Thus,CIRP may be an important factor related to early brain injury in SAH;RBM3 may be an important factor related to secondary brain injury in the later stages of SAH.Moreover,from the experimental results of different dosage groups of C23,we found that the highest dose of C23 protein after SAH showed the best inhibitory effect on CIRP.Conclusions:The expression of CIRP and RBM3 in neurons was increased after hypothermia and SAH in early brain injury and late secondary brain injury stages,thereby triggering changes in the expression of apoptosis-related proteins and inducing apoptosis of neurons.However,low temperature treatment of SAH rats can decrease the expression of CIRP and RBM3,and reduce the expression of apoptosis-related proteins and neuronal apoptosis.In the SAH model of rats,a high dose of C23 can reduce CIRP-induced brain inflammation,reduce apoptosis and improve motor functions.