The Effects Of Hypothermia On Promoting Neurite Sprouting And Regulating Suppressor Of Cytokine Signaling 3 Expression After Traumatic Brain Injury

Objectives: To investigate the effects of hypothermia on promoting neurite sprouting of the rat primary neuron after injury and the rat ipsilateral cortex after moderate TBI and its mechanisms.Methods: The primary rat neurons in BioFlex 6 well plates were randomly divided into seven groups: SNG(n=10),SHG(n=10),TNG(n=10),THG(n=10),the control group(n=5),the negative control lentiviral group(n=5),the SOCS3 knockdown lentiviral group(n=5).SNG cells were placed in the 37? incubator.SHG cells were placed in the 33? incubator and maintained for 3 h.TNG cells received stretchinduced injury and placed in the 37? incubator.THG cells received stretch-induced injury and placed in the 33? incubator and maintained for 3 h.The control group,the negative control lentiviral group and the SOCS3 knockdown lentiviral group received stretch-induced injury and placed in the 37? incubator.Immunofluorescence assay,TUNEL staining,dynamic observation of living cells and Western blot assay were performed.The primary rat neurons in 6 well plates were randomly divided into seven groups: SNG(n=5),SHG(n=5),TNG(n=5),THG(n=5),the control group(n=5),the negative control lentiviral group(n=5),the SOCS3 knockdown lentiviral group(n=5).SNG cells were placed in the 37? incubator.SHG cells were placed in the 33? incubator and maintained for 3 h.TNG cells received scratch wound injury and placed in the 37? incubator.THG cells received scratch wound injury and placed in the 33? incubator and maintained for 3 h.The control group,the negative control lentiviral group and the SOCS3 knockdown lentiviral group received scratch wound injury and placed in the 37? incubator.Immunofluorescence and western blot assay were performed.One hundred twenty rats were randomly divided into four groups: SNG(n=30),SHG(n=30),TNG(n=30),THG(n=30).SNG rats only received craniotomies and maintained the body temperature at 37?.SHG rats only received craniotomies and their temperature was decreased to 33? in 30 min and maintained for 3 h.TNG rats received FPI and maintained the body temperature at 37?.THG rats received FPI and their temperature was decreased to 33? in 30 min and maintained for 3 h.The rats(n=6)were perfused at 1 day after moderate TBI and the brain samples were collected.HE staining and Immunohistochemistry were performed.The rats(n=18)were perfused at 7 days after moderate TBI and the brain samples were collected.The levels of SOCS3 and GAP43 expression in the tissues around 2mm injured area were assessed using western blot and Immunofluorescence.RT-PCR assay was performed to observe the mRNA expression changes in ipsilateral cortex after moderate TBI.The rats(n=6)underwent Rotarod and Morris water maze test.Results: In the stretch-induced injury model,the neurite structures were obviously broken,and the positive rate of TNG was higher than that of THG at 1 day after injury in TUNEL assay.Dynamic observation of living cells,immunofluorescence and western blot assay showed that hypothermia promoted the neurite regeneration and the GAP43 expression after injury,and knockdown SOCS3 also promoted the neurite regeneration and the GAP43 expression after injury in scratch wound injury model and stretch-induced injury model.The gross morphological observation and HE staining assay showed that there were obvious cerebral hemorrhage,edema and defect at 1 day after moderate TBI,and the damage of THG were slighter than that of TNG.The immunofluorescence assay showed that APP positive number in TNG was more than that in TNG.The Rotarod and Morris water maze test showed hypothermia improved the motor function and memory function of the rats after moderate TBI.Immunofluorescence and western blot assay showed that hypothermia increased the GAP43 expression and decreased the SOCS3 expression after moderate TBI.Microarray screening found that SOCS3 mRNA increased in the ipsilateral hippocampus after moderate TBI.RT-PCR assay showed that hypothermia inhibited SOCS3 mRNA expression in the injured area.Conclusions: Hypothermia significantly prevents the morphological abnormality and neuronal apoptosis after stretch-induced injury.Hypothermia promotes the neurite regeneration and the GAP43 expression after injury in the stretch-induced injury model and the scratch wound model,and increases the expression level of GAP43 in the ipsilateral cortex after FPI in vivo.The mechanism may be associated with the reduction of SOCS3 expression.