Effects Of Focal Irrigation By ACSF And Focal Mild Hypothermia On Brain Edema And Apoptosis After Traumatic Brain Injury

Objective: To observe the effects of focal irrigation by ACSF and focal mild hypothermia on brain edema and protein expression of Caspase-3, Cyto C, Bcl-2, Bax and degree of apoptosis in impact site after traumatic brain injury in rats. To identify the effect of focal mild hypothermia on regulation of apoptosis. To identify the effect of focal irrigation by ACSF on TBI. To compare the therapeutic effect of focal irrigation by ACSF, focal hypothermia and focal irrigation combined with focal hypothermia. Whether these ways have cooperative efficacy to secondary brain injury or not. TO provide useful clues for the treatment of patients with severe TBI in clinical practice.Methods: Three hundred and sixty healthy adult Sprague-Dawley rats were randomly separated to sham operation group(SO group),traumatic brain injury animal model group (TBI group), focal mild hypothermia group (FH group), focal irrigation group (FI group), focal hypothermia combined with focal irrigation group (FIH). Except SO group, the others were subjected to traumatic brain injury by the Feeney's method. The animals were sacrificed at 4, 8, hrs and 1, 3, 5, 7 days posttrauma. Groups were divided into seven subgroups according to the six time points (n=12 in each subgroup). The content of water was measured by the wet and dry weight method; the content of sodium (Na~+), potassium (K~+) was measured by a flame photometer, ultrastructural were observed by electron- microscopy respectively in 7 rats chosen randomly from each subgroup. Apoptosis in impact site were detected by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), and protein expression of Caspase-3, Cyto C, Bcl-2, Bax were detected by immunohistochemistry respectively.Results:1. The content of Water, sodium and potassium in injured brain tissue : Water content of brain tissue in other four groups was significantly higher than SO group at 4h, 8h, 1d,3d and 5d posttrauma (P＜0.05), and the peak value appeared at 1-3d posttrauma , and went back to normal levels compared with SO group at 7d posttrauma (P＞0.05). Water content of FIH group, FH group, FI group was markedly less than that of the TBI group from 8h to 5d posttrauma (P＜0.05). Water content of FH group was less than that of FI group (P＜0.05). Water content of FIH group was less than that of FH group or FI group (P＜0.05). The changes of sodium content in brain tissue had the same pattern to that of water content, and that of potassium content was reverse.2.TUNEL-positive cells in injured brain tissue : TUNEL-positive cells were seldom in SO group. TUNEL-positive cells in other four groups were significantly higher than SO group at 4h, 8h, 1d,3d,5d and 7d posttrauma (P＜0.05), and the peak value appeared at 1d posttrauma. TUNEL-positive cells of FIH group, FH group, FI group were markedly less than those of TBI group (P＜0.05). TUNEL-positive cells of FH group were less than those of FI group (P＜0.05). TUNEL-positive cells of FIH group were less than those of FH group or FI group (P＜0.05).3. Caspase-3 protein Expression in injured brain tissue : Caspase-3 protein expression was low in SO group. Caspase-3 protein expression in other four groups was higher than SO group at 8h, 1d,3d,5d and 7d posttrauma (P＜0.05), and the peak value appeared at 1d posttrauma . Caspase-3 protein expression in FIH group, FH group, FI group was markedly less than that of TBI group (P＜0.05). Caspase-3 protein expression of FH group was less than that of FI group (P＜0.05). Caspase-3 protein expression of FIH group was less than that of FH group or FI group (P＜0.05).4. Cyto C protein Expression in injured brain tissue : Cyto C protein expression was low in SO group. Cyto C protein expression in other four groups was higher than SO group at 4h, 8h, ld,3d,5d and 7d posttrauma (P＜0.05), and the peak value appeared at ld posttrauma. Cyto C protein expression in FIH group, FH group, FI group was markedly less than that of TBI group (P＜0.05). Cyto C protein expression of FH group was less than that of FI group (P＜0.05). Cyto C protein expression of FIH group was less than that of FH group or FI group (P＜0.05).5. Bcl-2 protein Expression in injured brain tissue : Bcl-2 protein expression was high in SO group. Bcl-2 protein expression in other four groups was lower than SO group at 4h, 8h, 1d,3d,5d and 7d posttrauma (P＜0.05), and the lowest value appeared at ld posttrauma. Bcl-2 protein expression in FIH group, FH group, FI group was markedly higher than that of TBI group (P＜0.05). Bcl-2 protein expression of FH group was higher than that of FI group (P＜0.05). Bcl-2 protein expression of FIH group was higher than that of FH group or FI group (P＜0.05).6. Bax protein Expression in injured brain tissue : Bax protein expression was low in SO group. Bax protein expression was not significantly difference in all groups at 4h, 8h posttrauma(P＞0.05). Bax protein expression in other four groups was higher than SO group at 1d,3d,5d and 7d posttrauma (P＜0.05), and the peak value appeared at 1d posttrauma. Bax protein expression in FIH group, FH group, FI group was markedly less than that of TBI group at ld and 3d posttrauma (P＜0.05). Bax protein expression of FH group was less than that of FI group at 1d and 3d posttrauma (P＜0.05). Bax protein expression of FIH group was less than that of FH group or FI group at ld and 3d posttrauma (P＜0.05).7. Bcl-2/Bax ratio : Bcl-2/Bax ratio was high in SO group. Bcl-2/Bax ratio in other four groups was lower than SO group at 4h, 8h, 1d,3d,5d and 7d posttrauma (P＜0.05), and the lowest value appeared at 1d posttrauma. Bcl-2/Bax ratio in FIH group, FH group, FI group was markedly higher than that of TBI group (P＜0.05). Bcl-2/Bax ratio of FH group was higher than that of FI group (P＜0.05). Bcl-2/Bax ratio of FIH group was higher than that of FH group or FI group (P＜0.05).8. Pathological changes: Electron microscopic changes showed that the degree of brain edema and apoptosis were most obvious in TBI group. FIH group, FH group and FI group were less obvious than TBI group.Conclusion :1. Focal irrigation by ACSF can alleviate the degree of brain edema and apoptosis posttrauma. Focal irrigation by ACSF has therapeutic efficacy on TBI.2. Focal mild hypothermia can alleviate the degree of brain edema and apoptosis posttrauma. Focal mild hypothermia has therapeutic efficacy on TBI.3. Focal hypothermia has better therapeutic efficacy than focal irrigation on TBI.4. Focal hypothermia combined with focal irrigation has therapeutic efficacy on TBI.5. Focal hypothermia combined with focal irrigation has better therapeutic efficacy than focal hypothermia or focal irrigation alone on TBI.