摘要目的 探讨亚低温能否通过减轻海马神经元细胞凋亡促进创伤性脑损伤(TBI)后海马齿状回区神经再生及认知功能恢复,并探讨其可能机制.方法 健康成年SD大鼠66只,按随机数字表法分为假手术组、TBI组及TBI+亚低温组,每组22只.采用液压冲击仪建立大鼠TBI模型,TBI+亚低温组在伤后立即接受亚低温(33.5℃)治疗4 h.5-溴脱氧尿嘧啶核苷(BrdU)腹腔注射标记有丝分裂细胞.Morris水迷宫试验评估大鼠空间学习和记忆能力,免疫荧光染色观察伤后7 d及28 d大鼠海马齿状回区BrdU、微管相关蛋白(DCX)、神经元特异性核蛋白(NeuN)、半胱氨酸天冬氨酸蛋白酶3(caspase-3)及剪切活化caspase-3表达情况,Western blot检测大鼠海马凋亡相关蛋白——自杀相关因子(FAS)/自杀相关因子配体(FASL)、B淋巴细胞瘤-2(Bcl-2)、caspase-3及剪切活化caspase-3表达情况.结果 与TBI组比较,TBI+亚低温组伤后28 d水迷宫试验的逃避潜伏期显著缩短[(24.2±5.9)s:(18.0±4.1)s],目标象限停留时间、平台穿越次数明显增加[(24.9±6.5)s:(31.7±5.2)s、(1.9±0.8)次:(3.5±1.2)次](P<0.05).与假手术组比较,TBI组及TBI+亚低温组伤后7 d海马齿状回区BrdU+新生细胞数[(9.4±4.1)个:(33.4±3.8)个、(9.4±4.1)个:(45.8±5.6)个]、伤后7 d BrdU+/DCX+新生神经元细胞数[(2.0±0.6)个:(9.6±1.6)个、(2.0±0.6)个:(19.2±3.7)个]及伤后28 d BrdU+/NeuN+成熟神经元细胞数[(2.6±1.0)个:(17.2±3.9)个、(2.6±1.0)个:(33.6±9.1)个]均显著增加,且TBI+亚低温组较TBI组增加更为明显(P均<0.01).与伤后7 d比较,伤后28 d TBI+亚低温组海马齿状回区BrdU+新生细胞数进一步增加,但TBI组BrdU+新生细胞数减少[(45.8±5.6)个:(58.8±9.2)个、(33.4±3.8)个:(22.0±3.5)个](P<0.05或<0.01).与假手术组比较,TBI组伤后7 d损伤侧海马齿状回区剪切活化caspase-3+NeuN+及caspase-3+NeuN+凋亡神经元细胞数显著增多[(2.0±0.9)个:(11.6±2.6)个、(2.6±1.0)个:(10.2±2.9)个],而TBI+亚低温组较TBI组剪切活化caspase-3+NeuN+凋亡神经元细胞数减少[(6.6±2.0)个:(11.6±2.6)个](P均<0.05).与TBI组比较,TBI+亚低温组损伤侧海马FAS、FASL、剪切活化caspase-3及caspase-3表达均下调,Bcl-2表达上调(1.54±0.15:1.14±0.12、1.06±0.04:0.80±0.09、0.84±0.03:0.62±0.08、0.93±0.06:0.86±0.09、0.71±0.01:1.58±0.18](P均<0.05).结论 亚低温可能通过剪切活化caspase-3、FAS/FASL及Bcl-2途径减轻TBI后海马神经元细胞凋亡,促进TBI后海马齿状回区神经元细胞再生及成熟,进而促进大鼠认知功能恢复.提示亚低温诱导的抗海马细胞凋亡及促神经元细胞再生及成熟作用可能对TBI患者预后具有潜在预测作用.

abstractsObjective To investigate whether mild hypothermia can promote neurogenesis in the dentate gyrus of hippocampus and cognitive function recovery after traumatic brain injury ( TBI) through inhibiting apoptosis of hippocampal neurons. Methods A total of 66 healthy adult Sprague-Dawley rats were randomly divided into sham group, TBI group and TBI+hypothermia group, with 22 rats in each group. The rat TBI model was established using the fluid percussion device. The rats in TBI +hypothermia group received 4-hour hypothermia therapy immediately after injury, with the target temperature of 33. 5℃. Bromodeoxyuridine (BrdU) was injected into the rats' abdominal cavity to label the mitotic cells. The test of Morris water maze was used to evaluate the rats' spatial learning and memory capabilities. Immunofluorescence staining was used to observe the expression levels of BrdU, doublecortin (DCX), neuron specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3) and cleaved caspase-3 expressions in dentate gyrus of hippocampus at 7 days and 28 days after injury. Expressions apoptosis-related proteins including the factor associated suicide ( FAS )/factor associated suicide ligand (FASL), B-cell lymphoma-2 (Bcl-2), caspase-3 and cleaved caspase-3 expressions were detected by Western blot assay. Results The water maze tests at 28 days after injury showed that compared with TBI group, the escape latency in TBI+hypothermia group was significantly shorter [(24. 2 ± 5. 9)s:(18 ± 4. 1)s], and both the time in the target quadrant and the number of platform crossing were increasedsignificantly[(24.9±6.5)s:(31.7±5.2)s; (1.9±0.8) times:(3.5±1.2)times](P<0. 05). Compared with the sham group, in TBI group and TBI+hypothermia group, the BrdU+ new-born cells in the dentate gyrus of hippocampus were significantly increased at 7 days after injury [(9. 4 ± 4. 1):(33. 4 ± 3. 8);(9. 4 ± 4. 1):(45. 8 ± 5. 6)], the BrdU+ /DCX+ new-born neurons were increased at 7 days after injury [(2. 0 ± 0. 6):(9. 6 ± 1. 6);(2. 0 ± 0. 6):(19. 2 ± 3. 7)], and the BrdU+ /NeuN+mature neurons were increased at 28 days after injury [(2. 6 ± 1. 0) :(17. 2 ± 3. 9); (2. 6 ± 1. 0) :(33. 6 ± 9. 1)] (P<0. 01). TBI group showed more obvious increase than the TBI+hypothermia group (P<0. 01). Moreover, compared with 7 days after injury, the number of BrdU+ cells at 28 days after injury was further increased in TBI +hypothermia group but decreased in TBI group [(45. 8 ± 5. 6) :(58. 8 ± 9. 2);(33. 4 ± 3. 8):(22. 0 ± 3. 5)](P<0. 05 or <0. 01). Compared with the sham group, the caspase-3 +NeuN+ and caspase-3 +NeuN+ apoptotic neurons were significantly increased at 7 days after injury in TBI group [(2. 0 ± 0. 9):(11. 6 ± 2. 6); (2. 6 ± 1. 0):(10. 2 ± 2. 9)] (P<0. 05). Compared with the TBI group, the cleaved caspase-3 +NeuN+ apoptotic neurons were decreased in TBI+hypothermia group [(6. 6 ± 2. 0):(11. 6 ± 2. 6)](P<0. 05). Furthermore, compared with the TBI group, mild hypothermia might down-regulate the expression of FAS, FASL, cleaved caspase-3 and caspase-3 and up-regulate the expression of Bcl-2 in the hippocampus [(1. 54 ± 0. 15) :(1. 14 ± 0. 12);(1. 06 ± 0. 04):(0. 80 ± 0. 09); (0. 84 ± 0. 03):(0. 62 ± 0. 08); (0. 93 ± 0. 06):(0. 86 ± 0. 09);(0. 71 ± 0. 01):(1. 58 ± 0. 18)](P<0. 05). Conclusions Mild hypothermia might inhibit apoptosis of hippocampal neurons through cleaved caspase-3, FAS/FASL and Bcl-2 pathways, thus improving the neurogenesis and maturation of neurons in the dentate gyrus of hippocampus and facilitating cognitive function recovery in rats. It indicates that the function of hypothermia in anti-apoptosis and neurogenesis and maturity of hippocampal neurons may have a potential role in predicting the prognosis of TBI patients.