颅脑创伤模型小鼠海马水通道蛋白1表达及作用

Expression and roles of aquaporin 1 in hippocampus of mice model with traumatic brain injury

研究背景颅脑创伤后继发性脑损伤包括脑组织缺血、缺氧和脑水肿,可进一步加重原发性损伤,影响预后。作为选择性易损区,海马对缺血和水肿尤为敏感,易出现不可逆性损伤。水通道蛋白1(AQP1)与脑水肿的发生关系密切,但迄今尚无颅脑创伤后海马AQP1表达变化及其相关作用的报道。本研究采用闭合性颅脑创伤小鼠模型对海马水肿过程进行观察,以探讨AQP1在相关病理生理学过程中的作用机制。方法采用改良自由落体法建立BALB/c系小鼠闭合性颅脑创伤模型,于创伤后不同观察时间点(1、6、24和72 h)进行神经功能缺损程度评价和脑组织含水量测定,并通过TUNEL法观察海马神经元凋亡率、免疫组织化学染色和Western blotting法检测AQP1表达变化。结果成功制备闭合性颅脑创伤小鼠模型,并经神经功能评价和脑组织含水量测定证实存在重型颅脑创伤和脑水肿。TUNEL检测显示,模型组小鼠伤后6 h海马神经元凋亡率即升高[(44.26±15.18)%对(8.61±8.25)%;t=-9.676,P=0.002],至72 h达峰值水平[(61.62±26.55)%对(10.17±6.08)%;t=-5.018,P=0.015];免疫组织化学染色和Western blotting法观察,模型组小鼠创伤后各观察时间点海马AQP1表达水平均高于假手术组(P<0.05),以伤后24 h表达水平最高(0.69±0.32对0.15±0.07,t=-4.335,P=0.023;0.46±0.19对0.14±0.04,t=-4.113,P=0.004)。结论颅脑创伤后小鼠海马AQP1表达上调可能参与了脑水肿和迟发性神经元凋亡等病理生理学过程,AQP1可能成为继发性脑损伤机制研究的新靶点。

Background The ＂secondary brain insult＂ including ischemia, hypoxia and edema after primary traumatic brain injury （TBI） may deteriorate the brain damages and greatly influence the prognosis. As a selective vulnerable region, the hippocampus is especially sensitive to ischemia, bypoxia or edema and yields irreversible sequelae. Aquaporin 1 （AQP1） has been reported to be related to cerebral edema, but the expression and role of AQP1 in hippocampal edema after TBI have seldomly been investigated. In this study, we established BALB/c mouse closed craniocerebral injury models and investigated the changes of AQP1 expression in hippocampus of mouse models after TBI, thereby discussing its effects on relevant patbophysiological processes. Methods Seventy-five BALB/c mice were used to establish experimental closed TBI models with a free-falling weight drop device, and the equal numbers of mice were subject to sham operation and categorized as sham group. The neurological function of each mouse in either TBI group or sham group was scored at different time points （1, 6, 24 and 72 h） after TBI or sham operation, and brain edema formation of the mice in both groups was also evaluated accordingly at 6, 24 and 72 h. The apoptotic hippocampal cells dUTP-biotin nick end labeling （TUNEL） method were stained in situ and detected using TdT-mediated at different time points （6, 24 and 72 h）, then AQP1 expression in hippocampus was also correspondingly detected using immunohistochemistry and Western blotting. All the data were finally compared with those in sham operation group and analyzed. Results Experimental TBI models were successfully established and confirmed by the neurological function score and hippoeampal edema evaluation. Six hours after eraniocerebral injury, the apoptotic cells increased significantly in the hippocampus of mice in TBI group compared with those in sham group [（44.26 ± 15.18）% vs （8.61 ± 8.25）%, t = - 9.676, P = 0.002]. The apoptotic rate increased gradually with the prolonging of time and was highest at 72 h [（61.62 ± 26.55）% vs （10.17 ± 6.08）%; t = - 5.018, P = 0.015]. Determined at different time points by immunohistochemistry and Western blotting assays, the AQP1 expression in mouse hippocampus was constantly higher in TBI group than in sham group （P 〈 0.05）, and reached the peak at 24 h （0.69±0.32 vs 0.15±0.07, t =-4.335, P=0.023 and 0.46±0.19 vs 0.14±0.04, t= - 4.113, P = 0.004, respectively）. Conclusions The up-regulation of AQP1 in mouse hippocampus after TBI perhaps participates in edema formation and delayed apoptosis of hippocampus, and AQP1 may be a new therapeutic target to protect hippoeampus from secondary iniury after TBI.