创伤性脑损伤对大鼠海马骨架蛋白及学习记忆功能影响

创伤性脑损伤（traumatic brain injury，TBI）是极为常见的外伤性疾病，致死率和致残率很高。存活者伴随的空间认知功能障碍，给患者家庭和社会造成了极大的负担。目前，对TBI造成的空间记忆障碍缺乏系统研究。脑损伤后海马组织与记忆有关的分子以及组成神经元骨架的分子如何变化研究甚少。本研究采用Wistar大鼠为研究对象，并随机将其分为假手术（sham）组和创伤性脑损伤（TBI）组。TBI组再按致伤后时间长短分为6 h、12 h、24 h、72 h、15 d五个亚组。TBI组应用PinPointTM颅脑撞击器撞击而致伤，sham组不撞击。采用Morris水迷宫评价实验动物空间记忆能力；干湿重法测定脑含水量，评估脑水肿与海马水通道蛋白4（aquaporin-4，AQP-4）的相关性；海马神经元特异性核蛋白（neuron specific nuclear protein，NeuN）标记和免疫荧光检测评估TBI致大鼠神经元丢失情况；通过Western印迹检测TBI致海马骨架相关蛋白质和记忆相关蛋白质含量变化。本研究证实，与sham组相比，TBI组大鼠潜伏期明显增加［（61.98±12.82) s vs.(28.32±8.52) s，n=5，P<0.01，day 15］，探索时间明显缩短［（36.98±0.37) s vs. (73.68±5.09) s，n=5，P<0.01，day15］，表明脑创伤损害了动物的空间参考记忆能力和空间工作记忆能力。与sham组相比，TBI组大鼠海马AQP-4在蛋白质水平上的表达和脑含水量持续升高，15 d恢复正常；在12 h［（3.78±0.74)，(83.78±0.35)%］和72 h［（3.49±0.85)，(82.28±0.63)%］均形成两个波峰，n=5，P均<0.01，表明继发性脑损伤与持续脑水肿和海马AQP-4在蛋白质上的高表达有关。与sham组相比，NeuN标记和免疫荧光检测发现，TBI后24 h 致大鼠海马神经元丢失严重［（198.2±8.002) vs.(297.2±6.866) cells/mm2, n=5，P<0.01］,表明TBI动物的海马功能受损。与sham相比，TBI组海马神经元树突标志物微管结合蛋白2（microtubule associated proein 2，MAP2）和突触前终末特异性标记物突触素（synaptophysin，SYN）在蛋白质水平均伤后逐步降低（n=5，P均<0.01），72 h［（0.55±0.05) vs.(1.27±0.08), (0.52±0.14) vs.(1.06±0.16), n=5，P均<0.01］降低最明显；TBI组形成神经元纤维缠结主要成分的过度磷酸化tau（ser404）,伤后逐步升高，72 h［（1.25±0.11)vs. (0.33±0.07), n=5，P<0.01］升高最明显。 MAP2、SYN和过度磷酸化的tau（ser404）检测指标的改变，表明脑损伤致神经元受损，神经元生长和损伤修复能力减弱，最终导致神经元骨架破环，TBI损害了动物的海马空间记忆能力。与sham组相比，TBI组大鼠海马环磷酸腺苷反应元件结合蛋白（cAMP response element binding protein，CREB）和磷酸化CREB ser133(phosphorylated CREB Ser133, pCREB Ser133)含量降低明显（n=5，P均<0.05），表明脑损伤动物海马的存储记忆能力减弱；TBI组大鼠海马一般调控阻遏蛋白激酶2(general control nonderepressible 2 kinase,GCN2)蛋白质升高明显（n=5，P均<0.05），表明脑损伤动物海马将新信息转化成长期记忆能力下降。本研究提示，创伤性脑损伤可使大鼠海马神经元骨架破坏，进而导致在学习记忆过程中起重要作用的分子蛋白质下调，抑制记忆储存的蛋白质（GCN2）上调，促使学习记忆功能障碍。

Effects of Traumatic Brain Injury on Skeletal Proteins in Hippocampal Neurons and Learning and Memory Function of Rats

Traumatic brain injury (TBI) is a very common traumatic disease with high mortality and disability. Cognitive impairment has a great burden on the family and society of the patients. Currently, there is no systematic study on the spatial memory impairment caused by TBI. It is not known how the molecules that promote and inhibit memory in the hippocampus and the molecules that make up the skeleton of neurons change after brain injury. Wistar rats were randomly divided into a sham-operated group and traumatic brain injury (TBI) group. The TBI group was further divided into five subgroups according to the length of time after injury: 6 h, 12 h, 24 h, 72 h and 15 d. The TBI group was injured by the impact of PinPointTM craniocerebral impactor, while the sham-operated group was not impacted. The Morris water maze was used to evaluate the spatial memory ability of experimental animals; the brain water content was measured by the dry-wet gravimetric method to evaluate the correlation between brain edema and hippocampal aquaporin-4 (AQP-4)；Neun specific nuclear protein (NeuN) labeling and immunofluorescence assays were used to evaluate neuron loss induced by TBI in rats. Western blotting was used to detect the changes of skeleton-related proteins and memory-related proteins in hippocampus induced by TBI. This study confirmed that compared with the sham-operated group, the escape latency of rats in TBI groups increased significantly ［(61.98 ±12.82) seconds vs. (28.32 ±8.52) seconds, n=5, P< 0.01, day15］, and the searching time shortened significantly ［(36.98 ±0.37) seconds vs. (73.68 ±5.09) seconds, n=5, P < 0.01, day15］, suggesting that brain trauma impaired the spatial reference memory ability and spatial working memory ability of animals. Compared with the sham-operated group, the expression of AQP-4 in hippocampus and brain water contents in TBI groups increased continuously and returned to normal after 15 days; two peaks were formed at 12 h ［(3.78 ±0.74), (83.78 ±0.35)%］ and 72 h ［(3.49±0.85), (82.28 ±0.63)%, (n=5, all P < 0.01), indicating that the secondary brain injury was associated with persistent brain edema and the high expression of AQP-4 in hippocampus. Compared with the sham-operated group, NeuN labeling and immunofluorescence detection showed that the loss of hippocampal neurons was serious ［(198.2 ±8.002) vs. (297.2 ±6.866) cells/mm2, n=5, P < 0.01］ at 24 hours after TBI, indicating that the hippocampal function of TBI animals was impaired. Compared with the sham-operated group, microtubule associated protein 2 (MAP2) and synaptophysin (SYN) in TBI groups decreased gradually at the protein level (n=5, all P< 0.01). There was the most significant reduction at the 72 hours（0-55±0-05 vs.1.27±0.08， 0-52±0.14 vs.1.06±0.16，n=5，all P<0.01); compared with the sham-operated group, tau (Ser404), the main component of neurofibrillary tangles, was over phosphorylated in the TBI group, and increased gradually after injury, with the most obvious increase at 72 h ［(1.25 ±0.11) vs. (0.33± 0.07), n=5, P < 0.01］. This study confirms that changes in the detection indices of MAP2, SYN and over-phosphorylated tau (ser404) indicate that brain injury leads to damage of dendrites of neurons, weakening of neuron growth and repair ability, eventually leading to the breakage of neuronal skeleton, and TBI impairs the spatial memory energy of hippocampus in animals. Compared with the sham-operated group, the contents of cAMP response element binding protein (CREB) and phosphorylated CREB Ser133 (pCREB Ser133) in hippocampus of rats in TBI groups were significantly decreased (n=5, P < 0.05), indicating that the storage and memory ability of hippocampus of rats with brain injury was weakened; in TBI groups, the hippocampus general control nonderepressible 2 kinase (GCN2) protein increased significantly (n=5, all P < 0.05), indicating that the ability of hippocampus to transform new information into long-term memory decreased after brain injury. This study suggests that traumatic brain injury can destroy the skeleton of hippocampal neurons in rats, leading to the down-regulation of molecular proteins that play an important role in learning and memory, upregulation of GCN2, a protein that inhibits memory storage, promote learning and memory dysfunction.