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目的:探讨金刚烷胺(amantadine,AMA)长期注射对创伤性脑损伤(traumatic brain injury,TBI)幼鼠脑内酪氨酸羟化酶(tyrosine hydroxylase,TH)的影响,为深入探讨AMA药理学效应和指导临床儿童颅脑外伤的治疗提供实验学依据。方法:昆明幼年小鼠(P10)80只,性别不拘,随机分为对照组、AMA组、TBI组及TBI+AMA组。TBI模型在第10 d(P10)应用自由落体重力打击制作成轻中度闭合型TBI模型,再连续给药10 d,停药后第10 d(P30)进行自发活动观察和被动回避性跳台实验检测;其中停药后第1 d(P21)和第3 d(P23)取脑;应用Western blot技术检测前脑内相关脑区TH的含量变化;应用冰冻切片的免疫组织化学染色技术检测小鼠脑内酪氨酸羟化酶(TH)的表达变化。结果:(1)行为学检测:旷场实验中,AMA组和TBI组幼鼠自发活动均明显低于对照组(P<0.05),但TBI+AMA组幼鼠自发活动增加,与TBI组的差别有统计学意义(P<0.05)。被动回避性跳台实验中,AMA组与对照组之间的差别无统计学意义(P>0.05),但TBI组比对照组幼鼠学习记忆时间明显延长,差别有统计学意义(P<0.05);TBI+AMA组与TBI组幼鼠学习记忆能力无明显改变。(2)Western Blot结果显示:AMA组幼鼠杏仁核+杏仁周皮质、前额皮质内TH蛋白的含量在1 d时高于对照组(P<0.05),3 d时AMA组与对照组之间TH蛋白含量的差异无统计学意义(P>0.05)。TBI+AMA组幼鼠隔区和前额皮质TH蛋白的含量在1 d时高于TBI组(P<0.05),3 d时仅TBI+AMA组隔区TH蛋白含量高于TBI组(P<0.05)。(3)免疫组织化学染色结果显示:TBI+AMA组幼鼠黑质、腹侧被盖区、蓝斑的TH阳性神经元数量明显高于TBI组,光密度值分析两组间差异有统计学意义(P<0.05)。结论:AMA长期注射可提高TBI幼鼠脑内杏仁核和前额皮质等脑区多巴胺的浓度,进而改善创伤性脑损伤幼鼠的行为学症状。
Objective: To investigate the effect of long-term injection of amantadine (AMA) on the brain tyrosine hydroxylase (TH) in traumatic brain injury (TBI) Effect and guidance of clinical children with traumatic brain injury treatment to provide experimental evidence. Methods: Eighty Kunming young mice (P10) were randomly divided into control group, AMA group, TBI group and TBI + AMA group without any gender. The TBI model was made into a mild to moderate closed TBI model by free-fall gravity attack on the 10th day (P10), and then continuously administered for 10 days. On the 10th day (P30), the spontaneous activity observation and passive avoidance jumping test (P21) and P23 (P23) were taken out from the rats. Western blot was used to detect the content of TH in the relevant forebrain brain regions. Immunohistochemical staining was used to detect the expression of TH Brain tyrosine hydroxylase (TH) expression changes. Results: (1) Behavioral testing: During the open-field test, the spontaneous activities of pups in AMA group and TBI group were significantly lower than those in control group (P <0.05). However, spontaneous activity of pups in TBI + AMA group was increased, The difference was statistically significant (P <0.05). There was no significant difference between the AMA group and the control group in the passive avoidance platform (P> 0.05), but the learning and memory time in the TBI group was significantly longer than that in the control group (P <0.05) There was no significant change in learning and memory abilities in TBI + AMA group and TBI group. (2) The results of Western Blot showed that the content of TH protein in amygdala + almond cortex and prefrontal cortex in AMA group was higher than that in control group (P <0.05) on the 1st day, and between AMA group and control group TH protein content of the difference was not statistically significant (P> 0.05). The content of TH protein in the parietal and prefrontal cortex in TBI + AMA group was higher than that in TBI group at 1 d (P <0.05), and the content of TH protein in TBI + AMA group was higher than that in TBI group only at 3 d ). (3) The results of immunohistochemical staining showed that the number of TH-positive neurons in substantia nigra, ventral tegmental area and locus coeruleus of TBI + AMA group was significantly higher than that of TBI group. The difference of optical density between the two groups was statistically significant Significance (P <0.05). Conclusion: Long-term AMA injection can increase brain dopamine concentration in the brain such as amygdala and prefrontal cortex in TBI rats and further improve behavioral symptoms in traumatic brain injury rats.