梓醇增强缺血灶周围大脑皮质神经元轴突芽生及突触新生

Catalpol promotes axon growth and synaptogenesis in peri-infarct cortex in rats

目的观察梓醇对局灶脑缺血大鼠缺血灶周围区大脑皮质(peri-infarct cortex,PIC)神经元轴突芽生及其形成突触的影响,揭示梓醇促神经修复作用的形态学基础。方法SD大鼠36只,随机分为假手术组、模型组、生理盐水组、梓醇低、中、高剂量组(剂量分别为1、5、10 mg.kg-1)和胞磷胆碱组(阳性药物对照组,剂量为0.5 g.kg-1)。开颅电凝右侧大脑中动脉制备局灶永久性脑缺血模型,造模后24 h,首次经腹腔注射梓醇或胞磷胆碱进行干预,每日1次,连续7 d。于造模后15 d断头取脑,采用免疫荧光双标组织化学染色技术检测PIC区生长相关蛋白-43(growth-associated protein,GAP-43)与突触素(synaptophysin,P38)共定位信号,了解梓醇对PIC区芽生轴突形成突触的影响;采用透射电镜结合体视学方法观察梓醇对PIC区神经毡内突触数密度(number density,Nv)和面密度(surface density,Sv)的影响,明确梓醇对突触重构的可能作用。结果免疫荧光双标组织化学染色显示,绿色荧光显示P38标记的突触前末端,红色荧光显示GAP-43表达阳性的芽生轴突,GAP-43/P38共定位呈现黄色,显示芽生轴突形成的突触末端。Pearson相关系数反映GAP-43/P38共定位频度,Pearson相关系数越大表明芽生轴突形成突触连接的数量越多。结果模型组和生理盐水组Pearson相关系数明显大于假手术组(P<0.05),提示脑缺血后存在自发性轴突芽生,并形成新的突触连接;梓醇各剂量组Pearson相关系数均比模型组明显增大(P<0.05),且梓醇中、高剂量组明显大于胞磷胆碱组(P<0.05),提示梓醇可促进PIC区芽生轴突形成突触连接,增强突触新生。突触超微结构分析显示,梓醇中剂量组PIC区神经毡内突触Nv和Sv较模型组和生理盐水组明显增加(P<0.05),但与胞磷胆碱组比较差异无显著性(P>0.05),提示梓醇对脑缺血后突触重构有明显促进作用。结论梓醇能促进局灶脑缺血大鼠PIC区神经元芽生轴突形成新的突触连接,增强突触结构可塑性。

Aim To observe the effects of catalpol on the axon sprouting and synaptogenesis within the peri- infarct cortex（PIC） in rats with focal cerebral ischemi- a, so as to explore the morphological bases for catalpol promoting neurorepairment after stroke. Methods 36 adult Sprague-Dawley rats were randomized into 7 groups, including sham operation group, model group, normal saline group, low dose, middle dose and high dose of catalpol treatment group （1, 5 and 10 mg kg - 1 , respectively） and citicoline treatment group （0.5 g kg-1）. Rats were subjected to permanent occlu- sions of the right middle cerebral artery （pMCAO） and were intraperitoneally administered with either catal- pol, citcoline or saline 24 h after pMCAO and daily for 7 days. The immunohistochemical double staining technique was employed to detect the co-localisation of growth associated protein 43 （ GAP-43 ） with synapto- physin （P38） protein in PIC, and stereological meth- ods and transmission electron microscopy were then used to quantify synaptic density in PIC. Results The distribution of P38 and GAP-43 in PIC region were de- termined with double immunofluorescence. P38 was la- beled with FITC and shown in green, GAP-43 was la- beled with cy3 and shown in red, and the co-localisati- on of P38/GAP-43 was shown in yellow. The Pearson＇s correlation coefficient of GAP-43 with P38 was calculated. The more the Pearson＇ s correlation co- efficient was, the more new presynaptic terminus was formed. It was shown that the Pearson＇ s correlation coefficient was significantly higher in the model group than in the sham operation group （ P 〈 0. 05 ） , indica- ting spontaneous axon sprouting and new synapse con- nection formed after cerebral ischemic stroke. The Pearson＇s correlation coefficient in the catalpol group increased remarkably, compared with the model group and the citicoline group （ P 〈 0.05 ）. It showed that catalpol promoted the new axon sprouting to form new synapse connection effectively. Synaptic uhrastructural analysis showed that catalpol at dose of 5 mg~ kg-1 in- creased the synaptic number density and the surface density in the neuropil within the PIC obviously （P 〈 O. 05）, when compared with the model group and the normal saline group, but there was no difference be- tween the catalpol and citicoline group （ P 〉 O. 05 ）. It demonstrated that catalpol could enhance the synaptic reorganization after cerebral ischemia. Conclusion Catalpol can promote CNS axonal sprouting and in- crease synaptic structure plasticity by synaptogenesis.