Cdk5-CRMP通路在七氟醚抑制新生大鼠前额叶皮层树突发育中的作用

Role of Cdk5-CRMP pathway in sevoflurane-induced dendritic developmental disorder of neurons in prefrontal cortex of neonatal rats

目的:探究七氟醚对新生大鼠前额叶皮层(prefrontal cortex,PFC)树突发育的影响及与细胞周期依赖性蛋白激酶5(cyclin dependent kinase 5,Cdk5)-坍塌反应调节蛋白(collapsin response mediator protein,CRMP)通路的关系。方法:88只出生后7 d的SD大鼠随机分为4组,每组22只:空白对照组(Air+NS组),Cdk5抑制剂roscovitine(Ros)对照组(Air+Ros组),Sevo+NS组,Sevo+Ros组。前2组吸入空气,后2组吸入2.8%七氟醚4 h。Air+Ros组和Sevo+Ros组在麻醉前15 min经腹腔注射10 mg/kg的Cdk5抑制剂roscovitine。其余2组则在麻醉前15min经腹腔注射150μL生理盐水。麻醉结束后,每组各取5只幼鼠,取出新鲜皮质组织,Western blot检测P35、P25、Cdk5,CRMP1、2、4的蛋白表达以及CRMP2 Ser 522的磷酸化水平。剩余幼鼠继续饲养,出生后30 d每组各取6只幼鼠,取脑进行高尔基染色,制备冰冻切片,镜下观察神经元形态。其余幼鼠在出生后25~27 d进行旷场试验,31~32 d进行情景依赖性恐惧记忆检测。结果:(1)与Air+NS组比较,Sevo+NS组的P35减少了47.32%,同时P25增加了173.77%(P<0.05);Sevo+Ros组的P35比Sevo+NS组增加了78.81%,其P25则减少了60.22%(P<0.05)。Cdk5表达组间差异尚无统计学意义。(2)与Air+NS组比较,Sevo+NS组的总CRMP1、2和4表达分别下降了36.22%、53.74%和51.28%(P<0.05),同时p-CRMP2 Ser522/CRMP2比值增加了96.20%(P<0.05);Sevo+Ros组的总CRMP1和4与Sevo+NS组比较分别增加了56.95%和88.91%(P<0.05),而pCRMP2 Ser522/CRMP2比值下降了43.58%(P<0.05)。(3)与Air+NS组比较,Sevo+NS组的树突总长度、第二级树突长度以及60和80μm直径圆环上的交点数均显著下降(P<0.01)。Sevo+Ros组的树突总长度、第二级树突长度以及60和80μm直径圆环上的交点数均比Sevo+NS组显著增加(P<0.05)。(4)在恐惧记忆实验中,与Air+NS组比较,Sevo+NS组大鼠的"木僵"时间比Air+NS组减少约21%(P<0.05)。Sevo+Ros组的"木僵"时间比Sevo+NS组显著增加(P<0.05)。各组幼鼠在旷场实验的结果均无统计学差异。结论:七氟醚抑制了新生大鼠前额叶皮层神经元树突的正常生长发育和幼鼠学习记忆能力,而Cdk5-CRMP通路激活可能是七氟醚引起神经发育毒性的机制之一。

AIM ： To investigate the effect of sevoflurane （Sevo） on the dendritic development in prefrontal cortex （PFC） of neonatal rats and the role of cyclin dependent kinase 5 （Cdk5）- collapsin response mediator protein （CRMP） pathway in it. METHODS： Eighty-eight postnatal day 7 Sprague Dawley （SD） rats were randomly divided into 4 groups （n = 22） ： Air ＋ NS group, Air ＋ roscovitine （Ros） group, Sevo ＋ NS group and Sevo ＋ Ros group. The rats inAir ＋ NS group and Air ＋ Ros group were exposed to the air for 4 h, while the rats in the other 2 groups were exposed to 2. 8% sevofurane for 4 h. The rats received intraperitoneal injection of 150 μL normal saline 15 rain before exposure in the Air ＋ NS group and Sevo ＋ NS group, while the rats in the Air ＋ Ros group and Sevo ＋ Ros group received intraperitoneal injection of 150 I^L roscovitine （in DMSO solution, 10 mg/kg） 15 min before exposure. At the end of exposure, the corti- ces of the rat brain were collected and the protein levels of P35, P25, Cdk5, CRMP1, CRMP2, CRMP4 and p-CRMP2 Ser522 in PFC were detected by Western blot. On the postnatal day 30, the rat brains were sectioned for Golgi-Cox staining and morphological analysis of dendrites in the PFC neurons. Open-field test and contextual fear conditioning test were per- formed on postnatal days 25 ~ 27 and 31 ~ 32, respectively. RESULTS： Compared with Air ＋ NS group, the expression of P35 in the Sevo ＋ NS group was significantly decreased, and the expression of P25 was dramatically increased （ P 〈 0. 05 ）, whereas roscovitine partly reversed the changes above induced by sevoflurane （ P 〈 0.05 ）. The expression of Cdk5 was not significantly different among all groups. Compared with the Air ＋ NS group, the expression of CRMP1, 2, and 4 in the Se- vo ＋ NS group were decreased, and the protein level of p-CRMP2 Ser522/CRMP2 was increased （ P 〈 0. 05 ）, whereas roseovitine partly reversed the changes above induced by sevoflurane （ P 〈 0.05 ） , except for the expression of CRMP2. Compared with Air ＋ NS group, the total dendrite length, secondary dendritic length and interactions on 60 and 80 μm shells in the Sevo ＋ NS group were decreased （P 〈 0. 01 ）, whereas roscovitine partly reversed the changes above induced by sevoflurane （P 〈 0. 05 ）. Compared with Air ＋ NS group, the percentage of freezing time in the Sevo ＋ NS group was de- creased （P 〈 0. 01 ）, whereas roscovitine partly reversed the changes induced by sevoflurane （P 〈 0.05 ）. No significant difference among groups in the open-feld test was observed. CONCLUSION： Sevoflurane exposure disturbed dendritic de- velopment of neurons in PFC, learning and memory ability of neonatal rats, which may be mediated by Cdk5-CRMP pathway.