METHODS: We administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified using ELISA.
RESULTS: We observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1β levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1β, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway.
CONCLUSIONS: Our findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes.
方法:我们在出生后第7天(PND7)对新生大鼠进行1.5%异氟烷麻醉,持续时间为4小时。在PND28使用旷场测试评估焦虑水平,而在PND31和PND34之间使用Morris水迷宫测试评估认知功能。NKCC1、KCC2、BDNF、通过蛋白质印迹分析测量海马中的磷酸化ERK(P-ERK)。促炎细胞因子IL-1β,使用ELISA定量IL-6和TNF-α。
结果:我们观察到,与CON幼崽相比,ISO组中心区域内的运动轨迹减少,总距离明显缩短,表明异氟烷诱导焦虑样行为。在莫里斯水迷宫(MWM)测试中,暴露于异氟烷的大鼠在平台上表现出延长的逃避潜伏期。此外,在PND34的MWM实验中,异氟烷给药导致杂交时间减少,提示记忆功能长期受损.此外,我们发现异氟烷触发了促炎细胞因子IL-1β的激活,IL-6和TNF-α;下调PND7大鼠海马中KCC2/BDNF/P-ERK的表达;并增加NKCC1/KCC2的比率。布美他定(NKCC1特异性抑制剂)通过抑制TNF-α激活逆转异氟醚诱导的新生大鼠认知损伤和有效障碍,使IL-6和IL-1β水平正常化,恢复KCC2表达水平以及BDNF和ERK信号通路。基于这些发现,可以推测BDNF,P-ERK,IL-1β,IL-6和TNF-α可能作用于NKCC1/KCC2通路的下游。
结论:我们的发现提供了证据,即新生大鼠的异氟醚给药通过阳离子-氯化物协同转运蛋白NKCC1和KCC2,BDNF的失调导致持续的认知缺陷,p-ERK蛋白,以及神经炎症过程。