■周围神经损伤后的功能恢复仍不令人满意。本研究旨在对盐酸法舒地尔治疗大鼠坐骨神经横断损伤的疗效及其机制进行综合评价。
■在动物实验中,将75只接受右坐骨神经横切和修复的SpragueDawley大鼠分为对照组,法舒地尔,和Fas+LY组,每天腹腔注射生理盐水,盐酸法舒地尔(10mg/kg),和盐酸法舒地尔加LY294002(5mg/kg),分别。手术后第3天,ROCK2、p-PI3K、在L4-5DRG中和p-AKT,并使用蛋白质印迹法测定腰骶部肿大。在第7天和第14天,使用抗神经丝-200抗体用免疫染色评估远端残端中的轴突密度。在第30天,通过在远端残端注射荧光金来进行逆行追踪。手术三个月后,使用抗MPZ抗体和透射电子显微镜进行免疫染色分析。此外,运动诱发电位,用神经监测仪评估感觉运动功能的恢复,足迹,热板和VonFrey长丝,分别。Moveover,对腓肠肌进行称重,然后进行H&E染色,并使用α-银环蛇毒素对神经肌肉接头进行染色,以评估腓肠肌终板的萎缩和变性程度。体外,培养脊髓运动神经元(SMNs)和背根神经节(DRG),以检查盐酸法舒地尔和LY294002对轴突生长的影响。
■受伤三天后,ROCK2的表达明显增加(P<0.01),应用法舒地尔可显著增加L4-6DRG中p-PI3K和p-AKT的表达及腰骶部增大(P<0.05)。手术后第7天和第14天,法舒地尔组轴突密度较高(P<0.05)。手术后第30天,法舒地尔组术后3个月可观察到大量的运动和感觉神经元吸收荧光金(P<0.01),法舒地尔组髓鞘厚度较大(P<0.05)。电生理测试显示,法舒地尔组可触发较大幅度的运动诱发电位(P<0.01)。行为测试表明,法舒地尔组可以测量到较高的坐骨功能指数和较低的对热和机械刺激的反应阈值。(P<0.01)。法舒地尔组腓肠肌湿重比及其肌原纤维横截面面积较大(P<0.01),这也表明轴突-端板连接的比例更大,端板的尺寸更大(P<0.05)。上述指标在对照组和Fas+LY组之间差异无统计学意义(P>0.05).体外研究表明,法舒地尔可以显著促进DRG和SMNs的轴突生长,增加p-PI3K和p-AKT的表达,可被LY294002废除(P<0.05)。
■法舒地尔可以促进轴突再生和髓鞘再生,并通过激活PI3K/AKT通路恢复坐骨神经损伤后的功能。
■本文的翻译潜力是我们首次报道盐酸法舒地尔通过ROCK/PI3K/AKT通路在改善大鼠右坐骨神经横断损伤后轴突再生和髓鞘再生方面具有显著功效,具有转化潜力,可用于临床治疗周围神经损伤。
UNASSIGNED: The functional recovery after peripheral nerve injury remains unsatisfactory. This study aims to perform a comprehensive evaluation of the efficacy of Fasudil Hydrochloride at treating the sciatic nerve transection injury in rats and the mechanism involved.
UNASSIGNED: In animal experiments, 75 Sprague Dawley rats that underwent transection and repair of the right sciatic nerve were divided into a control, Fasudil, and Fas + LY group, receiving daily intraperitoneal injection of saline, Fasudil Hydrochloride (10 mg/kg), and Fasudil Hydrochloride plus LY294002 (5 mg/kg), respectively. At day 3 after surgery, the expression of ROCK2, p-PI3K, and p-AKT in L4-5 DRG and the lumbosacral enlargement was determined using Western blotting. At day 7 and 14, axon density in the distal stump was evaluated with immunostaining using the anti-Neurofilament-200 antibody. At day 30, retrograde tracing by injecting Fluoro-gold in the distal stump was performed. Three months after surgery, remyelination was analyzed with immostaining using the anti-MPZ antibody and the transmission electron microscope; Moreover, Motion-Evoked Potential, and recovery of sensorimotor functions was evaluated with a neuromonitor, Footprint, Hot Plate and Von Frey Filaments, respectively. Moveover, the Gastrocnemius muscles were weighed, and then underwent H&E staining, and staining of the neuromuscular junction using α-Bungarotoxin to evaluate the extent of atrophy and degeneration of the endplates in the Gastrocnemius. In vitro, spinal motor neurons (SMNs) and dorsal root ganglia (DRG) were cultured to examine the impact of Fasudil Hydrochloride and LY294002 on the axon outgrowth.
UNASSIGNED: Three days after injury, the expression of ROCK2 increased significantly (P<0.01), and Fasudil application significantly increased the expression of p-PI3K and p-AKT in L4-6 DRG and the lumbosacral enlargement (P < 0.05). At day 7 and 14 after surgery, a higher axon density could be observed in the Fasudil group(P < 0.05). At day 30 after surgery, a larger number of motor and sensory neurons absorbing Fluoro-gold could be observed in the Fasudil group (P < 0.01) Three months after surgery, a greater thickness of myelin sheath could be observed in the Fasudil group (P < 0.05). The electrophysiological test showed that a larger amplitude of motion-evoked potential could be triggered in the Fasudil group (P < 0.01). Behavioral tests showed that a higher sciatic function index and a lower threshold for reacting to heat and mechanical stimuli could be measured in the Fasudil group. (P < 0.01). The wet weight ratio of the Gastrocnemius muscles and the area of the cross section of its myofibrils were greater in the Fasudil group (P < 0.01), which also demonstrated a higer ratio of axon-endplate connection and a larger size of endplates (P < 0.05). And there were no significant differences for the abovementioned parameters between the control and Fas + LY groups (P>0.05). In vitro studies showed that Fasudil could significantly promote axon growth in DRG and SMNs, and increase the expression of p-PI3K and p-AKT, which could be abolished by LY294002 (P < 0.05).
UNASSIGNED: Fasudil can augment axon regeneration and remyelination, and functional recovery after sciatic nerve injury by activating the PI3K/AKT pathway.
UNASSIGNED: The translation potential of this article is that we report for the first time that Fasudil Hydrochloride has a remarkable efficacy at improving axon regeneration and remyelination following a transection injury of the right sciatic nerve in rats through the ROCK/PI3K/AKT pathway, which has a translational potential to be used clinically to treat peripheral nerve injury.