Supplementary Material for: Neuronal Nitric Oxide Synthase Inhibition Prevents Cerebral Palsy following Hypoxia-Ischemia in Fetal Rabbits: Comparison between JI-8 and 7-Nitroindazole

Cerebral palsy and death are serious consequences of perinatal hypoxia-ischemia (HI). Important concepts can now be tested using an animal model of cerebral palsy. We have previously shown that reactive oxygen and nitrogen species are produced in antenatal HI. A novel class of neuronal nitric oxide synthase (nNOS) inhibitors have been designed, and they ameliorate postnatal motor deficits when administered prior to the hypoxic-ischemic insult. This study asks how the new class of inhibitors, using JI-8 (K_i for nNOS: 0.014 µ<i>M</i>) as a representative, compare with the frequently used nNOS inhibitor 7-nitroindazole (7-NI; K_i: 0.09 ± 0.024 µ<i>M</i>). A theoretical dose equivalent to 75 K_i of JI-8 or equimolar 7-NI was administered to pregnant rabbit dams 30 min prior to and immediately after 40 min of uterine ischemia at 22 days gestation (70% term). JI-8 treatment resulted in a significant decrease in NOS activity (39%) in fetal brain homogenates acutely after HI, without affecting maternal blood pressure and heart rate. JI-8 treatment resulted in 33 normal kits, 2 moderately and 13 severely affected kits and 5 stillbirths, compared with 8 normal, 3 moderately affected and 5 severely affected kits and 10 stillbirths in the 7-NI group. In terms of neurobehavioral outcome, 7-NI was not different from saline treatment, while JI-8 was superior to saline and 7-NI in its protective effect (p &lt; 0.05). In the surviving kits, JI-8 significantly improved the locomotion score over both saline and 7-NI scores. JI-8 was also significantly superior to saline in preserving smell, muscle tone and righting reflex function, but 7-NI did not show significant improvement. Furthermore, a 100-fold increase in the dose (15.75 µmol/kg) of 7-NI significantly decreased systolic blood pressure in the dam, while JI-8 did not. The new class of inhibitors such as JI-8 shows promise in the prevention of cerebral palsy and is superior to the previously more commonly used nNOS inhibitor.

脑瘫与死亡是围产期缺氧缺血（perinatal hypoxia-ischemia, HI）引发的严重不良结局。如今可借助脑瘫动物模型对关键科学概念进行验证。本团队此前已证实，产前缺氧缺血过程中会产生活性氧与活性氮。研究人员已设计出一类新型神经元型一氧化氮合酶（neuronal nitric oxide synthase, nNOS）抑制剂，该类抑制剂可在缺氧缺血损伤前给药，以改善出生后运动功能缺陷。本研究以JI-8（nNOS的抑制常数K_i：0.014 μM）为代表，探讨这类新型抑制剂与当前常用的nNOS抑制剂7-硝基吲唑（7-nitroindazole, 7-NI；K_i：0.09 ± 0.024 μM）的药效差异。实验于妊娠22天（约为足月的70%）的孕兔子宫缺血40分钟前30分钟及缺血即刻，分别给予相当于75倍K_i剂量的JI-8，或等摩尔剂量的7-NI。结果显示，JI-8给药可显著降低缺氧缺血后急性胎脑匀浆中的一氧化氮合酶活性（降幅达39%），且不会对孕兔的血压与心率造成影响。JI-8给药组共产下33只健康幼兔、2只轻度受损幼兔、13只重度受损幼兔以及5只死胎；而7-NI给药组则为8只健康幼兔、3只轻度受损幼兔、5只重度受损幼兔以及10只死胎。神经行为结局评估结果表明，7-NI给药组与生理盐水对照组无显著差异，而JI-8的神经保护效果显著优于生理盐水对照组与7-NI给药组（p < 0.05）。在存活幼兔中，JI-8的运动评分显著高于生理盐水组与7-NI组。此外，JI-8在保留嗅觉功能、肌张力与翻正反射方面均显著优于生理盐水组，但7-NI未表现出显著改善效果。进一步研究发现，将7-NI剂量提升至原剂量的100倍（15.75 μmol/kg）时，可显著降低孕兔的收缩压，而JI-8则无此不良反应。这类新型nNOS抑制剂（以JI-8为代表）在脑瘫预防方面展现出良好应用前景，且其药效优于此前广泛使用的7-NI。