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

Abstract

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 µM) as a representative, compare with the frequently used nNOS inhibitor 7-nitroindazole (7-NI; K_i: 0.09 ± 0.024 µM). 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 < 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.