Effect of concurrent administration of apoptotic inhibitors and hypothermia on post hypoxic cerebral injury in the newborn.

Neonatal hypoxic-ischemic encephalopathy is known to cause long-term neurodevelopmental impairment. Experimental studies and clinical trials demonstrated that treatment with hypothermia after hypoxic-ischemic insults reduced brain injury. As a result of these data, hypothermia has emerged as the standard of care for treatment of neonatal hypoxic-ischemic encephalopathy. However up to 40% of newborns with hypoxic-ischemic encephalopathy who are treated with hypothermia have significant neurocognitive deficits on follow-up. Obviously, there remains a need to further optimize cooling strategies and to identify adjuvant therapies that could potentially augment the neuroprotective effects and accentuate neuroprotection by hypothermia. As the occurrence of hypoxia in the newborn brain can not be predicted beforehand, the only opportunity we have to improve outcomes after hypoxic-ischemic encephalopathy is to pursue neuroprotective strategies that can be used as an adjunct to therapeutic hypothermia in the post-hypoxia-ischemia period, with special emphasis on mechanism mediating the early stages of hypoxic injury. Previously, we have demonstrated in the newborn piglet that within one hour of exposure to hypoxia, there is increased activation of the enzyme Ca++/calmodulin kinase (CaM Kinase) IV localized in the nucleus, a key regulator of transcription of apoptotic genes. We have also demonstrated that the hypoxia-induced enzyme CaM kinase IV activation is mediated by activation of two protein tyrosine kinases, Src kinase and EGFR kinase and by increased Ca++ influx into the nucleus. Inhibition of Src kinase by the selective inhibitor PP2 and of EGFR kinase by the selective inhibitor PD168393 at the onset of hypoxia prevented CaM kinase IV activation and decreased subsequent hypoxia-induced neuronal death. The aim of this study was to test the hypothesis that the combined treatment with hypothermia and PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a highly selective inhibitor of Src kinase, immediately after the hypoxic insult may augment the beneficial effect of hypothermia on hypoxia/ischemia- induced neuronal necrosis. To this aim we assessed the levels of CaM Kinase IV activity as well as the levels of Na+-K+-ATPase in the Cerebral Cortex of Newborn Piglets exposed experimental hypoxia that were treated with hypothermia with or without concomitant PP2 administration. 2-3 day old piglets were anesthetized and ventilated. In conclusion, our preliminary data show that concurrent administration of Src kinase inhibitor in combination with induction of whole body hypothermia results in augmented neuroprotection as indicated by further attenuation of hypoxic-ischemic induced CaM kinase IV activation and improvement in neuronal membrane integrity compared to hypothermia alone.