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.