An innovative model of asphyxic cardiac arrest in piglets will be used in which the pattern of selective neuronal vulnerability simulates term neonatal hypoxic-ischemic (HI) encephalopathy, a major cause of morbidity and mortality in human newborns. Although hypothermia holds promise as a treatment, the delay in initiating hypothermia typical in clinical trials may not protect striatum, where neurons can die within 6 h of reoxygenation. This proposal focuses on therapies that are tailored for rapid striatal neuroprotection and that can extend the therapeutic window for hypothermia to protect delayed neurodegeneration in other regions. Mechanisms of injury specific to striatum have not been well studied in neonatal HI. Striatonigral neurons are enriched with dopamine D1 receptors, and striatopallidal neurons are enriched with adenosine A2A receptors, both of which act via PKA and the phosphorylation regulatory protein DARPP-32. D1 receptor activation amplifies NMDA channel calcium currents by NR1 phosphorylation and decreases Na,K-ATPase activity. In previous work, D1 dopamine receptor antagonist treatment ameliorated HI-induced phosphorylation at PKA- sensitive sites on DARPP-32, NR1 and Na,K-ATPase at 3 h of recovery, improved Na,K-ATPase activity, and selectively protected D1 neurons in piglet striatum. Preliminary data indicate that A2A antagonist post- treatment protects a portion of striatal neurons and attenuates HI-induced phosphorylation at PKA-sensitive sites on DARPP-32, NR1, and Na,K-ATPase. The arachidonic acid metabolite 20-HETE is also known to decrease Na,K-ATPase activity, but by PKC-dependent phosphorylation. Preliminary data with 20-HETE synthesis inhibitor post-treatment indicate partial neuroprotection and selective blockage of phosphorylation at PKC-sensitive sites on Na,K-ATPase and NR1. By targeting different phosphorylation sites of key proteins involved in excitotoxicity and multiple cell types, 20-HETE synthesis inhibition could complement D1 and A2A antagonism.
In Aim 1, the effect of treatment with an A2A antagonist after cardiac resuscitation from HI will be studied on DARPP-32, NR1, and Na,K-ATPase phosphorylation, Na,K-ATPase activity, superoxide production and markers of oxidative stress, neuronal viability, and preservation of A2A immunoreactivity in piglet striatum.
Aim 2 will determine whether post-treatment with combined A2A and D1 antagonists provides additive protection of distinct neuronal populations in striatum.
Aim 3 will determine if post-treatment with a 20-HETE synthesis inhibitor reduces phosphorylation at PKC-sensitive sites on NR1 and Na,K-ATPase, improves Na,K-ATPase activity, and protects both D1 and A2A striatal neurons.
Aim 4 will determine whether combined post-treatment with drugs found to be effective in Aims 1 3 extends the therapeutic window for delayed hypothermia. These novel neuroprotective studies, using a large animal model of whole body asphyxia, will render both unique mechanistic insights into the cause of rapid neurodegeneration in immature basal ganglia and therapies that can be readily translated for treatment of neonatal HI encephalopathy.

Public Health Relevance

Available treatments are limited for newborns who experience periods of low oxygenation that damages their brains during labor and delivery, and after birth, and that leads to long-term disabilities, such as spastic muscle control, cognitive deficits, seizures, and developmental delays. The mechanisms of injury are multifactorial and differ among the specific regions of immature brain. Using a newborn animal model of asphyxic cardiac arrest to simulate the brain injury in term human newborns, the goal of this application is to investigate these mechanisms in selectively vulnerable brain regions and to formulate a rational design of combination therapies that involve drugs and cooling the body for ameliorating the progression of brain injury that leads to life-long devastating consequences.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Developmental Brain Disorders Study Section (DBD)
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Koenig, James I
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Johns Hopkins University
Schools of Medicine
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