398 - Elevation of mitochondrial electrical conductance precedes brain damage and is attenuated with hypothermia in neonatal mice with hypoxia-ischemia (HI).

Poster Number: 398

Maria A. Neginskaya, New York University, New York, NY, United States; Sergey Sosunov, Rutgers, Robert Wood Johnson Medical School, New Brunswick, NJ, United States; Zoya Niatsetskaya, Rutgers State University, New Brunswick, NJ, United States; Evgeny Pavlov, New York University, New York, NY, United States; Vadim Ten, Rutgers/RWJMS, Staten Island, NY, United States

Background: Activation of mitochondrial permeability transition (mPT) has been implicated in the evolution of post-ischemic brain injury. Mechanistic significance of this event has been demonstrated by neuroprotective effect of mPT inhibition. However, in contrast to the mature brain, the same strategy has failed to protect neonatal HI-brain.

Objective: To determine if in the neonatal HI, the mPT activates and contributes to cellular death in reperfusion.

Design/Methods: In neonatal (p10) mice subjected to the Rice-Vannucci model, at the end of HI (no reperfusion), at 30 minutes and at four hours of reperfusion, the ischemic hemisphere underwent mitochondrial isolation, electron microscopy (EM) and immunostaining for activation of caspase 3, a loss of microtubule associated protein 2 (MAP2) and nuclear pyknosis/rrhexis. Using excised patch-clamp, isolated mitochondria were probed for their inner membrane ion-permeability (mPT activation) and were examined under EM. A separate cohorts were exposed to hypothermia (HT, rectal t = 31°C) for the initial 30 minutes of reperfusion and 30 minutes of rewarming to a rectal t = 37°C or recovered under normothermia (NT, rectal t = 37°C). In randomly selected HT and NT mice, an ischemic brain mitochondria were isolated and probed for mPT activation.

Results: In NT mice, at 30 min. of reperfusion, mitochondrial electrical conductance was significantly elevated compared to naives and to that of prior-to reperfusion. EM of the same mitochondria showed only minimal swelling, and no cellular damage was detected by immunostaining. At 4 hours of reperfusion, mitochondria lost their matrix integrity. The same changes were detected in the tissue obtained from the same cerebral hemisphere. Immunostaining detected extensive cerebral injury evidenced by the loss of MAP2, nuclear pyknosis, but without extensive caspase 3 activation. Compared to NT-mice, HT-mice exhibited preserved mitochondrial electrical conductance and significantly decreased extent of their brain injury.Conclusion(s): Neonatal HI-reperfusion injury results in ion/proton leak across the inner mitochondrial membrane. This leak is consistent with mPT which activates within 30 minutes of reperfusion and precedes histopathological signs of cellular damage. Prevention of mPT activation afforded by the neuroprotective hypothermia highlights mechanistic contribution of this event to HI brain injury.