Effect of Concurrent Src Kinase Inhibition with Short-Duration Hypothermia on Ca2+/Calmodulin Kinase IV Activity and Neuropathology after Hypoxia-Ischemia in the Newborn Swine Brain

Abstract

Background: Hypoxia-ischemia (HI) results in increased activation of Ca2+/calmodulin kinase IV (CaM kinase IV) mediated by Src kinase. Therapeutic hypothermia ameliorates neuronal injury in the newborn.

Hypothesis: Inhibition of Src kinase concurrently with hypothermia further attenuates the hypoxia-induced increased activation of CaM kinase IV compared with hypothermia alone.

Design/methods: Ventilated piglets were exposed to HI, received saline or a selective Src kinase inhibitor (PP2), and were cooled to 33°C. Neuropathology, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations, and CaM kinase IV activity were determined.

Results: The neuropathology mean score (mean ± SD) was 0.4 ± 0.43 in normoxia-normothermia (p < 0.05 vs. hypoxia-normothermia), 3.5 ± 0.89 in hypoxia-normothermia (p < 0.05 vs. normoxia-normothermia), 0.7 ± 0.73 in hypoxia-hypothermia (p < 0.05 vs. normoxia-normothermia), and 0.5 ± 0.70 in normoxia-hypothermia (p < 0.05 vs. hypoxia-normothermia). The CaM kinase IV activity in cerebral tissue (pmol Pi/mg protein/min; mean ± SD) was 2,002 ± 729 in normoxia-normothermia, 1,704 ± 18 in normoxia-hypothermia, 6,017 ± 2,510 in hypoxia-normothermia, 4,104 ± 542 in hypoxia-hypothermia (p < 0.05 vs. normoxia-hypothermia), and 2,165 ± 415 in hypoxia-hypothermia with PP2 (p < 0.05 vs. hypoxia-hypothermia). The hypoxic groups with and without hypothermia or Src kinase inhibitor were comparable in the levels of ATP and PCr, indicating that they were similar in their degree of energy failure prior to treatments. Hypothermia or Src kinase inhibitor (PP2) did not restore the ATP and PCr levels.

Conclusions: Hypothermia and Src kinase inhibition attenuated apoptotic cell death and improved neuropathology after hypoxia. The combination of short-duration hypothermia with Src kinase inhibition following hypoxia further attenuates the increased activation of CaM kinase IV compared to hypothermia alone in the newborn swine brain.

Keywords: CaM kinase IV; Cerebral hypoxia; Hypothermia; Src kinase.

Figure 1

Representative H&E-stained hippocampal sections illustrating a. a neuronal apoptotic profile (arrow) and b. normal neuronal cells (arrow heads). The photos were taken at × 600 magnification with oil immersion.

Figure 2

CaM kinase IV activation during normoxia and hypoxia and treatment with hypothermia and with or without Src kinase inhibition. The hypoxia induced increase in CaM kinase IV activation was reduced by hypothermia and reduced further when hypothermia was combined with Src kinase inhibition (PP2).

Figure 2

CaM kinase IV activation during normoxia and hypoxia and treatment with hypothermia and with or without Src kinase inhibition. The hypoxia induced increase in CaM kinase IV activation was reduced by hypothermia and reduced further when hypothermia was combined with Src kinase inhibition (PP2).

Diagram 1

Mechanism of apoptotic cell death following cerebral hypoxia. Note the point of action (marked with X) of hypothermia and the Src kinase inhibitor (PP2). Following inhibition of Src kinase, the activation of other enzymes, such as CaM kinase IV and Caspase 9 and 3 have been shown to be decreased.