Expression of P-selectin mRNA and Protein on Hypoxic-Ischemic Brain Injury in Neonatal Rat

Kim, Kyung Burm; Lim, Ji Hye; Pee, Dae Hun; Eun, Baik Lin; Tockgo, Young Chang

Abstract

PURPOSE: The selectin family of adhesion molecules plays a role in the initiation of endothelium-leukocyte interaction of inflammation and ischemia-reperfusion. P-selectin, a rapidly expressed endothelial cell adhesion molecule, is essential for both neutrophil rolling after endothelial stimulation and neutrophil transmigration. P-selectins were expressed after brain injury and could play an important role in the pathogenesis of ischemic brain injury in adult animal. However, the mechanisms leading to post-hypoxic-ischemic injury in immature brain are unknown yet. We hypothesize that P-selectin might mediate post-hypoxic-ischemic injury in immature rat brain. We evaluated the expression of mRNA and protein of P-selectin in post-hypoxic-ischemic immature rat brain.
METHODS
In isoflurane-anesthetized P7(Postnatal day 7) Sprague-Dawley rats(n=81), the right carotid artery was isolated and coagulated. 1-2 h later animals were exposed to 8% oxygen(balanced with nitrogen) for 2 h in glass chambers, in a warm air incubator (temperature maintained at 36.5 degrees C). Control included carotid artery coagulation alone, hypoxia alone, and normal(neither hypoxia nor coagulation). For RNA extraction, the rats were decapitated at 0, 2, 4, 8, 12, 24 and 48 h after hypoxic-ischemic injury. For Western blot analyses with P-selectin, rats were decapitated at 0, 15, 30 min, 1, 2, 4, 8, 12, 24 and 48 h after hypoxic-ischemic injury. Control or hypoxia alone rats were sacrificed 8 h after the respective intervention.
RESULTS
There was no expression of P-selectin mRNA in control groups(carotid artery coagulation alone, hypoxia alone, or normal). P-selectin mRNA expression in the ipsilateral(right) hemisphere reached a peak at 8 h after hypoxia-ischemia and then barely detected after 24 h. Expression of P-selectin protein was not observed in brain tissue of control rats. P-selectin protein was detected as early as 15 min and 30 min at both hemisphere in experimental rats and decreased at 1 h. P-selectin protein increased in right hemisphere at 4 h post-hypoxia-ischemia, peaked at 8 h and no longer detectable at 24 h.
CONCLUSION
Hypoxic-ischemic injury leads to P-selectin expression in neonatal rats brain. The temporal profiles of post-hypoxic-ischemic P-selectin mRNA and protein expression are consistent with a role in the evolution of subsequent brain injury.