Myeloperoxidase Assay on Hypoxic-Ischemic Brain Injury in Immature Rats

Yang, Hae Joung; Pee, Dae Hun; Lim, Ji Hye; Choi, Byoung Min; Lee, Kee Hyoung; Eun, Baik Lin

Abstract

PURPOSE: Neutrophils found around an infarcted area in the brain was once considered as only the physiologic response following the brain injury, but recent studies have shown that inflammatory responses by neutrophils play an important role in the reperfusion injury. The presence of polymorphonuclear leukocytes(PML) is proven by biochemical assay of myeloperoxidase(MPO) secreted in the cytoplasmic granules. We observed the process of PML infiltration on hypoxic-ischemic brain injury of immature rats by the assay of MPO activity and changes of the MPO activity after the administration of fucoidin, inhibitor of P- and L-selectin.
METHODS
We used a well characterized model of the brains of 7 day-old-rats, which had unilateral hypoxic and ischemic injuries(HI). Those injuries were induced by unilateral carotid artery ligation followed by timed exposure to hypoxic inspiratory gas mixture(8% O2). MPO activity was measured in the brain tissue homogenates of HI rats(n=18) at 0, 2, 8, 24 and 48 hrs and in rats that received fucoidin immediately before and again after hypoxia(50 mg/kg, n=6) at 8 and 24 hrs. Controls(n=2) were rats with neither hypoxia nor ischemia. The brain samples were homogenized in 20 mM potassium phosphate buffer(pH 7.4) for 50 secs. The homogenate was centrifuged at 14,000 g at 4degrees C for 15 mins and the supernatant was discarded. The tissue was pulverized, weighed, and suspended in 1 mL of 50 mM potassium phosphate buffer solution(pH 6.0) containing 0.5% cetylditrimethylammonium bromide(wt/vol). The tissue was sonicated and centrifuged at 10,000 g for 15 mins. 200 micro L of the supernatant was mixed with 1 mL of 50 mM potassium phosphate buffer solution(pH 6.0) containing 10 micro L of 1.325 mM o-dianisidine hydrochloride and 170 micro L of 3% hydrogen peroxide(vol/vol). Changes in absorbance at 460 nm were measured for 5 mins by using microplate reader. One unit of MPO activity was defined as that degrading 1 micro mol peroxide/min at 25degrees C, and the result was expressed as units of MPO/100 mg tissue.
RESULTS
In HI rats, MPO activity increased at 2 hrs after HI and peaked at 24 hrs in the right hemisphere. In rats with fucoidin treatment immediately before and again after hypoxia, the MPO activity significantly decreased in both hemispheres compared with HI rats(P<0.05). MPO activity in the tissue of control rats was insignificant.
CONCLUSION
The dynamic changes of the MPO activity suggest the important role of PMN on hypoxic-ischemic brain injuries in immature rats. MPO activity could be used as an index of the severity of injuries of hypoxic-ischemic brains.