Reperfusion increases neutrophils and leukotriene B4 receptor binding in rat focal ischemia

Neutrophils are critically involved with ischemia and reperfusion injury in many tissues but have not been studied under conditions of reperfusion after focal cerebral ischemia. The present studies were conducted to confirm our previous observations quantifying neutrophils in rat permanent focal stroke using a myeloperoxidase activity assay and to extend them to transient ischemia with reperfusion. In addition, leukotriene B4 receptor binding in ischemic tissue was evaluated as a potential marker for inflammatory cell infiltration.

Histological, enzymatic, and receptor binding techniques were used to evaluate neutrophil infiltration and receptor binding in infarcted cortical tissue 24 hours after permanent middle cerebral artery occlusion (n = 25) or temporary occlusion for 80 (n = 12) or 160 (n = 22) minutes followed by reperfusion for 24 hours in spontaneously hypertensive rats.

Sham surgery (n = 26) produced no changes in any parameter measured. After permanent middle cerebral artery occlusion, neutrophil accumulation was observed histologically, but the infiltration was moderate and typically within and adjacent to blood vessels bordering the infarcted cortex. After temporary middle cerebral artery occlusion with reperfusion, marked neutrophil infiltration was observed throughout the infarcted cortex. Myeloperoxidase activity was increased (p less than 0.05) after permanent occlusion and to a greater extent after temporary occlusion with reperfusion. Myeloperoxidase activity (units per gram wet weight) in ischemic cortex was increased over that in nonischemic (control) cortex 32.2-fold, 54.6-fold, and 92.1-fold for permanent occlusion and 80 and 160 minutes of temporary occlusion with reperfusion, respectively (p less than 0.05). Sham surgery produced no changes in myeloperoxidase activity. Leukotriene B4 receptor binding also was increased (p less than 0.05) after focal ischemia and paralleled the increases in myeloperoxidase activity. Ischemic cortex-specific receptor binding (femtomoles per milligram protein) was 3.87 +/- 0.63 in sham-operated rats and 4.57 +/- 0.98, 8.98 +/- 1.11, and 11.12 +/- 1.63 for rats subjected to permanent occlusion and 80 and 160 minutes of temporary occlusion with reperfusion, respectively (all p less than 0.05 different from sham-operated). Cortical myeloperoxidase activity was significantly correlated with the degree of cortical leukotriene B4 receptor binding (r = 0.66 and r = 0.79 in two different studies, p less than 0.01).

These data indicate that neutrophils are involved in focal ischemia and that there is a dramatic accumulation of neutrophils in infarcted tissue during reperfusion that can be quantified using the myeloperoxidase activity assay. Leukotriene B4 receptor binding increases in infarcted tissue in a parallel manner, which suggests that the increased leukotriene B4 binding is to receptors located on the accumulating neutrophils.