Barone F C, Schmidt D B, Hillegass L M, Price W J, White R F, Feuerstein G Z, Clark R K, Lee E V, Griswold D E, Sarau H M
Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pa. 19406.
Stroke. 1992 Sep;23(9):1337-47; discussion 1347-8. doi: 10.1161/01.str.23.9.1337.
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.
中性粒细胞在许多组织的缺血和再灌注损伤中起关键作用，但在局灶性脑缺血后的再灌注条件下尚未得到研究。本研究旨在通过髓过氧化物酶活性测定法证实我们之前对大鼠永久性局灶性卒中中性粒细胞定量的观察结果，并将其扩展至短暂性缺血再灌注情况。此外，对缺血组织中的白三烯B4受体结合情况进行评估，作为炎症细胞浸润的潜在标志物。
采用组织学、酶学和受体结合技术，对自发性高血压大鼠在永久性大脑中动脉闭塞24小时后（n = 25），或短暂闭塞80分钟（n = 12）或160分钟（n = 22）后再灌注24小时的梗死皮质组织中的中性粒细胞浸润和受体结合情况进行评估。
假手术（n = 26）未使所测任何参数发生变化。永久性大脑中动脉闭塞后，组织学观察到中性粒细胞聚集，但浸润程度中等，通常位于梗死皮质周边血管内及血管旁。短暂性大脑中动脉闭塞再灌注后，整个梗死皮质均观察到明显的中性粒细胞浸润。永久性闭塞后髓过氧化物酶活性增加（p < 0.05），短暂性闭塞再灌注后增加程度更大。缺血皮质中的髓过氧化物酶活性（每克湿重单位）在永久性闭塞、80分钟和160分钟短暂性闭塞再灌注时，分别比非缺血（对照）皮质增加了32.2倍、54.6倍和92.1倍（p < 0.05）。假手术未使髓过氧化物酶活性发生变化。局灶性缺血后白三烯B4受体结合也增加（p < 0.05），且与髓过氧化物酶活性增加平行。假手术大鼠缺血皮质特异性受体结合（每毫克蛋白飞摩尔数）为3.87 ± 0.63，永久性闭塞、80分钟和160分钟短暂性闭塞再灌注的大鼠分别为4.57 ± 0.98、8.98 ± 1.11和11.12 ± 1.63（与假手术相比均p < 0.05）。皮质髓过氧化物酶活性与皮质白三烯B4受体结合程度显著相关（两项不同研究中r分别为0.66和0.79，p < 0.01）。
这些数据表明中性粒细胞参与局灶性缺血，且再灌注期间梗死组织中有大量中性粒细胞聚集，可通过髓过氧化物酶活性测定法进行定量。梗死组织中白三烯B4受体结合以平行方式增加，这表明白三烯B4结合增加是由于聚集的中性粒细胞上的受体。
