Grosche A, Morton A J, Polyak M M R, Matyjaszek S, Freeman D E
Island Whirl Equine Colic Research Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA.
Equine Vet J. 2008 Jun;40(4):393-9. doi: 10.2746/042516408X302500.
The cytosolic protein complex, calprotectin, is abundant in neutrophils and could be used to improve the ability to localise and assess neutrophil infiltration in the equine intestine during ischaemia and reperfusion (I/R), but further study is required.
To assess the number of calprotectin-containing cells by immunohistochemistry in correlation with direct counting and scoring of neutrophils in the equine colon during I/R.
One and 2 h ischaemia of the left dorsal colon were induced, followed by 30 min reperfusion under general anaesthesia or by 18 h reperfusion after anaesthetic recovery. Biopsies were processed for light microscopy and stained with H/E for detection of neutrophils. To identify calprotectin-containing cells, immunohistochemistry was performed on formalin-fixed tissues with the murine MAC 387 antibody and a biotin-free peroxidase staining procedure. The number of neutrophils within submucosal venules and the colonic mucosa were calculated and compared with the number of calprotectin-positive cells.
The number of calprotectin-positive cells within submucosal venules and within the colonic mucosa correlated significantly with the accumulation of neutrophils within the corresponding tissue segments. Within the submucosal venules, both calprotectin-positive cells and H/E-stained neutrophils increased with duration of ischaemia and peaked after 30 min of reperfusion. After 18 h reperfusion the number of these cells declined within the vessels. After 2 h ischaemia, neutrophils started to migrate into the mucosa towards the epithelium, with a significant increase over time during reperfusion, and peak infiltration after 18 h reperfusion.
Neutrophil infiltration into the colon after I/R is a time-dependent process, involving migration through the submucosa towards the epithelium.
胞质蛋白复合物钙卫蛋白在中性粒细胞中含量丰富，可用于提高对马肠道缺血再灌注（I/R）期间中性粒细胞浸润进行定位和评估的能力，但仍需进一步研究。
通过免疫组织化学评估含钙卫蛋白细胞的数量，并与马结肠在I/R期间中性粒细胞的直接计数和评分进行相关性分析。
诱导左背侧结肠缺血1小时和2小时，然后在全身麻醉下进行30分钟再灌注或在麻醉恢复后进行18小时再灌注。取活检组织进行光学显微镜检查，并用苏木精-伊红（H/E）染色以检测中性粒细胞。为鉴定含钙卫蛋白的细胞，对福尔马林固定的组织采用鼠MAC 387抗体和无生物素过氧化物酶染色程序进行免疫组织化学检测。计算黏膜下小静脉和结肠黏膜内中性粒细胞的数量，并与钙卫蛋白阳性细胞的数量进行比较。
黏膜下小静脉和结肠黏膜内钙卫蛋白阳性细胞的数量与相应组织段内中性粒细胞的积聚显著相关。在黏膜下小静脉内，钙卫蛋白阳性细胞和H/E染色的中性粒细胞数量均随缺血时间延长而增加，并在再灌注30分钟后达到峰值。再灌注18小时后，血管内这些细胞的数量减少。缺血2小时后，中性粒细胞开始向黏膜上皮迁移，在再灌注期间随时间显著增加，并在再灌注18小时后达到浸润峰值。
I/R后中性粒细胞向结肠的浸润是一个时间依赖性过程，包括通过黏膜下层向上皮迁移。
