Reason for performing study: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.Summary
Objectives: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.Summary
Methods: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.Summary
Results: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.Summary
Conclusions:Neutrophil infiltration into the colon after I/R is a time-dependent process, involving migration through the submucosa towards the epithelium.Summary
Potential relevance:Immunohistochemical staining for calprotectin can be used to demonstrate the pattern of neutrophil accumulation and migration following I/R.