Direct measurement of collagenase in colonic anastomosis

Abstract

Collagenase has been implicated in colonic anastomotic dehiscence but the enzyme has not previously been specifically measured in colonic healing. A 72 h tissue culture method for colonic tissue and a radiochemical assay for collagenase were adapted to measure the enzyme in healing rabbit colon, with specificity of the assay confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis. Normal and postoperative colon secreted collagenase, predominantly in a latent form, in the first 24 h of culture. Total activity reached a plateau after 48 and 72 h in culture, when 50-70 per cent of the enzyme was in an active form. At these times in culture, activity was significantly higher than after 24 h (P less than 0.001). One day after anastomosis the total amount of collagenase secreted in culture was higher than normal but the increase did not achieve significance. Three days after anastomosis the colon secreted more collagenase than explants from 1 day postoperative tissue (P less than 0.002). The proportion of active enzyme in the first 24 h in culture was also increased. Since active collagenase can be measured in culture medium from both normal and postoperative colon, the tissue may be secreting plasminogen activator which allows plasmin to activate the enzyme. The increase in collagenase after operation coincided with a decrease in collagen concentration in the colon wall, measured by hydroxyproline. This supports previous suggestions that collagenase contributes to anastomotic dehiscence. However, the findings must be interpreted with caution as the variance of the results was shown to be predominantly due to time in culture, suggesting this could be a bigger influence than the operation itself. In addition, our previously reported immunohistochemical study of this system indicated that collagenase only occurred in a localized region, restricted to the everted portion of the anastomosis, with the activity being tightly controlled by its inhibitor, tissue inhibitor of metalloproteinases.

Effects of Two Conventional Preoperative Radiation Schedules on Anastomotic Healing in the Rat Colon

“…Leakage after colorectal anastomosis mainly occurs between the 5th and the 7th postoperative days, which represents the beginning of the reparative phase of wound healing, at which time the maturation of collagen is the leading process [23] . The extracellular matrix, particularly collagen metabolism and its potential disturbance, is assumed to be an important factor influencing the outcome of intestinal anastomotic healing [9,24] . The process of collagen cleavage, another fundamental branch that determines connective tissue quality, is mainly regulated by proteases, specifically MMPs, which are involved in various processes during wound healing, such as debridement, angiogenesis, and matrix remodeling [5] .…”

“…Several MMPs such as collagenase (MMP-1), the gelatinases (MMP-2 and MMP-9) and stromelysin (MMP-3) have been shown to be up-regulated in the direct vicinity of the healing anastomosis [6][7][8] . An increased presence of the active forms of these MMPs could lead to localized matrix degradation and transient loss of anastomotic strength [9,10] . In normal healing, restriction of the MMPs is consistent with a role in the remodeling of colonic anastomosis, but when healing is compromised, these enzymes are more widespread and may contribute to anastomotic dehiscence [11] .…”

Reinforcement of Colonic Anastomoses with a Collagenous Double-Layer Matrix Extracted from Porcine Dermis

“…The balance of de novo collagen synthesis versus collagenolysis at the anastomotic site is translocated towards collagenolysis because of induction of colonic collagenase by colonic injury during creation of the anastomosis. The collagenolytic activity peaks on days 3 and 4 and returns to normal values on day 7 [25] . In order to examine the anastomoses in this fragile period, early follow-up was done on the 4th postoperative day.…”

Role of Single-Dose Clostridiopeptidase A Collagenase in Peritoneal Adhesions

“…Multiple doses of CAC may reduce PPA formation and/or the number of previously formed PPAs. Multiple doses may cause intra-abdominal organ and/or vascular wounding and may thus represent overdosing of CAC [33,34]. …”