Perioperative pain relief by a COX-2 inhibitor affects ileal repair and provides a model for anastomotic leakage in the intestine.

The authors examined the potential of the cyclooxygenase 2 (COX-2) inhibitor carprofen to reproducibly induce anastomotic leakage. In experiment 1, an anastomosis was constructed in both ileum and colon of 20 rats, and they were given carprofen (5 mg/kg subcutaneously every 24 hours) or buprenorphine (0.02 mg/kg subcutaneously every 12 hours). In another 20 rats an anastomosis was constructed in either ileum or colon, and all received carprofen (experiment 2). Animals were sacrificed after 3 days. In experiment 1, the ileal dehiscence rate was 60% in the carprofen group and 0% in the buprenorphine group (P = .0108). Colonic anastomoses in both groups remained patent. In experiment 2, the anastomotic leakage rate was 80% in ileum and 0% in colon. Thus, COX-2 inhibitors can severely interfere with intestinal healing, particularly in the ileum. Perioperative administration of carprofen yields a unique model for anastomotic leakage, which allows translational research on the effectiveness of perisuture line reinforcement.

Early anastomotic repair in the rat intestine is affected by transient preoperative mesenteric ischemia.

INTRODUCTION: During bowel surgery, perioperative blood loss and hypotension can lead to transient intestinal ischemia. Recent preclinical studies reveal that the strength of intestinal anastomoses can be compromised after reperfusion. So far, this phenomenon has not been investigated in the very first days of healing when wound strength is lowest. MATERIAL AND METHOD: Ischemia was induced in rats by clamping both the superior mesenteric artery and ileal branches for 30 min. Immediately after declamping, anastomoses were constructed in both terminal ileum and descending colon. The same was done in control groups after sham-ischemia. Anastomotic bursting pressure and breaking strength were measured immediately after operation (day 0) and after 1, 2, or 3 days. Anastomotic hydroxyproline content, gelatinase activity, and histology were analyzed. RESULTS AND DISCUSSION: In ileal anastomoses, at day 1, both the breaking strength and bursting pressure were significantly (p < 0.05) lower in the ischemic group, while at day 2, this was the case for the bursting pressure only. In the colon, the bursting pressure in the ischemic group was lower at day 1. Anastomotic hydroxyproline content remained unchanged. Increased presence of the various gelatinase activities was found in ileum only at day 0 and in colon at days 1 and 2. Histological mucosal damage was found in ischemia-reperfusion groups. CONCLUSION: Transient mesenteric ischemia can negatively affect anastomotic strength during the very first days of healing, even if the tissue used for anastomotic construction looks vital.

Selective cyclo-oxygenase 2 inhibition affects ileal but not colonic anastomotic healing in the early postoperative period.

BACKGROUND: Selective cyclo-oxygenase 2 (COX-2) inhibitors are increasingly prescribed in the perioperative period. Recent recognition of a possible role for COX-2 in wound healing has raised concerns about the safety of their use in surgical practice. Therefore, the influence of celecoxib, a selective COX-2 inhibitor, on early anastomotic healing was investigated. METHODS: Celecoxib, in doses of 15, 50 or 200 mg per kg per day, was given daily from the day before operation onwards to male Wistar rats that received both ileal and colonic anastomoses. Anastomotic strength was assessed by measuring the breaking strength and bursting pressure on the third day after operation. A second group received a dose of 50 mg per kg per day and a colonic anastomosis only, and healing was assessed on the third and fifth day after surgery. RESULTS: Expression of COX-2 protein was upregulated in the anastomotic area. Administration of celecoxib, at all doses tested, resulted in a significantly higher ileal dehiscence rate than in control rats (P = 0.002). In contrast, colonic anastomoses healed normally within the same animals. The latter was confirmed in rats with colonic anastomoses only. CONCLUSION: In this model, administration of the COX-2 inhibitor celecoxib affected ileal but not colonic anastomotic healing in the early postoperative period.

No detrimental effects of repeated laparotomies on early healing of experimental intestinal anastomoses.

BACKGROUND: Little is known about the impact of repeated laparotomies on intestinal anastomotic healing. While experimental data are completely lacking, the sparse data available from clinical studies report high anastomotic failure rates, suggesting a negative effect in this respect. Since the unequivocal determination of such an effect may have important consequences for choosing the optimal treatment strategy for patients suffering from intra-abdominal infection, an experimental study has been performed in an established rodent model. METHODS: Intestinal anastomoses were constructed in healthy Wistar rats (ileal and colonic anastomoses) or 24 h after peritonitis was induced by caecal ligation and puncture (colonic anastomosis only). Rats were then scheduled to undergo no, one (after 24 h) or two relaparotomies (after 24 and 48 h). Anastomotic strength was assessed 3 and 5 days after anastomotic construction. On the third post-operative day anastomotic hydroxyproline levels, matrix metalloproteinase activity and myeloperoxidase activity were measured. RESULTS: No negative impact of repeated laparotomies was measured on any of the parameters measured. Under non-infectious conditions even an improvement in breaking strength (+48%, p=0.017) but not bursting pressure was found after two relaparotomies, but only in the ileum on the third post-operative day. CONCLUSIONS: In this experimental setting, early anastomotic healing is not adversely affected by repeated laparotomies.

Colonic anastomotic strength and matrix metalloproteinase activity in an experimental model of bacterial peritonitis.

BACKGROUND: Clinical studies report conflicting results on the safety of primary intestinal anastomoses in the presence of peritonitis, and comprehensive experimental data are lacking. The present study investigated whether the strength of experimental colonic anastomoses is affected if surgery is performed in the presence of pre-existing bacterial peritonitis. METHODS: Colonic anastomoses were constructed in Wistar rats 24 h after caecal ligation and puncture or a sham procedure. Anastomotic strength was assessed by measuring breaking strength and bursting pressure during the first 5 days after operation. Anastomotic hydroxyproline levels were measured and matrix metalloproteinase (MMP) activity was analysed by quantitative gelatin zymography. RESULTS: Anastomotic strength was lowered in the presence of bacterial peritonitis but in a minor and transient way. The breaking strength was lower only immediately after construction of the anastomosis (- 15 per cent, P = 0.011) and the bursting pressure only on the third postoperative day (- 33 per cent, P = 0.038); no anastomotic dehiscence was observed. At 3 days after operation increased levels of MMP activity were observed but anastomotic hydroxyproline content was not affected by bacterial peritonitis. CONCLUSION: The influence of bacterial peritonitis on the development of anastomotic strength is limited. This experimental finding lends support to recent clinical studies that have demonstrated the feasibility of constructing a primary anastomosis under these conditions.

The matrix metalloproteinase inhibitor BB-94 improves the strength of intestinal anastomoses in the rat.

BACKGROUND AND AIMS: The strength of intestinal anastomoses is relatively low in the first days after operation, possibly as a result of localized degradation of the supporting matrix by enzymes from the matrix metalloproteinase (MMP) family. This study examined whether BB-94, a broad spectrum inhibitor of MMP activity, could enhance anastomotic strength. MATERIALS AND METHODS: Male Wistar rats received anastomoses in both ileum and colon. From the day before operation onwards, animals were treated daily with BB-94 intraperitoneally at a dose of 30 mg/kg or with saline only. Rats were killed 1, 3, or 7 days after operation, and anastomotic bursting pressure and breaking strength were measured. On day 3 anastomotic hydroxyproline levels were measured, and MMP (gelatinase) activity was analyzed by gelatin zymography. RESULTS: BB-94 strongly enhanced wound strength, but only on day 3, when it was at its lowest. Daily administration increased median colonic and ileal breaking strength by 27% and 108%, respectively; colonic and ileal bursting pressure were increased by 54% and 58%, respectively. MMP activities were significantly lowered in anastomotic extracts from the rats treated with BB-94. CONCLUSION: Administration of BB-94 enhances anastomotic strength. Specific inhibition of MMP activity should be investigated further as a means to preserve anastomotic integrity.

Hyaluronic acid-based agents do not affect anastomotic strength in the rat colon, in either the presence or absence of bacterial peritonitis.

BACKGROUND: Hyaluronic acid (HA) agents reduce postsurgical adhesion formation. The effect of their perioperative administration on early anastomotic healing is unknown. This study investigated the influence of two HA-containing agents on the development of strength in colonic anastomosis during the first postoperative week, both in normal rats and in rats with bacterial peritonitis. METHODS: In 90 male Wistar rats a 1-cm segment was resected from the descending colon and an end-to-end anastomosis was constructed. In 108 rats a bacterial peritonitis was induced using caecal ligation and puncture (CLP). Some 24 h after CLP the abdomen was reopened, the caecum was taken out and, after resection of a 1-cm segment, an anastomosis was made. Animals in both groups were randomized to receive either an HA-carboxymethylcellulose (CMC) bioresorbable membrane, 0.4 per cent HA solution or no treatment. One-third of each group was killed at day 1, 3 and 7 after operation. Cultures were taken from the abdominal cavity for microbiological analysis in half of the animals. Subsequently, both bursting pressure and breaking strength were determined as parameters for anastomotic strength. RESULTS: No differences in anastomotic bursting pressure or breaking strength were found between the experimental groups and their controls. In addition, there was no significant difference in the number of bacteria cultured from the abdominal cavity between rats treated with HA and controls. CONCLUSION: Neither HA-CMC bioresorbable membrane nor 0.4 per cent HA solution interferes with the development of early anastomotic strength in the colon, and can therefore be safely used to prevent intra-abdominal adhesion formation after performing bowel anastomosis.

Interleukin 10 mitigates the development of the zymosan-induced multiple organ dysfunction syndrome in mice.

We investigated the effect of interleukin 10 on the development of zymosan-induced multiple organ dysfunction syndrome (MODS) and on plasma concentrations and production capacity of tumour necrosis factor (TNF)-alpha by peritoneal cells. Groups of C57BL/6 mice received a single intraperitoneal injection with zymosan, a cell wall component of Saccharomyces cerevisiae, at day 0. Daily doses of human recombinant interleukin 10 (IL-10: 10 or 50 microg/kg) were given intraperitoneally either starting directly before administration of zymosan (day 0), or 5 or 8 days after administration of zymosan. The animals were monitored for survival, condition, body weight and temperature. On day 12 all surviving animals were killed to obtain plasma, organs and peritoneal cells. Plasma concentrations of TNF-alpha and lipopolysaccharide-stimulated production of TNF-alpha by peritoneal cells were measured; organ weights were registered as an indicator for organ damage. IL-10 improves survival and clinical condition and also reduces organ damage, but only at the highest dose used and only when started simultaneously with the administration of zymosan. Circulating TNF-alpha concentrations 12 days after zymosan are not affected by any of the IL-10 schedules used. However, lipopolysaccharide-stimulated production of TNF-alpha by peritoneal cells is increased, in a dose- and time-dependent fashion. The anti-inflammatory cytokine IL-10 is able to attenuate the development of MODS in this model, but only when given simultaneously with zymosan, and in high dosages.

Is early post-operative treatment with 5-fluorouracil possible without affecting anastomotic strength in the intestine?

Early post-operative local or systemic administration of 5-fluorouracil (5-FU) is under investigation as a means to improve outcome after resection of intestinal malignancies. It is therefore quite important to delineate accurately its potentially negative effects on anastomotic repair. Five groups (n = 24) of rats underwent resection and anastomosis of both ileum and colon: a control group and four experimental groups receiving daily 5-FU, starting immediately after operation or after 1, 2 or 3 days. Within each group, the drug (or saline) was delivered either intraperitoneally (n = 12) or intravenously (n = 12). Animals were killed 7 days after operation and healing was assessed by measurement of anastomotic bursting pressure, breaking strength and hydroxyproline content. In all cases, 5-FU treatment from the day of operation or from day 1 significantly (P<0.025) and severely suppressed wound strength; concomitantly, the anastomotic hydroxyproline content was reduced. Depending on the location of the anastomosis and the route of 5-FU administration, even a period of 3 days between operation and first dosage seemed insufficient to prevent weakening of the anastomosis. The effects of intravenous administration, though qualitatively similar, were quantitatively less dramatic than those observed after intraperitoneal delivery. Post-operative treatment with 5-FU, if started within the first 3 days after operation, is detrimental to anastomotic strength and may compromise anastomotic integrity.

Moderate doses of intraoperative radiation severely suppress early strength of anastomoses in the rat colon.

Intraoperative irradiation appears to be a valuable addition to the modalities available to treat patients with large bowel cancer. However, its potential effect on healing of anastomoses has not been investigated extensively. For this purpose, male Wistar rats underwent colonic resection. Subsequently, 1 cm of each bowel end was irradiated with doses of 10, 15, 20 or 25 Gy and intestinal continuity was restored. After 3 or 7 days, animals were killed and the anastomoses were analyzed for bursting pressure (intraluminal force), breaking strength (longitudinal force) and hydroxyproline content. Intraoperative irradiation led to a massive (40-70%) and significant (P < 0.025) reduction in bursting pressure 3 days after operation compared to the control group for every dose used. After 7 days, the bursting site was outside the area of the anastomosis in all groups. The breaking strength at day 3 was also reduced, even after 10 Gy. At day 7, when tearing still occurred in the wound area, the breaking strength was still significantly lower in the 15- and 25-Gy groups than in the control group. The hydroxyproline content of the anastomoses was significantly reduced only after irradiation with the higher doses. Thus intraoperative irradiation constitutes a threat to early strength of anastomoses in the rat colon, and even at moderate doses it may threaten the integrity of the anastomosis.

Inhibition of fibroblast collagen synthesis and proliferation by levamisole and 5-fluorouracil.

Experimental studies indicate that anastomotic healing in the intestine is compromised by the immediate postoperative administration of 5-fluorouracil and levamisole. Since fibroblast functions are crucial to healing, we investigated the effects of (combinations of) both drugs on proliferation and collagen synthesis of rat skin fibroblasts in vitro. Proliferation was measured in actively dividing cells by cellular [3H]thymidine uptake and collagen synthesis in non-dividing cells by [3H]proline incorporation into collagenase-digestible protein. 5-Fluorouracil strongly and significantly (P < 0.05) reduced DNA synthesis and collagen synthesis at concentrations of 1 microM or more. The latter effect was not specific for collagen since total protein production was affected similarly. Both effects depended on the duration of exposure to the drugs. Levamisole also inhibited fibroblast proliferation dose-dependently, but less effectively than 5-fluorouracil: 50% inhibition was observed at approximately 0.1 mM. Collagen synthesis was unaffected by levamisole. If levamisole was added together with a low (0.1 microM) concentration of 5-fluorouracil, which in itself did not decrease thymidine incorporation, levamisole's antiproliferative effects became apparent at concentrations as low as 1 microM. A similar effect, but at a much higher concentration (1 mM) was noted on fibroblast collagen synthesis. These results indicate that levamisole potentiates 5-fluorouracil effects in fibroblast cultures and that direct effects of these drugs, alone or in combination, on fibroblast proliferation and collagen synthesis may be responsible for their negative influence on wound repair.

A semiquantitative histological analysis of repair of anastomoses in the rat colon after combined preoperative irradiation and local hyperthermia.

Hyperthermia is a promising method for increasing the efficacy of radiation therapy of colorectal cancer. To study the histological aspects of healing of an anastomosis in the colon, after combined preoperative (sham) irradiation and (sham) hyperthermia treatment, 48 male Wistar rats were divided randomly into four groups. In each animal, a segment of the colon was treated successively by (sham) irradiation (single dose of 25 Gy X rays) and/or (sham) hyperthermia (44 degrees C, 30 min). After 5 days, a resection of the colon was performed by construction of an anastomosis: The distal limb consisted of (sham-) irradiated and/or (sham-) hyperthermia-treated bowel. Rats were killed 3 or 7 days after the surgical procedure. Evaluation of healing of the anastomosis was made by: (1) histological analysis of sections stained with hematoxylin and eosin, (2) semiquantitative measurement of collagen in the area of the anastomosis and (3) semiquantitative analysis of the number of macrophages by immunocytochemistry. Healing of the anastomoses in animals receiving irradiation or hyperthermia alone and in control animals was relatively uneventful. There were no differences between groups in formation of collagen or infiltration by macrophages in the area of the anastomosis. Animals treated with both radiation and hyperthermia showed marked necrosis, infiltration by polymorphonuclear leukocytes and rupture of the anastomosis. It is concluded that preoperative irradiation with a single dose of 25 Gy in combination with local hyperthermia at 44 degrees C for 30 min leads to disturbed repair of anastomoses.

Intra-operative irradiation prolongs the presence of matrix metalloproteinase activity in large bowel anastomoses of the rat.

There exists a growing interest in intra-operative radiation therapy as a treatment modality for large bowel cancer. In a previous experimental study we showed that high-dose intra-operative irradiation delays the healing of colonic anastomoses. However, the contribution of proteases is unknown. In the present study, the gelatinolytic and collagenolytic activity in the healing anastomoses is investigated. After a resection of a 1-cm length of colon (uninjured colon), the rats were irradiated with a single dose of 25 Gy, either to the proximal limb, referred to as the proximal group, or to both proximal and distal limbs of the bowel, referred to as the combined group, before anastomotic construction. Both groups were compared to a control group with anastomoses which were sham-irradiated. The animals were killed 1, 3 or 7 days after operation. The gelatinolytic activity in uninjured and anastomotic tissue was quantified by gelatin zymography and the collagenolytic activity by an assay using a fibrillar rat collagen substrate. Compared with resected uninjured colon, most of the gelatinolytic activities were markedly increased in anastomotic tissue of all groups during the first postoperative week, and new additional activities were detected. The additional metalloproteinases (the 95-kDa family) of both irradiated groups were significantly elevated compared to the anastomoses of the sham-irradiated control group at 7 days after operation. In anastomotic tissue of all groups, the collagenolytic activity of the tissue was also significantly increased at 1 and 3 days after construction with respect to the resected, uninjured colon. After 7 days this effect had disappeared for the sham-irradiated anastomoses, but the activity in the anastomoses in both the proximal and combined groups remained significantly elevated. The findings provide evidence that intra-operative irradiation prolongs the presence of elevated gelatinolytic and collagenolytic activities in colon anastomoses. It may contribute to a reduced or delayed accumulation of collagen and other matrix proteins that supply anastomotic strength.

The effects of 5-fluorouracil and interferon-alpha on early healing of experimental intestinal anastomoses.

The continuing search for effective adjuvant therapy after resection of intestinal malignancies has prompted a growing interest in both immediate post-operative regional chemotherapy and the combination of 5-fluorouracil (5-FU) and interferon-alpha as drugs of choice. We have compared the effects of both compounds, alone and together, on early healing of intestinal anastomoses. Four groups (n = 26 each) of rats underwent resection and anastomosis of both ileum and colon: a control group and three groups receiving intraperitoneal 5-FU, interferon-alpha or both on the day of surgery and the next 2 days. Animals were killed 3 or 7 days (n = 10 each) after operation in order to measure anastomotic strength and hydroxyproline content. The remaining six animals in each group were used to study anastomotic collagen synthetic capacity at day 3. Three days after operation, ileal anastomotic bursting pressure was lowered by 37% in the 5-FU/interferon-alpha group (P = 0.0104). At day 7, anastomotic breaking strength was reduced significantly in ileum (P = 0.0221) and colon (P = 0.0054) of the 5-FU/interferon-alpha group and in colon of the interferon-alpha group (P = 0.0221). Collagen synthetic capacity was strongly suppressed by 5-FU but not by interferon-alpha. However, no differences in anastomotic hydroxyproline content were observed between groups at both days 3 and 7. Thus, post-operative use of interferon-alpha, in particular in combination with 5-FU, may be detrimental to anastomotic repair in the intestine.

Intraoperative irradiation delays anastomotic repair in rat colon.

BACKGROUND: There exists a growing interest in intraoperative radiation therapy as a treatment modality for large-bowel cancer. Since such therapy could interfere with wound repair, we investigated its effects on early healing of colonic anastomoses. METHODS: After resection of 1 cm of colon, rats were irradiated with a single dose of 25 Gy, either to the proximal limb (P group) or to both proximal and distal limbs of the bowel (PD group) before anastomotic construction. Both groups were compared with a sham-irradiated control group. Animals were killed 3, 7, or 14 days after operation, and healing was assessed by mechanical and biochemical (collagen) parameters. RESULTS: Three days after operation, bursting pressure was significantly lowered in the P group, whereas in the PD group both bursting pressure and breaking strength were strongly reduced. At day 7, the breaking strength was still reduced in the PD group, but not significantly so in the P group. The collagen synthetic capacity of the anastomotic segments was significantly lowered in both irradiated groups at day 3, resulting in a diminished collagen concentration in the actual wound area after 7 days. At 14 days after operation, no differences in strength were found between control and irradiated groups, while anastomotic hydroxyproline levels were significantly higher in both the P and PD groups than in the control group. CONCLUSIONS: High-dose intraoperative radiation therapy delays the healing of colonic anastomoses; it transiently reduces strength, probably as a result of a diminished accumulation of collagen.

Post-operative levamisole may compromise early healing of experimental intestinal anastomoses.

There exists growing interest in immediate post-operative local adjuvant therapy after resection of intestinal malignancies. It is therefore necessary to assess it potential effect on the healing of intestinal anastomoses. Five groups (n = 20) of rats underwent resection and anastomosis of both ileum and colon: a control group and four experimental groups receiving intraperitoneal 5-fluorouracil (5-FU), 5-FU plus leucovorin, 5-FU plus levamisole or levamisole alone, on the day of surgery and the next 2 days. Animals were killed 3 or 7 days after operation. Another three groups (n = 6) of animals were used to compare anastomotic collagen synthetic capacity in control rats or rats receiving 5-FU or 5-FU plus levamisole. On the third post-operative day, the average anastomotic bursting pressure in the 5-FU/levamisole group was reduced by 36% as compared with the control group, both in ileum (P = 0.02) and in colon (P = 0.01). Values in the other groups were similar to those in the control group. Anastomotic breaking strength was significantly (P < 0.025) lowered in the ileum from the levamisole group at both days 3 and 7. Anastomotic collagen synthetic capacity was strongly reduced in the 5-FU and 5-FU/levamisole groups. However, there was no significant difference between the control group and the four experimental groups with regard to anastomotic hydroxyproline concentration and content, either 3 or 7 days after operation. Thus, limited use of levamisole, alone or in combination with intraperitoneal 5-FU, may compromise intestinal healing.

Serum lipofuscin as a prognostic indicator of adult respiratory distress syndrome and multiple organ failure.

Toxic oxygen free radical damage is thought to play an important role in events such as trauma and sepsis, in which adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF) are the major causes of late death. Serum lipofuscin concentration has been proposed as an indicator of lipid peroxidation caused by toxic oxygen free radicals. Serum lipofuscin level was measured in 75 healthy controls and sequentially in 66 patients in the intensive care unit; the latter included 18 patients who had undergone elective major vascular surgery, 20 after repair of ruptured abdominal aortic aneurysm and 28 with severe blunt trauma. Fifteen of the 66 patients died within 2 days. Ten of the remaining 51 patients developed ARDS and/or MOF and 41 had an uncomplicated postoperative course. Serum lipofuscin concentration in controls showed a positive correlation with age. Compared with controls, all three patient groups had significantly increased lipofuscin concentrations during the first day after major vascular surgery, trauma or shock. In addition, the ten patients who subsequently developed ARDS and/or MOF showed significantly increased lipofuscin levels on day 1, compared with those in the 41 who had an uncomplicated clinical course. The concentration of serum lipofuscin, which may act as a simple and valuable measure of grading oxidative stress, is positively related to the incidence of subsequent ARDS and/or MOF in patients at risk of these syndromes.

Collagen synthetic capacity throughout the uninjured and anastomosed intestinal wall.

The capacity to synthesize collagen was measured throughout the intestinal tract of the rat, using an in vitro technique. In addition, the effect of anastomotic construction in either the ileum or the colon on collagen synthetic capacity at specific distant locations both 1 and 4 days after operation was investigated. Collagen synthetic capacity is relatively uniform throughout the large bowel. However, in the small bowel in which synthesis is lower than in the large bowel, synthesis is significantly higher in the most proximal and distal segments than in the intermediate tissue. Anastomotic construction in either part of the bowel strongly increases collagen synthetic capacity at the immediate wound site. The synthetic response is not restricted to the anastomotic site but appears to be more generalized and is, in the small bowel, to some extent specific for collagen since the collagen synthetic capacity is increased far more than the capacity to produce noncollagenous protein. Anastomotic construction in the colon has only minor, although sometimes significant, effects on collagen synthetic capacity in the ileum, and vice versa.

Collagenolytic activity in experimental intestinal anastomoses. Differences between small and large bowel and evidence for the presence of collagenase.

Collagen degradation is thought to be an integral part of the healing sequence of intestinal anastomoses, but almost nothing is known about the enzyme activities involved. We have studied collagenolytic activities, extracted from 1 day-old intestinal anastomoses in the rat. Using either soluble type I collagen or fibrillar type I or type III collagen as a substrate, activities measured in extracts from anastomotic segments were compared to those in extracts from uninjured intestine, removed at operation: in all cases, the collagenolytic activity in anastomotic extracts was significantly higher. This increase was significantly more pronounced in large bowel than in small bowel. The activities were strongly inhibited by serum and metallo-chelating compounds. Analysis, by means of SDS-polyacrylamide gel electrophoresis, of the reaction products of the degradation of fibrillar type I collagen by the extracts revealed the presence of a multitude of fragments, amongst them TcA fragments characteristic for the activity of mammalian collagenase. Thus, the degradative capacity towards various collagen substrates is enhanced in the anastomotic area during the first postoperative period and a true mammalian collagenase is one of the enzymes present.

Collagenases from human and rat skin fibroblasts purified on a zinc chelating column reveal marked differences in latency as a result of serum culture conditions.

1. Fibroblasts from both human and rat skin were grown in the presence or absence of serum and the collagenase activity in the medium was partially purified on zinc-Sepharose. 2. During chromatography, using a discontinuous elution gradient, the rat collagenase elutes at different pH and ionic strength than the human collagenase. Both latent and active collagenases of both species are retarded by the affinity matrix. 3. Latency of collagenase in media obtained from fibroblast cultures appears to be influenced by the presence of a serum component in the culture medium. 4. The results demonstrate that collagenases secreted by fibroblast cultures established from the same tissue but obtained from different species are biochemically diverse and that, within one species, the amount of active enzyme depends on the presence of a serum factor.