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.

The effects of adjuvant experimental radioimmunotherapy and hyperthermic intraperitoneal chemotherapy on intestinal and abdominal healing after cytoreductive surgery for peritoneal carcinomatosis in the rat.

BACKGROUND: Cytoreductive surgery (CS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) results in limited survival benefit and high morbidity and mortality rates in patients with peritoneal carcinomatosis (PC). Radioimmunotherapy (RIT) after CS of experimental PC has been shown to increase survival and compare favorably to HIPEC. The effects of RIT and HIPEC on wound healing after CS need to be determined. METHODS: PC was induced by intraperitoneal inoculation of CC-531 colon carcinoma cells in Wag/Rij rats. Animals were subjected to CS and anastomotic construction only or followed by RIT or HIPEC. RIT consisted of 74 MBq (177)lutetium-labeled anti-CC531 antibody MG1. HIPEC was performed by a closed abdominal perfusion technique using mitomycin-C during 60 minutes. Anastomotic and abdominal wall strength measurements were performed 3 and 5 days after surgery. RESULTS: At day 5, bursting pressure in ileum and colon anastomoses in the CS + HIPEC group, but not in the CS + RIT group, was lower (P < .01) than in the CS group. In the CS group, the colonic bursting site was more often outside the true anastomotic area (8 of 12 animals) than in the CS + HIPEC (1 of 12) and CS + RIT (5 of 12) groups. Abdominal wall strength in the CS + HIPEC group was significantly (P < .01) lower, at both measuring points, than that in both the CS group and the CS + RIT group. There was no difference between the latter. CONCLUSION: As adjuvant to CS, HIPEC showed a decrease in anastomotic and abdominal wall wound strength in a model of PC of CRC, whereas RIT did not.

Protein folding and aggregation studied by isoelectric focusing across a urea gradient and isoelectric focusing in two dimensions.

Isoelectric focusing across a concentration gradient of urea was used to study the folding-unfolding and association-dissociation processes of proteins. Myoglobulin, albumin, RNase, papain, beta L- and alpha-crystallin were analyzed with this technique, and examples are given of visualized dissociation steps and of equilibrium-unfolding intermediates. Furthermore, a two-dimensional isoelectric focusing technique is presented that is useful to deduce whether a transition of a protein aggregate observed upon urea-gradient isoelectric focusing must be attributed to a change in the protein's tertiary or quaternary structure.

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.

Combined preoperative irradiation and direct postoperative 5-fluorouracil without negative effects on early anastomotic healing in the rat colon.

BACKGROUND AND PURPOSE: Preoperative irradiation with direct postoperative chemotherapy could benefit patients undergoing surgery for colorectal cancer. This study was designed to examine, in an experimental model, if such treatment is feasible without detrimental effects on early anastomotic healing. MATERIAL AND METHODS: A colonic segment was irradiated (25 Gy) in 3 groups (n = 10 each) of male Wistar rats. After 5 days, a colonic resection was performed with anastomotic construction; only the distal limb consisted of irradiated bowel. Postoperatively, animals received daily intraperitoneal 5-fluorouracil (5-FU, group I/CH: 17.5 mg/kg; group I/CL: 12.5 mg/kg) or saline (group I). Three additional groups were treated similarly, but with sham-irradiation: CH, CL and C, respectively. All rats were killed 7 days postoperatively. Parameters measured were: weight, serum albumin and protein, and anastomotic bursting pressure, breaking strength and hydroxyproline content. RESULTS: Body weight was diminished significantly in rats receiving chemotherapy. Serum albumin and protein was significantly lower in irradiated groups. At sacrifice, 40% of I/CH rats had functional rectal stenosis. The average bursting pressure (P = 0.0005) and the average breaking strength (P = 0.012) were only reduced significantly in the CH group. The anastomotic hydroxyproline content was significantly higher in the I/CH and I/CL groups vs. the control group. CONCLUSION: High-dose direct postoperative 5-FU leads to reduced anastomotic strength. Although the combination of preoperative irradiation (25 Gy) and direct postoperative high-dose 5-FU does not reduce early anastomotic strength, some stenosis may occur. The combination of preoperative irradiation and low-dose 5-FU has no such effect.

Chondrocyte behaviour within different types of collagen gel in vitro.

In cartilage repair experiments chondrocytes are transplanted into osteochondral defects. Biological substances are used as cell vehicles and are likely to play an important role in the outcome of these studies. Collagen gel is formed by polymerization of type I collagen and is used in plastic surgery and for three-dimensional culture systems. To test collagen gel as a potential vehicle for transplantation, we evaluated chondrocyte behaviour in vitro in different collagen gels. Collagen type I was extracted and purified from rat tail tendon and fetal calf skin and compared with commercially available collagen type I. After suspension of bovine chondrocytes, five different collagen gels were cultured for 14 days and evaluated by light and electron microscopy. Cells proliferated within all gels and synthesized proteoglycans as assessed by 35S incorporation; 40-90% of cells maintained a chondrocyte-like morphology after 1 week in culture depending on the type of collagen gel. Synthetic and secretory activity was confirmed by electron microscopy. Based on these results, calf skin collagen is recommended for culturing chondrocytes for implantation.

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.

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.

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.

Combined preoperative irradiation and local hyperthermia delays early healing of experimental colonic anastomoses.

OBJECTIVE: To determine if a combination of preoperative irradiation and local hyperthermia of a colonic segment is detrimental to subsequent early anastomotic healing. DESIGN: A prospective randomized experimental trial. SETTING: An animal research laboratory. INTERVENTIONS: Eighty male Wistar rats were randomly divided into 4 groups. In each animal, a segment of the colon was treated successively by (sham) irradiation and (sham) hyperthermia. After 5 days, a colonic resection was performed and an anastomosis was constructed; the distal limb consisted of (sham) irradiated, (sham) hyperthermia-treated bowel. The rats were killed 3 or 7 days after surgery. MAIN OUTCOME MEASURES: Body weight, serum albumin and protein levels, anastomotic bursting pressure, breaking strength, and hydroxyproline content. RESULTS: All animals tolerated (sham) treatment well. Weight was diminished, though not notably, in treated animals vs the control group. After combined preoperative irradiation and hyperthermia, the frequency of local anastomotic complications increased: 4 of 20 animals had a covered perforation when they were killed. In this group, the bursting pressure was lower 3 days after the operation (P = .008). The breaking strength was also lower but not notably. The serum albumin level was significantly lower in this group vs the control group (P = .006); the serum protein level was not decreased. After 7 days, no differences existed between the groups. The hydroxyproline content of the anastomotic tissue was notably higher in rats treated with radiation plus hyperthermia vs control rats (in both the 3- and 7-day groups). The anastomotic hydroxyproline concentration did not differ between the groups. CONCLUSIONS: The combination of preoperative irradiation and hyperthermia results in increased local anastomotic complications. Anastomotic strength is at risk in the first days after the anastomotic reconstruction. Preoperative irradiation or hyperthermia alone does not lead to impaired anastomotic healing in the early phase.

Are changes in myocardial integrated backscatter restricted to the ischemic zone in acute induced ischemia? An in vivo animal study.

Integrated backscatter (IB) from a myocardial region, calculated from radiofrequency echocardiographic data, has been proposed as a useful parameter for investigating changes in myocardial tissue induced by ischemia. In 10 closed-chest dogs, 5 minutes of myocardial ischemia was induced by either a proximal occlusion of the circumflex coronary artery (CX) (5 dogs), resulting in extensive ischemia in the posterior wall, or by occluding the distal CX vessel (5 dogs) to produce a small localized ischemic zone in the posterior wall. High-resolution digital radiofrequency data from the whole left ventricular myocardium, in the imaging plane during one complete heart cycle, were acquired with a whole-image real-time acquisition approach. Regions in the septum and posterior wall (both ischemic tissue and, in the case of distal occlusions, tissue surrounding the ischemic zone) were chosen for analysis, and IB and cyclic variation (CV) of IB were calculated. Post occlusion, an increase in mean IB values was found in the ischemic segment. However, an increase in CV was also observed in the peri-ischemic zone for the distal CX occlusion and in the septum after proximal CX occlusion. These findings show that changes in CV are not restricted to the ischemic zone but may also occur in distal myocardium. This may be explained by changes in the regional contractile state and loading conditions of the "normal" myocardium, which are altered in response to the distal ischemia.

An iterative maximum-likelihood polychromatic algorithm for CT.

A new iterative maximum-likelihood reconstruction algorithm for X-ray computed tomography is presented. The algorithm prevents beam hardening artifacts by incorporating a polychromatic acquisition model. The continuous spectrum of the X-ray tube is modeled as a number of discrete energies. The energy dependence of the attenuation is taken into account by decomposing the linear attenuation coefficient into a photoelectric component and a Compton scatter component. The relative weight of these components is constrained based on prior material assumptions. Excellent results are obtained for simulations and for phantom measurements. Beam-hardening artifacts are effectively eliminated. The relation with existing algorithms is discussed. The results confirm that improving the acquisition model assumed by the reconstruction algorithm results in reduced artifacts. Preliminary results indicate that metal artifact reduction is a very promising application for this new algorithm.

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.

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.

Iterative reconstruction for helical CT: a simulation study.

Iterative reconstruction algorithms for helical CT are presented. The algorithms are derived from two-dimensional reconstruction algorithms, by adapting the projector/backprojector to the helical orbit of the source, and by constraining the axial frequencies with a Gaussian sieve. Simulations have been carried out and the performance of the iterative algorithms is compared to that of filtered backprojection of synthetic (interpolated) two-dimensional sinograms. The iterative algorithms produce superior bias-noise curves. Axial resolution is superior, but disturbing edge-artefacts are introduced.

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.

The effects of lathyrogens on intestinal anastomoses in the rat.

Submucosal collagen provides strength to the intestinal wall. In order to assess the importance of collagen fibers for the developing strength of intestinal anastomoses we have sought to prevent postoperative collagen crosslinking by administration of lathyrogens. Rats, receiving both an ileal and a colonic anastomosis, were treated with either D-penicillamine or beta-aminopropionitrile from 1 day before operation. Animals were sacrificed 7 days postoperatively and bursting pressures, bursting sites, and anastomotic collagen (hydroxyproline) content and solubility were determined. D-Penicillamine, in a dose of 500 mg/kg/day and administered orally, had no effect at all. beta-Aminopropionitrile, in a dose of 625 mg/kg/day and given orally or intraperitoneally, significantly increased the acid solubility of anastomotic hydroxyproline in both ileum and colon without affecting total hydroxyproline content or concentration. Bursting pressures of the anastomotic segments were lowered, more significantly in colon than in ileum. Also, the bursting site was found more frequently in the anastomotic area in these animals. By inhibiting the formation of crosslinks in intestinal wounds with beta-aminopropionitrile, the anastomotic strength was reduced. These results demonstrate the importance of collagen in maintaining anastomotic integrity and at the same time emphasize that not only the quantity but also, and perhaps even more so, the quality of the collagen should be taken as an index of healing.