The effect of fibrin glue on the early healing phase of intestinal anastomoses in the rat.

PURPOSE: Protecting the anastomotic integrity using suture or staple line reinforcement remains an important goal for ongoing research. The present comprehensive study aims to establish the effects of fibrin glue on the early phase of anastomotic healing in the rat intestine. METHODS: One hundred and eight young adult male Wistar rats underwent resection and anastomosis of both the ileum and colon. In half, fibrin glue was applied around the anastomoses. Parameters for repair included wound strength, both bursting pressure and breaking strength at days 1, 3, and 5 after operation; hydroxyproline content; and histology, the latter also after 7 days. RESULTS: A transient colonic ileus was observed in the experimental group. Anastomotic breaking strength was always similar in both the control and fibrin glue groups. Anastomotic bursting pressures remained low at days 1 and 3, without any differences between the groups. In both groups, the bursting pressure increased sharply (p < 0.001) between days 3 and 5. At day 5, the bursting pressure in the fibrin glue group remained below than that in the controls, although only significantly (p = 0.0138) so in the ileum. At day 5, but not at day 7, the wounds in the fibrin glue group contained less collagen. Other aspects of microscopic wound architecture appeared to be the same. CONCLUSIONS: There is no justification for using fibrin glue on patent anastomoses constructed under low-risk conditions. Its potential benefit under conditions where chances for anastomotic leakage are enhanced needs further investigation.

Everolimus-induced loss of wound strength can be prevented by a short postoperative delay in its administration.

The use of mammalian target of rapamycin inhibitors coincides with an increased incidence of surgical complications. In previous experiments, serious negative effects of postoperative everolimus on anastomotic strength were found. This study aims to investigate if delayed drug administration can prevent loss of wound strength. Ten groups of Wistar rats each received daily oral doses of 1.0 or 2.0 mg/kg everolimus, starting the day of anastomotic construction in both ileum and colon, or 1, 2, 3, or 4 days later. The 11th group received saline. Seven days later, wound strength in anastomoses and in the abdominal wall and wound hydroxyproline levels were measured. Mean wound strength was significantly and dose-dependently reduced if everolimus was started on the day of operation. In ileum and colon, strength was not affected if drug administration was delayed until the third or second day, respectively. In abdominal fascia, this was the case only if everolimus was withheld until day 4. In general, changes in wound hydroxyproline content showed similarities to changes in wound strength. Thus, delaying administration of everolimus for 2-4 days after operation can prevent a serious loss of wound strength, both in the intestine and in the abdominal fascia.

Reduction of oxygenation and blood flow in pedicled bowel segments in the rat and its consequences for anastomotic healing.

PURPOSE: Experimental studies indicate that perioperative hypoperfusion impairs anastomotic healing. In bowel surgery, the part of bowel that will be anastomosed is often pedicled, leaving the blood supply dependent on the marginal artery only. Little is known about the blood supply in such a segment, and whether anastomotic strength is affected when flow would be reduced. This study describes oxygenation and blood flow in pedicled bowel segments in the rat and investigates whether early anastomotic strength changes with variations in blood flow. METHODS: In rats, pedicled segments were created in ileum and colon by successive ligation of the feeding arteries. Oxygenation and blood flow were measured in the distal part of this segment by use of near-infrared spectroscopy with indocyanine green as an intravascular tracer. In a second experiment, a short pedicled colonic segment was created and, after flow measurements, an anastomosis was constructed. Wound strength and hydroxyproline content were analyzed 2 and 5 days after operation. RESULTS: After creation of a pedicled segment, the concentration of oxygenated hemoglobin decreased significantly. Blood flow also significantly decreased to even less than 10% of baseline. A very large variation was observed between animals, in particular, after ligation of the first arteries. The strength of colonic anastomoses was not significantly correlated with the blood flow in the pedicled segment before anastomotic construction. CONCLUSIONS: The creation of a pedicled bowel segment greatly reduces tissue oxygenation and blood flow to its distal part. Such impaired perioperative flow does not significantly affect early wound strength after anastomotic construction.

Persistent effects of everolimus on strength of experimental wounds in intestine and fascia.

The introduction of mTOR-inhibitors in transplantation surgery has been associated with an increase in wound complications. We have previously reported a massive negative effect of everolimus on anastomotic strength in rat intestine at 7 days postoperatively. Because it is clinically important to know if this effect persists and occurs generally, repair in both intestine and abdominal wall has been investigated over a period of 4 weeks. Wistar rats received a daily dose of 1 or 2 mg/kg everolimus orally, from the operation day onwards. Controls received saline. In each rat a resection of ileum and colon was performed, and end-to-end anastomoses were constructed. On day 7, 14, and 28 the animals were killed and anastomoses and abdominal wall wounds were analyzed, wound strength being the primary parameter. Breaking strength of ileum, colon, and fascia was consistently and significantly reduced in the experimental groups at all time points. Anastomotic bursting pressures followed the same pattern. Loss of strength was accompanied by a decrease in hydroxyproline content after 7 days. Thus, the negative effect of everolimus on wound repair persists for at least 4 weeks after operation in this rodent model. This protracted effect may have clinical consequences and cause surgical morbidity.

Everolimus interferes with healing of experimental intestinal anastomoses.

BACKGROUND: Although clinical data suggest its existence, little is known about the effect of rapamycin derivatives on wound repair. This study aims to delineate the influence of the mammalian target of rapamycin inhibitor everolimus on wound healing in the rat intestine. METHODS: Four groups of 26 male Wistar rats received everolimus in daily oral dosages of 0 (controls), 0.5 (group E-0.5), 1.0 (group E-1), and 3.0 (group E-3) mg/kg every 24 hours, respectively, starting four hours before the operation until killing. After resection of 1-cm segments of colon and ileum, intestinal anastomoses were constructed. The animals were killed at days three or seven after operation. Wound healing was assessed by mechanical (bursting pressure, breaking strength), biochemical (collagen content, gelatinase activity), and histologic parameters. RESULTS: No differences between groups were recorded for any of the parameters on day three. On day seven, a dose-dependent reduction in breaking strength (P<0.05) was measured. The largest effects were found in group E-3 in which the breaking strength was reduced by 56% and 73% in colonic and ileal anastomoses, respectively. A similar pattern was observed with the bursting pressure. Loss of strength was accompanied by a reduction in hydroxyproline content and by a lessened collagen deposition in the wound area but not by an increased gelatinase activity. No further histologic abnormalities were found. CONCLUSION: Everolimus causes a massive reduction in anastomotic strength such as normally observed in the proliferative phase of repair. The data suggest this to be caused by an impaired deposition of collagen in the anastomotic area.

The Effect of Mycophenolate Mofetil on Early Wound Healing in a Rodent Model.

BACKGROUND: Immunosuppressant agents are inevitable for solid organ recipients, but may have a negative effect on wound healing that is difficult to measure because of clinical use of a polydrug regime. The evidence on mycophenolate mofetil (MMF) is scarce and contradictory. This study aims to investigate the effect of MMF administration on wound healing. METHODS: Ninety-six male Wistar rats divided into 4 groups underwent anastomotic construction in ileum and colon at day 0. Three groups received daily oral doses of 20 or 40 mg/kg MMF or saline (control group) from day 0 until the end of the experiment. Half of each group was analyzed after 3 days and half after 7 days. Another group started the medication 3 days after the laparotomy and was analyzed after 7 days, half of this group received 20 mg/kg and half 40 mg/kg MMF. Wound strength in anastomoses and in the abdominal wall was measured using bursting pressure, breaking strength, and histology. Trough levels were measured. RESULTS: Significant differences in wound strength were seen in ileum tissue after 3 days, which surprisingly showed a stronger anastomosis in the experimental groups. Bursting pressure as well as breaking strength was higher in the low-dose and high-dose MMF group compared with the control group. A negative effect was measured in abdominal wall tissue for the highest-dose group, which disappeared when the medication was delayed for 3 days. Histology showed poorer bridging of the submucosal layer and more polymorphonuclear cell infiltration in the ileum specimens of the control group compared with the treatment groups. CONCLUSIONS: As a single agent in a preclinical wound healing model in the rat, MMF has no negative effect on healing of bowel anastomoses but might have a negative effect on the healing of abdominal wall.

Doxycycline improves wound strength after intestinal anastomosis in the rat.

BACKGROUND: 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. The aim of this study was to examine whether doxycycline, a drug known to inhibit MMP activity, could enhance anastomotic strength. METHODS: Male Wistar rats received anastomoses in both ileum and colon. From the day before operation onwards, animals were treated daily with doxycycline (orally or subcutaneously) in a dose of 10 mg/day or with saline only. Rats were killed 1, 3, or 5 days after operation, and anastomotic bursting pressure and breaking strength were measured. At day 3, anastomotic hydroxyproline levels were measured, MMP (gelatinase) activity was analyzed by gelatin zymography, and anastomotic histology was examined. RESULTS: Doxycycline enhanced wound strength, but only at day 3, when it was at its lowest. Subcutaneous administration of 10 mg/day increased median colonic and ileal breaking strength by 27% (P =.0019) and 104% (P =.0376), respectively. Colonic bursting pressure was increased by 93% (P =.0002). Wound histology was similar in experimental and control groups. CONCLUSIONS: Administration of doxycycline enhances anastomotic strength and should be investigated further as a means to preserve anastomotic integrity.

Peritoneal cytokines predict mortality after surgical treatment of secondary peritonitis in the rat.

BACKGROUND: The study aimed to analyze if peritoneal cytokine levels can predict survival in an experimental model for peritonitis. Early identification of patients most at risk for adverse outcomes would facilitate the decision for aggressive therapy in order to maximally exploit their chance for survival. STUDY DESIGN: Peritonitis was induced by intraperitoneal injection of a feces/bacteria mixture in 175 rats. Surgical debridement was performed after 1 hour. Abdominal fluid samples were taken after 24 and 72 hours for the measurement of interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha. Surviving animals were sacrificed after 5 days and correlations between cytokine levels and survival were analyzed. RESULTS: Altogether, 60 animals died prematurely, 12 before the first sampling of cytokines. So, 48 nonsurvivors and 115 survivors were analyzed. Peritoneal cytokine levels were much higher (p < 0.0001) in nonsurvivors than in survivors. At 24 hours there were strong correlations between cytokine levels, especially between IL-6 and IL-10 (r = 0.93). Peritoneal cytokines at 24 hours also discriminated between animals dying within the next 24 hours and those dying later. A strongly (p < 0.0001) increased mortality was observed if IL-6, IL-10, or TNF-alpha levels exceeded 2, 1, or 0.2 ng/mL, respectively. Receiver operating characteristic curves were promising for all 3, but IL-10 showed the best characteristics, with an area under the curve of 0.94 and 67% sensitivity at 95% specificity, obtained at a cut-off value of 1.26 ng/mL. CONCLUSIONS: These data should generate renewed interest to examine the peritoneal cytokines as early markers for adverse outcomes in patients with secondary peritonitis. Possibly, combinations of peritoneal cytokines with other markers can lead to much needed, reliable early prediction of disease severity.

Tissue-type plasminogen activator prevents abscess formation but does not affect healing of bowel anastomoses and laparotomy wounds in rats with secondary peritonitis.

BACKGROUND: Intra-abdominal application of recombinant tissue-type plasminogen activator (rtPA) can decrease the rate of abscess formation in a rat peritonitis model. Before using rtPA clinically, its effects on healing of bowel anastomoses and laparotomy wounds should be investigated. METHODS: Peritonitis was induced in 148 male Wistar rats via intra-abdominal injection of a feces/bacteria mixture. Laparotomy, operative debridement and construction of a colo-colostomy after a limited colectomy or ileo-ileostomy after a limited ileal resection were performed after 1 hour. All animals received antibiotics (ceftriaxone plus metronidazole). In addition to untreated controls, other animals received rtPA in 1 of 3 dosing schemes, starting immediately after operation or 24 hour afterwards. Wound strength and hydroxyproline content of the wound were analyzed after 3 or 7 days. RESULTS: Mortality was 2% and manifestations of excessive bleeding were virtually absent. RtPA significantly decreased the rate of abscess formation. Neither bursting pressure nor breaking strength of the anastomoses was affected by any of the rtPA protocols. The same was true for wound strength in the abdominal fascia. Additionally, wound hydroxyproline content and architecture remained unchanged after rtPA administration. CONCLUSION: Intraperitoneal rtPA administration consistently and significantly decreased the rate of abscess formation, but did not affect wound healing. Clinical studies investigating its potential as an adjunct in the treatment of secondary peritonitis may be warranted.

Intercellular adhesion molecule-1 and gelatinase expression in human peritoneal mesothelial cells during propagation in culture.

Mesothelial cells are involved in a variety of biological processes, which include the formation of peritoneal adhesions. The cultures of human peritoneal mesothelial cells comprise an important tool to investigate the behavior and functions of mesothelial cells. Very little is known about the differences among mesothelial cells isolated from different sources and about the changes in specific functions as caused by cell propagation in vitro or that result from storage of cells at low temperatures. This study aims to characterize 2 particular cellular activities relevant for tissue repair, which include the expression of intercellular adhesion molecule-1 (ICAM-1) and the gelatinase activity; in addition, this study will assess the effect of hyaluronan, which is an antiadhesive agent, on these cellular activities. Viable cell lines were established from both omentum and peritoneal lavage fluid from 7 patients. Both ICAM-1 expression, which was measured by enzyme-linked immunosorbent assay (ELISA), and matrix metalloproteinase (MMP) bioactivity, which was measured by zymography, were measured in the 2nd and 4th passage; the latter also was measured after freezing and storing of cells in liquid nitrogen. The effects of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), phorbol myristate acetate (PMA), and hyaluronan were analyzed. ICAM-1 was constitutively expressed and stimulated by IL-1beta, TNF-alpha, and PMA. All cell lines produced both MMP-2 and MMP-9. Only the latter activity was affected by TNF-alpha and, especially so, PMA. Differences were found between the 2nd and 4th passage, as well as between cells of different lineage, mostly so if the relative stimulation by the various agents was compared. The addition of sodium hyaluronate either to control cultures or to cultures together with any of the 3 stimuli examined did not significantly change either ICAM-1 expression or gelatinase activity. The freezing and storage of cells did not affect their functions. Both the human omentum and peritoneal lavage fluid are good sources to establish mesothelial cell lines, which can be propagated also after freezing without qualitative changes in their ability to express ICAM-1 and produce the gelatinases. For omental cells, a differential effect of stimulation occurs depending on whether the cells have been passaged 2 or 4 times. The presence of hyaluronan did not affect the expression of ICAM-1 or the gelatinases.

Intestinal anastomoses from diabetic rats contain supranormal levels of gelatinase activity.

PURPOSE: Early postoperative strength in intestinal anastomoses is reduced in diabetic rats, whereas collagen deposition is essentially unchanged, suggesting that increased matrix degradation may be the cause of diminished wound strength. The aim of this study was to investigate whether (gelatin-degrading) matrix metalloproteinase activity is enhanced in intestinal anastomoses from diabetic rats. METHODS: Sixty male young adult Wistar rats underwent resection and anastomosis of both ileum and colon. In half the animals diabetes was induced seven days before operation by streptozotocin injection (50 mg/kg intravenously). Gelatinase activities in extracts from uninjured intestine and anastomoses at one, three, or seven days after surgery were measured by quantitative gelatin zymography. RESULTS: After surgery, profound changes were observed with time for gelatinase activities with molecular weights of 50 and 60 kDa, thought to represent matrix metalloproteinase-2, and of 66, 80, 105, 140, 220, and 260 kDa, thought to represent various forms of matrix metalloproteinase-9. In many cases, specific activities were significantly (P < 0.05) higher in the anastomotic extracts from diabetic rats. Total anastomotic activities present at Day 7 were strongly elevated for most matrix metalloproteinase forms in ileum and colon from diabetic animals. CONCLUSION: Experimental diabetes leads to a sustained and elevated presence of gelatinase activity in intestinal anastomoses. Increased local matrix degradation may contribute significantly to impaired anastomotic strength in the intestine observed under this condition.