Methylene blue prevents surgery-induced peritoneal adhesions but impairs the early phase of anastomotic wound healing.

OBJECTIVES: Adhesion formation continues to be an important problem in gastrointestinal surgery. In recent years, methylene blue (MB) has been reported to be an effective agent for preventing peritoneal adhesions. However, its effects on the wound healing process are unknown. In the present study, we investigated the effects of MB on the early and late phases of anastomotic wound healing and on adhesion formation. METHODS: We randomly categorized 92 rats into 2 groups in bursting pressure measurements and 50 rats into 3 groups in the adhesion model. We divided the animals into saline-treated (n = 46) or MB-treated (n = 46) groups. Bursting pressures of the anastomoses were measured on postoperative days 3 and 7. In biochemical studies, tissue hydroxyproline levels, total nitrite/nitrate levels and nitric oxide synthase activity were measured on postoperative days 3 and 7. In the adhesion model, we randomly categorized rats into sham (n = 10), saline-treated (n = 20) and MB-treated (n = 20) groups, and the formation of intraperitoneal adhesions was scored on postoperative day 14. We compared the measurement of bursting pressure and biochemical measurements of tissue hydroxyproline levels, total nitrite/nitrate levels and nitric oxide synthase activity. Histopathological findings of specimens were presented. RESULTS: During the early phase of wound healing (postoperative day 3), bursting pressures, tissue hydroxyproline, total nitrite/nitrate levels and nitric oxide synthase activity in the MB-treated group were significantly lower than those of the saline-treated group. On postoperative day 7, there was no significant difference in these parameters between MB and saline-treated groups. In the adhesion model, MB caused a significant reduction in the formation of peritoneal adhesions. CONCLUSION: MB prevents peritoneal adhesions but causes a significant impairment of anastomotic bursting pressure during the early phase of the wound healing process by its transient inhibitory effect on the nitric oxide pathway.     

Efficacy of bioactive polypeptides on bleeding and intra-abdominal adhesions

BACKGROUND: Perioperative bleeding and postoperative adhesions are two problems encountered in abdominal surgery. Commercial products are available that decrease both bleeding and development of abdominal adhesions, but no products are effective in both situations. The combination of differently charged bioactive polypeptides, administered intraperitoneally, has previously been shown effective in decreasing postoperative adhesions. The present study is a pilot examination of the effects on perioperative bleeding and postoperative adhesions, applying the polypeptide concept. METHODS: Standardized wounds in the liver and spleen were induced in 52 NMRI mice. The amounts of bleeding and postoperative adhesions were measured after 1 and 7 days, respectively. Separate animals were examined after 8 weeks for long-term healing of the parenchymal wounds. RESULTS: Both parenchymal bleeding and the extent of adhesions significantly decreased (p = 0.001 and p = 0.029, respectively) as compared to controls. Histology after 8 weeks showed no clear signs of impaired or altered healing. CONCLUSION: Intraperitoneal administration of differently charged polypeptides significantly decreased postoperative bleeding and postoperative adhesions. Bioactive polypeptides appear promising in the promotion of peritoneal healing and merits further studies.     

β—七叶皂甙钠预防术后肠粘连的实验研究

目的:观察β七叶皂甙钠预防术后肠粘连作用及对切口愈合的影响。方法:选用Wistar 大鼠40 只,分别刮伤盲肠浆膜,创面滴无水酒精,短暂钳夹盲肠系膜动脉,术后随机分成2 组,分别静脉注射生理盐水和β七叶皂甙钠。术后第7 d 进行大体粘连程度分级,并分别测定盲肠壁及腹壁切口组织的羟脯氨酸(OHP)水平。结果:与对照组相比,β七叶皂甙钠能明显减轻术后肠粘连程度和盲肠壁OHP水平( P<0 .01),而不影响腹壁切口OHP水平( P>0.05)。结论:β七叶皂甙钠能有效预防术后肠粘连。     

High glucose levels inhibit focal adhesion kinase-mediated wound healing of rat peritoneal mesothelial cells

BACKGROUND: The peritoneum is progressively denuded of its mesothelial cell monolayer in patients on continuous ambulatory peritoneal dialysis (CAPD). These alterations of the mesothelium cause membrane dysfunction and progressive peritoneal fibrosis. Integrins regulate cell motility and play an important role in wound healing. We investigated the effects of high glucose on the regeneration process of the peritoneal mesothelial cell monolayer using cultured rat peritoneal mesothelial cells (RPMC). METHODS: The effects of glucose or mannitol on the regeneration of RPMC and formation of focal adhesions were examined by in vitro wound healing assay and immunocytochemistry, respectively. Activities of focal adhesion kinase (FAK) and its downstream p130Cas were examined by Western blotting. Effects of wild-type and dominant-negative FAK on RPMC migration were examined by a transient transfection assay. RESULTS: Cell migration over fibronectin (FN) was clearly inhibited in culture media containing high glucose (28 to 140 mmol/L). RPMC formed focal adhesions on FN in the presence of a regular glucose concentration (5.6 mmol/L); however, tyrosine phosphorylation of FAK and p130Cas and formation of focal adhesions observed by FAK and vinculin staining were substantially inhibited by high glucose. Mannitol also induced significant inhibitory effects, but these were milder than those of glucose. Transfection of dominant-negative FAK inhibited cell migration in a regular glucose concentration, whereas overexpression of wild-type FAK abrogated glucose-induced inhibition of cell migration. CONCLUSIONS: Our results demonstrate that high glucose concentrations as well as high osmolarity inhibit FAK-mediated migration of mesothelial cells, and suggest that dialysates containing high glucose concentrations may cause peritoneal damage by inhibiting wound healing of the mesothelial cell monolayer.     

The effect of vitamin E on experimentally induced peritoneal adhesions in mice

Previous studies in our laboratory demonstrated that dietary supplementation with vitamin A enhances peritoneal adhesion formation in mice. Other researchers have shown that vitamin E antagonizes some effects of vitamin A in various systems, eg, wound healing. We investigated our hypothesis that dietary supplementation with vitamin E would decrease peritoneal adhesion formation. Adult mice were divided into the following groups: group 1, which ate a standard chow containing 65 IU of vitamin E per kilogram diet (twice the National Research Council's recommended daily allowance for normal mice); and group 2, which ate the same chow supplemented with vitamin E at 300 IU/kg diet (a nontoxic level). Following peritoneal ligation, all mice were killed on the tenth postoperative day and their peritoneal cavities examined for the presence and extent of adhesions. There was a statistically significant decrease in the incidence and degree of adhesions in the vitamin E-supplemented animals; these data supported our hypothesis.     

Effect of transforming growth factor beta on postoperative adhesion formation and intact peritoneum.

Transforming growth factor beta (TGF beta) is an extremely potent chemoattractant for macrophages, mononuclear leukocytes, and fibroblasts. It also acts as a potent stimulant for collagen and fibronectin synthesis and inhibits epithelial cell growth. TGF beta plays an important role in healing many types of wounds, but its role in peritoneal adhesion formation is not known. These studies were performed to determine if TGF beta could affect postoperative wound healing in a rat model. In the first experiment, 20 rats were divided into two groups and received either 2 micrograms TGF beta or control diluent IP daily for 5 days after surgical injury to the uterine horns. The severity of the adhesions were graded 2 weeks postoperatively using a score of 0-3. The TGF beta group showed a higher adhesion score at 2 weeks compared to control, 2.9 +/- 0.34 and 1.6 +/- 0.61, respectively (P less than 0.001). On H&E stained sections of the adhesions, there was an increase in the number of both inflammatory cells and fibroblasts in the TGF beta-treated animals. A comparison trial of bone-derived TGF beta (a gift from Collagen Corporation, Palo Alto, CA) versus recombinant TGF beta (a gift from Oncogen, Seattle, WA) versus control using the same protocol as above showed that both sources of TGF beta were more effective in promoting postoperative adhesions when compared to controls, and there was no difference between TGF beta groups, 3.0 +/- 0 for both TGF beta groups, and 2.2 +/- 0.91 for control (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lexipafant inhibits postsurgical adhesion formation

BACKGROUND: PAF and its antagonists have been studied in the pathophysiology of various inflammatory conditions. This study investigates the effects of a platelet activating factor antagonist, lexipafant, on peritoneal adhesion formation and wound healing. MATERIALS AND METHODS: Forty-eight Wistar albino rats (300-350 g) were divided into four equal groups; adhesion-induced lexipafant (AL), adhesion-induced saline (AS), sham-operated lexipafant (SL), and sham-operated saline (SS). All rats underwent a midline laparotomy under sterile conditions. The anterior wall of the left uterine horn was scraped to cause hemorrhages in adhesion-induced groups. Following peritoneal injections of either saline or lexipafant, the incisions were closed in layers. On the 14th day, the rats were killed and adhesions were scored from 0 (none) to 4 (dense). Tissue samples from the adhesions and the left horn of uterus were examined biochemically for hydroxyproline content, and serum IL-6 levels were determined. RESULTS: The adhesion formation score was significantly increased in the AS group compared to the SL and AL groups (P < 0.001). The IL-6 levels of the AS group were higher than those of the other groups (P < 0.05). There was no significant difference in hydroxyproline content between groups (P > 0.05). CONCLUSIONS: Lexipafant plays a role in the prevention of adhesion formation without affecting wound healing.     

Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors

To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 microg MMP Eq/ml) compared to acute wounds (median 0.76 microg MMP Eq/ml) (p < 0.001). The addition of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase-9 with smaller elevations of matrix metalloproteinase-2. In addition tissue inhibitor of metalloproteinase-1 levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase-1 levels were compared to protease levels there was an inverse correlation (p = 0.02, r = - 0.78). In vitro degradation of epidermal growth factor was measured by addition of 125I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.1%) compared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0. 001, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collected from 15 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median 12%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers (p < 0.01). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.     

The influence of suturing and sepsis on the development of postoperative peritoneal adhesions.

Postoperative peritoneal adhesions are a major cause of morbidity. The purpose of this study was to investigate the potential contributions of suturing and sepsis to their formation in animals undergoing laparotomy. Suturing the peritoneum with plain catgut was associated with a high incidence of adhesions to the wound at 8 days (11/15), but this was significantly less at 25 days (5/15, P less than 0.04). Use of monofilament nylon, or non-suture, were each associated with a low incidence of adhesions. Wound strength was significantly greater at 25 days than at 8 days (P less than 0.0005), but did not differ between groups. In a separate experiment, bacterial infection, even in the absence of a particulate carrier, proved to be a potent cause of postoperative peritoneal adhesions (8/9, P = 0.02) compared with uninfected controls (3/10). Suturing the peritoneum in the presence of infection caused an especially high incidence of adhesions to the wound (8/9, P = 0.004 vs 2/10 unsutured). It is concluded that the lowest incidence of adhesions to the wound is likely to be obtained, both in uninfected and in infected cases, if the peritoneum is not sutured during closure of abdominal wounds, and that such an approach does not compromise wound strength.     

Activated protein C prevents inflammation yet stimulates angiogenesis to promote cutaneous wound healing

Activated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation, and has more recently been shown to be a potent anti-inflammatory mediator. Using cultured human cells, we show here that APC up-regulates the angiogenic promoters matrix metalloproteinase-2 in skin fibroblasts and umbilical vein endothelial cells, vascular endothelial growth factor in keratinocytes and fibroblasts, and monocyte chemoattractant protein-1 in fibroblasts. In the chick embryo chorioallantoic membrane assay, APC promoted the granulation/remodeling phases of wound healing by markedly stimulating angiogenesis as well as promoting reepithelialization. In a full-thickness rat skin-healing model, a single topical application of APC enhanced wound healing compared to saline control. APC-treated wounds had markedly more blood vessels on day 7 and a significantly lower infiltration of neutrophils at days 4 and 7. The broad spectrum matrix metallo-proteinase, GM6001, prevented the ability of APC to promote wound healing. In summary, our results show that APC promotes cutaneous wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation. These unique properties of APC make it an attractive therapeutic agent to promote the healing of chronic wounds.     

The effectiveness of a single intraperitoneal infusion of a neurokinin-1 receptor antagonist in reducing postoperative adhesion formation is time dependent

BACKGROUND: Current methods to prevent intraabdominal adhesions are not uniformly effective. We recently showed in rats that a neurokinin-1 receptor (NK-1R) antagonist is capable of reducing adhesion formation. To determine the clinical feasibility of using an NK-1R antagonist to reduce adhesions, this study examined the time dependence for the effectiveness of NK-1R antagonist administration and its effects on wound healing. METHODS: Adhesions were surgically induced in rats receiving a single intraperitoneal infusion of the NK-1R antagonist, CJ-12,255, during or 1, 5, 12, or 24 hours after surgery. Adhesion formation was assessed 7 days later. In a subset of animals, tissue plasminogen activator (tPA) activity, which is a measure of peritoneal fibrinolytic activity, was determined in peritoneal fluid 24 hours after surgery (48 hours for animals infused at 24 hours). The tPA activity was also determined in nonoperated animals 24 hours after peritoneal injection of the NK-1R antagonist. Colonic burst pressures were measured 7 days after creation of anastomoses in rats that were administered the antagonist at surgery. RESULTS: The NK-1R antagonist significantly reduced (P=.003) intraabdominal adhesions when administered during or 1 hour after surgery, only moderately reduced (P=.08) adhesions when administered at 5 hours, and had no effect at 12 or 24 hours. Peritoneal tPA activity was significantly increased (P<.05) in peritoneal fluid 24 hours after administration of the NK-1R antagonist regardless of the surgical procedure. The NK-1R antagonist did not alter colonic anastomotic healing. CONCLUSIONS: These data show that some of the events critical to adhesion formation occur within the first 5 hours following an abdominal operation in this model. The fact that the NK-1R antagonist does not impair colonic anastomotic healing enhances its usefulness as a therapeutic agent to inhibit adhesion formation.     

Enteric bacteria and their antigens may stimulate postoperative peritoneal adhesion formation

BACKGROUND: Intraabdominal sepsis causes exuberant inflammation, which results in dense adhesions. Translocation of enteric bacteria and/or their antigens after laparotomy may therefore also affect peritoneal healing by promoting local release of proinflammatory cytokines. Our hypothesis was that targeted counter therapy could be beneficial if such contamination was to augment postoperative adhesion formation. METHODS: Two endotoxin-hyposensitive mouse strains (C3H/HeJ and C57BL/10ScCr) and their syngeneic counterparts (C3H/HeN and C57BL10/ScSn, respectively) underwent reproducible adhesion-inducing operation (AIO) (n=10/group) with sacrifice and blinded adhesion grading 14 days later. In addition, CD-1 mice were gavaged with fluorescein isothiocyanate labeled-lipopolysaccharide (FITC-LPS) prior to either AIO or sham laparotomy and had both peritoneal macrophages and circulating monocytes assessed by flow cytometry afterward. The cytokine-release response of resident peritoneal cells to LPS stimulation was assessed in vitro (murine peritoneal mast cell cultures) and in vivo (unoperated CD-1 mice administered LPS intraperitoneally [10 & 50 microg/mouse]). Finally, CD-1 mice (n=10/group) had AIO and received either bactericidal/permeability increasing protein (rBPI, 2 mg/mouse) or vehicle solution in the early postoperative period with assessment of adhesion formation 2 weeks later. RESULTS: Both HeJ and ScCr mice had less adhesions than their controls (P=.0015 and .0001, respectively, Mann Whitney U test). FITC-LPS uptake by peritoneal macrophages was striking after AIO. Intraperitoneal LPS provoked significant local vascular endothelial growth factor (VEGF) release as did the process of AIO. In vitro, LPS induced significant interleukin-(IL)-6 release from isolated mast cells. Intraperitoneal administration of rBPI to CD-1 mice early after AIO markedly attenuated subsequent adhesion formation (P=.0003). CONCLUSIONS: Peritoneal adhesion formation is exacerbated by peritoneal contamination due to translocation after laparotomy and may be attenuated by therapeutic antagonism.     

Role of mast cells and myofibroblasts in human peritoneal adhesion formation

OBJECTIVE: To study fibroblasts and mast cells in human peritoneal adhesions and to evaluate whether their interaction plays a role in adhesion development. SUMMARY BACKGROUND DATA: Myofibroblasts play a critical role in wound repair/fibrosis. Mast cells influence the formation of peritoneal adhesions in a rat model, and they are modulators of fibroblast functions. METHODS: Peritoneal adhesion biopsies were processed for either histology (H&E, toluidine blue) or immunohistochemistry (tryptase, laminin, collagen type IV and VIII, and alpha-SMA) or grown as explants for obtention of fibroblasts. The effects of mast cell (HMC-1) sonicate and selected mast cell mediators and cytokines on fibroblast proliferation ([ (3)H]thymidine) and collagen synthesis ([ (3)H]proline) and on fibroblast contractile activity (tridimensional collagen lattice) were evaluated. Mast cell mediators influencing fibroblast proliferation were partially characterized by enzymatic susceptibility and FPLC gel filtration column chromatography. RESULTS: Most of the fibroblasts in peritoneal adhesions were identified as alpha-SMA-positive myofibroblasts. Mast cell hyperplasia was observed and more than one third of the mast cells were degranulated. Few mast cells showed a faint staining for laminin or collagen type IV and VIII. Mast cell sonicate increased fibroblast proliferation and contractile activity while decreasing collagen synthesis. Mast cell sonicate proliferating activities were found to be proteinase-sensitive with a molecular weight of more than 158 kd, of approximately 40 kd, and of less than 10 kd. TGF-beta and tryptase enhanced collagen synthesis; TNF-alpha and chymase decreased it. None of the selected mediators increased fibroblast proliferation. CONCLUSIONS: Myofibroblasts are the main connective tissue cells present in human peritoneal adhesions, and mast cells play a direct role in peritoneal adhesion formation.     

Remote Efficacy for Two Different Forms of Hyaluronate-Based Adhesion Barriers

ABSTRACT

Background: Chemically modified sodium hyaluronate and carboxymethylcellulose (HA/CMC) membrane clinically reduces adhesion formation following surgery but was not designed for laparoscopic use. HA/CMC powder of identical chemical composition has been developed to allow for application laparoscopically. We compared the adhesion reduction efficacy of HA/CMC powder and film when applied directly to or remote from sites of surgical trauma. We also investigated the effect of the powder on wound healing. Materials and Methods: Two animal models of adhesion formation were used to evaluate efficacy: a rat peritoneal sidewall defect model and a rabbit cecal abrasion/sidewall defect model. The products were applied directly to the defect or the contralateral sidewall. Adhesions were examined seven days after surgery. In a separate study, the effect of the powder on healing was evaluated at 5, 7, and 28 days using a rat incisional wound strength model. Results: HA/CMC powder and film, when applied directly to the peritoneal defect, significantly reduced adhesions relative to the untreated control in both models. Remote applications of HA/CMC powder also reduced adhesions. In contrast, remote applications of HA/CMC film had no effect. HA/CMC powder did not significantly alter incisional wound strength at any of the timepoints tested. Conclusion: In our preclinical models, HA/CMC powder had similar adhesion reduction efficacy to HA/CMC film when applied directly to sites of trauma. In addition, HA/CMC powder reduced adhesions remote from the application site. Importantly, HA/CMC powder did not impair incisional wound healing. On the basis of these results, future investigation of HA/CMC powder is warranted.     

Ventral Hernia Repair: a Practical Point of View

Purpose: To evaluate the effect of PVP-I liposome hydrogel on intraperitoneal postoperative adhesions. Material and Methods: Thirty Wistar-Albino male rats were randomly divided into three groups. After midline laparotomy, a 1 cm² area of the caecum was abraded with a sterile gauze until subserosal haemorrhage had developed. A 1 χ 1 cm patch of peritoneum located opposite of caecal abrasion was completely dissected. In group 1 (control group, C) adhesion induction was performed and nothing was applied to the wounds. In group 2 and 3, PVP-I solution (3%) (group 2, PI) and PVP-I liposome hydrogel (group 3, PIL) were applied to the caecal abrasion areas and peritoneal defects. Adhesions were classified according to a classification system based on the evaluation of the appearance, extent and strength of the adhesions on postoperative 21^st day.

Results: There was no significant difference of the adhesion scores between the groups (U1 = 45, p > 0.05; U2 = 48, p > 0.05; U3 = 47.5, p > 0.05).

Conclusions: We found that PVP liposome hydrogel did not influence postoperative intraabdominal adhesions and should be further explored for its potential use in various intraabdominal procedures.