Evaluation of absorbable and permanent mesh fixation devices: adhesion formation and mechanical strength.

PURPOSE: Laparoscopic ventral hernia repair is commonly performed with mesh prostheses; however, there is no standard for fixation devices used to secure mesh to the abdominal wall. This study is a functional comparison of novel, screw-type absorbable and permanent fixation devices with a traditional titanium fixation device. METHODS: Fifteen pigs each underwent the laparoscopic placement of two 11 × 14-cm mesh prostheses and were randomized for mesh fixation with either titanium spiral tacks (TS), absorbable screw-type fasteners (SF), or permanent screw-type fasteners (PF) (n = 10 mesh prostheses for each fixation group). Adhesions were assessed laparoscopically at 4 weeks. The fixation devices were also embedded in porcine abdominal rectus muscle for ex vivo mechanical testing along with partial thickness polypropylene suture (PR) as a control group (n = 40 for each group). Maximum pull-off forces were measured. All statistical tests were two-tailed, and a P-value < 0.05 was considered to be significant. RESULTS: The mean tenacity adhesion scores were 1.40 ± 0.52 (PF), 1.7 ± 0.82 (SF), and 2.6 ± 1.07 (TS). Adhesions in the PF group were significantly less tenacious compared with the TS group (P = 0.01). Quantitative adhesion scores were not significantly different among groups. The maximum pull-off forces, measured in Newtons, were 28.61 N ± 4.89 N (TS), 22.71 N ± 7.86 N (SF), 16.98 N ± 7.59 N (PF), and 20.83 N ± 6.25 N (PR). The pull-off force in the TS group was higher than all of the other groups (P < 0.001). The SF group also had a higher pull-off force compared with the PF group (P < 0.001). CONCLUSIONS: The screw-type absorbable and permanent fixation devices provided adequate fixation and were associated with decreased adhesions in this porcine model.

Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer.

Dysfunction in the physiological pathways of programmed cell death may promote proliferation of malignant cells, and correction of such defects may selectively induce apoptosis in cancer cells. We measured the levels of ceramide, a candidate lipid mediator of apoptosis, in human metastatic colorectal cancer and tested in vitro and in vivo effects of various ceramide analogues in inducing apoptosis in metastatic colon cancer. Human colon cancer showed a > 50% decrease in the cellular content of ceramide when compared with normal colon mucosa. Application of ceramide analogues and ceramidase inhibitors induced rapid cell death through activation of various proapoptotic molecules, such as caspases and release of cytochrome c. Ceramidase inhibition increases the ceramide content of tumor cells, resulting in maximum activation of the apoptotic cascade. Normal liver cells were completely resistant to inhibitors of ceramidases. Treatment of nude mice with B13, the most potent ceramidase inhibitor, completely prevented tumor growth using two different aggressive human colon cancer cell lines metastatic to the liver. Therefore, B13 and related analogues of ceramide and inhibitors of ceramidases offer a promising therapeutic strategy with selective toxicity toward malignant but not normal cells. These studies also suggest that the ceramide content in cancer cells might be involved in the pathogenesis of tumor growth in vitro and in vivo.

Mechanisms of ischemic injury are different in the steatotic and normal rat liver.

Hepatic steatosis is associated with significant morbidity and mortality after liver resection and transplantation. Although apoptosis is a key mechanism of reperfusion injury in the normal liver, the pathway leading to cell death in steatotic hepatocytes is unknown. A model of hepatic ischemia and reperfusion injury in fatty and lean Zucker rats was used. Fatty animals had increased aspartate aminotransferase (AST) release and decreased survival after 60 minutes of ischemia compared with lean animals. Apoptosis was the predominant form of cell death in the lean rats (82%), whereas necrosis was minimal. In contrast, fatty animals developed only moderate amounts of apoptosis but showed massive necrosis (73%) after 24 hours of reperfusion. Intracellular mediators of apoptosis, such as caspase 8, caspase 3, and cytochrome c, were significantly lower in the steatotic than in the lean liver indicating dysfunction in activation of the apoptotic pathway. The high percentage of necrosis in the steatotic rats was associated with renal acute tubular necrosis after 24 hours of reperfusion in the fatty, but not in lean rats. Caspase inhibition significantly decreased reperfusion injury in lean animals, but was ineffective in fatty animals. The results indicate that the increased susceptibility of fatty livers to reperfusion injury is associated with a change from an apoptotic form of cell death to necrosis. We conclude that new therapeutic strategies are necessary in the fatty liver.

Prolonged rewarming time during allograft implantation predisposes to recurrent hepatitis C infection after liver transplantation.

The majority of patients undergoing orthotopic liver transplantation (OLT) have end-stage liver disease secondary to hepatitis C virus (HCV) infection. Although OLT does not cure the disease and recurrent virus is present in all patients, relatively few patients with recurrent viremia develop clinical disease. When the disease recurs, however, the results can be devastating. Factors associated with increased risk for recurrent HCV disease remain controversial. We hypothesized that preservation injury may predispose to the severity of HCV disease after OLT. We reviewed our series of OLTs performed for HCV cirrhosis between January 1994 and December 1998 (n = 56; 62 transplants). Patients were grouped according to the severity of recurrent hepatitis C. Group 1 had no or mild HCV disease (n = 36), and group 2 had moderate to severe HCV disease (n = 20). The duration of ischemic rewarming during graft implantation was significantly associated with the severity of recurrent hepatitis C (P <.04). The estimated chances of severe disease within the first year post-OLT after 30, 60, or 90 minutes of ischemic rewarming time were 19%, 40%, and 65%, respectively. Cold ischemia time, transaminase levels, and prothrombin time did not correlate with the severity of hepatitis C. In conclusion, our data suggest that the duration of ischemic rewarming predisposes to severe recurrent hepatitis C. This finding warrants the investigation of the pathogenesis of recurrent HCV disease after ischemic injury. Reduction of rewarming time should be stressed in OLT, particularly in patients with HCV cirrhosis.

Protective effects of ischemic preconditioning for liver resection performed under inflow occlusion in humans.

OBJECTIVE: To determine whether ischemic preconditioning protects the human liver against a subsequent period of ischemia in patients undergoing hemihepatectomy, and to identify possible underlying protective mechanisms of ischemic preconditioning, such as inhibition of hepatocellular apoptosis. SUMMARY BACKGROUND DATA: Ischemic preconditioning is a short period of ischemia followed by a brief period of reperfusion before a sustained ischemic insult. Recent studies in rodents suggest that ischemic preconditioning is a simple and powerful protective modality against ischemic injury of the liver. The underlying mechanisms are thought to be related to downregulation of the apoptotic pathway. METHODS: Twenty-four patients undergoing hemihepatectomy for various reasons alternatively received ischemic preconditioning (10 minutes of ischemia and 10 minutes of reperfusion) before transection of the liver performed under inflow occlusion for exactly 30 minutes. Liver wedge and Tru-cut biopsy samples were obtained at the opening of the abdomen and 30 minutes after the end of the hepatectomy. Serum levels of aspartate transferase, alanine transferase, bilirubin and prothrombin time were determined daily until discharge. Hepatocellular apoptosis was evaluated by in situ terminal deoxynucleotidyl transferase mediated d-UTP nick end-labeling (TUNEL) assay and electron microscopy. Caspase 3 and 8 activities were measured in tissue using specific fluorometric assays. RESULTS: Serum levels of aspartate transferase and alanine transferase were reduced by more than twofold in patients subjected to ischemic preconditioning versus controls. The analysis of a subgroup of patients with mild to moderate steatosis indicated possible increased protective effects of ischemic preconditioning. In situ TUNEL staining demonstrated a dramatic reduction in the number of apoptotic sinusoidal lining cells in the ischemic preconditioning group. Electron microscopy confirmed features of apoptosis present in control but not in ischemic preconditioning patients. There was no significant difference in caspase 3 and 8 activity when patients with ischemic preconditioning were compared with controls. CONCLUSIONS: Ischemic preconditioning is a simple and effective modality protecting the liver against subsequent prolonged periods of ischemia. This strategy may be a more attractive technique than intermittent inflow occlusion, which is associated with increased blood loss during each period of reperfusion.

Platelets induce sinusoidal endothelial cell apoptosis upon reperfusion of the cold ischemic rat liver.

BACKGROUND & AIMS: Sinusoidal endothelial cell (SEC) apoptosis is a central feature of reperfusion injury in liver transplantation. Platelet sequestration occurs after transplantation with possible deleterious effects. We tested the hypothesis that platelets mediate SEC apoptosis. METHODS: Livers were perfused after 24 hours of cold preservation in University of Wisconsin solution in an isolated perfused rat liver model. The perfusate contained isolated syngeneic red blood cells and purified platelets. Effects of inhibiting platelet adhesion on SEC apoptosis was tested using sialyl Lewis-X oligosaccharide (sLe(x)), a natural ligand of selectin adhesion molecules. Reperfusion injury was assessed by established markers of injury. Apoptosis was determined by TUNEL and electron microscopy. RESULTS: A third of the circulating platelets was rapidly sequestered in the liver after reperfusion. This was associated with increased graft injury. Single platelets were adherent to sinusoidal lining without morphological or dynamic evidence of impairment of microcirculation. TUNEL staining revealed a 6-fold increase in the number of apoptotic SECs at 1 hour of reperfusion. No hepatocyte death or evidence of necrosis was detected up to 3 hours of reperfusion. Addition of sLe(x) inhibited adhesion and significantly reduced SEC apoptosis. CONCLUSIONS: Platelets cause SEC apoptosis upon reperfusion of liver grafts. Prevention of adhesion is protective.

Ischemic preconditioning protects the mouse liver by inhibition of apoptosis through a caspase-dependent pathway.

A short period of ischemia and reperfusion, called ischemic preconditioning, protects various tissues against subsequent sustained ischemic insults. We previously showed that apoptosis of hepatocytes and sinusoidal endothelial cells is a critical mechanism of injury in the ischemic liver. Because caspases, calpains, and Bcl-2 have a pivotal role in the regulation of apoptosis, we hypothesized that ischemic preconditioning protects by inhibition of apoptosis through down-regulation of caspase and calpain activities and up-regulation of Bcl-2. A preconditioning period of 10 minutes of ischemia followed by 15 minutes of reperfusion maximally protected livers subjected to prolonged ischemia. After reperfusion, serum aspartate transaminase (AST) levels were reduced up to 3-fold in preconditioned animals. All animals subjected to 75 minutes of ischemia died, whereas all those who received ischemic preconditioning survived. Apoptosis of hepatocytes and sinusoidal endothelial cells, assessed by in situ TUNEL assay and DNA fragmentation by gel electrophoresis, was dramatically reduced with preconditioning. Caspase activity, measured by poly (adenosine diphosphate ribose) polymerase (PARP) proteolysis and a specific caspase-3 fluorometric assay, was inhibited by ischemic preconditioning. The antiapoptotic mechanism did not involve calpain-like activity or Bcl-2 expression because levels were similar in control and preconditioned livers. In conclusion, ischemic preconditioning confers dramatic protection against prolonged ischemia via inhibition of apoptosis through down-regulation of caspase 3 activity, independent of calpain-like activity or Bcl-2 expression.

Calpain inhibition prevents sinusoidal endothelial cell apoptosis in the cold ischemic rat liver.

BACKGROUND: Cold preservation of the liver followed by reperfusion results in sinusoidal endothelial cell (SEC) apoptosis. Calpain-like activity is dramatically increased during reperfusion and inhibition of calpains results in lower graft injury and longer survival. Recently, calpains have been implicated in inducing apoptosis. Our aim was to determine the effect of calpain inhibition on SEC apoptosis. METHODS: Livers were stored in the University of Wisconsin solution for 24 hr (survival conditions) and 40 hr (nonsurvival conditions) and ex vivo reperfused for 1 hr at 37 degrees C. Calpain-like activity was inhibited in some experiments using an i.p. injection of a selective inhibitor 2 hr before explantation. Apoptosis was quantified using the terminal deoxynucleotidyl trans. ferase-mediated dUTP nick end-labeling assay. Cross-inhibition by the inhibitor was determined for caspases 1 and 3. RESULTS: Apoptosis of exclusively the SEC was a key feature of reperfusion injury after both storage periods in University of Wisconsin solution after 1 hr normothermic reperfusion. Inhibition of calpain activity with Cbz-Val-Phe methyl ester resulted in a 50% reduction of apoptotic SEC in the 40-hr preserved liver, and an almost complete abrogation of SEC apoptosis after 24 hr preservation. Only minimal cross-inhibition of caspases was determined at high concentrations in vitro by the calpain inhibitor. CONCLUSION: Apoptosis of exclusively SEC is a key feature of reperfusion injury partially mediated through calpain-dependent processes. Calpain inhibition reduces the number of apoptotic SEC. Based on these data and our previous work, calpain inhibition may prove to be useful in clinical transplantation.