Vanadate ingestion increases the gain in wound breaking strength and leads to better organized collagen fibers in rats during healing.

Repair of incision wounds closed by suturing is evaluated by the progressive gain in wound breaking strength. Previously the closure of open wounds in rats ingesting vanadate, an inhibitor of tyrosine phosphate phosphatases, was shown to occur with deposition of more uniformly organized collagen fiber bundles. The hypothesis of this study was that deposition of more uniformly organized collagen fibers would enhance the gain in wound breaking strength of incisional wounds. Six adult rats received vanadate-supplemented saline drinking water for 1 week before placement of two 6-cm, parallel, suture-closed wounds on their backs. Six control rats received identical wounds and were given saline drinking water. The drinking water regimen was continued for 1 week after wounding, and then wound strength was tested with a tensiometer and tissue samples were obtained for histologic evaluation. Wound breaking strength doubled in vanadate-treated rats compared with controls. Bright-field and polarized light microscopy showed that the connective tissue matrix of granulation tissue from control rats was oriented perpendicular to the surface of the skin. In contrast, the connective tissue matrix of granulation tissue from vanadate-treated rats was oriented parallel to the skin surface. The gap in granulation tissue between the edges of the wounds in the vanadate-treated rats was greater than that in controls. Electron microscopy showed that wounds in the vanadate-treated contained uniform collagen fibers that were 20 percent greater in diameter and more evenly spaced than they were in controls. It is proposed that these changes in the organization of collagen fibers within incisional wounds were responsible for the increased wound breaking strength observed in rats ingesting vanadate.

Effects of tumor necrosis factor-binding protein on hepatic protein synthesis during chronic sepsis.

BACKGROUND: Cytokines are thought to play a role in the stimulation of protein synthesis in liver during inflammation and sepsis. We previously showed that administration of tumor necrosis factor-binding protein (TNFbp) prevents the sepsis-induced inhibition of protein synthesis in skeletal muscle. The purpose of the present set of experiments was to investigate the effect of TNFbp on hepatic protein synthesis and its ability to modulate the mechanisms responsible for increased hepatic protein synthesis during chronic (5-day) intraabdominal sepsis. MATERIALS AND METHODS: We examined the effects of TNFbp on hepatic protein synthesis during sepsis in four groups of rats: control, control + TNFbp, septic, and septic + TNFbp. Saline (1.0 ml) or TNFbp (1 mg/kg, 1.0 ml) was injected daily starting 4 h prior to the induction of sepsis. The effect of sepsis and TNFbp administration on hepatic protein synthesis in vivo was examined 5 days later. RESULTS: Sepsis increased the rate of protein synthesis by 35% relative to controls. Accelerated rates of protein synthesis were accompanied by increased total RNA content, eukaryotic initiation factor (eIF) 2alpha content, and phosphorylation of p70S6 kinase. Injection of TNFbp into septic rats for 5 days did not diminish the sepsis-induced stimulation of hepatic protein synthesis. Compared with controls, septic rats treated with TNFbp also showed elevated total RNA content, elF2alpha content, and phosphorylation of p70S6 kinase. No significant differences in any of the parameters measured were observed between untreated and TNFbp-treated septic rats. Treatment of control animals with TNFbp for 5 days was without effect on any of the parameters examined. CONCLUSIONS: TNFbp did not prevent the sepsis-induced stimulation of hepatic protein metabolism or modulate the septic-induced changes in factors regulating protein synthesis. Global rates of protein synthesis in livers from septic rats are accelerated by increases in the abundance or activity of components of translational apparatus.

Growth hormone does not attenuate the inhibitory effects of sepsis on wound healing.

Chronic abdominal sepsis is associated with impaired tissue repair. Treatment of burn patients with growth hormone results in improved healing of skin graft donor sites. The goal of this study was to determine whether administration of growth hormone could attenuate the inhibitory effects of sepsis on cutaneous wound healing. Four groups of male Sprague Dawley rats were studied: control, control + growth hormone, sepsis, and sepsis + growth hormone. Sepsis was caused by implantation of a bacterial focus in the peritoneal cavity. Control animals underwent sham laparotomy, and polyvinyl alcohol sponge implants were placed in subdermal pockets in all animals. Saline or growth hormone (400 microg) was injected subcutaneously every 12 hours. On day 5, the incisional wounds and polyvinyl alcohol sponge implants were harvested. The breaking strength of abdominal incisions was measured. Granulation tissue penetration and quality were determined by scoring polyvinyl alcohol sponge implant histology from 1 to 4 in a blinded fashion. Collagen deposition in polyvinyl alcohol sponge implants was quantitated by hydroxyproline assay. Septic mortality was not altered by growth hormone administration. Septic animals showed a reduction in food consumption for 2 days after surgery (p < 0.05 vs. controls), which was not affected by growth hormone administration. The breaking strength of incisional wounds and hydroxyproline content of polyvinyl alcohol sponge implants was reduced in septic rats (p < 0.001 vs. controls) but administration of growth hormone for 5 days did not improve breaking strength or collagen deposition in either group. We conclude that the administration of growth hormone for 5 days did not improve collagen deposition or breaking strength in cutaneous wounds from control or septic animals. The results suggest that growth hormone treatment is unlikely to improve tissue repair in sepsis-induced catabolic illness.

Interleukin-1 receptor antagonist attenuates tumor necrosis factor-induced alterations in wound breaking strength.

BACKGROUND: Tumor necrosis factor (TNF) is an important mediator of impaired wound healing during sepsis. To determine whether the inhibitory effects of systemic TNF on wound healing are mediated directly by TNF or by means of the induction of interleukin-1 (IL-1), we investigated the effects of TNF and interleukin- receptor antagonist (IL-1ra) on wound healing in healthy rats. METHODS: Male Sprague-Dawley rats were anesthetized, and jugular catheters were placed. After recovery of 48 hours, osmotic minipumps were inserted into the peritoneal cavity and polyvinyl alcohol implants were placed subcutaneously. Control rats were infused with saline (24 microL/day, i.p., and 15 mL/day, i.v.). TNF rats received TNF i.p. (100 microg/kg per day) and saline i.v. (15 mL/day). TNF+IL-1ra rats received TNF i.p. (100 microg/kg per day) and IL-1ra i.v. (2 mg/kg per day;15 mL/day). All animals were pair fed to the TNF group. On day 6, the wounds were harvested. The breaking strength of the abdominal incision was measured. Granulation tissue penetration and quality were determined by scoring polyvinyl alcohol implant histology from 1 to 4 in a blinded manner. Collagen deposition in polyvinyl alcohol implants was quantified by hydroxyproline assay. RESULTS: TNF decreased the breaking strength of incisional wounds to 40% of control levels (p < 0.001). IL-1ra restored the breaking strength of incisions from TNF-infused animals to 80% of control levels. Similar reductions in granulation tissue penetration, quality, and hydroxyproline content were observed in TNF-treated animals and were partially ameliorated by IL-1ra. CONCLUSION: IL-1ra significantly attenuates the inhibitory effects of systemic TNF on wound healing. These results suggest that the inhibitory effects of TNF on cutaneous tissue repair are mediated in part by IL-1.

Chronic infusion of interleukin 1 induces hyperlactatemia and altered regulation of lactate metabolism in skeletal muscle.

BACKGROUND: Hyperlactatemia is observed commonly in patients with severe inflammation syndrome or sepsis. Elevated plasma lactate concentrations may be caused by cytokine-mediated alterations in specific organ systems responsible for lactate homeostasis. The role of interleukin 1 (IL-1) in inducing hyperlactatemia and derangements in skeletal muscle and hepatic lactate metabolism was investigated by examining the consequences of infusing IL-1 continuously into normal rats. METHODS: Male Sprague-Dawley rats were anesthetized, and catheters were placed in the jugular vein. Rats were allowed to recover for 48 hours and were infused subsequently with either saline (control) or human recombinant IL-1alpha (20 microg/kg/d) for 6 days. On day 6, plasma, liver, and muscle samples were extracted and assayed for lactate and pyruvate dehydrogenase (PDH) activity. RESULTS: Plasma glucose concentrations were not different in the two groups. IL-1 infusion resulted in a twofold (p < .05) increase in the plasma lactate concentration compared with controls. IL-1 infusion also resulted in an elevated lactate content in skeletal muscle (p < .05) but not in liver. The proportion of PDH in the active form (PDHa) was reduced significantly (p < .05) in the skeletal muscle of animals infused with IL-1 compared with controls. In contrast to muscle, hepatic PDHa did not differ between the two groups. Total PDH complex activity was not affected in either liver or skeletal muscle. CONCLUSIONS: IL-1 infusion results in hyperlactatemia, increased skeletal muscle lactate, and a reduced PDHa in skeletal muscle. We conclude that IL-1 is a potential mediator of the derangements in lactate metabolism in skeletal muscle but not in liver.

Mechanism of IL-1 induced inhibition of protein synthesis in skeletal muscle.

Chronic interleukin (IL)-1 administration is associated with negative nitrogen balance and the loss of lean body mass. To elucidate the molecular mechanism(s) by which IL-1 modulates protein metabolism in muscle, we investigated the effects of chronic (6 day) IL-1alpha infusion on protein synthesis in Individual muscles (gastrocnemius, soleus, heart) compared with saline-infused control rats. IL-1 significantly decreased muscle weight, protein content, and the rate of protein synthesis in gastrocnemius (fast-twitch muscle). IL-1 had no effect on these parameters in the heart, whereas only the rate of protein synthesis was reduced in soleus (slow-twitch muscle). The reduction in gastrocnemius protein synthesis was not the result of a decrease in total RNA content, but was associated with a diminished translational efficiency. The diminished translational efficiency correlated with a 40% reduction in the epsilon-subunit of eukaryotic initiation factor 2B (elF2Bepsilon) in gastrocnemius from IL-1 -treated animals. However, the content of the alpha-subunit of elF2 (elF2alpha) was unaffected. In contrast, the elF2alpha content in heart was increased by IL-1, although elF2Bepsilon levels were unchanged. Reductions in skeletal muscle protein synthesis were not associated with a concomitant reduction in circulating or tissue content of insulin-like growth factor I. In summary, the IL-1-induced decrease in gastrocnemius protein synthesis appears to be regulated at the level of RNA translation via a reduction in elF2Bepsilon. These findings support a regulatory role for IL-1 as a mediator of muscle protein synthesis and the alterations in body composition observed in catabolic states where this cytokine is overexpressed.

TNF-binding protein ameliorates inhibition of skeletal muscle protein synthesis during sepsis.

We examined the effects of TNF-binding protein (TNFBP) on regulatory mechanisms of muscle protein synthesis during sepsis in four groups of rats: Control; Control+TNFBP; Septic; and Septic+TNFBP. Saline (1. 0 ml) or TNFBP (1 mg/kg, 1.0 ml) was injected daily starting 4 h before the induction of sepsis. The effect of TNFBP on gastrocnemius weight, protein content, and the rate of protein synthesis was examined 5 days later. Sepsis reduced the rate of protein synthesis by 35% relative to controls by depressing translational efficiency. Decreases in protein synthesis were accompanied by similar reductions in protein content and muscle weight. Treatment of septic animals with TNFBP for 5 days prevented the sepsis-induced inhibition of protein synthesis and restored translational efficiency to control values. TNFBP treatment of Control rats for 5 days was without effect on muscle protein content or protein synthesis. We also assessed potential mechanisms regulating translational efficiency. The phosphorylation state of p70(S6) kinase was not altered by sepsis. Sepsis reduced the gastrocnemius content of eukaryotic initiation factor 2Bepsilon (eIF2Bepsilon), but not eIF2alpha. The decrease in eIF2Bepsilon content was prevented by treatment of septic rats with TNFBP. TNFBP ameliorates the sepsis-induced changes in protein metabolism in gastrocnemius, indicating a role for TNF in the septic process. The data suggest that TNF may impair muscle protein synthesis by reducing expression of specific initiation factors during sepsis.

TNF binding protein prevents hyperlactatemia and inactivation of PDH complex in skeletal muscle during sepsis.

BACKGROUND: Hyperlactatemia is a metabolic complication of hypermetabolic, hyperdynamic sepsis. An important mechanism responsible for elevating plasma lactate concentrations in sepsis is altered regulation of the pyruvate dehydrogenase complex (PDH) in skeletal muscle. We investigated the ability of a specific tumor necrosis factor binding protein, TNFbp, to modulate lactate concentrations and skeletal muscle PDH activity in a rodent model of chronic abdominal sepsis. MATERIALS AND METHODS: We examined the regulation of lactate metabolism in four groups of animals: Control, Control + TNFbp, Septic, and Septic + TNFbp. Chronic (5 days) sepsis was induced by the creation of a stable intraabdominal abscess using a sterile fecal-agar pellet inoculated with E. coli plus B. Fragilis as the foreign body nidus. TNFbp (1 mg/kg/day) was injected subcutaneously daily. RESULTS: Sepsis increased plasma and skeletal muscle lactate concentrations 2-fold compared with control. In septic rats treated with TNFbp, plasma and skeletal muscle lactate concentrations were significantly decreased compared with untreated septic rats. In skeletal muscle, sepsis resulted in a 70% decrease in the proportion of the PDH in the active form compared with controls. The sepsis-induced inhibition in the PDH complex activity was prevented by TNFbp. PDH kinase was enhanced 1.8-fold in sepsis, and the increase in PDH kinase activity was prevented by treatment with TNFbp. TNFbp treatment did not have any effects on plasma lactate or the proportion of active skeletal muscle PDH activity in control animals. CONCLUSIONS: TNFbp prevents the sepsis-induced hyperlactatemia and derangements in skeletal muscle lactate concentrations and PDH activity. These observations suggest that TNF is an important mediator responsible for lactate dyshomeostasis during sepsis.

Tumor necrosis factor binding protein improves incisional wound healing in sepsis.

BACKGROUND: Sepsis is associated with poor wound healing; however, the exact role of tumor necrosis factor (TNF) as a mediator of sepsis-induced alterations in different types of tissue repair is unknown. This study examines the effects of a specific TNF antagonist (TNFbp) on the healing of intestinal anastomoses, incisional wounds, and polyvinyl (PVA) sponge implants in chronic abdominal sepsis. METHODS: Three groups of male Sprague-Dawley rats were studied: control, sepsis, and sepsis + TNFbp. Jejunal resection and anastomosis were performed through a 4-cm upper midline incision on day 1. On day 3, sepsis was induced by creation of a chronic abdominal abscess. Saline (0.1 ml) or TNFbp (1.0 mg/kg, 0.1 ml) was injected subcutaneously every day starting 4 h prior to sepsis. On day 7, the wound-breaking strength (WBS) of the skin incision and intestinal anastomoses was determined using a tensiometer. Wound histology and collagen deposition were evaluated by comparison of Sirius red-stained sections. The hydroxyproline content of PVA sponges was used to quantitate collagen content under the different experimental conditions. RESULTS: Septic mortality (20% vs 26%) was not significantly altered by TNFbp. Septic animals demonstrated a reduction in food consumption on days 3 to 5 that was not affected by TNFbp administration. Neither sepsis nor TNFbp altered the breaking strength or histologic appearance of intestinal anastomoses. However, the breaking strength of incisional wounds was decreased by 40% in septic rats (P < 0.001 vs controls). Administration of TNFbp to septic rats significantly improved incisional WBS (P < 0.01 vs sepsis), but not to control levels. Serius red staining of incisional wounds and PVA sponges demonstrated a decrease in collagen organization and deposition in septic rats that was ameliorated by TNFbp. Similarly, the reduction in hydroxyproline content of PVA sponges from septic animals was prevented by TNFbp. CONCLUSIONS: The process of tissue repair in intestine and skin wounds appears to be significantly different following the septic insult. The healing of jejunal anastomoses was refractory to the catabolic effects of sepsis. In contrast, collagen deposition and organization are significantly decreased in cutaneous wounds during chronic sepsis. TNFbp significantly ameliorated the inhibitory effects of sepsis on cutaneous wound healing. These results suggest that TNF is an important mediator of the decrease in collagen deposition observed in cutaneous wounds during the septic state.

Tumor necrosis factor mediates impaired wound healing in chronic abdominal sepsis.

BACKGROUND: The role of systemic tumor necrosis factor (TNF) as a mediator of impaired wound healing in sepsis is unclear. The purpose of this study was to examine the effects of a specific TNF antagonist (TNFbp) on wound healing during chronic abdominal sepsis. METHODS: Male Sprague-Dawley rats were divided into four groups: control, control + TNFbp, sepsis, and sepsis + TNFbp. Saline (1.0 mL) or TNFbp (1 mg/kg, 1.0 mL) was injected subcutaneously daily, polyvinylalcohol (PVA) sponge implants were placed in subcutaneous pockets, and sepsis was induced by creation of a chronic, intra-abdominal abscess. Sponge implants were removed on day 5 and examined histologically. Granulation tissue infiltration and quality (connective tissue, cellularity, vascularity) were scored on a scale from 1 to 4 in a blinded fashion. RESULTS: Septic mortality (19 vs. 25%) was not influenced by TNFbp. Granulation tissue penetration and quality were decreased in septic animals. The administration of TNFbp significantly attenuated the effects of sepsis on granulation tissue histology, but not to control levels. CONCLUSIONS: These studies provide evidence that TNF contributes to the impaired wound healing observed in this model of chronic abdominal sepsis.