Gretchen M. Ahrendt

Diabetes-impaired healing and reduced wound nitric oxide synthesis : A possible pathophysiologic correlation

BACKGROUNDnNitric oxide (NO) is synthesized in wounds, but its role in the healing process is not fully understood. The inhibition of NO production during wound healing is accompanied by decreased wound reparative collagen deposition. To further define the role of NO in reparative collagen accumulation, we studied its production during diabetes-induced wound healing impairment.nnnMETHODSnMale Sprague-Dawley rats (290 to 310 gm) were rendered diabetic by intraperitoneal streptozotocin administration. Seven days after induction of diabetes (blood glucose greater than 300 mg/dl), the rats underwent dorsal skin incision and subcutaneous implantation of polyvinyl alcohol sponges. Beginning on the day of wounding, 21 diabetic animals were treated with 3 units/day insulin via intraperitoneally implanted miniosmotic pumps. Ten days after injury, wound breaking strength was determined, and wound collagen accumulation and types I and III collagen gene expression were measured in subcutaneously implanted polyvinyl alcohol sponges. NO-synthesis, as measured by nitrite/nitrate accumulation, was determined in wound fluid and in supernatants of wound cell cultures.nnnRESULTSnStreptozotocin-induced diabetes markedly impaired wound breaking strength and collagen deposition. A parallel decrease occurred in wound NO synthesis as reflected by decreased nitrite/nitrate concentration in wound fluid and in diminished ex vivo NO production by wound cells. Insulin treatment partially but significantly improved wound mechanical strength (p < 0.01) and collagen accumulation (p < 0.001). Decreased wound NO accumulation and ex vivo NO production by wound cells were also partially restored by insulin treatment.nnnCONCLUSIONSnImpaired diabetic wound healing is paralleled by decreased wound NO synthesis, supporting the hypothesis that NO plays a significant role in wound reparative collagen accumulation.

Intra-abdominal sepsis impairs colonic reparative collagen synthesis

BACKGROUNDnIntra-abdominal infection is generally considered a contraindication to primary colon anastomosis. In order to elucidate the mechanisms by which sepsis affects colonic healing, we studied anastomotic new collagen and protein synthesis and collagen gene expression in a relevant animal model.nnnMETHODSnForty male Sprague-Dawley rats (240 to 260 g) underwent sham laparotomy (SHAM, n = 18) or cecal ligation and single puncture (CLP, n = 22). After 24 hours, animals underwent single-layer left colon anastomosis. Animals were sacrificed either 1 or 4 days postanastomosis. Anastomotic segments of colon were excised, minced, and incubated with 4.5 muCi 3H-proline. After 3 hours, tissue 3H-proline incorporation was quantitated as an index of total new protein synthesis. The protein fraction was then digested with purified collagenase enzyme to determine 3H-proline incorporation into collagenase-digestible protein, an index of new collagen synthesis. Total RNA was extracted from anastomotic tissue samples and subjected to Northern blot analysis for type I and type III collagen genes.nnnRESULTSnIntra-abdominal sepsis resulted in markedly less new collagen synthesis 1 day postanastomosis (9,163 +/- 1,234 versus 3,744 +/- 444 disintegrates per minute 3H-proline/mg of protein, P < 0.0001) and 4 days postanastomosis (8,462 +/- 956 versus 5,708 +/- 802 dpm/mg of protein P < 0.05). Noncollagenous protein synthesis was also impaired in anastomotic tissue from CLP rats on postanastomosis day 1 (37,497 +/- 3,740 versus 18,593 +/- 2,695 dpm/mg of of protein, P < 0.001) and postanastomosis day 4 (28,238 +/- 834 versus 17,784 +/- 1,415 dpm/mg of of protein, P < 0.0001). The expression of type I and type III collagen was altered relative to the normal temporal sequence observed in SHAM animals.nnnCONCLUSIONnIntra-abdominal infection impairs colonic reparative collagen and protein synthesis. In addition, regulation of type I and type III collagen genes is altered by intra-abdominal sepsis, and the alteration likely contributes to impaired new collagen synthesis and decreased colonic mechanical strength.

Stimulation of fibroblast proliferation and matrix contraction by wound fluid

Fibroblast proliferation and fibroblast-mediated matrix contraction are critical to wound healing. Different cytokines have been shown to modulate fibroblast functions but little is known about the physiological role of these soluble factors during wound repair. In these experiments we characterized a fibroblast stimulating factor in wound fluid. Wound fluid was obtained from subcutaneously implanted polyvinyl alcohol sponges harvested 10 days post-wounding (pool of 100 Lewis rats). Normal dermal fibroblasts were obtained from Lewis rats by an explant technique, while wound fibroblasts were isolated from sponges harvested 10 days post-wounding. Proliferation in response to 0.5% and 10% fetal bovine serum was assessed by [3H]-thymidine incorporation. A fibroblast-populated collagen lattice was used for assaying contractile properties. Wound fibroblasts demonstrated markedly diminished proliferative and enhanced contractile properties compared to normal dermal fibroblasts. 10% wound fluid (v/v) stimulated proliferation of normal dermal fibroblasts (119%, p < 0.001) and wound fibroblasts (103%, p < 0.001). Wound fluid also stimulated collagen gel contraction by normal dermal fibroblasts (24% at 24 hr and 16% at 72 hr, p < 0.01), but not by wound fibroblasts. Separation by Sephadex G-100 gel filtration identified the active factor in wound fluid to have a molecular weight of about 100 kDa. Characterization of the soluble factor showed it to be a protein (ammonium sulfate precipitation), sensitive to trypsin digestion, heat resistant (56 degrees C, 30 min), and neuraminidase resistant. The isoelectric point appeared to be 7.0, as determined by ion exchange chromatography. Mitogenic proliferation of thymic lymphocytes was not affected by the active factor, suggesting cell target specificity. These data demonstrate that the wound environment contains high molecular weight protein(s) that promote fibroblast functions, essential for the healing process.

Hypercalcemia decreases bile flow and increases biliary calcium in the prairie dog

BACKGROUNDnBiliary calcium is known to play an important role in the pathogenesis of gallstones. Calcium salts are present in all pigment gallstones and are also present in the core of most, if not all, cholesterol gallstones.nnnMETHODSnThe effects of acute hypercalcemia on bile flow and biliary calcium secretion were examined in 22 prairie dogs during intravenous taurocholate infusion (0, 1.0, 2.25, and 4.5 mumol/kg/min).nnnRESULTSnBile flow was linearly correlated with bile acid output in both control (y = 7.62x + 13.5, r = 0.98) and hypercalcemic (y = 7.00x + 10.4, r = 0.96) animals. At lower bile acid outputs (< 3.0 mumol/kg/min), biliary ionized calcium output per increment bile acid output was significantly increased in hypercalcemic animals (0.016 versus 0.011 mumol Ca++ mumol taurocholate, p < 0.001). Bile ionized calcium concentrations approximated Gibbs-Donnan predicted values only at low bile flow rate.nnnCONCLUSIONSnHypercalcemia decreases bile flow and increases biliary ionized calcium concentration in the prairie dog. These effects favor the precipitation of calcium salts in bile.

Nutrition and multiple organ dysfunction syndrome

Multiple organ dysfunction syndrome (MODS) is seen with increasing frequency in intensive care units as a result of our ability to support single-organ systems and prolong survival in patients with severe illness. One hypothesis of MODS is that altered intestinal permeability during periods of stress allows translocation of bacteria and endotoxin to sterile tissues, thereby initiating this inflammatory process. Nutritional therapy aims to maintain gastrointestinal structure and function to prevent translocation and allow absorption and metabolism of nutrients. Organ-specific nutrient therapy for the gut includes supplementation of either enteral or parenteral formulas with glutamine, arginine, specific fatty acids, and fiber. Ideally, these nutrients would provide stimulus to the gut mucosa to preserve important immune and barrier function as well as modulate the hosts immune response to limit cytokine toxicity.

STICKSTOFFMONOXID IST BEI DER DIABETISCHEN WUNDHEILUNG VERMINDERT

Stickstoffmonoxid (NO) ist ein kurzlebiger biologischer Mediator, der in einem enzymatischen Prozes aus Sauerstoff und Arginin entsteht. In Wunden wird NO von immunkompetenten Entzundungszellen und Fibroblasten gebildet [1, 2]. Obwohl die genaue Bedeutung des NO bei der Heilung nicht bekannt ist, scheint es an der Kaskade zellularer Interaktionen beteiligt zu sein [2]. Die systemische Hemmung der NO-Synthese fuhrt zur verminderten Kollagenbildung und mechanischen Festigkeit von Wunden [3]. Experimentell kommt es bei der Wundheilung unter diabetischen Stoffwechselbedingungen zu einer gestorten Kollagenformation, die durch fruhzeitige Insulingaben teilweise wieder ausgeglichen werden kann [4]. Inwieweit NO bei der diabetischen Wundheilung eine Rolle spielt, ist nicht bekannt.