Michael D. Caldwell

Modulatory activities of wound fluid on fibroblast proliferation and collagen synthesis.

Several different cell types play a role in the regulatory mechanisms involved in wound healing. A rat wound model was used to evaluate temporal changes in the cellular infiltrate, histology, and effects of wound fluid (WF) on fibroblast growth and collagen synthesis in vitro. Polyvinyl alcohol sponges were implanted in male Sprague/Dawley rats and harvested after 1, 3, 5, 7, 10, and 15 days. Rat wound fibroblasts were cultured in different media with 10 or 20% WF pooled from five or more rats from each time interval, and then pulsed with [3H]thymidine. Days 1 through 5 WF stimulated proliferation, whereas Days 10 and 15 WF inhibited proliferation. Stimulatory activity was found in the greater than 300 kDa molecular weight fraction; inhibitory activity was in the less than 10 kDa molecular weight fraction. Ten percent WF from both Day 1 and Day 15 sponges exerted a stimulatory effect in incubated fibroblasts on collagen production, measured as protein-bound [3H]hydroxyproline. Fibroblast proliferation and collagen synthesis appeared to be independently regulated functions. Fibroblasts were stimulated by the wound environment to proliferate for about 1 week after injury, at which point further growth was inhibited, while collagen production was maintained.

Continuity between wound macrophage and fibroblast phenotype: analysis of wound fibroblast phagocytosis

Analysis of phagocytic activity in wound fibroblasts was chosen as a means to assess the possible continuity between macrophage and fibroblast phenotypes. Fibroblast phagocytosis of uncoated, IgG-coated, or collagen-coated fluorescent beads was analyzed by flow cytometry in vivo and in vitro. Phagocytosis of fluorescent beads by procollagen I-positive cells (fibroblasts) was evaluated in vivo by injecting beads into subcutaneously implanted sponge wounds in anesthetized Fisher rats. Phagocytic activity of a purified population of wound fibroblasts was measured in vitro and correlated with oxidation state using hydroethidium. In the wound environment, 50-60% of the cells that engulfed uncoated, IgG-coated, or collagen-coated beads were procollagen I-positive cells (i.e., fibroblasts). Procollagen I-positive cells engulfed uncoated and IgG-coated beads in preference to collagen-coated beads in vivo. Cultured wound fibroblasts engulfed uncoated, IgG-coated, and collagen-coated particles. The majority of fibroblasts that engulfed beads were in an elevated oxidation state. We conclude that substantial fibroblast phagocytosis occurs in the wound, but scavenger receptor-mediated fibroblast phagocytosis is different from that of macrophages. Additional markers will be helpful in defining the macrophage fibroblast continuum.Analysis of phagocytic activity in wound fibroblasts was chosen as a means to assess the possible continuity between macrophage and fibroblast phenotypes. Fibroblast phagocytosis of uncoated, IgG-coated, or collagen-coated fluorescent beads was analyzed by flow cytometry in vivo and in vitro. Phagocytosis of fluorescent beads by procollagen I-positive cells (fibroblasts) was evaluated in vivo by injecting beads into subcutaneously implanted sponge wounds in anesthetized Fisher rats. Phagocytic activity of a purified population of wound fibroblasts was measured in vitro and correlated with oxidation state using hydroethidium. In the wound environment, 50-60% of the cells that engulfed uncoated, IgG-coated, or collagen-coated beads were procollagen I-positive cells (i.e., fibroblasts). Procollagen I-positive cells engulfed uncoated and IgG-coated beads in preference to collagen-coated beads in vivo. Cultured wound fibroblasts engulfed uncoated, IgG-coated, and collagen-coated particles. The majority of fibroblasts that engulfed beads were in an elevated oxidation state. We conclude that substantial fibroblast phagocytosis occurs in the wound, but scavenger receptor-mediated fibroblast phagocytosis is different from that of macrophages. Additional markers will be helpful in defining the macrophage fibroblast continuum.

Blunt injury augments interleukin-6 but not tumor necrosis factor in isolated, perfused rat hindlimbs.

An isolated, perfused hindlimb model in rats was used to examine the immediate inflammatory response after blunt tissue injury. A femur-fracture degloving model was used in isolated rat hindlimbs perfused with a modified Krebs buffer (pH 7.4) containing albumin, washed human red blood cells (RBCs), amino acids, and glucose at 37 degrees C. Arterial and venous perfusate was sampled at 5, 20, and 80 minutes of perfusion. Initial experiments were conducted in perfusate void of white blood cells (WBCs), group 1 (-inj/-WBC, n = 6) and group 2 (+inj/-WBC). Subsequent experiments were conducted in perfusate containing activated WBCs, group 3 (-inj/+WBC, n = 6) and group 4 (+inj/+WBC, n = 7). Hindlimb muscle was analyzed for adenylate energy charge (EC) and lactate-to-pyruvate ratios (LPR) at the end of each perfusion. This preparation appeared metabolically stable in that oxygen consumption and lactate remained stable during the 80-minute perfusion and muscle EC and LPR indicated aerobic metabolism. Tumor necrosis factor (TNF) and thromboxane B2 (TXB2) were measured in all four groups while prostaglandin F (PGF1 alpha), IL-6, myeloperoxidase, and 8-isoprostane were measured in groups 3 and 4. Initial perfusions in the -WBC hindlimbs indicated no change in TNF release after injury. The TXB2 level increased during perfusion irrespective of injury. The PGF1 alpha was elevated at 80 minutes in both groups 3 and 4, however at 20 minutes PGF1 alpha levels were higher in group 4 compared with group 3. Interestingly, the IL-6 level was significantly elevated at 80 minutes in group 4 but not in group 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage interaction with skeletal muscle: a potential role of macrophages in determining the energy state of healing wounds

A decrease in ATP and creatine phosphate (CP) is characteristic of local injury to skeletal muscle. Recent data have suggested adequate potential for high-energy phosphate production in the wounded tissue. Thus, an adequate explanation for the deficit in the high-energy tissue content in wounds was lacking. Since the wound has multiple components (muscle + cellular infiltrate), the tissue content represents the summation of these components. Therefore, a technique to separate these components was designed. Using a 0.5% solution of lambda-carrageenan as the wounding agent, the extensor digitorum longus muscles (EDL) of male Fisher rats were unilaterally wounded with intramuscular injections. Five days later, both wounded and contralateral nonwounded muscles were incubated in a standardized fashion. The groups of EDL were: wounded as described, contralateral nonwounded, or contralateral nonwounded with the addition to the incubate of 6 X 10(6)/ml lambda-carrageenan elicited peritoneal macrophages. Following incubation, the individual component parts of the system (muscle and macrophage) were rapidly frozen and assayed for high-energy phosphate and DNA content. Examination of the high-energy phosphate content of the separate components of a wound demonstrated that macrophages increased the ATP and CP content of normal skeletal muscle. Yet when total high-energy phosphate content was normalized for total DNA (muscle + macrophages) in the reconstituted system, the values approximated those of wounded muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor Effects on Gluconeogenesis in the Isolated Perfused Rat Liver

Alterations in metabolism in the tumor-bearing host can be explained by: 1) alterations of metabolic processes in the tumor itself, and/or 2) tumor effects on host metabolism. Tumor effects on host liver metabolism were studied using an isolated perfused rat liver preparation. The livers of fasted female Lewis Wistar rats with and without transplanted subcutaneous mammary tumors were perfused for 1 hr with medium containing 5 mM glucose and physiological levels of amino acids. The rate of gluconeogenesis, as measured by conversion of 14C-lactate to 14C-glucose, showed a significant increase in the rate of glucose production from lactate in tumor-bearing rats (2.40 vs 2.00 mumol/min/100 gm). Hepatic glycogen and 14C-glycogen content were not significantly different between the two groups. In order to evaluate whether this tumor model exhibits characteristic changes in metabolism previously reported in other animal tumor models, serum lactate, triglyceride, glucose, and blood urea nitrogen were measured in non-perfused animals. The serum concentration of lactate and triglycerides were significantly higher in tumor-bearing rats (0.9 mM vs 2.7 mM lactate; 244 mg % vs 365.5 mg % triglycerides). Serum glucose and blood urea nitrogen were not significantly different in the two groups. An effect of tumor on host energy metabolism and serum metabolite levels is demonstrated. A method for the study of host-tumor metabolic interactions is described.

The temporal characteristics of the metabolic and endocrine response to injury.

The neuroendocrine and substrate responses immediately after injury have been extensively investigated in man and animals. The purpose of the present study was to examine simultaneously, the temporal, metabolic and endocrine consequences of a single uniform injury induced by the injection of lambda-carrageenan into the hindlimbs of male Sprague-Dawley rats and to compare this response to that observed in semistarved pair-fed control animals. Immediately after injury there was a decrease in the plasma hematocrit, increase in tissue water and peripheral vasoconstriction that suggested hypovolemia. This was followed by a restoration of the blood volume by 1 day as reflected in hemodilution. Alterations in insulin, glucagon, ACTH, corticosterone, epinephrine, norepinephrine, and dopamine in wounded animals occurred during the first 5 days. However, similar changes were observed in pair-fed control animals from days 1 to 5. These findings implied that the early endocrine response observed from 0 to 24 hours after injury arises, primarily as a result of hypovolemia, whereas the response observed from 1 to 5 days appeared to be the result of semistarvation. In contrast to the endocrine alterations observed, alterations in the plasma concentrations of lactate, acetoacetate and beta-hydroxybutyrate persisted for up to 15 days. The presence of these substrate alterations in the absence of hormonal stimuli suggest that nonendocrine mechanisms exist to induce these alterations. The possibility is raised that these substrate alterations may be, at least in part, the result of the inflammatory infiltrate.

Effect of starvation on the local and systemic metabolic effects of the λ-carrageenan wound☆☆☆

Abstract The contribution that starvation makes to the altered glucose metabolism in injured rats was evaluated. Food intake, weight change, nitrogen balance, and muscle tissue concentrations of glycogen, glucose, and the glycolytic intermediates were determined in these animals. This study concluded that the wounded and pair fed control groups presented adequately represent the metabolic states associated with injury and semistarvation in experimental animals, decreased food intake plays a major role in the weight loss and nitrogen balance in this wound model, wounding overrides two of the controlling steps of glycolysis (hexokinase and phosphofructokinase) in skeletal muscle during starvation, the finding of similar pyruvate dehydrogenase activity after wounding and starvation as demonstrated by tissue lactate to pyruvate ratios and lactate and pyruvate concentrations suggest that lactate production in wounded tissue may not be simply a manifestation of an altered redox state secondary to anaerobic conditions.

Effects of Hypocaloric Dextrose and Amino Acids on Body Composition and Nitrogen Balance in Rats

Studies were conducted to compare the effects of isocaloric intake of glucose, amino acids, and combinations of these nutrients on nitrogen balance, body composition, and selected metabolic parameters of adult rats. Diets were administered either orally or intravenously at 20% (semi-starvation) of the daily caloric requirement. Although nitrogen balance was improved when all calories were supplied as amino acids when compared to glucose alone, one-half the amino acids could be replaced with glucose without sacrificing nitrogen balance. Animals from the semi-starvation treatment showed similar weight and body composition changes, there was no apparent difference whether the diets were administered orally or intravenously. These results indicate that replacement of at least one-half the calories of an all amino acid hypocaloric diet with glucose does not adversely affect body composition weight change or nitrogen balance, but may reduce blood urea nitrogen, ketosis, and cost of nutritional support.

Endogenous Growth Factors and Nutrients in the Healing Wound

This chapter focuses on the temporal sequence of endogenous growth factors and nutrients in the healing wound. We propose that the function and proliferation of wound cells are dependent on the concentration of substrates, hormones, growth factors, cytokines, matrix, etc. in the local wound environment and less dependent on serum factors. The consistent proliferative effect of early wound fluid and inhibitory effect of late wound fluid is supportive of this hypothesis (1).