精氨酸促进糖尿病伤口愈合的机制

目的 观察精氨酸促进糖尿病伤口愈合的机制。方法 成年雄性Lewis大鼠，以链脲佐菌素复制糖尿病模型，7d后在大鼠背部制作伤口，置入PVA海绵，每天腹腔注射L-精氨酸1g／kg体重，10d后动物安乐死取样，观察伤口液精氨酸、鸟氨酸、瓜氨酸、NOx、尿素氮及蛋白含量的变化。结果与对照组大鼠比较，糖尿病大鼠伤口液总蛋白、NOx水平显著升高，精氨酸、鸟瓜氨酸、瓜氨酸及尿素氮水平无明显变化。结论 精氨酸促进糖尿病伤口愈合的机制可能与iNOS途径有关，与精氨酸酶途径无关。     

Supplemental dietary arginine enhances wound healing in normal but not inducible nitric oxide synthase knockout mice

BACKGROUND: Although generation of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) has been shown to be required for cutaneous wound healing, no differences have been noted in incisional healing between iNOS knockout (iNOS-KO) and wild type (WT) mice. Because supplemental dietary arginine enhances cutaneous healing in normal rodents and is the sole substrate for NO synthesis, we studied whether arginine can enhance cutaneous wound healing in iNOS-KO mice. METHODS: Twenty iNOS-KO and 20 WT mice, all on a C57BL/6 background, were divided into 4 groups of 10 animals each. Ten animals with each trait were randomized to receive either normal food and tap water or food and water each supplemented with 0.5% arginine (w/w). All animals underwent a 2.5-cm dorsal skin incision with implantation of four 20-mg polyvinyl alcohol sponges into subcutaneous pockets. On postoperative day 14 the animals were killed. The dorsal wound was harvested for breaking strength determination and the wound sponges were assayed for hydroxyproline content and total wound fluid nitrite/nitrate concentration. RESULTS: Dietary arginine supplementation enhanced both wound breaking strength and collagen deposition in WT but not iNOS-KO mice. Wound fluid nitrite/nitrate levels were higher in WT than iNOS-KO animals but were not significantly influenced by additional arginine. CONCLUSIONS: These data demonstrate that supplemental dietary arginine enhances wound healing in normal mice. The loss of a functional iNOS gene abrogates the beneficial effect of arginine in wound healing. This suggests that the metabolism of arginine via the NO pathway is one mechanism by which arginine enhances wound healing.     

Characterization of incisional wound healing in inducible nitric oxide synthase knockout mice

BACKGROUND: Excisional wound healing in inducible nitric oxide synthase knockout (iNOS-KO) mice has been previously shown to be impaired compared with their background strain controls. Incisional wounds were created in this experiment in both types of animals and paradoxically were found to heal with the same rapidity and breaking strength in both groups. METHODS: Dorsal 2.5 cm incisional wounds were created in iNOS-KO mice, as well as their parental strain controls (C57BL/6J). Standardized polyvinyl alcohol sponges were implanted in the wounds to allow for measurement of collagen deposition. Animals were harvested on postoperative days (PODs) 3, 5, 7, 10, 14, and 28, and their wounds subjected to tensiometric breaking strength analysis. Nonisotopic in situ hybridization quantitative analysis for iNOS, endothelial NOS (eNOS), basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and interleukin-4 (IL-4) expression in the wounds was performed. Hydroxyproline levels were quantitated in the harvested polyvinyl alcohol sponges. Data were analyzed with the Students t test. RESULTS: No significant differences were found in breaking strengths or levels of hydroxyproline (and thus collagen) in iNOS-KO versus wild-type wounds at all tested time points. Flawed iNOS expression levels in iNOS-KO animals were similar to (functional) iNOS expression in wild-types. eNOS and bFGF expression nearly doubled on POD 7 in iNOS-KO incisions (P =.002, and.002), respectively and remained 200% to 300% elevated thereafter. TGF-beta1 expression was increased approximately 50% to 100% in iNOS-KO wounds on PODs 5 and 7 (P =.006 and.01, respectively). VEGF and IL-4 expression was elevated by 25% to 100% in wild-type compared with iNOS-KO animals at all time points (P <.01). CONCLUSIONS: The overexpression of TGF-beta1 and eNOS may represent mechanisms in iNOS-KO mice to compensate for their loss of functional iNOS, resulting in incisional wound healing equivalent to controls. Their impaired expression of VEGF and IL-4, on the other hand, may partially explain the delayed excisional wound healing noted in these animals.     

Nitric oxide enhances experimental wound healing in diabetes

BACKGROUND: Diabetes is characterized by a nitric oxide deficiency at the wound site. This study investigated whether exogenous nitric oxide supplementation with the nitric oxide donor molsidomine (N-ethoxycarbomyl-3-morpholinyl-sidnonimine) could reverse the impaired healing in diabetes. METHODS: Wound healing was studied by creating a dorsal skin incision with subcutaneous polyvinyl alcohol sponge implantation in diabetic and non-diabetic rats. Half of each group was treated with molsidomine. Collagen metabolism was assessed by wound breaking strength, hydroxyproline (OHP) content, RNA expression for collagen type I and III, and matrix metalloproteinase (MMP) 2 activity in wound sponges. Wound fluid, plasma and urinary nitric oxide metabolite levels, and the number of inflammatory cells were assessed. RESULTS: OHP content and wound breaking strength were significantly increased by molsidomine. MMP-2 activity in wound fluid was decreased in diabetes and upregulated by nitric oxide donors. The impaired inflammatory reaction in diabetes was unaffected by nitric oxide donor treatment and ex vivo nitric oxide synthesis was no different between wound macrophages from control and diabetic animals, suggesting that the nitric oxide deficiency in the wound is due to a smaller inflammatory reaction in diabetes. CONCLUSION: The nitric oxide donor molsidomine can at least partially reverse impaired healing associated with diabetes.     

Supplemental l-arginine enhances wound healing following trauma/hemorrhagic shock

To determine whether parenteral L-arginine supplementation enhances the impaired wound healing of rats subjected to trauma/hemorrhagic shock. Impaired wound healing after trauma and shock has been documented experimentally and clinically. L-arginine has been shown to enhance wound strength and collagen synthesis in rodents and humans. Its efficacy under conditions of impaired wound healing is less well defined. Forty-eight male Lewis rats were used in this study. Using a well-defined model, 24 rats underwent trauma/hemorrhagic shock before wounding. Twenty-four untreated rats served as controls. All animals underwent a dorsal skin incision with implantation of polyvinyl-alcohol sponges. Half of the animals in each group were assigned to receive 1 g/kg/day of L-arginine by intraperitoneal injection in three divided doses, while the other half received saline injections only. Animals were sacrificed 10 days postwounding, and wound-breaking strength (WBS) and wound sponge total hydroxyproline (OHP) and nitrite/nitrate (NO(x)) content were determined. Wound sponge RNA was collected and subjected to Northern blot analysis for procollagens I and III. Trauma/hemorrhage greatly decreased WBS with a concomitant diminution in collagen (OHP) deposition. L-arginine significantly enhanced WBS (19%) and increased OHP (21%) levels in control animals as well as in rats subjected to trauma/hemorrhage (WBS +29%, OHP 40%) compared with their saline-treated counterparts. Procollagen I and III mRNA levels were elevated by L-arginine treatment in both trauma/hemorrhage and control rats. Arginine treatment had no effect on wound fluid and plasma NO(x). The data demonstrate that the impaired healing subsequent to trauma/hemorrhage can be greatly alleviated by L-arginine supplementation.     

Inhibition of nitric oxide synthesis in wounds: pharmacology and effect on accumulation of collagen in wounds in mice.

OBJECTIVE: To investigate the effect of systemic inhibition of nitric oxide (NO) synthesis in wounds on collagen accumulation. DESIGN: Randomised experimental study. SETTING: Teaching hospital, USA. MATERIAL: 240 Balb/C mice divided into groups of 10 animals each. INTERVENTIONS: Polyvinyl alcohol sponges were inserted subcutaneously through a dorsal skin incision. Beginning on the day of wounding, N omega-nitro-L-arginine-methylester (L-NAME), NG-L-monomethyl-arginine (L-NMMA), aminoguanidine hemisulphate (AGU), and S-methyl isothiouronium (MITU) were given orally or intraperitoneally. The mice were killed 10 days later. MAIN OUTCOME MEASURES: Nitrite and nitrate concentrations, both stable end products of NO, were measured in wound fluid. Sponge hydroxyproline content was assayed as an index of reparative collagen deposition. RESULTS: NOS inhibitors given orally in the drinking water or by daily intraperitoneal injection had no effect on wound nitrite/nitrate concentrations or deposition of collagen in wounds. When given continuously through intraperitoneally-placed osmotic pumps, AGU (500 mg/kg/day) (p < 0.001) and MITU (p < 0.01) significantly reduced wound fluid nitrite/nitrate concentrations in a dose dependent manner. Inhibition of wound nitric oxide synthase by 500 mg AGU/kg/day and 100 mg MITU/kg/day was paralleled by lowered accumulation of collagen in wounds (p < 0.01). CONCLUSION: NO is beneficial in wound healing.     

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.     

Thymic inhibition of wound healing: Abrogation by adult thymectomy

It has been previously observed that the thymus and wound respond in a similar manner, i.e., agents that enhance thymic function increase wound healing, while factors which decrease thymic function impair healing. In order to elucidate if the thymus has a direct influence on wounds, we have studied wound healing in adult rats who have undergone thymectomy at 4–8 weeks of age. In three separate experiments we found that thymectomized rats had fresh wound breaking strengths significantly greater than sham-thymectomized rats. There were no differences noted in the amount of reparative collagen accumulated in subcutaneously implanted polyvinyl alcohol sponges or in the breaking strength of wound strips fixed in 10% formalin, which maximally cross-links the collagen present; the ratios of fixed to fresh wound breaking strengths were significantly greater in sham-thymectomized rats. Rats who had undergone thymectomy with immediate intraperitoneal placement of Millipore chambers containing autologous thymic fragments had wound breaking strengths similar to sham-thymectomized or intact animals. We conclude that thymectomy at 4–8 weeks of age increases wound maturation and collagen cross-linking. This suggests that the thymus normally has an inhibitory effect on wound healing and a role of T-suppressor cells on this process is postulated.     

The effect of testosterone propionate on wound healing in normal and castrate rats

The dependence of wound healing on testosterone was studied in normal and castrate rats by determination of wound breaking strength (WBS) in dermal wounds, by implantation of subcutaneous polyvinyl sponges (PVS) and by [³H]proline tracer studies. The level of testosterone achieved with various doses of testosterone propionate (TP) was assessed using the androgenic effect of this hormone on prostate and seminal vesicle weights. Exogenous testosterone propionate (0.25 – 3.0 mg/day) produced no acceleration of wound healing as measured by WBS on 14- and 21-day wounds. In castrate rats a mild inhibition of healing (15% decrease in WBS) was found in 14-day wounds but no difference was found between castrate and control in 21-day wounds. The rate of wound collagen synthesis was assessed by measuring the conversion of [³H]proline to [³H]hydroxyproline, a process essentially limited to procollagen synthesis. It was not altered by castration, or by administration of testosterone propionate (0.0625 – 1.0 mg/day) to castrate rats. Similarly, deposition of tissue in polyvinyl sponges whether measured as added dry weight or total hydroxyproline did not differ significantly between control and castrate rats receiving testosterone propionate (0–1.0 mg/day). As a method of assessing wound healing, WBS measurements produced the most consistent results. In conclusion, no longterm dependence of wound healing on testosterone was identified in the testosterone-depleted (castrate) rat although some early depression was noted, and no acceleration of the normal process resulted from exogenous testosterone administration in the normal or testosterone-depleted rat.     

Sirolimus impairs wound healing

Background and aims Clinically, the immunosuppressive drug sirolimus, used in organ transplantation, appears to impair wound healing. Little is known about the mechanisms of action. We investigated the effect of sirolimus on wound healing, and we analyzed the expression of stimulating mediators of angiogenesis (VEGF, vascular endothelial growth factor) and collagen synthesis (nitric oxide) in wounds. Materials and methods Groups of ten rats underwent dorsal skin incision, and polyvinyl alcohol sponges were implanted subcutaneously. Beginning at the day of wounding, rats were treated with 0.5, 2.0, or 5.0 mg sirolimus/kg/day. Animals were killed 10 days later to determine wound breaking strength and reparative collagen deposition. Expression of VEGF and nitric oxide was studied in wounds. Results Splenic lymphocyte proliferative activity was significantly decreased by sirolimus (p < 0.05). Sirolimus levels in wound fluid were found to be approximately two- to fivefold higher than blood levels (p < 0.01). Sirolimus (2.0 and 5.0 mg kg⁻¹ day⁻¹) reduced wound breaking strength (p < 0.01) and wound collagen deposition (p < 0.05). This was paralleled by decreased expression of VEGF and nitric oxide in wounds. Conclusion Experimentally, our data show that sirolimus impairs wound healing, and this is reflected by diminished expression of VEGF and nitric oxide in the wound. Best abstracts — Surgical Forum 2007.     

Effect of supplemental ornithine on wound healing

BACKGROUND: Supplemental arginine has been shown to enhance wound healing, in particular collagen synthesis. Ornithine is the main metabolite of arginine in the urea cycle and shares many of the biopharmacologic effects of arginine. The present study examines the effect of ornithine supplementation on wound healing and attempts to describe its possible mechanism. METHODS: Wild type (WT) and iNOS knockout (KO) mice were randomized to receive either normal chow and tap water or chow and water each supplemented with 0.5% ornithine (w/w). All animals underwent a midline dorsal skin incision with implantation of polyvinyl alcohol sponges into subcutaneous pockets. On postoperative day 14 the animals were sacrificed. The dorsal wound was harvested for breaking strength determination while the wound sponges were assayed for hydroxyproline content, total wound fluid amino acid, and nitrite/nitrate (NOx) concentration. RESULTS: Dietary ornithine supplementation enhanced wound breaking strength and collagen deposition in both WT and KO mice. This was accompanied by increased wound fluid proline and ornithine levels but not arginine, citrulline, or NOx levels. CONCLUSIONS: The results from this study demonstrate that ornithine supplementation enhances wound healing in both WT and KO mice. This suggests that ornithine's effect on wound healing is independent of the iNOS pathway.     

Effects of Hibiscus rosa sinensis L (Malvaceae) on wound healing activity: a preclinical study in a Sprague Dawley rat

Hibiscus rosa sinensis (H rosa sinensis), a plant product, has been used for the treatment of a variety of diseases as well as to promote wound healing. The wound-healing activity of the ethanol extract of H rosa sinensis flower was determined in rats, using excision, incision, and dead space wound models and is presented in this report. The animals were randomly divided into 2 groups of 6 each in all the models. Test group animals in each model were treated with the ethanol extract of H rosa sinensis orally by mixing in drinking water (120 mg kg(-1) day(-1)), and the control group animals were maintained with plain drinking water. Healing was assessed by the rate of wound contraction, period of epithelialization, tensile strength (skin breaking strength), granulation tissue weight, and hydroxyproline content. The antimicrobial activity of the flower extract against selected microorganisms that infect the wounds was also assessed. Animals treated with the extract exhibited an 86% reduction in the wound area compared with controls, who exhibited a 75% reduction. The extract-treated animals were found to epithelize their wounds significantly faster than controls (P < .002) and have shown significantly higher skin-breaking strength than controls (P < .002). The dry and wet weight of granulation tissue and hydroxyproline content were also increased significantly when compared with controls. The reported observations suggest H rosa sinensis aids wound healing in the rat model.     

Regulation of nitric oxide synthesis in wounds by IFN-gamma depends on TNF-alpha.

Macrophage-derived nitric oxide is a critical mediator in wound healing. Its regulation in vivo, however, remains unclear. We hypothesized that interferon (IFN)-gamma plays an important role in the regulation of nitric oxide in wounds. Groups of 12 male IFN-gamma -knockout mice and wild-type controls underwent dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. Mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Synthesis of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and IFN-gamma was measured in the wound. Wound-derived macrophages were tested for NO synthesis in the presence or absence of IFN-gamma, TNF-alpha, and anti-TNF-alpha antibody. In a separate experiment, IFN-gamma -knockout mice and wild-type controls were treated with molsidomine, a nitric oxide donor. It was found that wound collagen deposition and wound breaking strength were impaired in IFN-gamma-knockout mice (p < .05). Impaired healing was reflected in diminished synthesis of TNF-alpha and NO in wounds (p < .05). In vivo treatment with molsidomine reversed impaired healing in IFN-gamma-deficient mice. Ex vivo, addition of IFN-gamma stimulated the synthesis of TNF-alpha and NO in wound-derived macrophages. IFN-gamma -induced NO synthesis by wound-derived macrophages was abolished by anti-TNF-alpha-antibody-treatment, which could be fully reversed by exogenous TNF-alpha. Thus we conclude that IFN-gamma-deficiency impairs wound healing and diminishes NO synthesis in wound-derived macrophages. The stimulatory effect of IFN-gamma on macrophage NO production depends on endogenous TNF-alpha synthesis.     

Regulation of Nitric Oxide Synthesis in Wounds by IFN-γ Depends on TNF-α

Macrophage-derived nitric oxide is a critical mediator in wound healing. Its regulation in vivo, however, remains unclear. We hypothesized that interferon (IFN)-γ plays an important role in the regulation of nitric oxide in wounds. Groups of 12 male IFN-γ -knockout mice and wild-type controls underwent dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. Mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Synthesis of nitric oxide (NO), tumor necrosis factor (TNF)-α, and IFN-γ was measured in the wound. Wound-derived macrophages were tested for NO synthesis in the presence or absence of IFN-γ, TNF-α, and anti-TNF-α antibody. In a separate experiment, IFN-γ -knockout mice and wild-type controls were treated with molsidomine, a nitric oxide donor. It was found that wound collagen deposition and wound breaking strength were impaired in IFN-γ-knockout mice (p <. 05). Impaired healing was reflected in diminished synthesis of TNF-α and NO in wounds (p <. 05). In vivo treatment with molsidomine reversed impaired healing in IFN-γ-deficient mice. Ex vivo, addition of IFN-γ stimulated the synthesis of TNF-α and NO in wound-derived macrophages. IFN-γ -induced NO synthesis by wound-derived macrophages was abolished by anti-TNF-α-antibody-treatment, which could be fully reversed by exogenous TNF-α. Thus we conclude that IFN-γ-deficiency impairs wound healing and diminishes NO synthesis in wound-derived macrophages. The stimulatory effect of IFN-γ on macrophage NO production depends on endogenous TNF-α synthesis.     

Evaluation of wound healing activity of ferulic acid in diabetic rats

In diabetic patients, there is impairment in angiogenesis, neovascularisation and failure in matrix metalloproteineases (MMPs), keratinocyte and fibroblast functions, which affects wound healing mechanism. Hence, diabetic patients are more prone to infections and ulcers, which finally result in gangrene. Ferulic acid (FA) is a natural antioxidant found in fruits and vegetables, such as tomatoes, rice bran and sweet corn. In this study, wound healing activity of FA was evaluated in streptozotocin‐induced diabetic rats using excision wound model. FA‐treated wounds were found to epithelise faster as compared with diabetic wound control group. The hydroxyproline and hexosamine content increased significantly when compared with diabetic wound control. FA effectively inhibited the lipid peroxidation and elevated the catalase, superoxide dismutase, glutathione and nitric oxide levels along with the increase in the serum zinc and copper levels probably aiding the wound healing process. Hence, the results indicate that FA significantly promotes wound healing in diabetic rats.     

Single local instillation of Staphylococcus aureus peptidoglycan prevents diabetes-induced impaired wound healing

Diabetes-induced impaired wound healing is characterized by inhibition of the inflammatory response to wounding, macrophage infiltration, angiogenesis, fibroplasia, reparative collagen accumulation, and wound breaking strength. Because all of these processes are accelerated in normal rats by a single local application at operation of Staphylococcus aureus peptidoglycan, we hypothesized that S. aureus peptidoglycan would prevent diabetes-induced impaired wound healing, despite persistent, untreated hyperglycemia, polydipsia, glycosuria, and polyuria. Sprague-Dawley male rats were divided into two groups. One group received an intraperitoneal injection of streptozotocin (65 mg/kg) in citrate solution; the other group received an intraperitoneal injection of an equivalent volume of citrate solution. Seventeen days after the injections, the diabetic and control rats received aseptically two 5.5-cm paravertebral incisions and subcutaneous implantation of six polyvinyl alcohol sponges, three on each side. On one side, each sponge contained 0.5 mg S. aureus peptidoglycan in 50 µl saline solution, and the incision was inoculated along its length with 4.7 mg S. aureus peptidoglycan in 157 µl saline solution (860 µg/S. aureus peptidoglycan/cm incision); on the other side, the same respective volumes of saline were used. During the preoperative and postoperative periods, diabetic rats lost a small amount of weight (2%), were hyperglycemic (363 ± 10 mg/100 ml blood), polydipsic, glycosuric, and polyuric, whereas the controls gained weight (25%) and were normoglycemic (104 ± 5 mg/100 ml blood); these differences were significantly different (p < .001 in each case). In controls, S. aureus peptidoglycan inoculation increased wound breaking strength (by a factor of 2.0) and hydroxyproline content (by a factor of 1.4; p < .001 in each case); in diabetics, there were significant decreases in wound breaking strength (by a factor of 1.7) and hydroxyproline content (by a factor of 1.3) of saline solution-inoculated incisions and sponges compared with the wound breaking strength and hydroxyproline content of saline solution-inoculated incisions and sponges in controls (p < .02 and p < .001, respectively). These decreases were completely prevented when the incisions and polyvinyl alcohol sponges had been inoculated at operation with S. aureus peptidoglycan; S. aureus peptidoglycan inoculation in the diabetic rats increased wound breaking strength by a factor of 2.2 and sponge reparative tissue hydroxyproline by a factor of 1.6 (p < .001 in each case). Thus, diabetes-induced impaired wound healing was prevented completely by a single local instillation at operation of S. aureus peptidoglycan, despite persistent, untreated hyperglycemia, polydipsia, polyuria, and glycosuria.     

Interleukin 2 enhances wound healing in rats

Antigen-stimulated lymphocytes secrete lymphokines which have been shown to enhance in vitro fibroblast migration, proliferation, and protein synthesis. In the present experiments, the effect of human recombinant interleukin 2 (RIL-2) on wound healing was assessed in vivo. Groups of male Lewis rats, 225-250 g, underwent intraperitoneal insertion of osmotic pumps and a 7-cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges under anesthesia. The dorsal wounds were closed with stainless-steel sutures. The dose of RIL-2 administered was 60,000 u/rat/day for 7 days in experiment I, and 140,000 u/rat/day for 7 days in experiment II. Controls received equal volumes of excipient. Animals were sacrificed 10 days post wounding and wound healing was assessed by fresh breaking strength, fixed breaking strength (following 72 hr of Formalin fixation which maximally crosslinks the collagen present), and sponge hydroxyproline content (an index of reparative collagen accumulation). In vivo RIL-2 administration significantly augmented wound fresh and fixed breaking strength and wound collagen synthesis. Higher doses of RIL-2 (experiment II) did not result in further increases in the parameters studied. The data suggest that lymphocytes participate directly in the process of wound healing.     

Enhancement of wound repair with a topically applied nitric oxide-releasing polymer

Nitric oxide is an important cytotoxic agent for host defense which also regulates gene expression, signal transduction, and vasodilation. In normal wounds, nitric oxide synthesis and metabolism are significantly increased during inflammation and tissue remodeling. However, nitric oxide production is suppressed in wounds where healing is impaired by diabetes or steroid-treatment. Topical delivery of nitric oxide in therapeutic amounts may alleviate this deficiency and thereby enhance wound repair. Consequently, we developed polyethyleneimine cellulose NONOate polymer, a nonsoluble, nontoxic, polymer-based NONOate--one of a new class of compounds that spontaneously release nitric oxide in a controlled fashion in aqueous media. Polyethyleneimine cellulose NONOate polymer was synthesized from polyethyleneimine cellulose to provide extended nitric oxide release with a half-life of 16 hours. Polyethyleneimine cellulose NONOate polymer or a control polymer was applied topically on full-thickness dermal wounds of rats at the time of wounding and days 3, 7, 10, 14, 17, and 21. Nitric oxide delivery was determined indirectly by measuring urinary nitrate. The first two polyethyleneimine cellulose NONOate polymer applications increased urinary nitrate output twofold to fourfold, whereas urinary nitrate output of control rats did not significantly increase. Nitrate output in polyethyleneimine cellulose NONOate polymer-treated rats was elevated compared with controls after each application, although this was attenuated in later applications. Rate of wound closure was measured with computer-based video imaging. Polyethyleneimine cellulose NONOate polymer-treated wounds were significantly smaller (p < 0.05) on days 7, 10, and 17 relative to controls, based on percentage of wound open relative to initial wound area. In a second experiment, telemetry-implanted rats were wounded to detect potential hypotensive effects as a result of polyethyleneimine cellulose NONOate polymer application. Topical polyethyleneimine cellulose NONOate polymer application to wounds showed no prolonged hypotensive effects, in contrast to a soluble NONOate which suppressed systolic blood pressure for over 6 hours. These results show that a nonsoluble, polymeric NONOate can provide topical nitric oxide delivery to wounds in a controlled manner, which may enhance wound healing. Further studies are in progress with other promising NONOate candidates to establish dose-response effects and therapeutic limits of exogenous nitric oxide release in impaired wound models.     

Wound healing in nude mice: a study on the regulatory role of lymphocytes in fibroplasia

In order to understand the role of T cells in postinjury fibroplasia, we have studied wound healing in congenitally athymic nude mice that lack a normally developed T cell system. Healing of incisional wounds, as assessed by wound breaking strength, was significantly stronger in nude mice compared with normal thymus-bearing animals. This was accompanied by a marked increase in the amount of reparative collagen synthesized at the wound site, as assessed by the hydroxyproline content of subcutaneously implanted sponges. Because nude mice have some extrathymic T cell maturation, we used an anti-Thy-1.2 (30H12) monoclonal antibody to selectively deplete T cells in vivo. Although such treatments impaired wound healing in normal mice, they had no effect on any wound healing parameter in nude mice. In a separate experiment, T cell reconstitution of nude mice, sufficient to significantly enhance in vivo delayed hypersensitivity responses, led to a decrease in both wound breaking strength and hydroxyproline deposition in subcutaneously implanted polyvinyl sponges. The data suggest that T cells play a dual role in wound healing: an early stimulatory role on macrophages, endothelial cells, and fibroblasts, and a late counterregulatory role, which may be responsible for the orderly completion of wound repair.     

L-arginine improves wound healing after trauma-hemorrhage by increasing collagen synthesis

BACKGROUND: Several studies indicate impaired wound healing after trauma and shock. Wound immune cell dysfunction seems to be responsible for altered wound healing after trauma-hemorrhage (T-H). In this respect, administration of the amino acid L-arginine normalized wound immune cell function under those conditions. It remains unknown, however, whether L-arginine improves impaired wound healing after T-H. METHODS: To study this, male C3H/HeN mice were subjected to a midline laparotomy (i.e., soft tissue trauma induced), and polyvinyl sponges were implanted subcutaneously at the wound site before hemorrhage (35 +/- 5 mm Hg for 90 minutes) or were subjected to sham operation. During resuscitation, mice received 300 mg/kg body weight L-arginine or saline (vehicle). Seven days thereafter, hydroxyproline (OHP), a metabolite of collagen synthesis, was measured in the wound fluid using high-performance liquid chromatography. Collagen types I and III were determined in the wound by Western blot analysis. In addition, wound breaking strength was measured 10 days after T-H or sham operation. RESULTS: The results indicate that OHP was significantly decreased in T-H mice. L-arginine, however, restored depressed OHP in the wound fluid in the T-H animals. Similarly, L-arginine treatment prevented a significant depression of collagen I synthesis after T-H. Collagen III was not significantly affected by T-H or L-arginine. Most important, L-arginine increased maximal wound breaking strength after severe blood loss. Therefore, L-arginine improves wound healing after T-H by increasing collagen synthesis. CONCLUSION: Because L-arginine improves wound healing, the results suggest that L-arginine might represent a novel and useful adjunct to fluid resuscitation for decreasing wound complications after trauma and severe blood loss.