Biomedical subjects
Adrian Barbul
Publications and source records attributed to Adrian Barbul.
Nutrition and wound healing.
The relationship between host nutrition and wound healing has been the subject of study and experimentation for centuries. Despite the many years of study and a substantial knowledge base of the specific processes and factors involved, wound healing remains enigmatic. There is still much to learn about the wound-specific nutritional interventions that are available to improve wound healing. Nutrition profoundly influences the process of wound healing. Nutritional depletion exerts an inhibitory effect, and nutritional supplementation with such positive effectors as arginine can stimulate wound healing. Within this paradigm, the physician should be able to recognize patients who may be expected to have wound healing difficulties and offer early intervention to avoid wound failure.
Vessel injury and capillary leak.
OBJECTIVE: To understand the mechanism of pathologic capillary leak in the critically ill patient. DESIGN: Review of normal and altered physiology of the microvasculature. Review of recent literature describing pathogenesis, mediators, and interventions influencing capillary leak and microvascular repair. SETTING: In vitro and in vivo studies, the latter including animal and human subjects. MEASUREMENTS AND MAIN RESULTS: Capillary leak with resultant edema develops in the critical care setting on the basis of perturbations in Starling's equation, primarily as a result of increased capillary permeability to larger molecules. This process is most likely fueled by inflammatory mediators or mechanical stress. Attempts to prevent or treat this process remain largely unsuccessful; resuscitation is more often symptomatic than therapeutic. Models of microvascular repair focus on discrete injury and may not be applicable to the recovery of capillary damage secondary to a systemic leak CONCLUSIONS: Our understanding of capillary leak syndrome remains fragmented and weighted toward specific mediators contributing to the leak. The implications of extensive edema and the mechanism by which it resolves continue to be the subject of speculation rather than study.
Repair of full-thickness bowel injury.
OBJECTIVE: Colon surgery is more and more often performed in complex situations such as after trauma, under immunosuppression, or in the elderly. Even under optimal conditions, anastomosis fails in certain situations. The objective of this study was to demonstrate the normal phases of bowel healing and to review the local and systemic factors affecting healing with special attention to critical care variables such as major surgery, acute hemorrhage, and infections. DATA SOURCE: MEDLINE cited and/or published articles. DESIGN: Review analysis. RESULTS: Colon healing is a structured cascade of different phases that can be affected by a multitude of local (infection, ischemia) and systemic (diabetes, malnutrition, anemia, hypothermia, trauma) factors. The normal phases of repair, the resulting bursting pressure as an experimental index of healing, and the available published data on local and systemic factors affecting healing are summarized. CONCLUSION: Several local and systemic factors negatively affect bowel healing; there is still a small portion of patients who fail to heal, suggesting that intrinsic factors need to be analyzed.
Upregulation of arginase expression in wound-derived fibroblasts.
BACKGROUND: Wound-derived fibroblasts (WFBs) are phenotypically different from normal dermal fibroblasts (NFBs). We have previously shown that the wound phenotype correlates with expression of the inducible isoform of nitric oxide synthase (iNOS) in fibroblasts. l-Arginine is the sole substrate for iNOS. Arginase is an alternative pathway of l-arginine metabolism in wounds. To clarify the role of l-arginine in wound healing, we investigated arginase expression and activity in WFB. METHODS: Male Lewis rats underwent dorsal skin incisions and subcutaneous PVA sponge implantation. WFBs were harvested from sponges retrieved at different days postimplantation. Normal fibroblasts were obtained from uninjured skin by an explant technique. Arginase activity was measured by newly formed urea (nmol/min/mg protein) and protein expression was detected by Western blotting using specific antibodies for type I (AI) and type II (AII). The effect of transforming growth factor beta1 (TGF-beta1), interleukin-4 (IL-4), lipopolysaccharide, and wound fluid on arginase activity was also investigated. RESULTS: WFB arginase activity was significantly elevated compared with NFB activity at all times postwounding. This was paralleled by increased AI protein expression by Western blotting. AII was not detectable. TGF-beta and IL-4 significantly increased arginase activity and protein expression whereas lipopolysaccharide and wound fluid did not affect it. CONCLUSIONS: The upregulation of the arginase expression in WFB underlines the distinct regulation of l-arginine metabolism in WFBs. Further work is needed to elucidate the functional implications.
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.
Role of nitric oxide in wound repair.
After injury, wound healing is essential for recovery of the integrity of the body. It is a complex, sequential cascade of events. Nitric oxide (NO) is a small radical, formed from the amino acid L-arginine by three distinct isoforms of nitric oxide synthase. The inducible isoform (iNOS) is synthesized in the early phase of wound healing by inflammatory cells, mainly macrophages. However many cells participate in NO synthesis during the proliferative phase after wounding. NO released through iNOS regulates collagen formation, cell proliferation and wound contraction in distinct ways in animal models of wound healing. Although iNOS gene deletion delays, and arginine and NO administration improve healing, the exact mechanisms of action of NO on wound healing parameters are still unknown. The current review summarizes what is known about the role of NO in wound healing and points out path for further research.
L-Arginine supplementation enhances diabetic wound healing: involvement of the nitric oxide synthase and arginase pathways.
Diabetes impairs wound healing and there are few therapeutic options to reverse it. Previous work has demonstrated the importance of nitric oxide for successful wound healing. In diabetes, NO synthesis is reduced in the wound milieu. The amino acid L-arginine is the only substrate for NO synthesis. We hypothesized that L-arginine supplementation would enhance wound healing by restoring NO synthesis. Thirty-six male Sprague-Dawley rats (body weight, 225 to 250 g) were separated in 4 groups: 20 rats were rendered diabetic 7 days prior to wounding by intraperitoneal streptozotocin (STZ) injection (70 mg/kg). Sixteen rats served as controls. Half of the animals of each group received 1 g/kg supplemental L-arginine administered by gavage twice daily. Control rats were gavaged with water. Treatment was started 3 days before wounding. All rats underwent a dorsal skin incision and subcutaneous implantation of polyvinyl alcohol (PVA) sponges. The rats were killed 10 days post wounding and wound breaking strength, hydroxyproline content of the sponges, nitrite/nitrate (NO(x)) concentration, arginase activity, and amino acid composition of the wound fluid and plasma were analyzed. Wound fluid NO(x) concentrations and wound breaking strength were significantly reduced in the diabetic group compared to the controls. L-Arginine treatment restored diabetic NO(x) levels toward normal values and significantly enhanced wound breaking strength. Wound fluid arginase activity and ornithine concentrations were significantly lower in the diabetic animals but unaffected by treatment. The data demonstrate that the impaired NO synthesis in the diabetic wound milieu can at least partially be reversed by arginine supplementation. In view of previous results on the importance of NO for wound healing, the data suggest that arginine supplementation restores impaired healing in this acute wound model by normalizing the NO pathway but without affecting arginase activity.
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.
Effect of a specialized amino acid mixture on human collagen deposition.
OBJECTIVE: To examine the effect of arginine, beta-hydroxy-beta-methylbutyrate (HMB), and glutamine supplementation on wound collagen accumulation in a double-blind, randomized study. SUMMARY BACKGROUND DATA: Control of wound collagen synthesis has been an elusive goal for clinicians and scientists alike. In many clinical instances, it is desired to increase collagen deposition as a means of enhancing wound strength and integrity. Arginine, a semiessential amino acid, has been shown to increase wound collagen accumulation in rodents and humans. HMB, a metabolite of leucine, regulates muscle proteolysis in animals and humans and increases collagen deposition in rodents. METHODS: Thirty-five healthy, nonsmoking human volunteers 70 years or older were enrolled and underwent subcutaneous implantation of two small, sterile polytetrafluoroethylene (PTFE) tubes into the deltoid region under strict aseptic techniques. The tubes were 1 mm in diameter and 6 cm in length with pore size of 90 to 120 microm to allow optimal ingrowth of fibroblasts and the deposition of matrix. Eighteen volunteers (mean age 75.4 years; 2 men, 16 women) were randomized to receive daily supplementation of 14 g arginine, 3 g HMB, and 14 g glutamine (total nitrogen 3.59 g) in two divided doses. The control group (n = 17; mean age 75.3 years; 6 men, 11 women) received an isonitrogenous, isocaloric supplementation of nonessential amino acids. Catheters were removed at 7 and 14 days postimplantation and analyzed for hydroxyproline (OHP, nmol/cm catheter, an index of collagen accumulation) and alpha-amino nitrogen (alpha-AN, mmol/cm, an index of total protein deposition). RESULTS: Supplements were well tolerated, without any reported side effects. Supplementation with the specialized amino acid mixture led to a significant rise in plasma arginine and ornithine levels. The specialized amino acid supplement led to a significant increase in collagen deposition (as reflected by OHP content) in the PTFE tubes without an effect on total protein accumulation. CONCLUSIONS: Collagen synthesis is significantly enhanced in healthy elderly volunteers by the oral administration of a mixture of arginine, HMB, and glutamine. This provides a safe nutritional means for increasing wound repair in patients.
Modulation of growth factor and cytokine expression by nitric oxide during rat colon anastomotic healing.
We have previously shown that inhibition of nitric oxide generated by inducible nitric oxide synthase (iNOS) results in impaired colon anastomotic healing. Therefore, we proceeded to assess whether disruption of iNOS activity alters the normal pattern of growth factor expression during anastomotic healing. Two groups of male Sprague-Dawley rats underwent distal colonic division and anastomosis, jugular venous catheterization and subcutaneous placement of polyvinyl alcohol sponges. The first group (n = 10) received q8 hour intravenous injections of 10 mg/kg L-N-iminoethyl-lysine (L-NIL, a selective inhibitor of iNOS), while the second group (n = 12) received equal volumes of saline. On postoperative day 5, animals were sacrificed and anastomotic bursting pressure was determined. Histologic sections of the anastomosis were subjected to in situ hybridization versus mRNA of the proteins listed below. Positive controls were reacted with a poly-thymidine (poly-T) probe versus ubiquitous mRNA poly-adenine tails. Positively stained cells were quantified using a calibrated optical grid encompassing 0.5 mm(2) area centered over the anastomosis. Results are reported as the number of positive cells per 1000 cells positive for poly-T. L-NIL treated animals demonstrated an 18% decrease in wound fluid NO(X) compared to controls (29.2 +/- 1.2 vs. 34.6 +/- 2.0 microM, mean +/- SEM; P = 0.035). This corresponded to a 17% decrease in anastomotic bursting pressure (153 +/- 4 vs. 182 +/- 8 mm Hg, mean +/- SEM; P < 0.05). L-NIL also markedly increased the number of cells expressing transforming growth factor-beta, tumor necrosis factor-alpha, vascular endothelial growth factor, and both inducible and endothelial forms of nitric oxide synthase. L-NIL had no effect on the expression of basic fibroblast growth factor. The data demonstrate that iNOS inhibition markedly disrupts the profile of cytokine and growth factor mRNA normally expressed during anastomotic healing. This provides in vivo evidence that NO modulates gene expression during anastomotic healing.
Tacrolimus enhances colon anastomotic healing in rats.
Tacrolimus inhibits T-cell function and neutrophil chemotaxis during inflammation. We hypothesized that tacrolimus would enhance healing of a rat colon anastomosis by reducing the inflammatory response. Fifty-five male Sprague Dawley rats, 230-260 g body weight, underwent identical surgical manipulation consisting of a single-layer, inverted colon anastomosis and the implantation of osmotic pumps subcutaneously in the left flank area. The animals were randomly assigned to receive tacrolimus, at a dose of 0.01, 0.1, or 1.0 mg/kg/day, or only the control solvent solution. The animals were euthanized 4 days after surgery. Colon-bursting pressure (mmHg), anastomotic collagen content ( micro g hydroxyproline/mg wet tissue), and anastomotic type IV collagenase activity (mU/mg protein) were measured. Tacrolimus significantly increased colon-bursting pressure at all doses used (146 +/- 9, 158 +/- 10, 151 +/- 6 mmHg; 0.01, 0.1, and 1.0 mg/kg/day, respectively) vs. control (119 +/- 7 mmHg, p < 0.01). There was no effect on collagen accumulation except at a dose of 0.01 mg/kg/day, which significantly decreased anastomotic collagen content (p < 0.05). Tacrolimus at a dose of 0.01 mg/kg/day increased anastomotic collagenase activity, which was not changed by treatment with the higher doses. Microscopic examination revealed the preservation of the multilayered structure, including the mucosal muscle, a thickened submucosa, and the proper muscle of the anastomotic site in the tacrolimus-treated groups. These data suggest that tacrolimus enhances wound strength during acute anastomotic healing despite a reduction in collagen content.
Supplemental L-arginine enhances wound healing in diabetic rats.
L-arginine has been shown to enhance wound strength and collagen deposition in rodents and humans. Diabetes mellitus, which impairs wound healing, is accompanied by a reduction in nitric oxide at the wound site. The amino acid L-arginine is the only substrate for nitric oxide synthesis. We sought to determine whether supplemental L-arginine can restore the impaired wound healing of diabetic rats. Fifty-six male Lewis rats were used in this study, of which twenty-nine rats were rendered diabetic 7 days prior to surgery with intraperitoneal streptozotocin. Twenty-seven untreated rats served as controls. Animals underwent a dorsal skin incision with implantation of polyvinyl-alcohol sponges. Sixteen diabetic and 14 normal rats received 1 g/kg/day of L-arginine by injection, while the remainder received saline injections only. Animals were euthanized 10 days postwounding, and their wounds were analyzed for breaking strength. The wound sponges were assayed for total hydroxyproline and nitrite/nitrate content. Plasma and wound fluid concentrations of L-arginine, ornithine, and citrulline were determined. Wound sponge RNA was extracted and subjected to Northern blot analysis for procollagen I and III. Diabetic wounds had greatly decreased breaking strengths compared with controls. L-arginine significantly enhanced wound breaking strengths in both control (+23%) and diabetic animals (+44%), and also increased wound hydroxyproline levels in both diabetic (+40%) and control animals (+24%) as compared to their saline-treated counterparts. mRNA for procollagen I and III were elevated by L-arginine treatment in both diabetic rats and controls. Treatment with L-arginine significantly increased wound fluid nitrite/nitrate levels in diabetic animals. The data show that the impaired healing of diabetic wounds can be partially corrected by L-arginine supplementation, and that this effect is accompanied by enhanced wound nitric oxide synthesis.