A Novel Model of Chronic Wounds: Importance of Redox Imbalance and Biofilm-Forming Bacteria for Establishment of Chronicity

<div><p>Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109848#pone.0109848-Sen1" target="_blank">[1]</a>. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that <i>chronic wounds</i> can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.</p></div

Histological evaluation of chronic wounds.

<p>(A) Representative picture of H&E-stained sections of a LIGHT^−/− chronic wounds from an animal treated with catalase and GPx inhibitors and the application of bacteria. The epithelium does not cover the wound tissue and the granulation tissue is poorly formed. Scale bar 500 µm. (B) Higher magnification of the boxed area in (A). Epithelial tongue is outlined with a dotted line (compare with Figure S4A). Scale bar 100 µm. (C) Immunolabeling for Collagen IV delineates the presence of basement membrane; dotted line marks where basement membrane is missing in the migrating tongue. (D) propidium iodide staining identifies cell nuclei. (E) Merger of (C) & (D). (F) Immunolabeling for F4/80, a marker for macrophages, to illustrate the presence of inflammation; (G) propidium iodide staining identifies cell nuclei. (H) Merger of (F) & (G). Inserts are high magnifications of a single macrophage. (I) Representative Masson-trichrome (blue color) stained section illustrating loss of collagen bundles; scale bar 100 µm. (J,K) SHIM analysis of a similar section (J) confirms results in (I) and, for comparison, collagen in the granulation tissue of a normal wound similarly analyzed by SHIM (K) showing filamentous collagen (red arrow); scale bar 10 µm.</p

Manipulating redox parameters leads to development of chronic wounds.

<p>(A) C57BL/6 and LIGHT^−/− mice were wounded and immediately treated with inhibitors for GPx and catalase followed by the application of biofilm-forming bacteria 24 hrs later. The wounds were covered with sterile tegaderm to maintain a moist wound environment and prevent external infection. The LIGHT^−/− wounds became chronic and remained open for more than 30 days. <i>n = 30</i>. (B) Wound areas were traced using ImageJ and % open wound area was calculated. The LIGHT^−/− wounds remained open for significantly longer time than the C57BL/6 wounds with similar treatment. <i>n = 8</i>. (C-F) SOD activity (C); H₂O₂ levels (D); Catalase activity (E); and GPx Activity (F) were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109848#pone-0109848-g001" target="_blank">Figure 1</a>. All were greatly different from controls. For all tests <i>n = 6</i> at minimum. <i>Time zero in C-F represents unwounded skin. All data are Mean ± SD. *p<0.05,**p<0.01,***p<0.001.</i></p

Morphological characterization of biofilm present in LIGHT^−/− wounds.

<p>Scanning electron microscopy (SEM) images of the Au/Pd sputtered, fixed and dried, chronic wound samples were captured using an FEI XL30 FEG SEM. (A) Image shows the presence of bacterial rods (b) in the wound bed. (B) High magnification image of bacteria embedded in a biofilm-associated matrix (m) in a well-defined niche (n). (C) Matrix beneath the biofilm showing the presence of matrix (m) and of cocci bacteria (b). A Lymphocyte (L/arrow) was highlighted for size references. Scale bars 5 µm (A,C) and 1 µm (B).</p

Identification and characterization of the microflora that colonizes the LIGHT^−/− chronic wounds.

<p>(A) Biofilm production was quantified by measuring the optical densities of stained bacterial films adherent to plastic tissue culture plates. Biofilm forming capacity of <i>S. epidermidis</i> was seen throughout the time course of chronic wounds. <i>n = 7</i>. (B) Bacterial identification was carried out by growing bacteria on tryptic soy agar. Gram-negative rods were characterized using the API 20E identification kit. <i>n = 7</i>. (C) Biofilm quantification of exudate obtained from wounds was performed at OD570 nm. The dynamics of the polymicrobial community in the wounds does not seem to affect the overall degree of biofilm production during the later stages of healing. Controls used were biofilm-negative (OD570 nm<0.125) <i>S. hominis</i> SP2 and biofilm-positive <i>S. epidermidis</i> C2. <i>n = 8</i>. (D) Antibiotic challenge on wound exudates collected from LIGHT^−/− mice was done using Amoxicillin. The CMIC of amoxicillin on the bacteria found in the chronic LIGHT^−/− wound exudate at day 22/24 was 50 µg/ml, much higher than exudate collected at day 5 when biofilm is not yet abundant. (E) Bacterial burden was evaluated by colony forming unit counts. The CFU/mL was relatively low during the early phases of healing and was highest during the impaired and chronic stages of healing. <i>n = 7</i>. (F) Normal skin swabs were collected from LIGHT and C57BL/6 mice to evaluate resident organisms. The microbiota of the skin was similar in both C57BL/6 and LIGHT^−/− mice.</p

Early oxidative and nitrosative stress in LIGHT^−/− wounds have damaging effects on proteins, lipids and DNA and increased cell death.

<p>(A) Protein modification measurements were based on a competitive enzyme immunoassay; nitrotyrosine levels in the LIGHT^−/− mice were significantly different from control throughout healing. (B) Lipid peroxidation levels were measured fluorometrically at an Ex/Em of 540 nm/590 nm using thiobarbituric acid reactive substances (TBARS); the MDA levels were significantly elevated throughout the course of wound healing in LIGHT^−/− mice. <i>n = 6</i>. (C, D) F₂ isoprostanes, were measured using the approach described in the M&M section; levels of 8- and 5-isoprostanes detected in LIGHT^−/− mice were much higher than those in the control mice at early times. This correlates with the MDA levels that are the stable byproducts of lipid peroxidation. <i>n = 5</i>. (E) Levels of 8-OH-dG, were based on a competitive enzyme immunoassay; the samples were read spectrophotometrically at 412 nm using Ellman's reagent. 8-OH-dG levels were found to be significantly elevated during the course of healing in LIGHT^−/− mice. <i>n = 4</i>. (F) Cell death by apoptosis and necrosis was determined by staining with Annexin V-FITC and propidium iodide, respectively, followed by FACS analysis. Cell death was increased significantly in the LIGHT^−/− mice. The greatest difference occurred with necrosis, which showed to be much higher in LIGHT^−/− mice. <i>Time zero represents unwounded skin. All data are Mean ± SD</i>. <i>*p<0.05,**p<0.01,***p<0.001.</i></p

Oxidative stress is elevated in LIGHT^−/− wounds.

<p>(A) SOD activity was measured using a tetrazolium salt that converts into a formazan dye detectable at 450 nm. SOD activity remains significantly elevated in LIGHT^−/− mice in the first 48 hrs post-wounding. <i>n = 6</i>. (B) Resofurin formation, detected at 590 nm, was used to determine H₂O₂ levels. Significant increases in H₂O₂ very shortly post-wounding were seen. <i>n = 8</i>. (C) Enzymatic reaction of catalase and methanol in the presence of H₂O₂ gives rise to formaldehyde, spectrophotometrically detected with purpald chromogen, at 540 nm. Catalase activity in adult LIGHT^−/− and control wounds was similar. <i>n = 6</i>. (D) GPx detoxifying activity was measured indirectly at 340 nm by a coupled reaction with glutathione reductase where GPx activity was rate-limiting. The level of GPx activity in the adult LIGHT^−/− wounds was essentially identical to that of the controls. <i>n = 6</i>. (E-H) The findings in old LIGHT^−/− mice were exacerbated in all four parameters when compared to adult LIGHT^−/− mice. <i>n = 6. Time zero represents unwounded skin. All data are Mean ± SD. *p<0.05,**p<0.01,***p<0.001.</i></p

A Randomized Clinical Trial Testing the Anti-Inflammatory Effects of Preemptive Inhaled Nitric Oxide in Human Liver Transplantation

<div><p>Decreases in endothelial nitric oxide synthase derived nitric oxide (NO) production during liver transplantation promotes injury. We hypothesized that preemptive inhaled NO (iNO) would improve allograft function (primary) and reduce complications post-transplantation (secondary). Patients at two university centers (Center A and B) were randomized to receive placebo (n = 20/center) or iNO (80 ppm, n = 20/center) during the operative phase of liver transplantation. Data were analyzed at set intervals for up to 9-months post-transplantation and compared between groups. Patient characteristics and outcomes were examined with the Mann-Whitney U test, Student t-test, logistic regression, repeated measures ANOVA, and Cox proportional hazards models. Combined and site stratified analyses were performed. MELD scores were significantly higher at Center B (22.5 vs. 19.5, p<0.0001), surgical times were greater at Center B (7.7 vs. 4.5 hrs, p<0.001) and warm ischemia times were greater at Center B (95.4 vs. 69.7 min, p<0.0001). No adverse metabolic or hematologic effects from iNO occurred. iNO enhanced allograft function indexed by liver function tests (Center B, p<0.05; and p<0.03 for ALT with center data combined) and reduced complications at 9-months (Center A and B, p = 0.0062, OR = 0.15, 95% CI (0.04, 0.59)). ICU (p = 0.47) and hospital length of stay (p = 0.49) were not decreased. iNO increased concentrations of nitrate (p<0.001), nitrite (p<0.001) and nitrosylhemoglobin (p<0.001), with nitrite being postulated as a protective mechanism. Mean costs of iNO were $1,020 per transplant. iNO was safe and improved allograft function at one center and trended toward improving allograft function at the other. ClinicalTrials.gov with registry number 00582010 and the following URL:<a href="http://clinicaltrials.gov/show/NCT00582010" target="_blank">http://clinicaltrials.gov/show/NCT00582010</a>.</p></div

Effects of iNO on reperfusion induced injury and PMN accumulation.

<p>Liver biopsy samples were collected pre- (LB1, □) and 1 hr post dual reperfusion (LB2, ▪) and assessed for injury by histopathologic evaluation (Panel A, B) and infiltration of PMN (panel C, D). Data from both UAB and UW cohorts are combined (n = 38). P-values indicated on graph are by paired t-test for panels A and C or by * unpaired t-test (panels B and D).</p

The Distribution, Frequency and Type of Hepatobiliary Complications.

<p>Center A.</p><p>Placebo: allograft dysfunction (1), primary graft non-function (1), reduced portal vein flow (1), hepatic artery bleeding (2).</p><p>iNO: allograft dysfunction (1), hepatic artery bleeding (1).</p><p>Center B.</p><p>Placebo: 1-month:primary graft dysfunction (1), reduced hepatic artery blood flow (1), biliary leak (1), 6-months:biliary stricture (2), 9-months:rejection (1), hepatic artery stenosis (1), death (1).</p><p>iNO: 9-months:hepatic artery stenosis (1), death (1).</p