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A novel model of chronic wounds: importance of redox imbalance and biofilm-forming bacteria for establishment of chronicity.

Dhall S, Do D, Garcia M, Wijesinghe DS, Brandon A, Kim J, Sanchez A, Lyubovitsky J, Gallagher S, Nothnagel EA, Chalfant CE, Patel RP, Schiller N, Martins-Green M - PLoS ONE (2014)

Bottom Line: 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 chronic wounds 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell Biology and Neuroscience, University of California Riverside, Riverside, California, United States of America; Bioengineering Interdepartmental Graduate Program, University of California Riverside, Riverside, California, United States of America.

ABSTRACT
Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone. 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 chronic wounds 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.

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Histological evaluation of chronic wounds.(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.
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pone-0109848-g005: Histological evaluation of chronic wounds.(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.

Mentions: Histological examination of chronic LIGHT−/− wounds showed that the migrating tongue of the epidermis was blunted and tortuous (Figure 5A,B) rather than thin and linear as in the control (Figure S4A in File S1). Also, the granulation tissue was poorly developed (Figure 5A) when compared to normal granulation tissue (Figure S4B,C in File S1). Collagen IV, a component of the basal lamina, was well-formed behind the migrating tongue but was absent under the tortuous migrating edge (Figure 5C-E). We also found that these wounds contain macrophages, indicating that inflammation has not been resolved (Figure 5F,H; inserts show higher magnification of one macrophage). Furthermore, the interstitial collagen deposition and organization were abnormal in the LIGHT−/− chronic wounds as revealed by Masson trichrome staining (Figure 5I) and by second harmonic generation imaging microscopy (SHIM) (Figure 5J,K). Although interstitial collagen was present, the collagen fibers were not clearly visible and did not form proper bundles (Figure 5J). This is similar to the finding we published on the impaired wounds of LIGHT−/− wounds [21] but much more exaggerated.


A novel model of chronic wounds: importance of redox imbalance and biofilm-forming bacteria for establishment of chronicity.

Dhall S, Do D, Garcia M, Wijesinghe DS, Brandon A, Kim J, Sanchez A, Lyubovitsky J, Gallagher S, Nothnagel EA, Chalfant CE, Patel RP, Schiller N, Martins-Green M - PLoS ONE (2014)

Histological evaluation of chronic wounds.(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.
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pone-0109848-g005: Histological evaluation of chronic wounds.(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.
Mentions: Histological examination of chronic LIGHT−/− wounds showed that the migrating tongue of the epidermis was blunted and tortuous (Figure 5A,B) rather than thin and linear as in the control (Figure S4A in File S1). Also, the granulation tissue was poorly developed (Figure 5A) when compared to normal granulation tissue (Figure S4B,C in File S1). Collagen IV, a component of the basal lamina, was well-formed behind the migrating tongue but was absent under the tortuous migrating edge (Figure 5C-E). We also found that these wounds contain macrophages, indicating that inflammation has not been resolved (Figure 5F,H; inserts show higher magnification of one macrophage). Furthermore, the interstitial collagen deposition and organization were abnormal in the LIGHT−/− chronic wounds as revealed by Masson trichrome staining (Figure 5I) and by second harmonic generation imaging microscopy (SHIM) (Figure 5J,K). Although interstitial collagen was present, the collagen fibers were not clearly visible and did not form proper bundles (Figure 5J). This is similar to the finding we published on the impaired wounds of LIGHT−/− wounds [21] but much more exaggerated.

Bottom Line: 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 chronic wounds 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell Biology and Neuroscience, University of California Riverside, Riverside, California, United States of America; Bioengineering Interdepartmental Graduate Program, University of California Riverside, Riverside, California, United States of America.

ABSTRACT
Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone. 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 chronic wounds 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.

Show MeSH
Related in: MedlinePlus