The alpha-1A adrenergic receptor regulates mitochondrial oxidative metabolism in the mouse heart.

AIMS: The sympathetic nervous system regulates numerous critical aspects of mitochondrial function in the heart through activation of adrenergic receptors (ARs) on cardiomyocytes. Mounting evidence suggests that α1-ARs, particularly the α1A subtype, are cardioprotective and may mitigate the deleterious effects of chronic ß-AR activation by shared ligands. The mechanisms underlying these adaptive effects remain unclear. Here, we tested the hypothesis that α1A-ARs adaptively regulate cardiomyocyte oxidative metabolism in both the uninjured and infarcted heart. METHODS: We used high resolution respirometry, fatty acid oxidation (FAO) enzyme assays, substrate-specific electron transport chain (ETC) enzyme assays, transmission electron microscopy (TEM) and proteomics to characterize mitochondrial function comprehensively in the uninjured hearts of wild type and α1A-AR knockout mice and defined the effects of chronic ß-AR activation and myocardial infarction on selected mitochondrial functions. RESULTS: We found that isolated cardiac mitochondria from α1A-KO mice had deficits in fatty acid-dependent respiration, FAO, and ETC enzyme activity. TEM revealed abnormalities of mitochondrial morphology characteristic of these functional deficits. The selective α1A-AR agonist A61603 enhanced fatty-acid dependent respiration, fatty acid oxidation, and ETC enzyme activity in isolated cardiac mitochondria. The ß-AR agonist isoproterenol enhanced oxidative stress in vitro and this adverse effect was mitigated by A61603. A61603 enhanced ETC Complex I activity and protected contractile function following myocardial infarction. CONCLUSIONS: Collectively, these novel findings position α1A-ARs as critical regulators of cardiomyocyte metabolism in the basal state and suggest that metabolic mechanisms may underlie the protective effects of α1A-AR activation in the failing heart.

Decellularized Fetal Matrix Suppresses Fibrotic Gene Expression and Promotes Myogenesis in a Rat Model of Volumetric Muscle Loss.

BACKGROUND: Traumatic muscle loss often results in poor functional restoration. Skeletal muscle injuries cannot be repaired without substantial fibrosis and loss of muscle function. Given its regenerative properties, the authors evaluated outcomes of fetal tissue-derived decellularized matrix for skeletal muscle regeneration. The authors hypothesized that fetal matrix would lead to enhanced myogenesis and suppress inflammation and fibrosis. METHODS: Composite tissue composed of dermis, subcutaneous tissue, and panniculus carnosus was harvested from the trunk of New Zealand White rabbit fetuses on gestational day 24 and from Sprague-Dawley rats on gestational day 18 and neonatal day 3, and decellularized using a sodium dodecyl sulfate-based negative-pressure protocol. Six, 10-mm-diameter, full-thickness rat latissimus dorsi wounds were created for each treatment, matrix was implanted (excluding the defect groups), and the wounds were allowed to heal for 60 days. Analyses were performed to characterize myogenesis, neovascularization, inflammation, and fibrosis at harvest. RESULTS: Significant myocyte ingrowth was visualized in both allogeneic and xenogeneic fetal matrix groups compared to neonatal and defect groups based on myosin heavy chain immunofluorescence staining. Microvascular networks were appreciated within all implanted matrices. At day 60, expression of Ccn2, Col1a1, and Ptgs2 were decreased in fetal matrix groups compared to defect. Neonatal matrix-implanted wounds failed to show decreased expression of Col1a1 or Ptgs2, and demonstrated increased expression of Tnf, but also demonstrated a significant reduction in Ccn2 expression. CONCLUSIONS: Initial studies of fetal matrices demonstrate promise for muscle regeneration in a rat latissimus dorsi model. Further research is necessary to evaluate fetal matrix for future translational use and better understand its effects.

The sodium channel NaX : Possible player in excitation-contraction coupling.

The sodium channel NaX (encoded by the SCN7A gene) was originally identified in the heart and skeletal muscle and is structurally similar to the other voltage-gated sodium channels but does not appear to be voltage gated. Although NaX is expressed at high levels in cardiac and skeletal muscle, little information exists on the function of NaX in these tissues. Transcriptional profiling of ion channels in the heart in a subset of patients with Brugada syndrome revealed an inverse relationship between the expression of NaX and NaV 1.5 suggesting that, in cardiac myocytes, the expression of these channels may be linked. We propose that NaX plays a role in excitation-contraction coupling based on our experimental observations. Here we show that in cardiac myocytes, NaX is expressed in a striated pattern on the sarcolemma in regions corresponding to the sarcomeric M-line. Knocking down NaX expression decreased NaV 1.5 mRNA and protein and reduced the inward sodium current (INa+ ) following cell depolarization. When the expression of NaV 1.5 was knocked down, ~85% of the INa+ was reduced consistent with the observations that NaV 1.5 is the main voltage-gated sodium channel in cardiac muscle and that NaX likely does not directly participate in mediating the INa+ following depolarization. Silencing NaV 1.5 expression led to significant upregulation of NaX mRNA. Similar to NaV 1.5, NaX protein levels were rapidly downregulated when the intracellular [Ca2+ ] was increased either by CaCl2 or caffeine. These data suggest that a relationship exists between NaX and NaV 1.5 and that NaX may play a role in excitation-contraction coupling.

Local Application of Statins Significantly Reduced Hypertrophic Scarring in a Rabbit Ear Model.

BACKGROUND: We previously showed that intradermal injection of statins is a successful treatment for hypertrophic scarring. Topical application has many advantages over intradermal injection. In this study, we demonstrate the efficacy of topical statin treatment in reducing scar in our validated rabbit ear scar model. METHODS: Twenty New Zealand White rabbits were divided into 2 study groups, with 6 rabbits receiving 10 µm pravastatin intradermally at postoperative days 15, 18, and 21, and 14 rabbits receiving 0.4%, 2%, and 10% simvastatin topical application at postoperative days 14-25. Four or 6 full-thickness circular dermal punches 7 mm in diameter were made on the ventral surface of the ear down to but not including the perichondrium. Specimens were collected at 28 days to evaluate the effects of statins on hypertrophic scarring. RESULTS: Treatment with pravastatin intradermal administration significantly reduced scarring in terms of scar elevation index. Topical treatment with both medium- and high-dose simvastatin also significantly reduced scarring. High-dose simvastatin topical treatment showed a major effect in scar reduction but induced side effects of scaling, erythema, and epidermal hyperplasia, which were improved with coapplication of cholesterol. There is a dose response in scar reduction with low-, medium- and high-dose simvastatin topical treatment. High-dose simvastatin treatment significantly reduced the messenger ribonucleic acid (mRNA) expression of connective tissue growth factor, consistent with our previously published work on intradermally injected statins. More directly, high-dose simvastatin treatment also significantly reduced the mRNA expression of collagen 1A1. CONCLUSIONS: Topical simvastatin significantly reduces scar formation. The mechanism of efficacy for statin treatment through interference with connective tissue growth factor mRNA expression was confirmed.

Thermal injury model in the rabbit ear with quantifiable burn progression and hypertrophic scar.

Hypertrophic scar is a major clinical outcome of deep-partial thickness to full thickness thermal burn injury. Appropriate animal models are a limitation to burn research due to the lack of, or access to, animal models which address the endpoint of hypertrophic scar. Lower species, such as rodents, heal mainly by contracture, which limits the duration of study. Higher species, such as pigs, heal more similarly to humans, but are associated with high cost, long duration for scar development, challenges in quantifying scar hypertrophy, and poor manageability. Here, we present a quantifiable deep-partial thickness burn model in the rabbit ear. Burns were created using a dry-heated brass rod for 10 and 20 seconds at 90 °C. At the time of eschar excision on day 3, excisional wounds were made on the contralateral ear for comparison. Burn wound progression, in which the wound size expands over time is a major distinction between excisional and thermal injuries, was quantified at 1 hour and 3 days after the injuries using calibrated photographs and histology and the size of the wounds was found to be unchanged from the initial wound size at 1 hour, but 10% in the 20 seconds burn wounds at 3 days. A quantifiable hypertrophic scar, measured by histology as the scar elevation index, was present in both 20 seconds burn wounds and excisional wounds at day 35. ImageJ measurements revealed that the 20 seconds burn wound scars were 22% larger than the excisional wound scars and the 20 seconds burn scar area measurements from histology were 26% greater than in the excisional wound scar. The ability to measure both burn progression and scar hypertrophy over a 35-day time frame suits this model to screening early intervention burn wound therapeutics or scar treatments in a burn-specific scar model.

Topical Administration of Oxygenated Hemoglobin Improved Wound Healing in an Ischemic Rabbit Ear Model.

BACKGROUND: Localized oxygen deficiency plays a central role in the pathogenesis of chronic wounds; thus, rectifying localized ischemia with oxygen therapy has been postulated to be an integral aspect of the management of chronic wounds. The efficacy of a novel approach for oxygen therapy on chronic wound healing was evaluated. METHODS: Oxygen was delivered to ischemic wounds by means of the topical application of oxygenated, chemically modified bovine hemoglobin (IKOR 2084) in a validated rabbit ear ischemic wound model. The wound healing was evaluated histologically by measuring epithelial gap and neo-granulation tissue area. In situ expression of endothelial cells (CD31) and proliferative cells (Ki-67) was examined by immunohistochemistry analysis. The mRNA of vascular endothelial growth factor, endothelial nitric oxide synthase, and matrix metalloproteinase-9 was quantified by real-time reverse-transcriptase polymerase chain reaction. The collagen was detected by Sirius red staining. RESULTS: In comparison with topical application of saline, the administration of oxygenated IKOR 2084 increases wound reepithelialization and formation of neo-granulation tissue in a dose-dependent manner, and cellular proliferation (Ki-67). Conversely, the administration of deoxygenated IKOR 2084 aggravated the ischemic wound healing process. Moreover, the topical administration of oxygenated IKOR 2084 induces angiogenesis as evidenced by concomitant increases in CD31 protein and vascular endothelial growth factor and endothelial nitric oxide synthase mRNA expression in treated wounds. Oxygenated IKOR 2084 administration also increased collagen deposition in wounds, with decreases in the expression of matrix metalloproteinase-9 mRNA. CONCLUSION: This study suggests that the topical application of oxygenated IKOR 2084 ameliorates the reparative progress of ischemic wounds through enhanced angiogenesis, cellular proliferation, and collagen deposition.

S100A8 and S100A9 Are Induced by Decreased Hydration in the Epidermis and Promote Fibroblast Activation and Fibrosis in the Dermis.

The most critical function of the epidermis is to prevent water loss and maintain skin homeostasis. Disruption of the functional skin barrier causes delayed wound healing, hypertrophic scarring, and many skin diseases. Herein, we show that reduced hydration increases the expression of S100 protein family members, S100A8/S100A9, in stratified keratinocyte culture and human ex vivo skin culture. Immunohistological analyses show that S100A8/A9 are highly expressed in the epidermis of human hypertrophic scar and keloid tissues. Reduced hydration demonstrates activation of fibroblasts in the keratinocyte-fibroblast co-culture. In contrast, knockdown of S100A8 or S100A9 by RNA interference in keratinocytes failed to activate fibroblasts. Pretreatment with pharmacological blockers of S100A8/A9 receptors, Toll-like receptor 4 and receptor for advanced glycation end products, inhibits fibroblast activation induced by recombinant S100A8/A9 proteins. Moreover, we observe that local delivery of S100A8 protein results in a marked increase in hypertrophic scarring in the in vivo rabbit ear scar model. Our results indicate that hydration status promotes fibroblast activation and fibrosis by directly affecting the expression of inflammatory signaling in keratinocytes, thereby strongly suggesting S100A8/A9 to be novel targets in preventing scarring.

Impact of a novel, antimicrobial dressing on in vivo, Pseudomonas aeruginosa wound biofilm: quantitative comparative analysis using a rabbit ear model.

The importance of bacterial biofilms to chronic wound pathogenesis is well established. Different treatment modalities, including topical dressings, have yet to show consistent efficacy against wound biofilm. This study evaluates the impact of a novel, antimicrobial Test Dressing on Pseudomonas aeruginosa biofilm-infected wounds. Six-mm dermal punch wounds in rabbit ears were inoculated with 10(6) colony-forming units of P. aeruginosa. Biofilm was established in vivo using our published model. Dressing changes were performed every other day with either Active Control or Test Dressings. Treated and untreated wounds were harvested for several quantitative endpoints. Confirmatory studies were performed to measure treatment impact on in vitro P. aeruginosa and in vivo polybacterial wounds containing P. aeruginosa and Staphylococcus aureus. The Test Dressing consistently decreased P. aeruginosa bacterial counts, and improved wound healing relative to Inactive Vehicle and Active Control wounds (p < 0.05). In vitro bacterial counts were also significantly reduced following Test Dressing therapy (p < 0.05). Similarly, improvements in bacterial burden and wound healing were also achieved in polybacterial wounds (p < 0.05). This study represents the first quantifiable and consistent in vivo evidence of a topical antimicrobial dressing's impact against established wound biofilm. The development of clinically applicable therapies against biofilm such as this is critical to improving chronic wound care.

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.

Dermal wound transcriptomic responses to Infection with Pseudomonas aeruginosa versus Klebsiella pneumoniae in a rabbit ear wound model.

BACKGROUND: Bacterial infections of wounds impair healing and worsen scarring. We hypothesized that transcriptome analysis of wounds infected with Klebsiella pneumoniae (K.p.) or Pseudomonas aeruginosa (P.a.) would indicate host-responses associated with the worse healing of P.a.- than K.p.-infected wounds. METHODS: Wounds created on post-operative day (POD) 0 were infected during the inflammatory phase of healing on POD3 and were harvested on POD4 for microarray and transcriptome analysis. Other wounds received topical antibiotic after infection for 24 hours to promote biofilm development, and were harvested on POD6 or POD12. RESULTS: Wounds infected for 24 hours, relative to uninfected wounds, elevated transcripts of immune-response functions characteristic of infiltrating leukocytes. But P.a.-infected wounds elevated many more transcripts and to higher levels than K.p.-infected wounds. Coincidently, suppressed transcripts of both wounds enriched into stress-response pathways, including EIF2 signaling; however, this was more extensive for P.a.-infected wounds, including many-fold more transcripts enriching in the 'cell death' annotation, suggesting resident cutaneous cell toxicity in response to a more damaging P.a. inflammatory milieu. The POD6 wounds were colonized with biofilm but expressed magnitudes fewer immune-response transcripts with no stress-response enrichments. However, elevated transcripts of P.a.-infected wounds were inferred to be regulated by type I interferons, similar to a network unique to P.a.-infected wounds on POD4. On POD12, transcripts that were more elevated in K.p.-infected wounds suggested healing, while transcripts more elevated in P.a.-infected wounds indicated inflammation. CONCLUSIONS: An extensive inflammatory response of wounds was evident from upregulated transcripts 24 hours after infection with either bacterium, but the response was more intense for P.a.- than K.p.-infected wounds. Coincidently, more extensive down-regulated transcripts of P.a.-infected wounds indicated a stronger "integrated stress response" to the inflammatory milieu that tipped more toward cutaneous cell death. Unique to P.a.-infected wounds on POD4 and POD6 were networks inferred to be regulated by interferons, which may result from intracellular replication of P.a. These data point to specific downregulated transcripts of cells resident to the wound as well as upregulated transcripts characteristic of infiltrating leukocytes that could be useful markers of poorly healing wounds and indicators of wound-specific treatments for improving outcomes.

The expression of proinflammatory genes in epidermal keratinocytes is regulated by hydration status.

Mucosal wounds heal more rapidly, exhibit less inflammation, and are associated with minimal scarring when compared with equivalent cutaneous wounds. We previously demonstrated that cutaneous epithelium exhibits an exaggerated response to injury compared with mucosal epithelium. We hypothesized that treatment of injured skin with a semiocclusive dressing preserves the hydration of the skin and results in a wound healing phenotype that more closely resembles that of mucosa. Here we explored whether changes in hydration status alter epidermal gene expression patterns in rabbit partial-thickness incisional wounds. Using microarray studies on injured epidermis, we showed that global gene expression patterns in highly occluded versus non-occluded wounds are distinct. Many genes including IL-1ß, IL-8, TNF-α (tumor necrosis factor-α), and COX-2 (cyclooxygenase 2) are upregulated in non-occluded wounds compared with highly occluded wounds. In addition, decreased levels of hydration resulted in an increased expression of proinflammatory genes in human ex vivo skin culture (HESC) and stratified keratinocytes. Hierarchical analysis of genes using RNA interference showed that both TNF-α and IL-1ß regulate the expression of IL-8 through independent pathways in response to reduced hydration. Furthermore, both gene knockdown and pharmacological inhibition studies showed that COX-2 mediates the TNF-α/IL-8 pathway by increasing the production of prostaglandin E2 (PGE2). IL-8 in turn controls the production of matrix metalloproteinase-9 in keratinocytes. Our data show that hydration status directly affects the expression of inflammatory signaling in the epidermis. The identification of genes involved in the epithelial hydration pathway provides an opportunity to develop strategies to reduce scarring and optimize wound healing.

Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic, biofilm-containing chronic wounds.

Diabetic patients exhibit dysregulated inflammatory and immune responses that predispose them to chronic wound infections and the threat of limb loss. The molecular underpinnings responsible for this have not been well elucidated, particularly in the setting of wound biofilms. This study evaluates host responses in biofilm-impaired wounds using the TallyHo mouse, a clinically relevant polygenic model of type 2 diabetes. No differences in cytokine or Toll-like receptor (TLR) expression were noted in unwounded skin or noninoculated wounds of diabetic and wild-type mice. However, diabetic biofilm-containing wounds had significantly less TLR 2, TLR 4, interleukin-1ß, and tumor necrosis factor-α expression than wild-type wounds with biofilm (all p < 0.001). Both groups had similar bacterial burden and neutrophil infiltration after development of biofilms at 3 days postwounding, but diabetic wounds had significantly less neutrophil oxidative burst activity. This translated into a log-fold greater bacterial burden and significant delay of wound epithelization for biofilm-impaired diabetic wounds at 10 days postwounding. These results suggest that impaired recognition of bacterial infection via the TLR pathway leading to inadequate cytokine stimulation of antimicrobial host responses may represent a potential mechanism underlying diabetic susceptibility to wound infection and ulceration.

Excisional wound healing is delayed in a murine model of chronic kidney disease.

BACKGROUND: Approximately 15% of the United States population suffers from chronic kidney disease (CKD), often demonstrating an associated impairment in wound healing. This study outlines the development of a surgical murine model of CKD in order to investigate the mechanisms underlying this impairment. METHODS: CKD was induced in mice by partial cauterization of one kidney cortex and contralateral nephrectomy, modifying a previously published technique. After a minimum of 6-weeks, splinted, dorsal excisional wounds were created to permit assessment of wound healing parameters. Wounds were harvested on postoperative days (POD) 0, 3, 7, and 14 for histological, immunofluorescent, and quantitative PCR (qPCR). RESULTS: CKD mice exhibited deranged blood chemistry and hematology profiles, including profound uremia and anemia. Significant decreases in re-epithelialization and granulation tissue deposition rates were found in uremic mice wounds relative to controls. On immunofluorescent analysis, uremic mice demonstrated significant reductions in cellular proliferation (BrdU) and angiogenesis (CD31), with a concurrent increase in inflammation (CD45) as compared to controls. CKD mice also displayed differential expression of wound healing-related genes (VEGF, IL-1ß, eNOS, iNOS) on qPCR. CONCLUSIONS: These findings represent the first reported investigation of cutaneous healing in a CKD animal model. Ongoing studies of this significantly delayed wound healing phenotype include the establishment of renal failure model in diabetic strains to study the combined effects of CKD and diabetes.

Noncontact, low-frequency ultrasound as an effective therapy against Pseudomonas aeruginosa-infected biofilm wounds.

Bacterial biofilms, a critical chronic wound mediator, remain difficult to treat. Energy-based devices may potentially improve healing, but with no evidence of efficacy against biofilms. This study evaluates noncontact, low-frequency ultrasound (NLFU) in the treatment of biofilm-infected wounds. Six-millimeter dermal punch wounds in rabbit ears were inoculated with 10(7) colony-forming units of Pseudomonas aeruginosa or left as sterile controls. A biofilm was established in vivo using our published model. NLFU treatment was carried out every other day or every day, with contralateral ear wounds acting as internal, untreated controls. Wounds were harvested for several quantitative endpoints and scanning electron microscopy to evaluate the biofilm structure. The P. aeruginosa biofilm consistently impaired wound epithelialization and granulation. NLFU, both every other day and every day, improved healing and reduced bacterial counts relative to untreated controls (p < 0.05). Scanning electron microscopy confirmed a qualitative decrease in bacteria after both treatments. NLFU also reduced inflammatory cytokine expression (p < 0.05). Our study suggests that NLFU is an effective therapy against P. aeruginosa wound biofilm. This represents the first in vivo evidence of energy-based modalities' impact on wound biofilm, setting the foundation for future mechanistic studies. Continued wound care technology research is essential to improving our understanding, and treatment, of biofilm-infected chronic wounds.

Bacteriophage therapy for Staphylococcus aureus biofilm-infected wounds: a new approach to chronic wound care.

BACKGROUND: Bacterial biofilms, which are critical mediators of chronic wounds, remain difficult to treat with traditional methods. Bacteriophage therapy against biofilm has not been rigorously studied in vivo. The authors evaluate the efficacy of a species-specific bacteriophage against Staphylococcus aureus biofilm-infected wounds using a validated, quantitative, rabbit ear model. METHODS: Six-millimeter dermal punch wounds in New Zealand rabbit ears were inoculated with wild-type or mutant, biofilm-deficient S. aureus. In vivo biofilm was established and maintained using procedures from our previously published wound biofilm model. Wounds were left untreated, or treated every other day with topical S. aureus-specific bacteriophage, sharp débridement, or both. Histologic wound healing and viable bacterial count measurements, and scanning electron microscopy were performed following harvest. RESULTS: Wild-type S. aureus biofilm wounds demonstrated no differences in healing or viable bacteria following bacteriophage application or sharp débridement alone. However, the combination of both treatments significantly improved all measured wound healing parameters (p < 0.05) and reduced bacteria counts (p = 0.03), which was confirmed by scanning electron microscopy. Bacteriophage treatment of biofilm-deficient S. aureus mutant wounds alone also resulted in similar trends for both endpoints (p < 0.05). CONCLUSIONS: Bacteriophages can be an effective topical therapy against S. aureus biofilm-infected wounds in the setting of a deficient (mutant) or disrupted (débridement) biofilm structure. Combination treatment aimed at disturbing the extracellular biofilm matrix, allowing for increased penetration of species-specific bacteriophages, represents a new and potentially effective approach to chronic wound care. These results establish principles for biofilm therapy that may be applied to several different clinical and surgical problems.

Comparative analysis of single-species and polybacterial wound biofilms using a quantitative, in vivo, rabbit ear model.

INTRODUCTION: The recent literature suggests that chronic wound biofilms often consist of multiple bacterial species. However, without appropriate in vivo, polybacterial biofilm models, our understanding of these complex infections remains limited. We evaluate and compare the effect of single- and mixed-species biofilm infections on host wound healing dynamics using a quantitative, in vivo, rabbit ear model. METHODS: Six-mm dermal punch wounds in New Zealand rabbit ears were inoculated with Staphylococcus aureus strain UAMS-1, Pseudomonas aeruginosa strain PAO1, or both, totaling 10/6 colony-forming units/wound. Bacterial proliferation and maintenance in vivo were done using procedures from our previously published model. Wounds were harvested for histological measurement of wound healing, viable bacterial counts using selective media, or inflammatory cytokine (IL-1ß, TNF-α) expression via quantitative reverse-transcription PCR. Biofilm structure was studied using scanning electron microscopy (SEM). For comparison, biofilm deficient mutant UAMS-929 replaced strain UAMS-1 in some mixed-species infections. RESULTS: Bacterial counts verified the presence of both strains UAMS-1 and PAO1 in polybacterial wounds. Over time, strain PAO1 became predominant (p<0.001). SEM showed colocalization of both species within an extracellular matrix at multiple time-points. Compared to each monospecies infection, polybacterial biofilms impaired all wound healing parameters (p<0.01), and increased expression of IL-1ß and TNF-α (p<0.05). In contrast, mixed-species infections using biofilm-deficient mutant UAMS-929 instead of wild-type strain UAMS-1 showed less wound impairment (p<0.01) with decreased host cytokine expression (p<0.01), despite a bacterial burden and distribution comparable to that of mixed-wild-type wounds. CONCLUSIONS: This study reveals that mixed-species biofilms have a greater impact on wound healing dynamics than their monospecies counterparts. The increased virulence of polybacterial biofilm appears dependent on the combined pathogenicity of each species, verified using a mutant strain. These data suggest that individual bacterial species can interact synergistically within a single biofilm structure.

In vivo modeling of biofilm-infected wounds: a review.

Chronic wounds continue to represent a difficult and complex problem for both patients and healthcare providers. Bacterial biofilms represent a critical component of nonhealing wounds, utilizing several different mechanisms to inhibit innate inflammatory pathways and resist traditional therapeutics. Although in vitro biofilm systems have been well described and studied, understanding the intricacies of wound biofilm pathology requires appropriate in vivo models to understand the interactions between bacteria and host. In an effort to clarify the available literature, this review describes and critically evaluates all of the in vivo wound biofilm models currently published to-date, including model advantages and clinical applicability. We will also address the need for continued therapeutic development and testing using these currently available in vivo models.

Quantitative comparison and analysis of species-specific wound biofilm virulence using an in vivo, rabbit-ear model.

BACKGROUND: Although bacterial biofilm is recognized as an important contributor to chronic wound pathogenesis, differences in biofilm virulence between species have never been studied in vivo. STUDY DESIGN: Dermal punch wounds in New Zealand white rabbit ears were inoculated with Klebsiella pneumoniae, Staphylococcus aureus, or Pseudomonas aeruginosa, or left uninfected as controls. In vivo biofilm was established and maintained using procedures from our previously published wound biofilm model. Virulence was assessed by measurement of histologic wound healing and host inflammatory mediators. Scanning electron microscopy (SEM) and bacterial counts verified biofilm viability. Extracellular polymeric substance (EPS)-deficient P aeruginosa was used for comparison. RESULTS: SEM confirmed the presence of wound biofilm for each species. P aeruginosa biofilm-infected wounds showed significantly more healing impairment than uninfected, K pneumoniae, and S aureus (p < 0.05), while also triggering the largest host inflammatory response (p < 0.05). Extracellular polymeric substance-deficient P aeruginosa demonstrated a reduced impact on the same quantitative endpoints relative to its wild-type strain (p < 0.05). CONCLUSIONS: Our novel analysis demonstrates that individual bacterial species possess distinct levels of biofilm virulence. Biofilm EPS may represent an integral part of their distinct pathogenicity. Rigorous examination of species-dependent differences in biofilm virulence is critical to developing specific therapeutics, while lending insight to the interactions within clinically relevant, polybacterial biofilms.

Understanding the host inflammatory response to wound infection: an in vivo study of Klebsiella pneumoniae in a rabbit ear wound model.

Wound infection development is critically dependent on the complex interactions between bacteria and host. Klebsiella pneumoniae has become an increasingly common wound pathogen, but its natural history within wounds has never been studied. Using a validated, in vivo rabbit ear model, wounds were inoculated with K. pneumoniae at different concentrations (10²-107 colony-forming units) with measurement of viable and nonviable bacterial counts, histological wound-healing parameters, and host inflammatory gene expression at multiple time points postinoculation (48, 96, and 240 hours). Bacteria and wound morphologies were evaluated with scanning electron microscopy. Comparable experiments were performed in ischemic ears to model immune response impairment. All wounds, despite different inoculants, equilibrated to similar bacterial concentrations by 96 hours. With a 106 colony-forming units inoculant, wounds at 240 hours showed decreased bacterial counts (p < 0.01), with a corresponding improvement in healing (p < 0.01) and a decrease in inflammatory response (p < 0.05). In contrast, ischemic wounds revealed impaired inflammatory gene expression (p < 0.05) resulting in higher steady-state bacterial concentrations (p < 0.01), impaired healing (p < 0.05), and biofilm formation on scanning electron microscopy. We conclude that a normal inflammatory response can effectively stabilize and overcome a K. pneumoniae wound infection. An impaired host cannot control this bacterial burden, preventing adequate healing while allowing bacteria to establish a chronic presence. Our novel study quantitatively validates the host immune response as integral to wound infection dynamics.