Staphylococcus aureus Biofilm Infection Compromises Wound Healing by Causing Deficiencies in Granulation Tissue Collagen.

OBJECTIVE: The objective of this work was to causatively link biofilm properties of bacterial infection to specific pathogenic mechanisms in wound healing. BACKGROUND: Staphylococcus aureus is one of the four most prevalent bacterial species identified in chronic wounds. Causatively linking wound pathology to biofilm properties of bacterial infection is challenging. Thus, isogenic mutant stains of S. aureus with varying degree of biofilm formation ability was studied in an established preclinical porcine model of wound biofilm infection. METHODS: Isogenic mutant strains of S. aureus with varying degree (ΔrexB > USA300 > ΔsarA) of biofilm-forming ability were used to infect full-thickness porcine cutaneous wounds. RESULTS: Compared with that of ΔsarA infection, wound biofilm burden was significantly higher in response to ΔrexB or USA300 infection. Biofilm infection caused degradation of cutaneous collagen, specifically collagen 1 (Col1), with ΔrexB being most pathogenic in that regard. Biofilm infection of the wound repressed wound-edge miR-143 causing upregulation of its downstream target gene matrix metalloproteinase-2. Pathogenic rise of collagenolytic matrix metalloproteinase-2 in biofilm-infected wound-edge tissue sharply decreased collagen 1/collagen 3 ratio compromising the biomechanical properties of the repaired skin. Tensile strength of the biofilm infected skin was compromised supporting the notion that healed wounds with a history of biofilm infection are likely to recur. CONCLUSION: This study provides maiden evidence that chronic S. aureus biofilm infection in wounds results in impaired granulation tissue collagen leading to compromised wound tissue biomechanics. Clinically, such compromise in tissue repair is likely to increase wound recidivism.

The cost of self-imposed regulatory burden in animal research.

U.S. federal regulations and standards governing the care and use of research animals enacted in the mid- to late 1980s, while having positive effects on the welfare and quality of the animals, have resulted in dramatic increases in overall research costs. In addition to the expenses of housing and caring for animals according to the standards, establishing the requisite internal compliance bureaucracies has markedly driven up costs, in both institutional monetary expenditures and lost research effort. However, many institutions are increasing these costs even further through additional self-imposed regulatory burden, typically characterized by overly complex compliance organizations and unnecessary policies and procedures. We discuss the sources of this self-imposed burden and recommend strategies for avoiding it while preserving an appropriate focus on animal well-being and research success.

Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function.

In chronic wounds, biofilm infects host tissue for extended periods of time. This work establishes the first chronic preclinical model of wound biofilm infection aimed at addressing the long-term host response. Although biofilm-infected wounds did not show marked differences in wound closure, the repaired skin demonstrated compromised barrier function. This observation is clinically significant, because it leads to the notion that even if a biofilm infected wound is closed, as observed visually, it may be complicated by the presence of failed skin, which is likely to be infected and/or further complicated postclosure. Study of the underlying mechanisms recognized for the first time biofilm-inducible miR-146a and miR-106b in the host skin wound-edge tissue. These miRs silenced ZO-1 and ZO-2 to compromise tight junction function, resulting in leaky skin as measured by transepidermal water loss (TEWL). Intervention strategies aimed at inhibiting biofilm-inducible miRNAs may be productive in restoring the barrier function of host skin.

Serum Concentrations of a Long-acting Cat Formulation of Transdermal Buprenorphine in C57BL/6 Mice.

Long-acting analgesics such as extended-release buprenorphine are desirable in rodent medicine because they reduce need for administration of additional medication and provide stable drug levels. We measured the serum concentrations of buprenorphine after topical administration of a long-acting transdermal buprenorphine (LAT-bup) solution to female C57BL/6 mice. We hypothesized that LAT-bup dosed topically at 40mg/kg would achieve serum drug concentrations of greater than 1ng/mL, which is considered the therapeutic level for adequate analgesia in rodents. LAT-bup administered at 40mg/kg resulted in serum drug concentrations above 1ng/mL for all mice at time points 2, 4, 24, 48, 72, and 96 h (n = 3/time point), as assessed by liquid chromatography-mass spectrometry. No adverse effects were noted when LAT-bup was dosed at either 30mg/kg or 40mg/kg. We conclude that LAT-bup is easily administered to mice and achieves adequate blood levels for 96 h. Further studies evaluating analgesic efficacy are indicated.

Comparison of Floor Cleaning and Disinfection Processes in a Research Animal Facility.

Floor cleaning and disinfection are essential components of maintaining animal health status and meeting regulatory requirements in research vivaria. However, best practices for method, frequency, and evaluation techniques have not been established. Reuse of cotton string mop and bucket systems has been implicated in spreading contamination in the human hospital setting. We evaluated 4 different combinations of disinfectant and mop systems commonly used in rodent vivaria. Eight housing rooms were mopped a total of 4 times using one of the following methods: quaternary ammonium compound (QUAT) and cotton string mop (QC), QUAT and microfiber mop (QM), hydrogen peroxide disinfectant (HPD) and cotton string mop (HC), or HPD and microfiber mop (HM). ATP and RODAC samples of the floor were taken before and after mopping. The time to mop each room, floor drying time, and the amount of disinfectant used were recorded. The QC method was associated with significantly more bacterial contamination while all other methods significantly reduced bacterial contamination. The QC method performed significantly worse in reducing bacterial contamination as compared with all other methods when cotton mop heads were reused. All methods except QC significantly reduced ATP levels, with the HC and HM methods being significantly more effective at reducing ATP levels than the QC and QM methods. Costs were similar for the QC, QM, and HM methods. The results of this study indicate that reuse of cotton string mop heads with QUAT increases floor contamination while HPD is effective for up to 3 reuses. Single use microfiber mops were effective with both QUAT and HPD but did not result in more effective cleaning or disinfection than cotton string mops.

Platelet-rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation.

The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.

Evaluation of Rodent Cage Processing Using Reduced Water Temperatures.

Studies published in 1994 and 2000 established a temperature range of 143-180 °F for effective cage sanitization in animal facilities. These 2 studies were, respectively, theoretical and based on experiments using hot water to sanitize bacteria-coated test tubes. However, such experimental methods may not capture the practical advantages of modern washing technology or account for the routine use of detergent in cage wash. Moreover, these methods may not translate to the challenges of removing adhered debris and animal waste from the surfaces being sanitized. A sample of highly soiled cage bottoms, half of which were autoclaved with bedding to create challenging cleaning conditions, were processed at 6 combinations of wash and rinse cycles with 125 °F, 140 °F, and 180 °F water with detergent. All cycles were equipped with a data logging device to independently verify temperatures. After washing, cages underwent visual inspection and microbial sampling consisting of organic material detection using ATP detection and Replicate Organism Detection and Counting (RODAC) plates. Cages with any amount of visible debris failed inspection, as did cages that exceeded institutional sanitization thresholds. Results indicate that wash and rinse temperatures of 140 °F for a programmed wash duration of 450 s and rinse of 50 s effectively clean and disinfect both highly soiled and autoclaved cages. Accounting for both steam and electrical energy, these parameters result in an annual savings of $21,867.08 per washer on an equivalent run basis using the current institutional standard of 180 °F.

Reuse of Disposable Isolation Gowns in Rodent Facilities during a Pandemic.

Reuse of disposable personal protective equipment is traditionally discouraged, yet in times of heightened medical applications such as the SARS CoV-2 pandemic, it can be difficult to obtain. In this article we examine the reuse of disposable gowns with respect to still providing personnel protection. XR7, a fluorescent powder, was used to track contamination of gowns after manipulation of rodent cages. Mouse cages were treated with XR7 prior to manipulations. Disposable gowns were labeled for single person use and hung in common procedure spaces within the vivarium between usages. A simulated rack change of 140 cages was completed using XR7-treated cages. One individual changed all cages with a break occurring after the first 70 cages, requiring the gown to be removed and reused once. To simulate research activities, 5 individuals accessed 3 XR7-treated cages daily for 5 d. Each mouse in the XR7-treated cages was manipulated at least once before returning cages to the housing room. Disposable gowns were reused 5 times per individual. Gowns, gloves, clothing, bare arms, and hands were scanned for fluorescence before and after removing PPE. Fluorescence was localized to gloves and gown sleeves in closest contact with animals and caging. No fluorescence was detected on underlying clothing, or bare arms and hands after removing PPE. Fluorescence was not detected in procedure spaces where gowns were hung. The lack of fluorescence on personnel or surfaces indicate that gowns can be reused 1 time for routine husbandry tasks and up to 5 times for research personnel. A method for decontamination of used gowns using Vaporized Hydrogen Peroxide (VHP) was also validated for use in areas where animals are considered high risk such as quarantine, or for fragile immunocompromised rodent colonies.

Regulation of scar formation by vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF-A) is known for its effects on endothelial cells and as a positive mediator of angiogenesis. VEGF is thought to promote repair of cutaneous wounds due to its proangiogenic properties, but its ability to regulate other aspects of wound repair, such as the generation of scar tissue, has not been studied well. We examined the role of VEGF in scar tissue production using models of scarless and fibrotic repair. Scarless fetal wounds had lower levels of VEGF and were less vascular than fibrotic fetal wounds, and the scarless phenotype could be converted to a scar-forming phenotype by adding exogenous VEGF. Similarly, neutralization of VEGF reduced vascularity and decreased scar formation in adult wounds. These results show that VEGF levels have a strong influence on scar tissue formation. Our data suggest that VEGF may not simply function as a mediator of wound angiogenesis, but instead may play a more diverse role in the wound repair process.

Micro-field evoked potentials recorded from the porcine sub-dural cortical surface utilizing a microelectrode array.

A sub-dural surface microelectrode array designed to detect micro-field evoked potentials has been developed. The device is comprised of an array of 350-microm square gold contacts, with bidirectional spacing of 150 microm, contained within a polyimide Kapton material. Cytotoxicity testing suggests that the device is suitable for use with animal and human patients. Implementation of the device in animal studies revealed that reliable evoked potentials could be acquired. Further work will be needed to determine how these micro-field potentials, which demonstrate selectivity for one eye, relate to the distribution of the ocular dominance columns of the occipital cortex.

Institutional Animal Care and Use Committee Considerations Regarding the Use of Virus-Induced Carcinogenesis and Oncolytic Viral Models.

The use of virus-induced carcinogenesis and oncologic experimental animal models is essential in understanding the mechanisms of cancer development to advance prevention, diagnosis, and treatment methods. The Institutional Animal Care and Use Committee (IACUC) is responsible for both the complex philosophical and practical considerations associated with animal models of cancer. Animal models of cancer carry their own unique issues that require special consideration from the IACUC. Many of the considerations to be discussed apply to cancer models in general; specific issues related to viral carcinogenesis or oncolytic viruses will be specifically discussed as they arise. Responsible animal use integrates good science, humane care, and regulatory compliance. To meet those standards, the IACUC, in conjunction with the research investigator and attending veterinarian, must address a wide range of issues, including animal model selection, cancer model selection, humane end point considerations, experimental considerations, postapproval monitoring, reporting requirements, and animal management and personnel safety considerations.

Effect of Ventilated Caging on Water Intake and Loss in 4 Strains of Laboratory Mice.

Food availability, temperature, humidity, strain, and caging type all affect water consumption by mice. Measurement of transepidermal water loss (TEWL) is a new technique for the quantification of water turnover in mice. To understand water turnover in common strains of adult mice, male and female SCID, SKH, C57BL/6, and FVB mice were housed in same-sex groups of 5 animals in static cages or IVC. Body weight, TEWL, urine osmolality, and water consumption of mice and intracage temperature and humidity were measured every 48 h for comparison. Static cages were monitored for 7 d and IVC for 14 d before cage change. Female SCID, FVB, and C57 mice drank less water than did their male counterparts. Male and female SCID, SKH, and FVB mice in IVC drank less water and had higher urine osmolality than did those in static cages. In SCID and SKH mice, TEWL paralleled water consumption. C57 mice in static cages drank less water, had lower urine osmolality, and had less TEWL than did those in IVC. Temperature and humidity within the cage was higher than the macroenvironmental levels for all housing conditions, mouse strains, and sexes. Temperatures within IVC ranged from 76.6 to 81.4 °F compared with 69±0.4 °F in the room. Humidity within IVC ranged from 68% to 79% compared with 27.o%±2.7% within the room. These data demonstrate that mouse strain and housing conditions significantly influence water balance and indicate that macroenvironmental measurements do not always reflect the intracage environment.

Performance analysis of exam gloves used for aseptic rodent surgery.

Aseptic technique includes the use of sterile surgical gloves for survival surgeries in rodents to minimize the incidence of infections. Exam gloves are much less expensive than are surgical gloves and may represent a cost-effective, readily available option for use in rodent surgery. This study examined the effectiveness of surface disinfection of exam gloves with 70% isopropyl alcohol or a solution of hydrogen peroxide and peracetic acid (HP-PA) in reducing bacterial contamination. Performance levels for asepsis were met when gloves were negative for bacterial contamination after surface disinfection and sham 'exertion' activity. According to these criteria, 94% of HP-PA-disinfected gloves passed, compared with 47% of alcohol-disinfected gloves. In addition, the effect of autoclaving on the integrity of exam gloves was examined, given that autoclaving is another readily available option for aseptic preparation. Performance criteria for glove integrity after autoclaving consisted of: the ability to don the gloves followed by successful simulation of wound closure and completion of stretch tests without tearing or observable defects. Using this criteria, 98% of autoclaved nitrile exam gloves and 76% of autoclaved latex exam gloves met performance expectations compared with the performance of standard surgical gloves (88% nitrile, 100% latex). The results of this study support the use of HP-PA-disinfected latex and nitrile exam gloves or autoclaved nitrile exam gloves as viable cost-effective alternatives to sterile surgical gloves for rodent surgeries.

Exenatide pretreatment improved graft function in nonhuman primate islet recipients compared to treatment after transplant only.

The GLP-1 receptor agonist, exenatide, has previously been shown to improve insulin secretion, protect beta cells from apoptosis, and promote beta cell regeneration. We propose that pretreatment with exenatide will promote islet graft survival and improve graft function. Pancreatectomized cynomolgus monkeys underwent islet allotransplantation and were treated with exenatide beginning on day 0 or day -2. A third group of animals was treated with an immunosuppressive regimen while a fourth group remained untreated. Fasting blood glucose (FBG) was used to evaluate graft function along with intravenous glucose tolerance tests (IVGTTs) performed at study endpoint (day 10 for untreated and posttransplant exenatide or day 90 for pretreatment exenatide and immunosuppression). The average FBG for pre-treated animals day 5 following transplant was 52.7 ± 14.8 mg/dl, compared to 154.3 ± 105.5 mg/dl for animals treated only following transplant, 59.4 mg/dl ±12.1 for animals treated with immunosuppression, and 265.5 ± 172.3 mg/dl for untreated animals. IVGTTs performed at study endpoint showed normal glucose and insulin curves in the pre-treated exenatide and immunosuppression groups only, with beta cell function actually improving after transplant in the pre-treated group. We conclude, therefore, that exenatide pre-treatment can successfully maintain islet graft survival in nonhuman primates.

Processing and treatment of corncob bedding affects cage-change frequency for C57BL/6 mice.

The goal of this study was to evaluate the effectiveness of a new proprietary processed corncob bedding material (PCC)compared with standard corncob in ventilated and static mouse housing systems. Intracage ammonia levels, bacterial growth, and absorptive capacity of bedding were measured for cages of C57BL/6 mice under nonautoclaved and autoclaved conditions on static and ventilated racks in a barrier facility. Ammonia concentration was measured daily, and cages were removed from the study when measurements reached or exceeded 25 ppm. Bacterial growth in bedding was quantified and speciated before exposure to mice and at the time of cage removal. The absorptive capacity of all bedding material was determined under autoclaved and nonautoclaved conditions. Ventilated cages with PCC or autoclaved corncob took longer to reach ammonia concentrations of 25 ppm than did those with corncob or autoclaved PCC; PCC-filled cages remained below 25 ppm NH3 for at least 3 wk. The type of bedding material did not affect the number of days required to reach 25 ppm in static cages. Compared with other bedding types in the absence of mice, 1/4-in. PCC had a lower and 1/8-in. corncob a higher bacterial load. Autoclaving altered the absorptive capacity of 1/4-in. bedding materials, and for 1/8-in. bedding, corncob was more absorptive than PCC regardless of autoclaving. The results of this study indicate that PCC is comparable to autoclaved corncob in controlling intracage ammonia levels, and a cage-change interval of 3 wk is possible when ventilated cages are used with this bedding.

Effectiveness of shoe covers for bioexclusion within an animal facility.

The personal protective equipment (PPE) required for entry into rodent barrier rooms often includes a hair bonnet, face mask, disposable gown, gloves, and shoe covers. Traditionally, shoe covers have been considered essential PPE for maintaining a 'clean' animal room. The introduction of microisolation caging and ventilated rack housing prompted us to reevaluate the contribution of shoe covers to bioexclusion. Contamination powder that fluoresces under black light was to track particle dispersal on the floor and personnel. The test mouse room contained a ventilated microisolation rack and biosafety cabinet. Powder was applied directly inside or outside the animal room doorway. PPE with or without shoe covers was donned outside of the animal room doorway and discarded on exiting. Participants either were scanned on entry into the room for the presence of florescence or asked to complete a simulated standard animal room activity while wearing full PPE. Animal rooms were scanned for florescence after exit of participants. All participants donning shoe covers fluoresced in multiple areas, primarily on gloves and gowns. Shoe covers had no effect on the spread of powder in normal traffic patterns, with no powder detected within caging. Powder also was used to determine the distance substances could be carried on the floor from building entry points. Results indicate that shoe covers do not improve (and actually may compromise) bioexclusion. Donning of shoe covers offers a potential for contamination of personnel from contact with shoe bottoms.

Tocotrienol vitamin E protects against preclinical canine ischemic stroke by inducing arteriogenesis.

Vitamin E consists of tocopherols and tocotrienols, in which α-tocotrienol is the most potent neuroprotective form that is also effective in protecting against stroke in rodents. As neuroprotective agents alone are insufficient to protect against stroke, we sought to test the effects of tocotrienol on the cerebrovascular circulation during ischemic stroke using a preclinical model that enables fluoroscopy-guided angiography. Mongrel canines (mean weight=26.3±3.2 kg) were supplemented with tocotrienol-enriched (TE) supplement (200 mg b.i.d, n=11) or vehicle placebo (n=9) for 10 weeks before inducing transient middle cerebral artery (MCA) occlusion. Magnetic resonance imaging was performed 1 hour and 24 hours post reperfusion to assess stroke-induced lesion volume. Tocotrienol-enriched supplementation significantly attenuated ischemic stroke-induced lesion volume (P<0.005). Furthermore, TE prevented loss of white matter fiber tract connectivity after stroke as evident by probabilistic tractography. Post hoc analysis of cerebral angiograms during MCA occlusion revealed that TE-supplemented canines had improved cerebrovascular collateral circulation to the ischemic MCA territory (P<0.05). Tocotrienol-enriched supplementation induced arteriogenic tissue inhibitor of metalloprotease 1 and subsequently attenuated the activity of matrix metalloproteinase-2. Outcomes of the current preclinical trial set the stage for a clinical trial testing the effects of TE in patients who have suffered from transient ischemic attack and are therefore at a high risk for stroke.

An endovascular canine middle cerebral artery occlusion model for the study of leptomeningeal collateral recruitment.

OBJECTIVES: This work aimed to refine a large animal in minimally invasive reversible middle cerebral artery (MCA) occlusion (MCAO) model to account for leptomeningeal collateral formation. MATERIALS AND METHODS: An angiographically based methodology allowed for transient MCA and carotid terminus occlusion in 12 mongrel dogs and assessment of pial collateral recruitment. Outcome measures included 1- and 24-hour magnetic resonance imaging-based infarct volume calculation, a behavioral scale and histopathologic sections. RESULTS: MCAO succeeded in 8 of 12 dogs (67% efficiency). One-hour postreperfusion infarct volume predicted 24-hour postreperfusion infarct volume (r = 0.997, P < 0.0001). Pial collateral recruitment varied with time and reproducibly assessed predicted infarct volume on 1-hour postreperfusion mean diffusivity maps (P < 0.0001; r = 0.946) and 24-hour fluid-attenuated inversion recovery FLAIR magnetic resonance imaging (P = 0.0033; r = 0.961). The canine stroke scale score correlated with infarct volumes and pial collateral score. CONCLUSION: This canine MCAO model produces defined cerebral infarct lesions whose volumes correlate with leptomeningeal collateral formation and canine behavior.

Ribonucleotide reductase inhibitors reduce atherosclerosis in a double-injury rabbit model.

Atheroproliferative disorders such as atherosclerosis are an important health problem and one of the leading causes of morbidity and mortality in the United States. Minimally invasive therapeutic procedures, including angioplasty with stent deployment, are used frequently for obstructive coronary artery disease. However, restenosis, a proliferative vascular response, is a common sequela to this procedure. The current study investigated the effect of inhibiting ribonucleotide reductase (RR), an enzyme necessary for cellular proliferation, in an attempt to ameliorate the proliferative response. Two RR inhibitors, didox and hydroxyurea, were chosen for their potent antiproliferative properties. Studies were carried out by using a double-injury rabbit model, in which endothelial denudation was followed by the administration of a high-fat diet. At 4 wk after initial endothelial denudation, the developing atherosclerotic lesion was subjected to transluminal balloon dilation to simulate clinical intervention with percutaneous transluminal angioplasty. The degree of restenosis and atheroproliferation was assessed at 8 wk. Histologic evaluation of the lesion demonstrated that treatment with didox and hydroxyurea significantly decreased lesion area and lumen loss. These results suggest that RR inhibition may be an effective new tool for the treatment of atheroproliferative disorders.