The authors would like to thank EPSRC (EP/R513313/1) for funding. The authors would like to also thank R.B Elliot and Son Abattoir in Calow, Chesterfield, for providing lambs&#39; heads and for being so accommodating in the early stages of the project, Kasia Emery for her support throughout the development of this protocol, and Fiona Wright from the Department of Infection, Immunity and Cardiovascular Disease at the University of Sheffield for processing the histology samples and being so incredibly helpful throughout this project.

Lambs&#39; heads from the R.B Elliott and Son Abattoir were used as the source of skin samples in this project. All lambs were slaughtered for consumption as food. Instead of discarding the heads, these were repurposed for research. Ethics approval was not required as the tissue was sourced from waste discarded from abattoirs.
1. Sterilization
<ol>
	<li>Disinfect forceps prior to collection of the heads by taking clean forceps and performing dry heat sterilization in an oven at 200 &#176;C for 1 h. Autoclave all glassware at 121 &#176;C for 15 min before use.</li>
	<li>Carry out all described work within a microbiology class 2 cabinet. Prepare all reagents as per the manufacturer&#39;s instructions.</li>
</ol>
2. Sample collection
<ol>
	<li>In the abattoir, Swaledale lambs were slaughtered by stunning using electricity or a captive-bolt pistol and exsanguinated. Collect lamb heads no more than 4 h post-slaughter.</li>
</ol>
3. Preparation of the heads
<ol>
	<li>Disinfect the forehead section of the lamb by pouring approximately 100 mL of 200 ppm chlorine dioxide solution onto the sample area. Shave the forehead section of the head using electric clippers and wash the area with 200 mL of 200 ppm chlorine dioxide solution.</li>
	<li>Wipe the area with ethanol and blue roll and cover the sample area with hair removal cream for 35 min. Gently scrape off the hair removal cream using a scraping tool and assess the sample area. If a significant amount of hair remains, repeat the hair removal process.</li>
	<li>Use a further 200 mL of chlorine dioxide solution to rinse the area, and then rinse with ethanol and wipe with a blue roll.</li>
	<li>Using a sterile 8 mm biopsy punch, cut out 8 mm skin samples from the prepared area. Remove the samples using sterile forceps and a 15-blade scalpel, ensuring that all cutaneous fat is removed.</li>
	<li>Place the samples in a sterile 0.5 L jar filled with sterile phosphate-buffered saline (PBS), and then transfer them to sterile 50 mL tubes with 50 mL of 200 ppm chlorine dioxide solution, invert twice, and leave to sterilize for 30 min.</li>
	<li>Remove the samples from the chlorine dioxide solution and wash them by placing them in a 50 mL tube filled with 40 mL of sterile PBS. Once washed, place each individual skin sample in a separate well of a 24-well plate.</li>
	<li>Add 350 &#181;L of pre-warmed medium while maintaining the sample at the air-liquid interface. The composition of the media is as follows: MK media (Medium 199 with Hanks&#39; salts, L-glutamate, and 1.75 mg/mL sodium bicarbonate) and Ham&#39;s F12 in a 1:1 ratio, with added FBS (10% v/v), EGF (10 ng/mL), insulin (5 &#181;g/mL), penicillin-streptomycin (100 U/mL), and amphotericin B (2.5 &#181;g/mL).</li>
	<li>Seal the 24-well plates with a gas permeable plate seal and incubate at 37 &#176;C in a humidified 5% CO2 tissue incubator for up to 24 h.</li>
</ol>
4. Maintenance of skin samples
<ol>
	<li>After incubation, remove the culture medium and rinse the samples in 500 &#181;L of sterile PBS. Add antibiotic-free media to each sample and incubate at 37 &#176;C in a humidified 5% CO2 tissue incubator for 24 h to remove residual antibiotics in the sample.</li>
	<li>If turbidity or fungal infection develop in the antibiotic-free media after 24 h, then discard the sample.</li>
</ol>
5. Preparation of the inoculum
<ol>
	<li>Prepare a 50 mL tube with 10 mL of sterile tryptic soy broth. Take a fresh agar plate of S. aureus and use a swab to transfer several colonies into the broth. Incubate for 18 h at 37 &#176;C at 150 rpm.</li>
	<li>Centrifuge at 4,000 x g for 3 min. Remove the supernatant and resuspend the cell pellet in 10 mL of sterile PBS. Repeat twice to ensure adequate washing of the cells.</li>
	<li>Adjust the inoculum to 0.6 OD600 in sterile PBS. Confirm the inoculum load by undertaking a manual viable plate count.</li>
</ol>
6. Infection of skin samples
<ol>
	<li>Prepare a fresh 24-well plate with 400 &#181;L of pre-warmed antibiotic-free media and add in the 24-well inserts using sterile forceps.</li>
	<li>Remove the media from the skin samples, wash with 500 &#181;L of sterile PBS, and remove the wash. Use sterile forceps to gently hold the sample to the bottom of the well.</li>
	<li>Use a 4 mm punch biopsy to make a central wound flap, piercing through to a rough depth of 1-2 mm. Then, use a 15-blade scalpel and sterile toothed allis tissue forceps to remove the top layer of the wound flap. Variability in the wound dimensions may affect the outcome of the infection and the endpoint colony forming units (CFU).</li>
	<li>Once all the samples have been wounded, transfer them to the 24-well inserts using sterile forceps. Pipette 15 &#181;L of the bacterial inoculum into the wound bed. Then, incubate for 24 h at 37 &#176;C in a humidified 5% CO2 tissue incubator.</li>
	<li>If longer incubation periods are necessary, remove the media and replace it with fresh media every 24 h and incubate in the same conditions.</li>
</ol>
7. Determination of the bacterial load
<ol>
	<li>Remove the media from the bottom of the wells. With sterile forceps, transfer each sample into a separate 50 mL tube filled with 1 mL of sterile PBS.</li>
	<li>Use a fine-tipped homogenizer to homogenize the surface of the sample. Take care to ensure that the wound bed is in direct contact with the tip of the homogenizer.</li>
	<li>Homogenize each sample for 35 s on medium/high. The homogenizer detaches the bacteria from the surface of the wound bed to allow for enumeration of the bacterial load.</li>
	<li>Once all the samples are processed, vortex each sample, in turn, prior to pipetting. This is to ensure the bacterial homogenate is mixed.</li>
	<li>Pipette 20 &#181;L of vortexed homogenate into the corresponding well of a 96-well plate containing 180 &#181;L of sterile PBS.</li>
	<li>Serially dilute each sample homogenate to 1 x 10&#8722;7 and pipette 10 &#181;L of the diluted homogenate onto a tryptic soy agar plate in triplicate.</li>
	<li>Incubate the agar plate for 18 h at 37 &#176;C. Then, count the number of colonies to determine the CFU for each sample.</li>
</ol>

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The authors have nothing to disclose

The development of antimicrobials is an important but expensive venture that is estimated to cost around $1 billion and take around 15 years to complete. Over 90% of antimicrobial drug discovery and preclinical studies of antimicrobial drug efficacy are carried out by academic researchers and small to medium companies with typically less than 50 employees22. These teams are very financially constrained, which makes the failure of lead molecules in later stages of translational research calamitous....

Skin infections are an important global issue, with large economic costs to healthcare providers around the world. The development of multidrug resistance and biofilm formation by pathogens plays a key role in the prevalence of non-healing wounds1,2,3,4. As a result of this, skin and soft tissue infections are one of the more common reasons for extended hospitalization and subsequent readmission5. Delays in wound healing are costly for both the patient and healthcare providers, with some estimates suggesting around 6.5 million patients are affected annually in the US. In the UK, skin infections and associated complications result in approximately 75,000 deaths annually2,4,6.
Staphylococcus aureus (S. aureus) is a formidable wound pathogen frequently isolated from patient wounds2,7. The rate of emergence of multidrug resistance increased drastically in the 2000s. During this time, around 60% of acute bacterial skin and skin structure infections were culture positive for methicillin-resistant S. aureus1. The increasing number of multidrug-resistant strains among Staphylococci, and indeed other pathogens, within the last 2 decades indicates an urgent need for the rapid development of antibiotics with new modes of action that can overcome resistance.
However, since the early 2000s, antibiotic discovery programs have been dominated by longer developmental times and low success rates, with only 17% of novel antibiotics entering clinical trials in the US achieving market approval8. This suggests a disparity between results from in vitro testing of emerging antibiotics and their clinical outcomes. It can be contended that this disparity is largely due to differences in bacterial physiology during infections in vivo and during conventional microbiological methods when testing the efficacy of antibiotics in the in vitro preclinical stages. Therefore, novel laboratory methods that are more representative of bacterial physiology during infection are needed to improve the success rates in antibiotic discovery programs.
Current methods for studying skin infections include studies in live animals (e.g., mice), ex vivo skin models (e.g., porcine), and 3D tissue-engineered skin models (e.g., human)9,10,11,12. Studies in live animals are strictly regulated and have relatively low throughput. In animal models, wounding and infection cause significant distress to the animals and raise ethical concerns. Human skin models, ex vivo or tissue-engineered, require ethical approval, compliance with local and global legislation (the Human Tissue Act, the Declaration of Helsinki), and there is difficulty in acquiring tissues, with some requests taking years to fulfil13,14. Both model types are labor intensive and require significant expertise to ensure experimental success. Some current ex vivo skin infection models require pre-inoculated discs and additives for the wound bed to enable infection; although these models are incredibly useful, there are limitations in the infection process as additives limit the utilization of the wound bed as a nutrient source10,15,16,17. The model described in this study uses no additives to the wound bed, which ensures that the pathology of infection and viable cell counts are a result of direct utilization of the wound bed as the only nutrient source.
Given the need for new laboratory methods, a novel high-throughput ex vivo ovine model of skin infections for use in evaluating the efficacy of emerging antibiotics has been developed. Skin infection studies face many challenges-high costs, ethical concerns, and models that do not show a full picture20,21. Ex vivo models and 3D explant models allow for better visualization of the disease process and the impact treatments can have from a more clinically relevant model. Here, the setup of a novel ovine skin model is described, which&#160;is simple, reproducible, and clinically relevant and has high throughput. Ovine skin was chosen as sheep are one of the large mammals commonly used to model responses to infections in vivo. Moreover, they are readily available from abattoirs, ensuring a steady supply of skin for research, and their carcasses are not scalded, ensuring good tissue quality. This study used S. aureus as the exemplar pathogen; however, the model works well with other microorganisms.

<table><tbody><tr><td>24 Well Companion Plate</td><td>SLS&amp;nbsp;</td><td>353504</td><td></td></tr><tr><td>4 mm Biopsy Punch</td><td>Williams Medical</td><td>D7484</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>Fisher Scientific&amp;nbsp;</td><td>10788561</td><td></td></tr><tr><td>8 mm Biopsy Punch</td><td>Williams Medical</td><td>D7488</td><td></td></tr><tr><td>Amphotericin B solution, sterile</td><td>Sigma&amp;nbsp;</td><td>A2942</td><td></td></tr><tr><td>Colour Pro Style Cordless Hair Clipper</td><td>Wahl</td><td>9639-2117X</td><td>Hair Clippers</td></tr><tr><td>Dual Oven Incubator</td><td>SLS</td><td>OVe1020</td><td>Sterilising oven</td></tr><tr><td>Epidermal growth factor&amp;nbsp;</td><td>SLS</td><td>E5036-200UG</td><td></td></tr><tr><td>Ethanol</td><td>Honeywell</td><td>458600-2.5L</td><td></td></tr><tr><td>F12 HAM</td><td>Sigma</td><td>N4888</td><td></td></tr><tr><td>Foetal bovine serum&amp;nbsp;</td><td>Labtech International</td><td>CA-115/500</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>15307805</td><td></td></tr><tr><td>Hair Removal Cream</td><td>Veet</td><td>Not applicable</td><td></td></tr><tr><td>Heracell VIOS 160i</td><td>Thermo Scientific</td><td>15373212&amp;nbsp;</td><td>Tissue culture incubator</td></tr><tr><td>Heraeus Megafuge 16R</td><td>VWR</td><td>521-2242</td><td>Centrifuge</td></tr><tr><td>Homogenizer 220, Handheld</td><td>Fisher Scientific</td><td>15575809</td><td></td></tr><tr><td>Homogenizer 220, plastic blending cones</td><td>Fisher Scientific&amp;nbsp;</td><td>15585819</td><td></td></tr><tr><td>Insert Individual 24 well 0.4um membrane</td><td>VWR International</td><td>353095</td><td></td></tr><tr><td>Insulin, recombinant Human</td><td>SLS</td><td>91077C-1G</td><td></td></tr><tr><td>Medium 199 (MK media)</td><td>Sigma</td><td>M0393</td><td></td></tr><tr><td>Microplate, cell culture Costar 96 well</td><td>Fisher Scientific</td><td>10687551</td><td></td></tr><tr><td>Multitron</td><td>Infors</td><td>Not applicable</td><td>Bacterial incubator</td></tr><tr><td>PBS tablets</td><td>Sigma&amp;nbsp;</td><td>P4417-100TAB</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>SLS&amp;nbsp;</td><td>P0781</td><td></td></tr><tr><td>Plate seals</td><td>Fisher Scientific</td><td>ESI-B-100</td><td></td></tr><tr><td>Safe 2020</td><td>Fisher Scientific</td><td>1284804</td><td>Class II microbiology safety cabinet</td></tr><tr><td>Scalpel blade number 15</td><td>Fisher Scientific</td><td>O305</td><td></td></tr><tr><td>Scalpel Swann Morton</td><td>Fisher Scientific</td><td>11849002</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761-1KG</td><td></td></tr><tr><td>Toothed Allis Tissue Forceps</td><td>Rocialle</td><td>RSPU500-322</td><td></td></tr><tr><td>Tryptic Soy Agar</td><td>Merck Life Science UK Limited</td><td>14432-500G-F</td><td></td></tr><tr><td>Tryptic Soy Broth</td><td>Merck Life Science UK Limited</td><td>41298-500G-F</td><td></td></tr><tr><td>Vimoba Tablets</td><td>Quip Labs</td><td>VMTAB75BX</td><td></td></tr></tbody></table>

a novel high-throughput ex vivo ovine skin wound model for testing emerging antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus&#160;is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen&#39;s ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

The identification of a route to sterilize the skin before setting up the wound infection model was challenging. The challenge lay in sterilizing the skin without damaging the different skin layers, which may then go on to have unintended consequences in the outcome of infection. To identify an appropriate sterilization regime, different treatments were tried for varying lengths of time, as outlined in Table 1. Contamination was recorded as the development of turbidity after 48 h in the MK medium used to...

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A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery ...

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Research

JoVE Journal

Immunology and Infection

1.9K Views.  University of Sheffield. The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials.

Video: A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicine

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Bioengineering

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Developmental Biology

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

Developing a Long-Term Swine Model for Biofilm-Infected Burn Wound Studies

Swine Model of Biofilm Infection and Invisible Wounds

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the involvement of the host immune system. Iterative changes in the host and pathogen during the formation of such clinically relevant biofilm can only occur in vivo. The swine wound model is recognized for its advantages as a powerful pre-clinical model. There are several reported approaches for studying wound biofilms. In vitro and ex vivo systems are deficient in terms of the host immune response. Short-term in vivo studies involve acute responses and, thus, do not allow for biofilm maturation, as is known to occur clinically. The first long-term swine wound biofilm study was reported in 2014. The study recognized that biofilm-infected wounds may close as determined by planimetry, but the skin barrier function of the affected site may fail to be restored. Later, this observation was validated clinically. The concept of functional wound closure was thus born. Wounds closed but deficient in skin barrier function may be viewed as invisible wounds. In this work, we seek to report the methodological details necessary to reproduce the long-term swine model of biofilm-infected severe burn injury, which is clinically relevant and has translational value. This protocol provides detailed guidance on establishing an 8 week wound biofilm infection using P. aeruginosa (PA01). Eight full-thickness burn wounds were created symmetrically on the dorsum of domestic white pigs, which were inoculated with (PA01) at day 3 post-burn; subsequently, noninvasive assessments of the wound healing were conducted at different time points using laser speckle imaging (LSI), high-resolution ultrasound (HUSD), and transepidermal water loss (TEWL). The inoculated burn wounds were covered with a four-layer dressing. Biofilms, as established and confirmed structurally by SEM at day 7 post-inoculation, compromised the functional wound closure. Such an adverse outcome is subject to reversal in response to appropriate interventions.

Author Spotlight: Studying Host-Microbe Interactions in Wound Biofilm Formation 

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the ...

A Murine Skin Wound Infection Model to Study the Immune Response against a Bacterial Pathogen

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates a technique to generate a murine model of skin wound infection. Upon infecting mice &#8212; engineered to express a fluorescent protein in neutrophils &#8212; with a genetically modified bioluminescent Staphylococcus aureus &#8212; a pathogenic bacteria. The bacterial burden at the infection site and the neutrophil recruitment dynamics can be studied using whole animal imaging.

Source: Anderson, L. S., et al. A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection. J. Vis. Exp. (2019).
This video ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

Source: Okurowska, K., et al. <a href="https://www.jove.com/v/61156">Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis.</a> J. Vis. Exp (2020).
This video describes a protocol to set up an ex vivo model for bacterial keratitis using a corneoscleral button dissected from the porcine eye. Inoculated bacteria traverse the corneal epithelial incisions to invade the underlying stroma and establish an infection within the corneal tissues that mimic the in vivo infection.

Source: Okurowska, K., et al. Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis. J. Vis. Exp (2020).
...

Biology

Large Animal Models

Porcine

Generating an Ex Vivo Ovine Wounded Skin Model Infected with a Bacterial Pathogen

Source: Regan, H. C., et al. <a href="https://app.jove.com/v/64041">A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics.</a> J. Vis. Exp. (2022).
The video demonstrates the generation of an ex vivo ovine skin model infected with Staphylococcus aureus. This entails treating a lamb&#39;s forehead skin biopsy section with antibiotics, establishing a wound bed, positioning it on a permeable membrane in a multiwell plate with antibiotic-free medium, and establishing bacterial infection in the skin wound.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Immunotherapy

Model Development and Disease Study

Porcine Ex Vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

Remove the media from the Petri dish, and rinse the corneas twice with one milliliter of sterile PBS. Hold the cornea with forceps and squeeze gently. Use a 10A scalpel to make four cuts - two vertical, two horizontal - in the central section of the corneoscleral button through the epithelial layer to the underlying stroma.
Place a sterile glass mold in a six-well plate with the wide part up. Then, place the cornea in the middle of the glass mold, with the epithelium side facing down, and the wounded part of the cornea centered in the glass mold.
Fill the glass mold completely by adding one milliliter of agar solution. After allowing the agar to set, invert the glass mold so that the corneal epithelium is facing upwards.
The glass mold has to be sealed with agar up to the brim to prevent leaking of the inoculum or drug solution.
Pipette 200 microliters of the bacterial culture directly into a cut area. Also, for each experiment, set up a control by adding 200 microliters of sterile PBS to one cornea, instead of adding the bacterial culture.
Next, add 185 microliters of PBS to the top of each cornea to keep the epithelium moist. Then, add one milliliter of DMEM without antibiotics to the bottom of each well. Incubate the six-welled plate at 37 degrees Celsius with humidity and 5% carbon dioxide, for up to 24 hours.
Discard the DMEM from the six-welled plate and add one milliliter of sterile PBS to each well to rinse it. Remove the PBS gently, without touching the central part of the corneoscleral button. Remove the glass mold using sterile forceps, and place it in 5% Distel. Gently rinse the top of the corneoscleral button twice with one milliliter of PBS.
Use fine-tip forceps to lift the edge of the corneoscleral button, detach it from the agar underneath, and transfer it to a 50-milliliter tube filled with one to two milliliters of ice-cold PBS. To help detach bacteria from the corneal epithelium and the cut area, add PBS to the tube and use a fine-tip homogenizer to shear the top of the infected cornea. The tissue does not have to be completely liquidized.
Vortex to resuspend settled bacteria, and add 20 microliters of the homogenized cornea to 180 microliters of PBS. Then, perform serial dilutions of the homogenate in a 96-well plate.
Pipette 10 microliters of the diluted homogenate onto a blood agar plate.
After incubating the plate for 16 hours, count the number of colony-forming units.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

Summary

Explore More Videos

Chapters in this video

Murine Model of Wound Healing

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

Swine Model of Biofilm Infection and Invisible Wounds

A Murine Skin Wound Infection Model to Study the Immune Response against a Bacterial Pathogen

Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

Generating an Ex Vivo Ovine Wounded Skin Model Infected with a Bacterial Pathogen

Porcine Ex Vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells