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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 fetal bovine serum (FBS, 10% v/v), epidermal growth factor (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 phosphate-buffered saline (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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 Well Companion Plate</td>
			<td>SLS</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</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</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</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</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</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>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</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>SLS</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 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>

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.

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

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

Take a pre-sterilized skin biopsy section obtained from a lamb&#39;s forehead in an antibiotic-free medium.
The section comprises an outer epidermis and an underlying dermis layer.
Remove the medium and use a buffer to wash the section.
Make a circular cut in the section and remove the top layer. This breaks the barrier, generating a wound bed in the skin.
Transfer the wounded sections onto a permeable membrane insert in a multiwell plate containing an antibiotic-free medium.
Introduce a suspension of pathogenic bacteria onto the wound bed and incubate.
During incubation, the wound bed provides a tissue surface for bacterial attachment and the nutrients to support bacterial growth.
This allows the multiplication and spreading of the bacteria across the wound bed, establishing an infection.

generating-an-ex-vivo-ovine-wounded-skin-model-infected-with

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JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

37 Views. Source: Regan, H. C., et al. A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics. 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'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. 

Video: Generating an Ex Vivo Ovine Wounded Skin Model Infected with a Bacterial Pathogen - Concept

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

JoVE Journal

Immunology and Infection

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

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 ...

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

Transcript

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

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

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