generating an excisional skin wound in a mouse model via punch biopsy

Generating an Excisional Skin Wound in a Mouse Model via Punch Biopsy

For excisional skin wounding of the recipient animals, confirm a lack of response to toe pinch before shaving a 1- by 2-inch section on the back of the mouse. Gently wipe to remove any stray hairs, and disinfect the exposed skin with sequential 10% povidone-iodine and 70% ethanol-soaked gauze applications, moving outward from the center of the surgical area in a spiral pattern.
After allowing the skin to dry for about one minute, firmly press a sterile 6-millimeter punch biopsy at the center of the prepared surgical area. Twist the punch biopsy to create a circular outline on the skin that fully cuts through the skin tissue in at least one section of the outline, taking care not to cut into the underlying fascia or tissue. Then, use sterile scissors and forceps to cut through the epidermis and dermis, following the circular pattern imprinted by the punch biopsy.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

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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. Mouse Source and Housing
<ol>
	<li>Derive Lysozyme M- enhanced green fluorescent protein (LysM-eGFP) mice on a C57BL/6J genetic background as described previously. Derive LysM-EGFP&#215; myeloid differentiation primary response 88 (MyD88)-/- mice by crossing LysM-EGFP mice with MyD88-/- mice on a C57BL/6J background.</li>
	<li>House mice in a vivarium. For these studies, animals were housed at the University of California, Davis in groups prior to surgery and singly housed following surgery. Use mice between the ages of 10-16 weeks.</li>
</ol>
2. Excisional Skin Wounding 
<ol>
	<li>Administer 100 &#181;L of 0.03 mg/mL buprenorphine hydrochloride (~0.2 mg/kg) to each mouse via intraperitoneal injection.</li>
	<li>Twenty minutes post-injection, place 2-4 mice in a chamber with 2-3 liters per minute (LPM) oxygen with 2-4% isoflurane. Once mice are fully anesthetized, transfer the mice one at a time to a nose cone connected to 2-3 LPM oxygen with 2-4% isoflurane. Verify mice are fully anesthetized by firmly pinching each rear paw between a thumb and forefinger. Proceed to the next step if the animal does not respond to the pinch.</li>
	<li>Shave a 1-inch by 2-inch section on the back of the mouse with electric clippers and clear the area of fur clippings using a clean wipe or gauze. Avoid using depilatory lotion because it may cause excess inflammation.</li>
	<li>Clean the back of the mouse first with 10% povidone-iodine-soaked gauze and then with 70% ethanol-soaked gauze. Clean the area in a spiral pattern, moving outward from the center of the surgical area. Wait approximately 1 minute for the surgical area to dry prior to surgery.</li>
	<li>Hold the shaved back of the mouse loosely between two fingers and firmly press a sterile 6 mm punch biopsy at the center of the prepared surgical area. Do not pull the skin taut.
	<ol>
		<li>Twist the punch biopsy to create a circular outline on the skin that fully cuts through the skin in at least one section of the outline. Be careful not to cut into the underlying fascia or tissue.</li>
		<li>Use sterile scissors and forceps to cut through the epidermis and dermis following the circular pattern imprinted by the punch biopsy.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6mm Disposable Biopsy Punch</td>
			<td>Integra Miltex</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenoprhine hydrochloride injectable</td>
			<td>Western Medical Supply</td>
			<td>7292</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>Jackson Labratory</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>LysM-eGFP Mice</td>
			<td>Thomas Graff Albert Einstein College of New York</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MyD88 KO Mice</td>
			<td>Jackson Labratory</td>
			<td>9088</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-woven sponges</td>
			<td>AMD- Ritmed Inc</td>
			<td>A2101-CH</td>
			<td>5 cm x 5 cm</td>
		</tr>
		<tr>
			<td>Povidone Iodine 10% Solution</td>
			<td>Aplicare</td>
			<td>697731</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 punch biopsy procedure to create a precise wound in a mouse model. The excision exposes the subcutaneous layer of the skin, providing a suitable site for studying immune responses to localized pathogen infection.

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

To create an excisional skin wound, begin by placing an anesthetized mouse in a prone position and shaving its back to expose the epidermis, the outermost skin layer.
Apply a disinfectant solution to reduce the contamination risk. Take a punch biopsy tool &#8212; a hollow metal tool with a circular blade at the edge, to create a precise cut.
Position the punch biopsy tool perpendicularly at the center of the pre-prepared surgical area. Apply downward pressure on the epidermis to create a circular incision.
Twist the punch biopsy tool while penetrating the epidermis and dermis &#8212; the middle layer of the skin. This generates a complete circular cut on the epidermis and dermis without damaging the underlying fascia which is a fibrous connective tissue layer encasing the muscle.
Dissect the layers of skin following a circular pattern and remove them, creating an excisional skin wound with an exposed subcutaneous layer, ready for immunological studies.

375 vistas. Generación de una herida cutánea por escisión en un modelo de ratón mediante biopsia por punción

Video: Generación de una herida cutánea por escisión en un modelo de ratón mediante biopsia por punción - Experimento

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

Demostración de la rata isquémica de la piel herida Modelo

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Investigación

JoVE Journal

Medicina

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

La denervación quirúrgica cutánea: Un método para probar el requisito de nervios en el ratón modelos de enfermedad de la piel

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Un modelo murino de una herida de quemadura reconstruida con un injerto de piel alogénico

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Generating an Excisional Skin Wound in a Mouse Model via Punch Biopsy

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