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A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft
Chapters
Summary August 8th, 2020
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.
Transcript
The overall goal of this procedure is to create a reproducible murine model of full thickness burn wound reconstructed with an allergic skin graft. Skin is divided into two components the epidermis and the dermis. Beneath the dermis is subcutaneous fat, which covers bone, tendon or fascia.
The thickness classification of burn injury is often used to define the extent of burn injury depth. Superficial burns involve only epidermis, Superficial partial thickness burns involve epidermis and only pillory or upper dermis. Deep dermal partial thickness burns involve epidermis and dermis, the reticular dermis containing blood vessels, hair follicles, oil and sweat glands.
Full thickness burns are characterized by evidenting involvement of the whole thickness of the skin and possibly subcutaneous tissue. This type of wound can only heal by contraction or with skin grafting. The overall goal of this procedure was to create a full thickness burn wound and reconstructed with a full thickness skin graft.
This was achieved by putting a circular brass block preheated to 80 degrees Celsius and applied to mouth skin for the duration of 20 seconds. Necrotic tissue was surgically excised 24 hours after the burn injury. Full thickness skin grafts harvested from the tail of a donor mouse were laid over the excised burn wounds.
The grafts will then secured with a surgical adhesive. Both donor and recipient animals come from the same inbred strain of mice. Biopsy mice are used during the course of the study.
However, also strain of mice may also be used. This includes immunocompetent hairless mice, which are particularly useful in wound healing studies. As hair removal and associated inflammation a hobo did and hair regrowth does not interfere with digital image analysis, when area measurements and other types of microscopic wound assessment.
This is especially true in models of infection, where our suspension of bio-organism and bacteria is applied to this your face of the wound and bio-organism signal is measured to indicate the rate of bacterial growth. The thermal burn injury device consists of a custom-made circular brass block measuring 10 millimeters in diameter preheated to 80 degrees Celsius. The temperature of the brass block is verified using an infrared thermal imaging camera.
To create a full thickness burn wound, the brass block is applied for 20 seconds using a constant pressure of 0.15 Newtons which is monitored by digital manometer. Multimodal analgesia is the best practice for pain management in invasive animal research procedures. The ideal administration regime of analgesia includes pre-emptive or pre-procedural analgesic administration.
Paracetamol at three milligrams per meal was added to drinking water and supplied 12 hours before and for 72 hours after the procedure. Buprenorphine at 0.05 microgram per gram was administered subcutaneously 30 minutes before the procedure and every six hours for the first 72 hours after the procedure. Lidocaine at five milligrams per kilogram was administered subcutaneously around the dorsum of each mouse 15 minutes before the procedure, a total volume of 200 microliters of Lactated Ringers Solution at 5%dextrose was injected subcutaneously before the procedure, and six hours after the procedure to prevent dehydration.
Mice were nisertide using an intraperitoneal injection of Xylazine at 10 milligrams per kilogram, and Ketamine at 100 milligrams per kilogram. Anesthetic depth can be assessed via a toe pinch. Anesthesia abolishes the blink reflex, preventing the refreshment of the tear film required for the maintenance of corneal hydration and eye continuously exposed to air quickly becomes dehydrated, a lubricant was applied on the eyes to maintain hydration of the cornea.
The dorsum of each mouse is clipped and depilating cream is applied for one minute to remove hair. The nisertide animal is positioned prone on a flat table and thermal burn is created by applying a circular brass block preheated to 80 degrees Celsius for the duration of 20 seconds. The mouse, is placed back on the heating pad to keep it warm and prevent hypothermia, a frequent side effect of the anesthetic agents.
Once recovered from anesthesia, the mouse may be placed into an individual cage. 24 hours after the induction of burn injury. Necrotic tissue is surgically excites.
Four sickness skin graft are harvested from euthanize donor mouse tail. Although other side so there's under tail may also be using the mouse model. So degree of human response may vary depending on the origin of donor skin graft.
For example, ear and back skin have no longer in sales in comparison to tail skin and provoke a more robust human response, which may lead to graft rejection. Skin grafts thickness may also be taken into account as it may play a determining factor in successful engraftment of skin graft take. Secure skin such as that from the back has higher probability of failure due to the higher metabolic demands of the tissue.
Consequently, the reported success of allergenic skin graft engraftment for back skin is lower than the reported success for the tail skin graft. Anesthesia was induced by inhalation of 5%Isoflurane in 100%oxygen at a flow rate of four liters per minute, followed by maintenance of anesthesia using 2%Isoflurane at two liters per minute for the maintenance during surgery. Buprenorphine was administered at the initiation of the procedure to provide postoperative analgesia.
Burn wounds were prepped with Povidone-iodine, followed by 70%alcohol. Necrotic Burn is excised with scissors to remove the circular full thickness skin defect, the panniculus carnosus layer of the hypodermis is removed to create a stable skin graft recipient bed some authors suggest preserving the panniculus carnosus layer of the hypodermis as it is believed to be necessary for revascularization of the graft. However, in this case, the mobility of this layer and the resultant potential shear forces between the graft and recipient bed may become problematic.
Removal of the panniculus carnosus layer did not adversely impact the success of the grafting procedure. Full thickness grafts, are harvested from the tail of euthanized donor mouse. The tail is incised longitudinally, allowing the skin to be separated from the underlying soft tissue.
The tail skin is then spread on a Petri dish filled with sterile, normal saline and cut into circular patches using a ruler. The grafts are then transferred to a Petri dish with a sterile gauze soaked in normal saline and kept the four degrees until ready to be grafted. The grafts are then secured to the recipient bed with surgical glue.
The gaps between donor skin graft and recipient wound bed are gently press to align the skin edges. The wounds are dressed within inert paraffin gauze and adhesive secondary dressing. Digital photographs, of grafted wounds were taken at one, three and seven days post burn injury.
At the post-mortem the dorsal burn wounds were surgically excised to the fascia, wounds were bisected with one half fixed in 10%buffered formalin, and processed for histology and immunohistochemistry. Skin grafts provide a scaffold system for cellular migration. Successful graft integration depends on efficient fibroblasts and keratinocyte migration from the recipient bid into the donor graft which is slowly degraded and replaced by cells capable of producing the extracellular matrix.
Histological section of mass wounds was subjected to hematoxylin and eosin staining. Representative images of hematoxylin and eosin stained sections of wounds, showed that the length of near epidermis highlighted in yellow is significantly increased on day seven, compared to day three after burn injury. To estimate the rate of re-epithelization, the area of the wound that was covered with near epidermis was expressed as a percentage of the entire wound.
The extracellular matrix is the non cellular component of the skin, which provides scaffolding for the cellular components. The extracellular matrix is composed of fibrous proteins, such as Collagen and Fibronectin. These molecules enhance the maturity of wounds and maintain the structural integrity of the skin semiquantitative immunohistochemistry for collagen type one, and histological stains such as Mason's trichrome may be used to assist the extracellular matrix integrity from photo micrograph.
More sophisticated methods such as second harmonic generation microscopy have emerged as a powerful tool for quantitative analysis of collagen structure. Murine skin grafting provides an excellent potential preclinical model to investigate the integration of graphs into the recipient side. This study demonstrates a reliable method to successfully perform skin grafts in a mouse model as well as the histological assessment of the healing wounds.
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