The authors would like to acknowledge funding support from the University of South Florida, Department of Surgery (Mack Wu, MD, PhD).

NOTE: Approval for all animal procedures presented below was obtained from the University of South Florida&#8217;s Animal Care Committee (IACUC) and abide by all requirements of the Animal Welfare Act and the Guide for Care and Use of Laboratory Animals.
1. Preparation of Silicone Sheets and Surgical Instruments
<ol>
	<li>Precut strips (10.5 cm x 3.0 cm) of non-reinforced 0.01 thickness, medical grade silicone sheeting and sterilize using an autoclave.</li>
	<li>Clean and sterilize suitable surgical instruments (scissors, forceps and drapes or towels to create a sterile field during surgery).</li>
</ol>
2. Experimental Animals
<ol>
	<li>Use adult male or female rats weighing in range from 250-350 g obtained from a commercial breeder. If aged rats are utilized, they should be &#8805;350 g in order to ensure better survival post-surgery. Prior to the start of any experiments acclimate all animals for at least 7 days under standard conditions of a 12 hr light-dark cycle with food and water ad libitum.</li>
</ol>
3. Anesthesia, Pre-operative Analgesia and Operative Preparation
<ol>
	<li>Induce general anesthesia by using isoflurane at 3%-4% via an induction chamber and maintain (via use of a nose cone) at 1%-2% with O2 during skin preparation and surgery. Monitor the depth of anesthesia by observation of the rate and depth of respiration, interdigital pinch or palpebral blink reflex. 
	NOTE: At this time, a vet ointment can be placed on the eyes to prevent dryness while the animal is under anesthesia.</li>
	<li>In a location remote from the sterile surgical area, place the rat in the prone position and shave the dorsum with clippers from the base of the neck down approximately 11 cm. Stencil with permanent marker, the outline for the 3.0 cm x 10.5 cm flap (see Figure 1A).</li>
	<li>Move the rat to a clean, designated surgical area equipped with an approved heating pad and sterile surgical drapes or towels. Inject 5 mg/kg Ketoprofen subcutaneously prior to the first surgical incisions for pain management. Additional fluids (saline) can be given (up to 5 cc) subcutaneously as needed.</li>
	<li>Prepare the skin further by swabbing first with 70% isopropyl alcohol and second with 0.2% chlorhexidine, then apply sterile drapes to create a sterile field. 10% povidone-iodine (Betadine) can also be used. 
	NOTE: An antibiotic (ampicillin at 15 mg/kg) can be administered subcutaneously, but if good aseptic techniques are used it is not required.</li>
</ol>
4. Creating Excisional Wounds and Bipedicled Flap
<ol>
	<li>Using a sterile, disposable 6 mm biopsy punch tool, create two circular &#8220;ischemic&#8221; wounds in the center of the designated flap area (Figure 1B). The depth of the wound should be down to (not through) the underlying fascia of the panniculus carnosus muscle (Figure 1B inset).</li>
	<li>Using forceps lift the skin in the middle of the wound outline created by the punch biopsy and then use iris scissors (with curved tips) to excise the circular piece of tissue (including the panniculus carnosus muscle). The result will be a full-thickness wound with the fascia as the base of the wound. 
	NOTE: The excised tissue (wound plugs) can be snap frozen in liquid nitrogen or fixed in 10% buffered-formalin O/N for later processing as control, normal skin.</li>
	<li>Create a bipedicled flap by making incisions with a sterile scalpel on each side of the ischemic wounds along the pre-drawn lines (Figure 1C) that are 10.5 cm in length and 3.0 cm apart. The depth of the incisions should be down to the paraspinous muscles. Using iris scissors, separate the panniculus carnosus fascia from the paraspinous muscles, being careful to keep the fascia intact as the &#8220;base&#8221; of the 6 mm punches (Figure 1D).</li>
	<li>Take 1 sterile pre-cut silicone sheet and place it in between the panniculus carnosus fascia and the paraspinous muscles (Figure 1E) ensuring that the sheet does not buckle or fold. Using black, non-absorbable sutures (size 4.0) close both incisions by anchoring the silicone sheet to the skin with at least 8 interrupted stitches on each side, along the length of the flap (Figure 1F and 1G).</li>
	<li>Using a sterile, disposable biopsy punch tool, create two internal control &#8220;non-ischemic&#8221; wounds (down to the anterior fascia of the panniculus carnosus muscle) 1 cm lateral to the ischemic wounds on either side of the flap (Figure 1G).</li>
	<li>Place a ruler below the wounds and take digital photos for wound measurement purposes (see Figure 3A). At this time, blood flow (perfusion) can be monitored by use of laser Doppler or other manipulations (topical drug placement) performed.</li>
	<li>Apply an approved liquid adhesive both cranial and caudal to the wounds and a transparent film dressing to keep the wound environment moist and clean (sterile). An additional dressing may be placed at the caudal end of the flap to prevent the animal from removing the most caudal sutures.</li>
</ol>
5. Post-operative Procedures
<ol>
	<li>Place animals in cages (singly housed) equipped with shallow feeders so as to prevent the surgical site from rubbing against the feeder. Animals should not be left unattended or returned to the company of other animals until they regain sufficient consciousness to maintain sternal recumbency and exhibit purposeful movement. Heating mats should be placed under half of the cage for up to 2 days during recovery.</li>
	<li>To manage pain post-operatively, administer Ketoprofen (5 mg/kg) subcutaneously to animals the following morning and 1x per day for up to 48 hr post-surgery. Animals should also be monitored daily for prolonged signs of pain, weight loss or surgical site infections.</li>
</ol>
6. Subsequent Wound Measurements and Dressing Changes
<ol>
	<li>Measure ischemic and non-ischemic wounds frequently under general anesthesia using isoflurane at 3%-4% via an induction chamber and maintained (via a nose cone) at 1%-2% with O2 as in step 3.1.</li>
	<li>Remove the dressing gently as to not pull the adhesive from the skin. At this time additional digital photos are taken for wound measurements, topical treatments re-applied, laser Doppler imaging (LDI) or other manipulations performed to suit the investigator&#8217;s needs.</li>
	<li>Apply adhesive and a clean dressing and allow the animal to recover as in step 5.1.</li>
</ol>
7. Wound Collection and Euthanasia
<ol>
	<li>Harvest ischemic and non-ischemic wounds (on days the investigator deems appropriate) while the animal is under general anesthesia using isoflurane at 3%-4% via an induction chamber and maintained (via a nose cone) at 1%-2% with O2 as in step 3.1.</li>
	<li>Using a scalpel, make a square shaped excision around the wound to include some healthy tissue around the wound. Place the excision into a 1.5 ml snap cap tube and snap freeze in liquid nitrogen (store at -80 &#176;C) for future molecular analysis or incubate in 10% buffered formalin O/N at RT for histological processing. 
	NOTE: The wound excisions can also be cut in half to provide more samples for analysis.</li>
	<li>After wound tissue removal, euthanize the animal using the approved method of CO2 inhalation.</li>
</ol>

<ol><li>Ansell, D. M., Holden, K. A., Hardman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+wound+repair%3A+Are+they+cutting+it%5BTitle%5D)+AND+%22Experimental+dermatology%22%5BJournal%5D)">Animal models of wound repair: Are they cutting it.</a> Experimental dermatology. 21 (8), 581-585 (2012).</li>
<li>Dorsett-Martin, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+models+of+skin+wound+healing%3A+a+review%5BTitle%5D)+AND+%22Wound+repair+and+regeneration+%3A+official+publication+of+the+Wound+Healing+Society+%5Band%5D+the+European+Tissue+Repair+Society%22%5BJournal%5D)">Rat models of skin wound healing: a review.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 12 (6), 591-599 (2003).</li>
<li>Davidson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+for+wound+repair%5BTitle%5D)+AND+%22Arch+Dermatol+Res%22%5BJournal%5D)">Animal models for wound repair.</a> Arch Dermatol Res. 290 (S1-11), (1998).</li>
<li>Finn, G., Magnus, S. A., Tonny, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Models+for+use+in+wound+healing+research%3A+A+survey+focusing+on+in+vitro+and+in+vivo+adult+soft+tissue%29%5BTitle%5D)+AND+%22Wound+Repair+and+Regeneration%22%5BJournal%5D)">Models for use in wound healing research: A survey focusing on in vitro and in vivo adult soft tissue).</a> Wound Repair and Regeneration. 8, (2000).</li>
<li>Gould, L. J., Leong, M., Sonstein, J., Wilson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+and+validation+of+an+ischemic+wound+model%5BTitle%5D)+AND+%22Wound+repair+and+regeneration+%3A+official+publication+of+the+Wound+Healing+Society+%5Band%5D+the+European+Tissue+Repair+Society%22%5BJournal%5D)">Optimization and validation of an ischemic wound model.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 13 (6), 576-582 (2004).</li>
<li>Loots, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differences+in+cellular+infiltrate+and+extracellular+matrix+of+chronic+diabetic+and+venous+ulcers+versus+acute+wounds%5BTitle%5D)+AND+%22The+Journal+of+investigative+dermatology%22%5BJournal%5D)">Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds.</a> The Journal of investigative dermatology. 111 (5), 850-857 (1998).</li>
<li>Schwartz, D. A., Lindblad, W. J., Rees, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+collagen+metabolism+and+delayed+healing+in+a+novel+model+of+ischemic+wounds%5BTitle%5D)+AND+%22Wound+Repair+Regen%22%5BJournal%5D)">Altered collagen metabolism and delayed healing in a novel model of ischemic wounds.</a> Wound Repair Regen. 3 (2), 204-212 (1995).</li>
<li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+and+mechanistic+validation+of+delayed+healing+rat+wounds+as+a+model+for+human+chronic+wounds%5BTitle%5D)+AND+%22Wound+Repair+and+Regeneration%22%5BJournal%5D)">Molecular and mechanistic validation of delayed healing rat wounds as a model for human chronic wounds.</a> Wound Repair and Regeneration. 7, (1999).</li>
<li>Zhang, Q., Chang, Q., Cox, R. A., Gong, X., Gould, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperbaric+oxygen+attenuates+apoptosis+and+decreases+inflammation+in+an+ischemic+wound+model%5BTitle%5D)+AND+%22The+Journal+of+investigative+dermatology%22%5BJournal%5D)">Hyperbaric oxygen attenuates apoptosis and decreases inflammation in an ischemic wound model.</a> The Journal of investigative dermatology. 128 (8), 2102-2112 (2008).</li>
<li>Zhang, Q., Gould, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperbaric+oxygen+reduces+matrix+metalloproteinases+in+ischemic+wounds+through+a+redox-dependent+mechanism%5BTitle%5D)+AND+%22The+Journal+of+investigative+dermatology%22%5BJournal%5D)">Hyperbaric oxygen reduces matrix metalloproteinases in ischemic wounds through a redox-dependent mechanism.</a> The Journal of investigative dermatology. 134 (1), 237-246 (2013).</li>
<li>Moor, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Consequences+of+age+on+ischemic+wound+healing+in+rats%3A+altered+antioxidant+activity+and+delayed+wound+closure%5BTitle%5D)+AND+%22Age+%28Dordrecht%2C+Netherlands%29%22%5BJournal%5D)">Consequences of age on ischemic wound healing in rats: altered antioxidant activity and delayed wound closure.</a> Age (Dordrecht, Netherlands). 36 (2), 733-748 (2014).</li>
<li>Roy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+preclinical+model+of+chronic+ischemic+wound%29%5BTitle%5D)+AND+%22Physiological+genomics%22%5BJournal%5D)">Characterization of a preclinical model of chronic ischemic wound).</a> Physiological genomics. 37 (3), 211-224 (2009).</li>
<li>Biswas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+inducible+microRNA+210+attenuates+keratinocyte+proliferation+and+impairs+closure+in+a+murine+model+of+ischemic+wounds%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Hypoxia inducible microRNA 210 attenuates keratinocyte proliferation and impairs closure in a murine model of ischemic wounds.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (15), 6976-6981 (2010).</li>
<li>Winocour, S., Vorstenbosch, J., Trzeciak, A., Lessard, L., Philip, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD109%2C+a+novel+TGF-beta+antagonist%2C+decreases+fibrotic+responses+in+a+hypoxic+wound+model%5BTitle%5D)+AND+%22Exp+Dermatol%22%5BJournal%5D)">CD109, a novel TGF-beta antagonist, decreases fibrotic responses in a hypoxic wound model.</a> Exp Dermatol. 23 (7), 475-479 (2014).</li>
</ol>

The authors have nothing to disclose.

Wound healing in rats has often been the subject of debate due to their ability to heal infected wounds and high rate of interanimal variability5. One of the original goals of the model during its development was to decrease this variation. Modifications to the width of the flap, reducing the number of wounds with specific placement (centered on the flap with consistent cranio-caudal location) and introduction of a silicone sheet has accomplished this goal. Wound healing by contraction has also been reduced and healing by epithelialization, as in humans, is the measured outcome. Adaptation of the model to a different strain of rat, i.e., the F344, has also proven successful and reproduces the degree of ischemia observed using Sprague Dawley rats. Overall the surgical techniques (biopsy, suturing and wound excision) required for this model are easily acquired by most students and technicians with limited surgical experience.
To achieve consistency with this model while performing multiple surgeries, it was found that it is important to create the ischemic wounds prior to elevation of the flap for silicone sheet placement5. Additionally, not punching through the panniculus carnosus fascia is critical to provide a viable wound bed to remain over the silicone. The silicone acts not only to prevent vascular regrowth but also as a &#8220;splint&#8221; that encourages wound re-epithelialization. The application of the adhesive and dressings to prevent infection and maintain a moist environment for wound healing is also important. Product choice can be what is preferred or used in the researcher&#8217;s animal facility. However, it is not uncommon for some of the animals to be able to remove their dressings, no matter what type of adhesive/dressing combination is used.
The bipedicled flap should remain viable throughout a time course of healing which is approximately 28 days, depending on rat strain and other co-morbidities present. Rarely, abscesses can form in the flap (particularly near sutures) and seromas may form under the flap. Fluid can be drained and antibiotics administered if necessary. However, if the flap loses viability and becomes necrotic it is recommended that that animal no longer be used. Wound excision for biochemical analysis does introduce variability due to (1) some normal tissue must be retained for support (2) the choice of tissue homogenization and preparation for isolation of RNA, DNA or protein and (3) inherent interanimal variability5,10,11. One could consider this last point a limitation to the model and it was found that reducing the size of the flap (&#60;2.0 cm) or flap trauma can cause necrosis, indicating that minor variations in factors such as temperature or stress levels, may also lead to biochemically detectable variation between wound samples from one rat to another5.
In summary, this model, with a longitudinal, bipedicled flap ranging from 2.0-3.0 cm in width and a strategically placed silicone sheet, is a reliable model of prolonged tissue ischemia. Once the user is adept at using the techniques to create a consistent ischemic wound, they should be able to adapt it to additional ages and species of rodents (mice included). The excisional wounds can be treated topically, or systemic treatments utilized to further explore the mechanism(s) involved in chronic wound formation, exacerbated inflammatory responses, aberrant angiogenesis and delayed wound closure.

Effective drug development and other wound healing therapeutics require appropriate in vivo models, despite known problems in translating findings in animal models to human therapies1. What follows is a description of a detailed protocol for the use of a rat model of ischemic skin wound healing to investigate mechanisms that further the understanding of pathological wound healing. The rat species, often employed due to its wide availability, size and docile nature is used for wound healing studies as it is large enough to provide a suitable skin area for incisional and excisional wounding, imaging and tissue collection2. However it should be taken into careful consideration that the skin of a rat and a human are different anatomically, with rats being referred to as loose-skinned animals. This distinct characteristic allows for wound contraction, rather than epithelialization to contribute significantly to the closing of rat skin wounds2. Additionally, the presence of a subcutaneous panniculus carnosus muscle in rats, contributes to healing by both contraction and collagen formation3,4. These very important anatomical distinctions were considered in the development of the rat ischemic skin wound model and specific modifications were implemented to decrease wound contraction and reduce the influence of the panniculus carnosus muscle5.
In diabetic foot ulcers, venous leg ulcers, and pressure ulcers, healing is delayed and these wounds are considered chronic. The wounds are characterized by excessive inflammation, which prevents the wound from progressing to the next phases of wound repair6. One of the main factors in the development of a chronic wound is localized tissue ischemia (reduced blood flow)5 contributing to the inability to clear inflammation. At the time during which this model was being developed and validated (in 2003-4), there were no standardized animal models that could provide enough tissue to test induction of angiogenesis in the wound bed, a key stage during normal wound healing and the motivation for developing this model5. That said, the model presented here is a modification of the ischemic wound model originally described by Schwartz et al.7 and subsequently used in modified form by Chen et al.8
In the modified ischemic wound model, changes have been made to circumvent the above mentioned anatomical characteristics of the rat that lead to healing by contraction rather than epithelialization: (1) Two full-thickness excisional wounds are created within a bipedicled dorsal skin flap and the panniculus carnosus muscle is removed from the wound bed by dissecting just above the muscle fascia. (2) The flap itself has more narrow dimensions, ensuring that blood supply is random and the wounds located at the midpoint of the flap are ischemic. (3) A silicone sheet is inserted beneath the flap, preventing readherence and reperfusion of the flap from underlying tissue. Wound contraction is limited (not eliminated) by anchoring or suturing the flap to the silicone sheet5.
The model has recently been used in studies ranging from hyperbaric oxygen effects on ischemic wound healing9,10 to ischemic wound healing in young versus aged rats11 and has proven to be a reliable model of prolonged tissue ischemia. The dimensions of the bipedicled flap have also been adapted to different rat strains, including Sprague Dawley (11 cm long by 2 cm wide) and F344 rats (10.5 cm long by 3.0-3.5 cm wide) and other species, including swine12 and mice13,14 . This video utilizes the F344 inbred rat strain in the demonstration of the ischemic skin wound model.
Approval for all animal procedures presented below was obtained from the University of South Florida&#8217;s Animal Care Committee (IACUC) and abide by all requirements of the Animal Welfare Act and the Guide for Care and Use of Laboratory Animals.

<table><tbody><tr><td>Sil-Tec medical grade sheeting</td><td>Technical Products Inc.</td><td>500-3</td><td>nonreinforced, 0.01 inches in thickness</td></tr><tr><td>Mini Iris scissors, 8 cm, curved, SS</td><td>World Precision Instruments</td><td>#503671</td><td></td></tr><tr><td>Ethilon Nylon Sutures</td><td>Ethicon</td><td>1964G</td><td>black, size 4.0, PC-3 16&amp;nbsp;mm needles (3/8 circle)</td></tr><tr><td>Laser Doppler Imager</td><td>Moor Instruments</td><td>moorLDI2-IR</td><td>Standard blood flow imager: http://us.moor.co.uk/product/moorldi2-laser-doppler-imager/8</td></tr><tr><td>ImageJ</td><td>NIH</td><td>free download</td><td>http://rsb.info.nih.gov/ij/</td></tr><tr><td>Mastisol</td><td>Henry Schein</td><td>Cat # 7289210</td><td>Fisher Scientific NC9774929</td></tr><tr><td>Tegaderm</td><td>Medical Specialties&amp;nbsp;</td><td>3M1624W</td><td>Fisher Scientific NC9922128</td></tr></tbody></table>

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 ischemic wound healing model protocol should take approximately 20 min&#160;per animal if performed efficiently. Prior to application of a dressing the model should appear as represented in Figure 1G. It will be important to verify that the bipedicled flap and wounds therein are ischemic. Subcutaneous oxygen tension (PscO2) at the level of the wounds has been measured during validation of this model5 by placing a polarographic electrode in the subcutaneous tissue between the two ischemic wounds. PscO2 values were in the critically ischemic range (20-40 mmHg). Since the development of this model the use of LDI has increasingly been used to measure blood perfusion and this technique will give adequate information about the ischemic state of the bipedicled flap.
Briefly, the LDI technique is based on the emission of a beam of laser light carried by a fiber-optic probe. The measuring depth depends on the tissue properties and wavelength of the laser light. In normal skin, an instrument with a probe with standard fiber separation (0.25 mm) and a 780 nm wavelength laser, measuring depth will be on the order of 0.5-1 mm. A representative Doppler image (left) for a dorsal bipedicled flap showing both a perfused area (left of the suture line) and the non-perfused area (right of the suture line) is shown in Figure 2.
In addition to measuring subcutaneous oxygen tension, one can also use probes or common biochemical markers to establish that the wounds in the flap are ischemic. PECAM-1 or CD31 normally found on vascular endothelial cells is a marker for new vessel formation in the wounds. Various markers for reactive oxygen species, found to be elevated in ischemic wounds, are available commercially often as fluorescent-tagged antibodies or superoxide indicators such as dihydroethidium (DHE).
Wound area measurements to track wound closure may be represented in a variety of ways. Commonly, wound area is quantified from digital images of the wounds over the time course of healing11 using a formula such as (6 mm punch biopsy area = (&#960;) r2 = 3.14 x 9 = 28.26 mm2 at day 0) with data presented as percentage of initial wound area or wound surface area on a particular day can be quantified as in Gould et al.5 For the purposes of this demonstration, wound closure progression is represented as percent of initial wound area over a time course of 28 days. Using the free software ImageJ, a digital picture is opened and the scale is set using 10 mm on the ruler in the image (Figure 3A). A line drawn 10 mm in length equates to a pixel count (inset in Figure 3A) which can be converted into a unit of choice, in this case (mm). Next, the circumference of the wound is traced on the image (Figure 3B) and once the measure command is given, the area is presented in mm (inset in Figure 3B). Data can then be presented as percentage of initial wound area on the y-axis and days on the x-axis (Figure 3C).
<img alt="Figure 1" src="/files/ftp_upload/52637/52637fig1.jpg" /> 
Figure 1. Photographs depicting steps during surgery to create ischemic wounds, the bipedicled flap, and non-ischemic wounds. (A) Pre-surgical hair removal and skin preparation of an anesthetized rat receiving a pre-surgical dose of analgesic (Ketoprofen) subcutaneously for pain management. (B and inset) Ischemic wounds are created inside the 2 incision markers by utilizing a sterile punch biopsy tool. (C) Incisions are made along the marked lines down to the paraspinous muscle and (D) the bipedicled flap is raised to show the separation of the flap (with the panniculus carnosus fascia intact) from the muscle layer below. (E) A sheet of sterile silicone (black arrow) is placed between the fascia of the panniculus carnosus and the paraspinous muscle. (F) Black, non-absorbable sutures (size 4.0) close both incisions by anchoring the silicone sheet to the skin with multiple interrupted stitches along the length of the flap (G). Two non-ischemic wounds (black arrows) are created using a sterile punch biopsy tool lateral to the bipedicled flap on both sides. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52637/52637fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52637/52637fig2.jpg" /> 
Figure 2. Representative laser Doppler image of blood perfusion post-surgery. The right panel shows a black and white image of 2 ischemic wounds (black arrows) in the middle of the bipedicled flap and 1 non-ischemic wound (single black arrow) lateral to the flap. The suture line has been highlighted in white. The left panel shows the Doppler image of the same area depicted in the right panel. Areas with brighter colors are more perfused than areas with dark blue. This difference in perfusion between the non-ischemic area (left) and the ischemic area (right) is clear and can be followed along the entire length of the suture line. Note that the wounds on both sides appear bright, as blood cells are still present to some degree. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52637/52637fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52637/52637fig3.jpg" /> 
Figure 3. Wound measurement and data representation. (A) Digital photographs depicting wound area measurement using software (ImageJ), conversion of pixels to (mm) (black arrows) and the method to capture an accurate circumference (single black arrow) of the wound (B). Statistical data (mean &#177; SEM of wound areas on a given day) can then be presented as percentage of initial wound area (day 0) on the y-axis and days on the x-axis (C). The line graph presents data analyzed using a 2-way ANOVA with Sidak&#8217;s multiple comparisons test, *** represents significantly higher (P = 0.0004) than non-ischemic at the same time point (day 10), N = 8 wounds per group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52637/52637fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Demonstration of the Rat Ischemic Skin Wound Model at JoVE.com

Demonstration of the Rat Ischemic Skin Wound Model

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.

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

demonstration-of-the-rat-ischemic-skin-wound-model

Nanyang Technological University

Research

JoVE Journal

Medicine

21.3K Views.  University of South Florida.  De rat, door zijn omvang, de beschikbaarheid en vrij volgzaam gedrag, is gebruikt als een onderzoeksmodel voor vele jaren. Het doel van dit protocol is om de rat als een ischemische huid wondgenezing model te gebruiken om waardevolle inzichten in de pathofysiologie van chronische wonden te bieden. 

Video: Demonstration of the Rat Ischemic Skin Wound Model

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

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. ...

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

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 ...

Immunology and Infection

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary to perform reproducible burn research. The present protocol describes a simple and reproducible method to create ~20% total body surface area (TBSA) full-thickness burns in rats. Here, a 22.89 cm2 (5.4 cm diameter) copper rod heated at 97&#160;&#176;C in a water bath was applied to the rat skin surface to induce the burn injury. A copper rod with a high thermal conductivity was able to dissipate the heat deeper in the skin tissue to create a full-thickness burn. Histology analysis shows attenuated epidermis with coagulative damage to the full-thickness extent of the dermis and the subcutaneous tissue. Additionally, this model is representative of the clinical situations observed in hospitalized burn patients following burn injury such as immune dysregulation and bacterial infections. The model can recapitulate the systemic bacterial infection by both Gram-positive and Gram-negative bacteria. In conclusion, this paper presents an easy-to-learn and robust rat burn model that mimics the clinical situations, including immune dysregulation and bacterial infections, which is of considerable utility for the development of new topical antibiotic drugs for burn wound and infections.

A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments.

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal models of type 1 diabetic wounds. However, improper manipulation can lead to model instability and high mortality in rats. Unfortunately, there are few existing guidelines on type 1 diabetic wound modeling, and they lack detail and do not present specific reference strategies. Therefore, this protocol details the complete procedure for constructing a type 1 diabetic wound model and analyzes the progression and angiogenic characteristics of the diabetic wounds. Type 1 diabetic wound modeling involves the following steps: preparation of the streptozotocin injection, induction of type 1 diabetes mellitus, and construction of the wound model. The wound area was measured on day 7 and day 14 after wounding, and the skin tissues of the rats were extracted for histopathological and immunofluorescence analysis. The results revealed that type 1 diabetes mellitus induced by 55 mg/kg streptozotocin was associated with lower mortality and a high success rate. The blood glucose levels were relatively stable after 5 weeks of induction. The diabetic wound healing rate was significantly lower than that of normal wounds on day 7 and day 14 (p &#60; 0.05), but both could reach more than 90% on day 14. Compared with the normal group, the epidermal layer closure of diabetic wounds on day 14 was incomplete and had delayed re-epithelialization and significantly lower angiogenesis (p &#60; 0.01). The type 1 diabetic wound model constructed based on this protocol has the characteristics of chronic wound healing, including poor closure, delayed re-epithelialization, and decreased angiogenesis compared to normal rat wounds.

The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds.

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal ...

Demonstration of the Rat Ischemic Skin Wound Model

Samenvatting

Meer video's verkennen