This study was supported in part by NIH grants 1RO1DK74566 and 1RO1AM52984. The authors would like to thank Ms. Jacqueline McCarty in RRC for her help during surgery, Drs. Jianpu Wang and Harshini Sarojini for their help in surgery and postoperative care of some rabbits, Dr. Rong Wan for her histology work, and Ms. Ming Li for her HPLC measurements of high-energy phosphates.

1. Anatomic Basis
The rabbit ear has been a favored study material for wound care since the early 20th century. The ear is supplied by three vascular bundles (central, cranial, and caudal); the central bundle is the largest, while the vessels in the cranial and caudal bundles vary in size. Occasionally, the caudal artery may be absent.1 All of these vessels are easily identified on the dorsal side after the ear is shaved (Fig. 1). On the ventral side, the distal skin does not receive blood supply from the proximal vessels. The three vessel bundles on the dorsal ear have numerous branches that penetrate through the cartilage to provide blood supply to the distal ventral skin (Fig. 2). In some rabbits, a very small subcutaneous vessel also occurs on the ventral side, which supplies blood to no more than 1/3 of the ventral skin. In an open circumferential skin incision, there is essentially no bleeding from a ventral incision.
2. Surgical Procedure
<ol>
	<li>The rabbit is weighed and the ears are shaved, along with the back of the neck.</li>
	<li>A premedication of Buprenorphine (0.01 mg/kg) is given IM one hour before surgery.</li>
	<li>Anesthesia is induced by a mixture of ketamine (50 mg/kg) and Xylazine (5 mg/kg) IM.</li>
	<li>The rabbit is protected with a heating pad during surgery, the eyes are protected with ointment, and its vital signs are monitored during surgery.</li>
	<li>The surgical field is prepared aseptically with a surgical scrub and draped so that the two ears are exposed.</li>
	<li>One ear is rendered ischemic, while the other ear serves as a paired non-ischemic control.</li>
	<li>The three vascular pedicles are identified by the arteries and veins.</li>
	<li>Using a #15 blade, three small vertical incisions (1-1.5 cm) are made on the vascular pedicles, about 1-2 cm from the ear base.</li>
	<li>The central vein is isolated from the surrounding tissues and a suture is used to encircle it for retraction. The central artery is ligated with 4-0 sutures and divided.</li>
	<li>The accompanying central nerve is also cut.</li>
	<li>The whole cranial pedicle is ligated and divided.</li>
	<li>The caudal pedicle is preserved.</li>
	<li>A subcutaneous tunnel is made through the 3 incisions. This is accomplished by blunt dissection with a clamp and sharp dissections with a knife and scissors. All of the subcutaneous tissues, muscles, nerves, and small vascular branches are discontinued. The small vasculatures underneath the dermis are totally divided. All of the subcutaneous tissues are cut to the level of the cartilage with the blade.</li>
	<li>Further sharp and blunt dissections are used to clean up the base, leaving a bare cartilage about 5-10 mm wide.</li>
	<li>This procedure interrupts all of the subcutaneous vascular branches to prevent them from rapidly establishing collaterals.</li>
	<li>Bleeding is minimal. If there is any, it is easily controlled by gauze compression, without the need for electrocautery.</li>
	<li>The skin inside the ear does not carry much blood supply and thus is not incised.</li>
	<li>The 3 skin incisions are closed using 4-0 or 5-0 sutures, and covered with sterile gauze.</li>
	<li>The procedure results in 3 small skin incisions and incurs much less skin damage than an open surgery.</li>
	<li>Four circular full-thickness skin wounds are created on the ventral side of each ear with a 6-mm stainless steel punch. The distance between the wounds is at least 20-30 mm. The skin inside the punch wound is removed from the cartilage. The perichondrium is also removed, with the skin or separately. The base on which granulation and epithelization take place is the cartilage, but the cartilage is not perforated (Fig. 3).</li>
	<li>When the wounds on both ears are completed, testing drug is applied to one side of two wounds while the control drug is applied to the wounds on the other side. An occlusive dressing (TegaDerm, 3M) is used to cover the wound site. This prevents the wounds from becoming desiccated.</li>
	<li>The wounds at the bottom portion of the ear normally heal faster than the wounds at the top of the ear. For wound healing comparison study, it is recommended to compare the two wounds on one side with the two wounds on the other side, or to compare the top and bottom wounds separately.</li>
</ol>
3. Postoperative Care
<ol>
	<li>A Duragesic patch is attached to the back skin to release Fentanyl (25 &mu;g/hour) for 2-3 days to reduce possible pain.</li>
	<li>The rabbits are allowed free access to food and drink.</li>
	<li>Dressing changes are carried out daily. The old dressings are removed and the wounds are cleaned with sterile cotton swabs. Digital photos are taken for comparison. New dressings are applied, and the wounds are covered again by TegaDerm. No anesthetic is needed during dressing changes.</li>
	<li>Skin temperature is measured daily on each ear for comparison.</li>
	<li>Additional ear blood circulation can be measured with a transcutaneous oxygen tension meter or a laser Doppler flowmeter.</li>
	<li>The sutures may be removed after one week.</li>
	<li>For non-diabetic animals, most of the ear wounds usually heal within 2-3 weeks.</li>
</ol>
4. Potential Complications and How to Avoid Them
This minimally invasive surgery is safe and complications are rare. Except for a transitional postoperative mild ear edema, none of our rabbits has had any severe surgical complications. Nevertheless, there are several potential complications for this procedure:
<ol>
	<li>Bleeding. Bleeding may occur in the incisions if hemostasis is incomplete or if a larger blood vessel (such as one of the main pedicle vessels) is damaged. This is easily avoided by careful dissection. In addition to the three major vascular bundles, there are many minor subcutaneous vascular branches. When the subcutaneous tunnel is made, these vessels are being cut but no tying is necessary. Hemostasis can be achieved simply by gauze pressing. This is easily done by inserting a corner of gauze into the tunnel for a few minutes. Before closing the incision, make sure no active bleeding is present.</li>
	<li>Incision infection and dehiscence: These complications may occur if the incisions are contaminated. This can be avoided by use of stringent aseptic technique and no antibiotic is needed. However, if infection does occur, appropriate drainage and dressing changes are needed.</li>
	<li>Ear edema: Due to vascular ligations and disruption of lymph circulation, mild ear edema may present for a few days. However, it disappears by itself and no treatment is necessary.</li>
</ol>
5. Representative Results
Since 2007, we have used this procedure in more than 70 rabbits, including 23 diabetic animals. In the diabetic group, surgery was performed at different diabetic times: seven rabbits had two weeks of diabetic time, four of them had two months, ten had 12 months, and 2 had 24 months of diabetic time.
Immediately after surgery, the ischemic ear appeared cool and cyanotic, with mild edema. The most important ear artery-the central artery-had a strong pulse in the normal ear, but this pulse was not present in the ischemic ear. Ear movement was reduced, but did not totally disappear. The skin showed clear depressions over the tunnel, but no ischemia or necrosis was found in any animal (Fig. 4).
The skin temperatures of the normal and ischemic ears were measured with a digital thermometer (Omron by Omron Healthcare, Bannockburn, IL, or a Type K infrared spot thermometer by Fisher Scientific, Pittsburgh, PA). The temperature differences between the non-ischemic and ischemic ear ranged from 1 to 10 &deg;C, with most rabbits falling within the range of 2-6 &deg;C. Fig. 5 shows the average temperature differences from 30 rabbits. The temperature difference was higher in the early days after surgery, decreased gradually after day 15, but was still maintained at the end of one month.
Tissue high-energy phosphate contents were measured in some animals after they were sacrificed. Six tissue samples were taken from each ear and high-energy phosphate contents were measured by HPLC. Both ATP and total energy concentrations were higher in the normal ears than in the ischemic ears. Fig. 6 shows an example of a rabbit sacrificed 23 days after surgery. The mean tissue ATP contents were 0.349&plusmn;0.047 &mu;Mol/g (mean&plusmn;SD) in the normal ear vs. 0.237&plusmn;0.059 &mu;Mol/g in the ischemic ear (p&lt;0.005). Mean total energy (ATP+ADP+AMP) was 0.882&plusmn;0.137 &mu;Mol/g in the normal ear vs. 0.556&plusmn;0.115 &mu;Mol/g in the ischemic ear (p&lt;0.001).
Wound healing times were always longer for the ischemic ear than for the non-ischemic ear in all groups (non-diabetic or diabetic). Fig. 7 shows a comparison of healing between 26 pairs of wounds on ischemic and non-ischemic ears. Healing times ranged from 13 to 18 days (mean 14.9&plusmn;1.6 days) on the normal ears vs. 17 to 27 days (mean 20.5&plusmn;3.4 days) on the ischemic ears (p=0.001). Wound healing times in aged and diabetic rabbits were much longer, especially when diabetic time was more than 12 months.
In these rabbits, all the incisions healed normally without any complications, including the rabbits with 24 months of diabetic time. Figure 8 is a histology sample showing the healed subcutaneous tunnel in which muscle is absent and is replaced by fibrous tissues. However, the skin is intact.
<img alt="Figure 1" src="/files/ftp_upload/3341/3341fig1.jpg" /> 
Figure 1. The rabbit ear is supplied by three vascular bundles. The central is the largest, the cranial is smaller, and the caudal bundle is the smallest. These vessels are easily seen on the surface after the ear is shaved.
<img alt="Figure 2" src="/files/ftp_upload/3341/3341fig2.jpg" /> 
Figure 2. The three vessel bundles on the dorsal ear supply blood not only to the dorsal skin, but also to the ventral skin through penetrating vessels. The arrow shows the penetrating vessels through the cartilage. The inset shows cross section of these vessels.
<img alt="Figure 3" src="/files/ftp_upload/3341/3341fig3.jpg" /> 
Figure 3. Four 6-mm wounds are created on the ventral side of the ear, and the perichondrium is also removed. The base on which granulation and epithelization take place is the cartilage, but the cartilage is not perforated. The 3 small skin incisions are closed with very little skin disruption.
<img alt="Figure 4" src="/files/ftp_upload/3341/3341fig4.jpg" /> 
Figure 4. After the skin incisions are closed, the indention over the subcutaneous tunnel is clearly seen.
<img alt="Figure 5" src="/files/ftp_upload/3341/3341fig5.jpg" /> 
Figure 5. The average skin temperature difference between the normal and ischemic ear in 30 rabbits. The early difference ranges from 7 &deg;C to 10 &deg;C after surgery. The difference decreases gradually but still maintained at the end of one month.
<img alt="Figure 6" src="/files/ftp_upload/3341/3341fig6.jpg" /> 
Figure 6. Ear tissue high-energy phosphate contents are reduced in the ischemic ear as compared to non-ischemic ear at 23 days after surgery (p&lt;0.001).
<img alt="Figure 7" src="/files/ftp_upload/3341/3341fig7.jpg" /> 
Figure 7. Wound healing time is longer in the ischemic ears than the non-ischemic ears (26 wounds each). Healing is defined as the restoration of integrity to injured tissue, i.e., the complete reepithelialization of wound sites.
<img alt="Figure 8" src="/files/ftp_upload/3341/3341fig8.jpg" /> 
Figure 8. Histology sample showing the ear portion containing subcutaneous tunnel. The healed tissue is thinner than normal ear, the original skeletal muscle is absent and is replaced by fibrous tissues. However, the skin is intact.

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There is no financial conflict of interest involved in this study.

Diabetes affects 23.6 million people in the US,2,3 and 15-20% of these patients develop foot ulcers in their lifetime,4,5 at an annual treatment cost of more than $25 billion.3,6 However, even the best available treatments achieve only a 50% healing rate and this is often temporary,7 with recurrence rates as high as 66% in diabetics.3,6 When ulcers occur in diabetic patients, major complications such as infection and amputation increase dramatically. Despite almost a...

For monitoring during surgery
<ol>
 <li> A heating pad to maintain normal temperature. </li>
 <li>A transcutaneous oxygen monitor. </li>
 <li> A temperature monitor. </li>
</ol>
For surgery
<ol>
 <li> A #3 knife with #15 blade. </li>
 <li>Two pairs of forceps (one for skin and another for other soft tissues). </li>
 <li> 2-3 pairs of small clamps. </li>
 <li> One or two pairs of micro scissors (for tissue cutting). </li>
 <li>One pair of small scissors (for cutting sutures). </li>
 <li>A needle holder. </li>
 <li>A few stainless steel punches (for making wounds). </li>
</ol>

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.

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A Simplified Technique for Producing an Ischemic Wound Model

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 ...

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17.1K Views.  University of Louisville. 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.

Video: A Simplified Technique for Producing an Ischemic Wound Model

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

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. ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

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.

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

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.