Name: an epithelial abrasion model for studying corneal wound healing
Uploaded: 2021-12-29T14:00:00
Description: Watch this Scientific Journal Video about An Epithelial Abrasion Model for Studying Corneal Wound Healing at JoVE.com

Funding: Supported by: NIH EY018239 (A.R.B., C.W.S., and R.E.R.), P30EY007551 (A.R.B.), and Sigma Xi Grant in Aid of Research (P.K.A.). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health, or Sigma Xi.

All animal protocols were approved by the Institutional Animal Care and Use Committees at the University of Houston and Baylor College of Medicine. The guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in vision and ophthalmic research were followed in handling and using the mice.
1. Preparation
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	<li>Preparation of fluorescein solution
	<ol>
		<li>Prepare 1% fluorescein solution by dissolving 10 mg of ...

The purpose of this methods paper was to describe a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud. This murine model has been used to study corneal inflammation and its contribution to wound healing. This type of model can be used to study corneal wound healing mechanisms under normal physiological conditions and in pathologies17,28,29,<sup c...

The cornea is the transparent anterior one-third of the eye. The cornea serves several functions including protecting the inner structures of the eye and forming a structural barrier that protects the eye against infections1. More importantly, the cornea is critical for vision, providing about two-thirds of the refractive power of the eye2,3. Crucial to the role of the cornea in vision is its transparency. However, due to its outward position, the cornea is exposed to a wide variety of injuries on a day-to-day basis that can lead to disruption of its barrier function, loss of transparency, and eventual blindness. Loss of corneal transparency is a leading cause of visual impairment worldwide4,5. Corneal abrasions are a common reason for visits to the emergency room (ER), accounting for half of the eye-related cases presented at the ER6. Over 1 million individuals are estimated to suffer from eye-related injuries annually in the United States7. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of its transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring8,9. Also, the increasing popularity of refractive surgeries places a unique traumatic challenge on the cornea10. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease.
To that end, several animal models of corneal wound healing have been developed11,12,13,14,15. Murine models of corneal wound healing have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Different types of corneal wounds have been employed in studying corneal wound healing, each suitable for investigating different aspects of the wound healing process. The commonest types of wound models used in corneal wound healing studies are the mechanical and chemical wound models. Chemical corneal wounds, mostly involving the creation of alkaline burns on the cornea, are useful for studying corneal ulcers, opacification, and neovascularization13. Mechanical corneal wounds involve debridement (abrasion) wounds and keratectomy wounds14,15,16. An intact or breached corneal epithelial basement membrane defines debridement and keratectomy wounds, respectively. In debridement wounds, the epithelial basement membrane remains intact, while in keratectomy wounds, the basement membrane is breached with penetration mostly into the anterior stroma. Debridement wounds are most useful for studying re-epithelialization, epithelial cell proliferation, immune response, and nerve regeneration following corneal wounding. Keratectomy wounds, on the other hand, are most useful for studying corneal scarring14,15.
Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease17.

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			<td>Anti-CD31 antibody</td>
			<td>BD Bioscience, Pharmingen</td>
			<td>550274</td>
			<td></td>
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		<tr>
			<td>Anti-CD41 antibody</td>
			<td>BD Bioscience, Pharmingen</td>
			<td>553847</td>
			<td></td>
		</tr>
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			<td>Anti-Ly6G antibody</td>
			<td>BD Bioscience, Pharmingen</td>
			<td>551459</td>
			<td></td>
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			<td>Bovine serum albumin (BSA)</td>
			<td>ThermoFisher scientific</td>
			<td>B14</td>
			<td></td>
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		<tr>
			<td>C57BL/6 mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td></td>
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			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D8417</td>
			<td></td>
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			<td>DeltaVision wide-field deconvolution fluorescence microscope</td>
			<td>GE Life Sciences</td>
			<td></td>
			<td></td>
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			<td>Dissecting microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td></td>
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			<td>Electronic Toploading Balances (Weighing scale)</td>
			<td>Fisher Scientific</td>
			<td></td>
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			<td>Ethanol</td>
			<td>ThermoFisher scientific</td>
			<td>T038181000CS</td>
			<td></td>
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			<td>Golf-club spud</td>
			<td>Stephens instruments</td>
			<td>S2-1135</td>
			<td></td>
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			<td>Iris curve scissors</td>
			<td>Fisher Scientific</td>
			<td>31212</td>
			<td></td>
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			<td>Isoflurane</td>
			<td>Patterson veterinary</td>
			<td>07-893-1389</td>
			<td></td>
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			<td>Ketamine</td>
			<td>Patterson veterinary</td>
			<td>07-890-8598</td>
			<td></td>
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			<td>Phospate buffered saline (PBS)</td>
			<td>ThermoFisher scientific</td>
			<td>AM9624</td>
			<td></td>
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		<tr>
			<td>Sodium fluorescein salt</td>
			<td>Sigma Aldrich</td>
			<td>46970</td>
			<td></td>
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		<tr>
			<td>Surgical blade (scapel blade)</td>
			<td>Fine Science tools</td>
			<td>10022-00</td>
			<td></td>
		</tr>
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			<td>Trephine</td>
			<td>Integra Miltex</td>
			<td>33-31</td>
			<td></td>
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			<td>TritonX -100</td>
			<td>Fisher Scientific</td>
			<td>50-295-34</td>
			<td></td>
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			<td>Forcep</td>
			<td>Fine Science tools</td>
			<td>11923-13</td>
			<td></td>
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			<td>Xylazine</td>
			<td>Patterson veterinary</td>
			<td>07-808-1947</td>
			<td></td>
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an epithelial abrasion model for studying corneal wound healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Figure 3 shows a transmission electron micrograph of a corneal wound created with the blunt golf club spud, demonstrating that the epithelial&#160;basement membrane is indeed intact after injury.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63112/63112fig03.jpg" /> 
Figure 3: Epithelial basement membrane remains intact after corneal abrasion. Transmission electron mi...

Watch this Scientific Journal Video about An Epithelial Abrasion Model for Studying Corneal Wound Healing at JoVE.com

An Epithelial Abrasion Model for Studying Corneal Wound Healing

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

This protocol is significant because it is an excellent model for studying corneal inflammation and its contribution to wound healing under normal and pathological conditions. The main advantage of this technique is that it creates a precise and reproducible epithelial wound without breaching the basement membrane. This model of corneal wounding is straightforward and quick to perform.Demonstrating the procedure will be Prince Akowuah, a PhD student from my laboratory. To begin, prepare a 1%fluorescein solution by dissolving 10 milligrams of sodium fluorescein salt in one milliliter of sterile saline or PBS. Next, for anesthesia, prepare 10 milliliters of a ketamine-xylazine cocktail by mixing two milliliters of ketamine, one milliliter of xylazine, and seven milliliters of sterile PBS.After anesthetizing an 8-to 12-week-old C57 black 6 wild-type mouse, evaluate the depth of anesthesia by assessing the pedal reflex after toe pinch. To create the epithelial corneal wound, place the anesthetized mouse under a dissection microscope. Wound the right or left eye only and maintain consistency with the eye being wounded when moving from mouse to mouse.Keep the eye wide open by holding the eyelids with the thumb and index finger. Then, using a two-millimeter-diameter sterile trephine, demarcate the center of the cornea and gently twirl the trephine to make an impression on the corneal epithelium. Do not apply excessive pressure, as this can result in corneal perforation.Next, holding the sterile blunt golf club spud at an approximately 45-degree angle from the cornea's surface, debride the epithelium by careful and continuous scraping within the demarcated area using the spud. Do not apply excessive force and maintain hydration using sterile PBS if necessary. For monitoring wound closure and re-epithelization, pipette 1 to 1.5 microliters of 1%fluorescein solution onto the wounded surface and image the cornea using a digital microscope with a blue light source.Image the wounded cornea at specific times after wounding. Take images within the first minute of fluorescein solution addition to avoid spreading of the solution to the surrounding epithelium, which can lead to an overestimation of the wound size. Next, using an image analysis software, trace the wound area and express the wound area at each time point as a percentage of the original wound area at zero hour.A transmission electron micrograph of the corneal wound demonstrates that the epithelium basement membrane remains intact even after the injury. In 8-to 12-week-old wild-type mice, wound closure is complete 24 hours after wounding. Inflammatory response to wounding is characterized by cell imaging at the limbus.Shown here is the limbal vasculature of an unwounded cornea with extra-vascular neutrophils, and that of a wounded cornea with extravascular platelets and neutrophils 30 hours after abrasion. To successfully reproduce this protocol, the same strain in each range of mouse must be used, and the wound size and depth must be created as described.

an-epithelial-abrasion-model-for-studying-corneal-wound-healing

Research

JoVE Journal

Medicine

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. ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...