Name: inhibition of wound epidermis formation via full skin flap surgery during axolotl limb regeneration
Uploaded: 2020-06-24T14:30:00.000+00:00
Duration: 4 min 24 s
Description: Watch this Scientific Journal Video about Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration at JoVE.com

The author thanks Doug for his constant encouragement and unwavering support, as well as the members of the Melton lab for their helpful feedback and comments on the manuscript. The author would also like to thank the Harvard Office of Animal Resources (OAR) for their dedicated animal care.

All animal experiments were performed in accordance with IACUC (Protocol #: 11-32) and AAALAC guidelines at Harvard University.
1. Preparing solutions and setup for anesthesia and recovery
<ol>
	<li>Prepare fresh 0.1% tricaine solution for anesthesia and 0.5% sulfamerazine sodium salt solution for recovery. Make the solutions using water suitable for axolotl husbandry37 according to approved IACUC protocols at the relevant research institution (modified Holtfreter&#8217;s solution, for instance). Make sure that the solutions are well-mixed and that enough volume is prepared in order to submerge the entire axolotl.
	<ol>
		<li>To prepare 0.1% tricaine solution, mix 1 g of tricaine and 1 g of sodium bicarbonate with 1 L of water. The solution can be scaled up according to this recipe.</li>
		<li>To prepare 0.5% sulfamerazine sodium salt solution, mix 5 g of sulfamerazine sodium salt with 1 L of water. The solution can be scaled up according to this recipe. Sulfamerazine solution is an anti-biotic that will prevent bacterial infection during surgical recovery.</li>
	</ol>
	</li>
	<li>Sanitize the surgical area by spraying it down with Clidox-S or 70% ethanol. Sterilize surgical tools (forceps, dissecting scissors, spring scissors) by autoclaving. If performing multiple surgeries, make sure to sterilize the surgical tools with a hot bead sterilizer between animals.</li>
	<li>To set up the recovery area, place a 15 cm Petri dish or any container that will fit the axolotl on top of a bucket filled with wet ice. Fill the Petri dish with a low level of 0.5% sulfamerazine sodium salt solution, enough such that the axolotl would not be fully submerged. The recovery on ice post-surgery will slow the animal&#8217;s movement while it awakens from anesthesia, allowing the sutured area to heal relatively undisturbed. 
	NOTE: This setup can be customized by researchers depending on what materials they have available.</li>
</ol>
2. Performing the full skin flap surgical procedure
<ol>
	<li>Anesthetize the axolotl by submersing it in a container of 0.1% tricaine solution. This will take approximately 15-20 minutes. Ensure that the axolotl is indeed fully anesthetized by performing a tail pinch. If there is no response from the axolotl, proceed with the surgery. 
	NOTE: Use older, larger axolotls for this surgery (at least 15 cm in size). Make sure that the axolotl remains well hydrated throughout the surgery by wetting the skin periodically with axolotl system water using a plastic pipette. Make sure to aseptically prepare the surgical site by irrigating the area with sterile PBS prior to surgery. The animal should also be placed on a sterile surgical drape for the procedure.</li>
	<li>Perform a limb amputation at the distal end of the zeugopodial skeletal elements using the dissecting scissors (Figure 1.1).</li>
	<li>Using the spring scissors, make a small incision (approximately 2 mm) on the ventral portion of the skin (Figure 1.2).</li>
	<li>Using the forceps, carefully peel back the skin to approximately the midline of the zeugopodial skeletal elements, exposing the underlying limb tissues (muscle, bone, etc.) (Figure 1.3). Make sure not to damage the skin. See note after step 2.8.</li>
	<li>Amputate the exposed underlying limb tissues at the midline of the zeugopod using surgical scissors (Figure 1.4).</li>
	<li>Push back the muscle tissue with the surgical scissors and trim the exposed bone. 
	NOTE: This is necessary to ensure improved healing and also to increase the success of the surgery as protruding bone can be jagged against the sutured flap and disrupt the integrity of an intact skin flap later on.</li>
	<li>Using the forceps, carefully pull the extra full thickness skin over the amputation plane to cover the exposed underlying tissues and suture in place by connecting with the ventral full thickness skin (Figure 1.5).</li>
	<li>Suture the remaining right and left sides of the skin flap into the underlying ventral portions of intact skin. This can be done by either suturing the sides of the flap in a &#8220;criss-cross&#8221; manner (recommended) (Figure 1.6-1.9), or simply suturing straight into the ventral skin. Use the forceps and curved spring scissors for suturing. Make sure no exposed underlying tissues can be seen and that sutures are tied tight (knotted at least three times). 
	NOTE: It is critical that the intact skin is not damaged in steps 2.4, 2.7-2.8. We have found that damage to the full thickness skin has been correlated with unsuccessful surgeries, as the areas of damage may still form a small wound epidermis. If possible, try to use a duller pair of forceps when handing the full thickness skin flap.</li>
	<li>Perform an amputation on the contralateral limb (optional internal animal control) by amputating it at mid-zeugopod level with surgical scissors. Push back the muscle tissue with surgical scissors and trim the exposed bone. 
	NOTE: An internal contralateral limb control can be done to better assess the success of the surgery during step 4 in the same animal. However, amputation of the same limb in a separate animal can also be used to serve as a control.</li>
</ol>
3. Post-operative recovery and care
<ol>
	<li>Once the surgery is complete, place a Kimwipe or sterile paper towel at the bottom of the container or Petri dish to wet it. Place the animal into the container on wet ice and gently wrap the exposed ends of the Kimwipe or paper towel around the top of the animal to keep it well-hydrated with sulfamerazine solution. Leave on wet ice for 30 minutes to 1 hour to ensure minimal movement during recovery from anesthesia.</li>
	<li>Place the animal into a static housing container with 0.5% sulfamerazine solution. Axolotls must remain in this solution for the first 24 hours in order to prevent infection.</li>
	<li>Place the axolotl into normal system water and monitor health daily. Ensure that no sutures fall out each day as this can result in a small wound epidermis forming which will confound results. 
	NOTE: Make sure that the housing container has ample room for the axolotl to move around and minimize the chances that the sutured limb on the axolotl can come into contact with the sides of the container. This will help to ensure that the sutures stay in place, especially during the first week post-surgery.</li>
</ol>
4. Assessing success of the surgery under a stereo microscope
NOTE: We recommend checking animals under a stereo microscope at least once a week to assess the integrity of the full skin flap and success of the surgery.
<ol>
	<li>Anesthetize the axolotl in 0.1% tricaine as in Step 2.1. Make sure there is ample room in the container for the axolotl to move around.</li>
	<li>If inspecting during the first two weeks post-surgery, inspect the sutured limb using a stereomicroscope to make sure no sutures have popped out and that a clear thin wound epidermis is not visible anywhere. If inspecting on the third week post-surgery or later, make sure a blastema has not formed and compare with how the normal control amputated limb (either from the same animal or a different animal) has progressed during regeneration (i.e., whether a blastema has formed).</li>
	<li>When done, return the axolotl to normal system water and husbandry conditions.</li>
</ol>

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The authors have nothing to disclose.

This article describes a protocol for performing full skin flap surgeries in axolotl limbs to inhibit wound epidermis formation. While this surgery is relatively simple and technically reproducible compared to other methods of inhibiting wound epidermis formation, there are several critical steps that can impact the success of the surgery. First, when pulling the intact full skin flap over the exposed underlying tissues, it is paramount that the full thickness skin not be damaged in any way. Damage to the skin flap can still lead to the formation of a small wound epidermis, which can result in a small blastema-like outgrowth. Second, ensure sutures do not fall out during post-operative care as this can also lead to the formation of a small wound epidermis. To this point, minimizing the potential contact between the sutured limb and any surfaces is important, especially during the first week post-surgery. Several ways to prevent this entail housing and anesthetizing the axolotl in a large enough container such that the axolotl has plenty of room to move around post-surgery.
This surgery also has several limitations. Perhaps the most notable is that the success of surgeries can only be assessed in two ways: using the dissecting scope during the first two weeks of surgery to search for an absence of a wound epidermis and/or checking whether a blastema forms within 3 weeks. While these methods are effective, they are relatively low throughput. The development of future transgenic reporter axolotls for wound epidermis-specific markers may aid in quicker screening for successful versus unsuccessful surgeries. Furthermore, this surgery is more difficult to perform on younger animals as the intact skin is more fragile. Using sub-adult or adult axolotls is thus recommended.
While this surgery was originally developed in N. viridiscens19, it has been easily adapted for axolotls25,39 and can likely be applied to other salamander species as well. In sum, applying this technique to future limb regenerative studies will empower researchers to both develop more tools to address wound epidermis biology and identify the underlying mechanisms driving its function in initiating blastema formation.

Since Lazzaro Spallanzani reported it in 17681, salamander limb regeneration has been one of the most well-studied natural regenerative phenomenon that has enamored biologists for centuries. Successful limb regeneration hinges on the formation, outgrowth, and subsequent patterning of an undifferentiated cellular structure known as the blastema. Researchers have made significant strides in understanding the cellular composition of the blastema as well as which supportive tissues and cell types are necessary for its formation2,3,4,5,6,7,8,9,10,11,12,13. Yet, the coordinated signaling mechanisms between different tissues and cell types that lead to the initiation of blastema formation remain poorly understood.
A key requirement for successful blastema formation and regeneration is the wound epidermis, a transient and specialized epithelium that covers the amputation plane within 12 hours post-amputation10. Following amputation, epithelial cells from the intact skin proximal to the injury rapidly migrate over the amputation plane to form a thin wound epithelium14. As the blastema forms in the following weeks, the early wound epidermis develops into a thicker epithelial signaling structure called the apical epithelial cap (AEC)15. While normal full thickness skin contains both an epithelial and dermal layer separated by a basal lamina, the wound epidermis/AEC consists only of an epithelial layer and lacks a basal lamina16,17. The absence of the basal lamina and dermis allows for direct contact between the wound epithelial cells and the underlying tissues, which facilitates bi-directional signaling between the two compartments that is critical for both blastema formation and maintenance17,18.
Classical experimental studies devised various innovative surgical methods to probe wound epidermis/AEC function and necessity via inhibiting its formation. These methods included suturing19 or grafting full thickness skin20,21 over the amputation plane, immediately suturing the amputated limb into the body cavity22, and continuous daily removal or irradiation of the early wound epidermis and AEC23,24. Altogether, these experiments not only established the importance of the wound epidermis/AEC, but also further determined its roles in early tissue histolysis as well as maintaining progenitor cell proliferation and blastemal outgrowth13 throughout regeneration.
However, these previous studies were largely limited to histological staining as well as tritiated thymidine pulses to track cell proliferation. In fact, revisiting these classical experiments with modern sequencing technologies and functional techniques in salamanders has only recently been done and has led to the discovery of additional roles for the wound epidermis in modulating inflammation and ECM degradation/deposition during early stages of regeneration25. With the release of various salamander genome and transcriptome sequences26,27,28,29,30,31,32,33,34, as well as the burgeoning number of functional methods available in salamander species11,35,36,37,38, researchers are now well-positioned to begin to unravel the molecular mechanisms driving wound epidermis formation, function, and AEC development.
Unfortunately, several of these classical methods used to inhibit wound epidermis formation are technically challenging, presenting difficulties for reproducibility between biological replicates in the same experiment. For instance, maintaining skin grafts can be challenging as grafts may eventually fall off the host limb and removal of the wound epidermis/AEC daily is difficult without damaging the underlying tissues. Furthermore, suturing the amputated limb into the body cavity is challenging and also requires additional injury at the site of insertion. On the other hand, suturing full thickness skin immediately over the amputation plane is relatively simple, technically reproducible, and introduces minimal tissue damage. This full skin flap (FSF) surgical method was previously developed by Anthony Mescher in 1976 in adult newts (Notophthalmus viridiscens). He demonstrated that the FSF surgery inhibited wound epidermis formation and function by prohibiting both epithelial cell migration over the amputation plane and direct contact between epithelial cells and the underlying tissues.
Here, this surgical procedure is shown step-by-step using the axolotl limb. Coupled with modern day molecular and sequencing technologies, this technique may prove to be very helpful for researchers to deepen our understanding of wound epidermis/AEC formation and function during limb regeneration.

<table><tbody><tr><td>Curved spring scissors</td><td>Fine Scientific Tools</td><td>15009-08</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td><td>Sigma-Aldrich</td><td>886-86-2</td><td></td></tr><tr><td>Forceps</td><td>Fine Scientific Tools</td><td>11252-40</td><td>Need two pairs</td></tr><tr><td>Nylon monofilament sutures (9-0)</td><td>Roboz</td><td>SUT-1000-21</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Sulfamerazine sodium salt</td><td>Sigma-Aldrich</td><td>127-58-2</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Scientific Tools</td><td>14002-14</td><td></td></tr></tbody></table>

inhibition of wound epidermis formation via full skin flap surgery during axolotl limb regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This surgical protocol will allow for the complete inhibition of wound epidermis formation (Figure 1) and ultimately, limb regeneration. A successful surgery results in no blastema formation in approximately 2-3 weeks depending on the size of the animal, while control regenerating limbs should form a blastema normally.
Researchers should inspect the sutured limb by naked eye every 2-3 days to make sure that the sutures have not popped out and that a blastema is not forming. If one or more of the sutures pop out, a wound epidermis can still form resulting in either a small or large blastema and an unsuccessful surgery (Figure 2). Additionally, researchers should inspect the sutured limb at least once every week under a stereomicroscope to make sure that a thin wound epidermis is not evident anywhere on the amputation surface. For comparison, researchers should also examine the control regenerating limb which should have a wound epidermis over the amputation plane and form a blastema over 2-3 weeks. The wound epidermis will appear thin and clear, while the normal skin will appear more opaque and pale pink (almost white), light yellow, or dark green in leucistic, albino, or wildtype axolotls, respectively.
If researchers wish to collect tissue prior to the blastema formation stages at 2-3 weeks, they should inspect the sutured limbs prior to sample collection to make sure the sutures remained in place and that a small wound epidermis did not form. Additionally, sectioning sagitally through the sutured limb tissue and performing histological analyses at any time point can also verify the presence of the dermis from the full skin flap encircling the entire amputation plane and the absence of a wound epidermis (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61522/61522fig01.jpg" /> 
Figure 1: Schematic of the steps of the full skin flap surgery. 
The steps of the protocol are numbered and diagrammed here. The dotted lines denote the planes of amputation at steps 1 and 3 of the protocol. <a href="https://www.jove.com/files/ftp_upload/61522/61522fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61522/61522fig02.jpg" /> 
Figure 2: Examples of successful and unsuccessful full skin flap surgeries. 
Representative brightfield image of a limb that has undergone a successful surgery (left), an unsuccessful surgery (right), and a control regenerating limb (no surgery) at 25 days post-amputation (dpa). The successful surgery has a flat amputation plane where the full skin flap was sutured over, whereas the unsuccessful surgery has a small blastema developing. Arrowheads denote the amputation plane and white dotted lines are there to aid visualization of the absence of a blastema in the successful surgery and presence of blastemas in the unsuccessful surgery and control regenerating limbs. <a href="https://www.jove.com/files/ftp_upload/61522/61522fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61522/61522fig03.jpg" /> 
Figure 3: Histological staining of normal regenerating and FSF sutured limbs. 
(A-B&#8217;) Representative brightfield images of picro-mallory stained sections from regenerating (A-A&#8217;) and sutured axolotl limbs (B-B&#8217;) at 7 dpa. Insets in A and B are shown in A&#8217; and B&#8217;, respectively. The collagen-heavy dermal layer lines and covers the entire amputation plane in sutured limbs. Amputation plane is denoted by arrowheads in A-B. Scale bars represent 500 &#181;m. This figure was adapted from Tsai et al.25. <a href="https://www.jove.com/files/ftp_upload/61522/61522fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration at JoVE.com

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

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Biology

3.0K Views.  Harvard University. This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Video: Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Developmental Biology

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

Genetics

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.

This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques.

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as ...

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

Medicine

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl

The purpose of this study is to establish a standardized and reproducible regenerative blunt spinal cord injury model in the axolotl (Ambystoma mexicanum). Most clinical spinal cord injuries occur as high energy blunt traumas, inducing contusion injuries. However, most studies in the axolotl spinal cord have been conducted with sharp traumas. Hence, this study aims to produce a more clinically relevant regenerative model. Due to their impressive ability to regenerate almost any tissue, axolotls are widely used as models in regenerative studies and have been used extensively in spinal cord injury (SCI) studies. In this protocol, the axolotls are anesthetized by submersion in a benzocaine solution. Under the microscope, an angular incision is made bilaterally at a level just caudal to the hind limbs. From this incision, it is possible to dissect and expose the spinous processes. Using forceps and scissors, a two-level laminectomy is performed, exposing the spinal cord. A custom trauma device consisting of a falling rod in a cylinder is constructed, and this device is used to induce a contusion injury to the spinal cord. The incisions are then sutured, and the animal recovers from anesthesia. The surgical approach is successful in exposing the spinal cord. The trauma mechanism can produce contusion injuries to the spinal cord, as confirmed by histology, MRI, and neurological examination. Finally, the spinal cord regenerates from the injury. The critical step of the protocol is removing the spinous processes without inflicting damage to the spinal cord. This step requires training to ensure a safe procedure. Furthermore, wound closure is highly dependent on not inflicting unnecessary damage to the skin during incision. The protocol was performed in a randomized study of 12 animals.

This manuscript presents protocols for surgically inflicting controlled blunt and sharp spinal cord injuries to a regenerative axolotl (Ambystoma mexicanum).

The purpose of this study is to establish a standardized and reproducible regenerative blunt spinal cord injury model in the axolotl (Ambystoma ...

An Apical Resection Model in the Adult Xenopus tropicalis Heart

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, which&#160;can be employed to elucidate the mechanisms of adult heart regeneration.

An Apical Resection Model in the Adult Xenopus tropicalis Heart

Xenopus tropicalis is an ideal model for regenerative research as many of its organs possess a remarkable regenerative capacity. Here, we present a method for constructing a heart injury model in X. tropicalis via apex resection.

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. ...

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

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

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

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

Using Plate Fixation to Study Bone Healing and Limb Regeneration in Axolotls

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). &#8805;20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.

Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapies

A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Summary

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Chapters in this video

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl

An Apical Resection Model in the Adult Xenopus tropicalis Heart

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

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum