Design and Standardization of a Mandibular Injury Model for the Study of Craniofacial Tissue Regeneration in the Ambystoma mexicanum Model

Summary

The objective of this study was to standardize a safe and reproducible surgical approach for performing a complete transverse amputation of the mandible in Ambystoma mexicanum for the study of craniofacial tissue regeneration.

Abstract

The craniofacial complex is an anatomical region that, in humans, can be affected by developmental malformations, trauma, and pathologies such as cancer, leading to the loss of affected tissues. This creates a need to seek strategies for regenerating these lost tissues. This has promoted the study of cellular and molecular mechanisms that orchestrate the regenerative response of injured craniofacial structures in regenerating species, with Ambystoma mexicanum (A. mexicanum) being a species of great interest.

In accordance with the above, a reproducible surgical lesion model was designed and standardized in the jaw of axolotls to study the regenerative response of injured tissues and simulate a critical defect like those performed in humans. Thus, juvenile/adult animals between 12-15 cm were anesthetized with 0.1% tricaine for 20-30 min. Subsequently, a complete transverse amputation was performed using a four-handed technique on the distal third of the mandible, including the symphyseal and parasymphyseal region, removing skeletal structures corresponding to the dentary bone and Meckel's cartilage, as well as soft tissues such as muscle, connective tissue, nerves, and skin. Subsequently, bone remnants were regularized, and 0.5% sulfamerazine was applied. The tissue response was evaluated both macroscopically and by diaphanization. All procedures performed were approved by the animal experimentation committee of the University of Antioquia, Colombia. The results show that the amputated animals were able to regenerate the removed craniofacial structures morphologically, structurally, and functionally.

Of great relevance, this work provides a reproducible surgical technique with 100% survival of amputated and reamputated animals when the collection of regenerating tissues was required. Therefore, this surgical design represents a basic input for conducting comparative analyses with other vertebrates capable of regenerating craniofacial structures and for studying the cellular and molecular mechanisms underlying this regenerative response in the future.

Introduction

The ability to regenerate tissues, organs, and complex structures has been widely described throughout the animal kingdom^1,2,3. Thus, interest in understanding how these species can activate and regulate the regenerative response after injury or amputation of various structures within appendages, such as limbs and tails, has been the focus of attention in the study of the biology of regeneration^4,5,6,7,8.

Of great relevance to this work, the study of craniofacial tissue regeneration has also been an area of significant interest worldwide, given that this anatomical region is highly affected by facial trauma, craniofacial developmental disorders, infectious diseases, and tumor pathologies, including oral cancer⁹. In the presence of tumors, the excision of structures or large areas of tissue is required, either in the jaws or other facial regions, which subsequently generates the need to reconstruct and promote the repair of the affected area^10,11,12. Consequently, in recent decades, the study of craniofacial tissue regeneration has grown, given the need to explore and identify the cellular and molecular mechanisms that can enhance the regenerative response of these tissues in species with low regenerative capacity, such as humans^13,14. This has led to the use of animal models with the ability to regenerate craniofacial structures, such as the jaw, to explore and elucidate the mechanisms involved in the regenerative response following the creation of small lesions or critical defects on the jaw¹⁵.

Previous studies on mandibular amputation have highlighted the design of various types of surgical approaches that challenge the ability of different species to regenerate this structure¹⁶. In the case of zebrafish, transverse amputations in the distal third of the jaw and lateral approaches to the jaw have revealed a regenerative response in which the amputated segment is morphologically and histologically restored^17,18. On the other hand, pioneering studies in amphibians, such as Notophthalmus viridescens, have shown that after transverse amputation of the mandible, which includes structures like the tongue and pharyngeal apparatus in the amputation plane, lost tissues, including the dentary bone, muscle tissue, and teeth, can regenerate. However, the lingual structure and pharyngeal apparatus cannot be regenerated^19,20. Nevertheless, when the tongue and pharyngeal apparatus are not included in the amputation plane in this same species, all regenerated tissues recapitulate the shape and structure of the amputated tissues^21,22. In addition, transverse amputations in the species Cynops pyrrhogaster that do not include the tongue in the amputation plane show a regenerative response of the amputated mandibular structures²³. Other studies show that complete transverse amputations in the proximal third of the mandible and lateral recessions of the mandible in species such as Ambystoma maculatum, Desmognathus fuscus, Plethodon cinereus, Eurycea bislineata, and Ambystoma opacum promote a regenerative response in the injured tissue^24,25. This shows that in amphibians, the success of the regenerative response can vary depending on the design and extent of the tissues involved in the amputation plane.

On the other hand, one of the model species among amphibians used as a reference for studying the mechanisms underlying the regenerative response is the urodele amphibian A. mexicanum^26,27. Previous reports have demonstrated that this is an exceptional model capable of regenerating craniofacial structures, including the soft tissues of the floor of the mouth, the dentary bone, Meckel's cartilage, and muscular and dental structures^28,29,30,31. These findings have resulted from various surgical approaches, including punch injury to the floor of the mouth, dentectomy of teeth along with a portion of the dentary bone, and a broader approach characterized by lateral amputation of a portion of the mandible^28,29,30,31. However, to date, no complete transverse surgical approach has been described that creates a critical defect, simultaneously including the distal region of the left and right hemimandibles, as well as the dentary bone, Meckel's cartilage, the complete mandibular symphysis, and dental and muscular tissues bilaterally.

Therefore, this paper describes the step-by-step process for performing a complete transverse amputation of the jaw of A. mexicanum, which allows the regenerative response of the amputated tissues to be monitored with a 100% viability rate in the animals operated on. Furthermore, given the lack of details on how to perform this type of amputation, this surgical guide aims to ensure the reproducibility of this type of surgical approach for any researcher planning to perform comparative analyses at the morphological, tissue, cellular, and molecular levels in this and other species exposed to this same type of surgical approach, as well as other approaches, including lateral amputation of the mandible. Ultimately, this work provides a foundation for identifying future molecules that promote the regenerative response of these structures, with potential applications in craniofacial tissue regenerative medicine.

Protocol

The model organism used in this project was the salamander A. mexicanum, which is endemic to Xochimilco, Mexico. This colony of animals was legally established in 2014 with the importation of parent salamanders from the Ambystoma Genetic Stock Center at the University of Kentucky, and currently has 32 parents and over 200 juvenile animals. All procedures were approved by the Animal Experimentation Ethics Committee of the University of Antioquia (UdeA) under Act No. 151 of March 27, 2023, taking into account national and international standards of ethics in animal experimentation, such as Chapter VI of Law 84 of 1989 and Title V of Resolution 8430 of 1993 of the Ministry of Health, Colombia^32,33.

1. Animal handling

NOTE: Animals measuring approximately 12 to 15 cm in length with a wild phenotype were used to standardize this protocol. However, smaller animals and animals with other phenotypes are not excluded.

1. Select axolotls approximately 12-15 cm in length (from snout to tail).
2. Place the axolotls individually in a container large enough to allow them to move easily, ideally transparent and rectangular.
3. Keep the animals at a temperature between 19 °C and 21 °C in 20% Holtfreter's solution before the procedure and light-dark cycles (12:12).
   NOTE: Whenever possible, avoid overfeeding the animals 24 h prior to performing the procedure. This is to prevent episodes of emesis.

2. Complete transverse amputation in the distal third of the mandible

1. Preparation of anesthesia and pre-surgery for animals
   1. Prepare a 0.1% tricaine methanesulfonate anesthetic solution: mix 1 g of tricaine methanesulfonate, 100 µL of 1 M ethylenediaminetetraacetic acid (EDTA), pH 7.5, 50 µL of 0.5% phenol red, and make up to 1 L with 40% Holtfreter's solution. Adjust the pH to 7.5 with NaOH.
   2. Prepare all sterilized microsurgical instruments (scissors, forceps, tweezers, 7A spatula, 1.5 mL tubes, dentimeter, Pasteur pipette, Petri dish, and a plastic tray for the surgical procedure). Wear new disposable nitrile gloves before handling the instruments and the animal.
   3. Clean the work area with 70% antiseptic alcohol, adjust and position the direct light over the surgical field appropriately.
2. Anesthesia
   1. Place the axolotl in a container with approximately 500 mL of 0.1% tricaine methanesulfonate anesthetic solution for 20-30 min to ensure deep anesthesia.
      NOTE: Ensure that the animal is completely submerged in the anesthetic solution.
   2. Check for signs of general anesthesia by verifying the complete absence of gill movements and response to tactile stimuli.
      NOTE: To monitor jaw regeneration, once the animal has been anesthetized, take preoperative photographs using the stereomicroscope, following the steps in section 4.
3. Complete transverse amputation of the jaw
   NOTE: The amputation should be performed as carefully and quickly as possible using a four-handed technique, i.e., two surgeons.
   1. Carefully place the animal in the supine position (face up) on the plastic tray.
   2. Measure the region to be amputated with a calliper (endodontic dental meter) from the outer edge of the jaw toward the proximal end of this structure, approximately 2.5 mm in the chin/symphysis region (Figure 1A).
      NOTE: The measurement with a calliper is perfomed over the animal's skin (extraoral).
   3. Make a mark with a red wax pencil to identify the trajectory of the amputation plane and the extent of the surgical defect.
   4. Carefully lift the axolotl and hold it firmly with one hand so that the animal does not slip out of the operator's grasp.
      NOTE: This action should be performed by one of the operators (operator 1), while the other operator (operator 2) will be responsible for handling the surgical instruments and making the incision.
   5. Position a direct light over the head region of the axolotl, specifically over the anatomical region to be operated on.
   6. With the other hand, use the rounded, wider side of a 7A spatula to separate the tongue from the floor of the mouth and the jaw region to be operated on. (Figure 1B).
      NOTE: Make sure that the tongue is slightly superimposed on the upper jaw.
   7. First, make (operator 2) an initial cut with the surgical forceps, from the outer edge of the jaw over the skin and hard tissues (tooth bone and Meckel's cartilage) (Figure 1C).
      NOTE: Ensure that the instruments are sharpened adequately for greater control when cutting and manipulating tissue.
   8. Continue cutting transversely with fine surgical scissors so that the incision crosses the entire width of the jaw from the site of the first cut to the contralateral side until the distal tissue of the jaw is completely removed, following the planned amputation plane (complete transverse amputation) (Figure 1D).
      NOTE: In the case of larger axolotls with more robust jaws, it is recommended to finish the cutting plane with forceps for a cleaner and less traumatic cut of the skeletal structures (Figure 1E).
   9. Observe the immediate retraction of the remaining soft tissue (Figure 1F).
   10. Collect the amputated tissue with dissecting forceps and immerse it in a 1.5 mL tube with the desired solution according to the procedure to be performed: 4% paraformaldehyde (PFA) for fixation and subsequent histotechnology or immunofluorescence, or in Trizol or RNA later for RNA, DNA, or protein extraction, among other techniques (Figure 1F).
       NOTE: This tissue can be used as a non-regenerating control for comparison with regenerated tissue from a histological perspective, for basal gene expression, or for the detection of markers of final tissue differentiation.
   11. Place the axolotl in a Petri dish, observe under the stereomicroscope, and remodel the exposed bone and cartilage tissue edges with forceps.
   12. Carefully place the axolotl back in the plastic tray and apply 0.5% Sulfamerazine antibiotic baths for 12 h.

3. Postoperative recovery of A. mexicanum

NOTE: No postoperative analgesics were administered because there is currently no validated postoperative analgesic regimen specifically established for Ambystoma mexicanum. Experimental studies evaluating opioid analgesics in axolotls have not demonstrated consistent analgesic efficacy. Therefore, all procedures were performed under deep MS-222 anesthesia, and animals were closely monitored during postoperative recovery in accordance with the approved institutional animal care and use protocol.

1. After the surgical procedure, place the axolotl in a transparent plastic container containing 40% Holtfreter's solution and monitor recovery from general anesthesia by assessing gill movements, righting reflex, and responses to tactile stimuli. Full recovery typically occurs within 30–60 min. Continue daily postoperative monitoring, paying particular attention to swimming behavior, jaw movements, feeding after food is reintroduced (24–48 h postoperatively), and any signs of impaired recovery or distress.
   NOTE: Keep the 40% Holtfreter solution cold, at a temperature between approximately 4 °C and 8 °C.
2. Change the cold 40% Holtfreter solution every 24 h for at least 8 days.
   NOTE: The first 48 h are the most critical in the animal's recovery, so it must be under daily observation.
3. After 24-48 h post-procedure, feed the axolotl soft-textured food that is easier to catch and less traumatic for the animal.
   NOTE: Do not feed the axolotl for the first 24 h after the surgical procedure.

4. Image acquisition and monitoring of regenerative response

NOTE: All images should be taken as quickly as possible to avoid prolonged handling of the recently operated animal.

1. Follow the steps described in section 2.2. Once the animal is fully anesthetized, carefully pick it up and take it to the stereomicroscope and digital camera in a Petri dish to acquire pre- and post-amputation images.
   NOTE: Clean and disinfect the stereomicroscope with 70% alcohol beforehand.
2. Use an incident light that points at the axolotl's head to observe the jaw area better.
3. Hold the axolotl's body securely with one hand to prevent it from slipping out of the image acquisition field.
   NOTE: Avoid taking photos in an aqueous medium to prevent distortions of the actual size; use a Petri dish without liquid. Lightly moisten the axolotl occasionally with 40% Holtfreter's solution.
4. Properly identify the jaw area and focus on it appropriately.
   NOTE: For the days following (follow-up) the procedure, keep in mind the need for proper focus, as regenerating tissue in the early stages is often very subtle and, in some cases, imperceptible.
5. Optimize the parameters for image acquisition by adjusting the program tools (contrast, brightness, exposure, gain, white balance, etc.).
6. Acquire images using a stereomicroscope (0.7x) to take measurements and monitor the regeneration of the structure.
7. Once the regeneration period was complete, the regenerated tissues were stained using alcian blue and alizarin red according to the previously reported protocol³⁴.

Results

Considering the importance and relevance of studying the potential post-amputation regenerative response of craniofacial structures in species such as A. mexicanum²⁹, it is necessary to have a safe and reproducible surgical technique that allows not only the evaluation of the regenerative response of tissues, but also offers a low or zero mortality rate in the animals undergoing surgery. Consequently, this study presents the standardization of a successful surgical approach involving comp...

Discussion

This paper presents the successful surgical approach to complete transverse amputation of the mandible in the A. mexicanum animal model, along with several recommendations necessary to reproduce this surgical model for monitoring the regenerative response of these structures and the potential identification of factors regulating this regenerative process.

In this study, a complete transverse amputation was performed on the distal third of the mandible of A. mexicanum, involvi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Axolotl (Ambystoma mexicanum)	Ambystoma Genetic Stock Center	N/A	15-18 cm and 16 g - 22 g wildetype
Calcium Chloride dihydrate	Duksan	1098	CaCl2 2H2O
Digital camera MotiCAM 5	MotiCAM	https://www.motic.com/upload/File/201407/2014071115335993.pdf
EDTA - Sodium salt, dihydrate	Biobasic	EB0185	C10H14N2O8Na2 . H2O
Glass petri dish	any maker	N/A	Borosilicate glass
Magnesium Sulfate 7-hydrate	ITW Reagents	131404.1211	MgSO4 7H2O
Microsurgical instruments	N/A	N/A	Forceps, clamp, 7A spatula, dentimeter and scissors
Pasteur pipette	any maker	N/A	Low-density polyethylene
Potassium Chloride	Duksan	372	KCl
Red wax pencil	any maker	N/A
Sodium Chloride	Merk	K48297604 717	NaCl
Software Motic Images Plus-Versión 2.0	Motic	https://www.motic.com/As_Support_Download/d42.html
Stereo microscope	Olympus SZX16	N/A
Sulfamerazine sodium salt	Sigma- Aldrich	S0800	4-Amino-N-(4-methyl-2-pyrimidinyl) benzenesulfonamide
Tricaine methanesulfonate	Sigma- Aldrich	E10521	Ethyl 3-aminobenzoate methanesulfonate
Tubes 1.5 mL	Axygen	MCT150C	Pure polypropylene