Belfran Carbonell-M

Axolotls ( Ambystoma mexicanum ) are neotenic salamanders widely used as experimental models in regenerative biology, developmental research, toxicology, and pharmacology. Despite their extensive use in laboratory research and conservation programs, standardized necropsy procedures for this species remain limited and poorly documented. The absence of systematic protocols limits the consistency of postmortem examinations and complicates the interpretation of pathological findings in both experimental and diagnostic contexts. Here, we present a detailed and reproducible necropsy protocol for the neotenic axolotl based on 17 systematic necropsies performed under controlled laboratory conditions. The procedure describes step‐by‐step methods for external examination, access to the cranial cavity, exposure of the spinal cord, and systematic exploration of the coelomic cavity. Particular emphasis is placed on anatomical orientation, preservation of organ relationships, and strategies to minimize tissue damage during dissection. The protocol also includes guidelines for tissue sampling, fixation, and transport for downstream laboratory analyses such as histopathology and microbiology. Protocol refinement revealed practical considerations not previously described for this species, including variability in hepatic size and the absence of coelomic fat bodies in examined specimens, which simplified visceral exposure and influenced dissection strategy. These findings highlight the need to adapt necropsy techniques to species‐specific anatomical characteristics. This protocol provides a practical reference for veterinarians, pathologists, and researchers working with A. mexicanum , facilitating standardized postmortem examinations and improving the reliability and reproducibility of diagnostic and research findings in this important amphibian model. © 2026 Wiley Periodicals LLC. Basic Protocol 1 : External examination Basic Protocol 2 : Organs of the head and spinal cord examination Basic Protocol 3 : Organs of the coelomic cavity examination Support Protocol : Collection, preservation and transport of specimens for laboratory analysis

Current therapeutic strategies aimed at promoting the regeneration of craniofacial tissues do not fully address all physiological and aesthetic needs. This highlights the need to investigate the regenerative response of craniofacial tissues in regenerative species like Ambystoma mexicanum ( A. mexicanum ), particularly concerning the mandible. However, although previous studies have demonstrated the regenerative response after lateral cuts of the mandible in A. mexicanum , it is unknown whether it can regenerate the mandible after complete transverse amputation, a type of injury used to remove tumors and extensive lesions that involve the symphyseal and parasymphyseal. Furthermore, it remains uncertain whether there is a limit to the regenerative response along the mandible proximal-distal axis. In light of this, we assessed the regenerative response following axolotl mandible complete transverse amputation through macroscopic and histological analyses and skeletogenic assessment via diaphanization. We also evaluated proliferation and the expression of genes associated with blastema formation. The results indicate that following mandibular injury, a blastema formed from which all amputated tissues were regenerated by 180 days post-amputation (dpa) and integrated seamlessly with the remaining structures. However, morphological alterations were noted in the regenerated skeletal elements. Additionally, we identified the expression of genes characteristic of epimorphic regeneration, including Prrx1, Kazald1, Msx2, Pax7 , and Sox2 . Interestingly, regenerative failure was observed after a second, more proximal amputation. Together, our findings suggest that the regenerative response to complete transverse amputation of the mandible in axolotls is fundamentally epimorphic; however, this regenerative capacity appears to have limits along the mandible proximal-distal axis.

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.

Salamanders are known for their incredible regenerative abilities, but translating findings to mammals is complicated by unannotated genes and unclear orthology. An example highlighting this difficulty was a discovery in the axolotl ( Ambystoma mexicanum ) of a regeneration-associated gene that has been identified as either Kazald1 or Kazald2 . Since orthology inference of genes across species is crucial to identifying gains and losses of gene functions, and thus if gene usage is likely to be consistent across species, we investigated the evolution of the axolotl genes using an extensive cross-species analysis. Molecular phylogeny inference conclusively identified the regeneration-associated gene as Kazald2 , but also revealed an undescribed four-member Kazald gene family in jawed vertebrates. Moreover, synteny comparisons demonstrated that this family originated in the ancestral two-rounds of whole genome duplication. Additionally, we performed vertebrate-wide comparisons of Kazald gene expression profiles, employing available RNA-Seq which we validated in whole tissues of axolotl, zebrafish, and sharks. This uncovered seemingly ancestral connections conserved over jawed vertebrate evolution, such as Kazald1 with skeletogenesis and odontogenesis and Kazald2 with regeneration. It also suggested novel putative roles within specific lineages, including Kazald3 in teleost fish skeletogenesis and Kazald4 within avian brains. Our study thus demonstrates the establishment of a Kazald gene quartet in the jawed vertebrate ancestor, and elucidates the asymmetry of gene fates of its members, including deeply ancestral roles and comparably recent innovations. This provides a comprehensive report of this formerly undescribed gene family, offering a solid foundation for future studies of these genes in diverse species.

Sirtuins are a family of lysine deacetylases that regulate cellular homeostasis and energy sensing. Regeneration is the process that restores structural and functional homeostasis at the cellular, tissue, organ, and appendage levels. Several cellular processes, such as epithelial–mesenchymal transition (EMT), proliferation, migration, and differentiation, contribute to restoration after an injury. This review highlights the role of sirtuins in tissue, organ, and anatomical structure regeneration, showing how sirtuins modulate signalling pathways by deacetylating targets such as transcription factors. Furthermore, understanding the role of this protein family could help elucidate the molecular and cellular mechanisms underlying tissue regeneration, which may hold significant potential for fields such as regenerative medicine. The review compiles evidence suggesting that sirtuins are emerging factors in the regeneration of various organs (e.g., skin, liver, heart) and tissues (e.g., bone, muscle, cornea, spinal cord).

Background/Objectives: Antimicrobial peptides are essential molecules in the innate immunity of various organisms and possess a broad spectrum of antimicrobial, antitumor, and immunomodulatory activities. Due to their multifunctionality, they are seen as an alternative for controlling bacterial infections. Although conventional antibiotics have improved health worldwide, their indiscriminate use has led to the emergence of resistant microorganisms. To discover new molecules with antimicrobial activity that could overcome the limitations of traditional antibiotics, this study aimed to identify antimicrobial peptides in Ambystoma mexicanum. Methods: In this study, hypothetical proteins encoded in the Ambystoma mexicanum transcriptome were predicted. These proteins were aligned with peptides reported in the Antimicrobial Peptide Database (APD3) using the Fasta36 program. After identifying peptide sequences with potential antibacterial activity, their expression was confirmed through conventional polymerase chain reaction (PCR) and then chemically synthesized. The antibacterial activity of the synthesized peptides was evaluated against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. Results: A new antimicrobial peptide named AXOTL-13 was identified. AXOTL-13 is an amphipathic cationic alpha-helical peptide with the ability to inhibit the growth of Escherichia coli without causing hemolysis in red blood cells, with its action likely directed at the membrane, as suggested by morphological changes observed through scanning electron microscopy. Conclusions: This research is pioneering in evaluating the activity of antimicrobial peptides present in Ambystoma mexicanum and in specifically identifying one of these peptides. The findings will serve as a reference for future research in this field.

The discovery and improvements of antimicrobial peptides (AMPs) have become an alternative to conventional antibiotics. They are usually small and heat-stable peptides, exhibiting inhibitory activity against Gram-negative and Gram-positive bacteria. In this way, studies on broad-spectrum AMPs found in amphibians with the remarkable capability to regenerate a wide array of tissues are of particular interest in the search for new strategies to treat multidrug-resistant bacterial strains. In this work, the use of bioinformatic approaches such as sequence alignment with Fasta36 and prediction of antimicrobial activity allowed the identification of the Ramosin peptide from the de novo assembled transcriptome of the plethodontid salamander Bolitoglossa ramosi obtained from post-amputation of the upper limb tissue, heart, and intestine samples. BLAST analysis revealed that the Ramosin peptide sequence is unique in Bolitoglossa ramosi. The peptide was chemically synthesized, and physicochemical properties were characterized. Furthermore, the in vitro antimicrobial activity against relevant Gram-positive and Gram-negative human pathogenic bacteria was demonstrated. Finally, no effect against eukaryotic cells or human red blood cells was evidenced. This is the first antibacterial peptide identified from a Colombian endemic salamander with interesting antimicrobial properties and no hemolytic activity.

Introduction: Reactive oxygen species (ROS) represent molecules of great interest in the field of regenerative biology since several animal models require their production to promote and favor tissue, organ, and appendage regeneration. Recently, it has been shown that the production of ROS such as hydrogen peroxide (H₂O₂) is required for tail regeneration in Ambystoma mexicanum. However, to date, it is unknown whether ROS production is necessary for limb regeneration in this animal model. Methods: forelimbs of juvenile animals were amputated proximally and the dynamics of ROS production was determined using 2′7- dichlorofluorescein diacetate (DCFDA) during the regeneration process. Inhibition of ROS production was performed using the NADPH oxidase inhibitor apocynin. Subsequently, a rescue assay was performed using exogenous hydrogen peroxide (H₂O₂). The effect of these treatments on the size and skeletal structures of the regenerated limb was evaluated by staining with alcian blue and alizarin red, as well as the effect on blastema formation, cell proliferation, immune cell recruitment, and expression of genes related to proximal-distal identity. Results: our results show that inhibition of post-amputation limb ROS production in the A. mexicanum salamander model results in the regeneration of a miniature limb with a significant reduction in the size of skeletal elements such as the ulna, radius, and overall autopod. Additionally, other effects such as decrease in the number of carpals, defective joint morphology, and failure of integrity between the regenerated structure and the remaining tissue were identified. In addition, this treatment affected blastema formation and induced a reduction in the levels of cell proliferation in this structure, as well as a reduction in the number of CD45⁺ and CD11b + immune system cells. On the other hand, blocking ROS production affected the expression of proximo-distal identity genes such as Aldha1a1, Rarβ, Prod1, Meis1, Hoxa13, and other genes such as Agr2 and Yap1 in early/mid blastema. Of great interest, the failure in blastema formation, skeletal alterations, as well as the expression of the genes evaluated were rescued by the application of exogenous H₂O₂, suggesting that ROS/H₂O₂ production is necessary from the early stages for proper regeneration and patterning of the limb.

Ambystoma mexicanum is a urodele amphibian endemic to Xochimilco Lake in Mexico, it belongs to the salamander family Ambystomatidae. This species has frequently been used as model organism in developmental biology and regeneration laboratories around the world due to its broad regenerative capacities and adaptability to laboratory conditions. In this review we describe the establishment of the first colony of axolotls in Colombia to study tissue regeneration and our perspectives on the use A. mexicanum as a model organism in Colombia are discussed emphasizing its possible uses in regeneration and developmental biology

Background: Hydrogen peroxide (H₂O₂) is a key reactive oxygen species (ROS) generated during appendage regeneration among vertebrates. However, its role during tail regeneration in axolotl as redox signaling molecule is unclear.Results: Treatment with exogenous H₂O₂rescues inhibitory effects of apocynin‐induced growth suppression in tail blastema cells leading to cell proliferation. H₂O₂also promotes recruitment of immune cells, regulate the activation of AKT kinase andAgr2expression during blastema formation. Additionally, ROS/H₂O₂regulates the expression and transcriptional activity of Yap1 and its target genesCtgfandAreg.Conclusions: These results show that H₂O₂is necessary and sufficient to promote tail regeneration in axolotls. Additionally, Akt signaling andAgr2were identified as ROS targets, suggesting that ROS/H₂O₂is likely to regulate epimorphic regeneration through these signaling pathways. In addition, ROS/H₂O₂‐dependent‐Yap1 activity is required during tail regeneration.