Descripción morfométrica de la anatomía externa e interna del Ambystoma mexicanum / Morphometric description of the external and internal anatomy of Ambystoma mexicanum

RESUMEN: El Ambystoma mexicanum o comúnmente llamado axolote, es un anfibio miembro del género Ambystoma que comprende 32 especies presentes desde el sur de Canadá hasta la región central de México. Actualmente esta especie se encuentra en peligro de extinción debido a cambios fisicoquímicos en su medio ambiente y la depredación por organismos introducidos en su hábitat. Dentro de las múltiples estrategias para su conservación se encuentran el desarrollo de programas de investigación, educación y desarrollo del manejo clínico. El objetivo del presente trabajo estuvo enfocado en obtener y analizar datos morfométricos acompañados por una descripción de la anatomía externa e interna de la especie Ambystoma mexicanum con el propósito de contribuir a su conservación. Por lo anterior, cinco ejemplares de la especie Ambystoma mexicanum de 2 años, criados en cautiverio, fueron estudiados para obtener valores morfométricos externos e internos utilizando un calibrador vernier, balanzas analíticas y rayos X. Los resultados de la observación externa mostraron el dimorfismo sexual característico de estas especies presentes en la zona peri cloacal, además, el análisis radiográfico permitió observar y señalar algunas estructuras óseas del esqueleto axial y apendicular. Los ejemplares presentaron un peso promedio de 31,6 g y una longitud de 15,7 cm. Los miembros anteriores mostraron una longitud de 2,92 cm y 2,8 cm en los miembros posteriores. El análisis de los órganos internos mostró que el corazón tiene un peso de 0,036 g y una longitud de 0,75 cm, los pulmones un peso de 0,019 g y una longitud de 2,6 cm, el estómago arrojó una longitud de 4,86 cm y el intestino 10,88 cm. En conclusión, los valores presentados en el presente trabajo sirven de referencia en futuros trabajos de investigación clínica veterinaria y manejo zootécnico del axolote para su conservación y preservación.

SUMMARY: The Ambystoma mexicanum commonly known as axolotl, is an amphibian and member of the genus Ambystoma which includes 32 species that can be found from southern Canada to central Mexico. Currently this species is in danger of extinction due to physicochemical changes in its environment and predation by organisms introduced into its habitat. Among the multiple strategies to aid in its conservation are the development of research programs, education and development of clinical management. The objective of this work is focused on obtaining and analyzing morphometric data accompanied by a description of the external and internal anatomy of the species Ambystoma mexicanum with the purpose of contributing to the conservation of this species. Therefore, five 2-year-old specimens of the Ambystoma mexicanum species, raised in captivity, were studied to obtain external and internal morphometric values using a vernier caliper, analytical balances and X-rays. The results of external observation showed the characteristic sexual dimorphism of these species present in the pericloacal zone. Furthermore, the radiographic analysis allowed to observe and point out some bony structures of the axial and appendicular skeleton. The specimens presented an average weight of 31.60 g and a length of 15.70 cm. The forelimbs showed a length of 2.92 cm and 2.8 cm in the hindlimbs. Analysis of the internal organs showed that the heart had a weight of 0.036 g and a length of 0.75 cm, the lungs a weight of 0.019 g and a length of 2.6 cm, the stomach had a length of 4.86 cm and the intestine 10.88 cm. In conclusion, the values presented in this work serve as a reference for future veterinary clinical research and zootechnical management of the axolotl for its conservation and preservation.

Celiotomia e gastrotomia para remoção cirúrgica de corpos estranhos em axolote (Ambystoma mexicanum): relato de caso / Celiotomy and gastrotomy for surgical removal of foreign bodies in axolotl (Ambystoma mexicanum): case report

A ingestão de corpos estranhos em aquários artificiais é uma ocorrência frequente observada na clínica cirúrgica de anfíbios criados como pets, o que inclui os axolotes (Ambystoma mexicanum). O presente relato descreve um caso de ingestão de sete corpos estranhos em um axolote macho, de cinco meses de idade, com histórico de abaulamento irregular e irredutível da superfície corpórea ventral, de consistência firme. Na anamnese foi informado que o animal habitava um aquário com substrato de cascalhos. Ao exame físico, o axolote apresentou bom estado geral e parâmetros vitais dentro da normalidade para a espécie. Os materiais deglutidos foram identificados em região gástrica por meio de exame radiográfico corpóreo total, e suas características condiziam com o substrato utilizado no aquário do paciente. Como protocolo anestésico, foi priorizada a imersão em Isofluorano e gás oxigênio, com o objetivo de atingir a via branquial e, ocasionalmente, transdérmica. A remoção cirúrgica foi feita através de celiotomia e gastrotomia em ambiente aquático com temperatura, pH e coleção bacteriana controlados, conforme literatura disponível e tendo em consideração a natureza, diâmetro e localização dos corpos estranhos. Após cinco e quinze dias do procedimento, acompanhou-se a cicatrização da ferida cirúrgica, sendo possível constatar bom restabelecimento da continuidade dos tecidos e bom estado geral do paciente.

The ingestion of foreign bodies in artificial aquariums is a frequent occurrence observed in the surgical clinic of amphibians raised as pets, which includes axolotls (Ambystoma mexicanum). The present report describes a case of ingestion of seven foreign bodies in a male axolotl, five months old, with a history of irregular and irreducible bulging of the ventral body surface, with firm consistency. In the anamnesis, it was reported that the animal inhabited an aquariums with gravel substrate. On physical examination, the axolotl showed good general condition and vital parameters within the normal range for the species. The swallowed materials were identified in the gastric region by means of total body radiographic examination, and their characteristics were consistent with the substrate used in the patient’s aquarium. As anesthetic protocol, immersion in Isofluorane and oxygen gas was prioritized, in order to reach the branchial and, occasionally, transdermal route. Surgical removal was performed through celiotomy and gastrotomy in an aquatic environment with controlled temperature, pH and bacterial collection, according to available literature and taking into account the nature, diameter and location of foreign bodies. After five and fifteen days of the procedure, the healing of the surgical wound was monitored, showing a good restoration of tissue continuity and a good general condition of the patient.

Celiotomia e gastrotomia para remoção cirúrgica de corpos estranhos em axolote (Ambystoma mexicanum): relato de caso / Celiotomy and gastrotomy for surgical removal of foreign bodies in axolotl (Ambystoma mexicanum): case report

A ingestão de corpos estranhos em aquários artificiais é uma ocorrência frequente observada na clínica cirúrgica de anfíbios criados como pets, o que inclui os axolotes (Ambystoma mexicanum). O presente relato descreve um caso de ingestão de sete corpos estranhos em um axolote macho, de cinco meses de idade, com histórico de abaulamento irregular e irredutível da superfície corpórea ventral, de consistência firme. Na anamnese foi informado que o animal habitava um aquário com substrato de cascalhos. Ao exame físico, o axolote apresentou bom estado geral e parâmetros vitais dentro da normalidade para a espécie. Os materiais deglutidos foram identificados em região gástrica por meio de exame radiográfico corpóreo total, e suas características condiziam com o substrato utilizado no aquário do paciente. Como protocolo anestésico, foi priorizada a imersão em Isofluorano e gás oxigênio, com o objetivo de atingir a via branquial e, ocasionalmente, transdérmica. A remoção cirúrgica foi feita através de celiotomia e gastrotomia em ambiente aquático com temperatura, pH e coleção bacteriana controlados, conforme literatura disponível e tendo em consideração a natureza, diâmetro e localização dos corpos estranhos. Após cinco e quinze dias do procedimento, acompanhou-se a cicatrização da ferida cirúrgica, sendo possível constatar bom restabelecimento da continuidade dos tecidos e bom estado geral do paciente.

The ingestion of foreign bodies in artificial aquariums is a frequent occurrence observed in the surgical clinic of amphibians raised as pets, which includes axolotls (Ambystoma mexicanum). The present report describes a case of ingestion of seven foreign bodies in a male axolotl, five months old, with a history of irregular and irreducible bulging of the ventral body surface, with firm consistency. In the anamnesis, it was reported that the animal inhabited an aquariums with gravel substrate. On physical examination, the axolotl showed good general condition and vital parameters within the normal range for the species. The swallowed materials were identified in the gastric region by means of total body radiographic examination, and their characteristics were consistent with the substrate used in the patient's aquarium. As anesthetic protocol, immersion in Isofluorane and oxygen gas was prioritized, in order to reach the branchial and, occasionally, transdermal route. Surgical removal was performed through celiotomy and gastrotomy in an aquatic environment with controlled temperature, pH and bacterial collection, according to available literature and taking into account the nature, diameter and location of foreign bodies. After five and fifteen days of the procedure, the healing of the surgical wound was monitored, showing a good restoration of tissue continuity and a good general condition of the patient.

A double fluorescence chimeric limb regeneration model reveals muscle fiber reconnection during axolotl limb regeneration / Modelo de regeneración quimérica de doble fluorescencia del miembro revela la reconexión de la fibra muscular durante la regeneración del miembro axolotl

SUMMARY: Axolotl limb regeneration is a fascinating characteristic that has attracted attention for several decades. Our previous studies on axolotl limb regeneration indicated that the satellite cells in the remnant muscles move distally into the blastema to regenerate new muscles that are separated by a gap from remnant muscles. Thereafter, the regenerative muscle fibers start to reconnect with remnant ones. In this study, the reconnection at the individual muscle fiber level was elucidated to test the hypothesis that this reconnection happens synchronously among involved muscles. Three pairs of EGFP+ mid-bud stage blastemas were transplanted onto freshly amputated stumps of RFP+ axolotls at the same thigh position to generate double fluorescence chimeric regenerative hindlimbs. These regenerative limbs were harvested very late far beyond they had reached the late differentiation stage. Fluorescence imaging of these limbs in cross sections revealed that in the proximal remnant part of the muscle fiber, reconnection occurred at a different pace among the muscles. In the major thigh muscle gracilis, the reconnection started from the periphery before it was completed. Furthermore, RFP+ muscle fibers contributed to muscle regeneration in the distal regenerative parts. Intriguingly, this red cell contribution was limited to ventral superficial muscles of the calf. This kind of double fluorescence chimeric limb regeneration model may help increase the understanding of the patterning of axolotl limb regeneration in late stages.

RESUMEN: La regeneración del miembro de Axolotl es una característica fascinante que ha llamado la atención durante varias décadas. Nuestros estudios previos sobre la regeneración del miembro del Axolotl indicaron que las células satélite en los músculos remanentes se mueven distalmente hacia el blastema para regenerar nuevos músculos que están separados por una brecha de músculos remanentes. A partir de entonces, las fibras musculares regenerativas comienzan a reconectarse con las restantes. En este estudio, se aclaró la reconexión a nivel de fibra muscular individual para probar la hipótesis de que esta reconexión ocurre sincrónicamente entre los músculos involucrados. Se trasplantaron tres pares de blastemas EGFP+ en la etapa de yema media en tocones recién amputados de axolotls RFP+ en la misma posición del muslo para generar miembros posteriores regenerativos quiméricos de fluorescencia doble. Estos miembros regenerativos se cosecharon muy tarde mucho más allá de haber alcanzado la etapa de diferenciación tardía. Las imágenes de fluorescencia de estos miembros en secciones transversales revelaron que en la parte remanente proximal de la fibra muscular, la reconexión se produjo a un ritmo diferente entre los músculos. En el músculo grácil, la reconexión comenzó desde la periferia antes de completarse. Además, las fibras musculares RFP+ contribuyeron a la regeneración muscular en las partes regenerativas distales. Curiosamente, esta contribución de glóbulos rojos se limitó a los músculos superficiales ventrales de la pantorrilla. Este tipo de modelo de regeneración quimérica de doble fluorescencia del miembro puede ayudar a aumentar la comprensión del patrón de la regeneración del miembro del Axolotl en etapas tardías.

A comparative study of fixatives for axolotl blastema tissue / Estudio comparativo de fijadores para el tejido blastema del axolotl

Regeneration is defined as tissue renewal and functional restoration process of the damaged parts of the body after an injury. Ambystoma mexicanum, commonly named the Axolotl, is one of the unique vertebrates, which has a remarkable ability to regenerate their extremities following the amputation. Although the process of regeneration includes several periods, it can be divided into two main phases; blastema formation and dedifferentiation. In the couple of hours following the amputation, wound closure occurs by migration of epithelial cells around the amputation site followed by macrophage infiltration and dedifferentiation of cells to turn into stem cells. Accumulated stem cells form a very authentic tissue type called blastema, which is crucial for successful regeneration. In order to evaluate this exceptional tissue and acquire high quality images, it is crucial to employ specific procedures to prepare the tissue for imaging. Here, in this study, we aimed to investigate success of various fixative solutions (Carnoy's, Bouin's, % 10 NBF, Clarke's, Alcoholic Formaline and AFA) to monitor the fixed blastema. Our data reveals that integrity of the blastema tissue differs among used fixatives and a significant difference is observed between the samples in terms of staining quality.

La regeneración se define como la renovación del tejido y el proceso de restauración funcional de las partes dañadas del cuerpo después de una lesión. Ambystoma mexicanum, comúnmente llamado Axolotl, es uno de los únicos vertebrados que tiene una notable capacidad para regenerar sus miembros después de una amputación. Aunque el proceso de regeneración incluye varios períodos, se puede dividir en dos fases principales: formación del blastema y desdiferenciación. En el par de horas después de la amputación, el cierre de la herida ocurre por la migración de células epiteliales alrededor del sitio de la amputación seguido por una infiltración de macrófagos y la desdiferenciación de las células para convertirse en células madre. Las células madre acumuladas forman un tipo de tejido muy diferenciado denominado blastema, que es crucial para una exitosa regeneración. Para evaluar este tejido y adquirir imágenes de alta calidad, es crucial emplear procedimientos específicos para la obtención de imágenes. En este estudio, se intentó investigar el éxito de varias soluciones fijadoras (Carnoy, Bouin, % 10 NBF, Clarke, Formalina Alcohólica y AFA) para monitorear la fijación del blastema. Nuestros datos revelan que la integridad del tejido del blastema difiere entre los fijadores utilizados y una diferencia significativa observada entre las muestras se da en términos de la calidad de tinción.

Functional and structural regeneration in the axolotl heart (Ambystoma mexicanum) after partial ventricular amputation / Regeneración funcional y estructural del corazón de axolotl (Ambystoma mexicanum) después de amputación parcial del ventrículo

In the present study we evaluated the effect of partial ventricular amputation (PVA) in the heart of the adult urodele amphibian (Ambystoma mexicanum) in vivo on spontaneous heart contractile activity recorded in vitro in association to the structural recovery at one, five, 30 and 90 days after injury. One day after PVA, ventricular-tension (VT) (16 ± 3%), atrium-tension (AT) (46 ± 4%) and heart rate (HR) (58 ± 10%) resulted lower in comparison to control hearts. On days five, 30 and 90 after damage, values achieved a 61 ± 5, 93 ± 3, and 98 ± 5% (VT), 60 ± 4, 96 ± 3 and 99 ± 5% (AT) and 74 ± 5, 84 ± 10 and 95 ± 10% (HR) of the control values, respectively. Associated to contractile activity recovery we corroborated a gradual tissue restoration by cardiomyocyte proliferation. Our results represent the first quantitative evidence about the recovery of heart contractile activity after PVA in an adult urodele amphibian, indicating that the heart of A. mexicanum restores its functional capacity concomitantly to the structural recovery of the myocardium by proliferation of cardiomyocytes after PVA. These properties make the heart of A. mexicanum a potential model to study the mechanisms underlying heart regeneration in adult vertebrates in vivo.

En el presente estudio evaluamos el efecto de la amputación parcial del ventrículo (APV) del corazón de un anfibio urodelo adulto (Ambystoma mexicanum) in vivo, sobre la actividad contráctil espontánea del corazón registrada in vitro, a diferentes tiempos después de APV, en asociación a su recuperación estructural. Un día después del daño, los valores de tensión ventricular (TV) (16 ± 3%), tensión auricular (TA) (46 + 4%) y frecuencia cardiaca (FC) (58 + 10%), resultaron ser menores respecto al control. En los días cinco, 30 y 90 después del daño, los valores alcanzaron 61 ± 5, 93 ± 3 y 98 ± 5% (TV), 60 ± 4, 96 ± 3 y 99 ± 5% (AT) y 74 ± 5, 84 ± 10 y 95 ± 10% (FC) de los valores control, respectivamente. Además de la recuperación de la actividad contráctil, corroboramos la recuperación estructural y gradual del tejido miocárdico por proliferación de cardiomiocitos. Nuestros resultados representan la primera evidencia cuantitativa de la recuperación de la actividad contráctil del corazón de un anfibio urodelo adulto después de APV; indicando que el corazón de A. mexicanum recupera su capacidad funcional concomitantemente con la recuperación estructural del miocardio por proliferación de cardiomiocitos. El corazón de A. mexicanum es un modelo potencial para el estudio de los mecanismos de la regeneración miocárdica de vertebrados adultos in vivo.

Regeneración miocárdica en Ambystoma mexicanum después de lesión quirúrgica / Myocardial regeneration in Ambystoma mexicanum after surgical injury

Se realizó resección ventricular en el corazón de Ambystoma mexicanum, se evaluó si la restitución del tejido resulta de hipertrofia o de hiperplasia. Por medio de una tinción tricrómica se encontró que 5 días después del daño en el espacio de la resección se encontró un coágulo rodeado de fibras de colágena (83 ± 6%), músculo (10 ± 3%) y zonas sin tejido (7 ± 2%). Una proporción de 50 ± 4 y 90 ± 2% correspondió a tejido muscular 10 y 30 días después de la lesión. La tinción con bis-Benzimida indicó que en la zona lesionada hay proliferación celular. En tanto que la inmunohistoquímica doble para actina sarcomérica a y antígeno nuclear de proliferación celular mostró que el tejido que restituyó el espacio se produjo por proliferación de cardiomiocitos con un valor máximo de 68%, 5 días después de la lesión. Nuestros resultados indican que el miocardio de A. mexicanum recupera su estructura mediante la hiperplasia de cardiomiocitos y sugieren que su capacidad regenerativa es mayor que la informada para mamíferos adultos (1%), y para otros vertebrados no mamíferos (32%). Esto hace de A. mexicanum un modelo idóneo para estudiar los mecanismos reguladores de la regeneración miocárdica per se, en vertebrados adultos in vivo.

Ventricular resection of the heart of Ambystoma mexicanum was performed and the type of tissue that restored the lesion and if it is by hypertrophy or hyperplasia of myocardium, were evaluated. Masson's trichrome stain indicated that 5 days after resection, the gap was occupied with a blood clot surrounded by collagen fibres (83 ± 6%) and muscle (10 ± 3%) and the rest of area (7 ± 2%) free of tissue. A proportion of 50 ± 4 and 90 ± 2% was muscular tissue, 10 and 30 days after injury. The evaluation with bis-Benzimide indicated cell proliferation in the injured area. The double immunohistochemistry for a-sarcomeric actin and proliferating cell nuclear antigen indicated that the tissue that occupied the injury-produced gap was originated by cardiomyocyte proliferation, which presented a maximum of 68%, 5 day after injury. Our results indicate that the myocardium of A. mexicanum recovers its structure through cardiomyocyte hyperplasia and suggest that the myocardial regenerative capacity is higher than the reported for adult mammals (1%) and other non-mammalian vertebrates (32%). This characteristic makes A. mexicanum a suitable model to study the mechanisms that regulate per se, the myocardial regeneration in adult vertebrates in vivo.

Neuroepithelial organs of the lateral line system in urodele amphibians [Ampystoma, Axolotl]

10 full-term larvae and adult urodeles [Ambystoma, axolotl] were used in this study. Serial Epon sections were prepared and stained with toluidine blue. The lateral line receptors in the larval and adult animals consisted of 2 types of organs, the ordinary neuromasts and the ampullary organs. The organs were arranged in different lines in the head and along the whole side of the body. The ampullary organs were restricted only to the head. The 2 types of organs were histologically different in their constituent cells, especially th sensory cells. In the neuromast, the sensory cell showed a single hair, while in the ampullary organ it showed a single striated protrusion. The significances of the distribution, morphology and histological structure of these sense organs were discussed