The Axolotl Regeneration

The majority of its organs in the axolotl salamander have an amazing capacity for repair and regeneration. It has certain peculiar characteristics, such as extraordinary wound healing and limb regeneration abilities (Jill 42-48). In contrast to mammals, axolotls typically do not generate scar tissue as part of their wound healing process. Instead, the tissues around an amputated limb will transition back to an embryonic stem-like condition and generate new tissue to replace what was lost. Many amphibians start off as aquatic larvae after emerging from eggs, and then they go through metamorphosis, which restructures their anatomy and physiology to adapt their bodies for life on land. Not all salamanders, however, mature into land-dwelling animals, and axolotls are one such exception.

Animal Description

The axolotls have an external gill. Three branchlike projections extend from the neck region on each side of their heads. Covering each branch are feathery filaments used for gas exchange. They have lungs too, and will occasionally swim to the surface to gulp air, but they spend much of the time underwater with their branches splayed open to increase the exposure of the filaments to water. Observers will see the animal flex the branches every few minutes to move fresh water across the filaments. Because they produce a large number of eggs, mature females are typically the more massive of the two sexes, averaging ∼170–180 g. Males are usually smaller, averaging ∼125–130 g. They have increased activity Many people during the evenings. When uncomfortable, they roll, rubbing on plants, floating, or darting about rapidly.

Housing

The axolotl’s skin and gills are delicate and can be easily damaged by poor water quality, rough handling, or chemicals left on a handler’s gloves or hands. Holtfreter’s solution contains a mixture of several salts that reduce bacterial loads in the animal’s environment and also help support the animals’ electrolyte levels. Most tiny larvae will die from chlorine exposure in a few hours.chlorine, and chloramine is removing agent have a ‘stress-coat support’ additive that will help the axolotl maintain the protective mucus skin-coat that makes healthy axolotl skin feel slippery.

Healthcare

In the interest of minimizing antibiotic resistance, there are several options available before resorting to traditional pharmaceuticals, should illness occur. Depending on how far an infection has progressed, sometimes it helps to refrigerate the animal merely. Placing an axolotl in a refrigerator or cold room (∼7–8 °C) slows down their basal metabolism enough to inhibit most movement and nonvital processes, inducing an artificial torpor. The cold temperature presumably helps to create an inhospitable environment for the infectious agent.

Axolotls can be kept in this state for a month or more if necessary, but most infections that can be resolved by cold treatment will begin to show improvement after about a week. Regular water changes, frequent feedings, and a reliable quarantine program can frequently minimize skin parasites and other ectopic infections. For recurring skin and gill infections, animals can live in a dilute formalin– malachite green mixture for several months with no apparent side effects. To make the dilute solution, add 0.015 ml 10% formalin and 0.05 mg malachite green per liter of Holtfreter’s solution17. It is not uncommon to find juvenile axolotls floating upside-down with distended midsections full of air. Presumably, the most common cause is gas produced by the immature gut during digestion of their high-protein diet. Often, reducing their portion size when feeding helps to alleviate this problem. Two smaller meals are sometimes easier to digest, and the gas is less troublesome. Lowering the animal’s water level benefits as well, so they do not have to fight the extra buoyancy created by the air bubble. As the animal’s gut matures, this problem usually resolves itself. Tiny rupture(s) in the pulmonary system allows air to escape into the extrapulmonary space, causing distention. A series of antibiotic injections may prevent an infection, and one can remove the air with a syringe or allow it to resolve itself on its own18. Whatever the reason for the gas bubble’s presence, under no circumstances should an animal be anesthetized and ‘burped’ by squeezing the air out. Depending on where the air bubble is located, this manipulation can cause damage to the skin, connective tissue, internal organs, or all of these (Seifert, Ashley W., et al. 1-19).

Injections of enrofloxacin (Baytril) or amikacin often produce marked effects on most bacterial infections, whereas tetracycline and other ‘cyclic’ antibiotics like oxytetracycline and doxycycline are not good choices because they can cause severe skin irritation at the injection site. Although commercially available, over-the-counter fish medications may be used; one must usually calculate higher dosages. The concentrations necessary to treat fish are too low; therefore, many of these preparations will be ineffective axolotls if used per the package directions.

Ambystoma tigrinum virus (ATV), a ranavirus, has in the past infected laboratory-reared axolotls20. The initial presentation shows milky-white skin blisters the size of a pinhead. As the infection progresses, these skin blisters can break open and bleed profusely. In advanced cases, the animals’ limbs and neck region may appear bloated. As is the fact with all viruses, antibiotics are ineffective in treatment, and the infection must run its course in affected animals. Because ATV is always fatal, liberal use of bleach on surfaces and early euthanasia of symptomatic animals is recommended, to prevent the possibility of spread to nearby creatures. It is essential, though, to mention that skin blisters are not automatically indicative of ATV. Poor water quality and ectopic skin parasites can also cause blistering to occur.

Skin regeneration

The skin is injured more when it comes to injuries in mammals because it acts as the first line of external defense (Ashley et al. 1-19). Huge populations across the world world get scars each year in response to trauma and surgery, and the result is a spectrum of pathologies from thin line surgical scars to hypertrophic and chronic non-healing wounds. Both aquatic and terrestrial axolotls are capable skin regeneration (Ashley et al. 1-19). Adult axolotls (Ambystoma mexicanum) are aquatic and paedo-morphic, exhibiting several juvenile features as adults (e.g.retention of Leydig cells, pseudo-stratified epidermis, external gills).Although the dermis of adult axolotl skin is typical for amphibians, the epidermis is pseudo-stratified and lacks a well-defined stratum corneum(Seifert, Ashley W., et al. 1-19). Epithelial cells intercept with Leydig cells (specialized cells containing highly granulated cytoplasm) above the stratum germinativum that are characteristic of larval amphibian skin. The dermis contains epidermally-derived mucous and granular glands that are embedded within the stratum spongiosum, a loose network of thin collagen fibers and fibroblasts that lies above the stratum compactum. The stratum compactum develops a thickened sheet of compressed collagen fibers that sits atop hypodermis and separates the skin from the underlying muscle. It is argued that wounds made outside of regeneration fields (i.e., limbs, tail, head) heal with a scar but no proof has been done (Seifert, Ashley W., et al. 1-19).

In response to injury, blood flows into the wound bed, clotted, but did not form a scab. Epithelial cells emanating from the wound margins move across the underlying muscle and accumulate plasma and re-epithelialized entirely the wound within 24hrs (Ashley et al. 1-19). Fourteen days after the injury, fibroblasts appear within the wound bed with the newly deposited extracellular matrix. Twenty-one days post-injury muscle fibers continued to fragment liberating mono-nucleate cells into the surrounding tissue and a robust ECM form between the epidermis and muscle (Seifert, Ashley W., et al. 1-19). Forty-seven days after injury new epidermal organs were present in the regenerated stratum spongiosum however stratum compactum does not wholly regenerated, and this is evident at its margins. Although the timing to regeneration varies among individuals, full thickness skin, including epidermal organs and underlying muscle, completely regenerate by 80 days (Seifert, Ashley W., et al. 1-19). Previous work in restoring new limbs suggested that reformation of the basement membrane (BM) facilitates dermal regeneration and its delayed formation permits blastema formation (Godwin 1). The BM is appeared as a thick fibrous band in damaged skin separating epidermis from the dermis and is continuous except where mucous glands interject into the epidermis. Staining depicts a thin, immature structure under the new epidermis. The BM continues to mature and is wholly regenerated at least 47 days after wounding. Complete recovery of the BM corresponds to the regeneration of the dermis (except for stratum spongiosum) (Seifert, Ashley W., et al. 1-19).

Lamina lucida and lamina densa are both present detectable in the BM of uninjured skin, and surrounding glands and muscle fibers. Basal epidermal cells are negative for laminin and collagen IV (Seifert, Ashley W., et al. 1-19). Continuous and robust laminin staining is detected beneath the epidermis seven days post-injury indicating lamina Lucida reformation. Collagen IV is not detected continuously beneath the skin until day 14 (Seifert, Ashley W., et al. 1-19). In addition to protein localization, there is epidermal gene expression of the BM components collagen type IV and laminin alpha1 (Lama1), laminin beta 1 (Lamb1), laminin gamma 1 (Lamc1), which together form the protein laminin-111. Expression of alpha1 and alpha2 chains were down-regulated following injury, remained below baseline levels until day 3, after which they returned to baseline levels at day seven post-injury(Seifert, Ashley W., et al. 1-19).

Metamorphic Axolotls have a delay in Skin Regeneration as compared to Paedomorphic Axolotls. Seven days after the injuries, the wound bed contained plasma beneath the skin, and within the fragmented muscle, along with large numbers of erythrocytes and leukocytes, ECM deposition commences as it had in the paedomorphic. Approximately10–14 days after the injury and we noted that it appeared to extend deep into the muscle Twenty-one days post-injury the ECM was dense within the wound bed. Complete dermal regeneration was delayed in metamorphs (Seifert, Ashley W., et al. 1-19). While epidermal organs regenerated in both forms after 40 days, the wound bed and underlying muscle still contained densely compacted extracellular matrix in metamorphs. After 80 + days the stratum spongiosum regenerates, but the stratum compactum remains incomplete. After 120 days, the wound site resembles an 80-day regenerating wound in paedomorphs, and a few collagen deposits persisted in the underlying muscle). (Seifert, Ashley W., et al. 1-19).

The findings suggest that flank skin in adult metamorphic axolotls can completely restore following FTE wounding, but the time required to regenerate both the stratum compactum and mature granular glands is lengthened compared to paedomorphic (Seifert, Ashley W., et al. 1-19).Necrotic cryo-injuries are regenerated in axolotls. Damage to the heart ventricle by puncture or resection in both newt and axolotl salamanders results in transient ECM synthesis and functional replacement of missing or damaged cardiomyocytes, epicardium, endothelium and connective tissue cells. Regeneration relies on cardiomyocyte proliferation in a temporal sequence of activation.

Heart regeneration

Heart regeneration in the salamander involves the activation of cardiomyocyte proliferation (Godwin et al. 2017). Macrophage depletion before cardiac cryo-injury results in impaired cardiac function and reduced survival (Godwin et al. 2017). The effect of cryo-injuring the axolotl heart in the absence of macrophages was dramatic, with progressive scarring occurring in macrophage-depleted hearts evident at 25, 50 and 90-days post-injury accompanied by a change in ventricular function assessed by ultrasound analysis. Macrophage depletion results in disruption of the regeneration program by induction of fi fibroblast activation and alternative ECM profile (Godwin et al. 2017).

Lens regeneration

Among all tissues that can be regenerated, the lens is a special case. The lens can be removed in its entirety (Suetsugu-Maki et al. 10-103). Research shows that newts can regenerate the lens very faithfully no matter how many repeated lens tectomies are performed or the age of the animal. The lens has developed a stage 44 in the developing stages f axolotls. Lens regeneration was possible within a particular time window after lens removal in animals up to 14 days after hatching After that window of time; axolotls were found incompetent of regenerating their lenses. The process of regeneration is swift. Within a few days after lentectomy, a well-differentiated lens was present. The lens restores from the iris; however, it could be formed from either the dorsal or the ventral iris (Suetsugu-Maki et al. 10-103).

Limb regeneration

Upon injury, the axolotl generates a population of regeneration-competent limb progenitor cells known as the blastema (McCusker and David 565-571). Many cell types from the mature limb stump contribute to the blastema at different stages of regeneration. Limb blastema represents vertebrate regeneration (Kragl et al. 60). Blastema formation is the event leading to the formation of lost structures. Once it forms it represents all the characters of the limb bud that formed the limb during embryogenesis (Jones and Jeffrey 649-662). Blastema formation requires adequate nerve supply, permissive wound epithelium and cells connecting tissue origin (Roy and Mathieu 12-25). Blastema cells with positional information interact with each other and with cells in the stump to control pattern formation in ensuring that regenerated tissue form at the right place. A blastema forms during this event (Carlson, 202-208). An ectopic blastema may be induced on the side of the arm by making a small thickness skin wound and surgically deviating the brachial never to the wound site (McCusker and David 565-571).

Aging regeneration

Although most of what is understood about regenerative mechanisms pertain to the repair of acute injuries, we assume that these same mechanisms could be utilized therapeutically to slow or even reverse chronic damage associated with aging (McCusker and David 565-571). The cells and tissues of the axolotl undergo regeneration whether induced or natural, and since the cells are the building blocks of living things, the amphibians thus undergoes age regeneration ( McCusker et al. 54-71)

Conclusion

Axolotls can undergo various kind of organ regeneration due to the particular characteristics that the amphibian poses, they are unable to under complete metamorphosis giving them the unique features. They remain juvenile internally while mature sexually makes them cable of rejuvenating various organs such as the heart, the skin he lens and the limbs, they are also capable of regenerating their age to remain a bit younger.















Works Cited

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Carlson, Bruce M. "Multiple regeneration from axolotl limb stumps bearing cross-transplanted minced muscle regenerates." Developmental biology 45.1 (1975): 203-208.

Godwin J. W., R. Debuque, E. Salimova and N. A. Rosenthal"Heart regeneration in the salamander relies on macrophage-mediated control of fibroblast activation and the extracellular landscape." NPJ Regenerative Medicine 2 (2017): 1.

Jill Gresens Bs, Alat. "An introduction to the Mexican axolotl (Ambystoma mexicanum)." Lab animal 33.9 (2004): 41.

Jones, Jay E., and Jeffrey T. Corwin. "Regeneration of sensory cells after laser ablation in the lateral line system: hair cell lineage and macrophage behavior revealed by time-lapse video microscopy." Journal of Neuroscience 16.2 (1996): 649-662.

Kragl, Martin, et al. "Cells keep a memory of their tissue origin during axolotl limb regeneration." Nature 460.7251 (2009): 60.

McCusker, Catherine, and David M. Gardiner. "The axolotl model for regeneration and aging research: a mini-review." Gerontology57.6 (2011): 565-571.

McCusker, Catherine, Susan V. Bryant, and David M. Gardiner. "The axolotl limb blastema: cellular and molecular mechanisms driving blastema formation and limb regeneration in tetrapods." Regeneration 2.2 (2015): 54-71.

Rinako Suetsugu-Maki, Nobuyasu Maki, Kenta Nakamura, Saulius Sumanas Jie Zhu, Katia Del Rio-Tsonis and Panagiotis A Tsonis. Lens regeneration in axolotl: new evidence of developmental plasticity." BMC biology 10.1 (2012): 103.

Roy, Stéphane, and Mathieu Lévesque. "Limb regeneration in axolotl: is it superhealing?." The Scientific World Journal 6 (2006): 12-25.