Ultrastructural features of the process of wound healing after tail and limb amputation in lizard

Alibardi, L. 2010. Ultrastructural features of the process of wound healing after tail and limb amputation in lizard.—Acta Zoologica (Stockholm)  91 : 306–318 Wound healing and re‐epitelization after amputation of tail and limb in lizard have been studied by electron microscopy to understand the cytological base of immunity to infection in this species. After 2 days post‐amputation in both limb and tail stumps, numerous granulocytes are accumulated over the stump, and participate to the formation of the scab. Bacteria remain confined to the scab or are engulfed by leukocytes and migrating keratinocytes located underneath the scab. Bacteria are degraded within lysosomes present in these cells and are not observed among mesenchymal cells or in blood vessels of the regenerative blastema. Granulocytes, migrating keratinocytes, and later macrophages form an effective barrier responsible for limiting microbe penetration. The innate immunity in lizard is very effective in natural (dirty) condition and impedes the spreading of infection to inner tissues. While the complete re‐epitelization of the tail stump underneath the scab requires 4–7 days, the same process in the limb requires 8–18 or more days post‐amputation, depending from the level of amputation and the persistence of a protruding humerus or femurs on the stump surface. This delay produces the permanence of inflammatory cells such as granulocytes and macrophages in the limb stump for a much longer period than in the tail stump, a process that stimulates scarring.     

Dynamics of Mara–Serengeti ungulates in relation to land use changes

Caudal autotomy, the ability to shed the tail, is common in lizards as a response to attempted predation. Since Arnold's substantial review of caudal autotomy as a defence in reptiles 20 years ago, our understanding of the costs associated with tail loss has increased dramatically. In this paper, we review the incidence of caudal autotomy among lizards (Reptilia Sauria) with particular reference to questions posed by Arnold. We examine tail break frequencies and factors that determine occurrence of autotomy in natural populations (including anatomical mechanisms, predation efficiency and intensity, microhabitat preference, sex and ontogenetic differences, as well as intraspecific aggression). We also summarize the costs associated with tail loss in terms of survivorship and reproduction, focusing on potential mechanisms that influence fitness (i.e. locomotion costs, behavioural responses and metabolic costs). Finally, we examine the factors that may influence the facility with which autotomy takes place, including regeneration rate, body form and adaptive behaviour. Taking Arnold's example, we conclude with proposals for future research.     

Purification and kinetic properties of skeletal muscle lactate dehydrogenase from the lizard Agama stellio stellio

Lactate dehydrogenase isoenzyme LDH-5 (M4) was purified to homogeneity from the skeletal muscle of lizard Agama stellio stellio as a poikilothermic animal, using colchicine-Sepharose chromatography and heat inactivation. The purified enzyme showed a single band after SDS-PAGE, corresponding to a molecular weight of 36 kD. The K _m values for pyruvate, NADH, lactate, and NAD⁺ were 0.020, 0.040, 8.1, and 0.02 mM, respectively. Pyruvate showed maximum activity at about 180 M, with a decline at higher concentrations. The enzyme was stable at 70°C for 30 min, but was rapidly inactivated at 90°C. The optimum pH for the forward reaction (pyruvate to lactate) was 7.5, and for the reverse reaction (lactate to pyruvate) was 9.2. Oxalate, glutamate, Cu²⁺, Co²⁺, Mn²⁺, and Mg²⁺ were inhibitory in both forward and reverse reactions.     

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis

The claw of lizards is largely composed of beta‐keratins, also referred to as keratin‐associated beta‐proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta‐keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha‐keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha‐keratins resembled that of beta‐keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Ultrastructural features of skin pigmentation in the lizard Heloderma suspectum with emphasis on xanto‐melanophores

The morphological origin of the dark and pink‐orange areas in the skin of the venomous lizard Heloderma suspectum is not known. Histology and electron microscopy show that dark‐grey areas of the skin derived from dermal chromatophores localized in specific areas present underneath the epidermis. A dynamic chromatophoric unit in the dermis is absent. In the darkest areas of the skin, the accumulation of melanosomes in cells of the beta‐layer contributes to increase the black intensity. In the orange‐pink areas, the superficial dermis contains xantophores storing numerous carotenoid vesicles, rare or absent lamellated pterinosomes and a variable number of melanosomes. These xanto‐melanophores predominate over the remaining chromatophores and form a continuous stratum underneath the epidermis. Beneath this lipoid‐rich stratum, iridophores are infrequent and do not form a continuous layer in the dermis. In the paler areas of the skin, melanophores are sparse in both superficial and deeper part of the dermis where irregularly oriented bundles of collagen fibrils are present. The prevalent xanto‐melanophores localized in the pink‐orange areas of the skin contribute to an effective sunlight protection in desert conditions in addition to the darker regions occupied by melanophores.     

A new tale of lost tails: Correlates of tail breakage in the worm lizard Amphisbaena vermicularis

Predator–prey interactions are important evolutionary drivers of defensive behaviors, but they are usually difficult to record. This lack of data on natural history and ecological interactions of species can be overcome through museum specimens, at least for some reptiles. When facing aggressive interactions, reptile species may exhibit the defensive behavior of autotomy by losing the tail, which is also known as “urotomy”. The inspection of preserved specimens for scars of tail breakage can reveal possible ecological and biological correlates of urotomy. Herein, we investigated how the probability of urotomy in the worm lizard Amphisbaena vermicularis is affected by sex, body size, temperature, and precipitation. We found higher chances of urotomy for specimens with larger body size and from localities with warmer temperatures or lower precipitation. There was no difference in urotomy frequency between sexes. Older specimens likely faced – and survived – more predation attempts through their lifetime than smaller ones. Specimens from warmer regions might be more active both below‐ and aboveground, increasing the odds to encounter predators and hence urotomy. Probability of urotomy decreased with increased precipitation. Possibly, in places with heavier rainfall worm lizards come more frequently to the surface when galleries are filled with rainwater, remaining more exposed to efficient predators, which could result in less survival rates and fewer tailless specimens. This interesting defensive behavior is widespread in squamates, but yet little understood among amphisbaenians. The novel data presented here improve our understanding on the correlates of tail breakage and help us to interpret more tales of lost tails.     

Distribution of energy reserves in a viviparous skink: Does tail autotomy involve the loss of lipid stores?

Abstract Caudal autotomy is an effective defensive strategy used by many lizards to facilitate escape during predatory encounters. However, it has several potentially severe consequences, including a range of energetic costs that are believed to result from the depletion of caudal lipid reserves during tail loss. In this study we examined the possible effect of caudal autotomy on the energetic reserves of a small viviparous skink, Niveoscincus metallicus (O'Shaughnessy 1874). Animals of each sex were collected on three occasions to assess the distribution of lipid stores. In addition, the frequency and position of naturally occurring tail breaks were determined. Both abdominal and caudal lipid stores are present in N. metallicus; however, caudal fat bodies comprise the majority (55–78%) of these fat reserves. Temporal variation in fat body mass, both abdominal and caudal, was evident. There was a significant relationship between the two fat stores, which was distorted in pregnant females, when relatively more fat was stored in the tail. Examination of the distribution of caudal fat in the tail revealed that the majority (90–95%) occurs within the proximal third of the tail. The remainder is located in the middle portion of the tail, with no reserves in the most distal tail section. During late pregnancy, females store relatively more fat closer to the body. The frequency of tail loss in a natural population of N. metallicus was extremely high (78%). Tail breaks were normally distributed along the length of the tail (i.e. most near the middle and fewer distal and proximal breaks). Thus there was a relatively high chance of some lipid depletion as a result of tail loss, but because 76% of breaks occur in the middle and distal thirds of the tail, there is a high probability that tail loss results in only minimal (i.e. <10%) lipid depletion. This is the first instance where both the energetic ‘value’ of the tail and the likelihood of lipid depletion during tail loss have been determined in a lizard. Overall, the combination of the aggregation of caudal fat reserves and position of naturally occurring tail breaks may enable N. metallicus to combine caudal fat storage and tail autotomy with minimal conflict.     

Immunolocalization of Wnts in the lizard blastema supports a key role of these signaling proteins for tail regeneration

A highly upregulated gene during tail regeneration in lizards is Wnt2b, a gene broadly expressed during development. The present study examines the distribution of Wnt proteins, most likely wnt2b, by western blotting and immunofluorescence in the blastema-cone of lizards using a specific antibody produced against a lizard Wnt2b protein. Immunopositive bands at 48–50 and 18 kDa are present in the regenerative blastema, the latter likely as a degradation product. Immunofluorescence is mainly observed in the wound epidermis, including in the Apical Epidermal Peg where the protein appears localized in intermediate and differentiating keratinocytes. Labeling is more intense along the perimeter of keratinocytes, possibly as a secretory product, and indicates that the high epidermal proliferation of the regenerating epidermis is sustained by Wnt proteins. The regenerating spinal cord forms an ependymal tube within the blastema and shows immunolabeling especially in the cytoplasm of ependymal cells contacting the central canal where some secretion might occur. Also, regenerating nerves and proximal spinal ganglia innervating the regenerating blastema contain this signaling protein. In contrast, the blastema mesenchyme, muscles and cartilage show weak immunolabeling that tends to disappear in tissues located in more proximal regions, close to the original tail. However, a distal to proximal gradient of Wnt proteins was not detected. The present study supports the hypothesis that Wnt proteins, in particular Wnt2b, are secreted by the apical epidermis covering the blastema and released into the mesenchyme where they stimulate cell multiplication.     

Short-term thermal acclimation induces adaptive changes in the inner mitochondrial membranes of fish skeletal muscle

In the oxidative muscles (musculi laterales superficiales) of crucian carp Carassius carassius acclimated for 6 weeks to either 5 or 25° C, the volume density and the surface density of fibres per tissue did not differ significantly between the control and experimental groups. The correlation ratio (μ²) for these values was below 50, 39·3 and 43·9 respectively. After acclimation to 5° C, the surface density of outer mitochondrial membrane per fibre increased significantly from 0·93 to 1·23m² cm⁻³ in the summer population but dropped from 0·94 to 0·67 m² cm⁻³ in the winter population. The surface density of outer mitochondrial membrane per mitochondrion increased from 3·24 to 4·52 m² cm⁻³ in summer fish. After acclimation to 25° C, the surface density of inner mitochondrial membranes per muscle fibre decreased from 4·04 to 1·79 m² cm⁻³ in summer fish and from 3·86 to 1·07 m² cm⁻³ in winter fish. The surface density of inner mitochondrial membranes per mitochondrion increased from 14·17 to 15·60 m²cm⁻³ in summer fish but dropped from 13·91 to 10·67 m² cm⁻³ in winter fish. Correlation matrices demonstrate a negative correlation of the surface density of outer mitochondrial membrane per mitochondrion with the volume density of mitochondria per fibre and temperature, suggesting cold-induced proliferation of small mitochondria. It was concluded that short-term cold acclimation increased surface area of the inner mitochondrial membranes in summer fish.     

Helminths of the exotic lizard Hemidactylus mabouia from a rock outcrop area in southeastern Brazil

The helminth fauna of 291 Hemidactylus mabouia (Lacertilia: Gekkonidae) from a rock outcrop area in the state of S?o Paulo, Southeastern Brazil, was studied. Five species were recovered, namely one unidentified species of centrorhynchid acanthocephalan (present only as cystacanths) and the nematodes Parapharyngodon sceleratus, P. largitor (Oxyuroidea: Pharingodonidae), Physaloptera sp. (Spiruroidea: Physalopteridae) and one indeterminate species of Acuariidae (Acuaroidea), with the latter two forms present only as larvae. Infection rates tended to increase with host size, but appeared to be unaffected by season. Hemidactylus mabouia shared most of its helminth fauna with two other sympatric lizard hosts, Mabuya frenata and Tropidurus itambere. The helminth assemblage of the H. mabouia population appears to have been entirely acquired by this exotic gecko from the local helminth species pool, rather than possessing any species from the parasite faunas of the original African populations.     

Immunolocalization of c‐myc‐positive cells in lizard tail after amputation suggests cell activation and proliferation for tail regeneration

After tail amputation in lizard, a regenerative response is elicited leading to the formation of a new tail. The stimulation of the proliferation process may involve the proto‐oncogene c‐myc. The immunocytochemical analysis detects the c‐myc protein few days after wound in free cells accumulating over the injured tissues of the tail stump. Western blot detects a protein band at 68–70 kDa that is more intense in the regenerating blastema than in normal tail tissues. Nuclei positive for the c‐myc protein are seen in mesenchymal‐like cells located among muscles, connectives and fat tissues of the tail stump 4 days postamputation. Proliferating cells labelled for 5BrdU are seen at 4 days postamputation and are sparse in the mesenchyme of the regenerating blastema formed at 12 days postamputation. Fine immunolocalization of the c‐myc protein shows it is mainly located over euchromatin or poorly condensed chromatin to indicate gene activation. The study correlates the detection of the c‐myc protein with activation of cell division in the injured tissues leading to the formation of the regenerative blastema. The lizard c‐myc protein probably activates a controlled proliferation process through a mechanism that can give information on the uncontrolled process occurring in cancer.     

Immunohistochemical localization of a proto-cadherin fat tumour-suppressor homolog in the regenerating tail of lizard suggests a role in apical growth control

The present immunohistochemical and western blotting study evaluates the localization of a proto-cadherin which gene is overexpressed in the regenerating blastema of the lizard Podarcis muralis. Bioinformatic analysis suggests that the antibody recognizes FAT1/2 proteins. Western blot indicates a main band around 50 kDa, a likely fragment derived from the original membrane-bound large protein. Immunofluorescence shows main labelling in differentiating wound keratinocytes, lower in ependyma, mesenchyme and extracellular matrix of the blastema. The apical epidermal peg contains keratinocytes with labelled peripheral cytoplasm, as confirmed using ultrastructural immunogold that also reveals most labelling located along the cell surface of mesenchymal cells. Myoblasts and differentiating myotubes of regenerating muscles are less intensely labelled. The regenerating cartilaginous tube contains sparse labelled chondroblasts, especially in external and internal perichondria. In regenerating scales, differentiating beta-cells appear immunofluorescent mainly along the cell perimeter. In more differentiated muscle, cartilage and connective tissues of the new tail, the labelling lowers or disappears. The observations indicate that FAT1/2 proto-cadherins are present in the apical blastema where an intense remodelling takes place for the growth of the new tail but where also a tight control of cell division and migration is active and may regulate potential tumorigenic process.     

When one tail isn't enough: abnormal caudal regeneration in lepidosaurs and its potential ecological impacts

Abnormal caudal regeneration, the production of additional tails through regeneration events, occurs in lepidosaurs as a result of incomplete autotomy or sufficient caudal wound. Despite being widely known to occur, documented events generally are limited to opportunistic single observations – hindering the understanding of the ecological importance of caudal regeneration. Here we compiled and reviewed a robust global database of both peer‐reviewed and non‐peer reviewed records of abnormal regeneration events in lepidosaurs published over the last 400 years. Using this database, we qualitatively and quantitatively assessed the occurrence and characteristics of abnormal tail regeneration among individuals, among species, and among populations. We identified 425 observations from 366 records pertaining to 175 species of lepidosaurs across 22 families from 63 different countries. At an individual level, regenerations ranged from bifurcations to hexafurcations; from normal regeneration from the original tail to multiple regenerations arising from a single point; and from growth from the distal third to the proximal third of the tail. Species showing abnormal regenerations included those with intra‐vertebral, inter‐vertebral or no autotomy planes, indicating that abnormal regenerations evidently occur across lepidosaurs regardless of whether the species demonstrates caudal autotomy or not. Within populations, abnormal regenerations were estimated at a mean ± SD of 2.75 ± 3.41% (range 0.1–16.7%). There is a significant lack of experimental studies to understand the potential ecological impacts of regeneration on the fitness and life history of individuals and populations. We hypothesised that abnormal regeneration may affect lepidosaurs via influencing kinematics of locomotion, restrictions in escape mechanisms, anti‐predation tactics, and intra‐ and inter‐specific signalling. Behaviourally testing these hypotheses would be an important future research direction.     

Immunolabelling for RhoV and actin in early regenerating tail of the lizard Podarcis muralis suggests involvement in epithelial and mesenchymal cell motility

Immunolabelling for RhoV and actin in early regenerating tail of the lizard Podarcis muralis suggests involvement in epithelial and mesenchymal cell motility. Acta Zoologica, Stockolm. Immunolabelling for RhoV and α‐smooth muscle actin, genes that are highly expressed in the regenerating tail of lizards, shows that a main protein band immunolabelled for RhoV is seen at 65–70 kDa and only a weak band at 22–24 kDa. This suggests that alteration occurred during extraction or is due to biochemical processing of the protein. RhoV immunolabelled cells are present in apical and proximal regenerating epidermis during scale neogenesis. The apical ependyma is labelled but labelling fades and disappears in medial‐proximal regions, near the original spinal cord. Differentiating muscles and cartilage show low labelling. Ultrastructural immunolocalization of RhoV in wound keratinocytes shows labelling in regions containing actin filaments that associate with tonofilaments and desmosomes while a low labelling is present in mesenchymal cells. Filamentous regions of the nucleus, nuclear membrane and the nucleolus are immune‐labelled for RhoV. Similar localization is seen for actin that is present along the perimeters of keratinocytes associated with tonofilaments, in elongations of mesenchymal cells, in muscle satellite cells, endothelial and pericytes of blood vessels. It is suggested that RhoV and actin are associated in the dynamic cytoskeleton needed for the movements of epidermal and mesenchymal cells and in endothelial cells forming new blood vessels.     

Satellite cells in mature,uninjured skeletal muscle of the lizard tail

The uninjured caudal skeletal muscle of two lizards, Lygosoma and Anolis, contains satellite cells. The satellite cell nuclei constitute 7.5% and 4.8% of the combined satellite and muscle nuclei, in Lygosoma and Anolis, respectively.     

Age- and training-related changes in the collagen metabolism of rat skeletal muscle

The effects of ageing and life-long endurance training on the collagen metabolism of skeletal muscle were evaluated in a longitudinal study. Wistar rats performed treadmill running 5 days a week for 2 years. The activities of collagen biosynthesis enzymes, prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, were highest in the muscles of the youngest animals, decreased up to the age of 2 months and from then on remained virtually unchanged. The enzyme activity in young animals was higher in the slow collagenous soleus muscle than in the rectus femoris muscle. The enzyme activity in the soleus muscle was higher for older trained rats than older untrained rats. The relative proportion of type I collagen increased and that of type III collagen decreased with age, suggesting a more marked contribution by type I collagen to the age-related accumulation of total muscular collagen. The results show that collagen biosynthesis decreases with maturation and that life-long endurance training maintains a higher level of biosynthesis in slow muscles.