Growth associated protein 43 and neurofilament immunolabeling in the transected lumbar spinal cord of lizard indicates limited axonal regeneration

Previous cytological studies on the transected lumbar spinal cord of lizards have shown the presence of differentiating glial cells,few neurons and axons in the bridge region between the proximal and distal stumps of the spinal cord in some cases.A limited number of axons(20-50)can cross the bridge and re-connect the caudal stump of the spinal cord with small neurons located in the rostral stump of the spinal cord.This axonal regeneration appears to be related to the recovery of hind-limb movements after initial paralysis.The present study extends previous studies and shows that after transection of the lumbar spinal cord in lizards,a glial-connective tissue bridge that reconnects the rostral and caudal stumps of the interrupted spinal cord is formed at 11-34 days post-injury.Following an initial paralysis some recovery of hindlimb movements occurs within 1-3 months post-injury.Immunohistochemical and ultrastructural analysis for a growth associated protein 43(GAP-43)of 48-50 k Da shows that sparse GAP-43 positive axons are present in the proximal stump of the spinal cord but their number decreased in the bridge at 11-34 days post-transection.Few immunolabeled axons with a neurofilament protein of 200-220 k Da were seen in the bridge at 11-22 days post-transection but their number increased at 34 days and 3 months post-amputation in lizards that have recovered some hindlimb movements.Numerous neurons in the rostral and caudal stumps of the spinal cord were also labeled for GAP43,a cytoplasmic protein that is trans-located into their axonal growth cones.This indicates that GAP-43 biosynthesis is related to axonal regeneration and sprouting from neurons that were damaged by the transection.Taken together,previous studies that utilized tract-tracing technique to label the present observations confirm that a limited axonal re-connection of the transected spinal cord occurs 1-3 months post-injury in lizards.The few regenerating-sprouting axons within the bridge reconnect the caudal with the rostral stumps of the spinal cord,and likely contribute to activate the neural circuits that sustain the limited but important recovery of hind-limb movements after initial paralysis.The surgical procedures utilized in the study followed the regulations on animal care and experimental procedures under the Italian Guidelines(art.5,DL 116/92).

Tail regeneration reduction in lizards after repetitive amputation or cauterization reflects an increase of immune cells in blastemas

Lizards are key amniote models for studying organ regeneration. During tail regeneration in lizards, blastemas contain sparse granulocytes, macrophages, and lymphocytes among the prevalent mesenchymal cells. Using transmission electron microscopy to examine scarring blastemas after third and fourth sequential tail amputations, the number of granulocytes, macrophages, and lymphocytes increased at 3-4 weeks in comparison to the first regeneration. An increase in granulocytes and agranulocytes also occurred within a week after blastema cauterization during the process of scarring Blood at the third and fourth regeneration also showed a significant increase in white blood cells compared with that under normal conditions and at the first regeneration. The extracellular matrix of the scarring blastema, especially after cauterization, was denser than that in the normal blastema and numerous white blood cells and fibroblasts were surrounded by electron-pale, fine fibrinoid material mixed with variable collagen fibrils. In addition to previous studies, the present observations support the hypothesis that an increase in inflammation and immune reactions determine scarring rather than regeneration. These new findings verify that an immune reaction against mesenchymal and epidermal cells of the regenerative blastema is one of the main causes for the failure of organ regeneration in amniotes.

Immunodetection of ephrin receptors in the regenerating tail of the lizard Podarcis muralis suggests stimulation of differentiation and muscle segmentation

Ephrin receptors are the most common tyrosine kinase effectors operating during development. Ephrin receptor genes are reported to be up-regulated in the regenerating tail of the Podarcis muralis lizard. Thus, in the current study, we investigated immunolocalization of ephrin receptors in the Podarcis muralis tail during regeneration. Weak immunolabelled bands for ephrin receptors were detected at 15–17 kDa, with a stronger band also detected at 60–65 kDa. Labelled cells and nuclei were seen in the basal layer of the apical wound epidermis and ependyma, two key tissues stimulating tail regeneration. Strong nuclear and cytoplasmic labelling were present in the segmental muscles of the regenerating tail, sparse blood vessels, and perichondrium of regenerating cartilage. The immunolocalization of ephrin receptors in muscle that gives rise to large portions of new tail tissue was correlated with their segmentation. This study suggests that the high localization of ephrin receptors in differentiating epidermis, ependyma, muscle, and cartilaginous cells is connected to the regulation of cell proliferation through the activation of programs for cell differentiation in the proximal regions of the regenerating tail. The lower immunolabelling of ephrin receptors in the apical blastema, where signaling proteins stimulating cell proliferation are instead present, helps maintain the continuous growth of this region.

Regeneration of adhesive tail pad scales in the New Zealand gecko (Hoplodactylus maculatus) (Reptilia, Squamata; Lacertilia) can serve as an experimental model to analyze setal formation in lizards generally

During the regeneration of the tail in the arboreal New Zealand gecko （Hoplodactylus maculatus） a new set of tail scales, modified into pads bearing setae 5-20 μm long, is also regenerated. Stages of the formation of these specialized scales from epidermal pegs that invaginate the dermis of the regenerating tail are described on the basis of light and electron microscopic images. Within the pegs a differentiating clear layer interfaces with the spinulae and setae of the Oberh^utchen according to a process similar to that described for the digital pads. A layer of clear cytoplasm surrounds the growing tiny setae and eventually comities around them and their spatular ends, later leaving the new setae free- standing on the epidermal surface. The fresh adhesive pads help the gecko to maintain the prehensile function of its regenerated tail as together with the axial skeleton （made of a cylinder of elastic cartilage） the pads allow the regenerated tail to curl around twigs and small branches just like the original tail. The regeneration of caudal adhesive pads represents an ideal system to study the cellular processes that determine setal formation under normal or experimental manipulation as the progressive phases of the formation of the setae can be sequentially analyzed.

Ultrastructure of Bat (Pipistrellus kuhlii) Epidermis with Emphasis on Terminal Differentiation of Corneocytes

The ultrastrncture of the skin of air-adapted mammals （bats） is not known. The study at the electron microscope of the skin of the back and the flying membrane of Pipistrellus kuhlii showed that the thickness of the epidermis is very low （10- 12μm）, and that 1 - 2 flat spinosus cells are present beneath the stratum corneum which is formed by very thin corneoeytes that resemble those of avian apteric epidermis. The stratum granulosum is discontinuous and few small （less than 0.3μm large） keratohyalin granules are present. The epidermis is reduced to one flat basal layer in contact with the stratum corneum in many areas of the flying membrane. Transitional corneoeytes are almost absent suggesting that the process of eornification is very rapid. In the basement membrane numerous hemidesmosomes are present and form attachment points for the dense dermis underneath. Numerous collagen fibrils directly contact with the hemidesmosomes and the dense lamella of the basement membrane. Sparse elastic fibrils allow the stretching of the epidermis during flight and the rapid folding of the epidermis after flying without damaging the epidermis. Like in avian epidermis, the production of lipids is high in bat keratinocytes, and multilamellar bodies discharge lipids extra- and intra-cellularly. This may compensate the lack of a thick fat layer in the dermis of the flying membrane as lipids may help in thermical insulation against the cooling air currents flowing on the bat skin during flight. Fur hairs are very thin （4 - 7 μm）, and they have an elaborated cuticle made of pointed expansions similar in texture with that of the cortex. Cuticle ceils form hook-like grasping points that allow to keep hairs stuck together. In this way the pelage remains compact in order to maintain body temperature.

Ultrastructural analysis shows persistence of adhesion and tight junction proteins in mature human hair

The differentiation of cells composing mature human hairs produces layers with different corneous characteristics that would tend to flake away one from another,as in the corneous layer of the epidermis,without anchoring junctions.It is likely that cell junctions established in the forming cells of the hair bulb are not completely degraded like in the corneous layer of the epidermis but instead remain in the hair shaft to bind mature cuticle,cortex,and medulla cells into a compact hair shaft.During cell differentiation in hairs,cell junctions seem to disappear,and little is known about the fate of junctional proteins present in the mature human hair shaft.The present ultrastructural immunogold study has detected some marker proteins of adhesion junction(cadherin and beta-catenin)and tight junctions(occludin and cingulin)that are still present in cornified hairs where numerous isopeptide bonds are detected,especially in the medulla.This qualitative ultrastructural study indicates that aside from the cell membrane complex,a long corneo-desmosome bonding cortex and cuticle cells,also sparse adherens and tight junction remnants are present.It is suggested that the cornification of these junctions with the incorporation of their proteins within the mature corneous material of the hair shaft likely contributes to maintaining the integrity of the mature hair.This information will also allow us to evaluate the effects of different chemical components present in hair formulations and stains on these junctional proteins and the consequent integrity of the hair shaft.