Histochemical Identification of Sulfated and Non-sulfated Mucopolysaccharides in Regenerating Forelimbs of Adult Urodeles

Abstract

A precise, sequential staining procedure which demonstrates sulfated and non-sulfated mucopolysaccharides and glycogen in the urodele forelimb is described. The process, a combination of the alcian and PAS techniques, has proven valuable in following histogenic events during forelimb regeneration. Non-sulfated mucopolysaccharides are present in apical cap epithelium, sarcoplasm, and bone matrix. Both alcian blue and PAS responses are apparent in dedifferentiating cartilage. Glycogen was apparent in late blastema cells, mature and to a lesser extent in maturing muscle, and wound epithelium.

Hyaluronic acid in the tail and limb of amphibians and lizards recreates permissive embryonic conditions for regeneration due to its hygroscopic and immunosuppressive properties

“…In the blastema and elongating cone of the newt limb at 10–20 days after amputation, an intense blue staining is seen to reach the apical‐most regions of the blastema, located underneath the wound epidermis (Figures E, F and 5A, B; Schmidt, ; Procaccini and Doyle, ; Alibardi and Sala, ; Mescher and Cox, ). The meshwork of alcianophilic fibrils reaches the basement membrane of the apical wound epidermis, including the apical epidermal cap (Figure A–C).…”

“…In vertebrates, where tail or appendages regeneration occur, in fish and amphibians, the regenerative blastema has a soft consistence due to high hydration and HA content, resembling that of embryonic limb or tail buds. The average content in GAGs in normal and regenerating appendages, largely represented by HA in the blastema (Procaccini and Doyle, ; Toole and Gross, ; Alibardi and Sala, ; Mescher and Munaim, ), is much higher in amphibian tail or limb tissues than in lizard tissues (and in other amniote tissues) (Figure A and B; Alibardi, ). Therefore, in addition to the 80% water limit to organ regeneration, there is also a GAGs limits of 40–45 μg/mg dry weight of GAGs (with HA representing large part of these molecules), under which regeneration is unlikely (Figure B).…”

“…Histochemical, biochemical, immunohistochemical, and molecular studies on fish (Govindan and Iovine, ; Ouyang et al., ), amphibians (Procaccini and Doyle, ; Toole and Gross, ; Alibardi and Sala ; Mescher and Munaim, ; Mescher and Cox, ; Calve et al., ), and lizards (Shah and Hiradhar, ; Alibardi and Sala, ; Nambiar et al., ; Alibardi, ) have shown the increment in the production and localization of GAGs, in particular of HA, during wound healing and blastema formation, a large molecule that is later coated with other sulfated GAGs and matrix glycoproteins. Using Alcian blue at pH 2.5 on sections of the tail in the tadpole of R. dalmatina and R. esculenta at all stages of development, it appears that most of the loose connective tissues present around the cord and along the dorsal and ventral fins are stained forming a blue meshwork (Figure A).…”

The Inhibition of Limb Regeneration in Adult <i>Diemictylus viridescens </i>Treated with Streptomycin

“…Serial 10 ¡¿m sections were stained with a combination alcian blue-PAS stain [Procaccini and Doyle, 1970a].…”

Ultrastructural immunolocalization of hyaluronate in regenerating tail of lizards and amphibians supports an immune‐suppressive role to favor regeneration

“…Previous biochemical and a recent transcriptome study have shown that the lizard blastema appears immuno-suppressed in comparison to normal tissues, a condition that likely favors tail regeneration while this is not the case for the limb that instead forms scars (Alibardi, 2014(Alibardi, , 2017bVitulo et al, 2017b). Several mechanisms may be involved, including the immunoregulatory activity of mesenchymal cells (Wang, Ding, & Xu, 2016), but here we have focused on hyaluronate, a large macromolecule produced in high amount during wound healing and blastema formation in amphibians (Contreras et al, 2009;Mescher & Munaim, 1986;Procaccini & Doyle, 1970;Toole, 1997;Toole & Goss, 1971) and lizards (Alibardi, 2014(Alibardi, , 2017bAlibardi & Sala, 1983;Nambiar et al, 2008;Shah & Hiradhar, 1975). In fact, this nonsulfated glycosaminoglycan of high molecular weight (> 500 kDa) has also an immune-suppressive and anti-inflammatory function (Aya & Stern, 2014;Csoka & Stern, 2013;Lee-Sayer et al, 2015;Petrey & de la Motte, 2014;Reiter & Lepperdinger, 2013).…”