Revista nº 804

74 Óscar García García Hyaluronidase pre-treatment for collagen staining Actualidad Médica · Número 804 · Mayo/Agosto 2018 Páginas 72 a 75 DISCUSSION The characterization of ECM components has always been crucial to reveal tissue origin or development. Nowadays the search of new biological collagenous matrices as biomate- rial to be used in tissue engineering is still a challenge. Collag- enous tissues in no-studied species of vertebrates could pres- ent some troubles when conventional histological techniques are used. The current study aimed to determine the collagen pres- ence in sturgeon notochord and its fiber distribution on ECM trough a novel and modified PR staining method. Thus, a hy- aluronidase pre-treatment was tested as enhancer of the dy- ing molecular interactions with collagen fibers in the useful PR histological technique. In relation to characteristic birefringence of collagen fibers in PR staining, many studies relied on this staining to identify collagen types according to their colors under polar- ized light until few years ago (6, 12, 13). Controversially, other authors reported that the polarized colors of PR staining de- pend only on the thickness of the collagen fibers, the density of their packing and spatial arrangement, not on the compo- sition of the specific collagen type within collagen bundles (14-16). Also, Raed Lattouf et al. (2014) study illustrates the inability of PR staining to differentiate collagen types, since the absorbed amount of polarized light by this dye strictly de- pends on the orientation of the collagen bundles due to pic- rosirius red-stained reconstructed connective tissue sections (collagen lattice) were observed under polarized light before and after rotation of the microscope stage (17). In this study, polarized images of same sections of notochord tissue were tested, presenting a more intense signal of yellow, red or green birefringence in the marked areas of HP group that matched with specific dyed areas observed in light microscopy. This fact suggests that these more intense definite areas stained in HP group were collagen fibers and not an unspecific signal. The term “hyaluronidase” was introduced to denote spe- cifically the enzymes that degrade HA (18). Hyaluronidase has been used for numerous clinical applications, such as adjuvant therapy in cancer and to expedite the dispersion and absorp- tion of drugs, as well as it has been used in ophthalmological procedures in combination with local anesthetics, reduction of dermatological aging, among others (19, 20). This is the first study that uses hyaluronidase enzyme as pre-treatment of a histological method such as PR or AB stainings, with the purpose of splitting the glucosaminidic bond between C1 of the N-acetylglucosamine section and C4 of a glucuronic acid in HA (21) producing density decrease and increasing the perme- ability of sturgeon notochord ECM. AB staining contains four tetramethylisithioronium groups that react binding to carboxyl and sulphate-ester groups at acid medium (pH 2.5 or 1) in car- bohydrates molecules (3). Observing AB sections, the loss of expression observed in HP group suggests that the cleavage (at least partially) of HA (one of the major GAGs in ECM) was effective. This fact may decrease the viscosity and increase the permeability of ECM and produce the more intense dye on PR method together with a mildly loss of AB staining. In this milieu, histochemical methods play a key role in the characterization of ECM in native and artificial tissues (22) and, in consequence, an adequate customization of these his- tochemical techniques should be carried out in accordance to the specimen to be studied, specially in those samples with high ECM density, as sturgeon notochord, in order to improve the accuracy of the obtained expression. In this sense, a HP could be used for a better detection of collagen fibers in such tissues. Moreover, further research focused on deeper character- ization of molecular composition by immunostaining should be carried out as a complement that could provide more in- formation about molecular reactions taking place between tis- sues and dyes. CONCLUSIONS As we hypothesized, the large size and high presence of HA in the collagenous matrices could obstruct the interactions be- tween molecules of dying and target sites producing not as much specific PR stain of collagen fibers. The hyaluronidase pre-treat- ment permits to degrade, at least partially, HA in sturgeon noto- chord and shows a more intense and define pattern of collagen network in PR histological in light and polarized microscopy. Final- ly, further studies are still required in others collagenous tissues to assess the potential use of hyaluronidase as a pre-treatment enhancer of PR staining for collagen fibers. Acknowledgments The authors are grateful to Caviar de Riofrío SL Fish Farm (Spain) for providing the sturgeon specimen. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the authorship and publication of this article. REFERENCES 1. Gartner LP, Hiatt JL, Gartner LP. Color atlas and text of histology. 6th ed. Philadelphia: Wolters Kluwer Health/ Lippincott Williams & Wilkins; 2014. xviii, 525 p. p. 2. Varki A. Essentials of glycobiology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1999. xvii, 653 p. p. 3. 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