Revista nº 804

73 Óscar García García Hyaluronidase pre-treatment for collagen staining Actualidad Médica · Número 804 · Mayo/Agosto 2018 Páginas 72 a 75 INTRODUCTION Connective tissue is composed by cells and extracellular matrix (ECM), which is a complex network composed by proteoglycans (PG), glycoproteins and fibrillar proteins such as collagen and elastin (1) . The glycosaminoglycans (GAGs) are the main components of PG and they are long unbranched and polar polysaccharides composed by re- peating disaccharide unit (1). One of the repeating units consists of an amino sugar (N-acetylglucosamine or N-acetylgalactosamine) along with an uronic sugar (glucuronic acid or iduronic acid) (2). As a general rule GAGs are sulfated, such as heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate between others, what makes them bind co- valently with proteins forming glycoproteins. The only non-sulfated GAG is the hyaluronic acid which is a large macromolecule that can- not form covalent bounds with proteins by itself, and therefore bind- ing proteins are needed to stablish the interaction with PG (1). In relation to the fibers ECM, collagens are one of the most abundant proteins present in the ECM and are known to be rich in basic amino acids, concretely glycine, hydroxyproline and proline (3). These amino acids strongly interact with acidic dyes and col- lagen network are traditionally stained with Van Gieson and differ- ent trichrome methods with variable results (4). In this sense, the Picrosirius red method (PR) for collagen fibers, in which a solution composed by Sirius red F3BA dissolved in a satured picric acid solu- tion, offer some advantages as compared to traditional Van Gieson and trichrome methods (5). Sirius red F3BA is an elongated anionic sulfonatedazo dye that colors collagen by reacting, via its sulfonic acid groups, with basic groups present in the collagen molecule. The elongated dye molecules are attached to the collagen fiber in such a way that their long axes are parallel. The parallel organiza- tion of the dye in the collagen surface results in an enhanced of the natural birefringence of these fibers making them selectively visible by polarizing microscopy (6-8). Despite the many advantages offered by PR histochemical method some dense connective does not allow Sirius red to prop- erly bind to the collagen molecules and it could be a problem in the characterization of new natural collagenous matrices that may be used in tissue engineering. The high density of the ECM in some species could affect colorant penetration and it could obstruct the reactions of dye molecules with the target binding sites. In order to solve this problem, previous studies have used pre-treatments before the stain procedure trying to facilitate the specific molecu- lar groups’ interactions, such as papain or EDTA (7). The sturgeons emerged as potential source of natural biomaterials for biomedical applications (9). However, the tissue organization and molecular composition is poorly understood. The current study aimed to de- sign a histological method to determine the presence and organiza- tion of the collagen network in sturgeon notochord. MATERIALS &METHODS This observational study was conducted on notochord tis- sue of one specimen of Acipenser naccarii which were provided by Caviar de Riofrío SL fish farm Riofrío, Granada, Spain. The live sturgeon (weight about 9 kg) was sacrificed and tissues were im- mediately frozen at -20 ºC until its processing. Tissues were de- frosted at room temperature and the notochord was dissected and cut transversally in 40-50 mm sections. Histological analysis Samples of notochord were fixed for 24 h in 10% neutral buffered formalin solution in PBS (pH 7.4) at room temperature. They were washed in distilled water, dehydrated in graded alco- hol and embedded in paraffin following a conventional protocol (10). All samples were cut in 5 μm thick sections for morphologi- cal and histochemical analyses. Serial sections were used and assigned to two different experi- mental groups: Hyaluronidase pre-treatment (HP), and control (CTR) group, without pre-treatment. In HP group, firstly samples sections were immerse in an al- cohol acid solution (1% HCl in 70% alcohol solution) for 15 min to enhance the negative charge of GAG units. Secondly, hyaluronidase from bovine testes (Sigma-Aldrich, St Louis, USA) in a 2 µg/ ml con- centration (pre-heated at 37º C) was applied to the sections that were incubated at 37ºC for 1 hour. Then, the ECM was assessed by two histochemical methods: PR staining for 30 min with Sirius red (0.1% of Sirius red in saturated aqueous picric acid), for collagen bundle staining (8, 11), and AB staining as previously described to determine reticular and collagen fibers and GAGs, respectively (8). The sections were examined under a Nikon Eclipse 90i micro- scope, and images were captured with a Nikon Digital Camera DXM 1200c and NIS Elements software (Nikon, Tokyo, Japan) for light and polarized light microscopy. RESULTS Histology All samples analyzed in this study showed positive histochemi- cal reaction for collagen fibers. In the CTR group a larger and homoge- neous reaction was observed, but it reaction differed in comparison to the pattern, intensity and definition observed in the HP samples ( Figure 1.1). Curiously, the HP allowed to identify well-defined colla- gen fibers which were intensely stained ( Figure 1.C,D). On the other hand, polarized light microscopy revealed the typi- cal birefringence of the collagen fibers (red, yellow, orange and green) in all caseswith some differences ( Figure 1.2) . Interestingly, the yellow and red birefringence was considerably enhanced in specific areas of the tissues sections which were subjected to HP ( Figure 1.2) . These findings were confirmed by using the surface plot analysis, in which the areas with higher intensity were easily identified ( Figure 1.3 ). Concerning Alcian blue staining (Figure 1.4), a homogeneous and intense signal was obtained in CTR group in the entire samples (Figure 1M, N). In the case of HP group the Alcian blue histochemical method was less intense with a heterogeneous pattern ( Figure 1O,P ) confirming the extraction of hyaluronic acid-based PGs. Figure 1. Illustrative sections of sturgeon notochord (A–P). CTR: group control using conventional histochemical techniques; HP: Hyaluronidase pre-treatment group; PR: Picrosirius Red staining in light microscopy; PR-PL: Picrosirius Red staining under polarized light; PR-SCP: surface color plot of the PR-PL images; AB: Alcian Blue staining; Referring to PR staining, the CTR group presented a larger and homogeneous reaction was observed in the entire samples, whereas HP group presented a more definite and intense pattern of collagen network. Also, this more intense signal in HP group matched with an increase of birefringence in PR-PL. This fact is corroborated by PR-SCP. However, HP group showed a lower intense and more heterogeneous signal when was compared with CTR group in AB staining. Bar = 200 μm.