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Differentiation of oxytalan fibres from elastic fibres with reagents for detection of magnesium

Wilhelm MiiDer and Raimund Firsching*

Institute of Pathology, University of Cologne, loseph-Stelzmann-StraBe 9, and *Neuro­surgical University Clinic, D-W -5000 Koln 41, Germany

Summary. Elastic fibres in various human and animal tissues revealed positive results with reagents for the detec­tion of magnesium. By contrast, no Mg could be detected in oxytalan fibres or in zonular fibres of the ciliary body, which are composed of microfibrils not containing elastin. Mg is therefore concluded to be associated with the elastin core of elastic fibres. Its antagonism to calcium is speculated to play a protective role in maintaining the extensibility of elastin.

Key words: Oxytalan fibres - Zonular fibres - Elastic fibres - Magnesium - Calcium

Introduction

Elastic fibres may clearly be demonstrated with reagents for the detection of magnesium (Muller and Firsching 1991). As Mg could no longer be detected after pretreatment with elastase, it was concluded to be closely associated with elastin. The purpose of this study is a light microscopical analysis to support this conclusion.

Material and methods

Specimens were obtained from the inferior segment of the human esophagus at autopsy, and, immediatly after killing the animals, from the tongue of rabbits and from the ciliary body of bovine eyes. All specimens were fixed in formalin and embedded in paraffin. Deparaffinised sections of 10 [.tm were used for the following methods.

Reagents used for the detection of Mg were Magneson II (Merck Art. 6774) and titan yellow (C. I. 195490). Details of the techni­ques have been presented earlier (Muller and Firsching 1991). In case of a positive Magneson reaction, the colour is a bright cornflower blue, while the titan yellow reaction is a flame red colour, which emits a fluorescent orange colour with UV light

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(Cane 1969). Some swelling of the tissue cannot be avoided, as these reactions take place in an alkaline medium. Oxytalan fibres were demonstrated as recommended by Fullmer and Lillie (1958) and Gawlik and larucinska (1964), the only modification being potasium peroxymonosulphate in place of peracetic acid for oxida­tion (Rannie 1963). Further sections were stained with Weigert's resorcin fuchsin, orcein, and HE.

Results

Elastic fibres within connective tissue and the walls of blood vessels in sections from tongue and esophagus were clearly stained with resorcin fuchsin and orcein, with stainings for the demonstration of oxytalan fibres without oxidation and with both reactions for detection of Mg (Fig. 1). Subepithe­lial oxytalan fibres were impressively displayed with par­aldehyde fuchsin and cresyl violet after oxidation in both specimens (Fig. 2). Oxytalan fibres, however, showed no reaction with reagents for detection of Mg.

Elastin stainings also revealed the elastic fibres of vessels and the scanty elastic fibres in the stroma of the ciliarly body. The same fibres reacted with reagents for the detection of Mg, while the abundant zonular fibres between the ciliary processes did not stain at all. Zonular fibres were prominent with elastin stainings after oxidation, especially with par­aldehyde fuchsin. By contrast, zonules did not react with Mg reactions after oxidation, while the elastic fibres reacted the same as without oxidation (Fig. 3).

Discussion

Oxytalan fibres were first described by Fullmer and Lillie (1958) in a study of periodontal membranes. Oxytalan fibres have also been encountered in various other tissues, details

of their occurrence in the tongue and esophagus have been reported by Demmel et al. (1979) and Ferraz de Carvalho and Konig (1982). Further ultrastructural, histochemical, and biochemical investigations (Fullmer 1960; Carmichael and Fullmer 1966; Cotta-Pereira et al. 1976; Bock 1978) of these fibres disclosed bundles of tubular microfibrils. The elastin core of the typical elastic fibre is enclosed by this very same type of microfibril (Ross and Bomstein 1969; Gibson and Cleary 1987). For histological identification of oxytalan fibres, oxidation is a prerequisit for staining. Neither with, nor without oxidation, however, did oxytalan fibres react with Mg reagents. The absence of elastin within oxytalan fibres could be an explanation for this phenome­non. Another possibility could be the insufficient sensitivity of the reagents to detect these delicate fibres.

Ocular zonular fibres were included in this study, as these ligaments, which are located between the ciliary body and lens with a diameter of 7 !lm on the average (Petersen 1935), are solely composed of bundles of microfibrils identical with the microfibrils of the oxytalan fibres according to Raviola (1971), Streeten and Licari (1983), and Gamer and Alexan­der (1986). In our previous investigation Mg was no longer found in elastic tissues after treatment with elastase. Mg was therefore concluded associated with elastin. Since neither the

Fig. 1. Rabbit, tongue. Conspicuously fluorescent elastic lamina and elastic fibres of the tunica adventitia of a medium-sized muscular artery, Titan yellow in UV light. Bar 40 11m,

Fig. 2. Rabbit, tongue. Subepithelial oxytalan fibres (A). Cresyl violet after oxidation. Bar 10 !Am.

Fig. 3. Bovine ciliary body. Left: Impres­sively stained' zonular fibres. In the stroma oxytalan fibres particularly from the base of pigment epithelium (PE) and elastic fibres are demonstrated. Paraldehyde fuchsin after oxidation. Bar 40 /lm. Right: Only the scanty elastic fibres in the stroma are demonstrated. Often they show a dot­ted appearance (s. Streeten and Licari 1983). Magneson. Bar 100 /lm.

larger zonular fibres, nor oxytalan fibres reacted with rea­gents for detection of Mg, this assumption is further sup­ported. Zonular fibres exhibit a certain degree of elasticity too, which is superior to the elastic quality of collagen fibres, but it never reaches the elasticity of elastic structures (Rohen 1964). The effect of Mg on enzyme reactions (Gunther 1981) and its antagonism to Ca (Anders and Flajs 1981; Olhaberry et al. 1983) illustrate the essential role of this divalent cation. Mg not only competes with Ca by blocking Ca channels in the plasma membrane of excitable cells (Lansman et al. 1986; Audesirk 1990), but also by inhibiting the precipitation of Ca-carbonates and -phosphates (Boulet et al. 1962; Bachra and Fischer 1969; Tomazic et al. 1975). Considering the calcification of elastin bearing sub­stances (Martin et al. 1963; Urry et al. 1976; Ito et al. 1986) and the protective effect of Mg against this fundamental reduction of function (Selye 1958; Britton and Stokstad 1970; Altura and Altura 1981), the crucial importance ofMg for elastic tissues becomes evident. The exact site of Mg in components of elastic fibres, however, remains uncertain as of now. The molecular components of elastin may be specu­lated to be the binding site of the antagonizing Mg, as they are the favorite location for calcification (Weissmann and Weissmann 1960; Schiffman et al. 1964; Stadler and

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Orfanos 1978; Rayssiguier 1984). Based on Urry's (1981) conception of the molecular structure of elastin filaments, we believe the physico-chemical properties of Mg are essential in maintaining the hydratation of the polypentapeptide spirales of elastin, which, according to Gotte et aI. (1963), is a decisive prerequisite for the elastic quality of these fibres.

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Accepted January 16, 1992

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