Experimental Formation of 100 Nm Periodic Fibrils in the Mouse Corneal Stroma and Trabecular Meshwork

Experimental Formation of 100 nm Periodic Fibrils in the Mouse Corneal Stroma and Trabecular Meshwork

Koji Hirono,* Miya Kobayashi.f Kunihiko Kobayashi.t Takeshi Hoshino.f and Shinobu Awoyo*

The so-called long-spacing collagen that exhibits approximately 100 nm periodicity has been observed in the interstitial connective tissue of various organs under normal and pathological conditions. Al- though we, as well as many other investigators, have reported an increasing amount of these structures with age in the human trabecular meshwork, the pathological significance and the mechanism of its formation are still unknown. We incubated mouse ocular tissues in culture medium containing 20 mM ATP and prepared them for electron microscopic observation according to the method of Bruns et al (J Cell Biol 103:393,1986). After the incubation, abundant 100 nm periodic fibrils were observed in the corneal stroma and the region of the trabecular meshwork, both of which show no structure of 100 nm periodicity under normal conditions. For the experimental formation of 100 nm periodic fibrils, ATP, acidic condition and temperature around 37°C are necessary. The 100 nm periodic fibrils observed in our experiment were very similar to long-spacing collagen, in that the dark transverse bands have 100 nm intervals and very fine filaments of 6-7 nm diameter axially connect the bands. Long-spacing collagen is not usually observed in the human cornea, even in aged persons. The results of our study suggest that the occurrence of long-spacing collagen is related to special conditions developing in the trabecular meshwork with age but not in the corneal stroma. Experimental studies of 100 nm periodic fibril formation in mice offer a useful model for the age-related increase of long-spacing collagen in the trabecular meshwork of the human eye. Invest Ophthalmol Vis Sci 30:869-874, 1989

The so-called long-spacing collagen1 is character- VI collagen the same authors and Linsenmayer et al12 ized by its periodicity of 100 nm or more, compared showed that type VI collagen is a major component with the 64 nm periodicity of type I collagen fibrils. of the 100 nm periodic fibrils. An occurrence of the long-spacing collagen has been We have reported an increase of long-spacing col- reported in Descemet's membrane of Fuchs' corneal lagen with age in the human trabecular meshwork by dystrophy,2-3 or Chandler's syndrome,4 in the trabec- electron microscopy.13 Long-spacing collagen, how- ular meshwork of the patient with chronic simple ever, is not usually observed in the corneal stroma of glaucoma,5"7 and in the extracellular matrix of lacri- aged persons.14 Therefore, to explain the differences mal gland tumors.1 This structure is also observed in in the formation of 100 nm periodic structures be- normal human tissues; it is commonly found espe- tween the trabecular meshwork and the cornea would cially in the trabecular meshwork of aged persons.8'10 be important in searching for age-related changes of The pathological significance and the mechanism of extracellular components in the connective tissue of its formation are, however, still unknown. the eye. The purpose of our study is to experimentally The formation of 100 nm periodic fibrils, which produce 100 nm periodic fibrils and to analyze the resembled the long-spacing collagen mentioned mechanism of their formation in the mouse cornea above, was reported by Bruns et al" in human skin and trabecular meshwork, both of which show no 100 fibroblast culture subjected to prolonged incubation nm periodic fibrils under normal conditions. in culture medium, or in adult rat tail tendon treated with ATP. Using monoclonal antibodies against type Materials and Methods Eyeballs were enucleated from adult mice (strains: From the Departments of 'Ophthalmology and fAnatomy, Na- dd and beige) under ether anesthesia and cut into goya University School of Medicine, Nagoya, Japan. halves in the meridional direction. Some pieces were Presented in part at the 92nd Annual Congress of the Japanese fixed with 4% paraformaldehyde immediately after Ophthalmological Society at Kyoto, Japan, March, 1988. enucleation and prepared for electron microscopic Submitted for publication: June 16, 1988; accepted November 4, observation. Other pieces were incubated in the ex- 1988. perimental media, as detailed in Table 1. Care of Reprint requests: Koji Hirano, MD, Department of Ophthal- mology, Nagoya University School of Medicine, Showa-ku, Tsu- animals in this investigation conformed to the ARVO rumai-cho 65, Nagoya 466, Japan. Resolution on the Use of Animals in Research.

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Table 1. Experimental conditions and results of 100 nm periodic fibril formation by ATP incubation

Plus A TP experiments Minus A TP controls (no incubation) Exp. no. / 2 3 4 5 6 7 8 9 10 11 12 13 Medium F12 F12 PBS PBS PBS PBS F12 F12 F12 F12 PBS PBS 20 mM ATP + + + + + + ------— PH 4.7 4.7 3.0 3.0 6.6 6.6 7.4 7.4 7.4 4.7 3.1 3.1 — Temperature (°C) 37 4 37 37 37 37 37 37 4 37 37 37 — Incubation time (hr) 2 72 2 24 2 24 2 24 72 2 2 24 0 100 nm periodic fibril formation observed in: cornea ++ — +++ +++ - — — - — + — + — trabecular meshwork + - + + - - - - NE - - + - Pieces of mouse eyeballs were incubated under conditions indicated in the +++, abundant; ++, moderately present; +, present; -, absent; NE, not Table. After incubation, tissues were prepared for electron microscopic ob- examined. servation. Presence or absence of 100 nm periodic fibrils is expressed as

As human material, an eyeball was taken at au- 4°C for 2 hr, and postfixed in 1% osmium tetroxide topsy from a 79-year-old woman who had no history in the same buffer for 90 min at room temperature. of ophthalmic problems other than senile cataract. They were dehydrated with graded concentrations of Pieces of the enucleated globe were immediately fixed ethanol and embedded in Quetol 812 (Nissin EM, and processed for electron microscopy. Tokyo, Japan). Ultrathin sections were cut on a Porter-Blum MT-1 ultramicrotome using a diamond ATP Incubation knife. The sections were stained with uranyl acetate and lead citrate and examined in a H-800 electron ATP-2Na (Sigma A-5394; Sigma Chemical Co., microscope (Hitachi, Tokyo, Japan). St. Louis, MO) was added to F12 medium (GIBCO, Grand Island, NY) or phosphate-buffered saline (PBS) at a concentration of 20 mM in experiments Results 1-6 (Table 1). Experiment 1 was performed by the In the stroma of the cornea and trabecular mesh- method described by Bruns et al." In experiment 2, work fixed immediately after enucleation, fine fila- the incubation was carried out at 4°C for 72 hr. We ments were dispersed in the space among periodically also used PBS to avoid the effects of serum compo- striated collagen fibrils of 30-50 nm diameter (Fig. 1). nents (experiments 3-6). In addition, we tried a pro- There were no periodic structures other than the col- longed incubation of 24 hr at 37°C (experiment 4) or lagen fibrils in the mouse corneal stroma. The fine incubation at a low temperature of 4°C (experiment filaments often exhibited beaded structures at ap- 2). As the pH decreased when ATP • 2Na was added proximately 100 nm intervals, and the diameter of to PBS (experiments 3, 4), we neutralized the media the filamentous portion was 6-7 nm. They were with NaOH (experiments 5, 6). mostly associated with the collagen fibrils. Short fila- In experiments 7-12, tissues were incubated in the ments of 6-7 nm diameter were also interspersed media without ATP, as indicated in Table 1. When among the collagen fibrils. In the region of the ante- ATP was added to the media, the pH decreased to 4.7 rior chamber angle that corresponds to human tra- in F12 medium (experiments 1, 2) and 3.0 in PBS becular meshwork, no periodic structures like long- (experiments 3, 4). We used media at the same pH spacing collagens were observed, but fine filaments adjusted with HC1 in place of ATP to check the sole resembling those of the cornea were dispersed in the effect of an acidic condition (experiments 10-12). We extracellular space where striated collagen fibrils were measured the pH of media before and after the incu- loosely arranged. bation; pH did not change during incubation period. After 2 hr incubation at 37°C with 20 mM ATP (Exps. 1, 3), numerous fibrillar structures with dark Electron Microscopic Observations cross-bands or nodes of about 100 nm periodicity After incubation, each tissue was fixed with 4% were observed in the mouse corneal stroma (Fig. 2, paraformaldehyde in 0.1 M cacodylate buffer (pH Exp. 3). These fibrillar complexes, ranging in width 7.4) at 4°C for a week, followed by fixation with 2% from 25 to 150 nm, mostly paralleled the collagen glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at fibrils. At higher magnification fine filaments of'6-7

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nm diameter axially connected the cross-bands or nodes of approximately 35 nm in axial width (Fig. 3). We tentatively call these experimentally formed fibrils "100 nm periodic fibrils (100 nm PF)" in this article. The incubation for 24 hr with ATP (Exp. 4) also produced numerous 100 nm PF. There were no morphological differences between 100 nm PF in the 2 hr and 24 hr incubations (Exp. 3 vs. Exp. 4) or in the incubations in F12 medium and PBS (Exp. 1 vs. Exp. 3) (data not shown). In the region of the trabecular meshwork, 100 nm PF were also observed under the same conditions (Exps. 1, 3, 4). Compared with the corneal stroma, the 100 nm periodic fibrils were slenderer and shorter, and dispersed in the extracellular space of the trabecular meshwork (Exp. 1, Fig. 4). When the tissues were incubated with ATP in PBS neutralized to pH 6.6 by NaOH, no 100 nm PF were observed (Exps. 5, 6). Incubations in the F12 medium without ATP (pH 7.4) produced no 100 nm PF in either the cornea or the trabecular meshwork (Exps. 7, 8). However, small amounts of 100 nm PF were observed in the cornea incubated in F12 medium acidified to pH 4.7 for 2 hr (Exp. 10) as well as in the PBS acidified to pH 3.1 for 24 hr (Exp. 12, Fig. 5), but not in the cornea incubated in the same PBS for 2 hr (Exp. 11). Most of the fibrils formed in these experiments were thinner than those observed in the ATP incubations, and they often showed a beaded structure. Incubations at 4°C, with (Exp. 2) or without (Exp. 9) ATP caused no 100 nm PF formation, even though the incubation was prolonged up to 72 hr. Naturally occurring long-spacing collagen in the trabecular meshwork of a 79-year-old woman is shown in Figure 6. It has a very similar appearance to Fig. 1. Mouse corneal stroma fixed immediately after enucle- the 100 nm PF formed under experimental condi- ation (Exp. 13). Fine filaments are dispersed among collagen fibrils. tions in mice; it consists of dark periodic cross-bands, Some of the filaments exhibit beaded structures (arrows) (X58,OOO). Scale bar = 0.1 fim (in all Figs.) unless otherwise stated. which were axially connected by fine filaments. There were, however, differences in the details of morphology. The periodicity ranged from 100 to 120 media containing ATP at 37CC both in the cornea nm in the long-spacing collagen of human trabecular and trabecular meshwork of mice, where 100 nm PF meshwork, as opposed to 90 to 100 nm in the 100 nm are usually not found under normal conditions. We PF of mice. In the mouse cornea, the dark cross- suppose that some precursors that are dispersed bands were larger in axial width and more electron- among the collagen fibrils aggregate to form 100 nm dense, and the fibrils were thinner but longer and PF in the presence of ATP. One of the candidates for straight-running. The long-spacing collagen in the the precursors seems to be fine filaments with a human trabecular meshwork is often associated with beaded appearance of 100 nm periodicity. Lateral ag- amorphous material that constitutes an electron- gregation of these filaments at their beads may result dense background. in the formation of 100 nm PF. However, the amounts of these filaments observed in the unincu- bated control tissues are not enough to explain the Discussion amounts of 100 nm PF formed in the experiments. The current study showed that 100 nm periodic Short filaments of 6-7 nm diameter interspersed fibrils appear after an incubation of tissue in acidic among collagen fibrils may also be candidates. The

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Fig. 2. Mouse corneal stroma incubated with 20 mM ATP in PBS at 37°C for 2 hr (Exp. 3). Fibrillar structures with dark cross-bands of approximately 100 nm intervals (100 nm PF) are observed among collagen fibrils. Most of them parallel the collagen fibrils(X60,000) .

Fig. 3. Higher magnification of corneal stroma incubated with 20 mM ATP in PBS at 37°C for 2 hr (Exp. 3). The 100 nm PF consist of dark cross-bands of approximately 35 nm axial width (large arrows) and fine filaments axially connecting the cross-bands. The cross-bands appear as nodes (small arrows) in thinner 100 nm PF (x90,000).

Fig. 4. Slenderer and shorter periodic fibrils (arrows) are dispersed in the extracellular space of trabecular meshwork incubated with 20 mM ATP in F12 medium at 37°C for 2 hr (Exp. 1) (X60.000).

Fig. 5. Mouse corneal stroma incubated in PBS acidified to pH 3.1, at 37°C for 24 hr (Exp. 12). Thinner 100 nm PF (large arrows) as compared with those observed in the ATP incubation (Fig. 2) were found. There are also thin filaments with a beaded appearance (small arrows) (X6O,OOO). 4

amount of all these observable precursors still seems We and some other investigators reported1013 that to be smaller than that of experimentally formed 100 in the human trabecular meshwork, the structures of nm PF. There may be precursors difficult to identify long-spacing collagen increase with age. These unless they are artificially aggregated by an incuba- banded structures are more abundantly observed in tion with ATP. The results of Exps. 5 and 6 indicate that ATP is effective for the formation of 100 nm PF only under acidic conditions. It is suggested, therefore, that, as in the case of segment-long spacing forms aggregating from type I collagen molecules,15 polyanionic sites of ATP interact with surface cations of precursors, resulting in the formation of 100 nmPF. Incubation in an ATP-free acidic medium (Exps. 10, 12) resulted in the appearance of 100 nm periodic fibrillar structures, although the amount was smaller and the fibrils were thinner than in the ATP incubations. Since the presence of ATP or similar active substances cannot be excluded from the F12 medium or corneal tissues, smaller amounts of such substances could mediate a low rate of 100 nm PF formation. In the tissue incubated at 4°C with or without ATP, the 100 nm PF were not detected within 72 hr. These results suggest that some enzymatic reactions are involved in the formation of 100 nm PF. Some proteolytic reactions might be necessary to free the precursors from their connections with other structures such as collagen fibrils. Zimmerman et al16 reported that type VI collagen is a major component of the extracellular matrix of the human cornea and showed by an indirect immu- nofluorescence technique that this collagen is distrib- uted throughout the corneal stroma. Our result showing that the experimental formation of 100 nm PFs was remarkable in the mouse corneal stroma corresponds well to the hypothesis of Bruns et al" and Linsenmayer et al,12 that this periodic structure is made from type VI collagen. We assume that precursors, including type VI collagens, aggregate to form "beaded filaments" that have 100 nm periodicity, and furthermore, the beaded filaments laterally as- Fig. 6. Long-spacing collagen in the trabecular meshwork of a 79-year-old woman. Dark periodic cross-bands are connected by semble to produce 100 nm periodic fibrillar struc- thin filaments. The background of the long-spacing collagen is dark tures. with amorphous material (X59.000).

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the trabecular meshwork of patients with chronic Fuchs' combined dystrophy: I. Posterior portion of the cornea. simple glaucoma.5"7 On the other hand, long-spacing Invest Ophthalmol 10:9, 1971. collagen does not usually occur in the human corneal 3. Akiya S, Oshima T, and Wada M: The electron microscopic study of Fuchs' dystrophy: The first primary case in Japan. stroma, where type VI collagens have been shown to Acta Soc Ophthalmol Jpn 82:887, 1978. 16 be present. Our study has demonstrated that pre- 4. Richardson TM: Corneal decompensation in Chandler's syn- cursors, possibly including type VI collagens, can be drome: A scanning and transmission electron microscopic aggregated into 100 nm periodic structures under study. Arch Ophthalmol 97:2112, 1979. suitable conditions. Special conditions causing the 5. Rohen JW and Witmer R: Electron microscopic studies on the trabecular meshwork in glaucoma simplex. Graefes Arch Klin formation of long-spacing collagen may develop Exp Ophthalmol 183:251, 1972. gradually with age in the stroma of the trabecular 6. Rohen JW: Why is intraocular pressure elevated in chronic meshwork, where aqueous humor continuously per- simple glaucoma? Ophthalmology 90:758, 1983. meates. 7. Liitjen-Drecoll E, Shimizu T, Rohrbach, M, and Rohen JW: Quantitative analysis of 'plaque material' in the inner and We morphologically compared the experimentally outer wall of Schlemm's canal in normal and glaucomatous formed 100 nm PF in mice with the naturally occur- eyes. Exp Eye Res 42:443, 1986. ring long-spacing collagen in the human trabecular 8. Garron LK, Feeney ML, Hogan MJ, and McEwen WK: Elec- meshwork. Periodic, dark cross-bands and axial fine tron microscopic studies of the human eye: 1. Preliminary in- filaments connecting the bands were observed in both vestigations of the trabeculas. Am J Ophthalmol 46:27,, 1958. 9. Leeson TS and Speakman JS: The fine structure of extracellu- cases. The periodicity ranged from 90 to 100 nm in lar material in the pectinate ligament (trabecular meshwork) of the mouse cornea, and from 100 to 120 nm in the the human iris. Acta Anat 46:363, 1961. human trabecular meshwork. There may be some 10. Horstmann HJ, Rohen JW, and Sames K: Age-related changes differences in size of precursor molecules between in the composition of proteins in the trabecular meshwork of mouse and man. A standardization of specimen the human eye. Mech Ageing Dev 21:121, 1983. 11. Bruns RR, Press W, Engvall E, Timpl R, and Gross J: Type VI shrinkage, if any, will now be undertaken. The back- collagen in extracellular, 100-nm periodic filaments and fibrils: ground of human long-spacing collagen in aged per- Identification by immunoelectron microscopy. J Cell Biol sons was dark with electron-dense, amorphous mate- 103:393, 1986. rial that was not detected in mice. Components other 12. Linsenmayer TF, Bruns RR, Mentzer A, and Mayne R: Type than 100 nm PF will also be studied in relation to VI collagen: Immunohistochemical identification as a filamentous component of the extracellular matrix of the developing age-related changes of the extracellular matrix of avian corneal stroma. Dev Biol 118:425, 1986. the eye. 13. Hirano K, Awaya S, Kobayashi M, and HoshinoT: Ultrastruc- Key words: long-spacing collagen, 100 nm periodic fibrils, tural and quantitative studies on age-related changes of extracellular matrices in the human trabecular meshwork (in Japa- trabecular meshwork, corneal stroma, type VI collagen nese). Folia Ophthalmol Jpn 39:1680, 1988. 14. Hogan MJ, Alvarado JA, and Weddell JE: Histology of the References Human Eye. Philadelphia, W. B. SaundersCo., 1971. 15. Kiihn K: Segment-long-spacing crystallites, a powerful tool in 1. McDonald P, Jakobiec FA, Hornblass A, and Iwamoto T: collagen research. Coll Relat Res 2:61, 1982. Benign peripheral nerve sheath tumors (neurofibromas) of the 16. Zimmermann DR, Trueb B, Winterhalter K.H, Witmer R, and lacrimal gland. Ophthalmology 90:1403, 1983. Fischer RW: Type VI collagen is a major component of the 2. Iwamoto T and DeVoe AG: Electron microscopic studies on human cornea. FEBS Lett 197:55, 1986.

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