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Vitreous effects on scar tissue formation
Exp. Eye Res. (1981) 32, 39-50
Vitreous Effects on Scar Tissue Formation I. J .
CONSTABLE, R. HORNE, D. V. GRE]~R AND J . P A P A D I M I T R I O U
University of Western Australia and Royal Perth Hospital, Wellington Street, Perth 6000, Western Australia (Received 4 December 1978 and in revised form 13 Feburary 1980, New York) The effects of vitrectomy and replacement with hyaluronic acid or saline on vitreous scar tissue formation were examined in rabbits. A scar tissue bridge was grown across the vitreous cavity along a transcleral suture. In one group of eyes, the bridge was grown across normal vitreous and non-banded collagen of 15 nm diameter was laid down. In a second group in which vitrectomy was done 2-8 months previously, the fluid cavity was replaced with saline. In these eyes, striated collagen of 30-60 nm diameter was deposited. In a third group after vitrectomy and replacement with purified hyaluronic acid, a mixture of fine and thicker fibrils was seen. In both the latter two groups, patches of enormous collagen fibrils of up to 350 nm diameter (tactoids) were found. No differences in the proliferating cell lines were found in any of the three groups. It is suggested that normal vitreous and its glycosaminoglycan component (hyaluronic acid) may influence the extracellular macromolecular organization of vitreous collagen. Key words: vitreous; scar tissue; collagen; vitrectomy ; hyaluronic acid.
1. I n t r o d u c t i o n C o n t r a c t i o n o f v i t r e o u s or p r e r e t i n a l scar tissue l e a d i n g to r e t i n a l d i s t o r t i o n or d e t a c h m e n t a c c o u n t s for a m a j o r p r o p o r t i o n o f blindness, for e x a m p l e , in d i a b e t e s mellitus, p e n e t r a t i n g injuries a n d c o m p l i c a t e d r e t i n a l d e t a c h m e n t s . D e s c r i p t i o n s o f v i t r e o u s or p r e r e t i n a l scar tissue in h u m a n s (Foos, 1974; Bellhorn, F r i e d m a n , W i s e a n d H e n k i n d , 1975; Zinn, C o n s t a b l e a n d Schepens, 1976; Clarkson, Green a n d Massof, 1977; V a n H o r n , A a b e r g , M a c h e m e r a n d Fenzl, 1977; K e n y o n a n d Michels, 1977), m o n k e y s ( L a q u a a n d Machemer, 1975; M a c h e m e r a n d L a q u a , 1975), r a b b i t s (Rentsch, 1973; F o o s a n d Gloor, 1975) a n d c a t s (Gloor a n d D a i c k e r , 1975; G y w a t , D a i c k e r a n d Gloor, 1978) h a v e s u g g e s t e d t h a t r e t i n a l glial cells a n d p o s s i b l y r e t i n a l p i g m e n t epithelial cells are t h e principal e l e m e n t s c a p a b l e o f i n v a d i n g t h e v i t r e o u s c a v i t y , p r o l i f e r a t i n g a n d d e p o s i t i n g collagen. T h e collagen d e p o s i t e d in h u m a n s so far described lacks m a c r o p e r i o d i c i t y a n d is m o r p h o l o g i c a l l y s i m i l a r to n o r m a l v i t r e o u s collagen ( d i a m e t e r 10-20 nm) (Zinn et al., 1976; K e n y o n a n d Michels, 1977). U n d e r e x p e r i m e n t a l c o n d i t i o n s in animals, new collagenous fibrils o f d i a m e t e r 30-80 n m w i t h 64 n m p e r i o d i c i t y m a y be laid d o w n in t h e v i t r e o u s (Mandelcorn, M a c h e m e r , F i n e b e r g a n d Hersch, 1975; Constable, 1975). F o r e x a m p l e , f i b r o b l a s t i c tissue i n d u c e d in r a b b i t s b y v i t r e o u s p e n e t r a t i o n a n d b l o o d i n j e c t i o n or b y a t r a n s c l e r a l silk s u t u r e c o n t a i n s b o t h the a b o v e m o r p h o l o g i c a l l y d i s t i n c t t y p e s o f collagen. I t is n o t k n o w n w h e t h e r i n d i v i d u a l cell lines are p r o g r a m m e d to p r o d u c e a d i s t i n c t m o r p h o l o g i c a l collagen t y p e , or w h e t h e r t h e e x t r a c e l l u l a r m a t r i x influences t h e e x t e n t o f a g g r e g a t i o n o f fibrils a n d therefore t h e i r m o r p h o l o g i c a l characteristics. The s t u d y was u n d e r t a k e n to assess t h e effects o f v i t r e c t o m y a n d r e p l a c e m e n t w i t h saline or h y a t u r o n i c acid on t h e d e p o s i t i o n of s c a r tissue across t h e v i t r e o u s c a v i t y in r a b b i t s . Reprint requests to: Professor I. J. Constable, University Department of Ophthalmology, Royal Perth Hospital. GPO Box X2213, Perth 600t, Western Australia. 0014-4835/81/010039+ 12 $01.00/0
9 1981 Academic Press Inc. {London) Limited 39
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I . J . CONSTABLE ET AL.
2. Materials and M e t h o d s Twenty adult Dutch rabbits with normal eyes were used. Thirty eyes were subjected to closed transcleral vitrectomy under general anaesthesia with intravenous phenobarbitone sodium 25 mg/kg. Pupils were dilated with tropicamide 0'5 % and neosynephrine l0 %. As much vitreous as possible was systemically removed under microscopic control using a vitreous infusion suction cutter (Machemer, Paret and Buettner, 1970). Physiological saline was used as the vitreous infusion fluid. In the first seven eyes, the adequacy of vitreous removal was evaluated at the end of the procedure by temporary replacement of the resulting fluid vitreous space with 0"8-I ml air. Thereafter the presence of indirect traction on the retina while cutting vitreous was used as a guide to residual vitreous. Post-operatively, eyes were treated with atropine 1 ~o and chloramphenicol eye-drops. After 6 weeks all eyes were examined by indirect ophthalmoscopy and 10 were excluded from the experiments because of minor damage to lens or retina, infection or continued signs of inflammation. The l0 normal eyes and 20 undamaged post-vitrectomy eyes were then used to produce scar tissue with a single transcleral 4-0 silk suture as previously described (Numata, Constable and Whitney, 1975; Constable, 1975). In l0 of the post-vitrectomy eyes, 0"7-0.9 ml of the vitreous cavity fluid was exchanged with an equal volume of purified rooster comb hyaluronic acid, 6 mg/ml. The hyaluronic acid (Hyvisc, Med. Chem. Products, Boston) contained less than 0"2 % protein and was of average molecular weight 100 000. In another 10 eyes, vitreous fluid was exchanged with physiological saline. In all eyes the transvitreal suture was.removed after 10-14 days. The resulting bridge of scar tissue was observed for periods up to eight months. Fluid vitreous samples (0"3 ml) were aspirated through a 27-gauge needle at the time of suture placement and either once or twice again before killing at intervals up to 20 weeks. These samples were analysed for hyaluronie acid (Bitter and Muir, 1962) and total protein (Lowry, Rosebrough, F a r r and Randall, 1951). Animals were killed at 1, 2, 4, 6, 8 and 20 weeks after the suture was placed across the vitreous. These eyes were therefore 2-14 months post-vitrectomy at the time of killing. Eyes were fixed prior to enueleation by intravitreal injection of buffered glutaraldehyde 2"5 %. The anterior segments of the eyes were removed under an operating microscope. Samples of vitreous scar tissue were taken from the center of the vitreous cavity and on the vitreous side of the sites of retinal perforation. Specimens were washed in cacodylate buffer, post-fixed in 1 ~o osmium tetroxide and embedded in Araldite resin. Sections were cut on a L.K.B. Ultratome I I I , stained with lead citrate and viewed in a Siemens 102 electron microscope.
3. Results
Effects of vitrectomy V i t r e c t o m y in t h e absence of definite lens or r e t i n a l d a m a g e caused no visible clinical reaction. D a m a g e d eyes were e x c l u d e d from f u r t h e r e x p e r i m e n t s . A s p i r a t i o n o f 0"3 ml o f fluid v i t r e o u s s a m p l e s on two or t h r e e occasions u p to 20 weeks p o s t - v i t r e c t o m y resulted in no visible scar tissue f o r m a t i o n a t the t i m e of killing. T h e r e was a t r a n s i e n t 10-fold increase in t o t a l v i t r e o u s p r o t e i n following v i t r e c t o m y (Table I), H y a l u r o n i c acid was found a f t e r v i t r e c t o m y in v e r y low c o n c e n t r a t i o n s (Table I) n o t significantly different from n o r m a l r a b b i t eyes. On gross dissection, t h e whole v i t r e o u s base a n t e r i o r l y a n d r e m n a n t s Of p o s t e r i o r cortical gel r e m a i n e d in all eyes. I n m o s t v i t r e c t o m i z e d eyes in which a t r a n s v i t r e a l silk s u t u r e was placed, a firm, w h i t e scar tissue cord f o r m e d across t h e p a t h of t h e s u t u r e (Fig. 1). W h e r e t h e cord was i n c o m p l e t e centrally, t h e i n n e r m o s t edge was s a m p l e d a n d c o m p a r e d to t h e b a s e a t t h e site of r e t i n a l perforation.
Suture-induced scar in non-vitrectomized eyes Scar tissue s a m p l e s t a k e n from t h e v i t r e a l side o f t h e p e r f o r a t i o n sites c o n t a i n e d masses of p r o l i f e r a t i n g cells, m o s t l y w i t h glial cell c h a r a c t e r i s t i c s . These c h a r a c t e r i s t i c s
VITREOUS SCAR TISSUE
41
TABLE I
Effect of vitrectomy on mean concentration of macromolecules in rabbit vitreous Post vitrectomy (weeks)
No. of eyes sampled
Total protein (/~g/ml)
Hyaluronic acid (#g/ml)
3 12
5 8
1448--+520 453-+316
16-+ 12 32-+ 16
20
5
193-+68
10-+8
Normal
12
145 -+54
16 -+12
FIG. 1. Solid cord of scar tissue across vitreous 4 months after transvitreal suture in normal rabbit eye. included b a s e m e n t m e m b r a n e formation, intercellular j u n c t i o n a l complexes, microvillar projections a n d i n t r a c e l l u l a r filaments a n d m i c r o t u b u l e s (Fig. 2). Scattered clumps of p i g m e n t c o n t a i n i n g cells a n d c o m m o n fibroblasts were also seen. The collagen found a t the base of these m e m b r a n e s was u s u a l l y a m i x t u r e of 30-50 n m d i a m e t e r fibrils with p r o m i n e n t 64 n m m a c r o p e r i o d i c i t y a n d 10-20 n m d i a m e t e r fibrils w i t h o u t cross striations (Fig. 2). Samples of the scar tissue cord t a k e n from the central vitreous of n o n - v i t r e c t o m i z e d eyes c o n t a i n e d few cells a n d consisted p r e d o m i n a n t l y of sheets of 10-20 n m d i a m e t e r fibrils w i t h o u t m a c r o p e r i o d i c i t y (Fig. 3). The thicker fibrils were only rarely f o u n d towards the center a n d e v e n t h e n were few in n u m b e r .
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I.J. CONSTABLE ET AL.
Suture-induced scar in vitrectomized eyes with saline replacement As in non-vitrectomized eyes both fine and thick fibrils were deposited at the perforation sites either side by side in patches (Fig. 4) or intermingled. The central areas of the cords were moderately cellular up to 2 months after suture removal and showed definite orientation of the cells (/Fig. 5). The more superficial cells were seen to extend longitudinally along the cord, while central cells were more obliquely orientated. The collagen deposited in the central scar tissue after vitrectomy and saline replacement was invariably the thicker 30-40 nm diameter material. I n two eyes after vitreous replacement with saline, patches of grossly thickened fibrils (160-300 nm) were found (Fig. 6). These tactoids retained the typical 640 nm macroperiodicity of collagen and were accompanied by electron-dense amorphous material.
TABLE II
Hyaluronic acid concentration in vitrectomized rabbit eyes following 0"6-D'9 ml vitreous substitution at time of suture placement Hyaluronic acid (HA) ~ag/ml) Weeks
No. of eyes sampled
Suture + HA
Suture + saline
1 2 4 6
5 5 2 3
1096+427 812_386 480___60 129___80
8
3
42_+28
20
3
10_10
8_+_4 14___8 10_+6 8___6 12+8 16-+9
Suture-induced scar in vitrectomized eyes with hyaluronic acid replacement After vitrectomy and vitreous replacement with 0-6-0-9 ml hyaluronie acid (6 mg/ml), concentrations persisted above the normal range for approximately 2 months (Table II). The pattern of cellular proliferation along the suture track was not significantly different in the presence of hyaluronic acid from saline replaced eyes after vitrectomy. However, in comparison to saline replaced vitreous, fine unbanded fibrils were much more prevalent. Occasional patches of 30-60 nm diameter banded collagen were also found centrally in the majority of specimens examined. Grossly thickened fibrils (tactoids) were also seen in isolated patches in the presence of hyaluronic acid in one eye (Fig. 7). 4. D i s c u s s i o n
The cellular invasion along a suture across the vitreous cavity consisted of a mixture of common fibroblasts, cells with glial characteristics and cells containing melanin granules. In this respect this model of scar tissue formation was similar to previous reports in animals and humans. Prior vitrectomy did not appear to have any influence on the type or appearance of invading cells. The appearance of the collagenous fibrils deposited along discrete cords of scar tissue, however, was different. In normal vitreous almost all new fibrils laid down
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49
except at the surface were of small diameter, 10-20 nm, without visible macroperiodicity. After subtotal vitrectomy and vitreous replacement with saline, only thicker fibrils, of mainly 30-60 nm diameter, were found in the cords. Patches of grossly thickened collagen fibrils with electron-dense amorphous material were also found in the new scar tissue cords formed after vitrectomy. Similar thickened material has been observed under conditions of corneal destruction (Rowsey, Nisbet, Swedo and Katona, 1976; Van Horn, Davis, Hyndiuk and Alpren, 1978) and in experimental osteoarthritis (Kuhn and yon der Mark, 1978). I t has long been known that grossly thickened fibrils or tactoids can be reprecipitated from solubilized collagen under a variety of in vitro conditions. These conditions include precipitation at high pH (Orekhovitch, Tustanovski, Orekhovitch and Plotnikova, 1948) low pH (Bard and Chapman, 1968) and the presence in vitro of other maeromolecules such as heparin or DNA (Nemetschek, 1965). That the deposition of these polymorphic fibrils is dependent on local biochemical conditions rather than on cellular production is suggested by the fact that in vitro they can be readily redissolved and reprecipitated at neutral pH to once again form native fibrils. The polymorphism of collagen deposited in new scar tissue cords after vitrectomy may be due to some such changes in the local environment. The effects of vitreous replacement with hyaluronic acid were inconclusive, since all three collagen fibril morphologies were observed. However, since most of the fibrils found in the central cords with hyaluronic acid were of the fine variety it is tempting to speculate that this macromoleeule could influence the diameter of new formed vitreous fibrils. Certainly in most of the experimental and all of the human pathological conditions described, the fibrils deposited in the presence of intact vitreous gel have been of the fine variety. Further experiments will be necessary to elucidate this point. ACKNOWLEDGMENTS This work was supported by Grant No. RGO 99/6086/78 of the National Health and Medical Research Council of Australia, the Ophthalmic Research Institute of Australia, the Lions Save-Sight Foundation (W.A.) Inc., the Australian Foundation for the Prevention of Blindness (W.A. Division) Inc., and the Royal Perth Hospital Research Center. Rhonda Scrivener provided expert technical assistance. REFERENCES Bard, J. B. C. and Chapman, J. A. (1968). Polymorphism in collagen fibrils precipitated at low pH. Nature (London) 219, 1279-80. Bellhorn, M. B., :Friedman, A. H., Wise, G. N. and Henkind, P. (1975). Ultrastructure and clinicopathologic correlation of idiopathic preretinal macular fibrosis. Am. J. Ophthalmol. 79, 366-73. Bitter, T. and Muir, H. M. (1962). A modified uronic acid carbazole reaction. Analyt. Biochem. 4, 330-34. Clarkson, J. G., Green, W. R. and Massof, D. (1977). A histopathologic review of 168 cases of preretinal membrane. Am. J. Ophthalmol. 84, 1-17. Constable, I. J. (1975). Pathology of vitreous membranes and the effect of haemorrhage and new vessels on the vitreous. Trans. Ophthalmol. Soe. U.K. 95, 382-96. Foos, R. Y. (1974). Vitreoretinal juncture - simple epiretinal membranes. Albrecht yon Graefes Arch. Klin. exp. Ophthalmol. 189, 231-50. Foos, R.Y. and Gloor, B.P. (1975). Vitrcoretinal iuncture-Healing of experimental wounds. Albrecht yon Graefes Arch. Klin. exp. Ohthalmol. 196, 213-30. Gloor, B. P. and Daicker, B. C. (1975). Pathology of the vitreoretinal border structure. Trans. Ophthalmol. Soc. U.K. 95, 387-90.
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Gywat, L. J., Daicker, B. C. and Gloor, B. P, (1978). l~etinale Wundheilung nach mechanischem Trauma bei der Hauskatze. Albrecht von Graefes Arch. Kiln. exp, Ophthalmol. 2{}6, 269-80. Kenyon, K. R. and Michels, R, G. (1977). Ultrastructure of epiretinal membrane removed by pars plana vitreoretinal surgery. Am. J. Ophthalmol. 83, 815-23. Kuhn, K. and vonder Mark, K. (1978). The influence of proteoglyeans on the macromolecular structure of collagen, In Collagen Platelet Interaction. Proceedings of the First Munich Symposium on the Biology of Connective Tissue, 1976. (Ed. H. Gaspar). Pp. 123-7. F. K. Schattauer Verlag, Stuttgart, New York. Laqua, H. and Machemer, R. (1975). Clinical-pathological correlation in massive preretinal proliferation. Am. J. Ophthalmol. 80, 913-29. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-75. Machemer, R. and Laqua, H. (1975). Pigment epithelium proliferation in retinal detachment (massive periretinal proliferation). Am. J. Ophthalmol. 80, 1-23. Machemer, R., Parel, J. M. and Buettner, H. (1970). A new concept for vitreous surgery. I. Instrumentation. Am. J. Ophthalmol. 73, 1-7. Mandelcorn, M. S., Machemer, R., Fineberg, E. and Hersch, S. B. (1975). Proliferation and metaplasia of intravitreal retinal pigment epithelium cell auto transplants. Am. J. Ophthalmol. 80, 227-37. Nemetschek, T. (1965). Zur Frage typiseher Querstreifenmuster der Kollagens. Naturwissenschaften. 52, 478-9. Numata, T., Constable, I. J. and Whitney, D. E. (1975). Physical properties of experimental vitreous membranes. I. Tensile strength. Invest. Ophthalmol. 14, 148-52. Orekhovitch, V. H., Tustanovski, A., Orekhovitch, K. D. and Plotnikova, N. E. (1948). Brokhimiya 13, 55-58. Rentsch, F. J. (1973). Preretinal proliferation of glial cells after mechanical injury of the rabbit retina. Albrecht yon Graefes Arch. Klin. exp. Ophthalmol. 188, 79-90. Rowsey, J. J., Nisbett, R. M., Swedo, J. L. and Katona, L. (1976). Corneal eollagenolytie activity in rabbit polymorphonuclear leukocytes. J. Ultrastruct. Res. 58, 10-21. Swann, D. A., Constable, I. J. and Harper, E. (1972). Vitreous structure. III. Composition of bovine vitreous collagen. Invest. Ophthalmol. 11, 735-8. Van Horn, D. L., Aaberg, T. M., Machemer, R. and Fenzl, R. (1977). Glial cell proliferation in human retinal detachment with massive periretinal proliferation. Am. J. Ophthalmol. 84, 383-93. Van Horn, D. L., Davis, S. D., Hyndiuk, R. A. and Alpren, T. U. P. (1978). Pathogenesis of experimental pseudomonas keratitis in the guinea pig: bacteriologic, clinical and microscopic observation. Invest. Ophthalmol. 17, 1082-6. Zinn, K. M., Constable, I. J. and Schepens, C. L. (1977). The fine structure of human vitreous membranes. In Vitreous Surgery and Advances in Fundus Diagnosis and Treatment (Eds Freeman, H. M. et al.). Pp. 39-49, Appleton-Century-Crofts, New York.
Vitreous Effects on Scar Tissue Formation I. J .
CONSTABLE, R. HORNE, D. V. GRE]~R AND J . P A P A D I M I T R I O U
University of Western Australia and Royal Perth Hospital, Wellington Street, Perth 6000, Western Australia (Received 4 December 1978 and in revised form 13 Feburary 1980, New York) The effects of vitrectomy and replacement with hyaluronic acid or saline on vitreous scar tissue formation were examined in rabbits. A scar tissue bridge was grown across the vitreous cavity along a transcleral suture. In one group of eyes, the bridge was grown across normal vitreous and non-banded collagen of 15 nm diameter was laid down. In a second group in which vitrectomy was done 2-8 months previously, the fluid cavity was replaced with saline. In these eyes, striated collagen of 30-60 nm diameter was deposited. In a third group after vitrectomy and replacement with purified hyaluronic acid, a mixture of fine and thicker fibrils was seen. In both the latter two groups, patches of enormous collagen fibrils of up to 350 nm diameter (tactoids) were found. No differences in the proliferating cell lines were found in any of the three groups. It is suggested that normal vitreous and its glycosaminoglycan component (hyaluronic acid) may influence the extracellular macromolecular organization of vitreous collagen. Key words: vitreous; scar tissue; collagen; vitrectomy ; hyaluronic acid.
1. I n t r o d u c t i o n C o n t r a c t i o n o f v i t r e o u s or p r e r e t i n a l scar tissue l e a d i n g to r e t i n a l d i s t o r t i o n or d e t a c h m e n t a c c o u n t s for a m a j o r p r o p o r t i o n o f blindness, for e x a m p l e , in d i a b e t e s mellitus, p e n e t r a t i n g injuries a n d c o m p l i c a t e d r e t i n a l d e t a c h m e n t s . D e s c r i p t i o n s o f v i t r e o u s or p r e r e t i n a l scar tissue in h u m a n s (Foos, 1974; Bellhorn, F r i e d m a n , W i s e a n d H e n k i n d , 1975; Zinn, C o n s t a b l e a n d Schepens, 1976; Clarkson, Green a n d Massof, 1977; V a n H o r n , A a b e r g , M a c h e m e r a n d Fenzl, 1977; K e n y o n a n d Michels, 1977), m o n k e y s ( L a q u a a n d Machemer, 1975; M a c h e m e r a n d L a q u a , 1975), r a b b i t s (Rentsch, 1973; F o o s a n d Gloor, 1975) a n d c a t s (Gloor a n d D a i c k e r , 1975; G y w a t , D a i c k e r a n d Gloor, 1978) h a v e s u g g e s t e d t h a t r e t i n a l glial cells a n d p o s s i b l y r e t i n a l p i g m e n t epithelial cells are t h e principal e l e m e n t s c a p a b l e o f i n v a d i n g t h e v i t r e o u s c a v i t y , p r o l i f e r a t i n g a n d d e p o s i t i n g collagen. T h e collagen d e p o s i t e d in h u m a n s so far described lacks m a c r o p e r i o d i c i t y a n d is m o r p h o l o g i c a l l y s i m i l a r to n o r m a l v i t r e o u s collagen ( d i a m e t e r 10-20 nm) (Zinn et al., 1976; K e n y o n a n d Michels, 1977). U n d e r e x p e r i m e n t a l c o n d i t i o n s in animals, new collagenous fibrils o f d i a m e t e r 30-80 n m w i t h 64 n m p e r i o d i c i t y m a y be laid d o w n in t h e v i t r e o u s (Mandelcorn, M a c h e m e r , F i n e b e r g a n d Hersch, 1975; Constable, 1975). F o r e x a m p l e , f i b r o b l a s t i c tissue i n d u c e d in r a b b i t s b y v i t r e o u s p e n e t r a t i o n a n d b l o o d i n j e c t i o n or b y a t r a n s c l e r a l silk s u t u r e c o n t a i n s b o t h the a b o v e m o r p h o l o g i c a l l y d i s t i n c t t y p e s o f collagen. I t is n o t k n o w n w h e t h e r i n d i v i d u a l cell lines are p r o g r a m m e d to p r o d u c e a d i s t i n c t m o r p h o l o g i c a l collagen t y p e , or w h e t h e r t h e e x t r a c e l l u l a r m a t r i x influences t h e e x t e n t o f a g g r e g a t i o n o f fibrils a n d therefore t h e i r m o r p h o l o g i c a l characteristics. The s t u d y was u n d e r t a k e n to assess t h e effects o f v i t r e c t o m y a n d r e p l a c e m e n t w i t h saline or h y a t u r o n i c acid on t h e d e p o s i t i o n of s c a r tissue across t h e v i t r e o u s c a v i t y in r a b b i t s . Reprint requests to: Professor I. J. Constable, University Department of Ophthalmology, Royal Perth Hospital. GPO Box X2213, Perth 600t, Western Australia. 0014-4835/81/010039+ 12 $01.00/0
9 1981 Academic Press Inc. {London) Limited 39
40
I . J . CONSTABLE ET AL.
2. Materials and M e t h o d s Twenty adult Dutch rabbits with normal eyes were used. Thirty eyes were subjected to closed transcleral vitrectomy under general anaesthesia with intravenous phenobarbitone sodium 25 mg/kg. Pupils were dilated with tropicamide 0'5 % and neosynephrine l0 %. As much vitreous as possible was systemically removed under microscopic control using a vitreous infusion suction cutter (Machemer, Paret and Buettner, 1970). Physiological saline was used as the vitreous infusion fluid. In the first seven eyes, the adequacy of vitreous removal was evaluated at the end of the procedure by temporary replacement of the resulting fluid vitreous space with 0"8-I ml air. Thereafter the presence of indirect traction on the retina while cutting vitreous was used as a guide to residual vitreous. Post-operatively, eyes were treated with atropine 1 ~o and chloramphenicol eye-drops. After 6 weeks all eyes were examined by indirect ophthalmoscopy and 10 were excluded from the experiments because of minor damage to lens or retina, infection or continued signs of inflammation. The l0 normal eyes and 20 undamaged post-vitrectomy eyes were then used to produce scar tissue with a single transcleral 4-0 silk suture as previously described (Numata, Constable and Whitney, 1975; Constable, 1975). In l0 of the post-vitrectomy eyes, 0"7-0.9 ml of the vitreous cavity fluid was exchanged with an equal volume of purified rooster comb hyaluronic acid, 6 mg/ml. The hyaluronic acid (Hyvisc, Med. Chem. Products, Boston) contained less than 0"2 % protein and was of average molecular weight 100 000. In another 10 eyes, vitreous fluid was exchanged with physiological saline. In all eyes the transvitreal suture was.removed after 10-14 days. The resulting bridge of scar tissue was observed for periods up to eight months. Fluid vitreous samples (0"3 ml) were aspirated through a 27-gauge needle at the time of suture placement and either once or twice again before killing at intervals up to 20 weeks. These samples were analysed for hyaluronie acid (Bitter and Muir, 1962) and total protein (Lowry, Rosebrough, F a r r and Randall, 1951). Animals were killed at 1, 2, 4, 6, 8 and 20 weeks after the suture was placed across the vitreous. These eyes were therefore 2-14 months post-vitrectomy at the time of killing. Eyes were fixed prior to enueleation by intravitreal injection of buffered glutaraldehyde 2"5 %. The anterior segments of the eyes were removed under an operating microscope. Samples of vitreous scar tissue were taken from the center of the vitreous cavity and on the vitreous side of the sites of retinal perforation. Specimens were washed in cacodylate buffer, post-fixed in 1 ~o osmium tetroxide and embedded in Araldite resin. Sections were cut on a L.K.B. Ultratome I I I , stained with lead citrate and viewed in a Siemens 102 electron microscope.
3. Results
Effects of vitrectomy V i t r e c t o m y in t h e absence of definite lens or r e t i n a l d a m a g e caused no visible clinical reaction. D a m a g e d eyes were e x c l u d e d from f u r t h e r e x p e r i m e n t s . A s p i r a t i o n o f 0"3 ml o f fluid v i t r e o u s s a m p l e s on two or t h r e e occasions u p to 20 weeks p o s t - v i t r e c t o m y resulted in no visible scar tissue f o r m a t i o n a t the t i m e of killing. T h e r e was a t r a n s i e n t 10-fold increase in t o t a l v i t r e o u s p r o t e i n following v i t r e c t o m y (Table I), H y a l u r o n i c acid was found a f t e r v i t r e c t o m y in v e r y low c o n c e n t r a t i o n s (Table I) n o t significantly different from n o r m a l r a b b i t eyes. On gross dissection, t h e whole v i t r e o u s base a n t e r i o r l y a n d r e m n a n t s Of p o s t e r i o r cortical gel r e m a i n e d in all eyes. I n m o s t v i t r e c t o m i z e d eyes in which a t r a n s v i t r e a l silk s u t u r e was placed, a firm, w h i t e scar tissue cord f o r m e d across t h e p a t h of t h e s u t u r e (Fig. 1). W h e r e t h e cord was i n c o m p l e t e centrally, t h e i n n e r m o s t edge was s a m p l e d a n d c o m p a r e d to t h e b a s e a t t h e site of r e t i n a l perforation.
Suture-induced scar in non-vitrectomized eyes Scar tissue s a m p l e s t a k e n from t h e v i t r e a l side o f t h e p e r f o r a t i o n sites c o n t a i n e d masses of p r o l i f e r a t i n g cells, m o s t l y w i t h glial cell c h a r a c t e r i s t i c s . These c h a r a c t e r i s t i c s
VITREOUS SCAR TISSUE
41
TABLE I
Effect of vitrectomy on mean concentration of macromolecules in rabbit vitreous Post vitrectomy (weeks)
No. of eyes sampled
Total protein (/~g/ml)
Hyaluronic acid (#g/ml)
3 12
5 8
1448--+520 453-+316
16-+ 12 32-+ 16
20
5
193-+68
10-+8
Normal
12
145 -+54
16 -+12
FIG. 1. Solid cord of scar tissue across vitreous 4 months after transvitreal suture in normal rabbit eye. included b a s e m e n t m e m b r a n e formation, intercellular j u n c t i o n a l complexes, microvillar projections a n d i n t r a c e l l u l a r filaments a n d m i c r o t u b u l e s (Fig. 2). Scattered clumps of p i g m e n t c o n t a i n i n g cells a n d c o m m o n fibroblasts were also seen. The collagen found a t the base of these m e m b r a n e s was u s u a l l y a m i x t u r e of 30-50 n m d i a m e t e r fibrils with p r o m i n e n t 64 n m m a c r o p e r i o d i c i t y a n d 10-20 n m d i a m e t e r fibrils w i t h o u t cross striations (Fig. 2). Samples of the scar tissue cord t a k e n from the central vitreous of n o n - v i t r e c t o m i z e d eyes c o n t a i n e d few cells a n d consisted p r e d o m i n a n t l y of sheets of 10-20 n m d i a m e t e r fibrils w i t h o u t m a c r o p e r i o d i c i t y (Fig. 3). The thicker fibrils were only rarely f o u n d towards the center a n d e v e n t h e n were few in n u m b e r .
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I.J. CONSTABLE ET AL.
Suture-induced scar in vitrectomized eyes with saline replacement As in non-vitrectomized eyes both fine and thick fibrils were deposited at the perforation sites either side by side in patches (Fig. 4) or intermingled. The central areas of the cords were moderately cellular up to 2 months after suture removal and showed definite orientation of the cells (/Fig. 5). The more superficial cells were seen to extend longitudinally along the cord, while central cells were more obliquely orientated. The collagen deposited in the central scar tissue after vitrectomy and saline replacement was invariably the thicker 30-40 nm diameter material. I n two eyes after vitreous replacement with saline, patches of grossly thickened fibrils (160-300 nm) were found (Fig. 6). These tactoids retained the typical 640 nm macroperiodicity of collagen and were accompanied by electron-dense amorphous material.
TABLE II
Hyaluronic acid concentration in vitrectomized rabbit eyes following 0"6-D'9 ml vitreous substitution at time of suture placement Hyaluronic acid (HA) ~ag/ml) Weeks
No. of eyes sampled
Suture + HA
Suture + saline
1 2 4 6
5 5 2 3
1096+427 812_386 480___60 129___80
8
3
42_+28
20
3
10_10
8_+_4 14___8 10_+6 8___6 12+8 16-+9
Suture-induced scar in vitrectomized eyes with hyaluronic acid replacement After vitrectomy and vitreous replacement with 0-6-0-9 ml hyaluronie acid (6 mg/ml), concentrations persisted above the normal range for approximately 2 months (Table II). The pattern of cellular proliferation along the suture track was not significantly different in the presence of hyaluronic acid from saline replaced eyes after vitrectomy. However, in comparison to saline replaced vitreous, fine unbanded fibrils were much more prevalent. Occasional patches of 30-60 nm diameter banded collagen were also found centrally in the majority of specimens examined. Grossly thickened fibrils (tactoids) were also seen in isolated patches in the presence of hyaluronic acid in one eye (Fig. 7). 4. D i s c u s s i o n
The cellular invasion along a suture across the vitreous cavity consisted of a mixture of common fibroblasts, cells with glial characteristics and cells containing melanin granules. In this respect this model of scar tissue formation was similar to previous reports in animals and humans. Prior vitrectomy did not appear to have any influence on the type or appearance of invading cells. The appearance of the collagenous fibrils deposited along discrete cords of scar tissue, however, was different. In normal vitreous almost all new fibrils laid down
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VITREOUS SCAR TISSUE
49
except at the surface were of small diameter, 10-20 nm, without visible macroperiodicity. After subtotal vitrectomy and vitreous replacement with saline, only thicker fibrils, of mainly 30-60 nm diameter, were found in the cords. Patches of grossly thickened collagen fibrils with electron-dense amorphous material were also found in the new scar tissue cords formed after vitrectomy. Similar thickened material has been observed under conditions of corneal destruction (Rowsey, Nisbet, Swedo and Katona, 1976; Van Horn, Davis, Hyndiuk and Alpren, 1978) and in experimental osteoarthritis (Kuhn and yon der Mark, 1978). I t has long been known that grossly thickened fibrils or tactoids can be reprecipitated from solubilized collagen under a variety of in vitro conditions. These conditions include precipitation at high pH (Orekhovitch, Tustanovski, Orekhovitch and Plotnikova, 1948) low pH (Bard and Chapman, 1968) and the presence in vitro of other maeromolecules such as heparin or DNA (Nemetschek, 1965). That the deposition of these polymorphic fibrils is dependent on local biochemical conditions rather than on cellular production is suggested by the fact that in vitro they can be readily redissolved and reprecipitated at neutral pH to once again form native fibrils. The polymorphism of collagen deposited in new scar tissue cords after vitrectomy may be due to some such changes in the local environment. The effects of vitreous replacement with hyaluronic acid were inconclusive, since all three collagen fibril morphologies were observed. However, since most of the fibrils found in the central cords with hyaluronic acid were of the fine variety it is tempting to speculate that this macromoleeule could influence the diameter of new formed vitreous fibrils. Certainly in most of the experimental and all of the human pathological conditions described, the fibrils deposited in the presence of intact vitreous gel have been of the fine variety. Further experiments will be necessary to elucidate this point. ACKNOWLEDGMENTS This work was supported by Grant No. RGO 99/6086/78 of the National Health and Medical Research Council of Australia, the Ophthalmic Research Institute of Australia, the Lions Save-Sight Foundation (W.A.) Inc., the Australian Foundation for the Prevention of Blindness (W.A. Division) Inc., and the Royal Perth Hospital Research Center. Rhonda Scrivener provided expert technical assistance. REFERENCES Bard, J. B. C. and Chapman, J. A. (1968). Polymorphism in collagen fibrils precipitated at low pH. Nature (London) 219, 1279-80. Bellhorn, M. B., :Friedman, A. H., Wise, G. N. and Henkind, P. (1975). Ultrastructure and clinicopathologic correlation of idiopathic preretinal macular fibrosis. Am. J. Ophthalmol. 79, 366-73. Bitter, T. and Muir, H. M. (1962). A modified uronic acid carbazole reaction. Analyt. Biochem. 4, 330-34. Clarkson, J. G., Green, W. R. and Massof, D. (1977). A histopathologic review of 168 cases of preretinal membrane. Am. J. Ophthalmol. 84, 1-17. Constable, I. J. (1975). Pathology of vitreous membranes and the effect of haemorrhage and new vessels on the vitreous. Trans. Ophthalmol. Soe. U.K. 95, 382-96. Foos, R. Y. (1974). Vitreoretinal juncture - simple epiretinal membranes. Albrecht yon Graefes Arch. Klin. exp. Ophthalmol. 189, 231-50. Foos, R.Y. and Gloor, B.P. (1975). Vitrcoretinal iuncture-Healing of experimental wounds. Albrecht yon Graefes Arch. Klin. exp. Ohthalmol. 196, 213-30. Gloor, B. P. and Daicker, B. C. (1975). Pathology of the vitreoretinal border structure. Trans. Ophthalmol. Soc. U.K. 95, 387-90.
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Gywat, L. J., Daicker, B. C. and Gloor, B. P, (1978). l~etinale Wundheilung nach mechanischem Trauma bei der Hauskatze. Albrecht von Graefes Arch. Kiln. exp, Ophthalmol. 2{}6, 269-80. Kenyon, K. R. and Michels, R, G. (1977). Ultrastructure of epiretinal membrane removed by pars plana vitreoretinal surgery. Am. J. Ophthalmol. 83, 815-23. Kuhn, K. and vonder Mark, K. (1978). The influence of proteoglyeans on the macromolecular structure of collagen, In Collagen Platelet Interaction. Proceedings of the First Munich Symposium on the Biology of Connective Tissue, 1976. (Ed. H. Gaspar). Pp. 123-7. F. K. Schattauer Verlag, Stuttgart, New York. Laqua, H. and Machemer, R. (1975). Clinical-pathological correlation in massive preretinal proliferation. Am. J. Ophthalmol. 80, 913-29. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-75. Machemer, R. and Laqua, H. (1975). Pigment epithelium proliferation in retinal detachment (massive periretinal proliferation). Am. J. Ophthalmol. 80, 1-23. Machemer, R., Parel, J. M. and Buettner, H. (1970). A new concept for vitreous surgery. I. Instrumentation. Am. J. Ophthalmol. 73, 1-7. Mandelcorn, M. S., Machemer, R., Fineberg, E. and Hersch, S. B. (1975). Proliferation and metaplasia of intravitreal retinal pigment epithelium cell auto transplants. Am. J. Ophthalmol. 80, 227-37. Nemetschek, T. (1965). Zur Frage typiseher Querstreifenmuster der Kollagens. Naturwissenschaften. 52, 478-9. Numata, T., Constable, I. J. and Whitney, D. E. (1975). Physical properties of experimental vitreous membranes. I. Tensile strength. Invest. Ophthalmol. 14, 148-52. Orekhovitch, V. H., Tustanovski, A., Orekhovitch, K. D. and Plotnikova, N. E. (1948). Brokhimiya 13, 55-58. Rentsch, F. J. (1973). Preretinal proliferation of glial cells after mechanical injury of the rabbit retina. Albrecht yon Graefes Arch. Klin. exp. Ophthalmol. 188, 79-90. Rowsey, J. J., Nisbett, R. M., Swedo, J. L. and Katona, L. (1976). Corneal eollagenolytie activity in rabbit polymorphonuclear leukocytes. J. Ultrastruct. Res. 58, 10-21. Swann, D. A., Constable, I. J. and Harper, E. (1972). Vitreous structure. III. Composition of bovine vitreous collagen. Invest. Ophthalmol. 11, 735-8. Van Horn, D. L., Aaberg, T. M., Machemer, R. and Fenzl, R. (1977). Glial cell proliferation in human retinal detachment with massive periretinal proliferation. Am. J. Ophthalmol. 84, 383-93. Van Horn, D. L., Davis, S. D., Hyndiuk, R. A. and Alpren, T. U. P. (1978). Pathogenesis of experimental pseudomonas keratitis in the guinea pig: bacteriologic, clinical and microscopic observation. Invest. Ophthalmol. 17, 1082-6. Zinn, K. M., Constable, I. J. and Schepens, C. L. (1977). The fine structure of human vitreous membranes. In Vitreous Surgery and Advances in Fundus Diagnosis and Treatment (Eds Freeman, H. M. et al.). Pp. 39-49, Appleton-Century-Crofts, New York.