Fine structure of rat corneal vessels in advanced stages of wound healing

Transcription

1 Fine structure of rat corneal vessels in advanced stages of wound healing Jeanne Szalay and G. D. Pappas The present work is an examination of the fine structure of corneal vessels in advanced stages of wound healing in the Sprague-Dawley rat. Several weeks after burning the central cornea with silver nitrate or a cautery iron, vascularized corneas classified as ''healed" mildly wounded, or moderately to severely wounded were selected for electron microscopic observation. In all cases the blood vessels peripheral to the wound were mature. In contrast to newly formed blood vessels, they showed no frank gaps between endothelial cells. They had a low continuous endothelial sheet, a continuous basement membrane, one or more layers of pericytes, and tended to develop greatly thickened basement membranes. The surrounding stroma closely resembled that of control avascular corneas except that stromal cells sometimes showed increased amounts of rough-surfaced endoplasmic reticulum, and occasional phagocytes were present. The vessels within the wound of "healed" corneas and mildly icounded corneas were also mature vessels of the low continuous endothelial variety, but they did not contain greatly thickened basement membranes. While vessels from the wound of moderately to severely wounded corneas possessed some properties of immature vessels (i.e., rounded and irregularly shaped endothelial cells, and little or no basement membrane) the profile of organelles within the endothelial cell and the close approximation of endothelial cells at cell junctions were characteristic of mature blood vessels. In all corneas, the stroma from wound areas showed an irregular distribution of collagen and a trend toward increased numbers of activated stromal cells, phagocytes, and blood vessels in the more severely wounded corneas. Qualitative differences such as the presence of lymph channels and the presence of numerous electron opaque particles in the stroma and in phagocytic vacuoles of phagocytes only appeared in the wound areas of moderately to severely wounded corneas. Electron-opaque particles were observed only in corneas burned by silver nitrate and probably represent the morphological basis of argyria. Key words: corneal vessels, corneal wound healing, corneal burns, corneal vascularization, corneal endothelium, corneal basement membrane, corneal stroma cell, ultrastructure histopathology, rats, fine structure of blood vessels. From the Albert Einstein College of Medicine, New York, N. Y. Address correspondence to: Dr. Jeanne Szalay, Department of Anatomy, Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, N. Y This work is being supported by Grant NB National Institutes of Health, Bethesda, Md. Manuscript submitted Nov. 13, 1969; revised manuscript accepted Jan. 5, he fine structure of newly formed and developing blood vessels has been described by Schb'efl in vascularizing rat corneas, 1 and by Cliff using a rabbit ear chamber. 2 The vessels were found to possess distinct structural characteristics such as open gaps between endothelial cells and a thin or incomplete basement membrane. These properties appeared to be the

2 Volume 9 Number 5 Structure of rat corneal vessels in wound healing 355 Fig. 1. Light micrograph of a "healed" vascularized cornea. Note the lack of a definite wound area. (Final magnification x24.0.) morphological correlate of the high permeability and fragility of newly formed vessels. 1 However, the fine structure of vessels persisting for several months has not been described. It was our intention to compare the fine structure of the newly formed corneal vessels with those showing various stages of healing in order to gain information concerning the structure of well-established vessels in a tissue that is normally a vascular and the fine structure of these "mature" blood vessels in wounds of varying severity. Material and methods Experimental system. Interstitial corneal vascularization was induced by burning the center of the cornea with silver nitrate or a cautery iron. In general, wounds produced by silver nitrate were more extensive, induced a greater degree of vascularization, and healed more slowly than wounds from cautery-burned corneas. Male rats (Sprague-Dawley strain), weighing 250 to 350 Gm. were anesthetized with ether and the central cornea of the right eye was gently touched with a pointed silver nitrate crystal or a finely tapered hot cautery iron. Several dozen vascularized corneas were examined under a dissecting microscope over a period of several months (usually 2-3 although occasionally longer. After 2 to 3 months and 1 to 2 days prior to each experiment, the corneas were classified according to the degree of vascnlarization and extent of the wound as healed (Fig. 1), mildly wounded, or moderately to severely wounded (Fig. 2). At this time, approximately 40 per cent of the cautery-burned eyes appeared to have healed completely and were again avascular. The remaining 60 per cent had healed in varying degrees. Some contained a few Fig. 2. Light micrograph of a moderately to severely wounded cornea showing a relatively dense growth of blood vessels, especially in the wound area. (Final magnification x24.0.) vessels and were transparent, except for the occasional presence of a small white scar or faint opacity. These eyes, although vascular, appeared to be completely healed. Most of the vascularized corneas resulting from cautery-iron burns had wounds classified as mild. The wound was approximately 2.0 mm. in diameter, opaque, and fairly well vascularized, but the remaining cornea was clear. Occasionally wounds from cauteryburned eyes were classified as moderate to severe. The wound which could be 4.0 mm. wide sometimes had a green, yellow, or gray hue, and was heavily vascularized. Adhesions of iridial vessels to the cornea were apparent. On the other hand, the great majority of silver nitrate-burned corneas fell into this category and showed more extensive vascularization of the wound area than the moderately to severely wounded, cauteryburned eyes. More severely wounded corneas were not selected for experimentation. Five vascularized corneas from each category (healed, mildly wounded, and moderately to severely wounded) were examined. Some were taken from rats that had received intravenous injections of electron-opaque marker particles immediately prior to removal of the eye. The detailed results of these permeability studies will be treated in another report. Removal and fixation of the eye. The eye was flushed with cold fixative (1 per cent osmium tetroxide in M/15 phosphate buffer ph 7.3). A fine needle was used to make a drainage hole in the sclera, and cold fixative was injected through a second hole into the anterior chamber. The eye was rapidly removed, placed in cold fixative, dissected bilaterally, and the lens removed. It was flushed with cold fixative and kept in the osmium solution for 2.5 horns. Dehydration and embedding. Tissue was dehydrated in a series of graded ethyl alcohol

3 *.> «356 Szalay and Pappas Investigative Ophthalmology May 1970 solutions, and the corneas were dissected into strips in 95 per cent alcohol. Corneal strips often extended from the sclera to the wound. They were immersed in 1.1 Epon:Propylene (one day), 2:1 Epon:Propylene (one day), 3:1 Epon:Propylene (2 days), pure Epon (several hours), and then flat embedded. Light and electron microscopy. Blood vessels were visualized by examination of flat embedded specimens in a Leitz microscope at low power, and thick sections were made to localize selected vessels. Thin sections were made using a Porter- Blum microtome equipped with a diamond knife. Sections were picked up on uncoated copper grids, 300 mesh, or Formvar-carbon coated grids, 150 mesh, stained with lead citrate and/or uranyl acetate, and examined in an RCA-EMU-3G or in a Philips 200 electron microscope. Observations Fine structure of vascularized healed corneas. For the most part, the stroma of vascularized regions of healed corneas resembles that of the normal avascular cornea, collagen appearing to be evenly and compactly distributed within lamellae. However, fibroblasts may contain conspicuous amounts of rough-surfaced endoplasmic reticulum in contrast to the avascular cornea, and an occasional cell displaying phagocytic activity may be present adjacent to a blood vessel. The epithelium and mesothelium do not differ from those of avascular corneas. The blood vessels present are distinctly mature (for criteria of the properties of mature as opposed to immature blood vessels see Schoefl 1 or Cliff 2 ) with diameters of 3 to 30 fi. The capillaries possess a low continuous endothelium, 3 and the cytoplasm is evenly distributed although irregular areas and attenuated regions with fenestrations are occasionally seen. The endothelial cells contain numerous small vesicles approximately 600 A in diameter, numerous fine filaments, small amounts of rough-surfaced endoplasmic reticulum, free >.*. B small vessel from a "healed" cornea 12 weeks after injury inflicted by a cautery iron. Animal received an intravenous injection of thorium dioxide 8 minutes prior to death. (E) = endolial cell, (P) pericyte, (C) = collagen. (xl4,000.)

4 Volume 9 Number 5 Structure of rat corneal vessels in wound healing 357 ribosomes, and few mitochondria and do not show significant "bubbling" 1 of the lateral surface. An unusually thick basement membrane showing several lamellae characterizes these corneal vessels (Fig. 3). These basement membranes may be as thick as 0.7 JX, greater than the width of the endothelial cell. The thickest basement membranes are found surrounding vessels with an incomplete layer of pericytes and a very small lumen (4 to 10 n in diameter). Higher magnifications show that the basement membrane material is fuzzlike and not clearly fibrillar. This is true even when corneas are fixed in glutaraldehyde and postfixed in osmium. In addition to the endothelium and basement membrane, the vessel wall typically contains many pericytes. In a few of the vessels studied, these pericytes make frequent contact with each other and show dense bodies within the cytoplasm similar to those observed in smooth muscle cells. 4 However, in most cases the ultrastructure of the pericytes is indistinguishable from that of the endothelial cell, although the cytoplasm appears to be less dense. The area originally designated as a scar may or may not be vascularized. Collagen fibrils are often erratically distributed and numerous patches of dense amorphous material may be present (Fig. 4). These patches are usually found in a vascular areas within the opacity. Anteriorly, the epithelium often shows slight irregularities of the basal cell layer (Fig. 4); posteriorly, < Fig. 4. This micrograph is taken from the opacity of a "healed" cornea 8 weeks after injury inflicted by a cautery iron and shows some slight irregularities of the basal laver of the epithelium (Ep) and abundant patches of dense amorphous material (Pa), (Sj stromal cell. (xlo 3 OOO.)

5 358 Szalay and Pappas Investigative Ophthalmology May 1970 r B Ph Ph Fig. 5. A typical vascularized area within the opaque region of a "healed" cornea 12 weeks after injury inflicted by a cautery iron. Several phagocytic cells (Ph) are present, collagen is irregularly distributed (arrows), blood vessels contain at least one layer of pericytes (P) and a well-developed basement membrane (B), in addition to the endothelial cells (E). (A) = adventitial cell. This animal received an intravenous injection of thorium dioxide 8 minutes prior to death. (x6,000.) the mesothelium may also show signs of injury such as incomplete synthesis of Descemet's membrane. The latter may contain collagen fibrils and cells of unknown origin. When the opacity is vascularized, the blood vessels resemble those described above, but have only a thin basement membrane (Fig. 5). Fine structure of the mildly wounded cornea. The fine structure of the cornea and corneal blood vessels in mildly wounded corneas is the same as described above for healed vascular corneas. The difference is only one of degree. The obvious opaque area, which in vivo had the discrete appearance of a wound, is always vascularized. Within a typical wound area collagen is not always evenly distributed within layers or lamellae, fibroblasts are abundant, and phagocytes are present. The vessel wall is mature and contains 1 to 2 layers of pericytes and a distinct basement membrane. The fine structure of the blood vessels and the surrounding cornea, mesothelium, and epithelium bear a striking resemblance to those of the limbal-corneal junction. The obvious exceptions to such a comparison are the smaller diameter of the collagen fibers within the wound, and the presence of myelinated nerve fibers, mast cells, and leukocytes in the Strom a of the limbus. Peripheral to the wound, the fine structure of the cornea and corneal vessels is similar to transparent areas of the healed corneas. Fine structure of a moderately to severely wounded cornea. The wound region is the most prominent characteristic of mod-

6 Volume 9 Number 5 Structure of rat corneal vessels in wound healing 359 v Fig. 6. From the wound of a moderately to severely wounded cornea 12 weeks after injury inflicted by a cautery iron. Note the irregular contour of endothelial cells (E), and the small amount of basement membrane material (B), Stromal cells (S) appear to be activated, many phagocytic cells (Ph) are present and collagen (C) is irregularly distributed. (L) = lumen of blood vessel. (x8,700.) erately to severely wounded corneas. Fig. 6 is taken from a wound of a cornea burned with the cautery iron and which showed the greatest disruption of the normal structure of the stroma observed in our experiments. Fig. 8 is from a silver nitrate-burned eye. In addition to the presence of many blood vessels, these wounds are characterized by abundant activated fibroblasts, phagocytes, and very loosely packed collagen fibers. In some blood vessels, the endothelial cell has an irregular contour and appears short and wide (Fig. 6). The vessel shown in Fig. 6 represents the most extreme example of this phenomenon and superficially resembles a newly formed blood vessel. However, higher magnifications of this vessel (Fig. 7) and other vessels from wound areas (Figs. 9 and 10) show numerous fine filaments in the cytoplasm, few mitochondria, some rough-surfaced endoplasmic reticulum, moderate amounts of free ribosomes, and vesicles approximately 600 A in diameter. These vesicles sometimes appear to fuse, forming intracellular channels (Fig. 9). Fig. 10 shows a channel covered by a "diaphragm" at both ends. In addition to the endothelium, the vessel wall typically contains an incomplete layer of pericytes and little or no basement membrane. Occasional lymphatic vessels may also be found. In these vessels (Fig. 11), the endothelium is very thin and

7 1nt.rstigrrtit.c. Ojhtl~nlit~olopy May 1970 Fig. 7. IIigh ~nagnification of the insert in Fig. 6, showing ultrastructure of the t~ndothelial cell (E). R'u~nerous vtssicles (I,), mitochondria (At), srnnll nnlounts of rough-surft~cetl endopl;lsmic reticdr~m (RS), free polysomes (Po), and numerous fine filaments (F) are present. (J) = jmc tion between entlothelinl cells. ( ~44,000. )

8 Volume 9 Number 5 Structure of rat corneal vessels in loound healing 361 Fig. 8. A typical micrograph from the wound of a moderately to severely wounded cornea 11 weeks after injury inflicted by silver nitrate. Stromal cells (S) are numerous and collagen (C) is irregularly distributed. Blood vessels are fairly abundant and endothelial cells possess little or no basement membrane making positive identification of adventitial cells (A) difficult. This animal received an intravenous injection of thorium dioxide 8 minutes prior to death. (x5,500.) abluminal flaps appear to extend into the adjacent stroma. The basement membrane may be thin or inconspicuous, no pericytes are present, and no blood cells are found in the lumen. Light-microscopic evidence for the invasion of lymphatics into heavily vascularized rabbit corneas has been reported elsewhere. 5 In the posterior cornea, adhesions of iridial vessels may be seen. Anteriorly, the basal layer of the epithelium may be thrown into folds, but it remains covered by basement membrane. When moderately to severely wounded corneas were taken from animals which had been burned 'with silver nitrate, but had not received intravenous injections of electron opaque marker particles, the corneal stroma in the woimd area contained numerous electron-opaque particles. These particles were usually associated with patches of dense amorphous material (Fig. 12). Particles could also be seen accumulated within vacuoles of phagocytes. Discussion Jakus 6 found that 2 months after an injury made by a small penetrating cut into the cornea, healing was complete although an opacity or scar remained. In the present study, some vascularized corneas resulting from chemical or physical burns had faint opacities, but appeared

9 362 Szalay and Pappas Investigative Ophthalmology May 1970 "'ftft."wip* ^ -.k, *** ShC L : A? & * B Fig. 9. High magnification of part of a vessel wall from another area of the wound described in the legend of Fig. 8. Free ribosomes (R), vesicles which appear to have fused (V), junction (J) between endothelial cells 1 and 2, (B) = basement membrane, (A) = possible adventitial cell. (x58,000.) Fig. 10. Another micrograph from the wound described in the legend of Fig. 8. Endothelial cell (E) containing fine filaments (F), vesicles (V), and an intracellular channel covered by a diaphragm on both sides (arrows). (C) = collagen fibers. (x78/700.) under the dissecting microscope to be completely healed. However, examination of the opaque region with the electron microscope showed that in addition to the irregular distribution of collagen typical of a scar, 7 fibroblasts are activated, phagocytes are present, and the epithelium and mesothelium have not returned to normal. In addition, when the opaque region of an apparently healed cornea is vascularized, its fine structure is similar to that of the wound from mildly wounded corneas. These findings strongly suggest that healing is still in progress. Under the present experimental conditions the healing of the cornea is always accompanied by a decrease in the number of vessels present and it would appear that the only

10 Volume 9 Number 5 Structure of rat corneal vessels in wound healing 363 " Fig. 11. Possible lymph vessel from a moderately to severely wounded cornea 11 weeks after injury inflicted by silver nitrate. Note the attenuated endothelial cells (E), small vesicles (V), and abluminal flaps of the endothelium (Ab). No basement membrane is evident. (L) lumen of vessel. (x8,400.) completely healed corneas are those which have again become avascular. As described in the text, the fine structure of all corneas and comeal vessels peripheral to the wound is the same, and the fine structure within the opaque regions o corneas originally classified as "healed" or mildly wounded differs only in degree. It is only in the injured area of f V moderately to severely wounded corneas that qualitatively different characteristics appear. These include adhesions of iridial vessels to the posterior cornea, vessels characteristic of lymph channels, blood vessels with some properties of immature vessels, and only in the case of silver nitrate-injured eyes, the presence of electron-opaque particles in the stroma. The last two phenomena warrant special attention. The electron-opaque particles observed in unstained sections prepared from moderately to severely wounded silver nitrate-burned corneas removed from animals which had not been injected with electron-opaque particles may well be the morphological basis of argyria. It has long been known that prolonged or extensive penetration of silver compounds into the skin results in permanent pigmentation of the connective tissue. s Since we have nitrate-burned eyes examined, we may only observed deposition of opaque particles in the most severely injured silver assume that extensive penetration of silver nitrate occurred during the initial insult. Since the more severely cautery-burned corneas healed within 6 months, while the more severely silver nitrate burned corneas often remained wounded for 1.5 years or more, one should consider the possibility that the electron-opaque deposits observed and presumed to be silver compounds may interfere with completion of the healing process. The vessels in the woimd area of moderately to severely wounded corneas sometimes appear immature; the shape of the endothelial cell can be very irregular and the basement membrane may be thin or inconspicuous. However, the profile of cell organelles in the cytoplasm of these cells is characteristic of differentiated endothelial cells. The frank open gaps between endothelial cells reported for immature vessels 1 and in either locally induced 9 or systemically induced 10 inflammation were not observed, nor was there any evidence of the diapedesis of leukocytes and erythrocytes.

11 364 Szalay and Pappas Investigative Ophthalmology May 1970 Jfig. iu. Wound area ot a cornea burned with silver nitrate shows electron opaque particles (EO) 1 year later in the stroma, and in phagocytic vacuoles (PhV) of stromal cells (S). (C) = collagen. Unstained grid; x24,000.) In summary then, all the corneal vessels sampled in the wound and peripheral to it are mature with the characteristics of capillaries and venules and/or arterioles. 1 ' 3 They have a continuous endothelial sheet and in this respect resemble the capillaries of striated muscle, smooth muscle, subcutaneous tissue, and the limbus. It is of considerable interest to determine whether these structural similarities reflect similar functional (i.e., permeability) properties. At present, we may infer that they no longer possess the high permeability described for immature corneal vessels. The tendency of the mature corneal vessels peripheral to the wound to develop greatly thickened basement membranes may indicate special permeability properties of these vessels. Vessels with greatly thickened basement membranes also are found surrounding blood vessels in tissues of patients suffering from diabetes mellitus. 11 These tissues are notably prone to gangrene, and Banson 11 has speculated that the thickened basement membrane may hinder the inflammatory reaction by interfering with the exchange of nutrients and the migration of leukocytes. If this is correct, the mature vessels in healed areas of the cornea may also be less permeable and/or responsive to mediators of the inflammatory reaction than vessels in tissues such as skeletal muscle. In fact, we have data indicating a lack of response of these vessels to systemically induced histamine liberation. 12 ' 13 Vessels containing greatly thickened basement membranes in healed areas of the cornea may represent the ultimate development of mature vessels in a tissue with the properties of the cornea. It has been suggested by Ma]no 3 that capillaries with the thickest basement membranes are in organs where the blood pressure is

12 Volume 9 Number 5 Structure of rat corneal vessels in wound healing 365 high. Vessels with thickened basement membranes have also been reported in association with hypertension 14 ; it is not clear whether the thickened basement membranes of the arterioles are the cause or the effect of high blood pressure. Thickening of the basement membrane also occurs in aging 15 and in various pathologic conditions, 11 ' 14> 1G including diabetes mellitus. In addition to the above considerations, we would like to suggest that vessels with thickened basement membranes in the cornea may result from vessel regression, since we have found that the thickest basement membranes are associated with vessels having small lumens and few pericytes. Perhaps, as the lumen of the vessel gradually narrows, the endothelial cells continually produce more basement membrane and the pericyte processes recede. In this event, the numerous dense amorphous patches of material seen in once heavily vascular but now avascular regions of the wound area in "healed" corneas (Fig. 4) could be remnants of basement membrane left behind after withdrawal of the cellular components of the vessel wall. We wish to thank Miss Michele Fouchard for her excellent technical assistance, and Mr. Martin Besen for the publication prints. REFERENCES 1. Schoefl, G. I.: Studies on inflammation. III. Growing capillaries: Their structure and permeability, Virchow. Arch. Path. Anat. 337: 97, Cliff, W. J.: Observations on healing tissue: A combined light and electron microscopic investigation, Trans. Roy. Soc, Series B. 246: 305, Majno, G.: Ultrastructure of the vascular membrane, in Fenn, W. O., and Rahn, H.: Handbook of physiology, Circulation III, Baltimore, 1965, The Williams & Wilkins Company, p Rhodin, J. A. G.: Fine structure of vascular walls in mammals, Physiol. Rev. 42: 48, Collin, H. B.: Endothelial cell lined lymphatics in the vascularized rabbit cornea, INVEST. OPHTHAL. 5: 337, Jakus, M. A.: Retina Foundation, monographs and conferences; Boston, 1964, Little, Brown & Company, vol. I. 7. Jakus, M. A.: Further observations on the fine structure of the cornea, INVEST. OPHTHAL. 1: 202, Morehead, R. P.: Human pathology, New York, 1965, McGraw-Hill Book Company, Inc. 9. Majno, G., and Palade, G. E.: Studies on inflammation I. The effect of histamine and serotonin on vascular permeability: An electron microscopic study, J. Biophys. Biochem. Cytol. 11: 571, Pappas, G. D., and Tennyson, V. M.: An electron microscopic study of the passage of colloidal particles from the blood vessels of the ciliary processes and choroid plexus of the rabbit, J. Cell Biol. 15: 227, Banson, B. B.: Diabetic microangiopathy in human toes, with emphasis on the ultrastructural change in dermal capillaries, Amer. J. Path. 45: 41, Szalay, J., and Pappas, G.: Ultrastructure and permeability of mature blood vessels in rat cornea, Anat. Rec. 163: 272, Szalay, J., and Pappas, G. D.: Fine structure of rat corneal vessels in advanced stages of wound healing. I. Permeability to intravenously injected thorium dioxide. In preparation. 14. Ashworth, C. T., and Grollman, A.: Electron microscopy in experimental hypertension, Arch. Path. 68: 148, Ashworth, C. T., Erdmann, R. R., and Arnold, N. J.: Age changes in the renal basement membrane in rats, Amer. J. Path. 36: 165, McGee, W. G., and Ashworth, C. T.: Fine structure of chronic hypertensive arteriopathy in human kidney, Amer. J. Path, 43: 273, 1963.