An Evaluation of the Role of Leukocytes in the Pathogenesis of Experimentally Induced Corneal Vascularization I. Comparison of Experimental Models of Corneal Vascularization Carl H. Fromer, BS and Gordon K. Klintworth, MD, PhD Studies on corneal explants in the hamster cheek pouch chamber have demonstrated that blood vessels invade the cornea only if the tissue is first infiltrated by leukocytes. In view of this observation, a comparative study of the events that precede and accompany corneal vascularization was undertaken in various experimental models. A variety of established methods were used to induce corneal vascularization, including exposure of the cornea to noxious agents, intracorneal injection of antigens into sensitized animals, as well as maintaining animals on diets deficient in vitamin A or riboflavin. In all models studied, the corneal vascularization was a manifestation of the reparative phase of the inflammatory response. A conspicuous leukocytic infiltrate of the cornea preceded and accompanied the corneal vascularization in all of the models. Although the lesions varied in several respects in the different models, all models displayed three phases with regard to vascularization: an early prevascular phase of leukocytic infiltration, a second phase where both leukocytes and blood vessels were in the cornea, and a third phase where blood vessels persisted in the cornea in the absence of leukocytes. The latent period that preceded vascularization was directly related to the time of the initial leukocytic infiltration. The models in which a delay occurred in the leukocytic invasion displayed a subsequent delay in the vascular ingrowth. Conversely, in experiments where there was a rapid and extensive leukocytic invasion, there was also an early and enhanced corneal vasoproliferative response. In the various models investigated, the sites of the leukocytic infiltration and subsequent vascular ingrowth into the cornea paralleled each other. The data further support the hypotheses that leukocytes are a prerequisite to corneal vascularization and that leukocytes produce one or more factors which stimulate directional vascular growth. (Am J Pathol 79:537-554, 1975)
BLOOD VESSELS invade the normallv avascular cornea in many pathologic states, including chemical and traumatic injuries and inFrom the Department of Pathology, Duke University Medical Center, Durham, NC 27710. Supported in part by Grants EY-00146-03, EY-CA-00881-03, and 2-TO1-GM-00726-13 from the US Public Health Service; Dr. Klintworth is the recipient of Research Career Development Award EY-44795-04 from the National Eve Institute and an R. P. B. Louis B. MIayer Scholarship; Mr. Fromer is in the Medical Scientist Training Program at Duke University. Presented in part at the Seventv-first Annual Meeting of the American Association of Pathologists and Bacteriologists, San Francisco, Calif, 'March 1974. Accepted for publication Januarv 25, 1975. Address reprint requests to Dr. G. K. Klintworth, Department of Pathology, Box 3712, Duke University Miedical Center, Durham, NC 27710. 537
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fections. The pathogenesis of corneal vascularization has remained uncertain despite the fact that numerous investigators have studied the phenomenon experimentally.1-32 In a recent study on corneal vascularization in the hamster cheek pouch chamber, it was observed that corneal tissue became vascularized provided that it first provoked a leukocytic infiltration into itself.20 If cells did not penetrate the corneal tissue under investigation, it consistently remained avascular even if in a markedly edematous state. The data suggested that leukocytes might induce directional vascular growth into the cornea. In order to learn more about the generality of this observation, other experimental models of corneal vascularization were studied. This paper reviews these observations. Materials and Methods Corneal vascularization was provoked in New Zealand albino rabbits (2 to 3 kg) and Brown-Norwegian rats (100 to 125 g) of either sex according to established technics. The rabbits were anesthetized with intravenously administered sodium pentobarbital (1 ml injected at a concentration of 1 g/ml) or else Ophthaine (Squibb, New York, NY) was applied topically to the eye. Ether was used to anesthetize the rats. At regular intervals the eyes were observed with a dissecting microscope, and sequential photographs were taken. The animals were sacrificed at 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 days after injury. The eyes were processed for light microscopic analysis of the sequence of events that preceded and accompanied corneal vascularization. Light microscopic quantitation of cell populations was performed by counting cells in 8-I -thick tissue sections per high-power field with a Zeiss binocular microscope (40 x objective, 15 x ocular). The following experimental models of corneal vascularization were studied. Silver Nitrate Cauterization
To produce corneal lesions, a silver nitrate applicator tip (Graham-Field Surgical Company, New Hyde Park, NY) was applied to the corneas of rabbits approximately 2 to 3 mm from the superior region of the limbus for 3 seconds. In rats the lesion was produced in the center of the cornea. Topical Administration of Alloxan Approximately 4 ml of a freshly prepared aqueous solution of alloxan (2.4 g/ 100 ml, pH 4.5, J. T. Baker Chemical Co, Phillipsburg, NJ) was poured into a leucite eye cup which was held on the exposed surface of the cornea. The solution was allowed to remain in contact with the cornea for 30 minutes, with the contents being changed at 5-minute intervals. Alkali Injury A cotton pledget saturated with 0.5 N NaOH was
applied to the cornea of rabbits
for 30 seconds. Intracorneal Injection of Antigens
Rabbits were sensitized to bovine serum albumin (BSA, Miles Laboratories, Kanakee, Ill) or keyhole limpet hemocyanin (KLH, Calbiochem, San Diego, Calif).
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These antigens were incorporated into Freund's complete adjuvant (Difco Laboratories, Detroit, Mich) at a concentration of 20 mg of antigen/'ml. Two milliliters of this preparation were injected weekly into the muscles of the scapula region for up to 2 months. Sensitization was confirmed by finding a 2-mm or greater area of erythema after the intradermal injection of the same antigen. Sensitized and nonsensitized animals were injected intracomeally with 2 mg of antigen in a volume of 0.1 ml sterilized normal saline. In these experiments the corneas were studied in the same manner as in the other models. In addition, immunofluorescent microscopy was performed by the method of Coons and Kaplan33 to determine the localization of immunoglobulins (IgG) or complement (C3). In control studies to determine the effect of antigen-antibody complexes on nonsensitized rabbit comeas, equivalent volumes of BSA were injected into comeas. Antigen-antibody complexes were prepared by suspending the antigen in sterile normal saline, then serially diluting it and mixing it with serum that contained antibody. The maximum antigen-antibody precipitate was prepared immediately pnor to injection and was washed three times with 0.15 saline at less than 1 C and then injected into the cornea in a volume of 0.1 ml saline. Vitamin Deficiencies
Brown-Norwegian rats (100 g) were placed on a riboflavin- (30 rats) or vitaminA-deficient diet (15 rats) (Nutritional Biochemicals, Cleveland, Ohio). Control rats were maintained on a diet of Purina Lab Chow (Ralston Purina Co, St. Louis, Mo). Animals were separately housed in wire-bottomed cages to minimize the risk of injury. These rats were killed biweekly until the termination of the experiment at 24 weeks.
Results Silver Nitrate Cauterization
Initially the site of cauterization was white in the rabbit, but within 24 hours of cauterizing the corneas with silver nitrate a dark brown circular opacity (2 to 3 mm in diameter) appeared at the site of injury. By 2 days most of the corneas had become opaque around the area of discoloration, and the major limbal vessels were hyperemic. At Day 4 a conspicuous vascular invasion of the peripheral cornea was evident. The new vessels entered the cornea from the corneoscleral limbus nearest to the injured site and gradually extended towards the region of cauterization. Bv Day 9 the vessels usually reached the discolored area and invaded it (Figure 1). This vasoproliferative phase was followed by a period of approximately 1 to 13i months during which time the vessels persisted. Over a 6-month period of observation the vessels progressively diminished in caliber but never completely disappeared. Twenty,-four hours after corneal cauterization, light microscopy disclosed a) a denuded corneal epithelium overlving the site of cauterization, which was manifested by numerous pigmented granules extending throughout the entire thickness of the cornea and b) polymorphonuclear
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leukocytes in the corneal stroma, which included the discolored site where the silver nitrate was applied. Extravascular mononuclear cells (4 to 5 per high-power field) and a few erythrocytes were evident at the corneoscleral limbus at Days 2 and 3. By Day 3, capillaries containing erythrocytes could be seen extending into the corneal stroma from the limbus. Petechiae were occasionally discerned around the tips of these channels. With time the growth of vessels continued into the anterior segment of the cornea, and by Day 9 the vascular channels reached and penetrated the area of injury. By 1 month the vessels were still present in the corneal stroma, although leukocytes were now virtually absent. In the rat, by Day 1 corneal opacification and limbal congestion were pronounced. At Day 3 a centripetal growth of vessels extending into the cornea from the entire limbus was evident and was sufficiently pronounced to be seen with the naked eye. By light microscopy these vessels extended through the entire thickness of the corneal stroma, as did leukocytes. This vascular invasion ultimately reached the site of injury by 5 to 6 days. Microscopically the corneal lesion in the rat resembled that observed in the rabbit, with numerous leukocytes extending throughout the cornea at Day 1. By Day 2 microscopic vessels were seen extending into the peripheral corneal stroma. At about Day 6 a radiating system of freely communicating vessels permeated the entire cornea between the limbus and the site of injury. At 2 weeks leukocytes were no longer evident. Vascular channels were still appreciable at 1 month when the observations were discontinued. Topical Application of Alloxan
Within 24 hours of the topical application of alloxan the entire cornea appeared opaque. This was accompanied by limbal congestion. In this model corneal vascularization was only evident on gross examination after a time lag of 6 to 9 days. Vascular growth was circumferential in nature and progressed for 3 weeks (Figure 2). By 1 month the cornea had partially regained its transparency, but several vessels could still be appreciated in it. On microscopic examination, the corneal stroma was almost totally acellular within 24 hours due to the loss of fibroblasts within its matrix. Polymorphonuclear leukocytes (30 to 35 per high-power field) were seen in the peripheral corneal stroma within 72 hours of the application of alloxan. The vascular ingrowth into the corneal stroma was first observed at 5 days. By 2 weeks, the leukocytes were detected throughout the depth of the cornea at a maximum concentration of 160 to 180
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per high-power field. Vessels invaded the periphery of the entire cornea and were noted in the deeper segments of the cornea at 2 weeks. By 1 month, the leukocytic infiltration of the cornea disappeared, but the vascularization persisted. Alkali Injury
In the rabbit, sodium hydroxide produced a pronounced opacity in the area of alkali application within 24 hours. The limbal blood vessels, particularly those closest to the site of corneal injury, became engorged prior to corneal vascularization. By Day 5, capillaries began to extend into the cornea towards the lesion in a brush-like fashion from the limbus closest to the site of injury. These vessels eventually extended across the margin of the lesion and into it (Figure 3). By the second week, alkali-injured corneas frequently exhibited central or paracentral ulcers which often progressed to perforation. Less than 24 hours after exposure of the eye to sodium hydroxide, microscopic examination revealed a denuded corneal epithelium, faintly staining or unstainable corneal fibroblasts, and polymorphonuclear leukocytes (30 to 50 per high-power field) primarily in the anterior corneal stroma. Reepithelialition and vascular ingrowth was observed by 4 days. At this time, in the areas of corneal vascularization, many stromal fibroblasts could be appreciated, generally parallel to the vascular ingrowth. Light microscopy disclosed the vascularization to be in the anterior half of the comeal stroma. Unlike leukocytes, fibroblasts were rarely observed central to the advancing neovascularization. With time, the leukocyte population diminished, and by 3 weeks and thereafter vascular channels persisted in the corneal stroma unaccompanied by leukocytes. Intracomeal Injection Injection of Antigens or Antigen-Antibody Complexes Into Sensitized Rabbits
Within 24 hours of injection of BSA or KiLH into the center of sensitized rabbits, an opaque ring developed in the periphery of the cornea ("immune ring"). When the antigens were instilled eccentrically into the superior portion of the cornea, the ring extended into the corneoscleral limbus where focal hemorrhage and hyperemia formed a conspicuous arc in that portion of the eye superior to the arc. The ring corresponded to the line of antigen-antibody precipitation first reported by Wesseley.34 When the complete immune ring was located in the cornea, blood vessels entered the cornea in 2 to 3 days from the entire corneoscleral limbus and grew towards the precipitate. However, when
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the antigen-antibody precipitate formed an arc across the cornea, the blood vessels only invaded the cornea from the limbus into the inferior portion of the cornea (Figure 4). The vessels grew toward the opaque arc. By Day 6 the immune precipitate could no longer be appreciated, and the dense ingrowth of blood vessels (which were too numerous to count) reached the site of the previously identifiable ring. Later (8 days) the blood vessels entered that portion of the cornea internal to the precipitate and extended an additional 1 to 2 mm toward the center of the cornea during the second week (Figure 5). Thereafter, the vessels became less evident to the naked eye, and by 1 month the cornea had regained much of its clarity and only a few fine vessels were still observable by gross examination (Figure 6). At the time that the immune ring was present, immunofluorescent microscopy disclosed the presence of immunoglobulin (IgG) and complement (C3) as heavy granular precipitates deposited in a linear fashion throughout the cornea at the site of the ring. Neither immunoglobulin (IgG) nor complement could be detected in the regions central to the immune ring. In hematoxylin- and eosin-stained tissue sections the immune ring appeared as a sharply demarcated eosinophilic line traversing the cornea from the epithelium to the endothelium. A dense accumulation of polymorphonuclear leukocytes extended throughout the thickness of the ring (Figure 12). Numerous leukocytes were present between the limbus and the antigen-antibody ring and abutted against the band on its limbal side. Many polymorphonuclear leukocytes and lymphocytes preceded and accompanied the proliferating vessels into the cornea. The leukocytic and vascular invasion was more extensive in this model than in any of the other models investigated. Light microscopy disclosed that leukocytes and blood vessels frequently extended throughout the depth of the cornea (Figure 11). By 3 weeks the leukocytes could no longer be appreciated by light microscopy. At 1 month the corneal stroma still exhibited vascular channels but otherwise appeared normal. Injection of Antigens Into Nonsensitized Animals
In control experiments in which BSA or KLH was inoculated intracorneally into nonsensitized rabbits in amounts equivalent to what was injected into corneas of sensitized rabbits, neither leukocytes nor blood vessels invaded the cornea up to 14 days, after which time observations ceased. Injection of Antigen-Antibody Complexes Into Nonsensitized Animals
Injection of protein in the form of antigen-antibody complexes pro-
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duced a slight vascular ingrowth from the limbus which was grossly first observed at 9 days. Histologic examination revealed a mixed population of leukocytes extending into the central corneal stroma and into the injection site. The latter appeared as an eosinophilic deposit which was infiltrated by leukocytes. Vitamin Deficiencies Riboflavin Deficiency
Rats on a riboflavin-deficient diet failed to gain weight and developed an arched back, unkempt appearance, alopecia, greasy hair, and hyperactivity by the seventh week. The corneas of these rodents appeared unremarkable by gross and microscopic examination until they had been on the riboflavin-deficient diet for 12 weeks. Thereafter, a slight polymorphonuclear leukocytic infiltrate was observed beneath the corneal epithelium, with no appreciable changes in the other parts of the cornea. By 14 weeks leukocytes (15 to 20 per high-power field) extended into the deeper corneal stroma. Capillaries were identified for the first time in the superficial corneal stroma at the sixteenth week. The vascular ingrowth was appreciated in all of the animals which were studied after this time. In these riboflavin-deficient rats, corneal vascularization only occurred in severely debilitated animals following the leukocytic infiltration into the cornea. The leukocytes gradually diminished in number, and by 24 weeks they were no longer observed, but the blood vessels persisted. Vitamin A Deficiency
Rats placed on a vitamin-A-deficient diet failed to grow or thrive, unlike the controls. After becoming very emaciated, the animals died by the twelfth week. No corneal changes were observed grossly with the naked eye or with the dissecting microscope. Beginning at 4 weeks, microscopic examination revealed a substitution of the normal epithelium by a keratinized squamous epithelium. Neither leukocytes nor blood vessels were observed in any of the corneas of rats fed the vitaminA-deficient diet. Discussion
As in the previous studies in the hamster cheek pouch, the corneal vascularization in the present model was invariably a manifestation of the inflammatory response. All of the models manifested three distinct phases with respect to the presence of leukocytes and blood vessels within the corneal stroma. Leukocytes initially infiltrated the avascular
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cornea from the corneoscleral limbus (prevascular phase, Figure 7). Subsequently, capillaries invaded the tissue, and both leukocytes and vascular proliferation coexisted in the cornea simultaneously (vasoproliferative phase, Figure 8). Later, leukocytes disappeared from the corneal stroma, while blood vessels remained (established phase, Figure 9). The different models varied with respect to the localization, extent, depth of stromal involvement, and the latent period between the time of corneal injury and the onset of vascularization (Table 1). The localization, the depths of stromal involvement, and the direction of the vascular invasion from the limbus in the different models paralleled the pattern of the leukocytic infiltration which these lesions induced. Focal corneal injuries which provoked a localized leukocytic infiltration into the corneal stroma produced a localized vascular ingrowth into the damaged area from the limbus closest to the site of the lesion. On the other hand, models which caused a diffuse, circumferential leukocytic infiltration into the cornea stimulated a vascular invasion into the periphery of the entire cornea. When leukocytes infiltrated the entire thickness of the corneal stroma (as when antigen was injected into corneas of sensitized animals), so did the vascular ingrowth (Figure 11). In some models, leukocytes and blood vessels infiltrated primarily the superficial corneal stroma (Figure 10). The onset of corneal vascularization was also temporally related to the preceding leukocytic infiltration. For instance, injection of antigen into sensitized animals produced an early leukocytic infiltration and corneal vascularization (Table 1). This was in sharp contrast to the alloxan model in which a longer latent period anteceded both the leukocytic and vascular invasion. Other investigators have studied corneal vascularization in the models we investigated from different standpoints. Reports on alloxan-induced corneal vascularization have stressed the hydration, metabolic, and permeability changes preceding the growth of new vessels 3,21 or the effect of cortisone and other drugs in inhibiting the vascularization produced by this chemical.1'2 With regard to rats on a riboflavindeficient diet, our observations are at a variance in one respect with those reported by Bessey and Wolbach in 1939.4 These investigators reported leukocytes in the cornea within a week or two after the vessels had penetrated the tissue but did not discuss histologic findings prior to that time. In the present investigation a leukocytic invasion clearly anteceded the vascular ingrowth. In their study as well as in ours, the vascular invasion occurred in severely debilitated animals. Although none of our rats developed corneal vascularization while on a vitamin-
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BLOOD VESSELS invade the normallv avascular cornea in many pathologic states, including chemical and traumatic injuries and inFrom the Department of Pathology, Duke University Medical Center, Durham, NC 27710. Supported in part by Grants EY-00146-03, EY-CA-00881-03, and 2-TO1-GM-00726-13 from the US Public Health Service; Dr. Klintworth is the recipient of Research Career Development Award EY-44795-04 from the National Eve Institute and an R. P. B. Louis B. MIayer Scholarship; Mr. Fromer is in the Medical Scientist Training Program at Duke University. Presented in part at the Seventv-first Annual Meeting of the American Association of Pathologists and Bacteriologists, San Francisco, Calif, 'March 1974. Accepted for publication Januarv 25, 1975. Address reprint requests to Dr. G. K. Klintworth, Department of Pathology, Box 3712, Duke University Miedical Center, Durham, NC 27710. 537
538
FROMER AND KLINTWORTH
American Journal of Pathology
fections. The pathogenesis of corneal vascularization has remained uncertain despite the fact that numerous investigators have studied the phenomenon experimentally.1-32 In a recent study on corneal vascularization in the hamster cheek pouch chamber, it was observed that corneal tissue became vascularized provided that it first provoked a leukocytic infiltration into itself.20 If cells did not penetrate the corneal tissue under investigation, it consistently remained avascular even if in a markedly edematous state. The data suggested that leukocytes might induce directional vascular growth into the cornea. In order to learn more about the generality of this observation, other experimental models of corneal vascularization were studied. This paper reviews these observations. Materials and Methods Corneal vascularization was provoked in New Zealand albino rabbits (2 to 3 kg) and Brown-Norwegian rats (100 to 125 g) of either sex according to established technics. The rabbits were anesthetized with intravenously administered sodium pentobarbital (1 ml injected at a concentration of 1 g/ml) or else Ophthaine (Squibb, New York, NY) was applied topically to the eye. Ether was used to anesthetize the rats. At regular intervals the eyes were observed with a dissecting microscope, and sequential photographs were taken. The animals were sacrificed at 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 days after injury. The eyes were processed for light microscopic analysis of the sequence of events that preceded and accompanied corneal vascularization. Light microscopic quantitation of cell populations was performed by counting cells in 8-I -thick tissue sections per high-power field with a Zeiss binocular microscope (40 x objective, 15 x ocular). The following experimental models of corneal vascularization were studied. Silver Nitrate Cauterization
To produce corneal lesions, a silver nitrate applicator tip (Graham-Field Surgical Company, New Hyde Park, NY) was applied to the corneas of rabbits approximately 2 to 3 mm from the superior region of the limbus for 3 seconds. In rats the lesion was produced in the center of the cornea. Topical Administration of Alloxan Approximately 4 ml of a freshly prepared aqueous solution of alloxan (2.4 g/ 100 ml, pH 4.5, J. T. Baker Chemical Co, Phillipsburg, NJ) was poured into a leucite eye cup which was held on the exposed surface of the cornea. The solution was allowed to remain in contact with the cornea for 30 minutes, with the contents being changed at 5-minute intervals. Alkali Injury A cotton pledget saturated with 0.5 N NaOH was
applied to the cornea of rabbits
for 30 seconds. Intracorneal Injection of Antigens
Rabbits were sensitized to bovine serum albumin (BSA, Miles Laboratories, Kanakee, Ill) or keyhole limpet hemocyanin (KLH, Calbiochem, San Diego, Calif).
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These antigens were incorporated into Freund's complete adjuvant (Difco Laboratories, Detroit, Mich) at a concentration of 20 mg of antigen/'ml. Two milliliters of this preparation were injected weekly into the muscles of the scapula region for up to 2 months. Sensitization was confirmed by finding a 2-mm or greater area of erythema after the intradermal injection of the same antigen. Sensitized and nonsensitized animals were injected intracomeally with 2 mg of antigen in a volume of 0.1 ml sterilized normal saline. In these experiments the corneas were studied in the same manner as in the other models. In addition, immunofluorescent microscopy was performed by the method of Coons and Kaplan33 to determine the localization of immunoglobulins (IgG) or complement (C3). In control studies to determine the effect of antigen-antibody complexes on nonsensitized rabbit comeas, equivalent volumes of BSA were injected into comeas. Antigen-antibody complexes were prepared by suspending the antigen in sterile normal saline, then serially diluting it and mixing it with serum that contained antibody. The maximum antigen-antibody precipitate was prepared immediately pnor to injection and was washed three times with 0.15 saline at less than 1 C and then injected into the cornea in a volume of 0.1 ml saline. Vitamin Deficiencies
Brown-Norwegian rats (100 g) were placed on a riboflavin- (30 rats) or vitaminA-deficient diet (15 rats) (Nutritional Biochemicals, Cleveland, Ohio). Control rats were maintained on a diet of Purina Lab Chow (Ralston Purina Co, St. Louis, Mo). Animals were separately housed in wire-bottomed cages to minimize the risk of injury. These rats were killed biweekly until the termination of the experiment at 24 weeks.
Results Silver Nitrate Cauterization
Initially the site of cauterization was white in the rabbit, but within 24 hours of cauterizing the corneas with silver nitrate a dark brown circular opacity (2 to 3 mm in diameter) appeared at the site of injury. By 2 days most of the corneas had become opaque around the area of discoloration, and the major limbal vessels were hyperemic. At Day 4 a conspicuous vascular invasion of the peripheral cornea was evident. The new vessels entered the cornea from the corneoscleral limbus nearest to the injured site and gradually extended towards the region of cauterization. Bv Day 9 the vessels usually reached the discolored area and invaded it (Figure 1). This vasoproliferative phase was followed by a period of approximately 1 to 13i months during which time the vessels persisted. Over a 6-month period of observation the vessels progressively diminished in caliber but never completely disappeared. Twenty,-four hours after corneal cauterization, light microscopy disclosed a) a denuded corneal epithelium overlving the site of cauterization, which was manifested by numerous pigmented granules extending throughout the entire thickness of the cornea and b) polymorphonuclear
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leukocytes in the corneal stroma, which included the discolored site where the silver nitrate was applied. Extravascular mononuclear cells (4 to 5 per high-power field) and a few erythrocytes were evident at the corneoscleral limbus at Days 2 and 3. By Day 3, capillaries containing erythrocytes could be seen extending into the corneal stroma from the limbus. Petechiae were occasionally discerned around the tips of these channels. With time the growth of vessels continued into the anterior segment of the cornea, and by Day 9 the vascular channels reached and penetrated the area of injury. By 1 month the vessels were still present in the corneal stroma, although leukocytes were now virtually absent. In the rat, by Day 1 corneal opacification and limbal congestion were pronounced. At Day 3 a centripetal growth of vessels extending into the cornea from the entire limbus was evident and was sufficiently pronounced to be seen with the naked eye. By light microscopy these vessels extended through the entire thickness of the corneal stroma, as did leukocytes. This vascular invasion ultimately reached the site of injury by 5 to 6 days. Microscopically the corneal lesion in the rat resembled that observed in the rabbit, with numerous leukocytes extending throughout the cornea at Day 1. By Day 2 microscopic vessels were seen extending into the peripheral corneal stroma. At about Day 6 a radiating system of freely communicating vessels permeated the entire cornea between the limbus and the site of injury. At 2 weeks leukocytes were no longer evident. Vascular channels were still appreciable at 1 month when the observations were discontinued. Topical Application of Alloxan
Within 24 hours of the topical application of alloxan the entire cornea appeared opaque. This was accompanied by limbal congestion. In this model corneal vascularization was only evident on gross examination after a time lag of 6 to 9 days. Vascular growth was circumferential in nature and progressed for 3 weeks (Figure 2). By 1 month the cornea had partially regained its transparency, but several vessels could still be appreciated in it. On microscopic examination, the corneal stroma was almost totally acellular within 24 hours due to the loss of fibroblasts within its matrix. Polymorphonuclear leukocytes (30 to 35 per high-power field) were seen in the peripheral corneal stroma within 72 hours of the application of alloxan. The vascular ingrowth into the corneal stroma was first observed at 5 days. By 2 weeks, the leukocytes were detected throughout the depth of the cornea at a maximum concentration of 160 to 180
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per high-power field. Vessels invaded the periphery of the entire cornea and were noted in the deeper segments of the cornea at 2 weeks. By 1 month, the leukocytic infiltration of the cornea disappeared, but the vascularization persisted. Alkali Injury
In the rabbit, sodium hydroxide produced a pronounced opacity in the area of alkali application within 24 hours. The limbal blood vessels, particularly those closest to the site of corneal injury, became engorged prior to corneal vascularization. By Day 5, capillaries began to extend into the cornea towards the lesion in a brush-like fashion from the limbus closest to the site of injury. These vessels eventually extended across the margin of the lesion and into it (Figure 3). By the second week, alkali-injured corneas frequently exhibited central or paracentral ulcers which often progressed to perforation. Less than 24 hours after exposure of the eye to sodium hydroxide, microscopic examination revealed a denuded corneal epithelium, faintly staining or unstainable corneal fibroblasts, and polymorphonuclear leukocytes (30 to 50 per high-power field) primarily in the anterior corneal stroma. Reepithelialition and vascular ingrowth was observed by 4 days. At this time, in the areas of corneal vascularization, many stromal fibroblasts could be appreciated, generally parallel to the vascular ingrowth. Light microscopy disclosed the vascularization to be in the anterior half of the comeal stroma. Unlike leukocytes, fibroblasts were rarely observed central to the advancing neovascularization. With time, the leukocyte population diminished, and by 3 weeks and thereafter vascular channels persisted in the corneal stroma unaccompanied by leukocytes. Intracomeal Injection Injection of Antigens or Antigen-Antibody Complexes Into Sensitized Rabbits
Within 24 hours of injection of BSA or KiLH into the center of sensitized rabbits, an opaque ring developed in the periphery of the cornea ("immune ring"). When the antigens were instilled eccentrically into the superior portion of the cornea, the ring extended into the corneoscleral limbus where focal hemorrhage and hyperemia formed a conspicuous arc in that portion of the eye superior to the arc. The ring corresponded to the line of antigen-antibody precipitation first reported by Wesseley.34 When the complete immune ring was located in the cornea, blood vessels entered the cornea in 2 to 3 days from the entire corneoscleral limbus and grew towards the precipitate. However, when
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FROMER AND KLINTWORTH
American Journal of Pathology
the antigen-antibody precipitate formed an arc across the cornea, the blood vessels only invaded the cornea from the limbus into the inferior portion of the cornea (Figure 4). The vessels grew toward the opaque arc. By Day 6 the immune precipitate could no longer be appreciated, and the dense ingrowth of blood vessels (which were too numerous to count) reached the site of the previously identifiable ring. Later (8 days) the blood vessels entered that portion of the cornea internal to the precipitate and extended an additional 1 to 2 mm toward the center of the cornea during the second week (Figure 5). Thereafter, the vessels became less evident to the naked eye, and by 1 month the cornea had regained much of its clarity and only a few fine vessels were still observable by gross examination (Figure 6). At the time that the immune ring was present, immunofluorescent microscopy disclosed the presence of immunoglobulin (IgG) and complement (C3) as heavy granular precipitates deposited in a linear fashion throughout the cornea at the site of the ring. Neither immunoglobulin (IgG) nor complement could be detected in the regions central to the immune ring. In hematoxylin- and eosin-stained tissue sections the immune ring appeared as a sharply demarcated eosinophilic line traversing the cornea from the epithelium to the endothelium. A dense accumulation of polymorphonuclear leukocytes extended throughout the thickness of the ring (Figure 12). Numerous leukocytes were present between the limbus and the antigen-antibody ring and abutted against the band on its limbal side. Many polymorphonuclear leukocytes and lymphocytes preceded and accompanied the proliferating vessels into the cornea. The leukocytic and vascular invasion was more extensive in this model than in any of the other models investigated. Light microscopy disclosed that leukocytes and blood vessels frequently extended throughout the depth of the cornea (Figure 11). By 3 weeks the leukocytes could no longer be appreciated by light microscopy. At 1 month the corneal stroma still exhibited vascular channels but otherwise appeared normal. Injection of Antigens Into Nonsensitized Animals
In control experiments in which BSA or KLH was inoculated intracorneally into nonsensitized rabbits in amounts equivalent to what was injected into corneas of sensitized rabbits, neither leukocytes nor blood vessels invaded the cornea up to 14 days, after which time observations ceased. Injection of Antigen-Antibody Complexes Into Nonsensitized Animals
Injection of protein in the form of antigen-antibody complexes pro-
Vol. 79, No. 3 June 1975
CORNEAL VASCULARIZATION
543
duced a slight vascular ingrowth from the limbus which was grossly first observed at 9 days. Histologic examination revealed a mixed population of leukocytes extending into the central corneal stroma and into the injection site. The latter appeared as an eosinophilic deposit which was infiltrated by leukocytes. Vitamin Deficiencies Riboflavin Deficiency
Rats on a riboflavin-deficient diet failed to gain weight and developed an arched back, unkempt appearance, alopecia, greasy hair, and hyperactivity by the seventh week. The corneas of these rodents appeared unremarkable by gross and microscopic examination until they had been on the riboflavin-deficient diet for 12 weeks. Thereafter, a slight polymorphonuclear leukocytic infiltrate was observed beneath the corneal epithelium, with no appreciable changes in the other parts of the cornea. By 14 weeks leukocytes (15 to 20 per high-power field) extended into the deeper corneal stroma. Capillaries were identified for the first time in the superficial corneal stroma at the sixteenth week. The vascular ingrowth was appreciated in all of the animals which were studied after this time. In these riboflavin-deficient rats, corneal vascularization only occurred in severely debilitated animals following the leukocytic infiltration into the cornea. The leukocytes gradually diminished in number, and by 24 weeks they were no longer observed, but the blood vessels persisted. Vitamin A Deficiency
Rats placed on a vitamin-A-deficient diet failed to grow or thrive, unlike the controls. After becoming very emaciated, the animals died by the twelfth week. No corneal changes were observed grossly with the naked eye or with the dissecting microscope. Beginning at 4 weeks, microscopic examination revealed a substitution of the normal epithelium by a keratinized squamous epithelium. Neither leukocytes nor blood vessels were observed in any of the corneas of rats fed the vitaminA-deficient diet. Discussion
As in the previous studies in the hamster cheek pouch, the corneal vascularization in the present model was invariably a manifestation of the inflammatory response. All of the models manifested three distinct phases with respect to the presence of leukocytes and blood vessels within the corneal stroma. Leukocytes initially infiltrated the avascular
544
FROMER AND KLINTWORTH
American Journal of Pathology
cornea from the corneoscleral limbus (prevascular phase, Figure 7). Subsequently, capillaries invaded the tissue, and both leukocytes and vascular proliferation coexisted in the cornea simultaneously (vasoproliferative phase, Figure 8). Later, leukocytes disappeared from the corneal stroma, while blood vessels remained (established phase, Figure 9). The different models varied with respect to the localization, extent, depth of stromal involvement, and the latent period between the time of corneal injury and the onset of vascularization (Table 1). The localization, the depths of stromal involvement, and the direction of the vascular invasion from the limbus in the different models paralleled the pattern of the leukocytic infiltration which these lesions induced. Focal corneal injuries which provoked a localized leukocytic infiltration into the corneal stroma produced a localized vascular ingrowth into the damaged area from the limbus closest to the site of the lesion. On the other hand, models which caused a diffuse, circumferential leukocytic infiltration into the cornea stimulated a vascular invasion into the periphery of the entire cornea. When leukocytes infiltrated the entire thickness of the corneal stroma (as when antigen was injected into corneas of sensitized animals), so did the vascular ingrowth (Figure 11). In some models, leukocytes and blood vessels infiltrated primarily the superficial corneal stroma (Figure 10). The onset of corneal vascularization was also temporally related to the preceding leukocytic infiltration. For instance, injection of antigen into sensitized animals produced an early leukocytic infiltration and corneal vascularization (Table 1). This was in sharp contrast to the alloxan model in which a longer latent period anteceded both the leukocytic and vascular invasion. Other investigators have studied corneal vascularization in the models we investigated from different standpoints. Reports on alloxan-induced corneal vascularization have stressed the hydration, metabolic, and permeability changes preceding the growth of new vessels 3,21 or the effect of cortisone and other drugs in inhibiting the vascularization produced by this chemical.1'2 With regard to rats on a riboflavindeficient diet, our observations are at a variance in one respect with those reported by Bessey and Wolbach in 1939.4 These investigators reported leukocytes in the cornea within a week or two after the vessels had penetrated the tissue but did not discuss histologic findings prior to that time. In the present investigation a leukocytic invasion clearly anteceded the vascular ingrowth. In their study as well as in ours, the vascular invasion occurred in severely debilitated animals. Although none of our rats developed corneal vascularization while on a vitamin-
Vol. 79, No. 3 June 1975
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CORNEAL VASCULARIZATION
545
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