laser photocoagulation for corneal stromal vascularization

LASER PHOTOCOAGULATION FORCORNEAL STROMAL VASCULARIZATION

BY Verinder S. Nirankari, MD

INTRODUCTION

CORNEAL STROMAL VASCULARIZATION IS A MAJOR RISK FACTOR FOR COR-neal graft rejection and can also cause edema, inflammation, scarring, andlipid keratopathy. This thesis evaluates the role of laser photocoagulationin treating corneal stromal vascularization. Current knowledge about thecauses and significance of corneal vascularization, its pathogenesis, andthe different types of treatment that have been used for it are reviewed. Arabbit model in which we have consistently been able to induce cornealstromal vascularization by injecting sodium hydroxide into the stroma isdescribed. The effects of laser photocoagulation in reducing corneal stro-mal vascularization in this model is elaborated. Histopathologic studiesare included. Our experience with the treatment of corneal stromalvascularization with laser photocoagulation in different categories of pa-tients is described. Finally, all the evidence is combined, and the role oflaser photocoagulation as an effective tool for the treatment of cornealstromal vascularization is discussed. Comparison is also made with theother methods that have been used for the treatment of corneal vascular-ization.

HISTORICAL REVIEW

As early as 1841 it was recognized by Toynbeel that the human cornea isnormally avascular but can become vascular in certain pathologic condi-tions. During the early 1900s, studies of human angiogenesis were doneusing the cornea,2-4 and corneal vascularization was studied in animals.5-9Detailed investigation of the pathogenesis of corneal vascularization be-gan around the mid 20th century with studies by Campbell and Michael-son10 and by Cogan. "

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CAUSES AND SIGNIFICANCE

Corneal vascularization is a "response to a call for help by a tissue indifficulty."12 In humans, this can occur in a variety of conditions, includ-ing corneal graft rejection, infections, contact lens wear, trauma, burns,vasculitides, metabolic disorders, toxins, and nutritional deficiency states.Although corneal vascularization represents a defense mechanism againstdisease or injury, its presence can lead to a decrease in vision due to theloss of corneal transparency.12 The presence of corneal vascularizationincreases the chance of corneal graft rejection.13-'5 Vascularized corneasmay also be more susceptible to lipid deposition resulting in cornealopacity.16 Loss of globlet cells and transdifferentiation of migrating con-junctival epithelium during healing of corneal epithelial defects may behindered ifthe cornea is vascularized. 17-20 This lack oftransdifferentiationcan lead to impaired barrier function of the corneal epithelium.21 In thisbackground, persistent corneal vascularization is an undesirable event.Many experimental models have been investigated in order to better

understand corneal vascularization. Growth ofblood vessels in the corneahas been induced by numerous agents in these experimental models. Thelist of these vascularization inducers is extensive and includes chemicalcompounds, deficiency states, immune reactions, intoxication, microor-ganisms and their products, physical injury, and the placement of cells ortissues in the cornea. Among the chemical compounds, experimentalcorneal vascularization can be induced by acids (including acetic,22 hyal-uronic,23 hydrochloric,"l 22 lactic,22 and pyruvic22 acids), adenosine di-phosphonate,24 25 alloxan,2640 biogenic amines,41 ceruloplasmin,4245chloroquine,46 colchicine,32,47,48 Disprin,49 enzymes (including collagen-ase,50 hyaluronidase,51 and plasminogen activator52), ethanol,5 fibrin,5growth factors, 24-4-66 heparin,24,43-45 iodinle,67 leukocyte attractants,24,49,68lymphokines,62,69-74 mustard gas,75 nicotinamide,76 nitrogen,27 peptidecomplexed with copper,44,45 phorbol esters,77 prostagIdins,42A4450,54,66,78,79saline,27,80 selenomethionine,225 serum,27,80,81 silica,82 silver nitrate,39.83-93sodium hydroxide,11"39'94-104 and water. 11 Deficiency states of amino acids(including histidine,105 lysine,106-110 methionine,109-111 and trypto-phanl06'09"112), protein,"13 total nutrition,110 vitamins (including ascorbicacid,'14 riboflavin,39,115-117 and vitamin A117,118), and zinc119,120 can in-duce blood vessel growth in the cornea under experimental conditions.Immune reaction to antigens,6-8,39,121-126 antibodies,127 and corneal graftrejection15,124,128 have also induced corneal vascularization experimen-tally. Intoxication with thallium'07 and tyrosinel29-131 has similarly in-duced blood vessel growth in the cornea. Microorganisms that can inducecorneal vascularization experimentally include Aspergillus fumigatus,132

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herpes simplex, I, 13, 14 Mycobacterium tuberculosis,35W37 and staphylo-coccus.'18 Products of microorganisms such as bacterial nucleoprotein7and endotoxinl39-141 can also induce blood vessel growth in the cornea.Physical injury with heat,10,o',s,88, radiation,'49"15 and trauma27'12" 151has induced corneal vascularization in some experimental models. Amongthe cells and tissues that can induce experimental blood vessel growthwhen placed in the cornea are leukocytes,69"152-156 macrophages,157-161neoplasms,42,44,61,82,162-173 platelets,53 and tissue from the brain,55174eye27,80,143,175176 kidney,177 lymph node,82"5 muscle,'76 and omen-tum. 178-180 Contact lens use, 181-185 corneal grafts,27 ischemia to the ante-rior segment of the eye,186-188 and suture placement in the cornea189-194are the other means that can induce blood vessel growth in the corneaunder experimental conditions. Some of the aforementioned corneal vas-cularization-inducing agents are associated with inflammation, and othersare not. A model of spontaneous corneal vascularization in mice has alsobeen reported.'85 Most animal models of corneal vascularization haveused rabbits, rats, or mice for the experiments.

In spite of the numerous agents that can induce corneal vascularization,it has been difficult to obtain consistent results in experimental models.Our model, described later, where we inject sodium hydroxide into thecorneal stroma of rabbits, has produced relatively consistent and stableblood vessel growth in the corneal stroma.

It has been suggested that the normal avascular nature of the corneaconfers a relative immune privilege from donor graft rejection.'86"97Avascular corneal grafts have an 85% to 95% chance of a successfuloutcome.198-200 The major factors that reduce the chance of a successfulcorneal graft are previous graft rejection and vascularization. 13-15,199,200Vascularization may reduce the rate of successful corneal grafts to as lowas 35%.199,200 During 1990, 40,631 corneal grafts were done in the UnitedStates.201 It is estimated that 10% to 20% of the corneal grafts fall into thehigh-risk category for failure,202 thereby making it a significant problem.The question of whether matching for HLA antigens and/or crossmatch-ing for lymphocytotoxic antibodies can reduce the incidence of cornealgraft rejection in high-risk patients is being investigated by the Collabora-tive Corneal Transplantation Studies.202'203 Another approach that mayreduce the risk of corneal graft failure in high-risk patients with vascular-ization prior to grafting, and in those with growth ofblood vessels into thegraft who have not responded to conventional means such as sutureremoval and maximal corticosteroid therapy, is to ablate the blood vesselswith laser photocoagulation. The rationale for this is that destruction ofthe blood vessels may restore the relative immune privilege of the cornea

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by interrupting both the afferent and efferent limbs of the immuneresponse that leads to graft rejection. Blood vessel growth into a previ-ously avascular corneal graft can lead to "donor recognition" and anincreased risk of graft rejection. This thesis addresses the question ofwhether corneal vascularization can be treated effectively with laserphotocoagulation.

PATHOGENESIS

The exact basis for the pathogenesis of corneal vascularization is notunderstood at present. Cogan" proposed in 1949 that swelling of thecorneal stroma causes reduction in the tissue compactness, which leads toingrowth of blood vessels. However, it was later argued that althoughedema ofthe corneal stroma does play a role, it does not elicit vasculariza-tion.12'204 Klintworth205 has recently proposed that the evidence so farsuggests that corneal vascularization is initiated and controlled by numer-ous cells and molecules involved in the inflammatory response. Evidencesuggesting a role for angiogenic factor(s) in corneal vascularization hasbeen gradually getting stronger.10.22.27,39,41,89,143,152,163,2062 Other hy-potheses for corneal vascularization that have been considered but haverelatively less support at present include destruction of antiangiogenicsubstance and hypoxia.204The cells involved in the inflammatory response that may play a role in

corneal vascularization include lymphocytes,-54153-156,209-213 polymorpho-nuclear leukocytes,50'89"188'207 macrophages,58,63,70157,158,161211,214 andmast cells.67'215 Among the molecules involved in the inflammatory re-sponse that may participate in corneal vascularization are prostaglan-dins 42,44,50,54,66,78,79lymphokines,62'69-74 biogenic amines,41 plasminogenactivator,52 and fibrin.53 The nature ofthe angiogenic factor(s) that may beinvolved in corneal vascularization has not been elucidated, but variouskinds of growth factors are possible candidates.216

According to Klintworth,217 the sequence of events in corneal vascular-ization involves a latency period after corneal injury, followed by dilata-tion and increased permeability of blood vessels, corneal edema, vascularendothelial cell retraction and decreased junctions, degradation of endo-thelial cell basal lamina, endothelial cell migration and replication, vascu-lar sprouting, lumen formation and anastomoses, basal lamina formationin new vessels, capillary regression, and vascular maturation.

Since the basic mechanisms involved in the pathogenesis of cornealvascularization are not well understood at present, it is not currentlypossible to prevent corneal graft rejection by treating the underlyingcause of vascularization. Therefore, when suture removal and maximal

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corticosteroid therapy have either failed to reverse vascularization in thecorneal graft or are contraindicated, destruction of blood vessels mayminimize the immune response leading to corneal graft rejection. De-struction of blood vessels in vascularized corneas being considered forreplacement with graft may also reduce the chances of graft rejection byminimizing the immune response. Laser photocoagulation of the bloodvessels may be one way to achieve this end, as evidence presented later inthis thesis illustrates.

TREATMENT

Many different types of treatment have been tried previously for cornealvascularization. None has proven to be consistently successful in theclinical setting. Effective treatment of corneal vascularization could de-crease the number of corneal graft rejections associated with growth ofblood vessels into the graft and the grafts done on previously vascularizedcorneas. Traditionally, sutures are removed if they are thought to beinciting blood vessel growth, and maximal corticosteroid therapy, includ-ing topical, subconjunctival, and even oral and intravenous, is given forvascularization into the corneal graft. Often, these conventional treat-ments are not fully effective.

CorticosteroidsFor about 40 years it has been known that topical corticosteroids suppresscorneal vascularization.26 In both the clinical and experimental settings,the effect of topical corticosteroids in suppressing corneal vascularizationhas been well documented, but this suppression is incomplete.26,4049,68,78,89,91,122,127,142, 146-148,151,190,207,218-220 Topical corticosteroids that have a cor-

neal vascularization suppression effect include cortisone,26,151 dexameth-asone, 68,78,91,127,190 methylprednisolone, 49,89 prednisolone, 40,78,91,127,146-148ticabesone propionate,91 and triamcinolone.127 The antiangiogenic effectof corticosteroids is not limited to the cornea; it has been shown in othertissues as well. 221222

Although the basis for the antiangiogenic effect of corticosteroids in thecornea is not fully clear, it is thought to be related to their anti-inflamma-tory effect.223 Heparin has been shown to enhance the effect of cortisonein suppressing corneal vascularization.78 151 This effect of heparin may berelated to its "carrier molecule" property, which may allow better accessof cortisone at the site(s) of its antiangiogenic effect.'40

Because of incomplete suppression of corneal vascularization by cor-ticosteroids, and also because of the side effects of local corticosteroids(including cataract, glaucoma, superinfection, herpes simplex recurrence)

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and those of systemic corticosteroids, more effective means of treatingcorneal vascularization are being sought.

Nonsteroidal Anti-Inflammatory DrugsThe nonsteroidal anti-inflammatory drugs (NSAIDs) that have been re-ported to suppress corneal vascularization include flurbiprofen,40,68,183,187'224indomethacin42,99,147,180,190,224,225 and ketorolac.91'224 The corneal anti-angiogenic effect of NSAIDs has been very variable, and therefore theyare not usually used clinically for this purpose.

Cyclosporin ACyclosporin A has been reported to promote corneal graft survival afterretrobulbar injection in rabbits with heavily vascularized corneas. 226 Topi-cal application of cyclosporin A solution to rabbit eyes with corneal graftsin which vascularization was induced has also been reported to prolonggraft survival.227'228 In a human study, topical application of 2% cyclo-sporin A in olive oil in 11 high-risk human corneal transplants resulted in10 clear grafts at a follow-up ranging between 6 and 24 months.229Significant circulating whole blood levels of cyclosporin A (14 to 64 ng/dlwere noted in this study after topical use.229 Systemic cyclosporin A canhave serious side effects, such as nephrotoxicity and increased incidenceof lymphoma.230'231 Because of the potential serious toxicity of cyclo-sporin A, it is not being routinely used at present for treatment of cornealvascularization to promote graft survival.

IrradiationPreviously, total-body irradiation has been shown to reduce corneal vas-cularization under various experimental conditions.89,172'232'233 Recently,however, total-body irradiation of Fischer 344 rats after silver-potassiumnitrate cautery of the cornea did not show a decrease in vascularization 2,3, or 4 days postcautery as compared with controls.23 Thiotepa and betaradiation have been reported to be somewhat successful in reducingcorneal vascularization in rabbits.23'5236

Clinically, partial success has been reported in the treatment of cornealvascularization with beta radiation.237 This has not been accepted as aroutine means of treating corneal vascularization, because the success islimited and radiation can cause serious complications.

Cryotherapy, Heat Cautery, Scar Tissue Barrier, Excision of VesselsThe other methods reported to have a short-lived success in treatingcorneal vascularization under experimental conditions include cryother-apy, heat cautery, creation of scar tissue barrier to the growth of blood

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vessels, and surgical excision of blood vessels at the limbus.236 Thesemethods are not used clinically because their effect is variable and tempo-rary. One clinical study238 did not find any beneficial effect ofcryotherapyin treating corneal vascularization.

Laser PhotocoagulationIn 1973 Cherry and associates239 reported the results of argon lasertreatment of corneal vascularization in four patients. Two of these patientshad chemical burns of the cornea, and the other two had herpes simplexkeratitis. In 1976 Cherry and Garner240 referred to these previous resultsof argon laser treatment as two failures, one success, and one partialsuccess, which prompted them to conduct a controlled trial in rabbits inan attempt to define the role of argon laser in the treatmemt of cornealvascularization.

Meanwhile, in 1975 Reed and associates241 reported that argon lasertreatment was effective in treating corneal vascularization in rabbits.These investigators induced vascularization by applying silver nitrate 3 to4 mm from the limbus for 3 seconds at two to three sites in each eye of 15albino rabbits. Blood vessels reached the point of injury within 2 to 3weeks. The vessels extending to one lesion in each eye were treated withargon laser, starting with those at the limbus; those extending to the otherlesions were not treated, so that they could serve as controls. The mosteffective laser settings were found to be a power of 450 to 500 mW with abeam diameter of 100 ,um. One- to two-second pulses were used to treatthe entire length of the blood vessel(s). Subjective grading of the amountof the vascularization was done. Argon laser photocoagulation was foundto be quite effective in reducing the amount ofcorneal vascularization. Nosignificant side effects were noted.

In their study, Cherry and Garner240 induced corneal vascularizationby placing black silk sutures at the 2- and 10-o'clock positions 3 mm fromthe limbus in one eye of each of 11 pigmented Dutch rabbits. Vasculariza-tion was established in about 3 weeks, after which the sutures were re-moved. There were two sectors of vascularization due to the two sites ofsuture placement in each cornea. One sector of vessels was treated withthe argon laser while the other was used as a control. The laser settingswere usually a power of 150 mW, beam spot size of50 ,um, and pulse timeof 0.2 second. Intravenous fluorescein was usually given immediately be-fore the laser treatment because, according to the investigators, thiswould enable more energy to be absorbed by the vessels. The laser treat-ment was started at the limbus so that the trapped blood in the cornealvessels would allow better absorption of the argon laser energy. The in-

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vestigators state that argon laser treatment was effective in obliteratingcorneal blood vessels, provided the stimulus for vascularization was notcontinuously present. The most obvious complication noted was damageto the iris in these pigmented rabbits.

In 1982 Marsh and Marshall242 reported the results of argon laserphotocoagulation of feeder blood vessels in patients with lipid keratopa-thy. The feeder vessels were treated with argon laser settings of200 to 800mW of power, 50 to 100 ,um beam aperture, and 0.1-second pulses. Ac-cording to the investigators, among the 19 patients followed up for at leasta year, recurrence of corneal vascularity was not a problem in 8 patients,easily handled in 4 patients, and troublesome in 7 patients. These patientswith troublesome recurrence were mostly the ones with severe cornealvascularization to begin with. The density and extent of lipid depositionin the cornea were diminished in 50% of the cases. The commonestcomplications were bleeding into the lipid keratopathy and iris damage.The only serious problem was a disciform type of lipid keratopathy thatflared up after laser treatment in some patients. Marsh reported again in1982 the encouraging results of argon laser photocoagulation in reducingvascularity and lipid deposits in 41 patients with lipid keratopathy fol-lowed up for at least 9 months.243

In 1986 Mendelsohn and associates192 reported the results of argonlaser photocoagulation of feeder vessels in lipid keratopathy in a rabbitmodel. This model consisted of rendering New Zealand white strain rab-bits hypercholesterolemic by feeding them a high-cholesterol diet and thesimultaneous placement of corneal suture to induce vascularization.191Mendelsohn and associates192 used argon laser settings of 700 mW ofpower, 100-I,m beam spot size, and 0.1-second pulses to treat the limbalvessels, and then reduced the beam spot size to 50 ,um to treat thenarrower corneal vessels. They found that 1 week after laser treatmentthe vascularization in the lasered eyes was about 50% as severe as in thenonlasered eyes, but it was comparable in the lasered and nonlasered eyes3 weeks after laser treatment. They also found that the corneal cholesterolcontent increased after the laser treatment. Therefore, argon laser photo-coagulation may not be a preferred mode oftreatment in this rabbit modelof lipid keratopathy.

In 1986 Nirankari and Baer244 reported the results of argon blue-greenlaser photocoagulation in 13 patients with deep corneal stromal vascular-ization. In 8 of these patients, deep stromal vessels into the corneal grafthad developed after penetrating keratoplasty, and there were signs ofgraft rejection that did not reverse with suture removal and corticosteroidtreatment. After argon laser photocoagulation there was marked regres-

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sion of the corneal vascularization and reversal of graft rejection in alleyes. Three additional patients with vascularized corneas, referred forpenetrating keratoplasty, underwent argon laser photocoagulation preop-eratively. The corneal grafts in these patients have remained avascularand clear. Two other patients with corneal injury causing progressiveopacification and vascularization were also treated with argon laser photo-coagulation, resulting in significant clearing of opacity and vessels andimprovement in vision. The argon laser settings used in this study were200 to 700mW ofpower (depending on the vessel patency), 50- to 100-,umbeam spot size, and 0.1 to 0.2-second pulse duration. The afferent vesselswere treated first, if possible, and the treatment was started paracentrallyand carried peripherally.

In 1989 Baer and Foster245 reported good success in treating cornealvascularization with the 577-nm yellow dye laser in patients with refrac-tory graft rejection, in high-risk patients with vascularization prior tocorneal grafting, and in patients with keratitis resulting in vascularizationthreatening the visual axis. Recently, Nirankari and associates246 reportedthat corneal stromal vascularization induced in New Zealand Albino rab-bits with intrastromal injection ofsodium hydroxide can be treated favora-bly-with yellow dye laser photocoagulation. The dye laser settings used inthis study were 577-nm wavelength, 400 mW of power, 50-,um beam spotsize, and 0.05-to 0.2-second pulse duration.

Photochemical ThrombosisRecently, Mendelsohn and associates247 reported success in reducingcorneal vascularization and corneal cholesterol content by injecting aphotosensitizing dye, rose bengal, intravenously in their previously men-tioned rabbit model of lipid keratopathy followed by argon laser treatmentof corneal feeder vessels. Huang and associates248249 have also attemptedto apply this method to treat corneal vascularization. In their studies onNew Zealand Albino rabbits, these investigators induced corneal vascular-ization by removing corneal and limbal epithelium, along with someconjunctival epithelium, with surgical scraping and n-heptanol debride-ment. Corneal vascularization was observed 2 to 3 weeks after the epithe-lium removal. Intravenous rose bengal was injected, 40 mg/kg bodyweight, followed by treatment of the corneal blood vessels by argon laserat the settings of 514.5-nm wavelength, 130 mW of power, 63-,um beamspot size, and 0.2-second pulse duration. The treated blood vessels werefound to be occluded throughout the 4-month study period. Transientelevations in serum urea nitrogen, aspartate aminotransferase, alanineaminotransferase, alkaline phosphatase, and total bilirubin levels and a

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decrease in serum phosphorus level were noted after intravenous injec-tion of rose bengal.248More recently, Corrent and associates250 have studied corneal graft

survival after treatment of corneal vascularization with photochemicalthrombosis. Silk sutures were placed in the corneas of New ZealandAlbino rabbits to induce vascularization. These sutures were removed 14to 18 days after placement. Within 48 to 56 hours after suture removal,intravenous rose bengal was injected, 20 to 30 mg/kg body weight,followed by argon laser treatment of corneal blood vessels at settings of514.5-nm wavelength, 75 to 90 mW of power, 63-,um beam spot size, and0.2-second pulse duration. Penetrating keratoplasty was performed 36 to42 hours after photochemical thrombosis. During the 61/2- to 181/2-weekfollow-up period, six of eight corneal grafts in eyes which had undergonephotochemical thrombosis were clear, compared with vascularization andopacity in seven of eight grafts in fellow control eyes that did not undergophotochemical thrombosis. According to the authors, photochemical throm-bosis produced temporary occlusion of corneal blood vessels in this mod-el. They noted damage to the anterior corneal stroma in some eyes afterphotochemical thrombosis, which they attribute to possible leakage ofrose bengal from the vessels. Another adverse effect was sectoral areas ofchorioretinal scarring in some eyes, probably caused by absorption ofsome laser energy by the photosensitized choroidal and retinal bloodvessels.Although laser photocoagulation of corneal vascularization after photo-

sensitization of the vessels with a dye has a theoretical advantage oversimple laser photocoagulation for obliteration of these vessels, it hascertain practical disadvantages. As already noted, photosensitization canalso result in undesirable chorioretinal scarring, and leakage of the dye inthe corneal stroma may cause damage by photochemical reaction.250Transient abnormalities in some liver enzymes and electrolytes have alsobeen noted after intravenous injection of rose bengal in rabbits.248 Forthese reasons, photochemical thrombosis has not been used in humans forthe treatment of corneal vascularization.

Photodynamic TherapyVery recently, Epstein and associates251 reported success in reducingcorneal vascularization in albino A/J mice with photodynamic therapyusing aron laser after injecting the photosensitizing dye dihematopor-phyrin ether intravenously. Corneal vascularization was induced by intra-corneal injection of interleukin-2. Dihematoporphyrin ether, 10 mg/kgbody weight, was injected intravenously. Seventy-two hours later argon

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laser treatment was applied to the cornea at settings of 514-nm wave-length, 800 mW of power, 1000-pum beam spot size, and 2-second pulseduration. Eight such laser spots were applied to each cornea to cover mostof the corneal surface area. Significant reduction in the amount of cornealvascularization was found at 4-, 8-, and 12-weeks intervals as comparedwith the controls. Complications included iris damage in 18% of thetreated eyes and blepharitis in 9%. The treated eyes also showed astriking decrease in the number of stromal keratocytes. Basophilia andvacuoles were seen in the anterior portion of the lens in 10% of thetreated eyes sampled. None of these changes were seen in the dihe-matoporphyrin ether-only or laser-only controls.

It remains to be seen whether photodynamic therapy for corneal vas-cularization will be safe in humans. Photosensitization may lead to ab-sorption of laser energy by blood vessels at sites other than the cornealvascularization, which may lead to undesirable damage. Chorioretinalscarring has been noted with the previously discussed model of photo-chemical thrombosis for corneal vascularization using a photosensitizingdye.250 Corneal vascularization consists of leaky blood vessels. This isreadily evident from leakage of the fluorescein dye into the cornealstroma seen in the late phase of anterior-segment fluorescein angiographydone in patients with corneal vascularization (Fig 1A and B). Leakage ofthe photosensitizing dye into the corneal stroma could lead to nonspecificdamage on application of laser treatment. It is also not clear whetherphotodynamic therapy will be effective in treating focal corneal vascular-ization, which is often the case in the human clinical setting, since lasertreatment was applied to the whole cornea in the study of Epstein andassociates.251

ANIMAL STUDIES

We chose the rabbit for our studies because its cornea is similar to that ofthe human, the main histological difference between the two being thelack of a significant Bowman's layer in the rabbit cornea.252 The rabbitcornea with an average horizontal diameter of about 15.0 mm and verticaldiameter of about 13.5 to 14.0 mm,253 is slightly bigger than the humancornea, which has an average horizontal diameter of 12.6 mm and verticaldiameter of 11.7 mm.2-i The rabbit cornea is 0.35 to 0.45 mm thick, withthe periphery slightly thicker than the center according to some investi-gators.2-3 The average thickness of the human cornea is 0.52 mm centrallyand 0.65 mm peripherally.254 Apart from these slight differences, therabbit a human corneas are comparable in anatomy and function to a large

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FIGURE 1

A: Anterior segment fluorescein angiograms in a 37-year old woman with corneal stromalvascularization due to herpes simplex keratitis. Early phase (25 seconds after injection) Notethat corneal stromal vessels are well delineated. B: Late phase (161 seconds after injection).

Note diffuse leakage of dye around corneal stromal vessels.

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extent.252-254 Therefore, the rabbit has served well for experimental studyof the cornea and has been used extensively in the study of cornealvascularization. Other animals that have been used in the study of cornealvascularization include the rat and mice, whose corneas are much smallerand thinner than the human cornea.

RABBIT MODEL OF CORNEL STROMAL VASCULARIZATION

Most published experimental studies on the effect of laser treatment oncorneal vascularization have used the rabbit model.192,M,241,24r>2 Inthese studies, new blood vessel growth in the rabbit cornea has beeninduced by silver nitrate application on the cornea,241 silk suture place-ment in the cornea,192Z4O240272 surgical scraping and n-heptanol de-bridement of the corneal and conjunctival epithelium,2'48249 and injectionof sodium hydroxide into the corneal stroma.246 Most of these studieshave not discussed how deep in the cornea the new blood vessel growthWas seen in the respective models. We feel that in order to study theeffect of laser treatment, the induction of corneal stromal vascularizationin the rabbit model is an important issue, because in humans the cornealgraft rejection is associated with stromal vascularization, and the othersequelae of corneal vascularization, such as edema and scarring, are moreextensive with stromal than with superficial vascularization. Rabbit mod-els that have superficial corneal vascularization may not be adequatelyrepresentative of the human clinical entity of serious concern, that is,corneal stromal vascularization. Therefore, we have developed a model ofcorneal stromal vascularization in the New Zealand Albino rabbit, whichinvolves injection of sodium hydroxide into the corneal stroma.

Our ModelOur protocol for the treatment of rabbits has been approved by theAnimal Care and Use Committee of the University of Maryland School ofMedicine. We anesthetize the rabbit with 35 mg/kg body weight ofketamine hydrochloride and 5 mg/kg body weight of xylazine hydro-chloride mixed in the same syringe, and inject it intramuscularly into thegluteus maximus of the rabbit. This anesthetizes the rabbit within 10 to 15minutes, and the anesthesia lasts for 45 to 60 minutes. The rabbit is thenplaced under an operating microscope, and the appropriate eye is prop-tosed by gently pushing back the eyelids with the help of a latex sleeveand a tongue depressor after giving a drop of proparacaine hydrochloride0.5% topically as local anesthetic. The corneal injection site is chosen inbetween the attachment of two of the extraocular muscles in order toavoid ingrowth of vasculature from the muscles to the injection site.

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FIGURE 2A: Injection of sodium hydroxide into corneal stroma of rabbit eye. Placement of 30-gaugeneedle attached to tuberculin syringe containing 0.1 N sodium hydroxide. Arrow indicatespoint of entry into cornea. B: Sodium hydroxide injected gradually into corneal stroma.Note spreading haziness in cornea caused by disruption of arrangement of collagen fibrils.

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FIGURE 2 (Cont'd)C: Continued injection of sodium hydroxide, causing further spread of haziness in cornea.

D: Completion of injection of 50-1.l 0.1 N sodium hydroxide.

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While looking through the operating microscope, the surgeon marks theexact injection site on the cornea 2 mm inside the limbus with the help ofcalipers. A 30-gauge needle attached to a tuberculin syringe containing0.1 N sodium hydroxide is introduced into the corneal stroma at themarked site with the bevel facing up (Fig 2A). Then 50 ,ul of sodiumhydroxide is injected gradually into the corneal stroma while the spread-ing haziness in the cornea is carefully observed (Fig 2B through D).Special care is taken to avoid entering the anterior chamber ofthe eye andspillage of sodium hydroxide on the corneal surface. After the injectionthe needle is immediately withdrawn and the cornea irrigated with bal-anced salt solution. The eyelids are then brought forward so that the eyecan go back in the socket, and a drop of gentamicin sulfate 0.3% is giventopically.The rabbit eye is examined at least once every week after the sodium

hydroxide injection. Corneal stromal vascularization has been observedto stabilize in 4 to 5 weeks in our model and is documented with photogra-phy (Fig 3A) and anterior segment fluorescein angiography (Fig 3B) afterinjecting 1.5 ml of 10% fluorescein dye in the central ear vein of therabbit. Fluoresccin angiography has been found to be quite useful inelucidating anterior segment vasculature.255 Histologic study has shownthat the new blood vessels in our model are present in the anterior andmid stroma of the cornea (Fig 4). Follow-up in the early phase of ourstudies showed that the stromal corneal vascularization induced in ourmodel was stable for at least 6 months. The data presented in the resultspart of the animal studies section support this observation.

Measurement of VascularizationWe have tried three methods for measuring the extent ofcorneal vascular-ization: grid, calipers, and computerized image analysis.A grid consisting of 1 x 1 mm squares is superimposed on a slide

showing corneal vascularization (Fig 5). The number of squares that are atleast half filled with blood vessels are counted, and their area added toobtain the total area of corneal vascularization after talking the magnifica-tion factor into account.We use calipers to measure the area of corneal vascularization on slides

by the following method. Almost all areas of corneal vascularization arewedge-shaped. One measurement is made at the base ofthe wedge on thelimbus, and one parallel to the first measurement at the other base of thewedge toward the center of the cornea. Perpendiculars are then drawnfrom the two edges of the other base to the limbal base. This divides thearea of corneal vascularization into a central rectangle and two right-

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FIGURE 3A: Stable corneal stromal vascularization in rabbit eye 5 weeks after intrastromal injection ofsodium hydroxide. B: Anterior segment fluorescein angiogram of corneal stromal vessels of

same eye 34 seconds after dye injection.

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FIGURE 4Cross-section of rabbit cornea 5 weeks after intrastromal injection of sodium hydroxide.Arrows indicate patent blood vessels containing erythrocytes in anterior and mid corneal

stroma. Also note fibrosis in anterior and mid corneal stroma.

angled triangles on each side. The total area of corneal vascularization isthen calculated by adding the areas of the rectangle and the two trianglesusing standard formulas.We have also attempted to use an image analysis system to calculate the

area of corneal vascularization. This system can acquire a digital image ofthe photograph showing corneal vascularization with a resolution of 480x 512 pixels. Then by trial-and-error method, a brightness threshold on ascale of 0 to 256 can be set such that all pixels in a given area that aredarker than this threshold indicate blood vessels. Using conversion fac-tors, the area covered by these pixels can then be calculated. In ourexperience this method of calculating the area of corneal vascularizationwas not very useful because there was not enough contrast between thetransparent cornea and the blood vessels for the image analysis system tomake reliable measurements. A computerized image analysis system hasbeen successfully used to measure the area occupied by carbon-filledblood vessels in the rat cornea on a fixed mount.93 The limitation of thismethod is that serial measurements on the Same cornea cannot be madebecause the animal has to be sacrificed for making the corneal mount, andfor obvious reasons this method Cannot be used in humans.

612


FIGURE 5Measurement of extent of corneal vascularization. Grid of 1 x 1 mm squares is superim-posed on slide (magnification, 1.6) showing corneal stromal vascularization in same rabbiteye as in Fig 3. Each grid square covers an area of 0.4 mm2 on slide. Nineteen squares weremore than half filled with area of corneal stromal vascularization, which represented a total

area of 7.6 mm2.

We found the grid method of measuring the area of corneal vasculariza-tion simple, yet reasonably reliable according to measurements by twomasked observers. Therefore, we used this as the primary method in ourquantitative studies.

LASER TREATMENT

In our earlier experiments we used argon laser with 514.5-nm wavelengthfor the treatment of corneal vascularization. Later we switched to theyellow dye laser with 577-nm wavelength for this treatment because of therelatively higher absorption of this wavelength by oxyhemoglobin andreduced hemoglobin.256 It was reported that destruction of retinal vascu-lar abnormalities required 15% to 20% lower power density with theyellow laser to create the same coagulation appearance as with the argongreen laser.256We study the fluorescein angiogram before applying the laser treat-

ment in order to get a better idea of the blood vessels present in thecornea. We use the Abraham iridectomy laser lens for laser treatment

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because it helps in stabilization of the globe, magnification of the cornealvessels, and focusing laser energy on the corneal vessels. We start thelaser treatment paracentrally and proceed peripherally. Ifthe afferent andefferent vessels can be distinguished, laser treatment is first applied tothe afferent and then to the efferent vessels. We avoid applying lasertreatment in the central corneal region to prevent any possible damage inthe visual axis. The central vessels regress on their own because bloodsupply to them is cut offwhen the paracentral and peripheral vasculariza-tion is photocoagulated.

Corneal stromal vascularization was induced in both eyes of seven NewZealand Albino rabbits with intrastromal injection ofsodium hydroxide bythe method described earlier. The vascularization stabilized 5 weeks afterthe injection of sodium hydroxide. The extent ofnew blood vessel growthwas documented at this time with photography and anterior segmentfluorescein angiography. One eye of each rabbit was chosen randomly forlaser treatment with the yellow dye laser set at 577-nm wavelength, 400mW of power, 50-,um beam spot size, and 0.05- to 0.2-second pulseduration. Between 300 and 600 laser spots were applied depending on theextent of vascularization with the technique described earlier. The un-lasered eye of each rabbit was taken as the control. Both eyes of eachrabbit underwent photography and anterior segment fluorescein angiog-raphy after the laser treatment. Repeat photographs of both eyes of eachrabbit were taken at intervals of 1 to 4 weeks during a follow-up period of6 months. The grid method was used to measure the total area of cornealvascularization on all photographs prior to laser treatment and during thefollow-up period after laser treatment.

RESULTS

Just prior to laser photocoagulation the area of corneal vascularization inthe eyes randomized for laser treatment was 24.6 + 3.5 mm2 (mean +standard error of the mean [SEM]), range 12.0 to 40.0 mm2. The compa-rable area in the control eyes was 19.3 + 4.1 mm2 (mean + SEM), range10.0 to 40.0 mm2. This difference between the two groups ofeyes was notstatistically significant (P = 0.14, paired t-test).The percentage change from baseline in the area of corneal vasculariza-

tion was compared between the lasered and control eyes at 2, 4, and 6months after laser photocoagulation. The percent decrease from baselinein the area of corneal vascularization was more in the lasered eyes than inthe control eyes at all of the three time intervals (Fig 6): at 2 months: 40.7± 5.0 (mean ± SEM) decrease in lasered eyes, 11.1 + 6.2 (mean +SEM) increase in control eyes (P = 0.0002, paired t-test); at 4 months:

614


c0

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a1 0 9 --_ Lasered eyes

40s p =0.0002 p=0.04 p=0.05

co~

2 20-

0S

coA0..00 0I4p 00

2 4 6

Time after Laser Treatment (Months)

FIGURE 6Percentage change in area of corneal stromal vascularization in seven control and sevenlaser-treated rabbit eyes over 6 months' follow-up. Vertical bars represent 1 SEM on each

side of mean. P values are obtained by using paired t-test.

45.3 ± 3.3 (mean ± SEM) decrease in lasered eyes, 16.9 ± 8.2 (mean +SEM) decrease in control eyes (P = 0.04, paired t-test); at 6 months: 34.9+ 5.2 (mean ± SEM) decrease in lasered eyes, 3.0 ± 11.2 (mean +SEM) decrease in control eyes (P = 0.05, paired t-test). The last of thesecomparisons had only borderline statistical significance, probably becauseof the high variance in the control group.One of the rabbits had an unexplained massive increase in the area of

corneal vascularization in both eyes 4 months after laser photocoagula-tion: 100% and 194% increase from baseline in the lasered and controleyes, respectively. No obvious reason could be found for these unusualfindings. At this time another laser treatment was given to the previouslylasered eye. Two months later there was a 25% decrease in the area ofcorneal vascularization in the lasered eye, while the area remained almostthe same in the control eye. In our understanding the findings in botheyes of this rabbit 4 months after laser photocoagulation represented an"outlier," and hence these data were not included in the analysis for the 4-and 6-month intervals. The role of excluding obvious outliers from dataanalysis has been discussed earlier.257

HISTOPATHOLOGY

Paraffin sections of the cornea were stained with hematoxylin and eosin,periodic acid-Schifl Masson trichrome, and van Gieson stains at various

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FIGURE 7Cross-section of rabbit cornea 1 hour after laser photocoagulation for corneal stromalvascularization. Note presence offewer blood vessels than in unlasered control eye in Fig 4.Arrows indicate neutrophils in corneal stroma. Note marked disorganization of collagen andfibroblastic reaction in anterior and mid stroma. Deep stroma and endothelium are intact.

stages after the intrasomal injection of sodium hydroxide, and after thelaser treatment for study under light microscopy in a masked fashion.Epoxy resin sections of the cornea were studied under transmissionelectron microscopy.

Light MicroscopyInfiltration ofthe corneal stroma with leukocytes, mainly neutrophils, wasnoted at about 8 hours after the sodium hydroxide injection. Increasedvascularity of the anterior and mid stroma was noted within a few days ofthe sodium hydroxide injection. Infiltration of the corneal stroma withleukocytes was noted to have cleared within about 2 to 3 weeks of thesodium hydroxide injection. Blood vessel growth was found to havestabilized within 4 to 5 weeks after the sodium hydroxide injection. Aphotomicrograph of a corneal section 5 weeks after the sodium hydroxideinjection shows some fibrosis and many patent blood vessels containingerythrocytes in the anterior and mid stroma (Fig 4).We do the laser treatment 5 weeks after the sodium hydroxide injec-

tion. The presence of fewer patent blood vessels, pronounced fibroblastic

616


FIGURE 8Cross-section of a rabbit cornea 4 hours after laser photocoagulation for corneal stromalvascularization. Inflammatory cells are seen in anterior corneal stroma. Hemorrhage is alsoseen in anterior stroma. Note marked disorganization of collagen and fibroblastic reaction in

anterior and mid stroma.

proliferation, and inflammatory cells in the anterior and mid cornealstroma of lasered eyes enabled all seven of these studied to be distin-guished from the controls by one masked observer, and five out of theseseven to be distinguished by another masked observer.The corneal section at 1 hour after laser treatment (Fig 7) showed

stromal edema and relatively fewer blood vessels than in the unlaseredcontrol cornea (Fig 4). Few neutrophils were also seen at 1 hour after lasertreatment (Fig 7). More inflammatory cells were seen at 4 hours afterlaser treatment (Fig 8), and still more at 8 hours after laser treatment (Fig9). The corneal section at 4 hours after laser treatment showed engorgedand ruptured blood vessels (Fig 8). This may be related to our techniqueof starting the laser photocoagulation paracentrally and proceeding pe-ripherally. This may lead to temporary engorgement of the central bloodvessels and their subsequent rupture. Since this hemorrhage was seenonly in one specimen, it is thought to be an uncommon occurrence. Asdiscussed earlier, the central vessels regress gradually because theirblood supply has been cut off.

617

618 Nirankari

vi,-~~~~~~~~~~~, i

0|11 E0 1

FIGURE 9Cross-section of rabbit cornea 8 hours after laser photocoagulation for corneal stromalvascularization. Many inflammatory cells are seen in anterior and mid corneal stroma. Notemarked disorganization of the collagen and fibroblastic reaction in anterior and mid stroma.

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FIGURE 10Cross-section of rabbit cornea 24 hours after laser photocoagulation for corneal stromalvascularization. Inflammatory cells persist in anterior and mid corneal stroma. Note marked

disorganization of collagen and fibroblastic reaction in anterior and mid stroma.


FIGURE 11Cross-section of rabbit cornea 48 hours after laser photocoagulation for corneal stromalvascularization. Inflammatory cells still persist in anterior and mid corneal stroma, but areless than at 24 hours (Fig 10). Arrows indicate empty ghost vessels, those without erythro-cytes. Note marked disorganization of collagen and fibroblastic reaction in anterior and mid

stroma.

At 24 hours (Fig 10) and 48 hours (Fig 11) after laser treatment theinflammatory cells persisted in the stroma. A few ghost vessels, oneswithout erythrocytes and therefore thought to be nonfunctioning, wereseen in the corneal sections at 48 hours and 6 days after laser treatment(Figs 11 and 12). The presence of inflammatory cells had diminished inthe corneal section at 6 days after laser treatment (Fig 12). The bloodvessels were fewer and the fibroblastic proliferation pronounced in all thelasered eyes as compared with the control unlasered eyes. No damage wasseen to the deep corneal stroma or the endothelium in any of the laser-treated eyes (Figs 7 through 12).

Transmission Electron MicroscopyBlood vessels with a smooth contour lined with normal endothelial cells,with regular nucleus and cytoplasmic organelles, and containing manyerythrocytes were seen in the corneal stroma of control eyes (Fig 13). Theorganized pattern of the collagen fibrils in the corneal stroma could alsobe appreciated in the control eyes (Figs 13 and 14). No inflammatory cellswere seen in the corneal stroma of control eyes.

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FIGURE 12Cross-section of rabbit cornea 6 days after laser photocoagulation for corneal stromalvascularization. Inflammatory cells in corneal stroma are less than at 48 hours in Fig 11.Arrows indicate empty ghost vessels, those without erythrocytes. Note marked disorganiza-

tion of collagen and fibroblastic reaction in anterior and mid stroma.

Laser-treated eyes showed damaged endothelial cells, with cytoplasmicedema and irregular nucleus, lining the blood vessels in the cornealstroma (Fig 15). Granulocytes, other inflammatory cells, extravasatederythrocytes, and edema were seen in the corneal stroma (Fig 15). Neu-trophils were seen in the blood vessel lumen (Fig 15). A haphazard arrayof collagen fibrils was noticed in the corneal stroma of the laser-treatedeyes (Figs 15 and 16). Thin and empty ghost vessels were also seen in thecorneal stroma (Fig 17). One section showed the presence of granularfibrinous material around closely packed erythrocytes in the lumen of ablood vessel, suggesting thrombus formation after laser photocoagulation(Fig 18). Presence of distorted erythrocytes was also noticed in onesection of the corneal stroma of a laser-treated eye (Fig 16).

CLNICAL STUDIES

We have used laser photocoagulation for about 7 years to prevent thesequelae of corneal vascularization in patients.

620


FIGURE 13Electron micrograph of unlasered rabbit cornea 5 weeks after intrastromal injection ofsodium hydroxide. Blood vessel shows uniform lining of endothelial cells, and lumencontains erythrocytes. Surrounding collagen displays uniform arrangement of fibrils. No

inflammatory cells are seen in or around vessel (original magnification, x 3300).

RATIONALE

It is believed that the normal avascularity of the cornea makes it relativelyimmune to donor graft rejection.196,197 Avascular corneal grafts have asuccess rate of 85% to 95%, but vascularization may drop this rate to aslow as 35%.198-200 In high-risk patients with corneal vascularization priorto grafting, and in those with blood vessel growth into the graft who havenot responded to conventional treatment including suture removal andmaximal steroid therapy, elimination of the blood vessels with laserphotocoagulation may restore the relative immune privilege of the corneato graft rejection.

Corneal vascularization secondary to infection or trauma can causeedema, scarring, and lipid keratopathy. If vascularization in these casesdoes not respond to conventional treatment, laser photocoagulation oftheblood vessels can prevent the undesirable sequelae.

CATEGORIES OF PATIENTS

We have used laser photocoagulation to treat corneal vascularization inconsecutive series of patients in the following categories:

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FIGURE 14Electron micrograph of unlasered rabbit cornea 5 weeks after intrastromal injection ofsodium hydroxide. Endothelial cell lining blood vessel shows normal regular nucleus andcytoplasmic organelles. There is an adjacent fibroblast (arrow). Surrounding collagen fibrils

are uniformly arranged (original magnification, x 9000).

1. Patients with corneal graft rejection who have not responded tosuture removal and maximal steroid therapy.

2. Patients with previously unresolving corneal vascularization beingconsidered for graft.

3. Patients with corneal vascularization due to infection, trauma, orcontact lens wear who have not responded to conventional therapy.

4. Patients with corneal vascularization into the corneoscleral lamellargraft or epikeratoplasty graft who have not responded to sutureremoval and maximal steroid therapy.

RESULTS OF LASER TREATMENT

In the beginning we used argon laser with 514.5-nm wavelength to treatcorneal vascularization. But later we changed over to the yellow dye laserwith 577-nm wavelength because of the relatively higher absorption ofthis wavelength by oxyhemoglobin and reduced hemoglobin.256 Depend-ing upon the blood vessel patency judged through the laser slit lamp, thelaser settings were 200 to 700 mW of power, 50- to 100-pum beam spot

622

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FIGURE 15Electron micrograph of rabbit cornea 24 hours after laser photocoagulation for cornealstromal vascularization. Endothelial cells lining blood vessel display cytoplasmic edema andirregular nucleus, indicating damage. Neutrophils occupy most of vessel lumen. There arescattered stromal granulocytes (arrows) and extravasated erythrocyte (arrowhead). Collagen

fibrils in stroma are haphazardly arranged (original magnification, x 4300).

size, and 0.1- to 0.2-second pulse duration with the 514.5-nm laser, and200 to 500 mW of power, 50-,um beam spot size, and 0.05- to 0.1-secondpulse duration with the 577-nm laser. The number of laser applicationsranged between 50 and 600, depending upon the extent of corneal vas-cularization. We use the Abraham iridectomy laser lens to stabilize theglobe, magnify the corneal vessels, and focus the laser energy on thecorneal vessels. We start the laser treatment paracentrally and proceedperipherally (Fig 19). Afferent vessels, if identifiable, are treated first,followed by the efferent vessels. We avoid laser treatment in the centralcorneal region. The central blood vessels, if any, regress on their ownonce their blood supply is cut off.The data on the four groups of patients treated with laser photocoagula-

tion for corneal vascularization are summarized in Tables I through IV.Groups 1, 2, 3, and 4 included 8, 7, 12, and 3 patients, respectively.We have not attempted to treat corneal stromal vascularization involv-

ing the entire cornea with laser photocoagulation. We use laser photo-coagulation for the treatment of corneal stromal vascularization only if it

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FIGURE 16Electron micrograph of rabbit cornea 4 hours after laser photocoagulation for cornealstromal vascularization. Corneal stroma displays marked disarray of collagen fibrils. Fibro-blasts (curved arrow) and neutrophils (thick arrow) are scattered in stroma. There is anaccumulation of distorted extravasated erythrocytes (asterisk) (original magnification, x

3300).

involves two or less quadrants. Many times, one session of laser photo-coagulation was adequate for the treatment ofcorneal stromal vasculariza-tion. But if the vessels persisted, repeat laser photocoagulation was doneat 3- to 4-week intervals.

In group 1, patients were still using topical steroids for acute graftrejection when laser photocoagulation was done for corneal stromal vas-cularization, and continued to use the topical steroids for 3 to 4 weeksafter the laser treatment.

In group 1, laser photocoagulation was effective in treating cornealstromal vascularization and reversing acute graft rejection 9 of 11 times(82%) after conventional therapy, including suture removal and maximaltreatment with periocular and hourly topical steroids, had been unsuc-cessful (Table I). Vision improved from 20/200 + 20/40 (mean + SEM)before laser photocoagulation to 20/100 + 20/40 (mean + SEM) at 37 +10 (mean ± SEM) months, range 3 to 93 months, after laser photo-coagulation (P = 0.02, paired t-test). Examples of some of the patients inthis group follow.

624


FIGURE 17Electron micrograph of rabbit cornea 24 hours after laser photocoagulation for cornealstromal vascularization. Blood vessel lumen is empty. There are periluminal fibroblasts(curved arrow) and adjacent granulocytes (thick arrow). Collagen fibrils in stroma are

haphazardly arranged (original magnification, x 3300).

FIGURE 18Electron micrograph of rabbit cornea 4 hours after laser photocoagulation for cornealstromal vascularization. Endothelial cell lining blood vessel has cytoplasmic edema andirregular nucleus. Vessel lumen is packed with erythrocytes and granular fibrinous material(asterLsk) suggesting thrombus formation. Two fibroblasts are seen touching vessel (original

magnification, x 9000).

625

Nirankari

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FIGURE 19(Group 1, patient 1): Laser photocoagulation of corneal stromal vascularization due to acutegraft rejection in 68-year-old man. Laser treatment is started paracentrally (vertical arrows)and carried peripherally (horizontal arrow). Interruptions in vessels indicate sites of laser

treatment.

Patient 1 is a 68-year-old man who underwent penetrating keratoplastyfor scarring due to herpes simplex keratitis. Twenty-nine months after thepenetrating keratoplasty, he had an episode of acute graft rejection indi-cated by corneal stromal vascularization at the 1-o'clock position, cornealedema and haziness, and endothelial keratic precipitates (Fig 20A). Therejection episode did not respond to suture removal and treatment withperiocular and hourly topical steroids. Two sessions of 514.5-nm corneallaser photocoagulation resulted in regression of the corneal stromal vas-cularization and reversal of the rejection episode (Fig 20B). Forty-ninemonths later he had another episode of acute graft rejection with cornealstromal vascularization, which did not respond to maximal steroid thera-py (Fig 21A and B). The corneal stromal vascularization was treated withone session of 514.5-nm laser photocoagulation (Fig 19). This resultedagain in regression of the corneal stromal vascularization and reversal ofgraft rejection (Fig 22A and B). Similarly, another episode of acute graftrejection 41 months later was reversed successfully by treating the cor-neal stromal vascularization with one session of 577-nm laser photo-coagulation. This patient's corneal graft is clear at 122 months after the

628

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FIGURE 20(Group 1, patient 1). A: Acute graft rejection 29 months after penetrating keratoplasty.Corneal stroma vascularization at 1-o'clock position, corneal edema, and endothelial keraticprecipitates are seen. B: Regression of corneal stromal vascularization and reversal of acute

graft rejection after two sessions of 514.5-nm laser photocoagulation.

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Nirankari

FIGURE 21(Group 1, patient 1). A: Acute graft rejection 78 months after penetrating keratoplasty.Corneal stromal vascularization at 10-o'clock position and corneal edema are seen. B:Anterior segment angiogram showing corneal stromal vascularization at 2- and 10-o'clock

positions.

630


FIGURE 22(Group 1, patient 1). A: Regression of corneal stromal vascularization and reversal of acutegraft rejection after one session of 514.5-nm laser photocoagulation. B: Anterior-segmentangiogram showing regression of corneal stromal vascularization at 2- and 10-o'clock positions.

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Nirankari

penetrating keratoplasty, and his vision is 20/30.Patient 6 is a 30-year-old woman who underwent penetrating kerato-

plasty for severe keratoconus. Forty-eight months after the penetratingkeratoplasty, corneal stromal vascularization, lipid keratopathy, and cen-tral corneal edema with keratic precipitates developed, indicating acutegraft rejection (Fig 23A and B). Discontinuation of contact lenses, sutureremoval, and treatment with periocular and hourly topical steroids wasunsuccessful. Two sessions of514.5-nm corneal laser photocoagulation re-sulted in regression of the stromal vascularization and lipid keratopathy,and reversal of the rejection episode (Fig 24A and B). She had some irisatrophy at the site of laser photocoagulation (Fig 24A). This patient hasresumed wearing her rigid gas permeable contact lenses and has a visionof 20/20 at 54 months after the penetrating keratoplasty.

Patient 8 is another 30-year-old woman who underwent keratoplasty forsevere keratoconus. Fifteen months after the penetrating keratoplasty,corneal stromal vascularization at the 3- to 4 o'clock position, and acutegraft rejection developed (Fig 25A and B). Suture removal and maximalsteroid therapy could not reverse the stromal vascularization and graftrejection. One session of 577-nm corneal laser photocoagulation causeddiminution of the stromal vascularization and reversal of the graft rejec-tion, although some superficial vessels persisted in the corneal periphery(Fig 26). At 19 months' follow-up after the keratoplasty, she has a vision of20/20.

In group 2, consisting of seven eyes with corneal stromal vasculariza-tion that did not respond to maximal conventional therapy, laser photo-coagulation was done before penetrating keratoplasty. Previously vas-cularized corneas are known to be at high risk for graft failure.199 In ourpatients in this group, there were only two acute graft rejections in theseven eyes followed up for 28 ± 7 (mean + SEM) months after penetrat-ing keratoplasty (Table II). Only one of these two rejections was associatedwith corneal stromal vascularization (patient 4). Treatment with corneallaser photocoagulation caused regression of this stromal vascularizationand reversed the graft rejection. No corneal stromal vascularization oc-curred into any of the six other grafts.

Patient 1 is a 44-year-old woman who had persistent corneal stromalvascularization and scarring due to herpes simplex keratitis (Fig 27A andB). She was referred for corneal grafting. She underwent one session of514.5-nm corneal laser photocoagulation, which reduced the stromalvascularization markedly. Two months later she underwent penetratingkeratoplasty. At a follow-up of 60 months, no signs or symptoms of acutegraft rejection developed and her graft is clear (Fig 28). She has onecorneal stromal vessel in the periphery. Her vision is 20/25.

632


FIGURE 23(Group 1, patient 6). A: Acute graft rejection 48 months after penetrating keratoplasty.Corneal edema, lipid keratopathy, and corneal stromal vascularization are seen. B: Angio-

gram showing corneal stromal vascularization.

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Nirankari

FIGURE 24(Group 1, patient 6). A: Regression of corneal stromal vascularization and lipid keratopathy,and reversal of acute graft rejection after two sessions of 514.5-nm laser photocoagulation.Some iris atrophy is seen at site of laser photocoagulation (arrow). B: Anterior segmentangiogram of same showing regression of corneal stromal vascularization. Only one small

vessel going to graft-host junction is present.

634


FIGURE 25(Group 1, patient 8). A: Acute graft rejection 15 months after penetrating keratoplasty.Corneal stromal vascularization at 3- to 4-o'clock position and corneal haziness are seen. B:Anterior-segment angiogram showing corneal stromal vascularization at 3- to 4-o'clock

position.

635

Nirankari

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FIGURE 26(Group 1, patient 8). Diminution of corneal stromal vascularization and reversal of acutegraft rejection after one session of 577-nm laser photocoagulation. Some superficial vessels

persist in corneal periphery.

Patient 2 is a 37-year-old woman who had persistent corneal scarringand stromal vascularization due to herpes simplex keratitis (Fig 29A andB). She underwent two sessions of 514.5-nm corneal laser photocoagula-tion resulting in regression of the stromal vascularization (Fig 30). Pene-trating keratoplasty was performed 6 months later. She wears rigid gaspermeable contact lenses. She has not had any graft rejection or recur-rence ofcorneal stromal vascularization over a follow-up of35 months (Fig31). Her vision is 20/25.Group 3 consisted of 12 patients with corneal stromal vascularization

due to infection, trauma, or contact lens wear that did not respond tomaximal conventional therapy. These patients were treated with laserphotocoagulation, resulting in marked reduction or complete resolutionof the corneal stromal vascularization in all except one eye (Table III).Vision in these patients improved from 20/60 ± 20/15 (mean + SEM)before laser photocoagulation to 20/25 ± 20/1 (mean ± SEM) at 22 ± 6(mean ± SEM) months follow-up after laser photocoagulation (P = 0.01,paired t-test).

Patient 1 is a 56-year-old man who had an alkali eye burn. He wastreated with topical steroids, but his inferior corneal stromal vasculariza-

638


FIGURE 27(Group 2, patient 1). A: Persistent corneal stromal vascularization and scar due to herpessimplex keratitis. B: Anterior segment angiogram showing corneal stromal vascularization

extending into center of cornea. Corneal stromal vascularization is more evident.

I

F

639

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FIGURE 28(Group 2, patient 1). Clear graft at 60 months' follow-up after penetrating keratoplastyperformed 2 months after one session of514.5-nm laser photocoagulation for corneal stromal

vascularization. One deep vessel is seen in corneal periphery at 8-o'clock position.v~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......

FIGURE 30(Group 2, patient 2). Regression of the corneal stromal vascularization after two sessions of

514.5-nm laser photocoagulation.

640


FIGURE 29(Group 2, patient 2). A: Persistent corneal stromal vascularization and scar due to herpessimplex keratitis. B: Anterior segment angiogram showing corneal stromal vascularization.

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FIGuRE 31(Group 2, patient 2). Clear graft at 35 months' follow-up after penetrating keratoplastyperformed 6 months after two session of 514.5 nm laser photocoagulation for corneal stromal

vascularization.

tion did not resolve (Fig 32A). He underwent one session of 514.5-nmcorneal laser photocoagulation (Fig 32B). His corneal stromal vasculariza-tion regressed and has not recurred in over 76 months of follow-up (Fig33). His vision before laser photocoagulation was 20/200 and is now 20/25.He has a faint persistent inferior corneal scar and also has inferior irisatrophy (Fig 33).

Patient 7 is an aphakic 70-year-old male who had corneal stromalvascularization and scar in the visual axis secondary to hard contact lenswear (Fig 34A). Discontinuation of contact lens wear and application oftopical steroids did not cause regression of the corneal stromal vasculari-zation. He underwent one session of 514.5-nm corneal laser photocoagu-lation (Fig 34B). This caused resolution of the stromal vascularization andreduction in the scar as well as improvement in vision from 20/40 to 20/25.Corneal stromal vascularization did not recur during 8 months of follow-up (Fig 35A and B). This patient has inferior iris atrophy secondary tocorneal laser photocoagulation (Fig 35B).

Patient 8 is a 57-year-old man who had corneal stromal vascularizationand keratouveitis secondary to presumed herpes simplex infection. Treat-ment with topical antivirals and steroids was not successful in reducing

642


FIGURE 32(Group 3, patient 1). A: Persistent inferior corneal stromal vascularization and scarring dueto alkali eye burn. B: Laser photocoagulation at 514.5 nm. Laser treatment is startedparacentrally and carried out peripherally. Interruptions in vessels indicate sites of laser

treatment.

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FIGURE 33(Group 3, patient 1). Absence of corneal vascularization 76 months after corneal laser

photocoagulation. Iris atrophy due to laser treatment is seen inferiorly.

the corneal stromal vascularization, corneal edema, and uveitis (Fig 36Aand B). He underwent two sessions of577-nm corneal laser photocoagula-tion, which caused regression of the stromal vascularization and resolu-tion of the keratouveitis (Fig 37A and B). His vision improved from 20/50to 20/25. At 6 months' follow-up, he has had no recurrence of the cornealstromal vascularization and has discontinued all topical ophthalmic medi-cations.

In group 4, laser photocoagulation was used to treat corneal stromalvascularization at the interface of corneoscleral lamellar graft in twopatients and epikeratoplasty graft in another patient after suture removaland maximal steroid therapy failed. Corneal stromal vascularization re-solved in two of these patients and regressed markedly in the third, andvision improved in all three patients (Table IV). No recurrence of cornealstromal vascularization has occurred at a follow-up of 118, 11, and 4months in these three patients.

Patient 2 is a 63-year-old woman who had a superiorly dislocatedposterior chamber intraocular lens, resulting in uveitis, glaucoma, andcystoid macular edema. During slit-lamp photography, pressure causedby lifting of the upper eyelid resulted in bleeding from the posterior iris

6466


FIGURE 34(Group 3, patient 7). A: Corneal stromal vascularization and scar in visual axis due to aphakichard contact lens wear. B: Laser photocoagulation at 514.5-nm. Laser treatment is startedparacentrally and carried out peripherally. Interruptions in vessels indicate sites of laser

treatment.

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FIGURE 35(Group 3, patient 7). A: Regression of previous corneal stromal vascularization 8 monthsafter corneal laser photocoagulation. B: Absence of corneal vascularization 8 months aftercorneal laser photocoagulation. Iris atrophy due to laser treatment is seen in 4- to 6-o'clock

sector.

648


FIGURE 36(Group 3, patient 8). A: Persistent corneal stromal vascularization and keratouveitis due topresumed herpes simplex infection. B: Slit-lamp microphotograph showing keratouveitis

and corneal stromal vascularization.

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FIGURE 37(Group 3, patient 8). A: Regression of corneal stromal vascularization and reduction inkeratouveitis after two sessions of 577-nm laser photocoagulation for corneal stromal vas-cularization. B: Slit-lamp photomicrograph showing reduction in keratouveitis and regres-sion of corneal stromal vascularization after two sessions of 577-nm laser photocoagulation

for corneal stromal vascularization.

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and ciliary body, and therefore the dislocated intraocular lens was notremoved for fear ofcausing massive bleeding. Visual acuity was 20/40 withan aphakic correction. The patient underwent epikeratoplasty for refrac-tive correction. She also had Nd-YAG laser capsulotomy for opacity in theposterior lens capsule. Nine months later lipid keratopathy and cornealstromal vascularization developed at the interface of the graft and thecornea. This did not resolve after suture removal and maximal topicalsteroid therapy (Fig 38A). She underwent one session of 577-nm corneallaser photocoagulation (Fig 38B). This resulted in resolution of the stro-mal vascularization and lipid keratopathy (Fig 39A and B). Her graft isclear at 11 months' follow-up. Her vision has not improved beyond 20/40because of persistent cystoid macular edema.

COMPLICATIONS

Mild uveitis was seen in most patients immediately after corneal laserphotocoagulation, but this resolved by itself within a few days. The othertwo complications of corneal laser photocoagulation noted in our patientswere iris atrophy and intracorneal hemorrhage. Iris atrophy was seen inmost patients treated with argon blue-green 514.5-nm laser photocoag-ulation (Figs 24A, 33, and 35B). None of the patients treated with yellowdye 577-nm laser photocoagulation had iris atrophy. This may be due tothe relatively lower total energy used with the 577-nm treatment thanwith the 514.5-nm treatment to bring about similar photocoagulation ofblood vessels in our patients. It has been reported earlier that the de-struction of retinal vascular abnormalities required 15% to 20% lowerpower density with the yellow laser to create the same coagulationappearance as with the green argon laser.256 Intracorneal hemorrhage wasseen in one patient only. This resolved gradually over 3 weeks. None ofthese complications had serious consequences.

Corneal thinning, descemetocele, or corneal perforation was not seenin any of our patients treated with laser photocoagulation for cornealstromal vascularization. None of these patients had increase in cornealstromal vascularization immediately after laser treatment or recurrence ofcorneal stromal vascularization later.

DISCUSSION

The conditions that may cause corneal vascularization in humans includecorneal graft rejection, infections, trauma, contact lens wear, burns,vasculitides, metabolic disorders, toxins, and nutritional deficiency states.Persistent corneal stromal vascularization is undesirable for various rea-

652


FIGURE 38(Group 4, patient 2). A: Anterior segment angiogram showing persistent corneal stromalvascularization at interface of epikeratoplasty graft and cornea. Eye also had persistent lipidkeratopathy. B: Laser photocoagulation at 577 nm. Laser treatment is started paracentrallyand carried out peripherally. Interruptions in vessels indicate sites of laser treatment.Opacity in posterior lens capsule was treated earlier with Nd-YAG laser capsulotomy.

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FIGURE 39(Group 4, patient 2). A: Resolution of corneal stromal vascularization and lipid keratopathyafter one session of 577-nm laser photocoagulation for corneal stromal vascularization. B:Anterior segment angiogram showing resolution of corneal stromal vascularization at inter-face of epikeratoplasty graft and cornea after one session of 577-nm laser photocoagulation

for corneal stromal vascularization.

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sons. The immediate detrimental effect of corneal vascularization is de-crease in vision due to the loss of corneal transparency.12 Vascularizationof the cornea is a major risk factor for corneal graft rejection.13-15 Thenormal avascular nature of the cornea gives it relative immune privilegefrom donor graft rejection.196-197 Vascularization of the cornea providesthe afferent and efferent limbs for the immune rejection of the graft totake place. This compromises the immune protection of the normallyavascular cornea from donor graft rejection.196,197 Vascularization mayreduce the success rate ofcorneal grafts drastically to as low as 35% from arate of 85% to 95% in avascular grafts. 198-200 A total of 40,631 cornealgrafts were done in the United States during 1990.201 About 10% to 20%of all corneal grafts are estimated to fall into the high-risk category forfailure.202 A large proportion of these grafts are high-risk due to persis-tent vascularization of the recipient cornea. Therefore, persistent vas-cularization of a cornea being considered for grafting or vascularizationinto a donor corneal graft are both undesirable. Apart from graft rejec-tion, persistent corneal vascularization can cause edema, scarring, andlipid keratopathy, leading to decrease in vision. 12,16 Vascularization of thecornea may also lead to impaired barrier function of the corneal epitheli-um.21

It has been suggested that matching for two or more HLA antigens maydecrease the failure rate in high-risk corneal grafts.258 259 However, manybasic issues regarding the benefits ofHLA antigen matching for high-riskcorneal grafts, including the question whether the increase in cost wouldbe justified by the benefits, are not well understood.2' The CollaborativeCorneal Transplantation Studies are investigating whether matching forHLA antigens and/or crossmatching for lymphocytotoxic antibodies canreduce the incidence of corneal graft rejection in high-risk patients.202.203It has also been suggested that use of topical cyclosporin A may reducethe failure rate of high-risk corneal grafts.229 However, because of the lackof adequate understanding about the role of cyclosporin A in high-riskcorneal grafts, and the risk of its toxicity, it is not being used routinely.Therefore, no definite way to reduce the risk of corneal graft failure inhigh-risk patients is known at present.

Topical, and sometimes periocular, corticosteroids are presently themainstay of therapy for corneal stromal vascularization. However, treat-ment with corticosteroids may not always be successful. Another concernis that long-term use of corticosteroids locally in the eye may have sideeffects, including cataract, glaucoma, superinfection, and herpes simplexrecurrence. Other topical medical treatments that have been tried forcorneal stromal vascularization, but are not used clinically because of lack

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of consistent effectiveness or serious side effects or both, include non-steroidal anti-inflammatory drugs and cyclosporin A. Invasive treatmentsfor corneal vascularization that have been investigated but were found tobe clinically inapplicable include irradiation, cryotherapy, heat cautery,scar tissue barrier, and excision of vessels.

Laser treatment for corneal vascularization after intravenous injectionof photosensitizing dyes, rose bengal and dihematoporphyrin ether, hasbeen investigated.247-25' However, this approach has not yet been appliedto humans because of the potential complications. Photosensitization ofchoroidal and retinal vessels may lead to laser energy absorption by thesevessels, too, while ablation of the photosensitized corneal vessels is beingattempted. Probably because of this reason, sectoral areas of chorioretinalscarring have been noticed in some rabbit eyes after intravenous injectionof rose bengal followed by laser photocoagulation of corneal vasculariza-tion.250 Transient abnormalities in some liver enzymes and electrolyteshave also been noted after intravenous injection of rose bengal in rab-bits.248 Corneal vascularization consists ofimmature vessels that are leaky(Fig 1A and B). Therefore, leakage of a photosensitizing dye into thecorneal stroma could lead to photochemical reaction and nonspecificdamage on application of laser energy. Anterior corneal stromal damagewas noticed in some rabbit eyes with corneal vascularization treated withlaser photocoagulation after intravenous injection of rose bengal.250 Astriking decrease in the number of stromal keratocytes, and basophiliaand vacuoles in the lens anteriorly, was seen in mouse eyes that had argonlaser treatment for corneal vascularization after intravenous injection ofdihematoporphyrin ether.25' For the preceding reasons, photosensitiza-tion of the corneal vessels before laser photocoagulation has not beenused in humans so far.

However, effective treatment of corneal stromal vascularization is need-ed because it poses a serious problem in a variety of clinical settings,including the following: patients with corneal graft rejection not respond-ing to suture removal and maximal steroid therapy, patients with unre-solving vascularization being considered for corneal grafting, patientswith vascularization due to infection or trauma or contact lens wear notresponding to conventional therapy, and patients with vascularization intoepikeratoplasty or corneoscleral lamellar grafts not responding to sutureremoval and maximal steroid therapy. At this time, the basic mechanismsinvolved in the pathogenesis of corneal stromal vascularization are notwell understood. Hence, a therapy aimed at suppressing the basic mecha-nisms causing corneal stromal vascularization is not possible at present.Under these circumstances, we investigated the effectiveness of laser

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photocoagulation, without prior injection of any photosensitizing dye, inthe treatment of corneal stromal vascularization.We developed a rabbit model of corneal stromal vascularization. We

found that injection of 50-,ul 0.1 N sodium hydroxide into the cornealstroma of New Zealand Albino rabbits induces stromal vascularization,which stabilizes at about 5 weeks after injection. Presence of stromalblood vessels in this model was established by anterior-segment fluores-cein angiography and histologic study. This induced corneal stromal vas-cularization was found to remain stable for at least 6 months. In a ran-domized masked study, we found that photocoagulation of the cornealstromal vessels in our rabbit model with the 577-nm yellow dye lasercauses significant reduction in the amount ofvascularization in the treatedeyes as compared with the control eyes over a follow-up of 6 months.

Histopathologic study of the rabbit corneas with light and transmissionelectron microscopy within 6 days after laser treatment for stromal vas-cularization revealed damage to the endothelial cells lining the stromalvessels and presence of thrombus in the vessel lumen. All laser-treatedcorneas had fewer patent stromal blood vessels than the control corneas.Laser-treated corneas also revealed the presence of edema, inflammatorycells, fibroblastic proliferation, extravasated erythrocytes, and a disarrayof collagen fibrils in the stroma. The stromal edema and inflammatorycells began diminishing at about 48 hours after the laser treatment. Noneof the laser-treated corneas showed any damage to the deep cornealstroma or the endothelium.We have treated persistent corneal stromal vascularization with laser

photocoagulation in the previously mentioned four categories of patientsfor about 7 years. We started by using the argon blue-green 514.5-nmwavelength for corneal laser photocoagulation. But later we switched tothe yellow dye 577-nm wavelength because it was reported to be ab-sorbed relatively more by oxyhemoglobin and reduced hemoglobin.256Destruction of retinal vascular abnormalities was found to require 15% to20% lower power density with the yellow laser than with the green argonlaser.256 Therefore, theoretically it appears that less total energy would beneeded for ablation of the corneal stromal vessels with the 577-nm laserthan with the 514.5-nm laser, and thereby damage to the surroundingtissue could be minimized.

In our patient series, out of the 11 acute graft rejections that did notrespond to suture removal and periocular and hourly topical steroidtherapy, 9 were reversed after treatment of the corneal stromal vascular-ization with laser photocoagulation. No recurrence of corneal stromalvascularization or acute graft rejection occurred over a mean follow-up of

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37 months in the corneas that were successfully treated with laser photo-coagulation. It appears that when conventional means of treating cornealstromal vascularization in acute graft rejection have failed, an 82% successrate with laser photocoagulation is very useful.

In seven eyes with corneal stromal vascularization and scarring requir-ing penetrating keratoplasty, laser photocoagulation was done to reducevascularization prior to grafting. Vascularized corneas undergoing pene-trating keratoplasty are at very high risk of graft failure. 199 Only two ofthese seven eyes had acute graft rejection over a mean follow-up of 28months. Only one of these two acute graft rejections was associated withcorneal stromal vascularization, and that, too, was successfully treatedwith laser photocoagulation and the acute graft rejection reversed. Thenumber of patients in this group is small, and therefore no definiteconclusions can be made. However, the lack of recurrence of cornealstromal vascularization in six of seven grafts in previously vascularizedcorneas after laser photocoagulation appears encouraging.Twelve patients with persistent corneal stromal vascularization that did

not respond to maximal conventional therapy were treated with laserphotocoagulation. In all except one eye, there was marked reduction orcomplete resolution of the corneal stromal vascularization and significantimprovement in vision. There was no recurrence of corneal stromalvascularization in these patients over a mean follow-up of 22 months.Therefore, laser photocoagulation of corneal stromal vascularization seemsuseful in these patients.Three patients with corneal stromal vascularization at the interface of

epikeratoplasty or corneoscleral lamellar graft and host cornea, which didnot respond to suture removal and maximal steroid therapy, underwentcorneal laser photocoagulation. Corneal stromal vascularization resolvedin two patients and regressed markedly in the third. Vision improved inall three patients. No recurrence of vascularization has occurred in thesepatients at a mean follow-up of 44 months.No serious consequences after corneal laser photocoagulation were

seen in any of the patients in our series. None of these patients hadcorneal thinning, descemetocele, or corneal perforation. None of thepatients had an increase in corneal stromal vascularization immediatelyafter laser treatment or recurrences later.Our animal data and clinical experience suggest that laser photocoagu-

lation is a useful adjunctive tool in the treatment of corneal stromalvascularization when conventional therapy has failed. Laser photocoagu-lation was found to be successful and safe in the treatment of refractorycases of corneal stromal vascularization not responding to other treat-

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ments. There was significant reduction in the amount of corneal stromalvascularization in our rabbit studies after laser photocoagulation over 6months' follow-up. In our clinical experience, laser photocoagulation hashelped maintain corneal avascularity for as long as 6 years. No seriouscomplications of laser photocoagulation for corneal stromal vasculariza-tion have been noted in our clinical experience and in our experimentaland histopathologic studies on rabbits. This lack of serious complicationsmay be because it is possible to apply focal treatment to the cornealvessels with laser photocoagulation. Therefore, we propose that laserphotocoagulation is a valuable adjunctive tool in the treatment of cornealstromal vascularization.

REFERENCES

1. Toynbee J: Researches, tending to prove the non-vascularity and the peculiar uniformmode oforganization and nutrition ofcertain animal tissues, viz articular cartilage, andthe cartilage of the different classes of fibrocartilage: The cornea, the crystalline lens,and the vitreous humour; and the epidermoid appendage. Philos Trans R Soc Lond(Biol) 1841; 131:159-192.

2. Augstein XX: Gefass-Studien an der Hornhaut und Iris: A. Hornhaut-Gefasse. ZAugenheilkd 1902; 8:317.

3. Bruckner A: Klinische studien uber Hornhaut-Gefasse. Arch Augenheilkd 1909; 62:17-41.

4. Kreiker A: Ueber die Entwicklung der Gefaissbildung in der Hornhaut, aufGrund vonSpaltlampenbeobachtungen. Magy Orvosi Arch 1923; 24:65. Abstracted Zentralbl GesOphthalmol 1924; 11:288.

5. Ehlers H: Some experimental researches on corneal vessels. Acta Ophthalmol 1927;5:99-112.

6. Julianelle LA, Morris MC, Harrison RW: An experimental study of corneal vasculari-zation. Am J Ophthalmol 1933; 16:962-966.

7. Julianelle LA, Lamb HD: Studies on vascularization of the cornea: V. Histologicalchanges accompanying corneal hypersensitiveness. Am J Ophthalmol 1934; 17:916-921.

8. Julianelle LA, Bishop GH: The formation and development of blood vessels in thesensitized cornea. Am J Anat 1936; 58:109-125.

9. Swindle PF: Events of vascularization and devascularization seen in corneas. ArchOphthalmol 1938; 20:974-995.

10. Campbell FW, Michaelson IC: Blood vessel formation in the cornea. BrJ Ophthalmol1949; 33:248-255.

11. Cogan DG: Vascularization of the cornea: Its experimental induction by small lesionsand a new theory of its pathogenesis. Arch Ophthalmol 1949; 41:406-416.

12. Duke-Elder S, Leigh AG: Corneal vascularization, in S Duke-Elder (ed): System ofOphthalmology. Vol VIII. Diseases ofthe Outer Eye, Part 2. St Louis, CV Mosby, 1965,pp 676-691.

13. Khodadoust AA, Silverstein AM: Transplantation and rejection of individual cell layersof the cornea. Invest Ophthalmol 1969; 8:180-195.

14. Langston RH, Pavan-Langston D: Penetrating keratoplasty for herpetic keratitis:Decision-making and management. Int Ophthalnol Clin 1975; 15:125-140.

15. Herbort CP, Matsubara M, Nishi M, et al: Penetrating keratoplasty in the rat: A modelfor the study of immunosuppressive treatment of graft rejection. Jpn J Ophthalnol1989; 33:212-220.

659

Nirankari

16. Cogan DG, Kuwabara T: Lipogenesis of cells of the cornea. Arch Pathol 1955; 59:453-456.

17. Friedenwald JS: Growth pressure and metaplasia of conjunctival and corneal epitheli-um. Doc Ophthalmol 1951; 56:184.

18. Thoft RA, Friend J, Murphy HS: Ocular surface epithelium and corneal vascularizationin rabbits: I. The role of wounding. Invest Ophthalmol Vis Sci 1979; 18:85-92.

19. Tseng SCG, Hirst LW, Farazdaghi M, et al: Goblet cell density and vascularizationduring conjunctival transdifferentiation. Invest Ophthalmol Vis Sci 1984; 25:1168-1176.

20. Tseng SCG, Farazdaghi M, Rider AA: Conjunctival transdifferentiation induced bysystemic vitamin A deficiency in vascularized rat corneas. Invest Ophthalmol Vis Sci1987; 28:1497-1504.

21. Huang AJW, Tseng SCG, Kenyon KR: Alteration of epithelial paracellular permeabil-ity during corneal epithelial wound healing. Invest Ophthalmol Vis Sci 1990; 31:429-435.

22. Imre G: The mechanism ofcorneal vascularization. Acta MorpholAcad Sci Hung 1966;14:99-104.

23. Talman EL, Harris JE: Ocular changes induced by polysaccharides: III. Paper chroma-tographic fractionation of a biologically active hyaluronic acid sulfate preparation. AmJOphthalmol 1959; 48:560-572.

24. McAuslan BR, Reilly WG, Hannan GN, et al: Angiogenic factors and their assay:Activity of formyl methionyl leucyl phenylalanine, adenosine diphosphate, heparin,copper, and bovine endothelium stimulating factor. Microvasc Res 1983; 26:323-338.

25. McAuslan BR, Reilly W: Selenium-induced cell migration and proliferation: Relevanceto angiogenesis and microangiopathy. Microvasc Res 1986; 32:112-120.

26. Ashton N, Cook C, Langham M: Effect of cortisone on vascularization and opacifica-tion of the cornea induced by alloxan. Br J Ophthalmol 1951; 35:718-724.

27. Ashton N, Cook C: Mechanism of corneal vascularization. Br J Ophthalmol 1953;37:193-209.

28. Langham M: Observations on the growth of blood vessels into the cornea: Applicationof a new experimental technique. Br J Ophthalmol 1953; 37:210-222.

29. Ashton N, Cook C: Direct observation of the effect of oxygen on developing bloodvessels: Preliminary report. Br J Ophthalmol 1954; 38:433-440.

30. Smelser GK, Ozanics V: The effect of vascularization on the metabolism of the sulfatedmucopolysaccharides and swelling properties of the cornea. Am J Ophthalmol 1959;48:418-426.

31. Langham ME: The inhibition of corneal vascularization by triethylene thiophosphor-amide. Am J Ophthalmol 1960; 49:1111-1117.

32. Henkind P: Migration of limbal melanocytes into the corneal epithelium of guineapigs. Exp Eye Res 1965; 4:42-47.

33. Ahuja OP, Nema HV: Experimental corneal vascularization and its management. AmJOphthalmol 1966; 62:707-710.

34. Collin HB: Corneal lymphatics in alloxan vascularized rabbit eyes. Invest Ophthalmol1966; 5:1-13.

35. Graymore C, McCormick A: Induction of corneal vascularization with alloxan. Br JOphthalmol 1968; 52:138-140.

36. Obenberger J: Calcification in corneas with alloxan-induced vascularization. Am JOphthalmol 1969; 68:1113-1119.

37. Sugiura S, Matsuda H: Electron microscopic observations on the corneal neovascular-ization. Acta Soc Ophthalmol Jpn 1969; 73:1208-1221.

38. Matsuda H, Sugiura S: Ultrastructure of "tubular body" in the endothelial cells of theocular blood vessels. Invest Ophthalmol 1970; 9:919-925.

39. Fromer CH, Klintworth GK: An evaluation of the role of leukocytes in the patho-genesis of experimentally induced corneal vascularization: I. Comparison of experi-mental models of corneal vascularization. Am J Pathol 1975; 79:537-554.

660

Corneal Scleral Vascularization 661

40. Cooper CA, Bergamini MVW, Leopold IH: Use of flurbiprofen to inhibit cornealneovascularization. Arch Ophthalmol 1980; 98:1102-1105.

41. Zauberman H, Michaelson IC, Bergman F, et al: Stimulation of neovascularization ofthe cornea by biogenic amines. Exp Eye Res 1969; 8:77-83.

42. Ziche M, Jones J, Gullino PM: Role of prostaglandin E1 and copper in angiogenesis. JNatl Cancer Inst 1982; 69:475-482.

43. Alessandri G, Raju K, Gullino PM: Mobilization of capillary endothelium in vitroinduced by effectors of angiogenesis in vivo. Cancer Res 1983; 43:1790-1797.

44. Raju KS, Alessandri G, Gullino PM: Characterization of a chemoattractant for endo-thelium induced by angiogenesis effectors. Cancer Res 1984; 44:1579-1584.

45. Alessandri G, Raju KS, Gullino PM: Interaction of gangliosides with fibronectin in themobilization of capillary endothelium: Possible on the growth of metastasis. InvasionMetastasis 1986; 6:145-165.

46. Francois J, Maudgal MC: Experimental chloroquine keratopathy. Am J Ophthalmol1965; 60:459-464.

47. McCracken JS, Klintworth GK: Ultrastructural observations on experimentally pro-duced melanin pigmentation of the corneal epithelium. AmJ Pathol 1976; 85: 167-182.

48. Kupriianov W, Gurina 01: Angiogennyi effekt kolkhitsina. BiuU Eksp Biol Med 1987;104:347-349.

49. McAuslan BR, Gole GA: Cellular and molecular mechanisms in angiogenesis. TransOphthalmol Soc UK 1980; 100:354-358.

50. Lin MT, Chen YL, Lue CM: The involvement of collagenase from polymorphonuclearleucocytes (PMN) in PGE1 induced corneal neovascularization. Fed Proc 1988; 2:A1715.

51. Meyer K, Chaffee E: The mucopolysaccharide acid of the cornea and its enzymatichydrolysis. Am J Ophthalmol 1940; 23:1320-1325.

52. Berman M, Winthrop S, Ausprunk D, et al: Plasminogen activator (urokinase) causesvascularization of the cornea. Invest Ophthalmol Vis Sci 1982; 22:191-199.

53. Knighton DR, Hunt TK, Thakral KK, et al: Role of platelets and fibrin in the healingsequence: An in vivo study of angiogenesis and collagen synthesis. Ann Surg 1982;196:379-388.

54. BenEzra D: Neovasculogenic ability of prostaglandins, growth factors, and syntheticchemoattractants. Am J Ophthalmol 1978; 86:455-461.

55. Gospodarowicz D, Mescher AL, Birdwell CR: Control of cellular proliferation byfibroblast and epidermal growth factors. Nati Cancer Inst Monogr 1978; 48:109-130.

56. Gospodarowicz D, Bialecki H, Thakral TK: The angiogenic activity of the fibroblastand epidermal growth factor. Exp Eye Res 1979; 28:501-514.

57. Waterbury LD, Zagotta MT, Shott LD: The effect of dexamethasone on cornealneovascularization induced by epidermal growth factor. Proc West Pharmacol Soc1981; 24:153-158.

58. Wissler JH, Renner H: Inflammation, chemotropism and morphogenesis: Novel leuko-cyte-derived mediators for directional growth of blood vessels and regulation of tissueneovascularization. Z Physiol Chem 1981; 362:244.

59. Lobb RR, Alderman EM, Fett JW: Induction of angiogenesis by bovine brain derivedclass 1 heparin-binding growth factor. Biochemistry 1985; 24:4969-4973.

60. Fett JW, Strydom DJ, Lobb RR, et al: Isolation and characterization of angiogenin, anangiogenic protein from human carcinoma cells. Biochemistry 1985; 24:5480-5486.

61. Shing Y, Folkman J, Haudenschild C, et al: Angiogenesis is stimulated by a tumor-derived endothelial cell growth factor. J CeU Biochem 1985; 29:275-287.

62. Frater-Schroder M, Risau W, Hallmann R, et al: Tumor necrosis factor type alpha, apotent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc NatlAcad Sci 1987; 84:5277-5281.

63. Hockel M, Sasse J, Wissler JH: Purified monocyte-derived angiogenic substance(angiotropin) stimulates migration, phenotypic changes, and "tube formation" but notproliferation of capillary endothelial cells in vitro. J CeU Physiol 1987; 133:1-13.

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64. Herbert JM, Laplace MC, Maffrand JP: Effect of heparin on the angiogenic potency ofbasic and acidic fibroblast growth factors in the rabbit cornea assay. Int 1 Tssue React1988; 10:133-139.

65. Connolly DT, Heuvelman DM, Nelson R, et al: Tumor vascular permeability factorstimulates cell growth and angiogenesis. J Clin Invest 1989; 84:1470-1478.

66. Ziche M, Alessandri G, Gullino PM: Gangliosides promote the angiogenic response.Lab Invest 1989; 61:629-634.

67. Smith RS: The development ofmast cells in the vascularized cornea. Arch Ophthalmol1961; 66:383-390.

68. Parke A, Bhattacheijee P, Palmer RMJ, et al: Characterization and quantification ofcopper sulfate-induced vascularization of the rabbit cornea. Am J Pathol 1988; 130:173-178.

69. Epstein RJ, Stulting RD, Hendricks RL, et al: Corneal neovascularization: Patho-genesis and inhibition. Cornea 1987; 6:250-257.

70. Leibovich SJ, Polverini PJ, Shepard HM, et al: Macrophage-induced angiogenesis ismediated by tumor necrosis factor-alpha. Nature 1987; 329:630-632.

71. Fiegel VD, Knighton DR: Transforming growth factor-beta CTGFb) causes indirectangiogenesis by recruiting monocytes. Fed Proc 1988; 2:A1601.

72. Dinarello CA: Interleukin-1 and its biologically related cytokines. Adv Immunol 1989;44:153-205.

73. BenEzra D, Hemo I, Maftzir G: In vivo angiogenic activity of interleukins. ArchOphthalmol 1990; 108:573-576.

74. Epstein RJ, Hendricks RL, Stulting RD: Interleukin-2 induces corneal neovasculariza-tion in A/J mice. Cornea 1990; 9:318-323.

75. Mann I, Pirie A, Pullinger BD: An experimental and clinical study of the reaction ofthe anterior segment of the eye to chemical injury, with special reference to chemicalwarfare agents. Br J Ophthalmol (Suppl) 1948; 13:5-171.

76. Kull FC Jr, Brent DA, Parikh I, et al: Chemical identification of a tumor-derivedangiogenic factor. Science 1987; 236:843-845.

77. Morris PB, Hida T, Blackshear PJ, et al: Tumor-promoting phorbol esters induceangiogenesis in vivo. Am J Physiol 1988; 254:C318-322.

78. Chang CT, Chen YL, Lee SH, et al: The inhibition of prostaglandin El-inducedcorneal neovascularization by steroid eye drops. J Formosan Med Assoc 1989; 88:707-711.

79. Soubrane G, Jerdan J, Karpouzas I, et al: Binding of basic fibroblast growth factor tonormal and neovascularized rabbit cornea. Invest Ophthalmol Vis Sci 1990; 31:323-333.

80. Eliason JA: Angiogenic activity of the corneal epithelium. Exp Eye Res 1985; 41:721-732.

81. Breebaart AC, James-Whitte J: Studies on experimental corneal allergy. AmJ Ophthal-mol 1959; 48:3747.

82. Taylor S, Folkman J: Protamine is an inhibitor of angiogenesis. Nature 1982; 297:307-312.

83. Schoefl GI: Studies on inflammation: III. Growing capillaries: Their structure andpermeability. Virchows Arch Pathol Anat 1963; 337:97-141.

84. : Electron microscopic observations on the regeneration of blood vessels afterinjury. Ann NY Acad Sci 1964; 116:789-802.

85. Colin HB: Lymphatic drainage of 13II-albumin from the vascularized cornea. InvestOphthalnol 1970; 9:146-155.

86. : Ultrastructure oflymphatic vessels in the vascularized rabbit cornea. Exp EyeRes 1970;10:207-213.

87. Szalay J, Pappas GD: Fine structure of rat corneal vessels in advanced stages ofwoundhealing: I. Permeability of intravenously injected thorium dioxide. Microvasc Res1970; 2:319-329.

662

Corneal Scleral Vascularization 663

88. Szalay J, Pappas GD: Fine structure of rat corneal vessels in advanced stages ofwoundhealing. Invest Ophthalmol 1970; 9:354-365.

89. Fromer CH, Klintworth GK: An evaluation of the role of leukocytes in the patho-genesis of experimentally induced corneal vascularization: II. Studies on the effect ofleukocyte elimination on corneal vascularization. Am J Pathol 1975; 81:531-544.

90. Sholley MM, Ferguson GP, Seibel HR, et al: Mechanisms of neovascularization:Vascular sprouting can occur without proliferation of endothelial cells. Lab Invest1984; 51:624-634.

91. Mahoney JM, Waterbury LD: Drug effects on the neovascularization response to silvernitrate cauterization of the rat cornea. Curr Eye Res 1985; 4:531-535.

92. Form DM, Pratt BM, Madri JA: Endothelial cell proliferation during angiogenesis: Invitro modulation by basement membrane components. Lab Invest 1986; 55:521-530.

93. Proia AD, Chandler DB, Haynes WL, et al: Methods in laboratory investigation:Quantitation of corneal neovascularization using computerized image analysis. LabInvest 1988; 58:473479.

94. Leopold IH, Purnell JE, Cannon EJ, et al: Local and systemic cortisone in oculardisease. Am J Ophthalmol 1951; 34:361-371.

95. Lazar M, Lieberman TW, Leopold IH: Hyperbaric oxygenation and corneal neovascu-larization in the rabbit. Am J Ophthalmol 1968; 66:107-110.

96. Ey RC, Hughes WF, Bloome MA, et al: Prevention of corneal vascularization. Am JOphthalmol 1968; 66:1118-1131.

97. Brown SI, Wassermann HE, Dunn MW: Alkali burns of the cornea. Arch Ophthalmol1969; 82:91-94.

98. Kaiser RJ, Klopp DW: Hyperbaric air and corneal vascularization. Ann Ophthalmol1973; 5:44-47.

99. Frucht J, Zauberman H: Topical indomethacin effect on neovascularization of thecornea and on prostaglandin E2 levels. Br J Ophthalmol 1984; 68:656-659.

100. Reim M, Kaufhold FM, Kehrer T, et al: Zur Differenzierung der Therapie beiVeratzungen. Fortschr Ophthalmol 1984; 81:583-587.

101. Tabatabay CA, Baumgartner B, Leuenberger PM: Cellules de Langerhans et neo-vascularisation corneenne dans les bruilures alclines experimentales. J Fr Ophtalmol1987; 10:419-423.

102. Nakayasu K: Origin of pericytes in neovascularization of rat cornea. JpnJ Ophthalmol1988; 32:105-112.

103. Nirankari VS, Bauer SA, Rodrigues MM, et al: Corneal stromal vascularization (CSV)in albino rabbits. Invest Ophthalmol Vis Sci (Suppl) 1989; 30:3.

104. Ormerod LD, Abelson MB, Kenyon KR: Standard models of corneal injury usingalkali-immersed filter discs. Invest Ophthalmol Vis Sci 1989; 30:2148-2153.

105. Maun ME, Cahill WM, Davis RM: Morphological studies of rats derived of essentialamino acids: III. Histidine. Arch Pathol 1946; 41:25-31.

106. Totter JR, Day PL: Cataract and other changes resulting from tryptophane deficiency. JNutr 1942; 24:159-166.

107. Buschke W: Classification of experimental cataracts in the rat: Recent observations oncataract associated with tryptophan deficiency and with some other experimentalconditions. Arch Ophthalmol 1943; 30:735-762.

108. Albanese AA: Corneal vascularization in rats on a tryptophane deficient diet. Science1945; 101:619.

109. Hock CW, Hall WK, Pund ER, et al: Vascularization of the cornea as a result of lysinedeficiency. Fed Proc 1945; 4:155-156.

110. Sydenstricker VP, Hall WK, Hock CW, et al: Amino acid and protein deficiencies ascauses of corneal vascularization: A preliminary report. Science 1946; 103:194-196.

111. Berg JL, Pund ER, Sydenstricker VP, et al: The formation of capillaries and othertissue changes in the cornea of methionine-deficient rat. J Nutr 1947; 33:271-285.

112. Albanese AA, Buschke W: On cataract and certain other manifestations oftryptophanedeficiency in rats. Science 1942; 95:584-586.

664 Nirankari

113. Hall WK, Sydenstricker VP, Hock CW, et al: Protein deprivation as a cause ofvascularization of the cornea in the rat. J Nutr 1946; 32:509-524.

114. Sulkin DF, Sulkin NM, Nushan H: Corneal fine structure in experimental scorbutus.Invest Ophthalmol 1972; 11:633-643.

115. Bessey OA, Wolbach SB: Vascularization of the cornea of the rat in riboflavin deficien-cy, with a note on corneal vascularization in vitamin A deficiency. J Exp Med 1939;69:1-12.

116. Wise G: Ocular rosacea. Am J Ophthalmol 1943; 26:591-609.117. Bowles LL, Allen L, Sydenstricker VP, et al: The development and demonstration of

corneal vascularization in rats deficient in vitamin A and in riboflavin. J Nutr 1946;32:19-35.

118. Wolbach SB, Howe PR: Tissue changes following deprivation of fat soluble A vitamin. JExp Med 1925; 42:753-777.

119. Follis RH Jr, Day HG, McCollum EV: Histological studies of the tissues of rats fed adiet extremely low in zinc. J Nutr 1941; 22:223-238.

120. Leure-Dupree AE: Vascularization of the rat cornea after prolonged zinc deficiency.Anat Rec 1986; 216:27-32.

121. Germuth FG, Maumenee AE, Senterfit LB, et al: Immunohistological studies onantigen-antibody reactions in the avascular cornea: I. Reactions in rabbits activelysensitized to foreign protein. J Exp Med 1962; 115:919-928.

122. Olson CL: Subconjunctival steroids and corneal hypersensitivity. Arch Ophthalmol1966; 75:651-658.

123. Honegger H: Hornhautvascularisation: Experimentelle Untersuchung fiber die Rollevon Hornhautquellung und Entzuindung. Albrecht von Graefes Arch Klin Exp Oph-thalmol 1968; 176:239-244.

124. Inomata H, Smelser GK, Polack FM: Corneal vascularization in experimental uveitisand graft rejection: An electron microscopy study. Invest Ophthalmoll971; 10:840-850.

125. Rootman J, Bussanich N, Gudauskas G, et al: Effects of subconjunctivally injectedantineoplastic agents on three models of corneal inflammation. Can J Ophthalmol1985; 20:142-146.

126. Verbey NLJ, van Haeringen NJ, de Jong PTVM: Modulation of immunogenic keratitisin rabbits by topical administration of polyunsaturated fatty acids. Curr Eye Res 1988;7:549-556.

127. Meyer RF, Smolin G, Hall JM, et al: Effect oflocal corticosteroids on antibody-formingcells in the eye and draining lymph nodes. Invest Ophthalmol 1975; 14:138-144.

128. Aronson SB, Fish MB, Pollycove M, et al: Altered vascular permeability in ocularinflammatory disease. Arch Ophthalmol 1971; 85:455-466.

129. Hueper WC, Martin GJ: Tyrosine poisoning in rats. Arch Pathol 1943; 35:685-694.130. Burns RP, Beard ME, Weimar VL, et al: Modification of 1-tyrosine-induced keratopa-

thy by adrenal corticosteroids. Invest Ophthalmol 1974; 13:39-45.131. Gipson IK, Burns RP, Wolfe-Lande JD: Crystals in corneal epithelial lesions of ty-

rosine-fed rats. Invest Ophthalmol 1975; 14:937-941.132. Fons A, Garcia-de-Lomas J, Noqueira JM, et al: Histopathology of experimental

AspergiUus fumigatus keratitis. Mycopathologia 1988;101:129-131.133. Lass JH, Berman MB, Campbell RC, et al: Treatment of experimental herpetic

interstitial keratitis with medroxyprogesterone. Arch Ophthalmol 1980; 98:520-527.134. Hendricks RL, Epstein RJ, Tumpey T: The effect of cellular immune tolerance to

HSV-1 antigens on the immunopathology ofHSV-1 keratitis. Invest Ophthalnol Vis Scm1989; 30:105-115.

135. Lewis PA, Montgomery CM: Experimental tuberculosis of the cornea. J Exp Med1914; 20:269-281.

136. Long ER, Holley SW, Vorwald AJ: A comparison ofthe cellular reaction in experimen-tal tuberculosis of the cornea in animals of varying resistance. Am J Pathol 1933;9:329-336.


137. Rich AR, Follis RH Jr: Studies on the site of sensitivity in the Arthus phenomenon.Johns Hopkins Med J 1940; 66:106-122.

138. Haessler FH: A function of blood in corneal vascularization. TransAm Ophthalmol Soc1927; 25:412-417.

139. Howes EL, Cruse VK, Kwok MT: Mononuclear cells in the corneal response toendotoxin. Invest Ophthalmol Vis Sci 1982; 22:494-501.

140. Folkman J, Weisz PB, Joullie MM, et al: Control of angiogenesis with synthetic heparinsubstitutes. Science 1989; 243:1490-1493.

141. Moticka EJ, She SC: Comparison of failure rates of orthoptic corneal grafts using threedifferent grafting procedures. Curr Eye Res 1989; 8:813-820.

142. Lister A, Greaves DP: Effect of cortisone upon the vascularization which followscorneal burns: I. Heat burns. Br J Ophthalmol 1951; 35:725-729.

143. Folca PJ: Corneal vascularization induced experimentally with corneal extracts. Br JOphthalmol 1969; 53:827-832.

144. Collin HB: Limbal vascular response prior to corneal vascularization. Exp Eye Res1973; 16:443-455.

145. Schanzlin DJ, Cyr RJ, Friedlaender MH: The histopathology of corneal neovasculariza-tion. Arch Ophthalmol 1983; 101:472474.

146. Phillips K, Arffa R, Cintron C, et al: Effects of prednisolone and medroxyprogesteroneon corneal wound healing, ulceration, and neovascularization. Arch Ophthalmol 1983;101:640-643.

147. Robin JB, Regis-Pacheco LF, Kash RL, et al: The histopathology of corneal neo-vascularization: Inhibitor effects. Arch Ophthalmol 1985; 103:284-287.

148. Hoban BP, Collin HB: Effects of salicylate and steroid on neutrophil migration andcorneal blood vessel growth. Am J Optom Physiol Opt 1986; 63:271-276.

149.. Fine BS, Fine S, Feigen L, et al: Corneal injury threshold to carbon dioxide laserirradiation. Am J Ophthalmol 1968; 66:1-15.

150. Nunziata B, Smith RS, Weimar V: Corneal radiofrequency burns: Effects of prostaglan-dins and 48/80. Invest Ophthalmol Vis Sci 1977; 16:285-291.

151. Nikolic L, Friend J, Taylor S, et al: Inhibition of vascularization in rabbit corneas byheparin: Cortisone pellets. Invest Ophthalmol Vis Sci 1986; 27:449-456.

152. Fromer CH, Klintworth GK: An evaluation of the role of leukocytes in the patho-genesis of experimentally induced corneal vascularization: III. Studies related to thevasoproliferative capability of polymorphonuclear leukocytes and lymphocytes. Am JPathol 1976; 82:157-167.

153. Muthukkaruppan VR, Auerbach R: Angiogenesis in the mouse cornea. Science 1979;205:1416-1418.

154. Epstein RJ, Hughes WF: Lymphocyte-induced corneal neovascularization: A morpho-logic assessment. Invest Ophthalmol Vis Sci 1981; 21:87-94.

155. Lutty GA, Thompson DC, Gallup JY, et al: Vitreous: An inhibitor of retinal extract-induced neovascularization. Invest Ophthalmol Vis Sci 1983; 24:52-56.

156. Epstein RJ, Stulting RD: Corneal neovascularization induced by stimulated lympho-cytes in inbred mice. Invest Ophthalmol Vis Sci 1987; 28:1505-1513.

157. Polverini PJ, Cotran RS, Gimbrone MA Jr, et al: Activated macrophages inducevascular proliferation. Nature 1977; 269:804-806.

158. Polverini PJ, Leibovich SJ: Induction of neovascularization in vivo and endothelialproliferation in vitro by tumor-associated macrophages. Lab Invest 1984; 51:635-642.

159. Moore JW, Sholley MM: Comparison of the neovascular effects of stimulated macro-phages and neutrophils in autologous rabbit corneas. Am J Pathol 1985; 120:87-98.

160. Jensen JA, Hunt TK, Scheuenstuhl H, et al: Effect of lactate, pyruvate, and pH onsecretion of angiogenesis and mitogenesis factors by macrophages. Lab Invest 1986;54:574-578.

161. Koch AS, Polverini PJ, Leibovich SJ: Stimulation of neovascularization by humanrheumatoid synovial tissue macrophages. Arthritis Rheum 1986; 29:471-479.

1665

666 Nirankari

162. Gimbrone MA Jr, Leapman SB, Cotran RS, et al: Tumor angiogenesis: Iris neovascular-ization at a distance from experimental intraocular tumors. J Natl Cancer Inst 1973;50:219-228.

163. Gimbrone MA Jr, Cotran RS, Leapman SB, et al: Tumor growth and neovasculariza-tion: An experimental model using the rabbit cornea. J Natl Cancer Inst 1974;52:413-427.

164. Auerbach R, Arensman R, Kubai L, et al: Tumor-induced angiogenesis: Lack ofinhibition by radiation. Int J Cancer 1975; 15:241-245.

165. Brem H, Folkman J: Inhibition of tumor angiogenesis mediated by cartilage. J ExpMed 1975; 141:427439.

166. Brem S: The role of vascular proliferation in the growth of brain tumors. Clin Neuro-surg 1975; 23:440-453.

167. Brem S, Brem H, Folkman J, et al: Prolonged tumor dormancy by prevention ofvascularization in the vitreous. Cancer Res 1976; 36:2807-2812.

168. Langer R, Brem H, Falterman K, et al: Isolation ofa cartilage factor that inhibits tumorneovascularization. Science 1976; 193:70-72.

169. Finkelstein D, Brem S, Patz A, et al: Experimental retinal neovascularization inducedby intravitreal tumors. Am J Ophthalmol 1977; 83:660-664.

170. Preis I, Langer R, Brem H, et al: Inhibition ofneovascularization by an extract derivedfrom vitreous. Am J Ophthalmol 1977; 84:323-328.

171. Felton SM, Brown GC, Felberg NT, et al: Vitreous inhibition oftumor neovasculariza-tion. Arch Ophthalmol 1979; 97:1710-1713.

172. Ryu S, Albert DM: Evaluation of tumor angiogenesis factor with the rabbit corneamodel. Invest Ophthalmol Vis Sci 1979; 18:831-841.

173. Alderman EM, Lobb RR, Fett JW, et al: Angiogenic activity of human tumor plasmamembrane components. Biochemistry 1985; 24:7866-7871.

174. Gospodarowlcz D, Thakral KK: Production of a corpus luteum angiogenic factorresponsible for proliferation of capillaries of neovascularization of the corpus luteum.Proc Natl Acad Sci 1978; 75:847-851.

175. Chen CH, Chen SC: Angiogenic activity of vitreous and retinal extract. Invest Oph-thalmol Vis Sci 1980; 19:596-602.

176. Federman JL, Brown GC, Felberg NT, et al: Experimental ocular angiogenesis. AmJOphthalmol 1980; 89:231-237.

177. Risau W, Ekblom P: Production of a heparin-binding angiogenesis factor by theembryonic kidney. J CeU Biol 1986; 103:1101-1107.

178. Goldsmith HS, Griffith AL, Kupferman A, et al: Lipid angiogenic factor from omen-tum. JAMA 1984; 252:2034-2036.

179. Proia AD, Chung SM, Klintworth GK, et al: Cholinergic stimulation of phosphatidicformation by rat cornea in vitro. Invest Ophthalmol Vis Sci1986; 27:905-908.

180. Silverman LT, Lund DP, Zetter BR, et al: Angiogenic activity of adipose tissue.Biochem Biophys Res Commun 1988; 153:347-352.

181. Dixon JM, Lawaczeck E: Corneal vascularization due to contact lenses. Arch Ophthal-mol 1963; 69:72-75.

182. Dixon JM: Ocular changes due to contact lenses. AmJ Ophthalmol 1964;58:424-443.183. Duffin RM, Weissman BA, Glasser DB, et al: Flurbiprofen in the treatment of corneal

neovascularization induced by contact lenses. Am J Ophthalmol 1982; 93:607-614.184. Katz HR, Duffin RM, Glasser DB, et al: Complications ofcontact lens wear after radial

keratotomy in an animal model. Am J Ophthalmol 1982; 94:377-382.185. Katz HR, Aizuss DH, Mondino BJ: Inhibition of contact lens-induced corneal neo-

vascularization in radial keratotomized rabbit eyes. Cornea 1984; 3:65-72.186. Radnot M, Jobbagyi P: Vascularization of the cornea in ischemia of the anterior

segment. Klin Monatsbl Augenheilkd 1968; 153:840-844.187. Miki H, Yamane A, Tokura T, et al: Corneal neovascularization after anterior uveal

ischemia by occlusion of both long ciliary arteries in rabbit's eye. Proc Int Soc Eye Res1986; 4:17.


188. Yamane A, Tokura T, Nishikawa M, et al: Morphological study of experimental cornealneovascularization after anterior uveal ischemia. Acta Soc Ophthalmol Jpn 1989;93:315-321.

189. Paque J, Poirer RH: Corneal allograft reaction and its relationship to suture siteneovascularization. Ophthalmic Surg 1977; 8:71-74.

190. Harvey PT, Cherry PMH: Indomethacin v. dexamethasone in the suppression ofcorneal neovascularization. Can J Ophthalmol 1983; 18:293-295.

191. Stock EL, Mendelsohn AD, Lo GG, et al: Lipid keratopathy in rabbits: An animalmodel system. Arch Ophthalmol 1985; 103:726.

192. Mendelsohn AD, Stock EL, Lo GG, et al: Laser photocoagulation of feeder vessels inlipid keratopathy. Ophthalmic Surg 1986; 17:502-508.

193. Roth SI, Stock EL, Siel JM, et al: Pathogenesis of experimental lipid keratopathy.Invest Ophthalmol Vis Sci 1988; 29:1544-1551.

194. Corrent G, Roussel TJ, Tseng SC, et al: Promotion of graft survival by photothrombot-ic occlusion of corneal neovascularization. Arch Ophthalmoll989; 107:1501-1506.

195. Epstein RJ, Stulting RD, Rodrigues MM: Corneal opacities and anterior segmentanomalies in DBA/2 mice: Possible models for corneal elastosis and the iridocornealendothelial (ICE) syndrome. Cornea 1986; 5:95-105.

196. Billingham RE, Boswell T: Studies on the problem of corneal homografts. Proc R SocLond (Biol) 1953; 141:392-406.

197. Khodadoust AA, Silverstein AM: Studies on the nature of the privilege enjoyed bycorneal allografts. Invest Ophthalmol 1972; 11:137-148.

198. Polack FM: Editorial on recent advances: Corneal transplantation. Invest Ophthalmol1973; 12:85-86.

199. Khodadoust AA: The allograft rejection reaction: The leading cause of late failure ofclinical corneal grafts, in R Porter, J Knight (eds): Corneal Graft Failure. CibaFoundation Symposium 15. Amsterdam, Elsevier, 1973, pp 151-167.

200. Batchelor JR, Casey TA, Werb A, et al: HLA matching and corneal grafting. Lancet1976; 1:551-554.

201. Eye Banking Statistics, Washington, DC, Eye Bank Association of America, 1991, p 1.202. Klein PE, Stark WJ, Maguire MG, et al: Donor-recipient crossmatching and typing to

avoid corneal allograft rejection, in HD Cavanaugh (ed): The Cornea: The WorldCongress on the Cornea III. New York, Raven Press, 1988, pp 395-398.

203. Collaborative Corneal Transplantation Studies (CCTS), in Clinical Trials Supported bythe National Eye Institute. US Department of Health and Human Services, PublicHealth Service, National Institutes of Health, Bethesda NIH publication 90-2910,1990, pp 63-65.

204. Klintworth GK: Hypotheses about the pathogenesis of corneal vascularization, in GKKlintworth (ed): Corneal Angiogenesis: A Comprehensive Critical Review. New York,Springer-Verlag, 1991, pp 23-29.

205. Current concept of corneal angiogenesis, in GK Klintworth (ed): CornealAngiogenesis: A Comprehensive Critical Review. New York, Springer-Verlag, 1991, pp51-53.

206. Maurice DM, Zauberman H, Michaelson IC: The stimulus to neovascularization in thecornea. Exp Eye Res 1966; 5:168-184.

207. Klintworth GK: The hamster cheek pouch: An experimental model ofcorneal vascular-ization. Am J Pathol 1973; 73:691-710.

208. : The contribution of morphology to our understanding of the pathogenesis ofexperimentally produced corneal vascularization. Invest Ophthalmol Vis Sci 1977;16:281-285.

209. Auerbach R, Kubai L, Sidky Y: Angiogenesis induction by tumors, embryonic tissuesand lymphocytes. Cancer Res 1976; 36:3435-3440.

210. Auerbach R, Sidky YA: Nature of the stimulus leading to lymphocyte-induced angio-genesis. J Immunol 1979; 123:751-754.

667

668 Nirankari

211. Mostafa LK, Jones DB, Wright DH: Mechanism of the induction of angiogenesis byhuman neoplastic lymphoid tissue: Studies employing bovine aortic endothelial cellsin vitro. J Pathol 1980; 132:207-216.

212. Watt SL, Auerbach R: A mitogenic factor for endothelial cells obtained from mousesecondary mixed leukocyte cultures. J Immunol 1986; 136:197-202.

213. Kaminski M, Auerbach R: Angiogenesis induction by CD4 positive lymphocytes. ProcSoc Exp Biol Med 1988; 188:440-443.

214. Knighton DR, Hunt TK, Scheuenstuhl H, et al: Oxygen tension regulates the expres-sion of angiogenesis factor by macrophages. Science 1983; 221:1293-1295.

215. Smith SS, Basu PK: Mast cells in corneal immune reaction. Can J Ophthalmol 1970;5:175-183.

216. Klintworth GK: Nature of angiogenic factor, in GK Klintworth (ed): Corneal Angio-genesis: A Comprehensive Critical Review. New York, Springer-Verlag, 1991, pp 45-50.

217. Klintworth GK: Sequence of events in corneal neovascularization, in GK Klintworth(ed): Corneal Angiogenesis: A Comprehensive Critical Review. New York, Springer-Verlag, 1991, pp 5-10.

218. Michaelson IC: Effect of cortisone upon corneal vascularization produced experimen-taly. Arch Ophthalmol 1952; 47:459-464.

219. Hughes WF, Bloome MA, Tallman CB: Prevention of corneal vascularization.Am JOphthalmol 1968; 66:1118.

220. Williams KA, Grutzmacher RD, Roussel TJ, et al: A comparison of the effects of topicalcyclosporine and topical steroid on rabbit corneal allograft rejection. Transplant 1985;39:242-244.

221. Beck DW, Olson JJ, Linhardt RJ: Effect of heparin, heparin fragments, and cor-ticosteroids on cerebral endothelial cell growth in vitro and in vivo. J Neuropathol ExpNeurol 1986; 45:503-512.

222. Hase S, Nakazawa S, Tsukamoto Y, et al: Effects ofprednisolone and human epidermalgrowth factor on angiogenesis in granulation tissue of gastric ulcer induced by aceticacid. Digestion 1989; 42:135-142.

223. Zurier RB, Weissman G: Anti-immunologic and anti-inflammatory effects of steroidtherapy. Med Clin North Am 1973; 57:1295-1307.

224. Haynes WL, Proia AD, Klintworth GK: Effect of inhibitors ofarachidonic acid metabo-lism on corneal neovascularization in the rat. Invest Ophthalmol Vis Sci 1989; 30:1588-1593.

225. Deutsch TA, Hughes WF: Suppressive effects of indomethacin on thermally inducedneovascularization of rabbit corneas. Am J Ophthalmol 1979; 87:536-540.

226. Salisbury JD, Gebhardt BM: Suppression of corneal allograft rejection by cyclosporinA. Arch Ophthalmol 1981; 99:1640-1643.

227. Hunter PA, Wilhelmus KR, Rice NSC, et al: Cyclosporin-A applied topically to therecipient eye inhibits corneal graft rejection. Clin Exp Immunol 1981; 45:173-177.

228. Kana JH, Hoffman F, Buchen R, et al: Rabbit corneal allograft survival followingadministration of cyclosporin-A. Invest Ophthalmol Vis Sci 1982; 22:686-690.

229. Belin MW, Bouchard CS, Frantz S, et al: Topical cyclosporine in high-risk cornealtransplants. Ophthalmology 1989; 96:1144-1150.

230. Thiru S, Calne RY, Nagington J: Lymphoma in renal allograft patients treated withCyclosporin-A as one of the immunosuppressive agents. Transplant Proc 1981; 13:359-364.

231. Sweny P, Farrington K, Younis F, et al: Sixteen months experience with Cyclosporin-Ain human kidney transplantation. Transplant Proc 1981; 13:365-367.

232. Eliason JA: Leukocytes and experimental corneal vascularization. Invest OphthalmolV's Sri 1978; 17:1087-1095.

233. Sholley MM, Gimbrone MA, Cotran JS: The effects of leukocyte depletion on cornealneovascularization. Lab Invest 1978; 38:32-40.

234. Scroggs MW, Proia AD, Smith CF, et al: The effect of total-body irradiation on cornealneovascularization in the Fischer 344 rat after chemical cauterization. Invest Ophthal-nml Vis Sci 1991; 32:2105-2111.


235. Lavergne G, Colmant IA: Comparative study of the action of thiotepa and triam-cinolone on corneal vascularization in rabbits. Br J Ophthalmol 1964; 48:416-422.

236. Ey RC, Hughes WF, Bloome MA, et al: Prevention of corneal vascularization. Am JOphthalmol 1968; 66:1118-1131.

237. Ainslie D, Snelling MD, Ellis RE: Treatment of corneal vascularization by strontium90 beta plaque. Clin Radiol 1962; 13:29.

238. Mayer W: Cryotherapy in corneal vascularization. Arch Ophthalmol 1967; 77:637-641.239. Cherry PMH, Faulkner JD, Shaver RP, et al: Argon laser treatment of corneal neo-

vascularization. Ann Ophthalmol 1973; 5:911-920.240. Cherry PMH, Garner A: Corneal neovascularization treated with argon laser. Br J

Ophthalmol 1976; 60:464-472.241. Reed JW, Fromer C, Klintworth GK: Induced corneal vascularization remission with

argon laser therapy. Arch Ophthalmol 1975; 93:1017-1019.242. Marsh RJ, Marshall J: Treatment of lipid keratopathy with the argon laser. Br J

Ophthalmol 1982; 66:127-135.243. Marsh RJ: Lasering of lipid keratopathy. Trans Ophthalmol Soc UK 1982; 102:154-156.244. Nirankari VS, Baer JC: Corneal argon laser photocoagulation for neovascularization in

penetrating keratoplasty. Ophthalmology 1986; 93:1304-1309.245. Baer JC, Foster JC: Corneal laser photocoagulation (CLP) for treatment ofneovascular-

ization (NV): Efficacy of577 nm yellow dye laser. Ophthalmology (Suppl) 1989; 96:106.246. Nirankari VS, Gross SC, Bauer SA, et al: Corneal laser photocoagulation (CALP) of

stromal vascularization: A prospective study in rabbits. Invest Ophthalmol Vis Sci(Suppl) 1991; 32:996.

247. Mendelsohn AD, Watson BD, Alfonso EC, et al: Amelioration of experimental lipidkeratopathy by photochemically induced thrombosis of feeder vessels. Arch Ophthal-mol 1987; 105:983-988.

248. Huang AJW, Watson BD, Hernandez E, et al: Photothrombosis ofcorneal neovascular-ization by intravenous rose bengal and argon laser irradiation. Arch Ophthalmol 1988;106:680-685.

249. Huang AJW, Watson BD, Hernandez E, et al: Induction of conjunctival transdifferen-tiation on vascularized corneas by photothrombotic occlusion of corneal neovascular-ization. Ophthalmology 1988; 95:228-235.

250. Corrent G, Roussel TJ, Tseng SCG, et al: Promotion of graft survival by photothrom-botic occlusion of corneal neovascularization. Arch Ophthalmol 1989; 107:1501-1506.

251. Epstein RJ, Hendricks RL, Harris DM: Photodynamic therapy for corneal neovascular-ization. Cornea 1991; 10:424432.

252. Prince JH: Introduction: The rabbit eye related to that of man, in JH Prince (ed): TheRabbit in Eye Research. Springfield, Charles C Thomas, 1964, pp ix-xiv.

253. Cornea, trabecular region, and sclera, in JH Prince (ed): The Rabbit in EyeResearch. Springfield, Charles C Thomas, 1964, pp 86-139.

254. Klyce SD, Beuerman RW: Structure and function of the cornea, in HE Kaufman, BABarron, MB McDonald, et al, (eds): The Cornea. New York, Churchill Livingstone,1988, pp 3-54.

255. Billings EE, Nirankari VS: Corneal angiography of ghost vessels useful in evaluatingrisks of corneal surgery. J Ophthalmic Photog 1981; 4:6-10.

256. L'Esperance FA: Clinical photocoagulation with the organic dye laser: A preliminarycommunication. Arch Ophthalmol 1985; 103:1312-1316.

257. Dandona L, Morrison JC, Robin AL, et al: "Outlier" intraocular pressure readings:Valid or invalid for statistical analysis? Ophthalmic Surg 1989; 20:737-743.

258. Batchelor JR, Casey TA, Werb A, et al: HLA matching and corneal grafting. Lancet1976; 1:551-554.

259. Sanfilippo F, MacQueen JM, Vaughn WK, et al: Reduced graft rejection with goodHLA-A and B matching in high-risk corneal transplantation. N Engl J Med 1986;315:29-35.

260. Stark WJ, Stulting RD, Thoft R: HLA matching and corneal transplantation. N EnglIJMed [Letter] 1987; 317:1476.

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laser photocoagulation for corneal stromal vascularization

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