An Experimental Steroid Responsive Model of OcularInflammation in Rabbits Using an SLT Frequency

Doubled Q Switched Nd:YAG Laser

Vikas Gulati, Sandhya Pahuja, Shan Fan, and Carol B. Toris

Abstract

Purpose: To develop a minimally invasive rabbit model of postoperative anterior chamber (AC) inflammationusing a commercially available frequency doubled Nd:YAG laser [intended for selective laser trabeculoplasty(SLT)].Methods: Escalating laser energy was applied to the iris of male Dutch-belted rabbits and the subsequentinflammatory response was observed to determine the laser dose required to generate self-limiting inflammationof at least 3 days’ duration. In subsequent experiments, 10 eyes of 10 male Dutch-belted rabbits underwentbaseline slit lamp examination, intraocular pressure (IOP), and AC flare meter readings. Starting 1 day beforelaser application, 5 animals received topical 20 mL dexamethasone 1% to 1 eye 4 times daily for 5 days. Fivecontrol animals were treated with saline. Masked assessments of flare, cells, and IOP were made daily for 7 days.Histopathologic changes were assessed in enucleated eyes.Results: Compared to controls, dexamethasone-treated rabbits had less postlaser AC flare on postoperativeday (POD)2 (19 – 5 vs. 44 – 21photons/ms, P = 0.03) and POD3 (16 – 9 vs. 33 – 11 photons/ms, P = 0.03). Indexamethasone-treated rabbits, clinically graded flare (on POD1) and cells (on POD1 and 2) were lower thancontrols, but did not reach statistical significance. In the control group, IOP was significantly lower than thedexamethasone-treated group on POD2 (14.1 – 3.4 vs. 19.8 – 1.1 mmHg, P = 0.03) and POD3 (14.2 – 2.2 vs. 19.0 –2.2 mmHg, P = 0.01). Histopathology showed pigment clumping and changes limited to anterior layers of the iris.Conclusions: Commercially available SLT laser can be used to create a minimally invasive, steroid-responsiveanimal model of anterior uveitis with the potential for use in the evaluation and comparison of drugs intended totreat AC inflammation.

Introduction

Anterior uveitis is a common1 clinical presentation thatcan leave visually blinding sequelae if not treated

promptly and effectively.2 Anterior uveitis presents itself asthe breakdown of the blood–aqueous barrier manifested asthe appearance of leukocytes and leakage of proteins (flare)in the aqueous humor and changes in intraocular pressure(IOP). Animal models of uveitis play a critical role in drugdevelopment and the understanding of the pathophysiologyof uveitis.

A number of experimental animal models of uveitis havebeen reported. Several retinal antigens have been used fortheir uveitogenic properties, to immunize various animals,and thereby induce experimental autoimmune uveoretinitis(EAU).3,4 Another model called the experimental melanin

protein-induced uveitis uses bovine uveal tissue antigens forimmunization to induce inflammation that is more limited tothe uveal tissues.5,6 Other procedures to generate uveitisinclude, topical application of prostaglandin E2 (PGE2),7 aparacentesis procedure,8 subcutaneous injection of the my-cobacterium tuberculosis H37Ra antigen,9,10 intravitreal in-jections of bovine serum,11 horse serum,12 or egg albumin,13

intracameral injection of streptococcus beta-hemolytic groupA,14 and intravenous, intraperitoneal, or footpad injection ofendotoxin in rats.15

Some methods, such as the use of topical PGE2 for in-ducing anterior segment inflammation are fairly short induration, providing limited opportunity for observing theeffect of a therapeutic agent. Most of the above methods areinvasive, have limited repeatability because of dependenceon the individual animal’s variable immune response and

Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.

JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICSVolume 29, Number 7, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/jop.2012.0223

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can leave permanent blinding sequelae. These immunogenicprocedures can cause severe inflammation for months, andrequire sacrifice of the study animals. Also, immunologicallyinduced inflammation differs significantly from postoperativeinflammation. In the former, the source of inflammation re-mains in place until the inciting antigen is cleared by thebody’s defense mechanisms. In autoimmunity-mediatedmechanisms, the pathological process is ongoing despite theremoval of the inciting antigen from the body. On the otherhand, postoperative inflammation is likely a one-time trau-matic insult that leads to the breakdown of the blood–aqueousbarrier. Hence, antigenically induced inflammation may notbe the best model to study postoperative anti-inflammatorytherapies. Also, models that involve inadvertent posteriorsegment inflammation, along with the desired anterior seg-ment inflammation, may not be the best for evaluation oftopical therapies with excellent anterior segment penetrationand potency, but poor posterior segment penetration.

A new, less invasive and more predictable uveitismodel will be a valuable tool in the development of anti-inflammatory therapies. Also, a transient inflammation of afew days duration may allow adequate time for study ofanti-inflammatory therapy with limited consequences for thestudy animal.

Selective laser trabeculoplasty (SLT) is used clinically as atreatment for elevated IOP.16 A frequency doubled 532 nm QSwitched Nd:YAG laser is applied as fixed spots of 400mm insize and energy of 0.4–2.0 millijoules (mJ) for 3 ns each to thetrabecular meshwork. The IOP usually is reduced for monthsto years after the procedure, with minimal histopathologicchanges.17 The low fluence, short duration laser energy, basedon the principles of selective photothermolysis, targets intra-cellular melanin, causing no coagulative damage, and littlestructural change to the surrounding tissues.18–21 Applicationof SLT spots to the pigmented iris tissue offers an opportunityto create anterior segment inflammation with limited tissuedamage. To the best of our knowledge, this method of creatingexperimental anterior uveitis has not been reported previously.

The goal of this study was to evaluate the possibility ofgenerating self-limited anterior segment inflammation in arabbit model using application of SLT spots directly to theiris tissue. This report details the preliminary work involvedin determining the laser energy required to generate suchinflammation. The histopathologic consequences of laserapplications were studied. The steroid responsiveness ofthe model was tested to demonstrate a potential role for themodel in the study of anti-inflammatory therapies for thetreatment of anterior uveitis.

Methods

The study was conducted in 3 phases. All animal experi-ments described herein were approved by the InstitutionalAnimal Care and Use Committee of the University of Ne-braska Medical Center before the study start. The researchdescribed here adhered to the ARVO Statement for the Useof Animals in Ophthalmic and Visual Research. In Phase 1,the irides of 2 enucleated rabbit eyes were subjected to theescalating power of SLT laser ranging from 0.4 to 1.2 mJ. Thiswas done to confirm the absence of any gross coagulativeand disruptive changes to ocular tissues.

The purpose of Phase 2 of the study was to determine theenergy level and number of spots required to produce

inflammation of a few days duration. The objective was tocreate inflammation that was self-limiting without significantdamage to ocular tissues, yet long enough to allow the studyof uveitis treatment modalities. The specific power andnumber of spots were chosen empirically given the lack ofavailable data from similar experiments. Male Dutch-beltedrabbits, 37 to 40 weeks of age, were used in this phase of thestudy. While the animal was anesthetized with a cocktail ofketamine (15 mg/kg) and xylazine (3.75 mg/kg) adminis-tered intramuscularly, a slit lamp examination was per-formed and baseline measurements were made. After atopical drop of proparacaine (0.5%) anesthetic to the cornea,ocular measurements included IOP by pneumatonometry(Classic 30; Reichert Ophthalmic Instruments, Inc., Depew,NY) and flare readings with a laser flare-cell meter (KOWAmodel FM 600). The cells and flare were graded according tothe SUN Working Group Grading Scheme.22 Laser treat-ments commenced immediately following baseline exami-nations and measurements. Escalating doses were used toestablish the power and number of nonoverlapping spotsneeded to generate a mild inflammation that persisted for atleast 3 days. A doubling of the baseline flare value wasconsidered significant. Measurements were continued for upto 3 days with a follow-up measurement at 7 days to confirma return of flare to baseline levels. Initially, 0.6 and 1.0 mJ ofenergy were used and 30 spots were placed along the pe-ripheral iris of 1 eye of 1 rabbit each. Postoperatively, ani-mals were examined for the presence of flare for up to 3days. Subsequent protocols included 50 and 100 spots of1 mJ, 50 spots of 2.0 mJ, 100 spots of 1.5 mJ, and 100 and 150spots of 2.0 mJ. The maximum laser power (200 spots of 2 mJ)was limited by the maximum available energy from thecommercial SLT instrument and the number of spots waslimited by the area available for laser spot placement on theiris tissue. Laser spots were uniformly applied over the iristissue starting at the most peripheral iris and covering themore central tissue as the number of spots increased. Ininitial experiments, spots were at least 1 spot width apart.However, experiments with a greater number of spots re-quired a more confluent placement of laser spots. The central1–2 mm of the iris was not treated because of the potential ofinjury to the iris sphincter and native lens. Pneumatono-metry, slit lamp biomicroscopy, and laser flare meter read-ings were performed on all study days. The endpoints usedfor the assessment of inflammation thus generated wereclinically observed cells and flare, and objective flare mea-surements by an aqueous flare meter.

In phase 3 of the study, the response of the observed in-flammation to topical steroids was evaluated. Ten rabbitswere divided into 2 groups of 5 each; Group A was ad-ministered one 20mL drop of dexamethasone 1%, 4 timesdaily (9am, 11am, 2pm, and 5pm) to one randomly choseneye for 5 days starting 1 day before SLT. The same admin-istration schedule was applied to Group B, which was dosedwith 20mL sterile saline (Eye Stream; Alcon, Ft. Worth, TX)instead of dexamethasone. Based on the results of Phase 2, aregimen of 200 spots of 2 mJ each was selected for lasertreatment. Tonometry, slit lamp examination, and laser flaremeter readings were repeated as described in Phase 2.Measurements were made by an observer masked to theassigned treatment of each rabbit. All rabbits used in Phase 3had been lasered at least once before during phase 2 of thestudy. Animals were reused for this phase after a return to

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baseline (as assessed by cells, flare, and IOP). All values arereported as mean – standard deviation unless otherwise no-ted. Data means were compared using paired t-tests forcontinuous variables and nonparametric equivalents fordiscrete ordinal variables. Seven days after the laser treat-ment for Phase 3, the study animals were sacrificed with anoverdose of intravenous sodium pentobarbital (150 mg/kg)and histopathologic examinations were performed on oculartissues.

Results

Phase 1

No obvious tissue shrinkage or disruption was observedduring the application of SLT to enucleated eyes. The ob-served tissue response comprised of microcavitation bubbleformation, minimal flattening of the iris surface corre-sponding to the area of the laser spot, and tissue blanching inthe area of the laser spot. Greater blanching was observedwith higher laser powers and in the eye with subjectivelygreater pigmentation.

Phase 2

Phase 2 of this study involved observations at increasinglaser power and number of spots to produce a consistentflare response. Values after total laser application in paren-theses represent laser energy per spot times the number ofspots applied. In 1 rabbit treated with 18 mJ (0.6 mJ · 30spots), no significant increase in flare was observed on day 1.At 30 mJ (1 mJ · 30 spots), significant flare was observed onday 1, which returned to baseline by day 2 (n = 1). In 3 of the4 rabbits treated with 50 mJ (1 mJ · 50 spots), 1 rabbit treatedwith 75 mJ (1.5 mJ · 50 spots), and 1 rabbit treated with100 mJ (2 mJ · 50 spots), no significant increase in flare wasobserved on day 1. Two rabbits treated with 112.5 mJ(1.5 mJ · 75 spots) had significant flare on days 1 and 2 re-turning to baseline by day 3. At higher doses, a more con-sistent and sustained response was observed (Fig. 1). At400 mJ (2 mJ · 200 spots), 4 of 4 rabbits treated had increasedflare in the anterior chamber (AC) that persisted for at least 3

days and returned to baseline values by day 7. Based onthese experiments, 200 spots of 2 mJ each was chosen as thelaser power for Phase 3.

Phase 3

This phase of the study evaluated the steroid responsive-ness of the inflammation generated by the laser. Rabbitstreated with dexamethasone had an increase in flare froma baseline of 9.7 – 2.9 photons/ms to a peak of 92.0 – 68.5photons/ms on day 1, which progressively reduced to 23.0 –3.8 photons/ms by day 5 (Fig. 2). In the control group, flareincreased from a baseline of 15.7 – 6.5 photons/ms to a peakof 85.5 – 42.6 photons/ms on day 1, subsequently, progres-sively resolving by day 5, to 18.8 – 7.8 photons/ms. Flare wassignificantly higher in the control group as compared to thedexamethasone-treated group on day 2 (43.7 – 21.2 photons/msvs. 19.1 – 4.8 photons/ms, P = 0.03) and day 3 (33.4 – 11.0photons/ms vs. 16.1 – 8.7 photons/ms, P = 0.03) after laser.There was no difference in observed flare between the 2groups at baseline or on postlaser days 1, 4, and 5.

No cells or flare were detected in either group by the slitlamp examination at baseline. Clinically graded flare peakwas 1.8 – 0.4 on days 1 and 2 in the dexamethasone groupand 2.4 – 0.5 on day 1 in the control group (P = 0.09) with asubsequent progressive resolution in either group (Fig. 3).Cells peaked on day 2 in both the dexamethasone (2.2 – 0.8)and control group (2.6 – 0.5) with a subsequent resolution byday 5 (Fig. 4).

At most observation times (except in the control group onday 4), IOP was significantly lower in both groups comparedto their respective baselines. Compared to the treatmentgroup, IOP was significantly lower in the control group ondays 2 (14.1 – 3.4 mmHg vs. 19.8 – 1.1 mmHg, P = 0.03) and 3(14.1 – 2.2 mmHg vs. 19.0 – 2.2 mmHg, P = 0.01), coincidingwith the days of significantly higher flare in the controlgroup (Fig. 5). The significance of IOP lowering on thesedays is limited by the lack of IOP matching at baseline where

FIG. 1. Flare response of laser-treated eyes at increasinglaser energy levels. Laser spots were applied immediatelyfollowing baseline flare measurements. A dose–response re-lationship was observed between the laser energy appliedand the ensuing anterior chamber flare.

FIG. 2. Comparison of flare by a laser flare meter in thedexamethasone- and saline-treated groups after laser treat-ment to the iris. Laser spots were applied immediately fol-lowing baseline measurements. Error bars represent SEM.P-values were obtained using the unpaired t-test comparingthe 2 groups. Asterisks indicate a statistically significantdifference.

RABBIT MODEL OF LASER-INDUCED ANTERIOR UVEITIS 665

the control group had a lower baseline IOP than the treat-ment group (24.6 – 2.1 vs. 28.7 – 2.1 mmHg, respectively,P < 0.01).

Histopathology

On gross examination, tissue changes in the enucleatedspecimens were limited to the iris tissue with large areas ofpatchy depigmentation observed on the anterior iris surface,corresponding to the application of laser spots (Fig. 6a, b).Histopathologic changes were limited to the anterior layersof the iris and comprised of localized flattening of the surfacestroma with underlying large clumps of extra and intracel-

lular pigment (Fig. 7a, b). The localized flattening did notshow any evidence of adjacent coagulative changes. The fi-brovascular structures of the iris and the posterior layersappeared fairly well preserved in the lasered eyes. Nochanges were observed in the cornea, ciliary body, lens,retina, or choroid when comparing the lasered eye to thecontralateral control eye.

Discussion

This study describes a novel model of ocular inflammationin rabbits utilizing a commercially available frequency dou-bled Nd:YAG laser. Anterior segment inflammation canresult from a myriad of causes with postoperative inflam-mation being one of the most common encountered in anyclinical practice. The inflammation observed in our study ismore akin to surgically induced inflammation rather thanone produced by the introduction of foreign antigens. Thelatter is more likely to simulate inflammation from auto-immune and infectious causes.

The laser paradigm provides a longer window of obser-vation of the inflammatory response from a single insult, ascompared to the much shorter duration of inflammationgenerated using topical prostaglandins.7 The model is lessinvasive than one that requires injections of antigens into theeye; the intraocular placement of the needle alone can gen-erate ocular inflammation.8 Because no needle is insertedinto the eye, the current model avoids any accidental intro-duction of microorganisms and ocular infection. Infectioncan increase the amount of inflammation observed and likelywill not be responsive to an anti-inflammatory drug beingevaluated in a study. This potentially could make the drugunder study appear less efficacious in controlling inflam-mation and the results would be harder to interpret.

Dutch-belted rabbits tolerate the laser treatment well,based on behavior and the return to baseline of all observedparameters by 1 week. The changes observed on histopa-thology were very limited. Given the clinical experience with

FIG. 3. Comparison of clinically graded flare (grades of 0 to4) in the dexamethasone- and saline-treated groups after la-ser treatment. Laser spots were applied immediately fol-lowing baseline measurements. Error bars represent SEM.The P-value was obtained using the Mann–Whitney test.Asterisk indicates a borderline statistically significantdifference.

FIG. 4. Comparison of clinically graded cells (grades of 0 to4) in the dexamethasone- and saline-treated groups after la-ser treatment. Laser spots were applied immediately fol-lowing baseline measurements. Error bars represent SEM.The difference between the groups did not reach statisticalsignificance.

FIG. 5. Comparison of intraocular pressure in the dexa-methasone- and saline-treated groups after laser treatment.Error bars represent SEM. The difference between the groupswas significant at baseline and postoperative days 2 and 3.P-values were obtained using the unpaired t-test. Asterisksindicate a statistically significant difference.

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SLT and the observed minimal histopathologic changes tothe tissues,20 the generation of inflammation can be repeatedin the same animal.

Effects of laser application to iris tissue with lasers otherthan the SLT have been reported previously.23–28 To thebest of our knowledge, this is the first report of the effects ofapplication of SLT laser directly to the iris tissue. Comparedto argon laser, SLT laser is devoid of coagulative and me-chanical tissue damage when applied to the trabecularmeshwork of eye bank eyes.20 SLT laser uses a small frac-tion of the energy needed by argon laser in performing lasertrabeculoplasty. SLT laser selectively targets the pigmentedcells in the target tissues leading to cracking of in-tracytoplasmic pigment granules and disruption of endo-thelial cells.19 This may explain why minimal changes wereobserved in the histopathology sections despite large areasof depigmentation observed grossly on the tissue surface.The depigmenting properties of Q switched frequencydoubled Nd:YAG laser have been used for the treatment ofpigmented hypertrophic scars in the field of dermatology.29

When used clinically to perform trabeculoplasty, the 400 mmspot size of the SLT laser can lead to some inadvertent

overlap with tissues anterior or posterior to the trabecularmeshwork. The histopathologic findings of this study sug-gest that some overlap of laser spots on the iris tissueduring trabeculoplasty at clinically used energy levels maybe innocuous. However, the application of laser spots to thehuman iris tissue has not been studied and cannot be con-sidered safe without additional investigation.

The exact mechanism by which the laser generated in-flammation in our model has several possible explanations.The inflammation may have been a consequence of themechanical stimulation of the iris (without photocoagula-tion) and melanocyte disruption secondarily leading to aninflammatory response. Alternatively, the breakdown of theblood–aqueous barrier may have been a direct effect of thelaser spots. The peak absorption of hemoglobin is close tothe 532 nm wavelength of frequency doubled Nd:YAGlaser.30 Absorption of such energy by blood vessels formsthe basis of treatment of port wine stains with Nd:YAGlaser.31 A highly vascular tissue like the iris may absorbenergy by the circulating red blood cells that in turn inducea breakdown of the blood–aqueous barrier.

The reduction in measured flare by dexamethasone sup-ports the steroid responsiveness of our model. The lack of

FIG. 7. Photomicrograph of hematoxylin and eosin stainediris cross-section of a rabbit eye, (a) treated with laser spotsand (b) a contralateral control eye. The control eye showsnormal iris architecture. The lasered eye shows surface flat-tening with underlying pigment clumps. The fibrovascularstructure of the iris appears fairly well preserved.

FIG. 6. The iris tissue of the enucleated rabbit eye, (a) la-sered with 200 spots of 2 mJ and (b) the contralateral un-treated eye. The laser-treated eye showed large areas ofpatchy depigmentation corresponding to the areas of appli-cation of laser spots.

RABBIT MODEL OF LASER-INDUCED ANTERIOR UVEITIS 667

difference in flare between the 2 groups on day 1, but thesignificantly reduced flare in steroid-treated animals on days2 and 3, suggest that steroid pretreatment did not alter theimpact of the initial laser insult to the tissues. However, eyestreated with topical steroid had less flare and higher IOPduring resolution of the inflammation than control eyes.These results suggest that the model may be useful forcomparison or preclinical evaluation of anti-inflammatorytherapies used to control postoperative inflammation. Apredictable, simple model such as the one currently de-scribed can facilitate a quick screening of drug moleculesbeing evaluated for potential therapies.

The measurement of flare using a flare meter is a validatedtechnique for the study of ocular inflammation.32 Clinical as-sessment of cells and flare is a subjective technique at best,with significant inter- and intra- observer variability despitewell-described grading scales.33 The flare meter can circum-vent this problem by providing a measure of inflammation ona continuous scale, thereby significantly reducing the samplerequired to obtain statistically significant differences. For ex-ample, even though the difference in flare meter readingsreached statistical significance on days 2 and 3 with a samplesize of 5, the sample size required for the difference in cells tobe significant between the 2 groups in our study (with apower of 80%, and alpha error of 0.05) will be 42 rabbit eyes.

Our study and model have several limitations. The samplesize was small. This study was planned as a proof-of-conceptstudy and no ad hoc sample calculations were made. This wasdone to minimize the number of animals affected during theexecution of the study. Even with the limited sample size, thegoals of the study were accomplished. The limited samplenumber, with reuse of some of the study animals after returnto baseline, was sufficient to establish the dose of laser neededto generate clinically useful inflammation. A statistically sig-nificant difference in objectively measured flare and borderlinesignificant difference in subjectively graded flare could bedemonstrated with the use of a potent steroid. The data ob-tained from this study potentially can be used for sample sizecalculations of future planned studies. Because of technicallimitations of the commercially available laser, we were unableto establish the peak of the dose–response curve for the laserapplication. The highest response was seen with the highestpower of the laser used. It may be possible to generate moreand longer lasting flare with even higher laser energy levels. Inthe future, a custom-designed laser with higher available en-ergy levels may be able to accomplish this. Nevertheless, useof the commercially available version of the laser generatedclinically useful flare without significant trauma to the studyanimals. As mentioned above, this model likely representssurgically induced inflammation and is not likely to be rep-resentative of other endogenous infectious and noninfectiousuveitis encountered in clinical practice. The mean flare gen-erated on day 1 was significantly lower in phase 3 of the studyas compared to phase 2 for an identical dose of the laser. Allstudy animals used in phase 3 had been lasered at least oncepreviously in the same eye in phase 2 of the study, withvarying amount of laser applied to each individual rabbit.Repetition of laser in the same animal, after a return of flarereading to baseline, was done to minimize the number ofanimals sacrificed for this pilot study. This may explain alower amount of flare seen in phase 3 with 400 mJ of laserenergy used as compared to phase 2, where repeated appli-cation of laser to the same animal may be associated with

diminishing returns in terms of the flare generated. This issimilar to a lower initial efficacy of SLT laser therapy uponrepeat application, as compared to initial application, whenused for trabeculoplasty in humans.34 However, this shouldnot affect the conclusions from phase 3 of the study as both thecontrol and dexamethasone-treated eyes had received priorlaser and the purpose of this phase was to determine the ste-roid responsiveness of the inflammation generated. As anadditional limitation, as with any animal model, the resultsobtained from this model may or may not apply to humansituations. The blood–aqueous barrier in rabbits is known tobe less robust than that in primates.35 However, there areseveral reasons to use rabbits. They are relatively easy tohandle and maintain and may be the species of choice for drugscreening experiments before proceeding with more costly androbust primates. Dutch-belted rabbits are preferred over al-bino rabbits because of the pigmented iris, which may benecessary for the needed tissue response to laser.

In summary, this study describes a clinically useful, ste-roid responsive AC model of inflammation in Dutch-beltedrabbits by using a commercially available Nd:YAG laser.Future studies involving larger numbers, other species likenonhuman primates, and additional anti-inflammatorymedications will help determine the validity of the model ina wider variety of experimental and clinical circumstances.

Acknowledgments

The authors extend a special thanks to Tara Rudebush,Stacey Wenthur, and Brooke Dworak, for their help withanimal handling, and to Dr. Gerald Christensen andDr. William West for their expertise in interpreting thehistology slides. Support: Research to Prevent Blindness,Allergan Horizon Grant, Pfizer.

Author Disclosure Statement

No competing financial interests exist.

References

1. Linssen, A., Rothova, A., Valkenburg, H.A., et al. The life-time cumulative incidence of acute anterior uveitis in anormal population and its relation to ankylosing spondylitisand histocompatibility antigen HLA-B27. Invest. Ophthalmol.Vis. Sci. 32:2568–2578, 1991.

2. Menezo, V., and Lightman, S. The development of compli-cations in patients with chronic anterior uveitis. Am. J.Ophthalmol. 139:988–992, 2005.

3. Wacker, W.B., and Lipton, M.M. Experimental allergicuveitis: homologous retina as uveitogenic antigen. Nature.206:253–254, 1965.

4. Adamus, G., and Chan, C.C. Experimental autoimmuneuveitides: multiple antigens, diverse diseases. Int. Rev. Im-munol. 21:209–229, 2002.

5. Broekhuyse, R.M., Kuhlmann, E.D., Winkens, H.J., and VanVugt, A.H. Experimental autoimmune anterior uveitis(EAAU), a new form of experimental uveitis. I. induction bya detergent-insoluble, intrinsic protein fraction of the retinalpigment epithelium. Exp. Eye Res. 52:465–474, 1991.

6. Chan, C.C., Hikita, N., Dastgheib, K., Whitcup, S.M., Gery,I., and Nussenblatt, R.B. Experimental melanin-protein-induced uveitis in the lewis rat. immunopathologic processes.Ophthalmology. 101:1275–1280, 1994.

668 GULATI ET AL.

7. Yanagisawa, S., Hayasaka, S., Zhang, X.Y., Hayasaka, Y.,Nagaki, Y., and Kitagawa, K. Effect of topical betaxolol onacute rise of aqueous flare induced by prostaglandin E(2) inpigmented rabbits. Jpn. J. Ophthalmol. 45:669–671, 2001.

8. Rankin, A.J., Khrone, S.G., and Stiles, J. Evaluation of fourdrugs for inhibition of paracentesis-induced blood-aqueoushumor barrier breakdown in cats. Am. J. Vet. Res. 72:826–832, 2011.

9. Alm, A. Prostaglandin derivates as ocular hypotensiveagents. Prog. Retin. Eye Res. 17:291–312, 1998.

10. Adamus, G., Schmied, J.L., Hargrave, P.A., Arendt, A., andMoticka, E.J. Induction of experimental autoimmune uveitiswith rhodopsin synthetic peptides in lewis rats. Curr. EyeRes. 11:657–667, 1992.

11. Jamieson, L., Meckoll-Brinck, D., and Keller, N. Characterizedand predictable rabbit uveitis model for antiinflammatorydrug screening. J. Pharmacol. Methods. 21:329–338, 1989.

12. Tessler, H.H., and Weinberg, R.S. Aqueous and serum ly-sozyme values in experimental uveitis in rabbits. Invest.Ophthalmol. 14:953–956, 1975.

13. Stainer, G.A., Peyman, G.A., Berkowitz, R., and Tessler, H.H.Intraocular lysozyme in experimental uveitis in rabbits: Aqu-eous and vitreous assay. Invest. Ophthalmol. 15:312–315, 1976.

14. Dogan, D., Farah, C., and Teofil, M. Experimental animalstudies in uveitis. Oftalmologia. 48:102–110, 2004.

15. Rosenbaum, J.T., McDevitt, H.O., Guss, R.B., and Egbert,P.R. Endotoxin-induced uveitis in rats as a model for humandisease. Nature. 286:611–613, 1980.

16. Latina, M.A., Sibayan, S.A., Shin, D.H., Noecker, R.J., andMarcellino, G. Q-switched 532-nm nd:YAG laser trabeculo-plasty (selective laser trabeculoplasty): a multicenter, pilot,clinical study. Ophthalmology. 105:2082–2088; discussion2089–2090, 1998.

17. Murthy, S., and Latina, M.A. Pathophysiology of selectivelaser trabeculoplasty. Int. Ophthalmol. Clin. 49:89–98, 2009.

18. Anderson, R.R., and Parrish, J.A. Selective photothermolysis:precise microsurgery by selective absorption of pulsed ra-diation. Science. 220:524–527, 1983.

19. Latina, M.A., and Park, C. Selective targeting of trabecularmeshwork cells: in vitro studies of pulsed and CW laser in-teractions. Exp. Eye Res. 60:359–371, 1995.

20. Kramer, T.R., and Noecker, R.J. Comparison of the mor-phologic changes after selective laser trabeculoplasty andargon laser trabeculoplasty in human eye bank eyes. Oph-thalmology. 108:773–779, 2001.

21. Hollo, G. Argon and low energy, pulsed nd:YAG laser tra-beculoplasty. A prospective, comparative clinical and mor-phological study. Acta Ophthalmol. Scand. 74:126–131, 1996.

22. Jabs, D.A., Nussenblatt, R.B., Rosenbaum, J.T., and Stan-dardization of Uveitis Nomenclature (SUN) WorkingGroup. Standardization of uveitis nomenclature for report-ing clinical data. Results of the first international workshop.Am. J. Ophthalmol. 140:509–516, 2005.

23. Pollack, I.P., and Patz, A. Argon laser iridotomy: an exper-imental and clinical study. Ophthalmic Surg. 7:22–30, 1976.

24. Perkins, E.S. Prostaglandins and ocular trauma. Adv. Oph-thalmol. 34:149–152, 1977.

25. Unger, W.G., Cole, D.F., and Bass, M.S. Prostaglandin andneurogenically mediated ocular response to laser irradiationof the rabbit iris. Exp. Eye Res. 25:209–220, 1977.

26. Dannheim, F., and Rassow, B. Laser lesions of the anteriorsegment of the rabbit eye. Studies of cornea, iris, lens andsclera using an argon, ruby and YAG-laser. Albrecht VonGraefes Arch. Klin. Exp. Ophthalmol. 205:175–205, 1978.

27. van der Zypen, E., Fankhauser, F., and Bebie, H. On the effectsof different laser energy sources upon the iris of the pig-mented and the albino rabbit. Int. Ophthalmol. 1:39–48, 1978.

28. Huber, G.K., van der Zypen, E., and Fankhauser, F. Mor-phology of the primary damage caused by the argon-ion laserto the iris of the pigmented rabbit (author’s transl). AlbrechtVon Graefes Arch. Klin. Exp. Ophthalmol. 211:94–112, 1979.

29. Bowes, L.E., Nouri, K., Berman, B., et al. Treatment of pig-mented hypertrophic scars with the 585 nm pulsed dye laserand the 532 nm frequency-doubled nd:YAG laser in theQ-switched and variable pulse modes: a comparative study.Dermatol. Surg. 28:714–719, 2002.

30. Horecker, B.L. The absorption spectra of hemoglobin and itsderivatives in the visible and near infra-red regions. J. Biol.Chem. 148:173–183, 1943.

31. Dummer, R., Graf, P., Greif, C., and Burg, G. Treatment ofvascular lesions using the VersaPulse variable pulse widthfrequency doubled neodymium:YAG laser. Dermatology.197:158–161, 1998.

32. Sawa, M., Tsurimaki, Y., Tsuru, T., and Shimizu, H. Newquantitative method to determine protein concentration andcell number in aqueous in vivo. Jpn. J Ophthalmol. 32:132–142,1988.

33. Ladas, J.G., Wheeler, N.C., Morhun, P.J., Rimmer, S.O., andHolland, G.N. Laser flare-cell photometry: methodology andclinical applications. Surv. Ophthalmol. 50:27–47, 2005.

34. Hong, B.K., Winer, J.C., Martone, J.F., Wand, M., Altman, B.,and Shields, B. Repeat selective laser trabeculoplasty.J. Glaucoma. 18:180–183, 2009.

35. Bill, A. The blood-aqueous barrier. Trans. Ophthalmol. Soc. UK.105 (Pt 2):149–155, 1986.

Received: October 19, 2012Accepted: March 9, 2013

Address correspondence to:Dr. Vikas Gulati

Department of Ophthalmology and Visual SciencesUniversity of Nebraska Medical Center

985540 Nebraska Medical CenterOmaha, NE 68198-5540

E-mail: vgulati@unmc.edu

RABBIT MODEL OF LASER-INDUCED ANTERIOR UVEITIS 669