morphological study

British Joumnal of Ophthalmology 1996; 80: 451-457

Effects of allopurinol and steroids oninflammation and oxidative tissue damage inexperimental lens induced uveitis: a biochemicaland morphological study

A J Augustin, M Spitznas, W Sekundo, F Koch, J Lutz, D Meller, F H Grus, A Wegener,S H-H Blumenroder

AbstractAims-To evaluate the effects of allo-purinol in lens induced uveitis (LIU) bymorphological methods and to comparethese effects with those of steroids and acombination of both drugs biochemicallyand morphologically.Methods-Lipid peroxides (LPO) of theretinal tissue were determined by two dif-ferent methods (thiobarbituric acid assay(TBA) and high performance liquidchromatography expressed as malondi-aldehyde-like substances). Myeloperoxi-dase (MPO) activity in the iris/ciliarybody complex was analysed spectro-photometrically. Histological changes onthree morphological levels of LIU eyeswere evaluated.Results-Both allopurinol and the combi-nation of allopurinol/prednisolone led toa significant reduction in the increasedretinal LPO values. Prednisolone onlyrevealed significant effects on retinal LPOwhen being measured with the TBAmethod. MPO activity in hris and ciliarybody was significantly reduced in alltherapy groups. The morphologicalevaluation of the sections by two maskedinvestigators revealed a significant reduc-tion (p<OO5) in the inflammation score inall therapy groups. Morphometric studiesusing the QUANTimE system (Leica,Cambridge) showed significantly reducedvalues (p<O05) in the allopurinol groupand in the group receiving prednisoloneand allopurinol. Prednisolone alone did notlead to a significant reduction in the values.Conclusions-The findings show that bothallopurinol and steroids exert positiveeffects on the variables determined inLIU. The effects ofsteroids are believed tobe mostly due to their direct action oninflammatory cells. The recently reportedscavenging effects of methylprednisoloneshould play a minor role in this diseasemodel. Allopurinol and oxypurinol act asdirect scavengers of free radicals andhypochlorous acid, which is produced viaMPO catalysis, thus leading to a reductionin tissue inflammation and tissue damage.(BrJ Ophthalmol 1996; 80: 451-457)

In direct relation to the dose administeredallopurinol was shown to countercheck the

oxidative tissue damage following lens induceduveitis (LIU). I This is believed to be due to itsfree radical scavenging activity and the scav-enging of hypochlorous acid by its majormetabolite oxypurinol. On the other hand,steroids applied as standard therapy foruveitis are known to reduce the synthesis ofprostaglandins and thromboxanes by inhibi-tion of the liberation of arachidonic acid fromphospholipids. Moreover, several steroidssuch as methylprednisolone have scavengeractivities that are dose dependent.2 3

In the present study, biochemical measure-ments were performed to compare thetherapeutic effects of allopurinol with those ofsteroids under the same disease conditions.In order to evaluate whether steroids andallopurinol show additive therapeutic effectsthe two drugs were given together.

Additionally, the therapeutic effects wereanalysed histologically, using two methods.

Materials and methods

ANIMAL EXPERIMENTSThe treatment of animals conformed to theARVO resolution on the use of animals inresearch. The experiments were performedwith male Wistar rats (n=37). The animalswere divided randomly into seven groups (twogroups, five animals each for the biochemicalstudy; five groups, five or six animals each forthe slit-lamp/histological study). The values ofthe basic tissue levels, controls and the valuesof the allopurinol group (biochemical analysisonly) were obtained from previous studies.'These values are established laboratory stan-dards. The animals were anaesthetised usingether and the tissue samples were taken asdescribed below.

Experimental uveitis was induced accordingto Rao et al.4 The animals were sensitised every2 weeks over a 2 month period with foursubcutaneous injections of bovine lens protein10 mg in complete Freund's adjuvant (Sigma,Deisenhofen, Germany). One week after thelast injection the lens of one eye was rupturedusing the tip of a 30 gauge needle. At the timeof lens rupture the therapy groups weregiven allopurinol (50 mg/kg body weight;Henning, Berlin, Germany), methylpred-nisolone (7.5 mg/kg body weight; Hoechst,Frankfurt, Germany), or a combination of thetwo, intravenously.

University EyeHospital, University ofBonn, Bonn, GermanyA J AugustinM SpitznasW SekundoF KochD MellerF H GrusS H-H Blumenroder

Department ofPhysiology, UniversityofWuerzburg,Wuerzburg, GermanyJ Lutz

Division ofExperimentalOphthalmology,University ofBonn,Bonn, GermanyA Wegener

Correspondence to:A J Augustin, University EyeHospital, University of Bonn,Sigmund-Freud-Strasse,53105 Bonn, Germany.Accepted for publication7 February 1996

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Figure 1 Lipid peroxide (LPO) levels in retinal tissue as base values (base), untreatedlens induced uveitis (control) and after application of allopurinol (50 mg/kg body weight),methylprednisolone (75 mg/kg body weight), and a combination of the two drugs.Untreated uveitis controls are statistically compared with base levels. All treatment valuesare statistically compared with untreated uveitis controls. Methylprednisolone showed asignificant difference from untreated controls with the thiobarbituric acid method only(*p<0 05).

BIOCHEMICAL STUDYThe biochemical values of the basic tissuelevels, the control group, and the therapy groupreceiving allopurinol at a dose of 50 mg/kg bodyweight were obtained from previous studies.1Twenty four hours after lens rupture theanimals were anaesthetised. Iris, ciliary body,and neurosensory retina were surgicallyremoved using microforceps and an operatingmicroscope. Thereafter, biochemical analysiswas performed. All animals were killed after-wards by exsanguination. Determination oflipid peroxide (LPO) levels in retinal tissuesamples, expressed as 'malondialdehyde-likesubstances' (MDA), was accomplished by highperformance liquid chromatography (HPLC)according to the method of Esterbauer et al.5Determination of LPO with the thiobarbituricacid (TBA) assay, expressed as TBA reactivesubstances (TBARS), was performed accord-ing to Ohkawa et al,6 as modified by Augustinand Lutz.7Both methods (MDA, TBARS) were com-

pared and a correlation was calculated.

Malondialdehyde-like substances (MDA)HPLC was performed using an instrumentby Bio-Rad (Munich, Germany). The retinasample was suspended in acetonitrile,homogenised using an Ultra Turrax blender(Janke & Kunkel, Staufen, Germany) and cen-trifuged at 3000 g for 5 minutes.

Clear supematant 20 jil was injected intothe HPLC apparatus. The following columnswere used for direct determination of MDA:(a) 250X4 5 mm S5 NH2 spherisorb(aminophase); (b) precolumn 50X4-6 mm(aminophase) (Promochem, Wesel, Germany).The upper pressure limit of the high pressurepump was set at 5000 psi. An injection valvewith a 20 ,ul loop and a 50 ,ul syringe wereused. The ultraviolet detector was set at270 nm. Values were calculated automaticallywith the aid of a computer and correlated withthe protein content of the retinal sample, watersoluble protein/ml. MDA values of the retina

samples are expressed as means of MDA/mgwater soluble protein (SEM). MDA valuesof the aqueous humour are expressed asmeans/ml.

Thiobarbituric acid reactive substances (TBARS)Tissue samples were homogenised in 0-9 ml1 15 O/o KC1 for 30 seconds with an UltraTurrax blender and centrifuged at 3000 g for 5minutes. The assay mixture consisted of 0-1 mlof the supematant, 2 ml of 0 9% NaCl, 0-2 mlof sodium dodecylsulphate, and 3 ml of TBA,containing equal parts of 0O8% aqueous TBAand acetic acid. TBARS values are reported asmeans of TBARS/mg water soluble protein(SEM).8

Myeloperoxidase (MPO)MPO activity in the iris and ciliary bodywas determined using the method of Bradleyet al.9 To free MPO from primary granules ofneutrophilic leucocytes, specimens werehomogenised with an Ultra Turrax blender for5 seconds in 3 ml of hexadecyl trimethylam-monium bromide (HTBA) solution (0-5%HTBA in 50 mM phosphate buffer, pH 6-0).The homogenate was sonicated for 10 seconds,freeze thawed three times, and centrifuged at40 000 g for 15 minutes at 4°C, resulting in theformation of a stable pellet. The supernatantwas assayed for MPO activity by spectropho-tometry using a mixture of 01 ml of super-natant and 2-9 ml of 50 mM phosphate buffer,pH 6-0, containing 0d167 mg/ml of o-diani-sidine hydrochloride and 0-00050/o hydrogenperoxide. The change in light absorbance at460 nm was measured with a spectrophoto-meter. MPO values are expressed as U/mgwater soluble protein (SEM).8

HISTOLOGICAL STUDYFor the histological study the animals weredeeply anaesthesised using ether. The lateral

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Base Control Pred Allo+pred AlloFigure 2 Myeloperoxidase (MPO) activity in iris andciliary as base values (base), untreated lens induced uveitis(control) and after application of allopurinol (50 mg/kgbody weight), methylprednisolone (7 5 mg/kg bodyweight), and a combination of the two drugs. Untreateduveitis controls are statistically compared with base levels.All treatment values are statistically compared withuntreated uveitis controls (*p<0 05).

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Effects of allopuninol and steroids on inflammation and oxidative tissue damage in experimental lens induced uveitis

Table 1 Scoring and QUANTIMED evaluation of cyclitis, iritis, and calculated uveitis values (means (SD)). The therapy values are compared withuntreated controls

Control Allopurinol Methylprednisolone Combination

Cyclitis Irtis Uveitis Cyclitis Iritis Uveitis Cyclitis Iritis Uveitis Cyclitis Iritis Uveitis

Scoring 2-97 (0-74) 3-11 (0-84) 3-04 (0-77) 1-75 (0-80)* 2-02 (0-80)* 1-89 (0-66)* 1-83 (0-50)* 1-88 (0-58)* 1-86 (0-52)* 1-56 (0-39)* 1-79 (0-45)* 1-67 (0-4)*QUANTIMED 644 (128) 444 (190) 544 (151) 461 (173) 208 (77)* 334 (117)* 626 (55) 258 (78) 442 (62) 435 (180) 175 (67)* 314 (100)*

(NS) (NS) (NS) (NS) (NS)

*p<0-05. NS=not significant.

aspects of the globes were marked by suture.Immediately thereafter a non-traumatic enu-cleation was performed and the animals werekilled by exsanguination. The eyes were fixedby immersion in cold Karnovsky's fixative(4% paraformaldehyde, 2-5% glutaraldehydebuffered in 0G1 M Na2HPO4, and 0O1 MKH2PO4).10 Numbers were randomlyassigned to the enucleated globes by a maskedtechnician. Then the globes were divided inthe horizontal plane. One half of the specimenwas embedded in paraffin. Sections of 4 ,umwere cut on a sliding microtome. In total,three levels (at the distance of 20 and 10 serialsection, respectively) were prepared. The

Fig 3A

* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.~~.......... ....... .....Fig 3B

Figure 3 Control group. (A) Slit-lamp photograph shows a diffuse haze in the anteriorchamber obscuring the entire iris structure. (B) Histologically, there is gross inflammatoryinfiltration of the limbus (limbitis=Li) and of the peripheral cornea. The ciliary body (CB)and the iris (I) also exhibit distinct infiltration with round cells. The anterior chamber isfilled with an eosinophilic proteinaceous exudate (Ex), lymphocytes, and leucocytes. Notecongested iris vessels, which correlate with inflammatory induced hyperaemia.L=crystalline lens. (Haematoxylin and eosin, magnification X 75.)

sections were stained with haematoxylin andeosin.Two independent, masked investigators

(DM, WS) examined the numbered sectionsusing a 1+ to 4+ score, depending on thedegree of intraocular inflammation, which wasmorphologically seen as an accumulation oflymphocytes and polymorphonuclear granulo-cytes. The evaluation was done separately foriris and ciliary body tissue in the describedmanner. Thereafter, the histological numberswere reassigned to the original therapeuticgroups.

In an independent, parallel setting a totalarea of the cell nucleus within a standardisedregion of the iris and the ciliary body of eachlevel was calculated in a standardised mannerusing a modified QUANTIMED 500C imaginganalysis system (Leica, Cambridge) connectedto an Olympus light microscope (IMT-2,Olympus Optical Co (Europa) GmbH,Hamburg, Germany).With the investigator scores as well as with

the QUANTIMED scores a combined uveitisscore was calculated from the average of theiritis and the cyclitis scores in each sample.

BIOMICROSCOPYBefore enucleation, both eyes of each animalwere inspected with a slit-lamp microscope(Zeiss photo slit-lamp microscope) and exemp-lary cases were photographed on AgfachromeRS 100 colour slide film. For this inspection,dilatation of the pupils was not attempted,because inflammation induced miosis waspresent in all LIU eyes. All optical examina-tions were performed in a dark room.

STATISTICAL ANALYSIS

Biochemical analysisStatistical analysis of MDA/TBARS values inretinal tissue samples and MPO values wasperformed by Student's t test. Normal distri-bution was assumed.

Tissue evaluation by scoring and by theQUANTIMED systemFor each type of tissue investigated (iris andciliary body), a separate statistical analysis ofthe scores was performed. The average of thehistological iritis score and the cyclitis scorewas calculated as the combined uveitis score.This calculation was applied both to thequantitative results of the QUANTIMED Sys-tem and to the scores determined by thetwo masked investigators. To determine the

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Fig 4A

L-Fig 4B

Figure 4 Methylprednisolone group. (A) Slit-lamp photograph shows a moderate haze.However, the small iris vessels are not discernible. (B) HistologicaIly, there is virtualy nolimbitis, but a distinct congestion oflimbal vessels (arrows). The ciliary body (CB) and theiris (I) exhibit moderate inflammatory infiltration. Only afew scattered inflammatory cellsare seen in the chamber angle. AC=antenior chamber. Compare with Figure 3.(Haematoxylin and eosin, magnification X 75.)

difference between groups, the Mann-Whitney-Wilcoxon test (U test) included instandard statistical software (spcc-Pc, SPSSChicago, IL, USA) was used. The exact limitvalues of the U test were used. The p valuesgiven in the text are two sided values of thistest.

Results

BIOCHEMICAL STUDYRetinal lipid peroxide values increased signifi-cantly in untreated uveitis controls as com-pared with base values (p<0 05; valuesobtained from Augustin et all). A significantdecrease in comparison with untreated uveitiscontrols was achieved with a dose of 50 mg/kgbody weight of allopurinol (p<0 05; valuesobtained from Augustin et a1l).When given alone, prednisolone resulted in

a slight, not significant reduction in the valuesin comparison with those obtained by theHPLC method. However, a significant reduc-tion (p<0 05) was achieved when the results

obtained with the TBA method were com-pared with each other.A combination of the two drugs resulted in a

significant reduction in the LPO values (Fig 1).The correlation ofthe two methods was 0-897.

Iris and ciliary body showed a significantincrease in myeloperoxidase activity (p<0 05)as compared with base values (values obtainedfrom Augustin et a1l). A significant reductioninMPO (p<0 05) was achieved using allopuri-nol (values obtained from Augustin et all),prednisolone, and a combination of the twodrugs. The combination of allopurinol andprednisolone did not produce any additionalsignificant changes compared with what wereobtained by the single administration of eachsubstance (Fig 2).

MORPHOLOGICAL STUDY (TABLE 1)

Scoring by two investigatorsIn all therapy groups the tissue inflammation(uveitis) was significantly reduced as comparedwith untreated controls (p<0 05).

QUANTIMED evaluationThe tissue inflammation (uveitis) evaluated bythe QUANTIMED system revealed significantlyreduced values (p<0 05) in the allopurinolgroup and the group receiving steroid andallopurinol as compared with untreatedcontrols. In the present study, the steroid alonedid not lead to a significant reduction in thevalues (p=0 18). The therapy groups (allo-purinol, prednisolone, and allopurinol/prednisolone) did not differ significantly withrespect to the uveitis, iritis, or cyclitis scores.

Figures 3 to 6 show representative slit-lampphotographs of the uveitis eye (A) and thecorresponding microphotograph of a histo-logical section (B) of each study group. In thecontrol group anterior chamber structures werebarely seen, because of an extensive flare (Fig3A). On histology the flare correlated with amassive proteinaceous exudate that is rich ininflammatory cells derived from grosslyinflamed anterior chamber structures (Fig 3B).In contrast, in the methylprednisolone groupthe slit-lamp view ofthe iris structure was muchbetter (Fig 4A) and the histology showed amoderate, mainly polymorphonuclear, inflam-matory infiltration of the iris and the ciliarybody and a congestion of the limbal vessels. Inthe vast majority of the allupurinol treatedanimals the anterior chamber was clinicallyclear (Fig 5A). This observation was confirmedby microscopy. Here, only minor sequelae ofthe severe Arthus reaction, as shown in the con-trol group, could be seen (Fig 5B). Clinical andlight microscopic changes in the allopurinol/methylprednisolone group were, to a certainextent, comparable with those seen in theallopurinol treated animals (Figs 6A and 6B).

DiscussionThe morphological results confirm previously

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Fig 5A

Fig SB

Figure S Allopurinol group. (A) Slit-lamp photograph clearly shows details of the irisvasculature. (B) The limbus (Li) and the ciliary body (CB) are almostfree ofinflammatory cels. The latter are scattered through the iris stroma (I) and to a minorextent within the anterior chamber (AC). Inflammatory cells are also seen adjacent to thesite of the anterior lens capsule rupture, where the lens cortex is protrudingforward (arrow).Compare with Figure 3. (Haematoxylin and eosin, magnification x 75.)

reported biochemical data regarding thebeneficial effects of allopurinol on the inflam-matory processes in LIU.' The effects ofallopurinol at a dose of 50 mg/lkg body weightwere comparable with those of steroids whengiven at a dose of 7'5 mg/kg body weight usingthe scoring evaluation. A combination of thetwo drugs did not show significant additiveeffects either biochemically or morphologic-ally.

BIOCHEMICAL RESULTSIn the present study the variables of inflamma-tion (MPO) and oxidative tissue damage(LPO) of the affected ocular structures wereevaluated systematically.

Inflammation and inflammation associatedreactions are believed to be the major source offree radicals leading to oxidative tissue damagefollowing uveitis.4 11-14

In the present study the oxidative tissuedamage was evaluated by two methods. Thegreat advantage of the thiobarbituric acidmethod is its high sensitivity in detecting per-oxides. However, the main disadvantage of

this method is its lower specificity. Therefore,the two methods were used together and acorrelation was calculated. The problem ofjust using the TBA method is documented inthe results of the steroid group. In particular,values such as the steroid values bordering thethreshold of significance may give differentresults with each of the two methods. Onereason for this difference might be the highersensitivity of the TBA method, which maylead to a higher variation of the values. Thus,it is conceivable that the steroid results can beregarded as a less pronounced improve-ment when compared with allopurinol and/orcombination therapy. This fact is also docu-mented in the morphological data, whichdemonstrate a significant improvement whenusing the score system and no significantimprovement when using the QUANTIMEDevaluation.

MethylprednisoloneSteroids influence several mechanisms leadingto oxidative metabolites and reduce theaccumulation of neutrophils and macrophagesin the inflamed tissue."1 15-18 Steroids do notprevent the synthesis of antibodies. Theirimmunological effects are based on the reduc-tion of the inflammatory response to immuno-logical processes and the prevention of thepassage of antigen-antibody complexesthrough basal membranes. Apart from thereduction of the absolute number of phago-cytic cells in the inflamed tissue steroidsinfluence the arachidonic acid cascade thatmight be one source of oxidative metabolites inthe LIU model.

However, there are additional sources ofoxidative reactions in activated inflammatorycells, such as the MPO derived production ofhypochlorous acid and the respiratory burstleading to powerful radicals such as thehydroxyl radical and a singlet oxygen, bothbeing capable oftissue oxygenation.4 One mightassume that the antioxidative effects of steroidsare only the result of a reduction in the absolutenumber of inflammatory cells and the specificaction on different pathways capable ofproduc-ing oxidative metabolites. Another explanationfor the minor effects of steroids when comparedwith those obtained by powerful scavengerssuch as allopurinol and/or oxypurinol, which areable to influence inflammatory induced freeradicals and radicals from other sources, isoffered by the recently reported free radicalscavenging activity of methylprednisolone.2 3Using a model of spinal cord injury this studyshowed that the corticosteroid methyl-prednisolone possesses significant antioxidantefficacy when administered in doses exceedingthose given in the present study.2 3The free radical scavenging activity of

methylprednisolone is in no way related to thesteroid's glucocorticoid receptor mediatedactions.23 Structurally similar drugs such aslazaroids are also known to be both potentscavengers of hydroxyl radical/superoxideanion radicals and inhibitors of activatedphagocytic cells.'9

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Fig 6A

Figure 6 Allopurinol/methyIprednisolone group. (A) Slit-lamp photograph shows afairlyclear view of the anterior chamber structures similar to Figure SA. An incidental hyphaema isdue to a paracentesis at the time of lens rupture. (B) While there is moderate limbitis (Li),the iris (I) and ciliary body (CB) exhibit barely discernible signs ofinflammation. A fewinflammatory precipitates are adjacent to the corneal endothelium (arrows). L=crystallinelens. Compare with Figure 3. (Haematoxylin and eosin, magnification X 75).

Thus, it is conceivable that when given indoses below the scavenger dose of 30 mg/kgbody weight methylprednisolone acts only on

inflammation and thus only prevents theoxidative damage originating directly from theinflammation. The radicals already beinggenerated and those originating from othersources such as iron are not influenced becausethe steroid dose is too low. These facts are con-firmed by our results, which showed a signifi-cant reduction in MPO activity, but lesspronounced effects on lipid peroxides in theretinal tissue.The aim of this study was to compare the

action of allopurinol with steroids at a doserange given in uveitis therapy. The dosesnecessary for scavenging activities will not begiven in ophthalmology. Nevertheless, the LIUmodel might be suitable for a pharmacodynamicstudy of the scavenging effects of steroids.

Allopurinol und oxypurinolIn a previous study' we demonstrated thatallopurinol reaches the ocular tissues at levelsnecessary for a scavenger activity of this drug.

The scavenging effects of both allopurinol andoxypurinol in LIU were discussed in detail. Inthe present study we confirm the biochemicalresults achieved by morphological evaluationwith a score system and with the QUANTIMEDevaluation. Our findings indicate the necessityfor additional investigations on the effects ofallopurinol and oxypurinol on inflammatoryophthalmic diseases. The topical application ofallopurinol particularly shows promise as analternative to steroids in several diseases.The beneficial effects of these drugs on

oxidative tissue damage and inflammatoryresponse alone do not provide enough infor-mation regarding the beneficial effects on thewhole disease process. Therefore, to drawconclusions about the contribution of tissueoxidation to the disease process and to evaluatethe effects of the drugs investigated, it wasnecessary to obtain additional information,especially quantitative morphological data.

HISTOLOGYThe intraocular inflammation could easily beseen by light microscopy as an accumulation ofpolymorphonuclear granulocytes and/or lym-phocytes within the anterior segment struc-tures. The fellow eyes of the LIU eyes did notshow any significant inflammatory reactionand were therefore given a score of 0. In con-trast, the LIU eyes exhibited inflammatoryinfiltration of the iris, the ciliary body, and thelimbus, as well as the presence of a protein-aceous and inflammatory cellular anteriorchamber exudate, depending on the mode oftreatment (Figs 3-6). Although a subjectiveimpression cannot be entirely ruled out in anymorphological study, we attempted tominimise this influence not only by randomisa-tion of the enucleated globes, but also by (1)masking the investigators, (2) contributingexaminations at three different levels, and (3)defining strict score criteria, ranging from 0 ineyes with no inflammation to 4+ in eyes inwhich the above mentioned structures exhib-ited pronounced inflammatory alterations.Indeed, after reassignment, only the untreatedeyes yielded the maximum score. The mainadvantage of the morphological method is thatit examines the entire globe using differentmagnifications, delivering a comprehensiveimpression. It is not surprising that the treatedeyes contained fewer inflammatory cells thanuntreated ones.The morphometric studies using the modi-

fied image analyser QUANTIMED were per-formed in an attempt to gain better objectivityofthe histological data. Again, the main advan-tage of this system is an investigator indepen-dent calculation of the nuclear area, as definedby the basophilic colour of the nuclei onhaematoxylin and eosin stained sections.However, the disadvantages of the methodwere as follows: (1) the calculations werecarried out on a defined area of the iris and theciliary body irrespective of the degree of theinflammatory changes. Thus, this selection didnot always contain a representative area; (2) inparticular, both the ciliary body and the iris

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possess epithelial and stromal cells withbasophilic nuclei whose area was also calcu-lated. Although we assumed these values to beequal in all eyes, there are probably differencesbetween individual eyes. This could be one

explanation as to why the prednisolone treatedgroup did not show a significant effect withregard to anterior uveitis and cyclitis alone, butwas significant in the iritis group. In fact, thecellular density of the ciliary body is muchhigher than that of the iris. Therefore, theinflammatory cell infiltration of the iris stromacalculated as an area value is not as high inabsolute numbers as is the infiltration of theciliary body.Supported by Deutsche Forschungsgemeinschaft (DFG),AU 126/1-1

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2 Hall ED. Lipid antioxidants in acute central nervous systeminjury. Ann Emerg Med 1993; 22: 1022-7.

3 Hall ED. The effects of glucocorticoid and nonglucocorti-coid steroids on acute neuronal degeneration. Adv Neurol1993; 59: 241-8.

4 Rao NA, Calandra AJ, Sevanian A, Bowe B, Delmage JM,Marak GE. Modulation of lens induced uveitis by super-oxide dismutase. Ophthalmic Res 1986; 18: 41-6.

5 Esterbauer H, Lang J, Zadravec S, Slater TF. Detection ofmalonaldehyde by high-performance liquid chromato-graphy. Methods Enzymol 1984; 105: 319-28.

6 Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides inanimal tissues by thiobarbituric acid. Anal Biochem 1979;95: 351-8.

7 Augustin AJ, Lutz J. Intestinal, hepatic and renal productionof thiobarbituric acid reactive substances and myeloper-oxidase activity after temporary aortic occlusion andreperfusion. Life Sci 1991; 49: 961-8.

8 Schacterle GR, Pollack RL. A simplified method for thequantitative assay of small amounts of protein in biologicmaterial. Anal Biochem 1973; 51: 654-5.

9 Bradley PP, Priebat DA, Christensen RD, Rothstein G.Measurement of cutaneous inflammation: estimation ofneutrophil content with an enzyme marker. 7 InvestDermatol 1982; 78: 206-9.

10 Karnovsky M. A formaldehyde-glutaraldehyde fixative ofhigh osmolarity for use in electron microscopy. Jf CeUl Biol1965; 27: 137-8.

11 Bazan NG, Abreu MT de, Bazan HEP, Belfort R.Arachidonic acid cascade and platelet activating factorin the network of eye inflammatory mediators: therapeu-tic implications in uveitis. Int Ophthalmol 1990; 14:335-44.

12 Rao NA. Role of oxygen free radicals in retinal damageassociated with experimental uveitis. Trans AmOphthalmol Soc 1990; 88: 797-850.

13 Wu GS, Sevanian A, Rao NA. Detection of retinal lipidhydroperoxides in experimental uveitis. Free Rad Biol Med1992; 12: 19-27.

14 Goto H, Wu GS, Gritz DC, Atalla LR, Rao NA.Chemotactic activity of the peroxidized retinal membranelipids in experimental autoimmune uveitis. Curr Eye Res1991; 10: 1009-14.

15 Weissmann G, Sessa G, Bevans V. Effect of DMSO on thestabilization of lysosomes by cortisone and chloroquine invitro. Ann NYAcad Sci 1967; 141: 326-32.

16 Gadelata D, Verma M, Davis JM. Inhibition of neutrophilleukotriene generation by the 21 aminosteroid, U-74389F. Jf Surg Res 1994; 57: 233-7.

17 Borgeat P, Samuelsson B. Transformation of arachidonicacid by rabbit polymorphonuclear leukocytes. J Biol Chem1979; 254: 2643-6.

18 Ward PA. The chemosuppression of chemotaxis. J Exp Med1966; 124: 209-26.

19 Thomas PD, Mao GD, Rabinovitch A, Poznanzky MJ.Inhibition of superoxide-generating NADPH oxidase ofhuman neutrophils by lazaroids (2 1-aminosteroids and 2-methylaminochromans). Biochem Pharmacol 1993; 45:241-51.

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