Download Free radical tissue damages in the anterior segment of the

Free Radical Tissue Damages in the Anterior Segment of
the Eye in Experimental Autoimmune Uveitis
Sei-ichi Ishimoto, Guey-Shuang Wu, Seiji Hayashi, Jie Zhang, and Narsing A. Rao
Purpose. To investigate increased free radical activity and the accumulation and localization
of lipid peroxidation in the anterior segment of the eye with uveitis.
Methods. Experimental autoimmune uveitis (EAU) was induced in rats using human S-antigen
peptide. Conjugated dienes (CD) and keto-dienes (KD) were then extracted from the cornea,
iris-ciliary body and lens of the EAU eyes. The quantity of CD and KD were determined by
measuring ultraviolet absorption and estimating by means of a molar extinction coefficient.
Frozen sections of EAU eyes were reacted with 3-hydroxy-2-naphthoic acid hydrazide (NAH),
and NAH-carbonyl compounds were detected using a confocal laser scanning microscope.
Statistical comparisons of CD and KD products between the EAU groups and controls were
performed using the Student's t-test.
Results. Compared to controls, CD and KD were significantly increased in the cornea and
iris-ciliary body of EAU eyes. Lenses of EAU eyes showed a tendency to elevated levels of
CD and KD. In EAU, primarily the anterior border layer and the posterior epithelium of the
iris—and, to a lesser extent, the trabecular meshwork and corneal endothelium—revealed
positive fluorescence staining for peroxidized carbonyl products. No staining was observed
on the ciliary epithelium.
Conclusions. Free radicals and lipid peroxidation products are generated in the anterior segment of the eye in EAU. Because the individual tissues in the anterior segment are composed
of various levels of fatty acids and different concentrations of antioxidants, the extent of tissue
damage from lipid peroxidation may represent a balance between the fatty acid composition
and the antioxidant distribution in each of the tissues. Invest Ophthalmol Vis Sci.
1996; 37:630-636.
V-Jxygen-free radicals, primarily the hydroxyl radical
(•OH), hydrogen peroxide (H 2 O 2 ), and superoxide
anion (O 2 - ~), are known to be toxic to tissue components. 1 In an inflammatory process, phagocytic cells,
such as polymorphonuclear cells (PMNs) and macrophages, produce these oxygen-free radicals, which, in
turn, induce a series of cytotoxic effects responsible
for tissue damage. 2
In human uveitis, inflammation of ocular tissues
often results in pathologic conditions such as retinal
degeneration, secondary glaucoma, complicated cataract, iris atrophy, and corneal degeneration. It has
From the Doheny Eye Institute, University of Southern California, School of
Medicine, Los Angeles, California.
Supported by National Institute of Health grant EY10212
Submitted for publication August 30, 1995; revised November 29, 1995; accepted
December 1, 1995.
Proprietary interest category: N.
Reprint requests: Narsing A. Rao, Doheny Eye Institute, 1355 San Pablo Street, Los
Angeles, CA 90033.
630
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been demonstrated that lipid peroxidation of the retina subsequently induced retinal degeneration in experimental autoimmune uveitis (EAU).3 There have
also been reports of the free radical-induced tissue
damage in the anterior segment of the eye in a variety
of pathogenic ocular conditions. Spector and Garner4
and Bhuyan and Bhuyan5 found that endogenous hydrogen peroxide and lipid peroxidation products
were significantly increased in aqueous humor in senile cataract. Babizhaye and Bunin6 found that trabecular meshwork tissues in primary open angle glaucoma contained a high proportion of lipid peroxidation products. Artola et al7 demonstrated that
injection of exogenous hydrogen peroxide into the
anterior chamber of the rabbit induced lipid peroxidation in the iris epithelial cell membranes. These
reports suggest that oxygen-free radical-mediated peroxidation may play a causative role in subsequent
pathologic conditions of the anterior segment of the
Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No. 4
Copyright © Association for Research in Vision and Ophthalmology
Free Radical Damage in Uveitis
631
eye in uveitis. Oxygen radical-derived tissue damage
in the anterior segment has not been studied in uveitis. In the current study, we investigated the generation of lipid peroxidation products and their localization in the anterior segment, including the cornea,
the trabecular meshwork, the iris-ciliary body, and
the lens, in EAU.
solved in 1 ml ethanol (anhydrous "photrex" grade;
J.T. Baker Chemical, Phillisburg, NJ) for ultraviolet
measurement. Absorptions for CD and KD were measured at 233 nm and 280 nm (Shimadzu [Kyoto, Japan] spectrophotometer, UV-160), respectively. The
quantity of CD and KD products was estimated using
molar extinction coefficients of 25,200 and 20,000,
respectively.
MATERIALS AND METHODS
Detection of Peroxidized Carbonyl Products
Peroxidized carbonyl products were detected histochemically by the method previously described.10
Briefly, eyes were embedded in optimum cutting temperature compound (Miles Laboratory, Naperville,
IL) and immediately snap frozen in liquid nitrogen.
Ten-micrometer frozen sections were prepared, fixed
in 5% trichloracetic acid, and reacted with 3-hydroxy2-naphthoic acid hydrazide (NAH) catalyzed by a trace
of p-toluenesulfionic acid. The fluorescent products
also were coupled with Fast Blue B (FBB) for color
visualization. The fluorescent NAH-carbonyl compounds were detected by Zeiss confocal laser scanning
microscope (Carl Zeiss, Thornwood, NY) using an excitation at 488 nm and an emission at 520 nm.
Induction of Experimental Autoimmune Uveitis
Human S-antigen peptide, DTNLASSTIIKEGIDKLG,
was synthesized on 4-hydroxymethylphenoxymethylresin using an automated peptide synthesizer (model
430A; Applied Biosystems, Foster City, CA) and was
desalted on a Sephadex G-10 column (Sigma, St.
Louis, MO).8 The purity was assessed by a reversephase high-pressure liquid chromatography (Bio-Rad
RP304 column; Bio-Rad, Richmond, CA).
Twenty-eight female Lewis rats, each weighing 150
to 175 g, were obtained from Charles River Laboratory
(Wilmington, MA). Fourteen rats were immunized
with 100 fig human S-antigen peptide in an 1:1 emulsion with complete Freund's adjuvant containing 4
mg/ml Mycobacterium tuberculosis strain H37RA (Difco Statistical Analysis
Laboratory, Detroit, MI) in a total of 0.2 ml into one
footpad. At the time of immunization, animals also
received an intravenous injection in the tail vein with
1 fig pertussis toxin (List Biological Laboratory, Campbell, CA) in 0.3 ml sterile saline. Another 14 nonimmunized female rats were used for control. All animals
were treated in accordance with the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research.
Light Microscopic Study
At the peak of inflammation, between 14 and 16 days
after immunization, the eyes were enucleated under
anesthesia and fixed in formalin for paraffin embedding. Seven-micrometer sections were stained with hematoxylin and eosin.
Measurement of Conjugated Dienes and KetoDienes
The cornea, iris-ciliary body, and lens were dissected
from the eyes. Lipids were extracted by the method
previously reported.9 Briefly, each determination used
either the cornea or the lens from one eye or the irisciliary body complex from two eyes. The cornea and
iris-ciliary body tissues were homogenized and extracted in 1 ml chloroformrmethanol (2:1), and the
pooled extracts were washed with 0.2 ml water. Lens
tissues underwent the same procedure with 2 ml solvent and washing with 0.4 ml water. Solvents were
evaporated under nitrogen, and the residue was dis-
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Statistical comparisons of CD and KD products between the EAU groups and controls were performed
using the Student's Mest. The null hypothesis was rejected for P values < 0.05.
RESULTS
All 14 Lewis rats sensitized with human S-antigen peptide developed uveitis, characterized by conjunctival
hyperemia, corneal edema, and anterior chamber
cells and flare. Some of these animals developed hypopyon and hyphema. Clinically, the ocular inflammation reached its peak between 14 and 16 days after
immunization. Controls showed none of these clinical
signs.
Light Microscopic Findings
In the eyes of EAU group, massive inflammatory cells,
mostly PMNs and mononuclear cells, have infiltrated
the iris, ciliary body, and trabecular meshwork (Fig.
1). A large number of PMNs also were evident in the
anterior and posterior chamber, and proteinaceous
material and fibrin debris were present in the anterior
chamber. Some PMNs were adherent to the corneal
endothelium and surface of the lens capsule (Fig. 2).
The retina and choroid also were infiltrated with
PMNs and mononuclear cells.
Conjugated Dienes and Keto-Dienes Products
Results for CD and KD, expressed as mean ± SD in
nmoles, are shown in Table 1. A statistically significant
632
Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No. 4
FIGURE l. Anterior segments of the experimental autoimmune uveitis eye. Massive inflammatory cells, mostly polymorphonuclear cells (PMNs) and mononuclear cells, infiltrate the iris and ciliary body. Note the disorganized trabecular meshwork and die presence of PMNs and mononuclear
cells in die trabecular meshwork. AC = anterior chamber;
TM = trabecular meshwork. Hematoxylin and eosin, X100.
increase of CD was found in the experimental eyes
compared to control eyes in the cornea (P < 0.001),
the iris-ciliary body (P < 0.001), and the lens (P =
0.005). A significant increase of KD was found in experimental animals in the cornea (P = 0.004) and the
iris-ciliary body (P = 0.01). Compared to the control
group, KD products within the lens tended to increase
in the experimental group, but the increase was not
statistically significant.
tion products and by the detection of peroxidized carbonyi products. For the confirmation and quantification of in vivo lipid peroxidation, we used two parameters in this study, CD and KD, because any one
parameter may not be sufficient to provide an accurate
measure of complex lipid peroxidation processes.11
Conjugated dienes represent the primary formation
of monomeric fatty acid hydroperoxides, as well as any
secondary dimeric and polymeric hydroperoxides that
retain the diene conjugation in the molecule. Ketodienes reflect the fraction of hydroperoxide rearranged to ketodiene structure. These CD and KD
are parameters as consequential products of free radical-initiated lipid peroxidation, indicating oxygen-dependent degradative processes of polyunsaturated
fatty acids (PUFAs).
In the current study, the cornea and iris-ciliary
body showed a significant increase of CD and KD,and
the lens showed slightly elevated levels of lipid peroxidation products in EAU (Table 1). Although all cellular constituents, including DNA strands, enzymes, iron
channels, structural proteins, and membrane lipids
are potential targets for these reactive free radicals,
cell membranes and structural lipoproteins are particularly susceptible to lipid peroxidation because of
their abundant PUFAs.12"14 Therefore, the elevation
of lipid peroxidation products within the cornea and
iris-ciliary body in EAU indicates the presence of destructive processes of these tissues mediated by oxygen-free radicals. In contrast, the accumulation of
lipid peroxidation products in lens tissue is less in
acute inflammations such as EAU, because the lens
fiber membranes contain fewer than 1% PUFAs.15 Be-
Localization of Peroxidized Carbonyi Products
Positive fluorescence stainings were observed in the
experimental group, primarily in the iris and, to a
lesser extent, in the trabecular meshwork (Figs. 3, 4).
In the iris, fluorescence staining appeared as small
clumps spread throughout the entire surface of the
anterior border layer and the posterior epithelium
of the iris. No staining was observed on the ciliary
epithelium. In the cornea, granular fluorescence
stainings were present along the endothelium (Fig.
4). These results were supported by visualization of
bluish-purple carbonyl-NAH-FBB products. The control group was negative for fluorescence and NAHFBB products.
DISCUSSION
We demonstrated biochemical and histochemical evidence of oxygen-free radical-mediated tissue damage
in the anterior segment of the eye in EAU. The presence and localization of free radical tissue damage
were confirmed by the measurement of lipid peroxida-
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2. Anterior segments of die experimental autoimmune uveitis eye. Numerous cellular infiltrates are evident
in the anterior and posterior chamber. Proteinaceous material and fibrin debris are present in the anterior chamber.
Some polymorphonuclear cells adhere to die corneal endothelium and die anterior lens capsule. C = cornea; IR =
iris; L = lens. Hematoxylin and eosin, X80.
FIGURE
633
Free Radical Damage in Uveitis
TABLE l. Conjugated Diene and Keto-Diene Production in Experimental
Autoimmune Uveitis
KD
CD
Tissue
Mean ± SD
(nmol)
P Value
Mean ± SD
(nmol)
P Value
Cornea
EAU
Control
Iris-ciliary body
EAU
Control
Lens
EAU
Control
2.32 ± 0.29
1.28 ± 0.21
<0.001
2.91 ± 0.45
0.98 ± 0.25
0.004
1.42 ± 0.32
0.71 ± 0.20
<0.001
0.52 ± 0.19
0.27 ± 0.12
0.01
3.08 ± 0.61
2.32 ± 0.33
0.005
0.88 ± 0.35
0.65 ± 0.17
CD = conjugated diene; KD = keto-diene; SD = standard deviation; n = number of determinations; EAU = experimental autoimmune
uveitis.
cause of this unique proportion of PUFAs, oxidative
damage to the lens may require a longer exposure to
oxygen-free radicals, as is the case in chronic uveitis.16
Histochemically, peroxidized carbonyl products
are located primarily on the anterior border layer and
posterior epithelium of the iris and, to a lesser extent,
on the trabecular meshwork, and the corneal endothelium in EAU. Numerous PMNs were seen in the iris
and the trabecular meshwork. Inflammatory cell infiltration also was seen in the anterior and posterior
chamber surrounding the lens capsule as well as adherent to the corneal endothelium. Such PMNs are
capable of inducing oxidation of PUFAs of these tissues. Generally, PUFAs of cell membranes consist primarily of linoleic acid (18:2), linolenic acid (18:3),
arachidonic acid (20:4), and docosahexaenoic acid
(22:6), all of which are potential targets for lipid peroxidation, their susceptibility increasing as the number of double bonds increases.14 Table 2 shows PUFA
composition reported for ocular tissues. In the anterior segment, the proportion of PUFAs ranges from
34% for the iris to less than 1% for the lens. 1517 " 19 It
is evident that the extent of pathologic peroxidation
in the anterior segment in EAU appears to correlate
with PUFA content of these tissues. The photoreceptor membranes contain a higher proportion of PUFAs
(more than 50% of 22:6) than other organs in the
body.20'21 Therefore, the retinal outer photoreceptor
layer, by virtue of its structure, is extremely susceptible
to peroxidation. 22 ' 23 In fact, peroxidized carbonyl
products were found localized primarily within the
outer photoreceptor layer in the retina in EAU.24
In contrast to the iris epithelium, no oxidized carbonyl products were detected on the ciliary epithelium. Under normal and inflammatory conditions, the
eye is well protected from the damaging effects of
oxygen and its metabolites by the control of antioxidant enzymes and other antioxidant agents, which are
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widely distributed in the eye.25 29 Particularly, antioxidant enzymes, such as superoxide dismutase, glutathione peroxide, and catalase, are found in abundance,
predominantly in the ciliary epithelium, corneal epithelium and endothelium, and retinal pigment epithelium.25"28 Therefore, the ciliary epithelium and corneal endothelium seem to be well protected against
oxygen metabolites in comparison to the iris and trabeculum. In addition, the ciliary body secretes aqueous humor that contains antioxidants, including ascorbate, as normal constituents.4'29 Therefore, the aqueous humor, which is closer to the ciliary body by its
aqueous dynamics, contains a higher concentration of
antioxidants and, thus, a lower accumulation of oxygen metabolites. Consequently, the exposure of oxygen-free radicals to the ciliary epithelium may be
lower. It is supposed that damage to individual tissue
correlates with a balance between the exposure to oxygen and its metabolites and the protection of the tissues afforded by the antioxidants contained in each.
Treatment for ocular inflammatory disease has
been based on suppressing a presumed immune response and reducing its inflammatory tissue damage.
Although corticosteroids and immunosuppressive
drugs such as cyclosporine have been effective in treating patients with uveitis, serious side effects have limited their use.30 3I Hence, a search for new therapeutic
approaches for uveitis is fostered. The possible therapeutic advantage of antioxidant treatment for inflammatory disease has been focused. Bhuyan et al5
showed that one of the antioxidant agents, vitamin E,
could prevent the peroxidative degeneration of lens
lipid in vitro. Wu et al32 also demonstrated an efficient
systemic antioxidant therapy for free radical damage
of the retina in EAU. Alio et al33 reported the beneficial effects of topical antioxidant therapy on the evolution of infiltration in experimental keratitis. Therefore, it is reasonable to speculate that antioxidant
634
Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No, 4
FIGURE 3. {tap). False color, contocal imaging of fluorescence aistrmution in iNAn-reaciea
anterior segments of the experimental autoimmune uveitis eye. Positive fluorescence stainings primarily locate in the iris {arrowhead) and the trabeculum {arroiv). The false colors
refer to increasing fluorescence values, from blue (lowest), green, yellow, and red to pale
pink (highest). AC = anterior chamber; IR = iris; TM = trabecular meshwork. Magnification,
X500.
FIGURE 4. {bottom). False color, confocal imaging of fluorescence distribution in NAH-reacted
the anterior segments of the experimental autoimmune uveitis eye. Positive fluorescence
stainings appear as the lesions spread throughout the anterior border layer of the iris. Note
granular fluorescence stainings along the corneal endothelium {arrow). False colors indicate
the same fluorescence values in Figure 3. C = cornea; AC = anterior chamber; IR = iris.
Magnification, X500.
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Free Radical Damage in Uveitis
TABLE 2.
635
Polyunsaturated Fatty Acid Composition in Ocular Tissues
Proportion ofPUFAs (%)
Tissue
18:2
18:3
Cornea
Epithelium
Stroma
Endothelium
Trabeculum
Iris-ciliary body
Lens
Retina
—
1
2
4
12
—
—
—
—
—
5
—
20:4
22:5
22:6
2
—
9
11
12
17
—
4
2
—
—
2
—
1
—
—
2
Others
Total
Species
2
Rabbit
Rabbit
Rabbit
Bovine
Rabbit
Rabbit
Bovine
16
13
18
34
Cl
61
1
—
—
51
Reference Number
19
19
19
18
17
15
20
PUFAs = polyunsaturated fatty acids.
drugs may be useful for the treatment of ocular inflammation in humans. Probably in concert with conventional anti-inflammatory drugs, antioxidant therapy would be able to reduce the currently irreversible
tissue damage—such as iris atrophy, secondary glaucoma, corneal degeneration, and complicated cataract— that occurs with persistent anterior uveitis.
9.
10.
Key Words
anterior segment, experimental autoimmune uveoretinitis,
free radicals, lipid peroxidation, uveitis
11.
Acknowledgments
The authors thank David Stanforth for his expert technical
assistance.
12.
13.
References
1. Blake DR, Allen RE, Lunec J. Free radicals in biological systems: A review orientated to inflammatory processes. BrMedBull. 1987; 43:371-385.
2. Rossi F, Bellative P, Berton G, Grzeskowiak M, Papini
E. Mechanism of production of toxic oxygen radicals
by granulocytes and macrophages and their function
in the inflammatory process. Pathol Res Pract.
1985; 180:136-142.
3. Goto H, Wu GS, Chen F, Kristeva M, Sevanian A, Rao
NA. Lipid peroxidation in experimental uveitis: Sequential studies. CurrEyeRes. 1992; 6:489-499.
4. Spector A, Garner WH. Hydrogen peroxide and human cataract. Exp Eye Res. 1981;33:673-681.
5. Bhuyan KC, Bhuyan DK. Molecular mechanism of cataractogenesis: III: Toxic metabolites of oxygen as initiators of lipid peroxidation and cataract. Curr Eye Res.
1984;3:67-81.
6. Babizhayev MA, Bunin AY. Lipid peroxidation in
open-angle glaucoma. Ada Ophthalmol. 1989; 67:371377.
7. Artola A, Alio JL, Bellot JL, Ruiz JM. Lipid peroxidation in the iris and its protection by means of viscoelastic substances (sodium hyaluronate and hydroxypropylmethylcellulose). Ophthalmic Res. 1993; 25:172176.
8. Donoso LA, Merryman CF, Sery TW, et al. S-antigen:
Downloaded From: http://iovs.arvojournals.org/ on 05/04/2017
14.
15.
16.
17.
18.
19.
20.
21.
Characterization of pathogenic epitope which mediates experimental autoimmune uveitis and pinealitis
in Lewis rats. CurrEyeRes. 1987;6:1151-1159.
Folch J, Lees M, Sloan-Stanley GH. A simple method
for the isolation and purification of total lipides from
animal tissues. / Biol Chem. 1957; 226:497-509.
Pompella A, Comporti M. The use of 3-hydroxy-2naphthoic acid hydrazide and fast blue B for the histochemical detection of lipid peroxidation in animal
tissues: A microphotometric study. Histochemistry.
1991;95:255-262.
Halliwell B, Gutteridge JMC. Free Radicals in Biology
and Medicine. Oxford: Clarendon Press; 1989:139170.
Rice-Evans CA, Diplock AT. Current status of antioxidant therapy. Free Radic Biol Med. 1993; 15:77-96.
Floyd RA, Carney JM. Free radical damage to protein
and DNA: Mechanisms involved and relevant observations on brain undergoing oxidative stress. Ann Neurol.
1992;32:S22-S27.
Cheeseman KH. Mechanisms and effects of lipid peroxidation. Mol Aspects Med. 1993; 14:191-197.
Zelenka PS. Lens lipids. Curr Eye Res. 1984;3:13371359.
Yugay MT, Pereira PC, Mota MC, Cunha-Vaz JG. Possible role of free radical oxidation in postuveal cataract pathogenesis. ARVO Abstracts. Invest Ophthalmol
Vis Sri. 1995;36:S605.
Zhang Y, Yousufzai SYK, Abdel-Latif AA. Comparative
studies on fatty acid composition and phospholipases
A2 and C activities in rabbit and bovine iris-ciliary
body. Exp Eye Res. 1993;56:151-155.
Nawar WW, Kim SK, Yates-Berg D, Anderson PJ. Fatty
acid composition of total lipid from TM of calf and cow.
ARVO Abstracts. Invest Ophthalmol Vis Sri. 1990; 31:357.
Bazan HEP, Bazan NG. Composition of phospholipids
and free fatty acids and incorporation of labeled arachidonic acid in rabbit cornea: Comparison of epithelium, stroma and endothelium. Curr Eye Res.
1984;3:1313-1319.
Stone WL, Farnsworth CC, Dratz EA. A reinvestigation
of the fatty acid content of bovine, rat and frog retinal
rod outer segments. Exp Eye Res. 1979;28:387-397.
Wheeler TG, Benolken RM, Anderson RE. Visual
Investigative Ophthalmology & Visual Science, March 1996, Vol. 37, No. 4
636
22.
23.
24.
25.
26.
27.
membranes: Specificity of fatty acid precursors for the
electrical response to illumination. Science. 1975; 188:
1312-1314.
Rao NA. Role of oxygen-free radicals in retinal damage associated with experimental uveitis. Trans Am
Ophthalmol Soc. 1990;88:797-850.
Anderson RE, Rapp LM, Wiegand RD. Lipid peroxidation and retinal degeneration. Curr Eye Res. 1984; 3:
223-227.
Wu GS, Stanforth DA, Rao NA. Localization of EAUmediated oxidative damage in retina by fluorescent
derivatization of cellular carbonyls. ARVO Abstracts.
Invest Ophthalmol Vis Sri. 1994; 35:1517.
Armstrong D, Santangelo G, Connole E. The distribution of peroxide regulating enzymes in the canine eye.
Curr Eye Res. 1981; 1:225-242.
Rao NA, Thaete LG, Delmage JM, Sevanian A. Superoxide dismutase in ocular structures. Invest Ophthalmol
Vis Sri. 1985;26:1778-1781.
Atalla L, Fernandez MA, Rao NA. Immunohistochemi-
Downloaded From: http://iovs.arvojournals.org/ on 05/04/2017
28.
29.
30.
31.
cal localization of catalase in occular tissue. Curr Eye
Res. 1987;6:1181-1187.
Attala LR, Sevanian A, Rao NA. Immunohistochemical
localization of glutathione peroxidase in ocular tissue.
Curr Eye Res. 1988; 7:1023-1027.
Bode AM, Green E, Yavarow CR, et al. Ascorbic acid
regeneration by bovine iris-ciliary body. Curr Eye Res.
1993; 12:593-601.
Whitcup SM, Nussenblatt RB. Treatment of autoimmune uveitis. AnnNY Acad Sri. 1993;696:307-318.
Masuda K, Nakajima A. A double-masked study of
cyclosporine in Behcet's disease. In: Schindler R, ed.
Cyclosporine Treatment in Behcet's Disease. Berlin:
Springer-Verlag; 1985:162-164.
32. Wu GS, Walker J, Rao NA. Effect of Deferoxamine
on retinal lipid peroxidation in experimental uveitis.
Invest Ophthalmol Vis Sri. 1993; 34:3084-3089.
33. Alio JL, Artola A, Serra A, Ayala MJ, Mulet ME. Effect
of topical antioxidant therapy on experimental infectious keratitis. Cornea. 1995; 14:175-179.