Autoreactive Antibodies and Loss of Retinal Ganglion Cells ... Autoreactive Antibodies and Loss of Retinal Ganglion Cells in Rats Induced by Immunization with Ocular Antigens Panagiotis

Autoreactive Antibodies and Loss of Retinal GanglionCells in Rats Induced by Immunization withOcular Antigens

Panagiotis Laspas,1 Oliver W. Gramlich,1 Harald D. Muller,2 Clemens S. Cuny,1

Philip F. Gottschling,1 Norbert Pfeiffer,1 H. Burkhard Dick,3 Stephanie C. Joachim,*,3,4

and Franz H. Grus*,1,4

PURPOSE. In an experimental autoimmune animal model, retinalganglion cell (RGC) loss was induced through immunization withglaucoma-related antigens. The target of this study was to investi-gate the pathomechanism behind this decline and the serumantibody reactivity against ocular and neuronal tissues after im-munization with glaucoma- and non–glaucoma-associated anti-gens.

METHODS. Rats immunized with optic nerve antigen homoge-nate (ONA) or keratin (KER) were compared to control rats(CO). Intraocular pressure (IOP) was measured, and the fundiwere examined regularly. Four weeks afterward, cells werecounted in retinal flat mounts. Retina, optic nerve, and brainsections from healthy animals and optic nerve sections fromimmunized animals were incubated with serum collected atdifferent time points. The occurrence of autoreactive antibod-ies was examined. Signs of antibody deposits, microglia activa-tion, and demyelination were sought in optic nerves of immu-nized animals. Brain sections were examined for abnormalities.

RESULTS. No IOP or fundus changes were observed. Animalsimmunized with ONA showed a significant cell loss comparedwith the CO group. Elevated autoreactive antibodies againstretina, optic nerve, and brain were observed. Animals immu-nized with KER, despite their immunologic response againstKER, demonstrated neither RGC loss, nor increased develop-ment of autoreactive antibodies. Optic nerve from animalsimmunized with ONA demonstrated antibody accumulation,glia activation, and demyelination. No such observations were

made in the KER or CO groups. Brain sections were withoutpathologic findings.

CONCLUSIONS. Systemic autoimmunity against ocular and neuro-nal epitopes, mediated by accordant autoreactive antibodies, isinvolved in the inflammatory processes that cause RGC degen-eration in this experimental animal model. (Invest OphthalmolVis Sci. 2011;52:8835–8848) DOI:10.1167/iovs.10-6889

Glaucoma is one of the most common causes of irreversibleblindness worldwide. By 2020, approximately 80 million

people affected by glaucoma are expected to be bilaterallyblind.1 However, the pathogenesis of the disease is not fullyunderstood. Elevated intraocular pressure (IOP) is still consid-ered to be one of the most important risk factors, but cannotexplain all cases of glaucoma.2

Today, many research groups support the theory that im-munity plays an important role in glaucoma pathogenesis.3,4

Previous studies have shown that glaucoma patients developantibody alterations against specific retina and optic nerveproteins.5 Despite these results, it is still unclear whether thechanges in antibody patterns have a causal connection withglaucoma development or are epiphenomena of the disease.

In vitro experiments have already shown that RGC survivalcan be impaired by the presence of exogenously applied anti-bodies—for example, those against heat shock proteins.6 In anattempt to further investigate in vivo the role of antibodies inglaucoma, we used an animal model in which retinal cytotox-icity can be provoked through antigen immunization. As thismodel uses no methods of direct damage of the retina or theoptic nerve, such as chronic ocular hypertension,7 acute reti-nal ischemia,8 or optic nerve crush,9 it is suitable to analyze therole of the immune system during RGC death. A significantdecline of RGCs in the retina of rats after immunization withspecific ocular antigens (heat shock proteins) was recentlypresented by other studies.10,11 Wax et al.11 observed infiltra-tion of activated T-cells on retinal flat mounts from immunizedanimals. Joachim et al.10,12 showed that immunization withheat shock proteins leads to RGC loss and causes alterations inthe antibody profile of animals, including up- and downregu-lation of specific retinal proteins,13 similar to those observed inpatients with glaucoma.

The main objective of this study was to analyze the devel-opment and the time course of antibody autoreactivity againstocular tissues (retina and optic nerve), as well as against centralnervous system (CNS) tissues (brain) in animals, after immuni-zation with glaucoma- or non–glaucoma-associated antigens. Inaddition, histologic examinations of brain and optic nervesections were conducted to search for antibody accumulationand signs of a possible inflammatory process, such as demyeli-nation, cellular infiltrates, and glia activation, allowing a deeper

From 1Experimental Ophthalmology, Department of Ophthalmol-ogy and the 2Department of Neuropathology, University Medical Cen-ter, Johannes Gutenberg University, Mainz, Germany; and the 3Exper-imental Eye Research Institute, Ruhr University Eye Hospital, Bochum,Germany.

4These authors contributed equally to the work presented hereand should therefore be regarded as equivalent authors.

Supported in part by Deutsche Forschungsgemeinschaft Grant JO886/1-1 (SCJ) and a scholarship from the German Academic ExchangeService (DAAD) (PL).

Submitted for publication November 13, 2010; revised May 20 andSeptember 25, 2011; accepted October 9, 2011.

Disclosure: P. Lapas, None; O.W. Gramlich, None; H.D. Muller.None; C.S. Cuny, None; P.F. Gottschling, None; N. Pfeiffer, None;H.B. Dick, None; S.C. Joachim, None; F.H. Grus, None

*Each of the following is a corresponding author: Franz H. Grus,Department of Ophthalmology, Augenklinik der Universitatsmedizinder Johannes-Gutenberg-Universitat, Langenbeckstr. 1, 55131 Mainz,Germany; [email protected] C. Joachim, Experimental Eye Research Institute, Ruhr Uni-versity Eye Hospital, In der Schornau 23-25, 44892 Bochum, Germany;[email protected].

Glaucoma

Investigative Ophthalmology & Visual Science, November 2011, Vol. 52, No. 12Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 8835

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insight into the pathomechanism of the observed neuronaldecline in retina in this model.

METHODS

Animals

Twenty adult male Lewis rats were obtained from Charles River (Sul-zfeld, Germany). All animals were handled according to the ARVOStatement for the Use of Animals in Ophthalmic and Vision Research,and the project was approved by the Animal Care Committee ofRhineland-Palatinate, Germany.

The animals were kept in a 12:12 hour light–dark cycle environ-ment under standardized conditions. Food and water were provided adlibitum to the rats as usual. Clinical examinations of all animals, includ-ing ophthalmic examinations, were performed daily.

Immunization

All animals were immunized with a single intraperitoneal injectioncontaining the antigen together with incomplete Freund’s adjuvant

and pertussis toxin (both Sigma-Aldrich, Munich, Germany), as de-scribed previously.14,15 Four weeks later, all animals were euthanized.

Animals were divided in four groups (n � 5 each). Two groups (ONAI and ONA II) were immunized with homogenate of bovine optic nerve.The animals in group ONA I received 8 mg homogenate plus 500 �Lincomplete Freund’s adjuvant and 120 �L (3 �g) pertussis toxin. Theanimals in group ONA II were immunized with half this dose, consistingof 4 mg optic nerve homogenate, 250 �L incomplete Freund’s adjuvant,and 60 �L (1.5 �g) pertussis toxin. A third group (KER) received KER (MPBiomedicals, Solon, OH), an antigen currently not associated with glau-coma. The immunization contained 1 mg KER diluted in 500 �L incom-plete Freund’s adjuvant together with 120 �L (3 �g) pertussis toxin. KERsare a family of fibrous structural proteins that form hard but unmineralizedstructures, such as hair and nails. Such proteins are contained mainly inthe intracytoplasmic cytoskeleton of epithelial tissue and are essentialin the maintenance of its mechanical stability and integrity.16 In the eye,only the corneal epithelium contains KERs in high quantities. KER 3 and12 are expressed in all corneal epithelial cell layers. An exception is thelimbus area, where only suprabasal cells are KER positive, whereas thebasally located corneal stem cells are negative.17 Mutations in the genes ofthese KERs are responsible for Meesmann’s corneal dystrophy.18 The lackof KER 12 in genetically engineered mice results in a mechanically fragile,easily detachable corneal epithelium.19 For immunization in animals, KERsare used to obtain anti-KER sera or to specify and isolate anti-KER anti-bodies.20,21 Therefore, they have been shown to be effective immuno-gens. The CO group received 1 mL sodium chloride, together with 500 �Lincomplete Freund’s adjuvant and 120 �L (3 �g) pertussis toxin.

Measurement of IOPRats were subjected to IOP measurement before immunization, as wellas 2 and 4 weeks afterward. Measurements were conducted with a

TABLE 1. IOP in the Experimental and Control Groups

Baseline 2 Weeks 4 Weeks

CO 12.8 � 0.3 12.6 � 0.3 12.4 � 0.2ONA I 12.9 � 0.2 12.2 � 0.2 12.3 � 0.1ONA II 12.5 � 0.2 12.7 � 0.2 12.5 � 0.2KER 12.9 � 0.2 12.7 � 0.3 12.5 � 0.2

IOP of all groups before immunization and 2 and 4 weeks after-wards. Data are expressed as the mean � SEM.

FIGURE 1. (A) Intraocular pressureof all study groups before as well as 2and 4 weeks after immunization. (B)Fundus photographs from a rat beforeand 2 weeks after immunization withoptic nerve homogenate (ONA I).

8836 Laspas et al. IOVS, November 2011, Vol. 52, No. 12


hand-held tonometer (TonoPen; Medtronic, Baseweiler, Germany), asdescribed previously.22–24 During each examination, 10 measurementsper eye were performed, and mean counts were calculated (Table 1;Fig. 1A).

Funduscopy

Fundi were examined directly after IOP was measured. During thisprocedure, the animals were anesthetized with gaseous isoflurane. Thefundi were inspected and photographed through a binocular surgicalmicroscope (Carl Zeiss, Jena, Germany). Images obtained at the threetime points were compared at the end of the study (Fig. 1B).

Retinal Flat Mounts and Cell CountsEyes were enucleated and fixed in 4% paraformaldehyde solution(VWR, West Chester, PA), and retinal flat mounts were prepared.Cells were stained with cresyl blue according to standard proto-cols.25–27 After de- and rehydration by increasing and decreasingconcentrations (70%–100%) of ethanol, respectively, the flat mountswere placed in distilled water and stained with 2% cresyl blue(Merck, Darmstadt, Germany). After staining, they were differenti-ated, dehydrated in ethanol, incubated in xylene, and fixed inquick-hardening mounting medium (Eukitt; Sigma-Aldrich, Munich,Germany). To quantify the different neurons in the superficial

FIGURE 2. (A) Retina flat mount af-ter cresyl blue stain. Sixteen areaswere photographed per flat mount atfour eccentricities from the opticnerve: (1) central, (2) mid-central,(3) mid-peripheral, and (4) periph-eral. (B) Micrographs of retinal flatmounts from the different studygroups. The density of the neuronalcells stained with cresyl blue is re-duced in the ONA group comparedwith the CO and the KER group. Be-tween the KER and the CO groups,no difference was noted. (C) Percent-ages of surviving neuronal cells inthe ONA I, ONA II, and KER groupsin relation to the neuronal cells ofthe CO group. *Significantly lowercell percentages were counted in theONA I and ONA II groups, whereasin the KER group, nearly all of theRGCs survived. (D) Percentage ofRGCs preserved in the ONA I, theONA II, and the KER groups, thistime in correlation with the differentareas of the flat mounts: (1) central,(2) mid-central, (3) mid-peripheral,and (4) peripheral. It can be ob-served that the differences betweenthe ONA groups and the KER groupbecome greater as we move from thecentral to the peripheral areas of theflat mounts, showing a geographicpreference of RGC decline in the pe-ripheral retina Scale bar, 10 �m.

IOVS, November 2011, Vol. 52, No. 12 Autoreactive Antibodies 8837


retinal ganglion cell layer, micrographs were taken as in formerstudies in 16 predefined areas, four in each quadrant with 40�magnification28,29 (Figs. 2A, 2B). As shown in Figure 2A, these areaswere located in the central, mid-central, mid-peripheral, and periph-eral areas of the flat mount in relation to the optic nerve head (1– 4).An epifluorescence microscope (Axio Imager M1; Carl Zeiss)equipped with a digital camera (AxioCam HRc; Carl Zeiss) was usedfor photography and analysis of retinal flat mounts. All cells in thesuperficial retinal ganglion cell layer were manually counted by anexperienced examiner masked to the protocol, who was not in-volved in previous parts of the study. The cells were subdivided intothree cell types by morphologic criteria, such as shape, location,structure, and size of the Nissl substance using the cell counterplug-in iImage J software (developed by Wayne Rasband, NationalInstitutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html).30 Neurons rich in Nissl substance with a prom-inent nucleolus have a diameter above 8 �m, and their shape ischaracterized by irregular outlines. Glial cells (GCs), diameter �8�m, are usually round with regular outlines and acquire a darkerstain. Endothelial cells, easy to see due to their more longitudinalshape, were excluded from further analysis. The classification ofretinal cells according to morphologic criteria after staining withcresyl violet is an established method already used in previousstudies in different species.25,31–33

Collection of Blood Samples

Blood samples from all animals were obtained via tail vein puncturebefore and 2 weeks after immunization.34,35 At the end of the study,blood was collected via heart puncture. All blood samples weretransferred into reaction tubes (Eppendorf, Hamburg, Germany)immediately after collection. After 20 minutes of clotting, they werecentrifuged at 12,000 rpm for 20 minutes at 4°C (Biofuge Heraeus,Hanau, Germany). Serum samples were obtained and stored at�80°C for later analyses.

Quantification of Antibody Reactivity againstOcular Tissues

To evaluate the antibody reactivity of serum against retina and opticnerve, tissues of healthy Lewis rats embedded in paraffin were used.Retinal and optic nerve cross sections were cut in 1-�m slices on amicrotome (Reichert-Jung, Depew, NY), mounted on glass slides (Su-perfrost Plus; Menzel, Braunschweig, Germany), and subsequentlystained.36,37 The tissues were initially pretreated for 10 minutes with0.3% hydrogen peroxide (Roth, Karlsruhe, Germany) in phosphate-buffered saline solution (PBS; Invitrogen, Carlsbad, CA) to decreaseendogenous peroxidase activity. To increase their binding sensitivitywith the primary antibody, they were immersed in preheated targetretrieval solution (Dako, Carpinteria, CA) and incubated for 45 min-utes. Afterward, they were incubated with 1% bovine serum albumin in0.5% Triton X-100 (both Sigma-Aldrich) to prevent nonspecific bind-ing. Subsequently, they were incubated with serum samples (dilution1:200 for retina and 1:750 for optic nerve sections). Serum samplesfrom all groups (CO, ONA I, ONA II, and KER) collected at three timepoints during the study (0, 2, and 4 weeks after immunization) wereused. A monoclonal secondary anti-IgG antibody (H�L, 1:500; PierceBiotechnology, Rockford, IL) was subsequently applied. Color wasdeveloped through application of 3,3-diaminobenzidine tetrahydro-

chloride (DCS, Hamburg, Germany) used as a co-substrate. Finally, allslides were counterstained with hematoxylin (Merck) and mounted.Micrographs of stained sections were taken with the same epifluores-cence microscope with digital camera (Figs. 3, 4) as that used for cellcounts. All cross sections were examined and evaluated microscopi-cally by three independent examiners. The scores ranged from 0, nostaining, up to 3, intense staining.37,38

To evaluate endogenous changes in tissues of immunized animalsfavoring antibody binding, as well as a possible already present anti-body autoreactivity in their serum, the same immunohistochemistryprocess was followed on longitudinal optic nerve sections from immu-nized animals of all groups of the study (Fig. 5).

Detection of Antibody Reactivity against Brainand Dermal Tissues

To test whether autoreactive antibodies can be detected againstneuronal tissues besides the eye, brains were obtained from healthyrats and embedded in paraffin. Brain cross sections of 3-�m thick-ness were prepared and pretreated according to a modified protocolof the one described earlier. After de- and rehydration, the tissueswere incubated in a water bath (99°C) with target retrieval solution(Dako) for 1 hour, followed by incubation in 1% bovine serumalbumin with 0.1% Triton X-100 and 1% goat serum in PBS solution(1 hour). Afterward, the slides were incubated with the differentserum samples obtained from the study animals as previously de-scribed for the ocular tissues. Sera from all time points (before theimmunization, as well as 2 and 4 weeks afterward) were used.Serum incubation continued overnight, followed by incubationwith goat anti-rat IgG immune fluorescence FITC-labeled antibodyas a secondary antibody. Finally, the sections were mounted withDAPI.

Several slides of dermal tissue from the ear obtained from healthyrats underwent the same procedure.

All slides were examined directly afterward through a fluorescencemicroscope (Eclipse TE 2000; Nikon; Dusseldorf, Germany) equippedwith a CCD camera (Fig. 6). Two independent examiners, masked tothe protocol, scored all brain sections, as described above. Scoresranged from 0, no staining, to 3, intense staining.37,38

Histopathology of Optic Nerve

Optic nerve tissues were obtained from all groups. They were cut at adistance of 2 mm from optic chiasm, fixed in 4% paraformaldehyde,and embedded in paraffin. Longitudinal sections (5 �m) were stainedwith hematoxylin-eosin (H&E) and Luxol fast blue (LFB), with orwithout Nissl (LFB/Nissl), to detect possible pathologic changes, suchas inflammation, aberration, or demyelination. Demyelination wasgraded by two examiners masked to the protocol and using a scoringsystem from 0 to 3 (0, no demyelination; 1, rare foci of demyelination;2, small areas of demyelination; and 3, large areas of demyelination).39

IgG Antibody Accumulation and Microglia inOptic Nerves

Optic nerve sections were prepared for detection of possible IgGantibody deposits and microglia activation.30 Sections were transferredonto slides, deparaffinated, and rehydrated. The slides were initiallypretreated with target retrieval solution for 45 minutes (Dako), fol-

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FIGURE 3. (A) Retina cross sections after incubation with rat sera. Sera collected before immunization triggered no IgG antibody reactivity. Twoweeks after immunization, some serum samples showed weak signs of antibody reactivity. Four weeks after immunization, serum from ONAimmunized rats produced a much stronger staining, whereas no staining was detectable in the CO and the KER groups. (B) Example of retina crosssections incubated with ONA and CO serum. The basic retinal layers are labeled: RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, innernuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PHR, photoreceptors. The autoreactive antibodies bound particularly to theretinal ganglion cell layer and the nerve fiber layer. On the other side, no antibody binding can be detected in the different retinal layers afterincubation with serum from the CO group. (C) IgG antibody reactivity against retina before the immunization, as well as 2 and 4 weeks afterward.Scale bars, 50 �m.







lowed by blocking with a solution containing 0.5% normal goat serum(Vector Laboratories, Burlingame, CA), 1% BSA, and 0.1%Triton X-100dissolved in PBS. For detection of IgG depositions, tissues were incu-bated overnight with an FITC-labeled anti-rat IgG antibody (1:500;GenWay Biotech, San Diego, CA) and then mounted with antifademedium (Vectashield; Vector Laboratories).

As a microglia marker, an antibody against the ionized calciumbinding adaptor molecule 1 (rabbit anti-Iba 1, 1:500; Wako PureChemical Industries, Osaka, Japan) was used. Tissues were incu-bated with this antibody overnight, followed by secondary antibodyincubation with a Cy3-conjugated goat anti-rabbit IgG antibody(1:500; Linaris, Wertheim-Bettingen, Germany) for 3 hours. Imageswere taken via a fluorescence microscope (Fig. 8B). For microgliaanalysis, the number of nuclei with Iba1-positive perikarya wascounted in a 214 � 173-�m region of interest (ROI, 37.02 mm2)after background subtraction by ImageJ software. Activated andramified microglia were distinguished based on morphologic crite-ria such as major branches with their ramified processes or anamoeboid shape (activated).30,40 The ROI quadrate was alwaysplaced axially in the longitudinal sections approximately 1 mmdistal to the cutting edge to the optic chiasm, representing themiddle part of the optic nerve.

Histologic Processing of Brain

The brains for all rats were immersion-fixed in 4% buffered formalin,paraffin-embedded, and 4-�m-thick sections were prepared for H&E

and Luxol fast blue with periodic acid Schiff staining (LFB/PAS)staining, as well as for immunohistochemistry. An autostainingdevice (Autostainer Plus; Dako) was used for immunostaining ofhorizontal sections of the brains as well as transverse sections of thespinal cord with monoclonal antibodies to CD3 (polyclonal, dilution1:150; Dako), which is a marker for T cells; CD20 (clone L26,dilution 1:500; Dako), which is a B-cell marker; and CD68 (clonePG-M1, dilution 1:600; Dako), which is a marker for monocytes andmacrophages. Processing by the autostaining program consisted of5 minutes in peroxidase block (S2023; Dako), a 30-minute incuba-tion in primary antibody, followed by a 30-minute incubation insecondary reagent (Histofine; Dako) and 10 minutes in diaminoben-zidine substrate. After completion of the staining procedure, thestained slides were removed from the device and counterstained for5 minutes in hemalum. All slides (Fig. 9) were examined microscop-ically (Vanox-T; Olympus, Tokyo, Japan).

Statistical Analysis

All data collected from IOP measurements, retinal cell counts, IgGantibody autoreactivity, demyelination, and microglia activation scor-ing were transferred to the software (Statistica, ver. 8.0; StatSoft, Tulsa,OK) for statistical analysis. Results of all animal groups were comparedusing two-way ANOVA analysis and are presented in mean counts. TheTukey test for multiple comparisons was applied, and differencesreaching P � 0.05 were considered significant.

RESULTS

Intraocular Pressure

IOP was found to be stable in all groups throughout the study(Table 1, Fig. 1 A). The IOP within each study group showedno significant alterations between the different points in time(P � 0.05), and no significant difference was found betweenstudy groups (P � 0.05).

Funduscopy

No animal developed pathologic fundus changes during thestudy (Fig. 1B). Fundus examinations revealed neither ab-normalities in blood vessels nor neoangiogenesis or retinalbleedings. The optic discs and blood vessels were clearlyvisible at all points in time, and no differences were notedbetween study groups. Corneas were also inspected micro-scopically, and no signs of opacity or other pathologicfindings were observed.

Evaluation of Retinal Flat Mounts

To evaluate the possible cell loss in rats after immunization, theCO group was used as a point of reference and comparison. Asthe CO group demonstrated the highest cell counts in thesuperficial retinal ganglion cell layer (Table 2A), the othergroups kept only a percentage of these numbers (Fig. 2C). Inthe two ONA groups, only 69.7% (ONA I) and 70.1% (ONA II)of the RGCs survived after immunization. The mean counts forthe ONA groups were significantly lower than those of the COgroup (P � 0.000008 for both ONA groups). On the otherhand, in the animals that received KER, a higher percentage(95.1%) of RGCs was preserved. The mean counts of the KER

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FIGURE 4. (A) Optic nerve cross sections after IgG antibody detection. Before and 2 weeks after immunization, there was no or very little detectablestaining. Four weeks after immunization a strong binding of autoreactive antibodies was observed regarding ONA samples. The CO and KER groupsrevealed no such signs. (B) Micrograph of optic nerve cross sections incubated with serum from the ONA and the CO group. Staining can be seenon the whole mass of the section around the nuclei of the Schwann cells. On the other hand, no staining is detected on the neuronal tissue afterincubation with serum from the CO group. (C) IgG antibody reactivity against optic nerve related to the time points before and after immunization.Scale bar, 50 �m.

FIGURE 5. Left: Longitudinal optic nerve sections from healthy ratsincubated with serum samples from the different groups of the study.Right: longitudinal optic nerve sections from study rats (ONA, KER,and CO groups) incubated with their own serum. The incubation ofhealthy tissues with serum from the CO or KER groups led to noantibody binding. When optic nerve tissues from these animals areincubated with probes of their own serum, a very moderate staining isobserved. On the other hand, intense antibody reactivity is seen inserum of animals of the ONA group against healthy optic nerve andwas even higher against optic nerve tissues of the same animals. Scalebar, 10 �m.



group were not significantly different from those of the COgroup (P � 0.28). Moreover, when the counts of the ONA I andII groups were compared with those of the KER group, significantdifferences were also observed (P � 0.000008 for both ONAgroups). Notable is the fact that despite the different antigendoses they received, the ONA I and II groups showed no statisti-cally significant difference from each other (P � 0.28).

Examination of four areas of the retinal flat mounts re-vealed a predominant neuronal cell loss in the peripheralsections of the retina (Table 2C, Fig. 2D). Regarding thenumber of glia measured in the surface of the retinal gan-glion cell layer, only a slight elevation of the cell count wasobserved in animals immunized with ocular antigens and itwas not statistically significant.

FIGURE 6. (A) Brain cross sectionsof the cerebellum after IgG antibodydetection through immunofluores-cence staining. Before the immuniza-tion, only slight staining was ob-served on brain tissue. Four weeksafter immunization, there was con-siderable immunofluorescence stain-ing on brain sections incubated withserum from ONA animals. The stain-ing was most intense around the Pur-kinje cells and their dendrites. Onthe other hand, no staining was de-tected on brain sections incubatedwith KER serum. (B) IgG antibodyreactivity (score) against brain tis-sues in relationship to the timepoints of the study. Scale bar, 10 �m.



Autoreactive Antibodies against Ocular Tissuesfrom Naïve and Immunized Animals of the Study

Incubation of cross sections with serum obtained from allanimals before immunization showed no detectable autoreac-tive IgG antibodies against ocular tissues (Table 3). Meanscores for retina and optic nerve sections in all groups wereapproximately 0. Hence, no significant differences were ob-served between the study groups.

After 2 weeks, some signs of autoreactivity against theretina were observed after incubation with ONA sera (Table 3;Fig. 3). In both ONA groups, scores were significantly higherthan the mean score of the CO group (P � 0.02 for both ONAgroups). The KER group showed no significant signs of auto-reactivity, with a mean score not significantly different from

that of the CO group (P � 0.8). The comparison between ONAgroups and the KER group revealed no significant difference(P � 0.1 for both ONA groups). Similar observations weremade for the optic nerve cross sections (Table 3, Fig. 4). Incomparison to the CO group, elevated antibody reactivity wasfound in the ONA I group (P � 0.001). The ONA II and KERgroups showed levels of autoreactive antibodies similar to thatof the CO group (ONA II: P � 0.4; KER: P � 0.99). Of the ONAgroups, only ONA I showed increased reactivity in comparisonto the KER group (ONA I: P � 0.0008; ONA II: P � 0.45).

Four weeks after immunization, the antibody reactivityagainst retina (Table 3, Fig. 3) increased further in the animalsimmunized with ONA than in the CO group (ONA I; P �0.0003; ONA II: P � 0.0008). The KER group continued to

TABLE 2. Neuronal Cell Data

A. Count of Neuronal Cells on Retinal Flat Mounts

Total Flat Mount Central Mid-central Mid-peripheral Peripheral

CO 297 � 37 313 � 35 301 � 35 301 � 36 273 � 32ONA I 207 � 51 247 � 44 225 � 38 183 � 40 172 � 42ONA II 220 � 42 239 � 36 241 � 32 215 � 36 184 � 40KER 284 � 28 295 � 17 296 � 22 281 � 25 263 � 36

B. P Values of Comparisons between the Animal Groups Regarding the Neuronal Counts in the Whole Retinal Flat Mounts

CO ONA I ONA II KER

CO — 0.000008 0.000008 0.28ONA I 0.000008 — 0.28 0.000008ONA II 0.000008 0.28 — 0.000008KER 0.28 0.000008 0.000008 —

C. Percentage of Surviving Neuronal Cells in the Study Groups in Relation to CO Group Counts

Total Flat Mount Central Mid-central Mid-peripheral Peripheral

ONA I 69.7 � 17.1 79.1 � 14.1 74.6 � 11.2 60.3 � 13.4 63.3 � 15.4ONA II 74.1 � 14.2 76.3 � 11.3 80.2 � 10.6 71.6 � 11.8 67.5 � 14.8KER 95.6 � 9.6 84.6 � 5.3 98.3 � 7.1 93.5 � 8.4 96.6 � 13.1

(A) Multiple comparisons between all animal gtroups were performed by two-way ANOVA, followed by Tukery’s post hoc test. Values areexpressed as mean counts � SD. Results are presented for all examined retinal areas together, as well as for the four retinal areas separately. (B)P values from Tukey’s post hoc test after two-way ANOVA of all comparisons between the study groups of RGC counts in the whole retinal flatmounts. Only P � 0.05 was regarded as statistically significant and is shown in bold. (C) Percentages of RGCs that were preserved in the ONA I,the ONA II, and the KER groups (% � SD), in the whole retina flat mounts and in the different areas of the flat mounts.

TABLE 3. Scores of Autoreactive IgG Antibodies

CO ONA I ONA II KER

Retina Cross Sections

Baseline 0.4 � 0.3 0.3 � 0.1 0.5 � 0.2 0.5 � 0.22 Weeks 0.1 � 0.1 1.3 � 0.4 1.3 � 0.3 0.4 � 0.24 Weeks 0.1 � 0.1 2.3 � 0.2 2.1 � 0.4 0.6 � 0.3

Optic Nerve Cross Sections

Baselline 0.00 0.00 0.00 0.002 Weeks 0.1 � 0.1 1.6 � 0.4 0.5 � 0.2 0.1 � 0.14 Weeks 0.1 � 0.1 2.5 � 0.2 1.7 � 0.5 0.3 � 0.3

Brain Cross Sections

Baseline 0.1 � 0.1 0.1 � 0.1 0.1 � 0.1 0.1 � 0.12 Weeks 0.2 � 0.1 1.6 � 0.3 0.8 � 0.2 0.4 � 0.14 Weeks 0.3 � 0.1 1.5 � 0.3 1.7 � 0.1 0.2 � 0.1

Data are expressed as the mean � SEM.



demonstrate low levels of autoreactive antibodies not signifi-cantly different from that of the CO group (P � 0.6). At thistime point, the antibody reactivity was significantly higher inthe ONA groups than in the KER group (ONA I: P � 0.002;ONA II: P � 0.007). The examination using optic nerve crosssections revealed similar results (Table 3, Fig. 4). Both ONAgroups had significantly higher antibody reactivity than the COgroup (ONA I: P � 0.0003; ONA II: P � 0.005). Again, the KERgroup showed no significant difference from the CO group(P � 0.9). The comparison between the ONA groups and theKER group revealed significant differences (ONA I: P � 0.0005,ONA II: P � 0.018).

Regarding the antibody autoreactivity against ocular tissuesfrom immunized animals of the study, a very moderate stainingwas observed in optic nerves of animals of the CO and KERgroups after incubation with their own serum (obtained after 4weeks). A much more intense staining was seen when opticnerves from the ONA groups were incubated with serum fromthese animals (Fig. 5).

Autoreactive Antibodies against Brain Tissue

Almost no immunofluorescence staining was detected on allbrain cross sections incubated with serum taken before immu-nization (Table 3; Fig. 6). Therefore no difference betweengroups was observed.

Two weeks after immunization, antibody reactivity wasdetected in sera from some animals immunized with ONA. Thedifference between the ONA I group and the CO group wasstatistically significant (P � 0.0001). The ONA II and the KERgroups showed no significant difference from the CO group(ONA II: P � 0.29; KER: P � 0.99). Of the ONA groups, onlyONA I had significantly higher antibody reactivity than the KERgroup (ONA I: P � 0.0003; ONA II: P � 0.12).

Four weeks after immunization, the antibody reactivityagainst brain tissues was increased in ONA groups. The othergroups demonstrated almost no signs of antibody reactivity.The differences between both ONA groups and the CO groupwas statistically significant (ONA I: P � 0.0003; ONA II: P �0.0001). There was no significant group difference betweenKER and CO (P � 0.99), but significant differences were noted

between the KER group and the two ONA groups (ONA I: P �0.00016; ONA II: P � 0.00013).

Optic Nerve Demyelination

Analysis of the LFB-stained longitudinal optic nerve sectionsrevealed moderate demyelination in both ONA groups 4 weeksafter immunization (Fig. 7A). Demyelination was characterizedby disruption or alterations in the organization of the neuronmyelin sheaths, whereas the optic nerve sections from animalsof the CO and the KER group showed no or few such signs.Moreover, several cellular infiltrates were seen in longitudinaloptic nerve sections of the ONA groups. After scoring andstatistic analysis, the differences between both ONA groupsand the CO (ONA I: P � 0.0005; ONA II: P � 0.013) and KER(ONA I: P � 0.0005; ONA II: P � 0.013) groups were found tobe significant. No significant difference (P � 1.0) was foundbetween the CO and KER groups (Fig. 7B).

IgG Deposition and Activation of Microglia inOptic Nerves

Several IgG accumulations were noted in almost all opticnerves of ONA animals. Beside focal diffuse depositions, stron-ger staining patterns were observed on single axons (Fig. 8A).Optic nerves of CO and KER animals revealed only staining atthe blood vessels and some unspecific background fluores-cence staining, but no axon-guiding IgG deposits.

Ramified microglia decreased in the immunized groups (Fig.8B), whereas a significant increase in activated microglia wasseen. In optic nerve tissues from the CO and the KER groupsno such increase was noted. After scoring and statistical anal-ysis, the differences between the ONA and the CO groups(ONA I: P � 0.00017; ONA II: 0.00018) as well as betweenthe ONA groups and the KER group (ONA I: P � 0.00017;ONA II: P � 0.00076) were statistically significant. Betweenthe CO and the KER groups, no difference was noted (P �0.64; Fig. 8C).

Brain Sections

Brains from all animals were also examined 4 weeks afterimmunization. Contrary to the optic nerve cross sections, in

FIGURE 7. (A) Longitudinal opticnerve section from the CO and theKER groups stained with LFB/Nisslrevealed normal myelin patternswith no infiltrates. In sections fromONA immunized animals, rare foci(arrowheads) of moderate demyeli-nation with disruption of the lamellarstructure are shown. Noticeable aredarker stained nuclei of cellular infil-trates beside the demyelination (ar-rows). (B) *Both ONA groupsshowed a significantly increased de-myelination of the optic nerve incomparison to the CO and the KERgroups. Scale bar, 50 �m.



these cases, neither demyelinated lesions nor mononuclear cellinfiltrates consisting of macrophages and/or lymphocytes weredetectable in the rat brains by routine histology (H&E andLFB/PAS staining; Fig. 9) and immunohistochemistry withmonoclonal antibodies to CD3, CD20, and CD68 (not shown).

DISCUSSION

In previous studies, complex antibody profiles and alteredlevels of antibodies against specific ocular antigens were de-tected in serum samples of glaucoma patients.5,41–47 However,it is still unclear whether these antibodies are involved in theinitial pathogenesis of glaucoma or they develop during thecourse of the disease as an accessory phenomenon. To furtherinvestigate the role of antibodies, we used an animal model ofexperimental autoimmune RGC loss.

A decrease in RGCs in rats after immunization with heatshock proteins was recently shown.10,11 Wax et al.11 reporteda decrease in RGC density 1 and 4 months after immunization.Joachim et al.10 observed similar results 5 weeks after immu-nization. In our study, we showed that immunization with anoptic nerve homogenate, a complex mixture of neuronal anti-

gens, triggered neuronal cell loss. In contrast, the immuniza-tion with KER, a non–glaucoma-associated antigen, left thesecells predominantly unaffected. These results suggest that theimmune response triggered through the immunization couldbe target-specific and lead to RGC death. Increased IgG anti-body reactivity against ear cross sections (structures rich inKER) was detected in sera of animals immunized with KER(data not shown). In the eye, however, KERs can be found inhigh quantities only in corneal tissue.16 As the retina containsno KER, the RGCs remain unaffected by an immunologic re-sponse, which would include the development of antibodiesagainst such molecules. This fact could be the reason that theKER injection did not affect neuronal cells in these animals.

We also noted that the immunization with different doses ofthe same antigen had a similar effect on the RGCs. Differentdoses of immunization have been tested in studies of experi-mental autoimmune uveitis.48,49 Results were not uniform, butdepended on the antigen used for immunization. Broekhuyseet al.48 were able to induce a mild posterior retinitis in Lewisrats with opsin, which was not influenced by the amount of theinjected antigen. On the other hand, the same study revealed astronger dose-dependent uveitis caused by immunization with

FIGURE 8. (A) Longitudinal opticnerve sections stained for IgG depos-its. Optic nerves from the CO andKER groups showed no IgG accumu-lation, apart from nonspecific back-ground staining. On the contrary, inboth ONA groups, several axonswere positive for IgG deposits (ar-rowheads). (B) Microglia cells de-tected and visualized by anti-Iba 1immunostaining in optic nerve of ananimal immunized with ONA. Ar-rows: ramified form of the microglia,characterized by the presence ofmore than one major branch. Arrow-heads: the round, compact or amoe-boid shape is typical of activated mi-croglia. (C) Mean values of activatedand ramified microglia. *The ONAgroups showed a significantly in-creased number of activated micro-glia and simultaneously a moderatereduction in the ramified forms.Scale bar, 10 �m.



interphotoreceptor retinoid-binding protein or retinal S-anti-gen. We hypothesize that the doses of optic nerve homogenateused in our study were above a crucial level that did not allowsignificant distinction of the intensity of RGC decline. Futurestudies, including a greater range of immunization doses,should be conducted to investigate in depth the possible cor-relation between the antigen quantity and cell loss.

Furthermore, we found that the animals immunized withONA developed antibodies that were autoreactive against oc-ular tissues, more specifically against retina and optic nerve.These results agree with findings of Joachim et al.,12,13 whoidentified complex alterations in antibody patterns in rats afterimmunization with heat shock proteins. We assume that, in ourstudy, immunization with the homogenate of optic nerve led toa complex systemic immune response, which included theincrease of antibody reactivity in serum against several retinaland neuronal antigens. The animals in our model immunizedwith optic nerve antigens also developed autoreactive antibod-ies against retinal epitopes, predominantly in the nerve fiberlayer and the RGC layer. Since optic nerve tissue includes theRGC axons, retina and optic nerve do form a certain kind ofentity, sharing a large number of antigens and proteins.50–52

Apart from these, much more increased antibody reactivitywas detected when optic nerve tissues from immunized ani-mals were incubated with their own serum. The detected IgGantibodies are expected in this case to be endogenous autoan-tibodies, already bound on their targets, as well as antibodiescontained on the serum probes used for incubation. Concern-ing the ONA groups we assume a basal presence of endoge-nous antibodies, as well as an elevated aggressivity of theserum antibodies against the neuronal epitopes, possibly be-cause of molecular changes happening after immunization.Regarding the CO and the KER groups a moderate endogenousantibody presence was also confirmed, apparently for the samereasons, the more so as also these animals were handled withpertussis toxin and Freund’s adjuvant. In any event, endoge-nous autoreactive antibodies against neuronal tissues in naïverats have already been reported.53,54

Moreover, in animals of the ONA group, elevated antibodyautoreactivity against neuronal (brain) tissues was noted.Rosenmann et al.55 detected autoreactive antibodies againstthe tau protein in serum of mice immunized with neuronal tauprotein,55 accompanied by axonal damage and gliosis in cen-tral nervous system. Tau protein is an antigen found mostly inthe central nervous system and elsewhere, such as the opticnerve and retina, and is associated with neuronal injury andneurodegenerative diseases—among them, glaucoma.56 Ani-mals of our model demonstrated elevated antibody reactivity,not only against retina and optic nerve, but also, similar to thefindings of Rosenmann et al.,55 against brain tissues, after theirimmunization with a neuronal homogenate, which was actu-ally expected to include several neuronal antigens common inthe nervous system, among them glaucoma-associated antigenssuch as tau protein.

On the other hand, animals of the KER group showed noalteration of their antibody autoreactivity against ocular orneuronal tissues throughout the study. This applies also to thecontrol group of our study which showed no autoimmunereactivity.

According to these results, both groups with no signs of aspecific autoimmune reactivity, which targets neuronalepitopes in eye and brain, retained their retinal neuronal cellsthrough the study. Since only animals with high levels ofantibody autoreactivity against such tissues suffered addition-ally from RGC loss, it allows us to propose that these twofindings are connected.

Results of other studies demonstrate that antibodies areindeed able to induce neuronal cell death through a variety ofpathways. Matus et al.57 showed that antiribosomal P-proteinautoantibodies cause apoptotic cell death in brain cellularcultures by increasing the calcium influx into neurons signifi-cantly. A decline in neuronal cultures of the rat cortex was alsoinduced by GluR3 autoantibodies via complement and mem-brane attack complex activation.58 In vitro experiments by Luet al.59 revealed that autoantibodies against the intermediateneurofilament �-internexin can mediate neuronal damage byinhibiting axonal elongation.

Therefore, we suggest that in our model, elevated antibodyautoreactivity against neuronal antigens in eye and brain tissuesimpaired the survival of RGCs through similar mechanisms.Since evidence of the presence of autoreactive antibodiesagainst retina, optic nerve, and brain tissue in serum of immu-nized animals was found in vitro, a possible in vivo binding ofsuperficial or intracellular (after endocytosis) antigens couldtrigger mechanisms that lead to the observed RGC death.

In vitro experiments in a study by Tezel and Wax6 havealready shown that the survival of retinal cells can be impairedby the presence of exogenously applied antibodies—for exam-ple, against heat shock proteins. Moreover, this study offeredevidence of later presence of these antibodies in cytoplasmicand nuclear structures. Of course, such a pathogenic mecha-nism in experiments in vivo, like those conducted in our study,would have as an essential prerequisite the capability of thedeveloped autoantibodies to pass through the blood–retinalbarrier.60 Examination of tissues from immunized animals byelectron microscopy may reveal whether antibodies are indeedable to enter RGCs and on which epitopes they may bind. Inour model, antibody accumulation could be detected on opticnerve tissue from animals immunized with ocular antigens. Nodetection was possible in animals of the CO or the KER group,allowing us once more to connect these findings with theretinal decline observed in the ONA groups.

Even if these antibodies do not directly trigger cell death,autoreactive antibodies could indirectly lead to RGC degener-ation through anatomic alterations or through the activation ofcellular mechanisms, whose role in the pathogenesis of glau-

FIGURE 9. (A) Representative brain stem sections of a CO, an ONA I,and a KER animal after H&E, as well as LFB/PAS, staining. (B) Corre-sponding hippocampus sections of animals from three groups (CO,ONA I, and KER) stained with H&E and LFB/PAS 4 weeks after immu-nization. Neither demyelinated lesions nor mononuclear cell infiltratesconsisting of macrophages and/or lymphocytes were detectable in therat brains. Scale bar, 100 �m.



coma has been discussed.61 In our model, pathologic altera-tions in the anatomy of the optic nerve with a significantreduction of the myelin sheaths of optical neurons were ob-served in animals immunized with ocular antigens. Further-more, cellular infiltration of the neuronal tissue was detected inthe same animals, whereas animals from the CO and KERgroups demonstrated no such signs. Demyelination and acti-vated cytotoxic T cells are known to be pathogenic factors inanimal models of experimental autoimmune diseases, such asexperimental autoimmune uveitis62 and experimental autoim-mune encephalitis.63 On the other hand, no such observationswere made in the brain tissues examined in the study, indicat-ing an inflammatory process targeting specifically the opticnerve. Beside the possible contribution of T cells, the involve-ment of microglia has to be discussed also.64,65 Qualitativechanges were observed regarding the glia cells detected in ourmodel, as the normal ramified glia cells were in large scalereplaced from activated cells in animals of the ONA groups, incomparison to the CO and the KER groups.

According to all these results, only the animals immunizedwith ONA demonstrated fully developed pathology, with ab-normalities in anatomic (demyelination), cellular (cellular infil-tration, glia activation, and molecular (antibody reactivity) lev-els. This pathology probably led to the reduction of RGCs,which was also observed only in animals immunized withONA. How these anatomic, cellular, and molecular mecha-nisms combine to lead to neuronal death in the retina in ourmodel remains for further investigation. For example, an acti-vation of T cells via autoreactive antibodies could result in therelease of smaller molecules, which can easily pass the blood–retina barrier and induce RGC apoptosis66 (e.g., soluble Fasligand, whose potential role in RGC death has already beenproposed in a model of autoimmune glaucoma),11 or ligandsassociated with the tumor necrosis factor death receptor fam-ily.67 On the other hand, microglia are the resident immuno-competent and phagocytic cells in the CNS, and their activa-tion is closely linked to neurodegenerative processes inducedby various stimuli.68 In this regard, microglia could be stimu-lated, as described above for T cells or by antibody cross-linking of retinal and optic nerve proteins.68–70

Further studies should be performed to investigate the roleof these autoreactive antibodies in cell death in more detail.Can a transfer of serum antibodies from immunized to naïveanimals cause cell death in these animals?

In summary, it is known that cellular and chemical autoim-munity are critical for RGC survival.4 Regulatory errors, such asover- or underactivity of these complex mechanisms, could befatal for RGCs. Based on the results of this study, we assumethat specific autoantibodies against neuronal tissues in eye orbrain could be involved in the RGC apoptosis in this experi-mental autoimmune animal model.

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Autoreactive Antibodies and Loss of Retinal Ganglion Cells ... Autoreactive Antibodies and Loss of Retinal Ganglion Cells in Rats Induced by Immunization with Ocular Antigens Panagiotis