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Originally published In Press as doi:10.1167/iovs.08-2348 on August 29, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:5458-5465.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.08-2348
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The Clinical Time-Course of Experimental Autoimmune Uveoretinitis Using Topical Endoscopic Fundal Imaging with Histologic and Cellular Infiltrate Correlation

David A. Copland,1 Michael S. Wertheim,1,2 W. John Armitage,1 Lindsay B. Nicholson,1,3 Ben J. E. Raveney,*,3,4 and Andrew D. Dick*,1,2,3

1From the Academic Unit of Ophthalmology, Department of Clinical Sciences at South Bristol, and the 3Department of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom; 2Bristol Eye Hospital, Lower Maudlin Street, Bristol, United Kingdom; and the 4Department of Immunology, National Institute of Neuroscience NCNP, Tokyo, Japan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. EAU is an established preclinical model for assessment of immunotherapeutic efficacy toward translation of therapy for posterior uveitis. Reliable screening of clinical features that correlate with underlying retinal changes and damage has not been possible to date. This study was undertaken to describe, validate, and correlate topical endoscopic fundus imaging (TEFI) with histologic features of murine experimental autoimmune uveoretinitis (EAU), with the intent of generating a rapid noninvasive panretinal assessment of ocular inflammation.

METHODS. EAU was induced in B10.RIII mice by immunization with the peptide RBP-3161-180. The clinical disease course (days 0–63) was monitored and documented using TEFI. Disease severity and pathology were confirmed at various time points by histologic assessment. The composition of the cell infiltrate was also examined and enumerated by flow cytometry.

RESULTS. TEFI demonstrated the hallmark features of EAU, paralleling many of the clinical features of human uveitis, and closely aligned with underlying histologic changes, the severity of which correlated significantly with the number of infiltrating retinal leukocytes. Leukocytic infiltration occurred before manifestation of clinical disease and clinically fulminant disease, as well as cell infiltrate, resolved faster than histologic scores. During the resolution phase, neither the clinical appearance nor number of infiltrating retinal leukocytes returned to predisease levels.

CONCLUSIONS. In EAU, there is a strong correlation between histologic severity and the number of infiltrating leukocytes into the retina. TEFI enhances the monitoring of clinical disease in a rapid and noninvasive fashion. Full assessment of preclinical immunotherapeutic efficacy requires the use of all three parameters: TEFI, histologic assessment, and flow cytometric analysis of retinal infiltrate.

Experimental autoimmune uveoretinitis (EAU) is a suitable correlate to the spectrum of clinicopathologic features of human uveitides and, as a result, is a successful preclinical model for translation of immunotherapies.1 2 Furthermore, the model serves to dissect immunopathogenic mechanisms relating to immune-mediated tissue damage, which in turn highlight avenues for future immunotherapies.3 4 5 6 Murine EAU is generated after systemic activation of ocular-specific CD4+ T cells that are frequently located within or around photoreceptor segments.7 8 9 10 11 In particular, EAU can be induced via administration of dominant peptides from retinoid binding protein (RBP)-3 (previously called interphotoreceptor retinoid binding protein [IRBP]) in an appropriate adjuvant.12 Disease occurs subsequent to T cell infiltration into the target organ that recruits and activates macrophages into the eye, generating structural damage via mechanisms including secretion of nitric oxide (NO).13

To quantify the extent and severity of disease, which is clearly essential for validating the efficacy of preclinical therapies, two approaches have been used to date: nonvalidated clinical scoring and semiquantitative histologic scoring and grading. Clinical EAU assessment involves in vivo examination of the eye using indirect slit lamp biomicroscopy and scoring the features of retinal, anterior chamber, and pupil appearance during disease.11 In this regard, fundus photography has until now been limited by technical difficulties and the poor resolution of existing techniques for disease assessment.14 Immunohistochemical assessment of retinal sections, with grading according to the degree of inflammatory infiltrate and structural damage, has been used for assessment of disease severity,15 but this technique has inherent limitations, such as the fact that only a small proportion of the whole retina can be examined. Therefore, a new easy-to-use imaging system that facilitates rapid, reproducible, live clinical assessment of the whole fundus, closely correlating with histologic changes is required as an approach to monitor progression of retinal disease in experimental models, including EAU.

Topical endoscopic fundus imaging (TEFI) is a recently described compact system that allows high-resolution in vivo color photography of the retina in rodents and was developed in normal eyes of mice.16 TEFI is based on the use of an endoscope with parallel, lateral, crescent-shaped illumination connected to a digital camera. This technique facilitates rapid assessment and capture of high-quality images of the whole fundus, including the peripheral retina and ciliary body, without distress to the mouse or the requirement for general anesthesia.

The objectives of this study were to validate a platform by using the TEFI system for assessment of the clinical disease time course of RBP-3161-180–induced EAU in B10.RIII mice, and correlate clinical features to both matched published histologic severity scores and the extent of inflammatory retinal cell infiltrate determined by flow cytometric analysis.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice
B10.RIII mice were originally obtained from Harlan UK, Ltd. (Oxford, UK) and a breeding colony established within the Animal Services Unit at Bristol University (Bristol, UK). All mice were housed in specific pathogen-free conditions with continuously available food and water. Female mice, immunized for disease induction, were aged between 6 and 8 weeks. Treatment of animals conformed to UK legislation and to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Reagents
The peptide RBP-3161-180 (SGIPYIISYLHPGNTILHVD) was synthesized by Sigma-Genosys Ltd. (Poole, UK). Peptide purity was >95% as determined by HPLC.

EAU Induction and Scoring
B10.RIII mice were immunized SC in one flank with 50 µg/mouse RBP-3161-180 peptide in PBS (2% DMSO), in Complete Freud’s Adjuvant (CFA; 1 mg/mL; 1:1 vol/vol) supplemented with 1.5 mg/mL Mycobacterium tuberculosis complete H37 Ra (BD Biosciences, Oxford, UK), and 1.5 µg Bordetella pertussis toxin (Sigma-Aldrich, Poole, UK) was given intraperitoneally. At various time points after immunization, the eyes were enucleated, oriented in optimal cutting temperature (OCT) compound (R. Lamb Ltd., East Sussex, UK), and carefully snap frozen. Serial 12-µm sections were cut and stored at –80°, before thawing at room temperature and fixation in acetone for 10 minutes. Sections were stained with rat anti-mouse CD45 monoclonal antibody (Serotec, Oxford, UK), counterstained with hematoxylin (ThermoShandon, Pittsburgh, PA), and then scored for inflammatory infiltrate (presence of CD45-positive cells) and structural disease (disruption of morphology). Cellular infiltrate was scored within the ciliary body, vitreous, vessels, rod outer segments, and choroid, whereas structural disease was scored within the rod outer segments, neuronal layers, and retinal morphology. Both scores were added together to calculate a final disease total (Table 1) .


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  		TABLE 1. Summary of EAU Disease Scoring

 
Topical Endoscope Fundus Imaging
Using a method adapted from Paques et al.,16 we connected an endoscope with a 5-cm-long tele-otoscope with a 3-mm outer diameter (1218AA; Karl Storz, Tuttlingen, Germany) a digital camera (D80 with a 10-million-pixel charge-coupled device [CCD] image sensor and AF 85/F1.8 D objective (Nikkor; all from Nikon, Tokyo, Japan), with a additional +4.00-D magnifying lens. The settings of the camera were as follows: large and superfine image, manual focus; operating mode S (shutter speed priority), shutter set at 1/100 s, and white balance set at fluorescent. A xenon lamp (201315-20; Karl Storz) connected through a flexible optic fiber to the endoscope was used as the light source.

The pupils of the mice were dilated with topical tropicamide 1% and phenylephrine 2.5% (Minims; Chauvin Pharmaceuticals, Romford, UK), then topical oxybuprocaine 0.4% (Minims) and eye gel (Novartis Pharmaceuticals, Camberley, UK), were applied for corneal anesthesia and endoscope contact, respectively. For imaging, the camera with endoscope was attached to a bench-clamp, and the mouse was slowly moved toward the tip of the endoscope. Once contact with the gel covering the cornea was obtained, focus and illumination were adjusted by using the camera, and the fundus was examined and the image was captured. Images were transferred to computer for processing (Photoshop; Adobe Systems, Mountain View, CA). Images were cropped to a size of 6 x 4.85 in. The blue curves tool was used to render the image a natural color. We did not use RAW imaging, as no image manipulation (other than color adjustment) was required. We found that the superfine setting was more than adequate for our purposes and each image was around 3 MB in size. After numerous trials, we found that using the fluorescent light white balance setting generated the best image detail after further blue curve adjustment in the image analysis software.

Isolation of Retinal Infiltrating Cells
Infiltrating retinal cells were isolated by using a previously described method.17 In brief, the eyes were enucleated and the retinas (including the ciliary body) of each animal were dissected microscopically and washed in wash media (complete RPMI supplemented with 10% [vol/vol] FCS and 1 mM HEPES; all from Invitrogen, Paisley, UK). Retinas were then cut into small pieces and digested in 1 mL wash medium, supplemented with 0.5 mg/mL collagenase D (Roche, Welwyn Garden City, UK) and 750 U/mL DNase I (Sigma-Aldrich) for 20 minutes at 37°C. An additional 0.5 mg/mL collagenase D and DNase 750 U/mL was added before incubation for a further 10 minutes at 37°C. Cell suspensions were forced through a 40-µm cell strainer (BD-Falcon, Cambridge, UK), with a syringe plunger, and the cell suspensions were stained for flow cytometric analysis.

Flow Cytometry
The cell suspensions were incubated with 24G2 cell supernatant for 5 minutes at 4°C. For cell counting, retinal cell suspensions were stained with PE-Cy5-conjugated anti-mouse CD4 monoclonal antibody (mAb), APC-Cy7-conjugated anti-mouse CD11b mAb, and PE-Cy7-conjugated anti-mouse CD45 mAb (all BD Pharmingen, Oxford, UK), at 4°C for 20 minutes. Cell suspensions were acquired with a flow cytometer (LSR-II; BD Cytometry Systems, Oxford, UK). Analysis was then performed (FlowJo software; TreeStar, San Carlos, CA). The number of cells counted was calculated by reference to a known standard.

Statistical Analyses
Partial correlation was performed (SPSS Inc, ver. 14; SPSS, Chicago, IL) and used to explore the relationship between the number of CD45+ cells (after square root transformation) and histologic score, while controlling for time (days) after immunization.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
TEFI Imaging of the Retina during EAU
TEFI is a system that allows high resolution in vivo color photography of the retina in rodents and was developed in the normal eyes of C57BL/6 and BALB/c mice.16 We found that high-quality panretinal images, with clear visualization of the peripheral retina could also be obtained in the B10.RIII mouse strain, when the pupil was suitably dilated. The optimal pupil dilation was achieved by using a drop combination of phenylephrine 2.5% and tropicamide 1%, instilled at least 5 minutes before initiating TEFI.

Using this adapted TEFI method, we sought to monitor the clinical changes in the retina that occur during disease progression in mice immunized for EAU. We used the highly susceptible B10.RIII mouse strain, in which the immunizing regimen generates reliable disease induction and consistent moderate disease severity in our hands, thus ensuring that any clinical changes to the retina would be clearly evident.

Mice were immunized SC with 50 µg RBP-3161-180 emulsified in CFA, and pertussis toxin was coadministered intraperitoneally. In the initial experiment, 10 mice were immunized and the disease progression was monitored from days 0 to 63. The TEFI method enabled us to capture a variety of clinical images (Fig. 1) . Clinical features of EAU were clearly observed, including vasculitis and optic nerve swelling (Fig. 1A) ; exudative retinal detachment (Fig. 1B) ; retinal folds, observed as retinal flecks (Fig. 1E) ; and choroidal lesions, analogous to chorioretinal lesions in uveitis in humans (Fig. 1F) . The periphery of the retina could also be visualized, demonstrating the anatomy of the ciliary body and the drainage angle (Figs. 1C 1D) . Figure 1D demonstrates how we were able to increase the magnification of views of the ciliary body and drainage angle by virtue of imaging through the mouse lens.


Figure 1
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  		FIGURE 1. Clinical observations of EAU in B10.RIII mice using topical endoscopic fundus imaging. Shown are examples of clinical disease observed in a representative cohort of B10.RIII mice immunized for EAU using RBP-3161-180 in CFA. Images show raised and swollen optic nerve, with typical perivascular cuffing and caliber changes to vessels (arrowhead, A), and an inferior exudative retinal detachment (B), at day 20 pi. Images including the ciliary body demonstrate peripheral chorioretinal inflammation and inflammatory vascular changes of the marginal vein (C, D). Scattered flecks which correlate to histologic features of retinal folds, are typically observed after day 15 pi (E). Multiple choroidal lesions (arrowhead) associated with inflammatory vascular changes (perivascular cuffing) and swollen optic nerve persisted at day 28 pi (F).

 
The time-course of EAU in the right eye of a representative individual mouse demonstrates the significant changes that occurred during disease progression (Fig. 2) . The retina and vasculature remained normal in appearance, with no clinical evidence of disease from day 0 to 10 postimmunization (pi). However, by day 13 pi, we recorded swelling of the optic nerve that increased in severity to include the central retinal vasculature (analogous to retinal vasculitis in humans). With time (days 14–18 pi), vitritis (cellular infiltrate within the vitreous gel) made the fundus increasingly indistinct (vitreous haze). Despite this vitreous haze, large exudative retinal detachments were documented from day 17 pi onward and resolved after day 21 pi. From day 15 pi onward, white retinal flecks were observed uniformly throughout the retina, which are presumed to be clinical evidence of small retinal folds (described later). Accompanying resolution of the exudative detachments was clinical resolution of features of retinal vessel involvement (vasculitis) and optic nerve swelling. Over the time period examined, the retina did not regain its normal appearance, as clinical images from day 28 pi onward demonstrated persistence of retinal flecks throughout the resolution phase and up to and including day 63 pi. Examination of the contralateral eye in selected mice verified that there was similar clinical appearance between eyes at all time points (data not shown).


Figure 2
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  		FIGURE 2. Clinical features during the study time course in a B10.RIII mouse immunized for EAU. At each time point from days 10 to 63 pi, the right eyes were dilated and photographed.

 
Comparison of TEFI, Histologic Features, and Composite of Cellular Infiltrate during EAU
Given the relative ease and reproducibility of TEFI when used to monitor disease in immunized mice, we wanted to determine whether clinical features would correlate with histologic changes and the kinetics of cellular infiltrate.

We immunized 40 B10.RIII mice, and on days 12, 13, 14, 15, 18, 19, 21, 28, 35, 42, and 63 pi, TEFI images of the right eye were obtained from four mice at each time point (three mice on days 42 and 63) before death. The right eyes were enucleated and sections prepared for immunohistochemical staining with anti-CD45 antibody. Three sections per retina per time point were scored for inflammatory infiltrate and structural damage, as described previously (Table 1) .

Figure 3 shows our findings as a representative comparison of TEFI and histology images taken from the same eye. Observations from days 0 to 12 pi demonstrated a normal retinal appearance by TEFI, which was confirmed histologically in sections that displayed normal morphology and no inflammatory infiltrate. By days 13 and 14 pi, clinical changes that included a raised appearance of the optic nerve were observed in 75% of the mice, although at this stage there was no clinical or histologic evidence of altered retinal morphology. The increase in histologic disease score was secondary to infiltrate that arose at the ciliary body and scleral–choroidal interface in that area (Fig. 3 , inset).


Figure 3
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  		FIGURE 3. Comparison of clinical and histologic images during the EAU time course. Forty female B10.RIII mice were immunized for EAU using RBP-3161-180 in CFA. At the days after immunization indicated, clinical images were obtained before the mice were killed. The right eyes were enucleated, sectioned, and stained for CD45+ infiltrate. A 12-µm retinal section from the right eye with total disease score is shown next to the corresponding clinical image from the right eye at each day, representative of each group (n = 4). Histology images from days 13 and 14 include insets showing the ciliary body–ciliary marginal zone and surrounding CD45+ perivascular infiltrate.

 
From days 15 to 19 pi, exudative retinal detachments and signs of cellular infiltrate (white lesions) and perivascular sheathing (vasculitis) were evident in all (100%) animals examined at these times. Where severe vitritis in the mice prevented clear visualization of the retina, histologic assessment confirmed the characteristics of clinical disease. This result correlates with extensive retinal disruption and folding, vasculitis, and perivascular infiltrate associated with increased CD45+ infiltrate observed by histology. However, by day 21 pi, retinal detachments were reduced clinically, whereas perivascular infiltrate persisted and by histology, both infiltrate and retinal morphologic disruption remained clearly evident. The overall clinical appearance improved from days 28 to 63 pi in all (100%) animals, with reduced inflammation of the optic nerve and retina (as observed as reduced optic nerve head swelling, reduced perivascular infiltrate and reduced creamy chorioretinal deep presumed infiltrative lesion), although during the resolution phase (postpeak disease), white, worm-like retinal flecks persisted. Histologic assessment of the eyes, corroborated such findings and demonstrated markedly reduced infiltrate, but persistent small retinal folds, likely to represent flecks observed by TEFI, were still apparent. Such folds are similar to those previously documented in other models.18

Analysis of the inflammatory infiltrate and structural scores throughout the time course by histology exhibited the classic monophasic disease course of EAU in B10.RIII mice (Fig. 4A) . From days 12 to 14 pi, increased levels of CD45+ cell infiltrate were detected, while little or no structural damage was observed within the retina. Disease progressed from day 15 pi onward, with a peak of disease at day 19 pi, as reflected by high scores for inflammatory infiltrate and associated structural damage. The period from day 21 pi onward is often termed the resolution phase, and although disease scores are reduced, morphologic changes (structural damage) and CD45+ cellular infiltration persists through to day 63 pi. Although this infers a level of regulation and repair, neither the number of CD45+ cells nor retinal morphology returned to normal predisease levels.


Figure 4
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  		FIGURE 4. Comparison of histologic scores and retinal cellular infiltrate during the EAU time course. (A) Right eyes were enucleated at the post immunization day indicated. Eyes were snap frozen before cryosectioning and staining for immunohistochemical analysis of CD45+ infiltrate. Average disease score ± SD of inflammatory infiltrate and structural disease is shown (n = 4/time point). Histology demonstrated that the EAU was a monophasic disease that peaked at day 19 pi, but did not fully resolve or return to normal levels. (B) Left eyes were simultaneously enucleated at each time point, and the retina and ciliary body excised and digested with collagenase. The number of immune cells per eye was measured by flow cytometry. The total number of immune cells is detailed as follows: CD45+CD11b+ (CD11b), CD45+CD4+ (CD4), and CD45+CD11b–CD4– (CD45) (n = 4/time point). Elevated levels of retinal infiltrate were observed from day 12 pi, with an expansion of CD45+ cells including macrophages and T cells seen from day 15 pi, peaking at day 18 pi. From days 19 to 21 pi, the level of infiltrate was reduced but the cells persisted throughout the resolution phase.

 
Inflammatory Cell Infiltrate
Considering the clinical pictures obtained using TEFI and the close relationship we observed to the underlying histologic changes, we wished to determine whether the clinical features also related to the kinetics and levels of inflammatory cell infiltrate present in the eye during the course of EAU. The isolation and analysis of retinal infiltrate using flow cytometric methods has been used to determine the normal immune status of the eye,19 to quantify and monitor the kinetics of inflammatory infiltrate in the retina, and also evaluate the effects and efficacy of potential new immunomodulatory agents in EAU.17 Therefore, at the same time points described earlier, the left eye was also enucleated (as we noted synchronous bilateralism of clinical features during EAU), dissected, and single cell suspensions were prepared from the retina and the ciliary body. Cells were then stained with fluorochrome-conjugated monoclonal antibodies against CD11b (macrophages), CD4 (T cells), and CD45 (leukocytes) surface markers and analyzed by flow cytometry to enable quantification of total cell number and phenotype (Fig. 4B) .

We observed elevated levels of leukocytes compared with normal retina from day 12 pi forward. The number of CD11b+ and CD4+ cells increased steadily from day 13 pi onward, with the main expansion of both cell types occurring after day 15 pi, and at a maximum on day 18 pi. Furthermore, during this time CD4+ cells were present at lower levels, with a predominance of CD11b+ cells. Of note was the fact that an increased number of cells was detected before any evidence of clinical (TEFI images) or histologic disease. From day 19 to 21 pi, the level of inflammatory cell infiltrate reduced and CD45+ cell numbers remained throughout (to day 63 pi) at levels equivalent to those on day 13 pi. The number of cells never returned to normal predisease levels, indicating that CD45+ infiltrate persists and may contribute to the clinical changes observed during the resolution phase. The ratio of CD11b+ to CD4+ cells during this phase is also reduced with both cell types present in equal amounts at the later time points.

Correlation between CD45 Infiltrate and Histology
Figure 5A shows the change in the number of CD45+ cells compared with the change in histologic score with time after immunization. The data suggest an association between these variables with both staying low at days 12 to 13 pi, increasing between days 15 and 20 pi and then reducing again thereafter. Although the number of cells fell to levels similar to those at days 12 to 13 pi, the histologic score remained somewhat elevated, albeit lower than the peak scores. Partial correlation was used to explore the relationship between histologic score and numbers of CD45+ cells while controlling for time (days) after immunization. This confirmed that there was a strong, positive partial correlation (r = 0.78, df = 32, P < 0.001), with high histologic scores being associated with high cell counts (Fig. 5B) . The zero order correlation (r = 0.73) suggests that time has little influence on the strength of the association between these two variables.


Figure 5
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  		FIGURE 5. Correlation of infiltrate and histologic disease score. (A) Changes in the number of CD45+ cells and histologic score with time after immunization. (B) Scatterplot of total histologic score against the square root of total number of CD45+ cells. Partial correlation (r = 0.78, df = 32, P < 0.001) shows a strong, positive association between these variables while controlling for time after immunization.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
TEFI has been an effective technique that permits high resolution in vivo imaging of the clinical changes that occur during EAU disease progression in mice. Previous techniques were limited, and TEFI now offers an improved, rapid, no-anesthesia approach to generating detailed panfundal images in mice. It also facilitates the reduction, replacement, and refinement goals now favored by ethics committees in animal experimentation. With TEFI, when comparisons and correlation are made with histologic scoring and flow cytometric assessment of retinal infiltrate, several important unrecognized features of this model become apparent. First, significant retinal cell infiltrate is observed whereas clinically the retina appears largely unaffected; second, the clinical resolution of peak disease is much faster than resolution of histologic disease, and finally, in the resolution phase of EAU, neither the clinical appearance or the extent and composition of CD45+ cells within the retina return to predisease levels (up to 63 days pi). Although we noted a correlation between histologic scoring and flow cytometric assessment of infiltrating leukocyte numbers, cell counts resolved faster than severity of histologic changes; and, with the use of TEFI, our results emphasize that significant changes may occur that are not always clinically manifest.

The objectives of this study were to validate a platform that uses the TEFI system for assessment of clinical time course of RBP-3161-180–induced EAU in B10.RIII mice and correlate clinicopathologic features to both histologic severity and the extent of inflammatory cell infiltration of the retina. The clinical images obtained using the TEFI approach overall closely associate to the pathologic features of disease observed by histology. Histologic assessment demonstrated that the disease in this model of EAU followed the classic monophasic profile, with peak disease severity observed at day 19. Analysis of the dynamics and kinetics of retinal infiltrate demonstrated that an expansion of CD45+ cells, including CD11b+ and CD4+ populations, was present before this peak. Although the number of infiltrating cells was reduced during the later resolution phase, histologic disease scores and infiltrate levels never returned to normal; this finding has not been appreciated in this model of autoimmune destruction of the retina. Statistical correlation analyses demonstrated a positive association between the number of infiltrating CD45+ cells and the resulting histologic disease severity.

By using TEFI, it is now possible to record and monitor the dramatic clinical changes that occur in B10.RIII mice during the normal disease course of RBP-3161-180–induced EAU. From the time of immunization until day 12 pi, the retina and vasculature appeared normal and healthy, followed by a series of clinical changes from days 13 to 18, including raised optic nerve, perivascular infiltration developing that manifests the perivascular infiltrate and vitritis normally appreciated as hallmarks of this model. The development of large exudative retinal detachments from day 17, which resolve, along with the other clinical features of perivascular infiltrate and vitritis, can also be observed. The emergence of retinal flecks, uniformly distributed across the retina is demonstrated from day 15 pi. The retinal flecks correspond to the retinal folds we observed histologically. Clinically and histologically, retinal integrity never normalizes to the predisease state during the EAU time course. We also noted with TEFI that clinical features of EAU were constant between contralateral eyes.

EAU serves as a model for the spectrum of human posterior uveitis including sympathetic ophthalmia and Vogt-Koyanagi-Harada syndrome (VKH; particularly in relation to exudative retinal detachments), multifocal choroiditis, ocular sarcoidosis, and other forms of idiopathic disease.1 20 For example, the clinical features seen in this study correlate well with clinical features of VKH, in which resolving exudative retinal detachments are observed. After resolution of acute VKH, the classic clinical features of sunset-glow retina with its appreciated degenerative features are seen, again correlating with our TEFI images from day 28 onward.

Flow cytometric analysis of cells isolated from the retina demonstrated that the elevated levels of inflammatory infiltrate observed from day 12 onward during the time course of EAU, consisted of macrophages, T cells, and other CD45+ leukocytes. Infiltration of cells at this time has been examined by histology, which demonstrates the perivascular accumulation of CD45+ cells in the retina,21 although this static analysis cannot fully assess the dynamics of infiltration. The infiltration kinetics revealed that the main expansion of cells occurred after day 15 pi, culminating in a peak at day 18, and during this time, the proportion of CD4+ cells present was reduced compared to the number of CD11b+ cells. After the infiltrative peak, total CD45+ cells were greatly reduced over the remainder of the time course, but never returned to predisease levels. During this resolution phase, both the main CD11b+ and CD4+ populations were present at equal levels. Persistence of elevated levels of infiltrate in the eye would suggest that resolution and recovery do not equate to normal leukocyte counts, and may further suggest that certain regulatory mechanisms are maintained in the eye after inflammation.22 23 Similarly assessment of immunotherapeutic agents, given our current findings of temporal disparity between clinical appearance and cell infiltrate in the earlier stages of disease, and together with previous observations of maintained cellular infiltrate despite reduced histologic scores,24 shows that it is plausible that changes in constituents and number of infiltrating cells are not appreciated in the face of normal clinical phenotype and may conversely not always indicate preservation of function.

Nevertheless, TEFI is a method that allows confirmation of disease status and severity. It will aid in the design of experimental protocols according to clinical observations. TEFI will also greatly assist with current approaches to preclinical testing of experimental eye models, as it allows direct observation and assessment of therapeutic efficacy of new potential ocular therapy. It will also provide a rapid assessment to determine potential adverse effects incurred due to invasive procedures including intravitreous or subretinal injections.

Although, unlike experimental autoimmune encephalomyelitis (EAE),25 in which we are unable to ascribe directly functional deficit (paralysis) to histologic change or with the more technically demanding imaging of cellular infiltrate in the CNS,26 we are now able in EAU to directly correlate and assess clinical changes with histologic and flow cytometric analysis of cellular infiltrate. In both models, we now understand that significant cellular infiltrate occurs before the onset of clinical signs in the fundus of EAU and clinically in EAE.

Furthermore, the current published clinical grading of disease17 27 28 in both B10.RIII and/or C57BL/6 mouse models have been developed without incorporating evolution of clinical phenotype and comparison of such temporal characteristics with respect to the extent and timing of leukocytic infiltration (e.g., by flow cytometry analysis) and contemporaneous histopathologic appearances throughout the course of EAU. Although these scores may still be used, and indeed clinical features we show can mirror underlying histologic change, ascribing scoring of clinical severity or damage in light of this new data necessitates further investigation of EAU progression with larger groups of mice and in other strains (C57BL/6) to generate and then validate such a proposed grading system. The most recent report29 in C57BL/6 model of TEFI grading of clinical changes in chronic EAU supports our findings in this model of EAU. The advantage of adapting TEFI is therefore highlighted in both models and serves to assess more reproducibly the signs of inflammatory disease and correlate with underlying histologic and flow cytometric data.

Arguably, to fully assess preclinical immunotherapeutic efficacy requires the use of all three parameters: TEFI, histologic assessment, and flow cytometric analysis of retinal infiltrate. Combined TEFI and histologic methods enable the observation of clinical features and severity of disease, but information regarding the dynamics, phenotype, function and quantity of cellular traffic through the eye is only provided through detailed analysis of cell populations present in the eye at various stages of disease progression.


   Footnotes
 
Supported by a National Eye Research Centre grant.

Submitted for publication May 28, 2008; revised July 17 and August 13, 2008; accepted October 24, 2008.

Disclosure: D.A. Copland, None; M.S. Wertheim, None; W.J. Armitage, None; L.B. Nicholson, None; B.J.E. Raveney, None; A.D. Dick, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

* Each of the following is a corresponding author: Andrew D. Dick, Academic Unit of Ophthalmology, Department of Clinical Sciences at South Bristol, University of Bristol, Bristol BS8 1TD, UK; a.dick{at}bristol.ac.uk. Ben J. E. Raveney, Department of Immunology, National Institute of Neuroscience NCNP, Tokyo, Japan; ben{at}raveney.co.uk. Back


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Forrester JV, Liversidge J, Dua HS, Towler H, McMenamin PG. Comparison of clinical and experimental uveitis. Curr Eye Res. 1990;9(suppl)75–84.[CrossRef][Medline][Order article via Infotrieve]
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B. J. E. Raveney, D. A. Copland, L. B. Nicholson, and A. D. Dick
Fingolimod (FTY720) as an Acute Rescue Therapy for Intraocular Inflammatory Disease
Arch Ophthalmol, October 1, 2008; 126(10): 1390 - 1395.
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