Assessment and In Vivo Scoring of Murine Experimental Autoimmune Uveoretinitis Using Optical Coherence Tomography

et al. (2013) Assessment and In Vivo Scoring of Murine Experimental Autoimmune Uveoretinitis Using Optical Coherence Tomography. PLoS ONE 8(5): e63002. doi:10.1371/journal.pone.0063002 Assessment and In Vivo Scoring of Murine Experimental Autoimmune Uveoretinitis Using Optical Coherence Tomography Colin J. Chu 0 Philipp Herrmann 0 Livia S. Carvalho 0 Sidath E. Liyanage 0 James W. B. Bainbridge 0 Robin R. Ali 0 Andrew D. Dick 0 Ulrich F. O. Luhmann 0 Celia Oreja-Guevara, University Hospital La Paz, Spain 0 1 Department of Genetics, UCL Institute of Ophthalmology , London , United Kingdom , 2 NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital , London , United Kingdom , 3 Unit of Ophthalmology, School of Clinical Sciences, University of Bristol , Bristol , United Kingdom Despite advances in clinical imaging and grading our understanding of retinal immune responses and their morphological correlates in experimental autoimmune uveoretinitis (EAU), has been hindered by the requirement for post-mortem histology. To date, monitoring changes occurring during EAU disease progression and evaluating the effect of therapeutic intervention in real time has not been possible. We wanted to establish whether optical coherence tomography (OCT) could detect intraretinal changes during inflammation and to determine its utility as a tool for accurate scoring of EAU. EAU was induced in C57BL/6J mice and animals evaluated after 15, 26, 36 and 60 days. At each time-point, contemporaneous Spectralis-OCT scanning, topical endoscopic fundal imaging (TEFI), fundus fluorescein angiography (FFA) and CD45immunolabelled histology were performed. OCT features were further characterised on retinal flat-mounts using immunohistochemistry and 3D reconstruction. Optic disc swelling and vitreous opacities detected by OCT corresponded to CD45+ cell infiltration on histology. Vasculitis identified by FFA and OCT matched perivascular myeloid and T-cell infiltrates and could be differentiated from unaffected vessels. Evolution of these changes could be followed over time in the same eye. Retinal folds were visible and found to encapsulate mixed populations of activated myeloid cells, T-cells and microglia. Using these features, an OCT-based EAU scoring system was developed, with significant correlation to validated histological (Pearson r2 = 0.6392, P,0.0001, n = 31 eyes) and TEFI based scoring systems (r2 = 0.6784, P,0.0001). OCT distinguishes the fundamental features of murine EAU in vivo, permits dynamic assessment of intraretinal changes and can be used to score disease severity. As a result, it allows tissue synchronisation with subsequent cellular and functional assessment and greater efficiency of animal usage. By relating OCT signals with immunohistochemistry in EAU, our findings offer the opportunity to inform the interpretation of OCT changes in human uveitis. - Funding: CJC and SEL are joint MRC and Fight for Sight clinical research training fellows (G1100383 and MR/K003003/1 respectively). PH is a fellow of the German Research Foundation (DFG He 6175/1- 1). RRA is partly funded by the Department of Healths National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital. ADD is partly funded as the theme lead for inflammation and immunotherapy at the NIHR Moorfields Biomedical Research Centre. JB is supported by a NIHR Research Professorship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. As an animal model with features resembling those of human intraocular autoimmune inflammatory disease, murine experimental autoimmune uveoretinitis (EAU) has been central to many of the advances and translational studies in ocular immunology over the last decade [1,2]. Spontaneous and inducible EAU can be obtained using a variety of wildtype and transgenic mouse strains to achieve different disease kinetics, severity and polarity of immune responses [3]. More recently, laboratories have advanced our knowledge of EAU and thus human disease through assays such as retinal multiparameter flow cytometric analysis. This has facilitated the dissection of molecular mechanisms that determine disease phenotype and the function, for example, of recruited macrophages [4,5]. To date however, the inability to identify histological changes in real time has limited our ability to correlate cellular infiltrate and function with morphological changes. Despite increasing sensitivity of flow cytometric analysis within the retina, a robust readout of disease status would improve the utility of EAU for preclinical translational studies. Currently, morphological assessments are only possible with histology and semi-quantitative scoring systems using either haematoxylin/eosin [6] or CD45 (pan-leukocyte) immunolabelling [7]. Whilst histology and flow cytometric assessment of cell infiltrate provide detailed information at a single time-point, the terminal nature of these analyses intrinsically precludes repeated scoring of the same animal. This limitation is compounded by variation in EAU development, amplitude and dynamics, with asymmetry even between the eyes of the same animal [8]. To circumvent this, data from large cohorts of mice are normally pooled, sacrificing information on individual variation and amalgamating results into potentially misleading averages. Progress towards overcoming these limitations culminated in the introduction of topical endoscopic fundal imaging (TEFI) as a scoring platform for murine EAU [9]. Using this technique, repeated detailed images of the retina from the same eye could be obtained over the entire timecourse of disease. By scoring these fundus photographs for disc, vessel and structural changes, a surrogate measure can be obtained, which has been shown empirically to approximate to infiltration and histological scores [10]. Thereby it has been possible to correlate disease changes with cellular infiltration and confirm the effects of novel treatments e.g. of drugs that affect cell trafficking [11,12]. Whilst a substantial advance, TEFI requires high levels of light exposure and corneal instrumentation, that with cumulative use may have the potential to cause damage to the retina and ocular surface [13]. Disease can also be overestimated, predominantly by failing to define normal optic disc appearance or in later stages confusing perivascular scarring as active vasculitis. Furthermore TEFI is unable to quantify vitreous infiltration or resolve intraretinal changes and thus remains an inadequate surrogate score for the gold standard of histology (using 2D colour images to approximate 3D intraretinal tissue changes). For these reasons the pursuit of improved systems has continued. Optical coherence tomography (OCT) has revolutionised the diagnosis, monitoring and treatment of huma (...truncated)