January 2017
Volume 58, Issue 1
Open Access
Anatomy and Pathology/Oncology  |   January 2017
Murine Autoimmune Optic Neuritis Is Not Phenotypically Altered by the Retinal Degeneration 8 Mutation
Author Affiliations & Notes
  • Aleksandar Stojic
    Department of Neurology, University Clinic Heidelberg, Heidelberg, Germany
  • Richard Fairless
    Department of Neurology, University Clinic Heidelberg, Heidelberg, Germany
  • Susanne C. Beck
    Division of Ocular Neurodegeneration, Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany
  • Vithiyanjali Sothilingam
    Division of Ocular Neurodegeneration, Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany
  • Petra Weissgerber
    Department of Experimental and Clinical Pharmacology & Toxicology, Saarland University, Homburg, Germany
  • Ulrich Wissenbach
    Department of Experimental and Clinical Pharmacology & Toxicology, Saarland University, Homburg, Germany
  • Valerie Gimmy
    Department of Neurology, University Clinic Heidelberg, Heidelberg, Germany
  • Mathias W. Seeliger
    Division of Ocular Neurodegeneration, Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany
  • Veit Flockerzi
    Department of Experimental and Clinical Pharmacology & Toxicology, Saarland University, Homburg, Germany
  • Ricarda Diem
    Department of Neurology, University Clinic Heidelberg, Heidelberg, Germany
  • Sarah K. Williams
    Department of Neurology, University Clinic Heidelberg, Heidelberg, Germany
  • Correspondence: Sarah K. Williams, Department of Neurology, University Clinic Heidelberg, Otto-Meyerhof-Zentrum (OMZ), Im Neuenheimer Feld 350, 69120 Heidelberg, Germany; [email protected]
Investigative Ophthalmology & Visual Science January 2017, Vol.58, 318-328. doi:https://doi.org/10.1167/iovs.16-20419
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      Aleksandar Stojic, Richard Fairless, Susanne C. Beck, Vithiyanjali Sothilingam, Petra Weissgerber, Ulrich Wissenbach, Valerie Gimmy, Mathias W. Seeliger, Veit Flockerzi, Ricarda Diem, Sarah K. Williams; Murine Autoimmune Optic Neuritis Is Not Phenotypically Altered by the Retinal Degeneration 8 Mutation. Invest. Ophthalmol. Vis. Sci. 2017;58(1):318-328. https://doi.org/10.1167/iovs.16-20419.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To investigate whether the presence of the retinal degeneration 8 (rd8) mutation in C57BL/6 mice alters the phenotype of autoimmune optic neuritis (AON).

Methods: C57BL/6J and C57BL/6N mice were genotyped for the rd8 mutation and fundus analyses and examination of retinal layer morphology were performed in vivo by scanning laser ophthalmoscopy and optical coherence tomography. Visual function was assessed by recording electroretinographs, and visual evoked potentials and retinae and optic nerves were assessed histopathologically. Retinal ganglion cell numbers were determined by retrograde labeling with fluorogold. Mice were then immunized with myelin oligodendrocyte glycoprotein 35-55 to induce AON before assessment of retinal ganglion cell degeneration, inflammatory infiltration of retinae and optic nerves, and demyelination. Furthermore, visual function was assessed by visual evoked potentials.

Results: All C57BL/6N mice were homozygous for the mutation (Crb1rd8/rd8) and had pathology typical of the rd8 mutation; however, this was not seen in the C57BL/6J (Crb1wt/wt) mice. Following induction of AON, no differences were seen between the Crb1rd8/rd8 and Crb1wt/wt mice regarding disease parameters nor regarding inner retinal degeneration either in the retina as a whole or in the inferior nasal quadrant.

Conclusions: The presence of the rd8 mutation in C57BL/6 mice does not affect the course of AON and should not provide a confounding factor in the interpretation of experimental results obtained in this model. However, it could be dangerous in other models of ocular pathology.

The retinal degeneration 8 (rd8) mutation is a spontaneous single-base deletion found to occur in several mouse strains resulting in the truncation of the transmembrane crumbs family member (CRB) 1 protein1,2 CRB1 is an important part of the outer limiting membrane of the retina where it is involved in maintaining adherens junctions between the Müller glia and photoreceptors.2,3 Mice homozygous for this mutation have retinal folds and pseudorosettes involving the photoreceptors, leading to distorted inner and outer nuclear layers as well as retinal thinning.1,2 Pathologic alterations are focal in nature and occur predominantly within the inferior nasal quadrant (INQ) of the retina.2 Degeneration can be detected as early as 2 weeks after birth2 and progresses with age.4 Interestingly, mutations in CRB1 also lead to a number of heritable human retinal disorders, including retinitis pigmentosa5 and Leber's congenital amaurosis.6 
It was previously reported that the rd8 mutation is present in all C57BL/6N mice from major vendors in the United States, but not in C57BL/6J mice,7 and that the presence of the rd8 mutation may account for some of the phenotypes observed in models of ocular disease. For example, in a model of macular degeneration,8 and also birdshot chorioretinopathy, a form of uveitis,9 the phenotype was subsequently demonstrated to be a result of the rd8 mutation.7,10 Therefore, to correctly interpret results in other models of retinal disease, it is imperative to discover the impact of this mutation in the model of interest. 
One such model that should be assessed is autoimmune optic neuritis (AON). AON commonly occurs in patients suffering from multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system, where it is often one of the first presenting symptoms. It is defined as inflammation of the optic nerve that results in a decrease in visual acuity through both demyelination and axonal loss within the optic nerves as well as a loss of retinal ganglion cell (RGC) bodies in the retina. AON can be modeled in both mice1113 and rats14,15 through immunization with immunogens, such as myelin oligodendrocyte glycoprotein. This results in a reproducible disease that recapitulates many of the features of the human disease, helping to overcome the problem of limited tissue availability from the acute phase of MS. As an experimental model, it has also proven itself to be a valuable tool for assessing the efficacy of therapeutic strategies prior to translation into clinical trials.1619 Furthermore, the ability to model AON in rodents means that genetically engineered mouse strains can be used to provide further insights into neurodegeneration in MS.11,19,20 However, because of the potential complication of the rd8 mutation arising in such strains, an assessment is necessary. In particular, because many genetically modified mouse lines have been derived using a C57BL/6N background21 and studies of AON have already been performed on some of these lines,11 or even on C57BL6 mice of an undefined substrain identity,20,22,23,24 such an assessment would allow readers to know whether one can have confidence in data obtained from such studies. 
Here we show that the presence of the rd8 mutation does not affect any of the parameters that have been previously described as key features of AON, including RGC degeneration, axonal loss, inflammatory infiltration, and demyelination. However, caution should be exercised when choosing mice strains for future studies. 
Materials and Methods
Animals
Female C57BL/6N (strain code 027) and C57BL/6J mice (stock number 000664) of 6 to 8 weeks of age were obtained from specific pathogen free rooms of Charles River (Sulzfeld, Germany) and kept under environmentally controlled conditions in the absence of pathogens. Animals were taken into experiments without further breeding or intercrossing. All experiments involving animal use were approved by the authorities of Baden-Württemberg, Germany, and performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Genotyping
In this study, 1 μl of DNA, isolated by standard procedures from mouse tail biopsies, was used as a template to amplify the 203 bp fragment of the mouse crumbs family member 1 (mCrb1) gene carrying the rd8 mutation7 by PCR using the primers Crb1_forward 5′-GCA TGG AGG AAA CTG TGA AGA CAG CTA CAG TTG ATA T-3′ and mCrb1_reverse 5′-TGT CTA CAT CCA CCT CAC AG-3′. In the Crb1_forward primer, the nucleotides (nt) CT of the mouse crb1 gene were replaced by nt GA (indicated in italic in the sequence above) to generate a new Escherichia coli strain RY13 enzyme 5 (EcoRV) restriction site in the wild-type allele. PCR reactions were performed in parallel: 94°C for 5 minutes, followed by 38 cycles (95°C [20 seconds], 60°C [30 seconds], and 72°C [30 seconds]) and an extension step (72°C for 5 minutes). Two aliquots of each PCR reaction were incubated in the absence (not cut or “NC” in Fig. 1) or presence of EcoRV (“cut” in Fig. 1), run on a 3% agarose gel and stained with Ethidium bromide. The amplified wild-type allele is cut by EcoRV and corresponds to the 168 bp fragment (Fig. 1) and a smaller fragment (35 bp) not visible on the gel system; the amplified 202 bp mutated (rd8) allele is not cut (Fig. 1). In parallel DNA samples were sequenced on both strands. 
Figure 1
 
Genotyping. Sample gel showing separation of wild-type (wt/wt) mice (n = 30) and homozygous rd8-mutant (rd8/rd8) mice (n = 31) as described in the Materials and Methods section. Each PCR reaction was incubated in the absence (not cut or “NC”) or presence of EcoRV (“cut”).
Figure 1
 
Genotyping. Sample gel showing separation of wild-type (wt/wt) mice (n = 30) and homozygous rd8-mutant (rd8/rd8) mice (n = 31) as described in the Materials and Methods section. Each PCR reaction was incubated in the absence (not cut or “NC”) or presence of EcoRV (“cut”).
In Vivo Morphologic and Functional Characterization of the rd8 Mutation
Electroretinography (ERG), optical coherence tomography (OCT) and confocal scanning-laser ophthalmoscopy (SLO) were performed consecutively in the same session. Full-field ERG was performed with C57BL/6N and C57BL/6J mice (n = 4) aged 14 weeks according to previously published procedures.25 After an overnight dark adaptation, the mice were anesthetized with ketamine (66.7 mg/kg) and xylazine (11.7 mg/kg), and the pupils were dilated with tropicamide (Mydriaticum Stulln, Pharma Stulln, Stulln, Germany). Functional analyses included single-flash ERGs under dark-adapted (no background illumination, 0 cd/m2) and light-adapted (with a background illumination of 30 cd/m2, starting 10 minutes before recording) conditions. Single white flash intensity series ranged from −4.0 to 1.5 log cd*s/m2. A total of 10 responses were averaged with interstimulus intervals of 5 or 17 seconds (for 0–1.5 log cd*s/m2). For all ERG recordings, band-pass cut-off frequencies were 0.3 and 300 Hz. 
OCT analyses were performed with a commercially available Spectralis Heidelberg Retinal Angiography (HRA)+OCT device (Heidelberg Engineering, Heidelberg, Germany) featuring a broadband superluminescent diode low coherent light source.26,27 Each 2-dimensional B-scan (set to 30° field of view) consisted of 1536 A-scans acquired at 40,000 scans/second. Imaging was performed using the proprietary software package Eye Explorer (version 5.3.3.0.; Heidelberg Engineering). 
The ocular fundus and vasculature were visualized via SLO imaging with an HRA 2 (Heidelberg Engineering) according to previously described procedures.28 Briefly, the HRA 2 system features lasers in the short (visible) wavelength range (488 nm and 514 nm) and also in the long (infrared) wavelength range (785 nm and 815 nm). Native fundus imaging was performed at 514 nm; autofluorescence was detected at 488 nm, and 488 and 785 nm lasers were used for fluorescein and indocyanine green angiography, respectively. 
Visual Evoked Potential (VEP) Measurements
At 2 weeks prior to immunization, the animals were anesthetized with ketamine (150 mg/kg) and xylazine (10 mg/kg). VEPs were measured using the UTAS Visual Diagnostic System (LKC Technologies, Gaithersburg, MD, USA) on mice maintained at 37°C. Pupils were dilated with 0.5% atropine (Ursapharm, Saarbrücken, Germany), and the animals were dark adapted for 5 minutes. Needle-type electrodes were placed in the primary visual cortex, 3 mm lateral to lambda. Reference and ground electrodes were placed subcutaneously in the neck and tail, respectively. The animals were placed in the dome equipped with a light-emitting diode (LED) wholefield stimulator. Flash stimuli were presented at 0 dB intensity (2.5 cd-s/m2 luminance) and at 2 Hz. A total of 100 sweeps were averaged per recording, during which eye desiccation was prevented by application of Liquifilm O.K. (Allergan, Westport, Ireland). Each eye was recorded separately and repeated at the indicated disease time points. The signal amplitude (μV) and latency (ms) were calculated from the first negative to the second positive peak of the response using software provided by the UTAS Visual Diagnostic System. 
Retrograde Labeling of RGCs, Induction, and Evaluation of Experimental Autoimmune Encephalomyelitis (EAE)
In healthy controls and prior to immunization, RGCs were retrogradely labeled with fluorogold (FG; Fluorochrome LLC, Denver, CO, USA), and disease was induced as previously described.11,19 Briefly, the mice were immunized with 200 μg myelin oligodendrocyte glycoprotein 35-55 and weighed and scored on a daily basis, with disease severity assessed using a scale ranging from 0 to 5. EAE experiments were performed on 2 separate occasions. 
Optic Nerve Histopathology
Both healthy mice and those at day 12 of EAE received an overdose of ketamine/xylazine and were transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Optic nerves were dissected and processed for paraffin-embedding, and 0.5 μm transverse sections were cut. Luxol fast blue staining and Bielschowsky's silver impregnation were performed to assess demyelination and axonal pathology, respectively, as previously described.29 Antibodies against Mac-3 (1:200, BD Biosciences, San Jose, CA, USA) and CD3 (1:150, Dako, Glostrup, Denmark) were used to detect activated microglia/macrophages and T cells, respectively, using previously described protocols.11,19 For all histopathologic and immunohistochemical investigations, a minimum of 10 sections taken throughout the length of each optic nerve were quantified. 
Quantification of Retinal Ganglion Cell Density
Retinae were dissected, fixed, and flat mounted onto glass slides. RGCs were visualized by fluorescence microscopy (Nikon Eclipse 80i; Nikon GmbH, Düsseldorf, Germany) using a UV filter (365/397 nm), and the densities were determined by counting labeled cells in 3 areas (62,500 μm2) per retinal quadrant at 3 different eccentricities of 1/6, 3/6, and 5/6 of the retinal radius. 
Retinal Immunohistochemistry and TUNEL Labeling
Following perfusion, the eyes were enucleated, postfixed in 4% paraformaldehyde for 2 hours, and cryoprotected in 30% sucrose overnight. The eyes were then embedded in a mounting medium (Tissue-Tek O.C.T. Compound, Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands) and frozen and 12 μm coronal sections were cut. For the TUNEL assay, a previously described protocol was followed.15 For immunohistochemistry, sections were incubated with primary antibodies against CD3 (1:300, Dako) and ionized calcium-binding adaptor molecule 1 (Iba-1) (1:500, Abcam, Cambridge, UK) again using previously described protocols.15 
Statistical Analyses
All data are presented as mean ± SEM. Statistical comparisons were made using SigmaPlot 12 (Systat Software, San Jose, CA, USA). Data were assessed for normality using the Shapiro-Wilk test and subsequently analyzed using either a Mann-Whitney or 2-tailed t-test. A P value of < 0.05 was considered statistically significant. 
Results
Female C57BL/6 mice, of both N and J substrains, were obtained commercially and genotyped for the rd8 mutation (Fig. 1), confirming previous findings that all C57BL/6N mice examined carry the mutation (Crb1rd8/rd8).7 The mice were between 7 and 9 weeks of age at the start of the experiment and approximately 14 weeks at the end. As a result of the progressive nature of the pathology, healthy mice of the same age (14 weeks) were used as controls. 
Identification of the Retinal rd8 Phenotype
Retinal morphology was assessed in C57BL/6J (wt/wt) and C57BL/6N (rd8/rd8) mice at 14 weeks of age by in vivo confocal SLO and spectral-domain OCT (Fig. 2). C57BL/6J (wt/wt) animals showed no abnormalities in fundus appearance (Fig. 2A), fundus autofluorescence (Fig. 2B), retinal vasculature (Fig. 2C), or retinal layer morphology (Figs. 2D, 2E). However, in C57BL/6N mice, the typical Crb1rd8/rd8 phenotype30,31 could be observed. Fundus autofluorescence imaging revealed bright spots (Fig. 2G, arrow) in the inferior part of the retina corresponding to sites of retinal disorganization as shown by OCT (Figs. 2I, 2K). In these half rosettes, the integrity of the outer limiting membrane was lost (Figs. 2I, 2K) through cellular mislocalization into the outer plexiform layer.30,31 In between these local disturbances, the retinal layering appeared normal. Furthermore, at this time point, the overall retinal thickness was unaffected by the presence of these half rosette structures (Figs. 2I, 2K). As evident from in vivo angiography, the appearance and integrity of the retinal vasculature was also unaffected by the rd8 mutation (Fig. 2H). 
Figure 2
 
Visualization of the pathology typical of the rd8 mutation. Healthy mice of 14 weeks of age were examined with confocal scanning laser ophthalmoscopy (cSLO) imaging (A, F), fundus autofluorescence (B, G), fluorescein angiography (C, H), and spectral domain optical coherence tomography (OCT) (E, D, I, K). The wt/wt (C7BL6/J) mice showed a normal fundus appearance and retinal layering. In native SLO imaging, no differences were detected between both C57BL/6 substrains (A, F). However, in the autofluorescence mode, bright spots could be detected within the inferior nasal quadrant (INQ) in rd8/rd8 (C57BL/6N) mice (G, arrow), which correlated to the typical half-rosette structures of the rd8 mutation detected by OCT imaging (I, magnification K). wt/wt and rd8/rd8, n = 4. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane, I/OS inner/outer segment border; RPE/CC, retinal pigmented epithelium/choriocapillaris complex; t, temporal; n, nasal.
Figure 2
 
Visualization of the pathology typical of the rd8 mutation. Healthy mice of 14 weeks of age were examined with confocal scanning laser ophthalmoscopy (cSLO) imaging (A, F), fundus autofluorescence (B, G), fluorescein angiography (C, H), and spectral domain optical coherence tomography (OCT) (E, D, I, K). The wt/wt (C7BL6/J) mice showed a normal fundus appearance and retinal layering. In native SLO imaging, no differences were detected between both C57BL/6 substrains (A, F). However, in the autofluorescence mode, bright spots could be detected within the inferior nasal quadrant (INQ) in rd8/rd8 (C57BL/6N) mice (G, arrow), which correlated to the typical half-rosette structures of the rd8 mutation detected by OCT imaging (I, magnification K). wt/wt and rd8/rd8, n = 4. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane, I/OS inner/outer segment border; RPE/CC, retinal pigmented epithelium/choriocapillaris complex; t, temporal; n, nasal.
The Inner Retina Shows No Global Pathologic Changes in Mice Carrying the rd8 Mutation
AON is characterized by a degeneration of RGCs, accompanied by inflammation and demyelination of the optic nerve. Initially, we sought to determine the effect of the rd8 mutation on the retinae of healthy, age-matched control mice. To determine whether the rd8 mutation has any effect on the inflammatory environment of the retina, immunohistochemistry against CD3 and Iba-1 to detect the presence of T cells and activated microglia/macrophages, respectively, was performed. Although a small number of CD3-positive cells were observed in both wt/wt and rd8/rd8 retinae, there was no significant difference between the groups (Figs. 3A, 3B, 3Q). Similarly, small numbers of Iba-1-positive cells were seen in both groups, but again with no significant difference (Figs. 3E, 3F, 3R). 
Figure 3
 
Global retinal pathology in healthy mice and during AON. Immunohistochemistry was performed on frozen retinal sections from both healthy, age-matched mice and 12 days after the onset of spinal cord symptoms. No difference was seen between the number of CD3-positive cells in wt/wt (A) and rd8/rd8 healthy mice (B, Q) or in wt/wt (C) and rd8/rd8 (D, Q) mice during AON. No difference was also seen between the number of Iba-1-positive cells in healthy wt/wt (E) and rd8/rd8 mice (F, R) or in wt/wt (G) and rd8/rd8 (H, R) mice during AON. TUNEL staining was performed on retinal sections to investigate apoptosis; however, no TUNEL-positive cells were seen in the GCL of either wt/wt (I) or rd8/rd8 (J) healthy mice. TUNEL positive cells were seen within the GCL layer during AON; however, there was no difference in the number quantified between wt/wt (K) and rd8/rd8 (L) mice (S). Fluorogold (FG)-positive RGC densities were quantified in flat-mounted retinae from healthy wt/wt (M) and rd8/rd8 mice (N); however, no difference in the number of labeled cells was observed (T). RGC densities were also quantified during AON, but no differences were seen in the number of surviving FG-labeled RGCs in wt/wt (O) and rd8/rd8 mice (P, T). Q, R, S: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt and rd8/rd8, n = 9 from 5 animals. T: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt, n = 8 from 5 animals; rd8/rd8, n = 12 from 7 animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; n.s., not significant; OPL, outer plexiform layer; ONL, outer nuclear layer. *P < 0.05, **P < 0.01. Scale bars = 50 μm.
Figure 3
 
Global retinal pathology in healthy mice and during AON. Immunohistochemistry was performed on frozen retinal sections from both healthy, age-matched mice and 12 days after the onset of spinal cord symptoms. No difference was seen between the number of CD3-positive cells in wt/wt (A) and rd8/rd8 healthy mice (B, Q) or in wt/wt (C) and rd8/rd8 (D, Q) mice during AON. No difference was also seen between the number of Iba-1-positive cells in healthy wt/wt (E) and rd8/rd8 mice (F, R) or in wt/wt (G) and rd8/rd8 (H, R) mice during AON. TUNEL staining was performed on retinal sections to investigate apoptosis; however, no TUNEL-positive cells were seen in the GCL of either wt/wt (I) or rd8/rd8 (J) healthy mice. TUNEL positive cells were seen within the GCL layer during AON; however, there was no difference in the number quantified between wt/wt (K) and rd8/rd8 (L) mice (S). Fluorogold (FG)-positive RGC densities were quantified in flat-mounted retinae from healthy wt/wt (M) and rd8/rd8 mice (N); however, no difference in the number of labeled cells was observed (T). RGC densities were also quantified during AON, but no differences were seen in the number of surviving FG-labeled RGCs in wt/wt (O) and rd8/rd8 mice (P, T). Q, R, S: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt and rd8/rd8, n = 9 from 5 animals. T: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt, n = 8 from 5 animals; rd8/rd8, n = 12 from 7 animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; n.s., not significant; OPL, outer plexiform layer; ONL, outer nuclear layer. *P < 0.05, **P < 0.01. Scale bars = 50 μm.
To determine if the rd8 mutation caused any apoptosis within the ganglion cell layer (GCL) of the retina, TUNEL staining was performed. No TUNEL-positive cells were observed in the GCLs of either wt/wt or rd8/rd8 retinae (Figs. 3I, 3J, 3S), nor differences in the number of FG-labeled RGCs (Figs. 3M, 3N, 3T). 
Retinal Histopathology Is Unaltered During Optic Neuritis With rd8 Mutation
Following the characterization of healthy mice, AON was induced. AON is a common manifestation of the animal model EAE, which is characterized by ascending spinal cord paralysis. Following induction, no differences were seen in either the day of disease onset or the severity of spinal cord symptoms between the 2 genotypes (data not shown) as followed until 12 days after disease manifestation. 
Immunohistochemistry was performed to determine the effect of the rd8 mutation on inflammatory infiltration within the retina during AON. Significant infiltration of both CD3-positive T cells (Figs. 3C, 3D, 3Q) and Iba-1 positive activated microglia/macrophages (Figs. 3G, 3H, 3R) was seen at day 12 of EAE in comparison to healthy controls; however, again no difference was seen between wt/wt and rd8/rd8 retinae. 
To determine whether the degeneration of RGCs during AON was affected by the rd8 mutation, TUNEL staining was performed at day 12 of EAE. TUNEL-positive cells were seen within the GCL of both EAE groups, but no significant difference was seen between the groups (Figs. 3K, 3L, 3S). Furthermore, although both wt/wt and rd8/rd8 retinae had highly significantly reduced numbers of surviving FG-labeled RGCs at day 12 of EAE when compared with healthy controls, the numbers between the 2 groups were not significantly different (Figs. 3O, 3P, 3T). 
The rd8 Mutation Does Not Affect Inner Retinal Pathology in the Inferior Retina
As has been previously reported, mice homozygous for the rd8 mutation show degeneration predominantly in the INQ of the retina.1,2 We therefore prepared retinae from both wt/wt and rd8/rd8 mice to study retinal pathology within this specific region. Initially, we compared inflammatory infiltration in the INQ between wt/wt and rd8/rd8 healthy control mice. We observed the presence of a small number of T cells within both genotypes in this region; however, no significant differences were found (Figs. 4A, 4B, 4M). We then looked at the number of Iba-1-positive microglia/macrophages and, as expected, a slight elevation in the number of positive cells within the INQ of rd8/rd8 mice was found (Figs. 4C, 4D, 4N). We then examined if there was increased RGC apoptosis in the INQ of healthy rd8/rd8 mice. However, no TUNEL-positive cells were seen within the GCL of either wt/wt or rd8/rd8 mice (Figs. 4I, 4J, 4O). 
Figure 4
 
Retinal pathology within the inferior nasal quadrant (INQ) in healthy mice and during AON. Frozen retinal sections from both healthy, age-matched mice and 12 days after the onset of spinal cord symptoms were generated in which the orientation of the retina was maintained to investigate region of the inferior nasal quadrant (INQ) specifically. No difference was seen between the number of CD3-positive T cells in wt/wt (A) and rd8/rd8 healthy mice (B, M) or in wt/wt (C) and rd8/rd8 (D, M) mice during AON in the INQ. No difference was also seen between the number of Iba-1-positive activated microglia/macrophages cells in healthy wt/wt (E) and rd8/rd8 mice (F, N) or in wt/wt (G) and rd8/rd8 (H, N) mice during AON in the INQ. TUNEL staining was performed to investigate the presence of apoptotic cells; however, no TUNEL-positive cells were seen in the GCL of either wt/wt (I) or rd8/rd8 (J) healthy mice in the INQ. TUNEL positive cells were seen within the GCL layer during AON; however, there was no difference in the number quantified between wt/wt (K) and rd8/rd8 (L) mice (O) in the INQ. M, N, O: healthy wt/wt and rd8/rd8, n = 5 from 3 animals; AON wt/wt and rd8/rd8, n = 6 from 3 animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; n.s., not significant; OPL, outer plexiform layer; ONL, outer nuclear layer. *P < 0.05, **P < 0.01. Scale bars = 50 μm.
Figure 4
 
Retinal pathology within the inferior nasal quadrant (INQ) in healthy mice and during AON. Frozen retinal sections from both healthy, age-matched mice and 12 days after the onset of spinal cord symptoms were generated in which the orientation of the retina was maintained to investigate region of the inferior nasal quadrant (INQ) specifically. No difference was seen between the number of CD3-positive T cells in wt/wt (A) and rd8/rd8 healthy mice (B, M) or in wt/wt (C) and rd8/rd8 (D, M) mice during AON in the INQ. No difference was also seen between the number of Iba-1-positive activated microglia/macrophages cells in healthy wt/wt (E) and rd8/rd8 mice (F, N) or in wt/wt (G) and rd8/rd8 (H, N) mice during AON in the INQ. TUNEL staining was performed to investigate the presence of apoptotic cells; however, no TUNEL-positive cells were seen in the GCL of either wt/wt (I) or rd8/rd8 (J) healthy mice in the INQ. TUNEL positive cells were seen within the GCL layer during AON; however, there was no difference in the number quantified between wt/wt (K) and rd8/rd8 (L) mice (O) in the INQ. M, N, O: healthy wt/wt and rd8/rd8, n = 5 from 3 animals; AON wt/wt and rd8/rd8, n = 6 from 3 animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; n.s., not significant; OPL, outer plexiform layer; ONL, outer nuclear layer. *P < 0.05, **P < 0.01. Scale bars = 50 μm.
Following this characterization in healthy mice, we proceeded to examine the INQ during AON. During AON, no significant difference in either CD3-positive T cells (Figs. 4C, 4D, 4M) or Iba-1-positive microglia/macrophages (Figs. 4G, 4H, 4N) was seen between the 2 groups in the INQ. We also investigated whether rd8/rd8 mice had increased apoptosis within the GCL of the INQ during AON when compared with wt/wt mice, however, this was not observed (Figs. 4K, 4L, 4O). 
Healthy Optic Nerves Are Unaltered by the rd8 Mutation
Because AON is characterized by the inflammatory demyelination of the optic nerves, we sought to determine whether the rd8 mutation led to demyelination in healthy mice. In both wt/wt and rd8/rd8 mice, all optic nerves were found to be fully myelinated (Figs. 5A, 5B, 5Q). AON is also accompanied by degeneration of RGC axons, which comprise the optic nerves. Axonal densities in the presence and absence of the rd8 mutation were determined using Bielschowsky's silver impregnation; however, no difference was seen between the 2 groups (Figs. 5E, 5F, 5R). As for retinae, inflammatory cells within the optic nerves were assessed by immunohistochemistry. No differences were seen in either the number of CD3-positive T cells (Figs. 5I, 5J, 5S) or the number of Mac-3-positive activated microglia/macrophages (Figs. 5M, 5N, 5T) between wt/wt and rd8/rd8 mice. 
Figure 5
 
Optic nerve pathology in healthy mice and during AON. Optic nerves from healthy, age-matched mice and at day 12 of EAE were assessed histopathologically. Neither healthy wt/wt (A) nor rd8/rd8 (B) mice showed any optic nerve demyelination (Q). During AON, EAE optic nerves from both wt/wt (C) and rd8/rd8 mice (D) showed a similar level of demyelination (Q). As assessed by Bielschowsky's silver impregnation, wt/wt (E) and rd8/rd8 (F) optic nerves had a similar axonal density (R) in healthy optic nerves as well as at day 12 of EAE (G, H, R). A small number of CD3-positive cells were observed in both wt/wt (I) and rd8/rd8 (J) optic nerves, although there was no statistical difference (S). During AON, no difference was seen in the presence of CD3-positive cells between wt/wt (K) and rd8/rd8 mice (L, S) Furthermore, there was no difference in the numbers of Mac-3-positive activated microglia/macrophages in healthy optic nerves (M, N, T) or during AON (O, P, T). Q, R, S, T: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt, n = 10 from 5 animals; rd8/rd8, n = 12 from 6 animals. n.s., not significant. **P < 0.01. Scale bars in AD, IP = 50 μm; EH = 10 μm.
Figure 5
 
Optic nerve pathology in healthy mice and during AON. Optic nerves from healthy, age-matched mice and at day 12 of EAE were assessed histopathologically. Neither healthy wt/wt (A) nor rd8/rd8 (B) mice showed any optic nerve demyelination (Q). During AON, EAE optic nerves from both wt/wt (C) and rd8/rd8 mice (D) showed a similar level of demyelination (Q). As assessed by Bielschowsky's silver impregnation, wt/wt (E) and rd8/rd8 (F) optic nerves had a similar axonal density (R) in healthy optic nerves as well as at day 12 of EAE (G, H, R). A small number of CD3-positive cells were observed in both wt/wt (I) and rd8/rd8 (J) optic nerves, although there was no statistical difference (S). During AON, no difference was seen in the presence of CD3-positive cells between wt/wt (K) and rd8/rd8 mice (L, S) Furthermore, there was no difference in the numbers of Mac-3-positive activated microglia/macrophages in healthy optic nerves (M, N, T) or during AON (O, P, T). Q, R, S, T: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt, n = 10 from 5 animals; rd8/rd8, n = 12 from 6 animals. n.s., not significant. **P < 0.01. Scale bars in AD, IP = 50 μm; EH = 10 μm.
Histopathology Is Unaltered During AON In Optic Nerves Carrying the rd8 Mutation
To confirm that the rd8 mutation did not affect any further parameters associated with AON, histopathology of the optic nerves was investigated at day 12 of EAE. Both wt/wt and rd8/rd8 optic nerves exhibited demyelination; however, there was no difference in its extent between the 2 groups (Figs. 5C, 5D, 5Q) nor in the degree of axonal loss (Figs. 5G, 5H, 5R). Furthermore, although numbers of both CD3-positive T cells (Figs. 5K, 5L, 5S) and Mac-3-positive activated microglia/macrophages (Figs. 5O, 5P, 5T) were significantly elevated in both wt/wt and rd8/rd8 optic nerves during disease, no significant differences were seen. 
Function of the Visual System Is Unaffected by the rd8 Mutation
ERGs were performed to examine whether the rd8 mutation affects either photoreceptor function or bipolar cell activity. Full-field, single-flash ERGs are characterized by a negative deflection (a-wave, generated by photoreceptors) occurring at higher intensities, followed by a larger positive deflection (b-wave, mainly derived from ON-bipolar cell activity). Both dark-adapted and light-adapted single-flash ERG analysis of wt/wt (Figs. 6A, 6B; left) and rd8/rd8 (Figs. 6A, 6B; right) mice did not show any differences in retinal function. This is consistent for the entire group because the b-wave amplitude analysis of wt/wt mice (Fig. 6C, open boxes) overlapped very well with the rd8/rd8 values (Fig. 6C, filled boxes). This is not surprising as the retinal degeneration observed was very localized to the inferior retina, whereas the remainder of the retina appeared grossly normal (Figs. 2I, 2K). 
Figure 6
 
Visual function in the presence of the rd8 mutation. Electroretinographic data observed in wt/wt and rd8/rd8 mice (AD). Dark-adapted (DA; A) and light-adapted (LA; B) single-flash ERG intensity series of wt/wt (A, B, left) and rd8/rd8 (A, B, right) mice show no differences in both groups in shape and size. Quantification of the b-wave amplitude: box-and-whisker plots reflecting the 5th, 25th, 50th, 75th, and 95th percentiles (C). Superpositions of selected single-flash ERG traces (D) representing the pure rod system function (DA, −2 log*s/m2), the mixed rod-cone system function (DA, 1 log*s/m2), and the exclusive cone system function (LA, 1 log*s/m2) point out that both photoreceptor systems were unaffected in wt/wt and rd8/rd8 mice, respectively. To determine the electrophysiological functioning of the optic nerves, VEPs were recorded in response to flash stimuli in healthy mice. No differences were seen between wt/wt (E) and rd8/rd8 (F) in either the latency (G) or amplitude (H). Further VEPS were recorded on both days 1 and 12 of EAE. wt/wt and rd8/rd8 mice had similar increases in latency and reduction in amplitude at both day 1 and day 12 of EAE (G, H). AD: wt/wt and rd8/rd8, n = 4; EG: wt/wt healthy, n = 13 from 7 animals; d1 and d12 EAE, n = 8 from 5 animals; rd8/rd8 healthy, n = 14 from 7 animals; d1 EAE, n = 10 from 6 animals; d12 EAE, n = 8 from 5 animals. n.s., not significant.
Figure 6
 
Visual function in the presence of the rd8 mutation. Electroretinographic data observed in wt/wt and rd8/rd8 mice (AD). Dark-adapted (DA; A) and light-adapted (LA; B) single-flash ERG intensity series of wt/wt (A, B, left) and rd8/rd8 (A, B, right) mice show no differences in both groups in shape and size. Quantification of the b-wave amplitude: box-and-whisker plots reflecting the 5th, 25th, 50th, 75th, and 95th percentiles (C). Superpositions of selected single-flash ERG traces (D) representing the pure rod system function (DA, −2 log*s/m2), the mixed rod-cone system function (DA, 1 log*s/m2), and the exclusive cone system function (LA, 1 log*s/m2) point out that both photoreceptor systems were unaffected in wt/wt and rd8/rd8 mice, respectively. To determine the electrophysiological functioning of the optic nerves, VEPs were recorded in response to flash stimuli in healthy mice. No differences were seen between wt/wt (E) and rd8/rd8 (F) in either the latency (G) or amplitude (H). Further VEPS were recorded on both days 1 and 12 of EAE. wt/wt and rd8/rd8 mice had similar increases in latency and reduction in amplitude at both day 1 and day 12 of EAE (G, H). AD: wt/wt and rd8/rd8, n = 4; EG: wt/wt healthy, n = 13 from 7 animals; d1 and d12 EAE, n = 8 from 5 animals; rd8/rd8 healthy, n = 14 from 7 animals; d1 EAE, n = 10 from 6 animals; d12 EAE, n = 8 from 5 animals. n.s., not significant.
To further assess the downstream processing of visual stimuli from the retina to higher brain areas, VEPs were recorded in healthy mice from the primary visual cortices in response to flash stimuli. Neither the latency (Fig. 6G) nor the amplitude (Fig. 6H) was altered in rd8/rd8 mice when compared with wt/wt mice. 
To determine the visual acuity of the mice during AON, VEPs were recorded on both the first day and day 12 of EAE. In comparison to healthy mice, latencies were significantly increased in both groups starting on the first day of EAE symptoms (Fig. 6G). However, no differences were seen between the groups at either time point (Fig. 6G). VEP amplitudes were also found to be decreased in both groups, starting on the first day of EAE, in comparison to controls, but again no significant differences between the genotypes were observed (Fig. 6H). 
Discussion
Interpreting results from models of ocular disease may be confounded by the presence of the rd8 mutation in background mice strains.710 Here we show that murine AON, a commonly used model in the study of MS, is unaffected by the presence of this mutation. 
AON is often used to study many of the pathologic features of MS and to develop novel therapies. The noninvasive nature of visual electrophysiological methods, such as VEPs and ERGs, allows compounds to be tested for functional efficacy. Also, because of the compartmentalization of the optic system, with RGC bodies located in the retina projecting their axons to higher visual areas, AON can give insights into neurodegenerative processes typical of an autoimmune inflammatory environment such as MS. 
C57BL/6 mice, of both J and N substrains, have become one of the main strains for EAE studies32 both because of the ability to induce disease reliably and because most genetically engineered mice are generated with C57BL/6-derived ES cells. It is therefore imperative to ascertain whether the rd8 mutation impacts the characteristic pathologic features of AON. 
Pathologic alterations in rd8 mice can be observed as early as 2 weeks after birth.2 AON, in common with most models of EAE, is induced in female mice of approximately 8 weeks of age because older mice are less susceptible to disease induction.33,34 In this study, mice were approximately 14 weeks of age at the end of the experiment. At a similar age, mice homozygous for the rd8 mutation have been reported to have spot-like fundus abnormalities and alterations in retinal layering, photoreceptor degeneration, and Müller glia activation and an increase in subretinal microglia.2,3,35,36 Through in vivo analyses, we observed a typical rd8 mutation phenotype with disorganization of the retinal layering and displacement of photoreceptor nuclei into the outer plexiform layer, appearing as half-rosette structures, proposed to be initiated by a loss of adhesion between the Müller glia and photoreceptors.2,3,31 Consistent with previous findings,30,31 the retinal disorganizations observed were localized to the inferior retina, whereas the remainder of the retinae appeared grossly normal. This is reflected in the absence of functional disturbances in photoreceptor and bipolar cells as demonstrated by ERGs. Although the pathology of the rd8 mutation has been reported to be progressive in nature and to be more severe in mice aged 10 months and older,4,31,35 the susceptibility of animals to EAE induction is vastly reduced in animals more than 1 year old33,34; thus we restricted our analysis to that of younger mice (3–4 months old). 
Our finding that RGC degeneration is unaltered by the rd8 mutation is similar to findings in other models of retinal degeneration. RGCs were preserved in mice homozygous for the rd1 mutation, which have early-onset photoreceptor degeneration as a result of a nonsense mutation in the β subunit of the rod phosphodiesterase gene 6, (Pde6b).37 Furthermore, RGCs in rd10 mice, which have a mis-sense mutation in the Pde6b gene resulting in widespread photoreceptor degeneration, also showed no pathologic changes.38 
In our study, we observed evidence of retinal folds within the inner and outer nuclear layers, but perhaps because of their localized restriction, these did not affect any of the more global parameters of AON. However, on closer analysis of the INQ, we still did not observe any influence on inner retinal degeneration or inflammation. Therefore it appears that, although the rd8 mutation affects outer retinal pathology, this does not translate to a perturbation in inner retinal parameters commonly assessed in AON. However, it should be acknowledged that in MS, outer retinal pathology as a feature is being increasingly recognized.39,40 How this is represented in rodent models of optic neuritis is still unclear, although there is some evidence for trans-synaptic degeneration to the outer retinal layers.15 Whether such trans-synaptic degeneration occurs in the other direction is at present unclear, but from this study and others,37,38 it appears not to be the case. 
In conclusion, the presence of the rd8 mutation does not affect the course of AON in young WT mice and should not provide a confounding factor in the interpretation of results obtained in this model. However, the relevance of the rd8 mutation to other ocular diseases must still be assessed by investigators. 
Acknowledgments
The authors thank Marika Dienes and Stefanie Buchholz for excellent technical assistance. Electrophysiological support was provided by the Interdisciplinary Neurobehavioral Core (INBC) facility of the Medical Faculty Heidelberg. 
Supported by the Deutsche Forschungsgemeinschaft (FOR 2289; DI935/12-1, DI935/13-1, WI3488/1-1, FL135/10-1). 
Disclosure: A. Stojic, None; R. Fairless, None; S.C. Beck, None; V. Sothilingam, None; P. Weissgerber, None; U. Wissenbach, None; V. Gimmy, None; M.W. Seeliger, None; V. Flockerzi, None; R. Diem, None; S.K. Williams, None 
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Figure 1
 
Genotyping. Sample gel showing separation of wild-type (wt/wt) mice (n = 30) and homozygous rd8-mutant (rd8/rd8) mice (n = 31) as described in the Materials and Methods section. Each PCR reaction was incubated in the absence (not cut or “NC”) or presence of EcoRV (“cut”).
Figure 1
 
Genotyping. Sample gel showing separation of wild-type (wt/wt) mice (n = 30) and homozygous rd8-mutant (rd8/rd8) mice (n = 31) as described in the Materials and Methods section. Each PCR reaction was incubated in the absence (not cut or “NC”) or presence of EcoRV (“cut”).
Figure 2
 
Visualization of the pathology typical of the rd8 mutation. Healthy mice of 14 weeks of age were examined with confocal scanning laser ophthalmoscopy (cSLO) imaging (A, F), fundus autofluorescence (B, G), fluorescein angiography (C, H), and spectral domain optical coherence tomography (OCT) (E, D, I, K). The wt/wt (C7BL6/J) mice showed a normal fundus appearance and retinal layering. In native SLO imaging, no differences were detected between both C57BL/6 substrains (A, F). However, in the autofluorescence mode, bright spots could be detected within the inferior nasal quadrant (INQ) in rd8/rd8 (C57BL/6N) mice (G, arrow), which correlated to the typical half-rosette structures of the rd8 mutation detected by OCT imaging (I, magnification K). wt/wt and rd8/rd8, n = 4. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane, I/OS inner/outer segment border; RPE/CC, retinal pigmented epithelium/choriocapillaris complex; t, temporal; n, nasal.
Figure 2
 
Visualization of the pathology typical of the rd8 mutation. Healthy mice of 14 weeks of age were examined with confocal scanning laser ophthalmoscopy (cSLO) imaging (A, F), fundus autofluorescence (B, G), fluorescein angiography (C, H), and spectral domain optical coherence tomography (OCT) (E, D, I, K). The wt/wt (C7BL6/J) mice showed a normal fundus appearance and retinal layering. In native SLO imaging, no differences were detected between both C57BL/6 substrains (A, F). However, in the autofluorescence mode, bright spots could be detected within the inferior nasal quadrant (INQ) in rd8/rd8 (C57BL/6N) mice (G, arrow), which correlated to the typical half-rosette structures of the rd8 mutation detected by OCT imaging (I, magnification K). wt/wt and rd8/rd8, n = 4. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane, I/OS inner/outer segment border; RPE/CC, retinal pigmented epithelium/choriocapillaris complex; t, temporal; n, nasal.
Figure 3
 
Global retinal pathology in healthy mice and during AON. Immunohistochemistry was performed on frozen retinal sections from both healthy, age-matched mice and 12 days after the onset of spinal cord symptoms. No difference was seen between the number of CD3-positive cells in wt/wt (A) and rd8/rd8 healthy mice (B, Q) or in wt/wt (C) and rd8/rd8 (D, Q) mice during AON. No difference was also seen between the number of Iba-1-positive cells in healthy wt/wt (E) and rd8/rd8 mice (F, R) or in wt/wt (G) and rd8/rd8 (H, R) mice during AON. TUNEL staining was performed on retinal sections to investigate apoptosis; however, no TUNEL-positive cells were seen in the GCL of either wt/wt (I) or rd8/rd8 (J) healthy mice. TUNEL positive cells were seen within the GCL layer during AON; however, there was no difference in the number quantified between wt/wt (K) and rd8/rd8 (L) mice (S). Fluorogold (FG)-positive RGC densities were quantified in flat-mounted retinae from healthy wt/wt (M) and rd8/rd8 mice (N); however, no difference in the number of labeled cells was observed (T). RGC densities were also quantified during AON, but no differences were seen in the number of surviving FG-labeled RGCs in wt/wt (O) and rd8/rd8 mice (P, T). Q, R, S: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt and rd8/rd8, n = 9 from 5 animals. T: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt, n = 8 from 5 animals; rd8/rd8, n = 12 from 7 animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; n.s., not significant; OPL, outer plexiform layer; ONL, outer nuclear layer. *P < 0.05, **P < 0.01. Scale bars = 50 μm.
Figure 3
 
Global retinal pathology in healthy mice and during AON. Immunohistochemistry was performed on frozen retinal sections from both healthy, age-matched mice and 12 days after the onset of spinal cord symptoms. No difference was seen between the number of CD3-positive cells in wt/wt (A) and rd8/rd8 healthy mice (B, Q) or in wt/wt (C) and rd8/rd8 (D, Q) mice during AON. No difference was also seen between the number of Iba-1-positive cells in healthy wt/wt (E) and rd8/rd8 mice (F, R) or in wt/wt (G) and rd8/rd8 (H, R) mice during AON. TUNEL staining was performed on retinal sections to investigate apoptosis; however, no TUNEL-positive cells were seen in the GCL of either wt/wt (I) or rd8/rd8 (J) healthy mice. TUNEL positive cells were seen within the GCL layer during AON; however, there was no difference in the number quantified between wt/wt (K) and rd8/rd8 (L) mice (S). Fluorogold (FG)-positive RGC densities were quantified in flat-mounted retinae from healthy wt/wt (M) and rd8/rd8 mice (N); however, no difference in the number of labeled cells was observed (T). RGC densities were also quantified during AON, but no differences were seen in the number of surviving FG-labeled RGCs in wt/wt (O) and rd8/rd8 mice (P, T). Q, R, S: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt and rd8/rd8, n = 9 from 5 animals. T: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt, n = 8 from 5 animals; rd8/rd8, n = 12 from 7 animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; n.s., not significant; OPL, outer plexiform layer; ONL, outer nuclear layer. *P < 0.05, **P < 0.01. Scale bars = 50 μm.
Figure 4
 
Retinal pathology within the inferior nasal quadrant (INQ) in healthy mice and during AON. Frozen retinal sections from both healthy, age-matched mice and 12 days after the onset of spinal cord symptoms were generated in which the orientation of the retina was maintained to investigate region of the inferior nasal quadrant (INQ) specifically. No difference was seen between the number of CD3-positive T cells in wt/wt (A) and rd8/rd8 healthy mice (B, M) or in wt/wt (C) and rd8/rd8 (D, M) mice during AON in the INQ. No difference was also seen between the number of Iba-1-positive activated microglia/macrophages cells in healthy wt/wt (E) and rd8/rd8 mice (F, N) or in wt/wt (G) and rd8/rd8 (H, N) mice during AON in the INQ. TUNEL staining was performed to investigate the presence of apoptotic cells; however, no TUNEL-positive cells were seen in the GCL of either wt/wt (I) or rd8/rd8 (J) healthy mice in the INQ. TUNEL positive cells were seen within the GCL layer during AON; however, there was no difference in the number quantified between wt/wt (K) and rd8/rd8 (L) mice (O) in the INQ. M, N, O: healthy wt/wt and rd8/rd8, n = 5 from 3 animals; AON wt/wt and rd8/rd8, n = 6 from 3 animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; n.s., not significant; OPL, outer plexiform layer; ONL, outer nuclear layer. *P < 0.05, **P < 0.01. Scale bars = 50 μm.
Figure 4
 
Retinal pathology within the inferior nasal quadrant (INQ) in healthy mice and during AON. Frozen retinal sections from both healthy, age-matched mice and 12 days after the onset of spinal cord symptoms were generated in which the orientation of the retina was maintained to investigate region of the inferior nasal quadrant (INQ) specifically. No difference was seen between the number of CD3-positive T cells in wt/wt (A) and rd8/rd8 healthy mice (B, M) or in wt/wt (C) and rd8/rd8 (D, M) mice during AON in the INQ. No difference was also seen between the number of Iba-1-positive activated microglia/macrophages cells in healthy wt/wt (E) and rd8/rd8 mice (F, N) or in wt/wt (G) and rd8/rd8 (H, N) mice during AON in the INQ. TUNEL staining was performed to investigate the presence of apoptotic cells; however, no TUNEL-positive cells were seen in the GCL of either wt/wt (I) or rd8/rd8 (J) healthy mice in the INQ. TUNEL positive cells were seen within the GCL layer during AON; however, there was no difference in the number quantified between wt/wt (K) and rd8/rd8 (L) mice (O) in the INQ. M, N, O: healthy wt/wt and rd8/rd8, n = 5 from 3 animals; AON wt/wt and rd8/rd8, n = 6 from 3 animals. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; n.s., not significant; OPL, outer plexiform layer; ONL, outer nuclear layer. *P < 0.05, **P < 0.01. Scale bars = 50 μm.
Figure 5
 
Optic nerve pathology in healthy mice and during AON. Optic nerves from healthy, age-matched mice and at day 12 of EAE were assessed histopathologically. Neither healthy wt/wt (A) nor rd8/rd8 (B) mice showed any optic nerve demyelination (Q). During AON, EAE optic nerves from both wt/wt (C) and rd8/rd8 mice (D) showed a similar level of demyelination (Q). As assessed by Bielschowsky's silver impregnation, wt/wt (E) and rd8/rd8 (F) optic nerves had a similar axonal density (R) in healthy optic nerves as well as at day 12 of EAE (G, H, R). A small number of CD3-positive cells were observed in both wt/wt (I) and rd8/rd8 (J) optic nerves, although there was no statistical difference (S). During AON, no difference was seen in the presence of CD3-positive cells between wt/wt (K) and rd8/rd8 mice (L, S) Furthermore, there was no difference in the numbers of Mac-3-positive activated microglia/macrophages in healthy optic nerves (M, N, T) or during AON (O, P, T). Q, R, S, T: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt, n = 10 from 5 animals; rd8/rd8, n = 12 from 6 animals. n.s., not significant. **P < 0.01. Scale bars in AD, IP = 50 μm; EH = 10 μm.
Figure 5
 
Optic nerve pathology in healthy mice and during AON. Optic nerves from healthy, age-matched mice and at day 12 of EAE were assessed histopathologically. Neither healthy wt/wt (A) nor rd8/rd8 (B) mice showed any optic nerve demyelination (Q). During AON, EAE optic nerves from both wt/wt (C) and rd8/rd8 mice (D) showed a similar level of demyelination (Q). As assessed by Bielschowsky's silver impregnation, wt/wt (E) and rd8/rd8 (F) optic nerves had a similar axonal density (R) in healthy optic nerves as well as at day 12 of EAE (G, H, R). A small number of CD3-positive cells were observed in both wt/wt (I) and rd8/rd8 (J) optic nerves, although there was no statistical difference (S). During AON, no difference was seen in the presence of CD3-positive cells between wt/wt (K) and rd8/rd8 mice (L, S) Furthermore, there was no difference in the numbers of Mac-3-positive activated microglia/macrophages in healthy optic nerves (M, N, T) or during AON (O, P, T). Q, R, S, T: healthy wt/wt and rd8/rd8, n = 6 from 3 animals; AON wt/wt, n = 10 from 5 animals; rd8/rd8, n = 12 from 6 animals. n.s., not significant. **P < 0.01. Scale bars in AD, IP = 50 μm; EH = 10 μm.
Figure 6
 
Visual function in the presence of the rd8 mutation. Electroretinographic data observed in wt/wt and rd8/rd8 mice (AD). Dark-adapted (DA; A) and light-adapted (LA; B) single-flash ERG intensity series of wt/wt (A, B, left) and rd8/rd8 (A, B, right) mice show no differences in both groups in shape and size. Quantification of the b-wave amplitude: box-and-whisker plots reflecting the 5th, 25th, 50th, 75th, and 95th percentiles (C). Superpositions of selected single-flash ERG traces (D) representing the pure rod system function (DA, −2 log*s/m2), the mixed rod-cone system function (DA, 1 log*s/m2), and the exclusive cone system function (LA, 1 log*s/m2) point out that both photoreceptor systems were unaffected in wt/wt and rd8/rd8 mice, respectively. To determine the electrophysiological functioning of the optic nerves, VEPs were recorded in response to flash stimuli in healthy mice. No differences were seen between wt/wt (E) and rd8/rd8 (F) in either the latency (G) or amplitude (H). Further VEPS were recorded on both days 1 and 12 of EAE. wt/wt and rd8/rd8 mice had similar increases in latency and reduction in amplitude at both day 1 and day 12 of EAE (G, H). AD: wt/wt and rd8/rd8, n = 4; EG: wt/wt healthy, n = 13 from 7 animals; d1 and d12 EAE, n = 8 from 5 animals; rd8/rd8 healthy, n = 14 from 7 animals; d1 EAE, n = 10 from 6 animals; d12 EAE, n = 8 from 5 animals. n.s., not significant.
Figure 6
 
Visual function in the presence of the rd8 mutation. Electroretinographic data observed in wt/wt and rd8/rd8 mice (AD). Dark-adapted (DA; A) and light-adapted (LA; B) single-flash ERG intensity series of wt/wt (A, B, left) and rd8/rd8 (A, B, right) mice show no differences in both groups in shape and size. Quantification of the b-wave amplitude: box-and-whisker plots reflecting the 5th, 25th, 50th, 75th, and 95th percentiles (C). Superpositions of selected single-flash ERG traces (D) representing the pure rod system function (DA, −2 log*s/m2), the mixed rod-cone system function (DA, 1 log*s/m2), and the exclusive cone system function (LA, 1 log*s/m2) point out that both photoreceptor systems were unaffected in wt/wt and rd8/rd8 mice, respectively. To determine the electrophysiological functioning of the optic nerves, VEPs were recorded in response to flash stimuli in healthy mice. No differences were seen between wt/wt (E) and rd8/rd8 (F) in either the latency (G) or amplitude (H). Further VEPS were recorded on both days 1 and 12 of EAE. wt/wt and rd8/rd8 mice had similar increases in latency and reduction in amplitude at both day 1 and day 12 of EAE (G, H). AD: wt/wt and rd8/rd8, n = 4; EG: wt/wt healthy, n = 13 from 7 animals; d1 and d12 EAE, n = 8 from 5 animals; rd8/rd8 healthy, n = 14 from 7 animals; d1 EAE, n = 10 from 6 animals; d12 EAE, n = 8 from 5 animals. n.s., not significant.
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