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. 2018 Jun 14;15(1):183.
doi: 10.1186/s12974-018-1208-3.

Laquinimod protects the optic nerve and retina in an experimental autoimmune encephalomyelitis model

Anna T Wilmes  1 , Sabrina Reinehr  1 , Sandra Kühn  1 , Xiomara Pedreiturria  2 , Laura Petrikowski  2 , Simon Faissner  2 , Ilya Ayzenberg  2 , Gesa Stute  1 , Ralf Gold  2 , H Burkhard Dick  1 , Ingo Kleiter  3 , Stephanie C Joachim  4
Affiliations

Laquinimod protects the optic nerve and retina in an experimental autoimmune encephalomyelitis model

Anna T Wilmes et al. J Neuroinflammation. .

Abstract

Background: The oral immunomodulatory agent laquinimod is currently evaluated for multiple sclerosis (MS) treatment. Phase II and III studies demonstrated a reduction of degenerative processes. In addition to anti-inflammatory effects, laquinimod might have neuroprotective properties, but its impact on the visual system, which is often affected by MS, is unknown. The aim of our study was to investigate potential protective effects of laquinimod on the optic nerve and retina in an experimental autoimmune encephalomyelitis (EAE) model.

Methods: We induced EAE in C57/BL6 mice via MOG35-55 immunization. Animals were divided into an untreated EAE group, three EAE groups receiving laquinimod (1, 5, or 25 mg/kg daily), starting the day post-immunization, and a non-immunized control group. Thirty days post-immunization, scotopic electroretinograms were carried out, and mice were sacrificed for histopathology (HE, LFB), immunohistochemistry (MBP, Iba1, Tmem119, F4/80, GFAP, vimentin, Brn-3a, cleaved caspase 3) of the optic nerve and retina, and retinal qRT-PCR analyses (Brn-3a, Iba1, Tmem119, AMWAP, CD68, GFAP). To evaluate the effect of a therapeutic approach, EAE animals were treated with 25 mg/kg laquinimod from day 16 when 60% of the animals had developed clinical signs of EAE.

Results: Laquinimod reduced neurological EAE symptoms and improved the neuronal electrical output of the inner nuclear layer compared to untreated EAE mice. Furthermore, cellular infiltration, especially recruited phagocytes, and demyelination in the optic nerve were reduced. Microglia were diminished in optic nerve and retina. Retinal macroglial signal was reduced under treatment, whereas in the optic nerve macroglia were not affected. Additionally, laquinimod preserved retinal ganglion cells and reduced apoptosis. A later treatment with laquinimod in a therapeutic approach led to a reduction of clinical signs and to an improved b-wave amplitude. However, no changes in cellular infiltration and demyelination of the optic nerves were observed. Also, the number of retinal ganglion cells remained unaltered.

Conclusion: From our study, we deduce neuroprotective and anti-inflammatory effects of laquinimod on the optic nerve and retina in EAE mice, when animals were treated before any clinical signs were noted. Given the fact that the visual system is frequently affected by MS, the agent might be an interesting subject of further neuro-ophthalmic investigations.

Keywords: Demyelination; EAE; Electroretinogram; Glia response; Inflammation; Laquinimod; Multiple sclerosis; Optic nerve; Protection; Retinal degeneration.

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Conflict of interest statement

Ethics approval and consent to participate

Not applicable.

Competing interests

SF received travel grants from Biogen Idec and Genzyme, none related to this manuscript.

RG serves on scientific advisory boards for Teva Pharmaceutical Industries Ltd., Biogen Idec, Bayer Schering Pharma, and Novartis; has received speaker honoraria from Biogen Idec, Teva Pharmaceutical Industries Ltd., Bayer Schering Pharma, and Novartis; serves as an editor for Therapeutic Advances in Neurological Diseases and on the editorial boards of Experimental Neurology and the Journal of Neuroimmunology; and receives research support from Teva Pharmaceutical Industries Ltd., Biogen Idec, Bayer Schering Pharma, Genzyme, Merck Serono, and Novartis, which are not related to this manuscript.

IK received honoraria for consultancy or speaking and travel reimbursement from Bayer Healthcare, Chugai, Merck, Roche, and Shire and grant support from Affectis, Biogen, Chugai and Diamed, all not related to this manuscript. The other authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Clinical effects of laquinimod treatment. a Mean clinical EAE scores after immunization with MOG35–55 peptide. Green arrowhead: onset of symptoms. Red arrowhead: symptoms’ peak. b Electroretinograms were measured at the end of the experiment at day 30. A-wave amplitudes illustrate conductivity of photoreceptors. c B-wave amplitudes illustrate conductivity of the inner nuclear layer. Values represent mean ± SD. One-way ANOVA and Tukey post hoc. N = 6–7/group in a and n = 5/group in b, c. Comparison to control group: #p < 0.05, ###p < 0.001. Comparison to untreated EAE group: *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 2
Fig. 2
Structural preservation of the optic nerve in laquinimod-treated mice. a HE-stained healthy optic nerves show a linear formation of nuclei in a bead-like manner (green arrow), which in EAE is disrupted (white arrows) and might comprise cellular conglomerates (orange arrowheads). The usual structure of LFB-stained myelin sheaths in optic nerve tissue resembles combed bundles in a steady parallel arrangement (control group). In EAE, brightening in structure represents an interruption of this arrangement (star). Labeling of MBP, a main component of myelin sheaths, shows specific changes in myelin structure. b Cellular infiltration measured via HE score. c Demyelination measured via LFB score. d Demyelination measured via MBP staining. Values represent median, interquartile range, range in b and c, and mean ± SD in d. Kruskal-Wallis plus Dunn’s test for b and c and one-way ANOVA plus Tukey post hoc for d. N = 6–7/group. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 40 μm in HE and LFB, 20 μm in MBP
Fig. 3
Fig. 3
Less microglia and recruited phagocytes in the optic nerve under laquinimod treatment. a Iba1 antibody was used to label all phagocytes and combined with Tmem119 antibody to distinguish between microglia (Tmem+ and Iba1+, turquoise arrowheads) and recruited phagocytes (Tmem− and Iba1+, white arrows). b Numbers of phagocytes/mm2. c Numbers of microglia/mm2. d Numbers of infiltrating phagocytes/mm2. All groups showed more infiltrating phagocytes than microglia. e Iba1 and F4/80 antibody to select cells with macrophage function (F4/80+ and Iba1+, pink arrowheads). f Numbers of cells with macrophage function/mm2. Values represent mean ± SD. One-way ANOVA and Tukey post hoc. N = 6–7/group. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 20 μm in a, 10 μm in e
Fig. 4
Fig. 4
No change of macroglia in the optic nerve under laquinimod treatment. a Macroglia were labeled with GFAP antibody. b Macroglia signal area as percentage. Values represent mean ± SD. One-way ANOVA plus Tukey post hoc. N = 6–7/group. *p < 0.05. Scale bar: 20 μm
Fig. 5
Fig. 5
Protection of retinal ganglion cells under laquinimod treatment. a Retinal ganglion cells were marked with Brn-3a antibody (green), apoptotic cells via cleaved caspase 3 antibody (red). b Numbers of retinal ganglion cells/mm. c Percentage of apoptotic retinal ganglion cells. d Brn-3a expression compared to the control group. e Brn-3a expression compared to the EAE group. Values represent mean ± SD in b and c and median, interquartile range, range in d and e. One-way ANOVA plus Tukey post hoc for b and c and pairwise fixed reallocation and randomization test for d and e. N = 6–7/group in a–c and n = 5/group in d and e. *p < 0.01, **p < 0.01, ***p < 0.001. Scale bar: 10 μm. GCL = ganglion cell layer, IPL = inner plexiform layer
Fig. 6
Fig. 6
Less retinal microglia and macrophages under laquinimod treatment. a Exemplary staining of retinal Tmem119 (microglia) and Iba1 (phagocytes). b Iba 1 antibody (red) and F4/80 antibody (green) were co-stained to select cells with macrophage function. c Numbers of phagocytes/mm. d Numbers of cells with macrophage function/mm. e Iba1 expression compared to the control group. f Iba1 expression compared to the EAE group. g Tmem119 expression compared to control. h Tmem119 expression compared to EAE. i Expression of active microglia marker AMWAP compared to the control group. j AMWAP expression compared to EAE. k Macrophage marker CD68 expression compared to the control group. l CD68 expression compared to EAE. Values represent mean ± SD in c and d and median, interquartile range, range in e–l. One-way ANOVA plus Tukey post hoc for c and d; pairwise fixed reallocation and randomization test for e–l. N = 6–7/group in b–d and n = 5/group in e–l. *p < 0.01, **p < 0.01, ***p < 0.001. Scale bars: 20 μm. NFL = nerve fiber layer, GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer
Fig. 7
Fig. 7
Less retinal macroglia response under laquinimod treatment. a GFAP antibody was applied to mark retinal macroglia (green). Retinal Müller glia was investigated via vimentin antibody (red). b Macroglia signal area as percentage. c Müller glia signal area as percentage. d GFAP expression compared to the control group. e GFAP expression compared to EAE. Values represent mean ± SD in b and c and median, interquartile range, range in d and e. One-way ANOVA plus Tukey post hoc for b and c; pairwise fixed reallocation and randomization test for d and e. N = 6–7/group in a–c and n = 5/group in d and e. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 20 μm. GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer
Fig. 8
Fig. 8
Clinical effects of therapeutic laquinimod treatment. a Mean clinical EAE scores after immunization with MOG35–55 peptide. Yellow arrowhead: start of laquinimod treatment. b Electroretinograms were measured at day 30. A-wave amplitudes illustrate conductivity of photoreceptors. c B-wave amplitudes illustrate conductivity of the inner nuclear layer. Values represent mean ± SD in a, mean ± SD ± SEM in b and c. Mann-Whitney U test for a; Student’s t test for b and c. N = 3–5/group in a–c. *p < 0.05, **p < 0.01
Fig. 9
Fig. 9
Therapeutic laquinimod treatment had no effect on optic nerve structure and retinal ganglion cells. a Optic nerves of the EAE and 25 mg/kg group were stained with HE and LFB. b Cellular infiltration was measured via HE score. c Demyelination was analyzed via LFB score. d Retinal ganglion cells were marked with Brn-3a antibody (green). e Numbers of retinal ganglion cells/mm. Values represent mean ± SD ± SEM in b, c, and e. Mann-Whitney U test for b and c; Student’s t test for e. N = 3–5/group in a–c. Scale bars: 20 μm in a; 10 μm in c. GCL = ganglion cell layer, IPL = inner plexiform layer
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References

    1. Kawachi I, Lassmann H. Neurodegeneration in multiple sclerosis and neuromyelitis optica. J Neurol Neurosurg Psychiatry. 2017;88:137–145. doi: 10.1136/jnnp-2016-313300. - DOI - PubMed
    1. Runia TF, van Pelt-Gravesteijn ED, Hintzen RQ. Recent gains in clinical multiple sclerosis research. CNS Neurol Disord Drug Targets. 2012;11:497–505. doi: 10.2174/187152712801661239. - DOI - PubMed
    1. Olsson T, Barcellos LF, Alfredsson L. Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis. Nat Rev Neurol. 2017;13:25–36. doi: 10.1038/nrneurol.2016.187. - DOI - PubMed
    1. Stadelmann C, Wegner C, Bruck W. Inflammation, demyelination, and degeneration - recent insights from MS pathology. Biochim Biophys Acta. 2011;1812:275–282. doi: 10.1016/j.bbadis.2010.07.007. - DOI - PubMed
    1. Compston A, Coles A. Multiple sclerosis. Lancet. 2008;372:1502–1517. doi: 10.1016/S0140-6736(08)61620-7. - DOI - PubMed

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