Anti-inflammatory effects of FTY720 do not prevent neuronal cell loss in a rat model of optic neuritis - PubMed (original) (raw)

Anti-inflammatory effects of FTY720 do not prevent neuronal cell loss in a rat model of optic neuritis

Christian R Rau et al. Am J Pathol. 2011 Apr.

Abstract

In multiple sclerosis, long-term disability is caused by axonal and neuronal damage. Established therapies target primarily the inflammatory component of the disease, but fail to prevent neurodegeneration. Fingolimod (codenamed FTY720) is an oral sphingosine 1-phosphate (S1P) receptor modulator with promising results in phase II trials in multiple sclerosis patients and is under further development as a novel treatment for multiple sclerosis. To evaluate whether FTY720 has neuroprotective properties, we tested this drug in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. FTY720 exerted significant anti-inflammatory effects during optic neuritis and reduced inflammation, demyelination, and axonal damage; however, FTY720 treatment did not prevent apoptosis of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. Consistent with this lack of effect on RGC survival, FTY720 treatment did not improve visual function, nor did it prevent apoptosis of RGCs in vitro. We observed a persistent activation of apoptotic signaling pathways in RGCs under FTY720 treatment, a possible underlying mechanism for the lack of neuroprotection in the presence of strong anti-inflammatory effects, Furthermore, FTY720 shifted the remaining inflammation in the optic nerve toward neurotoxicity by modest up-regulation of potential neurotoxic cytokines. We conclude that FTY720-induced anti-inflammation and axon protection did not of itself protect neurons from apoptotic cell death.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Clinical score. Daily oral application of FTY720 started at day 7 after immunization significantly suppresses incidence of MOG-EAE. Severity of disease was also substantially reduced. Mean clinical score under treatment with FTY720 during disease course was compared with vehicle-treated animals (n = 10 for each group); all animals are included, with or without clinical symptoms. The clinical score represents primarily spinal cord lesions; visual function is not included.

Figure 2

Figure 2

Visual evoked potentials (VEP) and RGC survival. Electrophysiological measurement of the visual system at day 28 after immunization showed no improvement in VEP recordings in FTY720-treated animals, compared with vehicle-treated controls. A–C: Representative pattern VEP recordings of the left eye and the right eye of a sham-immunized control animal (A), FTY720-treated animal (B), and a vehicle-treated animal (C). D: Mean VEP score was 3.8 in the control rats immunized with complete Freund's adjuvant (ctrl), 2.4 in the vehicle group, and 2.6 in the FTY720-treated group (n = 10 in each group; P = 0.91466 for FTY720- vs. vehicle-treated animals). E: Mean density of RGCs in sham-immunized control (ctrl), FTY72-treated, and vehicle-treated animals at 28 days after immunization. There was no significant difference in RGC survival between the FTY720 treatment and vehicle treatment groups (P = 0.71256).

Figure 3

Figure 3

Optical nerve (ON) histopathology. A and B: Representative Luxol Fast Blue-stained cross-section of ONs of an FTY720-treated rat (A) shows only small areas of demyelination, with mainly intact myelin (blue); in contrast, a cross-section from a vehicle-treated control animal (B) shows extensively demyelinated areas (purple). C and D: Representative examples of the substantially higher number of ED1+ macrophages/activated microglia detected in the ON of a vehicle-treated control animal (D), compared with an animal that had received FTY720 (C). Inset: Higher-magnification view of ED1+ cells. E and F: Representative ON cross-sections showing reduced β-APP+ axons (arrows) under FTY720 treatment on EAE day 8 (E), compared with the vehicle-treated control (F). Appearance on day 28 after immunization was essentially the same as shown here (E) for day 8 of MOG-EAE. F, Inset: Higher-magnification view of a β-APP+ axon. G and H: Bielschowsky's silver impregnation of an ON cross-section reveals different densities of axons on EAE day 8 in FTY720-treated animals (G) and vehicle-treated control animals (H). Scale bars: 100 μm (A–F); 20 μm (G and H).

Figure 4

Figure 4

Nitrotyrosine (NT) expression. In Western blot analysisthe nitrotyrosine level in the retina (A) was unchanged, but in the optic nerve (B) the level was decreased in FTY720-treated animals (+), compared with vehicle-treated animals (−). β-Tubulin serves as a housekeeping protein. C: Quantitation of nitrotyrosine expression level in the optic nerve. *P < 0.05, compared with vehicle-treated animals.

Figure 5

Figure 5

Immunohistochemistry for the S1P1-receptor. In comparison of S1P1-receptor expression pattern between healthy and MOG-EAE animals, no expression was found in optic nerve (A) or retina (C) of healthy animals, but strong S1P1 expression was evident in diseased optic nerve (B), as well as in both healthy (D) and diseased (E) spinal cord. Scale bars: 100 μm (A–C); 20 μm (D and E). Insets: matching higher magnification view of S1P1-receptor-negative (A) and S1P1-receptor-positive (B, D and E) cells.

Figure 6

Figure 6

Western blot analysis. Western blots of retinal protein lysates obtained on the day of clinical manifestation of MOG-EAE revealed that FTY720 treatment (+) resulted in unchanged protein levels of Bcl-2 and Bax, compared with treatment with vehicle alone (−). Analyses of phosphorylation levels of Akt and MAPK 1/2 also revealed unchanged protein expression. β-Tubulin serves as a housekeeping protein.

Figure 7

Figure 7

Enzyme-linked immunosorbent assay analysis of the expression pattern of neuroprotective cytokines and neurotrophins in the ON under influence of FTY720. Levels of IL-12, TGF-β1, and TNF-α were similar in ON lysates of the FTY720- and vehicle-treated animals (n = 10 in each group).

Figure 8

Figure 8

pFTY720 had no direct influence on RGC survival in vitro in cell culture with (A) or without (B) growth factor withdrawal. The numbers of surviving immunopurified RGCs treated with different concentrations of pFTY720 are expressed as a percentage of the matching control.

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