Cathepsin-B inhibitor CA-074 attenuates retinopathy and optic neuritis in experimental autoimmune encephalomyelitis induced in SJL/J mice

Abstract

The complicated multiple sclerosis (MS) can exhibit subacute sight deterioration and can lead to total deprivation of vision. In the current work, we explored the therapeutic outcome of Cathepsin B inhibitor (CA-074) against retinopathy and optic neuritis (ON) caused by experimental autoimmune encephalomyelitis (EAE) induced by proteolipid protein peptide (PLP) in female SJL/J mice. A daily dose of 10 mg/kg CA-074 was administered to the EAE mice intraperitoneally for 14 days from day 14 post-immunization until day 28. The Western blot and immunofluorescence analyses show inflammation in the optic nerve through the elevation of iNOS and NFkB markers in EAE mice. Optic neuritis was reported which is a consequence of demyelination and axon injury, estimated with the reduction in myelin basic protein (MBP). The glial fibrillary acidic protein (GFAP) expression level was found to be elevated in the retina of EAE mice which confirmed the retinopathy. The administration of CA-074 ameliorated optic neuritis and retinopathy by reducing inflammation. The treatment with CA-074 also reduced the demyelination and axonal injuries in the EAE mice. The findings of this study have shown the protective effect of CA-074 in the case of retinopathy and ON inflicted by EAE in SJL/J mice.

Keywords: Demyelination; Multiple sclerosis; Neurodegeneration; Optic Neuritis; Retinopathy.

Fig. 1

Western blot analysis of inducible nitric oxide synthase (iNOS) expression has shown that the treatment with CA-074 attenuated the expression of iNOS in the optic nerve of the EAE Mice. (A)Western blot analysis of iNOS expression. (B) NF-kB expression. The Data are shown as mean ± S.E.M, n = 6. p < 0.05 is accepted as the level of significance; ∗p < 0.05. *p < 0.05 compared to the SJL/J control group; **p < 0.05 compared to EAE group.

Fig. 2

Western blot analysis of NF-kB expression has shown that the treatment with CA-074 downregulated the activation of NFkB in the optic nerve of the EAE Mice. The Data are shown as mean ± S.E.M, n = 6. p < 0.05 is accepted as the level of significance; ∗p < 0.05. *p < 0.05 compared to the SJL/J control group; **p < 0.05 compared to EAE group.

Fig. 3

The CA-074 reduces the activation of glial cells in the inner retina in EAE mice. Western blot analysis. The treatment of CA-074 downregulated the expression of GFAP (A) and IBA1 (B). The Data were shown as mean ± S.E.M, n = 6. p < 0.05 is accepted as the level of significance; ∗p < 0.05. *p < 0.05 compared to the SJL/J control group; **p < 0.05 compared to EAE group.

Fig. 4

The activation of Glial cells occurred in the retinas of EAE mice, the treatment with CA-074 decreases the activation of glial cells in EAE mice. The analyses of GFAP immunofluorescence in the retina (n = 6), of control SJL/J (A), The increased GFAP + glia cells in EAE mouse (B) The reduced staining intensity of GFAP in EAE + CA-074 treated mice (C). The Data were shown as mean ± S.E.M, n = 6. p < 0.05 is accepted as the level of significance; ∗p < 0.05. *p < 0.05 compared to the SJL/J control group; **p < 0.05 compared to EAE group. Scale bars represent 20 μm.

Fig. 5

The activation of glial cells occurred in the optic nerves of the EAE mice, the treatment with the CA-074 reduces the activation of glial cells in the optic nerve of EAE mice. The estimation of GFAP immunofluorescence in the optic nerve (n = 6), of control, the increased GFAP positive glia cells in EAE mouse and reduced staining intensity of GFAP in EAE + CA-074 treated mice. The Data were shown as mean ± S.E.M, n = 6. p < 0.05 is accepted as the level of significance; ∗p < 0.05. *p < 0.05 compared to the SJL/J control group; **p < 0.05 compared to EAE group. Scale bars represent 20 μm.

Fig. 6

The Demyelination and axonal injury occurred in the optic nerves of the EAE mice, the treatment with CA-074-protected axonal demyelination is shown by estimation of MBP immunofluorescence in the optic nerve (n = 6), of control, the demyelination in EAE mice and reduced demyelination in EAE + CA-074 treated mice. The Data were shown as mean ± S.E.M, n = 6. p < 0.05 is accepted as the level of significance; ∗p < 0.05. *p < 0.05 compared to the SJL/J control group; **p < 0.05 compared to EAE group. Scale bars represent 20 μm.