Timing of corticosteroid therapy is critical to prevent retinal ganglion cell loss in experimental optic neuritis

Abstract

Purpose: Acute vision loss from optic neuritis typically resolves; however, recovery is often not complete. Permanent vision loss from retinal ganglion cell (RGC) death occurs in 40% to 60% of patients. Current therapy (high-dose corticosteroids) speeds recovery but does not change final visual outcomes. Here the authors examined whether corticosteroids administered early in the disease course can prevent RGC loss in experimental optic neuritis.

Methods: RGCs were retrogradely labeled with fluorogold in SJL/J mice. Experimental autoimmune encephalomyelitis (EAE) was induced by immunization with proteolipid protein peptide. Optic neuritis began 9 days after immunization. Mice were treated daily with dexamethasone, methylprednisolone, or PBS from days 0 to 14 or days 10 to 14 and then were killed on day 14, 18, or 22.

Results: Corticosteroid treatment initiated before optic neuritis onset (days 0-14) suppressed EAE and reduced optic neuritis incidence through day 14. In the few eyes that developed optic neuritis, inflammation was mild, and RGC loss was attenuated. After treatment was stopped on day 14, mice rapidly developed EAE and optic neuritis by day 18, but RGC loss was still reduced. By day 22, RGC loss increased to levels similar to those of untreated optic neuritis eyes. Corticosteroid treatment after optic neuritis onset (days 10-14) slowed EAE progression and showed a trend toward suppression of optic neuritis and RGC loss on day 14 that was lost by day 18.

Conclusions: Corticosteroids can suppress optic neuritis and prevent RGC loss if treatment is initiated before optic nerve inflammation onset. Treatment is less effective after inflammation begins. Results suggest that chronic immunomodulation may prevent recurrent optic neuritis and RGC damage.

Figure 1.

Dexamethasone treatment beginning on the day of immunization suppresses EAE and optic neuritis and attenuates RGC loss. RGCs were retrogradely labeled by injection of fluorochrome into the superior colliculi of 7-week-old female SJL/J mice. Mice were immunized 1 week later (day 0) with PLP in CFA to induce EAE; controls were sham immunized with PBS in CFA. Mice were treated daily from days 0 to 14 with 200 mg/kg subcutaneous dexamethasone or an equal volume of PBS, then killed on day 14. (A) EAE mice treated with dexamethasone developed significantly less neurologic signs of EAE than mice treated with PBS alone. (B) The percentage of eyes that developed detectable optic nerve inflammation was lower in dexamethasone-treated EAE mice than in PBS-treated mice. (C) Longitudinal section through an optic nerve from a control mouse stained by hematoxylin and eosin demonstrates normal cellularity. (D) Optic nerve from 1 of 3 eyes from dexamethasone-treated EAE mice that did develop optic neuritis shows the relatively mild level of inflammation observed, with just a few small pockets of inflammatory cells (arrowheads) present. (E) Moderate to severe inflammation with numerous infiltrating cells within the optic nerve of a PBS-treated EAE mouse. (F) RGCs were photographed and counted in 12 standardized fields of flat-mounted retinas. Eyes with optic neuritis from PBS-treated mice had significantly fewer RGCs than eyes from control mice, eyes from EAE mice that did not develop optic neuritis, or the three eyes from dexamethasone-treated EAE mice that did develop optic neuritis (*P < 0.0001). Optic neuritis eyes from dexamethasone-treated mice had RGC numbers equivalent to those of control mice. Scale bars, 50 μm (C–E).

Figure 2.

Withdrawal of dexamethasone treatment leads to a flare of EAE and optic neuritis. Control and EAE mice were treated daily from days 0 to 14 with 200 mg/kg dexamethasone or PBS, then continued to be observed daily after treatment was stopped until kill on day 18 or day 22. (A) Dexamethasone treatment significantly suppressed EAE through day 14 when mice were still being treated, but clinical EAE developed rapidly when treatment was discontinued after day 14, with disease severity equal to that found in PBS-treated EAE mice by day 16 and maintained through day 18. (B) The percentage of eyes with detectable optic nerve inflammation was no different 18 days after immunization between EAE mice treated with dexamethasone and those treated with PBS alone. (C) At day 18, the number of RGCs in optic neuritis eyes from PBS-treated EAE mice was significantly lower than in control eyes or optic neuritis eyes from dexamethasone-treated EAE mice (*P = 0.0018). (D) At day 22, RGC numbers were significantly lower in optic neuritis eyes from both PBS- and dexamethasone-treated EAE mice than in eyes from control mice (*P = 0.0001), but there was no significant difference between PBS-treated and dexamethasone-treated optic neuritis eyes.

Figure 3.

Methylprednisolone treatment beginning on the day of immunization suppresses EAE and reduces optic neuritis at higher doses. Control and EAE mice were treated daily from days 0 to 14 with 20 to 80 mg/kg intraperitoneal methylprednisolone or an equal volume of PBS, then killed on day 14. (A) 20 mg/kg methylprednisolone treatment significantly reduced signs of EAE compared with mice treated with PBS. (B) There was no significant difference in the percentage of eyes developing optic neuritis between PBS-treated and 20 mg/kg methylprednisolone-treated EAE mice. (C) Optic neuritis eyes from PBS-treated EAE mice had significantly fewer RGCs than control eyes (*P = 0.0175), with a trend toward fewer RGCs than optic neuritis eyes from 20 mg/kg methylprednisolone-treated EAE mice that was not significant. (D) 40 mg/kg methylprednisolone treatment prevented RGC loss in optic neuritis eyes, with RGC numbers equivalent to those in control eyes. At 80 mg/kg, methylprednisolone almost completely blocked optic nerve inflammation, with just 1 of 12 eyes developing optic neuritis; that eye had normal RGC numbers.

Figure 4.

Corticosteroid treatment beginning after disease onset slows the progression of EAE. (A) Mice immunized with PLP on day 0 were treated daily with 200 mg/kg dexamethasone or an equal volume of PBS from days 10 to 14 and were observed daily through day 18 for signs of EAE. Typical early signs of EAE were observed by days 9 to 10. After treatment was initiated, EAE severity was significantly reduced in dexamethasone-treated mice compared with PBS-treated mice (P = 0.0304 for repeated measures). (B) Mice immunized with PLP on day 0 were treated daily with 20 mg/kg methylprednisolone or an equal volume of PBS from days 10 to 14 and observed daily through day 18 for signs of EAE. Typical early signs of EAE were observed by days 9 to 10. Methylprednisolone significantly suppressed the peak of EAE disease on days 12 to 14.

Figure 5.

Corticosteroid treatment beginning after disease onset does not suppress optic neuritis. (A) Dexamethasone treatment daily from days 10 to 14 led to a trend toward decreased incidence of optic neuritis at day 14 in EAE mice compared with PBS treatment. This effect was not significant (P = 0.2377). (B) No difference in optic neuritis incidence was found at day 18 between EAE mice treated daily from days 10 to 14 with dexamethasone compared with PBS. (C, D) Similarly, no difference in optic neuritis incidence was found at day 14 (C) or day 18 (D) between EAE mice treated daily from days 10 to 14 with methylprednisolone compared with PBS.

Figure 6.

RGC loss is not attenuated by dexamethasone or methylprednisolone when treatment begins after disease onset. RGCs were labeled with fluorochrome, and mice were immunized 1 week later (day 0) with PLP in CFA to induce EAE, or controls were sham immunized with PBS in CFA. Mice were treated daily from days 10 to 14 with 200 mg/kg dexamethasone, 20 mg/kg methylprednisolone, or an equal volume of PBS. Mice were killed on day 14 or day 18, and RGCs were counted by fluorescence microscopy. (A) 14 days after immunization, significant loss of RGCs was detected in eyes with optic neuritis compared with control mouse eyes in all treatment groups (*P < 0.05). (B) 18 days after immunization, significant loss of RGCs was detected in eyes with optic neuritis compared with control mouse eyes in the PBS and dexamethasone treatment groups (*P < 0.05), with a similar trend toward decreased RGC numbers in optic neuritis eyes from methylprednisolone-treated EAE mice.