Intracranial Inoculation Is More Potent Than Intranasal Inoculation for Inducing Optic Neuritis in the Mouse Hepatitis Virus-Induced Model of Multiple Sclerosis

Singh, Manmeet; Khan, Reas S.; Dine, Kimberly; Das Sarma, Jayasri; Shindler, Kenneth S.

Proteins rapidly accumulate at high concentrations in optic nerve and in the eye following intranasal administration (Khan et al., 2017) . Thus, the potential for MHV viruses to similarly spread FIGURE 2 | Comparative optic nerve demyelination study between intracranially-and intranasally-infected mice. Representative optic nerve sections from chronic stage (day 26 p.i.) mock-infected (n = 6) (A), RSA59 intrancranially-infected (n = 6) (B), RSA59 intranasally-infected (n = 5) (C), and RSMHV2 intranasally-infected (n = 5) (D) mice stained with LFB show demyelination only in RSA59 intracranially-infected mice (scale bar = 100 µm). The relative level of demyelination scored by a blinded investigator showed significant demyelination in optic nerves of mice inoculated intracranially with RSA59, but not in mock-infected (control) mice nor in mice infected intranasally with either RSA59 or RSMHV2 (*p < 0.05 vs. all other groups) (E). Data comparisons were done by one-way ANOVA and Tukey's Multiple Comparison post-hoc testing with GraphPad Prism 6.0 software. to optic nerve and retina was assessed 1 day following intranasal inoculation. Four-week-old, MHV-free, C57BL/6J mice were inoculated intranasally with 50% of the LD50 dose of RSA59 or RSMHV2, and mice were euthanized 1 day later. Retinas and optic nerves were isolated, sectioned, and immunostained with anti-viral nucleocapsid antisera to detect viral spread. No significant staining was observed in any of the retinas or optic nerves from mock-infected (n = 6), RSA59-infected (n = 6), or RSMHV2-infected (n = 6) mice (Figure 4) . As shown in prior studies (Shindler et al., 2011) , viral antigen does not reach the retina within 1 day following intracranial inoculation with RSA59 (data not shown). Viral antigen is found in the retina 6 days after intrancranial inoculation (Figure 4) , while no antigen is detectable in retina following intranasal inoculation at day 1 (Figure 4 ) or any later time points (data not shown). To further confirm that intranasal RSA59 administration fails to induce optic neuritis, acutely, optic nerve sections were immunostained for the microglial/macrophage marker Iba1. Previously, it has been observed that intracranial inoculation with RSA59 induces acute optic nerve inflammation containing almost entirely activated microglia/macrophages (Shindler et al., 2011) 3-6 days post-inoculation. To study whether intranasal FIGURE 3 | RSA59 infection induces RGC loss. Representative photos illustrate the decreased RGC numbers in eyes of mice inoculated intracranially with RSA59 (B) compared to mock-infected control mice (A). Mice inoculated intranasally with RSA59 (C) or RSMHV2 did not show RGC loss (D) (scale bar = 50 µm). The total number of labeled RGCs present in 12 standardized retinal fields was counted. The average number of surviving RGCs/eye (n = 6/group) shows intracranial RSA59 induced a significant decrease in RGC numbers compared to control mice (**p < 0.01). Neither RSA59 nor RSMHV2 induced RGC loss compared to control mice when administered intranasally. RGC numbers in RSMHV2-infected mice were significantly higher than in mice intracranially inoculated with RSA59 (*p < 0.05) (E). Data comparisons were done by one-way ANOVA and Tukey's Multiple Comparison post-hoc testing with GraphPad Prism 6.0 software. inoculation rapidly induces similar optic nerve inflammation, optic nerve sections were stained with anti-Iba1 antibody. Sections from mock-infected mice wer