Fig 1.
ST266 increases Schwan cell proliferation following 24-hours starvation conditions.
Media of Schwan cells cultured in normal growth media (NGM) was replaced by minimal starvation medium for 24-hours and subsequently replaced by either NGM, IMDM, STM100 (base medium), ST266 or filtrate of ST266 below 50- or 30- kDa cutoff. (a) After 24-hours treatment, cell proliferation was determined by extracellular reduction of WST-8, with results shown as a ratio of sample absorbance to the NGM value for each plate. (b) Representative light micrograph images of cultured murine Schwann cells following starvation and a 24-hours treatment demonstrate relative presence of healthy, elongated Schwann cells at 20X (scale bars = 200 μm) and 40X (scale bars = 100 μm) magnification. Images illustrating the development of color from CCK-8 assay in Schwann cell cultures. Corresponding cell viability is listed below each representative set of images. Data shown as mean + SEM from three separate experiments with total of n = 12 per group (**p < 0.0001 and *p < 0.05).
Fig 2.
Mild EAE induced by MOG peptide immunization.
(a) Mice immunized with MOG peptide to induce EAE were treated daily with intranasal ST266 (n = 8 mice), <50 kDa ST266 (n = 8) or PBS (vehicle) (n = 8) from days 15–42 post-immunization, and control, non-EAE mice (n = 6) were treated with PBS. Mice were scored daily based on clinical signs of ascending paralysis. Mild EAE disease developed in all immunized groups with no difference in EAE scores between treatment groups (one-way repeated measures ANOVA p>0.05). (b) To estimate visual function, OKR testing was performed. Measurements were taken at baseline and then weekly for 6 weeks. In this cohort of mice with mild EAE disease, no significant vision loss developed compared with control mice, and no difference was detected in mean scores between treatment groups over time (one-way repeated measures ANOVA p>0.05) or at the final measurement on day 42 (one way ANOVA p >0.05). Data shown as mean +SEM.
Fig 3.
RGC neuroprotection by ST266 in mild EAE optic neuritis.
(a) Representative photos of Brn3a immunolabeled RGCs in flat mounted retinas isolated 42 days post-immunization from the same cohorts of mice shown in Fig 2. Lower magnification images (top; original magnification X 10) demonstrate the density of RGCs. White boxes indicate areas shown at higher magnification (bottom) demonstrating the ability to identify individual Brn3a positive cells. (b) Total number of RGCs across 12 standardized fields for each eye show a trend toward loss of RGCs in eyes from PBS-treated EAE mice (n = 16 eyes) compared with eyes from control mice (n = 12 eyes) (student t-test †p = 0.155). Eyes from EAE mice treated with ST266 (n = 16) showed greater RGC counts than vehicle-treated EAE animals (ANOVA *p<0.05), whereas the trend toward increased RGC numbers in eyes from <50 kDa ST266-treated EAE mice (n = 16) compared with PBS-treated EAE mice was not significant. (c) Diagram shows the eccentricity of standardized photos of RGCs, including a central (c), mid-peripheral (m) and peripheral (p) photo which were obtained in each retinal quadrant. (d) Average RGC numbers present in each retinal region show that eyes from mice treated with ST266 have greater numbers of surviving RGCs than eyes from PBS-treated EAE mice (one-way ANOVA *p<0.05) in all three retinal regions. The mid-peripheral region showed the greatest difference in RGC numbers between eyes from control mice and eyes from PBS-treated EAE mice, although this was not significant by ANOVA but was significant in direct comparison between these two groups (student t-test †p = 0.05). Data shown as mean +SEM.
Fig 4.
Optic nerve demyelination is significantly reduced by both ST266 and <50 kDa ST266 in mild EAE optic neuritis.
(a) Representative photos of optic nerve sections are shown from the same cohorts of mice shown in Fig 2, prepared following sacrifice on day 42 post-immunization. H&E staining shows the relative cellularity within the optic nerves, and LFB staining shows the relative level of myelination. Scale bar = 50 microns in lower magnification images (top two rows) and scale bar = 5 microns in higher magnification images (bottom two rows). (b) Masked scoring demonstrates increased inflammatory cell infiltration present in nerves from PBS-treated EAE mice (n = 16 optic nerves) as compared with nerves from control, non-EAE mice (n = 12) (*p<0.05). Daily intranasal ST266 (n = 16) or <50 kDa ST266 (n = 16) treatment both show a strong trend towards reducing optic nerve inflammation that is not statistically significant as compared with PBS-treated EAE mice. Masked scoring of LFB staining demonstrates increased levels of myelin loss present in nerves from PBS-treated EAE mice (n = 16 optic nerves) as compared with nerves from control, non-EAE mice (n = 12) (*p<0.05). Daily intranasal ST266 (n = 16) or <50 kDa ST266 (n = 16) treatment both show significant reduction in optic nerve demyelination as compared with PBS-treated EAE mice (*p<0.05). Comparisons analyzed by one-way ANOVA with Tukey post-hoc testing. Data shown as mean +SEM.
Fig 5.
ST266 preserves visual responses in mice with mild-moderate EAE optic neuritis.
Mice immunized with MOG peptide to induce EAE were treated daily with intranasal ST266 (n = 24 mice), <50 kDa ST266 (n = 24) or PBS (n = 24) from days 15–42 post-immunization, and control, non-EAE mice (n = 22) were treated with PBS across three identical experiments. Eyes from mice that developed at least minimal EAE paralysis (cumulative EAE score greater than 0 over 42 days post-immunization) but without severe disease (cumulative EAE score not more than 60) were included for further analysis of visual function. OKR scores measured weekly across 42 days showed a non-significant trend towards improved responses in eyes from EAE mice treated with ST266 as compared with eyes from PBS-treated EAE mice when compared by ANOVA of repeated measures. At day 42 post-immunization, a significant decrease in OKR responses in eyes from PBS-treated EAE mice (n = 23 eyes) was observed as compared to eyes from control, non-EAE mice (n = 44) (*p<0.05), and this decrease was significantly improved in eyes from ST266-treated EAE mice (n = 28) (*p>0.05), while a trend towards improvement in eyes of <50 kDa ST266-treated mice (n = 24) did not reach statistical significance. Data shown as mean +SEM.
Fig 6.
ST266 attenuates RGC loss in mice with mild-moderate EAE optic neuritis.
Retinas were isolated from the eyes of control mice and EAE mice with mild-moderate EAE disease and immunolabeled with Brn3a. Data are from the same eyes shown in Fig 5, with the exception of one eye from the vehicle (PBS)-treated EAE mouse cohort in which the retina was damaged during dissection and therefore could not be quantified. (a) The average normalized RGC count across the entire retina shows a decrease in RGC numbers in vehicle-treated EAE mouse eyes (n = 22 eyes) compared to eyes from non-EAE control mice (n = 44 eyes) (*p<0.05). RGC numbers were significantly higher in eyes from ST266-treated EAE mice (n = 28) as compared with PBS-treated EAE mice (*p<0.05), whereas treatment with <50 kDa ST266 (n = 24 eyes) led to a non-significant trend towards increased RGCs. (b) RGC numbers in central, mid-peripheral and peripheral regions of the retina showed a significant decrease in all regions in eyes of PBS-treated EAE mice as compared with control mouse eyes, and treatment with daily intranasal ST266 improved RGC survival in both the mid-peripheral and peripheral retinal regions (*p<0.05). Data shown as mean +SEM.