Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies

Abstract

T-cell autoimmunity to myelin basic protein was recently shown to be neuroprotective in injured rat optic nerves. In the present study, using the mouse optic nerve, we examined whether active immunization rather than passive transfer of T-cells can be beneficial in protecting retinal ganglion cells (RGCs) from post-traumatic death. Before severe crush injury of the optic nerve, SJL/J and C3H.SW mice were actively immunized with encephalitogenic or nonencephalitogenic peptides of proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG), respectively. At different times after the injury, the numbers of surviving RGCs in both strains immunized with the nonencephalitogenic peptides pPLP 190-209 or pMOG 1-22 were significantly higher than in injured controls treated with the non-self-antigen ovalbumin or with a peptide derived from beta-amyloid, a non-myelin-associated protein. Immunization with the encephalitogenic myelin peptide pPLP 139-151 was beneficial only when the disease it induced, experimental autoimmune encephalomyelitis, was mild. The results of this study show that survival of RGCs after axonal injury can be enhanced by vaccination with an appropriate self-antigen. Furthermore, the use of nonencephalitogenic myelin peptides for immunization apparently allows neuroprotection without incurring the risk of an autoimmune disease. Application of these findings might lead to a promising new approach for treating optic neuropathies such as glaucoma.

Fig. 1.

Clinical course of EAE, induced by either pMOG 35–55 in C3H.SW mice (n = 4) (a) or pPLP 139–151 in SJL/J mice (n = 5) (b). For induction of disease, mice were injected with pMOG 35–55 or pPLP 190–209 in CFA supplemented with M. tuberculosis, as described in Materials and Methods. EAE was evaluated according to a neurological paralysis scale. The mean ± SEM daily clinical score is shown for each mouse strain. Note that, in the C3H.SW mice, the induced EAE had a chronic course, whereas in the SJL/J mice, it was relapsing–remitting in nature.

Fig. 2.

Negative correlation between the number of surviving RGCs of injured nerves from mice immunized with PLP 139–151 and EAE disease severity. Each point indicates the mean number of labeled RGCs per square millimeter 2 weeks after optic nerve injury. Two weeks before injury, mice were injected with pPLP 139–151 or OVA. The neurotracer dye FluoroGold was applied stereotactically 3 d before injury. Two weeks after the injury, the retinas were excised and flat-mounted, and the EAE score was determined. Labeled RGCs from three to five randomly selected fields in each retina (all located 1 mm from the optic disk) were counted by fluorescence microscopy. In mice immunized with PLP 139–151, in which no protection was observed, the average number of RGCs (563 RGCs/mm²; n = 3) was similar to that found in OVA-immunized mice (613 RGCs/mm²;n = 4). The correlation coefficient (r) was −0.96 according to linear regression; the p value was 0.009 according to one-way ANOVA.

Fig. 3.

Immunization with pPLP 190–209 slows down death of RGCs. The histograms record the mean number of labeled RGCs 2 weeks after optic nerve injury. Two weeks before injury, SJL/J mice were injected with pPLP 190–209, OVA, or βAP. The neurotracer dye FluoroGold was applied stereotactically 3 d before injury. Two weeks after the injury, the retinas were excised and flat-mounted. Labeled RGCs from three to five randomly selected fields in each retina (all located 1 mm from the optic disk) were counted by fluorescence microscopy. Survival in each group of injured nerves was expressed as the mean ± SEM number of labeled RGCs per square millimeter. The neuroprotective effect of pPLP 190–209 was significant compared with that of OVA (p < 0.001; one-way ANOVA). βAP did not differ significantly from OVA in its protective effect on neurons that had escaped the primary injury (p > 0.05; one-way ANOVA). The results are a summary of three experiments.

Fig. 4.

Immunization with pMOG 1–22 slows down RGC degeneration. The histograms record the mean ± SEM number of labeled RGCs per square millimeter. a, The number of labeled RGCs assessed 2 weeks after injury in retinas of mice before or after injury immunized with pMOG 1–22. For preinjury immunization, C3H.SW mice were injected with pMOG 1–22 or OVA, 14 d before optic nerve injury. Dye application, preparation, and counting of RGCs, as well as calculation of RGC survival, were as described for Figure 3. The number of labeled RGCs in the retinas of mice pretreated with pMOG 1–22 was significantly higher (p< 0.05; Student's t test) than in mice pretreated with OVA. The results are a summary of three experiments. For postinjury immunization, FluoroGold was applied stereotactically 3 d before optic nerve injury, and the mice were injected with either pMOG 1–22 or OVA immediately after the injury. Two weeks later, the retinas were prepared and counted as before. The number of labeled RGCs in the retinas of mice inoculated with pMOG 1–22 after the injury was significantly higher (p < 0.02; Student'st test) than in the mice inoculated with OVA before injury. The results shown are from one experiment only. Each group contained four to five mice. b, The number of labeled RGCs in preinjury immunized mice assessed 1 and 4 weeks after the injury. The histograms record the mean ± SD number of labeled RGCs per square millimeter. The RGC number in pMOG 1–22-immunized mice was significantly higher than that of PBS control mice (p < 0.0001; Student's ttest) either 1 or 4 weeks after the injury. The insetshows the ratio between the number of surviving RGCs in the retinas of MOG-immunized mice and the control mice, at each of the time points tested. The results shown are from one experiment only. Each group contained five to nine mice.

Fig. 5.

Confocal microscopy pictures showing T-cell accumulation at the optic nerve injury site. One week after the injury, the optic nerves of C3H.SW mice immunized with pMOG 1–22 before injury were excised and labeled immunocytochemically. Serial optic nerve sections immunolabeled for GFAP (a) delineate the site of injury (indicated by the arrows). Immunolabeling for CD3 (b) indicates the presence of T-cells at the injury site.