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R. Seitz, S. Hackl, E. R. Tamm, A. Ohlmann; Norrin Mediates Neuroprotective Effects on Retinal Ganglion Cells via Induction of Neurotrophic Factors in Müller Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3224.
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© ARVO (1962-2015); The Authors (2016-present)
Ndp(y/-) mutant mice that are deficient in the Norrie disease gene (Ndp) show a continuous loss of retinal ganglion cells (RGC), a phenotype that is completely rescued in mixed (beta)B1-norrin/Ndp(y/-) mice with transgenic overexpression of norrin, the secreted protein product of Ndp (Ohlmann et al., J. Neurosci. 2005). To analyze, if norrin has neuroprotective properties, we investigated its effects on RGC survival in mouse eyes following N-methyl-D-aspartate (NMDA) induced RGC damage.
Recombinant human norrin was isolated and purified from conditioned cell culture medium of HEK 293-EBNA cells. To induce RGC death, 3 µl NMDA [10 mM] were injected into the vitreous body of C57/Black6 mice while the fellow eye received 3 µl of a combination of NMDA [10 mM] and norrin [5 ng/µl]. To determine the degree of RGC damage, the number of axons in cross sections of the optic nerve and of RGC in meridional semithin sections of each eye was quantified. In addition, TUNEL labelling was performed on meridional sections, and the number of labelled nuclei was quantified. The expression of mRNA for several neurotrophic factors was investigated by quantitative real-time RT-PCR of treated retinae and of cultured retinal Müller cells after a 7 h incubation with norrin [40 ng/ml].
Three weeks after injection of NMDA, the number of optic nerve axons was almost two-fold higher in eyes injected with combined NMDA/norrin (20,383 ± 3,101; mean ± SEM) as compared to eyes that received NMDA only (11,150 ± 1,013), an effect that was statistically significant (p<0.05). Similarly, perikarya of surviving RGC were significantly more numerous in norrin/NMDA injected eyes (3.6 ± 0.5 RGC per 100 µm) when compared to eyes injected with NMDA only (2.3 ± 0.2 RGC per 100 µm; p<0.025). Comparable results were obtained by TUNEL labelling. By real-time RT-PCR, an increase in mRNA for bFGF (4.4 ± 0.6 -fold; p<0.05) and CNTF (1.4 ± 0.2 -fold; p<0.05) was observed in eyes injected with combined NMDA/norrin as compared to retinae that received NMDA only. The results correlated with those obtained in cultured Müller cells which showed a significant increase in mRNA for bFGF (2.7 ± 0.4 -fold; p<0.05) and CNTF (3.5 ± 0.5 -fold; p<0.05) after incubation with norrin.
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