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Adam Morgan Thompson, Martin Berry, Ann Logan, Zubair Ahmed; Bone morphogenetic protein-4 promotes retinal ganglion cell neuroprotection and axon regeneration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4967. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
It is established that bone morphogenetic protein-4 (BMP-4) and its downstream signalling pathways promote the survival of retinal ganglion cells (RGC) and here we test the hypothesis that BMP-4 also stimulates the regeneration of RGC axons after optic nerve (ON) crush injuries.
Recombinant BMP-4 was added to dissociated retinal cells including RGC, cultured from adult Sprague-Dawley rats. After 3d in culture, cells were fixed in 4% paraformaldehyde and RGC immunostained with βIII-tubulin and the number of surviving RGC, mean RGC neurite length and number of RGC with neurites quantified. The ON was also crushed in adult Sprague-Dawley rats, 2mm from the lamina cribrosa, and a graded concentration of recombinant BMP-4 intravitreally injected at 0d, 8d and 16d. Animals were killed at 24d, intracardially perfused with 4% paraformaldehyde, the eyes embedded in OCT and 15μm thick frozen radial sections prepared. Sections were immunostained with Brn3a and the number of surviving Brn3a RGC counted across 4 standard sections at the optic disc. The numbers of GAP-43+ axons regenerating in the distal ON were quantified in 4 longitudinal sections/ON. ANOVA was used to compare statistical differences with post hoc testing by Dunnetts Method.
There was a dose-dependent increase in the survival and neurite outgrowth of cultured RGC and at 200ng/ml an optimum concentration of BMP-4 promoted the survival of 70% RGC (30% > observed in control cultures) whilst 26% of surviving RGC grew neurites (compared to < 1% in control cultures) and neurite lengths were 230±10μm compared to neurite lengths of 35±2μm in control cultures. Increasing the concentration of BMP-4 above the optimum concentration significantly reduced the proportion of RGC surviving, the proportion and those with neurites and the mean neurite length (P<0.001, ANOVA). After optic nerve crush in adult Sprague-Dawley rats, intravitreal recombinant BMP-4 injection of 5μg/eye/injection of promoted significant RGC survival and RGC axon regeneration.
Our results are consistent with the hypothesis that recombinant BMP-4 promotes RGC survival and axon regeneration. Thus, intravitreal BMP-4 injections may constitute a potential treatment for the promotion of RGC survival and axon regeneration, thus restoring/protecting vision loss after optic nerve trauma and disease.
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