Purchase this article with an account.
Yuko Shinohara, Masayoshi Nakatani, Nobuharu Asai, Kan Ohtsuki; Brain-Derived Neurotrophic Factor-Secreting Müller Cells Increase Survival and Neurite Outgrowth of Rat Retinal Ganglion Cells in Vitro. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2000.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We examined the efficacy of brain-derived neurotrophic factor (BDNF)-secreting Müller cells in supporting the survival and neurite outgrowth of rat retinal ganglion cells (RGCs).
A Müller cell line (BD-TM4) stably expressing BDNF was established by transfecting a BDNF gene into an immortalized rat Müller cell line (TR-MUL). RGCs were isolated from 6-day-old rats and provided for co-culture on a porous membrane insert, avoiding direct contact with BD-TM4 or TR-MUL. After culture for 2 days in Neurobasal medium under conditions of neurotrophic factor deficiency, the cells were exposed to 200 μM glutamate for 24 h or left unexposed. RGC survival was evaluated by counting calcein AM-stained RGCs. Retinal explants from adult rats 7 days after optic nerve crush were cultured in BD-TM4- or TR-MUL-conditioned medium (CM) for 6 days. As a positive control, recombinant human BDNF (rhBDNF) was added to the explant cultures. Numbers of outgrowing neurites and surviving RGCs were counted by using immunohistochemical techniques.
The amount of BDNF released from BD-TM4 cells was approximately 1.0 ng/1x105 cells/day. Under conditions of neurotrophic factor deficiency without glutamate exposure, BD-TM4 cells enhanced RGC survival (to 300% of that in the control culture [i.e. culture of RGC cells alone]) significantly more than did TR-MUL cells (to 150% of that in the control culture). Similarly, under glutamate toxicity, more RGCs survived in the presence of BD-TM4 cells than in the presence of TR-MUL cells. Methionine sulfoximine, a glutamine synthetase inhibitor, significantly inhibited the protective effect of BD-TM4 (P < 0.05) or TR-MUL cells (P < 0.01). TR-MUL-CM had little effect on neurite outgrowth in retinal explants, whereas BD-TM4-CM significantly increased (by 320%) the number of neurite outgrowths compared with those in unconditioned Neurobasal medium. Similarly, rhBDNF-positive control medium significantly increased (by 380%) the number of neurite outgrowths compared with those in unconditioned medium. After optic nerve crushing, more surviving RGCs were observed in retinal explants cultured in BD-TM4-CM than in unconditioned medium.
Factors released from the BD-TM4 cells not only enhanced RGC survival but also stimulated neurite outgrowth, suggesting that BDNF secretion enhances the rescue effect of RGCs by the original cell line.
This PDF is available to Subscribers Only