Abstract
Purpose: :
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).
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: nutritional factors • Muller cells • neuroprotection