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C.J. Engelman, H.A. Fishman, P. Huie, D.H. Kim, K. Singh; Effect of Clonidine and Timolol on Purified Retinal Ganglion Cells in Culture after Withdrawal of Neurotrophic Growth Factors . Invest. Ophthalmol. Vis. Sci. 2003;44(13):151.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine whether clonidine (alpha-2-agonist) and/or timolol (non-selective beta-blocker) enhance retinal ganglion cell survival in purified cultures after withdrawal of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF). Methods: Purified retinal ganglion cells were isolated from postnatal day 7 whole rat retinas and inoculated in laminin coated 24-well culture plates. Between 7-10,000 cells per well were incubated at 37 degrees centigrade in 500 micro liters of growth media and varying concentrations of clonidine and timolol (10,50,100,250,500 micro molar) for 3 days. Three wells per drug treatment group and control were included. The growth media consisted of Pen/Strep, B27, L-glutamine, BDNF, CNTF, Forskolin, Neurobasal, Insulin, Sodium Pyruvate, Sato Stock, Thyroxine and N-acetylcysteine. Following the initial incubation period, the growth media in each well was exchanged maintaining drug concentrations but removing BDNF and CNTF. Molecular Probes LIVE/DEAD Viability/Cytotoxicity Kit TM was then applied to each well 10 days after neurotrophic withdrawal to assess cell viability. Bright field and Fluorescent micrographs with appropriate excitation/emission filters for the live/dead stains were acquired at 10 standardized locations per well using 10X magnification. Manual counting of total numbers of live and dead cells was performed in a masked fashion and differences between mean percent of living cells were compared by two-tailed Student's t-tests. Results: Ten days following neurotrophic withdrawal, 24.3% of control cells were alive. Survival in wells treated with 10, 50, 100, 250 and 500 micro molar concentrations of each drug were 41.8%, 22.6%, 44.4%, 58.3% and 61% for timolol and 38.5%, 27.9%, 42.1%, 41.8% and 37.1% for clonidine, respectively. Compared to control, survival for cells cultured in 250 and 500 micro molar concentrations of both drugs was significantly enhanced (p < .001) as was the cell viability in the 100 micro molar clonidine group (p < .04). Significantly higher survival rates were present for timolol when compared to clonidine at each of the 250 and 500 micro molar concentrations (p < .01). Conclusions: Survival of purified retinal ganglion cells in culture after withdrawal of BDNF and CNTF is significantly improved by co-culture with both clonidine (100,250, 500 um) and timolol (250, 500 um) with the latter showing a slightly greater protective effect.
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