Purchase this article with an account.
S Azadi, AR Caffe, Y Zhang, I Holmqvist, T van Veen; Cntf+bdnf Treatment Of Rd Mouse Retinal Explants Increases Ngf And Gdnf Gene Expression . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1441.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: CNTF+BDNF treatment rescues retinal photoreceptors in postnatal day 2 (PN2) rd/rd mouse retinal explants (RE) cultured till PN28. This treatment activates intracellular signaling pathways that change activity of transcription factors which, in turn, affects gene expression. Prime candidates for a role in photoreceptor rescue are proteins of the neurotrophic family. We started to identify changes in expression of the genes encoding for these proteins in response to CNTF+BNDF application. Methods: C3H rd/rd and congenic wild type (WT) mice were used to generate RE and in vivo retinal samples; PN2+div9 (9 days in vitro), PN11, and PN2+div9 rd/rd RE exposed either for the first two days or continuously to CNTF+BDNF (10ng/ml). Semi-quantitative RT-PCR was performed. Six retinas or RE per condition were pooled and total RNA extracted by the guanidium thyocianate procedure. RNA amounts were determined using spectrophotometry and gel electrophoresis (GE). Densitometry verified equal amounts of sample RNA. Using random primers first strand cDNA was synthesized from same amounts of sample RNA. The calibration (standard) curve for each specific primer pair was determined from pooled cDNA. Amplification was carried out using specific primer sets for NGF and GDNF. The PCR products were separated by GE, visualized by ethidium bromide, quantified by densitometry, and normalized against internal control (18S rRNA). Data was analyzed statistically. Results: In both WT and rd/rd PN11 retinas low basal levels of NGF and GDNF gene expression was observed. Culturing in control medium significantly increased both NGF and GDNF transcript levels in WT but not in the rd/rd RE. Exposure of rd/rd RE to 10ng/ml CNTF+BDNF for the first two days significantly increased NGF and GDNF transcript levels. Continuous treatment of rd/rd RE for 9 days also resulted in significantly elevated NGF and GDNF gene expression. Conclusion: The results show that PN2 WT RE upregulate NGF and GDNF gene expression (injury response) during 9 days culturing whereas the rd/rd RE is not able to perform this response. Transient and continuous treatments with CNTF+BDNF generate this injury response in rd/rd RE. One week after interruption of the treatment NGF and GDNF gene expression in rd/rd RE is still higher than in control tissue. These observations implicate NGF and GDNF gene expression in the molecular mechanisms underlying photoreceptor rescue. Gene expression of FGF2, BDNF, CNTF and caspases 3 and 9 is also being examined.
This PDF is available to Subscribers Only