Gene Expression Profiling of the Retina after Transcorneal Electrical Stimulation in Wildtype Brown Norway Rats

ABSTRACT: Purpose: To investigate the effect of transcorneal electrical stimulation (TES) on the retina of wildtype Brown Norway (BN) rats by gene expression profiling. Methods: TES was applied to BN adult wildtype rat retina in vivo for 1h (1ms biphasic pulses at 20Hz; current: 200 M-BM-5A). RNA was isolated, processed and used for microarray-based expression profiling 4hrs after initial TES. An expression profile was generated for genes differentially expressed at 4hrs after TES vs. sham stimulated animals using a fold change cutoff of 1.2. We validated the profile by real-time quantitative reverse transcription-polymerase chain reaction (qPCR). In addition, the application of TES was verified at the structural and functional level. Results: Transcriptome changes associated with TES vs. sham stimulated BN wildtype retina were identified. 490 genes were differentially expressed in TES and included well-known genes as well as a large number of novel genes. Electrophysiological recordings showed physiological retinal function after TES and structural in vivo and ex vivo studies revealed intact retinal layers. Conclusion: Our results demonstrate that TES applied to the retina of wildtype BN rats induce a variety of transcriptome level changes and may help to understand the mechanisms underlying TES. In addition TES has no negative effect on structure and function of wildtype BN retina 24hrs after application. RNA isolation and Microarray studies 4 animals were euthanized 4h after TES or respective sham treatment. Extraction of total RNA was performed using the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. QIAshredder mini-spin columns (Qiagen) as well as needle and syringe homogenization were applied for tissue homogenization. To ensure proper integrity all RNA used in our experiments showed single peaks for the 18S and 28S bands as determined by the Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip Kit (both Agilent Technologies Inc., CA, USA) following the manufacturer's instructions. The purity and concentration of total RNA was determined by absorbance measurements at 260 and 280nm using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). All RNA that did not have a 260/280 ratio between 1.9 and 2.1 was discarded to ensure high quality of samples. Respective RNAs from stimulated and sham samples were used to generate double-stranded cDNA with the T7-oligo (dT) primer according to GeneChipM-BM-. (Affymetrix Inc., Santa Clara, CA, USA) two cycle target labeling protocol as described by the manufacturer. The processed cDNA was hybridized to Affymetrix GeneChipM-BM-. Rat Gene 1.0 ST arrays for 18M-bM-^@M-^S24h (Affymetrix Inc.). Four TES and four sham replicates were used for hybridization to Microarray GeneChipsM-BM-.. Each microarray was washed and stained with streptavidinM-bM-^@M-^Sphycoerythrin and scanned at a 6M-NM-

ORGANISM(S): Rattus norvegicus

SUBMITTER: Gabriel Willmann 

PROVIDER: E-GEOD-26174 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress