Optic nerve crush induces spatial and temporal gene expression patterns in optic nerve of BALB/cJ mice. Mus musculus

Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of cellular and molecular events resulting in neuronal apoptosis and regenerative failure. The pathogenic mechanisms and gene expression changes associated with these detrimental events can be effectively studied using a rodent optic nerve crush (ONC) model. The purpose of this study was to use a mouse ONC model to: (a) evaluate changes in optic nerve (ON) gene expression, (b) identify neurodegenerative pathogenic pathways and (c) discover potential new therapeutic targets. Affymetrix Mouse Gene 1.0 ST arrays were utlized to detail the global gene expression profile following optic nerve crush (ONC) in the ON of BALB/cJ mice at six different days post crush (dpc) (naive, 3 dpc, 7 dpc, 14 dpc, 21 dpc and 28 dpc) to understand the pathogenic responses in relation to neuronal loss and regenerative failure. Overall design: BALB/cJ mice aged 2-4 months were utilized for all the experiments and were obtained from the Jackson Laboratories (Bar Harbor, ME). The mice were housed and maintained in a 12-hour light/dark cycle and fed ad libitum. All procedures were performed in accordance with Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research and the University of North Texas Health Science Center (UNTHSC) Institutional Animal Care and Use Committee regulations. The ON of the left eye was crushed 0.5mm posterior from the globe for 4 seconds using the Nickell's technique. Briefly, mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and then an incision was made along the superior orbital margin. The left ON was exposed and crushed using a self-closing jeweler’s forceps to ensure reproducible and constant force. For the microarray studies, five mice were used per time-point and tissue harvested at six different times (naive, 3 dpc, 7 dpc, 14 dpc, 21 dpc and 28 dpc). After the ON tissues were harvested, RNA was extracted and samples were all pooled for each experimental (left ON) and control (right ON) eye group to a total of 500ng of RNA/pool. Microarray hybridizations were performed at the University of Iowa DNA Core Facility. Total RNA (50ng) was converted to SPIA (Single Primer Isothermal Amplification) amplified cDNA using the WT-Ovation Pico RNA Amplification System. Biotin-labeled cDNA was placed onto Affymetrix Mouse Gene 1.0 ST arrays (Part No. 901168), and incubated at 45º C for 18 h and signals detected according to maufacturer's protocol. Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade, and data were collected using the using the GeneChip operating software (GCOS) v1.4. Microarray data were imported into the Partek Genomics Suite 6.6 software (Partek Inc., Louis, MO) and normalized based on the robust multi-array average (RMA). The ONC samples were contrasted against the control samples to calculate the microarray ratios and log2 fold values.