Gene Expression Microarray Analysis Of Spinal Nucleus Of Trigeminal Nerve In Migraine With Aura Rats

Objective:1. To observe the gene expression profiles in the spinal nucleus of trigeminal nerve of the rats after cortical spreading depression (CSD) which may induce migraine with aura, and to find the differential expressed genes.2. To approach the possible molecular mechanism of induction of migraine with aura by CSD.Methods:1. Establishment of migraine with aura model: 6 male Sprague-Dawley rats, weighing 220～250g, were randomly divided into two groups: the control (n=3) and operative group (n=3). After exposure of the skull, two burr holes of 1.8mm diameter were drilled over the left hemisphere as follows: 5mm lateral, 6mm posterior to bregma; and 2mm lateral, 3mm anterior to bregma. Great care was taken to keep the dura intact. A glass recording electrode filled with 0.5M sodium acetate solution was placed into the cortex 1mm at the anterior position and connected to a high impedance amplifier, and a microinjector was placed into the cortex 1mm at the posterior postion. CSD was induced in the rat brain by application of 1M KCL in operative group, and 1M NaCL in control group. Then the rats were sacrificied after six injections at 10-minute intervals. Powerlab system was used to record the electrical activity in succession.2. The gene expression profiles in the spinal nucleus of trigeminal nerve of the migraine with aura rats: We studied the gene expression profiles using Affymetrix Rat Genome 230 2.0 Oligo microarrays. Rats were decapitated and the bulb and high cervical cord (to C2) were removed from the skull. We extracted RNA samples using Trizol, determining the quantity and quality of RNA applying optical density and denaturing gel electrophoresis. We synthetized and purified cDNA, then cRNA was synthetized in vitro transcription. The biotin labeled cRNA was purified, quantified, precipitated and fragmented. After hybridization of the cRNA to the Affymetrix Rat Genome 230 2.0 gene chip, the chip was washed, stained and scanned on an GeneChip Scanner 3000. GeneChip operating software (GCOS) was used to analysis the presentation. Then SAM software was used to find the differential expressed genes between each group. We performed following functional classification for differential genes by Molecule Annotation System.3. Functional analysis of differential expressed genes in spinal nucleus of trigeminal nerve of the migraine with aura rats: 4 gene expression validation were executed by Real-Time quantitative polymerase chain reaction (PCR). Functional analysis of differential expressed genes were performed by bioinformatics analysis. Mechanism of CSD inducing migraine with aura was approached.Results:1. The model of migraine with aura was established through CSD induced by microinjection of KCL in cortex. In operative group the potentials were suppressed and inverted transparently.2. Using Affymetrix Rat Genome array 230 2.0, we studied the differential rat gene expression. According to the "Present" by detection p-value < 0.04, there is 20025 transcripts detected among the 31099 genomic probe sets in control group, in proportion to 64.39% for total transcripts; meanwhile 20718 transcripts detected in operative group and have a proportion of 66.62%. Then we screened the differential expressed genes between two groups using GCOS, SAM and MAS system. Compared to control group, there is 2 down-regulated genes and 7 up-regulated genes in operative group, which are involved in cell migration and movement, ubiquitin degradation, biochemistry enzymes and transcription factor. It is very important that we find the genes related to cell migration and movement are enriched.3. Of the 4 genes analysed by RT-PCR (Myh1,My11,Mylpf and Acta1), the direction and degree of change matched perfectly to the microarray data. They all were upregulated.Conclusions:1. The model of migraine with aura was established through CSD induced by microinjection of KCL in cortex.2. Cortical spreading depression caused some genes expression changes of spinal nucleus of trigeminal nerve, among which were several related with cell migration and movement, ubiquitin degradation, biochemistry enzymes and transcription factor, and cell migration and movement genes expression of spinal nucleus of trigeminal nerve may contribute to headache during attack of migraine with aura.3. RT-PCR confirmed the microarray results for several of the transcripts. Function analysis of differential expressed genes was carried out.