Regulation of gene expression: Transcriptional profiling in small and large DRG neurons from a neuropathic pain model using LCM

Abstract

Dorsal root ganglion (DRG) neurons play an important role in detecting, signaling and transmitting normal and noxious stimuli from the periphery to the central nervous system. In particular, small diameter neurons mediate nociceptive responses whereas large neurons facilitate mechanosensation. Understanding the molecular composition of both neuronal subpopulations could provide insights into how pain pathways function and are altered in neuropathic pain states. Traditional techniques (e.g. immunohistochemistry) to evaluate these neurons are relatively slow and only allow a limited number of genes to be examined. In the present study laser capture microdissection (LCM), together with Affymetrix GeneChip® arrays, has permitted the profiling of a large number of genes efficiently from both cell populations. To identify neurons, frozen tissue sections (10 μm) from rat spinal nerve ligated (SNL) and sham L5/L6 DRG were Nissl-stained. 500 small (<25 μm) and 500 large (>40 μm) diameter cells were captured using the PixCell II instrument. Total RNA was isolated from each sample using PicoPure reagents (Arcturus). First round aRNA was amplified with RiboAmp (Arcturus) and the second amplification labeled with biotin (Enzo). The aRNA was hybridized to the rat U34 A GeneChip® array, and the data analyzed using Rosetta Resolver®. Microarray analysis has shown altered expression patterns between SNL and sham animals especially in large DRG neurons. Although some genes showed a common expression signature across both small and large neurons, a number of genes were dysregulated in only one neuronal subtype; examples include NPY5 receptor, Kir 6.2 and Nell I in small neurons and P2X5, PKCϵ and Nav1.1 in large neurons. These findings demonstrate that LCM in conjunction with microarray analysis provides a valuable approach to profiling gene expression and will enable further understanding of the genomic landscape of these neuronal subpopulations in neuropathic pain states. Dorsal root ganglion (DRG) neurons play an important role in detecting, signaling and transmitting normal and noxious stimuli from the periphery to the central nervous system. In particular, small diameter neurons mediate nociceptive responses whereas large neurons facilitate mechanosensation. Understanding the molecular composition of both neuronal subpopulations could provide insights into how pain pathways function and are altered in neuropathic pain states. Traditional techniques (e.g. immunohistochemistry) to evaluate these neurons are relatively slow and only allow a limited number of genes to be examined. In the present study laser capture microdissection (LCM), together with Affymetrix GeneChip® arrays, has permitted the profiling of a large number of genes efficiently from both cell populations. To identify neurons, frozen tissue sections (10 μm) from rat spinal nerve ligated (SNL) and sham L5/L6 DRG were Nissl-stained. 500 small (<25 μm) and 500 large (>40 μm) diameter cells were captured using the PixCell II instrument. Total RNA was isolated from each sample using PicoPure reagents (Arcturus). First round aRNA was amplified with RiboAmp (Arcturus) and the second amplification labeled with biotin (Enzo). The aRNA was hybridized to the rat U34 A GeneChip® array, and the data analyzed using Rosetta Resolver®. Microarray analysis has shown altered expression patterns between SNL and sham animals especially in large DRG neurons. Although some genes showed a common expression signature across both small and large neurons, a number of genes were dysregulated in only one neuronal subtype; examples include NPY5 receptor, Kir 6.2 and Nell I in small neurons and P2X5, PKCϵ and Nav1.1 in large neurons. These findings demonstrate that LCM in conjunction with microarray analysis provides a valuable approach to profiling gene expression and will enable further understanding of the genomic landscape of these neuronal subpopulations in neuropathic pain states.