Peripheral nerve injury is accompanied by chronic transcriptome-wide changes in the mouse prefrontal cortex

Abstract

Background: Peripheral nerve injury can have long-term consequences including pain-related manifestations, such as hypersensitivity to cutaneous stimuli, as well as affective and cognitive disturbances, suggesting the involvement of supraspinal mechanisms. Changes in brain structure and cortical function associated with many chronic pain conditions have been reported in the prefrontal cortex (PFC). The PFC is implicated in pain-related co-morbidities such as depression, anxiety and impaired emotional decision-making ability. We recently reported that this region is subject to significant epigenetic reprogramming following peripheral nerve injury, and normalization of pain-related structural, functional and epigenetic abnormalities in the PFC are all associated with effective pain reduction. In this study, we used the Spared Nerve Injury (SNI) model of neuropathic pain to test the hypothesis that peripheral nerve injury triggers persistent long-lasting changes in gene expression in the PFC, which alter functional gene networks, thus providing a possible explanation for chronic pain associated behaviors.

Results: SNI or sham surgery where performed in male CD1 mice at three months of age. Six months after injury, we performed transcriptome-wide sequencing (RNAseq), which revealed 1147 differentially regulated transcripts in the PFC in nerve-injured vs. control mice. Changes in gene expression occurred across a number of functional gene clusters encoding cardinal biological processes as revealed by Ingenuity Pathway Analysis. Significantly altered biological processes included neurological disease, skeletal muscular disorders, behavior, and psychological disorders. Several of the changes detected by RNAseq were validated by RT-QPCR and included transcripts with known roles in chronic pain and/or neuronal plasticity including the NMDA receptor (glutamate receptor, ionotropic, NMDA; grin1), neurite outgrowth (roundabout 3; robo3), gliosis (glial fibrillary acidic protein; gfap), vesicular release (synaptotagmin 2; syt2), and neuronal excitability (voltage-gated sodium channel, type I; scn1a).

Conclusions: This study used an unbiased approach to document long-term alterations in gene expression in the brain following peripheral nerve injury. We propose that these changes are maintained as a memory of an insult that is temporally and spatially distant from the initial injury.

Figure 1

Direction and nature of transcriptome expression alterations in the prefrontal cortex six months after injury in SNI and Sham animals. (A) Distribution of exonic, intergenic and intronic elements within the mouse genome. (B) Distribution of transcribed exonic, intergenic and intronic elements in SNI PFC transcriptome relative to Sham PFC. (C) Distribution of differentially expressed RNAs in the PFC associated with peripheral nerve injury.

Figure 2

Validation of transcript mRNA expression. Quantitative PCR validation of downregulated transcripts CLCA1 (A) and GFAP (B) and upregulated transcripts XLR4B (C) and KRT20 (D) relative to GAPDH. *=p<0.05. n=8/group. Error bars = S.E.M.

Figure 3

Functional pathway analysis. Nerve injury affects transcriptional programs unique to neurological disease, skeletal and muscular disorders, psychological disorders and changes in behavior scored and ranked according to Ingenuity Pathway Analysis using Fisher’s exact test. The dotted line indicates the threshold value of p<0.05.

Figure 4

Cellular growth and differentiation. Nerve injury results in distinct changes in transcription in pathways involved in cellular growth and proliferation. (A) RNAseq and IPA identified interacting networks affecting cell cycle, cell proliferation and cellular development. Up-regulated transcripts are marked with red while downregulated transcripts are marked in blue. SYT2 (B) SCN1A (C) transcripts were validated by qPCR. *=p<0.05. n=8/group. Error bars = S.E.M.

Figure 5

Cell cycle and growth. SNI causes distinct changes in transcription in pathways involved in cell cycle and growth. (A) RNA sequencing and IPA identified interacting networks affecting cell cycle, cellular growth and proliferation and cellular development. Upregulated transcripts are marked with red while downregulated transcripts are marked in blue. ROBO3 (B) and KRT20 (C) transcripts were validated by qPCR. *=p<0.05. n=8/group. Error bars = S.E.M.

Figure 6

Neuronal development. SNI causes distinct changes in transcription in pathways involved neuronal development. (A) RNA sequencing and IPA identified interacting networks affecting cellular assembly and organization and nervous system development and function. Upregulated transcripts are marked with red while downregulated transcripts are marked in blue. (B) GRIN1 transcript was validated by qPCR. *=p<0.05. n=8/group. Error bars = S.E.M.