DNA Microarray Analysis of Differential Gene Expression in the Dorsal Root Ganglia of Four Different Neuropathic Pain Mouse Models

Abstract

Purpose: Pathological stimuli or injury to the peripheral nervous system can trigger neuropathic pain with common clinical features such as allodynia and hypersensitivity. Although various studies have identified molecules or genes related to neuropathic pain, the essential components are still unclear. Therefore, in this study, we investigated the molecular and genetic factors related to neuropathic pain.

Methods: We extracted candidate genes in the dorsal root ganglion (DRG) from three nerve injury mouse models and a sham-operated model (sciatic nerve ligation and resection, sural nerve resection, spared nerve injury [SNI], and sham) using DNA microarray to elucidate the genes responsible for the neuropathic pain mechanism in the SNI model, which exhibits hypersensitivity in the hindpaw of the preserved sural nerve area. We eliminated as many biases as possible. We then focused on an upregulated endogenous vasopressin receptor and clarified whether it is closely associated with traumatic neuropathic pain using a knockout mouse and drug-mediated suppression of the gene.

Results: Algorithm analysis of DNA microarray results identified 50 genes significantly upregulated in the DRG of the SNI model. Two independent genes-cyclin-dependent kinase-1 (CDK-1) and arginine vasopressin receptor 1A (V1a)-were subsequently identified as candidate SNI-specific genes in the DRG by quantitative PCR analysis. Administration of V1a agonist to wild-type SNI mice significantly alleviated neuropathic pain. However, V1a knockout mice did not exhibit higher hypersensitivity to mechanical stimulation than wild-type mice. In addition, V1a knockout mice showed similar pain behaviors after SNI to wild-type mice.

Conclusion: Through the DNA microarray analysis of several neuropathic models, we detected specific genes related to chronic pain. In particular, our results suggest that V1a in the DRG may partially contribute to the mechanism of neuropathic pain.

Keywords: arginine vasopressin 1a; dorsal root ganglion; microarray; molecular target; neuropathic pain; peripheral nerve injury.

Figure 1

Injury sites of each peripheral nerve injury model. The sciatic nerve branches into the tibial nerve, peroneal nerve, and sural nerve. The sural nerve includes only sensory fibers. In model 1, the sciatic nerve was ligated and resected (blue cross). In model 2, the sural nerve was resected (yellow cross). In model 3 (the spared nerve injury [SNI] model), both motor nerves were resected and the sensory nerve was preserved (red cross). In model 4, the animals underwent a sham operation. Only the SNI models showed mechanical hypersensitivity. Three weeks after the surgery, we isolated lumbar dorsal root ganglia (L3, 4, and 5) and compared DNA expression patterns among the groups.

Figure 2

Venn diagram of nerve injury models. The right oval area subtracted from the center circle was extracted as spared nerve injury-specific genes ((A), upregulated; (B), downregulated). SNI, spared nerve injury. (C) Of the 50 SNI-specific genes showing an increase, 2 were validated using real-time PCR. Cdk1 and Avpr1a were significantly increased compared with the sham model.

Figure 3

V1a expression in the dorsal root ganglia (L3, 4, and 5) 3 weeks and 6 weeks after SNI surgery. The SNI model showed a significant 2.23±0.8-fold increase in expression at 3 weeks. The SNI model also showed a significant 1.53±0.34-fold increase at 6 weeks.

Figure 4

Dorsal skin sensitivity changes. Changes in the mechanical flexor reflex withdrawal response to stimulation of the dorsal surface of the hindpaw (sural nerve territory) after spared nerve injury (SNI) (n = 8) and sham (n = 8) procedures. The withdrawal threshold of the dorsal skin had a higher threshold in the control period and a smaller reduction after the SNI. This change was similar in vasopressin receptor 1A knockout SNI mice.

Figure 5

Changes in mechanical stimulation hypersensitivity with vasopressin receptor 1A (V1a) agonist. The groups intraperitoneally administered 0.5 mg/kg and 1.0 mg/kg V1a selective agonist showed a significant increase in the threshold of the mechanical stimulation response 30 min after administration compared with the phosphate-buffered saline group for both males (A) and females (B). The change in the threshold 30 and 60 min after administration tended to be volume-dependent. Similar results were seen in spared nerve injury models 6 weeks after injury in both males (C) and females (D). There were no significant changes in the response to mechanical stimulation in male (E) or female (F) V1a knockout mice at any dose.

Figure 6

Changes in mechanical stimulation hypersensitivity induced by V1a antagonist. Wild-type (WT) sham (A) and spared nerve injury (SNI) mouse (B) groups 3 weeks after surgery. Neither the WT SNI nor sham mice of either sex showed any apparent mechanical hypersensitivity 30, 60, 90, or 120 min after administration.

Figure 7

Immunohistochemistry of the lumbar dorsal root ganglia with vasopressin receptor 1A (V1a) antibody. (A) V1a was mainly expressed in the rim of DRG cells in both SNI and sham-operated animals. (B) A significant difference was found in the cross-sectional area of V1a expression between SNI mice (2445±46.2 μm²) and the sham-operated model (1582 ± 50.7 μm², p<0.001). (C) Double-immunostaining assay showed that V1a colocalized with S-100 (arrowheads), but not with CGRP.