Changes in expression of sensory organ-specific microRNAs in rat dorsal root ganglia in association with mechanical hypersensitivity induced by spinal nerve ligation

Abstract

Chronic neuropathic pain caused by peripheral nerve injury is associated with global changes in gene expression in damaged neurons. To understand the molecular mechanisms underlying neuropathic pain, it is essential to elucidate how nerve injury alters gene expression and how the change contributes to the development and maintenance of chronic pain. MicroRNAs are non-protein-coding RNA molecules that regulate gene expression in a wide variety of biological processes mainly at the level of translation. This study investigated the possible involvement of microRNAs in gene regulation relevant to neuropathic pain. The analyses focused on a sensory organ-specific cluster of microRNAs that includes miR-96, -182, and -183. Quantitative real-time polymerase chain reaction (qPCR) analyses confirmed that these microRNAs were highly enriched in the dorsal root ganglion (DRG) of adult rats. Using the L5 spinal nerve ligation (SNL) model of chronic neuropathic pain, we observed a significant reduction in expression of these microRNAs in injured DRG neurons compared to controls. In situ hybridization and immunohistochemical analyses revealed that these microRNAs are expressed in both myelinated (N52 positive) and unmyelinated (IB4 positive) primary afferent neurons. They also revealed that the intracellular distributions of the microRNAs in DRG neurons were dramatically altered in animals with mechanical hypersensitivity. Whereas microRNAs were uniformly distributed within the DRG soma of non-allodynic animals, they were preferentially localized to the periphery of neurons in allodynic animals. The redistribution of microRNAs was associated with changes in the distribution of the stress granule (SG) protein, T-cell intracellular antigen 1 (TIA-1). These data demonstrate that SNL induces changes in expression levels and patterns of miR-96, -182, and -183, implying their possible contribution to chronic neuropathic pain through translational regulation of pain-relevant genes. Moreover, SGs were suggested to be assembled and associated with microRNAs after SNL, which may play a role in modification of microRNA-mediated gene regulation in DRG neurons.

Figure 1

The paw withdrawal thresholds for naïve, sham-operated, and ligated rats used in the qPCR, in situ hybridization, and immunohistochemistry analyses. Squares depict the threshold for the indicated hindpaw of each rat. Circles with error bars are the mean ± S.E.M. for each treatment group. Symbols with an asterisk indicate rats that did not show the expected pain behavior. n = the number of animals in each group.

Figure 2

Expression of miR-183 family microRNAs in tissues from the adult rat. (A) Amounts of miR-96, miR-182, and miR-183 in various adult rat tissues relative to that in the dorsal root ganglia. Data are the mean ± S.E.M. of determinations in 3 rats. (DRG = dorsal root ganglion; SC = spinal cord; Br St = brain stem; Cer = cerebellum; Cor = cortex; Hrt = heart; Int = intestine; Kid = kidney; Liv = liver; Lun = lung). (B) Data represented as fg of miR-96, miR-182, and miR-183 in 1 ng of total RNA extracted from adult rat dorsal root ganglia.

Figure 3

Relative expression of microRNAs in the L5 DRG of ligated and sham-operated rats determined by qPCR for miR-96, miR-182, miR-183, and miR-23b. Data are the mean ± S.E.M. of eight determinations. Data for the ipsilateral L5 DRG of sham rats and ipsilateral and contralateral L5 DRG of ligated rats are expressed relative to average microRNA amount (fg/ng total RNA) in L5 DRG of naive rats. Symbols above error bars indicate significant difference for ligated ipsilateral L5 DRG compared to naïve ipsilateral L5 DRG (^; ANOVA, P ≤ 0.05) or ligated ipsilateral L5 DRG compared to naïve and sham operated ipsilateral L5 DRG and ligated contralateral L5 DRG (*;ANOVA, P ≤ 0.05). The remaining comparisons were not significantly different (ANOVA, P > 0.05).

Figure 4

Expression patterns of miR-96, -182, and 183 in L5 DRGs from naïve rats. In situ hybridization staining for microRNAs (A, C, E) was performed in combination with immunohistochemical labeling (B, D, F) for myelinated (N52-positive, red) and unmyelinated (IB4-positive, greens) neurons. Large and small arrows indicate microRNA/N52 co-labeling and microRNA/IB4 co-labeling, respectively. Arrow heads indicate microRNA-positive/N52 and IB4-negative neurons. (A, B) miR-96, (C, D) miR-182, (E, F) miR-183.

Figure 5

Distribution of miR-96 in situ hybridization signal in ipsilateral L5 DRG from rats that underwent sham surgery (A, C) or L5 spinal nerve ligation (B, D). Spinal nerve ligation resulted in an overall reduction in the intensity of labeling and a redistribution of label within cytoplasm to the periphery of the neuron. Arrow in panel C indicates a neuron with weakly staining nucleus but uniform staining across cytoplasm, which can be differentiated from ligated neurons with strong staining on the edge of the cell with a weakly staining inner cytoplasmic area.

Figure 6

Representative examples of the distribution of in situ hybridization signal for miR-182 (A, B), miR-183 (C, D), let-7a (E, F), miR-23b (G, H), and miR-124a (I, J) in the ipsilateral L5 DRG two weeks after either sham surgery (A, C, E, G, I) or ligation of the L5 spinal nerve (B, D, F, H, J) in the rat.

Figure 7

Co- in situ hybridization and immunohistochemistry for TIA-1 (A, C) and miR-96 (B, D) in ipsilateral L5 DRG two weeks after sham surgery (A, B) or ligation of the L5 spinal nerve (C, D) in the rat. TIA-1 and miR-96 costaining in large diameter and small diameter neurons indicated by large and small arrows, respectively. Large arrows in panel C and D indicate a large diameter neuron with TIA-1 and miR-96 staining colocalizing to edge of cell. In sham operated DRGs, TIA-1 is present in the nucleus of both large and small diameter neurons (A; large and small arrows, respectively). In ligated DRGs, TIA-1 is present in the nucleus of large diameter neurons and absent in the nucleus small diameter neurons (C; large and small arrows, respectively).