Chronic stress and peripheral pain: Evidence for distinct, region-specific changes in visceral and somatosensory pain regulatory pathways

Abstract

Chronic stress alters the hypothalamic-pituitary-adrenal (HPA) axis and enhances visceral and somatosensory pain perception. It is unresolved whether chronic stress has distinct effects on visceral and somatosensory pain regulatory pathways. Previous studies reported that stress-induced visceral hyperalgesia is associated with reciprocal alterations of endovanilloid and endocannabinoid pain pathways in DRG neurons innervating the pelvic viscera. In this study, we compared somatosensory and visceral hyperalgesia with respect to differential responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress. We found that chronic stress induced reciprocal changes in the endocannabinoid 2-AG (increased) and endocannabinoid degradation enzymes COX-2 and FAAH (decreased), associated with down-regulation of CB1 and up-regulation of TRPV1 receptors in L6-S2 DRG but not L4-L5 DRG neurons. In contrast, sodium channels Nav1.7 and Nav1.8 were up-regulated in L4-L5 but not L6-S2 DRGs in stressed rats, which was reproduced in control DRGs treated with corticosterone in vitro. The reciprocal changes of CB1, TRPV1 and sodium channels were cell-specific and observed in the sub-population of nociceptive neurons. Behavioral assessment showed that visceral hyperalgesia persisted, whereas somatosensory hyperalgesia and enhanced expression of Nav1.7 and Nav1.8 sodium channels in L4-L5 DRGs normalized 3 days after completion of the stress phase. These data indicate that chronic stress induces visceral and somatosensory hyperalgesia that involves differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in DRGs innervating the pelvic viscera and lower extremities. These results suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively at different levels of the spinal cord.

Keywords: CB1; Chronic stress; Endocannabinoid; Pain; Sodium channel; Somatosensory hyperalgesia; Trpv1; Visceral hyperalgesia.

Fig. 1

Western blot analysis revealed region-specific alterations of TRPV1 and CB1 receptors in DRGs in WA stressed rats compared with controls. (A) Typical blotting bands demonstrating the expression levels of TRPV1 and CB1 in T5-T6, L4-L5 and L6-S2 DRG regions. (B) Graph depicting the percentage changes in the receptor expression in DRGs from control and stressed animals (n = 5). Data are expressed as mean ± standard error. *, P < 0.05.

Fig. 2

Colocalization of TRPV1 in neuronal subpopulations in L6-S2 DRGs. (A) Double immunofluorescence staining of TRPV1 (red) and small-sized nociceptive C-fiber marker, peripherin (green), in L6-S2 DRGs from control and WA stressed rats. (B) Statistical data of the double immuoreactivity-positive neurons for TRPV1 and peripherin in L4-L5 and L6-S2 DRGs (n = 4). (C) Double immunofluorescence staining of TRPV1 (red) and large-sized Aβ-fiber marker, NF200 (green), in L6-S2 DRGs from control and WA stressed rats. (D) Statistical data of the double immunoreactivity-positive neurons for TRPV1 and NF200 in L4-L5 and L6-S2 DRGs (n = 4). Scale bar, 60 μm. Data are expressed as mean ± standard error. *, P < 0.05.

Fig. 3

Decreased expression of CB1 in both small-sized and large-sized L6-S2 DRG neurons in WA stressed rats. (A) Double immunofluorescence staining of CB1 (red) and peripherin (green) in L6-S2 DRG neurons in control and WA stressed rats. (B) Statistical data of the double immuoreactivity-positive neurons for CB1 and peripherin in L4-L5 and L6-S2 DRGs (n = 4). (C) Double immunofluorescence staining of CB1 (red) and NF200 (green) in L6-S2 DRG neurons in control and WA stressed rats. (D) Statistical data of the double immunoreactivity-positive neurons for CB1 and NF200 in L4-L5 and L6-S2 DRGs (n = 4). Scale bar, 60 μm. Data are expressed as mean ± standard error. **, P < 0.01.

Fig. 4

WA stressed induced differential alterations of the endocannabinoid 2-AG content in L4-L5 and L6-S2 DRGs. (A) Representative chromatograph from L6-S2 DRG extracts depicting the relative abundance of endocannabinoid 2-AG in control and WA stressed rats. (B) The bar graph illustrated the increase in 2-AG content in L6-S2 but not L4-L5 DRGs in rats following chronic WA stress compared to controls (n = 5). Data are expressed as mean ± standard error. *, P < 0.05.

Fig. 5

Differential changes in endocannabinoid degradation enzymes in L4-L5 and L6-S2 DRGs. (A) Western blot analysis showed preferentially decreased expression levels of COX2 and FAAH protein in L6-S2 but not L4-L5 DRGs in WA stressed rats compared with controls. (B) Statistical analysis of the percentage change of COX2 and FAAH proteins in DRGs in control and WA stressed rats (n = 4). (C) Repeated corticosterone treatment of healthy control rats induced down-regulation of COX2 and FAAH in L6-S2 DRGs but not L4-L5 DRGs compared to untreated controls. (D) Bar graph depicting the percentage change of COX2 and FAAH proteins in L6-S2 and L4-L5 DRGs in the rats treated with corticosterone (n = 4). Data are expressed as mean ± standard error. *, P < 0.05.

Fig. 6

Effect of chronic WA stress on visceral motor response (VMR) in response to colorectal distention (CRD) at different time duration after WA stress. (A) Representative EMG recordings depicting the VMR to CRD at the distention pressure of 40 mmHg in control rats, WA stress rats, and WA stress rats with recovery for 3 days and 6 weeks, respectively, after completing the stress procedure. (B) Electromyographic amplitude expressed as area under curve (AUC) of the raw electromyographic response was significantly increased following chronic WA stress on day 11 (WA) and day 14 (WA with 3-day recovery), and returned to baseline level 6 weeks after stress (WA with 6-week recovery). Data are expressed as mean ± standard error, n = 6-8 in each group. *, P < 0.05 between the following groups: control and WA, control and WA with 3-day recovery, WA or WA with 3-day recovery and WA with 6-week recovery.

Fig. 7

Line graphs show acute development of somatosensory pain in WA stressed rats compared with controls. (A) Comparison the differences in pain-related behaviors in response to mechanical (Von Frey test) stimuli in the hind paws of the control, WA and WA with 3-day recovery (WA-Rec) rat groups. (B) Comparison the differences in pain-related behaviors in response to thermal (Hargreaves test) stimuli in the hind paws of the control, WA and WA with 3-day recovery (WA-Rec) rat groups. Data are expressed as mean ± standard error, n = 6 in each group. **, P < 0.01.

Fig. 8

Double immunofluorescence staining of DRG neurons with C-type fiber marker peripherin (green) and sodium channel (red) antibodies. The neurons of double immunoreactivity positive are shown in yellow. (A) Increased expression of TTX-S Nav1.7 and TTX-R Nav1.8 sodium channels in peripherin-positive neurons in L4-L5 DRGs from WA stressed rats (middle panel) compared with the control (left panel). After 3-day recovery following the stress procedure (right panel), the enhanced expression of Nav1.7 returned to control level. Scale bar, 50 μm. (B) Percentage changes of the number of double-positive neurons for sodium channels and C-fiber marker peripherin in L4-L5 DRG neurons in WA stressed rats compared to the controls. (C) The number of double-positive neurons for Na_v1.7 / Na_v1.8 and peripherin in L6-S2 DRG neurons in control and stressed rats. Data are expressed as mean ± standard error, n = 4 in each group. *, P < 0.05; **, P < 0.01.

Fig. 9

Corticosterone treatment induced up-regulation of Na_v1.7 and Na_v1.8 sodium channels in L4-L5 DRGs in vitro. (A) Western blot analysis showed significant increases in the expression levels of Na_v1.7 and Na_v1.8 proteins after corticosterone (CORT) treatment in vitro in isolated L4-L5 DRGs from control rats. Two replicates from different animal groups were shown. (B) Statistical analysis of the percentage change of Na_v1.7 and Na_v1.8 proteins in L4-L5 DRGs after corticosterone treatment. (C) Western blot analysis of Na_v1.7 and Na_v1.8 protein expression after corticosterone treatment in vitro in isolated L6-S2 DRGs from control rats. (D) No significant changes in Na_v1.7 and Na_v1.8 protein levels were observed in L6-S2 DRGs treated with corticosterone in vitro. Data are expressed as mean ± standard error, n = 4 in each group. *, P < 0.05.