Neonatal Colonic Inflammation Increases Spinal Transmission and Cystathionine β-Synthetase Expression in Spinal Dorsal Horn of Rats with Visceral Hypersensitivity

Abstract

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by chronic abdominal pain and alteration of bowel movements. The pathogenesis of visceral hypersensitivity in IBS patients remains largely unknown. Hydrogen sulfide (H₂S) is reported to play an important role in development of visceral hyperalgesia. However, the role of H₂S at spinal dorsal horn level remains elusive in visceral hypersensitivity. The aim of this study is designed to investigate how H₂S takes part in visceral hypersensitivity of adult rats with neonatal colonic inflammation (NCI). Visceral hypersensitivity was induced by neonatal colonic injection of diluted acetic acid. Expression of an endogenous H₂S synthesizing enzyme cystathionine β-synthetase (CBS) was determined by Western blot. Excitability and synaptic transmission of neurons in the substantia gelatinosa (SG) of spinal cord was recorded by patch clamping. Here, we showed that expression of CBS in the spinal dorsal horn was significantly upregulated in NCI rats. The frequency of glutamatergic synaptic activities in SG was markedly enhanced in NCI rats when compared with control rats. Application of NaHS increased the frequency of both spontaneous and miniature excitatory post-synaptic currents of SG neurons in control rats through a presynaptic mechanism. In contrast, application of AOAA, an inhibitor of CBS, dramatically suppressed the frequency of glutamatergic synaptic activities of SG neurons of NCI rats. Importantly, intrathecal injection of AOAA remarkably attenuated visceral hypersensitivity of NCI rats. These results suggest that H₂S modulates pain signaling likely through a presynaptic mechanism in SG of spinal dorsal horn, thus providing a potential therapeutic strategy for treatment for chronic visceral pain in patients with IBS.

Keywords: excitatory post-synaptic current; hydrogen sulfide; irritable bowel syndrome; spinal cord dorsal horn; visceral pain.

FIGURE 1

Upregulated expression of CBS in spinal cord (SC) of NCI rats. (A) NCI treatment significantly increased AWR scores to CRD at age of 6 weeks compared with the age-matched control rats (n = 8). (B) Increase in expression of CBS in SC at T13-L2 levels from NCI rats at age of 6 week compared with control (CON) rats (n = 5 for control group and n = 8 for NCI group). (C) No difference of CBS mRNA expression in SC between CON and NCI rats at age of 6 weeks (n = 5). (D) No difference of CSE expression in SC at T13-L2 level between CON and NCI rats at age of 6 weeks (n = 5). (E) No difference of CBS expression in SC at T7-T10 level between CON and NCI rats at age of 6 weeks (n = 4). (F) No difference of CBS expression in dorsal root ganglion (DRG) at L4-L6 level between CON and NCI rats at age of 6 weeks (n = 4). ^∗P < 0.05, ^∗∗P < 0.01 when compared with CON.

FIGURE 2

NCI increased the excitation of SG neurons. (A) Representative traces of APs caused by three different depolarizing current stimulation (40, 80, and 120 pA) in SG neurons from CON and NCI rats. (B) NCI greatly increased the frequency of APs elicited by current injection of 40, 80, and 120 pA compared with controls. n = 17 for control group and n = 18 for NCI group, ^∗P < 0.05 vs. CON, ^∗∗P < 0.01 vs. CON. (C) The mean resting membrane potential (RP) had no significant difference between CON and NCI rats (n = 59 for control group and n = 33 for NCI group). (D) There was significant hyperpolarization in action potential (AP) threshold between CON and NCI rats. n = 35 for control group and n = 20 for NCI group, ^∗∗P < 0.01. (E) NCI significantly decreased the rheobase when compared with CON. n = 35 for control group and n = 20 for NCI group, ^∗∗P < 0.01.

FIGURE 3

NCI increased the frequency of sEPSCs in SG neurons. (A) Representative traces of spontaneous excitatory post-synaptic currents (sEPSC) recorded from SG neurons hold at –70 mV in voltage clamp from control (CON) and NCI rats. (B) NCI increased the frequency of sEPSC (left, ^∗P < 0.05, compared with CON) but failed to change the amplitude of sEPSC (right) when compared with controls (n = 29 for control group and n = 35 for NCI group). (C) The administration of CNQX (10 μM) blocked all the sEPSCs in SG neurons from control rats. a: An expanded trace for sEPSC recorded in the absence of CNQX. b: An expanded trace for sEPSC recorded in the presence of CNQX.

FIGURE 4

NaHS enhanced the frequency of sEPSCs in control group. (A) Representative current traces of sEPSCs recorded in the presence and absence of NaHS (2.5 μM). a: An expanded trace for sEPSC recorded in the absence of NaHS; b: a expanded trace for sEPSC recorded in the presence of NaHS. (B) Cumulative distribution analysis of amplitude and inter-event intervals from one representative SG neuron. NaHS had no discernible effect on the amplitude, but NaHS caused a notably shift toward shorter inter-event intervals (n = 10, P < 0.05, Kolmogorov–Smirnov test). (C) Bar plot showing NaHS significantly increased mean frequency of sEPSC without change in peak amplitude. n = 9, ^∗P < 0.05.

FIGURE 5

NaHS enhanced the frequency of mEPSCs in control group. (A) Representative current traces of mEPSCs recorded in the presence and absence of NaHS (2.5 μM). Na⁺ channel blocker TTX (1 μM) was used in Kreb solution. a: An expanded trace for mEPSC recorded in the absence of NaHS; b: an expanded trace for mEPSC recorded in the presence of NaHS. (B) Cumulative distribution analysis of peak amplitude and inter-event intervals from the same neuron showed that NaHS reduced the inter-event intervals of mEPSC (n = 10, P < 0.01, Kolmogorov–Smirnov test) but had no effect on the amplitude. (C) Bar plot showing NaHS significantly increased mean frequency of sEPSCs but not amplitude. n = 10, ^∗P < 0.05.

FIGURE 6

AOAA decreased the frequency of sEPSCs in SG neurons and attenuated visceral pain of NCI-rats. (A) Representative traces of sEPSCs recorded from SG neurons holding at –70 mV in voltage clamp from NCI and AOAA-treated rats. (B) AOAA treatment didn’t change the amplitude of sEPSC compared with NCI rats. The frequency of sEPSC was decreased after AOAA treatment compared with NCI rats. n = 10 for NS, n = 9 for AOAA, ^∗P < 0.05. (C) Bar plot showing that intrathecal injection of AOAA significantly decreased AWR scores of NCI rats. n = 5, ^∗P < 0.05. (D) Bar plot showing that intrathecal injection of NaHS significantly increased AWR scores of control rats. n = 6, ^∗P < 0.05.