Background
Abdominal pain and discomfort are amongst the most vexing symptoms for patients suffering from inflammatory bowel diseases such as Crohn’s ileitis and present a major therapeutic challenge for physicians. The underline mechanisms are still unknown. Therefore, it is essential to identify the detailed molecular mechanisms that are involved in inflammatory/infectious events leading to visceral hypersensitivity both for understanding underlying mechanisms and developing new therapies.
In the visceral sensory neurotransmission process, the neurons that relay sensory information from the intestine to the spinal cord are the dorsal root ganglia (DRG) neurons. These pseudounipolar neurons have a peripheral axon that travels to the intestine and a central axon that project to sensory neurons in the dorsal horn of the spinal cord. Taken together, current knowledge suggests that changes in gene expression in DRGs may contribute to the generation and development of inflammatory visceral hypersensitivity. Proposed mechanisms that could account for the sensitization of primary sensory afferents include increased expression of receptor molecules involved in nociceptive pathways or alteration of ionic channel properties. Here, we took a broader approach, a cDNA array, to gain a comprehensive overview of the alteration of gene expression in DRGs after TNBS induced visceral inflammation.
Voltage-gated calcium channels (VGCC) regulate rapid and transient changes in intracellular calcium within excitable cells. Calcium entering the cell through these channels serves as a second messenger, initiating intracellular events such as contraction, secretion, protein phosphorylation, gene expression, neurotransmitter release and action potential firing patterns [
1]. Within neurons of the DRG the activity of calcium channels has been shown to play an important role in nociceptive processing [
2,
3] and regulation of calcium channel activity has been shown to modulate pain in clinical and experimental settings [
2,
4,
5]. More recently, agents which modulate the activity of the calcium channel α2δ subunit have also been shown to exhibit experimentally and clinically important pain relief [
6]. However, a link between changes in voltage-gated calcium channel of DRG and the occurrence of postinflammatory hypersensitivity symptoms still has to be established. The mechanism, for example, whether a specific subtype involves, or how or where they contribute to the postinflammatory visceral pain, has remained to be fully clarified, and the subtype channels could be viewed as potential therapeutic targets for the treatment of postinflammatory hypersensitivity symptoms.
Thus the aim of this study was to identify marked changes in the expression of genes in DRG neurons innervating the inflamed intestine. Then among these diverse changed molecular, we are try to developing successful therapies to target ion channels that promote the sensitization of primary sensory afferents.
Discussion
In this study, we perform a microarray screening for genes with different expression in rat models of inflammatory visceral pain and demonstrate that Cav1.2 and Cav2.3 participates in the visceral hyperalgesia induced by TNBS-colitis. The result revealed that the level of Cav1.2 and Cav2.3 protein in L6-S2 DRG were significantly up-regulated following colitis and L-type and R-type calcium currents significantly increased in colonic specific DRG neurons, while visceral sensitivity to mechanical stimuli also increased significantly. Intrathecal administration of the specifically inhibits nimodipine and SNX482 reduced the colonic hyperalgesia to balloon distention.
Genes regulated in the L6-S2 DRG with TNBS treated may contribute to a variety of biologic processes such as inflammatory visceral pain signaling. In this study, we found that a large number of ion channels strongly regulated in DRG after TNBS treated such as VGCC (Cav1.2, Cav2.3), inositol 1,4,5-trisphosphate receptor 2, ryanodine receptors 1(RyR1), P2X7, P2Y and so on. Some studies have reported about the role of these channel in pain sensation such as RyR1 [
7] P2X7 [
8‐
13] and P2Y1 [
14]. But these channels haven’t been studied in visceral sensation. In addition, P2X7 and P2Y1 receptors involved in the neuronal soma-satellite cell interaction [
13,
15]. Maybe the up-regulation of P2X7 and P2Y1 can improve the neuron–glia communication. We also found that a large number of neurotransmitter and receptor strongly regulated in DRG after TNBS treated such as tumor necrosis factor (TNF, member 2 and 4), PGER(EP2), 5-HTR 1A, 5-HTR 2A and FGFR3, which upregulated in DRG of TNBS treated rat. Recently, several studies provided evidence that TNF-α may act on primary afferent neurons and induces hypersensitivity [
12,
16,
17]. Whether the up-regulation of EP2 in L6-S2 DRGs of TNBS treated rats, mediating the visceral hypersensitivity, needed further study. The previous studies have suggested that 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3 and 5-HT4 receptor subtypes in the DRG play important roles in facilitating peripheral nociceptive transmission [
18‐
20]. Besides the ion channels and receptors, signal transduction molecules were also significantly changed. These changes could be associated with the rearrangement of pain sensory pathways that take place during the development of visceral inflammatory pain. All these changed genes maybe establish a positive feedback loop between neuron-neuron and neuron-satellite cell, thus contributing to exaggerated responses under pathological conditions which still need further experimental research proof.
In our study, we found that both the expression and the function of L- and R- type VGCC were upregulated in colonic DRG neurons of TNBS treated rats compare with control group. Kania has reported L-type VGCC blockers (nifedipine, diltiazem verapamil) can be useful in controlling acute visceral pain in the model of visceral pain or hypersensitivity of sheeps, which suggests that L-type VGCC play a crucial role in the modulation of acute visceral hyperalgesia [
21‐
23]. They only use the pharmacology method in the behavioral test lacking the studies of molecular mechanism. In addition to visceral pain, neuronal L-type VGCCs Cav1.2 and Cav1.3 have reported to be up- or down regulated in the model of neuropathic pain in rats [
24,
25]. R-type Ca
2+ channels located at primary synapses [
26] and can contribute to neurotransmitter release [
27]. R-type VGCCs are associated with nociceptive processing in various pain conditions. Mathews reported that spinal SNX-482, an R-type VGCC blocker, inhibited noxious C-fiber and Aδ-fiber-mediated dorsal horn neuronal responses in conditions of neuropathy, not in sham operated rats and that non-noxious Aδ-mediated responses did not affect by SNX-482 [
28]. In another study, mice lacking the α1E Ca
2+ channel subunit exhibited normal pain behavior against acute mechanical, thermal, and chemical stimuli; however, they showed reduced responses to somatic inflammatory pain [
29]. So far, there has been no definitive study that shows that the role of R-type VGCC in visceral sensation. Our results revealed that upregulated Cav1.2 (L-type) and Cav2.3 (R-type) maybe involved in the inflammatory visceral hypersensitivity induced by TNBS treated.
The VGCCs manage and adjust neurotransmitter release and Ca
2+-dependent depolarization that contribute to the characteristic firing patterns of most neurons, including those in pain pathways. As far, the role of VGCC in pain was not consensus. Because a change in [Ca
2+]
i can affect neuronal properties via changes in ion channel activity [
30,
31], enzymatic activity [
32] and/or gene expression [
33], the physiological impact of the inflammation induced changes in evoked increases in [Ca
2+]
i will depend on where in the neuron they are manifest. On the other hand, an increase in [Ca
2+]
i close to functional Ca
2+ dependent K
+ channels should increase K
+ channel activity resulting in a decrease in afferent excitability and the transmission of nociceptive information, while a change in the dynamics of Ca
2+ signaling in the cell body may result in changes in gene expression that are either pro- or anti-nociceptive [
34]. Thus, the colon inflammation-induced changes in the regulation of [Ca
2+]
i may contribute to the visceral inflammatory hyperalgesia or serve as a feedback inhibitory mechanism functioning as a “break” to a host of pro-nociceptive inflammatory processes. We further detected the visceral pain after intrathecal injection of specific calcium channel inhibitors. Nimodipine specifically inhibits Ca
v1.2 α1C and to a lesser extent T-type VGCC in central and peripheral neurons. But the concentration for T-type Ca
2+ channel inhibition is two orders of magnitude higher than for L-type Ca
2+ channel [
35]. The SNX-482 is specific inhibitor for Cav2.3. Cav1 (L-type) can be partially inhibited by saturating concentrations of SNX-482 [
36]. SNX-482 is a large peptide and the concentration of SNX-482 applied was 5 μg/Kg. The concentration in the tissue were limited and this concentration can’t inhibit the Cav1 [
28]. As the intrathecal injection of nimodipine or SNX-482 significantly reduced the visceral inflammatory hyperalgesia in this study, we supposed that Cav1.2 and Cav2.3 may contribute to the visceral inflammatory hypersensitivity. However, additional experiments will be needed to study the direct consequence of the increase of [Ca
2+]
i on function of the sensory neurons.
In summary, our data revealed that there are many genes marked changed in L6-S2 DRGs of visceral inflammatory model, which encompass a large number of distinct family members including neuropeptides, receptors, ion channels, signal transduction molecules and others. Among these changed genes, the up-regulation of Cav1.2 and Cav2.3 contributed to visceral inflammatory hyperalgesia, which maybe the potential therapeutic targets for visceral pain.
Materials and methods
Experimental colitis
Experiments were performed on male Sprague–Dawley (SD, 200 g-250 g) rats. All animal procedures used in this study were approved by Medicine Animal Care and Use Committee of Shanghai Jiaotong University School and were in accordance with the guidelines of the International Association for the Study of Pain. To induce inflammation in the distal colon, fasted rats were anaesthetized and TNBS (0.6 ml of 30 mg/ml TNBS in 50%Ethanol) was instilled into the lumen of the colon through a syringe-attached polyethylene catheter via the rectum 6 cm proximal to the anus. Animals that received an equal volume of 50% EtOH enema served as controls throughout these studies. All colonic instillations were performed under isoflurane (2%) anaesthesia.
Agilent DNA microarray analysis
After the 4-day recovery period, four rats from each group were terminally anesthetized with CO2, and then the L6-S2 DRGs was rapidly removed and stored at −80°C. Total RNA of DRG (L6-S2) from each group was extracted using Trizol (Invitrogen Inc., Carlsbad, CA, USA) and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA used for microarray analysis was prepared by the SuperAmpTM PCR based amplification method performed according to Miltenyi Biotec’s undisclosed protocol. Probe labelling was performed according to Miltenyi Biotec’s undisclosed protocol. The cDNA was denatured for 4 min at 70°C and cooled down to room temperature. Afterwards, 11 μL 10× control target solution, 11 μL 10× blocking agent solution (Agilent), and 55 μl 2× Gene Expression hybridization buffer (Agilent) was added. The entire volume of the reaction was subsequently hybridized overnight (17 h, 65°C) on Agilent Whole Rat Genome Oligo Microarrays (4 × 44 K format) using Agilent’s recommended hybridization chamber and oven. The microarrays were washed once with 6× SSPE buffer containing 0.005% Nlauroylsarcosine for 1 min, followed by a second wash with 0.06× SSPE containing 0.005% Nlauroylsarcosine for 1 min, and a final wash step with acetonitrile for 30 s. The fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent).
Data were imported into Genedata Expressionist Analyst (Genedata AG, Basel, Switzerland), and the Cyanine 3 fluorescence intensities normalized using lowest weighted linear regression (LOWESS). Normalized data in tab delineated text format were uploaded into NIA array analysis (
http://lgsun.grc.nia.nih.gov/ANOVA); this allowed a pairwise comparison of gene signal intensity (expression) with fold difference of two or greater, as determined by single factor analysis of variance with multiple hypothesis correction by the false discovery rate. The fold change was calculated for each sample to represent a ratio of expression between TNBS treated and control samples.
Validation of gene transcription by real-time PCR
RT- PCR was carried out on the same set of samples that were analyzed by the microarray approach. Three μg total RNA of each tested sample was used for reverse transcription. PCR primers were as follows:
Cav1.2 forward: TCAAAGGCTACCTGGACTGGAT
reverse: CCATGCCCTCGTCCTCATT
Cav2.3 forward: GCACTACATCTCTGAGCCCTATCTG
reverse: TCTCCTCCTCGCCACAGTCT
β-actin forward: TCTGTGTGGATTGGTGGCTCT,
and reverse: AGAAGCATTTGCGGTGCAC.
Quantitative PCR was performed in 25 μL with the ABI Power SYBR Green PCR Master Mix (ABI, USA) on the 7300 Sequence Detection System (ABI, USA). Thermal cycling was initiated with incubation at 50°C for 2 min and 95°C for 10 min. After this initial step, 40 cycles of PCR were performed. Each PCR cycle consisted of heating at 95°C for 15 s for melting and 60°C for 1 min for annealing and extension. All RT-PCRs were performed in triplicate, and the results were normalized with the Ct values of β-actin.
Western blot
The western blot for protein expression of DRGs was done as previous reported [
37]. Animals (5 rats of each group) were terminally anesthetized with CO2, the L6-S2 DRGs-containing colon afferent rapidly removed, and stored at −80°C. Protein extracts from pooled DRG (L6-S2) were prepared in SDS buffer: 50 mM Tris–HCl, 133 mM NaCl, 2%SDS, 1 mM DTT, 1 mM PMSF, 1:100 dilution of protease inhibitor cocktail (sigma), pH = 8. Twenty-five micrograms (25 μg) of protein were loaded onto 9% SDS-PAGE, and electrophoretically transferred to PVDF membrane (Bio-Rad, Hercules, CA) at 100 V for 2 h. The membranes were blotted with antibodies against Cav1.2 and Cav2.3 (sigma, dilution 1:200), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Dako Cytomation, Denmark). Bands were visualized using ECL (Amersham) kit and appropriate exposure to Kodak X-ray film. Films were scanned and band intensities measured using Gel-pro Analyzer 4.0 software (Media Cybernetics). Cav1.2 and Cav2.3 protein expression were normalized to β-actin.
Isolation and identification of distal colon projecting DRG neurons
Colon specific DRG neurons were labelled by injection of 1,1
′-dioleyl-3,3,3
′,3-tetramethylindocarbocyanine methanesulfonate (DiI, Invitrogen) into the colon wall, which has been described previously report [
37]. After 1 wk of DiI labeling, TNBS colitis was induced as previous. 4 days later, colonic DRG neurons from both groups were used for recording voltage-gated calcium current. Isolation of DRG neurons from these adult SD rats has been described previously report [
37].
Electrophysiological recordings
The whole cell patch clamp recording of DiI
+ has been described previously report [
37]. Patch electrodes with a resistance of 2–4 MΩ were pulled from borosilicate glass capillaries (ID 0.86 mm, OD 1.5 mm, Length 10 cm, Sutter Instruments) using a micropipette puller (P-97 Sutter Instruments, Novato, CA). Neurons were patched in the whole cell configuration and recorded using an EPC-10 amplifier (HEKA Instruments, Lambrecht, Germany). Seals (1–10 GΩ) between the electrode and the cell were established. After whole cell configuration was established, the cell membrane capacitance and series resistance were electronically compensated. Recordings were only made when access resistance fell to <15 MΩ. Up to 80% of the series resistance was compensated electronically. Leak currents were subtracted using the on-line P/4 protocol. Barium currents (
I Ba) flowing through calcium channels were recorded using extracellular solution consisting of (in mM): 140 TEA-Cl, 2 MgCl2, 3 BaCl2, 10 glucose, 10 HEPES (pH 7.4 adjusted with TEA-OH, osmolarity 320). The pipette solution contained (in mM): 120 CsCl, 1 MgCl2, 10 HEPES, 10 EGTA, 4 Mg-ATP and 0.3 Na-GTP (pH 7.2 adjusted with CsOH, osmolarity 300 mOsm). To minimize the “run-down” associated with the whole-cell recording, GTP and ATP were included in the pipette solution. The total
I Ba was elicited by a 300 ms voltage step from −80 to 50 mV with 10 mV increments in 3 s intervals (holding potential, −100 mV). The high voltage actived
I Ba was elicited by a 240 ms voltage step from −60 to 0 mV (holding potential, −60 mV). ω-conotoxin GVIA and ω-agatoxin IVA were dissolved in distilled water at 1000 times the final concentration and kept frozen in aliquots. Nimodipine was prepared as a stock solution dissolved in DMSO. To distinguish the L-, N-, and P/Q-type calcium currents in DiI
+ DRG neurons, the corresponding selective channel blockers nimodipine (5 μM, L-type), ω-conotoxin GVIA (1 μM, N-type), ω-agatoxin IVA (400 nM, P/Q-type) and Cd
2+ (300 μM, R-type) were applied to the same recording neurons. The stock solutions were diluted in extracellular solution just before use and held in a series of independent syringes connected to corresponding fused silica columns (ID 200 μm). Each drug solution was delivered to the recording chamber by gravity flow, and rapid solution exchange was achieved by controlling the corresponding valve switch (World Precision Instruments). All drugs and chemicals were all purchased from Sigma.
Intrathecal catheters
Chronic intrathecal catheters were implanted three days before TNBS induced colitis. Following anaesthetized with 1% pentobarbital sodium 100 mg/kg intraperitoneally. The L3 and L4 vertebrae were exposed. A 32-gauge polyimide catheter was then threaded into the subarachnoid space and passed in a caudal direction for approximately 3 cm. The external end of the catheter was connected to a length of PE-10 polyethylene tubing which was tunnelled and exteriorised over the forehead. Animals were then caged individually. Any animal developing motor impairment following catheter placement was excluded from the study. All the drugs were given intrathecally in a total volume of 10 μL. The channel blockers were injected twice daily for 4 d, starting from the TNBS treated day. The nimodipine was dissolved in DMSO and SNX-482 was dissolved in sterile saline. Nimodipine (20 μg/kg) and SNX-482(5 μg/kg) were administered as a bolus via the intrathecal catheter in volumes of 10 μl. The catheter was flushed afterwards with 10 μl saline. The same amount of vehicle (DMSO or 0.9%NaCl) was injected into some TNBS treated rats as vehicle control. Behavioral testing was performed after the last injection on the fourth day of TNBS treatment as described below.
Behavioral testing for nocifensive response
We used a grading system based on the abdominal withdrawal reflex (AWR) in response to colorectal distention (CRD) to evacuate the visceral sensitivity. Briefly, under sedation with aether, a flexible latex balloon (5 cm) attached to the perforated end of an arterial embolectomy probe (LeMaitre Embolectomy Catheter 6F; LeMaitre Vascular) was inserted 6–8 cm into the descending colon and rectum via the anus and held in place by taping the tubing to the tail. CRD was performed by rapidly inflating the balloon to a constant volumn. The balloon was inflated to 1, 2, 3 and 4 ml, for 20 s followed by 2 min rest. Behavioral response to CRD was measured by visual observation of the AWR by an observer blind to the experimental conditions, and AWR was scored as: 1 (normal behavior), 2 (contraction of abdominal muscles), 3 (lifting of abdominal wall), or 4 (body arching and lifting of pelvic structures).
Statistical analysis
Data were statistically analyzed by analysis of variance (ANOVA) and sequential differences between means were tested by Tukey contrast analysis at P < 0.05.
Competing interest
The authors declare that they have no competing interests.
Authors’ contributions
AQ carried out the path clamp studies; performed the statistical analysis and drafted the manuscript. DS and DT carried out rat behavior studies and molecular genetic studies. YL and XL were responsible for technical support; WY carried out western blot studies and was responsible for critical revision of the manuscript for important intellectual content. YY conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.