Essential Role of Mast Cells in the Visceral Hyperalgesia Induced by T. spiralis Infection and Stress in Rats

Mast cells (MCs) deficient rats (Ws/Ws) were used to investigate the roles of MCs in visceral hyperalgesia. Ws/Ws and wild control (+/+) rats were exposed to T. spiralis or submitted to acute cold restraint stress (ACRS). Levels of proteinase-activated receptor 2 (PAR2) and nerve growth factor (NGF) were determined by immunoblots and RT-PCR analysis, and the putative signal pathways including phosphorylated extracellular-regulated kinase (pERK1/2) and transient receptor potential vanilloid receptor 1 (TRPV1) were further identified. Visceral hyperalgesia triggered by ACRS was observed only in +/+ rats. The increased expression of PAR2 and NGF was observed only in +/+ rats induced by T. spiralis and ACRS. The activation of pERK1/2 induced by ACRS occurred only in +/+ rats. However, a significant increase of TRPV1 induced by T. spiralis and ACRS was observed only in +/+ rats. The activation of PAR2 and NGF via both TRPV1 and pERK1/2 signal pathway is dependent on MCs in ACRS-induced visceral hyperalgesia rats.


Introduction
Irritable bowel syndrome (IBS) is a common gastrointestinal disorder seen by gastroenterologists. Patients classically present with chronic abdominal pain, associated with an alteration in bowel habits, and visceral hyperalgesia which is generally considered to be a hallmark of IBS by their lowered thresholds for pain, increased intensity of sensations, and/or exaggerated visceromotor response to colorectal distension (CRD) [1,2]; however, the underlying pathogeneses of visceral hyperalgesia are still unknown.
At present, growing evidences have indicated that mast cells (MCs) play an important role in visceral hyperalgesia [3,4]. MCs take part of host defense against parasitic and bacterial infections. A subset of patients with IBS have an increased number of MCs in the colonic mucosa [5]. It has also been shown that MCs infiltration and release of mediators in the close proximity of mucosal innervation may contribute to abdominal pain perception in IBS patients [6]. Accumulated evidences have indicated that MCs tryptase is known to be involved in promoting pain and visceral hyperalgesia by activating the proteinase-activated receptor 2 (PAR2) which is expressed on primary afferent nociceptive neurons [7]. Nerve growth factor (NGF) can be released from MCs due to stimulus-induced degranulation, which also plays a pivotal role in colonic hyperalgesia [8].
On the processing of signal transduction of pain sensation, the transient receptor potential vanilloid receptor 1 (TRPV1) is also expressed and colocalized with PAR2 on Cfiber primary sensory afferent neurons [9]. Previous studies have shown that upregulation of PAR2 and NGF enhance the activation of TRPV1 channel [10]. Moreover, as a sensor for thermal and acidic nociception, TRPV1 plays critical roles in the processing of visceral inflammatory pain [11]. It has been demonstrated that pancreatic pronociceptive stimuli with PAR2 agonists cause extracellular-regulated kinase (ERK 1 and ERK2) phosphorylation in the spinal dorsal horn through activation of TRPV1 channels [12]. Noxious stimuli cause phosphorylation of ERK (pERK) in the afferent neuron that contributes to facilitation of pain sensation and is often used as an immediate marker for excitation of afferent neuron following colonic nociception [12]. It has been revealed that NGF activates ERK1/2 and pERK1/2 inhibition decreases excitability in DRG neurons 2 Mediators of Inflammation in culture [13]. Although the relationship between visceral hyperalgesia and MCs in IBS animal models and patients has been reported from several laboratories, the essential role of MCs in the progress of various stimulate is not well understood. In the present study, on the basis of using MCs deficient rats, we attempted to identify colonic (PAR2 and NGF) and peripheral sensory neuronic alterations (pERK1/2 and TRPV1) that can be involved in the visceral hyperalgesia triggered by both intestinal infection and stress.

Animals.
Male MCs deficient rats (Ws/Ws) and their normal wild-type littermates (+/+) were obtained from TGC Inc. (Kanagawa, Japan). Rats were housed in standard polypropylene cages containing 2.5 cm of wood chip bedding material, which was maintained at 22 • C with an automatic 12 hour light/dark cycle. Rats received a standard laboratory diet and tap water ad libitum. The experiments were conducted when the rats reached approximately 12 weeks of age. All procedures were aimed to minimize both animal number and suffering of the animals and were approved by the Animal Care Committee of Peking University.

T. spiralis Induced Colitis.
Rats were induced by administering 1.0 mL of 0.9% saline solution containing 1500 T. spiralis larvae by gavage. An equivalent volume of vehicle (saline) was administered into control rats. The postinfection (PI) rats were allowed to have a recovery during 100 days period following administration.

Acute Cold Restraint Stress Procedure (ACRS).
Briefly [14], 100 days after recovery, one half of the control and one half of the PI rats were restrained in individual polymethyl methacrylate restraint cages, and these animals were designated as ACRS and PI + ACRS groups. The animals were then placed in their cold home cages at 4 • C for 2 hour. ACRS was routinely performed between 10:00 AM and 12:00 AM.

Visceromotor
Response to CRD. Sensitivity to CRD was determined using the abdominal withdrawal reflex (AWR) as previously widely described. The rats received a standard CRD procedure, and the first balloon dilation used was 1 mL, and then increasing phases of distension (0.2 mL ascending increments) were applied for 20 second every 5 minutes until the AWR score reached 3. This evaluation was performed by three independent observers, and the AWR score was assigned as follows: 0: no behavioral response to distension; 1: brief head movements followed by cessation of movement; 2: contraction of abdominal muscle without lifting of abdomen; 3: lifting of abdomen; 4: body arching and lifting of pelvic structure.

2.5.
Immunofluorescence. L6S1 DRGs segments were removed and fixed for overnight in 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer saline (PBS) at 4 • C and then cryoprotected overnight in 30% sucrose in PBS. The tissue was embedded in Tissue-Tek OCT compound medium (Sakura Finetek, USA) and frozen in isopentane at −45 • C. Cryostat sections (10 μm) were postfixed with acetone (10 minute, −20 • C), and permeabilised with 0.3% Triton X-100 for 2 hours, and then blocked with 10% normal goat serum in PBS with 0.3% Triton X-100 for 30 minutes at room temperature. Sections were incubated overnight at 4 • C with rat anti-TRPV1 (1 : 500; Chemicon) and rabbit anti-phospho (p) ERK1/2 (pERK; 1 : 1000; Cell Signaling Technology). Sections were then washed with PBS and incubated for 30 minutes at 37 • C with FITC-conjugated goat anti-rat (1 : 100) and FITC-conjugated goat anti-rabbit (1 : 100) antibodies (Sigma). Sections were used to counterstain with DAPIfluoromount (SouthernBiotech, USA). Digital images of five slices per individual DRG per animal were captured under the same parameters in the fluorescence microscope (Leica DM3000, Leica Microsystems, Germany) at lower magnification (×200 objective). The mean gray level intensity for a region of interest of the images was determined by using Image Pro Plus 6.0 image analysis software system (Media cybernetics, Silver Spring, MD, USA).

RT-PCR.
The RNA extraction from colonic tissues was carried out using the RNeasy Micro Kit (QIAGEN) according to manufacturer's instructions. RNA concentration was determined by absorbance at 260 nm, and its integrity was verified by electrophoresis. The first-strand cDNA was synthesized from 2 μL of total RNA by using SuperScript II RNase H reverse transcription (Invitrogen) and oligo-(dT) 12-18 primers according to its protocol. Quantitative PCR was carried out using following primer while using Mediators of Inflammation 3 18S ribosomal RNA as an endogenous control. Samples were amplified in duplicate using the following thermal cycling conditions: 95 • C for 3 minutes followed by 40 cycles of amplification at 95 • C for 15 seconds and then 60 • C for 1 minute to allow for denaturation and annealing-extension. After amplification, a dissociation curve was plotted against melting temperature to ensure amplification of a single product. Comparative cycle threshold values were recorded, and the relative expression of mRNA species was then quantified in duplicate using the 2 −ΔΔCT method using iCycler optical system interface software (v2.0, Bio-Rad).
Primers for target genes included the followings:

Visceral Hyperalgesia Induced by ACRS Dependent on
MCs. In response to CRD, the distension volume to reach AWR score = 3 was significantly lower in +/+ rats triggered by transient T. spiralis intestinal infection (P < 0.01) and ACRS (P < 0.01). Although T. spiralis intestinal infection decreases visceral threshold of pain sensitivity to CRD in Ws/Ws rats (P < 0.01), it seems to be inoperative for Ws/Ws rats triggered by ACRS (Figure 1).

The Increased Transcription and Expression of PAR2 Induced by T. spiralis Infection and ACRS in Distal Colon
Dependent on MCs. The changes in PAR2 expression in rats were confirmed using western blot analysis. As shown in Figures 2(a) and 2(b), the protein level of PAR2 increased to 1.38 ± 0.03 (P = 0.001) and 1.32 ± 0.04 (P = 0.004) in distal colon in +/+ rats induced by ACRS, and T. spiralis intestinal infection, respectively, compares with controls (1.07 ± 0.03) by quantitative densitometry of the immunoblots. Although we only see a significant difference (P = 0.036) between the PI group (1.82 ± 0.15) and control group (1.00 ± 0.09) in +/+ rats (Figure 2(c)) in PAR2 mRNA levels, neither ACRS nor T. spiralis intestinal infection had an effect on both PAR2 protein and mRNA levels in Ws/Ws rat.
ERK phosphorylation was further confirmed by western blot with a phospho-ERK1/2 antibody. The mean immunoblot band density for pERK1/2 was greatly increased not only in +/+ rats induced by ACRS (P < 0.01) and T. spiralis intestinal infection (P < 0.05) but also in Ws/Ws rats induced by

The Increased TRPV1 Expression Induced by T. spiralis Intestinal Infection and ACRS in L6S1 DRGs in +/+, but
Not Ws/Ws, Rats. Immunofluorescence was used to assess the expression of TRPV1 in L6S1 DRG neurons. As shown in Figures 5(a)-5(g), in +/+ rats, the cytoplasmic TRPV1 IR signal was (0.047 ± 0.002) in L6S1 DRGs from control animals, whereas it was significantly increased to 0.09±0.007 (P = 0.004) in ACRS group and 0.100 ± 0.016 (P = 0.001) in PI group. No changes were observed in TRPV1 IR signal in Ws/Ws rats. The TRPV1 expression was further confirmed by western blot analysis. Compared with control group, the mean immunoblot band density for TRPV1 was also greatly increased not only in the ACRS group (P < 0.05) but also in the PI group (P < 0.05) in +/+ rats. In contrast, there is no significant difference observed in TRPV1 protein levels in Ws/Ws rats (Figures 5(h) and 5(i)).

Discussion
In  noticed that, in the wild control rats, the MCs of the control group and infection group retained a full complement of electron-dense secretory granules, while those of the stress groups underwent piecemeal degranulation [15]. Thus we deduced that visceral hyperalgesia induced by ACRS is dependent on MCs, although the accurate pathogenesis is still unknown. We investigated the role of PAR2 and NGF in rats due to their close relationship with MCs. MCs are some important proinflammatory cells, which not only participate in hostdefense immune responses but also regulate the functions of peripheral nerves and smooth muscles in the gastrointestinal tract [16]. Upon activation, mucosal MCs released act on PAR2 to sensitize sensory afferents in the proximity [17]. PAR2, a G-protein-coupled receptor for trypsin and MCs tryptase, has been identified in colonic myocytes, enterocytes, enteric neurons, terminals of mesenteric afferent nerves, and immune cells [18]. It has been demonstrated that activation of PAR2 on the plasma membrane of nociceptive DRG neurons innervating the mouse colon leads to sustained hyperexcitability, and ERK1/2 mediates the PAR2-induced hyperexcitability [19]. Intracolonic administration of the synthetic selective PAR2 agonist in rats increases paracellular permeability and produces visceral hyperalgesia [17]. PAR2mediated dysfunction of colonic epithelial barrier and subsequent allodynia or hyperalgesia may play an important role  in the pathogenesis of IBS [20]. Our results demonstrated that the increased expression of PAR2 was induced by both T. spiralis infection and ACRS in distal colon in +/+ but not Ws/Ws rats. If the results of visceral pain threshold detected by AWR score are taken into account as shown in Figure 1, we concluded the upregulation of PAR2 induced by ACRS in visceral hyperalgesia dependent on the activation of MCs. Moreover, the activation of MCs plays an important role in the upregulation of PAR2 induced by T. spiralis infection in visceral hyperalgesia. NGF may be produced in part by MCs and recognized as a potent immunomodulator, behaving like a bridge between neuronal and immune cells [21]. The peripheral stress mediator norepinephrine induces visceral hypersensitivity to CRD in response to heterotypic chronic stress by increasing the expression of NGF in the colon wall [22]. NGF can further sensitize afferent nociceptors directly by binding to the high-affinity receptor tyrosine kinase A (trkA) expressed on primary afferent neurons and indirectly by triggering MCs degranulation [23]. Moreover, there are interactions among them; that is, the increased NGF production can be induced by activation of MCs, whereas NGF induces histamine release from MCs [24,25]. In addition, anti-NGF treatments were effective in preventing the motor alterations induced by the T. spiralis infection, that is, inhibited increased spontaneous motor activity and reversed Mediators of Inflammation 7 altered response to cholecystokinin (CCK) [26]. In this study we observed that the upregulation of NGF mRNA in intestine of +/+ rats, but not Ws/Ws rats, was induced by T. spiralis infection and stress. If the results of visceral pain threshold detected by AWR score are taken into account as shown in Figure 1, we concluded the upregulation of NGF induced by ACRS in visceral hyperalgesia dependent on the activation of MCs. At least we can infer that the interaction between MCs and NGF plays an important role in visceral hyperalgesia. Moreover, the activation of MCs may also be involved in the upregulation of NGF induced by T. spiralis infection in visceral hyperalgesia. As the NGF protein level is undermeasured with western blot method, we only detected the NGF mRNA by RT-PCR. This is the deficiency in our studies.
DRG is the primary afferent neuron in the information transmission of visceral sensation. Nociceptive processing in the visceral afferent neuronal pathways is thought to be mediated primarily via ERK and TRPV1 signal pathway. Thus, in our studies the changes of ERK and TRPV1 were investigated in rats. Visceral stimuli cause prompt phosphorylation of ERK in the spinal dorsal horn that contributes to facilitation of pain sensation and is often used as an immediate marker for excitation of spinal neurons following colonic nociception [27]. ERK1/2 is involved in the transduction of NGF neurotrophic signals by interactions with TrkA [28]. ERKs are phosphorylated in the nervous system after visceral stimulation or inflammation and play roles in central sensitization and pain hypersensitivity [29]. Our results demonstrated that phosphorylation of ERK1/2 can be induced by T. spiralis infections; however, the ACRS evokes phosphorylation of ERK1/2 in L6S1 DRGs dependent on MCs.
The TRPV1 pathway may also plays an important role in the process of information transfer from the peripheric receptor to central nervous system. Several studies indicated that TRPV1 plays a critical effect in visceral hyperalgesia and pain in IBS which may be associated with the increased MCs [30]. And evidences indicated that stress-induced visceral hyperalgesia in rats occurs in the absence of overt inflammation and dependents on MCs degranulation and subsequent TRPV1 activation [5]. PAR2 activation has been proven to sensitize several downstream TRPV1 channels via its G-protein-coupled receptor on sensory afferent to induce hyperalgesia [31]. If the results of visceral pain threshold detected by AWR score are taken into account as shown in Figure 1, we concluded the increased TRPV1 expression in DRGs induced by ACRS in visceral hyperalgesia dependent on the activation of MCs. Moreover, the activation of MCs also plays an important role in the increased TRPV1 expression in DRGs induced by T. spiralis infection in visceral hyperalgesia.

Conclusions
Our results have shown that the visceral hyperalgesia can not been triggered by stress in MCs deficient rats, although both stress and infection play an important role in visceral hyperalgesia in wild control rats. We also found that the upregulation of mediators (PAR2 and NGF) and signal proteins (pERK1/2 and TRPV1) has a close relationship with the presence of MCs. Our studies provide new evidence that the activation of PAR2 and NGF via both TRPV1 and pERK1/2 signal pathway is dependent on MCs in stressinduced visceral hyperalgesia rats. And MCs also play an important role in infection-induced visceral hyperalgesia rats.