The gastrointestinal tract is innervated by intrinsic and extrinsic visceral afferent and visceral motor afferent neurons (Niu et al. 2020). Visceral afferent neurons convey gastrointestinal information to the brain through the vagal and spinal afferents, with their cell bodies located within the NG and DRG, respectively. To the best of our knowledge, this is the first report to compare the expression patterns of the thermosensitive TRPV2 and TRPV1 in the rat distal colon, DRG, and NG in normal and TNBS-induced colitis model.
We previously reported that TRPV2 was expressed in intrinsic primary afferent neurons and inhibitory motor neurons in the normal myenteric plexus of the rat esophagus (Matsumoto et al. 2021). Mihara et al. have demonstrated that TRPV2 was expressed in CGRP-positive intrinsic/extrinsic sensory and nNOS-positive intrinsic inhibitory motor neurons in murine small intestines and stomachs (Mihara et al. 2010 and 2013). In this study, we clearly identified subpopulations of intrinsic TRPV2 neurons, approximately half of the TRPV2-positive neurons were intrinsic primary afferent neurons and half were inhibitory motor neurons in the myenteric plexus. Although several studies have demonstrated that TRPV1 is limited to extrinsic primary afferent neurons, some studies have argued for intrinsic expression of TRPV1 in the enteric nervous system (Buckinx et al. 2013). In this study, TRPV1-imuunoreactive cell bodies were not detected in the myenteric plexus of normal and colitis model, suggesting that TRPV1-positive nerve fibers are not of intrinsic origin in the rat distal colon.
TRPV2 is expressed in both neuronal and non-neuronal cells such as mast cells, aortic smooth muscle cells, and monocytes/macrophages (Perálvarez et al. 2013). TRPV2 expression in macrophages has mainly been demonstrated in resident populations in peripheral organs, such as the liver, skin, lung, and testis (Eubler et al. 2021). In the gastrointestinal tract, non-neuronal TRPV2 is predominantly expressed in macrophages in the esophagus and oral cavity (Matsumoto et al. 2021; Shimohira et al. 2009). We confirmed that TRPV2-immunoreactive cells were mainly co-localized with ED2-positive resident macrophages in the mucosa of normal and colitis rats. Non-neuronal TRPV2 regulates innate and adaptive immune responses (Santoni et al. 2013). TRPV2 is essential for phagocytosis and chemotaxis in macrophages (Nagasawa et al. 2007; Link et al. 2010). Issa et al. have reported less severe intestinal inflammation in TRPV2-deficient mice than wild-type mice in a murine colitis model because of the reduced infiltration of macrophages, suggesting that the TRPV2 pathway plays a key role in the development of colitis (Issa et al. 2014). Further studies are required to elucidate the role of TRPV2 in the progression of intestinal inflammation in macrophages.
Spinal afferents innervating the distal colon originate from the lumbosacral region and play a major role in visceral hypersensitivity following colitis (Abdullah et al. 2020; Robinson et al. 2004). Vagal afferents may play a complex role in visceral pain processes (Chen et al. 2008). As previously reported, TRPV1 is expressed in the spinal and vagal afferents innervating the distal colon in retrograde labeling and/or functional experiments (Hong et al. 2011; Christianson et al. 2006). However, the origin of TRPV2 positive primary afferent innervation in the rat distal colon has not been analyzed in detail. Consistent with these previous reports, the retrograde tracer-labeled colonic neurons were immunopositive for TRPV1, with approximately 40% and 46% of TRPV1 neurons in the DRG and NG innervating the distal colon, respectively. Meanwhile, we observed that approximately 23% and 65% of TRPV2 neurons in the DRG and NG innervate the distal colon, respectively. The immunohistochemical results of the myenteric plexus and extrinsic primary afferent neurons suggest that TRPV2 and TRPV1 immunopositive nerve fibers in the distal colon are exogenous primary afferent neuronal projections from the DRG and NG.
TRPV1 immunopositive cells have small cell bodies, and highly co-localized with the C-fiber marker IB4 but not the A-fiber marker NF200 in normal rat DRG (Tonelotto et al. 2019). As previously reported, almost all middle- and large-sized TRPV2 neurons labelled with NF200 in the normal rat DRG (Lewinter et al. 2004). We confirmed that TRPV2 immunopositive cells had large- and middle-sized cell bodies and highly co-localized with NF200 but not with IB4 in normal and TNBS-treated rat DRG. Meanwhile, TRPV1 positive cells were small and highly co-localized with IB4 in normal and TNBS-treated rat DRG. Hence, TRPV2 and TRPV1 are expressed in the Aδ-/Aβ- and C-fibres of spinal afferent neurons, respectively.
Primary sensory neurons contain neuropeptides including CGRP and SP, which contribute to pain transduction. TRPV1 can promote the release of CGRP and SP from spinal afferent nerve terminals of DRG neurons under TNBS-induced visceral hypersensitivity conditions (Patapoutian A et al. 2009). Here, we confirmed that TRPV1 neurons were highly co-localized with SP and CGRP in normal and TNBS-treated rat DRG. As previously reported, approximately 40% of TRPV2 neurons co-localized with CGRP in the normal mouse DRG (Hsieh et al. 2012). Middle-sized TRPV2 neurons highly co-localized with SP (90%), but partially with CGRP (50%). SP and CGRP are the principal peptides released mainly from Aδ- and C-fiber axons associated with the DRG (Feng et al. 2012). These results suggest that both TRPV2-positive Aδ- and TRPV1-positive C-fibers are peptidergic neurons that contribute to the release of CGRP and SP during intestinal inflammation.
It has been reported that vagal afferent neuron, mainly consist of NG, is associated with visceral hypersensitivity in rats (Gschossmann et al. 2002; Chen et al. 2008; Kupari et al. 2019). Low-intensity electrical vagal stimulation of Aδ-fibers reduced the VMR to CRD, but high-intensity electrical vagal stimulation of C-fibers had no effect in normal rats (Chen et al. 2008). TRPV1 neurons co-localized with IB4 and NF200 in rat NG (Sun et al. 2009). However, TRPV2-neurons in the NG remain uncharacterized with respect to distribution and co-localization with IB4 and NF200. Double immunofluorescence analysis has suggested that co-expression of TRPV2 and TRPV1 is abundant in the NG, but not in the DRG (Ichikawa and Sugimoto 2003). However, we could not conduct co-expression experiments of TRPV2 and TRPV1 because the primary antibodies of TRPV2 and TRPV1 were raised in the same host species. We identified that both TRPV2 and TRPV1 co-localized with IB4-positve unmyelinated C-fibers and NF200-positive myelinated A-fibers in normal and TNBS-treated rats. By contrast, TRPV2 and TRPV1 expressed neurons were clearly different in the normal and TNBS-treated rat DRG. Thus, TRPV2 and TRPV1 have demonstrated different properties in spinal afferent neurons, but similar properties in vagal afferent neurons.
Peripheral inflammation has been reported to increase TRPV2 and TRPV1 expression in the rat DRG (Shimosato et al. 2005; De Schepper et al. 2008; Miranda et al. 2007). Here, TNBS-induced intestinal inflammation caused a significant increase in TRPV2 and TRPV1 expression in DRG and NG. However, the subpopulations of TRPV2 and TRPV1 expressed neurons did not change. These results suggest that TNBS-induced intestinal inflammation affects the number of TRPV2 and TRPV1 neurons, but not their characteristics. ERK1/2 activation in afferent neurons has been suggested to be involved in peripheral sensitization in TNBS-induced experimental colitis. ERK1/2 activation also mediates upregulation of TRP expression (Van den Eynde C et al. 2021). We examined the phosphorylation of ERK1/2 in the DRG and NG five days after intracolonic TNBS treatment. The number of p-ERK1/2-immunoreactive neurons that co-localized with TRPV2 and TRPV1 in TNBS-treated rats was significantly higher than that in normal rats in the DRG and NG. Therefore, DRG and NG neurons innervating the rat distal colon probably respond to various noxious stimuli in their peripheral nerve endings through TRPV1 and TRPV2 activation, and transduce nociceptive information to the central nervous system.
TRPV1 has attracted international attention as a therapeutic target for visceral hypersensitivity because of its high expression in C-fibers, which play an important role in visceral sensation (Balemans et al. 2017); a TRPV1 antagonist attenuates visceral hypersensitivity in TNBS-treated rats (Miranda et al. 2007). Consistent with previous findings, the TRPV1 selective antagonist BCTC significantly attenuated visceral hypersensitivity in TNBS-treated rats, indicating a role of TRPV1 in exacerbating visceral hypersensitivity in pathological conditions. However, no reports have been conducted on the role of TRPV2 in visceral sensing in humans and rodents. TRPV2-expressing afferents convey nociceptive mechanical information to the spinal cord and participate in the development of mechanical hyperalgesia and allodynia in rats (Petitjean et al. 2014). TRPV2 on primary afferent neurons has also been reported to contribute to mechanosensitive mechanisms in TRPV2-defiecient mice (Katanosaka et al. 2018). Myelinated Aδ-fibers that respond to nociceptive mechanical and thermal stimuli also transmit visceral sensations to the center, although the role of Aβ-fibers that detect low-threshold mechanical stimuli remains unclear. Thus, we used tranilast to inhibit TRPV2-expressing Aδ-/Aβ-fibers of the spinal primary afferents/intrinsic primary afferents and evaluated their effects on TNBS-induced visceral hypersensitivity. We observed that tranilast attenuated TNBS-induced visceral hypersensitivity compared to the control (normal vehicle-treated group) at all pressures.
This study had several limitations. First, TRPV2 is expressed not only in spinal/vagal primary afferent neurons but also in macrophages, intrinsic primary afferent neurons, and inhibitory motor neurons. However, we could not clearly determine the contribution of each TRPV2-expressed site to TNBS-induced visceral hypersensitivity. Second, further mechanistic studies including TRPV2 knockout mice might be needed to determine the involvement of TRPV2 in visceral hypersensitivity because selective TRPV2 antagonists are still not commercially available. Third, we did not identify a difference between TRPV2 and TRPV1 in vagal afferent neurons in the development of visceral hypersensitivity.
Our findings reveal that TRPV2 is expressed in resident macrophages, intrinsic primary afferent neurons, inhibitory motor neurons, and Aδ-/Aβ-fibers of spinal afferent and vagal afferent neurons innervating the distal colon. In contrast, TRPV1 is expressed in the C-fibers of spinal afferent and vagal primary afferent neurons innervating the distal colon but not intrinsic neurons. Sensitization and upregulation of TRPV2 and TRPV1 contributes to visceral hypersensitivity in an experimental colitis model.