Strong epidemiological data indicate that chemotherapy-induced gut toxicity and pain occur in parallel, indicating common underlying mechanisms. We have recently outlined evidence suggesting that TLR4 signaling may contribute to both side effects. We therefore aimed to determine if genetic deletion of TLR4 improves chemotherapy-induced gut toxicity and pain. Forty-two female wild-type (WT) and 42 Tlr4 null (−/−) BALB/c mice weighing between 18 and 25 g (10–13 weeks) received a single 270 mg/kg (i.p.) dose of irinotecan hydrochloride or vehicle control and were killed at 6, 24, 48, 72, and 96 hours. Bacterial sequencing was conducted on cecal samples of control animals to determine the gut microbiome profile. Gut toxicity was assessed using validated clinical and histopathologic markers, permeability assays, and inflammatory markers. Chemotherapy-induced pain was assessed using the validated rodent facial grimace criteria, as well as immunologic markers of glial activation in the lumbar spinal cord. TLR4 deletion attenuated irinotecan-induced gut toxicity, with improvements in weight loss (P = 0.0003) and diarrhea (P < 0.0001). Crypt apoptosis was significantly decreased in BALB/c-Tlr4−/−billy mice (P < 0.0001), correlating with lower mucosal injury scores (P < 0.005). Intestinal permeability to FITC-dextran (4 kDa) and LPS translocation was greater in WT mice than in BALB/c-Tlr4−/−billy (P = 0.01 and P < 0.0001, respectively). GFAP staining in the lumbar spinal cord, indicative of astrocytic activation, was increased at 6 and 72 hours in WT mice compared with BALB/c-Tlr4−/−billy mice (P = 0.008, P = 0.01). These data indicate that TLR4 is uniquely positioned to mediate irinotecan-induced gut toxicity and pain, highlighting the possibility of a targetable gut/CNS axis for improved toxicity outcomes. Mol Cancer Ther; 15(6); 1376–86. ©2016 AACR.

Tissue injury can initiate bidirectional signaling between neurons, glia and immune cells that creates and amplifies pain. While the ability for neurotransmitters, neuropeptides, and cytokines to initiate and maintain pain has been extensively studied, recent work has identified a key role for reactive oxygen and nitrogen species (nitroxidative species), including superoxide, peroxynitrite, and hydrogen peroxide. In this review, we describe how nitroxidative species are generated after tissue injury, and the mechanisms by which they enhance neuroexcitability in pain pathways. Finally, we discuss potential therapeutic strategies for normalizing nitroxidative signaling, which may also enhance opioid analgesia, to help to alleviate the enormous burden of pathological pain.

Purine receptors have been implicated in central neurotransmission from nociceptive primary afferent neurons, and adenosine 5'tri-phosphate (ATP)-mediated currents in sensory neurons have been shown to be mediated by both P2X 3 and P2X 2/3 receptors. The aim of the present study was to quantitatively examine the distribution of P2X 2 and P2X 3 receptors in primary afferent cell bodies in the rat trigeminal ganglion, including those innervating the dura. In order to determine the classes of neurons that express these receptor subtypes, purine receptor immunoreactivity was examined for colocalisation with markers of myelinated (neurofilament 200; NF200) or mostly unmyelinated, nonpeptidergic fibres (Bandeiraea simplicifolia isolectin B4; IB4). 40 % of P2X 2 and 64 % of P2X 3 receptor-expressing cells were IB4 positive, and 33 % of P2X 2 and 31 % of P2X 3 receptor-expressing cells were NF200 positive. Approximately 40 % of cells expressing P2X 2 receptors also expressed P2X 3 receptors and vice versa. Trigeminal ganglion neurons innervating the dura mater were retrogradely labelled and 52 % of these neurons expressed either P2X 2 or P2X 3 or both receptors. These results are consistent with electrophysiological findings that P2X receptors exist on the central terminals of trigeminal afferent neurons, and provide evidence that afferents supplying the dura express both receptors. In addition, the data suggest specific differences exist in P2X receptor expression between the spinal and trigeminal nociceptive systems. Keywords purine receptors; trigeminal ganglia; sensory neuron; migraine; pain Purine receptors have been widely implicated in nociceptive processing (Burnstock, 2000;Khakh, 2001;Liu and Salter, 2005). The receptors are of two types: ionotropic (P2X) and metabotropic (P2Y), both of which are stimulated by adenosine 5'tri-phosphate (ATP). Seven P2X receptor subtypes have been cloned, and some occur as heteromultimers (e.g. P2X 2/3 , P2X 1/5 and P2X 4/6 receptors). In the spinal somatosensory system, all P2X receptor subtypes with the exception of P2X 7 are expressed in the spinal dorsal horn, the dorsal root ganglion (DRG), and the central terminals of primary afferent neurons (Burnstock, 2000;Khakh, 2001;North, 2002). In vivo and in vitro electrophysiological recordings from single dorsal horn neurons have implicated P2X receptors on primary afferent terminals in spinal nociceptive transmission (Nakatsuka and Gu, 2001;Nakatsuka et al., 2002;Nakatsuka et al., 2003; Correspondence details: Dr. Ernest Jennings Dept. Anatomy & Cell Biology The University of Melbourne Parkville, Victoria, 3010, Australia Tel: +61 3 8344 5802 Fax: +61 3 9347 9619 Email: e.jennings@unimelb.edu.au. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form...

Background: 5-HT3 antagonists, such as ondansetron (Zofran), retard colonic transit and provide effective relief of symptoms of diarrhea-predominant irritable bowel syndrome (IBS), but the mechanism by which ondansetron retards transit is unclear. What is clear is that the frequency of colonic migrating motor complexes (CMMCs) is reduced by ondansetron, which could account for reduced transit. Our aim was to determine whether acute depletion of 5-HT from enteric neurons would disrupt spontaneous CMMCs; and determine whether the sensitivity of ondansetron to reduce CMMC frequency would change in a 5-HT depleted preparation. Methods: Mice were injected with reserpine 24 hours prior to euthanasia to deplete neuronally synthesized 5-HT. Mechanical recordings were made from proximal and mid-distal regions of isolated whole mouse colon. Immunohistochemical staining for 5-HT was used to detect neuronal 5-HT. Key Results: Reserpine depleted all detectable 5-HT from enteric nerves. In whole colons, with mucosa and submucosal plexus removed, the frequency and amplitude of spontaneous CMMCs was no different between groups treated with or without reserpine. Surprisingly, in mucosa and submucosal plexus-free preparations, ondansetron was equally, or, significantly more effective at inhibiting CMMC frequency when compared to control preparations (containing 5-HT). Reserpine pretreatment had no effect on the sensitivity of ondansetron to inhibit CMMCs. Conclusions & Inferences: Endogenous 5-HT in enteric neurons (or the mucosa) is not required for the spontaneous generation or propagation of CMMCs. Furthermore, the primary mechanism by which ondansetron inhibits CMMC frequency is not mediated via the mucosa, submucosal plexus, or 5-HT in myenteric neurons.

Primary afferent neurons transduce distension of the bladder wall into action potentials that are relayed into the spinal cord and brain, where autonomic reflexes necessary for maintaining continence are coordinated with pathways involved in sensation. However, the relationship between spinal circuits involved with physiological and nociceptive signalling from the bladder has only been partially characterised. We used ex vivo bladder afferent recordings to characterise mechanosensitive afferent responses to graded distension (0-60 mm Hg) and retrograde tracing from the bladder wall to identify central axon projections within the dorsal horn of the lumbosacral (LS) spinal cord. Labelling of dorsal horn neurons with phosphorylated-MAP-kinase (pERK), combined with labelling for neurochemical markers (calbindin, calretinin, gamma aminobutyric acid, and parvalbumin) after in vivo bladder distension (20-60 mm Hg), was used to identify spinal cord circuits processing bladder afferent input. Ex vivo bladder distension evoked an increase in primary afferent output, and the recruitment of both low- and high-threshold mechanosensitive afferents. Retrograde tracing revealed bladder afferent projections that localised with pERK-immunoreactive dorsal horn neurons within the superficial laminae (superficial dorsal horn), dorsal gray commissure, and lateral collateral tracts of the LS spinal cord. Populations of pERK-immunoreactive neurons colabelled with calbindin, calretinin, or gamma aminobutyric acid, but not parvalbumin. Noxious bladder distension increased the percentage of pERK-immunoreactive neurons colabelled with calretinin. We identified LS spinal circuits supporting autonomic and nociceptive reflexes responsible for maintaining continence and bladder sensations. Our findings show for the first time that low- and high-threshold bladder afferents relay into similar dorsal horn circuits, with nociceptive signalling recruiting a larger number of neurons.

; CNS-central nervous system; CSF-cerebrospinal fluid; CCI-chronic constriction injury of the sciatic nerve; DH-SC-dorsal horn of the spinal cord; DTT-dithiothreitol; EDTA-ethylenediaminetetraacetic acid; EGTA-ethylene glycol-bis(β-aminoethyl ether)-tetraacetic acid; ELISA-enzymelinked immunosorbent assay; HEPES-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;

Mechanisms involved in the generation of spontaneous uterine contractions are not fully understood. Kit-expressing interstitial cells of Cajal are pacemakers of contractile rhythm in other visceral organs, and recent studies describe a role for Ca(2+)-activated Cl(-) currents as the initiating conductance in these cells. The existence and role of similar specialized pacemaker cells in the nonpregnant uterus remains undetermined. Spontaneous contractility patterns were characterized throughout the estrous cycle in isolated, nonpregnant mouse uteri using spatiotemporal mapping and tension recordings. During proestrus, estrus, and diestrus, contraction origin predominated in the oviduct end of the uterus, suggesting the existence of a dominant pacemaker site. Propagation speed of contractions during estrus and diestrus were significantly slower than in proestrus and metestrus. Five major patterns of activity were predominantly exhibited in particular stages: quiescent (diestrus), high-frequency phasic (proestrus), low-frequency phasic (estrus), multivariant (metestrus), and complex. Kit-immunopositive cells reminiscent of pacemaking ICCs were not consistently observed within the uterus. Niflumic acid (10 μM), anthracene-9-carboxylic acid (0.1-1 mM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (10 μM) each reduced the frequency of spontaneous contractions, suggesting involvement of Cl(-) channels in generating spontaneous uterine motor activity. It is unlikely that this conductance is generated by the Ca(2+)-activated Cl(-) channels, anoctamin-1 and CLCA4, as immunohistochemical labeling did not reveal protein expression within muscle or pacemaker cell networks. In summary, these results suggest that spontaneous uterine contractions may be generated by a Kit-negative pacemaker cell type or uterine myocytes, likely involving the activity of a yet-unidentified Cl(-) channel.

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