Background and PurposeOxaliplatin is a platinum‐based chemotherapeutic drug used as a first‐line therapy for colorectal cancer. However, its use is associated with severe gastrointestinal side‐effects resulting in dose limitations and/or cessation of treatment. In this study, we tested whether oxidative stress, caused by chronic oxaliplatin treatment, induces enteric neuronal damage and colonic dysmotility.Experimental ApproachOxaliplatin (3 mg·kg−1 per day) was administered in vivo to Balb/c mice intraperitoneally three times a week. The distal colon was collected at day 14 of treatment. Immunohistochemistry was performed in wholemount preparations of submucosal and myenteric ganglia. Neuromuscular transmission was studied by intracellular electrophysiology. Circular muscle tone was studied by force transducers. Colon propulsive activity studied in organ bath experiments and faeces were collected to measure water content.Key ResultsChronic in vivo oxaliplatin treatment resulted in increased formation of reactive oxygen species (O2ˉ), nitration of proteins, mitochondrial membrane depolarisation resulting in the release of cytochrome c, loss of neurons, increased inducible NOS expression and apoptosis in both the submucosal and myenteric plexuses of the colon. Oxaliplatin treatment enhanced NO‐mediated inhibitory junction potentials and altered the response of circular muscles to the NO donor, sodium nitroprusside. It also reduced the frequency of colonic migrating motor complexes and decreased circular muscle tone, effects reversed by the NO synthase inhibitor, Nω‐Nitro‐L‐arginine.Conclusion and ImplicationsOur study is the first to provide evidence that oxidative stress is a key player in enteric neuropathy and colonic dysmotility leading to symptoms of chronic constipation observed in oxaliplatin‐treated mice.

There are differences in the structure and function between regions of the colon. In patients with slow transit constipation the activity of all regions is markedly slowed. Counts of colonic neurones in slow transit constipation have been semiquantitative and led to varying results. We have applied new methods of quantification of markers in whole mounts of the colonic myenteric plexus to compare density of innervation between regions and between normal patients and those undergoing resection for severe constipation. Whole mounts of colonic myenteric plexus were made from specimens removed for cancer treatment (controls) and cases of severe constipation. All neurones were labelled by anti-human neuronal protein antibodies. Neurones synthesizing acetyl choline were labelled for choline acetyltransferase (ChAT) and those for nitric oxide by antisera to nitric oxide synthase (NOS). Four populations of neurones were distinguished and quantified according to the two selective markers, ChAT and NOS. In the normal major populations were NOS alone (51% of ascending colon neurones and 44% of descending colon neurones) and ChAT alone (41% ascending colon, 48% descending colon). Nitric oxide synthase/ChAT and NOS-/ChAT-comprised only small populations. In all regions in severe constipation, the percentage of NOS-only colonic myenteric neurones was raised (54% ascending colon, 49% descending colon) and ChAT only was reduced (36% ascending colon, 42% descending colon). The other populations were not changed. Accurate quantification of neuronal populations in whole mounts of human colon reveals inter-regional differences in innervation and marked changes in innervation in cases of very severe constipation.

Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gαi/oand β-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain.

Robinson AM, Sakkal S, Park A, Jovanovska V, Payne N, Carbone SE, Miller S, Bornstein JC, Bernard C, Boyd R, Nurgali K. Mesenchymal stem cells and conditioned medium avert enteric neuropathy and colon dysfunction in guinea pig TNBS-induced colitis. Am J Physiol Gastrointest Liver Physiol 307: G1115-G1129, 2014. First published October 9, 2014 doi:10.1152/ajpgi.00174.2014.-Damage to the enteric nervous system (ENS) associated with intestinal inflammation may underlie persistent alterations to gut functions, suggesting that enteric neurons are viable targets for novel therapies. Mesenchymal stem cells (MSCs) offer therapeutic benefits for attenuation of neurodegenerative diseases by homing to areas of inflammation and exhibiting neuroprotective, anti-inflammatory, and immunomodulatory properties. In culture, MSCs release soluble bioactive factors promoting neuronal survival and suppressing inflammation suggesting that MSC-conditioned medium (CM) provides essential factors to repair damaged tissues. We investigated whether MSC and CM treatments administered by enema attenuate 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced enteric neuropathy and motility dysfunction in the guinea pig colon. Guinea pigs were randomly assigned to experimental groups and received a single application of TNBS (30 mg/kg) followed by 1 ϫ 10 6 human bone marrow-derived MSCs, 300 l CM, or 300 l unconditioned medium 3 h later. After 7 days, the effect of these treatments on enteric neurons was assessed by histological, immunohistochemical, and motility analyses. MSC and CM treatments prevented inflammation-associated weight loss and gross morphological damage in the colon; decreased the quantity of immune infiltrate in the colonic wall (P Ͻ 0.01) and at the level of the myenteric ganglia (P Ͻ 0.001); prevented loss of myenteric neurons (P Ͻ 0.05) and damage to nerve processes, changes in ChAT, and nNOS immunoreactivity (P Ͻ 0.05); and alleviated inflammationinduced colonic dysmotility (contraction speed; P Ͻ 0.001, contractions/min; P Ͻ 0.05). These results provide strong evidence that both MSC and CM treatments can effectively prevent damage to the ENS and alleviate gut dysfunction caused by TNBS-induced colitis.

This is the first study to provide analyses of intestinal transit and whole colon motility in an animal model of spontaneous chronic colitis. We found that cholinergic and purinergic neuromuscular transmission, as well as the smooth muscle cell responses to cholinergic and nitrergic stimulation, is altered in the chronically inflamed Winnie mouse colon. The changes to intestinal transit and colonic function we identified in the Winnie mouse are similar to those seen in inflammatory bowel disease patients.