Background & Aims Altered gastrointestinal motility is associated with significant morbidity and health care costs. Toll-like receptors (TLR) regulate intestinal homeostasis. We examined the roles of TLR4 signaling in survival of enteric neurons and gastrointestinal motility. Methods We assessed changes in intestinal motility by assessing stool frequency, bead expulsion, and isometric muscle recordings of colonic longitudinal muscle strips from mice that do not express TLR4 (Tlr4^Lps-d or TLR4^−/−) or Myd88 (Myd88^−/−), in wild-type germ-free mice or wild-type mice depleted of the microbiota, and in mice with neural crest-specific deletion of Myd88 (Wnt1Cre^+/−/Myd88^fl/fl). We studied the effects of the TLR4 agonist lipopolysaccharide (LPS) on survival of cultured, immortalized fetal enteric neurons and enteric neuronal cells isolated from wild-type and Tlr4^Lps-d mice at embryonic day 13.5. Results There was a significant delay in gastrointestinal motility and reduced numbers of nitrergic neurons in TLR4^Lps-d, TLR4^−/−, and Myd88^−/− mice compared with wild-type mice. A similar phenotype was observed in germ-free mice, mice depleted of intestinal microbiota, and Wnt1Cre^+/−/Myd88^fl/fl mice. Incubation of enteric neuronal cells with LPS led to activation of the transcription factor nuclear factor (NF)-κB and increased cell survival. Conclusions Interactions between enteric neurons and microbes increases neuron survival and gastrointestinal motility in mice. LPS activation of TLR4 and NF-κB appears to promote survival of enteric neurons. Factors that regulate TLR4 signaling in neurons might be developed to alter gastrointestinal motility.

Diabetes can result in loss of enteric neurons and subsequent gastrointestinal complications. The mechanism of enteric neuronal loss in diabetes is not known. We examined the effects of hyperglycemia on enteric neuronal survival and the effects of glial cell line–derived neurotrophic factor (GDNF) on modulating this survival. Exposure of primary enteric neurons to 20 mM glucose (hyperglycemia) for 24 hours resulted in a significant increase in apoptosis compared with 5 mM glucose (normoglycemia). Exposure to 20 mM glucose resulted in decreased Akt phosphorylation and enhanced nuclear translocation of forkhead box O3a (FOXO3a). Treatment of enteric neurons with GDNF ameliorated these changes. In streptozotocin-induced diabetic mice, there was evidence of myenteric neuronal apoptosis and reduced Akt phosphorylation. Diabetic mice had loss of NADPH diaphorase–stained myenteric neurons, delayed gastric emptying, and increased intestinal transit time. The pathophysiological effects of hyperglycemia (apoptosis, reduced Akt phosphorylation, loss of inhibitory neurons, motility changes) were reversed in diabetic glial fibrillary acidic protein–GDNF (GFAP-GDNF) Tg mice. In conclusion, we demonstrate that hyperglycemia induces neuronal loss through a reduction in Akt-mediated survival signaling and that these effects are reversed by GDNF. GDNF may be a potential therapeutic target for the gastrointestinal motility disorders related to diabetes.

Background Gastrointestinal dysfunction is very common in diabetic patients. We assessed the changes in the colonic enteric nervous system using colectomy specimens and intestinal biopsies from diabetic subjects and age-matched controls. Methods In control and diabetic colons, we determined the total ganglion area (hematoxylin–eosin staining), changes in neuronal markers–protein gene product 9.5, peripherin, neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), choline acetyl transferase (ChAT) and vasoactive intestinal peptide (by immunostaining), apoptosis (cleaved caspase-3 staining) and reduced glutathione levels. Superoxide dismutase mRNA was determined in enteric ganglia isolated by laser capture micro dissection. Isometric muscle recording was used to assess contraction and relaxation responses of colonic circular muscle strips. Apoptosis in enteric neurons under hyperglycemia in vitro was determined by cleaved caspase-3 Western blotting and protective effects of lipoic acid were evaluated. Key Results Diabetic subjects had higher incidence of lower gastrointestinal symptoms like constipation and diarrhea at baseline prior to surgery. Diabetic ganglia displayed significant decrease in ganglion size due to enhanced apoptosis and loss of peripherin, nNOS, NPY, and ChAT neurons. Reduced glutathione levels in the diabetic colon (HbA1C > 7%) were significantly less than the control, indicating increased oxidative stress. Colonic circular muscle strips from diabetic subjects showed impaired contraction and relaxation responses compared with the healthy controls. Hyperglycemia-induced cleaved caspase-3 in enteric neurons was reversed by lipoic acid. Conclusions & Inferences Our data demonstrate loss of enteric neurons in the colon due to increased oxidative stress and apoptosis which may cause the motility disturbances seen in human diabetes. Antioxidants may be of therapeutic value for preventing motility disorders in diabetes.