Cipriani, Gianluca

Abstract Background Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal ( ICC ) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying, whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. Methods Cultures of gastric ICC were treated with conditioned medium ( CM ) from activated bone marrow‐derived macrophages ( BMDM s) and the effect of CM was quantified by counting ICC per high‐powered field. Key Results Bone marrow‐derived macrophages were activated to a M1 or M2 phenotype confirmed by qRT ‐ PCR . Conditioned medium from M1 macrophages reduced ICC numbers by 41.1%, whereas M2‐ CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 that were significantly increased in M1‐ CM , including tumor necrosis factor alpha ( TNF ‐α). ELISA detected 0.697±0.03 ng mL −1 TNF ‐α in M1‐ CM . Recombinant mouse TNF ‐α reduced Kit expression and ICC numbers in a concentration‐dependent manner ( EC 50 = 0.817 ng mL −1 ). Blocking M1‐ CM TNF ‐α with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z‐ VAD .fmk partly preserved ICC numbers (cells/field; 6.63±1.04, 9.82±1.80 w/Z‐ VAD .fmk, n=6, P <.05). Conclusions & Inferences This work demonstrates that TNF ‐α secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase‐dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis.

AbstractBackgroundOtilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well‐characterized, little is known about the effects of chronic administration ofOBon enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function.MethodsAdult Sprague–Dawley rats were treated withOBin drinking water at a dose of 2 mg/kg for 30 days. The colons ofOB‐treated and age‐matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology.Key ResultsUsing immunohistochemistry, chronicOBadministration did not change total neuron number, assessed by anti‐HuIR, but resulted in a significant increase inNK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. ChronicOBadministration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonicICC, detected by KitIR, were not altered nor were slow waves or smooth muscle membrane potential.Conclusions & InferencesChronic treatment withOBcaused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.