Identification of intrinsic primary afferent neurons in mouse jejunum.

BACKGROUND: The gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut-brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum. METHODS: Advillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin-expressing enteric neurons using confocal microscopy, calcium imaging, and whole-cell patch-clamp electrophysiology. KEY RESULTS: Advillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene-related peptide (CGRP), but not nNOS. CONCLUSIONS AND INFERENCES: Advillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.

Change in Populations of Macrophages Promotes Development of Delayed Gastric Emptying in Mice.

BACKGROUND & AIMS: Muscularis propria macrophages lie close to cells that regulate gastrointestinal motor function, including interstitial cells of Cajal (ICC) and myenteric neurons. In animal models of diabetic gastroparesis, development of delayed gastric emptying has been associated with loss of macrophages that express cytoprotective markers and reduced networks of ICC. Mice with long-term diabetes and normal gastric emptying have macrophages that express anti-inflammatory markers and have normal gastric ICC. Mice homozygous for the osteopetrosis spontaneous mutation in the colony-stimulating factor 1 gene (Csf1op/op) do not have macrophages; when they are given streptozotocin to induce diabetes, they do not develop delayed gastric emptying. We investigated whether population of the gastric muscularis propria of diabetic Csf1op/op mice with macrophages is necessary to change gastric emptying, ICC, and myenteric neurons and investigated the macrophage-derived factors that determine whether diabetic mice do or do not develop delayed gastric emptying. METHODS: Wild-type and Csf1op/op mice were given streptozotocin to induce diabetes. Some Csf1op/op mice were given daily intraperitoneal injections of CSF1 for 7 weeks; gastric tissues were collected and cellular distributions were analyzed by immunohistochemistry. CD45+, CD11b+, F4/80+ macrophages were dissociated from gastric muscularis propria, isolated by flow cytometry and analyzed by quantitative real-time polymerase chain reaction. Cultured gastric muscularis propria from Csf1op/op mice was exposed to medium that was conditioned by culture with bone marrow-derived macrophages from wild-type mice. RESULTS: Gastric muscularis propria from Csf1op/op mice given CSF1 contained macrophages; 11 of 15 diabetic mice given CSF1 developed delayed gastric emptying and had damaged ICC. In non-diabetic Csf1op/op mice, administration of CSF1 reduced numbers of gastric myenteric neurons but did not affect the proportion of nitrergic neurons or ICC. In diabetic Csf1op/op mice given CSF1 that developed delayed gastric emptying, the proportion of nitrergic neurons was the same as in non-diabetic wild-type controls. Medium conditioned by macrophages previously exposed to oxidative injury caused damage to ICC in cultured gastric muscularis propria from Csf1op/op mice; neutralizing antibodies against IL6R or TNF prevented this damage to ICC. CD45+, CD11b+, and F4/80+ macrophages isolated from diabetic wild-type mice with delayed gastric emptying expressed higher levels of messenger RNAs encoding inflammatory markers (IL6 and inducible nitric oxide synthase) and lower levels of messenger RNAs encoding markers of anti-inflammatory cells (heme oxygenase 1, arginase 1, and FIZZ1) than macrophages isolated from diabetic mice with normal gastric emptying. CONCLUSIONS: In studies of Csf1op/op and wild-type mice with diabetes, we found delayed gastric emptying to be associated with increased production of inflammatory factors, and reduced production of anti-inflammatory factors, by macrophages, leading to loss of ICC.

Hyperglycemia Increases Interstitial Cells of Cajal via MAPK1 and MAPK3 Signaling to ETV1 and KIT, Leading to Rapid Gastric Emptying.

BACKGROUND & AIMS: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Leprdb/db), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc). METHODS: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase. RESULTS: In obese, hyperglycemic, hyperinsulinemic female Leprdb/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female KitK641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc. CONCLUSIONS: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.

Carbon-yl[tris-(3,5-diphenyl-pyrazol-1-yl-κN)methane]copper(I) hexa-fluorido-phosphate-dichloro-methane-diethyl ether (4/3/1).

In the title compound, [Cu(C(46)H(34)N(6))(CO)]PF(6)·0.75CH(2)Cl(2)·0.25C(4)H(10)O, the Cu(I) atom is coordinated by three N atoms from the tridentate chelating tris-(3,5-diphenyl-pyrazol-1-yl)methane ligand (average Cu-N distance = 2.055 Å) and the C atom from a carbon monoxide ligand in a distorted tetra-hedral coordination geometry. The average N-Cu-N angle between adjacent pyrazole-ring-coordinated N atoms is 88.6°, while the average N-Cu-C angle between the pyrazole-bound N atom and the C atom of carbon monoxide is 126.3°. One of the 3-phenyl rings of the tris-(pyrazol-yl)methane ligand is disordered over two sites each with an occupancy factor of 0.50. The structure also exhibits disorder of the monosolvate that has been modeled with 0.75 CH(2)Cl(2) and 0.25 Et(2)O occupancy.