Toll-like receptor 4 (TLR4) can activate pro-inflammatory cascades in the gastrointestinal tract. Our aim was to determine TLR4 expression in myenteric neurons of different gut regions using a type 1 diabetic model. Ten weeks after the onset of hyperglycemia, myenteric whole-mount preparations from the duodenum, ileum and colon of streptozotocin-induced diabetic, insulin-treated diabetic and control rats were prepared for TLR4/peripherin double-labelling fluorescent immunohistochemistry. Immunogold electron microscopy was applied to evaluate TLR4 expression in the myenteric perikaryon and neuropil. Tissue TLR4 levels were measured by enzyme-linked immunosorbent assay. In controls, the number and proportion of the TLR4-immunoreactive myenteric neurons showed an increasing tendency to aboral direction. These values were significantly higher in diabetics compared to controls in the duodenum and ileum, but were significantly lower in the colon. In diabetics, the distribution of TLR4-labelling gold particles between the perikaryon and neuropil of myenteric neurons varied in a different way by intestinal segment. TLR4 tissue concentration changed only in the diabetic duodenum, and it decreased in muscle/myenteric plexus-containing homogenates, while it increased in mucosa/submucosa/submucous plexus-containing samples relative to controls. Insulin had beneficial effects on TLR4 expression. These findings support that chronic hyperglycemia has segment-specific effects on TLR4 expression, contributing to gastrointestinal disorders in diabetic patients.

Interleukin 1β (IL1β) is a pro-inflammatory cytokine that may play a crucial role in enteric neuroinflammation in type 1 diabetes. Therefore, our goal is to evaluate the effects of chronic hyperglycemia and insulin treatment on IL1β immunoreactivity in myenteric neurons and their different subpopulations along the duodenum–ileum–colon axis. Fluorescent immunohistochemistry was used to count IL1β expressing neurons as well as the neuronal nitric oxide synthase (nNOS)- and calcitonin gene-related peptide (CGRP)-immunoreactive myenteric neurons within this group. Tissue IL1β level was measured by ELISA in muscle/myenteric plexus-containing homogenates. IL1β mRNA was detected by RNAscope in different intestinal layers. The proportion of IL1β-immunoreactive myenteric neurons was significantly higher in the colon than in the small intestine of controls. In diabetics, this proportion significantly increased in all gut segments, which was prevented by insulin treatment. The proportion of IL1β-nNOS-immunoreactive neurons only increased in the diabetic colon, while the proportion of IL1β-CGRP-immunoreactive neurons only increased in the diabetic ileum. Elevated IL1β levels were also confirmed in tissue homogenates. IL1β mRNA induction was detected in the myenteric ganglia, smooth muscle and intestinal mucosa of diabetics. These findings support that diabetes-related IL1β induction is specific for the different myenteric neuronal subpopulations, which may contribute to diabetic motility disturbances.

Background/Objectives: Type 1 diabetes affects cytokines as potential inducers of NFκB signalling involved in inflammation and neuronal survival. Our goal was to assess the expression of NFκB p65 and its negative regulator, Nrf2, in myenteric neurons and adjacent smooth muscle of different gut segments after chronic hyperglycaemia and immediate insulin treatment. Methods: After ten weeks of hyperglycaemia, intestinal samples of control, streptozotocin-induced diabetic and insulin-treated diabetic rats were prepared for fluorescent immunohistochemistry, immunogold electron microscopy, ELISA and qPCR. Results: In the diabetic rats, the proportion of NFκB p65-immunoreactive myenteric neurons decreased significantly in the duodenum and increased in the ileum. The density of NFκB p65-labelling gold particles increased in the ileal but remained unchanged in the duodenal ganglia. Meanwhile, both total and nuclear Nrf2 density increased in the myenteric neurons of the diabetic duodenum. In smooth muscle, NFκB p65 and Nrf2 density increased in the small intestine of diabetic rats. While on the mRNA level, NFκB p65 and Nrf2 were induced, on the protein level, NFκB p65 increased and Nrf2 decreased in muscle/myenteric plexus homogenates. Insulin treatment had protective effects. Conclusions: Our findings reveal a segment-specific NFκB and Nrf expression in myenteric neurons and ganglionic muscular environments, which may contribute to regional neuronal survival and motility disturbances in diabetes.

Background/Objectives: Enteric glial cells (EGCs) are key players in regulating enteric neurons and gastrointestinal functions including disturbed gut motility in diabetic patients. Enteric neuronal damage has been shown in type 1 diabetes, but EGCs’ vulnerability to hyperglycaemic insults requires more investigation. Therefore, we aimed to study the quantitative changes in the EGC network enmeshing enteric plexuses, intestinal smooth muscle and mucosa in streptozotocin-induced acute (1-week) and chronic (10-weeks) diabetic rat models. Methods: Fluorescent immunohistochemistry using Sox10 glial and HuC/HuD pan-neuronal markers, immunogold electron microscopy and ELISA were performed on different gut segments. Results: In the submucosal ganglia of the ileum and colon, the density of Sox10-immunoreactive EGCs was significantly reduced in acute and increased in chronic hyperglycaemic rats without any changes in the duodenum. In the myenteric ganglia, regionally distinct alterations of glial density were noted in acute hyperglycaemia; however, a remarkable decrease was observed in chronic animals. Alterations of neuronal density did not follow the pattern of glial changes, resulting in shifts in the glia/neuron ratio. The presence of Sox10-HuC/HuD-immunoreactive cells and their diabetes-related quantitative changes were also revealed in enteric plexuses. The density of Sox10-labelling gold particles was significantly increased in the duodenal myenteric glia of diabetic rats. Muscular EGC density increased only in the colon after acute hyperglycaemia and changed in all segments after chronic hyperglycaemia. Glial fibrillary acidic protein levels decreased in the small intestine of chronic hyperglycaemic rats. Conclusions: Our present findings reveal time-dependent and regionally distinct changes in the EGC network in response to hyperglycaemia, contributing to diabetic enteric neuropathy and gut motility disturbances.

Background/Objectives: Glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT3) are essential neurotrophic factors that continue to be expressed in the mature intestine. Myenteric neurons are damaged by hyperglycemia to varying degrees in the different gut segments. Therefore, our aims were to identify the gut-segment-specific expression patterns of GDNF and NT3 in acute and chronic hyperglycemic rat models. Methods: Segments of the duodenum, ileum, and colon were dissected one week (acute experiment) or ten weeks (chronic experiment) after the streptozotocin-induced hyperglycemia. GDNF–HuC/HuD fluorescent immunohistochemistry, NT3 enzyme-linked immunosorbent assay, and NT3 immunogold electron microscopy were performed in rat myenteric ganglia. Results: The proportion of GDNF–immunoreactive myenteric neurons did not change in gut segments in acute hyperglycemia. However, during chronic diabetes, it was elevated in the colon and remained unchanged in the duodenum and ileum. Expressional alterations of NT3 were investigated in the small intestine. In duodenal myenteric ganglia, acute hyperglycemia induced a significant increase in the number of NT3-labeling gold particles, and its density was markedly increased in chronic diabetes, which was partially prevented by insulin treatment. In the ileum, NT3 density increased in acute hyperglycemic rats, while it decreased in chronic diabetic rats. Conclusions: Induction of neurotrophic factors was specific to different gut segments during acute and chronic hyperglycemia. These results suggest that increased colonic GDNF and duodenal NT3 expression may reflect a compensatory response of myenteric neurons to hyperglycemic states.