Introduction/Aim: Delayed gastric emptying and constipation related to neurodegeneration are major complications of poorly controlled diabetes. Using scRNAseq, we identified a neuron-associated muscularis macrophage (NA-MM) population expressing F11r (CD321). Upon loss of this population a reduction of myenteric neurons is observed in adult mice (Viola et al., 2023), suggesting a crucial role of NA-MM in the maintenance of the enteric nervous system. Here, we hypothesized that gastrointestinal dysfunction in diabetes results from changes in phenotype and/or function of the NA-MM population, leading to neurodegeneration.
Methods: Diabetes was induced in 10-week old C57/BL6J mice by injection of streptozotocin (50mg/kg). Gastric emptying was measured using MRI at t=60min as previously described (Chavero-Pieres and Viola et al., 2022). Colonic transit was measured as previously described (Anitha et al., 2016). Neuronal density (HuC/D+) was quantified by immunohistochemistry. Gastric and colonic single cells were isolated and stained for flow cytometry. NA-MM were identified as CD45+CD11b+CD64+CD163-CD321+ cells
Results: In diabetic mice with delayed gastric emptying, but not in those with normal gastric emptying, the number of myenteric neurons was significantly decreased compared to controls (control: 602±63 neurons/mm2 , n=5 vs diabetic normal emptying: 537±118 neurons/mm2 , n=10 ; p=0.450 ; vs diabetic delayed emptying 454±72 neurons/mm2 , n=7 ; p=0.040). Similarly, tthere was a significant loss of myenteric neurons in the colon of diabetic mice compared to controls (control: 523±65 neurons/mm2 , n=4 vs diabetic: 384±51 neurons/mm2 , n=10 ; p=0.001). Of interest, there was a significant reduction in the percentage of NA-MM in the stomach (control: 15.1±2.2 % , n=10 vs diabetic: 11.2±3.2 % of CD64+ MM, n=12 ; p=0.004) and a trend towards a reduced frequency of NA-MM in the colon compared to controls (control: 7.5±2.0 % , n=11 vs diabetic: 6.1±1.3 % of CD64+ MM, n=12 ; p=0.056). Of note, a stronger reduction in NA-MM was observed in the colon of mice with a delayed colonic transit compared to controls or diabetic mice with a normal colonic transit (control: 7.5±2 % of CD64+ MM, n=11 vs diabetic normal colonic transit: 6.6±1.5 % of CD64+ MM, n=7 , p=0.537 ; vs diabetic delayed colonic transit: 5.3±0.8 % of CD64+ MM, n=5 ; p=0.056).
Conclusions: Our results indicate that diabetes leads to a reduction in NA-MM in both the gastric and colonic muscularis externa. The reduction of NA-MM is associated with a reduction in enteric neurons, resulting in delayed gastric emptying and constipation. Taken together, these data suggest a role for NA-MM in diabetic intestinal neurodegeneration. Further studies identifying the transcriptional changes and pathways involved may ultimately lead to the identification of novel therapeutic targets.

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