Gastric electrical stimulation ameliorates antral myenteric neurons in streptozotocin-induced diabetic beagle canines

Figure 1 Experimental protocol and intervention. GES: Gastric electrical stimulation; SGES: Sham gastric electrical stimulation.

Figure 2 Representative hematoxylin-eosin staining of pancreatic tail tissues in the diabetic models and controls. A: Diabetes mellitus; B: Control. Scale bar = 50 µm. DM: Diabetes mellitus.

Figure 3 Representative immunofluorescence images of myenteric choline acetyltransferase-immunoreactive positive neurons in full-thickness sections of canine gastric antrum (200 × magnification, scale bar = 50 μm). A: Representative images of anti-human neuronal protein HuC/HuD-immunoreactive (anti-Hu-IR) labeled neurons in diabetes mellitus (DM) + gastric electrical stimulation (GES); B: Representative images of choline acetyltransferase-immunoreactive (ChAT-IR) positive neurons in DM + GES; C: Representative merged images of anti-Hu-IR/ChAT-IR in DM + GES; D: Representative images of anti-Hu-IR labeled neurons in DM + sham GES (SGES); E: Representative images of ChAT-IR positive neurons in DM + SGES; F: Representative merged images of anti-Hu-IR/ChAT-IR in DM + SGES; G: Representative images of anti-Hu-IR labeled neurons in control + GES; H: Representative images of ChAT-IR positive neurons in control + GES; I: Representative merged images of anti-Hu-IR/ChAT-IR in control + GES; J: Representative images of anti-Hu-IR labeled neurons in control + SGES; K: Representative images of ChAT-IR positive neurons in control + SGES; L: Representative merged images of anti-Hu-IR/ChAT-IR in control + SGES; M: Number of anti-Hu-IR positive neurons; N: Number of ChAT-IR positive neurons; O: Proportion of ChAT-IR positive neurons relative to total neurons. The proportion of ChAT-IR neurons was higher in the diabetes mellitus + GES compared with the diabetes mellitus + SGES subgroup (^bP < 0.01). Within sham-stimulated subgroups, the proportion of ChAT-IR neurons was less in the diabetic canines compared with the controls (^bP < 0.01). Anti-Hu: Anti-human neuronal protein HuC/HuD; ChAT: Choline acetyltransferase; DM: Diabetes mellitus; GES: Gastric electrical stimulation; SGES: Sham gastric electrical stimulation.

Figure 4 Representative immunofluorescence images of myenteric vasoactive intestinal peptide-immunoreactive positive neurons in full-thickness sections of canine gastric antrum (200 × magnification, scale bar = 50 μm). A: Representative images of anti-human neuronal protein HuC/HuD-immunoreactive (anti-Hu-IR) labeled neurons in diabetes mellitus (DM) + gastric electrical stimulation (GES); B: Representative images of vasoactive intestinal peptide-immunoreactive (VIP-IR) positive neurons in DM + GES; C: Representative merged images of anti-Hu-IR/VIP-IR in DM + GES; D: Representative images of anti-Hu-IR labeled neurons in DM + sham GES (SGES); E: Representative images of VIP-IR positive neurons in DM + SGES; F: Representative merged images of anti-Hu-IR/VIP-IR in DM + SGES; G: Representative images of anti-Hu-IR labeled neurons in control + GES; H: Representative images of VIP-IR positive neurons in control + GES; I: Representative merged images of anti-Hu-IR/VIP-IR in control + GES; J: Representative images of anti-Hu-IR labeled neurons in control + SGES; K: Representative images of VIP-IR positive neurons in control + SGES; L: Representative merged images of anti-Hu-IR/VIP-IR in control + SGES; M: Number of anti-Hu-IR positive neurons; N: Number of VIP-IR positive neurons; O: Proportion of VIP-IR positive neurons relative to total neurons. The proportion of VIP-IR neurons was significantly higher in the diabetes mellitus canines compared with controls, whether in GES or SGES subgroups (^cP < 0.001). Anti-Hu: Anti-human neuronal protein HuC/HuD; DM: Diabetes mellitus; GES: Gastric electrical stimulation; IR: Immunoreactive; SGES: Sham gastric electrical stimulation; VIP: Vasoactive intestinal peptide.

Figure 5 Representative immunofluorescence images of myenteric neuronal nitric oxide synthase-immunoreactive positive neurons in full-thickness sections of canine gastric antrum (200 × magnification, scale bar = 50 μm). A: Representative images of anti-human neuronal protein HuC/HuD-immunoreactive (anti-Hu-IR) labeled neurons in diabetes mellitus (DM) + gastric electrical stimulation (GES); B: Representative images of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) positive neurons in DM + GES; C: Representative merged images of anti-Hu-IR/nNOS-IR in DM + GES; D: Representative images of anti-Hu-IR labeled neurons in DM + sham GES (SGES); E: Representative images of nNOS-IR positive neurons in DM + SGES; F: Representative merged images of anti-Hu-IR/nNOS-IR in DM + SGES; G: Representative images of anti-Hu-IR labeled neurons in control + GES; H: Representative images of nNOS-IR positive neurons in control + GES; I: Representative merged images of anti-Hu-IR/nNOS-IR in control + GES; J: Representative images of anti-Hu-IR labeled neurons in control + SGES; K: Representative images of nNOS-IR positive neurons in control + SGES; L: Representative merged images of anti-Hu-IR/nNOS-IR in control + SGES; M: Number of anti-Hu-IR positive neurons; N: Number of nNOS-IR positive neurons; O: Proportion of nNOS-IR positive neurons relative to total neurons. The proportion of nNOS-IR neurons was increased in the GES subgroups compared with sham GES in both diabetes mellitus (^bP < 0.01) and control canines (^aP < 0.05). Within sham-stimulated groups, the proportion of nNOS-IR neurons was less in the diabetes mellitus compared with the controls (^bP < 0.01). Anti-Hu: Anti-human neuronal protein HuC/HuD; DM: Diabetes mellitus; GES: Gastric electrical stimulation; nNOS: Neuronal nitric oxide synthase; SGES: Sham gastric electrical stimulation.

Figure 6 Representative immunofluorescence images of myenteric calretinin-immunoreactive positive neurons in full-thickness sections of canine gastric antrum (200 × magnification, scale bar = 50 μm). A: Representative images of anti-human neuronal protein HuC/HuD-immunoreactive (anti-Hu-IR) labeled neurons in diabetes mellitus (DM) + gastric electrical stimulation (GES); B: Representative images of calretinin-immunoreactive (CALR-IR) positive neurons in DM + GES; C: Representative merged images of anti-Hu-IR/CALR-IR in DM + GES; D: Representative images of anti-Hu-IR labeled neurons in DM + SGES; E: Representative images of CALR-IR positive neurons in DM + SGES; F: Representative merged images of anti-Hu-IR/CALR-IR in DM + SGES; G: Representative images of anti-Hu-IR labeled neurons in control + GES; H: Representative images of CALR-IR positive neurons in control + GES; I: Representative merged images of anti-Hu-IR/CALR-IR in control + GES; J: Representative images of anti-Hu-IR labeled neurons in control + SGES; K: Representative images of CALR-IR positive neurons in control + SGES; L: Representative merged images of anti-Hu-IR/CALR-IR in control + SGES; M: Number of anti-Hu-IR positive neurons; N: Number of CALR-IR positive neurons; O: Proportion of CALR-IR positive neurons relative to total neurons. Anti-Hu: Anti-human neuronal protein HuC/HuD; CALR: Calretinin; DM: Diabetes mellitus; GES: Gastric electrical stimulation; IR: Immunoreactive; SGES: Sham gastric electrical stimulation.