Tail clamping induces anxiety-like behaviors and visceral hypersensitivity in rat models of non-erosive reflux disease

Figure 1 Schematic diagram of the animal intervention. SD: Sprague-Dawley; OVA: Ovalbumin.

Figure 2 General condition comparisons of the animals. A: Comparison of body weight gain rate in Sprague-Dawley (SD) rats before and after tail clamping; B: Comparison of body weight gain rate in Wistar rats before and after tail clamping; C: Blood glucose levels in Wistar rats at the end of modeling; D: Esophageal pH values in Wistar rats at the end of modeling. ^aP < 0.01 vs post-SD-modified; ^bP < 0.05 vs pre-Wistar-model; ^cP < 0.01 vs pre-Wistar-modified; ^dP < 0.01 vs post-Wistar-sham; ^eP < 0.01 vs Wistar-sham. SD: Sprague-Dawley.

Figure 3 Behavioral assessment of rats after model establishment. A: Sucrose preference test; B: Animal movement trajectories; C: Total distance traveled; D: Average velocity; E: Immobility time (%); F: Time in center zone; G: Number of center zone entries; H: Abdominal withdrawal reflex score. ^aP < 0.01 vs Sprague-Dawley (SD)-modified, ^bP < 0.01 vs Wistar-sham, ^cP < 0.05 vs SD-modified, ^dP < 0.05 vs Wistar-sham, ^eP < 0.01 vs pre-SD-sham, ^fP < 0.05 vs pre-SD-modified, ^gP < 0.05 vs pre-Wistar-model. SD: Sprague-Dawley; AWR: Abdominal withdrawal reflex.

Figure 4 Histopathological and ultrastructural changes in the esophageal mucosa. A: Hematoxylin and eosin staining of esophageal mucosa (scale bar: 50 μm); B: Esophageal mucosa under transmission electron microscopy (scale bar: 2 μm). SD: Sprague-Dawley.

Figure 5 Multi-level comparison of neuroimmune metrics. A: Serum mast cell tryptase (MCT) levels in rats; B: Serum substance P (SP) levels in rats; C: Serum corticotropin-releasing hormone (CRH) levels in rats; D: Serum calcitonin gene-related peptide (CGRP) levels in rats; E: Serum 5-hydroxytryptamine (5-HT) levels in rats; F: Central amygdala (CeA) CRH mRNA levels in rats; G: CeA CRH-R1 mRNA levels in rats; H: Z-score normalized serum levels of MCT, SP, CRH, CGRP, and 5-HT in healthy volunteers (HV), non-erosive reflux disease patients, and experimental groups; I: MCT, SP, CRH, CGRP, and 5-HT, Δ% change of serum markers relative to HV. ^aP < 0.01 vs Sprague-Dawley (SD)-sham, ^bP < 0.01 vs Wistar-sham, ^cP < 0.01 vs Wistar-model, ^dP < 0.05 vs SD-sham, ^eP < 0.05 vs SD-model, ^fP < 0.01 vs SD-model, ^gP < 0.05 vs Wistar-sham, ^hP < 0.05 vs Wistar-model. SD: Sprague-Dawley; MCT: Mast cell tryptase; SP: Substance P; CRH: Corticotropin-releasing hormone; CGRP: Calcitonin gene-related peptide; 5-HT: 5-hydroxytryptamine; CeA: Central amygdala; NERD: Non-erosive reflux disease.