Anti-inflammatory Effects of Abdominal Vagus Nerve Stimulation on Experimental Intestinal Inflammation

Figures

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  FIGURE 1. Abdominal vagus nerve anatomy, electrode design and experimental schedule. (A) Schematic anatomical diagram shows the cervical and abdominal branches of the vagus nerve. Off-target effects in response to cervical stimulation (Cervical Stim indicated by arrow) and abdominal stimulation (Abdom Stim indicated by arrrow) were evaluated. For the VNS efficacy experiment, the electrode array was implanted onto the anterior abdominal vagus nerve, below the diaphragm and above the hepatic and celiac vagal branches. (B) The cuff electrode array had two platinum electrode pairs (E1–E2; E3–E4) that stimulated and recorded evoked neural responses. The array was anchored by suturing the Dacron tab to the esophagus. (C) Image of the abdominal vagus nerve electrode array in vivo. (D) Experimental schedule for the VNS efficacy experiment. Scale bar in (C) 1 mm.

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  FIGURE 2. Off-target effects during cervical and abdominal VNS. Representative recordings from from one animal show a decrease in heart rate (baseline heart rate indicated by red dotted line) (A), respiratory rate (Ai) and blood pressure (Aii) during cervical VNS (1.6 mA, 200 μs/phase). (B–Bii) No changes in heart rate (baseline heart rate indicated by red dotted line) (B), respiratory rate (Bi) and blood pressure (Bii) were seen during abdominal VNS (1.6 mA, 200 μs/phase). Scale bar in (A,B) represents 5 s.

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  FIGURE 3. Electrically-evoked C-fiber responses from the abdominal vagus nerve were recorded over the implantation period. Representative ECAPs from one animal are shown when taken immediately following implantation surgery (A, T = -14), prior to the injection of TNBS (B, T = 0) and at the conclusion of the experiment (C, T = 4). Note, the amplitude of the neural response recorded immediately post-implantation (T = -14) were consistently smaller than those at later time points green traces indicate the neural threshold (200 μA) and arrows indicate the elecrically evoked C-fiber response (Latency range: 5.3–5.6 ms). Current amplitude (in μA) is indicated to the right of each set of ECAPs.

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  FIGURE 4. (A) Vagus nerve stimulation improves clinical measurements of inflammation following TNBS injection. (A) The quality of stool produced from unstimulated (TNBS; n = 8) and stimulated (TNBS+VNS; n = 7) rats were scored following the TNBS injection. (B) Blood in stool produced from unstimulated (TNBS; n = 5) and stimulated (TNBS + VNS; n = 4) rats was scored following TNBS injection. (C) CRP content in blood plasma was measured in unstimulated (TNBS; n = 6) and stimulated (TNBS+VNS; n = 6) on day 0 (pre-TNBS control) and days 1, 2, and 4 following TNBS injection. All data show mean ± SEM. Significant differences from control (day 0, pre-TNBS) are indicated by “∗”, while significant differences between unstimulated (TNBS) and stimulated (TNBS+VNS) groups are indicated by “O”.

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  FIGURE 5. Vagal nerve stimulation reduced histological damage of small intestine tissue following TNBS injection. (A) Control small intestine (A,Ai) had undamaged, intact surface epithelium (arrows, Ai). (B,Bi) At 4 h following TNBS injection (the time that VNS was applied), extensive epithelial cell loss (arrow, Bi) and leukocyte infiltration (yellow circle) was observed. (C,Ci) At 4 days following TNBS injection, there was extensive damage to villus architecture (Ci, arrows) and leukocyte infiltration in venules (circle) in tissue from unstimulated rats (TNBS). (D,Di) In tissue from stimulated rats (TNBS+VNS), histological damage was less severe than tissue taken from unstimulated (TNBS) rats. Surface epithelial cells were intact (arrows), and villi were mostly undamaged, although some inflammation-induced irregularities remained. (E) The histological score was quantified in tissue taken from control animals, at 4 h following TNBS injection, and from unstimulated (TNBS) and stimulated animals (TNBS+VNS). Data show raw histological score (0–9) of each animal, median and interquartile range. Significant differences of P < 0.05 are indicated by “∗.” Scale bars in (A–D): 100 μm; in (Ai–Di):…

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  FIGURE 6. VNS reduces resident leukocyte populations in mucosal and submucosal layers following TNBS injection. (A–C) Quantification of eosinophils (A), MPO+ cells (neutrophils, B) and CD3+ cells (T cells; C) within the mucosal layer. (D–F) Quantification of eosinophils (D), MPO+ cells (E) and CD3+ cells (F) within the submucosal layer. Data show raw data from each animal, median and interquartile range. Significant differences of P < 0.05 are indicated by “∗.”

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  FIGURE 7. VNS reduces resident leukocyte populations in muscle layers following TNBS injection. (A–C) Images show myeloperoxidase (MPO) positive cells (neutrophils) within circular and longitudinal muscle layers of tissue taken from control (A), TNBS rats (B) and TNBS+VNS rats (C). (D–F) Quantification of eosinophils (D), MPO+ cells (E), and CD3+ cells (F) within the circular and longitudinal muscle layers. Data show mean count/animal, median and interquartile range. Significant differences of P < 0.05 are indicated by “∗.”

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  FIGURE 8. Histopathology of the anterior abdominal vagus nerve following implantation. (A,Ai) H&E stained vagus nerve that was stimulated for 4 days shows a benign foreign body tissue response (A, indicated by ∗), which extended into the electrode channel (Ai, indicated by ∗∗). There were no acute inflammatory cells within the epineurium, the two fascicles of the vagus nerve (outlined by a dotted line) appeared undamaged, and blood vessels (example indicated by “BV”) appeared patent. Tissue dye was used to identify the implanted nerve upon dissection. (B) Quantification of total fascicle area (example indicated by dotted lines in Ai) between non-implanted neural tissue, taken proximal to implantation site, and implanted tissue adjacent to electrodes E1–E4. Data show mean fascicle area (μm2) + SEM, and there were no significant differences between locations. Scale bar in (A) 500 μm Scale bar in (Ai) 100 μm.