Optogenetic stimulation of the brainstem dorsal motor nucleus ameliorates acute pancreatitis

Figures

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  Figure 1. Selective activation of DMN cholinergic neurons invokes efferent vagus nerve activity, reducing heart rate. (A) Breeding strategy used to generate ChAT-ChR2-YFP mice which express a photosensitive channelrhodopisin in ChAT-positive cells, including cholinergic neurons. (B–D) Optogenetic stimulation of DMN cholinergic neurons produced a significant decrease in heart rate in ChAT-ChR2-eYP mice. A fiber optic cannula was placed in the left DMN of ChAT-ChR2-YFP mice and animals were then subjected to (B) blue light (473nm, 20Hz, 25% duty cycle, 8-12 mW, 5 minutes), (C) no light, or (D) yellow light (593.5nm, 20Hz, 25% duty cycle, 8-12 mW, 5 minutes) stimulation. A decrease in heart rate was observed during blue laser stimulation. Representative heart rate data is shown for each condition. (E) Stimulation of ChR2-expressing DMN cholinergic neurons with blue light, but not with yellow light, induces bradycardia in ChAT-ChR2-eYFP mice. Data are represented as individual mouse data points, which represent an average heart rate over the stimulation period, with mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test, ****P ≤ 0.0001, ns, not significant.

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  Figure 2. Selective activation of DMN cholinergic neurons reduces circulating and pancreatic markers of acute pancreatitis Acute pancreatitis was induced in ChAT-ChR2-YFP mice by two intraperitoneal injections of caerulein (50 µg/kg). A fiberoptic cannula was placed in the left DMN and mice were then subjected to blue light stimulation (473 nm, 20 Hz, 25% duty cycle, 8-12 mW, 5 minutes), yellow light stimulation (593.5 nm, 20 Hz, 25% duty cycle, 8-12 mW, 5 minutes), or no laser stimulation. Mice were euthanized 4 hours after the final dose of caerulein. (A) Compared to non-caerulein-injected controls, caerulein-injected mice show significantly increased serum amylase levels consistent with the induction of acute pancreatitis. Optogenetic stimulation with blue light, but not with yellow light, significantly decreases serum amylase levels. Data are represented as individual mouse data points with mean ± SEM. One-way ANOVA: no caerulein vs yellow laser vs no laser (****P ≤ 0.0001, n = 7-11). (B) Optogenetic stimulation with blue light decreases pancreatic levels of (D) IL-6, (E) CXCL-1, and (F) MCP-1, but not (C) IL-1β. Data are represented as individual mouse data points with mean ± SEM.…

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  Figure 3. Selective activation of DMN cholinergic neurons reduces the histological severity of acute pancreatitis (A–I) Representative images of pancreatic tissue stained with H&E at 4x (left panels), 10x (middle panels), and 20x (right panels) from (A–C) non-caerulein-injected and non-stimulated controls, (D–F) caerulein-injected and yellow-light-stimulated, and (G–I)) caerulein-injected and blue light-stimulated mice. Bar = 200 μm. Histological scoring for (J) hemorrhage and fat necrosis, (K) acinar necrosis, (L) inflammation and perivascular infiltration, (M) edema, and (N) total severity. Data are represented as individual mouse data points with mean ± SEM. One way ANOVA with Kruskal-Wallis, *P P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n = 9-11, ns, not significant.

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  Figure 4. Selective activation of DMN cholinergic neurons fails to reduce serum amylase levels in mice subjected to subdiaphragmatic vagotomy. (A) A bilateral subdiaphragmatic vagotomy and transgastric pyloric dilation or sham surgery, consisting of celiotomy and transgastric pyloric dilation only, were performed on ChAT-ChR2-YFP mice. After one week of recovery, pancreatitis was induced with two intraperitoneal injections of caerulein (50 µg/kg). The mice were subjected to optogenetic stimulation with blue light or sham stimulation. The mice were euthanized 4 hours after the final dose of caerulein. (B) Optogenetic stimulation with blue light significantly decreases serum amylase levels in sham-operated mice (Sham), but not in mice subjected to subdiaphragmatic vagotomy (Vgx). Data are represented as individual mouse data points with mean ± SEM. One-way ANOVA with Kruskal-Wallis test, (*P ≤ 0.05, n = 3-6), ns, not significant.

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  Figure 5. Selective activation of DMN cholinergic neurons reduces serum amylase levels during pancreatitis through a nAChR-dependent mechanism. (A) One hour prior to induction of acute pancreatitis, ChAT-ChR2-YFP mice underwent intraperitoneal injection of vehicle or mecamylamine (1 mg/kg) was injected one hour prior followed by the induction of pancreatitis and optogenetic stimulation. The mice were euthanized 4 hours after the final dose of caerulein. (B) Optogenetic stimulation with blue light (473 nm, 20 Hz, 25% duty cycle, 8-12 mW, 5 minutes) of the DMN significantly decreases the level of serum amylase compared to no stimulation in the vehicle-treated mice but not in animals receiving mecamylamine. Data are represented as individual mouse data points with mean ± SEM. One-way ANOVA, (**P ≤ 0.01, ***P ≤ 0.001, n = 10, ns, not significant.).