TITLE: Effects of Vagus Nerve Stimulation/Gastric Electrical Stimulation on Gastric Emptying and Motility Assessed with Magnetic Resonance Imaging
AUTHORS: Kun-Han Lu, Zhongming Liu, Jiayue Cao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the methods used to evaluate the effects of vagus nerve stimulation (VNS) and gastric electrical stimulation (GES) settings on gastric emptying and motility in rats. Briefly, a gadolinium‐based contrast agent was mixed with the animal’s meal in order for chyme to appear “bright” in MRI scans, thereby delineating the gastric and intestinal volume. A multi‐slice MRI sequence was used to scan gastrointestinal (GI) functions and physiology with high spatial and temporal resolution.</div></div>

[STEPS]
?. [Acute effects of vagus nerve stimulation settings on gastric motility assessed with MRI]
Study titleAcute effects of vagus nerve stimulation settings on gastric motility assessed with MRIDescriptionThis protocol describes the methods used to evaluate the effects of VNS settings on gastric motility in rats. Briefly, a gadolinium‐based contrast agent was mixed with the animal’s meal in order for chyme to appear “bright” in MRI scans, thereby delineating the gastric and intestinal volume. A multi‐slice MRI sequence was used to scan antral contractions with high spatial and temporal resolution. The configuration of VNS was varied in terms of pulse parameters (amplitude, width, and frequency) and polarity (proximal or distal coil of a bipolar electrode connected as the cathode, which could be biased for afferent or efferent VNS).
?. [Acute effects of vagus nerve stimulation settings on gastric motility assessed with MRI]
Animal protocolTwelve rats(Male), ranging from 266 to 338g body weight were used in this study. Meal preparation, animal preparation, surgical steps, imaging sequence, data analysis were performed using our previously published protocol. Note that no Carprofen was administered to the animals in this study.
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?. [Acute effects of vagus nerve stimulation settings on gastric motility assessed with MRI]
Experiment DesignAfter obtaining about 5 minutes of stable, baseline dynamic MRI images, cervical VNS was delivered simultaneously with MRI acquisition. The 12 rats were allocated into 2 groups as follows: (i) a group of rats (N=7) that received afferent VNS (i.e. cathode placed proximally to the anode) and (ii) a group of rats (N=5) that received efferent VNS (i.e. cathode placed distally to the anode). In addition to evaluating the effect of afferent versus efferent VNS on gastric motility, we further applied bi-directional VNS (i.e. combined afferent and efferent VNS) to the rats in the afferent VNS group, immediately after which afferent VNS was performed. The bi-directional VNS was achieved by delivering monophasic pulses of current in alternating directions between the two electrodes.For each VNS group, the stimulus parameters were varied in terms of pulse amplitude (PA: 0.13, 0.25, 0.5, 1mA), width (PW: 0.13, 0.25, 0.5ms), and frequency (PF: 5, 10Hz). The frequency of afferent or efferent VNS was defined as the number of electrical pulses delivered per second. The frequency of bidirectional VNS was defined as the number of paired cathodal and anodal pulses delivered per second. Low, medium and high values were selected for each parameter settings, all of which are frequently used in clinical and preclinical settings. Each stimulation setting comprised of a duty cycle of 1-minute ON and 1-minute OFF, and different VNS settings were performed in a randomized order to eliminate any causal effect of one setting on the other. No stimulus was delivered during the OFF period. As a result, every rat in each group underwent VNS with 24 different sets of parameters.
?. [Effects of nodose ganglion blockade on gastric motility during cervical vagus nerve stimulation measured with MRI]
Study titleEffects of nodose ganglion blockade on gastric motility during cervical vagus nerve stimulation measured with MRIDescriptionThis protocol describes the methods used to evaluate the effects of cervical VNS on gastric motility in rats. Gastric MRI was performed during cervical VNS with and without afferent blockade at the nodose ganglion.
?. [Effects of nodose ganglion blockade on gastric motility during cervical vagus nerve stimulation measured with MRI]
Animal protocolEight rats(Male), ranging from 283 to 376g body weight were used in this study. Meal preparation, animal preparation, surgical steps, imaging sequence, data analysis were performed using our previously published protocol. Note that no Carprofen was administered to the animals in this study.
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?. [Effects of nodose ganglion blockade on gastric motility during cervical vagus nerve stimulation measured with MRI]
Experiment DesignThe 8 rats were allocated into 2 groups as follows: (i) a group of rats (N=4) that did not receive injection 1.5uL of 2% Lidocaineinto the left nodose ganglion and (ii) a group of rats (N=4) that received injection 1.5uL of 2% Lidocaine into the left nodose ganglion. After obtaining about 4 minutes of stable, baseline dynamic MRI images, cervical VNS (0.3mA, 0.2ms, 10Hz, 20s ON & 40s OFF) was delivered for 5 minutes simultaneously with MRI acquisition. The cathode was set cranial to the cuff to favor activation of afferent signaling to the brain. MRI images were collected for an additional 20 minutes after the offset of VNS.
?. [Acute effects of gastric electrical stimulation settings on gastric motility assessed with MRI]
Study titleAcute effects of gastric electrical stimulation settings on gastric motility assessed with MRIDescriptionThis protocol describes the methods used to evaluate the effects of GES settings on gastric motility in rats. Briefly, a gadolinium‐based contrast agent was mixed with the animal’s meal in order for chyme to appear “bright” in MRI scans, thereby delineating the gastric and intestinal volume. A multi‐slice MRI sequence was used to scan antral contractions with high spatial and temporal resolution. The configuration of VNS was varied in terms of pulse parameters (amplitude, width, and frequency).
?. [Acute effects of gastric electrical stimulation settings on gastric motility assessed with MRI]
Animal protocolFour rats(Male), ranging from 272 to 306g body weight were used in this study. All animals underwent an abdominal surgery for implantation of a patch electrode on the forestomach. Each animal was briefly anesthetized with 5% Isoflurane for 5 min, followed by 2% isoflurane to maintain a surgical plane of anesthesia.  Following a toe-pinch test, a ∼3 cm incision was made starting at 1 cm  caudal to the xiphoid and moving 3 cm caudally. After separating and retracting the skin and muscle layers, the ventral stomach was exposed such that the intersection of the greater curvature and the limiting  ridge could be identified. A pair of customized electrodes which consisted of Pt/Ir foils adhered to a thin perylene substrate (a rectangular shape of 4 mm-by-2mm size and 0.015 mm thickness), was directly sutured onto the forestomach wall. This patch electrode was placed along the greater curvature at about 4 mm proximal  to the limiting ridge. Next, the muscle and skin layers at the incision site were closed with sutures. The leads of the implanted electrodes were tunneled  subcutaneously to the back. The  animals were allowed at least 1 week for post-surgical recovery. To minimize pain and inflammatory responses  after the surgery, all animals were given (one tablet per day, 2 mg/Tablet) two days before the surgery and (0.01 mg/kg, subcutaneous) immediately before the surgery. Meal preparation, animal preparation, imaging sequence, data analysis were performed using our previously published protocol. Note that no Carprofen was administered to the animals in this study.
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?. [Acute effects of gastric electrical stimulation settings on gastric motility assessed with MRI]
Experiment DesignAfter obtaining about 5 minutes of stable, baseline dynamic MRI images, gastric electrical stimulation was delivered simultaneously with MRI acquisition. Monophasic pulses of current was delivered in alternating directions between the two electrodes.The stimulus parameters were varied in terms of pulse amplitude (PA: 0, 0.2, 0.4, 0.6, 0.8, 1mA), width (PW: 0.1, 0.2, 0.4ms), and frequency (PF: 2, 5, 10, 20Hz). The frequency of GES was defined as the number of paired cathodal and anodal pulses delivered per second. Low, medium and high values were selected for each parameter settings, all of which are frequently used in clinical and preclinical settings. Each stimulation setting comprised of a duty cycle of 20s ON and 40s OFF, and different GES settings were performed in a randomized order to eliminate any causal effect of one setting on the other. No stimulus was delivered during the OFF period. As a result, every rat underwent GES with 72 different sets of parameters.
?. [Assessment of gastric emptying and motility with MRI under gastric electrical stimulation]
Study titleAssessment of gastric emptying and motility with MRI under gastric electrical stimulationDescriptionThis protocol describes the methods used to evaluate the effects of GES on gastric emptying and motility in rats.
?. [Assessment of gastric emptying and motility with MRI under gastric electrical stimulation]
Animal protocolEleven rats(Male), ranging from 272 to 407g body weight were used in this study. All animals underwent an abdominal surgery for implantation of a patch electrode on the forestomach. Each animal was briefly anesthetized with 5% Isoflurane for 5 min, followed by 2% isoflurane to maintain a surgical plane of anesthesia.  Following a toe-pinch test, a ∼3 cm incision was made starting at 1 cm  caudal to the xiphoid and moving 3 cm caudally. After separating and retracting the skin and muscle layers, the ventral stomach was exposed such that the intersection of the greater curvature and the limiting  ridge could be identified. A pair of customized electrodes which consisted of Pt/Ir foils adhered to a thin perylene substrate (a rectangular shape of 4 mm-by-2mm size and 0.015 mm thickness), was directly sutured onto the forestomach wall. This patch electrode was placed along the greater curvature at about 4 mm proximal  to the limiting ridge. Next, the muscle and skin layers at the incision site were closed with sutures. The leads of the implanted electrodes were tunneled  subcutaneously to the back. The  animals were allowed at least 1 week for post-surgical recovery. To minimize pain and inflammatory responses  after the surgery, all animals were given (one tablet per day, 2 mg/Tablet) two days before the surgery and (0.01 mg/kg, subcutaneous) immediately before the surgery. Meal preparation, animal preparation, imaging sequence, data analysis were performed using our previously published protocol. Note that no Carprofen was administered to the animals in this study.
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?. [Assessment of gastric emptying and motility with MRI under gastric electrical stimulation]
Experiment DesignThe 11 rats were allocated into 3 groups as follows: (i) a group of rats (N=5) that did not receive GES, (ii) a group of rats (N=2) that received GES with stimulus setting being pulse amplitude=0.3mA, pulse width=0.2ms, pulse frequency=10Hz, and (iii) a group of rats (N=4) that received GES with stimulus setting being pulse amplitude=0.6mA, pulse width=0.2ms, pulse frequency=5Hz. Monophasic pulses of current was delivered in alternating directions between the two electrodes. Gastric electrical stimulation was delivered simultaneously with MRI acquisition. Volumetric and motility MRI imaging were performed in an interleaved manner continuously for 4 hours.
?. [Effects of vagal afferent blockade on gastric motility during cervical vagus nerve stimulation measured with MRI]
Study titleEffects of vagal afferent blockade on gastric motility during cervical vagus nerve stimulation measured with MRIDescriptionThis protocol describes the methods used to evaluate the effects of cervical VNS on gastric motility in rats. The aim is to selectively activate or block afferent pathway to disentangle their differential effects on gastric responses.
?. [Effects of vagal afferent blockade on gastric motility during cervical vagus nerve stimulation measured with MRI]
Animal protocolFourteen rats(Male), ranging from 242 to 327g body weight were used in this study. Meal preparation, animal preparation, surgical steps, imaging sequence, data analysis were performed using our previously published protocol. In addition, a cuff catheter was placed cranial to the stimulation cuff electrode.Note that no Carprofen was administered to the animals in this study.
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?. [Effects of vagal afferent blockade on gastric motility during cervical vagus nerve stimulation measured with MRI]
Experiment DesignThe 14 rats were allocated into 2 groups as follows: (i) a group of rats (N=7) that did not receive infusion of 2% Lidocaineonto the left cervical vagus and (ii) a group of rats (N=7) that received infusion of 2% Lidocaine onto the left cervical vagus. The infusion protocol consists an initial fast infusion of 2% Lidocaine at 0.3mL/h for 6 minutes, followed by continuous infusion at a rate of 0.03ml/h. As soon as the initial fast infusion was done, baseline dynamic MRI images were acquired for 15 minutes. Then, cervical VNS (0.25mA, 0.5ms, 5Hz, 20s ON & 40s OFF) was delivered for 15 minutes simultaneously with MRI acquisition. The cathode was set cranial to the cuff to favor activation of afferent signaling to the brain. MRI images were collected for an additional 50 minutes after the offset of VNS.
?. [Effects of vagal efferent blockade on gastric motility and emptying during cervical vagus nerve stimulation measured with MRI]
Study titleEffects of vagal efferent blockade on gastric motility and emptying during cervical vagus nerve stimulation measured with MRIDescriptionThis protocol describes the methods used to evaluate the effects of cervical VNS on gastric emptying and motility in rats. The aim is to selectively activate afferent pathway (with and without blockade of efferent pathway) to disentangle their differential effects on gastric responses.
?. [Effects of vagal efferent blockade on gastric motility and emptying during cervical vagus nerve stimulation measured with MRI]
Animal protocolNine rats(Male), ranging from 252 to 366g body weight were used in this study. Meal preparation, animal preparation, surgical steps, imaging sequence, data analysis were performed using our previously published protocol. In addition, a cuff catheter was placed caudal to the stimulation cuff electrode. Note that no Carprofen was administered to the animals in this study.
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?. [Effects of vagal efferent blockade on gastric motility and emptying during cervical vagus nerve stimulation measured with MRI]
Experiment DesignThe 9 rats were allocated into 2 groups as follows: (i) a group of rats (N=5) that did not receive infusion of 2% Lidocaineonto the left cervical vagus and (ii) a group of rats (N=4) that received infusion of 2% Lidocaine onto the left cervical vagus. The infusion protocol consists an initial fast infusion of 2% Lidocaine at 0.3mL/h for 6 minutes, followed by continuous infusion at a rate of 0.03ml/h. As soon as the initial fast infusion was done, volumetric and motility MRI imaging were performed in an interleaved manner continuously for 4 hours. Cervical VNS (0.25mA, 0.5ms, 5Hz, 20s ON & 40s OFF) was continuously with MRI acquisition. The cathode was set cranial to the cuff to favor activation of afferent signaling to the brain.
?. [In vivo mapping of gastric electrical activation with manganese-enhanced magnetic resonance imagingSPARC - OT2OD023847 - SA2.1b - Y3Q4]
Study titleIn vivo mapping of gastric electrical activation with manganese-enhanced magnetic resonance imagingDescriptionThis protocol describes the use of manganese-enhanced MRI to map gastric electrical activation following meal consumption. The main classes of ion channels expressed in GI smooth muscle mirror those in vascular smooth muscle cells (SMCs) and include similar K+ channel families that set the resting membrane potentials and buffer contraction and the voltage-gated calcium channels and cationic TRPC channels that mediate SMC contraction. To assess intracellular calcium, one possible technique is to use manganese ion that has physical properties similar to calcium that can be handled similarly in many biological systems. Manganese ion is also an excellent T1 contrast agent. Imaging manganese with MRI showed elevated calcium activity confined to the antrum and corpus.
?. [In vivo mapping of gastric electrical activation with manganese-enhanced magnetic resonance imagingSPARC - OT2OD023847 - SA2.1b - Y3Q4]
Animal protocolOne rat(Male, 342g) was used in the study according to procedures approved by the Purdue Animal Care and Use Committee. Rats were housed individually in ventilated cages with elevated stainless steel wire floors during all time to prevent the animals from accessing their feces. The environment was maintained on a 12:12 hour light‐dark cycle (lights on at 6 am and lights off at 6 pm). The animal was trained to consume a fixed quantity of palatable Dietgel.The diet training took about 7 days. In the first 2 days, the animal was supplied with both regular rat chow and ∼10g Dietgel (the Dietgel was put in a dish in the cage at 11AM) to accustom itself to the Dietgel. In the following days, the animal was fasted for 18 hours (5PM to 11AM)  and then was fed with the Dietgel only at 11AM. The animal was given 30 minutes to consume 10g Dietgel; then regular meals were supplied to the animal afterward regardless of whether it finished the Dietgel or not. After the diet training (∼2 to 3 repetitions), the animal was able to naturally consume the Dietgel following overnight food restriction. On the day for manganese-enhanced magnetic resonance imaging (MEMRI), the animal was given 5g of the test meal. Animal preparation and imaging protocol were performed using our previously published protocol.
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?. [In vivo mapping of gastric electrical activation with manganese-enhanced magnetic resonance imagingSPARC - OT2OD023847 - SA2.1b - Y3Q4]
Experiment DesignPre-contrast volumetric gastric MRI images were collected prior to the onset of MnCl2 infusion. Then, 100mM MnCl2 was systemically infused through the tail vein at a rate of 0.5ml/h for 20 minutes. Dynamic contrast-enhanced gastric MRI images were acquired continuously for 80 minutes.