Use of a bio-electronic device comprising of targeted dual neuromodulation of the hepatic and celiac vagal branches demonstrated enhanced glycemic control in a type 2 diabetic rat model as well as in an Alloxan treated swine model

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  FIGURE 1. The sub-diaphragmatic Vagus nerve consists of two major trunks, the anterior and posterior. The pancreas is primarily innervated by the celiac branch which stems from the posterior Vagal trunk. The liver is innervated by the hepatic branch which stems from the anterior Vagal trunk. The aim of this study was to apply stimulation to celiac fibers (green arrow) and apply high frequency alternating current (HFAC)-induced blockade to hepatic fibers (red arrow). Figure created by www.biorender.com.

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  FIGURE 2. Depiction of experimental groups and animal numbers/experimental condition.

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  FIGURE 3. High frequency alternating current waveform and electrode configuration for the isolated nerve electrophysiology studies. (A) The high frequency alternating current (HFAC) was a 5,000 Hz signal with 90 us square wave pulses with a 10 us shorting period between the charge and recharge phases and delivered at 8 mA. This HFAC signal was used in the isolated nerve electrophysiology experiments as well as the in vivo rat and swine experiments. (B) The electrode configuration used in the isolated vagus nerve electrophysiology experiments consisted of a distal stimulation electrode (Sd), an electrode delivering HFAC, a proximal control stimulation electrode (Sp) and a recording electrode (R). The segment of nerve between the proximal stimulation and the recording electrodes was submerged in oxygenated SIF. The proximal stimulation electrode was used to test for HFAC-induced excitation which may produce fatigue following cessation of the HFAC signal; as opposed to a prolonged depolarization block at the site of the HFAC electrode.

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  FIGURE 4. Study timeline for Alloxan treated swine experiments. Each arrow indicates an experiment. Each experiment was conducted in 1 day. There were 2 days of separation between Sham oral glucose tolerance tests (OGTTs) as well as 2 days of separation between high frequency alternating current (HFAC) + stimulation OGTTs and fasting plasma glucose (FPG) experiments. In Sham and OGTT experiments FPG was the baseline blood sample (swine were fasted 18 h prior to all experiments). *Rest involves recovery from OGTTs. **Depicts rest period prior to washout OGTT.

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  FIGURE 5. Alloxan treated Yucatan swine wore a specially designed jacket to house two of the ReShape Lifesciences Inc. Viking mobile chargers. Transmitter coils above the layer of the skin were connected to the mobile chargers to charge the implanted neuroregulators with a RF signal between experiments. Settings for stimulation and high frequency alternating current (HFAC) parameters were also programmed into the neuroregulators using a laptop computer and application software via the mobile chargers and transmitter coils. Swine were trained for 7 days to wear the jacket. During the charging sessions the swine were not restrained and there was no apparent stress to the animals.

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  FIGURE 6. High frequency alternating current applied to the hepatic branch of the vagus nerve with simultaneous stimulation of the celiac branch of the vagus nerve reversibly decreased plasma glucose (PG) following an intravenous glucose tolerance test (IVGTT) in control and Zucker obese (fatty) male (ZDF) rats compared to sham. (A) As a positive control the hepatic nerve was ligated [in place of application of high frequency alternating current (HFAC)] with concurrent stimulation of the celiac nerve. This produced increased glycemic control compared to sham following an IVGTT. Standalone celiac nerve stimulation or hepatic nerve ligation had the same response to the IVGTT as sham. (B) Similar to vagotomy + stimulation, the application of HFAC + stimulation increased glycemic control following the IVGTT compared to sham. A subsequent IVGTT 15 min following the cessation of HFAC + stimulation demonstrated decreased glycemic control; suggesting reversibility. (C) Control Sprague Dawley rats also demonstrated increased glycemic control with HFAC + stimulation and vagotomy + stimulation. Error bars are SEM.

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  FIGURE 7. Isolated swine sub-diaphragmatic vagus nerve electrophysiology demonstrated conduction block and recovery at a high frequency alternating current (HFAC) amplitude of 8 mA. (A) Representative compound action potential (CAP) elicited by electrical stimulation at the level below the diaphragm and recorded at a segment below the heart. The length of the isolated vagus nerve was approximately 35 mm. (B) Current-effect curve of CAP amplitude elicited from the proximal and distal stimulation electrodes immediately following the cessation of HFAC. CAP amplitude was normalized to baseline. (C) Recovery of CAP amplitude following full block. Error bars are SEM.

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  FIGURE 8. Following Alloxan treatment swine demonstrated glucose intolerance. Graph depicts plasma glucose levels during an intravenous glucose tolerance test (IVGTT) prior to and following the administration of Alloxan to the swine. n = 6 swine. Error bars are SEM.

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  FIGURE 9. Application of high frequency alternating current (HFAC) to the hepatic branch of the vagus nerve with simultaneous stimulation of the celiac branch of the vagus nerve increased performance on an oral glucose tolerance test (OGTT) compared to pre-device initiation OGTTs in Alloxan treated swine. (A) Results from an OGTT conducted prior to implant of the device and following the implant of device were similar; suggesting that the implant itself did not change glycemic control. n = 3 swine. (B) Application of HFAC + stimulation 5 min following the initiation of the OGTT significantly improved performance on the OGTT in Alloxan treated swine n = 6 swine. Green arrow depicts when HFAC + stimulation was turned on and red arrow depicts when HFAC + stimulation was tuned off. n = 6 swine. Error bars are SEM.

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  FIGURE 10. The insulin response was not attenuated following six applications of high frequency alternating current (HFAC) + stimulation (Post-HFAC + stimulation Experiments) vs. Sham (Pre-HFAC + stimulation Experiments). n = 3 swine Error bars are SEM.

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  FIGURE 11. Nerves and surrounding tissue were healthy after 5 of high frequency alternating current (HFAC) + stimulation applications. (A) A 10× view of the Vagus nerve surrounded by space formally occupied by electrode delivering HFAC. Scale bar 0.5 mm (B) Expanded view of nerve facials taken from the dashed box in panel (A). (C) Normal nerve surrounded by a minimally thick fibrous capsule with moderate fibroblasts and 5–10 vascular profiles present per field of view at 200× total magnification that were fine new blood vessels. There was minimal subacute hemorrhage in the capsule and mild amounts of necrosis along the margin of the capsule. Moderate numbers of lymphocytes and macrophages and mild numbers of polymorphonuclear cells infiltrated the capsule. Scale bar 0.05 mm. Abbreviations: F, fibrous capsule; N, neovascularization; L, lymphocytes; PMN, polymorphonuclear cells; M, macrophages. (D) A 10× view of the Vagus nerve surrounded by space formally occupied by electrode delivering stimulation. Scale bar 0.5 mm. (E) Expanded view of nerve facials taken from the dashed box in panel (D). (F) The slide consisted of normal nerve surrounded by a minimally thick fibrous capsule with mild…