Bioelectronic modulation of carotid sinus nerve activity in the rat: a potential therapeutic approach for type 2 diabetes

Aims/hypothesis A new class of treatments termed bioelectronic medicines are now emerging that aim to target individual nerve fibres or specific brain circuits in pathological conditions to repair lost function and reinstate a healthy balance. Carotid sinus nerve (CSN) denervation has been shown to improve glucose homeostasis in insulin-resistant and glucose-intolerant rats; however, these positive effects from surgery appear to diminish over time and are heavily caveated by the severe adverse effects associated with permanent loss of chemosensory function. Herein we characterise the ability of a novel bioelectronic application, classified as kilohertz frequency alternating current (KHFAC) modulation, to suppress neural signals within the CSN of rodents. Methods Rats were fed either a chow or high-fat/high-sucrose (HFHSu) diet (60% lipid-rich diet plus 35% sucrose drinking water) over 14 weeks. Neural interfaces were bilaterally implanted in the CSNs and attached to an external pulse generator. The rats were then randomised to KHFAC or sham modulation groups. KHFAC modulation variables were defined acutely by respiratory and cardiac responses to hypoxia (10% O2 + 90% N2). Insulin sensitivity was evaluated periodically through an ITT and glucose tolerance by an OGTT. Results KHFAC modulation of the CSN, applied over 9 weeks, restored insulin sensitivity (constant of the insulin tolerance test [KITT] HFHSu sham, 2.56 ± 0.41% glucose/min; KITT HFHSu KHFAC, 5.01 ± 0.52% glucose/min) and glucose tolerance (AUC HFHSu sham, 1278 ± 20.36 mmol/l × min; AUC HFHSu KHFAC, 1054.15 ± 62.64 mmol/l × min) in rat models of type 2 diabetes. Upon cessation of KHFAC, insulin resistance and glucose intolerance returned to normal values within 5 weeks. Conclusions/interpretation KHFAC modulation of the CSN improves metabolic control in rat models of type 2 diabetes. These positive outcomes have significant translational potential as a novel therapeutic modality for the purpose of treating metabolic diseases in humans. Electronic supplementary material The online version of this article (10.1007/s00125-017-4533-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.


Experimental design for animal tests Insulin Tolerance Test
Insulin sensitivity was evaluated through an insulin tolerance test (ITT) in conscious animals. The ITT is one of the earliest methods developed to assess insulin sensitivity in vivo and provides an estimate of overall insulin sensitivity, correlating well with the 'gold standard' hyperinsulinaemic-euglycaemic clamp [23]. The ITT consists in the administration of an intravenous insulin (Humulin® R 100IU/ml, Lilly) bolus of 0.1 U/kg body weight in the tail vein, after an overnight fast (approx. 16 hours), followed by the measurement of the decline in plasma glucose concentration over a 15 minute period. The constant rate for glucose decline (K ITT ) was calculated using the formula 0.693/t 1/2 [22,23].

Whole-body plethysmography recordings of ventilation
Ventilation was measured in conscious freely moving rats by whole-body plethysmography. The system (Emka Technologies, Paris, France) consisted of 5-litre methacrylate chambers continuously fluxed (2 l/min) with gases. Tidal volume (VT; ml) respiratory frequency (f; breaths/min; bpm) and minute ventilation (VE; ml/min/Kg) were monitored. Each rat was placed in the plethysmography chamber and allowed to breathe room air for 20 min to allow adaptation to chamber environment and to acquire a standard resting behavior.
Animal acclimatized well during this period and enabled subsequent ventilatory parameters to be recorded according to the protocol used. The protocol consisted in submitting the animals to 20 mins acclimatization followed by 10 min normoxia (20% O 2 balanced N 2 ) followed by 10 min hypoxia (10% O 2 balanced N 2 ), followed by 10 min normoxia, followed by 10 min hypercapnia (20% O 2 + 5% CO 2 balanced N 2 ) and then 10 min normoxia. The pressure change within the chamber reflecting tidal volume (VT) was measured with a high-gain differential pressure transducer. Ideally, the frequency of pressure fluctuations is identical to breathing movements; spurious fluctuations of the pressure due to animal movements were electronically rejected. The amplitude of the pressure oscillations is proportionally related to VT; a calibration of the system by injections of 0.2 to 0.5 ml air into the chamber allowed a direct estimation of VT. Pressure signals were fed to a computer for visualization and storage for later analysis with EMKA software (Emka Technologies, Paris, France).

Measurement of electrode impedance
Impedance was measured at 0 and 1 days post-implantation and just prior to animal's sacrifice using a handheld potentiostat (pocketSTAT, Ivium Technologies B.V., Eindhoven, Netherlands) connected to a computer running the electrochemical impedance spectroscopy (EIS) software (IviumSoft, Ivium Technologies). The impedances were measured using a two-pole setup: working and sense electrodes were connected to form the first pole, while the counter and reference electrodes were connected to form the second pole, ground electrode not connected. The EIS was performed at frequencies from 10 to 10,000Hz (ESM Fig. 4).

Histology
For histology analysis, the carotid artery (CA) area and the cuff electrodes were bilaterally dissected and immersion-fixed in PFA 4%. Samples were embedded into paraffin for routine H&E staining (4μm thick coronal sections). Some samples were instead frozen and cut with a cryostat (20-30μm thick) for H&E or toluidine blue staining. Some samples were used for electron microscopy analysis. Fixed samples were post fixed in 1% aqueous osmium tetroxide and processed into Agar 100 resin.1 mm toluidine blue stained survey sections were prepared and examined by light microscopy to locate the areas of interest.
Ultra-thin sections (1-µm thick) were stained with uranyl acetate and lead citrate and examined in a Hitachi H7500 transmission electron microscope. The AMT XR41 Digital Camera System v600.202 was used to capture TEM digital images. Representative digital images were taken.

ESM Tables
ESM Table 1 -Effect of carotid sinus nerve (CSN) bilateral resection on fasting blood glucose and insulin and C-peptide in control (CTL) and early-type 2 diabetes (HFHSu) animals.
Data are means ± SEM of 8/10 animals.