Rat Vagus Research Articles

The role of the vagus nerve on dexmedetomidine promoting survival and lung protection in a sepsis model in rats

BackgroundSepsis often results in acute lung injury (ALI). Dexmedetomidine (Dex) was reported to protect cells and organs due to its direct cellular effects. This study aims to investigate the role of vagus nerves on Dex induced lung protection in lipopolysaccharide (LPS)-induced ALI rats. MethodsThe bilateral cervical vagus nerve of male Sprague-Dawley rats was sectioned or just exposed as sham surgery. After LPS administration, Dex antagonist yohimbine (YOH) and/or Dex was injected intraperitoneally to rats with or without vagotomy. The severity of ALI was determined with survival curve analysis and lung pathological scores. The plasma concentrations of interleukin 1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), catecholamine and acetylcholine were measured with enzyme-linked immunosorbent assay. ResultsThe median survival time of LPS-induced ALI rats was prolonged by Dex (22 h, 95% CI, [24.46, 92.20]) vs. 14 h, 95% CI, [14.60, 89.57] of the LPS control group, P < 0.05), and the ALI score was reduced by Dex (6.5, 95% CI, [5.23, 8.10] vs. 11.5, 95% CI, [10.23, 13.10] in the LPS group, P < 0.01). However, these protective effects were significantly decreased by either YOH administration or vagotomy. Dex decreased LPS-induced IL-1β, TNF-α, and catecholamine but increased acetylcholine in blood serum; these effects of Dex was partially abolished by vagotomy. ConclusionsOur data suggested that Dex increased vagal nerve tone that partially contributed to its anti-inflammatory and lung-protective effects. The indirect anti-inflammation and direct cytoprotection of Dex are likely through high vagal nerve tone and α2-adrenoceptor activation, respectively.

Vagus nerve bundle stimulation using 1505-nm laser irradiation in an in-vivo rat model.

Laser nerve stimulation using near-infrared laser irradiation has recently been studied in the peripheral nervous system as an alternative method to conventional electrical nerve stimulation. Bringing this method to the vagus nerve model could leverage this emerging stimulation approach to be tested in broader preclinical applications. Here, we report the capability of the laser nerve stimulation method on the rat vagus nerve bundle with a 1505-nm diode laser operated in continuous-wave mode. Studies of the stimulation threshold and laser-induced acute thermal injury to the nerve bundle were also performed to determine a temperature window for safe, reliable and reproducible laser stimulation of the rat vagus nerve bundle. The results show that laser stimulation of the vagus nerve bundle provides reliable and reproducible nerve stimulation in a rat model. These results also confirm a threshold temperature of >42°C with acute nerve damage observed above 46°C. A strong correlation was obtained between the laser time required to raise the nerve temperature above the stimulation threshold and the mean arterial pressure response. Advantages of the method such as non-contact delivery of external stimulus signals at mm scaled distance in air, enhanced spatial selectivity and electrical artefact-free measurements may indicate its potential to counteract the side effects of conventional electrical vagus nerve stimulation.

In Vivo Evaluation of Fractal Microelectrodes Towards a More Targeted and Energy‐Efficient Vagus Nerve Stimulation

Vagus nerve stimulation (VNS) is a moderately effective treatment option for intractable epilepsy, depression, and pain, with promising applications in the treatment of gastrointestinal motility disorders like gastroparesis. The wide-ranging potential indications of VNS suggest an opportunity for a better understanding of underlying neural circuits that can be targeted using microscale electrodes. However, microscale Platinum (Pt) electrodes often have limited charge injection capacity due to their high impedance. Recently, we experimentally demonstrated that microscale Pt electrodes with Vicsek fractal geometry can facilitate electrochemical charge transfer more efficiently with 57% higher cathodic charge storage capacity than conventional circular microelectrodes. Here we are investigating whether the fractal electrode may be more effective in eliciting small fibers in the vagus nerve with less energy consumption due to its enhanced charge injection capability. To evaluate the in vivo performance of these novel microelectrode designs and to determine whether these electrodes are capable of stimulating small, unmyelinated C fibers that supply the stomach, we designed a cuff electrode with an array with four pairs of fractal and circular microelectrode to explore the possibility of radial selective stimulation of vagus nerve. We determined the dimension of the cuff to be 760 μm in diameter to wrap around a rat's vagus nerve so that it can withstand the swelling of the nerve from chronic implantation. We patterned fractal microelectrode to have a geometrical surface area equivalent to that of a circle with 100 μm of diameter. Using a rat model, we have stimulated the right cervical vagus nerve with fractal/circular electrode and recorded the compound action potential (CAP) distal from the stimulation site with 5-7 mm of conduction distance or the dorsal trunk of the abdominal vagus (conduction distance: 70 mm). We used an autonomous neural control (ANC) system, which can stimulate and record from the nerve with randomized stimulus parameter combinations (pulse amplitude: 0-0.4 mA, pulse width: 0.02-1 ms). We adapted a cathode-first, alternating monophasic stimulation for the reliable suppression of stimulus artifact (pulse repetition frequency: 5 Hz). Results from right cervical VNS experiments in anesthetized rats showed that both the fractal and the circular electrodes can recruit small, myelinated Ad and B fibers to a similar degree (pulse amplitude: 0-0.4 mA, pulse width: 0.1 ms) (Figure 1.), but fractal electrodes produce a more uniform and consistent fiber recruitment profile (Figure 2.). We observed volleys within the slow conduction velocity range (< 3 m/s), which we suspect to be signals from the C-fibers. Our next plan is to obtain more experimental data to statistically identify the efficiency of various electrode designs in stimulating small fibers and to investigate the electrical potential profile of these electrodes in a biophysical model.

Essential Oil of Croton zehntneri Prevents Conduction Alterations Produced by Diabetes Mellitus on Vagus Nerve.

Autonomic diabetic neuropathy (ADN) is a complication of diabetes mellitus (DM), to which there is no specific treatment. In this study, the efficacy of the essential oil of Croton zehntneri (EOCz) in preventing ADN was evaluated in the rat vagus nerve. For the two fastest conducting myelinated types of axons of the vagus nerve, the conduction velocities and rheobase decreased, whilst the duration of the components of the compound action potential of these fibers increased. EOCz completely prevented these DM-induced alterations of the vagus nerve. Unmyelinated fibers were not affected. In conclusion, this investigation demonstrated that EOCz is a potential therapeutic agent for the treatment of ADN.

Analysing vagus nerve spontaneous activity using finite element modelling

Objective. Finite element modelling has been widely used to understand the effect of stimulation on the nerve fibres. Yet the literature on analysis of spontaneous nerve activity is much scarcer. In this study, we introduce a method based on a finite element model, to analyse spontaneous nerve activity with a typical bipolar electrode recording setup, enabling the identification of spontaneously active fibres. We applied our method to the vagus nerve, which plays a key role in refractory epilepsy. Approach. We developed a 3D model including dynamic action potential (AP) propagation, based on the vagus nerve geometry. The impact of key recording parameters—inter-electrode distance and temperature—and uncontrolled parameters—fibre size and position in the nerve—on the ability to discriminate active fibres were quantified. A specific algorithm was implemented to detect and classify APs from recordings, and tested on six rat in-vivo vagus nerve recordings. Main results. Fibre diameters can be discriminated if they are below 3 μm and 7 μm, respectively for inter-electrode distances of 2 mm and 4 mm. The impact of the position of the fibre inside the nerve on fibre diameter discrimination is limited. The range of active fibres identified by modelling in the vagus nerve of rats is in agreement with ranges found at histology. Significance. The nerve fibre diameter, directly proportional to the AP propagation velocity, is related to a specific physiological function. Estimating the source fibre diameter is thus essential to interpret neural recordings. Among many possible applications, the present method was developed in the context of a project to improve vagus nerve stimulation therapy for epilepsy.

Activation of central adenosine A2B receptors mediate brain ghrelin-induced improvement of intestinal barrier function through the vagus nerve in rats

Leaky gut that is a condition reflecting intestinal barrier dysfunction has been attracting attention for its relations with many diseases such as irritable bowel syndrome or Alzheimer dementia. We have recently demonstrated that ghrelin acts in the brain to improve leaky gut via the vagus nerve. In the present study, we tried to clarify the precise central mechanisms by which ghrelin improves intestinal barrier function through the vagus nerve. Colonic permeability was estimated in vivo by quantifying the absorbed Evans blue in colonic tissue in rats. Adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), blocked the intracisternal ghrelin-induced improvement of intestinal hyperpermeability while dopamine, cannabinoid or opioid receptor antagonist failed to prevent it. Since DPCPX can block adenosine A1 and adenosine A2B receptors, we examined which subtype is involved in the mechanism. Intracisternal injection of adenosine A2B agonist but not adenosine A1 agonist improved colonic hyperpermeability, while peripheral injection of adenosine A2B agonist failed to improve it. Intracisternal adenosine A2B agonist-induced improvement of colonic hyperpermeability was blocked by vagotomy. Adenosine A2B specific antagonist, alloxazine blocked the ghrelin- or central vagal stimulation by 2-deoxy-d-glucose-induced improvement of intestinal hyperpermeability. These results suggest that activation of adenosine A2B receptors in the central nervous system is capable of improving intestinal barrier function through the vagal pathway, and the adenosine A2B receptors may mediate the ghrelin-induced improvement of leaky gut in a vagal dependent fashion. These findings may help us understand the pathophysiology in not only gastrointestinal diseases but also non-gastrointestinal diseases associated with the altered intestinal permeability.

Improvement in colonic inflammatory injury in rats via activating dorsal cholinergic neurons of vagus with electroacupuncture at sensitized acupoints

To observe the effect of electroacupuncture (EA) at sensitized acupoints on choline acetyltransferase positive (ChAT+) neurons in dorsal motor nucleus (DMV) of brainstem vagus in the colitis model rats and explore the mechanism of the improvement in colonic inflammatory injury in the rats. A total of 79 male SD rats were randomized into five groups, i.e. a normal group (20 rats), a normal plus sensitized acupoint group (5 rats), a model group (34 rats), an EA-1 group (15 rats) and an EA-2 group (5 rats). In the model group and the EA groups, 5% dextran sulfate sodium (DSS) was adopted for 6-day free drinking to establish colitis model rats. By injecting Evans blue (EB) into the caudal vein in the model rats, the sensitized acupoints were determined. Afterwards, in the normal plus sensitized acupoint group, the EA-1 group and the EA-2 group, EA was exerted at the sensitized acupoints, with disperse-dense wave, 2 Hz/ 15 Hz in frequency and 2 mA in intensity, for 30 min of intervention each day. The intervention lasted for 6 days in the EA-1 group and for 1 day in both the normal plus sensitized acupoint group and the EA-2 group. On day 0, 7 and 13 of experiment, successively, the score of disease activity index (DAI), the score of colonic histological damage, as well as the changes in the mechanical paw withdrawal threshold and thermal paw withdrawal latency were evaluated in the normal group, the model group and the EA-1 group. On day 7 of experiment, using immunofluorescence staining, the activation of different lamina neurons of spinal dorsal horn and ChAT+ neurons in DMV was observed in the normal group, the normal plus sensitized acupoint group, the model group and the EA-2 group separately. The EB extravasating areas were distributed in the segments from T12 to S1 on the body surface of colitis model rats, mainly focusing at L2 and L5. Therefore, "Shangjuxu" (ST 37) was taken as the sensitized acupoint. Compared with the normal group on day 7 and 13 of experiment, the mechanical paw withdrawal threshold were reduced (P<0.001), DAI scores and the scores of colonic histological damage were increased (P<0.001) in the model group. Compared with the normal group on day 7 of experiment, thermal paw withdrawal latency in the model group was reduced (P<0.001). Compared with the model group on day 13 of experiment, the mechanical paw withdrawal threshold was increased (P<0.001), DAI score and the score of colonic histological damage were reduced (P<0.01, P<0.05) in the EA-1 group. Compared with the normal group, the activated numbers of the neurons in superficial laminae (Ⅰand Ⅱ) at spinal dorsal horn of L4 to L6 and ChAT+ neurons in DMV were increased in the normal plus sensitized acupoint group and the model group (P<0.05, P<0.01). Compared with the normal plus sensitized acupoint group and the model group, the activated numbers of the neurons in superficial laminae at spinal dorsal horn of L4 to L6 and ChAT+ neurons in DMV were increased in the EA-2 group (P<0.001). The segmental dominance (acupoints) from T12 to S1 on the body surface of colitis rats is sensitized. EA at sensitized acupoints effectively relieves colonic inflammatory injury, which is probably by activating superficial lamina neurons of spinal dorsal horn and ChAT+ neurons of DMV.

Performance of Titanium Nitride Electrodes Used for Chronic Peripheral Nerve Stimulation

Electrical stimulation of the nervous system is used clinically to treat diseases such as epilepsy and there is increasing research using electrical stimulation to treat diseases and disorders where traditional pharmaceuticals are not effective. The performance of a device used for neural stimulation depends on its ability to reliably transfer the necessary charge to elicit a physiological response in a manner that is safe for both the tissue and electrode.Electrode materials that transfer charge via faradaic and non-faradaic mechanisms are used for neural stimulation. Electrodes that deliver charge via faradaic processes can accommodate the high amplitudes required for stimulation, but the faradaic reactions change the chemical composition of the surrounding tissue. Electrodes that deliver charge by charging and discharging the capacitive double layer typically deliver less charge than faradaic electrodes, however, they are attractive for neural stimulation because they do not change the chemical composition of the surrounding tissue. Titanium nitride (TiN) is an electrode material that has been used for non-faradaic charge transfer. This work evaluates the performance of TiN electrodes used for chronic peripheral nerve stimulation. To discriminate between electrode changes due to the chronic implantation versus stimulation, a separate set of TiN electrodes were subjected to continuous current-controlled biphasic pulsing in vitro. Methods: Nerve cuffs having four 0.39 mm2 rectangular TiN electrode sites were implanted around the cervical vagus nerve of male Brown Norway rats. The experimental procedures complied with the guidelines for the care and use of laboratory animals and were approved by the University of Florida Institutional Animal Care and Use Committee. Animals were chronically stimulated using current-controlled, charge-balanced biphasic cathode leading pulses. All electrochemical measurements were made with an Autolab potentiostat (PG- STAT12, Eco Chemie, Utrecht, The Netherlands). Electrochemical Impedance Spectroscopy (EIS) was measured using a cuff electrode site as the working electrode, and a bone screw as the reference and counter electrode. The Open Cell Potential (OCP) was measured prior to each measurement and was used as the pseudo zero for the EIS 20 mV peak-to-peak sinusoidal perturbation (wave type = 15 sines). The frequency was swept logarithmically downward from 100kHz to 50Hz. The in vitro electrical aging was performed on a separate set of TiN electrodes with the same specifications that were used in vivo. The electrodes were subjected to continuous current-controlled biphasic pulsing to replicate the amount of charge delivered over the lifetime of an implanted electrode. EIS and cyclic voltammetry (CV) were measured periodically throughout the pulsing. Results: The impedance magnitude increased over the duration of implantation for all animals. However, there appeared to be no relation between the impedance magnitude and amount of stimulation applied.The electrode potential was measured throughout the pulsing and showed a gradual increase over time in the electrode polarization during the anodic and cathodic phases. Electrochemical impedance spectroscopy and cyclic voltammetry were taken periodically throughout the pulsing and showed progressive changes in charge transfer for stimulated electrodes (figure 1B,C) , whereas electrode sites not subjected to stimulation had minimal changes (figure 1A). Stimulated electrodes also showed signs of electrode damage, such as delamination, that were not observed for unstimulated electrodes. Conclusion: Future work will focus on additional analysis of explanted cuffs to directly measure the influence of the in vivo environment. Figure 1

A Computational Model of Functionally-distinct Cervical Vagus Nerve Fibers.

Cervical vagus nerve stimulation (VNS) is a neuromodulation therapy used in the treatment of several chronic disorders. In order to maximize the therapeutic effectiveness of VNS, it has become increasingly important to deliver fiber-specific neurostimulation, so that undesired effects can be minimized. Assessing the activation of different vagal fiber types through electrical stimulation is therefore essential for developing fiber-selective VNS therapies. Towards this goal, we conducted in silico investigations using a generic model of functionally distinct nerve fibers and clinically relevant cuff electrodes using COMSOL. Our model is constrained by histological observations from rat cervical vagus nerves and its outputs are validated against averaged compound nerve action potentials (CNAPs) obtained from rat vagus nerve recordings. We propose this model as an effective tool to design fiber-specific stimulation protocols before testing them in experimental animals.

Employment of Different Waveform Shapes for Electrical Stimulation of the Vagus Nerve in Rats

The electrical stimulation of neural pathways has gained therapeutic relevance over the past decades. A wide range of clinical conditions benefits from vagus nerve stimulation (VNS), such as depression, epilepsy, heart failure, rheumatoid arthritis, cluster headache, traumatic brain injury, among others. Considering the relevance of experimental protocols aimed at a better understanding of mechanisms underlying the effects of VNS, proper adjustment of stimulation parameters such as frequency, pulse width, electric tension and waveform shape is crucial. The current study was intended to evaluate the effects of VNS on bradycardic response using three different waveform shapes. Wistar male rats were anesthetized with a ketamine/xylazine mixture and implanted with an electrode for electrocardiogram (ECG) recording and a bipolar stainless‐steel electrode into the cervical portion of the vagus nerve for VNS. Anesthetized animals had the ECG recorded and heart rate (HR) calculated on a beat‐to‐beat basis. Baseline HR was obtained during 5 minutes, followed by sessions of electrical stimulation of the vagus nerve. Taking into consideration the shape of the waveform used into VNS, rats were assigned into three groups: monopolar pulse (MP), depolarizing prepulse (DPP) and bipolar pulse (BP). Stimulation parameters were similar for all groups (frequency: 5 Hz; pulse width: 100 microseconds; electric tension: 1 V). Each VNS session lasted 30 seconds and sessions were 3 minutes apart. HR response during VNS was calculated to ensure the reliability of the preparation and was taken as the difference between average HR preceding VNS onset and maximum bradycardic response during the 30 seconds session. The magnitude of bradycardic response following the first VNS session was higher among DPP rats (Δ115 ± 23 bpm) when compared to MP (Δ37 ± 9 bpm) and BP (Δ83 ± 14 bpm) groups. Also, since our experimental setting contributes for better establishment of neural pathway longevity following exposure to electrical stimulation, the averaged bradycardic response among the three successive sessions of VNS was assessed. In a similar fashion to the findings regarding one single VNS session, it was observed a trend towards higher bradycardic response in DPP rats (Δ90 ± 32 bpm), when compared to MP (Δ56 ± 24 bpm) and BP (Δ70 ± 5 bpm) groups. From our findings, we can conclude that depolarizing prepulse waveform elicits a higher bradycardic response when VNS is conducted using parameters that match other waveform shapes. It is noteworthy to mention that the employment of VNS using techniques gentle (i.e. diminished damage) to neural pathways could contribute to nerve longevity.Support or Funding InformationBarao de Maua University Center: 344/18; FAPESP: 2017/05163‐6

SPARC: Acute Glucose Regulation Recordings from the Rat Vagus Nerve Using Carbon Fiber Microelectrode Arrays

Glucose regulation is a vital continuous process for maintaining a healthy biological state. This requires complicated coordination between the endocrine and nervous systems. An important pathway in the peripheral nervous system for glucose regulation is the vagus nerve, as it innervates the liver and pancreas, which are the main organs for glucose regulation. However, limited information is available on the neural activity for glucose regulation that propagates through the vagus nerve. An electrode that has shown promising neural recording abilities with minimal tissue damage in the brain is the carbon fiber microelectrode array (CFMA). The objective of this study was to identify glucose regulation neural signals in the vagus nerve using CFMAs.In this study, we performed non‐survival experiments on anesthetized, fasted Sprague‐Dawley rats. Through a midline ventral cervical incision, the left vagus nerve was isolated and placed on a custom 3D‐printed nerve‐holder. A CFMA (10–16 fibers; 200–250 μm in length; 132 μm pitch; 2‐row layout) was inserted in the vagus nerve and connected to a neural interface processor that recorded at a sampling rate of 30 kHz. A bolus of glucose (1 g) or insulin (20 U) was administered intraperitoneally to modulate blood glucose levels, or saline (1 mL) for control. Measurements of blood glucose concentrations from the tail were obtained every 5 minutes using a glucometer. Neural data was analyzed using Plexon Offline Sorter and MATLAB.Glucose administration elevated blood glucose levels up to 114% above baseline. Observed neural units with firing rates up to 5 spikes/sec and peak‐to‐peak amplitudes of 31–43 μV decreased their firing rates after administration of glucose. These units may represent afferent signaling that were driven from fasting the animal, and the firing rates of these units decreased after glucose injection. Insulin administration decreased blood glucose levels by 34% from baseline. Approximately 2–5 minutes after insulin administration, units with amplitudes of 78–96 μV peak‐to‐peak were firing at rates up to 21 spikes/sec. These units may represent an increase in afferent activity to request glucose intake. In control experiments, observed units with amplitudes of 44–62 μV peak‐to‐peak maintained their firing rates of 5–9 spikes/s before and after administration of saline. These units may represent continuous afferent signaling to increase food intake or signaling that is not related to glucose regulation.Overall, our results demonstrate that CFMA is a viable electrode for recording neural unit activity related to glucose regulation in the vagus nerve. Furthermore, this work suggests that the CFMA can be used to monitor neural unit activity in peripheral nerves to help better understand the neural regulation pathways in various physiological and pathophysiological conditions.Support or Funding InformationThis research was supported by the National Institute of Health SPARC Program (Award OT2OD024907) and the National Science Foundation (Award 1707316).

SPARC: Parameters of Electrical Excitation and Block of the Rat Vagus Nerve

There is growing interest in bioelectronic medicines, where diseases are treated through electrical stimulation and block of the peripheral autonomic nervous system. Given the widespread connections of the vagus nerve from the brainstem to most truncal organs, applications include treating epilepsy, depression, obesity, heart failure, and rheumatoid arthritis. However, designing and programming the parameters of bioelectronic medicines require understanding the biophysical responses of small myelinated and unmyelinated autonomic fibers. Using in vivo electrophysiology, we quantified strength‐duration properties, activity‐dependent slowing (ADS), and responses to kilohertz frequency (KHF) signals for rapidly conducting (&gt;2 m/s) and slowly conducting (&lt;2 m/s) fibers in the rat vagus nerve.We stimulated the cervical vagus nerve and recorded compound action potential (CAP) input‐output curves from the abdominal vagus nerve for pulse widths from 20 to 1000 μs. Thresholds were largest for the slowest fibres (0.5 to 1 m/s), especially at shorter pulse widths. Fits to the data using standard strength‐duration equations were qualitatively similar, but the estimates of chronaxie and rheobase varied substantially.ADS describes the slowed conduction in peripheral axons resulting from persistent low frequency activity. Using a novel cross‐correlation CAP‐based analysis method, we measured ADS of ~2.3% after 3 min of 2 Hz stimulation, approximately constant across fiber conduction speeds. This is comparable to that reported for sympathetic efferents in somatic nerves, but much smaller than ADS in cutaneous nociceptors. We found greater ADS with higher stimulation frequency and non‐monotonic changes in conduction speed in select cases.KHF signals can block neural conduction in peripheral axons, and we used CAP recordings to quantify the effects of KHF signals from 10 to 80 kHz on small autonomic fibers. Block thresholds were higher for more slowly conducting fibers, and block thresholds increased monotonically with frequency, in contrast to published findings indicating that block thresholds of unmyelinated axons vary non‐monotonically with frequency. Further, there are varied reports on the time for recovery of neural conduction after KHF block; we found that the carryover effect could last tens of seconds following 25 s of KHF signal.The quantification of mammalian autonomic nerve responses to conventional and kilohertz frequency signals provides essential information for development of bioelectronic medical devices and for understanding mechanisms of action.Support or Funding InformationThis work was supported by Fulbright Canada (15122811), the Natural Sciences and Engineering Research Council of Canada (PGS M‐425353‐2012 and PGS D3‐437918‐2013), and Duke University (University Scholars Program, James B. Duke Fellowship, and Pratt School of Engineering Faculty Discretionary Fund).

SPARC: A Hybrid Computational Approach to Classify Vagal C Fiber Functions

Neuroscience conventionally categorizes all unmyelinated fibers into an undifferentiated C fiber classification, implying a common function and impeding further classification. This is highly problematic, especially for autonomic nerves like the abdominal vagus, which contains &gt;99% unmyelinated C fibers. We require a modern classification framework to discriminate differences among these important, underappreciated messengers. One important and easily measurable property of C fibers is their conduction speed, which relates directly to their morphology or caliber. We present a hybrid computational modeling approach, integrating biophysical (HH‐type) and classical (e.g., Gasser, Erlanger and Grundfest) methods, to enable the classification of C fiber function according to fiber caliber/speed.The Hybrid Nerve Response Modeling and Classification System is a tool to classify C fiber functions with respect to fiber caliber and radial location within a nerve. Biophysical models of HH axons contribute a bottom‐up model of fiber activation that explains the influence of the electrode configuration and other factors on fiber activation threshold and the measured compound action potential (CAP) profile. Alternatively, modeled CAPs can be generated through superposition of individual axon contributions (single fiber action potentials; SFAPs), relating fiber caliber distribution data to SFAP shape and conduction properties; this classical model of the CAP accurately predicts the experimentally measured maximal CAP shape. Using published and novel nerve ultrastructure data, we unified the biophysical and classical modeling approaches in a new simulation framework that predicts graded CAP profiles in response to bipolar, constant‐current stimuli delivered through various cuff electrode configurations.In this work, we demonstrate how the Hybrid Model can be used to discern the spatial and size distribution of unmyelinated C fibers from the CAP of the ventral gastric branch of the rat vagus nerve. Through our web‐based user interface (Fig. 1), we will show how our system can predict the activation threshold of unmyelinated fibers in any nerve cross section as a function of the individual fiber size and spatial location within a common set of electrode geometries. We will provide examples of how this information may be used to accelerate our delineation of the various functions of C fibers using measured associations between nerve and organ responses to stimulation.Support or Funding InformationFunding AcknowledgmentsThis work was supported by NIH SPARC OT2 OD023847 (PI: Powley), NIH SPARC OT2 OD025340 (PI: Grill) and NIH SPARC OT2 OD026585 01 (PI: Havton)[LEFT] Web‐based interface for the Hybrid Model. [FAR RIGHT] Segmented electron micrograph of n = 4776 C fibers from a rat ventral vagal gastric branch. The user interface lets users track the location (e.g., spatial location in the nerve cross section and temporal location in the CAP response) and relative activation thresholds of C fibers with respect to observed changes in the downstream physiology of the organs and tissues they supply.Figure 1

In vivo quantification of excitation and kilohertz frequency block of the rat vagus nerve

Objective. There is growing interest in treating diseases by electrical stimulation and block of peripheral autonomic nerves, but a paucity of studies on the excitation and block of small-diameter autonomic axons. We conducted in vivo quantification of the strength-duration properties, activity-dependent slowing (ADS), and responses to kilohertz frequency (KHF) signals for the rat vagus nerve (VN). Approach. We conducted acute in vivo experiments in urethane-anaesthetized rats. We placed two cuff electrodes on the left cervical VN and one cuff electrode on the anterior subdiaphragmatic VN. The rostral cervical cuff was used to deliver pulses to quantify recruitment and ADS. The caudal cervical cuff was used to deliver KHF signals. The subdiaphragmatic cuff was used to record compound action potentials (CAPs). Main results. We quantified the input–output recruitment and strength-duration curves. Fits to the data using standard strength-duration equations were qualitatively similar, but the resulting chronaxie and rheobase estimates varied substantially. We measured larger thresholds for the slowest fibres (0.5–1 m s−1), especially at shorter pulse widths. Using a novel cross-correlation CAP-based analysis, we measured ADS of ~2.3% after 3 min of 2 Hz stimulation, which is comparable to the ADS reported for sympathetic efferents in somatic nerves, but much smaller than the ADS in cutaneous nociceptors. We found greater ADS with higher stimulation frequency and non-monotonic changes in CV in select cases. We found monotonically increasing block thresholds across frequencies from 10 to 80 kHz for both fast and slow fibres. Further, following 25 s of KHF signal, neural conduction could require tens of seconds to recover. Significance. The quantification of mammalian autonomic nerve responses to conventional and KHF signals provides essential information for the development of peripheral nerve stimulation therapies and for understanding their mechanisms of action.

Mechanisms underlying the stimulatory effect of inhaled sulfur dioxide on vagal bronchopulmonary C-fibres.

Brief inhalation of SO2 of concentration >500p.p.m. triggered a pronounced stimulatory effect on vagal bronchopulmonary C-fibres in anaesthetized rats. This stimulatory effect was drastically diminished by a pretreatment with NaHCO3 that raised the baseline arterial pH, suggesting a possible involvement of acidification of airway fluid and/or tissue generated by inhaled SO2 . The stimulation was completely abolished by pretreatment with antagonists of both acid-sensing ion channels and transient receptor potential vanilloid type-1 receptors, indicating that this effect was caused by acid activation of these cation channels expressed in airway sensory nerves. This conclusion was further supported by the results obtained from studies in isolated rat vagal bronchopulmonary sensory neurones and also in the cough response to SO2 inhalation challenge in awake mice. These results provide new insight into the underlying mechanism of harmful irritant effects in the respiratory tract caused by accidental exposure to a high concentration of SO2 . Inhalation of sulfur dioxide (SO2 ) triggers coughs and reflex bronchoconstriction, and stimulation of vagal bronchopulmonary C-fibres is primarily responsible. However, the mechanism underlying this stimulatory effect is not yet fully understood. In this study, we tested the hypothesis that the C-fibre stimulation was caused by SO2 -induced local tissue acidosis in the lung and airways. Single-unit activities of bronchopulmonary C-fibres in response to inhalation challenges of SO2 (500-1500p.p.m., 10 breaths) were measured in anaesthetized rats. Inhalation of SO2 reproducibly induced a pronounced and sustained stimulation (lasting for 15-60s) of pulmonary C-fibres in a concentration-dependent manner. This stimulatory effect was significantly attenuated by an increase in arterial pH generated by infusion of sodium bicarbonate (NaHCO3 ), and completely abrogated by a combined pretreatment with amiloride (an antagonist of acid-sensing ion channels, ASICs) and AMG8910 (a selective antagonist of the transient receptor potential vanilloid type-1 receptor, TRPV1). Furthermore, in isolated rat vagal pulmonary sensory neurones, perfusion of an aqueous solution of SO2 evoked a transient increase in the intracellular Ca2+ concentration; this response was also markedly diminished by a pretreatment with amiloride and AMG8910. In addition, inhalation of SO2 consistently evoked coughs in awake mice; responses were significantly smaller in TRPV1-/- mice than in wild-type mice, and almost completely abolished after a pretreatment with amiloride in TRPV1-/- mice. These results suggested that the stimulatory effect of inhaled SO2 on bronchopulmonary C-fibres was generated by acidification of fluid and/or tissue in the lung and airways, which activated both ASICs and TRPV1 expressed in these sensory nerves.

Flat electrode contacts for vagus nerve stimulation.

The majority of available systems for vagus nerve stimulation use helical stimulation electrodes, which cover the majority of the circumference of the nerve and produce largely uniform current density within the nerve. Flat stimulation electrodes that contact only one side of the nerve may provide advantages, including ease of fabrication. However, it is possible that the flat configuration will yield inefficient fiber recruitment due to a less uniform current distribution within the nerve. Here we tested the hypothesis that flat electrodes will require higher current amplitude to activate all large-diameter fibers throughout the whole cross-section of a nerve than circumferential designs. Computational modeling and in vivo experiments were performed to evaluate fiber recruitment in different nerves and different species using a variety of electrode designs. Initial results demonstrated similar fiber recruitment in the rat vagus and sciatic nerves with a standard circumferential cuff electrode and a cuff electrode modified to approximate a flat configuration. Follow up experiments comparing true flat electrodes to circumferential electrodes on the rabbit sciatic nerve confirmed that fiber recruitment was equivalent between the two designs. These findings demonstrate that flat electrodes represent a viable design for nerve stimulation that may provide advantages over the current circumferential designs for applications in which the goal is uniform activation of all fascicles within the nerve.

Extraction of Evoked Compound Nerve Action Potentials from Vagus Nerve Recordings.

Cervical vagus nerve stimulation (VNS) is a neuromodulation therapy for the treatment of several chronic disorders. The effects of VNS are mediated by activation of nerve fibers of different types. In order to maximize the desired and minimize the undesired effects of VNS, assessing activation of vagal fiber types by VNS is essential. Evoked compound nerve action potentials (CNAPs) are commonly used as a method to estimate vagal fiber activation in the context of neurostimulation. However, vagal CNAPs are frequently contaminated by signals from non-neural sources, like electrocardiography (ECG), stimulus artifacts and evoked electromyographic (EMG) activity. In this study, we present a systematic methodology for suppressing non-neural signals in CNAP recordings from the rat vagus. The methodology involves intravenous infusion of vecuronium under ventilation, for suppressing EMG, and digital and analog signal processing, for suppressing ECG and stimulus artifacts, respectively. We compiled A-, B- and C-type fiber activation profiles with and without this methodology and found that our method significantly increased the reliability of CNAPs. We found that the A-component is obscured by the stimulus artifact, whereas the B- and C-components are frequently contaminated by evoked EMG. We extracted CNAPs evoked by square pulses of different polarities and amplitudes and documented effects consistent with well-established biophysical attributes of VNS.

The regulating effect of auricle electrical stimulation on the cardiac vagus nerve in rats

Objective To compare the regulating effect of electrically stimulating different parts of the auricle on the cardiac vagus nerve in rats, and to explore the basic neural mechanism. Methods The tragus, concha auriculae and helix of 24 male Sprague-Dawley rats were stimulated at different intensities (0-16 mA) and with different durations (0-15 min) and any changes in the heart rate were observed. One week later, the rats were randomized into a tragus injection group, a concha auriculae injection group, a helix injection group and a control group, each of 6. The rats of the first three groups were injected with 2 μL of cholera toxin subunit B conjugate AF555 (CTB-AF555) at the right auricle, while the control group was injected with the same amount of aseptic phosphate-buffered saline at the right tragus. Five days later, all of the rats were sacrificed and their right superior and inferior ganglia and the whole bulbus medullae were resected to observe the fluorescent labeling sites. Results The rats′ heart rate declined with longer and more intense stimulation of the tragus or concha auriculae, but not with stimulation of the helix. With stimulation of the same duration, a significant decrease was observed in the heart rate when the tragus and concha auriculae were stimulated at 10, 12, 14 or 16 mA compared with when the helix was stimulated at the same intensities. The heart rate when the concha auriculae was stimulated at 12 mA was significantly slower than when the tragus was stimulated at the same intensity. At identical stimulus intensities, the heart rate slowed significantly more when the tragus was stimulated for 6 to 15 minutes and the concha auriculae for 4 to 15 minutes compared with stimulating the helix for the same length of time. And compared with stimulating the tragus for 6 to 10 minutes, the heart rate decreased significantly more when the concha auriculae was stimulated for the same length of time. All of the rats in the tragus and concha auriculae injection groups displayed nerve tracer in their superior and inferior ganglia. In the tragus injection group, CTB-AF555 was observed in the nucleus tractus solitarius (NTS) of 3 of the 6 rats. In the concha auriculae injection group it was observed in 4 of the 6. In the helix injection group, CTB-AF555 was observed in the nucleus of the spinal tract in 5 of the 6 rats, but no nerve tracer was found in their superior or inferior ganglia or in the NTS. Conclusion Electrical stimulation of the tragus and concha auriculae can regulate the functioning of the cardiac vagus nerve, but stimulating the helix cannot. This is partly because the nerve signals in tragus or concha auriculae stimulation and the cardiac sensory nerve signal are integrated in the inferior ganglion and then analyzed and processed in the bulbar center to monitor the heart. Key words: Electrical stimulation; Auricle; Vagus nerve; Heart rate

Effect of dispersion in nerve on compound action potential and impedance change: a modelling study

Objective: Electrical impedance tomography (EIT) is capable of imaging fast compound electrical activity (compound action potentials, or CAPs) inside peripheral nerves. The ability of EIT to detect impedance changes (dZ) which arise from the opening of ion channels during the CAP is limited by the dispersion with distance from the site of onset, as fibres have differing conduction velocities. The effect is largest for autonomic nerves mainly formed of slower conducting unmyelinated fibres where signals cannot be recorded more than a few cm away from the stimulation. However, as CAPs are biphasic, monophasic dZ are expected to be detectable further than them; testing this hypothesis was the main objective of this study. Approach: An anatomically accurate FEM model and simplified statistical models of 50-fibre Hodgkin–Huxley and C-nociceptor nerves were developed with normally distributed conduction velocities; the statistical models were extended to realistic nerves. Main results: Fifty-fibre models showed that dZ could persist further than biphasic CAPs, as these then cancelled. For realistic nerves consisting of Aα or Aβ fibres, significant dZ could be detected at 50 cm from the onset site with signal-to-noise ratios (SNR, mean ± s.d.) of 2.7 ± 0.2 and 1.8 ± 0.1 respectively; Aδ and rat sciatic nerve—at 20 cm (1.6 ± 0.03 and 1.6 ± 0.06), rat vagus—at 10 cm (1.6 ± 0.05); C fibres—at 1–2 cm (2.4 ± 0.02). Significance: This study provides a basis for determining the distance over which EIT may be used to image fascicular activity in electroceuticals and suggests dZ will persist further than CAPs if biphasic.

Influence of Cholecystokinin-8 on Compound Nerve Action Potentials from Ventral Gastric Vagus in Rats.

Vagus Nerve Stimulation (VNS) has shown great promise as a potential therapy for a number of conditions, such as epilepsy, depression and for Neurometabolic Therapies, especially for treating obesity. The objective of this study was to characterize the left ventral subdiaphragmatic gastric trunk of vagus nerve (SubDiaGVN) and to analyze the influence of intravenous injection of gut hormone cholecystokinin octapeptide (CCK-8) on compound nerve action potential (CNAP) observed on the same branch, with the aim of understanding the impact of hormones on VNS and incorporating the methods and results into closed loop implant design. The cervical region of the left vagus nerve (CerVN) of male Wistar rats was stimulated with electric current and the elicited CNAPs were recorded on the SubDiaGVN under four different conditions: Control (no injection), Saline, CCK1 (100[Formula: see text]pmol/kg) and CCK2 (1000[Formula: see text]pmol/kg) injections. We identified the presence of A[Formula: see text], B, C1, C2, C3 and C4 fibers with their respective velocity ranges. Intravenous administration of CCK in vivo results in selective, statistically significant reduction of CNAP components originating from A and B fibers, but with no discernible effect on the C fibers in [Formula: see text] animals. The affected CNAP components exhibit statistically significant ([Formula: see text] and [Formula: see text]) higher normalized stimulation thresholds. This approach of characterizing the vagus nerve can be used in closed loop systems to determine when to initiate VNS and also to tune the stimulation dose, which is patient-specific and changes over time.