Objective.Electrical stimulation of the baroreceptors pathways at the carotid sinus bulb-known as baroreflex activation therapy (BAT)-is intended to change autonomic tone and ultimately reduce blood pressure (BP) and heart rate. BAT is pre-market approved by the United States Food and Drug Administration (FDA) for the treatment of heart failure and received an FDA humanitarian device exemption for drug resistant hypertension. However, responder rates are limited by side-effects including numbness in the head and neck, altered speech, respiratory constriction, dry cough, vomiting, and altered sensory and motor function of the tongue (TN). We hypothesized that these side-effects are driven by activation of other nearby nerve fibers of similar or lower threshold than the carotid sinus nerve. In this study, we sought to identify the neural sources responsible for off-target muscle activation contributing to these side-effects. These sources would inform strategies mitigating off-target activation in BAT therapy.Approach.Domestic swine were used in this work as the diameter and thickness of the swine carotid artery are closer to human than those of canine models. A monopolar disk electrode mimicking the clinical CVRx® Neo electrode was surgically placed proximal to the carotid bifurcation with the position optimized for stimulation dose responsive changes in BP. Evoked responses were recorded during dose response testing from multiple neck muscles, and the corresponding off-target nerve pathways were identified by sequential transection of nearby nerves.Main results.The following activated off-target muscle groups and their corresponding nerve pathways were verified which included (1) the cricoarytenoid via recurrent laryngeal nerve, (2) cricothyroid via superior laryngeal nerve, (3) sternocleidomastoid via accessory nerve, and (4) the TN via hypoglossal nerve. The constrictor muscle group was also activated through a more complex neural pathway.Significance.We identified multiple sources of therapy-limiting side-effects of BAT in the swine model. These results will help guide the design of improved stimulation electrodes, surgical placement, and parameter programming to reduce BAT-associated side-effects while increasing on-target activation in patients.

Objective.Electrical stimulation of the vagus nerve (VNS) is an Food and Drug Administration approved therapy for epilepsy, depression and rehabilitation after stroke, with recent clinical trials to treat heart failure and inflammation. VNS is often assumed to activate either parasympathetic efferents projecting to visceral organs, and/or sensory afferents projecting from these organs, for its therapeutic effects. Recent studies in humans, swine and dogs have shown that sympathetic nerve fibers from the sympathetic trunk (ST) can frequently be found within the cervical vagus nerve (VN). However, the prevalence and functional consequence of sympathetic fibers on VNS have yet to be elucidated in the most common high throughput animal model to study disease, the rodent.Approach.We carefully traced ST from superior cervical ganglion (SCG) to find its location in the carotid sheath with reference to the VN in a cohort of Long Evans rats. We then assessed the prevalence of ST fibers with the cervical VN across the cohort using micro-computer tomography and immunohistochemistry. Finally, we stimulated the VN and the ST in isolation, and where they were conjoined, to evaluate the ST contribution to changes in heart rate (HR). VNS induced HR changes are a commonly used surrogate for changes in sympathetic/parasympathetic tone.Main results.The ST frequently runs in very close proximity to the VN in rats when traced caudally from the SCG. The ST is even conjoined with the VN for stretches within the carotid sheathe at the most common location to place an epineural cuff. Cross-connecting branches were found between the ST and the VN. VNS performed at locations where there was minimal ST crossover induced dose-dependent bradycardia (decrease in HR) across the cohort, with detectable bradycardia across the cohort beginning at 50μA (n= 8 right,n= 3 left). Conversely, stimulation of the isolated ST induced tachycardia (increase in HR) across the cohort beginning at ∼200μA (n= 7 right,n= 3 left).Significance.These data suggest that studies of VNS in the rodent model may also be stimulating sympathetic fibers from the ST in addition to canonical VN pathways. Concurrent sympathetic activation has profound implications for dissecting mechanisms of VNS for a host of diseases/disorders. As such, careful post-mortem assessment of the presence of 'hitchhiking' sympathetic fibers within the VN is critical for understanding sources of variability in VNS outcomes.

Objective. Evoked compound action potentials (ECAPs) measured during epidural spinal cord stimulation (SCS) can help elucidate fundamental mechanisms for the treatment of pain and inform closed-loop control of SCS. Previous studies have used ECAPs to characterize neural responses to various neuromodulation therapies and have demonstrated that ECAPs are highly prone to multiple sources of artifact, including post-stimulus pulse capacitive artifact, electromyography (EMG) bleed-through, and motion artifact. However, a thorough characterization has yet to be performed for how these sources of artifact may contaminate recordings within the temporal window commonly used to determine activation of A-beta fibers in a large animal model.Approach. We characterized sources of artifacts that can contaminate the recording of ECAPs in an epidural SCS swine model using the Abbott Octrode™ lead.Main results. Spinal ECAP recordings can be contaminated by capacitive artifact, short latency EMG from nearby muscles of the back, and motion artifact. The capacitive artifact can appear nearly identical in duration and waveshape to evoked A-beta responses. EMG bleed-through can have phase shifts across the electrode array, similar to the phase shift anticipated by propagation of an evoked A-beta fiber response. The short latency EMG is often evident at currents similar to those needed to activate A-beta fibers associated with the treatment of pain. Changes in CSF between the cord and dura, and motion induced during breathing created a cyclic oscillation in all evoked components of recorded ECAPs.Significance. Controls must be implemented to separate neural signal from sources of artifact in SCS ECAPs. We suggest experimental procedures and reporting requirements necessary to disambiguate underlying neural response from these confounds. These data are important to better understand the framework for epidural spinal recordings (ESRs), with components such as ECAPs, EMG, and artifacts, and have important implications for closed-loop control algorithms to account for transient motion such as postural changes and cough.

Electrical stimulation of the vagus nerve (VNS) is an FDA approved therapy for epilepsy, depression and rehabilitation after stroke, with recent clinical trials to treat heart failure and inflammation. VNS is often assumed to activate either parasympathetic efferents projecting to visceral organs, and/or sensory afferents projecting from these organs, for its therapeutic effects. Recent studies in humans, swine and dogs have shown that sympathetic nerve fibers from the sympathetic trunk (ST) can frequently be found within the cervical vagus nerve (VN). However, the prevalence and functional consequence of sympathetic fibers on VNS have yet to be elucidated in the most common high throughput animal model to study disease, the rodent. We carefully traced ST from sympathetic cervical ganglion (SCG) to find its location in the carotid sheath with reference to the VN in a cohort of Long Evans rats. We then assessed the prevalence of ST fibers with the cervical VN across the cohort using microCT and immunohistochemistry. Finally, we stimulated the VN and the ST in isolation, and where they were conjoined, to evaluate the ST contribution to changes in heart rate. VNS induced heart rate changes are a commonly used surrogate for changes in sympathetic/parasympathetic tone. The ST frequently runs in very close proximity to the VN in rats when traced caudally from the SCG. The ST is even conjoined with the VN for stretches within the carotid sheathe at the most common location to place an epineural cuff. Cross-connecting branches were found between the ST and the VN. VNS performed at locations where there was minimal ST crossover induced dose-dependent bradycardia (decrease in heart rate) across the cohort, with detectable bradycardia across the cohort beginning at 50 μA (n=8 right, n=3 left). Conversely, stimulation of the isolated ST induced tachycardia (increase in heart rate) across the cohort beginning at ∼200 μA (n=7 right, n=3 left). These data suggest that studies of VNS in the rodent model may also be stimulating sympathetic fibers from the ST in addition to canonical VN pathways. Concurrent sympathetic activation has profound implications for dissecting mechanisms of VNS for a host of diseases/disorders. As such, careful post-mortem assessment of the presence of ‘hitchhiking’ sympathetic fibers within the VN is critical for understanding sources of variability in VNS outcomes.

Background and PurposeSpinal cord stimulation (SCS) is approved by the Food and Drug Administration for treating chronic intractable pain in the back, trunk, or limbs through stimulation of the dorsal column. Numerous studies have used swine as an analog of the human spinal cord to better understand SCS and further improve its efficacy. We performed high-resolution imaging of the porcine spinal cord with intact dura mater using micro-computed tomography (mu CT) to construct detailed 3-dimensional (3D) visualizations of the spinal cord and characterize the morphology of the dorsal and ventral rootlets.MethodsWe obtained spinal cords from Yorkshire/Landrace crossbred swine (N = 7), stained samples with osmium tetroxide, and performed mu CT imaging of the T12-T15 levels at isotropic voxel resolutions ranging from 3.3 to 50 mu m. We measured the anatomical morphology using the 3D volumes and compared our results to measurements previously collected from swine and human spinal cords via microdissection techniques in prior literature.ResultsWhile the porcine thoracic-lumbar spinal cord is a popular model for SCS, we highlight multiple notable differences compared to previously published T8-T12 human measurements including rootlet counts (porcine dorsal/ventral: 12.2 +/- 2.6, 26.6 +/- 3.4; human dorsal/ventral: 5.3 +/- 1.3, 4.4 +/- 2.4), rootlet angles (porcine ventral-rostral: 161 +/- 1 degrees, ventral-caudal: 155 +/- 6 degrees, dorsal-rostral: 148 +/- 9 degrees, dorsal-caudal: 142 +/- 6 degrees; human ventral-rostral: 170 +/- 3 degrees, ventral-caudal: 22 +/- 10 degrees, dorsal-rostral: 171 +/- 3 degrees, dorsal-caudal: 15 +/- 7 degrees), and the presence and count of dorsal rootlet bundles.ConclusionsDetailed measurements and highlighted differences between human and porcine spinal cords can inform variations in modeling and electrophysiological experiments between the two species. In contrast to other approaches for measuring the spinal cord and rootlet morphology, our method keeps the dura intact, reducing potential artifacts from dissection.

BACKGROUND:Spinal cord stimulation (SCS) has demonstrated multiple benefits in treating chronic pain and other clinical disorders related to sensorimotor dysfunctions. However, the underlying mechanisms are still not fully understood, including how electrode placement in relation to the spinal cord neuroanatomy influences epidural spinal recordings (ESRs). To characterize this relationship, this study utilized stimulation applied at various anatomical sections of the spinal column, including at levels of the intervertebral disc and regions correlating to the dorsal root entry zone. METHOD:Two electrode arrays were surgically implanted into the dorsal epidural space of the swine. The stimulation leads were positioned such that the caudal-most electrode contact was at the level of a thoracic intervertebral segment. Intraoperative cone beam computed tomography (CBCT) images were utilized to precisely determine the location of the epidural leads relative to the spinal column. High-resolution microCT imaging and 3D-model reconstructions of the explanted spinal cord illustrated precise positioning and dimensions of the epidural leads in relation to the surrounding neuroanatomy, including the spinal rootlets of the dorsal and ventral columns of the spinal cord. In a separate swine cohort, implanted epidural leads were used for SCS and recording evoked ESRs. RESULTS:Reconstructed 3D-models of the swine spinal cord with epidural lead implants demonstrated considerable distinctions in the dimensions of a single electrode contact on a standard industry epidural stimulation lead compared to dorsal rootlets at the dorsal root entry zone (DREZ). At the intervertebral segment, it was observed that a single electrode contact may cover 20-25% of the DREZ if positioned laterally. Electrode contacts were estimated to be ~0.75 mm from the margins of the DREZ when placed at the midline. Furthermore, ventral rootlets were observed to travel in proximity and parallel to dorsal rootlets at this level prior to separation into their respective sides of the spinal cord. Cathodic stimulation at the level of the intervertebral disc, compared to an 'off-disc' stimulation (7 mm rostral), demonstrated considerable variations in the features of recorded ESRs, such as amplitude and shape, and evoked unintended motor activation at lower stimulation thresholds. This substantial change may be due to the influence of nearby ventral roots. To further illustrate the influence of rootlet activation vs. dorsal column activation, the stimulation lead was displaced laterally at ~2.88 mm from the midline, resulting in variances in both evoked compound action potential (ECAP) components and electromyography (EMG) components in ESRs at lower stimulation thresholds. CONCLUSION:The results of this study suggest that the ECAP and EMG components of recorded ESRs can vary depending on small differences in the location of the stimulating electrodes within the spinal anatomy, such as at the level of the intervertebral segment. Furthermore, the effects of sub-centimeter lateral displacement of the stimulation lead from the midline, leading to significant changes in electrophysiological metrics. The results of this pilot study reveal the importance of the small displacement of the electrodes that can cause significant changes to evoked responses SCS. These results may provide further valuable insights into the underlying mechanisms and assist in optimizing future SCS-related applications.

Supplementary Materials and Methods from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Recording evoked compound action potentials (ECAPs) during spinal cord stimulation (SCS) can be used to inform dosing for treatment of chronic pain. Our previous results identified a short latency signal that distorted the appearance of evoked a-beta response at low levels of stimulation, suggesting the activation of a local muscle group creating electromyography (EMG) bleed-through into the spinal cord recordings. In this study, we recorded EMG from the paraspinal muscles of the back and intercostal muscle, along with intercostal nerve recordings during SCS, to confirm this hypothesis.

Spinal cord epidural stimulation (SCS) is a safe and effective therapy of chronic pain. The most common complication for SCS is lead migration, resulting in inefficient stimulation. In addition, the shunting of current or activation of the dorsal rootlets may result in contraction of back muscles, via activation of motor fibers recruited at lower stimulation amplitudes. Activation of these rootlets could cause electromyography (EMG) bleed-through into the evoked compound action potential (ECAPs) recordings, as well as cause side effect during SCS therapy. In some cases, motor activation may limit the range of stimulation to levels below what is required for pain therapy.

Objective. Peripheral neural signals recorded during neuromodulation therapies provide insights into local neural target engagement and serve as a sensitive biomarker of physiological effect. Although these applications make peripheral recordings important for furthering neuromodulation therapies, the invasive nature of conventional nerve cuffs and longitudinal intrafascicular electrodes (LIFEs) limit their clinical utility. Furthermore, cuff electrodes typically record clear asynchronous neural activity in small animal models but not in large animal models. Microneurography, a minimally invasive technique, is already used routinely in humans to record asynchronous neural activity in the periphery. However, the relative performance of microneurography microelectrodes compared to cuff and LIFE electrodes in measuring neural signals relevant to neuromodulation therapies is not well understood. Approach. To address this gap, we recorded cervical vagus nerve electrically evoked compound action potentials (ECAPs) and spontaneous activity in a human-scaled large animal model—the pig. Additionally, we recorded sensory evoked activity and both invasively and non-invasively evoked CAPs from the great auricular nerve. In aggregate, this study assesses the potential of microneurography electrodes to measure neural activity during neuromodulation therapies with statistically powered and pre-registered outcomes (https://osf.io/y9k6j). Main results. The cuff recorded the largest ECAP signal (p < 0.01) and had the lowest noise floor amongst the evaluated electrodes. Despite the lower signal to noise ratio, microneurography electrodes were able to detect the threshold for neural activation with similar sensitivity to cuff and LIFE electrodes once a dose-response curve was constructed. Furthermore, the microneurography electrodes recorded distinct sensory evoked neural activity. Significance. The results show that microneurography electrodes can measure neural signals relevant to neuromodulation therapies. Microneurography could further neuromodulation therapies by providing a real-time biomarker to guide electrode placement and stimulation parameter selection to optimize local neural fiber engagement and study mechanisms of action.

Electrical stimulation of the cervical vagus nerve using implanted electrodes (VNS) is FDA-approved for the treatment of drug-resistant epilepsy, treatment-resistant depression, and most recently, chronic ischemic stroke rehabilitation. However, VNS is critically limited by the unwanted stimulation of nearby neck muscles—a result of non-specific stimulation activating motor nerve fibers within the vagus. Prior studies suggested that precise placement of small epineural electrodes can modify VNS therapeutic effects, such as cardiac responses. However, it remains unclear if placement can alter the balance between intended effect and limiting side effect. We used an FDA investigational device exemption approved six-contact epineural cuff to deliver VNS in pigs and quantified how epineural electrode location impacts on- and off-target VNS activation. Detailed post-mortem histology was conducted to understand how the underlying neuroanatomy impacts observed functional responses. Here we report the discovery and characterization of clear neuroanatomy-dependent differences in threshold and saturation for responses related to both effect (change in heart rate) and side effect (neck muscle contractions). The histological and electrophysiological data were used to develop and validate subject-specific computation models of VNS, creating a well-grounded quantitative framework to optimize electrode location-specific activation of nerve fibers governing intended effect versus unwanted side effect.

Recently, evoked compound action potentials (ECAPs) has gained attention as both a research tool and to deliver closed-loop targeted spinal cord stimulation (SCS). Historically, neural components of ECAPs are very prone to misinterpretation outside of tightly controlled experimental settings, due to confounding variables such as motion artifact, extended capacitive decay of the stimulation artifact, filter ringing, and activated muscles causing electromyographic (EMG) bleed-through. In this study, we characterized how the relative position of the recording and stimulating electrodes with respect to the root and rootlets entering the spinal cord, impacts direct and indirect efferent activation of the back muscles and capacitive decay of the stimulation artifact, distorting the calculation of the ECAP components in spinal recordings.

The results obtained from imaging fine neural structures can potentially inform improvements in analyzing evoked compound action potentials (ECAPs) in SCS recordings. Traditional clinical imaging techniques (CT and MRI) cannot provide the high resolution which is necessary to investigate neural micro-anatomy. Histology and electron microscopy provide high resolution insights to micro-anatomy, but only with a limited field of view. In this study, we imaged stained swine cadaver spinal cord tissues with microCT to investigate the dorsal root organization, to further elucidate the anatomical substrate upon which spinal cord stimulation acts.

Supplementary Figure 4 from Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase Seven in Absentia Homologue

Appropriately designed electrodes to make in vivo recordings of neural activity have been essential to understanding the nervous system. Recording electrode design has been extensively explored for use in measurement of naturally occurring neural activity in the brain. Recently, recordings of evoked compound action potentials (ECAPs) in the periphery have garnered interest due to their relevance in neuromodulation therapies for determining target engagement and enabling closed-loop operation. Here, we compare three types of recording electrodes - microelectrodes, cuff electrodes, and intrafascicular electrodes - and their ability to make in vivo recordings of electrically ECAPs, naturally occurring neural activity, and sensory evoked neural activity in the peripheral nerves of a human-relevant large animal model. The great auricular nerve, innervating the auricle, and cervical vagus nerve of domestic pigs were instrumented with the three recording electrodes. Naturally occurring neural activity, sensory evoked neural activity induced by gentle brushing of the auricle, and electrically ECAPs initiated by non-invasive and invasive stimulation electrodes were recorded. The study was pre-registered (https://osf.io/y9k6j). We showed that the recording cuff had superior performance in measuring invasive electrically ECAPs (p < 0.05) while the tungsten microelectrode, routinely used for microneurography in humans, had superior performance in measuring naturally occurring action potentials (p < 0.05). The microelectrode was still able to record electrically ECAPs. Further, we show how choice of reference electrode position may be optimized per application. Microneurography recordings paired with the deployment of a non-invasive neuromodulation therapy can be used to functionally map sensory innervation areas in patients, allowing individualized placement of stimulation electrodes to achieve on-target neural fiber activation. These findings provide a basis to select recording electrode type and configuration for measurement of neural signals in the development and deployment of neuromodulation therapies.

Neuromodulation approaches like spinal cord stimulation (SCS) are increasingly being applied as alternatives to the traditional pharmacologic treatments of chronic pain1. Tonic electrical stimulation of the spinal cord is most often applied through epidural electrodes, but in addition to putatively exciting the dorsal columns, it can induce paresthesia in many patients sometimes limiting therapeutic efficacy1. As a result, alternative, ‘paresthesia-free’ stimulation paradigms such as burst2 and high frequency stimulation are gaining popularity. Unfortunately, the exact mechanism of action for different SCS paradigms is not well understood. One important distinction between SCS methodologies may be lie in differences in activity evoked within the lateral and medial pain pathways which encode the discriminatory and motivation/affective/attentional aspects of pain, respectively2-6. Here, we present a combination of stimulation and recording methods that enable an interrogation of the effects of different SCS paradigms on the medial and lateral pain pathways based on evoked activity in the cortex.

Megan L Settell, Nicole A Pelot, Bruce E Knudsen, Aaron M Dingle, Andrea L McConico, Evan N Nicolai, James K Trevathan, J Ashley Ezzell, Erika K Ross, Kenneth J Gustafson, Andrew J Shoffstall, Justin CWilliams, Weifeng Zeng, Samuel O Poore, Luis C Populin, Aaron J Suminski, Warren M Grill and Kip A Ludwig1,4,∗ 1 Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, United States of America 2 Mayo Clinic, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN, United States of America 3 Department of Neurologic Surgery, Mayo Clinic, Rochester, MN, United States of America 4 Department of Neurosurgery, University of Wisconsin-Madison, Madison, WI, United States of America 5 Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, United States of America 6 Louis Stokes Cleveland VA Medical Center, Cleveland, OH, United States of America 7 Division of Plastic Surgery, Department of Surgery, University of Wisconsin-Madison, Madison, WI, United States of America 8 Abbott Neuromodulation, Plano, TX, United States of America 9 Department of Biomedical Engineering, Duke University, Durham, NC, United States of America 10 Department of Electrical and Computer Engineering, Duke University, Durham, NC, United States of America 11 Department of Neurobiology, Duke University, Durham, NC, United States of America 12 Department of Neurosurgery, Duke University, Durham, NC, United States of America 13 Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, United States of America 14 Department of Surgery, University of Wisconsin-Madison, Madison, WI, United States of America 15 University of Wisconsin School of Medicine and Public Health, Madison, WI, United States of America 16 Histology Research Core, University of North Carolina School of Medicine, Chapel Hill, NC, United States of America 17 Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC, United States of America ∗ Author to whom any correspondence should be addressed.

Background: Placement of the clinical vagus nerve stimulating cuff is a standard surgical procedure based on anatomical landmarks, with limited patient specificity in terms of fascicular organization or vagal anatomy. As such, the therapeutic effects are generally limited by unwanted side effects of neck muscle contractions, demonstrated by previous studies to result from stimulation of (1) motor fibers near the cuff in the superior laryngeal and (2) motor fibers within the cuff projecting to the recurrent laryngeal.Objective: Conventional non-invasive ultrasound, where the transducer is placed on the surface of the skin, has been previously used to visualize the vagus with respect to other landmarks such as the carotid and internal jugular vein. However, it lacks sufficient resolution to provide details about the vagus fascicular organization, or detail about smaller neural structures such as the recurrent and superior laryngeal branch responsible for therapy limiting side effects. Here, we characterize the use of ultrasound with the transducer placed in the surgical pocket to improve resolution without adding significant additional risk to the surgical procedure in the pig model.Methods: Ultrasound images were obtained from a point of known functional organization at the nodose ganglia to the point of placement of stimulating electrodes within the surgical window. Naïve volunteers with minimal training were then asked to use these ultrasound videos to trace afferent groupings of fascicles from the nodose to their location within the surgical window where a stimulating cuff would normally be placed. Volunteers were asked to select a location for epineural electrode placement away from the fascicles containing efferent motor nerves responsible for therapy limiting side effects. 2-D and 3-D reconstructions of the ultrasound were directly compared to post-mortem histology in the same animals.Results: High-resolution ultrasound from the surgical pocket enabled 2-D and 3-D reconstruction of the cervical vagus and surrounding structures that accurately depicted the functional vagotopy of the pig vagus nerve as confirmed via histology. Although resolution was not sufficient to match specific fascicles between ultrasound and histology 1 to 1, it was sufficient to trace fascicle groupings from a point of known functional organization at the nodose ganglia to their locations within the surgical window at stimulating electrode placement. Naïve volunteers were able place an electrode proximal to the sensory afferent grouping of fascicles and away from the motor nerve efferent grouping of fascicles in each subject (n = 3).Conclusion: The surgical pocket itself provides a unique opportunity to obtain higher resolution ultrasound images of neural targets responsible for intended therapeutic effect and limiting off-target effects. We demonstrate the increase in resolution is sufficient to aid patient-specific electrode placement to optimize outcomes. This simple technique could be easily adopted for multiple neuromodulation targets to better understand how patient specific anatomy impacts functional outcomes.