Human-relevant near-organ neuromodulation of the immune system via the splenic nerve.

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve-induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

Immunohistochemical characteristics of local sites that trigger atrial arrhythmias in response to high-frequency stimulation.

AIMS: The response to high frequency stimulation (HFS) is used to locate putative sites of ganglionated plexuses (GPs), which are implicated in triggering atrial fibrillation (AF). To identify topological and immunohistochemical characteristics of presumed GP sites functionally identified by HFS. METHODS AND RESULTS: Sixty-three atrial sites were tested with HFS in four Langendorff-perfused porcine hearts. A 3.5 mm tip quadripolar ablation catheter was used to stimulate and deliver HFS to the left and right atrial epicardium, within the local atrial refractory period. Tissue samples from sites triggering atrial ectopy/AF (ET) sites and non-ET sites were stained with choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH), for quantification of parasympathetic and sympathetic nerves, respectively. The average cross-sectional area (CSA) of nerves was also calculated. Histomorphometry of six ET sites (9.5%) identified by HFS evoking at least a single atrial ectopic was compared with non-ET sites. All ET sites contained ChAT-immunoreactive (ChAT-IR) and/or TH-immunoreactive nerves (TH-IR). Nerve density was greater in ET sites compared to non-ET sites (nerves/cm2: 162.3 ± 110.9 vs. 69.65 ± 72.48; P = 0.047). Overall, TH-IR nerves had a larger CSA than ChAT-IR nerves (µm2: 11 196 ± 35 141 vs. 2070 ± 5841; P < 0.0001), but in ET sites, TH-IR nerves were smaller than in non-ET sites (µm2: 6021 ± 14 586 vs. 25 254 ± 61 499; P < 0.001). CONCLUSIONS: ET sites identified by HFS contained a higher density of smaller nerves than non-ET sites. The majority of these nerves were within the atrial myocardium. This has important clinical implications for devising an effective therapeutic strategy for targeting autonomic triggers of AF.

A method to identify, dissect and stain equine neuromuscular junctions for morphological analysis.

Morphological study of the neuromuscular junction (NMJ), a specialised peripheral synapse formed between a lower motor neuron and skeletal muscle fibre, has significantly contributed to the understanding of synaptic biology and neuromuscular disease pathogenesis. Rodent NMJs are readily accessible, and research into conditions such as amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy (SMA) has relied heavily on experimental work in these small mammals. However, given that nerve length dependency is an important feature of many peripheral neuropathies, these rodent models have clear shortcomings; large animal models might be preferable, but their size presents novel anatomical challenges. Overcoming these constraints to study the NMJ morphology of large mammalian distal limb muscles is of prime importance to increase cross-species translational neuromuscular research potential, particularly in the study of long motor units. In the past, NMJ phenotype analysis of large muscle bodies within the equine distal pelvic limb, such as the tibialis cranialis, or within muscles of high fibrous content, such as the soleus, has posed a distinct experimental hurdle. We optimised a technique for NMJ location and dissection from equine pelvic limb muscles. Using a quantification method validated in smaller species, we demonstrate their morphology and show that equine NMJs can be reliably dissected, stained and analysed. We reveal that the NMJs within the equine soleus have distinctly different morphologies when compared to the extensor digitorum longus and tibialis cranialis muscles. Overall, we demonstrate that equine distal pelvic limb muscles can be regionally dissected, with samples whole-mounted and their innervation patterns visualised. These methods will allow the localisation and analysis of neuromuscular junctions within the muscle bodies of large mammals to identify neuroanatomical and neuropathological features.

Cultured dissociated primary dorsal root ganglion neurons from adult horses enable study of axonal transport.

Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movement was detected in cultured cells and was significantly slower in glial cells compared to DRG-derived neurons. In addition, mitochondria were more elongated in glial cells than those in neurons. Our culture model will be a useful tool to study the contribution of axonal transport defects to specific neurodegenerative diseases in horses as well as comparative studies aimed at evaluating species-specific differences in axonal transport and survival.

Ex vivo biomechanical evaluation of polyester and polyblend suture techniques to perform equine laryngoplasty.

OBJECTIVE: To validate the use of a polyblend tape suture in equine laryngoplasty (PL). STUDY DESIGN: Experimental study. ANIMALS: Thirty-two cadaveric larynges. METHODS: Each larynx was randomly assigned to 1 of 4 groups: PL with polyblend tape suture (TigerTape), without (TT) or with a cannula (TTC) in the muscular process of the arytenoid cartilage, and PL with polyester suture (Ethibond), without (EB) or with a cannula (EBC). Construct stiffness, total migration, creep, and drift values were measured after 3000 cycles. The specimens were then loaded to failure to assess their residual properties: load at failure, total energy, displacement, and 2 stiffness coefficients. RESULTS: After cyclic testing, the total migration and creep were lower in TTC (6.36 ± 1.20 mm; 1.35 ± 0.38 mm/s) than in EB (11.12 ± 4.20 mm; 3.39 ± 2.68 mm/s) and in the TT constructs (11.26 ± 1.49 mm; 3.20 ± 0.54 mm/s); however, no difference was found with EBC (9.19 ± 3.18 mm; 2.14 ± 0.99). A correlation was found between total migration and creep (R = .85). The TTC constructs failed at higher loads (129.51 ± 33.84 N) than EB (93.16 ± 18.21 N) and EBC (81.72 ± 13.26 N) whereas the EB and EBC constructs were less stiff than TT and TTC (P < .001). CONCLUSION: Biomechanical properties were generally superior for the TTC constructs tested under cyclical loading. The TT and TTC constructs failed at a higher load than EB and EBC constructs. The cannula in TTC and EBC reduced the failure at the muscular process. CLINICAL SIGNIFICANCE: These results provide evidence to support the in vivo evaluation of the polyblend tape suture with or without a cannula in the muscular process for laryngoplasty in horses.

Imaging of focal seizures with Electrical Impedance Tomography and depth electrodes in real time.

Intracranial EEG is the current gold standard technique for localizing seizures for surgery, but it can be insensitive to tangential dipole or distant sources. Electrical Impedance Tomography (EIT) offers a novel method to improve coverage and seizure onset localization. The feasibility of EIT has been previously assessed in a computer simulation, which revealed an improved accuracy of seizure detection with EIT compared to intracranial EEG. In this study, slow impedance changes, evoked by cell swelling occurring over seconds, were reconstructed in real time by frequency division multiplexing EIT using depth and subdural electrodes in a swine model of epilepsy. EIT allowed to generate repetitive images of ictal events at similar time course to fMRI but without its significant limitations. EIT was recorded with a system consisting of 32 parallel current sources and 64 voltage recorders. Seizures triggered with intracranial injection of benzylpenicillin (BPN) in five pigs caused a repetitive peak impedance increase of 3.4 ± 1.5 mV and 9.5 ± 3% (N =205 seizures); the impedance signal change was seen already after a single, first seizure. EIT enabled reconstruction of the seizure onset 9 ± 1.5 mm from the BPN cannula and 7.5 ± 1.1 mm from the closest SEEG contact (p<0.05, n =37 focal seizures in three pigs) and it could address problems with sampling error in intracranial EEG. The amplitude of the impedance change correlated with the spread of the seizure on the SEEG (p <<0.001, n =37). The results presented here suggest that combining a parallel EIT system with intracranial EEG monitoring has a potential to improve the diagnostic yield in epileptic patients and become a vital tool in improving our understanding of epilepsy.

Thyrohyoideus muscle innervation in the horse.

OBJECTIVE: To describe the innervation of the thyrohyoideus (TH) muscle and to confirm our findings with stimulation of first cervical (C1) nerve branches. STUDY DESIGN: Ex vivo phase 1 and clinical phase 2. ANIMALS: Fourteen head and neck specimens and 17 client-owned horses. METHODS: In phase 1, the cranial nerve (CN) XII and the C1 nerve were dissected with their branches in 20 dissections were performed on 14 specimens (6 left and right side and 8 only left or right) Anatomy was noted. Samples of nerve bifurcations were collected for histological confirmation of anatomical findings. First cervical nerve branches were stimulated in horses undergoing cervical nerve graft to treat laryngeal hemiplegia. RESULTS: The nerve innervating the TH muscle arose directly from the C1 nerve in 17 of 20 dissections, from an anastomotic branch between CN XII and the C1 nerve in two of 20 dissections, and from the C1 nerve and the anastomotic branch in one of 20 dissections. No direct connection between the TH muscle and CN XII was found. Histological examination revealed that the anastomosis was composed of C1 nerve fibers passing over to CN XII. First cervical stimulation resulted in TH muscle contraction in 16 of 17 horses. CONCLUSIONS: The innervation of the TH muscle originated from the C1 nerve according to dissection, histological, and conduction studies, with variation in the branching pattern. CLINICAL SIGNIFICANCE: Care should be taken to preserve the C1 nerve during prosthetic laryngoplasty. The surgical technique for C1 nerve grafts should be reconsidered in light of these findings, along with new options to treat dorsal displacement of the soft palate..

Development of a pro-arrhythmic ex vivo intact human and porcine model: cardiac electrophysiological changes associated with cellular uncoupling.

We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far-field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting. Porcine (n = 9) and human (n = 4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. A total of 1 mM of carbenoxolone was administered at 5 ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed. We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9 ± 4.1-67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and carbenoxolone were fractionation + 185.3%, endpoint amplitude - 106.9%, S-endpoint gradient + 54.9%, S point - 39.4%, RS ratio + 38.6% and (-dV/dt)max - 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.

Assessing pathological changes within the nucleus ambiguus of horses with recurrent laryngeal neuropathy: An extreme, length-dependent axonopathy.

INTRODUCTION: Equine recurrent laryngeal neuropathy (RLN) is a naturally occurring model of length-dependent axonopathy characterized by asymmetrical degeneration of recurrent laryngeal nerve axons (RLn). Distal RLn degeneration is marked, but it is unclear whether degeneration extends to include cell bodies (consistent with a neuronopathy). METHODS: With examiners blinded to RLN severity, brainstem location, and side, we examined correlations between RLN severity (assessed using left distal RLn myelinated axon count) and histopathological features (including chromatolysis and glial responses) in the nucleus ambiguus cell bodies, and myelinated axon count of the right distal RLn of 16 horses. RESULTS: RLN severity was not associated with RLn cell body number (P > .05), or degeneration. A positive correlation between the left and right distal RLn myelinated axon counts was identified (R2 = 0.57, P < .05). DISCUSSION: We confirm that RLN, a length-dependent distal axonopathy, occurs in the absence of detectable neuronopathy.

Functional electrical stimulation following nerve injury in a large animal model.

INTRODUCTION: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis. METHODS: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined. RESULTS: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p = 0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation. DISCUSSION: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. Muscle Nerve 59:717-725, 2019.

Anatomy of the vestibulum esophagi and surgical implications during prosthetic laryngoplasty in horses.

OBJECTIVE: To describe the anatomy of the entry to the equine esophagus (vestibulum esophagi) and to assess the risk of penetrating its adventitia and/or lumen during laryngoplasty. STUDY DESIGN: Ex vivo cadaveric study. SAMPLE POPULATION: Five isolated equine larynges and 39 equine head and neck specimens. METHODS: The anatomy of the vestibulum esophagi was studied by dissection of 5 cadaver specimens. Then, a bilateral laryngoplasty was performed, including 5 suture placements through the muscular processes, caudal, rostral, and sagittal, with straight and curved needles. Two of the 3 surgeons performing the implantations were unaware of the goals of the study. Suture positions and iatrogenic trauma to the lumen and/or adventitia of the vestibulum esophagi were identified during dissection of the specimens. Risk factors for penetrating the adventitia were evaluated with a multivariate regression model. RESULTS: The vestibulum esophagi spans between both wings of the thyroid cartilage over the entire width of the larynx, covering the rostral spine (arcuate crest) of the arytenoid cartilages. It is covered by the thyropharyngeus and cricopharyngeus muscles. Masked surgeons were associated with a significantly higher number of adventitia penetrations (72%) compared to the nonmasked surgeon (9%). The lumen of the vestibulum esophagi was penetrated in 4.6% of suture placements and only by the 2 masked surgeons. CONCLUSION: Penetration of the adventitia was more common when surgeons were unaware of the anatomical extent of the vestibulum esophagi. CLINICAL SIGNIFICANCE: Anatomical knowledge of the extent of the vestibulum esophagi reduces the risk of penetrating its lumen or adventitia during suture placement on the muscular process of the arytenoid cartilage.

Ex vivo biomechanical stability of 5 cricoid-suture constructs for equine laryngoplasty.

OBJECTIVE: To determine the biomechanical properties of 5 suture constructs in the equine cricoid under cyclic loading and load to failure testing. STUDY DESIGN: Ex vivo study. SAMPLES: Seventy-five equine cadaver larynges. METHODS: Each larynx was implanted with 1 of 5 cricoid-suture constructs. The standard laryngoplasty, where a suture is passed once through the cricoid, including its caudal edge, was used in 2 constructs: 1 with 5 USP Ethibond (ES) and 1 with 2 mm Fibertape (FS). In the third construct, the 2 mm Fibertape was passed twice through the cricoid including its caudal edge (Double Loop-DL). Constructs 4 and 5 used 2 mm Fibertape in a U-shaped loop passed through the cricoid but excluding its caudal edge. One construct was supported with a metallic button (MB) on the caudo-ventral aspect of the cricoid while the other included only the U-shaped loop (U). Constructs were subjected to cyclic loading and to single cycle to failure. Reduction of the left-to-right arytenoid angle quotient (LRQ), suture migration, and load at failure were compared. RESULTS: LRQ reduction after cyclic loading was lower in MB and U than ES constructs. During cyclic loading, suture migration was reduced in MB, U, and DL compared to ES constructs. Mean load at failure was lower in FS and U than in ES constructs. CONCLUSION: Loss of abduction after equine laryngoplasty may be reduced and pullout forces increased by applying a MB construct in the cricoid cartilage. In vivo testing is required to verify these results.

Ultrasonography detects early laryngeal muscle atrophy in an equine neurectomy model.

INTRODUCTION: A unilateral neurectomy model was used to study the relationship between histologic and ultrasonographic tissue characteristics during muscle atrophy over time. METHODS: This investigation was an in vivo experimental study in an equine model (n = 28). Mean pixel intensity of ultrasonographic images was measured, a muscle appearance grade was assigned weekly, and muscles were harvested from 4 to 32 weeks. Minimum fiber diameter, fiber density per unit area, percent collagen, percent fat, and fiber type profile were measured from muscle cryosections and correlated with the ultrasonographic parameters. RESULTS: A significant relationship was identified between collagen content, minimum fiber diameter, and ultrasonographic muscle appearance by as early as 8 weeks. There was no apparent association between fat content of muscle and the ultrasonographic appearance of atrophy before 28 weeks. CONCLUSIONS: Early muscle atrophy before fatty infiltration is detectable with ultrasound. The effect of muscle collagen content on echointensity may be mediated by reduced fiber diameter.

COMPUTED TOMOGRAPHIC APPEARANCE OF MELANOMAS IN THE EQUINE HEAD: 13 CASES.

Melanomas are one of the most common neoplasms in the horse and are frequently found in the head region. There is a genetic predisposition in horses with a gray hair coat. Computed tomography (CT) is frequently used in referral practice to evaluate the equine head but there are few reports describing the CT appearance of melanomas in this location. The aim of this retrospective, case series study was to describe characteristics in a group of horses with confirmed disease. Case records from two referral hospitals were reviewed, and 13 horses were identified that had undergone CT of the head, with a diagnosis of melanoma based on cytology, histopathology, or visual assessment of black (melanotic) tissue. A median of 11 melanomas was identified per horse (range 3-60), with a total of 216 masses. Melanomas were found most frequently in the parotid salivary gland, guttural pouches, surrounding the larynx and pharynx and adjacent to the hyoid apparatus. In noncontrast CT images, all melanomas were hyperattenuating (median; 113.5 Hounsfield units (HU), IQR; 26 HU) compared to masseter musculature (median; 69 HU, IQR; 5.5 HU). Fifty-six (25.9%) masses were partially mineralized and 41 (19.4%) included hypoattenuating areas. Histopathological assessment of these melanomas suggested that the hyperattenuation identified was most likely a result of abundant intracytoplasmic melanin pigment. Melanomas of the equine head appeared to have consistent CT features that aided detection of mass lesions and their distribution, although histopathological analysis or visual confirmation should still be obtained for definitive diagnosis.

Effects of Functional Electrical Stimulation on Denervated Laryngeal Muscle in a Large Animal Model.

Bilateral vocal fold paralysis (BVCP) is a life-threatening condition that follows injury to the Recurrent Laryngeal nerve (RLn) and denervation of the intrinsic laryngeal musculature. Functional electrical stimulation (FES) enables restoration and control of a wide variety of motor functions impaired by lower motor neuron lesions. Here we evaluate the effects of FES on the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle in a large animal model of RLn injury. Ten horses were instrumented with two quadripolar intramuscular electrodes in the left PCA muscle. Following a 12-week denervation period, the PCA was stimulated using a once-daily training session for 8 weeks in seven animals. Three animals were used as unstimulated controls. Denervation produced a significant increase in rheobase (P < 0.001). Electrical stimulation produced a 30% increase in fiber diameter in comparison with the unstimulated control group (33.9 ± 2.6 µm FES+, 23.6 ± 4.2 µm FES-, P = 0.04). A trend toward a decrease in the proportion of type 1 (slow) fibers and an increase in type 2a (fast) fibers was also observed. Despite these changes, improvement in PCA function at rest was not observed. These data suggest that electrical stimulation using a relatively conservative set of stimulation parameters can reverse the muscle fiber atrophy produced by complete denervation while avoiding a shift to a slow (type 1) fiber type.

Bilateral Functional Electrical Stimulation for the Treatment of Presbyphonia in a Sheep Model.

OBJECTIVES: The aim of the study was to increase muscle volume and improve phonation characteristics of the aged ovine larynx by functional electrical stimulation (FES) using a minimally invasive surgical procedure. METHODS: Stimulation electrodes were placed bilaterally near the terminal adduction branch of the recurrent laryngeal nerves (RLN). The electrodes were connected to battery powered pulse generators implanted subcutaneously at the neck region. Training patterns were programmed by an external programmer using a bidirectional radio frequency link. Training sessions were repeated automatically by the implant every other day for 1 week followed by every day for 8 weeks in the awake animal. Another group of animals were used as sham, with electrodes positioned but not connected to an implant. Outcome parameters included gene expression analysis, histological assessment of muscle fiber size, functional analysis, and volumetric measurements based on three-dimensional reconstructions of the entire thyroarytenoid muscle (TAM). RESULTS: Increase in minimal muscle fiber diameter and an improvement in vocal efficiency were observed following FES, compared with sham animals. CONCLUSION: This is the first study to demonstrate beneficial effects in the TAM of FES at molecular, histological, and functional levels. FES of the terminal branches of the RLN reversed the effects of age-related changes and improved vocal efficiency. LEVEL OF EVIDENCE: NA Laryngoscope, 134:848-854, 2024.

Long-term stimulation by implanted pacemaker enables non-atrophic treatment of bilateral vocal fold paresis in a human-like animal model.

A wide variety of treatments have been developed to improve respiratory function and quality of life in patients with bilateral vocal fold paresis (BVFP). One experimental method is the electrical activation of the posterior cricoarytenoid (PCA) muscle with a laryngeal pacemaker (LP) to open the vocal folds. We used an ovine (sheep) model of unilateral VFP to study the long-term effects of functional electrical stimulation on the PCA muscles. The left recurrent laryngeal nerve was cryo-damaged in all animals and an LP was implanted except for the controls. After a reinnervation phase of six months, animals were pooled into groups that received either no treatment, implantation of an LP only, or implantation of an LP and six months of stimulation with different duty cycles. Automated image analysis of fluorescently stained PCA cross-sections was performed to assess relevant muscle characteristics. We observed a fast-to-slow fibre type shift in response to nerve damage and stimulation, but no complete conversion to a slow-twitch-muscle. Fibre size, proportion of hybrid fibres, and intramuscular collagen content were not substantially altered by the stimulation. These results demonstrate that 30 Hz burst stimulation with duty cycles of 40% and 70% did not induce PCA atrophy or fibrosis. Thus, long-term stimulation with an LP is a promising approach for treating BVFP in humans without compromising muscle conditions.

Organotopic organization of the porcine mid-cervical vagus nerve.

Introduction: Despite detailed characterization of fascicular organization of somatic nerves, the functional anatomy of fascicles evident in human and large mammal cervical vagus nerve is unknown. The vagus nerve is a prime target for intervention in the field of electroceuticals due to its extensive distribution to the heart, larynx, lungs, and abdominal viscera. However, current practice of the approved vagus nerve stimulation (VNS) technique is to stimulate the entire nerve. This produces indiscriminate stimulation of non-targeted effectors and undesired side effects. Selective neuromodulation is now a possibility with a spatially-selective vagal nerve cuff. However, this requires the knowledge of the fascicular organization at the level of cuff placement to inform selectivity of only the desired target organ or function. Methods and results: We imaged function over milliseconds with fast neural electrical impedance tomography and selective stimulation, and found consistent spatially separated regions within the nerve correlating with the three fascicular groups of interest, suggesting organotopy. This was independently verified with structural imaging by tracing anatomical connections from the end organ with microCT and the development of an anatomical map of the vagus nerve. This confirmed organotopic organization. Discussion: Here we show, for the first time, localized fascicles in the porcine cervical vagus nerve which map to cardiac, pulmonary and recurrent laryngeal function (N = 4). These findings pave the way for improved outcomes in VNS as unwanted side effects could be reduced by targeted selective stimulation of identified organ-specific fiber-containing fascicles and the extension of this technique clinically beyond the currently approved disorders to treat heart failure, chronic inflammatory disorders, and more.

The cholinergic anti-inflammatory pathway inhibits inflammation without lymphocyte relay.

The magnitude of innate inflammatory immune responses is dependent on interactions between peripheral neural and immune cells. In particular, a cholinergic anti-inflammatory pathway (CAP) has been identified in the spleen whereby noradrenaline (NA) released by splenic nerves binds to ß2-adrenergic receptors (ß2-AR) on CD4+ T cells which, in turn, release acetylcholine (ACh). The binding of ACh to α7 acetylcholine receptors (α7-AChR) expressed by splenic macrophages inhibits the production of inflammatory cytokines, including tumor necrosis factor (TNF). However, the role of ACh-secreting CD4+ T-cells in the CAP is still controversial and largely based on the absence of this anti-inflammatory pathway in mice lacking T-cells (nude, FoxN1-/-). Using four conscious, non-lymphopenic transgenic mouse models, we found that, rather than acting on CD4+ T-cells, NA released by splenic nerve terminals acts directly onto ß2-AR on splenic myeloid cells to exert this anti-inflammatory effect. We also show that, while larger doses of LPS are needed to trigger CAP in nude mouse strain compared to other strains, TNF production can be inhibited in these animals lacking CD4+ T-cell by stimulating either the vagus or the splenic nerve. We demonstrate that CD4+ T-cells are dispensable for the CAP after antibody-mediated CD4+ T-cell depletion in wild type mice. Furthermore, we found that NA-mediated inhibition of in vitro LPS-induced TNF secretion by human or porcine splenocytes does not require α7-AChR signaling. Altogether our data demonstrate that activation of the CAP by stimulation of vagus or splenic nerves in mice is mainly mediated by direct binding of NA to ß2-AR on splenic macrophages, and suggest that the same mechanism is at play in larger species.

Overcoming temporal dispersion for measurement of activity-related impedance changes in unmyelinated nerves.

Objective.Fast neural electrical impedance tomography is an imaging technique that has been successful in visualising electrically evoked activity of myelinated fibres in peripheral nerves by measurement of the impedance changes (dZ) accompanying excitation. However, imaging of unmyelinated fibres is challenging due to temporal dispersion (TP) which occurs due to variability in conduction velocities of the fibres and leads to a decrease of the signal below the noise with distance from the stimulus. To overcome TP and allow electrical impedance tomography imaging in unmyelinated nerves, a new experimental and signal processing paradigm is required allowing dZ measurement further from the site of stimulation than compound neural activity is visible. The development of such a paradigm was the main objective of this study.Approach.A finite element-based statistical model of TP in porcine subdiaphragmatic nerve was developed and experimentally validatedex-vivo. Two paradigms for nerve stimulation and processing of the resulting data-continuous stimulation and trains of stimuli, were implemented; the optimal paradigm for recording dispersed dZ in unmyelinated nerves was determined.Main results.While continuous stimulation and coherent spikes averaging led to higher signal-to-noise ratios (SNRs) at close distances from the stimulus, stimulation by trains was more consistent across distances and allowed dZ measurement at up to 15 cm from the stimulus (SNR = 1.8 ± 0.8) if averaged for 30 min.Significance.The study develops a method that for the first time allows measurement of dZ in unmyelinated nerves in simulation and experiment, at the distances where compound action potentials are fully dispersed.