Vagus nerve stimulation (VNS) is an approved therapy for patients suffering from refractory epilepsy. While VNS is currently an open loop system, making the system closed loop can improve the therapeutic efficacy. Electrical recording of peripheral nerve activity using a nerve cuff electrode is a potential long-term solution for implementing a closed-loop controlled VNS system. However, the clinical utility of this approach is significantly limited by various factors, such as poor signal-to-noise ratio (SNR) of the recorded electroneurogram (ENG). In this study, we investigated the effects of (1) modifying the electrode contact dimensions, (2) implementing an external shielding layer on the nerve cuff electrode and (3) exploring shielded bipolar nerve cuff designs on the recorded ENG. Findings from both computer simulations and animal experiments suggest that significant improvements in peripheral nerve recordings can be achieved.

Vagus nerve stimulation (VNS) is an approved therapy for patients suffering from refractory epilepsy. While VNS is currently an open loop system, making the system closed loop can improve the therapeutic efficacy. Electrical recording of peripheral nerve activity using a nerve cuff electrode is a potential long-term solution for implementing a closed-loop controlled VNS system. However, the clinical utility of this approach is significantly limited by various factors, such as poor signal-to-noise ratio (SNR) of the recorded electroneurogram (ENG). In this study, we investigated the effects of (1) modifying the electrode contact dimensions, (2) implementing an external shielding layer on the nerve cuff electrode and (3) exploring shielded bipolar nerve cuff designs on the recorded ENG. Findings from both computer simulations and animal experiments suggest that significant improvements in peripheral nerve recordings can be achieved.

abstract: Stroke remains a leading cause of adult disability in the United States. In recent studies, chronic vagus nerve stimulation (VNS) has been proven to enhance functional recovery when paired with motor rehabilitation training after stroke. Other studies have also demonstrated that delivering VNS during the onset of a stroke may elicit some neuroprotective effects as observed in remaining neural tissue and motor function. While these studies have demonstrated the benefits of VNS as a treatment or therapy in combatting stroke damage, the mechanisms responsible for these effects are still not well understood or known. The aim of this research was to further investigate the mechanisms underlying the efficacy of acute VNS treatment of stroke by observing the effect of VNS when applied after the onset of stroke. Animals were randomly assigned to three groups: Stroke animals received cortical ischemia (ET-1 injection), VNS+Stroke animals received acute VNS starting within 48 hours after cortical ischemia and continuing once per day for three days, or Control animals which received neither the injury nor stimulation. Results showed that stroke animals receiving acute VNS had smaller lesion volumes and larger motor cortical maps than those in the Stroke group. The results suggest VNS may confer neuroprotective effects when delivered within the first 96 hours of stroke. / Dissertation/Thesis / Masters Thesis Electrical Engineering 2019

Altered gut microbial diversity has been associated with several chronic disease states, including heart failure. Stimulation of the vagus nerve, which innervates the heart and abdominal organs, is proving to be an effective therapeutic in heart failure. We hypothesized that cervical vagus nerve stimulation (VNS) could alter fecal flora and prevent aberrations observed in fecal samples from heart failure animals. To determine whether microbial abundances were altered by pressure overload (PO), leading to heart failure and VNS therapy, a VNS pulse generator was implanted with a stimulus lead on either the left or right vagus nerve before creation of PO by aortic constriction. Animals received intermittent, open-loop stimulation or sham treatment, and their heart function was monitored by echocardiography. Left ventricular end-systolic and diastolic volumes, as well as cardiac output, were impaired in PO animals compared with baseline. VNS mitigated these effects. Metagenetic analysis was then performed using 16S rRNA sequencing to identify bacterial genera present in fecal samples. The abundance of 10 genera was significantly altered by PO, 8 of which were mitigated in animals receiving either left- or right-sided VNS. Metatranscriptomics analyses indicate that the abundance of genera that express genes associated with ATP-binding cassette transport and amino sugar/nitrogen metabolism was significantly changed following PO. These gut flora changes were not observed in PO animals subjected to VNS. These data suggest that VNS prevents aberrant gut flora following PO, which could contribute to its beneficial effects in heart failure patients.

Chronic heart failure (HF) is estimated to affect 23 million people worldwide, and many patients show minimal improvement after treatment with high-potency medications. HF with reduced left ventricular ejection fraction makes up approximately half of cases and is associated with high mortality: a 5-year survival rate of only 25% after hospitalization. This disease is marked by autonomic and cardiac dysfunction, as well as increased inflammatory markers both in the brain and microbiota of the gastrointestinal tract. As a main component of the autonomic nervous system, the vagus nerve has been identified as a potential treatment target for HF. Vagus nerve stimulation (VNS) is thought to help re-balance the autonomic system and has shown promising results in clinical trials for treatment of HF. Although the mechanism of action for VNS remains partially understood, anti-inflammatory pathways have been shown to play a significant role, and these pathways may be enhanced by microbiota signaling via the vagus nerve. The goal of the current study is to provide insight into VNS treatment for HF with reduced ejection fraction via a pressure overload (PO) model. Male Sprague-Dawley rats were randomly divided into age-matched control (n=7), PO (n=6), and PO+VNS (n=11). PO rats underwent aortic constriction (~40%) to induce HF, and a subset of these had VNS leads implanted around the left cervical vagus nerve. Treatment was initiated for PO+VNS rats after reaching a 20% drop in left ventricular relative ejection fraction (EF, p<0.001). VNS was delivered using 1.0 mA pulses at 20 Hz, with 14 sec on-time followed by 66 sec off-time for 2 months to model settings used in successful clinical studies. Echocardiography to image the heart and fecal samples to assess microbiota were collected at regular intervals for all rats. Hearts were weighed at termination for a final heart to body weight ratio, and brains were processed to assess neuroinflammation. Findings indicate that while PO reduced EF ~40% at termination (p<0.05), VNS treatment restored EF back to control levels (p<0.0001 compared to study midpoint). Further, the heart/body weight ratio was increased for PO rats (p<0.05) compared to controls and PO+VNS rats. These data demonstrate that physiological markers of heart failure can be mitigated using these VNS settings. Notably, 66% of microbiota populations altered by PO were prevented with VNS treatment. Further, prolonged VNS significantly affected microbiota populations involved in inflammatory processes. Neuroinflammation was assessed in two key autonomic nuclei: paraventricular nucleus of the hypothalamus and locus coeruleus. PO displayed increased neuroinflammation as measured by microglial density in both regions, and VNS attenuated this effect (p<0.001). These findings indicate relevant contributions of inflammatory mechanisms and microbiome alterations for beneficial VNS effects leading to improved cardiac function in HF.

Non-invasive transcutaneous auricular vagus nerve stimulation (taVNS) is a neuromodulatory technique used to activate vagal afferent fibers located in the concha of the outer ear. Vagal afferents project to the nucleus of the solitary tract (NTS) where information is processed and propagated to higher brain regions. Widespread NTS connections provide a mechanism through which taVNS can be used to influence multiple systems and be a potential treatment for many disorders including heart failure, gastric motility disorders, and migraines. Recent studies are now investigating taVNS as an alternative treatment option to invasive cervical vagus nerve stimulation (cVNS) which is FDA approved to treat drug-resistant epilepsy and depression but has limited patient availability due to the invasiveness of the procedure. Migraine and epilepsy clinical studies have shown therapeutic taVNS benefits and human fMRI studies have demonstrated comparable brain activation between cVNS and taVNS. However, questions remain regarding optimal taVNS parameters, and no study has compared the direct mechanisms responsible for cVNS and taVNS effects. In this study, a high-impedance tungsten electrode was stereotaxically placed into NTS in 10 chloralose-anesthetized rats, and 40-70 neurons were interrogated using electrophysiological methods. Firing rate changes during stimulation on-times were compared to activity levels during stimulation off-times. Neurons were classified as positive responders if they displayed consistent firing rate increases during stimulation, negative responders if they displayed consistent decreases, and non-responders if there was no consistency using a mathematical cosine similarity score. Six taVNS stimulation parameters were investigated using three frequencies (20, 100, 250Hz) at two intensities (0.5, 1.0mA) to identify parameter-specific effects on NTS neurons. Additionally, neuronal activity was evaluated following cVNS at 20 and 250Hz at the bradycardic intensity (lowest intensity to generate a transient 5% decrease in heart rate, BI) and compared to taVNS effects at the corresponding frequencies. Our data shows that taVNS at 20Hz, 1.0mA yields the greatest number of positive responders and 100Hz, 1.0mA yields the greatest number of negative responders (p<0.05) suggesting different taVNS parameters can differentially influence NTS activity. Comparisons between the number of responders generated with cVNS and taVNS revealed significantly fewer negative responders with cVNS at 20Hz compared to taVNS at 20Hz regardless of intensity (p<0.01) but yielded comparable positive responders between cVNS at 20Hz, BI and taVNS at 20Hz, 1.0mA. No significant differences were observed between the number of cVNS and taVNS responders at 250Hz. Interestingly, individual neuronal responses were different between both methods of stimulation, suggesting that they could work through different neuronal pathways.

Vagus nerve stimulation (VNS) is a promising neuromodulatory therapy under investigation for a range of disorders, including heart failure, gastric dysmotility, and migraine. Two primary forms of VNS are currently investigated: cervical VNS (cVNS), involving surgically implantation to activate vagal afferents in the cervical branch in the neck and transcutaneous auricular VNS (taVNS) which subcutaneously stimulates the auricular branch in the outer ear. The nucleus of the solitary tract (NTS) serves as a relay-station receiving 90% of vagal afferents, enabling connections with higher-order brain regions and other brainstem nuclei like the spinal trigeminal nucleus (Sp5) and locus coeruleus (LC), facilitating neuromodulation through VNS. Research has established the efficacy of VNS at 20–30 Hz for disorders like depression, but the impact of alternative stimulation parameters on medullary nuclei neuromodulation remains unclear. These studies used anesthetized rats to extracellularly record neuronal activity across varying VNS parameters within NTS, Sp5, and LC. Neuronal responses were classified as positive (increased activity), negative (decreased activity), or non-responders (no response). In LC, cVNS at standard paradigms (≥ 10 Hz) and bursting paradigms with shorter interburst intervals or increased pulses induced more positive responders, while standard 5 Hz generated more negative responders. Additionally, a build-up effect was observed in LC, with increased responders over consecutive VNS cycles. In NTS and Sp5, taVNS evoked comparable activation, with more positive responders at 20 Hz and 100 Hz and stronger responses at higher intensities. However, Sp5 responses were twice as strong compared to NTS. Furthermore, comparative analysis between taVNS and cVNS revealed similar overall activation in NTS, but distinct activation profiles in individual neurons indicate different pathways. Finally, the therapeutic efficacy of VNS therapy was evaluated in heart failure using a pressure-overload rat model. A 60-day cVNS treatment restored adverse cardiac remodeling and dysfunction, mitigated cardiac molecular changes, and prevented neuroinflammatory responses within brainstem nuclei. The findings presented herein demonstrated differential parameter-specific and nuclei-specific responses to taVNS and cVNS, investigated the mechanisms responsible for taVNS modulation, and confirmed that VNS therapy, when initiated early, can mitigate heart failure development and restore multiorgan homeostasis in a PO model.

<p>Current challenges in neuronal tract tracing include sacrificing the animal, detailed sectioning of the brain, and cumbersome reconstruction of slices to gather information, which are very tedious, time consuming, and have low-throughput. In this regard, Manganese-enhanced Magnetic Resonance Imaging (MEMRI) has been an emerging methodology for fiber tract tracing <i>in vivo</i>. <i></i>The manganese ion (Mn²⁺) is paramagnetic and is analogous to calcium ions (Ca²⁺), which allows it to enter excitable cells through voltage-gated calcium channels, thereby reporting cellular activity in T₁-weighted MR images<i>. </i>Moreover, once the Mn²⁺enters the cell, it will move along the axon by microtubules, release at the synapse, and then uptake by post-synaptic neurons, hence revealing the pathway of Mn²⁺transportation. While most MEMRI neuronal tracing studies have focused on mapping circuitries within the brain, MEMRI has rarely been applied to trace peripheral nerve projections into the brain. </p><p>In this thesis, I will propose the use of MEMRI to trace vagal nerve projections into the central nervous system by showing enhancement of neuronal pathways with an optimized protocol. This protocol demonstrates <i>in vivo </i>monitoring of manganese transport into the brain from the nodose ganglion and shows how the enhancement in MR images can be promoted with vagus nerve stimulation (VNS). Additionally, I will present preliminary findings, for the very first time, that show the downstream projection of the sympathetic pathway from the brainstem. In sum, the technique presented in this thesis will shed light on the use of MEMRI to study the functional results of using clinically-based VNS settings</p>

Studies have indicated that approximately 30% of people with heart disease experience major depressive disorder (MDD). Despite strong clinical evidence of a link between the two diseases, the neurobiological processes involved in the relationship are poorly understood. A growing number of studies are revealing similar neuroanatomical and neurochemical abnormalities resulting from both depression and heart disease. The locus coeruleus (LC) is a group of neurons in the pons that synthesize and release norepinephrine, and that is known to play a significant role in depression pathobiology. For example, there is evidence that tyrosine hydroxylase (TH) is elevated in the LC in depression. In addition, there is evidence that the LC plays a role in cardiovascular autonomic regulation. The hippocampus is another region that exhibits abnormalities in both depression and heart disease. In this study, the levels of TH in the hippocampus and LC were examined in the guinea pig pressure-overload model of heart disease. TH levels were also measured in the pressure-overload model treated with vagal nerve stimulation, a new investigational therapeutic intervention in heart disease. This study found that there were no changes in TH levels in the LC or the hippocampus of the pressure-overload model or in the pressure-overload model treated with vagal nerve stimulation.

Infectious diseases are the worldwide leading cause of morbidity and mortality. Sepsis is the major cause of death in infectious diseases. It is one of the most serious and also one of the most difficult treatable conditions of contemporary medicine. Sepsis is the main cause of death in intensive care units. Causal therapy of sepsis does not yet exist. With a far better understanding of patho/physiological mechanism of sepsis, it is possible to model new preclinical experiments to verify the efficiency and security of new therapeutic procedures. Large animal experiments in progressive sepsis, with the use of domestic porcine, play a vital role. Long-standing experience with this model and similarity to human facilitate the realization of more complex experiments with potential for the relevant translation of results into the subsequent clinical studies on human subjects. The objective of this doctoral dissertation was to assess on the clinically relevant model: 1) the efficiency and security of extracorporeal membrane oxygenation in the event of vasoplegic septic shock; and 2) the benefit of two innovative therapeutic approaches to treatment of sepsis: a) the intravenous administration of mesenchymal stem cells; and b) the activation of the neuro-inflammatory reflex through the vagus nerve stimulation.