An Ultrastructural Model to Test Microburst Stimulation of Nerves

Some patients that suffer from epilepsy may become refractory to pharmaceutical treatment. An option with these patients is vagus nerve stimulation (VNS) therapy with neuro-cybernetic medical devices. The purpose of this research is two-fold: 1.) to determine if a recovery technique can be used with formalin-fixed samples of nerve tissue for transmission electron microscopy (TEM) and 2.) to determine if there is an ultrastructural difference in tissue exposed to the neuro-cybernetic device. If successful, the study will reduce animal studies and expense by establishing a mechanism to perform retrospective TEM studies on formalin-fixed tissue. Additionally, TEM allows examination of specimens in much greater detail than light microscopy. Therefore, using TEM to compare ultrastructural differences in tissue that was exposed to the medical device and healthy tissue will help determine with precision if any damage is caused by the medical device. To complete these objectives, formalin-fixed vagus nerve tissue from goats that were exposed to the medical device is collected, recovered, evaluated by TEM, and compared to healthy traditionally fixed vagus nerve tissue. Results show that the recovery technique makes it possible to achieve quantitative data from formalin-fixed tissue samples. This method establishes a mechanism to execute retrospective TEM studies on formalin-fixed tissue, thereby reducing future animal studies. Results also show that there are some differences in goat nerve that has been exposed to the medical device. These subtle ultrastructural changes (potentially reversible) do not appear to have clinical impact

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

OBJECTIVE: Fast neural Electrical Impedance Tomography (FnEIT) 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 EIT 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 FEM-based statistical model of temporal dispersion in porcine subdiaphragmatic nerve was developed and experimentally validated ex-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 (SNR) 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 minutes. 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

Imaging fascicular organisation in mammalian vagus nerve for selective VNS

Nerves contain a large number of nerve fibres, or axons, organised into bundles known as fascicles. Despite the somatic nervous system being well understood, the organisation of the fascicles within the nerves of the autonomic nervous system remains almost completely unknown. The new field of bioelectronics medicine, Electroceuticals, involves the electrical stimulation of nerves to treat diseases instead of administering drugs or performing complex surgical procedures. Of particular interest is the vagus nerve, a prime target for intervention due to its afferent and efferent innervation to the heart, lungs and majority of the visceral organs. Vagus nerve stimulation (VNS) is a promising therapy for treatment of various conditions resistant to standard therapeutics. However, due to the unknown anatomy, the whole nerve is stimulated which leads to unwanted off-target effects. Electrical Impedance Tomography (EIT) is a non-invasive medical imaging technique in which the impedance of a part of the body is inferred from electrode measurements and used to form a tomographic image of that part. Micro-computed tomography (microCT) is an ex vivo method that has the potential to allow for imaging and tracing of fascicles within experimental models and facilitate the development of a fascicular map. Additionally, it could validate the in vivo technique of EIT. The aim of this thesis was to develop and optimise the microCT imaging method for imaging the fascicles within the nerve and to determine the fascicular organisation of the vagus nerve, ultimately allowing for selective VNS. Understanding and imaging the fascicular anatomy of nerves will not only allow for selective VNS and the improvement of its therapeutic efficacy but could also be integrated into the study on all peripheral nerves for peripheral nerve repair, microsurgery and improving the implementation of nerve guidance conduits. Chapter 1 provides an introduction to vagus nerve anatomy and the principles of microCT, neuronal tracing and EIT. Chapter 2 describes the optimisation of microCT for imaging the fascicular anatomy of peripheral nerves in the experimental rat sciatic and pig vagus nerve models, including the development of pre-processing methods and scanning parameters. Cross-validation of this optimised microCT method, neuronal tracing and EIT in the rat sciatic nerve was detailed in Chapter 3. Chapter 4 describes the study with microCT with tracing, EIT and selective stimulation in pigs, a model for human nerves. The microCT tracing approach was then extended into the subdiaphragmatic branches of the vagus nerves, detailed in Chapter 5. The ultimate goal of human vagus nerve tracing was preliminarily performed and described in Chapter 6. Chapter 7 concludes the work and describes future work. Lastly, Appendix 1 (Chapter 8) is a mini review on the application of selective vagus nerve stimulation to treat acute respiratory distress syndrome and Appendix 2 is morphological data corresponding to Chapter 4

An Ultrastructural Model to Test Microburst Stimulation of Nerves

Some patients that suffer from epilepsy may become refractory to pharmaceutical treatment. An option with these patients is vagus nerve stimulation (VNS) therapy with neuro-cybernetic medical devices. The purpose of this research is two-fold: 1.) to determine if a recovery technique can be used with formalin-fixed samples of nerve tissue for transmission electron microscopy (TEM) and 2.) to determine if there is an ultrastructural difference in tissue exposed to the neuro-cybernetic device. If successful, the study will reduce animal studies and expense by establishing a mechanism to perform retrospective TEM studies on formalin-fixed tissue. Additionally, TEM allows examination of specimens in much greater detail than light microscopy. Therefore, using TEM to compare ultrastructural differences in tissue that was exposed to the medical device and healthy tissue will help determine with precision if any damage is caused by the medical device. To complete these objectives, formalin-fixed vagus nerve tissue from goats that were exposed to the medical device is collected, recovered, evaluated by TEM, and compared to healthy traditionally fixed vagus nerve tissue. Results show that the recovery technique makes it possible to achieve quantitative data from formalin-fixed tissue samples. This method establishes a mechanism to execute retrospective TEM studies on formalin-fixed tissue, thereby reducing future animal studies. Results also show that there are some differences in goat nerve that has been exposed to the medical device. These subtle ultrastructural changes (potentially reversible) do not appear to have clinical impact

Anodal block permits directional vagus nerve stimulation

© 2020, The Author(s). Vagus nerve stimulation (VNS) is a bioelectronic therapy for disorders of the brain and peripheral organs, and a tool to study the physiology of autonomic circuits. Selective activation of afferent or efferent vagal fibers can maximize efficacy and minimize off-target effects of VNS. Anodal block (ABL) has been used to achieve directional fiber activation in nerve stimulation. However, evidence for directional VNS with ABL has been scarce and inconsistent, and it is unknown whether ABL permits directional fiber activation with respect to functional effects of VNS. Through a series of vagotomies, we established physiological markers for afferent and efferent fiber activation by VNS: stimulus-elicited change in breathing rate (ΔBR) and heart rate (ΔHR), respectively. Bipolar VNS trains of both polarities elicited mixed ΔHR and ΔBR responses. Cathode cephalad polarity caused an afferent pattern of responses (relatively stronger ΔBR) whereas cathode caudad caused an efferent pattern (stronger ΔHR). Additionally, left VNS elicited a greater afferent and right VNS a greater efferent response. By analyzing stimulus-evoked compound nerve potentials, we confirmed that such polarity differences in functional responses to VNS can be explained by ABL of A- and B-fiber activation. We conclude that ABL is a mechanism that can be leveraged for directional VNS

Neural and humoral factors related to diaphragm fatigue

The aim of project was to gain an insight into the neural control of breathing during the development of diaphragm fatigue. In phase one, the role of vagal feedback in the control of breathing during the development of diaphragm fatigue was examined by comparing the ventilatory responses to inspiratory resistive loading (IRL) in vagally intact and vagally denervated rabbits. The results indicate that vagal inputs probably have no significant role to play in the control of breathing during the development of diaphragm fatigue. In phase two, the effects of IRL on arterial blood chemistry were examined to identify noxious chemicals generated during fatiguing IRL. This was necessary to identify potential chemical stimuli of small-phrenic afferent fibres. Potassium was identified as one such stimulus. The increase in arterial potassium concentration ([Ka+]) during IRL was associated with a combined metabolic and respiratory acidosis. On the basis of theoretical considerations, the increase in [K+] could have precipitated diaphragm fatigue. The effects of metabolic and respiratory acidoses on [Ka+] were independently assessed. Both produced a rise in [Ka+], but the sum was less than the rise in [Ka+] produced by IRL. In the final phase, it was established that activation of small-phrenic afferents either by electrical stimulation or by K+ applied to the abdominal surface of the diaphragm caused an increase in minute ventilation and a transient decrease in mean arterial blood pressure. In addition, K+ was shown to excite phrenic afferents. Two patterns of discharge were observed; one was rapidly adapting characteristic of group III fibres, the other was slowly adapting characteristic of group IV fibres

C-tactile afferents: Cutaneous mediators of oxytocin release during affiliative tactile interactions?

Low intensity, non-noxious, stimulation of cutaneous somatosensory nerves has been shown to trigger oxytocin release and is associated with increased social motivation, plus reduced physiological and behavioural reactivity to stressors. However, to date, little attention has been paid to the specific nature of the mechanosensory nerves which mediate these effects. In recent years, the neuroscientific study of human skin nerves (microneurography studies on single peripheral nerve fibres) has led to the identification and characterisation of a class of touch sensitive nerve fibres named C-tactile afferents. Neither itch nor pain receptive, these unmyelinated, low threshold mechanoreceptors, found only in hairy skin, respond optimally to low force/velocity stroking touch. Notably, the speed of stroking which c-tactile afferents fire most strongly to is also that which people perceive to be most pleasant. The social touch hypothesis posits that this system of nerves has evolved in mammals to signal the rewarding value of physical contact in nurturing and social interactions. In support of this hypothesis, in this paper we review the evidence that cutaneous stimulation directly targeted to optimally activate c-tactile afferents reduces physiological arousal, carries a positive affective value and, under healthy conditions, inhibits responses to painful stimuli. These effects mirror those, we also review, which have been reported following endogenous release and exogenous administration of oxytocin. Taken together this suggests C-tactile afferent stimulation may mediate oxytocin release during affiliative tactile interactions