Feasibility of kilohertz frequency alternating current neuromodulation of carotid sinus nerve activity in the pig

Recent research supports that over-activation of the carotid body plays a key role in metabolic diseases like type 2 diabetes. Supressing carotid body signalling through carotid sinus nerve (CSN) modulation may offer a therapeutic approach for treating such diseases. Here we anatomically and histologically characterised the CSN in the farm pig as a recommended path to translational medicine. We developed an acute in vivo porcine model to assess the application of kilohertz frequency alternating current (KHFAC) to the CSN of evoked chemo-afferent CSN responses. Our results demonstrate the feasibility of this approach in an acute setting, as KHFAC modulation was able to successfully, yet variably, block evoked chemo-afferent responses. The observed variability in blocking response is believed to reflect the complex and diverse anatomy of the porcine CSN, which closely resembles human anatomy, as well as the need for optimisation of electrodes and parameters for a human-sized nerve. Overall, these results demonstrate the feasibility of neuromodulation of the CSN in an anesthetised large animal model, and represent the first steps in driving KHFAC modulation towards clinical translation. Chronic recovery disease models will be required to assess safety and efficacy of this potential therapeutic modality for application in diabetes treatment

Dependence of excitability indices on membrane channel dynamics, myelin impedance, electrode location and stimulus waveforms in myelinated and unmyelinated fibre models

Neuronal excitability is determined in a complex way by several interacting factors, such as membrane dynamics, fibre geometry, electrode configuration, myelin impedance, neuronal terminations This study aims to increase understanding in excitability, by investigating the impact of these factors on different models of myelinated and unmyelinated fibres (five well-known membrane models are combined with three electrostimulation models, that take into account the spatial structure of the neuron). Several excitability indices (rheobase, polarity ratio, bi/monophasic ratio, time constants) are calculated during extensive parameter sweeps, allowing us to obtain novel findings on how these factors interact, e.g. how the dependency of excitability indices on the fibre diameter and myelin impedance is influenced by the electrode location and membrane dynamics. It was found that excitability is profoundly impacted by the used membrane model and the location of the neuronal terminations. The approximation of infinite myelin impedance was investigated by two implementations of the spatially extended non-linear node model. The impact of this approximation on the time constant of strength-duration plots is significant, most importantly in the Frankenhaeuser-Huxley membrane model for large electrode-neuron separations. Finally, a multi-compartmental model for C-fibres is used to determine the impact of the absence of internodes on excitability

Enhancing selectivity of minimally invasive peripheral nerve interfaces using combined stimulation and high frequency block: from design to application

The discovery of the excitable property of nerves was a fundamental step forward in our knowledge of the nervous system and our ability to interact with it. As the injection of charge into tissue can drive its artificial activation, devices have been conceived that can serve healthcare by substituting the input or output of the peripheral nervous system when damage or disease has rendered it inaccessible or its action pathological. Applications are far-ranging and transformational as can be attested by the success of neuroprosthetics such as the cochlear implant. However, the body’s immune response to invasive implants have prevented the use of more selective interfaces, leading to therapy side-effects and off-target activation. The inherent tradeoff between the selectivity and invasiveness of neural interfaces, and the consequences thereof, is still a defining problem for the field. More recently, continued research into how nervous tissue responds to stimulation has led to the discovery of High Frequency Alternating Current (HFAC) block as a stimulation method with inhibitory effects for nerve conduction. While leveraging the structure of the peripheral nervous system, this neuromodulation technique could be a key component in efforts to improve the selectivity-invasiveness tradeoff and provide more effective neuroprosthetic therapy while retaining the safety and reliability of minimally invasive neural interfaces. This thesis describes work investigating the use of HFAC block to improve the selectivity of peripheral nerve interfaces, towards applications such as bladder control or vagus nerve stimulation where selective peripheral nerve interfaces cannot be used, and yet there is an unmet need for more selectivity from stimulation-based therapy. An overview of the underlying neuroanatomy and electrophysiology of the peripheral nervous system combined with a review of existing electrode interfaces and electrochemistry will serve to inform the problem space. Original contributions are the design of a custom multi-channel stimulator able to combine conventional and high frequency stimulation, establishing a suitable experimental platform for ex-vivo electrophysiology of the rat sciatic nerve model for HFAC block, and exploratory experiments to determine the feasibility of using HFAC block in combination with conventional stimulation to enhance the selectivity of minimally-invasive peripheral nerve interfaces.Open Acces

Long-Term Activity-Dependent Plasticity of Action Potential Propagation Delay and Amplitude in Cortical Networks

Background: The precise temporal control of neuronal action potentials is essential for regulating many brain functions. From the viewpoint of a neuron, the specific timings of afferent input from the action potentials of its synaptic partners determines whether or not and when that neuron will fire its own action potential. Tuning such input would provide a powerful mechanism to adjust neuron function and in turn, that of the brain. However, axonal plasticity of action potential timing is counter to conventional notions of stable propagation and to the dominant theories of activity-dependent plasticity focusing on synaptic efficacies. Methodology/Principal Findings: Here we show the occurrence of activity-dependent plasticity of action potentia

A bioresorbable peripheral nerve stimulator for electronic pain block

Local electrical stimulation of peripheral nerves can block the propagation of action potentials, as an attractive alternative to pharmacological agents for the treatment of acute pain. Traditional hardware for such purposes, however, involves interfaces that can damage nerve tissue and, when used for temporary pain relief, that impose costs and risks due to requirements for surgical extraction after a period of need. Here, we introduce a bioresorbable nerve stimulator that enables electrical nerve block and associated pain mitigation without these drawbacks. This platform combines a collection of bioresorbable materials in architectures that support stable blocking with minimal adverse mechanical, electrical, or biochemical effects. Optimized designs ensure that the device disappears harmlessly in the body after a desired period of use. Studies in live animal models illustrate capabilities for complete nerve block and other key features of the technology. In certain clinically relevant scenarios, such approaches may reduce or eliminate the need for use of highly addictive drugs such as opioids

Optimal strategies for electrical stimulation with implantable neuromodulation devices

Electrical stimulation (ES) is a neuromodulation technique that uses electrical pulses to modulate the activity of excitable cells to provide a therapeutic effect. Many past and present ES applications use rectangular current waveforms that have been well studied and are easy to generate. However, an extensive body of scientific literature describes different stimulation waveforms and their potential benefits. A key measure of stimulation performance is the amplitude required to reach a certain percentual threshold of activation, as it directly influences important ES parameters such as energy consumption per pulse and charge density. The research summarized in this thesis was conducted to re-examine some of the most-commonly suggested ES waveform variations in a rodent in-vivo nerve-muscle preparation. A key feature of our experimental model is the ability to test stimulation with both principal electrode configurations, monopolar and bipolar, under computer control and in randomized order. Among the rectangular stimulation waveforms, we investigated the effect of interphase gaps (IPGs), asymmetric charge balanced pulses, and subthreshold conditioning pre-pulses. For all these rectangular waveforms, we surprisingly observed opposite effects in the monopolar compared to the bipolar stimulation electrode configuration. The rationale for this consistent observation was identified by analyzing electroneurograms (ENGs) of the stimulated nerve. In the monopolar configuration, biphasic pulses first evoked compound action potentials (eCAPs) as a response to the first field transition. In the bipolar electrode configuration, that is the mode in which many contemporary ES devices, including the envisioned miniaturized electroceuticals, operate, eCAPs were first elicited at the return electrode in response to the middle field transition of biphasic pulses. As all rectangular waveform variations achieve their effect by modulating the amplitude and timing of cathodic (excitatory) and anodic (inhibitory) field transitions, the inverted current profile at the bipolar return electrode explains these observed opposite effects. Further we investigated the claimed benefits of non-rectangular, Gaussian stimulation waveforms in our animal model. In our study only moderate energy savings of up to 17% were observed, a finding that is surprising in light of the predicted range of benefits of up to 60% energy savings with this novel waveform in question. Additionally, we identified a major disadvantage in terms of substantially increased maximum instantaneous power requirements with Gaussian compared to rectangular stimuli. We examined physiological changes in fast twitch muscle following motor nerve injury, and optimal stimulation strategies for activation of denervated muscle. While a high frequency doublet has previously been identified to enhance stimulation efficiency of healthy fast twitch muscle, an effect that has been termed “doublet effect”, we here show that this benefit is gradually lost in muscle during denervation. Lastly, the effect of long duration stimulation pulses, that are required to activate denervated muscle, on nerve is examined. We show that these long pulses can activate nerves up to three times when the three field transition within the biphasic pulses are separated by more than (i.e., when the phase width is above) the refractory period of that nerve. This observation challenges state-of-the-art computational models of extracellular nerve stimulation that do not seem to predict such multiple activations. Further, an undesired up to threefold co-activation of innervated structures nearby the denervated stimulation target warrants further research to study whether these co-activations can be lessened with alternative stimulation waveforms such as ramped sawtooth pulses

Restoring Upper Extremity Mobility through Functional Neuromuscular Stimulation using Macro Sieve Electrodes

The last decade has seen the advent of brain computer interfaces able to extract precise motor intentions from cortical activity of human subjects. It is possible to convert captured motor intentions into movement through coordinated, artificially induced, neuromuscular stimulation using peripheral nerve interfaces. Our lab has developed and tested a new type of peripheral nerve electrode called the Macro-Sieve electrode which exhibits excellent chronic stability and recruitment selectivity. Work presented in this thesis uses computational modeling to study the interaction between Macro-Sieve electrodes and regenerated peripheral nerves. It provides a detailed understanding of how regenerated fibers, both on an individual level and on a population level respond differently to functional electrical stimulation compared to non-disrupted axons. Despite significant efforts devoted to developing novel regenerative peripheral interfaces, the degree of spatial clustering between functionally related fibers in regenerated nerves is poorly understood. In this thesis, bioelectrical modeling is also used to predict the degree of topographical organization in regenerated nerve trunks. In addition, theoretical limits of the recruitment selectivity of the device is explored and a set of optimal stimulation paradigms used to selectively activate fibers in different regions of the nerve are determined. Finally, the bioelectrical model of the interface/nerve is integrated with a biomechanical model of the macaque upper limb to study the feasibility of using macro-sieve electrodes to achieve upper limb mobilization

Spinal cord stimulation in chronic pain: evidence and theory for mechanisms of action.

Well-established in the field of bioelectronic medicine, Spinal Cord Stimulation (SCS) offers an implantable, non-pharmacologic treatment for patients with intractable chronic pain conditions. Chronic pain is a widely heterogenous syndrome with regard to both pathophysiology and the resultant phenotype. Despite advances in our understanding of SCS-mediated antinociception, there still exists limited evidence clarifying the pathways recruited when patterned electric pulses are applied to the epidural space. The rapid clinical implementation of novel SCS methods including burst, high frequency and dorsal root ganglion SCS has provided the clinician with multiple options to treat refractory chronic pain. While compelling evidence for safety and efficacy exists in support of these novel paradigms, our understanding of their mechanisms of action (MOA) dramatically lags behind clinical data. In this review, we reconstruct the available basic science and clinical literature that offers support for mechanisms of both paresthesia spinal cord stimulation (P-SCS) and paresthesia-free spinal cord stimulation (PF-SCS). While P-SCS has been heavily examined since its inception, PF-SCS paradigms have recently been clinically approved with the support of limited preclinical research. Thus, wide knowledge gaps exist between their clinical efficacy and MOA. To close this gap, many rich investigative avenues for both P-SCS and PF-SCS are underway, which will further open the door for paradigm optimization, adjunctive therapies and new indications for SCS. As our understanding of these mechanisms evolves, clinicians will be empowered with the possibility of improving patient care using SCS to selectively target specific pathophysiological processes in chronic pain

High Fidelity Bioelectric Modelling of the Implanted Cochlea

Cochlear implants are medical devices that can restore sound perception in individuals with sensorineural hearing loss (SHL). Since their inception, improvements in performance have largely been driven by advances in signal processing, but progress has plateaued for almost a decade. This suggests that there is a bottleneck at the electrode-tissue interface, which is responsible for enacting the biophysical changes that govern neuronal recruitment. Understanding this interface is difficult because the cochlea is small, intricate, and difficult to access. As such, researchers have turned to modelling techniques to provide new insights. The state-of-the-art involves calculating the electric field using a volume conduction model of the implanted cochlea and coupling it with a neural excitation model to predict the response. However, many models are unable to predict patient outcomes consistently. This thesis aims to improve the reliability of these models by creating high fidelity reconstructions of the inner ear and critically assessing the validity of the underlying and hitherto untested assumptions. Regarding boundary conditions, the evidence suggests that the unmodelled monopolar return path should be accounted for, perhaps by applying a voltage offset at a boundary surface. Regarding vasculature, the models show that large modiolar vessels like the vein of the scala tympani have a strong local effect near the stimulating electrode. Finally, it appears that the oft-cited quasi-static assumption is not valid due to the high permittivity of neural tissue. It is hoped that the study improves the trustworthiness of all bioelectric models of the cochlea, either by validating the claims of existing models, or by prompting improvements in future work. Developing our understanding of the underlying physics will pave the way for advancing future electrode array designs as well as patient-specific simulations, ultimately improving the quality of life for those with SHL

Neuropathic pain in neuropathy: a combined clinical, neurophysiological and morphological study

Neuropathic pain (NP) is a major symptom which may be intractable in common neurological disorders such as neuropathy, spinal cord injury, multiple sclerosis and stroke. Pain is a complex sensation strongly modulated by cognitive influences, and understanding the underlying pathophysiological mechanisms in patients remains a challenge for pain specialists. The aim of my Phd-research was to show in according with present evidence-based studies the correlation between clinical manifestations of neuropathic pain and the underlying alteration of the different groups of fibers (Aβ, Aδ or C). In the second chapter I revised the previous guidelines about neuropathic pain assessment. History and clinical examination are a requirement to confirm the presence of a NP, and also an important step in reaching an aetiological diagnosis for NP. History and bedside examination are still fundamental to a correct diagnosis, while screening tools and questionnaires are useful in indicating probable NP. I argued in particular a recent technique, skin biopsy; I approached it at the beginning of my Phd during my stage at the I.R.C.S.S. C. Besta in Milan; then, I imported this procedure in our laboratory (Department of Pathological Anatomy, Sapienza University). We are now able to process skin biopsies and immunoassayed them with polyclonal anti-protein-gene-product 9.5 antibodies (specific for nerve fibers) using immunohistochemistry or immunofluorescence, which allowed demonstrating the extensive innervations of the epidermidis. In the following chapters I approached some common conditions of neuropathic pain. The third chapter is dedicated to the post-herpetic neuralgia, an exceptionally drug-resistant neuropathic pain. To investigate the pathophysiological mechanisms underlying postherpetic neuralgia we clinically investigated sensory disturbances, pains and itching, with an 11-point numerical rating scale in 41 patients with ophthalmic postherpetic neuralgia. In all the patients we recorded the blink reflex, mediated by non-nociceptive myelinated Aβ-fibers, and trigeminal laser evoked potentials (LEPs) related to nociceptive myelinated Aδ- and unmyelinated C-fiber activation. We also sought possible correlations between clinical sensory disturbances and neurophysiological data. Neurophysiological testing yielded significantly abnormal responses on the affected side compared with the normal side. The blink reflex delay correlated with the intensity of paroxysmal pain, whereas the Aδ- and C-LEP amplitude reduction correlated with the intensity of constant pain . Allodynia correlated with none of the neurophysiological data. Our study shows that postherpetic neuralgia impairs all sensory fiber groups. The neurophysiological-clinical correlations suggest that constant pain arises from a marked loss of nociceptive afferents, whereas paroxysmal pain is related to Aβ-fiber demyelination. These findings might be useful for a better understanding of pain mechanisms in postherpetic neuralgia. In the fourth chapter I treated the differential involvement of Aδ and Aβ fibers in neuropathic pain related to carpal tunnel syndrome (CTS). We studied 70 patients with a diagnosis of CTS (117 CTS hands). We used the DN4 questionnaire to select patients with neuropathic pain, and the Neuropathic Pain Symptom Inventory (NPSI) to assess the intensity of the various qualities of neuropathic pain. All patients underwent a standard nerve conduction study (NCS) to assess the function of non-nociceptive Aβ-fibres, and the cutaneous silent period (CSP) after stimulation of the IIIrd and Vth digits, to assess the function of nociceptive Aδ-fibres. In 40 patients (75 CTS hands) we also recorded LEPs in response to stimuli delivered to the median nerve territory and mediated by nociceptive Aδ-fibres. We sought possible correlations between neurophysiological data and the various qualities of neuropathic pain as assessed by the NPSI. We found that the median nerve sensory conduction velocity correlated with paroxysmal pain and abnormal sensations, whereas LEP amplitude correlated with spontaneous constant pain. Our findings suggest that whereas paroxysmal pain and abnormal sensations reflect demyelination of non-nociceptive Aβ-fibres, spontaneous constant pain arises from damage to nociceptive Aδ-fibres. In the fifth chapter I treated the mechanisms of pain in multiple sclerosis. In this clinical and neurophysiological study we sought information on the clinical characteristics and underlying mechanisms of neuropathic pain related to the disease. A total of 302 consecutive patients with multiple sclerosis were screened for neuropathic pain by clinical examination and the DN4 tool. In patients selected for having ongoing extremity pain or Lhermitte’s phenomenon, we recorded somatosensory evoked potentials, mediated by Aβ non-nociceptive fibres, and LEP, mediated by Aδ nociceptive fibres. Of the 302 patients, 92 had pain (30%), and 42 (14%) neuropathic pain. Patients with neuropathic pain had more severe multiple sclerosis, as assessed by the expanded disability severity score, than those without pain. Whereas in patients with ongoing neuropathic pain laser evoked potentials were more frequently abnormal than somatosensory evoked potentials we found the opposite in patients with Lhermitte’s phenomenon. Our data underline the clinical importance of pain in multiple sclerosis and indicate that a more severe disease is associated with a higher risk of developing neuropathic pain. The prevalence of pain we found, lower than that reported in previous studies, may reflect the lower disease severity in our patients. Neurophysiological data show that whereas ongoing extremity pain is associated with spinothalamic pathway damage, Lhermitte’s phenomenon is related to damage of non-nociceptive pathways. These findings may be useful in designing a new therapeutic approach to neuropathic pain related to multiple sclerosis. The sixth chapter is dedicated to the mechanisms of pain in distal symmetric neuropathy. I and my colleagues performed a clinical, neurophysiological and histomorphological study on patients with neuropathic pain in distal symmetric neuropathy. In patients with distal symmetric polyneuropathy we assessed non-nociceptive Aβ- and nociceptive Aδ- and C-afferents to investigate their role in the development of neuropathic pain. We screened 2240 consecutive patients with sensory disturbances and collected 269 patients with distal symmetric polyneuropathy (57% with pain and 43% without). All patients underwent the Neuropathic Pain Symptom Inventory to rate ongoing, paroxysmal and provoked pains, a standard NCS to assess Aβ-fibre function, LEPs to assess Aδ-fibre function, and skin biopsy to assess the unmyelinated innervations of the epidermidis. Patients with pain had the same age, but a longer delay since symptom onset than those without . Loss of intraepidermal innervation did not correlate with the presence of neuropathic pain. Whereas the LEP amplitude was significantly lower in patients with pain than in those without , NCS and intraepidermal fibre nerves data did not differ between groups. LEPs were more severely affected in patients with ongoing pain than in those with provoked pain. Our findings indicate that the impairment of Aβ-fibres has no role in the development of ongoing or provoked pain. In patients with ongoing pain the severe LEP suppression and the correlation between pain intensity and LEP attenuation may indicate that this type of pain reflects damage to nociceptive axons. The partially preserved LEPs in patients with provoked pain suggest that thistype of pain is related to the abnormal activity arising from partially spared and sensitised nociceptive terminals. Because clinical and neurophysiological abnormalities followed similar patterns regardless of aetiology, pain should be classified and treated on mechanism-based grounds. In the seventh chapter I treated the mechanisms of allodynia in distal symmetric polyneuropathy allodynia. Patients with painful neuropathy frequently complain of allodynia, i.e. pain in response to a normally non-painful stimulus. Many authors consider allodynia to be generated by sensitization of the second-order nociceptive neurons to Aβ-fibre input (central sensitization). With the hypothesis that patients suffering from this type of pain probably have a relative sparing of Aβ-fibres in comparison with patients with ongoing pain only, we sought aimed at seeking information on mechanisms underlying allodynia. In 200 patients with distal symmetric polyneuropathy (114 with pain, 86 without) we assessed non-nociceptive Aβ- and nociceptive Aδ-afferents to investigate their role in the development of allodynia. After a detailed clinical examination and pain questionnaires patients underwent a standard nerve conduction study (NCS) to assess Aβ-fibre function, and LEPs to assess Aδ-fibre function. Forthy-four out of 114 patients with painful neuropathy suffered from allodynia. While NCS data did not differ between patients with and without allodynia, LEP amplitude was higher in patients with allodynia than in those without. Our data argue against a role of Aβ-fibres and central sensitization as the main mechanism for the development of allodynia in distal symmetric polyneuropathy. The partially preserved LEPs in patients with allodynia suggests that this type of pain might be related to the abnormal reduction of mechanical threshold of nociceptive terminals (peripheral sensitization). In the eighth chapter I treated neuropathic pain in patient with crioglobulinemia. The study aimed at gaining information on peripheral neuropathy and neuropathic pain in patients with cryoglobulinaemia. We collected 48 consecutive patients with cryoglobulinaemia. All patients underwent a standard NCS to assess A-fibre function, LEPs to assess A-fibre function, and skin biopsy to assess C-fibre terminals. We used DN4 questionnaire to diagnose neuropathic pain, and the Neuropathic Pain Symptom Inventory to rate the intensity of the different qualities of neuropathic pain. Thirty patients had a peripheral neuropathy. Twenty-three had neuropathic pain as assessed by the DN4 questionnaire. NPSI questionnaire showed that the most frequent type of pain was the burning pain. Patients with peripheral neuropathy had an older age than those without . The duration of the disease correlated with the density of epidermal innervation as assessed by skin biopsy. The severity of the ongoing burning pain correlated with the amplitude of LEPs, but not with the density of epidermal innervation . Our findings showed that an older age is associated with the development of peripheral neuropathy, and a longer duration of disease with a more severe peripheral nerve damage, as assessed by skin biopsy. The correlation between the intensity of ongoing pain and LEP attenuation indicate that neuropathic pain reflects damage to nociceptive axons. In the ninth chapter I discussed the research on a peptide, the kiss-peptine, whose antagonist could be a new analgesic drug. More studies should be perform in the next future about it . Kisspeptin is a neuropeptide known for its role in the hypothalamic regulation of the reproductive axis. Following the recent description of kisspeptin and its 7-TM receptor, GPR54, in the dorsal root ganglia and dorsal horns of the spinal cord, we examined the role of kisspeptin in the regulation of pain sensitivity in mice. Immunofluorescent staining in the mouse skin showed the presence of GPR54 receptors in PGP9.5-positive sensory fibers. Intraplantar injection of kisspeptin (1 or 3 nmol/5 μl) induced a small nocifensive response in naive mice, and lowered thermal pain threshold in the hot plate test. Both intraplantar and intrathecal (0.5 or 1 nmol/3 μl) injection of kisspeptin caused hyperalgesia in the first and second phases of the formalin test, whereas the GPR54 antagonist, p234 (0.1 or 1 nmol), caused a robust analgesia. Intraplantar injection of kisspeptin combined with formalin enhanced TRPV1 phosphorylation at Ser800 at the injection site, and increased ERK1/2 phosphorylation in the ipsilateral dorsal horn as compared to naive mice and mice treated with formalin alone. These data demonstrate for the first time that kisspeptin regulates pain sensitivity in rodents and suggest that peripheral GPR54 receptors could be targeted by novel drugs in the treatment of inflammatory pain. In the tenth last chapter I gathered all the conclusion of the single studies. Here I tried to associate each quality of pain to an underling pathophysiological alteration, since the aim of y studies was to show the correlation between clinical manifestations of neuropathic pain and the underlying alteration of the different groups of fibers (A-β, A-δ or C)