System and device for treating neurodegenerative disorders using vagus nerve stimulation

Abstract

Systems, devices, and methods are described that use vagus nerve stimulation to treat demyelinating disorders and / or disorders of the blood-brain barrier. The vagus nerve stimulation therapies described herein are configured to reduce or prevent demyelination and / or promote remyelination, thereby treating various disorders related to demyelination, such as multiple sclerosis. A stimulation regimen with a low duty cycle having a relatively short on-time and a relatively long off-time can be used.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 833,631, filed April 12, 2019, entitled “VAGUS NERVE STIMULATION TO TREATNEURODEGENERATIVE DISORDERS,” which is incorporated herein by reference in its entirety.

[0003] This patent application may relate to U.S. Patent Application No. 16 / 158,222, filed October 11, 2018, entitled “VAGUS NERVE STIMULATION TO TREAT NEURODEGENERATIVE DISORDERS,” and claims priority to U.S. Provisional Patent Application No. 62 / 572,374, filed October 13, 2017, entitled “VAGUS NERVE STIMULATION TO TREAT NEURODEGENERATIVE DISORDERS,” and U.S. Provisional Patent Application No. 62 / 576,547, filed October 24, 2017, entitled “VAGUS NERVE STIMULATION TO TREAT NEURODEGENERATIVE DISORDERS,” each of which is incorporated herein by reference in its entirety.

[0004] References merged

[0005] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference. Technical Field

[0006] Embodiments of the present invention generally relate to apparatus (e.g., devices, systems) and methods for treating neurodegenerative and neuroinflammatory disorders by vagus nerve stimulation, and more specifically, to apparatus and methods for stimulating the vagus nerve to reduce demyelination (e.g., by preventing immune cell infiltration into the CNS) and / or promote myelin regeneration to treat various neurodegenerative and neuroinflammatory disorders, such as multiple sclerosis. Background Technology

[0007] Many central nervous system (CNS) demyelinating disorders, including multiple sclerosis, acute disseminated encephalomyelitis, and neuromyelitis optica spectrum disorders, are difficult to treat effectively. For example, multiple sclerosis (MS) is a neurodegenerative and neuroinflammatory disease characterized by demyelination of nerves in the central nervous system. Although the underlying cause of demyelination is unclear, it is generally associated with myelin sheath lesions and inflammation. Currently, there is no known cure for MS. Current treatments have achieved some success, primarily targeting the management of acute attacks and reducing disease relapses—either alleviating the frequency of attacks in a subtype or managing symptoms. However, current therapies can at best slow disease progression, and to date, no therapy has been shown to regenerate nerve myelin sheaths.

[0008] Therefore, there will be a need for additional therapies and systems that can be used independently or in combination with other therapies to reduce the rate or amount of demyelination. Furthermore, it will be desirable to provide a therapy that regenerates the myelin sheath and reverses the progression of demyelination. Summary of the Invention

[0009] The present invention generally relates to vagus nerve stimulation for the treatment of neurodegenerative and neuroinflammatory disorders, and more specifically, to the treatment of various neurodegenerative and neuroinflammatory disorders, such as multiple sclerosis, by vagus nerve stimulation to reduce demyelination and / or promote myelin regeneration.

[0010] For example, this document describes devices (e.g., instruments and / or systems) for reducing demyelination and / or increasing myelin regeneration by stimulating the vagus nerve. These devices may be implants or implanted in a patient. Any of these devices may include: a biosensor configured to detect one or more biomarkers; a stimulator configured to apply stimulation to the vagus nerve; and a controller coupled to the biosensor and the stimulator, configured to apply stimulation from the stimulator to the vagus nerve sufficient to reduce demyelination and / or increase myelin regeneration in the patient when the biosensor detects a biomarker indicating demyelination. In some variations, these devices include implants comprising a stimulator (e.g., a waveform and / or pulse generator, oscillator, power supply and / or power regulation circuitry, etc.), a stimulation applicator (e.g., one or more electrodes, a mechanical transducer, etc.), and a controller. The controller may be configured as a microcontroller and may be electrically communicated with the stimulator to control the operation of the stimulator. The controller may include one or more processors, a memory, and / or a timer. The stimulator and / or controller may be electrically communicated with one or more stimulation applicators. In some variations, the controller may include or communicate with wireless communication circuitry for wireless communication with one or more remote processors. The remote processor may be a handheld device (e.g., a smartphone, wearable electronic device, etc.). The controller may optionally communicate with one or more biosensors, which may be included in an implant or can be located remotely from an implant (e.g., wearable, disposable, etc.). In some variations, the biosensors are wirelessly connected to the device.

[0011] In some variations, the device can be used without a biosensor. For example, the device can be configured to periodically and / or apply VNS treatment as needed to prevent or reduce demyelination. The device can be configured to apply a VNS treatment dose multiple times a day (e.g., once a day, twice a day, three times a day, four times a day, five times a day, six times a day) or every other day or every three days. In some variations, the device can be configured to automatically apply a VNS treatment dose at predetermined and / or adjustable predetermined times, and to provide a VNS treatment dose based on input from the user (e.g., patient, physician, etc., including “on-demand” doses) and / or based on the detection of biomarkers indicating an actual or potential increase in demyelination.

[0012] In any of these variations, the biosensor can be configured to detect one or more markers (e.g., biomarkers) from the patient's body, including from the patient's blood and / or cerebrospinal fluid. Examples of biomarkers can be found herein. The biosensor can be part of an implantable device, or it can be connected to the device (e.g., a controller) via wired or wireless communication. The biosensor can be configured to detect any biomarker, including chemical markers (e.g., proteins, nucleotides, such as RNA, DNA, microRNA, etc., lipids, carbohydrates, etc.), as well as functional markers (neural conduction, etc.), body temperature, etc. For example, in some variations, the biosensor is configured to detect temperature.

[0013] Generally, the device described herein can be configured to be inserted into or implanted in the body. For example, the device can be configured to be implanted. The device may include a stimulation applicator (also simply referred to as a stimulator or VNS treatment stimulator), which may be a mechanical and / or electrical stimulator. The mechanical stimulator may be a piezoelectric actuator that can vibrate and / or apply pressure to tissue (including the vagus nerve) at VNS treatment parameters, for example, mechanically stimulating the vagus nerve at a frequency between 1 and 2 kHz for a treatment time (e.g., between 1 millisecond and 5 minutes, such as 10 milliseconds to 10 seconds, etc.). Alternatively or additionally, the stimulation applicator may be an electrical stimulation applicator and may include one or more (e.g., two or more) electrodes configured to apply electrical stimulation to the vagus nerve. For example, electrical stimulation of about 0.1 mA to 10 mA (e.g., between 1 mA and 5 mA) at a frequency between about 1 Hz and about 2 kHz (e.g., between about 1 and 100 Hz), wherein the applied pulse has a pulse width of about (50-500 µsec, such as about 100-300 µsec). The controller can be configured to enforce a “shutdown time” after a VNS treatment dose of approximately 10 minutes to 12 hours (e.g., between approximately 2 hours and 10 hours, between approximately 3 hours and 6 hours, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, etc.). For example, the stimulator may include electrodes configured to apply electrical energy to the vagus nerve.

[0014] In some variations, the device is configured to apply VNS treatment to a patient, wherein the VNS treatment is electrical stimulation. For example, the VNS treatment may include applying electrical energy in the range of approximately 1-100 Hz (e.g., approximately 1-50 Hz, approximately 1-20 Hz, approximately 5-30 Hz, approximately 5-15 Hz, approximately 5 Hz, approximately 10 Hz, approximately 15 Hz, etc.). The energy may have a peak amplitude between approximately 0.1 mA and approximately 2 mA (e.g., approximately 0.2 mA and approximately 1.8 mA, approximately 0.5 mA and approximately 1.5 mA, approximately 0.5 mA and approximately 1 mA, approximately 0.1 mA and approximately 1 mA, approximately 0.5 mA, approximately 0.75 mA, approximately 1 mA, etc.). Alternatively, the applied energy may have an average amplitude between about 0.1 mA and about 2 mA (e.g., between about 0.2 mA and about 1.8 mA, about 0.5 mA and about 1.5 mA, about 0.5 mA and about 1 mA, about 0.1 mA and about 1 mA, about 0.5 mA, about 0.75 mA, about 1 mA, etc.). The applied energy is typically pulsed and can be a pulsed square wave, sine wave, triangle wave, etc. The applied energy can be biphasic or monophasic. For example, the applied energy can be biphasic. The applied VNS processing can be a constant biphasic pulse sequence with a frequency between 1 and 100 Hz (e.g., 10 Hz) and a peak amplitude between about 0.5 mA and 2 mA (e.g., about 0.75 mA). Any processing method described herein can be configured to apply this type of VNS processing.

[0015] Any device (e.g., apparatus, system, etc.) described herein can be configured to be implanted onto the vagus nerve. Therefore, any of these devices can be implanted via a nerve sheath or cuff configured to secure the device to the nerve and / or prevent movement of the device relative to the nerve and / or insulate the device from other tissues. The implanted device can be implanted at any suitable location on the nerve, including on or around the vagus nerve in the upper chest, or on or around the vagus nerve in the subdiaphragmatic location. The implant may be a wireless implant connected to the vagus nerve (see, for example, US 8412338, US 8612002, US 8886339, and US 8788034, each of which is incorporated herein by reference in its entirety). For example, any of these devices may include a nerve cuff configured to secure a stimulator to the vagus nerve. Alternatively, any of these devices may include wires connecting a microstimulator and / or other components to a stimulation applicator on / around the vagus nerve via one or more wires.

[0016] As described above, any of these devices can be configured to apply VNS treatment to the vagus nerve, comprising electrical stimulation with a low duty cycle between approximately 0.25 mA and approximately 5 mA for less than approximately 2 minutes. The device can be configured to provide at least... x Shutdown time in minutes per hour (e.g., 10 minutes, 20 minutes, 30 minutes, 40 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, etc.).

[0017] Any device described herein may be configured to perform a method for reducing demyelination in a patient diagnosed with or at risk of developing a disorder involving demyelinating nerves (e.g., including, but not limited to, methods for treating demyelinating-related disorders and / or diseases, such as multiple sclerosis). For example, a method for reducing demyelination (and / or increasing myelin regeneration) may include detecting markers for demyelination and stimulating the vagus nerve to reduce nerve demyelination in the patient.

[0018] Stimulation of the vagus nerve includes applying VNS treatment and may include, for example, applying electrical stimulation between about 0.25 and about 5 mA to the vagus nerve for less than about 2 minutes. In some variations, this may include a waiting-off time (e.g., a waiting-off time of at least 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, etc.).

[0019] Any of these methods may include applying non-invasive stimulation to the vagus nerve. For example, stimulation may be performed on a portion of the vagus nerve via a percutaneous route (e.g., via surface electrodes and / or mechanical stimulation, including ultrasound). The vagus nerve comprises multiple branches or extensions that can be accessed and / or targeted from outside the body mechanically and / or electrically. For example, non-invasive application may include ultrasound stimulation of the vagus nerve. Any of these methods may include applying transcutaneous electrical nerve stimulation (TENS), etc.

[0020] Any method described herein may include, for example, periodic, on-demand, and / or continuous monitoring of one or more markers (e.g., biomarkers) for demyelination or demyelination risk. As described above, any suitable method or device for monitoring demyelination or demyelination risk may be used. For example, any of these methods may include detecting demyelination markers, including monitoring the patient's body temperature. Changes in core body temperature (including elevations) have been linked to an increase in symptoms of demyelinating disorders (including, but not limited to, MS).

[0021] Any methods and apparatus described herein can be used in conjunction with, or in connection with, biomarkers for blood-brain barrier integrity. The methods and apparatus described herein generally improve blood-brain barrier integrity. Therefore, any biomarker associated with leakage or loss of integrity of the blood-brain barrier can be used to trigger VNS therapy as described herein. Examples of biomarkers may include serum S100β, and imaging modalities such as contrast-enhanced magnetic resonance imaging, CT scans, and lumbar puncture.

[0022] Detection of one or more markers for demyelination (e.g., biomarkers) may include determining the level of tumor necrosis factor in a blood or cerebrospinal fluid sample.

[0023] For example, the methods described herein (e.g., methods for treating demyelinating disorders, such as but not limited to MS, and / or methods for reducing or reversing demyelinating) include: detecting demyelinating in the patient and applying stimulation to the vagus nerve to increase myelin regeneration in the patient's nerves.

[0024] For example, any of these methods may include repeatedly applying electrical stimulation to the patient’s vagus nerve for less than about 2 minutes at a low duty cycle of about 0.25 and about 5 mA, followed by a shutdown period (e.g., between about 10 minutes and about 48 hours) before the next stimulation.

[0025] Any of these methods and apparatus may also include or be adapted to include treatment with one or more agents simultaneously (immediately before, during, or after, including systemic and / or local), particularly those agents considered helpful for demyelinating diseases (e.g., but not limited to, MS). For example, any of these methods may include simultaneous treatment with one or more of the following agents: interferon beta-1a, interferon beta-1b, glatiramer acetate, glatiramer acetate, pegylated interferon beta-1a, dazumab, teriflunomide, fingolimod, dimethyl fumarate, alenmab, mitoxantrone, ozogluconate, natezumab.

[0026] As described above, any methods and apparatus described herein may include continuous monitoring of a patient for demyelination or the condition involved in demyelination. For example, any methods and apparatus described herein may include monitoring a patient for disease-related biomarkers selected from the group consisting of neurodegenerative diseases, neuroinflammatory diseases, and neuropathy. In some examples, the method includes detecting demyelination in a patient by detecting biomarkers associated with MS. For example, biomarkers (e.g., biomarkers) may be selected from the group consisting of neurofilaments, glial fibrillary acidic proteins, the monocyte / macrophage biomarker CD163, the glial cell activation biomarker YKL-40, the B cell chemokine CXCL13, miRNA, mRNA, myelin-reactive T cells, Kir4.1 antibody, osteopontin, and microbiome-associated lipopeptides.

[0027] In particular, this document describes methods and apparatus for reducing or preventing demyelination and / or increasing myelin regeneration by stimulating the vagus nerve. For example, an apparatus (e.g., a system, device, component, etc., including implants) may include: a vagus nerve stimulator configured to be implanted above or near the vagus nerve; one or more electrodes on the vagus nerve stimulator configured to apply electrical stimulation to the vagus nerve; and a controller coupled to the vagus nerve stimulator configured to apply electrical stimulation to the vagus nerve from the one or more electrodes, wherein the controller is limited to applying a charge between 2.5 nC and 7.5 mC per day to reduce demyelination and / or increase myelin regeneration in the patient. The apparatus may be a system.

[0028] The system may include inputs configured to receive one or more biomarker level indicators, wherein the controller is configured to adjust the applied charge based on one or more biomarker level indicators. For example, the system may include a biosensor configured to detect biomarkers from a patient's blood and / or cerebrospinal fluid and determine biomarker level indicators.

[0029] The controller can be configured to deliver electrical stimulation during one or more dose periods of about 5 minutes or less (e.g., 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, etc.). The controller can be configured to apply charge daily at a frequency between 1 and 20 Hz. In some variations, the controller is configured to apply charge daily at a frequency between 1 and 12 Hz.

[0030] In any of these devices, the system is configured to be implanted.

[0031] Any of these systems may include a neural cannula configured to attach a vagus nerve stimulator to the vagus nerve. The controller may be configured to apply electrical charge daily at two different frequencies between 1 and 20 Hz. The controller may be configured to apply a first dose of electrical stimulation at a first frequency between 1 and 20 Hz to reduce demyelination, and a second dose of electrical stimulation at a second frequency higher than the first frequency to increase myelin regeneration in the patient. For example, the first dose of electrical stimulation may have a frequency less than 10 Hz, and the second dose of electrical stimulation may have a frequency ranging from 10 Hz to 30 Hz. In some variations, the first dose of electrical stimulation has a frequency ranging from 1 Hz to 5 Hz, and the second dose of electrical stimulation has a frequency ranging from 10 Hz to 30 Hz.

[0032] This article also describes a method for increasing the removal of myelin fragments in patients diagnosed with or at risk of developing disorders involving demyelinating nerves, comprising applying vagal nerve stimulation to the patient at a frequency between 2.5 nC and 7.5 mC daily. Application may include applying vagal nerve stimulation to the vagus nerve at frequencies between 0.1 and 20 Hz. In some variations, application includes applying vagal nerve stimulation for less than about 5 minutes per day (e.g., less than about 4 minutes per day, less than about 3 minutes per day, less than about 2 minutes per day, less than about 1 minute per day, etc.). Application may include applying stimulation to the vagus nerve from an implanted neurostimulator attached to or adjacent to the vagus nerve.

[0033] Any of these methods may include applied vagal nerve stimulation adjusted based on the level of a biomarker. For example, the method may include detecting demyelination biomarkers in blood, sputum, and / or cerebrospinal fluid samples.

[0034] A system for reducing demyelination and / or increasing myelin regeneration by stimulating the vagus nerve may include: a vagus nerve stimulator configured to be implanted above or near the vagus nerve; one or more electrodes on the vagus nerve stimulator configured to apply electrical stimulation to the vagus nerve; and a controller coupled to the vagus nerve stimulator configured to apply electrical stimulation to the vagus nerve from the one or more electrodes, wherein the controller is limited to applying low duty cycle electrical stimulation for a duration between 1 second and 5 minutes per day, the electrical stimulation including a first dose of electrical stimulation at a first frequency between 1 and 20 Hz for reducing demyelination, and a second dose of electrical stimulation at a second frequency above the first frequency for increasing myelin regeneration.

[0035] The controller can be configured to apply electrical stimulation between 1 and 24 times per day. The frequency of the first dose of electrical stimulation can be between 1 Hz and 10 Hz. The frequency of the second dose of electrical stimulation can be between 1 Hz and 5 Hz. The frequency of the first dose of electrical stimulation can be between 10 Hz and 30 Hz. The first dose of electrical stimulation can have a frequency less than 5 Hz, and the second dose of electrical stimulation can have a frequency ranging from 10 Hz to 30 Hz.

[0036] As described above, the controller can be configured to modulate electrical stimulation based on feedback from the user or based on one or more biomarkers associated with demyelination. The controller can be configured to reduce the frequency of electrical stimulation based on feedback. For example, the controller can be configured to adjust the duration of the first and second doses based on feedback.

[0037] Methods for reducing demyelination and / or increasing myelination in patients with disorders involving demyelinating nerves may include: applying low-duty-cycle electrical stimulation for a total duration of between 1 second and 5 minutes per day, comprising a first dose of electrical stimulation at a first frequency between 1 and 20 Hz for reducing demyelination, and a second dose of electrical stimulation at a second frequency higher than the first frequency for increasing myelination. Low-duty-cycle electrical stimulation may be applied, for example, from 1 to 24 times per day.

[0038] The first frequency can range from 1 Hz to 10 Hz. In some variations, the first frequency ranges from 1 Hz to 5 Hz. The second frequency can range from 10 Hz to 30 Hz. The first dose of electrical stimulation can have a frequency less than 5 Hz, and the second dose of electrical stimulation can have a frequency ranging from 10 Hz to 30 Hz.

[0039] Any of these methods may also include modulating electrical stimulation based on feedback from the user or based on one or more biomarkers associated with demyelination. The controller can be configured to adjust the duration of the first and second doses based on feedback.

[0040] As described herein, any of these methods may include simultaneous administration of one or more of interferon beta drugs, glatiramer acetate, and dazumab, combined with the application of low-duty-cycle electrical stimulation to target interferon beta-1a and 1b receptors and T cell activation to reduce central nervous system inflammation and demyelination. For example, any of these methods may include administration of one or more of fingolimod, teriflunomide, and dimethyl fumarate, combined with the application of low-duty-cycle electrical stimulation to target lymphocyte migration or activation to reduce central nervous system inflammation and demyelination. Any of these methods may include administration of one or more of mitoxantrone, alenzab, oligrizumab, and natezumab, combined with the application of low-duty-cycle electrical stimulation to induce DNA breaks, CD52 to induce cell lysis, depleted B cell CD20 antigen, and / or integrin receptors to alter leukocyte migration to reduce central nervous system inflammation and demyelination. In some variations, these approaches may include administering one or more of chlormastine, selective estrogen receptor modulators (SERMs), and other drugs that target oligodendrocyte precursor cells to promote the maturation of myelin-producing oligodendrocytes, thereby enhancing myelin regeneration and clinical recovery from central nervous system injury. Attached Figure Description

[0041] The novel features of the invention are set forth in the following claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description, which sets forth illustrative embodiments utilizing the principles of the invention, and the accompanying drawings, wherein:

[0042] Figure 1 The illustration shows a typical example of four stages following the injection of lysophosphatidylcholine into the spinal cord in a model used to study multiple sclerosis.

[0043] Figure 2A and Figure 2B The diagram illustrates the experimental protocol used to study demyelination and myelin regeneration.

[0044] Figure 3A The illustration shows a cross-section of a healthy spinal cord.

[0045] Figure 3B The illustration shows a stained cross section of the spinal cord with lesions induced by lysophosphatidylcholine injection (which can be considered as demyelination).

[0046] Figures 4A-4D This is a diagram showing the reduction in demyelination caused by vagal nerve stimulation and lysophosphatidylcholine injection. Figure 4AFigure 1 shows the effect of vagus nerve stimulation (VNS) at different stimulation levels (0 mA, 0.25 mA, and 0.75 mA) on demyelination four days after induction of demyelinating lesions. Figure B shows the increase in myelin regeneration within two weeks after induction of demyelinating lesions with and without VNS treatment (0 mA), demonstrating rapid myelin regeneration upon VNS application. Figure 4C This is a 3D image showing the change in lesion size with depth four days after induced demyelinating lesions with and without VNS treatment. Figure 4D This is a 2D projection of the median lesion four days after induction of demyelinating lesions, comparing the sham surgery group (Sham) (without VNS treatment) and the VNS treatment group.

[0047] Figure 5A-5G This is a diagram showing the rate and / or amount of myelin regeneration increased by vagal nerve stimulation. Figure 5A This is a graph showing the changes in demyelination after induction with and without VNS treatment (determined by changes in induced lesion volume), showing the lesion volume (mm) of the induced region. 3 (This is a reduction of approximately 65% ​​per day.) Figure 5B It is a 3D representation of the size of demyelination (lesion) with depth with and without VNS treatment (sham surgery group) and with VNS treatment. Figure 5C It is a 2D projection of the median lesion volume eight days after induction of demyelination (e.g., lesion) in the cases with and without VNS treatment (sham surgery group). Figure 5D This is a 3D representation of the change in demyelination (lesion size) with depth on day 14 after induction of demyelination, with and without VNS treatment (sham surgery group). Figure 5E It is a 2D projection of median demyelination (lesion) two weeks after induction of demyelination in the presence of VNS treatment (VNS) and without VNS treatment ("sham surgery group"). Figure 5F This is a 3D representation of the changes in demyelination (lesion size) with depth on day 21 after induction of demyelination (day 14 after induction) with VNS treatment (VNS) and without VNS treatment (sham surgery group). Figure 5G It is a 2D projection of median demyelination (lesion) three weeks after induction of demyelination in the presence of VNS treatment (VNS) and without VNS treatment ("sham surgery group").

[0048] Figure 6A An experimental protocol for demonstrating the effect of VNS treatment as described herein on demyelinating induced vascular leakage is shown.

[0049] Figure 6BThe illustration shows the use of VNS treatment, as described herein, to reduce blood-brain barrier leakage after induced demyelination. VNS treatment prior to demyelination prevention prevented the dye (Evans blue) from crossing the blood-brain barrier in a rat model. VNS treatment after demyelination prevention reduced and reversed leakage. VNS treatment on day 0 (after LPC induction) significantly reduced leukocyte infiltration 24 hours post-stimulation, while VNS treatment on day 4 post-LPC induction significantly reduced leukocyte infiltration 24 hours post-stimulation.

[0050] Figure 7A The figure illustrates the role of α-7 nicotinic acetylcholine receptor (α7 nAChR) in preventing VNS treatment-induced demyelination and myelin regeneration (compared to the sham surgery group without VNS treatment).

[0051] Figure 7B The figure illustrates the role of α-7 nicotinic acetylcholine receptors in myelin regeneration induced by increased VNS treatment (compared to the sham-operated group without VNS treatment).

[0052] Figure 8 The study demonstrates the effectiveness of VNS treatment, as described herein, in preventing or reversing blood-brain barrier leakage compared to the sham-operated group (without VNS treatment). Figure 9 In the study, compared with the sham-operated group, CD3+ T cell infiltration in the VNS group was significantly reduced by 50% on day 3 after LPC induction.

[0053] Figure 9 The figure illustrates that, compared with the sham-operated group (without VNS treatment), macrophage infiltration across the blood-brain barrier was significantly reduced by 55% 24 hours after demyelination induction (e.g., via LPC) with VNS treatment.

[0054] Figure 10A and Figure 10B The figure illustrates the effect of regressor lipid peroxidation factor D1 (RvD1) after induced demyelination in animals with and without VNS treatment (VNS) (sham-operated group). It shows that RvD1 was increased in VNS animals on day 4 after LPC induction compared to the sham-operated group, and remained elevated for 14 days after LPC induction. At day 21 after LPC induction, the level decreased to below that of the sham-operated group, at which point no visible lesions were detected in the VNS animals.

[0055] Figure 11 An example of a device for reducing demyelination (e.g., increasing myelin regeneration and / or reducing leakage across the blood-brain barrier) is illustrated schematically, as described herein.

[0056] Figure 12The illustration shows that VNS at frequencies below approximately 100 Hz (e.g., 75 Hz or lower, 50 Hz or lower, 40 Hz or lower, 30 Hz or lower, 25 Hz or lower, etc.) are more effective than VNS at higher frequencies (e.g., 1.8 kHz and above).

[0057] Figure 13 The illustration shows significantly more myelin regeneration in the lesion area after stimulation at 10Hz for 1 or 5 minutes. In general, stimulation for less than 20 minutes (e.g., less than 15 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, etc.) is more effective than stimulation for longer periods.

[0058] Figure 14 and Figure 15 The illustration shows that vagal nerve stimulation (VNS) significantly increased myelin fragment uptake when the stimulation was within the prescribed treatment range.

[0059] Figure 16 A stained section of tissue from animals in the sham-operated group is shown.

[0060] Figure 17 Images of animals treated with VNS on day 4 (after VNS) of treatment paradigm A are shown.

[0061] Figure 18 and Figure 19 The analysis of the staining is shown to quantify the intensity and extent.

[0062] Figure 20 The figure shows that the lesion volume in the VNS group was reduced by 34% compared to the sham-operated group on day 10 after induction in aged mice.

[0063] Figure 21 The figure shows that on day 10 in aged mice, the VNS group showed a 2.4-fold increase in Oil Red O staining as lesion percentage compared to the sham-operated group (p<0.05).

[0064] Figure 22 This is a graph showing how VNS enhances myelin clearance, as evidenced by increased myelin uptake by macrophages after VNS (e.g., showing an increase in the percentage of cells phagocytizing myelin after VNS).

[0065] Figure 23 This is a graph comparing the clinical scores of rats treated with chronic low duty cycle VNS and sham VNS, showing that the clinical scores of implanted VNS were lower in the MS model compared to those of implanted sham VNS.

[0066] Figure 24The percentage of CD4+ T cells in rat spinal cord samples treated with VNS is shown compared to the sham-operated group. VNS also shows a reduction in pathogenic CD3+ / CD4+ / IFNg+TH1 cells in the spinal cord one to two days after the procedure.

[0067] Figure 25 The percentage of CD8+ T cells in rat spinal cord samples treated with VNS is shown compared to the sham-operated group. VNS also shows a reduction in pathogenic CD3+ / CD8+ / IFNg+TH1 cells in the spinal cord one to two days after VNS treatment. Specific Implementation

[0069] Electrical and / or mechanical stimulation of the cholinergic anti-inflammatory pathway (NCAP) by stimulating the carotid vagus nerve has been well described. See, for example, US 6,838,471, US 8,914,114, US 9,211,409, US 6,610,713, US 8,412,338, US 8,996,116, US 8,612,002, US 9,162,064, US 8,855,767, US 8,886,339, US 9,174,041, US 8,788,034, and US 9,211,410, each of which is incorporated herein by reference in its entirety. There has been no prior suggestion that vagus nerve stimulation can be used to prevent or reduce demyelination and / or improve myelination regeneration. Vagus nerve stimulation that activates both efferent and afferent pathways (or primarily through one of these pathways) may reduce inflammation associated with inflammatory diseases and disorders, thereby alleviating symptom severity and / or slowing, halting, or reversing disease progression. The applicant has surprisingly found that the devices (e.g., systems, instruments, etc.) and methods described herein can be used to stimulate the vagus nerve to reduce demyelination and / or increase or promote myelin regeneration. Furthermore, while modulating inflammation using the VNS is thought to involve the afferent pathway, myelin regeneration and demyelination may involve the efferent pathway or both the afferent and efferent pathways.

[0070] Diseases of the VNS that may benefit from the methods and devices described herein (e.g., diseases and disorders of myelination) include, but are not limited to, multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), chronic inflammatory demyelinating polyneuropathy (CIDP), and Barton's disease. Other neuroinflammatory disorders may include acute disseminated encephalomyelitis (ADEM), acute optic neuritis (AON), transverse myelitis, and neuromyelitis optica (NMO). Neuropathies that may benefit from the VNS include peripheral neuropathy, cranial neuropathy, and autonomic neuropathy. Therefore, any methods and devices described herein are applicable to (and suitable for) treating any of these diseases and neuropathies.

[0071] Vagus nerve stimulation systems and devices

[0072] In some variations, the device described herein is an implantable electrical stimulation device that can be activated to apply a current for a specified duration, followed by a period of time during which stimulation is not applied. As illustrated in the following examples, the stimulation protocol may include a very limited stimulation period (e.g., conduction time less than 5 minutes, 2 minutes, 1 minute, etc.), followed by a long off-duration period (e.g., greater than 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 12 hours, greater than 20 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, etc.) during which no stimulation is applied and may be protected from stimulation. The applied energy can be electrical energy, which is a fixed current with a frequency ranging from about 0.5 mA to 5 mA (e.g., about 2 mA), between about 1 Hz and about 1000 Hz (e.g., between 1 Hz and 100 Hz, 1 Hz and 30 Hz, 10 Hz and 200 Hz, etc.), wherein the applied pulse has a pulse width of about (50-500 µsec, e.g., a 200 µsec pulse). Therefore, the duty cycle of the applied current can be extremely low, and the duty cycle can refer to the ratio of on time to (on time plus off time). Stimulation is applied with an extremely low duty cycle, where the duty cycle can refer to the percentage of the on time of the ongoing treatment to the total on time and off time. For example, a low duty cycle can be less than about 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the total on time and off time. The effect may appear relatively quickly and may persist throughout the off time.

[0073] In particular, the methods and apparatus described herein may be applied as needed, for example, when a patient exhibits or may exhibit an increased risk of demyelination and / or is experiencing (or has experienced) demyelination. Alternatively or additionally, the methods and apparatus may be applied as needed when a patient exhibits or may exhibit and / or is experiencing (or has experienced) leakage across the blood-brain barrier.

[0074] For example, we demonstrate in this paper that low-level, low-duty-cycle stimulation protocols (as described herein) reduce demyelination and / or increase myelination, and prevent and / or reduce leakage across the blood-brain barrier. Even with low-level, low-duty-cycle vagal nerve stimulation (VNS therapy) administered within a single day, its effectiveness results in reduced demyelination and myelination that can be seen over a two- to three-week course. This type of stimulation contrasts with the use of high-duty-cycle stimulation, which is used to modulate vagal nerve-mediated functions (e.g., heart rate) or manage disorders (e.g., epilepsy and depression). A key finding here is the reduction in demyelination, and even more surprisingly, the increase in myelination. This effect was confirmed by examining the histology of the spinal cord under these low-duty-cycle parameters, as described below. While low-duty-cycle vagal nerve stimulation is effective and efficient in reducing inflammation, in some embodiments, higher duty-cycle stimulation can be used, for example, with a duty cycle greater than approximately 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50% of the total conduction and deactivation times.

[0075] MS patients may experience symptoms of circadian rhythm disturbances, which may be related to or partially caused by the circadian rhythm pattern of IL-6 levels. Optionally, medications, such as steroids, can be used in conjunction with VNS to suppress the nocturnal peak of IL-6. Similarly, VNS can be modulated by altering the timing of stimulation, for example, to more effectively suppress the nocturnal peak of IL-6. However, one advantage of VNS is the relatively long duration of action after a single stimulation, which may allow for suppression of IL-6 levels both at night and during the day, potentially eliminating the need for supplemental drug treatment or alternative timing. In some embodiments, VNS can be administered at bedtime, such as 15, 30, 45, 60, 90, 120, 150, or 180 minutes before bedtime, or during sleep at night, to ensure nocturnal suppression of IL-6 levels. In some embodiments, the stimulation amplitude during sleep can be reduced (e.g., less than 2, 1.5, or 1 mA) to avoid waking the patient. In some embodiments, IL-6 levels can be measured and / or monitored, and VNS can be modulated based on the measured and / or monitored IL-6 levels. Other cytokines, such as IL-1, TNF, IFN-γ, IL-12, IL-18, and GM-CSF, can also be measured and / or monitored. These other cytokines can be used in combination or alone to replace IL-6 or in addition to IL-6.

[0076] The methods, devices, and systems described herein are particularly well-suited for treating any impairment where reduced demyelination and / or increased myelination would be beneficial. For example, electrodes (e.g., cannulated electrodes, microstimulators) are described herein that can be placed around the vagus nerve and can communicate with one or more stimulators configured to apply appropriate vagal nerve stimulation to modulate demyelination and / or myelination. The stimulators can be implanted. In some variations, the stimulator is integrated with the electrode and can be externally charged. The extremely low duty cycle of the techniques described herein allows for device miniaturization, making them smaller than previously suspected devices for treating chronic impairments via implantable devices.

[0077] Typically, devices or systems for modulating demyelination and / or myelination regeneration may include stimulator elements (e.g., electrodes, actuators, etc.) and controllers for controlling the stimulation applied by the stimulator elements. Stimulator elements may be configured for electrical stimulation (e.g., electrodes, such as cannulated electrodes, needle electrodes, spatula electrodes, non-contact electrodes, arrays or multiple electrodes, etc.), mechanical stimulation (e.g., mechanical actuators, such as piezoelectric actuators, etc.), ultrasonic actuators, thermal actuators, etc. In some variations, the system and / or device is implantable. In some variations, the system and / or device is non-invasive. Typically, the controller may include control logic (hardware, software, firmware, etc.) to control the activation and / or intensity of the stimulator element. The controller may control timing (e.g., on-time, off-time, stimulation duration, stimulation frequency, etc.). In variations where the applied energy is electrical energy, the controller may control the applied waveform (amplitude, frequency, burst duration / duration between bursts, etc.). Other components may also be included as part of any of these devices or systems, such as power sources (e.g., batteries, inductors, capacitors, etc.), transmitting / receiving elements (e.g., antennas, encoders / decoders, etc.), signal generators (e.g., for modulating or shaping the applied signal waveform), etc. In some embodiments, a rechargeable battery that can be inductively charged allows the stimulator to deliver a large amount of electrical stimulation before needing to be recharged. In other embodiments, one or more capacitors that can also be inductively charged may be used to store a limited amount of energy, which may be sufficient to deliver a single stimulus or a daily stimulation dose. This greatly reduces the size and cost of the stimulator, but requires the user to charge the stimulator daily or before each use.

[0078] In one example, an implantable device for modulating demyelination and / or myelin regeneration (and / or reducing or preventing leakage across the blood-brain barrier) includes electrodes for electrically stimulating the vagus nerve. For example, the electrodes may be cannulated electrodes. The electrodes may be connected (directly or via connectors) to a controller and a signal generator. The signal generator may be configured to provide an electrical signal to one or more electrodes. For example, the electrical signal may be an electrical waveform with a frequency between about 0.1 Hz and about 1 kHz (e.g., 10 Hz), wherein the applied pulse has a pulse width of about (50-500 µsc, e.g., a 200 µsc pulse). The signal generator may be battery-powered (and / or inductively powered), and the electrical signal may be amplitude- and / or voltage-controlled. For example, in some variations, the device or system may be configured to apply a current between about 0.05 mA and 25 mA (e.g., about 0.5 mA, 1 mA, 2 mA, 3 mA, etc.). The electrical signal may be sinusoidal, square wave, random, etc., and may be charge-balanced. Typically, a controller (which can be embodied in a microcontroller, such as a programmable ASIC) can regulate the on and off states of the stimulus. For example, the on-time of the applied stimulus can range from approximately 0.1 seconds to 10 minutes (e.g., between 1 second and 5 minutes, between 1 second and 2 minutes, approximately 1 minute, etc.); the stimulus can be configured to repeat automatically every x hours or days, for example, every other day (approximately 48 hours off-time), once a day (e.g., approximately 24 hours off-time), twice a day (approximately 12 hours off-time), three times a day (approximately 8 hours off-time), four times a day (approximately 6 hours off-time), etc. In some variations, the implant can be configured to receive control information from a communication device. The communication device can allow modification of stimulus parameters (including off-time, on-time, waveform characteristics, etc.). The communication device can be worn, such as a collar around the neck or a handheld device.

[0079] In use, the implant can be configured to be inserted such that the electrodes contact or approach the vagus nerve or a portion thereof. In one variant, the implant includes a sheath at least partially surrounding the vagus nerve (e.g., near the carotid artery region). A controller and / or signal generator (including any power source) can be formed as part of the sheath or connected via a connector (e.g., a wire).

[0080] In some variations, the device can be non-invasive. For example, the device can be worn externally and can trigger stimulation of the vagus nerve from external sites such as the ear, neck, torso, etc. Non-invasive devices can include mechanical devices (e.g., configured to apply vibrational energy). In some variations, the device is configured to apply ultrasound that can specifically target the vagus nerve and apply energy to activate the vagus nerve. In some variations, transcutaneous magnetic stimulation of the vagus nerve can be used.

[0081] In any of the variations described herein, devices, systems, and methods may be configured to prevent signal desensitization in a manner that reduces or inhibits modulation of demyelination and / or myelination regeneration. For example, in some variations, “overstimulation” of the vagus nerve, such as excessive intensity or duration of application, or simulation outside the frequency range described herein, may lead to desensitization of the effect, thus limiting or inhibiting further modulation. Therefore, in some embodiments, the stimulation amplitude may be limited to not exceeding (i.e., less than) about 3 mA, 4 mA, or 5 mA, and / or the duty cycle may be limited to not exceeding about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25%. In some embodiments, the amplitude is also at least 0.25 mA, 0.5 mA, 0.75 mA, or 1.0 mA.

[0082] The examples above provide an in-depth understanding of devices, systems, and methods for stimulating the vagus nerve to modulate demyelination and / or myelination regeneration. These methods and devices can be used to treat any indication where modulation of demyelination and / or myelination regeneration would be beneficial. Non-limiting examples of indications include neurodegenerative and neuroinflammatory diseases such as multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and Barton's disease. Other examples include peripheral neuropathy, cranial neuropathy, and autonomic neuropathy. In general, these devices can provide alternative and, in some respects, better treatments than pharmacological interventions designed to modulate demyelination and / or myelination regeneration, and therefore can be used for any indication for which such pharmacological treatment is recommended or indicated. In some embodiments, the VNS treatments described herein can be used in combination with pharmacological treatments, particularly when the pharmacological treatments have a different mechanism of action than the VNS, which may result in synergistic outcomes.

[0083] Therefore, the methods for modulating demyelination and / or myelination regeneration as described herein can be used in combination with one or more pharmacological interventions, particularly those treating diseases associated with demyelination, neurodegeneration, or neuroinflammation. For example, when treating subjects receiving vagal nerve stimulation to modulate demyelination and / or myelination regeneration, it may also be beneficial to provide agents such as: intravenous corticosteroids (e.g., methylprednisolone), oral corticosteroids, interferon β-1a and β-1b, monoclonal antibodies (e.g., natezumab, alenzab, dazumab, and oligrizumab), and immunomodulators (e.g., glatiramer acetate, mitoxantrone, fingolimod, teriflunomide, and dimethyl fumarate).

[0084] Therefore, this document describes devices (VNS devices) for treating neurodegenerative and / or neuroinflammatory disorders. Such devices are typically configured to apply low-duty-cycle stimulation to the vagus nerve of a subject, as described in any variant (or sub-combination) of these variations. In some embodiments, prior to implantation of the VNS device and treatment, the patient is first diagnosed or identified as having a neurodegenerative and / or neuroinflammatory disorder, particularly characterized by demyelination or a condition requiring myelination.

[0085] In use, any of the methods described herein may include steps for monitoring demyelinating or demyelinating-related disorders, which can be determined by detecting biomarkers from blood and / or cerebrospinal fluid and / or by medical imaging techniques such as MRI or CT scans. For example, the determination of inflammatory cytokines (e.g., tumor necrosis factor) can be used to detect acute inflammatory episodes. Monitoring can be continuous or discrete (e.g., once or multiple times, or at time intervals). Additionally or alternatively, depending on the disease being treated, biomarkers associated with multiple sclerosis or other neurodegenerative diseases and / or neuroinflammatory diseases or neuropathy may be used for monitoring. See Housley, WJ, D. Pitt, and DA Hafler (2015). "Biomarkers in multiple sclerosis." Clin Immunol 161 (1): 51-58; and Katsavos, S., and M. Anagnostouli (2013). "Biomarkers in Multiple Sclerosis: An Up-to-Date Overview." Mult Scler Int 2013: 340508. For example, biomarkers found in serum and cerebrospinal fluid in MS include markers of neurodegeneration, including neurofilaments and GFAP, glial fibrillary acidic protein, the monocyte / macrophage marker CD163, the glial cell activation marker YKL-40, the B cell chemokine CXCL13, miRNAs and mRNAs, myelin-responsive T cells, Kir4.1 antibody, osteopontin, and microbiome-associated lipopeptides. Any of these biomarkers can be monitored and / or measured individually or in combination, and can be used as feedback for regulating VNS. Other biomarkers for treating MS patients are listed in Table 1.

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Table 1. Biomarkers in Multiple Sclerosis

[0097] The information described for the first time herein illustrates how stimulation of the vagus nerve modulates demyelination and / or myelination regeneration and / or leakage across the blood-brain barrier. The examples provided herein are not intended to be exhaustive, but are merely illustrative and reflective of certain variations of the invention. Those skilled in the art will understand and apply the invention as described herein without requiring extensive experimentation.

[0098] Example 1

[0099] To investigate the effects of VNS on neurodegeneration and neuroinflammation, a lysophosphatidylcholine (LPC)-induced MS model can be used. Lysophosphatidylcholine is a bioactive pro-inflammatory lipid and a detergent-like membrane solubilizer. A 1% LPC solution can be injected into the spinal cord white matter to induce localized demyelinating lesions. Four distinct phases were observed 14 days after injection: (1) demyelination; (2) oligodendrocyte precursor (OPC) recruitment; (3) differentiation; and (4) myelin regeneration. Figure 1 The illustrations illustrate typical examples of four phases, in which demyelination occurs at approximately 0–3 days, OPC recruitment at approximately 3–7 days, OPC differentiation at approximately 7–10 days, and myelin regeneration at approximately 10–14 days.

[0100] To induce self-limiting demyelinating lesions, 1% LPC (0.5 µL, 0.25 µL / min) was injected into the spinal cord of female BALB / c mice between T3 and T5. The procedure for injecting LPC into the mice was as follows: The mice were anesthetized and fixed in a stereotactic frame. A midline incision was made between the scapulae. The underlying fat pad was directly dissected, and the spinous process of the T2 vertebra was identified and a laminectomy was performed. The syringe was advanced 0.3 mm into the spinal cord, and 0.5 µL of LPC was injected at a rate of 0.250 µL / min for 2 minutes. The muscle and adipose tissue were sutured, and the skin was sutured with a surgical U-shaped stapler.

[0101] VNS was performed on day 0 or day 4 after LPC induction, as previously described (Olofsson, Levine et al., 2015. Bioelectronic Medicine: 37-42). More specifically, to investigate the effect of VNS on demyelination, VNS (0.75-1 mA, 250 µS pulse, 10 Hz) or sham-operated (0 mA) was performed immediately after LPC administration, and mice were euthanized on the day of the expected peak lesion volume (day 4 after induction; J Neurocytol 24(10): 775-81). The demyelination experimental protocol is summarized in Figure 2A middle.

[0102] The volume / area of ​​spinal cord lesions was quantified by myelin loss, as assessed from 15µm serial sections stained with Luke blue. Figure 3A An illustration of a typical cross-section of the spinal cord is shown, and Figure 3B This image shows a cross-section of the spinal cord with LPC-induced lesions in the anterior column of the white matter, 5 days after LPC injection, stained with Luk's blue. To investigate the effect of VNS on myelin regeneration, mice were treated with VNS or sham VNS 4 days post-induction, euthanized on days 8, 14, or 21 post-induction, and the nerves were treated as described above. The experimental protocol for myelin regeneration is summarized in... Figure 2B The mean lesion volume between groups was compared using a t-test.

[0103] result: Figure 2A The demyelinating protocol shown indicates that VNS inhibited the progression of demyelinating lesions compared to the sham-operated group. Mice were euthanized on day 4 post-induction, and spinal cord sections around the LPC injection site were prepared and stained with Lukl blue. Figures 4A-4D As shown, the mean lesion volume in the VNS group (0.75 mA) was significantly lower than that in the sham surgery group (p = 0.0023 by t-test). A VNS of 0.25 mA resulted in a mean lesion volume similar to that in the sham surgery group.

[0104] Figure 2B The myelin regeneration protocol shown illustrates that myelin regeneration occurs at a significantly accelerated rate in the VNS group. As... Figure 5A-5G As shown, the mean lesion volume decreased in the VNS group on day 8 post-induction. On day 14 post-induction, the mean lesion volume in the VNS group was significantly lower than that in the sham-operated group. On day 14, 11 out of 12 VNS animals had no detectable lesions. By day 21, the mean lesion volume in the sham-operated group had almost returned to baseline. Figure 5A The area under the curve (AUC) between day 4 and day 21 was shown to have decreased by approximately 65% ​​under vagal nerve stimulation.

[0105] Conclusion: VNS reduced demyelination and accelerated myelin regeneration, demonstrating a significant effect after a single dose in this model. Repeated stimulation of the vagus nerve using an implanted neurostimulator can further reduce the rate of demyelination and / or further accelerate myelin regeneration. This will be tested in an experimental autoimmune encephalomyelitis model to further evaluate the potential of VNS in treating MS.

[0106] Example 2

[0107] Another study was conducted to determine the effect of VNS on vascular leakage 24 hours after induction and stimulation. The lesion was induced using LPC injection as described above, and VNS was performed immediately after induction. At 24 hours, 0.15 mL of 1% Evans blue dye was administered intravenously via retro-orbital injection 1 hour after anesthesia. Figure 6A As shown. One hour later, the animals were euthanized by cervical dislocation. Extravasation in the spinal cord (SC) was measured by extracting and weighing the wet SC. The SC was then dried at 56°C for 24 hours and weighed. Evans blue dye was extracted using formamide solvent at 56°C for 48 hours. The supernatant was measured by spectrophotometry at 620 nm, and the amount of Evans blue dye was determined by interpolation using a reference curve. The amount of Evans blue dye was normalized to the dry weight of the SC. Figure 6B As shown, less Evans blue dye was extracted from the spinal cord of mice receiving VNS, providing evidence that VNS reduces vascular leakage 24 hours after induction and stimulation. Furthermore, the amount of Evans blue dye extracted from mice receiving VNS was similar to that extracted from native mice (without LPC-induced lesions).

[0108] Leakage of the blood-brain barrier can allow immune cells, inflammatory cytokines, and chemokines to pass through and lead to persistent inflammation in the brain and / or spinal cord. Therefore, the VNS can reduce vascular leakage around the central nervous system (CNS), thereby reducing the recruitment of pro-inflammatory cells (such as lymphocytes (e.g., T cells)) and macrophages to the brain and spinal cord, thus reducing inflammation in the CNS and the amount of demyelination caused by the inflammatory attack of the immune system.

[0109] Example 3

[0110] Devices and methods typically used in VNS therapy can also be used to prevent or treat increased leakage across the blood-brain barrier, such as... Figure 6B As shown.

[0111] Methods: 1% LPC was injected into the spinal white matter of BALB / c mice. For the first intervention time point, VNS therapy or sham-operated treatment was administered immediately after injection. Twenty-four hours later, mice (VNS, sham-operated, and native mice (without LPC)) were injected with 1% Evans blue dye, which binds to albumin in the blood and is allowed to circulate for 1 hour. The spinal cord was then harvested and dried in pre-weighed tubes at 60°C for 24 hours. The dried tissue was then incubated in formamide for 48 hours. The supernatant was then extracted from the tubes and spectrally read at 620 nm. For the second intervention time point, VNS therapy or sham-operated treatment occurred on day 4 after LPC induction. On day 5 after LPC induction, Evans blue extravasation was performed in the same manner as described in the demyelination experiment. Evans blue concentrations (ng / mg tissue) were compared to native mice and normalized.

[0112] Results: LPC increased hematospinal extravasation. Compared with the sham-operated group 24 hours after LPC induction, VNS therapy significantly reduced Evans blue extravasation into the spinal cord (by 81%). Figure 6B Furthermore, compared to the sham surgery group, VNS therapy performed on day 4 after LPC significantly reduced Evans blue extravasation on day 5 (by 52%).

[0113] Conclusion: VNS therapy increases the integrity of the blood-spinal barrier and thus reduces the extravasation of proteins / Evans blue and other circulating substances (including antibodies, DAMPS / PAMPS and immune cells) into the central nervous system.

[0114] Example 4

[0115] Another experiment was conducted to determine whether the effect of VNS on demyelination depends on the α7 nicotinic acetylcholine receptor (nAChR). Two mouse strains were used in the study. One strain was C57 Black subtype 6 (C57BL / 6), a common wild-type strain that expresses the α7 receptor and is indicated as α7+ / +. The second strain was an α7 knockout strain of the C57BL / 6 strain that lacks the α7 receptor and is indicated as α7- / -. Each mouse strain was injected with LPC in both the sham-operated group (without VNS) and the VNS group. Tissue extraction was performed 4 days post-injection. This procedure was essentially the same as the Balb / c mouse demyelination experiment described in Example 1 above.

[0116] like Figure 7AAs shown, the protective effect of VNS against demyelination is dependent on α7 nAChR. Compared with the sham-operated group, VNS treatment with α7 nAChR in mice showed a reduction in lesion volume, while VNS treatment in mice without α7 nAChR showed no reduction in lesion volume compared with the sham-operated group. Similarly, the myelin regeneration effect of VNS treatment may be dependent on α7 nAChR, as... Figure 7B As shown. In this example, the effect of VNS treatment on myelin regeneration in the presence (+ / +) and absence (- / -) of α7 nAChR, due to either sham surgery (without VNS treatment) or VNS treatment, was examined, showing a significant reduction in lesion volume, a marker of myelin regeneration following induced demyelination events (e.g., application of LPC).

[0117] exist Figures 7A-7B In this study, 1% LPC was injected into the spinal white matter of α7 nAChR knockout mice and C57BL / 6 (wild-type) mice. For the demyelination experiment, VNS treatment or sham VNS treatment, tissue collection, processing, and analysis were the same as described above. Figure 4A The same as mentioned above. Regarding myelin regeneration, the occurrence of interventions in the VNS and sham-operated groups was similar to... Figure 4B The experiment was the same as described in [the previous section]. Spinal cord was harvested only on day 8 after LPC induction. The procedures and analyses performed were the same as [previous section]. Figures 4A-4B The same as described in [the original text].

[0118] Results: VNS therapy reduced demyelination in wild-type C57BL / 6 mice. VNS therapy did not reduce demyelination in α7 KO animals. Figure 7A VNS therapy increased myelin regeneration in wild-type animals but not in knockout animals. Figure 7B Therefore, the effects of VNS on demyelination and myelin regeneration are dependent on α7.

[0119] Example 5

[0120] Generally, the devices and methods used for VNS therapy can also be used to prevent or treat the homing of increased immune cells to the central nervous system, such as... Figure 8 and Figure 9 As shown.

[0121] exist Figure 8 and Figure 9 In the VNS treatment group, CD3+ T cell infiltration across the blood-brain barrier model was significantly reduced. Figure 8 As shown, compared with the sham-operated group, CD3+ T cell infiltration across the blood-brain barrier was reduced by 50% on day 3 after LPC induction. Figure 9In the study, compared with the sham-operated group (without VNS treatment), the macrophage infiltration was significantly reduced by 55% 24 hours after LPC induction in the VNS-treated group.

[0122] Methods: Surgical procedures and VNS / dummy VNS treatment and Figure 4A Same procedure. Spinal cord from VNS-treated, sham-operated, and native mice was harvested on day 1 or 3 after LPC induction. The tissue was then dissolved in an enzyme mixture at 37°C for 20 minutes, followed by milling and filtration through a 100 μM sieve. The single-cell suspension was then passed through a density gradient to remove myelin fragments from glial cells and immune cells. After separation, the cells were blocked in FACS buffer and CD32 / CD19 for 30 minutes to prevent non-specific antibody staining. Cells were counted and their viability was examined using a hemocytometer. The cells were then placed in test tubes for staining with T cells (CD3+) or macrophages (CD11b+, CD45hi) and then analyzed by flow cytometry. Cell population was quantified using the FlowJo program.

[0123] Results: Compared with native tissue, LPC increased CD3+ T cell and macrophage infiltration in the spinal cord. Figure 8 and Figure 9 Compared with the sham-operated group, animals treated with VNS therapy showed a significant reduction (50%) in CD3+ T cell infiltration on day 3 after LPC induction. Figure 9 Furthermore, compared to the sham-operated group 1 day after LPC induction, VNS therapy resulted in a significant reduction (55%) in macrophage infiltration. Figure 9 Therefore, in this lysophosphatidylcholine-induced MS model, the VNS significantly reduced the infiltration of peripheral immune cells into the CNS.

[0124] like Figures 10A-10B As shown, VNS therapy also enhanced myelin regeneration following reduced myelination. Blood was also collected via cardiac puncture during spinal cord extraction in all previous experiments (see Examples 1-4 above). Blood was centrifuged at 8,000 x g for 5 minutes, serum was collected, and stored at -80°C. RvD1 levels were measured spectrally from the serum of VNS and sham-operated mice using the Resolvin D1 ELISA kit for demyelination (D4 harvest) and myelin regeneration (D8, D14, and D21 harvest) experiments. RvD1 levels (pg / mL) were analyzed and expressed as a percentage per day relative to the sham-operated group.

[0125] Result: As Figure 10A As shown, VNS therapy on day 0 (LPC induction) increased serum RvD1 levels on day 4. Figure 10BAs shown, VNS also showed increased serum RvD1 levels on day 4 after LPC induction, with the highest concentration occurring on day 14 after LPC induction. On day 21 after LPC induction, serum RvD1 levels in VNS were lower compared to the sham-operated group, possibly due to earlier regression in the VNS group.

[0126] Therefore, compared with the sham surgery group, VNS therapy increased the serum regression lipid mediator RvD1, which may help increase the speed of LPC-induced lesion regression.

[0127] Example: System

[0128] Figure 11 An example of a system 1100 for treating demyelination (e.g., for treating MS or any other demyelinating disorder) is schematically illustrated. In some variations, the system for reducing demyelination and / or increasing myelin regeneration by stimulating the vagus nerve includes a controller 1103, a stimulator 1105, and a pulse generator 1101. The pulse generator and stimulator may be connected to and controlled by the controller. In some variations, all or part of the system may be implanted in a patient. All or part of the device may be enclosed in a housing (e.g., an implant housing). Generally, the system may also include one or more biosensors 1107 configured to detect one or more biomarkers. The biosensors may be coupled to the rest of the system (e.g., the implant), or they may be standalone and communicate via wired or wireless connections. For example, biosensors may be implanted in the body to sample blood, cerebrospinal fluid, etc.; in some variations, the biosensors are located outside the body and may be for single use or configured for limited reusability. In some variations, the biosensors may include sensors for determining the patient's physical condition (e.g., body temperature, nerve conduction, etc.). In some variations, a biosensor can be an immunochemical sensor configured to detect the binding of one or more analytes and / or to provide concentration.

[0129] The stimulator can be configured to apply stimulation to the vagus nerve. The stimulator can be configured for electrical stimulation, mechanical stimulation, or both. For example, the stimulator may include a pulse generator 1101 (e.g., a waveform and / or pulse generator, oscillator, etc.) or coupled to a pulse generator 1101. The stimulator may include one or more stimulation applicators 1121 (e.g., one or more electrodes, mechanical transducers, etc.) for contacting tissues, including the vagus nerve.

[0130] Any device may also include one or more power supplies 1115 and / or power regulation circuits, etc.

[0131] The controller is typically functionally coupled to one or more biosensors (e.g., receiving data from one or more biosensors) and controls the stimulator, and can be configured to apply stimulation from the stimulator to the vagus nerve sufficient to reduce demyelination and / or increase myelin regeneration of the nerve in the patient when the biosensor detects a biomarker indicating demyelination (including detecting active demyelination or a marker indicating impending active demyelination).

[0132] For example, the system may include an implant comprising a stimulator (e.g., a waveform and / or pulse generator, oscillator, power supply and / or power regulation circuitry, etc.), a stimulation applicator (e.g., one or more electrodes, a mechanical transducer, etc.), and a controller. The controller may be configured as a microcontroller and may communicate electrically with the stimulator to control its operation. The controller may include one or more processors, a memory, and / or a timer. The stimulator and / or controller may communicate electrically with one or more stimulation applicators. In some variations, the controller may include or communicate with wireless communication circuitry 1117 for wireless communication with one or more remote processors 1131. The remote processor may be a handheld device (e.g., a smartphone, a wearable electronic device, etc.). The controller may optionally communicate with one or more biosensors, which may be included in the implant or can be located remotely from the implant (e.g., wearable, disposable, etc.). In some variations, the biosensors are wirelessly connected to the device.

[0133] Electronic devices (e.g., smartphones, wearable electronic devices, etc.) that communicate with a controller can be configured to receive input from a user and / or sense one or more biomarkers for triggering changes in electrical stimulation parameters. For example, a user may experience an episodic attack or relapse, where the user experiences an onset or worsening of symptoms (e.g., pain, muscle spasms or stiffness and / or fatigue). The electronic device can be configured to receive input from the user indicating that they are experiencing an episodic attack, which can cause the controller to adjust the stimulation parameters accordingly (e.g., increase or decrease frequency and / or current). Similarly, the electronic device can be configured to receive input from the user indicating that the user is recovering from an episodic attack (where the user experiences a reduction or disappearance of symptoms), which can cause the controller to adjust the stimulation accordingly (e.g., increase or decrease frequency and / or current). In some cases, the electronic device is configured to receive a score or rating indicating the severity of the episodic attack, and the controller can adjust the stimulation parameters based on the score or rating. Alternatively or additionally, the electronic device can be configured to sense one or more biomarkers indicating the onset of an episodic attack. In some embodiments, the electronic device is configured to non-invasively detect multiple biomarkers, such as using multiple renal biomarkers and / or optic nerve biomarkers. In some embodiments, the electronic device is additionally or alternatively configured to detect multiple biomarkers from a fluid sample. The electronic device may be configured to detect the severity of an attack based, for example, measurements of the sensed multiple biomarkers. The controller may be configured to (e.g., automatically) adjust stimulation parameters based on detected multiple biomarkers indicating the onset and / or attenuation of an attack and / or based on the severity of the attack.

[0134] Increased removal

[0135] The methods and apparatus described above describe the treatment of multiple sclerosis (MS). More specifically, the methods and apparatus described herein can be modified to increase the clearance of cellular debris that may be associated with MS and other neurodegenerative disorders and / or neuroinflammatory disorders, as well as acute neuronal injury. Thus, in some variations, the methods and apparatus described herein can increase the clearance of neuronal cellular debris by applying a charge to a vagal nerve target. Applying a charge to a vagal nerve target can enhance the clearance of cellular debris by modulating the activity of one or more microglia and / or other macrophages, particularly in patients with multiple sclerosis.

[0136] Endothelial cells lining small blood vessels may facilitate the clearance of myelin debris, a common and detrimental consequence of demyelinating diseases such as multiple sclerosis (MS). Activity may also decrease due to age or other disease states. In healthy subjects, macrophages / microglia may engulf myelin debris and may otherwise perform homeostatic activity in the normal CNS, a function associated with their highly motile branching processes and continuous phagocytic clearance of cellular debris. In some cases, including in MS, microglia recruitment or activation for clearing myelin debris may be reduced. The inadequate clearance by microglia, prevalent in several neurodegenerative and / or neuroinflammatory diseases and declining with age, is associated with insufficient regenerative responses. The methods and devices described herein can enhance the activity of microvascular endothelial cells and / or macrophages and / or microglia. Specifically, applying vagus nerve stimulation (VNS) within defined value ranges may result in increased endothelial-mediated clearance of tissue debris and / or macrophage / microglia-mediated clearance of tissue debris.

[0137] For example, this article describes a method for treating neurodegenerative disorders and / or neuroinflammatory disorders and / or acute CNS injury by increasing the activity of microglia and / or other macrophages over repetitive and / or sustained time periods. This can be achieved by applying vagus nerve stimulation within a charge range of approximately 2.5 nC / day (e.g., approximately 0.1 mA and 0.1 msec pulses – using the VNS) to approximately 7.5 mC / day (e.g., in the nanocoulomb to microcoulomb range). Pulses can be applied between 0.1 and 50 Hz (e.g., between 1 and 20 Hz, etc.). The charge can be delivered directly, e.g., via an implantable device, or indirectly, such as via a percutaneous delivery device. Outside these ranges (e.g., application below 2.5 nC / day) generally has little or no effect on most patients. Similarly, application greater than approximately 7.5 mC / day may have no additional effect and, in some cases, may lead to inhibition of the effect. Therefore, it may be beneficial to limit the daily charge application to between approximately 2.5 nC and 7.5 mC per day (e.g., between approximately 5 nC and 7 mC, between approximately 10 nC and approximately 6.5 mC, between approximately 50 nC and approximately 6 mC, between approximately 100 nC and approximately 6 mC, etc.).

[0138] The applicant has found that applying VNS within the said charge range increases the clearance of debris. For example, images of neuronal tissue from animals treated within the effective charge range (e.g., adjusted according to animal size and stimulation location) stained for myelin debris show increased intracellular breakdown products, indicating increased myelin debris uptake after stimulation. This increase can persist for a period of time after stimulation (e.g., hours and days). Surprisingly, this effect is also seen in older animals (e.g., other normal mice) compared to controls, showing enhancement compared to controls that exhibit a normal aging effect that reduces myelin clearance.

[0139] Any of the methods described herein can target endothelial cell and / or microglia and / or macrophage activity. For example, in some variations, the methods and / or devices may include examining one or more biomarkers for endothelial cell and / or macrophage and / or microglia activity. These biomarkers may be particularly useful for more precise targeting of treatment, whether used alone with VNS treatment or in combination with one or more compositions for treating disorders involving demyelinating neurons, such as (but not limited to) MS.

[0140] Figure 14 and Figure 15 The illustration shows an increase in regenerated myelin fragments following VNS within the effective dose range described herein. Generally, tissue regression of damage may require the clearance of damaged cells and debris. In particular, in MS, the process of myelin regeneration may heavily depend on the clearance of damaged cells and myelin fragments by endothelial, microglia, and macrophages (e.g., burial and phagocytosis) before oligodendrocytes can place new myelin on damaged axons. In MS, cells responsible for clearing cellular and extracellular debris may have become senescent and inefficient in general endocytosis; similar effects may be seen in older adults and aged animals, leading to delayed healing and rapid progression of disease (including, but not limited to, MS).

[0141] Electrical stimulation of the vagus nerve (e.g., VNS) can increase the rate of phagocytosis and necrolysis, thereby increasing the rate of disease regression, as demonstrated by myelin regeneration in rodent models of MS, as above and Figure 14 and Figure 15 This is discussed in [the literature]. Similar processes enable repair in the context of many other diseases. For example, electrical stimulation of the vagus nerve can induce more efficient general endocytosis in older rodents. This finding may extend to humans, where the vagus nerve can reverse cellular aging, including in older individuals, to improve repair rates and the regression of disease and damage, and potentially extend lifespan.

[0142] exist Figure 14In the first treatment paradigm (“Paradigm A”), data were obtained from an animal model (BALB / c mice) in which 1% lysophosphatidylcholine was injected into the spinal cord white matter (e.g., 0.5 uL, 0.25 uL / min), and either VNS (0.75 mA, 10 Hz in this example) or sham surgery was performed immediately after injection. Animals were euthanized on day 4 post-induction, and the intact spinal cord was harvested. In the second treatment paradigm (“Paradigm B”), similar mice were treated with VNS (0.75 mA, 10 Hz) or underwent sham surgery on day 4 post-induction. Animals were euthanized on days 5, 8, 10, or 14 post-induction, and the intact spinal cord was harvested.

[0143] Spinal cord sections were cut into 20 μM sections and stained with Oil Red O. Oil Red O indicates the presence of fat or lipids in fresh and frozen tissue sections and is primarily used to measure lipid accumulation. Oil Red O is a lipid-soluble diazo dye that can also be used as an oil-soluble staining agent (it produces a strong red-orange color when staining droplets). Oil Red O staining of myelin degradation products serves as a marker of phagocytosis. The stained area within the lesion was quantified as a percentage of the lesion.

[0144] like Figure 14 and Figure 15 As shown, when the stimulation is within the range of treatments described herein, vagal nerve stimulation (VNS) significantly increases myelin fragment uptake. Figure 14 In the study, compared with the sham-operated control group, the area of ​​positive staining increased, indicating that phagocytosis in this neural tissue region increased more than twofold. Specifically, when observing animals treated according to treatment paradigm A, the percentage of Oil Red O staining as lesions in the VNS group increased by approximately 2.5 times on day 4 compared with the sham-operated group (p<0.05). Figure 15 The treatment timeline is shown when using treatment paradigm B. In this example, Oil Red O staining as a percentage of lesions increased by approximately 2.5-fold and 5.5-fold, respectively, in the VNS group on days 5 and 8 compared to the sham-operated group (p<0.05). Oil Red O staining was increased in the sham-operated group on day 14 compared to days 4 and 8. No detectable myelin uptake was observed in the VNS group because no detectable lesions were present.

[0145] Therefore, as Figure 14 and Figure 15 As demonstrated by Oil Red O staining, VNS significantly increased the uptake of myelin fragments by macrophages and microglia in this model. The accelerated myelin fragment uptake was evident compared to the sham-operated group. This can also be seen from... Figure 16-19 The raw data shown in the figure reveals representative O Red O staining patterns from the tissue. Figure 16 and Figure 18 A stained section of tissue from animals in the sham-operated group is shown. Figure 17 and Figure 19 The image shown is of an animal processed with VNS on day 4 in processing paradigm A, and the image after VNS ( Figure 17 ) or the image on day 8 after paradigm B induction ( Figure 19 ). Figure 16 and Figure 17 The boxed areas indicate lesions. Significantly more staining is present in the VNS-treated areas. Figure 18 and Figure 19 The illustration area at the bottom shows the analysis of the staining to quantify the intensity and extent.

[0146] Similar results were observed in aged mice, where myelin regeneration normally declines. For example, 1% lysophosphatidylcholine was injected into the spinal white matter of aged (19-month-old) C57Black6 mice using treatment paradigm B. For example, VNS (0.75 mA, 10 Hz) or sham-operated groups were performed on day 4 post-induction, and the animals were euthanized on day 10 post-induction, with intact spinal cord harvested. The spinal cord was sectioned into 20 μM sections, stained with Luke's Fast Blue or Oil Red O, and the stained area within lesions was quantified. Figure 20 and Figure 21 As observed, VNS accelerates myelin regeneration ( Figure 20 And enhanced myelin fragment clearance () Figure 21 ).like Figure 20 As shown, on day 10 post-induction, the lesion volume in the VNS group was reduced by 34% compared to the sham surgery group. Figure 21 As shown, on day 10, the percentage of lesions stained with Oil Red O staining was 2.4-fold higher in the VNS group compared to the sham-operated group (p<0.05). Therefore, in aged mice, as assessed by Oil Red O staining, VNS increases myelin sheath uptake by macrophages and microglia, and in the VNS group, induced myelin regeneration of lesions was significantly accelerated.

[0147] Example 6: Evidence of enhanced phagocytosis

[0148] As described in this article, tissue regression following inflammatory events typically requires the clearance of damaged cells and debris. In multiple sclerosis (MS), myelin regeneration is thought to be heavily dependent on the clearance of damaged cells and myelin debris by endothelial, microglia, and macrophages (cytotoxicity and phagocytosis) before oligodendrocytes can place new myelin on damaged axons. Cells responsible for clearing cellular and extracellular debris become senescent and inefficient in general endocytosis; in older adults and aged animals, this leads to delayed healing and rapid progression of the disease (including, but not limited to, MS).

[0149] Surprisingly, as this article demonstrates, electrical stimulation of the vagus nerve can increase the rate of phagocytosis, which in turn increases the rate of disease regression. Figure 22 The results of the in vitro uptake study demonstrate evidence of enhanced myelin uptake by macrophages in mice following VNS treatment. Mice were treated with either the VNS dose or sham VNS. Macrophages were harvested from the treated mice, and myelin uptake was quantified. Primary macrophages isolated from mice treated with VNS showed more efficient myelin uptake compared to the sham-operated group. Figure 22 As shown, macrophages from mice treated with VNS doses exhibited accelerated myelin fragment uptake compared to macrophages from sham-stimulated mice. These results indicate that electrical stimulation of the vagus nerve can induce more efficient general endocytosis in rodents. These results suggest that VNS intervention in humans can reverse cellular senescence, thereby increasing repair rates and the regression of disease and damage, and consequently extending lifespan. Similar processes may be crucial for enabling repair in the context of many other diseases.

[0150] Figure 22 The results were based on an in vitro uptake study using the following method: BALB / c mice (from the Charles River Laboratory) were acclimated for 7 days, anesthetized, and treated with either a VNS dose (60-second pulse sequence, 10 Hz frequency, 250 μS pulse width, 0.75 mA) or sham VNS. Four hours later, the mice were euthanized by CO2 asphyxia, and peritoneal macrophages were extracted. A suspension of peritoneal macrophages was cultured in 24-well plates containing glass coverslips and incubated overnight at 37°C and 5% CO2. Cells were washed with phosphate-buffered saline (PBS) and incubated with CFSE-labeled myelin (100 μg) in complete RPMI medium at 37°C for 10 minutes. Cells were washed with PBS and fixed with 4% PFA for 10 minutes. Fixed cells were washed with PBS and stained with DAPI for 30 minutes. Stained cells were washed with PBS, and coverslips were mounted with ProLong anti-fading mounting medium and imaged using a Zeiss ApoTome fluorescence microscope. The total number of macrophages in the field of view was counted using DAPI staining. Myelin uptake was also quantified using CFSE-positive cells. The percentage of phagocytes in the entire population was then calculated.

[0151] Example 7 EAE Model

[0152] Figure 23-25 The results of an experimental autoimmune encephalomyelitis (EAE) model study are presented, comparing low duty cycle VNS treatment with sham surgery. Figure 23 Clinical symptom scores in rats treated with chronic low-duty-cycle VNS versus sham VNS were compared. These results show that low-duty-cycle VNS can delay the onset of significant disease symptoms and eliminate disease severity. Figure 24 and Figure 25 The percentages of CD4+ T cells or CD8+ T cells in rat samples treated with acute VNS were compared. Results showed that a single dose of VNS at 2Hz or 10Hz prevented the entry of pathogenic CD4+ and CD8+IFNg+ Th1 cells. In MS, disease progression is causally linked to the infiltration of immune cells, including T helper 1 (Th1) cells. Therapies targeting the infiltration of these cells have been found to be effective and are clinically approved for the treatment of MS (e.g., nitazolizumab). As demonstrated in this data, low duty cycle stimulation of the vagus nerve essentially prevented the infiltration of Th1 cells (CD4+ and CD8+IFNg-producing cells).

[0153] Obtain using the following methods Figure 23 Results: Female Lewis rats (from Charles River Laboratories) were acclimatized for 7 days. A pulse generator (Rodent-MR, SetPoint Medical) was implanted in the rats, with the cannulated electrode positioned around the left cervical vagus nerve. EAE was induced by subcutaneous injection of 0.1 mL of a mixture of gpMBP (69-88) antigen and CFA emulsion (Cat#EK-3110, Hooker Laboratories, Massachusetts) on both sides of the lower back. Rats were weighed and clinical scores were monitored according to the Hooker Laboratories EAE scoring guidelines. 60 seconds of VNS (0.3 mA, 10 Hz QD) daily or sham-operated group was started on day 8 post-induction.

[0154] Obtain using the following methods Figure 24 and Figure 25Results: Female Lewis rats (from the Charles River Laboratories) were acclimatized for 7 days and induced for EAE by subcutaneous injection of 0.1 mL of a mixture of gpMBP (69-88) antigen and CFA emulsion (Cat#EK-3110, Hooker Laboratories, Massachusetts) into both sides of the lower back. One rat was held uninduced and untreated. Rats were weighed daily and clinical scores were monitored according to the Hooker Laboratories EAE scoring guidelines. On day 9 post-induction, rats underwent VNS (30 seconds, 0.3 mA, 2 Hz or 10 Hz) or sham surgery under anesthesia. Rats were euthanized by CO2 asphyxiation 24–48 hours later, and blood was collected directly via cardiac puncture. Spinal cord tissue was collected in chilled hibernation medium. Tissue was washed with PBS (1x) and then dissolved in neural basal medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C on a rotary shaker for 1 hour with 1 µg / ml collagenase / dispersin (Hoffmann-Roche, Germany). After dissolution, spinal cord tissue was washed with HBSS (Gibco™ HBSS), homogenized using a fire-polished glass Pasteur pipette (Thermo Fisher Scientific, Waltham, MA), and filtered through a 70 µm mesh filter. The cell suspension was plated on 15% BSA in HBSS and centrifuged at ×129 g for 20 min without interruption. The cell pellet containing infiltrating cells was resuspended in FACS buffer (1x PBS, 1% FBS in 0.5 mM EDTA) containing a CD32 mouse anti-rat (Fc block, 1:100) blocking antibody and incubated on ice for 30 min. On ice, cells were stained (external surface staining) for 30 minutes using eFluor 455UV Fix viable dyes in FACS blocking buffer; eBioscience (1:2000), CD45-PB; Biolegend (1:100), CD11b PE-Cy7 (1:100), CD3 APC-Cy7; Novus Biologicals (1:100), CD4Alexa Fluor 488; Bio-Rad (1:100), CD8 BUV805; BD Biosciences (1:100). Cells were washed with PBS and fixed on ice for 10 minutes with CYTO-PERM fixation buffer for intracellular staining for 30 minutes on ice using IFN-γ eFluor 660; Thermo Fisher Scientific (1:100). Cells were washed and rinsed with PBS (1X), resuspended in FACS buffer, and transferred to FACS tubes. The stained cells were analyzed using BD FACSymphony.Therefore, stimulating the vagus nerve prevents IFNg+ T cell infiltration into the central nervous system, delays disease symptoms, and eliminates disease severity in the EAE rodent model of MS.

[0155] Stimulation regimen - dosage

[0156] Return to Figure 12 and Figure 13 These figures illustrate data from animal (mouse) models, showing the range of applied dose parameters. Figure 12 and Figure 13 In this study, mice were treated with LPC in precise vertebral regions, and VNS was administered from implanted electrodes after allowing the development of demyelinating lesions. For example, mice were anesthetized and fixed in a stereotactic frame, and a midline incision was made between the scapulae to access the spinous processes of the T2-T5 vertebrae; 0.5 μL of LPC was injected into the spinal cord at a rate of 0.250 μL / min for 2 minutes, and the incision site was sutured. Unless otherwise specified, VNS was delivered on day 4 after LPC induction (when peak lesion size was expected), using a charge-balanced, biphasic square wave pulse with a pulse width of 200–250 μs for 60 seconds at 10 Hz. The lesion was quantified by the area of ​​myelin loss, as assessed by nuclear firm red counterstaining on Lukl blue-stained serial sections.

[0157] like Figure 12 As shown, applying VNS at frequencies less than approximately 100 Hz (e.g., 75 Hz or lower, 50 Hz or lower, 40 Hz or lower, 30 Hz or lower, 25 Hz or lower, etc.) is more effective than applying VNS at higher frequencies (e.g., 1.8 kHz and above). In this exemplary figure, no current was delivered to the sham-operated group animals. All other mice were stimulated with a current of 0.75 mA and a pulse sequence duration of 60 seconds. The 5 kHz group was stimulated with a 5 kHz sinusoidal pulse; all other mice were stimulated with a 0.25 ms biphasic charge-balanced pulse. Figure 12 As shown, significantly more lesion areas underwent myelin regeneration after stimulation at 1, 10, or 20 Hz, and high-frequency stimulation did not modulate myelin regeneration. Generally, the stimulation frequency range of 1-10 Hz is more effective in enhancing myelin regeneration than 20 Hz and higher frequencies.

[0158] like Figure 13As shown, significantly more myelin regeneration occurred in the lesion area after stimulation at 10 Hz for 1 or 5 minutes, while generally, stimulation for less than 20 minutes (e.g., less than 15 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, etc.) was more effective than longer stimulation. Surprisingly, 1 minute of stimulation was more effective than 5 minutes. In this example, no current was delivered to the sham-operated group animals; all other mice were stimulated with a 0.75 mA, 10 Hz, 0.25 ms biphasic charge-balanced pulse. As shown, the pulse sequence duration was varied.

[0159] Based on preliminary work, similar trends are expected in human patients. For example, relatively low-frequency stimulation (e.g., between 0.1 and 20 Hz) and shorter pulse sequence durations (e.g., less than 5 minutes) may be more effective than higher-frequency stimulation with longer pulse sequence durations. This result is surprising and, combined with findings from the daily total charge survey, suggests that the optimal daily total charge applied to the vagus nerve is between approximately 2.5 nC and 7.5 mC to modulate endothelial cells, microglia, and / or macrophages, thereby increasing the clearance of cellular debris (e.g., myelin sheath fragments) and thus reducing lesion volume in diseases and disorders of myelination, including MS.

[0160] The distribution of the VNS dose to be delivered can be a single dose (e.g., once daily, every other day, every three days, every four days, every five days, every six days, once a week, etc.), or it can be distributed daily or over several days (e.g., 2x daily, 3x daily, 4x daily, 5x daily, etc.). The total charge delivered daily can be less than about 7.5 mC (e.g., about 7 mC / day, about 6.5 mC / day, about 6 mC / day, about 5.5 mC / day, about 5 mC / day, etc., and in some variants, between about 2.5 nC / day and about 7.5 mC / day, between about 2.5 nC / day and about 7 mC / day, between about 5 nC / day and about 6.5 mC / day, etc.).

[0161] Dosage can be varied or adjusted. In particular, dosage can be adjusted based on time since treatment began. In variants where VNS is administered via an implanted device, the dosage may be gradually reduced over time. For example, the device may reduce the amount and / or intensity of administered VNS over time, e.g., from once daily to once every two or three days, then to once weekly, etc. In some variants, dosage can be adjusted based on concurrently delivered medications or therapies. When medications treating diseases or disorders of myelination are taken concurrently, the dosage of VNS may be reduced.

[0162] As described above, in some variants, the dose can be frequency-optimized at around 10 Hz (e.g., between 0.1 Hz and 20 Hz, between about 1 Hz and 15 Hz, between about 1 Hz and 12 Hz, etc.). In some cases, frequencies above 30 Hz have been found to be essentially ineffective. Preliminary data also suggest that stimulation of 1 minute or less may be sufficient to achieve complete myelin regeneration per dose. Therefore, in some variants, the VNS dose used to treat diseases or disorders affecting myelination can be limited to between about 0.1 seconds and 120 seconds, e.g., less than about 120 seconds, less than 100 seconds, less than 90 seconds, less than 80 seconds, less than 70 seconds, less than 60 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 35 seconds, less than 30 seconds, etc. Any device described herein can be configured to limit stimulation to these effective ranges, which prevents exceeding the effective range.

[0163] Animal data suggest that there may be a lower limit of intensity (around 0.25 mA) in mice, below which VNS are ineffective. In humans, this level may be lower or comparable (e.g., 0.05 mA, 0.1 mA, 0.15 mA, 0.2 mA, 0.25 mA). Generally, the effective range of VNS for achieving myelin regeneration as described herein appears to differ from the range of VNS application parameters used to achieve other effects, including the previously mentioned modulation of inflammation. For example, applying VNS to modulate myelination (myelination / demyelination) appears to be more limited than its anti-inflammatory effects. For instance, administration at energy levels outside this range (e.g., below the minimum level, above the maximum level) may have a potent anti-inflammatory effect with little or no effect on myelination (e.g., between 2.5 nC and 7.5 mC per day).

[0164] Preliminary work in animal models (e.g., dogs) has shown that gradually reducing the applied dose (e.g., from daily to weekly) may be effective because (surprisingly) the effects of stimulation within the effective parameter range may persist for longer periods over time.

[0165] Multimodal stimulation

[0166] As described herein, the VNS treatment described herein has been found to reduce demyelination and promote myelination, proving effective in treating various neurodegenerative and / or neuroinflammatory disorders (e.g., multiple sclerosis). While treatments used to reduce demyelination may also promote myelination, and vice versa, the underlying mechanisms of demyelination and myelination may differ. Furthermore, the optimal frequency of VNS may vary and may span different (and non-overlapping) ranges. Therefore, the methods and apparatus described herein may include applying different “doses” of VNS at different frequencies (e.g., different frequency ranges) to improve either or both of demyelination (e.g., clearance) and myelination. In some variations, the methods and apparatus may adjust the duration of one frequency component relative to another frequency component to improve demyelination or myelination at different times during patient treatment. For example, in some variations, the method or device may be configured to apply stimulation at a first frequency between 1 and 20 Hz (e.g., between 1 and 10 Hz, between 1 and 7 Hz, between 1 and 5 Hz, etc.) to reduce or prevent demyelination, and to apply a second dose of electrical stimulation at a second frequency higher than the first frequency (e.g., between 10 and 30 Hz, between 15 and 30 Hz, etc.) to increase myelin regeneration. In some variations, the method or device may be configured to apply a lower dose (a lower frequency range to prevent or reduce demyelination), but may switch to applying a lower frequency range (first frequency) and a higher frequency range (e.g., second frequency) when a triggering event is detected. The triggering event may be triggered by a user (e.g., a patient and / or physician or other healthcare practitioner), for example, in response to a demyelination event. In some variations, the triggering event may be triggered by detecting one or more demyelination biomarkers, as described herein.

[0167] Specifically regarding MS, the methods and apparatus described herein can be used to specifically treat relapsing-remitting MS (RRMS). As described above, these methods and apparatus can be used to prevent or reduce the effects during periods of active inflammation (“relapses”) and / or the intervals between active inflammation. Alternatively, in some variations, the apparatus and methods described herein can be specifically used to treat primary-progressive MS (PPMS). In addition to or in lieu of patient-specific markers (e.g., biomarkers), parameters (e.g., controllers, feedback, etc., specifically including frequency and / or dosing regimens for treatment) can be adjusted based on the type of MS. These methods and apparatus can also, or alternatively, be applied to treat secondary-progressive MS (SPMS) and / or progressive-relapsing MS (PPMS).

[0168] Demyelination is typically associated with immune responses and pro-inflammatory mechanisms. Myelination is a regenerative process associated with immune regression. As described herein, some types of VNS stimulation (e.g., lower frequencies, such as between approximately 1–20 Hz, or between approximately 1–10 Hz, 1–7 Hz, 1–5 Hz, etc.) are more preventative and can reduce the severity of demyelination, for example, by preventing IFNg+ T cell infiltration (e.g., by immune cells, including Th1 cells) and myelin breakdown. Other types of VNS stimulation (e.g., higher frequencies, such as between 10–30 Hz, or between 12–30 Hz, 15–30 Hz, 20–30 Hz, etc.) can enhance myelination. Therefore, as mentioned above, VNS treatment protocols can be optimized to include different stimulation modalities based on whether the stimulation parameters are more effective / efficient in reducing demyelination or promoting myelination. For example, a first stimulation modality can be optimized to target a reduction in demyelination, and a second stimulation modality can be optimized to target an increase in myelination. In some embodiments, the VNS treatment scheme includes a combination of a first-mode stimulus and a second-mode stimulus. In other embodiments, the VNS treatment scheme includes only one or more first-mode stimuli, or only one or more second-mode stimuli. Any of these systems and methods can be configured to switch between the first and second modes manually, semi-automatically, or automatically based on feedback (user and / or biomarker feedback). In some variations, the amount of first-mode and second-mode stimulation can be adjusted, for example, by increasing the percentage of total stimulation, as described herein, which can be limited to the total daily charge transferred (e.g., between 2.5 nC and 7.5 mC) and / or the total daily stimulation time (e.g., between 1 minute and 5 minutes per day, etc.). The percentage of stimulation in the first and second modes can be approximately equal (e.g., about half the stimulation frequency of the first mode and about half the stimulation frequency of the second mode, i.e., 50% / 50%, or about 40% / 60%, about 30% / 70%, about 20% / 80%, about 10% / 90%, about 60% / 40%, about 70% / 30%, about 80% / 20%, about 80% / 10%, etc.).

[0169] In some cases, the treatment protocol includes a series of individually applied stimuli, comprising a first-mode stimulus (targeting demyelination reduction) and a second-mode stimulus (targeting increased myelination). For example, each first-mode stimulus may be followed directly by a second-mode stimulus. Alternatively, a set of multiple (e.g., 2, 3, 4, 5, 6, 10, or 20) first-mode stimuli may be applied, followed by a set of multiple (e.g., 2, 3, 4, 5, 6, 10, or 20) second-mode stimuli. In some cases, a first set of multiple first-mode stimuli (e.g., 2, 3, 4, 5, 6, 10, or 20) may be applied, followed by a single second-mode stimulus, and then a second set of multiple first-mode stimuli (e.g., 2, 3, 4, 5, 6, 10, or 20). In some cases, a first set of multiple second-mode stimuli (e.g., 2, 3, 4, 5, 6, 10, or 20) may be applied, followed by a single first-mode stimulus, and then a second set of multiple second-mode stimuli (e.g., 2, 3, 4, 5, 6, 10, or 20). The number of stimulations and / or total stimulation time for a specific pattern can be selected based on whether the patient's condition requires attention to reduce demyelination or promote myelination regeneration.

[0170] In some cases, treatment options are chosen based on the patient's symptoms. For example, in some variations, if a patient is experiencing a sudden onset of symptoms, one or more first-mode stimuli (targeting a reduction in demyelination) can be applied to counteract the inflammatory process. Once the inflammation has subsided sufficiently, one or more second-mode stimuli (targeting an increase in myelination) can be applied to promote restorative cellular processes and immune remission.

[0171] In some cases, VNS treatment protocols are based on specific neurodegenerative conditions or diseases, and / or neuroinflammatory conditions or diseases. For example, a treatment protocol involving a first-mode stimulation that targets and reduces demyelination may be administered to patients whose conditions are associated with higher levels of inflammatory or pro-inflammatory cellular processes. A treatment protocol involving a second-mode stimulation that targets and increases myelination may be administered to patients whose conditions are associated with lower levels of inflammation, thereby promoting healing.

[0172] In some variations, the treatment protocol is modulated based on biomarker feedback. For example, in response to the presence of one or more biomarkers indicating demyelination, the stimulation protocol may be modified to include a first modal stimulation that targets a greater degree (or alone) reduction in demyelination. Similarly, in response to the presence of one or more biomarkers indicating a lower level of demyelination, the stimulation protocol may be modified to include a second modal stimulation that targets a greater degree (or alone) myelination regeneration. In some cases, the intensity of the first and second modal stimulations is based on a threshold level of one or more biomarkers.

[0173] In some implementations, the stimulation parameters for reducing demyelination (first mode stimulation) include lower frequencies compared to the stimulation parameters used to promote myelin regeneration (second mode stimulation). In some embodiments, the demyelination reduction (first mode) frequency is less than about 10 Hz (e.g., <10 Hz, <8 Hz, <6 Hz, <4 Hz, <3 Hz, or <2 Hz). In some embodiments, the demyelination reduction (first mode) frequency ranges from about 1 Hz to about 9 Hz (e.g., 1 Hz–9 Hz, 1 Hz–8 Hz, 1 Hz–6 Hz, 1 Hz–5 Hz, 1 Hz–3 Hz, 2 Hz–9 Hz, 2 Hz–6 Hz, 2 Hz–4 Hz, or 2 Hz–3 Hz). In some embodiments, the myelin regeneration promoting (second mode) frequency is about 10 Hz or higher (e.g., 10 Hz or higher, 11 Hz or higher, 15 Hz or higher, 20 Hz or higher, or 25 Hz or higher). In some embodiments, the frequency range of myelin regeneration promotion (second mode) is from about 10 Hz to about 30 Hz (e.g., 10 Hz-30 Hz, 10 Hz-25 Hz, 11 Hz-20 Hz, 10 Hz-15 Hz, 15 Hz-30 Hz, 15 Hz-2 Hz or 20 Hz-30 Hz).

[0174] Either demyelination reduction (mode 1) or myelination regeneration promotion (mode 2) stimulation can be characterized as having the low current and / or low duty cycle stimulation properties described herein. In some implementations, demyelination reduction (first mode) and myelination regeneration promotion (second mode) stimulation are characterized by a current ranging from about 0.1 mA to about 5 mA (e.g., 0.1-4 mA, 0.1-3 mA, 0.1-2 mA, 0.1-1 mA, 0.25-1 mA, 0.1-0.75 mA, 0.25-0.75 mA, etc.), a stimulation duration ranging from about 1 second to about 5 minutes (e.g., 1 second-5 minutes, 1 second-3 minutes, 1 second-2 minutes, 1 second-1 minute, 30 seconds-1 minute, or 30 seconds-2 minutes, 30 seconds-5 minutes, 30 seconds-4 minutes, 30 seconds-3 minutes, etc.), and / or a shutdown time between stimulations ranging from about 10 minutes to about 24 hours (e.g., 10 minutes-24 hours, 10 minutes-6 hours, 30 minutes-6 hours, 6 hours-24 hours, or 30 minutes-24 hours).

[0175] Combined with drugs

[0176] The methods and apparatus described herein can be used in combination with or simultaneously with one or more drugs (e.g., in combination), including drugs used to treat myelination disorders or impairments.

[0177] It is well known that it can be difficult to combine or use multiple drugs to treat diseases or disorders of myelination, such as multiple sclerosis (MS), because these drugs may interact in undesirable and potentially dangerous ways. The methods and devices described herein can be used in combination with one or more drugs, and there are no negative interactions between the VNS methods and devices described herein and other drug-based therapies. The VNS methods and devices described herein for treating diseases or disorders of myelination can be used in combination with one or more of the following: Avonex (interferon beta-1a), Betaseron (interferon beta-1b), Copaxone (glatiramer acetate), Extavia (interferon beta-1b), Glatopa (glatiramer acetate-Copaxone injection, a universal equivalent of glatiramer acetate-Copaxone 20 mg and 40 mg doses), Glatopa (glatiramer acetate-Copaxone). Generic equivalents for 20 mg and 40 mg doses), Pplegridy (pegylated interferon β-1a), Rebif (interferon β-1a), Aubagio (teriflunomide), Gileadia (fingolimod), Tecfidera (dimethyl fumarate), Mayzent (simpomod), Mavenclad (cladribine), Lemtrada (alemumab), Novantrone (mitoxantrone), Ocrevus (orreizumab), Tysabri (nateizumab), Solu-Medrol (methylprednisolone), Deltasone (prednisolone), HPActhar Gel (ACTH), Ampyra (davalidinyl).

[0178] Therefore, the methods and devices described herein for applying VNS can be combined with drug treatments with low risk of additional side effects, possibly because VNS leads to an enhanced primary immune response, such as increased microglia and / or macrophage activity, which is typically a different pathway than that of drug formulations.

[0179] In particular, the VNS methods and devices described herein can be used with one or more drugs that enhance myelin regeneration. For example, the methods and devices described herein can be used with receptor muscarinic type 3 (M3R) modulating drugs. The VNS methods and devices described herein can be used alternatively or additionally with B cell-targeting drugs, such as drugs that target cell surface markers on B cells and T cells, such as cladribine and alemtuzumab.

[0180] The immune cell-modulating effects of VNS can be enhanced by additional medications, or can be used to enhance the effects of additional medications in the treatment of patients with neuroinflammatory disorders such as multiple sclerosis (MS).

[0181] In some cases, the VNS treatments described herein can be used in combination with interferon beta drugs and generics, glatiramer acetate and generics, and dazumab. The modes of action targeting interferon beta-1a and 1b receptors and T-cell activation may be complementary to, and can be additional to, VNS. These combinations can reduce central nervous system inflammation and demyelination.

[0182] In some cases, the VNS treatments described herein can be used in combination with fingolimod, teriflunomide, and dimethyl fumarate. Targeting lymphocyte migration or activation can be an adjunct to VNS. These combinations can reduce central nervous system inflammation and demyelination.

[0183] In some cases, the VNS treatments described herein can be used in combination with mitoxantrone, alenmab, oligrizumab, and natezumab. Targeted mechanisms of action that induce DNA breaks, CD52 to induce cell lysis, depleted B-cell CD20 antigen, and / or integrin receptors to alter leukocyte migration can be complementary to, and may be adjuncts to, VNS. These combinations should reduce central nervous system inflammation and demyelination.

[0184] In some cases, the VNS treatment described herein can be used in combination with clomastine, selective estrogen receptor modulators (SERMs) (e.g., badoxifene), and other drugs targeting oligodendrocyte precursor cells to enhance the maturation of myelin-producing oligodendrocytes. This enhanced maturation mechanism targeting myelin-producing oligodendrocytes can complement the effects of VNS on phagocytosis and can be an adjunct to VNS. These combinations should enhance myelin regeneration and clinical recovery from central nervous system injury.

[0185] In some variations, the therapy may include implanting an internal VNS stimulator, administering medication to the patient after they have stabilized and recovered, and maintaining the VNS indefinitely. VNS, used alone or in combination with other therapies (e.g., medications), can be used to reduce demyelination and / or increase myelin regeneration. In some variations, VNS, used alone or in combination with other therapies (e.g., medications), may delay the onset of MS, prevent disease progression, and, in some cases, reverse disease progression in MS patients. In some cases, patients who do not respond to conventional therapies may respond to VNS therapy used to treat diseases or disorders of myelination.

[0186] When a VNS as described herein is used in combination with a drug therapy (e.g., medication), the medication used can be adjusted, for example, by reducing the dosage of the medication, thereby significantly reducing the administration of additional MS medications compared to the dosage without a VNS. In some variations, a treatment method may include titrating the dosage of the drug therapy when used in combination with a VNS as described herein.

[0187] VNS implantation location

[0188] When the VNS therapy described herein includes implantable (e.g., surgically implanted) microstimulator devices, in some variations, the implantable device may be attached to or implanted on or near the vagus nerve in the neck region (cervical spine location). Alternatively, the microstimulator device may be implanted in the subdiaphragmatic location, on the subdiaphragmatic vagus nerve, the splenic nerve, etc.

[0189] Transcutaneous stimulation

[0190] Alternatively, in some variations, the VNS is applied externally via one or more transcutaneous electrical stimulators. Energy can be applied externally to one or more locations, such as the neck (including both sides of the neck, below the ears), chest (e.g., the midline of the chest), abdomen, ears, etc. Non-invasive (e.g., external) stimulation can be configured such that the stimulation received by the vagus nerve is within the parameters described for implantable systems as described herein. For example, a handheld or wearable application can be applied to the patient's neck or other areas of skin covering the vagus nerve region, and electrical energy in the range of 0.1–30 Hz (e.g., 1–10 Hz) can be applied such that, at the configured power settings, the total charge received by the vagus nerve is between 2.5 nC and 7.5 mC per day.

[0191] Feedback from MS biomarkers

[0192] Any apparatus and method described herein can also be modulated using one or more biomarkers, and particularly biomarkers targeting myelination (including myelination fragments, myelination clearance, etc.) and / or biomarkers targeting the activity of microglia and / or other macrophages. For example, biomarkers may include microglia biomarkers, such as antibody biomarkers (e.g., antibodies against irregular chemokine receptor (Cx3cr1), TMEM119, CD11b and CD45, ion-calcium binding adaptor molecule 1 (Iba1), CX3CR1 being an irregular chemokine receptor, F4 / 80, CD68, CD40, etc.). Furthermore, biomarkers may specifically target myelin, including antibodies directly targeting myelin.

[0193] For example, biomarkers may include qualitative and quantitative measurements of elevated immunoglobulins (IgG) in CSF and / or blood. Isoelectric focusing (IEF) is a qualitative method for detecting oligoclonal bands (OCBs) used to track MS and has been shown to have a sensitivity of over 95% in MS15, and a specificity generally considered to be over 86%. Any of the methods and devices described herein can track OCBs in an individual patient and adjust therapy (e.g., VNS therapy and / or VNS therapy plus medication) based on changes in this biomarker. Other similar biomarkers may include the detection of oligoclonal IgM bands, which may predict a more severe disease course with a shorter time to the next relapse.

[0194] Alternatively or additionally, antibodies against myelin-oligodendrocyte-glycoprotein (MOG) and / or myelin basic protein (MBP) can be measured / or tracked to modulate VNS therapy as described herein. MOG is typically located on the surface of myelin and oligodendrocytes, while MBP accounts for 30% of total central myelin proteins.

[0195] Any biomarker described herein may be specific to the myelination disorder or disease to be treated. For example, neuromyelitis optica (NMO) is an inflammatory demyelinating disorder that selectively affects the spinal cord and optic nerve. The presence of NMO-specific autoantibodies, NMO IgG, can be used as a biomarker. Furthermore, aquaporins (aquaporin-4), located in the foot processes of astrocytes at the blood-brain barrier, are a target antigen.

[0196] Other biomarkers may include interferon-β (IFN-β). Interferon-β levels, including neutralizing antibodies (NAbs) against IFN-β, can track the progression or state of myelination disorders and can be used to modulate VNS therapy. An increase in NAb titer (or a high standardized level) may indicate an increase in VNS dosage.

[0197] Natalizumab is a humanized monoclonal antibody that binds to very late activation antigen 4 (VLA-4) (an α4β1 integrin) and thereby prevents leukocytes from migrating across the blood-brain barrier. Therefore, natalizumab (and / or its NAb) can be used as a biomarker for modulating VNS processing in patients.

[0198] Biomarkers can be tested by acutely and periodically (during outpatient visits) and / or directly through the implant using one or more sensors within the device. In some variants, biomarkers can be sampled non-invasively by examining, for example, retinal biomarkers, optic nerve biomarkers, etc.

[0199] As described above, any system described herein can be configured to modulate stimulation based on the onset of a sudden attack, in which a patient experiences the onset or worsening of symptoms such as pain, muscle spasms or stiffness, and / or fatigue. In some variations, for example, the patient provides input indicating the onset of a sudden attack via a portable (e.g., wearable) electronic device. In some variations, measurements of one or more biomarkers can indicate whether a patient is experiencing or about to experience a sudden attack. Treatment can then be based on whether a sudden attack is occurring, about to occur, and in some cases, on the severity of the sudden attack. In some implementations, an increase in one or more biomarkers associated with demyelination and / or inflammation may indicate the onset of a sudden attack, and stimulation parameters can be specifically modulated to counteract demyelination and / or inflammation. For example, stimulation parameters can be adjusted to those found to reduce demyelination (e.g., frequencies less than 10 Hz, less than 8 Hz, less than 5 Hz, or less than 3 Hz).

[0200] When a feature or element is referred to herein as being “on” another feature or element, it may be directly on the other feature or element, or there may be intermediate features and / or elements present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intermediate features or elements. It should also be understood that when a feature or element is referred to as being “connected,” “attached,” or “coupled” to another feature or element, it may be directly connected, attached, or coupled to the other feature or element, or there may be intermediate features or elements present. In contrast, when a feature or element is referred to as being “directly connected,” “directly attached,” or “directly coupled” to another feature or element, there are no intermediate features or elements. Although one embodiment has been described or illustrated, the features and elements thus described or illustrated may be applicable to other embodiments. Those skilled in the art will also understand that references to structures or features disposed “adjacent” to another feature may have portions overlapping with or below the adjacent feature.

[0201] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. For example, as used herein, the singular forms “a / an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should be further understood that, when used in this specification, the term “comprises and / or comprising” specifies the presence of the stated features, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated to “ / ”.

[0202] For ease of description, spatial relative terms such as “under,” “below,” “lower,” “over,” and “upper” are used herein to describe the relationship of one element or feature to one or more other elements or features, as illustrated in the figures. It should be understood that, in addition to the orientation depicted in the figures, spatial relative terms are intended to cover different orientations of the device in use or operation. For example, if the device in the figure were reversed, the element described as “below” or “under” other elements or features would be “oriented” above the other elements or features. Thus, the exemplary term “under” can cover both above and below orientations. Devices may be oriented in other ways (rotated 90 degrees or in other orientations), and the spatial relative descriptors used herein shall be interpreted accordingly. Similarly, unless explicitly stated otherwise, the terms “up,” “down,” “vertical,” “horizontal,” etc., are used herein for illustrative purposes only.

[0203] Although the terms "first" and "second" may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context otherwise requires. These terms may be used to distinguish one feature / element from another. Therefore, without departing from the teachings of the invention, the first feature / element discussed below may be referred to as the second feature / element, and similarly, the second feature / element discussed below may be referred to as the first feature / element.

[0204] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” mean that various components may be commonly employed in methods and articles (e.g., compositions and apparatuses that include devices and methods). For example, the term “comprising” will be understood to imply the inclusion of any of the stated elements or steps, but does not exclude any other elements or steps.

[0205] As used herein and in the claims, including as in the examples, and unless explicitly stated otherwise, all numbers may be read as if they begin with the word “about or approximately”, even if the term is not explicitly stated. The phrase “about or approximately” may be used when describing magnitude and / or location to indicate that the described value and / or location is within a reasonably expected range of the value and / or location. For example, a numerical value may have values ​​such as: + / - 0.1% of the value (or range of values), + / - 1% of the value (or range of values), + / - 2% of the value (or range of values), + / - 5% of the value (or range of values), + / - 10% of the value (or range of values), etc. Unless the context otherwise requires, any numerical value given herein should also be understood to include “about or approximately”. For example, if the value “10” is disclosed, “about 10” is also disclosed. Any numerical range referenced herein is intended to include all subranges contained therein. It should also be understood that when a disclosed value is “less than or equal to”, “greater than or equal to” and the possible range between the value are also disclosed, as would be reasonably understood by one of ordinary skill in the art. For example, if a value “X” is disclosed, then “less than or equal to X” and “greater than or equal to X” are also disclosed (e.g., where X is a numerical value). It should also be understood that throughout this application, data is provided in several different forms, and the data represents endpoints and starting points, as well as a range of any combination of data points. For example, if specific data point “10” and specific data point “15” are disclosed, then it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed and between 10 and 15. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0206] Although various illustrative embodiments have been described above, any of a number of changes may be made to the various embodiments without departing from the scope of the invention as set forth in the claims. For example, in alternative embodiments, the order in which the various described method steps are performed may often be changed, and in other alternative embodiments, one or more method steps may be skipped together. Optional features of the various device and system embodiments may be included in some embodiments but not in others. Therefore, the foregoing description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as set forth in the claims.

[0207] The examples and illustrations included herein show specific embodiments in which the subject matter can be practiced by way of illustration and not limitation. As mentioned, other embodiments can be utilized and derived therefrom, allowing for structural and logical substitutions and changes without departing from the scope of this disclosure. These embodiments of the subject matter of the invention may be referred to herein individually or collectively by the term "invention" merely for convenience and not intended to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is disclosed in fact. Therefore, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may substitute for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of the various embodiments. Combinations of the above embodiments and other embodiments not specifically described herein will be apparent to those skilled in the art upon reading the above description.

Claims

1. A system for reducing demyelination and / or increasing myelin regeneration by stimulating the vagus nerve, the system comprising: A vagus nerve stimulator is configured to be implanted above or near the vagus nerve. One or more electrodes on the vagus nerve stimulator are configured to apply electrical stimulation to the vagus nerve; as well as A controller, coupled to the vagus nerve stimulator and configured to apply electrical stimulation to the vagus nerve from the one or more electrodes, wherein the controller is limited to applying a charge between 2.5 nC and 7.5 mC per day to reduce demyelination and / or increase myelin regeneration in the patient.

2. The system of claim 1, further comprising being configured to receive inputs of one or more marker level indicators, wherein the controller is configured to adjust the applied charge based on the one or more marker level indicators.

3. The system of claim 2 further includes a biosensor configured to detect biomarkers from the patient's blood and / or cerebrospinal fluid and determine biomarker level indices.

4. The system of claim 1, wherein the controller is configured to deliver the electrical stimulation during one or more dose phases of 5 minutes or less.

5. The system of claim 1, wherein the controller is configured to apply the charge per day at a frequency between 1 and 20 Hz.

6. The system of claim 5, wherein the controller is configured to apply the charge at a frequency between 1 and 12 Hz according to the date.

7. The system of claim 1, wherein the system is configured to be implanted.

8. The system of claim 1, further comprising a neural cannula configured to attach the vagus nerve stimulator to the vagus nerve.

9. The system of claim 1, wherein the controller is configured to apply the charge daily at two different frequencies between 1 and 20 Hz.

10. The system of claim 1, wherein the controller is configured to apply a first dose of electrical stimulation at a first frequency between 1 and 20 Hz to reduce demyelination, and to apply a second dose of electrical stimulation at a second frequency higher than the first frequency to increase myelin regeneration in the patient.

11. The system of claim 10, wherein the first dose of electrical stimulation has a frequency of less than 10 Hz, and the second dose of electrical stimulation has a frequency ranging from 10 Hz to 30 Hz.

12. The system of claim 10, wherein the first dose of electrical stimulation has a frequency ranging from 1 Hz to 5 Hz, and the second dose of electrical stimulation has a frequency ranging from 5 Hz to 30 Hz.

13. An apparatus for increasing the clearance of myelin sheath fragments in a patient diagnosed with or at risk of having a disorder involving demyelinating nerves, the apparatus comprising: The controller is configured to perform operations, the operations including: Vagus nerve stimulation was applied to the patient at a rate between 2.5 nC and 7.5 mC per day.

14. The apparatus of claim 13, wherein the application includes applying the vagus nerve stimulation to the vagus nerve at a frequency between 0.1 and 20 Hz.

15. The device of claim 13, wherein the application includes applying the vagus nerve stimulation for less than 10 minutes per day.

16. The device of claim 13, wherein applying stimulation to the vagus nerve includes applying stimulation to the vagus nerve from an implanted neurostimulator attached to or adjacent to the vagus nerve.

17. The apparatus of claim 13, wherein the operation further comprises the applied vagus nerve stimulation based on a level adjustment of the marker.

18. The apparatus of claim 17, wherein the operation further comprises detecting markers for demyelination in a blood, sputum, or cerebrospinal fluid sample.

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