Stimulation of nerve dispersed in spleen

Abstract

To provide an improved treatment method for inflammation disorders, the method minimizing off-target effects.SOLUTION: Provided is a treatment method for inflammation disorders by stimulating neural activity in nerves dispersed in the spleen associated with a neurovascular bundle to adjust inflammatory and anti-inflammatory molecule level, thereby reducing inflammation.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to neuromodulation of nerves innervating the spleen, in particular to devices, systems and methods for stimulating neural activity in the nerves, and more particularly to signal parameters and electrode designs. The present invention also relates to devices, systems and methods for stimulating neural activity in the nerves for the treatment of inflammatory disorders. [Background technology]

[0002] Inflammation plays a fundamental role in host defense and the progression of immune-mediated diseases (reviewed in [1]). Inflammatory responses are initiated in response to injury and / or infection by chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., leukocytes). A controlled inflammatory response is useful, for example, in initiating the repair of damaged tissues, providing removal of harmful agents and protection against infection. However, if unregulated, the inflammatory response can become harmful and lead to a variety of inflammatory disorders, such as rheumatoid arthritis, osteoarthritis, asthma, allergies, septic shock syndrome, atherosclerosis, and inflammatory bowel disease, Crohn's disease, ulcerative colitis, and other clinical conditions mediated by chronic inflammation.

[0003] The spleen contains half of the body's monocyte population, making this organ a major contributor to inflammation. It is known that this organ is innervated by different nerve branches (reviewed in [2]). The parasympathetic innervation of the spleen has been a subject of controversy since the isolation of acetylcholine (ACh) from the spleen by Dale [2]. Although Buij et al. proposed a parasympathetic innervation of the spleen in rodents [3,4], the human correlate of this nerve is unclear. The traditional picture of the splenic innervation is proposed to be 98% sympathetic, as indicated by neuroanatomical and neurochemical evidence [2]. Summary of the Invention [Problem to be solved by the invention]

[0004] From a functional point of view, vagus nerve stimulation (reviewed in [5]) as well as the nerve plexus surrounding the splenic artery, referred to herein as the splenic nerve, inhibits LPS-induced TNF release in mice [6]. According to Tracey et al., splenic nerve activity is directly controlled by the cholinergic anti-inflammatory pathway (CAP) originating from the efferent vagus nerve branch [5]. Although vagal control of inflammatory tone and inflammatory reflexes has received considerable attention, other researchers have disputed the connection between the vagus nerve and the splenic nerve. Some authors have shown that denervation of the splenic nerve in mice resulted in inhibition of the CAP [6]. However, Martelli et al. have questioned this idea by showing that the splenic nerve does not connect directly to the vagus nerve [7], but rather arises as an independent branch of the great splenic nerve that controls splenic nerve activity [8,9]. These authors also argue against the idea that neural sensing of inflammatory markers is humoral and not neural

[10] . Furthermore, there is debate as to whether the efferent arm of the inflammatory reflex response is sympathetic or parasympathetic.

[0005] Electrical stimulation of the vagus nerve has been shown to relieve the symptoms of rheumatoid arthritis in clinical trials.

[11] However, because the vagus nerve is composed of several bundles that innervate most of the organs, including the heart, liver, and gastrointestinal tract, there are concerns that stimulation of the vagus nerve may produce unwanted nonspecific CNS effects.

[0006] Electrical stimulation of the splenic nerve has been associated with vascular responses in the spleen.

[12] References [6, 13, 14, 15] describe electrical stimulation of the splenic artery nerve for the treatment of inflammatory disorders. However, the electrical parameters used in these references to induce stimulation of nerve activity may result in off-target effects, such as changes in splenic arterial and venous blood flow, as well as changes in systemic arterial blood pressure and heart rate.

[0007] Thus, there is a need for alternative and improved methods of stimulating neural activity in nerves innervating the spleen for the treatment of inflammatory disorders, such as autoimmune disorders (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis, spondyloarthropathy, ankylosing spondylitis, psoriasis, lupus erythematosus, multiple sclerosis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis) and sepsis. [Means for solving the problem]

[0008] The inventors have found that stimulation parameters for neural stimulation of nerves innervating the spleen (e.g., the splenic artery nerve) associated with the neurovascular bundle can be optimized by measuring blood flow changes in the spleen, splenic artery, or splenic vein. In particular, the inventors have found that the neurophysiological properties of the splenic artery nerve can be used to determine optimal stimulation patterns that enhance immunosuppressive effects while reducing possible systemic effects (e.g., changes in systemic arterial blood pressure and heart rate). The inventors have also used histological and electrophysiological data to find optimal electrode and neural interface designs for electrical stimulation of the splenic artery nerve.

[0009] Accordingly, the present invention provides a system for stimulating neural activity of a nerve (e.g., the splenic artery nerve) innervating the spleen associated with the neurovascular bundle. The system includes at least one electrode in signal transducing contact with the nerve, and at least one controller electrically coupled to the at least one electrode. The at least one controller is configured to apply an electrical signal to the nerve by controlling operation of the at least one electrode. The electrical signal has a frequency of ≦300 Hz and is applied in a periodic on-off pattern, or has a frequency of ≦50 Hz and is applied continuously. On-off pattern stimulation can also be referred to as burst stimulation or periodic stimulation.

[0010] The invention also provides a method of treating an inflammatory disorder in a subject, the method comprising providing a system of the invention, positioning at least one electrode in signal transducing contact with a nerve innervating the spleen associated with the neurovascular bundle (e.g., the splenic artery nerve), and controlling operation of the at least one electrode with at least one controller to apply an electrical signal to the nerve to stimulate neural activity.

[0011] The invention also provides a method of reversibly stimulating neural activity in a nerve innervating the spleen associated with a neurovascular bundle (e.g., the splenic artery nerve), comprising providing a system of the invention, positioning at least one electrode in signal transducing contact with the nerve, and controlling operation of the at least one electrode with at least one controller to apply an electrical signal to the nerve to stimulate neural activity.

[0012] The invention also provides a method of verifying whether a neural interface is properly placed in signaling contact with a nerve (e.g., the splenic artery nerve) innervating the spleen associated with a neurovascular bundle, the method comprising providing a system of the invention, positioning a neural interface in signaling contact with the nerve, applying an electrical signal to the nerve by controlling the movement of the at least one electrode with at least one controller, verifying (determining) that a change in blood flow velocity in the spleen, splenic artery, or splenic vein, a decrease in splenic volume, an increase in neural activity in the nerve, or a change in impedance of at least one electrode is detected, and indicating to an operator that the neural interface has been properly placed in signaling contact with the nerve.

[0013] That is, the present invention provides stimulation parameters for different functions. More specifically, continuous stimulation can be used to induce blood flow changes in the splenic vasculature that can be detected and used on-table or perioperatively as indicators of good electrode placement, good target (nerve) engagement, and / or amplitude determination; and rapid-fire or low-frequency continuous stimulation (≦1 Hz) can be used as the preferred treatment paradigm, thereby avoiding such blood flow changes and / or other possible systemic cardiovascular effects while maintaining medical efficacy.

[0014] Thus, the electrical signal may have a frequency of ≦300 Hz and be applied in an on-off pattern for therapy, or may have a frequency of >1 Hz and ≦50 Hz and be applied continuously for electrode placement indication, target (nerve) engagement and / or amplitude determination. Given these different functions, the continuous application may be used first for electrode placement indication, target (nerve) engagement and / or amplitude determination, and then, once electrode placement, target (nerve) engagement and / or amplitude have been confirmed, a burst on-off pattern stimulation may be used.

[0015] For example, continuous stimulation < 10 Hz can be used for efficacy and / or treatment; and / or continuous stimulation < 30 Hz and > 5 Hz can be used for blood flow detection; and / or rapid-fire stimulation > 10 Hz (optionally > 5 Hz) or continuous low frequency (< 1 Hz) can be used for efficacy and / or treatment.

[0016] The invention also provides a computer-implemented method of treating an inflammatory disorder in a subject, the method comprising applying a signal to a nerve innervating the spleen that is associated with a neurovascular bundle (e.g., the splenic artery nerve) to stimulate neural activity by controlling the operation of at least one electrode of a system of the invention.

[0017] The invention also provides a computer-implemented method for reversibly stimulating neural activity in a nerve innervating the spleen associated with a neurovascular bundle (e.g., the splenic artery nerve), comprising controlling the operation of at least one electrode of a system of the invention to apply a signal to said nerve to stimulate neural activity.

[0018] The invention also provides a computer-implemented method for verifying whether a neural interface is properly placed in signaling contact with a nerve (e.g., the splenic artery nerve) innervating the spleen associated with a neurovascular bundle, comprising applying an electrical signal to said nerve by controlling the operation of at least one electrode of the system of the invention, verifying that a change in blood flow in the spleen, splenic artery, or splenic vein, a decrease in splenic volume, an increase in neural activity in the nerve, or a change in impedance of at least one electrode is detected, and indicating to an operator that the neural interface is properly placed in signaling contact with said nerve.

[0019] The invention also provides a neurostimulating electrical signal for use in treating an inflammatory disorder in a subject, said electrical signal being any electrical signal according to the invention.

[0020] The invention also provides a modified nerve (e.g., splenic artery nerve) supplying the spleen associated with a neurovascular bundle with which the system of the invention is in signaling contact, whereby said at least one electrode is in signaling contact with said nerve such that said nerve can be differentiated from the nerve in its native state; said modified nerve being in a subject suffering from or at risk of an inflammatory disorder.

[0021] The invention also provides a modified nerve (e.g., the splenic artery nerve) supplying the spleen, which is associated with a neurovascular bundle surrounded by a nerve membrane, comprising a distribution of potassium and sodium ions movable across the nerve membrane to propagate an action potential along the nerve under normal conditions by altering the membrane potential of the nerve; at least a portion of the nerve is subjected to application of a transient external electric field which, under perturbed conditions, causes depolarization of the nerve membrane and transient generation of an action potential across that portion de novo by altering the concentration of potassium and sodium ions within the nerve; and when the external electric field is removed, the nerve returns to its normal state.

[0022] The invention also provides modified nerves innervating the spleen (eg, the splenic artery nerve) that are associated with a neurovascular bundle obtained by stimulating neural activity of the nerve by the methods of the invention.

[0023] The present invention also provides a neural interface suitable for placement around a plurality of splenic artery nerves, said neural interface completely surrounding said plurality of splenic artery nerves, and including at least one electrode.

[0024] The present invention also provides a neural interface suitable for placement around at least one splenic artery nerve and the splenic artery, said neural interface surrounding at least 50%, preferably at least 75%, of the splenic artery and said at least one splenic nerve, and comprising at least one electrode.

[0025] The present invention also provides a method of controlling a system of the present invention placed in signal transmitting contact with a nerve (e.g., a splenic artery nerve) supplying the spleen associated with a neurovascular bundle, comprising sending a control instruction to the system, in response to which the system applies a signal to the nerve. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Nerves surrounding the spleen The innervation of the spleen is primarily sympathetic or noradrenergic, with peptidergic neurons likely representing the majority of the remaining neurons. The human spleen is traditionally thought to be innervated by the splenic plexus, which surrounds only the splenic artery. The splenic artery is covered with nerve tissue that originates from the celiac plexus and continues with the splenic artery as the splenic plexus. The splenic plexus enters the spleen at the hilum, and the splenic artery branches into peripheral branches that continue with the splenic plexus into the splenic parenchyma.

[0027] The splenic plexus includes several nerve bundles, which surround the main splenic artery from the celiac trunk to the spleen, with each bundle containing a small bundle of nerve fibers. The nerve bundles surrounding the splenic nerve (also called periarterial nerve bundles) are referred to herein as the splenic nerve.

[0028] The present invention relates to applying electrical signals to nerves innervating the spleen and thereby modulating the neural activity of nerves innervating the spleen that are associated with the neurovascular bundle, preferably the splenic artery nerve.

[0029] In some embodiments, the nerve is a sympathetic nerve.

[0030] In some embodiments, the invention may involve applying a signal to one splenic nerve, while in other embodiments, the invention may involve multiple (i.e., bundles) of splenic nerves.

[0031] In other embodiments, the invention may involve applying an electrical signal to at least one of the splenic nerve and the splenic artery, hi other embodiments, the invention may involve applying an electrical signal to all of the splenic nerve and the splenic artery.

[0032] Stimulation of the nerves supplying the spleen The present invention relates to applying an electrical signal to a nerve (e.g., splenic artery nerve) that innervates the spleen, which is associated with the neurovascular bundle, to stimulate the neural activity in said nerve. Stimulation refers to an increase in signaling activity in at least a portion of the nerve compared to the baseline neural activity in that portion of the nerve, where baseline neural activity is the signaling activity of the nerve in a subject before intervention. In other words, stimulation creates neural activity, which increases the total neural activity in that portion of the nerve.

[0033] "Neural activity" of a nerve refers to the signaling activity of the nerve, such as the amplitude, frequency and / or pattern of action potentials in that nerve. As used herein in the context of action potentials in a nerve, the term "pattern" includes one or more of local field potentials, compound action potentials, population action potentials, as well as amplitudes, frequencies, areas under the curve, and other patterns of action potentials in the nerve or subgroups of neurons therein (e.g., nerve bundles).

[0034] Stimulation is typically accompanied by an increase in neural activity, e.g., the generation of an action potential across the point of stimulation in at least a portion of the nerve. At any point along the axon, a functional nerve has a distribution of potassium and sodium ions across the nerve membrane. The distribution at one point along the axon determines the membrane potential of the axon at that point, which in turn determines the distribution of potassium and sodium ions at adjacent points, which in turn determine the membrane potential of the axon at that point. This is a normally operating nerve, where action potentials propagate from one point to an adjacent point along the axon, which can be observed using conventional experiments.

[0035] One way to characterize stimulation of neural activity is the distribution of potassium and sodium ions at one or more locations in an axon that is caused by the application of a temporary external electric field, rather than by the membrane potential at an adjacent location or locations in the nerve as a result of a propagating action potential. The temporary external electric field artificially alters the distribution of potassium and sodium ions within a location in the nerve, causing a depolarization of the nerve membrane that would not otherwise occur. The depolarization of the nerve membrane caused by the temporary external electric field generates a de novo action potential across that location. This is a nerve operating in a perturbed state, which can be observed by the distribution of potassium and sodium ions at a location in the axon (the location that was being stimulated) that has a membrane potential that is not influenced or determined by the membrane potential of adjacent locations.

[0036] Thus, stimulation of neural activity is understood to enhance neural activity by continuing beyond the site of signal application. Thus, the nerve at the site of signal application is modified in that the nerve membrane is reversibly depolarized by the electric field, thereby generating and propagating a de novo action potential through the modified nerve. Thus, the nerve at the site of signal application is modified in that a de novo action potential is generated.

[0037] When the signal is an electrical signal, stimulation is based on the effect of an electric current on the distribution of ions across the nerve membrane (e.g., one or more electrons at an electrode in signal transducing contact with the nerve, or charged particles which may be, for example, one or more ions outside or within the nerve).

[0038] Stimulation of neural activity includes complete stimulation of neural activity in a nerve, i.e., includes embodiments in which total neural activity is increased throughout the nerve.

[0039] The stimulation of neural activity may be a partial stimulation. Partial stimulation may be such that the total signaling activity of the entire nerve may be partially increased, or the total signaling activity of a subset of nerve fibers of the nerve may be completely increased (i.e., there is no neural activity in that subset of fibers of the nerve), or the total signaling of a subset of nerve fibers of the nerve may be partially increased compared to the baseline neural activity in that subset of fibers of the nerve. For example, an increase in neural activity of ≦5%, ≦10%, ≦15%, ≦20%, ≦25%, ≦30%, ≦35%, ≦40%, ≦45%, ≦50%, ≦60%, ≦70%, ≦80%, ≦90% or ≦95%, or an increase in neural activity in a subset of nerve fibers of the nerve. Neural activity may be measured by methods known in the art, for example, by the number of action potentials propagating through the axon and / or the amplitude of local field potentials, which reflect the total activity of action potentials.

[0040] The stimulation of neural activity can be a change in the pattern of action potentials. It will be clear that the pattern of action potentials can be modulated without necessarily changing the overall frequency or amplitude. For example, the stimulation of neural activity can be (at least partially) corrective. As used herein, "corrective" is intended to mean that the modulated neural activity changes the pattern of neural activity in a healthy subject, which is referred to as axonal modulation therapy. That is, when the signal application is stopped, the neural activity in the nerve becomes more similar (ideally substantially completely similar) to the pattern of action potentials in the nerve observed in a healthy subject compared to before the signal application. Such a corrective stimulation can be any stimulation defined herein.

[0041] For example, application of a signal can result in increased neural activity, and when application of the signal is terminated, the pattern of action potentials in the nerve resembles the pattern of action potentials seen in a healthy subject. By way of further example, application of a signal can result in neural activity that resembles the pattern of action potentials seen in a healthy subject, and when the signal is terminated, the pattern of action potentials in the nerve remains the pattern of action potentials seen in a healthy subject.

[0042] Stimulation of neural activity may include altering neural activity in various other ways, such as increasing specific portions of baseline neural activity and / or stimulating new activity components, such as time intervals, particularly in specific patterns, frequency bands, etc.

[0043] One advantage of the present invention is that the stimulation of neural activity is reversible. Thus, the modulation of neural activity is not permanent. For example, when the signal application is stopped, the neural activity in the nerve substantially returns to baseline neural activity within 1-60 seconds, or within 1-60 minutes, or within 1-24 hours (e.g., within 1-12 hours, 1-6 hours, 1-4 hours, 1-2 hours), or within 1-7 days (e.g., 1-4 days, 1-2 days). In some examples of reversible stimulation, the neural activity substantially completely returns to baseline neural activity. That is, the neural activity after the cessation of the signal application is substantially the same as the neural activity before the signal application. Thus, the nerve or part of the nerve has regained its normal physiological ability to propagate action potentials.

[0044] In other embodiments, the stimulation of neural activity can be substantially continuous. As used herein, "continuous" is taken to mean that the neural activity has a long-term effect. For example, when the application of a signal is stopped, the neural activity in the nerve remains substantially the same as when the signal was applied, i.e., the neural activity during and after the application of the signal is substantially the same. Reversible modulation is preferred.

[0045] Preferred form of electrical signal The present invention employs an electrical signal applied via at least one electrode placed in signaling contact with a nerve (e.g., the splenic artery nerve) that innervates the spleen associated with the neurovascular bundle. As used herein, "signaling contact" means that at least a portion of the electrical signal applied via the at least one electrode is received by the nerve.

[0046] The electrical signals applied by the present invention are ideally non-destructive. As used herein, a "non-destructive signal" is a signal that, when applied, does not irreversibly destroy the underlying nerve signaling capacity of the nerve. That is, application of a non-destructive signal is such that the nerve or its fibers or other nerve tissue to which the signal is applied maintains the ability to conduct action potentials when the signal application ceases, even if conduction is in fact artificially stimulated as a result of application of the non-destructive signal.

[0047] The electrical signal applied in accordance with the present invention may be a voltage or current waveform.

[0048] An electrical signal can be characterized by one or more electrical signal parameters, including waveform, frequency, and amplitude.

[0049] Alternatively or additionally, the electrical signal may be characterized by a pattern of application of the electrical signal to the nerve. The pattern of application refers to the timing of the application of the electrical signal to the nerve. The pattern of application may be continuous or periodic and / or episodic.

[0050] Episodic application refers to application of an electrical signal to a nerve for a discrete number of episodes throughout the day, where each episode may be defined by a set duration or a set number of repetitions of the electrical signal.

[0051] Continuous application refers to applying the electrical signal to the nerve continuously. When applying the electrical signal continuously or episodically, it means that the signal is applied continuously for each episode of application. In an embodiment where the electrical signal is a series of pulses, the gap between those pulses (i.e., between the pulse width and phase duration) does not mean that the signal is not applied continuously.

[0052] Periodic application refers to an electrical signal being applied to a nerve in a repeating pattern (e.g., an on-off pattern). When an electrical signal is applied periodically and episodically, it means that the signal is applied periodically for each episode of application.

[0053] The inventors have discovered preferred electrical signal parameters and patterns of signal application for stimulating neural activity in nerves (e.g., the splenic artery nerve) that innervate the spleen, which are associated with the neurovascular bundle, thereby enhancing the immunosuppressive effect while reducing possible systemic effects when stimulating neural activity in the splenic artery nerve. The preferred signal parameters and patterns of application are discussed in detail below.

[0054] waveform The modulation (e.g., stimulation) of the nerves (e.g., splenic artery nerve) innervating the spleen, which are associated with the neurovascular bundle, can be achieved using an electrical signal that helps to reproduce the normal neural activity of the nerve. The waveform of the electrical signal thus includes one or more pulse trains, each having a defined pulse width. The pulses are preferably square pulses. However, other pulse waveforms, such as sawtooth, sinusoidal, triangular, trapezoidal, quasi-trapezoidal, or complex waveforms, can also be used in the present invention.

[0055] The pulses may be biphasic in nature. The term "biphasic" refers to a pulse that applies both positive and negative charges (anodal and cathodal phases) to a nerve over time. Biphasic pulses are preferably charge balanced.

[0056] The pulse may be charge-balanced, which refers to a pulse that applies equal (or nearly equal) amounts of positive and negative charge to the nerve over the duration of the pulse.

[0057] Pulses can be symmetric or asymmetric. A symmetric pulse is one in which the waveform when applying a positive charge to the nerve is symmetric to the waveform when applying a negative charge to the nerve. An asymmetric pulse is one in which the waveform when applying a positive charge to the nerve is not symmetric to the waveform when applying a negative charge to the nerve.

[0058] The pulses may have a pulse width (for each phase) of 250-1000 μs, preferably 400-1000 μs (this is applicable for both the positive and negative phases of the pulse in case of a biphasic pulse). For example, the pulse width may be ≦500 μs, ≦600 μs, ≦700 μs, ≦800 μs, ≦900 μs, or ≦1000 μs. Additionally or alternatively, the pulse width may be ≧400 μs, ≧500 μs, ≧600 μs, ≧700 μs, ≧800 μs, or ≧900 μs. Any combination of the above upper and lower limits is possible. The pulse width may further be limited by the frequency.

[0059] Pulse width refers to the width (or duration) of the dominant phase of a waveform. If a pulse includes a first, dominant phase and a second, recovery phase, e.g., an anodic and / or cathodic phase, pulse width refers to the width (or duration) of the first phase.

[0060] If the biphasic pulse is asymmetric but remains charge balanced, the areas of the opposing phases must be equal. The amplitude (see below) may be reduced, but the pulse width would need to be increased to ensure that the areas under the curve are matched.

[0061] In an exemplary embodiment, the waveform is a pulse train having biphasic, asymmetric, charge-balanced rectangular pulses.

[0062] amplitude In the context of the present invention, amplitude is referred to herein in terms of per-phase charge density. The per-phase charge density applied to a nerve by an electrical signal is defined as the integral of the current over one phase (e.g., over one phase of a biphasic pulse in the case of a charge-balanced biphasic pulse). Thus, the per-phase charge density applied to a nerve by an electrical signal is the charge per unit contact area between at least one electrode and the nerve, as well as the integral of the current density over one phase of the signal waveform. In other words, the per-phase charge density applied to a nerve by an electrical signal is the per-phase charge applied to a nerve by an electrical signal divided by the contact area between at least one electrode (typically the cathode) and the nerve.

[0063] The charge density per phase required by the present invention represents the amount of energy required to stimulate neural activity in nerves innervating the spleen (e.g., the splenic artery nerve) that are associated with the neurovascular bundle to enhance the immunosuppressive effect.

[0064] The inventors have demonstrated that the charge density per phase required to stimulate neural activity in the porcine splenic artery nerve using an extravascular cuff ranges from 5 μC to 150 μC / cm 2 / phase, or in some cases, 5μC to 180μC / cm 2 / phase (values ​​may be affected slightly by electrode design). For example, the charge density per phase applied by the electrical signal is ≦10 μC / cm 2 / phase, ≦15μC / cm 2 / phase, ≦20μC / cm 2 / phase, ≦25μC / cm 2 / phase, ≦30μC / cm 2 / phase, ≦40μC / cm 2 / phase, ≦50μC / cm 2 / phase, ≦75μC / cm 2 / phase, ≦100μC / cm 2 / phase, ≦125μC / cm 2 / phase, ≦150μC / cm 2 / phase, or ≦180μC / cm 2 Additionally or alternatively, the charge density per phase applied by the electrical signal may be ≥ 5 μC / cm2 / phase, ≧10μC / cm 2 / phase, ≧15μC / cm 2 / phase, ≧20μC / cm 2 / phase, ≧25μC / cm 2 / phase, ≧30μC / cm 2 / phase, ≧40μC / cm 2 / phase, ≧50μC / cm 2 / phase, ≧75μC / cm 2 / phase, ≧100μC / cm 2 / phase, ≧125μC / cm 2 / phase, or ≧150μC / cm 2 Any combination of the above upper and lower limits is also possible.

[0065] The inventors further demonstrated that the estimated per-phase charge density required to stimulate neural activity in the human splenic nerve is approximately 70-1300 μC / cm 2 For example, the charge density per phase applied by the electric signal was found to be ≦80 μC / cm 2 / phase, ≦140μC / cm 2 / phase, ≦170μC / cm 2 / phase, ≦230μC / cm 2 / phase, ≦250μC / cm 2 / phase, ≦300μC / cm 2 / phase, ≦350μC / cm 2 / phase, ≦400μC / cm 2 / phase, ≦450μC / cm 2 / phase, ≦500μC / cm 2 / phase, ≦1100μC / cm 2 / phase, or ≦1300μC / cm 2 Additionally or alternatively, the charge density per phase applied by the electrical signal may be ≥ 70 μC / cm 2 / phase, ≧140μC / cm 2 / phase, ≧170μC / cm 2 / phase, ≧230μC / cm 2 / phase, ≧250μC / cm 2 / phase, ≧300μC / cm 2 / phase, ≧350μC / cm 2 / phase, ≧400μC / cm2 / phase, ≧450μC / cm 2 / phase, ≧500μC / cm 2 / phase, ≧1100μC / cm 2 / phase, or ≧1300μC / cm 2 The total charge applied to a nerve by an electrical signal in any given period of time is a result of the charge density per phase of the signal as well as the frequency of the signal, the pattern of signal application, and the area of ​​contact between the at least one electrode and the nerve, as discussed further herein.

[0066] It will be apparent to one of skill in the art that the amplitude of the applied electrical signal required to achieve a given stimulation of neural activity will depend on the positioning of the electrodes and the associated electrophysiological properties (e.g., impedance). It is within the ability of one of skill in the art to determine the appropriate current amplitude to achieve the desired modulation of neural activity in a given subject.

[0067] Of course, it is understood in the art that the electrical signal applied to the nerve is within a clinical safety margin (e.g., suitable for maintaining nerve signaling function, suitable for maintaining nerve integrity, and suitable for maintaining subject safety). Electrical parameters within the clinical safety margin are typically determined by preclinical testing.

[0068] Episodic Impression Episodic application refers to application of an electrical signal to a nerve for a discrete number of episodes throughout the day. The electrical signal according to the present invention can be applied for up to 6 episodes per day. For example, the number of episodes of signal application per day can be 1, 2, 3, 4, 5 or 6.

[0069] The electrical signal can be applied episodically every 2 to 3 hours. For example, the electrical signal can be applied episodically once every 2 hours, 2 hours 15 minutes, 2 hours 30 minutes, 2 hours 45 minutes, or 3 hours.

[0070] Each episode may be defined by a set duration or a set number of repetitions of the electrical signal. In some embodiments, each episode may include applying 50-10,000, e.g., 60-3,000 pulses of the electrical signal, 100-2,400 pulses of the electrical signal, 200-1,200 pulses of the electrical signal, 400-600 pulses of the electrical signal, etc., to the nerve. For example, each episode may include applying ≦400, ≦800, ≦1,200, ≦1,600, ≦2,000, ≦2,400, ≦3,000, or ≦10,000 pulses of the electrical signal. In another example, each episode may include applying ≦200, ≦400, ≦600, ≦800, ≦1,000, or ≦1,200 pulses of the electrical signal. In yet another example, each episode may include applying ≦400, ≦425, ≦450, ≦475, ≦500, ≦525, ≦550, ≦575, or ≦600 pulses of the electrical signal.

[0071] In other embodiments, each episode includes 20-40 repetitions of the periodic pattern. For example, each episode includes applying 20, 25, 30, 35, or 40 repetitions of the periodic pattern, or any number in between. The higher the frequency, the lower the number of repetitions.

[0072] In some embodiments, the episodes can be based on the subject's sleep-wake cycle, and in particular, the episodes can be while the subject is asleep. In some such embodiments, the episodes can be applied between 10 PM and 6 AM. The sleep-wake cycle can be measured by known methods by detecting a circadian rhythm phase marker of the subject (e.g., cortisol level, melatonin level, or core body temperature) and / or by a detector to detect the subject's movement.

[0073] periodic application Periodic application refers to applying an electrical signal to a nerve in a repeating pattern. A preferred repeating pattern is an on-off pattern, where the signal is applied for a first period (referred to herein as an "on" period), then stopped for a second period (referred to herein as an "off" period), then applied again for the first period, then stopped again for the second period, etc.

[0074] The periodic on-off pattern preferably has an on period of 0.1-10 seconds and an off period of 0.5-30 seconds. For example, the on period (referred to as the time during which pulses of a certain frequency and amplitude are sent to the nerve) can be ≦0.2 seconds, ≦0.5 seconds, ≦1 second, ≦2 seconds, ≦5 seconds, or ≦10 seconds. Alternatively or additionally, the on period can be ≧0.1 seconds, ≧0.2 seconds, ≧0.5 seconds, ≧1 second, ≧2 seconds, or ≧5 seconds. Any combination of the above upper and lower limits for the on period is possible. For example, the off period (referred to as the time during which no pulses are sent to the nerve during the on period) can be ≦1 second, ≦3 seconds, ≦5 seconds, ≦10 seconds, ≦15 seconds, ≦20 seconds, ≦25 seconds, or ≦30 seconds. Alternatively or in addition, the off period can be ≧0.5 seconds, ≧1 second, ≧2 seconds, ≧5 seconds, ≧10 seconds, ≧15 seconds, ≧20 seconds, or ≦25 seconds. Any combination of the above upper and lower limits for the off period is also possible.

[0075] In an exemplary embodiment, the periodic on-off pattern has an on period of 0.5 seconds on and 4.5 seconds off. In another example, the periodic on-off pattern has an on period of 0.5 seconds on and 5 seconds off for a maximum 10 Hz pulse. For frequencies higher than 10 Hz (e.g., 30 Hz), an exemplary periodic on-off pattern has an on period of 0.1 seconds on or an off period of 3 seconds. That is, the ratio of on period:off period can be 1:5, and more preferably, the ratio is 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, or 1:30. The ratio of on period:off period can be 1:10 for pulse frequencies of 10 Hz or less, and the ratio of on period:off period can be 1:30 for pulse frequencies higher than 10 Hz.

[0076] When an electrical signal is applied periodically and episodically, it is meant that the signal is applied periodically for each episode of application.

[0077] Periodic application can also be referred to as duty cycle application. Duty cycle represents the percentage of time that a signal is applied to a nerve in a cycle of a periodic pattern. For example, a 20% duty cycle can represent a periodic pattern with an on-period of 2 seconds and an off-period of 10 seconds. Alternatively, a 20% duty cycle can represent a periodic pattern with an on-period of 1 second and an off-period of 5 seconds. That is, periodic application can also be referred to as on-off pattern stimulation or burst stimulation.

[0078] The duty cycle suitable for the present invention is between 0.1% and 100%.

[0079] frequency Frequency is defined as the inverse of the phase duration of the electrical waveform (ie, 1 / phase).

[0080] The inventors have found preferred frequencies for stimulation of nerves innervating the spleen that are associated with the neurovascular bundle (e.g., the splenic artery nerve), and in particular, the inventors have found preferred frequencies for embodiments in which the electrical signal is applied periodically and for embodiments in which the electrical signal is applied continuously.

[0081] As previously mentioned, embodiments in which electrical signals are applied periodically and continuously provide different capabilities using different stimulation parameters: Continuous stimulation can be used to induce blood flow changes in the splenic vasculature that can be detected and used as indicators of good electrode placement and / or amplitude determination, either on-table or perioperatively; cyclic stimulation can be used as a preferred therapeutic paradigm, whereby such blood flow changes and / or other possible systemic cardiovascular effects are mitigated or avoided while maintaining therapeutic efficacy.

[0082] In embodiments where the electrical signal is applied periodically, the electrical signal has a frequency of ≦300 Hz, preferably ≦50 Hz, more preferably ≦10 Hz. For example, the frequency of the electrical signal can be ≦50 Hz, ≦100 Hz, ≦150 Hz, ≦200 Hz, ≦250 Hz, or ≦300 Hz. In other examples, the frequency of the electrical signal can be ≦10 Hz, ≦15 Hz, ≦20 Hz, ≦25 Hz, ≦30 Hz, ≦35 Hz, ≦40 Hz, ≦45 Hz, or ≦50 Hz. In further examples, the frequency can be ≦1 Hz, ≦2 Hz, ≦5 Hz, or ≦10 Hz. Additionally or alternatively, the frequency of the electrical signal can be ≧10 Hz, ≧15 Hz, ≧20 Hz, ≧25 Hz, ≧30 Hz, ≧35 Hz, ≧40 Hz, ≧45 Hz, or ≧50 Hz. In other examples, the frequency of the electrical signal can be ≧0.1 Hz, ≧0.2 Hz, ≧0.5 Hz, ≧1 Hz, ≧2 Hz, or ≧5 Hz. Any combination of the above upper and lower limits is also possible.

[0083] In embodiments where the electrical signal is applied continuously, the electrical signal has a frequency of ≦50 Hz, preferably ≦10 Hz, more preferably ≦2 Hz, and even more preferably ≦1 Hz. For example, the frequency can be ≦1 Hz, ≦2 Hz, ≦5 Hz, or ≦10 Hz. In other examples, the frequency can be ≦0.1 Hz, ≦0.2 Hz, ≦0.3 Hz, ≦0.4 Hz ≦0.5 Hz, ≦0.6 Hz ≦0.7 Hz, ≦0.8 Hz, or ≦0.9 Hz. Additionally or alternatively, the frequency of the electrical signal can be ≧0.1 Hz, ≧0.2 Hz, ≧0.5 Hz, ≧1 Hz, ≧2 Hz, or ≧5 Hz. Any combination of the above upper and lower limits is possible.

[0084] If the signal waveform includes a pulse train, the pulses are applied to the nerve at intervals according to the frequency, e.g., a frequency of 50 Hz results in 50 pulses being applied to the nerve per second.

[0085] Electrode and Neural Interface Design An electrical signal is applied to a nerve innervating the spleen associated with the neurovascular bundle (e.g., the splenic artery nerve) via at least one electrode in signal transmitting contact with the nerve, which at least one electrode can be disposed on the neural interface 10.

[0086] In some embodiments, the electrodes and / or neural interface are configured to be placed around at least one splenic artery nerve and / or around the splenic artery, in such embodiments, the neural interface can be a cuff-type interface, although other interfaces that partially or completely surround the nerve can be used.

[0087] In other embodiments, the neural interface 10 is configured to be placed over at least one splenic nerve and / or over the splenic artery, hi such embodiments, the neural interface 10 may be a patch-type or clip-type interface.

[0088] In other embodiments, the neural interface 10 is configured to be placed within the splenic artery, hi such embodiments, the neural interface may be a catheter or probe type interface.

[0089] In other embodiments, the neural interface 10 is configured to be placed within at least one splenic artery nerve, hi such embodiments, the neural interface may be a pin-type interface.

[0090] The neural interface includes at least one electrode, which may be made from or partially or entirely coated with a high charge capacity material, such as platinum black, iridium oxide, titanium nitride, tantalum, poly(ethylenedioxythiophene), and suitable combinations thereof.

[0091] The at least one electrode may be a flexible flat interface electrode, particularly in embodiments where the neural interface is configured to be placed on or around at least one splenic nerve and / or splenic artery such that when the neural interface 10 is secured over the nerve it surrounds the nerve and / or splenic artery, although other electrode types are suitable for use with the present invention.

[0092] Other electrode types suitable for the present invention include cuff electrodes (e.g., spiral cuffs, helical cuffs, or flat interfaces); half-cuff electrodes; mesh, wire-and-rod, paddle-lead, or disc contact electrodes (such as multi-disc contact electrodes); hook electrodes; sling electrodes; intrafascicular electrodes; glass suction electrodes; paddle electrodes; and percutaneous cylinder electrodes.

[0093] The at least one electrode may include a first electrode 11 and a second electrode 12, referred to herein as a bipolar electrode configuration. Figure 1 is a schematic diagram of an exemplary bipolar electrode configuration in which electrodes are placed in signaling contact with at least one splenic nerve and / or splenic artery. As described elsewhere herein, suitable signaling contact may be achieved by placing electrodes around (i.e., partially or completely surrounding), on, or within the splenic nerve or artery.

[0094] As shown in Figure 1, a first electrode 11 and a second electrode 12 are positioned along the long axis of the nerve. An electrical signal can be applied to the electrodes such that the first electrode 11 is the anode and the second electrode 12 is the cathode. Alternatively, the first electrode 11 can be the cathode and the second electrode 12 can be the anode.

[0095] In other embodiments, the at least one electrode can include a first electrode, a second electrode, and a third electrode, referred to herein as a tripolar electrode configuration.

[0096] Similar to the bipolar configuration, the first, second and third electrodes can be positioned along the long axis of the nerve, and in one example, the second electrode can be positioned between the first electrode 11 and the third electrode 13.

[0097] The electrodes may be at least partially insulated from one another by a non-conductive biocompatible material, to which end the neural interface may include non-conductive biocompatible material spaced laterally along the nerve when the device is in use.

[0098] The present inventors have found a suitable electrode size for applying an electrical signal to at least one splenic artery nerve. The total surface area of ​​the electrode is 0.1 to 0.3 mm. 2 Preferably, the total surface area of ​​the electrodes is less than 0.2 cm 2 is less than.

[0099] In a preferred electrode configuration, the width of each of the first electrode 11 and the second electrode 12 may be 1 to 4 mm. For example, the width may be 1 mm to 3 mm, or 2 mm to 4 mm, or 2 mm to 3 mm.

[0100] Control device (controller) 1, the system 50 of the present invention, which may include a neural interface, may also include at least one controller, e.g., a microprocessor 60, electrically coupled to at least one electrode of the neural interface 10 and configured to control the operation of said at least one electrode. The at least one controller may be responsible for triggering the initiation and / or termination of a signal sent to a nerve by the at least one electrode. Optionally, the at least one controller may also be responsible for generating and / or controlling signal parameters.

[0101] The at least one controller can be configured to operate in an open loop fashion, delivering a predetermined signal (as described above) to the nerve in a predetermined application pattern (also described above), with or without an external trigger and without a control or feedback mechanism, or the at least one controller can be configured to operate in a closed loop fashion, applying a signal based on a control or feedback mechanism.

[0102] The at least one controller is preferably constructed to generate, in use, a preset and / or operator selectable signal that is independent of inputs at system 50. The preset and / or operator selectable signal may be any one of the electrical signals described above. In other embodiments, the at least one controller is responsive to an external signal, more preferably information (e.g., data) relating to one or more physiological parameters of the subject, yet still within the scope of the signals described above.

[0103] The at least one control device may be a microprocessor 60 in a system 50 suitable for implantation in a subject.

[0104] Alternatively or additionally, the at least one controller may be a controller external to the subject.

[0105] The at least one controller may be triggered upon receipt of a signal generated by an operator, e.g., a physician or a subject having device 106 implanted therein. To that end, system 50 may further include an external system 80 that includes controller 101. An example of such a system is described below with reference to FIG.

[0106] The external system 80 of the broader system 100 is external to the system 50 and to the subject and includes a controller 101. The controller 101 can be used to control and / or power the system 50. To that end, the controller 101 may include a power unit 102 and / or a programming unit 103. The external system 80 may further include a power transmitting antenna 104 and a data transmitting antenna 105, which are further described below.

[0107] The at least one controller, such as microprocessor 60 and controller 101, may be a processor connected to a memory (i.e., a non-transitory computer readable storage medium) having an executable computer program including code portions that, when loaded into the processor and operated, cause the processor to at least control the operation of the at least one electrode. By controlling the operation, it is meant that the at least one controller causes the at least one electrode to apply an electrical signal to a nerve using any of the signal parameters and patterns of application described above.

[0108] Neurostimulation Systems In addition to the neural interface 10 and the at least one controller 60, the system 50 may include a signal generator 113 configured to send the above-mentioned electrical signal to the at least one electrode in response to a control operation from the at least one controller. The signal generator may include at least one current source or voltage source.

[0109] The signal generator 113 can be electrically coupled to the at least one control device and the at least one electrode. In some embodiments, the at least one electrode can be connected to the signal generator 113 via conductors 107. In some embodiments, the conductors can be connected to the aforementioned interconnect. Alternatively, the signal generator 113 can be directly integrated into the at least one electrode without conductors. In either case, the system 50 can include a device 106 that can be implanted in the subject and can include a DC direct current blocking output circuit (or an AC direct current blocking output circuit) based on all output channels (e.g., output to at least one electrode or physiological sensor 111), optionally based on capacitors and / or inductors.

[0110] In addition to the neural interface 10, at least one electrode, at least one controller, and signal generator 113, system 50 may include one or more of the following components: an implantable transceiver 110, a power source 112, a memory 114 (alternatively referred to as a non-transitory computer readable storage device), a physiological sensor 111, and a physiological data processing module 115. The physiological sensor 111 and the physiological data processing module 115 are referred to herein as detectors.

[0111] The various components of system 50 are preferably part of a single physical device, either sharing a common housing or being a physically separate collection of interconnected components connected by electrical leads, as shown in FIG. 2. Alternatively, however, the invention may employ a system in which the components are physically separate and communicate wirelessly. Thus, for example, the at least one electrode and the implantable device (e.g., implantable device 106) may be part of a unitary device or together may form a system (e.g., system 50). In both cases, additional components may be present to form a wider system (e.g., system 100).

[0112] For example, in some embodiments, the implantable device 106 may include one or more of the following components: a power source 112; a memory 114; and a physiological data processing module 115.

[0113] The power source 112 may include a current source and / or a voltage source for providing power to the signal generator 113. The power source 112 may also provide power to the implantable device 106 and / or other components of the system 50, such as the microprocessor 60, the memory 114, and the implantable transceiver 110. The power source 112 may include a battery, which may be rechargeable.

[0114] It will be apparent that the availability of power is limited in implantable devices, and the present invention was conceived with this limitation in mind. The implantable device 106 and / or system 50 can be powered by an inductive or rechargeable power source.

[0115] Memory 114 may store power data and data relating to one or more physiological parameters. For example, memory 114 may store data relating to one or more signals indicative of the one or more physiological parameters detected by a detector (e.g., the one or more corresponding physiological parameters determined via physiological sensor 111 and / or via physiological data processing module 115). Additionally or alternatively, memory 114 may store power data and data relating to the one or more physiological parameters from external system 80 via implantable transceiver 110. To this end, implantable transceiver 110 may form part of a communication subsystem of broader system 100, discussed further below.

[0116] The physiological data processing module 115 is configured to process one or more signals indicative of one or more physiological parameters detected by the physiological sensor 111 to determine one or more corresponding physiological parameters. The physiological data processing module 115 can be configured to reduce a size of data associated with the one or more physiological parameters for storage in the memory 114 and / or transmission to an external system via the implantable transceiver 110. The implantable transceiver 110 can include one or more antennas. The implantable transceiver 100 can use any suitable signaling process, such as RF, wireless, infrared, etc., to transmit signals from outside the body to, for example, a wider system 100 of which the system 50 is a part.

[0117] Alternatively or additionally, the physiological data processing module 115 may be configured to process the signal indicative of one or more physiological parameters and / or process the measured one or more physiological parameters to determine the evolution of a disease in the subject. In such a case, the system 50, and in particular the implantable device 106, will include the capability to calculate and adjust the signal parameters based on said one or more physiological parameters of the subject and the measured progression of the disease in the subject.

[0118] The physiological data processing module 115 and the at least one physiological sensor 111 can form a physiological sensor subsystem, also referred to herein as a detector, either as part of the system 50, as part of the implantable device 106, or external to the system.

[0119] There may be at least one detector configured to detect one or more physiological parameters related to the treatment of an inflammatory disorder, such as a decrease in proinflammatory cytokines and chemokines, an increase in anti-inflammatory cytokines (e.g., IL-10) and / or convergent mediators (e.g., resolvins, lipoxins, eicosanoids, maresins, and protectins), an increase in catecholamines and acetylcholine, changes in hematology and cell counts, such as changes in immune cell populations or immune cell surface costimulatory molecules, a decrease in factors involved in the inflammatory cascade, and / or a decrease in immune response mediators, as discussed further below. For example, the detector may be configured to detect biomolecule concentrations using electrical, RF, or optical (visible, infrared) biochemical sensors.

[0120] There may be at least one detector configured to detect other physiological parameters, such as blood flow velocity in the spleen, blood flow velocity in the splenic artery, blood flow velocity in the splenic vein, splenic volume, neural activity in at least one splenic artery nerve, or impedance of at least one electrode.

[0121] For example, the detector can be configured to detect blood flow within an artery or vein or using surrounding intravascular or perivascular flow tubes, or the detector can detect splenic artery contraction and blood flow changes using electrical impedance tomography, electrical impedance, stimulator voltage compliance, Doppler blood flow, splenic tissue perfusion, ultrasound, strain measurements, or pressure.

[0122] In another example, the detector can be configured to detect neural activity of at least one splenic artery nerve using an electrical sensor. If the detector is configured to detect neural activity of a single splenic artery nerve, the detector can detect an action potential. If the detector is configured to detect neural activity of multiple splenic artery nerves, the detector can detect a compound action potential.

[0123] In a further example, the detector can be configured to detect spleen volume using ultrasound.

[0124] In another example, the detector may be configured to detect the impedance of the at least one electrode using an impedance meter, preferably a low current AC (e.g., 1 kHz) impedance meter. In particular, the detector may detect the impedance between the at least one electrode and ground and / or between an electrode pair (if there are multiple electrodes) of the at least one electrode. In such an example, the at least one electrode is suitable for placement on or around a nerve.

[0125] In another example, the detector can be configured to detect movement of the subject using an accelerometer to ascertain when the subject is asleep by determining whether the subject is lying down, i.e., whether there has been an extended period (e.g., >70 minutes) in which the subject has maintained a substantially lying position, the measurement being based on the direction and acceleration experienced and measured by the accelerometer.

[0126] The physiological parameters measured by the detectors can be used to trigger the microprocessor 60 to send signals of the types described above to the nerves using at least one electrode. Upon receiving signals indicative of the physiological parameters received from the physiological sensors 111, the physiological data processor 115 can perform calculations according to techniques known in the art to ascertain the physiological parameters and disease progression of the subject. For example, if a signal indicative of excessive cytokine (e.g., TNF) concentrations in the circulation is detected, the processor can trigger the sending of a signal to reduce secretion of a particular signaling molecule, as described elsewhere herein.

[0127] The memory 114 may store physiological data relating to normal levels of one or more physiological parameters, the data being specific to the subject implanted with the system 50 and may be collected from various tests known in the art. Upon receiving a signal indicative of a physiological parameter received from the physiological sensor 111, or periodically or upon request from the physiological sensor 111, the physiological data processor 115 may compare the physiological parameter determined from the signal received from the physiological sensor 111 to the data relating to normal levels of the physiological parameter stored in the memory 114 and determine whether the received signal indicates insufficiency or excess of a particular physiological parameter, thereby indicating the development of a disease in the subject.

[0128] The system 50 and / or the implantable device 106 can be configured such that if and when an insufficient or excessive level of a physiological parameter is measured by the physiological data processor 115, the physiological data processor 115 triggers the transmission of a signal by at least one electrode to the nerve in a manner described elsewhere herein. For example, if a physiological parameter indicative of a worsening of any of the physiological parameters and / or disease is measured, the physiological data processor 115 can trigger the transmission of a signal to reduce the secretion of a particular biochemical, as described elsewhere herein. Particular physiological parameters relevant to the present invention are described above. When the physiological data processor 115 receives one or more signals indicative of one or more of these physiological parameters, a signal can be applied to the nerve via the at least one electrode.

[0129] In some embodiments, the controller 101 can be configured to make adjustments to the operation of the system 50. For example, it can transmit physiological parameter data related to normal levels of signaling molecules secreted by the spleen via the communications subsystem (discussed further below). The data can be specific to the subject in which the device is implanted. The controller 101 can also be configured to adjust the signals sent to the nerves by the neural interface 10 by making adjustments to the operation of the power source 112, the signal generator 113, and the processing elements 60, 115 and / or the electrodes.

[0130] As an alternative or in addition to the ability of system 50 and / or implantable device 106 to respond to physiological parameters of the subject, microprocessor 60 may be triggered upon receipt of a signal generated by an operator (e.g., a physician or the subject in which system 50 is implanted). To that end, system 50 may be part of a broader system 100 that includes external system 80 and controller 101, discussed further below.

[0131] Beyond Neurostimulation Systems The neurostimulation system 50 may be part of a broader system 100 that includes a number of subsystems, such as an external system 80 (see FIG. 2 ). The external system 80 may be used to power and program the neurostimulation system 50 through the human skin and underlying tissue.

[0132] In addition to the controller 101, the external subsystem 80 may include one or more of: a power supply unit 102 for wirelessly recharging a battery of a power source 112 used to power the implantable device 106; and a programming unit 103 configured to communicate with the implantable transceiver 110. The programming unit 103 and the implantable transceiver 110 may form a communications subsystem. In some embodiments, the power supply unit 102 is housed together with the programming unit 103. In other embodiments, they may be housed in separate devices.

[0133] The external subsystem 80 may also include one or more of a power transmitting antenna 104; and a data transmitting antenna 105. The power transmitting antenna 104 may be configured to transmit an electromagnetic field at a low frequency (e.g., 30 kHz to 10 MHz). The data transmitting antenna 105 may be configured to transmit data for programming or reprogramming the implantable device 106 and may be used in addition to the power transmitting antenna 104 to transmit an electromagnetic field at a high frequency (e.g., 1 MHz to 10 GHz). During operation of the power transmitting antenna 104, the skin temperature is not more than 2° C. higher than the surrounding tissue. The at least one antenna of the implantable transceiver 110 may be configured to receive power from an external electromagnetic field generated by the power transmitting antenna 104, which may be used to charge a rechargeable battery of the power source 112.

[0134] The power transmitting antenna 104, the data transmitting antenna 105, and the at least one antenna of the implantable transceiver 110 have certain characteristics such as a resonant frequency and a quality factor (Q). One example of an antenna is a coil of wire with or without a ferrite core that forms an inductor with a defined inductance. The inductor may be coupled with a resonant capacitor and resistive losses to form a resonant circuit. The frequency is set to match the frequency of the electromagnetic field generated by the power transmitting antenna 105. A second antenna of the at least one antenna of the implantable transceiver 110 may be used in the system 50 for receiving and transmitting data from / to the external system 80. When multiple antennas are used in the system 50, the antennas may be rotated by 30° with respect to each other to achieve a better degree of power transfer efficiency when slightly misaligned with the power transmitting antenna 104.

[0135] The external system 80 may include one or more externally worn physiological sensors 121 (not shown) for detecting signals indicative of one or more physiological parameters. The signals may be transmitted to the system 50 via at least one antenna of the implantable transceiver 110. Alternatively or additionally, the signals may be transmitted to the external system 50 and then to the system 50 via at least one antenna of the implantable transceiver 110. Similar to the signals indicative of the one or more physiological parameters detected by the implantable physiological sensor 111, the signals indicative of the one or more physiological parameters detected by the external sensor 121 may be processed by the physiological data processing module 115 to determine one or more physiological parameters and / or stored in the memory 114 to operate the system 50 in a closed loop manner. The physiological parameters of the subject determined via signals received from the external sensor 121 may be used in addition to or instead of the physiological parameters determined via signals received from the implantable physiological sensor 111.

[0136] For example, in certain embodiments, detectors external to the implanted device may include non-invasive blood flow monitors, such as ultrasonic flow meters and / or non-invasive blood pressure monitors, and measurements of changes in physiological parameters, particularly those physiological parameters mentioned above, etc. As explained above, in response to measurements of one or more of these physiological parameters, the detectors may trigger the transmission of a signal by at least one electrode to the splenic artery nerve, or may alter parameters of the signal sent or the signal to be sent to the nerve by at least one electrode in the future.

[0137] System 100 may include safety protection functions that discontinue electrical stimulation of a nerve upon the following example events: abnormal operation of system 50 (e.g., excessive voltage); abnormal readings from implanted physiological sensor 111 (e.g., temperature rise of more than 2° C. or excessively high or low electrical impedance at the electrode-tissue interface); abnormal readings from externally worn physiological sensor 121 (not shown); or an abnormal response to stimulation detected by an operator (e.g., physician or subject). The safety protection functions may be implemented via controller 101 and communicated to system 50 or internally within system 50.

[0138] The external system 80 may include an actuator 120 (not shown) that, when pressed by an operator (e.g., a physician or subject), sends a signal via the control device 101 and a respective communication subsystem to trigger the microprocessor 60 of the system 50 to send a signal to a nerve via at least one electrode.

[0139] The external system 80 may include a display 109 for the microcontroller 60 or controller 101 to alert an operator (e.g., a physician or a subject) about the status of the system or the subject. The display 109 may be a monitor, such as an LED monitor, or may be a visual indicator, such as an LED.

[0140] The system 100 of the present invention, including the external system 80 but not specifically the system 50, is preferably made from or coated with a biostable and biocompatible material. This means that the system is protected from damage due to exposure to the body's tissues, while at the same time reducing the risk that the system will cause an undesirable reaction by the host that may ultimately lead to rejection. The materials used to construct or coat the system should ideally be resistant to biofilm formation. Suitable materials include, but are not limited to, poly(p-xylylene) polymers (referred to as parylenes) and polytetrafluoroethylene.

[0141] An implantable device 106 of the present invention will typically weigh less than 50 g.

[0142] Determining correct placement of the neural interface The present invention also provides a method for determining whether the neural interface 10 is properly placed in signaling contact with a nerve (e.g., the splenic artery nerve) that innervates the spleen associated with the neurovascular bundle, the method comprising the steps of: A. providing a system of the present invention; B. Positioning an electrode and / or neural interface in signal transducing contact with a nerve; C. applying an electrical signal to the nerve by controlling operation of the at least one electrode with at least one controller; D. determining that a change in blood flow velocity in the spleen, splenic artery, or splenic vein, a decrease in splenic volume, a decrease in splenic perfusion, a change in systemic arterial blood pressure and heart rate, a decrease in electrical impedance or voltage compliance, an increase in neural activity at the nerve, or a change in impedance of at least one electrode is detected; E. Indicating to an operator that the neural interface is properly placed in signal transducing contact with the nerve. Includes.

[0143] Referring to step A of the method, a system of the invention may be provided that includes a neural interface having at least one electrode, at least one controller, and at least one detector. Any other features of the systems described herein may be provided.

[0144] In step B, the electrodes and / or neural interface are preferably placed in signal transducing contact with the nerve. In some embodiments of the method, step B can be omitted. For example, step B can be omitted if it has been determined whether a previously implanted electrode and / or neural interface has migrated over time.

[0145] In step C, the at least one control device applies an electrical signal to the nerve by controlling the operation of the at least one electrode. The electrical signal may be similar to the electrical signal used to stimulate neural activity described above in that it may have the same waveform and also apply the same charge density per phase to the nerve. However, the total charge applied to the nerve is preferably higher. This may be achieved by using a continuous signal application instead of a periodic signal application and / or by using a higher frequency than that described for the continuous signal application above.

[0146] In particular, the signal can be applied continuously at a frequency at the rate previously described for periodic signal application. Thus, the electrical signal can have a frequency of ≦300Hz, preferably ≦50Hz, more preferably ≦10Hz. For example, continuous stimulation ≦10Hz can be used for efficacy and / or treatment; and / or continuous stimulation ≦30Hz and ≧5Hz can be used for splenic artery blood flow change detection; and / or periodic stimulation ≧10Hz (or optionally ≧5Hz) can be used for efficacy and / or treatment. For example, the frequency of the electrical signal can be ≦50Hz, ≦100Hz, ≦150Hz, ≦200Hz, ≦250Hz, or ≦300Hz. In other examples, the frequency of the electrical signal can be ≦10Hz, ≦15Hz, ≦20Hz, ≦25Hz, ≦30Hz, ≦35Hz, ≦40Hz, ≦45Hz, or ≦50Hz. In further examples, the frequency may be ≦1 Hz, ≦2 Hz, ≦5 Hz, or ≦10 Hz. Additionally or alternatively, the frequency of the electrical signal may be ≧10 Hz, ≧15 Hz, ≧20 Hz, ≧25 Hz, ≧30 Hz, ≧35 Hz, ≧40 Hz, ≧45 Hz, or ≧50 Hz. In other examples, the frequency of the electrical signal may be ≧0.1 Hz, ≧0.2 Hz, ≧0.5 Hz, ≧1 Hz, ≧2 Hz, or ≧5 Hz. Any combination of the above upper and lower limits is possible.

[0147] The electrical signal can be applied continuously in one of the aforementioned application episodes (e.g., 400 to 600 pulses). Alternatively, the electrical signal can be applied continuously in episodes defined by a set period. Preferably, the set period of electrical signal application is ≦3 hours. For example, the set period can be ≦1 min, ≦2 min, ≦5 min, ≦10 min, ≦20 min, ≦30 min, ≦1 hour, ≦2 hours, or ≦3 hours. Additionally or alternatively, the set period can be ≧1 min, ≧2 min, ≧5 min, ≧10 min, ≧20 min, ≧30 min, ≧1 hour, or ≧2 hours. Any combination of the above upper and lower limits is possible.

[0148] In step D, the at least one detector may detect one or more of blood flow velocity in the spleen, blood flow velocity in the splenic artery, blood flow velocity in the splenic vein, splenic volume, neural activity in a nerve, or impedance of the at least one electrode. Detectors suitable for detecting these physiological parameters are described above.

[0149] When the detector is configured to detect blood flow velocity in the spleen, blood flow velocity in the splenic artery, and / or blood flow velocity in the splenic vein, the electrode and / or neural interface can be determined to be correctly placed in signal transmitting contact with the nerve if the detected blood flow differs from the baseline blood flow.

[0150] If the detector is configured to detect neural activity at the nerve, the electrode and / or neural interface can be determined to be in signaling contact with the nerve and correctly positioned when the detected neural activity is higher than baseline neural activity.

[0151] If the detector is configured to detect a spleen volume, the electrode and / or neural interface may be determined to be in signaling contact with the nerve and correctly placed when the detected spleen volume is less than the baseline spleen volume, in other words, when the spleen is contracting.

[0152] If the detector is configured to detect the impedance of the at least one electrode, the electrode and / or neural interface can be determined to be in signaling contact with the nerve and correctly placed when the detected impedance differs from a baseline impedance. The detected impedance is the impedance between a first electrode and a second electrode of the at least one electrode measured while applying a signal to the nerve. The "baseline impedance" is the impedance between the first electrode and the second electrode prior to applying a signal to the nerve.

[0153] According to step E, once the at least one controller has determined that the neural interface is properly placed on the nerve, the at least one controller may indicate so to an operator. The indication to the operator may take the form of a notification on the system's display 109, a flashing LED, or the like.

[0154] If the detector determines that the electrodes and / or neural interface are not properly placed in signaling contact with the nerve, the at least one controller may indicate to the operator via the display 109 that the neural interface is not properly placed in signaling contact with the nerve.

[0155] If the neural interface is not properly placed, steps B through E can be repeated until the neural interface is properly placed in signal transducing contact with the nerve.

[0156] Steps C, D and E of the method are executable by at least one controller including a processor connected to a memory (i.e., a non-transitory computer readable storage medium) having an executable computer program including code portions that, when loaded into and operated by the processor, cause the processor to execute steps C, D and E.

[0157] The method for verifying neural interface placement can also be used to track the success of the system of the invention (i.e., implanted in a subject) post-operatively throughout the patient's life (e.g., using external transcutaneous splenic blood flow measurements or systemic arterial blood pressure as markers of good nerve-electrode intercation). Thus, in addition to ensuring correct placement, the method provides a way to determine proper efficacy (where cytokine readout would not be possible).

[0158] Inflammatory disorders The present invention is useful for treating conditions associated with an imbalance of pro- and anti-inflammatory cytokine profiles compared to physiological homeostatic states, such as inflammatory disorders (eg, chronic inflammatory disorders).

[0159] Inflammatory disorders are typically characterized by an imbalance in pro- and anti-inflammatory cytokine profiles compared to normal physiological homeostatic conditions, e.g., elevated levels of pro-inflammatory cytokines and / or decreased levels of anti-inflammatory cytokines compared to normal physiological homeostatic conditions.

[0160] Thus, the present invention is useful in treating subjects suffering from or at risk of developing an inflammatory disorder. The present invention can treat or improve the effectiveness of inflammatory disorders by reducing inflammation. This can be achieved by reversibly electrically stimulating nerves innervating the spleen that are associated with the neurovascular bundle, thereby decreasing the production and release of inflammatory cytokines from the spleen and / or increasing the production and release of anti-inflammatory cytokines and / or mediators with converging effects (e.g., proresolvins).

[0161] Inflammatory disorders include autoimmune disorders such as arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Graves' disease, myasthenia gravis, thyroiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcet's syndrome, allograft rejection, graft versus host disease, ankylosing spondylitis, Buerger's disease, diabetes such as type I diabetes, Reiter's syndrome, spondyloarthropathy psoriasis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, Addison's disease, autoimmune-mediated hair loss (e.g., alopecia areata), and ulcerative colitis.

[0162] Certain examples of inflammatory disorders include diseases involving the digestive tract and associated tissues, such as appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute and ischemic colitis, diverticulitis, cholangitis, cholecystitis, Crohn's disease, Whipple's disease, hepatitis, abdominal obstruction, intestinal volvulus, postoperative ileus, intestinal obstruction, celiac disease, periodontal disease, pernicious anemia, amebiasis and enteritis.

[0163] Further examples include systemic or local inflammatory diseases and conditions such as asthma, allergies, anaphylactic shock, immune complex disease, sepsis, septicemia, endotoxic shock, eosinophilic granuloma, granulomatous disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, cachexia, hyperexia, septic abortion, HIV infection, herpes infection, allograft rejection, disseminated bacteremia, dengue fever, malaria, and sarcoidosis.

[0164] Other examples include diseases involving the genitourinary system and associated tissues, such as epididymitis, vaginitis, orchitis, urinary tract infections, kidney stones, prostatitis, urethritis, pelvic inflammatory colitis, contrast induced nephropathy, reperfusion renal injury, acute kidney injury, infected kidney stones, herpes infections and candidiasis.

[0165] Other examples include the respiratory system and associated tissues, such as bronchitis, asthma, hay fever, ventilator-induced lung injury, cystic fibrosis, adult respiratory distress syndrome, pneumonia, alveolitis, epiglottitis, rhinitis, achlasia, respiratory syncytial virus, pharyngitis, sinusitis, pneumonia, alveolitis, influenza, pulmonary embolism, hydatid cyst and / or bronchiolitis.

[0166] Further examples are skin diseases and conditions (e.g., burns, dermatitis, dermatomyositis, burns, cellulitis, abscesses, contact dermatitis, dermatomyositis, warts, wheals, sunburn, urticaria warts, and wheals); diseases involving the cardiovascular system and related tissues (e.g., myocardial infarction, cardiac tamponade, vasculitis, aortic dissection, coronary artery disease, peripheral vascular disease, abdominal aortic aneurysm, vasculitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, congestive heart failure, periarteritis nodosa, and rheumatic fever, filariasis, thrombophlebitis, deep vein thrombosis); as well as various cancers, tumors and proliferative disorders (e.g., Hodgkin's disease), hospital-acquired infections; and, in any case, the inflammatory or immune host response to the primary disease.

[0167] Other examples of inflammatory disorders include diseases involving the central or peripheral nervous system and associated tissues, such as Alzheimer's disease, depression, multiple sclerosis, cerebral infarction, cerebral embolism, carotid artery disease, cerebral concussion, subdural hematoma, epidural hematoma, transient ischemic attack, temporal arteritis, spinal cord injury without imaging findings (SCIWORA), spinal cord compression, meningitis, encephalitis, cardiac arrest, Guillain-Barré, spinal cord injury, cerebral venous thrombosis, and paralysis.

[0168] Inflammatory disorders also include immune or inflammatory response-related conditions (i.e., acute inflammatory episodes), including injury to nerves and other tissues and pain associated with nerves or other tissues. Injury can be due to physical, chemical, or mechanical trauma. Non-limiting examples of injury include acute trauma, burns, whiplash, musculoskeletal contusion, and postoperative complications such as DVT, arrhythmia, ventilator-induced lung injury, and postoperative ileus.

[0169] Conditions relating to specific organs such as the eye or ear may also be due to immune or inflammatory responses, such as conjunctivitis, iritis, glaucoma, episcleritis, acute retinal occlusion, cataract, otitis media, otitis externa, uveitis and Meniere's disease.

[0170] Another example of an inflammatory disorder is postoperative ileus (POI). POI is experienced by the majority of patients undergoing abdominal surgery. POI is characterized by temporary impairment of gastrointestinal (GI) function along the GI tract, as well as pain and discomfort for the patient and increased hospitalization costs.

[0171] Impairment of GI function is not limited to the surgical site; for example, patients undergoing laparotomy may experience colon or ruminal dysfunction. POI is mediated, at least in part, by elevated levels of inflammatory cytokines and infiltration of leukocytes at the surgical site. In response to inflammation, neural inhibitory pathways are activated, contributing to paralysis of secondary GI organs distal to the surgical site. Thus, stimulation of neural activity as described herein may be effective in treating or preventing POI.

[0172] The present invention is particularly useful in treating autoimmune disorders (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis, spondyloarthropathy, ankylosing spondylitis, psoriasis, systemic lupus erythematosus (SLE), multiple sclerosis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis) and sepsis.

[0173] The present invention is particularly useful in the treatment of B cell mediated autoimmune disorders, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

[0174] The present invention is particularly useful in treating inflammatory conditions associated with bacterial infections, for example, inflammatory conditions caused or exacerbated by infection with Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitides, Streptococcus pneumoniae, Methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella or Enterobacter.

[0175] Treatment of inflammatory disorders Treatment of an inflammatory disorder can be assessed in a variety of ways, but typically involves measuring an improvement in one or more physiological parameters of the subject.

[0176] Useful physiological parameters of the present invention may be one or more of the group consisting of levels of inflammatory cytokines, levels of anti-inflammatory cytokines and / or mediators with a converging effect, levels of catecholamines, levels of immune cell populations, levels of red blood cells, levels of immune cell surface costimulatory molecules, levels of factors involved in the inflammatory cascade, levels of immune response mediators, and clinical observations and scoring systems (e.g., disease activity score or other suitable methods).

[0177] As used herein, "improvement in a measured physiological parameter" is understood to mean that for a given physiological parameter, the improvement is a change in the value of that parameter in a subject toward the normal value or normal range for that value, i.e., toward the expected value in a healthy subject. As used herein, "deterioration in a measured physiological parameter" is understood to mean that for a given physiological parameter, the deterioration is a change in the value of that parameter in a subject away from the normal value or normal range for that value, i.e., away from the expected value in a healthy subject.

[0178] Improvements in measured physiological parameters according to the present invention are indicated by one or more of the group consisting of reduction in inflammatory cytokines, increase in anti-inflammatory cytokines and / or mediators with convergent action, increase in catecholamines, levels of red blood cells, changes in immune cell populations, changes in immune cell surface costimulatory molecules, reduction in factors involved in the inflammatory cascade, and changes in levels of immune response mediators. The present invention may not result in changes in all of these parameters.

[0179] Stimulation of the splenic artery nerve can cause the spleen to (a) decrease secretion of proinflammatory cytokines compared to basal secretion; and / or (b) increase secretion of anti-inflammatory cytokines and / or mediators with convergent effects compared to basal secretion. For example, the decrease in proinflammatory cytokine secretion can be ≦5%, ≦10%, ≦15%, ≦20%, ≦25%, ≦30%, ≦35%, ≦40%, ≦45%, ≦50%, ≦60%, ≦70%, ≦80%, ≦90%, or ≦95%. The increase in anti-inflammatory cytokine secretion can be ≦5%, ≦10%, ≦15%, ≦20%, ≦25%, ≦30%, ≦35%, ≦40%, ≦45%, ≦50%, ≦60%, ≦70%, ≦80%, ≦90%, ≦95%, ≦100%, ≦150%, ≦200%, or ≦500-1000%.

[0180] When a cytokine is secreted into the circulation, its concentration in the circulation is diluted. Stimulation of the splenic artery nerve can (a) reduce the level of inflammatory cytokines in plasma or serum by ≦5%, ≦10%, ≦15%, ≦20%, ≦25%, ≦30%, ≦35%, ≦40%, ≦45%, ≦50%, ≦60%, ≦70%, ≦80%, ≦90%, or ≦95%; and / or (b) increase the level of anti-inflammatory cytokines and / or mediators with resolving effects in plasma or serum by ≦5%, ≦10%, ≦15%, ≦20%, ≦25%, ≦30%, ≦35%, ≦40%, ≦45%, ≦50%, ≦60%, ≦70%, ≦80%, ≦90%, ≦95%, ≦100%, ≦150%, or ≦200%. Preferably, the level in serum is measured.

[0181] By stimulating the splenic artery nerve, levels of catecholamines (e.g., norepinephrine or epinephrine), e.g., in the spleen or splenic vein, can be increased by, for example, ≦5%, ≦10%, ≦15%, ≦20%, ≦25%, ≦30%, ≦35%, ≦40%, ≦45%, ≦50%, ≦60%, ≦70%, ≦80%, ≦90%, ≦95%, ≦100%, ≦150% or ≦200%.

[0182] For example, the inventors have found that stimulation of the splenic artery nerve can reduce the levels of inflammatory cytokines (e.g., TNFα) in serum by 30%-60%.

[0183] Inflammatory cytokines are known in the art, and examples include tumor necrosis factor (TNF; also called TNFα or cachectin), interleukin (IL)-1α, IL-1β, IL-2, IL-5, IL-6, IL-8, IL-15, IL-18, interferon gamma (IFN-γ), platelet activating factor (PAF), thromboxane, soluble adhesion molecules, vasoactive neuropeptides, phospholipase A2, plasminogen activator inhibitor (PAI-1), free radical generation, neopterin, CD14, prostacyclin, neutrophil elastase, protein kinases, monocyte chemotactic protein 1 and 2 (MCP-1, MCP-2), macrophage migration inhibitory factor (MIF), high mobility group box protein 1 (HMGB-1), and other known factors.

[0184] Anti-inflammatory cytokines are known in the art, examples of which include IL-4, IL-10, IL-17, IL-13, IL-1α, and TNFα receptors.

[0185] Pro-resolving mediators are also known in the art. In particular, specialized pro-resolving mediators are a class of molecules produced during the metabolism of polyunsaturated fatty acids through the action of several enzymes (e.g., lipoxygenase, cyclooxygenase, cytochrome P450, and others). They resolve acute and chronic inflammation. Examples include lipoxins, resolvins, protectins, and maresins.

[0186] It will be apparent that some pro-inflammatory cytokines can act as anti-inflammatory cytokines in certain circumstances, and vice versa. Such cytokines are typically referred to as multifunctional cytokines.

[0187] Factors involved in the immune response can be measurable parameters useful in the present invention, for example, TGF, PDGF, VEGF, EGF, FGF, I-CAM, nitric oxide.

[0188] Chemokines can also be measurable parameters useful in the present invention, such as 6cKine and MIP3β, and chemokine receptors, such as the CCR7 receptor.

[0189] Changes in immune cell populations (Langerhans cells, dendritic cells, lymphocytes, monocytes, macrophages) or immune cell surface costimulatory molecules (major histocompatibility, CD80, CD86, CD28, CD40) may also be measurable parameters useful in the present invention. Application of a signal to a nerve in accordance with the present invention may cause a reduction in the total number of circulating or tissue-specific (e.g., joint-specific in the case of rheumatoid arthritis) white blood cells (e.g., monocytes and macrophages, lymphocytes, neutrophils, etc.).

[0190] Factors involved in the inflammatory cascade may also be measurable parameters useful in the present invention. For example, the signal transduction cascade includes factors such as NFκ-B, Egr-1, Smads, Toll-like receptors, and MAP kinases.

[0191] Methods for assessing these physiological parameters are known in the art. Detection of any of the above measurable parameters can occur before, during, and / or after modulation of neural activity in the nerve.

[0192] For example, cytokines, chemokines, or catecholamines (e.g., norepinephrine or epinephrine) can be detected directly, for example, by ELISA. Alternatively, the presence or amount of a nucleic acid, such as a polyribonucleotide, encoding a polypeptide described herein can serve as a measure of the presence or amount of the polypeptide. Thus, it will be understood that detecting the presence or amount of a polypeptide includes detecting the presence or amount of a polynucleotide encoding the polypeptide. Specialized pro-convergence mediators can also be measured and quantified directly using methods described and available in the art. Such methods include liquid chromatography and tandem mass spectrometry.

[0193] Quantitative changes in biomolecules (e.g., cytokines and pro-resolving mediators) can be measured in biological samples such as urine or plasma. Detection of the biomolecules can be performed directly in the sample taken from the subject, or the sample can be processed between taking from the subject and analysis. For example, a blood sample can be processed by adding an anticoagulant (e.g., EDTA) and then removing cells and cell debris to leave plasma containing the relevant molecules (e.g., cytokines and pro-resolving mediators) for analysis. Alternatively, the blood sample can be allowed to clot and then cells and various clotting factors can be removed to leave serum containing the relevant molecules (e.g., cytokines and pro-resolving mediators) for analysis.

[0194] In embodiments where the signal is applied while the subject is asleep, the invention may involve measuring the subject's circadian phase markers, such as the level of cortisol (or its metabolites), the level of melatonin (or its metabolites), or core body temperature. Cortisol or melatonin levels may be measured in blood (e.g., plasma or serum), saliva, or urine. Methods for measuring the levels of these markers are known in the art, for example, by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay. If the measurement of the subject's circadian phase markers indicates a diurnal oscillation of inflammatory markers that can be effectively controlled by application of a signal using the system of the invention, application of the signal late at night / early in the morning (when cortisol levels begin to rise) with a suitable periodicity according to the subject's circadian rhythm may be appropriate.

[0195] As used herein, a physiological parameter is not affected by the modulation (e.g., stimulation) of splenic nerve activity if the parameter does not change (in response to neuromodulation) from the normal value or normal range for that parameter that the subject exhibits, or the subject exhibits if no intervention is performed, i.e., it does not deviate from the baseline value for that parameter. Such physiological parameters can be arterial pressure, heart rate, or glucose metabolism. Suitable methods for measuring changes in these physiological parameters will be apparent to those skilled in the art.

[0196] It will be apparent to one of skill in the art that the baseline for any neural activity or physiological parameter in a subject need not be a fixed or specific value, but rather may vary within a normal range or may be an average value with associated error and confidence intervals. Suitable methods for measuring baseline values ​​are known to those of skill in the art.

[0197] As used herein, a physiological parameter is measured in a subject when measuring a value for that parameter exhibited by the subject at the time of detection. A detector (e.g., a physiological sensor subsystem, a physiological data processing module, a physiological sensor, etc.) is any element capable of making such a measurement.

[0198] Thus, in certain embodiments, the invention further includes measuring one or more physiological parameters of the subject, and applying the signal only if the measured physiological parameters meet or exceed a predetermined threshold. In such embodiments measuring multiple physiological parameters of the subject, the signal can be applied only if any one of the measured physiological parameters meets or exceeds its threshold, or if all of the measured physiological parameters meet or exceed their threshold. In certain embodiments applying the signal via a system of the invention, the system further includes at least one detector configured to measure one or more physiological parameters of the subject.

[0199] In certain embodiments, the physiological parameter is an action potential or a pattern of action potentials in a nerve of the subject, the action potential or pattern of action potentials being associated with the condition to be treated.

[0200] It will be apparent that any two physiological parameters can be measured in parallel embodiments and combined with a controller to detect a pattern of action potential resistance in a subject.

[0201] A predefined threshold for a physiological parameter is a minimum (or maximum) value for the parameter that must be exhibited by the subject or subjects before a specified intervention is applied. For any predefined parameter, the threshold may be defined as a value indicative of a pathological or disease state. The threshold may be defined as a value indicative of the onset of a pathological or disease state. Thus, depending on the predefined threshold, the present invention can be used as a treatment. Alternatively, the threshold may be defined as a value indicative of the physiological state of the subject (e.g., the subject is asleep, has eaten, or is exercising). Appropriate values ​​for any predefined physiological parameter are believed to be easily determined by the skilled person (see, e.g., medical practice standards).

[0202] A threshold is exceeded for a given physiological parameter if the value exhibited by the subject exceeds such threshold, i.e., the exhibited value is a greater deviation from the normal or healthy value for that physiological parameter compared to the pre-defined threshold.

[0203] In addition to having an implant, a subject of the present invention may receive medication for the condition. For example, a subject with an implant according to the present invention may receive anti-inflammatory medication (which would typically continue the medication they were taking before implanting the implant). Such medications include nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, 5ASAs, disease-modifying anti-inflammatory drugs (DMARDs) such as azathioprine, methotrexate and cyclosporine, biologics such as infliximab and adalimumab, and newer oral DMARDs such as Jak inhibitors. Thus, the present invention provides for the use of these drugs in combination with the system of the present invention.

[0204] General The methods described herein may be implemented by software in machine-readable form on a tangible storage medium, e.g., in the form of a computer program (which computer program may be embodied on a computer readable medium) including computer program code means adapted to perform all the steps of any of the methods described herein when the program is running on a computer. Examples of tangible (or non-transitory) storage media include disks, thumb drives, memory cards, etc., and do not include propagated signals. The software may be suitable for running on a parallel or serial processor, whereby the method steps may be performed in any suitable order or simultaneously. It is evidence that firmware and software may be valuable, separately exchangeable goods. It encompasses software that runs on or controls "dumb" or standard hardware to perform desired functions. It also encompasses software that "describes" or defines the configuration of hardware, e.g., HDL (Hardware Description Language) software, used to design silicon chips or configure general purpose programmable chips to perform desired functions.

[0205] Those skilled in the art will appreciate that storage devices used to store program instructions can be distributed over a network. For example, a remote computer can store an example of a process described as software. A local or terminal computer can access the remote computer and download some or all of the software to run the program. Alternatively, the local computer can download multiple pieces of software as needed, or execute some software instructions on the local terminal computer and some on the remote computer (or computer network). Those skilled in the art will also appreciate that all or some of the software instructions can be executed by dedicated circuitry, e.g., DSPs, programmable logic arrays, etc., using conventional techniques known to those skilled in the art.

[0206] Each embodiment described herein can be combined with another embodiment described herein unless otherwise stated. The term "comprising" encompasses "including" as well as "consisting." For example, a composition "comprising" X can consist exclusively of X or can include something else, e.g., X+Y.

[0207] It will be apparent that the benefits and advantages described above may relate to one embodiment or to several embodiments, and the embodiment is not limited to one that solves any or all of the problems described above or has any or all of the benefits and advantages described above.

[0208] The above description of preferred embodiments is provided for purposes of illustration only, and it will be understood that various modifications may be made by those skilled in the art. Although various embodiments have been described above in some detail, or with reference to one or more specific embodiments, it is contemplated that those skilled in the art may make numerous modifications to the disclosed embodiments without departing from the scope of the present invention. [Brief description of the drawings]

[0209] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: [Figure 1] FIG. 1 illustrates a neurostimulation system. [Diagram 2] FIG. 2 illustrates a broader system that includes a neurostimulation system. [Figure 3-1] Figure 3 shows the histological and electrophysiological characteristics of the porcine splenic nerve. Figure 3A is a photomicrograph of a semi-thin section (0.5 μm thick) of the SpA (splenic artery) / SpN (splenic nerve) stained with toluidine blue. No myelinated axons are visible in the image. [Figure 3-2]Figure 3B is a representative trace of evoked compound action potentials (eCAPs) recorded from a periarterial splenic nerve bundle dissected from an artery when stimulated at 1 Hz with a periarterial cuff (around the entire SpN plexus) or a small cuff around a few of the SpN bundles. The traces are the average of 10 responses. Figure 3C shows the range of conduction velocities of the different components of the eCAP. [Figure 3-3] Figures 3D and 3E show the intensity-duration curves of SpNs obtained by stimulating the whole plexus (Figure 3D) or several dissected bundles (Figure 3E). These graphs also show the relative charge density to obtain threshold eCAP at different stimulation amplitudes. All stimulations were performed at 1 Hz to limit stimulation-evoked action potential conduction slowing in the nerve. [Figure 4-1] Figure 4 shows the stimulus intensity-dependent temporal changes in mSpA BF, mSpV BF, sMABP and HR evoked by SpN stimulation. Figure 4A shows the mean (n=8) changes in mSpA BF (-30 to +180 s relative to stimulus onset) during 1 min stimulation (symmetric square biphasic pulse, 400 μs PW at 10 Hz) of the SpN plexus at different current amplitudes (3.5 to 20 mA). [Figure 4-2] Figure 4B shows the maximum decrease in mSpA BF reached during 1 min stimulation of the SpN plexus (symmetrical biphasic pulse, 400 μs PW at 10 Hz) at different current amplitudes. Each line represents a subject animal. Figure 4C shows the average (n≧3) maximum decrease in mSpA BF reached during 1 min stimulation of the SpN plexus (symmetrical biphasic pulse, 400 μs or 200 μs PW at 10 Hz) at different current amplitudes and two different PWs: 400 (filled circle) and 200 (filled square) μs. [Figure 4-3] Figure 4D shows the changes in mSpV BF (-30 to +180 s, relative to stimulation onset) during 1 min stimulation (symmetric biphasic pulses, 400 μs PW at 10 Hz) of the SpN plexus with different current amplitudes (3.5 to 12 mA). [Figure 4-4] Figure 4E shows the mean (n = 3) changes in sMABP and HR (-30 to +180 s, relative to stimulation onset) during 1 min stimulation (symmetric biphasic pulses, 400 μs PW at 10 Hz) of the SpN plexus at different current amplitudes (3.5 to 20 mA). [Figure 4-5] Figures 4F and 4G summarize the mean (n=3) maximum changes in mSpA BF, sMABP, HR and RR during 1 min stimulation (symmetric biphasic pulse, 400 μs PW at 10 Hz) of the SpN plexus (Figure 4F) or several dissected SpN bundles (Figure 4G) at different current amplitudes. Both graphs show the recorded eCAP amplitude (measured as the area under the curve of the response) (expressed as % of the maximum response). SpA BF changes are expressed as maximum drop from baseline in %, HR changes are expressed as breaths per minute (bpm), sMABP changes are expressed as mmHg and RR changes are expressed as breaths per minute (bpm). The two graphs also report the charge density per phase versus the stimulation amplitude used. [Figure 4-6] Figures 4F and 4G summarize the mean (n=3) maximum changes in mSpA BF, sMABP, HR and RR during 1 min stimulation (symmetric biphasic pulse, 400 μs PW at 10 Hz) of the SpN plexus (Figure 4F) or several dissected SpN bundles (Figure 4G) at different current amplitudes. Both graphs show the recorded eCAP amplitude (measured as the area under the curve of the response) (expressed as % of the maximum response). SpA BF changes are expressed as maximum drop from baseline in %, HR changes are expressed as breaths per minute (bpm), sMABP changes are expressed as mmHg and RR changes are expressed as breaths per minute (bpm). The two graphs also report the charge density per phase versus the stimulation amplitude used. [Figure 5a] Figure 5 shows that changes in mSpA BF, mSpV BF, sMABP and HR during SpN stimulation were frequency dependent. Figure 5A shows the mean (n=3) changes in mSpA BF (-30 to +180 sec relative to stimulation) during 1 min stimulation (symmetric biphasic pulse, 400 μs PW at approximately 36.9 μC / cm2 / phase) of the SpN plexus at different frequencies (0.25 to 100 Hz). [Figure 5b] Figure 5B shows the mean (n=3) maximal decrease in mSpA BF observed during 1 min stimulation of the SpN plexus (symmetric biphasic pulse, 400 μs PW at approximately 36.9 μC / cm2 / phase) at different frequencies (0.25-100 Hz). [Figure 5c] In Figures 5C-5D, the graphs show the changes in mSpV BF, sMABP, HR (expressed as % of pre-stimulation baseline) during 1 min stimulation (symmetric biphasic pulse, 400 μs PW at approximately 36.9 μC / cm2 / phase) of the SpN plexus at different frequencies (0.25-100 Hz). Data in Figure 5A are expressed as mean ± standard deviation. In Figures 5A and 5C-5D, boxes represent the stimulation time window. [Figure 5d] In Figures 5C-5D, the graphs show the changes in mSpV BF, sMABP, HR (expressed as % of pre-stimulation baseline) during 1 min stimulation (symmetric biphasic pulse, 400 μs PW at approximately 36.9 μC / cm2 / phase) of the SpN plexus at different frequencies (0.25-100 Hz). Data in Figure 5A are expressed as mean ± standard deviation. In Figures 5A and 5C-5D, boxes represent the stimulation time window. [Figure 6] Figure 6 shows the local and systemic effects of several dissected SpN bundles at different frequencies. In particular, Figure 6 shows representative experimental recordings of local and systemic changes associated with stimulation with different frequencies of several SpN bundles dissected from an artery. HR, sMABP, stimulation input, eCAP, SpA BF raw and mSpA BF data are shown from a representative experiment where frequencies ranged from 3 to 300 Hz. [Figure 7] Figure 7 shows the SpA blood flow changes monitored via intraoperative splenic ultrasound. The images in Figure 7 were obtained from two different animals during SpN stimulation. Note the Doppler trace depression during stimulation (middle panel) and before and after stimulation (top and bottom panels, respectively). [Figure 8-1]Figure 8 shows activity-dependent variations in SpN eCAP amplitude and conduction velocity. Figure 8A shows eCAP recorded from an SpN during 1 min of stimulation at different frequencies (1, 10 and 30 Hz, left to right). Each image shows a superposition of all evoked responses. For 1 Hz stimulation, 60 responses were superimposed. For 10 and 30 Hz, each trace represents the average of 5 consecutive responses. It is noteworthy that the responses at 10 and 30 Hz are shifted to the right over time and are smaller in amplitude over time. [Figure 8-2] Figure 8B shows eCAP recorded from an SpN during 1 min of stimulation delivered at 10 Hz in a train of 5 or 10 pulses separated by a 5 or 10 sec off-period. Each image shows a shift to the right over time, with decreasing amplitude. [Figure 8-3] Figure 8B shows eCAP recorded from an SpN during 1 min of stimulation delivered at 10 Hz in a train of 5 or 10 pulses separated by a 5 or 10 sec off-period. Each image shows a shift to the right over time, with decreasing amplitude. [Figure 8-4] Figure 8C shows the quantification of the area under the curve (AUC) of each recorded eCAP for the different stimulation paradigms. In particular, Figure 8C shows a comparison of values ​​between 1 pulse and 60 pulses delivered with the different paradigms. [Figure 8-5] FIG. 8D shows eCAP latency (expressed as % of the latency of the first response) for 600 consecutive pulses with different patterns of stimulation shown in FIG. 8A-C. Data are shown as means (N≧3). Dotted lines represent 95% confidence intervals. FIG. 8E shows eCAP amplitude (expressed as % of the amplitude of the first response) for 600 consecutive pulses with different patterns of stimulation shown in FIG. 8A-C. Data are shown as means (N≧3). Least squares regression curves were fitted to the latency data (shown in FIG. 8D) and amplitude data (shown in FIG. 8E). Dotted lines represent 95% confidence intervals. [Figure 8-6]FIG. 8F shows changes in SpA mBF (circles) and sMABP (triangles) during 60 sec stimulation of the porcine splenic neurovascular bundle (NVB) with 400 μs PW and 12 mA (symmetric, biphasic square pulses) delivered at 10 Hz (black), 1 Hz (light grey) or burst stimulation (10 Hz, 5 pulses every 10 sec, grey). Data are from a representative stimulation within the same animal. FIG. 8G shows the SpA mBF and sMABP maximum changes recorded during 60 sec stimulation delivered with the different stimulation patterns shown in FIG. 8F. Values ​​are expressed as % of the maximum change obtained at 10 Hz. Data are presented as mean (N=4)±standard deviation. Statistical analysis in FIG. 8C was performed using one-way ANOVA with Tukey's post-hoc correction for multiple comparisons. *, P≦0.05; **, P≦0.005; ***, P≦0.001; ****, P≦0.0001. [Figure 9] Figure 9 shows that burst and 1 Hz stimulation produced minimal changes in mSpA BF. In particular, Figure 9 shows the maximum change in mSpA BF expressed as % of the change obtained during 60 s stimulation with biphasic symmetric pulses delivered at 10 Hz (black). Different stimulation paradigms delivered with the same current amplitude are compared: continuous 10 Hz, continuous 1 Hz and burst stimulation (5 pulses at 10 Hz every 5 s) with symmetric or asymmetric biphasic pulses. [Figure 10] Figure 10 is a schematic diagram of the left flank of a pig highlighting the anatomical features of the splenic plexus (spleen, nerves, arteries and veins). The location of cuff placement during peri-arterial SpN stimulation experiments is indicated. Nerves are depicted in black, arteries and veins in grey. [Figure 11-1] Figure 11 shows that stimulation of SpN in pigs causes a decrease in LPS-induced cytokine production in vivo. Figures 11A and 11B show the dynamic changes in TNFα and IL-6 measured directly from plasma collected from terminally anesthetized pigs administered 0.25 μg E. coli LPS. [Figure 11-2]Figures 11C and 11E show the peak levels of TNFα and IL-6 measured in plasma after LPS challenge in the different groups. Figures 11D and 11F show the quantification of the area under the curve (AUC) for both TNFα and IL-6. Data are presented as mean + / - SD (Sham: N=6, Dexamethasone: N=2, LVNS: N=5, eLVNS: N=5, SpNS: N=6). [Figure 12-1] Figure 12 shows the anatomical and histological analysis of the SpN along with the main SpA and short gastric and omental arteries. Figure 12A is a schematic representation of the splenic nerve structure highlighting (dotted line) the area where histological analysis was performed. Figures 12B-12D show sections of the SpN at different levels, the main splenic artery (Figure 12B), the short gastric (SG) artery (Figure 12C) and the gastroepiploic (GEP) artery (Figure 12D) stained with hematoxylin and eosin (H&E). [Figure 12-2] Figures 12B-12D show sections of different levels of SpN, main splenic artery (Figure 12B), short gastric (SG) artery (Figure 12C) and gastroepiploic (GEP) artery (Figure 12D) stained with hematoxylin and eosin (H&E). Nerves in Figures 12C and 12D are indicated by arrows. In Figure 12D, the inset shows a high magnification caption of one nerve bundle. [Figure 12-3] Figure 12E shows box plots reporting the quantification of the number of SpN bundles at different locations (upper panel) and the mean diameter distribution of the same bundles at different locations (lower panel). Figure 12F shows the number of bundles at different locations and their relative mean diameters. [Figure 13-1] Figure 13 shows that the human splenic nerve is a plexus of periaortic bundles containing slow-conducting axons. Figure 13 includes the following subsections: A) Human splenic neurovascular bundle (NVB) freshly excised from a donor, including SpA, SpN, connective tissue, pancreas and lymph nodes. Two small cuff electrodes (650 μm diameter) were placed on certain dissected bundles. A schematic of the preparation shows the location of the stimulation and recording cuffs (a and b). The dotted lines indicate the areas where sections were taken, indicated in B and C. [Figure 13-2](B) Section of human NVB stained with hematoxylin and eosin (H&E), with the SpN bundle circled; (C) Section of the excised nerve bundle for electrophysiological studies, stained with H&E and showing the nerve bundle (circled) and fat / connective tissue. [Figure 13-3] (D) eCAP recorded upon monopolar monophasic stimulation of a human SpN at 1 Hz and 400 μs PW before (upper panel) and after (lower panel) crushing the nerve between the stimulation cuff and the recording cuff. The box on the left indicates the stimulation artifact, the larger box on the right indicates the area where eCAP should be observed, and the arrow indicates the eCAP; (E) Recruitment curve of a human SpN quantifying eCAP amplitude (expressed as % of maximum response) versus stimulation amplitude. Each point represents the mean amplitude of 8 consecutive monopolar monophasic pulses delivered at 1 Hz and 400 μs PW. [Figure 13-4] (F) Conduction velocity of all eCAP components recorded from human, porcine and rat SpNs; (G) Strength-duration relationship (filled circles) of a human SpN obtained by stimulating a dissected bundle. The data represent the minimum current required to trigger a detectable eCAP for the different PWs examined. The graph also shows the corresponding charge density (filled triangles) of the different stimuli (referred to as the right Y-axis). Least-squares regression curves were plotted for the strength-duration and charge density data. [Figure 13-5] (H) Charge density required to stimulate SpNs of three different species at different PWs. Data were fitted with linear regression. Scale bars: B = 2 mm; C = 100 μm. [Figure 14-1] FIG. 14 shows A) an example of a human spleen specimen with sutures showing the proximal end close to the abdominal cavity, (B) a conceptual representation of tissue sections in blocks for histology, (C) a hematoxylin and eosin (H&E) stained slide from one of the blocks, and (D) histomorphometric estimation method. [Figure 14-2]FIG. 14 shows A) an example of a human spleen specimen with sutures showing the proximal end close to the abdominal cavity, (B) a conceptual representation of tissue sections in blocks for histology, (C) a hematoxylin and eosin (H&E) stained slide from one of the blocks, and (D) histomorphometric estimation method. [Figure 14-3] FIG. 14 shows A) an example of a human spleen specimen with sutures showing the proximal end close to the abdominal cavity, (B) a conceptual representation of tissue sections in blocks for histology, (C) a hematoxylin and eosin (H&E) stained slide from one of the blocks, and (D) histomorphometric estimation method. [Figure 15] FIG. 15 shows (left) bundle diameter, (center) bundle extension around the adventitia (outer splenic artery wall) for the proximal, middle and distal portions of the splenic neurovascular bundle (NVB), and (right) percent bundle-distance from adventitia. [Figure 16-1] Figure 16 shows in vivo data from porcine splenic neurovascular bundle stimulation; (A) group recruitment curves, (B) intensity-duration curves. [Figure 16-2] Figure 16 shows in vivo data from porcine splenic neurovascular bundle stimulation; (A) group recruitment curves, (B) intensity-duration curves. [Figure 17-1] FIG. 17 shows (A) mobilization curves from in silico modeling in pigs where the x-axis represents estimated charge injection at 400 μs pulses, (B) the same mobilization curve where the x-axis reflects stimulation amplitude, (C) mobilization curves from in silico modeling in humans where the x-axis represents estimated charge injection at 400 μs (blue) and 1 ms pulses (red), and (D) the same mobilization curve where the x-axis reflects stimulation amplitude (mA). [Figure 17-2] FIG. 17 shows (A) mobilization curves from in silico modeling in pigs where the x-axis represents estimated charge injection at 400 μs pulses, (B) the same mobilization curve where the x-axis reflects stimulation amplitude, (C) mobilization curves from in silico modeling in humans where the x-axis represents estimated charge injection at 400 μs (blue) and 1 ms pulses (red), and (D) the same mobilization curve where the x-axis reflects stimulation amplitude (mA).

[0210] Pig Data Electrical stimulation of the splenic artery nerve in pigs Materials and Methods A total of eight pigs (body weight: 40–50 kg) were used for histological and electrophysiological characterization of the splenic nerve.

[0211] On the day of the experiment, animals were sedated with ketamine (1.5 mg / kg) and midazolam (0.5 mg / kg) administered intramuscularly. An intravenous catheter was placed in one auricular vein and anesthesia was achieved with intravenous propofol (2 mg / kg). An endotracheal tube was placed and anesthesia was maintained with sevoflurane combined with a continuous rate infusion (CRI) of fentanyl (0.2 μg / kg / min).

[0212] After administering general anesthesia, the animals were placed in a dorsal position for placement of bilateral indwelling tail vein catheters and one femoral artery catheter under ultrasound guidance. The animals undergoing SpN cuff implantation were then placed in a right lateral lying position.

[0213] The surgical approach for SpN cuff implantation was as follows: the thoracic-lumbar junction was supported and slightly elevated with sandbags. After appropriate surgical preparation (clipping and aseptic scrubbing with chlorhexidine gluconate and alcohol), the left flank was draped to expose a 20 × 25 cm area centered on the penultimate rib. A 15 cm skin incision was made at the penultimate intercostal space with a monopolar electrocautery. The incision was continued through the subcutaneous tissue and intercostal musculature until the peritoneum was exposed. Two Finochet rib retractors were placed retroperitoneally, taking care to engage the ribs. Over the next few minutes, the retractors were gradually opened to expose the left flank in an area of ​​approximately 10 × 8 cm. The retractor blades were covered with gauze sponges soaked in carboxymethylcellulose (CMC). The peritoneum was incised longitudinally and the skin was sutured (Vicryl 2-0; Ford interlocking suture pattern) to cover the blades of the retractor to reduce the risk of splenic laceration during handling. Using careful finger manipulation, the spleen was exteriorized and the splenic artery (SpA) was identified along its visceral surface. A short portion of the SpA was carefully dissected free of surrounding soft tissue for placement of a 1 mm ultrasound flow probe (Transonic) at the mid-spleen portion proximal to the SpA branching into the left gastroepiploic artery. After placement of the probe, the spleen was returned to the abdomen.

[0214] By slightly rotating the splenic visceral base relative to the operator and applying gentle abdominal traction to the spleen, the gastrosplenic ligament at the hilum of the spleen was dissected with Metzenbaum scissors to expose the SpA. The artery was followed dorsally to its origin (i.e., the bifurcation of the left gastric artery (LGA) and the celiac artery to the SpA). Directly distal to this bifurcation, a 1 cm segment of the SpA with intact periarterial SpN network was circumferentially removed by blunt dissection with Metzenbaum scissors. While grasping one flap of a 2.5 mm diameter CorTec cuff introduced into the surgical field with straight microdissection forceps, a curved Mixter artery forceps was inserted under the artery from caudal to cranial. The cuff was placed around the SpA and the intact periarterial SpN network by reversing the movement of the Mixter forceps, taking care to appose the two flaps of the cuff when properly placed. The tension on the spleen and artery was then released, the SpA blood flow readings were taken, and finally the rib retractors were partially closed and the exposed incision was covered with saline soaked gauze sponges.

[0215] Electrophysiological experiments usually involved dissection and cuffing (using 500 μm diameter bipolar or tripolar CorTec cuffs) of one or several separate SpN bundles several centimeters distal to the stimulation cuff (closer to the spleen) to allow evoked compound action potential (eCAP) recordings upon stimulation of the whole SpN plexus or several bundles (see FIG. 3). In addition, different combinations of nerve signaling blockade (e.g., using local administration of local anesthetics or transection of SpN bundles) upstream or downstream of the stimulation site were performed.

[0216] Recorded eCAPs were amplified and filtered (100-1000 Hz) using an 1800 2-channel microelectrode AC amplifier (AM Systems). Neural activity was continuously monitored using an oscilloscope and recorded on a computer using a 16-channel PowerLab (AD Instruments) acquisition system and LabChart 8 software using a sampling rate of 20 kHz. eCAPs were averaged (8-10 pulses) and the peak-to-peak or area under the curve (AUC) of the average response was quantified. Conduction velocity of the eCAP component of the SpN was calculated from the distance between the stimulation and recording sites and the latency of the eCAP signal.

[0217] Electrocardiogram (ECG), heart rate (HR), arterial blood pressure, respiratory rate (RR), pulse oximetry, capnography, and lung capacity were monitored throughout the procedure. Temperature was recorded continuously via an intranasal probe. Arterial blood gases were analyzed throughout the experiment to measure pH, glucose, pO2 and pCO2, K + Levels were monitored. All physiological parameters as well as the levels of sevoflurane used were recorded on a record sheet (every 5-10 min). Physiological data were also digitized using a Powerlab acquisition system and LabChart software. All parameters were sampled at frequencies between 0.1 and 2 kHz.

[0218] Depth of anesthesia was assessed by eyelid reflex, corneal reflex, medioventral eye position and jaw tone.

[0219] Additionally, the level of anesthesia was adjusted using physiological parameters as well as a bispectral index monitoring system (levels between 30 and 60). In some cases, a bolus of propofol was used.

[0220] In some cases, intraoperative ultrasound of the spleen was used for real-time monitoring of SpA blood flow changes during SpN stimulation, using an intraoperative probe (i12L-RS linear intraoperative transducer 4-10 MHz, 29 × 10 mm footprint, 25 mm field of view; GE Vivid-i) for the procedure.

[0221] SpA blood flow changes were assessed by color Doppler and continuous wave spectral tracing. After color Doppler identification of SpA within the splenic parenchyma 2-3 cm distal to the splenic hilum, continuous wave spectral tracing of SpA flow was obtained by directing the window cursor to the center of the SpA lumen. After obtaining a representative signal, SpN stimulation was initiated with the ultrasound probe and cursor window in the appropriate position.

[0222] All statistical analyses were performed using commercially available statistical software (JMP Pro 13.0.0 or GraphPad Prism 5.0).

[0223] result Recordings of eCAPs produced during SpN stimulation of the whole SpN plexus with a periarterial cuff or stimulation of a few bundles with a smaller cuff produced eCAPs with specific latencies depending on the distance between the stimulation and recording sites (Figure 3B). The range of conduction velocities of the different components of the eCAP is shown in Figure 3C. Stimulation of either the whole plexus or a few bundles produced eCAPs with average velocities below 1 m / s (Figure 3C). This conduction velocity is consistent with the histological findings in the characterization data below, which show that SpNs are unmyelinated nerves. The relationship between current amplitude and pulse duration required to elicit eCAPs stimulating the whole plexus or a few bundles is shown in Figures 3D and 3E (respectively). When stimulating the whole plexus with a periarterial cuff, the threshold for the nerve response ranged from 7.692 to 15.58 μC / cm 2 When stimulating a small number of incision bundles with a smaller cuff, the thresholds were found to be 5.796–11.594 μC / cm 2 In both cases, the threshold current density for eCAP recordings was lower with shorter pulse widths (PW).

[0224] SpN biphasic stimulation for 1 min at 10 Hz and 400 μs PW above a specific current threshold consistently induced a transient blood flow decrease in the distal SpA measured via a perivascular flow probe. There was a clear dose-response relationship between delivered current and blood flow decrease; the higher the amplitude, the greater the blood flow decrease observed (Figure 4A). Blood flow change thresholds, defined as a 5% change in mean SpA blood flow (mSpA BF) compared to pre-stimulus baseline, were found at approximately 4.5 mA (with 400 μs PW) and approximately 12 mA (with 200 μs pulse width) (Figures 10B and 10C). Calculation of the threshold per-phase charge density to induce blood flow change showed that the values ​​were very similar, approximately 13.8 μC / cm at 400 μs. 2 / phase and 18.46μC / cm at 200μs 2 / phase. 12mA and 400μs PW (36.9μC / cm 2 Stimulation with 100 / phase induced a mean maximum BF decrease in SpA of approximately 40% from baseline values.

[0225] In parallel, blood flow in the splenic vein (SpV) was recorded using a Doppler flow probe placed at the base of the spleen where the vein exits the splenic hilum. Interestingly, stimulation (symmetric biphasic pulse, 400, 10 Hz, 1 min) produced an increase in mean SpV blood flow (mSpVBF) that was current amplitude dependent. 2 Stimulation with 100 / phase (100 / phase) produced a maximum increase of approximately 200% when compared to baseline mSpV BF. The transient decrease in mSpA BF was accompanied by a transient increase in systemic mean arterial blood pressure (sMABP). This increase from baseline (average 1-6 mmHg) also correlated with stimulation intensity (Figure 4E). Consistent sMABP changes were observed with stimulation, producing a 20-30% decrease in SpA blood flow. In contrast, HR was only slightly affected (<3 bpm change, increase or decrease) and was more consistent only with high stimulation amplitudes (>45 μC / cm 2 / phase, resulting in changes of 3–10 bpm (Figure 4G). SpN stimulation did not affect respiratory rate (RR) under the conditions examined.

[0226] Different current amplitudes (1-50mA, 3.076-153.8μC / cm 2 The changes observed in mSpA BF, sMABP, HR, RR during 1 min of stimulation (symmetric biphasic pulses, 10 Hz, 400 μsPW) with 100 Hz pulses (corresponding to 100 / phase) are summarized in Figure 4F. In Figure 4F it can be observed how well the magnitude of these changes correlated with the recordings of eCAP (black lines or circles) from the SpNs. The greater the number of recruited fibers (measured as % eCAP relative to the maximum recorded response), the greater the decrease in mSpA BF and other associated changes.

[0227] Direct stimulation of individual SpN bundles dissected from the SpA (with a 500 μm diameter cuff) induced similar changes in mSpA BF, sMABP and HR. 2 These changes, occurring over a period of 1000 s / phase, are summarized in Figure 4G. Even in this case, the associated changes were dependent on the proportion of fibers recruited by the stimulation (eCAP, shown in black). The maximum eCAP (and therefore the maximum change) was approximately 153 μC / cm when the whole plexus was stimulated. 2 / phase, and about 70 μC / cm 2 The magnitude of changes obtained when stimulating a few bundles was lower than that obtained when stimulating the entire plexus, as expected given the lower total number of fibers stimulated and the lower frequency.

[0228] Blood flow changes in mSpA were also affected by different frequencies of stimulation. Stimulation at different frequencies (0.25–100 Hz) (symmetric biphasic pulse, approximately 36.9 μC / cm 2At 400 μs PW for 1 min in each phase, 30–50 Hz reliably induced the strongest blood flow reduction in SpA (Fig. 5A). Above 50 Hz (70–100 Hz), the BF reduction was actually smaller, in the range of the reduction obtained with 10 Hz stimulation (Fig. 5B). Changes in mSpV BF, sMABP, and HR were also found to be dependent on the frequency of the applied stimulation. The strongest effect was again observed at 30–50 Hz (Fig. 5C–5D).

[0229] Again, only a few bundles of the artery were excised, and the maximum (approximately 70 μC / cm 2 A stronger decrease in mSpA BF occurred already at relatively low frequencies (1 Hz and below) due to the higher nerve fiber recruitment compared to the stimulation amplitude used for the whole plexus during frequency analysis. Consistently, however, the lowest decrease was observed at 30-50 Hz (Figure 5D).

[0230] To further confirm that the observed changes in SpABF were due to direct neuronal activation (and not smooth muscle stimulation), lidocaine (2% lidocaine hydrochloride solution) was applied locally around the implanted SpN cuff (periarterial cuff or dissected bundle cuff). Lidocaine inhibited fast voltage-dependent Na +Lidocaine was able to block the changes in SpA BF. Moreover, mechanical occlusion of the SpA, which can reduce BF by up to 80%, did not cause any changes in sMABP or HR. Moreover, transection of the central end of the SpN (proximal to the cuff) did not abolish the stimulatory effects on SpA blood flow, sMABP, and HR. Furthermore, transection of SpNs within the GEP and SG fragments did not prevent these changes. Interestingly, all these effects were abolished only by cutting the peripheral end of the SpN (distal to the cuff). All these data suggest that the changes in SpA BF and SpV BF were neuronally driven and related to the constriction of SpA as well as the contraction of the splenic capsule. On the other hand, the changes in sMABP and HR were probably due to increased blood outflow from the spleen towards the heart, rather than due to activation of neuronal pathways towards the brain.

[0231] In a small number of animals, SpA blood flow changes upon stimulation were also monitored using intraoperative ultrasound at the splenic hilum. After identifying SpA by color Doppler, changes in BF were monitored as Doppler signals as shown in Figure 7. Upon stimulation at 10 Hz, a decrease in BF could be easily observed as indicated by the altered amplitude and shape of the blood flow trace.

[0232] Consideration Splenic nerve stimulation was associated with transient local changes in mSpA and mSpV BF and splenic contraction. These changes were due to direct activation of SpNs, not direct stimulation of the smooth muscle of the SpA. Splenic contraction upon SpN stimulation has been reported in other species

[16] . The observed changes in mSpA BF were highly consistent between animals. The variability was probably mainly due to different attachments of the cuff around the SpN plexus in different animals. The changes in SpA BF could be easily monitored by non-invasive ultrasound and could therefore be used as a marker to evaluate effective stimulation of SpNs in a clinical setting.

[0233] The temporal changes observed upon SpN stimulation were found to be amplitude and frequency dependent. During 1 min of stimulation with different current amplitudes, the strongest mSpA BF drop was observed at the highest current amplitude examined, which corresponds to the peak of the recorded eCAP. This was true when stimulating the entire SpN plexus (with a periarterial cuff) or when stimulating only a few bundles in a smaller cuff. The difference in the total charge density required to obtain maximum eCAP from the SpN plexus and from the SpN bundles could be explained by the partial coverage of the plexus by the 2.5 mm cuff used. Indeed, in most pigs, this cuff provided only 270-300° of circumferential coverage. When cuffing only a few bundles of SpNs dissected from the artery, coverage was almost total. Thus, optimal circumferential coverage of the artery would be necessary to limit the charge density required to obtain optimal recruitment of the SpN bundles.

[0234] The strongest changes (in mSpA BF and sMABP) were observed at frequencies between 30 and 50 Hz. The total number of pulses delivered may be an important factor in determining the magnitude of this change, but certainly the range of 30–50 Hz produced the strongest changes when comparing the changes produced by the same number of pulses delivered at different frequencies. This may be explained by previous published data showing that the maximum release of NA from cat spleen was observed at 30 Hz [17,18]. The relatively large release of NA may explain the relatively large magnitude of changes observed in this stimulation range.

[0235] Optimization of signal parameters Materials and Methods To develop an optimized stimulation paradigm, we investigated several signal parameter settings in the pigs using the materials and methods described above.

[0236] Parameter optimization focused on reducing target effects and increasing potency of the SpN response. In particular, since the systemic changes caused by SpN stimulation were related to local narrowing of SpA and contraction of the spleen, parameters that could minimize these changes would represent the optimal paradigm to be transferred to chronic and clinical studies where systemic effects (e.g., changes in sMABP and HR) are undesirable.

[0237] result It was found that repeated stimulation of SpNs caused fatigue in nerve fibers at certain frequencies. This effect consisted of two characteristics: i) a decrease in SpN eCAP amplitude and ii) a decrease in SpN conduction velocity. Both of these characteristics were observed at frequencies higher than 1 Hz, with the fatigue effect increasing with increasing frequency. Indeed, stimulation of SpNs at 10 Hz for 1 min in a row caused an adaptation in the recorded response, decreasing the eCAP amplitude over time (Fig. 8A) and the conduction velocity of each of the recorded eCAP peaks. This effect was stronger (in magnitude) and more rapid at higher frequencies. For example, stimulation of SpNs at 30 Hz for 1 min in a row caused a more rapid and stronger decrease in both eCAP amplitude (Fig. 8A, 8B) as well as conduction velocity. After 60 s of stimulation, 10 Hz (total of 600 pulses) pulses caused a decrease of about 60% of the eCAP maximum amplitude, while 30 Hz pulses (total of 1800 pulses) caused a decrease of about 80% (Fig. 8C, 8E). Instead, when the SpNs were stimulated for 1 min at 1 Hz, the decrease in eCAP over time (Figs. 8A and 8C) was very small and no significant decrease in conduction velocity was observed. Comparing the decrease in eCAP amplitude with the same number of pulses, 10 Hz and 30 Hz again produced a more rapid and stronger decrease (Fig. 8C). This fatigue effect of repeated stimulation of the SpNs could be mitigated by periodically switching the stimulation off and on. When the SpNs were stimulated with a rapid-fire paradigm, providing, for example, 5 pulses delivered at 10 Hz every 5 s, the decrease in eCAP and conduction velocity was abolished (Figs. 8C-8F).

[0238] Furthermore, when stimulated continuously at 10 and 30 Hz, a rapid increase in eCAP amplitude was observed within the first 5 to 20 pulses, which was preceded by a progressive decrease in amplitude and rate (Figures 8C and 8E).

[0239] As mentioned above, the frequency of stimulation not only influenced the neural response but also affected the physiology in different ways. Frequencies between 30 and 50 Hz produced the strongest changes in mSpA BF, driving the changes in sMABP. In comparison, frequencies ≦1 Hz produced little changes in mSpA BF. Frequencies between 1 and 30 Hz produced larger changes in mSpA BF. By choosing a high current amplitude (to recruit most SpN fibers), a 10 Hz, biphasic, symmetric 60 s stimulation was sufficient to produce at least a 50% maximal decrease in mSpA BF. Stimulation of SpNs with the same biphasic symmetric pulses and current amplitudes but at 1 Hz frequency produced a decrease in mSpA BF that was about 40% lower (almost 70% to almost 50% decrease) than that produced by 10 Hz stimulation (Figure 9, Figures 8F and 8G).

[0240] The same was observed when rapid-fire stimulation (5 pulses at 10 Hz every 5 seconds) was applied using biphasic pulses and the same current amplitude (Figure 9). Importantly, when stimulation was applied as biphasic, asymmetric pulses (and the same current amplitude), each of the stimulation paradigms produced a lower mSpA BF decrease compared to their respective biphasic, symmetric paradigms (Figure 9). Even in this case, 1 Hz and rapid-fire stimulation produced minimal changes in mSpA BF.

[0241] Described herein is the discovery of an opportunity to provide optimal neural activation without "neural fatigue" while providing the option to effectively stimulate (high frequency) or avoid (low or burst frequency) physiological changes depending on the target profile of the therapy. During surgery, it can be very effective to use blood flow changes to induce an easily visualized target engagement profile, ensuring that devices and therapies are properly positioned and of suitable amplitude. Switching to burst or low frequency stimulation then ensures ideal neural engagement for efficacy in splenic engagement, while avoiding the clinically ongoing effects of blood flow in awake patients.

[0242] Consideration The parameters for optimal stimulation (and therefore efficacy) should essentially be: i) to generate a highly consistent and sustained nerve amplitude response, ii) to deliver as many pulses as necessary (to obtain a therapeutic effect) in the shortest possible time window, reducing energy requirements and discomfort to the patient, and iii) to have the lowest spectrum of off-target effects. Stimulation of SpNs at frequencies higher than 1 Hz showed clear activity-dependent variations in response (eCAP) amplitude and conduction velocity. This effect has been observed previously in other unmyelinated nerves in both rodents and humans [19, 20, 21, 22]. During conventional stimulation with 0.25 Hz conditioning pulses intermittently inserted with varying interstimulus intervals, unmyelinated fibers showed a progressive decrease in conduction velocity that depended on the number of inserted stimuli

[20] . The same effect was observed with stimulation of SpNs, where repeated stimulation caused a slowing of conduction velocity as well as a reduction in eCPA amplitude. This effect was termed a "subnormal" action potential conduction.

[0243] When SpNs were stimulated continuously at 10 Hz, a short period of increased response was observed, followed by a delayed and reduced response phase. This other period has been reported previously and was termed "supernormal". The supernormal and subnormal action potential conduction is probably due to a period of post-depolarization of the membrane and a subsequent period of hyperpolarization when sent within a short time window (<1000 ms from each other) [20,21,22]. When eCAP was recorded, this membrane change in the axon causing an increase in the current activation threshold reduced the amplitude of the recorded signal. This was not simply an effect of a slower conduction velocity; the recorded eCAP shifted to longer latencies and reduced amplitude without overall width broadening. In fact, the measured AUC became smaller over time. However, it is still possible that the observed decrease in eCAP amplitude is due to desynchronization of the action potential, which is then offset when the compound response is recorded. Single-unit recordings would be ideal to show that SpNs do indeed undergo this activity-dependent change.

[0244] Here, we show that low-frequency stimulation (<1 Hz) or rapid-fire stimulation (10 Hz, 5 pulses every 5 seconds) caused limited to no fatigue in the SpNs and produced the lowest off-target effects. To date, these two paradigms delivered with biphasic asymmetric pulses represent optimal stimulation patterns of the SpNs for the treatment of immune-mediated conditions (e.g., inflammatory disorders). Frequencies higher than 1 Hz induce changes in blood flow and blood pressure and may be useful in confirming nerve-target engagement during electrode positioning.

[0245] Effect of continuous electrical stimulation on an in vivo LPS porcine model Materials and Methods animal A total of 23 pigs (body weight: 65-70 kg) were used in this section of the study.

[0246] General Design Pigs were terminally anesthetized and divided into five groups: Sham (electrodes implanted but not stimulated), Dexamethasone (SpNs were accessed and animals were then injected with dexamethasone at -2 and 0 hours), LVNS (pigs implanted with cervical LVNs), eLVNS (pigs implanted with cervical LVNs ligated and cut distally (to the heart) to the cuff electrodes, only the efferent stump stimulated), and SpNS (pigs implanted in peri-arterial SpNs).

[0247] LVNS, eLVNS and SpNS pig implants were continuously stimulated at 1 Hz from -2 hours to +1 hour (relative to LPS injection). E. coli-derived LPS was administered at 0 hours to all groups at a dose of 0.25 μg / kg. Dexamethasone was used as a positive control.

[0248] Peripheral venous blood was collected 2 hours before LPS injection (baseline) and then every half hour up to 4 hours after injection, and cytokine analysis (TNFα and IL-6) was performed by commercial ELISA at all these time points.

[0249] result Cytokine quantification. For all groups, LPS injection resulted in a significant increase in TNFα levels in all post-injection samples compared to baseline (pre-LPS injection), with the peak response observed at approximately 1 hour post-injection (FIG. 11A). A similar trend was observed for IL-6. The IL-6 response peaked at approximately 2.5 hours post-injection.

[0250] Cytokine levels were compared between groups by calculating the area under the curve (AUC) from -2 hours to +4 hours post-injection. TNFα levels were reduced by nearly 30% at 1.5 hours in the SpN, LVNS and eLVNS groups when compared to sham. There was a reduction in IL-6 in both peak response values ​​(Figure 11E) and AUC (Figure 11F) in the SpNS group. Similar reductions were also observed in the LVNS and eLVNS groups compared to sham controls.

[0251] Consideration In vivo LPS administration to mimic inflammatory responses provided a good model to examine the efficacy of SpN stimulation. Administration of LPS (0.25 μg / kg) in 65-70 kg pigs resulted in an increase in cytokines (TNFα and IL-6) in the blood of all subjects. In particular, TNFα reached a peak value of approximately 5 ng / mL 1 h after injection, whereas IL-6 peaked at approximately 0.5 ng / mL 2.5 h after LPS injection.

[0252] Thus, this model provides evidence that SpN stimulation can modulate responses to inflammatory stimuli, and shows that chronic stimulation of SpNs reduces levels of inflammatory cytokines, as seen in particular by a reduction in IL-6, which may be useful in reducing inflammatory responses in subjects, especially given recent evidence showing that vagus nerve stimulation is useful in the treatment of autoimmune disorders.

[0253] summary In summary, we found that stimulation of nerves innervating the spleen, particularly the splenic artery nerve, exhibited survival-promoting effects in an in vivo LPS animal model, suggesting that stimulation of splenic nerve activity may be particularly useful for the treatment of inflammatory disorders.

[0254] Characterization of the splenic artery nerve in pigs Materials and Methods Gross anatomical examination of the spleen along with associated organs was performed in 12 female pig cadavers (body weight 22-120 kg) within 1 hour of slaughter. The following measurements were made: spleen length and width; celiac artery length (from the aorta to the bifurcation into the left gastric artery and splenic artery); splenic artery (SpA) length (from the bifurcation of the celiac artery to the splenic parenchyma entrance); SpA diameter measured 1 cm distal to the celiac artery and at the splenic hilum; distance from the pancreas to the spleen; distance from the pancreas to the splenic lymph nodes. In addition, the number and course of abdominal vagus nerve branches, celiac ganglion, splanchnic nerves, and splenic nerve were recorded. The SpA with associated splenic nerve was processed for hematoxylin and eosin (H&E) histological examination.

[0255] Spleens with intact vasculature and innervation were collected from 12 female pig cadavers (body weight 22 kg, n=6; body weight 45 kg, n=6). All tissues were collected within 1 hour of sacrifice and immediately fixed in 10% neutral buffered formalin. The SpA with intact perivascular neural network was sectioned every 5 mm from its origin at the bifurcation of the celiac artery to the splenic hilum. This resulted in five sections defined as the bifurcation; proximal SpA; mid SpA; distal SpA and splenic hilum location. The proximal SpA section corresponds to the location of cuff positioning for the following electrical stimulation test discussed above.

[0256] At each of these five sites, sections were processed for routine H&E staining. Proximal, mid, and distal SpA sections were also processed for immunohistochemistry, and for semithin sections and staining with osmium tetroxide and toluidine blue.

[0257] Digital images of H&E stained sections were acquired at 2x magnification and appropriate software (Image J 1.50i) was used for histomorphometric analysis as detailed below. After manual selection of all single nerve bundles by using the ROI manager function, the number of periaortic nerve bundles was counted and bundle size was assessed by measuring the minimum ferret diameter (μm).

[0258] Total nerve area (unit: μm 2 ) was calculated and periarterial bundle distribution was quantified by assessing the percent of the arterial periphery in which bundles were identified, with 360° distribution defined as 100%. The distance from each bundle to the outer arterial wall was measured by drawing the shortest perpendicular line from each bundle to the arterial wall. The outer and inner diameters of the splenic artery were measured at the proximal, mid, and distal SpA locations.

[0259] Double staining with tyrosine hydroxylase (TH) and acetylcholine transferase (ChAT) was used to assess neuronal phenotype. By counterstaining with Neurofilament200 (NF200) and the nuclear stain 4',6-diamidino-2-phenylindole (DAPI), NF200-TH double positive nerves were considered sympathetic, and NF200-ChAT double positive nerves were considered parasympathetic. To determine the proportion of efferent and afferent nerves, the same locations were double stained with the efferent marker TH and the afferent marker calcitonin gene-related peptide (GCRP). Two different digital images were randomly captured from each nerve at 20x magnification, and pseudocolor composites were generated using the appropriate software (AxioVision LE64).

[0260] Myelination of SpN axons was assessed by immunofluorescence staining and from semi-thin sections. Different parts of SpA and SpN were stained with antibodies against neurofilament and β-III tubulin and myelin basic protein (MBP). Pseudo-color composite images were generated using the appropriate software as described above. Semi-thin sections were stained with osmium and toluidine blue. Digital images were acquired at 100x magnification and the number of myelinated and unmyelinated axons was manually counted in an area of ​​100x100 μm. This procedure was repeated three times per nerve and the average of these was used for further analysis. Furthermore, this procedure was used to calculate axon density (number of axons / mm 2 ) was used to derive

[0261] All statistical analyses were performed by commercial statistical software (JMP Pro 13.0.0). Due to non-normal distribution, all histomorphometric measurements were compared between different pig sizes and SpA locations using the Wilcoxon rank sum test. Statistical significance was defined as P<0.05.

[0262] result Neurovascular structures enter and exit the spleen only along the visceral surface: the first major abdominal branch of the aorta, the celiac artery, its branches to the hepatic artery, the SpA, and the LGA (Figure 10). The SpA enters the spleen at the hilum, a few centimeters distal to the base of the spleen. At the hilum, the SpA immediately bifurcates into one dorsal branch that runs toward the base of the spleen and one abdominal branch that runs along the visceral surface toward the apex of the spleen. The left gastroepiploic artery originates from this abdominal SpA branch, approximately at the junction between the middle and distal third of the spleen.

[0263] At the base of the spleen, the dorsal SpA branch divides into several relatively small arteries, the short gastric arteries, which run toward the greater curvature of the stomach. Although these arteries are considered to be terminal branches of the SpA, they can provide collateral circulation to the spleen by anastomosis with branches of the LGA and left gastroepiploic artery. The SpV runs parallel to the SpA along the visceral surface of the spleen from the apex to the splenic hilum. After leaving the splenic hilum, the SpV runs intimately with the SpA for a short distance before running medially into the hepatic portal vein, which then drains into the posterior aorta. This leaves a small space where the artery and vein run separated by a few millimeters of soft tissue. This area, directly distal to the bifurcation of the celiac trunk into the SpA and LGA, has been identified as the optimal interface location in the functional tests described below. At this location, the SpA diameter is 1.5-3 mm in 30 kg animals; 2-4 mm in 60 kg animals, and 5-8 mm in 110 kg animals.

[0264] The SpNs consist of a plexus of fibers that run along the SpA toward the splenic hilum. The origin of these nerves is difficult to determine, except that fibers arise from the CG, which is directly caudal to the branching of the celiac artery into the SpA and LGA. Data from previous studies, mainly performed in rodents, have confirmed that most of the SpNs originate from the celiac and adrenal ganglia. This has not yet been demonstrated in larger animal species.

[0265] It has also been hypothesized that, despite the presence of the celiac ganglion, a direct connection exists between the vagus nerve (VN) and the SpN. The subdiaphragmatic VN was seen to cross the lateral side of the celiac artery and run toward the CG and the left adrenal gland. Some fibers from the subdiaphragmatic VN were also seen to merge with fibers in the SpN plexus. These fibers are thought to continue to innervate the pancreas, but a direct connection between the VN and the spleen cannot be excluded based on these findings. In rodents, direct connections have been shown to exist between the VN and the SpN, and between the splanchnic nerves and the SpN via the CG. In pigs, parts of the splanchnic nerves were seen to run toward the adrenal gland and the CG.

[0266] In rodents, in addition to the periaortic SpNs, other nerves have been reported to innervate the spleen, more specifically, the apical nerve has been reported to be present within the gastrosplenic ligament in rats and mice. This is a sympathetic nerve (TH+) that probably originates from the paraspinal sympathetic nerve and runs within the gastrosplenic ligament towards the apex of the spleen.

[0267] All histological measurements are shown in Table 1. The SpN-SpA distance was the only measurement that was significantly greater in the 45 kg pigs versus the 22 kg pigs (at the mid-SpA and distal SpA locations; P<0.001). Therefore, for all other measurements, data from all pigs were combined for statistical analysis. There was a decrease in the number of periarterial nerve bundles along the SpA from proximal to distal. There were statistically significantly more bundles at the branching points versus all other locations (P<0.0001). At the hilus, nerve bundles were significantly larger than at other locations (P<0.0001). The SpA outer diameter was significantly larger at the proximal SpA location versus the mid- and distal SpA locations (P=0.0162 and P=0.0158, respectively). The SpN / SpA distance also decreased from proximal to distal. In the 45 kg pigs, the distance was significantly greater at the branching points versus all other locations (P<0.001). Even in 45 kg pigs, SpN / SpA distance was significantly greater at the hilum versus proximal, central, and distal SpA locations (P<0.008).

[0268] [Table 1]

[0269] Circumferential SpN distribution was significantly higher in proximal versus central and distal SpA locations (P = 0.02 and P = 0.15, respectively). Furthermore, bundles were more evenly distributed circumferentially around the SpA proximally, whereas in central and distal SpA, the distribution pattern was more bimodal, with bundles clustered on opposite sides of the artery.

[0270] In pigs, nerves are found along both the short gastric and gastro-epiploic arteries within the gastrosplenic ligament (Figure 12). These nerves appear to be a continuation of the main periaortic SpN plexus, running toward (and away from) the stomach. At this location, immunohistochemical analysis was performed and SpNs at both locations were found to be TH+ and ChAT-. Interestingly, along the main SpA, nerve fibers positive for calcitonin gene-related peptide (CGRP), which is commonly used as an afferent neuron marker, were identified.

[0271] The number of nerve bundles and the size of the bundles observed in these two regions are considerably smaller compared to those observed along the main SpA. Quantification of the number and relative diameter of nerve bundles along the main SpA and along other different anatomical locations in a 45-50 kg farm pig is shown in Figures 12E and 12F.

[0272] Further histochemical and immunohistochemical analyses revealed that the SpNs were composed of >99.9% unmyelinated fibers. Toluidine blue staining of semithin sections showed virtually no myelinated axons. Consistently, staining for myelin basic protein (MBP) revealed very few positive axons (<0.01%). Both techniques to assess myelination revealed an almost complete absence of myelin in the examined areas of the SpNs, as shown in FIG. 9.

[0273] Consideration The histological analysis performed here revealed that the SpNs constitute a neurovascular plexus along the main SpA and the short gastric and gastroepiploic arteries. The number of bundles is unexpectedly high. Considering the average size of the SpN axons (diameter about 2 μm), it can be calculated that the SpN plexus should contain (at most) a total of about 150 k axons at the level of the main SpA (central part). Some of these axons will innervate the SpA endothelium, and some of these axons will instead enter the spleen, forming synaptic connections with either smooth muscle or immune cells at the level of the border zone between the white pulp and the red pulp, as well as within the white pulp as previously reported in other species [23, 24, 25, 26, 27]. The number of axons seems high if it is considered that the human vagus nerve (which has the same size as the porcine vagus nerve), which targets several organs throughout the body, is expected to contain about 100 k axons. The large number of axons in the SpNs may be related to the size of the spleen in pigs, which has a volume approximately 2-3 times that of the human spleen, and the length of the arteries that the SpNs are expected to innervate. The number of bundles and axons in human SpNs may be different.

[0274] The spleen of pigs (and other mammals such as dogs) is also thought to contain a higher proportion of smooth muscle cells compared to the human spleen.

[28] However, several studies have demonstrated that the human spleen can contract under stressful conditions, such as apnea and physical exercise.[29,30]

[0275] The vascular configuration of the splenic artery and vein differs slightly between pigs and humans. In pigs, SpA and SpV run closely toward and away from the spleen. Furthermore, SpV and SpA do not show similar loops and convolutions as observed in humans. Therefore, only a short (approximately 1-1.5 cm) section of SpA close to the trifurcation of the celiac trunk is better separated from SpV. This section of the artery was selected as the best intervention point in the stimulation test described above. Access to the neurovascular bundle at this location is indeed safer, thus reducing the chance of damaging the nerves and arteries and veins during dissection.

[0276] Human Data Electrophysiological Characterization of the Human Splenic Nerve: Materials and Methods Human SpN specimens One freshly harvested tissue from a donor patient containing the splenic neurovascular bundle NVB was stored in a solution suitable for organ transplantation on ice for transport. Upon arrival, the specimen was placed in ice-cold Krebs solution under a dissecting microscope and a minimum of one individual SpN bundle per sample was carefully separated from the SpA, followed by attachment of two bipolar circumferential cuff electrodes (0.65 mm diameter, 5.5 mm length; CorTec GmbH) approximately 10 mm apart to elicit and record CAP. Bundle electrode coverage was estimated to be 100% in all transplants.

[0277] record Neural activity was continuously monitored using an oscilloscope and digitally recorded via a 1401 digital acquisition system and Spike2 software (Cambridge Electronic Design Ltd) with a sampling rate set at 20 kHz. Evoked CAPs were averaged (8 pulses) and the peak-to-peak amplitude of the average response was quantified. The conduction velocity of the eCAP component was calculated from the measured distance between the stimulation and recording sites and the latency of the eCAP signal (measured from the peak of the stimulation artifact to the peak of the eCAP).

[0278] result Compared to the porcine samples, the human SpA showed a more convoluted course as previously reported (Michels 1942). Furthermore, the splenic NVB was embedded in a significant amount of connective tissue and fat (Fig. 13A), allowing recordings from the entire circumference of the structure. However, using a dissecting microscope, some nerve bundles were visible to the naked eye and subsequently confirmed by histological sections of the specimens (Fig. 13B). After attaching stimulating and recording cuff electrodes to some of these bundles (Fig. 13A, top and bottom images), stimulation yielded clear eCAPs (Fig. 13D, top trace). To verify the validity of the recordings after the experiment was completed, the bundles were crushed between the stimulating and recording electrodes and re-recordings were attempted (Fig. 13D, bottom trace). Representative recruitment curves were obtained when stimuli were applied at specific pulse widths (e.g., 100, 200, 400, 800 and 1000 μs; PW) and increasing amplitudes (Fig. 13E).

[0279] Calculated conduction velocity showed values ​​representative of unmyelinated fibers, with a range and mean of conduction velocity of 0.49 m / s compared to porcine (0.7 m / s) and rat (0.72 m / s) SpNs (Figure 13F). Furthermore, eCAP recordings of human SpNs showed a representative strength-duration relationship between current amplitude and pulse width for neural recruitment (Figure 13G). Linear regression of calculated charge density values ​​for eCAP threshold recordings showed a slope significantly different from zero (P = 0.0084), with a minimum PW (100 μs) of 13.44 μC / cm 2 The maximum PW (2000μs) is 14.7μC / cm 2 Importantly, the gradient in charge density for human SpN bundles was found to be similar to that for porcine bundles (Figure S13H). Moreover, the charge density requirement for neural activation of dissected human bundles was approximately 1.5-2 times the charge density required for activation of porcine SpN bundles at any PW (Figure S13H).

[0280] Consideration Human SpNs have similar anatomical, morphological and electrophysiological characteristics to other mammals (pig and rodent). Human SpNs are composed of unmyelinated axons as confirmed by conduction velocity. Therefore, it is reasonable to assume that stimulation parameters (frequency and waveform) optimized in pigs are also suitable for the human splenic nerve. However, charge requirements need to be calculated from the entire NVB.

[0281] Histomorphometric characterization of human splenic anatomy The purpose of this study was to improve understanding of human splenic anatomy and to estimate the approximate dimensions of the splenic neurovascular bundle (NVB) using histology (see Table 2). The study was performed on splenic tissue obtained from transplant patients. Histomorphometric estimates of luminal diameter, arterial wall, bundle diameter (mean ferret diameter) and approximate distance of each bundle from the adventitia (splenic artery outer wall) were calculated.

[0282] Materials and Methods: Five human splenic NVBs were donated by transplant patients at Addenbrooke's hospital, Cambridge, UK. As soon as possible after excision, the tissues were immersed in 10% neutral buffered formalin (NBF). Photographs of the tissues were taken and a ruler was placed for gross measurements (see Fig. 14A). Samples were cut into serial blocks of 0.5-1.5 cm for histological examination (see Fig. 14B). The tissue surrounding the arteries was retained for inclusion in the blocks. Sections were embedded and cut to sample the same face of each block (i.e., proximal or distal to the spleen) at each time. The sections were usually 4-5 μm thick and stained with hematoxylin and eosin stain (H&E) (see Fig. 14C). Finally, a pathologist performed a quality check of the tissues and the slides were scanned at ×20. It should be noted that, as in the literature, 10% tissue shrinkage was taken into account. However, the arterial diameters are representative of zero pressure. All samples from transplant patients contained a high amount of adipose tissue, with the bundles found to be embedded in a thick layer of adipose tissue.

[0283] [Table 2]

[0284] For quantification, the splenic tissue was divided into three parts: proximal, middle, and distal. Each of these parts consisted of several sections. The proximal end was closer to the abdominal cavity, as shown in FIG. 14A with the sutures and pins, and the distal was closer to the spleen. Both of these would not be suitable intervention sites for neural interface placement. The middle part with the loop was considered a possible intervention site.

[0285] In summary, the fascicle diameters ranged from 20 to 400 μm, as shown in Figure 15. Regarding the extent of the fascicles, approximately half of the nerve fibers were found in the 0–1 mm region, 30% in the 1–2 mm region, 15% in the 2–3 mm region, and the remainder in the approximately 3–4 mm region.

[0286] Transformational charge requirements from porcine to human splenic neurovascular bundles Materials and Methods: Histological data from porcine and human spleen histology studies were used to create a 3D Finite Element Model computer simulation. It essentially consists of the splenic artery (lumen + arterial wall) and extravascular tissue. The "extravascular tissue" consists of "adipose tissue" and "connective tissue" with nerves embedded in the tissue. For the pig, a model with a split in the Cortec cuff (representative of the in vivo cuff) was used. For the human model, a cuff with a 3-arm structure was used. The diameter of the cuff used was 9 mm.

[0287] Consideration of the differences between porcine and human histology: a) the bundles in pigs are evenly distributed around the artery and are very close together, whereas in humans the bundles appear more dispersed, and b) the histology in pigs shows very little adipose tissue outside the vessels, versus a significant amount in humans.

[0288] To convert the estimated stimulation parameters from pig to human, modeling was performed in two phases as follows. Phase (a): Development of a 3D Finite Element Model (FEM) in Sim4Life simulation tool Using Sim4Life, representative nerve and artery models (based on histology and image quantification), cuffs and electrodes (CAD defined specifications) and 3D voltage fields were developed. Phase (b): Analysis of the FEM solution with the same tools. Using Sim4Life, we interpolated voltages along the axon using the Sundt nerve model

[31] and estimated the strength-duration and population recruitment curves by axonal simulation.

[0289] result Figure 16A represents in vivo acute data from the porcine splenic neurovascular bundle from five animals. The range from five animals for charge requirements was approximately 20-160 μC / cm at <50 mA, 400 μs and 10 Hz. 2 For the third animal, represented in grey, the charge requirement is estimated to be approximately 100 μC / cm at 30 mA, 400 μs and 10 Hz. 2 which correlates well with the in silico simulation data (see Figure 17A). Using the in silico vs. in vivo correlation as validation for the computational model in pigs, the charge requirements were translated to the human splenic neurovascular bundle using histology sections for the two pulse widths. The data are provided in Figure 17C-D and Table 3.

[0290] [Table 3]

[0291] The charge requirement in human acute models for 100% mobilization is approximately 80-1300 μC / cm 2 (400μS pulse width, 12mm 2 surface area) and 70-1100μC / cm 2 (using a 1 ms pulse width). Approximately 70% of the requirement is estimated to be 350 μC / cm 2An additional 30% recruitment requires an exponential increase in charge requirements beyond those that can be accommodated by the implanted device. For example, a 100% requirement would be 70-1300 μC / cm 2 For 80% mobilization, the range is 70-450 μC / cm 2 , 70-250μC / cm for 50% mobilization 2 , 70-170μC / cm for 30% mobilization 2 It can be seen that.

[0292] Consideration Nerve fibers in humans are more dispersed compared to pigs. The extent of the bundle spread around the splenic artery as shown by histology profiling can range from approximately 1-3 mm. Histomorphometric data was further used to optimize stimulation parameters and computational modeling tools were used to translate charge requirements from pig to human. Using the Sundt C fiber model, charge requirements in humans were estimated to be approximately 70-1000 μC / cm for 100% recruitment. 2 It has been shown that the range is

[0293] References [1]Medzhitov, Nature 454, 428-435(24 July 2008). [3]DM Nance, VM Sanders, Brain Behav Immun 21, 736. [4]HH Dale, HW Dudley, J Physiol 68, 97. [5] C. Cairotto et al., Neurogastroenterol Motil 24, 191. [6] M. Rosas-Ballina, KJ Tracey, Neuron 64, 28. [7] G. Vida, G. Pena, EA Deitch, L. Ulloa J Immunol 186, 4340. [8]BO Bratton et al., Exp Physio l97, 800. [9]D. Martelli, S. T. Yao, M. J. McKinley, R. M. McAllen, J Physiol 592(7), 1677.

[10] D. Martelli, S. T. Yao, J. Mancera, M. J. McKinley, R. M. McAllen, Am J Physiol Regul Integr Comp Physiol 307, R1085.

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[17] G. L. Brown, J. S. Gillespie J S, J Physiol, 138:81-102, 1957.

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[23] F. Tischendorf F, Biol Lat 9:307-342, 1956.

[24] M. Fillenz, Proc R Soc Lond B Biol Sci 174:459-468, 1970.

[25] IG Zelenova, Arkh Anat Gistol Embryo l60:88-90, 1971.

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Claims

1. 1. A system for stimulating neural activity of a neurovascular bundle, preferably a nerve innervating the spleen associated with the splenic artery nerve, comprising: at least one electrode for being in signal transmitting contact with the nerve; and at least one control device electrically coupled to the at least one electrode; wherein the at least one controller is configured to apply an electrical signal to the nerve by controlling operation of the at least one electrode, the electrical signal having a frequency of ≦300 Hz and applied in a periodic on-off pattern or having a frequency of ≦50 Hz and applied continuously.

2. 2. The system of claim 1, wherein the electrical signal has a frequency of ≦300 Hz and is applied in an on-off pattern, preferably the electrical signal has a frequency of ≦50 Hz, more preferably ≦10 Hz.

3. 3. The system of claim 2, wherein the periodic on-off pattern has an on period of 0.1 to 10 seconds and an off period of 0.5 to 30 seconds, preferably the ratio of the on period:off period is 1:5, more preferably the ratio is 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, or 1:30; optionally the on period is 2 seconds and the off period is 2 seconds, and further optionally the on period is 0.5 seconds and the off period is 10 seconds.

4. 2. The system of claim 1, wherein the electrical signal has a frequency of < 50 Hz and is applied continuously, preferably the electrical signal has a frequency of < 10 Hz, more preferably < 2 Hz, even more preferably < 1 Hz.

5. The system of any one of claims 1 to 4, wherein the electrical signal comprises a pulse train, the pulse train comprising a plurality of pulses.

6. The system of claim 5 , wherein the pulse is a square pulse.

7. The system of any one of claims 5 to 6, wherein the pulses are at least one of: charge-balanced, biphasic, symmetric, and asymmetric, preferably: biphasic, charge-balanced, and asymmetric.

8. The system according to any one of claims 5 to 7, wherein the electrical signal has a pulse width of 250 to 1000 μs, preferably 400 to 1000 μs.

9. 9. The system of any one of claims 1 to 8, wherein the electrical signal causes an improvement in a physiological parameter in the subject, the improvement in the physiological parameter being one or more of the group consisting of a decrease in pro-inflammatory cytokines, an increase in anti-inflammatory cytokines and / or pro-resolved mediators, an increase in catecholamines, changes in immune cell populations or immune cell surface costimulatory molecules, a decrease in factors involved in the inflammatory cascade, and / or a decrease in immune response mediators.

10. The system of claim 9 , wherein the system is for treating an inflammatory disorder in a subject.

11. A system according to any one of claims 5 to 10 when dependent on claim 5, wherein the electrical signal is applied episodically throughout the day, each episode comprising 50 to 10000, preferably 60 to 3000, optimally 100 to 2400 pulses of the electrical signal.

12. 12. The system of claim 11, wherein the electrical signal is applied to the subject episodically, once every 2-3 hours, up to 6 times per day.

13. 13. The system of any one of claims 1 to 12, further comprising a neural interface for suitable placement around the nerve, the neural interface comprising the at least one electrode.

14. 14. The system of claim 13, wherein the neural interface has at least two opposing ends that, when brought together, form a cuff for anchoring the neural interface on the nerve.

15. 15. The system of any one of claims 1 to 14, further comprising at least one detector configured to detect one or more of systemic arterial blood pressure, blood flow velocity in the spleen, blood flow velocity in the splenic artery, blood flow velocity in the splenic vein, splenic volume, splenic tissue perfusion, neural activity in a nerve, impedance of at least one electrode, or stimulator voltage compliance.

16. 16. The system of claim 15, wherein the at least one control device is configured to apply an electrical signal to the nerve to confirm that a neural interface in signal transmitting contact with the nerve is properly placed prior to applying the electrical signal to the nerve, the electrical signal having a frequency of < 300 Hz and applied continuously for a period of < 3 hours.

17. 17. The system of claim 16, wherein the detector is configured to detect one or more of a systemic arterial blood pressure, a blood flow velocity in the spleen, a blood flow velocity in a splenic artery, and a blood flow velocity in a splenic vein, and the at least one controller is further configured to determine whether the detected blood flow differs from a baseline blood flow, and if so, to indicate to an operator that the neural interface is properly placed in signaling contact with the nerve.

18. 20. The system of claim 17, wherein the detector is configured to measure blood flow using one or more of electrical impedance tomography, Doppler blood flow, ultrasound, strain measurement, pressure, and electrical impedance.

19. 17. The system of claim 16, wherein the detector is configured to detect neural activity in the nerve, and the at least one controller is further configured to determine whether the detected neural activity is higher than baseline neural activity, and if so, to indicate to an operator that the neural interface is properly placed in signaling contact with the nerve.

20. The system of claim 19 , wherein the detector comprises one or more recording electrodes.

21. 17. The system of claim 16, wherein the detector is configured to detect a spleen volume, and the at least one control device is further configured to verify whether the detected spleen volume is less than a baseline spleen volume, and if so, to indicate to an operator that the neural interface is properly placed in signaling contact with the nerve.

22. 22. The system of claim 21, wherein the detector is configured to measure spleen volume using ultrasound.

23. 17. The system of claim 16, wherein the detector is configured to detect impedance of the at least one electrode, and the at least one controller is further configured to determine whether the detected impedance differs from a baseline impedance and, if so, to indicate to an operator that the neural interface is properly placed in signal transmitting contact with the nerve.

24. 24. The system of claim 23, wherein the detector is configured to measure impedance using an impedance meter.

25. 25. The system of any one of claims 17 to 24, wherein the system further comprises a display, and the at least one control device is further configured to indicate to an operator via the display that the neural interface is correctly installed.

26. The charge density per phase applied to the nerve by the electrical signal is between 5 μC and 1100 μC / cm ２ / phase, optionally 5μC to 450μC / cm ２ / phase, optionally 5μC to 150μC / cm ２ / phase, optionally 50μC to 450μC / cm ２ / phase, and optionally from 50 μC to 160 μC / cm ２ 26. The system according to any one of claims 1 to 25, wherein the first phase is a first phase.

27. 27. The system of any one of claims 1 to 26, wherein the system includes a signal generator, the signal generator configured to send an electrical signal to the at least one electrode in response to a control operation from the control device.

28. 28. The system of claim 27, wherein the signal generator comprises at least one of a current source or a voltage source.

29. The system of any one of claims 1 to 28, wherein the at least one electrode comprises a first electrode and a second electrode.

30. 30. The system of claim 29, wherein the first electrode is an anode and the second electrode is a cathode.

31. 31. The system of claim 29 or 30, wherein the second electrode is configured to be in signal transmitting contact with the nerve and the first electrode is configured not to be in signal transmitting contact with the nerve, optionally the first electrode is grounded, and optionally the first and second electrodes form a monopolar configuration.

32. 31. The system of claim 29 or 30, wherein the at least one electrode further comprises a third electrode, the second electrode being positioned between the first electrode and the third electrode along the longitudinal axis of the nerve.

33. 33. The system of claim 32, wherein the third electrode is an anode.

34. 34. The system of any one of claims 29 to 33, wherein the width of the first electrode and the third electrode is from 0.5 to 4 mm, optionally from 0.5 to 2 mm, optionally from 0.5 to 1.5 mm, further optionally from 0.7 to 1 mm; optionally from 1 to 4 mm, optionally from 1 to 3 mm, optionally from 2 to 4 mm, optionally from 2 to 3 mm.

35. 35. A system according to any one of claims 29 to 34, wherein the distance between the first electrode and the second electrode and / or the distance between the second electrode and the third electrode is between 5mm and 7mm, optionally between 5.5mm and 6.5mm, further optionally between 6.2mm and 6.4mm, when dependent on claim 33, 34 or 35.

36. 36. The system of any one of claims 1 to 35, wherein the at least one control device includes a processor and a non-transitory computer readable storage medium having an executable computer program including code portions that, when loaded into the processor and operated, cause the processor to at least control operation of the at least one electrode.

37. 1. A method for reversibly stimulating neural activity in a nerve innervating the spleen associated with a neurovascular bundle, preferably the splenic artery nerve, comprising: Providing a system according to any one of claims 1 to 36; positioning at least one electrode in signal transmitting contact with the nerve; and controlling operation of the at least one electrode with at least one controller to apply an electrical signal to the nerve to stimulate neural activity. The method includes:

38. 38. The method of claim 37, wherein the method is for treating an inflammatory disorder in a subject.

39. 1. A method for verifying whether a neural interface is properly placed in signaling contact with a nerve supplying the spleen associated with a neurovascular bundle, preferably the splenic artery nerve, comprising: providing a system according to claim 13 or any one of claims 14 to 36 when dependent on claim 13; positioning the neural interface about the nerve; applying an electrical signal to the nerve by controlling operation of the at least one electrode with at least one controller; determining at least one of a change in blood flow velocity or pressure in the spleen, splenic artery, or splenic vein, a decrease in splenic volume, an increase in neural activity in the nerve, a change in heart rate, a change in systemic arterial blood pressure, a decrease in impedance of the at least one electrode, and a decrease in stimulator voltage compliance; and indicating to an operator that the neural interface has been properly placed in signal transducing contact with the nerve. The method includes:

40. 37. A computer-implemented method for reversibly stimulating neural activity in a nerve innervating the spleen associated with the neurovascular bundle, preferably the splenic artery nerve, comprising controlling the operation of at least one electrode of the system of claim 36 to apply a signal to said nerve to stimulate neural activity.

41. 41. The method of claim 40, wherein the method is for treating an inflammatory disorder in a subject.

42. 1. A computer-implemented method for verifying whether a neural interface is properly placed in signaling contact with a nerve innervating the spleen associated with a neurovascular bundle, preferably the splenic artery nerve, comprising: applying a signal to the nerve by controlling operation of at least one electrode of the system of claim 36; determining at least one of a change in blood flow velocity or pressure in the spleen, splenic artery, or splenic vein, a decrease in splenic volume, an increase in neural activity in the nerve, a change in heart rate, a change in systemic arterial blood pressure, a decrease in impedance of the at least one electrode, or a decrease in stimulator voltage compliance; and indicating to an operator that the neural interface has been properly placed in signal transducing contact with the nerve. The method includes:

43. A neurostimulating electrical signal for use in treating an inflammatory disorder in a subject, said electrical signal being the electrical signal of any one of claims 1 to 10.

44. A modified nerve, preferably the splenic artery nerve, supplying the spleen which is associated with the neurovascular bundle, said nerve being in signalling contact with the system of any one of claims 1 to 36, said at least one electrode being in signalling contact with said nerve so that said nerve can be distinguished from said nerve in its native state, said modified nerve being in a subject suffering from or at risk of an inflammatory disorder.

45. A modified nerve innervating the spleen associated with the neurovascular bundle, preferably the splenic artery nerve, which can be obtained by stimulating the neural activity of the nerve according to the method of any one of claims 37 to 42.

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