Treatment of inflammatory disorders

Abstract

Devices and methods for the stimulation of neural signaling of an apical splenic nerve, the device having a transducer for placement on or around the apical splenic nerve, and a signal generator to generate a signal that stimulates or inhibits the neural activity of the apical splenic nerve to produce a physiological response. The transducer has at least one electrode, and the signal generator is a voltage or current source. The stimulation electrical signal has a frequency of between 1 Hz and 50 Hz.

Claims

1. A device or system for stimulating neural activity of an apical splenic nerve, the device or system comprising: at least one transducer configured for placement on or around the apical splenic nerve, wherein the apical splenic nerve is a non-arteriolar associated nerve located at an apex of a spleen, wherein the apical splenic nerve enters a superior pole of the spleen, memory for storing patient specific physiological data pertaining to levels of signaling molecules secreted from the spleen, and a signal generator configured for generating at least one signal to be applied to the apical splenic nerve via the at least one transducer wherein the at least one signal stimulates or inhibits the neural activity of the apical splenic nerve to produce a physiological response in a subject, wherein the physiological response is one or more of the group consisting of: a reduction in pro-inflammatory cytokines, an increase in anti-inflammatory cytokines, an increase in catecholamines, changes in immune cell population or immune cell surface co-stimulatory molecules, a reduction in factors involved in an inflammation cascade or a reduction in immune response mediators; and wherein the at least one transducer is at least one electrode, and the signal generator is a voltage or current source configured to generate an electrical signal to be applied to the apical splenic nerve via the at least one electrode, and wherein the electrical signal has a frequency of between 1 and 50 Hz.

2. The device or system of claim 1, wherein the at least one transducer is configured to attach onto the apical splenic nerve.

3. The device or system of claim 1, wherein the electrical signal is an AC signal.

4. The device or system of claim 1, wherein the electrical signal comprises one or more pulse trains, each comprising a plurality of square or sawtooth pulses, wherein the plurality of pulses are delivered at a frequency in a range of 1 to 50 Hz.

5. The device or system of claim 4, wherein the plurality of pulses are delivered at a frequency of 1 Hz, 5 Hz or 10 Hz.

6. The device or system of claim 4, wherein the square or sawtooth pulses have a duration of between 10 μs and 5 ms.

7. The device or system of claim 4, wherein the square or sawtooth pulses are bipolar pulses.

8. The device or system of claim 4, wherein the square or sawtooth pulses have a constant current of between 200 μA and 5 mA.

9. The device or system of claim 8, wherein the square or sawtooth pulses have a constant current of 600 μA.

10. The device or system of claim 4, wherein the at least one signal is delivered for between 30 seconds and 5 minutes.

11. The device or system of claim 10, wherein the signal is delivered for 2 minutes.

12. The device or system of claim 4, wherein the square or sawtooth pulses have a duration of between 20 μs and 4 ms.

13. The device or system of claim 4, wherein the square or sawtooth pulses have a duration of between 50 μs and 2 ms.

14. The device or system of claim 4, wherein the square or sawtooth pulses have a duration of between 100 μs and 1 ms.

15. The device or system of claim 4, wherein the square or sawtooth pulses have a duration of between 200 μs and 500 μs.

16. The device or system of claim 4, wherein the electrical signal comprises one or more pulse trains, each comprising a plurality of square or sawtooth pulses, wherein the plurality of pulses are delivered at a frequency between 1 and 30 Hz.

17. The device or system of claim 1, further comprising a detection subsystem for detecting one or more sensory signals indicative of excessive or insufficient levels of a cytokine and, upon detection of the one or more sensory signals, cause the at least one signal to be applied to an apical splenic nerve via the at least one electrode.

18. The device or system of claim 17, further comprising a memory for storing data pertaining to sensory signals indicative of normal, excessive or insufficient levels of a cytokine, the detection subsystem configured to compare the one or more detected sensory signals with the data.

19. The device or system of claim 1, further comprising a signaling subsystem for receiving a control signal from a controller and, upon detection of the one or more control signals, cause the electrical signal to be applied to the apical splenic nerve via the at least one electrode.

20. The device or system of claim 1, wherein the signal generator is configured to apply the electric signal periodically.

21. The device or system of claim 1, wherein the device is configured to be attached to the apical splenic nerve and wherein the device is positioned such that the at least one transducer is in signaling contact with the apical splenic nerve, so the apical splenic nerve can be distinguished from the apical splenic nerve in its natural state, and wherein the apical splenic nerve is located in a patient who suffers from an inflammatory disorder.

22. The device or system of claim 1, wherein the device is configured to be attached to the apical splenic nerve and wherein the device is positioned such that a nerve membrane is reversibly depolarised or hyperpolarised by an electric field, such that an action potential is generated de novo in a modified nerve.

23. The device or system of claim 1, wherein the device is configured to be attached to the apical splenic nerve and wherein the device is positioned such that an action potential is propagated along the apical splenic nerve in a normal state; wherein at least a portion of the apical splenic nerve is subject to an application of a temporary external electrical field which modifies a concentration of potassium and sodium ions within the apical splenic nerve, causing depolarization or hyperpolarization of a nerve membrane, thereby, in a disrupted state, temporarily generating an action potential de novo across that portion; wherein the apical splenic nerve returns to its normal state once the temporary external electrical field is removed.

24. The device or system of claim 1, wherein the device is configured to be attached to the apical splenic nerve and wherein the device is positioned such that a portion of the apical splenic nerve is subject to application of a temporary external electrical field forms.

25. The device or system of claim 1, wherein the device modulates neural activity of the apical splenic nerve.

26. A method of reducing inflammation in a subject by reversibly stimulating neural activity of an apical splenic nerve, comprising: (i) implanting a device configured for stimulating neural activity of an apical splenic nerve into a patient, the device including memory for storing patient specific physiological data pertaining to levels of signaling molecules secreted from a spleen, wherein the apical splenic nerve is a non-arteriolar associated nerve located at an apex of the spleen, wherein the apical splenic nerve enters a superior pole of the spleen; (ii) positioning a transducer in signaling contact with an apical splenic nerve; and (iii) activating the device.

27. The method of claim 26, wherein said subject suffers from an inflammatory disorder.

28. A method of reversibly stimulating neural activity in an apical splenic nerve, comprising: (i) implanting a device configured for stimulating the neural activity of an apical splenic nerve into a patient, the device including memory for storing patient specific physiological data pertaining to levels of signaling molecules secreted from a spleen; (ii) positioning a transducer in signaling contact with an apical splenic nerve, wherein the apical splenic nerve is a non-arteriolar associated nerve located at an apex of the spleen, wherein the apical splenic nerve enters a superior pole of the spleen; and (iii) activating the device.

29. A method of treating in a subject who suffers from, or is at risk of, inflammatory disorder, comprising (i) implanting a device configured for stimulating neural activity of an apical splenic nerve into a patient, the device including memory for storing patient specific physiological data pertaining to levels of signaling molecules secreted from a spleen; (ii) positioning a transducer in signaling contact with an apical splenic nerve, wherein the apical splenic nerve is a non-arteriolar associated nerve located at an apex of the spleen, wherein the apical splenic nerve enters a superior pole of the spleen; and (iii) activating the device.

30. A method of controlling a device configured for stimulating the neural activity of an apical splenic nerve, the device including memory for storing physiological data pertaining to normal levels of signaling molecules secreted from the spleen, wherein the device is in signaling contact with an apical splenic nerve, comprising steps of: storing in memory patient specific physiological data pertaining to levels of signaling molecules secreted from the spleen; sending control instructions to the device; applying a signal to the apical splenic nerve, wherein the apical splenic nerve is a non-arteriolar associated nerve located at an apex of the spleen, wherein the apical splenic nerve enters a superior pole of the spleen; detecting a signal received from one or more sensors; and comparing the signal received from the one or more sensors with the physiological data stored in the memory to determine whether the signals are indicative of insufficient or excessive levels of a signaling molecule secreted from the spleen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows a representative image of the nerves innervating the spleen in a mouse.

(2) FIG. 1B shows representative confocal microscopy images of different branches of the spleen innervation in transgenic TH-Ires-Cre C57/B16 mice. Positive staining: tdTomato+, representing catecholaminergic fibers.

(3) FIG. 2 shows the splenic norepinephrine (NE) levels compared between mice that had and had not been electrostimulated at the apical splenic nerve. A Mann-Whitney test was applied (*=statistically significant).

(4) FIG. 3 shows the LPS-induced TNF and IL-6 levels in serum compared between mice that had and had not been electrostimulated at the apical splenic nerve or at the arterial splenic nerve. One representative experiment is shown in FIG. 3A (n=4), and a pool of 7 independent experiments are shown in FIGS. 3B (TNF) and 3C (IL-6) (each experiment: 4-7 animals/group). A one-way (A) or two-way (B and C) ANNOVA with Bonferroni multiple comparison test was applied (*=statistically significant).

(5) FIG. 4 shows the LPS-induced TNF and IL-6 levels in serum compared between apical splenic nerve resected mice and apical splenic nerve intact mice. A representative experiment is shown in FIG. 4A for each cytokine (n=4-5), and a pool of 5 (TNF) and 3 (IL-6) independent experiments are shown in FIGS. 4B and 4C, respectively (each experiment: 3-5 animals/group). A one-way (A) or two-way (B and C) ANNOVA with Bonferroni multiple comparison test was applied (*=statistically significant).

(6) FIG. 5A shows a representative recording of the arterial blood pressure in three anesthetized mice. “Start” denotes the beginning of electrostimulation.

(7) FIG. 5B shows the percentage of HR, mean BP, maximum BP (max BP) and minimum BP (min BP) in anaesthetized mice post-electrostimulation relative to pre-electrostimulation. The data were pooled from three independent mice. The apical splenic nerve or the vagus nerve was stimulated.

(8) FIG. 6 shows various LPS-induced cytokine levels in serum compared between mice that had and had not been electrostimulated at the apical splenic nerve (n=5-6 animals/group). (A) TNF-α, (B) IL-10, (C) IL-1β, (D) IL-6, (E) IFN-γ, (F) IL-12, (G) CXCL1.

(9) FIG. 7 shows the LPS-induced TNF levels in serum compared between mice that had and had not been electrostimulated at the apical splenic nerve at various conditions. One representative experiment is shown with at least 3 mice/group per condition. Amplitude tested at 10 Hz, t=0: 200 μA (B), 600 μA (A), and 1 mA (C). Frequency tested at 600 μA, t=0: 1 Hz (E), 10 Hz (D) and 20 Hz (F). Number of sessions tested at 10 Hz and 600 μA: one session at t=0 (G), two sessions at t=−10 min and t=+10 min (H), and three sessions at t=−10 min, t=0 min and t=+20 min (I). Starting times of electrostimulation are indicated in minutes relative to LPS injection.

(10) FIG. 8 shows the LPS-induced TNF levels in serum compared between conscious mice that had and had not been electrostimulated at apical or periarteriolar branches of the splenic nerve (arterial splenic nerve 1 or 2), or the vagus nerve. In FIG. 8A, the TNF levels (pg/ml) are indicated per group/mouse. Number of experiments (N) and total number of mice (n) are indicated, each group consisted of n=4-7 mice. FIG. 8B expresses the data in FIG. 8A as percent of inhibition of TNF compared to control mice.

(11) FIG. 9A shows the LPS-induced TNF levels in serum compared between conscious mice that had and had not been electrostimulated at the apical splenic nerve. Mice were injected ip with a lethal dose of LPS (400 μg/animal) or a sublethal dose of LPS (100 μg/animal).

(12) FIG. 9B shows the percentage survival of apical nerve stimulated mice compared to non-stimulated mice over time. One experiment is presented with at least 4 mice/group.

(13) FIG. 10 is an image showing the location of the apical splenic nerve in humans relative to the superior pole of the spleen and the phrenicosplenic ligament.

MODES FOR CARRYING OUT THE INVENTION

(14) Materials and Methods

(15) Mice and Reagents

(16) TH-Ires-Cre and Ai14 mice were used in the experiments. A sublethal (5 mg/kg) or lethal (20 mg/kg) dose of LPS from E. Coli 0127:B8 (Sigma Aldrich) was given intraperitoneally to the mice as indicated in the legend of the figures.

(17) Electrodes

(18) For acute electrostimulations, animals were anesthetized with a mixture of Ketamine (75 mg/kg) and Xylazine (60 mg/kg) i.p. The animal's trunk was shaved, the splenic nerve was exposed by a ventrolateral approach and the nerve was either cut or placed on a hook electrode from Harvard Apparatus (Holliston, USA). Animals were injected with a sublethal dose of LPS and kept anesthetized until blood sampling. Supplementary doses of anesthetic were given as needed to maintain anesthesia. For chronic implantation, animals were anesthetized by 2% isofluorane. The apical splenic nerve was exposed as described above and a bipolar electrode from Cortec (Freiburg, Germany) was implanted either onto the apical splenic (sling, 1 mm length, 100 μm diameter), the arterial splenic- (sling, 2 mm length, 100 μm diameter) or the vagus (tunnel, 2 mm length, 200 μm diameter) nerve. The wires were maintained in place by a stitch point placed on the abdominal muscles and exited abdominally. To avoid animal scraping, the abdomen was wrapped with bandages. The total duration of the procedure was about 20 minutes per animal. A morphinic derivative was given before and after the surgery (Buprecare®, 0.1 mg/kg, i.p. 30 minutes before surgery and 0.05 mg/kg, s.c. after surgery and the following 2 days). Five days later, animals were injected with a sublethal dose of LPS, electrostimulated and blood samples were collected 90 minutes later.

(19) Electrostimulation

(20) Electrostimulation was performed using a PlexStim V2.3 from Plexon (Dallas, Tex., USA). Unless specified, the set-up of the electrostimulation were rectangular charged-balanced biphasic pulses with 650 μA pulse amplitude, 2 ms pulse width (positive and negative) at 10 Hz frequency.

(21) Detection of Norepinephrine, TNF and IL-6

(22) Norepinephrine (NE) levels in biopsies were determined using a competitive ELISA for the quantitative determination of NE according to the manufactures protocol (DLD Diagnostika GmbH, Hamburg, Germany).

(23) Tumor Necrosis factor (TNF) and interleukin 6 (IL-6) sera were diluted 10-fold with PBS, and cytokine concentrations were assessed in duplicate by ELISA (R&D DuoSet, Minneapolis, Minn., USA) according to the manufacturers protocol.

(24) For multicytokine measurement (FIG. 6), the Meso Scale Discovery (MSD) multiplex assay kits (Rockville, Mass., USA) were used, and the kits allow quantitation of multiple analytes in the same sample with wide range of detection. V-PLEX Proinflammatory panel 1 mouse kit was used in this experiment.

(25) Statistics

(26) The student T-test or logrank test were used to calculate statistical differences. Mann-Whitney (FIG. 2) and Annova test were performed for small size groups. The survival curve gives a p=0.188 (LogRank test).

(27) Blood Pressure Recording

(28) Blood pressure was recorded on acutely electrostimulated animals by inserting a catheter into the common branch of the carotid. The catheter was connected to a blood pressure transducer TSD104A-MRI and to a data acquisition unit.

(29) Septic Shock

(30) Animals were operated as above-mentioned for chronic implantation. A lethal dose of LPS (400 μg/animal) was administered after five days recovery and electrostimulation (650 μA, 10 Hz, 2 ms pulse width) was applied for 2 minutes starting at −10, and +20 minutes relative to LPS challenge and again at 16, 20, 24, 30, 34 and 38 h post-LPS challenge for 2 min. Survival was monitored by three times daily examination of the cages.

(31) Results

(32) The Three Nerves that Project to the Mouse Spleen Contain Catecholaminergic Fibers

(33) Mice (n=4) were dissected to assess the anatomical organization of the splenic nerves, and FIG. 1A shows a representative image. It can be seen that the spleen is innervated by at least two distinct arteriolar associated nerves and a non-arteriolar associated nerve located at the apex of the spleen near the stomach. The latter is referred to as an apical splenic nerve.

(34) It was then investigated whether the apical splenic nerve in mice was of catecholaminergic nature. A transgenic mouse strain was generated to express a fluorescent reporter (tdTomato+) in the catecholaminergic fibers. TH-Ires-Cre C57/B16 mice in which the site-specific Cre recombinase was selectively expressed in cells that express the Tyrosine Hydroxylase (TH) were crossed with Ai14 mice that carry a loxP-flanked STOP cassette that prevented transcription of the downstream red fluorescent protein variant (tdTomato) inserted into the Gt(ROSA)26Sor locus. Because TH was selectively expressed in catecholaminergic fibers, the STOP sequence was deleted and tdTomato expression was observed only in these cells in the double mutant offspring.

(35) The different branches of the spleen innervation and artery were imaged, and representative images from TH-Ires-Cre x Ai14 mice (n=3) are shown in FIG. 1B.

(36) It was noted that, as expected, cellular bodies and axons from the celiac ganglion were tdTomato+(data not shown).

(37) FIG. 1B shows that the arteriolar splenic nerves were tdTomato+, as expected. Interestingly, the apical splenic nerve also showed tdTomato expression suggesting that it was also catecholaminergic.

(38) One of the limitations of the Cre/LoxP system is that the reporter gene may still be expressed in adults due to genetic recombination whereas the activity of the promoter may be lost in the early phase of the development. The functional ability of apical splenic nerve to release norepinephrine (NE) was therefore investigated.

(39) C57/B16 (N=2, n=3-7 mice/group) mice were anesthetized and a hook electrode is placed onto the apical splenic nerve. Electrostimulation (650 μA, 2 ms pulse width, 2-minutes duration, 10 Hz) was applied to mice. The control mice were not electrostimulated. Spleen biopsies were collected immediately after electrical stimulation and snap frozen in liquid nitrogen for NE ELISA measurements. A Mann-Whitney test was applied.

(40) The results are shown in FIG. 2. It can be seen that a two-fold increase of the NE splenic content following stimulation was observed, confirming the catecholaminergic nature of these fibers.

(41) Electrical Stimulation of the Splenic Nerves on LPS-Induced Inflammation in Anesthetized Mice

(42) The functional role of the splenic apical nerve on LPS-induced inflammation in anesthetized mice was investigated.

(43) C57/B16 mice were anesthetized and a hook electrode is placed onto either the apical splenic nerve or the arterial splenic nerves. These mice were injected ip with 100 μg of LPS and electrostimulation (650 μA, 2 ms pulse width, 2-minutes duration, 10 Hz) were applied at the same time. Control mice did not receive electrostimulation. Sera were collected 90 minutes after LPS injection and assessed for TNF and IL-6. The results are shown in FIG. 3. FIG. 3A shows one representative experiment (n=4), and FIGS. 3B and 3C show a pool of 7 independent experiments, respectively, and in each experiment 4-7 animals/group. A one-way (A) or two-way (B and C) ANNOVA with Bonferroni multiple comparison test was applied.

(44) FIG. 3 shows that, as expected, electrostimulation of the arterial splenic nerves in anesthetized animals resulted in markedly reduced serum levels of TNF and IL-6 after LPS injection. Interestingly, apical nerve electrostimulation also significantly reduced LPS-induced TNF and IL-6 secretions.

(45) Resection of the Apical Nerve to the Spleen Results in Increased Inflammatory Cytokines Secretion in Anesthetized Mice

(46) In order to confirm that electrostimulation of the apical splenic nerve was contributing to the effect, and that it is not mediated by current leakage to other nerves or organs (e.g. spleen), the apical splenic nerve was resected.

(47) C57/B16 mice were anesthetized and apical nerve of the spleen was cut or sham operated. These animals were injected ip with 100 μg of LPS. Sera was collected 90 minutes after LPS injection and assessed for TNF and IL-6 cytokine levels by ELISA.

(48) The results are shown in FIG. 4. FIG. 4A shows a representative experiment for each cytokine (n=4-5). FIGS. 4B and 4C show a pool of 5 (TNF) and 3 (IL-6) independent experiments, respectively, with in each experiment 3-5 animals/group. A one-way (A) or two-way (B and C) ANNOVA with Bonferroni multiple comparison test was applied.

(49) FIG. 4 shows that ablation of the apical splenic nerve resulted in increased levels of LPS-induced inflammatory cytokine release in anesthetized animals. Therefore, the apical splenic nerve contributed to the physiological anti-inflammatory reflex.

(50) Impact of Electrical Stimulation of the Apical Splenic Nerve on Arterial Blood Pressure

(51) Stimulation of the vagus nerve was known to cause a drop in blood pressure. It was therefore investigated whether apical splenic nerve stimulation might have an impact on systemic arterial blood pressure.

(52) C57/B16 mice were anesthetized and a hook electrode was placed onto the apical splenic nerve. Cardiovascular parameters (heart rate (HR), blood pressure (BP)) were recorded before (pre-stim.) and after (post-stim.) electrical stimulation (2 ms pulse width, 2-minutes duration). Frequencies tested: 5, 10, and 20 Hz. Amplitudes tested: 0.3, 0.6, 1, and 5 mA. Stimulation of the vagus nerve (VNS, 10 Hz, 600 μA, 2 ms, 2 min) was used as control.

(53) One representative recording is presented in FIG. 5A, and the HR, mean BP, maximum BP and minimum BP is pooled from three independent mice, as shown in FIG. 5B.

(54) It was found that electrostimulation of the apical splenic nerve had minimal impact on arterial blood pressure or heart rate, irrespective of the electrical parameters applied. In contrast, electrostimulation of the vagus nerve significantly decreased the arterial blood pressure and heart rate.

(55) Impact of Electrical Stimulation of the Apical Splenic Nerve on Cytokines and Chemokine Secretion

(56) The impact of electrical stimulation of the apical nerve on other cytokines and chemokine secretion in the blood was also investigated.

(57) C57/B16 mice (4 mice/group) were anesthetized and a hook electrode is placed onto the apical splenic nerve. The animals were injected iv with 100 μg of LPS and electrostimulation (350 μA, 2 ms pulse width, 2-minutes duration, 1 Hz or 10 Hz) was applied at the same time. The control mice did not receive electrostimulation. Sera was collected at 60, 90 and 120 minutes after LPS injection and assessed for cytokine and chemokine levels. Pro-inflammatory cytokines (TNF, IL-12, IL-1β, CXCL1 and IL-6) and anti-inflammatory cytokine (IL-10) levels were assessed. FIG. 6 shows one representative experiment out of 2 (n=5-6 animals/group).

(58) It was found that inflammatory cytokines (TNF, IL-12, IL-1β, CXCL1 and IL-6) secreted in the blood were dramatically reduced by electrical stimulation of the apical splenic nerve. In contrast, the anti-inflammatory cytokine IL-10 was increased at 120 min post electrical stimulation of the apical splenic nerve using 10 Hz.

(59) Optimization of Electrical Stimulation Parameters in Anesthetized Mice

(60) To evaluate the therapeutic potential of apical nerve electrical stimulation in conscious animals, the electrical stimulation was optimized in these animals.

(61) C57/B16 mice were anesthetized and a Cortec electrode was implanted onto the apical splenic nerve. Five days following surgery, these animals were injected ip with 100 μg of LPS and electrostimulation (2 ms pulse width) was applied with various amplitude, frequency and number of session. Amplitude tested: 200 μA, 600 μA, and 1 mA. Frequency tested: 1 Hz, 10 Hz and 20 Hz. Number of sessions tested: one session at t=0, two sessions at t=−10 min and t=+10 min, and three sessions at t=−10 min, t=0 min and t=+20 min. Starting times of electrostimulation are indicated in minutes relative to LPS injection. Sera was collected at 90 minutes after LPS injection and assessed for TNF levels. Implanted but non-stimulated animals were used as control. One representative experiment is shown in FIG. 7 with at least 3 mice/group per condition.

(62) Interestingly, three sessions of stimulation of 2 minutes applied −10 minutes, 0, and +20 minutes after LPS injection resulted in over 50% reduction of TNF secretion after LPS challenge (FIG. 7J). This inhibition was confirmed in seven independent experiments and was compared to the stimulation of periarteriolar-associated nerves.

(63) Electrical Stimulation of Apical and Arterial Splenic Nerves Inhibits LPS-Induced Inflammation in Conscious Animals.

(64) C57/B16 mice were anesthetized and Cortec electrodes were implanted either onto the apical or onto the periarteriolar splenic nerves (1 and 2). Five days following surgery, these animals were injected ip with 100 μg of LPS and electrostimulation (650 μA, 10 Hz, 2 ms pulse width, 2 min) was applied starting at −10, 0 and +20 minutes relative to LPS injection. Sera was collected at 90 minutes after LPS injection and assessed for TNF levels. Controls consist of fully Cortec implanted mice, which did not receive electrical stimulation.

(65) The results are shown in FIG. 8. FIG. 8A shows the TNF levels (pg/ml) per group/mouse. Number of experiments (N) and total number of mice (n) are indicated, each group consisted of n=4-7 mice. FIG. 8B shows the same data expressed as percent of inhibition of TNF compared to control mice. It was found that all nerves tested were efficient in inhibiting LPS-induced TNF secretion, with the arterial splenic nerve 1 providing the best inhibition, followed by apical splenic nerve. The apical splenic nerve provided ˜45% inhibition of LPS-induced TNF secretion.

(66) Electrical Stimulation of Apical Splenic Nerve Improves Survival Following Endotoxemic Shock

(67) It was investigated whether mice having its apical splenic nerve implanted with Cortec electrodes would survive after injection of a lethal dose of LPS by intraperitoneal route.

(68) C57/B16 mice were anesthetized and Cortec electrodes were implanted onto the apical splenic nerve. Five days following surgery, the animals were injected i.p. with a lethal dose of LPS (400 μg/animal) and electrostimulation (650 μA, 10 Hz, 2 ms pulse width) was applied for 2 minutes at −10, 0, +20 minutes relative to LPS challenge. Sera were collected at 90 min after LPS injection and assessed for TNF levels. The animals were then electrically stimulated with the same parameters at 16, 20, 24, 30, 34 and 38 hours after LPS injection. Survival was followed over 4 days. Controls consist of Cortec implanted mice, which did not receive electrical stimulation. One experiment is presented in FIG. 9 with at least 4 mice/group.

(69) FIG. 9A shows that LPS-induced TNF secretion was decreased when the mice were electrically stimulated at its apical splenic nerve. Although to a lesser extent, electrical stimulation of the apical splenic nerve still led to decreased TNF secretion following LPS lethal dose (400 μg/mouse) administration compared to LPS sublethal dose (100 μg/mouse).

(70) FIG. 9B shows that three out of five mice (60%) survived in the electrostimulated group compared to one out of six (16.7%) in the control group following LPS administration.

CONCLUSION

(71) In summary, the inventors found that an apical splenic nerve in mice was catecholaminergic. When electrically stimulated, this nerve was effective in inhibiting LPS-induced cytokine release in both anesthetized and conscious animals, and this effect was as potent as the periarteriolar branches of the splenic nerve. Interestingly, electrostimulation of the apical splenic nerve had minimal impact on the arterial pressure or heart rate. Electrical stimulation of the apical splenic nerve was also able to improve the survival following endotoxemic shock. Therefore, the inventors found that electrical stimulation of the splenic nerves, in particular, an apical splenic nerve, would be effective in treating inflammatory conditions, including autoimmune disorders (e.g. rheumatoid arthritis) and sepsis.

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