Treatment of Inflammatory Disorders

Abstract

Modulation of the neural activity of a nerve adjacent to the left gastro epiploic artery (LGEA) and/or a nerve adjacent to a short gastric artery (SGA) can modulate the neural activity of the sympathetic nerves that impact splenic function. This is useful for reducing inflammation and providing ways of treating inflammatory disorders.

Claims

1-27. (canceled)

28. A system for modulating neural activity in a subject's nerve adjacent to a left gastro epiploic artery (LGEA) and/or a subject's nerve adjacent to a short gastric artery (SGA), the system comprising: at least one electrode, in signaling contact with the nerve; and a voltage or current source configured to generate at least one electrical signal with a charge density to be applied to the nerve via the at least one electrode such that the charge density per phase applied to the nerve by the electrical signal is between 5 μC per cm.sup.2 per phase to 150 μC per cm.sup.2 per phase, wherein the electrical signal comprises a pulse train having a pulse width >1 ms, and wherein the electrical signal modulates the neural activity of the nerve to produce a change in a physiological parameter in the subject, wherein the change in the physiological parameter is one or more of the group consisting of: a reduction in a pro-inflammatory cytokine, an increase in an anti-inflammatory cytokine, an increase in a catecholamine, a change in an immune cell population, a change in an immune cell surface co-stimulatory molecule, a reduction in a factor involved in the inflammation cascade, a change in the level of an immune response mediator, a decrease in splenic blood flow, and an increase in systemic blood pressure.

29. The system of claim 28, wherein the pulse width is ≤5 ms.

30. The system of claim 28, wherein the pulse width is between 1.5 and 2.5 ms.

31. The system of claim 28, wherein the pulse width is ≤3 ms.

32. The system of claim 28, wherein the pulse train has an interphase delay of ≤0.3 ms.

33. The system of claim 32, wherein the interphase delay is ≥0.1 ms.

34. The system of claim 33, wherein the interphase delay is between 0.2 ms and 0.25 ms.

35. The system of claim 28, wherein the at least one electrode has a surface area of 0.1-0.3 cm.sup.2.

36. The system of claim 28, wherein the at least one electrode has a surface area of ≤0.2 cm.sup.2.

37. The system of claim 28, wherein the system modulates neural activity in a nerve adjacent to the LGEA, and the at least one electrode is placed on or around both the nerve adjacent to the LGEA and the LGEA.

38. The system of claim 28, wherein the system modulates neural activity in a nerve adjacent to the LGEA, wherein the at least one electrode is placed on or around the nerve adjacent to the LGEA.

39. The system of claim 28, wherein the system modulates neural activity in a nerve adjacent to the SGA, and the at least one electrode is placed on or around both the nerve adjacent to the SGA and the SGA.

40. The system of claim 28, wherein the system modulates neural activity in a nerve adjacent to the SGA, and the at least one electrode is placed on or around the nerve adjacent to the SGA.

41. The system of claim 28, wherein the voltage or current source is configured to apply the at least one electrical signal episodically.

42. The system of claim 28, wherein the voltage or current source is configured to apply the at least one electrical signal periodically.

43. The system of claim 28, comprising a detector configured to: detect one or more signals indicative of one or more physiological parameters; determine from the one or more signals one or more physiological parameters; determine the one or more physiological parameters indicative of worsening of the physiological parameter; and causing the at least one electrical signal to be applied to the nerve via the at least one electrode, wherein the physiological parameter is one or more of the group consisting of: a level of a pro-inflammatory or an anti-inflammatory cytokine, a level of a catecholamine, a level of an immune cell population, a level of an immune cell surface co-stimulatory molecule, a level of a factor involved in the inflammation cascade, a level of an immune response mediator, splenic blood flow, and systemic blood pressure.

44. The system of claim 43, further comprising a memory configured to store data pertaining to the physiological parameters in a healthy subject, wherein determining the one or more physiological parameters indicative of worsening of the physiological parameter comprises comparing the one or more physiological parameters with the data.

45. The system of claim 28, further comprising: a communication subsystem configured to receive a control signal from a controller and, upon detection of said control signal, cause the at least one electrical signal to be applied to the nerve via the at least one electrode.

46. A method of reducing inflammation in a subject by reversibly modulating neural activity of the subject's nerve adjacent to an LGEA and/or the subject's nerve adjacent to an SGA, comprising: (i) implanting in the subject a system of claim 28; (ii) positioning the at least one electrode in signaling contact with the nerve; and (iii) activating the system.

47. A method for treating an inflammatory disorder, comprising: applying an electrical signal with a charge density to a subject's nerve adjacent to a left gastro epiploic artery (LGEA) and/or a subject's nerve adjacent to a short gastric artery (SGA) via at least one electrode, in signaling contact with the nerve, wherein the electrical signal comprises a pulse train having a pulse width >1 ms, wherein the charge density per phase applied to the nerve by electrical signal is between 5 μC to 150 μC per cm.sup.2 per phase, such that the electrical signal reversibly modulates neural activity of the nerve to produce a change in a physiological parameter in the subject, wherein the change in the physiological parameter is one or more of the group consisting of: a reduction in a pro-inflammatory cytokine, an increase in an anti-inflammatory cytokine, an increase in a catecholamine, a change in an immune cell population, a change in an immune cell surface co-stimulatory molecule, a reduction in a factor involved in the inflammation cascade, a change in the level of an immune response mediator, a decrease in splenic blood flow, and an increase in systemic blood pressure.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0233] FIG. 1A is a ventral view of splenic vascularization in relation to the stomach and pancreas, where “CT” is the coeliac trunk, “GA” is the gastric artery, “LGEA” is the left gastroepiploic artery, “OA” is the omental artery, “PA” is pancreatic artery, “SA” is splenic artery, “RGEA” is right gastroepiploic artery, “SGA” is short gastric artery, and “TB” is terminal branch. Dash-dotted line outlines the blood vessels.

[0234] FIG. 1B is a transversal section through the upper abdomen illustrating the course of the splenic artery and a short gastric artery, where “GS ligament” is gastrosplenic ligament, “SGA” is short gastric artery, “SR ligament” is splenorenal ligament, “SA” is splenic artery, “V” is ventral, “D” is dorsal, “L” is left, and “R” is right. Dashed line outlines the peritoneum. Dash-dotted line outlines blood vessels.

[0235] FIG. 2 is a block diagram illustrating elements of a system for performing electrical modulation in the nerve according to the present invention.

[0236] FIG. 3 shows a schematic overview of the splenic artery (SA) and its branches, including the SGA and the LGEA, in relation to the pancreas and the spleen. This images was created to serve as a schematic support for branching pattern, sample location, distances and diameters, and does not represent realistic dimension.

[0237] FIGS. 4A and 4B are fluorescent tile images of LGEA (A) and SGA (B) samples of cadaver III. The arrows indicate nerve bundles (5).

[0238] FIG. 5 is a fluorescent tile images of the LGEA and five surrounding nerves. PGP: Protein Gene Product 9.5, which is a general nerve marker. CGRP: Calcitonin gene-related peptide, which is a sensory marker. TH: tyrosine hydroxylase which is a sympathetic nerve marker.

[0239] FIGS. 6A and 6B are images of gross anatomy of SG and GE artery, vein, and nerves in Yucatan pigs.

[0240] FIG. 7 is a histological image of the SGAs and nerves in Yucatan pigs.

[0241] FIG. 8A is a diagram showing the locations of the cuff-electrodes around the SGA and LGEA in Yucatan pigs for stimulation and recording. FIGS. 8B and 8C are contrast angiography of the pig spleen showing the locations of these cuff-electrodes.

[0242] FIG. 9 shows, in FIG. 9A, the percentage change of serum level of TNFα following LPS challenge, in FIG. 9B, systolic arterial blood pressure (SAP) and splenic arterial blood flow (SpABF), and, in FIG. 9C compound action potentials (CAPs) observed in the level of splenic hilum (n=6) following stimulation of the nerves surrounding the SGA in Yucatan pigs. The A-range shows the region of A-fiber action potentials and the C-range shows the region of C-fiber action potentials. M is the marker for the start of stimulation and X is a mark from the start of stimulation at which the peak on the neurogram is measured.

[0243] FIG. 10 shows, in FIG. 10A, the percentage change of serum level of TNFα following LPS challenge, in FIG. 10B, systolic arterial blood pressure (SAP), splenic arterial blood flow (SpABF), and neural activity recorded in the splenic nerve at the hilum, and in FIG. 10C, compound action potential (CAP) observed in the level of splenic hilum (n=5) following stimulation of the nerves adjacent to LGEA in Yucatan pigs. The A-range shows the region of A-fiber action potentials and the C-range shows the region of C-fiber action potentials. X is a mark from the start of stimulation at which the peak on the neurogram is measured.

[0244] FIG. 11 shows a decrease in splenic artery blood flow in all animals and that denervation abolished stimulation induced decrease in splenic blood flow. More specifically, FIG. 11 shows the percent change in splenic artery blood flow and mean arterial blood pressure (mean BP) during stimulation (10 Hz, 400 us/phase, biphasic, 12 mA for 1 minute) delivered through a cuff on the gastroepiploic nerve (GE) prior to (panel GE Stimulation) and after GE nerve transection (panel GE-X Transection+Stimulation). Prior to transection of the GE nerve stimulation for 1 minute (represented by the line with 12 mA) decreased splenic artery blood flow measured using a transit time flow probe placed on the splenic artery along the hilum of the spleen by approximately 15%. Mean BP did not change during stimulation. After ligating and cutting the GE both afferently and efferently the same stimulation parameters splenic artery blood flow was abolished (panel GE-X).

[0245] FIG. 12 shows (A) an example of the human splenic tissue. The dark stained spots on the sample indicate the splenic artery with aorta towards the left end, and spleen on the right end of the sample (for orientation). (B) shows placement of a pen-arterial cuff around the neurovascular bundle (I) and placement of a smaller diameter cuff around a few nerves (III). The nerve is dissected, placed in a bath with Kreb's solution, and traced all along till the end of the sample, where the hooks are placed to record compound action potentials (C, III). (D) shows a conceptual sketch of tissue with the cuff, and hook placement, and (E) shows an example of an eCAP observed on the oscilloscope.

[0246] FIG. 13 shows results from an ex-vivo electrophysiological study of the human splenic samples. (A) shows current amplitude-pulse width and charge density-pulse width curves. The error bars demonstrates the range, and the lower bar of the range is not presented on the graph. (B), (C), and (D) show recruitment graphs for 0.4 ms, 1 ms and 2 ms pulse widths respectively.

[0247] FIG. 14 shows predictions of recruitment curves for a human splenic nerve in chronic scenarios based on human ex-vivo data at 2 ms pulse width. The y-axis represents the eCAP amplitude as a percentage of maximum response and the x-axis represents the total charge (μC) injected into the human splenic nerve.

[0248] FIG. 15 shows comparisons of recruitment curves calculated for the human model for acute and chronic stimulations with different parameterisations of biphasic pulse waveforms, in particular different pulse widths (0.4 ms, 1 ms) and different interphase delays (0 ms, 0.1 ms, 0.2 ms). In the key (e.g. ‘Chronic1m0ms’), the word represents the type of stimulation (e.g. ‘Chronic’), first number represent the pulse width in ms (e.g. ‘1’ ms), and the second number represents the interphase delay in ms (e.g. ‘0’ ms).

[0249] FIG. 16 shows the charge required to stimulate neural activity per pulse width in a human splenic nerve based on in-silico modelling data. Simulations are based on electrical signals with pulse trains having biphasic pulses with a 0 ms interphase delay (“Biphasic”), biphasic pulses with a 0.1 ms interphase delay (“Biphasic (0.1 ms interp. delay”), and monophasic pulses (“Monophasic”).

[0250] FIG. 17 shows unmyelinated fiber pulse height thresholds verses interphase delay normalised to a 100 μs interphase delay. The y-axis represents the threshold relative to an interphase delay of 100 μs and the x-axis represents the interphase delay (μs).

[0251] FIG. 18 shows comparison of frequency. An increase in frequency from 1 Hz to 10 Hz indicates a reduction in eCAP amplitude and is indicative of nerve fatigue, thus re-confirming porcine data assumptions on frequency.

MODES FOR CARRYING OUT THE INVENTION

Study 1: Neurovascular Structures Going to the Spleen

[0252] The neurovascular structures going to the spleen in humans were investigated. In particular, next to the main splenic artery (SA) and nerve plexus, the area around the gastro splenic ligament, including the SGAs and the LGEA, were analyzed.

[0253] Six formaldehyde preserved cadavers were studied. The donors gave informed consent for the use of their tissues. Tissue blocks of the spleen, stomach, pancreas, greater omentum, gastrosplenic ligament and if present the phrenic splenic ligament were removed as a whole. The tissues were dissected and then tissue samples of the SA and its branches and of both ligaments were isolated and processed for histology. Different immunohistochemical stainings for nervous tissue were performed on adjacent slides, by means of antibodies raised against Protein Gene Product 9.5 (PGP9.5), Tyrosine Hydroxylase (TH) and Calcitonin Gene-Related Peptide (CGRP), respectively staining general, sympathetic and afferent nervous tissue. A specific substrate to visualize the bound antibodies was used to perform both brightfield and fluorescent microscopy on the same samples.

Materials and Methods

Collection of Material; Macroscopic Dissection

[0254] Tissue blocks of six cadavers that were embalmed by arterial perfusion with 4% formaldehyde were collected including the spleen, stomach, pancreas, greater omentum, gastrosplenic ligament and if present the phrenic splenic ligament.

Dissection

[0255] Dissection was performed mostly macroscopically and occasionally with a surgical microscope. During the dissection a photographic log was kept.

Histology

[0256] After extraction of all descriptive and quantitative dissection parameter data, samples of the gastrosplenic ligament, the phrenic splenic ligament and several places of the SA and its branches were removed for histological examination. All samples were degreased in 100% acetone for one hour and arterial samples were treated with a decalcifying agent (12.5% EDTA in distilled water, pH 7.5) for six days. After these pretreatments, all samples were further processed for paraffin embedding and sequentially placed in increasing percentages of ethanol, xylene and finally liquid paraffin. Sample blocks were cut on a microtome and 5 μm thick slices were alternately placed on glass slides. Subsequently, the sample slices were stretched and dried by placing the glass slides on a 60° C. plate for two hours.

[0257] Adjacent slides of each sample were stained with a PGP9.5, a TH, and a CGRP staining. First, the samples were deparaffinated by placing tissue slides sequentially in xylene, decreasing percentages of ethanol and distilled water, after which the slides were incubated with citrate buffer (room temperature) for five minutes. Next, the slides were placed in citrate buffer with a temperature of 95° C. for antigen retrieval (20 minutes). After cooling down and several washing steps with distilled water and Tris-buffered saline (TBS)+tween, tissue slides were pre-incubated with 5% Normal Human Serum in TBS-buffer for ten minutes, followed by incubation with primary antibodies (Rabbit anti-PGP (DAKO) (1:2000) 48 hours (40 C.), rabbit anti-TH (PelFreez) (1:1500) overnight (RT) or mouse anti-CGRP (Sigma) (1:1500) overnight (40 C.)) in TBS-buffer+3% BSA. Thereafter, tissue slides were washed with TBS-buffer +tween several times and incubated for 30 minutes with Brightvision Poly-AP Goat-anti-Rabbit (ImmunoLogic) (PGP and TH) or Brightvision Poly-AP Goat-anti-Mouse (ImmunoLogic) (CGRP). After washing with TBS-buffer several times, the samples were incubated with Liquid Permanent Red (LPR) (DAKO) for ten minutes, resulting in a pinkish precipitation reaction at the side of the antibodies-tissue complex. The slides were washed with distilled water and dipped in hematoxylin for counterstaining. Finally, the slides were placed in flowing tap water and rinsed in distilled water one last time after which they were placed in the 600 C. stove for 90 minutes. Subsequently, the slides were enclosed with entellan (diluted with xylene) and coverslipped. In addition, for each marker a negative control without the primary antibody was included. Samples of the vagus nerve were included as a positive control for afferent nervous tissue (CGRP staining). Intrinsic vessel wall innervation was used as a positive control for general and sympathetic nervous tissue (resp. PGP and TH staining).

Image Analysis

[0258] Both brightfield and fluorescent single images and tile scans were captured using a Leica DM6 microscope with a motorized scanning stage, a Leica DFC7000 T camera and Leica LASX software. For fluorescent images of the LPR substrate, the 13 fluorescent filter (band pass excitation at 450-490 nm and long pass suppression at 515 nm) of Leica was used. The image quality was set to 8-bit and the image format to Bin 2×2. The settings for the brightfield images were; intensity: 255, aperture: 27, field diaphragm: 33, exposure: 3.73 ms, gain: 1.0. The settings for the fluorescent images were; FIM: 100%, II-Fld: 6, exposure: 300 ms, gain: 1.1. Of each artery sample with surrounding nerve bundles, tile scans were made using the microscope. Multiple images were captured with a 20× magnification and automatically stitched to make a tile scan. Tile scans were made with a 20× magnification and were saved as jpg files. Tile scans of TH stained samples were analyzed using FIJI (ImageJ with additional plugins) and several parameters were extracted according to a predefined image analysis protocol. Nerve bundles with an area less than 400 μm2 were excluded, since this is most likely representing nervous tissue supplying the vessel wall itself (van Amsterdam et al, 2016).

Results

Left Gastric Epiploic Artery (LGEA) and the Adjacent Nerves

[0259] All six cadavers presented a single LGEA. The LGEA emerged as a branch directly from the SA in two out of six cadavers and from a lower terminal branch (LTB) in four out of six cadavers. Table 1 shows a summary of the collected quantitative data on dissection parameters concerning the LGEA of each cadaver, followed by the average value. The average diameter of the proximal LGEA was 0.2 cm (ranging from 0.15-0.28 cm), which slightly reduced during its course in the greater omentum. The average diameter of the SA before the branching LGEA was 0.31 cm (0.2-0.5). On average, the LGEA originated 9.43 cm (8.1-12.5) from the origin of the SA. While continuing its course in the greater omentum, the LGEA gave off branches to the stomach (gastric branches (GBs)) and to the greater omentum. The LGEA was mostly closely related with surrounding adipose tissue and connective tissue, but again relatively easily dissected from these tissues. FIG. 3 is a schematic representation of arteries going to the spleen, including the LGEA, in one of the cadavers.

TABLE-US-00001 TABLE 1 Quantitative data on dissection parameters concerning the LGEA and adjacent nerve bundles of each cadaver, followed by the average value. Cadaver number III IV VII VIII IX X Origin LTB LTB SA SA LTB LTB Average Distance 8.5 12.5 9.5 8.1 8.5 9.5 9.43 (81- from 12.5) origin SA (cm) Diameter 0.18 0.15 0.22 0.24 0.21 0.28 0.21 (1537- 2772) Diameter 0.25 0.2 0.5 0.4 0.2 0.3 0.21 SA (0.2-0.5) before LGEA (cm) Diameter 53 51 80 62 46 44 56 (14- of nerve (47- (14- (17- (23- (25- (19- 214) bundles 59) 89) 214) 145) 97) 86) (μm)

[0260] As shown in Table 1, the average amount of nerve bundles around the LGEA is 7 (ranging from 3 to 11 nerve bundles), and the average diameter of nerve bundles around the LGEA is 56 μm (ranging from 14-214 μm).

[0261] FIG. 4A shows an exemplary tile scan of the LGEA sample with surrounding 5 TH-IR nerve bundles. FIG. 5 shows that the nerves where mainly tyrosine hydroxylase (TH) positive indicating that the nerves were mainly sympathetic. No sensory, afferent, nerves were observed (absence of CGRP staining).

Short Gastric Arteries (SGAs) and the Adjacent Nerves

[0262] The average amount of SGAs branching from the SA was 3.33 (ranging from 1 to 6 SGAs). Table 2 shows a summary of the collected quantitative data on dissection parameters concerning the SGAs of each cadaver, followed by the average value. The average diameter of the SGAs was 0.15 cm (ranging from 0.08-0.4 cm) and the average diameter of the SA before the branching SGA was 0.28 cm (0.1-0.6). They originated 10.19 cm (6.0-16.0) from the origin of the SA, but this is dependent on the length of the SA. The SGAs originated either from the SA itself, or from a terminal branch of the SA. The most SGAs originated from the SA or a terminal branch relatively close to the hilum of the spleen and run in the gastrosplenic ligament to the stomach, but the SA also gave off early branching SGAs. All SGAs run in the gastrosplenic ligament, but parts of the SGAs were closely related with surrounding adipose tissue and connective tissue, although in most cases relatively easily dissected from these surrounding tissues. Some white fibrous strands seemed to go with the SGAs to the stomach, which could be nerve bundles.

[0263] FIG. 4B shows an exemplary tile scan of a SGA sample with surrounding give TH-IR nerve bundles. The average amount of nerve bundles around a SGA is 4.6 (ranging from 1 to 8 nerve bundles). The average diameter of a nerve bundle around a SGA is about 55 μm (ranging from 12-173 μm).

TABLE-US-00002 TABLE 2 Quantitative data on dissection parameters concerning the SGAs and adjacent nerve bundles of each cadaver, followed by the average value. III IV VII VIII IX X Average Amount 2 5 6 4 1 2 3.33 (1-6) Distance from 1: 8.5 1: 8.5 1: 8.5 1: 6.3 8.5 1: 6.0 10.19 (6.0-16.0) origin SA (cm) 2: 10.0 2: 12.5 2: 9.5 2: 7.9 2: 9.5 3: 12.5 3: 10.5 3: 9.7 4: 12.5 4: 10.5 4: 12.0 5: 16.0 5: 11.9 6: 12.5 Diameter (cm) 1: 0.23 1: 0.14.1 1: 0.15 1: 0.14 0.17 1: 0.12 0.15 (0.08-4.0) 2: 0.40 2: 0.14 2: 0.1 2: 0.14 2: 0.1 3: 0.15 3: 0.16 3: 0.1 4: 0.17 4: 0.11 5: 0.22 5: 0.08 6: 0.08 Diameter SA 1: 0.25 1: 0.5 1: 0.6 1: 0.4 0.25 1: 0.15 0.28 (0.1-0.6) before SGA 2: 0.4 2: 0.2 2: 0.5 2: 0.4 2: 0.15 (cm) 3: 0.2 3: 0.1 3: 0.3 4: 0.2 4: 0.1 4: 0.25 5: 0.2 5: 0.15 6: 0.3 Diameter of 1: 143 1: 79 1: 50 1: 44 1: 59 1: 35 55 nerve bundles 2: 44 2: 24 2: 73 2: 54 2: 30 (μm) 3: 63 3: 55 3: 31 4: 57 4: 63 5: 37 5: 71 6: 32

Study 2: Modulation of the Nerves Adjacent to the LGEA and the SGAs in Pigs

[0264] The nerves adjacent to the LGEA and SGAs in pigs were electrically stimulated, and the level of LPS-induced TNFα in an ex vivo whole blood assay, the splenic blood flow and systolic pressure were measured.

Dissection

[0265] The SGAs and the adjacent nerves were identified during gross postmortem observation and dissection in 10 Yucatan pigs. The SGA and the adjacent nerves were consistently located in the gastrosplenic ligament running from the proximal portion of the spleen to the greater curvature of the stomach. The SGAs and the adjacent nerves were commonly paired (n=8/10) and the nerves were located adjacent to the artery. The SGA originated from the cranial branch of the splenic artery (in all specimens).

[0266] The LGEA and the adjacent nerves were identified and isolated in 7 Yucatan pigs. The LGEA and the adjacent nerves were consistently located in a ligament that course between the distal spleen and the greater curvature of the stomach. The LGEA originated from the distal splenic artery along the hilum of the spleen (all specimens).

[0267] Gross anatomy of the SG and the LGE arteries, veins and nerves in the Yucatan pigs is shown in FIGS. 6A and 6B.

Histology

[0268] Initial histology from yucatan pigs (n=2, additional samples and TH pending) suggested that 2-3 nerves ranging from 100-150 microns course adjacent to the SGAs, which are approximately 200-400 microns in diameter. This is shown in FIG. 7.

Stimulation of the Nerves Adjacent to the SGAs

[0269] CorTec O-ring cuffs (bipolar; 800-2000 μm) of appropriate size were used to place around both the nerve adjacent to the SGA and the SGA (N=6). See FIGS. 8A, 8B and 8C for the cuff locations.

[0270] The stimulation parameters used were a current amplitude between 4-14 mA, a frequency of 10 Hz of 200 μS. The stimulation was performed for 1 minute. Stimulation parameters not optimized.

Stimulation of the Nerves Adjacent to the LGEA

[0271] CorTec O-ring cuffs (bipolar; 400-800 μm) of appropriate size were used to place around the GE nerve (no artery) (N=3). See FIGS. 8A, 8B and 8C for the cuff locations.

[0272] The stimulation parameters used were a current amplitude between 4-14 mA, a frequency of 10 Hz of 200 μS. The stimulation was performed for 1 minute. Stimulation parameters not optimized.

Results

[0273] The following measurements were performed: LPS-induced TNF production at baseline prior to stimulation and then 30, and 60 minute after stimulation, splenic arterial blood flow, systolic blood pressure, and Compound Action Potentials (CAPs; n=3)) at the level of the hilum of the spleen.

[0274] The responses following the stimulation of the nerves adjacent to the SGAs are shown in FIG. 9. After stimulation, a reduction of approximately 24% after 30 min and 15% after 60 min compared to base line was seen in LPS-induced TNF release in a whole blood assay (see FIG. 9A). Splenic arterial blood flow (SpABF) decreased by 0-15% and systolic arterial blood pressure (SAP) increased in by 0-15% during SG stimulation (see FIG. 9B). CAPs were observed in the level of splenic hilum (see FIG. 9C, n=3).

[0275] The responses following the stimulation of the nerves adjacent to the LGEA are shown in FIG. 10. After stimulation, a reduction of approximately 40% after 30 min and 32% after 60 min compared to base line was seen in LPS-induced TNF release in a whole blood assay (see FIG. 10A). Splenic arterial blood flow (SpABF) decreased consistently by 10% and systolic arterial blood pressure (SAP) changed little during SG stimulation (see FIG. 10B). Compound action potentials (CAPs) were observed in the level of splenic hilum (see FIG. 10C, n=3). Additionally cutting the nerve near the cuff abolished the decrease in splenic blood flow and CAP (n=2).

CONCLUSION

[0276] The effects of electrically stimulating the nerves adjacent to the SGAs or the LGEAs were similar to the effects of electrically stimulating the nerves adjacent to the SA. In particular, stimulating the nerves adjacent to the SGAs and LGEAs led to a decrease in LPS induced TNF, a decrease in splenic blood flow, and an increase in systolic pressure. In addition, by denervating the nerves adjacent to the LGEA it was shown that the effect was caused by a specific stimulation of the nerves and was not due to a specific current leakage.

Discussion

[0277] Histological analysis of the white fibers in the human gastrosplenic ligament revealed that these white strands were no nerves, but small nerve bundles were observed using different methods of staining. These nerves are run around the LGEA and the SGAs.

[0278] The LGEA and SGAs were visible by eye in a Yucatan pig. Usually two arteries surrounded by nerves were present in the gastrosplenic ligament. Histological analysis confirmed the presence of arteries and nerves in the gastro splenic ligament of the pig. Stimulation of the nerves adjacent to the LGEA and the nerves adjacent to the SGAs at the proximal part of the nerves near the spleen with a neural interface in acute experiments in pigs resulted in a systemic reduction in pro-inflammatory cytokines, including TNFα. These arteries therefore represent a stimulation target that is different from the splenic arterial nerve plexus and is useful for electric neuro-immunomodulation therapy in chronic inflammatory diseases.

[0279] It is more advantageous to stimulate the nerves adjacent to the LGEA and SGAs compared to the nerves adjacent to the SA. Some of the advantages are summarized as follows: [0280] I. The nerve plexuses surrounding the LGEA and SGA are surgically easier site to access compared to the nerve plexus surrounding the SA. [0281] II. Reduced safety issues; May represent less artery/vascular risk than encircling main splenic artery: [0282] a. Easily removable from the gastrosplenic ligament as needed; Loss of artery may have less severe impact. (surgical procedures exist in which the gastrosplenic ligament is removed [16]); [0283] b. SGA and LGEA not in proximity of pancreas; Avoids dissection adjacent to pancreas; and [0284] c. Surgical procedure shorter. [0285] III. Development of neural interface is easier: [0286] a. Pulsation of artery minimal; [0287] b. Potentially an existing neuromodulation device might be used; and [0288] c. Patch or clip neural interface might be used.

Key Findings

[0289] I. Nerves around arteries were detected in human and porcine specimens of the gastrosplenic ligament. [0290] II. The nerves in human and pig were similar in size and numbers. [0291] III. Stimulation delivered using a neural interface cuff around one of the nerves and artery, of either the LGEA or SGA, resulted in a reduction in pro-inflammatory cytokines. [0292] IV. Stimulating the nerve bundles surrounding LGEA without cuffing the artery resulted in a reduction in pro-inflammatory cytokines. [0293] V. Sites other than main nerve plexus along SA may be sites for intervention to modulate immune responses. [0294] VI. Effects of stimulating the nerves adjacent to the SGAs and the LGEA are similar to stimulation of nerves adjacent to the SA. [0295] VII. More than 98% of the nerves are sympathetic efferent nerves. [0296] VIII. SGAs and LGEA are present in 100% of the human cadavers investigated.

Ex-Vivo Electrophysiological Study of Human Splenic Nerves

[0297] The objective of this study was to estimate indicative stimulation parameters of human splenic nerves in order to de-risk and optimize the biological efficacy and reproducibility of stimulation parameters of the electrical signal for use in humans, in particular for stimulation of a human splenic nerve. The study was performed using ex-vivo using human splenic samples. It is expected that similar effects are produced when stimulating neural activity in the nerve adjacent to the LGEA and/or the nerve adjacent to a SGA with the indicative stimulation parameters.

Materials and Methods

[0298] FIG. 12A shows an example of fresh splenic sample from a 63-year-old female donor (it is noted that the range of age of donors making up the data described below is 23-63 years). The sample, approximately 15 cm in length, was placed in a petri dish, and the splenic neurovascular bundle (SNVB) was then carefully surgically isolated from excess adipose tissue and splenic vein under a microscope. The dots on the sample indicates the top part of the splenic artery used in order to maintain the orientation of the sample. The sample was tortuous and seemed to have loops. A few splenic nerves were carefully isolated distally for the purpose of recording eCAPs.

[0299] An isolated fascicle was used as a control and cuffed with a smaller diameter Cortec Cuff electrode (500 μm diameter) for recording and stimulation, as shown in FIG. 12B, (II). A bigger periarterial cuff of approximately 6 mm diameter was placed on the neurovascular bundle (see FIG. 12B, (I)). Subsequently, the tissue with the cuff was moved into the recording chamber which was constantly circulated with fresh, oxygenated and warm Kreb's solution (34-36 degrees Celsius). The stimulation cuffs were connected to a DS5 instrument (current stimulator) and recording cuff was connected to a bioamplifier (CWE, USA) as indicated in the schematics (see FIG. 12C, FIG. 12D). For stimulation, a bipolar configuration with monophasic pulses were used. The schematics of the evoked compound action potential is represented in FIG. 12E.

Results

[0300] The results from stimulation in isolated nerves with 500 μm cuff electrodes, which was used as a control, indicates a pulse height threshold of 1.5 mA with charge density of 100 μC/cm.sup.2, and 100% recruitment is indicated at a pulse height of approximately 5 mA with charge densities of 300 μC/cm.sup.2. The current strength-pulse width results from stimulation in eight human SNVB samples stimulated with 6 mm cuff demonstrates that the use of a 2 ms pulse width permits a 2.5- to 3-fold reduction of the stimulation threshold of pulse height for a 2.5-fold increase of pulse width i.e. from 0.4 to 2 ms (see FIG. 13A).

[0301] Interestingly, 400 ps pulse width, which seems to be an optimum stimulation parameter in the porcine in-vivo study, did not experimentally prove optimum in the case of human ex-vivo and in-silico tissue preparations. The mean pulse height from N=6 in acute porcine study was approximately 3.5 mA (see FIG. 3D), whereas in humans it was found to be at an average seven-eight times higher at approximately 25 mA. The reason why trade-off between pulse width and pulse height is important is to inform an optimum output level for implantable stimulator design and electrode charge injection capacities. With reference to FIG. 13A, 3 ms also seems a suitable pulse width, however, there is an increase in charge density with negligible decrease in pulse-duration. A significant increase in charge density is observed at and above 5 ms.

[0302] An increase in frequency from 1 Hz to 10 Hz indicates a reduction in eCAP amplitude and is indicative of nerve fatigue (see FIG. 18). Thus in this instance re-confirming porcine data assumptions on frequency. Nerve recruitment curves from individual donor samples at different pulse width of 0.4, 1 and 2 ms are illustrated in FIG. 13B, 13C, and 13D respectively. The compound action potentials are normalised with respect to the maximum eCAP amplitude response recorded on the oscilloscope. DS5 instrument has a limitation of 50 mA in amplitude, which was not enough to recruit 100% nerves at 0.4 ms (as seen in FIG. 13B). Thus, moving to 1 m and 2 ms pulse width effectively proves to be a more ideal trade-off. It is estimated that the charge requirements in human ex-vivo sample for 100% can be as high as 400 μC/cm.sup.2 (assuming a 0.12 cm.sup.2 total electrode surface area) as can be seen in FIG. 13D.

[0303] Based on assumptions of fibrotic encapsulation modelling, and the effects we have seen in pre-clinical animal models, a right shift effect is observed (as also seen in literature such as in [22]) by factors of x1.5, x2 and x3, for example, on the charge requirements in chronic. This can be seen in FIG. 14, where our estimation of charge requirements in chronic clinical scenario could be as high as approximately 100 μC (850 μC/cm.sup.2). A similar trend of charge requirements is observed from in-silico results for both 0.4 and 1 ms pulse width.

Discussion

[0304] It was found that for increasing pulse width, particularly pulse widths greater than 1 ms, a decrease in the pulse height threshold needed to trigger an action potential in a human splenic nerve is observed. This is a surprise based on the porcine model which showed the optimum pulse width to be far lower, at 0.4 ms. Lower pulse height thresholds are generally desirable because the biological efficacy and reproducibility of the stimulation parameters for use in humans is improved.

[0305] It has also been found that at a pulse width of 3ms or above (3-5 ms shown in data) there is no further decrease in pulse height, whereas there is an increase in charge density. Therefore, the strain of the electrodes outweighs the benefits seen in the IPG beyond a pulse width of 3 ms. Between 2 ms and 3 ms, there is a negligible decrease in pulse height threshold but the amount of charge density required increases. Therefore it may be desirable to use a pulse width of less than 3 ms in humans. Pulse width around 2 ms offer an optimal trade-off between ensuring a low charge density being required, and a low pulse height being required for the stimulation of a human splenic nerve.

[0306] It is estimated that the charge density per phase requirements in human ex-vivo sample for 100% nerve recruitment can be as high as 400 μC/cm.sup.2. However, it is expected that for chronic stimulation, the formation of scar tissue may reduce the nerve recruitment by a factor of between 1.5 and 3. FIG. 14 shows the 2 ms pulse width human ex-vivo data multiplied by a factor of 1.5×, 2×and 3×, and the change in recruitment based on the charge injected into the human splenic nerve. FIG. 14 suggests that up to 100 μC charge may need to be injected for recruitment of 100% nerves in humans in chronic scenario. This equates to a charge density per phase of approximately 850 μC/cm.sup.2 based on a 0.12 cm.sup.2 total electrode surface area. Accordingly, the charge density per phase required in order to achieve 100% recruitment of the human splenic nerve is expected to be up to approximately 850 μC/cm.sup.2 for a pulse width of 2 ms.

Human Chronic Model Stimulations

[0307] The purpose of this study was to determine the biological effect varying of interphase delay and pulse width. The study was conducted using a human chronic model simulation.

Materials and Methods

[0308] Hybrid electromagnetic (EM) and neuronal simulations were used to predict axonal recruitment in two representative image-based and 3D computational neurostimulation models of human and porcine splenic neurovascular bundle, for multiple variations of dielectric parameters of the nerve bundles, stimulus waveforms (0.4 ms, 1 ms and 2 ms biphasic pulses), and fibre diameters (0.5-1 mm). One representative cross section histological image of splenic neurovascular bundle for each species was segmented using iSEG within Sim4Life platform. Tissues were differentiated to identify vessel wall, blood, extra fascicular medium—internal and external to the electrode—and the endoneurium tissue within fascicles. The segmented tissue surfaces were extruded in 3D using extrusion functionalities. The bundle models were combined with cuff electrodes geometries, were surrounded by saline solution tissue to mimic experimental conditions, and fascicles were populated with multiple parallel axonal trajectories randomly distributed within each fascicle cross section.

[0309] EM simulations were performed using a FEM solver in the quasi-static approximation that handles anisotropic electric tensors conductivity and support thin layer settings. FEM calculations were executed on unstructured meshes created on the model geometries, built within Sim4Life using adaptive criteria and mesh quality adjustment. The meshes were edited to extract patches at the electrode surface to assign flux density boundary conditions, and at the interfaces between fascicles and interfascicular tissues to define thin layers mimicking the perineurium. In order to execute transient neuroelectric simulations for a given set of stimulation conditions (fibre diameters, pulse waveform, temperature), the range of parametrised axon electrophysiology in Sim4Life was extended by a c-fibre model (Sundt Model) completing the functionality required to stimulate nerves featuring distribution of unmyelinated c-fibres with arbitrary fibre diameters. Sim4Life functionalities such as the automatic sweeping and titration procedure were used to quantify stimulation thresholds (e.g. the pulse height threshold), investigate strength-duration (SD) curves and perform sensitivity analysis e.g. with respect to dielectric properties of tissues or pulse parameters. The creation of neuroelectric models, the creation and the setup of hybrid EM-neuronal simulations, and the post-processing of the results was assisted by 1) Python scripts facilitating the flexible, parametrised generation of functionalised nerve models, 2) the assignment of heterogeneous tissue properties and anisotropic electrical conductivities, 3) the creation of mesh and its editing, 4) the distribution of fibre models within fascicles, 5) the assignment of electrophysiological behaviour as well as for automised post-processing analysis, e.g. the quantification of stimulation thresholds, extraction of recruitment curves, identify location of spike initiation and latencies (time of first spikes) with respect to stimulus pulse-shape.

[0310] The image-based models of neurovascular bundles developed were adapted to include fibrotic tissue surrounding the electrodes and the insulating silicone to mimic the presence of a post-implantation fibrotic tissue. Hybrid EM-neuronal simulations were used to calculate the neuroelectric responses of electrophysiological models of individual unmyelinated C-fiber axons inserted within the fascicles of the bundles to quantify the stimulation thresholds (e.g. pulse height threshold) for initiation of the action potentials. From the calculated thresholds, recruitment curves were plotted for both acute and the chronic scenarios based on biphasic waveforms with different pulse durations (τdur) and interphase delays (τinter). The results are based on the following principal assumptions: (i) the dielectric properties, the structure, and the composition of the fibrotic tissue are uniform across all simulations; (ii) the fibrotic tissue is homogeneous and isotropic; (iii) there is no distinction between the fibrotic tissue formed around the electrodes vs. the silicone; (iv) the position of the fascicles is kept constant moving from acute to chronic scenario. The diameter of the neurovascular bundle is also kept constant and 0.5 mm of interfascicular tissue has been replaced by fibrotic tissue layer.

Results

[0311] FIG. 15 shows comparisons of the recruitment curves calculated for the human model for acute and chronic stimulations with different parameterisations of the biphasic pulse waveforms. For the chronic case, it was found that the presence of the fibrotic encapsulation increases the pulse height threshold required to trigger the creation of an action potential, with the increase for a fixed pulse duration being smaller for larger interphase delays. The increase in pulse height threshold is dependent on the specific parameters of the biphasic pulse waveform. For instance τdur=1 ms, the pulse height threshold increase is 37% when τinter=0 ms (simulations Acute1ms0ms vs. Chronic1ms0ms) but is 29% when tinter=0.2 ms (simulations Acute1ms0ms vs. Chronic1ms02ms). Similar results were found for τdur=0.4 ms: the pulse height threshold increase is 49% (simulation Acute04ms0ms vs. Chronic04ms0ms) vs. 27% with τinter=0.2 ms (Acute04ms0ms vs. Chronic04ms02ms). The results for 0.1 ms interphase have also been demonstrated in the graph for both the pulse durations (Chronic04ms0.1ms and Chronic1ms0.1ms). The impact of the pulse duration on pulse height threshold increase is large, ranging from 133% for the comparison of biphasic pulses of 0.4 ms vs. 1 ms in the acute case (Acute1ms0ms vs. Acute04ms0ms). Importantly, these results are for fibre diameter 1 μm. The variations in pulse height threshold due to acute vs. chronic stimulations were also investigated for dependence on fibre diameter for fibers of 0.5 μm vs. 1 μm. It was found for the acute scenario, thresholds increase by approximately 80-90% for a fiber of diameter 0.5 μm compared to one of 1 μm fiber. The studies have indicated that the pulse height threshold increases with decreasing fiber diameters and the pulse height threshold may be decreased by increasing the pulse duration. In particular, the interphase delay of 0.2 ms demonstrated a potential advantage of 5-10% over a 0 ms interphase delay. FIG. 17 shows the ex-vivo validation of these in-silico calculations, and beyond 0.3 ms no further improvement in threshold reduction is noted, thereby further illustrating 0.2 ms as an optimal interphase parameter.

[0312] The findings on pulse width in the ex-vivo preparations are further supported by this in-silico modelling data, as shown in FIG. 16. In particular, this figure shows that as the pulse width increases beyond 1 ms for a biphasic pulse train, the charge required to stimulate neural activity is reduced. Then, for pulse widths of 3 ms or higher, the charge required significantly increases.

Discussion

[0313] It was found that effects of interphase delay and pulse width are prominent. In particular, the interphase delay of 0.2 ms demonstrated a potential advantage of 5-10% over 0 ms interphase delay.

[0314] It is noted that these findings are supported by in-silico modelling data, as shown in FIG. 16. In particular, FIG. 16 shows that as the interphase delay of a biphasic pulse train is increased from 0 ms to 0.1 ms, the charge required to stimulate neural activity is reduced. It is further expected that as the interphase delay is increased beyond 0.1 ms, that the charge required to stimulate neural activity will reduce further and become closer to that required by a monophasic pulse train. Since it is not desirable to stimulate the nerve with a monophasic pulse train, a biphasic pulse train with an interphase delay greater than 0.1 ms is preferable.

[0315] Other ex-vivo studies in unmyelinated fibers have found that for interphase delays greater than 300 μs, no further reduction in pulse amplitude threshold is found. This is depicted in FIG. 17. Accordingly, the optimum interphase delay for stimulation of a human splenic nerve is likely to be between 100 μs and 300 μs, more particularly between 200 μs and 250 μs.

[0316] It is expected that similar effects are produced when stimulating neural activity in the nerve adjacent to the LGEA and/or the nerve adjacent to a SGA.

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