Splenic Nerve Neuromodulation Reduces Inflammation and Promotes Resolution in Chronically Implanted Pigs

Neuromodulation of the immune system has been proposed as a novel therapeutic strategy for the treatment of inflammatory conditions. We recently demonstrated that stimulation of near-organ autonomic nerves to the spleen can be harnessed to modulate the inflammatory response in an anesthetized pig model. The development of neuromodulation therapy for the clinic requires chronic efficacy and safety testing in a large animal model. This manuscript describes the effects of longitudinal conscious splenic nerve neuromodulation in chronically-implanted pigs. Firstly, clinically-relevant stimulation parameters were refined to efficiently activate the splenic nerve while reducing changes in cardiovascular parameters. Subsequently, pigs were implanted with a circumferential cuff electrode around the splenic neurovascular bundle connected to an implantable pulse generator, using a minimally-invasive laparoscopic procedure. Tolerability of stimulation was demonstrated in freely-behaving pigs using the refined stimulation parameters. Longitudinal stimulation significantly reduced circulating tumor necrosis factor alpha levels induced by systemic endotoxemia. This effect was accompanied by reduced peripheral monocytopenia as well as a lower systemic accumulation of CD16+CD14high pro-inflammatory monocytes. Further, lipid mediator profiling analysis demonstrated an increased concentration of specialized pro-resolving mediators in peripheral plasma of stimulated animals, with a concomitant reduction of pro-inflammatory eicosanoids including prostaglandins. Terminal electrophysiological and physiological measurements and histopathological assessment demonstrated integrity of the splenic nerves up to 70 days post implantation. These chronic translational experiments demonstrate that daily splenic nerve neuromodulation, via implanted electronics and clinically-relevant stimulation parameters, is well tolerated and is able to prime the immune system toward a less inflammatory, pro-resolving phenotype.

Twenty seconds before the start of all experimental stimulations, 10 ml blood was taken over 10 s. SpNS was then delivered for 120 s. Following the initial 10 s of stimulation, blood was sampled (10 ml) every 20 s from stimulation onset to offset. A total of 7 blood samples (1 pre; 6 during) were taken for each stimulation intensity.
All blood samples were transferred to EDTA-coated tubes and then immediately centrifuged at 4 °C, 2000 xG for 5 mins. The total plasma was then collected and placed in labelled tubes and immediately frozen on dry ice. Tubes were then stored at -80 °C prior to analysis.
Frozen plasma aliquots were thawed and immediately analyzed by ELISA for quantification of NA using the Noradrenaline Sensitive ELISA (DLD Diagnostika, cat. no. ea633/96), according to manufacturer's instructions. Plates were analyzed using the Infinite® 200 PRO spectrophotometer and iControl software (Tecan Group Ltd.). NA was quantified as AUC during stimulation and for mABP and SpA BF the maximum change which occurred during stimulation was plotted. Data were assessed by one-way ANOVA using Tukey post-hoc correction for multiple comparison. Statistical significance was defined as P ≤ 0.05 and analyses were performed with commercially available statistical software (JMP Pro 13.0.0 or GraphPad Prism 8.4.2).

Chronic Conscious Neuromodulation Study
This study developed a minimally invasive, laparoscopic technique in a translational porcine model to implant a cuff electrode around the splenic NVB and an IPG to enable delivery of chronic neuromodulation. The tolerability of SpNS in conscious, freely behaving pigs was then evaluated, prior to quantification of multiple immunological parameters in both naïve and endotoxin-challenged inflammatory conditions. These parameters included quantification of cytokines, flow cytometry and SPM analyses before, during and after LPS challenge. Under terminal anesthesia, contrast angiography, electrophysiology and histopathology demonstrated the integrity of the splenic NVB.

Animals
Due to the exploratory nature of the study and logistical difficulties associated with large-animal studies, animals progressed in 4 cohorts in weekly blocks. Each cohort contained animals receiving SpNS and non-stimulated sham animals to allow for any differences arising from different batches of LPS. Cohorts were used as a blocking factor in data analyses. They were individually housed in close apposition to allow visual and physical contact through slatted fencing, on straw bedding with environmental enrichment. Water was provided ad libitum and they were fed a commercial pelleted sow and weaner diet based on minimum basal and metabolic energy requirements.

Neuromodulation Device.
Stimulation Lead. The stimulation lead consists of a lead body with a distal end cuff electrode applied to the splenic NVB, and a proximal connector connected to the IPG manufactured from implant-grade silicone and metals. The design is able to support laparoscopic implantation for cuff placement around the splenic NVB. The cuff electrode is designed to interface with nerves located around the periphery of the splenic artery and contains two electrically active electrode arms and one inert middle arm for retention. Implantable Pulse Generator. A commercially available implantable pulse generator (IPG; 5cc mStim IPG, Integer CCC, Uruguay; customized by Galvani Bioelectronics) was connected to the stimulation lead to electrically stimulate the SpN . Two versions of the IPG were used on the current study. In cohorts 1 and 2, the IPG was capable of stimulation up to 15 µC (15.3 mA, 980 µs pulse width). In cohorts 3 and 4, the IPG was upgraded to provide output up to 40 µC (20.0 mA, 1980 µs). All other stimulation parameters remained identical and are detailed above. Differences in IPG output are noted in the relevant sections.

Anesthesia
Animals were started on a course (9 days) of antibiotics (amoxicillin 15 mg/kg i.m.) and anti-ulcer medication (omeprazole 40 mg p.o.) 24 h before surgery and then continued as prescribed afterwards. Food, but not water, was withheld for 18 h prior to surgery and animals received a veterinary examination to ensure health status. After induction of anesthesia as below, analgesia and antiinflammatory meloxicam (0.4 mg/kg i.m.) was administered and continued in the recovery period as needed.

Laparoscopic surgery
A minimally-invasive laparoscopic surgical procedure was used. Animals were placed in right lateral recumbency, and the left thorax and lateral abdomen were aseptically prepared and draped in a routine fashion. A total of 7 trocars were used for each surgical procedure. The first trocar (15 mm diameter) was initially placed in the paralumbar region between the last rib and wing of the ilium using the Hasson technique. The abdomen was then insufflated with gas at a constant pressure ranging between 10-16 mmHg. At the same time the lead was prepared, inspected and tested in saline for electrical continuity (impedance). The laparoscopic camera was then inserted and used to select the ideal location for successive trocars. A second trocar (5 mm) was then placed along the cranio-ventral axis of the abdomen to provide an access port for stomach retraction tools. A third trocar (12 mm) was placed approximately 20 cm caudal from the second trocar for spleen retraction. Three additional trocars (12 mm) were placed approximately 15-20 cm dorsal from the retraction trocars to form a triangle just ventral to the last and second to last ribs. The two trocars at the base of the triangle (n.4 and 5) were used for dissection and cuff implantation, while the other (n.6) was used for the laparoscope.
The head of the spleen was retracted caudally by fixing a self-retaining laparoscopic Overholt clamp at the base of the renosplenic ligament. The stomach was then retracted ventrally by using an articulating cobra liver retractor to provide access to the splenic NVB. Initial dissection of the peritoneum overlying the distal (closer to the spleen) splenic NVB was performed using a harmonic laparoscopic scalpel to prevent bleeding. The splenic NVB was then isolated by blunt dissection using a Maryland tool, 60 and 90-degree Overholt dissecting instruments and scissors. A region of approximately 2 cm of NVB was freed from the connective tissue and separated from the splenic vein. The stimulation lead was then prepared for deployment into the abdomen. A seventh trocar (5 mm) (n.7) was placed between the two access ports used for dissection. The lead was then introduced into the abdomen using atraumatic graspers to manipulate the lead. The lead was exteriorized by pulling the lead cap through trocar n.7. The circumferential cuff electrode was then implanted around the splenic NVB and impedance was checked using a Minirator (MR-PRO, NTI Audio, Switzerland) to confirm electrical continuity.
Stimulation was then performed with an external pulse generator (EPG; DS5, Digitimer, UK) to confirm electrical integrity and physiological functionality by a measured increase in mABP. Intraoperative splenic nerve neuromodulation (10 Hz continuous; 60 s) consistently induces changes in physiological biomarkers [4] which enabled robust confirmation of nerve-target activation via the test system. Specifically, systolic, diastolic and mean arterial blood pressure (mABP) are increased during splenic nerve stimulation under anesthesia; these are caused by smooth muscle contraction within the artery and spleen. These changes are directly correlated with the amplitude and frequency of stimulation, and such changes are resolved upon the cessation of stimulation.
A 3 min stable period of no stimulation was performed to obtain baseline values, including mABP. Stimulation (see Table S5 for stimulation parameters) was applied via the IPG; the ability to evoke an increase in mABP was used to demonstrate function of the implanted lead and effects on target physiology. A period of no stimulation (minimum 120 s) was performed to allow recovery of cardiovascular parameters (mABP) to ± 10% of pre-stimulation values between each stimulation.
A subcutaneous pocket was created to accommodate the implantable pulse generator (IPG; Integer, CCC, Uruguay) dorsally, approximately above the third to last rib and in line with the position of the n.7 trocar. An incision of 5-8 cm was performed and pocket created by blunt dissection between subcutaneous fat and muscle layer. The n.7 trocar was carefully removed, and the lead tunneled subcutaneously to the IPG pocket on the lateral thorax.
Subsequently, the lead connector was attached to the IPG and the IPG implanted into the subcutaneous pocket on the lateral thorax. Instruments and trocars were removed. A series of stimulations were then applied via the IPG to confirm functionality by comparison to changes evoked with the EPG, and IPG communication and charging were confirmed. The trocar locations were then sutured closed. Stimulation was then delivered via the IPG up to either 15 or 40 µC using a 10 Hz continuous paradigm as detailed in main methods.

Vascular access port (VAP) implantation
Following implantation and stimulation, animals were placed in dorsal recumbency and the neck and scapula region prepared and draped for aseptic surgery. An intravenous catheter was placed, as below, in the left external jugular vein using a minimally invasive ultrasound-guided approach and terminated with a subcutaneous vascular access port (Le Grand CompanionPort (CP305K) -Norfolk Vet Products, Skokie, IL, USA). The VAP consisted of a titanium port with a silicone septum and an attachable rounded tip silicone catheter (7 French). Both the port and catheter were flushed with 0.9% saline prior to insertion.
A 1 cm incision was made in the skin overlying the left external jugular vein in the mid-cervical region. An 8 Fr catheter introducer was placed into the jugular vein under ultrasound guidance and the catheter for the VAP passed through this introducer and advanced 9-10 cm distally. Tip localization was confirmed using fluoroscopy to ensure location was proximal to the heart. For placement of the port, a 5-to 6-cm curvilinear incision was made dorso-cranial to the scapula and 5 cm lateral to midline dorsal neck. Subcutaneous tissues overlying trapezius muscle were undermined to create a pocket for the port. The catheter was tunneled dorsally between the skin and subcutaneous tissues and attached to the port. The port was secured to the underlying musculature by using 3-0 polydioxanone suture at 2 anchor points on the port. Catheter patency was confirmed intraoperatively through withdrawal of a blood sample. The port and catheter were flushed with 5 to 6 mL 0.9% saline and locked with 5 mL heparinized saline (500 IU/mL, Hospira). VAPs were maintained as per below until blood sampling.

VAP maintenance and use
VAPs were maintained in all animals during the study period; they were accessed either for experimental procedures or minimally every 2 weeks for maintenance. Briefly, the animal was restrained in a crate to which it was habituated, and aseptic technique used throughout. Topical local anesthetic cream (lignocaine 2.5% and prilocaine 2.5%; EMLA 5% cream; Aspen medical) was rubbed in the skin over the access port, palpable under the skin, and left for a minimum of 1 h. After final skin preparation, the port was located and stabilized by holding the edge and a right angled Huber needle (22G 1"; Norfolk Vet Products, Skokie, IL, USA), attached to an extension set and syringe containing 0.9% saline flush solution, was inserted through the skin and into the dome of the port. Flush solution (5 mL) was introduced into the catheter, and then a syringe used to withdraw at least 3 times the volume of the VAP system, including the flush, which was discarded. Following this, either blood samples were taken or LPS injected. The catheter was then flushed again with 0.9% saline (3 times volume VAP system), followed by lock solution. If further samples were to be taken within 24 h, the needle remained indwelling in the port, attached to a sealed extension set and secured around the animal. When the system was accessed within 24 h and the needle left in place, it was flushed and locked with 5 mL heparinized saline (100 U/mL); when samples were not taken within that time prior to removing the needle, a lock solution of 5 mL heparinized saline (500 U/mL) was placed in the VAP.

Tolerability to SpNS as determined by behavioral responses
After a 14-day recovery period from surgery, in those pigs assigned to the neuromodulation group, tolerability to SpNS (as per the conscious stimulation paradigm; table S5), was determined. Impedance was measured via the IPG before, during and after each stimulation session. Animals in the sham group did not receive any neuromodulation. Behavioral responses indicative of perception to SpNS were scored by two independent observers. Responses identified as most severe: abdominal wall contractions, distressed vocalization and excessive agitation/psychomotor activity, warranted immediate cessation of stimulation if seen individually and only on a single occasion. The animal was allowed to recover for five minutes, and following veterinary assessment, stimulated again at the next lowest intensity before attempt at step-up made again. Other less severe behaviors (startle response, scratching/rubbing, nose bumping against a solid object, kneeling, squatting, stomping and stretching) were scored as absent or present; and if present whether as a single occasion, intermittent or continuous. If a combination of two or more these behaviors was seen at greater than a single frequency, stimulation was ceased and after a five-minute recovery, retested at the same intensity. If the response was observed during the second stimulation at the same level, no further increase in stimulation would occur and effects of stimulation reassessed at the next lowest intensity to confirm absence of limiting behavioral changes. If no response was observed during the second stimulation at the same level, the stimulation would be increased in 1 mA levels until either an observed response or IPG maximum was reached.

Blood Sampling at Baseline in Naïve Animals
Animals were then left for a further 14 days with no stimulation. Blood samples were then taken from all animals for baseline blood testing at Days -2, -1 and 0 and then following initiation of SpNS, on Days 2 and 7 from the VAP (as per above protocol). Bloods for ex vivo LPS cytokine assay and flow cytometry were collected in sodium heparin-coated tubes. Bloods for clinical biochemistry were collected in lithium heparin-and non-coated tubes. Blood for hematology was collected in EDTAcoated tubes.

LPS preparation methods
A 1 mg vial of LPS (Purified lipopolysaccharides from the cell membrane of Escherichia coli O111:B4; Sigma Aldrich) was reconstituted with 1 mL sterile saline to give 1 mg/mL solution. The vial was vortexed for 20 s and then sonicated for 5 min. Stock solution aliquots of 500 µg/mL were then made by adding 100 µL of 1 mg/mL LPS to 100 µL saline in individual tubes. These were again vortexed for 20 s and sonicated for 5 min and stored for up to 12 h at 4°C. Within 20 min of use, working solutions of LPS were made (50, 5 or 0 µg/mL): tubes were again vortexed and sonicated as above, before serial dilutions with sterile saline. Vortex and sonication were done between each dilution step. Working solutions were sonicated again immediately prior to incubation with blood samples.

Ex vivo LPS Cytokine Assay
Within 15 min of blood collection, blood tubes were inverted to resuspend cells and 20 µL of working LPS dilutions (50, 5 or 0 µg/mL) were added to culture tubes, followed by addition of 980 µL of blood to each tube to achieve final concentrations of 1000, 100 or 0 ng/mL LPS. Two replicates were performed per final concentration. Samples were mixed by 3 inversions and transferred to a 37°C incubator, flat on a rocker for 4 h. Plasma was then separated by centrifugation for 10 min at 2000 xG to pellet cells and then removed by pipette and stored in cryovials at -80°C.
Samples were thawed and TNF-α analyzed by commercially available ELISA kits (Porcine TNF-α; DY690B; DuoSet Solid Phase Sandwich ELISA, R&D Systems), run as per the manufacturer's instruction. All samples were run as technical replicates (n=3) for each time-point and LPS concentration.

Flow cytometry methods
Data recorded by the flow cytometer was analyzed using FlowJo™ software (version 10.6.2). Each blood sample was divided in 7 aliquots to generate 7 different panels: 1) unstained; 2) isotype control for panel 3; 3) antibodies against CD4 and CD8; 4) isotype control for panel 5; 5) antibodies against CD14/CD16; 6) isotype control for panel 7; 7) antibodies against CD172a/CD163. Gates were applied according to the gating strategy ( fig. S4). Identical gates were applied for all samples (all experimental animals, days and cohorts). Panel 3 was used to distinguish T cell subsets based on their CD4 and CD8 expression; Panels 5 and 7 were used to analyze the monocyte population. Simple gating on all CD14 + , CD16 + or CD172 + cells resulted in inclusion of granulocyte and lymphocyte populations ( fig. S4). Because our primary interest in Panels 5 and 7 was in the monocyte population, gating was done in a view where the marker was plotted against Side Scatter. The position of monocyte population on the Side Scatter axis was, as expected, between lymphocytes and granulocytes. Although there is a slight overlap with these populations, using side scatter in combination with the marker resulted in a reliable separation of these three subsets. This strategy was therefore used as the final gating strategy. Results are expressed as a percentage of its parent population. In the case of CD14 expression on CD16 + monocytes, median fluorescence intensity was measured along with percentage of CD14 low and CD14 high cells.

LPS preparation for in vivo experiments
LPS stock solution of 1 mg/mL was prepared as above. A 1:10 dilution of this was made by diluting 100 µL of stock solution into 900 µL sterile saline, which was then vortexed for 20 s. The final concentration of LPS to be given based on the animal's body weight was calculated (0.025 µg/kg) and a 1 mL solution of 2x final LPS concentration solution made into a glass vial, vortexed for 20 s and kept on ice until use (within 30 min). Just prior to use, the vial was sonicated for 5 min, 500 µL were then diluted in 9.5 mL sterile saline and the solution injected IV over 2 min into each animal.

In vivo LPS and cytokines assays
Immediately prior to LPS, and following LPS injection, blood samples were drawn in the following tubes: flow cytometry in sodium heparin-coated; cytokine analysis, SPM analysis and hematology in EDTA-coated; clinical biochemistry in lithium heparin and non-coated.

Monitoring of LPS-Induced Clinical Reactions
Clinical reactions to LPS were carefully monitored to ensure (as predicted from the preliminary study) that only mild to moderate clinical signs occurred, such as shivering and lethargy; animals were predicted to remain responsive and appetent throughout.

Targeted lipid mediator profiling
Prior to sample extraction, deuterated internal standards, representing each region in the chromatographic analysis were added to facilitate quantification. Samples were kept at -20° C for a minimum of 45 mins to allow protein precipitation. Supernatants were subjected to solid phase extraction, methyl formate and methanol fraction collected, dried and suspended in phase (methanol/water, 1:1, vol/vol) for injection on a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector, paired with a QTrap 6500+ (Sciex). An Agilent Poroshell 120 EC-C18 column (100 mm x 4.6 mm x 2.7 µm) was kept at 50° C and mediators eluted using a mobile phase consisting of methanol/water/acetic acid. QTrap 6500+ was operated using a multiple reaction monitoring method (see Dalli, J., et al., Lipid Mediator Metabolomics Via LC-MS/MS Profiling and Analysis. Methods Mol Biol, 2018. 1730: p. 59-72). Each lipid mediator was identified using established criteria including matching retention time to synthetic or authentic standards, an AUC >2000 counts and matching of at least 6 diagnostic ions in the MS/MS. Calibration curves were obtained for each mediator using lipid mediator mixtures at 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg that gave linear calibration curves with an r2 values of 0.98-0.99.

Terminally-Anesthetized Procedures
Anesthesia was performed using the same induction protocol as described previously for the chronic neuromodulation study.

Contrast Angiography
Contrast angiography was performed in an aseptic CT procedure room. Animals were positioned in dorsal recumbency and maintained under anesthesia (as described above). The skin over the right femoral artery was prepared in an aseptic manner and sterility maintained throughout the imaging procedure. An introducer was placed in the femoral artery under ultrasound guidance and secured for the duration of the imaging procedure. A catheter (6 or 7 Fr Avanti + Catheter; Cordis, Florida, USA) was placed through the introducer and advanced to the celiac artery for contrast angiography. Contrast agent (Iohexol, Omnipaque™ 300 mgI/mL; GE Healthcare, Amersham, UK) was injected through the catheter into the celiac artery to obtain scans from the dorsal, ventral, lateral, and oblique views/angles. Imaging was performed at a minimum along the long axis of the SpA. Upon completion of angiography, the intravascular catheter and introducer were removed and pressure applied to ensure hemostasis.

Histopathology
Five minutes prior to euthanasia 5000 IU heparin IV was administered to prevent post-mortem clotting.
Following gross pathology examination, tissues were harvested as follows: • The entire segment of splenic artery including the cuff and surrounding muscle/fat tissue. The entire block of tissue was attached to a cork board to maintain orientation during fixation and the proximal and distal ends labelled. The cuff was left in situ. • Tissues from lead and IPG regions: One section of pancreas closest to the neural interface (NI) site, one section proximal (by approx. 5 cm) and one section distal (by approx. 5cm). • One section of spleen from an area close to the NI implantation site and one section lateral to the first section (by approx. 5 cm). • One section of liver from the left lateral lobe, in the immediate vicinity of the implant site.
All tissues were immersed in 10% neutral buffered formalin solution (minimum 10x volume of tissue sample) for 48 hours prior to immersion in 70% alcohol.

Histology
For chronically implanted and surgical sham animals, four tissue blocks (fixed tissue) were prepared. Following fixation, the proximal and distal margins of the neural interface were marked with tissue marking ink and/or sutures and the cuff was carefully removed. The implant region was trimmed to generate four tissue blocks (P, MA, MB and D, as indicated in fig. S14). Three to four sequential 4-5 µm thick sections were taken from each block at the approximate location of the blue dashed arrows. One section at each level was stained with hematoxylin and eosin (H&E) and one with Gomori's elastin trichrome.
Sections of the splenic artery were assessed for microscopic changes including, but not limited to, degeneration of nerves, inflammatory infiltrates, necrosis, fibrosis, hemorrhage, neo-intimal hyperplasia/stenosis, and medial degeneration. Three H&E-stained tissue sections of the spleen and pancreas in the vicinity of the neural interface and 5 cm proximal and distal to the implant site and one section of the liver were also examined microscopically.
All changes were graded for severity using a semi-quantitative four or five-point grading scheme, based on the relative extent of each lesion within a section. Grading criteria for changes to nerve fascicles and surrounding connective tissue are listed in table S7. All tissue sections were examined microscopically by a board-certified veterinary pathologist.            Tables   Table S1 -Naïve Phase Hematology and Biochemistry -excel file 75%-100% of the area of interest is affected by the change.

5
Severe Denotes a very pronounced microscopic change with greater than 75% of the tissue affected.