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. Author manuscript; available in PMC: 2024 Apr 1.
Published in final edited form as: Anesthesiology. 2023 Apr 1;138(4):372–387. doi: 10.1097/ALN.0000000000004516

GABAergic Signaling During Spinal Cord Stimulation Reduces Cardiac Arrhythmias in a Porcine Model

Kimberly Howard-Quijano 1,2, Yuki Kuwabara 1, Tomoki Yamaguchi 1, Kenny Roman 1, Siamak Salavatian 1, Bradley Taylor 1, Aman Mahajan 1,2
PMCID: PMC9998372  NIHMSID: NIHMS1867468  PMID: 36724342

Abstract

Background:

Neuraxial modulation, including spinal cord stimulation, reduces cardiac sympathoexcitation and ventricular arrhythmogenesis. There is an incomplete understanding of the molecular mechanisms through which spinal cord stimulation modulates cardiospinal neural pathways. We hypothesize that spinal cord stimulation reduces myocardial ischemia/reperfusion induced sympathetic excitation and ventricular arrhythmias through γ-aminobutyric acid (GABA) mediated pathways in the thoracic spinal cord.

Methods:

Yorkshire pigs were randomized to Control (n=11), Ischemia/reperfusion (n=16), Ischemia/reperfusion+Spinal cord stimulation (n=17), Ischemia/reperfusion+Spinal cord stimulation +GABAA or GABAB receptor antagonist (GABAA, n=8, GABAB, n=8, and Ischemia/reperfusion+GABA transaminase inhibitor (GABAculine, n=8). A 4-pole spinal cord stimulation lead was placed epidurally (T1-T4) GABA modulating pharmacologic agents were administered intrathecally. Spinal cord stimulation at 50 Hertz was applied 30-min prior to ischemia. A 56-electrode epicardial mesh was used for high-resolution electrophysiological recordings, including activation recovery intervals and ventricular arrhythmia scores.. Immunohistochemistry and Western blots were performed to measure GABA receptor expression in the thoracic spinal cord.

Results:

Cardiac ischemia led to myocardial sympathoexcitation with reduction in activation recovery interval (mean±SD: −42 ± 11%), which was attenuated by spinal cord stimulation (−21 ± 17%, P=0.001). GABAA and GABAB receptor antagonists abolished spinal cord stimulation attenuation of sympathoexcitation (GABAA −9.7±9.7%, P=0.043 vs Ischemia/reperfusion + Spinal cord stimulation. GABAB −13±14%, P=0.012 vs Ischemia/reperfusion + Spinal cord stimulation), while GABAculine alone caused a therapeutic effect similar to spinal cord stimulation (−4.1±3.7%, P=0.038 vs Ischemia/Reperfusion). The ventricular arrhythmia score supported the above findings. Spinal cord stimulation during ischemia/reperfusion increased GABAA receptor expression with no change in GABAB receptor expression.

Conclusions:

Thoracic spinal cord stimulation reduces ischemia/reperfusion-induced sympathoexcitation and ventricular arrhythmias through activation of GABA signaling pathways. These data support the hypothesis that spinal cord stimulation-induced release of GABA activates inhibitory interneurons to decrease primary afferent signaling from superficial dorsal horn to sympathetic output neurons in the intermediolateral nucleus.

Introduction

Autonomic nervous system imbalances play a major role in the pathophysiology of myocardial ischemia induced ventricular arrhythmias and sudden cardiac death 1–3,4. Following myocardial ischemia, cardiac afferent sympathetic nerves are activated and synapse in the dorsal horn of the thoracic spinal cord initiating a complex cardiospinal neural circuit and reflex efferent sympathoexcitation5. The increased spinal sympathetic nervous system output leads to acute physiologic changes in cardiac electrophysiology as well as, long-term neuronal remodeling of the intrathoracic, extracardiac ganglia and the intrinsic cardiac nervous system6–8.

Neuromodulation therapy with spinal cord stimulation of the high-thoracic spinal cord has been shown to have cardiac antiarrhythmic effects9–12. Spinal cord stimulation therapy is postulated to reduce sympathetic afferent neural signaling induced by myocardial ischemia in the dorsal horn and stabilize efferent outflows to cardiac tissues, thus reducing ventricular arrhythmias during ischemia13. We and others have previously reported that spinal cord stimulation therapy can improve ventricular arrhythmias and cardiac function through a reduction in local sympathetic nerve activation in ischemic myocardium and reactive gliosis in the spinal cord12,14. However, the mechanisms through which spinal cord stimulation modulates neural signaling and cardiac sympathoexcitation have not been elucidated.

One possible mechanism through which spinal cord stimulation may affect neural signaling in the spinal cord is through γ-aminobutyric acid (GABA) mediated pathways15–19. GABA functions through activation of GABAA and GABAB receptors. Both receptor subtypes are found in the spinal cord, however, there are important differences in structure, anatomic location, and function between the receptor subtypes20,21 In a rodent model of neuropathic pain, spinal cord stimulation was found to work through GABA release in the dorsal horn22 and clinically, the use of intrathecal GABAB receptor agonists, enhanced the response to spinal cord stimulation for non-responder subjects23. Further studies investigating the role of GABAergic pathways in pain literature found differential effects of peripheral nerve injury and spinal cord stimulation models on GABAA vs GABAB receptors21,24,25,26.

Thus, the goal of this study is to determine the role of GABA in the therapeutic effects of spinal cord stimulation to reduce sympathetic excitation and ventricular arrhythmias during myocardial ischemia/reperfusion. We hypothesize that spinal cord stimulation reduces ventricular arrhythmias through GABA mediated pathways in the thoracic spinal cord. Our primary aim was to determine the effect of GABA on cardiac sympathoexcitation and arrhythmias during Ischemia/reperfusion, with and without spinal cord stimulation, through a series of functional experiments in which GABA receptors in the intrathecal space were pharmacologically blocked and augmented while the effects of spinal cord stimulation on cardiac sympathoexcitation and ventricular arrhythmogenesis were quantified in a translational large animal porcine model. Secondarily, given the possible differential effect of spinal cord stimulation on GABA receptor mediated pathways, we investigated changes in GABAA and GABAB receptor expression in the thoracic spinal cord with spinal cord stimulation. This data could provide mechanistic insight in the protective role of spinal cord stimulation on ventricular arrhythmias, thus helping clinical translation of spinal cord stimulation therapy.

Materials and Methods

Study protocol was approved by Institutional Animal Research Committee. All experiments were performed in compliance with the National Institution of Health Guide for the Care and Use of Laboratory Animals. All experiments were performed during daylight hours 6:30 and 19:00. Our report and study followed the appropriate Arrive guidelines (Supplemental document).

Experimental Protocols

Overview of experimental approach and timeline of experimental protocols are shown in Figure 1. Yorkshire pigs (n=68, 34 Males and 34 Females, mean age 4 months) were used in this study. In phase one of our experimental protocols, animals were first randomly assigned into three groups control, Ischemia/Reperfusion, and Ischemia/Reperfusion+ spinal cord stimulation to establish model of acute ischemia with and without spinal cord stimulation. Then in phase two animals were randomized into all six experimental groups to yield final sample sizes of; Control (n=11, 46 ± 9 kg), Ischemia/reperfusion (n=16, 41 ± 5 kg), Ischemia/reperfusion +Spinal cord stimulation (n=17, 44 ± 6 kg), Ischemia/reperfusion + Spinal cord stimulation +GABAA receptor antagonist (GABAA, n=8, 40 ± 5 kg), Ischemia/reperfusion + Spinal cord stimulation + GABAB receptor antagonist (GABAB, n=8, 40 ± 4 kg) and Ischemia/reperfusion + GABA transaminase inhibitor GABAculine (GABAculine, n=8, 49 ± 3 kg). There was one animal death in Ischemia/reperfusion +Spinal cord stimulation + GABAB receptor antagonist group prior to protocol completion, data is reported on n=7 animals in this group. Animals in the control group underwent the same surgical preparation and time course as experimental groups, however no cardiac ischemia or spinal cord stimulation was performed. In the Ischemia/reperfusion and Ischemia/reperfusion + Spinal cord stimulation groups, the animals had a spinal cord stimulation catheter placed, and cardiac ischemia performed but only the Ischemia/reperfusion + Spinal cord stimulation group had the catheter turned on during the protocol. In the GABAA and GABAB antagonist groups, the animals had intrathecal and spinal cord stimulation catheters placed, with spinal cord stimulation therapy on. GABA antagonists were applied, and cardiac ischemia performed as mentioned above. In the GABAculine group, the animals had intrathecal and spinal cord stimulation catheters placed, spinal cord stimulation was not turned on, GABA transaminase inhibitor was applied, and cardiac ischemia was performed as mentioned above.

Figure 1: Central Illustration and Timeline of experimental protocol.

Figure 1:

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A. Proposed pathway for ischemia induced sympathoexcitation and 1–3 are proposed sites for spinal cord stimulation GABA mediated inhibition of cardiac sympathoexcitation. Sub P: Substance P. NPY: Neuropeptide Y. GAD: Glutamate decarboxylase. ABAT: GABA-Transaminase. SSA: Succinic semialdehyde. TCA: Tricyclic antidepressants. SPN: Sympathetic preganglionic neurons. IML: Intermediolateral nucleus.

B. Experimental protocol and treatment groups for SCS mechanistic investigation Yorkshire pigs were randomized to six groups; Control, Ischemia/reperfusion, Ischemia/reperfusion +Spinal cord stimulation (IR +SCS), Ischemia/reperfusion+ Spinal cord stimulation +GABAA antagonist Bicuculline (GABAA), Ischemia/reperfusion + Spinal cord stimulation + GABAB antagonist CGP55845 (GABAB), and Ischemia/reperfusion + GABA transaminase inhibitor GABAculine (GABAculine) groups. Spinal cord stimulation was initiated 30min before ischemia. The intrathecal GABA antagonists were each applied 5 minutes before the start of spinal cord stimulation and reapplied every 60 minutes after that. Intrathecal Gabaculine was applied after baseline control measures and reapplied every 60 minutes. BL; Baseline, CON; control

Animal preparation

Animal experimental preparation was conducted as previously described12. Animals were sedated with telazol (4 to 8 mg/kg, intramuscular), intubated, and mechanically ventilated with oxygen. General anesthesia was induced and maintained with inhaled isoflurane (1 to 3%) during surgical preparation. Heart rate (HR) and surface electrocardiogram (ECG) were monitored throughout the experiment using a Prucka CardioLab recording system (GE Healthcare, USA). The carotid and femoral arteries were catheterized for blood pressure monitoring. In addition, jugular and femoral veins were cannulated for intravenous saline infusion (10 ml/kg) and drug administration. To maintain acid-base equilibrium, arterial blood gas was tested hourly with adjustment of ventilation as necessary. Body temperature was maintained by an external warmer. Animals were placed in the prone position and underwent partial laminectomy to expose the spinal cord. They were then placed in the supine position for median sternotomy to expose the heart. After the completion of surgical preparation, animals were placed in left lateral decubitus position and general anesthesia was transitioned to intravenous alpha-chloralose (50 mg/kg initial bolus followed by a 20 mg/kg/h continuous infusion). Use of intravenous alpha-chloralose as an anesthetic has been previously shown to be least disruptive of autonomic nervous system activity and has been used extensively in investigational studies27. The depth of anesthesia was assessed throughout the experiments by monitoring corneal reflexes, jaw tone, and hemodynamic indices. In the end, animals were euthanized by injection of potassium chloride.

Acute myocardial ischemia

We created acute myocardial ischemia as previously described27–29. Briefly, a prolene suture was placed around the left anterior descending coronary artery (LAD) below the second diagonal branch of the LAD. The suture was led through a short polyethylene tubing segment, which was then used to ligate the coronary artery to induce cardiac ischemia for one hour. Ischemia was confirmed by the presence of ST-segment elevations. After 1 hour of ischemia or when the pig had non-resuscitable pulseless ventricular tachycardia or ventricular fibrillation (defined as the lack of conversion to a perfusing rhythm after defibrillation 10 times) the suture was removed, and reperfusion was permitted for 2 hours. When pulseless ventricular tachycardia or ventricular fibrillation occurred during ischemia, resuscitation efforts were applied to the animal in accordance with ACLS (Advanced Cardiac Life Support) guideline.

Spinal cord stimulation

A four-pole spinal cord stimulating lead was inserted in the epidural space, with the lead located at the thoracic spinal cord level 1–4 and the most cranial pole of the lead at thoracic spinal cord level 1. Current controlled stimulation (model S88 stimulator, Grass Instruments) was delivered at 50 Hertz and 0.4 milliseconds pulse duration starting 30 minutes before ischemia and was continued throughout the ischemia-reperfusion protocol. Stimulation currents were set at 90% of motor threshold which was determined by increasing stimulus intensity with 2 Hertz of frequency and 0.4 milliseconds of pulse duration until muscle contractions were observed in the shoulder. The mean (± SD) motor threshold was 1.3 ± 1.0 milliamps.

Intrathecal administration GABAA/B receptor antagonists and GABA transaminase inhibitor

GABA receptor antagonists and GABA transaminase inhibitor were delivered via an intrathecal catheter placed at thoracic T1 to T4 spinal level inserted through a small incision in dura mater at thoracic spinal level 5. The lowest therapeutic dose was chosen based on literatures30–33. For the GABAA/B receptor antagonists 1000 microgram of GABAA antagonist Bicuculline (Sigma-Aldrich) or 3000 microgram of GABAB antagonist CGP55845 (Sigma-Aldrich) were dissolved in 2 milliliters of normal saline and warmed to 37° Celsius, were infused over 5 minutes using a syringe pump. Given that the peak dorsal horn drug concentration occurs 30 minutes after intrathecal administrations, each antagonist was applied 30 minutes before starting Ischemia/reperfusion and reapplied at 60 minute intervals. GABA transaminase inhibitor, GABAculine, was used as inhibition of GABA transaminase reduces the degradation of GABA leading to increased neuronal GABA concentrations34. 5 milligrams of GABAculine (Enzo) was dissolved in 1milliliter DMSO and 4 milliliter saline, 2 milligrams GABAculine was infused over 5 minutes using a syringe pump, 30 minutes prior to Ischemia/reperfusion, with no spinal cord stimulation, and reapplied at 60 minutes interval.

Hemodynamic assessment and surface ECG recordings

We performed hemodynamic assessment and ECG recording as previously described14. To measure left ventricular end-systolic and end-diastolic pressure throughout the experiment, a 12-pole conductance, high-fidelity pressure monitoring pigtail catheter (5 French) was inserted into the left ventricle via the left carotid artery and connected to an MPVS Ultra Pressure Volume Loop System (Millar Instruments, Houston, TX). Left ventricular systolic function was evaluated by end-systolic pressure (LVESP) and maximum rate of pressure change (dP/dt max) and left ventricular diastolic function was evaluated by end-diastolic pressure and minimum rate of chamber pressure change (dP/dt min). ECG data were continuously recorded on Prucka CardioLab system (GE Healthcare, Fairfield, CT). Precordial lead electrodes (V1-V6) were positioned posteriorly in a manner that reflects standard anterior precordial lead electrode placement and records the horizontal plane.

Electrophysiological recordings and analysis

A 56-electrode nylon mesh was placed around the heart and unipolar electrograms (0.05–500 Hertz) were measured using a Prucka CardioLab electrophysiology mapping system (GE Healthcare, Fairfield, CT) (Figure 2 A, B). All physiological measures were recorded at baseline; during spinal cord stimulation, during acute ischemia (or until pulseless ventricular tachycardia or ventricular fibrillation requiring > 10 internal cardiac defibrillation episodes), and throughout 2 hours of reperfusion. We assessed activation recovery interval which has been shown as a surrogate of local action potential duration (Figure 2 C). Activation recovery intervals were calculated with customized software (iScalDyn, University of Utah, Salt Lake City, UT) as previously described14. Sympathetic stimulation is associated with shortened activation recovery interval duration. In this study, activation recovery interval was analyzed by whole heart and regionally in the ischemic and non-ischemic zones of the myocardium, as defined by whether the distribution of the left anterior descending coronary artery was perfused. The percentage of ischemic myocardium was calculated as the area at risk within the ventricles. To ensure accuracy of activation recovery interval measurement, each electrogram with ST segment changes was both measured by semiautomated accepted software and then checked by hand following the guidelines described by Haws and Lux for activation recovery interval measurement in ischemia and carefully measured across four to five beats14. All electrophysiological and hemodynamic measurements were made off-line by investigators blinded to experimental group. Measurements were calculated every 15 minutes from baseline to end of recording in blinded fashion.

Figure 2. 56-electrode epicardial polar mapping.

Figure 2.

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(A) 56-electrode high-fidelity epicardial polar mapping. (B) The polar mapping sock was placed over the heart representing the electrode position and heart orientation. (C) An activation recovery interval (ARI) was measured from the electrograms. LAD: Left anterior descending artery. Right ventricle. Left ventricle

ECG-based arrhythmia scoring system and individual arrhythmias

Ventricular arrhythmias, which include premature ventricular contractions, ventricular tachycardia, ventricular fibrillation- were counted using Prucka CardioLab system. Premature ventricular contractions were identified by the presence of a premature QRS complex and ventricular tachycardia was classified as three or more consecutive Premature ventricular contractions in accordance with the recommendations of the Lambeth Conventions35. An arrhythmia score was calculated for each animal throughout ischemia and reperfusion. To calculate the arrhythmia score, a clinically based ECG scoring method was used, which was adapted from Curtis and Walker36. We evaluated the following components and formulated the score as described; “0: No Premature ventricular contractions, ventricular tachycardia, or ventricular fibrillation”, “1: Premature ventricular contractions”, “2: 1–5 episodes of ventricular tachycardia”, “3: >5 episodes of ventricular tachycardia or 1 episode of ventricular fibrillation”, “4: 2–5 episodes of ventricular fibrillation”, “5: >5 episodes of ventricular fibrillation”. The scoring system assigns a numeric value based upon the severity of arrhythmia with larger scores representing greater severity37.

Heart staining and measurement of area at risk (area at risk)

To determine potential ischemic insults, Evans Blue was used for the heart staining as previously described38. At the end of the experiment after the animal was sacrificed, the LAD ligation was tightened again, and a cross clamp was placed on the aorta just above the base of the heart with the animal in the supine position. We made sure that the aorta was completely sealed in order to prevent the dye from leaking out of the heart. Evans Blue dye was injected via the needle punctured right below the cross clamp. Area at risk was defined as the area where it was not stained by Evans Blue.

Immunohistochemistry and image analysis

Sections from spinal thoracic segment T3 were used for double labeling with NeuN + GABAA and GABAB receptor antibodies to measure the change in GABA + neurons in the thoracic spinal cord during Ischemia/reperfusion with and without spinal cord stimulation. Immediately upon collection, spinal cord tissues were placed in 4% paraformaldehyde (PFA, Thermo Scientific, USA) at 4 °C for about 48 hours, followed by a 30% buffered sucrose solution which contained ~ 0.01% sodium azide. After the tissues sank in sucrose, they were embedded in O.C.T. compound (Fisher, USA) and stored in −80 °C freezer before use. The frozen samples were cut at 35 micrometers thickness by using a cryostat (CryoStar NX 50; Thermo Fisher Scientific, USA) and washed in phosphate-buffered saline (PH 7.4) prior to blocking in a 5% normal goat or donkey blocking serum (phosphate-buffered saline 0.3% Triton x-100) blocking buffer at room temperature for 1 hour. The slices were firstly incubated in anti-GABAA or anti-GABAB antibody (Supplemental Table) in a phosphate-buffered saline 0.3% Triton x-100 solution overnight at 4 °C, and then transferred to anti-NeuN antibodies solution for another overnight at 4 °C before the secondary antibodies in phosphate-buffered saline solution applied (1 hour, at room temperature; Supplemental Table). The slices were rinsed 3–4 times (5 min per time) after each incubation of antibodies. After mounted and cover slipped with mounting medium (with DAPI; H-1500, Vector Laboratories, USA), the slices were imaged by using a Nikon Eclipse Ti2 Inverted Microscope Systems and NIS-Elements AR Imaging Software V 5.10.01 (Nikon Instruments Inc., USA). The spinal cord sections were imaged with the x20 objective. All exposure times and processing procedures were identical across samples and treatment groups.

Image analysis was performed with the investigator blinded to the experimental group spinal cord section during the entire analysis and protocols were standardized to avoid potential experimental bias. Analysis of images was completed using NIS-Elements AR Analysis Software v 5.10.01 (Nikon Instruments Inc). A minimum of two spinal cord slices were used per animal. The spinal cord was divided into left and right regions of interest and the number of immunoreactive cells were counted based on uniformly set thresholds across groups. Data were averaged for each animal and analyzed by group and anatomic region.

Western blot

For further quantitative evaluation of GABA receptor expression in the spinal cord, we extracted the proteins from T3 dorsal horn and examined the expression of GABA receptors by subtypes, GABAAα receptor, GABAAβ receptor, GABAB receptor 1, GABAB receptor 2, using Western blot. The fresh spinal cord tissues of pig were dissected on wet ice, flash-frozen in liquid nitrogen and stored in −80 °C freezer before use. Dorsal part of the frozen tissues was homogenized mechanically in ice-cold RIPA lysis buffer (Thermo Fisher Scientific, USA) containing 1X Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, USA). Homogenates were centrifuged at 4°C for 10 minutes at 14,000 revolutions per minute, and the supernatant fraction was used to measure protein concentration by a Bradford Assay kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. A total protein concentration of 10 micrograms was applied to 4–20% Tris-Glycine eXtended precast protein gels (Bio-rad Laboratories, California, USA) using Tris-Glycine Sodium Dodecyl Sulfate running buffer (Thermo Fisher Scientific, USA) at 200 voltage on ice for 1 hour, and then transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific, USA) at 100 voltage for 30 minutes at 4°C. Membranes were blocked with SuperBlock blocking buffer (Thermo Fisher Scientific, USA) with Tween 0.05% for 1.5 hours at room temperature, and then incubated at 4°C overnight with primary antibodies (Supplemental Table). To detect the primary antibody signals, horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection reagent (RPN2235, GE Healthcare Buckinghamshire, UK) were applied before imaging. Membranes were then stripped by a western blot stripping buffer (Thermo Fisher scientific, USA) for 10 min, blocked and incubated for 1 hour with GAPDH antibody. The expression of GAPDH was considered as a control and used to normalize the intensity levels of the target proteins. The membranes were captured in an image analysis system (ChemiDoc XRS+ System, Bio-rad, California, USA) and the bands were quantified by densitometry using an image analysis program (Image Lab; Bio-rad, California, USA).

Statistical Analysis

All data were examined for normality using the Shapiro-Wilk test. Data with normal distribution are expressed as means ± standard deviation (SD) and data with non-normal distribution is presented as median and interquartile range. One-way repeated measures ANOVA with post hoc Tukey test was used for all within group cardiac electrophysiological (activation recovery interval) and hemodynamic measures. Mixed-effect models were used to assess cardiac electrophysiological (activation recovery interval, arrhythmia score) and hemodynamic variables between experimental groups. We employed mixed effects models to examine the effect of time point (repeated measure) on raw values within testing condition wherein subject number was treated as a random effect and % area at risk and sex were treated as fixed effects. We employed mixed effects models to compare % change in measures from baseline between conditions controlling for sex, % area at risk wherein condition was entered as a class variable in the model. For immunohistochemistry and western blot analysis, one-way ANOVA with post hoc Tukey test was performed to compare the percentage of positive GABAA/B neurons and GABAA/B receptor subunit concentration between the groups. For all results, P value < 0.05 was considered statistically significant. All figures were created using GraphPad Prism software (version 8, GraphPad Software Inc, San Diego, CA). Calculation for sample size was based off preliminary data with a mean activation recovery interval of 450 millisecond, change of 20% from this mean, standard deviation of 65, two-tailed alpha 0.05, and power 80% during acute ischemia between control and spinal modulation, which determined sample size n=8 per experimental group.

Results

Ischemia-Reperfusion decreases global activation recovery interval

Cardiac electrophysiologic measures are reported at baseline, control 30 minutes, and control 60 minutes or spinal cord stimulation 30 minutes and LAD 30 minutes. Ischemia data is reported at 30 min as multiple animals had irretractable ventricular tachycardia/ventricular fibrillation requiring resuscitation as ischemia proceeded resulting in incomplete data sets at 60 minutes. Comparing cardiac electrophysiologic measures across time points, within each group; myocardial ischemia led to expected cardiac sympathetic excitation as demonstrated by activation recovery interval duration shortening, in all experimental groups except for the control group, which did not undergo cardiac Ischemia/reperfusion (Table 1). Comparing hemodynamic parameters within each group, HR increase was seen after ischemia in GABAA group and spinal cord stimulation decreased maximal rate of rise of left ventricular pressure (dP/dt max) during ischemia, as compared to baseline. All other hemodynamics did not have any significant changes (Table 2).

Table 1.

Global Activation Recovery Intervals

Control Ischemia/Reperfusion Ischemia/Reperfusion + Spinal Cord Stimulation GABAA receptor antagonist GABAB receptor antagonist GABA transaminase inhibitor
Baseline 376 ± 61 378 ± 65 405 ± 62 412 ± 101 437 ± 76 359 ± 27
Control 30 minutes or Spinal cord stimulation 30 minutes 391 ± 66 389 ± 76 406 ± 71 407 ± 70 432 ± 75 360 ± 35
LAD occlusion 30 minutes 395 ± 78 306 ± 63* 363 ± 71* 311 ± 36* 355 ± 69* 316 ± 43*
P value, Baseline vs LAD occlusion 0.353 <0.0001 0.002 0.029 0.036 0.008
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Data are expressed as mean SD. Global activation recovery intervals in all groups, except control, reduced in ischemia compared to baseline (* statistically significant, each P value shown in the table) Control (n=11), Ischemia Reperfusion (n = 16). Ischemia Reperfusion + spinal cord stimulation(n=17), GABAA receptor antagonists: ischemia/reperfusion + spinal cord stimulation with intrathecal GABAA antagonist (n=8), GABAB receptor antagonists: ischemia/reperfusion + spinal cord stimulation with intrathecal GABAB antagonist (n=7). GABA transaminase inhibitor: ischemia/reperfusion + intrathecal GABA transaminase inhibitor (n=8).

Table 2.

Hemodynamics

HR, beats/min SBP, mmHg Left Ventricular End Systolic Pressure, mmHg Maximal Rate of Rise Left Ventricular Pressure (dP/dt max), mmHg/s
Control Baseline 88 ± 26 110 ± 16 94 ± 18 2308 ± 659
Control 30 minutes 82 ± 18 109 ± 14 97 ± 21 2250 ± 802
Control 60 minutes 87 ± 22 112 ± 11 89 ± 19 2144 ± 546
Ischemia/Reperfusion Baseline 88 ± 21 125 ± 17 102 ± 18 2568 ± 919
Control 30 minutes 86 ± 22 125 ± 17 100 ± 18 2559 ± 959
LAD Occlusion 30 minutes 92 ± 26 117 ± 18 95 ± 19 2228 ± 775
Ischemia/Reperfusion + Spinal Cord Stimulation Baseline 82 ± 13 128 ± 21 106 ± 19 2609 ± 1207
Spinal Cord Stimulation 30 minutes 82 ± 16 127 ± 22 103 ± 23 2281 ± 708
LAD Occlusion 30 minutes 78 ± 12 122 ± 18 104 ± 17 2085 ± 682**
GABAA receptor antagonist Baseline 87 ± 17 125 ± 19 92 ± 5 24755 ± 413
Spinal Cord Stimulation 30 minutes 97 ± 19 125 ± 25 90 ± 3 2326 ± 302
LAD Occlusion 30 minutes 107 ± 16* 107 ± 23 84 ± 9 2013 ± 438
GABAB receptor antagonist Baseline 79 ± 13 112 ± 11 94 ± 9 2116 ± 435
Spinal Cord Stimulation 30 minutes 75 ± 17 108 ± 10 99 ± 19 2712 ± 1595
LAD Occlusion 30 minutes 78 ± 8 105 ± 12 106 ± 18 3020 ± 1265
GABA transaminase inhibitor Baseline 84 ± 7 144 ± 15 123 ± 15 1923 ± 642
Sham 30 minutes 82 ± 8 148 ± 16 117 ± 18 1902 ± 460
LAD Occlusion 30 minutes 85 ± 9 141 ± 21 120 ± 18 1689 ± 337
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Data are expressed as mean SD. Within-group analysis showed that HR decreased during ischemia/reperfusion compared to 30 minutes into spinal cord stimulation in GABAA receptor antagonist group (*P=0.029) and maximal rate of rise on left ventricular pressure reduced in ischemia/reperfusion + spinal cord stimulation group(** P=0.038). All other P>0.05 for within-group analysis. Control (n=11), Ischemia Reperfusion (n = 16). Ischemia Reperfusion + spinal cord stimulation(n=17), GABAA receptor antagonists: ischemia/reperfusion + spinal cord stimulation with intrathecal GABAA antagonist (n=8), GABAB receptor antagonists: ischemia/reperfusion + spinal cord stimulation with intrathecal GABAB antagonist (n=7). GABA transaminase inhibitor: ischemia/reperfusion + intrathecal GABA transaminase inhibitor (n=8).

Ischemia/reperfusion decreases Percent change in activation recovery interval in ischemic myocardium

Activation recovery interval was analyzed regionally in the ischemic and non-ischemic zones of the myocardium and the changes in cardiac EP measures during cardiac Ischemia/reperfusion were compared between groups the 5 experimental groups of: Ischemia/reperfusion alone, Ischemia/reperfusion + Spinal cord stimulation, Ischemia/reperfusion + Spinal cord stimulation +GABAA receptor antagonist (GABAA), Ischemia/reperfusion + Spinal cord stimulation +GABAB receptor antagonist (GABAB), and Ischemia/reperfusion + GABA transaminase inhibitor (GABAculine). The magnitude of cardiac ischemic insult was measured by the area at risk in heart. There were no differences in area at risk, between the groups (Ischemia/reperfusion 22 ± 12%, Ischemia/reperfusion + Spinal cord stimulation 26 ± 13%, GABAA 31 ± 10%, GABAB 25 ± 8%, GABAculine 22 ± 9%, All P>0.207, data presented as mean ±SD). The magnitude of ischemia induced sympathoexcitation, as determined by the change in activation recovery interval from baseline to 30 minutes LAD ligation, was compared between groups to see the effects of spinal cord stimulation alone as compared to spinal cord stimulation + GABA antagonists and GABAculine during cardiac ischemia. Cardiac ischemia decreased activation recovery interval in the ischemic region, and this activation recovery interval reduction was mitigated by spinal cord stimulation (Figure 3A). The effect of spinal cord stimulation on activation recovery interval reduction in the ischemic myocardium was abolished by both intrathecal GABA receptor antagonists’ applications (GABAA; P=0.043 vs spinal cord stimulation, GABAB; P=0.012 vs spinal cord stimulation). While intrathecal GABA transaminase inhibitor GABAculine application alone produced a similar effect on activation recovery interval as spinal cord stimulation, and lessened activation recovery interval reduction during LAD ischemia (Figure 3A). No activation recovery interval changes were seen in non-ischemic region between groups (Figure 3B).

Figure 3: Ischemia-Reperfusion decreases percent change in activation recovery interval in ischemic myocardium.

Figure 3:

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Within the ischemic region (A) Ischemia/reperfusion + spinal cord stimulation and GABAculine reduced %activation recovery interval change compared to Ischemia/reperfusion alone (*P= 0.001 Ischemia/reperfusion vs Ischemia/reperfusion +spinal cord stimulation, **P=0.038 Ischemia/reperfusion vs GABAculine, Ischemia/reperfusion vs GABAA receptor antagonists P=0.523 Ischemia/reperfusion vs GABAB receptor antagonist. P=0.261). Within the non-ischemic region (B) No significant differences were seen across the groups (All P>0.05). Values expressed as means ± SD. Ischemia/reperfusion (n = 16). Ischemia/reperfusion + Spinal cord stimulation (n=17), GABAA: Ischemia/reperfusion + Spinal cord stimulation with intrathecal GABAA receptor antagonist (n=8), GABAB: Ischemia/reperfusion + Spinal cord stimulation with intrathecal GABAB receptor antagonist (n=7). GABAculine: Ischemia/reperfusion + GABA transaminase inhibitor (n=8).

For hemodynamic parameters, SBP and maximal rate of rise of left ventricular pressure (dP/dt max), there were no differences between groups at 30 minutes after cardiac ischemia. However, the HR in GABAA group was greater than spinal cord stimulation and GABAB groups (P=0.018 vs spinal cord stimulation, P=0.019 vs GABAB). In addition, maximal rate of rise of left ventricular pressure (dP/dt max) max in GABAculine was greater than control and GABAA groups (P=0.043 vs control, P=0.022 vs GABAA) (Table1).

Ventricular Arrhythmia Score changes during ischemia-reperfusion

Ventricular arrhythmia scores were calculated throughout ischemia and reperfusion and compared across all experimental groups. A higher arrhythmia score indicates greater arrhythmia severity. Ischemia/reperfusion was associated with an elevation in arrhythmia score whereas spinal cord stimulation during ischemia, decreased cardiac arrhythmias. GABAculine treatment during Ischemia/reperfusion also reduced the arrhythmia score, similar to that of spinal cord stimulation. On the other hand, spinal cord stimulation + both intrathecal GABA receptor antagonists abolished the spinal cord stimulation reduction in cardiac arrhythmia score during myocardial Ischemia/reperfusion (Figure 4).

Figure 4. Ventricular Arrhythmia Score changes during ischemia and reperfusion.

Figure 4.

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Ischemia/reperfusion + spinal cord stimulation + intrathecal GABA transaminase inhibitor GABAculine reduced ventricular arrhythmias as compared to Ischemia/reperfusion alone. Values are expressed as median and interquartile range. * P<0.001, ** P=0.003. Ischemia/reperfusion (n = 16), Ischemia/reperfusion + Spinal cord stimulation (n=17), GABAA: Ischemia/reperfusion + Spinal cord stimulation with intrathecal GABAA receptor antagonist (n=8), GABAB: Ischemia/reperfusion+ Spinal cord stimulation with intrathecal GABAB receptor antagonist (n=7). GABAculine: Ischemia/reperfusion + GABA transaminase inhibitor (n=8).

Ischemia-Reperfusion decreases GABAA receptor expression and this reduction was less with spinal cord stimulation

Immunohistochemistry.

As demonstrated in Figure 5, cardiac Ischemia/reperfusion significantly reduced GABAA receptor+ neurons (quantified as the percentage of NeuN positive cells colocalized with GABAA receptor). Spinal cord stimulation during Ischemia/reperfusion showed a greater percentage of GABAA + neurons than Ischemia/reperfusion alone, however the expression was still lesser than control condition. In contrast, there was no change in expression of GABAB receptor + neurons during Ischemia/reperfusion with or without spinal cord stimulation (Figure 6). The anatomical distribution of GABA receptors was also investigated in the three regions; superficial dorsal horn laminae (I-II), deep laminae (III-VII, X), and the intermediolateral cell column (IML) as shown in Figure 8. There was greater expression of GABAA+ neurons with Ischemia/reperfusion + spinal cord stimulation, as opposed to Ischemia/reperfusion alone, in all anatomical regions (Figure 7). No differences were seen in %GABAB+ neurons per anatomical region in Ischemia/reperfusion vs Ischemia/reperfusion + Spinal cord stimulation (Ischemia/Reperfusion: Superficial 25% (15, 51) Deep 14 %(4, 37) Intermediolateral nucleus 11% (5, 35) vs Ischemia/reperfusion + Spinal cord stimulation : Superficial 26% (12, 35) Deep 18% (4, 31), Intermediolateral nucleus 9 %(5, 14); all p>0.05), data presented as median and interquartile range).

Figure 5. Ischemia-Reperfusion decreases GABAA receptor expression in the thoracic spinal cord and this reduction was less in the presence of spinal cord stimulation.

Figure 5.

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(A-C) Representative images from superficial dorsal horn laminae segment thoracic spinal cord T3. Magnification is 20x and scale bar = 50 micrometers. (D) Ischemia/reperfusion reduced the percentage of GABAA+ NeuN+ neurons as compared to the control group with no injury; this reduction was less in the presence of spinal cord stimulation. Values expressed as means ± SD. **P =0.003, ****P < 0.001. Control (n=5), Ischemia/reperfusion (n=5), Ischemia/reperfusion with Spinal Cord Stimulation (n=5)

Figure 6. GABAB receptor expression is unchanged in ischemia-reperfusion with or without spinal cord stimulation.

Figure 6.

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A-C) Representative images from superficial dorsal horn laminae segment T3. Magnification is ×20x and scale bar = 50 micrometers. (D)There was no change in percentage of GABAB+ NeuN+ neurons during ischemia with or without spinal cord stimulation. Values are expressed as means ± SD. Control (n=5), IR: Ischemia/reperfusion (n=5), Ischemia/reperfusion + Spinal cord stimulation (n=5).

Figure 8. Ischemia-Reperfusion with spinal cord stimulation increases GABAA but not GABAB receptor expression in dorsal horn.

Figure 8.

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(A-B) The expression of GABAAα and GABAAβ was significantly greater in the dorsal horn of Ischemia/reperfusion + Spinal cord stimulation group than Ischemia/reperfusion and Control groups. (C-D) There was no significant difference in the expression of GABAB receptor 1 and GABAB receptor 2 among the three groups. (E-H) Representative images of each membrane used for analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. * (A) P=0.004 vs control, (B) P=0.048 vs control and P=0.031 vs Ischemia/reperfusion. Control (n=5), Ischemia/reperfusion (n=5), Ischemia/reperfusion + Spinal cord stimulation (n=5).

Figure 7. Regional Analysis of GABAA+ Neurons in the thoracic spinal cord.

Figure 7.

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(A) Representative image of spinal cord regions of interest; superficial laminae (I-II), deep Laminae (III-VII, X), and intermediolateral nucleus (IML). Magnification is ×4. Scale bar =100 micrometers. (B) Percentage of GABAA+ neurons (expressing neuronal marker NeuN) in each regional area for Ischemia/reperfusion vs. +spinal cord stimulation. Data displayed as mean ± SD. * P<0.05 for regional comparison Ischemia/reperfusion vs Ischemia/reperfusion +spinal cord stimulation. Control (n=5), Ischemia/reperfusion (n=5), Ischemia/reperfusion + Spinal Cord Stimulation (n=5).

Western Blot

Myocardial ischemia alone did not affect the expression of GABA receptor subtypes. During Ischemia/reperfusion with spinal cord stimulation however, the expression of GABAAα receptor, GABAAβ receptor subtypes were greater than the control and Ischemia/reperfusion (Figure 8). Spinal cord stimulation did not affect the expression of either GABAB receptor subtypes.

Discussion

In this pre-clinical translational porcine model of cardiac ischemia/reperfusion injury with thoracic spinal cord stimulation, we show that 1) spinal cord stimulation therapy during cardiac ischemia reduced myocardial sympathoexcitation and ventricular arrhythmias, 2) intrathecal GABAA and GABAB receptor blockade during spinal cord stimulation therapy abolished the protective myocardial effects of spinal cord stimulation and increased sympathetic excitation and arrhythmias, 3) intrathecal administration of GABA transaminase inhibitor (GABAculine) reduced myocardial sympathoexcitation and ventricular arrhythmias during cardiac ischemia/reperfusion with similar magnitude to spinal cord stimulation, and 4) spinal cord stimulation neuromodulation during cardiac ischemia was associated with a significant increase in GABAA receptor expression with no significant change in GABAB receptor expression. Thus, these results importantly show that spinal cord stimulation is likely reducing ischemia/reperfusion induced sympathoexcitation and cardiac arrhythmias through activation of spinal GABAergic pathways.

The cardiac electrophysiological results from this study support our model of ischemia induced activation of cardiospinal neural reflexes and the therapeutic effect of thoracic spinal cord stimulation in modulating sympathetic output and reducing cardiac arrhythmias. The cell bodies of ischemia-sensitive cardiac neurons are located in the thoracic dorsal root ganglion and project back to the dorsal column of the thoracic spinal cord where they activate a complex cardiospinal neural reflex circuit which results in increased efferent output from sympathetic preganglionic neurons 1,4,5,39. As demonstrated by the results of this study and prior reports, neuromodulation via spinal cord stimulation can interrupt the cardiospinal reflex circuit, thus reducing local sympathoexcitation in ischemic myocardium and decreasing lethal ventricular arrhythmias14,39.

While GABA mediated pathways have been implicated in spinal cord stimulation’s analgesic mechanisms, far less is known about the role of GABA signaling in spinal cord stimulation therapy for the reduction of myocardial ischemia induced sympathoexcitation and cardiac arrhythmias. GABA inhibitory signaling in the spinal cord is primarily achieved through activation of either GABAA or GABAB receptors, which have important differences in structure, anatomic location, and function24,40,41. Structurally, the GABAA receptor is a ligand-gated chloride channel while the GABAB receptor is a G-protein coupled receptor. Anatomically, GABAA receptors are evenly distributed throughout the spinal cord, while GABAB receptors are concentrated in dorsal horn laminae I-III and can function as autoreceptors, presynaptic to GABA containing interneurons synapsing on primary afferent fibers 21,24,41(Figure 1).

Functionally, both receptor types mediate presynaptic inhibition of primary afferent fibers and interneuron regulation of spinal cord reflexes. GABAA receptors have been found to mediate shorter duration components of GABA-induced inhibition while GABAB receptors mediate the longer duration spinal reflexes. Additionally, there is evidence of differential GABAA and GABAB receptor expression in response to nerve injury and spinal cord stimulation18,21,26,41. Therefore to determine the role of GABA signaling pathways in spinal cord stimulation we evaluated 1) functional effect of pharmacologic blockade of GABA A and GABAB receptors, as well as, 2) individual changes in GABA receptor subtype expression during spinal cord stimulation.

Our results show that the reduction in myocardial sympathoexcitation and ventricular arrhythmias seen with spinal cord stimulation during cardiac ischemia/reperfusion were abolished by intrathecal administration of both GABAA and GABAB receptor antagonists. Additionally, intrathecal administration of the GABA transaminase inhibitor (GABAculine) alone provided cardiac protection similar to spinal cord stimulation therapy. Bicuculine is a GABA transaminase inhibitor and as such, reduces the degradation of GABA leading to increased neuronal GABA concentrations34. These results showing loss of spinal cord stimulation therapeutic effect with GABA antagonists and gain of therapeutic effect with a GABA transaminase inhibitor, provide strong evidence that spinal cord stimulation neuromodulation is working through activation of GABAergic signaling mechanisms within the spinal neural network to attenuate ischemia induced sympathoexcitation.

Interestingly, while we found no difference in the functional effect of pharmacological blockade of GABAA vs GABAB receptors, immunohistochemistry analysis showed a difference in GABAA vs GABAB receptor protein expression with cardiac ischemia and spinal cord stimulation. We found that cardiac ischemia was associated with a reduction in GABAA+ neurons, whereas ischemia + spinal cord stimulation resulted in an increase in GABAA+ neurons. No differences were seen in GABAB + neurons with either ischemia/reperfusion alone or ischemia/reperfusion +spinal cord stimulation. Additional quantification of GABAA and GABAB receptor subtype expression was performed using western blot analysis, and the results further supported the differences seen with immunohistochemistry. Ischemia/reperfusion +spinal cord stimulation increased both GABAAa and GABAAb subunits expression while there was no difference seen in either GABABR1 or GABABR2 during ischemia/reperfusion with or without spinal cord stimulation.

Investigation into the anatomic distribution of upregulated GABAA neurons revealed that GABAA neurons were increased throughout the superficial and deep laminae of the dorsal horn, as well as in the intermediolateral cell column during ischemia with spinal cord stimulation as compared to ischemia alone. GABAA receptors in the superficial dorsal horn laminae likely inhibit pre-synaptic cardiac ischemia sensitive primary afferent neurotransmitter release19,21,40. While the upregulation of GABAA neurons in the deeper laminae and intermediolateral cell column may represent activation of inhibitory GABAergic interneurons that are presynaptic to sympathetic preganglionic neurons 17,19,40. This study builds upon our previous work using Cfos for neuronal activation where we reported that spinal cord stimulation activates interneurons in the deep laminae of the thoracic dorsal horn12. Inhibitory interneurons in these deep laminae (V, VIII, and X) have been shown to synapse on sympathetic preganglionic neurons regulating efferent sympathetic outflow to the heart19,42.

Our finding of differential GABA receptor subtype expression with no difference in functional effects of GABAA or GABAB receptor antagonists is similar to previous studies investigating GABA signaling in nocioceptive pathways. While Castro-Lopes reported a down-regulation in GABAB receptor binding and an up-regulation in GABAA receptor binding in a rodent model of peripheral injury,21 follow -up studies by Gwak and Malan found similar functional responses to both GABAA and GABAB receptor agonists and antagonists25,31. Both GABA subtype receptor agonists induced analgesia and both GABAA and GABAB antagonists caused hyperalgesia and allodynia during nerve injury.

The ability of both GABAA and GABAB receptor antagonists to reverse the therapeutic effect of spinal cord stimulation in ischemia, suggests that spinal cord stimulation may be decreasing efferent sympathetic output through an increase in endogenous GABA tone in the spinal cord. This is further supported by our finding that GABAculine alone, which inhibits GABA degradation and increases neuronal GABA concentrations, caused a reduction in ischemia induced cardiac sympathoexcitation and ventricular arrhythmias similar to that seen with spinal cord stimulation. Alternatively, the similar functional response with both GABA receptor subtype antagonists could also be due to: 1)intrathecal pharmacologic receptor antagonists work to block GABA receptors that are already expressed and as such the effect of spinal cord stimulation on GABA receptor expression may be independent from the effect that blockade of GABA receptors may have, or 2) GABA receptor expression is affected by changes in neurotransmitter levels and therefore, we cannot determine if the changes seen in GABA receptor expression are due to a direct effect of spinal cord stimulation on receptor upregulation, or through changes in GABA neurotransmitter release/uptake.

Clinical Implications

The data from this study provides important new mechanistic insight into how spinal cord stimulation is reducing cardiac ischemia/reperfusion induced sympathoexcitation and ventricular arrhythmias. The majority of work investigating the mechanisms underlying spinal cord stimulation has focused on nociceptive pathways15,16,22,26. Our study uniquely focuses on the mechanisms through which spinal cord stimulation is decreasing cardiac autonomic sympathoexcitation since the mechanisms through which spinal cord stimulation reduces pain may not be the same as the those for reduction in cardiac sympathoexcitation. In fact, several studies suggest that spinal cord stimulation for analgesia in peripheral nerve injury has a greater effect via GABAB receptors, whereas in this study we are showing opposing results in that the autonomic modulation effects of spinal cord stimulation may be having a greater effect through GABAA receptors16,33.

While studies in animal models have demonstrated the cardiac protective effects of neuraxial modulation, the clinical application of spinal cord stimulation in humans with heart disease has been equivocal43. The limited clinical translation is likely due to our incomplete understanding of the mechanisms through which spinal neural signaling controls cardiac sympathoexcitation, and how spinal cord stimulation modulates cardiospinal neural pathways. Therefore, these data have possible wide reaching clinical implications as they can allow future studies to be aimed at maximizing the therapeutic effects of spinal cord stimulation specifically on autonomic modulation and reduction of sympathoexcitation induced cardiac arrhythmias. It is important to understand how we can best optimize spinal cord stimulation as it is an invasive therapy that carries its own procedural risks, especially in the setting of acute cardiac ischemia and revascularization procedures where patients may be anticoagulated.

Limitations

Although this study provides new insights into the mechanisms behind spinal cord stimulation neuromodulatory effects during cardiac ischemia, there are limitations. In this study acute ischemia was performed on normal hearts to determine the impact of GABA signaling pathways on spinal cord stimulation in a structurally normal central nervous system. These results may not be applicable in hearts with chronic infarction or heart failure in which there may be adverse remodeling in the cardiospinal neural network28,29. Additionally, given the open chest model that was used, continuous anesthesia was necessary throughout experimental protocols. As previously reported, many general anesthesia drugs are mediated by the GABA pathways, including alpha-chloralose, which is known to have less impact on the autonomic nervous system and used in many animal experiments44.Therefore, while it may be possible that alpha-chloralose affected GABA receptor expression, this study was conducted with alpha-chloralose used in the same concentration during surgical preparation across all experimental groups, so any effects seen would be similar for all experimental groups, and the differences shown between groups for GABA receptor expression changes would likely be unaffected

In conclusion, we demonstrate that thoracic spinal cord stimulation during acute cardiac ischemia/reperfusion reduces myocardial sympathoexcitation and ventricular arrhythmias through activation of GABAA signaling pathways, which may function to reduce primary afferent signaling in the superficial dorsal horn and activate inhibitory interneurons to decrease sympathetic output from sympathetic preganglionic neurons in thoracic spinal cord. These findings help shed light on the pathways through which spinal cord stimulation neuromodulation reduces cardiac ischemia induced sympathetic output and can aid in future studies to improve the efficacy of clinical spinal cord stimulation to reduce myocardial arrhythmogenesis.

Supplementary Material

Supplemental table

Digital Content 2- Supplemental Table: Primary and Secondary Antibodies

Supplemental document

Digital Content 1- Supplemental document: Arrive Guidelines Checklist

Funding

Dr. Mahajan is supported by NIH RO1 HL136836 and NIH R44 DA049630. Dr. Howard-Quijano is supported by NIH K08 HL135418

Abbreviations

SCS

spinal cord stimulation

GABA

γ-aminobutyric acid

Footnotes

Conflicts of Interest

The authors declare no competing interests.

References

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