Thoracic Dorsal Root Ganglion Application of Resiniferatoxin Reduces Myocardial Ischemia-Induced Ventricular Arrhythmias.

BACKGROUND: A myocardial ischemia/reperfusion (IR) injury activates the transient receptor potential vanilloid 1 (TRPV1) dorsal root ganglion (DRG) neurons. The activation of TRPV1 DRG neurons triggers the spinal dorsal horn and the sympathetic preganglionic neurons in the spinal intermediolateral column, which results in sympathoexcitation. In this study, we hypothesize that the selective epidural administration of resiniferatoxin (RTX) to DRGs may provide cardioprotection against ventricular arrhythmias by inhibiting afferent neurotransmission during IR injury. METHODS: Yorkshire pigs (n = 21) were assigned to either the sham, IR, or IR + RTX group. A laminectomy and sternotomy were performed on the anesthetized animals to expose the left T2-T4 spinal dorsal root and the heart for IR intervention, respectively. RTX (50 µg) was administered to the DRGs in the IR + RTX group. The activation recovery interval (ARI) was measured as a surrogate for the action potential duration (APD). Arrhythmia risk was investigated by assessing the dispersion of repolarization (DOR), a marker of arrhythmogenicity, and measuring the arrhythmia score and the number of non-sustained ventricular tachycardias (VTs). TRPV1 and calcitonin gene-related peptide (CGRP) expressions in DRGs and CGRP expression in the spinal cord were assessed using immunohistochemistry. RESULTS: The RTX mitigated IR-induced ARI shortening (-105 ms ± 13 ms in IR vs. -65 ms ± 11 ms in IR + RTX, p = 0.028) and DOR augmentation (7093 ms2 ± 701 ms2 in IR vs. 3788 ms2 ± 1161 ms2 in IR + RTX, p = 0.020). The arrhythmia score and VT episodes during an IR were decreased by RTX (arrhythmia score: 8.01 ± 1.44 in IR vs. 3.70 ± 0.81 in IR + RTX, p = 0.037. number of VT episodes: 12.00 ± 3.29 in IR vs. 0.57 ± 0.3 in IR + RTX, p = 0.002). The CGRP expression in the DRGs and spinal cord was decreased by RTX (DRGs: 6.8% ± 1.3% in IR vs. 0.6% ± 0.2% in IR + RTX, p < 0.001. Spinal cord: 12.0% ± 2.6% in IR vs. 4.5% ± 0.8% in IR + RTX, p = 0.047). CONCLUSIONS: The administration of RTX locally to thoracic DRGs reduces ventricular arrhythmia in a porcine model of IR, likely by inhibiting spinal afferent hyperactivity in the cardio-spinal sympathetic pathways.

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

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. The authors 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 plus spinal cord stimulation (n = 17), ischemia-reperfusion plus spinal cord stimulation plus γ-aminobutyric acid type A (GABAA) or γ-aminobutyric acid type B (GABAB) receptor antagonist (GABAA, n = 8; GABAB, n = 8), and ischemia-reperfusion plus GABA transaminase inhibitor (GABAculine, n = 8). A four-pole spinal cord stimulation lead was placed epidurally (T1 to T4). GABA modulating pharmacologic agents were administered intrathecally. Spinal cord stimulation at 50 Hz was applied 30 min before ischemia. A 56-electrode epicardial mesh was used for high-resolution electrophysiologic 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 plus spinal cord stimulation; GABAB, -13 ± 14%, P = 0.012 vs. ischemia-reperfusion plus 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 these 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.

Thoracic dorsal root ganglion stimulation reduces acute myocardial ischemia induced ventricular arrhythmias.

Background: Dorsal root ganglion stimulation (DRGS) may serve as a novel neuromodulation strategy to reduce cardiac sympathoexcitation and ventricular excitability. Objective: In this pre-clinical study, we investigated the effectiveness of DRGS on reducing ventricular arrhythmias and modulating cardiac sympathetic hyperactivity caused by myocardial ischemia. Methods: Twenty-three Yorkshire pigs were randomized to two groups, which was control LAD ischemia-reperfusion (CONTROL) or LAD ischemia-reperfusion + DRGS (DRGS) group. In the DRGS group (n = 10), high-frequency stimulation (1 kHz) at the second thoracic level (T2) was initiated 30 min before ischemia and continued throughout 1 h of ischemia and 2 h of reperfusion. Cardiac electrophysiological mapping and Ventricular Arrhythmia Score (VAS) were assessed, along with evaluation of cFos expression and apoptosis in the T2 spinal cord and DRG. Results: DRGS decreased the magnitude of activation recovery interval (ARI) shortening in the ischemic region (CONTROL: -201 ± 9.8 ms, DRGS: -170 ± 9.4 ms, p = 0.0373) and decreased global dispersion of repolarization (DOR) at 30 min of myocardial ischemia (CONTROL: 9546 ± 763 ms2, DRGS: 6491 ± 636 ms2, p = 0.0076). DRGS also decreased ventricular arrhythmias (VAS-CONTROL: 8.9 ± 1.1, DRGS: 6.3 ± 1.0, p = 0.038). Immunohistochemistry studies showed that DRGS decreased % cFos with NeuN expression in the T2 spinal cord (p = 0.048) and the number of apoptotic cells in the DRG (p = 0.0084). Conclusion: DRGS reduced the burden of myocardial ischemia-induced cardiac sympathoexcitation and has a potential to be a novel treatment option to reduce arrhythmogenesis.

Neuromodulation With Thoracic Dorsal Root Ganglion Stimulation Reduces Ventricular Arrhythmogenicity.

Introduction: Sympathetic hyperactivity is strongly associated with ventricular arrhythmias and sudden cardiac death. Neuromodulation provides therapeutic options for ventricular arrhythmias by modulating cardiospinal reflexes and reducing sympathetic output at the level of the spinal cord. Dorsal root ganglion stimulation (DRGS) is a recent neuromodulatory approach; however, its role in reducing ventricular arrhythmias has not been evaluated. The aim of this study was to determine if DRGS can reduce cardiac sympathoexcitation and the indices for ventricular arrhythmogenicity induced by programmed ventricular extrastimulation. We evaluated the efficacy of thoracic DRGS at both low (20 Hz) and high (1 kHz) stimulation frequencies. Methods: Cardiac sympathoexcitation was induced in Yorkshire pigs (n = 8) with ventricular extrastimulation (S1/S2 pacing), before and after DRGS. A DRG-stimulating catheter was placed at the left T2 spinal level, and animals were randomized to receive low-frequency (20 Hz and 0.4 ms) or high-frequency (1 kHz and 0.03 ms) DRGS for 30 min. High-fidelity cardiac electrophysiological recordings were performed with an epicardial electrode array measuring the indices of ventricular arrhythmogenicity-activation recovery intervals (ARIs), electrical restitution curve (Smax), and Tpeak-Tend interval (Tp-Te interval). Results: Dorsal root ganglion stimulation, at both 20 Hz and 1 kHz, decreased S1/S2 pacing-induced ARI shortening (20 Hz DRGS -21±7 ms, Control -50±9 ms, P = 0.007; 1 kHz DRGS -13 ± 2 ms, Control -46 ± 8 ms, P = 0.001). DRGS also reduced arrhythmogenicity as measured by a decrease in Smax (20 Hz DRGS 0.5 ± 0.07, Control 0.7 ± 0.04, P = 0.006; 1 kHz DRGS 0.5 ± 0.04, Control 0.7 ± 0.03, P = 0.007), and a decrease in Tp-Te interval/QTc (20 Hz DRGS 2.7 ± 0.13, Control 3.3 ± 0.12, P = 0.001; 1 kHz DRGS 2.8 ± 0.08, Control; 3.1 ± 0.03, P = 0.007). Conclusions: In a porcine model, we show that thoracic DRGS decreased cardiac sympathoexcitation and indices associated with ventricular arrhythmogenicity during programmed ventricular extrastimulation. In addition, we demonstrate that both low-frequency and high-frequency DRGS can be effective neuromodulatory approaches for reducing cardiac excitability during sympathetic hyperactivity.

Spinal Cord Stimulation Reduces Ventricular Arrhythmias by Attenuating Reactive Gliosis and Activation of Spinal Interneurons.

OBJECTIVES: This study investigated spinal cord neuronal and glial cell activation during cardiac ischemia-reperfusion (IR)-triggered ventricular arrhythmias and neuromodulation therapy by spinal cord stimulation (SCS). BACKGROUND: Myocardial ischemia induces changes in cardiospinal neural networks leading to sudden cardiac death. Neuromodulation with SCS decreases cardiac sympathoexcitation; however, the molecular mechanisms remain unknown. METHODS: Yorkshire pigs (n = 16) were randomized to Control, IR, or IR+SCS groups. A 4-pole SCS lead was placed in the T1-T4 epidural space with stimulation for 30 minutes before IR (50 Hz, 0.4-ms duration, 90% motor threshold). Cardiac electrophysiological mapping and Ventricular Arrhythmia Score (VAS) were recorded. Immunohistochemistry of thoracic spinal sections was used to map and identify Fos-positive neuronal and glial cell types during IR with and without SCS. RESULTS: IR increased cardiac sympathoexcitation and arrhythmias (VAS = 6.2 ± 0.9) that were attenuated in IR + SCS (VAS = 2.8 ± 0.5; P = 0.017). IR increased spinal cellular Fos expression (#Fos+ cells Control = 23 ± 2 vs IR = 88 ± 5; P < 0.0001) in T1-T4, with the greatest increase localized to T3, and the greatest %Fos+ cells being microglia and astrocytes. Fos expression was attenuated by IR + SCS (62 ± 4; P < 0.01), primarily though a reduction in Fos+ microglia and astrocytes, as SCS also led to increase in Fos+ neurons in deep dorsal laminae. CONCLUSIONS: In a porcine model, cardiac IR was associated with astrocyte and microglial cell activation. Our results suggest that preemptive thoracic SCS decreased IR-induced cardiac sympathoexcitation and ventricular arrhythmias through attenuation of reactive gliosis and activation of inhibitory interneurons in the dorsal horn of spinal cord.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells.

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells.

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Bone-targeting phospholipid polymers to solubilize the lipophilic anticancer drug.

Current chemotherapy methods have limited effectiveness in eliminating bone metastasis, which leads to a poor prognosis associated with severe bone disorders. To provide regional chemotherapy for this metastatic tumor, a bone-targeting drug carrier was produced by introducing the osteotropic bisphosphonate alendronate (ALN) units into an amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate). The polymer can form nanoparticles with a diameter of less than 30 nm; ALN units were exposed to the outer layer of the particle. A simple mixing procedure was used to encapsulate a hydrophobic anticancer drug, known as docetaxel (DTX), in the polymer nanoparticle, providing a uniform solution of a polymer-DTX complex in the aqueous phase. The complex showed anticancer activities against several breast cancer cell lines, and the complex formation did not hamper the pharmacological effect of DTX. The fluorescence observations evaluated by an in vivo imaging system and fluorescence microscopy showed that the addition of ALN to the polymer-DTX complex enhanced bone accumulation. Bone-targeting phospholipid polymers are potential solubilizing excipients used to formulate DTX and deliver the hydrophobic drug to bone tissues by blood administration.

Expression and subcellular localization of apical junction proteins in canine duodenal and colonic mucosa.

OBJECTIVE: To examine the expression and distribution of tight junction (TJ) and adherens junction (AJ) proteins in canine duodenal and colonic mucosa. SAMPLE: Mucosa obtained from 4 healthy Beagles. PROCEDURES: Biopsy specimens of the duodenum and colon were obtained via endoscopy from 4 healthy dogs. The expression patterns and subcelluar localization of claudin-1, -2, -3, -4, -5, -7, and -8; E-cadherin; and ß-catenin in the duodenum and colon were analyzed by use of immunoblotting and immunofluorescence microscopy. RESULTS: In the duodenum, there was clear expression of claudin-3 and -5, E-cadherin, and ß-catenin proteins and weak expression of claudin-7 protein. In contrast, there was clear expression of claudin-2 and -3, E-cadherin, and ß-catenin proteins and weak expression of claudin-5 and -7 proteins in the colon, as determined by use of immunoblotting. As determined by the use of immunofluorescence microscopy, the duodenum and colon had staining for claudin-3 and -5, E-cadherin, and ß-catenin in the most apical region and staining for claudin-7 in the basolateral region. Staining for claudin-2 was also observed in the colon. CONCLUSIONS AND CLINICAL RELEVANCE: Information was provided about the expression patterns of TJ and AJ proteins in the duodenum and colon of clinically normal dogs. These results may provide valuable information for use in evaluating the importance of these TJ and AJ proteins in the pathogenesis of inflammatory bowel disease in dogs.