STELLATE GANGLION BLOCK REVERSES PHSML-INDUCED VASCULAR HYPOREACTIVITY THROUGH INHIBITING AUTOPHAGY-MEDIATED PHENOTYPIC TRANSFORMATION OF VSMCs.

ABSTRACT: Posthemorrhagic shock mesenteric lymph (PHSML) return-contributed excessive autophagy of vascular smooth muscle cells (VSMCs) is involved in vascular hyporeactivity, which is inhibited by stellate ganglion block (SGB) treatment. The contractile phenotype of VSMCs transforms into a synthetic phenotype after stimulation with excessive autophagy. Therefore, we hypothesized that SGB ameliorates PHSML-induced vascular hyporeactivity by inhibiting autophagy-mediated phenotypic transformation of VSMCs. To substantiate this hypothesis, a hemorrhagic shock model in conscious rats was used to observe the effects of SGB intervention or intravenous infusion of the autophagy inhibitor 3-methyladenine (3-MA) on intestinal blood flow and the expression of autophagy- and phenotype-defining proteins in mesenteric secondary artery tissues. We also investigated the effects of intraperitoneal administration of PHSML intravenous infusion and the autophagy agonist rapamycin (RAPA) on the beneficial effect of SGB. The results showed that hemorrhagic shock decreased intestinal blood flow and enhanced the expression of LC3 II/I, Beclin 1, and matrix metalloproteinase 2, which were reversed by SGB or 3-MA treatment. In contrast, RAPA and PHSML administration abolished the beneficial effects of SGB. Furthermore, the effects of PHSML or PHSML obtained from rats treated with SGB (PHSML-SGB) on cellular contractility, autophagy, and VSMC phenotype were explored. Meanwhile, the effects of 3-MA on PHSML and RAPA on PHSML-SGB were observed. The results showed that PHSML, but not PHSML-SGB, incubation decreased VSMC contractility and induced autophagy activation and phenotype transformation. Importantly, 3-MA administration reversed the adverse effects of PHSML, and RAPA treatment attenuated the effects of PHSML-SGB incubation on VSMCs. Taken together, the protective effect of SGB on vascular reactivity is achieved by inhibiting excessive autophagy-mediated phenotypic transformation of VSMCs to maintain their contractile phenotype.

Chronic Expression of Interleukin-10 Transgene Modulates Cardiac Sympathetic Ganglion Resulting in Reduced Ventricular Arrhythmia.

The cardiac autonomic nervous system (CANS) is intimately connected to the regulation of electrophysiology and arrhythmogenesis in cardiac systems. This work aimed at investigating whether interleukin-10 (IL-10) could effectively modulate CANS and suppress ischemia-induced ventricular arrhythmia (VA) through chronically acting on the cardiac sympathetic ganglion (CSG). Using an adeno-associated virus (AAV), we achieved local chronic overproduction of IL-10 in the CSG, left stellate ganglion (LSG). As a result, in the IL-10 group, we observed a decreased number of tyrosine hydroxylase-positive (TH+) cells in the LSG. IL-10 markedly downregulated the nerve growth factor, synaptophysin, as well as growth-associated protein 43 expression. In vivo, results from ambulatory electrocardiography showed that IL-10 overexpression significantly inhibited the cardiac sympathetic nervous system activity and improved heart rate variability. Meanwhile, we observed decreased LSG function as well as prolonged ventricular effective refractory period and suppressed VA after myocardial infarction (MI) in the IL-10 group. In addition, IL-10 overexpression attenuated inflammation and decreased norepinephrine levels in the myocardium after acute MI. In conclusion, our data suggest that chronic IL-10 overexpression modulates cardiac sympathetic nerve remodeling and suppresses VA induced by MI. Neuromodulation through AAV-mediated IL-10 overexpression may have the characteristics of and advantages as a potential neuroimmunotherapy for preventing MI-induced VAs.

The potential role of CpG oligodeoxynucleotides on diabetic cardiac autonomic neuropathy mediated by P2Y12 receptor in rat stellate ganglia.

Cardiac autonomic neuropathy has a high prevalence in type 2 diabetes, which increases the risk of cardiovascular system disorders. CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) ligand, has been shown to have cardioprotection and cellular protection. Our previous work showed that P2Y12 in stellate ganglia (SG) is involved in the process of diabetic cardiac autonomic neuropathy (DCAN). Here, we aim to investigate whether CpG-ODN 1826 plays a protective role in DCAN and whether this beneficial protection involves regulation of the P2Y12-mediated cardiac sympathetic injury. Our results revealed that CpG-ODN 1826 activated TLR9 receptor, improved the abnormal blood pressure (BP), heart rate (HR), heart rate variability (HRV) and sympathetic nerve discharge (SND) activity in diabetic rats and reduced the up-regulated NF-κB, P2Y12 receptor, TNF-α and IL-1ß in SG. Meanwhile, CpG-ODN 1826 significantly decreased the elevated ATP, nuclear receptor coactivator 4 (NCOA4), iron, ROS and MDA levels and increased GPX4 and GSH levels. In addition, CpG-ODN 1826 contributes to maintain normalization of mitochondrial structure in SG. Overall, CpG-ODN 1826 alleviates the sympathetic excitation and abnormal neuron-glial signal communication via activating TLR9 receptors to achieve a balance of autonomic activity and relieve the DCAN in rats. The mechanism may involve the regulation of P2Y12 receptor in SG by reducing ATP release and NF-κB expression, which counteract neuroinflammation and ferroptosis mediated by activated P2Y12 in SG.

Tiered sympathetic control of cardiac function revealed by viral tracing and single cell transcriptome profiling.

The cell bodies of postganglionic sympathetic neurons innervating the heart primarily reside in the stellate ganglion (SG), alongside neurons innervating other organs and tissues. Whether cardiac-innervating stellate ganglionic neurons (SGNs) exhibit diversity and distinction from those innervating other tissues is not known. To identify and resolve the transcriptomic profiles of SGNs innervating the heart, we leveraged retrograde tracing techniques using adeno-associated virus (AAV) expressing fluorescent proteins (GFP or Td-tomato) with single cell RNA sequencing. We investigated electrophysiologic, morphologic, and physiologic roles for subsets of cardiac-specific neurons and found that three of five adrenergic SGN subtypes innervate the heart. These three subtypes stratify into two subpopulations; high (NA1a) and low (NA1b and NA1c) neuropeptide-Y (NPY) -expressing cells, exhibit distinct morphological, neurochemical, and electrophysiologic characteristics. In physiologic studies in transgenic mouse models modulating NPY signaling, we identified differential control of cardiac responses by these two subpopulations to high and low stress states. These findings provide novel insights into the unique properties of neurons responsible for cardiac sympathetic regulation, with implications for novel strategies to target specific neuronal subtypes for sympathetic blockade in cardiac disease.

Stellate ganglion block alleviates postoperative cognitive dysfunction via inhibiting TLR4/NF-κB signaling pathway.

Postoperative cognitive dysfunction (POCD) is common in aged patients after major surgery and is associated with increased risk of long-term morbidity and mortality. However, the underlying mechanism remains largely unknown and the clinical management of POCD is still controversial. Stellate ganglion block (SGB) is a clinical treatment for nerve injuries and circulatory issues. Recent evidence has identified the benefits of SGB in promoting learning and memory. We thus hypothesize that SGB could be effective in improving cognitive function after surgery. In present study, we established POCD model in aged rats via partial liver resection surgery. We found that the development of POCD was associated with the activation of toll-like receptor 4/nuclear factor kapa-B (TLR4/NF-κB) signaling pathway in the microglia in dorsal hippocampus, which induced the production of pro-inflammatory mediators (TNF-α, IL-1ß, IL-6) and promoted neuroinflammation. More importantly, we showed evidence that preoperative treatment with SGB could inhibit microglial activation, suppress TLR4/NF-κB-mediated neuroinflammation and effectively attenuate cognitive decline after the surgery. Our study suggested that SGB may serve as a novel treatment to prevent POCD in elderly patients. As SGB is safe procedure widely used in clinic, our findings can be easily translated into clinical practice and benefit more patients.

Stellate ganglion block suppresses hippocampal ferroptosis to attenuate cerebral ischemia-reperfusion injury via the Hippo pathway.

Ischemic stroke is a disabling and fatal disease caused by the insufficient blood supply to the brain. Stellate ganglion block (SGB) is a type of anesthesia commonly used to relieve pain. Here, we sought to identify the effects of SGB on cerebral ischemia-reperfusion (I/R) injury. The middle cerebral artery occlusion (MCAO) model was established in rats. The brain injury was assessed using the 2,3,5-triphenyl-tetrazolium-chloride (TTC) staining assay and neurological score. Ferroptosis was analyzed by detecting cell death, Fe2+ content, glutathione (GSH), malonic dialdehyde (MDA), superoxide dismutase (SOD), and ferroptosis-related factors. The mechanisms of SGB were assessed using the western blot. The results showed that I/R increased brain infarction and damaged neurological function. SGB decreased I/R-induced infarction and improved neurological function. Meantime, SGB inhibited ferroptosis of the hippocampus induced by I/R via the Hippo pathway. and the Yes1 associated transcriptional regulator (YAP) of this pathway was positively correlated with the ferroptosis-related solute carrier family 7 member 11 (SLC7A11). Inhibition of the Hippo pathway reversed the effects of SGB on brain injury and ferroptosis. In conclusion, SGB inhibited ferroptosis of hippocampal neurons via activating the Hippo pathway and thereby alleviated I/R injury. The data provide a novel insight into the treatment of ischemic stroke and even other ischemic encephalopathies.

Stellate ganglion block ameliorated central post-stroke pain with comorbid anxiety and depression through inhibiting HIF-1α/NLRP3 signaling following thalamic hemorrhagic stroke.

BACKGROUND: Central post-stroke pain (CPSP) is an intractable and disabling central neuropathic pain that severely affects patients' lives, well-being, and socialization abilities. However, CPSP has been poorly studied mechanistically and its treatment remains challenging. Here, we used a rat model of CPSP induced by thalamic hemorrhage to investigate its underlying mechanisms and the effect of stellate ganglion block (SGB) on CPSP and emotional comorbidities. METHODS: Thalamic hemorrhage was produced by injecting collagenase IV into the ventral-posterolateral nucleus (VPL) of the right thalamus. The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Behavioral tests were carried out to examine depressive and anxiety-like behaviors including the open field test (OFT), elevated plus maze test (EPMT), novelty-suppressed feeding test (NSFT), and forced swim test (FST). The peri-thalamic lesion tissues were collected for immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA). Genetic knockdown of thalamic hypoxia-inducible factor-1α (HIF-1α) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) with microinjection of HIF-1α siRNA and NLRP3 siRNA into the VPL of thalamus were performed 3 days before collagenase injection into the same regions. Microinjection of lificiguat (YC-1) and MCC950 into the VPL of thalamus were administrated 30 min before the collagenase injection in order to inhibited HIF-1α and NLRP3 pharmacologically. Repetitive right SGB was performed daily for 5 days and laser speckle contrast imaging (LSCI) was conducted to examine cerebral blood flow. RESULTS: Thalamic hemorrhage caused persistent mechanical allodynia and anxiety- and depression-like behaviors. Accompanying the persistent mechanical allodynia, the expression of HIF-1α and NLRP3, as well as the activities of microglia and astrocytes in the peri-thalamic lesion sites, were significantly increased. Genetic knockdown of thalamic HIF-1α and NLRP3 significantly attenuated mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage. Further studies revealed that intra-thalamic injection of YC-1, or MCC950 significantly suppressed the activation of microglia and astrocytes, the release of pro-inflammatory cytokines, the upregulation of malondialdehyde (MDA), and the downregulation of superoxide dismutase (SOD), as well as mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage. In addition, repetitive ipsilateral SGB significantly restored the upregulated HIF-1α/NLRP3 signaling and the hyperactivated microglia and astrocytes following thalamic hemorrhage. The enhanced expression of pro-inflammatory cytokines and the oxidative stress in the peri-thalamic lesion sites were also reversed by SGB. Moreover, LSCI showed that repetitive SGB significantly increased cerebral blood flow following thalamic hemorrhage. Most strikingly, SGB not only prevented, but also reversed the development of mechanical allodynia and anxiety- and depression-like behaviors induced by thalamic hemorrhage. However, pharmacological activation of thalamic HIF-1α and NLRP3 with specific agonists significantly eliminated the therapeutic effects of SGB on mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage. CONCLUSION: This study demonstrated for the first time that SGB could improve CPSP with comorbid anxiety and depression by increasing cerebral blood flow and inhibiting HIF-1α/NLRP3 inflammatory signaling.

Imperatorin Improves Obesity-Induced Cardiac Sympathetic Nerve Injury Mediated by P2X4 Receptor in Stellate Sympathetic Ganglion.

Obesity can activate the inflammatory signal pathway, induce in the body a state of chronic inflammation, and increase the excitability of the sympathetic nervous system, which may induce sympathetic neuropathic injury. The stellate sympathetic ganglia (SG) can express the P2X4 receptor, and the abnormal expression of the P2X4 receptor is related to inflammation. Imperatorin (IMP) is a kind of furan coumarin plant which has anti-inflammatory effects. This project aimed to investigate whether IMP can affect the expression of P2X4 receptors in the SG of obese rats to display a protective effect from high-fat-triggered cardiac sympathetic neuropathic injury. Molecular docking through homology modelling revealed that IMP had good affinity for the P2X4 receptor. Our results showed that compared with the normal group, the administration of IMP or P2X4 shRNA decreased sympathetic excitement; reduced the serum levels of triglyceride, total cholesterol, and lactate dehydrogenase; downregulated the expression of P2X4 receptors in SG; and inhibited the expression of inflammatory factors in the SG and serum of obese rats significantly. In addition, the expression of factors associated with the cell pyroptosis GSDMD, caspase-1, NLRP-3, and IL-18 in obese rats were significantly higher than those of the normal rats, and such effects were decreased after treatment with IMP or P2X4 shRNA. Furthermore, IMP significantly reduced the ATP-activated currents in HEK293 cells transfected with P2X4 receptor. Thus, the P2X4 receptor may be a key target for the treatment of obesity-induced cardiac sympathetic excitement. IMP can improve obesity-induced cardiac sympathetic excitement, and its mechanism of action may be related to the inhibition of P2X4 receptor expression and activity in the SG, suppression of cellular pyroptosis in the SG, and reduction of inflammatory factor levels.

Stellate ganglion block relieves acute lung injury induced by severe acute pancreatitis via the miR-155-5p/SOCS5/JAK2/STAT3 axis.

Acute lung injury (ALI), a prevalent complication of severe acute pancreatitis (SAP), is also a leading contributor to respiratory failure and even death of SAP patients. Here, we intended to investigate the function and mechanism of stellate ganglion block (SGB) in ameliorating SAP-induced ALI (SAP-ALI). We engineered an SAP-ALI model in rats and treated them with SGB. HE staining and the dry and wet method were implemented to evaluate pathological alterations in the tissues and pulmonary edema. The rats serum changes of the profiles of TNF-α, IL-6, IL-1ß, and IL-10 were examined. The profiles of miR-155-5p and SOCS5/JAK2/STAT3 were detected. Functional assays were performed for confirming the role of miR-155-5p in modulating the SOCS5/JAK2/STAT3 pathway in pulmonary epithelial cells. Our findings revealed that SGB vigorously alleviated SAP rat lung tissue damage and lung edema and lessened the generation of pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß. SGB enhanced SOCS5 expression, hampered miR-155-5p, and suppressed JAK2/STAT3 pathway activation. As evidenced by mechanism studies, miR-155-5p targeted the 3'UTR of SOCS5 and repressed its expression, hence resulting in JAK2/STAT3 pathway activation. During animal trials, we discovered that SGB ameliorated SAP-ALI, boosted SOCS5 expression, and mitigated the levels of pro-inflammatory factors and miR-155-5p in the plasma. In vitro, miR-155-5p overexpression substantially facilitated pulmonary epithelial cell apoptosis, inflammation, and JAK2/STAT3 pathway activation and restrained SOCS5 expression. All in all, our work hinted that SGB could modulate the miR-155-5p/SOCS5/JAK2/STAT3 axis to alleviate SAP-ALI.

Stellate Ganglion Block Improves Postoperative Cognitive Dysfunction in aged rats by SIRT1-mediated White Matter Lesion Repair.

Postoperative cognitive dysfunction (POCD) is a common complication of the central nervous system after surgery, especially in elderly patients. Many factors can influence POCD, one of which is white matter lesion. Nowadays, stellate ganglion block (SGB) is considered as an effective intervention for postoperative cognitive dysfunction and SIRT1 may play a role in that, but the exact mechanism remains unclear. Therefore, the underlying mechanisms that SGB improves postoperative cognitive dysfunction through SIRT1 in aged rats and its association with white matter lesion are yet to be elucidated. The role of SIRT1 in the process that stellate ganglion block improves the cognitive impairment, and its association with white matter lesion was investigated using splenectomy-induced POCD model. To investigate this result further, we performed transection of the cervical sympathetic trunk on the basis of POCD model, and the role of SIRT1 was then verified again by intraperitoneal injection of EX527 (5 mg/kg) five min before surgery. Data show that SGB treatment has neuroprotective effects in POCD rats. SGB treatment can ameliorate cognitive impairment, neuroinflammation and neuronal apoptosis in white matter. Moreover, SGB treatment enhanced the expression of SIRT1 in the hippocampus and white matter, decreased NF-κB activity in the hippocampus and white matter. It also increased the levels of inflammatory factor in serum and white matter, primarily at the level of anti-inflammatory factor. These findings indicated that SIRT1-mediate white matter repair could be a new therapeutic target for neurodegenerative illnesses.

Macrophage depletion in stellate ganglia alleviates cardiac sympathetic overactivation and ventricular arrhythmogenesis by attenuating neuroinflammation in heart failure.

Cardiac sympathetic overactivation is involved in arrhythmogenesis in patients with chronic heart failure (CHF). Inflammatory infiltration in the stellate ganglion (SG) is a critical factor for cardiac sympathoexcitation in patients with ventricular arrhythmias. This study aims to investigate if macrophage depletion in SGs decreases cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. Surgical ligation of the coronary artery was used for induction of CHF. Clodronate liposomes were microinjected into bilateral SGs of CHF rats for macrophage depletion. Using cytokine array, immunofluorescence staining, and Western blot analysis, we found that macrophage expansion and expression of TNFα and IL-1ß in SGs were markedly increased in CHF rats. Flow cytometry data confirmed that the percentage of macrophages in SGs was higher in CHF rats than that in sham rats. Clodronate liposomes significantly reduced CHF-elevated proinflammatory cytokine levels and macrophage expansion in SGs. Clodronate liposomes also reduced CHF-increased N-type Ca2+ currents and excitability of cardiac sympathetic postganglionic neurons and inhibited CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-h, continuous telemetry recording in conscious rats demonstrated that clodronate liposomes not only restored CHF-induced heterogeneity of ventricular electrical activities, but also decreased the incidence and duration of ventricular tachycardia/fibrillation in CHF. Macrophage depletion with clodronate liposomes attenuated CHF-induced cardiac sympathetic overactivation and ventricular arrhythmias through reduction of macrophage expansion and neuroinflammation in SGs.

Inhibition of N-type calcium channels in cardiac sympathetic neurons attenuates ventricular arrhythmogenesis in heart failure.

AIMS: Cardiac sympathetic overactivation is an important trigger of ventricular arrhythmias in patients with chronic heart failure (CHF). Our previous study demonstrated that N-type calcium (Cav2.2) currents in cardiac sympathetic post-ganglionic (CSP) neurons were increased in CHF. This study investigated the contribution of Cav2.2 channels in cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. METHODS AND RESULTS: Rat CHF was induced by surgical ligation of the left coronary artery. Lentiviral Cav2.2-α shRNA or scrambled shRNA was transfected in vivo into stellate ganglia (SG) in CHF rats. Final experiments were performed at 14 weeks after coronary artery ligation. Real-time polymerase chain reaction and western blot data showed that in vivo transfection of Cav2.2-α shRNA reduced the expression of Cav2.2-α mRNA and protein in the SG in CHF rats. Cav2.2-α shRNA also reduced Cav2.2 currents and cell excitability of CSP neurons and attenuated cardiac sympathetic nerve activities (CSNA) in CHF rats. The power spectral analysis of heart rate variability (HRV) further revealed that transfection of Cav2.2-α shRNA in the SG normalized CHF-caused cardiac sympathetic overactivation in conscious rats. Twenty-four-hour continuous telemetry electrocardiogram recording revealed that this Cav2.2-α shRNA not only decreased incidence and duration of ventricular tachycardia/ventricular fibrillation but also improved CHF-induced heterogeneity of ventricular electrical activity in conscious CHF rats. Cav2.2-α shRNA also decreased susceptibility to ventricular arrhythmias in anaesthetized CHF rats. However, Cav2.2-α shRNA failed to improve CHF-induced cardiac contractile dysfunction. Scrambled shRNA did not affect Cav2.2 currents and cell excitability of CSP neurons, CSNA, HRV, and ventricular arrhythmogenesis in CHF rats. CONCLUSIONS: Overactivation of Cav2.2 channels in CSP neurons contributes to cardiac sympathetic hyperactivation and ventricular arrhythmogenesis in CHF. This suggests that discovering purely selective and potent small-molecule Cav2.2 channel blockers could be a potential therapeutic strategy to decrease fatal ventricular arrhythmias in CHF.

Glucokinase in stellate ganglia cooperates with P2X3 receptor to develop cardiac sympathetic neuropathy in type 2 diabetes rats.

Glucokinase (GCK) may be involved in inflammatory pathological changes, while the P2X3 receptor in the stellate ganglia (SG) is related to diabetic cardiac autonomic neuropathy. In this study, we explored the relationship between the upregulated GCK in SG and diabetic cardiac sympathy. The expression and location of GCK and P2X3 in SG of type 2 diabetes mellitus (T2DM) rats were assessed. Changes in cardiac function were determined by measuring blood pressure, sympathetic nerve activity, heart rate, and heart rate variability. P2X3 agonist-activated currents in isolated stellate ganglion neurons and cultured human embryonic kidney 293 (HEK293) cells were recorded using whole-cell patch clamp techniques. The upregulated expression of GCK in SG of T2DM rats was decreased after treatment with GCK short hairpin RNA (shRNA). GCK shRNA treatment also improved the blood pressure, sympathetic nerve activity, heart rate, and heart rate variability in T2DM rats. By contrast, the expression of P2X3 and tumor necrosis factor α (TNF-α) was lessened by GCK shRNA treatment. In addition, adenosine triphosphate (ATP)-activated currents in stellate ganglion neurons and HEK293 cells co-transfected with GCK and P2X3 receptor plasmids were reduced after GCK shRNA treatment. In T2DM rats, knockdown of GCK relieved the diabetic cardiac sympathy mediated by P2X3 receptor-involved upregulation of GCK in SG.

Identification of NPB, NPW and Their Receptor in the Rat Heart.

Members of neuropeptide B/W signaling system have been predominantly detected and mapped within the CNS. In the rat, this system includes neuropeptide B (NPB), neuropeptide W (NPW) and their specific receptor NPBWR1. This signaling system has a wide spectrum of functions including a role in modulation of inflammatory pain and neuroendocrine functions. Expression of NPB, NPW and NPBWR1 in separate heart compartments, dorsal root ganglia (DRG) and stellate ganglia was proven by RT-qPCR, Western blot (WB) and immunofluorescence. Presence of mRNA for all tested genes was detected within all heart compartments and ganglia. The presence of proteins preproNPB, preproNPW and NPBWR1 was confirmed in all the chambers of heart by WB. Expression of preproNPW and preproNPB was proven in cardiac ganglionic cells obtained by laser capture microdissection. In immunofluorescence analysis, NPB immunoreactivity was detected in nerve fibers, some nerve cell bodies and smooth muscle within heart and both ganglia. NPW immunoreactivity was present in the nerve cell bodies and nerve fibers of heart ganglia. Weak nonhomogenous staining of cardiomyocytes was present within heart ventricles. NPBWR1 immunoreactivity was detected on cardiomyocytes and some nerve fibers. We confirmed the presence of NPB/W signaling system in heart, DRG and stellate ganglia by proteomic and genomic analyses.

Location, Dissection, and Analysis of the Murine Stellate Ganglion.

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG) - bilateral star-shaped structures of the sympathetic chain - are an important component of the sympathetic infrastructure. The SG are a recognized target for treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Angiotensin peptide synthesis and cyclic nucleotide modulation in sympathetic stellate ganglia.

Chronically elevated angiotensin II is a widely-established contributor to hypertension and heart failure via its action on the kidneys and vasculature. It also augments the activity of peripheral sympathetic nerves through activation of presynaptic angiotensin II receptors, thus contributing to sympathetic over-activity. Although some cells can synthesise angiotensin II locally, it is not known if this machinery is present in neurons closely coupled to the heart. Using a combination of RNA sequencing and quantitative real-time polymerase chain reaction, we demonstrate evidence for a renin-angiotensin synthesis pathway within human and rat sympathetic stellate ganglia, where significant alterations were observed in the spontaneously hypertensive rat stellate ganglia compared with Wistar stellates. We also used Förster Resonance Energy Transfer to demonstrate that administration of angiotensin II and angiotensin 1-7 peptides significantly elevate cyclic guanosine monophosphate in the rat stellate ganglia. Whether the release of angiotensin peptides from the sympathetic stellate ganglia alters neurotransmission and/or exacerbates cardiac dysfunction in states associated with sympathetic over activity remains to be established.

Cardiac sympathectomy and spinal cord stimulation attenuate reflex-mediated norepinephrine release during ischemia preventing ventricular fibrillation.

The purpose of this study was to define the mechanism by which cardiac neuraxial decentralization or spinal cord stimulation (SCS) reduces ischemia-induced ventricular fibrillation (VF). Direct measurements of norepinephrine (NE) levels in the left ventricular interstitial fluid (ISF) by microdialysis, in response to transient (15-minute) coronary artery occlusion (CAO), were performed in anesthetized canines. Responses were studied in animals with intact neuraxes and were compared with those in which the intrathoracic component of the cardiac neuraxes (stellate ganglia) or the intrinsic cardiac neuronal (ICN) system was surgically delinked from the central nervous system and those with intact neuraxes with preemptive SCS (T1-T3). With intact neuraxes, animals with exaggerated NE release due to CAO were at increased risk for VF. During CAO, there was a 152% increase in NE when the neuraxes were intact compared with 114% following stellate decentralization and 16% following ICN decentralization. During SCS, CAO NE levels increased by 59%. Risk for CAO-induced VF was 38% in controls, 8% following decentralization, and 11% following SCS. These data indicate that ischemia-related afferent neuronal transmission differentially engages central and intrathoracic sympathetic reflexes and amplifies sympathoexcitation. Differences in regional ventricular NE release are associated with increased risk for VF. Surgical decentralization or SCS reduced NE release and VF.

68Ga-DOTATATE Uptake by Cervicothoracic (Stellate) Ganglia.

Ga-DOTATATE PET/CT, a functional imaging modality for assessment of well-differentiated neuroendocrine tumors, targeting the somatostatin receptors, has essentially replaced the conventional gamma camera-based imaging with In-labeled octreotide. Physiologic distribution, normal variations, and common pitfalls with Ga-DOTATATE imaging have been well described in the literature. Here, we describe uptake of Ga-DOTATATE in 2 different patients at cervicothoracic junction within the stellate ganglia.

Interaction between Endothelin-1 and Left Stellate Ganglion Activation: A Potential Mechanism of Malignant Ventricular Arrhythmia during Myocardial Ischemia.

Endothelin-1 (ET-1) is synthesized primarily by endothelial cells. ET-1 administration in vivo enhances the cardiac sympathetic afferent reflex and sympathetic activity. Previous studies have shown that sympathetic hyperactivity promotes malignant ventricular arrhythmia (VA). The aim of this study was to investigate whether ET-1 could activate the left stellate ganglion (LSG) and promote malignant VA. Twelve male beagle dogs who received local microinjections of saline (control, n = 6) and ET-1 into the LSG (n = 6) were included. The ventricular effective refractory period (ERP), LSG function, and LSG activity were measured at different time points. VA was continuously recorded for 1 h after left anterior descending occlusion (LADO), and LSG tissues were then collected for molecular detection. Compared to that of the control group, local ET-1 microinjection significantly decreased the ERP and increased the occurrence of VA. In addition, local microinjection of ET-1 increased the function and activity of the LSG in the normal and ischemic hearts. The expression levels of proinflammatory cytokines and the protein expression of c-fos and nerve growth factor (NGF) in the LSG were also increased. More importantly, endothelin A receptor (ETA-R) expression was found in the LSG, and its signaling was significantly activated in the ET-1 group. LSG activation induced by local ET-1 microinjection aggravates LADO-induced VA. Activated ETA-R signaling and the upregulation of proinflammatory cytokines in the LSG may be responsible for these effects.

Sex differences in sympathetic gene expression and cardiac neurochemistry in Wistar Kyoto rats.

The stellate ganglia are the predominant source of sympathetic innervation to the heart. Remodeling of sympathetic nerves projecting to the heart has been observed in several cardiovascular diseases, and sympathetic dysfunction contributes to cardiac pathology. Wistar Kyoto rats are a common model for the study of cardiovascular diseases, but we lack a profile of the baseline transcriptomic and neurochemical characteristics of their cardiac sympathetic neurons. Most studies of cardiovascular disease have used male animals only, but in the future both male and female animals will be used for these types of studies; therefore, we sought to characterize the transcriptome of male and female stellate ganglia and to correlate that with catecholamine and acetylcholine content in the heart. We have generated a dataset of baseline RNA expression in male and female Wistar Kyoto rat stellate ganglia using RNA-seq, and have measured neurotransmitter levels in heart and stellate ganglia using HPLC and mass spectrometry. We identified numerous gene expression differences between male and female stellates, including genes encoding important developmental factors, receptors and neuropeptides. Female hearts had significantly higher neurotransmitter content than male hearts; however, no significant differences were detected in expression of the genes encoding neurotransmitter synthetic enzymes. Similarly, no statistically significant differences were identified between the sexes in cardiac tyrosine hydroxylase levels.