Sympathetic Neurotransmission Research Articles

Abstract 037: Neuromodulation Of Adipocyte Activity In Perivascular Adipose Tissue In Healthy Mice

Perivascular adipose tissue (PVAT) has emerged as a regulator of blood pressure by releasing anticontractile factors, which may be disrupted in disease. The production of adipocyte anticontractile factors is driven by norepinephrine (NE), a sympathetic neurotransmitter; however, the source of NE remains unknown. Here, we tested the hypothesis that neuronal innervation of PVAT stimulates adipocytes via NE. Mesenteric (mPVAT), aortic (aPVAT), and subcutaneous fat (sc) depots were studied in healthy male and female mice aged 8-12 weeks. Immunostaining with neuronal and vasculature markers was performed to assess anatomical evidence of neural innervation (n=3 region/sex). Adipocyte activity was studied in Adiponectin Cre;GCaMP 5g-tdT mice, which express the calcium indicator GCaMP5g in adipocytes (125-350 cells/group). Adipocytes were challenged with NE, a parasympathomimetic (bethanechol, 10uM, bath application), and nerve fibers were activated by electrical field stimulation (EFS; +100V, 0.1ms, 10 Hz). Immunolabeling data show that nerve fibers run adjacent to the larger blood vessels within adipose tissue, rather than radiating throughout. Stimulation with NE induced large responses (female: 0.91±0.73, 1.15±0.73, 1.45±0.9 dF/F 0 ; male: 0.75±0.57, 1.01±0.78, 0.73±0.53 dF/F 0 in aPVAT, mPVAT, sc, respectively). Both the parasympathomimetic and EFS induced minimal adipocyte responses compared to NE (% NE = [dF/F 0 bethanechol or EFS / dF/F 0 NE]*100%; bethanechol - female: 39.5%, 13.9%, 9.7%, male: 24.0%, 14.6%, 15.1% in aPVAT, mPVAT, sc, respectively; EFS - female: 31%, 12.2%, 9.7%, male: 28%, 13.9%, 13.7% in aPVAT, mPVAT, sc, respectively). These findings suggest that neural innervation within the PVAT, and functional regulation of adipocytes, is limited. Nerve fibers present within the PVAT are not the primary source of NE driving adipocyte production of anticontractile factors in the PVAT. Rather, this NE originates from other mechanisms including local production by non-neuronal cells or circulation of neurotransmitters.

Neuropeptide Y potentiates T-Lymphocyte inflammation after psychological trauma

Post-traumatic stress disorder (PTSD) is a psychiatric disorder characterized by severe behavioral alterations, but also demonstrates elevated levels of systemic inflammation, sympathoexcitation, and a potentiated risk for autoimmune and cardiovascular diseases. These physiological changes suggest perturbations to the immune system, however, the signaling mechanisms linking psychological trauma to the immune system are not well understood. Utilizing a pre-clinical mouse model of PTSD known as repeated social defeat stress (RSDS), we previously demonstrated that psychological trauma causes robust T-lymphocyte-driven inflammation in the spleen as evidenced by increased pro-inflammatory cytokine production (primarily interleukin 17A, IL-17A), inflammatory polarization, and enhanced mitochondrial reactive oxygen species. Moreover, targeted denervation of the spleen was able to completely ablate this T-lymphocyte inflammatory response after psychological trauma, suggesting the signal was autonomic and neural in nature. Given that the splenic nerve possesses only sympathetic neurons (no direct parasympathetic innervation), we hypothesized that psychological trauma mediates splenic T-lymphocyte inflammation via enhanced release of sympathetic neurotransmitters. To test this, we first exposed naïve primary T-lymphocytes to various sympathetic catecholamines, including dopamine, norepinephrine, and epinephrine. Contrary to our original hypothesis, we observed no significant changes in IL-17A expression or mitochondria reactive oxygen species. Next, we assessed the physiological response of naïve T-lymphocytes exposed to neuropeptide Y (NPY) given its role in both sympathoexcitation as well as in PTSD. Interestingly, naïve T-lymphocytes treated with NPY also demonstrated no effect, but when NPY was given in combination of norepinephrine, we observed both an increase in IL-17A gene expression (~2 fold) and intracellular redox (~2 fold). To further investigate this effect, we examined NPY levels in our RSDS mouse model over-time. Circulating NPY levels spiked immediately after only one day of RSDS (~4.5 fold, p=0.0317) but steadily declined over the remaining 10 day psychological trauma induction. Intriguingly, NPY levels were strongly correlated with both circulating IL-17A and T-lymphocyte mitochondrial reactive oxygen species levels after RSDS, supporting our in vitro observations. Overall, these data suggest NPY may be playing a critical role in shaping the inflammatory milieu after psychological trauma, which may enhance the likelihood of inflammatory comorbidities. NIH R01HL158521 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Local analgesia of electroacupuncture is mediated by the recruitment of neutrophils and released β-endorphins.

The efficacy of acupuncture in treating pain diseases has been recognized in clinical practice, and its mechanism of action has been a hot topic in academic acupuncture research. Previous basic research on acupuncture analgesia has focused mostly on the nervous system, with few studies addressing the immune system as a potential pathway of acupuncture analgesia. In this study, we investigated the effect of electroacupuncture (EA) on the β-endorphins (β-END) content, END-containing leukocyte type and number, sympathetic neurotransmitter norepinephrine (NE), and chemokine gene expression in inflamed tissues. To induce inflammatory pain, about 200 µL of complete Frester adjuvant (CFA) was injected into the unilateral medial femoral muscle of adult Wistar rats. Electroacupuncture treatment was performed for 3 days beginning on day 4 after CFA injection, with parameters of 2/100 Hz, 2 mA, and 30 minutes per treatment. The weight-bearing experiment and enzyme-linked immunosorbent assay showed that EA treatment significantly relieved spontaneous pain-like behaviors and increased the level of β-END in inflamed tissue. Injection of anti-END antibody in inflamed tissue blocked this analgesic effect. Flow cytometry and immunofluorescence staining revealed that the EA-induced increase in β-END was derived from opioid-containing ICAM-1 + /CD11b + immune cells in inflamed tissue. In addition, EA treatment increased the NE content and expression of β2 adrenergic receptor (ADR-β2) in inflammatory tissues and upregulated Cxcl1 and Cxcl6 gene expression levels. These findings provide new evidence for the peripheral analgesic effect of acupuncture treatment by recruiting β-END-containing ICAM-1 + /CD11b + immune cells and increasing the β-END content at the site of inflammation.

Activation of β2-adrenergic Receptor Alleviates Collagen-induced Arthritis by Ameliorating Th17/Treg Imbalance.

Recent research in our laboratory shows that CD4+ T cells express the β2 adrenergic receptor (β2-AR), and the sympathetic neurotransmitter norepinephrine regulates the function of T cells via β2-AR signaling. However, the immunoregulatory effect of β2-AR and its related mechanisms on rheumatoid arthritis is unknown. To explore the effects of β2-AR in collagen-induced arthritis (CIA) on the imbalance of T helper (Th) 17/ regulatory T (Treg) cells. In DBA1/J mice, collagen type II was injected intradermally at the tail base to prepare the CIA model. The specific β2-AR agonist, terbutaline (TBL), was administered intraperitoneally beginning on day 31 and continuing until day 47 after primary vaccination, twice a day. Magnetic beads were used to sort CD3+ T cells subsets from spleen tissues. In vivo, β2-AR agonist TBL alleviated arthritis symptoms in the CIA mice including histopathology of the ankle joints, four limbs' arthritis score, the thickness of ankle joints, and rear paws. After TBL treatment, in the ankle joints, the levels of proinflammatory factors (IL-17/22) notably decreased and the levels of immunosuppressive factors (IL-10/TGF-β) significantly increased. In vitro, ROR-γt protein expression, Th17 cell number, mRNA expression and the releasing of IL-17/22 from CD3+ T cells reduced following TBL administration. Moreover, TBL enhanced the anti-inflammatory responses of Treg cells. These results suggest that β2-AR activation exerts anti-inflammatory effects through the amelioration of Th17/Treg imbalance in the CIA disease.

Components of the sympathetic nervous system as targets to modulate inflammation – rheumatoid arthritis synovial fibroblasts as neuron-like cells?

BackgroundCatecholamines are major neurotransmitters of the sympathetic nervous system (SNS) and they are of pivotal importance in regulating numerous physiological and pathological processes. Rheumatoid arthritis (RA) is influenced by the activity of the SNS and its neurotransmitters norepinephrine (NE) and dopamine (DA) and early sympathectomy alleviates experimental arthritis in mice. In contrast, late sympathectomy aggravates RA, since this procedure eliminates anti-inflammatory, tyrosine hydroxylase (TH) positive cells that appear in the course of RA. While it has been shown that B cells can take up, degrade and synthesize catecholamines it is still unclear whether this also applies to synovial fibroblasts, a mesenchymal cell that is actively engaged in propagating inflammation and cartilage destruction in RA. Therefore, this study aims to present a detailed description of the catecholamine pathway and its influence on human RA synovial fibroblasts (RASFs).ResultsRASFs express all catecholamine-related targets including the synthesizing enzymes TH, DOPA decarboxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase. Furthermore, vesicular monoamine transporters 1/2 (VMAT1/2), dopamine transporter (DAT) and norepinephrine transporter (NET) were detected. RASFs are also able to degrade catecholamines as they express monoaminoxidase A and B (MAO-A/MAO-B) and catechol-O-methyltransferase (COMT). TNF upregulated VMAT2, MAO-B and NET levels in RASFs. DA, NE and epinephrine (EPI) were produced by RASFs and extracellular levels were augmented by either MAO, COMT, VMAT or DAT/NET inhibition but also by tumor necrosis factor (TNF) stimulation. While exogenous DA decreased interleukin-6 (IL-6) production and cell viability at the highest concentration (100 μM), NE above 1 μM increased IL-6 levels with a concomitant decrease in cell viability. MAO-A and MAO-B inhibition had differential effects on unstimulated and TNF treated RASFs. The MAO-A inhibitor clorgyline fostered IL-6 production in unstimulated but not TNF stimulated RASFs (10 nM-1 μM) while reducing IL-6 at 100 μM with a dose-dependent decrease in cell viability in both groups. The MAO-B inhibitor lazabemide hydrochloride did only modestly decrease cell viability at 100 μM while enhancing IL-6 production in unstimulated RASFs and decreasing IL-6 in TNF stimulated cells.ConclusionsRASFs possess a complete and functional catecholamine machinery whose function is altered under inflammatory conditions. Results from this study shed further light on the involvement of sympathetic neurotransmitters in RA pathology and might open therapeutic avenues to counteract inflammation with the MAO enzymes being key candidates.

Overactivation of Norepinephrine-β2-Adrenergic Receptor Axis Promotes Corneal Neovascularization.

To investigate the role of the sympathetic nervous system in corneal neovascularization (CNV) and to identify the downstream pathway involved in this regulation. Three types of CNV models were constructed with C57BL/6J mice, including the alkali burn model, suture model, and basic fibroblast growth factor (bFGF) corneal micropocket model. Subconjunctival injection of the sympathetic neurotransmitter norepinephrine (NE) was administered in these three models. Control mice received injections of water of the same volume. The corneal CNV was detected using slit-lamp microscopy and immunostaining with CD31, and the results were quantified by ImageJ. The expression of β2-adrenergic receptor (β2-AR) was stained with mouse corneas and human umbilical vein endothelial cells (HUVECs). Furthermore, the anti-CNV effects of β2-AR antagonist ICI-118,551 (ICI) were examined with HUVEC tube formation assay and with a bFGF micropocket model. Additionally, partial β2-AR knockdown mice (Adrb2+/-) were used to establish the bFGF micropocket model, and the corneal CNV size was quantified based on the slit-lamp images and vessel staining. Sympathetic nerves invaded the cornea in the suture CNV model. The NE receptor β2-AR was highly expressed in corneal epithelium and blood vessels. The addition of NE significantly promoted corneal angiogenesis, whereas ICI effectively inhibited CNV invasion and HUVEC tube formation. Adrb2 knockdown significantly reduced the cornea area occupied by CNV. Our study found that sympathetic nerves grow into the cornea in conjunction with newly formed vessels. The addition of the sympathetic neurotransmitter NE and activation of its downstream receptor β2-AR promoted CNV. Targeting β2-AR could potentially be used as an anti-CNV strategy.

Electroacupuncture activated local sympathetic noradrenergic signaling to relieve synovitis and referred pain behaviors in knee osteoarthritis rats.

Recent research has focused on the local control of articular inflammation through neuronal stimulation to avoid the systemic side effects of conventional pharmacological therapies. Electroacupuncture (EA) has been proven to be useful for inflammation suppressing and pain reduction in knee osteoarthritis (KOA) patients, yet its mechanism remains unclear. In the present study, the KOA model was established using the intra-articular injection of sodium monoiodoacetate (MIA) (1 mg/50 μL) into the knee cavity. EA was delivered at the ipsilateral ST36-GB34 acupoints. Hind paw weight-bearing and withdrawl thresholds were measured. On day 9, the histology, dep enrichment proteins, cytokines contents, immune cell population of the synovial membrane of the affected limbs were measured using HE staining, Masson staining, DIA quantitative proteomic analysis, flow cytometry, immunofluorescence staining, ELISA, and Western Blot. The ultrastructure of the saphenous nerve of the affected limb was observed using transmission electron microscopy on the 14th day after modeling. The result demonstrated that EA intervention during the midterm phase of the articular inflammation alleviated inflammatory pain behaviors and cartilage damage, but not during the early phase. Mid-term EA suppressed the levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the synovium on day 9 after MIA by elevating the level of sympathetic neurotransmitters Norepinephrine (NE) in the synovium but not systemic NE or systemic adrenaline. Selective blocking of the sympathetic function (6-OHDA) and β2-adrenergic receptor (ICI 118,551) prevented the anti-inflammatory effects of EA. EA-induced increment of the NE in the synovium inhibited the CXCL1-CXCR2 dependent overexpression of IL-6 in the synovial macrophages in a β2-adrenergic receptor (AR)-mediated manner. These results revealed that EA activated sympathetic noradrenergic signaling to control local inflammation in KOA rats and contributed to the development of novel therapeutic neurostimulation strategies for inflammatory diseases.

Abstract PR018: Neuropeptide Y as a metastatic factor

Abstract Although neuropeptide Y (NPY) is known mainly as a sympathetic neurotransmitter, growing evidence indicates its role in tumor biology. Previous studies from our laboratory and other groups suggested associations of high expression of NPY and its Y5 receptor (Y5R) with invasive and metastatic phenotypes of various tumor types; yet the direct evidence for the metastatic activity was lacking. Our recent work began to fill this gap. Using Ewing sarcoma (ES) as a model of NPY-rich tumor, we identified the NPY/Y5R axis as a crucial pathway responsible for hypoxia-induced ES bone metastasis. This osseous dissemination was driven by hypoxia-dependent over-activation of the NPY/Y5R/RhoA pathway, which led to cytokinesis defects and formation of the aneuploid ES cell population with high propensity to bone metastasis. Here, we sought to determine the impact of the Y5R signaling on overall, hypoxia-independent metastatic capabilities of ES cells. To this end, a doxycycline-inducible CRIPSR/Cas9 system was used to knockout Y5R in ES orthotopic xenografts and test its impact on metastasis and cell migration assessed by a transwell assay. The frequency of the NPY5R gene modifications was determined by sequencing and confirmed by immunohistochemistry. FISH was used to identify NPY5R gene gains in ES xenografts. RhoA activity was measured by immunocytochemistry and pull-down assay. As expected, ES/Y5R-sgRNA primary tumors in doxycycline-treated mice consisted of a heterogeneous ES cell population with variable Y5R levels. In contrast, metastases developing from these xenografts were primarily initiated by ES clones with an intact NPY5R gene. The frequency of NPY5R gene modifications in metastases from the doxycycline-treated group was markedly reduced, as compared to the primary tumors. No metastases arising from ES clones lacking intact NPY5R gene were detected. Similarly, metastasis from wild type ES xenografts was associated with a selection of clones with the NPY5R gene gain that was present in a small percent of the parental cells. Subsequent in vitro assays implicated Y5R-dependent ES cell motility driven by the activation of a key cytoskeleton regulator, RhoA, as the mechanism underlying the metastatic effects of NPY. The above results, along with our previously published data, provide direct evidence for the crucial role of the NPY/Y5R axis in metastasis. We have shown that under basal conditions the NPY/Y5R/RhoA axis promotes tumor cell motility and overall cancer cell dissemination, while over-activation of this pathway under hypoxic conditions leads to genomic instability and bone metastasis. While our study focused on ES due to its high endogenous expression of NPY and its receptors, our findings may be relevant to other malignancies rich in NPY and Y5R, such as neuroblastoma, breast and prostate cancer. Given availability of the FDA approved Y5R antagonist, NPY/Y5R pathway may be a promising target for therapies preventing cancer progression. Yet, further research is required to establish effectiveness of such pharmacological interventions. Citation Format: Mina Adnani, Sung-Hyeok Hong, Susana Galli, Akanksha Mahajan, Nouran Abualsaud, Lindsay Caprio, Jason U. Tilan, Olga Rodriguez, Hingkun Wang, Christopher Albanese, Luciane R. Cavalli, Joanna Kitlinska. Neuropeptide Y as a metastatic factor [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR018.

Norepinephrine/β2-Adrenergic Receptor Pathway Promotes the Cell Proliferation and Nerve Growth Factor Production in Triple-Negative Breast Cancer.

Invasive ductal carcinoma (IDC) accounts for 90% of triple-negative breast cancer (TNBC). IDC is mainly derived from the breast ductal epithelium which is innervated by the 4th to 6th thoracic sympathetic nerves. However, little is known about the contribution of the interactions between sympathetic nerves and breast cancer cells to the malignant progression of TNBC. The expression levels of the β2-adrenergic receptor (β2-AR, encoded by ADRB2 gene), nerve growth factor (NGF), and tropomyosin receptor kinase A (TrkA) were determined using immunohistochemistry (IHC). NGF expression levels in the serum were compared by enzyme-linked immunosorbent assay (ELISA). Cell proliferation was assessed using the Cell Counting Kit-8 assay. The β2-AR, NGF, p-ERK, and p-CERB expression levels were determined using western blotting. TNBC cells and neuronal cells of the dorsal root ganglion (DRG) in 2-day-old Sprague Dawley rats were co-cultured. Using norepinephrine (NE), NGF, and β2-AR, NGF/TrkA blocker pretreatments, the axon growth of each group of DRG neuron cells was detected by immunofluorescence analysis. The sympathetic adrenergic neurotransmitter NE activated the ERK signaling pathway in TNBC cells. NE/β2-AR signaling promotes NGF secretion. NGF further facilitates the malignant progression of TNBC by increasing sympathetic neurogenesis. In the co-culture assay, the sympathetic adrenergic NE/β2-AR signal pathway also enhanced NGF secretion. NGF binds TrkA in DRG neurons and promotes axonal growth. These results suggest that NE/β2-AR pathway promotes cell proliferation and NGF production in triple-negative breast cancer.

Influence of the Peripheral Nervous System on Murine Osteoporotic Fracture Healing and Fracture-Induced Hyperalgesia.

Osteoporotic fractures are often linked to persisting chronic pain and poor healing outcomes. Substance P (SP), α-calcitonin gene-related peptide (α-CGRP) and sympathetic neurotransmitters are involved in bone remodeling after trauma and nociceptive processes, e.g., fracture-induced hyperalgesia. We aimed to link sensory and sympathetic signaling to fracture healing and fracture-induced hyperalgesia under osteoporotic conditions. Externally stabilized femoral fractures were set 28 days after OVX in wild type (WT), α-CGRP- deficient (α-CGRP -/-), SP-deficient (Tac1-/-) and sympathectomized (SYX) mice. Functional MRI (fMRI) was performed two days before and five and 21 days post fracture, followed by µCT and biomechanical tests. Sympathectomy affected structural bone properties in the fracture callus whereas loss of sensory neurotransmitters affected trabecular structures in contralateral, non-fractured bones. Biomechanical properties were mostly similar in all groups. Both nociceptive and resting-state (RS) fMRI revealed significant baseline differences in functional connectivity (FC) between WT and neurotransmitter-deficient mice. The fracture-induced hyperalgesia modulated central nociception and had robust impact on RS FC in all groups. The changes demonstrated in RS FC in fMRI might potentially be used as a bone traumata-induced biomarker regarding fracture healing under pathophysiological musculoskeletal conditions. The findings are of clinical importance and relevance as they advance our understanding of pain during osteoporotic fracture healing and provide a potential imaging biomarker for fracture-related hyperalgesia and its temporal development. Overall, this may help to reduce the development of chronic pain after fracture thereby improving the treatment of osteoporotic fractures.

Neuroprotective Effects of Neuropeptide Y on Human Neuroblastoma SH-SY5Y Cells in Glutamate Excitotoxicity and ER Stress Conditions.

Neuropeptide Y (NPY), a sympathetic neurotransmitter, is involved in various physiological functions, and its dysregulation is implicated in several neurodegenerative diseases. Glutamate excitotoxicity, endoplasmic reticulum (ER) stress, and oxidative stress are the common mechanisms associated with numerous neurodegenerative illnesses. The present study aimed to elucidate the protective effects of NPY against glutamate toxicity and tunicamycin-induced ER stress in the human neuroblastoma SH-SY5Y cell line. We exposed the SH-SY5Y cells to glutamate and tunicamycin for two different time points and analyzed the protective effects of NPY at different concentrations. The protective effects of NPY treatments were assessed by cell viability assay, and the signalling pathway changes were evaluated by biochemical techniques such as Western blotting and immunofluorescence assays. Our results showed that treatment of SH-SY5Y cells with NPY significantly increased the viability of the cells in both glutamate toxicity and ER stress conditions. NPY treatments significantly attenuated the glutamate-induced pro-apoptotic activation of ERK1/2 and JNK/BAD pathways. The protective effects of NPY were further evident against tunicamycin-induced ER stress. NPY treatments significantly suppressed the ER stress activation by downregulating BiP, phospho-eIF2α, and CHOP expression. In addition, NPY alleviated the Akt/FoxO3a pathway in acute oxidative conditions caused by glutamate and tunicamycin in SH-SY5Y cells. Our results demonstrated that NPY is neuroprotective against glutamate-induced cell toxicity and tunicamycin-induced ER stress through anti-apoptotic actions.

Adrenergic signaling controls early transcriptional programs during CD8+ T cell responses to viral infection.

Norepinephrine is a key sympathetic neurotransmitter, which acts to suppress CD8 + T cell cytokine secretion and lytic activity by signaling through the β2-adrenergic receptor (ADRB2). Although ADRB2 signaling is considered generally immunosuppressive, its role in regulating the differentiation of effector T cells in response to infection has not been investigated. Using an adoptive transfer approach, we compared the expansion and differentiation of wild type (WT) to Adrb2-/- CD8 + T cells throughout the primary response to vesicular stomatitis virus (VSV) infection in vivo. We measured the dynamic changes in transcriptome profiles of antigen-specific CD8 + T cells as they responded to VSV. Within the first 7 days of infection, WT cells out-paced the expansion of Adrb2-/- cells, which correlated with reduced expression of IL-2 and the IL-2Rα in the absence of ADRB2. RNASeq analysis identified over 300 differentially expressed genes that were both temporally regulated following infection and selectively regulated in WT vs Adrb2-/- cells. These genes contributed to major transcriptional pathways including cytokine receptor activation, signaling in cancer, immune deficiency, and neurotransmitter pathways. By parsing genes within groups that were either induced or repressed over time in response to infection, we identified three main branches of genes that were differentially regulated by the ADRB2. These gene sets were predicted to be regulated by specific transcription factors involved in effector T cell development, such as Tbx21 and Eomes. Collectively, these data demonstrate a significant role for ADRB2 signaling in regulating key transcriptional pathways during CD8 + T cells responses to infection that may dramatically impact their functional capabilities and downstream memory cell development.

Elevated Vascular Sympathetic Neurotransmission and Remodelling Is a Common Feature in a Rat Model of Foetal Programming of Hypertension and SHR.

Hypertension is of unknown aetiology, with sympathetic nervous system hyperactivation being one of the possible contributors. Hypertension may have a developmental origin, owing to the exposure to adverse factors during the intrauterine period. Our hypothesis is that sympathetic hyperinnervation may be implicated in hypertension of developmental origins, being this is a common feature with essential hypertension. Two-animal models were used: spontaneously hypertensive rats (SHR-model of essential hypertension) and offspring from dams exposed to undernutrition (MUN-model of developmental hypertension), with their respective controls. In adult males, we assessed systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), sympathetic nerve function (3H-tritium release), sympathetic innervation (immunohistochemistry) and vascular remodelling (histology). MUN showed higher SBP/DBP, but not HR, while SHR exhibited higher SBP/DBP/HR. Regarding the mesenteric arteries, MUN and SHR showed reduced lumen, increased media and adventitial thickness and increased wall/lumen and connective tissue compared to respective controls. Regarding sympathetic nerve activation, MUN and SHR showed higher tritium release compared to controls. Total tritium tissue/tyrosine hydroxylase detection was higher in SHR and MUN adventitia arteries compared to respective controls. In conclusion, sympathetic hyperinnervation may be one of the contributors to vascular remodelling and hypertension in rats exposed to undernutrition during intrauterine life, which is a common feature with spontaneous hypertension.

The Role of Epicardial Adipose Tissue in the Development of Atrial Fibrillation, Coronary Artery Disease and Chronic Heart Failure in the Context of Obesity and Type 2 Diabetes Mellitus: A Narrative Review

Cardiovascular diseases (CVDs) are a significant burden globally and are especially prevalent in obese and/or diabetic populations. Epicardial adipose tissue (EAT) surrounding the heart has been implicated in the development of CVDs as EAT can shift from a protective to a maladaptive phenotype in diseased states. In diabetic and obese patients, an elevated EAT mass both secretes pro-fibrotic/pro-inflammatory adipokines and forms intramyocardial fibrofatty infiltrates. This narrative review considers the proposed pathophysiological roles of EAT in CVDs. Diabetes is associated with a disordered energy utilization in the heart, which promotes intramyocardial fat and structural remodeling. Fibrofatty infiltrates are associated with abnormal cardiomyocyte calcium handling and repolarization, increasing the probability of afterdepolarizations. The inflammatory phenotype also promotes lateralization of connexin (Cx) proteins, undermining unidirectional conduction. These changes are associated with conduction heterogeneity, together creating a substrate for atrial fibrillation (AF). EAT is also strongly implicated in coronary artery disease (CAD); inflammatory adipokines from peri-vascular fat can modulate intra-luminal homeostasis through an “outside-to-inside” mechanism. EAT is also a significant source of sympathetic neurotransmitters, which promote progressive diastolic dysfunction with eventual cardiac failure. Further investigations on the behavior of EAT in diabetic/obese patients with CVD could help elucidate the pathogenesis and uncover potential therapeutic targets.

Electroacupuncture promotes apoptosis and inhibits axonogenesis by activating p75 neurotrophin receptor for triple-negative breast xenograft in mice

PurposeThe aim of this study was to investigate the anti-tumor effect of electroacupuncture (EA) on mice bearing breast tumors by regulating p75 neurotrophin receptor (p75NTR) and remodelling intratumoral innervation. Methods: Female BALB/c mice were implanted with 4T1 breast tumor cells to establish a murine mammary cancer model. Tumor volume and weight were measured to evaluate tumor growth. Cell apoptosis was assessed by TUNEL assay. The relative expression of p75NTR, TrkA, TrkB, NGF and proNGF were detected by immunohistochemistry. Neurotransmitter and neurotrophin were detected by enzyme-linked immunosorbent assay. Intratumoral innervation was confirmed by β3-tubulin and TH labeling immunohistochemistry. The antagonist TAT-Pep5 was employed to determine if the effects of EA on tumor growth and cell apoptosis were mediated by p75NTR. Results: Peritumoral EA alleviated tumor growth especially after 14 days of intervention. Apoptosis index in the tumor tissue was obviously decreased after EA. Meanwhile, EA intervention significantly upregulated the expression of p75NTR and proNGF, along with a decline in the tumor growth and an increase in the cell apoptosis. Besides, EA reduced local sympathetic innervation and downregulated sympathetic neurotransmitter NE level in the local tumor. Furthermore, p75NTR antagonist alleviated EA-mediated cell apoptosis and intratumoral innervation. Conclusions: One mechanism of EA intervention for alleviating tumor progression is mediated by p75NTR to promote apoptosis and decrease intratumoral axonogenesis in the tumor microenvironment.

Association between T-182C polymorphism of the norepinephrine transporter SLC6A2(NET) gene and major depressive disorder in Iraqi patients

Major sympathetic nervous system neurotransmitter that is responsible for consciousness, attention, cognitive ability, as well as the function of the majority of body systems, glands, and the immune system, is called Norepinephrine (NE). Norepinephrine transporter (NET) and norepinephrine binding sites are decreased in the brains of MDD patients, showing that dysregulated synaptic NE plays a role in the pathophysiology of MDD. Indeed, different NET polymorphism combinations may be linked to different MDD sub-phenotypes. The aim of the current work is to investigate or detect if there is a relation between T-182C gene polymorphism of SLC6A2 gene with major depressive disorder (MDD) in Iraqi patients.Materials and Methods: Blood specimens were collected from 70 individuals with MDD and 20 healthy individuals as a control group (both patients and control ages ranged between 18-65 years). DNA extracted from white blood cells (WBC), and the T-182C gene was detected and amplified by the PCR-ARMS technique. Results: The distribution of TT, TC, and CC genotype ratio of MDD group was 22.9%, 27.1%, and 50.0%, respectively. While the control group show genotype distribution as 35.0% for TT, 55.0% for TC and 10.0% for CC ratio.

The Mechanism of Cardiac Sympathetic Activity Assessment Methods: Current Knowledge.

This review has summarized the methods currently available for cardiac sympathetic assessment in clinical or under research, with emphasis on the principles behind these methodologies. Heart rate variability (HRV) and other methods based on heart rate pattern analysis can reflect the dominance of sympathetic nerve to sinoatrial node function and indirectly show the average activity level of cardiac sympathetic nerve in a period of time. Sympathetic neurotransmitters play a key role of signal transduction after sympathetic nerve discharges. Plasma or local sympathetic neurotransmitter detection can mediately display sympathetic nerve activity. Given cardiac sympathetic nerve innervation, i.e., the distribution of stellate ganglion and its nerve fibers, stellate ganglion activity can be recorded either directly or subcutaneously, or through the surface of the skin using a neurophysiological approach. Stellate ganglion nerve activity (SGNA), subcutaneous nerve activity (SCNA), and skin sympathetic nerve activity (SKNA) can reflect immediate stellate ganglion discharge activity, i.e., cardiac sympathetic nerve activity. These cardiac sympathetic activity assessment methods are all based on the anatomy and physiology of the heart, especially the sympathetic innervation and the sympathetic regulation of the heart. Technological advances, discipline overlapping, and more understanding of the sympathetic innervation and sympathetic regulation of the heart will promote the development of cardiac sympathetic activity assessment methods.

Effect of sarpogrelate treatment on 5-HT modulation of vascular sympathetic innervation and platelet activity in diabetic rats

This study aimed to investigate whether the 5-HT2 receptor blockade alters the 5-HT effect on vascular sympathetic neurotransmission and platelet activation in type 1 diabetes. 28-day diabetes was obtained by alloxan (150 mg/kg; s.c.) in male Wistar rats, administering sarpogrelate (5-HT2 blocker; 30 mg/kg/day; p.o.) for 14 days. Blood glucose and body weight were monitored for 28 days. After 4 weeks of diabetes induction, food and drink intake, urine, plasma-platelet 5-HT, and platelet activation were determined in normoglycemic, non-treated diabetic and sarpogrelate-treated diabetic rats. Another set of diabetic rats were pithed to run the vascular sympathetic stimulation or exogenous noradrenaline administration, examining the induced vasoconstrictor responses. Sarpogrelate treatment significantly reduced drink intake and urine, whereas BW gain, hyperglycemia, and food intake were not modified in diabetic rats. The platelet activation and plasma 5-HT concentration were decreased (increasing the stored 5-HT platelet) by 5-HT2 blockade in diabetic animals. The sympathetic-induced vasoconstrictions were higher in non-treated than in sarpogrelate-treated diabetic rats. 5-HT inhibited these vasopressor responses, reproduced exclusively by the 5-HT1/5/7 receptor agonist, 5-CT. The 5-CT-produced inhibition was partly reversed by 5-HT1D or 5-HT7 antagonists (LY310762 or SB-258719, respectively), and totally annulled by the mixture of LY310762+SB‐258719. Noradrenaline-caused vasoconstrictions were also decreased by 5-CT. In conclusion, our results reveal that 14-day sarpogrelate treatment improves polydipsia and polyuria, reduces platelet hyperactivation, plasma 5-HT and the vascular sympathetic tone, and changes 5-HT receptors inhibiting noradrenergic drive in diabetic rats: pre and/or postjunctional 5-HT1D/7 are involved in the sympatho-inhibition.

208-LB: Sensory Nerve Neuropeptide Calcitonin Gene–Related Peptide (CGRP) in Adipose Tissue Changes According to Metabolic Status

White adipose tissue (WAT) is the chief energy storage site in the body and is densely innervated by sensory and sympathetic nerves that enable bi-directional neural communication with the central nervous system. The sympathetic neurotransmitter norepinephrine is the best-studied nerve product in WAT, and is known to stimulate lipolysis, browning (development of brown adipocytes in WAT) , thermogenesis, and other metabolically favorable processes. Conversely, sensory nerves in WAT secrete neuropeptides such as calcitonin gene related peptide (CGRP) , a vasodilatory neuropeptide that also impacts lipolysis, but has been vastly understudied. Circulating CGRP levels are known to be increased in obese humans, but given the beneficial metabolic effects of CGRP this may be a case of CGRP receptor resistance, similar to the observed leptin and insulin resistance with obesity. Therefore, a more comprehensive investigation into CGRP’s roles in adipose tissue and the importance of the sensory nerve supply for WAT function and insulin sensitivity is warranted. Using intravital calcium imaging in WAT of mice, we have observed activation of tissue nerves after application of lipids such as 13-HODE, which is an agonist of sensory nerves and leads to CGRP release. Thirty minutes after 13-HODE administration to WAT there was a significant increase in CGRP levels in WAT (by tissue ELISA) , which were reduced at 120min post-treatment. This suggests local lipid levels in WAT may trigger release of this sensory neuropeptide as an interoceptive response. With obesity, in both diet-induced and genetic ob/ob mice, we see increased WAT CGRP, which fits with reports in humans and may be due to chronically elevated tissue lipids. Exercise also increases tissue CGRP levels, perhaps due to an acute need for lipid fuels in this negative energy balance state. Taken together, CGRP is a novel factor in adipose that may be relevant for changing energy balance status and likely contributes to metabolic health. Disclosure G. Mishra: None. J. Willows: None.

Modeling the Progression of Cardiac Catecholamine Deficiency in Lewy Body Diseases.

BackgroundLewy body diseases (LBDs) feature deficiency of the sympathetic neurotransmitter norepinephrine in the left ventricular myocardium and sympathetic intra‐neuronal deposition of the protein alpha‐synuclein (αS). LBDs therefore are autonomic synucleinopathies. Computational modeling has revealed multiple functional abnormalities in residual myocardial sympathetic noradrenergic nerves in LBDs, including decreased norepinephrine synthesis, vesicular storage, and recycling. We report an extended model that enables predictions about the progression of LBDs and effects of genetic predispositions and treatments on that progression.Methods and ResultsThe model combines cardiac sympathetic activation with autotoxicity mediated by the dopamine metabolite 3,4‐dihydroxyphenylacetaldehyde. We tested the model by its ability to predict longitudinal empirical data based on cardiac sympathetic neuroimaging, effects of genetic variations related to particular intra‐neuronal reactions, treatment by monoamine oxidase inhibition to decrease 3,4‐dihydroxyphenylacetaldehyde production, and post‐mortem myocardial tissue contents of catecholamines and αS. The new model generated a triphasic decline in myocardial norepinephrine content. This pattern was confirmed by empirical data from serial cardiac 18F‐dopamine positron emission tomographic scanning in patients with LBDs. The model also correctly predicted empirical data about effects of genetic variants and monoamine oxidase inhibition and about myocardial levels of catecholamines and αS.ConclusionsThe present computational model predicts a triphasic decline in myocardial norepinephrine content as LBDs progress. According to the model, disease‐modifying interventions begun at the transition from the first to the second phase delay the onset of symptomatic disease. Computational modeling coupled with biomarkers of preclinical autonomic synucleinopathy may enable early detection and more effective treatment of LBDs.