Data highlighting the effects of spinal segmental stimulation of preganglionic sympathetic neurons on the electrophysiology of the rabbit heart.

This article presents data highlighting the functional selectivity of cardiac preganglionic sympathetic neurons in the rabbit heart. Specifically, the data draw attention to the role of each spinal segmental outflow on cardiac electrophysiology and the influence of each segment on cardiac excitability through investigating markers of arrhythmia such as electrical restitution. This data holds importance for exploring whether the preganglionic sympathetic neurons have functionally distinct pathways to the heart and whether some spinal segmental outflows have a greater potential for arrhythmia generation than others. Discussion of the data can be found in Chauhan et al. (2018) [1].

Electrophysiological effects of nicotinic and electrical stimulation of intrinsic cardiac ganglia in the absence of extrinsic autonomic nerves in the rabbit heart.

BACKGROUND: The intrinsic cardiac nervous system is a rich network of cardiac nerves that converge to form distinct ganglia and extend across the heart and is capable of influencing cardiac function. OBJECTIVE: The goals of this study were to provide a complete picture of the neurotransmitter/neuromodulator profile of the rabbit intrinsic cardiac nervous system and to determine the influence of spatially divergent ganglia on cardiac electrophysiology. METHODS: Nicotinic or electrical stimulation was applied at discrete sites of the intrinsic cardiac nerve plexus in the Langendorff-perfused rabbit heart. Functional effects on sinus rate and atrioventricular conduction were measured. Immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase, and/or neuronal nitric oxide synthase (nNOS) was performed using whole mount preparations. RESULTS: Stimulation within all ganglia produced either bradycardia, tachycardia, or a biphasic brady-tachycardia. Electrical stimulation of the right atrial and right neuronal cluster regions produced the largest chronotropic responses. Significant prolongation of atrioventricular conduction was predominant at the pulmonary vein-caudal vein region. Neurons immunoreactive (IR) only for ChAT, tyrosine hydroxylase, or nNOS were consistently located within the limits of the hilum and at the roots of the right cranial and right pulmonary veins. ChAT-IR neurons were most abundant (1946 ± 668 neurons). Neurons IR only for nNOS were distributed within ganglia. CONCLUSION: Stimulation of intrinsic ganglia, shown to be of phenotypic complexity but predominantly of cholinergic nature, indicates that clusters of neurons are capable of independent selective effects on cardiac electrophysiology, therefore providing a potential therapeutic target for the prevention and treatment of cardiac disease.

Functional selectivity of cardiac preganglionic sympathetic neurones in the rabbit heart.

BACKGROUND: Studies have shown regional and functional selectivity of cardiac postganglionic neurones indicating there might exist a similar heterogeneity in spinal segmental preganglionic neurones, which requires further investigation. METHODS: Right and left sympathetic chains were electrically stimulated from T6 to T1 in the innervated isolated rabbit heart preparation (n = 18). Sinus rate, left ventricular pressure, retrograde ventriculo-atrial conduction, monophasic action potential duration, effective refractory period, ventricular fibrillation threshold and electrical restitution were measured. RESULTS: Right sympathetic stimulation had a greater influence on heart rate (T1-T2: right; 59.9 ±â€¯6.0%, left; 41.1 ±â€¯5.6% P < 0.001) and left stimulation had greater effects on left ventricular pressure (T1-T2: right; 20.7 ±â€¯3.2%, left; 40.3 ±â€¯5.4%, P < 0.01) and ventriculo-atrial conduction (T1-T2: right; -6.8 ±â€¯1.1%, left; -15.5 ±â€¯0.2%) at all levels, with greater effects at rostral levels (T1-T3). Left sympathetic stimulation caused shorter monophasic action potentials at the base (T4-T5: right; 119.3 ±â€¯2.7 ms, left; 114.7 ±â€¯2.5 ms. P < 0.05) and apex (T4-T5: right; 118.8 ±â€¯1.2 ms, left; 114.6 ±â€¯2.6 ms. P < 0.05), greater shortening of effective refractory period (T4-T5: right; -3.6 ±â€¯1.3%, left; -7.7 ±â€¯1.8%. P < 0.05), a steeper maximum slope of restitution (T4-T5 base: right; 1.3 ±â€¯0.2, left; 1.8 ±â€¯0.2. P < 0.01. T4-T5 apex: right; 1.0 ±â€¯0.2, left; 1.6 ±â€¯0.3. P < 0.05) and a greater decrease in ventricular fibrillation threshold (T4-T5: right; -22.3 ±â€¯6.8%, left;-39.0 ±â€¯1.7%), with dominant effects at caudal levels (T4-T6). CONCLUSIONS: The preganglionic sympathetic efferent axons show functionally distinct pathways to the heart. The caudal segments (T4-T6) of the left sympathetic chain had a greater potential for arrhythmia generation and hence could pose a target for more focused clinical treatments for impairments in cardiac function.

Involvement of Corticotropin-Releasing Factor and Receptors in Immune Cells in Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder defined by ROME IV criteria as pain in the lower abdominal region, which is associated with altered bowel habit or defecation. The underlying mechanism of IBS is not completely understood. IBS seems to be a product of interactions between various factors with genetics, dietary/intestinal microbiota, low-grade inflammation, and stress playing a key role in the pathogenesis of this disease. The crosstalk between the immune system and stress in IBS mechanism is increasingly recognized. Corticotropin-releasing factor (CRF), a major mediator in the stress response, is involved in altered function in GI, including inflammatory processes, colonic transit time, contractile activity, defecation pattern, pain threshold, mucosal secretory function, and barrier functions. This mini review focuses on the recently establish local GI-CRF system, its involvement in modulating the immune response in IBS, and summarizes current IBS animal models and mapping of CRF, CRFR1, and CRFR2 expression in colon tissues. CRF and receptors might be a key molecule involving the immune and movement function via brain-gut axis in IBS.

Central control of autonomic function.

In this review, current understanding of the control of autonomic function is outlined and its development over the last 50 years highlighted. Using the control of the cardiovascular system as the primary tool, the importance of the patterning of autonomic outflows is shown to be crucial in both homeostasis and behaviour. Technical advances have made it possible to obtain a clearer idea of how the central nervous system evolves patterns of autonomic discharge that optimise autonomic changes to support motor and behavioural responses. The specific roles of sympathetic and parasympathetic preganglionic neurones and premotor neurones are surveyed and the importance of their roles in integrating afferent inputs that result from peripheral sensory inputs and drive from multiple levels of the neuraxis is outlined. The autonomic control of the viscera, including the urinogenital organs and other organs is discussed briefly. The current ability to use animal models to monitor and modulate autonomic neural discharge and simultaneously co-relate this with end-organ activity is shown to have translational potential. There is every prospect that these studies will lead to the identification of new therapies for pathophysiological conditions.

Systemic pro-inflammatory response facilitates the development of cerebral edema during short hypoxia.

BACKGROUND: High-altitude cerebral edema (HACE) is the severe type of acute mountain sickness (AMS) and life threatening. A subclinical inflammation has been speculated, but the exact mechanisms underlying the HACE are not fully understood. METHODS: Human volunteers ascended to high altitude (3860 m, 2 days), and rats were exposed to hypoxia in a hypobaric chamber (5000 m, 2 days). Human acute mountain sickness was evaluated by the Lake Louise Score (LLS), and plasma corticotrophin-releasing hormone (CRH) and cytokines TNF-α, IL-1ß, and IL-6 were measured in rats and humans. Subsequently, rats were pre-treated with lipopolysaccharide (LPS, intraperitoneal (ip) 4 mg/kg, 11 h) to induce inflammation prior to 1 h hypoxia (7000 m elevation). TNF-α, IL-1ß, IL-6, nitric oxide (NO), CRH, and aquaporin-4 (AQP4) and their gene expression, Evans blue, Na(+)-K(+)-ATPase activity, p65 translocation, and cell swelling were measured in brain by ELISA, Western blotting, Q-PCR, RT-PCR, immunohistochemistry, and transmission electron micrography. MAPKs, NF-κB pathway, and water permeability of primary astrocytes were demonstrated. All measurements were performed with or without LPS challenge. The release of NO, TNF-α, and IL-6 in cultured primary microglia by CRH stimulation with or without PDTC (NF-κB inhibitor) or CP154,526 (CRHR1 antagonist) were measured. RESULTS: Hypobaric hypoxia enhanced plasma TNF-α, IL-1ß, and IL-6 and CRH levels in human and rats, which positively correlated with AMS. A single LPS injection (ip, 4 mg/kg, 12 h) into rats increased TNF-α and IL-1ß levels in the serum and cortex, and AQP4 and AQP4 mRNA expression in cortex and astrocytes, and astrocyte water permeability but did not cause brain edema. However, LPS treatment 11 h prior to 1 h hypoxia (elevation, 7000 m) challenge caused cerebral edema, which was associated with activation of NF-κB and MAPKs, hypoxia-reduced Na(+)-K(+)-ATPase activity and blood-brain barrier (BBB) disruption. Both LPS and CRH stimulated TNF-α, IL-6, and NO release in cultured rat microglia via NF-κB and cAMP/PKA. CONCLUSIONS: Preexisting systemic inflammation plus a short severe hypoxia elicits cerebral edema through upregulated AQP4 and water permeability by TLR4 and CRH/CRHR1 signaling. This study revealed that both infection and hypoxia can cause inflammatory response in the brain. Systemic inflammation can facilitate onset of hypoxic cerebral edema through interaction of astrocyte and microglia by activation of TLR4 and CRH/CRHR1 signaling. Anti-inflammatory agents and CRHR1 antagonist may be useful for prevention and treatment of AMS and HACE.

Diving and exercise: the interaction of trigeminal receptors and muscle metaboreceptors on muscle sympathetic nerve activity in humans.

Swimming involves muscular activity and submersion, creating a conflict of autonomic reflexes elicited by the trigeminal receptors and skeletal muscle afferents. We sought to determine the autonomic cardiovascular responses to separate and concurrent stimulation of the trigeminal cutaneous receptors and metabolically sensitive skeletal muscle afferents (muscle metaboreflex). In eight healthy men (30 ± 2 yr) muscle sympathetic nerve activity (MSNA; microneurography), mean arterial pressure (MAP; Finometer), femoral artery blood flow (duplex Doppler ultrasonography), and femoral vascular conductance (femoral artery blood flow/MAP) were assessed during the following three experimental conditions: 1) facial cooling (trigeminal nerve stimulation), 2) postexercise ischemia (PEI; muscle metaboreflex activation) following isometric handgrip, and 3) trigeminal nerve stimulation with concurrent PEI. Trigeminal nerve stimulation produced significant increases in MSNA total activity (Δ347 ± 167%) and MAP (Δ21 ± 5%) and a reduction in femoral artery vascular conductance (Δ-17 ± 9%). PEI also evoked significant increases in MSNA total activity (Δ234 ± 83%) and MAP (Δ36 ± 4%) and a slight nonsignificant reduction in femoral artery vascular conductance (Δ-9 ± 12%). Trigeminal nerve stimulation with concurrent PEI evoked changes in MSNA total activity (Δ341 ± 96%), MAP (Δ39 ± 4%), and femoral artery vascular conductance (Δ-20 ± 9%) that were similar to those evoked by either separate trigeminal nerve stimulation or separate PEI. Thus, excitatory inputs from the trigeminal nerve and metabolically sensitive skeletal muscle afferents do not summate algebraically in eliciting a MSNA and cardiovascular response but rather exhibit synaptic occlusion, suggesting a high degree of convergent inputs on output neurons.

Cardiac contractility modulation increases action potential duration dispersion and decreases ventricular fibrillation threshold via ß1-adrenoceptor activation in the crystalloid perfused normal rabbit heart.

BACKGROUND/OBJECTIVES: Cardiac contractility modulation (CCM) is a new treatment being developed for heart failure (HF) involving application of electrical current during the absolute refractory period. We have previously shown that CCM increases ventricular force through ß1-adrenoceptor activation in the whole heart, a potential pro-arrhythmic mechanism. This study aimed to investigate the effect of CCM on ventricular fibrillation susceptibility. METHODS: Experiments were conducted in isolated New Zealand white rabbit hearts (2.0-2.5 kg, n=25). The effects of CCM (± 20 mA, 10 ms phase duration) on the left ventricular basal and apical monophasic action potential duration (MAPD) were assessed during constant pacing (200 bpm). Ventricular fibrillation threshold (VFT) was defined as the minimum current required to induce sustained VF with rapid pacing (30 × 30 ms). Protocols were repeated during perfusion of the ß1-adrenoceptor antagonist metoprolol (1.8 µM). In separate hearts, the dynamic and spatial electrophysiological effects of CCM were assessed using optical mapping with di-4-ANEPPS. RESULTS: CCM significantly shortened MAPD close to the stimulation site (Basal: 102 ± 5 [CCM] vs. 131 ± 6 [Control] ms, P<0.001). VFT was reduced during CCM (2.6 ± 0.6 [CCM] vs. 6.1 ± 0.8 [Control] mA, P<0.01) and was correlated (r(2)=0.40, P<0.01) with increased MAPD dispersion (26 ± 4 [CCM] vs. 5 ± 1 [Control] ms, P<0.01) (n=8). Optical mapping revealed greater spread of CCM induced MAPD shortening during basal vs. apical stimulation. CCM effects were abolished by metoprolol and exogenous acetylcholine. No evidence for direct electrotonic modulation of APD was found, with APD adaptation occurring secondary to adrenergic stimulation. CONCLUSIONS: CCM decreases VFT in a manner associated with increased MAPD dispersion in the crystalloid perfused normal rabbit heart.

Protein coated microcrystals formulated with model antigens and modified with calcium phosphate exhibit enhanced phagocytosis and immunogenicity.

Protein-coated microcrystals (PCMCs) were investigated as potential vaccine formulations for a range of model antigens. Presentation of antigens as PCMCs increased the antigen-specific IgG responses for all antigens tested, compared to soluble antigens. When compared to conventional aluminium-adjuvanted formulations, PCMCs modified with calcium phosphate (CaP) showed enhanced antigen-specific IgG responses and a decreased antigen-specific IgG1:IgG2a ratio, indicating the induction of a more balanced Th1/Th2 response. The rate of antigen release from CaP PCMCs, in vitro, decreased strongly with increasing CaP loading but their immunogenicity in vivo was not significantly different, suggesting the adjuvanticity was not due to a depot effect. Notably, it was found that CaP modification enhanced the phagocytosis of fluorescent antigen-PCMC particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen or soluble PCMCs. Thus, CaP PCMCs may provide an alternative to conventional aluminium-based acellular vaccines to provide a more balanced Th1/Th2 immune response.

Use of a dual reporter plasmid to demonstrate Bactofection with an attenuated AroA(-) derivative of Pasteurella multocida B:2.

A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP) from a constitutive prokaryotic promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP) from a constitutive eukaryotic promoter within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic bovine lung (EBL) cells and an attenuated AroA(-) derivative of Pasteurella multocida B:2 (strain JRMT12), harbouring the plasmid pSRG, showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously, indicating release of the plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria within these cells. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine.

Measurement of extravascular lung water following human brain death: implications for lung donor assessment and transplantation.

OBJECTIVES: The measurement of extravascular lung water could aid the assessment and guide the management of potential lung donors following brain death. We therefore sought to validate a single indicator thermodilution extravascular lung water index (EVLWI-T) measurement using gravimetry and to assess the impact and clinical correlates of elevated EVLWI-T in potential lung donors and transplant recipients. METHODS: In a prospective study, we measured serial EVLWI-T and haemodynamic and oxygenation data in 60 potential lung donors. To validate the EVLWI-T measurement, we measured in vivo thermodilution EVLWI (EVLWI-T) and gravimetric ex vivo EVLWI (EVLWI-G) in donor lungs rejected for transplant using the Holcroft and Trunkey modification of Pearce's method. We assessed the clinical correlates of elevated lung water and measured interleukin-8 and hepatocyte growth factor in bronchoalveolar lavage (BAL) fluid. RESULTS: The mean EVLWI-T (n = 60) was 9.7 (4.5) ml kg(-1), being >7-10 ml kg(-1) in 23/60 and >10 ml kg(-1) in 16/60 potential donors. Donor lungs with EVLWI >10 ml kg(-1) were more likely to be receiving norepinephrine (P = 0.04), have higher pulmonary capillary wedge pressures (P = 0.008), be unsuitable for transplantation (P = 0.007) and, if transplanted, have worse survival (P = 0.04). Lungs submitted to gravimetric analysis [n = 20 in 11 donors (9 double and 2 single)] had EVWLI-T of 10.8 (2.7) and EVLWI-G was 10.1 (2.5). There was a strong correlation between EVLW-T and EVLW-G (r = 0.7; P = 0.014), but EVLWI-T over-predicted the EVLWI-G by ≈ 1 ml kg(-1) (EVLW-T = 1.05 × EVLW-G). Cytokine levels in BAL fluid were elevated. CONCLUSIONS: Elevated lung water is found in >50% of potential lung donors, predicts lung suitability for transplant and may adversely affect recipient outcome. Although EVLWI-T intrinsically overestimates gravimetric lung water, its measurement may aid the assessment of organ suitability. Lung water accumulation and the proinflammatory response may both be targets for modifying therapy.

Switching off micturition using deep brain stimulation at midbrain sites.

Most of the time the bladder is locked in storage mode, switching to voiding only when it is judged safe and/or socially appropriate to urinate. Here we show, in humans and rodents, that deep brain stimulation in the periaqueductal gray matter can rapidly and reversibly manipulate switching within the micturition control circuitry, to defer voiding and maintain urinary continence, even when the bladder is full. Manipulation of neural continence pathways by deep brain stimulation may offer new avenues for the treatment of urinary incontinence of central origin.

Interaction between mammalian cells and Pasteurella multocida B:2. Adherence, invasion and intracellular survival.

A Pasteurella multocida B:2 strain from a case of bovine haemorrhagic septicaemia (HS) and a derivative, JRMT12, that was attenuated by a deletion in the aroA gene, were shown to adhere to, invade and survive within cultured embryonic bovine lung (EBL) cells. By comparison, bovine strains of Mannheimia haemolytica serotype A1 and P. multocida serotype A:3, although able to adhere to EBL cells, were not found intracellularly. The B:2 strains were viable intracellularly over a 7 h period, although a steady decline in viability was noted with time. Entry into the mammalian cells was inhibited by cytochalasin D, indicating that cell uptake was by an actin-dependent process. Viability assessment of EBL cells by trypan blue staining indicated that none of the bacterial strains was toxic for the EBL cells. Transmission electron microscopy (TEM) showed that, after entry into the mammalian cells, the B:2 strain resided in a vacuolar compartment. However, only a low percentage of mammalian cells appeared to contain one or more P. multocida B:2, suggesting that only certain EBL cells in the population were capable of being invaded by, or of taking up, the bacteria. TEM showed that P. multocida A:3 and M. haemolytica A:1 were found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. The cell-invasive capacity of P. multocida B:2 may be a virulence property related to its ability to translocate from the respiratory tract into the blood stream.

Differential cardiac responses to unilateral sympathetic nerve stimulation in the isolated innervated rabbit heart.

The heart receives both a left and right sympathetic innervation. Currently there is no description of an in vitro whole heart preparation for comparing the influence of each sympathetic supply on cardiac function. The aim was to establish the viability of using an in vitro model to investigate the effects of left and right sympathetic chain stimulation (LSS/RSS). For this purpose the upper sympathetic chain on each side was isolated and bipolar stimulating electrodes were attached between T2-T3 and electrically insulated from surrounding tissue in a Langendorff innervated rabbit heart preparation (n=8). Heart rate (HR) was investigated during sinus rhythm, whilst dromotropic, inotropic and ventricular electrophysiological effects were measured during constant pacing (250 bpm). All responses exhibited linear increases with increases in stimulation frequency (2-10 Hz). The change in HR was larger during RSS than LSS (P<0.01), increasing by 78±9 bpm and 49±8 bpm respectively (10 Hz, baseline; 145±7 bpm). Left ventricular pressure was increased from a baseline of 50±4 mmHg, by 22±5 mmHg (LSS, 10 Hz) and 4±1 mmHg (RSS, 10 Hz) respectively (P<0.001). LSS, but not RSS, caused a shortening of basal and apical monophasic action potential duration (MAPD90). We demonstrate that RSS exerts a greater effect at the sinoatrial node and LSS at the left ventricle. The study confirms previous experiments on dogs and cats, provides quantitative data on the comparative influence of right and left sympathetic nerves and demonstrates the feasibility of isolating and stimulating the ipsilateral cardiac sympathetic supply in an in vitro innervated rabbit heart preparation.