Autonomic neuro-cardiac profile of electrical, structural and neuronal remodeling in myocardial infarction-induced heart failure.

Aims: Heart failure is a clinical syndrome typified by abnormal autonomic tone, impaired ventricular function, and increased arrhythmic vulnerability. This study aims to examine electrophysiological, structural and neuronal remodeling following myocardial infarction in a rabbit heart failure model to establish its neuro-cardiac profile. Methods and results: Weight-matched adult male New Zealand White rabbits (3.2 ± 0.1 kg, n = 25) were randomized to have coronary ligation surgeries (HF group, n = 13) or sham procedures (SHM group, n = 12). Transthoracic echocardiography was performed six weeks post-operatively. On week 8, dual-innervated Langendorff-perfused heart preparations were set up for terminal experiments. Seventeen hearts (HF group, n = 10) underwent ex-vivo cardiac MRI. Twenty-two hearts (HF group, n = 7) were examined histologically. Electrical remodeling and abnormal autonomic profile were evident in HF rabbits with exaggerated sympathetic and attenuated vagal effect on ventricular fibrillation threshold, ventricular refractoriness and restitution curves, in addition to increased spatial restitution dispersion. Histologically, there was significant neuronal enlargement at the heart hila and conus arteriosus in HF. Structural remodeling was characterized by quantifiable myocardial scarring, enlarged left ventricles, altered ventricular geometry and impaired contractility. Conclusion: In an infarct-induced rabbit heart failure model, extensive structural, neuronal and electrophysiological remodeling in conjunction with abnormal autonomic profile provide substrates for ventricular arrhythmias.

Postcolonial control of Fiji soccer and the return of subjugated knowledges: From the 1970s to the 2010s.

The primary aim of this article is to use Foucault's idea of subjugated knowledges to search out areas and viewpoints within Fiji soccer which are suppressed by the governing authorities. To fulfill this aim, we explore and assess, via ethnographic research, the racial and ethnic aspects of Fiji soccer, from the 1970s to the 2010s, and how cultural hegemony facilitates continued Fiji Indian control and dominance within the sport. Next, and although we note the positive dimension of Fiji Football Association's 2014 Veterans' Dinner, we suggest that some ex-Ba players were apparently discriminated against by, puzzlingly, not being invited. The regulator was also unaware of, or insensitive to, ex-players' transportation needs as some were poor or invalid. We then look at the cases of Sweats Soccer Club (SSC) and Nadi Legends Football Club (NLFC) to show how, in the face of the regulator's indifference to the financial plight of an Indigenous village club (SSC), the ex-Nadi players set up instead a self-help organization (NLFC) to assist and encourage ex-players going through hard times. The latter was a cross-ethnic group/cross-class collaboration between ex-officials and ex-players and was largely outside the regulator's sphere of interest or intent.

The Effects of Vagus Nerve Stimulation on Ventricular Electrophysiology and Nitric Oxide Release in the Rabbit Heart.

Background: Abnormal autonomic activity including impaired parasympathetic control is a known hallmark of heart failure (HF). Vagus nerve stimulation (VNS) has been shown to reduce the susceptibility of the heart to ventricular fibrillation, however the precise underlying mechanisms are not well understood and the detailed stimulation parameters needed to improve patient outcomes clinically are currently inconclusive. Objective: To investigate NO release and cardiac electrophysiological effects of electrical stimulation of the vagus nerve at varying parameters using the isolated innervated rabbit heart preparation. Methods: The right cervical vagus nerve was electrically stimulated in the innervated isolated rabbit heart preparation (n = 30). Heart rate (HR), effective refractory period (ERP), ventricular fibrillation threshold (VFT) and electrical restitution were measured as well as NO release from the left ventricle. Results: High voltage with low frequency VNS resulted in the most significant reduction in HR (by -20.6 ± 3.3%, -25.7 ± 3.0% and -30.5 ± 3.0% at 0.1, 1 and 2 ms pulse widths, with minimal increase in NO release. Low voltage and high frequency VNS significantly altered NO release in the left ventricle, whilst significantly flattening the slope of restitution and significantly increasing VFT. HR changes however using low voltage, high frequency VNS were minimal at 20Hz (to 138.5 ± 7.7 bpm (-7.3 ± 2.0%) at 1 ms pulse width and 141.1 ± 6.6 bpm (-4.4 ± 1.1%) at 2 ms pulse width). Conclusion: The protective effects of the VNS are independent of HR reductions demonstrating the likelihood of such effects being as a result of the modulation of more than one molecular pathway. Altering the parameters of VNS impacts neural fibre recruitment in the ventricle; influencing changes in ventricular electrophysiology, the protective effect of VNS against VF and the release of NO from the left ventricle.

Association of Elevated Body Mass Index with Functional Outcome and Mortality following Acute Ischemic Stroke: The Obesity Paradox Revisited.

BACKGROUND: Previous literature has identified a survival advantage in acute ischemic stroke (AIS) patients with elevated body mass indices (BMIs), a phenomenon termed the "obesity paradox." OBJECTIVE: The aim of this study was to evaluate the independent association between obesity and clinical outcomes following AIS. METHODS: Weighted discharge data from the National Inpatient Sample were queried to identify AIS patients from 2015 to 2018. Multivariable logistic regression and Cox proportional hazards modeling were performed to evaluate associations between obesity (BMI ≥ 30) and clinical endpoints following adjustment for acute stroke severity and comorbidity burden. RESULTS: Among 1,687,805 AIS patients, 216,775 (12.8%) were obese. Compared to nonobese individuals, these patients were younger (64 vs. 72 mean years), had lower baseline NIHSS scores (6.9 vs. 7.9 mean score), and a higher comorbidity burden. Multivariable analysis demonstrated independent associations between obesity and lower likelihood of mortality (adjusted odds ratio [aOR] 0.76, 95% confidence interval [CI]: 0.71, 0.82, p < 0.001; hazard ratio 0.84, 95% CI: 0.73, 0.97, p = 0.015), intracranial hemorrhage (aOR 0.87, 95% CI: 0.82, 0.93, p < 0.001), and routine discharge to home (aOR 0.97, 95% CI: 0.95, 0.99; p = 0.015). Mortality rates between obese and nonobese patients were significantly lower across stroke severity thresholds, but this difference was attenuated among high severity (NIHSS > 20) strokes (21.6% vs. 23.2%, p = 0.358). Further stratification of the cohort into BMI categories demonstrated a "U-shaped" association with mortality (underweight aOR 1.58, 95% CI: 1.39, 1.79; p < 0.001, overweight aOR 0.64, 95% CI: 0.42, 0.99; p = 0.046, obese aOR 0.77, 95% CI: 0.71, 0.83; p < 0.001, severely obese aOR 1.18, 95% CI: 0.74, 1.87; p = 0.485). Sub-cohort assessment of thrombectomy-treated patients demonstrated an independent association of obesity (BMI 30-40) with lower mortality (aOR 0.79, 95% CI: 0.65, 0.96; p = 0.015), but not with routine discharge. CONCLUSION: This cross-sectional analysis demonstrates a lower likelihood of discharge to home as well as in-hospital mortality in obese patients following AIS, suggestive of a protective effect of obesity against mortality but not against all poststroke neurological deficits in the short term which would necessitate placement in acute rehabilitation and long-term care facilities.

Effects of sympatho-vagal interaction on ventricular electrophysiology and their modulation during beta-blockade.

AIMS: The effects of sympatho-vagal interaction on heart rate (HR) changes are characterized by vagal dominance resulting in accentuated antagonism. Complex autonomic modulation of ventricular electrophysiology may exert prognostic arrhythmic impact. We examined the effects of concurrent sympathetic (SNS) and vagus (VNS) nerve stimulation on ventricular fibrillation threshold (VFT) and standard restitution (RT) in an isolated rabbit heart preparation with intact dual autonomic innervation, with and without beta-blockade. METHODS AND RESULTS: Monophasic action potentials were recorded from left ventricular epicardial surface of dual-innervated isolated heart preparations from New Zealand white rabbits (n = 18). HR, VFT and RT were measured during different stimulation protocols (Protocol 1: VNS-SNS; Protocol 2: SNS-VNS) involving low- and high-frequency stimulations. A sub-study of Protocol 2 was performed in the presence of metoprolol tartrate. In both protocols, HR changes were characterized by vagal-dominant bradycardic component, affirming accentuated antagonism. During concurrent high-frequency VNS (HV), SNS prevails in lowering VFT in a frequency-sensitive manner during low (LS) or high (HS)-frequency stimulations (HV-LS: -2.8 ± 0.8 mA; HV-HS: -4.0 ± 0.9 mA, p < .05 vs. HV), with accompanying steepening of relative RT slope gradients (HV-LS: 223.54 ± 37.41%; HV-HS: 295.20 ± 60.86%, p < .05 vs. HV). In protocol 2, low (LV) and high (HV) vagal stimulations during concurrent HS raised VFT (HS-LV: 1.0 ± 0.4 mA; HS-HV: 3.0 ± 0.6 mA, p < .05 vs HS) with associated flattening of RT slopes (HS-LV: 32.40 ± 4.97%;HS-HV: 38.07 ± 6.37%; p < .05 vs HS). Metoprolol abolished accentuated antagonism in HR changes, reduced VFT and flattened RT globally during SNS-VNS. CONCLUSIONS: Accentuated antagonism is absent in ventricular electrophysiological changes during sympatho-vagal interaction with sympathetic effect prevailing, suggesting a different mechanism at the ventricular level from heart rate effects. Metoprolol nullified accentuated antagonism with additional anti-fibrillatory effect beyond adrenergic blockade during sympatho-vagal stimulations.

Epidemiology of bone stress injuries in Australian high performance athletes: A retrospective cohort study.

OBJECTIVES: To examine the epidemiology of bone stress injuries in an elite sports institute. DESIGN: Retrospective cohort study at the Australian Institute of Sport. METHODS: A retrospective analysis of the clinical records contained within the Australian Institute of Sport Athlete Management System electronic database was performed. Records with Orchard Sports Injury Classification System codes relating to bone stress injuries and stress fractures were reviewed and descriptive statistics relating to sport, site of injury, athlete age, sex and activity were analysed. RESULTS: In the three-year period January 2014-2017, 11,942 injuries were recorded across 48 sports. 181 bone stress injuries (0.15% of all injuries) were recorded across 16 sports. BSIs in the foot and lumbar spine were the most common accounting for 30% and 23% of all the reported BSIs respectively. Gymnasts had a high frequency of lumbar spine stress injuries (n=24, 51%) and rowers had a high frequency of rib stress injuries (n=22, 88%). The most common location for stress injuries, equally distributed across a variety of sports, were in the foot (n=54, 30%). Female athletes recorded more BSIs than males. CONCLUSION: Across a three-year period, 0.15% of injuries were related to bone stress injuries. Almost double the cases were recorded in female athletes. Sport specific injury sites were observed in the dataset.

Posttraumatic subgaleal herniation of an intracranial cerebral arterial segment.

A rare case of extracranial herniation of an intracranial arterial segment is described in an elderly patient presenting with loss of consciousness following mild head trauma. Cross-sectional imaging of the brain revealed a temporal skull fracture with associated intra-axial and extra-axial bleeding, and a computed tomography angiogram of the head and cerebral digital subtraction angiogram performed a few hours later displayed a rare finding. A small segment of a parietal branch of the right middle cerebral artery (M4 segment) herniated through the skull fracture, which coursed into the extracranial subgaleal space overlying the fracture site, before coursing back into the intracranial compartment. There was no evidence of cerebral ischemia or vascular compromise. The presence of a subgaleal artery on computed tomography angiogram may point to herniation of intracranial artery. In the absence of knowledge of this entity, even basic routine emergency care such as application of a scalp bandage or suturing of a scalp laceration could potentially result in devastating complications of vascular compromise and stroke.

Different paths, same destination: divergent action potential responses produce conserved cardiac fight-or-flight response in mouse and rabbit hearts.

KEY POINTS: Cardiac electrophysiology and Ca2+ handling change rapidly during the fight-or-flight response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac fight-or-flight response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca2+ handling responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca2+ handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological responses are fine-tuned across species to produce optimal cardiac fight-or-flight responses. ABSTRACT: Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+ handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca2+ -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9 ± 5.1 ms at t = 0 s vs. 60.6 ± 4.1 ms at t = 15 s, P < 0.05) followed by shortening (46.5 ± 9.1 ms at t = 60 s, P = NS vs. t = 0). We determined the biphasic APD response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by ß-adrenergic activation. Simulations with species-specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic responses. Thus, while the cardiac fight-or-flight response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly.

Updated pharmacological management of rheumatoid arthritis for women before, during, and after pregnancy, reflecting recent guidelines.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease which can cause significant disability, morbidity, mortality, and impaired fertility. It commonly affects women of childbearing age. Managing rheumatoid arthritis (RA) in the perinatal period poses challenges. There is concern about the teratogenic effects of many traditional disease-modifying anti-rheumatic drugs (DMARDs) and an ever-growing list of new therapeutic options with limited data in pregnancy and breastfeeding. AIMS: We aimed to create a standardized approach to pharmacological management of RA patients seen in our newly established Rheumatology and Reproductive Health Service. METHODS: We reviewed relevant publications on the use of anti-rheumatic drugs in pregnancy. These include recent guidelines from The British Society for Rheumatology (BSR) and British Health Professionals in Rheumatology (BHPR) and the European League Against Rheumatism (EULAR). RESULTS: After considering relevant publications, we developed a Saint Vincent's University Hospital/National Maternity Hospital consensus protocol for evidence-based medication in pregnancy in RA. CONCLUSIONS: RA tends to improve during pregnancy and flare postpartum. Several anti-rheumatic medication options during pregnancy and breastfeeding are now available including anti-tumor necrosis factor (anti-TNF) agents. Good disease control at all stages of reproduction is important to ensure best outcome for both mother and baby.

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.

The heart's 'little brain' controlling cardiac function in the rabbit.

NEW FINDINGS: What is the topic of this review? The topic of the review is the intrinsic cardiac nervous system in the rabbit. What advances does it highlight? The anatomy of rabbit intrinsic ganglia is similar to that of other species, including humans. Immunohistochemistry confirms the presence of cholinergic and adrenergic neurones, with a striking arrangement of neuronal nitric oxide synthase-positive cell bodies. Activation of atrial ganglia produces effects on local and remote regions. Heart disease is a primary cause of mortality in the developed world, and it is well recognized that neural mechanisms play an important role in many cardiac pathologies. The role of extrinsic autonomic nerves has traditionally attracted the most attention. However, there is a rich intrinsic innervation of the heart, including numerous cardiac ganglia (ganglionic plexuses), that has the potential to affect cardiac function independently as well as to influence the actions of the extrinsic nerves. To investigate this, an isolated, perfused, innervated rabbit Langendorff heart preparation was considered the best option. Although ganglionic plexuses have been well described for several species, there was no full description of the anatomy and histochemistry of rabbit hearts. To this end, rabbit intrinsic ganglia were located using acetylcholinesterase histology (n = 33 hearts). This revealed six generalized ganglionic regions, defined as a left neuronal complex above the left pulmonary vein, a right neuronal complex around the base of right cranial vein, three scattered in the dorsal right atrium and a region containing numerous ventricular ganglia located on the conus arteriosus. Using immunohistochemistry, neurons were found to contain choline acetyltransferase or tyrosine hydroxylase and/or neuronal nitric oxide synthase in differing amounts (choline acetyltransferase > tyrosine hydroxylase > neuronal nitric oxide synthase). The function of rabbit intrinsic ganglia was investigated using a bolus application of nicotine or electrical stimulation at each of the above sites whilst measuring heart rate and atrioventricular conduction. Nicotine applied to different ganglionic plexuses caused a bradycardia, a tachycardia or a mixture of the two, with the right atrial plexus producing the largest chronotropic responses. Electrical stimulation at these sites induced only a bradycardia. Atrioventricular conduction was modestly changed by nicotine, the main response being a prolongation. Electrical stimulation produced significant prolongation of atrioventricular conduction, particularly when the right neuronal complex was stimulated. These studies show that the intrinsic plexuses of the heart are important and could be crucial for understanding impairments of cardiac function. Additionally, they provide a strong basis from which to progress using the isolated, innervated rabbit heart preparation.

hERG potassium channel blockade by the HCN channel inhibitor bradycardic agent ivabradine.

BACKGROUND: Ivabradine is a specific bradycardic agent used in coronary artery disease and heart failure, lowering heart rate through inhibition of sinoatrial nodal HCN-channels. This study investigated the propensity of ivabradine to interact with KCNH2-encoded human Ether-à-go-go-Related Gene (hERG) potassium channels, which strongly influence ventricular repolarization and susceptibility to torsades de pointes arrhythmia. METHODS AND RESULTS: Patch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. Ih ERG was inhibited with an IC50 of 2.07 µmol/L for the hERG 1a isoform and 3.31 µmol/L for coexpressed hERG 1a/1b. The voltage and time-dependent characteristics of Ih ERG block were consistent with preferential gated-state-dependent channel block. Inhibition was partially attenuated by the N588K inactivation-mutant and the S624A pore-helix mutant and was strongly reduced by the Y652A and F656A S6 helix mutants. In docking simulations to a MthK-based homology model of hERG, the 2 aromatic rings of the drug could form multiple π-π interactions with the aromatic side chains of both Y652 and F656. In monophasic action potential (MAP) recordings from guinea-pig Langendorff-perfused hearts, ivabradine delayed ventricular repolarization and produced a steepening of the MAPD90 restitution curve. CONCLUSIONS: Ivabradine prolongs ventricular repolarization and alters electrical restitution properties at concentrations relevant to the upper therapeutic range. In absolute terms ivabradine does not discriminate between hERG and HCN channels: it inhibits Ih ERG with similar potency to that reported for native If and HCN channels, with S6 binding determinants resembling those observed for HCN4. These findings may have important implications both clinically and for future bradycardic drug design.

Rapid video-referenced ratings of reciprocal social behavior in toddlers: a twin study.

BACKGROUND: Reciprocal social behavior (RSB) is a developmental prerequisite for social competency, and deficits in RSB constitute a core feature of autism spectrum disorder (ASD). Although clinical screeners categorically ascertain risk of ASD in early childhood, rapid methods for quantitative measurement of RSB in toddlers are not yet established. Such measurements are critical for tracking developmental trajectories and incremental responses to intervention. METHODS: We developed and validated a 20-min video-referenced rating scale, the video-referenced rating of reciprocal social behavior (vrRSB), for untrained caregivers to provide standardized ratings of quantitative variation in RSB. Parents of 252 toddler twins [Monozygotic (MZ) = 31 pairs, Dizygotic (DZ) = 95 pairs] ascertained through birth records, rated their twins' RSB at two time points, on average 6 months apart, and completed two developmental measures, the Modified Checklist for Autism in Toddlers (M-CHAT) and the MacArthur Communicative Development Inventory Short Form (MCDI-s). RESULTS: Scores on the vrRSB were fully continuously distributed, with excellent 6-month test-retest reliability ([intraclass correlation coefficient] ICC = 0.704, p < .000). MZ twins displayed markedly greater trait concordance than DZ twins, (MZ ICC = 0.863, p < .000, DZ ICC = 0.231, p < .012). VrRSB score distributions were highly distinct for children passing versus failing the M-CHAT (t = -8.588, df = 31, p < .000), incrementally improved from 18-24 months, and were inversely correlated with receptive and expressive vocabulary on the MCDI-s. CONCLUSIONS: Like quantitative autistic trait ratings in school-aged children and adults, toddler scores on the vrRSB are continuously distributed and appear highly heritable. These ratings exhibited minimal measurement error, high inter-individual stability, and developmental progression in RSB as children matured from 18-24 months, supporting their potential utility for serially quantifying the severity of early autistic syndromes over time and in response to intervention. In addition, these findings inform the genetic-environmental structure of RSB in early typical development.

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures.

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

Innervation of sinoatrial nodal cardiomyocytes in mouse. A combined approach using immunofluorescent and electron microscopy.

Fluorescent immunohistochemistry on the cardiac conduction system in whole mount mouse heart preparations demonstrates a particularly dense and complex network of nerve fibres and cardiomyocytes which are positive to the hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4-positive cardiomyocytes) in the sinoatrial node region and adjacent areas around the root of right cranial vein. The present study was designed to investigate the morphologic and histochemical pattern of nerve fibres and HCN4-positive cardiomyocytes using fluorescent techniques and/or electron microscopy. Adrenergic and cholinergic nerve fibres together with HCN4-positive cardiomyocytes were identified using primary antibodies for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and the HCN4 channel respectively. Amid HCN4-positive cardiomyocytes, fluorescence and electron microscopy data demonstrated a dense distribution of nerve fibres immunoreactive for ChAT and TH. In addition, novel electron microscopy data revealed that the mouse sinoatrial node contained exclusively unmyelinated nerve fibres, in which the majority of axons possess varicosities with clear mediatory vesicles that can be classified as cholinergic. Synapses occurred without any clear terminal connection with the effector cell, i.e. these synapes were of "en passant" type. In general, the morphologic pattern of innervation of mouse HCN4-positive cardiomyocytes identified using electron microscopy corresponds well to the dense network of nerve fibres demonstrated by fluorescent immunohistochemistry in mouse sinoatrial node and adjacent areas. The complex and extraordinarily dense innervation of HCN4-positive cardiomyocytes in mouse sinoatrial node underpins the importance of neural regulation for the cardiac conduction system. Based on the present observations, it is concluded that the occurrence of numerous nerve fibres nearby atrial cardiomyocytes serves as a novel reliable extracellular criterion for discrimination of SA nodal cardiomyocytes using electron microscopy.