The Role Of Pinhole Size And Position In Confocal Imaging Systems

Abstract Background Sympathetic neurons (SNs) innervate the myocardium with a defined topology that allows physiological modulation of cardiac activity. Neurotrophins released by cardiac cells control SN viability and myocardial distribution, which are impaired in heart diseases with reduced (e.g. heart failure) or heterogenous sympathetic stimulation (e.g. arrhythmias). We previously demonstrated that SNs interact directly with cardiomyocytes (CMs) at neuro-cardiac junctions (NCJ), and such structured contact sites allow neurons to efficiently activate β-adrenoceptors on the myocyte membrane. Aims We here asked whether NCJs are functional for retrograde (myocyte to neuron) neurotrophic signaling. Methods and results Electron microscopy and immunofluorescence on mouse heart slices and SN/CM co-cultures showed that the NGF receptor, TrkA, is preferentially found in correspondence of the NCJ. Consistently, neurons taking structured contact with CMs showed fast TrkA activation and its retrograde transport to the soma, which was monitored using live confocal imaging in cells expressing TrkA-RFP. In accord with NGF dependent effects, CM-contacted SN showed larger synaptic varicosities and did not require NGF supplementation in the culture medium. In support that NGF locally released at NCJs sustains SN viability, the neurotrophin concentration in the culture medium was 1.61 pg/mL, and did not suffice to maintain neuronal viability, which was also perturbed (66% decrease of neuronal density) by silencing NGF expression in CMs. These results support that the NCJ is essential for intercellular neurotrophin signaling. Consistently, by applying competitive inhibition of TrkA with increasing doses of K252a, we estimated NGF concentration at the contact site to be about 1000-fold higher than that released by CM in the culture medium. To seek for the structural determinants of the NCJ, we focused on dystrophin, based on the finding that the protein accumulates on the CM membrane portion contacted by SNs, as observed in mouse heart slices, and co-cultured CMs. In support of a role of CM-expressed dystrophin in neurotrophic signaling, hearts from dystrophin-KO (mdx) mice showed 74.36% decrease of innervation, with no significant changes of NGF expression. In line with the purported role of NCJs, in co-cultures between wild type SNs and mdx CMs, TrkA activation (TrkA movements toward SN soma (%): WTCM-WTSN=18±4; MDXCM-WTSN= 12±3; p<0,05) and neuronal survival were reduced. Conclusions Taken together, our results suggest that NGF-dependent signaling to SNs requires a direct and specialized interaction with myocytes, and that loss of dystrophin at the CM membrane impairs retrograde signaling to the neurons leading to cardiac sympathetic dys-innervation. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): University of Padova

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Role of surface electrocardiogram in the era of high-resolution mapping and imaging systems—back to the future

Journal of Thoracic Disease ◽

10.21037/jtd.2019.01.11 ◽

2019 ◽

Vol 11 (S3) ◽

pp. S307-S310 ◽

Cited By ~ 1

Author(s):

Carlo Lavalle ◽

Marco Valerio Mariani ◽

Domenico Giovanni Della Rocca ◽

Andrea Natale

Keyword(s):

High Resolution ◽

Imaging Systems ◽

High Resolution Mapping ◽

The Future ◽

Resolution Mapping ◽

Surface Electrocardiogram

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The Changes in Ca2+ Sparks Associated with Measured Modifications of Intra-store Ca2+ Concentration in Skeletal Muscle

The Journal of General Physiology ◽

10.1085/jgp.200609545 ◽

2006 ◽

Vol 128 (1) ◽

pp. 45-54 ◽

Cited By ~ 16

Author(s):

Bradley S. Launikonis ◽

Jingsong Zhou ◽

Demetrio Santiago ◽

Gustavo Brum ◽

Eduardo Ríos

Keyword(s):

Skeletal Muscle ◽

Cardiac Muscle ◽

Confocal Imaging ◽

Frog Skeletal Muscle ◽

Average Increase ◽

Global Level ◽

Event Frequency ◽

Average Change ◽

Contractile Activation

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 μM on average in 10 experiments) and a high value (433 μM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The “strength” of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca2+]SR was limited, it reached up to 200% in individual fibers, without causing massive Ca2+ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle.

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Spectral characteristics of chromatic confocal imaging systems

Applied Optics ◽

10.1364/ao.53.007634 ◽

2014 ◽

Vol 53 (32) ◽

pp. 7634 ◽

Cited By ~ 14

Author(s):

Matthias Hillenbrand ◽

Beate Mitschunas ◽

Florian Brill ◽

Adrian Grewe ◽

Stefan Sinzinger

Keyword(s):

Spectral Characteristics ◽

Confocal Imaging ◽

Imaging Systems

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Antecedent hydrogen sulfide elicits an anti-inflammatory phenotype in postischemic murine small intestine: role of BK channels

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01229.2009 ◽

2010 ◽

Vol 299 (5) ◽

pp. H1554-H1567 ◽

Cited By ~ 33

Author(s):

Mozow Y. Zuidema ◽

Yan Yang ◽

Meifang Wang ◽

Theodore Kalogeris ◽

Yajun Liu ◽

...

Keyword(s):

Small Intestine ◽

Hydrogen Sulfide ◽

Ischemia Reperfusion ◽

Bk Channels ◽

Bk Channel ◽

Confocal Imaging ◽

Sodium Hydrosulfide ◽

Inflammatory Phenotype ◽

Anti Inflammatory

The objectives of this study were to determine the role of calcium-activated, small (SK), intermediate (IK), and large (BK) conductance potassium channels in initiating the development of an anti-inflammatory phenotype elicited by preconditioning with an exogenous hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS). Intravital microscopy was used to visualize rolling and firmly adherent leukocytes in vessels of the small intestine of mice preconditioned with NaHS (in the absence and presence of SK, IK, and BK channel inhibitors, apamin, TRAM-34, and paxilline, respectively) or SK/IK (NS-309) or BK channel activators (NS-1619) 24 h before ischemia-reperfusion (I/R). I/R induced marked increases in leukocyte rolling and adhesion, effects that were largely abolished by preconditioning with NaHS, NS-309, or NS-1619. The postischemic anti-inflammatory effects of NaHS-induced preconditioning were mitigated by BKB channel inhibitor treatment coincident with NaHS, but not by apamin or TRAM-34, 24 h before I/R. Confocal imaging and immunohistochemistry were used to demonstrate the presence of BKα subunit staining in both endothelial and vascular smooth muscle cells of isolated, pressurized mesenteric venules. Using patch-clamp techniques, we found that BK channels in cultured endothelial cells were activated after exposure to NaHS. Bath application of the same concentration of NaHS used in preconditioning protocols led to a rapid increase in a whole cell K+ current; specifically, the component of K+ current blocked by the selective BK channel antagonist iberiotoxin. The activation of BK current by NaHS could also be demonstrated in single channel recording mode where it was independent of a change in intracellular Ca+ concentration. Our data are consistent with the concept that H2S induces the development of an anti-adhesive state in I/R in part mediated by a BK channel-dependent mechanism.

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The Inhibitory Effect of WenxinKeli on H9C2 Cardiomyocytes Hypertrophy Induced by Angiotensin II through Regulating Autophagy Activity

Oxidative Medicine and Cellular Longevity ◽

10.1155/2017/7042872 ◽

2017 ◽

Vol 2017 ◽

pp. 1-11 ◽

Cited By ~ 5

Author(s):

Jie Li ◽

Yang Li ◽

Ying Zhang ◽

Dan Hu ◽

Yonghong Gao ◽

...

Keyword(s):

Angiotensin Ii ◽

Inhibitory Effect ◽

Cytoskeletal Proteins ◽

Confocal Imaging ◽

Control Group ◽

Cell Viability Assay ◽

Viability Assay ◽

H9c2 Cardiomyocytes ◽

Cardiomyocyte Autophagy

Objectives. We investigated the role of cardiomyocyte autophagy and its regulatory mechanisms by WenxinKeli (WXKL) in cells subjected to hypertrophy. Methods. H9C2 cardiomyocytes were divided into 8 groups. Cytoskeletal proteins as well as endogenously expressed autophagy marker proteins were studied by confocal imaging. Western blotting was used to assess the levels of light chain-3 (LC3) and mechanistic target of rapamycin (mTOR). The cell viability assay was used to detect the content of ATP. Flow cytometry was used to detect apoptotic cardiomyocytes. Results. (1) Compared with the control group, the length and width of cells in the Angiotensin II (AngII) group were significantly increased, while those in the 3-methyladenine (3-MA) and the WXKL groups were decreased. (2) Compared with AngII group, the expression of LC3 II/I protein in the 3-MA and WXKL groups was downregulated, while the expression of mTOR protein was upregulated. (3) Compared with the AngII group, the cardiomyocytes in the WXKL group showed increased ATP and decreased apoptosis rate and number of autophagosome. Conclusions. We propose a novel role of WXKL as a likely inhibitor of cardiac hypertrophy by regulation of pathological autophagy.

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Role of Nuclear Imaging to Understand the Neural Substrates of Brain Disorders in Laboratory Animals: Current Status and Future Prospects

Frontiers in Behavioral Neuroscience ◽

10.3389/fnbeh.2020.596509 ◽

2020 ◽

Vol 14 ◽

Author(s):

Annunziata D'Elia ◽

Sara Schiavi ◽

Andrea Soluri ◽

Roberto Massari ◽

Alessandro Soluri ◽

...

Keyword(s):

Small Animal Imaging ◽

Small Animal ◽

Nuclear Imaging ◽

Imaging Systems ◽

Current Status ◽

Laboratory Animals ◽

Brain Functioning ◽

Neuroscience Research ◽

Animal Imaging

Molecular imaging, which allows the real-time visualization, characterization and measurement of biological processes, is becoming increasingly used in neuroscience research. Scintigraphy techniques such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) provide qualitative and quantitative measurement of brain activity in both physiological and pathological states. Laboratory animals, and rodents in particular, are essential in neuroscience research, providing plenty of models of brain disorders. The development of innovative high-resolution small animal imaging systems together with their radiotracers pave the way to the study of brain functioning and neurotransmitter release during behavioral tasks in rodents. The assessment of local changes in the release of neurotransmitters associated with the performance of a given behavioral task is a turning point for the development of new potential drugs for psychiatric and neurological disorders. This review addresses the role of SPECT and PET small animal imaging systems for a better understanding of brain functioning in health and disease states. Brain imaging in rodent models faces a series of challenges since it acts within the boundaries of current imaging in terms of sensitivity and spatial resolution. Several topics are discussed, including technical considerations regarding the strengths and weaknesses of both technologies. Moreover, the application of some of the radioligands developed for small animal nuclear imaging studies is discussed. Then, we examine the changes in metabolic and neurotransmitter activity in various brain areas during task-induced neural activation with special regard to the imaging of opioid, dopaminergic and cannabinoid receptors. Finally, we discuss the current status providing future perspectives on the most innovative imaging techniques in small laboratory animals. The challenges and solutions discussed here might be useful to better understand brain functioning allowing the translation of preclinical results into clinical applications.

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Abstract 353: Triciribine, a Selective Akt Inhibitor, Inhibits Myofibroblast Differentiation and Ameliorates Pulmonary Vascular Remodeling in Pulmonary Hypertension

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.353 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Payaningal R Somanath ◽

Maha Abdalla

Keyword(s):

Extracellular Matrix ◽

Pulmonary Hypertension ◽

Vascular Remodeling ◽

Therapeutic Option ◽

Matrix Metalloproteases ◽

Confocal Imaging ◽

Extracellular Matrix Protein ◽

Myofibroblast Differentiation ◽

Akt Inhibitor

Objectives: Idiopathic pulmonary fibrosis (IPF) is an incurable, chronic and progressive disease with severely poor prognosis and often leads to pulmonary hypertension (PH). Persistent myofibroblast (MF) differentiation in response to TGFβ, marked by de novo expression of αSMA stress fibers, is the central orchestrator of tissue fibrosis and vasculopathy. Our previous studies indicated the integral role of Akt in myofibroblast differentiation and fibrosis. Here, we investigated the efficacy of Triciribine (TCBN), a selective Akt inhibitor, currently in clinical trials for cancer therapy, as a potential therapeutic option for IPF and PH. Methods: WT and Akt1 null mice, as well as those treated with TCBN were subjected to 3 week hypoxia along with normoxia control, and the lungs were used for imaging and western analysis to study the biochemical, structural and vascular changes in the lungs. Cutting edge techniques such as microfil-cast based vascular imaging, confocal imaging and analysis of extracellular matrix protein turnover was performed for the data collection and analysis. Results: Under hypoxia, WT mice exhibited robust vascular remodeling leading to thickening of the artery wall, reduction in lumen size and loss of capillaries in the lungs. This effect was significantly less in Akt1 null mice. Treatment with TCBN significantly ameliorated vascular remodeling in mice due to hypoxia, and resulted in normalization of the vasculature with increased lumen size, reduced wall thickness and increased number of capillary branches as compared to untreated controls. TCBN also affected expression of a number of different extracellular matrix proteins and matrix metalloproteases in addition to changes in the expression of contractile proteins alpha-actin, myocardin and serum response factor in the adventitial fibroblasts. Conclusions: Our results demonstrate the crucial role of Akt as an integral mediator of MF differentiation in IPF and PH. Further, TCBN could potentially be a therapeutic option for IPF and PH.

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