Neurochemical alterations of intrinsic cardiac ganglionated nerve plexus caused by arterial hypertension developed during ageing in spontaneously hypertensive and Wistar Kyoto rats.

The acknowledged hypothesis of the cause of arterial hypertension is the emerging disbalance in sympathetic and parasympathetic regulations of the cardiovascular system. This disbalance manifests in a disorder of sustainability of endogenous autonomic and sensory neural substances including calcitonin gene-related peptide (CGRP). This study aimed to examine neurochemical alterations of intrinsic cardiac ganglionated nerve plexus (GP) triggered by arterial hypertension during ageing in spontaneously hypertensive rats of juvenile (prehypertensive, 8-9 weeks), adult (early hypertensive, 12-18 weeks) and elderly (persistent hypertensive, 46-60 weeks) age in comparison with the age-matched Wistar-Kyoto rats as controls. Parasympathetic, sympathetic and sensory neural structures of GP were analysed and evaluated morphometrically in tissue sections and whole-mount cardiac preparations. Both the elevated blood pressure and the evident ultrasonic signs of heart failure were identified for spontaneously hypertensive rats and in part for the aged control rats. The amount of adrenergic and immunoreactive to CGRP neural structures was increased in the adult group of spontaneously hypertensive rats along with the significant alterations that occurred during ageing. In conclusion, the revealed chemical alterations of GP support the hypothesis about the possible disbalance of efferent and afferent heart innervation and may be considered as the basis for the emergence and progression of arterial hypertension and perhaps even as a consequence of hypertension in the aged spontaneously hypertensive rats. The determined anatomical changes in the ageing Wistar-Kyoto rats suggest this breed being as inappropriate for its use as control animals for hypertension studies in older animal age.

The distribution of sinoatrial nodal cells and their innervation in the pig.

The sinoatrial node (SAN) has been the object of interest of various studies. In experimental neurocardiology, the real challenge is the choice of the most appropriate animal model. Pig is routinely used animal due to its size and physiological features. Despite this, the anatomy and innervation of the pig SAN are not completely examined. This study analyses the distribution of SAN cells and their innervation in whole-mount preparations and the cross-sections of the pig right atrium. Our findings revealed the differences in the distribution of the SAN cells and their innervation pattern between pigs and other animals. The pig SAN myocytes were distributed around the root of the anterior vena cava. A meshwork of nerve fibers (NFs) in this area was four-fold denser compared to other right atrial areas and contained the adrenergic (positive for TH), cholinergic (positive for ChAT), nitrergic (positive for nNOS), and potentially sensory (positive for SP) NFs. The SAN area contained 98 ± 10 ganglia that involved 21 ± 2 neuronal somata per ganglion. The determined chemical phenotypes of ganglionic cells demonstrate their diversity in the pig SAN area as there were identified neuronal somata positive for ChAT, nNOS, TH, and simultaneously for ChAT/nNOS and ChAT/TH. Small intensively fluorescent cells were also abundant. The broad distribution of SAN cells, the chemical diversity, and the high density of neural components in the SAN area are comparable to the human one and, therefore, the pig may be considered as the appropriate animal model for experimental cardiology.

Comparative analysis of intracardiac neural structures in the aged rats with essential hypertension.

Persistent arterial hypertension initiates cardiac autonomic imbalance and alters cardiac tissues. Previous studies have shown that neural component contributes to arterial hypertension etiology, maintenance, and progression and leads to brain damage, peripheral neuropathy, and remodeling of intrinsic cardiac neural plexus. Recently, significant structural changes of the intracardiac neural plexus were demonstrated in young prehypertensive and adult hypertensive spontaneously hypertensive rats (SHR), yet structural alterations of intracardiac neural plexus that occur in the aged SHR remain undetermined. Thus, we analyzed the impact of uncontrolled arterial hypertension in old (48-52 weeks) SHR and the age-matched Wistar-Kyoto rats (WKY). Intrinsic cardiac neural plexus was examined using a combination of immunofluorescence confocal microscopy and transmission electron microscopy in cardiac sections and whole-mount preparations. Our findings demonstrate that structural changes of intrinsic cardiac neural plexus caused by arterial hypertension are heterogeneous and may support recent physiological implications about cardiac denervation occurring together with the hyperinnervation of the SHR heart. We conclude that arterial hypertension leads to (i) the decrease of the neuronal body area, the thickness of atrial nerves, the number of myelinated nerve fibers, unmyelinated axon area and cumulative axon area in the nerve, and the density of myocardial nerve fibers, and (ii) the increase in myelinated nerve fiber area and density of neuronal bodies within epicardiac ganglia. Despite neuropathic alterations of myelinated fibers were exposed within intracardiac nerves of both groups, SHR and WKY, we consider that the determined significant changes in structure of intrinsic cardiac neural plexus were predisposed by arterial hypertension.

Comparison of Microglial Morphology and Function in Primary Cerebellar Cell Cultures on Collagen and Collagen-Mimetic Hydrogels.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

Early structural alterations of intrinsic cardiac ganglionated plexus in spontaneously hypertensive rats.

Persistent arterial hypertension leads to structural and functional remodeling of the heart resulting in myocardial ischemia, fibrosis, hypertrophy, and eventually heart failure. Previous studies have shown that individual neurons composing the intracardiac ganglia are hypertrophied in the failing human, dog, and rat hearts, indicating that this process involves changes in cardiac innervation. However, despite a wealth of data on changes in intrinsic cardiac ganglionated plexus (GP) in late-stage disease models, little is known about the effects of hypertension on cardiac innervation during the early onset of heart failure development. Thus, we examined the impact of early hypertension on the structural organization of the intrinsic cardiac ganglionated plexus in juvenile (8-9 weeks) and adult (12-18 weeks) spontaneously hypertensive (SH) and age-matched Wistar-Kyoto (WKY) rats. GP was studied using a combination of immunofluorescence confocal microscopy and transmission electron microscopy in whole-mount preparations and tissue sections. Here, we report intrinsic cardiac GP of SH rats to display multiple structural alterations: (i) a decrease in the intracardiac neuronal number, (ii) a marked reduction in axonal diameters and their proportion within intracardiac nerves, (iii) an increased density of myocardial nerve fibers, and (iv) neuropathic abnormalities in cardiac glial cells. These findings represent early neurological changes of the intrinsic ganglionated plexus of the heart introduced by early-onset arterial hypertension in young adult SH rats.

Chemical phenotypes of intrinsic cardiac neurons in the newborn pig (Sus scrofa domesticus Erxleben, 1777).

Intrinsic cardiac neurons (ICNs) are crucial cells in the neural regulation of heart rhythm, myocardial contractility, and coronary blood flow. ICNs exhibit diversity in their morphology and neurotransmitters that probably are age-dependent. Therefore, neuroanatomical heart studies have been currently focused on the identification of chemical phenotypes of ICNs to disclose their possible functions in heart neural regulation. Employing whole-mount immunohistochemistry, we examined ICNs from atria of the newborn pigs (Sus scrofa domesticus) as ICNs at this stage of development have never been neurochemically characterized so far. We found that the majority of the examined ICNs (>60%) were of cholinergic phenotype. Biphenotypic neuronal somata (NS), that is, simultaneously positive for two neuronal markers, were also rather common and distributed evenly within the sampled ganglia. Simultaneous positivity for cholinergic and adrenergic neuromarkers was specific in 16.4%, for cholinergic and nitrergic-in 3.5% of the examined NS. Purely either adrenergic or nitrergic ICNs were observed at 13% and 3.1%, correspondingly. Purely adrenergic and nitrergic NS were the most frequent in the ventral left atrial subplexus. Similarly to neuronal phenotype, sizes of NS also varied depending on the atrial region providing insights into their functional implications. Axons, but not NS, positive for classic sensory neuronal markers (vesicular glutamate transporter 2 and calcitonin gene-related peptide) were identified within epicardiac nerves and ganglia. Moreover, a substantial number of ICNs could not be attributed to any phenotype as they were not immunoreactive for antisera used in this study. Numerous dendrites with putative peptidergic and adrenergic contacts on cholinergic NS contributed to neuropil of ganglia. Our observations demonstrate that intrinsic cardiac ganglionated plexus is not fully developed in the newborn pig despite of dense network of neuronal processes and numerous signs of neural contacts within ganglia.

Virus Mimetic Poly (I:C)-Primed Airway Exosome-like Particles Enter Brain and Induce Inflammatory Cytokines and Mitochondrial Reactive Oxygen Species in Microglia.

Viral infections induce extracellular vesicles (EVs) containing viral material and inflammatory factors. Exosomes can easily cross the blood-brain barrier during respiratory tract infection and transmit the inflammatory signal to the brain; however, such a hypothesis has no experimental evidence. The study investigated whether exosome-like vesicles (ELVs) from virus mimetic poly (I:C)-primed airway cells enter the brain and interact with brain immune cells microglia. Airway cells were isolated from Wistar rats and BALB/c mice; microglial cell cultures-from Wistar rats. ELVs from poly (I:C)-stimulated airway cell culture medium were isolated by precipitation, visualised by transmission electron microscopy, and evaluated by nanoparticle analyser; exosomal markers CD81 and CD9 were determined by ELISA. For in vitro and in vivo tracking, particles were loaded with Alexa Fluor 555-labelled RNA. Intracellular reactive oxygen species (ROS) were evaluated by DCFDA fluorescence and mitochondrial superoxide-by MitoSOX. ELVs from poly (I:C)-primed airway cells entered the brain within an hour after intranasal introduction, were internalised by microglia and induced intracellular and intramitochondrial ROS production. There was no ROS increase in microglial cells was after treatment with ELVs from airway cells untreated with poly (I:C). In addition, poly (I:C)-primed airway cells induced inflammatory cytokine expression in the brain. The data indicate that ELVs secreted by virus-primed airway cells might enter the brain, cause the activation of microglial cells and neuroinflammation.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Neuroanatomy of the Pig Cardiac Ventricles. A Stereomicroscopic, Confocal and Electron Microscope Study.

Although the pig is a model for heart disease, the neuroanatomy of cardiac ventricles (CV) in this species remains undetailed. We aimed to define the innervation pattern of pig CV, combining histochemistry for acetylcholinesterase, immunofluorescent labeling and electron microscopy. Forty nine examined pig hearts show that the major nerves supplying the ventral side of CV descend from the venous part of the heart hilum. Fewer in number and smaller in size, epicardial nerves supply the dorsal half of the CV. Epicardial nerves on the left ventricle are thicker than those on the right. Ventricular ganglia of various sizes distribute at the basal level of both CV. Averagely, we found 3,848 ventricular neuronal somata per heart. The majority of somata were cholinergic, although ganglionic cells of different neurochemical phenotypes (positive for nNOS, ChAT/nNOS, or ChAT/TH) were also observed. Large and most numerous nerves proceeded within the epicardium. Most of endocardium and myocardium contained a network of nerve bundles and nerve fibers (NFs). But, a large number of thin nerves extended along the bundle of His and its branches. The majority of NFs were adrenergic, while cholinergic NFs were scarce yet more abundant than nitrergic ones. Sensory NFs positive for CGRP were the second most abundant phenotype after adrenergic NFs in all layers of the ventricular wall. Electron microscopy elucidated that ultrastructure of nerves varied between different areas of CV. The described structural organization of CV provides an anatomical basis for further functional and pathophysiological studies in the pig heart. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1756-1780, 2017. © 2017 Wiley Periodicals, Inc.

Innervation of sinoatrial nodal cells in the rabbit.

In spite of the fact that the rabbit is being widely used as a laboratory animal in experimental neurocardiology, neural control of SAN cells in the rabbit heart has been insufficiently examined thus far. This study analyzes the distribution of SAN cells and their innervation pattern employing fluorescent immunohistochemistry on rabbit whole mount atrial preparations. A dense network of adrenergic (positive for TH), cholinergic (positive for ChAT), nitrergic (positive for nNOS) and possibly sensory (positive for SP) NFs together with numerous neuronal somata were identified on the RRCV where the main mass of SAN cells positive for HCN4 were distributed as well. In general, the area occupied by SAN cells comprised nearly the entire RRCV and possessed a three to four times denser network of NFs compared with adjacent atrial walls. Adrenergic NFs predominated noticeably in-between SAN cells. Solitary neuronal somata or somata gathered into small clusters were positive solely for ChAT or nNOS, respectively or simultaneously for both neuronal markers (ChAT and nNOS). Neuronal somata positive for nNOS were more frequent than those positive for ChAT. In conclusion, findings of the present study demonstrate a dense and complex ganglionated neural network of both autonomic and sensory NFs, closely related to SAN cells which spread widely on the RRCV and extend as sleeves of these cells toward the walls of the rabbit RA.

Innervation of the rabbit cardiac ventricles.

The rabbit is widely used in experimental cardiac physiology, but the neuroanatomy of the rabbit heart remains insufficiently examined. This study aimed to ascertain the architecture of the intrinsic nerve plexus in the walls and septum of rabbit cardiac ventricles. In 51 rabbit hearts, a combined approach involving: (i) histochemical acetylcholinesterase staining of intrinsic neural structures in total cardiac ventricles; (ii) immunofluorescent labelling of intrinsic nerves, nerve fibres (NFs) and neuronal somata (NS); and (iii) transmission electron microscopy of intrinsic ventricular nerves and NFs was used. Mediastinal nerves access the ventral and lateral surfaces of both ventricles at a restricted site between the root of the ascending aorta and the pulmonary trunk. The dorsal surface of both ventricles is supplied by several epicardial nerves extending from the left dorsal ganglionated nerve subplexus on the dorsal left atrium. Ventral accessing nerves are thicker and more numerous than dorsal nerves. Intrinsic ventricular NS are rare on the conus arteriosus and the root of the pulmonary trunk. The number of ventricular NS ranged from 11 to 220 per heart. Four chemical phenotypes of NS within ventricular ganglia were identified, i.e. ganglionic cells positive for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and biphenotypic, i.e. positive for both ChAT/nNOS and for ChAT/tyrosine hydroxylase. Clusters of small intensely fluorescent cells are distributed within or close to ganglia on the root of the pulmonary trunk, but not on the conus arteriosus. The largest and most numerous intrinsic nerves proceed within the epicardium. Scarce nerves were found near myocardial blood vessels, but the myocardium contained only a scarce meshwork of NFs. In the endocardium, large numbers of thin nerves and NFs proceed along the bundle of His and both its branches up to the apex of the ventricles. The endocardial meshwork of fine NFs was approximately eight times denser than the myocardial meshwork. Adrenergic NFs predominate considerably in all layers of the ventricular walls and septum, whereas NFs of other neurochemical phenotypes were in the minority and their amount differed between the epicardium, myocardium and endocardium. The densities of NFs positive for nNOS and ChAT were similar in the epicardium and endocardium, but NFs positive for nNOS in the myocardium were eight times more abundant than NFs positive for ChAT. Potentially sensory NFs positive for both calcitonin gene-related peptide and substance P were sparse in the myocardial layer, but numerous in epicardial nerves and particularly abundant within the endocardium. Electron microscopic observations demonstrate that intrinsic ventricular nerves have a distinctive morphology, which may be attributed to remodelling of the peripheral nerves after their access into the ventricular wall. In conclusion, the rabbit ventricles display complex structural organization of intrinsic ventricular nerves, NFs and ganglionic cells. The results provide a basic anatomical background for further functional analysis of the intrinsic nervous system in the cardiac ventricles.

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.

Neuroanatomy of the murine cardiac conduction system: a combined stereomicroscopic and fluorescence immunohistochemical study.

The mouse heart is a popular model to study the function and autonomic control of the specialized cardiac conduction system (CCS). However, the precise identity and anatomical distribution of the intrinsic cardiac nerves that modulate the function of the mouse CCS have not been adequately studied. We aimed at determining the organization and distribution of the intrinsic cardiac nerves that supply the CCS of the mouse. In whole mouse heart preparations, intrinsic neural structures were revealed by histochemical staining for acetylcholinesterase (AChE). Adrenergic, cholinergic and peptidergic neural components were identified, respectively, by immunohistochemical labeling for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), substance P (SP), and protein gene product 9.5 (PGP 9.5). Myocytes of the CCS were identified by immunolabeling of hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4). In addition, the presence of CCS myocytes in atypical locations was verified using fluorescent immunohistochemistry performed on routine paraffin sections. The results demonstrate that four microscopic epicardial nerves orientated toward the sinuatrial nodal (SAN) region derive from both the dorsal right atrial and right ventral nerve subplexuses. The atrioventricular nodal (AVN) region is typically supplied by a single intrinsic nerve derived from the left dorsal nerve subplexus at the posterior interatrial groove. SAN myocytes positive for HCN4 were widely distributed both on the medial, anterior, lateral and even posterior sides of the root of the right cranial (superior caval) vein. The distribution of HCN4-positive myocytes in the AVN region was also wider than previously considered. HCN4-positive cells and thin slivers of the AVN extended to the roots of the ascending aorta, posteriorly to the orifice of the coronary sinus, and even along both atrioventricular rings. Notwithstanding the fact that cholinergic nerve fibers and axons clearly predominate in the mouse CCS, adrenergic nerve fibers and axons are abundant therein as well. Altogether, these results provide new insight into the anatomical basis of the neural control of the mouse CCS.