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.

Saturation of visual responses explains size tuning in rat collicular neurons.

The receptive field of many visual neurons is composed of a central responsive area, the classical receptive field, and a non-classical receptive field, also called the "suppressive surround." A visual stimulus placed in the suppressive surround does not induce any response but modulates visual responses to stimuli within the classical receptive field, usually by suppressing them. Therefore, visual responses become smaller when stimuli exceed the classical receptive field size. The stimulus size inducing the maximal response is called the preferred stimulus size. In cortex, there is good correspondence between the sizes of the classical receptive field and the preferred stimulus. In contrast, in the rodent superior colliculus, the preferred size is often several fold smaller than the classical receptive field size. Here, we show that in the rat superior colliculus, the preferred stimulus size changes as a square root of the contrast inverse and the classical receptive field size is independent of contrast. In addition, responses to annulus were largely independent of the inner hole size. To explain these data, three models were tested: the divisive modulation of the gain by the suppressive surround (the "normalization" model), the difference of the Gaussians, and a divisive model that incorporates saturation to light flux. Despite the same number of free parameters, the model incorporating saturation to light performed the best. Thus, our data indicate that in rats, the saturation to light can be a dominant phenomenon even at relatively low illumination levels defining visual responses in the collicular neurons.

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.

Comparative gross anatomy of epicardiac ganglionated nerve plexi on the human and sheep cardiac ventricles.

This study aimed to examine the distribution and quantitative parameters of the epicardiac ventricular neural ganglionated plexus in the hearts of humans and sheep, highlighting the differences of this plexus in humans and large models. Five non-sectioned pressure distended whole hearts of the human newborns and 10 hearts of newborn German black-faced lambs were investigated applying a histochemical method for acetylcholinesterase to stain epicardiac neural structures with their subsequent stereomicroscopic examination. In humans, the ventricular nerves are spread by four epicardiac nerve subplexuses, that is, the left and right coronary as well as the left and middle dorsal. In sheep, the ventricular nerves are spread by five epicardiac nerve subplexuses, that is, the left and right coronary, the left and middle dorsal and the right ventral ones. The ventricular epicardium involved up to 129 ganglia in humans and up to 198-in sheep. The largest number of the ventricular ganglionic cells in humans were located on the ventral side, in front of the conus arteriosus, while on sheep ventricles, the most numerous neurons distributed on the dorsal wall of the left ventricle. This comparative study of the morphological patterns of the human and sheep ventricles demonstrates that the sheep heart is neuroanatomically distinct from the human one and this must be taking into consideration using the sheep model for the heart physiology experiments.

An efficient rAAV vector for protein expression in cortical parvalbumin expressing interneurons.

Recombinant adeno-associated viruses (rAAV) are extensively used in both research and clinical applications. Despite significant advances, there is a lack of short promoters able to drive the expression of virus delivered genes in specific classes of neurons. We designed an efficient rAAV vector suitable for the rAAV-mediated gene expression in cortical interneurons, mainly in the parvalbumin expressing cells. The vector includes a short parvalbumin promoter and a specialized poly(A) sequence. The degree of conservation of the parvalbumin gene adjoining non-coding regions was used in both the promoter design and the selection of the poly(A) sequence. The specificity was established by co-localizing the fluorescence of the virus delivered eGFP and the antibody for a neuronal marker. rAAV particles were injected in the visual cortex area V1/V2 of adult rats (2-4 months old). Neurons expressing the virus delivered eGFP were mainly positive for interneuronal markers: 66.5 ± 2.8% for parvalbumin, 14.6 ± 2.4% for somatostatin, 7.1 ± 1.2% for vasoactive intestinal peptide, 2.8 ± 0.6% for cholecystokinin. Meanwhile, only 2.1 ± 0.5% were positive for CaMKII, a marker for principal cells in the cortex. The efficiency of the construct was verified by optogenetic experiments: the expression of the virus delivered ChR2 channels was sufficient to evoke by blue light laser high frequency bursts of action potentials in putative fast spiking neurons. We conclude that our promoter allows highly specific expression of the rAAV delivered cDNAs in cortical interneurons with a strong preference for the parvalbumin positive cells.

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.

GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition.

BACKGROUND: Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically sympathetic nerve activity remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity and tonic drive from the peripheral chemoreceptors, the cause of which is unknown. We have previously shown hypertension to be critically dependent on the carotid body (CB) input in spontaneously hypertensive rat, a model that also exhibits a number of diabetic traits. CB overstimulation by insulin and leptin has been similarly implicated in the development of increased sympathetic nerve activity in metabolic syndrome and obesity. Thus, we hypothesized that in hypertensive diabetic state (spontaneously hypertensive rat), the CB is sensitized by altered metabolic signaling causing excessive sympathetic activity levels and dysfunctional reflex regulation. METHODS: Using a hypothesis-free RNA-seq approach, we investigated potential molecular targets implicated in energy metabolism mediating CB sensitization and its regulation of sympathetic outflow in experimental hypertension. Identified targets were characterized using molecular and functional techniques assessing peripheral chemoreflex sensitivity in situ and in vivo. RESULTS: We discovered GLP1R (glucagon-like peptide-1 receptor) expression in the CBs of rat and human and showed that its decreased expression is linked to sympathetic hyperactivity in rats with cardiometabolic disease. We demonstrate GLP1R to be localized to CB chemosensory cells, while targeted administration of GLP1R agonist to the CB lowered its basal discharge and attenuated chemoreflex-evoked blood pressure and sympathetic responses. Importantly, hyperglycemia-induced peripheral chemoreflex sensitization and associated basal sympathetic overactivity were abolished by GLP1R activation in the CB suggesting a role in a homeostatic response to high blood glucose. CONCLUSIONS: We show that GLP1 (glucagon-like peptide-1) modulates the peripheral chemoreflex acting on the CB, supporting this organ as a multimodal receptor. Our findings pinpoint CBs as potential targets for ameliorating excessive sympathetic activity using GLP1R agonists in the hypertensive-diabetic condition.

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.

Ultrastructural changes of the human enteric nervous system and interstitial cells of Cajal in diverticular disease.

BACKGROUND: In spite of numerous advances in understanding diverticular disease, its pathogenesis remains one of the main problems to be solved. We aimed to investigate the ultrastructural changes of the enteric nervous system in unaffected individuals, in asymptomatic patients with diverticulosis and in patients with diverticular disease. METHODS: Transmission electron microscopy was used to analyse samples of the myenteric, outer submucosal and inner submucosal plexuses from patients without diverticula (n=9), asymptomatic patients with diverticulosis (n=7) and in patients with complicated diverticular disease (n=9). We described the structure of ganglia, interstitial cells of Cajal and enteric nerves, as well as their relationship with each other. The distribution and size of nerve processes were analysed quantitatively. RESULTS: In complicated diverticular disease, neurons exhibited larger lipofuscin-like inclusions, their membranous organelles had larger cisterns and the nucleus showed deeper indentations. Nerve remodeling occurred in every plexus, characterised by an increased percentage of swollen and fine neurites. Interstitial cells of Cajal had looser contacts with the surrounding cells and showed cytoplasmic depletion and proliferation of the rough endoplasmic reticulum. In asymptomatic patients with diverticulosis, alterations of enteric nerves and ICC were less pronounced. CONCLUSIONS: In conclusion, the present findings suggest that most ultrastructural changes of the enteric nervous system occur in complicated diverticular disease. The changes are compatible with damage to the enteric nervous system and reactive remodeling of enteric ganglia, nerves and interstitial cells of Cajal. Disrupted architecture of enteric plexuses might explain clinical and pathophysiological changes associated with diverticular disease.

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.

Section's osseous slice biopsy during major amputation of lower extremity: preliminary results of prospective cohort study.

BACKGROUND: The purpose of this cohort study was to assess the incidence of positive cultures in section's osseous slice biopsy (SOB) taken at the level of major limb amputation. In case of positive cultures we sought whether the microorganisms present in SOB could take origin from the primary infection site necessitating the amputation. The impact of diabetes on culture results was also investigated. METHODS: This prospective cohort study, which aimed to confirm the results of the pilot study, analysed patients who underwent major limb amputation between 2012 and 2013 in three Lithuanian hospitals. SOBs at the amputation site (surgical bone biopsies) and percutaneous bone biopsies of the distal site were performed simultaneously during limb amputation. Tissue cultures were analysed by microbiologists, and species along with antibiograms were reported. Histopathological assessment and bacterial typing were also evaluated. A positive culture was defined as the identification of at least 1 bacteria not belonging to the skin flora, at least 2 bacteria belonging to the skin flora with the same antibiotic susceptibility profiles or the same bacteria belonging to the skin flora in two different sites. Fisher's exact test and Student's test were used to compare the populations and the microbiological results. The statistical significance level was set at P < 0.05. RESULTS: Sixty-nine patients (35 males/34 females), mean age 68.7 (S = 13.6) years, including 21 (30.4%) with diabetes underwent the major limb amputation. Forty-five amputations (65.2%) were done above the knee. In total, 207 SOBs and 207 percutaneous distal site biopsies were studied. SOB cultures were positive in 11 (15.9%) cases. In 5 (45.5%) cases the same microorganisms were identified in both SOB and distal biopsy cultures. No association between culture results and presence of diabetes was identified. CONCLUSIONS: Our results suggest that, independently of the diabetes status, foot infection may silently spread along the bone and can achieve the site of major limb amputation. Additional investigations aiming to confirm this hypothesis and to evaluate a prognostic value are in progress.

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.