A restudy of cardiac conduction pathways by techniques for visualization of cholinesterase reaction

Histochemical reactions which demonstrate cholinesterase reactions in tissues were used for slides of serial frozen sections of hearts of pigs, dogs, and rats to determine whether there are special types of modified muscle cells in continuous pathways from the SA (sinoartial) to the AV (atrioventricular) node. There were positive reactions for acetylcholinesterase with less reaction for butyrl cholinesterase in ganglion cells and nerve fibers. No continuous pathways of cholinesterase-reacting cardiac muscle fibers from the SA to the AV node were identified although the muscle fibers were in intimate relation with the nerve fibers. No cells of Purkinje type were demonstrated in the atria.     

A histochemical study of the esterases of the rat heart

The histochemistry of esterases was studied in the rat heart, using various methods of tissue preparation and a large number of substrates, inhibitors and activators. Non-specific esterase and cholinesterase was demonstrated in the fibers of the atrioventricular conduction system, in atrial and ventricular muscle fibers, in cardiac neurons and in some of the nerve fibers of the heart. The highest concentration of both types of enzymes was found in the conduction system and in the neural elements. Pericytes and macrophages showed only non-specific esterase activity. Two broad types of non-specific esterase activity, cytoplasmic and lysosomal, were distinguished in the pericytes, macrophages and cardiac muscle fibers by the use of selective inhibitors and activators. The cytoplasmic activity is due to the presence of B and C esterases and was better demonstrated with esters of a-naphthol than with naphthol AS derivatives or indigogenic esters. The esterase activity of lysosomes appears to be due to a group of fluoride- and organophosphate-resistant enzymes, some of which are probably cathepsins.     

Sensory components in the terminal innervation of the ovine cardiac conduction system

Neuromuscular relationships in the cardiac conduction system of the ovine heart were studied by means of light and electron microscopy. Fluorescence microscopy was used to demonstrate the distribution of adrenergic nerves. Histochemical methods demonstrated sites of acetylcholinesterase and nonspecific cholinesterase activity at both light and electron microscopic levels. Surgical denervation was used in an attempt to support the identification of different types of nerve endings. A dense plexus of nerves strongly positive for acetylcholinesterase was present in all parts of the conduction system. Nonspecific cholinesterase was found to be limited to large nerve bundles which were presumed to be preganglionic. A dense plexus of adrenergic nerve fibers was identified in both the SA and AV nodes, but such fibers were very sparsely distributed along the AV bundle and its branches. On the basis of morphology as demonstrated by electron microscopy, three types of nerve endings or varicosities associated with the specialized cells of the conduction system were identified. Cholinergic endings, presumably post-ganglionic parasympathetic, contained many nongranulated synaptic vesicles. Adrenergic or post-ganglionic sympathetic endings or varicosities contained granulated synaptic vesicles. Axonal enlargements containing numerous mitochondria and varying numbers of pleomorphic dense bodies were identified as sensory endings on the basis of their morphologic similarity to such endings found elsewhere in the animal body. These were found in 22% of the blocks examined by electron microscopy, were present in all parts of the conduction tissue, and were acetylcholinesterase positive. They did not show obvious signs of degeneration following vagotomies or thoracic sympathectomy, suggesting the possibility that they were endings of neurons intrinsic to the heart. No signs of degeneration were noted in the endomysial plexus following vagotomy either proximal or distal to the distal (nodose) ganglion. Degeneration of small fibers of the plexus was noted following thoracic sympathectomy. This study provides evidence suggesting a significant sensory innervation to the cardiac conduction system, with the cell bodies of the sensory neurons located in or near the heart.     

Cholinesterase activity in terminal Schwann cells associated with Ruffini endings in the periodontal ligament of rat incisors

A nonspecific cholinesterase activity was demonstrated in terminal Schwann cells associated with Ruffini endings in the periodontal ligament of rat incisors at the light and electron microscopic levels. The terminal Schwann cells are ultrastructurally characterized by a well-developed Golgi apparatus and rough endoplasmic reticulum. The cells in this study were positive for nonspecific cholinesterase, whereas ordinary Schwann cells associated with more proximal nerve fibers reacted negatively. The reaction products were densely deposited in the cisternae of the rough endoplasmic reticulum and along the nuclear envelop. A moderately intense labeling was found in the cytoplasmic extensions, in which the reaction products gathered in caveolae and vesicles. These findings indicate that nonspecific cholinesterase is a useful marker to distinguish terminal Schwann cells from ordinary Schwann cells and that the enzyme may be synthesized in the rough endoplasmic reticulum and conveyed toward the axon terminals. Since this enzyme has been known to be shared by the inner bulb of Pacinian corpuscles and the lamellar cells of Meissner's corpuscles, its possible involvement in mechanoreceptive functions in these specialized Schwann cells deserves further investigation.     

Immunolocalization of N-CAM in the heart of the early developing rat embryo

Background: Neural cell adhesion molecule (N-CAM) is important in the migration of neural crest cells and is expressed in the developing heart. The pattern of expression of N-CAM in the heart of early rat embryos was investigated to shed light on the potential role of N-CAM in cardiac neural crest cell migration. Methods: N-CAM expression was studied by immunohistochemistry in Sprague-Dawley rat hearts between embryonic days 11.5 and 15.5 HNK-1 immunoreactivity was also investigated for comparison with that of N-CAM. Results: A continuity of N-CAM immunoreactivity was transiently detected from the outflow tract to the recurrent nerve. N-CAM was also expressed around the sinus venosus, inferior vena cava, sinotrial septum, and coronary sinus, as well as on mesenchymal cells in the atrioventricular endocardial cushion tissues. Conclusions: The continuous N-CAM immunoreactivity from the outflow tract to the recurrent nerve appeared to represent the pathway along which cardiac neural crest cells migrate. N-CAM-immunoreactive sites around the sinus venosus may correspond to migrating neural crest cells that differentiate into nerve fibers or cardiac ganglia. Results indicate that N-CAM may play an important role in the migration, proliferation, and transformation of neural crest cells, thereby contributing to cardiac morphogenesis and to innervation around the heart and great arteries. © 1995 Wiley-Liss, Inc.     

Direct visualization by scanning electron microscopy of the preganglionic innervation and synapses on the true surfaces of neurons in the frog heart

A simple and flexible technique is reported for scanning electron microscopic observation of the true surfaces of neurons, and the distribution of glial cells, preganglionic nerve fibers and synapses on them. The interatrial septum of the frog was chosen as a convenient preparation, and connective tissue and glial cells were successively removed by incubation in collagenase and protease solutions. The three-dimensional relationships between presynaptic fibers and target neurons can be observed with high resolution over long distances. Some neurons have multiple innervation. Varicosities can be seen clearly, and where their interior has been exposed, synaptic vesicles can be discerned.     

Bradycardia during stimulation of the septum and somatic afferents in the rabbit.

Experiments were done in paralyzed rabbits anesthetized with either pentobarbital sodium or alpha-chloralose to test the possibility that the septum may alter the cardiovascular responses elicited by stimulation of somatic afferent fibers. Electrical stimulation of the lesser saphenous nerve (LSN), a branch of the sciatic nerve, at certain parameters elicited bradycardia, which could be abolished by bilateral vagotomy or intravenous injection of atropine methylbromide. Distinct and characteristic changes in mean arterial pressure and heart rate were elicited by electrical stimulation of histologically localized sites in five septal areas. Septal sites from which stimulation elicited bradycardia were chosen for the study of interaction between the septum and LSN. The cardiac slowing elicited by combined stimulation of the lateral septum and LSN was significantly greater than the sum of the responses elicited by separate stimulation of the two different structures. In contrast, the bradycardia elicited by combined stimulation of medial septal structures and LSN was significantly smaller than the sum of the individual responses. These experiments demonstrate that the magnitude of heart rate responses elicited by stimulation of somatic afferent fibers may be modified by the septum.     

Morphological study of the innervation pattern of the rabbit sinoatrial node

The pattern of nerves, ganglia, and fine nerve processes in the adult rabbit sinoatrial node, identified by microelectrode recording, was defined by staining histochemically for cholinesterase followed by silver impregnation. A generalized repeatable pattern of innervation was recognized, including (1) a large ganglionic complex inferior to the sinoatrial node; (2) two or three moderately large nerves traversing the sinoatrial node parallel to the crista terminalis; (3) nerves entering the region from the atrial septum, the superior vena cava, and the inferior vena cava; and (4) a fine network of nerve processes, particularly extensive in the morphologically dense small-cell part of the sinoatrial node. When the site of initial depolarization in the node was located and marked by a broken-off electrode tip, it was found, after cholinesterase staining, to be characterized by a cluster of cells enclosed in a nest or basket of fine nerves. Similar nested cell clusters were observed elsewhere in the sinoatrial node in this same preparation and in other hearts. A complex interweaving of atrial muscle fibers was observed medial and inferomedial to the sinoatrial node, which may form the anatomical basis for the lack of conduction through this region. The morphological pattern of nerves, ganglia, and myocardial cells described in this study emphasizes the complexity of innervation of the sinoatrial node, including its intrinsic neural elements. Cholinesterase/silver staining can be useful in the definition and comparison of electrophysiologically identified sites within the sinoatrial node.     

大鼠心瓣膜神经支配

本文通过对32只大鼠心瓣膜进行胆碱酯酶组织化学法、乙醛酸诱发荧光法及P物质免疫组化方法的观察,获得心瓣膜胆碱能、肾上腺素能及肽能(SP)神经支配全貌。P物质免疫反应阳性神经纤维在房室瓣自瓣膜基部向下及自腱索向上进入瓣膜;在动脉瓣自附着缘进入瓣膜。该纤维的性质及走向提示SP免疫反应阳性神经纤维可能是感受血流动力学变化的传入神经。     

Morphological studies of the altered pulps of the New Zealand white rabbit after resection of the inferior alveolar nerve and/or the superior cervical ganglion

This study was carried out to determine the effects of denervation of the pulp organs of teeth, particularly on the odontoblasts and their processes. Therefore, resection of the inferior alveolar, cervical sympathetic, or a combination of both types of nerves supplying the mandibular incisor teeth of New Zealand white rabbits was performed and the results compared to adjacent, unaltered teeth. Neural stains were used to evaluate the presence or absence of nerve elements, while histochemical and titrametric methods were employed to determine the presence, location and concentration of cholinesterase enzymes in these teeth. Dilation of blood vessels was noted after cervical sympathetic resection and larger nerve trunks as well as peripherally located fine fibers were absent in the pulps following inferior alveolar nerve resection. Further, irregular dentin formation and associated small openings in the tips of the teeth were observed after nerve resection. Titrametric analysis indicated a significant decrease in the concentration of cholinesterase in the pulp organ of incisors 15–19 days after resection of either the inferior alveolar nerve or both inferior alveolar and cervical sympathetic nerves. Only a slight decrease in the concentration of cholinesterase was noted after removal of the cervical sympathetic nerve alone. Histochemical results confirmed a decrease in cholinesterase after resection of the inferior alveolar, cervical sympathetic or both nerves. The concentration of the oxidative enzyme, succinic dehydrogenase, however, increased after all three types of nerve resection. This increase was apparent particularly in the odontoblastic cells underlying the predentin.     

新生大鼠脊神经节与心肌细胞联合培养的光镜观察

将新生大鼠的脊神经节与心肌细胞进行联合培养，用相差显微镜和Ｈｏｌｍｅｓ还原银染色观察了神经元的生长以及神经纤维与心肌细胞之间的关系。脊神经节与心肌细胞联合培养７２￣９６小时可观察到神经纤维终止于搏动的心肌细胞表面。神经节组织块周围有许多神经纤维在心肌细胞表面相互交织成网状。交叉的神经纤维相互粘连在一起，终止于搏动的心肌细胞表面的一条神经纤维移动将会牵动邻近的交叉神经纤维网。Ｈｏｌｍｅｓ还原银染色结     

Vagotomy induced changes in acetyl cholinesterase staining and substance P-like immunoreactivity in the gustatory lobes of goldfish

Summary In teleost fish, the visceral sensory nuclei of the medulla are clearly separated into gustatory lobes and a general visceral sensory nucleus. Those branches of the vagus nerve which innervate the orobranchial cavity terminate in the vagal gustatory lobe, while the general visceral component of the vagus nerve terminates in the separate general visceral nucleus. In goldfish, the vagal lobe is a complex, laminated structure containing both motor and sensory elements.Transection of the vagus nerve results in distinct changes in the pattern of acetylcholinesterase staining and substance-P-like (SPL) immunoreactivity in the vagal lobe of goldfish. Following vagotomy, cholinesterase activity is eliminated from layers 4 and 6, both being layers in which primary gustatory afferent fibers terminate. In addition, SPL immunoreactive fibers disappear from the capsular root of the vagus nerve. These results indicate that the primary afferent input to the gustatory lobe involves at least two cytochemically distinct fiber types, one containing substance-P-immunoreactive material and the other containing or inducing acetylcholinesterase activity.Vagotomy also affects immunostaining and cholinesterase activity of the motonuerons deep in the vagal lobe. Following nerve transection, acetylcholinesterase activity is diminished, and SPL-immunoreactivity increased in the affected motoneurons. Similar changes were observed in axotomized motoneurons of other cranial nerve nuclei.     

Significant Differences in Sympathetic Nerve Fiber Density Among the Facial Skin Nerves: A Histologic Study Using Human Cadaveric Specimens

Sympathetic nerve fibers in the skin nerves are connected with vasomotor, thermoregulatory, sensory input modulatory, and immunologic events; however, to our knowledge, no histological information is available for skin nerves in the human face. Using specimens from 17 donated cadavers (mean age, 86 years), we measured a sectional area of tyrosine hydroxylase (TH)‐positive fibers in (1) the frontal nerve (V1), (2) the infraorbital nerve (V2), (3) the mental nerve (V3), (4) the greater auricular nerve (C2), (5) the auriculotemporal nerve (ATN), and (6) the zygomatic branch of the facial nerve (VII). The V1, V2, and V3 were obtained at their entrances to the subcutaneous tissue from the bony canal or notch. The V1, C2, ATN, and/or VII usually contained abundant TH‐positive fibers (almost 3%–8% of the nerve sectional area), whereas the V2 and V3 consistently carried few TH‐positive fibers (<1%). The difference between these two groups was quite significant (P < 0.001). Thus, from the superior cervical ganglion, the sympathetic nerve fibers reached the forehead through the frontal nerve trunk, whereas artery‐bounded fibers came to the cheek, nose, and mouth. The sympathetic palsy caused by trigeminal nerve involvement is mainly characterized by the symptoms seen in the distribution of the ophthalmic division of the trigeminal nerve, such as in Horner's syndrome. It suggests that the forehead and the other facial areas are representative parts of those different sympathetic innervations that could be useful for evaluating the sympathetic function of the face in various diseases. Anat Rec, 299:1054–1059, 2016. © 2016 Wiley Periodicals, Inc.     

Resident Stem Cells in the Myocardium of Patients with Obstructive Hypertrophic Cardiomyopathy

Interventricular septum myocardium was studied in 40 patients with obstructive hypertrophic cardiomyopathy. Immunohistochemical assay revealed c-kit-positive resident cardiac stem cells in 82.5% patients. The content of the connective tissue and myofibrillar disarray zones and the degree of cardiomyocyte hypertrophy and myolysis were determined. In 30% cases, cardiomyocytes containing atrial natriuretic peptide were detected in the interventricular septum myocardium. The data were compared with clinical and functional parameters of patients. It was found that cardiac stem cells are present in patients, whose myocardium was characterized by increased density of the connective tissue, hypertrophy of mature cardiomyocytes, medium degree of myolysis in them, and accumulation of natriuretic peptide, a cardiac failure marker, in cardiomyocytes.     

Pathologic anatomy of the cardiomyopathies. Idiopathic dilated and hypertrophic types, infiltrative types, and endomyocardial disease with and without eosinophilia.

This presentation summarizes necropsy observations in patients with three types of cardiomyopathy: idiopathic, infiltrative, and endomyocardial disease. The idiopathic variety is subdivided into two types depending on the size of the ventricular cavity. In the dilated ventricular type the left ventricular wall is frequently less than 1.5 cm. thick, intracardiac thrombi are common, the atrioventricular valve rings usually are mildly dilated, and focal myocardial and endocardial scars are common. In the nondilated type (hypertrophic cardiomyopathy), the ventricular septum is usually thicker than the left ventricular free wall, which also is thick (greater than 1.5 cm.). When the septum is similar in thickness to the left ventricular free wall (symmetric), left ventricular outflow obstruction does not occur. When the septum is thicker than the left ventricular free wall (asymmetric), left or right ventricular outflow obstruction may or may not be present. The orientation of myocardial fibers one to another in the ventricular septum in the nondilated (hypertrophic) type is abnormal, whereas it is normal in the dilated ventricular type. Intracardiac thrombi are rare and atrioventricular valve rings are never dilated in the nondilated type of idiopathic cardiomegaly. The infiltrative types of cardiomyopathies include iron, calcium, lipids, mucopolysaccharides, granulomas, amyloid, and neoplasms. The first four usually are located within myocardial cells and the latter three, between myocardial cells. It is probable that all these myocardial infiltrates are capable of producing cardiac dysfunction, primarily on a restrictive basis. Endomyocardial disease may or may not be associated with eosinophilia. When the latter occurs, the eosinophils are structurally normal. Death is related to congestive cardiac failure. This category is actuality also in idiopathic.     

迷走神经结状神经节与心肌细胞联合培养的扫描电镜观察

用新生大鼠的迷走神经结状神经节与心肌细胞进行联合培养,用扫描电镜观察了神经纤维或神经终末与心肌细胞之间的关系。在低倍下可见结状神经节组织块长出的神经纤维呈放射状向四周延伸,到达分散的心肌细胞表面;在扫描电镜高倍下所观察到的神经纤维或神经终末的形态不同,神经纤维粗细不一,在单个心肌细胞表面相互交织成网状。有的神经纤维在行程中粗细基本均匀一致．而有的神经纤维则在行程中出现膨体样结构。终止于心肌细胞表面的神经终末的形态也不相同,有的末端膨大终止于心肌细胞表面,有的末端膨大并呈扇形展开后紧贴于心肌细胞表面,这种膨大的神经末梢与心肌细胞之间的关系似比在体时更为密切。     

Metallophilia in the Ranvier's nodes of nerve fibers in rats of different strains (histochemical study)

Myelinated nerve fibers formed by the processes of LV spinal ganglion neurons were studied in two lines of rats selected according to high and low thresholds of nerve system excitability to electric current. Before ultrastructural study the fibres have been treated with potassium pyroantimonate. It was demonstrated that specific dense precipitate was deposited in the nodes of Ranvier of the nerve fibers of rats with low excitability thresholds; this precipitate was not found in the fibers of rats with high thresholds. It is suggested that deposition of precipitate is indicative of a high density of sodium channels in neurilemma, i.e. of high functional activity of nerve fibers.     

Vagal afferent inhibition of primate thoracic spinothalamic neurons

Spinothalamic (ST) neurons in the C8-T5 segments of the spinal cord were examined for responses to electrical stimulation of the left thoracic vagus nerve (LTV). Seventy-one ST neurons were studied in 39 anesthetized monkeys (Macaca fascicularis). Each neuron could be excited by manipulation of its somatic field and by electrical stimulation of cardiopulmonary sympathetic afferent fibers. LTV stimulation resulted in inhibition of the background activity of 43 (61%) ST neurons. Nine (13%) were excited, 3 (4%) were excited and then inhibited, while 16 (22%) did not respond. There was little difference among these groups in terms of the type of somatic or sympathetic afferent input although inhibited cells tended to be more prevalent in the more superficial laminae. The degree of inhibition resulting from LTV stimulation was related, in a linear fashion, to the magnitude of cell activity before stimulation. LTV inhibition of background activity was similar among wide dynamic range, high threshold, and high-threshold cells with inhibitory hair input. Any apparent differences in LTV inhibitory effects among these groups were accounted for by the differences in ongoing cell activity as predicted by linear regression analysis. LTV stimulation inhibited responses of 32 of 32 ST cells to somatic stimuli. In most cases the stimulus was a noxious pinch; however, LTV stimulation also inhibited responses to innocuous stimuli such as hair movement. Bilateral cervical vagotomy abolished the inhibitory effect of LTV stimulation on background activity (six cells) or responses to somatic stimuli (seven cells). Stimulation of the cardiac branch of the vagus inhibited activity of three cells to a similar degree as LTV stimulation, while stimulation of the vagus below the heart was ineffective in reducing activity of 10 cells. We conclude that LTV stimulation alters activity of ST neurons in the upper thoracic spinal cord. Vagal inhibition of ST cell activity was due to stimulation of cardiopulmonary vagal afferent fibers coursing to the brain stem, which appear to activate descending inhibitory spinal pathways. Vagal afferent activity may participate in processing of somatosensory information as well as information related to cardiac pain.     

Morphologic evidence that the renal calyx and pelvis control ureteric activity in the rabbit

The structure of the renal calyx, pelvis and upper ureter has been investigated in the adult rabbit. The methods employed for light microscopy were designed to demonstrate smooth muscle, cholinesterase and catecholamine-containing nerves, elastic and collagenous fibers, and glycogen. Selected areas of the upper urinary tract were also examined using the electron microscope. The wall of the calyx and pelvis includes two layers of muscle cells the inner one of which appears to be continuous with the muscle coat of the ureter. The external layer extends across the renal pelvis from the calyx and ends abruptly in the pelvi-ureteric region. In some regions cells from this layer run towards and intermingle with the muscle of the inner layer. The muscle cells forming the outer layer are associated with many adrenergic nerves which run in the connective tissue separating individual muscle cells. Numerous elastic fibrils are closely related to these cells which usually contain glycogen. In contrast, the muscle cells forming the inner layer of the pelvis and those of the upper ureter have a relatively poor innervation, are closely packed together without intervening connective tissue, and are usually devoid of glycogen. These observations provide structural evidence in support of the concept that specific regions of the renal calyx and pelvis influence and possbily control the activity of the ureter.     

Physiological characteristics of the cardiac branches of the recurrent nerves

Summary The cardiac branches of recurrent nerves in 34 cats and 5 dogs were studied in acute experiments. The data obtained testify to the presence in recurrent nerves of afferent and efferent nerve fibers involved in the regulation of the arterial pressure and cardiac activity. Upon stimulation of recurrent nerves as well as their cardiac branches depressor and pressor effects were observed, this giving grounds to consider recurrent nerves as an intermediate type. This is confirmed by a number of anatomic studies of different authors.(Presented by Active Member AMN SSSR V. N. Shamov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 54, No. 9, pp. 14–18, September, 1962