Occurrence of adrenergic nerve fibers and of noradrenaline in thymus gland of juvenile and aged rats.

Adrenergic nerve fibers (ANF) were studied in juvenile, adult and old rats by biochemical and morphological methods and by quantitative analysis of images (QAI). After chemical sympathectomy with neurotoxin 6-OH dopamine, the greater part of ANF disappeared. The whole thymus was drawn in juvenile normal or sympathectomized rats, in adult normal or sympathectomized rats and in old normal or sympathectomized rats. Thymuses from the above-mentioned animals were weighed, measured and dissected. Thymic slices were stained with eosin orange for the detection of the microanatomical details and with Bodian's method for the recognition of the whole nerve fibers. Histofluorescence microscopy was used for staining of ANF while immunofluorescence microscopy was employed for staining of neuropeptide Y (NPY)-like immunoreactivity. Biochemical dosage of proteins and of noradrenaline amount was performed. Finally, all morphological results were subjected to QAI. Our results suggest that: (1) total innervation of the thymus increases with age; (2) ANF do not change with age; (3) the content of noradrenaline in the thymus increases with age; and (4) NPY-like immunoreactive structures in the thymus decrease with age. Biochemical results are in accordance with the morphological ones and both are confirmed by means of QAI. The probable function of sympathetic innervation of rat thymus is also discussed.     

Effect of interferon on human thymus microenvironment

Interferon, a thymic immunostimulator, was used with the aim of assessing the importance of adrenergic nerve fibers (ANF) and/or AChE-positive nerve fibers (AChENF) in the regulation of some immunological functions in humans. Thymic normal control fragments and/or thymic fragments of immunostimulated patients were removed during surgical biopsies. Thymic slices were stained with eosin-orange (for the recognition of microanatomical details of the microenvironment) and with Bodian's method for staining of nerve fibers. Histofluorescence microscopy was employed for staining ANF. AChENFs were detected by means of the direct-coloring thiocholine method. All images were submitted to quantitative morphometrical analysis and statistical comparisons of data. Moreover, the amount of proteins and noradrenaline was measured on thymic homogenates of the same patients. Treatment with interferon induces substantial changes in the thymic microenvironment, on ANF, on AChENFs and on the total amount of proteins and noradrenaline in thymic tissue homogenates. In conclusion immunostimulation with interferon induces substantial changes in the whole thymus and in its microenvironment, involving both sympathetic and parasympathetic nerve fibers.     

Histo- and immunohistochemical changes in acetylcholinesterase and choline acetyltransferase activities in the amygdaloid complex in aged rats.

Histo- and immunohistochemical distribution of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the amygdaloid nuclei of young adult (3 month old) and aged (26 month old) Wistar rats was compared. AChE staining and ChAT immunoreactivity showed the same regional variations in the amygdaloid nuclei of young adult rats. The density and staining of AChE- and ChAT-positive fibres, terminals, and nerve cells were reduced in aged rat amygdala. Moreover, heavily stained aberrant fibres and coarse terminals were located around the nerve cells, blood vessels, and occasionally in patches. In aged rats, atrophic AChE positive and ChAT immunoreactive nerve cells exhibited serpentine-like, thicker, and less extensively branched dendrites than those in young adult rats. These changes are similar to the age-related changes in the cholinergic enzymes in other brain regions which are targets to the basal forebrain.     

Monoamine oxidase enzymes and oxidative stress in the rat optic nerve: age‐related changes

In this study, age‐related changes in the monoamine oxidases (MAO) were studied in the optic nerve (ON) of both young and aged male rats. The aim of the study was to assess the role of MAO in age‐related changes in the rat ON and explain the mechanisms of neuroprotection mediated by MAO‐B‐specific inhibitors. Fifteen three month old and fifteen 26 month old Sprague–Dawley rats were used. The animals were killed by terminal anaesthesia. Staining of MAO, quantitative analysis of images, biochemical assays and statistical analysis of data were carried out. Samples of the ON were washed in water, fixed in Bowen fluid, dehydrated and embedded in Entellan. Histological sections were stained for MAO‐enzymatic activities. The specificity of the reaction was evaluated by incubating control sections in a medium either without substrate or without dye. The quantitative analysis of images was carried out at the same magnification and the same lighting using a Zeiss photomicroscope. The histochemical findings were compared with the biochemical results. After enzymatic staining, MAO could be demonstrated in the ON fibres of both young and aged animals; however, MAO were increased in the nerve fibres of the elderly rats. These morphological findings were confirmed biochemically. The possibility that age‐related changes in MAO levels may be attributed to impaired energy production mechanisms and/or represent the consequence of reduced energy needs is discussed.     

Connections between Adrenergic Nerves and other Tissue Components in the Eye

The connections between adrenergic nerve fibres and other ocular structures were studied in normal embryonic material (man, dogs, cats, guinea-pigs, and rats) as well as with a special vessel injection technique (adult rats, guinea-pigs, and rabbits). It was established that adrenergic fibres are a normal constituent of the cornea. The adrenergic nerves were more numerous in the embryo than in the adult, and also occurred within the embryonic corneal epithelium. These intraepithelial fibres disappear shortly after birth. Adrenergic fibres running in the connective tissue without connection to vessels were further found in the iris, the limbus region, the chamber angle (of the guinea-pig predominantly) and in the chorioid. It cannot be excluded that these fibres innervate some connective tissue component. In the sphincter pupillae, only a few adrenergic fibres were connected to the vessels, such as was the case also in the ciliary muscle of the guinea-pig. Under the ciliary epithelium of the ciliary body and the ciliary processes there was a thick and dense plexus of adrenergic fibres. Only a restricted number of them was associated with the vessels. The “capillaries” of the ciliary processes were remarkable in that they seemed to possess adrenergic fibres. No adrenergic innervation to the melanophores was apparent.     

Imaging myelinated nerve fibres by confocal fluorescence microscopy: individual fibres in whole nerve trunks traced through multiple consecutive internodes

Summary Current methods of morphological analysis do not permit detailed imaging of individual myelinated fibres over substantial lengths without disruption of neighbouring, potentially significant, cellular and extracellular relationships. We report a new method which overcomes this limitation by combining aldehyde-induced fluorescence with confocal microscopy. Myelin fluorescence was intense relative to that from other tissue components, enabling individual myelinated nerve fibres to be traced for distances of many millimeters in whole PNS nerve trunks. Images obtained with a Bio-Rad MRC-600 confocal laser scanning microscope clearly displayed features of PNS and CNS myelinated fibres including nodes of Ranvier; fibre diameter; sheath thickness and contour; branch points at nodes; as well as (in the PNS) Schmidt-Lanterman incisures and the position of Schwann cell nuclei. Direct comparisons using the same specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscopy in terms of contrast, apparent resolution and resistance to photobleaching. Development of the fluorophore was examined systematically in sciatic nerves of young adult rats. In separate experiments, animals were perfused systemically using (1) 5% glutaraldehyde; (2) Karnovsky's solution; (3) 4% paraformaldehyde; buffered with either 0.1 M sodium phosphate or sodium cacodylate (pH 7.4). The concentration of glutaraldehyde in the fixative solution was the principal determinant of fluorescence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovsky's. Fluorescence intensity increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The fluorophore was stable and resistant to fading during storage (15 months at least), enabling data collection over extended periods. To demonstrate application of the method in neuropathology, individual fibres in transected sciatic nerve trunks were traced through multiple successive internodes: Classical features of Wallerian degeneration (axonal swelling and debris; ovoid formation and incisure changes; variation among fibres in the extent of degeneration) were displayed. The method is compatible with subsequent ultrastructural examination and will complement existing methods of investigation of myelinated fibre anatomy and pathology, particularly where preservation of 3-dimensional relationships or elucidation of spatial gradients are required.     

Adrenergic and acetylcholinesterase-positive innervation of palatine tonsils in mammals

Innervation of human, feline and rabbit palatine tonsils was investigated. Adrenergic nerve components were visualized by formaldehyde-induced fluorescence of catecholamines and 5-HT, or by glyoxylic acid fluorescence, whereas acetylcholinesterase (AChE)-positive nerve structures were demonstrated by the direct thiocholine method. The largest density of adrenergic and AChE-positive nerve profiles was found in the adventitia of arterial branches in the fibrous capsula and septa, mainly in the form of periarterial nerve plexuses of different density. Fine nerve fibres lined the wall of small arteries which penetrated into extrafollicular lymphoid tissue and marginal layers of follicles. It is concluded that there are significant species-specific differences related to density, nature and topographic relations of adrenergic and AChE-positive nerve fibres in the various structural parts of palatine tonsils.     

On the sympathetic innervation to the cat's liver and its role for hepatic glucose release

Morphology and function of the adrenergic innervation of the liver were studied in cats. Fluorescence microscopy revealed a dense network of adrenergic nerve fibres in association with interlobular vessels and a sparse, but unequivocal innervation of the hepatocytes. These parenchymal adrenergic nerve fibres were more frequent in kittens (2 months old) than in adult cats. Electrical stimulation of the hepatic sympathetic nerves in the adult adrenalectomized cat evoked a small but insignificant increment (1–2 mM) of arterial plasma glucose concentration. When both hepatic and pancreatic sympathetic nerves were stimulated simultaneously, arterial plasma glucose concentration increased significantly by about 6 mM. We conclude that the pronounced hyperglycemic effect of activation of the sympathetic nervous system in the cat is mediated mainly via an adrenergic influence on the release of insulin and glucgon from the pancreas. The sympathetic innervation of the cat liver parenchyma seems to contribute to the hyperglycemia to a minor extent only.     

The effects of chronic stress on thymus innervation in the adult rat

Various stressors induce changes in the immune system. However, it has not yet been analyzed how stressors affect thymus innervation. To examine whether chronic stress alters the morphology of the thymus by changing the nerve components of the thymus, adult male rats, 9-weeks old, were exposed to forced swimming during 21 successive days. The animals were sacrificed by decapitation after the last session and their thymuses were used for analysis of (i) the thymus compartments, (ii) distribution patterns of monoamine-containing nerve profiles and (iii) distribution patterns of acetylcholinesterase (AChE)-containing nerve profiles. Our results show that chronic stress in rats reduces the volume of both thymus cortex and medulla, numbers of thymocytes in the deep cortex and medulla and the density of fluorescent nerve profiles, whereas it increases density of fluorescent cells. The distribution patterns of nerve profiles containing monoamine and AChE were not affected. These changes indicate that chronic stress affects thymus development and T cell maturation by altering the sympathetic nerve component.     

Pre- and postnatal development of catecholamine-containing and cholinesterase-positive nerves of the rat cornea and iris

Summary Glyoxylic acid-induced fluorescence technique and a copper thiocholine method were used to investigate the ontogenesis of the catecholamine-containing and cholinesterase-positive nerves of the rat iris and cornea.First fluorescent nerve fibres appeared in the iris on the 18th gestation day and in the cornea on the 19th day. A rapid increase in the density of the adrenergic nerve fibres of the iris continued to the age of three weeks, while the number of such fibres were small in the cornea.Acetylcholinesterase-positive fibres appeared both in the cornea and in the iris on the 19th gestation day. Their density increased more rapidly in the iris, especially in the sphincter muscle, than in the cornea.Non-specific cholinesterase activity was localized in the Schwann cells and the reaction was more intense during development than in the nerves of the cornea of adult rats.     

Innervation of internal female genital organs in the pig during prenatal development

This study investigated the innervation of internal genital organs in 5‐, 7‐ and 10‐week‐old female pig foetuses using single and double‐labelling immunofluorescence methods. The structure and topography of the organs was examined using a scanning electron microscope (SEM). The investigations revealed differences in the innervation between the three developmental periods. Immunostaining for protein gene product 9.5 (PGP; general neural marker) disclosed solitary nerve fibres in the external part of the gonadal ridge and just outside of the mesenchyme surrounding mesonephric ducts in 5‐week‐old foetuses. Double‐labelling immunohistochemistry revealed that nerve fibres associated with the ridge expressed dopamine β‐hydroxylase (DβH; adrenergic marker) or vesicular acetylcholine transporter (VAChT; cholinergic marker). In 7‐week‐old foetuses, the PGP‐positive nerve terminals were absent from the gonad but some of them ran outside and along, and sometimes penetrated into the mesenchyme surrounding the tubal and uterine segments of the paramesonephric ducts and uterovaginal canal. Few axons penetrated into the mesenchyme. DβH‐positive fibres were found in single nerve strands or bundles distributed at the edge of the mesenchyme. VAChT‐positive nerve terminals formed delicate bundles located at the edge of the mesenchyme, and the single nerves penetrated into the mesenchyme. DβH was also expressed by neurons which formed cell clusters comprising also DβH‐ or VAChT‐positive nerve fibres. In 10‐week‐old foetuses, PGP‐positive nerve fibres were still absent from the ovary but some were distributed in the mesenchyme associated with the uterovaginal canal and uterine and a tubal segment of the paramesonephric ducts, respectively. DβH‐ or VAChT‐positive nerve fibres were distributed at the periphery of the mesenchyme associated with the uterovaginal canal. Some DβH‐ and many VAChT‐positive nerve fibres were evenly distributed throughout the mesenchyme. The clusters of nerve cells comprised DβH‐positive perikarya and DβH‐ or VAChT‐positive nerve fibres. The investigations revealed no DβH/VAChT‐positive nerve fibres or neurons as well as no nerve structures stained for calcitonin gene‐related peptide and/or substance P (sensory markers) associated with the genital organs in the studied prenatal periods.     

Acetylcholinesterase activity in rat thymus after immunostimulation with interleukin beta.

The occurrence and distribution of Acetylcholinesterase (AChE) activity were examined in the thymus of normal and immuno-stimulated adult and aged rats using biochemical and enzymehistochemical methods. Specific AChE reactivity was found primarily in the arteries and, to a lesser extent, in the veins. Only a small amount of activity could be observed in association with the subcapsular and medullary part of the parenchyma and nerve fibers. Our findings indicate that AChE activity in the rat thymus increases after treatment with interleukin beta. In fact treatment with interleukin beta induces an increase of protein content, of the amounts of AChE biochemically assayed and at the levels of AChE histoenzymatically stained. Furthermore, staining of the different structures of the thymus in treated or untreated rats shows that the significant modifications concern the parenchyma, the structures resembling nerve fibers and the whole thymus, while only small changes are observed in AChE activity located in the walls of arteries, veins and lymphatic vessels.     

Histochemical demonstration of a concomitant reduction in neural vasoactive intestinal polypeptide, acetylcholinesterase, and noradrenaline of cat uterus during pregnancy

Noradrenaline, acetylcholinesterase, and vasoactive intestinal polypeptide (VIP) were visualized in uterine nerves of cats by histochemical techniques. Alterations were followed in different regions of the organs at various stages of pregnancy and compared with the situation in non-pregnant controls. Positively stained nerve fibres, the adrenergic type being particularly well developed, were found along the muscle bundles and around blood vessels in the smooth muscle layers, as well as in the mucosa, of both uterine horns and cervix. The nerve supply was especially prominent in the upper part of the cervix. The distribution of VIP-immunoreactive and acetylcholinesterase-positive nerve fibres resembled each other, but they were less numerous than the adrenergic fibres. In the course of pregnancy there was a marked reduction in the number of all positively reacting nerves, so that almost no fibres were visible in the uterine horns near term. A small number of positive nerve fibres was found to remain, however, in the wall of the sterile (empty) horn during unilateral pregnancy. The reduction was less prominent in the cervix, particularly its lower part. Distinct changes were encountered already during early and mid pregnancy in those parts of the uterine wall distended by the growing conceptus, where almost no fibres were seen. The nerve supply was more intact in the non-distended portions located between the fetuses, and especially in the empty horn of unilateral pregnancy. No overt reduction in the number of positively stained nerve fibres was found in the cervix at these pregnancy stages. The results show that marked alterations take place in the uterine autonomic innervation during such an entirely physiological event as pregnancy. There is reason to assume that the histochemical observations reflect both structural and functional alterations in the innervation related both to the type of nerves involved and to the localization of the conceptus.     

Rat ventral mesencephalon grown as organotypic slice cultures and co-cultured with striatum,hippocampus, and cerebellum

Summary Tissue slices of rat ventral mesencephalon (VM), striatum, hippocampus and cerebellum were prepared from late fetal (E21) to 7 day old (P7) rats and cultured for 3 to 60 days by the roller tube technique before they were stained immunocytochemically for tyrosine hydroxylase (TH), a marker of dopaminergic (DA) neurons and fibres. The TH immunoreactive (TH-i), DA neurons retained their morphological in vivo characteristics in the VM slice cultures consisting of the substantia nigra (SN) and the ventral tegmental area (VTA). The general morphology of the described neuronal cell types did not appear to change when the VM slices were cocultured with striatal tissue, a major normal target of the DA neurons, but an extensive innervation of the striatum by TH-i nerve fibres was observed. In co-cultures of VM and hippocampus, a minor target organ of DA fibres, growth of TH-i nerve fibres was observed mainly into the opposing edge of the hippocampal slice. In co-cultures of VM and cerebellum, which is normally devoid of DA fibres, no significant growth of TH-i nerve fibres into the cerebellar slices was observed. Besides suggesting a target orientated growth of ventral mesencephalic DA fibres, the results point to the further use of VM slice cultures in the study of the developmental, plastic and regenerative properties of DA neurons.     

The histological localization of noradrenaline in the cat spleen

1. The histological localization of noradrenaline in the cat spleen has been studied with a fluorescence technique and, after infusing DL-[(3)H] noradrenaline, both by the fluorescence technique and by autoradiography.2. In the normal cat spleen the specific fluorescence of catecholamines is confined to nerve fibres among the smooth muscle of the capsule, trabeculae, arteries and veins. These fluorescent fibres are not present after treating the animal with reserpine or after degeneration of the postganglionic splenic nerves.3. The splenic artery outside the spleen has few visible terminal adrenergic fibres. This is associated with the presence of an external elastic lamina. No terminal fibres were found in the walls of the main splenic vein.4. After infusing DL-[(3)H]noradrenaline 1.25 mug/min for 10 or 20 min fluorescence remained confined to the same sites as in normal cats, i.e. in fibres related to smooth muscle. The red and white pulp and the smooth muscle itself remained free of fluorescence. Autoradiography showed radioactivity in similar sites and with the same pattern as the fluorescent fibres. These results support the conclusion reached in the previous article that noradrenaline infused into the spleen is bound to adrenergic nerves.     

Effects of chronic deafferentation on adrenergic ganglion cells and small intensely fluorescent cells

Summary To determine the reaction of adrenergic ganglion cells and small intensely fluorescent (SIF) cells to chronic deafferentation, catecholamine fluorescence of the major pelvic ganglion (MPG) of the rat has been studied following section of the hypogastric nerve, pelvic nerve and sympathetic trunk. Only minor changes occurred following section of the hypogastric nerve; the fluorescence surrounding a few adrenergic ganglion cells became brighter. In contrast, pelvic neurectomy resulted in the appearance of numerous varicose fibres and an increase in the fluorescent intensity of fibres enclosing many ganglion cells. Varicose fibres seem to originate from adrenergic ganglion cells and SIF cells. In many instances, nests of SIF cells gave rise to radially oriented fibres. Removal of the sympathetic trunk appeared to have no effect on the MPG. It is suggested that the appearance of varicose fibres from SIF cells following deafferentation may be due to collateral sprouting of these cells or to the increased fluorescence of pre-existing processes.     

A comparative study of the spatial distribution of mast cells and microvessels in the foetal, adult human thymus and thymoma

Mast cells (MCs) are widely distributed in human and animal tissues and have been shown to play an important role in angiogenesis in normal and pathological conditions. Few data are available about the relationship between MCs and blood vessels in the normal human thymus, and there are virtually no data about their distribution and significance in thymoma. The aim of this study was to analyse the spatial distribution of MCs and microvessels in the normal foetal and adult thymus and thymoma. Twenty biopsy specimens of human thymus, including foetal and adult normal thymus and thymoma were analysed. Double staining with CD34 and mast cell tryptase was used to count both mast cells and microvessels in the same fields. Computer-assisted image analysis was performed to characterize the spatial distribution of MCs and blood vessels in selected specimens. Results demonstrated that MCs were localized exclusively to the medulla. Their number was significantly higher in thymoma specimens as compared with adult and foetal normal specimens respectively. In contrast the microvessel area was unchanged. The analysis of the spatial distribution and relationship between MCs and microvessels revealed that only in the thymoma specimens was there a significant spatial association between MCs and microvessels. Overall, these data suggest that MCs do not contribute significantly to the development of the vascular network in foetal and adult thymus, whereas in thymoma they show a close relationship to blood vessels. This could be an expression of their involvement not only in endothelial cells but also in tumour cell proliferation.     

Sympathetic innervation of fetal mouse thymus

This study was carried out to investigate the presence of sympathetic nerve fibers in the developing thymus. Thymic rudiments from 13 days of gestation up to birth and adult thymuses were examined by histofluorescence microscopy to detect the presence of these nerve fibers during ontogeny. Sympathetic nerve fibers were first visible around day 17 of gestation and increased in density and distribution by the time of birth and in the adult thymus. The appearance of immunoreactive cells about the same time as the presence of nerve fibers in the thymic rudiments has been discussed in light of a regulatory role for the sympathetic neuronal input on the maturation of thymocytes during ontogeny.     

Age-dependent changes of the sympathetic innervation of the rat kidney

The influence of aging on the sympathetic innervation of the kidney was studied in 3- (considered to be young), 12- (considered to be adult) and 24- (considered to be old) month-old male Sprague-Dawley rats by means of high pressure liquid chromatography with electrochemical detection, catecholamine histofluorescence and acetylcholinesterase (AChE) histochemistry. Body and kidney weights were significantly increased in adult in comparison with young rats. No further increase of either body or kidney weight was appreciated in old rats. Noradrenaline levels were increased by about 48% in adult rats and were decreased in old rats (by approx. 22% vs. young and 60% vs. adult). The density of perivascular noradrenergic fibres was significantly increased in adult rats and decreased in old animals. The percentage of kidney glomeruli supplied by AChE-positive nerve fibres is also remarkably increased in 12-month old rats and decreased in 24-month-old rats. The present data indicate that there is a striking increase in the expression of sympathetic innervation of rat kidney at 12 months of age followed by a significant decrease in the expression of innervation in old age. These changes are discussed in relation to the age-dependent impairment of renal function.