Histochemical and immunochemical studies on mammalian striated muscle fibres

Summary In 15 rabbit and 2 human distinct types of striated muscle, it was shown that all fibres classified as type I (red) by NADH-diaphorase and myofibrillar ATPase reaction exhibit intense fluorescence when treated with a FITC-labelled antibody against human pectoral actomyosin. Type II (white) fibres showing minimal fluorescence, an intermediate fibre type can be differentiated by all three methods. In the rabbit, all three fibre types are found, whereas, in the human muscles studied, only type I and intermediate type fibres were found.Abbreviations ATPase adenosintriphosphatase (EC 3.6.1.3) - anti-PAM anti-human-pectoralis actomyosin - FITC fluoresceine-isothiocyanate     

Fluorescence microscopic study of the architecture and structure of an adrenergic network in the plexus myentericus (Auerbach), plexus submucosus externus (Schabadasch) and plexus submucosus internus (Meissner) of the porcine small intestine

The distribution of adrenergic fibres in the ganglionated plexuses of the porcine small intestine has been made on air-dried stretch preparations using the glyoxylic acid fluorescence method. Adrenergic fluorescent fibres occur in the ganglia and internodal strands of the three fundamental ganglionated plexuses: the myenteric plexus (Auerbach) and the two superimposed meshworks of the plexus submucosus , i.e. the plexus submucosus externus ( Schabadasch ) and the plexus submucosus internus (Meissner). The plexus Auerbach consists of densely glyoxylic acid induced fluorescent (GIF) elongated ganglia with in general a longitudinal axis running parallel to the circular muscle layer and large dense interconnecting fibre tracts with primary, secondary and tertiary subdivisions. In the ganglia, the fibres are varicose, forming large fluorescent 'baskets' which might be related to the occurrence of well defined enteric neurones. The plexus Schabadasch can be distinguished from the plexus Meissner by its size, strongly fluorescent ganglia and broad densely fluorescent internodal strands. The pattern of fluorescing ring-like formations at the margin and out of the nodes, clearly present in the Auerbach and Schabadasch plexuses, completely lack in the plexus Meissner, the latter being narrow-meshed with smaller fluorescent 'baskets', indicating that the corresponding neurones are smaller in size. In the ganglionic nodes of all three plexuses the axons display comparatively more varicosities than in the fibre tracts. Each of the three main ganglionated enteric plexuses are quite different with regard to the pattern of the adrenergic network both in the ganglia and in the strands.     

Fluorescence microscopical visualization of elastic fibres using basic fuchsin

Summary We show that fluorescence microscopy after staining of tissue sections with basic fuchsin (BF) can be used successfully for the demonstration of elastic fibres. Using double staining with BF and antibodies reacting with microfibrils of elastic fibres (anti-SAP) we showed that BF reacts with the elastin core of elastic fibres and the elastin poor terminal branches of the subepidermal elastic fibre system. Small amounts of bound BF were easily seen by fluorescence microscopy (FL) but not by ordinary light microscopy. Both frozen sections and sections of paraffin embedded tissues could be stained. The BF-FL staining procedure is simple to perform and, due to its selectivity, it may be useful for detecting elastic fibres in various tissues at the light microscopical level.     

Fluorescence microscopical visualization of elastic fibres using basic fuchsin

We show that fluorescence microscopy after staining of tissue sections with basic fuchsin (BF) can be used successfully for the demonstration of elastic fibres. Using double staining with BF and antibodies reacting with microfibrils of elastic fibres (anti-SAP) we showed that BF reacts with the elastin core of elastic fibres and the elastin poor terminal branches of the subepidermal elastic fibre system. Small amounts of bound BF were easily seen by fluorescence microscopy (FL) but not by ordinary light microscopy. Both frozen sections and sections of paraffin embedded tissues could be stained. The BF-FL staining procedure is simple to perform and, due to its selectivity, it may be useful for detecting elastic fibres in various tissues at the light microscopical level.     

Morphogenesis of rat experimental pulmonary emphysema induced by intratracheally administered papain: changes in elastic fibres

The ultrastructural changes of elastic fibres in emphysematous lungs have been studied in men, but few works exist on this topic in experimental emphysematous animals. In this paper, the morphogenesis of emphysema and alterations of the elastic fibres produced by the instillation of papain are described by light and electron microscopy. Wistar rats were instilled through the trachea with papain at a rate of 3 mg/100 g animal weight. The animals were sacrificed 12 h, 3 days, 10 days and 60 days after enzyme instillation. The "Mean Linear Intercept" (MLI), the "Number of fenestrations/respiratory units" (NF) the "Number of macrophages per mm of alveolar wall" (NM) and the "Number of respiratory unit/mm2" (RU), both in the control and experimental groups were studied. Two months after treatment, the experimental group showed a strong increase in the MLI (p<0.001) and NF (p<0.001), and a diminished number of RU (p<0.05) compared with the control group. Partial correlation analysis showed a positive correlation only between MLI and NF. Twelve hours after papain instillation an inflammatory response was observed, the elastic fibres were ruptured, while the microfibrilar component remained. New formations of eulanin elastic fibres were observed three days post papain instillation. After ten days the interalveolar oedema had disappeared and the elastic fibres were of normal morphology although irregular groups of strips of elastic fibres were evident. A mixed pattern of panlobular, centrilobular and normal lung zones were observed. Two months after papain instillation abundant accumulations of elastic fibres of irregular outline were observed associated to collagen fibres. In conclusion, the morphometric parameters studied showed a significant progression of the emphysema. The strong correlation between NF and MLI suggested that papain-induced emphysema is principally caused by breaches of the alveolar walls. The results seem to point to a very abnormal remodelling process associated with elastic fibre regeneration, although there were no signs of destruction of these new fibres formed in emphysematous rat lung induced by papain.     

Routine oxygen consumption and characteristics of the myotomal muscle in tench: Effects of long-term acclimation to hypoxia

Summary Tench (Tinca tinca) were acclimated to either aerated (P _{O 2} 17.6 KPa) or hypoxic water (P _{O 2} 1.5 KPa) at 15° C. Fish acclimated to P _{O 2} 17.6 KPa had a routine oxygen consumption (mls O₂/Kg bodyweight/h) of 32.7 in aerated water. Upon acute exposure to P _{O 2} 1.5 KPa oxygen consumption decreased to 10.8 and 15.6 in fish acclimated to aerated and hypoxic water, respectively.On the basis of staining for glycogen and for the activities of myofibrillar ATPase and succinic dehydrogenase, three main fibre types can be differentiated in the myotomal muscle.Fibres have been classified as slow, fast aerobic and fast glycolytic. Fast aerobic fibres can be distinguished histochemically by their alkaline-stable Ca²⁺-activated myofibrillar ATPase activity and their intermediate levels of staining for glycogen and succinic dehydrogenase activity.The patterns of innervation of the fibre types have been investigated by staining neuromuscular endplates and peripheral axons for acetylcholinesterase activity. Motor axons to slow fibres branch extensively giving rise to a number of diffuse endplate formations on the same and adjacent fibres. Fast glycolytic fibres also have a complex pattern of innervation with 8–20 endplates per fibre. A large proportion of the endplates belonged to separate axons.Cross-sectional areas and perimeters of fibres, the number of capillaries/fibre and the lengths of contacts between capillaries and fibres were determined from low-magnification electron micrographs.Acclimation to hypoxia resulted in a decrease in the number of capillaries per fibre for both slow (1.8 to 1.0) and fast (0.8 to 0.2) muscles. The capillary perimeter supplying 1 m² of fibre cross-sectional area decreased by 43 % and 76 % for slow and fast fibres, respectively.     

The relationship between gastrin cells and bombesin-like immunoreactive nerve fibres in the gastric antral mucosa of guinea-pig,rat, dog and man

Summary The relationship between bombesin-like immunoreactive (bombesin-LI) nerve fibres and gastrin-LI G-cells was examined in gastric antral mucosa from guineapig, rat, dog and man using a double-labelling fluorescence immunohistochemical technique. The greatest density of bombesin-LI nerve fibres was found within the basal mucosa in all species and the density of innervation decreased towards the luminal surface. Most G-cells were in a band occupying approximately the middle third of the mucosa. The proportion of G-cells found within a distance of 2 m from bombesin-LI nerve fibres was low in all species (6% in the guinea-pig, 22% in the rat, 14% in the dog, and 9% in the human). It is proposed that the neuropeptide released from bombesin-LI antral mucosal nerve fibres traverses distances of greater than several m to reach the target G-cells. This may be achieved by passage through the mucosal microcirculation.     

The innervation and fine structure of paraneuronic cells in an amphibian pulmonary artery

Summary The pulmonary artery of Bufo marinus contains large numbers of bipolar cells situated in the tunica adventitia and in the outer layers of the media. These cells show a bright green-yellow fluorescence (emission spectra 485 nm) after formaldehyde pre-treatment suggesting that they contain a primary monoamine. The most characteristic fine-structural feature of these cells is the presence of numerous dense-cored vesicles (80—300 nm diameter) in their cytoplasm. The cells are in close contact (20 nm gap) with both agranular and granular nerve fibres. Both EM-cytochemical and formaldehyde-induced fluorescence tests indicate that the granule-containing nerve fibres are adrenergic. The agranular nerve fibres form discrete synaptic contacts with pre-and post-synaptic membrane thickenings on the cells. This was never observed with respect to the adrenergic fibres. Each process of the cells is about 45 m long. The processes do not bear any special relationship to either vessels of the arterial vasa vasorum or medial smooth muscle cells. Their location in the wall of the artery suggests that they are functionally significant with respect to activity of the arterial media.     

Effect of long-term alcohol intake on the innervation of the rat myocardium

The myocardium of rats kept on DeCarli and Lieber's liquid diet containing alcohol to 36% of the total calories for 8 weeks were examined with Falck-Hillarp's method for the histologic demonstration of catecholamines. Relative fluorescence intensity of the myocardial cells and nerves was measured with the Zeiss-Fluoval microphotometer. Under the effect of alcohol, density of the noradrenergic innervation (number of visualized nerve fibres) decreases and fluorescence of myocardial cells is intensified. The increased fluorescence of myocardial cells can be eliminated with reserpine. It is assumed that chronic alcohol intake induces continuous catecholamine release from monoaminergic nerve fibres of the heart.     

The hypothalamo-hypophysial system of the wild carp,Cyprinus carpio L.

Summary The posterior neurohypophysis (PNH)-pars intermedia complex of the wild and pond carp, Cyprinus carpio L., has been studied by light, fluorescence and electron microscopy. Gomori-positive neurosecretory fibres are abundant in the main trunk of the neurohypophysis as well as its roots penetrating the pars intermedia. Terminals of these fibres are in contact with capillaries of the general circulation and with glandular cells of the pars intermedia. Monoaminergic fibres with a weak green fluorescence, somewhat increasing after injection of nialamide into the pond carp, have largely the same distribution. Three types of neurosecretory fibres and their terminals have been recognized in the PNH-pars intermedia complex. Types-A₁ and -A₂ fibres, containing granules of 140–180 nm and 100–160 nm in diameter respectively, are peptidergic Gomori-positive. Type-A₂ fibres predominate in the PNH. The least frequent monoaminergic type-B fibres have granules of 60–100 nm in diameter. Numerous peptidergic and few monoaminergic neurosecretory terminals make contact with the capillaries located within the roots of the PNH as well as at the border between them and the pars intermedia. Both peptidergic and monoaminergic terminals make direct synaptoid contacts with the gland cells or end close to connective tissue septa, basal lamina or pituicytes. The PAS-positive gland cells and to a lesser degree the leadhaematoxylin-positive gland cells show these relationships with neurosecretory terminals. The question concerning the mode of interaction between peptidergic and monoaminergic structures in the dual control of the gland cells of the pars intermedia of teleosts is discussed.     

Loss of histochemically demonstrable catecholamines and acetylcholinesterase from sympathetic nerve fibres of the pineal body of the rat after chemical sympathectomy with 6-hydroxydopamine

Synopsis Newborn albino rats were injected daily for 8 days with 50 g/g of 6-hydroxydopamine. They were killed 3 weeks after the last injection together with untreated litter mate controls. Monoamines were demonstrated histochemically in the pineal body, in the iris and in the superior cervical ganglion with the formaldehyde-induced fluorescence method. Acetylcholinesterase was demonstrated in the pineal using acetylcholine as substrate and tetraisopropy-pyrophosphoramide (iso-OMPA) to inhibit non-specific cholinesterases.Treatment with 6-hydroxydopamine caused a complete disappearance of amine-containing fibres from the pineal, whereas some fluorescent ganglion cells remained in the superior cervical ganglion and in some rats a few amine-containing fibres in the iris. Acetylcholinesterase activity, located in fine nerve fibres of the pineal body, disappeared completely after treatment with 6-hydroxydopamine.Since 6-hydroxydopamine causes a selective destruction of the aminergic sympathetic fibres, it is concluded that the disappearance of the acetylcholinesterase activity indicates that in the pineal body this enzyme activity is located exclusively in truly aminergic nerve fibres.     

Simple Fluorescence Staining of Insect Central Nerve Fibres with Procion Yellow

Nerve fibres in the central nervous system of the cockroach Periplaneta ameri-cana can be displayed by staining whole ganglia for 1-2 hr in a saturated solution of Procion yellow M-4R in cockroach saline diluted to maintain isotonicity. Selected fibres are stained preferentially by cutting a peripheral nerve or interganglionic connective close to the ganglion, or damaging neuron cell-bodies. The ganglion is washed in saline, fixed in alcoholic Bouin, dehydrated and embedded. Under fluorescence microscopy, sections show stained fibres brilliant yellow against a green background. The method is simpler than intracellular injection and demonstrates even the finest fibres.     

The fluorescence of elastic fibres stained with Eosin and excited by visible light

Synopsis Fluorescent light of any wavelength, emitted by a microscopical specimen excited with visible light, can be observed using crossed polarizers and a dark-field condenser. The absorbance of Eosin is high in the green portion of the spectrum, so that visible light from a tungsten lamp is highly effective in exciting the fluorescence of this dye.Elastic fibres in routine sections stained with Haemalum and Eosin have been found to fluoresce rather strongly, while most other Eosin-stained structures fluoresce less or not at all. The reason for this relatively selective fluorescence of Eosin-stained elastic fibres is obscure, although the phenomenon may prove useful in routine histology. It is possible, however, that in most stained substrates the fluorescence of Eosin is largely quenched by aggregation of the dye molecules, but that this is inhibited inside the relatively dense (i.e. impermeable) elastic fibres.     

Remarkable adrenergic nerves in the exocrine pancreas

Summary The adrenergic nerves in the pancreas of mice, rats, guinea-pigs, rabbits, and cats were investigated with the fluorescence method of Falck and Hillarp. The relations between the adrenergic fibres and the vessels were studied by the injection of india ink into the vessels.Besides the normal manifestation of adrenergic fibres at the large vessels, some vessels of capillary size were also accompanied by adrenergic fibres. These fibres had a very weak fluorescence, and showed up regularly only when the animal had been treated with Nialamide and L-DOPA or dopamine to increase the catecholamine content of the adrenergic fibres. The weakness of the fluorescence is perhaps due to low transmitter concentration or to small size of the nerve fibres, or to both. A rough estimate indicated that either the transmitter concentration of the nerve fibre is at least approximately 100 times below that seen in adrenergic nerves in other tissues, or that the radius of the varicosities of the nerve fibres is less than 0.2 . Neither alternative has previously been recognized.The secretory acini of the pancreas seem to lack a direct adrenergic supply. In the intrapancreatic ganglia, non-fluorescent nerve cells were reached by adrenergic terminals. No adrenergic nerve cells were detected in the pancreas of rats and cats. Small intensely fluorescent catecholamine-containing cells were observed in connexion with the intrapancreatic ganglia of rats.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under grant AF EOAR 67-15 through the European Office of Aerospace Research (OAR), United States Air Force, by the United States Public Health Service (grant NB 06701-01) by the Swedish Medical Research Council (project B 67-12X-712-02A), and by the Faculty of Medicine, University of Lund, Sweden.     

On the ultrastructural localization of catecholamines in the beta lobes (corpora pedunculata) of Periplaneta americana

Summary In the brain of the cockroach Periplaneta americana, the beta lobes of the corpora pedunculata respond with an intense positive reaction to a specific fluorescence histochemical method for catecholamines. The fluorescence reaction disappears completely after prolonged treatment of the cockroaches with reserpine. An ultrastructural examination of the beta lobes in formaldehyde-glutaraldehyde-osmium fixed preparations reveals the presence of two types of fibres: 1) Fibres and nerve endings containing small clear vesicles and sligthly larger vesicles with a semi-dense content. The appearance and size distribution of these vesicles ist not affected by treatment with reserpine. 2) Fibres containing larger and denser vesicles, but practically no clear vesicles. The size distribution of these dense vesicles is only slightly affected by treatment of the cockroaches with reserpine.If brain slices are incubated in a medium containing noradrenaline or -methyl-noradrenaline and fixed in permanganate, small vesicles with electron-dense central cores show up, similar to those which have been described in vertebrate adrenergic nerve fibres (small granular vesicles). They are confined to one of the two types of fibres (a and b) visible in these preparations, namely to type b, whose correspondence with type 2 fibres of formaldehyde-glutaraldehyde-osmium fixed preparations is discussed.The authors wish to thank Mr. E. Chessa and Mr. F. Piccirilli for technical assistance in photography.     

State of the contractile apparatus during the development of a pathological process in the muscles. III. The nature and sequence of the structural changes in the contractile apparatus in Zenker's necrosis]

Using polarized ultraviolet (UV) fluorescence microscopy, it was shown that the local damage of muscle fibres causes in their morphologically unchanged parts the alternation of regions, being in different functional states referred to as "pseudocontraction" and "superrelaxation". The pattern of UV fluorescence anisotropy suggests that conformation of contractile proteins by "pseudocontraction" is similar to that at contraction, though changes in sarcomere length do not occur. The "superrelaxation" is characterized by a desorganization of myofilaments. During the spreading of Zenker's necrosis, the "pseudocontraction" is seen transferred first into "superrelaxation", and then into irreversible contracture and rigor. "The boundary of Zenker's necrosis" overlaps with the boundary of the self-propagating irreversible contracture. There is no proper boundary of Zenker's necrosis, because the destructive changes are observed over all the muscle fibre. Contraction nodules arise in the regions of "superrelaxation" and follow the changes of the contractile system, peculiar of contracture. The study of the influence of medium ionic composition on the development of Zenker's necrosis suggests that the arising and spreading of destruction are inseparably associated with the irreverrsible changes of intracellular membrane structures, and with the possibility of propogation of the damage signal by these structures along muscle fibres.     

Hints of a functional connection between the neuropeptidergic innervation of arteriovenous anastomoses and the appearance of epithelioid cells in the rabbit ear

Peripheral blood flow can be regulated by specialized vessel segments, the arteriovenous anastomoses. Their wall consists of a relatively thick layer of smooth muscle cells and so-called epithelioid cells. The epithelioid cell is a specialized myogenic cell phenotype expressing nitric oxide synthase. We studied the innervation of the different segments of arteriovenous anastomoses in the rabbit ear using antisera against neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P, as well as neuron-specific enolase, calbindin D and neurotubulin. The participation was especially examined of neuropeptidergic innervation and a possible morphological connection to the occurrence of epithelioid cells and a paracrine function. The NADPH diaphorase reaction and -smooth muscle actin immunoelectron microscopy served to distinguish epithelioid cells from smooth muscle cells. Using conventional fluorescence microscopy and confocal laser scanning microscopy, we found the most dense innervation pattern of pan-neuronal markers (neurotubulin, neuron-specific enolase), tyrosine hydroxylase-immunor eactive nerve fibres and neuro-peptidergic nerve fibres (neuropeptide Y, calcitonin gene-related peptide, substance P) around the intermediate segment in arteriovenous anastomoses, whereas the venous segment was barely marked. Single nerve fibres penetrated into the medial layer and reached the epithelioid cells. Using immunoelectron microscopy, we found intercellular contacts between epithelioid cells, but not the gap junction protein connexin 43. Here, we report for the first time a correlation of the innervation pattern with epithelioid cell type in arteriovenous anastomoses. Our findings suggest that epithelioid cells of the arteriovenous anastomoses are controlled by a dense network of neuropeptidergic nerve fibres in functional connection to their paracrine role as a nitric oxide producer. © 1998 Chapman & Hall     

Selective fluorescence of eosinophilic structures in grasshopper and mammalian testis stained with haematoxylin-eosin

After staining with Mayer's haematoxylin and eosin Y, paraffin sections of grasshopper and mouse testis were analysed by both transmitted light and fluorescence microscopy. Under violet-blue (436 nm) light excitation, a bright green emission was observed in all eosinophilic structures. Meiotic spindles (fibres and poles), mitochondrial aggregates, centriolar adjuncts in grasshopper spermatids, the basal lamina, flagellar bundles and remaining cytoplasmic droplets in the lumen of seminiferous tubules showed the most striking fluorescence induced by eosin Y. No emission was found in these structures after haemalum staining. Fluorescent microtubular components also revealed a positive immunoperoxidase reaction for -tubulin. All fixation and embedding procedures (Bouin, Zenker, formaldehyde alone or followed by dichromate or glutaraldehyde, freeze-substitution) were suitable for observation by fluorescence microscopy. Acetylation, deamination, and prolonged washing of stained sections with water, salt solution or ethanol strongly reduced eosin Y fluorescence, while it slightly increased after methylation. These results show that routine haematoxylin-eosin stained tissue sections can be routinely analysed by fluorescence microscopy. The emission of eosin Y allows easy and precise recognition of eosinophilic structures, which are poorly visible under bright field illumination.     

Pituitary monoamines of the cat with special reference to the presence of an unidentified monoamine-like substance in the adenohypophysis

Summary In the pituitary gland of the cat, dopamine (M.V. 0.78 g/g), noradrenaline (M.V. 0.29 g/g) and 5-HT (M.V. 0.94 g/g) have been found. With the histochemical fluorescence method, a rich system of delicate fluorescent varicose fibres, often provided with irregular swellings or droplets, was observed in the neural lobe and pars intermedia. Microspectrofluorimetrically, these fibre structures exhibit the spectral characteristics of catecholamines. Most cells in the pars intermedia and a large number of cells in pars distalis show a yellowish fluorescence, with microspectrofluorimetric characteristics which differ entirely from those of the catecholamines and 5-HT. In animals treated with reserpine, the pituitary dopamine, noradrenaline, and 5-HT are largely depleted. However, the intensity and the spectral properties of the cellular fluorescence are not affected by this treatment, whereas the fluorescent fibres can no longer be seen. Thus none — or only little — of the catecholamines and 5-HT but some other monoamine-like substance is stored in the fluorescent cells of the adenohypophysis. Preliminary studies suggest that this substance is closely related to or perhaps identical with tryptamine.Supported by grants from the Swedish Medical Research Council (No B 68-12X-712-03 B and B 68-14X-56-04 B), the Association for the Aid of Crippled Children, New York, and by the Faculty of Medicine, University of Lund, Sweden.     

Removal of the Schwann sheath from the giant nerve fiber of the squid: An electron-microscopic study of the axolemma and associated axoplasmic structures

Summary Surgical thyroidectomies were used as means of altering the thyroid state of adult recipients to study the possible influence of thyroid hormones on fibre formation in irides by immature noradrenaline neurons of the locus coeruleus grafted to the eye. Whole-mount preparations of irides were analysed using fluorescence histochemistry according to Falck-Hillarp, subjectively estimating on a blind basis the number of fibres, their pattern of distribution and individual morphology in the iris dilator plate. Neurones of the locus coeruleus formed nerve fibres in irides of thyroidectomized recipients to the same extent as in controls. Distribution and fine structure of these fibres, however, differed markedly. The numerous thick axon bundles from the attachment of the brain graft, normally seen to radiate out from locus coeruleus-neurones in oculo, were almost totally lacking in the thyroidectomized group. Also, the individual nerve fibres showed abundant peripheral accumulations of fluorescent material. This appearance of the outgrowth of fluorescent fibres in the experimental group, indicative of a disturbed formation of nerve fibres during development in oculo, was abolished by reversal of the thyroid hormone deficiency using daily injections of 1-thyroxin to the recipients throughout the experiment. This strongly indicates a role for thyroxin in the process of formation of nerve fibres originating from the neurones of the locus coeruleus during perinatal development. The present paper is supportive of recent reports claiming that during the development of the CNS thyroxin plays a crucial role in tubulin assembly, and thus presumably for the ability of neurones to form processes.