In vitro expression of n-cadherin adhesion molecule during early cardiac morphogenesis.

The aim of the present study was to investigate, "in vitro", the degree of organogenetic potentiality of the cells of the cardiogenic area during the early developmental stages of the chick embryo. Embryos from between the end of the presomitic stage to the 8 somite stage were studied. The subcephalic fold was cultured in liquid medium for up to 7 days. After 24 hs of culturing, an extended migration ring was observed. In the explants, from 3 somite stage, onwards, beating masses were noted, the shape and size of which suggested a vascular-like structure. Sections of the cultures were processed for the detection of the N-Cadherin adhesion molecule. The observations stated that the diffusion and intensity of expression of this receptor is related to the stage od development of the embryo. Cultures from the presomitic stage to 3 somite stage did not express the molecule. Instead, expression took place in those cultures of embryos at the 3 somite stage, onwards. In the cultures to which the antiserum against N-Cadherin had been to the medium, the formation of vascular-like structures was affected. The changes depended on the age of the embryos. These observations suggest that the expression of the N-Cadherin is related to the potentiality of the presumptive myocardic cells to organize themselves, at least "in vitro", to form a well-defined tridimensional structure. The expression of the adhesion molecule and the potentiality of the cells to build tubular structures were transient features, "in vitro" in our cultures. This suggests that "in vivo" the expression of the N-Cadherin must be aided by factors which, at present, are unidentified.

Ontogenesis of the structural elements of the heart.

A review of the birth locus and the first developmental stages of cardiomyocytes and Purkinje cells in the chick embryo is presented in the light of recent experimental results. Experiments done in vitro have shown that the stage of 2 somites is an important morphogenetic phase, characterized by mutual identification, selective adhesion and spatial organization of the putative cardiac cells of the splanchnopleural sheet in the cardiogenic area. Many experimental data suggest that the neural crest cells moving through the corridor of the dorsal mesocardium, as well as the cholinergic system related to them, may play a role of myogenic inductors on the mesothelial putative cardiac cells. In addition, other experimental findings suggest that the Purkinje cells of the conduction system may have a crestal origin. This hypothesis is well grounded, although the origin of Purkinje cells remains to be clarified.

"In vitro" expression of the cardiogenic potentiality of chick embryo heart-forming cells.

Chick embryos between final presomitic and 4 somites stage were studied. The subcephalic fold was handly severed from the embryos and cultivated in liquid medium for 7 days. Because of the embryo age, no heart anlage was observed at the moment of dissection. After 4 hours of culture the cells began to migrate from the explants. After 20 hours a very extended migration ring was observed in all of the cultures; in the explants, one or more newformed tubular or spherical masses of cells throbbed rhythmically. Their size and shape were related to the embryos age: from presomitic embryos, irregular clusters appeared, while starting from two somites embryos tubular, vascular-like structures were formed. The cells of the throbbing areas at submicroscopic observation showed organizing myofibrillar apparatus into the cytoplasm; junctional complexes between the cells and gap junctions in course of organization were present in the vascular-like structures. This suggests that very early, in the lateral mesoderm are the presumptive cardiac cells which can develop "in vitro" as myocardic elements even in absence of the interactions that occur during the development "in vitro"; the observed vascular-like structures may be considered as an attempt to form a sort of cardiac primordium "in vitro", and a further step in the expression of the cardiogenic potentiality, involving cell-cell communications. The serial sections of the embryos enhanced that into the cultivated areas, vessels from yolk sac are always present; this suggests that the vascular structures, i.e. the endothelium may be involved in the determination of the myocardic elements.

Embryogenesis of the avian iris sphincter muscle: in vivo and in vitro studies.

The histogenesis of iris sphincter muscle was studied in birds. Chick embryo iris "anlagen", ages from 3 days (st. 18 H.H.) to hatching, were examined. At the 4th day (st. 24 H.H.), nerve fibers were observed in the mesenchyme of the inferotemporal quadrant of the optic cup near the colobomic fissue. Among the mesenchymal cells, there were cells characterized by AChE activity, presence of desmin filaments, exhibiting ACh receptors, and ultrastructurally similar to the presumptive skeletal myoblasts. One day later (st. 27 H.H.), these myoblasts could be cultivated. The formation of myotubes began between 10 and 12 days. From 9 to 14 days, cells left the anterior epithelium of the iris to give rise to the smooth iris muscle; during this evolution some epithelial cells fused with the myotubes taking part in the histogenesis of striated muscle. The possibility of a neurogenic determination for the iris skeletal muscle is discussed.

The immunocytochemical distribution of seven peptides in the spinal cord and dorsal root ganglia of horse and pig.

The distribution of calcitonin gene-related peptide (CGRP), enkephalin, galanin, neuropeptide Y (NPY), somatostatin, tachykinins and vasoactive intestinal polypeptide (VIP) was compared in cervical, thoracic, lumbar and sacral segmental levels of spinal cord and dorsal root ganglia of horse and pig. In both species, immunoreactivity for the peptides under study was observed at all segmental levels of the spinal cord. Peptide-immunoreactive fibres were generally concentrated in laminae I-III, the region around the central canal, and in the autonomic nuclei. A general increase in the number of immunoreactive nerve fibres was noted in the lumbosacral segments of the spinal cord, which was particularly exaggerated in the case of VIP immunoreactivity. In the horse, some CGRP-, somatostatin- or tachykinin-immunoreactive cell bodies were present in the dorsal horn. In the pig, cells immunoreactive for somatostatin, enkephalin or NPY were noted in a similar location. In the ventral horn most motoneurones were CGRP-immunoreactive in both species. However, in pig many other cell types were CGRP-immunoreactive not only in the ventral horn, but also in laminae V-VI of the dorsal horn. With the exception of enkephalin and NPY immunoreactivity, which was not seen in pig dorsal root ganglia, all peptides studied were localised to neuronal cell bodies and/or fibres in the dorsal root ganglia. In both species, immunolabeled cell bodies were observed in ganglia from cervical, thoracic, lumbar and sacral levels, with the exception of VIP-immunoreactive cells that were detected only in the lumbosacral ganglia. Numerous CGRP- and tachykinin-immunoreactive cell bodies were visualised in both species, while the cells immunolabeled with other peptide antisera were much lower in number. In both species, immunostaining of serial sections revealed that a subset of CGRP-immunoreactive cells co-expressed tachykinin, galanin or somatostatin immunoreactivity. In the horse some enkephalin-immunoreactive cells were also CGRP positive and occasionally combinations of three peptides, e.g. CGRP, tachykinin and galanin or CGRP, tachykinin and enkephalin were identified. The results obtained suggest that the overall pattern of distribution of peptide immunoreactivities is in general agreement with that so far described in other mammals, although some species variations have been observed, particularly regarding the presence of immunoreactive cell bodies in the dorsal horn of the spinal cord.

Increasing potentiality in N.G.F. treated PC12 cells.

Somites from 9H. H. chick embryos and PC12 cells were co-cultivated in synthetic medium containing N.G.F., which induces the transformation of PC12 cells into neuron-like cells. During the first two days of culture, PC12 cells retained the spherical shape and tended to cluster. Somitic mesoderm cells exhibited a fibroblastic aspect. By the third day, PC12 cells extended long processes resembling nerve fibres which surrounded and penetrated the mesodermic explants. On the 10th day of culture, contractions, limited at first to a few cells were perceptible. Later, the contractions involved large cellular masses. Microscopic observations at 10 days revealed the presence of an increasing number of fusiform mononucleated cells. Later, long and narrow multinucleated elements appeared. Such elements never developed from cultures of only somites. Immunohistochemical observations revealed a desmin positivity in both mononucleated and multinucleated elements characterizing them as myogenic cells that are formed in and because of the presence of PC12 cells which were transformed by N.G.F. into nerve cells. After 10 days of culture, PC12 cells positive to antiserum antidesmin were noted. Desmin positivity of PC12 cells leads to the conclusion that newly-formed muscle cells exert an induction on Pheocromocytoma cells which, as derivatives of the neural crest, have a greater multipotentiality.

Ultrastructural studies on calcitonin gene-related peptide-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia: evidence for the colocalization of different peptides in single secretory granules.

Calcitonin gene-related peptide (CGRP)-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia have been studied by means of single and double immunogold labelling techniques. Peptide-immunoreactive neurones are generally B- or C-type cells of small size, with well developed rough endoplasmic reticulum and scanty neurofilaments. In neurones classifiable as A2-type cells, i.e. larger neurones with a lighter cytoplasm due to the presence of poorly developed Nissl bodies and numerous neurofilaments, only CGRP immunoreactivity was detected. Immunolabelled structures were identified as large (60-100 nm diameter), electron-dense, membrane-bounded p-type granules. They were observed only in neuronal cell bodies or in the intraganglionic portions of the axons. No granules immunoreactive to the antisera applied in this study were observed in non-neuronal cells. Immuno-staining experiments with different combinations of the antisera revealed, in some cells, the presence of double immunolabelled granules; in particular localization of CGRP and tachykinins, CGRP and somatostatin, and tachykinins and somatostatin to single secretory granules was demonstrated. The finding that more than one peptide is localized to the same secretory granule supports the postulate that peptides are co-released upon nerve stimulation providing morphological support for physiological and pharmacological data demonstrating an interaction between different peptides in the modulation of synaptic activity.

Histochemical, immunohistochemical, and ultrastructural observations on the iris muscles of Gallus gallus.

The distribution and typology of fibers in the two muscular systems (sphincter and dilator) of the iris in Gallus gallus were determined histochemically, immunohistochemically, and ultrastructurally. The sphincter muscle in proximity to the ciliary margin was composed predominantly of slow fibers. In the intermediate tract, a large group of fast oxidative fibers were evident and the pupillary margin was exclusively composed of slow fibers. The fast fibers had histochemical and immunohistochemical patterns similar to the alpha fibers in the skeletal control muscle (biventer cervicis). In contrast, the slow fibers were composed of at least three slow types, which were comparable to the isoforms of the different myosins in beta 1 and beta 2 skeletal fibers. In the dilator muscle, the oblique system was uniquely composed of fast oxidative fibers. The radial system was predominantly composed of slow fibers with isoforms of myosins different from the slow fibers of the sphincter and control muscles. Ultrastructural features (width of Z bands, extension of the sarcoplasmic reticulum and SR-T tubule junctions, and number of mitochondria) confirm the histochemical and immunohistochemical assessments of fiber types, even if some peculiar aspects in several fibers were observed. Smooth muscle cells separated from striated fibers were evident at the pupillary margin. The hypothesis of a mesenchymal origin for all irideal striated muscles is discussed.

Cryoanalgesia. Ultrastructural study on cryolytic lesion of sciatic nerve in rat and rabbit.

The sciatic nerve was exposed to cryoinjury at different freezing patterns in albino rats and rabbits and the frozen nerves were serially examined with electron microscopy from the time of cryolitic lesion (--60 degrees C for 3 minutes) for up to 28 days. The cryolesion was characterized by a total degeneration of the myelin fibers, while non-myelin fibers and vessels seemed less affected. Regeneration began 8 days after cryolysis. A peculiar pattern was the absence of Schwann cells, while the basal membrane around regenerating axons remained intact. The hypothesis that the basal membrane might play a role is discussed.

Effects at the periphery of the laser lesion in human brain and its tumors after CO2, Nd:YAG, and CO2 high-peak pulsed radiation.

The histological changes in various tissues irradiated with lasers are well known. Our own previous observations with the optical microscope confirm those already reported in the laser literature. If tissue is treated with various laser sources, the results are similar, with the characteristic three layers from the outside toward the inside of carbonization, coagulative necrosis, and edema. Otherwise, only the shapes and sizes of the lesions differ, with craters of different depths. In this paper, we report an ultrastructural study of the changes occurring in the periphery of the laser lesions in both normal human brain and neoplastic tissues (gliomas and meningiomas). Continuous-wave CO2 and Nd:YAG lasers were used at different exposure times and powers and the effects of high-peak pulsed CO2 laser radiation has also been investigated. The study, performed during neurosurgical procedures was mostly focused on microcirculation at 1.5-3 mm outside the area of coagulative necrosis, at the level of the edema zone. Only lesions of the blood brain barrier are produced in normal brain by CO2 radiation (power ranging from 40 to 80 W; exposure time from 3 to 10 seconds). The same results were achieved by Nd:YAG radiation of short duration (3 seconds) regardless of the power used (40 and 80 W). Long-duration Nd:YAG radiation (10 sec; power: 40-80 W) produces endoluminal phenomena leading to the complete occlusion of the capillaries. In neoplastic brain tissues, microcirculation does not seem to be impaired by CO2 radiation. More marked lesions are produced in tumors even after Nd:YAG short-time radiation. Endoluminal obliteration is observed in meningiomas and perivascular hemorrhage occurs in highly vascularized gliomas. According to these results, the risk of delayed post-operative hemorrhages, noticed in some patients with glioblastoma operated on by Nd:YAG lasers, suggests that residual tumor in the cavity should be treated by CO2 laser because of its minimal damage of microcirculation.

Histochemical and ultrastructural features of neuronal pigment in some encephalic nuclei of ruminants.

Neuronal pigment in red nucleus, locus coeruleus and substantia nigra from cattle, sheep and goat was examined with the aid of light and electron microscopy. In the red nucleus and locus coeruleus neurons of all the species examined the pigment shows the histochemical and ultrastructural features typical of lipofuscins. The ultrastructural observations indicated that the morphology of pigment granules is related to age and permitted identification of various stages of pigment evolution, which suggested a lysosomal origin of the pigment bodies. In bovine and sheep the substantia nigra is very reduced and contains no detectable pigment; while in goat, some neurons appear to contain discrete amounts of neuromelanin. Ultrastructurally this kind of pigment appears characterized by the features of a lipofuscin-like granule which stores highly electron dense material identifiable as melanin.

[Pigment in the neurons of some encephalic nuclei in Bos taurus]. / Il pigmento nei neuroni di alcuni nuclei encefalici in Bos taurus.

Pigment granules without the properties of melanin are present in neurons of several districts (Nucleus ruber, Locus coeruleus, Ala cinerea) in the C.N.S. in Bos Taurus. The distinctive features of this pigment are autofluorescence, specifity of staining with Oil red O, Sudan Black and a strong P.A.S. positivity. The early pigment granules appear about at the end of the first year of life, their amount increases with age. The most rich pigment neurons are in the Nucleus ruber; in the neurons of the Substantia nigra, the pigment is very scanty also in the very old animals.

Non-neural influences in the determination of chick skeletal muscle.

The technique of chicken-quail hybridization in "in vitro" cultures was used. Muscle differentiation of 9 H.H. chick embryo somites without the participation of nerve fibres was noted when they were cultured in the presence of quail pectoral muscle cells. It is suggested that this non-neural model may also be valid "in vivo", and that it probably interacts with the neurodependent mechanism demonstrated in a previous paper.