Increase in the number of NADPH-diaphorase-positive neurons in the lumbar dorsal root ganglia following lipopolysaccharide exposure of the sciatic nerve.

In the spinal cord, nitric oxide pathways are involved in hyperalgesia, and nitric oxide synthase, the enzyme responsible for its synthesis, is upregulated following several noxious and lesion stimuli. Since the histochemical reaction for NADPH-diaphorase colocalizes with NOS, we decided to study the effects of infusion of bacterial lipopolysaccharides close to the sciatic nerve on the expression of NADPH-d in the dorsal root ganglia and spinal cord of the rat. The percentage of NADPH-d-positive neurons in the L4 dorsal root ganglia increased 7-10 times on the treated side of LPS-treated rats (12.5-17.5%, compared to 0.5-2.5% of control side), whereas sham operation had no effects. The cross-sectional area of NADPH-d-positive neuronal profiles in all the dorsal root ganglia considered was consistently smaller than that of those which were negative to the histochemical reaction. In animals treated with LPS the NADPH-d-positive neurons were significantly (p = 0.02) smaller on the treated side (520 +/- 100 microns) than on the control one (679 +/- 135 microns), whereas those which were negative were of similar sizes on the two sides (1170 +/- 256 microns on the treated side vs 1214 +/- 371 microns on the control side). On the contrary, in control animals, there were no differences between untreated and sham operated sides, but differences between the sizes of NADPH-d-positive and negative neurons persisted. Therefore, LPS treatment on the sciatic nerve upregulates NADPH-d expression in the corresponding dorsal root ganglion, thus indicating an increased rate of NO production. Moreover, NADPH-d is upregulated mainly in small sized neurons, thus suggesting that it may be related with pain transmission.

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.

Sex steroids modulate NADPH-diaphorase expression in the postnatal adrenal neurons of the rat.

The rat adrenal gland contains nitric oxide-producing ganglion cells, contributing to its innervation. In a previous study postnatal number and morphology of these adrenal neurons were analyzed by NADPH-diaphorase histochemistry in the two sexes. A transient sex-related difference in the number of NADPH-diaphorase positive neurons per adrenal gland was found at postnatal day 10, when the number of stained neurons in males was nearly twice that found in females. In the present work we studied the effects of perinatal hormonal manipulation on the number of adrenal NADPH-diaphorase-positive neurons during the second postnatal week. The number of labeled adrenal neurons at postnatal day 10 was higher in females receiving perinatal androgen treatment than in control untreated females, and was similar to that of control untreated males. In contrast, in males that underwent perinatal deprivation of testosterone the number of labeled adrenal neurons was lower than in control males, and similar to that of control females. These differences were found in both the adrenal cortex and medulla. In males and in testosterone-treated females there was a higher proportion of stained multipolar neurons than in females and in androgen-deprived males. No intergroup differences were found in the size of stained neurons. Thus, we demonstrated that the postnatal difference in the number of NADPH-diaphorase-positive adrenal neurons in the two sexes is related to the epigenetic action of gonadal hormones during perinatal maturation.

NADPH-diaphorase-positive ganglion cells of the rat adrenal gland: age- and sex-related changes in their number, size, and distribution.

The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350-400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1-2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period.

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.

Models of neuronal plasticity and repair in the enteric nervous system: a review.

In the Fifties we developed an experimental model of neuronal plasticity in adulthood, involving the intestinal myenteric plexus. Causing an incomplete stenosis of the gut, the thickness of the wall and the diameter of the lumen underwent a massive increase on the oral side of the obstruction. The myenteric neurons innervating such hypertrophic and hyperplastic smooth muscle became hypertrophic and increased their number per ganglion, in absence of mitoses. Therefore we hypothesized the existence of a reserve pool of morphologically undifferentiated elements within the plexus, undergoing differentiation under conditions of functional hyperactivity. Some recent experiments suggest once again the existence in adulthood of a reserve pool of potential neurons. In fact, we put in evidence a subpopulation of NADPH-diaphorase positive myenteric neurons, very small in size orally to the stenosis and even smaller in the control gut. Following experimental ablation of the myenteric plexus in an intestinal segment and induction of hypertrophy in its smooth muscle layers, we found a two-five-fold increase in neuronal density along mesenteric nerves. This increase is probably due to the recruitment of cells, not readily identifiable as neurons, along mesenteric nerves in an attempt to reinnervate the damaged ileum. Moreover, it is demonstrated that hypertrophic smooth muscle cells may induce neuronal differentiation of transplanted PC12 cells. Finally, we explain the decreased total number of myenteric neurons in advanced age with the exhaustion of this reserve pool: in fact, NADPH-diaphorase positive small neurons in the myenteric plexus of old rats could not be found.

Quantitative study of the NADPH-diaphorase-positive myenteric neurons of the rat ileum.

The subpopulation of myenteric neurons able to synthesize nitric oxide was studied quantitatively in the adult rat, using the NADPH-diaphorase histochemical method on whole-mount preparations of distended distal ileum. The spatial density of NADPH-diaphorase-positive myenteric neurons was 2388 +/- 193/cm2 (S.D.; five rats), comprising about 27% of the nerve cell bodies per ganglion. Most neurons were intensely stained and displayed predominantly a Dogiel type I morphology; about 8% of the labelled nerve cells were ovoid neurons, exhibiting a pale cytoplasmic reaction product. The mean somatic size of all NADPH-diaphorase-positive myenteric neurons was 446 +/- 40 microns2, with a mean nuclear size of 96 +/- 18 microns2 (mean values +/- S.D.; five rats). Such values fell exactly within the range of neuronal sizes of the total myenteric population, marked by means of NADH-diaphorase histochemistry. Therefore, the morphometric analysis did not identify any peculiar cell size feature, characterizing this specific nerve cell subpopulation. Thus, the present study provides quantitative data on the size, density and proportion of those myenteric neurons that may synthesize nitric oxide in the distal ileum of the rat.

Mesenteric neurons in the adult rat are responsive to ileal treatment with benzalkonium chloride.

The application of a 2 mM solution of the cationic surfactant benzalkonium chloride (BAC) to an ileal segment produced a selective and extensive myenteric denervation. The aim of the present study was to investigate whether such a selective unbalance of the enteric nervous system in the adult rat elicited any plastic response within the mesenteric nervous structures contacting it. Acetylcholinesterase (AChE) staining, beta-nicotinamide adenine dinucleotide (NADH) and beta-nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and glyoxylic acid-induced fluorescence were performed on whole-mount preparations of myenteric plexus and mesenteric nerves. In both control and BAC-treated animals nervous elements were detected along the mesenteric nerves. Although rather similar in position, shape and size, these neurons displayed striking differences with regard to their arrangement and density per nerve. In the controls, few small aggregates of neurons could be detected; more commonly, isolated nerve cell bodies were scattered along the nerve trunks. In the BAC-treated animals, numerous spherical or spindle-shaped clusters of neurons were located along the nerves; the mean neuronal density per nerve displayed a two-five-fold increase over the control values. The observed changes within the mesenteric nerves might be involved in an attempt at reinnervation of the BAC-treated intestinal segments from extra-enteric sources.

"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.

Comparative study of Langerhans cells in normal and pathological human scars. I. Atrophic scars.

In the present study, we investigated Langerhans cells (LCs) in the epidermal component of human atrophic scars, comparing them with those in control skin and normotrophic scars. A preliminary analysis of the histological features was first carried out on vertical serial sections, stained with hematoxylin and eosin. The total epidermal thickness and the thickness of the single epidermal layers were then measured, by means of a digitizing tablet and a morphometric program run on an Apple IIe computer. These parameters were found to be significantly lower (40%) in atrophic scars, if compared to control skin and normotrophic scars (p less than 0.05). CDla-positive and HLA-DR-positive LCs were marked by indirect immunofluorescence. Their position among the epidermal layers, their dimensions, their density and their morphology were examined. In atrophic scars, LCs were densely and evenly distributed in all the epidermal layers. Their density was increased (about 1200 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both 300-400 cells/mm2 of epidermal area; p less than 0.001). The CDla-positive definite cell bodies, exhibiting an unstained nucleus, were as large as those evidentiated in the normotrophic scars and twice as much the control skin values (p less than 0.001). The present results provide morphological data that distinguish atrophic scars from control skin and normotrophic scars, and suggest an involvement of the Langerhans cells in this particular case of pathological scarring.

Comparative study of Langerhans cells in normal and pathological human scars. II. Hypertrophic scars.

Langerhans cells (LCs) seem to play a crucial role in the immune system of the skin. Changes in their density, distribution, phenotype and/or morphology have been described in a number of skin diseases, mostly immunologically mediated. For this reason, we investigated LCs in human hypertrophic scars, since these scars are presently believed to have an immunological basis. A preliminary analysis of the histological features was carried out on vertical serial sections, stained with hematoxylin and eosin. Both epidermal and dermal components of hypertrophic scar biopsies were examined. The total epidermal thickness and the thickness of the single epidermal layers were also measured; the values obtained were similar to those of control skin and normotrophic scars. Subsequently, CDla-positive LCs, revealed by indirect immunofluorescence and immunoperoxidase techniques, were studied to determine their position among the epidermal layers and within the dermis, their dimensions, their density and their morphology. According to these observations, two main types of hypertrophic scars were identified. In the first type (7 scars), LCs were widely clustered within both the whole epidermis and the dermis. Their density was increased (about 750 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both about 400 cells/mm2 of epidermal area; p less than 0.001). The epidermal cell profiles, nearly three times larger than those of control skin, exhibited a dense network of interconnected dendrites. Further analysis for the presence of HLA-DR molecules revealed an anomalous expression of these antigens on keratinocytes. In the second type (3 scars), LCs density within the stratum Malpighii was unchanged, relative to control skin and normal scars, while CDla-positive cell bodies remained numerous in basal position and within the subpapillary corion. Epidermal LCs, only slightly larger than those evidentiated in control skin, displayed short and retracted dendritic projections. The aberrant expression of HLA-DR antigens on keratinocytes was very weak and sparse. The present results strongly suggest an immunologically activated state of the tissues examined; they provide morphological data that support the involvement of the immune system in hypertrophic scarring.

Differentiation of cardiac conducting cells from the neural crest.

Some of the cells that migrate to the dorsal myocardium of the chick embryo in stage 21 H-H begin to synthesize desmin. They retain this property even when they reach the subendothelial layers of the heart bud, i.e. the characteristic site of the cardiac conducting cells.

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.

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.

Dynamics of soleplate formation.

Immunohistochemical techniques with anti-desmin, anti-acetylcholine receptor and anti-fibronectin antisera and autohistoradiography were used to determine the dynamics of neuromuscular synaptogenesis. Fast twitching muscles were taken from chick embryos at 5 to 14 days of incubation. "Primitive eminences" at terminal arborizations of motor neurons were composed of Karnowsky positive, anti-desmin and anti-acetylcholine receptor positive cells which contained sites bound to alpha-bungarotoxin. These cells, characterized as myoblasts, fused with the myotubes during formation of neuromuscular junctions in the sites of contact with terminal arborizations of motor neurons. Their nuclei and cytoplasmic organelles become the nuclei and organelles in the soleplate.

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.

DNA content in neurons of Auerbach's plexus under experimental conditions in adult rats.

Some nerve cells of the Auerbach's myenteric plexus of the intestine of the adult rat, which hypertrophied following a surgically induced stenosis, began DNA synthesis unrelated to mitotic division. The cytophotometric analysis confirmed and quantified the amount of synthesis revealed by autoradiography with tritiated thymidine uptake. Numerous nerve cells show a DNA content exceeding the diploid level. Only a few of these show twice the diploid content. The significance of the DNA synthesis is discussed.