Analysis of pre- and postsynaptic activity in the frog semicircular canal following ototoxic insult: differential recovery of background and evoked afferent activity.

Frogs were treated with a single dose of gentamicin administered intraotically to produce severe degeneration of posterior semicircular canal hair cells and to evaluate the time course of functional damage and recovery both at pre- and postsynaptic level. In isolated canal preparations the endoampullar potential, which reflects the summed receptor potentials of crista hair cells, was progressively reduced in amplitude and completely abolished 6 days after gentamicin treatment. At this time the crista epithelium was devoid of hair cells. The recovery of the endoampullar potential began around 9 days after the ototoxic insult and its amplitude progressively increased to reach, after 20 days, values close to those observed in control experiments. The endoampullar potential amplitude was related to the degree of hair cell regeneration in the crista epithelium. Consistent with the presynaptic damage, the slow generator potential (representing the summed miniature excitatory postsynaptic potential [mEPSP] activity of all posterior nerve fibres) and the resting and evoked spike discharge recorded from the whole ampullar nerve were abolished 6 days after gentamicin treatment. The recovery of the background and evoked afferent activity showed different behaviours. Background spike activity became detectable around 8 days after the ototoxic insult, but was not modulated by canal stimulation at this time, and no generator potential was detected. Moreover, the resting spike frequency fully recovered and reached control values around 15 days after gentamicin treatment, whereas the evoked activity attained normal values only 20 days after the ototoxic insult. These results were confirmed by intracellular recordings from single afferent fibres of the ampullar nerve in intact labyrinth preparations. Absence of any resting and evoked discharge was the most common pattern observed in the early period from 7 to 8 days after gentamicin treatment. Fifty-five percent of impaled afferents were silent while the others showed low resting frequencies of mEPSPs and spikes, and were unresponsive to canal rotation. In the intermediate period from 14 to 15 days after gentamicin treatment, background mEPSP and spike frequencies approached those evaluated in control experiments, but the frequencies of the evoked mEPSPs and spikes were clearly lower than in controls. In the late period, from 18 to 20 days after the ototoxic insult, the impaled afferents showed normal evoked mEPSP and spike frequencies. The present data indicate that the frog semicircular canal completely recovers its pre- and postsynaptic activity following severe ototoxic insult. During the regeneration process, the cytoneural junction regains function and the resting discharge reappears before recovery of mechanoelectrical transduction.

Isolated porcine bronchi provide a reliable model for development of bronchodilator anti-muscarinic agents for human use.

BACKGROUND AND PURPOSE: In human airways, muscarinic acetylcholine receptors (mAChRs) exert a predominant role in the control of airways resistance and anti-muscarinic agents are currently included in the pharmacological treatment of chronic obstructive pulmonary disease (COPD). However, the development of more effective mAChR antagonists is hampered by considerable species variability in the ultrastrucural and functional control of airway smooth muscle, making extrapolation of any particular animal model questionable. This study was designed to characterize the mAChRs in a bronchial preparation from pigs, animals considered to provide close models of human biology. EXPERIMENTAL APPROACH: Smooth muscle bronchial strips were examined by electron microscopy in order to compare their neuromuscular structure with that of human bronchi and used to study the affinity of a series of selective mAChR antagonists, estimated as pKis in competition binding assays with NMS and pA2, by Schild analysis, in contractile experiments. KEY RESULTS: Pharmacodynamic binding parameters and affinity profiles of a series of antagonists were consistent with the presence of a majority of M2 mAChRs along with a minor population of M3 mAChRs. Functionally, the highly significant correlation between postjunctional pA2 affinities and corresponding affinity constants at human recombinant M1-M5 subtypes indicated that smooth muscle contraction in porcine bronchi, as in human bronchi, was dependent on the M3 subtype. CONCLUSION AND IMPLICATIONS: Based on the characterization of mAChRs, isolated porcine bronchi provide an additional experimental model for development of mAChR antagonists for the treatment of human airway dysfunctions.

Selective impairment of noradrenergic transmission in the bisected rat vas deferens following photochemically-induced cerebral ischaemia.

BACKGROUND: Cerebrovascular disease may impair the autonomic control of peripheral organs including the male urogenital tract. This study investigates the effect of cortico-parietal focal ischaemia on the adrenergic and purinergic transmission in isolated epididymal and prostatic portion of rat vas deferens. METHODS: Focal brain ischaemia was induced in male rats by photochemical activation following rose bengal intravenous injection. Twenty-four hours following brain ischaemia, cumulative and non-cumulative concentration-response curves were obtained for noradrenaline and alpha,beta-methylene ATP in the right and left epididymal and prostatic portions of the vas deferens. Both portions were also stimulated by single-pulse or pulse trains at 2-30 Hz to produce isometric contractions. RESULTS: In both portions from ischaemic rats the response to exogenous noradrenaline was markedly depressed compared with controls. Acute cortico-parietal ischaemia almost completely abolished the adrenergic phase of the response to single-pulse stimulation in the epididymal portion of the vas deferens. In addition, brain ischaemia deeply depressed phasic and tonic contractions of the frequency-response curve in both portions of bisected vas deferens. CONCLUSIONS: Cortico-parietal ischaemia produces a selective noradrenergic impairment at the level of male sexual secondary organs that may contribute to sexual dysfunction after stroke.

Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration.

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogeneous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

Localization of Ca-ATPase in frog crista ampullaris.

The distribution of Ca-ATPase in frog crista ampullaris was mapped ultracytochemically by using a one-step lead citrate reaction. Electron-dense precipitates, as an expression of Ca-ATPase activity, were observed on the surface of stereocilia and on the apical membrane surrounding the cuticular plate of hair cells. Sensory cells of the isthmus region showed more reactivity than those of the peripheral regions of the crista. No reaction products were detectable on the basolateral membranes and in cytoplasmatic organelles. Supporting cells of the crista showed a quite variable Ca-ATPase reaction on microvilli and on basolateral membranes. The presence of an evident reactivity on the stereocilia is consistent with the existence of an apical calcium microdomain involved in the mechano-transduction process and supports the current view that calcium ions enter the stereocilia during natural stimulation. On the other hand, the lack of an observable reactivity on the basolateral membrane of hair cells suggests that in semicircular canals other mechanisms of active transport of calcium ions across the plasma membrane, such as Na-Ca exchange, may be involved in homeostasis of the ion.

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to beta-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.

Evidence of rat tail tendon proteoglycans at emission field scanning electron microscopy.

Alcian blue (AB) in critical electrolyte concentration (CEC) was used for scanning electron microscopy (SEM) on rat tail tendon. Segments with diameters of 4 to 9 nm are evident in the perifibrillar area with their lengths ranging from 180 to 400 nm. Frequently the segments adhere closely to collagen gap zones. Segments are not evident either in specimens fixed in glutaraldehyde or in specimens stained with AB in CEC solutions previously treated with glycanolytic enzymes. The segments suggestively correspond to electrodense filaments evident in transmission electron microscopy (TEM) if the rat tail tendon is similarly treated (Scott, 1981). It may be concluded that AB in CEC solutions can be utilized for SEM investigations and represents a good method for the detection of the three dimensional disposition of proteoglycans (PG) and their interaction with collagen.

The paracoronal (marginal) cells of fungiform papilla of Rana esculenta.

Paracoronal secretory cells can be observed outside the sensorial area of the fungiform papilla of Rana esculenta. The morphology of these cells, the type of secretion and their function have, to date, only been incidentally described. By scanning electron microscopy (SEM), the paracoronal cells appear as swallow's-nest-shaped formations with openings 10-15 microns in diameter. The walls of paracoronal cells are characterized by laminar processes subdividing the interior hollow. The cavity of these formations is occupied by amorphous material as demonstrated by light microscopy (LM) pictures. The secretory material fills 7/8 of the upper part of the cytoplasm and appears rather transparent. The secretory material is PAS-negative, unlike the secretory granules contained in laminar processes. By transmission electron microscopy (TEM), they appear as clear ovoid structures, the nucleus of which is situated in the deeper part of the cell, enveloped by a thin cytoplasmic layer and characterized by secretory apparatus and the presence of secretory granules of middle electron-opacity. The apical part of these cells presents large mucous droplets. These cells adhere both to ciliated and parietal cells. Following cytochalasin-B treatment, cells do not show any considerable ultrastructural modification, while after terbutaline treatment the profiles of secreted paracoronal cells increase greatly. Histochemical properties of their secretory products are similar to those of parietal cells and their particular anatomical localization may exclude the direct implication of these cells in taste transduction.

The role of proteoglycans in maintaining collagen fibril morphology.

The aortic wall contains various heterogenous proteoglycan populations which interact in different ways with other components of extracellular matrix. Proteoglycans (PGs) are known to provide structural support to the vessel wall as well as to influence specific physiological functions of the tissues. The aim of the present study was to investigate the effects of Chondroitinase AC (Chase), Streptococcal Hyaluronidase (Hyase) and Heparanase on human aortic wall collagen which had been treated previously with 4M GuHCl, in order to verify the effects of selective glycanolytic treatment on type I collagen fibril ultrastructure. Following 4M GuHCl treatment, collagen fibrils are seen to have a clearly visible period. Subsequent to GuHCl and Streptococcal Hyase treatment all collagen fibrils appear to be completely swollen in thin aperiodic filaments; the typical 64 nm collagen period is completely undetectable. After GuHCl and Chase treatment a small number of collagen fibrils are seen to be swollen in thin fibrils which are mainly localized at some distance from elastic fibres. Following GuHCl and Heparanase/Heparitinase III treatment a considerable number of collagen fibrils appear to be swollen in thin fibrils; the majority of which are situated in the vicinity of elastic fibrils. The swelling of collagen fibrils underlines the fundamental role of proteoglycans in maintaining collagen fibril integrity and periodicity. It is as yet impossible to precisely map interactions between these proteoglycans and collagen fibres. The role of Hyaluronic acid requires further investigation, although the nature of this interaction is undoubtedly a matter of considerable interest.

Morphological features of different regions in frog crista ampullaris (Rana esculenta).

The cellular organization of different regions of the crista epithelium from the frog posterior semicircular canal was studied by light, transmission and scanning microscopy. The sensory epithelium consists of hair cells surrounded by supporting cells and basal cells located close to the basement membrane. Three types of hair cells, namely club-like, cylindrical and pear-like cells differentially distributed along the crista could be recognized on the basis of their shape. Club-like cells are located only in the peripheral regions, cylindrical cells both in the central and in the peripheral regions, and pear-like cells appear segregated into the intermediate regions. Sensory cells of the central region are characterized by a ciliary apparatus consisting of stereocilia usually shorter--and in some cases less numerous--than those of cells of the other regions. The presence of large evaginations of the apical membrane of hair cells and of several vesicles of microexocytosis demonstrates that receptor cells have a considerable secretory activity. This secretory activity is also proven by the presence in the supranuclear region of hair cells of numerous Golgi complexes. Moreover, the presence of two kinds of Golgi complexes, one constituted by dilated cisternae containing a moderately electron-dense material and the other made up of flattened electron-transparent cisternae, suggests a diversified secretion of material by the hair cells. This heterogeneous material may provide substances important for cupula formation and the composition of the endolymph.

Benign idiopathic hypereosinophilia: a feeble masquerader or a smouldering form of the hypereosinophilic syndrome?

In a patient with long-standing idiopathic hypereosinophilia with no apparent organ damage we measured serum eosinophil cationic protein (ECP) and eosinophil protein X (EPX) titers, activated circulating eosinophil rates (by means of monoclonal antibodies EG1 and EG2), and the release of ECP and EPX in vitro by leukocytes at different cultures stages in order to detect possible functional abnormalities associated with hypereosinophilia. Our patient had elevated serum levels of both ECP and EPX, together with a high EG2 count, which would suggest eosinophil activation. However, serum levels of ECP and EPX were not significantly high in relation to the total number of eosinophil cells, although they were more numerous than in healthy controls. Moreover, the release of intracytoplasmic basic proteins by the patient's eosinophils was poor even after in vitro stimulation. Since hypereosinophilic syndrome (HES) with organ damage can appear as long as 8-9 years after the presence of a hypereosinophilic state, the absolutely benign nature of our patient's condition still cannot be defined. Thus, there is the possibility it could be slow-onset or smoldering HES.

Effects of cytochalasin B on the morphology of secretory cells of Rana esculenta fungiform papilla.

The effects of cytochalasin B on the three types of secreting cells (apical, paracoronal and parietal cells) present in the fungiform papilla of the frog's tongue were studied. Cytochalasin B induced morphological modifications only in the apical secreting cells, localized in the sensorial area, without affecting the paracoronal cells or the parietal cells. The morphological features of treated apical cells showed that the drug induces two different effects: a disappearance of microfilaments and an increase in the exocytotic process. The results are related to previous experimental findings which showed that cytochalasin B produces actin depolymerization and interferes with calcium transport.

The dependency of collagen fibrillogenesis in vitro on fibroblast culture conditions. Fibroblasts in mono- and multi-layers.

The extracellular matrix produced by monolayer and tridimensional cultures of fibroblasts was investigated using histochemical and ultrastructural methods. In monolayer cultures, collagen and proteoglycans produced by fibroblasts could not be organized into morphologically recognizable structures. Tridimensional fibroblast cultures produced a well organized matrix with periodic, parallel ordered collagen fibrils of 50 nm diameter, criss-crossed by alcianophylic segments 6-10 nm thick in diameter and 100-300 nm in length, parallel to each other, perpendicular to the collagen fibrils and spaced 67 nm from each other. Some alcianophylic segments lay perpendicular to the above described ones, with maximum lengths of 65-70 nm. Alcianophylic segments are the ultrastructural evidence of structural proteoglycans. These observations suggest that the culture conditions influence the collagen and proteoglycans secretion, so that the final organization of the matrix results quite different.

Morphology of epiphyseal apparatus of a ranid frog (Rana Esculenta)

Morphological, histochemical and ultrastructural investigations on epiphyseal apparatus of Rana Esculenta were made. The most important findings were the following: 1) metaphyseal cartilage is localized inside proximal diaphyseal compact bone as a plug; 2) metaphyseal cartilage do not reduce in thickness during ageing; 3) metaphyseal cartilage do not show vascular invasion and do not mineralize in degenerative zone; 4) trabecular bone was not at all evident in this animal; 5) external periosteum is well vascularized and proliferates in correspondence to marginal epiphyseal end of the diaphyseal. From these results the hypothesis that the ranid frog bone growth is not due to metaphyseal metabolism (as in avian and mammals) but to bone periosteal marginal mineralization is reached.

Scrotal angiokeratoma (Fordyce): histopathological and ultrastructural findings.

Bioptic findings related to four cases of scrotal angiokeratoma-Fordyce, were studied under light and electron microscopy. A particular heterogeneity of the structural and ultrastructural patterns typical of this lesion was thus observed. Light microscopy study pointed out, in particular, different degrees of dilation of papillary vessels, whereas ultrastructural study highlighted marked alterations of endothelial cells with structural and quantitative modifications of cytoplasmic organelles.

Fine structure of the fungiform papilla in a ranid frog (Rana esculenta).

The freetop of the fungiform papilla shows a sensorial area about 100 micron in diameter, surrounded by a ring of ciliated cells. Externally to the ciliated cells, i.e., in the lateral wall, numerous large goblet cells can be seen devoid of their mucous content. The sensorial area is composed by three types of cells: mucous, supporting, and neuroepithelial cells. Mucous cells form the most superficial layer, while the cell bodies of the other two are deep, and from them basal and apical processes arise. The above mentioned cells are connected by desmosomes preferentially located between the mucous and the supporting cells, rather than between the supporting and the neuroepithelial cells. The lateral wall of the papilla is made up of a multilayered epithelium that comprises two types of cells: the first type contains electron-dense granules and an abundant rough endoplasmic reticulum, the others are ciliated cells. In the connective axis of the papilla, numerous fenestrated capillaries with endothelial vesiculated cells and nerve fibers are found.

[The ciliary apparatus of the lingual mucosa in the frog]. / Gli apparati ciliari della mucosa linguale di rana.

By means of scanning and transmission microscopy it has been examined the ciliary system of the tongue mucosa. The scanning electronmicrographs of the fungiform papillae have revealed three ciliary apparatuses allocated respectively: at the papillary summit (corona ciliata and a narrow but separated paracoronal ciliary system) and on the peduncolar papillary stem. The cilia of both paracoronal and peduncolar groups have not been yet described. Also the filiform papillae are supplied with cilia but as irregularly distributed groups. The border of the tongue is a continuous and normal ciliary epithelium and finally groups of cilia are scattered also on the whole sublingual epithelium. At the transmission microscopy the cells of all the examined mucosal ciliary groups are showing a normal ultrastructural aspect.

[Paracoronal cavity system and papillary water uptake]. / Sistema cavitario paracoronale e richiamo idrico papillare.

In the research it has been carried out a morphological investigation of the paracoronal area of the fungiform papillae. By means of scanning, transmission and light microscopy it has been observed in this area a series of superficial openings around and external to the ciliary crown; and in the epithelium corresponding cavitary system. Each cavity on the other hand appears surrounded by extremely narrow epithelial cells and thus appears able to facilitate the papillary exchange activity. This paracoronal cavitary system is proposed as morphological candidate for the conspicuous water entry in the papillae during osmotic phenomena.