Induction of mild enterocolitis in zebrafish Danio rerio via ingestion of Vibrio anguillarum serovar O1.

Vibrio anguillarum is the etiological agent of a fatal hemorrhagic disease known as vibriosis that affects a wide range of fish species, causing severe economic losses. Several investigations have been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. The aim of our study was to evaluate the most effective way to induce mild enteritis in a fish model, in order to allow further applications. The experiments were carried out using 2 methods of administration of V. anguillarum serotype O1 to adult zebrafish Danio rerio: via intraperitoneal injection and via ingestion of infected Artemia nauplii. The results showed that the intraperitoneal administration often caused massive fish death due to severe systemic involvement. In our experiments, the effect of intraperitoneal infection was evident 48 h post infection, with cumulative mortality within 7 d post infection with severe histopathological changes in kidney hematopoietic tissue and in the intestine. In contrast, oral infection via Artemia did not show systemic involvement and only a moderate degree of inflammatory influx of the mucosa, a partial recovery at 12 d post infection, and no mortality. For these reasons, oral infection with live food appears to be the most effective method to induce mild enteritis with a local inflammatory response.

Localization of cholecystokinin in the zebrafish retina from larval to adult stage.

The peptide hormone cholecistokinin (CCK) plays a key role in the central and peripheral nervous system. It is known to be involved in the digestive physiology and in the regulation of food intake. Moreover, the CCK expression has also been detected in the retina of different vertebrates, including fish, although its biological activity in this tissue remains to be elucidated. In literature no data are yet available about the CCK-immunoreactivity in the zebrafish retina during development. Therefore, the aim of the study was to investigate the distribution of sulfated cholecystokinin octapeptide (CCK8-S) as a well preserved form during evolution in the zebrafish retina from 3days post hatching (dph) until adult stage, using immunohistochemistry in order to elucidate the potential role of this protein in the development and maintenance of normal retinal homeostasis. The cellular distribution of CCK in the retina was similar from 3 dph to 40days post fertilization (dpf) when immunoreactivity was found in the photoreceptors layer, in the outer plexiform layer, in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer (GCL). Immunohistochemical localization at 50 dpf as well as in the adult stage was observed in a subpopulation of amacrine cells in the proximal inner nuclear layer, in the inner plexiform layer, in displaced amacrine cells and in retinal ganglion cells in the GCL. Our results demonstrate for the first time the occurrence of CCK in the zebrafish retina from larval to adult stage with a different pattern of distribution, suggesting different roles of CCK during retinal cells maturation.

A new morpho-functional classification of the Fallopian tube based on its three-dimensional myoarchitecture.

The recent direct observations, under scanning electron microscopy (SEM), of the three-dimensional architecture of myosalpinx in different mammals allows us classify salpinxes according to the myoarchitecture of their tubo-uterine junction (TUJ) and isthmus segments. Based upon the myoarchitecture of the outer wall of the TUJ we could find barrier-like species (rat and sow), sphincter-like species type a (rabbit and ewe) and sphincter-like species type b (cow and woman). The different architecture of TUJ can be explained by the different nature of the mating process. Based upon the myoarchitecture of the isthmus we could distinguish type 1 (rat) and type 2 (rabbit, ewe, sow, cow and woman) salpinxes. In the latter the close fusion of musculature deriving from the meso (extrinsic musculature) with the musculature of salpinx (intrinsic musculature) suggests the existence of a unique mesosalpinx contractile system. The myosalpinx is mostly made up of a single network of muscular fibers. Such a plexiform structure, owing to the uneven distribution of fibers, rather than producing a series of regular contraction waves, is more likely to generate random contraction waves. The random propagation of muscular network contraction may deform the plexiform wall of the myosalpinx causing the stirring of tubal contents. By such a stirring movement the contact between hormones and nutrients and the eggs or embryos is intensified, thus favoring a correct fertilization and early embryo development. Taken all together, these systematic results probably suggest an additional and rather new function for the musculature of the tube, namely to increase fertility in a large number of species.

The three-dimensional architecture of the myosalpinx in the rat (Rattus norvegicus) as revealed by scanning electron microscopy.

The three-dimensional (3-D) architecture of myosalpinx in the rat has been investigated by means of scanning electron microscopy after microdissection and removing interstitial connective tissue with 6N NaOH digestion. In the extramural portion of tube-uterine junction the myosalpinx shows circularly arranged fibers originating from the uterus, together with oblique fibers typical for the salpinx, which occur more frequently in the deeper layers. As fibers approach the mucous folds they assume a plexiform arrangement, which is maintained through all tubal segments. In the isthmus surface fibers form wide muscle rings around the elbow of loops, peculiar to the rat tubal morphology. Surface fibers in the ampulla and pre-ampulla have an even circular course. Our 3-D results reveal that the muscular architecture of rat tube is mainly organized in concentric, monolayered shells with a plexiform arrangement tightly fastened together. Functionally, this muscular arrangement seems to be capable of stirring rather than pushing the embryo and gametes. Finally, such a plexiform network might work as a mechanism of "tube locking" in proximity of isthmic loops as well as at the level of the ampullary-isthmic junction.

Fine structure of spermatozoa in the common pandora (Pagellus erythrinus Linnaeus, 1758) (Perciformes, Sparidae).

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the Sparid fish Pagellus erythrinus L. The spermatozoon of pandora has a spherical head lacking an acrosome, a cone-shaped midpiece and a long tail. The midpiece houses a single mitochondrion. The centriolar complex lies inside the nuclear fossa and is composed of a proximal and a distal centriole which are arranged at right angles to each other. The flagellum is inserted medio-laterally into the head, contains the conventional 9+2 axoneme and possesses one pair of lateral fins. On the basis of its ultrastructural organization, the pandora sperm can be regarded as an evolved form of the primitive spermatozoon found in Teleosts. According to the morphological classification proposed by Mattei (1970), the sperm of pandora belongs to a "type I" designation, like that of the other Sparid fish.

Localization of vinculin and talin at perineurial cells of human sural nerve.

Immunolocalization of spectrin, vinculin, talin, desmin and titin was investigated in human sural nerve. No binding for spectrin and titin was seen in any structure of the nerve. Antibody against desmin immunostained sporadic epineurial vessels only. Endoneurial and epineurial vessels were intensely positive for vinculin and talin. We found expression of vinculin and talin at the perineurial cells, using immunocytochemistry and gold immunoelectron microscopy. Since vinculin and talin are known to be involved in cell-cell and cell-extracellular matrix transmembrane connections, we propose that they, possibly together with other cytoskeletal proteins, may be implicated in the permeability barrier property of the perineurium. In pathological conditions, perineurium plays an as yet unknown role. Future studies are needed to investigate expression of vinculin and talin in neuropathies.

Dp116, talin, vinculin and vimentin immunoreactivities following nerve transection.

The time course of the expression of Dp116, talin, vinculin and vimentin in rat sciatic nerve was investigated after experimental transection. Dp116 was still found at 5 days after experiment in some degenerating myelinated fibers of both proximal and distal stumps. The findings are consistent with the known preservation of electrical excitability of the distal nerve in the first days after injury. Some regenerating nerve fibers into the neuroma also expressed Dp116 at 25 and 40 days after nerve transection. Talin and vinculin markedly and diffusely immunostained the neuroma. Talin in the distal stump and vimentin in both proximal and distal stumps were found decreased during the time course of the experiment. Vinculin binding increased in the distal stump, due to a real overexpression or simply to a cross-reaction to degeneration products.

Experimental investigations on a model of cryogenic edema.

The role of mechanisms underlying formation and progression of vasogenic brain edema is investigated. On this purpose, cerebral edema was produced by cortical freezing in two different brain situations in rabbits (with or without replacement of bone flap). BBB (Blood-Brain Barrier) breakdown was evaluated by observation of Evans blue extravation, while a histopathological evaluation was carried out by light and transmission electron microscopy. Water content of brain tissue was determined by the wet/dry weight ratio method. Comparison of extension and intensity of cerebral edema between these two groups of animals shows a statistically significant difference: there was evidence of higher water content in the animals undergone replacement of bone flap. The Authors emphasize the importance of tissue pressure gradients in determining diffusion of cerebral edema.

Clinical, morphometric and ultrastructural aspects in a new model of spinal cord compression.

A new model of spinal cord compression is presented. Forty-two New Zealand albino rabbits weighing between 1.2 and 1.5 kg were submitted to spinal cord compression to about half of its normal diameter by vascular Sugita's clip to different compression time (sham, 5-10-20 minutes e 1-6-12 hours) and sacrified after 72 hours. The consequent neurological dysfunction size of lesion was graded by Tarlov's scale for voluntar motility. Ultrastructural and morphometrical studies were carried out to evaluate changes in shape, perimeters, axonal and myelin areas of the lateral and the posterior tract of spinal cord. A 10 minute decrease of 50% of the transverse diameter of the spinal cord causes a defined damage. The swelling of myelin is a useful index of the importance of the damage.

The architecture of the myosalpinx in the sow as revealed by scanning electron microscopy.

OBJECTIVE: To provide a definitive settlement of data on the architecture of myosalpinx in the sow in consideration of controversial data existing in literature. STUDY-DESIGN: To allow direct visualization of muscular architecture, segments of tube from fifteen sows were investigated by means of scanning electron microscopy after the removal of interstitial connective tissue with NAOH digestion. RESULTS: In the extramural portion of the tubo-uterine junction, in the isthmus and ampulla, the myosalpinx is mainly constituted by oblique bundles of variable length, which run around the tube and merge into the surrounding musculature, giving origin to a plexiform arrangement. In the ampulla the fibers join in short bundles variously oriented. CONCLUSION: The three-dimensional architecture of the sow myosalpinx consists of muscular bundles independent of one another which follow multiple spatial arrangements and form a complex network. Such a muscular structure is likely more suitable for stirring rather than pushing the embryo and gametes.

The three-dimensional architecture of the myosalpinx in the cow as revealed by scanning electron microscopy.

The three-dimensional architecture of the myosalpinx in the cow has been investigated by means of scanning electron microscopy after removal of interstitial connective tissue with NaOH digestion. In the extramural portion of the tubo-uterine junction, in the isthmus and ampulla, the myosalpinx is made up of oblique bundles of variable orientation and length, which are loosely distributed in the tubo-uterine junction and densely packed in both isthmus and ampulla. These bundles intersect and merge into the surrounding musculature. Our observations demonstrate how myosalpinx consists mainly of bundles of muscular fibers independent one of another, which show a multiple spatial arrangement and form a complex network. Such a muscular architecture is likely more suitable for stirring rather than pushing the embryos and gametes through the tube.

The three-dimensional architecture of the myosalpinx in the rabbit as revealed by scanning electron microscopy.

The three-dimensional (3-D) architecture of the myosalpinx in the rabbit has been investigated by means of scanning electron microscopy after microdissection and the removal of interstitial connective tissue with KOH digestion. In the extramural portion of the tubo-uterine junction, the myosalpinx presents outer longitudinal bundles which form a well-defined continuous muscular layer extending towards the ampulla. Underlying this layer there are single muscular bundles which follow an uneven circular arrangement. At a deeper level the same bundles present a plexiform arrangement. Several bundles reach the base of the mucous folds and follow a discontinuous, approximately longitudinal arrangement. In the isthmus, the myosalpinx shows superficial longitudinal strips that abruptly bend sideways as they approach the ampulla, thereby enveloping the underlying muscular structure. The latter is arranged in the same way as at the extramural portion of the tubo-uterine junction, except for the lack of the outer longitudinal layer originating from the uterus. Our 3-D results revealed that the architecture of the rabbit myosalpinx consists of muscular bundles independent of one another that follow multiple spatial arrangements and form a complex network. Such a type of structure seems to be capable of stirring rather than pushing the embryo and gametes.

[Morphometric analysis of the sciatic nerve and its principal branches in the pigeon (Columba livia)]. / Analisi morfometrica del nervo ischiatico e dei suoi rami principali nel colombo (Columba livia).

The methodological approach used in this study is to characterize the number, the density and the diameter distribution of myelinated fibers (MFs) and unmyelinated fibers (UMFs) in sciatic nerve and its main branches of pigeon. The results have shown that the fiber composition is quite variable because in pigeon there are relatively MF with thin myelin sheaths and MF with thicker sheaths. Our data suggest that morphometric analysis could represent a helpful methodological approach to better characterize these systems.

Fine structure of spermatozoa in the blackspot sea bream Pagellus bogaraveo (Brünnich, 1768) with some considerations about the centriolar complex.

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Pagellus bogaraveo. The spermatozoon of P. bogaraveo belongs, like that of the other sparid fish, to the teleostean "type I" spermatozoon with the flagellar axis insert perpendicular to the nuclear fossa. It has an ovoidal head, a short, cylindrically shaped midpiece and a long tail region. The nucleus reveals a deep invagination (nuclear fossa), in which the centriolar complex is located, and a satellite nuclear notch shaped like a golf club. The two centrioles are perpendicular to each other and show a conventional "9+0" pattern. The distal centriole is attached to the nuclear envelope by means of basal feet and radial fibers made of electron-dense material. Below the basal plate, plasma membrane pinches in, and the necklace, a specialized connection joining axonemal doublets to the plasma membrane, is visible. The short midpiece houses one mitochondrion. The flagellum is perpendicularly and eccentrically with respect to the nucleus and contains the conventional "9+2" axoneme.

Ontogeny of the digestive tract in sharpsnout sea bream Diplodus puntazzo (Cetti, 1777).

The ontogeny of the digestive tract was studied histologically and histochemically in sharpsnout sea bream Diplodus puntazzo from hatching (0 DAH, Days After Hatching) until day 57 (57 DAH). At hatching, the digestive tract appeared as a histologically undifferentiated straight tube lying dorsally to the yolk sac. When the mouth opened at 3 DAH, the digestive tract was differentiated into buccopharynx, oesophagus, incipient stomach and intestine. The pancreas, liver and gall bladder were also differentiated at this stage and both the bile and pancreatic duct had opened into the anterior intestine. Active feeding began in 50% of larvae at 4 DAH, although permanence of yolk reserves until 7 DAH suggests a period of both endogenous and exogenous feeding. Nutrient absorption was first visible from 5 DAH, as colourless supra- and infranuclear vacuoles in the anterior intestinal mucosa, suggesting a lipid content, as well as supranuclear, eosinophilic vacuoles, containing protein, in the posterior intestinal mucosa. Early caecal development could be detected from 10 DAH, whereas gastric glands appeared at 30 DAH, indicating the transition from larval to juvenile stage and the acquisition of an adult mode of digestion. Goblet cells appeared in the digestive tract of sharpsnout sea bream larvae shortly after first feeding. The mucus content of goblet cells varied with the digestive region and, in the buccal cavity and oesophagus, also with the developmental phase. This study provides knowledge for better husbandry practices in the aquaculture industry, as well as for the implementation of future nutritional studies.

Fine structure of spermatozoa in the gilthead sea bream (Sparus aurata Linnaeus, 1758) (Perciformes, Sparidae).

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Sparus aurata L. The mature spermatozoon of gilthead sea bream belongs, like that of the other sparid fish, to a "type I" as defined by Mattei (1970). It has a spherical head which lacks an acrosome, a short, irregularly-shaped midpiece and a long cylindrical tail. The nucleus reveals a deep invagination (nuclear fossa) in which the centriolar complex is located. The two centrioles are approximately perpendicular to each other and show a conventional "9+0" pattern. The proximal centriole is associated with a cross-striated cylindrical body lying inside a peculiar satellite nuclear notch which appears as a narrow invagination of the nuclear fossa. The distal centriole is attached to the nuclear envelope by means of a lateral plate and radial fibres made of an electron-dense material. The short midpiece houses one mitochondrion. The flagellum is inserted perpendicularly into the base of the nucleus and contains the conventional 9+2 axoneme.

Pored domes in adrenocortical capillaries. An ultrastructural study in mammals.

The adrenocortical gland is one of the most vascularized organs of the mammalian body. It undergoes continuous morphological changes dynamically dependent upon special permeability conditions related to various physiological and physiopathological events. The adrenal cortex of different adult mammals (3 pigs, 2 mice, 3 sheep) was studied by means of scanning (SEM) and transmission (TEM) electron microscopy. As seen by SEM it is formed of polyhedral cells which delimit a characteristic continuous labyrinth system of intercellular lacunae occupied by sinusoid-like capillaries. The capillary wall is fenestrated and is lined by flattened endothelial cells with their nuclear part bulging into the capillary lumen. The fenestrae are round or oval pores measuring 50-100 nm in diameter. They are usually clustered to form sieve plates and characteristically present a thin membranous diaphragm. Irregular microelevations and dome-like projections are often seen. These structures are made of thin cytoplasmic plates interrupted by numerous small pores which show a sieve plate profile. The pored-domes on the nuclear portion seem to be identical in structure to those found in the thinner endothelial part; some of them appear to detach from the endothelial cell. The pored-domes are structurally comparable to those reported both in the renal glomerular endothelium and in liver sinusoids. These endothelial structures may be an expression of the high rate of filtration of these tissues and may be also related to the final step of the replacement of the sieve plates during endothelial regeneration.

An ultrastructural study of pigeon bursa of Fabricius during involution.

The ultrastructure of the pigeon bursa of Fabricius during involution is described. According to the AA, the involutive process may be divided into 3 stages: an early involutive stage, a late involutive stage and a residual stage. The involutive process starts from epithelium later affecting the follicular medulla and all other structures of the organ. A mucoid degeneration can be observed, followed by an impressive fatty degeneration.

The collagen skeleton of the cat testis. A scanning electron microscopy study after 2N-NaOH maceration.

Testes from adult cats were studied by means of scanning electron microscopy (SEM) after 2N-NaOH maceration method which selectively digests cells and tissue compounds. As seen by this technique, the testis appears covered by a dense fibroconnective tunica albuginea that divides partially the organ by sending septa into the parenchyma. The interstitium is made up by a rich connective tissue composed by randomly-arranged fine collagen bundles that clearly outline the spaces occupied by the seminiferous tubules. The basement membrane of the seminiferous tubules consists in two rough layers of collagen fibers that in some places leave small fenestrations. The three-dimensional arrangement of collagen fibers in the interstitium outlines a system of cavities likely to be lacunar spaces for Leydig cells and vascular imprints. Especially, the above chemical technique followed by deep observations under the SEM allowed a clear and complete view of the real three-dimensional microarchitecture of the connective tissue of the testis. Therefore, the collagen component was revealed to actually form a unique and complex skeleton for the whole organ. This three-dimensional figure closely follows indeed the classical histological compartmentalization of the testis with a better insight of its spatial microtopographical features. Moreover, this structural pattern is also likely to give rise to very fine morphofunctional subcompartmentalization, especially regarding the basement membrane of the seminiferous tubules and the lacunar spaces for Leydig cells including satellite vessels.

Microarchitecture of the cat testis with special reference to Leydig cells: a three-dimensional study by alkali maceration method and scanning electron microscopy.

Testes from adult cats were studied by means of parallel transmission and scanning electron microscopy (SEM) after NaOH digestion technique, which selectively removed connective tissues or cells. The testis is covered by a dense fibroconnective tunica albuginea that partially divides the organ in lobules by sending septa into the parenchyma. The lamina propria of the seminiferous tubules consisted of one or two rows of cells. The interstitium was made up of randomly arranged collagen bundles. The most significant feature was the numerous Leydig cells rich in lipid droplets and displaying epithelioid features. Following alkali digestion and SEM these cells showed a cord-like arrangement. The cords were formed by one or two closely apposed cells, in between which some labyrinthine or canalicular-like spaces were left that in some areas opened in wide perivascular spaces. This particular arrangement of Leydig cells and the labyrinthine intercellular spaces is very likely designed to improve cell secretion of hormones, facilitating their transport into the blood, as well as the traffic of fluids and metabolites. The present techniques allowed the visualization of a real three-dimensional testicular microarchitecture and microtopography, not detectable with other methods. Such a study may help to better highlight the testicular morphophysiology.