Effect of light on the calretinin and calbindin expression in skin club cells of adult zebrafish.

In the last decade, zebrafish has been used as a model for the study of several human skin diseases. The epidermis of Danio rerio is composed of keratinocytes and two types of secretory cells: mucous cells and club cells. Club cells have multiple biological functions and among them may be important in the protection against ultraviolet damage through the proliferative response or through the increased production of protective substances. Calcium-binding proteins such as calbindin D28K and calretinin are used as markers of nervous and enteric nervous systems, but they are present in numerous other cells. These proteins are involved in a wide variety of cell activities, such as cytoskeletal organization, cell motility and differentiation, cell cycle regulation and neuroprotective function. In this study we demonstrated, for the first time, the presence of calretinin and calbindin D28K in skin club cells of Danio rerio exposed to different wavelengths by immunohistochemistry analysis. Exposure to white-blue light and blue light causes the expression and colocalization of calbindin-D28K and calretinin. These proteins were moderately expressed and no colocalization was observed in the club cells of the control fish. In zebrafish exposed to continuous darkness for 10 days, in the club cells the two antibodies did not detect any proteins specifically. These results demonstrate that calbindin and calretinin could be involved in the pathophysiology of skin injury due to exposure to short-wavelength visible light spectrums.

Expression of ghrelin and leptin in the chemosensory system of adult zebrafish.

Numerous data show that the chemosensory system seems to be modulated by changes in the circulating levels of different molecules such as ghrelin, orexin, leptin, NPY, CCK. The chemosensory system of the zebrafish is represented by the taste buds (skin, oral and oropharyngeal), the olfactory rosette and the solitary chemosensorial cells (SCCs). The purpose of our study was to analyze the distribution of two peripheral hormones such as ghrelin and leptin in the chemosensory organs of the zebrafish. Our results demonstrated the presence of immunoreaction for all antibodies used in the zebrafish chemosensory organs even if with different distribution. In particular, IR was observed for ghrelin in the olfactory rosette while IR for leptin was found in the olfactory rosette, in the skin and oropharyngeal taste buds and in the gills. Both these hormones were detected in the intestine, used as a control.

Melatonin treatment suppresses appetite genes and improves adipose tissue plasticity in diet-induced obese zebrafish.

PURPOSE: Overweight and obesity are important risk factors for diabetes, cardiovascular diseases, and premature death in modern society. Recently, numerous natural and synthetic compounds have been tested in diet-induced obese animal models, to counteract obesity. Melatonin is a circadian hormone, produced by pineal gland and extra-pineal sources, involved in processes which have in common a rhythmic expression. In teleost, it can control energy balance by activating or inhibiting appetite-related peptides. The study aims at testing effects of melatonin administration to control-fed and overfed zebrafish, in terms of expression levels of orexigenic (Ghrelin, orexin, NPY) and anorexigenic (leptin, POMC) genes expression and morphometry of visceral and subcutaneous fat depots. METHODS: Adult male zebrafish (n = 56) were divided into four dietary groups: control, overfed, control + melatonin, overfed + melatonin. The treatment lasted 5 weeks and BMI levels of every fish were measured each week. After this period fishes were sacrificed; morphological and morphometric studies have been carried out on histological sections of adipose tissue and adipocytes. Moreover, whole zebrafish brain and intestine were used for qRT-PCR. RESULTS: Our results demonstrate that melatonin supplementation may have an effect in mobilizing fat stores, in increasing basal metabolism and thus in preventing further excess fat accumulation. Melatonin stimulates the anorexigenic and inhibit the orexigenic signals. CONCLUSIONS: It seems that adequate melatonin treatment exerts anti-obesity protective effects, also in a diet-induced obesity zebrafish model, that might be the result of the restoration of many factors: the final endpoint reached is weight loss and stabilization of weight gain.

Neuromast hair cells retain the capacity of regeneration during heavy metal exposure.

The neuromast is the morphological unit of the lateral line of fishes and is composed of a cluster of central sensory cells (hair cells) surrounded by support and mantle cells. Heavy metals exposure leads to disruption of hair cells within the neuromast. It is well known that the zebrafish has the ability to regenerate the hair cells after damage caused by toxicants. The process of regeneration depends on proliferation, differentiation and cellular migration of sensory and non-sensory progenitor cells. Therefore, our study was made in order to identify which cellular types are involved in the complex process of regeneration during heavy metals exposure. For this purpose, adult zebrafish were exposed to various heavy metals (Arsenic, cadmium and zinc) for 72h. After acute (24h) exposure, immunohistochemical localization of S100 (a specific marker for hair cells) in the neuromasts highlighted the hair cells loss. The immunoreaction for Sox2 (a specific marker for stem cells), at the same time, was observed in the support and mantle cells, after exposure to arsenic and cadmium, while only in the support cells after exposure to zinc. After chronic (72h) exposure the hair cells were regenerated, showing an immunoreaction for S100 protein. At the same exposure time to the three metals, a Sox2 immunoreaction was expressed in support and mantle cells. Our results showed for the first time the regenerative capacity of hair cells, not only after, but also during exposure to heavy metals, demonstrated by the presence of different stem cells that can diversify in hair cells.

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.

Fine structure of the canal neuromasts of the lateral line system in the adult zebrafish.

The mechanosensory lateral line system of fish is responsible for several functions such as balance, hearing, and orientation in water flow and is formed by neuromast receptor organs distributed on head, trunk and tail. Superficial and canal neuromasts can be distinguished for localization and morphological differences. Several information is present regarding the superficial neuromasts of zebrafish and other teleosts especially during larval and juvenile stages, while not as numerous data are so far available about the ultrastructural characteristics of the canal neuromasts in adult zebrafish. Therefore, the aim of this study was to investigate by transmission electron microscopy the ultrastructural aspects of cells present in the canal neuromasts. Besides the typical cellular aspects of the neuromast, different cellular types of hair cells were observed that could be identified as developing hair cells during the physiological turnover. The knowledge of the observed cellular types of the canal neuromasts and their origin could give a contribution to studies carried out on adult zebrafish used as model in neurological and non-neurological damages, such as deafness and vestibular disorders.

Expression and distribution of leptin and its receptors in the digestive tract of DIO (diet-induced obese) zebrafish.

The expression and localization of leptin (A and B) and its receptor family in control and diet-induced obese (DIO) adult male zebrafish gut, after 5-weeks overfeeding, administering Artemia nauplii, as fat-rich food, were investigated. Recently, the obese adult zebrafish was considered an experimental model with pathophysiological pathways similar to mammalian obesity. Currently, there are no reports about leptin in fish obesity, or in a state of altered energy balance. By qRT-PCR, leptin A and leptin B expression levels were significantly higher in DIO zebrafish gut than in the control group (CTRL), and the lowest levels of leptin receptor mRNA appeared in DIO zebrafish gut. The presence of leptin and its receptor proteins in the intestinal tract was detected by western blot analysis in both control and DIO zebrafish. By single immunohistochemical staining, leptin and leptin receptor immunoreactive endocrine cells were identified in the intestinal tract either in DIO or control zebrafish. Moreover, leptin immunopositive enteric nervous system elements were observed in both groups. By double immunohistochemical staining, leptin and its receptor were colocalized especially in DIO zebrafish. Thus, our study represents a starting point in the investigation of a possible involvement of leptin in control of energy homeostasis in control and DIO zebrafish.

The Tongue Dorsal Surface in Fish: A Comparison Among Three Farmed Species.

The oral cavity of vertebrates has a very important role in many functions strictly related to the food processing. Two species, sea bass Dicentrarchus labrax and seabream Sparus aurata, are mainly involved in the aquaculture industry in the mediterranean area; moreover, the white seabream Diplodus sargus sargus was recently chosen as the best candidate for the diversification of species of commercial interest. This investigation was carried out, using the standard procedures for light and scanning electron microscopy, to analyse the morphology of the tongue dorsal surface in order to show whether relationships are present between the tongue morphology and the nutritional choices of these species. In all the three investigated species, three different areas were observed on the dorsal lingual surface: an apex, a body and a root. In the sea bass D. labrax, numerous caninelike teeth, organized in pads, were present along the dorsal surface. The presence of numerous taste buds, scattered on the tongue, was shown. In the seabream S. aurata, the apical part of the tongue is inserted in a pouch with a characteristic medial ridge on the body surface. Numerous taste buds were scattered on the whole surface. In the white seabream D. sargus sargus, a pouch partially covering the apex was clearly observed. In the pouch, filiform-like and foliate-like papillae can be distinguished. The results show the important role of the fish tongue in the food ingestion process, demonstrating the interaction of food processing, taste and morphological characteristics.

Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds.

Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds.

Acid-sensing ion channel immunoreactivities in the cephalic neuromasts of adult zebrafish.

The neuromasts are the morphofunctional unit of the lateral line system serving as mechanosensors for water flow and movement. The mechanisms underlying the detection of the mechanical stimuli in the vertebrate mechanosensory cells remain poorly understood at the molecular level, and no information is available on neuromasts. Mechanotransduction is the conversion of a mechanical stimulus into an electrical signal via activation of ion channels. The acid-sensing ion channels (ASICs) are presumably involved in mechanosensation, and therefore are expected to be expressed in the mechanoreceptors. Here we used immunohistochemistry to investigate the occurrence and distribution of ASICs in the cephalic neuromasts of the adult zebrafish. Specific immunoreactivity for ASIC1 and ASIC4 was detected in the hair cells while ASIC2 was restricted to the nerves supplying neuromasts. Moreover, supporting and mantle cells; i.e., the non-sensory cells of the neuromasts, also displayed ASIC4. For the first time, these results demonstrate the presence of the putative mechanoproteins ASIC1, ASIC2 and ASIC4 in neuromasts, suggesting a role for these ion channels in mechanosensation.

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.

Orexin A expression in the ovary of dog and cat.

Orexin A and B, also known as hypocretin A and B, are hypothalamic neuropeptides arising from a precursor to the 130 amino acid, called pre-pro orexin. They are synthesized mainly in lateral and posterior hypothalamus and are involved in different functions such as regulation of food intake and energy balance. Orexins and orexin receptors were previously described also in different tissues and organs outside the brain. The aim of this study was to demonstrate by means of the immunofluorescence technique, the presence of orexin A in the ovary of cat and dog, to support the hypothesis of the role of this substance also at the level of the female genital system. The presence of orexin A in the ovary either in dog or in cat is in agreement with previous data on the presence and role of orexins in the female genital system of other species.

Acid-sensing ion channel 2 (ASIC2) is selectively localized in the cilia of the non-sensory olfactory epithelium of adult zebrafish.

Ionic channels play key roles in the sensory cells, such as transducing specific stimuli into electrical signals. The acid-sensing ion channel (ASIC) family is voltage-insensitive, amiloride-sensitive, proton-gated cation channels involved in several sensory functions. ASIC2, in particular, has a dual function as mechano- and chemo-sensor. In this study, we explored the possible role of zebrafish ASIC2 in olfaction. RT-PCR, Western blot, chromogenic in situ hybridization and immunohistochemistry, as well as ultrastructural analysis, were performed on the olfactory rosette of adult zebrafish. ASIC2 mRNA and protein were detected in homogenates of olfactory rosettes. Specific ASIC2 hybridization was observed in the luminal pole of the non-sensory epithelium, especially in the cilia basal bodies, and immunoreactivity for ASIC2 was restricted to the cilia of the non-sensory cells where it was co-localized with the cilia marker tubulin. ASIC2 expression was always absent in the olfactory cells. These findings demonstrate for the first time the expression of ASIC2 in the olfactory epithelium of adult zebrafish and suggest that it is not involved in olfaction. Since the cilium sense and transduce mechanical and chemical stimuli, ASIC2 expression in this location might be related to detection of aquatic environment pH variations or to detection of water movement through the nasal cavity.

Tuberculosis: medico-legal aspects.

Tuberculosis is a diffusive infectious disease whose typical behaviour differentiates it from other infectious diseases spread by human-to-human transmission (flu, chicken pox, cholera, etc.) that follow a classic epidemic pattern. Indeed, in the presence of a known source of Koch bacilli that is capable of spreading the bacteria by air, not all exposed individuals inhale the bacteria, not all those who inhale them absorb them, not all those who absorb the bacteria are unable to eliminate them, not all who are able to eliminate them do so using delayed hypersensitivity, not all those who react with delayed hypersensitivity suffer lasting tissue damage (among other things, minor), not all who suffer tissue damage have anatomical sequelae, and not all those who have anatomical sequelae, however minimal, become carriers of bacilli in the latent period. The vast majority (90-95%) of the latter - which are in any case a portion, not the totality of those exposed - remain asymptomatic throughout their lives and never develop active tuberculosis. Based on these biological characteristics and the legal concepts of "epidemic" and "disease," it becomes highly problematic, if not impossible, to assert both that tuberculosis can cause events of sufficient magnitude to be associated with the crime of "epidemic," and that the mere diagnosis of a latent tuberculosis infection is sufficient to assume the presence of an illness legally prosecutable in criminal proceedings or a disability prosecutable in civil proceedings. Furthermore, clinically apparent tuberculosis is a temporarily-and in some cases permanently-disabling condition, and in certain work environments, even with the difficulties caused by the lack of available effective diagnostic tools and the insidious behaviour of the disease in the early stages, targeted monitoring to identify other persons who may become ill is appropriate.

Expression and anatomical distribution of TrkB in the encephalon of the adult zebrafish (Danio rerio).

Neurotrophins are a family of growth factor primarily acting in the nervous system, throughout two categories of membrane receptors on the basis of their high (Trk receptors) or low (p75NTR) affinity. Both neurotrophins and Trk receptors are phylogenetically conserved and are expressed not only in the central and peripheral nervous system but also in non-nervous tissues of vertebrates and some invertebrates. The brain-derived neurotrophic factor (BDNF)/TrkB system plays an important role in the development, phenotypic maintenance and plasticity of specific neuronal populations. Considering that this system is poorly characterized in the central nervous system of teleosts, the expression and anatomical distribution of TrkB in the brain of the adult zebrafish using reverse transcriptase-polymerase chain reaction (RT-PCR), Western-blot and immunohistochemistry were analysed. Both the riboprobe and the antibody used were designed to map within the catalytic domain of TrkB. RT-PCR detected specific TrkB mRNA in brain homogenates, while Western-blot identified one unique protein band with an estimated molecular weight of 145kDa, thus corresponding with the TrkB full-length isiform of the receptor. Immunohistochemistry showed specific TrkB immunoreactivity in restricted areas of the encephalon, i.e. the hypothalamus and a specific neuronal subpopulation of the reticular formation. The present results demonstrate, for the first time, that, as in mammals, the encephalon of adult zebrafish expresses TrkB in specific zones related to food intake, behaviour or motor activity.

Morphology of the tongue dorsal surface of gilthead seabream (Sparus aurata).

The gilthead seabream is a food fish, one of the most frequently used in aquaculture. In the species of commercial interest, feeding in captivity is very important and this process is strictly related to the morphological characteristics of the oral cavity. The aim of this study is, using the standard procedures for light and scanning electron microscopy, to analyze the morphology of the tongue dorsal surface to show if relationships are present between the tongue morphology and the nutritional habits and choices of this farmed species. The main characteristic of the gilthead seabream oral cavity floor is the presence of an apical pouch, with, probably, a protective role mainly for the apical, free part of the tongue. Three zones, like in other teleosts, an apex, a body and a root, can be clearly distinguished. In the pouch foliate-like papillae were observed, while the whole tongue is characterized by the presence of two types of papillae, respectively with a fungiform and cylindroid aspect, both randomly distributed throughout the whole dorsal surface of the tongue. Scattered and numerous taste buds, with the typical pear-onion shape, together with small and numerous taste pores are also present, distributed throughout the tongue surface. Our results demonstrate that in the gilthead seabream important mechanic and sensory roles are carried out by specific anatomical structures. Our anatomical data could give, together with further biochemical and physiological data, an important support with the aim of improving the nutrition of aquaculture species.

Ultrasound-guided vacuum-assisted breast biopsy: Use at the European Institute of Oncology in 2010.

Vacuum-assisted breast biopsy (VABB), which plays a fundamental role in the preoperative assessment of microcalcifications, can also be used (with ultrasound guidance) to obtain histology samples of breast lesions that are visible on ultrasonography. It is particularly useful when the lesion diameter is less than 1 cm. This technique has been used in our institute since 1998, and in this report we analyze our experience with it in 2010. This analysis shows that VABB is a safe, effective procedure that can markedly reduce the need for surgical biopsy.

Morphology of the lingual dorsal surface and oral taste buds in Italian lizard (Podarcis sicula).

The Italian lizard (Podarcis sicula) is the most diffused reptile in Italy, but it is also present in other European countries. This lizard belongs to the Lacertidae family, lives near walls, slants and along the borders of the paths; its diet includes bugs and aracnids. No data are so far available in literature about the three-dimensional morphology of the tongue of Podarcis sicula, therefore the aim of the present paper was to study by scanning electron and light microscopy the three-dimensional characteristics of the dorsal lingual surface and moreover the presence of chemosensory receptors like the taste buds in the oral cavity. Our results demonstrate that the Podarcis sicula tongue is a triangular muscular membranous organ, dorsoventrally flattened and that three different areas can be observed: a bifid apex, a body and a root. No papillae were observed in the apex, characterized by a flattened mucosa and by two deep median pouches. In the body cylindrical papillae with a flat surface are present, aborally gradually substituted by imbricated papillae. Foliate-like papillae were observed in the lateral parts of the tongue body. No sensory structures were showed on the lingual dorsal surface, while they were numerous in the oral cavity, particularly on the gingival epithelium. The light microscopy shows, on the dorsal surface, a stratified pavimentous not keratinized epithelium, conversely keratinized along the ventral surface. Many caliciform cells on the lateral parts of the papillae, deputed to the secretion of mucus, were also observed. Therefore, the results obtained in this paper could give a contribution to the knowledge of the tongue anatomy in a species widely diffused in different European countries and could be of help for clinical purposes in reptiles.

Sox-2 in taste bud and lateral line system of zebrafish during development.

The Sox-2 is a transcription factor involved in adult neurogenesis in different vertebrate species, including fishes. Sox-2 also participates in growth and renewal on sensory cells in neuromasts of the fish lateral line system, and it is essential for development of taste buds in mammals. Using immunohistochemistry and Western blot we have investigated the occurrence and localization of Sox-2 taste buds and neuromast of zebrafish from 10 days post-fertilization to adult stage (1 year). The antibody used identifies two protein bands with estimated molecular weights of 34 and 37kDa which are consistent with those predicted for Sox-2. Sensory cells in taste buds displayed Sox-2 immunoreactivity at all the ages sampled, whereas in the neuromasts Sox-2 expression was restricted to the basal non-sensory cells. Interestingly Sox-2 immunoreactivity was observed in epithelial cells associated with both taste buds and neuromasts. Present results demonstrate that Sox-2 expressed in taste buds and neuromasts of zebrafish during the whole lifespan. Nevertheless, whereas the role of Sox-2 in taste buds of zebrafish remains to be established, the results in neuromast suggest that Sox-2 could participate in cell renewal of the mechanosensory cells.