Biomedical subjects

L Alfei

Publications and source records attributed to L Alfei.

At least 19 recordsLinked to original sources

PubMed2001

Proliferative activity and motoneurone recruitment persist at the spinal cord central canal during larval and some postlarval stages in the rainbow trout (Oncorhynchus mykiss).

We previously found a linear relationship between the cross sectional myotomal area and the motoneurone number in the growing trout during postlarval stages. These neurones increased in number until a fish length of 150 mm, which prompted us to examine how motor neurones are recruited afterwards to meet the growth of their target myotomal muscle. Young adult (260 mm in length), fingerlings (F, 120-170 mm), fry (Fr, 70 mm) and eleutherembryos (Es, 20-30 mm) of rainbow trout (Oncorhyncus mykiss) were employed in this study. PCNA immunohistochemistry was used for monitoring the proliferative activity in the epithelium of the spinal cord central canal. This activity was quantified as the number of PCNA labelled cells for each spinal cord section. In Es and Fry, a mean value of 3-5 labelled cells for each section was found with a sharp decrease in young F (120 mm long). After this fish length, it was not possible to quantitatively evaluate the proliferative activity at the central canal. However, labelled cells were seldom found in the spinal cord sections until a fish length of 260 mm. From these data it is possible to conclude that motoneurone recruitment in the trout spinal cord is down-regulated at the F stage. Afterwards, we found that motoneurones increase in size to meet the growth of their target myotomal muscle.

Animals↗

PubMed1999

Hyaluronate receptor CD44 is expressed by astrocytes in the adult chicken and in astrocyte cell precursors in early development of the chick spinal cord.

The role of the hyaluronate receptor, CD44, is well known in adult mammal astrocytes where it modulates neuron-glia interactions. However, no data exist regarding its expression in other vertebrates during their development. In order to detect the expression of CD44 in the chicken and its possible involvement in glial precursor migratory patterns during spinal cord development, a monoclonal antibody (MoAb) against the mammalian standard isoform, CD44-H, was used in immunohistochemical and immunoblot assays. With these methods, CD44 hyaluronate receptors were found on mature astrocyte membranes of adult chicken spinal cord. Astrocytes were identified using a MoAb against GFAP. During development, small clusters of CD44 labelled cells were seen lining the central canal starting from embryonic stage E10. These labelled cells were dispersed in the dorsal, lateral and ventral funiculi of the spinal cord in the subsequent stages. After stage E15, the CD44 labelled cells were identified as astrocytes because of their GFAP immunoreactivity. We conclude that CD44 receptors on immature astrocyte precursors should be considered as early astrocyte markers which have a possible role during cell migratory dispersal.

Animals↗

PubMed1999

PCNA positivity in the telencephalic matrix areas in the adult of a lizard, Podarcis sicula.

In the present paper a reappraisal is made of the persistence and size of telencephalic matrix areas in normal adult specimens of a lizard, Podarcis sicula, using a recent in situ marker of cells in the S phase, the Proliferating Cell Nuclear Antigen (PCNA), belonging to the cyclin family, proteins that regulate the cell cycle and that can be immunocytochemically evidenced using monoclonal antibodies. Only a small number of positive PCNA elements, randomly arranged in the dorsal (medial and lateral) matrix areas, were found in the telencephalic ependymal tissue. These elements were found in larger numbers and distributed more uniformly in the ventral matrix areas, both opposite the ventricular lumen and outside the ependyma.

Animals↗

PubMed1999

PCNA positivity in the telencephalic matrix areas in the adult of a newt, Triturus carnifex.

The Proliferating Cell Nuclear Antigen (PCNA) is an auxiliary protein of the DNA polymerase delta, belonging to the cyclin family, which attains appreciable levels only in those phases of the cell cycle in which DNA synthesis occurs. Using an immunocytochemical method that exploits this proliferative marker, we observed a certain PCNA positivity in the telencephalon of normal adult individuals of Triturus carnifex. The elements that display this peculiarity occupy an ependymal and/or sub-ependymal site. They are located in the anterior portion of the telencephalon only at the dorsal level and tend to decrease in number until they disappear temporarily as the intermediate portion is approached. In the posterior portion of the telencephalon, the dorsal labelling patterns, the size of which is however smaller than that observed more anteriorly, reappear and in the ventral region a large population of labelled cells appears. Then, again proceeding in a caudal direction, while the PCNA-positive elements lying dorsally diminish and disappear, they persist in the ventral region and gradually taper off until they final disappear where the telencephalic ventricles come together. This immunocytochemical picture supports the findings of other authors in adult Urodeles under both normal and experimental conditions (with classical and autoradiographic histological techniques) concerning the persistence of the telencephalon in dorsal and ventral germinative areas responsible for physiological and plastic neurogenetic events.

Animals↗

PubMed1998

Phosphorylated and non-phosphorylated neurofilament epitopes are co-distributed in the fish Mauthner axon.

The neurofilament (NF) polypeptides of the fish giant Mauthner cell axons (MAs) and their degree of phosphorylation were investigated in the adult by means of immunoblot and immunohistochemical staining. Fasciculus longitudinalis medialis (FLM) axons of much smaller caliber, among which MAs run in the ventral spinal cord, were also analyzed in two teleost fish belonging to continuously growing species. To detect NF polypeptide subunits, commercially available monoclonal antibodies against mammalian NF-L (68 kDa), NF-M (160 kDa), phosphorylated (P) and non-phosphorylated (nP) NF-H (200 kDa) epitopes were used. These antibodies labelled bands of the identical molecular weight in fish cervical spinal cord total protein immunoblots. A peculiar NF composition of the MAs was observed, following immunohistochemical staining i.e. a low NF-M expression and a codistribution of P and (nP)-NF-H epitopes. Moreover, on the basis of immunoperoxidase staining in ultrathin longitudinal MA sections, we suggest that P NF-H are arranged in bundles, whilst (nP)-NF-H are likely to be free in the axoplasm. By contrast, FLM axons were found reactive with antibodies only against P NF-H. These results confirm that in carp and trout, NF have epitopes cross-reacting with monoclonal antibodies directed against the mammalian NF subunits. Furthermore, as regards the co-distribution of phosphorylated and non-phosphorylated NF-H epitopes in the M-cell axons, these might be considered as not yet completely mature axons taking into account that carp and trout belong to continuously growing species.

Animals↗

PubMed1994

PCNA/cyclin expression and BrdU uptake define proliferating myosatellite cells during hyperplastic muscle growth of fish (Cyprinus carpio L.).

The possibility of detecting in situ proliferating myosatellite cells during postlarval muscle growth in the carp (Cyprinus carpio, L.) by means of BrdU and PCNA (Cyclin) immunohistochemistry has been evaluated on paraffin embedded sections. Nine subadult stages were defined according to the body length and weight. The fish were injected intraperitoneally with BrdU and fixed one hour later. Adjacent cross sections mounted on glass slides were incubated with monoclonal antiBrdU (1:100) and antiPCNA (1:200) antibody. The proliferative rate, defined as the percentage of labelled cells for each stage, was correlated to the corresponding percentage of small fibers (area less than 200 microns 2) determined by morphometric analysis. Desmin expression, immunocytochemically detected, was aimed at discriminating between the postmitotic and stem myosatellite cells. A low myosatellite cell proliferative activity (2-7%) throughout the carp growth period considered was demonstrated. Quantitative analysis provided evidence that during growth stages in which small fibers are numerous, the myosatellite cell proliferative activity is low and it increases when the small fibers number decreases. It is suggested that an age dependence of myosatellite cell proliferative rate controls the recruitment of new fibers in the carp.

Animals↗

PubMed1993

Use of 5'-bromodeoxyuridine immunohistochemistry to examine proliferative activity of fish tissues.

The suitability of bromodeoxyuridine (BrdU) immunohistochemistry for the study of myosatellite cell proliferation in three subadult carp (Cyprinus carpio) stages (11, 15, 17 cm) was examined. They were injected intraperitoneally with BrdU and fixed one hour late. After fixation and dehydration, white myotomal muscle and small intestine samples were embedded in Histowax. Cross sections mounted on glass slides, were incubated with monoclonal antiBrdU antibody (1:100) after HCl denaturation. After washing twice in PBS, slides were incubated in goat antimouse IgG FITC secondary antibody (1:20). Single cell suspensions were obtained from gut samples. Cellular DNA partially denatured with 2 N HCl, were immunolabelled with monoclonal antibodies against BrdU. Bivariate distribution of BrdU/total cellular DNA content was measured by flow cytometry. Good visualization of BrdU labelled myosatellite cells (4-6%) and small intestine (8-9%) was obtained. Flow cytometric bivariate BrdU/DNA analysis gave evidence of the same proliferative rate in the gut samples. The applicability of these methods to fish tissues further extend the broad range of biological and biomedical investigation in which BrdU immunohistochemistry has been used.

Animals↗

PubMed1992

Morphometric analysis of Mauthner axon cytoskeletal components in adult and subadult fish.

A previous cytoskeletal analysis on trout MA during developmental stages demonstrated, during the subadult stages, neurofilaments (NF) as main components as expressed by the high values of neurofilament to microtubules (MT) ratio which was found to be of the order of 300:1. Since the MA cytoskeletal composition is not known in the adult fish, the MA cytoskeletal composition has been compared to other axons of much smaller diameter of the fasciculus longitudinalis medialis (flm) among which the MA run in the ventral spinal cord. The following parameters were measured on conventional electron microscopy in MA and flm axons cross sections micrographs by means of a computer linked graphic tablet (Apple II): axonal caliber, number of microtubules (MT), microtubular (MT/microns2) and neurofilament (NF/microns2) densities. The analysis of these parameters demonstrated that neurofilaments are the main architectural components in the adult and subadult fish MA and flm axons. However, MA cytoskeletal composition differs from the other flm axons because of its particular very high ratio of neurofilaments to microtubules. The inverse relationship of axonal caliber to microtubular density, previously found in the trout during developmental stages and suggested also for many other vertebrate species, was further confirmed for flm axons which, with calibers 10 times smaller than MA, exhibit a microtubular density 10 times larger.

Animals↗

PubMed1991

Cytoskeletal components and calibers in developing fish Mauthner axon (Salmo gairdneri Rich.).

In developing axons of many vertebrates, microtubular density is inversely correlated with fiber caliber. It is suggested that microtubules are causally related to axonal caliber. For this reason, cytoskeletal analysis during development of the fish Mauthner axon, which displays a giant caliber, is of particular interest. The Mauthner axon originates from the Mauthner cell in the medulla and runs in the fasciculus longitudinalis medialis in the spinal cord. At embryonic, larval and postlarval stages in trout (Salmo gairdneri Rich.), the following parameters were measured on conventional electron micrographs of Mauthner axon cross sections; axonal caliber, number of microtubules per axons, and microtubular and neurofilament densities. Results at each stage point to an inverse correlation between axonal caliber (x) and microtubular density (y) expressed by the equation y = axb (R = 0.932). Furthermore, three periods of Mauthner axon development are identified on the basis of the cytoskeletal content: (1) embryonic; the Mauthner axon has small caliber with a high microtubule density, (2) elongation period (larval stages); the axon enlarges and a transient peak of microtubules, corresponding to the caliber increment, is observed, and (3) postlarval; the axon enlarges still further (greater than 500 microns 2) but has the lowest microtubular content. During this period neurofilaments are the main axonal component.

Animals↗

PubMed1989

Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis.

The mechanism of postlarval fish myotomal growth was investigated in trout (Salmo gairdneri) by means of morphometric and cytofluorometric analysis. The mechanism by which new fibres are added during postlarval growth (hyperplasia) is not fully understood. In histological cross sections these new fibres have a small diameter which give the muscle a "mosaic" appearance. One hypothesis suggested that they could be derived from the proliferative activity of satellite cells. DNA cytofluorometric analysis of nuclei suspensions obtained from trout white myotomal muscle during different developmental stages (eleutherembyronic; alevin; yearling and adult) showed a consistently low S-cytometric phase during all stage in which myofibres of small diameters were present. The percentage of such small fibres, determined by morphometric analysis, suggested that satellite cells are the proliferative population. In fact, their percentages, as determined by morphometric analysis in histological section, bear a linear relationship with the S-cytometric phase percent nuclei (R = 0.927). Only in adults (67 cm in size) there was a significant decrease in the S-cytometric phase. At this stage, in histological sections, the myotomal muscle no longer had a "mosaic" appearance because of the disappearance of the small fibres. It may, therefore, be supposed that in the cm 67 adult specimens, the proliferative population is entering the G0 phase. It is known, in fact, that muscle growth proceeds only by fibre hypertrophy in trout longer than 70 cm in length (Stickland, 1983).

Aging↗

PubMed1988