Neuronal nitric oxide synthase positive neurons in the human corpus callosum: a possible link with the callosal blood-oxygen-level dependent (BOLD) effect.

Brain functions have been investigated in the past decades via the blood-oxygen-level dependent (BOLD) effect using functional magnetic resonance imaging. One hypothesis explaining the BOLD effect involves the Nitric Oxide (NO) gaseous neurotransmitter, possibly released also by cells in the corpus callosum (CC). The eventual presence of NO releasing neurons and/or glial cells in the CC can be assessed by immunohistochemistry. Serial sections both from paraffin-embedded and frozen samples of CC obtained from adult human brains autopsy were studied with immunohistochemistry and immunofluorescence analysis, using an antibody against the neuronal isoform of Nitric Oxide Synthase (nNOS), the enzyme synthetizing the NO. The staining revealed the presence of many nNOS-immunopositive cells in the CC, shown to be neurons with immunofluorescence. Neuronal NOS-positive neurons presented different morphologies, were more numerous 4 mm apart from the midline, and displayed a peak in the body of the CC. In some cases, they were located at the upper boundary of the CC, more densely packed in the proximity of the callosal arterioles. The significant presence of nNOS-immunopositive neurons within the commissure suggests their probable role in the CC neurovascular regulation in the adult brain and could explain the BOLD effect detected in human CC.

Scattered Tubular Cells Markers in Macula Densa of Normal Human Adult Kidney.

BACKGROUND: The scattered tubular cells (STCs) are a population of resident progenitor tubular cells with expansion, self-renewal and epithelial differentiation abilities. Although these cells are localized within the proximal (PTs) and distal (DTs) tubules in a normal adult kidney, their presence has never been demonstrated in human macula densa (MD). The purpose of the present study is to describe the presence of STCs in MD using specific markers such as prominin-1 (CD133), cytokeratin 7 (KRT7) and vimentin (VIM). METHODS: We analyzed two sets of three consecutive serial sections for each sample. The first sections of each set were immunostained for nNOS to identify MD, the second sections were immune-stained for CD133 (specific STCs marker) while the third sections were analyzed for KRT7 (another STCs specific marker) and VIM (that stains the basal pole of the STCs) in the first and second sets, respectively, in order to study the co-expression of KRT7 and VIM with the CD133 marker. RESULTS: CD133 was localized in some MD cells and in the adjacent DT cells. Moreover, CD133 was detected in the parietal epithelial cells of Bowman's capsule and in some proximal tubules (PT). KRT7-positive cells were identified in MD and adjacent DT cells, while KRT7 positivity was mostly confined in both DT and collecting ducts (CD) in the other areas of the renal parenchyma. CD133 and KRT7 were co-expressed in some MD and adjacent DT cells. Some of the latter cells were positive both for CD133 and VIM. CD133 was always localized in the apical part of the cells, whereas the VIM expression was evident only in the cellular basal pole. Although some cells of MD expressed VIM or CD133, none of them co-expressed VIM and CD133. CONCLUSIONS: The presence of STCs was demonstrated in human adult MD, suggesting that this structure has expansion, self-renewal and epithelial differentiation abilities, similar to all other parts of renal tubules.

Neuronal nitric oxyde synthase positive neurons in human indusium griseum.

The study was designed to analyze the nNOS positive neurons present in the indusium griseum by describing their distribution and morphology. To this purpose, sagittal serial sections from paraffin or frozen autopsy specimens of corpus callosum including the overlying indusium griseum were processed by immunohistochemistry and immunofluorescence, using an antibody against the neuronal form of the enzyme nitric oxyde synthase. To test the specificity of the antibody used, Western Blot was performed in the indusium griseum of the same specimens. The stainings revealed the presence of many neuronal nitric oxyde synthase-immunopositive neurons in human indusium griseum, located along both rostral-caudal and medio-lateral directions. In particular, they were more numerous 1 mm apart from the midline, and their number peaked over the body of the corpus callosum. They showed different morphologies; in some cases, they were located at the boundary between indusium griseum and corpus callosum, more densely packed in proximity to the pial arteries penetrating into the corpus callosum. The significant presence and distribution of neuronal nitric oxyde synthase-immunopositive neurons suggests that indusium griseum likely plays a functional role in the neurovascular regulation within the corpus callosum. Schematic representation of human adult IG and the neurovascular unit originating from sopracallosal artery (Sca) that branches into smaller arterioles (Br) (created in PowerPoint). The arterioles cross the three layers of IG (layers I, II and III) and penetrate into the CC separated from IG by the Virchow-Robin space (VRs). As the arterioles go deeper, this space disappears and the vascular basement membrane comes into direct contact with the astrocytic end-feets (intracallosal arterioles and capillaries). nNOS-immunopositive neurons (nNOSIP N) surround the arterioles and control the vasomotore tone secreting nitric oxyde (NO). Two morphological types of nNOSIP N can be appreciated by the use of different colors: fusiform (blue) and ovoidal (pink). Also NeuN-immunopositive neurons (N) and many astrocytes (As) are present, more numerous in IG than in CC.

Hypoxia-induced expression of neuronal nitric oxide synthase in astrocytes of human corpus callosum.

Nitric oxide (NO) is a gaseous neurotransmitter largely diffused in the brain; among other functions, it regulates the cerebral blood flow in response to hypoxia. NO can be synthetized by three different isoforms of the enzyme NO synthase: neuronal (nNOS), typical of neurons, endothelial and inducible. The aim of this study was to assess nNOS expression in human corpus callosum (CC) astrocytes, and its relationship with the hypoxia duration. Autoptic samples of CC from adult human subjects have been processed with immunohistochemistry and immunofluorescence using antibodies anti-nNOS and anti-glial fibrillary acidic protein (GFAP), the astrocyte marker. Results demonstrated for the first time the presence of nNOS-immunopositive astrocytes in the human CC. In particular, nNOS-positive astrocytes were absent in subjects deceased after a short hypoxia; their number and labeling intensity, however, increased with hypoxia prolongation. Neuronal NOS immunopositivity of CC astrocytes seems thus related to the hypoxia duration and the consequent brain damage.

Normal human macula densa morphology and cell turnover: A histological, ultrastructural, and immunohistochemical investigation.

The aim was to analyze the morphology of normal human macula densa (MD), evaluate the cells that may be responsible for its turnover, and collect quantitative data. Of four samples of normal human renal tissue, two were embedded in resin to measure the longitudinal extension and examine the ultrastructure of the MD, the other two were embedded in paraffin to study apoptosis and cell proliferation. The MD is composed of a monolayer tissue about 40 µm long, which includes 35-40 cells arranged in overlapping rows. Ultrastructurally, MD cells show two polarized portions: an apical end, with sensory features, and a basolateral aspect, with paracrine function. MD cells are connected apically by tight junctions, with/without adherens junctions, which form a barrier between the distal tubule lumen and the interstitium. Cells in degeneration, often associated with macrophages, and undifferentiated cells were found in the MD and adjacent distal tubule. A filamentous mat previously described in proximal tubule scattered tubular cells (STCs) was detected in the basal cytoplasm in undifferentiated cells. The tissue was consistently negative for the proliferation marker Ki67 and for the apoptotic markers caspase-3 and caspase-9. This work confirms our earlier morphological findings and provides new data: (a) MD cells display both apical adherens and tight junctions, the latter forming a tubulo-mesangial barrier; (b) the MD is a monolayer made up of about 40 cells arranged in rows; (c) the simultaneous presence of degenerating (8-13%) and undifferentiated (4-13%) cells reminiscent of STCs suggests a non-negligible cell turnover.

Postnatal development of the distribution of nitric oxide-producing neurons in the rat corpus callosum.

The postnatal development of nitric oxide (NO)-producing intracallosal neurons was studied in rats by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry from postnatal day 0 (P0) to P30. NADPH-d-positive neurons (NADPH-d+Ns) were detected already at P0, mainly in the rostral region of the corpus callosum (cc). Their location and the intensity of staining allowed them to be classified as type I NO-producing neurons. At P0, tufts of intensely labeled fibers, probably corresponding to the callosal septa described in the monkey and human cc, entered the ventral cc region and reached its dorsal portion. From P5, cell bodies and dendrites were often associated to blood vessels. The number of intracallosal NADPH-d+Ns rose in the first postnatal days to peak at P5, it declined until P10, and then remained almost constant until P30. Their size increased from P0 to P30, dramatically so (>65%) from P0 to P15. From P10 onward their distribution was adult-like, i.e. NADPH-d+Ns were more numerous in the lateral and intermediate portions of the cc and diminished close to the midline. In conjunction with previous data, these findings indicate that intracallosal NADPH-d+Ns could have a role in callosal axon guidance, myelination, refinement processes, and callosal blood flow regulation.

Insights on the seasonal variations of reproductive features in the Eastern Atlantic Bluefin Tuna.

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.

Macromolecular Characterization of Swordfish Oocytes by FTIR Imaging Spectroscopy.

During folliculogenesis, primary oocytes of teleosts grow by several orders of magnitude by-self synthesizing proteins and mRNA, or sequestering from blood specific macromolecular components, such as fatty acids and vitellogenin. All these materials are stored into cortical alveoli, yolk globules or oil droplets during oocyte development. The proper synthesis, storage and displacement of these macromolecular components inside the oocyte play a key role for a successful fertilization process and for the subsequently correct embryo development. In this study, for the first time, the FTIR Imaging (FTIRI) spectroscopy has been applied to characterize the chemical building blocks of several cellular components of swordfish oocytes at different developmental stages. In particular, the spectral features of previtellogenic (PV), vitellogenic (VTG), mature (M) and atretic (A) follicles as well as and of cortical alveoli (CA), yolk vesicles (YV), oil droplets (OD) and Zona Radiata (ZR) have been outlined, providing new insights in terms of composition and topographical distribution of macromolecules of biological interest such as lipids, proteins, carbohydrates and phosphates. The macromolecular characterization of swordfish oocytes at different developmental stages represents a starting point and a useful tool for the assessment of swordfish egg quality caught in different conditions, such as periods of the year or different fishing area.