Maria Angela Masini

Clinorotation-induced weightlessness influences the cytoskeleton of glial cells in culture

During and after spaceflight astronauts experience neurophysiological alterations. To investigate if the impairment observed might be traced back to cytomorphology, we undertook a ground based research using a random positioning machine (clinostat) as a simulation method for microgravity. The outcome of the study was represented by cytoskeletal changes occurring in cultured glial cells (C(6) line) after 15 min, 30 min, 1 h, 20 h and 32 h under simulated microgravity. Glia is fundamental for brain function and it is essential for the normal health of the entire nervous system. Our data showed that after 30 min under simulated microgravity the cytoskeleton was damaged: microfilaments (F-actin) and intermediate filaments (Vimentin, Glial Fibrillary Acidic Proteins GFAP) were highly disorganised, microtubules (alpha-tubulin) lost their radial array, the overall cellular shape was deteriorated, and the nuclei showed altered chromatin condensations and DNA fragmentation. This feature got less dramatic after 20 h of simulated microgravity when glial cells appeared to reorganise their cytoskeleton and mitotic figures were present. The research was carried out by immunohistochemistry using antibodies to alpha-tubulin, vimentin and GFAP, and cytochemical labelling of F-actin (Phalloidin-TRIC). The nuclei were stained with propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were observed at the conventional and/or the confocal laser scanning microscope. Samples were also observed at the scanning electron microscope (SEM). Our data showed that in weightlessness alterations occur already visible at the scale of the single cell; if this may lead to the neurophysiological problems observed in flight is yet to be established.

Feeding Behavior in Mammals Including Humans

The complex control of food intake and energy metabolism in mammals relies on the ability of the brain to integrate multiple signals indicating the nutritional state and the energy level of the organism and to produce appropriate responses in terms of food intake, energy expenditure, and metabolic activity. Central regulation of feeding is organized as a long‐loop mechanism involving humoral signals and afferent neuronal pathways to the brain, processing in hypothalamic neuronal circuits, and descending commands using vagal and spinal neurons. Sensor mechanisms or receptors sensitive to glucose and fatty acid metabolism, neuropeptide and cannabinoid receptors, as well as neurotransmitters and neuromodulators synthesized and secreted within the brain itself are all signals integrated in the hypothalamus, which therefore functions as an integrator of signals from central and peripheral structures. Homeostatic feedback mechanisms involving afferent neuroendocrine inputs from peripheral organs, like adipose tissue, gut, stomach, endocrine pancreas, adrenal, muscle, and liver, to hypothalamic sites thus contribute to the maintenance of normal feeding behavior and energy balance. In addition to transcriptional events, peripheral hormones may also alter firing and/or connection (synaptology) of hypothalamic neuronal networks in order to modulate food intake. Moreover, intracellular energy sensing and subsequent biochemical adaptations, including an increase in AMP‐activated protein kinase activity, occur in hypothalamic neurons. Understanding the regulation of appetite is clearly a major research effort but also seems promising for the development of novel therapeutic strategies for obesity.

IMMUNOCYTOCHEMICAL LOCALIZATION OF SOMATOSTATIN AND AUTORADIOGRAPHIC DISTRIBUTION OF SOMATOSTATIN BINDING SITES IN THE BRAIN OF THE AFRICAN LUNGFISH, PROTOPTERUS ANNECTENS

The anatomical distribution of somatostatin‐immunoreactive structures and the autoradiographic localization of somatostatin binding sites were investigated in the brain of the African lungfish, Protopterus annectens. In general, there was a good correlation between the distribution of somatostatin‐immunoreactive elements and the location of somatostatin binding sites in several areas of the brain, particularly in the anterior olfactory nucleus, the rostral part of the dorsal pallium, the medial subpallium, the anterior preoptic area, the tectum, and the tegmentum of the mesencephalon. However, mismatching was found in the mid‐caudal dorsal pallium, the reticular formation, and the cerebellum, which contained moderate to high concentrations of binding sites and very low densities of immunoreactive fibers. In contrast, the caudal hypothalamus and the neural lobe of the pituitary exhibited low concentrations of binding sites and a high to moderate density of somatostatin‐immunoreactive fibers. The present results provide the first localization of somatostatin in the brain of a dipnoan and the first anatomical distribution of somatostatin binding sites in the brain of a fish. The location of somatostatin‐immunoreactive elements in the brain of P. annectens is consistent with that reported in anuran amphibians, suggesting that the general organization of the somatostatin peptidergic systems occurred in a common ancestor of dipnoans and tetrapods. The anatomical distribution of somatostatin‐immunoreactive elements and somatostatin binding sites suggests that somatostatin acts as a hypophysiotropic neurohormone as well as a neurotransmitter and/or neuromodulator in the lungfish brain. J. Comp. Neurol. 388:337–353, 1997.

Low-intermediate dose testosterone replacement therapy by different pharmaceutical preparations improves frailty score in elderly hypogonadal hyperglycaemic patients

Abstract An open-label follow-up study of low-to-intermediate dose testosterone replacement therapy (TRT) was conducted in 64 overweight patients (aged 65–75 years) with late onset hypogonadism (LOH) and increased fasting plasma glucose (FPG). Patients were subdivided into four treatment groups: oral testosterone (T) (T undecanoate, 80 mg/d), transmucosal T (60 mg/d), transdermal T (30 mg/d) or no treatment (control), and evaluated at 0 and 6 months. FPG, hemoglobin (Hb), prostate-specific antigen (PSA) and total T were measured and the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) index was calculated. Body mass index (BMI), waist circumference, fitness level (6-min walking test), Aging Males’ Symptoms (AMS) scale, handgrip strength and energy expenditure with physical activity (Minnesota questionnaire for Leisure Time Physical Activity (LTPA)) were evaluated and a “frailty score” (based on: grip strength, gait speed and LTPA) was calculated. T levels increased in all treatment groups; the oral T group had values still in the hypogonadal range (5.9 ± 1.1 nmol/L). PSA and Hb concentrations did not change in any group. BMI, waist circumference, FPG and HOMA-IR improved in all T-treated groups after 6 months, with a greater effect seen with transmucosal and transdermal T compared with oral T. This study indicates that low-to-intermediate dose TRT may be safely utilized in LOH patients to ameliorate somatic and psychological frailty symptoms in association with improved anthropometric and glycometabolic parameters in aging, overweight men with LOH and impaired fasting glucose.

Immunocytochemical localization of atrial natriuretic factor and autoradiographic distribution of atria natriuretic factor binding sites in the brain of the African lungfish, Protopterus annectens

The localization of atrial natriuretic factor (ANF)‐immunoreactive elements was investigated in the brain of the African lungfish, Protopterus annectens, by using antisera raised against rat and human ANF(1–28). Concurrently, the distribution of ANF binding sites was studied by autoradiography using radioiodinated human ANF(1–28) as a tracer. In general, there was a good correlation between the distribution of ANF‐immunoreactive structures and the location of ANF binding sites in several areas of the brain, particularly in the ventral part of the medial subpallium, the rostral preoptic region, the preoptic periventricular nucleus, the caudal hypothalamus, the neural lobe of the pituitary, and the mesencephalic tectum. In contrast, mismatching was observed in the pallium (which contained a high density of binding sites and a low concentration of ANF immunoreactive elements) as well as in the lateral subpallium and the medial region of the ventral thalamus, in which a low concentration of binding sites but a high density of ANF‐immunoreactive fibers were detected.

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis. In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10−7M and 10−8M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains. In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. −90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules. Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.

Angiotensin II specific receptors in subcommissural organ

In the subcommissural organ of male rats, a circumventricular organ situated inside the blood-brain barrier, specific receptors for angiotensin II were demonstrated by binding studies on homogenated membranes and by autoradiography carried out on frozen sections using 125I-angiotensin II. The receptor sites were localized in the subnuclear region of cells of the subcommissural organ. A single class of binding sites was found whose capacity was modulated by changes in the sodium plasma concentration which led to variations in plasma volume.

Immunoreactive localization of vasoactive hormones (atrial natriuretic peptide and endothelin) in the heart of Protopterus annectens, an African lungfish

Abstract.The present study demonstrated, by immunohistochemistry and Western blotting, the presence of immunoreacting atrial natriuretic peptide (ANP) and endothelin in the heart of Protopterus annectens by both light and electron microscopy. The distribution of ANP granules was investigated. ANP granules were localised in myocytes from the atrium, ventricle and conus arteriosus; endothelin-1 (ET-1) was demonstrated in subendocardial myocytes of the atrium and the conus. No ET-1 immunoreactivity was observed in the ventricle wall. At the light-microscopical level, ET-1 appeared to occur in the endocardium, but at the electron-microscopical level no immunogold labelling was seen on the granules of the endocardial cells. It is suggested that ET-1 is produced and stored in the subendocardial cells and released into the subendocardial space to reach the ANP-producing myocytes and the endothelial cells.

Localization of interleukin-1β mRNA in the cerebral ganglion of the protochordate, Styela plicata

There is evidence that interleukin 1 beta (IL-1 beta) plays an important role in several biological functions in mammals where it is synthesized by cells of haematological, dermal and neural origin. Moreover, production of cytokine-like molecules has been demonstrated in some blood cells of non-mammalian vertebrates and invertebrates in which also nerve cells are demonstrated to be IL-1 beta immunoreactive. The purpose of the present study is to demonstrate the IL-1 beta mRNA expression in nerve cells of the ascidian Styela plicata by use of non-isotopic in situ hybridization technique. The expression of IL-1 beta messenger was demonstrated in monopolar neurons in the cortical layer of the cerebral ganglion. The neuronal expression of cytokine-like molecules in tunicates suggests that IL-1 beta is an ancestral and functionally conserved molecule, and that a neuroimmune axis appeared early during the metazoan phylogeny.

Immunoreactive atrial natriuretic-like peptide in antarctic teleosts

Abstract 1. 1. The presence of an atrial natriuretic-like peptide (ANP) has been investigated in the Antarctic fish Chionodraco hamatus (Channichthydae) and Pagothenia bernacchii (Notothenidae). 2. 2. By radioimmunoassay an ANP-like peptide has been detected in plasma and heart homogenates of both white blooded ( Chionodraco ) and red blooded ( Pagothenia ) species. 3. 3. By immunohistochemistry atrial nutriuretic factor (ANF) immunoreactive cardiocytes have been demonstrated in the atria of Chionodraco and Pagothenia using Ab to rat 99–126 ANF. Ventricular immunoreactivity is absent in both species. 4. 4. ANP binding sites have been visualized by autoradiography in the kidney, gills and heart of both Pagothenia and Chionodraco . 5. 5. Our data indicate that ANP concentration is greater in the white blooded than in the red blooded fish.