Roberto Toni

New paradigms in neuroendocrinology: relationships between obesity, systemic inflammation and the neuroendocrine system.

Obesity may be an independent risk factor for coronary artery disease and contribute to a chronic state of systemic inflammation leading to atherosclerosis and metabolic abnormalities, such as diabetes, insulin resistance, dyslipidemia and hypertension. Visceral fat, in fact, may act as an endocrine organ, synthesizing and releasing atherogenic inflammatory cytokines, whose circulating levels depend on the individual’s nutritional state, and the extent and anatomical location of fat stores. Unsuspected viral infections might also be involved in enhancing autocrine/ paracrine mechanisms of cytokine release from omental fat. Elevated levels of blood cytokines may interact with the neuroendocrine system, autonomic nerves and peripheral lymphatic organs. This may lead to local inflammatory reactions in many body compartments, in particular in the heart tissue, possibly affecting the process of circulatory recovery in obese subjects, and predisposing these patients to a greater risk of myocardial inflammatory disease than individuals with normal body mass index. Circulating levels of inflammatory cytokines might be considered to determine risk categories for development of cardiovascular complications in obese subjects. In addition, their reduction with pharmacological antagonists might prevent and/or control acute cardiovascular events and increase energy expenditure in obese patients, especially after surgical treatment, through reduction of cytokine inhibition of the hypothalamic-pituitary-thyroid axis.

Neuroendocrine regulation of thyrotropin-releasing hormone (TRH) in the tuberoinfundibular system

[...] It is now required to list each part needed for mucous excretion. They are two ducts in the brain substance, then a thin portion of membrane shaped as the infundibulum, then the gland that receives the tip of this infundibulum and the ducts that drive the mucus (pituita) from this gland to the palate and nares. [...] and I said that one (duct) [...] from the middle of the common cavity (third ventricle) descends [...] into the brain substance, and the end of this duct is [...] the sinus of the gland where the brain mucus is collected [...].

Anthropological Variations in the Anatomy of the Human Thyroid Arteries

Knowledge of anatomic variability of the superior (STA), inferior (ITA), and lowest accessory (IMA) thyroid arteries may be helpful in certain clinical conditions. However, details of this variability have not been thoroughly described. Specifically, whether the presence and site of origin of STA, ITA, and IMA are influenced by the anthropological group, to what extent their origin is symmetric or asymmetric, and the role played by this variability in visualizing each thyroid artery by nonselective thyroid angiography is not known. To clarify this we conducted a meta-analytical study on Caucasian and Asian subjects, the latter including only Japanese and Koreans. In Caucasians and Asians the presence of superior vessels compared to inferior vessels was more frequent and the probability of symmetric or asymmetric arterial origin for STA were equivalent. However, better recognition of inferior rather than superior vessels was achieved by nonselective angiography in Caucasians. Finally, different frequencies of presence and site of origin for each artery were identified in Caucasians compared to Asians. Our results suggest that the higher frequency of IMA in Asians than in Caucasians should result in a search for an IMA-dependent feeding artery of inferior parathyroid adenomas, primarily the mediastinal ones, especially in Asians both by imaging and transcatheter ablative approaches. In addition, we have found that a small percentage of Caucasian subjects lack an STA on the left side. Therefore, anatomic arterial compatibility should be carefully evaluated in the preoperative stage of laryngeal transplantation maintaining in situ the donors thyroid by terminal anastomoses between donor and recipient STAs. Finally, the lack of any individual thyroid artery in either Caucasians or Asians might influence the distribution of autonomic supply that runs with thyroid vessels to the thyroid parenchyma. This appears functionally relevant in cases of traumatic or surgical lesions of the cervical sympathetic chain involving thyroid nerves. In fact, a restricted local autonomic control of thyroid activity might be related to individual rami of thyroid nerves.

Thyrotropin-releasing-hormone-immunoreactive innervation of thyrotropin-releasing-hormone-tuberoinfundibular neurons in rat hypothalamus: anatomical basis to suggest ultrashort feedback regulation.

Thyrotropin-releasing-hormone (TRH)-synthesizing neurons in the medial and periventricular parvocellular subdivisions of the rat hypothalamic paraventricular nucleus (PVN) are involved in regulation of the anterior pituitary. Since ultrashort feedback regulation of TRH in the hypothalamus has been suggested by physiological studies, we sought to identify the presence of TRH synaptic contacts containing TRH on TRH tuberoinfundibular neurons in the PVN. An immunocytochemical study was performed at light- and electron-microscopic levels using antiserum directed to the N-terminal cryptic sequence of the TRH precursor, preproTRH 25-50. At the light-microscopic level, contacts between TRH immunoreactive (IR) fibers and the perikarya and processes of TRH-IR neurons were observed in medial and periventricular subdivisions of the PVN. At the ultrastructural level, TRH-neurons appeared either tightly juxtaposed to TRH-immunopositive perikarya and dendrites or to establish axodendritic and axosomatic contacts suggestive of synaptic associations. These data provide a morphologic basis to support a neuroendocrine role for TRH or processed forms of proTRH in the PVN and in particular suggest their involvement as neuromodulators in an ultrashort feedback regulation of TRH tuberoinfundibular neurons.

Thyroid Hormones in Neural Tissue

Publisher Summary This chapter reveals that the maintenance of normal thyroid function is recognized to be dependent on a complex interplay between the hypothalamus, anterior pituitary, and thyroid gland, as well as other factors that influence the function of these organ systems. The major hormone responsible for the secretion of thyroid hormone from the thyroid is thyroid-stimulating hormone (TSH), also termed thyrotropin. TSH is secreted from anterior pituitary thyrotropes, which comprise approximately 10% of the anterior pituitary cells, and in turn, is positively regulated by thyrotropin-releasing hormone (TRH), originating in the hypothalamus. The free fraction of thyroid hormone circulating in the bloodstream feeds back, both on the anterior pituitary and hypothalamus, to inhibit the secretion of TSH and TRH, respectively, thereby completing what is recognized as a classic example of a negative feedback loop system.

Effect of hypothyroidism on vasoactive intestinal polypeptide-immunoreactive neurons in forebrain-neurohypophysial nuclei of the rat brain

We have recently reported that hypothyroidism increases immunoreactive (IR)-vasoactive intestinal polypeptide (VIP) and VIP mRNA content in both parvocellular and magnocellular neurons of the rat, hypothalamic paraventricular nucleus (PVN). As VIP can stimulate vasopressin (AVP) secretion, we conducted an anatomical investigation to determine whether VIP-containing neurons in other regions of the brain that are involved with homeostatic mechanisms of water and salt conservation are also affected by hypothyroidism. The distribution and intensity of VIP immunostaining in neurons and fibers of the magnocellular-neurohypophysial system, including the hypothalamic PVN, supraoptic nucleus (SON) and accessory magnocellular cell groups, circumventricular subfornical organ (SFO), preoptic and anterior hypothalamus, midline thalamus, subthalamic zona incerta and posterior septal nuclei were studied using a highly sensitive immunocytochemical technique and unbiased neuronal counting methods, based on the optical dissector principle. Hypothyroidism increased the intensity of VIP immunostaining and/or the number/section, percentage and numerical density of IR-VIP neurons in the PVN, SON, nucleus circularis, periventricular preoptic nucleus of the hypothalamus and SFO. In addition, IR-VIP perikarya and/or fibers in the hypothalamic medial preoptic area and anterior periventricular nucleus, nucleus reuniens of the thalamus and dorsal fornix-triangular septal nucleus complex were also apparent in the hypothyroid animals while no immunostaining was seen in these areas in control animals. No quantitative and/or qualitative modifications in IR-VIP neurons and fibers were noted in the anterior hypothalamic area, suprachiasmatic nucleus, thalamic paraventricular nucles an subthalamic zona incerta between hypothyroid and control animals. These findings suggest an inverse relationship between thyroid hormone and VIP content and/or distribution of IR-VIP neurons in specific forebrain regions involved in the control of AVP release, extracellular fluid volume, thirst, blood pressure and anterior pituitary secretion. This raises the possibility that changes in fluid homeostasis and cardiovascular function occurring in hypothyroidism may be mediated, at least in part, by VIP-producing neurons in diverse regions of the brain.

1-naphthol-pyronin B as a novel substrate for silver intensification: application to light and electron microscopic immunocytochemistry of neuroendocrine systems.

We describe a modification of silver intensification of immunoperoxidase end-product using 1-naphthol (1N) and 1N enhanced by pyronin B after suppressing nonspecific tissue argyrophilia with a solution of penicillamine and merthiolate buffered near neutral pH. This approach facilitates the preservation of a second antigen sequentially labeled in the same tissue section for light microscopic double immunolabeling experiments and also allows retention of ultrastructural detail. Using this protocol, we obtained rapid and uniform silver intensification of somatostatin (SRIF)-immunoreactive (IR) neuronal perikarya and processes in the rat hypothalamic paraventricular nucleus (PVN). Ultrastructurally, 1N- and 1N-pyronin B-silver intensified reaction product was clearly recognized by the presence of a coarse intracellular precipitate of high electron density. Light microscopic double-immunolabeling studies demonstrated the association between SRIF- and thyrotropin-releasing hormone (TRH)-IR neuronal systems in the PVN. We propose that silver intensification of 1N and 1N-pyronin B is a useful alternative to standard methods of silver intensification of immunoperoxidase reaction product at both light and ultrastructural levels and may be particularly amenable for double-immunolabeling studies.

Growth on poly(l-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(l-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

Thyrogenic, adipogenic, and osteogenic differentiation of adult rat, thyroid stem cells enriched by long-term adherent subculture

We recently identified adult progenitor cells expressing multipotency markers in the rat thyroid (1). We have now studied these markers in primary cultures, thyrospheres, and adherent cells exhibiting features of side population / multilineage differentiation. Primary rat thyroid monolayers were immunolabeled / immunoblotted for ABCG2, Oct-3/4, HNF4a and Sca1. Thyrospheres were cytospinned and immunolabeled for Oct-3/4. Long-term subcultures were obtained by re-seeding monolayers at very low density, and growing them up to 5 months, using a starvation protocol to obtain colony forming unit (CFU)-like cultures. The latter were incubated with Hoechst (Hch) 33342 + the ABCG2 inhibitor, verapamil (VE), to identify a side population, and immunostained for ABCG2, vimentin (VIM), and cytokeratin (CYT). Thyroid monolayers and CFU-like cultures were differentiated using TSH, adipogenic, and osteogenic media. Up to 1/4 cells from primary monolayers and thyrospheres resulted either ABCG2-, Oct-3/4-, HNF4a-, or Sca-1-positive. In contrast, in CFU-like cultures ABCG2 was detected in up to 1/3 cells, whereas VIM was ubiquitous, and CYT disappeared. Consistently, a side population was revealed by the Hch-VE staining. Finally, CFU-like cultures differentiated to cells containing either thyroglobulin, or red oil-, or alizarin red-positive deposits. We conclude that multilineage differentiation of our CFU-like thyroid cultures reveals enrichment of a thyroid stem cell population.

Il segno di Dalrymple nell’ipertiroidismo

ipertiroidismo consiste nella retrazione bilaterale (anche asimmetrica e talvolta solo monolaterale) della palpebra superiore appena sotto, a livello, o sopra il limbo sclerocorneale (limite tra cornea e sclera), in quest’ultimo caso con esposizione della sclera superiore, mentre il paziente mantiene lo sguardo in avanti, senza sforzo (Figura 1A). Questa retrazione produce un aumento dell’ampiezza della rima palpebrale (spazio tra i margini liberi delle due palpebre), responsabile dell’espressione “ansiosa con sguardo fisso” tipica della facies ipertiroidea. L’eponimo semeiologico celebra l’oftalmologo, chirurgo e microscopista britannico John Dalrymple (1803-1852) cui si deve, nel 1846, la prima descrizione istologica dell’infiltrato costale plasmacellulare osservato all’autopsia del primo paziente (Thomas Alexander McBean, deceduto a 47 anni) dalle cui urine fu isolata, nel 1847, la proteina di Bence Jones (che prende il nome dal medico e chimico inglese Henry Bence Jones), ossia le catene leggere anticorpali nelle gammopatie monoclonali, come il plasmocitoma (Figura 1B). Il segno della retrazione palpebrale superiore fu pubblicato, per la prima volta, nel 1849 dall’oftalmologo Sir White Cooper, collega di Dalrymple al Il segno di Dalrymple nell’ipertiroidismo