Stefania Castrezzati

α-synuclein and synapsin III cooperatively regulate synaptic function in dopamine neurons.

ABSTRACT The main neuropathological features of Parkinsons disease are dopaminergic nigrostriatal neuron degeneration, and intraneuronal and intraneuritic proteinaceous inclusions named Lewy bodies and Lewy neurites, respectively, which mainly contain α-synuclein (α-syn, also known as SNCA). The neuronal phosphoprotein synapsin III (also known as SYN3), is a pivotal regulator of dopamine neuron synaptic function. Here, we show that α-syn interacts with and modulates synapsin III. The absence of α-syn causes a selective increase and redistribution of synapsin III, and changes the organization of synaptic vesicle pools in dopamine neurons. In α-syn-null mice, the alterations of synapsin III induce an increased locomotor response to the stimulation of synapsin-dependent dopamine overflow, despite this, these mice show decreased basal and depolarization-dependent striatal dopamine release. Of note, synapsin III seems to be involved in α-syn aggregation, which also coaxes its increase and redistribution. Furthermore, synapsin III accumulates in the caudate and putamen of individuals with Parkinsons disease. These findings support a reciprocal modulatory interaction of α-syn and synapsin III in the regulation of dopamine neuron synaptic function. Summary: Absence or aggregation of α-synuclein induces a selective increase and redistribution of synapsin III, reorganizing synaptic vesicle pools, which could have implications for dopamine neuron degeneration in Parkinsons disease.

Vascular endothelial cells and dysfunctions: role of melatonin.

Several pathological conditions, including hypertension, atherosclerosis, diabetes, ischemia/reperfusion injury and nicotine-induced vasculopathy, are associated with vascular endothelial dysfunction characterized by altered secretory output of endothelial cells. Therefore there is a search for molecules and interventions that could restore endothelial function, in particular augmenting NO production, reducing the generation of free radicals and vasoconstrictors and preventing undesired inflammation. The pineal hormone melatonin exhibits several endothelium protective properties: it scavenges free radicals, activates antioxidant defence enzymes, normalizes lipid and blood pressure profile and increases NO bioavailability. Melatonin improved vascular function in experimental hypertension, reducing intimal infiltration and restoring NO production. Melatonin improved the NO pathway also in animal models for the study of diabetes and prevented NO down-regulation and adhesive molecules up-regulation in nicotine-induced vasculopathy. The protection against endothelial damage, vasoconstriction, platelet aggregation and leukocyte infiltration might contribute to the beneficial effects against ischemia-reperfusion injury by melatonin. Therefore, melatonin administration has endothelium-protective potential in several pathological conditions. Nevertheless, it still needs to be established, whether melatonin is able to revert already established endothelial dysfunction in these conditions.

Melatonin reduces obesity and restores adipokine patterns and metabolism in obese (ob/ob) mice

The increasing incidence of obesity, leading to metabolic complications, is now recognized as a major public health problem. The adipocytes are not merely energy-storing cells, but they play crucial roles in the development of the so-called metabolic syndrome due to the adipocyte-derived bioactive factors such as adipokines, cytokines, and growth factors. The dysregulated production and secretion of adipokines seen in obesity is linked to the pathogenesis of the metabolic disease processes. In this study, we hypothesized that dietary melatonin administration would support an anti-inflammatory response and play an important role in energy metabolism in subcutaneous and visceral adipose tissues of obese mice and so may counteract some of the disruptive effects of obesity. Lean and obese mice (ob/ob) received melatonin or vehicle in drinking water for 8 weeks. Thereafter, they were evaluated for morphologic alteration, inflammatory cell infiltration, and the adipokine patterns in visceral and subcutaneous white fat depots. In obese mice treated with vehicle, we observed a significant increase in fat depots, inflammation, and a dysregulation of the adipokine network. In particular, we measured a significant reduction of adiponectin and an increase of tumor necrosis factor α, resistin, and visfatin in adipose tissue deposits. These changes were partially reversed when melatonin was supplemented to obese mice. Melatonin supplementation by regulating inflammatory infiltration ameliorates obesity-induced adipokine alteration, whereas melatonin administration in lean mice was unaffected. Thus, it is likely that melatonin would be provided in supplement form to control some of the disruptive effects on the basis of obesity pathogenic process.

Obesity-related dysfunction of the aorta and prevention by melatonin treatment in ob/ob mice

In this study, we hypothesized that melatonin administration can minimize alterations in aorta morphology in an animal model of obesity (ob/ob mice). The animals were divided into four groups: (i) control lean mice, (ii) control lean mice treated with melatonin, (iii) ob/ob mice and (iv) ob/ob mice treated with melatonin. The synthetic melatonin was dissolved in 1% ethanol and added to the drinking water from postnatal week 5-13 at a final dose of 100 mg/kg body weight/day. Compared with the obese mice, melatonin intake was associated with a significant decrease in body weight and water consumption. Histological analysis showed that the aortic wall of ob/ob mice had a high Tunica media/lumen ratio and that the elastic fibers in the media layer appeared disrupted and degraded. Moreover, the aorta of ob/ob mice displayed a higher degree of collagen accumulation in the Tunica media compared to the normal aorta. The aorta of ob/ob mice treated with melatonin had a lower Tunica media/lumen ratio and collagen accumulation in comparison with untreated ob/ob mice. Our results showed that whereas melatonin had no apparent histological effects on the aorta in lean mice with normal weight, its administration in ob/ob mice can lead to a reduction in body weight and can ameliorate aorta histopathological dysfunction. This experimental study indicates an apparent protective role for melatonin on the aorta in obesity and melatonin could possibly be an effective tool in the management of obesity-related vascular complications.

Biological Characterization and In Vitro Effects of Human ConcentratedGrowth Factor Preparation: An Innovative Approach to Tissue Regeneration

Scientific background: Platelet concentrates are nowadays widely applied in different clinical fields to improve soft tissue and bone regeneration. “Concentrated Growth Factors” (CGF) is a new generation of platelet concentrate products, which exhibits an interesting clinical and biotechnological application potential. Aim of the study: The aim of this study is to assess the biological rationale for the use of CGF, by evaluating blood cell localization, the in vitro cumulative release of seven growth factors (PDGF-AB, VEGF, TNF-α, TGF-β1, BDNF, BMP- 2 and IGF-1), its in vitro effects on cell proliferation and its mechanical behavior. Methods: CGFs were obtained from volunteer donors. Blood cell localization was evaluated after properly morphological staining and immunohistochemistry. The amount of growth factors release was measured at 5 hours, 1, 3, 6, 7 and 8 days, using ELISA assay. Cells were cultured with and without CGF and their proliferation were evaluated after 72 hours, performing the quantification of Ki-67, using flow cytometry (FACS). The mechanical response of CGF under compression was also attempted. Results: The results showed that platelets and leukocytes were found in a very thin space called “buffy coat”, localized between the white and red part of CGF. Each growth factor evaluated, had a specific kinetic release with a great variability among subjects. The in vitro cell proliferation was stimulated. CGF showed an “apparent plasticity” and its mechanical response was influenced by fibrin network structure. Conclusion: These findings support the CGF’s clinical use and will allow us to better understand and improve the clinical outcomes.

How the different material and shape of the blood collection tube influences the Concentrated Growth Factors production

Platelet concentrates, such as Concentrated Growth Factors (CGF), are autologous preparations obtained from the patients own blood and rich in platelets, growth factors and cytokines involved in the key processes of tissue regeneration. These autologous concentrates differ in the way of preparation and also in the content of platelets, growth factors and leucocytes, as well as in the fibrin network architecture. So it is difficult to have a standardized product. The aim of the present study was to evaluate how the use of test tubes of different material, for blood collection, could influence the CGF production. Three different test tubes were used and the obtained CGFs were subjected to histomorphological and immunohistochemical analyses. Results showed that the tube material and shape influenced the CGF composition. In fact, according to the type of tube used, the obtained CGFs showed differences in morphology, in the fibrin network architecture and in blood cell localization and distribution.

Platelet preparations in neuronal cell differentiation

Concentrated Growth Factors (CGF) is a platelet rich preparation that has the important feature of a tight fibrin network and containing a large number of growth factors possessing great regenerative potentialities [1]. The regeneration of nervous system is one of the mail goal of regenerative medicine. The aim of this study is to test the in vitro CGF effects on both differentiated and undifferentiated SH-SY5Y cells, derived from human neuroblastoma. To induce differentiation, SH-SY5Y cells have been treated with Retinoic Acid (RA) 10µM, in both basal and complete medium and in the presence and absence of CGF. After 72 hours, different parameters have been investigated: the morphological characteristics of the cells, the cell proliferation, the cellular vitality using the MTT test, the CGF and/or RA differentiation property and the immunocytochemical analysis of neuronal specific markers (NeuN, Sinaptophisine, β-III-tubulin, Nestin). Moreover the NGF (Nerve Growth Factor) and BDNF (Brain Derived Growth Factor) release have been assayed by ELISA test. Our results obtained suggest that treatment with CGF, also used alone, positively affects cell differentiation and neuronal phenotype regulating the expression of the neuronal markers and improving the outgrowth of neurites. Taken together these results seems to be promised into new approaches for neuronal regeneration using platelet preparations.

The anastomotic network around the anterior superior alveolar nerve: an anatomical and radiological study

Innervation of superior teeth is supplied by the posterior (PSAN), anterior (ASAN) and sometimes by middle superior alveolar nerve (MSAN). PSAN arises from the maxillary nerve and passes through the posterolateral maxillary wall towards the posterior teeth. ASAN arises from the anterior portion of the infraorbital nerve and courses within the infraorbital canal passing nearby the piriform aperture and premaxilla. When present, MSAN arises from the posterior portion of the infraorbital nerve and runs along the lateral maxillary wall. However, an additional nasopalatine or sublabial injection is frequently required to obtain a complete anesthesia of the maxillary teeth due to rich anastomotic network (1-2). With the aim to better describe the complexity of the superior alveolar nerve network, fifty-seven high-definition sinonasal cone-beam CT (CBCT) were analyzed. PSAN, ASAN and MSAN were detected by specific bony landmarks/canals and nervous anastomoses were accurately evaluated. In addition, medial anastomotic branches from the palatal and/or nasal nervous plexi were also considered. PSAN and ASAN were identified in 100% of cases whereas MSAN in 19.6% of cases. Anastomotic branch versus ASAN was identified in all cases from MSAN and in 60.3% from PSAN. Medial anastomotic branch was detected in 62.0% of cases from the nasal plexus and in 6.2% from the palatal plexus: the former passed through a bony defect in the floor of the piriform aperture or at the base of the nasal septum; the latter passed through a tiny canal in the interface between maxilla and premaxilla. These data confirm that maxillary teeth innervation, especially for incisor teeth, could be provided not only by alveolar nerves but also from palatal and nasal plexi via small branches running within maxillary bony canals. These results support the need of additional anesthetic injection to obtain adequate anesthesia of the maxillary teeth; moreover, the role of CBCT in the identification of the nervous pattern was underlined.

Effects of melatonin long-term treatment in aging mice

Aging is a complex and progressive process involving every organ in the body and it is the result of coordinated biology events that can span decades (1). At cardiovascular level, age-related degeneration and functional decline are quite heterogeneously, nevertheless oxidative stress is a well known pathological process involved. The aim of this study was to investigate the effects of a chronic and long-term treatment with melatonin on functional responses of small mesenteric arteries and on the expression of oxidative stress markers at aorta level of senescence-accelerated prone mice (SAMP8), a model of age-related vascular dysfunction and cognitive decline (2), respect relative controls, senescence-accelerated resistant mice (SAMR1). In the present study were investigated SAMP8 and SAMR1 mice orally treated or not treated for 10 months with melatonin (MelapureTM by Flamma S.p.A.). It was observed that the anticontractile effect of perivascular adipose tissue is impaired in untreated SAMP8, compared with SAMR1. On the contrary, the chronic treatment with melatonin decreased the contractile response to norepinephrine in mesenteric small arteries of SAMP8, restoring an anticontractile effect, probably through melatonin antioxidant mechanisms. In untreated SAMP8 mice was observed at aorta level also an overexpression of oxidative stress and inflammatory markers compared with controls; whereas the long-term treatment with melatonin in SAMP8 was able to increase the expression of some markers of vasculoprotection and to decrease oxidative stress and inflammation. A reduced expression of adiponectin and adiponectin receptor 1 was also observed at visceral fat level of untreated SAMP8 respect SAMR1, while a significant increase was observed after melatonin treatment. In conclusion, melatonin exhibited marked antioxidant and vasculoprotective effects, underlining its potential anti-aging properties at cardiovascular level. Sincere thanks to Flamma S.p.A.-Italy (www.flammagroup.com) for courteously providing the melatonin.

Symmetrical anatomical variation of the anterior belly of the digastric muscle

The digastric muscle is an important surgical landmark. Several anatomical varia- tions of the digastric muscle are reported in literature and the presence of accessory anterior bellies of the muscle are not uncommon (1,2). We reported a symmetrical variation of the digastric muscle that was found during a dissection of the suprahy- oid region. The dissection showed digastric muscles with an accessory anterior belly, which originated from the anterior belly of muscles in proximity and anterior to the intermediate tendon. The accessory bellies of both sides were fused together on the midline and were attached with a unique tendon to the inner surface of the mental symphysis, filling the submental triangle completely. This unreported anatomical var - iation could be considered an additional contribute in the description of the varia- tions of the digastric muscle, with several implications in head and neck pathology, diagnosis and surgery.