Possibile ruolo del sistema endocannabinoide nel disturbo d’alimentazione incontrollata (binge eating disorder): studi comportamentali, farmacologici e biochimici

According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-V), binge eating disorder (BED) is an eating disorder characterized by repetitive episodes of uncontrolled and excessive food consumption (binge eating), in a short period of time, without the inappropriate compensatory behaviors for limiting weight gain. BED is a stable condition that is associated with elevated psychiatric comorbidity, including depression and anxiety. A large body of evidence supports a contribution of the endocannabinoid system in the modulation of energy balance by controlling food intake through central and peripheral mechanisms. The cannabinoid CB1 receptor is abundantly expressed in the central nervous system and induces inhibition of neurotransmission through modulation of presynaptic neurotransmitter release. CB1 receptors inverse agonists inhibit food intake through both central and peripheral mechanisms while, in contrast, cannabinoid agonists stimulate food intake in humans and induce beneficial effects in acquired immune deficiency syndrome related to anorexia, suggesting altered endocannabinoid neurotransmission in anorectic conditions. A possible involvement of the endocannabinoid system in the pathogenesis of BED and other eating disorders has recently been supported by independent experimental evidence. Elevated plasma levels of anandamide were found in women affected by AN and BED. Moreover, anandamide levels were inversely correlated with plasma leptin concentrations. On the basis of these evidence, the aim of this thesis was to study whether and which elements of the endocannabinoid system might be correlated with the binge eating behavior, which brain areas are specifically involved, and if pharmacological treatments specific for the endocannabinoid system (i.e. agonists, antagonists, inverse agonists of cannabinoid receptors, inhibitors of endocannabinoid metabolisms) are be able, besides to modify the state of BED induced in laboratory animals, to restore a correct functionality of the endocannabinoid system. Furthermore an analysis of the behavioral profile of animals with BED has been evaluated before the start of drug treatment by means of different mazes in which is possible to study anxiety and depression. Binge eating behavior was induced in animals by giving them a sporadic (3 days/week) and limited (2h) access to a high fat diet (margarine) in addition to a continuous access to chow and water. In these animals, the intake of margarine becomes significantly greater than in animals with limited daily access to margarine and remains stable over prolonged periods of time. As revealed by forced swim test, animals with binge eating behavior did not show a depressive like behavior compared to control animals. However, using the elevated plus maze, an anxiety like behavior was highlighted in those animals before access to margarine (Pre - binge phase) that it was significantly reduced after the consumption of this palatable food (Post - binge phase). Results showed that an increase of the endocannabinoid signaling by CB1 agonists or by inhibitors of endocannabinoid metabolism did not modify the binge eating behavior presents in our animals. On the contrary, a decrease of the endocannabinoid signaling by CB1 receptors inverse agonist/antagonist rimonabant, was able to alter this behavior when given chronically. As regards the CB1 receptor density, no difference has been highlighted between animals showing binge eating behavior and control group. In conclusion, negative modulation of the endocannabinoid signal may represent an important strategy in the treatment of Binge Eating Disorder

Studio della variabilità genetica di specie animali autoctone della Sardegna

This work aimed to know the genetics state of four Sardinian autochthonous animal species: two mammalian species, the mouflon (Ovis gmelini musimon) and the Sardinian hare (Lepus capensis mediterraneus), and two birds, the European shag (Phalacrocorax aristotelis desmarestii) and the griffon (Gyps fulvus), in order to supply a method to monitories their welfare and the anthropic impact on these animal colonies and to manage to preserve their genetic diversity. Firstly we manage to construct a simple and atraumatic system to determine the sex in birds that do not show sexual dimorphism, such as griffon and European shag. We used DNA extracted from feather and blood and the polymerase chain reaction to assess a polymorphism of the sexual chromosom located Cromo Helicase DNA Binding 1 (CHDB1) gene. We evidenced a sex specific pattern in both bird species and evidenced a distorted sex ratio in the newborns of european shag populations of the north east of Sardinia. This phenomenon was due to the egg sex managements of European shag females induced by the high death of males during fishing near fishing nets. We used a molecular assessment of microsatellite markers to study population genetics of these species. Our results showed that in all studied species there are troubles due to inbreeding and genetic drift caused by decrease of animal numbers in each population and to the habitat fragmentation due to their interaction with human activities

Nuovi approcci per la valutazione della qualità dei gameti

The aim of our study was to use metabolomic and proteomic technologies to evaluate quality parameters of male and female gametes using ovine and equine as models. We evaluate 1) metabolomic factors in seminal plasma of subfertile rams pharmacologically treated with melatonin using capillary electrophoresis (CE) and mass spectrometry. We measured melatonin concentration in seminal plasma and evidenced its increase till 90 days after treatment. Parallelly to melatonin seminal plasma concentration it was affected carnitine and acetylcarnitine and some aminoacids that could be used as biomarkers of sperm quality; 2) Sperm quality and ATP concentration in stallions of different ages using capillary electrophoresys and UV determination. Our data did not evidence any difference between different experimental groups.3) Spermatozoa ATP concentration by CE with UV and seminal plasma proteomics using 2D-PAGE, in subfertile stallions carrying lower sperm viability and motility. Obtained data did not evidence differences in ATP concentration but it were showed differences in protein patterns; 3)ATP and protein patterns during maturation of ovine oocytes with different developmental competence using CE with LIF and 2D-PAGE respectively. ATP was showed a determining factor responsible to protein synthesis delay, meiotic cycle timing and embryo quality. Our study evidenced the reliable use of metabolomics and proteomics to determine male and female gametes quality

Evidence-based approach to medical equipment maintenance monitoring

Maintenance is a crucial part of the life cycle of medical equipment. We applied a specific set of codes to classify the corrective and scheduled maintenance work orders at the University Hospital of Careggi (Florence, Italy). A set of Key Performance Indicators (KPI) (financial, technological and organizational) helps in evaluating the performance of the medical equipment maintenance. The analysis of KPIs (related to costs, age and SM completion rate) revealed some problems in maintenance strategy. The results show, starting from the evidences (i.e. the failures), that the combination of these two methods can give a periodical cross-analysis of the maintenance performance and indicate the most appropriate procedure

Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick-transmitted micro-organisms in Sardinia, Italy

A total of 1485 adult ticks were collected from mammalian hosts in south-eastern Sardinia, Italy, during the years 2007–2008. Ticks were identified and tested by PCR analysis for presence of Rickettsia species of the spotted fever group, Ehrlichia canis, Anaplasma phagocytophilum, Coxiella burnetii, Bartonella species and Leishmania species. Among all tick species examined (Rhipicephalus sanguineus, Rhipicephalus turanicus, Rhipicephalus bursa, Rhipicephalus pusillus, Hyalomma marginatum marginatum, Haemaphysalis sulcata and Dermacentor marginatus), only Hyalomma marginatum marginatum produced negative results. A total of 22 pools belonging to the three tick species Rhipicephalus sanguineus (0.9 %), Rhipicephalus turanicus (4.5 %) and Rhipicephalus pusillus (100 %) were positive for Rickettsia species, while a total of five pools belonging to Rhipicephalus sanguineus (0.09 %), Haemaphysalis sulcata (16.7 %) and D. marginatus (7.8 %) were positive for E. canis. Five pools of Rhipicephalus turanicus (1.8 %) were positive for A. phagocytophilum. Positivity for C. burnetii was found in seven pools belonging to three tick species: Rhipicephalus sanguineus (0.5 %), Rhipicephalus turanicus (0.3 %) and Haemaphysalis sulcata (4.4 %). Finally, four pools belonging to Rhipicephalus sanguineus (0.09 %), Rhipicephalus turanicus (0.7 %) and Rhipicephalus bursa (1.1 %) were positive for Bartonella species. Leishmania species DNA was not detected in any of the tick pools examined. Data presented here increase our knowledge on tick-borne diseases in Sardinia, and provide a useful contribution to understanding their epidemiology

Neuropathological Characterization of a Dravet Syndrome Knock-In Mouse Model Useful for Investigating Cannabinoid Treatments

Dravet syndrome (DS) is an epileptic syndrome caused by mutations in the Scn1a gene encoding the alpha 1 subunit of the sodium channel Nav1.1, which is associated with febrile seizures that progress to severe tonic-clonic seizures and associated comorbidities. Treatment with cannabidiol has been approved to reduce seizures in DS, but it may also be active against these comorbidities. The aim of this study was to validate a new mouse model of DS having lower mortality than previous models, which may serve to further evaluate therapies for the long-term comorbidities. This new model consists of heterozygous conditional knock-in mice carrying a missense mutation (A1783V) in Scn1a gene expressed exclusively in neurons of the CNS (Syn-Cre/Scn1a(WT/A1783V)). These mice have been used here to determine the extent and persistence of the behavioral deterioration in different postnatal days (PND), as well as to investigate the alterations that the disease produces in the endocannabinoid system and the contribution of inflammatory events and impaired neurogenesis in the pathology. Syn-Cre/Scn1a(WT/A1783V) mice showed a strong reduction in hindlimb grasp reflex at PND10, whereas at PND25, they presented spontaneous convulsions and a greater susceptibility to pentylenetetrazole-induced seizures, marked hyperactivity, deficient spatial working memory, lower levels of anxiety, and altered social interaction behavior. These differences disappeared at PND40 and PND60, except the changes in social interaction and anxiety. The analysis of CNS structures associated with these behavioral alterations revealed an elevated glial reactivity in the prefrontal cortex and the dentate gyrus. This was associated in the dentate gyrus with a greater cell proliferation detected with Ki67 immunostaining, whereas double-labeling analyses identified that proliferating cells were GFAP-positive suggesting failed neurogenesis but astrocyte proliferation. The analysis of the endocannabinoid system of Syn-Cre/Scn1a(WT/A1783V) mice confirmed reductions in CB1 receptors and MAGL and FAAH enzymes, mainly in the cerebellum but also in other areas, whereas CB2 receptors became upregulated in the hippocampus. In conclusion, Syn-Cre/Scn1a(WT/A1783V) mice showed seizuring susceptibility and several comorbidities (hyperactivity, memory impairment, less anxiety, and altered social behavior), which exhibited a pattern of age expression similar to DS patients. Syn-Cre/Scn1a(WT/A1783V) mice also exhibited greater glial reactivity and a reactive response in the neurogenic niche, and regional changes in the status of the endocannabinoid signaling, events that could contribute in behavioral impairmentThis work was supported by grants from CIBERNED (CB06/05/0089) and MICIU (RTI2018-098885-B-100) to JF-R, Proyectos de Investigacion en Salud, Instituto de Salud Carlos III (PI20/00773) to OS, and Spanish Ministry of Economy and Competitiveness (MINECO; SAF-2015-70866-R, with FEDER Funds) to JE. These agencies had no further role in study design, collection, analysis and interpretation of the data, in the writing of the report, or in the decision to submit the paper for publication. CA is a predoctoral fellow supported by the FPU-Programme (FPU16/04768). The authors are indebted to Yolanda Garcia-Movellan for administrative assistanc

Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development

Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December – March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 μl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique

Preclinical investigation of β-caryophyllene as a therapeutic agent in an experimental murine model of Dravet syndrome

Dravet Syndrome (DS) is caused by mutations in the Scn1a gene encoding the α1 subunit of the sodium channel Nav1.1, which results in febrile seizures that progress to severe tonic-clonic seizures and associated comorbidities. Treatment with cannabidiol has been approved for the management of seizures in DS patients, but it appears to be also active against associated comorbidities. In this new study, we have investigated β-caryophyllene (BCP), a cannabinoid with terpene structure that appears to also have a broad-spectrum profile, as a useful therapy against both seizuring activity and progression of associated comorbidities. This has been studied in heterozygous conditional knock-in mice carrying a missense mutation (A1783V) in Scn1a gene expressed exclusively in neurons of the Central Nervous System (Syn-Cre/Scn1aWT/A1783V), using two experimental approaches. In the first approach, an acute treatment with BCP was effective against seizuring activity induced by pentylenetetrazole (PTZ) in wildtype (Scn1aWT/WT) and also in Syn-Cre/Scn1aWT/A1783V mice, with these last animals having a greater susceptibility to PTZ. Such benefits were paralleled by a BCP-induced reduction in PTZ-induced reactive astrogliosis (labelled with GFAP) and microgliosis (labelled with Iba-1) in the prefrontal cortex and the hippocampal dentate gyrus, which were visible in both wildtype (Scn1aWT/WT) and Syn-Cre/Scn1aWT/A1783V mice. In the second approach, both genotypes were treated repeatedly with BCP to investigate its effects on several DS comorbidities. Thus, BCP corrected important behavioural abnormalities of Syn-Cre/Scn1aWT/A1783V mice (e.g. delayed appearance of hindlimb grasp reflex, induction of clasping response, motor hyperactivity, altered social interaction and memory impairment), attenuated weight loss, and slightly delayed premature mortality. Again, these benefits were paralleled by a BCP-induced reduction in reactive astrogliosis and microgliosis in the prefrontal cortex and the hippocampal dentate gyrus typical of Syn-Cre/Scn1aWT/A1783V mice. In conclusion, BCP was active in Syn-Cre/Scn1aWT/A1783V mice against seizuring activity (acute treatment) and against several comorbidities (repeated treatment), in both cases in association with its capability to reduce glial reactivity in areas related to these behavioural abnormalities. This situates BCP in a promising position for further preclinical evaluation towards a close translation to DS patients

Livelli di inquinanti organici persistenti in stenelle (<i>Stenella coeruleoalba</i>) spiaggiate nel nord Sardegna = Concentration of persistent organic pollutants in striped dolphin (<i>Stenella coeruleoalba</i>) stranded in North Sardinia

Long-lived apex predators, such as marine mammals, are particularly at risk from effects of persistent organic pollutants (POPs), e.g. polychlorinated biphenyls (PCBs) and dichlorodiphenylethanes (e.g. DDT), due to bioaccumulation and biomagnification. POPs are lipophilic compounds that tend to accumulate in the lipid-rich blubber. In marine mammals, POPs enter the body almost exclusively through the diet. In the present study, the levels of PCBs and DDTs in the blubber of striped dolphin stranded in North Sardinia were determined. Our results showed that pollutant concentration are related to age and sex of the individuals. Considering the well known harmful consequences of bioaccumulation of POPs in marine mammals, further studies are needed to determine which POPs might be linked to effects on health