Automatic risk evaluation in elderly patients based on Autonomic Nervous System assessment

Dysfunction of Autonomic Nervous System (ANS) is a typical feature of chronic heart failure and other cardiovascular disease. As a simple non-invasive technology, heart rate variability (HRV) analysis provides reliable information on autonomic modulation of heart rate. The aim of this thesis was to research and develop automatic methods based on ANS assessment for evaluation of risk in cardiac patients. Several features selection and machine learning algorithms have been combined to achieve the goals. Automatic assessment of disease severity in Congestive Heart Failure (CHF) patients: a completely automatic method, based on long-term HRV was proposed in order to automatically assess the severity of CHF, achieving a sensitivity rate of 93% and a specificity rate of 64% in discriminating severe versus mild patients. Automatic identification of hypertensive patients at high risk of vascular events: a completely automatic system was proposed in order to identify hypertensive patients at higher risk to develop vascular events in the 12 months following the electrocardiographic recordings, achieving a sensitivity rate of 71% and a specificity rate of 86% in identifying high-risk subjects among hypertensive patients. Automatic identification of hypertensive patients with history of fall: it was explored whether an automatic identification of fallers among hypertensive patients based on HRV was feasible. The results obtained in this thesis could have implications both in clinical practice and in clinical research. The system has been designed and developed in order to be clinically feasible. Moreover, since 5-minute ECG recording is inexpensive, easy to assess, and non-invasive, future research will focus on the clinical applicability of the system as a screening tool in non-specialized ambulatories, in order to identify high-risk patients to be shortlisted for more complex investigations.

Spinal cord injury: assessment of autonomic state-dependent control of cardiovascular system and body core temperature

Spinal cord injury (SCI) results not only in paralysis; but it is also associated with a range of autonomic dysregulation that can interfere with cardiovascular, bladder, bowel, temperature, and sexual function. The entity of the autonomic dysfunction is related to the level and severity of injury to descending autonomic (sympathetic) pathways. For many years there was limited awareness of these issues and the attention given to them by the scientific and medical community was scarce. Yet, even if a new system to document the impact of SCI on autonomic function has recently been proposed, the current standard of assessment of SCI (American Spinal Injury Association (ASIA) examination) evaluates motor and sensory pathways, but not severity of injury to autonomic pathways. Beside the severe impact on quality of life, autonomic dysfunction in persons with SCI is associated with increased risk of cardiovascular disease and mortality. Therefore, obtaining information regarding autonomic function in persons with SCI is pivotal and clinical examinations and laboratory evaluations to detect the presence of autonomic dysfunction and quantitate its severity are mandatory. Furthermore, previous studies demonstrated that there is an intimate relationship between the autonomic nervous system and sleep from anatomical, physiological, and neurochemical points of view. Although, even if previous epidemiological studies demonstrated that sleep problems are common in spinal cord injury (SCI), so far only limited polysomnographic (PSG) data are available. Finally, until now, circadian and state dependent autonomic regulation of blood pressure (BP), heart rate (HR) and body core temperature (BcT) were never assessed in SCI patients. Aim of the current study was to establish the association between the autonomic control of the cardiovascular function and thermoregulation, sleep parameters and increased cardiovascular risk in SCI patients.

Innovative analytical methods for Central Nervous System Drug analysis in biological fluids

During recent years a consistent number of central nervous system (CNS) drugs have been approved and introduced on the market for the treatment of many psychiatric and neurological disorders, including psychosis, depression, Parkinson disease and epilepsy. Despite the great advancements obtained in the treatment of CNS diseases/disorders, partial response to therapy or treatment failure are frequent, at least in part due to poor compliance, but also genetic variability in the metabolism of psychotropic agents or polypharmacy, which may lead to sub-therapeutic or toxic plasma levels of the drugs, and finally inefficacy of the treatment or adverse/toxic effects. With the aim of improving the treatment, reducing toxic/side effects and patient hospitalisation, Therapeutic Drug Monitoring (TDM) is certainly useful, allowing for a personalisation of the therapy. Reliable analytical methods are required to determine the plasma levels of psychotropic drugs, which are often present at low concentrations (tens or hundreds of nanograms per millilitre). The present PhD Thesis has focused on the development of analytical methods for the determination of CNS drugs in biological fluids, including antidepressants (sertraline and duloxetine), antipsychotics (aripiprazole), antiepileptics (vigabatrin and topiramate) and antiparkinsons (pramipexole). Innovative methods based on liquid chromatography or capillary electrophoresis coupled to diode-array or laser-induced fluorescence detectors have been developed, together with the suitable sample pre-treatment for interference removal and fluorescent labelling in case of LIF detection. All methods have been validated according to official guidelines and applied to the analysis of real samples obtained from patients, resulting suitable for the TDM of psychotropic drugs.

Multifunctional nanocarriers encapsulating anti-Alzheimer drug for nasal delivery to central nervous system

Alzheimer's disease (AD) is a fatal neurodegenerative condition characterized clinically by progressive memory loss and irreversible cognitive deterioration. It has been shown that there is a progressive degeneration of the brain cholinergic neurons which leads to the appearance of cognitive symptoms of the disease. The aim of this work was the formulation of multifunctional nanocarriers for nasal administration of tacrine-HCl (THA). This route has many advantages; in particular is possible to convey the drug directly to the Central Nervous System, through the olfactory bulb. In particular, were prepared Albumin nanoparticles carrying beta cyclodextrin and two different beta cyclodextrin derivatives (hydroxypropyl beta cyclodextrin and sulphobutylether beta cyclodextrin), and Multifunctional liposomes, prepared using traditional excipients (cholesterol and phosphatidylcholine), partly enriched with α-tocopherol (Toc) and/or polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) (Ω3). Both nanosystems were characterized in terms of size, Zeta potential and encapsulation efficiency. Were also evaluated their functional properties such as mucoadhesion and permeability, using an ex-vivo assay based on nasal sheep mucosa. On Liposomes were also assessed drug neuronal uptake, cell toxicity, antioxidant and, cytoprotective activity in the human neuronal cell line SH-SY5Y and finally tocopherol trans-membrane diffusion. Both the nanocarriers produced presented excellent properties and a high potential as new systems for CNS-delivery of anti-Alzheimer drugs via the nasal route.

Interspecies study of the enteric nervous system and related pathologies

The enteric nervous system (ENS) modulates a number of digestive functions including well known ones, i.e. motility, secretion, absorption and blood flow, along with other critically relevant processes, i.e. immune responses of the gastrointestinal (GI) tract, gut microbiota and epithelial barrier . The characterization of the anatomical aspects of the ENS in large mammals and the identification of differences and similarities existing between species may represent a fundamental basis to decipher several digestive GI diseases in humans and animals. In this perspective, the aim of the present thesis is to highlight the ENS anatomical basis and pathological aspects in different mammalian species, such as horses, dogs and humans. Firstly, I designed two anatomical studies in horses:  “Excitatory and inhibitory enteric innervation of horse lower esophageal sphincter”.  “Localization of 5-hydroxytryptamine 4 receptor (5-HT4R) in the equine enteric nervous system”. Then I focused on the enteric dysfunctions, including:  A primary enteric aganglionosis in horses: “Extrinsic innervation of the ileum and pelvic flexure of foals with ileocolonic aganglionosis”.  A diabetic enteric neuropathy in dogs: “Quantification of nitrergic neurons in the myenteric plexus of gastric antrum and ileum of healthy and diabetic dogs”.  An enteric neuropathy in human neurological patients: “Functional and neurochemical abnormalities in patients with Parkinson's disease and chronic constipation”. The physiology of the GI tract is characterized by a high complexity and it is mainly dependent on the control of the intrinsic nervous system. ENS is critical to preserve body homeostasis as reflect by its derangement occurring in pathological conditions that can be lethal or seriously disabling to humans and animals. The knowledge of the anatomy and the pathology of the ENS represents a new important and fascinating topic, which deserves more attention in the veterinary medicine field.

Foetal heart rate recording: analysis and comparison of different methodologies

Monitoring foetal health is a very important task in clinical practice to appropriately plan pregnancy management and delivery. In the third trimester of pregnancy, ultrasound cardiotocography is the most employed diagnostic technique: foetal heart rate and uterine contractions signals are simultaneously recorded and analysed in order to ascertain foetal health. Because ultrasound cardiotocography interpretation still lacks of complete reliability, new parameters and methods of interpretation, or alternative methodologies, are necessary to further support physicians’ decisions. To this aim, in this thesis, foetal phonocardiography and electrocardiography are considered as different techniques. Further, variability of foetal heart rate is thoroughly studied. Frequency components and their modifications can be analysed by applying a time-frequency approach, for a distinct understanding of the spectral components and their change over time related to foetal reactions to internal and external stimuli (such as uterine contractions). Such modifications of the power spectrum can be a sign of autonomic nervous system reactions and therefore represent additional, objective information about foetal reactivity and health. However, some limits of ultrasonic cardiotocography still remain, such as in long-term foetal surveillance, which is often recommendable mainly in risky pregnancies. In these cases, the fully non-invasive acoustic recording, foetal phonocardiography, through maternal abdomen, represents a valuable alternative to the ultrasonic cardiotocography. Unfortunately, the so recorded foetal heart sound signal is heavily loaded by noise, thus the determination of the foetal heart rate raises serious signal processing issues. A new algorithm for foetal heart rate estimation from foetal phonocardiographic recordings is presented in this thesis. Different filtering and enhancement techniques, to enhance the first foetal heart sounds, were applied, so that different signal processing techniques were implemented, evaluated and compared, by identifying the strategy characterized on average by the best results. In particular, phonocardiographic signals were recorded simultaneously to ultrasonic cardiotocographic signals in order to compare the two foetal heart rate series (the one estimated by the developed algorithm and the other provided by cardiotocographic device). The algorithm performances were tested on phonocardiographic signals recorded on pregnant women, showing reliable foetal heart rate signals, very close to the ultrasound cardiotocographic recordings, considered as reference. The algorithm was also tested by using a foetal phonocardiographic recording simulator developed and presented in this research thesis. The target was to provide a software for simulating recordings relative to different foetal conditions and recordings situations and to use it as a test tool for comparing and assessing different foetal heart rate extraction algorithms. Since there are few studies about foetal heart sounds time characteristics and frequency content and the available literature is poor and not rigorous in this area, a data collection pilot study was also conducted with the purpose of specifically characterising both foetal and maternal heart sounds. Finally, in this thesis, the use of foetal phonocardiographic and electrocardiographic methodology and their combination, are presented in order to detect foetal heart rate and other functioning anomalies. The developed methodologies, suitable for longer-term assessment, were able to detect heart beat events correctly, such as first and second heart sounds and QRS waves. The detection of such events provides reliable measures of foetal heart rate, potentially information about measurement of the systolic time intervals and foetus circulatory impedance.

Caratterizzazione dei sintomi neurovegetativi e neuropsicologici nella malattia di Parkinson associata a mutazioni del gene glucocerebrosidasi

Pochi studi hanno indagato il profilo dei sintomi non-motori nella malattia di Parkinson associata al gene glucocerebrosidasi (GBA). Questo studio è mirato alla caratterizzazione dei sintomi non-motori, con particolare attenzione alla valutazione delle funzioni neurovegetativa, cognitiva e comportamentale, nel parkinsonismo associato a mutazione del gene GBA con la finalità di verificare se tali sintomi non-motori siano parte dello spettro clinico di questi pazienti. E’ stato condotto su una coorte di pazienti affetti da malattia di Parkinson che erano stati tutti sottoposti ad una analisi genetica per la ricerca di mutazioni in uno dei geni finora associati alla malattia di Parkinson. All’interno di questa coorte omogenea sono stati identificati due gruppi diversi in relazione al genotipo (pazienti portatori della mutazione GBA e pazienti non portatori di nessuna mutazione) e le caratteristiche non-motorie sono state confrontate nei due gruppi. Sono state pertanto indagati il sistema nervoso autonomo, mediante studio dei riflessi cardiovascolari e analisi dei sintomi disautonomici, e le funzioni cognitivo-comportamentali in pazienti affetti da malattia di Parkinson associata a mutazione del gene GBA. I risultati sono stati messi a confronto con il gruppo di controllo. Lo studio ha mostrato che i pazienti affetti da malattia di Parkinson associata a mutazione del gene GBA presentavano maggiore frequenza di disfunzioni ortosimpatiche, depressione, ansia, apatia, impulsività, oltre che di disturbi del controllo degli impulsi rispetto ai pazienti non portatori. In conclusione, i pazienti GBA positivi possono esprimere una sintomatologia non-motoria multidominio con sintomi autonomici, cognitivi e comportamentali in primo piano. Pertanto l’impostazione terapeutica in questi pazienti dovrebbe includere una accurata valutazione dei sintomi non-motori e un loro monitoraggio nel follow up clinico, allo scopo di ottimizzare i risultati e ridurre i rischi di complicazioni.

Sleep-related changes in blood pressure in hypocretin-deficient narcoleptic mice

Objectives. Blood pressure (BP) physiologically has higher and lower values during the active and rest period, respectively. Subjects failing to show the appropriate BP decrease (10-20%) on passing form diurnal activity to nocturnal rest and sleep have increased risk of target organ damage at the cardiac, vascular and cerebrovascular levels. Hypocretin (HCRT) releasing neurons, mainly located in the lateral hypothalamus, project widely to the central nervous system. Thus HCRT neurons are involved in several autonomic functions, including BP regulation. HCRT neurons also play a key role in wake-sleep cycle regulation, the lack of which becomes evident in HCRT-deficient narcoleptic patients. I investigated whether chronic lack of HCRT signaling alters BP during sleep in mouse models of narcolepsy. Methods. The main study was performed on HCRT-ataxin3 transgenic mice (TG) with selective post-natal ablation of HCRT neurons, HCRT gene knockout mice (KO) with preserved HCRT neurons, and Wild-Type control mice (WT) with identical genetic background. Experiments where replicated on TG and WT mice with hybrid genetic background (hTG and hWT, respectively). Mice were implanted with a telemetric pressure transducer (TA11PA-C10, DSI) and electrodes for discriminating wakefulness (W), rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Signals were recorded for 3 days. Mean BP values were computed in each wake-sleep state and analyzed by ANOVA and t-test with significance at p<0.05. Results. The decrease in BP between either NREMS or REMS and W was significantly blunted in TG and KO with respect to WT as well as in hTG with respect to hWT. Conclusions. Independently from the genetic background, chronic HCRT deficiency leads to a decreased BP difference between W and sleep potentially adverse in narcoleptic subjects. These data suggest that HCRT play an important role in the sleep-dependent cardiovascular control.

Involvement of the opioid system and BDNF in neuroplastic alterations occurring in neuropathic pain conditions

Chronic pain affects one in five adults, reducing quality of life and increasing risk of developing co-morbidities such as depression. Neuropathic pain results by lesions to the nervous system that alter its structure and function leading to spontaneous pain and amplified responses to noxious and innocuous stimuli. The Opioid System is probably the most important system involved in control of nociceptive transmission. Dynorphin and nociceptin systems have been suggested key mediators of some neuropathic pain aspects. An important role also for BDNF has been recently suggested since its involvement in the peripheral and central sensitization phenomena is known. We studied neuroplastic alterations occurring in chronic pain in mice subjected to the chronic constriction injury (CCI). We investigated gene expression alterations of both BDNF and Opioid System at spinal level at different intervals of time. A transient upregulation of pBDNF and pDYN was observed in spinal cord, while increasing upregulation of ppN/OFQ was found in the DRGs of injured mice. Development of neuropathic behavioral signs has been observed in ICR/CD-1 and BDNF+/+ mice, subjected to CCI. A different development of these signs was observed in BDNF+/-. We also studied gene expression changes of investigated systems in different brain areas fourteen days after surgery. We found pBDNF, pDYN, pKOP, ppN/OFQ and pNOP gene expression alterations in several areas of CCI mice. In the same brain regions we also determined bioactive nociceptin peptide levels, and elevated N/OFQ levels were observed in the amygdala area. Histone modifications studies have been performed in BDNF and DYN gene promoters of CCI animal spinal cord showing selected alterations in pDYN gene promoter. In addition, a preliminary characterization of the innovative NOP-EGFP mice was performed. Overall, our results could be useful to understand which and how neuropeptidergic systems are involved in neuroplastic mechanism occurring in neuropathic pain.