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.

Valutazione del rapporto urinario proteine totali/creatinina e albumina/creatinina in cani affetti da iperadrenocorticismo e da diabete mellito

INTRODUCTION – In human medicine, diabetes mellitus (DM), hypertension, proteinuria and nephropathy are often associated although it is still not clear whether hypertension is the consequence or the cause of nephropathy and albuminuria. Microalbuminuria, in humans, is an early and sensitive marker which permits timely and effective therapy in the early phase of renal damage. Conversely, in dogs, these relationships were not fully investigated, even though hypertension has been associated with many diseases (Bodey and Michell, 1996). In a previous study, 20% of diabetic dogs were found proteinuric based on a U:P/C > 1 and 46% were hypertensive; this latter finding is similar to the prevalence of hypertension in diabetic people (40-80%) (Struble et al., 1998). In the same canine study, hypertension was also positively correlated with the duration of the disease, as is the case in human beings. Hypertension was also found to be a common complication of hypercortisolism (HC) in dogs, with a prevalence which varies from 50 (Goy-Thollot et al., 2002) to 80% (Danese and Aron, 1994).The aim of our study was to evaluate the urinary albumin to creatinine ratio (U:A/C) in dogs affected by Diabetes Mellitus and HC in order to ascertain if, as in human beings, it could represent an early and more sensitive marker of renal damage than U:P/C. Furthermore, the relationship between proteinuria and hypertension in DM and HC was also investigated. MATERIALS AND METHODS – Twenty dogs with DM, 14 with HC and 21 healthy dogs (control group) were included in the prospective case-control study. Inclusion criteria were hyperglycaemia, glicosuria and serum fructosamine above the reference range for DM dogs and a positive ACTH stimulation test and/or low-dose dexamethasone test and consistent findings of HC on abdominal ultrasonography in HC dogs. Dogs were excluded if affected by urinary tract infections and if the serum creatinine or urea values were above the reference range. At the moment of inclusion, an appropriate therapy had already been instituted less than 1 month earlier in 12 diabetic dogs. The control dogs were considered healthy based on clinical exam and clinicopathological findings. All dogs underwent urine sample collection by cystocentesis and systemic blood pressure measurement by means of either an oscillometric device (BP-88 Next, Colin Corporation, Japan) or by Doppler ultrasonic traducer (Minidop ES-100VX, Hadeco, Japan). The choice of method depended on the dog’s body weight: Doppler ultrasonography was employed in dogs < 20 kg of body weight and the oscillometric method in the other subjects. Dogs were considered hypertensive whenever systemic blood pressure was found ≥ 160 mmHg. The urine was assayed for U:P/C and U:A/C (Gentilini et al., 2005). The data between groups were compared using the Mann-Whitney U test. The reference ranges for U:P/C and U:A/C had already been established by our laboratory as 0.6 and 0.05, respectively. U:P/C and U:A/C findings were correlated to systemic blood pressure and Spearman R correlation coefficients were calculated. In all cases, p < 0.05 was considered statistically significant. RESULTS – The mean ± sd urinary albumin concentration in the three groups was 1.79 mg/dl ± 2.18; 20.02 mg/dl ± 43.25; 52.02 mg/dl ± 98.27, in healthy, diabetic and hypercortisolemic dogs, respectively. The urine albumin concentration differed significantly between healthy and diabetic dogs (p = 0.008) and between healthy and HC dogs (p = 0.011). U:A/C values ranged from 0.00 to 0.34 (mean ± sd 0.02 ± 0.07), 0.00 to 6.72 (mean ± sd 0.62 ± 1.52) and 0.00 to 5.52 (mean ± sd 1.27 ± 1.70) in the control, DM and HC groups, respectively; U:P/C values ranged from 0.1 to 0.6 (mean ± sd 0.17 ± 0.15) 0.1 to 6.6 (mean ± sd 0.93 ± 1.15) and 0.2 to 7.1 (mean ± sd 1.90 ± 2.11) in the control, DM and HC groups, respectively. In diabetic dogs, U:A/C was above the reference range in 11 out of 20 dogs (55%). Among these, 5/20 (25%) showed an increase only in the U:A/C ratio while, in 6/20 (30%), both the U:P/C and the U:A/C were abnormal. Among the latter, 4 dogs had already undergone therapy. In subjects affected with HC, U:P/C and U:A/C were both increased in 10/14 (71%) while in 2/14 (14%) only U:A/C was above the reference range. Overall, by comparing U:P/C and U:A/C in the various groups, a significant increase in protein excretion in disease-affected animals compared to healthy dogs was found. Blood pressure (BP) in diabetic subjects ranged from 88 to 203 mmHg (mean ± sd 143 ± 33 mmHg) and 7/20 (35%) dogs were found to be hypertensive. In HC dogs, BP ranged from 116 to 200 mmHg (mean ± sd 167 ± 26 mmHg) and 9/14 (64%) dogs were hypertensive. Blood pressure and proteinuria were not significantly correlated. Furthermore, in the DM group, U:P/C and U:A/C were both increased in 3 hypertensive dogs and 2 normotensive dogs while the only increase of U:A/C was observed in 2 hypertensive and 3 normotensive dogs. In the HC group, the U:P/C and the U:A/C were both increased in 6 hypertensive and 2 normotensive dogs; the U:A/C was the sole increased parameter in 1 hypertensive dog and in 1 dog with normal pressure. DISCUSSION AND CONCLUSION- The findings of this study suggest that, in dogs affected by DM and HC, an increase in U:P/C, U:A/C and systemic hypertension is frequently present. Remarkably, some dogs affected by both DM and HC showed an U:A/C but not U:P/C above the reference range. In diabetic dogs, albuminuria was observed in 25% of the subjects, suggesting the possibility that this parameter could be employed for detecting renal damage at an early phase when common semiquantiative tests and even U:P/C fall inside the reference range. In HC dogs, a higher number of subjects with overt proteinuria was found while only 14% presented an increase only in the U:A/C. This fact, associated with a greater number of hypertensive dogs having HC rather than DM, could suggest a greater influence on renal function by the mechanisms involved in hypertension secondary to hypercortisolemia. Furthermore, it is possible that, in HC dogs, the diagnosis was more delayed than in DM dogs. However, the lack of a statistically significant correlation between hypertension and increased protein excretion as well as the apparently random distribution of proteinuric subjects in normotensive and hypertensive cases, imply that other factors besides hypertension are involved in causing proteinuria. Longitudinal studies are needed to further investigate the relationship between hypertension and proteinuria.

Development of Clinical Decision Support Systems based on Mathematical Models of Physiological Systems

In the last years of research, I focused my studies on different physiological problems. Together with my supervisors, I developed/improved different mathematical models in order to create valid tools useful for a better understanding of important clinical issues. The aim of all this work is to develop tools for learning and understanding cardiac and cerebrovascular physiology as well as pathology, generating research questions and developing clinical decision support systems useful for intensive care unit patients. I. ICP-model Designed for Medical Education We developed a comprehensive cerebral blood flow and intracranial pressure model to simulate and study the complex interactions in cerebrovascular dynamics caused by multiple simultaneous alterations, including normal and abnormal functional states of auto-regulation of the brain. Individual published equations (derived from prior animal and human studies) were implemented into a comprehensive simulation program. Included in the normal physiological modelling was: intracranial pressure, cerebral blood flow, blood pressure, and carbon dioxide (CO2) partial pressure. We also added external and pathological perturbations, such as head up position and intracranial haemorrhage. The model performed clinically realistically given inputs of published traumatized patients, and cases encountered by clinicians. The pulsatile nature of the output graphics was easy for clinicians to interpret. The manoeuvres simulated include changes of basic physiological inputs (e.g. blood pressure, central venous pressure, CO2 tension, head up position, and respiratory effects on vascular pressures) as well as pathological inputs (e.g. acute intracranial bleeding, and obstruction of cerebrospinal outflow). Based on the results, we believe the model would be useful to teach complex relationships of brain haemodynamics and study clinical research questions such as the optimal head-up position, the effects of intracranial haemorrhage on cerebral haemodynamics, as well as the best CO2 concentration to reach the optimal compromise between intracranial pressure and perfusion. We believe this model would be useful for both beginners and advanced learners. It could be used by practicing clinicians to model individual patients (entering the effects of needed clinical manipulations, and then running the model to test for optimal combinations of therapeutic manoeuvres). II. A Heterogeneous Cerebrovascular Mathematical Model Cerebrovascular pathologies are extremely complex, due to the multitude of factors acting simultaneously on cerebral haemodynamics. In this work, the mathematical model of cerebral haemodynamics and intracranial pressure dynamics, described in the point I, is extended to account for heterogeneity in cerebral blood flow. The model includes the Circle of Willis, six regional districts independently regulated by autoregulation and CO2 reactivity, distal cortical anastomoses, venous circulation, the cerebrospinal fluid circulation, and the intracranial pressure-volume relationship. Results agree with data in the literature and highlight the existence of a monotonic relationship between transient hyperemic response and the autoregulation gain. During unilateral internal carotid artery stenosis, local blood flow regulation is progressively lost in the ipsilateral territory with the presence of a steal phenomenon, while the anterior communicating artery plays the major role to redistribute the available blood flow. Conversely, distal collateral circulation plays a major role during unilateral occlusion of the middle cerebral artery. In conclusion, the model is able to reproduce several different pathological conditions characterized by heterogeneity in cerebrovascular haemodynamics and can not only explain generalized results in terms of physiological mechanisms involved, but also, by individualizing parameters, may represent a valuable tool to help with difficult clinical decisions. III. Effect of Cushing Response on Systemic Arterial Pressure. During cerebral hypoxic conditions, the sympathetic system causes an increase in arterial pressure (Cushing response), creating a link between the cerebral and the systemic circulation. This work investigates the complex relationships among cerebrovascular dynamics, intracranial pressure, Cushing response, and short-term systemic regulation, during plateau waves, by means of an original mathematical model. The model incorporates the pulsating heart, the pulmonary circulation and the systemic circulation, with an accurate description of the cerebral circulation and the intracranial pressure dynamics (same model as in the first paragraph). Various regulatory mechanisms are included: cerebral autoregulation, local blood flow control by oxygen (O2) and/or CO2 changes, sympathetic and vagal regulation of cardiovascular parameters by several reflex mechanisms (chemoreceptors, lung-stretch receptors, baroreceptors). The Cushing response has been described assuming a dramatic increase in sympathetic activity to vessels during a fall in brain O2 delivery. With this assumption, the model is able to simulate the cardiovascular effects experimentally observed when intracranial pressure is artificially elevated and maintained at constant level (arterial pressure increase and bradicardia). According to the model, these effects arise from the interaction between the Cushing response and the baroreflex response (secondary to arterial pressure increase). Then, patients with severe head injury have been simulated by reducing intracranial compliance and cerebrospinal fluid reabsorption. With these changes, oscillations with plateau waves developed. In these conditions, model results indicate that the Cushing response may have both positive effects, reducing the duration of the plateau phase via an increase in cerebral perfusion pressure, and negative effects, increasing the intracranial pressure plateau level, with a risk of greater compression of the cerebral vessels. This model may be of value to assist clinicians in finding the balance between clinical benefits of the Cushing response and its shortcomings. IV. Comprehensive Cardiopulmonary Simulation Model for the Analysis of Hypercapnic Respiratory Failure We developed a new comprehensive cardiopulmonary model that takes into account the mutual interactions between the cardiovascular and the respiratory systems along with their short-term regulatory mechanisms. The model includes the heart, systemic and pulmonary circulations, lung mechanics, gas exchange and transport equations, and cardio-ventilatory control. Results show good agreement with published patient data in case of normoxic and hyperoxic hypercapnia simulations. In particular, simulations predict a moderate increase in mean systemic arterial pressure and heart rate, with almost no change in cardiac output, paralleled by a relevant increase in minute ventilation, tidal volume and respiratory rate. The model can represent a valid tool for clinical practice and medical research, providing an alternative way to experience-based clinical decisions. In conclusion, models are not only capable of summarizing current knowledge, but also identifying missing knowledge. In the former case they can serve as training aids for teaching the operation of complex systems, especially if the model can be used to demonstrate the outcome of experiments. In the latter case they generate experiments to be performed to gather the missing data.

Studio della funzione cardiaca e della massa ventricolare sinistra mediante ecocardiografia convenzionale e tissue doppler imaging in una popolazione pediatrica con malattia renale cronica in terapia conservativa e sostitutiva

Background: Cardiovascular disease (CVD) is a common cause of morbidity and mortality in childhood chronic kidney disease (CKD). Left ventricular hypertrophy (LVH) is known to be one of the earliest events in CVD development. Left ventricular diastolic function (DF) is thought to be also impaired in children with CKD. Tissue Doppler imaging (TDI) provide an accurate measure of DF and is less load dependent than conventional ECHO. Aim: To evaluate the LV mass and the DF in a population of children with CKD. Methods: 37 patients, median age: 10.4 (3.3-19.8); underlying renal disease: hypo/dysplasia (N=28), nephronophthisis (N=4), Alport (N=2), ARPKD (N=3), were analyzed. Thirty-eight percent of the patients were on stage 1-2 of CKD, 38% on stage 3, 16% on stage 4. Three patients were on dialysis. The most frequent factors related to CVD in CKD have been studied. LVH has been defined as a left ventricular mass index (LVMI) more than 35.7 g/h2,7. Results: Twenty-five patients (81%) had a LVH. LVMI and diastolic function index (E’/A’) were significantly related to the glomerular filtration rate (p<0.003 and p<0.004). Moreover the LVMI was correlated with the phosphorus and the hemoglobin level (p<0.0001 and p<0.004). LVH was present since the first stages of CKD (58% of patients were on stages 1-2). Early-diastolic myocardial velocity was reduced in 73% of our patients. We didn’t find any correlation between LVH and systemic hypertension. Conclusion: ECHO evaluation with TDI is suggested also in children prior to dialysis and with a normal blood pressure. If LVH is diagnosed, a periodic follow-up is necessary with the treatment of the modifiable risk factors (hypertension, disturbances of calcium, phosphorus and PTH, anemia ).