Real-time optical manipulation of cardiac conduction in intact hearts.

KEY POINTS: Although optogenetics has clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies lack the capability to react acutely to ongoing cardiac wave dynamics. Here, we developed an all-optical platform to monitor and control electrical activity in real-time. The methodology was applied to restore normal electrical activity after atrioventricular block and to manipulate the intraventricular propagation of the electrical wavefront. The closed-loop approach was also applied to simulate a re-entrant circuit across the ventricle. The development of this innovative optical methodology provides the first proof-of-concept that a real-time all-optical stimulation can control cardiac rhythm in normal and abnormal conditions. ABSTRACT: Optogenetics has provided new insights in cardiovascular research, leading to new methods for cardiac pacing, resynchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies do not take into account cardiac wave dynamics in real time. Here, we developed an all-optical platform complemented by integrated, newly developed software to monitor and control electrical activity in intact mouse hearts. The system combined a wide-field mesoscope with a digital projector for optogenetic activation. Cardiac functionality could be manipulated either in free-run mode with submillisecond temporal resolution or in a closed-loop fashion: a tailored hardware and software platform allowed real-time intervention capable of reacting within 2 ms. The methodology was applied to restore normal electrical activity after atrioventricular block, by triggering the ventricle in response to optically mapped atrial activity with appropriate timing. Real-time intraventricular manipulation of the propagating electrical wavefront was also demonstrated, opening the prospect for real-time resynchronization therapy and cardiac defibrillation. Furthermore, the closed-loop approach was applied to simulate a re-entrant circuit across the ventricle demonstrating the capability of our system to manipulate heart conduction with high versatility even in arrhythmogenic conditions. The development of this innovative optical methodology provides the first proof-of-concept that a real-time optically based stimulation can control cardiac rhythm in normal and abnormal conditions, promising a new approach for the investigation of the (patho)physiology of the heart.

Effects of ultrasound and selenium on human neurons in vitro.

As the effects of ultrasound on human brain functions might bear therapeutic potential, in this study, we examined the effects of diagnostic, i.e. non-thermal, ultrasound, on morphology, networking, and metabolic activity of SH- SY5Y human neurons in culture, as well as on the expression of GAP-43, Hsp90 and VEGF proteins, with and without selenium in the culture medium. The rationale for studying selenium lays in the observation that selenium improves functional neurologic outcome in traumatic brain injury and, therefore, analysis of the interactions between ultrasound and selenium may be of clinical interest. In the presence of selenium, ultrasound increased the overall number and length of elongations arising from the neuron bodies, thus reflecting an increase in the complexity of neuronal networks and circuits. The expression of GAP-43, Hsp90 and VEGF and metabolic activity of SH-SY5Y neurons, studied as markers of cell damage, were not affected by ultrasound or selenium. This study suggests that ultrasound may modulate neuronal networking in vitro without inducing cellular or molecular damage and highlights the potential role of selenium in the ultrasound-elicited cellular responses.

Resonance artefacts in modern pressure monitoring systems.

Resonance in pressure monitoring catheters is a well-known problem which was studied several years ago. Current piezoelectric devices have mechanical properties providing a resonance frequency and damping factor that theoretically assure resonance-free data. However, in particular cases, the coupling between the device, the catheter, and the vascular compliance of the patient could introduce artefacts in clinical settings leading to wrong pressure waveforms and values displayed in the monitor. In this research work we study a laboratory model of a clinical setting to evaluate in which cases the compound (catheter and device) could cause resonances in an unacceptable range. The classical pop-test is expanded for analysing the effect of the catheter. Results indicate that the presence of different catheters may alter significantly the acquired signal, up to an unacceptable level. Particular care should be used in the selection of the appropriate catheter. In particular, smaller diameters introduce higher damping coefficient that could help in avoiding undesired oscillations.

Bioaccumulation of resveratrol metabolites in myocardial tissue is dose-time dependent and related to cardiac hemodynamics in diabetic rats.

BACKGROUND AND AIMS: Trans-resveratrol (RSV) is a natural compound occurring in different foods and plants, which in vivo is rapidly conjugated with glucuronic acid and sulfate. Despite its demonstrated cardioprotective activity, the bioaccumulation of RSV or its metabolites in cardiac tissue is still unknown. METHODS AND RESULTS: Diabetic rats were randomized to 1, 3 or 6 weeks of RSV treatment at two different doses (1 or 5 mg/kg/day). A dose and time-dependent accumulation was observed, with no detectable levels of RSV metabolites found in heart tissues after 1 week and significant concentrations of RSV-3-sulfate and RSV-3-glucuronide after 6 weeks of treatment (0.05 nmol/g of tissue and 0.01 nmol/g of tissue, respectively). Tissue accumulation of RSV metabolites was accompanied by an improvement of cardiac function in long-term diabetes, when myocardial morpho-functional damage is more evident, with an almost complete recovery of all hemodynamic parameters, at the highest RSV dose. CONCLUSION: Even if a higher concentration of RSV in tissues cannot be ruled out after constant oral administration, an accumulation coherent with what is usually evaluated in cell based mechanistic studies is largely unattainable and the RSV unconjugated form would not be present in this paradigm. The current investigation provides data on myocardial tissue concentrations of RSV metabolites, after short/medium term RSV treatment. This knowledge constitutes a basic requirement for future studies aimed at reliably defining the molecular pathways underlying RSV-mediated cardioprotective effects and opens up new perspectives for research focused on testing phenolic compounds as adjuvants in degenerative heart diseases.

Effect of local blood flow in thermal regulation in diabetic patient.

The presence of dysautonomia in diabetic neuropathy is correlated with impairment of vasomotor activity that drives blood microcirculation. Microcirculation, in turn, plays an important role in thermoregulation. In this work, we investigate the changes between two different physiological conditions of diabetic patients, induced by FREMS application, in the control of skin temperature, using a minimally invasive experiment. Skin is warmed up to a fixed temperature (44 °C) for a few minutes, then the heat source is turned off, letting the skin recover its physiological temperature. Both temperature and local blood flow, the latter measured with laser Doppler, are monitored during the experiment. A simple model of the cooling phase is used to evaluate the time constants involved in the process. Results indicate that significant differences exist in the model parameters between the two conditions.

Design of a wearable device for physiological parameter monitoring in a COVID setting.

The study focuses on the realization of an accurate device for the detection of different physiological parameters. It has been realized a simple portable system containing the necessary electronics and ensuring the monitoring of the blood oxygenation, the body temperature, the air quality, the respiratory rate and the ECG. The main processing unit consists in a Raspberry Pi Zero W connected to the Healthy Pi4. The latter provides the interface for the clinical pulse-oxymeter while the measures of temperature and quality air are provided using the I2C protocol. The Bluetooth module is finally used to provide the ECG and blood rate data. The collected data are elaborated using Matlab and Python. To evaluate the accuracy of the realized device some experimental tests have been conducted on different subjects, comparing subjects working in Covid area with others resting at home. In both cases the monitoring time was 4 hours. Results have shown good performances of the system, detecting accurately the differences of the parameters values between the two situations. The usability of the device was assessed by administering a questionnaire to the healthcare personnel involved in the experimentation. The outcome shows a good usability of the system as well as an acceptable dressing time.

Severe HDL deficiency due to novel defects in the ABCA1 transporter.

OBJECTIVES: The objective was the identification and functional characterization of mutations in the ABCA1 gene in four patients with severe HDL deficiency. SUBJECTS: Patients were referred to the clinic because of almost complete HDL deficiency. METHODS: The ABCA1 gene was sequenced directly. The analysis of the ABCA1 protein, ABCA1 mRNA and ABCA1-mediated cholesterol efflux was performed in cultured fibroblasts. Intracellular localization of ABCA1 mutants was investigated in transfected HEK293 cells. RESULTS: Two patients were homozygous for mutations in the coding region of the ABCA1 gene, resulting in an amino acid substitution (p.A1046D) and a truncated protein (p.I74YFsX76). The third patient was homozygous for a splice site mutation in intron 35 (c.4773 + 1g>a), resulting in an in-frame deletion of 25 amino acids (del p.D1567_K1591) in ABCA1. These patients had clinical manifestations of accumulation of cholesterol in the reticulo-endothelial system. The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). ABCA1-mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. A reduced ABCA1 protein content was observed in these cells, suggesting an increased intracellular degradation. The mutant p.W1699C was largely retained in the endoplasmic reticulum, when expressed in HEK293 cells. CONCLUSIONS: The homozygotes for mutations which abolish ABCA1 function showed overt signs of involvement of the reticulo-endothelial system. This was not the case in the compound heterozygote for missense mutations, suggesting that this patient retains some residual ABCA1 function that reduces cholesterol accumulation in the reticulo-endothelial system.

A comparison between internal and surface temperature measurement techniques during phacoemulsification cataract surgery: thermocamera versus thermocouple.

PURPOSE: Corneal and scleral burns, one of the main complications that can occur during a cataract operation, are produced by overheating due to the use of the phacoemulsifier. The temperature of the anterior chamber of the eye can be measured both invasively using thermocouples and non-invasively, but only superficially, using a thermocamera. METHODS: To compare the measures obtained from both techniques an in vitro experimental analysis was conducted on pigs' eyes. During a simulated phacoemulsification cataract operation both the surface temperature with a thermocamera and the temperature inside the anterior chamber with a thermocouple were recorded. For each procedure, the maximum temperature values measured by each technique were compared. RESULTS: The results of this research show that the difference between the maximum values measured with the two techniques is on average 0.5 degrees C. CONCLUSIONS: It is possible to employ a thermocamera technique instead of a thermocouple technique to provide an indication of the temperature inside the anterior chamber.

Shape analysis of microcalcifications using Radon transform.

Microcalcifications are one of the early signs of breast cancer, and they are of great importance for an early diagnosis. Moreover, the spatial distribution and the shape of the microcalcifications have a significant impact in medical practice to evaluate the probability of malignancy of the tumor. In this work a method, performing computer-aided classification of the shape of calcifications accordingly to the classification scheme proposed by Le Gal, is presented. In the first stage, in order to remove mammographic background, the image is preprocessed with a matched filter, designed by modeling the microcalcifications as Gaussian spots and the image as a Fractional Brownian Motion. Afterwards, morphology of spots has been evaluated using two different sets of parameters. The first set utilizes the moments of inertia of the second and third order to compute a set of features, which are invariant to rotations and translations of the image. The second set of parameters is derived from the evaluation of the Radon transform, as computed along eight axes. The results of the Radon transform are used to associate to each lesion a set of features, which are invariant to rotation and scaling of the image. In the final stage, a multilayer neural network has been used to assign each microcalcification to the classes introduced by Le Gal. The topology of the neural network is the same for both sets of descriptors, in order to allow comparison of the discriminative power of the two feature sets. Experimental results obtained with the proposed method from a set of digitized mammograms are reported and discussed.

[Anabolic effects and inhibition of interleukin 6 production induced by neridronate on human osteoblasts]. / Effetti anabolici e di inibizione della produzione di interleuchina 6 indotti dal neridronato in colture di osteoblasti umani.

UNLABELLED: Bisphosphonates (BPs) are pharmacological compounds widely used in the treatment of a variety of bone-related diseases, particularly where the bone-turnover is skewed in favour of osteolysis. The mechanisms by which BPs reduce bone-resorption directly acting on osteoclasts (OCs) are now largely clarified even at molecular level. The researches concerning the BPs effects on osteoblasts (OBs) have instead shown variable results. OBJECTIVES: We have investigated the efficacy of neridronate (NER), an amino-BP, as anabolic agent on human OBs. Moreover, we have tried to verify if NER is able to negatively modulate the production of IL-6 on OBs stimulated or not by the pro-inflammatory cytokine IL-1beta. METHODS: We have tested if different concentrations of NER (from 10-11 M to 10-3 M), added to primary human OB cultures, could affect the cells number, the endogenous cellular alkaline phosphatase (ALP) activity, the collagen I (COL-I) synthesis, the formation of mineralized nodules and the IL-6 production. Our experimental approach was performed testing a wide range of NER concentrations because, under physiological conditions, OBs seems to be exposed to variable and transient levels of the drug. RESULTS: Our results show that NER doesn't negatively affect in vitro the viability, proliferation and cellular activity of human OBs, even after 20 days of exposure to concentrations < or =10-5 M (therapeutic dose). In addition, NER seems to enhance the differentiation of cultured OBs in mature bone-forming cells. A maximum increase of COL-I synthesis (+25% after 4 days; p < 0.05), ALP activity (+50% after 10 days; p < 0.01) and mineralized nodules (+48% after 20 days; p < 0.05) was observed in cultures treated with NER 10-8 M. A maximal reduction of IL-6 secretion (-24% on IL-1beta stimulated cultures and -29% on unstimulated cultures) was observed for NER 10-9 M. CONCLUSIONS: These results encourage the use of neridronate in therapy of demineralizing metabolic bone disorders.

Monitoring the microcirculation at the bedside using hand-held imaging microscopes: Automatic tracking of erythrocytes.

In recent years, the development of portable microscopy devices that enable the noninvasive bedside evaluation of the mucosal microcirculation in critically ill patients has expanded the research on this level of the cardiovascular system. Several semi-quantitative scores have been defined to assess the peripheral perfusion, based on videos of the microcirculation. However, they are cumbersome and time-consuming processes, highlighting the need for the development of automated software techniques, to reduce physicians workload and ensure prompt and objective patient evaluation. We developed a rapid, automatic method for the quantitative assessment of erythrocyte velocities in Incident Dark Field (IDF) illumination videos of capillary flow. The technique, based on Kalman filter object tracking, was tested in 35 IDF videos against space-time diagrams, showing high correlation (r = 0.96) and agreement (bias = 3.3 µm/s) with space-time diagrams (STD), below 200 µm/s.

Shape analysis of the microcirculatory flow wave.

The cardiovascular system and its alterations are a crucial aspect of physiology and medicine. Non-invasive assessment of the functional properties of circulation is of considerable interest to clinicians and physiologists. In this work we investigate the possibility of detecting alterations of the flow waveform in microcirculation, using non-invasive measurements based on a laser Doppler flowmeter. As a test case, we focus on the effect of ageing. Skin is warmed up to a fixed temperature (44 °C) during measurement, to increase blood flow. The shape of the perfusion waveform during each heart beat after the flow was stabilized was used to estimate dynamic parameters of the microcirculatory system. Both the wave rise time, defined as the delay between the diastolic minimum and the following systolic maximum, and the oscillation fraction, defined as the normalized difference between the maximum and minimum flow, present significant variation with age.

Morphological analysis of neurons: Automatic identification of elongations.

Our study is focused on the development of a new method for the automatic analysis of cell images. We focused on neurons (cells line SH-SY5Y) treated/untreated with ultrasound and stained with Haematoxylin-Eosin. The aim of the algorithm is the automatic detection of the cell body as well as the determination of the number and the length of neuron elongations. Starting point of the algorithm was the convolution of an image with a bank of rotating Gaussian kernels and the construction of a module map. Then several strategies were implemented to detect cell bodies and to detect and extract data about cell elongations. We have also realized a graphical user interface allowing the loading, saving and processing of images. Results show that this method is able to properly and efficiently detect cell contours and elongations. The automated evaluation is in strong agreement with manual evaluation performed by an expert operator, with an average error of 11% with most parameter combinations. This tool constitutes an important support in biological research activities, where operators need to analyze a large number of images to investigate about cell morphology before and after a treatment.

Analysis of the microcirculatory pulse wave: age-related alterations.

Morphological analysis of the pulse wave of central blood pressure signal is commonly used for the study of cardiac and vascular properties, but very few attempts were performed for analyzing the peripheral pulse wave of blood flow. In this work, we analyzed this waveform using classical methods, based on the application of FFT, followed by principal components analysis, for assessing the properties of the blood flow. As a sample problem, we evaluated the capability of the proposed method of assessing the alterations correlated with the aging of the vascular system. Results show a good discrimination between the different age groups, confirming the validity of the approach.

Trends in the incidence of hip fracture in Siena, Italy, from 1980 to 1991.

There is general agreement that the crude incidence of proximal femur (hip) fractures is rising in conjunction with the ageing of the underlying population. To explore possible changes in hip fracture incidence over time we analysed all femoral fractures occurring in the county of Siena from 1980 to 1991. Data were collected from hospital record charts of the Department of Orthopaedics, recording all hip fractures occurring during the 12-year period. In this period, the mean resident population in Siena was 238,369 inhabitants (aged 0 to 90+ years) and the crude number of all femoral fractures was 2,238. However, in calculating incidence rates, only hip fractures occurring in the population aged over 50 years were considered. In this population, the number of hip fractures was 1,825 with a male/female ratio of 1:2.8. A time-series data analysis (temporal trend) of the incidence of hip fracture during the 12-year period revealed a linear and significant (p < 0.001) trend to increase, but only in males, with an annual increasing rate of 3.62 per 100,000 person-years. In the female population, the temporal analysis did not show any significant trend during the observation period.

Social stress, myocardial damage and arrhythmias in rats with cardiac hypertrophy.

In rat models of cardiac hypertrophy (moderate aortic coarctation: ACm, n=18; severe aortic coarctation: ACs, n=27; aging: OLD, n=25; spontaneous chronic hypertension: SHR, n=18) and properly matched control animals (C(ACm), n=17; C(ACs), n=19; C(OLD), n=24; C(SHR), n=22), we investigated the relative contribution of intense autonomic activity and cardiac structural damage to ventricular arrhythmogenesis. We used an "in vivo" to tissue level approach, by correlating in the same animal: (i) social stress-induced ventricular arrhythmias, telemetrically recorded, and (ii) left ventricular weights (LVW) and amount and geometrical properties of myocardial fibrosis (MF). Arterial blood pressure was significantly higher in ACm (+11%), ACs (+28%) and SHR (+34%) than in controls. LVW were approximately 20% greater in ACm, ACs and OLD and 50% greater in SHR. MF was about twice as great and characterized by more frequent occurrence of microscopic scarring in ACm and ACs, and eight times greater and associated with both a higher number and a larger size of fibrotic foci in OLD and SHR compared to controls. Social stress increased ventricular arrhythmia vulnerability in all models of cardiac hypertrophy, as well as in controls. The arrhythmogenic action of stress was facilitated in ACs, OLD and SHR. A correlation between structural cardiac remodeling and ventricular arrhythmias was found only in SHR and OLD, which exhibited the greatest increase in LVW and/or MF. Social stress proved to be a valuable tool for analyzing the combined effects of autonomic stimulation and altered myocardial substrate on the genesis of potentially life-threatening arrhythmias in social animals.

A new method of repair for quadriceps tendon ruptures. A case report.

Rupture of the quadriceps tendon is an uncommon injury observed predominantly in subjects over 40 years old. Multiple surgical techniques have been employed to repair fresh and neglected ruptures; methods that make use of allograft and augmentation with tissues harvested from around the knee have been reported. We describe a case of surgical repair of a tendon-bone junction rupture in a 64-year-old patient by use of suture anchors to attach the tendon to bone and improve fixation of the soft tissue elements. Clinical diagnosis of rupture was confirmed radiographically and echographically. Surgical repair was performed within 24 hours of injury. Active movement of the knee started after 3 weeks and the patient was permitted to walk without weight-bearing with a knee cage. Three weeks later, he was permitted to walk with full weight-bearing unassisted by crutches; the knee cage was removed 6 weeks after surgery. At his most recent follow-up 24 months postoperative, quadriceps strength was equal to that of the controlateral knee and the patient has returned to sports and daily activities. The surgical method presented here provides a suture of the tendon ends without putting excess stress on the suture line during the period of early knee mobilization. Advantages over other techniques include reduced operative time, easy access to the implantation site, and better resistance of the suture material: the patient is thus able to initiate physical therapy earlier and more aggressively.

Compression hip screw nail-plate system for intertrochanteric fractures.

BACKGROUND: The worldwide increased incidence in fractures of the proximal end of the femur is increasing with the parallel increase in the average age of the population. The advanced age and poor physical condition of these patients render surgery necessary. METHODS: In 1999, at the Orthopaedic Clinic of the University of Siena, 56 patients were treated with the compression hip nail-plate system, a new synthesis device for the treatment of peritrochanteric fractures. The system consists of a new dynamic cephalic screw that could be combined with a plate or a femoral nail. The dynamic cephalic screw of the BCM system has a limited sliding method, impeding excursions greater than 10 mm and preventing excessive collapse of the fracture fragments that can evolve into a shortening of the limb. The canulated nail has a diameter of only 9 mm, which permits insertion into the shaft canal without reaming. RESULTS: Four patients with a follow-up of less than 3 months, 2 patients lost to control, and 1 patient who died were excluded from the study. All 49 remaining patients healed clinically and radiographically within 3 months. CONCLUSIONS: The possibility of the double combination of the screw with a plate or with an intramedullary nail permits the surgeon to have a double solution with a single instrument at hand, and offers the opportunity of changing the choice of synthesis method to implant even during surgery.

The diagnosis of acute complete tears of the anterior cruciate ligament. Comparison of MRI, arthrometry and clinical examination.

We reviewed 38 patients with arthroscopically-proven complete ACL tears operated on less than three weeks after injury. Their average age was 26 years (16 to 43), with 27 males and 11 females. All patients had had MRI preoperatively. The same examiner performed the Lachman, anterior-drawer, and pivot-shift tests without anaesthesia, recording the differences between the injured and the normal knees. The patients then had KT-1000 arthrometry by the same examiner at 15 lb (6.8 kg) and 20 lb (9 kg), with active displacement, and with maximum manual displacement. All scored positive for differences greater than 3 mm. The results of physical examination, KT-1000 tests and MRI were analysed using McNemar's test for matched data with continuity correction and a 95% confidence interval for each test. The sensitivity of the KT-1000 manual maximum test was 97% for 3 mm and 100% for 2 mm; this was the most useful arthrometric result. The Lachman test gave 95% sensitivity, providing the best simple clinical assessment. MRI was 97% sensitive for the detection of all ACL injuries, but this fell to 82% with respect to complete rupture. We found no significant differences between the results of the Lachman test and the KT-1000 manual maximum test, but these were significantly better than all other tests. In an era of cost-containment, we have shown that inexpensive tests in the clinic can allow treatment to proceed rapidly and in the most economical manner without the routine use of MRI.

Titanium anchors for the repair of rotator cuff tears: preliminary report of a surgical technique.

We used titanium anchors for the surgical repair of rotator cuff tears in 34 selected patients, all of whom were < 60 years of age, had good bone quality, and had no known metabolic bone diseases. Nine tears were repaired within 6 months, 15 within 6-12 months, and 10 later than 12 months after injury. Tear size was graded as small (10 patients), medium (15 patients), and large (nine patients) during open operation. After 6-24 months of follow-up, 30 patients reported satisfactory pain relief, function, active forward flexion, and muscle strength [18 excellent and 12 good results based on the University of California at Los Angeles rating system (UCLA scores)]; there were no implant failures (p < 0.001). Two patients had unsatisfactory function but good relief of pain, whereas two patients were dissatisfied with their overall result (four poor results based on UCLA scores). Although trans-bone suturing is presently the most common and successful surgical technique for rotator cuff tears, we found that use of titanium anchors shortens operative time and has results comparable with the traditional technique. Titanium anchors should not be used when bone quality is poor or good patient compliance is doubtful. They are also contraindicated, as our four poor results indicate, when the tear is old (> 6 months) and large (diameter > 5 cm with significant tissue degeneration).