Assessment of cutaneous microcirculation by laser Doppler flowmetry in type 1 diabetes.

BACKGROUND: The burden of type 1 diabetes (T1D) is growing worldwide, stressing the requirement to limit the threat of its long-term complications. In this regard, the development of methods for the early diagnosis and non-invasive monitoring of vascular abnormalities is widely recognized as one of the greatest priorities of the clinical research in this field. OBJECTIVE: To assess the deterioration of physiological properties extracted from laser Doppler flowmetry (LDF) signals of microvascular perfusion and, secondly, to investigate their association with the quality of long-term metabolic control. METHODS: Microvascular perfusion was recorded at the hallux of 63 control subjects and 47 T1D patients, whose glycaemic control was characterized in terms of the annual average levels of glycosylated haemoglobin (HbA1c). Pulse Decomposition Analysis was applied to the LDF data, in order to derive non-invasive markers of vascular stiffness based on a multi-Gaussian representation of the peripheral pulse waveforms; furthermore, wavelet transform analysis was used to evaluate the microvascular myogenic vasomotion and, finally, a physiological model of the reactive hyperaemia to a local thermal stimulus at 43 was used to test the integrity of the neurovascular pathways. RESULTS: Compared to the control group, T1D patients showed a lower microvascular perfusion at baseline, and a larger vasodilatory reserve upon local heating, but no significant difference in myogenic activity. Moreover, the results of the PDA carried out on the LDF pulse waves, indicate the presence of a significant strong relation between large artery stiffness and the overall loss of glycaemic control over the past year.

Dynamic Contrast-Enhanced Ultrasound Identifies Microcirculatory Alterations in Sepsis-Induced Acute Kidney Injury.

OBJECTIVES: We developed quantitative methods to analyze microbubble kinetics based on renal contrast-enhanced ultrasound imaging combined with measurements of sublingual microcirculation on a fixed area to quantify early microvascular alterations in sepsis-induced acute kidney injury. DESIGN: Prospective controlled animal experiment study. SETTING: Hospital-affiliated animal research institution. SUBJECTS: Fifteen female pigs. INTERVENTIONS: The animals were instrumented with a renal artery flow probe after surgically exposing the kidney. Nine animals were given IV infusion of lipopolysaccharide to induce septic shock, and six were used as controls. MEASUREMENTS AND MAIN RESULTS: Contrast-enhanced ultrasound imaging was performed on the kidney before, during, and after having induced shock. Sublingual microcirculation was measured continuously using the Cytocam on the same spot. Contrast-enhanced ultrasound effectively allowed us to develop new analytical methods to measure dynamic variations in renal microvascular perfusion during shock and resuscitation. Renal microvascular hypoperfusion was quantified by decreased peak enhancement and an increased ratio of the final plateau intensity to peak enhancement. Reduced intrarenal blood flow could be estimated by measuring the microbubble transit times between the interlobar arteries and capillary vessels in the renal cortex. Sublingual microcirculation measured using the Cytocam in a fixed area showed decreased functional capillary density associated with plugged sublingual capillary vessels that persisted during and after fluid resuscitation. CONCLUSIONS: In our lipopolysaccharide model, with resuscitation targeted at blood pressure, contrast-enhanced ultrasound imaging can identify renal microvascular alterations by showing prolonged contrast enhancement in microcirculation during shock, worsened by resuscitation with fluids. Concomitant analysis of sublingual microcirculation mirrored those observed in the renal microcirculation.

Fiber enhancement and 3D orientation analysis in label-free two-photon fluorescence microscopy.

Fluorescence microscopy can be exploited for evaluating the brain's fiber architecture with unsurpassed spatial resolution in combination with different tissue preparation and staining protocols. Differently from state-of-the-art polarimetry-based neuroimaging modalities, the quantification of fiber tract orientations from fluorescence microscopy volume images entails the application of specific image processing techniques, such as Fourier or structure tensor analysis. These, however, may lead to unreliable outcomes as they do not isolate myelinated fibers from the surrounding tissue. In this work, we describe a novel image processing pipeline that enables the computation of accurate 3D fiber orientation maps from both grey and white matter regions, exploiting the selective multiscale enhancement of tubular structures of varying diameters provided by a 3D implementation of the Frangi filter. The developed software tool can efficiently generate orientation distribution function maps at arbitrary spatial scales which may support the histological validation of modern diffusion-weighted magnetic resonance imaging tractography. Despite being tested here on two-photon scanning fluorescence microscopy images, acquired from tissue samples treated with a label-free technique enhancing the autofluorescence of myelinated fibers, the presented pipeline was developed to be employed on all types of 3D fluorescence images and fiber staining.

Linear and Nonlinear Directed Connectivity Analysis of the Cardio-Respiratory System in Type 1 Diabetes.

In this study, we explored the possibility of developing non-invasive biomarkers for patients with type 1 diabetes (T1D) by quantifying the directional couplings between the cardiac, vascular, and respiratory systems, treating them as interconnected nodes in a network configuration. Towards this goal, we employed a linear directional connectivity measure, the directed transfer function (DTF), estimated by a linear multivariate autoregressive modelling of ECG, respiratory and skin perfusion signals, and a nonlinear method, the dynamical Bayesian inference (DBI) analysis of bivariate phase interactions. The physiological data were recorded concurrently for a relatively short time period (5 min) from 10 healthy control subjects and 10 T1D patients. We found that, in both control and T1D subjects, breathing had greater influence on the heart and perfusion with respect to the opposite coupling direction and that, by both employed methods of analysis, the causal influence of breathing on the heart was significantly decreased (p < 0.05) in T1D patients compared to the control group. These preliminary results, although obtained from a limited number of subjects, provide a strong indication for the usefulness of a network-based multi-modal analysis for the development of biomarkers of T1D-related complications from short-duration data, as well as their potential in the exploration of the pathophysiological mechanisms that underlie this devastating and very widespread disease.

Multimodal Characterization of Seizures in Zebrafish Larvae.

Epilepsy accounts for a significant proportion of the world's disease burden. Indeed, many research efforts are produced both to investigate the basic mechanism ruling its genesis and to find more effective therapies. In this framework, the use of zebrafish larvae, owing to their peculiar features, offers a great opportunity. Here, we employ transgenic zebrafish larvae expressing GCaMP6s in all neurons to characterize functional alterations occurring during seizures induced by pentylenetetrazole. Using a custom two-photon light-sheet microscope, we perform fast volumetric functional imaging of the entire larval brain, investigating how different brain regions contribute to seizure onset and propagation. Moreover, employing a custom behavioral tracking system, we outline the progressive alteration of larval swim kinematics, resulting from different grades of seizures. Collectively, our results show that the epileptic larval brain undergoes transitions between diverse neuronal activity regimes. Moreover, we observe that different brain regions are progressively recruited into the generation of seizures of diverse severity. We demonstrate that midbrain regions exhibit highest susceptibility to the convulsant effects and that, during periods preceding abrupt hypersynchronous paroxysmal activity, they show a consistent increase in functional connectivity. These aspects, coupled with the hub-like role that these regions exert, represent important cues in their identification as epileptogenic hubs.

Power-effective scanning with AODs for 3D optogenetic applications.

Two-photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub-micrometric-resolution light targeting into the brain. However, besides structural and functional imaging, 2P optogenetic stimulations are less routinary, especially in 3D. This is because of the adopted scanning systems, often feebly effective, slow and mechanically constricted. Faster illumination can be achieved through acousto-optic deflectors (AODs) although their applicability to large volumes excitation has been limited by large efficiency drop along the optical axis. Here, we present a new AOD-based scheme for 2P 3D scanning that improves the power delivery between different illumination planes. We applied this approach to photostimulate an optogenetic actuator in zebrafish larvae, demonstrating the method efficiency observing increased activity responses and uniform activation probabilities from neuronal clusters addressed in the volume. This novel driving scheme can open to new AOD applications in neuroscience, allowing more effective 3D interrogation in large neuronal networks.

Autofluorescence enhancement for label-free imaging of myelinated fibers in mammalian brains.

Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics. Different technologies try to address this goal; however, they are limited either by the ineffective labeling of the fibers or in the achievable resolution. The possibility of discriminating between different adjacent myelinated axons gives the opportunity of providing more information about the fiber composition and architecture within a specific area. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a tissue preparation method to perform label-free fluorescence imaging of myelinated fibers that is user friendly and easy to handle. We exploit the high axial and radial resolution of two-photon fluorescence microscopy (TPFM) optical sectioning to decipher the mixture of various fiber orientations within the sample of interest. We demonstrate its broad applicability by performing mesoscopic reconstruction at a sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in control and Reeler mouse's hippocampal sections. Our study provides a novel method for 3D label-free imaging of nerve fibers in fixed samples at high resolution, below micrometer level, that overcomes the limitation related to the myelinated axons exogenous labeling, improving the possibility of analyzing brain connectivity.

Pulse decomposition analysis in photoplethysmography imaging.

OBJECTIVE: Photoplethysmography imaging (PPGI) has gained immense attention over the last few years but only a few works have addressed morphological analysis so far. Pulse wave decomposition (PWD), i.e. the decomposition of a pulse wave by a varying number of kernels, allows for such analyses. This work investigates the applicability of PWD algorithms in the context of PPGI. APPROACH: We used simulated and experimental data to compare various PWD algorithms from the literature regarding their robustness against noise and motion artifacts while preserving morphological information as well as regarding their ability to reveal physiological changes by PPGI. MAIN RESULTS: Our experiments prove that algorithms that combine Gamma and Gaussian distributions outperform other choices. Further, algorithms with two kernels exhibit the highest robustness against noise and motion artifacts (improvement in [Formula: see text] of 14.09 %) while preserving the morphology similarly to algorithms using more kernels. Lastly, we showed that PWD can reveal physiological changes upon distal stimuli by PPGI. SIGNIFICANCE: This work proves the feasibility of pulse decomposition analysis in PPGI, particularly for algorithms with a low number of kernels, and opens up novel applications for PPGI. Not only for PPGI but for future research on PWD in general, our findings have importance as they elucidate differences between PWD algorithms and emphasize the importance of using initial values. To support such future research, we have released the algorithms and simulated data to the public.

Pulse decomposition analysis in camera-based photoplethysmography.

Imaging photoplethysmography (iPPG) is an interesting alternative to laser speckle contrast imaging for the analysis of spatio-temporal patterns in the cutaneous microcirculation. Recent years have witnessed the development of sophisticated techniques for the non-invasive extraction of vascular-related features. These techniques, referred to as pulse decomposition algorithms (PDA), most often involve the analysis of photoplethysmographic waves. This study validated the use of a multi-Gaussian (PDA) for the automatic mapping of iPPG pulse waveforms acquired with a standard camera. We show that iPPG-based PDA can reveal differences in skin perfusion in response to cold stimuli. The study thus proves the potential for morphological analyses of the iPPG pulse waveform.

Validation of a low-cost EEG device in detecting neural correlates of social conformity.

The study of conformity from a neurobiological point of view has interested many authors: among them, Shestakova and colleagues (2013) have showed how conformity can be assessed through the analysis of event related potentials (ERPs). More specifically, the P300 component of the ERP was shown to be sensitive to the behavioral adjustment that an individual makes when not agreeing with the majority of a group. Starting from these contributions, in the present study, the famous experiment of Solomon Asch [1] was replicated online. The experiment was conducted on a sample of university students, using an innovative and low-cost tool capable of recording the brain signal (a wireless headset equipped with fourteen electrodes, called Emotiv EPOC). The present research aims to demonstrate how cheap and little sensitive tools enable the detection of ERP components that characterize social conformity in an ecological context.

Glycemic Control Maintained over Time and Joint Stiffness in Young Type 1 Patients: What Is the Mathematical Relationship?

BACKGROUND: It is widely known that diabetes can induce stiffness and adversely affect joint mobility even in young patients with type 1 diabetes mellitus (T1D). The aim of this study was to identify a mathematical model of diabetes mellitus long-term effects on young T1D patients. METHODS: Ankle joint mobility (AJM) was evaluated using an inclinometer in 48 patients and 146 healthy, sex- BMI-, and age-matched controls. Assuming time invariance and linear superposition of the effects of hyperglycemia, the influence of T1D on AJM was formalized as an impulse response putting into relationship past supernormal HbA1c concentrations with the ankle total range of motion. The proposed model was identified by means of a nonlinear evolutionary optimization algorithm. RESULTS: AJM was significantly reduced in young T1D patients (P < .001). AJM in both plantar and dorsiflexion was significantly lower in subjects with diabetes than in controls (P < .001). The identified impulse response indicates that impaired metabolic control requires 3 months to bring out its maximum effect on the reduction of AJM, while the following long-lasting decay phase with the expected AJM recovery times, normally depends on the slow turnover of collagen. HbA1c concentration levels above 7.2% are sufficient to produce a reduction of ankle ROM. CONCLUSIONS: In young patients with T1D the lack of glycemic control over time affects AJM. HbA1c levels can serve as a relevant prognostic factor for assessing the progression of LJM in subjects with diabetes.

Detecting Vascular Age Using the Analysis of Peripheral Pulse.

Vascular ageing is known to be accompanied by arterial stiffening and vascular endothelial dysfunction, and represents an independent factor contributing to the development of cardiovascular disease. The microvascular pulse is affected by the biomechanical alterations of the circulatory system, and has been the focus of studies aiming at the development of non-invasive methods able to extract physiologically relevant features. OBJECTIVE: proposing an approach for the assessment of vascular ageing based on a support vector machine (SVM) learning from features of the pulse contour. METHODS: the supervised classifier was trained and validated over 20935 models of pulse wave, obtained with a multi-Gaussian decomposition algorithm, applied to laser Doppler flowmetry signals of 54 healthy, non-smoker subjects. RESULTS: the multi-Gaussian model showed a mean R2 of 0.98 and an average normalized root mean square error of 0.90, demonstrating the ability to reconstruct the pulse shape. Over 30 training and validation experiments, the SVM showed a mean Pearson's r of 0.808 between the rate of waves classified as old and the age of the subjects, along with an average area under the ROC curve of 0.953. CONCLUSION: the SVM showed the capability to discriminate differently aged individuals. SIGNIFICANCE: the proposed method might detect the ageing-related modifications of the vascular tree; furthermore, since diabetes promotes vascular alterations comparable to ageing, this approach may be also suitable for the screening of diabetic angiopathy.

Particle tracking for the assessment of microcirculatory perfusion.

In recent years the development of portable microscopes, which enable the noninvasive bedside evaluation of the sublingual microcirculation in critically ill patients, has expanded the clinical research on this level of the cardiovascular system. Several semi-quantitative scores have been defined in order to provide researchers with a standardized framework for the offline assessment of the microcirculation status. Among those, space-time diagrams (STDs) constitute an established method for obtaining an estimate of the red blood cells (RBCs) flow velocity in capillaries. However, STDs have the drawback of being time-consuming, inherently subjective, and difficult to manage when the flow is not regular. OBJECTIVE: In this work we propose an automated method for calculating erythrocyte flow speed, aiming to provide a fast and objective tool for the evaluation of peripheral blood perfusion. APPROACH: The proposed method exploits an image segmentation module for estimating the positions of candidate flowing cells. A multi-object tracking algorithm based on Kalman filters analyzes and matches the positions corresponding to specific erythrocytes within consecutive frames. Thus, the output of the filter enables to estimate the displacement of each cell, yielding their instantaneous speed. MAIN RESULTS: The method has been validated against the results obtained by the manual analysis of STDs, proving a good agreement for speeds up to 300 µm s-1. At higher speeds, RBC tracking becomes unstable due to the currently limited video acquisition rate (25 Hz) of state-of-the-art devices, that makes the matching between objects appearing in consecutive frames very challenging.

Spatial heterogeneity in the time and frequency properties of skin perfusion.

Pathological alterations of the microcirculatory system can be identified by measuring the temporal and spectral properties of laser Doppler flowmetry (LDF) signals acquired on the skin, and their changes following physiological stimulation. A wide range of stimulation protocols and measurement locations is observed in literature. Researchers often use non-invasive stimulation techniques, such as post-occlusive hyperaemia, cold tests, and local heating. As concerns the stimulation/recording sites, the forearm, fingers, and toes are typically selected to conduct microcirculation studies. However, recent clinical investigations showed that different anatomical sites present dissimilar blood flow patterns. Therefore, studies involving the comparison of LDF data, obtained from various anatomical locations, and thus subjected to the intrinsic heterogeneity of the microcirculation, may be methodologically inaccurate. At the moment, no consensus has been reached upon the optimal measurement location, the stimulation pattern, and the physiological parameters of interest. The aim of this study is to quantitatively characterize the heterogeneity of the peripheral perfusion at different anatomical locations: the index finger, the forearm, and the hallux. The skin microvascular system exhibits a complex vasodilatory response in the temporal domain, upon local heating. This physiological reactive hyperaemia comprises two effects: a fast transient response, correlated to neural activation, named axon reflex, followed by a slower hyperaemic plateau, mediated by the release of nitric oxide. In this work, we compare the vasodilatory reaction to heating at the different sites, based on a parametric representation of the perfusion signal. Moreover, skin blood flow is characterized by several components fluctuating at different time scales. Time-frequency decomposition of LDF signals allows to quantitatively evaluate the relative contribution of known physiological mechanisms to the regulation of the peripheral circulation. For this reason, we analyze the wavelet transform coefficients of LDF signals at baseline, to assess potential spatial heterogeneities of the perfusion power spectra among the aforementioned anatomical locations.