Can Pulse Rate Variability be Used to Monitor Compliance with a Breath Pacer?

Slow paced breathing has been demonstrated to provide significant health benefits for a person's health, and, during breathing sessions, it is desirable to monitor that a person is actually compliant with the breath pacer. We explore the potential use of pulse rate variability to monitor compliance with a breath pacer during meditation sessions. The study involved 6 human subjects each participating in 2-3 trials, where they are asked to follow or not to follow the breath pacer, where we collected data on how the magnitude of pulse rate variability changed. Two methods, logistic regression and a running standard deviation technique, were developed to detect non-compliance with the breath pacer based on pulse rate variability metrics. Results indicate that using pulse rate variability alone may not reliably detect non-compliance with the breath pacer. Both models exhibited limitations in terms of false positives and false negatives, with accuracy ranging from 67 to 65%. Existing methods involving visual, audio, and motion signals currently perform better for monitoring compliance with the breath pacer.

Dynamic Phase Extraction: Applications in Pulse Rate Variability.

Pulse rate variability is a physiological parameter that has been extensively studied and correlated with many physical ailments. However, the phase relationship between inter-beat interval, IBI, and breathing has very rarely been studied. Develop a technique by which the phase relationship between IBI and breathing can be accurately and efficiently extracted from photoplethysmography (PPG) data. A program based on Lock-in Amplifier technology was written in Python to implement a novel technique, Dynamic Phase Extraction. It was tested using a breath pacer and a PPG sensor on 6 subjects who followed a breath pacer at varied breathing rates. The data were then analyzed using both traditional methods and the novel technique (Dynamic Phase Extraction) utilizing a breath pacer. Pulse data was extracted using a PPG sensor. Dynamic Phase Extraction (DPE) gave the magnitudes of the variation in IBI associated with breathing [Formula: see text] measured with photoplethysmography during paced breathing (with premature ventricular contractions, abnormal arrhythmias, and other artifacts edited out). [Formula: see text] correlated well with two standard measures of pulse rate variability: the Standard Deviation of the inter-beat interval (SDNN) (ρ = 0.911) and with the integrated value of the Power Spectral Density between 0.04 and 0.15 Hz (Low Frequency Power or LF Power) (ρ = 0.885). These correlations were comparable to the correlation between the SDNN and the LF Power (ρ = 0.877). In addition to the magnitude [Formula: see text], Dynamic Phase Extraction also gave the phase between the breath pacer and the changes in the inter-beat interval (IBI) due to respiratory sinus arrythmia (RSA), and correlated well with the phase extracted using a Fourier transform (ρ = 0.857). Dynamic Phase Extraction can extract both the phase between the breath pacer and the changes in IBI due to the respiratory sinus arrhythmia component of pulse rate variability ([Formula: see text], but is limited by needing a breath pacer.

Action potential propagation recorded from single axonal arbors using multielectrode arrays.

We report the presence of co-occurring extracellular action potentials (eAPs) from cultured mouse hippocampal neurons among groups of planar electrodes on multielectrode arrays (MEAs). The invariant sequences of eAPs among coactive electrode groups, repeated co-occurrences, and short interelectrode latencies are consistent with action potential propagation in unmyelinated axons. Repeated eAP codetection by multiple electrodes was widespread in all our data records. Codetection of eAPs confirms they result from the same neuron and allows these eAPs to be isolated from all other spikes independently of spike sorting algorithms. We averaged co-occurring events and revealed additional electrodes with eAPs that would otherwise be below detection threshold. We used these eAP cohorts to explore the temperature sensitivity of action potential propagation and the relationship between voltage-gated sodium channel density and propagation velocity. The sequence of eAPs among coactive electrodes "fingerprints" neurons giving rise to these events and identifies them within neuronal ensembles. We used this property and the noninvasive nature of extracellular recording to monitor changes in excitability at multiple points in single axonal arbors simultaneously over several hours, demonstrating independence of axonal segments. Over several weeks, we recorded changes in interelectrode propagation latencies and ongoing changes in excitability in different regions of single axonal arbors. Our work illustrates how repeated eAP co-occurrences can be used to extract physiological data from single axons with low-density MEAs. However, repeated eAP co-occurrences lead to oversampling spikes from single neurons and thus can confound traditional spike-train analysis. NEW & NOTEWORTHY We studied action potential propagation in single axons using low-density multielectrode arrays. We unambiguously identified the neuronal sources of propagating action potentials and recorded extracellular action potentials from several positions within single axonal arbors. We found a surprisingly high density of axonal voltage-gated sodium channels responsible for a high propagation safety factor. Our experiments also demonstrate that excitability in different segments of single axons is regulated independently on timescales from hours to weeks.

Protosequences in human cortical organoids model intrinsic states in the developing cortex.

Neuronal firing sequences are thought to be the basic building blocks of neural coding and information broadcasting within the brain. However, when sequences emerge during neurodevelopment remains unknown. We demonstrate that structured firing sequences are present in spontaneous activity of human brain organoids and ex vivo neonatal brain slices from the murine somatosensory cortex. We observed a balance between temporally rigid and flexible firing patterns that are emergent phenomena in human brain organoids and early postnatal murine somatosensory cortex, but not in primary dissociated cortical cultures. Our findings suggest that temporal sequences do not arise in an experience-dependent manner, but are rather constrained by an innate preconfigured architecture established during neurogenesis. These findings highlight the potential for brain organoids to further explore how exogenous inputs can be used to refine neuronal circuits and enable new studies into the genetic mechanisms that govern assembly of functional circuitry during early human brain development.

Preliminary study: quantification of chronic pain from physiological data.

Introduction: It is unknown if physiological changes associated with chronic pain could be measured with inexpensive physiological sensors. Recently, acute pain and laboratory-induced pain have been quantified with physiological sensors. Objectives: To investigate the extent to which chronic pain can be quantified with physiological sensors. Methods: Data were collected from chronic pain sufferers who subjectively rated their pain on a 0 to 10 visual analogue scale, using our recently developed pain meter. Physiological variables, including pulse, temperature, and motion signals, were measured at head, neck, wrist, and finger with multiple sensors. To quantify pain, features were first extracted from 10-second windows. Linear models with recursive feature elimination were fit for each subject. A random forest regression model was used for pain score prediction for the population-level model. Results: Predictive performance was assessed using leave-one-recording-out cross-validation and nonparametric permutation testing. For individual-level models, 5 of 12 subjects yielded intraclass correlation coefficients between actual and predicted pain scores of 0.46 to 0.75. For the population-level model, the random forest method yielded an intraclass correlation coefficient of 0.58. Bland-Altman analysis shows that our model tends to overestimate the lower end of the pain scores and underestimate the higher end. Conclusion: This is the first demonstration that physiological data can be correlated with chronic pain, both for individuals and populations. Further research and more extensive data will be required to assess whether this approach could be used as a "chronic pain meter" to assess the level of chronic pain in patients.

Functional neuronal circuitry and oscillatory dynamics in human brain organoids.

Human brain organoids replicate much of the cellular diversity and developmental anatomy of the human brain. However, the physiology of neuronal circuits within organoids remains under-explored. With high-density CMOS microelectrode arrays and shank electrodes, we captured spontaneous extracellular activity from brain organoids derived from human induced pluripotent stem cells. We inferred functional connectivity from spike timing, revealing a large number of weak connections within a skeleton of significantly fewer strong connections. A benzodiazepine increased the uniformity of firing patterns and decreased the relative fraction of weakly connected edges. Our analysis of the local field potential demonstrate that brain organoids contain neuronal assemblies of sufficient size and functional connectivity to co-activate and generate field potentials from their collective transmembrane currents that phase-lock to spiking activity. These results point to the potential of brain organoids for the study of neuropsychiatric diseases, drug action, and the effects of external stimuli upon neuronal networks.

Extracellular detection of neuronal coupling.

We developed a method to non-invasively detect synaptic relationships among neurons from in vitro networks. Our method uses microelectrode arrays on which neurons are cultured and from which propagation of extracellular action potentials (eAPs) in single axons are recorded at multiple electrodes. Detecting eAP propagation bypasses ambiguity introduced by spike sorting. Our methods identify short latency spiking relationships between neurons with properties expected of synaptically coupled neurons, namely they were recapitulated by direct stimulation and were sensitive to changing the number of active synaptic sites. Our methods enabled us to assemble a functional subset of neuronal connectivity in our cultures.

In situ Materials Characterization using the Tissue Diagnostic Instrument.

An understanding of the mechanical behavior of polymers is critical towards the design, implementation, and quality control of such materials. Yet experiments and method for the characterization of material properties of polymers remain challenging due the need to reconcile constitutive assumptions with experimental conditions. Well-established modes of mechanical testing, such as unconfined compression or uniaxial tension, require samples with specific geometries and carefully controlled orientations. Moreover, producing specimens that conform to such specifications often requires a considerable amount of sample material. In this study we validate a micromechanical indentation device, the Tissue Diagnostic Instrument (TDI), which implements a cyclic indentation method to determine the material properties of polymers and elastomeric materials. Measurements using the TDI require little or no sample preparation, and they allow the testing of sample materials in situ. In order to validate the use of the TDI, we compared measurements of modulus determined by the TDI to those obtained by unconfined compression tests and by uniaxial tension tests within the limit of small stresses and strains. The results show that the TDI measurements were significantly correlated with both unconfined compression (p<0.001; r(2) = 0.92) and uniaxial tension tests (p<0.001; r(2)=0.87). Moreover, the measurements across all three modes of testing were statistically indistinguishable from each other (p=0.92; ANOVA) and demonstrate that TDI measurements can provide a surrogate for the conventional methods of mechanical characterization.

Significant correlation of bone material strength index as measured by the OsteoProbe with Vickers and Rockwell hardness.

The bone material strength index (BMSi), as measured by the OsteoProbe, is significantly correlated with Vickers hardness and Rockwell (RW) hardness measurements on conventional materials. The Vickers and RW measurements were carried out according to American Society for Testing and Materials standard test methods, and OsteoProbe measurements followed published standardized testing methods. The correlations between the BMSi and RW hardness, r = 0.93, and between the BMSi and Vickers hardness, r = 0.94, are comparable with the correlation between RW and Vickers hardness, r = 0.87. The correlation between the BMSi and RW is significant at p < 0.01, and the correlation between the BMSi and Vickers hardness is significant at p < 0.01. These results show that the indentation measurement performed by the OsteoProbe may be considered as a type of hardness measurement comparable to widely used conventional methods, with specific applications targeted by its portable and narrow design.

Non-contact monitoring of extra-cellular field potentials with a multi-electrode array.

Developing tools to enable non-invasive, high-throughput electrophysiology measurements of large functional-networks of electrogenic cells used as in vitro disease models for the heart and brain remains an outstanding challenge for preclinical drug discovery, where failures are costly and can prove to be fatal during clinical trials. Here we demonstrate, for the first time, that it is possible to perform non-contact monitoring of extra-cellular field potentials with a multi-electrode array (MEA). To do this preliminary demonstration we built a prototype with a custom mechanical stage to micro-position cells grown on conventional glass coverslips over the recording surface of a MEA sensor. The prototype can monitor extra-cellular fields generated by multi-cellular networks in a non-contact configuration, enabling a single MEA sensor to probe different cultures in succession, without fouling or degrading its sensitive electronic surface. This first demonstration with easy to culture cardiomyocyte cells and a prototype device points to the exciting possibility for instrument development leading to more efficient and cost-effective drug screening paradigms for cardiovascular and neurological diseases.

In vitro validation of in silico identified inhibitory interactions.

BACKGROUND: Understanding how neuronal signals propagate in local network is an important step in understanding information processing. As a result, spike trains recorded with multi-electrode arrays (MEAs) have been widely used to study the function of neural networks. Studying the dynamics of neuronal networks requires the identification of both excitatory and inhibitory connections. The detection of excitatory relationships can robustly be inferred by characterizing the statistical relationships of neural spike trains. However, the identification of inhibitory relationships is more difficult: distinguishing endogenous low firing rates from active inhibition is not obvious. NEW METHOD: In this paper, we propose an in silico interventional procedure that makes predictions about the effect of stimulating or inhibiting single neurons on other neurons, and thereby gives the ability to accurately identify inhibitory effects. COMPARISON: To experimentally test these predictions, we have developed a Neural Circuit Probe (NCP) that delivers drugs transiently and reversibly on individually identified neurons to assess their contributions to the neural circuit behavior. RESULTS: Using the NCP, putative inhibitory connections identified by the in silico procedure were validated through in vitro interventional experiments. CONCLUSIONS: Together, these results demonstrate how detailed microcircuitry can be inferred from statistical models derived from neurophysiology data.

Effect of Ca2+ ions on the adhesion and mechanical properties of adsorbed layers of human osteopontin.

Using an atomic force microscope and a surface force apparatus, we measured the surface coverage, adhesion, and mechanical properties of layers of osteopontin (OPN), a phosphoprotein of the human bones, adsorbed on mica. OPN is believed to connect mineralized collagen fibrils of the bone in a matrix that dissipates energy, reducing the risk of fractures. Atomic force microscopy normal force measurements showed large adhesion and energy dissipation upon retraction of the tip, which were due to the breaking of the many OPN-OPN and OPN-mica bonds formed during tip-sample contact. The dissipated energy increased in the presence of Ca(2+) ions due to the formation of additional OPN-OPN and OPN-mica salt bridges between negative charges. The forces measured by surface force apparatus between two macroscopic mica surfaces were mainly repulsive and became hysteretic only in the presence of Ca(2+): adsorbed layers underwent an irreversible compaction during compression due to the formation of long-lived calcium salt bridges. This provides an energy storage mechanism, which is complementary to energy dissipation and may be equally relevant to bone recovery after yield. The prevalence of one mechanism or the other appears to depend on the confinement geometry, adsorption protocol, and loading-unloading rates.

The bone diagnostic instrument II: indentation distance increase.

The bone diagnostic instrument (BDI) is being developed with the long-term goal of providing a way for researchers and clinicians to measure bone material properties of human bone in vivo. Such measurements could contribute to the overall assessment of bone fragility in the future. Here, we describe an improved BDI, the Osteoprobe IItrade mark. In the Osteoprobe IItrade mark, the probe assembly, which is designed to penetrate soft tissue, consists of a reference probe (a 22 gauge hypodermic needle) and a test probe (a small diameter, sharpened rod) which slides through the inside of the reference probe. The probe assembly is inserted through the skin to rest on the bone. The distance that the test probe is indented into the bone can be measured relative to the position of the reference probe. At this stage of development, the indentation distance increase (IDI) with repeated cycling to a fixed force appears to best distinguish bone that is more easily fractured from bone that is less easily fractured. Specifically, in three model systems, in which previous mechanical testing and/or tests reported here found degraded mechanical properties such as toughness and postyield strain, the BDI found increased IDI. However, it must be emphasized that, at this time, neither the IDI nor any other mechanical measurement by any technique has been shown clinically to correlate with fracture risk. Further, we do not yet understand the mechanism responsible for determining IDI beyond noting that it is a measure of the continuing damage that results from repeated loading. As such, it is more a measure of plasticity than elasticity in the bone.

MEA Viewer: A high-performance interactive application for visualizing electrophysiological data.

Action potentials can be recorded extracellularly from hundreds of neurons simultaneously with multi-electrode arrays. These can typically have as many as 120 or more electrodes. The brief duration of action potentials requires a high sampling frequency to reliably capture each waveform. The resulting raw data files are therefore large and difficult to visualize with traditional plotting tools. Common approaches to deal with the difficulties of data display, such as extracting spike times and performing spike train analysis, are useful in many contexts but they also significantly reduce data dimensionality. The use of tools which minimize data processing enable the development of heuristic perspective of experimental results. Here we introduce MEA Viewer, a high-performance open source application for the direct visualization of multi-channel electrophysiological data. MEA Viewer includes several high-performance visualizations, including an easily navigable overview of recorded extracellular action potentials from all data channels overlaid with spike timestamp data and an interactive raster plot. MEA Viewer can also display the two dimensional extent of action potential propagation in single neurons by signal averaging extracellular action potentials (eAPs) from single neurons detected on multiple electrodes. This view extracts and displays eAP timing information and eAP waveforms that are otherwise below the spike detection threshold. This entirely new method of using MEAs opens up novel research applications for medium density arrays. MEA Viewer is licensed under the General Public License version 3, GPLv3, and is available at http://github.com/dbridges/mea-tools.

Analyzing the effect of hydration on the wedge indentation fracture behavior of cortical bone.

Hydration directly affects the mechanical properties of bone. An initial and basic procedure shows both wedge indentation fracture experiments under plane strain conditions in cortical bone and numerical simulation with finite elements agree that dry bone fractures much more easily than fully hydrated bone submerged in an aqueous environment, such as in the body of an animal. The wedge indentation experiments were performed with high speed video microscopy, under dry and fully hydrated (submerged) conditions. The numerical simulation, specifically finite element analysis using cohesive elements to simulate fracture, was utilized to capture plasticity, fracture initiation and propagation, and to study the applicability of brittle material based indentation fracture theory. Experiment and theory give similar results for the dependence of depth of fracture initiation, and size of plastic zone, on hydration state. Comparison of fracture propagation characteristics between wet and dry bone are examined and discussed. This research demonstrates the ability to quantitatively assess the effect of hydration on the fracture initiation, propagation, and plastic zone size of cortical bone, through an approach using simple wedge indentation, with important implications for efforts in developing methods to understand clinical diagnostic testing and general fracture behavior of living bone in the ultimate interest of health care purposes.

Components for high speed atomic force microscopy.

Many applications in materials science, life science and process control would benefit from atomic force microscopes (AFM) with higher scan speeds. To achieve this, the performance of many of the AFM components has to be increased. In this work, we focus on the cantilever sensor, the scanning unit and the data acquisition. We manufactured 10 microm wide cantilevers which combine high resonance frequencies with low spring constants (160-360 kHz with spring constants of 1-5 pN/nm). For the scanning unit, we developed a new scanner principle, based on stack piezos, which allows the construction of a scanner with 15 microm scan range while retaining high resonance frequencies (>10 kHz). To drive the AFM at high scan speeds and record the height and error signal, we implemented a fast Data Acquisition (DAQ) system based on a commercial DAQ card and a LabView user interface capable of recording 30 frames per second at 150 x 150 pixels.

Effect of various testing conditions on results for a handheld reference point indentation instrument in horses.

OBJECTIVE: To compare results obtained with a handheld reference point indentation instrument for bone material strength index (BMSi) measurements in the equine third metacarpal bone for various testing conditions. SAMPLE: 24 third metacarpal bones. PROCEDURES: Third metacarpal bones from both forelimbs of 12 horses were obtained. The dorsal surface of each bone was divided into 6 testing regions. In vivo and ex vivo measurements of BMSi were obtained through the skin and on exposed bone, respectively, to determine effects of each testing condition. Difference plots were used to assess agreement between BMSi obtained for various conditions. Linear regression analysis was used to assess effects of age, sex, and body weight on BMSi. A mixed-model ANOVA was used to assess effects of age, sex, limb, bone region, and testing condition on BMSi values. RESULTS: Indentation measurements were performed on standing sedated and recumbent anesthetized horses and on cadaveric bone. Regional differences in BMSi values were detected in adult horses. A significant linear relationship (r(2) = 0.71) was found between body weight and BMSi values. There was no difference between in vivo and ex vivo BMSi values. A small constant bias was detected between BMSi obtained through the skin, compared with values obtained directly on bone. CONCLUSIONS AND CLINICAL RELEVANCE: Reference point indentation can be used for in vivo assessment of the resistance of bone tissue to microfracture in horses. Testing through the skin should account for a small constant bias, compared with results for testing directly on exposed bone.

The backbone conformational entropy of protein folding: experimental measures from atomic force microscopy.

The energy dissipated during the atomic force microscopy-based mechanical unfolding and extension of proteins is typically an order of magnitude greater than their folding free energy. The vast majority of the "excess" energy dissipated is thought to arise due to backbone conformational entropy losses as the solvated, random-coil unfolded state is stretched into an extended, low-entropy conformation. We have investigated this hypothesis in light of recent measurements of the energy dissipated during the mechanical unfolding of "polyproteins" comprised of multiple, homogeneous domains. Given the assumption that backbone conformational entropy losses account for the vast majority of the energy dissipated (an assumption supported by numerous lines of experimental evidence), we estimate that approximately 19(+/-2)J/(mol K residue) of entropy is lost during the extension of three mechanically stable beta-sheet polyproteins. If, as suggested by measured peak-to-peak extension distances, pulling proceeds to near completion, this estimate corresponds to the absolute backbone conformational entropy of the unfolded state. As such, it is exceedingly close to previous theoretical and semi-empirical estimates that place this value at approximately 20J/(mol K residue). The estimated backbone conformational entropy lost during the extension of two helical polyproteins, which, in contrast to the mechanically stable beta-sheet polyproteins, rupture at very low applied forces, is three- to sixfold less. Either previous estimates of the backbone conformational entropy are significantly in error, or the reduced mechanical strength of the helical proteins leads to the rupture of a subsequent domain before full extension (and thus complete entropy loss) is achieved.

Study of indentation of a sample equine bone using finite element simulation and single cycle reference point indentation.

In an attempt to study the mechanical behavior of bone under indentation, methods of analyses and experimental validations have been developed, with a selected test material. The test material chosen is from an equine cortical bone. Stress-strain relationships are first obtained from conventional mechanical property tests. A finite element simulation procedure is developed for indentation analyses. The simulation results are experimentally validated by determining (1) the maximum depth of indentation with a single cycle type of reference point indentation, and (2) the profile and depth of the unloaded, permanent indentation with atomic force microscopy. The advantage of incorporating in the simulation a yield criterion calibrated by tested mechanical properties, with different values in tension and compression, is demonstrated. In addition, the benefit of including damage through a reduction in Young's modulus is shown in predicting the permanent indentation after unloading and recovery. The expected differences in response between two indenter tips with different sharpness are predicted and experimentally observed. Results show predicted indentation depths agree with experimental data. Thus, finite element simulation methods with experimental validation, and with damage approximation by a reduction of Young's modulus, may provide a good approach for analysis of indentation of cortical bone. These methods reveal that multiple factors affect measured indentation depth and that the shape of the permanent indentation contains useful information about bone material properties. Only further work can determine if these methods or extensions to these methods can give useful insights into bone pathology, for example the bone fragility of thoroughbred racehorses.

Bone Tissue Properties Measurement by Reference Point Indentation in Glucocorticoid-Induced Osteoporosis.

Glucocorticoids, widely used in inflammatory disorders, rapidly increase bone fragility and, therefore, fracture risk. However, common bone densitometry measurements are not sensitive enough to detect these changes. Moreover, densitometry only partially recognizes treatment-induced fracture reductions in osteoporosis. Here, we tested whether the reference point indentation technique could detect bone tissue property changes early after glucocorticoid treatment initiation. After initial laboratory and bone density measurements, patients were allocated into groups receiving calcium + vitamin D (Ca+D) supplements or anti-osteoporotic drugs (risedronate, denosumab, teriparatide). Reference point indentation was performed on the cortical bone layer of the tibia by a handheld device measuring bone material strength index (BMSi). Bone mineral density was measured by dual-energy X-ray absorptiometry (DXA). Although Ca+D-treated patients exhibited substantial and significant deterioration, risedronate-treated patients exhibited no significant change, and both denosumab- and teriparatide-treated participants exhibited significantly improved BMSi 7 weeks after initial treatment compared with baseline; these trends remained stable for 20 weeks. In contrast, no densitometry changes were observed during this study period. In conclusion, our study is the first to our knowledge to demonstrate that reference point indentation is sensitive enough to reflect changes in cortical bone indentation after treatment with osteoporosis therapies in patients newly exposed to glucocorticoids.