A ketogenic diet affects brain volume and metabolome in juvenile mice.

Ketogenic diet (KD) is a high-fat and low-carbohydrate therapy for medically intractable epilepsy, and its applications in other neurological conditions, including those occurring in children, have been increasingly tested. However, how KD affects childhood neurodevelopment, a highly sensitive and plastic process, is not clear. In this study, we explored structural, metabolic, and functional consequences of a brief treatment of a strict KD (weight ratio of fat to carbohydrate plus protein is approximately 6.3:1) in naive juvenile mice of different inbred strains, using a multidisciplinary approach. Systemic measurements using magnetic resonance imaging revealed that unexpectedly, the volumes of most brain structures in KD-fed mice were about 90% of those in mice of the same strain but fed a standard diet. The reductions in volumes were nonselective, including different regions throughout the brain, the ventricles, and the white matter. The relative volumes of different brain structures were unaltered. Additionally, as KD is a metabolism-based treatment, we performed untargeted metabolomic profiling to explore potential means by which KD affected brain growth and to identify metabolic changes in the brain. We found that brain metabolomic profile was significantly impacted by KD, through both distinct and common pathways in different mouse strains. To explore whether the volumetric and metabolic changes induced by this KD treatment were associated with functional consequences, we recorded spontaneous EEG to measure brain network activity. Results demonstrated limited alterations in EEG patterns in KD-fed animals. In addition, we observed that cortical levels of brain-derived neurotrophic factor (BDNF), a critical molecule in neurodevelopment, did not change in KD-fed animals. Together, these findings indicate that a strict KD could affect volumetric development and metabolic profile of the brain in inbred juvenile mice, while global network activities and BDNF signaling in the brain were mostly preserved. Whether the volumetric and metabolic changes are related to any core functional consequences during neurodevelopment and whether they are also observed in humans need to be further investigated. In addition, our results indicate that certain outcomes of KD are specific to the individual mouse strains tested, suggesting that the physiological profiles of individuals may need to be examined to maximize the clinical benefit of KD.

Diffuse photon-remission associated with single-fiber geometry may be a simple scaling of that collected over the same area when under centered-illumination.

Robust models for single-fiber reflectance (SFR) are relatively complex [Opt. Lett.45, 2078 (2020)OPLEDP0146-959210.1364/OL.385845] due to overlapping of the illumination and collection areas that entails probability weighting of the spatial integration of photon-remission. We demonstrate, via analytical means for limiting cases and Monte Carlo simulation of broader conditions, that diffuse photon-remission collected by single-fiber geometry may be scaled over the center-illuminated photon-remission. We specify for a medium revealing Henyey-Greenstein (HG) scattering anisotropy that the diffuse photon-remission from a sub-diffusive area of a top-hat illumination is ∼84.9% of that collected over the same area when under a centered-illumination. This ratio remains consistent over a reduced-scattering fiber-size product of µs'dfib=[10-5,100], for absorption varying 3 orders of magnitude. When applied to hemoglobin oxygenation changes induced in an aqueous phantom using a 200 µm single-fiber probe, the center-illumination-scaled model of SFR produced fitting results agreeing with reference measurements.

Spatiotemporal dynamics of cortical perfusion in response to thalamic deep brain stimulation.

Deep brain stimulation (DBS) has revolutionized the treatment of movement disorders. The parameters of electrical stimulation are important to its therapeutic effect and remain a source of clinical controversy. DBS exerts its actions not only locally at the site of stimulation but also remotely through afferent and efferent connections, which are vital to its clinical effects. Yet, only a few studies have examined how cortical activity changes in response to various electrical parameters. Here, we investigated how the parameters of thalamic DBS alter cortical perfusion in rats using intrinsic optical imaging. We hypothesized that thalamic DBS will increase perfusion in primary motor cortex (M1), proportional to amplitude, pulse width, or frequency of the stimulation applied. We applied 45 different combinations of amplitude, pulse width and frequency in the ventro-lateral (VL) nucleus of the thalamus in anesthetized rats while measuring perfusion in M1. VL thalamic DBS reduced cortical reflectance, which corresponds to an increase in cortical perfusion. We computed the maximum change in reflectance (MCR) as well as the spatial spread of MCR in each trial. Both MCR and spatial spread increased linearly with increases in current amplitude or pulse width of stimulation; however, the effect of frequency was non-linear. Stimulation at 20 Hz was significantly different from that at higher frequencies while stimulation at higher frequencies did not differ significantly from each other. Moreover, the effect of pulse width on MCR was larger than the effect of amplitude. The proportional increase in M1 perfusion due to increase in amplitude or pulse width suggests that both activate more neural elements and increase the volume of tissue activated. These results should help clinicians set parameters of DBS. The use of optical imaging to monitor effects of DBS on M1 may not only help understand DBS mechanisms, but may also provide feedback for closed loop DBS devices.

A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy.

We report the development of an all-fiber-optic scanning endomicroscope capable of high-resolution second harmonic generation (SHG) imaging of biological tissues and demonstrate its utility for monitoring the remodeling of cervical collagen during gestation in mice. The endomicroscope has an overall 2.0 mm diameter and consists of a single customized double-clad fiber, a compact rapid two-dimensional beam scanner, and a miniature compound objective lens for excitation beam delivery, scanning, focusing, and efficient SHG signal collection. Endomicroscopic SHG images of murine cervical tissue sections at different stages of normal pregnancy reveal progressive, quantifiable changes in cervical collagen morphology with resolution similar to that of bench-top SHG microscopy. SHG endomicroscopic imaging of ex vivo murine and human cervical tissues through intact epithelium has also been performed. Our findings demonstrate the feasibility of SHG endomicroscopy technology for staging normal pregnancy, and suggest its potential application as a minimally invasive tool for clinical assessment of abnormal cervical remodeling associated with preterm birth.

Distinguishing motion artifacts during optical fiber-based in-vivo hemodynamics recordings from brain regions of freely moving rodents.

Significance: Motion artifacts in the signals recorded during optical fiber-based measurements can lead to misinterpretation of data. In this work, we address this problem during in-vivo rodent experiments and develop a motion artifacts correction (MAC) algorithm for single-fiber system (SFS) hemodynamics measurements from the brains of rodents. Aim: (i) To distinguish the effect of motion artifacts in the SFS signals. (ii) Develop a MAC algorithm by combining information from the experiments and simulations and validate it. Approach: Monte-Carlo (MC) simulations were performed across 450 to 790 nm to identify wavelengths where the reflectance is least sensitive to blood absorption-based changes. This wavelength region is then used to develop a quantitative metric to measure motion artifacts, termed the dissimilarity metric (DM). We used MC simulations to mimic artifacts seen during experiments. Further, we developed a mathematical model describing light intensity at various optical interfaces. Finally, an MAC algorithm was formulated and validated using simulation and experimental data. Results: We found that the 670 to 680 nm wavelength region is relatively less sensitive to blood absorption. The standard deviation of DM (σDM) can measure the relative magnitude of motion artifacts in the SFS signals. The artifacts cause rapid shifts in the reflectance data that can be modeled as transmission changes in the optical lightpath. The changes observed during the experiment were found to be in agreement to those obtained from MC simulations. The mathematical model developed to model transmission changes to represent motion artifacts was extended to an MAC algorithm. The MAC algorithm was validated using simulations and experimental data. Conclusions: We distinguished motion artifacts from SFS signals during in vivo hemodynamic monitoring experiments. From simulation and experimental data, we showed that motion artifacts can be modeled as transmission changes. The developed MAC algorithm was shown to minimize artifactual variations in both simulation and experimental data.

Markerless Mouse Tracking for Social Experiments.

Automated behavior quantification in socially interacting animals requires accurate tracking. While many methods have been very successful and highly generalizable to different settings, issues of mistaken identities and lost information on key anatomical features are common, although they can be alleviated by increased human effort in training or post-processing. We propose a markerless video-based tool to simultaneously track two interacting mice of the same appearance in controlled settings for quantifying behaviors such as different types of sniffing, touching, and locomotion to improve tracking accuracy under these settings without increased human effort. It incorporates conventional handcrafted tracking and deep-learning-based techniques. The tool is trained on a small number of manually annotated images from a basic experimental setup and outputs body masks and coordinates of the snout and tail-base for each mouse. The method was tested on several commonly used experimental conditions including bedding in the cage and fiberoptic or headstage implants on the mice. Results obtained without any human corrections after the automated analysis showed a near elimination of identities switches and a ∼15% improvement in tracking accuracy over pure deep-learning-based pose estimation tracking approaches. Our approach can be optionally ensembled with such techniques for further improvement. Finally, we demonstrated an application of this approach in studies of social behavior of mice by quantifying and comparing interactions between pairs of mice in which some lack olfaction. Together, these results suggest that our approach could be valuable for studying group behaviors in rodents, such as social interactions.

Redox-Concatenated Aptamer Integrated Skin Mimicking Electrochemical Patch for Noninvasive Detection of Cortisol.

This research focuses on developing and validating a wearable electrochemical biosensor called the concatenated aptamer integrated skin patch, also known as the Captain Patch. The main objective is to detect cortisol levels in sweat, which can provide valuable insights into an individual's health. The biosensor utilizes a corrugated surface that mimics the skin, allowing for better attachment and an improved electrochemical performance. The study demonstrates the successful application of Captain Patch on the human body by using artificially spiked sweat samples. The results indicate good measurement accuracy and conformity when the patch is worn on the body. However, for long-term usage, the patch needs to be changed every 3-4 h or worn three times a day to enable monitoring of cortisol levels. Despite the need for frequent patch changes, the cost-effectiveness and ease of operation make these skin patches suitable for longitudinal cortisol monitoring and other sweat analytes. By customization of the biorecognition probe, the developed biowearable can be used to monitor a variety of vital biomarkers. Overall, Captain Patch, with its capability of detecting specific health markers such as cortisol, hints at the future potential of wearables to offer valuable data on various other biomarkers. Our approach presents the first step in integrating a cost-effective wearable electrochemical patch integrated with a redox-concatenated aptamer for noninvasive biomarker detection. This personalized approach to monitoring can lead to improved patient outcomes and increased patient engagement in managing their health.

A clinically relevant selective ERK-pathway inhibitor reverses core deficits in a mouse model of autism.

BACKGROUND: Extracellular signal-regulated kinase (ERK/MAPK) pathway in the brain is hypothesized to be a critical convergent node in the development of autism spectrum disorder. We reasoned that selectively targeting this pathway could reverse core autism-like phenotype in animal models. METHODS: Here we tested a clinically relevant, selective inhibitor of ERK pathway, PD325901 (Mirdametinib), in a mouse model of idiopathic autism, the BTBR mice. FINDINGS: We report that treating juvenile mice with PD325901 reduced ERK pathway activation, dose and duration-dependently reduced core disease-modeling deficits in sociability, vocalization and repetitive behavior, and reversed abnormal EEG signals. Further analysis revealed that subchronic treatment did not affect weight gain, locomotion, or neuronal density in the brain. Parallel treatment in the C57BL/6J mice did not alter their phenotype. INTERPRETATION: Our data indicate that selectively inhibiting ERK pathway using PD325901 is beneficial in the BTBR model, thus further support the notion that ERK pathway is critically involved in the pathophysiology of autism. These results suggest that a similar approach could be applied to animal models of syndromic autism with dysregulated ERK signaling, to further test selectively targeting ERK pathway as a new approach for treating autism. FUNDING: This has beenwork was supported by Alberta Children's Hospital Research Foundation (JMR & NC), University of Calgary Faculty of Veterinary Medicine (NC), Kids Brain Health Network (NC), and Natural Sciences and Engineering Research Council of Canada (NC).

Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging.

We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence tomography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound lens suitable for simultaneous acquisition of en face OCT and TPF images. A fiber-optic wavelength division multiplexer was employed in the integrated platform to combine the low coherence OCT light source and the femtosecond two-photon excitation laser into the same optical path. Preliminary imaging results of cell cultures and mouse tissue ex vivo demonstrate the feasibility of simultaneous real-time OCT and TPF imaging in a scanning endomicroscopy setting for the first time.

Monitoring stimulus-evoked hemodynamic response during deep brain stimulation with single fiber spectroscopy.

Deep brain stimulation (DBS) is a revolutionary treatment for movement disorders. Measuring DBS-induced hemodynamic responses may be useful for surgical guidance of DBS electrode implantation as well as to study the mechanism and assess therapeutic effects of DBS. In this study, we evaluated the performance of a single fiber spectroscopic (SFS) system for measuring hemodynamic response in different cortical layers in a DBS animal model. We showed that SFS is capable of measuring minute relative changes in oxygen saturation and blood volume fraction in-vivo at a sampling rate of 22-33 Hz. During stimulation, blood volume fraction increased, while oxygen saturation showed both increases and decreases at different cortical depths across animals. In addition, we showed the potential of using SFS for measuring other physiological parameters, for example, heart rate, and respiratory rate.

Compensation-free, all-fiber-optic, two-photon endomicroscopy at 1.55 µm.

We present an all-fiber-optic scanning multiphoton endomicroscope with 1.55 µm excitation without the need for prechirping femtosecond pulses before the endomicroscope. The system consists of a 1.55 µm femtosecond fiber laser, a customized double-clad fiber for light delivery and fluorescence collection, and a piezoelectric scan head. We demonstrate two-photon imaging of cultured cells and mouse tissue, both labeled with indocyanine green. Free-space multiphoton imaging with near-IR emission has previously shown benefits in reduced background fluorescence and lower attenuation for the fluorescence emission. For fiber-optic multiphoton imaging there is the additional advantage of using the soliton effect at the telecommunication wavelengths (1.3-1.6 µm) in fibers, permitting dispersion-compensation-free, small-footprint systems. We expect these advantages will help transition multiphoton endomicroscopy to the clinic.

Induced pluripotency in the context of stem cell expansion bioprocess development, optimization, and manufacturing: a roadmap to the clinic.

The translation of laboratory-scale bioprocess protocols and technologies to industrial scales and the application of human induced pluripotent stem cell (hiPSC) derivatives in clinical trials globally presents optimism for the future of stem-cell products to impact healthcare. However, while many promising therapeutic approaches are being tested in pre-clinical studies, hiPSC-derived products currently account for a small fraction of active clinical trials. The complexity and volatility of hiPSCs present several bioprocessing challenges, where the goal is to generate a sufficiently large, high-quality, homogeneous population for downstream differentiation-the derivatives of which must retain functional efficacy and meet regulatory safety criteria in application. It is argued herein that one of the major challenges currently faced in improving the robustness of routine stem-cell biomanufacturing is in utilizing continuous, meaningful assessments of molecular and cellular characteristics from process to application. This includes integrating process data with biological characteristic and functional assessment data to model the interplay between variables in the search for global optimization strategies. Coupling complete datasets with relevant computational methods will contribute significantly to model development and automation in achieving process robustness. This overarching approach is thus crucially important in realizing the potential of hiPSC biomanufacturing for transformation of regenerative medicine and the healthcare industry.

µDrop: Multi-analyte portable electrochemical-sensing device for blood-based detection of cleaved tau and neuron filament light in traumatic brain injury patients.

Over 27 million individuals are affected every year worldwide with central nervous system (CNS) injuries. These injuries include but are not limited to traumatic brain injury (TBI) and spinal cord injury (SCI). CNS injuries remain a significant public health concern which demands reliable tools for rapid, on-sight, on-field, and point-of-care diagnostic (POC) solutions. To address these challenges, we developed a low-cost, open-source, hand-held, portable, and POC detection technology, termed as MicroDrop (µDrop), which can simultaneously detect up to eight target biomolecules and display results in both analog and digital formats. The data acquired is stored wirelessly in a cloud server for further investigation and statistical analysis. Multiplexing capability of µDrop and immuno-biosensors detects and quantifies Cleaved-Tau Protein (C-Tau) and Neuron-Filament (NFL) proteins in the blood of TBI patients. Immuno-biosensors rapidly sense the two target proteins in less than 30 min, with µDrop and a conventional potentiostat. C-Tau and NFL were selectively detected with µDrop within the dynamic range of 10 pg/mL - 100 ng/mL and the sensitivity range of 47 µA/pg mm2 - 65 µA/pg mm2. Comparing the biosensing performance with enzyme-linked immunosorbent assays (ELISA) shows that the immuno-biosensors combined with µDrop could successfully differentiate between clinical controls and injured patients.

OSERR: an open-source standalone electrophysiology recording system for rodents.

Behavioral assessment of rodents is critical for investigation of brain function in health and disease. In vivo neurophysiological recordings are powerful tools to mechanistically dissect neural pathways that underlie behavioral changes, and serve as markers for dynamics, efficacy and safety of potential therapeutic approaches. However, most in vivo recording systems require tethers or telemetry receivers, limiting their compatibility with some behavioral tests. Here, we developed an open-source standalone electrophysiology recording system for rodents (OSERR). It is a tether-free, standalone recording device with two channels, a reference and a ground, that acquires, amplifies, filters and stores data all in itself. Thus, it does not require any cable or receiver. It is also compact and light-weight, and compatible with juvenile mice, as well as multiple recording modalities and standard electrode implantation methods. In addition, we provide the complete design of hardware, and software for operation. As an example, we demonstrated that this standalone system, when configured with a bandwidth of 1-120 Hz and gain of 1000, successfully collected EEG signals during induced seizure, extended recording, anesthesia, and social interactions in mice. The design of this system is practical, economical, and freely available. Thus, this system could enable recording of brain activity during diverse behavioral assays in a variety of arenas and settings, and allow simultaneous recordings from multiple subjects to examine social behaviors. Importantly, with the open-source documentation, researchers could customize the design of the system to their specific needs.

Neurovascular coupling during deep brain stimulation.

BACKGROUND: Deep brain stimulation (DBS) is an effective treatment for movement disorders, yet its mechanisms of action remain unclear. One method used to study its circuit-wide neuromodulatory effects is functional magnetic resonance imaging (fMRI) which measures hemodynamics as a proxy of neural activity. To interpret functional imaging data, we must understand the relationship between neural and vascular responses, which has never been studied with the high frequencies used for DBS. OBJECTIVE: To measure neurovascular coupling in the rat motor cortex during thalamic DBS. METHOD: Simultaneous intrinsic optical imaging and extracellular electrophysiology was performed in the motor cortex of urethane-anesthetized rats during thalamic DBS at 7 different frequencies. We related Maximum Change in Reflectance (MCR) from the imaging data to Integrated Evoked Potential (IEP) and change in broadband power of multi-unit (MU) activity, computing Spearman's correlation to determine the strength of these relationships. To determine the source of these effects, we studied the contributions of antidromic versus orthodromic activation in motor cortex perfusion using synaptic blockers. RESULTS: MCR, IEP and change in MU power increased linearly to 60 Hz and saturated at higher frequencies of stimulation. Blocking orthodromic transmission only reduced the DBS-induced change in optical signal by ∼25%, suggesting that activation of corticofugal fibers have a major contribution in thalamic-induced cortical activation. CONCLUSION: DBS-evoked vascular response is related to both evoked field potentials as well as multi-unit activity.

Fiber photometry for monitoring cerebral oxygen saturation in freely-moving rodents.

Hemodynamic parameters, such as tissue oxygen saturation and blood volume fraction, are important markers of brain physiology. They are also widely used surrogate markers of electrophysiological activity. Here, we present a single fiber spectroscopic (SFS) system for monitoring cerebral oxygen saturation in localized, non-line-of-sight brain regions in freely-moving rodents. We adapted the implantation ferrule and patch cable design from commercialized optogenetics and fiber photometry systems, enabling stereotaxic fiber implantation, longitudinal tissue access and measurement from freely-moving animals. The optical system delivers and collects light from the brain through a 200 µm-core-diameter, 0.39NA multimode fiber. We robustly measured oxygen saturation from phantoms with different optical properties mimicking brain tissue. In mice, we demonstrated, for the first time, measurements of oxygen saturation from a highly-localized, targeted brain region over 31 days and continuous measurements from a freely-moving animal for over an hour. These results suggest that single fiber spectroscopy has enormous potential for functional brain monitoring and investigating neurovascular coupling in freely-moving animals. In addition, this technique can potentially be combined with fiber photometry systems to correct for hemodynamic artifacts in the fluorescence detection.

High spatiotemporal resolution imaging of the neurovascular response to electrical stimulation of rat peripheral trigeminal nerve as revealed by in vivo temporal laser speckle contrast.

Previous studies have implicated the abnormal activation of the trigeminal system to be a factor in the pathogenesis of migraine. The relationship between vascular changes and migraine, however, is under considerable debate. In this study, temporal laser speckle contrast imaging is combined with ridge tracking based vessel detection to obtain high resolution (6.7 microm x 6.7 microm), high contrast images of cerebral vascular structure. For the first time, the vasomotor and blood flow responses to electrical stimulation in rat peripheral trigeminal system were obtained simultaneously. The system is capable of picking up individual vessels with diameters down to 30 microm. The spatial spread of the blood velocity response relative to the point of stimulation was studied. Analysis of branching vessels showed a 50+/-5% vs. 30+/-5% change in mean peak magnitude and a 54% per second vs. 17% per second change in mean rate of increase for vessels proximal vs. distal to the stimulation site. The penetration depth of the laser used was proven to be sufficient to image dural as well as cortical vessels through a thinned skull preparation. Different responses were observed from cortical and dural vessels. While the diameter of cortical vessels did not change in response to the stimulation the blood velocity went up by 65+/-5% per second. Dural vessels enlarged by 40+/-8% and the blood velocity increased by 50+/-5%. The method described here could be very useful in understanding and studying disorders in the neurovascular system.

Which Photodiode to Use: A Comparison of CMOS-Compatible Structures.

While great advances have been made in optimizing fabrication process technologies for solid state image sensors, the need remains to be able to fabricate high quality photosensors in standard CMOS processes. The quality metrics depend on both the pixel architecture and the photosensitive structure. This paper presents a comparison of three photodiode structures in terms of spectral sensitivity, noise and dark current. The three structures are n(+)/p-sub, n-well/p-sub and p(+)/n-well/p-sub. All structures were fabricated in a 0.5 mum 3-metal, 2-poly, n-well process and shared the same pixel and readout architectures. Two pixel structures were fabricated-the standard three transistor active pixel sensor, where the output depends on the photodiode capacitance, and one incorporating an in-pixel capacitive transimpedance amplifier where the output is dependent only on a designed feedback capacitor. The n-well/p-sub diode performed best in terms of sensitivity (an improvement of 3.5 x and 1.6 x over the n(+)/p-sub and p(+)/n-well/p-sub diodes, respectively) and signal-to-noise ratio (1.5 x and 1.2 x improvement over the n(+)/p-sub and p(+)/n-well/p-sub diodes, respectively) while the p(+)/n-well/p-sub diode had the minimum (33% compared to other two structures) dark current for a given sensitivity.

Precocious myelination in a mouse model of autism.

Autism spectrum disorder (ASD) has been hypothesized to be a result of altered connectivity in the brain. Recent imaging studies suggest accelerated maturation of the white matter in young children with ASD, with underlying mechanisms unknown. Myelin is an integral part of the white matter and critical for connectivity; however, its role in ASD remains largely unclear. Here, we investigated myelin development in a model of idiopathic ASD, the BTBR mice. Magnetic resonance imaging revealed that fiber tracts in the frontal brain of the BTBR mice had increased volume at postnatal day 6, but the difference reduced over time, reminiscent of the findings in young patients. We further identified that myelination in the frontal brain of both male and female neonatal BTBR mice was increased, associated with elevated levels of myelin basic protein. However, myelin pattern was unaltered in adult BTBR mice, revealing accelerated developmental trajectory of myelination. Consistently, we found that signaling of platelet-derived growth factor receptor alpha (PDGFRα) was reduced in the frontal brain of neonatal BTBR mice. However, levels of microRNA species known to regulate PDGFRα signaling and myelination were unaltered. Together, these results suggest that precocious myelination could potentially contribute to increased volume and connectivity of the white matter observed in young children with ASD.