Electrical microstimulation provides high-resolution control of neural circuits for causal studies and restoration of impaired functions, yet how responses to artificial activation evolve with learning remains unclear. Here, we deploy a detection task and pair ultraflexible electrodes for stable intracortical microstimulation (ICMS) with longitudinal imaging and recordings to track single-cell and population responses across weeks of learning. Detection thresholds decreased with learning, indicating plasticity. Chronic imaging showed that stimulus-evoked recruitment expanded at a fixed current, while a consistent number of neurons continued to underlie behavioral responses. A subset of learning-sensitive cells enhanced modulation and reduced latency. Electrophysiological recordings further distinguished two forms of adaptation: Directly activated, pulse-locked neurons strengthened their excitability, whereas polysynaptically recruited neurons expanded in number and were predictive of behavioral outcomes. These results show that learning in an ICMS task reshapes cortical circuits through activation-mechanism-dependent plasticity, underscoring the need for stimulation paradigms that adapt to both cell-intrinsic and network dynamics.

We report a hybrid miniaturized achromatic objective for nonlinear endomicroscopy that combines two-photon polymerization (2PP)-fabricated polymer optics with a commercial glass lens. The objective achieves 0.6 numerical aperture with dual-wavelength correction at 775 and 860 nm and a 200 µm field of view, all within a 3 mm housing compatible with a hypodermic tube. Fabrication leverages sequential multi-step two-photon polymerization (2PP) to produce a monolithic multi-material triplet with submicron precision. The triplet incorporates two photoresists with different dispersion properties printed in successive steps with post-processing and marker-based realignment between steps, yielding a single monolithic optic that eliminates adhesives while correcting chromatic aberrations. Imaging tests with USAF resolution targets, biological samples, and two-photon fluorescence microscopy of pollen grains demonstrate near-diffraction-limited performance across the design wavelength range. The approach offers a practical method for compact optical systems in biomedical applications.

We present a fully 3D-printed, broadband endomicroscopic objective optimized for near-infrared two-photon fluorescence imaging, featuring a compact, alignment-free design. The objective provides a numerical aperture of 0.6 in water immersion over a 200 µm field of view and maintains near diffraction-limited performance across the 720–950 nm excitation band. Monolithically fabricated with multi-material via two-photon polymerization (2PP), the three-element refractive objective achieves a 2 mm lens mechanical diameter, a mechanical housing diameter of 3 mm, and a total length of 2.1 mm with an approximately 10-hour build time. A self-aligning architecture integrates the printed half-housing with the optics to minimize assembly error, yielding sub-10 µm element decenter and <0.1° element tilt. Optical performance was validated using a resolution target and biological specimens, with broadband excitation behavior assessed by two-photon imaging of pollen grains across multiple excitation wavelengths, and with autofluorescence imaging of fresh mouse liver confirming feasibility for in situ tissue imaging. This work demonstrates the potential of fully printed broadband objectives for compact, high-performance endomicroscopy and integrated biomedical optics.

American football has become the focus of numerous studies highlighting a growing concern that cumulative exposure to repetitive, sports-related head acceleration events (HAEs) may have negative consequences for brain health, even in the absence of a diagnosed concussion. In this longitudinal study, brain functional connectivity was analyzed in a cohort of high school American football athletes over a single play season and compared against participants in non-collision high school sports. Football athletes underwent four resting-state functional magnetic resonance imaging sessions: once before (pre-season), twice during (in-season), and once 34–80 days after the contact activities play season ended (post-season). For each imaging session, functional connectomes (FCs) were computed for each athlete and compared across sessions using a metric reflecting the (self) similarity between two FCs. HAEs were monitored during all practices and games throughout the season using head-mounted sensors. Relative to the pre-season scan session, football athletes exhibited decreased FC self-similarity at the later in-season session, with apparent recovery of self-similarity by the time of the post-season session. In addition, both within and post-season self-similarity was correlated with cumulative exposure to head acceleration events. These results suggest that repetitive exposure to HAEs produces alterations in functional brain connectivity and highlight the necessity of collision-free recovery periods for football athletes.

Neural stimulation has a variety of applications in neuroscience research and clinical therapies. Conventionally, stimulation is delivered by implanted electrodes, such as in intracortical microstimulation (ICMS) and deep brain stimulation (DBS). However, long-term reliability associated with the electrode-tissue interface remains a fundamental challenge [1], due to foreign body responses that can lead to glial scar formation and encapsulation of electrodes (Fig. 17.5.1). Magnetic stimulation has emerged as a promising alternative to overcome this challenge, as no direct contact is needed between the stimulation coil and tissues. Its efficacy has been clinically verified through FDA-approved transcranial magnetic stimulation (TMS) for treating neurological disorders. However, TMS devices are power-hungry and bulky (Fig. 17.5.1), which not only restricts patient accessibility but also limits the spatial resolution of the stimulation.

Intracortical microstimulation (ICMS) enables applications ranging from neuroprosthetics to causal circuit manipulations. However, the resolution, efficacy, and chronic stability of neuromodulation is often compromised by the adverse tissue responses to the indwelling electrodes. Here we engineer ultraflexible stim-Nanoelectronic Threads (StimNETs) and demonstrate low activation threshold, high resolution, and chronically stable ICMS in awake, behaving mouse models. In vivo two-photon imaging reveals that StimNETs remain seamlessly integrated with the nervous tissue throughout chronic stimulation periods and elicit stable, focal neuronal activation at low currents of 2 μA. Importantly, StimNETs evoke longitudinally stable behavioral responses for over eight months at markedly low charge injection of 0.25 nC/phase. Quantified histological analysis show that chronic ICMS by StimNETs induce no neuronal degeneration or glial scarring. These results suggest that tissue-integrated electrodes provide a path for robust, long-lasting, spatially-selective neuromodulation at low currents which lessen risks of tissue damage or exacerbation of off-target side-effects.

Reports estimate between 1.6-3.8 million sports-related concussions occur annually, with 30% occurring in youth male American football athletes. Many studies report neurophysiological changes in these athletes, but the exact reasons for these changes remain elusive. Investigation of injury mechanics highlights a need to address how player position might impact these changes. Here, 55 high school American football athletes (20 linemen; 35 non-linemen) underwent magnetic resonance spectroscopy four times over the course of a football season—once prior to the season (Pre), twice during (In1, In2), and once following (Post) to quantify metabolites (N-acetyl aspartate, choline, creatine, myo-inositol, and glutamate/glutamine) in the dorsolateral prefrontal cortex (DLPFC) and primary motor cortex (M1). Head acceleration events (HAEs) were monitored at each practice and game. Spectroscopic and HAE data were analyzed by imaging session and player position. Linear regression analyses were conducted between metabolite levels and HAEs, and metabolite levels in football athletes were compared with age-and gender-matched non-contact athletes. Across-season (i.e., between Pre and In1, In2, Post), different DLPFC and M1 metabolites decreased (p < 0.05) according to player position (i.e., linemen vs. non-linemen). The majority of regression results involved DLPFC metabolites in linemen, where metabolite levels were higher from Pre to Post, with increasing HAE load. Comparisons with control athletes revealed higher metabolite levels in football athletes both before and after the season. This study highlights the importance of player position when conducting analyses on American football athletes and demonstrates elevated DLPFC and M1 brain metabolites in football athletes compared with control athletes at both Pre and Post, suggesting potential HAE-related neurocompensatory mechanisms.

Observations of short-term changes in the neural health of youth athletes participating in collision sports (e.g., football and soccer) have highlighted a need to explore potential structural alterations in brain tissue volumes for these persons. Studies have shown biochemical, vascular, functional connectivity, and white matter diffusivity changes in the brain physiology of these athletes that are strongly correlated with repetitive head acceleration exposure. Here, research is presented that highlights regional anatomical volumetric measures that change longitudinally with accrued subconcussive trauma. A novel pipeline is introduced that provides simplified data analysis on standard-space template to quantify group-level longitudinal volumetric changes within these populations. For both sports, results highlight incremental relative regional volumetric changes in the subcortical cerebrospinal fluid that are strongly correlated with head exposure events greater than a 50-G threshold at the short-term post-season assessment. Moreover, longitudinal regional gray matter volumes are observed to decrease with time, only returning to baseline/pre-participation levels after sufficient (5-6 months) rest from collision-based exposure. These temporal structural volumetric alterations are significantly different from normal aging observed in sex- and age-matched controls participating in non-collision sports. Future work involves modeling repetitive head exposure thresholds with multi-modal image analysis and understanding the underlying physiological reason. A possible pathophysiological pathway is presented, highlighting the probable metabolic regulatory mechanisms. Continual participation in collision-based activities may represent a risk wherein recovery cannot occur. Even when present, the degree of the eventual recovery remains to be explored, but has strong implications for the well-being of collision-sport participants.

The goal of the study was to evaluate how repetitive head traumas sustained by athletes in contact sports depend on sport and level of play. A total of 16 middle school football players, 107 high school football players, and 65 high school female soccer players participated. Players were separated into levels of play: middle school (MS), freshman (FR), junior varsity (JV), junior varsity-varsity (JV-V), and varsity (V). xPatch sensors were used to measure peak translational and angular accelerations (PTA and PAA, respectively) for each head acceleration event (HAE) during practice and game sessions. Data were analyzed using a custom MATLAB program to compare metrics that have been correlated with functional neurological changes: session metrics (median HAEs per contact session), season metrics (total HAEs, cumulative PTA/PAA), and regressions (cumulative PTA/PAA versus total HAEs, total HAEs versus median HAEs per contact session). Football players had greater session (p<.001) and season (p<.001) metrics than soccer players, but soccer players had a significantly greater player average PAA per HAE than football players (p<.001). Middle school football players had similar session and season metrics to high school level athletes. In conclusion, sport has a greater influence on HAE characteristics than level of play.

Objective: The goal of this pilot study was to evaluate the number of head acceleration events (HAEs) based on position, play type, and starting stance. Design: Prospective cohort study. Setting: Postcollegiate skill development camp during practice sessions and 1 exhibition game. Participants: Seventy-eight male adult North American football athletes. Independent Variables: A position was assigned to each participant, and plays in the exhibition game were separated by play type for analysis. During the exhibition game, video data were used to determine the effects of the starting position (“up” in a 2-point stance or “down” in a 3- or 4-point stance) on the HAEs experienced by players on the offensive line. Main Outcome Measures: Peak linear acceleration and number of HAEs greater than 20 g (g = 9.81 m/s 2 ) were measured using an xPatch (X2 Biosystems, Seattle, WA). Results: Four hundred thirty-seven HAEs were recorded during practices and 272 recorded during the exhibition game; 98 and 52 HAEs, the greatest number of HAEs by position in the game, were experienced by the offensive and defensive linemen, respectively. Linebackers and tight ends experienced high percentages of HAEs above 60 g. Offensive line players in a down stance had a higher likelihood of sustaining a HAE than players in an up stance regardless of the type of play (run vs pass). Conclusions: Changing the stance of players on the offensive line and reducing the number of full-contact practices will lower HAEs.

Most fatal human skull injuries occur under impact loading conditions, such as car collisions, where the strain rates fall in the range of intermediate (1/s-10(2)/s) and high (10(2)/s-10(3)/s) rates. Therefore, knowledge of the mechanical behaviors of human cranial bone at higher strain rates, i.e., intermediate and high strain rates, may provide insight into the prevention of skull injuries and help the design of efficient head protection systems. In the present study, the compressive mechanical behaviors of human frontal skull bone along and perpendicular to its through-the-thickness direction were experimentally characterized at quasi-static (0.01/s), intermediate (30/s) and high (625/s) strain rates in this study. A total number of 75 specimens prepared from three male donors with ages of 70-74 were separated into three groups: quasi-static (N = 23), intermediate (N = 23), and high (N = 29) strain rates. Experiments at quasi-static and intermediate strain rates were performed using a hydraulically driven materials testing system (MTS), while a Kolsky compression bar was used to load the skull bone specimen at high strain rates. X-ray computed tomography was performed to obtain the structural parameters and visualize the microstructures of the skull bone. The in-situ failure processes of the specimens under high-rate loading were documented by a high-speed camera. The human skull exhibited a loading-direction dependent mechanical behavior, as higher ultimate strength and elastic modulus were found in the direction perpendicular to the thickness when compared with those along the thickness direction, exhibiting an increasing ratio as high as 2 and 3 for strength and modulus, respectively. High-speed images revealed that the specimens loaded along the thickness direction generally failed due to the crushing in diploe (the trabecular bone tissue) whereas separation of the entire architecture was observed as the main failure mode when compressed in the perpendicular direction. The effect of loading rate was also evident: the skull specimens were increasingly brittle as strain rate increased from quasi-static to high rate for both the loading directions. The elastic modulus increased by a factor of 4 in radial direction and it increased by a factor of 2.5 in the tangential direction across the quasi-static, intermediate and high strain rates. Significant differences were also found in ultimate strength and work to failure as loading rate increased from quasi-static to high rates. The results also suggested that the strength in the radial direction was mainly depended on the diploe porosity while the diploe layer ratio played the predominant role in tangential direction.

Implanted electrodes provide one of the most important neurotechniques for fundamental and translational neurosciences by permitting time-resolved electri-cal detection of individual neurons in vivo. However, conventional rigid electrodes typically cannot provide stable, long-lasting recordings. Numerous interwoven biotic and abiotic factors at the tissue-electrode interface lead to short-and long-term instability of the recording performance. Making neural electrodes flexible provides a promising approach to mitigate these challenges on the implants and at the tissue-electrode interface. Here we review the recent progress of ultraflexible neural electrodes and discuss the engineering principles, the material properties, and the implantation strategies to achieve stable tissue -electrode interface and reliable unit recordings in living brains.

Implanted neural probes are among the most important techniques in both fundamental and clinical neuroscience. Despite great successes and promise, neural electrodes are technically limited by their scalability. A recent study by Obaid et al. demonstrated an innovative way to greatly scale up the channel count and density of neural electrode arrays.

During chondrogenesis, tissue organization changes dramatically. We previously showed that the compressive moduli of chondrocytes increase concomitantly with extracellular matrix (ECM) stiffness, suggesting cells were remodeling to adapt to the surrounding environment. Due to the difficulty in analyzing the mechanical response of cells in situ, we sought to create an in silico model that would enable us to investigate why cell and ECM stiffness increased in tandem. The goal of this study was to establish a methodology to segment, quantify, and generate mechanical models of developing cartilage to explore how variations in geometry and material properties affect strain distributions. Multicellular geometries from embryonic day E16.5 and postnatal day P3 murine cartilage were imaged in three-dimensional (3D) using confocal microscopy. Image stacks were processed using MATLAB to create geometries for finite element analysis using ANSYS. The geometries based on confocal images and isolated, single cell models were compressed 5% and the equivalent von Mises strain of cells and ECM were compared. Our simulations indicated that cells had similar strains at both time points, suggesting that the stiffness and organization of cartilage changes during development to maintain a constant strain profile within cells. In contrast, the ECM at P3 took on more strain than at E16.5. The isolated, single-cell geometries underestimated both cell and ECM strain and were not able to capture the similarity in cell strain at both time points. We expect this experimental and computational pipeline will facilitate studies investigating other model systems to implement physiologically derived geometries.

We experimentally determined the tensile stress-strain response of human muscle along fiber direction and compressive stress-strain response transverse to fiber direction at intermediate strain rates (10(0)-10(2)/s). A hydraulically driven material testing system with a dynamic testing mode was used to perform the tensile and compressive experiments on human muscle tissue. Experiments at quasi-static strain rates (below 10(0)/s) were also conducted to investigate the strain-rate effects over a wider range. The experimental results show that, at intermediate strain rates, both the human muscle's tensile and compressive stress-strain responses are nonlinear and strain-rate sensitive. Human muscle also exhibits a stiffer and stronger tensile mechanical behavior along fiber direction than its compressive mechanical behavior along the direction transverse to fiber direction. An Ogden model with two material constants was adopted to describe the nonlinear tensile and compressive behaviors of human muscle.

Recent evidence of short-term alterations in brain physiology associated with repeated exposure to moderate intensity subconcussive head acceleration events (HAEs), prompts the question whether these alterations represent an underlying neural injury. A retrospective analysis combining counts of experienced HAEs and longitudinal diffusion-weighted imaging explored whether greater exposure to incident mechanical forces was associated with traditional diffusion-based measures of neural injury-reduced fractional anisotropy (FA) and increased mean diffusivity (MD). Brains of high school athletes (N = 61) participating in American football exhibited greater spatial extents (or volumes) experiencing substantial changes (increases and decreases) in both FA and MD than brains of peers who do not participate in collision-based sports (N = 15). Further, the spatial extents of the football athlete brain exhibiting traditional diffusion-based markers of neural injury were found to be significantly correlated with the cumulative exposure to HAEs having peak translational acceleration exceeding 20 g. This finding demonstrates that subconcussive HAEs induce low-level neurotrauma, with prolonged exposure producing greater accumulation of neural damage. The duration and extent of recovery associated with periods in which athletes do not experience subconcussive HAEs now represents a priority for future study, such that appropriate participation and training schedules may be developed to minimize the risk of long-term neurological dysfunction.

Perlecan, or heparan sulfate proteoglycan (Hspg2), is an extracellular matrix (ECM) protein localized to the pericellular matrix (PCM). When Hspg2 is knocked down, the phenotype mimics the skeletal defects observed in human Schwartz‐Jampel syndrome. We previously demonstrated, using atomic force microscopy, that perlecan knockdown significantly decreased the stiffness of the ECM and chondrocytes in developing cartilage; however, it is not clear what changes occur in ECM structure and organization to cause this decrease. To address this question, our goal is to develop computational models that incorporate geometries and material properties that will enable us to analyze the mechanical response of cells in situ. Therefore, we created 3D physiologically‐derived geometries that can generate repeatable simulations of cartilage mechanics. In parallel, we are using mass spectrometry (LC‐MS/MS) and transmission electron microscopy (TEM) to define how cartilage composition and organization changes as a function of development and perlecan knockdown.Multi‐cellular geometries from embryonic day (E)16.5 and postnatal day (P)3 murine cartilage were imaged in 3D using confocal microscopy. Image stacks were processed using MATLAB to create multicellular geometries for finite element analysis using ANSYS (Fig 1A,B). Geometries based on confocal images and isolated, single cell models were compressed 5% and cells and ECM strain were compared (Fig 1C,D). Our simulations indicated that cells had similar strains at both time points, even though cell and ECM stiffness at P3 was significantly higher than at E16.5. In addition, the ECM at P3 took on more strain than at E16.5. The isolated, single cell geometries underestimated both cell and ECM strain and were not able to capture the similarity in cell strain observed with physiologically‐derived geometries.A limitation of the current model is that cells and ECM were treated as homogenous materials, whereas native cartilage is a biphasic material comprised of type II collagen fibrils embedded in an amorphous proteoglycan matrix. To identify how ECM composition and organization changes, cartilage from distal humeri or femurs were dissected from E16.5 and P3 wildtype, heterozygous, and homozygous Hspg2 mice for analysis via LC‐MS/MS or TEM. Proteins were extracted with guanidine‐HCl and processed for LC‐MS/MS. Relative increases in the abundance of ECM and associated proteins, including collagen types II & IX and aggrecan highlighted expected developmental changes between E16.5 and P3. Multiple proteins were significantly more abundant in homozygous vs. wildtype P3 mice, including collagens type II & IX, fibronectin and matrilin‐3. Surprisingly, TEM studies indicate that collagen fibril density and diameter decreased between wildtype and homozygous mice, suggesting that perlecan knockdown leads to a decrease in cross‐linking and increased ECM extractability. We aim to integrate these changes in material properties with our simulations to generate a computational model that can describe and predict the mechanics of developing cartilage.Support or Funding InformationThis work was supported by the National Institutes of Health [R21 AR069248, R01 AR071359 and DP2 AT009833 to S.C.].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Peripheral artery disease (PAD) is a broad disorder encompassing multiple forms of arterial disease outside of the heart. As such, PAD development is a multifactorial process with a variety of manifestations. For example, aneurysms are pathological expansions of an artery that can lead to rupture, while ischemic atherosclerosis reduces blood flow, increasing the risk of claudication, poor wound healing, limb amputation, and stroke. Current PAD treatment is often ineffective or associated with serious risks, largely because these disorders are commonly undiagnosed or misdiagnosed. Active areas of research are focused on detecting and characterizing deleterious arterial changes at early stages using non-invasive imaging strategies, such as ultrasound, as well as emerging technologies like photoacoustic imaging. Earlier disease detection and characterization could improve interventional strategies, leading to better prognosis in PAD patients. While rodents are being used to investigate PAD pathophysiology, imaging of these animal models has been underutilized. This review focuses on structural and molecular information and disease progression revealed by recent imaging efforts of aortic, cerebral, and peripheral vascular disease models in mice, rats, and rabbits. Effective translation to humans involves better understanding of underlying PAD pathophysiology to develop novel therapeutics and apply non-invasive imaging techniques in the clinic.