Real-Time Automated Segmentation of Median Nerve in Dynamic Ultrasonography Using Deep Learning.

OBJECTIVE: The morphological dynamics of the median nerve across the level extracted from dynamic ultrasonography are valuable for the diagnosis and evaluation of carpal tunnel syndrome (CTS), but the data extraction requires tremendous labor to manually segment the nerve across the image sequence. Our aim was to provide visually real-time, automated median nerve segmentation and subsequent data extraction in dynamic ultrasonography. METHODS: We proposed a deep-learning model modified from SOLOv2 and tailored for median nerve segmentation. Ensemble strategies combining several state-of-the-art models were also employed to examine whether the segmentation accuracy could be improved. Image data were acquired from nine normal participants and 59 patients with idiopathic CTS. DISCUSSION: Our model outperformed several state-of-the-art models with respect to inference speed, whereas the segmentation accuracy was on a par with that achieved by these models. When evaluated on a single 1080Ti GPU card, our model achieved an intersection over union score of 0.855 and Dice coefficient of 0.922 at 28.9 frames/s. The ensemble models slightly improved segmentation accuracy. CONCLUSION: Our model has great potential for use in the clinical setting, as the real-time, automated extraction of the morphological dynamics of the median nerve allows clinicians to diagnose and treat CTS as the images are acquired.

Automated Segmentation of Median Nerve in Dynamic Sonography Using Deep Learning: Evaluation of Model Performance.

There is an emerging trend to employ dynamic sonography in the diagnosis of entrapment neuropathy, which exhibits aberrant spatiotemporal characteristics of the entrapped nerve when adjacent tissues move. However, the manual tracking of the entrapped nerve in consecutive images demands tons of human labors and impedes its popularity clinically. Here we evaluated the performance of automated median nerve segmentation in dynamic sonography using a variety of deep learning models pretrained with ImageNet, including DeepLabV3+, U-Net, FPN, and Mask-R-CNN. Dynamic ultrasound images of the median nerve at across wrist level were acquired from 52 subjects diagnosed as carpal tunnel syndrome when they moved their fingers. The videos of 16 subjects exhibiting diverse appearance and that of the remaining 36 subjects were used for model test and training, respectively. The centroid, circularity, perimeter, and cross section area of the median nerve in individual frame were automatically determined from the inferred nerve. The model performance was evaluated by the score of intersection over union (IoU) between the annotated and model-predicted data. We found that both DeepLabV3+ and Mask R-CNN predicted median nerve the best with averaged IOU scores close to 0.83, which indicates the feasibility of automated median nerve segmentation in dynamic sonography using deep learning.

Hydrostatic pressure promotes migration and filamin-A activation in fibroblasts with increased p38 phosphorylation and TGF-ß production.

Fibroblast migration is closely regulated by the mechanical characteristics in surrounding microenvironment. While increased interstitial hydrostatic pressure (HP) is a hallmark in many pathological and physiological conditions, little is known about how the HP affects fibroblast motility. Using cell-culture chips with elevated HP conditions, we showed that 20 cmH2O HP significantly accelerated fibroblast migration. The HP-induced migration acceleration was dependent on the augmentation of transforming growth factor-ß1, and correlated with the activation of filamin A via the phosphorylation of p38 mitogen-activated protein kinase. Our results suggest that interstitial HP elevation associated with various pathological states could significantly regulate fibroblast migration.

Effect of Perineural Injection with Different Dextrose Volumes on Median Nerve Size, Elasticity and Mobility in Hands with Carpal Tunnel Syndrome.

This study aimed to investigate the effect of different injectate volumes on ultrasonographic parameters and the correlation to clinical outcomes under perineural dextrose injection (PDI). In this post hoc analysis of the randomized, double-blinded, three-arm trial, ultrasound-guided PDI with either 1 mL, 2 mL, and 4 mL 5% dextrose water was administered, respectively, in 14, 14, and 17 patients. Ultrasound outcomes included mobility, shear-wave elastography (SWE), and cross-sectional area (CSA) of the median nerve; clinical outcomes were Visual Analog Scale (VAS) and Boston Carpal Tunnel Questionnaire (BCTQ) score. Outcomes were measured before injection, and after injection at the 1st, 4th, 12th, and 24th week. For ultrasound outcomes, CSA decreased significantly from baseline data at all follow-up time-points in the 2 mL group (p = 0.005) and the 4 mL group (p = 0.015). The mean change of mobility from baseline showed a greater improvement on the 4 mL group than the other groups at the 1st week post-injection. For clinical outcomes, negative correlation between the VAS and mobility at the 1st (p = 0.046) and 4th week (p = 0.031) post-injection in the 4 mL group were observed. In conclusion, PDI with higher volume yielded better nerve mobility and decreased CSA of median nerve, but no changes of nerve elasticity.

Pathogenic Hydrogel? A Novel-Entrapment Neuropathy Model Induced by Ultrasound-Guided Perineural Injections.

Entrapment neuropathy (EN) is a prevalent and debilitative condition caused by a complex pathogenesis that involves a chronic compression-edema-ischemia cascade and perineural adhesion that results in excessive shear stress during motion. Despite decades of research, an easily accessible and surgery-free animal model mimicking the mixed etiology is currently lacking, thus limiting our understanding of the disease and the development of effective therapies. In this proof-of-concept study, we used ultrasound-guided perineural injection of a methoxy poly(ethylene glycol)-b-Poly(lactide-co-glycoilide) carboxylic acid (mPEG-PLGA-BOX) hydrogel near the rat's sciatic nerve to induce EN, as confirmed sonographically, electrophysiologically, and histologically. The nerve that was injected with hydrogel appeared unevenly contoured and swollen proximally with slowed nerve conduction velocities across the injected segments, thus showing the compressive features of EN. Histology showed perineural cellular infiltration, deposition of irregular collagen fibers, and a possible early demyelination process, thus indicating the existence of adhesions. The novel method provides a surgery-free and cost-effective way to establish a small-animal model of EN that has mixed compression and adhesion features, thus facilitating the additional elucidation of the pathophysiology of EN and the search for promising treatments.

Evaluation of cytotoxic T lymphocyte-mediated anticancer response against tumor interstitium-simulating physical barriers.

Tumor antigen-specific cytotoxic T lymphocyte (CTL) is a promising agent for cancer therapy. Most solid tumors are characterized by increased interstitial fluid pressure (IFP) and dense collagen capsule, which form physical barriers to impede cancer treatment. However, it remains unclear how CTL-mediated anticancer response is affected at the presence of these obstacles. Using a microfluidic-based platform mimicking these obstacles, we investigated the migration characteristics and performance of anticancer response of CTLs targeting hepatic cancer cells via antigen-specific and allogeneic recognition. The device consisted of slit channels mimicking the narrow interstitial paths constrained by the fibrous capsule and increased IFP was simulated by applying hydrostatic pressure to the tumor center. We found that antigen-specificity of CTLs against the targeted cancer cells determined the cytotoxic efficacy of the CTLs but did not significantly affect the success rate in CTLs that attempted to infiltrate into the tumor center. When increased IFP was present in the tumor center, CTL recruitment to tumor peripheries was promoted but success of infiltration was hindered. Our results highlight the importance of incorporating the physical characteristics of tumor interstitum into the development of CTL-based cancer immunotherapy.

Two-Dimensional Laser-Align Device for Ultrasound-Guided Injection.

Ultrasound-guided injection is a widely used technique, however, it takes substantial amounts of time for novices to master the skill. The most critical issue to improve the accuracy of the injection is to align the needle with the scan plane of the ultrasound beam and orient the needle angle after piercing skin to aim at the targeted tissue. In the present study, we developed a two-dimensional laser align device to assist physicians to accurately position the needle in the scan plane and advance it at an angle correctly pointing to the target. The device is inexpensive, light-weighted, and easy to fabricate and accommodate for any types of ultrasound probe. Statistical analysis revealed that the assistance with our device significantly reduced the successful targeting time and times of retargeting in comparison with the traditional freehand approach or only with in-plane assistance for inexperience subjects. Our results indicate that the device exhibits great potential in effectively reducing the learning time to master the skill and speeding up the procedure for ultrasound-guided injection.

Effects of Extracorporeal Shock Wave-Mediated Transdermal Local Anesthetic Drug Delivery on Rat Caudal Nerves.

Cavitation plays a substantial role in the clinical effects of extracorporeal shock wave therapy (ESWT). It is also generally accepted as a major mechanism in sonophoresis. To identify the enhancing effect of extracorporeal shock wave-mediated transdermal drug delivery, 24 Wistar rats were randomly assigned to four groups: (i) topical application of a eutectic mixture of local anesthetics (EMLA); (ii) 1-MHz ultrasound; (iii) ESWT pre-treatment combined with EMLA application; (iv) ESWT concurrent with EMLA application on rat tails. The degree of anesthesia was assessed using the amplitude and latency of sensory nerve action potentials within 5 min after a 60-min EMLA application. The results indicated that ESWT pre-treatment and concurrent ESWT accelerated the anesthetic effects of the EMLA cream on the tail nerve (p < 0.05). This finding might indicate that shock wave-mediated transdermal drug delivery is possible during the ESWT period.

Shear-wave elasticity measurements of three-dimensional cell cultures for mechanobiology.

Studying mechanobiology in three-dimensional (3D) cell cultures better recapitulates cell behaviors in response to various types of mechanical stimuli in vivo Stiffening of the extracellular matrix resulting from cell remodeling potentiates many pathological conditions, including advanced cancers. However, an effective tool for measuring the spatiotemporal changes in elastic properties of such 3D cell cultures without directly contacting the samples has not been reported previously. We describe an ultrasonic shear-wave-based platform for quantitatively evaluating the spatiotemporal dynamics of the elasticity of a matrix remodeled by cells cultured in 3D environments. We used this approach to measure the elasticity changes of 3D matrices grown with highly invasive lung cancer cells and cardiac myoblasts, and to delineate the principal mechanism underlying the stiffening of matrices remodeled by these cells. The described approach can be a useful tool in fields investigating and manipulating the mechanotransduction of cells in 3D contexts, and also has potential as a drug-screening platform.

Shear Wave Measurements for Evaluation of Tendon Diseases.

This paper investigated the feasibility of using supersonic shear wave measurements to quantitatively differentiate normal and damaged tendons based on their mechanical properties. Five freshly harvested porcine tendons excised from pig legs were used. Tendon damage was induced by incubating the tendons with a 1% w/v collagenase solution. Values of shear modulus were derived both by a time-of-flight (TOF) approach and a transverse isotropic plate model (TI-model). The results show that as the preload applied to the tendon increased from 0 to 3 N, the mean shear modulus derived based on the TOF approach, the TI-model, and Young's modulus estimated from mechanical testing increased from 14.6 to 89.9 kPa, 53.9 to 348 kPa, and from 1.45 to 10.36 MPa, respectively, in untreated tendons, and from 8.4 to 67 kPa, 28 to 258 kPa, and from 0.93 to 7.2 MPa in collagenase-treated tendons. Both the TOF approach and the TI-model correlated well with the changes in Young's modulus. Although there is bias on the estimation of shear modulus using the TOF approach, it still provides statistical significance to differentiate normal and damaged tendons. Our data indicate that supersonic shear wave imaging is a valuable imaging technique to assess tendon stiffness dynamics and characterize normal and collagenase-damaged tendons.

Protective Effect of Vanillic Acid against Hyperinsulinemia, Hyperglycemia and Hyperlipidemia via Alleviating Hepatic Insulin Resistance and Inflammation in High-Fat Diet (HFD)-Fed Rats.

Excess free fatty acid accumulation from abnormal lipid metabolism results in the insulin resistance in peripheral cells, subsequently causing hyperinsulinemia, hyperglycemia and/or hyperlipidemia in diabetes mellitus (DM) patients. Herein, we investigated the effect of phenolic acids on glucose uptake in an insulin-resistant cell-culture model and on hepatic insulin resistance and inflammation in rats fed a high-fat diet (HFD). The results show that vanillic acid (VA) demonstrated the highest glucose uptake ability among all tested phenolic acids in insulin-resistant FL83B mouse hepatocytes. Furthermore, rats fed HFD for 16 weeks were orally administered with VA daily (30 mg/kg body weight) at weeks 13-16. The results show that levels of serum insulin, glucose, triglyceride, and free fatty acid were significantly decreased in VA-treated HFD rats (p < 0.05), indicating the protective effects of VA against hyperinsulinemia, hyperglycemia and hyperlipidemia in HFD rats. Moreover, VA significantly reduced values of area under the curve for glucose (AUCglucose) in oral glucose tolerance test and homeostasis model assessment-insulin resistance (HOMA-IR) index, suggesting the improving effect on glucose tolerance and insulin resistance in HFD rats. The Western blot analysis revealed that VA significantly up-regulated expression of hepatic insulin-signaling and lipid metabolism-related protein, including insulin receptor, phosphatidylinositol-3 kinase, glucose transporter 2, and phosphorylated acetyl CoA carboxylase in HFD rats. VA also significantly down-regulated hepatic inflammation-related proteins, including cyclooxygenase-2 and monocyte chemoattractant protein-1 expressions in HFD rats. These results indicate that VA might ameliorate insulin resistance via improving hepatic insulin signaling and alleviating inflammation pathways in HFD rats. These findings also suggest the potential of VA in preventing the progression of DM.

Modulating chemotaxis of lung cancer cells by using electric fields in a microfluidic device.

We employed direct-current electric fields (dcEFs) to modulate the chemotaxis of lung cancer cells in a microfluidic cell culture device that incorporates both stable concentration gradients and dcEFs. We found that the chemotaxis induced by a 0.5 µM/mm concentration gradient of epidermal growth factor can be nearly compensated by a 360 mV/mm dcEF. When the effect of chemical stimulation was balanced by the electrical drive, the cells migrated randomly, and the path lengths were largely reduced. We also demonstrated electrically modulated chemotaxis of two types of lung cancer cells with opposite directions of electrotaxis in this device.

Substrate stiffness regulates filopodial activities in lung cancer cells.

Microenvironment stiffening plays a crucial role in tumorigenesis. While filopodia are generally thought to be one of the cellular mechanosensors for probing environmental stiffness, the effects of environmental stiffness on filopodial activities of cancer cells remain unclear. In this work, we investigated the filopodial activities of human lung adenocarcinoma cells CL1-5 cultured on substrates of tunable stiffness using a novel platform. The platform consists of an optical system called structured illumination nano-profilometry, which allows time-lapsed visualization of filopodial activities without fluorescence labeling. The culturing substrates were composed of polyvinyl chloride mixed with an environmentally friendly plasticizer to yield Young's modulus ranging from 20 to 60 kPa. Cell viability studies showed that the viability of cells cultured on the substrates was similar to those cultured on commonly used elastomers such as polydimethylsiloxane. Time-lapsed live cell images were acquired and the filopodial activities in response to substrates with varying degrees of stiffness were analyzed. Statistical analyses revealed that lung cancer cells cultured on softer substrates appeared to have longer filopodia, higher filopodial densities with respect to the cellular perimeter, and slower filopodial retraction rates. Nonetheless, the temporal analysis of filopodial activities revealed that whether a filopodium decides to extend or retract is purely a stochastic process without dependency on substrate stiffness. The discrepancy of the filopodial activities between lung cancer cells cultured on substrates with different degrees of stiffness vanished when the myosin II activities were inhibited by treating the cells with blebbistatin, which suggests that the filopodial activities are closely modulated by the adhesion strength of the cells. Our data quantitatively relate filopodial activities of lung cancer cells with environmental stiffness and should shed light on the understanding and treatment of cancer progression and metastasis.

Contribution of creatine to protein homeostasis in athletes after endurance and sprint running.

PURPOSE: Few studies have focused on the metabolic changes induced by creatine supplementation. This study investigated the effects of creatine supplementation on plasma and urinary metabolite changes of athletes after endurance and sprint running. METHODS: Twelve male athletes (20.3 ± 1.4 y) performed two identical (65-70 % maximum heart rate reserved) 60 min running exercises (endurance trial) before and after creatine supplementation (12 g creatine monohydrate/day for 15 days), followed by a 5-day washout period. Subsequently, they performed two identical 100 m sprint running exercises (power trial) before and after 15 days of creatine supplementation in accordance with the supplementary protocol of the endurance trial. Body composition measurements were performed during the entire study. Plasma samples were examined for the concentrations of glucose, lactate, branched-chain amino acids (BCAAs), free-tryptophan (f-TRP), glutamine, alanine, hypoxanthine, and uric acid. Urinary samples were examined for the concentrations of hydroxyproline, 3-methylhistidine, urea nitrogen, and creatinine. RESULTS: Creatine supplementation significantly increased body weights of the athletes of endurance trial. Plasma lactate concentration and ratio of f-TRP/BCAAs after recovery from endurance running were significantly decreased with creatine supplementation. Plasma purine metabolites (the sum of hypoxanthine and uric acid), glutamine, urinary 3-methylhistidine, and urea nitrogen concentrations tended to decrease before running in trials with creatine supplements. After running, urinary hydroxyproline concentration significantly increased in the power trial with creatine supplements. CONCLUSIONS: The findings suggest that creatine supplementation tended to decrease muscle glycogen and protein degradation, especially after endurance exercise. However, creatine supplementation might induce collagen proteolysis in athletes after sprint running.

Increased hydrostatic pressure enhances motility of lung cancer cells.

Interstitial fluid pressures within most solid tumors are significantly higher than that in the surrounding normal tissues. Therefore, cancer cells must proliferate and migrate under the influence of elevated hydrostatic pressure while a tumor grows. In this study, we developed a pressurized cell culture device and investigated the influence of hydrostatic pressure on the migration speeds of lung cancer cells (CL1-5 and A549). The migration speeds of lung cancer cells were increased by 50-60% under a 20 mmHg hydrostatic pressure. We also observed that the expressions of aquaporin in CL1-5 and A549 cells were increased under the hydrostatic pressure. Our preliminary results indicate that increased hydrostatic pressure plays an important role in tumor metastasis.

Microfluidic device with dual mechanical cues for cell migration investigation.

Cell migration plays an important role in numerous physiological and pathological conditions, such as angiogenesis, wound healing and cancer metastasis. Understanding the fundamental mechanisms of cell migration is crucial to develop strategies for disease treatment and regenerative medicine. Several biomechanical cues have been well studied about their effects on guiding cell migration. However, the effects of dual or multiple cues on cell migration are barely addressed. In this work, we developed a microfluidic-based device to study the combinatory effects of osmotic and stiffness gradient on cell migration. Computer simulation and experimental validation showed that the device was capable of providing stable osmotic and stiffness gradient to cultured cells at the same time. Preliminary results suggest that our device has a valuable potential in studying cell migration in complex conditions which better recapitulate the complex environmental conditions in vivo.

Imaging monitored loosening of dense fibrous tissues using high-intensity pulsed ultrasound.

Pulsed high-intensity focused ultrasound (HIFU) is proposed as a new alternative treatment for contracture of dense fibrous tissue. It is hypothesized that the pulsed-HIFU can release the contracted tissues by attenuating tensile stiffness along the fiber axis, and that the stiffness reduction can be quantitatively monitored by change of B-mode images. Fresh porcine tendons and ligaments were adapted to an ex vivo model and insonated with pulsed-HIFU for durations ranging from 5 to 30 min. The pulse length was 91 µs with a repetition frequency of 500 Hz, and the peak rarefactional pressure was 6.36 MPa. The corresponding average intensities were kept around 1606 W cm(-2) for ISPPA and 72.3 W cm(-2) for ISPTA. B-mode images of the tissues were acquired before and after pulsed-HIFU exposure, and the changes in speckle intensity and organization were analyzed. The tensile stiffness of the HIFU-exposed tissues along the longitudinal axis was examined using a stretching machine. Histology examinations were performed by optical and transmission electron microscopy. Pulsed-HIFU exposure significantly decreased the tensile stiffness of the ligaments and tendons. The intensity and organization of tissue speckles in the exposed region were also decreased. The speckle changes correlated well with the degree of stiffness alteration. Histology examinations revealed that pulsed-HIFU exposure probably damages tissues via a cavitation-mediated mechanism. Our results suggest that pulsed-HIFU with a low duty factor is a promising tool for developing new treatment strategies for orthopedic disorders.

Myocyte shape regulates lateral registry of sarcomeres and contractility.

The heart actively remodels architecture in response to various physiological and pathological conditions. Gross structural change of the heart chambers is directly reflected at the cellular level by altering the morphological characteristics of individual cardiomyocytes. However, an understanding of the relationship between cardiomyocyte shape and the contractile function remains unclear. By using in vitro assays to analyze systolic stress of cardiomyocytes with controlled shape, we demonstrated that the characteristic morphological features of cardiomyocytes observed in a variety of pathophysiological conditions are correlated with mechanical performance. We found that cardiomyocyte contractility is optimized at the cell length/width ratio observed in normal hearts, and decreases in cardiomyocytes with morphological characteristics resembling those isolated from failing hearts. Quantitative analysis of sarcomeric architecture revealed that the change of contractility may arise from alteration of myofibrillar structure. Measurements of intracellular calcium in myocytes revealed unique characteristics of calcium metabolism as a function of myocyte shape. Our data suggest that cell shape is critical in determining contractile performance of single cardiomyocytes by regulating the intracellular structure and calcium handling ability.

The migration speed of cancer cells influenced by macrophages and myofibroblasts co-cultured in a microfluidic chip.

We employ a microfluidic chip with three culture chambers to investigate the interactions among lung cancer cells, macrophages and myofibroblasts. By mixing the conditioned media of macrophages and myofibroblasts in this chip, we confirm that these two stromal cells have synergistic effects in accelerating the migration of cancer cells. However, as the myofibroblasts are pretreated with the conditioned medium of macrophages, the myofibroblasts' ability to enhance the migration of cancer cells is lowered. The tumour necrosis factor-α produced by macrophages reduces the expression of α-smooth muscle actin and the secretion of transforming growth factor-ß1 in myofibroblasts. Once the tumour necrosis factor-α in the macrophage conditioned medium is neutralized, the macrophage medium-pretreated myofibroblasts can still accelerate the migration of cancer cells.

Electrophysiologic evidence of spinal accessory neuropathy in patients with cervical myofascial pain syndrome.

OBJECTIVE: To evaluate whether or not spinal accessory neuropathy exists in patients with cervical myofascial pain syndrome (MFPS). DESIGN: Prospective study. SETTING: A neurophysiologic laboratory in a university hospital. PARTICIPANTS: Patients with cervical MFPS (n=25) and healthy controls (n=20). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: We performed nerve conduction studies (NCSs) in bilateral spinal accessory nerves, and electromyography and stimulated single-fiber electromyography in the trapezius muscles of all patients and controls. Parameters including nerve conduction velocities (NCVs), amplitudes and areas of compound muscle action potentials (CMAPs), and mean consecutive differences (MCDs) in single-fiber electromyography were measured, analyzed, and compared with the disease durations of the patients. RESULTS: Spinal accessory NCSs showed normative NCVs but with prominently reduced CMAP amplitude in the patients with cervical MFPS, which is recognized as an axonal neuropathy of the spinal accessory nerves. Electromyography showed prominent evidence of denervation and reinnervation patterns in 48% of the MFPS patients. The abnormal MCDs in single-fiber electromyography indicated a synaptic delay of motor endplates in the motor units, and may signify evolving instability of neuromuscular transmission in the spinal accessory nerves innervating trapezius muscles of the patients. CONCLUSIONS: This study demonstrates electrophysiologic evidence of neuroaxonal degeneration and neuromuscular transmission disorder in a significant proportion of patients with cervical MFPS. We suggest that spinal accessory neuropathy may be associated with cervical MFPS.