Children and Adolescents with Early Treated Phenylketonuria: Cognitive Development and Fluctuations of Blood Phenylalanine Levels.

BACKGROUND: We assessed the relationship between the cognitive development of children and adolescents with phenylketonuria (PKU) and fluctuations in peripheral phenylalanine (Phe) levels. METHODS: We examined the neurocognitive performance of 33 children and adolescents with early treated PKU, of whom 18 were treated with sapropterin dihydrochloride, and 15 were on a classic diet. For 26 weeks, patients were assessed weekly for their blood phenylalanine (Phe) levels. Phe levels were analyzed for fluctuations indicated by the individual standard deviation. Fluctuations were compared to the standard deviation of 26 Phe level measurements before the study interval. We also assessed the concurrent IQ of the patients. This was repeated at one-, two-, and seven-year intervals. RESULTS: Full-scale IQ in patients treated with a classic diet did not change within the follow-up. In patients treated with Sapropterin dihydrochloride, however, there was a considerable gain in full-scale IQ. This was particularly true if blood Phe fluctuations increased in patients of this treatment group. CONCLUSIONS: Sapropterin dihydrochloride enhances Phe tolerance in patients with PKU. Increasing blood Phe fluctuations following enhanced Phe tolerance may indicate that the treatment not only allows patients to relax their Phe-restricted diet but also may support cognitive development in patients.

Differential roles of FOXC2 in the trabecular meshwork and Schlemm's canal in glaucomatous pathology.

Impaired development and maintenance of Schlemm's canal (SC) are associated with perturbed aqueous humor outflow and intraocular pressure. The angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and maintenance, whereas the molecular mechanisms of crosstalk between SC and the neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are poorly understood. Here, we show NC-specific forkhead box (Fox)c2 deletion in mice results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular pressure. Visible-light optical coherence tomography analysis further demonstrated functional impairment of the SC in response to changes in intraocular pressure in NC-Foxc2 -/- mice, suggesting altered TM biomechanics. Single-cell RNA-sequencing analysis identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM cell clusters, including increased matrix metalloproteinase expression, which can cleave the TIE2 ectodomain to produce soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC morphogenesis because of reduced TIE2 expression, which was rescued by deleting the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and morphogenesis via TM-SC crosstalk.

Adaptive spectroscopic visible-light optical coherence tomography for clinical retinal oximetry.

BACKGROUND: Retinal oxygen saturation (sO2) provides essential information about the eye's response to pathological changes that can result in vision loss. Visible-light optical coherence tomography (vis-OCT) is a noninvasive tool that has the potential to measure retinal sO2 in a clinical setting. However, its reliability is currently limited by unwanted signals referred to as spectral contaminants (SCs), and a comprehensive strategy to isolate true oxygen-dependent signals from SCs in vis-OCT is lacking. METHODS: We develop an adaptive spectroscopic vis-OCT (ADS-vis-OCT) technique that can adaptively remove SCs and accurately measure sO2 under the unique conditions of each vessel. We also validate the accuracy of ADS-vis-OCT using ex vivo blood phantoms and assess its repeatability in the retina of healthy volunteers. RESULTS: In ex vivo blood phantoms, ADS-vis-OCT agrees with a blood gas machine with only a 1% bias in samples with sO2 ranging from 0% to 100%. In the human retina, the root mean squared error between sO2 values in major arteries measured by ADS-vis-OCT and a pulse oximeter is 2.1% across 18 research participants. Additionally, the standard deviations of repeated ADS-vis-OCT measurements of sO2 values in smaller arteries and veins are 2.5% and 2.3%, respectively. Non-adaptive methods do not achieve comparable repeatabilities from healthy volunteers. CONCLUSIONS: ADS-vis-OCT effectively removes SCs from human images, yielding accurate and repeatable sO2 measurements in retinal arteries and veins with varying diameters. This work could have important implications for the clinical use of vis-OCT to manage eye diseases.

Numerous diseases that cause blindness are associated with disrupted oxygen consumption in the retina, the part of the eye that senses light. This highlights the importance of accurately measuring oxygen consumption in the clinic. To address this challenge, we developed a method to analyze images of the retina which have been collected using visible-light optical coherence tomography, a non-invasive imaging method. Our approach achieves accurate oxygen level measurements in blood samples and in healthy volunteers. With further testing, our approach may prove useful in the clinical management of several diseases that cause blindness, allowing clinicians to more accurately diagnose disease and monitor the health of the eye.

Recent advances in optical coherence tomography for anterior segment imaging in small animals and their clinical implications.

Anterior segment optical coherence tomography (AS-OCT) is a rapidly evolving area of OCT imaging, providing high-resolution and non-invasive volumetric imaging of the anterior segment. This review focuses on recent advances in AS-OCT imaging in small animals, which we categorize into ultrahigh-resolution, spectroscopic, magnetomotive, polarization-sensitive, and angiographic AS-OCTs. We summarize their technical foundations, review their applications to small animal imaging, and briefly discuss their current and future clinical applications.

Task and information conflicts in the numerical Stroop task.

Studies of the Stroop color-word task have provided evidence for the existence of two conflicts: (1) an early task conflict between noting the relevant color and reading afforded by the irrelevant word (or word-like stimuli), and (2) a late information conflict between the information provided by the word and the information provided by the color. Measurements of pupil changes, in addition to reaction time (RT), have extended understanding regarding these two conflicts. The current work examines the generalizability of such understanding. We ask whether similar processes work in the comparative judgment of numbers (e.g., in the numerical Stroop task). We present two experiments that support and extend the knowledge gained in the word-color context to numerical processing. Similar to results with the Stroop color-word task, we found a dissociation between RT and pupillometry and an early task conflict followed by an information conflict.

In vivo imaging of the inner retinal layer structure in mice after eye-opening using visible-light optical coherence tomography.

The growth of the mouse eye and retina after birth is a dynamic, highly regulated process. In this study, we applied visible-light optical coherence tomography (vis-OCT), a non-invasive imaging technique, to examine developing retinal layer structures after eye-opening. We introduced a resampled circumpapillary B-scan averaging technique to improve the inter-layer contrast, enabling retinal layer thickness measurements as early as postnatal day 13 (P13) - right after eye-opening. We confirmed vis-OCT measurements using ex vivo confocal microscopy of retinal sections at different ages. Our results demonstrate that vis-OCT can visualize the developmental murine retinal layer structure in vivo, which offers us new opportunities to better characterize the pathological alterations in mouse models of developmental eye diseases.

Super-resolution imaging of flat-mounted whole mouse cornea.

Super-resolution microscopy revolutionized biomedical research with significantly improved imaging resolution down to the molecular scale. To date, only limited studies reported multi-color super-resolution imaging of thin tissue slices mainly because of unavailable staining protocols and incompatible imaging techniques. Here, we show the first super-resolution imaging of flat-mounted whole mouse cornea using single-molecule localization microscopy (SMLM). We optimized immunofluorescence staining protocols for ß-Tubulin, Vimentin, Peroxisome marker (PMP70), and Histone-H4 in whole mouse corneas. Using the optimized staining protocols, we imaged these four intracellular protein structures in the epithelium and endothelium layers of flat-mounted mouse corneas. We also achieved simultaneous two-color spectroscopic SMLM (sSMLM) imaging of ß-Tubulin and Histone-H4 in corneal endothelial cells. The spatial localization precision of sSMLM in these studies was around 20-nm. This work sets the stage for investigating multiple intracellular alterations in corneal diseases at a nanoscopic resolution using whole corneal flat-mount beyond cell cultures.

Intravenous ketamine for long term anesthesia in rats.

Ketamine/xylazine anesthesia has been used primarily for short term procedures in animals, but two prior reports used intravenous ketamine/xylazine for experiments taking many hours. However, there is a discrepancy about the appropriate dose, which is resolved here. Adult Long-Evans rats were used for recording from the retina. Doses of Ketamine/xylazine were adjusted to minimize anesthetic in terminal experiments lasting 10 h. An allometric relation was fitted to the resulting data on doses as a function of body weight, and compared to prior work. The allometric relationship between the continuously infused specific dose and weight was: dose = 9.13 (weight)-1.213 (r2 = 0.73), where dose is in mg-kg-1-hr-1 and rat weight is in kg. The dose of xylazine was 3.3% of the ketamine dose. No attempt was made to explore different relative doses of xylazine and ketamine. Prior work is consistent with this relationship, showing that the earlier discrepancy resulted from using rats of different sizes. Ketamine at the doses used here still depressed the electroretinogram relative to historical controls using urethane. We conclude that intravenous ketamine dosing in rats should not use the same mg-kg-1-hr-1 dose for all rats, but take into account the strong allometric relationship between dose and rat weight. There is an advantage in using smaller doses in order to prevent unnecessary depression of neural responses.

In Vivo Imaging of Schlemm's Canal and Limbal Vascular Network in Mouse Using Visible-Light OCT.

Purpose: To validate the ability of visible-light optical coherence tomography (vis-OCT) in imaging the full Schlemm's canal (SC) and its surrounding limbal vascular network in mice in vivo through a compound circumlimbal scan. Methods: We developed an anterior segment vis-OCT system and a compound circumlimbal scanning method, which montages eight rotated raster scans. We calibrated the circumlimbal scan geometry using a three-dimensional printed phantom eyeball before imaging wild-type C57BL/6J mice. We measured SC size by segmenting SC cross sections from vis-OCT B-scan images and imaged the limbal microvascular network using vis-OCT angiography (vis-OCTA). To introduce changes in SC size, we used a manometer to adjust the intraocular pressure (IOP) to different levels. To create additional optical scattering contrast to enhance SC imaging, we surgically increased the episcleral venous pressure (EVP) and caused blood reflux into SC. Results: Using the compound circumlimbal scan, our anterior segment vis-OCT noninvasively imaged the full SC and limbal microvascular network in mouse for the first time. We observed an average 123% increase in SC volume when we decreased the IOP by 10 mm Hg from the baseline IOP of 7 to 10 mm Hg and an average 72% decrease in SC volume when the IOP level was elevated by 10 mm Hg from the baseline IOP. We also observed location-dependent SC size responses to IOP changes. Blood reflux caused by increased EVP enabled vis-OCTA to directly visualize SC, which matched well with the segmented SC. Conclusions: Vis-OCT and vis-OCTA can accurately image the entire SC and limbal microvascular network in vivo using the compound circumlimbal scan. Vis-OCT is also able to quantitatively measure SC responses to changing IOP levels.

Longitudinal deep-brain imaging in mouse using visible-light optical coherence tomography through chronic microprism cranial window.

We longitudinally imaged both the superficial and deep cortical microvascular networks in brains of healthy mice and in a mouse model of stroke in vivo using visible-light optical coherence tomography (vis-OCT). We surgically implanted a microprism in mouse brains sealed by a chronic cranial window. The microprism enabled vis-OCT to image the entire depth of the mouse cortex. Following microprism implantation, we imaged the mice for 28 days and found that that it took around 15 days for both the superficial and deep cortical microvessels to recover from the implantation surgery. After the brains recovered, we introduced ischemic strokes by transient middle cerebral artery occlusion (tMCAO). We monitored the strokes for up to 60 days and observed different microvascular responses to tMCAO at different cortical depths in both the acute and chronic phases of the stroke. This work demonstrates that the combined microprism and cranial window is well-suited for longitudinal investigation of cortical microvascular disorders using vis-OCT.

Speckle reduction in visible-light optical coherence tomography using scan modulation.

We present a technique to reduce speckle in visible-light optical coherence tomography (vis-OCT) that preserves fine structural details and is robust against sample motion. Specifically, we locally modulate B-scans orthogonally to their axis of acquisition. Such modulation enables acquisition of uncorrelated speckle patterns from similar anatomical locations, which can be averaged to reduce speckle. To verify the effectiveness of speckle reduction, we performed in-vivo retinal imaging using modulated raster and circular scans in both mice and humans. We compared speckle-reduced vis-OCT images with the images acquired with unmodulated B-scans from the same anatomical locations. We compared contrast-to-noise ratio (CNR) and equivalent number of looks (ENL) to quantify the image quality enhancement. Speckle-reduced images showed up to a 2.35-dB improvement in CNR and up to a 3.1-fold improvement in ENL with more discernable anatomical features using eight modulated A-line averages at a 25-kHz A-line rate.

Designing visible-light optical coherence tomography towards clinics.

BACKGROUND: The capabilities of visible-light optical coherence tomography (vis-OCT) in noninvasive anatomical and functional retinal imaging have been demonstrated by multiple groups in both rodents and healthy human subjects. Translating laboratory prototypes to an integrated clinical-environment-friendly system is required to explore the full potential of vis-OCT in disease management. METHODS: We developed and optimized a portable vis-OCT system for human retinal imaging in clinical settings. We acquired raster- and circular-scan images from both healthy and diseased human eyes. RESULTS: The new vis-OCT provided high-quality retinal images of both subjects without any known eye diseases and patients with various retinal diseases, including retinal occlusive disease and diabetic retinopathy (DR) over a broad range of ages. CONCLUSIONS: A newly designed vis-OCT system is sufficiently optimized to be suited for routine patients' examinations in clinics. Vis-OCT has the potential to add new anatomical and functional imaging capabilities to ophthalmic clinical care.

Visible-light optical coherence tomography oximetry based on circumpapillary scan and graph-search segmentation.

Visible-light optical coherence tomography (vis-OCT) enables retinal oximetry by measuring the oxygen saturation of hemoglobin (sO2) from within individual retinal blood vessels. The sO2 calculation requires reliable estimation of the true spectrum of backscattered light from the posterior vessel wall. Unfortunately, subject motion and image noise make averaging from multiple A-lines at the same depth position challenging, and lead to inaccurate sO2 estimation. In this study, we developed an algorithm to reliably extract the backscattered light's spectrum. We used circumpapillary scanning to sample the vessels repeatedly at the same location. A combination of cross-correlation and graph-search based segmentation extracted the posterior wall locations. Using measurements from 100 B-scans as a gold standard, we demonstrated that our method achieved highly accurate measures of sO2 with minimal bias. In addition, we also investigated how the number of repeated measurements affects the accuracy of sO2 measurement. Our method sets the stage for large-scale studies of retinal oxygenation in animals and humans.

Visible-light optical coherence tomography: a review.

Visible-light optical coherence tomography (vis-OCT) is an emerging imaging modality, providing new capabilities in both anatomical and functional imaging of biological tissue. It relies on visible light illumination, whereas most commercial and investigational OCTs use near-infrared light. As a result, vis-OCT requires different considerations in engineering design and implementation but brings unique potential benefits to both fundamental research and clinical care of several diseases. Here, we intend to provide a summary of the development of vis-OCT and its demonstrated applications. We also provide perspectives on future technology improvement and applications.

Optical coherence tomography angiography of retinal vascular occlusions produced by imaging-guided laser photocoagulation.

Retinal vascular occlusive diseases represent a major form of vision loss worldwide. Rodent models of these diseases have traditionally relied upon a slit-lamp biomicroscope to help visualize the fundus and subsequently aid delivery of high-power laser shots to a target vessel. Here we describe a multimodal imaging system that can produce, image, and monitor retinal vascular occlusions in rodents. The system combines a spectral-domain optical coherence tomography system for cross-sectional structural imaging and three-dimensional angiography, and a fluorescence scanning laser ophthalmoscope for Rose Bengal monitoring and high-power laser delivery to a target vessel. This multimodal system facilitates the precise production of occlusions in the branched retinal veins, central retinal vein, and branched retinal arteries. Additionally, changes in the retinal morphology and retinal vasculature can be longitudinally documented. With our device, retinal vascular occlusions can be easily and consistently created, which paves the way for futures studies on their pathophysiology and therapeutic targets.

Optimal electrode placement and frequency range selection for the detection of lung water using bioimpedance spectroscopy.

Bioimpedance Spectroscopy (BIS) is a non-invasive technique for the determination of human body composition such as water or fat content. Next to absolute body water determination, it enables also the detection of body water shifts. Therefore, it seems to be a good candidate to detect cardiac lung edema at an early stage and to monitor its medical treatment. However, stable long term BIS measurements are very difficult to make. The measurement conditions and hardware specifications of BIS devices must be well chosen in order to get correct and reproducible results. Thus, a Finite Element Simulation of the human thorax is implemented to find the most sensitive electrode position for a BIS measurement as well as a MatLab simulation of the optimal frequency range. Both simulations and results are presented in this article and shall contribute to increase the accuracy and significance of BIS thoracic measurements.