Perivascular neurons instruct 3D vascular lattice formation via neurovascular contact.

The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.

Comparison of Three Handheld Fundus Cameras for Assessment of the Vertical Cup-To-Disk Ratio.

Purpose To compare the quality of optic nerve photographs from three different handheld fundus cameras and to assess the reproducibility and agreement of vertical cup-to-disk ratio (VCDR) measurements from each camera. Methods Adult patients from a comprehensive ophthalmology clinic and an intravitreous injection clinic in northern Thailand were recruited for this cross-sectional study. Each participant had optic nerve photography performed with each of 3 handheld cameras: the Volk iNview, Volk Pictor Plus, and Peek Retina. Images were graded for VCDR in a masked fashion by two photo-graders and images with > 0.2 discrepancy in VCDR were assessed by a third photo-grader. Results A total of 355 eyes underwent imaging with three different handheld fundus cameras. Optic nerve images were judged ungradable in 130 (37%) eyes imaged with Peek Retina, compared to 36 (10%) and 55 (15%) eyes imaged with the iNview and Pictor Plus, respectively. For 193 eyes with gradable images from all 3 cameras, inter-rater reliability for VCDR measurements was poor or moderate for each of the cameras, with intraclass correlation coefficients ranging from 0.41 to 0.52. A VCDR ≥ 0.6 was found in 6 eyes on iNview images, 9 eyes on Pictor Plus images, and 3 eyes on Peek images, with poor agreement between cameras (e.g., no eyes graded as VCDR ≥ 0.6 on images from both the iNview and Pictor Plus). Conclusions Inter-rater reliability of VCDR grades from 3 handheld cameras was poor. Cameras did not agree on which eyes had large VCDRs.

Longitudinal Use of Telehealth During the COVID-19 Pandemic and Utility of Asynchronous Testing for Subspecialty-Level Ophthalmic Care.

Importance: Telehealth in ophthalmology has traditionally focused on preventive disease screening with limited use in outpatient evaluation. The unique conditions of the COVID-19 pandemic afforded the opportunity to evaluate different implementations of teleophthalmology at scale, providing insight into expanding teleophthalmology care. Objective: To compare telehealth use in ophthalmology with other specialties and assess the feasibility of augmenting ophthalmic telehealth encounters with asynchronous testing during the COVID-19 pandemic. Design, Setting, and Participants: This quality improvement study evaluated retrospective, longitudinal, observational data from the first 18 months of the COVID-19 pandemic (January 1, 2020, through July 31, 2021) for 881 080 patients receiving care from outpatient primary care, cardiology, neurology, gastroenterology, surgery, neurosurgery, urology, orthopedic surgery, otolaryngology, obstetrics/gynecology, and ophthalmology clinics of the University of California, San Francisco. Asynchronous testing was evaluated for teleophthalmology encounters. Interventions: A hybrid care model wherein ophthalmic testing data were acquired asynchronously and used to augment telehealth encounters. Main Outcomes and Measures: Telehealth as a percentage of total volume of ambulatory care and use of asynchronous testing for ophthalmic conditions. Results: The volume of in-person outpatient visits dropped by 83.3% (39 488 of 47 390) across the evaluated specialties at the onset of shelter-in-place orders for the COVID-19 pandemic, and the initial use of telehealth increased for these specialties before stabilizing over the 18-month study period. In ophthalmology, telehealth use peaked at 488 of 1575 encounters (31.0%) early in the pandemic and returned to mostly in-person visits as COVID-19 restrictions lifted. Elective use of telehealth was highest in gastroenterology, urology, neurology, and neurosurgery and lowest in ophthalmology. Asynchronous testing was combined with 126 teleophthalmology encounters, resulting in change of clinical management for 32 patients (25.4%) and no change for 91 (72.2%). Conclusions and Relevance: Telehealth increased across various specialties during the COVID-19 pandemic. Combining teleophthalmic visits with asynchronous testing suggested that this approach is feasible for subspecialty-level evaluation. Additional study is needed to evaluate whether asynchronous testing outside the same institution could provide an effective and lasting approach for expanding the reach of ophthalmic telehealth.

Diagnostic accuracy of handheld fundus photography: A comparative study of three commercially available cameras.

The objective of this study was to compare the sensitivity and specificity of handheld fundus cameras in detecting diabetic retinopathy (DR), diabetic macular edema (DME), and macular degeneration. Participants in the study, conducted at Maharaj Nakorn Hospital in Northern Thailand between September 2018 and May 2019, underwent an ophthalmologist examination as well as mydriatic fundus photography with three handheld fundus cameras (iNview, Peek Retina, Pictor Plus). Photographs were graded and adjudicated by masked ophthalmologists. Outcome measures included the sensitivity and specificity of each fundus camera for detecting DR, DME, and macular degeneration, relative to ophthalmologist examination. Fundus photographs of 355 eyes from 185 participants were captured with each of the three retinal cameras. Of the 355 eyes, 102 had DR, 71 had DME, and 89 had macular degeneration on ophthalmologist examination. The Pictor Plus was the most sensitive camera for each of the diseases (73-77%) and also achieved relatively high specificity (77-91%). The Peek Retina was the most specific (96-99%), although in part due to its low sensitivity (6-18%). The iNview had slightly lower estimates of sensitivity (55-72%) and specificity (86-90%) compared to the Pictor Plus. These findings demonstrated that the handheld cameras achieved high specificity but variable sensitivities in detecting DR, DME, and macular degeneration. The Pictor Plus, iNview, and Peek Retina would have distinct advantages and disadvantages when applied for utilization in tele-ophthalmology retinal screening programs.

Comparison of automated and expert human grading of diabetic retinopathy using smartphone-based retinal photography.

PURPOSE: The aim of this study is to investigate the efficacy of a mobile platform that combines smartphone-based retinal imaging with automated grading for determining the presence of referral-warranted diabetic retinopathy (RWDR). METHODS: A smartphone-based camera (RetinaScope) was used by non-ophthalmic personnel to image the retina of patients with diabetes. Images were analyzed with the Eyenuk EyeArt® system, which generated referral recommendations based on presence of diabetic retinopathy (DR) and/or markers for clinically significant macular oedema. Images were independently evaluated by two masked readers and categorized as refer/no refer. The accuracies of the graders and automated interpretation were determined by comparing results to gold standard clinical diagnoses. RESULTS: A total of 119 eyes from 69 patients were included. RWDR was present in 88 eyes (73.9%) and in 54 patients (78.3%). At the patient-level, automated interpretation had a sensitivity of 87.0% and specificity of 78.6%; grader 1 had a sensitivity of 96.3% and specificity of 42.9%; grader 2 had a sensitivity of 92.5% and specificity of 50.0%. At the eye-level, automated interpretation had a sensitivity of 77.8% and specificity of 71.5%; grader 1 had a sensitivity of 94.0% and specificity of 52.2%; grader 2 had a sensitivity of 89.5% and specificity of 66.9%. DISCUSSION: Retinal photography with RetinaScope combined with automated interpretation by EyeArt achieved a lower sensitivity but higher specificity than trained expert graders. Feasibility testing was performed using non-ophthalmic personnel in a retina clinic with high disease burden. Additional studies are needed to assess efficacy of screening diabetic patients from general population.

Remote corneal suturing wet lab: microsurgical education during the COVID-19 pandemic.

PURPOSE: To study the feasibility and efficacy of a new remote wet lab for microsurgical education using a corneal suturing task. SETTING: Department of Ophthalmology, University of California San Francisco, San Francisco, California, USA. DESIGN: Prospective randomized controlled study. METHODS: Ten ophthalmology residents were stratified by postgraduate year and randomized to perform a corneal suturing task consisting of placing the 4 cardinal sutures for a penetrating keratoplasty in porcine eyes with or without remote ophthalmology attending feedback. Subsequently, both groups repeated the same task without remote feedback to test whether initial remote feedback affected subsequent performance. Finally, the group without feedback was crossed over to repeat the same corneal suturing task with remote feedback. The effectiveness of the remote wet lab was assessed subjectively by survey and objectively by grading each suture pass. RESULTS: Resident-reported comfort with corneal suturing improved significantly after the remote wet lab for all residents. Residents and attendings rated the remote wet lab as equally or more effective compared with previous in-person wet labs and overall effective in corneal suturing. Attendings rated the remote wet lab as effective in multiple domains of microsurgical education using a modified microsurgical global rating scale. Objective corneal suturing performance was similar for both groups. CONCLUSIONS: The remote wet lab was feasible and effective for training ophthalmology residents in corneal suturing. This represents a new social distancing compliant platform for microsurgical education during the COVID-19 pandemic.

The Role of Telemedicine to Alleviate an Increasingly Burdened Healthcare System: Retinopathy of Prematurity.

Telemedicine-based remote digital fundus imaging (RDFI-TM) offers a promising platform for the screening of retinopathy of prematurity. RDFI-TM addresses some of the challenges faced by ophthalmologists in examining this vulnerable population in both low- and high-income countries. In this review, we studied the evidence on the use of RDFI-TM and analyzed the practical framework for RDFI-TM systems. We assessed the novel technological advances that can be deployed within RDFI-TM systems including noncontact imaging systems, smartphone-based imaging tools, and deep learning algorithms.

Smartphone-based fundus photography for screening of plus-disease retinopathy of prematurity.

BACKGROUND: Inadequate screening of treatment-warranted retinopathy of prematurity (ROP) can lead to devastating visual outcomes. Especially in resource-poor communities, the use of an affordable, portable, and easy to use smartphone-based non-contact fundus photography device may prove useful for screening for high-risk ROP. This study evaluates the feasibility of screening for high-risk ROP using a novel smartphone-based fundus photography device, RetinaScope. METHODS: Retinal images were obtained using RetinaScope on a cohort of prematurely born infants during routine examinations for ROP. Images were reviewed by two masked graders who determined the image quality, the presence or absence of plus disease, and whether there was retinopathy that met predefined criteria for referral. The agreement between image-based assessments was compared to the gold standard indirect ophthalmoscopic assessment. RESULTS: Fifty-four eyes of 27 infants were included. A wide-field fundus photograph was obtained using RetinaScope. Image quality was acceptable or excellent in 98% and 95% of cases. There was substantial agreement between the gold standard and photographic assessment of presence or absence of plus disease (Cohen's κ = 0.85). Intergrader agreement on the presence of any retinopathy in photographs was also high (κ = 0.92). CONCLUSIONS: RetinaScope can capture digital retinal photographs of prematurely born infants with good image quality for grading of plus disease.

Smartphone-Based, Rapid, Wide-Field Fundus Photography for Diagnosis of Pediatric Retinal Diseases.

PURPOSE: An important, unmet clinical need is for cost-effective, reliable, easy-to-use, and portable retinal photography to evaluate preventable causes of vision loss in children. This study presents the feasibility of a novel smartphone-based retinal imaging device tailored to imaging the pediatric fundus. METHODS: Several modifications for children were made to our previous device, including a child-friendly 3D printed housing of animals, attention-grabbing targets, enhanced image stitching, and video-recording capabilities. Retinal photographs were obtained in children undergoing routine dilated eye examination. Experienced masked retina-specialist graders determined photograph quality and made diagnoses based on the images, which were compared to the treating clinician's diagnosis. RESULTS: Dilated fundus photographs were acquired in 43 patients with a mean age of 6.7 years. The diagnoses included retinoblastoma, Coats' disease, commotio retinae, and optic nerve hypoplasia, among others. Mean time to acquire five standard photographs totaling 90-degree field of vision was 2.3 ± 1.1 minutes. Patients rated their experience of image acquisition favorably, with a Likert score of 4.6 ± 0.8 out of 5. There was 96% agreement between image-based diagnosis and the treating clinician's diagnosis. CONCLUSIONS: We report a handheld smartphone-based device with modifications tailored for wide-field fundus photography in pediatric patients that can rapidly acquire fundus photos while being well-tolerated. TRANSLATIONAL RELEVANCE: Advances in handheld smartphone-based fundus photography devices decrease the technical barrier for image acquisition in children and may potentially increase access to ophthalmic care in communities with limited resources.

Usability testing of a smartphone-based retinal camera among first-time users in the primary care setting.

Smartphone-based retinal photography is a promising method for increasing accessibility of retinal screening in the primary care and community settings. Recent work has focused on validating its use in detection of diabetic retinopathy. However, retinal imaging can be technically challenging and additional work is needed to improve ease of retinal imaging in the primary care setting. We therefore performed usability testing of a smartphone-based retinal camera, RetinaScope, among medical assistants in primary care who had never performed retinal imaging. A total of 24 medical assistants performed first-time imaging in a total of five rounds of testing, and iterative improvements to the device were made between test rounds based on the results. The time to acquire a single ~50 degree image of the posterior pole of a model eye decreased from 283 ± 60 seconds to 34 ± 17 seconds (p < 0.01) for first-time users. The time to acquire 5 overlapping images of the retina decreased from 325 ± 60 seconds to 118 ± 26 seconds (p = 0.02) for first-time users. Testing in the human eye demonstrated that a single wide-view retinal image could be captured in 65 ± 7 seconds and 5 overlapping images in 229 ± 114 seconds. Users reported high Systems Usability Scores of 86 ± 13 throughout the rounds, reflecting a high level of comfort in first-time operation of the device. Our study demonstrates that smartphone-based retinal photography has the potential to be quickly adopted among medical assistants in the primary care setting.

A Smartphone-Based Tool for Rapid, Portable, and Automated Wide-Field Retinal Imaging.

PURPOSE: High-quality, wide-field retinal imaging is a valuable method for screening preventable, vision-threatening diseases of the retina. Smartphone-based retinal cameras hold promise for increasing access to retinal imaging, but variable image quality and restricted field of view can limit their utility. We developed and clinically tested a smartphone-based system that addresses these challenges with automation-assisted imaging. METHODS: The system was designed to improve smartphone retinal imaging by combining automated fixation guidance, photomontage, and multicolored illumination with optimized optics, user-tested ergonomics, and touch-screen interface. System performance was evaluated from images of ophthalmic patients taken by nonophthalmic personnel. Two masked ophthalmologists evaluated images for abnormalities and disease severity. RESULTS: The system automatically generated 100° retinal photomontages from five overlapping images in under 1 minute at full resolution (52.3 pixels per retinal degree) fully on-phone, revealing numerous retinal abnormalities. Feasibility of the system for diabetic retinopathy (DR) screening using the retinal photomontages was performed in 71 diabetics by masked graders. DR grade matched perfectly with dilated clinical examination in 55.1% of eyes and within 1 severity level for 85.2% of eyes. For referral-warranted DR, average sensitivity was 93.3% and specificity 56.8%. CONCLUSIONS: Automation-assisted imaging produced high-quality, wide-field retinal images that demonstrate the potential of smartphone-based retinal cameras to be used for retinal disease screening. TRANSLATIONAL RELEVANCE: Enhancement of smartphone-based retinal imaging through automation and software intelligence holds great promise for increasing the accessibility of retinal screening.

Glaucoma Screening in Nepal: Cup-to-Disc Estimate With Standard Mydriatic Fundus Camera Compared to Portable Nonmydriatic Camera.

PURPOSE: To compare cup-to-disc ratio (CDR) measurements from images taken with a portable, 45-degree nonmydriatic fundus camera to images from a traditional tabletop mydriatic fundus camera. DESIGN: Prospective, cross-sectional, comparative instrument validation study. METHODS: Setting: Clinic-based. STUDY POPULATION: A total of 422 eyes of 211 subjects were recruited from the Tilganga Institute of Ophthalmology (Kathmandu, Nepal). Two masked readers measured CDR and noted possible evidence of glaucoma (CDR ≥ 0.7 or the presence of a notch or disc hemorrhage) from fundus photographs taken with a nonmydriatic portable camera and a mydriatic standard camera. Each image was graded twice. MAIN OUTCOME MEASURES: Effect of camera modality on CDR measurement; inter- and intraobserver agreement for each camera for the diagnosis of glaucoma. RESULTS: A total of 196 eyes (46.5%) were diagnosed with glaucoma by chart review; 41.2%-59.0% of eyes were remotely diagnosed with glaucoma over grader, repeat measurement, and camera modality. There was no significant difference in CDR measurement between cameras after adjusting for grader and measurement order (estimate = 0.004, 95% confidence interval [CI], 0.003-0.011, P = .24). There was moderate interobserver reliability for the diagnosis of glaucoma (Pictor: κ = 0.54, CI, 0.46-0.61; Topcon: κ = 0.63, CI, 0.55-0.70) and moderate intraobserver agreement upon repeat grading (Pictor: κ = 0.63 and 0.64, for graders 1 and 2, respectively; Topcon: κ = 0.72 and 0.80, for graders 1 and 2, respectively). CONCLUSIONS: A portable, nonmydriatic, fundus camera can facilitate remote evaluation of disc images on par with standard mydriatic fundus photography.

Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels.

Arteriovenous (AV) malformation (AVM) is a devastating condition characterized by focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. AVMs can form anywhere in the body and can cause debilitating ischemia and life-threatening hemorrhagic stroke. The mechanisms that underlie AVM formation remain poorly understood. Here, we examined the cellular and hemodynamic changes at the earliest stages of brain AVM formation by time-lapse two-photon imaging through cranial windows of mice expressing constitutively active Notch4 (Notch4*). AVMs arose from enlargement of preexisting microvessels with capillary diameter and blood flow and no smooth muscle cell coverage. AV shunting began promptly after Notch4* expression in endothelial cells (ECs), accompanied by increased individual EC areas, rather than increased EC number or proliferation. Alterations in Notch signaling in ECs of all vessels, but not arteries alone, affected AVM formation, suggesting that Notch functions in the microvasculature and/or veins to induce AVM. Increased Notch signaling interfered with the normal biological control of hemodynamics, permitting a positive feedback loop of increasing blood flow and vessel diameter and driving focal AVM growth from AV connections with higher blood velocity at the expense of adjacent AV connections with lower velocity. Endothelial expression of constitutively active Notch1 also led to brain AVMs in mice. Our data shed light on cellular and hemodynamic mechanisms underlying AVM pathogenesis elicited by increased Notch signaling in the endothelium.

Molecular identification of venous progenitors in the dorsal aorta reveals an aortic origin for the cardinal vein in mammals.

Coordinated arterial-venous differentiation is crucial for vascular development and function. The origin of the cardinal vein (CV) in mammals is unknown, while conflicting theories have been reported in chick and zebrafish. Here, we provide the first molecular characterization of endothelial cells (ECs) expressing venous molecular markers, or venous-fated ECs, within the emergent dorsal aorta (DA). These ECs, expressing the venous molecular markers Coup-TFII and EphB4, cohabited the early DA with ECs expressing the arterial molecular markers ephrin B2, Notch and connexin 40. These mixed ECs in the early DA expressed either the arterial or venous molecular marker, but rarely both. Subsequently, the DA exhibited uniform arterial markers. Real-time imaging of mouse embryos revealed EC movement from the DA to the CV during the stage when venous-fated ECs occupied the DA. We analyzed mutants for EphB4, which encodes a receptor tyrosine kinase for the ephrin B2 ligand, as we hypothesized that ephrin B2/EphB4 signaling may mediate the repulsion of venous-fated ECs from the DA to the CV. Using an EC quantification approach, we discovered that venous-fated ECs increased in the DA and decreased in the CV in the mutants, whereas the rest of the ECs in each vessel were unaffected. This result suggests that the venous-fated ECs were retained in the DA and missing in the CV in the EphB4 mutant, and thus that ephrin B2/EphB4 signaling normally functions to clear venous-fated ECs from the DA to the CV by cell repulsion. Therefore, our cellular and molecular evidence suggests that the DA harbors venous progenitors that move to participate in CV formation, and that ephrin B2/EphB4 signaling regulates this aortic contribution to the mammalian CV.

X-space MPI: magnetic nanoparticles for safe medical imaging.

One quarter of all iodinated contrast X-ray clinical imaging studies are now performed on Chronic Kidney Disease (CKD) patients. Unfortunately, the iodine contrast agent used in X-ray is often toxic to CKD patients' weak kidneys, leading to significant morbidity and mortality. Hence, we are pioneering a new medical imaging method, called Magnetic Particle Imaging (MPI), to replace X-ray and CT iodinated angiography, especially for CKD patients. MPI uses magnetic nanoparticle contrast agents that are much safer than iodine for CKD patients. MPI already offers superb contrast and extraordinary sensitivity. The iron oxide nanoparticle tracers required for MPI are also used in MRI, and some are already approved for human use, but the contrast agents are far more effective at illuminating blood vessels when used in the MPI modality. We have recently developed a systems theoretic framework for MPI called x-space MPI, which has already dramatically improved the speed and robustness of MPI image reconstruction. X-space MPI has allowed us to optimize the hardware for fi ve MPI scanners. Moreover, x-space MPI provides a powerful framework for optimizing the size and magnetic properties of the iron oxide nanoparticle tracers used in MPI. Currently MPI nanoparticles have diameters in the 10-20 nanometer range, enabling millimeter-scale resolution in small animals. X-space MPI theory predicts that larger nanoparticles could enable up to 250 micrometer resolution imaging, which would represent a major breakthrough in safe imaging for CKD patients.

Active cathepsins B, L, and S in murine and human pancreatitis.

Cathepsins regulate premature trypsinogen activation within acinar cells, a key initial step in pancreatitis. The identity, origin, and causative roles of activated cathepsins in pancreatic inflammation and pain are not defined. By using a near infrared-labeled activity-based probe (GB123) that covalently modifies active cathepsins, we localized and identified activated cathepsins in mice with cerulein-induced pancreatitis and in pancreatic juice from patients with chronic pancreatitis. We used inhibitors of activated cathepsins to define their causative role in pancreatic inflammation and pain. After GB123 administration to mice with pancreatitis, reflectance and confocal imaging showed significant accumulation of the probe in inflamed pancreas compared with controls, particularly in acinar cells and macrophages, and in spinal cord microglia and neurons. Biochemical analysis of pancreatic extracts identified them as cathepsins B, L, and S (Cat-B, Cat-L, and Cat-S, respectively). These active cathepsins were also identified in pancreatic juice from patients with chronic pancreatitis undergoing an endoscopic procedure for the treatment of pain, indicating cathepsin secretion. The cathepsin inhibitor K11777 suppressed cerulein-induced activation of Cat-B, Cat-L, and Cat-S in the pancreas and ameliorated pancreatic inflammation, nocifensive behavior, and activation of spinal nociceptive neurons. Thus pancreatitis is associated with an increase in the active forms of the proteases Cat-B, Cat-L, and Cat-S in pancreatic acinar cells and macrophages, and in spinal neurons and microglial cells. Inhibition of cathepsin activation ameliorated pancreatic inflammation and pain. Activity-based probes permit identification of proteases that are predictive biomarkers of disease progression and response to therapy and may be useful noninvasive tools for the detection of pancreatic inflammation.

Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals.

BACKGROUND: The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions. METHODOLOGY/PRINCIPAL FINDINGS: We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 µm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters. CONCLUSIONS/SIGNIFICANCE: To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies.

Notch4 normalization reduces blood vessel size in arteriovenous malformations.

Abnormally enlarged blood vessels underlie many life-threatening disorders including arteriovenous (AV) malformations (AVMs). The core defect in AVMs is high-flow AV shunts, which connect arteries directly to veins, "stealing" blood from capillaries. Here, we studied mouse brain AV shunts caused by up-regulation of Notch signaling in endothelial cells (ECs) through transgenic expression of constitutively active Notch4 (Notch4*). Using four-dimensional two-photon imaging through a cranial window, we found that normalizing Notch signaling by repressing Notch4* expression converted large-caliber, high-flow AV shunts to capillary-like vessels. The structural regression of the high-flow AV shunts returned blood to capillaries, thus reversing tissue hypoxia. This regression was initiated by vessel narrowing without the loss of ECs and required restoration of EphB4 receptor expression by venous ECs. Normalization of Notch signaling resulting in regression of high-flow AV shunts, and a return to normal blood flow suggests that targeting the Notch pathway may be useful therapeutically for treating diseases such as AVMs.