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Abstract In the course of our studies aiming to discover vascular bed‐specific endothelial cell (EC) mitogens, we identified leukemia inhibitory factor (LIF) as a mitogen for bovine choroidal EC (BCE), although LIF has been mainly characterized as an EC growth inhibitor and an anti‐angiogenic molecule. LIF stimulated growth of BCE while it inhibited, as previously reported, bovine aortic EC (BAE) growth. The JAK‐STAT3 pathway mediated LIF actions in both BCE and BAE cells, but a caspase‐independent proapoptotic signal mediated by cathepsins was triggered in BAE but not in BCE. LIF administration directly promoted activation of STAT3 and increased blood vessel density in mouse eyes. LIF also had protective effects on the choriocapillaris in a model of oxidative retinal injury. Analysis of available single‐cell transcriptomic datasets shows strong expression of the specific LIF receptor in mouse and human choroidal EC. Our data suggest that LIF administration may be an innovative approach to prevent atrophy associated with AMD, through protection of the choriocapillaris.

PurposeTo validate retinal capillary density and caliber associations with diabetic retinopathy (DR) severity in different clinical settings.MethodsThis cross-sectional study assessed retinal capillary density and caliber in the superficial retinal layer of 3-mm OCTA scans centered on the fovea. Images were collected from non-diabetic controls and subjects with mild or referable DR (defined DR worse than mild DR) between February 2016 and December 2019 at secondary and tertiary eye care centers. Vessel Skeleton Density (VSD), a measure of capillary density, and Vessel Diameter Index (VDI), a measure of vascular caliber, were calculated from these images. Discriminatory performance of VSD and VDI was evaluated using multivariable logistic regression models predicting DR severity with adjustments for sex, hypertension, and hyperlipidemia. Area under the curve (AUC) was estimated. Model performance was evaluated in two different cohorts.ResultsThis study included 594 eyes from 385 subjects. Cohort 1 was a training cohort of 509 eyes including 159 control, 155 mild non-proliferative DR (NPDR) and 195 referable DR eyes. Cohort 2 was a validation cohort consisting of 85 eyes including 16 mild NPDR and 69 referable DR eyes. In Cohort 1, addition of VSD and VDI to a model using only demographic data significantly improved the model's AUC for discrimination of eyes with any DR severity from controls (0.91 [95% CI, 0.88-0.93] versus 0.80 [95% CI, 0.76-0.83], p < 0.001) and eyes with referable DR from mild NPDR (0.90 [95% CI, 0.86-0.93] versus 0.69 [95% CI, 0.64-0.75], p < 0.001). The transportability of this regression model was excellent when implemented in Cohort 2 for the referable DR versus mild NPDR comparison. The odds ratio of having any DR compared to control subjects, and referable DR compared to mild DR decreased by 15% (95% CI: 12-18%), and 13% (95% CI: 10-15%), respectively, for every 0.001 unit increase in VSD after adjusting for comorbidities.ConclusionOCTA-derived capillary density has real world clinical value for rapidly assessing DR severity.

PurposeTo investigate the relationships between global and sectoral macular vascular microcirculation parameters in the ganglion cell-inner plexiform layer (GCIPL) assessed by optical coherence tomography angiography (OCTA), and global and sectoral visual field (VF) central mean sensitivity (CMS) assessed by standard automated perimetry.MethodsFifty-four eyes with open angle glaucoma were scanned using a swept-source OCTA (Plex Elite 9000, Zeiss, Dublin, CA) and macular vascular microcirculation was measured by calculating the overall flux and vessel area density (VAD) over the entire 6mm x 6mm area, excluding large retinal vessels. Central 10-degree VF CMS was calculated based on 24-2 VF. Pearson correlation was used to investigate the correlation between global and sectoral OCTA parameters and global and sectoral VF CMS.ResultsBoth global GCIPL flux and VAD were significantly correlated with VF CMS (pConclusionsGlobal and sectoral macular vascular microcirculation in the GCIPL, as determined by OCTA, was significantly correlated with global and sectoral VF CMS in glaucomatous patients. OCTA can aid in the understanding of the structure-function relationships of the macular region.

PurposeTo quantitatively assess choriocapillaris (CC) flow deficits in eyes with diabetic retinopathy (DR) using swept-source optical coherence tomography angiography (SS-OCTA).MethodsDiabetic subjects with different stages of DR and age-matched healthy subjects were recruited and imaged with SS-OCTA. The en face CC blood flow images were generated using previously published and validated algorithms. The percentage of CC flow deficits (FD%) and the mean CC flow deficit size were calculated in a 5-mm-diameter circle centered on the fovea from the 6×6-mm scans.ResultsForty-five diabetic subjects and 27 control subjects were included in the study. The CC FD% in diabetic eyes was on average 1.4-fold greater than in control eyes (12.34±4.14% vs 8.82±2.61%, P < 0.001). The mean CC FD size in diabetic eyes was on average 1.4-fold larger than in control eyes (2151.3± 650.8μm2 vs 1574.4±255.0 μm2, P < 0.001). No significant difference in CC FD% or mean CC FD size was observed between eyes with nonproliferative DR and eyes with proliferative DR (P = 1.000 and P = 1.000, respectively).ConclusionsCC perfusion in DR can be objectively and quantitatively assessed with FD% and FD size. In the macular region, both CC FD% and CC FD size are increased in eyes with DR. SS-OCTA provides new insights for the investigations of CC perfusion status in diabetes in vivo.

PURPOSE:To quantitatively evaluate the retinal microvasculature in human subjects with retinal venous occlusions (RVO) using optical coherence tomography angiography (OCTA). DESIGN:Retrospective, cross-sectional, observational case series. PARTICIPANTS:Sixty subjects (84 eyes) were included (20 BRVO, 14 CRVO, 24 unaffected fellow eyes, and 26 controls). METHODS:OCTA was performed on a prototype, spectral domain-OCTA system in the 3x3mm central macular region. Custom software was used to quantify morphology and density of retinal capillaries using four quantitative parameters. The vasculature of the segmented retinal layers and nonsegmented whole retina were analyzed. MAIN OUTCOME MEASURES:Fractal dimension (FD), vessel density (VD), skeletal density (SD), and vessel diameter index (VDI) within the segmented retinal layers and nonsegmented whole retina vasculature. RESULTS:Nonsegmented analysis of RVO eyes demonstrated significantly lower FD (1.64±0.01 vs 1.715±0.002; p

Purpose: To report longitudinal changes in choroidal thickness and the choroidal vasculature using SS-OCT imaging in a patient with superior ophthalmic vein thrombosis (SOVT). Observations: In a 93-year-old woman with a left-sided SOVT, the choroid in the left eye was thickened and the choroidal vessels were dilated both superiorly and inferiorly, with greater changes evident in the inferotemporal region of the choroid. After the superior ophthalmic vein was decompressed, a decrease in the choroidal thickness and choroidal vessel dilatation was observed both superiorly and inferiorly. Conclusions and importance: In an eye with thrombosis of the superior ophthalmic vein, longitudinal SS-OCT choroidal imaging showed a greater increase in choroidal thickness and choroidal vessel dilation away from the obstructed quadrant, which improved after treatment. These observations associated with outflow obstruction may be applicable to other choroidal diseases characterized by venous overload.

We report the use of a high-resolution optical coherence tomography (OCT) imaging platform to identify and quantify pressure-dependent aqueous outflow system (AOS) tissue relationships and to infer mechanical stiffness through examination of tissue properties in ex vivo human eyes. Five enucleated human eyes are included in this study, with each eye prepared with four equal-sized quadrants, each encompassing 90 degrees of the limbal circumference. In radial limbal segments perfusion pressure within Schlemm's canal (SC) is controlled by means of a perfusion cannula inserted into the canal lumen, while the other end of the cannula leads to a reservoir at a height that can control the pressure in the cannula. The OCT system images the sample with a spatial resolution of about 5 μm from the trabecular meshwork (TM) surface. Geometric parameters are quantified from the 2D OCT images acquired from the sample subjected to controlled changes in perfusion pressures; parameters include area and height of the lumen of SC, collector channel entrances (CCE) and intrascleral collector channels (ISCC). We show that 3D OCT imaging permits the identification of 3-D relationships of the SC, CCE and ISCC lumen dimensions. Collagen flaps or leaflets are found at CCE that are attached or hinged at only one end, whilst the flaps are connected to the TM by cylindrical structures spanning SC. Increasing static SC pressures resulted in SC lumen enlargement with corresponding enlargement of the CCE and ISCC lumen. Pressure-dependent SC lumen area and height changes are significant at the 0.01 levels for ANOVA, and at the 0.05 for both polynomial curves and Tukey paired comparisons. Dynamic measurements demonstrate a synchronous increase in SC, CCE and ISCC lumen height in response to pressure changes from 0 to 10, 30 or 50 mm Hg, respectively, and the response time is within the 50-millisecond range. From the measured SC volume and corresponding IOP values, we demonstrate that an elastance curve can be developed to infer the mechanical stiffness of the TM by means of quantifying pressure-dependent SC volume changes over a 2 mm radial region of SC. Our study finds pressure-dependent motion of the TM that corresponds to collagen leaflet configuration motion at CCE; the synchronous tissue motion also corresponds with synchrony of SC and CCE lumen dimension changes.

PURPOSE:To investigate optic disc perfusion differences in normal, primary open-angle glaucoma (POAG), and normal tension glaucoma (NTG) eyes using optical microangiography (OMAG) based optical coherence tomography (OCT) angiography technique. DESIGN:Cross-sectional, observational study. SUBJECTS:Twenty-eight normal, 30 POAG, and 31 NTG subjects. METHODS:One eye from each subject was scanned with a 68 kHz Cirrus HD-OCT 5,000-based OMAG prototype system centered at the optic nerve head (ONH) (Carl Zeiss Meditec Inc, Dublin, CA). Microvascular images were generated from the OMAG dataset by detecting the differences in OCT signal between consecutive B-scans. The pre-laminar layer (preLC) was isolated by a semi-automatic segmentation program. MAIN OUTCOME MEASURES:Optic disc perfusion, quantified as flux, vessel area density, and normalized flux (flux normalized by the vessel area) within the ONH. RESULTS:Glaucomatous eyes had significantly lower optic disc perfusion in preLC in all three perfusion metrics (p

Purpose: Spectral-domain OCT angiography (SD-OCTA) scans were used in an algorithm developed for swept-source OCT angiography (SS-OCTA) scans to determine if SD-OCTA scans yielded similar results for the measurement of hyperreflective foci (HRF) in intermediate age-related macular degeneration (iAMD). Design: Retrospective study. Participants: Forty eyes from 35 patients with iAMD. Methods: Patients underwent SD-OCTA and SS-OCTA imaging at the same visit using a 6 × 6 mm OCTA scan pattern. Hyperreflective foci were detected as hypotransmission defects on en face structural images generated from a custom slab positioned 64 to 400 μm beneath Bruch’s membrane and confirmed on corresponding B-scans by the presence of well circumscribed lesions within the neurosensory retina or along the retinal pigment epithelium (RPE) that are of equal or greater reflectivity than that of the RPE. Two independent graders evaluated the en face images and B-scans for the presence of these lesions. Outlines of HRF on en face images were generated using a published semiautomated algorithm developed for SS-OCTA scans and manually corrected by the graders when necessary. The total area measurements of the HRF within the 5-mm circle centered on the fovea were obtained from the algorithm using each imaging method. Main Outcome Measures: Agreement of the square root (sqrt) of the HRF total areas obtained from SS-OCTA and SD-OCTA. Results: The sqrt total areas of the HRF from both imaging modalities were highly concordant, with Lin’s concordance correlation coefficient (rc) of 0.94 (95% confidence interval: 0.86–0.97; P

Purpose: Spectral-domain OCT angiography (SD-OCTA) scans were tested in an algorithm developed for use with swept-source OCT angiography (SS-OCTA) scans to determine if SD-OCTA scans yielded similar results for the detection and measurement of persistent choroidal hypertransmission defects (hyperTDs). Design: Retrospective study. Participants: Forty pairs of scans from 32 patients with late-stage nonexudative age-related macular degeneration (AMD). Methods: Patients underwent both SD-OCTA and SS-OCTA imaging at the same visit using the 6 × 6 mm OCTA scan patterns. Using a semiautomatic algorithm that helped with outlining the hyperTDs, 2 graders independently validated persistent hyperTDs, which are defined as having a greatest linear dimension ≥250 μm on the en face images generated using a slab extending from 64 to 400 μm beneath Bruch’s membrane. The number of lesions and square root (sqrt) total area of the hyperTDs were obtained from the algorithm using each imaging method. Main Outcome Measures: The mean sqrt area measurements and the number of hyperTDs were compared. Results: The number of lesions and sqrt total area of the hyperTDs were highly concordant between the 2 instruments (rc = 0.969 and rc = 0.999, respectively). The mean number of hyperTDs was 4.3 ± 3.1 for SD-OCTA scans and 4.5 ± 3.3 for SS-OCTA scans (P = 0.06). The mean sqrt total area measurements were 1.16 ± 0.64 mm for the SD-OCTA scans and 1.17 ± 0.65 mm for the SS-OCTA scans (P < 0.001). Because of the small standard error of the differences, the mean difference between the scans was statistically significant but not clinically significant. Conclusions: Spectral-domain OCTA scans provide similar results to SS-OCTA scans when used to obtain the number and area measurements of persistent hyperTDs through a semiautomated algorithm previously developed for SS-OCTA. This facilitates the detection of atrophy with a more widely available scan pattern and the longitudinal study of early to late-stage AMD. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

In vivo imaging of mouse brain vasculature typically requires applying skull window opening techniques: open-skull cranial window or thinned-skull cranial window. We report non-invasive 3D in vivo cerebral blood flow imaging of C57/BL mouse by the use of ultra-high sensitive optical microangiography (UHS-OMAG) and Doppler optical microangiography (DOMAG) techniques to evaluate two cranial window types based on their procedures and ability to visualize surface pial vessel dynamics. Application of the thinned-skull technique is found to be effective in achieving high quality images for pial vessels for short-term imaging, and has advantages over the open-skull technique in available imaging area, surgical efficiency, and cerebral environment preservation. In summary, thinned-skull cranial window serves as a promising tool in studying hemodynamics in pial microvasculature using OMAG or other OCT blood flow imaging modalities.

Optical microangiography (OMAG) and Doppler optical microangiography (DOMAG) are two non-invasive techniques capable of determining the tissue microstructural content, microvasculature angiography, and blood flow velocity and direction. These techniques were used to visualize the acute and chronic microvascular and tissue responses upon an injury in vivo. A tissue wound was induced using a 0.5 mm biopsy punch on a mouse pinna. The changes in the microangiography, blood flow velocity and direction were quantified for the acute (

A synchronized dual-wavelength laser speckle contrast imaging (DWLSCI) system and a Doppler optical microangiography (DOMAG) system was developed to determine several ischemic parameters in the cochlea due to a systemic hypoxic challenge. DWLSCI can obtain two-dimensional data, and was used to determine the relative changes in cochlear blood flow, and change in the concentrations of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) in mice. DOMAG can obtain three-dimensional data, and was used to determine the changes in cochlear blood flow with single vessel resolution. It was demonstrated that during a hypoxic challenge there was an increase in the concentrations of Hb, a decrease in the concentrations of HbO and cochlear blood flow, and a slight decrease in the concentration of HbT. Also, the rate of change in the concentrations of Hb and HbO was quantified during and after the hypoxic challenge. The ability to simultaneously measure these ischemic parameters with high spatio-temporal resolution will allow the detailed quantitative analysis of several hearing disorders, and will be useful for diagnosing and developing treatments.

Mammalian hearing is refined by amplification of the sound-evoked vibration of the cochlear partition. This amplification is at least partly due to forces produced by protein motors residing in the cylindrical body of the outer hair cell. To transmit power to the cochlear partition, it is required that the outer hair cells dynamically change their length, in addition to generating force. These length changes, which have not previously been measured in vivo, must be correctly timed with the acoustic stimulus to produce amplification.Using in vivo optical coherence tomography, we demonstrate that outer hair cells in living guinea pigs have length changes with unexpected timing and magnitudes that depend on the stimulus level in the sensitive cochlea.The level-dependent length change is a necessary condition for directly validating that power is expended by the active process presumed to underlie normal hearing.

Oxygen availability is regarded as a critical factor to metabolically regulate systemic blood flow. There is a debate as to how peripheral blood flow (PBF) is affected and modulated during hypoxia and hyperoxia; however in vivo evaluating of functional PBF under oxygen-related physiological perturbation remains challenging. Microscopic observation, the current frequently used imaging modality for PBF characterization often involves the use of exogenous contrast agents, which would inevitably perturb the intrinsic physiologic responses of microcirculation being investigated. In this paper, optical micro-angiography (OMAG) was employed that uses intrinsic optical scattering signals backscattered from blood flows for imaging PBF in skeletal muscle challenged by the alteration of oxygen concentration. By utilizing optical reflectance signals, we demonstrated that OMAG is able to show the response of hemodynamic activities upon acute hypoxia and hyperoxia, including the modulation of macrovascular caliber, microvascular density, and flux regulation within different sized vessels within skeletal muscle in mice in vivo. Our results suggest that OMAG is a promising tool for in vivo monitoring of functional macro- or micro-vascular responses within peripheral vascular beds.

Abstract During the past 15 years, new treatment paradigms for neovascular age-related macular degeneration (nvAMD) have evolved due to the advent of intravitreal anti-vascular endothelial growth factor (VEGF) therapy and rapid advances in retinal imaging. Recent publications describe eyes with type 1 macular neovascularization (MNV) as showing more resistance to macular atrophy than eyes with other lesion types. We sought to explore whether the perfusion status of the native choriocapillaris (CC) surrounding type 1 MNV influences its pattern of growth. To evaluate this effect, we analyzed a case series of 22 eyes from 19 nvAMD patients with type 1 MNV exhibiting growth on swept-source optical coherence tomography angiography (SS-OCTA) over a minimum follow-up of 12 months. We observed an overall weak correlation between type 1 MNV growth and CC flow deficits (FDs) average size (τ = 0.17, 95% CI [− 0.20, 0.62]) and a moderate correlation with CC FD % (τ = 0.21, 95% CI [− 0.16, 0.68]). Type 1 MNV was located beneath the fovea in most of the eyes (86%) and median visual acuity was 20/35 Snellen equivalent. Our results support that type 1 MNV recapitulates areas of CC blood flow impairment while serving to preserve foveal function.

Abstract Background Persistent placoid maculopathy (PPM) is a rare idiopathic chorioretinopathy characterized by choriocapillaris (CC) hypoperfusion. In a case of PPM, we quantified CC flow deficits (FDs) over time and observed an increase in CC perfusion as the visual acuity and outer photoreceptor anatomy improved. Case presentation A 58-year-old man was diagnosed with PPM in both eyes based on the patient’s clinical presentation and imaging. He presented with sudden-onset central scotomas in both eyes for about two months. On referral, the best corrected visual acuity (BCVA) was 20/20 in the right eye and 20/100 in the left eye. Plaque-like yellowish macular lesions were observed bilaterally and autofluorescence imaging showed bilateral hyperautofluorescent lesions. Fluorescein angiography (FA) revealed early-phase hyper-fluorescent staining that intensified in the late phases, while indocyanine green angiography (ICGA) displayed persistent hypofluorescence in both eyes. Foveal centered swept source optical coherence tomography (SS-OCT) B-scans showed bilateral focal deposits on the level of retinal pigment epithelium (RPE) and disruption of outer photoreceptor bands. The CC FDs were quantified on SS-OCT angiography (SS-OCTA) images using a previously published algorithm that was validated. The CC FD% was 12.52% in the right eye and 14.64% in the left eye within a 5 mm circle centered on the fovea. After 5 months of steroid treatment, BCVA remained 20/20 in the right eye and improved to 20/25 in the left eye. On OCT imaging, the outer photoreceptor bands fully recovered in both eyes, while some focal deposits remained along the RPE in the left eye. The CC perfusion in both eyes improved, with CC FD% decreasing from 12.52% to 9.16% in the right eye and from 14.64% to 9.34% in the left eye. Conclusions Significant impairment of macular CC perfusion was detected after the onset of PPM. Improvement in central macular CC perfusion corresponded with improvements in BCVA and outer retinal anatomy. Our findings suggest that imaging and quantification of CC FDs could serve as a valuable imaging strategy for diagnosing PPM and for following disease progression.

In the original publication [...]

Purpose: The purpose of this study was to objectively evaluate the longitudinal changes observed in the retinal capillaries in eyes with macula-on rhegmatogenous retinal detachment (RRD), assessed with optical coherence tomography angiography (OCTA), and to assess the role of these microvascular measures as potential biomarkers of postoperative visual outcomes. Methods: This was a prospective, longitudinal study conducted on consecutive patients who underwent 25 G pars plana vitrectomy for primary RRD. The vessel area density (VAD), vessel skeleton density (VSD), and vessel diameter index (VDI) were assessed in the superficial (SCP) and deep (DCP) capillary plexuses. Results: Twenty-three eyes with macula-on RRD were included in the study. The mean preoperative VDI, VAD, and VSD of the RRD eye were 18.6 ± 1.1, 0.43 ± 0.02, and 0.17 ± 0.01 in the SCP; and 18.6 ± 0.6, 0.38 ± 0.03, and 0.15 ± 0.01 in the DCP, respectively. At month 6, eight (34.8%) eyes achieved a best-corrected visual acuity (BCVA) gain ≥ 0.1 LogMAR. A preoperative VDI > 17.80, VSD > 0.170, and VDI-change > −0.395 in the SCP; and VDI > 18.11 and VDI change > −0.039 in the DCP were predictors of achieving a BCVA-gain ≥ 0.1 LogMAR. Conclusions: The preoperative VDI in SCP and DCP, the preoperative VSD in SCP, and their changes from preoperative values were predictors of visual outcomes. A quantitative OCTA analysis of capillary density and morphology may represent a potential predictive tool for surgical outcomes in RRD. The results reported in this study suggest that OCTA is a useful ancillary test for clinical disease management.

Summary: Neurorehabilitation strategies for ischemic stroke have shown promise for functional recovery, yet minimal tools are available to study rehabilitation techniques in non-human primates (NHPs). Here, we present a protocol to study rehabilitation techniques in NHPs using a photothrombotic technique, a form of optical focal lesioning. We also describe steps for simultaneous neurophysiological recording and in vivo validation through vascular flow imaging. This interface can examine emerging neurorehabilitation strategies in the post-stroke environment in NHPs that are evolutionarily close to humans.For complete details on the use and execution of this protocol, please refer to Khateeb et al. (2022).6 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Purpose: To evaluate changes in the anisotropic elastic properties of ex vivo human cornea treated with ultraviolet cross-linking (CXL) using noncontact acoustic micro-tapping optical coherence elastography (AμT-OCE). Design: Acoustic micro-tapping OCE was performed on normal and CXL human donor cornea in an ex vivo laboratory study. Subjects: Normal human donor cornea (n = 22) divided into 4 subgroups. All samples were stored in optisol. Methods: Elastic properties (in-plane Young’s, E, and out-of-plane, G, shear modulus) of normal and ultraviolet CXL–treated human corneas were quantified using noncontact AμT-OCE. A nearly incompressible transverse isotropic model was used to reconstruct moduli from AμT-OCE data. Independently, cornea elastic moduli were also measured with destructive mechanical tests (tensile extensometry and shear rheometry). Main Outcome Measures: Corneal elastic moduli (in-plane Young’s modulus, E, in-plane, μ, and out-of-plane, G, shear moduli) can be evaluated in both normal and CXL treated tissues, as well as monitored during the CXL procedure using noncontact AμT-OCE. Results: Cross-linking induced a significant increase in both in-plane and out-of-plane elastic moduli in human cornea. The statistical mean in the paired study (presurgery and postsurgery, n = 7) of the in-plane Young’s modulus, E=3μ, increased from 19 MPa to 43 MPa, while the out-of-plane shear modulus, G, increased from 188 kPa to 673 kPa. Mechanical tests in a separate subgroup support CXL-induced cornea moduli changes and generally agree with noncontact AμT-OCE measurements. Conclusions: The human cornea is a highly anisotropic material where in-plane mechanical properties are very different from those out-of-plane. Noncontact AμT-OCE can measure changes in the anisotropic elastic properties in human cornea as a result of ultraviolet CXL. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references.