Comparative evaluation of trypsin and elastase digestion techniques for isolation of murine retinal vasculature.

Dysfunctional pericytes and disruption of adherens or tight junctions are related to many microvascular diseases, including diabetic retinopathy. In this context, visualizing retinal vascular architecture becomes essential for understanding retinal vascular disease pathophysiology. Although flat mounts provide a demonstration of the retinal blood vasculature, they often lack a clear view of microaneurysms and capillary architecture. Trypsin and elastase digestion are the two techniques for isolating retinal vasculatures in rats, mice, and other animal models. Our observations in the present study reveal that trypsin digestion impacts the association between pericytes and endothelial cells. In contrast, elastase digestion effectively preserves these features in the blood vessels. Furthermore, trypsin digestion disrupts endothelial adherens and tight junctions that elastase digestion does not. Therefore, elastase digestion emerges as a superior technique for isolating retinal vessels, which can be utilized to collect reliable and consistent data to comprehend the pathophysiology of disorders involving microvascular structures.

Clinical and genetic studies for a cohort of patients with Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is a group of early-onset retinal degenerative disorders, resulting in blindness in children. This study aimed to describe the clinical and genetic characteristics of a cohort of patients with LCA and to investigate the retinal vascular characteristics in LCA patients. METHODS: Fifty-two children with LCA were included in the study. All patients underwent detailed ocular examinations. Electroretinography (ERG) was used to evaluate the retinal function. Optical coherence tomography (OCT) was used to assess the structure change of the retina for those patients who were able to cooperate very well. Panel-based next-generation sequencing was performed to identify pathogenic variants in genes associated with LCA. Diameters of the retinal vessels were measured using the EVision AI screening system with an artificial intelligence (AI) technique. An ultrasound Doppler was used to evaluate hemodynamic parameters, including peak systolic velocity (PSV), resistive index (RI), and pulsatility index (PI), in the ophthalmic, central retinal, posterior ciliary, carotid, and internal carotid as well as external carotid arteries in 12 patients aged from 3 to 14 years. RESULTS: We detected 75 pathogenic variants from ten genes of RPGRIP1, CEP290, GUCY2D, LCA5, AIPL1, CRB1, RPE65, CRX, RDH12, and TULP1, including 29 novel and 36 previously reported variants in 52 affected children with LCA, with the highest detective rate in RPGRIP1 (26.9%). Fundus appearance is diverse in patients with LCA, ranging from normal to severe peripheral or central retinopathy. Retinal vasculature was evaluated in 12 patients with different gene variants, showing narrowed arteries with an average diameter of 43.6 ± 3.8 µm compared to that of 51.7 ± 2.6 µm in the normal controls (P < 0.001, n = 12). Meanwhile, their hemodynamic parameters were changed as well in the ophthalmic artery (OA), with a decreased PSV (P = 0.0132, n = 12) and slightly increased PI (P = 0.0488, n = 12) compared to the normal controls. However, the hemodynamic parameters did not change significantly in the other vessels. CONCLUSIONS: Blood supply to the eyeball is predicted to be reduced in patients with LCA, presumably due to photoreceptor cell degeneration. The novel identified variants will expand the spectrum of variants in LCA-related genes and be useful for studying the molecular mechanisms of LCA.

Retinal vascular remodeling in photoreceptor degenerative disease.

Abnormal vasculature in the retina, specifically tortuous vessels and capillary degeneration, is common in many of the most prevalent retinal degenerative diseases, currently affecting millions of people across the world. However, the formation and development of abnormal vasculature in the context of retinal degenerative diseases are still poorly understood. The FVB/N (rd1) and rd10 mice are well-studied animal models of retinal degenerative diseases, but how photoreceptor degeneration leads to vascular abnormality in the diseases remains to be elucidated. Here, we used advancements in confocal microscopy, immunohistochemistry, and image analysis software to systematically characterize the pathological vasculature in the FVB/N (rd1) and rd10 mice, known as a chronic, rapid and slower retinal degenerative model, respectively. We demonstrated that there was plexus-specific vascular degeneration in the retinal trilaminar vascular network paralleled to photoreceptor degeneration in the diseased retinas. We also quantitatively analyzed the vascular structural architecture in the wild-type and diseased retinas to provide valuable information on vascular remodeling in retinal degenerative disease.

Anatomy of the complete mouse eye vasculature explored by light-sheet fluorescence microscopy exposes subvascular-specific remodeling in development and pathology.

Eye development and function rely on precise establishment, regression and maintenance of its many sub-vasculatures. These crucial vascular properties have been extensively investigated in eye development and disease utilizing genetic and experimental mouse models. However, due to technical limitations, individual studies have often restricted their focus to one specific sub-vasculature. Here, we apply a workflow that allows for visualization of complete vasculatures of mouse eyes of various developmental stages. Through tissue depigmentation, immunostaining, clearing and light-sheet fluorescence microscopy (LSFM) entire vasculatures of the retina, vitreous (hyaloids) and uvea were simultaneously imaged at high resolution. In silico dissection provided detailed information on their 3D architecture and interconnections. By this method we describe successive remodeling of the postnatal iris vasculature, involving sprouting and pruning, following its disconnection from the embryonic feeding hyaloid vasculature. In addition, we demonstrate examples of conventional and LSFM-mediated analysis of choroidal neovascularization after laser-induced wounding, showing added value of the presented workflow in analysis of modelled eye disease. These advancements in visualization and analysis of the respective eye vasculatures in development and complex eye disease open for novel observations of their functional interplay at a whole-organ level.

Myopic Vascular Changes Revealed by Optical Tomography Angiography and Their Association with Myopic Fundus Manifestations.

INTRODUCTION: We aimed to quantify and evaluate fundal vascular changes at different severities of myopia using optical tomography angiography (OCTA) and explore their association with fundus changes captured by ultra-widefield (UWF) fundus cameras. METHODS: Seventy-four participants with myopia were enrolled in the study and underwent basic ophthalmic examination, OCTA, and UWF fundus photography. Multiple parameters were obtained using OCTA (flow area, structure thickness, and vessel density) and UWF fundus cameras (tessellation and parapapillary atrophy [PPA]). RESULTS: The right eye of 30 participants with low and moderate myopia and 44 participants with high myopia (HM) were included. Patients with HM had a larger flow area of the outer retina (FA-OR) and a smaller thickness of choroid (TC). Axial length was significantly correlated with retinal and choroidal flow area and thickness in the different zones. The PPA area was positively correlated with FA-OR and negatively correlated with TC. Tessellation exhibited different levels of correlation with OCTA parameters regarding the flow area, thickness, and vessel density of the fundal layers, mainly in the inner retina. CONCLUSION: FA-OR and TC exhibited sensitive changes in patients with HM and axial elongation; therefore, they could serve as predictive OCTA biomarkers. The PPA and tessellation were connected to the vascular and structural changes revealed by OCTA.

Retinal Venule Coverage by Pericytes Decreases in Multiparous Mice in a Time-Dependent Manner Post-Delivery.

Structural changes in the retinal vasculature have been linked to increased cardiovascular risks and also change as a function of age. Because multiparity has been associated with poorer cardiovascular health scores, we hypothesized that changes in retinal vascular caliber would be observed in multiparous, compared to nulliparous, females and retired breeder males. Age-matched nulliparous (n = 6) and multiparous (n = 11, retired breeder females with 4 ± 1 litters), and male breeder (n = 7) SMA-GFP reporter mice were included for assessment of retinal vascular structure. Multiparous females had higher body mass, heart weight, and kidney weight compared to nulliparous mice, with lower kidney and higher brain weight compared to male breeders. There was no difference in number of retinal arterioles or venules, or arteriole or venule diameter among groups; however, venous pericyte density (number per venule area) decreased in multiparous vs. nulliparous mice and was negatively associated with the time since last litter and with age. Our results suggest that the time elapsed since delivery is an important factor to be considered in multiparity studies. Taken together, changes in vascular structure and potentially function, are time- and age-dependent. Ongoing and future work will determine whether structural changes are associated with functional consequences at the blood-retinal barrier.

Acrolein Induces Retinal Abnormalities of Alzheimer's Disease in Mice.

It is reported that retinal abnormities are related to Alzheimer's disease (AD) in patients and animal models. However, it is unclear whether the retinal abnormities appear in the mouse model of sporadic Alzheimer's disease (sAD) induced by acrolein. We investigated the alterations of retinal function and structure, the levels of ß-amyloid (Aß) and phosphorylated Tau (p-Tau) in the retina, and the changes in the retinal vascular system in this mouse model. We demonstrated that the levels of Aß and p-Tau were increased in the retinas of mice from the acrolein groups. Subsequently, a decreased amplitudes of b-waves in the scotopic and photopic electroretinogram (ERG), decreased thicknesses of the retinal nerve fiber layer (RNFL) in the retina, and slight retinal venous beading were found in the mice induced by acrolein. We propose that sAD mice induced by acrolein showed abnormalities in the retina, which may provide a valuable reference for the study of the retina in sAD.

Cytochrome P450 1B1 Expression Regulates Intracellular Iron Levels and Oxidative Stress in the Retinal Endothelium.

Cytochrome P450 (CYP) 1B1 is a heme-containing monooxygenase found mainly in extrahepatic tissues, including the retina. CYP1B1 substrates include exogenous aromatic hydrocarbons, such as dioxins, and endogenous bioactive compounds, including 17ß-estradiol (E2) and arachidonic acid. The endogenous compounds and their metabolites are mediators of various cellular and physiological processes, suggesting that CYP1B1 activity is likely important in maintaining proper cellular and tissue functions. We previously demonstrated that lack of CYP1B1 expression and activity are associated with increased levels of reactive oxygen species and oxidative stress in the retinal vasculature and vascular cells, including retinal endothelial cells (ECs). However, the detailed mechanism(s) of how CYP1B1 activity modulates redox homeostasis remained unknown. We hypothesized that CYP1B1 metabolism of E2 affects bone morphogenic protein 6 (BMP6)-hepcidin-mediated iron homeostasis and lipid peroxidation impacting cellular redox state. Here, we demonstrate retinal EC prepared from Cyp1b1-deficient (Cyp1b1-/-) mice exhibits increased estrogen receptor-α (ERα) activity and expresses higher levels of BMP6. BMP6 is an inducer of the iron-regulatory hormone hepcidin in the endothelium. Increased hepcidin expression in Cyp1b1-/- retinal EC resulted in decreased levels of the iron exporter protein ferroportin and, as a result, increased intracellular iron accumulation. Removal of excess iron or antagonism of ERα in Cyp1b1-/- retinal EC was sufficient to mitigate increased lipid peroxidation and reduce oxidative stress. Suppression of lipid peroxidation and antagonism of ERα also restored ischemia-mediated retinal neovascularization in Cyp1b1-/- mice. Thus, CYP1B1 expression in retinal EC is important in the regulation of intracellular iron levels, with a significant impact on ocular redox homeostasis and oxidative stress through modulation of the ERα/BMP6/hepcidin axis.

Three Major Causes of Metabolic Retinal Degenerations and Three Ways to Avoid Them.

An imbalance of homeostasis in the retina leads to neuron loss and this eventually results in a deterioration of vision. If the stress threshold is exceeded, different protective/survival mechanisms are activated. Numerous key molecular actors contribute to prevalent metabolically induced retinal diseases-the three major challenges are age-related alterations, diabetic retinopathy and glaucoma. These diseases have complex dysregulation of glucose-, lipid-, amino acid or purine metabolism. In this review, we summarize current knowledge on possible ways of preventing or circumventing retinal degeneration by available methods. We intend to provide a unified background, common prevention and treatment rationale for these disorders and identify the mechanisms through which these actions protect the retina. We suggest a role for herbal medicines, internal neuroprotective substances and synthetic drugs targeting four processes: parainflammation and/or glial cell activation, ischemia and related reactive oxygen species and vascular endothelial growth factor accumulation, apoptosis and/or autophagy of nerve cells and an elevation of ocular perfusion pressure and/or intraocular pressure. We conclude that in order to achieve substantial preventive or therapeutic effects, at least two of the mentioned pathways should be targeted synergistically. A repositioning of some drugs is considered to use them for the cure of the other related conditions.

Structure and function of the retina of low-density lipoprotein receptor-related protein 5 (Lrp5)-deficient rats.

Loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), result in familial exudative vitreoretinopathy (FEVR), osteoporosis-pseudoglioma syndrome (OPPG), and Norrie disease. CRISPR/Cas9 gene editing was used to produce rat strains deficient in Lrp5. The purpose of this study was to validate this rat model for studies of hypovascular, exudative retinopathies. The retinal vasculature of wildtype and Lrp5 knockout rats was stained with Giffonia simplifolia isolectin B4 and imaged by fluorescence microscopy. Effects on retinal structure were investigated by histology. The integrity of the blood-retina barrier was analyzed by measurement of permeability to Evans blue dye and staining for claudin-5. Retinas were imaged by fundus photography and SD-OCT, and electroretinograms were recorded. Lrp5 gene deletion led to sparse superficial retinal capillaries and loss of the deep and intermediate plexuses. Autofluorescent exudates were observed and are correlated with increased Evans blue permeability and absence of claudin-5 expression in superficial vessels. OCT images show pathology similar to OCT of humans with FEVR, and retinal thickness is reduced by 50% compared to wild-type rats. Histology and OCT reveal that photoreceptor and outer plexiform layers are absent. The retina failed to demonstrate an ERG response. CRISPR/Cas9 gene-editing produced a predictable rat Lrp5 knockout model with extensive defects in the retinal vascular and neural structure and function. This rat model should be useful for studies of exudative retinal vascular diseases involving the Wnt and norrin pathways.

Evaluation of a Rapid and Simple Method for Assessing Retinal Vessel Structures in Adult Zebrafish.

The evaluation of retinal vascular structures is important for analyzing various ophthalmic diseases. Conventional trypsin digestion was used for separating retinal vasculatures in mouse, rat, and other animal models; however, the trypsin method alone is technically difficult to perform and has not been reported in zebrafish to date. In this study, we introduced a rapid and convenient method that allows the investigation of fine vessel structures at a cellular level in the relatively intact retinal vasculature of adult zebrafish. Using an anti-ZO-1 antibody, tight junction structures in retinal vessels were examined in detail and several different cell types constituting blood vessels in arterial and capillary areas were identified. In addition, using cell type-specific antibodies, we identified smooth muscle cells, blood cells, and endothelial cells in the retinal vasculature. Finally, using the hyperglycemic model, we observed the dilation of retinal vessels, the downregulation of tight junction proteins, and the reduction in smooth muscle cells. Based on these results, we provide a rapid and convenient method for the study of retinal vasculature disease in the zebrafish animal model.

Dysfunctional cGMP Signaling Leads to Age-Related Retinal Vascular Alterations and Astrocyte Remodeling in Mice.

The nitric oxide-guanylyl cyclase-1-cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway is integral to the control of vascular tone and morphology. Mice lacking the alpha catalytic domain of guanylate cyclase (GC1-/-) develop retinal ganglion cell (RGC) degeneration with age, with only modest fluctuations in intraocular pressure (IOP). Increasing the bioavailability of cGMP in GC1-/- mice prevents neurodegeneration independently of IOP, suggesting alternative mechanisms of retinal neurodegeneration. In continuation to these studies, we explored the hypothesis that dysfunctional cGMP signaling leads to changes in the neurovascular unit that may contribute to RGC degeneration. We assessed retinal vasculature and astrocyte morphology in young and aged GC1-/- and wild type mice. GC1-/- mice exhibit increased peripheral retinal vessel dilation and shorter retinal vessel branching with increasing age compared to Wt mice. Astrocyte cell morphology is aberrant, and glial fibrillary acidic protein (GFAP) density is increased in young and aged GC1-/- mice, with areas of dense astrocyte matting around blood vessels. Our results suggest that proper cGMP signaling is essential to retinal vessel morphology with increasing age. Vascular changed are preceded by alterations in astrocyte morphology which may together contribute to retinal neurodegeneration and loss of visual acuity observed in GC1-/- mice.

Vessel tech: a high-accuracy pipeline for comprehensive mouse retinal vasculature characterization.

Mouse retinal vasculature is a well-recognized and commonly used animal model for angiogenesis and microvascular remodeling. Morphological features of retinal vasculature reflect the vessel's biological functions, and are critical in understanding the physiological and pathological process of vascular development and disease. Here we developed a comprehensive software, Vessel Tech, using retinal vasculature images of postnatal mice. This pipeline can automatically process retinal vascular images, reconstruct vessel network with high accuracy and assess global and local vascular characteristics based on the recent machine-learning techniques. The development of Vessel Tech provides a powerful tool for vascular biologists.

Inflammatory resolution and vascular barrier restoration after retinal ischemia reperfusion injury.

BACKGROUND: Several retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration. METHODS: Using the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes. RESULTS: A 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C+ classical inflammatory monocytes, a slow accumulation of Ly6Cneg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45+ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1ß, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA. CONCLUSIONS: These results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.

The effect of AAV-mediated downregulation of Claudin-3 on the development of mouse retinal vasculature.

Retinal vascular development is a very tightly regulated and organized process of vessel formation and regression to generate the mature vasculature system. Claudin-3 has been found to be required for the normal development of the neural retina and its vessels in zebrafish in our recent study. In this study, we investigated whether Claudin-3 played a role in the development of mouse retinal vasculature. Immunofluorescent staining was performed to detect the expression and localization of Claudin-3 in the mouse retina. Intravitreal injection of a recombinant adeno-associated virus (AAV) expressing a short hairpin RNA targeting Claudin-3 mRNA was performed to down-regulate Claudin-3 expression in retina in neonatal (Postnatal Day 3, P3) C57BL/6J mice. Retinal vessels were examined by isolectin B4 immunofluorescent staining on the whole-mount retinas and frozen retinal sections at P10. The apoptotic retinal ganglion cells (RGCs) were measured by TdT-mediated dUTP nick-end labelling (TUNEL) staining. Vascular endothelial growth factor A (VEGF-A) expression was detected by immunofluorescent staining. The protein levels of Claudin-3, VEGF-A and B cell lymphoma 2 (Bcl-2) were evaluated by Western blot at P7, P10 and P14. We found that Claudin-3 mainly expressed in the RGCs and progressively increased during the retinal development. The AAV-mediated downregulation of Claudin-3 at P3 impeded the development of retinal deep vascularization of P10 mouse, but without effect on the development of the retinal superficial plexus. Claudin-3 knockdown increased RGC apoptosis and reduced the expression of VEGF-A and Bcl-2 in the retinas. These results suggested that the downregulation of Claudin-3 induced RGC apoptosis and impeded the mouse retinal vascular development by downregulating the levels of VEGF-A and Bcl-2.

Angiotensin II is a crucial factor in retinal aneurysm formation.

Retinal arterial macroaneurysms are characterized by the acquired fusiform or saccular dilatations of the retinal artery. Angiotensin II (Ang II) is a major signal molecule of the renin-angiotensin system, which exerts a range of pathogenic actions that are relevant to retinal vascular abnormalities. We aimed to study the effect of Ang II on retinal vessels and explore its relationship with retinal aneurysmal disease. C57BL/6J male mice were administered Ang II at 1000 ng/kg/min for 28 days, and the mice given saline served as controls. The mice in the treatment group were treated once daily by gastric gavage of candesartan cilexetil (an antagonist of Ang II type 1 (AT1) receptor) at 100 mg/kg/day. The in vivo imaging of murine retinas was performed using fundus photography, optical coherence tomography, fluorescein angiography, and indocyanine green angiography at 7th, 14th, and 28th days of infusion. At the end of the infusion and treatment, the morphological changes were evaluated by histopathological examination and electron microscopy; the levels of related proteins in murine retinas were examined by antibody array and Western blot analyses. We found that Ang II infusion induced aneurysm formation in mice retina, which presented as either solitary aneurysms or retinal arterial beading. The aneurysm formation was often accompanied with vessel leakage. Moreover, Ang II infusion itself may result in increased vascular permeability and ganglion cell and inner plexiform layer thickening. The blockade of AT1 receptors by systemic administration of candesartan cilexetil alleviated the Ang II-induced retinal vasculopathy. The protein level analysis further showed that Ang II upregulated IL-1ß, PDGFR-ß, and MMP-9 expression, and the expression of IL-1ß could be inhibited by AT1 receptor antagonist. Our study provides evidence that Ang II is a crucial factor in retinal aneurysm formation and vessel leakage. It is probably the combined effect of Ang II on vessel inflammatory response, pericyte function, and extracellular matrix remodeling that predisposes the retinal arterial wall to aneurysm formation and blood-retinal barrier breakdown.

Microvascular comparison in younger and older patients with retinal vein occlusion analyzed by OCT angiography.

BACKGROUND: To compare changes in retinal microvasculature of young and elderly patients with retinal vein occlusion (RVO) after anti-VEGF treatment. METHODS: RVO patients who underwent anti-VEGF treatment were retrospectively reviewed and categorized into two groups based on age. The OCT angiography images were obtained during each visit. Best corrected visual acuity (BCVA), vessel density (VD) and foveal avascular zone (FAZ) were measured and compared between the two groups. Vision improvements and retinal microvasculature changes were also correlated. RESULTS: Twenty patients with 20 eyes were enrolled in the younger group and 46 patients with 46 eyes were enrolled in the older group. Younger patients demonstrated better BCVA, higher VD and smaller FAZ than older patients at 12 months after the first anti-VEGF treatment. The improvement of VD was observed only in the younger group. A positive correlation between vision improvement and VD increase was noted. CONCLUSIONS: Young patients with RVO can achieve rapid rehabilitation of deep retinal vasculature which lead to a better visual outcome.

Impact of cardiometabolic factors on retinal vasculature: A 3 × 3, 6 × 6 and 8 × 8-mm ocular coherence tomography angiography study.

BACKGROUND: Ocular coherence tomography angiography (OCTA) is available in varying size and resolution. We sought to characterise associations of cardiometabolic factors with retinal microvascular changes using 3 × 3, 6 × 6 and 8 × 8-mm OCTA scans to determine differences in detection with varying scan size. METHODS: Cross-sectional study of 247 cardiovascular patients from a single-centre tertiary-care hospital. Demographic, comorbidity and medication data were obtained. Patients underwent 3 × 3, 6 × 6 and 8 × 8-mm macula OCTA scanning using Carl Zeiss CIRRUS HD-OCT Model 5000. Angioplex and AngioTool software was used to quantify vascular parameters in the superficial capillary plexus. RESULTS: Increasing age, hypertension, dyslipidaemia, diabetes, chronic kidney disease, coronary artery disease and peripheral vascular disease were associated with reductions in vessel density, vessel perfusion, average vessel length and/or junction density in 3 × 3-mm OCTA (P < .05 for all). Conversely, smoking was associated with increased vessel density, vessel length and junction density in 3 × 3-mm OCTA (P < .05 for all). Associations of vessel abnormalities with cardiometabolic factors were progressively weakened and statistically attenuated in 6 × 6 and 8 × 8-mm OCTA scans. In multivariate analyses, dyslipidaemia remained an independent predictor of reduced vessel density, average vessel length and junction density (P < .05). CONCLUSIONS: Cardiometabolic factors are associated with multiple retinal microvascular changes in 3 × 3-mm OCTA scans. These associations were weakened and progressively attenuated in OCTA scans of larger 6 × 6 and 8 × 8-mm size. These findings advance our understanding of microcirculatory dysfunction and may have future implications for the screening and management of patients with cardiometabolic risk factors. Additional studies are required to further investigate these important associations.

Single transient intraocular pressure elevations cause prolonged retinal ganglion cell dysfunction and retinal capillary abnormalities in mice.

Transient intraocular pressure (IOP) elevations are likely to occur in certain forms of glaucoma and after intravitreal injections to treat various retinal diseases. However, the impact of these transient IOP elevations on the physiology of individual retinal ganglion cells (RGCs) is unknown. In this report, we explore how transient IOP elevations in mice affect RGC physiology, RGC anatomy, and retinal arteriole and capillary structure. Transient IOP elevation was induced in 12-week old wild type C57BL6J mice by injecting sodium hyaluronate into the anterior chamber. IOP was measured immediately after the injection and again 1 and 7 days later. Average peak IOP after injection was ~50 mmHg and subsequent IOPs returned to normal. RGC physiology was assessed with a multielectrode array (MEA) by calculating a spike triggered average (STA) at the same time points. RGC counts and retinal vascular structure were assessed 14 days after injection with immunohistochemistry to label RGCs and blood vessels. Transient IOP elevation caused a marked reduction of scotopic STA presence and delayed center and surround STA peak times that did not recover. Transient IOP elevation also caused a reduced photopic receptive field size and spontaneous firing rate, both of which showed some recovery with time. Transient IOP elevation also induced vascular remodeling: the number of capillary branches was decreased within the superficial and intermediate vascular plexi. RGC counts, retinal arteriole diameter, and deep capillary plexus branching were unaffected. These previously unappreciated findings suggest that transient IOP elevation may cause unrecognized and potentially long-term pathology to RGCs and associated neurovascular units which should be accounted for in clinical practice.

The effects of endothelium-specific CYP2J2 overexpression on the attenuation of retinal ganglion cell apoptosis in a glaucoma rat model.

Glaucoma is a leading cause of irreversible blindness worldwide. Vascular factors play a substantial role in the pathogenesis of glaucoma. Expressed in the vascular endothelium, cytochrome P450 (CYP) 2J2 is one of the CYP epoxygenases that metabolize arachidonic acid to produce epoxyeicosatrienoic acids and exert pleiotropic protective effects on the vasculature. In the present study, we investigated whether endothelium-specific overexpression of CYP2J2 (tie2-CYP2J2-Tr) protects against retinal ganglion cell (RGC) loss induced by glaucoma and in what way retinal vessels are involved in this process. We used a glaucoma model of retinal ischemia-reperfusion (I/R) injury in rats and found that endothelium-specific overexpression of CYP2J2 attenuated RGC loss induced by retinal I/R. Moreover, retinal I/R triggered retinal vascular senescence, indicated by up-regulated senescence-related proteins p53, p16, and ß-galactosidase activity. The senescent endothelial cells resulted in pericyte loss and increased endothelial secretion of matrix metallopeptidase 9, which further contributed to RGC loss. CYP2J2 overexpression alleviated vascular senescence, pericyte loss, and matrix metallopeptidase 9 secretion. CYP2J2 suppressed endothelial senescence by down-regulating senescence-associated proteins p53 and p16. These 2 proteins were positively regulated by microRNA-128-3p, which was inhibited by CYP2J2. These results suggest that CYP2J2 protects against endothelial senescence and RGC loss in glaucoma, a discovery that may lead to the development of a potential treatment strategy for glaucoma.-Huang, J., Zhao, Q., Li, M., Duan, Q., Zhao, Y., Zhang, H. The effects of endothelium-specific CYP2J2 overexpression on the attenuation of retinal ganglion cell apoptosis in a glaucoma rat model.