Substance P and calcitonin gene-related peptide intrinsic choroidal neurons in human choroidal whole-mounts

To determine the presence in the human choroid of substance P (SP)-and calcitonin gene-related peptide (CGRP) positive intrinsic choroidal neurons (ICNs), choroidal whole-mounts were processed for indirect immunofluorescence. An antibody to a component of the neuronal cytoskeleton, neurofilament 200 kDa (NF-200), was combined with antibodies to SP and to CGRP (neuropeptides proper to the sensory nervous system). The human choroid possesses numerous SP(+) and CGRP(+) ICNs. These neurons were observed in the suprachoroid, both in isolation and forming microganglia. For both types of ICNs studied, neurons were more numerous in the temporal than in the nasal regions. In both locations, SP(+) and CGRP(+) ICNs were more abundant in the central choroid (the choroid underneath the macular area of the retina), with cell density diminishing outwards to the choroidal periphery. There were no appreciable differences between the two populations of ICNs studied in terms of size, morphology or immunostaining characteristics. In conclusion, given that peripheral sensory innervation could be involved in the regulation of both choroidal blood flow and vascular architecture, the SP(+) and CGRP(+) ICNs described for the first time in the present work may be involved in these mechanisms of vascular regulation.     

Three-dimensional segmentation and depth-encoded visualization of choroidal vasculature using swept-source optical coherence tomography

The choroid provides nutritional support for the retinal pigment epithelium and photoreceptors. Choroidal dysfunction plays a major role in several of the most important causes of vision loss including age-related macular degeneration, myopic degeneration, and pachychoroid diseases such as central serous chorioretinopathy and polypoidal choroidal vasculopathy. We describe an imaging technique using depth-resolved swept-source optical coherence tomography (SS-OCT) that provides full-thickness three-dimensional (3D) visualization of choroidal anatomy including topographical features of individual vessels. Enrolled subjects with different clinical manifestations within the pachychoroid disease spectrum underwent 15 mm × 9 mm volume scans centered on the fovea. A fully automated method segmented the choroidal vessels using their hyporeflective lumens. Binarized choroidal vessels were rendered in a 3D viewer as a vascular network within a choroidal slab. The network of choroidal vessels was color depth-encoded with a reference to the Bruch’s membrane segmentation. Topographical features of the choroidal vasculature were characterized and compared with choroidal imaging obtained with indocyanine green angiography (ICGA) from the same subject. The en face SS-OCT projections of the larger choroid vessels closely resembled to that obtained with ICGA, with the automated SS-OCT approach proving additional depth-encoded 3D information. In 16 eyes with pachychoroid disease, the SS-OCT approach added clinically relevant structural details, including choroidal thickness and vessel depth, which the ICGA studies could not provide. Our technique appears to advance the in vivo visualization of the full-thickness choroid, successfully reveals the topographical features of choroidal vasculature, and shows potential for further quantitative analysis when compared with other choroidal imaging techniques. This improved visualization of choroidal vasculature and its 3D structure should provide an insight into choroid-related disease mechanisms as well as their responses to treatment.     

Prevalence and Genetic Characteristics of Geographic Atrophy among Elderly Japanese with Age-Related Macular Degeneration

Objective

To investigate the prevalence and genetic characteristics of geographic atrophy (GA) among elderly Japanese with advanced age-related macular degeneration (AMD) in a clinic-based study.

Methods

Two-hundred and ninety consecutive patients with advanced AMD were classified into typical neovascular AMD, polypoidal choroidal vasculopathy (PCV), retinal angiomatous proliferation (RAP) or geographic atrophy (GA). Genetic variants of ARMS2 A69S (rs10490924) and CFH I62V (rs800292) were genotyped using TaqMan Genotyping Assays. The clinical and genetic characteristics were compared between patients with and without GA.

Results

The number of patients diagnosed as having typical neovascular AMD, PCV, RAP and GA were 98 (33.8%), 151 (52.1%), 22 (7.5%) and 19 (6.6%), respectively. Of 19 patients with GA, 13 patients (68.4%) had unilateral GA with exudative AMD in the contralateral eye. Patients with GA were significantly older, with a higher prevalence of reticular pseudodrusen, bilateral involvement of advanced AMD and T-allele frequency of ARMS2 A69S compared with those with typical AMD and PCV; although there were no differences in the genetic and clinical characteristics among patients with GA and RAP.

Conclusions

The prevalence of GA was 6.6% among elderly Japanese with AMD. Patients with GA and RAP exhibited genetic and clinical similarities.     

NPY and TH innervation in human choroidal whole-mounts

To determine the distribution of NPY and TH human choroidal innervation, choroidal whole-mounts were processed for indirect immunofluorescence. An antibody to a component of the neuronal cytoskeleton, neurofilament 200 kDa (NF-200) was used to identify neurons and axons. A double immunostaining was performed, antibodies against NF-200 being combined with antibodies against neuropeptide Y (NPY) and tyroxine hydroxylase (TH). Fibers containing both NPY and TH were distributed in three plexuses, one in the suprachoroid large-sized vessel layer, and two in the medium-sized vessel layer. Intrinsic choroidal neurons (ICNs) containing NPY and TH were observed in the suprachoroid. The TH(+) ICNs were located in the medium-sized vessel layer. Overall, NPY(+) and TH(+) ICNs were more frequent in the central temporal area, both in isolation and forming microganglia. We also detected small spindle elements intensely immunoreactive to TH(+) and distributed mainly in the suprachoroid from the equator to the periphery. In conclusion, the human choroid contains abundant NPY and TH nerve fibers related to chroroidal vascular structures; it further possesses NPY(+) and TH(+) ICNs which contribute to the choroidal self-regulation persisting after sympathetic denervation. Additionally, these ICNs may at least partially explain why the choroidal blood flow does not respond to the factors that influence systemic vascular control. The preferential location of these cells in the submacular area suggests that dysfunction or degeneration of these cells may be a factor in vascular pathologies found in ocular disease, such as diabetic macular edema or age-related macular degeneration.     

Perivascular Mural Cells of the Mouse Choroid Demonstrate Morphological Diversity That Is Correlated to Vasoregulatory Function

Objective

Perivascular mural cells of the choroid have been implicated in physiological functioning as well as in retinal disease pathogenesis. However details regarding their form and function are not well understood. We aim to characterize choroidal mural cells in the adult mouse choroid in terms of their distribution and morphology, and correlate these to their contractile behavior.

Methods

Sclerochoroidal flat-mounted explants were prepared from albino transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives the expression of green fluorescent protein (GFP). α-SMA-expressing smooth muscle cells and pericytes in the living choroid were thereby rendered fluorescent and imaged with confocal microscopy and live-cell imaging in situ.

Results

Choroidal perivascular mural cells demonstrate significant diversity in terms of their distribution and morphology at different levels of the vasculature. They range from densely-packed circumferentially-oriented cells that provide complete vascular coverage in primary arteries to widely-spaced stellate-shaped cells that are distributed sparsely over terminal arterioles. Mural cells at each level are immunopositive for contractile proteins α-SMA and desmin and demonstrate vasoconstrictory contractile movements in response to endothelin-1 and the calcium ionophore, A23187, and vasodilation in response to the calcium chelator, BAPTA. The prominence of vasoregulatory contractile responses varies with mural cell morphology and density, and is greater in vessels with dense coverage of mural cells with circumferential cellular morphologies. In the choriocapillaris, pericytes demonstrate a sparse, horizontal distribution and are selectively distributed only to the scleral surface of the choriocapillaris.

Conclusions

Diversity and regional specialization of perivascular mural cells may subserve varying requirements for vasoregulation in the choroid. The model of the α-SMA-GFP transgenic albino mouse provides a useful and intact system for the morphological and functional study of choroidal mural cells.     

The Association of Elastin Gene Variants with Two Angiographic Subtypes of Polypoidal Choroidal Vasculopathy

Objective

To compare the association of elastin (ELN) gene variants between two different angiographic phenotypes of polypoidal choroidal vasculopathy (PCV).

Methods

We included 411 treatment-naïve PCV patients and 350 controls in the present study. PCV was classified into two phenotypes (152 Type 1 and 259 Type 2) according to the presence or absence of feeding vessels found in indocyanine-green angiography. Single nucleotide polymorphisms (SNPs) in the ELN region including rs868005, rs884843, rs2301995, rs13239907 and rs2856728 were genotyped using TaqMan Genotyping Assays.

Results

In the allelic association analyses, rs868005 showed the strongest association with Type 2 PCV (allelic odds ratio 1.56; p = 7.4x10^-6), while no SNP was significantly associated with Type 1 PCV. Genotype association analyses revealed the significant association of rs868005 with Type 2 PCV in log additive model and predominant model (odds ratio 1.75; p = 1.5x10^-6 and odds ratio 1.60; p = 0.0044, respectively), but not with Type 1 PCV. These findings were further corroborated by another control group in the literature.

Conclusions

There may be significantly different associations in genetic variants of elastin between two angiographic phenotypes of PCV.     

A Pilot Study of Morphometric Analysis of Choroidal Vasculature In Vivo,Using En Face Optical Coherence Tomography

Purpose

To study the ability of volumetric spectral domain optical coherence tomography (SD-OCT) to perform quantitative measurement of the choroidal vasculature in vivo.

Methods

Choroidal vascular density and vessel size were quantified using en face choroidal scans from various depths below the retinal pigment epithelium (RPE) in 58 eyes of 58 patients with either epiretinal membranes (ERM), early age-related macular degeneration (AMD), or reticular pseudo-drusen (RPD). For each patient, we used the macular volume scan (6×6 mm cube) for vessel quantification, while high-definition (HD) cross-section raster scans were used to qualitatively assess vascularity of the choroidal sub-layers, and measure choroidal thickness.

Results

Of the 58 patients, more were female (66% versus 34% male), of whom 14 (24%) had ERM, 11 (19%) early AMD, and 33 (57%) RPD. Compared to intact choriocapillaris in all ERM (100%), none of the RPD and only 5/11 (45%) early AMD eyes had visible choriocapillaris on either cross section or C-scans (p-value<0.001). When comparing select regions from the most superficial C-scans, early AMD group had lowest vascular density and RPD had highest (p-value 0.04). Qualitative evaluation of C-scans from all three groups revealed a more granular appearance of the choriocapillaris in ERM versus increased stroma and larger vessels in the RPD eyes.

Conclusions

SD-OCT can be used to qualitatively and quantitatively assess choroidal vascularity in vivo. Our findings correlate to previously reported histopathologic studies. Lack of choriocapillaris on HD cross-sections or C-scans in all RPD and about half of early AMD eyes suggests earlier choroidal involvement in AMD and specifically, RPD.     

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).     

Hyperhomocysteinemia in Patients with Polypoidal Choroidal Vasculopathy: A Case Control Study

Purpose

To determine whether elevated plasma homocysteine and serum high sensitivity C-reactive protein (hsCRP) levels, two established risk factors of vascular diseases, are associated with polypoidal choroidal vasculopathy (PCV).

Design

Retrospective case-control study.

Methods

One hundred and nineteen consecutive patients with PCV and 119 matched controls were enrolled in a tertiary hospital from September 2008 to June 2013. Plasma homocysteine and serum hsCRP levels were measured. Associations among plasma homocysteine, serum hsCRP levels and PCV were further evaluated using multivariable logistic regression analysis.

Results

The median plasma homocysteine level was significantly higher in patients with PCV than in the controls (12.20 µmol/L vs. 9.80 µmol/L, p<0.001). The median serum hsCRP level was slightly higher in the PCV group (0.16 mg/dl vs. 0.11 mg/dl in control group, p = 0.07). After multivariable logistic regression analysis, each 1 µmol/L increase of plasma homocysteine was associated with a 1.5-fold increase in likelihood of having PCV (OR, 1.54; 95% confidence interval (CI), 1.33–1.79, p<0.001).

Conclusions

Hyperhomocysteinemia was associated with PCV and might play a role in the pathogenesis of PCV.     

Persistence of tight junctions and changes in apical structures and protein expression in choroid plexus epithelium of rats after short-term head-down tilt

Major alterations of choroidal cell polarity and protein expression were previously shown to be induced in rats by long-term adaptation to space flight (14 days aboard a space shuttle) or anti-orthostatic suspension (14 and 28 days) performed by tilting rats head-down (i.e. using a ground-based model known to simulate several effects of weightlessness). In rabbits, it was hypothesized that the blood-CSF barrier was opened in choroid plexus, after a short head-down suspension. To understand the early responses to fluid shifts induced by head-down tilts and evaluate the tightness of the choroidal junctions, we have investigated the effects of acute adaptations to anti-orthostatic restraints, using hindlimb-suspended Sprague-Dawley and Wistar rats. Ultrastructural and immunocytochemical studies were performed on choroid plexuses from lateral, third and fourth ventricles, after 30, 90 and 180 minutes of head-down tilt. Alterations were not perceptible at the level of choroidal tight junctions, as shown by freeze-fracture, claudin-1 and ZO-1 immunolocalizations and conventional electron microscopy, after intravenous injection of cytochrome C. The apical surface of choroidal cells was clearly more affected. Microvilli were longer and thinner and ezrin was over-expressed during all the periods of time considered, showing an early cytoskeletal response. Several proteins involved in the choroidal production of cerebrospinal fluid (sodium-potassium ATPase, carbonic anhydrase II, aquaporin 1) appeared first increased (30 minutes after the tilt), and then, returned to the control level or were lowered (after a 3-hour head-down suspension). Although head-down tilts do not seem to damage the blood-cerebrospinal fluid barrier in choroid plexus, it seemed that the expression of several apical proteins is affected very early.     

Measurement of Myopia and Normal Human Choroidal Thickness Using Spectral Domain Optical Coherence Tomography

Myopia is a common ophthalmic deficiency. The structure and function of choroid layer is assumed to be associated with myopia. In this study, a laboratory developed spectral domain optical coherence tomography scanning system is used to image human eyes. The axial resolution of the system is about 7 μm, and the acquisition rate is 100 kHz. Firstly, a cross-sectional image was acquired by averaging 100 images from imaging posterior segment of each eye. The choroid thickness was measured by 11 discrete points. The average thickness of normal human eyes was (0.296 ± 0.126) mm, whereas the average choroid thickness of myopic eyes was (0.220 ± 0.095) mm. Afterwards, the T test is used to calculate the data statistically. The analysis of the final result is based on the average thickness measured and the thickness of each measuring point. There was a significant difference in choroid thickness between myopia and normal eyes (P value < 0.01), which indicates that the choroid thickness of myopia was significantly thinner than that of normal eyes. Besides, there are findings that the choroidal thickness in nasal side is thinner than that in the fovea and temporal side in each eye. The choroidal thickness on temporal side in myopia eye has the most significant difference comparing with that in normal eye. The comprehensive evaluation of myopia and normal choroidal thickness using spectral domain optical coherence tomography may provide an important reference for the development of medical methods for diagnosis and treatment of myopia.     

Correlation of Aging and Segmental Choroidal Thickness Measurement using Swept Source Optical Coherence Tomography in Healthy Eyes

Purpose

To assess and compare choroidal thickness changes related to aging, we determined whether changes are due to thinning of the choriocapillaris plus Sattler''s (CS) layer and/or the large vessel layer in healthy eyes using swept-source optical coherence tomography (SS-OCT) at a wavelength of 1,050-nm.

Methods

We studied 115 normal eyes of 115 healthy volunteers, all with refractive errors of less than -6 diopters. All 115 eyes underwent analysis of choroidal thickness at the fovea, the CS layer and the large choroidal vessel layer. In 68 of the 115 eyes, choroidal thickness was determined at five sites (the fovea, and superior, inferior, nasal, and temporal sites) using SS-OCT with an Early Treatment of Diabetic Retinopathy grid scan.

Results

Total choroidal thicknesses at each of the five sites were related to subject age (P<0.0001). The choroid was thinnest at the nasal site, followed by the temporal, inferior, superior and finally the subfoveal site itself. The total choroidal thickness at the nasal site was significantly less than those at the other four sites (p<0.05). The CS layer showed thinning which correlated with age (P<0.0001). The thickness of the choroidal large vessel layer also decreased with age (p = 0.02). Subfoveal choroidal thickness was calculated as follows: 443.89–2.98×age (μm) (P<0.0001).

Conclusion

Subfoveal choroidal thickness decreases by 2.98 μm each year. Total choroidal thickness diminishes with age. The CS and large vessel layers of the choroid at the subfovea showed significant decreases, though only the former correlated strongly with age.     

和血明目片联合全视网膜激光光凝术对糖尿病视网膜病变患者视力状况、血液流变学及脉络膜厚度的影响

摘要 目的：探讨和血明目片联合全视网膜激光光凝术（PRP）对糖尿病视网膜病变（DR）患者视力状况、血液流变学及脉络膜厚度的影响。方法：选择2018年3月~2019年12月期间来安徽省第二人民医院接受治疗的60例DR患者,经计算机随机编号按奇偶顺序分为对照组（奇数,n=30）和研究组（偶数,n=30）,两组在接受常规降糖治疗的基础上,对照组患者接受PRP治疗,研究组患者接受和血明目片联合PRP治疗,对比两组临床总有效率,观察两组治疗前后的视力状况、血液流变学及脉络膜厚度变化。结果：研究组的临床总有效率高于对照组（P<0.05）。治疗后,两组裸眼视力、视野、视敏度均升高,研究组的升高程度大于对照组（P<0.05）。治疗后,两组血浆黏度（PV）、红细胞聚集指数（AI）、全血高切黏度（NBH）、全血低切黏度（NBL）、血沉（ESR）均下降,研究组的下降程度大于对照组（P<0.05）。治疗后,两组黄斑中心凹下脉络膜厚度（SFCT）、下方脉络膜厚度（ICT）、上方脉络膜厚度（SCT）、鼻侧脉络膜厚度（NCT）、颞侧脉络膜厚度（TCT）均下降,研究组的下降程度大于对照组（P<0.05）。两组均未见明显的不良反应发生。结论：和血明目片联合PRP可有效改善DR患者的视力状况、血液流变学及脉络膜厚度。     

Altered clearance of beta-amyloid from the cerebrospinal fluid following subchronic lead exposure in rats: Roles of RAGE and LRP1 in the choroid plexus

Formation of amyloid plaques is the hallmark of Alzheimer’s disease. Our early studies show that lead (Pb) exposure in PDAPP transgenic mice increases β-amyloid (Aβ) levels in the cerebrospinal fluid (CSF) and hippocampus, leading to the formation of amyloid plaques in mouse brain. Aβ in the CSF is regulated by the blood-CSF barrier (BCB) in the choroid plexus. However, the questions as to whether and how Pb exposure affected the influx and efflux of Aβ in BCB remained unknown. This study was conducted to investigate whether Pb exposure altered the Aβ efflux in the choroid plexus from the CSF to blood, and how Pb may affect the expression and subcellular translocation of two major Aβ transporters, i.e., the receptor for advanced glycation end-products (RAGE) and the low density lipoprotein receptor protein-1 (LRP1) in the choroid plexus. Sprague-Dawley rats received daily oral gavage at doses of 0, 14 (low-dose), and 27 (high-dose) mg Pb/kg as Pb acetate, 5 d/wk, for 4 or 8 wks. At the end of Pb exposure, a solution containing Aβ₄₀ (2.5 μg/mL) was infused to rat brain via a cannulated internal carotid artery. Subchronic Pb exposure at both dose levels significantly increased Aβ levels in the CSF and choroid plexus (p < 0.05) by ELISA. Confocal data showed that 4-wk Pb exposures prompted subcellular translocation of RAGE from the choroidal cytoplasm toward apical microvilli. Furthermore, it increased the RAGE expression in the choroid plexus by 34.1 % and 25.1 % over the controls (p < 0.05) in the low- and high- dose groups, respectfully. Subchronic Pb exposure did not significantly affect the expression of LRP1; yet the high-dose group showed LRP1 concentrated along the basal lamina. The data from the ventriculo-cisternal perfusion revealed a significantly decreased efflux of Aβ₄₀ from the CSF to blood via the blood-CSF barrier. Incubation of freshly dissected plexus tissues with Pb in artificial CSF supported a Pb effect on increased RAGE expression. Taken together, these data suggest that Pb accumulation in the choroid plexus after subchronic exposure reduces the clearance of Aβ from the CSF to blood by the choroid plexus, which, in turn, leads to an increase of Aβ in the CSF. Interaction of Pb with RAGE and LRP1 in choroidal epithelial cells may contribute to the altered Aβ transport by the blood-CSF barrier in brain ventricles.     

An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice

The study and treatment of age-related macular degeneration (AMD), a leading cause of blindness, has been hampered by a lack of animal models. Here we report that mice deficient either in monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) or its cognate C-C chemokine receptor-2 (Ccr-2) develop cardinal features of AMD, including accumulation of lipofuscin in and drusen beneath the retinal pigmented epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). Complement and IgG deposition in RPE and choroid accompanies senescence in this model, as in human AMD. RPE or choroidal endothelial production of Ccl-2 induced by complement C5a and IgG may mediate choroidal macrophage infiltration into aged wild-type choroids. Wild-type choroidal macrophages degrade C5 and IgG in eye sections of Ccl2(-/-) or Ccr2(-/-) mice. Impaired macrophage recruitment may allow accumulation of C5a and IgG, which induces vascular endothelial growth factor (VEGF) production by RPE, possibly mediating development of CNV. These models implicate macrophage dysfunction in AMD pathogenesis and may be useful as a platform for validating therapies.     

Scleral thickness in human eyes

Purpose

To obtain information about scleral thickness in different ocular regions and its associations.

Methods

The histomorphometric study included 238 human globes which had been enucleated because of choroidal melanomas or due to secondary angle-closure glaucoma. Using light microscopy, anterior-posterior pupil-optic nerve sections were measured.

Results

In the non-axially elongated group (axial length ≤26 mm), scleral thickness decreased from the limbus (0.50±0.11 mm) to the ora serrata (0.43±0.14 mm) and the equator (0.42±0.15 mm), and then increased to the midpoint between posterior pole and equator (0.65±0.15 mm) and to the posterior pole (0.94±0.18 mm), from where it decreased to the peri-optic nerve region (0.86±0.21 mm) and finally the peripapillary scleral flange (0.39±0.09 mm). Scleral thickness was significantly lower in the axially elongated group (axial length >26 mm) than in the non-axially elongated group for measurements taken at and posterior to the equator. Scleral thickness measurements of the posterior pole and of the peripapillary scleral flange were correlated with lamina cribrosa thickness measurements. Scleral thickness measurements at any location of examination were not significantly (all P>0.10) correlated with corneal thickness measurements. Scleral thickness was statistically independent of age, gender and presence of glaucoma.

Conclusions

In non-axially elongated eyes, the sclera was thickest at the posterior pole, followed by the peri-optic nerve region, the midpoint between posterior pole and equator, the limbus, the ora serrata, the equator and finally the peripapillary scleral flange. In axially elongated eyes, scleral thinning occurred at and posterior to the equator, being more marked closer to the posterior pole and the longer the axial length was. Within the anterior and posterior segment respectively, scleral thickness measurements were correlated with each other. Posterior scleral thickness was correlated with lamina cribrosa thickness. Scleral thickness measurements at any location of examination were not significantly correlated with corneal thickness or with age, gender and presence of absolute secondary angler-closure glaucoma.     

The Features of Inflammation Factors Concentrations in Aqueous Humor of Polypoidal Choroidal Vasculopathy

Purpose

To investigate the cytokine concentrations in the aqueous humor of patients with refractory polypoidal choroidal vasculopathy (PCV).

Methods

Three separate groups of patients were studied–refractory PCV (Group A, 41 eyes), stable PCV (Group B, 39 eyes) and senile cataract (Group C, 44 eyes). Aqueous humor samples were collected at two time points for Groups A and B–before the first intravitreal ranibizumab injection and before the last injection. Aqueous humor samples were collected prior to phacoemulsification in Group C. The cytokine concentrations of interleukin 2, 6, and 8 (IL-2, IL-6, and IL-8), tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and vascular endothelial growth factor (VEGF) were measured by cytometric bead array and flow cytometry.

Results

Before the first treatment, the MCP-1, VEGF, and TNF-α levels in Group A were significantly higher than those in Group C (P < 0.05), and the MCP-1 and VEGF levels in Group A were significantly higher than those in Group B (P < 0.05). Significantly higher MCP-1 and VEGF levels were seen in Group B compared to Group C (P < 0.05). Before the final treatment, the MCP-1, VEGF, and TNF-α concentrations in Group A were significantly higher than those in Group B (P < 0.05) and Group C (P < 0.05). IL-2 levels were significantly lower in Group A compared to Group B (P < 0.05) and Group C (P < 0.05).

Conclusion

Inflammatory cytokines such as MCP-1, VEGF, and TNF-α may be associated with the pathogenesis of both stable and refractory PCV.     

Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

Multiple genetic studies have suggested that high-temperature requirement serine protease (HTRA1) is associated with polypoidal choroidal vasculopathy (PCV). To date, no functional studies have investigated the biological effect of HTRA1 on vascular endothelial cells, essential vascular components involved in polypoidal vascular abnormalities and arteriosclerosis-like changes. In vitro studies were performed to investigate the effect of HTRA1 on the regulation of fibronectin, laminin, vascular endothelial growth factor (VEGF), platelet derived growth factor receptor (PDGFR) and matrix metalloparoteinases 2 (MMP-2) and the role of HTRA1 in choroid-retina endothelial (RF/6A) and human umbilical vein endothelial (HUVEC) cells. Lentivirus-mediated overexpression of HTRA1 was used to explore effects of the protease on RF/6A and HUVEC cells in vitro. HTRA1 overexpression inhibited the proliferation, cell cycle, migration and tube formation of RF/6A and HUVEC cells, effects that might contribute to the early stage of PCV pathological lesions. Fibronectin mRNA and protein levels were significantly down-regulated following the upregulation of HTRA1, whereas the expressions of laminin, VEGF and MMP-2 were unaffected by alterations in HTRA1 expression. The decreased biological function of vascular endothelial cells and the degradation of extracellular matrix proteins, such as fibronectin, may be involved in a contributory role for HTRA1 in PCV pathogenesis.     

Imaging Evidence of Diabetic Choroidopathy In Vivo: Angiographic Pathoanatomy and Choroidal-Enhanced Depth Imaging

Purpose

To describe the pathoanatomy of diabetic choroidopathy (DC) in pre-diagnosed diabetic retinopathy (DR) cases and to provide angiographic and optical evidence for DC using indocyanine green angiography (ICGA) and enhanced depth imaging spectral-domain optical coherence tomography (EDI SD-OCT).

Methods

A retrospective analysis of 80 eyes from 40 DR patients was conducted. In Group One, choroidal vascular abnormalities were evaluated by comparing angiographic findings from simultaneous ICGA with those from fundus fluorescein angiography (FFA). In Group Two, EDI SD-OCT was used to evaluate the subfoveal choroidal thickness (SFCT) and define the choroid boundary in order to acquire the bilateral and symmetric choroidal area (CA). Data were then analyzed by Image Pro Plus 6.0.

Results

In Group One, choroidal abnormalities that were evident using ICGA but not FFA included early hypofluorescent spots in 47 eyes (75.81%), late hyperfluorescent spots in 37 eyes (59.68%), and late choroidal non-perfusion regions in 32 eyes (51.61%). In particular, a significant difference between proliferative DR (PDR) in 17 of 23 eyes (73.91%) and non-PDR in 16 of 39 eyes (41.03%) was observed in late choroidal non-perfusion regions. Eighteen of 31 eyes (58.06%) also exhibited “inverted inflow phenomena.” In Group Two, both the SFCT and CA of eyes with diabetic macular edema and serous macular detachment were significantly greater than those in the other eyes. The CA in panretinal photocoagulation (PRP) treated cases was also greater than that in non-PRP treated cases.

Conclusions

Early hypofluorescent spots, late choroidal non-perfusion regions, inverted inflow phenomena, higher SFCT, and larger CA are qualitative and quantitative indexes for DC. Moreover, the late choroidal non-perfusion region is a risk factor for DC with DR. Our study suggests that the supplemental use of ICGA and EDI SD-OCT with FFA is a better choice for DR patients.