Age-related changes in murine limbal lymphatic vessels and corneal lymphangiogenesis

Important risk factors for graft rejection after corneal transplantation are pathologic corneal lymphangiogenesis and young recipient age. Purpose of this study was to investigate whether there are age-related differences in normal murine limbal and pathologic corneal lymphatic vessels, which could partly explain the unequal outcome of corneal transplantation in young versus old recipients. Furthermore, we investigated whether these observed differences correlate with changes in allograft survival in the murine model of corneal transplantation. Corneal whole mounts from untreated young (aged 6-8 weeks), untreated old (aged 9-15 months) and young and old mice after suture-induced, inflammatory corneal neovascularization were prepared and stained with LYVE-1 as a lymphendothelial marker. Angles of corneal parts with and without a main circumferential limbal lymphatic vessel were measured and then related to the total 360° of corneal circumference. Centrally directed vascular extensions from the main limbal lymphatic vessel (“sprouts”) of previously untreated old mice were counted. Concerning the outgrowth of pathologic lymphatic vessels after inflammatory corneal neovascularization, the area covered with pathologic lymphatic vessels was detected by an algorithm on digitized whole mounts using cell^F^® software. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed with one recipient group being young, the other group being old mice. In young, untreated mice, 70.5% of the total corneal circumference was covered by a main circumferential limbal lymphatic vessel versus 60.8% in old, untreated mice. Comparing the number of centripedal vascular extensions from the main limbal lymphatic vessel (“sprouts”), untreated old mice had significantly less extensions than young, untreated mice (p < 0.001). After an inflammatory stimulus, old mice had significantly less pathologic corneal lymphatic vessels than young mice (42% less, p < 0.001). Comparing the survival proportions after corneal transplantation, old recipient mice showed a significantly better graft survival 6 weeks after transplantation (65% versus 33%, p < 0.05). Thus, limbal lymphatic vascular sprouts and inflammation-induced pathologic corneal lymphangiogenesis decrease with age. The lower lymphangiogenic potency of older mice may explain the better outcome of corneal transplantations in old recipients, supporting the concept that lymphangiogenesis is an important risk-factor for corneal transplant rejection.     

Corneal lymphangiogenesis correlates closely with hemangiogenesis after keratoplasty

AIM: To examine the relationship between corneal lymphangiogenesis and hemangiogenesis after keratoplasty. · METHODS: Nineteen human corneas were obtained from 19 patients undergoing a second corneal transplantation in Zhongshan Ophthalmic Center in 2005. Blood and lymphatic vessels in human transplanted corneas were identified by lymphatic vessel endothelial receptor (LYVE-1) and platelet endothelial cell adhesion modecule-1 (PECAM-1) immunohi- stochemistry, and double enzyme-histochemistry; then the association of corneal blood vessel counting (BVC) with lymphatic vessel counting (LVC) was examined. · RESULTS: Corneal hemangiogenesis was present in 12 cases (63%), and lymphangiogenesis occurred in 5 cases (26%) human transplanted corneas. In addition, corneal lymphangiogenesis was only present in vascularized corneas. LVC was strongly and positively correlated with BVC(r=0.725, P <0.01). · CONCLUSION: Corneal lymphangiogenesis develops after keratoplasty and strongly associates with hemangiogenesis.     

角膜移植后角膜新生淋巴管与新生血管间的关联

目的:研究角膜移植后角膜新生淋巴管与新生血管间关联。方法:人角膜取自行二次角膜移植的19名患者。5核苷酸酶-碱性磷酸酶(5-nase-Alkaline phosphatase,5-NA-ALP)双重酶组织化学染色及淋巴内皮细胞受体(lymphatic vessel endothelial receptor,LYVE-1)、内皮细胞黏附因子-1(platelet endothelial cell adhesion modecule-1,PECAM-1)双重免疫组化法标记角膜中的新生血管和淋巴管,并进行淋巴管计数(lymphatic vessels counting,LVC)和血管计数(blood vessels counting,BVC),比较BVC与LVC之间的关联。结果:角膜中存在角膜新生血管12例(63%),存在角膜新生淋巴管5例(26%)。角膜新生淋巴管仅出现在血管化角膜中。角膜移植后BVC与LVC间呈显著性正相关(r=0.725;P<0.01)。结论:人角膜移植后角膜新生淋巴管与新生血管之间存在密切关联。     

Topical Ranibizumab inhibits inflammatory corneal hem‐ and lymphangiogenesis

Purpose: Ranibizumab (Lucentis^®) is a Fab‐Fragment of a recombinant, humanized, monoclonal VEGF (anti‐vascular endothelial growth factor) antibody. This study analyzed the ability of topical Ranibizumab to inhibit lymphangiogenesis in addition to hemangiogenesis after acute corneal inflammation in vivo. In addition, the effect of Ranibizumab on the proliferation of human lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) in vitro was studied. Methods: The inhibitory effect of Ranibizumab on LECs and BECs was studied in vitro using a proliferation enzyme‐linked immunosorbent assay (ELISA) assay. To study the in vivo effects of Ranibizumab, the mouse model of suture induced inflammatory corneal neovascularization was used. Study mice received topical Ranibizumab as eye drops. After 1 week excised corneas were stained with LYVE‐1 and CD31. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically by using a semiautomatic method based on the image analyzing program Cell^F. Results: An antiproliferative effect of Ranibizumab was seen in vitro on both human BECs and LECs with a significance of p < 0.0001 and p < 0.0004, respectively. In vivo experiments showed that topical application of Ranibizumab significantly inhibits both hemangiogenesis (p = 0.0026) and lymphangiogenesis (p = 0.0026) in the cornea. Conclusion: Ranibizumab is a potent inhibitor of inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo with a direct inhibitory effect on both endothelial cell types in vitro. This study for the first time demonstrates an inhibitory effect of Ranibizumab on lymphatic vessels which could have a wider range of clinical applications.     

Blockade of the VEGF isoforms in inflammatory corneal hemangiogenesis and lymphangiogenesis

Background

The VEGF-A family plays a crucial role in the induction of pathological corneal neovascularization. The role of the different VEGF-A isoforms during lymphangiogenesis is only little-known. Current anti-angiogenic therapies in the eye and other organs inhibit all VEGF-A isoforms, and have effects on both blood and lymphatic vessels. Here we investigate whether selective targeting of the isoform VEGF 165 is able to inhibit corneal lymphangiogenesis under inflammatory conditions.

Methods

The mouse model of suture-induced corneal neovascularization was used to assess the antihem- and antilymphangiogenic effect of topically applied pegaptanib. Corneal blood and lymph vascularized areas were analyzed morphometrically. Furthermore, we analyzed the proliferative effects of VEGF A 121, 165, and 189 on blood and lymphatic endothelial cells (BEC/LEC) via a cell-proliferation assay.

Results

Pegaptanib significantly inhibited inflammatory corneal hemangiogenesis (p?<?0.01), but not lymphangiogenesis in vivo (p?>?0.05), both topically as well as systemically, in the inflamed cornea. In vitro, BECs were more susceptible to pegaptanib than LECs.

Conclusions

Targeting VEGF-A 165 significantly inhibits hem- but not lymphangiogenesis, suggesting VEGF-A 165 to be critical for hem-, but dispensable for lymphangiogenesis, at least in the inflamed cornea.     

Bevacizumab as a potent inhibitor of inflammatory corneal angiogenesis and lymphangiogenesis

PURPOSE: To analyze whether bevacizumab can inhibit inflammatory angiogenesis and lymphangiogenesis in the cornea. Bevacizumab (Avastin; Roche, Welwyn Garden City, UK) is a recombinant, humanized, monoclonal antibody against VEGF-A that has been approved by the U.S. Food and Drug Administration for the treatment of colon carcinomas. METHODS: The mouse model of suture-induced corneal neovascularization was used to assess the antihemangiogenic and antilymphangiogenic effect of bevacizumab by systemic and topical application. Corneal flatmounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and CD31 as a pan-endothelial marker, and blood and lymph vascularized areas were analyzed morphometrically. The inhibitory effect of bevacizumab on lymphatic endothelial cells (LECs) was analyzed with a colorimetric (BrdU) proliferation ELISA. The binding ability of bevacizumab to murine VEGF-A was analyzed by Western blot, ELISA, and surface plasmon resonance. RESULTS: The systemic and topical applications of bevacizumab significantly inhibited the outgrowth of blood (P < 0.006 and P < 0.0001, respectively) and lymphatic (P < 0.002 and P < 0.0001, respectively) vessels. Inhibition of the proliferation of LECs was also significant (P < 0.0001). Western blot analysis, ELISA, and the surface plasmon resonance assay showed that bevacizumab binds murine VEGF-A. CONCLUSIONS: Topical or systemic application of bevacizumab inhibits both inflammation-induced angiogenesis and lymphangiogenesis in the cornea. This finding suggests an important role of VEGF-A in corneal lymphangiogenesis. Bevacizumab may be useful in preventing immune rejections after penetrating keratoplasty or tumor metastasis via lymphatic vessels.     

Blockade of VEGFR3-signalling specifically inhibits lymphangiogenesis in inflammatory corneal neovascularisation

Purpose Inflammatory corneal hem- and lymphangiogenesis occurring both prior to as well as after penetrating keratoplasty significantly increase the risk for subsequent immune rejections. The purpose of this study was to analyze whether the blocking anti-VEGFR3 antibody mF4-31C1 is able to inhibit the outgrowth of pathologic new lymphatic vessels in a mouse model of suture-induced, inflammatory corneal neovascularisation, and whether this antibody specifically inhibits lymphangiogenesis without affecting hemangiogenesis. Methods Three interrupted 11-0 nylon sutures were placed into the corneal stroma of BALB/c mice (6 weeks old) and left in place for 7 days to induce neovascularisation. The treatment group (n = 9) received the anti-VEGFR3 antibody mF4-31C1 intraperitoneally on the day of surgery and 3 days later (0.5 mg/mouse). Control mice received an equal amount of control IgG solution. For immunohistochemistry, corneal flat mounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and with CD31 as panendothelial marker. Morphometry was performed with the image analysis software analySIS^B (Soft Imaging Systems GmbH, Münster, Germany). To improve the objectivity and precision of the morphometrical analysis, we established a modified method using grey filter sampling on monochromatic pictures. Results The mF4-31C1 antibody-treated mice displayed nearly complete inhibition of lymphangiogenesis compared with IgG controls (p < 0.006). In contrast, there was no significant inhibitory effect observed with respect to blood vessel growth (p > 0.05). Conclusions Inflammatory corneal lymphangiogenesis seems to depend on VEGFR3-signalling. By blocking this receptor the ingrowths of lymphatic vessels can be inhibited almost completely, and specifically without affecting hemangiogenesis. This may open new treatment options to promote (corneal) graft survival without affecting hemangiogenesis. Interdisciplinary Centre for Clinical Research (IZKF) Erlangen (A9), ELAN Funds Erlangen, DFG (Cu 47/2-1).     

角膜移植后患者角膜新生淋巴管与新生血管和炎症的关联

目的:研究角膜移植后角膜新生淋巴管与新生血管和炎症的关联。方法:人角膜取自行二次角膜移植的患者19例。淋巴内皮细胞受体(lymphatic vessel endothelial hyaluronan receptor,LYVE-1)和内皮细胞黏附因子-1(platelet endothelial celladhesion modecule-1,PECAM-1)双重免疫组化法标记角膜中的新生血管和淋巴管,进行淋巴管计数(lymphatic ves-sels counting,LVC)和血管计数(blood vessels counting,BVC),比较BVC、炎症指数(inflammation index,IF)、移植历史(transplantation history,TH)与LVC之间的关联。结果:角膜移植后BVC,IF与LVC间均呈显著性正相关,而TH与LVC间呈显著性负相关。角膜移植后新生淋巴管、血管、眼表炎症间大致成平行发展,新生淋巴管最先退化,其次是眼表炎症,新生血管最后消退。结论:人角膜移植后角膜新生淋巴管与新生血管、眼表炎症之间存在着极为密切的关联。     

Processing of transient signals in the visual system of the European starling (Sturnusvulgaris) and humans

The double-pulse resolution (DPR) measures the processing performance for transient visual signals as the threshold duration for detecting a temporal gap between two light flashes in relation to gap duration. The DPR of four European starlings (Sturnus vulgaris) and four humans was measured in an operant Go/NoGo procedure. We applied the method of constant stimuli and determined thresholds using signal-detection theory. The starling DPR (22.2 ms ± 2.3 ms SE) was significantly shorter than human DPR (35.2 ms ± 1.3 ms SE; p < 0.01, t-test). The difference suggests that starlings have a higher temporal resolution for transient visual signals than humans.     

高危角膜移植大鼠排斥反应及VEGF-C/D表达的研究

目的：探讨鼠角膜碱烧伤后VEGF-C/D的表达和意义,以及新生淋巴管在高危角膜移植后排斥反应中的作用。
　　方法：制作角膜碱烧伤模型,取不同时间段角膜进行电镜观察,观察角膜血管化情况;采用免疫组织化学方法检测l、3、5、7、14、28 d角膜组织VEGF-C/D及VEGFR-3的表达;并在角膜中仅有血管(A组),同时存在新生血管及新生淋巴管(B组),新生淋巴管消退期(C组),角膜新生血管消退期(D组)以及正常组(N 组)进行穿透性角膜移植,比较不同角膜植片的排斥反应指数( rejection index, RI)值及存活时间。
　　结果：电镜观察发现,在碱烧伤后第7d时鼠角膜出现新生血管,未出现新生淋巴管,在碱烧伤2 wk时出现新生血管的同时出现淋巴管,5wk时无明显的新生淋巴管,8wk时新生血管逐渐消退;大鼠角膜组织中 VEGF-C/D 及VEGFR-3的表达从第3 d开始明显上升,并于第5 d达到最高峰。角膜移植后N、A、B、C、D组的植片平均存活时间分别为14.25±0.62、9.35±1.02、5.06±1.13、8.71±0.83、9.44±1.05d。组间比较发现,B组植片平均存活时间显著性缩短(P<0.05),A、C、D的存活时间均显著性延长(P<0.05)。
　　结论：角膜碱烧伤后存在VEGF-C/D及VEGFR-3的高表达,而且新生淋巴管能加速高危角膜移植后的免疫排斥反应。     

Corneal lymphangiogenesis facilitates ocular surface inflammation and cell trafficking in dry eye disease

Purpose

While the normal cornea has limited innervation by the lymphatic system, chronic immune-inflammatory disorders such as dry eye (DE) can induce lymphangiogenesis in the ocular surface. Using a conditional knock-down murine model, Lyve-1^Cre;VEGFR2^flox mice, this study investigated the role of lymphangiogenesis in the pathophysiology of DE.

Individual set-point and gain of emmetropization in chickens

During the developmental process of emmetropization evidence shows that visual feedback guides the eye as it approaches a refractive state close to zero, or slightly hyperopic. How this “set-point” is internally defined, in the presence of continuous shifts of the focal plane with different viewing distances and accommodation, remains unclear. Minimizing defocus blur over time should produce similar end-point refractions in different individuals. However, we found that individual chickens display considerable variability in their set-point refractive states, despite that they all had the same visual experience. This variability is not random since the refractions in both eyes were highly correlated - even though it is known that they can emmetropize independently. Furthermore, if chicks underwent a period of experimentally induced ametropia, they returned to their individual set-point refractions during recovery (correlation of the refractions before treatment versus after recovery: n = 19 chicks, 38 eyes, left eyes: slope 1.01, R = 0.860; right eyes: slope 0.85, R = 0.610, p < 0.001, linear regression). Also, the induced deprivation myopia was correlated in both eyes (n = 18 chicks, 36 eyes, p < 0.01, orthogonal regression). If chicks were treated with spectacle lenses, the compensatory changes in refraction were, on average, appropriate but individual chicks displayed variable responses. Again, the refractions of both eyes remained correlated (negative lenses, n = 18 chicks, 36 eyes, slope 0.89, R = 0.504, p < 0.01, positive lenses: n = 21 chicks, 42 eyes, slope 1.14, R = 0.791, p < 0.001). The amount of deprivation myopia that developed in two successive treatment cycles, with an intermittent period of recovery, was not correlated; only vitreous chamber growth was almost significantly correlated in both cycles (n = 7 chicks, 14 eyes; p < 0.05). The amounts of ametropia and vitreous chamber changes induced in two successive cycles of treatment, first with lenses and then with diffusers, were also not correlated, suggesting that the “gains of lens compensation” are different from those in deprivation myopia. In summary, (1) there appears to be an endogenous, possibly genetic, definition of the set-point of emmetropization in each individual, which is similar in both eyes, (2) visual conditions that induce ametropia produce variable changes in refractions, with high correlations between both eyes, (3) overall, the “gain of emmetropization” appears only weakly controlled by endogenous factors.     

Role of aquaporin-1 in trabecular meshwork cell homeostasis during mechanical strain

Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemm's canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24 h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to β-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8 h (n = 4, p < 0.05) and 24 h (n = 6, p < 0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n = 4, p < 0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r² = 0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.     

Axial myopia induced by a monocularly-deprived facemask in guinea pigs: A non-invasive and effective model

This study evaluated the efficacy of a facemask, a non-invasive and potentially more reliable method, in inducing axial myopia in guinea pigs. Thirty-six animals were randomly assigned to 3 groups: MDF (monocularly-deprived facemask, n=6), lid-suture (eyelids sutured monocularly, n=24) and normal control (free of form deprivation, n=6). All the groups underwent biometric measurement (refraction, corneal curvature and axial length) prior to the experiment. All animals in the MDF group underwent biometric measurement at each of the 4 timepoints (2, 4, 6 and 8 weeks of form deprivation). In the lid-sutured group, the animals were randomly assigned to 4 subgroups (n=6 each) and each subgroup underwent biometric measurement at one of the timepoints matching those of the MDF group. In the normal control group, all animals underwent biometric measurement at each of the timepoints matching those of the 2 experimental groups. Placement of a facemask on an animal took approximately 10 sec and all the facemasks remained in place at all timepoints. The procedure of lid-suture took at least 20 min for an animal and rupture of the sutures occurred in 50% of the animals after 4 weeks. The MDF eyes developed myopia from −2.21±2.11D (Mean±s.d.) at 2 weeks to −4.38±2.14 at 8 weeks (p<0.05 at all timepoints, compared to the contralateral eyes) with a lengthening of the vitreous chamber from 0.17±0.05 mm at 2 weeks to 0.29±0.12 mm at 8 weeks (p<0.01 at all timepoints, compared to the contralateral eyes). The lid-sutured eyes developed myopia from −2.38±1.21D at 2 weeks to −4.75±1.39D at 8 weeks (p<0.05 at all timepoints, compared to the contralateral eyes) with a lengthening of the vitreous chamber from 0.13±0.02 mm at 2 weeks to 0.30±0.10 mm at 8 weeks (p<0.05 at 2, 4, 8 weeks, but >0.05 at 6 weeks, compared to the contralateral eyes) and an increase in the radius of the corneal curvature (0.20±0.07 mm at 4 weeks, p<0.01; 0.17±0.05 mm at 8 weeks, p<0.05; compared to the contralateral eyes). Both the MDF and lid-sutured groups had a similar development in myopia and vitreous length (MDF vs lid-suturing: p>0.05 at all timepoints, one-way ANOVA with Bonferroni correction). This development was significantly faster than in the normal control group (MDF or lid-suture vs normal control: p<0.05 to <0.01 from 2 to 8 weeks, one-way ANOVA with Bonferroni correction). The radius of corneal curvature in the lid-sutured group was significantly greater than in either the MDF group or the normal control group since 4 weeks of form deprivation (p<0.05, one-way ANOVA with Bonferroni correction). Treatment with MDFs is as effective as the lid-suture in inducing axial myopia in guinea pigs. This method is non-invasive and allows evaluation of the same group of animals at different timepoints so that the number of animals required could be minimized without affecting the accuracy of the results.     

Investigating structural and biochemical correlates of ganglion cell dysfunction in streptozotocin-induced diabetic rats

The aim of this study was to determine whether inner retinal dysfunction in diabetic rats is correlated with structural and/or biochemical changes in the retina and optic nerve. Using the electroretinogram (ERG; −5.83 to 1.28 log cd.s.m⁻²) retinal function (photoreceptor, bipolar, amacrine and ganglion cell components) was measured in control (n = 13; citrate buffer) and diabetic (n = 13; streptozotocin, STZ, 50 mg kg⁻¹) rats, 12 weeks following treatment. Retinae and optic nerves were analyzed for structural changes and retinae were assessed for alterations in growth factor/cytokine expression using quantitative real-time PCR. We found that phototransduction efficiency was reduced 12 weeks after STZ-induced diabetes (−30%), leading to reduced amplitude of ON-bipolar (−18%) and amacrine cell (−29%) dominated responses; ganglion cell dysfunction (−84%) was more profound. In the optic nerve, nerve fascicle area and myelin sheath thickness were reduced (p < 0.05), whereas the ratio of blood vessels and connective tissue to total nerve cross-sectional area was increased (p < 0.05) in diabetic compared to control rats. In the retina, connective tissue growth factor (CTGF), transforming growth factor beta, type 2 receptor (TGFβ-r2) mRNA and platelet-derived growth factor B (PDGF-B) mRNA were increased (p < 0.035). Reduced ganglion cell function was correlated with increased CTGF and TGFβ-r2, but not PDGF-B mRNA. In summary, the ganglion cell component exhibited the greatest level of dysfunction within the ERG components examined after 12 weeks of STZ-induced diabetes; the level correlated with increased CTGF and TGFβ-r2 mRNA, but not with gross morphological changes in the retina or optic nerve.     

The effect of modulating ocular depth of focus upon accommodation microfluctuations in myopic and emmetropic subjects

The magnitude of accommodation microfluctuations increases in emmetropic subjects viewing low luminance targets or viewing a target through small artificial pupils. Larger microfluctuations reported in myopia may result from an abnormally large depth of focus (DoF). The effect of modulating the size of the DoF has not been investigated in myopic subjects and may help to explain the cause of the increased DoF. Accommodation microfluctuations were recorded under two experimental conditions. Firstly, 12 emmetropes (EMMs), and 24 myopes (MYOs) viewed a Maltese Cross target with luminance levels of 0.002, 0.2, 6 and 600 cd/m² and in darkness, and second, 14 EMMs and 16 MYOs viewed a Maltese Cross target through pupil diameters of 0.5, 1, 2, 3, 4 and 5 mm presented in Maxwellian view. The magnitude of the accommodation microfluctuations increased significantly with a target luminance of 0.002 cd/m² (p < .03) and pinhole diameters of <2 mm (p < .05). For all other luminance levels and pupil diameters the magnitude was constant. For both conditions, MYOs had significantly larger microfluctuations than EMMs (p < .01). Considerable inter-subject variability was observed in the degree to which the magnitude of the microfluctuations increased, for both the 0.002 cd/m² luminance and 0.5 mm pupils, however, this was not correlated with refractive error. The increase in the magnitude of the microfluctuations while viewing a low luminance target (0.002 cd/m²) may be due to a shallower contrast gradient in the cortical image, with a consequent increase in DoF. The microfluctuations also increase when viewing through small pupils (<2 mm), which increases the DoF without altering the contrast gradient. The larger microfluctuations found in the MYOs consolidates the theory that MYOs have a larger DoF than EMMs and therefore have a higher threshold for retinal image blur.     

The impact of macular pigment augmentation on visual performance in normal subjects: COMPASS

This study was conducted to investigate whether augmentation of macular pigment (MP) enhances visual performance (VP). 121 normal subjects were recruited. The active (A) group consumed 12 mg of lutein (L) and 1 mg of zeaxanthin (Z) daily. MP optical density (MPOD) was assessed by customized heterochromatic flicker photometry. VP was assessed as best corrected visual acuity (BCVA), mesopic and photopic contrast sensitivity (CS), glare disability, photostress, and subjective visual function. Subjects were assessed at baseline; 3; 6; 12 months (V1, V2, V3 and V4, respectively). Central MPOD increased significantly in the A group (p < 0.05) but not in the placebo group (p > 0.05). This statistically significant increase in MPOD in the A group was not, in general, associated with a corresponding improvement in VP (p > 0.05, for all variables), with the exception of a statistically significant time/treatment effect in “daily tasks comparative analysis” (p = 0.03). At V4, we report statistically significant differences in mesopic CS at 20.7 cpd, mesopic CS at 1.5 cpd under high glare conditions, and light/dark adaptation comparative analysis between the lower and the upper MP tertile groups (p < 0.05) Further study into the relationship between MP and VP is warranted, with particular attention directed towards individuals with low MP and suboptimal VP.     

Latanoprost protects rat retinal ganglion cells from apoptosis in vitro and in vivo

We investigated whether latanoprost has a direct anti-apoptotic effect in retinal ganglion cell (RGC) line and RGCs in the rat. RGC-5 cells were induced to undergo apoptosis by serum deprivation and exogenous glutamate. The level of cell death with or without latanoprost acid was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in the level of intracellular calcium ([Ca²⁺]i) were measured with fluo-4 fluorescence. The XTT assay revealed that latanoprost acid increased RGC-5 cell viability. Latanoprost acid significantly reduced caspase-3 positive cells and suppressed [Ca²⁺]i evoked by glutamate. U0126, a mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 inhibitor, partially blocked the rescue effect of latnanoprost acid (p = 0.013). In vivo, rat RGCs were degenerated by optic nerve crush. After topical instillation of latanoprost for 7 days, RGCs labeled with fluorogold were significantly. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells. TUNEL-positive cells were significantly decreased in eyes with topically instilled latanoprost (p = 0.015). These data suggest that latanoprost has an neuroprotective ability in RGCs.     

Genomic response of hypoxic Müller cells involves the very low density lipoprotein receptor as part of an angiogenic network

Müller cells have recently been found to produce select angiogenic substances. In choosing a more comprehensive approach, we wanted to study the genomic response of Müller cells to hypoxia to identify novel angiogenic genes. An established Müller cell line (rMC-1) was exposed to standard or hypoxic conditions. We analyzed gene expression with three independent microarrays and determined differential expression levels compared to normoxia. Selected genes were confirmed by real-time PCR (RTPCR). Subcellular localization of proteins was examined by immunocytochemistry. A network-based pathway analysis was performed to investigate how those genes may contribute to angiogenesis. We found 19?004 of 28?000 known rat genes expressed in Müller cells. 211 genes were upregulated by hypoxia 1.5 to 14.9-fold (p < 0.001, FDR ≤ 5%) and 220 genes were downregulated 1.5-4.6-fold (p < 0.001, FDR ≤ 5%). Unexpectedly, expression patterns of cell proliferation, differentiation and organogenesis were increased besides predictable declines in cell function. Very low density lipoprotein receptor (VLDLR) and tribbles 3 (TRIB3) were further analyzed because of recent implication in retinal neovascularization and macular degeneration (VLDLR) and in ocular mesodermal development and differentiation (TRIB3), respectively. VLDLR was upregulated 3.1-fold (p = 0.001, RTPCR 3.0-fold) and TRIB3 2.8-fold (p = 0.025, RTPCR 5.1-fold). VEGF was increased 3.1-fold (p = 0.003, RTPCR 8.3-fold) and apelin, a novel factor of retinal angiogenesis, 5.6-fold (p = 0.006, RTPCR 8.7-fold). A network of interacting angiogenic genes was identified in silico that included VLDLR as a surface receptor. VLDLR protein localized to the perinucleus, cytoplasm and cell membrane, while TRIB3 was found in nucleoli, the nucleus and cytoplasm. We conclude that hypoxia triggers an angiogenic network response in Müller cells with VLDLR as a novel node and gene expression patterns of proliferation, differentiation and organogenesis.