Inhibition of experimental corneal neovascularization by using subconjunctival injection of bevacizumab (Avastin)

PURPOSE: To evaluate the effect of subconjunctival bevacizumab (Avastin) administration on corneal neovascularization (NV) in rabbits. METHODS: NV was induced by placing a suture at the corneal periphery of the right eye of 20 rabbits. Immediately after suturing and again 1 week later, rabbits were divided into 2 groups and administered a subconjunctival injection of normal saline (control) or bevacizumab (Avastin; 5 mg/0.2 mL), respectively. On day 14, digital photographs of the cornea were taken and analyzed to determine the area of the cornea covered by NV. In addition, immunohistochemical analysis was used to determine CD31 and vascular endothelial growth factor (VEGF) expression in corneal tissue. RESULTS: Analysis of digital photographs showed that there was less corneal NV in bevacizumab-treated eyes than in controls (P < 0.001, Mann-Whitney U test). In addition, there was less staining for VEGF and CD31 in corneas from bevacizumab-treated eyes than in control eyes. Subconjunctival bevacizumab injections were not associated with any complications during observation. CONCLUSIONS: Subconjunctival bevacizumab administration decreased suture-induced corneal neovascularization in rabbits.     

Plasminogen kringle 5 inhibits alkali-burn-induced corneal neovascularization

PURPOSE: Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor. The purpose of the present study was to evaluate the therapeutic effect of K5 on alkali-burn-induced corneal neovascularization (NV) and to investigate its mechanism of action. METHODS: Corneal NV was induced in rabbits by NaOH. The rabbits received eye drops containing K5 or vehicle alone, four times per day. Corneal NV and inflammation were monitored every other day with a slit lamp microscope, and the length of the vessels in the cornea and the area of NV were measured. Vascular endothelial growth factor (VEGF) was determined by immunohistochemical and Western blot analyses. The TUNEL assay was used to assess the apoptosis of endothelial cells. The effects of K5 on primary bovine aortic endothelial cells (BAECs) were determined by MTT assay, flow cytometry, transmission electron microscopy, and DNA fragmentation assay. RESULTS: Alkali-burn-induced progressive corneal NV and inflammation in the cornea. K5 delayed the onset of corneal NV (P < 0.05) and decreased NV areas (P < 0.05) in a dose-dependent manner. K5 treatment, after the formation of corneal NV, induced regression of newly formatted vessels in the cornea. K5 decreased the inflammatory index in the corneas at different time points after the alkali burn. Corneal VEGF levels were reduced by K5 treatment. K5 inhibits proliferation and induces apoptosis in BAECs. CONCLUSIONS: Topical application of K5 may have therapeutic potential for the chemical burn-induced corneal NV and inflammation. The inhibitory effect of K5 on corneal NV may be by downregulation of VEGF expression.     

代谢性酸中毒诱导新生大鼠视网膜 新生血管的实验研究

目的了解代谢性酸中毒诱导新生大鼠视网膜病变的发展进程，探讨其发生与血管内皮生长因子（VEGF）的关系。方法实验组新生Sprague-Dawley大鼠425只。从大鼠出生后第2天开始按535 mg/kg的剂量管饲NH₄Cl(浓度为50 mg/ml)，每天2次，管饲6 d，然后进入恢复期。另选150只新生大鼠未进行管饲，作为对照组。两组大鼠分别于出生后3、5、8、10、13、20 d处死。取出双眼进行视网膜铺片和二磷酸腺苷酶染色，对视网膜血管进行评估；用酶联免疫分析法进行VEGF的测定。结果实验组鼠在出生后3、5、8、10、13、20 d的新生血管（NV）发生率分别为0%、9%、26%、55%、19%、0%。出生后第3天，实验组鼠VEGF的蛋白水平［（101.1±14.2 ） pg/mg］比对照组［（133.2±15.9） pg/mg下降（P＝0.004），出后生第8天，实验组鼠VEGF蛋白水平［(98.4±19.2) pg/mg］比对照组［（78.1±8.7）pg/mg］升高（P=0.028)；出生后第5、10、13、20天，两组差异无统计学意义（P>0.05）。结论代谢性酸中毒可能损害了正在发育的视网膜血管而引起新生血管；酸中毒引起的视网膜新生血管与VEGF有关。(中华眼底病杂志,2005,21:296-299)     

结膜下注射Avastin抑制角膜新生血管的实验研究

目的:观察结膜下注射Avastin对实验性兔眼角膜新生血管(neovascularization,NV)的抑制作用,初步探讨作用机制。方法:应用5mm直径的加样器(末端附有棉片)吸入1mol/LNaOH接触新西兰兔右眼(20眼)中央角膜区烧灼30s,制作碱烧伤兔眼角膜NV模型。将实验兔随机分成2组,10眼(A组)碱烧伤后立即结膜下注射Avastin 2.5mg;其余10眼为对照组(B组),结膜下注射等量生理盐水。烧灼后次日每天裂隙灯观察角膜NV、角膜水肿情况,分别于3,7,14,21,28d裂隙灯照相并计算NV面积及NV抑制率。伤后7,28d各组随即处死5只实验兔,取角膜组织做石蜡切片行组织病理学检查及VEGF免疫组织化学检测。结果:两组兔眼伤后第1d角膜缘血管网明显扩张充血,3d时血管开始侵入角膜,7～14d时NV达到高峰,14～21d后NV稳定并逐渐回退。两组角膜NV长度、NV面积及角膜水肿程度存在差异(P<0.05);A组各时间点角膜NV抑制率为44.2%～55%。A组角膜上皮及实质层水肿较轻,NV较少,后弹力层基本完整,VEGF表达明显弱于B组。结论:结膜下注射Avastin对碱烧伤诱导的兔眼角膜NV形成及生长具有明显的抑制作用,可能通过下调VEGF表达发挥作用。     

Topical dihydroartemisinin inhibits suture-induced neovascularization in rat corneas through ERK1/2 and p38 pathways

AIM: To determine if topical instillation of dihydroarte- misinin (DHA) inhibits corneal neovascularization (NV) in rats and to investigate the role of the extracellular regulated kinases (ERK) 1/2 and p38 pathways in this process. METHODS: Suture-induced corneal NV was produced in rats and the eyes were topically treated with different concentrations of DHA (20mg/L, 10mg/L or 5mg/L) or normal saline 4 times a day for 7 days. The corneal NV was quantified as the proportion of NV area to the whole cornea. Western blot was used to determine the expressions of vascular endothelial growth factor (VEGF) and the phosphorylation status of VEGF receptor-2, ERK1/2 and p38 in the corneas. Immunofluorescent staining was used to determine the expressions of phospho-ERK1/2 and phospho-p38 in the corneal tissues from the eyes treated with 20 mg/L DHA (DHA group) or normal saline (control group). RESULTS: The proportion of corneal NV area in the eyes treated with normal saline or DHA at dosages of 20mg/L, 10mg/L or 5mg/L was (23.74±3.00)%, (15.73±2.88)%, (19.53±2.42)%, and (23.38±2.79)%, respectively. In the eyes treated with 20mg/L or 10mg/L DHA, the corneal NV area was significantly reduced when compared to that in eyes with normal saline (P<0.05). Western blot analyses revealed that 20mg/L DHA significantly inhibited the expressions of VEGF and phospho-VEGFR-2. Both 20mg/L and 10mg/L DHA inhibited the expressions of phospho-ERK1/2 and phospho- p38. Immunofluorescent staining further demonstrated that 20mg/L DHA lowered the expression levels of phospho- ERK1/2 and phospho-p38 in the corneas with suture-induced NV. CONCLUSION: Suture-induced NV in rat corneas was significantly inhibited by topical treatment with 20mg/L and 10mg/L DHA. The results suggest that the effects could be partially dependent on the DHA-mediated inhibitions of the ERK1/2 and p38 pathways.     

Inhibitory effect of polysulfated heparin endostatin onalkali burn induced corneal neovascularization in rabbits

AIM: To investigate anti-angiogenic effects of polysulfated heparin endostatin (PSH-ES) on alkali burn induced corneal neovascularization (NV) in rabbits. METHODS: An alkali burn was made on rabbit corneas to induce corneal NV in the right eye of 24 rabbits. One day after burn creation, a 0.2 mL subconjunctival injection of 50 μg/mL PSH-ES, 50 μg/mL recombinant endostatin (ES), or normal saline was administered every other day for a total of 14d (7 injections). Histology and immunohistochemisty were used to examine corneas. Corneal NV growth was evaluated as microvessel quantity and corneal vascular endothelial growth factor (VEGF) expression was measured by immunohistochemical assay. RESULTS: Subconjunctival injection of ES and PSH-ES resulted in significant corneal NV suppression, but PSH-ES had a more powerful anti-angiogenic effect than ES. Mean VEGF concentration in PSH-ES treated corneas was significantly lower than in ES treated and saline treated corneas. Histological examination showed that corneas treated with either PSH-ES or ES had significantly fewer microvessels than eyes treated with saline. Additionally corneas treated with PSH-ES had significantly fewer microvessels than corneas treated with ES. CONCLUSION: Both PSH-ES and recombinant ES effectively inhibit corneal NV induced by alkali burn. However, PSH-ES is a more powerful anti-angiogenic agent than ES. This research has the potential to provide a new treatment option for preventing and treating corneal NV.     

Inhibition of corneal neovascularization by topical application of nintedanib in rabbit models

AIM: To evaluate the potential efficacy and mechanisms of nintedanib in corneal neovascularization (NV) in rabbit models. METHODS: Corneal NV was induced using 1 mol/L NaOH. Rabbits (n=21) were randomized to 3 groups: Group 1 were treated with 0.9% NaCl, Group 2 with Avastin (5 mg/mL), and Group 3 with nintedanib (1 mg/mL). All treatments started 1d after alkaline burns and were topically performed 3 times a day for 2wk. Photographs were taken on a slit lamp microscope on day 7 and 14. The NV area, the length of the vascularization and angiogenesis index (AI) were used to evaluate the corneal NV. On day 14, the immunohistochemical (IHC) studies of the cornea were examined. Western blot was performed to test the expression levels of vascular endothelial growth factor (VEGF), Akt, p-Akt, P38, p-P38, MMP-2 and MMP-9. RESULTS: The corneal NV area, vessel length and AI in Group 3 were significantly lower than Group 2, with both being lower than Group 1. IHC staining showed that VEGF was significantly overexpressed in the epithelium and stroma of cornea following alkaline burns. In contrast, the level of VEGF was significantly suppressed in both Group 2 and Group 3. Western blot results further confirmed that, compared with Group 1, Group 3 had significantly reduced expressions of VEGF, Akt, p-Akt, p-P38, MMP-2, and MMP-9 in corneal tissues. Trends of lower levels of MMP-2, AKT, and p-AKT in Group 3 than Group 2 were identified. CONCLUSION: Nintedanib and Avastin can effectively inhibit corneal NV, with P38 MAPK and AKT signaling pathways being possibly involved. Nintedanib seems more effective than Avastin and has the potential to be a novel therapy for preventing corneal NV.     

自体血清与小牛血去蛋白眼用凝胶治疗兔角膜碱烧伤的比较

目的:通过建立兔角膜碱烧伤模型,比较自体血清与小牛血去蛋白眼用凝胶在兔角膜碱烧伤治疗中的效果。方法:制作兔右眼角膜碱烧伤模型30眼,造模后碱烧伤分度全部为Ⅲ度烧伤,将其随机分为三组,分别采用生理盐水、小牛血去蛋白眼用凝胶及自体血清滴眼液4次/d,各组滴阿托品眼用凝胶1次/晚,氧氟沙星眼用凝胶1次/晚,连续2wk。治疗7、14d后观察角膜新生血管形态并计算面积;第14d处死各组实验动物后,摘除双眼角膜,其中左眼为正常对照,右眼为实验眼,分别进行常规组织病理学检查,角膜组织匀浆内CD45、IL-10、IFN-γ及VEGF浓度测定。结果:治疗7、14d后小牛血去蛋白眼用凝胶组角膜新生血管面积(29.48±2.27、34.19±2.67mm2)与自体血清组(34.19±2.67、33.89±2.74mm2)无差异(P>0.05)。治疗14d角膜组织匀浆测定:小牛血去蛋白眼用凝胶组(0.56±0.04ng/mL)和自体血清组(0.54±0.05ng/mL)CD45含量均小于生理盐水组(1.27±0.07ng/mL)(P<0.05);自体血清组(452.49±11.40pg/mL)和小牛血去蛋白眼用凝胶组(332.49±13.67pg/mL)IL-10含量均大于生理盐水组(111.05±6.95pg/mL)(P<0.05);小牛血去蛋白眼用凝胶组(23.20±2.89pg/mL)和自体血清组(22.61±2.72pg/mL)IFN-γ含量均小于生理盐水组(41.77±4.26pg/mL)(P<0.05);小牛血去蛋白眼用凝胶组(151.14±18.21pg/mL)和自体血清组浓度(149.11±14.75pg/mL)VEGF含量均小于生理盐水组(391.35±28.59pg/mL)(P<0.05)。结论:兔角膜碱烧伤后抑制炎症因子CD45、IFN-γ及VEGF释放及抑制角膜新生血管形成方面,自体血清与小牛血去蛋白眼用凝胶作用相当;在促进抗炎因子IL-10释放、抑制炎症细胞浸润方面,自体血清作用强,小牛血去蛋白眼用凝胶次之。     

Hyperglycemia affects flicker-induced vasodilation in the retina of healthy subjects

Flickering light stimulation of retinal photoreceptors induces retinal vessel dilation in humans. In the present study the effect of high blood glucose levels on this neuro-vascular mechanism was investigated in 12 healthy young male subjects. Blood glucose levels were consecutively increased during 30 min to 100, 200 and 300 mg/dl and kept at the respective level for the following 30 min using hyperglycemic insulin clamps. Eight Hertz flickering light was applied to the fundus at the end of each glucose plateau during continuous retinal vessel diameter measurements with the Zeiss retinal vessel analyser (RVA). During normoglycemia (100 mg/dl) flickering light induced a significant vasodilation of retinal arteries (+2.8+/-0.4%, p<0.0001) and veins (+2.6+/-0.4%, p<0.0001). At 300 mg/dl blood glucose the flicker response in retinal veins was significantly decreased by 55% (p=0.015 versus 100 mg/dl). The modified RVA employed in the present study provides high sensitivity and is capable of studying flicker-induced retinal vasodilation. Using this technique the present study confirms that flickering light stimulation of the human retina induces vasodilation in retinal vessels in healthy subjects. In addition, our data indicate that the retinal vessel response to flickering light stimulation is significantly reduced during hyperglycemia in humans. The relevance of this finding for diabetes-related eye disease remains to be shown.     

HbA1 and HbA1c determination in ophthalmology. New parameters for monitoring metabolism in diabetics

Determination of glycosylated hemoglobins HbA1 or HbA1c by column chromatography is used for long-term glucose control. In the study reported here the authors compared the advantages and disadvantages of this new method and tried to show the importance of these determinations for patients with eye diseases. In a control group (blood glucose less than 120 mg/dl) HbA1-values of 6.5 +/- 1.2% and HbA1c-values of 4.4 +/- 0.5% were found. Well-controlled diabetics (blood glucose 80-180 mg/dl) had an HbA1 of 8.1 +/- 1.6% and an HbA1c of 5.8 +/- 1.1%, while diabetics with poor control (blood glucose 160-420 mg/dl) had an HbA1 of 10.6 +/- 2.1% and an HbA1c of 7.7 +/- 1.7%. HbA1 and HbA1c, respectively, should be measured in addition to blood glucose determination and oral glucose tolerance test.     

Diurnal and long-term variations in corneal thickness in the normal and alloxan-induced diabetic rabbit

The variation of central corneal thickness with age was measured weekly at 10am by ultrasound pachometry in 10 normal (5 to 19 weeks of age) and 10 alloxan-induced diabetic (9 to 19 weeks of age) rabbits. The diurnal variation of corneal thickness was also measured at 2 hour intervals for a period of 24 hours on both groups of rabbits at 19 weeks of age. A nonlinear increase in corneal thickness with age was noted in both groups, with the diabetic cornea becoming significantly thicker (p less than 0.05) than the normal control after 7 weeks of hyperglycemia. At 19 weeks of age, the diabetic rabbit cornea (379 +/- 18um) was 5.2% thicker than the normal control (360 +/- 10um). Both normal (26um) and diabetic (26um) rabbits demonstrated a similar sinusoidal diurnal variation of corneal thickness, with the cornea being thinnest in both groups near midnight. A negative exponential function derived using nonlinear regression analysis was found to adequately describe the age data, and could be used to provide a number of possibly useful clinical indices of corneal thickness variation with age. The data from this study demonstrate that 1) the variation of rabbit corneal thickness with age can be modelled using a negative exponential function, 2) alloxan-induced hyperglycemia is associated with an increase in rabbit corneal thickness, 3) alloxan-induced hyperglycemia does not appear to influence the diurnal variation of rabbit corneal thickness and 4) care should be taken in the interpretation of in vivo corneal thickness studies using normal rabbits of under 12 weeks of age (2kg body weight).     

Vascular endothelial growth factor is increased in aqueous humor of glaucomatous eyes

PURPOSE: To assess the concentrations of vascular endothelial growth factor (VEGF) in aqueous humor in eyes with and without glaucoma. METHODS: Concentrations of VEGF were measured using a sandwich ELISA kit in aqueous humor aspirates taken during anterior segment surgery from 87 patients, of whom 54 had glaucoma (27 primary open-angle glaucoma, 8 angle-closure glaucoma, 16 exfoliative glaucoma) and 33 had cataract only. RESULTS: Vascular endothelial growth factor was detected in all samples. The concentration in eyes with cataract only without glaucoma was 102.4 +/- 29.7 pg/mL (mean +/- SD), which was significantly lower than that from eyes with glaucoma (146.7 +/- 51.8 pg/mL). There were no significant differences between primary open-angle glaucoma (140.4 +/- 51.0 pg/mL), angle-closure glaucoma (142.8 +/- 40.2 pg/mL), and exfoliative glaucoma (158.6 +/- 58.9 pg/mL). An unusually high VEGF concentration was detected in one eye with neovascular glaucoma (759 pg/mL) and two eyes with uveitic glaucoma (322 pg/mL). No effect of age, gender, or previous history of medical, laser, or surgical treatment of the aqueous humor VEGF concentration could be detected ( > 0.05). Aqueous humor and plasma VEGF concentrations were measured and compared in 46 patients. The aqueous humor VEGF concentration (144.2 +/- 107.9 pg/mL) was significantly higher ( < 0.01) than the plasma concentration (79.2 +/- 46.1 pg/mL). No significant correlation was found between aqueous humor and plasma VEGF concentrations. CONCLUSION: Aqueous VEGF concentration is increased in eyes with glaucoma.     

Autologous serum application in the treatment of neurotrophic keratopathy

OBJECTIVE: To evaluate the effect of autologous serum application for epithelial disorders in neurotrophic keratopathy (NK). DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: Fourteen eyes of 11 patients with NK seen at Tokyo Dental College, Ichikawa General Hospital, Department of Ophthalmology, were studied. INTERVENTION: Twenty percent topical autologous serum eye drops were applied 5 to 10 times daily until resolution of the NK. Patients underwent routine ophthalmic examinations, including slit-lamp examination, corneal fluorescein dye testing, Cochet-Bonnet corneal sensitivity (Luneau, France), and best-corrected visual acuity (BCVA) measurements before and at the end of the treatment. Moreover, serum samples from 10 healthy volunteers were studied for the levels of substance P (SP), insulinlike growth factor (IGF-1), and nerve growth factor (NGF) by using radioimmunoassay and enzyme-linked immunosorbent assay techniques. Tear samples from 3 healthy subjects also were analyzed for NGF and IGF-1 levels by the same techniques. MAIN OUTCOME MEASURES: The changes in corneal disease state, corneal sensitivity, and BCVA with treatment were evaluated. The levels of neural healing factors like SP, IGF-1, and NGF in serum as well as NGF and IGF-1 in tears of healthy subjects also were examined. RESULTS: The epithelial disorders healed completely in all eyes within 6 to 32 days (mean, 17.1+/-8.0 days), with a decrease in corneal scarring. The mean pretreatment corneal sensitivity was 11.8+/-11.6 mm, which increased to 30.0+/-22.9 mm after treatment at the last follow-up. Five eyes attained normal corneal sensitivity with treatment. The BCVA improved by >2 Landolt lines in 78.6% of the eyes. The mean concentrations of SP in diluted and undiluted serum were 31.4+/-8.4 pg/ml and 157.0+/-42.1 pg/ml, respectively. The mean respective concentrations of IGF-1 in diluted and undiluted serum were 31.4+/-14.8 ng/ml and 157.0+/-73.9 ng/ml. The mean concentrations for NGF were 93.6+/-63.5 pg/ml and 468.3+/-317.4 pg/ml in serum samples with and without dilution, respectively. The mean concentration of NGF in tears was found to be 54 pg/ml. Insulinlike growth factor 1 was not detected in tears in this study. CONCLUSIONS: Autologous serum harbors neurotrophic factors. Autologous serum treatment may provide neural healers to the compromised ocular surface and seems promising for the restoration of the ocular surface epithelial integrity in patients with NK.     

结膜下注射贝伐单抗对兔角膜新生血管的抑制作用

目的 观察碱烧伤后不同时间结膜下注射贝伐单抗（Bevacizumab）角膜新生血管（CNV）的形成与转归.方法 新西兰白兔54只,制成单眼碱烧伤模型,随机分为3组,每组18只眼,A组碱烧伤后结膜下立即注射贝伐单抗2.5 mg（0.1 ml）,B组碱烧伤后3d结膜下注射贝伐单抗2.5 mg（0.1 ml）,C组结膜下注射生理盐水0.1ml,为对照组.共观察28 d.裂隙灯显微镜下观察角膜新生血管生长情况,行眼前段照相并计算其面积,伤后7、14、28 d各组随机取6例角膜行共焦显微镜检查,观察角膜组织炎性细胞浸润情况及角膜新生血管形态学变化.结果 A、B及C组角膜新生血管开始出现的时间分别为（5.9＋0.8）d、（3.5＋0.6）d及（3.4＋1.1）d,其中A组明显较C组延长（P＜0.05）,B组与C组差异无统计学意义（P =0.068）.伤后各时间点A、B组角膜新生血管的生长面积均明显较C组减少（P＜0.05）,A组与B组角膜新生血管面积比较,差异有统计学意义（P＜0.05）.共焦显微镜检查可见C组烧伤区大量炎性细胞浸润及新生血管形成,而A组角膜炎性细胞较少,烧伤区无新生血管形成,B组见少量新生血管侵入烧伤区.3组基质层均可见纤维及瘢痕组织增生,其中治疗组纤维增生程度与瘢痕组织均较对照组轻.结论 结膜下注射贝伐单抗可抑制角膜炎性细胞形成,改善损伤角膜基质,促进角膜愈合,从而减少碱烧伤引起的角膜新生血管的生长,在早期注射能取得更好的疗效.     

Quantitative MR imaging study of intravitreal sustained release of VEGF in rabbits

PURPOSE: To determine whether sustained elevation of vascular endothelial growth factor (VEGF) in the vitreous cavity causes retinal hyperpermeability [blood-retinal barrier (BRB) breakdown] before the development of retinal neovascularization (NV) and to document the kinetics of the integrity of BRB breakdown versus time. METHODS: Poly(L-lactide-co-glycolide)based devices loaded with VEGF were implanted intravitreally in rabbit eyes. Contrast-enhanced magnetic resonance imaging (MRI) methods were used to identify and quantitate the retinal permeability at various time points after implantation. This was done with the newly developed MR tracer AngioMARK (Epix Medical, Boston, MA). After the MRI measurements, fundus photography and fluorescein angiography (FA) also were performed on the same set of animals. RESULTS: At 3 days after implantation, the MR images showed a significant retinal leakage into the vitreous cavity (BRB breakdown) of the VEGF-implanted eyes. To quantitate this leakage, the permeability surface area product (PS) was measured. At 3 days, the mean PS product was 1.25 +/-0.25 x 10(-5) cm3/min. Based on the VEGF in vitro release study, this 3-day BRB breakdown corresponded to a total sustained release of 7.42 +/- 0.54 microg/ml of VEGF. The fundus and FA photographs of these VEGF-implanted eyes taken at 4 days after implantation also showed a considerable level of retinal vascular dilation and tortuosity. By 12 days after implantation, the mean PS product decreased to 5.83 +/- 1.38 x 10(-6) cm3/min. However, the retinal NV was observed only after the second week after implantation. By this time, a total of 10.70 +/- 0.92 microg/ml of VEGF was released in a sustained fashion. Also, after the retinal NV development, retinal detachment also was observed. The control eyes, however, which were implanted with blank devices, remained unchanged and normal during the entire course of this study (PS = 5.57 +/- 0.66 x 10(-7) cm3/min). CONCLUSIONS. The findings indicate that sustained delivery of elevated amounts of VEGF in the vitreous cavity induces a BRB breakdown even earlier than 3 days after implantation. This was achieved after a total sustained release of 7.42 +/- 0.54 microg/ml of VEGF. This retinal leakage regressed by more than half by the time the retinal NV developed. Furthermore, a retinal detachment occurred after this retinal NV. These results are similar to proliferative diabetic retinopathy (PDR). The sustained elevation of VEGF in the vitreous cavity of rabbit eyes is potentially a good model to test VEGF antagonists to treat or prevent PDR in humans. The quantifiable change of BRB breakdown by the contrast-enhanced MRI method is ideal to assess the therapeutic intervention in vivo without killing the animal and may prove to be clinically useful in humans.     

Inhibitory effect of triamcinolone acetonide on corneal neovascularization

Background Corneal neovascularization (NV) plays an important role in the pathogenesis of corneal disorders. Recently, triamcinolone acetonide (TA) has been reported as a potential treatment for ocular angiogenesis. However, there are no reports on the inhibitory effect of TA on the corneal NV. Methods Triamcinolone acetonide (2 mg) was administered to four rabbits' eyes by a subconjunctival injection immediately after a basic fibroblast growth factor (bFGF)-pellet was placed into the cornea. As a control, four eyes received an injection of distilled water. Four weeks later, the inhibition of corneal NV was evaluated as the percentage ratio of the vessel invasion area to the area that was sandwiched between the pellet and the limbus cornea. To identify the characteristic appearance of new corneal vessels, the control cornea was examined by using the antibody of vascular endothelial growth factor (VEGF). To confirm TA concentration in TA-treated corneas, the TA level was measured using high-performance liquid chromatography. Results Neovascularization from the limbus to the pellet was detected in control eyes 4 weeks after the bFGF pellet implantation. TA-treated eyes demonstrated the inhibition of the neovascular response to the pellet. The severity of NV as compared between control and TA-treated eyes was statistically significant (P<0.05). Morphologically, new vessel growth was shown in the control cornea, and endothelial cells of new vessels were positively stained with the antibody of VEGF. TA concentration in TA-treated corneas at 2 weeks showed 63.5±42.8 μg/g (n=4, mean ± SD), while TA was not detected in control and TA-treated corneas at 4 weeks. The level of TA was effectively maintained for at least 2 weeks after the subconjunctival injection. Conclusion We have demonstrated that subconjunctival TA administration inhibited rabbit corneal NV. This agent may prove useful in the treatment of corneal angiogenic disorders. No human subjects are involved as experimental animals were used in this study     

Intravitreal bevacizumab (Avastin) for persistent new vessels in diabetic retinopathy (IBEPE study)

OBJECTIVE: To evaluate the short-term fluorescein angiographic and visual acuity effects of a single intravitreal injection of bevacizumab (Avastin) for the management of persistent new vessels (NV) associated with diabetic retinopathy. METHODS: A prospective, nonrandomized open-label study of diabetic patients with actively leaking NV refractory to laser treatment and best-corrected Early Treatment Diabetic Retinopathy Study visual acuity (BCVA) worse than 20/40. Standardized ophthalmic evaluation was performed at baseline and at weeks 1, 6, and 12 (+/-1) following intravitreal injection of 1.5 mg of bevacizumab. Main outcome measures include changes in total area of fluorescein leakage from active NV and BCVA. RESULTS: Fifteen consecutive patients (men, 9 [60%]; women, 6 [40%]) were included and all completed the 12-week follow-up period of the study. The mean +/- SD age of participants was 60.08 +/- 7.75 years (median, 59.5; range, 49-73 years). At baseline, mean +/- standard error of the mean (SEM) NV leakage area was 27.79 +/- 6.29 mm2. The mean +/- SEM area of active leaking NV decreased significantly to 5.43 +/- 2.18 mm2 and 5.50 +/- 1.24 mm2 (P < 0.05, Tukey multiple comparisons post-test) at 1 and 12 weeks postinjection, respectively; at week 6 no leakage was observed. The mean +/- SEM logMAR (Snellen equivalent) BCVA improved significantly from 0.90 (20/160) +/- 0.11 at baseline to 0.76 (20/125(+2)) +/- 0.12, 0.77 (20/125(+2)) +/- 0.11, and 0.77 (20/125(+2)) +/- 0.12 at weeks 1, 6, and 12, respectively (P < 0.05, Tukey multiple comparisons post-test). No major adverse events were observed. CONCLUSIONS: Intravitreal injection of bevacizumab achieved short-term reduction of fluorescein leakage from persistent active NV without loss of vision in patients with diabetic retinopathy. Further studies to investigate the role of anti-VEGF therapy with bevacizumab for the management of diabetic retinopathy refractory to laser treatment are warranted.     

Differential retinal angiogenic response to sustained intravitreal release of VEGF and bFGF in different pigmented rabbit breeds

PURPOSE: To determine if two different breeds of pigmented rabbits can demonstrate differences in the degree of inducible angiogenesis within the retina. METHODS: Non-biodegradable Hydron pellets approximately 1.5 mm in diameter containing both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were implanted intravitreally over the optic disk of either Dutch belt rabbits or New Zealand White/Black satin cross rabbits. Control animals from both groups were implanted with blank Hydron pellets. Animals were examined periodically over a 30-day period following implantation. Results were documented by fundus photography and flourescein angiography. Stages of neovascularization (NV) were graded between +1 (preproliferative) and +4 (total NV) with +5 for NV complicated by hemorrhage and/or retinal detachment. RESULTS: The angiogenic response in the retinas of pigmented NZW/Black satin cross rabbits (N = 5) following implantation of VEGF/bFGF-containing pellets varied extensively from the Dutch belt animals (N = 7). In the Dutch belt rabbits, grading of the angiogenic response demonstrated either +4 or +5 between day 20 and day 30 after implantation. In contrast, the NZW/Black satin cross animals gave a more muted response with a maximum grade of +2 following exposure to the same amount of VEGF and bFGF. Control eyes that received only blank pellets showed no evidence of retinal NV in either the Dutch belts (N = 5) or the NZW/Black satin cross rabbits (N = 5). Statistical analysis showed a significant interaction effect for breed and pellet type (F = 44.85 with 1 df, p < 0.00005), indicating a difference between the breeds in the angiogenic response to the pellet. Moreover, both the NZW/BSC and Dutch belt rabbits displayed a significant increase in angiogenesis with the VEGF/bFGF pellet in comparison to the blank pellet (p = 0.037 and p < 0.00005, respectively). CONCLUSIONS: These studies indicate that two different breeds of pigmented rabbits exhibit different angiogenic responses to the same amount of both VEGF and bFGF. Florid retinal NV leading to hemorrhage, fibrovascular membrane formation, and traction retinal detachment occurred in the Dutch belt rabbits while tortuosity and dilatation of existing blood vessels with subsequent regression occurred in the NZW/Black satin cross animals. Such differences in the angio-genic response may be due to differences in the genetic background of these animals. If genetic heteriogeneity exists for angiogenic responses, then understanding the genetic role in the regulation of angiogenesis will lead to the design of more effective anti-angiogenic agents and can provide predictive outcomes of individual responses to therapy.     

Effect of chronic hyperglycemia on intraocular pressure in patients with diabetes

PURPOSE: To investigate the effect of chronic hyperglycemia on intraocular pressure (IOP) in patients with diabetes. DESIGN: Cross-sectional study. METHODS: We prospectively measured the IOP by Goldmann applanation tonometry in patients with diabetes with mild hyperglycemia (glycosylated hemoglobin A1c (HbA1c) < or = 6.5%), moderate hyperglycemia (6.5% < HbA1c < 8.0), and severe hyperglycemia (HbA1c > or = 8.0%). None of the patients with diabetes had diabetic retinopathy, secondary glaucoma, ocular or laser therapy, or history of glaucoma. The HbA1c level determined within three months of the IOP measurement was used. Statistical analysis was performed by Mann-Whitney U test. P < .05 was considered significant. RESULTS: The mean IOP in the mild group (n = 58) was 15.5 +/- 2.5 mm Hg, which was statistically significantly lower than that of the severe group (16.6 +/- 2.4 mm Hg; P = .013; n = 60). CONCLUSIONS: Chronic hyperglycemia is associated with increased IOP in patients with diabetes.     

Concentration of vascular endothelial growth factor in the subretinal fluid of retinal detachment.

The purpose of this study was to investigate the release of vascular endothelial growth factor (VEGF) within the subretinal fluid in eyes with retinal detachment. Subretinal fluid was collected from patients with retinal detachment undergoing surgery for scleral buckling. Serum samples were also collected. The concentration of VEGF in the subretinal fluid and serum was investigated by enzyme-linked immunospecific assay. The average concentration of VEGF in serum samples was 168 +/- 153 pg/ml (mean +/- standard deviation). It was lower than the VEGF concentration in the subretinal fluid (485 +/- 570 pg/ml) in the same 18 patients with retinal detachment (p < 0.05). The average concentration of VEGF was 355 +/- 373 pg/ml in 31 samples of the subretinal fluid collected from simple rhegmatogenous retinal detachment. It was lower than the average concentration of 901 +/- 385 pg/ml in 8 samples of the subretinal fluid from retinal detachment with proliferative vitreoretinopathy (p < 0.05). The results suggest that the relative retinal ischemia in detached retina increases the release of VEGF into the subretinal space. Also, the concentration of VEGF within the subretinal fluid in proliferative vitreoretinopathy was higher than in simple rhegmatogenous retinal detachment.