Evaluation of viscoelastic poly(ethylene glycol) sols as vitreous substitutes in an experimental vitrectomy model in rabbits

The aim of this study was to employ an experimental protocol for in vivo evaluation of sols of 5 wt.% poly(ethylene glycol) (PEG) in phosphate-buffered saline as artificial vitreous substitutes. A 20 gauge pars plana vitrectomy and posterior vitreous detachment were performed in the right eye of eight pigmented rabbits. Approximately 1 ml of the viscoelastic PEG sols was then injected into the vitreous space of six eyes. PEG with an average molecular weight of 300,000 and 400,000 g mol(-1) was used in two and four eyes, respectively. Two eyes received balanced salt solution and served as controls. Full-field electroretinography was carried out and intra-ocular pressure (IOP, palpation) measured pre- and post-operatively at regular intervals up to 41 days. The rabbits were killed and the eyes examined by retinal photography, gross macroscopic examination and histology. The viscoelastic sols were successfully injected and remained translucent throughout the post-operative period, with some inferior formation of precipitates. None of the eyes displayed IOP elevation post-operatively, but in three of the PEG sol injected eyes transient hypotony was noted. One eye sustained retinal detachment during surgery and another two in the post-operative period. ERG recordings confirmed preservation of retinal function in three out of four eyes injected with 400,000 g mol(-1) PEG. Histological examination revealed up-regulation of glial acidic fibrillary protein in Müller cells in PEG sol injected eyes, but normal overall morphology in eyes with attached retinas. The viscosity of the sol was not retained throughout the post-operative period, indicating the demand for polymer cross-linking to increase residence time. The results provide promising preliminary results on the use of PEG hydrogels as a vitreous substitute.     

27G微创玻璃体切割术的临床应用

目的 探讨27G微创玻璃体切割手术治疗部分玻璃体视网膜疾病的初步临床疗效及安全性.方法 收集行27G微创玻璃体切割手术治疗的玻璃体视网膜疾病患者13例13眼.手术后随访6～12个月.对比观察术前及术后的视力、眼压变化情况,观察总体手术时间及单纯玻璃体切割时间、术后切口愈合状态及手术并发症的情况.结果 平均最佳矫正视力从术前的(1.26±0.66) logMAR(0.10±0.09)提高至末次随访的(0.63±0.52) logMAR(0.35±0.24),差异有统计学意义(t=2.743,P=0.018).术前、术后第1天、术后第5天、术后1个月及末次随访的平均眼压差异无统计学意义(F=0.593,P＞0.05).平均总体手术时间为(36.38±14.97) min,平均单纯玻璃体切割时间为(10.12±3.54)min.术后巩膜切口成线状闭合,未发现巩膜切口哆开、玻璃体嵌顿及切口结膜下积液.术中及术后未发现医源性视网膜裂孔、眼内炎、脉络膜脱离、视网膜脱离、玻璃体腔再出血等并发症.结论 27G微创玻璃体切割手术治疗玻璃体视网膜疾病可提高术后视力,且切口相关并发症少,是较为安全有效的手术方式.     

Contraction of scar tissue in the rabbit vitreous

Sheets of vitreous membrane (scar tissue) and associated retinal detachment were produced in the right eye of 86 adult New Zealand white rabbits by intravitreal injection of cultured autologous skin fibroblasts. The membranes were examined by light and electron microscopy and time-lapse cinephotomicrography. Immunohistochemistry was used to demonstrate alterations in the distribution of cytoplasmic contractile proteins. While retinal detachment and membrane contraction were taking place, there was pronounced increase in the numbers of fibroblasts with an elongated spindle shape. These spindle-shaped cells had some similarities to myofibroblasts including the presence of 'stress cables'. However, the myofibroblast-like cells stained much less avidly for cytoplasmic (actin) microfilaments than migratory fibroblasts seen at early stages of membrane development. The significance of migrating fibroblasts and myofibroblasts in scar contraction is discussed.     

Contraction of Scar Tissue in the Rabbit Vitreous

Sheets of vitreous membrane (scar tissue) and associated retinal detachment were produced in the right eye of 86 adult New Zealand white rabbits by intravitreal injection of cultured autologous skin fibroblasts. The membranes were examined by light and electron microscopy and time-lapse cinephotomicrography. Immuno-histochemistry was used to demonstrate alterations in the distribution of cytoplasmic contractile proteins. While retinal detachment and membrane contraction were taking place, there was pronounced increase in the numbers of fibroblasts with an elongated spindle shape. These spindle-shaped cells had some similarities to myofibroblasts including the presence of ‘stress cables’. However, the myofibroblast-like cells stained much less avidly for cytoplasmic (actin) microfilaments than migratory fibroblasts seen at early stages of membrane development. The significance of migrating fibroblasts and myofibroblasts in scar contraction is discussed.     

Vascular endothelial growth factor and hepatocyte growth factor levels are differentially elevated in patients with advanced retinopathy of prematurity

Although the roles of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in angiogenesis are well described, the putative roles of these factors in retinopathy of prematurity (ROP) remain unknown. We evaluated VEGF and HGF protein levels in subretinal fluid of eyes with ROP, and expression of their corresponding receptors in retrolental membranes associated with stage 5 ROP. We examined subretinal fluid samples from eyes using rhegmatogenous retinal detachment as a control. VEGF and HGF were differentially elevated in eyes with ROP. In Stage 5 ROP (n = 22), the mean VEGF and HGF levels were 14.77 +/- 14.01 ng/ml and 16.56 +/- 9.62 ng/ml, respectively. Interestingly, in patients with active stage 4 ROP, mean VEGF levels were highly elevated (44.16 +/- 18.72 ng/ml), whereas mean HGF levels remained very low (4.77 +/- 2.50 ng/ml). Next, we investigated in vivo expression of VEGF receptor-2 and HGF receptor in retrolental membranes from 16 patients with stage 5 ROP. Both VEGF receptor-2 and HGF receptor proteins were detected mainly in posterior portions of the membrane as well as in vessel walls and along the retinal interface where angiogenesis was active. These findings together suggest that VEGF and HGF play important roles in the pathogenesis of ROP.     

Acute postoperative endophthalmitis caused by Staphylococcus lugdunensis

Acute postoperative endophthalmitis caused by Staphylococcus lugdunensis is infrequently reported in clinical studies. Five cases of acute postcataract surgery endophthalmitis caused by S. lugdunensis were taken from a multicenter prospective study conducted in four university-affiliated hospitals in France (2004 to 2005). These cases were characterized by severe ocular inflammation occurring with a mean delay of 7.6 days after cataract surgery, severe visual loss (hand motions or less in three cases), and dense infiltration of the vitreous. Each of these patients was initially treated by using a standard protocol with intravitreal (vancomycin and ceftazidime), systemic, and topical antibiotics. Given the severity of the endophthalmitis, even though bacteria were sensitive to intravitreal antibiotics, pars plana vitrectomy was needed in four cases. The final visual prognosis was complicated by severe retinal detachment in three cases. The microbiological diagnosis was reached by using conventional cultures with specific biochemical tests and eubacterial PCR amplification followed by direct sequencing.     

Vitreous pharmacokinetics and electroretinographic findings after intravitreal injection of acyclovir in rabbits

OBJECTIVES:

Acute retinal necrosis is a rapidly progressive and devastating viral retinitis caused by the herpesvirus family. Systemic acyclovir is the treatment of choice; however, the progression of retinal lesions ceases approximately 2 days after treatment initiation. An intravitreal injection of acyclovir may be used an adjuvant therapy during the first 2 days of treatment when systemically administered acyclovir has not reached therapeutic levels in the retina. The aims of this study were to determine the pharmacokinetic profile of acyclovir in the rabbit vitreous after intravitreal injection and the functional effects of acyclovir in the rabbit retina.

METHODS:

Acyclovir (Acyclovir; Bedford Laboratories, Bedford, OH, USA) 1 mg in 0.1 mL was injected into the right eye vitreous of 32 New Zealand white rabbits, and 0.1 mL sterile saline solution was injected into the left eye as a control. The animals were sacrificed after 2, 9, 14, or 28 days. The eyes were enucleated, and the vitreous was removed. The half-life of acyclovir was determined using high-performance liquid chromatography. Electroretinograms were recorded on days 2, 9, 14, and 28 in the eight animals that were sacrificed 28 days after injection according to a modified protocol of the International Society for Clinical Electrophysiology of Vision.

RESULTS:

Acyclovir rapidly decayed in the vitreous within the first two days after treatment and remained at low levels from day 9 onward. The eyes that were injected with acyclovir did not present any electroretinographic changes compared with the control eyes.

CONCLUSIONS:

The vitreous half-life of acyclovir is short, and the electrophysiological findings suggest that the intravitreal delivery of 1 mg acyclovir is safe and well tolerated by the rabbit retina.     

Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice

Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-beta (TGF-beta) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-beta and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of alpha-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-beta signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR.     

大肠杆菌内毒素诱导的SD大鼠眼内炎

目的 观察细菌内毒素脂多糖(LPS)诱导的大鼠眼内炎性反应的临床、组织病理学和血眼屏障的特点.方法 SD大鼠玻璃体腔内注人大肠杆菌LPS(1μg)建立LPS诱导的眼内炎动物模型,对照组注入无菌生理盐水.在LPS注入后6h至7d的不同时点,分别对眼部炎性反应评分、浸润白细胞计数、前房水蛋白质浓度和玻璃体内LPS水平进行测定和评估.结果 在LPS注射后6～72 h眼内可见严重的炎性反应,至术后7d炎性反应基本消退.眼内白细胞的浸润数量在术后24h达到高峰[(1182.63±191.15)细胞/眼],至术后3 d浸润细胞数量迅速下降[(331.25±57.9)细胞/眼].在各观察时点,均可观察到眼内炎组房水蛋白质浓度较对照组显著增高(P<0.01). LPS注入眼内后前3 d,玻璃体腔内LPS含量迅速下降[前3d分别为(327.02±51.54)、(176.0±53.68)和(54.91±13.26)ng],在7d后玻璃体腔内LPS基本被清除.结论 大肠杆菌内毒素在SD大鼠可诱导出严重的实验性眼内炎,大量白细胞眼内浸润、血眼屏障破坏和玻璃体腔自发性细菌成分清除是本实验性眼内炎模型的主要病理特点.     

雌二醇玻璃体腔注射缓解大鼠玻璃体后脱离的研究

目的　探讨雌二醇缓解玻璃体后脱离（posterior vitreous detachment, PVD）的有效性和安全性。 方法　Sprague-Dawley雄性大鼠48只，随机分为A（正常对照组）、B（平衡盐液2 μL）、C（纤溶酶3 μg/2 μL）、D（纤溶酶3μg+雌二醇10-6 mol/L, 2 μL）组，每组12只，玻璃体腔内注射7 d后,各组大鼠用视觉电生理（flash electroretinography, F-ERG）、病理切片、免疫组织荧光法、蛋白质印迹法来观察和评价视网膜结构、玻璃体后脱离的情况以及各组玻璃体视网膜opticin蛋白的表达水平。 结果　各组视网膜形态结构较完整，A组和B组无PVD发生, C组发生完全性PVD，D组发生部分性PVD（8/12）或无PVD（4/12）。F-ERG显示各组均无视网膜毒性损伤。C组opticin蛋白表达较A、B组明显降低，D组opticin蛋白表达较C组增高，差异均有统计学意义（P<0.05）。 结论　玻璃体腔注射雌二醇对大鼠玻璃体后脱离具有修复作用。     

A two-compartment model of the human retina

PURPOSE: In this article we question a basic concept in retinal pathology, which views the retina as composed primarily of neural elements, in a single compartment. METHODS: We suggest an alternative approach, centering on the epithelial-glial elements of the retina, dividing the retina into two distinct compartments. The framework of these two compartments is composed of two epithelial-like monostratified cell layers facing each other by their apical surfaces. This model is in agreement with the embryological development of the retina. RESULTS: Each compartment is composed of a monostratified cell layer in which neural elements are embedded and each is supplied by a different blood supply. The inner compartment, also referred to as the Muller cell compartment, extends between the inner and outer limiting membranes. The outer, or RPE, compartment extends between the outer limiting and Bruch's membranes. The border between the two compartments is formed by the outer limiting membrane (OLM). One simplified example utilizing the two-compartment concept is as follows: inner compartment edema (inner blood-retinal barrier breakdown) may manifest as cystoid edema, but not as serous retinal detachment, while outer compartment edema (outer blood-retinal barrier breakdown) may manifest as serous retinal detachment but not as cystoid edema, as long as the integrity of the OLM is maintained. CONCLUSION: A two-compartment approach to the structure of the retina, centering on non-neural elements, may enhance our understanding of some retinal pathologies. Various retinal diseases, mainly of vascular origin, are limited to one of the two compartments.     

The pathogenesis of vitreoretinal proliferation and traction: a working hypothesis

Traction retinal detachment due to proliferative vitreoretinopathy (PVR) is a serious complication of ocular trauma, retinal detachment, and previous vitreoretinal surgery. The cause is the active proliferation of fibroblasts, glial cells, and retinal pigment epithelial cells in the periretinal spaces, leading to the formation of contractile cellular membranes. The generation of growth and mitosis stimulation for these cells has remained obscure. We postulate that invading macrophages and local microglia secrete growth factors, notably PDGF (platelet-derived growth factor), which in turn mediates the mitogenic effects of transferrin (TF), a protein present in huge amounts in native vitreous, in plasma and in intraocular proliferative tissue.     

Numerical simulation of the fluid dynamics in vitreous cavity due to saccadic eye movement

In this study, the flow dynamics of vitreous due to saccadic movements following vitreous liquefaction or in post-vitrectomy eyes is investigated. Using a dynamic mesh technique, the eye motion was modeled and unsteady three-dimensional forms of continuity and Navier-Stokes equations were solved numerically. Firstly, the numerical model was validated for a sphere as a model of vitreous chamber and agrees well with the results based on available analytic solutions and experimental data. Then, numerical simulation was performed based on a deformed sphere with an indentation representing the lens. This consists of a vitreous cavity filled with a liquid having the viscosity of liquefied vitreous and balanced salt solution. The wall shear stress on the retina was computed and compared for various saccade amplitudes. Also, the effect of variation in vitreous viscosity and the size of lens indentation are investigated. The results show that the secondary flow in the vertical direction in the vitreous cavity is much higher for the liquefied vitreous and balanced salt solution compared with that for silicone oil. The possible effect of shear stress on the retinal detachment for all studied cases is discussed. A semi-analytic correlation is also developed for maximum wall shear stress of the spherical domain that is subjected to sinusoidal rotations.     

In situ formation of hydrogels as vitreous substitutes: Viscoelastic comparison to porcine vitreous

The human vitreous is a gelatinous substance predominantly composed of water (97-99%). Vitreous substitutes are needed for treatment of retinal detachments by reapproximating the retina to the back of the eye, or during vitrectomies for maintenance of ocular volume. None of the current substitutes can be used long-term due to their short retention time, toxicity, or complications such as glaucoma or cataracts. In addition, all of the current compounds have a specific gravity less than water and so are not appropriate for inferior retinal detachments. The viscoelastic properties of the porcine vitreous were analyzed to develop a model for ideal substitutes. Synthetic polymers that form hydrogels in situ were researched for the development of artificial vitreous. In this study, the physical, mechanical, and optical properties of four self-gelling polyacrylamide copolymeric hydrogels were determined and compared with those of the porcine vitreous. The 2% formulation of high crosslink density, hydrophobically modified hydrogel most closely mimicked the porcine vitreous. The viscoelastic properties of hydrogels capable of being formed in situ were compared to those of the porcine vitreous to determine their efficacy as vitreous substitutes.     

Vitreous growth hormone and visual dysfunction

Growth factors have been found in vitreous fluid, in which they regulate retinal function and provide markers of ocular dysfunction. Since growth hormone (GH) has recently been discovered in the vitreous of human eyes, the possibility that vitreal GH concentrations might differ in different ocular disease states was assessed. GH-immunoreactivity in the vitreous of cadaver controls and in the vitreous of patients with ocular dysfunction was quantified by ELISA. In non-diabetics, vitreous GH concentrations were comparable in patients with subretinal hemorrhage (SH), vitreous hemorrhage (VH), vitreous debris (VD), retinal detachment (RD), central retinal vein occlusion (CRVO), macular hole (MH), dislocated crystalline lens (DCL) or epiretinal membrane (ERM). The GH concentration, corrected for protein content, in the vitreous of diabetic patients was, however, lower than that in cadaver controls with no history of ocular disease and lower than that in non-diabetic patients with ocular dysfunction. Vitreous GH concentrations in diabetic patients with proliferative diabetic retinopathy (PDR) did not differ from those without PDR. The presence of GH in the human vitreous suggests that vitreous GH may have roles in normal ocular function and be involved in the pathogenesis of ocular disease. Low GH concentrations in the vitreous of diabetic patients may correlate with retinal neurodegeneration and may provide a marker to follow its progression.     

The active transport of fluorescein by the retinal vessels and the retina

1. The movement of fluorescein across the retinal surface of the rabbit's eye was estimated by measuring the concentration gradient of the dye in the vitreous body. These measurements were made in vivo by means of a slit-lamp fluorophotometer, or were taken from frozen sections of enucleated eyes.2. In the normal eye, fluorescein does not pass from the blood to the vitreous body across any part of the retina. When injected into the vitreous body it passes rapidly out across the entire retinal surface, even against a very large concentration gradient.3. A variety of metabolic and competitive inhibitors, effective in blocking organic anion transport in the kidney and liver, tend to abolish this unidirectional movement of fluorescein across the retina.4. The region occupied by the retinal vessels is more sensitive to inhibition than other areas of the retina. Occlusion of the vessels by diathermy prevents the exchange of fluorescein in this region.5. It appears, then, that there is an active transport of organic anions out of the vitreous body, both by the retinal capillaries and by the retina itself. The latter system is probably located in the pigment epithelium and seems to be carried forward to the rear surface of the iris.6. Since the walls of the retinal vessels of the rabbit are freely in contact with the vitreous body, the active transport must take place across the capillary endothelial cells themselves. These vessels have structural and permeability characteristics found only in the central nervous system and it is to be presumed that the anion transport system is shared by the capillaries of the brain.7. The function of the transport in the retina may be to protect the nervous tissue from toxic materials by preventing their entry from the blood or by removing products of metabolism conjugated as organic anions. Alternatively, the mechanism may be concerned in maintaining the normal adhesion of the retina to the choroid, since retinal detachment was observed to follow its total inhibition.     

酵母多糖在Wistar大鼠真菌性眼内炎中的致病作用

目的 观察真菌酵母多糖诱导的Wistar大鼠眼内炎模型的组织病理学特征、血眼屏障的改变和玻璃体内肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的表达情况.方法 采用随机数字表法将大鼠分为生理盐水对照组(SC组,n=168)、眼内炎组(EO组,n=168).EO组玻璃体腔注射真菌酵母多糖溶液诱导眼内炎动物模型,SC组注入等量无菌生理盐水.注射后6、12 h及1、2、3、5、7 d,观察各组大鼠眼部炎性反应表现,并分别摘除眼球行组织病理学检查,同时取其房水检测蛋白质浓度.取玻璃体检测TNF-α、IL-1β水平.结果 注射后6 h～3 d,EO组眼内可见严重炎性反应,注射后7 d炎性反应基本消退.注射后1 d,EO组眼内白细胞浸润数量为(482.63±191.15)细胞/眼,达到高峰;注射后3 d浸润细胞数量迅速下降至(131.25±57.95)细胞/眼.注射后I d,EO组TNF-α和IL-1β浓度分别为(331.17±99.81)、(2 156.09±1 440.27)ng/L,均达到高峰并持续至注射后2 d,随后迅速下降;注射后7d,EO组TNF-α和IL-1β浓度分别为(5.55±5.27)、(43.66±18.73)ng/L.在各观察时点,均可观察到EO组房水蛋白质浓度较SC组注射后6 h显著增高(均P＜0.01).结论 真菌酵母多糖在Wistar大鼠可诱导出严重的实验性眼内炎,大量白细胞眼内浸润、血眼屏障破坏和玻璃体TNF-α、IL-1β的高水平表达是本实验性眼内炎模型的主要病理特点.     

Poly(I-vinyl-2-pyrrolidinone) hydrogels as vitreous substitutes: Histopathological evaluation in the animal eye

A homopolymer of 1-vinyl-2 pyrrolidinone and its copolymer with 2-hydroxyethyl methacrylate, both cross-linked with divinyl glycol, were produced as possible substitutes for the vitreous body of the eye. The hydrated polymers behaved like viscoelastic gels, displaying excellent physical and optical properties. The sterile gels (0.7-1.5 ml) were injected into the vitreous cavity of rabbits, which previously underwent gas-mediated vitrectomy. Clinically, the eyes were quiet, with the exception of transient opacities in the vitreous. After 4 weeks, the operated eyes were enucleated and subjected to histopathological analysis using light and transmission electron microscopy. The common feature in all sections was the invasion of inflammatory cells. Vacuoles containing granular material, assumed to be polymer, were seen in the intercellular spaces of the neural retina, in the retinal pigment epithelium cells, and in macrophages. These findings indicated the fragmentation and phagocytosis of synthetic gels. It appeared that the biodegradation of the internalized polymers did not proceed further, however, the fate of polymers and their usefulness as vitreous substitutes should be investigated through long-term experiments.     

冷凝联合光凝与单纯冷凝治疗孔源性视网膜脱离的比较观察

目的 比较观察冷凝联合激光光凝与单纯冷凝治疗孔源性视网膜脱离的疗效。方法 回顾性分析2001年6月-2005年6月因孔源性视网膜脱离进行手术治疗的患者108例109眼。其中一组66眼采用巩膜扣带术、冷凝联合激光光凝治疗，一组采用巩膜扣带术单纯冷凝治疗。结果 两组一次性手术复位率无显著性差异，术后复发率单纯冷凝组高于联合治疗组且有显著性差异（X^2=4．79，P〈0．05），术后联合治疗组视力改善高于单纯冷凝组有显著性差异（X^2=7，91，P〈0．05）。结论 对于孔源性视网膜脱离其增殖性病变在PVRC2级以下者，冷凝联合激光光凝治疗较单纯冷凝治疗效果好。     

Finite element modeling of drug distribution in the vitreous humor of the rabbit eye

Direct intravitreal injection of drug is a common method for treating diseases of the retina or vitreous. The stagnant nature of the vitreous humor and surrounding tissue barriers creates concentration gradients within the vitreous that must be accounted for when developing drug therapy. The objective of this research was to study drug distribution in the vitreous humor of the rabbit eye after an intravitreal injection, using a finite element model. Fluorescein and fluorescein glucuronide were selected as model compounds due to available experimental data. All required model parameters were known except for the permeability of these compounds through the retina, which was determined by fitting model predictions to experimental data. The location of the intravitreal injection in the experimental studies was not precisely known; therefore, several injection locations were considered, and best-fit retinal permeability was determined for each case. Retinal permeability of fluorescein and fluorescein glucuronide estimated by the model ranged from 1.94×10⁻⁵ to 3.5×10⁻⁵ cm s⁻¹ and from 0 to 7.62×10⁻⁷ cm s⁻¹, respectively, depending on the assumed site of the injection. These permeability values were compared with values previously calculated from other models, and the limitations of the models are discussed. Intravitreal injection position was found to be an important variable that must be controlled in both experimental and clinical settings.