Polymeric hydrogel as a vitreous substitute: current research, challenges, and future directions.

Vitreoretinal surgery is an essential approach to treat proliferative diabetic vitreopathy, retinal detachment, retinal tear, ocular trauma, and macular holes. The removal of the natural vitreous and the replacement with substitutes are critical steps for retina reattachment. Vitreous substitutes including silicone oil (SiO), air, sulfur hexafluoride (SF6), and perfluoropropane (C3F8), have been widely applied in clinical practice. However, these substitutes are reported to cause complications such as emulsification, high intraocular pressure, and lens opacification. Polymeric hydrogels are a kind of material with favorable physical, mechanical properties, and adaptable biocompatibility, thus being highly expected to be ideal vitreous substitutes. Despite years of research, very few polymeric hydrogels can be applied practically in the vitreous cavity. In this review, we focus on the development of polymeric natural-based hydrogels and synthetic hydrogels. Particularly, we pay attention to recent advances in the novel stimuli-response and self-assembly supramolecular hydrogels. Characterized by easy injectability and long residence time, this kind of hydrogel becomes the potentially promising candidates for ideal vitreous substitutes. Finally, we evaluate the current challenges and provide the future directions of vitreous substitutes.

Macro- and Microscale Properties of the Vitreous Humor to Inform Substitute Design and Intravitreal Biotransport.

Research on the vitreous humor and development of hydrogel vitreous substitutes have gained a rapid increase in interest within the past two decades. However, the properties of the vitreous humor and vitreous substitutes have yet to be consolidated. In this paper, the mechanical properties of the vitreous humor and hydrogel vitreous substitutes were systematically reviewed. The number of publications on the vitreous humor and vitreous substitutes over the years, as well as their respective testing conditions and testing techniques were analyzed. The mechanical properties of the human vitreous were found to be most similar to the vitreous of pigs and rabbits. The storage and loss moduli of the hydrogel vitreous substitutes developed were found to be orders of magnitude higher in comparison to the native human vitreous. However, the reported modulus for human vitreous, which was most commonly tested in vitro, has been hypothesized to be different in vivo. Future studies should focus on testing the mechanical properties of the vitreous in situ or in vivo. In addition to its mechanical properties, the vitreous humor has other biotransport mechanisms and biochemical functions that establish a redox balance and maintain an oxygen gradient inside the vitreous chamber to protect intraocular tissues from oxidative damage. Biomimetic hydrogel vitreous substitutes have the potential to provide ophthalmologists with additional avenues for treating and controlling vitreoretinal diseases while preventing complications after vitrectomy. Due to the proximity and interconnectedness of the vitreous humor to other ocular tissues, particularly the lens and the retina, more interest has been placed on understanding the properties of the vitreous humor in recent years. A better understanding of the properties of the vitreous humor will aid in improving the design of biomimetic vitreous substitutes and enhancing intravitreal biotransport.

Pressure dependent direct transtissue transmission of eyewall, sclera and vitreous body in the range of 350-1050nm.

PURPOSE: For transscleral application of optical radiation or light to the eye, it is important to know the transmission and absorbance of the tissue layers of the eyewall. The impact of photochemical energy to the retina located directly at the point of contact with the radiation emitter must be considered as well as the absorbance in choroid and retina. Therefore, the direct transmission of ex vivo porcine eyewalls and vitreous body were measured in this study. METHODS: At ex vivo porcine eyes (N=221) pressure dependent transmission measurements were performed with a pressure inducing setup. Pressure and wavelength dependent direct transmission of eyewall and vitreous body of porcine eyes were described for different applied pressures (23mmHg, 78mmHg and 132mmHg). The transmissions were investigated within the spectral range of 350-1050nm. In addition to the complete eyewall transmission measurements, the transmission of sclera, vitreous body and water was measured individually and the transmission of retina and choroid was calculated. RESULTS: With increasing wavelength and pressure, a significant transmission increase of the eyewall could be achieved (p<0.05). At 400nm and 132mmHg the transmission raised to 0.10%. At 1050nm it increased up to a value of 12.22%. In the visible spectrum, the direct transmission was always below 3.17%. The differences in eyewall transmission of eyes with different iris colors were significant at low pressure. With increasing pressure, the effect strength decreased and the differences lost their significance. CONCLUSIONS: The pressure and wavelength dependent direct transmission of the eyewall and the vitreous body significantly increased with increasing pressure. This results are benefical for technical and clinical safety, research and development of illumination devices. It is necessary to know the intraocular brightness in the eye during diaphanoscopy, photocoagulation application to determine hazards.

Intraocular pressure and axial length changes during altitude acclimatization from Beijing to Lhasa.

PURPOSE: To investigate changes in intraocular pressure (IOP) and axial length (AL) on the ascent to high altitude from Beijing to Lhasa. PATIENTS AND METHODS: Twenty volunteers (17 men, 3 women) who had been sent to work in Lhasa, Tibet for more than 6 months were enrolled. One of their journeys from Beijing to Lhasa was chosen as the time for the examination. IOP, AL, corneal curvature (K), and blood pressure (BP) were measured in Beijing (altitude 43 m) and Lhasa (altitude 3658 m). Their first examination was conducted at least 1 day before arriving in Lhasa and the second examination after they had stayed in Lhasa for 7 days. The data from the highland and lowland examinations were analyzed with a paired-sample T test and Pearson's correlation coefficient was calculated for the association between IOP and average BP. RESULTS: The mean IOP was 12.65±2.34 mmHg in Beijing and 14.85±3.1 mmHg in Lhasa. The mean AL was 24.61±1.50mm in Beijing, and 24.98±1.45 mm in Lhasa. The IOP and AL showed a significant elevation in highland compared with lowland (P<0.05). The mean K was 43.58±2.25 D in Beijing and 43.56±2.21 D in Lhasa and no significant difference was found in this study (P>0.05). A positive correlation between variance of IOP and ACD was found (r = 0.475, P<0.05) and no correlation between IOP and average BP was noted. CONCLUSIONS: High altitude may lead to a small but significant change in IOP and axial length. However, the shape of the corneal surface was not influenced by the hypobaric and hypoxic conditions.

ULTRASOUND IN VITRECTOMY: An Alternative Approach to Traditional Vitrectomy Techniques.

PURPOSE: To study a prototype of an ultrasound-based vitrector, and to try to understand the physical phenomena underlying this new technology. METHODS: We tested the ultrasound-based vitrector prototype (UV) (ultrasonically-driven handpiece obtained from a modified version of the Alcon CONSTELLATION Vision System [Alcon]) using an automatic experimental setup. Balanced saline solution (BSS) and vitreous (from fresh postmortem enucleated porcine eyes) flow rates were analyzed using three different tips. RESULTS: In general, BSS solution flow rates increased with increasing aspiration levels and decreased when we used % US power. Vitreous flow rates were influenced by aspiration levels, % US power, and ultrasound-related phenomena: cavitation phenomenon and "jet streaming." CONCLUSION: Ultrasound-based vitrectomy may represent an important alternative to traditional vitrectomy. Such a tool, capable of liquefying and excising the vitreous body using ultrasound, could overcome all the limits of the guillotine-based technique (GV). Knowledge of the physical phenomena underlying ultrasound-based technology is a necessary prerequisite for further development of this new technology.

Associations between anthropometric indicators and both refraction and ocular biometrics in a cross-sectional study of Chinese schoolchildren.

OBJECTIVE: To identify associations between anthropometric indicators (height, weight and body mass index (BMI)) and both refraction and ocular biometrics in Chinese schoolchildren in Tianjin, China. DESIGN: Cross-sectional study. PARTICIPANTS: A total of 482 (86.07%) students (6-15 years old) with no history of ocular or systemic pathologies were enrolled in this study. METHODOLOGY: Height and weight were measured using standardised protocols. Ocular biometrics (axial length (AL), vitreous chamber depth (VCD) and corneal curvature (CC)) were measured by a low-coherence optical reflectometry device. Cycloplegic refraction was measured using autorefraction. The AL/CC ratio and spherical equivalent refraction (SER) were calculated. Myopia was defined as SER ≤-0.50 dioptres (D). Multiple linear regression analysis was performed to explore the associations between anthropometric indicators (height, weight and BMI) and both refraction and ocular biometrics. RESULTS: The overall prevalence of myopia was 71.16%. Overall, only height was associated with ALs, VCDs, AL/CC ratios and refractions after controlling for age, gender, parental myopia, family income, reading and writing distance and time spent outdoors. Furthermore, age-specific results demonstrated that height and weight were independently associated with refraction in participants aged 6-8 years and 9-11 years participants. Higher heights in schoolchildren were associated with longer ALs (regression coefficient b=+0.25 for each 10 cm difference in height, p<0.01), deeper VCDs (b=+0.23, p<0.01), higher AL/CC ratios (b=+0.04, p<0.01) and more negative refractions (b=-0.48, p<0.01). Heavier weights were also associated with longer ALs (+0.29 mm, p<0.01), deeper VCDs (+0.29 mm, p<0.01), higher AL/CC ratios (+0.04, p<0.01) and more negative refractions (-0.48 D, p<0.01). CONCLUSIONS: Height and weight remained independently related to refraction and various ocular biometrics during the early adolescent growth period after adequately controlling for covariates, which could support the idea that a shared mechanism may regulate the coordinated growth of body and eye size in children.

Ex vivo biophysical characterization of a hydrogel-based artificial vitreous substitute.

PURPOSE: To characterize the biophysical properties of an artificial vitreous body substitute (VBS), which consists of a biocompatible, cross-linked, hyaluronic acid (HA)-based hydrogel, by analysing the VBS's influence on intraocular pressure (IOP) and retinal integrity in distinct ex vivo eye models in order to evaluate the its potential for in vivo biocompatibility testing. METHODS: Pig eyes were obtained immediately postmortem, and VBS was injected after core-vitrectomy. IOP was followed for 24 h (n = 5). VBS influence on retinal integrity was investigated using isolated bovine retinas superfused with an oxygen saturated nutrient solution. An electroretinogram (ERG) was recorded on explanted bovine retinae using silver/silver chloride electrodes; after application of VBS for 2 min, a washout period of 70 min was employed. The percentage of a-and b-wave reduction at the end of the washout phase was compared to baseline values (n = 5). Data were calculated throughout as the mean and the standard deviation. qRT-PCR (Bax/Bcl-2-ratio, GFAP- and PGP9.5-levels) or western blot analysis was used to test for toxicity of Princess Volume after 24 h (and ß-3 tubulin with GAPDH as a control gene). Significance was estimated by Student´s t-test; p ≤0.05 was considered to be statistically significant. RESULTS: The IOP increased non-significantly by 10% after 24 h. Short-term biocompatibility testing using isolated superfused bovine retinas showed neither significant reductions of the b-wave nor the a-wave amplitudes (b-wave reduction 14.2%, p>0.05; a-wave reduction 23.9%, p>0.05). qRT-PCR and western blot analysis did not reveal significant toxicity after 24 h. CONCLUSIONS: The manufactured HA-based hydrogel showed highly favourable biophysical characteristics in the explored ex vivo models, justifying in vivo studies enabling the assessment of biocompatibility.

A swarm of slippery micropropellers penetrates the vitreous body of the eye.

The intravitreal delivery of therapeutic agents promises major benefits in the field of ocular medicine. Traditional delivery methods rely on the random, passive diffusion of molecules, which do not allow for the rapid delivery of a concentrated cargo to a defined region at the posterior pole of the eye. The use of particles promises targeted delivery but faces the challenge that most tissues including the vitreous have a tight macromolecular matrix that acts as a barrier and prevents its penetration. Here, we demonstrate novel intravitreal delivery microvehicles-slippery micropropellers-that can be actively propelled through the vitreous humor to reach the retina. The propulsion is achieved by helical magnetic micropropellers that have a liquid layer coating to minimize adhesion to the surrounding biopolymeric network. The submicrometer diameter of the propellers enables the penetration of the biopolymeric network and the propulsion through the porcine vitreous body of the eye over centimeter distances. Clinical optical coherence tomography is used to monitor the movement of the propellers and confirm their arrival on the retina near the optic disc. Overcoming the adhesion forces and actively navigating a swarm of micropropellers in the dense vitreous humor promise practical applications in ophthalmology.

A Combined Approach for the Analysis of Ocular Fluid Dynamics in the Presence of Saccadic Movements.

One of the main ocular diseases is age-related macular degeneration, actually treated with antibodies injections into the eye. This problem has been faced by computational approaches, taking into account either the influence of the tissues surrounding the vitreous, or the saccades. The aim of this work is to propose a combined fluid dynamic model of the vitreous chamber that analyses the impact of the saccades on the fluid dynamic mechanisms. The ocular vitreous humor was modeled considering liquefaction occurring in presence of age-related macular degeneration. We identified two kinds of boundary conditions, one related to the physiological environment outside the chamber, and one related to the saccades. The scleral hydraulic conductivity was evaluated by means of experimental permeability tests. An exponential decay was used to describe the trend of the scleral hydraulic conductivity with the acting pressure drop. The streamline analysis shows two main stagnant regions on the equatorial plane and peculiar fluid dynamics in absence of saccades. This study demonstrates the major role played by the saccades in determining the fluid dynamic mechanisms inside the vitreous chamber of the eye and represents a powerful tool to investigate vitreous dynamics and its relation to clinical issues.

Algorithm for establishing the time of death of a dog based on temperature measurements in selected sites of the body during the early post-mortem period.

Post-mortem measurements were made of the body temperature of dogs. The aim of the study was to evaluate and verify a reliable mathematical model that can be used to establish the time elapsed since the death of a dog during the initial post-mortem period at room temperature, using the eye (vitreous body), internal organs (heart, liver, kidney and lung), and rectum as sites for temperature measurement. The measurements were performed at six points in the body using an electronic thermometer in conjunction with a temperature probe. The method of temperature measurement is simple and does not cause perceptible macroscopic changes or disfigure the carcass. Multiple regression analysis was shown to be suitable for estimating the time elapsed from death to the discovery of the body for a period up to 12h post-mortem. The proposed multiple regression equation using body weight and the temperature at a specific site reduces manipulation of the carcass to a minimum and thus reduces error in establishing the time of death. The multiple regression model makes it possible to precisely estimate the time elapsed since the death of the animal.

Real-Time Intraocular Pressure Measurements in the Vitreous Chamber of Rabbit Eyes During Small Incision Lenticule Extraction (SMILE).

PURPOSE: To investigate real-time intraocular pressure (IOP) during small incision lenticule extraction (SMILE) in rabbit eyes for myopia correction. METHODS: During SMILE, real-time IOP was measured in the vitreous cavity of rabbit eyes with an optic fiber pressure sensor (OFPS). Two groups (n = 6 for each) underwent surgery, one group for a -2.00 diopter (D) refractive spherical correction and the other for a -6.00 D correction. RESULTS: During surgery, the IOP increased once the glass contact attached to the cornea (Pre-suction), and peaked 83.94 mmHg (SD ± 23.87 mmHg) for the -2.00 D group and 89.17 mmHg (SD ± 22.66 mmHg) for the -6.00 D group, both average values were less than 110 mmHg when suction was initiated to fix the glass contact onto the cornea (Suction on). It then fell to 74.81 mmHg (SD ± 20.64 mmHg) and 76.94 mmHg (SD ± 27.43 mmHg), respectively, and remained stable during lenticule creation (Cutting). After suction stopped (Suction off), IOP fell steeply. During lenticule separation/extraction, the change in IOP was 32.26 mmHg (SD ± 2.91 mmHg). Notably, the average duration of elevated IOP during the surgery was 166.05 s (no longer than 3 min). CONCLUSIONS: The IOP fluctuations in the vitreous cavity using an OFPS in a rabbit model during SMILE showed that real-time IOP significantly was increased during Pre-suction, Suction on, Cutting, Suction off, and lenticule separation/extraction compared to baseline IOP, although, peaked at Suction on. Neither the degree of myopic correction nor central corneal thickness significantly affected these changes in IOP.

Does the Bursa Pre-Macularis protect the fovea from shear stress? A possible mechanical role.

Purpose of present study is to evaluate whether the Pre-Macular Bursa (PMB) modifies Wall Shear Stress (WSS) at the retinal surface during saccadic movements. We created a mathematical model consisting of 25,000 grid cells and simulated a horizontal saccade spanning 50° in 0.17s, both in absence and in presence of the PMB. Wall Shear Stress SS was computed throughout the retinal surface and the posterior pole was divided into 3 Zones comprising 400 nodes each: Zone 1 (radius 3.5 mm; 0°-17°) corresponding to the PMB area; Zone 2 (concentric annular area 5 mm in radius; 22°) and Zone 3 (concentric annular area 5.5 mm; 28°). The PMB reduced WSS significantly at the macula and increased it in the immediate surroundings. Average WSS in Zone 1 was 1.53 ±â€¯1.01 (max 4.23 Pa) with PMB Vs 6.94 ±â€¯9.23 (max 35.83 Pa) without. Zone 2 WSS was 9.39 ±â€¯10.33 (max 48.36 Pa) with PMB Vs 6.95 ±â€¯9.40 (max 38.60 Pa) without Zone 3 WSS was 8.41 ±â€¯10.03 (max 43.16 Pa) with PMB Vs 6.88 ±â€¯9.42 (max 39.43 Pa) without (p < 0.001 in all cases). The PMB significantly reduces WSS over the retinal surface underlying the bursa region; conversely, WSS slightly increases it in the immediate neighboring areas.

Introduction of the MDM2 T309G Mutation in Primary Human Retinal Epithelial Cells Enhances Experimental Proliferative Vitreoretinopathy.

Purpose: The murine double minute (MDM)2 is a critical negative regulator of the p53 tumor suppressor, and MDM2 SNP309G is associated with a higher risk of proliferative vitreoretinopathy (PVR); in addition, the MDM2 T309G created using clustered regularly interspaced short palindromic repeats (CRISPR)/associated endonuclease (Cas)9 enhances normal rabbit vitreous-induced expression of MDM2 and survival of primary human retinal pigment epithelial (hRPE) cells in vitro. The goal of this study was to determine whether this MDM2 T309G contributes to the development of experimental PVR. Methods: hRPE cells expressing MDM2 T309G or T309T only were treated with vitreous from human PVR donors (HV). The expression of MDM2 and p53 in the treated cells was examined by Western blot. The in vitro vitreous-induced cellular responses, such as contraction were assessed, and PVR was induced by intravitreal injection of the hRPE cells with MDM2 T309G or T309T only into rabbit eyes. Results: Western blot analyses indicated that treatment of hRPE cells with HV led to a significant increase (1.7 ± 0.2-fold) in the expression of MDM2 and a significant decrease in p53 in the cells expressing MDM2 T309G compared with those with MDM2 T309T. In addition, HV promoted contraction of the hRPE cells expressing MDM2 T309G significantly more than those with MDM2 T309T only. Furthermore, MDM2 T309G in the hRPE cells enhanced the development of PVR in a rabbit model. Conclusions: The MDM2 SNP309 in RPE cells enhances their potential of PVR pathogenesis.

Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells.

Purpose: In humans, vitrectomy is associated with development of nuclear cataracts. Iron catalyzes free radical formation causing oxidative damage, which is implicated in cataract formation. This study was designed to determine if vitreous humor, which can initiate differentiation of lens epithelial cells, would have an effect on iron-handling proteins. Methods: Cultured canine lens epithelial cells were treated with collected canine vitreous humor. Lysates of treated and control cells were separated by SDS-PAGE. Ferritin H- and L-chains, transferrin receptor 1, and aquaporin 0 were immunodetected and quantitated with specific antibodies. Morphologic changes in treated cells were assessed. Results: Treatment of lens epithelial cells with a 33% (vol/vol) solution of vitreous humor changed the morphology of lens cells and induced expression of aquaporin 0, a marker of fiber cell differentiation that was undetectable in control cells. Treatment did not modify the size of iron-handling proteins but significantly increased content of ferritin from 2.9- to 8.8-fold over control and decreased levels of transferrin receptor by 37% to 59%. Conclusions: Vitreous humor may significantly limit iron uptake by transferrin/transferrin receptor pathway, and by increasing ferritin levels could profoundly increase the iron-storage capacity of ferritin in lens cells. Vitreous humor may play a significant protective role against iron-catalyzed oxidative damage of lens epithelial cells and therefore in the formation of cataracts.

Lens density measurement with Scheimpflug camera in vitrectomised eyes. / Densidad critaliniana medida con cámara de Scheimpfug en ojos vitrectomizados.

OBJETIVE: To determine whether vitrectomised eyes have a higher lens optical density, when measured with a Scheimpflug camera, compared to non-vitrectomised eyes. METHOD: The peak and linear Scheimpflug optical density (DOS), and area of both eyes were measured in a sample size of 81 vitrectomised phakic patients. A comparison was made between the DOS of the vitrectomised eye lens and the contralateral non-vitrectomised eye using the Student-t test. RESULTS: A significantly higher linear DOS and area was obtained in the 81 vitrectomised phakic eyes when compared to the non-vitrectomised eyes (P<.001). The peak DOS is not significantly increased in respect to non-vitrectomised eyes (P=.59). CONCLUSIONS: The lens DOS in vitrectomised eyes is higher than in non-vitrectomised eyes. The importance of the vitreous in the maintenance of lens transparency is emphasised.

An evaluation of ocular elasticity using real-time ultrasound elastography in primary open-angle glaucoma.

OBJECTIVE: The aim of this study was to compare sonoelastographic findings in the retina-choroid-sclera (RCS) complex and vitreous in glaucomatous and healthy eyes. METHODS: For this cross-sectional comparative study, 20 patients with primary open-angle glaucoma and 20 healthy volunteers were recruited. Ultrasound elastography measurements were taken with a sonographic scanner of the RCS complex, anterior vitreous (AV), posterior vitreous (PV), retrobulbar fat tissue (RFT), optic disc (OD) and optic nerve (ON) in each eye. RESULTS: The elasticity index of the RCS complex, RFT, OD, ON, AV and PV was similar in both groups (p > 0.05), although the AV/PV strain ratio in the group of patients with glaucoma was significantly higher (p = 0.04). CONCLUSION: Glaucoma increases the AV/PV strain ratio. In providing reproducible and consistent values, the real-time elastography (RTE) technique may be helpful in elucidating the mechanisms of glaucoma in some aspects. ADVANCES IN KNOWLEDGE: This study can help to evaluate the elasticity of the RCS complex and vitreous in glaucomatous eyes with RTE.

Accommodative movements of the lens/capsule and the strand that extends between the posterior vitreous zonule insertion zone & the lens equator, in relation to the vitreous face and aging.

PURPOSE: To elucidate the dynamic accommodative movements of the lens capsule, posterior lens and the strand that attaches to the posterior vitreous zonule insertion zone and posterior lens equator (PVZ INS-LE), and their age-related changes. METHODS: Twelve human subjects (ages 19-65 years) and 12 rhesus monkeys (ages 6-27 years) were studied. Accommodation was induced pharmacologically (humans) or by central electrical stimulation (monkeys). Ultrasound biomicroscopy was used to image intraocular structures in both species. Surgical procedures and contrast agents were utilized in the monkey eyes to elucidate function and allow visualization of the intraocular accommodative structures. RESULTS: Human: The posterior pole of the lens moves posteriorly during accommodation in proportion to accommodative amplitude and ciliary muscle movement. Monkey: Similar accommodative movements of the posterior lens pole were seen in the monkey eyes. Following extracapsular lens extraction (ECLE), the central capsule bows backward during accommodation in proportion to accommodative amplitude and ciliary muscle movement, while the peripheral capsule moves forward. During accommodation the ciliary muscle moved forward by ~1.0 mm, pulling forward the vitreous zonule and the PVZ INS-LE structure. During the accommodative response the PVZ INS-LE structure moved forward when the lens was intact and when the lens substance and capsule were removed. In both the monkey and the human eyes these movements declined with age. CONCLUSIONS: The accommodative shape change of the central capsule may be due to the elastic properties of the capsule itself. For these capsule/lens accommodative posterior movements to occur, the vitreous face must either allow for it or facilitate it. The PVZ INS-LE structure may act as a 'strut' to the posterior lens equator (pushing the lens equator forward) and thereby facilitate accommodative forward lens equator movement and lens thickening. The age-related posterior restriction of the ciliary muscle, vitreous zonule and the PVZ-INS LE structure dampens the accommodative lens shape change. Future descriptions of the accommodative mechanism, and approaches to presbyopia therapy, may need to incorporate these findings.

Effects of mechanical stress and vitreous samples in retinal pigment epithelial cells.

In rhegmatogenous retinal detachment (RRD), scattered RPE cells from the basement membrane into the vitreous cavity undergo an epithelial mesenchymal transition (EMT) and form the intraocular fibrous membrane in response to vitreous fluid. We investigated whether exposure to vitreous samples was associated with EMT-associated signals and mesenchymal characters. Human vitreous samples were collected from patients with RRD, epiretinal membrane (ERM), or macular hole (MH). We evaluated the effects of vitreous on ARPE-19 cells in suspension cultures using poly 2-hydroxyethyl methacrylate-coated dishes and three-dimensional (3D) Matrigel cultures. We found that exposure to vitreous samples did not induce morphological changes or accelerate wound closure in monolayers. Several samples showed increased phosphorylation of Smad2 and nuclear translocation of nuclear factor-κB. Mechanical stress triggered an elevation of phosphorylation levels in Smad2. In addition, exposure to vitreous fluid increased the phosphorylation of p38 mitogen-activated protein kinase in cell suspension cultures after mechanical stress. Moreover, ARPE-19 cells showed a stellate invasive phenotype in 3D Matrigel cultures with vitreous samples. In this study, we demonstrated that mechanical stress and vitreous were associated with EMT-associated signals and invasive phenotypes in 3D cultures but not in monolayers. These results have important implications for the role of vitreous humor in the induction of EMT and intraocular fibrosis.

Retinal Biochemistry, Physiology and Cell Biology.

The vitreous, the vasculature of the retina, macular pigments, phototransduction, retinal pigment epithelium, Bruch's membrane and the extracellular matrix, all play an important role in the normal function of the retina as well as in diseases. Understanding the pathophysiology allows us to target treatment. As ocular angiogenesis, immunity and inflammation are covered elsewhere, those subjects will not be discussed in this chapter.