Rheology of the vitreous gel: effects of macromolecule organization on the viscoelastic properties

The macromolecular organization of vitreous gel is responsible for its viscoelastic properties. Knowledge of this correlation enables us to relate the physical properties of vitreous to its pathology, as well as optimize surgical procedures such as vitrectomy. Herein, we studied the rheological properties (e.g. dynamic deformation, shear stress-strain flow, and creep compliance) of porcine vitreous humor using a stressed-control shear rheometer. All experiments were performed in a closed environment with the temperature set to that of the human body (i.e. 37°C) to mimic in-vivo conditions. We modeled the creep deformation using the two-element retardation spectrum model. By associating each element of the model to an individual biopolymeric system in the vitreous gel, a distinct response to the applied stress was observed from each component. We hypothesized that the first viscoelastic response with the short time scale (~1 s) is associated with the collagen structure, while the second viscoelastic response with longer time scale (~100 s) is related to the microfibrilis and hyaluronan network. Consequently, we were able to differentiate the role of each main component from the overall viscoelastic properties.     

Vitreous Humor Rheology After Nd:YAG Laser Photo Disruption

This work aimed to consider the hazardous side effect of eye floaters treatment with Q-switched Nd:YAG laser on the protein and viscoelastic properties of the vitreous humor, and evaluate the protective role of vitamin C against laser photo disruption. Five groups of New Zealand rabbits were divided as follows: control group for (n = 3) without any treatment, the second group (n = 9) treated with Q-switched Nd:YAG laser energy of 5 mJ × 100 pulse delivered to the anterior, middle, and posterior vitreous, respectively (n = 3 for each). The third group (n = 9) received a daily dose of 25 mg/kg body weight vitamin C for 2 weeks, and then treated with laser as the previous group. The fourth group (n = 9) treated with 10 mJ × 50 pulse delivered to the anterior, middle, and posterior vitreous, respectively (n = 3 rabbits each). The fifth group (n = 9) received a daily dose of 25 mg/kg body weight vitamin C for 2 weeks, and then treated with laser as the previous group. After 2 weeks of laser treatment, the protein content, refractive index (RI), and the rheological properties of vitreous humor, such as consistency, shear stress, and viscosity, were determined. The results showed that, the anterior vitreous group exposed to of 5 mJ × 100 pulse and/or supplemented with vitamin C, showed no obvious change. Furthermore, all other treated groups especially for mid-vitreous and posterior vitreous humor showed increase in the protein content, RI and the viscosity of vitreous humor. The flow index remained below unity indicating the non-Newtonian behavior of the vitreous humor. Application of Q-switched Nd:YAG laser should be restricted to the anterior vitreous humor to prevent the deleterious effect of laser on the gel state of the vitreous humor.     

Proteomic Insight into the Molecular Function of the Vitreous

The human vitreous contains primarily water, but also contains proteins which have yet to be fully characterized. To gain insight into the four vitreous substructures and their potential functions, we isolated and analyzed the vitreous protein profiles of three non-diseased human eyes. The four analyzed substructures were the anterior hyaloid, the vitreous cortex, the vitreous core, and the vitreous base. Proteins were separated by multidimensional liquid chromatography and identified by tandem mass spectrometry. Bioinformatics tools then extracted the expression profiles, signaling pathways, and interactomes unique to each tissue. From each substructure, a mean of 2,062 unique proteins were identified, with many being differentially expressed in a specific substructure: 278 proteins were unique to the anterior hyaloid, 322 to the vitreous cortex, 128 to the vitreous base, and 136 to the vitreous core. When the identified proteins were organized according to relevant functional pathways and networks, key patterns appeared. The blood coagulation pathway and extracellular matrix turnover networks were highly represented. Oxidative stress regulation and energy metabolism proteins were distributed throughout the vitreous. Immune functions were represented by high levels of immunoglobulin, the complement pathway, damage-associated molecular patterns (DAMPs), and evolutionarily conserved antimicrobial proteins. The majority of vitreous proteins detected were intracellular proteins, some of which originate from the retina, including rhodopsin (RHO), phosphodiesterase 6 (PDE6), and glial fibrillary acidic protein (GFAP). This comprehensive analysis uncovers a picture of the vitreous as a biologically active tissue, where proteins localize to distinct substructures to protect the intraocular tissues from infection, oxidative stress, and energy disequilibrium. It also reveals the retina as a potential source of inflammatory mediators. The vitreous proteome catalogues the dynamic interactions between the vitreous and surrounding tissues. It therefore could be an indirect and effective method for surveying vitreoretinal disease for specific biomarkers.     

Traction on the retina induced by saccadic eye movements in the presence of posterior vitreous detachment

Posterior vitreous detachment is a fairly common condition in elderly people. Tractions exerted by the detached vitreous on the retina may result in retinal tears and detachments. We studied how these tractions can arise from saccadic eye movements. Numerical simulations have been performed on a two-dimensional model of the vitreous chamber within a rigid spherical sclera, subjected to prescribed finite-amplitude rotations about a vertical axis. The vitreous chamber was assumed to be split into two regions: one occupied by the detached vitreous, modeled as an elastic viscous solid, and the other occupied by the separated liquefied vitreous, modeled as a Newtonian fluid. At the interface between the two phases, we also considered the presence of the vitreous cortex, modeled as an elastic membrane. We tested several different configurations of the interface. In all cases, we found that eye rotations generate large tractions on the retina close to the attachment points of the membrane. Comparing them, we identified configurations of the vitreous detachment that exhibit higher tractions. We also investigated how the response to saccadic movements depends on some physical parameters, in particular on the rheological properties of the solid phase and the membrane. The numerical simulations show that the generated tractions may be of the same order of magnitude as the adhesive force between the retina and the pigment epithelium. Therefore, the model provides a sound physical justification for the hypothesis that saccadic movements, in the presence of posterior vitreous detachment, could be responsible for high tractions on the retina, which may trigger retinal tear formation.     

The nature and origin of the glycosaminoglycans of the embryonic chick vitreous body

Glycosaminoglycans of the embryonic chicken vitreous were characterized and then were used as markers to establish which tissues synthesize the vitreous humor during development. The glycosaminoglycans are predominantly chondroitin sulfates by several criteria. They are resistant to streptomyces hyaluronidase, an enzyme which degrades only hyaluronate, and are digested by testicular hyaluronidase and chondroitinase AC, enzymes which degrade hyaluronate plus chondroitin 4- and 6-sulfates. On electrophoresis on cellulose acetate in 0.15 M phosphate buffer, pH 6.7, the vitreous glycosaminoglycans migrate slightly slower than authentic chondroitin sulfate, but, in 0.1 N HCl, they migrate very close to chondroitin sulfate standards. Finally, the disaccharides produced by digestion of these radioactively labeled glycosaminoglycans with chondroitinases AC and ABC were identified as Δdi-4S and Δdi-6S, as expected for chondroitin 4- and 6-sulfate. By using incorporation of radioactive precursors into chondroitin sulfates in vitro, we than determined which tissues synthesize the vitreous humor in the developing eye. Late in development, on Day 12–13, the isolated vitreous is very active in chondroitin sulfate synthesis, while the neural retina, the lens, and the pecten are less active and produce a high proportion of enzyme-resistant GAG. The eye tissues isolated from embryos labeled in ovo retain similar amounts and types of glycosaminoglycans, indicating that cells within the vitreous synthesize the vitreous humor glycosaminoglycans at this time. Earlier in development, from Days 6 to 8, the isolated vitreous incorporates very low levels of radioactivity into GAG, but the neural retina incorporates high levels of radioactivity into chondroitin sulfate. When the embryos are labeled in ovo and the same tissues are isolated following incorporation, the vitreous retains more radioactive chondroitin sulfate than does the neural retina. Thus, the vitreous humour glycosaminoglycan is initially synthesized by the neural retina and is secreted into the vitreous space.     

Method of determining the colloidal status of the vitreous body

A new method for the quantitative estimation of the vitreous body colloidal state has been suggested. For realization of the method a fragment of the vitreous body and an equal volume of water are dried out, and the drying time is registered. The higher is the ratio between the drying time of the vitreous body fragment and water, the higher is the density of the vitreous body gel. The drying out of the vitreous body fragment and water on the analytical balance cups permits the registration of the drying out process in the time course. Thus some additional parameters are obtained that characterize the object studied more completely. The method can be used in experimental and clinical investigations of the vitreous body and other biological liquids.     

A computational study of the flow through a vitreous cutter

Vitrectomy is an ophthalmic microsurgical procedure that removes part or all of the vitreous humor from the eye. The procedure uses a vitreous cutter consisting of a narrow shaft with a small orifice at the end through which the humor is aspirated by an applied suction. An internal guillotine oscillates back and forth across the orifice to alter the local shear response of the humor. In this work, a computational study of the flow in a vitreous cutter is conducted in order to gain better understanding of the vitreous behavior and provide guidelines for a new vitreous cutter design. The flow of a Newtonian surrogate of vitreous in a two-dimensional analog geometry is investigated using a finite difference-based immersed boundary method with an algebraically formulated fractional-step method. A series of numerical experiments is performed to evaluate the impact of cutting rate, aspiration pressure, and opening/closing transition on the vitreous cutter flow rate and transorifice pressure variation during vitrectomy. The mean flow rate is observed to increase approximately linearly with aspiration pressure and also increase nearly linearly with duty cycle. A study of time-varying flow rate, velocity field, and vorticity illuminates the flow behavior during each phase of the cutting cycle and shows that the opening/closing transition plays a key role in improving the vitreous cutter's efficacy and minimizing the potential damage to surrounding tissue. The numerical results show similar trend in flow rate as previous in vitro experiments using water and balanced saline solution and also demonstrate that high duty cycle and slow opening/closing phases lead to high flow rate and minor disturbance to the eye during vitrectomy, which are the design requirements of an ideal vitreous cutter.     

Saccade movements effect on the intravitreal drug delivery in vitreous substitutes: a numerical study

In this study, the distributions of intravitreal injected drugs in post-vitrectomy human eyes, which are subjected to periodic saccade movements, are investigated. The computational model for the vitreous cavity of human eye is a sphere with one side truncated by the eye lens. A dynamic mesh technique was used to model the eye motion and the unsteady 3-D forms of continuity; Navier–Stokes and concentration transport of drug equations were solved numerically. The numerical model was validated earlier for the vitreous liquid flow field. The predicted drug concentration for idealized geometry was compared with the available analytic solution and excellent agreement was observed. The validated computer model was then used to simulate a real vitreous cavity filled with Balanced Salt Solution or aqueous humor as a vitreous substitute in order to obtain distribution of drugs in the post-vitrectomy eyes or liquefied vitreous. Additionally, effects of locations of drug injection, drug diffusion coefficients and saccade amplitude on the drug distribution and its uniformity were investigated. Although the earlier findings in the literature reported a day or a week as a needed time for drug uniform distribution in the vitreous substitutes, the present work depicts that saccade movements augment the transport of the drug in a way that the uniformity of the drug distribution can be achieved in a matter of minutes. Furthermore, in a vitreous cavity subjected to the saccade movements, the diffusion coefficient of drugs does not significantly affect their distribution after a few minutes. Even the injection location does not matter as uniform distribution is achieved after some time.     

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.^1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.^1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.⁵ This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.     

Insulin in the vitreous of the normal and streptozotocin-induced diabetic rat.

Insulin has been detected by ELISA in the vitreous of the normal and streptozotocin-diabetic rat at levels for both about 1% of those in serum. 131I-labeled insulin, administered to conscious rats via an indwelling cannula in the right atrium, was found to cross the blood-ocular barrier into the vitreous. Autoradiographic gel analysis showed the peptide was transferred as an intact molecule. Vitreous insulin levels reflected serum levels as seen in relatively constant vitreous-to-serum insulin ratios over a wide range of serum insulin concentrations. The rate of blood-to-vitreous passage of insulin was about the same in normal as in diabetic rats (fasting serum glucose greater than or equal to 21 mM). At least a portion of vitreous insulin is therefore of pancreatic origin, and retinal tissue in the normal and diabetic animal is thus accessible to circulating hormone. The blood-ocular barrier is unaltered in streptozotocin diabetes with regard to insulin passage.     

Attempts to demonstrate specific antibody responses in the vitreous body of the eye

Postmortem serum and vitreous humor specimens obtained from 31 autopsied human bodies were assayed for specific antibody responses to adenoviruses, RS virus and Mycoplasma pneumoniae using the complement-fixation (CF) test and the ELISA procedure (in 23 of the bodies examined). The antibody responses as measured by the CF test were negative in all vitreous body samples tested, with the ELISA five specimens gave a positive reaction at a titre 1 : 40 and one at 1 : 80. These positive antibody titres turned out to invariably coincide with the high-titre antibody levels in the serum. Implicitly, at high-titre levels in the serum these antibodies tend to penetrate in the vitreous body of the eye.     

In vivo measurements of the viscoelasticity of the human vitreous humor.

A point of source of light against a dark background is perceived by the human retina as a point image enhanced by off-axis points (rays if the source is polychromatic) of light scattered from objects along the optic axis. As a consequence of movement of the vitreous humor, scattering centers imbedded there impart of this scattering pattern a corresponding movement. A method has been devised to give the vitreous humor reproducible initial conditions and to record the observed relaxation of the scattering pattern to its new rest position. The vitreous humor is found to be overdamped, and the heretofore unreported shear elastic modulus has been determined. A striking gravitational effect is revealed by comparing observations along a horizontal optic axis with ones along a vertical optic axis. For the former, gravitational torque is found to dominate the elastic torque. The reason nature has developed a slow-responding gravitational sensor in the vitreous humor is not clear.     

Studies on the possible role of vitreous humor on protein synthesis and morphology of organ cultured adult rabbit lens. II. Epithelial cells.

The protein synthetic activities of epithelial cells of lenses organ-cultured without adhered vitreous humor manifest significant increase compared to the epithelial cells of lenses incubated with attached vitreous humor. This effect is not due to trauma of vitreous removal, as the addition of freeze-dried vitreous humor to the culture medium of lenses without attached vitreous humor could inhibit protein synthesis. However, the protein synthesis inhibitor in the vitreous humor has no visible effect on the lens morphology. It was also found that the factor from vitreous humor has no effect on mRNA production or cell-free protein synthesis. Thus, it seems that the effect on protein synthesis must be mediated via some other pathway.     

Monitoring in situ circular dichroism of the intact vitreous: a new approach

For the first time, an attempt has been made to study the vitreous humor in situ by circular dichroism (CD). The vitreous, an avascular and acellular gel-like tissue, is optically transparent and homogeneous, and, thus, light scattering is minimal. The macromolecular components of this tissue, hyaluronate (HA), collagen and noncollagenous protein (NC-P), appear to exist in the matrix in a nonoriented fashion. As a result, no linear dichroism was observed. A typical CD of the vitreous shows a minimum at 206 nm with a shoulder at 220 nm and one small positive peak at approximately 252 nm. Gaussian analysis resolves this spectrum into four component bands. CD analysis of individual components reveals that NC-P makes the major contribution to the dichroic strength of the vitreous; contributions of HA and collagen, on the other hand, are small. The positive peak arises largely from ascorbic acid in the vitreous. CD measurement of the intact vitreous appears to be a useful technique for assessing the structure and changes of the constituent molecules in the normal and diseased vitreous.     

Down-regulation of pigment epithelium-derived factor in uveitic lesion associates with focal vascular endothelial growth factor expression and breakdown of the blood-retinal barrier

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Identifying biological markers or pathways associated with this disease may allow the understanding of its pathogenesis at a molecular level. The vitreous is the body fluid closest to the disease-affected tissue and possibly also an effector of pathological processes relevant for ERU. Surgical removal of vitreous leads to cessation of relapses in spontaneous uveitis of both man and horse, therefore vitreous composites are likely to contribute to disease progression. Uveitic vitreous is likely to contain potential biomarkers in relatively undiluted quantities. With the goal to identify these markers, we systematically compared vitreous from healthy and disease-affected eyes by proteomic profiling. Nine differentially expressed proteins were identified, that are functionally related to immune response, inflammation, and maintenance of the blood-retinal barrier. One of these, pigment epithelium-derived factor, a protein involved in maintaining a proper blood-retina barrier as well as protecting from neoangiogenesis was additionally found to be down-regulated within uveitic retinal lesions whereas, conversely, vascular endothelial growth factor was found to be up-regulated at these sites. Together, these changes point to as of yet undiscovered biological pathways involved in the pathogenesis of this autoimmune disease.     

Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy

An understanding of the diabetes-induced alterations in vitreous protein composition in the absence and in the presence of proliferative diabetic retinopathy (PDR) may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of vitreous samples from individuals with diabetes but without diabetic retinopathy (noDR) or with PDR and nondiabetic individuals (NDM). Using preparative one-dimensional SDS-PAGE and nano-LC/MS/MS of 17 independent vitreous samples, we identified 252 proteins from human vitreous. Fifty-six proteins were differentially abundant in noDR and PDR vitreous compared with NDM vitreous, including 32 proteins increased and 10 proteins decreased in PDR vitreous compared with NDM vitreous. Comparison of noDR and PDR groups revealed increased levels of angiotensinogen and decreased levels of calsyntenin-1, interphotoreceptor retinoid-binding protein, and neuroserpin in PDR vitreous. Biological pathway analysis revealed that vitreous contains 30 proteins associated with the kallikrein-kinin, coagulation, and complement systems. Five of them (complement C3, complement factor I, prothrombin, alpha-1-antitrypsin, and antithrombin III) were increased in PDR vitreous compared with NDM vitreous. Factor XII was detected in PDR vitreous but not observed in either NDM or noDR vitreous. PDR vitreous also had increased levels of peroxiredoxin-1 and decreased levels of extracellular superoxide dismutase, compared with noDR or NDM vitreous. These data provide an in depth analysis of the human vitreous proteome and reveal protein alterations that are associated with PDR.     

Proteomic Interactions in the Mouse Vitreous-Retina Complex

Purpose

Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina.

Methods

Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software.

Results

We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor.

Conclusions

Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.     

Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic properties

The viscoelastic deformation of porcine aortic endothelial cells grown under static culture conditions was measured using the micropipette technique. Experiments were conducted both for control cells (mechanically or trypsin detached from the substrate) and for cells in which cytoskeletal elements were disrupted by cytochalasin B or colchicine. The time course of the aspirated length into the pipette was measured after applying a stepwise increase in aspiration pressure. To analyze the data, a standard linear viscoelastic half-space model of the endothelial cell was used. The aspirated length was expressed as an exponential function of time. The actin microfilaments were found to be the major cytoskeletal component determining the viscoelastic response of endothelial cells grown in static culture.     

Spatial Variations in Vitreous Oxygen Consumption

We investigated the spatial variation of vitreous oxygen consumption in enucleated porcine eyes. A custom made oxygen source was fabricated that could be localized to either the mid or posterior vitreous cavity and steady state vitreous oxygen tension was measured as a function of distance from the source using a commercially available probe. The reaction rate constant of ascorbate oxidation was estimated ex vivo by measuring the change in oxygen tension over time using vitreous harvested from porcine eyes. Vitreous ascorbate from mid and posterior vitreous was measured spectrophotometrically. When the oxygen source was placed in either the mid-vitreous (N = 6) or the posterior vitreous (N = 6), we measured a statistically significant decrease in vitreous oxygen tension as a function of distance from the oxygen source when compared to control experiments without an oxygen source; (p<0.005 for mid-vitreous and p<0.018 for posterior vitreous at all distances). The mid-vitreous oxygen tension change was significantly different from the posterior vitreous oxygen tension change at 2 and 3mm distances from the respective oxygen source (p<0.001). We also found a statistically significant lower concentration of ascorbate in the mid-vitreous as compared to posterior vitreous (p = 0.02). We determined the reaction rate constant, k = 1.61 M^-1s^-1 ± 0.708 M^-1s^-1 (SE), of the oxidation of ascorbate which was modeled following a second order rate equation. Our data demonstrates that vitreous oxygen consumption is higher in the posterior vitreous compared to the mid-vitreous. We also show spatial variations in vitreous ascorbate concentration.