Motor protein decoration of microtubules grown in high salt conditions reveals the presence of mixed lattices

We have used back-projection methods to obtain three-dimensional maps of motor-protein decorated nine and ten protofilament microtubules polymerized in the presence of high salt and preserved in vitreous ice. The resulting three-dimensional maps show that the vast majority of these microtubules have multiple seams, rather than being helical as would be expected according to the lattice accommodation model. These results indicate that microtubules should be analyzed by back-projection before using helical reconstruction approaches, and that nine and ten protofilament microtubules polymerized in high salt conditions are not suitable for helical analysis.     

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.     

Role of calcium ions in frequency accomodation of rat hippocampal neurons

Electrophysiological investigations of extracellular Ca ions the influence on the mechanism of frequency accommodation were carried out on isolated rat hippocampal neurones. It is established that interpulse interval during potential generation action linearly depended on quantity of Ca ions, which enter the cell through Ca2+ channels. Analysis of the results has shown that Ca ions influenced the forming and parameters of frequency accommodation in hippocampal neurones, probably via activation of Ca-dependent outward K-currents. Theoretical model that quantitatively describes the mechanisms of frequency adaptation is proposed. Adequacy of model to experimental data on frequency accommodation and electrical activity of hippocampal neurones is estimated.     

Identification of crystallin family proteins in vitreous body in rat endotoxin-induced uveitis: Involvement of crystallin truncation in uveitis pathogenesis

Endotoxin-induced uveitis (EIU) is an animal model of acute ocular inflammation. To characterize the mechanism of EIU, we analyzed the infiltration of proteins in the vitreous bodies of rats with EIU and normal rats using 2-DE and micro LC/LC-MS/MS. Twenty spots were identified in vitreous bodies of rats. Eighteen of these spots were members of the crystallin family. The truncated form of beta A4- and beta B2-crystallin were predominant in normal vitreous bodies, but there were intact form of crystallins in lipopolysaccharide-injected rats with EIU. These results suggest that crystallin family proteins are the major group of proteins involved in uveitic vitreous and that C-terminal truncation of beta-crystallins may play a role in EIU-related disease progression.     

蝮蛇抗栓酶（清栓酶）对实验性玻璃体积血的疗效探讨

玻璃体积血是眼外伤及某些眼病的常见并发症,且常严重损害视功能,为了寻找一种在早期能促进玻璃体积血吸收而又使用方便的药物,我们用~(51)铬标记红细胞之全血制成玻璃体积血模型,对蝮蛇抗栓酶(清栓酶)的疗效进行观察,结果证明蝮蛇抗栓酶对实验性玻璃体积血的清除有显著效果(P<0.001).     

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.     

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.     

New data on the microtubule surface lattice

The in vitro polymerisation of tubulin is a remarkable example of protein self-assembly in that several closely related microtubule structures coexist on the polymerisation plateau. Unfixed and unstained in vitro assembled microtubules were observed in vitreous ice by cryo-electron microscopy. New results are reported that considerably extend previous observations [47]. In ice, microtubule images have a distinctive contrast related to the number and skew of the protofilaments. The microtubules observed have from twelve to seventeen protofilaments. Comparison with thin sections of pelleted material allows a direct identification of images from microtubules with thirteen, fourteen and fifteen protofilaments. A surface lattice accommodation mechanism, previously proposed to explain how variable numbers of protofilaments can be incorporated into the basic thirteen protofilament structure, is described in detail. Our new experimental results are shown to be in overall agreement with the theoretical predictions. Only thirteen protofilament microtubules have unskewed protofilaments, this was confirmed by observations on axoneme fragments. The results imply that the microtubule surface lattice is based on a mixed packing which combines features of the standard A and B lattices.     

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.     

An ultrastructural study of histochemical staining of complex carbohydrates in the mouse posterior vitreous body

The vitreous body contains complex carbohydrates that can be demonstrated morphologically. Vitreous hyaluronic acid is very soluble but it can be precipitated by cetylpyridinium chloride (CPC) while being cross-linked by glutaraldehyde. Oligosaccharide chains of vitreous glycoproteins are fixed with glutaraldehyde alone. Mouse eyes were fixed with glutaraldehyde or glutaraldehyde and CPC and the complex carbohydrates of the posterior vitreous cortex were studied by electron microscopy. Cationic dyes were used in the fixative or for block-staining on most fixed tissue blocks to allow detailed observations of complex carbohydrates. Most blocks were postfixed with OsO4. The hyaluronic-acid domain on vitreous collagen fibrils sequentially contracted and expanded in size with various histochemical manipulations. Contraction of the domain of hyaluronic acid generally indicates an increased charge density. OsO4 contributes considerable charge density upon forming osmate esters, but tissue postfixed with OsO4 contained large globular forms of hyaluronic acid rather than the small globules observed in non-osmicated preparations. A model is proposed to explain the seemingly paradoxical findings by reference to suggested mechanisms of polysaccharide-ligand-OsO4 interactions.     

Comparative evaluation of possible ocular photochemical toxicity of fluoroquinolones meant for ocular use in experimental models

Fluoroquinolones (FQs) are extensively used in bacterial keratitis and other intraocular infections. Since eye is constantly exposed to light, incidence of ocular phototoxicity due to commonly used FQs is of great interest for their safe use. Phototoxicity of commonly used FQs (ciprofloxacin, lomefloxacin, pefloxacin, ofloxacin, sparfloxacin and gatifloxacin), has been evaluated by using HET-CAM-UV model (Photo Hen Egg Test-C Chorioallantoic Membrane model). This study was further extended by adding lomefloxacin dissolved in bovine vitreous (0.5 ml) on the chorioallantoic membrane (CAM). Using a standard scale, the phototoxic damage was assessed at different time intervals. Respective controls were kept in dark to distinguish the toxicity of the drugs per se. The results showed that the phototoxicity induced by lomefloxacin was very high followed by gatifloxacin and sparfloxacin and least for other drugs studied. Interestingly, lomefloxacin along with vitreous showed significantly low phototoxicity. This could be due to the antioxidant property of ascorbic acid present in the vitreous.     

Transient transport across an inhomogeneous blood-retina barrier

A mathematical model for transport across the blood-retina barrier and diffusion into the vitreous body of the human eye is formulated. The eye is modeled as a sphere, the surface of which represents the blood-retina barrier. The equations of the model are solved analytically, using an expansion in spherical harmonics and inversion of the Laplace transform in the time variable. The numerical properties of the solution are investigated and the applicability of the model to the analysis of data from three-dimensional vitreous fluorometry is discussed.     

Owl monkey vitreous: A novel model for hyaluronic acid structural studies

Circular dichroism (CD) studies have been made on hyaluronic acid (HA) obtained from dialyzed owl monkey vitreous. The protein content of these samples is low enough not to interfere with the CD measurements of hyaluronic acid. The ellipticity values of vitreous HA are higher than those of HA from other tissues, indicating a higher degree of preferred order. Since the purification procedures involve only dialysis, the owl monkey vitreous can be a model tissue for structural studies of HA close to its native state.     

Transient transport across the blood-retina barrier

A mathematical model of the transport of fluorescein across the blood-retina barrier in the transient state and the subsequent diffusion of fluorescein in the vitreous body is presented. The function of the barrier is lumped in a single parameter—the permeability. The sensitivity of this parameter due to changes in the other parameters of the model is given. This establishes the foundation for the quantitative assessment of the barrier function through vitreous fluorophotometry.     

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.     

Cataract induction by products of lipid peroxidation

Liposome suspension prepared from the unsaturated phospholipids exposed to lipid peroxidation (LPO) induced posterior subcapsular cataracts after injection into the posterior vitreous of rabbit eyes. In the background of this model lies a type of lens opacity formed during retinal degeneration when toxic peroxide substances diffuse anteriorly through the vitreous body resulting in vitreous opacities and complicated cataracts. Saturated liposomes (prepared from beta-oleoyl-gamma-palmitoyl) L-alpha-lecithin) did not induce lens opacities, which is the evidence that a lipid peroxidation mechanism may be responsible for the posterior cataracts. Along with cataract formation accumulation of LPO fluorescent products in vitreous, aqueous humor and lens was observed. It was followed by a decreased level of reduced glutathione in the lens. The obtained results strongly support the hypothesis of LPO initial role in cataracts.     

Effects of visually demanding near work on trapezius muscle activity

Poor visual ergonomics is associated with visual and neck/shoulder discomfort, but the relation between visual demands and neck/shoulder muscle activity is unclear. The aims of this study were to investigate whether trapezius muscle activity was affected by: (i) eye-lens accommodation; (ii) incongruence between accommodation and convergence; and (iii) presence of neck/shoulder discomfort. Sixty-six participants (33 controls and 33 with neck pain) performed visually demanding near work under four different trial-lens conditions. Results showed that eye-lens accommodation per se did not affect trapezius muscle activity significantly. However, when incongruence between accommodation and convergence was present, a significant positive relationship between eye-lens accommodation and trapezius muscle activity was found. There were no significant group-differences. It was concluded that incongruence between accommodation and convergence is an important factor in the relation between visually demanding near work and trapezius muscle activity. The relatively low demands on accommodation and convergence in the present study imply that visually demanding near work may contribute to increased muscle activity, and over time to the development of near work related neck/shoulder discomfort.     

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.     

Accommodation of the human lens capsule using a finite element model based on nonlinear regionally anisotropic biomembranes

Accommodation of the eyes, the mechanism that allows humans to focus their vision on near objects, naturally diminishes with age via presbyopia. People who have undergone cataract surgery, using current surgical methods and artificial lens implants, are also left without the ability to accommodate. The process of accommodation is generally well known; however the specific mechanical details have not been adequately explained due to difficulties and consequences of performing in vivo studies. Most studies have modeled the mechanics of accommodation under assumptions of a linearly elastic, isotropic, homogenous lens and lens capsule. Recent experimental and numerical studies showed that the lens capsule exhibits nonlinear elasticity and regional anisotropy. In this paper we present a numerical model of human accommodation using a membrane theory based finite element approach, incorporating recent findings on capsular properties. This study seeks to provide a novel perspective of the mechanics of accommodation. Such findings may prove significant in seeking biomedical solutions to restoring loss of visual power.