Chondroitin sulfate proteoglycans are structural renewable constituents of the rabbit vitreous body

Purpose: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. Methods: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [S-35]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. Results: the electron microscopic study revealed a network with hyaluronic acid ( HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous ( e. g., one heparan-and two chondroitin-sulfate ones). Conclusions: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.

Measuring localized viscoelasticity of the vitreous body using intraocular microprobes.

Vitrectomy is a standard ophthalmic procedure to remove the vitreous body from the eye. The biomechanics of the vitreous affects its duration (by changing the removal rate) and the mechanical forces transmitted via the vitreous on the surrounding tissues during the procedure. Biomechanical characterization of the vitreous is essential for optimizing the design and control of instruments that operate within the vitreous for improved precision, safety, and efficacy. The measurements are carried out using a magnetic microprobe inserted into the vitreous, a method known as magnetic microrheology. The location of the probe is tracked by a microscope/camera while magnetic forces are exerted wirelessly by applied magnetic fields. In this work, in vitro artificial vitreous, ex vivo human vitreous and ex vivo porcine vitreous were characterized. In addition, in vivo rabbit measurements were performed using a suturelessly injected probe. Measurements indicate that viscoelasticity parameters of the ex vivo human vitreous are an order of magnitude different from those of the ex vivo porcine vitreous. The in vivo intra-operative measurements show typical viscoelastic behavior of the vitreous with a lower compliance than the ex vivo measurements. The results of the magnetic microrheology measurements were validated with those obtained by a standard atomic force microscopy (AFM) method and in vitro artificial vitreous. This method allows minimally-invasive characterization of localized mechanical properties of the vitreous in vitro, ex vivo, and in vivo. A better understanding of the characteristics of the vitreous can lead to improvements in treatments concerning vitreal manipulation such as vitrectomy.

The vitreous body, its origin, development, and structure as observed in the eye of the pig,

"Contribution from the Biological Laboratory of the Catholic University of America, no. 4."

Sulfation of intrinsic glycoproteins of the rabbit vitreous

The experiments reported here were designed to characterize the intrinsic vitreous glycoproteins and to understand the process of their sulfation. Rabbits were injected intravitreally with S-35-sodium sulfate and killed at several time intervals after injection. In another series of experiments, rabbits were injected either with S-35-sodium sulfate, H-3-fucose or H-3-tyrosine, associated or not associated with tunicamycin administration. Vitreous from the control eyes was also digested with N-glycosidase.. Furthermore, ciliary bodies, the putative source of the intrinsic vitreous glycoproteins, were incubated with S-35-sodium sulfate in the presence or absence of the protein synthesis inhibitor cycloheximide, and the culture media recovered for analysis. These and the vitreous samples of the other experiments were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Except for serum albumin, practically all polypeptide bands of the vitreous and culture media were labeled with radioactive sulfate and were shown to undergo renewal. The experiments using tunicamycin or enzyme treatment suggest that radioactive sulfate was incorporated not only into the carbohydrate side chains of the glycoproteins but also into the amino acid tyrosine of the polypeptide backbone of these glycoproteins. (C) 1998 Academic Press.

Focal irradiation of perforating eye injuries

Plaques constructed with 125I were used to irradiate the sites of perforating ocular injuries in rabbits. An approximate dose of 16Gy given over a period of 6 days was shown to significantly reduce intraocular cellular proliferation when irradiation was commenced within 24 hours after injury. If irradiation was delayed until day 5, this reduction in cellular proliferation and intraocular membrane formation did not occur. Smaller radiation doses of approximately 6Gy given within 24 hours post-injury and administered over 6 days also reduced the extent of cellular proliferation but was not as effective as the 16Gy dose.

Does unidirectional vesicular transport occur in retinal vessels?

This paper challenges the hypothesis that the smooth 80 nm plasmalemmal caveolae found in abundance at the abluminal aspect of the endothelium in retinal blood vessels participate in a unidirectional vesicular transport mechanism. Evidence is presented which indicates that horseradish peroxidase, when introduced to the extracellular space of the retina via the vitreous body, may enter the intravascular compartment through junctional incompetence which occurs at or after enucleation of the eye. It is proposed that the plasmalemmal caveolae at the abluminal plasma membrane of endothelial cells in retinal blood vessels are static structures which facilitate the transport of small solutes and ions across the blood retinal barrier.

Is the formula of Traub still up to date in antemortem blood glucose level estimation?

According to the hypothesis of Traub, also known as the 'formula of Traub', postmortem values of glucose and lactate found in the cerebrospinal fluid or vitreous humor are considered indicators of antemortem blood glucose levels. However, because the lactate concentration increases in the vitreous and cerebrospinal fluid after death, some authors postulated that using the sum value to estimate antemortem blood glucose levels could lead to an overestimation of the cases of glucose metabolic disorders with fatal outcomes, such as diabetic ketoacidosis. The aim of our study, performed on 470 consecutive forensic cases, was to ascertain the advantages of the sum value to estimate antemortem blood glucose concentrations and, consequently, to rule out fatal diabetic ketoacidosis as the cause of death. Other biochemical parameters, such as blood 3-beta-hydroxybutyrate, acetoacetate, acetone, glycated haemoglobin and urine glucose levels, were also determined. In addition, postmortem native CT scan, autopsy, histology, neuropathology and toxicology were performed to confirm diabetic ketoacidosis as the cause of death. According to our results, the sum value does not add any further information for the estimation of antemortem blood glucose concentration. The vitreous glucose concentration appears to be the most reliable marker to estimate antemortem hyperglycaemia and, along with the determination of other biochemical markers (such as blood acetone and 3-beta-hydroxybutyrate, urine glucose and glycated haemoglobin), to confirm diabetic ketoacidosis as the cause of death.

Potentiel des liposomes pour l'injection intravitréenne de molécules thérapeutiques [Potential of liposomes for the intravitreal injection of therapeutic molecules].

Intravitreal administration has been widely used since 20 years and has been shown to improve the treatment of diseases of the posterior segment of the eye with infectious origin or in edematous maculopathies. This route of administration allows to achieve high concentration of drug in the vitreous and avoids the problems resulting from systemic administration. However, two basic problems limit the use of intravitreal therapy. Many drugs are rapidly cleared from the vitreous humor; therefore, to reach and to maintain effective therapy repeated injections are necessary. Repeated intravitreal injections increase the risk of endophthalmitis, damage to lens, retinal detachment. Moreover, some drugs provoke a local toxicity at their effective dose inducing side-effects and possible retinal lesions. In this context, the development and the use of new drug delivery systems for intravitreal administration are necessary to treat chronic ocular diseases. Among them, particulate systems such as liposomes have been widely studied. Liposomes are easily injectable and permit to reduce the toxicity and to increase the residence time of several drugs in the eye. They are also able to protect in vivo poorly-stable molecules from degradation such as peptides and nucleic acids. Some promising results have been obtained for the treatment of retinitis induced by cytomegalovirus in human and more recently for the treatment of uveitis in animal. Finally, the fate of liposomes in ocular tissues and fluids after their injection into the vitreous and their elimination routes begin to be more known.

Toxicidade da mitomicina C ao endotélio da córnea de coelhos

Purpose: To evaluate corneal endothelium alterations after applying mitomycin C to the sclera using transmission and scanning electron microscopy, correlating alterations with time, concentration, and evaluation methods. Methods: The corneal endothelium of both eyes of 32 albino rabbits was evaluated and distributed into four groups of 8. Mitomycin C was applied under a scleral flap in the right eye for 5 minutes. Mitomycin C concentrations were 0.5 mg/ml for G1 and G2 and 0.2 mg/ml for G3 and G4. Examinations were performed 15 days after application to G1 and G3, and 30 days after application to G2 and G4. Four cornea in each group were prepared for transmission electron microscopy and four for scanning electron microscopy. Left eyes of all animals were used as controls. Results: Transmission electron microscopy showed corneal endothelium alterations in all groups: rarefied cytoplasm, dilation and fragmentation of rough endoplasmic reticulum cisternae, Golgi apparatus with cisternal dilation, reduced vacuoles, and irregularities of internal membrane more noticeable in G1 and G2. Scanning electron microscopy revealed alterations in all groups except G1: changes in the shape and size of cells and longer filopodial projections. Conclusions: 1-Corneal endothelium alterations were seen at both 0.5 and 0.2 mg/ml concentrations and at 15 and 30 days after mytomicin C application; 2 - Alterations were more intense with higher mytomicin C concentration by transmission electron but not by scanning electron microscopy; 3 - The alterations correlated with time by scanning electron microscopy but not by transmission electron microscopy.

Sistemas biodegradáveis contendo acetato de prednisolona para administração orbitária

Purpose: The present study aimed to evaluate an injectable extended-release formulation of prednisolone acetate (PA) for orbital administration. Methods: Microspheres (MEs) of poly-ε-caprolactone (PCL) containing PA were developed by the method of solvent evaporation. The MEs obtained were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), encapsulation efficiency and in vitro release profile. The in vivo release profile was evaluated in rabbits after periocular injection of an aqueous suspension of MEs. The local biocompatibility of the system was verified by histopathologic analysis of the deployment region. Results: After MEs preparation, morphological analysis by SEM showed the feasibility of the employed method. The content of PA encapsulated was 43 ± 7% and can be considered as satisfactory. The system characterization by DSC technique, in addition to confirm the system stability, did not indicate the existence of interaction between the drug and the polymer. The in vitro release study showed the prolonged-release features of the developed system. Preliminary in vivo study showed the absence of local toxicity and confirmed the prolonged release profile of PA from MEs, suggesting the viability of the developed system for the treatment of orbital inflammatory diseases. Conclusion: The results obtained in this work are relevant and accredit the system developed as a possible alternative to the treatment of inflammatory orbitopathy.

Histopatologia e imunoistoquímica do bulbo do olho de equinos (Equus caballus, Linnaeus, 1758) soropositivos ou soronegativos para leptospirose

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Linkage mapping of ovine microphthalmia to chromosome 23, the sheep orthologue of human chromosome 18

PURPOSE: To characterize the phenotype and map the locus responsible for autosomal recessive inherited ovine microphthalmia (OMO) in sheep. METHODS: Microphthalmia-affected lambs and their available relatives were collected in a field, and experimental matings were performed to obtain affected and normal lambs for detailed necropsy and histologic examinations. The matings resulted in 18 sheep families with 48 cases of microphthalmia. A comparative candidate gene approach was used to map the disease locus within the sheep genome. Initially, 27 loci responsible for the microphthalmia-anophthalmia phenotypes in humans or mice were selected to test for comparative linkage. Fifty flanking markers that were predicted from comparative genomic analysis to be closely linked to these genes were tested for linkage to the disease locus. After observation of statistical evidence for linkage, a confirmatory fine mapping strategy was applied by further genotyping of 43 microsatellites. RESULTS: The clinical and pathologic examinations showed slightly variable expressivity of isolated bilateral microphthalmia. The anterior eye chamber was small or absent, and a white mass admixed with cystic spaces extended from the papilla to the anterior eye chamber, while no recognizable vitreous body or lens was found within the affected eyes. Significant linkage to a single candidate region was identified at sheep chromosome 23. Fine mapping and haplotype analysis assigned the candidate region to a critical interval of 12.4 cM. This ovine chromosome segment encompasses an ancestral chromosomal breakpoint corresponding to two orthologue segments of human chromosomes 18, short and long arms. For the examined animals, we excluded the complete coding region and adjacent intronic regions of ovine TGIF1 to harbor disease-causing mutations. CONCLUSIONS: This is the first genetic localization for hereditary ovine isolated microphthalmia. It seems unlikely that a mutation in the TGIF1 gene is responsible for this disorder. The studied sheep represent a valuable large animal model for similar human ocular phenotypes.

The eye and vision:an overview

The eye is the major organ of vision and highly specialized for photoreception. It focusses light from an object onto the light-sensitive retina. Changes in specialized neurons in the retina result in nerve action potentials which are relayed to the brain via the optic nerve. Visual processing by the brain results in ‘visual perception’, the construction of a sensory image which is consciously appreciated as vision. All other structures of the eye are subsidiary to this function, either by facilitating focusing of light rays or by supporting the tissues of the eye. This chapter is an introduction to the various parts of the eye including the eyelids and associated structures, conjunctiva, cornea, sclera, iris, lens, vitreous body, retina, optic disc and nerve, and orbit. This chapter describes the functions of these various structures and their importance in achieving a visual image.

Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.