Antonio Haddad

Radioautographic study of glycoprotein biosynthesis and renewal in the ovarian follicles of mice and the origin of the zona pellucida.

SummaryL-fucose-3H was injected intravenously into mice which were killed at several time intervals after injection and semi-thin sections of their ovaries were processed for radioautography and analysed quantitatively. At the same time the specific activity of serum glycoproteins was determined. Glycoprotein biosynthesis was demonstrated in the oocytes, granulosa and stromal cells. The silver grain density of the follicular fluid in large follicles reached a peak at 4 h, remained high at 8 h after injection and decreased steadily at the subsequent intervals. It was demonstrated that the labeling pattern of the follicular fluid depends on the secretory activity of the granulosa cells and also on the specific activity of serum glycoproteins. The collapsed zonae pellucidae which represent the highest degree of follicular atresia are able to take up glycoprotein macromolecules. Based on this finding and also on the labeling pattern of the large follicles it was shown that there is very little synthesis of specific glycoproteins for the zona pellucida in large follicles. A more specific labeling of the zona pellucida occurred in the medium follicles. Following the growth of these follicles having a previously labeled zona pellucida, it was demonstrated that this extracellular structure is secreted by the oocyte.

Intravitreal injection of dispase causes retinal hemorrhages in rabbit and human eyes

Purpose. To check the effects of intravitreally injected dispase in the vitreo-retinal region. Methods. Dispase, 0.05 to 2.5 units dissolved in 100µl of phosphate-buffered saline, was injected into the midvitreous of rabbits which were killed from 15 to 120 min afterwards. The enzyme was also injected into four human eyes of patients with orbital tumors 15 min before enucleation during orbital exenteration surgery. The eyes were examined in vivo as well as by light and electron microscopy. Results. Hemorrhages were detected by fundus observations and confirmed by microscopical analysis in nearly all rabbits and in half of the human eyes. The red blood cells were observed in the vitreous and retina. Breaches in the inner limiting membrane were visualized in human eyes and ruptures of small blood vessels in rabbit eyes. In spite of that, vitreous detachment was not verified. In fact, the cortical-vitreous collagen-fibril network was conspicuous on scanning electron micrographs. Conclusions. Retinal hemorrhages were evident as early as 11 min after injection. It is suggested that this enzyme degraded selectively basement membrane components without affecting other proteins involved in the vitreous-retinal junction.

The origin of the intrinsic glycoproteins of the rabbit vitreous body : an immunohistochemical and autoradiographic study

A cartilage matrix glycoprotein (CMGP), previously identified in human and bovine vitreous, now has been found in the vitreous body of rabbits aged 1-22 months by immunohistochemical techniques. Epithelial cells of the inner layer of the ciliary epithelium contain material that has immunologic cross-reactivity with a specific antibody to CMGP. These cells also secrete glycoproteins, as determined by autoradiography after intravitreal injection of [3H]fucose. Approximately 14 bands, representing intrinsic glycoproteins containing fucose residues, can be identified in fluorograms of SDS-polyacrylamide gels of vitreous bodies from 6- and 22-month-old rabbits. Fluorograms of gels of samples of vitreous and ciliary bodies from several time points after intravitreal injection of [3H]fucose reveal at least seven comigrating protein bands and also demonstrate turnover of the labeled ciliary body glycoproteins. These results suggest that the inner layer of the ciliary epithelium is the source of the glycoproteins of the vitreous body and that these glycoproteins undergo turnover, probably throughout the entire life of the animals.

Pharmacokinetic and toxicity investigations of a new intraocular lens with a dexamethasone drug delivery system : A pilot study

Aim: To investigate the short-term safety and pharmacokinetic behavior of a new intraocular lens containing a dexamethasone drug delivery system (IOL-DDS) in rabbit eyes. Methods: A modified polymethylmethacrylate IOL containing a biodegradable dexamethasone DDS was implanted into the posterior chamber of the right eyes of 9 New Zealand white rabbits. Serial slitlamp and indirect ophthalmoscopic examinations (including grading of intraocular inflammation) were performed. After 3, 6 and 9 days, the rabbits were euthanized and the globes were removed for histological examination and for determination of dexamethasone levels in the aqueous humor and in the vitreous. Analysis of dexamethasone concentrations was performed by ELISA. Results: Therapeutic concentrations of dexamethasone were detectable in the aqueous and vitreous of the study eyes throughout the 9-day period in all tested animals. The mean aqueous dexamethasone concentration (ng/ml, ±SD) was 1,015.42 (±43.05), 970.11 (±32.47) and 757.58 (±30.19) and the mean vitreous concentration (ng/ml, ±SD) was 399.82 (±38.05), 287.38 (±34.47) and 268.15 (±32.00) at 3, 6 and 9 days after the surgical procedure, respectively. No corneal or retinal histological changes were observed during the study period. Conclusion: The IOL-DDS is effective in delivering therapeutic concentrations of dexamethasone to the aqueous and vitreous, without acute damage to the cornea and retina. Further controlled studies in the same animal model are under way to determine the potential value of this lens in the prevention and treatment of inflammation following cataract surgery.

Partial characterization, origin and turnover of glycoproteins of the rabbit vitreous body.

L-[3H]fucose was injected intravitreally into rabbits that were killed from 1 hr to 28 days after injection. The vitreous bodies were processed for radiometric techniques, and electrophoresis followed by fluorography. Unbound [3H]fucose remained at a high level up to 1 day, whereas the peak of [3H]fucose bound to glycoproteins was observed at 3 days after injection with a continuous decrease afterwards. The turnover rate of vitreous glycoproteins was estimated at 4.37% per day and their turnover time at 22.85 days. Electrophoresis and fluorography combined revealed about 14 bands of glycoproteins with fucose residues and there were strong indications of differences in turnover rate among individual glycoproteins. The most prominent band in the Coomassie blue-stained gels was the one having a molecular weight of 69 kDa and it was not labeled with [3H]fucose. This band was tentatively identified as serum albumin. On the other hand, the 14 bands labeled with [3H]fucose were glycoproteins originating from within the eye, that is, they are intrinsic constituents of the vitreous body.

The lectin KM+ induces corneal epithelial wound healing in rabbits.

Neutrophil influx is essential for corneal regeneration ( Gan et al. 1999 ). KM+, a lectin from Artocarpus integrifolia, induces neutrophil migration ( Santos‐de‐Oliveira et al. 1994 ). This study aims at investigating a possible effect of KM+ on corneal regeneration in rabbits. A 6.0‐mm diameter area of debridement was created on the cornea of both eyes by mechanical scraping. The experimental eyes received drops of KM+ (2.5 μg/ml) every 2 h. The control eyes received buffer. The epithelial wounded areas of the lectin‐treated and untreated eyes were stained with fluorescein, photographed and measured. The animals were killed 12 h (group 1, n = 5), 24 h (group 2, n = 10) and 48 h (group 3, n = 5) after the scraping. The corneas were analysed histologically (haematoxylin and eosin and immunostaining for proliferation cell nuclear antigen, p63, vascular endothelial growth factor, c‐Met and laminin). No significant differences were found at the epithelial gap between treated and control eyes in the group 1. However, the number of neutrophils in the wounded area was significantly higher in treated eyes in this group. Three control and seven treated eyes were healed completely and only rare neutrophils persisted in the corneal stroma in group 2. No morphological distinction was observed between treated and control eyes in group 3. In treated corneas of group 2, there was an increase in immunostaining of factors involved in corneal healing compared to controls. Thus, topical application of KM+ may facilitate corneal epithelial wound healing in rabbits by means of a mechanism that involves increased influx of neutrophils into the wounded area induced by the lectin.

Rabbit Retinal Neovascularization Induced by Latex Angiogenic-Derived Fraction: An Experimental Model

Purpose: To create a retinal neovascularization experimental model using intravitreal injection of microspheres loaded with latex-derived angiogenic fraction. Methods: Thirty-two albino New Zealand rabbits, divided in 4 groups of 8 animals, were enrolled in this study. Rabbits in groups I, II, and III received one intravitreal injection of PLGA (L-lactide-co-glycolide) microspheres with 10, 30, and 50 µg of latex-derived angiogenic fraction into their right eyes, respectively, and group IV received 0.1 ml of microspheres without the angiogenic fraction. Weekly follow-up with ophthalmoscopy and fluorescein angiography was performed; the rabbits were sacrificed in the 4th week and their eyes processed for light microscopy. Results: All eyes from group I demonstrated increased retinal vascular tortuosity, observed from 14 days after injection and maintained for 28 days, otherwise without new vessels detection. All group II eyes showed vascular changes similar to group I. Fifty percent of the eyes from group II rabbits developed retinal neovascularization 21 days after injection. All eyes from group III demonstrated significant vascular tortuosity and retinal new vessels 2 weeks after injection, progressing to fibrovascular proliferation and tractional retinal detachment. No vascular changes or retinal new vessels were observed in group IV eyes. Light microscopy confirmed the existence of new vessels previously seen on fluorescein angiography, in retinal sections adjacent to the optic disc, not observed in sections at the same area in the control group. Conclusion: Thirty- and 50-µg microspheres containing latex-derived angiogenic fraction injected into the vitreous cavity induced retinal neovascularization in rabbits.

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 [35S]-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.

Synthesis and secretion of transferrin by isolated ciliary epithelium of rabbit.

It has been shown that the vitreous contains several intrinsic glycoproteins whose origin remains to be clarified. Isolated ciliary epithelium (CE) was assayed to verify its role in the synthesis and secretion of transferrin for the vitreous body. It was cultured in the presence of [35S]methionine and the incubation medium was processed for immunoprecipitation. Total RNA from CE was processed for RT-PCR and the amplification products were sequenced. Also, whole preparations of isolated CE were processed for immunolocalization of transferrin. From the incubation assays, a labeled peptide of about 80 kDa was immunopurified that is the expected size of transferrin. The RT-PCR and sequencing experiments detected the presence of transferrin mRNA. Both layers of the CE exhibited transferrin reactivity, following immunohistochemical processing. Taken altogether, these results indicate the CE as one of the possible sources of vitreous intrinsic transferrin.

ORIGIN AND RENEWAL OF THE INTRINSIC GLYCOPROTEINS OF THE AQUEOUS HUMOR

Rabbits were injected either intravitreally or intra-aqueously with l-[3H]-fucose and killed at several intervals after the administration of this marker for glycoproteins. The aqueous humor, the vitreous body and the ciliary body were processed for radiometry (liquid scintillation counting), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Light microscopic autoradiography was carried out on semi-thin sections of the ciliary body and revealed intense activity in terms of the synthesis, migration and renewal of glycoproteins in the ciliary epithelium. The amount of unbound [3H]-fucose in the aqueous humor decreased sharply by 4 h, and the labeled glycoproteins were present only in very small quantities at 1 day after the intra-aqueous injection. When [3H]-fucose was injected intravitreally, unbound radiolabel could be detected in the aqueous humor for >1 day and the labeled glycoproteins, for up to 21 days after injection. The amount of unbound or bound [3H]-fucose was higher in the vitreous than in the aqueous at any interval after the intravitreal injection. Following the intra-aqueous injection, the amount of label that reached the vitreous body was practically insignificant. The levels of radioactivity in the serum were extremely low, as they were in the contralateral eye when tritiated fucose was injected into one eye only. Most of the Coomassie blue-stained bands detected in SDS-PAGE contained labeled glycoproteins as revealed by fluorography of gels simultaneously containing aqueous and vitreous samples. One notable exception was the most prominent band in the stained gels; this unlabeled band had an apparent mol. wt. of 69 kDa and was assumed to represent serum albumin. The labeled aqueous bands detected by fluorography were practically the same ones identified in the vitreous body, except that the bands in the latter were more radioactive and appeared earlier than those in the aqueous humor. In conclusion, most of the aqueous proteins are intrinsic glycoproteins of the vitreous body, which, in turn, are secretory products of the inner epithelial layer of the ciliary body.