In vivo release and retinal safety of intravitreal implants of thalidomide in rabbit eyes and antiangiogenic effect on the chorioallantoic membrane.

PURPOSE: To evaluate the in vivo release, retinal safety and antiangiogenic effect of a thalidomide-loaded poly-lactide-co-glycolide intravitreal implant. METHODS: New Zealand white rabbits, divided into two groups, I and II, received an intravitreal implant containing or not thalidomide, respectively (n = 12). Intravitreal drug levels were determined during a six-week study period. The potential for toxicity associated with the implants was evaluated by electroretinography and light microscopy (n = 8). Twelve chorioallantoic membranes (CAMs) from chicken eggs were incubated with thalidomide dispersion, implants containing or not thalidomide and vitreous samples and analyzed after two days regarding the percentage of vessels regression. RESULTS: Intravitreal concentrations of thalidomide (ng/ml) were 690.21 ± 177.95, 372.51 ± 185.56, 240.59 ± 133.48, 327.54 ± 169.71, 294.26 ± 142.41 and 465.18 ± 157.51 at 1, 2, 3, 4, 5 and 6 weeks, respectively, after implantation in group I rabbits. No drug was detected in group II samples. Electroretinography and histological evaluations did not show any sign of retina toxicity. There was significant regression of vessels in CAM incubated with thalidomide dispersion, thalidomide-loaded implants and vitreous samples from group I when compared to control. CONCLUSION: The intravitreal implants delivered safe doses of thalidomide that were also effective to induce vessels regression in CAMs.

Vitreous pharmacokinetics and retinal safety of intravitreal preserved versus non-preserved triamcinolone acetonide in rabbit eyes.

PURPOSE: To compare the intravitreal pharmacokinetic profile of a triamcinolone acetonide formulation containing the preservative benzyl alcohol (TA-BA) versus a preservative-free triamcinolone acetonide formulation (TA-PF), and evaluate potential signs of toxicity to the retina. METHODS: A total of 60 New Zealand male white rabbits, divided into two groups, were studied. In the TA-BA group, 30 rabbits received an intravitreal injection of TA-BA (4 mg/0.1 ml) into the right eye. In the TA-PF group, 30 rabbits received an intravitreal injection of TA-PF (4 mg/0.1 ml) into the right eye. The intravitreal drug levels were determined in 25 animals from each group by high-performance liquid chromatography (HPLC). The potential for toxicity associated with the intravitreal triamcinolone injections was evaluated in five randomly selected animals from each group by electroretinography (ERG) and by light microscopy. RESULTS: Median intravitreal concentrations of TA-BA (µg/ml) were 1903.1, 1213.0, 857.8, 442.0, 248.6 at 3, 7, 14, 21 and 28 days after injection. Intravitreal concentrations of TA-PF (µg/ml) were 1032.9, 570.1, 516.6, 347.9, 102.8 at 3, 7, 14, 21 and 28 days after injection. The median intravitreal triamcinolone concentration was significantly higher in the TA-BA compared to the TA-PF group at 7 days post-injection (p < 0.05). There was no significant difference between the two groups in median triamcinolone concentration at the other time points evaluated. There was no evidence of toxic effects on the retina in either group based on ERG or histological analyses. CONCLUSIONS: Following a single intravitreal injection, the median concentration of triamcinolone acetonide is significantly higher in the TA-BA compared to the TA-PF group at 7 days post-injection. No toxic reactions in the retina were observed in either group.

Autoradiographic study on the regenerative capability of the epithelium lining the center of the cornea after multiple debridements of its peripheral region.

BACKGROUND: The epithelium lining the center of the cornea is assumed to lack stem cells.The purpose is to investigate by autoradiography the regenerative capability of the epithelium lining the central region of the rabbit cornea following seven scrapings of its peripheral lining, during several months. METHODS: After marking the center of the cornea with a 6 mm-diameter trephine, the epithelium outside this area was scraped until reaching the corneoscleral zone. This procedure was repeated seven times on the same eye at intervals of 20 days. One day after the last scraping, (3)H-thymidine was injected intravitreally and the corneas processed for autoradiography. RESULTS: At 2 days after injection, the corneal surface was entirely lined by an epithelium made up by two layers of squamous cells, most of them being labeled with the DNA precursor. A multilayered epithelium was visualized at the center with most of its basal cells also labeled. The limbal epithelium had at least two of its layers labeled with the precursor. At 9 days, the multilayered central unscraped epithelium exhibited labeled cells not only in the basal but also in its suprabasal layers. The labeling index (labeled nuclei/100 cells) for its basal stratum was very close to 100%. A similar feature was observed at 16 days, except that the mutilayered central epithelium was seen lining a larger area when compared to the precedent interval and that it exhibited evidences for vertical renewal. CONCLUSIONS: The epithelium lining the central region of the cornea--where it was assumed that stem cells do not exist--exhibited capability for regeneration and self-renewal in spite of seven consecutive debridements of its periphery. No evidence was found for transposition of limbal epithelial cells to the center of the cornea during the early merger of the epithelial sliding fronts.

Regeneration of the corneal epithelium after debridement of its central region: an autoradiographic study on rabbits.

PURPOSE: To investigate the proliferative behavior of the corneal and limbal epithelia after debridement on the central region of the rabbit cornea. METHODS: After scraping a circular epithelial area, 5 mm in diameter, in the center of the cornea, ([3]) H-thymidine ( ([3]) H-TdR) was injected intravitreally, and the rabbits killed from 1 to 49 days afterward. The cornea, together with the adjacent conjunctiva, was processed for autoradiography. RESULTS: The regenerating epithelium at the center of the cornea exhibited high frequencies of labeled nuclei when compared to controls. The mitotic indexes for the limbus were comparable in experimental and control eyes. The unique basal stratum of the limbal epithelium exhibited quick proliferation and vertical migration in all eyes. Cells that remained labeled for four weeks or more were observed throughout the corneal epithelium, including its basal stratum, and this did not depend on epithelial damage. CONCLUSION: Corneal epithelium wounds are healed by sliding and proliferation of cells surrounding the epithelial gap without any evidence for the participation of the limbal epithelium. Daughter cells labeled with ([3]) H-TdR were visualized in all layers of the corneal epithelium up to 7 weeks after the DNA precursor injection. However, at this long interval, the only labeled cells in the limbus were in the suprabasal layers.

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 microg/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.

Proliferation of the vascular endothelium of the iris following total debridement of the corneal epithelium and limbal excision of rabbits.

BACKGROUND: Damage to the corneal epithelium causes not only a reaction for its repair but also affects other parts of the cornea as well as different components of the anterior segment of the eye. The purpose of this investigation was to analyze the consequences, following epithelial and limbal damage, to the iris of rabbits (Oryctolagus cuniculus). METHODS: The corneal epithelium was thoroughly scraped followed by surgical excision of the limbus. Next, (3)H-thymidine ((3)H-TdR) was injected intravitreally both into the right (experimental) and left (control) eyes which had their anterior segments processed for autoradiography at intervals of 2, 7 and 21 days after surgery (three rabbits per interval). The irises were also examined with scanning-electron and confocal microscopy after Evans blue injection. RESULTS: There was a high frequency of labeling in the cells of the iris blood vessels in the experimental eye, particularly the endothelial ones. The ratio of labeled cells between experimental and control irises was 40:1, with a population of nuclei increasing by 25% and remaining labeled up to 21 days. There was also an increase in the volume of the iris vasculature as shown by confocal microscopy. The high labeling frequencies of the vascular cells were observed throughout the iris from the ciliary to the pupillary regions. CONCLUSIONS: The lesions on the corneal epithelium elicit proliferation of the iris vascular cells, mainly its endothelium, as well as an early breakdown of the blood-aqueous barrier. The daughter cells resulting from the damage to the eye surface were detected up to 21 days after a single injection of (3)H-TdR, most likely due to their slow turnover. As a consequence of this proliferation, the vasculature of the iris increased in volume.

Morphological and autoradiographic studies on the corneal and limbal epithelium of rabbits.

The investigation was centered on the morphological features of the conjunctiva-cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [(3)H]thymidine ([(3)H]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels, were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28-day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high-resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label-retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium.

Molecular analysis of neurolysin expression in the rat and bovine ciliary body.

PURPOSE: This paper deals with the capability of the ciliary epithelium to express neurolysin, involved in the inactivation of numerous neuropeptides. METHODS: Total RNAs from ciliary body (CB) were processed for RT-PCR, and the amplification products were sequenced. The whole-protein extracts of CBs were analyzed using the Western blot. The CBs were processed for neurolysin immunolocalization. RESULTS: The RT-PCR detected the presence of neurolysin mRNA in the ciliary body. The Western blot assays demonstrated immunochemical cross-reactivity with neurolysin. The immunoreactivity to neurolysin was observed in ciliary epithelium. CONCLUSIONS: These results indicate that the ciliary epithelium expresses neurolysin.

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.

Molecular and biochemical analysis of ceruloplasmin expression in rabbit and rat ciliary body.

PURPOSE: To verify the capability of rabbit and rat ciliary body to synthesize and secrete ceruloplasmin. METHODS: Isolated ciliary body (CB) was cultured in the presence of [35S]-methionine, and the incubation medium was processed for immunoprecipitation. Total RNA from CB was processed for RT-PCR, and the amplification products were sequenced. Also, sections of CB were immunostained for the localization of ceruloplasmin. RESULTS: A labeled peptide, having a molecular weight of about 135 kDa, the expected size of ceruloplasmin, was immunopurified in the incubation media from both animal species. The RT-PCR and sequencing experiments detected the presence of ceruloplasmin mRNA in rat samples. Both layers of rabbit and rat ciliary epithelium (CE) exhibited ceruloplasmin reactivity after immunohistochemical processing. CONCLUSIONS: Taken altogether, these results indicate the CB, particularly its epithelium, as one of the possible sources of the ocular intrinsic ceruloplasmin.

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.

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 100 microl 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.

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.

Aneurisma do segmento distal da artéria cerebral anterior: aspectos clínicos, anatômicos e cirúrgicos / Distal anterior cerbral artery aneurysms: the clinical, anatomical and surgical aspects

Säo revistos os aspectos clínicos, anatômicos e cirúrgicos relacionados aos aneurismas da porçäo distal da artéria cerebral anterior. Onze casos säo apresentados

Uso da nifedipina no pós-operatório de pacientes submetidos à cirurgia coronária com artéria mamária / The uso of nifedipine in the postoperative period of patients subjected to coronary surgery with mammary artery

Vinte pacientes consecutivos, submetidos à revascularizaçäo do miocárido através de anastomose de, no mínimo, uma artéria mamária e utilizando-se nifedipina no pós-operatório imediato, foram estudados. Eles foram avaliados clínica, eletrocardiograficamente e através da CK-MB, visando evitar espasmo da artéria mamária. Em nenhum doente houve manifestaçäo anginosa, alteraçäo eletrocardiográfica ou da curva enzimática, o que sugere a eficácia da nifedipina na prevençäo de uma grave complicaçäo