Transglutaminase binding fusion protein linked to SLPI reduced corneal inflammation and neovascularization.

BACKGROUND: To study the effect of topical administration of a fusion protein (PF-MC) made up of N-terminal portion of the protease inhibitor Trappin-2 (which is a substrate of transglutaminasa-2) and SLPI (protein with anti-inflammatory, anti-bacterial and anti-viral ability), in an animal model of corneal inflammation and angiogenesis. METHODS: An alkali injury was produced with a filter paper of 3 mm with 1 N NaOH during 40 seconds on the right cornea of 36 male Sprague Dawley rats, under general anesthesia. Animals were divided into three groups according to treatment. Group 1 was treated with 10 ul of PF-MC (200 ug/ml; n = 12), Group 2, with 10 ul of SLPI (200 ug/ml; n = 12) and Group 3 was treated with buffer (10 ul; n = 12) topically administered four times a day for up to 7 days. Half of the animals were sacrificed at day 3 before making a re-epithelialization time analysis with fluorescein staining at 18 and 24 hours. In the remaining animals corneal opacity was studied and digital photographs were taken at day 7 before doing euthanasia. Eyes were processed for histology and immunofluorescence. RESULTS: Corneal ulcerated area was significantly lower in PF-MC treated animals compared to SLPI and buffer-treated animals at 18 hours and 24 hours postinjury. A clear cornea and fundus red reflex was only found among PF-MC treated animals. Histological analysis revealed a stratified corneal epithelium with at least three layers in all PF-MC animals at day 7. In this group there was a reduced number of PMNs in the corneal stroma at 3 and 7 days of follow-up. Besides, corneal neovascularization was much more extended in SLPI and Buffer animals than in animals treated with PF-MC. CONCLUSIONS: The binding of SLPI with Cementoin to transglutaminase seems to be an effective strategy to treat corneal inflammation and angiogenesis.

Primary diffuse leptomeningeal gliomatosis in children: a clinical pathologic correlation.

PURPOSE: To describe a rare case of primary diffuse leptomeningeal gliomatosis (PDLG) presenting with progressive proptosis and direct involvement of the optic nerve sheath in a child and review of the relevant literature. METHODS: Retrospective review of a single case and systematic literature review of 26 biopsy-proven cases reported in the MEDLINE-indexed English literature. A 10-year-old girl developed proptosis and progressive visual loss associated with thickening of the optic nerve sheaths and dilation of the subarachnoid spaces with multilobulated appearance of the brain meninges and thickened peripheral nerve root sheaths. Biopsy of the optic nerve sheath was diagnostic. The patient underwent chemotherapy combined with oral temozolomide and conformational radiotherapy to the brain and spine. She died 3 years after the onset of the disease. An extensive review of the published literature using the key words "primary diffuse leptomeningeal gliomatosis" and "optic nerve" confirmed the case herein reported to be the first case of primary diffuse leptomeningeal gliomatosis in which direct optic nerve infiltration was demonstrated during the course of the disease. RESULTS: Immunohistochemistry demonstrated expression of CD56 and glial fibrillary acidic protein, and an elevated level of Ki-67; all the other markers were negative. CONCLUSIONS: According to a comprehensive literature review, we report the first case of PDLG that presented with bilateral proptosis and direct involvement of the optic nerve during the course of the disease. These new findings may explain an alternative mechanism of visual loss and proptosis in PDLG. We emphasize the importance of close collaboration between neurologists and ophthalmologists in all cases of visual symptoms associated with a neurologic condition. In case of optic nerve involvement, ophthalmologists could provide an easier route to achieve tissue specimen early in the course of this rare and fatal disease.

The cornea of Guinea pig: structural and functional studies.

OBJECTIVE: To describe the functional and structural characteristics of the cornea in healthy Guinea pigs. ANIMALS STUDIED: Healthy male and female pigmented and albino Guinea pigs (Caviaporcellus) aged 3-5 months old were used. PROCEDURES: The animals' corneas underwent different in vivo studies including: slit-lamp biomicroscopy, fluorescein staining (FS), break-up time test (BUT), confocal microscopy and pachymetry. The corneas were also studied histopathologically with light microscopy, immunohistochemistry and transmission electron microscopy. RESULTS: No significant differences were found between pigmented and albino animals, male and female, OD and OS in any study performed. The differences on corneal thickness values were not significant among central (227.85 +/- 14.09 microm) and upper and temporal peripheral regions (226.60 +/- 12.50 and 225.70 +/- 14.40 microm, respectively). All histological studies performed permitted identification and precise description of the different corneal structures in Guinea pigs: the stratified epithelium (45.52 +/- 5.26 microm), Bowman's layer (2.23 +/- 0.38 microm), stroma (163.69 +/- 4.90 microm), Descemet's membrane (3.96 +/- 0.46 microm) and the endothelium (5.09 +/- 0.71 microm). Combining results from all eyes mean and SD from corneal BUT values was 4.98 +/- 1.67 s. Corneas often showed discrete superficial erosions being the FS positive in both eyes from all the animals. CONCLUSION: This study provides a detailed in vivo and postfixed histological description of the Guinea pig's cornea and information about the physiological tests.

Sustained-release hydrogels of topotecan for retinoblastoma.

Treatment of retinoblastoma, the most common primary ocular malignancy in children, has greatly improved over the last decade. Still, new devices for chemotherapy are needed to achieve better tumor control. The aim of this project was to develop an ocular drug delivery system for topotecan (TPT) loaded in biocompatible hydrogels of poly(ε-caprolactone)-poly(ethyleneglycol)-poly(ε-caprolactone) block copolymers (PCL-PEG-PCL) for sustained TPT release in the vitreous humor. Hydrogels were prepared from TPT and synthesized PCL-PEG-PCL copolymers. Rheological properties and in vitro and in vivo TPT release were studied. Hydrogel cytotoxicity was evaluated in retinoblastoma cells as a surrogate for efficacy and TPT vitreous pharmacokinetics and systemic as well as ocular toxicity were evaluated in rabbits. The pseudoplastic behavior of the hydrogels makes them suitable for intraocular administration. In vitro release profiles showed a sustained release of TPT from PCL-PEG-PCL up to 7days and drug loading did not affect the release pattern. Blank hydrogels did not affect retinoblastoma cell viability but 0.4% (w/w) TPT-loaded hydrogel was highly cytotoxic for at least 7days. After intravitreal injection, TPT vitreous concentrations were sustained above the pharmacologically active concentration. One month after injection, animals with blank or TPT-loaded hydrogels showed no systemic toxicity or retinal impairment on fundus examination, electroretinographic, and histopathological assessments. These novel TPT-hydrogels can deliver sustained concentrations of active drug into the vitreous with excellent biocompatibility in vivo and pronounced cytotoxic activity in retinoblastoma cells and may become an additional strategy for intraocular retinoblastoma treatment.

Pharmacokinetics, Safety, and Efficacy of Intravitreal Digoxin in Preclinical Models for Retinoblastoma.

PURPOSE: To assess in vitro cytotoxic activity and antiangiogenic effect, ocular and systemic disposition, and toxicity of digoxin in rabbits after intravitreal injection as a potential candidate for retinoblastoma treatment. METHODS: A panel of two retinoblastoma and three endothelial cell types were exposed to increasing concentrations of digoxin in a conventional (72-hour exposure) and metronomic (daily exposure) treatment scheme. Cytotoxicity was defined as the digoxin concentration that killed 50% of the cells (IC50) and was assessed with a vital dye in all cell types. Induction of apoptosis and cell-cycle status were evaluated by flow cytometry after both treatment schemes. Ocular and systemic disposition after intravitreal injection as well as toxicity was assessed in rabbits. Electroretinograms (ERGs) were recorded before and after digoxin doses and histopathological examinations were performed after enucleation. RESULTS: Digoxin was cytotoxic to retinoblastoma and endothelial cells under conventional and metronomic treatment. IC50 was comparable between both schedules and induced apoptosis in all cell lines. Calculated vitreous digoxin Cmax was 8.5 µg/mL and the levels remained above the IC50 for at least 24 hours after intravitreal injection. Plasma digoxin concentration was below 0.5 ng/ml. Retinal toxicity was evident after the third intravitreal dose with considerable changes in the ERG and histologic damage to the retina. CONCLUSIONS: Digoxin has antitumor activity for retinoblastoma while exerting antiangiogenic activity in vitro at similar concentrations. Metronomic treatment showed no advantage in terms of dose for cytotoxic effect. Four biweekly injections of digoxin led to local toxicity to the retina but no systemic toxicity in rabbits.

Proliferative retinopathy and neovascularization of the anterior segment in female type 2 diabetic rats.

BACKGROUND: To examine the presence of diabetic retinopathy in a female rat model of type 2 diabetes fed on a high-fat diet (HFD). METHODS: Wistar rats were injected with streptozotocin (STZ) at the age of two days and fed on an HFD from eight weeks onwards. Five diabetic animals were euthanized at 110 weeks of disease, together with a control group of age-matched, non-diabetic animals. A group of diabetic animals at 57 weeks of disease was included for comparison. Cross sections of the rats' corneas, iris and retinas were histologically examined and analysed by immunohistochemistry and immunofluorescence, using glial-fibrillary-acidic-protein (GFAP), the vascular endothelial growth factor (VEGF) and the Von Willebrand factor (vWF). The trypsine digestive technique was used for the pericytes count. RESULTS: Neovascularization was only found in the retinas, irises and corneas of the diabetic animals of 110 weeks of disease. There was also a significantly lower number of pericytes in these animals than in the controls. CONCLUSION: The female rat model of type 2 diabetes fed on an HFD may prove useful in evaluating the mechanisms involved in diabetic retinopathy, together with strategies to reduce its severity.