Marcela Aldrovani

Corneal angiogenesis based on different protocols of alkaline cauterization in murine models

Purpose: To establish and compare protocols of alkaline cauterization for inducing corneal angiogenesis in murine models. Methods: Twenty-four adult Wistar rats were distributed into four groups (G1, G2, G3, and G4). The right eye cornea from each rat was cauterized using filter paper (3 mm), soaked in a solution of silver and potassium nitrates (3:1). Cauterization times were 10 (G1 and G4), or 20 seconds (G2 and G3). Cauterized corneas were washed with Ringers lactate solution. The filter paper was either removed before washing (G1 and G2), or kept on the corneas (G3 and G4). Corneas were photographed at multiple time points (2, 4, 6, 8, 11, 13, and 15 days after the procedure), and neovascularization parameters were assayed. Results: Neovascularization was observed in 66% of G1 corneas, and 100% of G2, G3, and G4 corneas. On day 15, G1 corneas showed smaller vascularized areas (12.63 ± 12.59%) compared to those in the G3 (41.95 ± 17.32%) and G4 (33 ± 11.74%) (P < 0.05) groups. Conclusions: The silver and potassium nitrate solution effectively induced corneal angiogenesis. The G2, G3, and G4 protocols showed excellent reproducibility, and induced vascularization in 100% of corneas.

Effects of morphine on the expression of cytokines and inflammatory mediators in a rabbit model of endotoxin-induced experimental uveitis.

PURPOSE To evaluate the effects of 1% morphine instillation on clinical parameters, aqueous humor turbidity, and expression levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1beta), prostaglandin E2 (PGE2), and myeloperoxidase (MPO) in rabbits with endotoxin-induced experimental uveitis. METHODS Twenty four New Zealand white rabbits were divided into four groups (n=6 each): control (CG), morphine (MG), naloxone (NG), and morphine-naloxone (MNG) groups. Under dissociative anesthesia, 0.1 mL of solution containing 0.2 µg of lipopolysaccharide (LPS) endotoxin from the Salmonella typhimurium cell wall was injected in the vitreous chamber. Clinical evaluations (conjunctical hyperemia, chemosis blepharospasm, and ocular discharge) and laser flaremetry were performed before (baseline), and 10 and 20 hours after induction of uveitis. Rabbits were subsequently euthanized and eyes were enucleated to quantify expression levels of TNF-α, IL-1 beta, PGE2, and MPO. RESULTS No significant differences in clinical parameters and flare values were observed between the study groups. TNF-α and IL-1 beta levels increased significantly in the CG, MG, NG, and MNG groups compared to baseline (P<0.05). Significant differences in PGE2 levels were observed between the MG and NMG groups (P<0.05). A trend toward increased MPO activity was observed in response to uveitis induction; however, this trend did not reach statistical significance (P>0.05). CONCLUSIONS Morphine has no effect on clinical parameters, flare, or expression levels of inflammatory mediators in a rabbit model of uveitis induced by intravitreal injection of LPS.

Ophthalmic parameters in adult lowland paca (Cuniculus paca) raised in captivity

OBJECTIVE To investigate the ophthalmic parameters of lowland pacas, including the anatomic features, tear production, intraocular pressure, central corneal thickness, and morphology of the corneal endothelium. ANIMALS STUDIED Thirteen adult, anesthetized Cuniculus paca. PROCEDURE Eyes were evaluated using slit-lamp biomicroscopy, the Schirmer tear test I, digital applanation tonometry, binocular indirect ophthalmoscopy, and noncontact specular microscopy. RESULTS The biomicroscopy findings showed blue/brown pigmented bulbar conjunctivae, well-developed cilia (only in the upper eyelid margin), superior and inferior lacrimal puncta, brown irides, round pupils, and vestiges of the nictitating membrane. The results of the Schirmer tear test I revealed (mean ± SD) a lacrimation rate of 4.10 ± 0.44 mm/min. The intraocular pressure was 6.34 ± 0.43 mmHg. Central corneal thickness measured by specular microscopy was 0.35 ± 0.01 mm. The mean values of density, hexagonality, and the area of the endothelial cells were 2083.15 ± 42.47 cells/mm2 , 67.07 ± 3.30%, and 486.30 ± 9.56 μm2 , respectively. CONCLUSIONS The ocular parameters defined in this study may be used for reference in future studies and might also contribute to therapeutic approaches appropriate to this species.

Effects of intracameral ascorbic acid on the corneal endothelium of dogs undergoing phacoemulsification

OBJECTIVE Cataracts are the most common ocular disorder in dogs. Phacoemulsification is the preferred treatment method among ophthalmologists, but the cellularity of the endothelium must be considered for its success, as endothelial lesions may produce permanent corneal decompensation. The objective of this study was to evaluate the effects of intracameral ascorbic acid, a known antioxidant, on the corneal endothelium of dogs undergoing phacoemulsification. ANIMAL STUDIED In all, 40 eyes from 20 dogs, males and females from 7 to 12 years of age, were assessed for mature cataracts. PROCEDURES Two groups were formed (n = 20): Group 1 (G1) received a balanced salt solution (BSS), whereas Group 2 (G2) received sterile ascorbic acid diluted in a BSS, at a final concentration of 0.001 m ascorbic acid. The corneal endothelium was assessed via non-contact specular microscopy at multiple time points before and after phacoemulsification. Cell density (cells/mm2 ) and area (mm2 ), corneal thickness (mm), hexagonality, and the coefficient of variation of cell size were all assessed. P values equal to or less than 0.05 were considered significant. RESULTS With respect to the density of endothelial cells, both groups showed losses, but they were less severe in G2. There were no differences in corneal thickness. Hexagonality decreased significantly in the postoperative period in G1. Also in G1, the coefficient of variation of cell size increased significantly. CONCLUSION According to the results obtained, ascorbic acid minimizes cellular losses in the corneal endothelium.

Clinical features and corneal optical anisotropies in a rabbit model of limbal stem cell deficiency

PURPOSES To investigate the intra-laboratory reproducibility of clinical features and to evaluate corneal optical anisotropies in a rabbit model of limbal stem cell deficiency. METHODS Limbal injury was induced in the right eye of 23 adult New Zealand White rabbits using a highly aggressive protocol that combined 360 degrees limbal peritomy, keratolimbectomy, alkaline chemical burn, and mechanical removal of the epithelium. Clinical evaluation of the injured eyes was performed for 28 days and included corneal impression cytology. Corneas with a severe clinical outcome set typical of limbal stem cell deficiency were then collected, subjected to a histopathological examination, and examined for optical anisotropies. Corneas from healthy rabbit eyes were used as controls. Differences in optical path due to stromal collagen birefringence, as well as linear dichroism related to the expression and spatial orientation of glycosaminoglycan chains from proteoglycans, were measured from cross-sections under a quantitative polarized light microscope. RESULTS One eye showed signs of hypopyon and was excluded. Signs of ocular inflammation were observed in all eyes studied (n=22). Corneal impression cytology did not detect goblet cells. Twelve of the 22 corneas presented a clinical outcome set typical of limbal stem cell deficiency, which is characterized by the presence of epithelial defects, inflammatory cells, moderate-to-severe opacity, and neovascularization. Microscopic studies under polarized light revealed that relative to controls, limbal stem cell deficiency caused a 24.4% increase in corneal optical path differences. Further, corneas with limbal stem cell deficiency were less dichroic than controls. CONCLUSIONS These results suggest that rabbit models of limbal stem cell deficiency must be rigorously screened for use in preclinical studies to ensure experimental homogeneity because protocols used to create limbal stem cell deficiency could be not associated with good intra-laboratory reproducibility of clinical features. Limbal stem cell deficiency, as induced herein, altered the optical anisotropic properties of the corneal stroma. Such alterations are indicative of changes in collagen packing and the spatial orientation of glycosaminoglycan chains from proteoglycans. Knowledge of these changes is important to potentiate strategies aimed at restoring the morphofunctional integrity of the corneal stroma affected by limbal stem cell deficiency.

Acoustic radiation force impulse elastography of the eyes of brachycephalic dogs

Eyes from brachycephalic dogs were studied by acoustic radiation force impulse (ARFI) elastography to establish quantitative and qualitative reference values related to the shape and rigidity of some ocular structures. Eighty-four eyes from 42 adult brachycephalic dogs were included in this study. Animals were subjected to physical, laboratory, and ophthalmic examinations. Following confirmation of good health, the dogs were subjected to ultrasonography and ARFI elastography, emphasizing the optic nerve, ciliary body, and lens. On qualitative elastography, homogeneous characteristics were observed on the ciliary body and optic nerve. Aqueous and vitreous humors were visualized as mosaic-like images. Quantitative elastography showed the mean optic nerve shear wave velocity (SWV) was 1.01±0.27m/s, temporal ciliary body was 0.91±0.24m/s, and nasal ciliary body was 0.91±0.3m/s. The SWV of the lens values were out of range, this value was not detectable by this software. Elastography has proved to be a non-invasive procedure and feasible in dogs. The establishmentof the parameters of degree of rigidity of ocular structures will serve as a baseline for animals with eye disorders. Results may be extrapolated to primary research on the applicability of ARFI in the evaluation of ocular bulb in humans.

A supramolecular look at microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny

Various approaches have been taken to improve our knowledge of the microenvironmental regulation of limbal epithelial stem cells. Researchers have extensively investigated the roles of growth factors, survival factors, cytokines, enzymes, and permeable molecules secreted by the limbal cells. However, recent evidence suggests that stem cell fate (i.e., self-renewal or differentiation) can also be influenced by biophysical and mechanical cues related to the supramolecular organization and the liquid crystalline (mesophase) nature of the stromal extracellular matrix. These cues can be sensed by stem cells and transduced into intracellular biochemical and functional responses, a process known as mechanotransduction. The objective of this review is to offer perspectives on the supramolecular microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny.

Supra-organization and optical anisotropies of the extracellular matrix in the amniotic membrane and limbal stroma before and after explant culture

In this research we evaluated the supramolecular organizations and the optical anisotropical properties of the de-epithelialized human amniotic membrane and rabbit limbal stroma, before and after explant culture. Birefringence, monochromatic light spectral absorption and linear dichroism of the main extracellular matrix biopolymers, that is, the fibrillar collagens and proteoglycans, were investigated by polarized light microscopy combined with image analysis. Our results demonstrated that the culture procedure-induced stimuli altered the supra-organizational characteristics (in terms of collagens/proteoglycans spatial orientation and ordered-aggregational state) of the amniotic and limbal extracellular matrix, which led to changes in optical anisotropical properties.