THE EYE OF CRAB-EATING FOX (CERDOCYON THOUS): ANATOMICAL CHARACTERISTICS AND NORMATIVE VALUES OF SELECTED DIAGNOSTIC TESTS, MORPHOMETRY OF CORNEAL TISSUE, AND ARRANGEMENTS OF CORNEAL STROMAL COLLAGEN FIBERS.

This study aimed to evaluate the ophthalmic parameters, morphometric features of corneal tissue, and arrangements of corneal stromal collagen fibers in crab-eating fox (Cerdocyon thous), a species of neotropical wild canid. We conducted the study on six juvenile crab-eating foxes (12 eyes), whilst 16 eyes were obtained post mortem from eight adult crab-eating foxes. The research was divided into two stages. In the first stage, eye anatomical characteristics, tear production (Schirmer 1 tear test, STT1), intraocular pressure (IOP), ocular echobiometry, and specular microscopy parameters related to morphology of corneal endothelium were studied in juvenile animals. In the second stage, morphometric features of corneal tissue (central corneal thickness [CCT] and corneal epithelium thickness) and arrangements of stromal collagen fibers were studied using eyes from adult animals. The main findings were that crab-eating fox eyes have vertical-slit pupils, holangiotic retina, and reference values (mean ± SD) of 13.37 ± 3.79 mm/min for STT1 and of 10.43 ± 3.84 mmHg for IOP. The ocular echobiometric features observed in crab-eating foxes are different from those reported for domestic dogs (Canis familiaris). Conversely, the corneal endothelial parameters are similar to those of domestic dogs. The CCT measured by tissue morphometry was 0.54 ± 0.06 mm, and the corneal epithelium thickness was 60.13 ± 8.71 µm. Mean coherency related to alignment of collagen fibers was 0.66 ± 0.12. The crab-eating fox cornea had predominantly thick collagen fibers. Crab-eating fox eyes have morphofunctional peculiarities. They resemble the eyes of domestic dogs in some aspects, but diverge in others.

Behavioural and physiological effects of methadone in the perioperative period on the Nile tilapia Oreochromis niloticus.

Through the analysis of behavioural changes, this study demonstrates that methadone has behavioural, but not analgesic, effects on Oreochromis niloticus. It provides information that suggests the drug has sedative abilities, as the recovery time was shorter in the fish receiving methadone. Future research, with different doses and stimuli, is required to provide more information about analgesia.

Clinical features and corneal optical anisotropies in a rabbit model of limbal stem cell deficiency / Fenótipos clínicos e anisotropias ópticas da córnea em coelhos com deficiência de células tronco limbais

ABSTRACT 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.

RESUMO Objetivos: Investigar a reprodutibilidade intra-la­boratorial dos fenótipos clínicos e avaliar anisotropias ópticas em córneas de coelhos com deficiência de células tronco limbais. Métodos: Lesões ao limbo foram feitas no olho direito de 23 coelhos adultos da Nova Zelândia Branco, usando um protocolo altamente agressivo, que envolveu peritomia limbal em 360 graus, ceratolimbectomia, cauterização por álcali, e remoção mecânica de epitélio remanescente. Os olhos foram clinicamente avaliados por 28 dias, inclusive por citologia de impressão corneal. As córneas que manifestaram um conjunto de alterações típicas de deficiência de células tronco limbais foram coletadas e submetidas à estudos em histopatologia e em anisotropias ópticas. Córneas saudáveis foram usadas como controles. Diferenças de caminho óptico de birrefringência relacionada à organização do colágeno estromal, e dicroísmo linear relacionado à expressão e à orientação das cadeias de glicosaminoglicanos dos proteoglicanos estromais, foram quantificados por microscopia de luz polarizada. Resultados: Um olho apresentou hipópio e foi excluído do estudo. Todos os olhos estudados (n=22) apresentaram sinais de inflamação ocular. A citologia de impressão não detectou células caliciformes na superfície corneal. Doze de 22 córneas manifestaram alterações clínicas típicas de deficiência de células tronco limbais, caracterizado por defeitos epiteliais, infiltrados inflamatórios, opacidade de moderada à severa, e neovascularização. Estudos por microscopia de luz polarizada mostraram que a deficiência de células tronco limbais aumentou a diferenças de caminho óptico corneal em 24,4% (versus controles). As cór­neas com deficiência de células tronco limbais foram menos dicroicas do que as córneas controle. Conclusões: Coelhos com deficiência de células tronco limbais, para aplicações em estudos pré-clínicos, devem ser rigorosamente selecionados para assegurar homogeneidade experimental, pois há evidências de que protocolos utilizados para indução de deficiência de células tronco limbais não estão associados com boa reprodutibilidade intra-laboratorial de fenótipos clínicos. A deficiência de células tronco limbais, como induzida aqui, alterou as propriedades ópticas anisotrópicas do estroma corneal. Tais alterações são indicativas de mudanças no empacotamento de colágeno e na orientação das cadeias de glicosaminoglicanos dos proteoglicanos. Conhecimentos nessas alterações são importantes para potencializar estratégias que visam a restabelecer a integridade morfofuncional do estromal corneal acometido pela deficiência de células tronco limbais.

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.

Reduction in Histone H3 Acetylation and Chromatin Remodeling in Corneas of Alloxan-Induced Diabetic Rats.

PURPOSE: To evaluate acetylation of histone H3, chromatin remodeling, nuclear size and shape, DNA ploidy, and distribution of nucleolus organizing regions (NORs) in corneal epithelial and stromal cells of diabetic and nondiabetic rats. METHODS: Diabetes was induced by a single intraperitoneal injection of alloxan. All diabetic rats (n = 20) included in the study had 4 weeks of moderate-to-severe hyperglycemia (plasma glucose levels >400 mg/dL). Acetylated histone H3 levels were quantified in corneal tissue using a colorimetric assay. Chromatin remodeling, nuclear sizes (area/perimeter) and shapes (circularity), and DNA ploidies were evaluated from Feulgen-stained tissue sections using video image analysis. Distributions of NORs were studied in tissue sections impregnated with silver ions. Ophthalmic clinical parameters, including corneal sensitivity, were investigated. Twenty nondiabetic rats were used as controls. RESULTS: Acetylation of histone H3 was reduced in the corneas of the diabetic rats. Nuclei in corneal epithelial cells of diabetic rats compacted chromatin, increased in size, modified their shapes, and elevated DNA ploidy. The only nuclear change observed in the corneal stromal cells of diabetic rats was chromatin decompaction. The size of the silver-stained NOR did not differ between the study samples. The corneal sensitivity in diabetic rats was 51.8% lower than that in nondiabetic rats. CONCLUSIONS: The results of this study show that alloxan-induced diabetes altered the histone H3 acetylation pattern and compromised the chromatin supraorganization in corneal tissue/cells. Continued research is needed to understand the clinical and morphofunctional significance of changes in corneal cell nuclei of diabetic individuals.

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.

Short-Term Effects of Y-27632, a Rho-Associated Protein Kinase Inhibitor, on Chromatin Supraorganization and DNA Amount in Epithelial Cells of the Rat Cornea and Limbus.

PURPOSE: To assess the short-term effects of instilling Y-27632, an inhibitor of Rho/Rho-associated protein kinases, on the chromatin supraorganization and DNA amount of corneal and limbal epithelial cells of healthy rats. METHODS: Longitudinal sections (7 µm) of enucleated eyes of healthy rats that received, by instillation, balanced salt solution with or without 10 mM of Y-27632 daily for 7 or 15 days, were subjected to the Feulgen reaction. Feulgen-stained nuclei of corneal and limbal epithelial cells were studied by microscopy and video image analysis to establish the nuclear size (area and perimeter), supraorganization of chromatin (texture and degrees of condensation), and the Feulgen-DNA amount. RESULTS: Instillation of Y-27632 for up to 15 days did not change the size of the nucleus or the chromatin texture of corneal and limbal epithelial cells. Samples treated with Y-27632 for 7 days showed condensed chromatin and a high Feulgen-DNA amount. Both corneal and limbal epithelium showed the presence of near-tetraploid nuclei corresponding to cells in the S and G2 phases of the cell cycle. The degrees of condensation and Feulgen-DNA amount of the nuclei of epithelial cells of the cornea and limbus of eyes from rats receiving Y-27632 for 15 days did not differ from control (no drug). CONCLUSIONS: Changes in chromatin supraorganization and DNA amount, such as seen in this study, are indicative of cell proliferation and do not seem to be associated with disturbances in gene activity and transcription of DNA.

Intraocular pressure, specular microscopy, and prostaglandin E2 concentration in dogs with mature and hypermature cataract.

This study aimed to evaluate and correlate intraocular pressure (IOP), endothelial cell density (CD), and hexagonality (HEX), and the aqueous humor prostaglandin E2 (PGE2 ) concentration in dogs with mature (MG, n = 8) and hypermature (HG, n = 8) cataracts. Eight laboratory beagles with no ocular abnormalities were included as a control group (CG). The IOP was measured using a digital applanation tonometer. Noncontact specular microscopy was used to evaluate CD and HEX. Samples of aqueous humor were used to determine prostaglandin E2 concentration using enzyme-linked immunoassay. Data were compared by anova and Bonferroni's multiple comparison test, and possible correlations among the PGE2 aqueous concentration and corneal endothelium cell parameters were assessed by Person's test (P < 0.05). Average values of IOP (P = 0.45) and CD (P = 0.39) were not significantly different between MG, HM, and CG. Average values of HEX were lower, and PGE2 concentration was increased in the MG and HG in comparison with CG (P < 0.05); however, such parameters did not change significantly between MG and HG (P > 0.05). PGE2 values did not correlate with IOP, CD, and HEX in any group (P > 0.05). Although there were a small number of dogs studied, our results demonstrated that cataract progression from mature to hypermature did not have a significant change in PGE2 aqueous concentration, IOP, corneal endothelial cell count, or morphology. In addition, PGE2 concentration was not correlated with parameters of the corneal endothelium or IOP in dogs with mature or hypermature cataracts.