Birefringence analysis of collagen supraorganization in cat corneas with tropical keratopathy.

OBJECTIVE: To evaluate the birefringent properties of the cornea and examine the supraorganizational aspects of collagen fibers in cats with tropical keratopathy. PROCEDURE: In this study, 10-micrometer-thick sections of corneal tissue from cats with tropical keratopathy were examined, both in the opaque and transparent areas of the anterior stroma. Control samples were obtained from healthy cat corneas. Polarized light microscopy was employed to evaluate the birefringent properties using two distinct methods. The first method involved measuring the optical retardation associated with corneal birefringence, while the second method assessed the alignment/waviness of the birefringent collagen fibers. Differences were significant when p < .05. RESULTS: Tropical keratopathy resulted in a significant rise (p < .05) in optical retardation in both opaque and transparent regions of the cat cornea. In the anterior stroma, both the opaque zones and transparent tissue exhibited a higher degree of collagen fiber packing than the control corneas. However, no significant differences (p > .05) in alignment were observed between the transparent tissue of the diseased cornea and the healthy corneas. CONCLUSION: Supraorganizational changes in collagen fiber packing are not restricted to lesion zones in cat corneas affected by tropical keratopathy. Such alterations also occur in the corneal tissue of the anterior stroma adjoining the lesions. Therefore, it is plausible that the transparent tissue of the anterior stroma in corneas affected by the disease may have functional abnormalities, despite its macroscopic healthy appearance. Additional investigations are required to clarify the implications of these potential defects and their conceivable contribution to tropical keratopathy.

Accuracy of ARFI elastography in the differentiation of cataract stages in dogs

ABSTRACT: Objective was to evaluate the accuracy of elastography in the differentiation between normal and cataract lenses One hundred forty-five eyes of 98 dogs were divided into groups according to cataract stage. Forty-twoeyes were submitted to phacoemulsification. Biometric parameters, echogenicity and echotexture patterns of the anterior, posterior and vitreous chambers, lens and retina-choroid-sclera complexes were evaluated by ocular ultrasound in modes A and B. Deformability, and color (blue color = indicated less rigid structures, color red = more rigid structures) of the lenses were evaluated by the elastogram. The shear wave velocity (SWV; m/s) was calculated in three regions of the lens, both in the cortex and in the nucleus. The SWV of nucleus was statistically different between the normal lenses and with cataracts, and between the stages of cataract (P 0.001). Healthy lenses and incipient cataracts had a more rigid nucleus. Mature cataracts presented lowest nuclear rigidity (P 0.001). On cortical region the SWV was significantly higher (P 0.01) in intumescent and incipient cataracts. SWV less than 2.67m/s indicates cataract with a sensitivity of 72% and specificity of 94%. Values lower than 2.23m/s suggest mature cataract, with sensitivity of 71% and specificity of 76%. SWV greater than 2.66 m/s are associated with normal lenses or incipient cataract, presenting sensitivity of 94% and specificity of 84%. Qualitative method allowed differentiation between healthy and affected lenses and the classification of evolutionary stages. There was a correlation between the degree of stiffness of lens in cortical and nuclear regions (p=00165, r=0.37) and between the balanced saline solution quantitative and surgical time (P 0.01, r=0.73). Degree of stiffness of lens did not correlate with parameters of phacoemulsification. Elastographic proved feasible for evaluating the lens of dogs, characterizing the types of cataracts, and demonstrating increased stiffness of the diseased lenses.

RESUMO: O objetivo foi avaliar a precisão da elastografia na diferenciação entre lentes normais e de catarata. Cento e quarenta e cinco olhos de 98 cães foram divididos em grupos de acordo com o estágio de maturação da catarata. Quarenta e dois olhos foram submetidos à facoemulsificação. Parâmetros biométricos, ecogenicidade e padrões de ecotextura das câmaras anterior, posterior e vítrea, lente e complexos retina-coróide-esclera foram avaliados por ultrassonografia ocular nos modos A e B. A deformabilidade e a coloração (cor azul = indicou estruturas menos rígidas, cor vermelha = estruturas mais rígidas) das lentes foram avaliadas pelo elastograma. A velocidade da onda de cisalhamento (SWV; m/s) foi calculada em três regiões da lente, tanto no córtex quanto no núcleo. A SWV do núcleo foi estatisticamente diferente entre as lentes normais e com catarata e entre os estágios da catarata (P 0,001). Lentes saudáveis e cataratas incipientes tinham um núcleo mais rígido. Cataratas maduras apresentaram menor rigidez nuclear (P 0,001). Na região cortical, a SWV foi significativamente maior (P 0,01) nas cataratas intumescentes e incipientes. Uma SWV menor que 2,67m/s indica catarata com sensibilidade de 72% e especificidade de 94%. Valores inferiores a 2,23m/s sugerem catarata madura, com sensibilidade de 71% e especificidade de 76%. Uma SWV superior a 2,66m/s está associada à catarata normal ou incipiente, apresentando sensibilidade de 94% e especificidade de 84%. O método qualitativo permitiu a diferenciação entre lentes normais de olhos saudáveis e afetadas e a classificação dos estágios evolutivos. A elastografia se mostrara uma ferramenta viável para avaliar as lentes de cães, caracterizando os tipos de catarata e demonstrando maior rigidez das lentes doentes.

Accuracy of ARFI elastography in the differentiation of cataract stages in dogs / Acurácia da elastografia ARFI na diferenciação dos estágios da catarata em cães

Objective was to evaluate the accuracy of elastography in the differentiation between normal and cataract lenses One hundred forty-five eyes of 98 dogs were divided into groups according to cataract stage. Forty-twoeyes were submitted to phacoemulsification. Biometric parameters, echogenicity and echotexture patterns of the anterior, posterior and vitreous chambers, lens and retina-choroid-sclera complexes were evaluated by ocular ultrasound in modes A and B. Deformability, and color (blue color = indicated less rigid structures, color red = more rigid structures) of the lenses were evaluated by the elastogram. The shear wave velocity (SWV; m/s) was calculated in three regions of the lens, both in the cortex and in the nucleus. The SWV of nucleus was statistically different between the normal lenses and with cataracts, and between the stages of cataract (P<0.001). Healthy lenses and incipient cataracts had a more rigid nucleus. Mature cataracts presented lowest nuclear rigidity (P<0.001). On cortical region the SWV was significantly higher (P<0.01) in intumescent and incipient cataracts. SWV less than 2.67m/s indicates cataract with a sensitivity of 72% and specificity of 94%. Values lower than 2.23m/s suggest mature cataract, with sensitivity of 71% and specificity of 76%. SWV greater than 2.66 m/s are associated with normal lenses or incipient cataract, presenting sensitivity of 94% and specificity of 84%. Qualitative method allowed differentiation between healthy and affected lenses and the classification of evolutionary stages. There was a correlation between the degree of stiffness of lens in cortical and nuclear regions (p=00165, r=0.37) and between the balanced saline solution quantitative and surgical time (P<0.01, r=0.73). Degree of stiffness of lens did not correlate with parameters of phacoemulsification. Elastographic proved feasible for evaluating the lens of dogs, characterizing the types of cataracts, and demonstrating increased stiffness of the diseased lenses.(AU)

O objetivo foi avaliar a precisão da elastografia na diferenciação entre lentes normais e de catarata. Cento e quarenta e cinco olhos de 98 cães foram divididos em grupos de acordo com o estágio de maturação da catarata. Quarenta e dois olhos foram submetidos à facoemulsificação. Parâmetros biométricos, ecogenicidade e padrões de ecotextura das câmaras anterior, posterior e vítrea, lente e complexos retina-coróide-esclera foram avaliados por ultrassonografia ocular nos modos A e B. A deformabilidade e a coloração (cor azul = indicou estruturas menos rígidas, cor vermelha = estruturas mais rígidas) das lentes foram avaliadas pelo elastograma. A velocidade da onda de cisalhamento (SWV; m/s) foi calculada em três regiões da lente, tanto no córtex quanto no núcleo. A SWV do núcleo foi estatisticamente diferente entre as lentes normais e com catarata e entre os estágios da catarata (P<0,001). Lentes saudáveis e cataratas incipientes tinham um núcleo mais rígido. Cataratas maduras apresentaram menor rigidez nuclear (P<0,001). Na região cortical, a SWV foi significativamente maior (P<0,01) nas cataratas intumescentes e incipientes. Uma SWV menor que 2,67m/s indica catarata com sensibilidade de 72% e especificidade de 94%. Valores inferiores a 2,23m/s sugerem catarata madura, com sensibilidade de 71% e especificidade de 76%. Uma SWV superior a 2,66m/s está associada à catarata normal ou incipiente, apresentando sensibilidade de 94% e especificidade de 84%. O método qualitativo permitiu a diferenciação entre lentes normais de olhos saudáveis e afetadas e a classificação dos estágios evolutivos. A elastografia se mostrara uma ferramenta viável para avaliar as lentes de cães, caracterizando os tipos de catarata e demonstrando maior rigidez das lentes doentes.(AU)

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.

Nuclear parameters and chromatin remodeling in epithelial cells and lymphocytes from the palpebral conjunctiva of dogs with keratoconjunctivitis sicca.

OBJECTIVE: To study parameters related to nuclear morphology and chromatin remodeling in epithelial cells and lymphocytes from the inferior palpebral conjunctiva of dogs with and without keratoconjunctivitis sicca (KCS). ANIMALS STUDIED: Thirty-two dogs (64 eyes) were included in the study. Based on the tear production measured by Schirmer tear test 1, the dogs were distributed into control and KCS groups. PROCEDURES: Epithelial cells and lymphocytes were collected by conjunctival brush cytology, fixed on glass slides, and subjected to the Feulgen reaction, a topochemical method specific for DNA/chromatin. Feulgen-stained cells were studied by microscopy and video image analysis to establish nuclear size (area and perimeter) and shape (relative nuclear roundness factor = RNRF), DNA content (ploidy), and compaction and texture of chromatin. RESULTS: Conjunctival samples in the KCS group showed infiltration of inflammatory and immune cells. Micronuclei, snake-like chromatin, aberrant chromosomes, and goblet cells were not detected. Compared with the controls, cells on the conjunctival surface of dogs with KCS showed altered nuclei. Conjunctival epithelial cells were more affected by KCS (changes in nuclear size, shape, DNA content, and chromatin compaction) than lymphocytes (changes in chromatin compaction, only). Significant chromatin decompaction was observed in both conjunctival epithelial cells and lymphocytes. CONCLUSIONS: Our results show that KCS promotes chromatin remodeling in epithelial cells and lymphocytes on the conjunctival surface of dogs. The changes described in this study are different from those reported for conjunctival cell nuclei of human KCS patients.

Effect of metronidazole ophthalmic solution on corneal neovascularization in a rat model.

PURPOSE: To evaluate the effect of metronidazole ophthalmic solutions on corneal neovascularization (CNV) in a rat model. METHODS: A chemical burn was created in the right central cornea of 40 rats. Animals were randomized and distributed into four study groups (n = 10 rats per group) designated Met_0.1%, Met_0.5%, sham, and untreated groups. Chemical-burned corneas in the Met_0.1% and Met_0.5% groups received ophthalmic solutions of 0.1 and 0.5% metronidazole, respectively. Corneas in the sham group received phosphate-buffered saline (metronidazole diluent). All treated eyes received ophthalmic solution at intervals of 6 h, for up to 30 days. Untreated corneas received no treatment. CNV was evaluated postinjury using corneal photographs at different evaluation time points. The main CNV outcome measures were: burn intensity, index of CNV, and percentage of vascularized corneal area. Five rats from each group were euthanized, on days 15 and 30; the samples were collected for histological analyses. Differences with P < 0.05 were considered significant. RESULTS: CNV was observed in the eyes from day 7 postinjury. However, the indices of CNV for the Met_0.1% and Met_0.5% groups were smaller than those for the sham and untreated groups (P < 0.05). Furthermore, corneas treated with 0.1 or 0.5% metronidazole had smaller vascularized areas compared to control corneas. On histological study, the presence of blood vessels confirmed clinical outcomes. CONCLUSIONS: Regular instillation of 0.1 or 0.5% metronidazole had a significant inhibitory effect for CNV on chemical burns induced in a rat model.

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.

Suture and venous traction test analysis in dogs fixed in alcohol and preserved in saline solution / Análise de sutura e de teste de tração venosa em cães fixados em álcool e conservados em solução salina

The objective of this research was to determine in necropsied dogs the best time for fixation in ethylic alcohol (EA) and preservation in 30% sodium chloride aqueous solution (SCAS 30%), aiming micro-surgical training. Five groups of necropsied dogs (G1 to G5) were fixed with EA, and put in boxes containing EA for 30 (G2), 60 (G3), 90 (G4) or 120 days (G5). After that, each group was preserved in SCAS 30% for 120 days. The control group (G1) was composed by cadavers without fixation/preservation. At the end of each period, two fragments of external jugular vein per cadaver were collected, for traction test. Immediately after the collection, the cadavers femoral veins were evaluated (by 2 people) regarding the suture quality in binocular surgical microscope, and attributed scores from 0 (bad) to 5 (excellent), regarding the fresh samples. The average at the maximum rupture strength of the G3 fixation end (21.51N), such as the average of the G2 preserving end (21.62N) remained closer to the control group (19.98N) and the G2 was the group with the best score for venous suture training. The EA was efficient as a fixative just like SCAS as a dog cadavers' preservative. The small change of the traction test values, together with the best suture score, indicated the group kept for 30 days in EA and SCAS (G2) as the best for venous micro-surgical training.(AU)

O objetivo deste trabalho foi determinar, em cadáveres de cães, o melhor tempo para fixação em álcool etílico (AE) e conservação em solução aquosa de cloreto de sódio (SACS) a 30% visando o treinamento microcirúrgico. Cadáveres de cinco grupos (G1 a G5) foram fixados com AE e colocados em caixas contendo AE por 30 (G2), 60 (G3), 90 (G4) ou 120 days (G5). Depois, cada grupo foi conservado em SACS 30% por 120 dias. O grupo controle (G1) foi composto de cães sem fixação/conservação. Ao final de cada período, 2 fragmentos da veia jugular externa por cadáver foram coletados para o teste de tração. Imediatamente após a coleta, as veias femorais foram avaliadas (por 2 pessoas) em relação à qualidade da sutura em microscópio cirúrgico binocular, e atribuídos escores de 0 (péssimo) à 5 (excelente), em relação às amostras frescas. A média da força máxima de ruptura no G3 no final da fixação (21,51N), assim como a média do G2 no final da conservação (21,62N) foram as que mais se mantiveram próxima do grupo controle (19,98N) e o G2 foi o grupo com o melhor escore para treinamento da sutura venosa. O AE foi eficiente como fixador assim como a SACS foi efetiva na conservação de cadáveres de cães. A pequena alteração nos valores do teste de tração, junto com o melhor escore para sutura, indicaram o grupo mantido por 30 dias em AE e SACS (G2) como o melhor para treinamento venoso microcirúrgico.(AU)

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.

Molecular characterization and potential sources of aqueous humor bacterial contamination during phacoemulsification with intraocular lens implantation in dogs.

Bacterial contamination of the anterior chamber during cataract surgery is one of the main responsible for endophthalmitis postoperative. Phacoemulsification is a less invasive technique for cataract treatment, although it does not exclude the possibility of contamination. In this study, bacterial contaminants of aqueous humor collected pre- and post-phacoemulsification with intraocular lens implantation (IOL) of twenty dogs were identified. As the conjunctival microbiota constitute a significant source of anterior chamber contamination, bacterial isolates from aqueous humor were genetically compared with those present in the conjunctival surface of the patients. Three dogs presented bacterial growth in both aqueous humor and conjunctival surface samples. Bacterial isolates from these samples were grouped according to their genetic profiles by repetitive-element PCR (rep-PCR) and their representatives were identified by 16S rRNA sequencing. Isolates from conjunctival surface were identified as Enterobacter spp., Staphylococcus spp. and S. aureus; and from aqueous humor samples as Enterobacter spp., Pantoea spp., Streptococcus spp. and Staphylococcus spp., respectively in decreasing order of prevalence. According to the rep-PCR analysis, 16.6% of Enterobacter spp. isolates from conjunctival surface were genetically similar to those from aqueous humor. The rest of isolates encountered in aqueous humor were genetically distinct from those of conjunctival surface. The significant genetic diversity of bacterial isolates found in the aqueous humor samples after surgery denoted the possibility of anterior chamber contamination during phacoemulsification by bacteria not only from conjunctival surface but also from different sources related to surgical environment.

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.

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.

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.

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 Ringer's 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.

Selected ophthalmic and physiologic parameters in rabbits (Oryctolagus cuniculus) submitted to retrobulbar block with lidocaine, morphine or ketamine / Avaliação de parâmetros oftálmicos e fisiológicos em coelhos (Oryctolagus cuniculus) submetidos a bloqueio retrobulbar com lidocaína, morfina ou cetamina

The aim of the present study was to evaluate selected ophthalmic and physiologic parameters in rabbits submitted to retrobulbar blockade with lidocaine, morphine or ketamine. Eighteen adult rabbits, seven males and eleven females, New Zealand White breed, weighing 3.9 ± 0.7 kg were randomly assigned to perform the retrobulbar block according to the groups: LID (2% lidocaine without a vasoconstrictor - 7 mg kg-1); MOR (1% morphine - 1 mg kg-1) or KET (10% Ketamine - 5 mg kg-1). Ophthalmic and physiologic parameters were assessed, including lacrimal production using Schirmer tear test (STT), corneal touch threshold (CTT), pupillary diameter, intraocular pressure (IOP), pulse rate (PR), respiratory rate (RR), oxyhemoglobin saturation (SpO2), rectal temperature (RT) and systolic, mean and diastolic blood pressures (SAP, MAP and DAP) and were evaluated every 10 minutes for 70 minutes. All drugs used in the present study promoted central positioning of the eyeball for up to one minute later the retrobulbar administration in all cases. There was a significant increase of STT values in MOR and LID, when compared to baseline, while the CTT values had a significant decrease in all groups. KET kept the IOP values unaltered at the time points and there was a significant decrease of pupillary diameter in MOR. There was no significant change in PR, RR and SpO2; however, LID presented significantly lower values of SAP. MOR had increased values for RT when compared to the other two groups. The established parameters may help in ophthalmic procedures using retrobulbar nerve blocks.

Objetivou-se com o presente estudo estabelecer parâmetros oftálmicos e hemodinâmicos em coelhos submetidos ao bloqueio retrobulbar com lidocaína, morfina ou cetamina. Dezoito coelhos da raça Nova Zelândia, adultos, sete machos e onze fêmeas, com peso de 3,9 ± 0,7 kg foram aleatoriamente distribuídos para realização de bloqueio retrobulbar de acordo com os seguintes grupos: LID (lidocaína 2% sem vasoconstrictor ­ 7 mg/kg); MOR (morfina 1% - 1 mg/kg) ou KET (cetamina 10% ­ 5 mg/kg). Os seguintes parâmetros oftálmicos e hemodinâmicos foram avaliados: produção lacrimal através do teste lacrimal de Schirmer (TLS), limiar de sensibilidade corneana ao toque, diâmetro pupilar, pressão intraocular (PIO), frequência de pulso (FP) e respiratória (FR), saturação da oxihemoglobina (SpO2), temperatura retal (TR) e pressão arterial sistólica, diastólica e média (PAS, PAD e PAM). Todos os fármacos utilizados no presente estudo promoveram centralização do bulbo do olho em até um minuto após a sua administração retrobulbar em todos os casos. Houve um aumento significativo do TLS no grupo MOR e LID, quando comparados aos valores basais, enquanto o limiar de sensibilidade corneana reduziu significativamente em todos os grupos. O grupo KET manteve os valores da PIO inalterados em todos os tempos e houve uma redução significativa do diâmetro pupilar no grupo MOR. Não houve alteração significativa dos valores de FP, FR e SpO2. No entanto, o grupo LID apresentou valores significativamente menores de PAS. O grupo MOR apresentou maiores valores de TR quando comparado aos demais grupos. Os parâmetros estabelecidos no presente estudo podem servir como base para a realização de procedimentos oftálmicos que requerem o uso de bloqueio retrobulbar.

Histological, morphometric, protein and gene expression analyses of rat retinas with ischaemia-reperfusion injury model treated with sildenafil citrate.

The aim of this study was to better understand the role of apoptosis in a retinal ischaemia-reperfusion injury model and to determine whether sildenafil citrate treatment can prevent retinal cell apoptosis. Thirty-six rats were divided into a control group (n = 6) and two experimentally induced ischaemia-reperfusion groups (7 and 21 days; n = 15 per group). The induced ischaemia-reperfusion groups were treated with sildenafil for 7 and 21 days (n = 10 per group), and 10 animals were treated with a placebo for the same period (n = 5 per group). Paracentesis of the anterior chamber was performed with a 30-G needle attached to a saline solution (0.9%) bag positioned at a height of 150 cm above the eye for 60 min. Intraocular pressure was measured by rebound tonometer (TonoVet® ). The eyes were analysed by histology and morphometry, and by immunohistochemistry and qRT-PCR for expression of Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. Sildenafil-treated animals showed lower levels of histopathological changes (inflammatory, cellular and tissue) than their placebo-treated counterparts at both 7 and 21 days. The retinal ganglion cell layer (RGC) was preserved in the sildenafil groups (SG), with a cell count closer to control than in the placebo groups (PG). Caspase-7 expression was significantly higher in both treated groups at 7 days compared to controls. Gene expression levels in both treatment groups differed from the controls, but in SG Bax and Caspase-6 expression levels were similar to control animals. These results suggest that the main mechanism of retinal cell death in this model is apoptosis, as there is an increase in pro-apoptotic factors and decrease in the anti-apoptotic ones. Also, sildenafil seems to protect the retinal ganglion cell layer from apoptosis. Cell survival was evident in the histological and morphometric analyses, and sildenafil treatment had a protective effect in the apoptosis pathways, with gene expression levels in SG similar to the controls.

A supramolecular look at microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny / Uma visão supramolecular sobre a regulação microambiental de células tronco epiteliais limbais e a diferenciação de sua progênie

ABSTRACT 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.

RESUMO Muitas abordagens têm sido utilizadas para ampliar entendimentos sobre a regulação microambiental das células tronco epiteliais limbais. Neste contexto, pesquisadores têm exaustivamente investigado a participação de fatores de crescimento, fatores de sobrevida, citocinas, enzimas e moléculas permeáveis secretadas pelas células limbais. Entretanto, evidências recentes sugerem que o destino (ie. autorrenovação ou recrutamento para a via de diferenciação) das células tronco também sofre influência de estímulos biofísicos ou mecânicos relacionados à organização supramolecular e à natureza liquido-cristalina (mesofases) da matriz extracelular estromal. Esses estímulos podem ser percebidos e traduzidos pelas células tronco em sinais bioquímicos que geram respostas funcionais, através de um processo designado de mecanotransdução. Objetiva-se, com a presente revisão, oferecer ao leitor perspectivas supramoleculares sobre a regulação microambiental das células tronco epiteliais limbais e a diferenciação de sua progênie.

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

Abstract 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 Ringer's 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.

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.