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ABSTRACT Purpose: To examine subbasal corneal nerve changes in patients with bacterial infectious keratitis using in vivo confocal microscopy. Methods: Thirteen patients (13 eyes) with unilateral bacterial keratitis and 12 healthy controls were prospectively enrolled in the study. In vivo confocal microscopy was performed in all the patients at 2 time points, in the acute phase of infectious keratitis and at 28 ± 0.6 months after resolution of the infection. Results: The subbasal nerve length was 5.15 ± 1.03 mm/mm2 during the acute phase of bacterial keratitis (compared with that of the controls: 19.02 ± 1.78 mm/mm2, p<0.05). Despite the significant corneal nerve regeneration over the interval of 28 months after the resolution of the infection, the nerve density was still significantly reduced as compared with that of the controls (9.73 ± 0.93 mm/mm2, p<0.05). Moreover, in vivo confocal microscopy images showed diffuse high-reflecting areas referring to the scar tissue areas with thin and tortuous nerve branches regenerating toward these areas. Conclusions: A partial corneal nerve regeneration of subbasal nerve plexus during the first 28 months after the acute phase of infectious keratitis was observed. Moreover, the regenerated nerves of the patients remained morphologically altered as compared with those of the healthy controls. These results may be relevant to the clinical follow-up and surgical planning for these patients.
RESUMO Objetivo: Relatar as alterações no plexo nervoso corneano subbasal em pacientes com ceratite infecciosa de origem bacteriana utilizando a microscopia confocal in vivo. Métodos: Treze olhos de 13 pacientes com ceratite bacteriana unilateral e 12 indivíduos saudáveis como grupo controle foram incluídos prospectivamente no estudo. A microscopia confocal in vivo foi realizada em todos os pacientes em 2 momentos: na fase aguda da ceratite infecciosa e após 28 ± 0,6 meses da resolução da infecção. Resultados: A densidade dos nervos no plexo subbasal foi de 5,15 ± 1,03 mm/mm2 na fase aguda da ceratite infecciosa (comparada com o grupo controle: 19,02 ± 1,78 mm/mm2, p<0,05). Apesar de significativa regeneração dos nervos corneanos ao longo de um intervalo de 28 meses após a resolução da infecção, a densidade dos nervos se manteve significativamente reduzida (9,73 ± 0,93 mm/mm2) quando comparada com o grupo controle (19,02 ± 1,78 mm/mm2, p<0,05). Além disso, as imagens obtidas com a microscopia confocal mostraram áreas de hiperreflectividade referente ao tecido corneano cicatricial com ramos de nervos, afinados e tortuosos, se regenerando nessas áreas. Conclusões: Foi observado regeneração parcial dos nervos do plexo corneano subbasal durante os primeiro 28 meses após a resolução da fase aguda da ceratite infecciosa. Além disso, os nervos corneanos regenerados se mantiveram morfologicamente alterados quando comparados ao grupo controle. Esses resultados podem ser relevantes para o acompanhamento clínico e planejamento cirúrgico desses pacientes.
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Background To understand the distribution and development of corneal nerve in animal or human has an important significance for clinical and basic research of corneal diseases.At present,some studies on cornea nerve development and location have been performed.However,the quantified study on innervation and distribution of corneal nerve fibers as embryonic development has not been reported.Objective This study attempted to understand the distribution of corneal nerve fibers in the development of chick embryo,and to evaluate the changes of the length and density of corneal nerve fibers with aging of chick embryo.Methods Whole chick corneas with limbus were obtained from chick embryo aged 6-20 days (E6-E20),and corneal nerve fiber was labeled using immunofluorescence technique by anti-neuron-specific β-Ⅲ tubulin antibody.The corneas were radially cut into 4 parts,and the integrate corneal flat mounts were prepared with the upward epithelium and mounting with anti-fade fluorescent quenching buffer glycerin containing DAPI.Fluorescence microscope was used to capture the nerve fiber images in cornea,and cornea area and the number of nerve fiber bundles were exhibited by using Photoshop CS4.Cornea nerve fiber density and total length were measured by Imaris x64 7.4.2 software.Results Total cornea flat mounts showed that the nerve bundles grew from temporal scleral forward cornea limbus at E6-E8,and the nerve fibers formed the ring surrounding by limbus during E9-E10.Then the fibers extended forward the central cornea in E11 to E15 and developed into nerve fiber plexus on the whole cornea in E16 to E20.During the period of E6-E20,the corneal surface area,the length and density of corneal nerve fibers were gradually increased with the aging of chick embryo,showing statistically significant differences among different time points (F =127.007,227.051,67.748,all at P＜0.01).The increase of the corneal area of the chick embryo presented a strong positive correlation with the extending of length of the corneal nerve fiber (r =0.863,P＜0.01).Chick corneal nerve fiber bundle appeared at E13,with a number of (59.00 ± 1.14)/mm2 and then increased to a peak of (576.75 ±29.16)/mm2 at E 18 and reduced to (299.67± 25.46)/mm2 at E20,with a significant difference among them (F =13.759,P=0.000).Conclusions Corneal nerve starts to develop in E9 of chick embryo,and the corneal surface area,the total length of the corneal nerve fibers and the density rapidly increase concurrently with the development of chick embryo.
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ABSTRACT Purpose: To quantify the morphological alterations in corneal nerve fibers and cells in patients with pseudoexfoliation syndrome (PEX) and their relationship with the presence of hyperreflective endothelial deposits observed using in vivo confocal microscopy. Methods: One eye each of 37 patients with PEX and 20 age-matched healthy control subjects was evaluated by in vivo corneal confocal microscopy. Patients with PEX were further classified into two groups: those with and without hyperreflective endothelial deposits. We evaluated the densities of basal epithelial cells, anterior and posterior stromal keratocytes, and endothelial cells and structure of sub-basal nerve fibers. Results: The mean anterior and posterior stromal keratocyte and endothelial cell densities and corneal sub-basal nerve plexus variables were significantly lower in patients with PEX compared with those in healthy control subjects. The mean basal epithelial cell density did not significantly differ. Conclusion: Eyes with PEX presented decreased corneal sub-basal nerve plexus variables and cell densities in all corneas, except for the mean basal epithelial cell density. Further, a trend of lower corneal sub-basal nerve plexus measurements in patients with hyperreflective endothelial deposits compared with those without endothelial deposits was observed.
RESUMO Objetivo: Quantificar as alterações morfológicas nas fibras nervosas e nas células da córnea em pacientes com síndrome de pseudoexfoliação (PEX) e sua relação com a presença de depósitos endoteliais hiper-refletivos, observados por meio da microscopia confocal in vivo. Métodos: Trinta e sete olhos de 37 pacientes portadores de PEX e 20 olhos de 20 indivíduos saudáveis, pareados por idade, foram avaliados por meio da microscopia confocal de córnea. Os pacientes com PEX foram classificados em dois grupos: pacientes sem depósitos endoteliais hiper-refletivos e pacientes com depósitos endoteliais hiper-refletivos. Células basais epiteliais, ceratócitos do estroma anterior e posterior, e densidades celulares endoteliais assim como a estrutura das fibras nervosas sub-basais foram avaliadas. Resultados: A média de ceratócitos do estroma anterior e posterior, as densidades celulares endoteliais, bem como as variáveis de plexo nervo sub-basal foram significativamente menores nos pacientes com PEX em comparação com indivíduos saudáveis. A densidade celular epitelial basal não diferiu significativamente. Conclusões: Os olhos com PEX apresentaram diminuição das variáveis do plexo do nervo sub-basal e das densidades celulares em toda a córnea, exceto na camada basal das células epiteliais. Além disso, foi notada uma tendência para valores mais baixos nas variáveis do plexo do nervoso sub-basal em pacientes com depósitos endoteliais hiper-refletivos em comparação àqueles sem depósitos endoteliais.