BackgroundTo objectively measure changes in nuclear morphology and cell distribution following induction of apoptosis.MethodsA spontaneously immortalized retinal pigment epithelial cell line (ARPE-19) was cultured for three days in DMEM/F12 with 10% fetal bovine serum followed by 24 hours incubation in staurosporine to induce apoptosis. Cells that were not incubated in staurosporine served as control. Caspase-3 expression in apoptotic cells was demonstrated by quantitative immunofluorescence. Nuclei were counterstained with DAPI. Assessments of nuclear morphology and cell distribution were performed using ImageJ software. Statistical analyses included Student’s t-test and Pearson’s correlation coefficient. Nearest neighbor analysis was used to assess cell nuclei distribution.ResultsCaspase-3 expression in staurosporine-incubated cells increased by 471% ± 182% compared to control (P = 0.014). Relative to the control, cells in the staurosporine-incubated cultures had smaller average nuclear area (68% ± 5%; P < 0.001) and nuclear circumference (78 ± 3%; P < 0.001), while nuclear form factor was larger (110% ± 1%; P < 0.001). Cell nuclei from the staurosporine-group (R = 1.12 ± 0.04; P < 0.01) and the control (R = 1.28 ± 0.03; P < 0.01) were evenly spaced throughout the cultures, thereby demonstrating a non-clustered and non-random cell distribution. However, the staurosporine-incubated group had a significantly lower R-value compared to the control (P = 0.002), which indicated a move towards cell clustering following induction of apoptosis. Caspase-3 expression of each individual cell correlated significantly with the following morphological indicators: circumference of the nucleus divided by form factor (r = -0.475; P < 0.001), nuclear area divided by form factor (r = -0.470; P < 0.001), nuclear circumference (r = -0.469; P < 0.001), nuclear area (r = -0.445; P < 0.001), nuclear form factor (r = 0.410; P < 0.001) and the nuclear area multiplied by form factor) (r = -0.377; P < 0.001).ConclusionsCaspase-3 positive apoptotic cells demonstrate morphological features that can be objectively quantified using freely available ImageJ software. A novel morphological indicator, defined as the nuclear circumference divided by form factor, demonstrated the strongest correlation with caspase-3 expression.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3271993311662947

The current study investigates whether microRNA (miRNA) regulators of epithelial-mesenchymal transition (EMT), tissue fibrosis, and angiogenesis are differentially expressed in human primary pterygium. Genome-wide miRNA and mRNA expression profiling of paired pterygium and normal conjunctiva was performed in the context of conventional excision of pterygium with autotransplantation of conjunctiva (n=8). Quantitative real time polymerase chain reaction (qRT-PCR) was used to validate the expression of key molecules previously detected by microarray. In pterygium, 25 miRNAs and 31 mRNAs were significantly differentially expressed by more than two-fold compared to normal conjunctiva. 14 miRNAs were up-regulated (miR-1246, −486, −451, −3172, −3175, −1308, −1972, −143, −211, −665, −1973, −18a, 143, and −663b), whereas 11 were down-regulated (miR-675, −200b-star, −200a-star, −29b, −200b, −210, −141, −31, −200a, −934, and −375). Unsupervised hierarchical cluster analysis demonstrated that members of the miR-200 family were coexpressed and down-regulated in pterygium. The molecular and cellular functions that were most significant to the miRNA data sets were cellular development, cellular growth and proliferation, and cellular movement. qRT-PCR confirmed the expression of 15 of the 16 genes tested and revealed that miR-429 was down-regulated by more than two-fold in pterygium. The concerted down-regulation of four members from both clusters of the miR-200 family (miR-200a/−200b/−429 and miR-200c/−141), which are known to regulate EMT, and up-regulation of the predicted target and mesenchymal marker fibronectin (FN1), suggest that EMT could potentially play a role in the pathogenesis of pterygium and might constitute promising new targets for therapeutic intervention in pterygium.

Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C–37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.

PurposeTo determine the measurement reliability of CorVis ST, a dynamic Scheimpflug analyser, in virgin and post-photorefractive keratectomy (PRK) eyes and compare the results between these two groups.MethodsForty virgin eyes and 42 post-PRK eyes underwent CorVis ST measurements performed by two technicians. Repeatability was evaluated by comparing three consecutive measurements by technician A. Reproducibility was determined by comparing the first measurement by technician A with one performed by technician B. Intraobserver and interobserver intraclass correlation coefficients (ICCs) were calculated. Univariate analysis of covariance (ANCOVA) was used to compare measured parameters between virgin and post-PRK eyes.ResultsThe intraocular pressure (IOP), central corneal thickness (CCT) and 1st applanation time demonstrated good intraobserver repeatability and interobserver reproducibility (ICC≧0.90) in virgin and post-PRK eyes. The deformation amplitude showed a good or close to good repeatability and reproducibility in both groups (ICC≧0.88). The CCT correlated positively with 1st applanation time (r = 0.437 and 0.483, respectively, p<0.05) and negatively with deformation amplitude (r = −0.384 and −0.375, respectively, p<0.05) in both groups. Compared to post-PRK eyes, virgin eyes showed longer 1st applanation time (7.29±0.21 vs. 6.96±0.17 ms, p<0.05) and lower deformation amplitude (1.06±0.07 vs. 1.17±0.08 mm, p<0.05).ConclusionsCorVis ST demonstrated reliable measurements for CCT, IOP, and 1st applanation time, as well as relatively reliable measurement for deformation amplitude in both virgin and post-PRK eyes. There were differences in 1st applanation time and deformation amplitude between virgin and post-PRK eyes, which may reflect corneal biomechanical changes occurring after the surgery in the latter.