Influence of temperature on corneas stored in culture medium. A comparative study using functional and morphological methods.

PURPOSE: To investigate the influence of storage temperature on corneal swelling and on endothelial morphology in cultured corneas. MATERIAL AND METHODS: Forty-eight rabbit corneas were separated into four groups of 12. The corneas were stored in culture medium at 37 degrees (group 37), 34 degrees (group 34), 31 degrees (group 31) and 23 degrees (room temperature) (group 23), respectively. All the corneas were monitored by weight recordings on days 0, 1, 2, 3, 4 and 7. On day 7, corneas were prepared for scanning electron microscopy and endothelial cell counts were performed. RESULTS: Lowering the temperature of the culture medium resulted in less swelling. Both temperature and storage time had significant effects on corneal swelling (p < 0.001). On day 7, the observed mean weight increase was 131.2%, 143.0%, 172.5% and 199.7% in groups 23, 31, 34 and 37, respectively. The estimated mean daily weight increase for the corneas were 2.6%, 4.0%, 9.1% and 16.0% in groups 23, 31, 34 and 37, respectively. Scanning electron microscopy showed an intact endothelial layer in all groups after 7 days and there were no statistically significant differences in endothelial counts between groups 23, 31 and 34. In group 37, the cell borders were difficult to distinguish after 7 days and no meaningful count could be performed. CONCLUSIONS: The swelling rate of cultured corneas is significantly less at 23 degrees and 31 degrees than it is at 34 degrees and 37 degrees during the first week. This is most likely the result of a greatly increased barrier effect at lower temperatures. Whereas weight recording revealed profound differences between the groups, scanning electron microscopy and endothelial cell counting did not. The results support the hypothesis that storage at 37 degrees is not optimal in culturing corneas. Lowering the temperature below body temperature, and even lower than 31 degrees, results in less corneal swelling.

Induction of antiproliferative effects in HaCaT cells by a synthetic hydroperoxide.

The effects of decylhydroperoxide (DHP) on proliferation were assessed in HaCaT keratinocytes. DHP, a model compound of lipid peroxidation, reduced proliferation dose dependently. The analyses of cell number, DNA synthesis and cell cycle distribution suggest a delayed progression through S-phase of DHP-treated HaCaT cells. It has been proven that necrotic and apoptotic cell death were also causes for the DHP-induced decrease in proliferation. Moreover, in HaCaT cultures treated with the highest DHP concentration, apoptosis occurred to a much greater extent thus playing an overproportional role in the decline in living cell number. Results with the radical scavenger ascorbic acid and the iron chelator deferoxamine support the conclusion that an iron-catalyzed radical formation from DHP may induce the DHP-mediated reduction in proliferation.

G2/M checkpoint is p53-dependent and independent after irradiation in five human sarcoma cell lines.

To determine the role of p53 in G2/M arrest, G2/M transition and apoptosis, we investigated five human sarcoma cell lines with different p53 gene status in their response to X-rays. The p53 status of the cell lines was mutant (US 8-93, LMS 6-93 and RD), null (SAOS-2) and wildtype (A-204). Clonogenic survival of the cell lines varied as the survival fraction at 2 Gy (SF2) ranged from 0.28 to 0.79. Compared with the mutated p53 cell lines (SF2 with a range from 0.46 to 0.79) the clonogenic survival of the wildtype p53 (wt-p53) cell line A-204 (SF2 = 0.34) was lower. The p53 null cell line (SAOS-2) was also sensitive to X-rays (SF2 = 0.28). We detected, in all cell lines a similar behavior in their response to irradiation with G2/M arrest and apoptosis. However, the maximal rate of apoptosis with a range from 7.0 to 18.0% was rather small. The decrease of G2/M cells was coupled with an increased percentage of apoptotic cells. However, a different delay in G2/M did not result in a change of radiation sensitivity. Western analyses showed an increased P53 level only for the cell line A-204 (wt-p53) after irradiation. Our results point out that there is not always a simple relationship between p53 gene status and radiation sensitivity. We suggest, that wt-p53 plays an active role in G2/M arrest and in decreasing the number of G2/M cells as a response to apoptosis. Therefore, p53-dependent regulation in G2/M may be as important as p53-independent mechanisms.

Radiation induced G2/M block and apoptosis in two human sarcoma cell lines with different p53 gene status.

We investigated a wild-type (wt) p53 rhabdomyosarcoma (A-204) and a mutated (mt p53) undifferentiated sarcoma cell line (US8-93) for their response to X-rays. The observation period was 0 to 96 h after irradiation. Both cell lines showed a strikingly delayed G2/M arrest and an induction of apoptosis after irradiation. Compared with the cell line A-204 (wt p53), the cell line US8-93 (mt p53) revealed a stronger G2/M arrest. In agreement with this, in terms of viability as well as the rate of apoptosis, A-204 (wt p53) showed a stronger response to irradiation than US8-93 (mt p53). We suggest that the different p53 gene status might be the cause for a different response to irradiation.