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. 1998 Apr;82(4):448–452. doi: 10.1136/bjo.82.4.448

Long term growth arrest of human Tenon's fibroblasts following single applications of β radiation

P Constable 1, J Crowston 1, N Occleston 1, M Cordeiro 1, P Khaw 1
PMCID: PMC1722552  PMID: 9640199

Abstract

AIMS/BACKGROUND—Antimetabolites are increasingly used to manipulate the healing response after filtration surgery, but problems with thin cystic blebs have been encountered with the liquid agents commonly used such as 5-fluorouracil and mitomycin C. β Radiation appears to be a useful adjuvant treatment for preventing scarring after trabeculectomy, resulting in diffuse rather than cystic bleb formation, but much of the basic cell biology of the ocular fibroblast response to β radiation remains unclear. The effects of β radiation on ocular fibroblast proliferation and cell cycling were investigated to determine the nature and duration of these effects on these cells.
METHODS—In vitro cell culture techniques were used to investigate fibroblast proliferation. Cell viability was studied using trypan blue dye exclusion. The effect of radiation on cell cycling was investigated using bromodeoxyuridine uptake. p53 expression was demonstrated using immunocytochemistry .
RESULTS—β Radiation inhibited fibroblast proliferation in a dose dependent manner. Early cell death was not a prominent feature, but irradiated fibroblasts demonstrated a rapid onset and sustained period of growth arrest. p53 expression was found to be increased in irradiated cells.
CONCLUSIONS—Single doses of β radiation significantly inhibit Tenon's capsule fibroblast proliferation in vitro over a 28 day period. This inhibition is the result of a rapid onset and sustained period of growth arrest in irradiated cells. Irradiated fibroblasts show an increase in p53 expression, a nuclear phosphoprotein which has been associated with control of the cell cycle. Single applications of β radiation may be an effective treatment for the prevention of bleb failure as a result of prolonged growth arrest of Tenon's capsule fibroblasts.

 Keywords: radiation; fibroblasts; growth arrest

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Figure 1  .

Figure 1  

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Effects of β radiation on fibroblast proliferation. Fibroblasts were grown in tissue culture, passaged, and used for proliferation experiments when in exponential phase growth. The effect of single doses of β radiation between 250 and 2000 cGy was investigated with control cells remaining unirradiated. At each time point, the mean number of human Tenon's fibroblasts (HTFs) in triplicate wells was determined for each dose of radiation studied. Single doses of β radiation significantly inhibited HTF proliferation relative to control cells at all time points (p<0.05 at each time point using one way ANOVA with Bonferroni's modification).

Figure 2  .

Figure 2  

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Cell cycling following β irradiation. Fibroblasts were cast into 96 well plates and allowed to settle overnight. Test wells were irradiated with 500, 1000, or 2000 cGy, and control cells remained unirradiated. At various time points following treatment, triplicate wells from each treatment group were pulsed in 1:1000 BRdU in DMEM/10% FCS for 24 hours, following which the incorporated BRdU was demonstrated by immunochemical antibody staining and DAB precipitation. The mean percentage number of labelled cells in each treatment group, together with the 95% confidence interval, was plotted at the various time points. Statistical analysis performed using a one way ANOVA test with Bonferroni's modification demonstrated that fibroblast proliferation was significantly greater in the control group than in all irradiated populations at all time points (p<0.05).

Figure 3  .

Figure 3  

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The effect of β radiation on p53 expression. (A) Irradiated fibroblasts at 24 hours (1000 cGy). Dark staining nuclei indicate positive p53 staining. (B) Unirradiated control fibroblasts at 24 hours. Bars = 50 µm.

Figure 4  .

Figure 4  

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Effects of β radiation on p53 expression. Monolayers of exponentially growing fibroblasts were prepared and treated as before. At each time point triplicate wells from each treatment group and control groups were fixed in 50:50 acetone/ethanol and then immunostained with Dako DO-7 anti-p53 antibody. Bound antibody was visualised using DAB immunoprecipitation and the percentage of positively staining cells was determined for each treatment and plotted against time.

Selected References

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