Ph Constable

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

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.

The effects of single doses of β radiation on the wound healing behaviour of human Tenon’s capsule fibroblasts

Aim: To determine the effects of single doses of β radiation on the wound healing functions of human Tenon’s capsule fibroblasts (hTf). Methods: hTf were grown in tissue culture and irradiated with β radiation using a strontium 90 source. The effects of β radiation on fibroblast migration was studied using microporous transwell membranes. The effects of radiation on fibroblast contraction was investigated using a fibroblast populated collagen gels model. Production of extracellular matrix molecules (collagen I, collagen III, and fibronectin) by monolayers of irradiated fibroblasts was quantified for 14 days following single doses of β radiation. Results: Growth inhibiting doses of β radiation did not inhibit fibroblast migration or contraction at any time point. Levels of soluble fibronectin from irradiated populations were significantly reduced after >500 cGy β radiation. Collagen I and III levels were not reduced after any dose of radiation, and increased following treatment with 1000 cGy β radiation. Conclusions: Growth arresting doses of β radiation have unique effects on the wound healing behaviour of human Tenon’s capsule fibroblasts. There was no significant effect on cellular migration or contraction, but ECM production was altered. Fibronectin production was inhibited following higher radiation doses, and collagen I and III production increased after 1000 cGy. The effects of single doses of β radiation on ocular fibroblast wound healing behaviour are very different from those of 5-fluorouracil and mitomycin C, and these differences may be exploited clinically in the regulation of wound healing after glaucoma filtration surgery.