The effects of single doses of beta radiation on the wound healing behaviour of human Tenon's capsule fibroblasts.

AIM: To determine the effects of single doses of beta radiation on the wound healing functions of human Tenon's capsule fibroblasts (hTf). METHODS: hTf were grown in tissue culture and irradiated with beta radiation using a strontium 90 source. The effects of beta 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 beta radiation. RESULTS: Growth inhibiting doses of beta 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 beta radiation. Collagen I and III levels were not reduced after any dose of radiation, and increased following treatment with 1000 cGy beta radiation. CONCLUSIONS: Growth arresting doses of beta 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 beta 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.

Does frequency doubling technology perimetry reliably detect neurological visual field defects?

AIM: To determine the ability of frequency doubling technology (FDT) perimetry to detect visual field defects of neurological origin. METHODS: A total of 15 eyes of nine patients who all had complete hemianopias or quadrantanopias underwent the FDT 20-5 screening mode test and Humphrey 24-2 SITA Fast visual field test (HFA). The FDT results were scored according to the number of abnormal test locations (out of a maximum of 4) in each affected quadrant. FDT locations showing a defect of P< 2% were considered abnormal. RESULTS: Of the 15 eyes, six showed complete superior quadrantanopic and nine complete hemianopic field defects on HFA. Of 96 FDT test locations in these quadrants or hemifields only 38 were abnormal on FDT testing (40%). For the quadrantanopic field defects, five out of 24 locations were abnormal (21%). For the hemianopic field defects, 33 out of 72 locations were abnormal (49%). In three eyes (two with quadrantanopias and one with complete hemianopia), FDT perimetry failed to demonstrate any corresponding abnormality. CONCLUSIONS: The FDT screening test can fail to demonstrate complete hemianopic and quadrantanopic field defects. Users should be aware of this deficiency when using FDT to screen for field defects.

Beta irradiation: new uses for an old treatment: a review.

Beta radiation has a long history as a treatment modality in ophthalmology. It is a convenient and practical method of applying radiation and has the advantage of minimal tissue penetration. There has been a recent resurgence in the use of beta radiation in other areas in medicine, such as the prevention of restenosis after coronary artery stenting. Beta radiation has been shown in vitro and in vivo to inhibit proliferation of human Tenon's fibroblasts, which enter a period of growth arrest but do not die. Effects on the cell cycle controller p53 have been shown to be important in this process. In ophthalmology, beta radiation has been used widely for the treatment of pterygium and is under evaluation for treatment of age-related macular degeneration and for controlling wound healing after glaucoma drainage surgery. In this latter role, beta radiation may be particularly appropriate for use in developing countries to improve the results of trabeculectomy while potentially avoiding some of the side effects of other antimetabolites.

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. beta 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 beta radiation remains unclear. The effects of beta 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: beta 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 beta 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 beta radiation may be an effective treatment for the prevention of bleb failure as a result of prolonged growth arrest of Tenon's capsule fibroblasts.

Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts.

PURPOSE: To determine whether treatment with mitomycin-c and 5-fluorouracil induces apoptotic death in cultured subconjunctival fibroblasts. METHODS: Cultured human subconjunctival Tenon's capsule fibroblasts were exposed to 5-minute applications of mitomycin-C (up to 1 mg/ml) or 5-fluorouracil (up to 50 mg/ml) or phosphate-buffered saline solution (PBS). Fibroblast apoptosis was determined by cell morphology, apoptosis-specific protein expression, and DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL). In addition, apoptosis was quantified by direct cell counts based on morphology or lactate dehydrogenase release. RESULTS: Morphologic changes characteristic of apoptosis included nuclear and cytoplasmic condensation and occasional nuclear fragmentation while the plasma membrane remained intact. Apoptosis-specific protein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibroblasts. The amount of apoptosis induced was dose dependent and partially inhibited by the addition of fetal calf serum to growth medium immediately after treatment. CONCLUSIONS: Mitomycin-C and high-dose 5-fluorouracil induce apoptosis in cultured Tenon's fibroblasts. Mitomycin-C-induced apoptosis is inhibited by fetal calf serum, indicating that exogenous factors influence the susceptibility of a fibroblast population to apoptosis. The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.

Effect of varying the mitomycin-C treatment area in glaucoma filtration surgery in the rabbit.

PURPOSE: To investigate the effect of varying the treatment area of subconjunctival mitomycin-C (MMC) using an adapted rabbit model of filtration surgery. METHODS: Twenty-four New Zealand White rabbits underwent filtration surgery, with random allocation to one of three treatments: 5-minute subconjunctival applications of MMC (0.4 mg/ml) with either a large (8 x 10 mm) or small (4 x 2 mm) sponge or no treatment (control). Drainage was achieved by placing an intravenous cannula through a scleral tunnel into the anterior chamber. Rabbits were examined at set intervals for up to 30 days after surgery. Measurements of appearance, size, height, and vascularity of blebs and of intraocular pressure and anterior chamber depth were made by a masked observer. Histologic analysis of eyes was performed at 3, 14, and 30 days. RESULTS: Statistical analysis showed a significant difference in bleb survival among all groups (log rank P = 0.0054, with 100% survival with large areas of MMC treatment). Comparison between large and small treatment area groups revealed significant differences in bleb survival (log rank P = 0.0388); bleb area (between-subject analysis, P = 0.009), and bleb height (between-subject analysis, P = 0.005). These differences were seen clinically, with large areas of MMC treatment producing diffuse and elevated blebs, small areas of treatment producing thin-walled and localized blebs with scarring at 21 days, and no treatment resulting in comparatively vascularized and scarred blebs before 14 days. Histologic analysis revealed clear differences among groups, with an increase in subconjunctival cellularity and scar tissue in eyes with failed blebs. CONCLUSIONS: The size of the area of subconjunctival MMC treatment significantly affects surgical outcome. Histologic features mirror differences observed clinically. Alteration of the size of the MMC treatment area may provide an alternative and more controllable approach to modulating the wound-healing response after drainage surgery and, more important in the clinical context, to modifying bleb morphology.

Use of topical diclofenac in association with external ocular compression.

This study investigated the effect of pretreatment with topical diclofenac 0.1% on the changes in intraocular pressure (IOP) associated with use of an external ocular compression device. The IOP reduction in eyes receiving pretreatment was significantly greater than in controls after 40 minutes of compression, and the recovery in IOP after removal of the compression device was significantly reduced. We conclude that the IOP rise after such compression is largely prostaglandin mediated, and suggest that use of ocular compression devices is associated with marked intraocular prostaglandin release.

Extraocular compression prior to cataract surgery: time course of reduction and subsequent recovery of intraocular pressure.

This study investigated the rate and degree of reduction in intraocular pressure (IOP) obtained with an external ocular compression device. Following removal of the device, the subsequent recovery in IOP was monitored. We aimed to establish the time course of IOP changes, and thereby to optimise our use of such devices prior to cataract surgery. A rapid initial reduction over the first 10 minutes of compression was followed by a more gradual reduction to a mean reduction of 6.97 mmHg at 40 minutes. Recovery of IOP was rapid and complete by 20 minutes. We conclude that compression of up to 40 minutes duration is beneficial, and suggest such devices should be left on until immediately prior to surgery to preserve the reduction achieved.