Determination of colony numbers in pig epidermis as an estimate for radiosensitivity. A rapid assay based on in vitro BrdU-labelling.

Abstract

Abstract. A rapid assay has been developed for the quantitation of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. The number of surviving clonogenic cells per unit area was related to the epidermal in vivo response of moist desquamation. After irradiation with single doses, ranging from 20 to 36 Gy, skin biopsies were taken and incubated in dispase for enzymatic separation of the epidermis and dermis. Full thickness epidermal sheets were labelled with bromodeoxyuridine (BrdU) in vitro. Proliferating cells were visualized using standard immunohistochemical procedures. Cell groups containing ¢16 cells were counted as colonies. These colonies were ®rst seen on day 14/15 after irradiation. The number of colonies per cm 2 , as a function of skin surface dose, yielded a cell survival curve with a D 0 (¡SE) of 3.87¡0.57 Gy. The ED 50 for the epidermal in vivo reaction of moist desquamation corresponded with a colony density of 2.7 colonies per cm 2 . After higher doses, abundant smaller colonies of 4±8 BrdUpositive cells were seen and these were more radioresistant, as represented by higher D 0 values.Radiation-induced epidermal injury of the skin is often assessed with the use of macroscopic endpoints such as erythema and moist desquamation. In pig skin these endpoints can be evaluated at 4±6 weeks after irradiation. However, the severity and incidence of this damage may be in¯uenced by environmental factors, such as infections, partially obscuring the dose±effect relationship. To avoid these possible confounding factors a more direct approach was used, by studying colony formation from surviving epidermal clonogenic cells. There is likely to be an inverse relationship between the incidence of moist desquamation and the survival of clonogenic cells. Colony formation can be used as an estimate of radiosensitivity [1±3]. In the past, several groups have attempted to determine radiation damage (more directly) using the number of colonies arising from surviving clonogenic cells as a parameter for epidermal radiosensitivity [4±7]. Various types of assays were used, such as (i) a macroscopic in vivo colony assay for rodent skin [5±7], (ii) autoradiographs of whole epidermal sheets for the detection of proliferating cells in mouse skin [4], (iii) crosssections of pig skin viewed by light microscopy [8], or (iv) blocking cells in mitosis followed by staining with Schiff's reagents using whole epidermal sheets from biopsies of pig skin [9]. A drawback of the macroscopic colony assay is the fact that smaller colonies are easily missed, while the use of autoradiographs and cross-sections is laborious and time consuming. The assay introduced by Chen et al [9] involved three injections of vincristine, resulting in the induction of a metaphase arrest, which allowed recognition of proliferating cells by the presence of metaphases. In this assay, separation of the epidermis from the dermis was accomplished by hydrolysis with 5 N HC1 followed by teasing apart with forceps.The objective of this pap...

Changes in epidermal radiosensitivity with time associated with increased colony numbers

“…More than 1000 BrdU-labelled cells were counted in biopsies from unirradiated skin in an area covering at least four ocular grids. The number of BrdU-positive cells was expressed per mm 2 .…”

“…The immunohistochemical procedure has been described in detail previously [2]. Briefly, biopsies were incubated in 0.5% dispase (Boehringer, Mannheim, Germany) to separate epidermis and dermis.…”

“…For this purpose, a quantitative assay was developed to determine colony formation in pig epidermis [1]. For our study, we adopted this assay and modified it using in vitro labelling with bromodeoxyuridine (BrdU) followed by standard immunohistochemistry [2]. In the past, various studies have been carried out to determine radiation damage to the epidermis using the number of colonies arising from surviving clonogenic cells as a parameter for radiosensitivity [3][4][5][6][7][8][9].…”