Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.

We assessed the effect of irradiation on COX-2 expression in blood vessels of the mouse bladder wall during the early and late radiation response phases. Vasodilatation was quantified as an additional marker of inflammation related to COX-2 activity. Female C3H/Neu mice were irradiated with a single dose of 20 Gy. The intensity of the COX-2 immunohistochemical staining signal was assessed using an arbitrary semiquantitative score of 0 to 3. To evaluate vasodilatation the percent of the bladder wall covered by the lumen of the blood vessels, depicted as vascular area, was determined. Constitutive COX-2 expression was found in the tunica intima and media of bladder blood vessels. During the early response significant biphasic changes were detected in the COX-2 staining signal as well as in the vascular area with peak values on days 4 and 16 after irradiation. A significant association was found between vascular area and COX-2 expression in blood vessels during the early radiation response (p <0.0001). No changes in COX-2 expression were observed during the late phase, that is between days 90 and 360 after irradiation. Minor vasodilatation seen during the late phase did not correlate with COX-2 activity. Irradiation resulted in pronounced COX-2 dependent inflammatory changes in the bladder wall during the early but not during the late radiation reaction. Therefore, a potential effect of COX-2 inhibition on early radiation side effects in the bladder can be proposed.

Complementary medicine (CM) use is common among cancer patients. However, little is known about CM products that are utilised during radiotherapy and/or chemotherapy. Out of 62 cancer patients who completed a specialised survey, 35 (56%) consumed some type of CM during active anti-cancer therapy. Cancer patients reported the use of herbal teas (52%), vitamins and other dietary supplements (45%), vegetables and juices (39%), special diets (19%), herbal medicines, including Chinese medicines (19%) and 'immunomodulators' (3%). Most of patients (86%) consumed CM products every day. However, nearly 47% of CM users did not admit this to their oncologists. Majority of CM users (85%) were convinced that supplementary products increase the efficacy of standard anti-cancer therapy and prolong their survival. Information about CM was mainly obtained through internet sources (36%), books and brochures (25%). Although most CM users (82%) trusted the received information, 73% of them admitted that additional information about CM methods would be necessary. Patients would like to receive additional information through a specialised consultation (60%), but also from brochures (44%) and the internet (20%). Adequate counselling of patients is of paramount importance since some CM methods may cause significant side effects and decrease the efficacy of radiotherapy and/or chemotherapy.

Purpose: We identified changes in vascular barrier function in relation to collagen deposition during the late radiation response of mouse bladders. In this study albumin leakage was assessed as a marker of blood vessel barrier disruption. Materials and Methods: Female C3H/Neu mice were irradiated with a single dose of 20 Gy and sacrificed for (immuno) histological studies after 90, 120, 180, 240 and 360 days. The fractional area of subepithelial tissue with extravascular albumin signal was defined. Moreover, the amount of collagen was determined after Masson’s trichrome staining on an arbitrary score of 0 to 3. Results: Subepithelial albumin infiltration due to leakage from the blood vessels was seen in irradiated animals during the entire late phase. It was most pronounced at days 120 and 180 after treatment (vs control p <0.0001). Similarly an increase in collagen deposition in the bladder wall was seen during the whole late radiation phase but with increasing values toward the end of the experiment (day 360 vs control p <0.0001). Conclusions: Irradiation of the bladder results in late damage to the barrier function of small subepithelial blood vessels, causing pronounced albumin leakage at the early steps of the chronic radiation reaction, clearly preceding excessive collagen deposition in the bladder wall. Current data suggest a vascular component in the development of late radiation induced changes in the bladder.

The aim was to assess the effect of irradiation on intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells of vessels in mouse urinary bladder and to compare endothelial ICAM-1 expression with changes in bladder function (storage capacity) during the early and late radiation response phases. Female C3H/Neu mice were irradiated with doses of either 20 or 0 Gy. For assessment of ICAM-1 expression, which was measured by the intensity of the immunohistochemical staining signal in bladder endothelium, an arbitrary semiquantitative score (0 – 3) was applied. Bladder storage function was assessed by transurethral cystotonometry. A positive functional radiation response, defined as a reduction in bladder capacity by > 50%, between days 0 and 15 or 16 and 30 was found in 40 and 64% of the animals, respectively. A late functional response was observed in 71% of the animals sacrificed after day 180. Minor constitutive expression of ICAM-1 was observed in bladder endothelial cells. After irradiation, an increase in staining signal by day 2, with a maximum on day 4, and on days 16 – 28 was found, which preceded the functional radiation effects. A permanent increase in ICAM-1 staining signal was observed in the late phase on top of an age-related rise. ICAM-1 expression was significantly higher in animals with a positive late response on day 90, i.e. during the initial late phase. Irradiation induces significant early and chronic variations in ICAM-1 expression in bladder endothelium, which preceded the functional response. This suggests that endothelial ICAM-1 is involved in the pathogenesis of both the early and late phases of radiation-induced urinary bladder effects.