Characterization and prevention of phototoxic effects in intravital fluorescence microscopy in the hamster dorsal skinfold model

Intravital microscopy is widely used to study the microcirculation. However , the use of fluorescent dyes can induce phototoxic effects which may affec t the measurements, particularly in tissue exposed to oxidative stress, The aim of the study was to determine the threshold light dose at which fluore scent microscopy is associated with phototoxic effects in the hamster dorsa l skinfold chamber under normal and pathological conditions. The extent of phototoxicity in the microcirculation in the hamster skinfold chamber was i nvestigated using intravital fluorescent microscopy during 60 min of illumi nation (1048 mW/cm(2)) applying two different concentrations of fluorescein isothiocyanate dextran under baseline conditions (groups A and B and follo wing 4 h of ischemia (groups C and D). In the second part of the study the microvasculature was analyzed regarding phototoxic effects during a standar dized intravital microscopic examination after 4 h of pressure induced isch emia. Groups I and II (n=7) were studied using epiillumination after injection of fluorescein isothiocyanate dextran plus rhodamine 6G or rhodamine 6G only. In group LII (n=7) only transillumination was used. Arteriolar vasospasm, microvascular perfusion failure, thrombus formation, and enhanced leukocyte endothelium interaction were observed as signs of a phototoxic effect in n ormal tissue. However, the light doses needed to induce these effects clear ly exceeded those during standard examinations. The induction of a 4-h isch emia and reperfusion further enhanced these effects. Despite the predamage by ischemia/reperfusion the comparison of epiillumination and transillumina tion microscopy using a standard protocol showed no differences regarding t he parameters analyzed at any time. This indicates that epiillumination and the fluorescent dyes per se did not affect the experimental results. These results show that ischemia/reperfusion studies in the dorsal skinfold cham ber of the Syrian golden hamster can be carried out safely without the risk of inducing phototoxic effects by fluorescent microscopy. Nevertheless eve ry laboratory using epiillumination and fluorescent dyes should take precau tions to avoid these effects by the use of sensitive cameras to lower the l ight dose.