Da. Bellnier et al., THE VALIDATION OF A NEW VASCULAR DAMAGE ASSAY FOR PHOTODYNAMIC THERAPY AGENTS, Photochemistry and photobiology, 62(5), 1995, pp. 896-905
The therapeutic effect of photodynamic therapy (PDT: photodynamic sens
itizer + light) is partly due to vascular damage. This report describe
s a new vascular photodamage assay for PDT agents and a validation of
the assay. The method described here quantitates changes in tissue blo
od perfusion based on the relative amount of injected fluorescein dye
in treated and untreated tissues. A specially designed fluorometer use
s chopped monochromatic light from an argon laser as a source for exci
ting fluorescein fluorescence. The fluorescent Light emitted from the
tissue is collected by a six element fiberoptic array, filtered and de
livered to a photodiode detector coupled to a phase-locked amplifier f
or conversion to a voltage signal for recording. This arrangement perm
its a rather simple, inexpensive construction and allows for the simul
taneous use of the argon laser by other investigators. The routine ass
ay for characterizing a specific photosensitizer at a standard dose co
nsists of the sequential allocation of eight mice to a set of differen
t light doses designed to span the dose-response range of fluorescein
fluorescence exclusion (measured 8-10 min after fluorescein injection)
. The assay validation experiment used an anionic photosensitizer, 2-[
1-hexyloxyethyl]-2-devinyl pyropheophorbide-a at a dose of 0.4 mu mol/
kg. The parameter estimates (n = 34 mice) from fitting the standard Hi
ll dose-response model to the data were: median fluorescence exclusion
light dose FE(50) = 275 +/- 8.3 J/cm(2) and Hill sigmoidicity paramet
er m = -3.66 +/- 0.28. Subsets of the full data set randomly selected
to simulate a standard eight mice experiment yielded similar parameter
estimates, The new assay provides reliable estimates of PDT vascular
damage with a frugal sequential experimental design.