shows typical, 5-wavelength spectra measured in the pilot group, including the sensitised, unsensitised and euthanised animals (50
). As expected for 1
luminescence, a clear 1270
nm peak was observed for the live sensitised animals. As will be demonstrated below, significant variability was observed both in terms of 1
generation and treatment response, even between animals receiving PDT under the same conditions. For this reason, representative data from a single sensitised animal are shown, as opposed to the average of the 1
luminescence observed from all of the live sensitised animals. A small but still significant peak at 1270
nm was observed in the unsensitised control animals, most likely due to 1
generated from naturally occurring porphyrins in the skin. No peak was observed at 1270
nm in the sensitised but euthanised animals, confirming the oxygen dependence of the signal. Hence, the system was capable of measuring 1
luminescence in this in vivo
Figure 2 Typical NIR spectra measured from single sensitised, unsensitised and hypoxic animals. For the sensitised animal, the individual spectra were measured at different times during treatment. For the control and hypoxic animals, error bars reflect the standard (more ...)
shows the average, cumulative 1
luminescence for sensitised animals treated with 15, 50 and 150
up to a total fluence of 50
, after subtracting the mean control (unsensitised) spectra at the same irradiances. The total 1
luminescence decreased with increasing irradiance. This trend was statistically significant between the three groups: specifically, one-tailed Student's t
-tests yielded P
=0.005 comparing the 15 and 50
treatments and P
=0.014 between the 50 and 150
treatments. Note that the error bars in represent the standard deviation between the six animals in each experiment. The error due to photon counting statistics was negligible compared to this systematic variability. The relevance of this observation is discussed below.
Similar curves were measured for all other treatment groups. summarises the mean total 1O2 luminescence observed in each group. As would be expected, the total 1O2 luminescence increased with radiant exposure (fluence) at a constant irradiance.
Total 1O2 luminescence as a function of total fluence for all treatment groups (means±1s.d. in six animals).
(inset) shows the skin scores for spots treated with 50
at varying irradiances, corresponding to the treatments in , as well as the unsensitised controls at 50
. The unsensitised animals had no observable response to light alone at 15 or 150
or to ALA alone (data not shown for brevity). For the sensitised animals, the skin response increased significantly with decreasing irradiance. This was statistically significant: P
=0.001 between 15 and 50
0.003 between 50 and 150
. This irradiance dependence was also observed by Robinson et al (1998)
, and similar effects have been reported by other authors in different in vitro
and in vivo
models (Feins et al, 1990
; Gibson et al, 1990
; Foster et al, 1993
; Sitnik et al, 1998
), and has usually been attributed to photochemical depletion of oxygen at high irradiances. The total skin scores as a function of fluence for all treatments are summarised in . These were defined as the sum of the 14 individual daily scores over the 2-week period in each case. Again, these are generally consistent with those obtained by Robinson et al (1998)
Total skin score (means±1s.d. in six animals) as a function of total fluence for all treatment conditions. Inset: skin score as a function of time following treatment in days (means±1s.d. in six animals).
A striking feature in , and is the relatively large variability in the measurements for nominally identical PDT treatments. As will be discussed, this variability was primarily due to animal-to-animal differences in ALA uptake and/or PpIX synthesis, local skin pO2 and relative photosensitivity of the skin, as opposed to true ‘experimental error'. The variability observed here was not atypically large, but serves to illustrate the difficulty inherent in predicting the outcome of PDT treatments based on administered light and photosensitiser dose alone.
The total skin score as a function of the total 1
luminescence for all treatments is shown in . There is a strong correlation with the 1
luminescence, regardless of the fluence or irradiance used. shows the individual data points that comprise , together with the best χ2
fit to a three-parameter sigmoidal curve (constrained to pass through the origin) of the form:
where TSS is the total skin score and L
is the total 1
luminescence measured. The use of this functional form for the response has no a priori
mechanistic basis at this time, but is a convenient way to summarise the data. This fit was performed with four outliers removed (dotted symbols in ) and this fit yielded A
=1.6 and C
000 and a reduced χ2
of 2.0 (χ2
/NDF=5.1 with all data points included).
Total skin score (means±1s.d.) as a function of total 1O2 luminescence (means±1s.d.).
Figure 7 Total skin score as a function of total 1O2 luminescence for all individual data points that comprise the figure. The curve is the fit to Equation (E) after removal of the four outliers (open points). The error bars indicate the assumed systematic uncertainty (more ...)