The detection of phthalocyanine fluorescence in normal rat bladder wall using sensitive digital imaging microscopy

Aj Pope,D Phillips,Aj Macrobert,Sg Bown

doi:10.1038/bjc.1991.417

Abstract

The ability to detect photosensitisers in tissue at a microscopical level is important when studying photodynamic therapy (PDT) in both normal and malignant tissue. We have studied the fluorescence distribution of aluminium sulphonated phthalocyanine (A1SPc) in the normal rat bladder using a cooled CCD (charge coupled device) imaging system with computerised image processing. This system makes it possible to carry out a quantitative assessment of photosensitiser fluorescence in the various layers of the bladder wall. The highest fluorescence intensities were obtained within 1 h of intravenous administration but there was little selectivity of uptake between layers. A1SPc was eliminated from the deeper muscle layers more quickly than from the superficial layers of the bladder wall so that by 24 h a 4:1 ratio of fluorescence intensity was apparent which persisted at least until 72 h, although the absolute amount of photosensitiser declined. Following irradiation by red light (675 nm), photobleaching of the sensitiser in the deeper layers further increased this ratio. Direct absorption of A1SPc by the bladder wall following intravesical administration proved unreliable.

Highlights

Fluorescence images are shown from representative sections of normal rat bladder together with the corresponding haematoxylin and eosin (H&E) stained slides
By 24 h after sensitisation a more marked gradient of between 3.5 and 4 had developed between the aluminium sulphonated phthalocyanine (AlSPc) fluorescence intensity in the superficial vs muscle layers (Figure 3), and this differential was maintained at 72 h. This gradient was due to more rapid elimination from the muscle layers; fluorescence readings from muscle were 45 ± 5% at 24 h and 40 ± 5% at 72 h of their value at 1 h compared with levels in the superficial layers of 70 ± 10% and 62 ± 8% respectively
We found the uptake of intravesical AlSPc mixture to be patchy and unpredictable which mirrored the results in our animal photodynamic therapy (PDT) studies (Pope & Bown, 1991)

Summary

Methods

Aluminium sulphonated phthalocyanine (AISPc) was obtained from Ciba-Geigy and used after dissolving in 0.9% saline. This preparation is a mixture of molecules containing on average three sulphonate groups. Normal bladder tissue was obtained from female Wistar rats (approximately 200 g) which received varying concentrations (0.5-5 mg kg-') of AlSPc (mixture) by tail vein injection. At intervals after injection the animals were sacrificed and the bladder catheterised with a fine cannula so that any urine which might contain photosensitiser could be gently washed out. Their bladders were distended to 0.3 ml with OCT

Results

Discussion

Conclusion