P. B. Boulos

The Contrasting Mechanisms of Colonic Collagen Damage Between Photodynamic Therapy and Thermal-Injury

Abstract The colon is protected from disruption and bursting pressures by the submucosal collagen layer. Photodynamic therapy with aluminium sulphonated phthalocyanine (AlSPc) does not cause perforation or reduction in the bursting strength of the rodent colon despite causing full thickness damage. Thermal injury also produces full thickness necrosis but causes perforation and considerably reduces the bursting strength of the colon. The differing mechanisms of damage were examined. Using transmission electron microscopy we examined collagen from undamaged normal rodent colon, colon damaged by photodynamic therapy and thermally injured colon. Following photodynamic therapy collagen maintained its architecture and periodicity. Thermally damaged collagen became grossly swollen and lost its fibrillary architecture. We have concluded that photodynamic therapy with AlSPc is collagen sparing.

Photodynamic therapy in the normal rat colon with phthalocyanine sensitisation

Photodynamic therapy (PDT) involves the interaction of light with an administered photosensitising agent to produce cellular destruction. It has promising potential for the local and endoscopic treatment of gastrointestinal cancer. There is however little data on the response of normal intestine to PDT. We have investigated the use of a new photosensitiser chloro aluminum sulphonated phthalocyanine (AlSPc) for colonic PDT. The peak concentration of AlSPc in the colon measured by alkali extraction occurred 1 h after i.v. injection. The cellular uptake demonstrated by laser fluorescence microscopy was greater in the mucosa than in the muscle. AlSPc was activated in the tissues by light from an argon ion pumped dye laser at 675 nm. The laser power was set at 100 mW and the fibre placed touching the mucosa. In control animals no macroscopic damage was seen. Temperature measurement using a microthermocouple array showed no temperature rise during light exposure. The energy (fluence), dose of sensitiser and time from sensitisation to phototherapy were altered and the area of necrosis measured. The geometry of the colon made theoretical analysis of the correlation between laser energy and size of lesion difficult. However, following direct measurement of the relative light intensity (fluence rate) in the colon we were able to confirm that there was a threshold fluence for colonic necrosis. The area of photodynamic damage seen 72 h after phototherapy fell with the fall in tissue concentration of AlSPc from 1 h to 1 month after i.v. injection. However, maximum tissue necrosis occurred when treatment was performed immediately after i.v. injection. In this situation, intense vascular spasm was seen and any light transmitted through the colon which fell on the small bowel mesentery caused a lethal ischaemic necrosis. The initial histological changes after PDT were vascular, followed by full thickness necrosis at 72 h. Healing by regeneration was complete by 2-3 weeks. Despite full thickness necrosis there was no reduction in the colonic bursting pressure at any time. Colon treated by hyperthermia had a reduced bursting pressure. Specific collagen stains showed that PDT did not alter the submucosal collagen architecture whereas hyperthermia did.

The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs), is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumour cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

COMPARISON OF DISTRIBUTION AND PHOTODYNAMIC EFFECTS OF DI- AND TETRA-SULPHONATED ALUMINIUM PHTHALOCYANINES IN NORMAL RAT COLON

Abstract— —We have previously reported photodynamic therapy of normal rat colon using aluminium sulphonated phthalocyanine (AlSPc). In that study, the AlSPc used was a mixture of phthalocyanines of different degrees of sulphonation. Phthalocyanines of defined degrees of sulphonation have recently become available and we compared the distribution of the di‐ and tetra‐sulphonates (AlS2Pc and AlS4Pc) in rat colon and colon wall structures employing both chemical extraction and fluorescence photometry using a charge coupled device imaging system. Also, the photodynamic effects produced by these components in rat colon were compared at various times after photosensitization. After intravenous photosensitizer administration using equimolar doses, the concentration of AlS2Pc in colon fell off more rapidly with time than AlS4Pc. Differences were noted in the microscopic distribution of these compounds, with the di‐sulphonate exhibiting peak fluorescence in colon wall structures by 1 h after photosensitization, while mucosal fluorescence with the tetra‐sulphonate peaked at 5 h. Fluorescence was also lost from the colon wall much more slowly with the tetra‐sulphonate, which tended to be retained in the submucosa. Maximum photosensitizing capability was seen at 1 h with AlS2Pc and no lesions could be produced with photodynamic therapy at 1 week, with up to 5.65 μmol/kg. With AlS4Pc (5.65 μmol/kg), while no lesions could be produced with light treatment at 1 h, photodynamic therapy at 1 week produced lesions only slightly smaller than those produced with treatment at 48 h (the time of maximum effect), and significant photosensitization was present at 2 weeks. It is proposed that differences between AlS2Pc and AlS4Pc with respect to pharmacokinetics, microscopic distribution and qualitative (related to the timing) and quantitative differences in photodynamic effects in normal rat colon are related to physico‐chemical characteristics of the phthalocyanines used, particularly lipid solubility. Similar studies are now required on tumour bearing animals.

Comparison of lasers for photodynamic therapy with a phthalocyanine photosensitizer

Three different lasers were compared under the same conditions for their effectiveness at producing photodynamic damage to normal colon following sensitization with aluminium sulphonated phthalocyanine (AlSPc). One laser was an argon ion pumped continuous wave (CW) dye laser and the other two were pulsed at 10 kHz (copper vapour laser pumped dye laser, and 5 Hz (flashlamp pumped dye laser). The CW and 10 kHz laser were equally effective at producing damage. The 5 Hz laser failed to produce a photodynamic effect, although occasionally caused a photomechanical effect when the laser fibre was placed touching the colonic mucosa. Quantitative analysis suggests that the high energy pulses of the flashlamp pumped dye laser saturate AlSPc, so very little of the available energy can be used to produce a photodynamic effect, in contrast to the two other lasers which do not produce saturation conditions.

The significance of the nature of the photosensitizer for photodynamic therapy: quantitative and biological studies in the colon.

Photodynamic therapy (PDT) depends on the interaction of light with an administered photosensitiser to produce a local cytotoxic effect. The most widely used photosensitiser is haematoporphyrin derivative (HpD), but newer photosensitisers such as aluminium sulphonated phthalocyanine (A1SPc) are promising. HpD and A1SPc have been compared as photosensitisers for colonic PDT in the rat. Quantitative analysis showed that following injection of a standard photosensitiser dose, A1SPc produced more damage than HpD with increasing energy (fluence). Alteration of the injected dose of photosensitiser did not produce a clear difference. There was a loss of reciprocity for photosensitiser/light combinations at low injected dose (0.5 mg kg-1), both HpD and A1SPc producing no damage. Similarly at high photosensitiser dosage (25 mg kg-1) there was no quantitative difference between A1SPc and HpD. Photosensitiser photodegradation at low photosensitiser doses, and light attenuation by high tissue concentrations of A1SPc account for these findings. PDT with either agent produced the same histological damage and full thickness necrosis produced no mechanical weakening of the colon measured by the bursting pressure. The submucosal collagen was preserved and healing was by regeneration.