PHOTODYNAMIC THERAPY OF ARTERIES: PRESERVATION OF MECHANICAL INTEGRITY

William E Grant, Colin Hopper, Giovanni Buonaccorsi, Paul M Speight, Alexander MacRobert, Kathy Fan, Stephen G 
Bown

National Medical Laser Centre, Department of Surgery, University College London Medical School, The Rayne 
Institute,
5 University Street, London WC1E 6JJ

ABSTRACT

Photodynamic therapy (PDT) of tumours, as a primary treatment or as an adjunctive intra-operative therapy, may 
expose major vascular structures to injury. PDT has also been proposed to prevent neointimal hyperplasia following 
angioplasty of stenotic arteries. This study aimed to determine the effect of PDT on the normal rabbit carotid artery, 
and to determine whether this injury resulted in weakening of the vessel wall. PDT of the carotid arteries of NZW 
rabbits, using either disulphonated aluminium phthalocyanine, or 5-aminolaevulinic acid induced protoporphyrin IX, as 
photosensitisers, was performed using a light dose of 100J/cm2. Histological examination of the carotids treated with 
both drugs demonstrated full thickness loss of cellularity 3 days following photodynamic therapy. Treated vessels all 
remained patent and no inflammatory infiltrate was observed. Elastin van Gieson staining showed preservation of inner 
and medial elastic laminae and medial and adventitial collagen. Further rabbits were similarly treated with PDT to I cm 
segments of both common carotids and sacrificed at 3, 7, and 21 days. The carotids were exposed and control and 
treated segments subjected to intraluminal hydrostatic distension until the vessels ruptured. No reduction in the 
pressure re~guired to rupture the vessels was evident in treated vessels compared with controls. It is concluded that in 
spite of full thickness cell death, PDT treated arteries are not at risk of thrombotic occlusion or haemorrhage.

Keywords: photodynamic therapy, arteries, mechanical integrity, aminolaevulinic acid, phthalocyanine

2. INTRODUCTION

PDT tumour destruction has resulled in fatal carotid haemorrhage in two cases,lZ2 and in major blood loss from a 
colorectal
carcinoma3. As direct invasion of large vessels by tumour in these cases is quite likely to have occurred, it is necessary 
to confirm the safety of PDT when used on tumours adjacent to major arteries. Intra-operative adjunctive PDT may 
offer the
prospect of decreased local recurrence due to the ablation of microscopic residual malignant disease495. Such 
applications may be of value for example in the treatment of the operative field following radical neck dissection for 
locoregional metastatic spread from upper aerodigestive tract malignancies. The normal carotid artery and its major 
branches would potentially be at risk in such procedures. Blood vessels have been shown to be susceptible to 
photodynamic injury. Star
observed capillary haemorrhage in rat ear observation chambers following PDT using haematoporphyrin derivative6 
and microvascular injury is accepted to be a major feature of PDT necrosis. In larger vessels, characteristic loss of 
endothelium and medial smoolh muscle cell death have been demonstrated7~9.

The possibility of weakening of larger vessel walls with loss of mechanical integrity and haemorrhage is a potential 
catastrophic outcome. The objective of this study was characterise the injury sustained by rabbit carotid arteries 
exposed to photodynamic injury, and to determine if this injury resulted in weakening of the vessel wall as detected by 
intraluminal hydrostatic distension to bursting poinL

3. MATERIALS AND METHODS:

New Zealand White rabbits were injected with either 200mg/kg aminolaevulinic acid (ALA) or lmg/kg disulphonated 
aluminium phthalocyanine (AlS2Pc). They were then anaesthetised, had their necks shaved and incision and dissection 
performed to expose the common carotid arteries in the paratracheal gutter on both sides. The carotids were gently 
dissected and optically isolated from surrounding structures by placing a piece of foil-lined opaque card deep to them. 
A I cm length of each artery was then exposed, at one hour for the AlS2Pc group and three hours for the ALA group, to 
100J/cm2 laser light at 630nm. We have previously demonstrated arterial injury in rat femoral arteries using both 
AlS2Pc and ALA induced PPIX9. In this study preliminary fluorescence quantification studies demonstrated peak 
sensitiser presence at one hour following intravenous administration of AlS2Pc and three hours following ALA.

Light was delivered by surface illumination, using a 400 micron fused silica optical fibre with a microlens attachment. 
Light doses of this order represent typical tumouricidal dosesl ~ll. A power density of 127mW/cm2~ below the 
threshold for thermal injury as confirmed in the control experiments described below was used, and each treatment 
lasted 785 seconds, the time required to deliver a total dose of 1001/cm2. The treated segments were marked by the 
placing of two 5:0 silk sutures in the adjacent fascia of the carotid sheath, care being taken not to damage the adventitia 
of the vessels. Following PDT the incisions were closed, the animals were recovered, and returned to their cages. No 
animal suffered any obvious adverse effect from the therapy.

To confirm that photodynamic therapy produced a vascular injury, four rabbits, two from each drug group, were 
sacrificed at three days, the carotids removed, fixed in buffered formaldehyde, and sectioned transversely for 
haematoxylin and eosin (HtE) and elastin van Gieson (EVG) staining. Sections were taken from treated segments and 
also from adjacent segments not exposed to light, to act as "drug + dissection but no light" controls. Two further rabbits 
had identical light irradiation but without prior photosensitisation to act as light only controls, and were also killed at 
three days.

A further eighteen rabbits underwent identical treatment, nine with AlS2Pc and nine with ALA. Three rabbits from 
each drug group were sacrificed on day 3, day 7 and day 21. The neck incisions were re-opened and the carotids 
re-exposed and the treated segments identified. A 4:0 silk ligature was tied at the midpoint of the treated segment, 
identified by the sutures placed at the time of treatment and by a slightly blanched appearance of the vessel. A second 
ligature was loosely placed just inside the marker for the end of the treatment segment. An incision was then made in 
the untreated artery wall about half a centimetre away from the treated segment, a blunted 18 gauge butterfly needle 
inserted to lie within the the treated zone, and the segment irrigated gently with normal saline. The loose ligature was 
then tied so that the needle tip was isolated in the half centimetre of treatment segmenL The fluid filled butterfly 
needle and tubing was connected to one "leg" of a three way tap. The remaining legs were connected to a pressure 
transducer capable of measuring pressures up to 10bar (7600mmHg) and to the output of a water filled pressurised 
vessel. The pressure transducer was connected a +IOV stabilised power supply and to a chart recorder and digital 
voltmeter. The input to the pressure vessel was connected to a compressed air cylinder through a manual pressure 
regulator capable of delivering up to 16bar (12000mmHg). When the butterfly needle was appropriately located and 
fixed in the prepared section of artery, the three way tap was rotated to the position shown and the gas supply opened. 
The pressure was manually increased until the isolated segment of vessel ruptured, usually after a period of several 
seconds. Rupture was indicated by a fluid leak and sudden dramatic fall in pressure which was noted and recorded on 
the flat bed recorder. The voltmeter output was monitored for cross-comparison with the reading from the Ratbed 
recorder. This procedure was repeated for the other half of the treated segment, and for proximal and distal normal 
segments of the same artery to act as controls. The whole procedure was repeated on the contralateral treated and non 
treated segments of carotid giving four treatment values and four controls per rabbit. Mean values for treated and 
control segments were calculated. Bursting pressure measurements from each treatment group were compared with 
controls and statistically analysed using Student's t-test.

4. RESULTS

All rabbits tolerated the treatment satisfactorily and showed no ill-effects in the post-operative period. On exposure of 
the treated and control arteries no evidence of dilatation, aneurysm formation or obvious diminished blood flow could 
be determined. Macroscopically the treated sections appeared a little paler than adjacent untreatred segments.

The treated arteries sampled at three days all demonstrated evidence of characteristic PDT injury. The same injury was 
observed in each drug group on HOE staining and comprised loss of endothelium and evidence of complete cell death 
throughout the media and adventitia. The adjacent segments of artery which had been sensitised and dissected but not 
exposed to laser light showed normal arterial features with an intact and cellular intima, media and adventitia. EVG 
staining for elastin and collagen demonstrated no discernible difference between treatment and control arteries. Both 
showed normal configuration of collagen in the media and adventitia as well as an intact inner elastic lamina and 
medial elastic laminae.

Control arterial segments (drug plus dissection but no light) were found to burst at a mean of 5200 mm Hg (+/- SD 990 
mm Hg.) There was no significant difference in the hydrostatic pressure required to burst treatment or control groups at 
three days. At 7 and 21 days however, considerably greater pressure was required to burst the treated segments than the 
controls in both groups. The pressure required to burst all vessels was vastly supra-physiological, being in the order of 
50 to 70 times that of arterial blood pressure. Both control and treated arteries tended to rupture in a linear split along 
the length of the isolated segment.

5. DISCUSSION

The histological effects of PDT using these sensitisers was as expected, with loss of endothelial cells from the intima 
and a complete loss of cellular structures in the media. Similar findings have been previously documented by ourselves 
and others7~9, in rat arteries. As well as confirming a similar response in the larger rabbit carotid, it was necessary to 
confirm that the timing of light exposure was appropriate for this model, and that the injury was circumferential and 
not just sustained by the surface of the vessel on which the light was directly incidenL All treated segments 
demonstrated that full circumferential injury was caused by the treatment parameters chosen. Control "drug + 
dissection only" groups, and "light only" groups demonstrated preservation of a normal cellular intima, media, and 
adventitia. The fact that bilateral common carotid PDT did not result in thrombotic occlusion is evidenced by the 
failure of any treated rabbit to show signs of stroke. Furthermore no histological evidence of mural thrombosis was 
found. The main purpose of this study was to determine and document any weakening of the vessel wall in the early 
weeks following PDT. This study demonstrates that in spite of full thickness cell death induced by PDT in the vessel 
wall no weakening or loss of mechanical integrity took place. It is not unreasonable to suppose that, in the presence of 
such extensive necrosis, an inflammatory response might be observed, and that the release of inflammatory mediators 
might contribute to weakening of the vessel wall. We have previously suggested that PDT in this situation may provoke 
a form of programmed cell death similar to apoptosis in which the inflammatory response is absent9. This would help 
explain the preservation of mechanical integrity of the vessels in this study. Furthermore it is apparent that PDT does 
not result in the degradation or denaturation of collagen and elastin12 and this is likely to be a contributing factor in 
functional preservation. Barr et al, in studies in PDT treated rat colon, and Smith et al, in studies in rat trachea have 
simlarly shown preservation of mural collagen and no reduction in the hydrostatic pressure required to rupture the 
treated organ13Xl4. A possible contributory factor is that PDT brings about cross-linking of collagen, through singlet 
oxygen reactions, which increases its resistance to deformation15~l7. The increase in pressure required to burst the 
vessels at 7 and 21 days might be explained further by the repopulation of the media, and to a lesser extent the intima, 
by proliferating smooth muscle cells, resulting in a minor degree of neointimal hyperplasia.

The photosensitising agenus used here offer advantages over currently used drugs such as Photofrin. Phthalocyanines 
absorb light at a longer wavelength than Photofrin, thus allowing greater penetration of tissues, and are less readily 
activated by sunlight thus diminishing cutaneous photosensitivity18. ALA induced protoporphyrin IX is cleared within 
approximately 24 hours of administration thus virtually eliminating the prolonged skin photosensitivity associated with 
Photofrin. Both ALA

and AlS2Pc have potential applications in tumour therapy. It is reassuring that major vessels, once photosensitised and
exposed to light will not rupture or thrombose. This has significance for the use of PDT in the treatment of tumours 
occurring in proximity to major vessels, as may occur in the head and neck, and also in the intraoperative adjunctive 
use of PDT to ablate microscopic residual disease. Such applications as the treatment of the neck field immediately 
following radical neclc dissection, or the illumination of the peritoneal cavity following resection of lower GI tumours, 
may reduce the incidence of recurrence following surgery.

A further potential application of PDT is in the treatment of vascular stenotic lesions, either on its own, or following
angioplasty. Porphyrins have been shown to selectively locate in atherosclerotic plaquesl9~22, and PDT has been 
proposed to treat such stenoses. Similarly following angioplasty, myointimal proliferation is responsible for re-stenosis 
in up to 50gs of cases. PDT ablation of medial smooth muscle cells has been shown to prevent early restenosis in 
experimental
models8w23f24. The preservation of mechanical integrity is obviously of vital importance in this situation.

6. CONCLUSION

This study demonstrates that in spite of full thickness vascular mural cell death following PDT using ALA or AIS2Pc, 
treated vessels do not undergo mechanical weakening of the wall. PDT is thus likely to be a safe treatment modality 
from the point of view of injury to major vessels in the treatment field.

7. ACKNOWLEDGEMENTS

Funding for this project was received from the Association for International Cancer Research. SG Bown acknowledges 
support from Imperial Cancer Research Fund. [

8. REFERENCES

1. Schuller DE, McCaughan JS, Rock RP. "Photodynamic therapy in head and neck cancer." Arch Otolaryngol 1985; 
111: 351 -357

2. Gluckman JL. "Hematoporphyrin photodynamic therapy: Is there truly a future in head and neck oncology? 
Reflections on a 5-year experience. " Laryngoscope 1991; 101: 36-42.

3. Barr H. Krasner N. Boulos PB, Chatlani P. Bown SG. "Photodynamic therapy for colorectal cancer: a quantitative 
pilot study" Br J Surg 1990; 77, 93-96.

4. Allardice JT, Abulafi AM, Dean R. Rowland AC, Williams NS. "Light delivery systems for adjunctive intraoperative 
photodynamic therapy." Lasers in Medical Science 1993; 8, 1-14.

5. Evrard S. Aprahamian M, Marescaux J. 'Intra-abdominal photodynamic therapy: from theory to feasability." Br J 
Surg 1993; 80: 298-303.

6. Chevretton EB, Berenbaum MC, Bonnett R. "The effect of photodynamic therapy on normal skeletal muscle in an 
animal model." Lasers Med Sci. 1992; 7: 103-110.

7. Ortu P. LaMuraglia GM, Roberts G. Flotte TJ, Hasan T. "Photodynamic therapy of arteries. A novel approach for 
treatment of experimental intimal hyperplasia" Circulation 1992; 85:3, 1189- 1196.

8. Grant WE, Speight PM, MacRobert AJ, Hopper C, Bown SG. Photodynamic therapy of normal rat arteries after 
phOtosensitisation using disulphonated aluminium phthalocyanine and 5-aminolaevulinic acid." Br J Cancer 1994, 70, 
7278.

9. Star WM, Marijnissen HPA, van den Berg-Block AE, Versteeg JAC, Franken KAP, Reinhold HS. 'Destruction of rat 
mammary tumour and normal tissue microcirculation by HPD photoradiation observed in vivo in sandwich observation 
chambers- Cancer Res 1986: 46, 2532-2540.

10. Grant WE, Hopper C, MacRobert AJ, Speight PM, Bown SG. "Photodynamic therapy of oral cancer: 
photosensitisation with systemic aminolaevulinic acid." Lancet 1993; 342: 147-148

11. Grant WE, Hopper C, Speight PM, MacRobert AJ, Bown SG. 'Photodynamic therapy of malignant and 
premalignant lesions in patients with "field cancerisation" of the oral cavity." J Laryngol Otol 1993; 107: 1140-1145.

12. Barr H. Tralau CJ, Boulos PB, MacRobert AJ, Tilley R. Bown SJ. "The contrasting mechanisms of collagen 
damage between photodynamic therapy and thermal injury.' Photochem. Photobiol. 1987; 46: 795-800.

13. Barr H. Tralau CJ, MacRobert AJ, Krasner N. Boulos PB, Clark CG. "Photodynamic therapy in the normal rat 
colon with phthalocyanine sensitisation." Br J Surg 1987, 56 111 - 118.

14. Smith SGT, Bedwell J. MacRobert AJ, Griffiths MH, Bown SG, Hetzel MR. "Experimental studies to assess the 
potential of photodynamic therapy for the treatment of bronchial carcinomas." Thorax 1993; 48: 474-480.

15. Spikes JD, "Effects of photodynamic treatrnent on the thermal -mechanical properties of collagen (rat tail tendon)." 
Abstracts of the 21st annual Meeting of the American Society for Photobiology. J Photochem Photobiol 1993; 5:965.

16. Spikes JD, Ghiron CA. "Photodynamic effects in biological systems." In: Augenstein L, Mason R. and Rosenberg 
B. eds, Physical Processes in Radiation Biology. Academic Press New York 1964, 309.

17 Verweij H. Dubbelman T. van Steveninck JV. "Photodynamic cross-linking." Biochimica et Biophysica Acta 1981; 
647:

18. Tralau CJ, Young AR, Walker NPJ, Vemon DI, MacRobert AJ, Brown SB, Bown SG. "Mouse skin photosensitivity 
with dihaematoporphyrin (DHE) and aluminium sulphonated phthalocyanine (AlSPc): a comparative study.' 
Photochem, Photobiol 1989; 49, 3:305-312.

19. Spears JR, Serur J. Shropshire D, Paulin S. "Fluorescence of experimental atheromatous plaques with 
haematoporphyrin derivative." J Clin Invest 1983; 71: 395-399.

20. Kessel D, Sykes E. "Porphyrin accumulation by atheromatous plaques of the aorta." Photochem Photobiol 1984; 40: 
5961.

21. Prevosti LG Wynne JJ, Becker CG, Linsker R. Shires GT. "Laser-induced fluorescence detection of atherosclerotic 
plaque with haematoporphyrin derivative used as an exogenous probe." J Vasc Surg 1988, 7: 500-506

22. Spokojny AM, Serur JR, Skillman J. Spears JR. "Uptake of haematoporphyrin derivative by atheromatous plaques: 
studies in human in vitro and rabbit in vivo". J Am Coll Cardiol 1986: 8: 1387-92

23. Mackie RW, Vincent GM, Fox J. Orme EC, Hammond MEH, Chang-Zong C, Johnson MD. "In-vivo canine 
coronary artery laser irradiation: Photodynamic therapy using dihaematoporphyrin ether and 632nm laser. A safety and 
dose response study.' Lasers in Surgery and Medicine 1991 11: 535-544.

24. Neave V, Giannotta S. Shigeyo H. Schneider J. "Haematoporphyrin uptake in atherosclerotic plaques: Therapeutic 
potentials. Neurosurgery 1988.23:3 307-312."