Wavelength and Fluence Effect on Vascular Damage with Photodynamic Therapy on Skin1  Maria M. Tsoukas, Salvador González, Thomas J. Flotte, R. Rox Anderson, Margaret E. Sherwood, Nikiforos Kollias  Journal of Investigative Dermatology  Volume 114, Issue 2, Pages 303-308 (February 2000) DOI: 10.1046/j.1523-1747.2000.00872.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 PDT with BPD produces pronounced vascular occlusion. (a) Normal skin, control. Hematoxylin and eosin, scale bar: 100 μm. (b) BPD treated skin exposed to MPuD (690 nm, immediately after exposure). Note the papillary dermal edema (asterisk) and stasis of blood vessels (arrows). Hematoxylin and eosin, scale bar: 100 μm. Inset: higher magnification of blood vessels showing intravascular congestion. Hematoxylin and eosin, scale bar, 50 μm. (c) BPD-treated skin exposed to MPuD (458 nm, immediately after exposure). Epidermis appears normal. Note vasodilatation of dermal capillaries (arrows). Hematoxylin and eosin, scale bar: 100 μm. Journal of Investigative Dermatology 2000 114, 303-308DOI: (10.1046/j.1523-1747.2000.00872.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Vascular stasis shows a strong dose-response for red light (690 nm) and is independent of dose for blue light (458 nm) for PDT with BPD. Vascular response immediately after irradiation at MPuD: 690 nm (▪) and 458 nm (□). The number of small capillaries demonstrating stasis per unit area (1 mm2) in papillary dermis – density of vascular congestion-is increasing according to an exponential as fluence increases, when light is delivered at 690 nm. No similar effect was obtained at 458 nm. Morphometric analysis was used to quantitate the phototoxic responses. This was accomplished by counting the number of congested capillaries per 1 mm2, seen right below the epidermis–papillary dermis. There was no vasocongestion seen below this level, immediately after irradiation. Vasocongestion was characterized by complete filling of capillary lumen by red blood cells (mean ± SD) Journal of Investigative Dermatology 2000 114, 303-308DOI: (10.1046/j.1523-1747.2000.00872.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 TEM of blood vessels of BPD-treated skin immediately after exposure at MPuD. (a) 690 nm: Endothelial cells demonstrating condensation of chromatin (C), perinuclear spaces (arrows), and swollen mitochondria (M). Pericytes show cytoplasmic vacuolization (V). (b) 458 nm: Endothelial cells show moderate swelling of mitochondria but no perinuclear spaces. Scale bar: 1 μm. Journal of Investigative Dermatology 2000 114, 303-308DOI: (10.1046/j.1523-1747.2000.00872.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 BPD-treated skin one day after exposure. (a, c) Epidermis and papillary dermis exposed to a dose equal to MPuD; (b, d) reticular dermis and muscle exposed to a dose equal to 40% greater than the MPuD. (a) 690 nm: Note numerous thrombosed vessels (arrows) and dermal edema (asterisk). Hematoxylin and eosin, scale bar: 200 μm. (b) 690 nm: the smooth muscle fibers in the deep dermis show significant damage (M). Note the numerous vessels with intravascular congestion (arrows). Hematoxylin and eosin, scale bar: 200 μm. (c) 458 nm: Note congestion of the small blood vessels in the upper dermis (arrows). Hematoxylin and eosin, scale bar: 200 μm. (d) 458 nm. Smooth muscle fibers in the deep dermis appear normal. Hematoxylin and eosin, scale bar: 200 μm. Journal of Investigative Dermatology 2000 114, 303-308DOI: (10.1046/j.1523-1747.2000.00872.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Fluence-depth effect for vascular damage on day 1. Fluences delivered at 690 nm (▪) have a steeper depth effect in skin compared with equivalent treatment at 458 nm (□). Exposures with red light beyond MPuD reached the level of muscle and adipose tissue (beyond 4 mm depth in dermis). Treatments with blue light even at much higher dose than threshold do not demonstrate the same potency for penetration in dermis (approximately 2 mm depth in dermis). LU = 0.1 mm (mean ± SD). Journal of Investigative Dermatology 2000 114, 303-308DOI: (10.1046/j.1523-1747.2000.00872.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Fluence depth effect for scar formation on day 15. At MPuD and at both wavelengths, 690 nm (▪) and 458 nm (□), the depth of scar (dotted line) in dermis may reach an average 0.8–1.5 mm, treatments with red light have a steeper dose–response in dermis compared with exposures at 458 nm. The extent of scar formation (dotted line) in dermis parallels stasis (bold line) observed on day 1, to a lesser depth. Phototoxicity monitoring is accurate on day 1 after exposure and predictive of scar at 15 d (LU = 0.1 mm) (mean ± SD). Journal of Investigative Dermatology 2000 114, 303-308DOI: (10.1046/j.1523-1747.2000.00872.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions