The effect of fluence rate on the acute response of vessel diameter and red blood cell velocity during topical 5-aminolevulinic acid photodynamic therapy.

BACKGROUND: In a previous study it is shown that for topically applied ALA-PDT, PpIX concentration correlates with vascular changes including vasoconstriction and/or vascular leakage of small vessels and arterioles in the mouse epidermis and dermis. In this study we report on vascular responses induced by ALA-PDT for different fluence rates, including both changes in vessel diameter and dynamics in RBC velocity in arterioles, imaged using intra-vital confocal microscopy in skinfold chambers in hairless mice. Our interest is in the dynamics of vascular changes in the early stages of illumination. METHODS: We have determined the total PDT dose to be relatively low, 13 J cm(-2), and fluence rates of 26, 65 and 130 mW cm(-2) were investigated. Local vascular effects occurred very soon after the start of the therapeutic illumination in ALA-PDT. RESULTS: In this study, we did not find a significant difference between fluence rates. Arterioles were particularly sensitive to vasoconstriction during low dose PDT, often resulting in complete vasoconstriction. When we observed complete vasoconstriction, this coincided with changes in RBC velocity. CONCLUSION: Since the therapeutic effects of PDT are dependent on a fine balance between the need for oxygen during illumination and disruption of the vasculature, the results of the present study add to our understanding of acute vascular effects during ALA-PDT and aid our efforts to optimize PDT using porphyrin pre-cursors.

The effect of light fractionation with a 2-h dark interval on the efficacy of topical hexyl-aminolevulinate photodynamic therapy in normal mouse skin.

BACKGROUND: Light fractionation with a 2-h dark interval increases the efficacy of topical aminolevulinic acid (ALA) photodynamic therapy (PDT). Hexyl-aminolevulinate (HAL) is the hexyl ester of ALA. Both HAL and ALA lead to protoporphyrin IX (PpIX) accumulation in endothelial cells and to vascular effects, which are important for light fractionation. We investigated light fractionation for HAL-PDT in a mouse skin model and compared this with ALA. METHODS: Three illumination schemes were studied: (a) 100 J cm(-2) in a single illumination; (b) 50+50 J cm(-2) in a twofold illumination; (c) a small first light fraction until 50% of PpIX was photobleached (ca. 3 J cm(-2)), followed by 97 J cm(-2) 2h later. PpIX fluorescence was measured continuously during illumination. Efficacy was evaluated by daily visual skin damage scoring up to 7 days after PDT. RESULTS: Light fractionation showed a trend towards increased efficacy for HAL-PDT. Both the initial PpIX synthesis and the PpIX resynthesis during the dark interval were higher for ALA, but these were not correlated with efficacy. Single HAL-PDT was more effective than single ALA-PDT. Photobleaching rates of HAL and ALA were similar indicating similar biodistributions at depth. CONCLUSION: Our results provide evidence to support that light fractionation may be beneficial for HAL-PDT. We are cautious because we found only a non-significant increase in response. However, combining our results with literature data suggest that the illumination scheme may be further optimized for HAL-PDT to potentially enhance the effect of light fractionation.

Topical hexylaminolevulinate and aminolevulinic acid photodynamic therapy: complete arteriole vasoconstriction occurs frequently and depends on protoporphyrin IX concentration in vessel wall.

Vascular responses to photodynamic therapy (PDT) may influence the availability of oxygen during PDT and the extent of tumor destruction after PDT. However, for topical PDT vascular effects are largely unknown. Arteriole and venule diameters were measured before and after hexylaminolevulinate (HAL) and aminolevulinic acid (ALA) PDT and related to the protoporphyrin IX (PpIX) concentration in the vessel wall. A mouse skin fold chamber model and an intravital confocal microscope allowed direct imaging of the subcutaneous vessels underlying the treated area. In both HAL and ALA groups over 60% of arterioles constricted completely, while venules generally did not respond, except for two larger veins that constricted partially. Arteriole vasoconstriction strongly correlated with PpIX fluorescence intensity in the arteriole wall. Total PpIX fluorescence intensity was significantly higher for HAL than ALA for the whole area that was imaged but not for the arteriole walls. In conclusion, complete arteriole vasoconstriction occurs frequently in both HAL and ALA based topical PDT, especially when relatively high PpIX concentrations in arteriole walls are reached. Vasoconstriction will likely influence PDT effect and should be considered in studies on topical HAL and ALA-PDT. Also, our results may redefine the vasculature as a potential secondary target for topical PDT.

In vivo quantification of the scattering properties of tissue using multi-diameter single fiber reflectance spectroscopy.

Multi diameter single fiber reflectance (MDSFR) spectroscopy is a non-invasive optical technique based on using multiple fibers of different diameters to determine both the reduced scattering coefficient (µs') and a parameter γ that is related to the angular distribution of scattering, where γ = (1-g2)/(1-g1) and g1 and g2 the first and second moment of the phase function, respectively. Here we present the first in vivo MDSFR measurements of µs'(λ) and γ(λ) and their wavelength dependence. MDSFR is performed on nineteen mice in four tissue types including skin, liver, normal tongue and in an orthotopic oral squamous cell carcinoma. The wavelength-dependent slope of µs'(λ) (scattering power) is significantly higher for tongue and skin than for oral cancer and liver. The reduced scattering coefficient at 800 nm of oral cancer is significantly higher than of normal tongue and liver. Gamma generally increases with increasing wavelength; for tumor it increases monotonically with wavelength, while for skin, liver and tongue γ(λ) reaches a plateau or even decreases for longer wavelengths. The mean γ(λ) in the wavelength range 400-850 nm is highest for liver (1.87 ± 0.07) and lowest for skin (1.37 ± 0.14). Gamma of tumor and normal tongue falls in between these values where tumor exhibits a higher average γ(λ) (1.72 ± 0.09) than normal tongue (1.58 ± 0.07). This study shows the potential of using light scattering spectroscopy to optically characterize tissue in vivo.

Laser speckle imaging of dynamic changes in flow during photodynamic therapy.

We present a study investigating the use of laser speckle imaging (LSI) for monitoring blood flow during photodynamic therapy (PDT) utilizing the therapeutic illumination radiation. The coherent nature of a laser source, often used in PDT, offers the possibility of obtaining information on the blood flow without interrupting treatment. We have found that in the rat skin-fold observation chamber, it is possible to monitor the vasculature response to PDT in individual arteries, veins and in tumour microvasculature with significantly higher spatial and temporal resolution than current methods. This illustrates the potential for LSI for monitoring PDT, in particular for vascular-localizing photosensitizers, where current non-invasive methods are difficult because of high absorption due to blood and the specific localization of photosensitizer within the vasculature. However, critical problems need to be further investigated and solved, like the influence of tissue sampling volume, changing of optical properties and movement artefacts from other vessels on the LSI signal. Until then, the real potential of LSI for monitoring blood flow remains of limited value.

Fractionated illumination after topical application of 5-aminolevulinic acid on normal skin of hairless mice: the influence of the dark interval.

We have previously shown that light fractionation during topical aminolevulinic acid based photodynamic therapy (ALA-PDT) with a dark interval of 2h leads to a significant increase in efficacy in both pre-clinical and clinical PDT. However this fractionated illumination scheme required an extended overall treatment time. Therefore we investigated the relationship between the dark interval and PDT response with the aim of reducing the overall treatment time without reducing the efficacy. Five groups of mice were treated with ALA-PDT using a single light fraction or the two-fold illumination scheme with a dark interval of 30 min, 1, 1.5 and 2h. Protoporphyrin IX fluorescence kinetics were monitored during illumination. Visual skin response was monitored in the first seven days after PDT and assessed as PDT response. The PDT response decreases with decreasing length of the dark interval. Only the dark interval of 2h showed significantly more damage compared to all the other dark intervals investigated (P<0.05 compared to 1.5h and P<0.01 compared to 1h, 30 min and a single illumination). No relationship could be shown between the utilized PpIX fluorescence during the two-fold illumination and the PDT response. The rate of photobleaching was comparable for the first and the second light fraction and not dependent of the length of dark interval used. We conclude that in the skin of the hairless mouse the dark interval cannot be reduced below 2h without a significant reduction in PDT efficacy.

In vivo nonlinear spectral imaging in mouse skin.

We report on two-photon autofluorescence and second harmonic spectral imaging of live mouse tissues. The use of a high sensitivity detector and ultraviolet optics allowed us to record razor-sharp deep-tissue spectral images of weak autofluorescence and short-wavelength second harmonic generation by mouse skin. Real-color image representation combined with depth-resolved spectral analysis enabled us to identify tissue structures. The results show that linking nonlinear deep-tissue imaging microscopy with autofluorescence spectroscopy has the potential to provide important information for the diagnosis of skin tissues.

Monitoring PDT by means of superficial reflectance spectroscopy.

Monitoring of relevant parameters during photodynamic therapy (PDT) and correlating these with treatment response is necessary to guarantee optimal and reproducible treatment outcome. In this paper we study the correlation between changes in the local tissue optical properties (absorption and scattering coefficients) during ALA-PDT and changes in PpIX fluorescence. The optical properties are measured extremely superficially by employing a single fiber for the delivery and collection of white light to and from the tissue. The measured reflectance spectrum is modeled in terms of four relevant parameters: blood saturation, relative blood volume fraction, scattering intensity and wavelength dependence of the scattering. All these parameters, except the relative blood volume fraction, are shown to correlate with the rate of photobleaching of PpIX, which in turn has previously been shown to correlate with the response of tissues to PDT. These results yield valuable insight in the behavior of these parameters during PDT and their suitability to predict PDT-response for other photosensitizers for which monitoring through photobleaching is not possible.