Comparison of normal piglet bladder damage after PDT with oral or intravesical administration of ALA.

5-Aminolaevulinic-acid (ALA) can be used as an alternative drug in photodynamic therapy of the bladder, since the selective formation of protoporphyrin IX (PpIX) in the tumour and the virtual absence of induced skin photosensitivity are theoretically advantageous for clinical use. A preclinical study was performed, using an in vivo normal piglet bladder model, in order to determine the maximum drug and light doses for reversible tissue damage. Various ALA doses were administered either orally or instilled in the bladder and different radiant exposures were applied. Bladder biopsies were taken at regular intervals and tissue damage was investigated histologically. After oral ALA-administration the PpIX concentration was determined in plasma, erythrocytes and various tissues. In the case of oral administration, reversible bladder damage was observed using 60-75 mg/kg ALA combined with a radiant exposure of 100 J/cm(2) (direct radiant exposure plus scattered 632 nm light) 5-7 h later. For an oral ALA dose of up to 150 mg/kg, the maximum PpIX concentration is reached at approximately 5 h following administration and in neither skin nor bladder tissue is PpIX present at 10-11 h after administration. This ALA dose combined with a radiant exposure of 200 J/cm(2) produces irreversible bladder damage (extensive necrosis and ulceration). In the case of intravesical instillation for 4-4.75 h, an ALA dose of 2.5 g in 50 ml phosphate buffered saline and a radiant exposure of 100 J/cm(2) are still too high to obtain reversible tissue damage; at this dose one of the 13 pigs developed a shrunken bladder with a fibrotic, thickened bladder wall. These drug and light combinations reported above should be regarded as upper limits in pigs and can serve as an indication for the toxicity of the treatment in a clinical setting.

Fractionated illumination for oesophageal ALA-PDT: effect on blood flow and PpIX formation.

The effect of fractionating the 633 nm illumination of 5-aminolaevulinic (ALA)-based photodynamic therapy (PDT) of the normal rat oesophagus was studied. Fractionation of the illumination could enhance the PDT effect in two ways: (a) delay of the vascular shutdown or relaxation of the vasoconstriction induced by ALA-PDT and (b) use of newly formed protoporphyrin IX (PpIX), produced during the dark interval. Forty rats were randomly allocated to two groups of 20 animals each. To study vascular effects, in group 1 illumination with 633 nm (100 mW/cm) was performed at 3 h after oral ALA administration (200 mg/kg) either continuously with 20 J/cm diffuser length (n = 5) or fractionated 2 x 10 J/cm with a 150 s interval (n = 5), five animals served as controls. Blood flow was measured with a laser Doppler flowmeter. To study the effect of renewed PpIX forming, animals in group 2 were illuminated continuously at 3 h after ALA with 20 J/cm (n = 5) or 40 J/cm (n = 5) or fractionated 2 x 20 J/cm with a 3 h interval (n = 5), five animals served as controls. In all animals the in vivo fluence rate and PpIX fluorescence were measured during illuminations and animals were killed at 48 h after PDT. ALA-PDT did not cause any significant vasoconstriction. Fluorescence measurements and dosimetric results in group 1 did not differ between animals illuminated continuously or fractionated with a 150 s interval. In group 2, during a 3 h dark interval, PpIX fluorescence increased and was bleached during the second illumination. The tissue optical properties changed during the 3 h dark interval, resulting in a lower in vivo fluence rate (p < or = 0.001). Fractionation did not result in more oesophageal damage. It was concluded that a 150 s interval during illumination in ALA-PDT does not increase oesophageal blood flow. During an interval of 3 h new PpIX is formed. In the present study, fractionated illumination using short or long time intervals did not result in more damage. Thus, this study shows no evidence for improved PDT effect with fractionated light delivery.

Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies.

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665-900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8-24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.

Fractionated illumination in oesophageal ALA-PDT: effect on ferrochelatase activity.

BACKGROUND AND OBJECTIVE: Administration of 5-aminolevulinic acid (ALA) induces accumulation of the photosensitive compound protoporphyrin IX (PpIX) in certain tissues. PplX can be used as photosensitizer in photodynamic therapy (PDT). More selective or higher PpIX accumulation in the area to be treated could optimize the results of ALA-PDT. Porphobilinogen deaminase (PBGD) is rate-limiting in PpIX formation whereas ferrochelatase converts PpIX into haem by chelation of ferrous iron into PpIX. This results in a moment of close interaction (ferrochelatase binding to PpIX) during which ferrochelatase could selectively be destroyed resulting in an increased PpIX concentration. The aim of the present study was to investigate whether illumination before PDT can selectively destroy ferrochelatase. and whether this results in higher PpIX accumulation and thereby increases the PDT effect. Furthermore, the effect of a second ALA dose was tested. STUDY DESIGN/MATERIALS AND METHODS: Oesophageal tissue of 60 rats were allocated to 2 groups of 30 animals each. In one group, enzyme and PpIX measurements were performed after ALA administration (200 mg/kg orally, n=20), or a second dose of 200 mg/kg ALA at 4 h (n=10), half of each group with and without illumination at 1 h with 12.5 J/cm diffuser length. In the second group, PDT was performed. Ten animals were illuminated at 3 h after ALA administration with 20 (n=5) or 32.5 J/cm (n=5), 10 animals were illuminated at 1 h (12.5 J/cm) and received intra-oesophageal PDT treatment (20 J/cm) at 3 h (n=5) or 4 h (n=5) after ALA. Additionally, 10 animals received a second dose of 200 mg/kg ALA at 4 h and were illuminated (20 J/cm) at 7 h after the first dose of ALA with (n=5) or without (n=5) illumination at 4 h (12.5 J/cm). RESULTS: Illumination with 12.5 J/cm at 1 h after ALA administration caused inhibition of the activity of ferrochelatase at 3 and 4 h after ALA (P=0.02 and P<0.001, respectively), but not at 7 h (P=0.3). In animals sacrificed at 4 h the ratio PBGD:ferrochelatase was higher in animals illuminated at 1 h compared to non-illuminated animals (P<0.001). PpIX concentration was highest (42.7 +/- 3.2 pmol/mg protein) at 3 h after ALA administration and did not increase by illumination at 1 h. Administration of a second dose of ALA did not result in higher PpIX accumulation. After PDT, no difference in epithelial or muscular damage was found between the various groups. CONCLUSION: Illumination at 1 h after ALA administration can cause selective destruction of ferrochelatase, resulting in a higher ratio of PBGD:ferrochelatase. This does not result in accumulation of more porphyrins, even when a second dose of ALA is given. Therefore, under the conditions used in this study fractionated illumination does not enhance ALA-PDT-induced epithelial ablation of the rat oesophagus.

Localisation and accumulation of a new carotenoporphyrin in two primary tumour models.

We have investigated the tumour-localising properties and in vivo fluorescence kinetics of a hexamethoxylated carotenqporphyrin (CP6) in two primary tumour models: UV-B-induced early skin cancer in hairless mice and chemically induced mucosal dysplasia in the rat palate. CP6 fluorescence kinetics are investigated by measuring in vivo fluorescence spectra and images of the mouse skin and the rat palate at different time points after injection. For the tumour-localising properties, microscopic phase-contrast and fluorescence images are recorded. The in vivo fluorescence kinetics in the mouse skin show localization of CP6 in the tumours. However, fluorescence microscopy images show that CP6 localises in the dermis and structures that are not related to the malignant transformation of the mouse skin. The fluorescence kinetics in the rat palate show a significant correlation between the degree of malignancy and the CP6 fluorescence build-up time in the palate. The microscopic images show that CP6 fluorescence localises in the connective tissue and not in the dysplastic epithelium. In conclusion, CP6 does not localise preferentially in (pre-) cancerous tissue in the two primary tumour models studied here, in contrast to reports about localisation of carotenoporphyrins in transplanted tumours. However, the CP6 build-up time in rat palates correlates with the degree of malignancy and this might possibly be a useful parameter in tumour detection.

Classification of clinical autofluorescence spectra of oral leukoplakia using an artificial neural network: a pilot study.

The performance of an artificial neural network was evaluated as an alternative classification technique of autofluorescence spectra of oral leukoplakia, which may reflect the grade of tissue dysplasia. Twenty-two visible lesions of 21 patients suffering from oral leukoplakia and six locations on normal oral mucosa of volunteers were investigated with autofluorescence spectroscopy (420 nm excitation, 465-650 nm emission). Pre-scaled spectra were combined with the corresponding visual and histopathological classifications in order to train artificial neural networks. A trained network is mapping input spectra to tissue characteristics, which was evaluated using a blind set of spectra. Abnormal tissue could be distinguished from normal tissue by a neural network with a sensitivity of 86% and a specificity of 100%. Also, classifying either homogeneous or non-homogeneous tissue performed reasonably well. Weak or no correlation existed between spectral patterns and verrucous or erosive tissue or the grade of dysplasia, hyperplasia and hyperkeratosis.

Photodynamic therapy for esophageal lesions: selectivity depends on wavelength, power, and light dose.

BACKGROUND: Photodynamic therapy with 5-aminolevulinic acid-induced photosensitization could selectively eliminate esophageal epithelial lesions. This study aimed at optimizing laser parameters for 5-aminolevulinic acid photodynamic therapy of the normal rat esophagus. METHODS: Sixty rats received 200 mg/kg 5-aminolevulinic acid orally and were illuminated 3 hours later with either 633 or 532 nm light (n = 30 for each group) through an endoesophageal balloon catheter. Rats received either 8.3 or 25 J/cm diffuser, applied with a 33, 100, or 300 mW/cm diffuser. During illumination, tissue fluorescence measurements and light dosimetry were done. Rats were sacrificed at 48 hours after photodynamic therapy. RESULTS: During illumination, protoporphyrin IX fluorescence declined faster when a higher power output was used. Fluence rate at the esophageal surface was highest for 633-nm light. At 532 nm, light caused less damage to the epithelium and muscle than 633-nm light. Illumination with 33 mW resulted in selective epithelial ablation, whereas illumination with 300 mW caused muscle damage with minor epithelial damage. CONCLUSIONS: The assumed selective epithelial damage of 5-aminolevulinic acid photodynamic therapy in the esophagus largely depends on the combination of wavelength, power, and light dose applied. Most selective epithelial damage was found when low-power 633-nm light was used.

Timing of illumination is essential for effective and safe photodynamic therapy: a study in the normal rat oesophagus.

5-Aminolaevulinic acid (ALA)-induced, protoporphyrin IX (PpIX)-mediated photodynamic therapy (PDT) is an experimental treatment modality for (pre)malignant oesophageal lesions. This study aimed to optimize the time of illumination after ALA administration. Six groups of eight rats received 200 mg kg(-1) ALA orally, eight rats served as controls. Illumination was performed at 1, 2, 3, 4, 6 or 12 h after ALA administration with a 1-cm cylindrical diffuser placed in a balloon catheter (laser parameters: 633 nm, 25 J radiant energy, power output 100 mW). During illumination, fluorescence measurements and light dosimetry were performed. Animals were sacrificed at 48 h (n = 4) or 28 days (n = 4) after PDT. At day 28, an oesophagogram was performed. Largest PpIX fluorescence was found at 3 h after ALA administration. In vivo fluence rate was three times higher than the calculated incident fluence rate. At 48 h after PDT, major epithelial damage was found in all animals illuminated at 2 h, whereas less epithelial damage was found at 3-6 h and none at 1 and 12 h. In animals illuminated at 4, 6 and 12 h, but not at 2 h, oesophagograms showed severe dilatations and histology showed loss of Schwann cells. These results demonstrate that the choice of time interval between ALA administration and illumination is critical for achieving epithelial damage without oesophageal functional impairment. A short interval of 2-3 h seems to be most appropriate.

The optical properties of lung as a function of respiration.

Lung consists of alveoli enclosed by tissue and both structures contribute to volume-dependent scattering of light. It is the purpose of this paper to determine the volume-dependent optical properties of lung. In vivo interstitial fibre measurements of the effective attenuation coefficient mu eff at 632.8 nm differed during inspiration (mu eff = 2.5 +/- 0.5 cm-1) from that during expiration (mu eff = 3.2 +/- 0.6 cm-1). In vitro measurements on a piglet lung insufflated with oxygen from 50 to 150 ml showed the effective attenuation coefficient at 632.8 nm decreases as a function of oxygen volume in the lung (at 50 ml mu eff = 2.97 +/- 0.11 cm-1, at 100 ml mu eff = 1.50 +/- 0.07 cm-1, and at 150 ml mu eff = 1.36 +/- 0.15 cm-1). This was explained by combining scattering of alveoli (Mie theory) with optical properties of collapsed lung tissue using integrating sphere measurements. Theory and measured in vitro values showed good agreement (deviation < or = 15%). Combination of these data yields the absorption coefficient and scattering parameters of lung tissue as a function of lung volume. We conclude that the light fluence rate in lung tissue should be estimated using optical properties that include scattering by the alveoli.

Bladder PDT with intravesical clear and light scattering media: effect of an eccentric isotropic light source on the light distribution.

BACKGROUND AND OBJECTIVE: Whole bladder wall photodynamic therapy (PDT) is sometimes performed with a light scattering medium in the bladder, as it is assumed that this will promote a more uniform illumination of the bladder wall. The influence of eccentric placement of an isotropic light emitting diffuser on the homogeneity of the light distribution at the bladder wall is assessed. STUDY DESIGN/MATERIALS AND METHODS: Whole bladder wall irradiations were performed at approximately 630 nm, and fluence rates were measured with and without controlled amounts of Intralipid in an ex vivo pig bladder and in vitro in a bladder phantom. Experimental values were compared to Monte Carlo simulations using in vitro bladder optical properties. RESULTS: An eccentric diffuser in a clear intravesical medium produces a better uniform illumination than in a light scattering intravesical medium. Also, intravesical light absorption, e.g., by urine, would lead to a substantial loss of the energy delivered in case of light scattering cavity contents. CONCLUSION: The use of a clear intravesical medium guarantees the highest and most uniform fluence rate at the bladder wall during optical irradiation with an isotropic light source in clinical PDT of nonspherical bladders, whereas an intravesical light scattering medium reduces both the magnitude and the uniformity of the fluence rate.

Integrating sphere effect in whole-bladder wall photodynamic therapy: III. Fluence multiplication, optical penetration and light distribution with an eccentric source for human bladder optical properties.

Whole-bladder-wall (WBW) photodynamic therapy (PDT) is performed using approximately 630 nm light emitted by an isotropic light source centered in the bladder cavity. The phenomenon of an increased fluence rate in this spherical geometry, due to light scattering, is denoted as the integrating sphere effect. The fluence rate and the optical penetration depth depend on a single tissue optical parameter, namely the reduced albedo. The optical properties of (diseased) human bladder tissue, i.e. absorption coefficient, scattering coefficient, anisotropy factor and refractive index, were determined in vitro in the wavelength range of 450-880 nm. The integrating sphere effect and optical penetration depth were calculated with diffusion theory and compared to Monte Carlo (MC) computer simulations using approximately 630 nm optical properties. With increasing albedo, the integrating sphere effect calculated with diffusion approximation is increasingly larger than that found with MC simulations. Calculated and simulated optical penetration depths are in reasonable agreement. The smaller the integrating sphere effect for a given tissue absorption, the larger the optical penetration depth into the bladder wall, as the effective attenuation coefficient decreases. Optical penetration depths up to approximately 7.5 mm (definition dependent) can be responsible for unintended tissue damage beyond the bladder tissue. MC simulations were also performed with an eccentric light source and the uniformity of the light distribution at the bladder wall was assessed. The simulations show that even for a small eccentricity, the extremes in deviation from the mean fluence rate are large. All these results indicate that WBW PDT should be performed with some kind of in situ light dosimetry.

Integrating sphere effect in whole-bladder-wall photodynamic therapy: II. The influence of urine at 458, 488, 514 and 630 nm optical irradiation.

Whole-bladder-wall (WBW) photodynamic therapy (PDT) performed with 458 nm instead of 630 nm wavelength might be advantageous. On the basis of Monte Carlo (MC) computer simulations using in vitro bladder optical properties, these wavelengths show an equally strong integrating sphere effect, while haematoporphyrin derivatives can be excited equally efficiently and more easily with an Ar+ laser at 458 nm. To test this, fluence rates were measured at the walls of two piglet bladders during in vivo and in vitro WBW optical irradiations at 458, 488, 514 and 630 nm. In the in vitro experiment, a controlled amount of urine with known absorption coefficient at the irradiation wavelengths was introduced in the bladder cavity. The optical absorption and scattering coefficients and anisotropy factor of the tissue of both piglet bladders were determined in vitro with a double integrating sphere set-up. MC simulations, using the in vitro optical properties, agree only partly with the measured bladder wall fluence rates. In the in vitro experiment with saline in the bladder cavity, the fluence rate at the bladder wall is lowest for 514 nm irradiation and highest for 458 and 630 nm irradiation. In the in vivo experiment and the in vitro experiment with light absorbing urine in the bladder cavity, which mimics the clinical situation, irradiation at 458 nm wavelength resulted in the lowest fluence rate for a given optical power emitted. It cannot be completely ruled out that in an in vivo bladder the light absorption by haemoglobin further reduces the integrating sphere effect at wavelengths shorter than 630 nm. Thus, WBW PDT with red light (630 nm) is technically more advantageous than that with green light (514 nm) or blue light (488 and 458 nm) as this gives the strongest integrating sphere effect.

Integrating sphere effect in whole bladder wall photodynamic therapy: I. 532 nm versus 630 nm optical irradiation.

The optical absorption, scattering and anisotropy coefficients of piglet bladder, with and without Photofrin, and of diseased human bladder were determined in vitro with a double integrating sphere set-up in the wavelength range 450-800 nm. Monte Carlo simulations were performed in a spherical geometry, representing the bladder, using the optical properties at 532 nm and 630 nm determined in vitro. The calculated fluence rates support the fluence rates that were measured at the bladder wall of a piglet during an in vivo whole bladder wall (WBW) irradiation at 532 nm and 630 nm. Fluence rates calculated and measured in vivo at 630 nm are in agreement with those measured previously in clinical photodynamic therapy (PDT) at 630 nm. WBW-PDT with red light (630 nm) will be technically more advantageous than with green light (532 nm) because of a stronger integrating sphere effect, which reduces the variations of the fluence rate at the bladder wall when the isotropic light source is moved away from the centre of the bladder. Since the optical properties show considerable variations from bladder to bladder, and since as a result the light fluence rate at the bladder wall can vary by a factor of 3 to 4 for the same non-scattered light fluence rate, we conclude that in situ light dosimetry during clinical WBW-PDT is a necessity.

Interstitial Nd:YAG laser coagulation with a cylindrical diffusing fiber tip in experimental liver metastases.

Interstitial laser coagulation as a means of destructing hepatic metastases was investigated. Colon carcinoma CC531 was implanted in the liver of 42 Wag/Rij rats; 20 days later, tumors (5.5 +/- 0.2 mm) were exposed to 1,064 nm Nd:YAG laser light at 4 W/cm and either 600, 1,200, 2,400, 3,400, or 4,800 J/cm from a 0.5 cm Helioseal coated cylindrical diffuser. Temperature and fluence rate were measured at the tumor boundary. Lesions were studied on day 2 and 36 posttreatment by light microscopy. Tumor proliferative activity was assessed by bromodeoxyuridine incorporation. Liver damage and function were determined by serum liver enzymes and antipyrine clearance. Fluence rate increased during laser treatment up to 170%; mean temperature increased logarithmically up to 69.7 degrees C. Short-term light microscopy showed coagulation necrosis of 7-11 mm without charring. Lesion size and liver enzymes increased logarithmically with laser energy applied. No deterioration in liver function was found. At 4,800 J/cm complete tumor remission occurred in three of four animals. This study shows the ability of interstitial laser coagulation to produce selective destruction of colonic tumor within the liver.

Light scattering in Intralipid-10% in the wavelength range of 400-1100 nm.

The absorption, scattering, and anisotropy coefficients of the fat emulsion Intralipid-10% have been measured at 457.9, 514.5, 632.8, and 1064 nm. The size and shape distributions of the scattering particles in Intralipid-10% were determined by transmission electron microscopy. Mie theory calculations performed by using the particle size distribution yielded values for the scattering and anisotropy coefficients from 400 to 1100 nm. The agreement with experimental values is better than 6%.