Cystoscopy is the standard procedure for clinical diagnosis of bladder cancer diagnosis. Bladder carcinoma in situ are often multifocal and spread over large areas. In vivo, localization and follow-up of these tumors and their nearby sites is necessary. But, due to the small field of view (FOV) of the cystoscopic video images, urologists cannot easily interpret the scene. Bladder mosaicing using image registration facilitates this interpretation through the visualization of entire lesions with respect to anatomical landmarks. The reference white light (WL) modality is affected by a strong variability in terms of texture, illumination conditions and motion blur. Moreover, in the complementary fluorescence light (FL) modality, the texture is visually different from that of the WL. Existing algorithms were developed for a particular modality and scene conditions. This paper proposes a more general on fly image registration approach for dealing with these variability issues in cystoscopy. To do so, we present a novel, robust and accurate image registration scheme by redefining the data-term of the classical total variational (TV) approach. Quantitative results on realistic bladder phantom images are used for verifying accuracy and robustness of the proposed model. This method is also qualitatively assessed with patient data mosaicing for both WL and FL modalities.
Objective: The objective of this study was two folds: firstly, we would like to investigate the efficiency of
bimodal spectroscopic technique in characterization of hypertrophic scarring tissue deliberately created on a
preclinical model (rabbit's ear); on the other hand, we evaluate the inhibition effect of an anti-inflammatory
medication (tacrolimus) on hypertrophic formation in scar by using our bimodal spectroscopic system.
Study design: This study was conducted on 20 New Zealand Rabbits receiving hypertrophic scarring treatment
on their ears. Fluorescence and Diffuse Reflectance spectra were collected from each scar, amongst which
some had received tacrolimus treatment. Features were extracted from corrected spectral data and analyzed to
classify the scarring tissues into hypertrophic or non-hypertrophic. Diagnostic algorithms were developed with
the use of k-NN classifier and validated by comparing to histological classification result with Leave-one- out
cross validation.
Results and discussion: The accuracy of our bimodal spectroscopy method for detecting hypertrophic
scarring scar tissue was good (sensibility: 90.84%, specificity: 94.44%). The features used for classification were
mainly extracted from the spectra exited at 360, 410 and 420 nm. This indicates that the difference between the
spectra acquired from hypertrophic and non-hypertrophic tissue may be due to the different intensity distribution
of several fluorophores (collagen,elastin and NADH) excited in this range, or to the change in proportion of tissue
layers (epidermis and dermis) explored by the CEFS in use.
KEYWORDS: Tumors, Photodynamic therapy, Bladder, Luminescence, Bladder cancer, Control systems, Animal model studies, Fluorescence correlation spectroscopy, Tissues, Green fluorescent protein
The prevalence of bladder cancer is very high, due to its high recurrence rate in superficial bladder
cancer (30 to 85%), which is the staging of approximately 80% of the patients at first diagnosis. Risk of
recurrence and progression is associated with grade, stage, presence of concomitant carcinoma in situ, size and
number of lesions, as well as time to first recurrence. Recurrences can be partly attributed to new occurrences
but also to residual tumors after resection. Incomplete tumor removal has been observed in 30 to 50% of TUR's,
especially when dealing with T1 or poorly visible malignant or pre-malignant disease1. Fluorescence guided
resection with 5 amino levulinic acid (ALA) or its hexyl ester derivative (Hexvix, has now unequivocally
been demonstrated to increase detection rate and a growing number of studies indicate this has a positive impact
on recurrence and progression ratesImplantation of viable tumor cells, dispersed during resection, is a third factor influencing bladder cancer recurrence. The aim of early intravesical therapy is to interfere with cell
viability and thus reduce implantation risks.
This work is first a description of a statistical simulation algorithm developed for simulating the spectral absorption and emission of several fluorophores in an absorbing and diffusing multilayer model. Second, a detailed experimental validation of the simulation program is conducted on two sets of liquid and solid multilayer phantoms, containing one, two, or three fluorophores, within absorbing and scattering media. Experimental spatially resolved reflectance spectra are acquired in the wavelength band 400 to 800 nm and compared to corresponding simulated spectra. The degree of similarity between experimentation and simulation data is quantified. The results obtained underline good correlations with mean errors varying from 2 to 10%, depending on the number of layers and on the complexity of the phantom's composition.
Histopathological analysis and in vivo optical spectroscopy were used to discriminate several histological stages of UV-irradiated mouse skin. At different times throughout the 30-week irradiation, autofluorescence (AF) and diffuse reflectance (DR) spectra were acquired in a bimodal approach. Then skin was sampled and processed to be classified, according to morphological criteria, into four histological categories: normal, and three types of hyperplasia (compensatory, atypical, and dysplastic). After extracting spectral characteristics, principal component analysis (data reduction) and the k-nearest neighbor classifying method were applied to compare diagnostic performances of monoexcitation AF (based on each of the seven excitation wavelengths: 360, 368, 390, 400, 410, 420, and 430 nm), multiexcitation AF (combining the seven excitation wavelengths), DR, and bimodal spectroscopies. Visible wavelengths are the most sensitive ones to discriminate compensatory from precancerous (atypical and dysplastic) states. Multiexcitation AF provides an average 6-percentage-point increased sensitivity compared to the best scores obtained with monoexcitation AF for all pairs of tissue categories. Bimodality results in a 4-percentage-point increase of specificity when discriminating the three types of hyperplasia. Thus, bimodal spectroscopy appears to be a promising tool to discriminate benign from precancerous stages; clinical investigations should be carried out to confirm these results.
Skin cancer full resection implies an evaluation of safety margins around the visible tumour. For melanomas such
margins are proportional to tumour's thickness also known as "Breslow Index". In order to see if Diffuse Reflectance
Spectroscopy (DRS) could be used to non-invasively evaluate Breslow Index, an in vitro study as well as numerical
simulations were performed. Bilayered phantoms were made : a lower layer mimicking dermis underneath an absorbing
layer mimicking a melanoma. Five groups of phantoms each having a specific top layer's thickness were made : 2, 3, 4, 5
or 6 mm. For wavelengths longer than 600 nm, Diffuse Reflectance spectra were significantly different (p<0.05) for each
thickness at every Collecting to Excitation Fibre Separations (CEFS) : 271, 536, 834, 1076 and 1341 &mgr;m. Monte Carlo
simulations were performed to check if DRS could detect smaller (i.e. 0.5 mm) thickness variations. Both experimental
and numerical results showed the DR signal intensity linearly (R2>0.9) decreases as CEFS increases. The thicker the
melanic layer was the smaller the slope (absolute value) was. These in vitro results will help setting up a clinical trial to
non invasively evaluate Breslow Index : the bandwidth should be the NIR one (wavelengths longer than 600 nm) and
CEFS should be shorter than 1 mm. Calibration will have to be made in order to relate slope to Breslow Index.
Gap junctional intercellular communication (GJIC) has been shown to be involved in the carcinogenesis process. Gap-FRAP (Fluorescence Recovery After Photobleaching) technique could be used to estimate gap junctions functionality and their potential involvement for distinguish normal and cancer cells. In this study, the gap-FRAP technique was used to analyse functional gap-junction-mediated communication for cell lines with different GJIC status. Gap-FRAP data and connexin 43 protein expression decreased for FaDu cancer cell line, in contrast to fibroblast and KB positives cell lines. To check the involvement and functionality of gap junctions in the restitution of the fluorescence after photobleaching, we used a gap junction channel inhibition assay with 18 α-glycyrrhetinic acid. Our results indicate that the degree of gap junctional intercellular communication could be estimated by this technique in vitro.
Cryopreservation is the only method for conserving blood vessels as future allografts with biological immunity controls. Although it affects vessels mechanical structure, no biomechanical integrity simple test is available today.
Biological tissues optical properties characterization by spectroscopic methods is of interest due to their types or natures variations. Collected data complementarity contributes to "photodiagnosis" applicative prospects (cancer, vascular...).
Pig carotid artery rings were tested after excision and after one month cryopreservation. An uniaxial mechanical testing device was used for ring stretching, and elongation and axial forces measurement. Circumferential large strains and stresses were calculated. Simultaneously, each artery ring optical characteristics was measured using fibered autofluorescence and elastic scattering spectrometers.
Mechanical results showed nonlinear strain/stress curves and large deformations in good agreement with other referenced works. Significant differences (p<0.05) between fresh and cryopreserved rings mechanical properties were noticed. Elastic scattering spectra intensity variations were well correlated with artery mechanical properties. The standardized autofluorescence spectra were more clearly correlated with anatomo-histological changes due to cryopreservation, providing rather accurate differentiation between fresh and cryopreserved samples.
This study offers a new perspective to detect changes of cryopreserved arterial samples mechanical properties. Coupling mechanical tests (uniaxial traction of arterial rings) and optical spectroscopic measurements (autofluorescence, elastic scattering) is the driving point: it allows correlating mechanical modifications and spectral variations of artery rings before and after cryopreservation. Ultimately, this new approach could help developping a device allowing non-invasive, atraumatic and contactless optical examinations of arterial graft to assess its mechanical state before reimplantation.
Several reports show that cancerous cells are linked to early decrease in gap-junction number and functionality diminution. This precancerous phenomenon may be accessed by different fluorescence techniques and particularly by gap-FRAP technique (gap-junction fluorescence recovery after photobleaching). Measurements at cell or tissue scale allowed by this method lead to consider its potential interest in endoscopic technics applied to early cancer detection. Experiments were performed on HT-29 (human colon adenocarcinoma), MCF-7 (human breast cancer cell) and CCD-1137Sk (human Fibroblasts) in the presence of 5.6CFDA. Dye was bleached by laser light (488nm) during few seconds depending on the region of interest (one or fewer cells). Fluorescence recovery kinetic after photobleaching was measured by imaging and spectral analysis with a confocal laser scanning microscope as reference technique. Then, a microspectrofluorimeter was used in order to evaluate the faisability on a fiber optics based system offering measurement condition close to the tissue clinical endoscopy conditions.Preliminary results obtained on the various cells lines show significant differences in kinetics for normal and cancerous cells. We have shown that CCD-1137Sk line cells possess functional communicating junctions, contrary to the carcinogenic HT-29 and MCF-7 cells. Results obtained by microspectrofluorimetry are related to confocal microscopes ones confirming the feasibility in endoscopy.
In this paper we describe a compact, portable light-emitting diode (LED)-based fiber-optic system allowing in vivo diffuse reflectance spectra registration in visible and near IR spectral range at two distances between illuminating and collecting fibers. The construction of fiber optic probe is adapted for endoscopy application. We further report the preliminary results of in vivo discrimination between of the benign and malignant tissues and different types of malignant tissues in the animal models.
KEYWORDS: Point spread functions, Luminescence, Deconvolution, Microscopy, 3D acquisition, 3D image processing, Monochromatic aberrations, Image processing, Data acquisition, Objectives
3-D optical fluorescence microscopy becomes now an efficient tool for volumic investigation of living biological samples. However, acquired raw data suffer from different distortions. In order to carry out biological analysis, restoration of raw data by deconvolution is mandatory. The system identification is useful to obtain the knowledge of the actual system and to quantify the influence of experimental parameters. High order centered moments are used as PSF descriptors. Oil immersion index, numerical aperture and specimen thickness are critical parameters for data quality. Furthermore, PSF identification is helpful to precise the experimental protocol. Application to 3-D anthracycline distribution in breast cancer cells is presented.
The present study evaluates the impact of the fluence rate of red light irradiation ((lambda) = 650 nm) on photodynamic efficacy in mTHPC-sensitized HT29 and Colo26 spheroids. Photocytotoxicity, computed as product of the cell yield and plating efficiency, increased progressively when the fluence rate was reduced from 90 to 30 mW cm-2 in both types of spheroids. The observed photocytotoxicity was mainly due to an immediate rather than a delayed cell death for HT29 spheroids. An opposite effect was observed for Colo26 spheroids. The plausible explanations could be the difference in penetration of mTHPC into the spheroids as well as their heterogeneous structure.
Lipid peroxidation (LP) is involved in cell damage induced by photodynamic treatment (PDT) sensitized by some lipophylic porphyrins. We investigated an effect of lipophylic antioxidant (alpha) -tocopherol and its water-soluble analog, trolox, on meta-tetra(hydroxyphenyl)chlorin (mTHPC) sensitized PDT (413 nm) of cultured human colon adenocarcinoma cells (HT29). Cell survival was measured by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion to farmazan (MTT assay). Both antioxidants in concentrations lower than 0.1 mM did not affect photokilling of HT29 cells. These data might suggest that LP is not of crucial importance in cell damage photosensitized by mTHPC. One mM (alpha) -tocopherol or trolox decreased cell survival by ca. 15 and 13% respectively. Both antioxidants increased PDT- induced damage of HT29. Potentiation was evident as the decrease in the initial shoulder part of fluence dependence curve. We propose that antioxidants at height, pro-oxidant concentrations can potentiate PDT induced killing of tumor cells.
Autofluorescence spectra of normal and pathological mucosa of the bladder were measured in vivo in five patients using 410 nm excitation light and ex vivo in five samples, within 30 minutes after removal, using both 364 and 400 nm excitation lights. Inflammatory and angiodysplasia post-radiotherapy lesions, and papilloma were differentiated from normal mucosa by the low autofluorescence intensity and the shape of spectra. The autofluorescence intensity ratio, at a common emission peak (515 - 520 nm) induced by 410 nm and 1 mW excitation light, between normal mucosa and pathological tissues for each patient was variable. A minimum of 5 and a maximum of 22.5 were observed. The autofluorescence intensity for certain inflammatory post-radiotherapy lesions was null. Furthermore, due to high absorption of hemoglobin, spectra of angiodysplasia post-radiotherapy lesions were characterized by two peaks at about 555 and 600 nm. The measured autofluorescence spectra in samples caused by two excitation wavelengths (360 & 400 nm) and 1 mW light power showed similar structures to in vivo results. Though, a NADH peak appeared in variable intensities in spectra of both normal and pathological tissues at the excitation light of 364 nm. In certain lesions, this emission was highly attenuated.
A tamoxifen resistant cell line (MCF7TAM) was established from tamoxifen sensitive MCF-7 human adenocarcinoma cells expressing estrogen receptors. The resistant cell line was found to express estrogen receptors to similar level as the parent cell line but the receptors were found to be altered, having lost their ability to bind estradiol or tamoxifen. The fluorescence of eosin-tamoxifen ionic association was used to investigate intracellular location of tamoxifen in both sensitive and resistant cell lines. Fluorescence emission spectra of eosin, tamoxifen and eosin-tamoxifen complex ((lambda) exc equals 480 nm) were analyzed and showed that maximal fluorescence intensity of the complex ((lambda) em equals 540 nm) was four times higher than that of eosin alone while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be diffusively located in the cytoplasm and nuclear fluorescence intensity was significantly lower. No difference was observed in fluorescence intensity or location in tamoxifen resistant cells, although it has been previously correlated with clinical responsiveness. Improvement of this fluorescence microscopy methodology appears necessary to provide accurate results taking into account the complexity of tamoxifen resistance molecular pathways.
Video epifluorescence microscopy and image analysis are used for studying anthracycline resistance in breast cancer cells. In order to perform a semi-quantitative image analysis, several deconvolution algorithms are tested and validated on model beads. The most performant algorithm is applied to fluorescent biological specimens. We show that deconvolution makes image segmentation easier. Semi-quantitative measurements on resulting images are correlated with results obtained by cytometry.
Preliminary results of clinical experiments using UV induced autofluorescence spectroscopy in 23 patients with Barrett's esophagus are reported in this paper. Excitation wavelengths of 351 nm and 330 nm were used to induce Barrett's mucosa autofluorescence. Autofluorescence acquisition and signal processing were performed using a CP200 Jobin-Yvon system coupled to a flexible three optical fiber sensor. Three distinct emission bands were observed in the measured spectra after normalization according to the backscattered light power. These emissions were attributed to collagen, elastin, NADH and flavin. Fluorescence intensities and ratios between the emission bands were used to discriminate high grade dysplasia and early stage cancer in Barrett's esophagus from normal surrounding tissues. A significant decrease of the overall fluorescence intensity was observed for the Barrett's mucosa compared to the normal esophageal one. Autofluorescence spectral shape was modified. Collagen and elastin contribution was found to decrease going from normal to tumoral tissue.
In order to study the contribution of endogenous and exogenous fluorophores that participate in the global fluorescence signal, a Gaussian analyzing model which can be used for the extraction of important clinical information about tissue metabolic-structural characterization has been developed. This model is intended to the detection of lesions that are characterized by a low or masqued fluorescence emission of a specific fluorophore. The model described here in is based on experimental data of fluorescence spectra measured in phantoms simulating tissue absorption, diffusion, autofluorescence and induced fluorescence (with known concentrations of MTHPC). Each fluorescence peak in the measured spectrum is supposed to be situated in the center of a Gaussian distribution. So, the fitted fluorescence model is composed of the sum of all the Gaussians representing principal and secondary peaks appeared in the measured spectrum. Every Gaussian is characterized by three parameters: bandwidth (Bw), central peak position ((lambda) p) and proportionality coefficient ((alpha) ). The built model can be then used to reconstruct the spectral shape of the fluorescence emission of a given phantom or tissue when only the proportionality coefficients are fitted and to separate effects due to fluorescence yield, tissue modulation, and fluorophores concentration. This approach permits us to identify tissue characteristics more accurately.
We have studied the influence of preirradiated by visible light hematoporphyrin derivative (HpD) solution in PBS on the production of reactive oxygen species (ROS) and phagocytosis of latex particles by rat peritoneal polymorphonuclear neutrophils (PMN), and also on the delayed type hypersensitivity reaction (DTH) to sheep red blood cells in mice. The release of ROS and phagocytic activity were observed by means of registration of the luminol- enhanced chemiluminescence (ChL) in the absence and the in the presence of latex particles. Non-irradiated HpD did not influence neither spontaneous ChL response, nor latex- activated. HpD preirradiated by 135 J/m2 did not affect spontaneous, but increased latex-activated ChL response by 20 percent. This fact indicates an increase in PMN phagocytic activity under the treatment with preirradiated HpD. Increase in preirradiation fluence up to 8.1 kJ/m2 resulted in significant enhancement of spontaneous ChL and inhibition of latex-activated ChL response of PMN. Results of spectroscopic analysis showed negligible decease in HpD Soret band after preirradiation of HpD by the highest fluences used in this study. We could not detect any significant photoproduct formation by differential absorption spectroscopy. Earlier, we have propose the photoinduced aggragation as one of the possible mechanisms of photodegradation of aqueous porphyrin solutions. In all probability, the increase in ROS production by PMN, treated with preirradiated HpD can be attributed to the phagocytosis of aggregates formed. It is possible that ROS can influence directly the DTH-effector cells leading to the observed decrease in DTH reaction level.
The PDT uses the LASER characteristics in order to treat locally some types of cancers.Its principle is to irradiate the tumoral zones after the injection of a photosensitizer. Two processes take place during the treatment: one of them is a photochemical process and the other is a photothermal one. The parameters of major interest involved in these processes are the light energy, the oxygen rate in the tumor and the temperature. the aim of the light dosimetry is to optimize the light dose delivered by the LASER in order to enhance the therapeutic yield of the treatment. A sensor dedicated to this task has been studied in our laboratory. Our aim is to perform the measurement of several light dosimetry parameters with a single sensor. These parameters are the light attenuation coefficient, the oxygen saturation and the temperature. The general principle of the instrumentation is based on the backscattering of the light emitted by the optical treatment fiber. The associated instrumentation is composed with three parts: an optical structure, an electronic system and a software for the digital treatment. Our goal is to use this sensor in order to define a general method to extract the different parameters from the single backscattered signal.
The imaging system we have developed for early cancers detection, acquires and displays in real time, two different spectral images representing the same site excited alternately with an Argon laser and a Xenon lamp. A step by step engine and an optical system are used to alternate and focalize the two excitation lights through the endoscope optical canal. Image acquisition proceeded via a high pass filtered camera, is synchronized with excitation lights thanks to signals exchange between four electronic boards. Inter-image processing is performed to eliminate undesirable information. In-vivo fluorescence images showing bladder's lesions more contrasted than white lighted ones, have been obtained with a previous version of this system. The first results of the final system were achieved on phantoms simulating healthy and cancerous tissues.
Superficial bladder tumor is a challenging indication for photodynamic therapy. Due to lack of specificity of the sensitizers, the light has to be precisely monitored over the bladder surface, illuminated by an isotropic source, to restrict the cytotoxic effect to the tumor without affecting the normal epithelium. In order to assist the surgeon while processing the therapy, an urothelium illumination model is proposed. It is computed through a spline interpolation, on the basis of 12 intravesical sensors. This paper presents the overall system architecture and details the modelization and visualization processes. With this model, the surgeon is able to master the source displacement inside the bladder and to homogenize the tissue exposure.
Photodynamic therapy (PDT) consists in administering a photosensitizer generating cytotoxic radical species when submitted to light irradiation. One of the difficulties encountered in PDT is to find a photosensitizer absorbing at a wavelength penetrating tissues deeply. Meso-tetra(m- hydroxyphenyl)chlorin(mTHPC) presents this characteristic since it is activated at 650 nm. The photodynamic efficiency of mTHPC (0.3 mg/kg) was evaluated 72 hrs after intraperitoneal injection in HT29 human tumor bearing mice. This interval has been determined by a biodistribution study using fluorescence spectroscopy and HPLC. Mice were irradiated at 650 nm, 10 J/cm2 using a dye laser. The photodynamic efficiency was evaluated by two methods: tumor growth after photodynamic treatment and macroscopic measurement of necrosis depth after tumoral resection using in vivo staining procedure with Evans blue dye. The normalization of the tumor volume (V equals 1/6 pi D3) to the initial volume showed no significant difference of control and treated mice, no regression was observed. Secondly the necrosis depth was determined 24 hrs after irradiation using Evans blue which circulates in vessels not damaged by the treatment. Only tumors from treated animals presented measurable necrosis area, mostly localized in surface around the irradiated site with a mean depth of 3.0 plus or minus 0.3 mm. No prolonged tumoral regression was observed. Unexpectedly, the photodynamic activity was higher when using a low irradiance (32 mW/cm2) than when using a higher one (160 mW/cm2). These results were not related to intratumoral mTHPC photodestruction. Tumor eradication may occur either in tumors measuring less than 3 mm, with a small light intensity, or through fractionated irradiation.
A spectroscopic system with flexible three optical fiber sensor had been developed to study tissue fluorescence for a clinical use. Autofluorescence spectra at 413 nm and 10 mW excitation light power from different tissues in oral cavity had been measured in vivo in 25 subjects. The correlation coefficient in spectral shape between individual spectra and the mean emission spectrum of each site was about 0.9 and fluorescence intensity variation ranged between 20% and 45% according to the examined site. The variation in fluorescence intensity of the main emission wavelength at about 520 nm between spectra of the lower part of tongue, gingiva, lips, floor of cavity, cheek and palate was not statistically significant. But the spectrum of the upper part of tongue had been characterized by an additional peak around 635 nm. Otherwise, autofluorescence spectra at 410 nm and 0.5 mW excitation light power of 8 carcinoma of buccal and lung tissues were measured. The fluorescence ratio at 520 emission peak between normal tissue and carcinoma was evaluated at a maximum value of 13 for a lung cancer (ex vivo measurement) and a minimum of 3.3 for a cancer of the oro-pharynx (in vivo measurement). On the other hand, a fluorescence peak at 635 nm had characterized the carcinoma of the floor of cavity and the upper part of tongue.
Photochemotherapy using I.V. administered porphyrin photosensitizers has been used to treat superficial bladder cancers. In order to avoid cutaneous photosensitivity, lasting 6 - 8 weeks, we instilled the photosensitizer intravesically. After first studying the diffusion and localization of HpD in aqueous phase (5 mg/ml) in vitro through the bladder wall of pig by spectrofluorimetry ((lambda) ex equals 392 nm and (lambda) em equals 612.8 nm) and fluorescence microscopy, we determined the biodistribution of HpD in vivo in the rat bladder wall, 2 and 4 hours after bladder instillation of 0.4 ml of HpD: (1) the controls show only a weak autofluorescence restricted to the urothelium after 2 hours (24 micrometers plus or minus 5 micrometers, n equals 3) as well as after 4 hours (29.5 micrometers plus or minus 5 micrometers, n equals 3); (2) on the test preparation a higher fluorescence was observed: after 2 hours, HpD was localized in the urothelium and a very small part of the chorion (55 micrometers plus or minus 9 micrometers, n equals 9) whereas after 4 hours, it penetrated almost completely in the bladder wall (960 micrometers plus or minus 118 micrometers, n equals 9). In conclusion, a bladder instillation of 2 hours seems to be the optimal time of application in the rat since superficial bladder cancers, like carcinoma in situ, particularly occur in the urothelium (stage 0, pTa) or in the chorion (stage 1, pT1).
The effects of a classic two-stage carcinogenesis protocol on the formation of skin tumors in hairless female SKH-1 mice were studied in order to carry out photochemotherapy on the mice bearing tumors later. Mice were initiated with a single application of 100 nmol of 7,12- dimethylbenz[a]anthracene in 0.1 ml acetone and promoted one week later, twice weekly with topical applications of 1.8 nmol (first protocol) or 5 nmol (second protocol) 12-o- tetradecanoylphorbol-13-acetate in 0.1 ml acetone. The first tumors occurred between 4 and 6 weeks after the beginning of the promotion process depending on the protocol and the percentage of mice bearing tumors increased and reached 41% and 100% at the end of the treatment respectively for the first and the second protocol. Depending on the protocol, the tumor yield was 0.8 for the first one and approximately 10 for the second one whereas we expected 3 tumors per mouse. Histology of some skin tumors revealed that all were papillomas, hence benign tumors. These papillomatous lesions seem characteristic of a viral attack as seen in other strains of mammals including humans.
Meso-tetrahydroxyphenylchlorin (mTHPC) was evaluated in vitro in HT29 human colon adenocarcinoma cell line and compared with hematoporphyrin derivative (HpD). The cellular distribution of mTHPC was analyzed by fluorescence microscopy and revealed that mTHPC distributed diffusively in the cytoplasm. A lower proportion of the photosensitizer (50%) was found in the nuclear area. In both areas, mTHPC fluorescence decay was monoexponential. The incorporation kinetics, evaluated by flow cytometry showed that mTHPC cellular uptake is related to the incubation time until 12 hours, then a plateau appeared. For 12 hr-incubation period, the cellular uptake of mTHPC was found to be linearly related to the extracellular concentration, suggesting passive diffusion mechanism. Cytotoxicity assays were performed using MTT assay after photoirradiation at 650 nm for mTHPC and 630 nm for HpD. The photodynamic activity of mTHPC was influenced by serum protein of the culture medium and time-delayed with a maximal activity 48 hrs after the photoirradiation. In optimized experimental conditions i.e. 2% serum protein-containing culture medium, 12 hr-incubation period, cytotoxicity measured 48 hrs after exposure to 10 J/cm2 light fluence, mTHPC appeared approximately 50-fold more active than HpD with IC50 values of 0.06 (mu) g/ml and 3.30 (mu) g/ml, respectively.
Photodynamic therapy (PDT) consists in the administration of a photosensitizer and subsequent irradiation of the tumor with visible light. Routinely, the photosensitizer is given intravenously (i.v.), but the major drawback of this procedure is the resulting skin photosensitivity. The goal of our study was to examine whether intravesical (i.b.) instillation of the photosensitizer for PDT of bladder cancer might be feasible in order to target the tumors and to avoid the photosensitization phenomenon. After studying the normal bladder histology of pig and rat, not much described so far, we studied the diffusion and localization of hematoporphyrin derivative (HpD) in vitro on the pig bladder and the biodistribution of HpD in vivo in the rat bladder, two and four hours after intravesical administration, by spectrofluorimetry and fluorescence microscopy. We have the following results: (1) no diffusion through the pig bladder wall was detected; (2) the penetration depth of HpD into the pig bladder wall was 450 plus or minus 44 micrometers (n equals 8), including urothelium and chorion in totality and a small part of the muscles; (3) the penetration depth of HpD into the rat bladder wall was 55 plus or minus 9 micrometer (n equals 9) after two hours and 960 plus or minus 118 micrometer (n equals 9) after four hours, corresponding respectively to the totality of the urothelium and a small part of the chorion or almost completely in the bladder wall, a small part of the adventicia being excluded. In conclusion, intravesical instillation is feasible and, as superficial bladder cancer, especially carcinoma in situ particularly occur in the urothelium or in the chorion, a bladder instillation of two hours should be advantageous.
Ultrasounds were described by a few authors as possibly inducing sonodynamic reaction, with singlet oxygen production, as photodynamic therapy. The aim of this project was to evidence this effect and to try to explain its different mechanisms. A specific device was developed with a strict control of temperature to avoid hyperthermia and of acoustical intensity: the characteristics of the US beam and the reproducibility of treatment conditions were strictly evaluated. We studied the frequency of 2.21 MHz using an antiresonance frequency of a transducer. US treatment was applied continuously or in pulsed mode. Human colorectal adenocarcinoma cells (HT-29) were used to test the cytotoxicity using trypan blue exclusion test. Analyses were performed using cell suspensions. Different intensities were studied ranging from 0 to 3.7 W/cm2. Moreover, fluorescence emission spectra of hematoporphyrine derivative (HpD) were recorded before and after US treatment. Results of viability showed a higher cytotoxicity with US alone or with HpD in cell suspensions from 3.7 W/cm2 (20% survival). These results show that cavitation alone can account for the cytotoxic effects of sonotherapy. In fact, cavitation is higher with continuous than with pulsed US treatment. No significant difference was found with or without HpD. HpD fluorescence spectra did not differ before and after US treatment suggesting that no modification of HpD structure was induced by US. Fluorescence spectra showed a very slow and small decrease in fluorescence intensity with time probably caused by the low interfering light used for the experiment. In conclusion, in our experiments, ultrasounds do not seem to induce any chemical reaction with photosensitizers, conversely to what was already reported. However, other photosensitizers, molecules and different cell lines (less resistant) must be studied in order to conclude about the absence of cytotoxicity of this technique.
The aim of a dosimetry for the photodynamic therapy (PDT) is to optimize the therapeutic index: destruction of the pathological areas without impairing surrounding healthy tissue. The sensitizer concentration (PS), the global extinction factor (Sigma) eff and the partial pressure of oxygen PO2 in the tumor are the parameters to be studied from the backscattered light during irradiation. A spectrofluorimeter, suitable for flexible endoscopes, has been designed for measurement of (PS) in situ; kinetics of fixation of the sensitizer in the tumor and in healthy tissues allow the user to choose the optimal schedule for treatment; follow-up of the variation of (PS) during irradiation gives an idea of the photochemical reactions. To measure (Sigma) eff in the situation of an interstitial treatment we use the backscattered signal of the excitating light collected through the emitting fiber itself and several contiguous receiving optical fibers. The prototype of this device is 2 mm in diameter. The calculation of the depth of penetration allows computation of light isodoses in the tumor. To have an idea of the available oxygen we measure the absorption of red light by reduced haemoglobin. A general organization chart of the dosimetry shows the various parameters and interactions working towards the therapeutic result.
The control of light dosimetry during photodynamic therapy requires the knowledge of all the optical coefficient in situ. Therefore, a sensor based upon the backscattering phenomenon has been conceived. It is described in the first part. The second part of the paper shows how the combination of the Kubelka and Munk's theory and an ARX modeling of light gives access to the required values. In the last part the results obtained in vitro on optical phantoms and in vivo on nude mice are analyzed.
The extent of tumor necrosis can be determined by the knowledge of the penetration depth of the light through tissue. So, we must determine the effective penetration depth `(delta) ' during treatment, depth at which the incident light intensity is reduced to 36.6%. Light transmission measurements through tissues is the experimental model usually used as a reference method to estimate the value of (delta) . In order to achieve subsequently on line and in situ light dosimetry during PDT, light backscattering has been studied in different concentrations of blue ink and intralipid-20%. All parameters which would influence the value of the back-attenuation coefficient (Sigma) ba have been reviewed separately. The results of transmission and backscattering measurements are compared. A correlation function `R' between (Sigma) eff and (Sigma) ba can be deduced, the knowledge of R allows us to determine the effective light penetration depth in tissue through backscattering measurement.
The hematoporphyrin derivative (HpD) is one of the most employed fluorescent markers for cancer diagnosis due to its ability to localize itself at a higher level in most tumor tissues and to emit a characteristic red fluorescence upon excitation with light at proper wavelength. This autofluorescence is responsible for a significant decrease of the fluorescence contrast between the tumor and the surrounding normal tissue. Consequently, there is a need to develop techniques to detect this fluorescence. This paper deals with the conception of a diagnosis system to detect early cancer at the surface of the hollow organs in the human body by endoscopy. The device and the principle of the image processing are detailed, and the limits of this system are discussed through the analysis of measurements carried out on phantoms and nude mice.
In the present study, the biological testing of the photodegradation of protoporphyrin IX (PP IX) is proposed. The method implies preirradiation of PP IX ((lambda) < 415 nm) in buffer solution (with or without serum) with the following adding to cells and irradiation of this mixture ((lambda) < 415 nm). The increase in survival with increase in preirradiation fluence was used for estimation of rate and quantum yield of PP IX photobleaching in solutions. The quantum yield estimated by the proposed method was compared with quantum yields of photodegradation of PP IX by decay in absorbance and decay in fluorescence. The highest quantum yield of photodegradation of PP IX was obtained by the test of cells photoinactivation, the lowest by the decay monitored by absorbance. The different values of quantum yield of photodegradation are discussed in the view of photobleaching of monomeric forms which are much more photolabile than aggregates and of photoproduct formation. Biological testing of photodegradation of monomeric species of PP IX seems to be the most specific.
In order to increase the penetration of HpD through the skin and to prevent photosensitization from occurring in normal skin after systemic administration, HpD was encapsulated into topical form of liposomes, composed of egg phosphatidylcholine and cholesterol 6.5-1 (molar ratio). Multilamellar vesicles (MLV) and vesicles prepared by dehydration rehydration procedure (DRV) were tested. The topical delivery of the liposomally encapsulated HpD was measured in vitro on Hairless female rat biopsies by a stripping technique and spectrofluorimetry. We have got the following results: (1) The encapsulation ration ranged from 5% to 12% according to the liposome's type. (2) The penetration of liposomal HpD into the 10th strip was better for the MLV than for the DRV (90 to 180%). It was more efficient when the application time increased but the augmentation of lipids concentration had no effect on it. (3) The cumulated quantity of HpD collected on 10 strips was increased, for each type of liposomes, with higher application times or lipid concentrations. The next experiments will be carried out with another composition of liposomes close to the lipidic skin composition, with human skin and with a model of rat cutaneous tumor.
Superficial tumors of the bladder or in situ carcinoma could be interesting indications of Photodynamic Therapy (PDT), since a total mutilating cystectomy could be avoided. The plurifocality of the lesions requires a treatment of the whole mucose; the quantity of light energy must be homogenous and sufficient to induce a therapeutic effect, still non toxic for normal tissues. We therefore developed a system of treatment and intravesical dosimetry control so that the operator can have precise information on the light repartition in the bladder in real time to enable him to optimize the positioning of the irradiation source. This intravesical device consists of twelve light sensors with optical fiber distributed symmetrically against the walls of the bladder; the emitting source is constituted of a scattering isotropic sphere. The signals emitted by the sensors are converted into tension. The acquisition part of the system values consists of two parts : an analogic part, the values are multiplexed on a same oscilloscope track to see in real time their evolution according to the position of the emitting source. The other part is constituted by the numeric acquisition of values for further analysis. We developed, from a mathematical modelisation of the bladder, a centering program of the diffusor that indicates its position in the bladder, as well as a cartography program where the bladder is re-built by interpolation with the different lighting levels.
Photodynamic therapy, developed since 1961 with Lipson''s studies, is now limited in its clinical applications by the lack of knowledge about light comportment and the action of hematoporphyrin in tissues. Using human tumor models in mice, the intratumoral light flux was measured during an interstitial illumination (cylindrical diffusor 5 mm of length) by an argon dye laser emitting continuously at 630 nm (Spectra-Physics 375 B). The flux measured was captured by a plane-cut fiber (400 micrometers ) linked with an optical power meter (Newport 815). The light decrease in tissue had an exponential shape, and k, the global attenuation coefficient, was easily calculated as well as the depth penetration (1/k). Control measurements were performed in beef muscle, and the k value was very consistent with published data. In small tumors (3), the results presented a good reproducibility for the same histology (ksarcoma equals 0.48 +/- 0.08 mm-1, kcholangiocarcinoma equals 0.67 +/- 0.01 mm-1). The intraperitoneal injection of hematoporphyrin derivative (HpD at 10 mg/kg) did not seem to significantly influence the light evolution in tissues compared with control measurements without HpD. The simplicity and the reproducibility of this technique raises hopes of a coming clinical application and a possible comparison between different studies with measurable references.
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