A novel spectral image-analysis system was used for tumor fluorescence and reflectance imaging in an animal model and in patients. Transcutaneous fluorescence imaging was carried out on Balb/c mice bearing subcutaneous C26 colon carcinoma after intraperitoneal (i.p.) administration of 5-aminolevulinic acid (ALA), a metabolic precursor of protoporphyrin-IX (PP), and of a novel photosensitizer tetrahydroporphyrin (THP). Tumors were clearly observable by fluorescence detection using green light excitation. Tumor versus normal tissue uptake of the photosensitizing agents was determined by monitoring fluorescence intensity. Maximal PP accumulation in tumor was observed 3 h after i.p. injection of ALA, whereas THP showed selective accumulation in tumor 24 h after administration. Reflectance spectroscopy was employed to study pigmented human skin lesions (nevus, pigmented BCC and pigmented melanoma). In the near-infrared region (800-880 nm) pigmented BCC and melanoma exhibited a differently shaped reflectance spectrum compared to normal skin and nevus. Spatially and spectrally resolved imaging, in combination with mathematical algorithms (such as normalization, spectral similarity mapping and division) allowed unambiguous detection of malignancies. Optical biopsy results from a total of 51 patients showed 45 benign nevi, 3 pigmented BCC and 3 malignant melanomas, as confirmed by histology.
Recent studies have shown spray cooling of the skin surface with millisecond cryogen spurts to be an effective method for protecting the epidermis from non-specific thermal injury during various laser mediated dermatological procedures. We have investigated the effects of ambient humidity level, spraying distance, and cryogen boiling point on the resulting radiometric surface temperature. Our findings indicate that: (1) decreasing the ambient humidity level results in less ice formation on the skin surface without altering the radiometric surface temperature during a cryogen spurt; (2) increasing the spraying distance to 85 mm lowers the radiometric surface temperature; and (3) boiling point of the cryogen does not directly affect the surface temperature in the geometries studied.
Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints causing pain deformities and disability. The highly vascular inflamed synovium has aggressive and destructive characteristics, it invades, erodes and gradually destroys cartilage and underlying bone. Photodynamic therapy (PDT) was performed using the chick chorioallantoic membrane (CAM) model to investigate the vitality of synovium and cartilage implanted on the CAM. Synovium, obtained from human patients, was grafted onto the CAM; gross microscopy and histology proved its vitality 7 days post grafting. Cartilage obtained from rabbit knee joint was also maintained on the CAM for 7 days. Its vitality was demonstrated by histology and by measuring metabolic and enzymatic activity of cartilage cells (chondrocytes) as well as the collagen and proteoglycans content. Selective PDT was performed using aluminum phthalocyanine tetrasulfonate (AlPcS4), a hydrophilic compound, soluble in biological solutions, as a photosensitizer. After irradiation with a diode laser (lambda equals 670 nm, 10 mW) damage was observed in vascularized synovium grafts, whereas avascular cartilage remained intact.
Cell damage in UV and NIR laser microscopes by highly focused micromanipulation and fluorescence excitation microbeams has been studied. Damage in erythrocytes, spermatozoa and Chinese hamster ovary cells was detected by monitoring morphology changes, autofluorescence detection, cloning assay, and viability screening. It was found that 364 nm/365 nm UVA radiation induced irreversible cell damage at radiant exposures as low as <10 J/cm2. NIR CW microradiation used in laser tweezers was also able to damage cells via a two-photon excitation process, in particular, when using <800 nm trapping beams. Non- destructive two-photon excitation in femtosecond NIR microscopes is possible within a narrow intensity window. The lower limit is determined by two-photon absorption coefficients and detector efficiency, the higher by intracellular optical breakdown in the extranuclear region. Above certain wavelength-dependent intensity thresholds in femtosecond microscopy, cells were completely destroyed by fragmentation concomitant with plasma generation. The influence of excitation and micromanipulation microbeams should be considered when studying physiology and metabolism of vital cells.
The clinical objective in laser treatment of hemangiomas is to photocoagulate the dilated cutaneous blood vessels, while at the same time minimizing nonspecific thermal injury to the overlying epidermis. We present an in-vivo experimental procedure, using a chicken comb animal model, and an infrared feedback system to deliver repetitive cryogen spurts during continuous Nd:YAG laser irradiation. Gross and histologic observations are consistent with calculated thicknesses of protected and damaged tissues, and demonstrate the feasibility of inducing spatially selective photocoagulation when using cryogen spray cooling in conjunction with laser irradiation. Experimental observation of epidermal protection in the chicken comb model suggests selective photocoagulation of subsurface targeted blood vessels for successful treatment of hemangiomas can be achieved by repetitive applications of a cryogen spurt during continuous Nd:YAG laser irradiation.
Porphyrins and porphine analogs have been shown to induce cytotoxic effects on cells and tissues after exposure to light, an effect which is currently being studied as a new modality for treatment of cancer, termed photodynamic therapy (PDT). One of the important factors in PDT is the preferential uptake of sensitizers by rapidly proliferating tissues. Previous studies showed that cytoskeletal structures are affected by porphyrin-induced PDT. In the present study we investigate the inhibitory efficiency of porphines on tubulin assembly in vitro. We analyze the efficiency of several sulfonated porphine isomers: tetraphenylporphine n-sulfonate (TPPSn) where n equals 4, 2a and 2o (a and o refer to adjacent and opposite substitution, respectively) and the structural isomers of tetra(o-,m-, and p-hydroxyphenyl)porphine (o-,m- ,p-THPP), in order to find a possible structure-activity relationship. The efficiency of the sensitizers was assayed by their capacity to inhibit microtubule assembly. Binding to monomeric tubulin is essential for effective inhibition of assembly, with or without exposure to light. Without exposure to light, TPPS2o was found to be the most potent inhibitor, followed by TPPS2a and to a much smaller extent by TPPS4. All THPP isomers have negligible inhibitory effect. Upon exposure to white light, microtubule assembly was inhibited in the same order:TPPS2o greater than TPPS2a greater than TPPS4 greater than THPP. All porphines were found to have high affinity to the same site on tubulin even those who had almost no dark effect on tubulin assembly (THPP). Addition of the porphines to assembled microtubules did not lead to their depolymerization even after prolonged irradiation. Since it was previously suggested that porphines may share the same binding site on tubulin as bis-ANS, a known tubulin assembly inhibitor, we performed competition studies with this inhibitor and the porphines. It was shown that bis-ANS does not share the same site on tubulin as the porphines and therefore their effects are additive.
The chick chorioallantoic membrane (CAM) is a convenient model for the study of photodynamic therapy (PDT). This membrane has a rich vasculature, which mimics the tumor neovasculature, and can also serve as a host for implanted tumors. The transparency of the CAM enables in-vivo monitoring of vascular changes during and post PDT, without the need to sacrifice test animals at each time point. Video documentation and analysis of events occurring during and after irradiation permit the quantification of changes in vessel morphology, blood perfusion and tumor development. The compounds tested in this study belong to a family of potential sensitizers -- the porphycenes. These are phorphyrin isomers based on a 16-membered macrocycle, in which the four methine moieties linking the pyrrole rings have been replaced by two direct bonds and two ethine bridges. Experiments were performed on blood vessels of the intact CAM and on recurrent human melanoma cells implanted on the CAM. Tumor selectivity was demonstrated by measuring drug uptake using fluorescence methods. A sensitizer injected systemically into the embryo yolk sac could be detected in the blood vessels 30 min after injection; 1 h later the sensitizer had preferentially accumulated in the tumor. Tumors were irradiated at the optimal uptake time (after 1 h) for 16 min with a 20 mW HeNe laser. Video image analysis showed that 96 h after irradiation tumors had decreased to 5% of their original size. In contrast, non-irradiated control tumors on the same CAM, continued to proliferate and grew to more than twice their original size. In addition, we observed a difference in the damage mechanism after systemic compared to topical administration. Topical application followed by irradiation caused fast necrosis of tumors, which might suggest direct damage to tumor cells, whereas after systemic administration, PDT damage was manifested by slower necrosis, presumably caused by vascular destruction.
The clinical objective in laser treatment of selected dermatoses such as port wine stain (PWS), hemangioma and telangiectasia is to maximize thermal damage to the blood vessels, while at the same time minimizing nonspecific injury to the normal overlying epidermis. 'Dynamic' cooling of skin, whereby a cryogen is sprayed onto the surface for an appropriately short period of time (on the order of tens of milliseconds), may offer an effective method for eliminating epidermal thermal injury during laser treatment. We present theoretical and experimental investigations of the thermal response of skin to dynamic cooling in conjunction with pulsed laser irradiation at 585 nm. Computed temperature distributions indicate that cooling the skin immediately prior to pulsed laser irradiation with a cryogen spurt of tetrafluoroethane is an effective method for eliminating epidermal thermal injury during laser treatment of PWS. Experimental results show rapid reduction of skin surface temperature is obtained when using tetrafluoroethane spurts of 20 - 100 ms duration. Successful blanching of PWS without thermal injury to the overlying epidermis is accomplished.
In vivo uptake of the natural porphyrins, uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), was monitored by fluorescence microscopy. Experiments were performed using the chick chorioallantoic membrane (CAM) model, which allowed video documentation of fluorescence both in real time and after integration over a chosen time interval (usually 2 s). Sensitizers at a concentration of 50 (mu) M (100 (mu) L) were injected into a medium-sized vein (diameter approximately 40 micrometer) using an ultra-fine 10 micrometer diameter needle. Fluorescence images were quantitated by subtracting the fluorescence intensity of surrounding CAM tissue (Fmatrix) from the intravascular fluorescence intensity (Fintravascular), after transformation of the video frames into digital form. The differential fluorescence intensity, Fintravascular - Fmatrix, is a measure of the biodistribution. Real time measurements clearly showed that CP and UP fluorescence is associated with moving erythrocytes and not with endothelial cells of the vessel wall. Fluorescence intensity was monitored, up to 60 minutes after injection, by averaging the fluorescence over time intervals of 2 s and recording the integrated images. The fluorescence intensity reached its maximum in about 20 - 30 min after injection, presumably after monomerization inside erythrocyte membranes. The results are interpreted in terms of physical-chemical characteristics (e.g. hydrophilicity) and correlated with the photodynamically induced hemostasis in CAM blood vessels.
The intracellular accumulation of a variety of photosensitizers in human (non-nucleated) and chicken (nucleated) erythrocytes, as well as the photodynamically induced hemolysis were studied using 488 nm laser microirradiation (15 (mu) W, 100X) and confocal laser scanning fluorescence microscopy. Cells incubated with the negatively charged hydrophilic compounds TPPS4 and Pd-TPPS4 exhibited no significant fluorescence before irradiation, but developed strong fluorescence in the cellular and nuclear membranes following photoinduced membrane damage. In contrast, microirradiation of Photofrin-incubated erythrocytes showed instantaneous fluorescence which decreased due to photodegradation. For the cationic, hydrophilic dye Methylene Blue, significant fluorescence was detected in the nucleus only. Following ALA incubation, large intercellular differences were observed in fluorescence in the red spectral region. These differences are probably due to the differential ability of individual erythrocytes to biosynthesize protoporphyrin IX. Photofrin was the most efficient photosensitizer to induce hemolysis. Higher radiant exposures were required for lysis of nucleated than of human red blood cells, except in the case of Methylene Blue. Irradiation was more efficient for unwashed cell suspensions than for washed suspensions, indicating the non-negligible role of extracellular photosensitizing molecules.
The clinical objective in treatment of port wine stains is to maximize thermal damage to the abnormal blood vessels without introducing thermal damage to the normal overlying epidermis. The rationale of dynamic cooling is to protect the epidermis from thermal damage by selectively cooling this layer down immediately before delivering the laser pulse. This work discusses the thermal dynamics of epidermal cooling by milliseconds cryogen spurts and melanosomal heating by the laser pulse.
Novel porphyrinoid photosensitizers are currently being considered for use in photodynamic therapy (PDT) of cancer. This class of sensitizers combines high absorption characteristics at the therapeutic wavelengths ((lambda) > 600 nm) and good tumor targeting properties. We have investigated the in-vivo uptake and photodynamic damage of several porphycenes. Our model system was the chick chorioallantoic membrane (CAM) which we have adapted for use in PDT studies. The CAM assay allows fast screening of novel drugs and obtaining statistically relevant results with minute quantities of the drug. Sensitizers were `trapped' in EPC (egg phosphatidylcholine) or in DPPC (dipalmitoyl phosphatidylcholine); their efficiencies were independent of the vehicle used for application of the sensitizer. The efficiencies of various porphycenes in PDT, as a function of drug and light dose, compare well with those of standard porphyrins and phthalocyanines.
Photodynamic therapy (PDT) was performed in the chorioallantoic membrane (CAM) of the chick embryo. This is a convenient model to study vascular effects. PDT-induced damage was monitored continuously during irradiation and recorded on a VCR driven by a PC. Time-lapse video documentation provided a detailed view of the entire process: tumor growth, angiogenesis, vascularization, PDT, and tumor regression. Our particular interest was in resolving the controversy regarding the primary mechanism in PDT, as to whether the predominant damage is to the parenchymal tumor cells or to the vascular endothelium. Image analysis techniques enabled us to follow quantitatively changes occurring during the process. These changes included alternations in blood vessels (color and morphology) and in tumors (tumor area).
Individual blood vessels in the chick chorioallantoic membrane (CAM) were selectively coagulated through photothermolysis, using pulsed laser irradiation at 585 nm. Pulse durations were chosen to be 0.45 ms and 10 ms, which correspond to the thermal relaxation times in blood vessels of 30 micrometers and 150 micrometers diameter, respectively. The dose vs diameter (D vs d) relationship for coagulation was calculated for the two pulse shapes. The energy deposited in a cylindrical absorber of diameter d by an optical field, incident perpendicular to the vessel, was expressed analytically and compared with the energy required to coagulate a blood vessel of the same lumen diameter. When thermal diffusion is incorporated into the model, our findings can be accounted for quantitatively. This information will be of use for improving the laser treatment of port wine stains and other vasculopathies.
The chick chorioallantoic membrane (CAM) model was used to study synergistic effects of photodynamic therapy (PDT) and hyperthermia (HPT). Since HPT is known, and PDT is believed, to involve a vascular mechanism, the CAM is an ideal medium to study the synergism of these modalities. Moreover, the CAM is a particularly convenient model to manipulate the PDT and HPT parameters and to monitor the modifications of the vasculature: (1) It is possible to view individual blood vessels in the CAM and to examine structural changes in real time. (2) The CAM is a closed system in which HPT can be performed quantitatively and to a selected depth, using different lasers. And (3) variations of surface temperature during PDT + HPT can be readily monitored by noninvasive radiometric techniques. A porphyrin-type photosensitizer solution was applied to areas of the CAM, defined by teflon O-rings placed on the surface. Uptake of the sensitizer into the CAM was determined by monitoring its fluorescence. Excitation light at 405 nm from a spectrofluorometer was directed onto the CAM surface using a bifurcated fiberoptic light guide which also transmitted the fluorescence from the CAM area. The fluorescence-emission spectrum (630-730 nm) and intensity at different times following sensitizer application was measured in vivo. This technique permitted the determination of the uptake dynamics of the sensitizer in the CAM and the establishment of the optimal time for irradiation. After an equilibrium time of 30 minutes, to allow for uptake of sensitizer in the CAM, the area was irradiated with a dual-wavelength system composed of a dye laser at 644 nm (to induce PDT) and a CO2 laser at 10.6 micrometers (to bring about HPT). Damage to the CAM vasculature, due to combined PDT + HPT, was compared to the outcome of the separate modalities. The observed synergistic effect of about 30 was interpreted by invoking various physiological processes. The egg, being a closed in vivo system, lends itself to mathematical modeling of the temporal and spatial temperature profile. The importance of heat dissipation due to diffusion, radiation, and blood perfusion was shown to be small compared to that of heat dissipation due to evaporation of water from the CAM.
The mechanism of photodynamic therapy (PDT) involves, as a primary step,
damage either directly to the tumor cells or to the surrounding vasculature
which, in turn, causes disruption of tumor blood flow and, ultimately, tissue
necrosis by anoxia. We report here a novel in-vivo model for investigating
vascular events during PDT. The chick chorioallantoic membrane (CAM) model was
chosen since it is an established model for studying biological processes such as
implantation and angiogenesis . The photosensitizers meso-tetraphenylporphine
tetrasulfonate (TPPSJ and chloro-aluminum phthalocyanine tetrasulfonate
(CAPcS) were topically applied onto the CAM. In all cases where sensitizer
plus radiation was administered, changes in the CAM microcirculation occurred, as
viewed through a stereoscopic microscope. With increasing light/drug dose we
observed capillary leakage, stasis, occlusion, hemorrhage, and engorgement. The
course of damage formation was documented in real time by video photography. All
controls (sensitizer alone or light alone) remained unchanged compared to treated
CAM. This work also describes preliminary experiments on tumor cells transplanted
onto the CAM.
Transcutaneous oxygen electrodes are used to non-invasively measure tissue oxygen tension during photodynamic therapy
(PDT). Measurements are performed on VX-2 skin carcinomas in rabbit ears. The degree of tumor oxygen tension reduction is
proportional to the applied light dose. In the absence of irradiation, oxygen tension returns to pre-irradiation levels until a
"damage threshold" has been reached. For 50mW/cm2 irradiations of Photofrmn II (at 630 nm) and tetraphenylporphine
tetrasulfonate (at 657 tim), the cumulative dose required to irreversibly deplete tumor iranscutaneous oxygen was approximately
300 kJ/M2 and 600 kJ/M2, respectively.
Continued iraprovement in the results of laser treatment of portwine
stain (PWS) , with reduction in scarring, will depend on the
ability to use lasers to induce selective injury of only the
abnormal blood vessels in the dennis . Photodynamic therapy (PDT),
using an exogenous photosensitizing drug specifically activated by
a certain wavelength of light, may be used to destroy selectively
certain targets in biological systems. The current study
demonstrates, in the chicken comb animal model, that PDT using
porphyrins and phthalocyanines activated by red light could be used
to treat hypervascular derinal tissues.
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