As part of an ongoing program to develop two-photon (2-) photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1- and 2- activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2- cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2- PDT of AMD.
We have developed a novel turbid polarimetry platform for characterization of biological tissues. Currently, we are
exploring the use of this platform for characterization of the extracellular matrix particularly for use in monitoring
regenerative treatments of myocardial infarctions. Collagen is a fibrous protein and exhibits birefringence due to
different refractive indices parallel and perpendicular to the direction of the fibers. As a result, changes in the collagen
content and organization in the tissue lead to changes in birefringence. We demonstrate our ability to measure these
extracellular changes in vivo using a mouse dorsal window chamber model. Collagenase was injected into a region of the
chamber to denature the extracellular matrix. Birefringence measurements show a large decrease in birefringence
associated with the destruction of collagen fibers. Birefringence measurements were also made through ex vivo
myocardial tissues from rats with induced myocardial infarctions including a number that had undergone regenerative
treatment with mesenchymal stem cells. Results show a decrease in birefringence from normal to infracted myocardium,
indicating a decrease in tissue organization associated with scar formation, however, an increase in birefringence was
seen in those myocardial tissues that had undergone regenerative treatment indicating reorganization of tissue structure.
We demonstrate the first in vivo use of a Mueller matrix decomposition method for polarization-based characterization of tissue. Collagenase is injected into a region of dermal tissue in a dorsal skin window chamber in a nude mouse to alter the structure of the extracellular matrix. Mueller matrices for polarized light transmitted through the window chamber in the collagenase-treated region, as well as a distal control region, are measured. From the measured matrices, the individual constituent polarization properties of the tissue are extracted through polar matrix decomposition. Large decreases in birefringence and depolarization are seen in the collagenase-treated region due to the destruction of collagen, showing the potential for this method to monitor the organization and structural anisotropy of tissue. This study represents the first in vivo demonstration of a Mueller matrix decomposition method for polarimetric tissue characterization.
Intravital imaging using confocal microscopy facilitates high-resolution studies of cellular and molecular events in vivo. We use this, complemented by Doppler optical coherence tomography (OCT), to assess blood flow in a mouse dorsal skin-fold window chamber model to image the response of individual blood vessels to localized photodynamic therapy (PDT). Specific fluorescent cell markers were used to assess the effect on the vascular endothelial cell lining of the treated vessels. A fluorescently tagged antibody against an endothelial transmembrane glycoprotein (CD31) was used to image endothelial cell integrity in the targeted blood vessel. A cell permeability (viability) indicator, SYTOX Orange, was also used to further assess damage to endothelial cells. A fluorescently labeled anti-CD41 antibody that binds to platelets was used to confirm platelet aggregation in the treated vessel. These optical techniques enable dynamic assessment of responses to PDT in vivo, at both the vascular endothelial cell and whole vessel levels.
Two-photon (2-γ) photodynamic therapy (PDT) as opposed to "standard" one-photon (1-γ) PDT with Visudyne has recently been suggested as a targeted treatment alternative for wet-form age-related macular degeneration (AMD) and other neovascular diseases. AMD is a major cause of severe vision loss in the older population. It occurs due to growth of new leaky blood vessels (neovasculature) from the choriocapillaris, which results in destruction of photoreceptors in the fovea and loss of central vision. Damage outside the diseased region is always a concern, due to photosensitizer accumulation and its 1-γ excitation. Highly targeted 2-γ excitation, due to its non-linear intensity dependence, intrinsically avoids out-of-focus damage to healthy tissues and so could be valuable for wet-AMD. We have previously developed a quantitative approach for comparing the 2-γ efficacy of photosensitizers in vitro. In this study, we report further the development of ex vivo and in vivo techniques. A mouse mesenteric vessel has been investigated as the ex vivo model of neovasculature. For the in vivo studies, we have explored a mouse dorsal skin-fold window chamber model. Two-photon PDT is delivered using tightly focused ~300 fs laser pulses from a Ti:sapphire laser operating at 850 nm with 90 MHz pulse repetition rate. Confocal microscopy coupled to the laser was used to visualize the vessel's/cell's response before, during and after the treatment. We are able to demonstrate quantitative biological techniques to evaluate efficacy of 2-γ PDT photosensitizers in vivo.
Photodynamic therapy (PDT) can be targeted toward different subcellular localizations and it is widely believed different
subcellular targets vary in their sensitivity to photobiological damage. In this study, PDT-generated near-infrared singlet
oxygen (1O2) luminescence was measured along with cell viability under two different incubation protocols: 5-
aminolevulinic acid (ALA) endogenously-induced protoporphyrin IX (PpIX) and exogenous PpIX, at different
incubation times. Confocal fluorescence microscopy indicated that ALA-induced PpIX (2 h) localized in the
mitochondria, whereas exogenous PpIX (1 h) mainly localized to the plasma membrane. Cell viability was determined at
several time points during PDT treatments using colony-forming assays, and the surviving fraction correlated well with
cumulative 1O2 luminescence counts under both incubation protocols. Preliminary results indicate the plasma membrane
is less sensitive to PDT-generated 1O2 than the mitochondria.
Photodynamic therapy (PDT) using verteporfin is widely used for treatment of age related macular degeneration (AMD).
Due to non-perfect selectivity of the drug accumulation in the neovasculature some collateral damage to healthy tissue
arises during the treatment. Damage to healthy structures in the eye is always a concern because of a high probability of
reducing visual acuity. Two-photon (2-&ggr;) photodynamic therapy potentially offers much higher treatment selectivity than
its one-photon (1-&ggr;) counterpart. By utilizing focused light for 2-&ggr; excitation, treatment volumes on the order of
microliters can be achieved thus maximizing localized insult to abnormal blood vessels and sparing healthy tissue. We
propose that 2-&ggr; photodynamic therapy will be valuable in the treatment of choroidal neovascularization secondary to
age related macular degeneration as well as other conditions. To ascertain feasibility of 2-&ggr; photodynamic therapy we
measured 2-&ggr; spectrum and cross sections of verteporfin (80 GM at 940 nm, 1 GM = 10-50 cm4s/photon), chlorin e6 (14
GM at 800 nm) and tetrasulfonated aluminum phthalocyanine (140 GM at 900 nm) and investigated their in vitro
efficiency under 2-&ggr; excitation. Only verteporfin demonstrated cell kill under the used irradiation parameters (average
light intensity 9.1 mW, wavelength 850 nm, total light dose 6900 J/cm2). Dorsal skinfold window chamber model in
mouse was used to test efficiency of 2-&ggr; PDT with verteporfin in vivo. Although we were able to induce photodynamic
damage to a blood vessel using 1-&ggr; excitation, 2-&ggr; excitation resulted in no visible damage to irradiated blood vessel. The most probable reason is low efficiency of verteporfin as a 2-&ggr; photosensitizer. We also report 2-&ggr; spectrum of new
photosensitizer, HCC4 (4300 GM at 830 nm), specifically designed for efficient 2-&ggr; excitation.
Bioluminescence Imaging (BLI) has been employed as an imaging modality to identify and characterize
fundamental processes related to cancer development and response at cellular and molecular levels. This
technique is based on the reaction of luciferin with oxygen in the presence of luciferase and ATP. A major
concern in this technique is that tumors are generally hypoxic, either constitutively and/or as a result of
treatment, therefore the oxygen available for the bioluminescence reaction could possibly be reduced to
limiting levels, and thus leading to underestimation of the actual number of luciferase-labeled cells during in
vivo procedures. In this report, we present the initial in vitro results of the oxygen dependence of the
bioluminescence signal in rat gliosarcoma 9L cells tagged with the luciferase gene (9Lluc cells).
Bioluminescence photon emission from cells exposed to different oxygen tensions was detected by a sensitive
CCD camera upon exposure to luciferin. The results showed that bioluminescence signal decreased at
administered pO2 levels below about 5%, falling by approximately 50% at 0.2% pO2. Additional experiments
showed that changes in BLI was due to the cell inability to maintain normal levels of ATP during the hypoxic
period reducing the ATP concentration to limiting levels for BLI.
This study investigates the efficacy of low level laser therapy (LLLT) in modulating inducible nitric oxide synthase (iNOS) expression as molecular marker of the inflammation signaling pathway. LLLT was mediated by different therapeutic wavelengths using transgenic animals with the luciferase gene under control of the iNOS gene expression. Inflammation in 30 transgenic mice (iNOS-luc mice, from FVB strain) was induced by intra-articular injection of Zymosan-A in both knee joints. Four experimental groups were treated with one of four different wavelengths (λ=635, 785, 808 and 905nm) and one not laser-irradiated control group. Laser treatment (25 mW cm-2, 5 J cm-2) was applied to the knees 15 minutes after inflammation induction. Measurements of iNOS expression were performed at multiple times (0, 3, 5, 7, 9 and 24h) post-LLLT by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera.
The responsivity of BLI was sufficient to demonstrate a significant increase in bioluminescence signals after laser irradiation of 635nm when compared to non-irradiated animals and the other LLLT treated groups, showing the wavelength-dependence of LLLT on iNOS expression during the acute inflammatory process.
Photodynamic Therapy (PDT) is a promising modality for tumor treatment that combines a photosensitizing agent and visible light resulting in the production of cytotoxic reactive oxygen species leading to cell death. Bioluminescence detection/imaging is a noninvasive technique that uses luciferase gene transfection together with administration of luciferin to generate detectable visible light. It can provide real-time assessment of tumor growth and therapeutic response. The aim of this study is to investigate the potential fo bioluminescence following animolevulinic acid (ALA)-mediated PDT. The in vitro results show a decrease of luminescence, with an excellent correlation to the number of viable cells. In vivo, the tumor growth was monitored using a cooled CCD camera, and ALA-PDT was performed 7-10 days post tumor implantation. The results show a decrease of the bioluminescence signal from the tumor that corresponds to a decrease of viable cells within the tumor, followed by re-growth at the sub-curative PDT doses used.
Fluorescence-guided brain tumor resection may help the neurosurgeon to identify tumor margins that merge imperceptibly into the normal brain tissue and are difficult to identify under white light illumination even using an operating microscope. We compared the amount of residual tumor after white light resection using an operating microscope versus that after fluorescnece-guided resection of an intracranial VX2 tumor in a preclinical model using our previously developed co-axial fluorscence imaging and spectroscopy system, exciting and detecting PpIX fluorescence at 405nm and 635nm respectively. Preliminary results: No fluorescence was present in 3 non-tumor-bearing animals. Fluorescence was present in all 15 tumor-bearing animals after white light resection was completed. To date in 4 rabbits, a decrease in residual tumor was found when using additional fluorescence guided resection compared to white light resection only. Conclusions: ALA induced PpIX fluorescence detects tumor margins not seen under an operation microscope using while light. Using fluorescence imaging to guide tumor resection resulted in a 3-fold decrease in the amount of residual timor. However, these preliminary results indicate that also an additional amount of normal brain is resected, which will be further investigated.
The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates over extended periods in order to increase selective tumor cell kill through apoptosis. The focus of the present work is on mPDT treatment of malignant brain tumors, in which selectivity between damage to tumor cells versus normal brain tissue is critical. Previous studies have shown that low-dose PDT using aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. In order to produce enough tumor cell kill to be an effective therapy, multiple PDT treatments, such as hyperfractionation or metronomic delivery, are likely requried, based on the levels of apoptosis achieved and model calculations of tumor growth rates. mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of interstitial devices for extended light delivery while allowing free movement. In rat models ALA administration via the drinking water has been accomplished at significant doses for up to 10 days, and ex vivo spectrofluorimetry of tumore, normal brain and other tissues post mortem demonstrates a 3-4 increase in the tumor-to-brain concentration of PpIX, without toxicity. Prototype light sources and delivery devices are also shown to be practical, either using a laser diode or light emitting diode (LED) coupled to an implanted optical fiber in the case of the rat model or a directly-implanted LED in rabbits. The combined delivery of both drug and light over an extended period, with survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.
Thermal damage in dental pulp during Nd:YAG laser irradiation have been studied by several researchers; but due to dentin inhomogeneous structure, laser interaction with dentin in the hypersensitivity treatment are not fully understood. In this work, heat distribution profile on human dentine samples irradiated with Nd:YAG laser was simulated at surface and subjacent layers. Calculations were carried out using the Crank-Nicolson's finite difference method. Sixteen dentin samples with 1,5 mm of thickness were evenly distributed into four groups and irradiated with Nd:YAG laser pulses, according to the following scheme: (I) 1 pulse of 900 mJ, (II) 2 pulses of 450 mJ, (III) 3 pulses of 300 mJ, (IV) 6 pulses of 150 mJ; corresponding to a total laser energy of 900 mJ. The pulse interval was 300ms, the pulse duration of 900 ms and irradiated surface area of 0,005 mm2. Laser induced morphological changes in dentin were observed for all the irradiated samples. The heat distribution throughout the dentin layer, from the external dentin surface to the pulpal chamber wall, was calculated for each case, in order to obtain further information about the pulsed Nd:YAG laser-oral hard tissue interaction. The simulation showed significant differences in the final temperature at the pulpal chamber, depending on the exposition time and the energy contained in the laser pulse.
Eduardo Moriyama, Renato Zangaro, Antonio G. J. Villaverde, Ii-Sei Watanabe, Egberto Munin, Luis Sasaki, Daniel Otsuka, Paulo Lobo, Marcos Pacheco, Durval Junior
Infrared lasers have been used for several clinical applications in dentistry, including laser ablation, oral surgeries and dentin hypersensitivity treatment. Despite of dentin low absorption coefficient in the near infrared spectrum, Nd:YAG laser radiation ((lambda) = 1064 nm) is able to melt the human dentin surface resulting in dentin tubules closure that can suppress the symptoms of dentin hypersensitivity pathology. Objectives: This study aims to analyze, through SEM technique, the morphological changes in dentin surface after Nd:YAG laser irradiation using different parameters in energy distribution. Materials and Methods: In this study sixteen human dentin samples were submitted to Nd:YAG laser radiation using a total energy of 900mJ distributed in one, two, three or six laser pulses with energy for each pulse of 900, 450, 300 or 150 mJ respectively. All the samples were irradiated with laser pulse width of 90ms, pulse intervals of 300 ms and spot size area of 0,005 cm2. Results: SEM analysis suggests that differences in energy distribution results in morphological differences even though the same energy is used for all the samples.
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