In vivo imaging of oral mucositis in an animal model using optical coherence tomography and optical Doppler tomography.

PURPOSE: To assess noninvasive optical coherence tomography (OCT) and optical Doppler tomography (ODT) for early detection and evaluation of chemotherapy-induced oral mucositis. EXPERIMENTAL DESIGN: Cheek pouches of 10 Syrian golden hamsters were imaged using OCT/ODT during development of chemotherapy-induced mucositis. I.p. injections of 5-fluorouracil and mechanical irritation induced oral lesions. At 2, 4, 7, and 11 days, one hamster was sacrificed and processed for histopathology. OCT images were visually examined; ODT results were semiquantified. Imaging data were compared with histologic findings. RESULTS: During the development of mucositis, OCT/ODT identified the following events: (a) change in epithelial thickness (beginning on day 2), (b) loss of surface keratinized layer continuity (beginning on day 4), (c) loss of epithelial (day 4 onwards) and submucosal integrity (day 7 onwards), (d) changes in axial blood flow velocity (increased on days 2 and 4; decreased on day 7), and (e) changes in blood vessel size (diameter doubled on day 2; quadrupled on day 4; unchanged on day 7). The semiquantitative imaging-based scoring system identified the severity of mucositis as defined by histopathology. The combination of imaging criteria used allowed for the detection of early, intermediate, and late mucositic changes. Imaging data gave higher scores compared with clinical scores early on, suggesting that the imaging-based diagnostic scoring was more sensitive to early mucositic change than the clinical scoring system. Once mucositis was established, imaging and clinical scores converged. CONCLUSION: OCT/ODT identified chemotherapy-induced oral changes before their clinical manifestation, and the proposed scoring system for oral mucositis was validated for the semiquantification of mucositic change.

In vivo optical coherence tomography-based scoring of oral mucositis in human subjects: a pilot study.

A preliminary study to assess noninvasive optical coherence tomography (OCT) for early detection and evaluation of chemotherapy-induced oral mucositis in five patients. In five patients receiving neoadjuvant chemotherapy for primary breast cancer, oral mucositis was assessed clinically, and imaged using noninvasive OCT. Imaging was scored using a novel imaging-based scoring system. Conventional clinical assessment using the Oral Mucositis Assessment Scale was used as the gold standard. Patients were evaluated on days 0, 2, 4, 7, and 11 after commencement of chemotherapy. OCT images were visually examined by one blinded investigator. The following events were identified using OCT: (1) change in epithelial thickness and subepithelial tissue integrity (beginning on day 2), (2) loss of surface keratinized layer continuity (beginning on day 4), (3) loss of epithelial integrity (beginning on day 4). Imaging data gave higher scores compared to clinical scores earlier in treatment, suggesting that the imaging-based diagnostic scoring was more sensitive to early mucositic change than the clinical scoring system. Once mucositis was established, imaging and clinical scores converged. Chemotherapy-induced oral changes were identified prior to their clinical manifestation using OCT, and the proposed scoring system for oral mucositis was validated for the semiquantification of mucositic change.

Detection of vesicant-induced upper airway mucosa damage in the hamster cheek pouch model using optical coherence tomography.

Hamster cheek pouches were exposed to 2-chloroethyl ethyl sulfide [CEES, half-mustard gas (HMG)] at a concentration of 0.4, 2.0, or 5.0 mg/ml for 1 or 5 min. Twenty-four hours post-HMG exposure, tissue damage was assessed by both stereomicrography and optical coherence tomography (OCT). Damage that was not visible on gross visual examination was apparent in the OCT images. Tissue changes were found to be dependent on both HMG concentration and exposure time. The submucosal and muscle layers of the cheek pouch tissue showed the greatest amount of structural alteration. Routine light microscope histology was performed to confirm the OCT observations.

Use of polar decomposition for the diagnosis of oral precancer.

The Mueller matrix describes all the polarizing properties of a sample and, therefore, the optical differences between noncancerous and precancerous tissue that may be present within the matrix elements. A high-speed polarimetry system that generates 16 (4x4) full Mueller matrices to characterize tissues is presented. Feature extraction is done on the Mueller matrix elements resulting in depolarization and retardance images by polar decomposition. These are used to detect and classify early oral cancers and precancerous changes in epithelium such as dysplasia. These images are compared with orthogonal polarization images and analyzed in an attempt to identity useful factors for the differentiation between cancerous lesions and their benign counterparts. Our results indicate that polarimetry has potential as a method for the in vivo early detection and diagnosis of oral premalignancy.

Fluorescence diagnostics of Helicobacter pylori-infected human gastric mucosa: establishing technique and validity.

OBJECTIVE: Bacterial factors, including strain type, anatomic distribution and density, and host responses are important determinants in the pathogenesis of erosive and neoplastic changes linked to gastric Helicobacter pylori (H. pylori) infection. The purpose of this study was to investigate the potential use of photodiagnostics in mapping H. pylori infection. The relationship between fluorescence in individual gastric pits of H. pylori(+) and H. pylori(-) subjects versus that in larger field views of the gastric mucosa and the use of fluorescence to determine H. pylori status in different gastric areas were studied. MATERIAL AND METHODS: Antrum, corpus and fundus biopsies from 8 H. pylori(+) and 4 H. pylori(-) subjects taken during two gastroscopies were used for autofluorescence (535 nm excitation) and aminolevulinic acid (ALA)-induced protoporphyrin IX fluorescence (405 nm excitation) determinations. RESULTS: In the antrum, corpus and fundus a close correlation between individual pit and full-field image (FFI) fluorescence was demonstrated for H. pylori status (R>0.85; R>0.75; R>0.80) and both excitation wavelengths (R>0.89; R>0.85; R>0.95), respectively. In the antrum, FFI in H. pylori(+) subjects exceeded that in H. pylori(-) subjects using 405 nm but not 535 nm excitation regardless of ALA treatment (pcorpus>antrum for both H. pylori(+) and H. pylori(-) subjects regardless of ALA treatment (p=0.03). CONCLUSIONS: Fluorescence-based identification of areas of H. pylori-infected gastric mucosa using 405 nm excitation combined with ALA treatment is feasible and, using a ratio of 535 nm:405 nm-excited fluorescence, it is possible to distinguish H. pylori status and the different areas of the stomach even without ALA.

Nonlinear optical microscopy of articular cartilage.

OBJECTIVE: To assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage. METHOD: Fresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination. RESULTS: NLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes. CONCLUSION: Thin images of living articular cartilage using NLOM may be obtained with (sub-)cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.

Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for photosensitizers administered intraperitoneally (IP) into the chick embryo.

The chick chorioallantoic membrane (CAM) assay was used to determine the photodynamic response (PDR) of blood vessels to Photofrin, 5-aminolevulinic acid (ALA), benzoporphyrin derivative monoacid ring A (BPD-MA) and lutetium texaphyrin (Lutex). The photosensitizers were administered systemically via intraperitoneal injection into the chick embryo. Forward stepwise regression analysis of the PDR results enabled the individual contributions of seven experimental variables to be ranked: drug dose, light dose, fluence rate, drug uptake time, vessel type (whether arterioles or venules), vessel diameter, and embryo age. The order of importance of the variables, the PDR profile, was determined for each photosensitizer. Relative contributions of the experimental variables from this study to the CAM PDR were compared with those from our previous study on PDR of CAM blood vessels following topical application of the same photosensitizers. PDR profiles were interpreted in terms of biophysical and biochemical characteristics of the individual photosensitizers and the variation in their interactions with the delivery/distribution environment.

Influence of wavelength on response to laser photothermolysis of blood vessels: implications for port wine stain laser therapy.

BACKGROUND AND OBJECTIVE: Treatment of port wine stains (PWS) by photothermolysis can be improved by optimizing laser parameters. We have studied the all-important role of wavelength (lambda) by performing pulsed laser photothermolysis in the vasculature of the chick chorioallantoic membrane (CAM) assay. STUDY DESIGN/MATERIALS AND METHODS: The CAM contains an extensive microvascular network ranging from capillaries with diameter D < 20 microm to blood vessels of D approximately 120 microm. The CAM assay enables observation and real-time video documentation of blood flow in pre-capillary arterioles (A) and post-capillary venules (V). The ScleroPlus trade mark laser (Candela Corp., Wayland, MA) has a smooth output over its fixed 1.5 ms pulse duration and allows the operator to vary several treatment parameters such as wavelength, spot size, and energy. Blood vessels in the CAM were irradiated at two clinically relevant wavelengths, lambda = 587 or 597 nm, constant spot size (7 mm), and at different exposures (E = 5-12 J/cm(2)). Threshold exposure (E(th)) (at which non-reversible damage occurred) were 5 J/cm(2) at 587 nm and 8 J/cm(2) at 597 nm. Mathematical modeling was developed to interpret initial (within 30 seconds) injury of arterioles and venules at both wavelengths as a function of D when varying E. RESULTS: Vessel injury was graded off-line from videotapes. For all combinations of lambda and E, measurable injury was evident within 30 seconds of irradiation and maximal damage was reached in less than 10 minutes. Vessel damage was found to depend strongly on lambda. Damage decreased with increasing vessel size. For all D, damage of arterioles was greater than for corresponding venules. CONCLUSIONS: Selection of the correct wavelength is crucial for successful laser therapy of hypervascular lesions and, ideally, should be based on knowledge of vessel diameters for a specific PWS lesion and of optical penetration depths in blood. As a general statement, smaller blood vessels (D = 10-60 microm) should be treated using shorter wavelengths such as 585 nm. To ensure deposition of sufficient laser energy, irradiation at 585 nm, characterized by lower optical penetration depth in blood, may be preferable for PWS therapy.

Vascular response to laser photothermolysis as a function of pulse duration, vessel type, and diameter: implications for port wine stain laser therapy.

BACKGROUND AND OBJECTIVE: Treatment of port wine stains (PWS) by photothermolysis can be improved by optimizing laser parameters on an individual patient basis. We have studied the critical role of pulse duration (t(p)) on the treatment efficacy. STUDY DESIGN/MATERIALS AND METHODS: The V-beam laser (Candela) allowed changing t(p) over user-specified discrete values between 1.5 and 40 milliseconds by delivering a series of 100 microsecond spikes. For the 1.5 and 3 millisecond pulses, three spikes were observed at intervals t(p)/2 and for t(p)> or =6 milliseconds, four spikes separated by t(p)/3. The ScleroPlus laser (Candela) has a smooth output over its fixed 1.5 milliseconds duration. Blood vessels in the chick chorioallantoic membrane (CAM) were irradiated at fixed wavelength (595 nm), spot size (7 mm), radiant exposure (15 Jcm(-2)), and at variable t(p). The CAM contains an extensive microvascular network ranging from capillaries with diameter D<30 microm to blood vessels of D approximately 120 microm. The CAM assay allows real-time video documentation, and observation of blood flow in pre-capillary arterioles (A) and post-capillary venules (V). Vessel injury was graded from recorded videotapes. Mathematical modeling was developed to interpret results of vessel injury when varying t(p) and D. A modified thermal relaxation time was introduced to calculate vessel wall temperature following laser exposure. RESULTS: Arterioles. For increasing t(p), overall damage was found to decrease. For fixed t(p), damage decreased with vessel size. Venules. For all D, damage was smaller than for corresponding arterioles. There was no dependence of damage on t(p). For given t(p), no variation of damage with D was observed. Photothermolysis due to spiked (V-beam) vs. smooth (Scleroplus) delivery of laser energy at fixed t(p) (1.5 milliseconds), showed similar vessel injuries for al values of D (P>0.05). CONCLUSIONS: The difference between initial arteriole and venule damage could be explained by the threefold higher absorption coefficient at 595 nm in (oxygen-poor!) arterioles. In human patients, PWS consist of ectatic venules (characterized by higher absorption), so that these considerations favor the use of 595-nm irradiation for laser photothermolysis. For optimal treatment of PWS it is proposed that t(p) be between 0.1 and 1.5 milliseconds. This is based on a modified relaxation time tau'(d), defined as the time required for heat conduction into the full thickness of the vessel wall, which is assumed to have a thickness DeltaD approximately 0.1D. The corresponding tau'(d) will be a factor of about six smaller than given in the literature. For vessels with D between 30 and 300 mum, tau'(d) ranges from 0.1 to 1.5 milliseconds.

Combined photodynamic and photothermal induced injury enhances damage to in vivo model blood vessels.

BACKGROUND AND OBJECTIVES: The degree of port wine stain (PWS) blanching following pulsed dye laser (PDL) therapy remains variable and unpredictable. Because of the limitations of current PDL therapy, alternative treatment approaches should be explored. The objective was to evaluate a novel methodology for selective vascular damage, combined photodynamic (PDT) and photothermal (PDL) treatment, using the in vivo chick chorioallantoic membrane (CAM) model. STUDY DESIGN/MATERIALS AND METHODS: Thirty microliters of benzoporphyrin derivative monoacid ring A (BPD) solution was administered intraperitoneally into chick embryos at day 12 of development. Study groups were: (1) control (no BPD, no light); (2) BPD alone; (3) continuous wave irradiation (CW) alone (576 nm, 60 mW/cm2, 125 seconds); (4) CW + PDL; (5) BPD+PDL; (6) PDT (BPD+CW); (7) PDL alone (585 nm, 4 J/cm(2)); and (8) PDT+PDL (BPD + CW followed immediately by PDL). Vessels were videotaped prior to, and at 1 hour post-intervention and then assessed for damage based on the following scale: 0, no damage; 1, coagulation; 1.5, vasoconstriction; 2.0, coagulation+vasoconstriction; 2.5, angiostasis; 3.0, hemorrhage. Damage scores were weighted by vessel "order." RESULTS: PDT + PDL resulted in significantly (P < 0.01) more severe vascular damage than was observed in any other study group: 127% more than PDT, 47% more than PDL alone. CONCLUSIONS: PDT + PDL is a novel and promising approach for selective vascular damage and may offer a more effective method for treatment of PWS and other vascular skin lesions.

Laparoscopic photodynamic diagnosis of ovarian cancer using 5-aminolevulinic acid in a rat model.

OBJECTIVE: The objective of this study was to determine the efficacy and sensitivity of laparoscopic photodynamic diagnosis to detect 5-aminolevulinic acid (ALA)-induced fluorescent tumors in an animal model. METHODS: Cancer cells were injected into the peritoneum of rats to induce peritoneal carcinomatosis. After 3-4 weeks, ALA was administered to establish fluorescence in tumor nodules. All intraperitoneal surfaces were inspected using fluorescence and white light laparoscopy. Suspicious lesions were then biopsied in vivo under either fluorescence or white light laparoscopic guidance. Fluorescence intensities of the cancerous lesions compared to normal tissues were determined. A pathologist blinded to our clinical impression analyzed all biopsied specimens. We compared the sensitivity of fluorescence and white light laparoscopic-guided detection of cancerous lesions and determined the clinical utility of fluorescent photodynamic diagnosis in detecting metastatic ovarian cancer. RESULTS: Forty-three biopsies were performed in vivo under laparoscopic fluorescent guidance and 42 biopsies were taken using white light in various regions of the peritoneal surface from nine rats. Ten biopsies were also removed from nonfluorescent regions as nontumor controls. Cancerous lesions showed significantly higher fluorescent intensity compared to noncancerous lesions. Cancerous lesions that were difficult to differentiate from normal surrounding tissue under white light conditions were clearly detected by ALA-induced fluorescence. The average size of these metastatic lesions biopsied under fluorescent light was 1.0 mm (range: 0.3-2.5) compared to 1.5 mm (range: 0.5-2.9) with white light illumination (P < 0.05). CONCLUSIONS: Fluorescent laparoscopic detection of micrometastatic ovarian cancer using ALA is significantly more sensitive than white-light laparoscopy in detecting smaller cancerous lesions in an ovarian cancer rat model. Human trials are indicated.