Marie J. Hammer-Wilson

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.

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.

Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for topically applied photosensitizers

The relative efficacy of Photofrin-based photodynamic therapy (PDT) has been compared with that of the second-generation photosensitizers 5-aminolevulinic acid (ALA), sulfonated chloro-aluminum phthalocyanine (AlPcSn), benzoporphyrin derivative monoacid ring A (BPD-MA), and lutetium texaphyrin (Lutex). PDT-induced vascular damage in the chick chorioallantoic membrane (CAM) is measured following topical application of the photosensitizers. In order to make meaningful comparisons, care is taken to keep treatment variables the same. These include light dose (5 and 10 J/cm2), power density (33 and 100 mW/cm2), and drug uptake time (30 and 90 min). The drug dose ranges from 0.1 microgram/cm2 for BPD to 5000 micrograms/cm2 for ALA. Results are also analyzed statistically according to CAM vessel type (arterioles versus venules), vessel diameter, and vessel development (embryonic age). For each photosensitizer, the order of importance for the various PDT parameters is found to be unique. The differences between the sensitizers are most likely due to variation in biophysical and biochemical characteristics, biodistribution, and uptake kinetics.

PHOTOSENSITIZATION OF EXPERIMENTAL ATHEROMAS BY PORPHYRINS

Arteriosclerotic arteries have been shown to fluoresce when treated with hematoporphyrin derivative. This study investigates the incorporation and distribution of a partially purified form of hematoporphyrin derivative (Photofrin II) in normal and arteriosclerotic rabbit aortas. A thoracoabdominal exploration was performed in 15 rabbits. Group I comprised normal rabbits, Group II normal rabbits given 5 mg/kg Photofrin II 48 hours before surgery, Group III arteriosclerotic rabbits and Group IV arteriosclerotic rabbits given 5 mg/kg Photofrin II 48 hours before surgery. Multiple aortic biopsy specimens for frozen section were taken from all rabbits. In addition, open laser endarterectomy (with an argon ion laser) was performed on Group III and Group IV rabbits. Frozen sections were studied by digital video fluorescence microscopy to determine the distribution of Photofrin II within the layers of the aortic wall. The fluorescence of the intima of Group IV rabbits was found to be significantly greater than that of the intima, internal elastic lamina, media or adventitia of the other groups (p less than 0.01) and significantly greater than that of the internal elastic lamina, media or adventitia of Group IV rabbits (p less than 0.01). When open laser endarterectomy was performed, Group III rabbits required 103 +/- 14 J/cm2 and Group IV required 33 +/- 3 J/cm2 (p less than 0.01). It is concluded that porphyrins are selectively localized within the intima of arteriosclerotic arteries. This localization sensitizes atheromas to argon ion laser light and facilitates laser endarterectomy.

MUTATION AND SISTER CHROMATID EXCHANGE INDUCTION IN CHINESE HAMSTER OVARY (CHO) CELLS BY PULSED EXCIMER LASER RADIATION AT 93 nm AND 308 nm AND CONTINUOUS UV RADIATION AT 254 nm

Abstract— We compared mutagenesis and sister chromatid exchange (SCE) induction by 193 nm and 308 nm pulsed excimer laser radiation with 254 nm low intensity continuous wave UV light in Chinese hamster ovary (CHO) cells in culture. The 254 nm radiation was most mutagenic of the radiations, in accordance with expectation, and also was most effective in increasing the level of SCEs. The 193 nm radiation was mutagenic at the ouabain resistance locus, but not at the HGPRT locus. However, 193 nm radiation was also strongly cytotoxic at energies producing measurable mutations. This radiation also caused a dose‐related increase in SCEs. Pulsed excimer radiation at 308 nm was mutagenic at both loci, and also increased the incidence of SCEs. Comparison of the ratio of mutants/surviving cells at the D37 after radiation showed similar values for 254 nm and 308 nm at the HGPRT locus, but at the ouabain resistance locus, the ratio for the 308 nm radiation was about 5 times that for 254 nm radiation. These results indicate that some risk for mutagenesis may accompany the use of excimer radiation in the UVA region in therapeutic applications.

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.

Systemic application of photosensitizers in the chick chorioallantoic membrane (CAM) model: Photodynamic response of CAM vessels and 5-aminolevulinic acid uptake kinetics by transplantable tumors

The aim of this study is to modify the chick chorioallantoic membrane (CAM) model into a whole-animal tumor model for photodynamic therapy (PDT). By using intraperitoneal (i.p.) photosensitizer injection of the chick embryo, use of the CAM for PDT has been extended to include systemic delivery as well as topical application of photosensitizers. The model has been tested for its capability to mimic an animal tumor model and to serve for PDT studies by measuring drug fluorescence and PDT-induced effects. Three second-generation photosensitizers have been tested for their ability to produce photodynamic response in the chick embryo/CAM system when delivered by i.p. injection: 5-aminolevulinic acid (ALA), benzoporphyrin derivative monoacid ring A (BPD-MA), and Lutetium-texaphyrin (Lu-Tex). Exposure of the CAM vasculature to the appropriate laser light results in light-dose-dependent vascular damage with all three compounds. Localization of ALA following i.p. injections in embryos, whose CAMs have been implanted with rat ovarian cancer cells to produce nodules, is determined in real time by fluorescence of the photoactive metabolite protoporphyrin IX (PpIX). Dose-dependent fluorescence in the normal CAM vasculature and the tumor implants confirms the uptake of ALA from the peritoneum, systemic circulation of the drug, and its conversion to PpIX.

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.

Genetic microsurgery by laser: establishment of a clonal population of rat kangaroo cells (PTK2) with a directed deficiency in a chromosomal nucleolar organizer.

An ultraviolet laser beam was focused to a submicron spot on one of the nucleolar organizer regions of mitotic chromosomes of rat kangaroo cells in tissue culture. The daughter cells were isolated and cloned into a viable population that maintained the directed nucleolar deficiency. It is concluded that the laser can be used to delete preselected genetic regions and the genetic deletion is maintained as a heritable deficiency in subsequent daughter cells.

Experimental arteriosclerosis treated by conventional and laser endarterectomy

Open laser endarterectomy was compared to standard surgical endarterectomy in the rabbit arteriosclerosis model. The aorta was exposed by a thoracoabdominal exploration in 16 rabbits. In Group I (8 rabbits), a conventional endarterectomy (CE) was performed with standard vascular instruments. In Group II (8 rabbits), laser endarterectomy (LE) was performed with an argon ion laser (488 nm and 514.5 nm) at a power of 1.0 W. Aortas were fixed, serially sectioned (6 micron) and stained (H +/- E) following each procedure. Gross and light microscopic examination revealed identical results for the endarterectomy surfaces of CE and LE. The proper cleavage plane within the media was developed with both techniques and the remaining arterial wall was not damaged with either procedure. The end points of LE were consistently superior to those of CE because of phototherapy fusion. The LE end points were tapered and the intima was fused. Intimal flaps were seen in 2/8 CE experiments and the remaining end points exhibited an uneven transition. LE required an average energy density of 124 +/- 9 J/cm2. We conclude that LE provides a satisfactory method for the in vivo evaluation of laser radiation upon arteriosclerotic arteries. LE may be the way to begin clinical laser trials.