Chen Sun

The response of human glioma spheroids to repetitive 5-aminolevulinic acid-mediated photodynamic therapy was investigated. In all cases, light fluences were kept below toxic thresholds to simulate conditions typically found at 1-2 cm depths in brain adjacent to tumor. Significant inhibition of spheroid growth was observed following multiple PDT treatments at sub-threshold light fluences. The effect appears to be insensitive to the treatment intervals investigated. In all cases, suppression of growth was observed for the duration of treatment. Low fluence rates appear to be more effective than high fluence rates. No evidence of PDT resistance was found in this investigation.

Many commercially available dermatologic lasers utilize cryogen spray cooling for epidermal protection. A previous tissue culture study demonstrated that single cryogen spurts of 80 ms or less were unlikely to cause cryo-injury in light-skinned individuals. More recently, multiple cryogen spurts have been incorporated into commercial devices, but the effects of MCS have not been evaluated. The aim was to study an in vitro tissue culture model and the epidermal and dermal effects of SCS vs patterns of shorter MCS with the same preset total cryogen delivery time and provide an explanation for noted differences. Four different spurt patterns were evaluated: SCS: one 40-ms cryogen spurt; MCS2: two 20-ms cryogen spurts; MCS4: four 10-ms cryogen spurts; MCS8: eight 5-ms cryogen spurts. Actual Δt c and total cooling time were measured for each spurt pattern. RAFT tissue culture specimens were exposed to cryogen spurt patterns and biopsies were taken immediately and at days 3 and 7. Actual Δt c was increased while Δt Total remained relatively constant as the preset Δt c of 40 ms was delivered as shorter MCS. Progressively more epidermal damage was noted with exposure to the MCS patterns. No dermal injury was noted with either SCS or MCS. For a constant preset Δt c of 40 ms, delivering cryogen in patterns of shorter MCS increased the actual Δt c and consequently the observed epidermal cryo-injury as compared to an SCS. © Springer-Verlag London Limited 2005.

The spectroscopic properties, ultrafast kinetics and utilization of a photochromic molecule as a bi-stable fluorescing sensor of polarity in live cells are described. This molecule is a photochromic fulgimide, 2,3-dialkylidenesuccinimide, which emits fluorescence that can be switched optically on and off. The fluorescence intensity is a function of the polarity of the molecular environment, namely it fluoresces strongly when the molecule is in its polar isomeric structure form. We demonstrate that this molecule enters live cells without inducing damage, it binds primarily to internal membranous organelles and its fluorescence can be switched optically "on" and "off" repeatedly while inside the living cell. A possible use as a bi-stable, on/off sensor is discussed. © 2004 Published by Elsevier B.V.

Background: Keloids and hypertrophic scars result from aberrant wound healing and remain a potential complication of any surgical procedure or trauma. Investigation of aberrant wound healing has been limited to the study of growth factors, collagen precursors, and DNA synthesis in simple in vitro systems, which necessitate removal or destruction of cells or factors in the growth environment of cell cultures. Multiphoton microscopy can use endogenous chromophores such as collagen and nicotinamide adenine dinucleotide hydrogenase to produce thin optical sections of thick living tissues without the use of dyes or stains. Endogenous second-harmonic-generation signals in collagen can be collected to form an MPM image. Objective: To present a novel wound-healing model used to investigate keloid-derived fibroblast activity and collagen production in the same intact tissue-engineered construct over time. Methods: Artificial tissue constructs called RAFTs were cultured at air-fluid interface. Multiphoton microscopy SHG images of collagen in the intact tissue constructs consisting of normal or keloid-derived fibroblasts were obtained. The constructs were then incised with a scalpel. Serial MPM and phase-contrast microscopy images were obtained to monitor changes in the extracellular matrix in response to wounding of the artificial skin construct over 8 days. Results: The tissue-engineered constructs formed a bilayer resembling the dermis and epidermis of human skin. Phase-contrast microscopy revealed migration of keratinocytes into the defect created by scalpel wounding. The constructs were found to contract with time after wounding. The MPM SHG images showed collagen deposition in the tissue constructs after wounding. Tissue constructs with keloid-derived fibroblasts were found to deposit collagen at a higher rate than those with normal fibroblasts. Conclusions: The MPM model described herein permits serial observation of the same intact specimens without the need for fixation or cytotoxic stains. Furthermore, it demonstrates the biologic activity of RAFT artificial tissue constructs. © 2004 American Medical Association. All rights reserved.

The structural origin of scattering contrast from single cells is examined by using a combined optical coherence and multiphoton microscope based on a 12 fs Ti:sapphire source and a 0.95 NA objective. High-resolution coherence-gated scattering images from single cells are coregistered and compared with two-photon-excited fluorescence images. Scattering contrast is observed from mitochondria, plasma membrane, actin filaments, and the boundary between cytoplasm and nucleus. There is little contribution to scattering from regions inside the nuclear core. These results confirm that light scattering signals from specific subcellular structures can be visualized by using coherent reflectance geometry. © 2007 Optical Society of America.

During photodynamic therapy both normal and pathological brain tissue, in close proximity to the light source, can experience significant temperature increases. The purpose of this study was to investigate the anti-tumor effects of concurrent 5-aminolevulinic acid -mediated PDT and hyperthermia in human and rat glioma spheroids. Human or rat glioma spheroids were subjected to PDT, HT, o r a combination of the two treatments. Therapies were given concurrently to simulate the conditions that will occur during patient PDT. Predictio ns of diffusion theory suggest that brain tissue immediately adjacent to a spherical light applicator may experience temperature increases approaching 8 °C for laser input powers of 2 W. In the in vitro model employed here, HT had no effect on spheroid survival at temperatures below 49 °C, while sub-threshold fluence PDT results in only modest decrease in survival. HT and PDT interact in a synergistic manner if the two treatments are given concurrently. The degree of synergism increases with increasing temperature and light fluence. Apoptosis is the primary mode of cell death following both low-fluence rate PDT and combined HT + PDT. © Kluwer Academic Publishers 2004.

The effects of photodynamic therapy in human glioma spheroids incubated in 5-aminolevulinic acid, or ALA esters, are investigated. Spheroid survival and growth are monitored following PDT at representative drug concentrations, light doses, and dose rates. The primary finding of this study is that the response of human glioma spheroids to PDT with lipophilic ester derivatives, such as benzyl-ALA and hexyl-ALA, is equivalent to that observed with ALA, however, this equivalency is obtained for ester concentrations 10-20 times lower than the parent compound. The enhanced efficiency of the esters is likely due to their increased membrane penetrance. Potential clinical advantages of using lipophilic esters in PDT of gliomas are discussed.

The effects of combined photodynamic therapy and ionizing radiation are studied in a human glioma spheroid model. The degree of interaction between the two modalities depends in a complex manner on factors such as PDT irradiation fluence, fluence rate and dose of ionizing radiation. It is shown that gamma radiation and PDT interact in a synergistic manner only if both light fluence and gamma radiation dose exceed approximately 25 J cm -2 and 8 Gy, respectively. Synergistic interactions are observed only for the lower fluence rate investigated. The degree of interaction appears to be independent of both sequence and the PDT or ionizing radiation time intervals investigated. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays show that low-fluence rate PDT is very efficient at inducing apoptotic cell death, whereas neither high-fluence rate PDT nor ionizing radiation produces significant apoptosis. Although the mechanisms remain to be elucidated, the data imply that the observed synergism is likely not due to gamma-induced cell cycle arrest or to PDT-induced inhibition of DNA repair.