In Vivo NADH Fluorescence Monitoring as an Assay for Cellular Damage in Photodynamic Therapy¶

Abstract

In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)–induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT‐treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 ± 25 nm emission band relative to the fluorescence intensity integrated over the entire 400–600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin‐based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT‐induced cellular death in the tissue being treated.

Time-Resolved Fluorescence in Photodynamic Therapy

“…These include reduced nicotinamide adenine dinucleotide (NADH), reduced NADH phosphate (NADPH), and flavin adenine dinucleotide (FAD) [104,105]. In vitro TRF measurement of photosensitizers focused more on the lifetime changes of the photosensitizers themselves; however, it is indispensable to take the interactions from abundant endogenous fluorophores into account for in vivo environment.…”

NAD(P)H Oscillates in Pollen Tubes and Is Correlated with Tip Growth

“…NAD(P)H is fluorescent and has been visualized by exciting at 340 to 365 nm and detecting emission at 400 to 500 nm (Lakowicz et al, 1992;Paul and Schneckenburger, 1996;Danø et al, 1999;Pogue et al, 2001;Schuchmann et al, 2001;Hu et al, 2002;Brachmanski et al, 2004;Blinova et al, 2005;Kasimova et al, 2006). To visualize the fluorescent signal, we used an inverted wide-field fluorescence microscope (Nikon TE-300), equipped with a CCD camera (CoolSnap HQ; Roper Instruments) mounted on the Kö hler port, and a Xenon excitatory light source (DG-4; Sutter Instruments).…”

“…Their high energy status and reducing power drive many key biosynthetic reactions and ATP production. Because NAD(P)H but not NAD(P) 1 possesses an endogenous fluorescence, it is possible to detect the reduced form in living cells; this property has been widely exploited (Lakowicz et al, 1992;Pogue et al, 2001;Schuchmann et al, 2001;Hu et al, 2002;Brachmanski et al, 2004;Blinova et al, 2005;Kasimova et al, 2006). Particularly pertinent are the oscillations in NADH that have been observed in yeast cells, which serve as a marker for changes in metabolism (Ghosh and Chance, 1964;Goldbeter, 1996;Danø et al, 1999).…”

Treatment-Induced Changes in Tumor Oxygenation Predict Photodynamic Therapy Outcome

“…The development of noninvasive means of implicit or explicit treatment dosimetry in PDT has expanded in recent years with reports on singlet oxygen monitoring (55), NADH fluorescence monitoring (56), 31 P magnetic resonance spectroscopy (57,58), and BOLD-contrast magnetic resonance imaging (59) providing evidence of potential utilization of each technique in PDT. Broadband reflectance spectroscopy is based on the diffusion model and photon migration inside scattering media, similar to near-infrared photon-migration spectroscopy reported by Pham et al (21).…”