Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy

Abstract

Abstract. Ex vivo human skin has been used extensively for cosmeceutical and drug delivery studies, transplantable skin allografts, or skin flaps. However, it has a half-life of a few days due to ischemic necrosis. Traditional methods of assessing viability can be timeconsuming and provide limited metabolic information. Using multiphoton tomography and fluorescence lifetime imaging ͑MPT-FLIM͒ we assess ischemic necrosis of ex vivo skin by NAD͑P͒H autofluorescence intensity and fluorescence lifetime. Ex vivo skin is stored in the presence and absence of nutrient media ͑Dulbecco Modified Eagle Medium͒ at −20, 4, and 37°C and room temperature over a 7-day time course to establish different rates of metabolic deterioration. At higher temperatures we observe a decrease in NAD͑P͒H autofluorescence, higher image noise, and a significant increase in the average fluorescence lifetime ͑ m ͒ from ϳ1000 to 2000 ps. Additionally, significant distortions in NAD͑P͒H fluorescence lifetime histograms correspond to the reduction in autofluorescence. Skin kept at 4°C, with or without media, showed the least change. Our findings suggest that MPT-FLIM enables useful noninvasive optical biopsies to monitor the metabolic state and deterioration of human skin for research and clinical purposes.

Intravital multiphoton microscopy can model uptake and excretion of fluorescein in hepatic ischemia-reperfusion injury

“…As a result, NAD(P)H may be recruited by similar proteins related to oxidative repair, causing the lifetime change we observe in both solar-exposed skin and the liver after I/R injury. 33 Interestingly, these lifetime changes are in sharp contrast to ischemic necrosis and apoptosis, which are associated with a significant increase in the fluorescence lifetime of NAD(P)H. 11,34,35 While these processes also involve oxidative damage, the outcome is ultimately cell death and a breakdown of internal compartmentalization within the cell, 36 which may alter protein recruitment of NAD(P)H and thus result in the lifetime changes we observe. Further studies are required to identify the protein-binding associated changes for NAD(P)H in response to I/R injury and cell death.…”

“…9 For example, autofluorescent nicotinamide adenine dinucleotide (NADH) is a direct measure of cellular metabolic state and can be used to study cellular damage. [10][11][12] The free and bound forms of NADH are involved in the anaerobic and aerobic production of adenosine triphosphate (ATP), respectively. 13,14 Accordingly, a change in the free/bound ratio gives insight into the metabolic state of a cell.…”

Delivery of drugs applied topically to the skin

“…They can provide morphological and biochemical information about the skin by detecting auto fluorescence from endogenous skin fluorophores with little damage to the tissue, particularly with multiphoton tomography [127,128]. Similar imaging studies can also be carried out with freshly excised skin for short periods following excision [129]. Fluorescence lifetime imaging is used in conjunction with multiphoton tomography to provide information about the state or environment of a particular fluorophore, being useful in assessing the effects of physical treatments [130,131].…”

Multiphoton microscopy can visualize zonal damage and decreased cellular metabolic activity in hepatic ischemia-reperfusion injury in rats

“…We have previously studied NAD(P)H in ischemic necrosis of the skin and showed that MPM-FLIM was useful for noninvasive imaging and to monitor the metabolic state of the skin. 11 Thus, the aim of this study was to investigate whether MPM and FLIM are useful tools in detecting disease progression in I/R injury, without the need for external markers.…”

“…9 The metabolic state can be determined in vivo through the recent advances in microscopy and the inherent fluorescent properties of NADH, as has been shown in assessing the viability of excised human skin stored under various conditions. 11,12 Multiphoton microscopy (MPM) has been utilized for the visualization of fluorescent molecules within the cell, 13 including measuring the absorption of nanoparticles into human skin. 14 MPM enables high-resolution imaging of physiology, morphology, and cell-cell interactions in live animals or intact tissue.…”