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Fluorescence Spectroscopy and Cryoimaging of Rat Lung Tissue Mitochondrial Redox State a

R. Sepehra, S. Audib,c, K. Staniszewskia, S Malekia, M. Ranji*a Department of Electrical Engineering, University of Wisconsin Milwaukee, 3200 N. Cramer St., Milwaukee, WI 5321; b Department of Biomedical engineering, Marquette University 1217 West Wisconsin Avenue, Milwaukee, WI, 53233. c Zablocki VA Medical Center, 5000 W. National Avenue Milwaukee, WI 53295 ABSTRACT

The objective of this study was to demonstrate the utility of optical cryoimaging and fluorometry to evaluate tissue redox state of the mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (Flavin Adenine Dinucleotide) in intact rat lungs. The ratio (NADH/FAD), referred to as mitochondrial redox ratio (RR), is a measure of the lung tissue mitochondrial redox state. Isolated rat lungs were connected to a ventilation-perfused system. Surface NADH and FAD fluorescence signals were acquired before and after lung perfusion in the absence (control perfusate) or presence of potassium cyanide (KCN, complex IV inhibitor) to reduce the mitochondrial respiratory chain (state 5 respiration). Another group of lungs were perfused with control perfusate or KCN-containing perfusate as above, after which the lungs were deflated and frozen rapidly for subsequent 3D cryoimaging. Results demonstrate that lung treatment with KCN increased lung surface NADH signal by 22%, decreased FAD signal by 8%, and as result increased RR by 31% as compared to control perfusate (baseline) values. Cryoimaging results also show that KCN increased mean lung tissue NADH signal by 37%, decreased mean FAD signal by 4%, and increased mean RR by 47%. These results demonstrate the utility of these optical techniques to evaluate the effect of pulmonary oxidative stress on tissue mitochondrial redox state in intact lungs. Keywords: Optical imaging, NADH, FAD, Mitochondrial redox, Lung tissue

1. INTRODUCTION Optical fluorescence techniques have the potential to diagnose tissue metabolic status in real time in a nondestructive manner in intact organs and in vivo [1-7]. These techniques are widely used in biomedical applications and have been shown to have a high sensitivity and specificity for discriminating between diseased and non-diseased tissue. The mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and FADH2 (Flavoprotein Adenine Dinucleotide) are the primary electron carriers in oxidative phosphorylation. NADH and FAD (oxidized form of FADH2) are autofluorescent and can be monitored without exogenous labels by noninvasive optical techniques [4]. The fluorescence signals of these intrinsic fluorophores have been used as indicators of tissue metabolism in injuries due to hypoxia, ischemia, and cell death [1]. We have demonstrated that the normalized ratio of these fluorophores, (NADH/ FAD), called the mitochondrial redox ratio (RR), is a marker of the mitochondrial redox and metabolic state of myocardial tissue in intact hearts and in vivo [1-3]. Rapid freeze trapping of organs in liquid nitrogen temperatures preserves the intact tissue metabolic state. Subsequent low temperature fluorescence imaging (cryoimaging) is advantageous since it provides high fluorescence quantum yield of NADH and FAD as compared with room temperature, and 3-D spatial distribution of tissue NADH and FAD fluorescence intensities [8]. The above optical and imaging techniques are widely used to probe tissue redox state and energy homeostasis in organs such as the heart [5], brain [7], kidney [6], liver [9] , skeletal muscles [10], cervix [11], and colon[12], but have not been fully used in lungs [13]. Thus, the objective of the study was to demonstrate the utility of these techniques to evaluate lung tissue mitochondrial redox state in intact rat lungs.

Clinical and Biomedical Spectroscopy and Imaging II, edited by Nirmala Ramanujam, Jürgen Popp, Proc. of SPIE-OSA Biomedical Optics, SPIE Vol. 8087, 80870A · © 2011 SPIE-OSA · CCC code: 1605-7422/11/$18 · doi: 10.1117/12.890019 SPIE-OSA/ Vol. 8087 80870A-1

2. MATERIALS AND METHODS 2.1 Isolated perfused rat lung: Male Sprague-Dawley rats (275-350 g; Charles River) were used for this study. The isolated perfused rat lung preparation has been previously described [14]. Briefly, each rat was anesthetized with pentobarbital sodium (40 mg/kg body wt i.p.), after which the trachea was clamped and the chest opened. Heparin (0.7 IU/g body wt.) was injected into the right ventricle. Cannulas were placed in the pulmonary artery and the trachea, and the pulmonary venous outflow was accessed via a cannula in the left atrium. The lungs were removed from the chest and attached to a ventilation and perfusion system. The perfusate was Krebs-Ringer bicarbonate solution containing 5% dextran [14]. The ventilation gas mixture was 15% O2, 6% CO2 in N2. The perfusate was pumped (at 10 ml/min) through the lung until it was clear of blood, after which surface NADH and FAD fluorescence signals were acquired first under resting conditions (control perfusate) and then under state 5 (low respiration), which was induced by perfusing lungs for 5 min with perfusate containing potassium cyanide (complex IV inhibitor; 1 mM). Another group of lungs were perfused with control perfusate or potassium cyanide containing perfusate as above, after which the lungs were deflated and frozen rapidly for subsequent 3D cryoimaging. 2.2 Fluorometer: The fluorometer that we designed for this study (Fig. 1a) is equipped with two synchronized filter wheels, one to accommodate 10 excitation wavelengths, the other one to house 10 emission filters, a bifurcated fiber optic bundle, and a photomultiplier tube (PMT) for signal detection. The filter wheels communication with the computer is through a control box (Lambda- 3, Sutter instrument, CA). To control PMT gain, wheels synchronization, and data acquisition, a LabView program was developed. The bifurcated fiber bundle consists of high grade fused silica fibers for UV transmission (Newport instrument, NJ) with distal end of 3 mm inner diameter. For fluorescence spectroscopy, the peak energy of the NADH and FAD excitation spectrum occurs at 365nm and 436nm respectively. The emission filters for NADH and FAD fluorescence are 455nm and 525nm, respectively. The fluorometer was used in a dark room to acquire lung surface NADH and FAD fluorescence signals by placing the fiber optic probe against the pleural surface of the right lobe (Fig. 1b.). At the beginning of each tissue experiment, the fluorescence from the surface of a standard block with fluorescent paper was measured with the light guide at a fixed position and distance from the block. The measurement from the standard is required for normalizing the corresponding measurements from lung surface to account for day-to-day variations in light intensity and PMT gain settings.

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Figure 1. a) Schematic of Fluorometer. b) Top: The tip of the probe is acquiring surface NADH and FAD fluorescence. Bottom: During data acquisition the lights are turned off; the blue light seen on the picture is the timeshared excitation light.

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2.3 Cryoimager: The Cryoimager (Fig. 2) was used for 3D fluorescence imaging of frozen lung tissue. This instrument is an automated image acquisition and analysis system consisted of hardware and software designed to acquire fluorescence images of tissue sections. A motor-driven microtome sequentially sections frozen tissue at the desired slice's thickness while mercury filtered light excites fluorophores in the exposed surface of the tissue block up to four different fluorophores. The excitation light source is a 300W Cermax Xenon lamp filtered at the excitation wavelength of NADH and FAD, 366 nm and 470 nm, respectively. The emission wavelengths are 520nm and 450nm for FAD and NADH, respectively. At each slice, a CCD video camera records fluorescence images of the tissue block in pixel dimensions of 27.1 µm × 27.1 µm to be later analyzed for fluorescence distribution display. The microtome is housed in a freezer unit that maintains the sample at -40oC during sample slicing and image acquisition. The resolution in the z direction of microtome slices can be as small as 5μm. For this study, we used a resolution of 25.4μm in the z direction, which resulted in ~ 1000 zslices per lung [15].

Figure 2. Schematic of Cryoimager [15].

2.4 Data Processing: The fluorometry data contains time shared sequence of NADH and FAD signals collected from the surface of the lung. The data from each channel was smoothed out using a median filter (n=4) followed by a moving average (n=4) for each channel. The results were then used to determine the mitochondrial redox ratio (RR = NADH/FAD). The FAD and NADH autofluorescence cryoimages of frozen lungs were acquired in 1000 slices and processed using Matlab software. For each lung, the composite images were created using all image slices. The normalized redox ratio (RR = NADH/FAD) was then calculated, voxel by voxel, for each lung volume and a histogram was extracted from the redox volume.

3. RESULTS Figure 3 shows surface fluorescence data from a lung before (control perfusate) and after perfusion with KCN-containing perfusate. The baseline data (control perfusate) shows that lung inflation and deflation added noise to both NADH and FAD data (top and middle panels). The response of both NADH and FAD to KCN in perfusate was relatively fast, with NADH signal increasing by 22% and FAD signal decreasing by 8%, and as a result the redox ratio (RR = NADH/FAD) increased by 31%. The effect of KCN on NADH and FAD is consistent with its inhibition of cytochrome c oxidase (complex IV) and reduction of the mitochondrial respiratory chain. The effect of KCN on lung surface NADH and FAD fluorescence was reversible by perfusing the lung with control perfusate. Rapid freeze trapping of intact lungs in liquid N2 preserves their tissue metabolic state (NADH and FAD redox status). Subsequent low temperature NADH and FAD fluorescence imaging (cryoimaging) is particularly advantageous since it provides a higher fluorescence quantum yield of NADH and FAD as compared to room temperature, and the 3D spatial

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distribution of tissue NADH and FAD fluorescence intensities within the tissue [8]. Figure 4 shows 3D rendering of NADH and FAD fluorescence signals and their ratio (RR = NADH/FAD) from two lungs perfused with control perfusate (top panel) or KCN-containing perfusate (middle panel). The bottom panel shows a histogram of RR values for both lungs. These data show that KCN increased mean tissue NADH signal by 37%, decreased mean tissue FAD signal by 4%, and as a result increased mean tissue RR by 47%.

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Figure 3. Lung surface NADH (top panel), FAD (middle panel), and mitochondrial redox ratio (RR) (bottom panel) before (baseline) and after lung perfusion with potassium cynide (KCN).

4. DISCUSSION AND CONCLUSION The surface fluorometry and cryoimaging results are consistent with the expected effects of lung treatment with KCN, which inhibits cytochrome c oxidase and reduce the respiratory chain (state 5 respiration). Both techniques demonstrate a KCN-induced increase in tissue NADH signal, decrease in tissue FAD signal, and a result an increase in tissue RR. Both techniques reveal a larger KCN-induced change in NADH signal (22-37%) than FAD signal (4-8%) from baseline values (control perfusate). The 22% change in lung surface NADH fluorescence signal in response to KCN treatment is larger than the 7% change reported by Fisher et al. [13] in 1974 in rat lungs, reflecting the improvement in our fluorometer design with more sensitive detectors, and higher fiber optic light collection efficiency. Work is in progress to evaluate the effect of lung treatment with a mitochondrial uncoupler, which oxidizes the mitochondrial chain, on tissue NADH and FAD signals, and tissue RR. The results of this study demonstrate the utility of cryoimaging and fluorometry for measuring lung tissue NADH and FAD redox state, and lung tissue mitochondrial redox state ( NADH/FAD) in intact lungs, and suggest that lung surface RR measurements are representative of mean lung tissue RR. These results will be important for future studies designed to evaluate the effect of pulmonary oxidative stress (e.g. chronic hyperoxia, ischemia-reperfusion) on lung tissue mitochondrial redox state [14].

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Figure 4. NADH, FAD and mitochondrial redox ratio (RR) of lung perfused with control perfusate (top panel) or KCN-containing perfusate (middle panel). Bottom panel: Histogram distribution of RR for both lungs.

5. ACKNOWLEDGEMENT The authors would like to acknowledge the support of UWM Startup Fund, UWM Research Foundation Research fellow award, NIH grant HL-24349, and the Department of Veterans’ Affairs References.

REFERENCES [1] M. Ranji, et al., "Fluorescence spectroscopy and imaging of myocardial apoptosis," J Biomed Opt, vol. 11, p. 064036, Nov-Dec 2006. [2] M. Ranji, et al., "Quantifying acute myocardial injury using ratiometric fluorometry," IEEE Trans Biomed Eng, vol. 56, pp. 1556-63, May 2009.

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[3] M. Matsubara, et al., "In vivo fluorometric assessment of cyclosporine on mitochondrial function during myocardial ischemia and reperfusion," Ann Thorac Surg, vol. 89, pp. 1532-7, May. [4] B. Chance and H. Baltscheffsky, "Respiratory enzymes in oxidative phosphorylation. VII. Binding of intramitochondrial reduced pyridine nucleotide," J Biol Chem, vol. 233, pp. 736-9, Sep 1958. [5] C. H. Barlow, et al., "Fluorescence mapping of mitochondrial redox changes in heart and brain," Crit Care Med, vol. 7, pp. 402-6, Sep 1979. [6] R. S. Balaban and L. J. Mandel, "Coupling of aerobic metabolism to active ion transport in the kidney," J Physiol, vol. 304, pp. 331-48, Jul 1980. [7] E. Meirovithz, et al., "Effect of hyperbaric oxygenation on brain hemodynamics, hemoglobin oxygenation and mitochondrial NADH," Brain Res Rev, vol. 54, pp. 294-304, Jun 2007. [8] B. Quistorff, et al., "High spatial resolution readout of 3-D metabolic organ structure: an automated, lowtemperature redox ratio-scanning instrument," Anal Biochem, vol. 148, pp. 389-400, Aug 1 1985. [9] B. Vollmar, et al., "A correlation of intravital microscopically assessed NADH fluorescence, tissue oxygenation, and organ function during shock and resuscitation of the rat liver," Adv Exp Med Biol, vol. 454, pp. 95-101, 1998. [10] S. Nioka, et al., "Simulation of Mb/Hb in NIRS and oxygen gradient in the human and canine skeletal muscles using H-NMR and NIRS," Adv Exp Med Biol, vol. 578, pp. 223-8, 2006. [11] N. Ramanujam, et al., "In vivo diagnosis of cervical intraepithelial neoplasia using 337-nm-excited laserinduced fluorescence," Proc Natl Acad Sci U S A, vol. 91, pp. 10193-7, Oct 11 1994. [12] M. RANJI, "Fluorescent images of mitochondrial redox states of in situ mouse hypoxic ischemic intestines," Journal of Innovative Optical Health Sciences (JIOHS), vol. 2, pp. 365-374, 2009 2009. [13] A. B. Fisher, et al., "Evaluation of redox state of isolated perfused rat lung," Am J Physiol, vol. 230, pp. 1198-1204, May 1976. [14] S. H. Audi, et al., "Coenzyme Q1 redox metabolism during passage through the rat pulmonary circulation and the effect of hyperoxia," J Appl Physiol, vol. 105, pp. 1114-26, Oct 2008. [15] S. L. Bernard, et al., "High spatial resolution measurements of organ blood flow in small laboratory animals," Am J Physiol Heart Circ Physiol, vol. 279, pp. H2043-52, Nov 2000.

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