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1.IntroductionIschemia-reperfusion (I/R) injury of the liver is a common phenomenon and a major cause of morbidity and mortality in many clinical situations, including hepatic resection and liver transplantation.1, 2 Cellular damage in I/R injury is intensified when the transplanted liver is reperfused with blood after prolonged period(s) of ischemia in the donated liver.3 During orthotopic liver transplantation, oxygenated blood is reintroduced to the liver resulting in oxidative stress through free radical production.4 Hepatic injury in the transplanted liver results in intracellular adenosine triphosphate (ATP) loss, increased microvascular permeability, inflammatory cell infiltration, and potentially, cell death in the forms of apoptosis and necrosis.5, 6 As a consequence, oxidative stress may lead to graft failure and rejection of the transplanted liver.6 The reduced form of nicotinamide adenine dinucleotide (NADH) is essential in the production of ATP as an electron donor and a coenzyme.7, 8, 9 ATP can be produced in two ways: 1. through glycolysis, an anaerobic process occurring in the cytoplasm; or 2. through oxidative phosphorylation, an aerobic process occurring in the mitochondria.8 NADH can be free or protein-bound where binding occurs to a mitochondrial membrane protein, which mediates electron transfer from NADH to oxygen and results in H+ being transported to the cytoplasm. This yields a gradient that is necessary for ATP synthesis and occurs only in the presence of oxygen.9 Thus, bound NADH is involved in ATP synthesis in aerobic conditions.9 The free form of NADH is mainly located in the cytoplasm, where glycolysis occurs, producing ATP without oxygen.10 Accordingly, free and protein-bound NADH are involved in the production of ATP and changes in the ratio between the two forms gives insight into the metabolic state of a cell.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.15 The effective resolution in MPM is far superior to confocal microscopy because fluorescence only arises from the focus.15 Photodamage in MPM is therefore limited to the focal plane, however, the possibility of damage in this region still remains.15 Confocal microscopy, however, uses single photon excitation resulting in broad exposure to high-energy UV and visible excitation sources in the specimen above the focus plane, which could damage photosensitive samples.16 MPM is also better suited for deep-tissue imaging compared to confocal, because the infrared light used to generate multiphoton excitation is less scattered in biological samples.16 This results in a tightly focused beam with high-photon flux enabling deeper imaging into thick tissue while restricting photobleaching and phototoxicity.17, 18 The two or more low energy photons in MPM are simultaneously absorbed in order to excite an electron from a fluorescent molecule.19, 20 The photons have similar energies and produce an excitation equivalent to single-photon absorption possessing twice the energy.15 The images originate at the beam focus with no out of focus background present.17 Fluorescence lifetime imaging microscopy (FLIM) is based on the average time it takes for an electron to return from the excited state to the ground state. It adds to the information from MPM by making it possible to resolve multiple lifetimes from decay data.21 The advantage of FLIM is that it is dependent on excited state reactions, but independent of the concentration of the fluorescent molecule.22 The concept of FLIM has been simply explained by Lakowicz Briefly, the fluorescence intensity cannot reveal any environmental changes, such as protein binding or pH, but FLIM can.23 Therefore, FLIM provides details about the structure and dynamics of fluorescent molecules12 and is sensitive to changes in conformation, molecular interactions, ion concentrations, pH9 and oxygen concentrations.24 Our previous results using MPM-FLIM focused on nanoparticle and solvent uptake in both excised14, 25, 26, 27 and in vivo 28, 29 skin. In addition, this method has been used to study fluorescein distribution and metabolism in the liver in vivo in rats.14, 30 In the present study, we utilized the autofluorescent properties of the liver cells to investigate cellular damage progression in I/R injury. NADH has fluorescent properties with peak absorption at 350 nm and peak emission of approximately 450 nm.10 The free and protein-bound forms of NADH have similar excitation and emission wavelengths, but can be separated using their distinct fluorescence lifetimes of 0.4 to 0.5 and 2.0 to 2.5 ns, respectively.10 Nicotinamide adenine dinucleotide phosphate (NADPH) is another reducing agent used in biosynthesis of fatty acids and steroids. NADH and NADPH have the same excitation and emission wavelengths and fluorescence lifetimes, and cannot be spectrally resolved.31 Hence, NADH and NADPH are referred to as NAD(P)H in this paper. However, NADPH has lower fluorescence intensity, lower concentration, and is roughly constant with respect to metabolic perturbations compared to NADH.32 The influence of NADPH is therefore considered very low, although not insignificant. 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. 2.Materials and Methods2.1.AnimalsMale Wistar rats, purchased from the Animal Resource Centre (Perth, Western Australia), weighing approximately 300 g were used in all experiments. Studies were approved by the Animal Ethics Committee of the University of Queensland and were carried out in accordance with the legislation of Australian authorities for the care and use of experimental animals. Animals were housed in the Biological Resource Facility at the Princess Alexandra Hospital, where the temperature is maintained at 20 ± 1°C and humidity at 60 to 75%, with artificial light for 12 h (7 am to 7 pm) daily. All animals had unlimited access to food and water. 2.2.Surgical ProceduresRats were anaesthetized by an initial intraperitoneal injection of xylazine 10 mg/kg and ketamine hydrochloride 80 mg/kg. Anaesthesia was maintained throughout the experiment by intraperitoneally administering ketamine (2.2 mg/100 g) and xylazine (0.25 mg/100 g). Body temperature was controlled by placing them on a heated pad set to 37°C. A midline laparotomy was performed with the left lobe of the liver exposed for imaging. 2.3.I/R Model70% ischemia was induced by clamping the portal vein, hepatic artery, and bile duct supplying the median and left lobes of the liver using a microvascular clamp. After 60 min of partial ischemia, the clamp was removed to allow reperfusion in the liver. Sham rats (controls) underwent the same procedures without clamping the vessels. 2.4.Tissue CollectionBlood (0.2 ml) was collected from the inferior vena cava using a 30-gauge needle before ischemia induction and at 2 and 4 h of reperfusion. After the animal was euthanized at the end of 4 h reperfusion, the left and median lobes were excised, portions were either snap frozen in liquid nitrogen and stored at −70°C for later analysis of glutathione (GSH), or immersed into 10% buffered formalin for histological assessment. 2.5.MPM-FLIMMPM was performed using a DermaInspect system (JenLab GmbH, Jena, Germany) equipped with an ultrashort (85 femtosecond pulse width) pulsed mode-locked 80-MHz Titanium:Sapphire MaiTai laser (Spectra Physics, Mountain View, California). For FLIM, a time correlated single photon counting 830 detector (Becker & Hickl, Berlin, Germany) was incorporated into the MPM system. The excitation wavelength was set to 740 nm for FLIM (emission range 350 to 450 nm) and 780 nm for imaging of autofluorescence. For imaging of the liver, the left lobe was placed on a small metal plate, attached to an adjustable stand that could be elevated or lowered as required. The plate was slightly raised above the intraperitoneal cavity to minimize pressure on the organs underneath. The laser power was 15 mW and a BG39 (350 to 650 nm) emission filter was used. The acquisition time for obtaining the images was 13.4 s per frame. Images were taken pre-ischemia induction, during ischemia, and at 0, 1, 2, and 4 h of reperfusion. 2.5.1.Data analysisFLIM images were analyzed using SPCImage (Becker & Hickl, Berlin, Germany). Lifetime distributions for NAD(P)H were obtained by fitting photon count F(t) profiles from each image to a bi-exponential decay function ( [TeX:] $F(t) = \alpha_1 e^{ - t/\tau _1 }\break + \alpha_2 e^{ - t/\tau _2 }$ ). The reduced chi-square coefficient was used to indicate goodness-of-fit. Two lifetimes, τ1 and τ2 represent the “fast” and “slow” lifetimes of free and protein-bound NAD(P)H, respectively. The amplitudes α1 and α2 represent the relative concentration fraction of NAD(P)H, where α1 + α2 = 100.12 The ratio of a 1 and a 2 is the best way to determine the free and protein-bound state of NAD(P)H, therefore showing the metabolic changes of the cell. In this study the actual ratio of α1 and α2 is shown. In addition, the ratio of α1 and α2 is also presented as pseudo color images exported from the analyzing software, where a change in color indicates a change in ratio. Actual changes in lifetimes, τ1 and τ2, are not shown in this study. For statistical analysis, student t-test for paired samples and one-way ANOVA with Tukey's adjustment for multiple comparisons were used. Results were considered statistically significant with a p-value ≤ 0.05. 2.6.Histopathological AnalysesStaining of haematoxylin and eosin (H&E), ApopTag (for apoptosis) and immunohistochemistry for ED1 (for macrophages) was performed according to standard procedure. Visualization of ApopTag and ED1 localization was facilitated with diaminobenzidine hydrochloride, which stained brown in positively-labeled cells. Fixed liver was sub-sectioned onto each slide (two to three sections at 5 μm from different slices of each liver), stained, and then scanned using an Aperio ScanScope XT slide scanning system (Aperio Technologies Inc., Vista, California) at 20× magnification. The Aperio ImageScope V10.2. software was used to visualize and score the slides. Severity of tissue damage was quantified in five areas per slide by two independent personnel using a previously described method.33 Three sets of parameters were studied: a. cellular degeneration defined as cellular swelling and vacuolation, and reversible changes in nuclear density; b. infiltration of neutrophils, monocytes, and macrophages; and c. necrosis defined by cells showing pyknosis, karyorrhexis, karyolysis, and cytolysis. These parameters were graded as follows. Grade 1: no change from normal; Grade 2: a limited number of isolated cells (up to 5% of cells); Grade 3: groups of cells (5 to 30% of the total cell number); and Grade 4: diffuse cell damage (higher than 30% of the total cell number). In addition, zonal localization of tissue damage was recorded. Using immunohistochemistry for ED1, the number of monocytes and macrophages were identified by counting ED1-positive cells in the sinusoids in 10 random fields per slide. Apoptotic cells were identified enzymatically using ApopTag labeling, and positive nuclei were counted in 10 random fields per region per liver slide. The three different regions were 1. periportal (cells surrounding the portal tract), 2. centrilobular (cells surrounding the central vein), and 3. midzonal (cells between the periportal and centrilobular zones). 2.7.Serum Biochemical MeasurementsPlasma concentration of alanine aminotransferase (ALT) was measured using a Hitachi 747 analyzer (Hitachi Ltd., Tokyo, Japan) in the Pathology Department at Princess Alexandra Hospital, Brisbane. 2.8.Determination of Total Glutathione (GSH)GSH was determined using the Glutathione assay kit (Cayman Chemical Company). The kit is based on the reaction between GSH and DTNB [5,5′-dinitro-bis-2-(nitrobenzoic acid)], which produces a yellow colored TNB (5-thio-2-nitrobenzoic acid). The production of TNB is directly proportional to the concentration of GSH. 3.Results3.1.Alanine Transaminase (ALT)The model of ischemia used resulted in extensive liver damage as measured by increased liver ALT. ALT was significantly (p < 0.05) increased at 2 (1120 ± 308 U/L) and 4 h (1820 ± 600 U/L) of reperfusion compared to sham (Table 1). Table 1Alanine aminotransferase before (pre) and after ischemia induction at 2 and 4 h of reperfusion. [* p < 0.05 versrus sham, # p < 0.05 versus pre. Mean (±SEM).]
3.2.Oxidative StressAs a measure of oxidative stress, GSH concentration at 4 h I/R was determined in excised livers. GSH concentration was significantly (p < 0.05) reduced in the I/R group compared to sham (Table 2). Table 2Levels of oxidative stress were measured by glutathione concentration in the liver at 4 h of reperfusion in I/R injury and compared to sham rats (±SEM). (* p < 0.05 versus sham.)
3.3.Histopathological FindingsMild to moderate structural alterations in the liver were found in the I/R injury group [Fig. 1a]. Cellular vacuolation was present in all sections from the I/R injury group, with scores from 2 to 4 for cell degeneration. Some inflammatory cell infiltration was present in sham and I/R rats at 4 h of reperfusion (Table 3). There was no statistical difference in the number of monocytes and macrophages, stained with ED1, as seen in Fig. 1b. The number of monocytes and macrophages tended to be slightly decreased in I/R injury (240 ± 50) compared to the sham group (310 ± 70), however this was not significantly different. Fig. 1Histology of liver tissue after I/R injury. (a) H&E stained liver tissue shows increased vacuolation in the I/R group. White arrow indicates vacuoles. (b) ED1 stained liver showing monocytes and macrophages stained in brown. Black arrows show inflammatory cells. (c) ApopTag stained liver showing apoptotic cells in the three different regions in the liver lobule, periportal, centrilobular, and midzonal. Black arrows indicate apoptotic cells. Magnification 20×, sham n = 5, I/R n = 6.  Table 3Semi-quantitative categorization of the morphological injury parameters in liver tissue after I/R injury. Scoring from 1 to 4, where 1 is no and 4 is a major change (±SEM). (* p < 0.05 versus sham.)
The number of apoptotic cells was statistically increased in the centrilobular and midzonal regions compared to the same regions in the sham group [Fig. 1c, Table 4] (p < 0.05). There was a slight increase in the number of apoptotic cells in the periportal region, however, not statistically significant (Table 4). Table 4Number of apoptotic cells in the three regions of the liver lobule. ApopTag-labeled nuclei were counted in 10 different fields per zone of the liver acinus (mean ± SEM). (* p < 0.05 versus I/R injury, # p < 0.05 versus periportal and centrilobular.)
3.4.MPM-FLIMThe autofluorescence of the liver in vivo was imaged with high resolution at 0, 1, 2, and 4 h of reperfusion. Figure 2 shows representative images from one rat in the I/R injury group and one in the sham group. A large dark area is seen at 4 h of reperfusion in the I/R group, which cannot be seen to the same extent in sham rats (Fig. 2). In addition, vacuoles were formed within the cells in some of the images taken after ischemia, and this was not seen in sham rats. Figure 3a shows an example of such a phenomenon at 1 h of reperfusion. The same experiment was done using a lower magnification objective to visualize the entire liver acinus. Figure 3b shows sample images from one rat before the start of ischemia (control) and at 4 h of reperfusion. Dark patches are evident in different areas around the acinus at 4 h of reperfusion that was not seen in the sham group. Fig. 2Multiphoton microscopy images of liver in vivo in I/R injury at various reperfusion time points compared to sham rats. The white arrow indicates a large dark area not seen in the sham group.  Fig. 3Multiphoton microscopy images of liver tissue on cellular and acinus levels. (a) Images recorded at 1 h of reperfusion in the I/R group using high and (b) at 4 h using low magnifications (40× and 10×, respectively). White opened arrow indicates vacuolation and solid arrows indicate increased dark areas.  FLIM measures the relative contribution of free/bound NAD(P)H in the liver in vivo in I/R injury. The relative concentrations of free (α1) and bound (α2) NAD(P)H were calculated and plotted against time (Fig. 4). The ratio of free/bound NAD(P)H was significantly increased after 2 and 4 h of reperfusion [p < 0.05; Fig. 4a]. This increase was due to a statistical increase and decrease in the relative concentration of free [Fig. 4b] and bound [Fig. 4c] NAD(P)H, respectively. Fig. 4Changes in free and bound levels of NAD(P)H recorded by fluorescence lifetime imaging microscopy. (a) Ratio of free/bound NAD(P)H was significantly increased at 2 and 4 h of reperfusion in the I/R group compared to sham. This increase was shown to be due to an increase in (b) free NAD(P)H and a decrease in (c) bound NAD(P)H, respectively. (d) Relative concentration of free/bound NAD(P)H is expressed in pseudo color images. The color of the cells in I/R injury shifts toward yellow over time indicating an increase in the ratio of free/bound NAD(P)H. This increase was not seen in sham rats. *p < 0.05 versus sham (n = 4).  Another way of showing the relative concentration of free/bound NAD(P)H is to false color the images obtained from the FLIM analysis. Figure 4d shows sample images from one rat in the I/R injury group and another from the sham group. The color of the images (from blue to red) indicates the ratio of free/bound NAD(P)H. At 1, 2, and 4 h of reperfusion there is a shift in the color to yellow, indicating an increase in the ratio as seen in the color bar. 4.DiscussionFluorescence microscopy can provide valuable information on tissue morphology without the need for histological assessment.34 The technique utilizes the autofluorescence of tissue components such as NADH,34 flavine adenine dinucleotide,35 and other flavoproteins.36 However, most previous studies have been conducted in vitro with only a few studies performed on live animals.34 External markers for liver damage such as rhodamine 123 and propidium iodide have previously been used to study mitochondrial permeability transition and necrosis, respectively.37, 38 However, the aims of this study were to investigate whether we could use the autofluorescent properties of the tissue to detect cell damage using MPM in combination with FLIM. MPM uses a high-pulsed laser, which provides low average energy received by the sample imaged.39 In addition, MPM restricts excitation of a fluorescent molecule to the focal plane of the microscope.39 Consequently, MPM significantly improves the quality of images acquired from biological tissue, by reducing photodamage and photobleaching of tissue compared to conventional fluorescence microscopy.17, 19, 20 The model of ischemia used resulted in extensive liver damage, as measured by increased liver ALT (Table 1) and depletion of one of the cellular defence mechanisms against free radicals (GSH) (Table 2). Early ischemia reperfusion injury has in previous studies resulted in similar increases of ALT and GSH levels.40, 41 It has been shown that this injury is mediated through a release in free radicals both within the cell and outside through Kupffer cell activation.42 It was evident that hepatocytes became vacuolated after 1 h of reperfusion, indicating cellular stress43 that was not evident in sham rats [Fig. 3a]. In addition to the fluorescence intensity images, traditional H&E staining of the liver also showed vacuolation in the hepatocytes [Fig. 1a], which confirms the findings of Sireli, who reported swollen hepatocytes with vacuolated cytoplasm during I/R injury.44 Additional immunohistological staining for monocytes and macrophages showed no increase in the number of these inflammatory cells in the I/R injury group. Since the liver has its own macrophages, called Kupffer cells, that line the sinusoids, it is probable that ED1 would stain those as well.45 The number of Kupffer cells should stay constant in the liver, which would explain why the number of stained monocytes and macrophages are similar in both groups.45 In the images recorded by MPM, large dark areas were noticed at 4 h of reperfusion in the I/R injury group that could not be seen in any of the sham rats. A decrease in fluorescence is most likely due to a decrease in NAD(P)H fluorescence, which occurs in cell death.46 Using high magnification, this dark area was seen in three out of four I/R animals. We, therefore, suspected a zonal spread in the liver damage and investigated this at lower magnification. While the results showed variation in intensity of light and dark, the dark areas of the liver were localized away from the periportal region. To confirm the heterogenic spread in the damage the harvested livers were histopathologically analyzed. ApopTag stained cells, staining DNA damaged nuclei,47 were significantly increased in the centrilobular and midzonal regions, but not in the periportal region. Early ischemia reperfusion injury induces a burst of free radicals,42 which in turn cause DNA strand breaks in the nuclei of the cells. Although the strand breaks may predetermine progress to apoptosis, they may also be repairable and so the injury is reversible. ApopTag enzymatic labeling, by its very nature,48 will label both repairable and apoptosis-inducing DNA strand breaks. The nuclei that stained positive with the ApopTag as shown in Fig. 1c, but had apparently normal structure after the (4 h) reperfusion, may ultimately take either pathway. Our group is now focusing on the long term damage of hepatic I/R injury. A review of the literature suggests that there is an altered distribution of zonal damage associated with different liver diseases. For example, hypoxia and free radical production have been reported to affect cells around the central vein in alcohol induced liver injury,49 whereas midzonal parenchymal cells were more susceptible to oxidative stress in cancer and cirrhosis.50 Furthermore, liver damage was more pronounced in centrilobular and midzonal regions after acetaminophen administration.51 The brighter parts of the images are likely explained by an increase in NAD(P)H concentration during I/R injury. Barbiro showed a significant increase in NADH levels in hepatic I/R injury,52 and similar results have been reported in hypoxic hepatocytes in vitro.53 NADH loses fluorescence during oxidation to NAD(P)+, thus NAD(P)H fluorescence can be used to monitor metabolism.54 However, measuring total concentration of NAD(P)H can be misleading because it is not possible to know whether the fluorescence is derived from free or protein-bound NAD(P)H.32 FLIM is, therefore, a better probe for studying changes in NAD(P)H because the lifetime of the molecule is significantly enhanced with binding.32 The amplitude (α) can be related to the concentration of NAD(P)H9, 54, 55 and the ratio of free/bound (α1/ α2) NAD(P)H was calculated at different reperfusion time points. A significant increase was seen at 2 and 4 h of reperfusion [Figs. 4a and 4d]. This was attributed to an increase in free and a decrease in bound NAD(P)H [Figs. 4b and 4c]. It is possible that the increase in free/bound NAD(P)H seen in this study is attributable to the Pasteur effect, where cellular respiration is shifted to glycolysis, producing ATP in anaerobic conditions.56, 57 An increase in glycolysis leads to an increase in lactic acid production,35 which was previously reported in pig liver during warm ischemia.58 In addition, increased levels of lactic acid were seen in patients undergoing hepatectomy, which further coincides with the theory of shift to glycolysis in I/R injury of the liver.59 Our group has previously shown an increase in free NAD(P)H in skin after zinc oxide application in vivo.28 In conclusion, liver damage associated with I/R injury, with up to 4 h of reperfusion, was structurally characterized by a zonal distribution of vacuolated and degenerating hepatocytes, increased apoptosis, and mild necrosis. The damage was mainly localized in the midzonal region. MPM was able to detect both cellular degeneration and the zonal spread of damage, by showing irregular fluorescence intensity of the hepatocytes. FLIM added to these results by detecting changes in cellular respiration, which was attributed by an increase in free NAD(P)H and a decrease in bound NAD(P)H in I/R injury. This is an important and novel finding in that it allows us to follow disease development without the administration of an external marker in vivo. AcknowledgmentsNational Health and Medical Research Council (NHMRC #569710) is acknowledged for funding this project. Clay Winterford and colleagues at Queensland Institute of Medical Research (QIMR) prepared the histology and immunohistochemistry. Goce Dimeski and colleagues at Queensland Pathology Services, Princess Alexandra Hospital, conducted ALT measurements. 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