Superoxide anion is the key radical that causes intracellular oxidative stress. The lack of a method to directly monitor superoxide concentration in vivo in real time has severely hindered our understanding on its pathophysiology. We made transgenic zebrafish to specifically express yellow fluorescent proteins, a reversible superoxide-specific indicator, in the liver and used a fiber-optic fluorescent probe to noninvasively monitor the superoxide concentration in real time. Several superoxide-inducing and scavenging reagents were administrated onto the fish to alter superoxide concentrations. The distinct biochemical pathways of the reagents can be discerned from the transient behaviors of fluorescence time courses. These results demonstrate the feasibility of this method for analyzing superoxide dynamics and its potential as an in vivo pharmaceutical screening platform.
©2013 Optical Society of America
Reactive oxygen species (ROS) are oxygen-derived molecules that have high chemical reactivity and can readily oxidize other molecules. They are essential for the regulation of a wide variety of cellular functions . However, excessive production of ROS leads to oxidative stress in cells and eventually results in apoptotic or necrotic cell death . Increasing evidence reveals the relation between high ROS level and a panel of distinct clinical disorders, such as cancer, aging, inflammation, etc [3, 4]. Most intracellular ROS are derived from superoxide anion (O2−), which is the primary oxygen free radical generated through the electron transport chain (ETC) in mitochondria [1, 5]. Therefore, there are great interests to quantify the concentration of superoxide in vivo in real time to reveal its physiological functions. Nevertheless, current superoxide analysis can only be done in vitro and real time monitoring is not possible due to the lack of a reversible superoxide-specific indicator [6, 7].
Current superoxide detection methods include electron paramagnetic resonance (EPR) , aconitase inhibition , and 2-hydroxyethidium quantification . These techniques require extraction and pulverization of tissues, renders them only suitable for in vitro analysis. Although fluorescent or luminescent probes, such as MitoSOX-red , 2′,7′-dihydrodichlorofluorescin (H2DCF) , and lucigenin , are developed for in vivo detection and are widely used, their reactions are not reversible. Therefore they cannot be used to investigate superoxide dynamics induced by treatments in real time. Some real time in vivo superoxide detection methods have been proposed, e.g., using a cytochrome-c-coated carbon electrode  and an electrochemical O2− sensor . However, they are all invasive.
Recently, a circularly permuted yellow fluorescent protein (cpYFP)  was developed as a reversible superoxide-specific indicator. This protein has been used to monitor superoxide production in the mitochondria of myocytes in real time . This cpYFP is ideal for the quantification of superoxide generation in animal model in vivo in real time. We have engineered the cpYFP to be specifically expressed in liver cells of transgenic zebrafish, an emerging animal model for human disease studies . The embryo and the juvenile zebrafish are almost transparent to visible light as shown in Figs 1(a) and 1(b). The fluorescent liver can be easily located using a fluorescent microscope. This makes zebrafish an ideal vertebrate animal model for in vivo pathological studies by optical means [18, 19]. Using zebrafish model to study the pathology of liver and intestinal organ diseases has been proven to be effective [20–22].
Oxidative stress has been implicated in the pathogenesis of chronic liver diseases, such as hepatitis and liver cancer . It has been known that hepatitis virus infection is the main cause of chronic hepatitis, which may progress into malignant liver disease. Current hypothesis is that the delivery of hepatitis virus core protein into hepatocyte may partially disable the ETC on the mitochondrial inner membrane, which alters the Ca2+ retention and leads to magnified ROS generation . However, the crucial link between chronic abnormal increase of ROS and hepatitis or hepatocellular carcinoma is still unresolved . The ability to do real time monitoring of the concentrations of ROS, especially superoxide, would greatly advance our understanding about the pathophysiology of ROS induced chronic liver diseases.
In this report, we demonstrate the use of a compact and flexible fiber-optic probe [25, 26] to monitor generation of superoxide in the liver of transgenic zebrafish in real time in vivo. The zebrafish are treated with various superoxide-inducing or scavenging reagents and the fluorescent signals from cpYFP are monitored accordingly. We use microfluidic channels to immobilize the fish and for reagents administration. The liver can be easily located using a microscope as it fluoresces when illuminated by the fiber as shown in Figs. 1(c) and 1(d). There are several advantages to use the compact fiber probe for optical investigation on zebrafish. First, the fiber probe can be inserted in the liquid where the fish is placed and attached on the skin of the fish. Therefore the air-liquid index mismatch and the scattering caused by the skin can be minimized. The signal collection efficiency is only limited by the numerical aperture (NA) of the fiber, which can be better than a bulky fluorescent microscope. Besides, the illuminated area is controlled by the size and the NA of the fiber probe. For the system presented here, the diameter of illumination is on the order of 200 μm, which is a perfect fit to the size of the liver. In addition, because only the liver area is illuminated, we have minimized influence from autofluorescence of surrounding tissues and organs. To the best of our knowledge, this is the first demonstration of real-time noninvasive investigation of superoxide dynamics in an intact animal model with the help of fluorescent proteins [13, 14].
2. Materials and methods
2.1 Fiber-optic fluorescence monitoring system
The optical setup is shown in Fig. 2(a). The wavelength of the excitation laser is 473 nm. The laser is focused into the inner cladding of the fiber through an objective lens and the fluorescent signal is collected back through the same fiber and the objective lens. A neutral density (ND) filter is used to control the laser power on the sample. The single-fiber configuration is compact, robust and most efficient in signal collection [27, 28].The fiber used here is a double-clad fiber (cladding diameter = 125μm, NA>0.46; core diameter = 10μm, NA = 0.08, P-10/125DC, nLight corp.). The double-clad fiber is adopted due to its high NA [29, 30]. After transmitting through the dichroic mirror (DM, MD499, Thorlabs Inc.), the signal is filtered (filter 1, MF 530-43, Thorlabs Inc.) and focused into a photodiode (detector 1, DET36A, Thorlabs Inc.). In order to make sure the measured subtle fluorescence intensity fluctuation is truly from variation of superoxide concentration, the excitation laser intensity is monitored simultaneously to exclude its influence. The stray laser light after the DM is monitored with a photodiode (detector 2, DET36A) after passing through a bandpass filter (filter 2, MF475-35, Thorlabs Inc.). The signals from the photodiodes are sent into a computer controlled data acquisition card (USB-6251, National Instruments) for recording and analysis.
Before the in vivo measurements, the quantification ability of the fiber probe was characterized. The fiber probe was dipped into fluorescein isothiocyanate (FITC) solutions of concentrations from 10−6 to 10−3 M. The measured fluorescence intensity closely followed a linear dependence on the concentrations (data not shown). The signal to noise ratio (SNR) was characterized to be 26 dB at the signal level of in vivo measurements. The laser power at the fiber probe end was maintained at 0.5 mW. During in vivo studies, the probe was placed directly on the skin of the fish with the assist of a stereo microscope as shown in Fig. 1(c), 1(d). Because only the liver fluoresces, it can be easily located by optimizing the fluorescent signal collected by the fiber probe.
2.2 Microfluidic channel
In order to maintain the zebrafish in an aqueous condition and for ease of reagents administration, we designed a Y-type microfluidic channel as shown in Fig. 2(b). The zebrafish was placed in the shaded area after sedation treatment. The microfluidic chip was made of polymethylmethacrylate (PMMA) and the microchannels were made by laser machining. The depth of the microfluidic channel is 1mm and the width varies from 0.6 to 0.8 mm to accommodate larvae of different sizes due to their age characterized by dpf (days post fertilization). Valve A and B control the flow of reagent A and B. Only one valve is open at a time. By continuous pumping the syringe pump, the concentration of the reagent around the fish is maintained and the fish is immobilized in the examining area.
2.3 Animal model and test reagents
The study protocol was approved by the Institutional Animal Care and Use Committee of the University. The transgenic zebrafish line with cpYFP specifically expressed in the mitochondrial matrix of liver cells was developed in the Free Radical Biotechnology Laboratory in the National Changhua University of Education. The zebrafish were maintained and cultured in accordance with the standard procedures . Juvenile zebrafish of 6 to 7 dpf were anesthetized with 0.1-0.2% 2-phenoxyethanol before loading into the microchannel. For each experimental setting, parallel measurements were performed on at least five fish.
In order to investigate the production and reduction of superoxide anions, we used superoxide-inducing reagents, such as paraquat  (PQ, 4 mM), thioacetamide [33, 34] (TAA, 32 mM), and copper ions  (Cu2+, in the form of copper sulfate, 10 ppm) to induce the generation of superoxide and used superoxide-scavenging reagents, such as ascorbic acid  (AA, 50 mM) and silymarin  (5000 ppm) to reduce superoxide concentrations.
3. Results and discussion
The measured fluorescence intensity time courses are shown in Fig. 3. The fluorescence intensity time courses of three cpYFP transfected zebrafish (control-1,2,3) and a wild-type zebrafish, which does not have cpYFP, are plotted together in Fig. 3(a) for comparison. The fluorescence intensity from the wild-type zebrafish is close to the basal level, while the fluorescence from the liver of cpYFP transfected zebrafish is more than two orders of magnitude brighter. Comparing the time courses of three control zebrafish, the fluorescence intensities, which reflect the concentrations of cpYFP and superoxide, are evidently all at a similar level. However, each time course has a different transient behavior and fluctuation range, which is common in in vivo measurements due to distinct physiological condition of individual fish. One common characteristic of the intensity measurements is that they all decay gradually. The reduction in the fluorescence intensity can be due to reduced superoxide or cpYFP concentration in the cells. More specifically, when the fish were treated with anesthetic drug and transported to the microchannel, the process might have raised the oxidative stress and stimulated more superoxide generation. The superoxide anions were then gradually scavenged in the cells as normally. In addition, because of the sedation treatment, the generation rate of the fluorescent proteins and the superoxide anions in the liver cells may decrease due to decelerated metabolism, contributing to the gradually decaying intensities. Although photobleaching might also contribute to the decaying of fluorescence intensity, because the low laser power and the large illumination area, the effect is negligible compared to the changes due to physiological conditions.
Representative fluorescent intensity time traces when PQ, Cu2+, or TAA was used as the superoxide-inducing reagent are shown in Figs. 3(b), 3(c), or 3(d), respectively. When treating PQ, a common superoxide-inducing reagent, to the zebrafish, the cpYFP fluorescence intensity increased immediately, indicating a generation of superoxide in liver cells. After reaching a maximum, the fluorescent signal decreased gradually due to self-scavenging of superoxide within the cells. One minute later, ascorbic acid (AA) solution was administrated onto the fish (Fig. 3(b-1)), causing the fluorescence intensity to decrease drastically as a result of its strong superoxide scavenging effect . When silymarin was applied as the scavenging agent (Fig. 3(b-2)), a similar reduction in the fluorescence intensity was observed. The significant quenching magnitude and fast decay rate reveal the strong antioxidant properties of silymarin . After another one minute (at about 160-170 second), PQ of the same concentration was treated again on the fish. The fluorescence intensity ceased decay and only a mild increase was observed. The fluorescence intensity stayed at a similar level until the superoxide scavenger was applied again. We observed similar dynamics irrespective of the superoxide-inducing reagent in subsequent experiments. Since the cpYFP is a reversible superoxide-specific protein, after the superoxide anions were scavenged, the remaining cpYFP should still be reactive to newly generated superoxide. Therefore one would expect to see the fluorescence intensity to rise back to a similar level as the first time since the fish is immersed in the reagent solution of the same concentration. One and the most possible reason for the lower fluorescence intensity is that the remaining antioxidants within the fish might prevent excessive production of superoxide. It is also possible that the cells might have been damaged when the superoxide inducer was applied for the first time as the concentrations of the reagents were relatively high compared to normal physiological conditions. Since this is the first study of superoxide dynamics in the liver of live animal in such a short time scale, we have not found relevant literature to elucidate the observed phenomenon. A study performed in a longer time scale is under way to look into this phenomenon. For extended time scale studies, the fish is released from the microfluidic device and revived back to the tank after the first run of treatment. After a certain period when the remaining reagents are cleared from the fish, it will be anesthetized and treated with the reagents again to evaluate the effects from multiple treatments.
Next, copper ions (Cu2+) were used as the superoxide-inducing agent. Copper ions can undergo Fenton reaction to directly generate superoxide anions in the cells , hence we expect to see an immediate response in the superoxide concentration . The representative time courses are shown in Fig. 3(c). The fluorescence intensities indeed increased immediately after the administration of copper sulfate to the fish. After reaching a plateau, the fluorescence intensities decay gradually as in the case of PQ. When administered AA or silymarin to the fish, the fluorescence intensities decay more rapidly due to their superoxide-scavenging abilities. However, it can be seen in the figures that the decay rates are not as high as those in the case of PQ. This might be due to the fact that Cu2+ is a more effective superoxide generating agent and the remaining Cu2+ ions in the fish continued generating superoxide anions. After about 2 minutes, the copper sulfate was administrated again to the fish. Similar to the case of PQ, the fluorescence intensity ceased decay, and stayed at a stable level. When the superoxide scavengers were treated again, the fluorescence intensities gradually decreased again. By looking into the effectiveness of AA and silymarin, one intriguing phenomenon was noticed. Almost every time when AA was applied, a unique sudden increase in fluorescence was observed preceding the decay, which is not observed for silymarin. Although the magnitude of increase might vary, this behavior is repeatable (Fig. 3(c-1)). This is believed to be due to the ability of AA to reverse photoionization on fluorophores, which results in the restoration of fluorescence .
For the long run, we plan to use this zebrafish model to study the impact of oxidative stress on chronic diseases in liver. Thioacetamide (TAA) is a highly-specific hepatotoxin, and is frequently used in animal studies to induce oxidative stress in liver . The fluorescence intensity time courses when TAA were treated are shown in Fig. 3(d). The TAA indeed increased the superoxide concentrations in liver cells as the time courses implied. However, the increase rates (slope) were obviously slower than those of PQ and Cu2+. This is because TAA stimulates oxidative stress in hepatocytes through an indirect way. The metabolites of TAA produced by liver deplete glutathione, a crucial element for ROS detoxification in mitochondria. Thus, mitochondrial metabolism is disturbed, which in turn generates excessive superoxide anions . Such cellular biochemistry is evidently revealed in the transient behaviors of fluorescence intensities. This demonstrates the unique advantage of our method. When AA was applied, fluorescence restoration caused by AA was noted and followed by fluorescence decay as previously (Fig. 3(d-0), 3(d-1)). The fluorescence restoration effect was more profound here, which might be due to the indirect mechanism of superoxide stimulation by TAA. Similar superoxide dynamics as in the previous cases were observed when the reagents were administrated subsequently. The antioxidants protected the cells from overly production of superoxides. The marked detoxification property of silymarin in liver is exhibited in its immediate effect and significantly reduced superoxide concentration .
Our results demonstrated the feasibility of using the compact fiber probe to monitor superoxide generation dynamics in the liver of zebrafish in a sub-second time scale. Because the fiber probe has superior signal collection efficiency and rejects autofluorescence background from surrounding tissues, we have excellent SNR. Therefore, the fluorescence intensity variations truly represent the effects of the reagents. Except for the advantage of low cost as compare to conventional fluorescent microscope, a real promise of fiber-optic technology is its ability to integrate multiple input/output into a compact monolithic system. By using an integrated monolithic system with multiple fiber probes, it is possible to simultaneously monitor multiple zebrafish treated with different drugs, establishing a high-throughput drug screening platform.
We have demonstrated the use of a high numerical aperture single-fiber fluorescent probe to conduct real time monitoring of superoxide dynamics in vivo in transgenic zebrafish. By measuring the fluorescence intensity of cpYFP in the liver of zebrafish, the concentration of superoxide anions can be quantified accordingly. The efficacy of superoxide-modulating reagents can be inferred from the magnitude and rate of change in fluorescence time courses, which reflect the dynamics of their profound biochemical actions. We envision this method to have fundamental impact on the studies of oxidative stress and related chronic diseases.
The project was supported by the National Science Council of Taiwan through grants 100-2628-E-018-002, 101-2628-E-018-001, and 99-2313-B-018-001-MY3. We thank the assistance from members at the Free Radicals Biotechnology Lab. We are also grateful to the discussions and technical suggestions from Prof. Kerwin Wang at the Department of Mechatronics.
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