Online Electrochemical Monitoring of Dynamic Change of

Sep 20, 2013 - This study demonstrates the validity of an online electrochemical system (OECS) for ascorbate detection as a platform for in vivo evalu...
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Online Electrochemical Monitoring of Dynamic Change of Hippocampal Ascorbate: Toward a Platform for In Vivo Evaluation of Antioxidant Neuroprotective Efficiency against Cerebral Ischemia Injury Kun Liu,†,‡ Ping Yu,† Yuqing Lin,† Yuexiang Wang,† Takeo Ohsaka,§ and Lanqun Mao*,† †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100190, P. R. China ‡ Capital University of Physical Education and Sports, Beijing 100191, P. R. China § Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan ABSTRACT: Effective monitoring of cerebral ascorbate following intravenous antioxidant treatment is of great importance in evaluating the antioxidant efficiency for neuroprotection because ascorbate is closely related to a series of ischemia-induced neuropathological processes. This study demonstrates the validity of an online electrochemical system (OECS) for ascorbate detection as a platform for in vivo evaluation of neuroprotective efficiency of antioxidants by studying the dynamic change of hippocampal ascorbate during the acute period of cerebral ischemia and its responses to intravenous administration of antioxidants including ascorbate and glutathione (GSH). The OECS consists of a selective electrochemical detector made of a thin-layer electrochemical flow cell integrated with in vivo microdialysis. With such a system, the basal level of hippocampal ascorbate is determined to be 5.18 ± 0.60 μM (n = 20). This level is increased by 10 min of two-vessel occlusion (2-VO) ischemia treatment and reaches 11.51 ± 3.43 μM (n = 5) at the time point of 60 min after the ischemia. The 2-VO ischemia-induced hippocampal ascorbate increase is obviously attenuated by immediate intravenous administration of ascorbate (2.94 g/kg) or glutathione (5.12 g/kg) within 10 min after ischemia and the ascorbate level remains to be 3.75 ± 1.66 μM (n = 4) and 5.30 ± 0.79 μM (n = 5), respectively, at the time point of 60 min after ischemia. To confirm if the attenuated hippocampal ascorbate increase is attributed to the antioxidant-induced oxidative stress alleviation, we further study the immunoreactivity of 8-hydroxy-2-deoxyguanosine (8-OHdG) in the ischemic hippocampus and find that the 8-OHdG immunoreactivity is decreased by the administration of ascorbate or GSH as compared to the ischemic brain without antioxidant treatment. These results substantially demonstrate that the OECS for ascorbate detection could be potentially used as a platform for evaluating the efficiency of antioxidant neuroprotection in cerebral ischemia treatment.

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in vivo evaluating the neuroprotective efficiency of antioxidants in acute phase after cerebral ischemia is highly required. Ascorbate is the monovalent anion form of ascorbic acid at physiological pH. It is one of the most abundant neuroprotective small-molecule antioxidants and free radical scavengers endogenously existing in the central nervous system. Moreover, ascorbate is also an important neuromodulator of glutamate-mediated neurotransmission in brain.5 According to the previous reports and our studies, ascorbate is highly concentrated in neural cells and the extracellular ascorbate level is extensively involved in the neurochemical processes in the acute phase within seconds to a few hours after cerebral ischemia.6 For instance, within a few seconds after cerebral

s one of the major causes of death and disability, cerebral ischemia induces irreversible neural injury and brain dysfunction. Global cerebral ischemia causes selective neural injury in vulnerable brain regions such as the hippocampus, which is highly sensitive to the ischemic hypoxia and thereby damages brain functions.1 The long-term functional and histological damage after cerebral ischemia could be induced by a series of acute neurochemical changes including excessive oxidative stress and glutamate neurotoxicity and so forth.2 These acute neurochemical processes trigger necrosis in acute phase after ischemia and further induce inflammatory responses in the subacute phase and apoptosis in the chronic phase.3 Aiming at the reduction of the acute excessive oxidative damage, antioxidant treatment has been used as an important neuroprotective method to alleviate oxidative stress and restrain necrosis spread.4 In this context, a platform that can be used for © 2013 American Chemical Society

Received: August 17, 2013 Accepted: September 20, 2013 Published: September 20, 2013 9947

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were freshly prepared prior to experiments. Artificial cerebrospinal fluid (aCSF) was prepared by dissolving NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into water, and the solution pH was adjusted to pH 7.4. All aqueous solutions were prepared with Milli-Q water. Singlewalled carbon nanotubes (SWNTs) were purchased from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China) and purified by refluxing the as-received SWNTs in 2.6 M nitric acid for 5 h prior to use. Animals and Surgery. Adult male Sprague−Dawley rats (3 months of age, weighing 300 ± 50 g at the time of surgery) were individually housed under 12 h light/dark cycle and had food and water ad libitum. The animals were anaesthetized with chloral hydrate (350 mg/kg i.p.) and were placed in a stereotaxic frame with the incisor-bar set at 3.3 mm below the interaural line for the flat skull position. A microdialysis guide cannula was implanted into the dorsal hippocampus (AP = −4.4 mm, L = 3.0 mm from bregma, V = 2.5 mm from the surface of the skull), according to standard stereotaxic procedures.12 Three screws were fixed into the skull and cemented with the cannula by dental acrylic. Throughout the surgery, the body temperature of the rats was maintained at 37 °C with a heating pad. After the surgery, the rats were placed into a warm incubator individually until they recovered from anesthesia. Animals were allowed to recover from surgery for 2 days before microdialysis sampling and online electrochemical detection. The surgical procedures of two-vessel occlusion (2VO) ischemia were performed during the period of in vivo microdialysis sampling and online electrochemical measurements. The 2-VO ischemia model was constructed with ligation of the bilateral common carotids arteries (CCAs) with a 3-0 suture to induce permanent forebrain ischemia, with the methods reported previously.11b To study the hippocampal ascorbate response induced by 2-VO global cerebral ischemia and by the following antioxidant treatment, rats were divided into four groups at random, i.e., 2-VO ischemia (2-VO) group (n = 5), 2-VO followed by ascorbate (2.94 g/kg) intravenous infusion (2-VO + ASC) group (n = 5), 2-VO followed by glutathione (5.12 g/kg) intravenous infusion (2-VO + GSH) group (n = 5), and 2-VO followed by saline intravenous infusion (2-VO + SAL) group (n = 5). The intravenous infusion of ascorbate or GSH was performed by putting a needle of a syringe which was filled with the solution of ascorbate or GSH into caudal vein of the rats and infusing the antioxidants into the vein. To demonstrate whether the hippocampal ascorbate will be influenced by intravenous infusion of antioxidants, the same doses of ascorbate, GSH, and saline were given to 3 groups of sham-operated rats as follows: ascorbate intravenous infusion (ASC) group (n = 3), GSH intravenous infusion (GSH) group (n = 3), and saline intravenous infusion (SAL) group (n = 3). All efforts were made to reduce both the animal suffering and the number of animals used. In Vivo Microdialysis and Online Electrochemical System for Ascorbate. Online electrochemical system (OECS) for the detection of extracellular ascorbate in rat brain was performed as reported previously in our studies.10,11 Briefly, highly selective electrochemical detection of ascorbate was achieved with a thin-layer electrochemical flow cell coupled with in vivo microdialysis. The thin-layer electrochemical flow cell (BAS) was equipped with a glassy carbon (GC) working electrode (6-mm diameter) modified with SWNTs, a Ag/AgCl

ischemia, ascorbate supply from blood plasma is shut down by interruption of brain blood flow. Then, anoxic depolarization caused by energy failure and adenosine triphosphate (ATP) depletion turns on ascorbate efflux from the neural cells after a few minutes after cerebral ischemia.6,7,3c In the same time, excessive glutamate is released into extracellular space and further promotes the spatial spread of depolarization.5,8 Meanwhile, the increased depolarization and glutamate reuptake through glutamate transporters located on the presynaptic terminals of neurons and on glial cells further facilitate ascorbate release through a heteroexchange mechanism between extracellular glutamate and intracellular ascorbate. Furthermore, excessive glutamate release also activates N-methyl-D-aspartate (NMDA) type glutamate receptors, resulting in expression of several preoxidant enzymes and eventually produces free radicals and ROS in neural cells. These overproduced oxidative species results in neural damage (necrosis) due to oxidative stress happening within minutes to hours after cerebral ischemia.9 Therefore, the homeostasis of extracellular ascorbate in the ischemic brain is violated by the following two reasons. On one hand, highly concentrated ascorbate (4−10 mM) stored in neural cells is poured out into extracellular space during the process of necrosis. On the other hand, extracellular ascorbate is consumed by oxidative species due to the antioxidant effect of ascorbate. While these results suggest that ascorbate is intensively involved in the pathological processes in the acute period after cerebral ischemia and could be possibly used as a potential biomarker to evaluate the neuroprotective efficiency of antioxidants, such a potentiality has not been explored so far. In this study, we demonstrate an effective platform for in vivo evaluating the neuroprotective efficiency of antioxidants by using online electrochemical system (OECS) for ascorbate detection to demonstrate that ascorbate could be used as a potential biomarker to indicate the efficiency of antioxidant treatment. In our early studies, we have developed an OECS for continuously monitoring ascorbate in rat brain by efficiently coupling in vivo microdialysis with an online selective electrochemical detector. We utilized the excellent electrochemical properties of carbon nanotubes for selective electrochemical oxidation of ascorbate.10 The system has been demonstrated to be independent of pH and O2 fluctuation caused by brain ischemia and well suited for studying the changes of cerebral ascorbate induced by different brain ischemia models.11 In this study, we use our OECS for ascorbate detection to in vivo investigate the dynamic changes of hippocampal ascorbate induced by cerebral ischemia and its response to the intravenous administration of antioxidants including ascorbate and glutathione (GSH) within 10 min (typically 5−6 min) after 2-VO cerebral ischemia, aiming at the development of an effective yet technically simple platform for in vivo evaluation of antioxidant efficiency in cerebral ischemia treatment. This platform is of great importance in understanding molecular mechanisms underlying cerebral ischemia and of in vivo drug screening for antioxidant treatment in preclinic and clinic research.

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EXPERIMENTAL SECTION Chemicals and Materials. Sodium ascorbate and glutathione (GSH, Cucurbita species, EC 1.10.3.3) were purchased from Sigma. Other chemicals of analytical grade or higher were purchased from Beijing Chemical Corporation (Beijing, China) and used as received. Aqueous solutions of ascorbate and GSH 9948

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corrected for multiple comparisons was used to analyze ascorbate data between different groups. Ascorbate levels after 2-VO cerebral ischemia were compared with their basal level by two-tailed Student’s t test. P < 0.05 was considered as significantly different.

reference electrode, and a stainless steel counter electrode. The GC electrode was modified with SWNTs by drop-coating 4 μL of the SWNT dispersion in N,N-dimethylformamide (2 mg/ mL) on the electrode and allowing the electrode (SWNTmodified electrode) to be dried under ambient temperature. The SWNT-modified electrode was polarized at +30 mV vs Ag/AgCl for the continuous monitoring of the microdialysate ascorbate. The current recorded by OECS shows a good linear response toward ascorbate from 0.5 μM to 100 μM with a negligible deviation. The linear equation was I (nA) = 5.81CAA (μM) + 5.93 with a linear coefficiency of 0.999. On the day of microdialysis experiments, the rats with guide cannula implantation in their brain were anesthetized with chloral hydrate (350 mg/kg i.p.). The microdialysis probe (2 mm active membrane; Bioanalytical Systems Inc. (BAS), BAS Carnegie Medicine) was implanted into the hippocampus through the guide cannula and was perfused with aCSF at a flow rate of 3 μL/min driven by a microinjection pump (CMA/ 100; CMA Microdialysis AB, Stockholm, Sweden). After 90 min continuous aCSF perfusion, the microdialysate was directly delivered into the thin-layer radial electrochemical flow cell through tetrafluoroethylene hexafluoropropene tubing for continuous measurements of ascorbate. Immunohistology. To immunohistologically evaluate the oxidative stress caused by 2-VO cerebral ischemia, rats were separated into the following groups at random: sham-operated group (n = 2), 2-VO ischemia (2-VO) group (n = 3), 2-VO ischemia followed by ascorbate treatment (2-VO + ASC) group (n = 3), and 2-VO ischemia followed by glutathione treatment (2-VO + GSH) group (n = 3). The oxidative DNA damage induced by 2-VO cerebral ischemia and the following antioxidant treatment was assessed with 8-OHdG immunoreactivity, as described in the previous studies.13 Briefly, at the time point of 60 min after 2-VO ischemia with or without antioxidant treatment within 10 min after ischemia, the animals were decapitated under deep anesthesia. The brains were quickly removed and frozen in powdered dry ice. Coronal sections of 10 μm thickness containing the hippocampus were cut on a cryostat at −22 °C and collected on glass slides covered with saline. For immunohistochemical analysis of 8OHdG, the sections were fixed in ice-cold acetone for 5 min and air-dried, followed by rinsing in phosphate-buffered saline (PBS). After blocking with 2% normal horse serum in PBS for 2 h, the slices were incubated with mouse monoclonal antibody against 8-OHdG at a concentration of 7.0 mg/mL in 2% normal horse serum and 0.3% polyoxyethelene octylphenyl ether (Triton-X) for 8 h at 4 °C. The slices were then washed and incubated for 3 h with biotinylated antimouse IgG (BA2000, Vector Laboratories, Burlingame, CA) at 1:200 dilution in PBS. Subsequently, they were incubated with avidin−biotinhorseradish peroxidase complex (PK-6102, Vector Laboratories) for 30 min and then developed using diaminobenzidine as a color substrate. The reaction was terminated by washing the slices with water. A set of sections was stained in a similar way without the first antibody. Statistics. For statistic analysis of the microdialysate ascorbate levels, the current responses recorded with the OECS were converted into the concentration (μM) of ascorbate according to the linear calibration equation described above. The levels of microdialysate ascorbate were reported as the percentage of their basal level. The data were presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by a post hoc Tukey test

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RESULTS AND DISCUSSION OECS for Brain Ascorbate Detection. To verify that the OECS for ascorbate could be used as a platform for studying the neuroprotective efficiency of antioxidants in cerebral ischemia, the change of the hippocampal ascorbate in the sham-operated rats induced by intravenous infusion of ascorbate (2.94 g/kg) or GSH (5.12 g/kg) have to be first studied. For this purpose, we continuously monitored the hippocampal ascorbate in sham-operated rats (i.e., without ischemia) with our OECS for ascorbate detection (Figure 1A). Figure 1B depicts typical current−time responses, and Figure 2 shows the statistic results of the percent change of the hippocampal ascorbate levels in the sham-operated rats after the intravenous infusion of ascorbate, glutathione, or saline as a control. As typically shown in Figure 1B (black), the intravenous infusion of ascorbate in the sham-operated rats

Figure 1. (A) Schematic illustration of experimental setup of online electrochemical system (OCES) for continuously monitoring hippocampal ascorbate change in sham-operated rats with intravenous infusion of ascorbate or GSH. (B) Typical current−time responses of ascorbate recorded in the microdialysates continuously sampled from the hippocampus after intravenous infusion of ascorbate (black), GSH (red), or saline (blue) in sham-operated rats. The conditions of the intravenous infusion are indicated in the figure. The microdialysis probe was perfused with aCSF with a flow rate of 3 μL/min. The working electrode was polarized at +30 mV (vs Ag/AgCl electrode). 9949

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Figure 2 (red). This result was consistent with the previous reports that GSH administration did not change brain extracellular ascorbate level in a short-term (60 min) after the GSH infusion.16 We have also studied the change of the hippocampal ascorbate caused by the intravenous infusion of saline and found that saline intravenous infusion did not cause a remarkable change in the hippocampal ascorbate at the time point of 60 min after the infusion either (t test, n = 3), as shown in Figure 1B (blue) and Figure 2 (blue). Moreover, in our previous works, we have found that the hippocampal ascorbate was not changed by the sham operation.11 These results demonstrate that neither intravenous infusion of antioxidants such as ascorbate and GSH nor sham operation could change the hippocampal extracellular ascorbate level in rats. This feature substantially provides the basis of our OECS for ascorbate detection for in vivo evaluating the neuroprotective efficiency of antioxidants through continuously monitoring hippocampal ascorbate in 2-VO ischemia rats with intravenous infusion of antioxidants including ascorbate and GSH, as demonstrated later. It is noteworthy that although extracellular ascorbate in brain has been measured with in vivo voltammetry17,5b or highperformance liquid chromatography (HPLC) coupled with offline electrochemical detection,18 the application of our OECS for ascorbate monitoring remains remarkable because of the chemical instability of ascorbate and the less technical demanding feature, the avoidance of sample collection, as well as the near real-time feature of the OECS.10,19 To be specific, although in vivo voltammetry has a good temporal resolution in detecting cerebral ascorbate, the interference from other kinds of electroactive species in the cerebral system such as dopamine, 3,4-dihydroxyphenylacetic acid, uric acid, and 5hydroxytryptamine remains a challenge.10 On the other hand, the off-line HPLC methods for cerebral ascorbate detection might be time-consuming and lacking of temporal resolution.18,11a As demonstrated in our earlier studies, the OECS established with the integration of a selective electrochemical detector with in vivo microdialysis exhibits a good accuracy, high specificity, and stability for continuous ascorbate monitoring.10 More remarkably, such a system bears a high tolerance against the variation of O2 and pH under pathological cerebral ischemia conditions.11a These properties potentially validate our OECS for ascorbate detection as an effective platform for in vivo investigation of neuroprotective efficiency of antioxidants in the acute period of cerebral ischemia. Hippocampal Ascorbate Change after 2-VO Ischemia and Antioxidant Infusion. Figure 3B depicts typical current−time responses and Figure 4 shows the statistic results of the percent ascorbate changes recorded with the OECS for ascorbate detection in the microdialysates continuously sampled from hippocampus in the 2-VO group, 2-VO + ASC group, 2-VO + GSH group, and 2-VO + SAL group. The basal ascorbate level in the hippocampus microdialysate was determined to be 5.18 ± 0.60 μM (n = 20), with no significant difference among different groups. As depicted in Figure 3B (purple) and Figure 4 (purple), after 2-VO cerebral ischemia the hippocampal ascorbate increases at 10 min and reaches 11.51 ± 3.43 μM (256.60 ± 81.22% of the basal level) at the time point of 60 min after ischemia (two-tailed Student’s t test, P < 0.05, n = 5). Similar increases in the hippocampal ascorbate have also been previously observed in 2-VO ischemia/ reperfusion,20 focal cerebral ischemia,21 and closed head injury models.22 The increase of the hippocampal ascorbate after

Figure 2. Statistical results of the hippocampus ascorbate level in ASC group (black), GSH group (red), or SAL group (blue) of shamoperated rats. The conditions for the intravenous infusion were indicated in the figure.

leads to about a 10.67% temporary increase in the current response for the hippocampal extracellular ascorbate compared with that for the basal ascorbate at 30 min after the infusion. This temporary increase turned back to the basal level within 60 min after the infusion. The statistic analysis, shown in Figure 2 (black), suggests the temporary ascorbate increase has no significant difference compared with the basal ascorbate level (t test, n = 3). Previous studies have demonstrated that ascorbate administration did not significantly increase extracellular ascorbate, although the ascorbate level in brain tissue homogenate was increased.14,5a,6 This could be understandable since the extracellular ascorbate is dynamically self-regulated through homeostasis, for example, excessive ascorbate could be transported into neural cells by sodium-dependent vitamin C transporter 2 (SVCT2). As reported previously, intravenously infused ascorbate could be transported into brain extracellular space by the SVCT2 located on brain capillary endothelial cells, resulting in the increase of the extracellular ascorbate.15,5a Meanwhile the increased extracellular ascorbate could be transported into cytoplasm by the SVCT2 located on neural cells to keep the extracellular ascorbate level in homeostasis, leading to the intracellular ascorbate level ten times higher than the extracellular one.5 Similar with the results obtained with the intravenous infusion of ascorbate, the intravenous infusion of GSH in the sham-operated rats does not induce any obvious change in the hippocampal extracellular ascorbate level within 60 min after the infusion (t test, n = 3), as displayed in Figure 1B (red) and 9950

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attenuation of the ischemia-induced hippocampal ascorbate increase caused by administration of ascorbate in such a shortterm after ischemia has not been reported so far. According to the previous reports, intravenously infused ascorbate could be transported into cerebral extracellular space and further transported into neural cells to perform their antioxidant neuroprotective function.5,6 Thus, the remarkably attenuated increase in the hippocampal ascorbate induced by 2-VO ischemia observed in our experiments could be understood by the neuroprotective role of the intravenously infused ascorbate. As a support to our hypothesis, the previous studies have demonstrated that 15 min or 3 h postischemia intravenous infusion of dehydro-ascorbic acid (DHA), an oxidized bloodbrain barrier transportable form of the ascorbate, elevated cerebral ascorbate in the whole brain homogenate within 2 h after middle cerebral artery occlusion (MCAO) models and further inhibited cerebral lipid peroxidation, decreased infarct volume and neurological deficits 24 h after the MCAO ischemia.24 Moreover, previous studies have also demonstrated that intravenous ascorbate administration after cerebral ischemia could increase ascorbate level in brain tissue homogenate and decreased brain infarct size due to antioxidant neuroprotection.25 According to these reports, the changes in the hippocampal ascorbate could be used to evaluate the neuroprotective efficiency of antioxidants in the acute phase after 2-VO ischemia. This feature in turn demonstrates that our OECS for ascorbate could be potentially used as a platform for in vivo evaluating the neuroprotective efficiency of antioxidants. To further demonstrate the potentiality described above, the other kind of antioxidant, i.e., GSH, was intravenously infused into the 2-VO ischemia rats within 10 min after ischemia with the same procedures employed for those for ascorbate. Quite similar to the effect of the intravenous infusion of ascorbate, no significant increase in the hippocampal ascorbate after 2-VO cerebral ischemia was observed upon the intravenous infusion of GSH; the hippocampal ascorbate remains at the basal level for at least 60 min after ischemia, which was 5.30 ± 0.79 μM (n = 5, 95.56 ± 12.70% of basal level) at the time point of 60 min after ischemia (Figure 3B (red) and Figure 4 (red)). This result suggests that GSH infusion also plays an important role in neuroprotection against cerebral ischemia injury.26,16 As a control, we have studied the effect of the intravenous infusion of saline on hippocampal ascorbate after cerebral ischemia, as displayed in Figure 3 (blue) and Figure 4 (blue). After the infusion of saline within 10 min after ischemia, a similar increase in the hippocampal ascorbate was observed and the ascorbate level reaches 12.44 ± 3.33 μM (n = 5) (235.67 ± 62.82% of basal level) at the time point of 60 min after ischemia (two-tailed Student’s t test, P < 0.05, n = 5). This result suggests that the infusion of saline does not affect the increase of the hippocampal ascorbate induced by 2-VO ischemia. For a convenient comparison, we summarized the hippocampal ascorbate levels before ischemia with those 60 min after 2-VO ischemia, with or without antioxidant infusion. As displayed in Figure 5, the hippocampal ascorbate levels recorded with our OECS significantly increased in both 2-VO ischemia and 2-VO + SAL groups, as compared with their basal levels. However, the hippocampal ascorbate levels after 60 min 2-VO ischemia in both 2-VO + ASC and 2-VO + GSH groups have no significant change, as compared with their basal levels. One-way ANOVA followed by a post hoc Tukey test indicated a significant difference among the four groups at the time point of 60 min after the ischemia and the antioxidant infusion (F =

Figure 3. (A) Schematic illustration of experimental setup of online electrochemical system for continuously monitoring hippocampal ascorbate change in 2-VO ischemia rats with intravenous infusion of antioxidants. (B) Typical current−time responses of ascorbate recorded in the microdialysates continuously sampled from the hippocampus of 2-VO ischemia group (purple), 2-VO + ASC group (black), 2-VO + GSH group (red), and 2-VO + SAL group (blue). Other conditions were the same as those in Figure 1.

cerebral ischemia was considered to be a comprehensive result of a series of neuropathological processes including anoxia depolarization, glutamate release and reuptake, glutamate excitotoxicity, accumulation of oxidative free radicals, and even neural cell necrosis, as reported previously.23 Although the previous studies have suggested that antioxidant treatment preor postcerebral ischemia could be beneficial to brain function recovery by reversing the brain ascorbate increase in subacute and chronic phase after ischemia, the effects of antioxidant treatment on the ischemia-induced change in the cerebral ascorbate remains unclear.24 As shown in Figure 3B (black) and Figure 4 (black), with the intravenous infusion of ascorbate within 10 min after ischemia, the hippocampal ascorbate shows almost no significant change and the ascorbate level was 3.75 ± 1.66 μM (n = 4, 80.83 ± 29.06% of basal level) at the time point of 60 min after ischemia. As far as we know, the 9951

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Figure 4. Statistical results of the hippocampus ascorbate levels in 2-VO ischemia group (purple), 2-VO + ASC group (black), 2-VO + GSH group (red), and 2-VO + SAL group (blue). The time interval for data acquisition was 100 s. The conditions in different groups were indicated in the figure.

cells in the sham-operated brain slices (Figure 6A). On contrast, at the time point of 60 min after 2-VO cerebral ischemia, immunoreactivity of 8-OHdG was moderately observed in hippocampal granule cells which was indicated in the panel B with red arrows (Figure 6B). However, as shown in Figure 6C,D, the ischemia-induced immunoreactivity of 8OHdG at the time point of 60 min after ischemia was significantly decreased by the intravenous infusion of ascorbate or glutathione. These results were consistent with the previous reports, in which 8-OHdG immunoreactivity was markedly increased in hippocampal neurons after ischemia27 and was attenuated by the antioxidant treatment.28 The decreased 8OHdG immunoreactivity in Figure 6C,D demonstrates a lower oxidative DNA damage, a lower glutamate exitocticity, and a decreased neural damage,27 indicating a good neuroprotective efficiency of ascorbate and GSH. The observed ischemiainduced increase in the hippocampal 8-OHdG immunoreactivity (Figure 6B) and its attenuation by the intravenous infusion of ascorbate (Figure 6C) and GSH (Figure 6D) were quite consistent with the 2-VO ischemia-induced increase in the hippocampal ascorbate (Figure 3B (purple) and Figure 4 (purple)) and its attenuation by the intravenous infusion of the ascorbate (Figure 3B (black) and Figure 4 (black)) and GSH (Figure 3B (red) and Figure 4 (red)). According to these results, the increase of the hippocampal ascorbate induced by 2VO ischemia reflects the ischemia-induced neural damage, which could be partially demonstrated by the increase of 8OHdG immunoreactivity. Meanwhile, the attenuated increase of the hippocampal ascorbate by the intravenous infusion of antioxidants after the ischemia suggests a decreased neural damage, which was also seen from the attenuated 8-OHdG immunoreactivity induced by the antioxidant infusion. Thus, the change of hippocampal extracellular ascorbate, synchronously occurring with the change of intracellular 8-OHdG immunoreactivity, could be potentially used as an important biomarker to indicate the neural damage and the antioxidant neuroprotection after cerebral ischemia. This consistency

Figure 5. Statistic results of hippocampal ascorbate levels recorded with our OECS for ascorbate before ischemia and 60 min after ischemia in the 2-VO group, 2-VO + ASC group, 2-VO + GSH group, or 2-VO + SAL group. Data presented as mean ± SD. The asterisks (*P < 0.05) indicate significant differences between ascorbate levels before ischemia and at the time point of 60 min after ischemia.

7.508, P < 0.01; P = 0.01, F(3,15) = 3.29). These results substantially demonstrate that the attenuation of the 2-VO ischemia-induced increase of the hippocampal ascorbate is mainly caused by the intravenous infusion of antioxidants including ascorbate and GSH. These results strongly demonstrate that our OECS for ascorbate detection could be used as an effective platform for in vivo evaluating the neuroprotective efficiency of antioxidants. Immunohistology. To further demonstrate the neuroprotective efficiency of the intravenously infused antioxidants including ascorbate and GSH, we tested the immunoreactivity of 8-hydroxy-2-deoxyguanosine (8-OHdG) in the hippocampus in the sham-operated group (n = 3), 2-VO ischemia group (n = 3), 2-VO + ASC group (n = 3), and 2-VO + GSH group (n = 3). Figure 6 displays typical immunohistological results in hippocampal slices. There were no detectable 8-OHdG positive 9952

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Figure 6. DNA damage assessed with 8-OHdG immunoreactivity in hippocampus at the time point of 60 min after the sham-operation or ischemia in the sham-operated group (A), 2-VO group (B), 2-VO + ASC group (C), and 2-VO + GSH group (D). Staining of positive immunoreactivity was mainly localized in nuclei and a lesser degree in cytoplasm of cells which were indicated with red arrows. Scale bar, 50 μm.

Notes

further validates our OECS as an effective platform for in vivo evaluating neuroprotective efficiency of antioxidant treatment after cerebral ischemia.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is financially supported by the NSF of China (Grants 21321003, 20935005, 21127901, 21210007, and 91213305 for L.M. and Grant 30900709 for K.L.), the National Basic Research Program of China (973 Programs, Grants 2010CB33502 and 2013CB933704), and The Chinese Academy of Sciences (Grant KJCX2-YW-W25). K.L. acknowledges the financial support from Beijing Municipality (Grants KZ201010029031, PHR201008179, and PXM2012_014206_000003).

CONCLUSIONS We have successfully demonstrated that a technically simple yet practically useful OECS consisting of a selective electrochemical detector made of a thin-layer electrochemical flow cell integrated with in vivo microdialysis can be used as an effective platform for in vivo evaluation of antioxidant efficiency. The evaluation could be simply accomplished by continuously monitoring the dynamic changes of the hippocampal extracellular ascorbate and their responses to the intravenous infusion of antioxidants in living rats. The OECS bears a good tolerance against the intravenous infusion of ascorbate or GSH and can near real-time and selectively monitor the continuous change of the hippocampal ascorbate in the 2-VO ischemia rats before and after antioxidant infusion. This study essentially offers a new platform for in vivo evaluating antioxidant neuroprotective efficiency in cerebral ischemia, which is believed to be able to find some interesting applications in drug screening and molecular mechanism investigations.

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REFERENCES

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dx.doi.org/10.1021/ac402620c | Anal. Chem. 2013, 85, 9947−9954

Analytical Chemistry

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dx.doi.org/10.1021/ac402620c | Anal. Chem. 2013, 85, 9947−9954