Ischemic Postconditioning Recovers Cortex Ascorbic Acid during

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Ischemic Postconditioning Recovers Cortex Ascorbic Acid during Ischemia/Reperfusion Monitored with an Online Electrochemical System Dalei Wang, Xianchan Li, Ying Jiang, Yanan Jiang, Wenjie Ma, Ping Yu, and Lanqun Mao ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00056 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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ACS Chemical Neuroscience

Ischemic Postconditioning Recovers Cortex Ascorbic Acid during Ischemia/Reperfusion Monitored with an Online Electrochemical System Dalei Wang,†,§ Xianchan Li,†,§ Ying Jiang,† Yanan Jiang,†,‡ Wenjie Ma,†,‡ Ping Yu,†,‡ Lanqun Mao*,†,‡ † Beijing

National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living

Biosystems, Institute of Chemistry, the Chinese Academy of Sciences (CAS), CAS Research/Education Center for Excellence in Molecule Science, Beijing 100190, China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

*Corresponding

Author. Fax: (+86)-10-6255-9373; E-mail: [email protected]

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ABSTRACT: As a promising therapeutic treatment, ischemic postconditioning has recently received considerable attention. Although the neuroprotection effect of postconditioning has been observed, a reliable approach that can evaluate the neuroprotective efficiency of postconditioning treatment during the acute period after ischemia remains to be developed. This study investigates the dynamics of cortex ascorbic acid during the acute period of cerebral ischemia before and after ischemic postconditioning with an online electrochemical system (OECS). The cerebral ischemia/reperfusion injury and the neuronal functional outcome are evaluated with triphenyltetrazolium chloride staining, immunohistochemistry and electrophysiological recording techniques. Electrochemical recording results show that cortex ascorbic acid sharply increases 10 min after middle cerebral artery occlusion, then reaches a plateau. After direct reperfusion following ischemia (i.e., without ischemic postconditioning), the cortex ascorbic acid further increases, and then starts to decrease slowly at a time point of about 40 min after reperfusion. In striking contrast, the cortex ascorbic acid drops and recovers to its basal level after ischemic postconditioning followed by reperfusion. With the recovery of cortex ascorbic acid, ischemic postconditioning concomitantly promotes the recovery of neural function and reduces the oxidative damage. These results demonstrate that our OECS for monitoring cortex ascorbic acid can be used as a platform for evaluating the neuroprotective efficiency of ischemic postconditioning in acute phase of cerebral ischemia, which is of great importance for screening proper postconditioning parameters for preventing ischemia damages.

KEYWORDS: ascorbic acid, ischemic postconditioning, ischemia/reperfusion, in vivo microdialysis, online electrochemical system, neuroprotective efficiency

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INTRODUCTION Ischemic stroke is one of the most common causes of death and disability in the world. Globally, about 15 million people suffer from stroke each year.1, 2 It is generally believed that, restriction of blood flow to the brain during ischemia stroke results in insufficient oxygen and glucose supply for maintaining the cellular homeostasis and thereby alters cellular functions at both neurochemical and electrophysiological levels, such as glutamate neurotoxicity, cellular depolarization, Ca2+ influx, and so forth.3-10 In addition, the dysfunction of the oxidative respiratory chain in mitochondria, combined with the activation of cytoplasmic oxidases happening during ischemia stroke, may generate excessive free radicals, inducing the necrotic and apoptotic cell death and ultimate damage to the brain function.11-14 In current clinical practice, dredging the occluded blood vessels with intravascular techniques or thrombolytic agents to allow the reperfusion timely is one of the guiding principles for the treatment of ischemic stroke.15, 16 However, like a double-edged sword, reperfusion generates a larger infarct in many animal ischemic models compared with that of permanent vessel occlusion.17 This is caused by the fact that reperfusion itself might generate overproduction of reactive oxygen species (ROS) and other free radicals that could induce the additional injuries.18-20 Hence, a more effective treatment for ischemic stroke is highly desired in clinical practices. One of the promising therapeutic treatments of ischemic stroke at research stage is ischemic postconditioning, which contains a series of brief, repetitive occlusion/release of the cerebral blood vessels immediately after the onset of reperfusion.21 Studies over the past few years have proved that ischemic postconditioning treatment could provide neuroprotection against the brain injuries induced by ischemiareperfusion for both focal and global cerebral ischemia models, and improve the functional outcome in animals.22, 23 Moreover, ischemic postconditioning could reduce the infarct size, mostly plausibly by blocking the overproduction of ROS and lipid peroxidation, and inhibiting apoptosis, in which protein kinase B (Akt) , mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and ATP-sensitive K+ (KATP) channel cell signaling pathways, cytochrome c/caspase-mediated apoptotic pathways, and inflammation are all involved.24 Although the neuroprotection effect of ischemic postconditioning has been demonstrated, its neuroprotective efficiency largely varies with the conditions employed in postconditioning. For example, previous report suggested that 10 cycles of 10 s-reperfusion / 10 s-reocclusion starting at 10 s after cerebral 3



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blood vessel occlusion could reduce the infarct size by 56%, whereas 10 cycles of 30 s-reperfusion/10 sreocclusion starting at 30 s after cerebral blood vessel occlusion showed no protective effect.25 Currently, electrophysiological recording is the main method used to evaluate the neuroprotective efficiency of ischemic postconditioning in acute phase. Previous studies showed that the electrophysiological recording could provide immediate information of the tissue and offer additional prognostication of neurological outcome in varied neurological disorders, thus, this technique is widely used for evaluating the neuronal function in cerebral ischemia study.26, 27 However, its accuracy is sometimes affected by the anesthetics treating animals and other factors.28 Therefore, a more reliable approach that can evaluate the neuroprotective efficiency of ischemic postconditioning treatment during the acute period after ischemia is highly desired. Previous studies have showed that, neurochemical changes from few seconds to minutes after the cerebral ischemia play critical roles in modulating the histological syndrome and functional damage occurring from several hours to days afterwards.29, 30 Many kinds of neurochemicals, such as neurotransmitters, neuromodulators, ROS, other free radicals and energy substances, have been demonstrated to associate with ischemia processes. Among them, ascorbic acid has been regarded as one of the most important neurochemicals in brain.31 Its enediol structure enables it as a vital antioxidant molecule and free radical scavenger in brain via losing two electrons. Generally, the level of ascorbic acid in brain is under homeostatic regulation with a turnover about 2% per hour. Even under some conditions that lead to the deficiency of ascorbic acid, the level of ascorbic acid in brain retains tenaciously with a decrease of less than 2% per day.32 In striking contrast to the precise regulation of ascorbic acid maintained in brain, severe disturbance of the extracellular level of ascorbic acid under some conditions, such as ischemia and hypoxia, makes it a sensitive biomarker for evaluating initial ischemic neural damage at very early stage of focal ischemia.33, 34 In our recent report, by using an online electrochemical system (OECS), we demonstrated that ascorbic acid could be used as a potential biomarker to evaluate both the neural damage and the neuroprotective efficiency of antioxidants in brain.35, 36 However, the potentiality of monitoring the extracellular ascorbic acid with the OECS to evaluate the neuroprotective efficiency of ischemic postconditioning, for instance, whether ischemic postconditioning could induce the change of extracellular ascorbic acid and whether this change of ascorbic acid could be detected by the OECS, has not been explored so far. In this study, by using the OECS for the measurement of ascorbic acid in vivo, we monitor the dynamics 4


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of cortex ascorbic acid in rat during the acute period of cerebral ischemia and after the ischemic postconditioning treatment. The cerebral ischemia/reperfusion injury and the neuronal functional outcome of ischemic postconditioning are also evaluated with triphenyltetrazolium chloride staining, immunohistochemistry and electrophysiological recording. These results essentially demonstrate the potentiality of OECS for monitoring ascorbic acid as a reliable platform to evaluate the neuroprotective efficiency of ischemic postconditioning treatment for ischemia stroke. RESULTS AND DISCUSSION Postconditioning Attenuates Focal Cerebral Ischemia/Reperfusion Injury. To observe the capability of ischemic postconditioning to reduce the damage caused by cerebral ischemia, we compared the difference in infarct volume, a typical approach to evaluate the damage of ischemia,23 between ischemia/reperfusion (I/R) and ischemic postconditioning (PostCond) groups. Here, the suture was removed 1 h after the occlusion and no further interruption of reperfusion was applied in the I/R group. While, in the PostCond group, 10 cycles of 10 s-reperfusion/10 s-reocclusion of middle cerebral artery was applied immediately after the removal of the suture. As typically shown in Figure 1A, TTC staining shows that ischemic postconditioning after ischemia clearly decreases the volume of infarcted tissue 24 h after reperfusion compared with that of the I/R group. Statistically, the percentage of infarct volume is 46.3% ± 7.8% (mean ± SD, n = 6) and 21.8% ± 4.5% (mean ± SD, n = 6) for the I/R and the PostCond groups, respectively, demonstrating a significant difference between the two groups (Figure 1B). Consistent with the studies reported previously,22 this result suggests the capability of the postconditioning treatment to reduce the damage of the cerebral ischemia. Postconditioning Treatment Recovers Cortex Ascorbic Acid in Ischemia/Reperfusion. In the previous studies, we developed an OECS consisting of in vivo microdialysis sampling and a selective electrochemical detector that can continuously monitor ascorbic acid in brain with high specificity, stability, and reproducibility (Figure 2A).36 More significantly, this OECS is independent of pH and O2 fluctuation that are usually accompanying during brain ischemia, validating its application in ischemia/reperfusion study.36-38 Here, using this OECS, we examined the effect of ischemic postconditioning on the cortex ascorbic acid in the ischemia/reperfusion model. Typical current-time responses recorded with the OECS for sham, I/R and PostCond groups were shown in Figure 2B, C and D, respectively. Similar to the previous reports,35, 36 the basal level of ascorbic acid (C0) in the dialysate from rat cortex was determined as 4.1 μM ± 0.35 μM (mean ± 5



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SD, n = 6), indicating again the OECS system is effective for the measurement of ascorbic acid in vivo. Ten minutes after the surgery of middle cerebral artery occlusion (MCAO), the concentration of ascorbic acid increases, then levels off and reaches 311.3% ± 42.2% (mean ± SD, n = 6) and 375.7% ± 47.5% (mean ± SD, n = 6) of the basal levels respectively in the I/R and the PostCond groups, at a time point of 60 min after MCAO (Figure 3). However, there is no statistical significance between these two groups. After removing the suture from the middle cerebral artery (i.e., reperfusion), the level of cortex ascorbic acid in the I/R group continuously increases and reaches its maximum, 419.3 % ± 49.9% (mean ± SD, n = 6), at a time point of about 35 min after reperfusion. This trend was consistent with the previous study.35 During the first few seconds of cerebral ischemia, the interruption of blood flow could decrease oxygen and glucose supplies in brain. In turn, the depletion of adenosine triphosphate (ATP) prevents Na+/K+-ATPase, inducing the influx of Na+ into the intracellular compartment.39 Consequently, the resulting anoxic depolarization of cell by extensive intracellular Na+ ultimately stimulates the release of ascorbic acid into extracellular fluid along with neurotransmitters in a Ca2+-independent mode.40 During reperfusion, the further increase of extracellular ascorbic acid may be caused by a glutamate/ascorbate hetero-exchange mechanism,40 that is, the release of ascorbic acid from intracellular sites occurs synchronously with the uptake of released glutamate into both neurons and glia by a Na+-dependent process.41 It has been reported that the intracellular ascorbic acid, rather than the extracellular one, is crucial for protection against oxidant stress.31, 42 Therefore, cerebral ischemia/reperfusion reduces intracellular ascorbic acid, which may not be adequate to scavenge excess reactive species, and makes cells vulnerable to oxidative damage that might be associated with stroke pathology.32 Conversely, reperfusion with postconditioning treatment induces a slight and not significant increase on the level of ascorbic acid at the beginning of the reperfusion. This is probably one reason for less injury happening in PostCond group than the I/R group (Figure 1). An observable decrease in the levels of cortex ascorbic acid gradually occurs in the I/R group at the time point of about 40 min after reperfusion, however, this decrease happens 10 min earlier and more sharply in the PostCond group (Figure 3). From 1 h to 2 h after reperfusion, the level of ascorbic acid in the PostCond group is much lower than that in the I/R group (P < 0.05). At the time point of 2 h after reperfusion, ascorbic acid in the PostCond group almost returns to its basal level (102.3% ± 30.6% of basal level) (Figure 3, blue), whereas in the I/R group it is still ~3 times of the basal level (306.9% ± 42.4%) (Figure 3, black). This result 6


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indicates that ischemic postconditioning followed by reperfusion could recover the extracellular cortex ascorbic acid after ischemia damage faster than the direct reperfusion (i.e., without ischemic postconditioning). To the best of our knowledge, this is the first demonstration of the recovery of the cortex ascorbic acid by the ischemic postconditioning in such a short-term after ischemia/reperfusion. This highlights the possibility of using OECS for the measurement of ascorbic acid as a platform for evaluating neuroprotective efficiency of the ischemic postconditioning. Ascorbic Acid Fluctuation Accompanies with Neuronal Function Change Monitored with Electrophysiological Recording. To investigate the relationship of ascorbic acid fluctuation with the neuronal function, we studied the electrophysiological activity before, during ischemia, and after reperfusion or postconditioning with the electrocorticography (ECoG) recording technique. Figure 4A displays the representative ECoG tracings for two groups. Obviously, ECoG signals become flatter, i.e., more isoelectric, within 15 seconds after MCAO and remain unchanged during ischemia until reperfusion is introduced, from where ECoG signals starts to recover with different paces for the I/R and the PostCond groups. The root mean square (RMS) of the amplitude of ECoG was analyzed because RMS is suitable for quantitatively evaluating the electrocortical activity in slow varying state, for example, sleeping and anesthesia.43 As shown in Figure 4B, the normalized RMS of the ECoG signals reduces to be 24.4% ± 5.1% and 28.2% ± 5.8% of the basal level respectively in both the I/R and the PostCond groups, showing no statistical difference between groups. This massive shutdown of neural activity is to adapt to the insufficient supply of energy at the onset of ischemia, through this neurons can maintain the minimal metabolism required for survival.43 However, when the ischemia is prolonged, persistent damage to synaptic activity may become the major reason for the observed reduction of neural activity.44 The recovery of RMS by reperfusion 1 h after MCAO was reported to be poor.44 Here, even 2 h after reperfusion in the I/R group, the recovery of RMS is still negligible, which may be relevant to the synaptic failure.43 Notably, ischemic postconditioning treatment after ischemia increases the RMS of ECoG signals more significantly from 30 min after the postconditioning treatment compared with that of the I/R group. This implies that the ischemic postconditioning can enhance functional recovery, which accompanies with the recovery of cortex ascorbic acid (Figure 4B). To further explore the neurofunctional changes accompanying the fluctuation of ascorbic acid, we analyzed the changes of the frequency composition of the ECoG (Figure 4C). Typically, ECoG can be 7



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divided into five frequency bands, δ (1-4Hz), θ (4-8Hz), α (8-13Hz), β (13-30Hz) and γ band (>30Hz).45 Previous studies have demonstrated that the relative power of δ frequency band (1 - 4 Hz) correlates negatively with the cerebral blood flow (CBF) during the ischemia and reperfusion.46, 47 In this study, the relative power of δ frequency band increases to 125.1% ± 4.0% and 120.1% ± 4.0% of basal levels, respectively, in both the I/R and the PostCond groups at 5 min after MCAO. Then, it increases very slightly during ~1 h of ischemia, reaching to 129.8% ± 5.0% and 125.4% ± 5.8% of the basal levels in the I/R and PostCond groups, respectively, at the time point of 60 min after MCAO. The increase of the relative power of δ frequency band during ischemia indicates a stimulation of slow-wave activities in ECoG, which might be relevant to the interruptions of the subcortical afferent input to the cortex neurons, the brain injury as well as the functional deficits.43 After reperfusion, the relative power of δ frequency band shows no significant change in the I/R group. However, it starts to decrease at the time of 15 min after postconditioning treatment and the value is statistically lower than the corresponding one in the I/R group from 60 min to 120 min after reperfusion. This result suggests that the ischemic postconditioning could increase CBF and recover the neuronal function 60 min after reperfusion, which was consistent with the previous study, in which CBF was measured with a laser Doppler flowmeter.48 When comparing the PostCond group in Figure 4 B and C, the relative power of δ frequency band of the ECoG signal changes synchronously with the recovery of cortex ascorbic acid, demonstrating significant changes from the time point of 60 min after postconditioning treatment, whereas the RMS value of ECoG signal changes significantly at 30 min after postconditioning treatment. As reported previously, a slight change of CBF led to 3-7 times of change on neural activity.49 This was consistent with the results displayed in Figure 4 that a moderate increase of CBF induces a significant recovery of the neuronal function. In other words, the significant recovery of neuronal function happens earlier than that of CBF. Moreover, the synchronization of the change of ascorbic acid and the relative power of δ frequency band may imply the close relationship between ascorbic acid and CBF after postconditioning treatment. Changes of RMS and the relative power of δ frequency band demonstrate the consistent effect of ischemia/reperfusion or postconditioning treatment on neuronal function. Moreover, the observation of the fluctuation of ascorbic acid accompanies with the change of neuronal function in both the I/R and the PostCond groups further demonstrates the potentiality of OECS for the detection of ascorbic acid as a 8


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platform for in vivo evaluating the neuroprotective efficiency of the postconditioning treatment. Oxidative Damage Tested with Immunohistochemistry. Ascorbic acid has been reported as a vital antioxidant and free radical scavenger in biological system.42 In order to further explore the relationship between the cortex ascorbic acid and the oxidative neural damage, we compared the oxidative damage to DNA by immunostaining of 8-hydroxy-2-deoxyguanosine (8-OHdG), one of the best markers for evaluating oxidative damage.36, 50 As shown in Figure 5A, very few 8-OHdG-expressing positive cells were detected in cortex slices in sham group, indicating the minimal oxidative neurodamage induced by sham surgery. However, 2 h after direct reperfusion in the I/R group, the staining of 8-OHdG turns strong, which suggests ischemia/reperfusion induces severe oxidative damage in cells (Figure 5B). It is worthy of note that the staining of 8-OHdG in the PostCond group is weaker than that in the I/R group (Figure 5C). Quantitatively, the number of 8-OHdG-expressing positive cells in PostCond group (84 ± 7 cells/mm2) was significantly lower than that of the I/R group (484 ± 15 cells/mm2), as displayed in Figure 5D. Although the number of 8OHdG-expressing positive cells of the PostCond group was slightly higher than the sham group, the substantial reduction of 8-OHdG expression in the PostCond group compared with that in the I/R group suggests the vital neuroprotective effects of postconditioning treatment with concomitant recovery of cortex ascorbic acid, in preventing the oxidative damage from ischemia/reperfusion. Taken all together, the change of cortex ascorbic acid monitored with OECS accompanies with the change of neural function, as well as oxidative neurodamage. This implies that cortex ascorbic acid could be treated as a biological marker to indicate the neural injury of cerebral ischemia/reperfusion and the neural protection of ischemic postconditioning treatment. Moreover, our platform with OECS for monitoring ascorbic acid in rat cortex is sufficient for evaluating the neuroprotective efficiency of postconditioning. CONCLUSIONS By using an online electrochemical system, we for the first time demonstrate that, postconditioning treatment recovers the level of cortex ascorbic acid in the acute phase after cerebral ischemia. The fluctuation of cortex ascorbic acid occurs synchronously with cerebral blood flow recovery, and accompanies with the neural activity recovery as well as the decreased intracellular oxidative damage. Moreover, glutamate/ascorbate hetero-exchange during ischemia and reperfusion could be potentially probed by monitoring extracellular ascorbic acid in our system. These results imply that cortex ascorbic acid could be 9



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treated as a biological marker to evaluate the neurodamage by direct ischemic reperfusion and the neuroprotective efficiency of postconditioning treatment in acute phase of cerebral ischemia in vivo. Furthermore, this validates the substantial practical applicability of OECS for the measurement of ascorbic acid for in vivo evaluating the neuroprotective efficiency of postconditioning, meanwhile diversifying analytical approaches in this area. This study may be of great importance in screening of proper postconditioning parameters and in bridging the translation of postconditioning treatment from basic research to clinical application for ischemic stroke. METHODS Chemicals and Solutions. Sodium ascorbate purchased from Sigma was used as received. Antibodies used for immunohistochemistry were all purchased from Beijing Biosynthesis Biotechnology Co., LTD (China). Other chemicals were of at least analytical grade and used as received. Aqueous solution of ascorbate (1.0 mM) was prepared just before the use. Artificial cerebrospinal fluid (aCSF) used for in vivo microdialysis 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 Milli-Q water and the solution pH was adjusted to 7.4. Single-walled carbon nanotubes (SWNTs) were purchased from Shenzhen Nanotech Port Co., Ltd. (China) and purified as reported before.37 Animals. Adult male Sprague-Dawley rats (250-280 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All rats were randomly divided into 6 groups: ischemia/reperfusion (I/R) for OECS recording, I/R + postconditioning for OECS, I/R for electrophysiological recording, I/R + postconditioning for electrophysiological recording, I/R for immunohistochemistry, and I/R + postconditioning for immunohistochemistry. Animals were maintained on a 12 h-light / 12 h-dark schedule with food and water ad libitum. All the animal experimental procedures were approved by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology of China. Focal Cerebral Ischemia and Ischemic Postconditioning. Rats were anesthetized with isoflurane (4% for induction, 2% for maintenance) through a gas pump (RWD R520, Shenzhen, China) and placed on a stereotaxic frame. Using aseptic stereotaxic surgical techniques, the microdialysis guide cannula with replaceable inner guide was slowly lowered into the right cortex (AP=0 mm and L=5.0 mm from the bregma, V=1.0 mm from the surface of the skull, using a flat skull position).51 Then the microdialysis cannula was 10


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secured with a bone screw and acrylic cement. After removal of the anesthesia, animals were allowed to recover for at least 24 h. Middle cerebral artery occlusion (MCAO) surgery was introduced according to procedures reported previously.52 Briefly, rats were anesthetized with isoflurane and then positioned supinely on a heating pad by which the rectal temperature was maintained at 37°C throughout the experiments. The right common carotid artery, internal carotid artery, and external carotid artery were exposed through a midline incision at the ventral surface of the neck. A 4-0 monofilament nylon suture with a rounded tip was inserted into the internal carotid artery through an incision made in external carotid artery. To induce MCAO, the suture was gently advanced through the internal carotid artery until resistance was felt, approximately 20 ±1 mm from the common carotid artery bifurcation. In I/R groups, the suture was removed 1 h after the occlusion and no further interruption of reperfusion was applied. In the ischemic postconditioning groups, however, 10 cycles of 10 s-reperfusion/10 s-reocclusion of middle cerebral artery was applied after the removal of the suture. OECS for Ascorbic Acid Monitoring. OECS for ascorbic acid monitoring was performed with in vivo microdialysis sampling coupled with an online selective electrochemical detection, as schematically shown in Figure 2A.36, 37 Briefly, a thin-layer electrochemical flow cell consisting of a glassy carbon (GC) working electrode (6 mm in diameter), an Ag/AgCl reference electrode and stainless steel counter electrode was used as the detector. To achieve the selectivity for ascorbic acid recording, the working electrode was modified with SWNTs and polarized at +30 mV for continuously monitoring the extracellular ascorbic acid. At a flow rate of 2 μL/min, the current (I) recorded from the OECS was linear with the concentration of ascorbic acid (CAA) with a linear equation of I (nA) = 4.36 CAA (μM) + 7.00 (r = 0.997) within the concentration range from 1 μM to 100 μM. To monitor the dynamics of ascorbic acid in rat cortex, rats were anesthetized with isoflurane and the microdialysis probe (2 mm in length; Microbiotech/se AB, Sweden) was implanted into the cortex through the microdialysis guide cannula. By using the microinjection pump (CMA/100, CMA Microdialysis AB, Sweden), the microdialysis probe was perfused with aCSF for 90 min at a flow rate of 2 μL/min. Then the focal cerebral ischemia and postconditioning treatment were carried out. Meanwhile, the microdialysate sampled was continuously delivered into a thin-layer radial electrochemical flow cell for the detection of ascorbic acid. Electrophysiological Recording. Rats were anesthetized with isoflurane and placed on a stereotaxic 11



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frame. Using aseptic stereotaxic surgical techniques, a tungsten electrode (90 μm in diameter) with only tip exposed was implanted into the right cortex (AP = 0 mm and L = 5.0 mm from the bregma, V = 1.0 mm from the surface of the skull, using a flat skull position).51 A stainless-steel wire was placed under the skin as the reference/ground electrode. Then the electrodes were secured with bone screws and acrylic cement. After removal of the anesthesia, animals were allowed to recover for at least 24 h. For electrophysiological recording, rats were anesthetized with isoflurane and the implanted electrodes were connected to the Electrocorticograms (ECoG) recording system (RM6240B, Chengdu Instrument Factory, China) through soft cables. Then rats were subjected to ischemic/reperfusion surgery or ischemic/reperfusion plus ischemic postconditioning as described above. The changes of ECoG signals before, during ischemia, and after reperfusion or ischemic postconditioning were all recorded with gain 8000×, sampling frequency 400 Hz, band-pass 0.1-100 Hz. Immunohistochemistry. Immunohistochemistry for 8-hydroxyl-2’-deoxyguanosine (8-OHdG) was used to examine the oxidative injury of DNA.36 Two hours after direct reperfusion (i.e., without ischemic postconditioning, I/R) or ischemic postconditioning (I/R + postconditioning) treatment, rats in the immunohistochemistry groups were treated with intracardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) under deep anesthesia. The brains were quickly removed and immersed in 4% paraformaldehyde for 12 h, then kept in 25% sucrose for 3 days at 4℃. Tissues were embedded in paraffin wax, then 6 μm-thick sections were cut and mounted onto polylysine-coated slides. For immunohistochemical analysis of 8-OHdG, slices were blocked with goat serum albumin for 20 min, then incubated overnight with the rabbit polyclonal anti-8-OHdG antibody (1:200, Biosynthesis Biotechnology, China) at 4℃. After washing with PBS, the slices were further incubated for 20 min with secondary antibody, biotin labeled goat antirabbit IgG (1:200, Biosynthesis Biotechnology, China), followed by reaction with avidin-biotin peroxidase complex (ABC) for 20 min at room temperature. Finally, the labeling was visualized using diaminobenzidine (DAB, Biosynthesis Biotechnology, China) for 5 min, and the slices were counterstained with hematoxylin. Infarct Volume Measurements. Twenty-four hours after direct reperfusion or ischemic postconditioning treatment, rats employed for the infarct volume measurements were anesthetized with an

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overdose of chloral hydrate followed by decapitation. Then the brains were rapidly removed and cut into 2mm-thick coronal sections by using a rat brain matrix (RWD 68710, Shenzhen, China). Each section of the brain was immersed in a solution of 2% 2,3,5-triphenyltetrazolium-chloride (TTC) for 25 min at 37 ℃. The normal tissue was stained red, whereas the infarcted tissue remained white. The TTC-stained brain sections were photographed by a digital camera, and then the infarct volume was analyzed by Image-Pro Plus (Media Cybernetics, version 6.0). To avoid the influence of edema, the percentage of infarct was calculated by dividing the area of the infarct by that of the contralateral, nonischemic cortex.23, 53 Data Analysis. Electrophysiological data was analyzed with Chart5 software (AD Instrument, Australia). Thirty seconds epochs of ECoG were selected every 5 minutes from the raw data and the root mean square (RMS), which is numerically equal to the standard deviation of the amplitude values, was calculated. percentage of the power value of delta (δ) frequency band (1-4 Hz) against that of the overall 1-30 Hz band was calculated by applying Fast Fourier Transform (FFT) with FFT size 512 by Welch’s method.45 For the measurement of ascorbic acid in the microdialysate, the current response recorded with OECS was converted to the concentration of ascorbic acid (CAA) according to the linear equation mentioned above. To minimize the difference between subject variations, the normalized concentration of ascorbic acid to the basal level was used for comparison. All the data were presented as mean ± SD. The statistical tests of infarct volume and 8OHdG positive cells were subjected to the two-tailed Student's t-test and one-way analysis of variance (1ANOVA) by IBM SPSS software (International Business Machines Corp), respectively. For the statistical analysis of normalized RMS, relative power of δ frequency band (1-4 Hz) and extracellular ascorbic acid level, 1-ANOVA with repeated measures was performed. P < 0.05 was considered to be statistically significant.

AUTHOR INFORMATION Corresponding Author * E-mail:

[email protected]. Fax: (+86)-10-62559373.

Author Contributions

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§ D.W.

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and X. L. contributed equally. L. M., D.W. and X. L. conceived and designed the experiments; D.W.

performed the experiments; X. L., Y.J. and Y-N.J. analyzed the data; D.W., X. L., P.Y. and L. M. proofed the writing; D.W., X. L. and L. M. wrote the paper. Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS We acknowledge ﬁnancial support from the National Natural Science Foundation of China (Grant Nos. 21790390, 21790391, and 21621062 for L.M.; 21790393, 21605147 for Y.J.; 21790392, 21705155 for W.M.), the National Basic Research Program of China (Grant Nos. 2016YFA0200104), the Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDB30000000), and the Chinese Academy of Sciences (QYZDJ-SSW-SLH030). REFERENCES (1) Jackson, S. P., and Calkin, A. C. (2007) The clot thickens-oxidized lipids and thrombosis. Nat. Med. 13, 1015. (2) Sporns, P. B., Hanning, U., Schwindt, W., Velasco, A., Minnerup, J., Zoubi, T., Heindel, W., Jeibmann, A., and Niederstadt, T. U. (2017) Ischemic stroke: what does the histological composition tell us about the origin of the thrombus? Stroke 48, 2206-2210. (3) Rossi, D. J., Oshima, T., and Attwell, D. (2000) Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature 403, 316-321. (4) Nakatsuka, N., Yang, K. A., Abendroth, J. M., Cheung, K. M., Xu, X., Yang, H., Zhao, C., Zhu, B., Rim, Y. S., Yang, Y., Weiss, P. S., Stojanović, M. N., and Andrews, A. M. (2018) Aptamer–field-effect transistors overcome Debye length limitations for small-molecule sensing. Science 362, 319-324. (5) Zhang B., Adams K. L., Luber S. J., Eves D. J., Heien M. L., and Ewing A. G. (2008) Spatially and temporally resolved single-cell exocytosis utilizing individually addressable carbon microelectrode arrays. Anal. Chem. 80, 1394. (6) Phan, N. T. N., Li, X., and Ewing, A. G. (2017) Measuring synaptic vesicles using cellular electrochemistry and nanoscale molecular imaging. Nat. Rev. Chem. 1, 0048. (7) Denno, M. E., Privman, E., Borman, R. P., Wolin, D. C., and Venton, B. J. (2016) Quantification of histamine and carcinine in drosophila melanogaster tissues. ACS Chem. Neurosci. 7, 407-414. (8) Sasaki, T., Takemori, H., Yagita, Y., Terasaki, Y., Uebi, T., Horike, N., Takagi, H., Susumu, T., Teraoka, H., and Kusano, K. I. (2011) SIK2 is a key regulator for neuronal survival after ischemia via TORC1-CREB. Neuron 69, 106-

14


Page 15 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


119. (9) Esenwa, C. C., and Elkind, M. S. (2016) Inflammatory risk factors, biomarkers and associated therapy in ischaemic stroke. Nat. Rev. Neurol. 12, 594. (10) Basso, M., and Ratan, R. R. (2013) Transglutaminase is a therapeutic target for oxidative stress, excitotoxicity and stroke: a new epigenetic kid on the CNS block. J. Cerebr. Blood F. Met. 33, 809-818. (11) Unal-Cevik, I., Kilinç, M., Can, A., Gürsoy-Ozdemir, Y., and Dalkara, T. (2004) Apoptotic and necrotic death mechanisms are concomitantly activated in the same cell after cerebral ischemia. Stroke 35, 2189-2194. (12) Zhao, X., Wang, K., Li, B., Wang, C., Ding, Y., Li, C., Mao, L., and Lin, Y. (2018) Fabrication of a flexible and stretchable nanostructured gold electrode using a facile ultraviolet-irradiation approach for the detection of nitric oxide released from cells. Anal. Chem. 90, 7158-7163. (13) Liu, X., Xiao, T., Wu, F., Shen, M. Y., Zhang, M., Yu, H. H., and Mao, L. (2017) Ultrathin cell-membrane-mimic phosphorylcholine polymer film coating enables large improvement for In vivo electrochemical detection. Angew. Chem. Int. Ed. 56, 11802-11806. (14) Bhowmick, S., Moore, J. T., Kirschner, D. L., Curry, M. C., Westbrook, E. G., Rasley, B. T., and Drew, K. L. (2017) Acidotoxicity via ASIC1a mediates cell death during oxygen glucose deprivation and abolishes excitotoxicity. ACS Chem. Neurosci. 8, 1204-1212. (15) Lee, J. J., Li, L., Jung, H. H., and Zuo, Z. (2008) Postconditioning with isoflurane reduced ischemia-induced brain injury in rats. Anesthesiology 108, 1055-1062. (16) Denorme, F., Langhauser, F., Desender, L., Vandenbulcke, A., Rottensteiner, H., Plaimauer, B., François, O., Andersson, T., Deckmyn, H., and Scheiflinger, F. (2016) ADAMTS13-mediated thrombolysis of t-PA resistant occlusions in ischemic stroke in mice. Blood 127, 2337. (17) Kalogeris, T., Bao, Y., and Korthuis, R. J. (2014) Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning. Redox Biol. 2, 702-714. (18) Chouchani, E. T., Pell, V. R., James, A. M., Work, L. M., Saeb-Parsy, K., Frezza, C., Krieg, T., and Murphy, M. P. (2016) A unifying mechanism for mitochondrial superoxide production during ischemia-reperfusion injury. Cell Metab. 23, 254-263. (19) Papadimitriou, K. I., Wang, C., Rogers, M. L., Gowers, S. A. N., Leong, C. L., Boutelle, M. G., and Drakakis, E. M. (2016) High-performance bioinstrumentation for real-time neuroelectrochemical traumatic brain injury monitoring. Front. Hum. Neurosci. 10, 212. (20) Pagkalos, I., Rogers, M., Boutelle, M. G., and Drakakis, E. M. (2018) A high performance application specific

15



Page 16 of 24

integrated circuit for electrical and neurochemical traumatic brain injury monitoring. Chemphyschem 19, 1215-1225. (21) Zhao, H., Sapolsky, R. M., and Steinberg, G. K. (2006) Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. J. Cerebr. Blood F. Met. 26, 1114-1121. (22) Elga, E., Kazuhide, H., Takakuni, M., Ken, A., and Lo, E. H. (2015) Effects of postconditioning on neurogenesis and angiogenesis during the recovery phase after focal cerebral ischemia. Stroke 46, 2691-2694. (23) Zhao, H., Wang, R., Tao, Z., Gao, L., Yan, F., Gao, Z., Liu, X., Ji, X., and Luo, Y. (2014) Ischemic postconditioning relieves cerebral ischemia and reperfusion injury through activating T-LAK cell-originated protein kinase/protein kinase B pathway in rats. Stroke 45, 2417-2424. (24) Heng, Z., Chuancheng, R., Xingmiao, C., and Jiangang, S. (2012) From rapid to delayed and remote postconditioning: the evolving concept of ischemic postconditioning in brain ischemia. Curr. Drug Targets 13, 173-187. (25) Gao, X., Ren, C., and Zhao, H. (2008) Protective effects of ischemic postconditioning compared with gradual reperfusion or preconditioning. J. Neurosci. Res. 86, 2505-2511. (26) Menyhárt, Á., Makra, P., Szepes, B. É., Tóth, O. M., Hertelendy, P., Bari, F., and Farkas, E. (2015) High incidence of adverse cerebral blood flow responses to spreading depolarization in the aged ischemic rat brain. Neurobiol. Aging 36, 3269-3277. (27) Geocadin, R. G., Sherman, D. L., Christian, H. H., Kimura, T., Niedermeyer, E., Thakor, N. V., and Hanley, D. F. (2002) Neurological recovery by EEG bursting after resuscitation from cardiac arrest in rats. Resuscitation 55, 193-200. (28) Aksenov, D. P., Li, L., Miller, M. J., Iordanescu, G., and Wyrwicz, A. M. (2015) Effects of anesthesia on BOLD signal and neuronal activity in the somatosensory cortex. J. Cerebr. Blood F. Met. 35, 1819-1826. (29) Petit-Pierre, G., Colin, P., Laurer, E., Déglon, J., Bertsch, A., Thomas, A., Schneider, B. L., and Renaud, P. (2017) In vivo neurochemical measurements in cerebral tissues using a droplet-based monitoring system. Nat. Commun. 8, 1239. (30) Zhou, L., Li, F., Xu, H. B., Luo, C. X., Wu, H. Y., Zhu, M. M., Lu, W., Ji, X., Zhou, Q. G., and Zhu, D. Y. (2010) Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nat. Med. 16, 1439. (31) Harrison, F. E., and May, J. M. (2009) Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2. Free Radical Biol. Med. 46, 719-730. (32) Rice, M. E. (2000) Ascorbate regulation and its neuroprotective role in the brain. Trends Neurosci. 23, 209-216. (33) Horn, J. D. V., Bhattrai, A., and Irimia, A. (2017) Multimodal imaging of neurometabolic pathology due to traumatic brain injury. Trends Neurosci. 40, 39–59. (34) Cherubini, A., Ruggiero, C., Polidori, M. C., and Mecocci, P. (2005) Potential markers of oxidative stress in stroke. Free Radical Biol. Med. 39, 841-852.

16


Page 17 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(35) Liu, K., Lin, Y., Yu, P., and Mao, L. (2009) Dynamic regional changes of extracellular ascorbic acid during global cerebral ischemia: Studied with in vivo microdialysis coupled with on-line electrochemical detection. Brain Res. 1253, 161-168. (36) Liu, K., Yu, P., Lin, Y., Wang, Y., Ohsaka, T., and Mao, L. (2013) Online electrochemical monitoring of dynamic change of hippocampal ascorbate: toward a platform for in vivo evaluation of antioxidant neuroprotective efficiency against cerebral ischemia injury. Anal. Chem. 85, 9947-9954. (37) Zhang, M., Liu, K., Gong, K., Su, L., Chen, Y., and Mao, L. (2005) Continuous on-line monitoring of extracellular ascorbate depletion in the rat striatum induced by global ischemia with carbon nanotube-modified glassy carbon electrode integrated into a thin-layer radial flow cell. Anal. Chem. 77, 6234-6242. (38) Xiao, T., Li, X., Wei, H., Ji, W., Yue, Q., Yu, P., and Mao, L. (2018) In vivo monitoring of oxygen fluctuation simultaneously at multiple sites of rat cortex during spreading depression. Anal. Chem. 90, 13783-13789. (39) Takano, T., Tian, G., W, Lou, N., Lovatt, D., Hansen, A., Kasischke, K., and Nedergaard, M. (2007) Cortical spreading depression causes and coincides with tissue hypoxia. Nat. Neurosci. 10, 754. (40) Yusa, T. (2001) Increased extracellular ascorbate release reflects glutamate re-uptake during the early stage of reperfusion after forebrain ischemia in rats. Brain Res. 897, 104-113. (41) Wang, S., Liu, X., and Zhang, M. (2017) Reduction of ammineruthenium(III) by sulfide enables in vivo electrochemical monitoring of free endogenous hydrogen sulfide. Anal. Chem. 89. (42) Abhaya, D., Rekha, G., Sreejata, C., Freek, A., Sujit Kumar, S., and Siva, U. (2015) Ascorbate protects neurons against oxidative stress: a raman microspectroscopic study. ACS Chem. Neurosci. 6, 1794-1801. (43) Zagrean, L., Moldovan, M., Munteanu, A. M., Spulber, S., Voiculescu, B., and Popescu, B. (2000) Early electrocortical changes consistent with ischemic preconditioning in rat. J. Cell. Mol. Med. 4, 215-223. (44) Bolay, H., and Dalkara, T. (1998) Mechanisms of motor dysfunction after transient MCA occlusion: persistent transmission failure in cortical synapses is a major determinant. Stroke 29, 1988-1993. (45) Latreille, V., Carrier, J., Gaudetfex, B., Rodriguesbrazète, J., Panisset, M., Chouinard, S., Postuma, R. B., and Gagnon, J. F. (2016) Electroencephalographic prodromal markers of dementia across conscious states in Parkinson’s disease. Brain 139, 1189. (46) Foreman, B., Albers, D., Schmidt, J. M., Falo, C. M., Velasquez, A., Connolly, E. S., and Claassen, J. (2017) Intracortical electrophysiological correlates of blood flow after severe SAH: a multimodality monitoring study. J. Cerebr. Blood F. Met. 38, 506-517. (47) Hartings, J. A., Watanabe, T., Bullock, M. R., Okonkwo, D. O., Fabricius, M., Woitzik, J., Dreier, J. P., Ava, P.,

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Page 18 of 24

Shutter, L. A., and Clemens, P. (2011) Spreading depolarizations have prolonged direct current shifts and are associated with poor outcome in brain trauma. Brain 134, 1529-1540. (48) Wang, J. Y., Shen, J., Gao, Q., Ye, Z. G., Yang, S. Y., Liang, H. W., Bruce, I. C., Luo, B. Y., and Xia, Q. (2008) Ischemic postconditioning protects against global cerebral ischemia/reperfusion-induced injury in rats. Stroke 39, 983990. (49) Gold, L., and Lauritzen, M. (2002) Neuronal deactivation explains decreased cerebellar blood flow in response to focal cerebral ischemia or suppressed neocortical function. Proc. Natl. Acad. Sci. USA. 99, 7699-7704. (50) Jeng, W., Loniewska, M. M., and Wells, P. G. (2013) Brain glucose-6-phosphate dehydrogenase protects against endogenous oxidative DNA damage and neurodegeneration in aged mice. ACS Chem. Neurosci. 4, 1123-1132. (51) Gao, X., Zhang, H., Takahashi, T., Hsieh, J., Liao, J., Steinberg, G. K., and Zhao, H. (2008) The Akt signaling pathway contributes to postconditioning’s protection against stroke; the protection is associated with the MAPK and PKC pathways. J. Neurochem. 105, 943-955. (52) Xing, B., Chen, H., Zhang, M., Zhao, D., Jiang, R., Liu, X., and Zhang, S. (2008) Ischemic postconditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury in the rat. Stroke 39, 2362-2369. (53) Schuhmann, M. K., Langhauser, F., Kraft, P., and Kleinschnitz, C. (2017) B cells do not have a major pathophysiologic role in acute ischemic stroke in mice. J. Neuroinflamm. 14, 112.

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B

A

* Infarct volume (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


I/R

PostCond

I/R

PostCond

Figure 1. Ischemic postconditioning attenuated focal cerebral ischemia/reperfusion injury. (A) Triphenyltetrazolium chloride staining of representative coronal sections 24 h after focal cerebral reperfusion. The suture was removed 1 h after the occlusion in ischemia/reperfusion (I/R) group, while 10 cycles of 10 sreperfusion/10 s-reocclusion of middle cerebral artery was applied after the removal of the suture in the postconditioning (PostCond) group. (B) Quantification of the infarct volume at the time point of 24 h after focal cerebral reperfusion in both groups (each group, n = 6). The asterisk (*) indicates significant difference between the I/R and the PostCond groups by the two-tailed Student's t-test (P < 0.05).

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A

Potentiostat Electrochemical and computer Flow Cell

H HOCH2

H

-2e-,-H+

O

O

OH O-

HOCH2

O

O H

O

OH

OH

HOCH2

O

H2O O O

OH O

OH

OH

Microdialysis Probe MCAO 1 h Reperfusion 2 h

B

20 nA

MCAO 1 h

Postconditioning +Reperfusion 2 h

sham surgery

aCSF

30 nA

C

Microinjection Pump

20 min

aCSF basal level

30 min

aCSF basal level

reperfusion

MCAO

aCSF

D 30 nA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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30 min

postconditioning

aCSF basal level

reperfusion

MCAO

aCSF

Figure 2. (A) Schematic illustration of experimental setup of the online electrochemical system (OCES) for continuously monitoring ascorbic acid in rat cortex. Typical current-time responses recorded for the ascorbic acid continuously sampled from rats subjected to sham surgery (B), ischemia/reperfusion surgery (C) and ischemia surgery and postconditioning treatment followed by reperfusion (D). The microdialysis probe was perfused with aCSF at a flow rate of 2 μL/min. The working electrode was poised at +30 mV (vs. Ag/AgCl electrode). 20


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basal level

MCAO 1h

500

Reperfusion 2h

postconditioning

400

CAA / C0 (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


300 *

200

*

100 0

*

I/R PostCond

0

30

60

90

120

150

*

##

#

# # #

180

# # #

210

T / min Figure 3. Statistical comparison of the cortex ascorbic acid in the I/R and the PostCond groups. C0 represents the basal level of ascorbic acid in rat cortex. The time interval for data acquisition is 5 min. n = 6. * and # represents significant differences compared with the I/R group with P < 0.05 and P < 0.01, respectively.

21



basal level MCAO 1h

reperfusion 2h

A 0.8 mV

PostCond I/R

I/R PostCond Ascorbic acid in PostCond

400

80 60

** 300 ***** ************

40

200

20

100 0

C

500

CAA / C0 (%)

Normalized root mean square (%)

100

30

60

90

160

120

150

180

210

I/R PostCond Ascorbic acid in PostCond

500

400

140

300 120

***** ******* *

100

CAA / C0 (%)

B

Normalized relative delta power (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

200

100 0

30

60

90

120

150

180

210

T / min

Figure 4. (A) Representative ECoG tracings for the I/R and the PostCond groups. (B) Corresponding changes of normalized root mean square (RMS) of the ECoG signals. Thirty seconds epochs of ECoG were selected every 5 min to calculate the RMS that was numerically equal to the standard deviation of the amplitude values. (C) Dynamics of normalized percent power values of the δ frequency band (1-4 Hz). The percent power value is the percentage of the power of δ frequency band (1-4 Hz) against that of the overall 1-30 Hz band. The average value of the percent power of the δ frequency band (1-4 Hz) before MCAO were regarded as 100% and values during and after MCAO were normalized against the basal value to eliminate the individual differences (n = 6). The red dotted line represents the dynamics of the average level of the cortex ascorbic acid in the PostCond group. Asterisks indicate the significant difference at the relative time point between the PostCond and the I/R group (P < 0.05).

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A

B

C

D **

8-OHdG positive cells (cells/mm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


**, # Sham

I/R

PostCond

Figure 5. Effect of ischemic postconditioning on oxidative injury of DNA in rat cortex. Representative photomicrographs of 8-OHdG immunostaining cells (brown color) in sham group (A), 2 h after reperfusion of the I/R group (B) and 2 h after postconditioning in the PostCond group (C). Arrows, examples of 8-OHdG positive cells. Scale bars, 50 μm. (D) Quantification of the density of 8-OHdG positive cells in three groups. Error bar, SD. ** P < 0.01 vs. sham group, # P < 0.05 vs. the I/R group (n = 3).

23



For Table of Content Only

postconditioning H HOCH2

O O

OH O-

OH

ascorbate

ischemia/reperfusion PostCond

Ascorbic acid

basal level

MCAO 1h

Reperfusion 2h

24


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Ischemic Postconditioning Recovers Cortex Ascorbic Acid during

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