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In Vivo Monitoring of Oxygen Fluctuation Simultaneously at Multiple Sites of Rat Cortex during Spreading Depression Tongfang Xiao, Xianchan Li, Huan Wei, Wenliang Ji, Qingwei Yue, Ping Yu, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04348 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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Analytical Chemistry

In Vivo Monitoring of Oxygen Fluctuation Simultaneously at Multiple Sites of Rat Cortex during Spreading Depression Tongfang Xiao,†,§ Xianchan Li,†,§ Huan Wei,†,‡ Wenliang Ji,† Qingwei Yue,†,‡ 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

§These

of Chinese Academy of Sciences, Beijing 100049, China.

authors contributed equally. 1

ACS Paragon Plus Environment

Analytical Chemistry 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

* Corresponding

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author. Fax: +86-10-62559373. E-mail: [email protected].

ABSTRACT Spreading depression (SD) is a common pathological process in brain featured as propagating neuronal depolarization followed by activity depression over brain and is closely related with migraine and epilepsy. Although O2 is known to fluctuate during SD, the difference of O2 responses at different sites in the same brain region remains unknown. In this study, we develop an in vivo electrochemical method with microelectrode arrays (MEAs) to real-time monitor O2 fluctuation at multiple sites of rat cortex during SD with high spatial/temporal resolution. Platinum nanoparticles are electrochemically deposited on the multiplexed electrodes of the MEAs to monitor O2 fluctuation simultaneously and selectively via a four-electron reduction process. Configuration of electrode arrays is designed rationally to exclude the probable crosstalk between neighbor recording electrodes during simultaneous measurements. With the MEAs, we find both the basal O2 levels and O2 fluctuations at different sites of cortex during SD exhibit significant differences, indicating the intensity of energy metabolism and oxidative stress vary at different sites even in the same brain region. Further studies prove that O2 fluctuation is mostly caused by the increase of brain blood flow and the consumption of neuronal O2 during SD. Our study reveals that energy metabolism varies at different sites in brain cortex during SD propagation, which may provide new understanding for SD-related pathological processes.

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Spreading depression (SD), firstly proposed by Leao in 1944, is a self-propagating wave of neural depolarization across the brain in a regenerative manner accompanied with electrical activity depression.1 SD is similar with the spreading waves of some pathological processes, such as migraine and epilepsy, thus making it an important model in evolution studies of these neurological disorders. SD could be induced by high concentration of K+, repetitive electrical stimulation and mechanical stimulation.2-4 Chaos of the distribution of trans-membrane neurochemicals induces a negative deflection of the prolonged extracellular direct-current (DC) potential during SD.5 It has been confirmed that large amounts of K+ and glutamate released from neurons into the interstitial fluid, and meanwhile, Na+ and Ca2+ flow into neurons thus causing the imbalance of ionic distribution.6 To recover the normal concentrations of intra- and extra-cellular species during SD, neuronal energy metabolism increases, which may bring considerable metabolic challenge and oxidative stress in the brain.7,8 As an indispensable substrate in energy metabolism, O2 plays an important role in various physiological and pathological processes including SD.9,10 The dynamic equilibrium of O2 in the brain microenvironment is premised to maintain normal physiological functions of brain and its distribution in the brain is balanced between the supply of arterial blood O2 and the cellular O2 consumption.11,12 Therefore, O2 tension remains a wide range depending on the heterogeneity of brain tissue. Moreover, O2 fluctuation reflects the change of neuronal metabolism in various neurochemical processes. During SD, the fast O2 consumption caused by the severe neuron activity disturbs the O2 balance, which may not be recovered by cerebral blood flow in a short time.13 The O2 deficiency in local tissue will induce energy metabolism disorders or even neuron death through the oxidative stress.8 In addition, the oxidative stress could spread across most regions of brain during SD propagation, thus further induce secondary brain injury in the brain. As an important biomarker, extracellular O2 in local microenvironment could reflect the neuronal energy metabolism and oxidative stress in various brain regions.14 Therefore, it is significant to evaluate the O2 3



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tension at different sites of the brain during SD propagation. Qualitative and quantitative methods for monitoring the O2 dynamics in different locations of brain, such as functional magnetic resonance imaging, near-infrared light spectroscopy and electron paramagnetic resonance, have been widely developed over these years, and their applications have led to some significant conclusions for the critical roles of O2 in the brain.15,16 However, it still remains difficult to monitor O2 dynamics precisely due to the low spatial/temporal resolution of these methods.17,18 In most case, the O2 tension monitored by these techniques is an average value in certain areas of brain over a certain time. Therefore, the conclusions made with these methods have been limited in terms of the microenvironment to which the individual neurons are exposed.12 The facts mentioned above, unfortunately, offers a barrier to the application of these methods to monitor O2 during SD, in which O2 tension changes rapidly and spreads across the brain. O2-selective microelectrodes have been used to monitor extracellular O2 fluctuation during SD.8,15 In such measurements, a single microelectrode is implanted and measures the O2 at specific location of brain although it has high spatial resolution at micrometer ranges.19,20 To the best of our knowledge, simultaneous measurements of O2 fluctuation at multiple locations in brain during SD wave propagation have not been reported so far. Microelectrode arrays (MEAs) with multiplexed electrodes have used demonstrated to be particularly useful to monitor neurochemicals simultaneously.21-26 MEAs-based in vivo monitoring of neurochemicals exhibit not only high temporal resolution but also high spatial resolution of the multiple detection sites benefited from the advanced microelectronics technology.27-37 In this study, we develop MEAs consisting of multiplexed electrodes for monitoring O2 fluctuation at multiple locations of rat cortex during SD propagation. After eliminating the overlap between the diffusion layers of neighbor recording sites, four microelectrodes at MEAs modified with platinum nanoparticles are employed to monitor O2 simultaneously at multiple sites of rat cortex with a high temporal/spatial resolution. The MEAs exhibit high stability and selectivity towards in vivo O2 measurement. With the O2 MEAs, we observe distinct differences of O2 basal level and O2 fluctuation at different locations of cortex at micrometer ranges during SD evoked by electrical stimulation. Further studies confirm that O2 change is mostly caused by the increase of blood flow and the neuronal O2 consumption during SD propagation. Therefore, monitoring of O2 tension at multiple sites of brain by MEAs can offer us an avenue to understanding energy metabolism and 4


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neurochemical processes during SD, which may advance the mechanistic study underlying SD-related pathological processes.

EXPERIMENTAL SECTION Reagents and Solutions. Dopamine (DA), ascorbate, uric acid (UA), 3, 4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), epinephrine (E) and norepinephrine (NE) were all purchased from SigmaAldrich and used as supplied. Chloroplatinic acid hexahydrate (H2PtCl6·6H2O) was purchased from Aladdin (China). Artificial cerebrospinal fluid (aCSF) was prepared by mixing 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. Other chemicals were of at least analytical grade and used as obtained. All aqueous solutions were prepared with Milli-Q water. Unless stated otherwise, all experiments were carried out at room temperature. Apparatus and Measurements. Electrochemical measurements were performed on a computer-controlled multi-channel electrochemical analyzer (Model 1030C, CHI Instruments, Shanghai, China). Electrical stimulation was performed on an A.M.P.I. Master-9 SDK system (A.M.P.I., Jerusalem, Israel). Local field potential and single unit discharge were recorded on an omniplex/128-D electrophysiological recording system (PLEXON, Dallas, America). For both in vitro and in vivo electrochemical measurements, MEAs were used as working electrodes, a platinum wire as counter electrode, and a tissue-implantable Ag/AgCl electrode as reference electrode. The reference electrode was prepared as described previously.38 MEAs were calibrated before and after the in vivo experiments in aCSF saturated with N2, ambient air, and O2, in which the O2 concentration was identified as 0, 200, and 1250 μM, respectively. Scanning electron microscopy (SEM) was performed on a Model S4300-F microscope (Hitachi, Inc., Tokyo, Japan). Preparation of Microelectrode Arrays. Gold-based MEAs were bought from Institute of Semiconductors, Chinese Academy of Sciences (Beijing, China). The fabrication process was reported previously.39 Briefly, a silicon-on-insulator (SOI) wafer was used as the substrate. Titanium / gold (100 nm / 300 nm, respectively) was evaporated and patterned to form the connecting lines. A dielectric SiO2 layer was deposited by plasma enhanced chemical vapor deposition to insulate the conductive layer from the substrate. Au nanoparticles were sputtered to 5



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form the planar recording sites on the contact holes. Subsequently, the SOI wafer was etched by inductive-coupled plasma (ICP) on a SiO2 etching equipment under the protection of the thick photoresist. Finally, a 16-recording-site microelectrode array with 30 µm in thickness was formed on four 5 mm-long shanks that were spaced 125 µm. The spacing distance between the recording sites in one shank was 100 µm and every Au recording site was 20 µm in diameter. To achieve the four-electron reduction pathway of O2, Pt nanoparticles were deposited electrochemically on Au recording sites to form Pt-based MEAs. For this purpose, MEAs were first rinsed thoroughly with ethanol and water and activated electrochemically in 0.5 M H2SO4 by scanning the potential between -0.2 V and 1.1 V for 20 cycles at 0.5 V/s. Then the electrochemical deposition was performed in 0.5 M HCl solution containing 19.3 mM H2PtCl6 and 0.3 mM (CH3COO)2Pb with MEAs as working electrodes, a Pt wire as counter electrode and an Ag/AgCl electrode as reference electrode. A potential of -0.3 V was simultaneously applied to the multiplexed electrodes for about 40 s with total deposition electricity at 1.5×10-5 C. After that, the MEAs were rinsed with water, dried at 60 °C, and then kept for subsequent use. In Vivo Experiments. Adult Sprague-Dawley rats (male, 300-350 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed on a 12 h : 12 h light-dark schedule with food and water ad libitum. All procedures were approved and directed by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology of China. The animals were anaesthetized with chloral hydrate (345 mg/kg, i.p.) and positioned onto a stereotaxic frame. The Pt/MEAs were implanted into rat auditory cortex (AP = -4 mm, L = 6.5 mm from bregma, V = 2.0 mm from dura) using standard stereotaxic procedures. The Ag/AgCl reference electrode was positioned into the dura of brain. A platinum wire embedded in subcutaneous tissue of the brain was used as counter electrode. Mild hyperoxia and hypoxia were induced by exposing the animal in O2 atmosphere for 100 s and N2 atmosphere for 50 s, respectively. O2 or N2 was stored in rubber bladders and delivered from a tube with the end placed close to the nose of the rats. All electrodes in the arrays were polarized at -0.5 V for in vivo amperometric measurements of O2 in rat brain. A bipolar stimulation electrode connected to a Master-9 SDK system was placed in the burr hole located in the cortex (AP = -2.6 mm, L = 4.0 mm from bregma, V = 1.5 mm from dura). A continuous monophasic current (600 μA, 5 s) was delivered at the stimulation electrode to induce SD in rat cortex. The electrophysiological signals were sampled at a rate of 25 6


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kHz. A low pass filter at 100 Hz was applied to record the local field potential (LFP), while a high pass filter at 250 Hz was applied to record the neural activity. Statistical Analysis. All statistical tests were subjected to one-way analysis of variance (1-ANOVA) and paired Student’s t-test by IBM SPSS software (International Business Machines Corp). p < 0.05 was considered as significantly different.

RESULTS AND DISCUSSION Multiplexed Electrodes of MEAs. When multiplexed electrodes of MEAs are employed for monitoring of neurochemicals simultaneously, electrode crosstalk is a common problem that can bias measurements significantly.40-42 Herein, to avoid the crosstalk from the neighbor microelectrodes that essentially occurs when multiplexed electrodes are employed for O2 measurement simultaneously, we did calculation to optimize the design of MEAs. Under simple mass-transport control, the current (i) at the micro-disk electrodes could be expressed with equation 1,43 𝑖=

4𝑛𝐹𝐴𝐷0𝐶0∗ 𝜋𝑟0

(1)

𝑓(𝜏)

𝑓(𝜏) = 0.7854 + 0.8862𝜏 ―1/2 +0.2146𝑒 ―0.7823𝜏

―1/2

(2)

in which n is the number of electrons in O2 reduction (4 here for O2 reduction at Pt nanoparticles-modified electrode), F is the Faraday’s constant (96500 C-1), A is the area of the micro-disk electrode, D0 is the diffusion coefficient of O2, 𝑐0∗ is the initial concentration of O2, 𝑟0 is the radius of micro-disk electrode, and 𝜏 is a non-dimensional parameter, another pattern of manifestation of time t. And 𝜏=

4𝐷0𝑡

(3)

𝑟20

The thickness of diffusion layer (l) of the micro-disk electrode within a short time can be readily expressed by the equation 4,43 1

(4)

𝑙 = 2(𝐷0𝑡)2 7



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Therefore, l can be expressed with the equation 5, 1

(5)

𝑙 = 𝑟0(𝜏)2

For microdisk electrodes, the thickness of the diffusion layer increases with increasing time until the electrode reaches its limiting current (𝑖ss). From equations 1 and 2, when τ reaches 100, 𝑓(𝜏) approximates its maximum constant 1, at which point the current of the microdisk electrode almost approaches 𝑖ss.43 Then, the equation 5 can be derived to l = 10 𝑟0.40 For the microdisk electrodes with 10 μm in radius, l is about 100 μm, so the distance between two recording sites for monitoring O2 simultaneously is at least 200 μm (Figure 1B). Therefore, we used every other electrode (labeled as E1-E4 in Figure 1A) on the commercial MEAs, instead of all the electrodes for subsequent Pt nanoparticles modification to monitor O2. Figure 1C displays SEM image of Pt nanoparticles deposited electrochemically on E1-E4. Benefited from the electrochemical deposition, Pt nanoparticles were distributed relatively uniformly on the microdisk electrode with high controllability. As shown in Figure 1D, cyclic voltammograms (CV) of the Pt/MEAs in acidic media exhibits the adsorption and desorption of hydrogen, which is characteristic of a clean electroactive surface of Pt. The application of Pt nanoparticles could avoid producing the intermediate H2O2, which is toxic in the central nervous system (CNS), because O2 could be reduced to H2O electrochemically on Pt electrodes through a four-electron reduction process.17 Note that, Pt nanoparticles should not exceed the edge of the Au subtract because the diffusion layer will be changed consequently otherwise. Figure 1E shows the electrochemical reduction of O2 at E1-E4 of Pt/MEAs recorded separately or simultaneously. At the Pt/MEAs, O2 reduction reaction occurs at an onset potential of ca. + 0.2 V, a typical value for catalysis of O2 reduction at Pt surface,17 suggesting the Pt/MEAs electrochemically active for O2 reduction. Obviously, CV responses of O2 recorded simultaneously at E1-E4 are almost the same with the respective CV response recorded separately, demonstrating that the diffusion layer of E1-E4 does not overlap, as calculated above. Although the differences exist in the electrochemical responses at the four recording electrodes, which are probably caused by the deviation of Au substrate and/or the electrochemical deposition processes, the Pt/MEAs could be employed for the simultaneous detection of O2 with calibration at each of the four recording electrodes.

8


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Figure 1. (A) Photo of the MEAs showing four recording electrodes labeled with E1, E2, E3 and E4. (B) Illustrated diffusion layers of two neighbor recording sites calculated with equation 5. (C) SEM image of the Pt nanoparticles deposited on the surface of the MEAs. (D) Typical cyclic voltammogram (CV) obtained at the Pt/MEAs in 0.5 M H2SO4 solution. Scan rate, 50 mV s-1. (E) Typical CVs obtained at E1-E4 in aCSF simultaneously (black solid curves) and separately (red curves). The black dashed curves represent CVs in aCSF saturated with N2. Scan rate, 50 mV s-1. Analytical Properties. We first studied the selectivity towards O2 reduction at the as-prepared Pt/MEAs. Compared to 30 μM O2, the presence of the electrochemically active species, including DA, NE, 5-HT, E, UA, DOPAC or AA, at their physiological levels in the cerebral system does not produce any significant current change when the potential was set at -0.5 V vs. Ag/AgCl (Figure 2A). This suggests these species do not interfere the O2 measurement at the selected conditions. Although H2O2 could be electrochemically reduced at Pt surface at the same potential as that for O2 reduction, the level of H2O2 in brain under normal conditions is much lower (< 1 μM) than that of O2 (30-80 μM) so that the interference of H2O2 to the detection of O2 in brain can be negligible in the subsequent studies.17 These observations validate the as-prepared Pt/MEAs for the selective measurement of O2 in brain.

9



A

B

O2

0

DA NE 5-HT E

I / nA

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UA DOPAC AA

-100

N2 air O2

20 nA -200

0

400

800

1200

1600

-0.6

-0.4

-0.2

0.0

0.2

E / V vs. Ag/AgCl

T/s

Figure 2. (A) Amperometric response of 10 μM DA, 10 μM NE, 10 μM 5-HT, 10 μM E, 50 μM UA, 20 μM DOPAC, 400 μM AA and 30 μM O2 in aCSF at the Pt/MEAs. The electrode was polarized at -0.5 V vs. Ag/AgCl (aCSF). (B) Typical CVs at the Pt/MEAs in aCSF (pH 7.4) saturated with N2 (black curve), ambient air (red curve), or O2 (blue curve). Scan rate, 50 mV s-1.

Next, we explored the linearity of O2 measurement at the Pt/MEAs. As shown in Figure 2B, the steady-state currents of the O2 reduction increase proportionally when the concentration of O2 in aCSF increases (0, 200, and 1250 μM O2 in aCSF saturated with N2, ambient air, and O2, respectively) at the as-prepared Pt/MEAs (γ = 0.9981). It suggests that the Pt/MEAs exhibits wide linearity for O2 in the range of 0 μM - 1250 μM, which covers the concentration of O2 (< 200 μM) in the CNS.27 To further validate the Pt/MEAs for monitoring of O2 in vivo, we performed the stability study of the Pt/MEAs in rat cortex. As shown in Figure 3A, no significant change appeared in amperometric response during continuously recording in rat cortex for 1.5 hrs., suggesting the good stability of the Pt/MEAs for measuring O2 in vivo. To minimize the deviation from electrode fouling by protein existing in cerebrospinal fluid, the Pt/MEAs were calibrated after animal experiments (i.e., postcalibration). Furthermore, the Pt/MEAs were employed to investigate the sensitivity towards O2 in rat cortex under mild hypoxia and hyperoxia. Figure 3B provides the representative dynamic current responses recorded at E1-E4 in rat cortex exposed to N2 for 50 s or O2 for100 s, a mimic of animal breathing in abnormal condition. Obviously, exposing to pure N2 (i.e., hypoxia) decreases the O2 level detected at E1-E4 immediately and simultaneously, while 10


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exposing to pure O2 gas (i.e., mild hyperoxia) increases the O2 level rapidly (n = 3). When the intervention terminates, the current quickly returns to the basal level, indicating the rapid metabolic regulation of the CNS and the fast response of the multiplexed electrodes towards O2 measurement. These unique electrochemical properties of the Pt/MEAs, especially its high temporal/spatial resolution, make the Pt/MEAs particularly attractive for in vivo monitoring of O2 at multiple locations of rat cortex simultaneously. A

1 nA 600 s

B

100% N2

E1 20 μM

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


E2

150 s

E3 E4 100% O2

Figure 3. (A) Amperometric response of O2 recorded at the Pt/MEAs implanted into rat cortex. (B) Representative amperometric responses of O2 at the four electrodes (E1-E4) of the Pt/MEAs in the cortex of anesthetized rat exposed to pure N2 for 50s or pure O2 for 100s. The electrodes were polarized at -0.5 V vs. Ag/AgCl (aCSF). Oxygen Fluctuation at Different Sites of Rat Cortex during SD Monitored Simultaneously. With the multiplexed electrodes of the Pt/MEAs, we explored O2 fluctuation at multiple sites of rat cortex during SD induced by electrical stimulation. A direct 600 µA electrical stimulation for 5 s arouses a transient change of O2 concentration at all the four recording sites in the cortex during SD. In most cases, O2 concentration at the four electrode sites exhibits a biphasic response with an initial decrease and a subsequent increase after electrical stimulation, as shown in Figure 4A. The propagation velocity of O2 fluctuation was in an agreement with the propagative rate of SD in brain tissue (i.e., 3-5 mm/min), as reported before.4 Moreover, the time delays of O2 response at the four electrodes from the cessation of electrical simulation were 58 ± 4 s, 62 ± 4 s,

11



A 40 μM

E1 E2

60 s

E3 E4

B

C

O2 basal level

* *

*

O2 fluctuation

*

*

100

CO2 change / %

120

CO2 / μM

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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80

40

50 0 -50

-100 0

E1

E2

E3

E4

E1

*

E2

*

E3

E4

Figure 4. (A) Typical amperometric responses of O2 at the four electrodes of MEAs in the cortex of single anesthetized rat during SD evoked by electrical stimulation (600 µA for 5 s, indicated with solid triangle). The electrodes were polarized at -0.5 V vs. Ag/AgCl (aCSF). (B) Comparison of the basal O2 level measured at E1-E4 in rat cortex. (C) O2 fluctuation measured at E1-E4 during SD. Data were presented as mean ± SD, n = 4. The asterisks (*) indicate significant differences, p < 0.05.

58 ± 2 s, 61 ± 7 s (n = 4), exhibiting no statistical difference between each other (Figure S1A). This is because the differences of the distances between the recording electrodes and the simulation site (max. 0.26 mm) could be negligible compared with that between MEAs and the simulation site (~3 mm), as illustrated in Figure S1B. Figure 4B compares the average basal levels of O2 measured at E1-E4 in rat cortex, which were 40 ± 8 µM, 90 ± 20 µM, 30 ± 10 µM, 90 ± 20 µM (n = 4), respectively. It is interesting that there are significant differences between each of electrodes with an exception of E1 and E3, and of E2 and E4. This result suggests that the basal level of O2 in rat cortex might exhibit more remarkable difference according to the depth than the horizontal orientation. Moreover, the O2 fluctuation at the four recording sites of cortex during SD also shows significant 12


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difference (Figure 4C). Compared with the basal level of O2 recorded at the corresponding electrode, the O2 levels at E1-E4 decreases by ca. 59% ± 16%, 26% ± 11%, 90% ± 14%, 89% ± 8% after electric simulation, and increases subsequently by ca. 92% ± 23%, 31% ± 8%, 31% ± 10%, 35% ± 11%, respectively (n = 4). Obviously, more O2 is consumed at E1, E3 and E4 sites compared with that at E2, and afterwards the O2 level at E1 increases more significantly than all the other sites. Although O2 fluctuation during SD has been studied in previous studies,6,9 the difference of O2 responses at different sites in the same functional region was not reported, unfortunately. O2 level usually represents the metabolism in the CNS and it can serve as a valuable biomarker of the metabolic state in specific brain region.10 Our results therefore indicate that O2 fluctuation varies at different sites of rat cortex, reflecting the different degree of depression and oxidative stress during SD even at different locations of the same brain region. It has been widely known that O2 concentration in the CNS is determined by the dynamic equilibrium between cereal blood flow (CBF) and cellular O2 consumption.44 To explain the reason for the O2 fluctuation during SD, we studied the changes of CBF and the neuron activity after electrical stimulation. As reported previously, the proportion of the intensity of low frequency signal (1-4 Hz) at local field potential (LFP) correlates inversely with the CBF.45-48 Figure 5 A displays the change of LFP in rat cortex after electrical stimulation. A significant deflection of LFP occurs at 50 s after the cessation of electrical stimulation, indicating a severe disruption of the ion distribution during SD. As shown in Figure 5B, the proportion of the

13



40 μV

C

4 mV

A

20 s

150

*

10 s

D

*

20 μV

B

50

1 ms

40

rfiring / s-1

100

P1-4 Hz / %

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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50

30 20 10

0

0

50

100

150

200

0

250

0

T/s

45

T/s

90

Figure 5. (A) The representative dynamics of the local field potential (LFP) during SD evoked by electrical stimulation (600 µA for 5 s, indicated with solid triangle). (B) The change of proportion of the intensity of low frequency signal (1-4 Hz) in LFP during SD. Data were presented as mean ± SD, n = 5. The asterisks (*) indicate significant difference, p < 0.05. Red curve was obtained with Gaussian Fitting. (C) The representative neuronal discharge during SD evoked by electrical stimulation (600 µA for 5 s, indicated with solid triangle). Inset, representative extracellular unit-spike waveform of a single neuron. (D) Histograms of firing rate measured during SD.

intensity of the low frequency signal (1-4 Hz) in LFP remains relatively constant before and around the electrical stimulation, however, it begins to decrease at ca. 50 s after the stimulation and reaches the minimum and then returns to the basal level at ca. 160 s after the stimulation. This indicates that the CBF goes through a significant increase after the electrical stimulation. The increase of CBF can deliver more energetic substrates, including O2 and glucose, to maintain the neuronal metabolism and restore the ion gradients during SD. We also investigated the neuronal activity during SD. As shown in Figure 5C, the neuron discharge starts to increase significantly at ca. 40 s after the electrical stimulation, and then lasts for ca. 2 s. This change during SD exhibits a significant difference compared with that before the stimulation (n = 5, Figure 5D). The abnormal 14


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excitation of neurons could transiently induce acute consumption of cellular O2 during SD, which might be another reason for inducing O2 fluctuation in the CNS. Moreover, the timing of the LFP change and the neuronal discharge were in an agreement with the O2 fluctuation after electrical stimulation-induced SD, suggesting again that O2 fluctuation during SD might be related to the change of CBF and the O2 consumption induced by neuron excitement. The difference of O2 fluctuation at the four recording sites in rat cortex may be caused by the different amount of neurons and blood vessels around the recording sites. Due to the difference of metabolic intensity in different sites, O2 deficiency could happen at the sites with massive neurons and rare blood capillaries, thus inducing oxidative stress or even cell death finally.8 CONCLUSION We have demonstrated implantable multiplexed microelectrodes arrays (MEAs) for simultaneous measurements of O2 at multiple locations of rat cortex during SD. Both in vitro and in vivo experiments show that the MEAs possess a high selectivity and stability for reliable measurements of O2 in rat cortex. We found that O2 fluctuation exhibited significant differences at different sites of rat cortex during SD evoked by the electrical stimulation. By monitoring the changes of local field potential and neuronal discharge, we speculate that the different level of O2 fluctuation at different recording sites of rat cortex during SD is related to the amount of neurons and blood vessels around the recording sites. Importantly, the development of this novel and reliable analytical method for simultaneous O2 measurements with high spatiotemporal resolution enables investigation and understanding of various physiological and pathological processes associated with O2 change at different sites of the same brain regain. In addition, our results also suggest that O2 fluctuation could be treated as a biomarker for evaluating the damage of SD on the brain.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications Website at DOI: xxxxxxxxxx.

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Comparison of time delay of O2 fluctuation at four recording sites E1-E4 during SD, and the effect of different pattern and intensity of electrical stimulation on O2 fluctuation. AUTHOR INFORMATION Corresponding Author *E-mail:

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

Author Contributions § These

authors contribute equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We acknowledge financial support from the National Natural Science Foundation of China (Grant Nos. 21790390, 21790391, 21435007, 21621062 for L.M., 21790053 and 21775151 for P.Y.), 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).

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In Vivo Monitoring of Oxygen Fluctuation ... - ACS Publications

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