Combined in Vivo Amperometric Oximetry and Electrophysiology in a

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Combined in vivo Amperometric Oximetry and Electrophysiology in a Single Sensor – a Tool for Epilepsy Research Ana Ledo, Cátia F. Lourenço, João Laranjinha, Greg A. Gerhardt, and Rui M. Barbosa Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03452 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 29, 2017

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Analytical Chemistry

Combined in vivo Amperometric Oximetry and Electrophysiology in a Single Sensor – a Tool for Epilepsy Research

Ana Ledo1,2,*, Cátia F. Lourenço1, João Laranjinha1,3, Greg A. Gerhardt4, Rui M. Barbosa1,3

1

Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga, 3004-504 Coimbra,

Portugal 2

BrainSense, Lda., Biocant Park, Cantanhede, Portugal

3

Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Coimbra 3000 Coimbra, Portugal

4

Center for Microelectrode Technology (CenMeT), Department of Neuroscience, University of

Kentucky Medical Center, Lexington, USA

*

Corresponding Author

[email protected] Center for Neuroscience and Cell Biology University of Coimbra Ed. Faculty of Medicine Rua Larga 3004-504 Coimbra, Portugal Phone: +351 918424289 Fax: +351 239 822776

1 ACS Paragon Plus Environment


Abstract 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

Seizures are paroxysmal events in which increased neuronal activity is accompanied by an increase in localized energetic demand. The ability to simultaneously record electrical and chemical events using a single sensor poses a promising approach to identify seizure onset zones in the brain. In the present work, we used ceramic-based platinum microelectrode arrays (MEA) to perform high-frequency amperometric recording of local pO2 and local field potential (LFP)-related currents during seizures in the hippocampus of chronically implanted freely-moving rats. Resting levels of O2 in the rodent brain varied between 6.6 ± 0.7 µM in the DG region of the hippocampus and 22.1 ± 4.9 µM in the cerebral cortex. We also observed an expected increase in hippocampal pO2 (15 ± 4 % from baseline) in response to tail pinch stress paradigm. Finally, induction of status epilepticus by intrahippocampal injection of pilocarpine induced biphasic changes in pO2 in the hippocampus. The initial dip at seizure onset (∆O2= -4.5 ± 0.7 µM) was followed by a prolonged hyperoxygenation phase (∆O2 = +10.4 ± 2.9 µM). By acquiring the amperometry signal with a high sampling rate of 100 Hz we decomposed the raw signal in an oximetry recording (1 Hz), demonstrating that each individual Pt site can simultaneously report changes in local pO2 and LFP-related currents during pilocarpine-induced seizure activity. This has high potential for translation into the clinical setting supported on intracranial grid or strip electrodes.


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Introduction 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

Temporal lobe epilepsy (TLE) is the most prevalent form of acquired epilepsy in adults, and is characterized as an electro-clinical syndrome in which seizures emanate from the limbic system, particularly the hippocampus, amygdala and entorhinal cortex1-2. The emergence of TLE is typically preceded by an epileptogenic event such as febrile seizure, brain trauma, or, on occasion, status epilepticus (SE), followed by a latent period during which unilateral hippocampal atrophy, typically caused by neuronal loss and gliosis, triggers spontaneous seizures 3. TLE is particularly disabling due to the unpredictable and recurrent nature of seizures as well as the high incidence of anti-epileptic drug resistance4, resulting many times in an indication for resection surgery5-6. Pre-surgical identification of the seizure onset zone should rely on a multimodal approach, using several methods based on different pathophysiological principles. The golden standard technique is long-term intracranial EEG7, an invasive approach through which the brain’s electrical activity is recorded. Non-invasive approaches include high resolution magnetic resonance imaging (MRI)8 to identify morphological abnormalities, and functional neuroimaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT)

9-10

as well as functional MRI (fMRI)

11

. These neuroimaging techniques

explore brain hemodynamics as a surrogate signal for brain activity, based on the phenomenon of neurovascular coupling, a mechanism through which changes in neuronal activity are met by a cerebrovascular response, allowing for adequate blood supply as a function of activity level 12-13. To date, no single sensor approach allows the simultaneous recording of electrical and hemodynamic response. Simultaneous EEG and fMRI (EEG-fMRI), combining the acquisition of epileptiform electrical brain activity and hemodynamic changes, has been employed as an experimental strategy to localize and track epileptic activity in the brain success

15-16

14

with demonstrated

. Poor spatial and temporal resolution of such neuroimaging techniques has driven

researchers and clinicians to explore alternative approaches to monitor pO2 and/or Hb-O2, 3 ACS Paragon Plus Environment


including optical recording of intrinsic signals (ORIS) 17-19, near infra-red spectroscopy (NIRS) 20-21, 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

and electrochemical oximetry using oxygen sensitive electrodes 22-25. These approaches have revealed that epileptiform activity produces a biphasic change in tissue pO2. In an initially phase and despite the rapid increase in focal cerebral blood flow (CBF), a decrease in pO2 is observed as a result of increased the cerebral metabolic rate for O2, which has been coined the epileptic dip 18-19, 21, 26-28 and has been shown to have predictive value, as it may precede the occurrence of an epileptic seizure

17

. The second phase is characterized by a

persistent increase in tissue pO2 resulting from a diffuse increase in CBF (hyperemia) and/or metabolic changes 23. Monitoring changes in tissue pO2 associated with both neurometabolic and neurovascular responses is critical to better understand the neuropathology of epilepsy and may be key in therapy development. In the present work, we have recorded changes in pO2 in the hippocampus of rats receiving a single intra-hippocampal injection of pilocarpine for SE induction 29-30. We have used multisite ceramic-based Pt microelectrode arrays (MEA) previously characterized in terms of their analytical performance towards oxygen reduction 31 to perform amperometric recordings of local pO2 at a high frequency sampling rate (100Hz). We then separated the electrochemical signal into the low (1Hz) frequency components through FFT filtering. This strategy revealed that a single sensor can simultaneously report chemical (pO2) and electrophysiological (local field potential related currents – LFPrc) information 32. In sum, each single sensor site can monitor rapid changes in electrical and neurovascular/neurometabolic activities.

Experimental Section Reagents and Solutions. All reagents were analytical grade and obtained from Sigma-Aldrich. Microelectrode in vitro evaluations were performed in PBS Lite 0.05 M pH 7.4 with the following composition (in mM): 10 Na2HPO4, 40 NaH2PO4, and 100 NaCl. Saturated O2 solutions for MEA 4 ACS Paragon Plus Environment

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calibration were prepared by bubbling PBS Lite with 95% O2 at 37ºC for 20 min, resulting in an O2 solution at 722 mm Hg (1.0 mM concentration33). Pilocarpine hydrochloride was obtained from Alfa Aesar. Pilocarpine solution was prepared fresh on the day of administration in sterile 0.9% NaCl at a concentration of 1.2 mg/µL. Microelectrode Arrays. We used ceramic-based multisite Pt microelectrode arrays (MEA) designed and developed at CenMet, University of Kentucky, Lexington, KY, USA. These MEAs are produced

by

photolithographic

techniques,

allowing

high

intra-

and

inter-electrode

reproducibility. The MEAs used were miniaturized S2 typology containing four 15x333 μm Pt sites side-by-side as shown in Fig. S1A. The MEA was mounted onto a pedestal for implantation (Fig. S1B) along with a Teflon coated Ag wire (200 μm o.d., Science Products GmbH, Hofheim, Germany) with an exposed tip, used to produce the pseudo-reference electrode, as described below. Electrochemical Instrumentation. Electrochemical recordings were performed using the FAST16mkIII potentiostat (Quanteon, KY, USA) in a 2-electrode electrochemical cell configuration, with the Pt MEA as a working electrode and an Ag/AgCl miniature pseudo-reference electrode. The later was produced by anodization of the exposed tip of the Teflon-coated Ag wire in 1 M HCl saturated with NaCl, which, when in contact with cerebrospinal fluid in the brain containing chloride ions, develops a Ag/AgCl half-cell. All in vitro and in vivo electrochemical recordings of pO2 were performed at a constant potential of -0.6 vs Ag/AgCl31. For dual potential recording, a CompactStat Bi-Potentiostat was used (Ivium Technologies, The Netherlands). Microelectrode Array Calibration. The MEAs were calibrated to assess their analytical performance in PBS Lite (20 mL) at 37 ºC with low speed (240 rpm) continuous stirring. Oxygen was removed by purging the buffer solution with argon for a minimum period of 30 min, after which the needle was removed from solution and kept above the surface to minimize O2 back-



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diffusion to the solution. Once a stable baseline was obtained, 5 µM aliquots of the O2 saturated solution were added in 5 repetitions (concentration range 0-25 µM). Animals. Six male wistar rats weighing 280-350 g (Charles-River Laboratories) were used in the experiments. Animals were housed in pairs in the local vivarium in a 12h light/dark cycle and with food and water available ad libitum. All animals implanted chronically for freely moving experiments were allowed soft food and palatable treats (cereals) in the 24h post-surgical period. These animals were housed individually following surgery. All procedures are in accordance with the European Community Council Directive for the Care and Use of Laboratory Animals (2010/63/EU) and received favorable approval from both the local ethics committee for animal experimentation (ORBEA, Ref. ORBEA_146_2016_31102016) and the Portuguese General Direction for Agriculture and Veterinary. In vivo recording of pO2 in the brain of freely moving rats. Following induction of anesthesia (5% isoflurane for 5 min) the surgical area was shaved and cleaned with iodine solution and 70% alcohol alternated 3 times. Animals were transferred onto the stereotaxic frame and anesthesia was thereon maintained at 2-3% isoflurane carried in medicinal oxygen (Conoxia, Linde). Animal temperature was maintained at 37 ºC with a heated pad coupled to a Gaymar Heating Pump (Braintree Scientific, Inc. USA). Artificial tears were applied to the rat’s eyes. Following a midline scalp incision, skin overlying the scull was reflected and bleeding was controlled with a Bovie® cautery. Following identification of bregma, 6 burr holes were drilled into the scull: one over the parietal cortex for the insertion of the MEA, one over the ipsilateral primary visual cortex for insertion of a guide cannula, one in the contralateral frontal bone for the insertion of the pseudoreference electrode, and 3 for the placement of anchoring screws (Stoelting Co., Ireland). Following the placement of the anchoring screws, the reference electrode was slid through its hole and placed between the scull surface and meninges. The MEA was then lowered into the granular cell layer of the dorsal hippocampus, using the target coordinates of AP: -4.1; ML: -2.2 for 6 ACS Paragon Plus Environment

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the MEA tip (all coordinates are relative to Bregma34). The guide cannula was inserted into the 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

polymorphic region of DG of the ventral hippocampus using the target coordinates of AP -6.4; ML: -4.5; DV: -5.0. The positioning of the MEA and dummy cannula are depicted in Fig. S1C. Dental acrylic (TAB2000, Kerr) was applied to fix the head-cap assembly to the ancor screws. Following surgery, the animal received 5 mg/kg carprofen and 1 mL/kg saline (sub-cutaneous) and was transferred to its box over a heating pad and allowed to recover for 24h before returning to local vivarium. Carprofen and saline administrations were repeated in the 3 days post-surgery when necessary. Amperometric recordings were initiated on 3rd day post-surgery. Animals were placed in a 50x50x50 cm behavior box. Recording of pO2 required connection of the head-cap to a 4channel pre-amplifier (20 mV/pA; mHat, Quanteon, KY, USA) connected to an 18-lead commutator and subsequently to the FAST16mkIII control box. Unless otherwise stated, the acquisition rate for amperometric recording in awake behaving rats was set at 100 Hz. Stress paradigm consisting of a 5-min tail pinch was used to evoke changes in pO2. A cloth peg was placed at the base of the animal’s tail as to produce increased locomotor activity, but not physical pain. For pilocarpineinduced SE, the dummy cannula was replaced by a microinjection cannula connected to a 10 µL Hamilton syringe mounted on a syringe pump (Pump 11 Elite Series, Harvard Apparatus, UK). Pilocarpine (1.2 mg/µL, total volume administered of 1 µL) was administered at a flow rate of 0.5 µL/min and microinjector was left in place for 1 min following administration. The rat was held gently throughout the administration period and then placed back into the behavior box. At 90 minutes following onset of SE, seizure was controlled by administration of diazepam, 4 mg/kg, administered through intraperitoneal injection. Data Analysis. Analyses was performed using FAST Analysis version 6.0, OriginPro 2016 and GraphPad 5.0 software. The numerical values presented are given in mean ± SD unless otherwise stated. The number of repetitions is indicated in each determination. Calculated parameters were statistically evaluated using one-way ANOVA followed by Tukey’s Post-hoc test. The sensitivity of 7 ACS Paragon Plus Environment


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the MEAs towards O2 was determined by linear regression for the concentration range of 0-25 µM. Conversion between pO2 (mmHg) and [O2] was performed in accordance to Henry’s Law, considering the KH value of 1.04 x 10-3 M atm-1 for 37ºC 33. The change in pO2 resulting from the tail pinch stress paradigm was determined as the % change from baseline value prior to tail pinch. Data are presented as mean ± SEM in 30 s bins obtained by averaging the signal in each successive 30 s period. Data shown for the change in pO2 during pilocarpine-induced SE was processed as follows: the raw signal (current) acquired at an averaged sampling rate of 100Hz was filtered using a low pass FFT with a cutoff of 1 Hz. This was then converted into pO2 considering the slope of the calibration curve previously obtained. Finally, the signal was smoothed by a 1000-point adjacentaveraging method. The frequency power spectra were obtained by applying a Short Time Fourier Transform to the 1-49 Hz FFT band-pass filtered raw signal (current), using a FFT length of 256, overlap of 128 and Rectangle window type. Data shown representing the high-frequency component corresponds to the FFT band-pass (1-49 Hz) of the raw signal.

Results and Discussion Resting levels of pO2 in different brain regions. We determined the mean tonic or basal pO2 in two sub-regions of the hippocampus as well as in the striatum and the cortex of freely moving rats. The resting levels of pO2 were calculated from the amperometric current at the end of a continuous 3h-recording period between days 3 and 7 post MEA implant. As can be observed in Fig. 1, the hippocampal DG showed lowest (p1Hz (inset in Fig. S2, panel A), the HFC showed the same profile at all channels (Fig. S2, panel C). To date, reports of simultaneous recordings of in vivo electrochemistry and electrophysiology are limited and typically required the use of two independent recording systems. One option has 12 ACS Paragon Plus Environment

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been to use a serial/sequential approach, alternating between the two recording modalities using 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

a single microelectrode

49-52

. An alternative has been to employ a parallel approach, using either

two microelectrodes placed in close proximity to each other arrays with different sites operating in each modality

53-54

24, 55-59

or combination microelectrode

. In a seminal paper, Zhang et al.

demonstrated that an electrochemical recording carries LFPrc information, establishing the basis for single-electrode simultaneous recording of neurochemical and neuronal activity

32, 60

.

Furthermore, the same authors demonstrated that the frequency fluctuations in the 1–20 Hz band were qualitatively and quantitatively similar to simultaneously LFP signals recorded in anesthetized rats and exhibited similar spectral features as LFPs regarding distinct brain states. Of particular relevance to the present work is that fact that these authors used the same Pt-MEAs and recording system (FAST16mkII, by Quanteon, LLC.) used here, although we have a bare surface MEA operating in reduction mode and an average sampling rate of 100 Hz, available in the latest version of the electrochemical system (FAST16mkIII). This experimental approach was later used with Pt/Ir wire-base choline biosensor 61 as well as using a dual glucose/lactate biosensor in a model of cortical spreading depolarization 62. Of mention, these previous works were performed in anesthetized rats, while we here show that this approach can be applied as well in awake and freely moving rats chronically implanted with MEAs. Furthermore, the methodology is robust enough to successfully recording during the highly-agitated state of pilocarpine-induced SE.

Conclusions To understand complex neurological disorders such as epilepsy, both basic researchers and clinicians require multimodal approaches which bring together information from different physiological phenomena. This may be even truer in the quest to design novel clinical tools that might allow optimal identification of seizure onset zones in patients suffering from refractory epilepsy indicated for resection surgery and/or monitor surgery outcome and afford seizure 13 ACS Paragon Plus Environment


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prediction. To date, no single sensor approach has achieved this goal. We present here the use of 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

multi-site Pt ceramic-based MEAs for the electrochemical oximetry coupled using high-frequency sampling as a suitable tool to follow changes in pO2 and neuronal activity simultaneously, from each individual site. Supported by the previous demonstration that the electrochemical recording carries LFP information, we validate in vivo the applicability and robustness of this approach in a model of SE by recording in awake freely-moving rats during pilocarpine-induced status epilepticus. This approach may be explored using enzyme-based biosensors to monitor neurotransmitters and neuromodulators such as glutamate, choline or metabolites such as glucose and lactate. Combining the study of changes in neurotransmission, the function of neuromodulatory systems or neurometabolic coupling with that of neuronal activity may contribute significantly to expand our understanding of the underlying mechanisms of pathological brain states such as epilepsy. Conflict of interest G.A.G. is the sole proprietor of Quanteon, LLC, which makes the Fast16 recording system used for control of the MEA technology. AL as received salary support from Quanteon, LLC. The remaining authors have no conflicts of interest. Acknowledgements This work was financed by the European Regional Development Fund (ERDF), through the Centro

2020

Regional

Operational

Program:

project

CENTRO-01-0145-FEDER-000012-

HealthyAging2020, the COMPETE 2020 - Operational Program for Competitiveness and Internationalization, and the Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia,

I.P.:

project

POCI-01-0145-FEDER-007440.

C.F.L.

SFRH/BPD/82436/2011 from FCT. Supporting Information


acknowledges

fellowship

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Experimental design used for recording pO2 in vivo in awake rats and pilocarpine inducted status epilepticus (Figure S1); plot showing level of redundancy of the 4-site recording in an S2 MEA (Figure S2); and dual potential in vivo recording in the DG of an anesthetized rat during pilocarpine-induced SE (Figure S3).



References 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

1. Bartolomei, F.; Khalil, M.; Wendling, F.; Sontheimer, A.; Regis, J.; Ranjeva, J. P.; Guye, M.; Chauvel, P. Epilepsia 2005, 46, 677-87. 2. McIntyre, D. C.; Gilby, K. L. Epilepsia 2008, 49 Suppl 3, 23-30. 3. French, J. A.; Williamson, P. D.; Thadani, V. M.; Darcey, T. M.; Mattson, R. H.; Spencer, S. S.; Spencer, D. D. Ann. Neurol. 1993, 34, 774-80. 4. Engel, J., Jr. Epilepsy Res. 1996, 26, 141-50. 5. Engel, J.; Wiebe, S.; French, J.; Sperling, M.; Williamson, P.; Spencer, D.; Gumnit, R.; Zahn, C.; Westbrook, E.; Enos, B., Neurology 2003, 60, 538-547. 6. Berg, A. T. Curr. Opin. Neurol. 2008, 21, 173-8. 7. Reif, P. S.; Strzelczyk, A.; Rosenow, F. Seizure 2016, 41, 191-5. 8. Stylianou, P.; Hoffmann, C.; Blat, I.; Harnof, S. J. Clin. Neurosci. 2016, 23, 14-22. 9. la Fougere, C.; Rominger, A.; Forster, S.; Geisler, J. Epilepsy Behav. 2009, 15, 50-5. 10. Peter, J.; Houshmand, S.; Werner, T. J.; Rubello, D.; Alavi, A. Nucl. Med. Commun. 2016, 37, 223-30. 11. Stylianou, P.; Kimchi, G.; Hoffmann, C.; Blat, I.; Harnof, S. J. Clin. Neurosci. 2016, 23, 23-33. 12. Huneau, C.; Benali, H.; Chabriat, H. Front. Neurosci. 2015, 9, 467. 13. Lauritzen, M.; Gold, L. J. Neurosci. 2003, 23, 3972-80. 14. van Graan, L. A.; Lemieux, L.; Chaudhary, U. J. Quant. Imaging Med. Surg. 2015, 5, 300-12. 15. van Houdt, P. J.; de Munck, J. C.; Leijten, F. S.; Huiskamp, G. J.; Colon, A. J.; Boon, P. A.; Ossenblok, P.P. NeuroImage 2013, 75, 238-48. 16. Pittau, F.; Dubeau, F.; Gotman, J. Neurology 2012, 78, 1479-87. 17. Zhao, M.; Suh, M.; Ma, H.; Perry, C.; Geneslaw, A.; Schwartz, T. H. Epilepsia 2007, 48, 2059-67. 18. Bahar, S.; Suh, M.; Zhao, M.; Schwartz, T. H. NeuroReport 2006, 17, 499-503. 19. Suh, M.; Bahar, S.; Mehta, A. D.; Schwartz, T. H. NeuroImage 2006, 31, 66-75. 20. Watanabe, E.; Nagahori, Y.; Mayanagi, Y. Epilepsia 2002, 43 Suppl 9, 50-5. 21. Moseley, B. D.; Britton, J. W.; Nelson, C.; Lee, R. W.; So, E. Epilepsia 2012, 53, e208-11. 22. Springett, R.; Swartz, H. M. Antioxid. Redox Signaling 2007, 9, 1295-301. 23. Zhao, M.; Ma, H.; Suh, M.; Schwartz, T. H. Neurosci. 2009, 29, 2814-23. 24. Thompson, J. K.; Peterson, M. R.; Freeman, R. D. Science 2003, 299, 1070-2. 16 ACS Paragon Plus Environment

Page 16 of 23

Page 17 of 23


25. Ivanov, A. I.; Bernard, C.; Turner, D. A. Neurobiol. Dis. 2015, 75:1-14. 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

26. Chakrabarti, D.; Byrappa, V.; Kamath, S. J. Neurosurg. Anesthesiol. 2015, 27, 269-70. 27. Ko, S. B.; Ortega-Gutierrez, S.; Choi, H. A.; Claassen, J.; Presciutti, M.; Schmidt, J. M.; Badjatia, N.; Lee, K.; Mayer, S. A. Arch. Neurol. 2011, 68, 1323-6. 28. Shariff, S.; Suh, M.; Zhao, M.; Ma, H.; Schwartz, T. H. Epilepsy Behav. 2006, 8, 363-75. 29. Furtado, M. A.; Castro, O. W.; Del Vecchio, F.; de Oliveira, J. A.; Garcia-Cairasco, N. Epilepsy Behav. 2011, 20, 257-66. 30. Castro, O. W.; Furtado, M. A.; Tilelli, C. Q.; Fernandes, A.; Pajolla, G. P.; Garcia-Cairasco, N. Brain Res. 2011, 1374, 43-55. 31. Ledo, A.; Lourenço, C. F.; Laranjinha, J.; Brett, C. M.; Gerhardt, G. A.; Barbosa, R. M. Anal. Chem. 2017, 89, 1674-1683. 32. Zhang, H.; Lin, S.-C.; Nicolelis, M. A. L. J. Neurosci. Methods 2009, 179, 191-200. 33. Sander, R. Atmos. Chem. Phys. 2015, 15, 4399-4981. 34. Paxinos, G.; Watson, C., The Rat Brain. Elsevier Inc.: Burlington, USA, 2007; Vol. 6th, p 456. 35. Dunn, J. F.; Khan, M. N.; Hou, H. G.; Merlis, J.; Abajian, M. A.; Demidenko, E.; Grinberg, O. Y.; Swartz, H. M. High. Alt. Med. Biol. 2011, 12, 71-7. 36. Dunn, J. F.; Grinberg, O.; Roche, M.; Nwaigwe, C. I.; Hou, H. G.; Swartz, H. M. J. Cereb. Blood Flow Metab. 2000, 20, 1632-5. 37. Gegentonglaga; Yoshizato, H.; Higuchi, Y.; Toyota, Y.; Hanai, Y.; Ando, Y.; Yoshimura, A. Life Sci. 2013, 93, 773-7. 38. Ortiz-Prado, E.; Natah, S.; Srinivasan, S.; Dunn, J. F. J. Neurosci. Methods 2010, 193, 217-25. 39. Kealy, J.; Bennett, R.; Lowry, J. P. J. Neurosci. Methods 2013, 215, 110-20. 40. Fellows, L. K.; Boutelle, M. G. Brain Res. 1993, 604, 225-31. 41. Lourenço, C. F.; Ledo, A.; Barbosa, R. M.; Laranjinha, J. Free Radic. Biol. Med. 2017, 108, 668682. 42. Ledo, A.; Barbosa, R.; Cadenas, E.; Laranjinha, J. Free Radic. Biol. Med. 2010, 48, 1044-1050. 43. Ledo, A.; Barbosa, R. M.; Laranjinha, J. Methods Mol. Biol. 2012, 810, 73-88. 44. Walton, L. R.; Boustead, N. G.; Carroll, S.; Wightman, R. M. ACS Chem. Neurosci. 2017, 8, 15981608. 45. Pinel, J. P.; Rovner, L. I. Exp. Neurol. 1978, 58, 190-202. 46. Racine, R. J. Electroencephalogr. Clin. Neurophysiol. 1972, 32, 281-94. 17 ACS Paragon Plus Environment


47. Suh, M.; Ma, H.; Zhao, M.; Sharif, S.; Schwartz, T. H. Mol. Neurobiol. 2006, 33, 181-97. 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

48. Desai, A.; Bekelis, K.; Thadani, V. M.; Roberts, D. W.; Jobst, B. C.; Duhaime, A. C.; Gilbert, K.; Darcey, T. M.; Studholme, C.; Siegel, A. Epilepsia 2013, 54, 341-50. 49. Cheer, J. F.; Aragona, B. J.; Heien, M. L.; Seipel, A. T.; Carelli, R. M.; Wightman, R. M. Neuron 2007, 54, 237-44. 50. Crespi, F.; England, T.; Ratti, E.; Trist, D. G. Neurosci. Lett. 1995, 188, 33-6. 51. Cheer, J. F.; Heien, M. L.; Garris, P. A.; Carelli, R. M.; Wightman, R. M. Proc. Natl. Acad. Sci. USA 2005, 102, 19150-5. 52. Hobbs, C. N.; Johnson, J. A.; Verber, M. D.; Mark Wightman, R. Analyst 2017, 142, 2912-2920. 53. Ewing, A. G.; Alloway, K. D.; Curtis, S. D.; Dayton, M. A.; Wightman, R. M.; Rebec, G. V. Brain Res. 1983, 261, 101-8. 54. Hefti, F.; Felix, D. J. Neurosci. Methods 1983, 7, 151-6. 55. Johnson, M. D.; Franklin, R. K.; Gibson, M. D.; Brown, R. B.; Kipke, D. R. J. Neurosci. Methods 2008, 174, 62-70. 56. Fan, X.; Song, Y.; Ma, Y.; Zhang, S.; Xiao, G.; Yang, L.; Xu, H.; Zhang, D.; Cai, X. Sensors (Basel) 2016, 17 ,61. 57. Viswanathan, A.; Freeman, R. D. Nat. Neurosci. 2007, 10, 1308-12. 58. Wei, W.; Song, Y.; Fan, X.; Zhang, S.; Wang, L.; Xu, S.; Wang, M.; Cai, X. Nanotechnology 2016, 27, 114001. 59. Zhang, S.; Song, Y.; Wang, M.; Zhang, Z.; Fan, X.; Song, X.; Zhuang, P.; Yue, F.; Chan, P.; Cai, X. Biosen., Bioelectron. 2016, 85, 53-61. 60. Zhang, H.; Lin, S. C.; Nicolelis, M. A. J. Neurosci. 2010, 30, 13431-40. 61. Santos, R. M.; Laranjinha, J.; Barbosa, R. M.; Sirota, A. Biosens. Bioelectron. 2015, 69, 83-94. 62. Lourenço, C. F.; Ledo, A.; Laranjinha, J.; Gerhardt, G. A.; Barbosa, R. Sci.Rep. 2017, 7. 63. Zhang, C.; Belanger, S.; Pouliot, P.; Lesage, F. PLoS One 2015, 10, e0135536. 64. Sakadzic, S.; Roussakis, E.; Yaseen, M. A.; Mandeville, E. T.; Srinivasan, V. J.; Arai, K.; Ruvinskaya, S.; Devor, A.; Lo, E. H.; Vinogradov, S. A.; Boas, D. A. Nat. Methods 2010, 7, 755-9. 65. Cater, D. B.; Garattini, S.; Marina, F.; Silver, I. A. Proc. R. Soc. London, Ser. B 1961, 155, 136. 66. Vovenko, E. Pflugers Arch. 1999, 437, 617-23. 67. Piilgaard, H.; Lauritzen, M. J. Cereb. Blood Flow Metab. 2009, 29, 1517-27. 68. Offenhauser, N.; Thomsen, K.; Caesar, K.; Lauritzen, M. J. Physiol. 2005, 565, 279-94.


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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


69. Liu, K. J.; Bacic, G.; Hoopes, P. J.; Jiang, J.; Du, H.; Ou, L. C.; Dunn, J. F.; Swartz, H. M. Brain Res. 1995, 685, 91-8. 70. Liu, S.; Shi, H.; Liu, W.; Furuichi, T.; Timmins, G. S.; Liu, K. J. J. Cereb. Blood Flow Metab. 2004, 24, 343-9. 71. Marina, N.; Ang, R.; Machhada, A.; Kasymov, V.; Karagiannis, A.; Hosford, P. S.; Mosienko, V.; Teschemacher, A. G.; Vihko, P.; Paton, J. F.; Kasparov, S.; Gourine, A. V. Hypertension 2015, 65, 775-83. 72. Nwaigwe, C. I.; Roche, M. A.; Grinberg, O.; Dunn, J. F. Brain Res. 2000, 868, 150-6. 73. Geneslaw, A. S.; Zhao, M.; Ma, H.; Schwartz, T. H. J. Cereb. Blood Flow Metab. 2011, 31, 1394402. 74. Freund, T. F.; Buzsaki, G.; Prohaska, O. J.; Leon, A.; Somogyi, P. Neuroscience 1989, 28, 539-49.



Figure Captions and Tables 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

Figure 1 – Average resting pO2 values in different regions of the rat brain. For the sake of comparison with values reported in the literature, both molar and partial pressure scales are shown. Values are represented as scatter and mean ± 95% CI (Number of animals: DG = 4; CA1 = 2; Striatum = 2; Cortex = 3).

Figure 2 – Monitoring phasic changes in pO2 in the hippocampus upon tail pinch stress paradigm in awakebehaving rats. Average of 12 recording sites from 3 animals. Data is presented as % change from baseline ± SEM for 30 s bins. Gray box represents period of tail pinch stimulus.


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Figure 3 – Representative recording of pO2 and LFPrc during SE induced in an awake freely-moving rat by a single intrahippocampal administration of Pilocarpine (H-Pilocarpine; grey box). (A) Representative recording of pO2 in the DG region of the dorsal hippocampus highlighting the epileptic dip in the initial phase (inset). Solid line shows signal following processing (FFT filtering and averaging) of the raw signal, shown in the shaded region (B) High-frequency component (1-49 Hz FFT band pass) of the amperometric recording – local field potential related currents (LFPrc). Highlighted above and below are discrete 5 s epochs from different moments of the recording. (C) Power spectrum analysis of the high-frequency component and mean (± SD) power density determined for 100 s periods at moment indicated by arrows and averaged from the 4 Pt recording sites (D) Behavioral alterations registered in a modified Racine Scale for seizure activity. DZP – Administration of diazepam (4 mg/kg, i.p.) for seizure control.



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Table 1 – Reported values of resting pO2 in the rodent brain

Animal Model

Methodology

Anesthetized Mouse

Phosphorescent quenching Two-photon–enhanced phosphorescent quenching Pt microelectrode Pt needle microelectrode

Anesthetized Rat

Pt microelectrode Clark-type microelectrode EPR Oximetry

Awake Rat** Freely Moving Rat

pO2 range (mm Hg)

Brain Structure

Optical Oxygen Sensor Fluorescent Oxygen Sensor EPR Oximetry (LiPc) Carbon Paste Microelectrode Clark-type microelectrode Fiber optic probe (fluorescent quenching)

Somatosensory cortex (38 sites close to an artery) Cortex (100 µm from surface; 29 sites) Cortex Cortex (surface of arterial/venous end of capillary) Hippocampus Neocortex Cerebellar Cortex Hippocampus Basal Ganglia Brain Stem Thalamus Cortex Frontal Cortex Hippocampus Hippocampus Cortex

Ref

6-25

63

6-25

64

19–40 57.9 ± 11.1 to 40.9 ± 11.5 20.3 18.0±1.9 12.7 - 64.4 34.1 ± 5.6 33.4 ± 6.0 26±4 26.0 ± 4.2 27.1 ± 7.5 26.7 ± 2.2 100.26 ± 5.76 µM (73 ± 4 mmHg *)

65

15.0 ± 4.3- 16.4 ± 4.2

37

66 65 67 68 69 70 71 72 36 35 39

30.2 ± 3.3 (mean ± S.E.)

38

*Calculated from published data, considering the KH = 1.04 x 10-3 M atm-1 (37ºC); ** restrained

Table 2 - Changes in pO2 during pilocarpine induced status epilepticus Animal model Whole hippocampus immature rats (P7)

Model

Electrical stimulation

Clark-type microelectrode (Unisense) Clark-type microelectrode (Unisense) Clark-type microelectrode Clark-type microelectrode (Unisense) Clark-type microelectrode (Unisense) Ottosensors electrode probe (Au-Ag/AgAgCl)

Pilocarpine

Pt-MEA

Low Mg2+

Bic 4-AP Bic

Anesthetized Rat

Freely Moving Rat

Methodology

4-AP

Region

Epileptic Dip

Hyperoxygenation Phase

Hippocampus (CA1)

114 ± 6 mmHg

-

25

Hippocampus (CA1)

76 ± 6 mmHg

-

25

Neocortex

64.3 ± 23.5% from baseline

-

18

Neocortex

5.51 ± 2.5 %

6.98 ± 2.58 %

73

Neocortex

22.8 ± 2.1%

32.5 ± 6.8 %

17

Dorsal hippocampus Hippocampus (DG)

Observed, not quantified 4.5 ± 0.7 µM (3.3 0.5 mmHg)


Ref

74

10.4 ± 2.9 µM (7.6 ± 2.1 mmHg)

Present study

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For TOC Only 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


Combined in Vivo Amperometric Oximetry and Electrophysiology in a

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