Photoacoustics can image spreading depolarization deep in gyrencephalic brain
Thomas Kirchner, Janek Gröhl, Mildred Herrera, Tim Adler, Adrián Hernández-Aguilera, Edgar Santos, Lena Maier-Hein
PPhotoacoustics can image spreading depolarizationdeep in gyrencephalic brain
Thomas Kirchner , Janek Gr¨ohl , Mildred A. Herrera , Tim Adler , Adri´an Hern´andez-Aguilera ,Edgar Santos , Lena Maier-Hein ‡ ‡ [email protected] Abstract
Spreading depolarization (SD) is a self-propagating wave of near-complete neuronal depolarization that isabundant in a wide range of neurological conditions, including stroke. SD was only recently documented inhumans and is now considered a therapeutic target for brain injury, but the mechanisms related to SD incomplex brains are not well understood. While there are numerous approaches to interventional imaging ofSD on the exposed brain surface, measuring SD deep in brain is so far only possible with low spatiotemporalresolution and poor contrast. Here, we show that photoacoustic imaging enables the study of SD and itshemodynamics deep in the gyrencephalic brain with high spatiotemporal resolution. As rapid neuronaldepolarization causes tissue hypoxia, we achieve this by continuously estimating blood oxygenation withan intraoperative hybrid photoacoustic and ultrasonic (PAUS) imaging system. Due to its high resolution,promising imaging depth and high contrast, this novel approach to SD imaging can yield new insights into SDand thereby lead to advances in stroke, and brain injury research.
Main
Spreading depolarization (SD) is a self-propagating wave of near-complete neuronal depolarization that occursabundantly [1] in individuals with progressive neuronal injury after stroke [2] and traumatic brain injury [3] aswell as subarachnoid hemorrhage [4], intracerebral hemorrhage [5], and migraine with aura [6, 7]. Sixty yearsafter the discovery of SD [8], many mechanisms related to SD have still not fully been understood while recentresearch increasingly finds SDs to be a therapeutic target in injured brain [9, 10].In order to increase understanding of SD, the morphologies of their wave fronts have been a subject of intensestudy [11–13]. In these, the gyrencephalic brain has been found to be capable of irregular SD propagation1/16 a r X i v : . [ phy s i c s . m e d - ph ] J a n atterns [13, 14] not found in lissencephalic brain. It remains to be studied if and how these patterns occurand evolve in depth. A better classification of the morphologies of these wave fronts could lead to a cleardefinition of a therapeutic target beyond mere occurrence of SD. The methods used to study SD can beclassified in electrophysiological and optical approaches. The current clinical state of the art for monitoringSD is electrocorticography (ECoG) using subdural electrodes placed directly on the cortex [15, 16] to recordelectrical activity. Because SDs propagate far from their point of origin, placing ECoG electrodes allowsfor remote monitoring of various brain injury. Characteristic patterns usually appear delayed for adjacentelectrodes, with an SD registering as a large near direct current (DC) shift in the electrodes signal, followedby persistent depression of spontaneous cortical activity registering as higher frequency alternating current(AC) signal components [15]. While ECoG is clinical practice for surface measurements, implanting electrodesdeep into the brain is the prime method of investigating SD beyond the brain surface. Doing so, SDs havebeen shown to occur in deep structures of the lissencephalic brain and in the brainstem, where they have beenassociated with sudden unexpected death in epilepsy [17]. How an SD, which originates on the cortex spreadsto deep structures without direct gray matter connection is unclear as the use of electrical monitoring does notyield sufficient spatial information.While optical techniques are not in routine clinical use, a range of them are used to study SD. Theycan yield high spatiotemporal resolution information usually related to the hemodynamic response to SD.These techniques include two photon microscopy (TPM) [12, 18–20], laser speckle (LS) imaging [11, 21, 22],intrinsic optical signal (IOS) imaging [13, 23, 24] and near infrared spectroscopy (NIRS) [25–27]. TPM has anexceptional, single cell spatial resolution using the fluorescence of reduced nicotinamide adenine dinucleotide(NADH) as contrast. It can achieve a temporal resolution in a seconds range for a sub-millimeter imagingfield [12] and has a sub-millimeter penetration depth. TPM is therefore mostly used in small animal models.LS imaging or LS flowmetry images changes in cerebral blood flow in single vessels [22]. It is complementaryto the larger field of view IOS imaging [21] which images reflectance changes of light in one [24] or two [23]narrow bands. IOS has approximately one second temporal resolution and micron spatial resolution, whileagain being diffusion limited to a sub-millimeter penetration depth and no depth resolution. NIRS, in contrastto the other optical techniques, is no imaging technique but employs point measurement probes [25] or optrodestrips [26] to monitor millimeter scale areas similar to electrodes. Like IOS, it indirectly measures reflectancescorrelated to relative concentration changes of the chromophores oxyhemoglobin (HbO) and deoxyhemoglobin(Hb), but its signal response is integrated over the area under the probe, leading to single spot measurementsand no spatial resolution.Functional magnetic resonance imaging (fMRI) with blood oxygen level dependent (BOLD) or diffusionweighted contrasts is the only modality that has been used to image the hemodynamic response of SDdeep in brain [7]. Substantial drawbacks besides the complex imaging setup are the poor spatiotemporalresolution [28, 29] and low contrast [7] when compared to optical or electrical measurements.Overall, it can be concluded that the imaging methods proposed to date either feature high spatiotemporalresolution (IOS, TPM, LS) or are capable to provide depth-resolved information on SD beyond the surface(fMRI, implanted electrodes), but cannot provide both. To address this bottleneck, we investigate photoacoustic(PA) imaging as a possible high-resolution imaging technique for measuring SD deep in the gyrencephalic2/16rain. Near infrared (NIR) light can penetrate deep into tissue, is scattered and gets diffused, thereby losingspatial information after a fraction of a millimeter. Photoacoustics [30] is capable of imaging beyond thissub-millimeter optical diffusion limit through the PA effect [31]; light is delivered as a nanosecond laser pulseand where it is absorbed, it causes sudden thermoelastic expansion which in turn gives rise to acoustic waves.The ultrasonic spectral component of these waves emitted by the PA effect scatter much less than NIR lightin tissue and can be detected by ultrasound (US) probes. Reconstructing their origin yields PA images. Amultispectral stack of such images can be processed to reconstruct images of estimated tissue oxygenationthat feature the spatiotemporal resolution and imaging depth of US combined with the optical contrast ofNIRS. Multispectral photoacoustic imaging has shown to image blood oxygenation and perfusion in a varietyof applications [32–36]. In the context of brain imaging, however, the application of PA has been restricted tolissencephalic brains [29] and its potential for monitoring SD remains to be investigated.Rapid neuronal depolarization and repolarization causes tissue hypoxia [12]. Therefore, our work is basedon the assumption that the imaging of hemodynamic changes with photoacoustics enables the monitoringof SD deep inside the tissue. We hypothesize that multispectral PA imaging is able to image SD inducedhemodynamic changes in the entirety of the cortical gray matter of a gyrencephalic brain. For the purposes ofthis study, we measure an estimation of blood oxygenation (sO ) and total hemoglobin (THb). Our imagingconcept, which is illustrated in Fig. 1, relies on a hybrid photoacoustic ultrasonic (PAUS) imaging systemwhich combines (1) an US research system featuring a linear US transducer with a center frequency of 7.5 MHzand broad acoustic response [37] with (2) a near infrared (NIR) fast tuning optical parametric oscillator (OPO)laser [38] (see Methods). The system operates in an interleaved PAUS imaging mode, acquiring multispectralPA sequences with corresponding US images for each PA image. This concurrent US imaging is used asanatomical reference for the physician (e.g. as guidance for the stimulation) and for motion compensation ofthe PA data (see Methods). Each multispectral PA image stack is converted into an image of estimated sO and by spectral unmixing. Laser systemUltrasound DAQKCl stimulation Stereotactic frameECoG electrodes
Figure 1.
Setup for characterizing spreading depolarization (SD)deep inside the gyrencephalic brain with a hybrid photoacousticultrasonic (PAUS) imaging system. The PAUS probe is placedon a gel pad to allow for PAUS-guided potassium chloride (KCl)stimulation in the imaging plane. Electrocorticography (ECoG)recordings serve as a clinical reference. xperiments & Results
Two experiments were performed with our PAUS system to investigate whether the monitoring of tissueoxygenation with PA enables the detection and monitoring of SDs in the entire depth of the cortical graymatter of a gyrencephalic brain. In both experiments, brain activity was monitored with ECoG using astandard subdural electrode strip (Fig. 1).The aim of the initial wave experiment was to investigate if the hemodynamic response of the brain toan induced SD can be imaged with multispectral PA. We performed the experiment in an uninjured brain.To analyze tissue hemodynamics before, during and after the occurence of SD, we took continuous PAUSmeasurements starting 24 min before the the first potassium chloride (KCl) stimulation (see Fig. 1) and endingone hour after the stimulation. After the experiment we cut sagittal surgical slices from the extracted brain torelate the acquired PA and US images to the brain morphology as seen on the exposed tissue. As shown inFig. 2 we were able to image PA signal up to a depth of approximately 1 cm, which allowed us to image theentire cortical gray matter in the field of view of the imaging plane.
Figure 2.
Hybrid photoacoustic ultrasonic (PAUS) imaging of aporcine brain. The dashed white line and boxes show correspondingsections of the swine cortex. (a)
Photograph of a sagittal surgicalslice segmented from the extracted brain, 1 cm from the midline.The shown segment is manually registered to (b) a representativephotoacoustic (PA) image with two regions of interest (ROI), and (c) the corresponding ultrasound (US) B-Mode image. (d)
Photo-graph of the exposed cortex after the craniotomy and dura materretraction with the dashed line marking the PAUS imaging plane.The electrocorticography (ECoG) electrodes are positioned in thelateral margins.
By estimating sO in each pixel of our reconstructed multispectral images we observed a single wave ofhypoxia spreading from the point of KCl stimulation through the tissue at a speed of approximately 5 mm/min.The estimated sO for two sub-surface regions of interest (ROI) is plotted in Fig. 3 a to illustrate this wave.sO in a wide field of view during the same time frame is shown in Supplemental Video 1 played at a factor4/16
40 min a sO [%] in ROI b DC/AC ECoG [mV] c d gfe3 mm
ΔsO [%] at stimulation site0+40-40 T KCl + 10 s c d e f g T KCl - 40 s T
KCl + 20 s T
KCl + 50 s T
KCl + 80 s
ROI 1ROI 2
Figure 3.
Results of initial wave experiment showing spreading de-polarization (SD) starting from an equilibrium state. (a)
Estimatedblood oxygenation (sO ) of two regions of interest (ROI) in the lefthemisphere (see Fig. 2 b). (b) Simultaneous electrocorticography(ECoG) monitoring. Data from two adjacent electrodes on theleft hemisphere is shown – the other three channels on the lefthemisphere and the five channels on the right hemisphere showedno change. The electrodes were placed on the lateral margins ofthe brain as to not interfere with hybrid photoacoustic ultrasonic(PAUS) imaging. (c) – (g) Absolute change in estimated sO ( ∆ sO ) in a region near the stimulation site (c) before potassiumchloride (KCl) stimulation and (d)–(g) 10-80 s after stimulation. In(d)–(f) spreading, intensifying hypoxia is measured followed by (g)an overcompensation in sO .
100 timelapse. The wave of hypoxia coincides with the ECoG measurements on two electrodes in the proximitywhose signals are plotted in Fig. 3 b; they clearly show a single SD wave moving through the cortex, while theother electrodes on both hemispheres showed no change in activity. Fig. 3 c–g shows the change in estimatedsO in the region around the stimulation as hypoxia propagating through the tissue followed by an increase insO over the baseline.The purpose of the cluster experiment was to investigate the hemodynamic changes during SD clusterswith PAUS. To this end, we repeatedly stimulated the brain with KCl until we observed the occurrence of ROI 1 ROI 2 sO [%] in ROIsO [%] estimation by PA a (T ) (T )(T ) 50 min0
15 16 17 18 19 20 21 min75507550
KCl stimulation
ΔsO [%] T after stimutation T = 200 sT = 230 sT = 260 s3 mm3 mm cb ROI 3 ROI 4ROI 1ROI 2
Figure 4.
Multispectral photoacoustic (PA) imaging of blood oxygenation (sO ) as part of the cluster experiment . After a 15 minbaseline scan, spreading depolarization (SD) was induced by potassium chloride (KCl) stimulation in the left hemisphere of a porcinebrain. The sagittal plane was continuously imaged for 51 min. (a) PA sO estimation before stimulation with marked regions of interest(ROI). Refer to Supplemental Video 2 – a time lapse video of the change of sO – for a complete view. The playback speed is 90 timesthe recording speed. (b) Time evolution of estimated sO in the two ROI (top: whole recording period; bottom: first wave). (c) Thechange in blood oxygenation ( ∆ sO ) relative to before KCl stimulation is shown for three example time steps 30 seconds apart (T , T and T ), corresponding to the dashed lines in (b). estimation inthe imaged sagittal plane in the left hemisphere during the baseline period. After KCl stimulation, we observedrepetitive waves of hypoxia propagating through the imaging plane, followed by an overcompensation in sO propagating through the cortex to up to a depth of approximately 2 to 5 mm below the brain surface. Fig.4c illustrates one such wave propagating from left to right during one minute as a change in sO estimation.The speed of the waves was measured as 3–9 mm/min between ROI 1 and 2. ECoG measurements on the lefthemisphere shown in Fig. 5 b indicate a SD cluster with the same frequency and speed of the sO changes(Fig. 5 a). As was the case in the initial wave experiment no change in ECoG activity in the right hemispherewas observed.In addition to the sO estimation from spectral unmixing we estimated the total hemoglobin (THb) for the cluster experiment ; this is visualized in Supplemental Video 3. The changes of THb in the ROI are shown inFig. 5 c, where ROIs 3 and 4 seem to exhibit low frequency vascular fluctuations (LF-VF) [39] which appear tobe depressed after SD [11, 40].
0 10
30 40 50 min a sO [%] in ROI b DC/AC ECoG [mV]
ROI 1ROI 2ROI 3ROI 4 KCl stimulation c THb [a.u.] in ROI
ROI 1ROI 2ROI 3ROI 4
Figure 5.
Monitoring of hemodynamic changes in four regionsof interest (ROI) (see Fig. 4 a) as part of the cluster experiment .Spreading Depolarization (SD) was induced 15 min after start ofrecording by potassium chloride (KCl) stimulation on the left hemi-sphere of a porcine brain. (a) Blood oxygenation (sO ) in fourROIs in the left hemisphere. (b) Simultaneous electrocorticography(ECoG) monitoring with five electrodes placed on the left hemi-sphere of the porcine brain. The occurrence of clustered SD isclearly visible as sudden direct current (DC) shifts spreading toneighboring channels, coinciding with spreading depression of thehigh frequency components. (c) Monitoring of total hemoglobin(THb) in four ROI. In ROI 3 and ROI 4 low frequency vascular fluc-tuations (LF-VF) can be observed which appear to be periodicallydepressed by SD. Discussion
We investigated the imaging of SDs based on the concept of PA imaging. Our approach involves simultaneousUS and multispectral PA imaging for time-resolved reconstruction of tissue oxygenation in sagittal image slices.6/16wo in vivo porcine experiments with our PAUS system provide the following evidence suggesting that ourconcept allows for the detection and monitoring of SD.(1)
Hypoxia consistent with ECoG:
By estimation of sO , we observed pronounced drops in estimated sO after KCl stimulation (cf. Fig. 3 a + 4 b). This local hypoxia lasted for around 30 seconds and was followed byan overcompensation or return to baseline sO . These changes were consistent with the occurrence of SD inECoG. The indicators we used to identify SD in ECoG were based on consensus [15]: A characteristic abruptDC shift followed by a longer lasting positivity, and a reduction in amplitudes of spontaneous AC activity.Both of which needed to spread with a speed of 1.5–9.5 mm/min between electrodes and not cross hemispheres.(2) Transient increase in blood volume:
By estimation of THb, we also observed a so-called normalhemodynamic response – a pronounced transient increase in blood volume (hyperemia) which was followed bya mild long-lasting oligemia (Fig. 5 c). Note that the results from THb estimation are less conclusive comparedto the sO based measurements as THb estimations are more susceptible to absolute changes in light fluenceover all measured wavelengths. This is caused by the change in illumination geometry due to swelling whichcauses a slow shift in the absolute THb signal as shown in Fig. 4 c. Lower fluence, generally in higher depthsalso cause the signal to noise ratio to deteriorate, which can be observed when comparing ROI 1 with ROI 3 inFig. 5 a.(3) Speed of wave propagation:
Both the changes in sO and THb propagated through the gray matter atspeeds of 3–9 mm/min. This is consistent with speed of SD reported in the literature as 1.7–9.2 mm/min [11]or 1.5–9.5 mm/min [15] in gyrencephalic brain (3–9 mm/min in porcine brain [13]).(4) Low-frequency vascular fluctuations:
We also observed changes in low-frequency vascular fluctuations(LF-VF) [40] (Fig. 5 c). The observed LF-VF ”display[ed] a spreading suppression in a similar fashion to thatof SDs” in ECoG (see [26]). Note that LF-VF were only visible in the vicinity of larger vessels, which was therationale for placing ROIs 3 and 4 in such regions.We conclude from these observations that our measurements clearly support our initial hypothesis andsuggest that PAUS is able to image SD as a change in sO . In contrast to all other methods proposed tomonitor SD to date, our approach has the unique advantage that it features both high resolution and highimaging depth. While it is not suitable for imaging the entire gyrencephalic brain, penetration depth issufficient to image the entire thickness of the cortical grey matter. Furthermore, the simultaneous PA and USimaging proved to be useful for anatomical orientation during the intervention (i.e. for needle guidance forKCl stimulation). Given these advantages, we see a potential use of PA imaging for SD characterization i.e.during pharmacological trials on the gyrencephalic brain. As the thickness of the human cerebral cortex iscomparable, usually averaging 2.5 mm and not exceeding 5 mm [41], PA imaging would be ideally suited forthe study of SD in patients, as well. While, PA imaging cannot currently penetrate through an intact humanskull [29], PA imaging could for example be used postoperative to study SD in stroke patients [42] with ahemicraniectomy [43].Our pilot study strongly suggests that photoacoustics could become a valuable tool for detection, imaging,and monitoring SD. Due to its high spatiotemporal resolution this approach can be used to more preciselystudy where (i.e. which neuron layer) SDs originate and how they propagate, thus adding to our understandingof the nature of SD and its contribution to brain injuries and disease progression. 7/16 ethods PAUS imaging system
The custom built hybrid photoacoustic ultrasonic (PAUS) imaging system is based on a 128 channel ultrasounddata acquisition system (DiPhAs, Fraunhofer IBMT, St. Ingbert, Germany) with a 128-element linear UStransducer operating on a center frequency of 7.5 MHz and broad acoustic response (L7-Xtech, Vermon, Tours,France). Due to its low level application programming interface (API) access, the system allows for raw dataaccess and an interleaved PAUS imaging mode. This interleaved mode acquires US data from several shotsafter each PA data acquisition. The data acquisition (DAQ) module is combined with a fast tuning opticalparametric oscillator (OPO) laser cart (Phocus Mobile, Opotek, Carlsbad, USA) which yields 690 nm – 950 nm,5 ns long laser pulses with a pulse repetition rate of 20 Hz and a per laser pulse power of up to 50 mJ. Thewavelength of each laser pulse can be tuned in between shots, allowing for real time multispectral acquisitionsequences. Laser fiber bundles ending in two line arrays are attached to the transducer by a 3D-printed frameincluding acrylic windows for the laser output. For each experiment the entire probe was wrapped in a sterileultrasound probe cover and gold leaf was placed between the US transducer and the probe cover to reduceartifacts created by light absorption in the US transducer. For live imaging and recording all APIs to thesystem were integrated in the Medical Imaging Interaction Toolkit (MITK) software framework and the MITKworkbench application was used throughout the intervention to control the PAUS system, configure the imageacquisition, and show live PA and US imaging streams. During our experiments we visualized both streamswith 15–20 fps using delay and sum (DAS) beamforming for an imaging depth of 4 cm with 256 reconstructedlines. For the initial wave experiment we imaged the wavelength sequencence (735 nm, 756 nm, 850 nm, 900 nm)selected to distinguish Hb and HbO [44]. Because we added an estimation of THb in the cluster experiment weinstead imaged the isosbestic point of Hb and HbO at 798 nm for further reference, leading to the wavelengthsequencence (760 nm, 798 nm, 858 nm).
Image reconstruction
The raw radiofrequency (rf) PA data acquired during the experiments was matched with the laser pulseenergies recorded by a pyroelectric sensor (Ophir PE25-C, Ophir Optronics, North Andover, USA) built inthe laser system (Phocus Mobile, Opotek, Carlsbad, USA) and matched with the wavelengths of the laserpulses measured by a spectrometer (HR2000+, Ocean Optics, Dunedin, USA). The wavelengths of the pulseswere measured independently of the imaging system to account for calibration errors. The rf PA slice wasthen corrected for the corresponding pulse energies. The recorded PAUS data was already beamformed liveduring the experiment to reduce the system load writing to disk. A single US slice was recorded after each PAslice. The US image was a compounded image averaged from US data acquired at five angles, equidistantfrom +10 deg to −
10 deg and beamformed to 256 lines using a delay and sum (DAS) algorithm with boxcarapodization. To convert the acquired rf PA slices into meaningful images suitable for multispectral analysis,the slices were beamformed with a reference DAS implementation [45] using Hanning apodization to 512 lines.B-Mode images with isotropic pixel spacing of 0.075 mm were formed with a Hilbert transform based envelope8/16etection filter. US B-Mode images were formed in the same way, only adding a subsequent logarithmiccompression.
Motion compensation
The PA images obtained after beamforming are corrected for inter-frame motion introduced by breathing,pulse or swelling (1) to enable a more stable spectral unmixing and (2) to assure that a given pixel locationcorresponds to the same physical regions of interest (ROI). To correct for the inter-frame motion an opticalflow based method is employed. The optical flow of each US image relative to the first US image in the entirerecording is estimated using an algorithm proposed by Farneb¨ack [46]. The flow estimated from the US B-modeimage is then used to warp the corresponding PA B-mode image.
Experimental Data Analysis
Because of the slow propagation of SD wave fronts we averaged over ten motion corrected frames of the samewavelength and still have the 1 s temporal resolution of IOS. Spectral unmixing of those image sequences wasthen performed using a non-negative constrained linear least squares solver ( scipy.optimize.nnls ). In allfigures and supplemental material plots and videos one PA datapoint is averaged over ten frames and thenaveraged over the ROIs. Speeds of SD wavefronts were obtained by measuring the time between the localminima of sO ROI 1 and ROI 2. The positions of ROIs 1 and 2 in both experiments were chosen at 1 mmdepth and 7.5 mm apart, in the center of the reconstructed image stream. ROIs 3 and 4 were chosen deeperand close to larger vessels to investigate the LF-VF effect which, as discussed, can only be observed there.ROIs are otherwise representative of the entire data set as can be seen in the supplemental videos.
Animals
Protocols for all experiments were approved by the institutional animal care and use committee in Karlsruhe,Baden-Wuerttemberg, Germany (Protocol No. 35-9185.81/G-174/16). Female German Landrace swines of 31and 33 kg were premedicated with Midazolam (Dormicum 0.5–0.7 mg/kg) and Azaperone (Stresnil 4 mg/kg)intramuscularly. After premedication, two venous lines were placed in the ear veins, and propofol (Disoprivan5–7 mg/kg) was administered intravenously to facilitate the intubation. The animals were then intubatedand mechanically ventilated and the pressure controlled ventilation was adapted to a respiration rate of12–20/min, a flow of 2.5 l O /min, 2.0 l air/min, FiO2 35 % and volume 7–10 ml per kg. The maintenance ofanesthesia required inhalational anesthesia with isoflurane (Isosthesia 0.6—1.0 %) and intravenous midazolamat a continuous dose of 0.5–0.7 mg/kg/h via perfusion and maintained throughout the entire experiment. If awakening reaction occurred, a bolus of propofol (Disoprivan 5–7 mg/kg) was administered. Temperature wasmonitored with a rectal probe. A 4-Fr catheter was placed in the right femoral artery for permanent monitoringof the mean arterial blood pressure (Raumedic AG, Helmbrechts, Germany). Capillary oxygen saturation(SpO ) was monitored from one ear. Arterial blood gases were obtained in the animal used for the initial waveexperiment . Ringer’s solution was given intravenously over 8—12 h, to compensate for intraoperative bleeding,9/16rinary output and insensible losses. The two animals used in this study were used primarily for this project.After finishing the protocol the animal used for the cluster experiment was used for other unrelated studies. Surgery
Animals were fixed in a stereotactic frame (Standard Stereotaxic Instruments, RWD Life Science, Shenzhen,China) and an extensive craniotomy with excision of the dura mater was performed, to view the subarachnoidalspace bilaterally. Initially, the brain surface was immersed for 30 to 40 min in a standard lactated Ringer’ssolution with an elevated K+ concentration (7 mmol/l), as preconditioning for SD induction, as proposed byBowyer et al. [47] for the KCl model of SD. EcoG was performed with two strips of 5 electrodes each (Ad-tech,Racine, Wisconsin, USA) that were placed at the lateral margins of the craniotomy below the dura mater andabove the parietal cortex. A camera for IOS imaging and its corresponding light sources were mounted abovethe stereotactic frame. After preconditioning a 5–10 mm deep paraffin pool was filled over the exposed cortex,to reduce the diffusion of the KCl stimulation. When necessary, paraffin was withdrawn and new paraffin wasadded. The preparation time was 4–5 h before the KCl stimulations started. A gel pad (Aquaflex UltrasoundGel Pad, Parker Laboratories, Fairfield, USA) was cut in shape of the exposed brain surface and placed in theparaffin pool. The custom designed PAUS probe (see Methods, PAUS Imaging system) was placed on topthe gel pad and fixed relative to the frame. For the initial wave experiment the gel pad and PAUS imagingsystem was placed before the initial stimulation. For the cluster experiment the gel pad and system was placedafter the initial KCl stimulations and the accompanying IOS imaging was performed. With the help of live USimaging, it was positioned to image a sagittal plane of the left hemisphere approximately 1 cm from the midline.For the cluster experiment we waited until any residual SD from prior stimulation subsided in the ECoGmonitoring. Only then did we start recording PAUS data in a sagittal plane for 15 min as a baseline. After asufficient baseline recording, spreading depolarization was triggered using 2–5 µ l of 1 mol/l KCl solution with aHamilton syringe. The stimulus needle was guided using the live PAUS image streams visualized in MITK. Monitoring
All relevant physiological parameters, such as mean arterial pressure, rectal temperature, heart rate, andarterial oxygen saturation, were continuously monitored. A mean systolic arterial pressure of 60 to 80 mmHg,a temperature between 36 and 37 ◦ C, SaO >
90 %, pCO between 35 and 45 mmHg, pO >
80 mmHg weremaintained.
Electrocorticography
Electrocorticography (ECoG) recording with the subdural electrodes was perfomed in 10 active channels, usingthe Powerlab 16/SP analogue/digital converter coupled with the LabChart-7 software (ADInstruments, NewSouth Wales, Australia) at a sampling frequency of 400 Hz. For visualization, in all figures and supplementalmaterial, ECoG data was post-processed in Python using a 45 Hz Butterworth low pass filter to filter alternatingcurrent (AC) noise. 10/16 ntrinsic optical signal imaging intrinsic optical signal (IOS) imaging is a functional neuroimaging technique that measures cortical reflectancechanges [24]. We imaged one band at a wavelength of 564 nm (14 nm FWHM) with a charge-coupled device(CCD) camera (Smartec GC1621M, MaxxVision GmbH, Stuttgart, Germany) which was mounted 25 cm abovethe exposed cortex. Images were acquired with static illumination and 2 s CCD integration time. Changesin tissue reflectance were registered using a method described in [24]. IOS was only used as an additionalreference to the ECoG in the animal corresponding to the cluster experiment to ensure that preconditioningwas sufficient and SDs were easily triggered.
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Acknowledgements
The authors would like to acknowledge support from the European Union through the ERC starting grantCOMBIOSCOPY under the New Horizon Framework Programme under grant agreement ERC-2015-StG-37960.The authors declare no conflict of interest.
Author Contributions
T.K. conceived the study, implemented the system, designed the experiments, performed the experiments,analyzed the data, drafted the initial manuscript; J.G. designed the experiments, performed the experiments,analyzed the data, edited the entire manuscript; M.A.H. helped plan the experiments, performed the experiments,edited the entire manuscript; T.A. performed the experiments, helped analyze the data, edited the entiremanuscript; A.H.-A. performed the experiments, edited the entire manuscript; E.S. conceived the study,designed the experiments, performed the experiments, supervised the neurosurgical aspects of the work, editedthe entire manuscript; L.M.-H. conceived the study, designed the experiments, supervised the biomedicalinformatics and engineering aspects of the work, edited the entire manuscript.
Supplemental Material
Supplemental Video 1
Initial wave experiment.
Blood oxygenation (sO ): link to online video Supplemental Video 2
Cluster experiment.
Blood oxygenation (sO ): link to online video Supplemental Video 3