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Brain Stimulation

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Brain Stimulation 7 (2014) 190e193

Short Communication

fMRI of Deep Brain Stimulation at the Rat Ventral Posteromedial Thalamus

Yen-Yu Ian Shih a,b,c,d,*, Tiwari V. Yash a,e, Bill Rogers a, Timothy Q. Duong a,f,**

aResearch Imaging Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USAbDepartment of Neurology, University of North Carolina, Chapel Hill, NC 27599, USAcBiomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC 27599, USAdDepartment of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599, USAeDepartment of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX 78249, USAf South Texas Veterans Health Care System, Department of Veterans Affairs, USA

a r t i c l e i n f o

Article history:Received 3 August 2013Received in revised form 25 September 2013Accepted 1 November 2013Available online 2 December 2013

Keywords:StriatumDeep brain stimulationfMRIThalamusRatIsoflurane

Grant support: This work was supported in part bciation (10POST4290091), Clinical Translational SciencUL1RR025767), and San Antonio Area Foundation toNIH/NINDS R01 NS45879 and VA MERIT to Dr. Timoth

Statement of financial disclosure/conflict of intereto disclose.* Corresponding author. Experimental Neuroimagin

Neurology and Biomedical Research Imaging Center,130 Mason Farm Road, CB# 7513, Chapel Hill, NC 27599fax: þ1 919 843 4456.** Corresponding author. Research Imaging InstituteScience Center at San Antonio, 8403 Floyd Curl Drive,Tel.: þ1 210 567 8120; fax: þ1 210 567 8152.

E-mail addresses: shihy@unc.edu (Y.-Y.I. Shih), duon

1935-861X/$ e see front matter � 2014 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.brs.2013.11.001

a b s t r a c t

Background: Functional magnetic resonance imaging (fMRI) of deep brain stimulation (DBS) haspotentials to reveal neuroanatomical connectivity of a specific brain region in vivo.Objective: This study aimed to demonstrate frequency and amplitude tunings of the thalamocortical tractusing DBS fMRI at the rat ventral posteromedial thalamus.Methods: Blood oxygenation level dependent (BOLD) fMRI data were acquired in a total of twelve rats at ahigh-field 11.7 T MRI scanner with modulation of nine stimulus frequencies (1e40 Hz) and sevenstimulus amplitudes (0.2e3.6 mA).Results: BOLD response in the barrel cortex peaked at 25 Hz. The response increased with stimulusamplitude and reached a plateau at 1 mA. Cortical spreading depolarization (CSD) was observed occa-sionally after DBS that carries >10% BOLD waves spanning the entire ipsilateral cortex.Conclusion: fMRI is sensitive to the frequency effect of DBS and has potential to investigate the function ofa particular neuroanatomical pathway.

� 2014 Elsevier Inc. All rights reserved.

Introduction

Deep brain stimulation (DBS) is clinically used to treat severalneurological symptoms such as Parkinson’s disease, tremor anddystonia [1,2]. The underlying mechanisms remain however largelyunexplored. DBS efficacy in the brain has been studied primarily byelectrophysiology recording, which does not provide depth-resolved mapping of DBS responses on a whole brain scale. Bloodoxygenation level dependent (BOLD) functionalmagnetic resonance

y the American Heart Asso-e Awards (CTSA, parent grantDr. Yen-Yu Ian Shih and they Q. Duong.st: The authors have nothing

g Laboratory, Department ofUniversity of North Carolina,, USA. Tel.: þ1 919 843 4729;

, University of Texas HealthSan Antonio, TX 78229, USA.

gt@uthscsa.edu (T.Q. Duong).

ll rights reserved.

imaging (fMRI) provides noninvasive, in vivo measurement of ce-rebral neurovascular function and has potentials to study wholebrain responses to real-time DBS.

In this study, we aimed to demonstrate thalamocortical con-nectivity under different frequency and amplitude tunings in ratsand establish a DBS fMRI protocol under isoflurane anesthesia thatallows longitudinal study. BOLD fMRI (n¼ 12) datawere acquired onan 11.7 TMRI scanner withmodulation of nine stimulus frequencies(1, 3, 7, 11, 15, 20, 25, 30, and 40 Hz fixed at 1 mA and correspondingpulse widths of 1/frequency ms) and seven stimulus amplitudes(0.2, 0.6, 1, 1.2, 1.4, 1.8, and 3.6 mA fixed at 7 Hz and 1/7 ms pulsewidth). Ventral posteromedial (VPM) thalamus was selected due toits rich innervation to the cortex. We hypothesized that the barrelcortex can be reliably activated by DBS and the responses shouldexhibit frequency and amplitude-dependent properties.

Material and methods

Subjects

Atotal of12adultmaleSpragueDawley rats (weighing250e300g;CharlesRiverLaboratories)were studied.All experimental procedures

Y.-Y.I. Shih et al. / Brain Stimulation 7 (2014) 190e193 191

were approved by the Institutional of Animal Care and UtilizationCommittee, UT Health Science Center at San Antonio.

Animal preparation

Rats were initially anesthetized with 3% isoflurane and oro-tracheally intubated for mechanical ventilation. After the animalwas secured in an MRI compatible rat stereotaxic headset, iso-flurane was reduced to 1e1.2%. End-tidal CO2 was continuouslymonitored via a capnometer. Non-invasive end-tidal CO2 valueswere previously calibrated against invasive blood-gas samplingsunder identical conditions [3,4]. Rectal temperature was main-tained at 37.0 � 0.5 �C with warm-water circulating pad. Heart rateand blood oxygen saturation level were continuously monitoredby a pulse oximeter. All recorded physiologic parameters weremaintained within normal ranges as reported previously [5,6]. Atwo-channel twisted platinum microelectrode (MS303/9C-B, Plas-ticsOne, Roanoke, VA) with 156 mm wire diameter was implantedinto the VPM thalamusa at 3 mm posterior to the bregma, 3 mmlateral to the midline, 6 mm below the cortical surface [7,8], andfixed with dental cement.

Stimulation

Stimulation pulses were generated by a custom-written soft-ware with a multifunctional USB module (USBe1208HS-2AO,Measurement Computing, Norton, MA) offering a digital-to-analogconversion rate of 1 MS/s to trigger an isolated current stimulator(A365, World Precision Instruments, Sarasota, FL). Stimulus cur-rent was carried by a 2 channel twisted cable (305-491/2 withmesh, PlasticsOne, Roanoke, VA) with a home-made silver wireextension to the DBS electrode. In the first study, modulation ofnine stimulus frequencies (1, 3, 7, 11, 15, 20, 25, 30, and 40 Hz fixedat 1 mA and corresponding pulse widths of 1/frequency ms) werestudied (number of subjects ¼ 12). The pulse-width was calibratedto ensure the total amount of charge delivered into the brain tissueper second was identical across different frequencies. In the sec-ond study, modulation of seven stimulus amplitudes (0.2, 0.6, 1,1.2, 1.4, 1.8, and 3.6 mA fixed at 7 Hz and 1/7 ms pulse width) werestudied (number of subjects ¼ 7). These parameters were chosenbased on a pilot study designed by the authors that explored po-tential frequency and amplitude dependent responses of VPM DBS.Frequency of 10e30 Hz has been widely used to study whiskersystem [9e11], by which VPM serves as an important relay. Thestimulus parameters were randomized for both studies. Stimula-tion paradigm was OFF-ON-OFF-ON-OFF, where OFF ¼ 100 s andON ¼ 50 s.

fMRI experiments

fMRI data were acquired on a 11.7 T, 16 cm bore magnet and a74 G/cm gradient insert (Bruker, Billerica, MA). A custom-madecircular surface coil (ID w 2 cm) was placed on the rat head.Magnetic field homogeneity was optimized using standard FAST-MAP shimming on an isotropic voxel of 7 � 7 � 7 mm. A T2-weighted pilot image was taken in the mid-sagittal plane tolocalize the anatomical position by identifying the anteriorcommissure (bregma �0.8 mm). BOLD fMRI was acquired with 4-shot gradient-echo EPI sequence using spectral width ¼ 200 kHz,TR/TE¼ 1250/12 ms, FOV¼ 2.56� 2.56 cm, slice thickness¼ 1 mm,matrix ¼ 128 � 128, and temporal resolution ¼ 5 s. Repeated scanswere performed when cortical spreading depolarization (CSD)-likesignal occurred.

Data analysis

Image analysis was performed using a custom-written graphicuser interface [12,13] in Matlab (Math-Works, Natick, MA). PercentBOLD activation map was generated by comparing the signal beforeand during stimulation. Region of interests (ROIs) were placed onthe barrel cortex to extract fMRI time-course data. Dynamic cineanalysis was used to delineate CSD propagation. The color-codedpixel values at each time frame were formed by subtracting themean map of the pre-stimulus period, and the threshold was set asa 3% BOLD signal increase from the baseline. Repeated-measuresANOVA with LSD post-hoc test was used to compare stimulus-evoked BOLD signal changes at different stimulus conditions. Thesignificance level for all the data analysis was set at P < 0.05.

Results

MR image with an overlaid of a brain atlas showed the electrodetract. The size of electrode artifactually appeared larger due tosusceptibility effect (Fig. 1A). Representative BOLD fMRI mapsevoked by 20 Hz DBS are shown in Fig. 1B. The activations aremainly located in the S1 barrel field and the upper lip region, whichare known to be densely innervated by VPM neurons. No apparentspatial shift was observed at different stimulus parametersthroughout the study. In the first study (modulation of nine fre-quencies), averaged BOLD fMRI time courses at 1e40 Hz stimuli areshown in Fig. 1C. BOLD responses in the barrel cortex exhibited abell-shaped tuning curve peaked at 25 Hz (Fig. 1D). Compared tothe 25 Hz data, significantly lower BOLD responses were observedat 1, 3, 7, and 40 Hz (P < 0.05), and no significant differences weredetected at 11, 15, 20, and 30 Hz (P> 0.05). BOLD responses showedrobust activation with high contrast-to-noise ratio (up to 8% BOLDat 11.7 T). In the second study (modulation of six amplitudes),averaged BOLD fMRI time courses at 0.2e3.6 mA stimuli are shownin Fig. 1E. BOLD responses increased with stimulus amplitude andreached a plateau at 1 mA.

CSD-like signal was observed occasionally after the DBS (12times out of 151 trials) that carries >10% BOLD waves spanning theentire ipsilateral cortex (Fig. 2), possibly due to hyperexcitation [14].This propagating wave usually initiated 1 min after the stimulusonset, propagated toward the midline, anterior, and posterior partof the cortex with an estimated speed at w4 mm/min. Among alltrials, the depolarization waves never crossed the hemisphere andonly stayed in the cortex.

Discussion

By systemically evaluating 9 frequencies and 7 pulse widths, weshowed that up to 8% BOLD signal changes can be detected at 11.7 T.No obvious lesion was observed in the T2*-weighted images up to3.6 mA. The subsequent trials also evoked robust and repeatablefMRI responses, indicating the VPM neurons were not functionallydamaged at this current amplitude when a short pulse width (1/7 ms) was applied. However, a single pulse of 0.1 mAwith 5 s pulsewidth produced observable lesion in the T2*-weighted images(data not shown), suggesting cautions on using DBS with long pulsewidth and highlighting the importance of DBS parameter calibra-tion. BOLD response in VPM was not observed because of the sus-ceptibility artifact induced by the commercial electrode. Withproper selection of electrode materials, local BOLD responses maybe seen [15]. Of note, the cortical BOLD response could originatefrom stimulating thalamocortical cells, or antidromically stimu-lating cortical thalamic afferent terminals [16]. Further elucidationusing c-fos or optogenetics is warranted.

Figure 1. DBS fMRI with stimulus frequency modulation. (A) Rat brain atlas overlaid on an anatomy (bregma �2.8 mm), showing the position of the microelectrode. (B) BOLDactivation maps of a representative subject. Responses were mainly located in the S1 barrel field/upper lip region. ROI was placed at �0.8 mm. (C) Grand-averaged BOLD responses to9 stimulus frequencies. Yellow-shaded area indicates stimulus epoch. (D) BOLD responses exhibited a frequency-dependent pattern peaked round 25 Hz, *P < 0.05, different frompeak value. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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One potential limitation of the present study is that animalswere anesthetized during DBS sessions. Anesthesia is known toalter peak frequency when peripheral sensory stimulus was applied[9,17]; however, a recent study showed that animals undera-chloralose and isoflurane anesthesia exhibit similar frequency-dependence and location of BOLD response, indicating that anes-thesia may have less impact on DBS-induced fMRI signals [15]. Apossible explanation is that DBS bypassed the anesthetic confoundson the peripheral nervous system and ascending neurons in thespinal cord.

In summary, the present study demonstrated thalamocorticalconnectivity by stimulating VPM thalamus in rats. This fMRIresponse was highly frequency-dependent. We also demonstratedthat DBS can induce CSD-like signals. This study demonstrates thefeasibility of measuring DBS frequency-dependent responses usingfMRI. The procedure established herein could serve as the basis of awide variety of system neuroscience applications, such as mappingDBS treatment effect in Parkinsonian animal models when target-ing different brain regions [6,18] or evaluating plasticity changes ofthalamocortical tract following injury [19]. This work also provides

Figure 2. DBS induced spreading depolarization. (A) The CSD initiated in the barrel cortex, propagated toward the midline and spread in both anterior and posterior directions. (B)fMRI time-course data from two ROIs from (A). The red window between two stimulus epochs indicates the temporal span of (A). (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

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important information regarding the selection of stimulus param-eters for future DBS fMRI studies in animal models.

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