Experimental Neurology 222 (2010) 42–50

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Experimental Neurology

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Intracranial electrode implantation produces regional neuroinflammation andmemory deficits in rats

Yafit (Kuttner) Hirshler a,b, Uri Polat c,d, Anat Biegon a,e,⁎a Joseph Sagol Neuroscience Center, Chaim Sheba Medical Center, Tel Hashomer, Israelb The Leslie and Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat Gan, Israelc Goldschleger Eye Research Institute, Chaim Sheba Medical Center, Tel Hashomer, Israeld Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israele Medical Department, Brookhaven National Lab, Upton, New York, USA

⁎ Corresponding author. Medical Department, BuildLab, Upton, NY 11973, USA. Fax: +1 631 344 5311.

E-mail address: Biegon@bnl.gov (A. Biegon).

0014-4886/$ – see front matter. Published by Elsevierdoi:10.1016/j.expneurol.2009.12.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 June 2009Revised 28 August 2009Accepted 5 December 2009Available online 21 December 2009

Keywords:Deep brain stimulationBrain injuryPeripheral Benzodiazepine ReceptorTranslocator proteinAutoradiographyNovel Object Recognition

Deep brain stimulation (DBS) is an established treatment for advanced Parkinson's disease (PD). Theprocedure entails intracranial implantation of an electrode in a specific brain structure followed by chronicstimulation. Although the beneficial effects of DBS on motor symptoms in PD are well known, it is oftenaccompanied by cognitive impairments, the origin of which is not fully understood. To explore the possiblecontribution of the surgical procedure itself, we studied the effect of electrode implantation in thesubthalamic nucleus (STN) on regional neuroinflammation and memory function in rats implantedbilaterally with stainless steel electrodes. Age-matched sham and intact rats were used as controls. Brainswere removed 1 or 8 weeks post-implantation and processed for in vitro autoradiography with [3H]PK11195,an established marker of microglial activation. Memory function was assessed by the novel objectrecognition test (ORT) before surgery and 2 and 8 weeks after surgery. Electrode implantation producedregion-dependent changes in ligand binding density in the implanted brains at 1 as well as 8 weeks post-implantation. Cortical regions showed more intense and widespread neuroinflammation than striatal orthalamic structures. Furthermore, implanted animals showed deficits in ORT performance 2 and 8 weekspost-implantation. Thus, electrode implantation resulted in a widespread and persistent neuroinflammationand sustained memory impairment. These results suggest that the insertion and continued presence ofelectrodes in the brain, even without stimulation, may lead to inflammation-mediated cognitive deficits insusceptible individuals, as observed in patients treated with DBS.

Published by Elsevier Inc.

Introduction

Deep brain stimulation (DBS) is an increasingly popular thera-peutic approach for diverse neurological disorders including Parkin-son's Disease (PD) (Benabid et al., 2005; Limousin and Martinez-Torres, 2008), depression (Marangell et al., 2007), obsessive compul-sive disorder (Lipsman et al., 2007) and epilepsy (Vonck et al., 2003,2007). This approach involves chronic implantation of an electrode ina specific brain structure followed by chronic electrical stimulation.

While DBS relieves motor symptoms, there is a steady increase inreports of cognitive impairments associated with this procedure in PD(Daniele et al., 2003; Dujardin et al., 2001; Funkiewiez et al., 2004;Saint-Cyr et al., 2000; Trepanier et al., 2000;Witt et al., 2008; York et al.,2008) as well as Huntington disease (Fasano et al., 2008), essentialtremor (Fields et al., 2003) and dystonia (Kiss et al., 2007). Previousstudies explained the cognitive impairment accompanying DBS in PD

ing 490, Brookhaven National

Inc.

patients as a result of the electrical stimulation of the subthalamicnucleus (Alegret et al., 2001; Ardouin et al., 1999; Dujardin et al., 2001;Saint-Cyr et al., 2000; Trepanier et al., 2000) which alters the basalganglia–anterior cingulate cortex circuit activity (Cilia et al., 2007a;Kalbe et al., 2009; Schroeder et al., 2003). In contrast, there is alsoevidence of cognitive decline after DBS surgery both “on” and “off”stimulation, compared to pre-surgical performance, suggesting effectsrelated to the surgical procedure rather than the stimulation (Daniele etal., 2003;Morrison et al., 2004; Pillon et al., 2000).However, the relativecontribution of the surgery would be hard to assess under theseconditions due to possible lingering effects of the chronic stimulation.

Postmortem analyses of brain biopsies from patients treated withDBS demonstrate a local brain tissue reaction to the electrodecharacterized by the presence of activated astrocytes (Boockvar etal., 2000; Burbaud et al., 2002; Haberler et al., 2000; Henderson et al.,2001; Henderson et al., 2002; Jarraya et al., 2003; Nielsen et al., 2007;Pilitsis et al., 2008) and activated microglia (Chou et al., 2004;Haberler et al., 2000; Henderson et al., 2002; Jarraya et al., 2003;Nielsen et al., 2007; Pilitsis et al., 2008). These findings were verysimilar, regardless of the disease, the electrode location and the

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duration of the implantation. Very similar inflammatory responseswere observed following implantation of cerebrospinal fluid shuntdevices (Del Bigio, 1998) and recording electrodes used for localiza-tion of epileptogenic tissue (Stephan et al., 2001) in humans, which donot involve electrical stimulation. Animal studies with variousintracranial implants similarly report activated astrocytes (Kim etal., 2004; Lenarz et al., 2007; Leung et al., 2008;McConnell et al., 2007;Mokry et al., 2000; Stice et al., 2007; Szarowski et al., 2003; Turner etal., 1999) and activatedmicroglia (Biran et al., 2007; Biran et al., 2005;Griffith and Humphrey, 2006; Kim et al., 2004; Leung et al., 2008;McConnell et al., 2007; Mokry et al., 2000; Szarowski et al., 2003) inclose proximity to the implant site.

Since neuroinflammation can cause cognitive impairment inhumans (Hoogman et al., 2007; Pikis et al., 1996; Schmidt et al.,2006) and in animals exposed to lipopolysaccharide (LPS) (Hauss-Wegrzyniak et al., 2000) or ischemia (Langdon et al., 2008; Liu et al.,2007), we set out to test the hypothesis that implantation-inducedneuroinflammation is not limited to the implantation site and mayspread through brain regions playing a critical role in cognitivefunctioning, thereby leading to memory deficits. To facilitatequantitative regional measurement of neuroinflammation, we choseto employ [3H]PK11195, an established marker of neuroinflammationwhich labels peripheral benzodiazepine receptors (PBR) on astrocytesand microglia (James et al., 2006; Lang, 2002).

Materials and methods

Electrodes

Three types of electrodes were used: (1) bipolar twisted electrodewith an insulated strand diameter of 0.28 mm (Plastics One, part no.MS303/1), referred to as “thick” electrodes throughout the text; (2)bipolar twisted electrode with an insulated strand diameter of0.15 mm (Plastics One, part no. MS303/3), referred to as “thin”electrodes throughout the text; and (3) bipolar concentric electrodewith an inner electrode projection of 1 mm, inner insulated electrodediameter of 0.15mm and outer electrode diameter of 0.4 mm (PlasticsOne, part no. MS303/8). These are referred to as concentric electrodesthroughout the text. All electrodes are constructed of stainless steeland coated with polyimide.

Animals

Four-month-old Sprague Dawley male rats were housed in theSheba animal facility under controlled light/dark cycle with food andwater available ad libitum. Animals were housed singly after surgeryto prevent them from removing each others' implants. Animals weremaintained for 1 or 8 weeks post-implantation surgery. Parallelgroups of age-matched non-surgical controls were maintained underthe same conditions.

Experiments were conducted in accordance with internationalstandards on animal welfare andwere approved by the ShebaMedicalCenter and Bar-Ilan University institutional animal care and usecommittees. Adequate measures were taken to minimize pain ordiscomfort.

Thirty eight animals were used for the study andwere divided intothe following experimental groups:

Experiment 1: Four-month-old male Sprague Dawley rats wereimplanted bilaterally with “thick” (n=7) or “thin” (n=5)electrodes in the subthalamic nucleus (STN). The control group(n=7) consisted of intact (n=3) and sham-operated (n=4) age-matched animals. Implanted and control animals were maintainedfor 1 week.Experiment 2: Four-month-old male Sprague Dawley rats (n=9)were implanted bilaterally with concentric electrodes in the

subthalamic nucleus. The control group (n=10) consisted ofsham treated (n=4) and intact (n=6) age-matched animals.Implanted and control animals were maintained for 8 weeks.These animals were tested in the object recognition test (ORT) pre-implantation (n=10) and 2 weeks (n=16) and 8 weeks (n=17)post-implantation.

Electrode implantation surgery

Rats were anesthetized with intraperitoneal (i.p.) equithesin(120 mg/kg chloral hydrate, 25.2 mg/kg nembutal) and placed in astereotaxic apparatus (Kopf Instruments, Tujunga, CA). A midlinelongitudinal incision was performed, the skin was retracted and theskull was exposed. Two holes for electrodes were drilled into the skullusing a dental drill (Elektrotechnisches, Leutkirch, Germany). Thecenter of the drilled holes was positioned at stereotaxic coordinates+3 mm forward of bregma, and ±2.4 mm lateral to bregmaaccording to the rat brain atlas (Paxinos and Watson, 1998). Thedura was exposed and stainless steel electrodes coated withpolyimide (Plastics One, Roanoke, VA) were inserted at an angle of38°, passing through cortex, striatum and thalamus and terminatingin the STN, mimicking the surgical path employed in patients with PD(Benabid et al., 2009). Three additional holes were drilled into theskull and were used for surgical bone screws (Small Parts, Inc.,Miramar, FL). Acrylic dental adhesive (Major Dental, Moncalieri, Italy)was applied as a slurry around the bone screws to cover the skull andused to firmly secure the electrodes. The skin was sutured and anantibiotic ointment was applied to the wound. Sham rats wereanesthetized, a midline longitudinal incision was performed, the skinwas sutured and an antibiotic ointment was applied to the wound.

Tissue processing

Rats were decapitated 1 or 8 weeks post-implantation surgery andthe brains were quickly removed, rinsed, and separated along themidline, and the two separated hemispheres were frozen in powdereddry ice. Long-term storage of brains was at −80 °C. The frozen brainswere sectioned in a cryostat (Leica, Nussloch, Germany) in the sagittalplane. Sections (20 μm) were produced at a cutting temperature of−15 °C and 10 consecutive series were collected at 200 μm intervalsby thawmounting onto coated glass slides, starting from the temporalcortex towards the midline for each hemisphere.

Histological staining

One series of brain sections was stained with hematoxylin & eosin.Electrode track verification was carried out using the stained sectionsand a rat brain atlas (Paxinos and Watson, 1998).

In vitro autoradiography

On the day of the assay, sections were removed from the −80 °Cfreezer and allowed to reach room temperature. Peripheral-typebenzodiazepine receptors were labeled with [3H]PK11195 (PerkinElmer,Waltham, MA; specific activity 84.8 Ci/mmol; 1mCi/mL) usinga methodology adapted from the literature (Guilarte et al., 1995;Raghavendra Rao et al., 2000). Briefly, sections were first pre-incubated in PBS, pH 7.4 (Sigma-Aldrich, Rehovot, Israel) for 15 minat room temperature, followed by 30 min incubation at roomtemperature with the radioactive ligand. Total binding was deter-mined with 1 nM [3H]PK11195, whereas nonspecific binding wasdetermined on consecutive slides in the presence of excess (20 μM)unlabeled PK11195 (Sigma-Aldrich, Rehovot, Israel). Sections werethen washed twice for 6 min in ice-cold (4 °C) PBS and dipped in ice-cold (4 °C) double distilled water prior to drying to remove buffer

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salts. The dried sections were exposed to Biomax MR films (Kodak,Rochester, NY) for 21 days alongside calibrated tritium microscales(Amersham, Buckinghamshire, England).

Quantitative image analysis

The films were scanned and digitized using an HP scanner. Theintensity and spread of the neuroinflammatory response wereassessed through regional density measurements of anatomicallydefined structures. Mean grey level of each structure was measuredusing the ROI manager tool in ImageJ and was translated toradioactivity level through a calibration standard curve using thetritium microscale measurements. Brain regions were identified inreference to a rat brain atlas (Paxinos andWatson, 1998). For analysis,the brains were divided into four consecutive anatomical levels fromlateral to medial as follows: level 1, temporal cortex; level 2,entorhinal cortex, insular cortex, occipital cortex, parietal cortex,perirhinal cortex, striatum, subiculum and ventral hippocampus; level3, dorsolateral prefrontal cortex, frontal cortex, cornu ammoni 1(CA1), cornu ammoni 3 (CA3) and dentate gyrus (DG) in thehippocampus, laterodorsal thalamic nucleus–ventrolateral part(LDVL), occipital cortex, parietal cortex, striatum, substantia nigra(SN), ventral pallidum/substantia innominata (VPSI) and ventralposteromedial thalamic nucleus (VPM); and level 4, frontal cortex andmedial prefrontal cortex (mPFC). Overall, [3H]PK11195 binding wasmeasured in 19 distinct anatomical regions which were previouslyfound to be differentially sensitive to LPS injection and traumaticbrain injury (Biegon et al., 2002; Grossman et al., 2003). The peri-lesional area was defined as an area around the center of theelectrode's track (0.5 mm in each direction) and was divided intothree sub regions: cortex, striatum and thalamus. Grey levels ofsections that represent non-specific binding were measured for allstructures together at the same level. Non-specific binding levels weresubtracted from the total binding to obtain the specific binding in eachbrain structure. In order to illustrate the spread of neuroinflammationwithin the cortex and within the striatum of implanted animals, plotprofiles of [3H]PK11195 density versus distance were obtained usingthe plot profile tool in ImageJ software. Lines were drawn on sagittal

Table 1Electrode implantation and PBR densities at 1 week after surgery.

Brain region Control Thick electrodeMean±SE Mean±SE

CA1 8.0±0.4 7.1±0.7CA3 6.4±0.4 5.8±0.6DG 10.9±0.5 9.5±0.8DLPFC 9.5±1.0 11.4±0.5#

Ent Cx 11.0±1.5 6.9±0.8⁎

Frontal Cx 8.5±0.6 23.6±2.9⁎⁎

Insular Cx 9.6±0.9 10.9±1.5LDVL 6.1±0.8 6.7±0.9mPFC 9.3±1.5 15.0±1.6⁎

Occipital Cx 7.6±0.5 6.7±0.5Parietal Cx 8.3±0.7 11.5±1.2⁎

PRh Cx 9.2±1.8 4.6±0.9⁎

Subiculum 9.4±0.8 7.5±0.5⁎

SN 8.3±0.8 8.9±0.8Striatum 7.9±0.6 10.3±0.6⁎⁎

Temporal Cx 7.7±0.7 9.0±1.1vHipp 9.8±0.8 8.6±0.8VPM 6.1±0.8 6.7±0.8VPSI 6.6±1.5 7.5±0.8

Animals were killed 1 week after bilateral implantation of thick or thin twisted electrodes. V(n=6 intact and 8 sham-operated hemispheres), 14 hemispheres implanted with thick elregion. CA, cornu ammoni; Cx, cortex; DG, dentate gyrus; DLPFC, dorsolateral prefrontal cmedial prefrontal cortex; PRh, perirhinal; SN, substantia nigra; vHipp, ventral hippocampinnominata. ⁎Pb0.05, ⁎⁎Pb0.01, #Pb0.1, electrode vs. control; aPb0.05, bPb0.01, cPb0.1, thicktreatment).

sections containing the electrode track. The line in the cortex wasdrawn from the parietal cortex (above the ventricle), through thefrontal cortex and prefrontal cortex, and terminated at the edge of thesection. The line in the striatumwas drawn from the ventricle throughthe striatum and terminated at the corpus callosum.

Object recognition test

The object recognition test was performed as originally described byEnnaceur andDelacour (1988). Briefly, in thefirst day of the test animalswere placed in the testing cage (a plastic box of 72×47×34 cm) for 1h habituation. On the following day (pre-implantation and 2 and8weeks post-implantation) rats were placed in the same cagewith twoidentical objects. The cumulative time spent by the rat exploring theobjects was recorded during a 5 min interval. Four hours later, theanimals were reintroduced into the cage for a 5min test. One of the twoidentical objects was replaced by a novel object. To offset location bias,thenovel objectwasplacedat the locationof theold objectwhich the ratspent less time exploring (less than 45%) during the familiarizationphase. The time (out of 5 min total) spent exploring each of the objectswas recorded. The outcome measure was percentage of time spentexploring the novel object during the testing phase, whereby normalhealthy rodentswill spend relativelymore time exploring a novel objectthan a familiar, i.e., “memorized” object. The proportion of time spentexploring thenovel object during the testingphasewas compared to theproportion of time spent exploring the object at the same locationduring the familiarization phase.

Statistical analysis

We used Q-Q plots to analyze the data distribution and it wasfound to be normal, justifying the use of analyses of variance. Theeffect of electrode implantation on regional density of PBR wasexamined using repeated measures ANOVA (repeated measure=region) including regions represented in all hemispheres (10 regionsin experiment 1 and 18 regions in experiment 2) and by two wayANOVA (treatment X region). Posthoc comparisons for all regionswere performed using Fisher's PLSD. The effect of electrode

Thick vs. control Thin electrode Thin vs. control% change Mean±SE % change

−11 10.2±1.7c 26−10 8.4±1.2#a 32−13 13.3±2.0a 22

21 13.1±1.0⁎ 39−37 12.4±1.2b 13178 23.6±2.2⁎⁎ 17714 13.7±1.4⁎⁎ 4410 6.8±1.1 1261 11.3±0.5 22

−12 10.6±0.8⁎⁎b 3939 16.2±1.6⁎⁎a 96

−50 8.7±0.8b −6−21 10.1±1.3a 7

7 11.4±2.5 3831 11.6±1.1⁎⁎ 4718 15.0±1.8⁎⁎b 96

−12 9.3±1.1 −510 7.9±1.1 2814 10.4±2.3 59

alues represent specifically bound radioactivity in nCi/mg from 14 control hemispheresectrode and 9 hemispheres implanted with thin electrode with 6–14 measurements/ortex; Ent, entorhinal; LDVL, laterodorsal thalamic nucleus–ventrolateral part; mPFC,us; VPM, ventral posteromedial thalamic nucleus; VPSI, ventral pallidum/substantiaelectrode vs. thin electrode by posthoc analysis following twoway ANOVA (by region ×

Fig. 2. Regional neuroinflammation lateral to the electrode track 1 week followingelectrode implantation. Autoradiograms of sagittal brain sections from a sham animal(A), an animal implanted with thick electrodes (B), and an animal implanted with thinelectrodes (C). Autoradiograms were pseudocolored using the rainbow spectrum (topleft). Note the decrease in PBR binding in the entorhinal cortex (red arrow), perirhinalcortex (white arrow) and subiculum (green arrow) with the thick, but not the thin,electrode. Scale bar: 0.5 cm.

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implantation on PBR density in peri-lesional areas was examined onlyin the implanted animals by one way ANOVA (by region). Significancewas preset at Pb0.05. The effect of electrode implantation on theperformance of the object recognition test was examined using pairedor unpaired T-test as appropriate.

Results

Effects of thick and thin electrode implantation on PBR density 1 weekpost-implantation (experiment 1)

There were no significant differences between hemispheres ofsham animals compared with hemispheres of intact animals byrepeated measures ANOVA; thus, results from these hemisphereswere pooled and considered as control hemispheres. Electrodeimplantations resulted in significant increases in the density of PBRindicative of neuroinflammation 1 week post-implantation (signifi-cant region × treatment interaction by two way ANOVA, F=4.62Pb0.0001). Changes observed in hemispheres implanted with thickelectrode compared to control hemispheres showed a region-dependent pattern with the largest and most significant increase(177%) detected in the frontal cortex. Smaller but statisticallysignificant increases were observed in the medial prefrontal cortex,parietal cortex and striatum (31–61%). Non-significant elevations inPBR density were seen in the dorsolateral prefrontal cortex, temporalcortex, insular cortex, ventral pallidum/substantia innominata,thalamic nuclei and substantia nigra. Significant decreases in PBRdensity were detected in the subiculum, entorhinal cortex and theperirhinal cortex (21–50%). Ventral hippocampus, occipital cortex,dentate gyrus, CA1 and CA3 showed trends towards a decrease indensity which were not statistically significant (Table 1; Figs. 1 and 2).

A similar pattern of increases in PBR density was observed inhemispheres implanted with thin electrode (compared to controlhemispheres) with the largest increases in the frontal cortex (177%).Smaller but significant increases were seen in parietal, temporal,insular, occipital and dorsolateral prefrontal cortex and in striatum(38–96%). Large increaseswere also observed in the ventral pallidum/substantia innominata (58%) and substantia nigra (38%) but did notreach significance due to higher measurement variability in thesesmall regions. Changes in the CA3, thalamic nuclei, CA1, dentate gyrus,medial prefrontal cortex, entorhinal cortex, subiculum, ventral

Fig. 1. Regional neuroinflammation at the level of the STN 1 week following electrodeimplantation. Autoradiograms of sagittal brain sections from an intact animal (A) andan animal implanted with thick electrodes (B). Autoradiograms were pseudocoloredusing the rainbow spectrum (top left) with red representing the highest value. Theelectrode insertion point is marked by an arrow. Scale bar: 0.5 cm.

hippocampus and perirhinal cortex were small and not statisticallysignificant (Table 1; Fig. 2).

Further characterization of the regional profile of the neuroinflam-mation revealed that cortical increases in PBR density are larger andspread further from the site of implantation compared to the striatumwhen measured 1 week post-implantation (Fig. 3). PBR density in theperi-lesional cortex was higher than the peri-lesional striatum (30%difference, Pb0.0001) and the peri-lesional thalamus (50% difference,Pb0.0001). In addition, PBR density in the striatum was higher thanthe peri-lesional thalamus (30% difference, P=0.0002) (Fig. 6).

Effects of electrode implantation on PBR density 8 weekspost-implantation (experiment 2)

Like in the short-term study above, there were no significantdifferences between hemispheres of sham animals compared withhemispheres of intact animals by repeated measures ANOVA, andresults from these hemispheres were pooled and considered ascontrol hemispheres. Increased density of PBR indicative of neuroin-flammation was observed in hemispheres implanted with concentricelectrodes compared with control (sham and intact) hemispheres8 weeks post-implantation (significant region × treatment interactionby two way ANOVA, F=5.56, Pb0.0001). The largest significantincrease was found in the frontal cortex (56%). Smaller but significantincreases were seen in the striatum, medial prefrontal cortex,substantia nigra, ventral posteromedial thalamic nucleus, parietaland insular cortex (10–29%). Non-significant changes were seen inthe ventral pallidum/substantia innominata (17%), entorhinal cortex(11%), and perirhinal cortex (−11%) (Table 2; Fig. 4).

Fig. 3. The spread of neuroinflammation within the cortex and striatum 1 weekfollowing electrode implantation. Line plot profile of [3H]PK11195 specific bindingversus distance in the cortex (A) and striatum (B) of an intact animal (red line) and animplanted animal (blue line). The line in the cortex was drawn from the parietal cortex(above the ventricle), through the frontal cortex and prefrontal cortex, and terminatedat the edge of the section. The line in the striatumwas drawn from the ventricle throughthe striatum and terminated at the corpus callosum.

Table 2Electrode implantation and PBR densities at 8 weeks after surgery.

Brain region Control Electrode Electrode vs. controlMean±SE Mean±SE % change

CA1 8.2±0.4 8.0±0.3 −2CA3 8.2±0.4 8.2±0.3 0DG 12.0±0.4 12.5±0.4 4DLPFC 10.2±0.4 10.8±0.5 7Ent Cx 14.9±0.7 16.5±0.6# 11Frontal Cx 10.3±0.4 16.1±1.2⁎⁎ 59Insular Cx 9.8±0.3 10.9±0.3⁎ 10LDVL 6.7±0.4 7.1±0.4 6mPFC 9.2±0.6 11.3±0.9⁎ 23Occipital Cx 9.5±0.3 9.5±0.2 0Parietal Cx 9.9±0.2 11.0±0.3⁎⁎ 11PRh Cx 12.7±0.8 11.3±0.6 −11SN 8.7±0.5 10.5±0.5⁎⁎ 21Striatum 7.9±0.2 10.2±0.3⁎⁎ 29Subiculum 10.5±0.4 10.2±0.3 −3Temporal Cx 9.3±0.3 9.2±0.5 0vHipp 10.2±0.4 9.7±0.5 −4VPM 6.3±0.3 7.4±0.3⁎ 18VPSI 5.4±0.3 6.3±0.4# 17

Animalswere killed 8weeks after bilateral implantation of concentric electrodes. Valuesrepresent specifically bound radioactivity in nCi/mg from 20 control hemispheres(n=11 intact and 9 sham-operated hemispheres) and 18 implanted hemispheres with8–20 measurements/region. CA, cornu ammoni; Cx, cortex; DG, dentate gyrus; DLPFC,dorsolateral prefrontal cortex; Ent, entorhinal; LDVL, laterodorsal thalamic nucleus–ventrolateral part;mPFC,medial prefrontal cortex; PRh, perirhinal; SN, substantia nigra;vHipp, ventral hippocampus; VPM, ventral postermedial thalamic nucleus; VPSI, ventralpallidum/substantia innominata. ⁎Pb0.05, ⁎⁎Pb0.01, #Pb0.1, electrode vs. control,posthoc analysis following two way ANOVA (by region × treatment).

Fig. 4. Regional neuroinflammation at the level of the STN 8 weeks following electrodeimplantation. Autoradiograms of sagittal brain sections from an intact animal (A) andan implanted animal (B). Autoradiograms were pseudocolored using the rainbowspectrum (top left) with red representing the highest value. Electrode insertion point ismarked by a white arrow. Scale bar: 0.5 cm.

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Density profile analysis 8 weeks post-implantation shows that thedistribution of PBR within the cortex is more restricted relative to thedistribution 1 week post-implantation, although it is still higher thanstriatum (Fig. 5).

PBR density in the peri-lesional cortex was higher than the peri-lesional striatum (20% difference, Pb0.0001) and the peri-lesionalthalamus (60% difference, Pb0.0001). PBR density in the peri-lesionalstriatum was significantly higher than in the peri-lesional thalamus(50% difference, P=0.0001) (Fig. 6).

Effects of electrode implantation surgery on ORT performance

When tested before randomization to treatment, the animalsspent 58.6% (±5.8) of total time exploring the novel object during thetesting phase in comparison to 35.7% (±2.6) of total time spentexploring the old object in the same location during the familiariza-

tion phase. This difference was statistically significant (T=4.2,Pb0.01) (Fig. 7A).

Control (sham and intact) animals showed the expected increasein exploration of the novel object when tested again 2 or 8 weekslater. Two weeks after the procedure, the control animals spent 57.2%(±2) of total time exploring the novel object during the testing phasein comparison to 44.8% (±3.5) of total time spent exploring the oldobject in the same location during the familiarization phase (T=3.9,Pb0.005). Eight weeks post-implantation the control rats spent54.2% (±4.2) of total time exploring the novel object during thetesting phase in comparison to 30.6% (±6.1) of total time spent

Fig. 5. The spread of neuroinflammation within the cortex and the striatum 8 weeksfollowing electrode implantation. Line plot profile of [3H]PK11195 specific bindingversus distance in the cortex (A) and striatum (B) of an intact animal (red line) and animplanted animal (blue line).

Fig. 6. Neuroinflammation in the peri-lesional areas 1 and 8 weeks following electrodeimplantation. Bars depict means±SEM of peri-lesional PBR density in cortex (n=11–16, black bars), striatum (n=10–16, white bars) and thalamus (n=6–15, hatchedbars). aPb0.0001, cortex compared to striatum; bPb0.0001 cortex compared tothalamus; cPb0.0002 striatum compared to thalamus, by one way ANOVA (by region).

Fig. 7. ORT performance 2 and 8 weeks following electrodes implantation. Bars depictmeans±SEM of percentage of time exploring the familiar (white bars) or novel (blackbars) object. (A) Performance in the ORT pre-implantation (n=10). (B) Performance ofcontrol animals 2 and 8 weeks post-sham procedure (n=9 at 2 weeks, n=8 at8 weeks). (C) Performance of implanted animals 2 and 8 weeks post-implantation(n=7 at 2 weeks, n=9 at 8 weeks). ⁎Pb0.05, ⁎⁎Pb0.005 by paired t-test.

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exploring the old object in the same location during the familiariza-tion phase (T=2.9, P=0.02) (Fig. 7B).

In contrast, the implanted animals did not show a significantpreference for the novel object at both time points: spending 46.6%(±5.9) with the novel object compared to 35.2% (±4.3) of total timeexploring the old object when tested 2 weeks post-implantation(T=1.4, P=0.2). Eight weeks post-implantation implanted animalsspent 47.3% (±9.3) of total time exploring the novel object comparedto 36.1% (±4) of total time spent exploring the old object (T=1.3,P=0.2) (Fig. 7C).

Total exploration time of implanted animals in the familiarizationphase was not significantly different from control rats at 2 weeks(29.8±3.3 and 37.8±5.6, respectively; T=1.23, P=0.23) and8 weeks (21.9±2.4 and 25.5±9, respectively; T=0.38, P=0.71)post-implantation, suggesting there was no significant motor deficitor general decrease in spontaneous exploration in the implantedanimals.

Discussion

The current study shows for the first time that chronicimplantation of electrodes in the STN produces a memory deficit

48 Y.(K.) Hirshler et al. / Experimental Neurology 222 (2010) 42–50

as well as persistent and widespread neuroinflammation in rats,which extends beyond the electrode track in a region-selectivemanner. Widespread neuroinflammation appears to be a generalfeature of the chronic implantation procedure since it was found inrats implanted with three different types of electrodes varying inthickness and shape. Unlike previous studies which used immuno-histochemistry and investigated only the local reaction to theimplant (Biran et al., 2005; Griffith and Humphrey, 2006; Kim et al.,2004; Leung et al., 2008; McConnell et al., 2007; Mokry et al., 2000;Stice et al., 2007; Szarowski et al., 2003; Turner et al., 1999), wemeasured the intensity of neuroinflammation in multiple regionsthroughout the brain using quantitative in vitro autoradiographywith [3H]PK11195 for PBR labeling (Dubois et al., 1988; Myers et al.,1991a,b; Pappata et al., 2000; Stephenson et al., 1995). Among thebrain regions traversed by the electrode, the cortex showed themost intense and widespread increase in PBR (nearly two fold) 1week after the surgery. This time point corresponds to the maximalmicroglial reaction and PBR density in other models of acute braininjury and neurotoxicity (Maeda et al., 2007; Miyazawa et al.,1995). Significant increases in PBR were found extending from thepoint of insertion in the frontal cortex anteriorly (to dorsolateralprefrontal and insular cortex), posteriorly (to parietal and occipitalcortex), laterally (to temporal cortex) and medially (to medialprefrontal cortex). Smaller though significant increases were seen inthe striatum (30–50%) and the thalamus demonstrated even smallerincreases (10–28%) which were not statistically significant. Thisregional pattern of sensitivity to inflammatory insult is similar tothe one we reported previously in acute models of pure globalneuroinflammation (LPS injection in the cisterna magna (Biegon etal., 2002) and closed head injury (Grossman et al., 2003)) which donot involve insertion of a foreign object into the brain, suggestingthat the vulnerability of the cortex and relative resistance of thestriatum and thalamus are the results of an inherent regionalsensitivity to neuroinflammatory challenge. Consistent with earlierreports, we found large elevations in PBR density in close proximityto the electrode track 1 week post-implantation (Biran et al., 2007;McConnell et al., 2007; Szarowski et al., 2003). Measurement of PBRdensity in peri-lesional areas also revealed an enhanced sensitivityof the cortex relative to the striatum and the thalamus.

A similar regional pattern of increases in PBR density was foundin brain hemispheres examined 8 weeks post-implantation, thoughthe effects were generally smaller than those observed 1 week post-implantation. The frontal cortex again showed the largest response(N50% increase) compared to striatum (b30%), with small thoughsignificant increases in remote cortical areas including medialprefrontal cortex and insular cortex. Two regions which showed atrend towards an increase at this time point were the substantianigra and substantia innominata/ventral pallidum. It is noteworthythat the substantia nigra and substantia innominata were amongthe regions most vulnerable to inflammation induced acutely bydelivering LPS non-invasively into the cisterna magna (Biegon et al.,2002) as well as after intraparenchymal injection to the substantianigra (Kim et al., 2000) and substantia innominata (Willard et al.,2000).

Our observations of persistent neuroinflammation near theelectrode are in agreement with previous observations of sustainedneuroinflammation adjacent to the implant site measured 4–12weeks post-implantation (Biran et al., 2007; Biran et al., 2005; Griffithand Humphrey, 2006; Kim et al., 2004; Lenarz et al., 2007; Leung et al.,2008; McConnell et al., 2007; Mokry et al., 2000; Stice et al., 2007;Szarowski et al., 2003; Turner et al., 1999). However our results showthat even along the electrode track, there is regional sensitivity(higher sensitivity of the peri-lesional cortex relative to striatum andthalamus) which is sustained through the later time point as well.

Interestingly, the implantation of a thick electrode (but not a thinelectrode) also resulted in reductions in PBR density compared to

control hemispheres in regions distant from the implant site such asentorhinal cortex, perirhinal cortex and subiculum 1 week post-implantation. A possible explanation for this finding could be themigration of microglia from remote regions towards the lesion site asshown by Carbonell et al. (2005) following focal brain injury, whichcould be more pronounced with a thicker electrode at short timeintervals (Szarowski et al., 2003).

On the behavioral level, the implanted animals did not show anygross pathology when examined 1 week or more after surgery andtheir spontaneous locomotion was similar to controls. However,performance in the ORT was disrupted relative to control rats at 2 aswell as 8 weeks post-implantation. These findings are in line with arecent study showing that chronic unilateral microdialysis cannula-tion of the striatum led to widespread reduction of cortical glucosemetabolism and memory deficits in the ORT at 3, 7, 14 and 56 daysafter implantationwhile general locomotionwas not affected at any ofthe time points (Frumberg et al., 2007). In addition, it was previouslyshown that neuroinflammation can lead to memory impairment(Hauss-Wegrzyniak et al., 2000; Langdon et al., 2008; Liu et al., 2007).Hence, we hypothesize that the presence of memory deficits andabsence of motor deficits observed in the implanted animals are aconsequence of the implantation-induced region-selective neuroin-flammation. Among the regions that showed high neuroinflammationas a result of the electrode implantation there are several regions thatmediate cognitive functions, e.g., the insular cortex, which is involvedin the object recognition test performance (Balderas et al., 2008;Bermudez-Rattoni et al., 2005). Importantly, the electrode trajectoryin brains of PD patients traverses the prefrontal cortex (Benabid et al.,2009) which is responsible for many cognitive functions in humans(Funahashi, 2001; Godefroy, 2003; Smith and Jonides, 1999). Indeed,the most prevalent cognitive deficits observed in PD patients treatedwith DBS are related to prefrontal dysfunctions (Campbell et al., 2008;Cilia et al., 2007b; De Gaspari et al., 2006; Dujardin et al., 2001). In fact,a path through the prefrontal (“non-eloquent”) cortex is favored inmost intracranial neurosurgical procedures. However, we do not havedirect evidence of cortical neuroinflammation from biopsies obtainedfrom patients treated with DBS, since published studies focused onlyon the area adjacent to the electrode's contacts, which are usuallylocated subcortically (Boockvar et al., 2000; Burbaud et al., 2002; Chouet al., 2004; Haberler et al., 2000; Henderson et al., 2001; Nielsen et al.,2007; Pilitsis et al., 2008).

Taken together, theseobservations suggest that the cognitive deficitsobserved in DBS patients may result at least in part from neuroin-flammation caused by the surgical procedure, irrespective of the targetregion and its direct stimulation. If this is indeed the case, it may alsoexplain the increased cognitive vulnerability of older patients to DBS(Saint-Cyr et al., 2000; Trepanier et al., 2000), since it was shown that aneuroinflammatory challenge in aged mice produces a disproportion-ately high induction of proinflammatory cytokines in microglia (Henryet al., 2009). In addition, it was shown by post-mortem analysis thatactivated microglia are present in the cortex of PD brains (Imamuraet al., 2003). Therefore, the disease itself may also contribute to theneuroinflammation seen in PD patients treated with DBS.

Perhaps more importantly, these observations support an investi-gation of the ability of inhibitors of microglial activation (e.g.,minocycline, a derivative of the antibiotic tetracycline (Kim et al.,2009; Liu et al., 2007; Yrjanheikki et al., 1998) to prevent or amelioratecognitive deficits associatedwith DBS (Cai et al., 2008; Liu et al., 2007).Further experiments in animal models and patients with DBS areneeded to validate and translate these hypotheses.

Acknowledgments

We would like to thank Dr. Spiegelman from the Department ofNeurosurgery in Sheba Medical Center for helpful discussions.Supported in part by NIH RO1 NS050285 to Anat Biegon.

49Y.(K.) Hirshler et al. / Experimental Neurology 222 (2010) 42–50

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