Vagus nerve stimulation during rehabilitative training improves functional recovery after intracerebral hemorrhage

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Neurobiology of Disease xxx (2013) xxx–xxx

YNBDI-03019; No. of pages: 10; 4C:

Contents lists available at SciVerse ScienceDirect

Neurobiology of Disease

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Vagus nerve stimulation during rehabilitative training improves forelimbstrength following ischemic stroke

FN. Khodaparast ⁎, S.A. Hays, A.M. Sloan, D.R. Hulsey, A. Ruiz, M. Pantoja, R.L. Rennaker II, M.P. KilgardThe University of Texas at Dallas, School of Behavioral Brain Sciences, 800 West Campbell Road, GR41, Richardson, TX 75080-3021, USA
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⁎ Corresponding author. Fax: +1 972 883 2491.E-mail address: [email protected] (N. KhAvailable online on ScienceDirect (www.sciencedire

0969-9961/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.nbd.2013.08.002

Please cite this article as: Khodaparast, N., eischemic stroke, Neurobiol. Dis. (2013), http

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Article history:Received 3 July 2013Revised 31 July 2013Accepted 7 August 2013Available online xxxx

Keywords:Vagus nerve stimulationPlasticityNeurorehabilitationMotor cortexStrokeIschemia

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RUpper limb impairment is a common debilitating consequence of ischemic stroke. Physical rehabilitation afterstroke enhances neuroplasticity and improves limb function, but does not typically restore normal movement.We have recently developed a novel method that uses vagus nerve stimulation (VNS) paired with forelimbmovements to drive specific, long-lasting map plasticity in rat primary motor cortex. Here we report that VNSpaired with rehabilitative training can enhance recovery of forelimb force generation following infarction ofprimary motor cortex in rats. Quantitative measures of forelimb function returned to pre-lesion levels whenVNS was delivered during rehab training. Intensive rehab training without VNS failed to restore function backto pre-lesion levels. Animals that received VNS during rehab improved twice as much as rats that received thesame rehabilitation without VNS. VNS delivered during physical rehabilitation represents a novel method thatmay provide long-lasting benefits towards stroke recovery.

© 2013 Elsevier Inc. All rights reserved.

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UNCO

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Stroke is the second most common cause of disability worldwide(Leary and Saver, 2003). Ischemic stroke causes neural death dueto inadequate blood flow, often resulting in movement impairmentson the opposite side of the body (Deb et al., 2010; Lo et al., 2003).Seventy-five percent of patients who survive an ischemic stroke contin-ue to have significant weakness in the upper extremities even after ex-tensive rehabilitative therapy (Harvey and Nudo, 2007; Kwakkel, 2009;Levine and Greenwald, 2009). Impaired limb function reduces the abilityto perform activities of daily living, reduces the quality of life, and in-creasesmedical costs (King, 1996;Whyte et al., 2004). The developmentof an effective therapy to restore motor function would fulfill a largeunmet clinical need.

Physical rehabilitation after stroke drives plasticity in the form ofreorganization of cortical circuitry in the motor system (Johansson,2000; Nudo, 2003; Rossini and Forno, 2004; Schaechter, 2004; Wardand Cohen, 2004). One common rehabilitative intervention, constraintinduced movement therapy (CIMT) causes reorganization of themotor cortex map of arm movement (Sawaki et al., 2008; Schaechteret al., 2002). Additionally, newmethods using virtual reality and electri-cal stimulation of motor cortex may also promote increased synapticplasticity and cortical reorganization within the motor cortex (Adkins-Muir and Jones, 2003; Lindenberg et al., 2012; You et al., 2005).The development of additional methods to increase neural plasticity

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t al., Vagus nerve stimulation://dx.doi.org/10.1016/j.nbd.20

may lead to improved recovery of motor function (Hallett, 2001;Nudo, 2003). We have recently developed a method to induce specificand long-lasting cortical map plasticity by pairing vagus nerve stimula-tion (VNS) with movements or sensory stimuli in intact rats (Engineeret al., 2011; Porter et al., 2011). Repeatedly delivering VNS with fore-limb movements resulted in movement-specific map plasticity withinthe primary motor cortex beyond training without VNS (Porter et al.,2011).We hypothesized that this enhancement in reorganization with-in the motor cortex may improve recovery of function after stroke.

Upper limb strength is one of the best prognostic indicators for armfunction and chronic disability following stroke (Harris and Eng, 2007;Mercier and Bourbonnais, 2004; Sunderland et al., 1989). Here, weevaluated whether the delivery of VNS during rehabilitative trainingcan enhance recovery of forelimb strength in amodel of ischemic stroke.Rats were trained to perform an isometric force task that quantitativelymeasures forelimb force generation (Hays et al., 2012). This task is fullyautomated, allowing the experimenter to test several animals simulta-neously and avoid the possibility of experimenter bias. Unilateralinjections of a peptide vasoconstrictor, endothelin-1, into primarymotor cortex caused an ischemic infarct and impaired function of thetrained forelimb (Fang et al., 2010; Gilmour et al., 2004; Hays et al.,2012). Rats underwent rehabilitative training for five weeks withor without the delivery of VNS. No VNS was delivered on week six toallow evaluation of persistent effects. VNS delivered during rehabilita-tive training restored pull force generation back to pre-lesion levels,whereas extensive rehabilitative training without VNS failed to restorefunction. These findings suggest that VNS paired with physical rehabil-itation may hold promise for enhancing recovery of upper extremityfunction after stroke.

during rehabilitative training improves forelimb strength following13.08.002

http://dx.doi.org/10.1016/j.nbd.2013.08.002

mailto:[email protected]


http://www.sciencedirect.com/science/journal/09699961


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2 N. Khodaparast et al. / Neurobiology of Disease xxx (2013) xxx–xxx

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Materials and methods

Subjects

Nineteen adult female Sprague–Dawley rats, approximately 4 monthsold andweighing approximately 250 gwhen the experiment began,wereused in this experiment. The rats were housed in a 12:12 h reversedlight cycle environment so that behavioral testing took place during thedark cycle in order to increase daytime activity levels. Rats were fooddeprived to no less than 85% of their normal body weight during trainingasmotivation for the food pellet rewards. This studywas designed to takeinto consideration the rapid hormonal cycle of female rats. To ensure thatthedata for each ratwas collectedduring every stage of the estrus cycle allanalyses were based on the average of a week's worth of behavioral data.All handling, housing, surgical procedures, and behavioral training ofthe rats were approved by the University of Texas at Dallas InstitutionalAnimal Care and Use Committee.

Behavioral apparatus and software

The behavioral chamber consisted of an acrylic box(10 × 12 × 4.75 in.) with a slot (2.5 × 0.4 in.) located in the frontright corner of the box through which the rats could access thepull handle. The slot location restricted access such that only theright forelimb could be used to perform the task. The aluminumpull handle was centered in the slot at a height of 2.5 in. fromthe cage floor and at lateral distances varying from 0.75 in. insideto 0.75 in. outside relative to the inner wall surface of the cage,depending on the training stage. The handle was affixed to a customdesigned force transducer (Motor Pull Device, Vulintus LLC, Sachse,TX) located outside the cage. The maximum load capacity of thetransducer was 2 kg, and the typical forces generated by the ratsfell within the linear range of measurement. Forces readings weresampled at 20Hz and measured with ±1 g accuracy. Force measure-ments were calibrated with a force meter at least once per week.

Custom software was used to control the task and collect data. Amotor controller board (Motor Controller, Vulintus LLC, Sachse, TX)sampled the force transducer every 50 ms and relayed information toa custom MATLAB software which analyzed, displayed, and stored thedata. Force values and corresponding timestamps were collected ascontinuous traces for each trial to allow for the analysis of force profilesover the course of a session. If a trial was successful, the software trig-gered an automated pellet dispenser (Vulintus LLC, Sachse, TX) to delivera sucrose pellet (45 mg dustless precision pellet, BioServ, Frenchtown,NJ) to a receptacle located in the front left corner of the cage.

Isometric force task training

The isometric force task was performed as previously described.Training sessions lasted 30 min and were conducted twice daily, fivedays a week, with sessions on the same day separated by at least 2 h.During early phases of training, experimentersmanually shaped animalsby using ground sucrose pellets to encourage interaction with the han-dle. Rats pulled the handle initially located 0.75 in. inside the trainingcage to receive a sucrose reward pellet. A trial was initiated when therat generated a force of at least 10 g on the handle. After trial initiation,the force was sampled for 4 s. If the force threshold was broken withina 2 second window following the initial contact, the trial was recordedas a success and a reward pellet was delivered. If the force did not exceedthresholdwithin the 2 secondwindow, the trialwas recorded as a failureandno rewardwas given. Hit ratewas calculated based on the number ofsuccessful trials over the total number of initiated trials: Hit rate =[(total successful trials / total trials) ∗ 100]. Force on the pull handlewas sampled for 2 additional seconds following the 2 second trialwindow, regardless of the trial outcome, to capture any late attemptswhich were unrewarded. Following the 4 s of data collection there was

Please cite this article as: Khodaparast, N., et al., Vagus nerve stimulationischemic stroke, Neurobiol. Dis. (2013), http://dx.doi.org/10.1016/j.nbd.20

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a 50 millisecond pause before rats could initiate another trial. If rats didnot receive 50 pellets in a single day, they were given 10 g of pelletsafter daily training sessions were complete. The task was made progres-sively more difficult as rats met the criterion for number of successfultrials within a session and progressed to the next stage. As the trainingstages increased, the handle was gradually retracted to 0.75 in. outsidethe cage and the force threshold progressively increased up to 120 g.If an animal exceeded criteria for a proceeding stage, they were auto-matically advanced to the stage that matched their performance. Theprescribed position and threshold values were strictly adhered to forpre- and post-lesion measurements.

Rats were held at the pre-lesion stage until they had 10 successivesessions averaging over 85% success rate. The pre-lesion data reportedin this study is compiled from these 10 sessions. After this point, therats were given an ischemic lesion followed by seven days of recovery,after which they returned for post-lesion behavioral testing withthe same parameters as pre-lesion allowing for a direct comparison ofperformance. All rats were tested until they had 4 sessions with greaterthan 10 trials each during the post-lesion assessment. Rats then pro-ceeded to the therapy stage where VNS was delivered on each success-ful trial for 25 days (Fig. S1). Following the therapy stage, all ratsunderwent an additional two days (week 6) of rehabilitative trainingonly, to allow assessment of the persistent effects of VNS pairing.

D PUnilateral motor cortex ischemic lesion

Unilateral ischemic lesions of primarymotor cortexwere performedsimilar to a previously described method (Fang et al., 2010; Gilmouret al., 2004; Hays et al., 2012, 2013). See Supplementary Methodssection for details.

Vagus nerve cuff implantation

Following ischemic lesion, all rats were implanted with a skull-mounted two-channel connector (headcap) and a bipolar stimulatingnerve cuff constructedwith platinum-iridium leads (5–6 kΩ impedance).Implantations were performed as previously described (Engineer et al.,2011; Porter et al., 2011). See Supplementary Methods sections fordetails.

Application of VNS

Behavioral training was identical for all rats. The VNS during Rehabgroup received approximately 9000 total stimulations over 25 days(i.e., fifty 30 min sessions). VNS was delivered within 50 ms of a suc-cessful pull attempt. VNS was delivered as a 500 ms train of 15 pulsesat 30 Hz (Fig. S1). Each biphasic pulse was 0.8 mA in amplitude and100 μs in phase duration. These parameters are identical to our earlierstudies (Engineer et al., 2011; Porter et al., 2011). Previous studiesusing the same parameters employed in this study have demonstratedchanges in electroencephalographic measures and neuronal spikingsynchrony during VNS, indicating that the nerve is successfully stimu-lated (Engineer et al., 2011; Nichols et al., 2011). No stimulationwas delivered during the post-lesion assessment stage. During thefirst day of post-lesion assessment, no rats had a stimulator cable con-nected to the headcap. For both the Rehab rats and the VNS + Rehabrats, the stimulator cable was first connected during the second day ofpost-lesion assessment and was connected every day until the end ofthe fifth week of therapy. The stimulation cable for the Rehab rats wasnot connected to a stimulator. During the sixth week, the stimulatorcable was not utilized for either group. Rats were perfused and brainsremoved following the sixth week of training to quantify lesion size(see Supplementary Methods for details).



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3N. Khodaparast et al. / Neurobiology of Disease xxx (2013) xxx–xxx

Statistics

All data are reported as the mean ± SEM. All comparisons wereplanned in the experimental design a priori, and significant differenceswere determined using one-way ANOVA, two-way ANOVA, and t-testswhere appropriate. Statistical tests for each comparison are noted inthe text. One tailed t-tests comparing individual subject performanceafter therapy (week 6) to baseline performance (PRE) were used todetermine which rats exhibited a significant impairment after therapy.All other t-tests were two-tailed. Paired t-tests were used to comparerepeated measures over time within groups. Alpha level was set at0.05 for all comparisons. Significant differences between the Rehaband VNS + Rehab groups are noted in the figures with an asterisk.See Table S1 for statistical values for t-test comparisons.

Results

Rats acquire skilled performance of the isometric force task

To assess forelimb function in the context of stroke, ratswere trainedto perform the isometric force task, a behavioral test that quantitativelyassesses multiple parameters of forelimb function (Hays et al., 2012).The task requires rats to reach out and grasp a handle attached to a

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Fig. 1. Acquisition of skilled performance on the isometric force task. (A) Depiction of a rat perfgrasp and pull. Frames are separated by 150 ms. Previously shown in Hays et al. (2012). (B) Exa120 g hit threshold. Open arrowheadsmark separate pulls. The black horizontal dashed linemapellet andVNS,when appropriate,were delivered. (C) Example data from a single unsuccessful treward or VNS was delivered. (D) Distribution of peak forces in newly trained and highly trainetrained rats generate a wide range of forces. Maximal peak forces in newly trained rats are cperformance is not due to muscle strengthening, but rather refinement of task performance.


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force transducer and apply 120 g of force to receive a food reward(Fig. 1). Rats became highly proficient at the task in 10.1 ± 0.6 days.Early in training, rats were able to generate forces up to 400 g. Highlytrained rats did not generate higher forces (Fig. 1D). The majority oftrials in highly trained were over 120 g. Early in training, pull forcewas often less than 80 g and varied substantially from trial to trial. Asignificant decrease in the variance was observed in highly trained ratscompared to newly trained rats (F test for equal variance, P b 0.001).This demonstrates that the increase in task performance with trainingis unlikely to be due to strengthening of forelimb muscles, but ratheris due to the acquisition of skilled forelimb use. Daily observations didnot reveal any obvious differences in the reach or grasp strategy usedto perform the pull task.

Unilateral ischemic lesion impairs task performance

Prior to the induction of ischemic damage, subjects were held untilthey achieved a pre-lesion baseline of five consecutive days exceeding85%hit rate performance. Performanceduring thebaseline did not differsignificantly across days (Day 1: 84.6 ± 2.1%, Day 5: 87.2 ± 1.6%, n =15, P = 0.36, paired t-test). Single trial examples matched to the bot-tom quartile of force show that pull force exceeded the 120 g thresholdon the vast majority of trials (Figs. 2A,B, left panel). Both groups were

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orming the task. The sequential images show the extension of the forelimb, followed by ample of force data collected from a single successful trial. The gray dashed line denotes therks themaximal force of the trial. The arrowmarks the threshold crossing, when a rewardrial. Symbols are the same as in B. Note that the force never exceeds the hit threshold, so nod rats. Highly trained rats tend to generate peak forces centered about 120 g, while newlyomparable to those observed in highly trained rats, suggesting that the improvement in



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Fig. 2. VNS paired with Rehab improves hit rate after ischemic lesion. (A, B) Single trial force profiles matched to the bottom quartile of force for each experimental group throughout thecourse of the experiment. The gray dashed line indicates the 120 g hit threshold. (C) Hit rate performance over the course of the experiment. VNS paired with Rehab improves recoverycompared to Rehab on most weeks. The increase in hit rate is still present at week 6, after the cessation of VNS therapy. N refers to number of rats in each group. * indicates significantdifference between Rehab and VNS + Rehab. (D) Correlation of individual subject performance prior to lesion and after the completion of therapy. Empty symbols denote a significantreduction after therapy compared to pre-lesion performance. Symbols on or above the line suggest recovery, while those below the line indicate impairment. Note the consistent recoveryin the VNS + Rehab group and the wide variability in recovery in the Rehab group.


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between groups (Fig. 2C, PRE, Rehab: 87.0 ± 0.7%; VNS + Rehab:87.7 ± 1.5%, n = 9,6, unpaired t-test, P = 0.63, also see Movie S1).

Induction of unilateral ischemic damage significantly worsenedperformance in both groups compared to pre-lesion (Fig. 2C, POST,Rehab: 37.8 ± 4.1%, paired t-test, P b 0.001; VNS + Rehab: 38.8 ± 7.5%,P b 0.001, also see Movie S2). No difference was observed betweengroups (unpaired t-test, P = 0.89). Single trial examples matched tothe bottom quartile of force after lesion illustrate the reduction in peakforce and increase in number of pulls per trial (Figs. 2A,B, center panel).

Physical rehabilitation is the most common intervention torestore motor function after stroke (Piernik-Yoder, 2013), so wesought to evaluate the effectiveness of rehabilitative training withoutVNS to improve motor outcomes. ANOVA of hit rate in this grouprevealed a significant effect (F[6,56] = 2.64, P = 0.025). Rehabilitativetrainingwithout VNS (Rehab) resulted in amodest recovery of forelimbfunction, but was unable to return performance to pre-lesion levels.Average hit ratewas significantly reduced compared to pre-lesion levelsthroughout the course of therapy (Fig. 2C, PRE vs. weeks 1–6, allP b 0.01, also see Table S1 and Movie S3). By week 6, the impairmentof hit rate had recovered 47.5 ± 15.4%. Performance of individual ratsvaried widely after therapy, ranging from a substantial impairment tofull recovery (Fig. 2D, also see Fig. S2A). 6 of 9 rats (67%) were signifi-cantly worse compared to pre-lesion performance after the therapy,suggesting that rehabilitative training without VNS is typically insuffi-cient to restore forelimb performance to pre-lesion levels.

We sought to evaluate if the addition of VNS pairedwith rehabilitativetraining enhanced recovery of forelimb function. ANOVA of hit rate


revealed a significant effect (F[6,35] = 11.83, P b 0.001). VNS pairedwith rehabilitative training (VNS + Rehab) fully restored forelimb per-formance to pre-lesion levels. No significant difference from pre-lesionwas observed between weeks 2 and 6 (all P N 0.05). The benefits ofVNS paired with physical rehabilitation were evident during weeksix after the cessation of VNS, suggesting a long-lasting benefit (week5 vs. week 6, within, P = 0.89). After the completion of therapy, hitrate was indistinguishable from pre-lesion performance (99.2 ± 9.6%recovery, also see Movie S4). Only 1 of 6 rats (17%) demonstrated astatistically significant impairment compared to pre-lesion performanceafter the therapy, suggesting that VNS paired with physical rehabilitationimproves recovery of forelimb function.

To determine if VNS paired with rehabilitative training confersan advantage beyond rehabilitative trainingwithout VNS, we comparedperformance across groups at each week of therapy. ANOVA of hit raterevealed a significant effect of treatment (F[1,83] = 33.21, P b 0.001)and time (F[5,83] = 3.14, P = 0.012). Post hoc comparisons demon-strated that VNS + Rehab displayed an increased hit rate comparedto Rehab on most weeks (Fig. 2C). The increase in hit rate persistedthroughout the remainder of therapy (unpaired t-test, P b 0.05 forweeks 2, 3, 5, 6). These results demonstrate that VNS paired with reha-bilitative training results in a significant increase in recovery comparedto rehabilitative training without VNS.

Unilateral ischemic lesion reduces forelimb strength

In addition to assessing hit rate performance, we sought to evaluateforelimb strength. Before ischemic lesion, peak force generated by the



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forelimbwas similar in both groups. On themajority of trials, peak forceexceeded 120 g (Figs. 3A,B, left panel). Averaged pre-lesion peak forcewas slightly, but significantly, higher in the VNS + Rehab group(Fig. 3C, PRE, Rehab: 144.1 ± 2.0 g; VNS + Rehab: 152.0 ± 2.2 g,unpaired t-test, P = 0.017). After ischemic lesion, peak force gener-ation was significantly reduced in both groups compared to pre-lesion (Fig. 3C, POST, Rehab: 103.6 ± 4.2 g, paired t-test, P b 0.001;VNS + Rehab: 99.0 ± 7.9 g, P b 0.001). No difference in peak forcewas observed between groups (unpaired t-test, P = 0.57). The distribu-tion of peak forces demonstrated a notable leftward shift with sig-nificantly fewer trials with peak forces above the 120 g threshold(Figs. 3A,B, center panel).

Rehabilitative training without VNS was insufficient to fully restoreforelimb strength. The distribution of peak forces after therapy revealssubstantial deficiencies compared to pre-lesion (Fig. 3B). Significantincreases are observed in bins 80–120 g (paired t-test, P b 0.05 foreach bin) and significant decreases are observed in bin 140–160 g(P b 0.05) after therapy. This increase in low force pulls and decreasein high force pulls is consistent with a deficit in forelimb strength.ANOVA on peak force revealed a significant effect of therapy for theRehab group (F[6,56] = 2.98, P = 0.014). Rehab resulted in a smallbut significant improvement in peak force by week 2 (Fig. 3C, POST v.

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Fig. 3.VNS pairedwith Rehab improves recovery of forelimb strength. (A, B) Peak force distributhe 120 g threshold. The numerical value indicates the cumulative percentage (± SE) of triaVNS pairedwith rehabilitative training significantly improvesmaximal force compared to rehabat week 6 after the cessation of VNS. N refers to number of rats in each group. * indicates signmaximal force prior to lesion and after the completion of therapy. Empty symbols denote(6 of 6) in the VNS + Rehab group demonstrate complete recovery, while only 2 of 9 subjects


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week 2, paired t-test, P b 0.05). However, peak force remained sig-nificantly reduced compared to pre-lesion levels throughout the courseof therapy (Fig. 3C, PRE vs. weeks 1–6, paired t-test, all P b 0.05). Onweek 6, peak force had recovered 56.8 ± 16.6% of the deficit relativeto pre-lesion levels. 7 of 9 rats (78%) demonstrated significant impair-ment of force generation after the completion of therapy (Fig. 3D, alsosee Fig. S2B).

VNS paired with physical rehabilitation resulted in notable recoveryof forelimb strength. After five weeks of therapy, the distribution ofpeak forces is highly similar to that observed pre-lesion, with no differ-ences observed between any bins (Fig. 3A, paired t-test, all P N 0.15).This indicates a complete restoration of forelimb strength. ANOVAon peak force revealed a significant effect of therapy for the VNSgroup (F[6,35] = 8.88, P b 0.001). Examination of group averagesover the course of therapy demonstrates that peak force increasedsignificantly compared to the post-lesion baseline during the firstweek of therapy (Fig. 3C, POST vs. week 1, paired t-test, P b 0.05).No significant difference from pre-lesion was observed between weeks1 and 6 (all P N 0.05). At the completion of therapy, peak force had recov-ered 104.3 ± 15.3% relative to the deficit andwas indistinguishable frompre-lesion levels. The restoration of peak force remained after the cessa-tion of VNS therapy at week 6, indicating that the recovery of forelimb

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tions for both groups at each time point. The gray dashed box indicates trials which exceedls exceeding the 120 g threshold. (C) Maximal force over the course of the experiment.ilitative training alone by the secondweek of therapy. The increase in force is still presentificant difference between Rehab and VNS + Rehab. (D) Correlation of individual subjecta significant reduction after therapy compared to pre-lesion performance. All subjectsin the Rehab group recover.



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function was long-lasting. None of the subjects (0 of 6) that receivedVNS + Rehab demonstrated an impairment of force generation atthe completion of therapy (Fig. 3D). These findings demonstrate thatVNS + Rehab fully restores forelimb force generation.

Consistent with the recovery observed in single subjects, averagepeak force in the VNS + Rehab group is significantly greater than theRehab group (Fig. 3C). ANOVA of peak force revealed a significant effectof treatment (F[1,83] = 47.92, P b 0.001) and time (F[5,83] = 3.74,P = 0.004). By week 2, maximal force was significantly higher inthe VNS + Rehab group. The improvement in maximal force was evi-dent throughout the remainder of therapy (unpaired t-test, P b 0.05forweeks 2–6). These results demonstrate that VNS pairedwith rehabil-itative training results in a significant increase in forelimb strength com-pared to rehabilitative training without VNS.

Intensity of training cannot account for the differences in recovery

Insufficient training intensity or motivation can limit the gains fromrehabilitation (Kwakkel et al., 1999; Sivenius et al., 1985). To confirmthat the Rehab group did not perform less intensive training, we com-pared the total number of pull attempts performed over the course oftherapy. The total number of pulls during therapy is significantly higherin the Rehab group compared to the VNS + Rehab group (Fig. 4A,Rehab: 67,653 ± 7379 total attempts, VNS + Rehab: 47,656 ± 4535total attempts, unpaired t-test, P = 0.050). Because the Rehab groupperforms more repetitions but displays worse functional outcomes,the intensity of the training cannot account for the difference betweenthe groups. These findings highlight the marked benefit of VNS pairedwith rehabilitative training beyond rehabilitative training without VNS.

Lesion size cannot account for the differences in recovery

We sought to determine if our stimulation parameters would conferneuroprotective effects that could reduce lesion size, and if any changescould account for differences in functional recovery. Lesion size spannedthe left caudal forelimb area through all layers of cortex (Fig. 4B, also seeFig. S5). The resulting infarct was primarily restricted to cortex, butminor white matter damage was observed in one subject in the Rehabgroup and one subject in the VNS + Rehab group. There was no differ-ence in lesion volumeobserved between groups (Fig. 4C, VNS + Rehab:9.64 ± 2.55 mm3, Rehab: 10.03 ± 2.41 mm3, n = 6,8, unpaired t-test,P = 0.78, also see Fig. S3). These findings demonstrate that the stimula-tion parameters used in this study did not confer any observableneuroprotective effects on lesion size (see Supplementary materials).

Discussion

This study tested whether delivering VNS during rehabilitativetraining could improve recovery of forelimb motor function followingcortical ischemic damage compared to rehabilitative training alone.Forelimb function was assessed using the automated isometric pull taskwith approximately 50,000 pull attempts collected per rat, resulting inunbiased data collection and high statistical power (Hays et al., 2012).Rats received rehabilitative training on an isometric force task (Hayset al., 2012) for five weeks with or without the delivery of VNS. Weeksof daily intensive rehabilitative training without VNS failed to restorepre-lesion function. Forelimb function recovered completely when briefbursts of VNS were delivered during rehabilitative training. VNS pairedwith rehabilitative training doubled recovery of hit rate performanceand forelimb strength compared to rehabilitative training without VNS.VNS did not alter the size of the lesion or increase the intensity of rehabil-itative training. The enhanced recovery facilitated by the delivery of VNSduring rehabilitative training may present an opportunity for reducingmotor impairments in stroke patients.

Stroke often results in deficits of skilled movement which persist inspite of extensive rehabilitation (Segura et al., 2006; Van Peppen et al.,


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2004). Rehabilitative training is focused on improving motor functionafter stroke, which is thought to be supported by reorganization of themotor cortex (Hallett, 2001; Kleim, 2011; Nudo, 2003). Rehabilitation-induced cortical plasticity is associated with the degree of recovery inanimal models (Castro-Alamancos and Borrell, 1995; Dijkhuizen et al.,2001; Frost et al., 2003; Ramanathan et al., 2006) and in stroke patients(Calautti and Baron, 2003; Lindenberg et al., 2012). A variety of factorsthat limit neural plasticity reduce recovery following brain damage(Boyeson et al., 1992; Conner et al., 2005; Goldstein et al., 1991;McHughen et al., 2010; Siironen et al., 2007; Sweetnam et al., 2012).Because of the association of plasticity and recovery, it was reasonableto expect that enhancement of plasticity would lead to gains in func-tional recovery after stroke. Vagus nerve stimulation paired withmotor training in unlesioned animals induced robust plasticity in themotor cortex, while similar amounts of motor training without VNSdid not drive observable plasticity (Porter et al., 2011). The improve-ment of recovery observed in this study in subjects that receivedVNS during rehabilitative training is likely due to the VNS-dependentenhancement of plasticity within motor cortex. However, the cellularand molecular mechanisms that underlie VNS-dependent recoveryremain unclear.

Stimulation of the vagus nerve engages multiple neuromodulatorysystems and results in the release of acetylcholine, norepinephrine,and brain-derived neurotrophic factor (Dorr and Debonnel, 2006;Follesa et al., 2007; Groves and Brown, 2005; Hassert et al., 2004;Nichols et al., 2011; Roosevelt et al., 2006). Individually, each of theseneuromodulators is known to enhance cortical plasticity and facilitaterecovery after brain damage (Boyeson et al., 1992; Conner et al., 2005;Goldstein et al., 1991; Ramanathan et al., 2009; Schäbitz et al., 2004,2007). There is considerable evidence that these neuromodulators,particularly acetylcholine and norepinephrine, operate synergisticallyto promote plasticity (Bear and Singer, 1986; Salgado et al., 2012; Seolet al., 2007).

The ability of vagus nerve stimulation to engage these neuro-modulatory systems arises from its unique anatomy. Eighty percent ofthe vagus nerve is comprised of afferent sensory fibers that projectinto the medulla (Foley and DuBois, 1937; George et al., 2000). Thesefibers synapse bilaterally on neurons within the nucleus of the tractussolitarius, which then project to the noradrenergic locus coeruleus(LC) and the cholinergic basal forebrain (BF) (Berntson et al., 1998;George et al., 2000; Henry, 2002; Semba et al., 1988). Stimulationof the vagus nerve drives activity within both the LC and BF regionsand consequently induces release of acetylcholine and norepinephrinethroughout the cortex (Follesa et al., 2007; Nichols et al., 2011;Roosevelt et al., 2006). Both of these regions are required for the effectsof VNS in the central nervous system (Krahl et al., 1998; Nichols et al.,2011). It is not yet knownwhether the release of these neuromodulatorsis required for the robust enhancement of recovery driven by VNS.

Our results provides a proof of concept demonstration that VNSduring rehabilitative training holds promise for improving recoveryof motor function after stroke. However, translating pre-clinical strokeresearch into effective therapies for patient has proven to be difficult(Lyden and Lapchak, 2012; O'Collins et al., 2006). Many therapies re-quire delivery soon after the onset of ischemic damage, either to inhibitneuronal death or to bolster the innate transient increase in plasticityafter damage (Adams et al., 1994; Savitz, 2007). As a result, many strat-egies are less effective once chronic deficits are in place. The discrepancybetween the timing of delivery of a treatment in animal studies andhuman trials is thought to be a contributing factor the failure of manypromising preclinical therapies (Cheng et al., 2004; Gladstone et al.,2002; Kahle and Bix, 2012). In this study, VNS paired with rehabilitativetraining was effective when initiated nine days after the stroke. Theability of VNS to confer a beneficial outcome when delivered at thistime scale is an improvement over interventions that must be deliveredshortly after (i.e., typically within six hours of) stroke to be effective (Ayet al., 2009; Hiraki et al., 2012; Yenari and Hemmen, 2010; Zivin, 1998).



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Fig. 4.VNS does not increase intensity of rehabilitative training or decrease lesion size. (A) Rehab rats perform slightly, but significantly, more pull attempts over the course of therapy thatVNS + Rehab rats. (B) Reconstructions demonstrating the extent of the smallest, representative, and largest lesions. Ischemic damage is primarily confined to the forelimb area. Numbersrefer to mm from bregma. (C) No difference in lesion size was observed between the Rehab and VNS + Rehab groups.


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may not be ineffective when delivered long after stroke. Laterdelivery of rehabilitation is associated with worse functional out-comes in animal models and patients (Biernaskie et al., 2004; Cifuand Stewart, 1999), potentially because the rehabilitation occursafter the transient upregulation of plasticity and growth-promoting fac-tors induced by brain damage (Carmichael et al., 2005; Murphy andCorbett, 2009; Wieloch and Nikolich, 2006).

In spite of this, two factors suggest that a delay of weeks or monthsmight not occlude the beneficial effects of VNS pairedwith rehabilitativetraining. First, VNS paired with physical training can generate motorcortical plasticity independent of brain damage (Porter et al., 2011).This suggests that VNS-dependent plasticity does not rely on the tran-sient period of enhanced growth and plasticity after injury; thereforeVNS may be successful when delivered later. Second, VNS drives aprecisely-timed release of neuromodulators that are normally increasedduring natural motor learning (Izaki et al., 1998; Orsetti et al., 1996).It has been predicted that the ability to achieve supranormal levels ofthese neuromodulators during rehabilitative trainingmay improve func-tional gains (Nadeau et al., 2004). Pharmacological interventions thatalter the levels of acetylcholine and norepinephrine during rehabilitativetraining improve motor outcomes in some studies (Adkins and Jones,2005; Gilmour et al., 2005; Kessler et al., 2000; Nadeau et al., 2004;Walker-Batson et al., 1995). If VNS improves rehabilitation by triggeringa consistent trial-by-trial burst of neuromodulators, then it is reasonableto expect that VNS might continue to improve rehabilitation that beginslong after stroke onset. However, since the current experiments do nottest this possibility, the potential utility of VNS during the chronic stageis speculative and needs to be tested.


There is considerable preclinical and clinical evidence that VNScould be safely delivered in stroke patients. VNS has been used totreat of a wide range of conditions, including refractory epilepsy (Ben-Menachem, 2002; Morris and Mueller, 1999), treatment-resistant de-pression (Rush et al., 2005; Sackeim et al., 2001), Alzheimer's disease(Sjogren et al., 2002), fibromyalgia (Lange et al., 2011), and bipolardisorder (Marangell et al., 2008). Over 60,000 patients have receivedVNS over the past twenty-five years (Englot et al., 2011). The clinicallyapproved therapy, which is typically delivers 100 times more dailycurrent than our therapy, is well-tolerated and usually continues formany years (Morris andMueller, 1999). Fewpatients report side effects,the most common of which are cough and hoarseness (Sackeim et al.,2001). Even at these high currents, no significant changes in heart rateor oxygen saturation are observed (Binks et al., 2001; Handforth et al.,1998). Additional studies will be needed to determinewhether deliveryof VNS during physical therapy would be safe in stroke patients.

This is the first study to show that VNS paired with rehabilitativetraining promotes recovery of strength in a model of stroke. However,the ability for VNS to enhance event-specific plasticity applies inother brain regions and may have therapeutic implications for otherdiseases (Kilgard, 2012; Lozano, 2011). VNS paired with tones drivestone-specific plasticity within auditory cortex (Engineer et al., 2011).A therapy based on specifically targeting plasticity employed VNSpairing with tones and successfully reversed pathological plasticityand eliminated the behavioral correlate of tinnitus in a rat model(Engineer et al., 2011). This same therapy is now undergoing clinicaltrials in chronic tinnitus patients, with promising preliminary results(Arns and De Ridder, 2011). The benefits of therapy for tinnitus appearto be long-lasting and seem to be blocked bymedications that interfere



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with acetylcholine and norepinephrine, providing further mechanisticsupport of a neuromodulatory basis of VNS-directed plasticity. In addi-tion to tinnitus and stroke, targeted plasticity represents a potential toolfor other neurological disorders, including aphasia, apraxia, dystonia,and pain (Lozano, 2011).

Conclusion/implications

This study provides a proof of concept demonstration that stimula-tion of the vagus nerve paired with rehabilitative training can improverecovery of forelimb function in a rat model of stroke. VNS deliveredduring rehabilitative training fully restored forelimb force generationto pre-lesion levels. A similar amount of rehabilitative training withoutVNS was insufficient to restore performance. These results suggestthat VNS paired with physical rehabilitation is a potentially viable newtherapy for enhancing recovery of motor function after stroke.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nbd.2013.08.002.

Funding

This work was supported by MicroTransponder, Inc.

Author contributions

N.K., S.A.H., andM.P.K. wrote themanuscript. N.K., M.P.K., R.L.R., andA.M.S. designed the study. N.K., S.A.H., D.R.H., A.R., and M.P. performedbehavioral testing. N.K., S.A.H., and A.M.S. analyzed the data. A.M.S.and R.L.R. provided software and hardware support. All authors discussedthe results and provided comments on the manuscript.

Uncited references

Atkinson and Santer, 1999Black-Schaffer and Winston, 2004Boyke et al., 2008Carey et al., 2002Dick et al., 1995Gallo, 2006Müller-Dahlhaus et al., 2008O'Donnell et al., 2010Pascual-Leone et al., 2011Paxinos and Watson, 2007Roger et al., 2012Sawaki et al., 2003Seidler, 2007Soltanpour and Santer, 1996

Acknowledgments

We would like to T. Fayyaz, N. Alam, F. Naqvi, D. Cao, R. Babu, R.Gattamaraju, V. Konduru, S. Burghul, and R. Joseph for help withbehavioral training.

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https://www.researchgate.net/publication/8513286_Schaechter_JDMotor_rehabilitation_and_brain_plasticity_after_hemiparetic_stroke_Prog_Neurobiol_7361-72?el=1_x_8&enrichId=rgreq-5f08299f-96f3-4ef1-81bd-0ddefb6d66d7&enrichSource=Y292ZXJQYWdlOzI1NTk3NDgyODtBUzoxMDI2NDU1NDUwNDYwMjJAMTQwMTQ4NDAxMzQzMQ==

https://www.researchgate.net/publication/8513286_Schaechter_JDMotor_rehabilitation_and_brain_plasticity_after_hemiparetic_stroke_Prog_Neurobiol_7361-72?el=1_x_8&enrichId=rgreq-5f08299f-96f3-4ef1-81bd-0ddefb6d66d7&enrichSource=Y292ZXJQYWdlOzI1NTk3NDgyODtBUzoxMDI2NDU1NDUwNDYwMjJAMTQwMTQ4NDAxMzQzMQ==

https://www.researchgate.net/publication/8215372_Neuronal_post-stroke_plasticity_in_the_adult?el=1_x_8&enrichId=rgreq-5f08299f-96f3-4ef1-81bd-0ddefb6d66d7&enrichSource=Y292ZXJQYWdlOzI1NTk3NDgyODtBUzoxMDI2NDU1NDUwNDYwMjJAMTQwMTQ4NDAxMzQzMQ==

https://www.researchgate.net/publication/8215372_Neuronal_post-stroke_plasticity_in_the_adult?el=1_x_8&enrichId=rgreq-5f08299f-96f3-4ef1-81bd-0ddefb6d66d7&enrichSource=Y292ZXJQYWdlOzI1NTk3NDgyODtBUzoxMDI2NDU1NDUwNDYwMjJAMTQwMTQ4NDAxMzQzMQ==

https://www.researchgate.net/publication/6234387_Seidler_RD_Older_adults_can_learn_to_learn_new_motor_skills_Behav?el=1_x_8&enrichId=rgreq-5f08299f-96f3-4ef1-81bd-0ddefb6d66d7&enrichSource=Y292ZXJQYWdlOzI1NTk3NDgyODtBUzoxMDI2NDU1NDUwNDYwMjJAMTQwMTQ4NDAxMzQzMQ==



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Vagus nerve stimulation during rehabilitative training improves functional recovery after intracerebral hemorrhage

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