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w w w . e l s e v i e r . c o m / l o c a t e / p a i n

Reactive oxygen species contribute to neuropathic pain and locomotordysfunction via activation of CamKII in remote segments followingspinal cord contusion injury in rats

0304-3959/$36.00 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.http://dx.doi.org/10.1016/j.pain.2013.05.018

⇑ Corresponding author. Address: Department of Neuroscience and Cell Biology,University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1043, USA. Tel.: +1 409 772 2939; fax: +1 409 772 3222.

E-mail address: cehulseb@utmb.edu (C.E. Hulsebosch).

Young S. Gwak, Shayne E. Hassler, Claire E. Hulsebosch ⇑Department of Neuroscience and Cell Biology, University of Texas Medical Branch at Galveston, TX, USA

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e i n f o

Article history:Received 4 February 2013Received in revised form 6 May 2013Accepted 9 May 2013

Keywords:Below-level central neuropathic painCamKIIHyperexcitabilityReactive oxygen speciesSpinal cord injury

a b s t r a c t

In this study, we examined whether blocking spinal cord injury (SCI)-induced increases in reactive oxy-gen species (ROS) by a ROS scavenger would attenuate below-level central neuropathic pain and promoterecovery of locomotion. Rats with T10 SCI developed mechanical allodynia in both hind paws and over-production of ROS, as assayed by Dhet intensity, in neurons in the lumbar 4/5 dorsal horn (⁄P < 0.05). Toscavenge ROS, phenyl-N-tert-butylnitrone (PBN, a ROS scavenger) was administered immediately afterSCI and for 7 consecutive days (early treatment) by either intrathecal (it; 1 and 3 mg) or systemic (ip;10, 50 and 100 mg) injections. In addition, the high doses of it (3 mg) or ip (100 mg) injections were per-formed at 35 days (delayed treatment) after SCI. High doses of PBN (ip, 100 mg, and it, 3 mg) significantlyattenuated mechanical allodynia in both hind paws at both early and delayed treatments, respectively(⁄P < 0.05). The abnormal hyperexcitability of wide dynamic range neurons after SCI was significantlyattenuated by both early and delayed PBN treatment (⁄P < 0.05). Early PBN treatment (100 mg, ip, and3 mg, it) attenuated overproduction of ROS in neurons in the lumbar 4/5 dorsal horn. In addition, itand ip t-BOOH (ROS donor) treatment dose-dependently produced mechanical allodynia in both hindpaws (⁄P < 0.05). Both SCI and t-BOOH treatment groups showed significantly increased phospho-CamKII(pCamKII) expression in neurons and KN-93 (an inhibitor of pCamKII) significantly attenuated mechan-ical allodynia (⁄P < 0.05). In addition, high doses of PBN significantly promoted the recovery of locomotion(⁄P < 0.05). In conclusion, the present data suggest that overproduction of ROS contribute to sensory andmotor abnormalities in remote segments below the lesion after thoracic SCI.

Published by Elsevier B.V. on behalf of International Association for the Study of Pain.

1. Introduction

Spinal cord injuries (SCI) produce direct and indirect spinal corddamage. Once the spinal cord is damaged, maladaptive intracellu-lar and extracellular biochemical signaling events contribute to en-hanced nociceptive transmission in the spinal dorsal horn viaoverexpression of receptors and ion channels [28,45,49], increasedrelease of neurotransmitters and proinflammatory cytokines[13,16], and activation of glial cells [24,25]. Subsequently, neuro-anatomical and neurochemical changes following SCI produce per-sistent hyperexcitable states in spinal dorsal horn neurons, oftencalled central sensitization, that result in persistent central neuro-

pathic pain [23]. However, the molecules that mediate the mal-adaptive nociceptive transmission in neuropathic pain after SCIare not well understood.

Reactive oxygen species (ROS) are particularly important mole-cules in signal transduction pathways in the spinal cord [33]. In thecentral nervous system, 2 groups of ROS are identified: (1) radicalgroups, which are superoxide anions, hydroxyl radicals and nitricoxides that contain at least 1 unpaired electron in the shell andcan exist independently; and (2) nonradical groups of ROS com-posed of hydrogen peroxides and peroxynitrites [37]. Radical andnonradical molecules are highly reactive and contribute signifi-cantly to cellular signaling pathways [20,22]. The intracellular lev-els of ROS are strictly controlled by the redox cycle of pro-oxidantsand anti-oxidants in the nervous system [19,53]. However, SCI re-sults in overproduction of ROS and in imbalance between the cells’natural production of oxidants and their normal ability to processthe oxidants into safer molecules. This is followed by structuraland physiological changes, such as lipid peroxidation, protein S-

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thiolation, and DNA damage via oxidation processes [2,44,54,57],which are collectively called oxidative damage.

In somatosensory spinal cord neurons, changes of protein/lipidarchitectural membrane structures and intracellular translationalprocessing are important to nociceptive transmission. For example,alterations can result in the development and the maintenance ofneuropathic pain, such as allodynia (non-noxious stimuli becomenoxious), hyperalgesia (noxious stimuli become enhanced), andneuronal hyperexcitability [11,12]. Recent studies report that re-moval of ROS by spin trap ROS scavengers in several pathophysio-logical conditions, such as peripheral neuropathy [34,36], visceralpain [62], capsaicin-induced hyperalgesia [55], and inflammation[32], significantly attenuated painlike behaviors. In addition, SCIstudies have suggested that prevention of ROS production contrib-uted to neuroprotection via reduction of tissue damage immedi-ately after injury [3,27]. Taken together, the overproduction ofROS must be an important contributor to sensory and motorabnormalities in several pathophysiological conditions. However,the role of ROS in the induction and maintenance of central neuro-pathic pain and recovery of locomotion following SCI is not fullyunderstood. More important, ROS-mediated intracellular signalingpathways following SCI are not clear.

Recently, we have suggested possible intracellular pathwaysthat contribute to SCI-induced neuropathic pain behaviors [10–12]. For example, SCI produces activation of calcium-calmodulin–dependent protein kinase II (CamKII) which contributes to neuro-pathic pain at the segmental level of injury. In addition, some stud-ies suggest that oxidative stress is one of the key factors in theactivation of CamKII in various pathophysiological conditions andorgans [15,56,61]. Consequently, in the present study, we exam-ined whether ROS modulate activation of CamKII and result in neu-ropathic painlike behaviors and recovery of locomotion in caudalregions remote from a thoracic spinal cord contusion injury in rats.Thus, we tested administration of a ROS scavenger with 2 routes ofadministration, intraperitoneal (ip) and intrathecal (it) followingSCI and attenuation of neuropathic painlike behavioral effects, dor-sal horn neuron hyperexcitability (substrate of mechanical allo-dynia), neuronal and glial production of ROS, and phospho-CamKII (pCamKII) upregulation. To test the specificity of the pro-posed pathway critically (SCI ? ROS ? pCamKII ? mechanismsof neuropathic pain), we tested inhibition of CamKII phosphoryla-tion and expression of pCamKII (activated by phosphorylation-p)on behavioral outcome and pCamKII expression in the presenceof a ROS donor. We report attenuation of mechanical allodynia,neuronal hyperexcitability, and ROS overproduction with improve-ment in locomotor recovery after administration of a ROS scaven-ger following SCI.

2. Materials and methods

2.1. Spinal cord injury

Male Sprague-Dawley (250 g) rats (N = 295) were obtainedfrom Harlan Sprague-Dawley (Houston, TX) and housed with a re-verse light/dark cycle of 12/12 h, and fed ad libitium. SCI at T10was performed using the Infinite Horizon Impactor machine (Pre-cision Systems, Des Plaines, IL) (150 kdyne with 1 s dwell time).Briefly, rats were anesthetized via ip injection of sodium pentobar-bital (60 mg/kg). Low thoracic laminectomy of the T8/9 vertebralsegment exposed the T10 spinal level with the aid of a surgicalmicroscope (KAPS International, Aalen, Germany). As a controlgroup, sham surgery (no spinal contusion) was performed withanesthesia, laminectomy and mounting on the Infinite HorizonImpactor frame with age-matched rats. Animal protocols were re-viewed by the University of Texas Medical Branch Institutional

Animal Care and Use Committee and were carried out in accor-dance with the National Institutes of Health Guide for the Careand Use of Laboratory Animals.

2.2. Experiment designs

Rats were randomly divided into 3 groups: (1) the SCI + PBN(ROS scavenger) group (n = 194); (2) the naive + t-BOOH (t-butylhydroperoxide) (ROS donor) group (n = 72); and (3) the SCI + p-CamKII inhibitor groups (n = 29), respectively. Behavior, electro-physiology and immunohistochemistry were performed toexamine the role of ROS for below-level neuropathic pain followingthoracic SCI. The experiment designs were as follows. First, wecompared mechanical allodynia, excitability of wide dynamicrange (WDR) dorsal horn neurons and production of ROS in thelumbar 4/5 dorsal horn between sham controls and the SCI groups.Second, we compared mechanical allodynia, WDR excitability andROS production by PBN treatment (both early and delayed treat-ment). Third, we compared expression of pCamKII in sham, SCI,SCI + ROS scavenger and naive + ROS donor groups, respectively,to examine the role of pCamKII in mechanical allodynia. Fourth,we compared the role of ROS in mechanical allodynia after treat-ment of ROS donor. Fifth, we compared the role of ROS on hind-limb locomotor function after SCI. To scavenge ROS, PBN (a potentspin trap free-radical scavenger) was administered ip (10, 50, and100 mg/kg/mL) and it (1 and 3 mg/kg/50 lL and 5 lL to flush outthe intrathecal tube) immediately after SCI and for 7 consecutived (early treatment) or on postoperation day (POD) 35 after SCI (de-layed treatment), respectively. These paradigms of it and ip admin-istration and dose selection were performed according topreviously reported methods [26,38]. To test causality, t-BOOH(ROS donor) was administered it (10, 50, 100, and 200 lg/kg/30 lL plus 5 lL to flush out the intrathecal tube) and ip (1, 10,50, and 100 mg/kg/mL) in naive groups, and mechanical allodyniawas evaluated.

2.2.1. Behavioral experiments2.2.1.1. Intrathecal implantation. Intrathecal implantation was per-formed by inserting an intrathecal catheter 3 d prior to spinal con-tusion. Briefly, under masked-inhalation anesthesia (isoflurane,induction 3% and maintenance 1.5%), a premeasured length ofCS-1 Intrathecal Catheter 32G (ReCathCo, Allison Park, PA) waspassed caudally from the cisterna magna near the medulla, and2 cm of the free end were left exposed at the nape of the neck. Sal-ine (30 lL) was injected daily to prevent clogging of the intrathecalcatheter. To protect against slipping or loss, the intrathecal cathe-ter tube was loosely tied using an overhand knot (0.5 cm diameter)fixed by Glass Ionomer Base Cement (Shofu Dental Corp., San Mar-cos, Calif.) and sutured to the surrounding paravertebral muscula-ture just proximal to the intrathecal entry site. Rats wereindividually housed to prevent the removal of intrathecal catheterby cage mates. The exposed intrathecal tube was sealed by sterilestainless steel wire to prevent infection. The wire was removed forsaline, PBN or t-BOOH administration and immediately replacedafter injections. After the last treatment, the it tubes wereremoved.

2.2.1.2. Measurements of mechanical allodynia. To test hind-pawwithdrawal responses to mechanical stimuli applied on the centerof glabrous surface of the paw, rats were individually housed inclear acrylic boxes (8 � 8 � 24 cm) above a metal mesh(0.5 � 0.5 cm) and acclimated for 15 m to avoid the stress of envi-ronmental change [25]. Mechanical allodynic behaviors, which arecharacterized by abrupt withdrawal responses accompanied bysupraspinal behaviors consistent with the receipt of a noxious

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stimuli, such as head turn, biting and licking, were measured aspaw withdrawal thresholds to von Frey filament stimuli. Beginningwith the 4.31 von Frey filament, 6 applications of calibrated vonFrey filaments were applied with 10-s intervals between stimuli.The series included filaments 3.61 (0.45 g)’ 3.84 (0.74 g); 4.08(1.26 g); 4.31 (2.04 g); 4.56 (3.31 g); 4.74 (5.50 g); 4.93 (8.32 g);and 5.18 (14.45 g). The paw-withdrawal response was determinedby a 50% withdrawal mechanical threshold using the formulalogð50%thresholdÞ ¼ 10ðXfþjdÞ=10000. The Xf = value of the finalvon Frey filament (log unit); j = correction factors (from calibra-tion table); and d = mean differences of log units between stimuli.The 18 g pressure of 50% withdrawal threshold measurement(according to the up-down method) was selected as the cutoff va-lue [7,14].

2.2.2. Electrophysiological experimentsThe electrophysiological responses of spinal lumbar 4/5 WDR

dorsal horn neurons to mechanical stimuli were investigated usingin vivo single extracellular recording techniques [24,25]. Briefly,rats were anesthetized by sodium pentobarbital (60 mg/kg, ip)and then a laminectomy of vertebral segments T12 through L1was performed to expose the lumbar enlargement (L4 throughL5). Tracheal and jugular vein cannulae were inserted for breathingand infusion of sodium pentobarbital (5 mg/h/300 g) to maintainthe physiological and anesthetic levels of the rat during the singleunit recording, respectively. The rats were held in place by a ste-reotaxic apparatus, and rectal temperature was maintained at37�C. A single-unit WDR recording was performed by using a car-bon-filament–filled single glass microelectrode (Kation Scientific,Minneapolis, MN) in the lumbar L4/5 dorsal horn. We soughtWDR neurons in the lumbar dorsal horn that displayed gradedactivity patterns in response to increased intensities of mechanicalstimuli given to the WDR receptive fields on the hind paw [9,25].After identification of single-unit activity, 3 graded mechanicalstimuli were applied to that unit’s peripheral receptive field to lo-cate and characterize WDR neurons. These were: (1) brush stimu-lation of the skin with a hairy brush; (2) pressure stimulation byapplying a large arterial clip (bulldog clamps; George Tiemann,Hauppauge, NY) with weak grip to a fold of the skin (firm pres-sure); and (3) pinch stimulation by applying a small arterial clip(Serrefines; Tiemann) with a strong grip to a fold of the skin (pain-ful pressure). The 3 mechanical stimuli were applied successivelyfor 10 s each, with an interstimulus interval of 20 s. The unit activ-ity was amplified and filtered (DAM80; World Precision Instru-ments, Sarasota, FL), fed directly into either an oscilloscope(World Precision Instruments) or the data acquisition unit (CED-1401; Cambridge Electronic Design, Cambridge, UK), and storedon computer in order to construct the waveforms or to plot peris-timulus time histograms (spikes/1 s bin width (Spike2 software,Cambridge Electronic Design) [24]. To test whether ROS influenceson WDR neuronal activity, high doses of PBN (3 mg/kg/50 lL byHamilton syringe) and vehicles (for control) were delivered to testthe changes in WDR activity. Responses were recorded for 10, 30,60, and 120 m after baseline recording. As a control, to ensure thata single and the same WDR unit were held for the duration of therecording experiment, we used the Spike2 program to confirm thesame action potential shape and amplitude.

2.2.3. Immunohistochemical experimentsTo test whether SCI influences the production of ROS and the

expression of pCamKII, double immunofluorescence staining wasperformed. We used 4 groups of rats (sham; SCI + vehicle;SCI + PBN; and naive + t-BOOH) to test changes in ROS productionand pCamKII expression in neurons, astrocytes and microglia,respectively. To test production of ROS, dihydroethidium, an auto-immunoflorescent agent for detection of ROS (Dhet) (Invitrogen,

Grand Island, NY), 50 lM it, was administered intrathecally 24 hprior to perfusion. Rats were deeply anesthetized with sodiumpentobarbital (80 mg/kg, ip) and perfused intracardially with hep-arinized physiological saline followed by 4% cold buffered parafor-maldehyde/0.1 M phosphate buffer (PB) solution. After perfusion,the lumbar spinal cord (L4/5) was removed immediately and post-fixed overnight in 4% paraformaldehyde/0.1 M PB, followed bycryoprotection in 30% sucrose in 4% paraformaldehyde/0.1 M PBover several days [24,25]. After postfixation, spinal cords wereembedded in O.C.T. compound (VWR International, Radnor, PA)individually and then sectioned at 20 lm. Antibodies for neurons(NeuN, 1:2000; Millipore, Billerica, MA); astrocytes (1:1000; GFAP;Millipore); microglia (OX-42 1:100; Serotec, Oxford, UK); andpCamKII (1:300; Santa Cruz Biotechnology, Santa Cruz, CA) wereincubated with a cocktail solution (0.1M PB, 0.15% Triton X-100and 1% NGS) at room temperature, overnight. After 4 washes (each10 m) with 0.1 M PB, sections were incubated with secondary anti-bodies (2 h, 1:600, Molecular Probes, Grand Island, NY). Sectionswere collected by free-floating methods and mounted on gel-coated slides with mounting media (DAPI, Vectashield; Vector Lab-oratories, Burlingame, CA). The specificity of the antibody forpCamKII was tested by Western blot analyses and is reported else-where [10]. Images were captured by Confocal microscopy (Radi-ance 2000, Bio-Rad, Hercules, CA) with Lasersharp 2000 imagingsoftware (Sunnyvale, CA) and were evaluated by measuring inten-sity using a computer-assisted image analysis program (MetaMor-ph 6.1; Molecular Devices, Sunnyvale, CA). Imaging captureparameters, such as offset, gain and iris settings, were kept con-stant during image capture for valid comparisons of differentialexpression within a study (neuronal, microglial and astrocytic)on between-SCI and SCI + treatment groups.

2.2.4. Measurement of locomotionLocomotor function was evaluated using the Basso, Beattie, and

Bresnahan (BBB) open field locomotor scale test [5]. The BBB scoresrange from 0 (no hind-limb movement) to 21 (normal movement,including coordinated gait with parallel paw placement). Briefly,the individual rat was placed in an open field area(90 � 60 � 10 cm) and acclimated for 3 to 5 min to avoid the stressof environmental changes. The scores were evaluated according tothe movement of each joint, each paw placement, coordination,and stepping; scores between 0 and 7 indicate the return of iso-lated movements in the 3 joints (hip, knee, and ankle). Scores be-tween 8 and 13 indicate the return of paw placement andcoordinated movements with the forelimbs. Scores between 14and 21 show the return of toe clearance during stepping, predom-inant paw position, trunk stability, and tail position.

2.2.5. Statistical analysisBehavioral statistical analyses were performed using repeated

2-way or 1-way ANOVA and using the Student-Newman-Keulsmethod for multiple comparisons. Electrophysiological and immu-nohistochemical statistical analyses were performed using the ttest for comparison, respectively. An alpha level of significancewas set at 0.05 for all statistical tests using the SigmaStat program(v 3.1, Setauket, NY). Data are expressed as means ± SE.

3. Results

3.1. ROS production increased following SCI and attenuated by PBN

To test whether SCI produces increased production of ROS inneurons, Dhet (a ROS indicator) intensity was measured using dou-ble immunofluorescence. Fig. 1 shows the Dhet intensity in thelumbar 4/5 spinal dorsal horn neurons following T10 contusion in-

Fig. 1. The ROS overproduction in the lumbar 4/5 spinal dorsal horn following T10contusion injury. Double immnuofluorescence staining for neurons (NeuN, green)and Dhet (ROS marker, red) in the lumbar 4/5 spinal dorsal horn at 35 d followingT10 contusion injury (top panel). Histogram of intensity measures of the 4 groupsused in this study (bottom panel). Compared to sham controls, SCI groups showedincreased intensity of Dhet that was attenuated by early PBN treatment, both byintraperitoneal (IP) and intrathecal (IT) administration (⁄P < 0.05). Small boxesshow representative neurons from laminae III to V to demonstrate neuronallocalization of Dhet at a higher magnification, not to demonstrate differences insoma size. Scale bar: 50 lm.

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jury. After normalization (100%) of sham control (n = 5), the meanintensity of Dhet in the SCI group (POD 35, no PBN group, n = 5)was 161.2 ± 12.9% and showed significant difference compared tosham controls (⁄P < 0.05, Fig 1). By contrast, the Dhet intensity afterearly PBN treatment (n = 4, 100 mg/kg, ip, and n = 4, 3 mg/kg, it)was 118.8 ± 13.8% and 128.5 ± 10.2, respectively, and showed sig-nificant differences compared to SCI groups (⁄P < 0.05, Fig 1).

3.2. Mechanical allodynia attenuated by blocking ROS following SCI

To test whether ROS is involved in mechanical allodynia follow-ing SCI, early and delayed PBN were administered. In sham controls(n = 5), the mean withdrawal threshold of hind paws to von Freyfilaments was 18 ± 0 g and did not show significant differences inthe entire test periods, whereas the mean withdrawal thresholdof SCI + vehicle groups significantly decreased between POD 28and 42 following SCI (⁄P < 0.05, Fig. 2A and B). In early PBN treat-ment (ip, 100 mg/kg, n = 7), the mean withdrawal threshold was6.4 ± 1.2 g (POD 28) and 5.2 ± 1.2 g (POD 35) and showed signifi-cant differences compared to 2.5 ± 0.1 g and 2.5 ± 0.3 g (SCI + vehi-cle groups, n = 6) (#P < 0.05, Fig. 2A), respectively. In early it PBNtreatment (it, 3 mg/kg, n = 8), the mean withdrawal thresholdwas 9.7 ± 1.1 (POD 28) and 7.8 ± 1.6 g (POD 35) and showed signif-icant differences compared to 4.1 ± 1.4 g and 2.8 ± 0.4 g (SCI + vehi-cle groups, n = 6, ⁄P < 0.05, Fig. 2A), respectively. The meanwithdrawal threshold after systemic (10 and 50 mg, n = 6 each)and intrathecal (1 mg, n = 6) treatment did not show significantdifferences compared to the SCI + vehicle groups. To test the effectof PBN to reverse the lowered mechanical thresholds after SCI, PBNwas administered ip (n = 8, 100 mg/kg) and it (n = 7, 3 mg/kg) atPOD 35 (delayed treatment). After 1 h following PBN treatment,the mean withdrawal thresholds were 9.1 ± 1.1 g (ip) and6.1 ± 0.8 g (it), and they showed significant differences comparedto SCI + vehicle groups (4.3 ± 0.4 g, ⁄P < 0.05, Fig. 2B), respectively.The PBN-induced increase in withdrawal thresholds, which weinterpret as attenuation of mechanical allodynia, lasted more than5 h.

3.3. Dorsal horn hyperexcitability after SCI attenuated by PBN

To test whether ROS is involved in neuronal hyperexcitabilityfollowing SCI, the changes in neuronal responsiveness to mechan-ical stimuli were measured. Fig. 3A and B show typical waveformsof activity patterns and histograms of WDR neurons in the lumbar4/5 dorsal horn in response to brush (Br), pressure (Pr) and pinch(Pi) stimuli in sham, vehicle and PBN groups, respectively. In shamgroups, the mean activity of WDR neurons (n = 23 from 6 rats) tobrush, pressure and pinch stimuli was 8.6 ± 2.1, 15.9 ± 1.5 and18.8 ± 1.8 spikes/s, respectively. In contusion groups (POD 35),the mean activity of WDR neurons (vehicle group, n = 55 from 14rats) to brush, pressure and pinch stimuli was 16.8 ± 0.8,29.4 ± 2.3 and 30.9 ± 2.1 spikes/s, respectively and showed signifi-cant differences compared to sham groups (⁄P < 0.05, Fig. 3C).However, the mean activity of WDR neurons after early PBN treat-ment (n = 45 from 14 rats, ip, 100 mg) to brush, pressure and pinchstimuli was 11.9 ± 0.9, 22.4 ± 1.6, and 24 ± 2 spikes/s and showedsignificant differences compared to SCI groups (#P < 0.05, Fig. 3C).In addition, delayed PBN treatment (n = 9 from 9 rats, it, 3 mg)on POD 35 significantly attenuated the WDR hyperexcitabilitycompared to SCI groups (⁄P < 0.05, Fig. 3D). The mean activity ofWDR neurons 60 m after topical 3 mg PBN treatment, to brush,pressure and pinch stimuli, was 10.5 ± 2.1, 13.9 ± 2.6, and15.7 ± 2.9 spikes/s, respectively, and showed significant differencescompared to vehicle (16.3 ± 1.2, 24 ± 3.5 and 26.3 ± 3.9 spikes/s,respectively, n = 4 from 4 rats) groups (⁄P < 0.05).

3.4. Overproduction of ROS in microglial but not astrocytic cells afterSCI

After normalization (100%) to sham controls, the microglialDhet intensity in SCI groups was 392.9 ± 99.1% and was signifi-cantly increased in the lumbar 4/5 dorsal horn compared to shamgroups (⁄P < 0.05, Fig. 4A). The intensity of Dhet after early PBNtreatment (3 mg, it) was 209.7 ± 38.8% and was significantly de-creased in the lumbar 4/5 dorsal horn compared to SCI groups(#P < 0.05, Fig. 4A). However, the astrocytic Dhet in SCI groupswas 101.5 ± 12.5% and was not significantly different from that insham groups (Fig. 4B).

3.5. Activation of CamKII in neurons by SCI attenuated by PB

To test whether ROS is involved in activation of CamKII in lumbar4/5 dorsal horn following SCI, activated CamKII (phosphorylated;pCamKII) intensity was measured using immunocytochemical ap-proaches to allow cellular localization, and it was quantitated byusing image analyses software. After normalization (100%) to shamcontrols (n = 5), the neuronal intensity of pCamKII in the SCI group(n = 5) was 301 ± 32.8% and was significantly increased in the lum-bar 4/5 dorsal horn compared to sham groups (⁄P < 0.05, Fig. 5). Theintensity of pCamKII after early PBN treatments (100 mg, ip, n = 5and 3 mg, it, n = 5) was 143.8 ± 18.3% and 196.6 ± 20.3% and wassignificantly decreased in the lumbar 4/5 dorsal horn neurons com-pared to SCI groups, respectively (#P < 0.05, Fig. 5).

3.6. Activation of CamKII modulates mechanical allodynia

To test whether activated CamKII, pCamKII, is involved inmechanical allodynia following SCI, an inhibitor of pCamKII andits inactive form was administered intrathecally (i.t.). The meanwithdrawal threshold of sham controls (n = 5) was 16.6 ± 0.7 g anddid not show any significant changes during the test periods(Fig. 6). However, the mean withdrawal threshold of SCI (POD 35)groups was 3.5 ± 0.4 g and was significantly decreased during thetest periods (⁄P < 0.05, Fig. 6). Thirty min after intrathecal KN-93

Fig. 2. Attenuation of mechanical allodynia by treatment of ROS scavenger after SCI. (A) In the early treatment paradigm, T10 SCI produced mechanical allodynia in both hindpaws (below-level) that was attenuated by 7 days of consecutive intraperitoneal (100 mg, ip) or intrathecal (3 mg, it) treatment. (B) In the delayed treatment paradigm, 35 dafter T10 SCI, single intraperitoneal (100 mg, IP; open circle) and intrathecal (3 mg, IT; reversed closed triangle) PBN treatments attenuated mechanical allodynia over 5 h(⁄P < 0.05, #P < 0.05).

Fig. 3. Attenuation of neuronal hyperexcitability by treatment of the ROS scavenger PBN after SCI. In vivo extracellular single unit recordings of lumbar 4/5 spinal dorsal hornneurons are shown as single-unit waveforms of activity patterns (A) and peristimulus time histograms (B) (bin with 1 s) of wide dynamic range (WDR) neurons from sham,SCI + vehicle and SCI + 3 mg PBN groups, respectively. Early PBN treatment (C) and delayed PBN treatment (D) significantly attenuated the hyperexcitability of WDR neuronsafter SCI (⁄P < 0.05) to brush (Br), pressure (Pr) and pinch (Pi) stimuli compared to SCI groups, respectively (#P < 0.05).

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treatment (15 lg, n = 6, and 30 lg, n = 6), the mean withdrawalthreshold was 6.2 ± 0.5 g and 7.3 ± 1 g, which is significantly differ-ent from the values before (B) treatment, respectively, whereas7.5 lg (n = 4) and 3.75 lg (n = 4) did not show significant changes(⁄P < 0.05, Fig. 5). As a control, KN-92 (n = 4, 30 lg, it) (an inactiveform of KN-93) treatment did not show significant differences com-pared to values before treatment in SCI groups.

3.7. Increased mechanical allodynia and pCamKII expression by ROSdonor

To test whether ROS is involved in mechanical allodynia, t-BOOH, a ROS donor, was given to naive controls. In the naive con-trol (vehicle) groups, the mean withdrawal threshold did not showsignificant changes in the test periods after both the ip (n = 6;

Fig. 4. Spinal cord injury modulates production of ROS in the lumbar dorsal horn glial cells. (A) Compared to sham controls, T10 SCI produced increased expression of ROS inthe lumbar 4/5 dorsal horn microglia (⁄P < 0.05) that was attenuated by early PBN treatment with intrathecal (IT) administration (#P < 0.05). (B) However, astrocytic ROS didnot show significant changes. Scale bar: 30 lm

Fig. 5. ROS modulates expression of pCamKII in the lumbar dorsal horn neurons following T10 contusion injury. Compared to sham controls, SCI produced increasedexpression of pCamKII in the lumbar 4/5 dorsal horn neurons (⁄P < 0.05) that was attenuated by early PBN treatment with intraperitoneal (IP) and intrathecal (IT)administration, respectively (#P < 0.05). Small boxes show the high magnifications. Scale bar: 300 lm.

Fig. 6. Inhibition of pCamKII attenuates mechanical allodynia after SCI. Pawwithdrawal thresholds in sham, SCI + KN-93 (a CamKII inhibitor) and SCI + KN-92(an inactive form of KN-93) groups were compared. Thirty min after intrathecal (i.t.)KN-93 treatments (3.5 to 30 lg/kg) demonstrated a dose-dependent attenuation ofmechanical allodynia compared to values before (B) treatment (⁄P < 0.05), whereasa high dose of KN-92 did not show significant changes.

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Fig. 7A) and the it (n = 5; Fig. 7B) groups. Values before the ipadministration of t-BOOH (10 and 50 mg/kg) of mean withdrawalthresholds were 17.3 ± 0.3 g, and 30 min after t-BOOH treatment,the mean withdrawal threshold was 12.2 ± 1.5 (n = 6, 10 mg/kg)and 12.3 ± 1.5 (n = 6, 50 mg/kg); the values were significantly de-creased compared to values before treatment (⁄P < 0.05, Fig. 7A).Intrathecal t-BOOH treatments (10, 50, 100, and 200 lg/kg)showed dose-dependent differences compared to vehicle groups(Fig. 7B). Thirty min after it t-BOOH treatment, the mean with-drawal threshold was 7.63 ± 1 g (n = 8, 200 lg/kg) and11.2 ± 1.7 g (n = 6, 100 lg/kg), respectively, and was significantlydecreased compared to values before the treatment (14.4 ± 1 gand 15.9 ± 0.8 g) (⁄P < 0.05, Fig. 7B). However, ip (1 mg/kg, n = 6,Fig. 6A) and it (10 lg/kg, n = 7 and 50 lg/kg, n = 8, Fig. 7B) didnot show significant differences compared to values before admin-istration, whereas 100 mg ip produced sedation effects (data notshown). In addition, ROS modulated the expression of pCamKII inthe lumbar 4/5 dorsal horn neurons. After normalization (100%)to naive controls, the intensity of pCamKII after it t-BOOH treat-ment (1 h, 200 lg/kg, n = 4) was 128.3 ± 5.8% and was significantly

Fig. 7. Mechanical allodynia and pCamKII expression by ROS donor treatment. In naive groups, the t-BOOH (ROS donor) treatment dose-dependently produced mechanicalallodynia by either intraperitoneal (ip; A) or intrathecal (it; B) treatments, respectively (⁄P < 0.05, #P < 0.05). (C) One hour after single t-BOOH treatment (it) in the naivegroups, lumbar 4/5 spinal dorsal horn neurons showed increased expression of pCamKII compared to vehicle groups (⁄P < 0.05).

Fig. 8. The recovery of locomotion by ROS scavenger treatment. T10 SCI groupsshowed complete loss of locomotion (POD1) and gradually recovered, whereassham groups did not show any changes during the entire test periods. In early PBNtreatment (ip, 100 mg/kg) showed significant differences compared to vehiclegroup ($P < 0.05). In early PBN treatment it (3 mg/kg), the mean BBB scores showedsignificant differences compared to vehicle (⁄P < 0.05) and 1 mg group (#P < 0.05),respectively. It is noteworthy that the 3 mg it group showed significant differences(POD 16 to POD 28) compared to 100 mg ip groups (^P < 0.05).

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increased in the lumbar 4/5 dorsal horn neurons compared to vehi-cle groups (⁄P < 0.05, Fig. 7C).

3.8. PBN promotes the recovery of locomotion

To test whether ROS is involved in the loss of locomotion, BBBscores were measured. In the sham groups (n = 4), the mean BBBscores were 21 ± 0 and did not change during the entire test period,whereas, the SCI group showed complete loss of locomotion (BBBscores 0 ± 0 on POD 1) and gradually recovered (vehicle groups,Fig. 8). With early PBN treatment by ip (100 mg/kg, n = 5), themean BBB scores were 5.5 ± 1.4 (POD 10) and 6.9 ± 1.3 (POD 13)and were significantly increased compared to vehicle groups(n = 7, 2.6 ± 0.5 and 3.2 ± 0.6, (⁄P < 0.05) and ($P < 0.05), Fig. 8).However, 10 (n = 6) and 50 (n = 5) mg PBN groups did not showsignificant differences (Fig. 8). In early PBN treatment by it(3 mg/kg, n = 9), the mean BBB scores were significantly increasedfrom POD 7 to POD 37 compared to vehicle (⁄P < 0.05) and fromPOD 7 to POD 28 compared to 1 mg groups (n = 6, #P < 0.05,Fig. 8), respectively. In addition, 3 mg it groups were significantlyincreased between POD 16 to POD 28 compared to 100 mg ipgroups (^P < 0.05, Fig. 8).

4. Discussion

In the present study, we report that ROS scavengers attenuatedboth the development (acute experiments) and the maintenance(chronic experiments) of mechanical allodynia, neuronal hyperex-citability and ROS overproduction in the lumbar dorsal horn neu-

rons as well as promoted the recovery of locomotion in hindlimbs following thoracic spinal contusion injury in rats. In addition,in SCI and ROS donor groups, induced activation of CamKII byphosphorylation was attenuated by ROS scavenger treatment. Ta-ken together, the present study suggests that overproduction ofROS contributes to activation of CamKII, which results in below-le-vel neuropathic pain and loss of locomotion following thoracic

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spinal contusion injury in rats. Thus, ROS are critical to both thedevelopment and the maintenance of neuropathic pain likebehavior.

To review, ROS serve as a key factor in synaptic transmission inthe central nervous system [17,34]. ROS is strictly controlled byantioxidant and/or redox reactions in physiological conditions[37]. However, overproduction of ROS, a result of pathophysiolog-ical conditions such as SCI, causes irreversible damages in neurons,such as lipid peroxidation, protein S-thiolation and nucleic aciddamage via oxidation processes, which collectively are called oxi-dative damage and neural cell death [21,52,60]. The neuroanatomi-cal and neurochemical changes in neurons directly or indirectlycontribute to the abnormality in cellular excitability that resultsin neural damage and enhanced nociceptive transmission [21].The early administration most likely contributed to neuroprotec-tion mechanisms, whereas later intervention altered intracellularsignaling mechanisms that contribute to neuronal hyperexcitabil-ity and/or glial activation.

Herein, we scavenged overproduced ROS by 2 routes (intrathe-cal and intraperitoneal) and by 2 timings of administration (earlyand delayed), and we observed attenuations in mechanical allo-dynia, neuronal excitability and pCamKII expression and promo-tion of locomotor recovery. Both early and delayed PBNadministration attenuated neuropathic pain following SCI. Phar-macologically, PBN is well characterized and is the most potentfree radical spin trap scavenging agent. PBN easily penetrates alltissues and inhibits inducible nitric oxide synthesis, protein phos-phorylation, cytokine, and transcription factor gene activation[29,46], all of which are strong candidates for maintaining persis-tent pain transmission after SCI. Specifically, the activation ofNMDA and other glutamate receptors and inducible nitric oxidesynthase via high extracellular glutamate concentrations followingSCI, initiates intracellular pathways such as activation of mitogen-activated protein kinases (MAPK), such as p-38 MAPK and ERK,that subsequently activate transcriptional factors, including CREBand Elk-1 [11,40]. The MAPK pathway is a predominant intracellu-lar downstream signaling pathway following SCI and potentiatesnociceptive transmission in spinal dorsal horn neurons [8,12,25].Recently, evidence that ROS modulate MAPK activation in painpathways on other models has been produced. For example,H2O2 contributes to the phosphorylation of p38 MAPK via transientreceptor potential vanilloid1 (TRPV1) activation [32]. Thus, inhibi-tion of MAPK pathways is one possible mechanism of PBN-inducedattenuation of neuropathic pain following SCI. In addition, produc-tion of proinflammatory cytokines, such as TNFa, IL-1 and IL-6,causes overproduction of ROS in the spinal dorsal horn immedi-ately following SCI [13,41,51,63]. It is known that activation ofTNFa and IL-1 contributes to activation of NOS and consistentlyproduces nitric oxide followed by activation of Rac-1, a smallGTP-binding protein that is important in contributing to persistinghyperexcitability of spinal dorsal horn neurons and central neuro-pathic pain behaviors after SCI [4,59].

It is of note that activation of glutamate receptors, increasedproinflammatory cytokines, MAP kinases and CREB are all consis-tently activated and contribute to the induction and maintenanceof neuropathic pain after SCI [12,13,40].

In this article, we present data showing that intrathecal andintraperitoneal administration of PBN attenuates neuropathic painafter SCI. The half-life of PBN is approximately 2 h and easily pen-etrates the blood-brain barrier [38]. Intraperitoneal administrationof PBN may produces its pharmacological effects by systemicallyeffecting changes in the sensitivity of peripheral nociceptors, dor-sal root ganglia and spinal cord circuits; whereas intrathecaladministration presumably would produce effects only in thespinal cord circuits, including the dorsal root entry zone. However,our behavioral data suggest that both ip and it administration pro-

duce similar PBN effects on the attenuation of neuropathic painand pCamKII expression, suggesting that administration of PBNmay produce attenuation of neuropathic pain via CNS mechanisms[36].

It is of interest that intrathecal administration of PBN producedgreater locomotor recovery than intraperitoneal administration. Itis reported that SCI produces immediate intracellular toxic levels ofROS through proinflammatory cytokines and activation of gluta-mate receptors [4,45]. Subsequently, the intracellular toxic levelsof ROS lead to neuronal cell death, including apoptosis, necrosisand maladaptive synaptic circuits [31] that affect both sensoryand motor function. The assessment of locomotor function, usingthe open field test or the BBB test was originally designed as anacute-outcome measure that correlated well with severity of injury[5]. Thus, several SCI laboratories use the BBB score as an indicatorof the severity of SCI and use the open field test with reports of theBBB rank score; these are routine behavioral assessments in mostSCI studies to allow interlaboratory comparisons of SCI.

In the present study, we report the novel finding that inhibitionof superoxide function, with a ROS scavenger, by several consecu-tive days of treatment improves locomotion, as determined by theBBB scores for different doses. However, Li and colleagues demon-strated that PBN, given before or after SCI, had no effect on locomo-tor function, using the inclined plane test, after spinal compressioninjury [41]. In the present study we demonstrate that PBN facili-tates the recovery of locomotor function. We suggest that the dif-ferences in the observed results may be differences in model(compression vs contusion in the present study); in the locomotortest used (inclined plane vs open field in the present study); and/orthe dose and duration of the PBN delivery. In the Li et al. study, PBNwas administered ip at 100 mg/kg prior to SCI and 50 mg/kg at 1.5and 3.5 h after SCI. By contrast, we administered PBN immediatelyafter SCI and for 7 consecutive d, at doses of 800 mg/kg ip or24 mg/kg it Although the differences in the model of SCI and/orthe locomotor behavioral tests used may account for the differ-ences in outcome after PBN administration, we think it more likelythat the differences in timing, dose and duration can account forthe different outcomes when comparing the 2 studies. Our findingsare consistent with other studies. ROS production is increased inthe dorsal horn at both early and delayed time phases followingSCI [57,8]. In addition, Yune et al. reported that long-lasting admin-istrations of PEP-1 SOD-1 (Cu, Zn superoxide dismutase expressionvector) produced significant improvement in locomotion recoveryafter SCI [64]. These studies suggest that both early and delayedscavenging of ROS would be a useful therapeutic strategy for thetreatment of neuropathic pain and recovery of locomotionfollowing SCI, and overproduction of ROS is one of the major com-ponents in neurotrauma and neurodegenerative diseases. There-fore, prevention of ROS overproduction is an important clue toconsider in therapeutic strategy, such as reduction in mitochon-drial dysfunction and enhancement of antiapoptotic kinasesactivity, to promote neurological recovery following SCI[30,42,43,58].

In this study, we suggest an important intracellular role for ROSin below-level neuropathic pain following thoracic SCI. Our immu-nohistochemistry results demonstrated that increased Dhet inten-sity in the L4/5 spinal dorsal horn neurons and microglia, but not inastrocytes, following T10 thoracic contusion injury, that was atten-uated by PBN treatment. In glial cells, microglial activation is alsoknown to produce high concentrations of ROS, particularly aftertrauma to the CNS [7]. We have confirmed that ROS productionis increased in microglia, not in astrocytes, in the lumbar dorsalhorn [57]. It is noted that astrocytes have less polyunsaturated lip-ids, low iron content and high concentrations of antioxidant en-zymes, including glutathione, that confer more resistance tooxidative stress [31,48,52]. In addition, activated CamKII expressed

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in the L4/5 spinal dorsal horn neurons but not in astrocytes ormicroglia, following T10 thoracic contusion injury, was attenuatedby PBN treatment. Therefore, the present study suggests that ROS-mediated activation of CamKII by phosphorylation (phosphory-lated CamKII, pCamKII) is an important intracellular pathway inneurons that results in below-level central neuropathic pain fol-lowing SCI. In addition, our previous data reported that T10 SCIproduced activation of CamKII in neurons but not in astrocytes ormicroglia. This model results in at-level neuropathic pain and neu-ronal hyperexcitability in thoracic dorsal horn neurons [10]. Thepresent data focus on mechanisms several segments remote fromthe injury site. The present data also demonstrate that ROS contrib-ute to the cellular mechanisms involved in activating CamKII-med-iated alterations in the lumbar regions and result in below-levelcentral neuropathic pain following T10 SCI. Activation of CamKIIinitiates activation of MAPK and transcriptional factors and alsomodulates the calcium-dependent signaling [6,47]. It is reportedthat oxidative stress induces activation of CamKII in various path-ophysiological conditions [61]. For example, ROS oxidize methio-nine 281/288 of the regulatory domain of CamKII, which resultsin autophosphorylation in cardiac cells [61]. However, little isknown about the specific pathways in which ROS modulate activa-tion of CamKII in sensory neurons after SCI. Herein we suggest thatoverproduction of ROS activate CamKII in neurons and that ROS-mediated CamKII activation is an important intracellular pathwayfor below-level neuropathic pain after SCI. ROS potentiate synaptictransmission via activation of CamKII in various pathophysiologicalconditions [50,61]. First, ROS oxidize the regulatory domains ofCamKII and induce oxidation-mediated phosphorylation of CamKII(autophosphorylation), which maintains the activation state ofCamKII [15,56]. Second, the activation of protein kinase C, whichis activated by ROS production induced by mitochondrial perme-ability transition pore dysfunction, initiates phosphorylation ofCamKII, followed by NMDA-receptor trafficking that results inthe potentiation of synaptic transmission [1,18]. Recently, Leeet al. reported that treatment of PBN attenuates neuropathic painvia NMDA receptor activation–dependent phosphorylation ofGluA1 and GluA2, which suggests that ROS is a key factor in thephosphorylation and cell-surface localization of AMPA receptors[39].

Although we did not characterize the specific sources of ROS inthe present study, it is known that mitochondrial dysfunction is amajor source of superoxide production that can contribute to theactivation of CamKII, neuropathic pain and motor dysfunction[35,64]. A good example of neuronal mitochondrial-ROS pathwaysin the production of hyperexcitability in neurons is the following:superoxide production and lowered calcium uptake by mitochon-drial dysfunction increases cytosolic calcium concentrations thatresult in activation of CamKII and facilitates activation of L-typecalcium channels and, therefore, results in the potentiation of neu-ronal excitation and synaptic transmission [29,34,50]. However, itis noted that microglia did not show activation of CamKII, consis-tent with our previous report [10]. It is well documented thatastrocytes and microglia contribute to neuropathic pain followingSCI [24,25]. Therefore, the present study suggests a different mech-anism of initiation and maintenance of neuropathic pain—one ofneuronal-glial and glial-neuronal interactions following SCI. Thespecific interaction of astrocytes and microglia in modulating neu-ronal intracellular pathways and hyperexcitability in neuropathicpain remains for future studies.

In summary, thoracic SCI produces below-level neuropathicpain via ROS-mediated CamKII activation and promoted recoveryof locomotion. The present data suggest that ROS scavengers maybe useful therapeutic methods for spinal cord injury–mediatedsensory and motor abnormalities.

Conflict of interest statement

None of the authors have conflicts of interest with respect tothis work.

Acknowledgement

Supported by National Institutes of Health grants NS11255 andNS39161.

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