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JOURNAL OF NEUROTRAUMAVolume 24, Number 7, 2007© Mary Ann Liebert, Inc.Pp. 1108–1118DOI: 10.1089/neu.2006.0254

The Novel Apolipoprotein E–Based Peptide COG1410Improves Sensorimotor Performance and Reduces Injury

Magnitude following Cortical Contusion Injury

MICHAEL R. HOANE,1 JEREMY L. PIERCE,1 MICHAEL A. HOLLAND,1NICHOLAS D. BIRKY,1 TAN DANG,1

MICHAEL P. VITEK,2,3,4 and SUZANNE E. MCKENNA2

ABSTRACT

It has previously been shown that small peptide molecules derived from the apolipoprotein E (ApoE)receptor binding region are anti-inflammatory in nature and can improve outcome following headinjury. The present study evaluated the preclinical efficacy of COG1410, a small molecule ApoE-mimetic peptide (1410 daltons), following cortical contusion injury (CCI). Animals were preparedwith a unilateral CCI of the sensorimotor cortex (SMC) or sham procedure. Thirty mins post-CCIthe animals received i.v. infusions of 0.8 mg/kg COG1410, 0.4 mg/kg COG1410, or vehicle. Start-ing on day 2, the animals were tested on a battery of behavioral measures to assess sensorimotor(vibrissae-forelimb placing and forelimb use-asymmetry), and motor (tapered balance beam) per-formance. Administration of the 0.8 mg/kg dose of COG1410 significantly improved recovery onthe vibrissae-forelimb and limb asymmetry tests. However, no facilitation was observed on the ta-pered beam. The low dose (0.4 mg/kg) of COG1410 did not show any significant differences com-pared to vehicle. Lesion analysis revealed that the 0.8 mg/kg dose of COG1410 significantly reducedthe size of the injury cavity compared to the 0.4 mg/kg dose and vehicle. The 0.8 mg/kg dose alsoreduced the number of glial fibrillary acid protein (GFAP�) reactive cells in the injured cortex.These results suggest that a single dose of COG1410 facilitates behavioral recovery and providesneuroprotection in a dose and task-dependent manner. Thus, the continued clinical development ofApoE based therapeutics is warranted and could represent a novel strategy for the treatment oftraumatic brain injuries.

Key words: apoE; behavioral recovery; GFAP; neuroprotection; recovery of function; TBI; trauma

1Restorative Neuroscience Laboratory, Center for Integrative Research in Cognitive and Neural Sciences, Department ofPsychology, Southern Illinois University, Carbondale, Illinois.

2Cognosci Inc., Research Triangle Park, North Carolina.3Division of Neurology, Department of Medicine, and 4Department of Neurobiology, Duke University Medical Center, Durham,

North Carolina.

INTRODUCTION

TRAUMATIC BRAIN INJURY (TBI) is a major publichealth issue. Conservative estimates for TBI in the

United States put the incidence rate at 200 per 100,000,yielding 500,000 cases, with 20% being classified by theGlasgow Coma Scale (GCS) as severe or moderate in-juries (Narayan et al., 2002). These disabilities includecognitive, sensory, motor, and emotional impairments.This major health issue is compounded by the limitednumber of pharmacological treatments that are currentlyavailable for patients suffering head injuries, which is sur-prising because of the amount of effort invested into find-ing such treatments (Statler et al., 2001; Narayan et al.,2002). Most treatments for TBI have used neuroprotec-tive strategies that attempt to prevent or reduce excito-toxicity following TBI (Clausen and Bullock, 2001).However, the role of inflammation following TBI iswidely known but there are currently no effective treat-ments targeting the inflammatory process following TBI(Tolias and Bullock, 2004; Lynch et al., 2005); however,some preclinical work has been done (Cernak et al., 2002;Gopez et al., 2005).

Apolipoprotein E (ApoE, protein; APOE, gene) is theprimary apolipoprotein synthesized in the brain in re-sponse to injury where it functions via anti-oxidant, anti-inflammatory, anti-excitotoxic, and neurotrophic mecha-nisms (Aono et al., 2002, 2003; Colton et al., 2002;Laskowitz et al., 1997, 2001; Lomnitsky et al., 1999;Lynch et al., 2001; Miyata and Smith 1996; Nathan etal., 1994). A vast literature implicates a role for APOEin modulating the susceptibility and/or severity of neu-rological disorders characterized by neuroinflammationincluding Alzheimer’s disease (AD), multiple sclerosis(MS), Parkinson’s disease, stroke, and cerebral hemor-rhage (Chapman et al., 2001; Corder et al., 1993; Craw-ford et al., 2002; McCarron et al., 1998; Zareparsi et al.,2002), although the most compelling data is in the set-ting of TBI (Friedman et al., 1999; Teasdale et al., 1999).

Previous work has demonstrated that a small moleculepeptide derived from amino acids 133–149 located in thereceptor-binding region of apoE (COG133) retains theneuroprotective, anti-oxidant, anti-excitotoxic and anti-inflammatory properties of the intact apoE holoprotein,in vitro and in vivo (Aono et al., 2003; Laskowitz et al.,2001; Lynch et al., 2003, 2005; McAdoo et al., 2005;Misra et al., 2001). The in vivo neuroprotective activityof COG133 has been demonstrated in several clinicallyrelevant models of neurological disorders including TBI,MS, and hypoxic-ischemic injury (HIE) (Lynch et al.,2005; Li et al., 2005; McAdoo et al., 2005). A single i.v.administration of COG133 at 30 mins following closedhead TBI resulted in statistically significant improve-

ments in mortality, short-term vestibulomotor function,long-term cognitive function, and survival of hippocam-pal neurons compared to head injured mice that receivedvehicle (Lynch et al., 2005). In a murine model of MS,treatment with COG133 significantly decreased clinicalsigns of disease and the number of spinal cord infiltrates,and increased the number of mice that achieved remis-sion compared to mice that received vehicle (Li et al.,2006). Finally, neuroprotective effects of intrathecal ad-ministration of COG133 has been demonstrated in a peri-natal rat model (McAdoo et al., 2005). These data, to-gether with the numerous reports establishing a role forapoE in modulating recovery from cerebral injury makesa compelling case for the development of a neuroprotec-tive therapy based on apoE.

COG133 retains a measurable degree of alpha helicitypresent in the receptor-binding region of the native apoEholoprotein from which it was derived (Aono et al., 2003;Gay et al., 2006; Laskowitz et al., 2001). Alanine scan-ning studies suggested that enhancing the alpha helicalcharacter of COG133 may enhance anti-inflammatory ac-tivity (Laskowitz et al., 2006). This led to the develop-ment of COG1410, a shorter apoE-mimetic composed ofapoE residues 138–149 with aminoisobutyric acid (Aib)substitutions at positions 140 and 145, which exhibitedsuperior anti-inflammatory efficacy and potency in vitro(Laskowitz, et al., 2006). COG1410’s in vivo efficacywas recently established in a model of subarachnoid he-morrhage (SAH) where treatment with COG1410 re-sulted in a dramatic reduction in mortality, vasospasm,cerebral edema, and functional deficits compared to ve-hicle treated mice (Gao et al., 2006).

The purpose of the present study was to examine thepreclinical efficacy of COG1410 in a standard rat modelof TBI. COG1410 is smaller (1410 daltons) than theCOG133 (2171 daltons), which may enhance passagethrough the blood–brain barrier (BBB). Following uni-lateral controlled cortical impact (CCI) of the sensori-motor cortex (SMC), animals were administeredCOG1410 at either low dose (0.4 mg/kg, i.v.) or highdose (0.8 mg/kg, iv) 30 mins post-CCI. Rats were testedon a battery of sensorimotor tests. Following completionof testing the brains were harvested for lesion analysisand glial fibrillary acid protein (GFAP) analysis. Thisstudy represents the first attempt to test preclinical effi-cacy of COG1410 in a rat TBI model. There is a grow-ing consensus that the most beneficial TBI treatmentwould be directed at multiple targets/mechanisms relatedto the pathological mechanisms associated with TBI(Narayan et al., 2002; Schmidt et al., 2005). ApoE-mimetic peptides which function via anti-oxidant, anti-inflammatory, anti-excitotoxic, and neurotrophic mecha-nisms represent a promising novel multi-mechanism

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therapeutic for the treatment of TBI (Aono et al., 2002,2003; Colton et al., 2002; Laskowitz et al., 1997, 2001;Lynch et al., 2001; Lomnitsky et al., 1999; Miyata andSmith 1996; Nathan et al., 1994).

METHODS

Subjects

Forty male Sprague-Dawley rats, approximately 3months old, were used for this experiment. All experi-mental procedures were reviewed and approved by theInstitutional Animal Care and Use Committee, and thestudy was conducted in a facility certified by the Amer-ican Association for the Accreditation of Laboratory An-imal Care. Rats were maintained on a standard 12-hlight/dark cycle with food and water available ad libitum.

Surgery

The surgical procedure was performed using asepticprocedures and conditions. The CCI model utilized inthe present study was based on previous studies (Hoaneet al., 2004; Barbre and Hoane, 2006). Rats were anes-thetized with isoflurane (3–5%) in O2 (0.8 L/min) andthen prepared for surgery. When the rat was unrespon-sive (no ocular or pedal reflexes), the head was shavedand then scrubbed with 70% alcohol, followed by Be-tadine and placed into a digital stereotaxic device(Stoelting Inst.). A midline incision was made in theskin and underlying fascia. A circular craniotomy (6.0mm) was performed using a Dremel motor tool and aspecially designed drill bit that prevented damage tothe meninges and cortex. The craniotomy was unilat-eral and centered over the sensorimotor cortex (0.0 a-p, �3.0 mm lateral to bregma. The contusion injurywas created with an electromagnetic contusion device(www.myneurolab.com) using a sterile, stainless steelimpactor tip (5.0 mm diameter) that was activated at avelocity of 2.78 m/sec. The impactor tip was positionedabove the cortex, and upon activation of the piston, theimpactor tip made contact with the cortex for 0.5 sec,which resulted in a 2.0-mm compression of the cortex.Following the contusion, any bleeding was controlledwith sterile sponges soaked in cold saline, and the in-cision was closed with nylon suture material. Thewound was treated with a triple antibiotic ointment.Normal body temperature (37°C) was maintained witha warm water recycling pump and bed system (EZ-Anesthesia, Inc.) during the surgical procedure and re-covery. Rats receiving sham surgeries were treated inan identical manner as those receiving injuries; how-ever, no CCI was performed.

Synthesis of Peptides

Peptides were synthesized by NeoMPS (San Diego,CA) to a purity of 95%. COG1410 is Ac- AS(Aib)-LRKL(Aib)KRLL-amide, which is derived from apoEresidues 138–149 with Aib substitutions at positions 140and 145. The peptides were reconstituted in sterile lac-tated Ringers solution at either the 0.8 or 0.4 mg/kg doseprior to administration.

Drug Administration

Rats were randomly assigned to one of two differentdoses of COG1410 (0.8 or 0.4 mg/kg, i.v.) or vehicle (lac-tated ringers; 1 ml/kg, i.v.). Injections were given 30 minfollowing injury via tail vein infusion. All testing andanalyses were conducted without knowledge of drug as-signment.

Vibrissae–Forelimb Placing Test

Sensorimotor function was evaluated by scoring thevibrissae mediated forelimb placing reaction (Hoane etal., 2000; Hoane and Barth, 2001; Schallert and Woodlee,2005; Barbre and Hoane, 2006). Each rat was held by thetrunk ensuring the forelimbs were free to move. One sideof the rat was oriented parallel to a Plexiglas surface andwas slowly moved until the vibrissae on one side touchedthe surface. In intact rats, a reliable lateralized placingresponse was elicited each time the vibrissae made con-tact with the surface. A successful forelimb placing re-sponse was recorded if the animal raised its forelimb andplaced it on the surface in response to stimulation of thevibrissae ipsilateral to the forelimb. Each rat was given10 trials for each forelimb. If a placing response was notelicited within 5 sec of vibrissae stimulation, the trial wasrecorded as unsuccessful. Baseline performance wasrecorded prior to injury. The animals were tested on days2, 4, 6, 8, 10, 12, and 14 post-CCI.

Limb-Use Asymmetry Test

This task measures somatosensory dysfunction follow-ing injury. For this task, an animal was placed in a glasschamber (25 cm � 25 cm � 25 cm) and was allowed toexplore for 5 min while being videotaped (Becerra et al.,2007). While exploring the chamber animals rear, press-ing one or both forepaws against the vertical surface(Schallert and Woodlee, 2005). Intact animals use theirforelimbs (right vs. left) equally, and do not show a limbuse asymmetry. During video analysis, the number oftimes each forelimb was placed on the vertical glass sur-face was recorded. The data are represented as the per-centage of preference for the contralateral limb, calculatedusing the following formula: [contralateral forelimb/ (con-

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tralateral � ipsilateral)] � 100. A derived score of 50%reflects equal use of both forelimbs, a score greater than50% represents greater preference for the limb ipsilateralto the site of injury, while a score less than 50% indicatesgreater use of the limb contralateral to the injury. Animalswere tested on post-CCI days 10 and 23.

Tapered Beam Walk Test

All animals were trained (two trials per day for 3 days)on this test of vestibulomotor function and motor coor-dination prior to injury. Animals were trained to escapea bright light and enter a darkened box by traversing atapered 165-cm-long elevated beam, with a 2-cm ledge(Schallert and Woodlee, 2005). Criterion performancewas assessed as the ability to traverse the beam with nomore than two foot slips on a single trial for 2 consecu-tive days. Following surgery, each rat was tested on twotrials/day on days 4, 12, and 23 post-CCI. Post-injury per-formance was rated on the number of full slips with thecontralateral hindlimb (paw was completely on theledge).

Histology

At 25 days post-CCI, the rats were anesthetized withurethane (3.0 g/kg, i.p.) and transcardially perfused with0.9% phosphate-buffered saline (PBS), followed by 10%phosphate-buffered formalin (PBF). Brains were post-fixed in PBF following removal from the cranium. A 30%sucrose solution was used to cryopreserve the brains for3 days prior to frozen sectioning. Serial sections (40 �mthickness) were sliced using a sliding microtome with anelectronic freezing stage and collected into PBS.

Lesion Analysis

Prior to sectioning, the dorsal surface of the brain wasimaged with an Olympus stereomicroscope (SZX9) and13.5 megapixel digital camera (DP-70). These images(4�) were analyzed with ImageTool to determine thearea of the dorsal surface of the injury cavity.

Following slicing, a series of coronal sections, mountedon gelatin-subbed microscope slides, were stained withcresyl violet, dehydrated and cover slipped. The extent ofthe lesion was analyzed with an Olympus microscope (BX-51) and DP-70 camera. Images of the sections throughoutthe extent of the injury were captured using the digital cap-turing system and area measurements of the lesioned tis-sue were determined using ImageTool software (Barbreand Hoane, 2006). The Calvalieri method was used to cal-culate the volumes of the ipsilateral cortex and the con-tralateral cortex (Coggeshall, 1992). The number of sec-tions and the section thickness (40 �m) were multiplied

by the mean area of the lesion cavity (calculated at fourstereotaxic coordinates surrounding the lesion: 3.00, 2.00,1.00, 0.00, and �1.00 relative to bregma) (Paxinos andWatson, 1998). The extent of cortical injury was measuredby calculating the percent reduction in the ipsilateral cor-tex compared to the contralateral cortex using the formula(1 � (ipsi/contra) � 100).

GFAP Immunohistochemistry

A second series of sections were stained using a free-floating protocol for GFAP immunoreactivity to deter-mine reactive gliosis expression following CCI (Hoaneet al., 2003, 2006). Tissue sections were incubated witha GFAP rabbit anti-cow primary antibody (DAKO, Z334,diluted 1:2000) for 48 h, rinsed in PBS � 0.4% Triton-X (TX) (6 � 5 min), and then incubated using anti-rab-bit IgG (BA-1000; Vector Labs, Burlingame, CA) for 90min. The sections were rinsed in PBS � 0.4% TX (6 �5 min) and then placed in ABC substrate (Vector; PK-6100) for 90 min. Sections were rinsed in PBS � 0.4%TX (3 � 5 min), followed by 0.1 PB (3 � 5 min) andthen reacted in a nickel-intensified chromagen solutioncontaining acetate-imidizole buffer, 2.5% nickel ammo-nium sulfate, 0.05% DAB, and 0.01% H2O2. Tissue sec-tions were mounted on subbed slides, dehydrated andcover-slipped. The number of GFAP� cells surroundingthe lesion was determined using cell counts from two dif-ferent slices through the injury cavity. In each slice, twosites randomly selected from the tissue surrounding thelesion—one from dorsal cortex and one from lateral cor-tex—were used for the analysis. The tissue was viewedwith an Olympus video microscope (BX-51) system at4�, and then increased to 40� and captured. The num-ber of GFAP� cells within the captured field of view(35.29 mm2) was counted using ImageTool software. Thetotal number of GFAP� cells counted from the four siteswas used for the cortical analyses.

Statistical Analysis

Analysis of variance (ANOVA) tests were performedusing procedures for general linear models (SPSS 14.0for Windows) with options for repeated measures whereappropriate for all behavioral measures. The between-group factor was group (0.8 mg/kg COG1410, 0.4 mg/kgCOG1410, vehicle, and sham injured) and the within-group factor was day of testing. Huynh-Feldt probabili-ties (HFP) and Fischer’s least significant difference tests(LSD) were used to correct for Type-1 error associatedwith repeated measures and post hoc means comparisons,respectively. For all HFP corrections, the actual correcteddegrees of freedom (i.e., 3.13 dof) were used and reportedinstead of the uncorrected degrees of freedom (i.e., 3 dof),

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this ensures correction for Type-1 errors. Anatomical datawere analyzed with one-way ANOVA procedures andFischer’s LSD. A significance level of p � 0.05 was usedfor all statistical analyses.

RESULTS

Vibrissae–Forelimb Placing

Forelimb placing responses were analyzed by two4 � 7 repeated-measures ANOVAs: for the ipsilateraland contralateral forelimbs. Group (0.8 mg/kgCOG1410, 0.4 mg/kg COG1410, vehicle, sham) and day(2, 4, 6, 8, 10, 12, 14 post-CCI) were used as the be-tween-group and within-group factors in the analysis.Unilateral CCI did not significantly impair the animals’ability to place with the ipsilateral forelimb (p � 0.05).Conversely, a significant group effect was observed inthe placing reaction on the contralateral forelimb [F(3,24) � 24.55, p � 0.001] (Fig. 1). A significant recov-ery was observed across time, as reflected in the maineffect for test days [F(3.13, 75.16) � 21.59, p � 0.001].The group � day interaction was also significant[F(9.40, 75.16) � 2.43, p � 0.02]. Post hoc compar-isons revealed a significant and persistent treatment ef-fect for the 0.8 mg/kg COG1410 group beginning asearly as day 4 post-CCI (p � 0.03) and lasting through

day 14 of testing (p � 0.001). The 0.4 mg/kg COG1410group showed an induction of recovery beginning ondays 6–8 and reached a level of greater than 60% re-covery by the end of testing; however, none of the testdays were significantly different compared to vehicle(p � 0.05). Both COG1410 groups showed recoverygreater than 60% by the last day of testing compared tovehicle (30%).

Limb Use Asymmetry

Post-CCI performance on limb use asymmetry was as-sessed with a 4 � 2 repeated-measures ANOVA, withgroup (0.8 mg/kg COG1410, 0.4 mg/kg COG1410, ve-hicle, sham) and day (10 and 23 post-CCI) as the factorsin the analysis. The rats limb-use asymmetries did notimprove with time following injury; the main effect forday was not significant [F(1.00, 24.00) � 0.64, p �0.43]. The group � day interaction effect was also notsignificant [F(3.00, 24.00) � 1.04, p � 0.39]. However,a significant group difference was observed [F(3, 24) �8.86, p � 0.0001]. Post hoc analysis of the group maineffect revealed that the 0.8 mg/kg COG1410 groupshowed a significantly different asymmetry compared tovehicle (p � 0.02) and the 0.4 mg/kg dose (p � 0.04). Inaddition, the 0.8 mg/kg COG1410 group was not signif-icantly different from the sham group (p � 0.06). The 0.4mg/kg COG1410 group was not significantly differentfrom vehicle (p � 0.05) (Fig. 2).

Tapered Beam Walk Test

The post-injury performance on the tapered beam wasanalyzed in a 4 � 3 repeated-measures ANOVA, withgroup (0.8 mg/kg COG1410, 0.4 mg/kg COG1410, ve-hicle, sham) and day (4, 12, and 23 post-CCI) as factorsin the analysis. Their ability to traverse the beam im-proved with time following injury; the main effect forday was significant [F(1.79, 42.98) � 52.41, p � 0.001].Significant group differences were also observed [F(3,24) � 15.56, p � 0.001]. However, the group � day in-teraction effect was not significant [F(5.37, 42.98) �1.97, p � 0.09]. Post hoc analyses revealed that all of theCCI-injured groups were significantly impaired com-pared to the sham-injured groups (p � 0.001) (Fig. 3).However, the 0.8 mg/kg COG14 group was not signifi-cantly better at traversing the beam (p � 0.34), comparedto the vehicle-treated animals. The 0.4 mg/kg COG1410group also showed a non-significant effect compared tovehicle (p � 0.54).

Lesion Analysis

The extent of dorsal cavity was analyzed in a one-fac-tor ANOVA, using treatment (0.8 mg/kg COG1410, 0.4

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FIG. 1. The effects of COG1410 (0.8 mg/kg and 0.4 mg/kg,iv) or vehicle administered following unilateral CCI or shamsurgery on the vibrissae-forelimb placing test. The graph showsthe plotted mean (�SEM) percentages of unsuccessful placingattempts. Treatment with 0.8 mg/kg COG1410 significantly re-duced the unsuccessful placing attempts compared to vehicletreatment (*p � 0.05) and were not significantly different fromsham on days 12–14 (^p � 0.05).

mg/kg COG1410, vehicle, sham) as the factor in theanalysis. A significant difference in remaining tissue vol-ume between groups was found [F(3, 24) � 29.64, p �0.001] (Fig. 4A). Post-hoc analysis revealed the 0.8

mg/kg COG1410 group was significantly different com-pared to vehicle (p � 0.05). A second ANOVA analysiswas performed on the percent reduction in cortical tissuevolume through the injury site There was a significantdifference in injury volume between groups [F(3, 24) �8.06, p � 0.001] (Fig. 4B). Post-hoc analysis revealed the0.8 mg/kg COG1410 group was significantly differentcompared to vehicle (p � 0.05). Comparisons with the0.4 mg/kg group were not significantly different from ve-hicle on either measure (p � 0.05). Representative his-tological images are provided in Figure 5.

GFAP Analysis

The number of GFAP� cells was analyzed in a one-factor ANOVA using treatment (0.8 mg/kg COG1410,0.4 mg/kg COG1410, vehicle, sham) as the factor in theanalysis. A significant difference in the number ofGFAP� cells was found following injury [F(3, 23) �5.50, p � 0.005] (Fig. 6). Post-hoc analysis revealed thatthe 0.8 mg/kg COG1410 group was significantly differ-ent compared to vehicle (p � 0.003). The 0.8 mg/kg dosewas also significantly different from the 0.4 mg/kg dose(p � 0.02). Additionally, no significant differences weredetected in the number of GFAP� in the injured cortexbetween the 0.8 mg/kg group and the sham group (p �0.97). Representative images showing the extent of pro-liferation and the size of the reactive astrocytes are shownin Figure 7.

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FIG. 2. The effects of COG1410 (0.8 mg/kg and 0.4 mg/kg, iv) or vehicle administered following unilateral CCI or sham surgeryon the limb-use asymmetry test. The graph shows the plotted mean (�SEM) forelimb-use asymmetries. Preference for the ipsilateralforelimb is represented in the range above 50%; contralateral forelimb preference is represented in the range below 50%. The 50%point indicates equal usage of both forelimbs. Injury significantly impaired the use of the contralateral forelimb. Treatment with 0.8mg/kg of COG1410 significantly reduced the limb use asymmetry compared to vehicle treatment.

FIG. 3. The effects of COG1410 (0.8 mg/kg and 0.4 mg/kg,iv) or vehicle administered following unilateral CCI or shamsurgery on the tapered-beam walk test. The graph shows theplotted mean (�SEM) percentages of impairments with the con-tralateral hindlimb. A severe and enduring injury effect was ob-served; however, neither COG1410 treatment significantly re-duced the limb impairments compared to vehicle treatment.

DISCUSSION

The results of this study have shown that a small mol-ecule peptide derived from apoE has therapeutic benefitwhen administered following induction of TBI. This

peptide, COG1410, had a dose-dependent effect on re-covery of function following unilateral CCI of the SMCin the rat. Specifically, the 0.8 mg/kg dose of COG1410induced recovery on the vibrissae-forelimb placing testbeginning on post-CCI day 4 and reached a plateau of

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FIG. 4. Lesion analysis. Analysis of the observable dorsal surface injury cavity is shown in (A) Plotted is the mean (�SEM)area of the dorsal surface cavity for each group. COG1410 (0.8 mg/kg) reduced the development of the dorsal surface injury cav-ity following unilateral CCI (*p � 0.05). Analysis of the percent reduction in injured cortical volume compared to contralateralcortex volume is shown in (B) Plotted is the mean (�SEM) percent reductions in cortical volume for each group. COG1410 (0.8mg/kg) reduced the amount of injury-induced reduction following unilateral CCI (*p � 0.05).

FIG. 5. Representative injury cavities. CCI produced marked cortical cavitation. Shown are representative images of sham,high-dose COG1410 (0.8 mg/kg), and vehicle through the extent of the injury (�0.44).

greater than 85% improvement of the deficit by day 12compared to the vehicle-treated group, which had im-proved less than 14% by day 12. The 0.8 mg/kg grouphad recovered to such an extent that, by day 12, theywere not significantly different from the sham controls.The lower dose of COG1410 (0.4 mg/kg) started to in-duce recovery on day 6 and reached a maximum recov-ery of greater than 64% by day 14; although this wasnot significantly different from vehicle, this dose showedstrong improvement.

A similar effect to that observed with the forelimb plac-ing test was also found on the limb use asymmetry test,when tested on post-CCI days 10 and 23. The rats ad-ministered the 0.8 mg/kg dose of COG1410 showed a re-duction in their forelimb asymmetry scores (day 10, meanof 63% unimpaired bias; day 23, mean of 60%) comparedto vehicle-treated controls (day 10, mean of 72% unim-paired bias; day 23, mean of 72%). For comparison pur-poses, the uninjured sham groups showed practically noasymmetry (day 10, mean of 51% unimpaired bias; day23, mean of 53%). Thus, this data suggests that treatmentwith the 0.8 mg/kg dose of COG1410 improved sensori-motor performance, resulting in reduced asymmetriescompared to injured, vehicle-controls.

In contrast to these positive results, neither dose ofCOG1410 improved performance on the tapered balancebeam task. This was surprising given that the 0.8 mg/kg

dose of COG1410 significantly improved sensorimotorperformance on both the vibrissae-forelimb and limb useasymmetry tests. The tapered beam is designed with aledge that reduces the compensatory motor changes thathave been associated with the traditional beam test, mak-ing this test a sensitive measure of recovery. However,the testing regimen chosen in the present study (two tri-als on 3 different post-CCI days) may not have beenenough trials to allow for recovery to occur. Given thatit was also found that the high dose of COG1410 pro-vided significant protection against injury-induced cav-ity development, it is likely that the animals were nottested enough to allow for recovery on this test. Furtherstudies utilizing the tapered beam as a sensitive measureof motor function and the effect of testing regimens areongoing.

In a similar manner to what was seen with the behav-ioral data, the anatomical data from the present study alsoshowed a dose-dependent effect. The 0.8 mg/kg dose ofCOG1410 significantly reduced cortical cavity develop-ment following CCI. Two measures of cortical cavity as-sessment were used in the present study. The dorsal areaof the contusion cavity was imaged prior to sectioningand an area analysis showed that the vehicle-treatedgroup had the largest CCI cavity (17.37 mm2) comparedto the 0.8 mg/kg (12.85 mm2) and the 0.4 mg/kg (19.00mm2) doses of COG1410. The high dose of COG1410group showed a significant reduction compared to the ve-hicle control group. The analysis of the percent reductionin injured cortex volume compared to the noninjured cor-tex showed that the vehicle-treated group had the great-est injury induced reduction (11.95%) compared to the0.8 mg/kg (5.08%) and the 0.4 mg/kg (10.80) doses ofCOG1410. Similarly, the high-dose treatment groups sig-nificantly improved compared to the vehicle controlgroup. Thus, on both measures of injury reduction, thehigh dose of COG1410 (0.8 mg/kg) group significantlyprevented cortical loss following CCI.

Although this is the first report of COG1410’s abilityto prevent tissue loss following injury, another small mol-ecule derived from apoE has shown neuroprotective abil-ity following TBI. Administration of COG133, a largermolecule than COG1410, has shown a significant reduc-tion in hippocampal neuron loss following closed headinjury in mice (Lynch et al., 2005).

The ability of apoE to reduce glial activation and mod-ulate CNS inflammation has been previously established(Laskowitz et al., 1997, 2001; Lynch et al., 2003). Thus,we examined the ability of COG1410 to modulate reac-tive gliosis following TBI. We have previously shownthat many neuroprotective compounds show loweredGFAP expression following injury, that correlate with be-havioral improvement and reductions in cortical loss

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FIG. 6. The effects of COG1410 (0.8 mg/kg and 0.4 mg/kg,iv) or vehicle administered following unilateral CCI or shamsurgery on GFAP expression. The graph shows the plotted mean(�SEM) cell counts of GFAP� cells in the cortex surroundingthe injury cavity. Treatment with the 0.8 mg/kg dose ofCOG1410 significantly reduced the number of GFAP� cellsaround the lesion compared to saline treatment (*p � 0.05).

(Hoane et al., 2003, 2005, 2006). In the present study, itwas found that administration of COG1410 followingunilateral CCI significantly reduced the number ofGFAP� cells in the area adjacent to cortical lesion de-velopment. This supports the findings that apoE-derivedcompounds (i.e., COG133 and COG1410) have glial andinflammatory modulating capabilities in vivo.

The results of this study indicate that COG1410 (anovel apoE-mimetic peptide) has substantial preclinicalefficacy in a rodent model of TBI. A single injection at30 min post-CCI significantly improved performance ontwo out of three behavioral measures of sensorimotorfunction, reduced tissue loss in two different measure-ments, and reduced the expression of GFAP. A dose-de-pendent effect was observed, with the high dose (0.8mg/kg) being the only effective dose. Although not ex-amined in the present study, COG1410 reduced cerebral

edema in a murine model of SAH (Gao et al., 2006).This is particularly relevant because cerebral edema rep-resents a significant source of neurological morbidity inthe clinical setting following TBI, as well as in neu-rodegenerative diseases such as AD and MS. The neu-roprotective effects of apoE mimetic peptides in pre-clinical models of TBI, SAH, HIE, and MS suggest thatapoE mimetic peptides represent a novel therapy for thetreatment of neurological diseases with neuroinflamma-tory components (Gao et al., 2006; Li et al., 2006; Lynchet al., 2005; McAdoo et al., 2005). In a similar manner,a recent study has shown that soluble amyloid precursorprotein a (sAPP�), administered following weight-dropinjury, was effective in improving motor performanceand reducing neuronal injury (Thornton et al., 2006). Al-though a neuroprotective mechanism of action is sus-pected with APP�,the fact that these endogenous pro-

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FIG. 7. Representative GFAP immunoreactivity. Shown are representative images of GFAP immunoreactivity in the injuredcortex: (A) whole section (�0.44); (B) CCI-vehicle treated (�40); (C) sham injured (�40); (D) CCI-0.8 mg/kg COG1410 treated(�40). Treatment with 0.8 mg/kg COG1410 significantly reduced the number of GFAP� cells following injury compared to ve-hicle treatment. There are also observable difference in the size and complexity of the GFAP� cells in B compared to D.

teins may provide a unique treatment avenue is inter-esting.

In the present study, COG1410 promoted significantfunctional recovery when administered as a single injec-tion 30 min post-TBI. These results suggest that a mul-tiple administration paradigm, which would provideCOG1410 coverage for a sustained period of the neu-roinflammatory cascade, may prove even more benefi-cial for the treatment of TBI. These data warrant furtherevaluation of the efficacy of this peptide in other TBImodels and species and a detailed assessment of the ther-apeutic window, pharmacokinetics and pharmacodynam-ics, optimal dosing paradigm, and continured assessmentof mechanisms of action (e.g., BBB breach, edema re-duction, and acute neuroprotection). In general, takingadvantage of the anti-inflammatory, anti-excitotoxic,anti-oxidant, and neurotrophic properties of apoE-mimetic peptides may be a unique avenue of treatmentfor TBI and other disorders characterized by neuroin-flammation.

ACKNOWLEDGMENTS

Special thanks go to Nicholas Kaufman and LisaHoane for their histological assistance. Research was sup-ported by Cognosci Inc. through grants R44NS048689and r44AG020473 from the NIH.

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Address reprint requests to:Michael R. Hoane, Ph.D.

Restorative Neuroscience LaboratoryDepartment of PsychologyLife Science II, MC 6502

Southern Illinois UniversityCarbondale, IL 62901

E-mail: mhoane@siu.edu

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