title: Imaging and serum biomarkers reflecting the functional efficacy of extended erythropoietin treatment in rats following infantile traumatic brain injury.
abstract: OBJECTIVE Traumatic brain injury (TBI) is a leading cause of death and severe morbidity for otherwise healthy full-term infants around the world. Currently, the primary treatment for infant TBI is supportive, as no targeted therapies exist to actively promote recovery. The developing infant brain, in particular, has a unique response to injury and the potential for repair, both of which vary with maturation. Targeted interventions and objective measures of therapeutic efficacy are needed in this special population. The authors hypothesized that MRI and serum biomarkers can be used to quantify outcomes following infantile TBI in a preclinical rat model and that the potential efficacy of the neuro-reparative agent erythropoietin (EPO) in promoting recovery can be tested using these biomarkers as surrogates for functional outcomes. METHODS With institutional approval, a controlled cortical impact (CCI) was delivered to postnatal Day (P)12 rats of both sexes (76 rats). On postinjury Day (PID)1, the 49 CCI rats designated for chronic studies were randomized to EPO (3000 U/kg/dose, CCI-EPO, 24 rats) or vehicle (CCI-veh, 25 rats) administered intraperitoneally on PID1-4, 6, and 8. Acute injury (PID3) was evaluated with an immunoassay of injured cortex and serum, and chronic injury (PID13-28) was evaluated with digitized gait analyses, MRI, and serum immunoassay. The CCI-veh and CCI-EPO rats were compared with shams (49 rats) primarily using 2-way ANOVA with Bonferroni post hoc correction. RESULTS Following CCI, there was 4.8% mortality and 55% of injured rats exhibited convulsions. Of the injured rats designated for chronic analyses, 8.1% developed leptomeningeal cyst-like lesions verified with MRI and were excluded from further study. On PID3, Western blot showed that EPO receptor expression was increased in the injured cortex (p = 0.008). These Western blots also showed elevated ipsilateral cortex calpain degradation products for αII-spectrin (αII-SDPs; p < 0.001), potassium chloride cotransporter 2 (KCC2-DPs; p = 0.037), and glial fibrillary acidic protein (GFAP-DPs; p = 0.002), as well as serum GFAP (serum GFAP-DPs; p = 0.001). In injured rats multiplex electrochemiluminescence analyses on PID3 revealed elevated serum tumor necrosis factor alpha (TNFα p = 0.01) and chemokine (CXC) ligand 1 (CXCL1). Chronically, that is, in PID13-16 CCI-veh rats, as compared with sham rats, gait deficits were demonstrated (p = 0.033) but then were reversed (p = 0.022) with EPO treatment. Diffusion tensor MRI of the ipsilateral and contralateral cortex and white matter in PID16-23 CCI-veh rats showed widespread injury and significant abnormalities of functional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD); MD, AD, and RD improved after EPO treatment. Chronically, P13-P28 CCI-veh rats also had elevated serum CXCL1 levels, which normalized in CCI-EPO rats. CONCLUSIONS Efficient translation of emerging neuro-reparative interventions dictates the use of age-appropriate preclinical models with human clinical trial-compatible biomarkers. In the present study, the authors showed that CCI produced chronic gait deficits in P12 rats that resolved with EPO treatment and that chronic imaging and serum biomarkers correlated with this improvement.
authors.affiliation: Brain Injury Center and |Departments of 2 Neurosurgery.|Neurology.|F. M. Kirby Center for Neurobiology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; and 
authors.forename: Shenandoah
authors.initials: S
authors.lastname: Robinson
authors.affiliation: Brain Injury Center and |Departments of 2 Neurosurgery.
authors.forename: Jesse L
authors.initials: JL
authors.lastname: Winer
authors.affiliation: Brain Injury Center and |Emergency Medicine.
authors.forename: Justin
authors.initials: J
authors.lastname: Berkner
authors.affiliation: Brain Injury Center and |Departments of 2 Neurosurgery.
authors.forename: Lindsay A S
authors.initials: LA
authors.lastname: Chan
authors.affiliation: Departments of 8 Pediatrics.
authors.forename: Jesse L
authors.initials: JL
authors.lastname: Denson
authors.affiliation: Departments of 8 Pediatrics.
authors.forename: Jessie R
authors.initials: JR
authors.lastname: Maxwell
authors.affiliation: Neurology, University of New Mexico School of Medicine, Albuquerque, New Mexico.
authors.forename: Yirong
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Neurology, University of New Mexico School of Medicine, Albuquerque, New Mexico.
authors.forename: Laurel O
authors.initials: LO
authors.lastname: Sillerud
authors.affiliation: Brain Injury Center and |Neurology.|Anesthesiology.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Tasker
authors.affiliation: Brain Injury Center and |Sports Medicine, and.
authors.forename: William P
authors.initials: WP
authors.lastname: Meehan
authors.affiliation: Brain Injury Center and |Emergency Medicine.
authors.forename: Rebekah
authors.initials: R
authors.lastname: Mannix
authors.affiliation: Departments of 8 Pediatrics.|Neurosciences, and.
authors.forename: Lauren L
authors.initials: LL
authors.lastname: Jantzie
mesh_terms: D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000097:blood*; D000070642:Brain Injuries, Traumatic / Q000097:blood* / Q000150:complications / Q000000981:diagnostic imaging* / Q000188:drug therapy*; D002154:Calpain / Q000378:metabolism; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D016207:Cytokines / Q000097:blood; D038524:Diffusion Magnetic Resonance Imaging; D004195:Disease Models, Animal; D000068817:Epoetin Alfa / Q000378:metabolism; D004921:Erythropoietin / Q000627:therapeutic use*; D005260:Female; D020233:Gait Disorders, Neurologic / Q000188:drug therapy / Q000209:etiology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008297:Male; D051381:Rats; D017467:Receptors, Erythropoietin / Q000378:metabolism; D018709:Statistics, Nonparametric; D027981:Symporters; D013997:Time Factors; D000096922:K Cl- Cotransporters
chemical_list: D015415:Biomarkers; D002154:Calpain; D016207:Cytokines; D000068817:Epoetin Alfa; D004921:Erythropoietin; D005904:Glial Fibrillary Acidic Protein; D017467:Receptors, Erythropoietin; D027981:Symporters; D000096922:K Cl- Cotransporters
keywords: AD = axial diffusivity; CCI = controlled cortical impact; CXCL1 = chemokine (CXC) ligand 1; DP = degradation product; DTI = diffusion tensor imaging; EP = echo planar; EPO = erythropoietin; EPOR = EPO receptor; FA = fractional anisotropy; GFAP = glial fibrillary acidic protein; IFNγ = interferon gamma; IL = interleukin; KCC2 = potassium chloride cotransporter 2; MD = mean diffusivity; MECI = multielectrochemiluminescence; P = postnatal day; PID = postinjury day; RD = radial diffusivity; ROI = region of interest; SWI = susceptibility-weighted imaging; TBI = traumatic brain injury; TNFα = tumor necrosis factor alpha; controlled cortical impact; diffusion tensor imaging; diffusivity; erythropoietin; infant; serum biomarker; trauma; traumatic brain injury; veh = vehicle; αII-SDPs = αII-spectrin DPs