Imaging and serum biomarkers reflecting the functional efficacy of extended erythropoietin treatment in rats following infantile traumatic brain injury

Abstract

<jats:sec> <jats:title>OBJECTIVE</jats:title> <jats:p>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.</jats:p></jats:sec> <jats:sec> <jats:title>METHODS</jats:title> <jats:p>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.</jats:p></jats:sec> <jats:sec> <jats:title>RESULTS</jats:title> <jats:p>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 &lt; 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.</jats:p></jats:sec> <jats:sec> <jats:title>CONCLUSIONS</jats:title> <jats:p>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.</jats:p></jats:sec>

Extended Combined Neonatal Treatment With Erythropoietin Plus Melatonin Prevents Posthemorrhagic Hydrocephalus of Prematurity in Rats

“…For each experiment with a statistical analysis, a power analysis was performed to estimate the required sample size (G ∗ Power 3.1.9.3). We used preliminary and published data to estimate means and standard deviations for each group for the primary outcomes of intra-aural distance, ventriculomegaly, neurodevelopmental assessment, qPCR, and DTI analyses ( Farkas et al, 2009 ; Yung et al, 2011 ; Baharnoori et al, 2012 ; Jantzie et al, 2014a , b , 2018 ; Robinson et al, 2016 ). Scanning electron microscopy qualitative analysis of EMC was set a priori as two per group.…”

“…The pluripotent cytokines, erythropoietin (EPO) and melatonin (MLT), are leading candidates for neurorepair in neonates with central nervous system (CNS) injury due to their safety profile ( Robertson et al, 2012 ), and multiple and complementary, mechanisms of action on developing neural cells ( Brines et al, 2000 ; Carloni et al, 2008 ; Mazur et al, 2010 ; Jantzie et al, 2018 ). Both EPO and MLT support the genesis, survival and maturation of neural cells ( Iwai et al, 2010 ; Mazur et al, 2010 ; Jantzie et al, 2013 ; Li et al, 2017 ; Zhang et al, 2018 ), reduce excess calpain degradation ( Samantaray et al, 2008 ; Jantzie et al, 2014b , 2016 ; Robinson et al, 2016 ), limit neuroinflammation and oxidative damage ( Carloni et al, 2016 ; Ramirez-Jirano et al, 2016 ; Dominguez Rubio et al, 2017 ; McDougall et al, 2017 ; Wang et al, 2017 ; Wei et al, 2017 ; Zhou et al, 2017 ), and suppress endoplasmic reticulum stress and mitochondrial dysfunction ( Hong et al, 2012 ; Das et al, 2013 ; Carloni et al, 2014 ; Fernandez et al, 2015 ; Hadj Ayed Tka et al, 2015 ; Zhao et al, 2015 ; Hu et al, 2016 ; Hardeland, 2017 ; Mendivil-Perez et al, 2017 ; Tang et al, 2017 ; Xue et al, 2017 ). Individually, EPO and MLT are in clinical trials to ameliorate CNS injury from extremely preterm birth, demonstrating their clinical viability as therapeutics in preterm infants ( Juul et al, 2008 ; Ohls et al, 2014 , 2016 ; Fauchere et al, 2015 ; Wu et al, 2016 ; Carloni et al, 2017 ).…”

Acute oligodendrocyte loss with persistent white matter injury in a third trimester equivalent mouse model of fetal alcohol spectrum disorder

“…For scanning, brains were embedded in 2% agarose containing 3 mM sodium azide. Images were acquired, as previously described (Jantzie et al 2015a; Robinson et al 2016), on a Bruker 4.7T BioSpec 47/40 Ultra-Shielded Refrigerated nuclear MRI system equipped with a quadrature RF coil (72 mm I.D.) and a small-bore (12 cm I.D.)…”

“…For scanning, brains were embedded in 2% agarose containing 3 mM sodium azide. Images were acquired, as previously described(Jantzie et al, 2015a;Robinson et al, Third trimester-equivalent ethanol exposure in nestin-CreER T2 :tdTomato mice Nestin-CreER T2 :tdTomato bitransgenic mice were used across all treatment groups to distinguish mature OLs and OPCs originating from the postnatal SVZ from those originating earlier in embryonic development.…”

Diffusion Tensor Imaging-Based Studies at the Group-Level Applied to Animal Models of Neurodegenerative Diseases

“…Finally, DTI-MRI has been used as read-out of treatment efficacy in rodents' models of TBI. In particular, DTI has revealed the beneficial effect on white matter integrity of activation of mitochondrial calcium fluxes (Parent et al, 2020 ), of autophagy modulators (Medina et al, 2017 ; Yin et al, 2018 ), estrogens (Kim et al, 2015 , 2017 ), metamphetamine (Ding et al, 2013 ), erythropoietin (Robinson et al, 2016 , 2018 ), tissue plasminogen activator (in mice; Xia et al, 2018 ), mGluR5 (in mice; Byrnes et al, 2012 ), and dietary modulations (Shultz et al, 2015 ; Schober et al, 2016 ; Tan et al, 2016 ). Notably, the detrimental effect of alcohol in TBI was also investigated by DTI (Kong et al, 2013 ).…”