Beneficial Effects Of Carbamylated Erythropoietin Against Traumatic Brain Edema: Proposed Molecular Mechanisms Of Action

Studies have shown that the hematopoietic agent erythropoietin (EPO)is also strongly neuroprotective; in vivo it lessens brain edema after stroke,traumatic brain injury, and subarachnoid hemorrhage. However, EPO is notan ideal drug candidate for treating diseases of the central nervous systembecause it leads to thromboembolic and hematopoietic complications. Thusresearch has focused on the development of chemical derivatives toseparate EPO’ s neuroprotective and erythropoietic properties.Carbamylated EPO (C-EPO), one of the EPO derivatives, retain anti-edemaand neuroprotective properties but is not hematopoietic. C-EPO is thereforemore useful and safer than EPO for treating diseases of the central nervoussystem, including serious brain edema. Yet, beneficial effects ofcarbamylated erythropoietin against traumatic brain edema and theproposed molecular mechanisms of action are unknown.This study investigated the intracellular and molecular mechanisms underlying the anti-edema property of C-EPO.We first studied the effects of C-EPO on traumatic brain edema in rats.168SD rats were randomly divided into four groups: Normal group, Shamgroup, TBI group, C-EPO treatment group. After suffering from TBI, ratswere intraperitoneal injected with C-EPO100ug/kg every day, or the samevolume of physiological saline. Neurologic function of rats was observed bya mNSS; Dry-wet weight method was used to detect brain tissue watercontent; E-B assay was used to detect blood brain barrier (BBB)permeability; HE staining was used to detect brain tissue pathologicalchanges. Using RT-PCR to detect levels of AQP4mRNA expression; UsingWestern Blot to detect phosphorylation levels of PKC and the expressionlevels of AQP4protein; with immunofluorescence staining method, AQP4protein expression was further detected.Results:(1) the brain water content: Brain water content of C-EPOtreatment group rats was significantly lower than the TBI group6h and1,3d after TBI (P <0.05).(2) EB staining: EB content in C-EPO treatmentgroup was significantly lower than the TBI group1,3d after TBI (P <0.05).(3) H E staining: The swelling, damage and necrosis of nerve cells in rhEPOgroup is quite lighten than TBI group.(4) mNSS score: mNSS score in EPOtreatment group reduced statistically significant compared with TBI group3,7, and14d after injury (P <0.05).(5) C-EPO therapy significantlyimproved the phosphorylation levels of PKC, and reduced the high expression levels of AQP4after TBI.An in vitro model of astrocyte swelling was then created by5h ofoxygen–glucose deprivation and subsequent reperfusion (OGD/Rep).Astrocyte cultures were then treated with C-EPO or left as control cells.Here we show that increases in astrocyte volume, morphological cellswelling, and changes in ultrastructure after OGD/Rep were significantlymitigated by treatment with C-EPO (10ng/ml). The decreases in AQP-4phosphorylation after OGD/Rep were remarkably recovered by C-EPOtreatment. The OGD/Rep-induced upregulations of AQP-4mRNA andprotein were also prevented by C-EPO treatment. Additional treatment withH-7, an inhibitor of PKC, blocked these protections. Our findings establishthat C-EPO effectively mitigates astrocyte swelling induced by ischemiaand reperfusion-like injury. The modulation of AQP-4phosphorylation andexpression via the PKC pathway is participated in the anti-edemaneuroprotective effects of C-EPO.