Comparison of the effects of erythropoietin and anakinra on functional recovery and gene expression in a traumatic brain injury model

Abstract

The goal of this study was to compare the effects of two inflammatory modulators, erythropoietin (EPO) and anakinra, on functional recovery and brain gene expression following a cortical contusion impact (CCI) injury. Dosage regimens were designed to provide serum concentrations in the range obtained with clinically approved doses. Functional recovery was assessed using both motor and spatial learning tasks and neuropathological measurements conducted in the cortex and hippocampus. Microarray-based transcriptional profiling was used to determine the effect on gene expression at 24 h, 72 h, and 7 days post-CCI. Ingenuity Pathway Analysis was used to evaluate the effect on relevant functional categories. EPO and anakinra treatment resulted in significant changes in brain gene expression in the CCI model demonstrating acceptable brain penetration. At all three time points, EPO treatment resulted in significantly more differentially expressed genes than anakinra. For anakinra at 24 h and EPO at 24 h, 72 h, and 7 days, the genes in the top 3 functional categories were involved in cellular movement, inflammatory response and cell-to-cell signaling. For EPO, the majority of the genes in the top 10 canonical pathways identified were associated with inflammatory and immune signaling processes. This was true for anakinra only at 24 h post-traumatic brain injury (TBI). The immunomodulation effects of EPO and anakinra did not translate into positive effects on functional behavioral and lesion studies. Treatment with either EPO or anakinra failed to induce significant beneficial effects on recovery of function or produce any significant effects on the prevention of injury induced tissue loss at 30 days post-injury. In conclusion, treatment with EPO or anakinra resulted in significant effects on gene expression in the brain without affecting functional outcome. This suggests that targeting these inflammatory processes alone may not be sufficient for preventing secondary injuries after TBI.

Keywords: anakinra; cortical contusion impact; erythropoietin; gene expression; recovery of function; traumatic brain injury.

Figure 1

Concentration-time curves after administration of (A) EPO 2500 mg/kg i.p. for the first dose, followed by s.c. every 12 h and (B) anakinra 100 mg/kg i.p. for the first dose, followed by s.c. every 12 h.

Figure 2

Sensorimotor Assessment. (A) The Locomotor Placing task showing the average fault scores (+SEM) for days 2, 4, 6, 8, and 10 post-CCI. No significant differences between the treated animals and vehicle were found. A trend toward worsening was seen in the anakinra-treated group. (B) The Rotorod test showing the average latency to fall (+SEM) off of the rotating cylinder for day's 8–12 post-CCI. No significant differences between the treated animals and vehicle were found.

Figure 3

Cognitive assessment. (A) The MWM reference memory task showing the average latency (+SEM) to reach the platform on day's 14–17 post-CCI. No significant differences between the treated animals and vehicle were found. (B) The MWM working memory task showing the average latency (+SEM) to reach the platform on day's 21–23 post-CCI. No significant differences between the treated animals and vehicle were found.

Figure 4

Lesion Analysis. (A) The average (+SEM) percent reduction in hemispherical, cortical, and hippocampal volumes between the ipsilateral and contralateral sides of the injury. No significant differences between the treated animals and vehicle were found. (B) Representative images of cresyl violet stained tissue throughout the injury coordinates; −0.08 mm, −1.8 mm, −2.8 mm, and −3.8 mm, relative to bregma. Scale bar = 2.0 mm.

Figure 5

The Venn diagrams show the number of genes whose expression was up or down regulated more than 1.5-fold (p < 0.05) in the EPO/Vehicle and Anakinra/Vehicle contrasts at the 24 h, 72 h, and 7 day time points. Venn diagrams were generated with the Bioconductorlimma package.

Figure 6

TaqMan based RT-PCR validation of the microarray data for the selected genes: Alox15 (arachidonate 15-lipoxygenase), Casp12 (caspase 12), CD68 (CD 68 molecule), Cyp1b1 (cytochrome P450 1b1), Gal (galanin), Hmox1 (hemeoxygenase 1), IGF2 (insulin like growth factor 2), IL1rn (interleukin 1 receptor antagonist), Mdk (midkine), Mmp9 (matrix metallopeptidase 9), Niacr1 (niacin receptor 1), S100a9 (S100 calcium binding protein A9), Xdh (xanthine dehydrogenase). The RT-PCR data was normalized to the housekeeping gene β-actin. Pearson's coefficient, r = 0.859.