Comparison of the effect of minocycline and simvastatin on functional recovery and gene expression in a rat traumatic brain injury model

Abstract

The goal of this study was to compare the effects of minocycline and simvastatin on functional recovery and brain gene expression after a cortical contusion impact (CCI) injury. Dosage regimens were designed to provide serum concentrations in a rat model in the range obtained with clinically approved doses; minocycline 60 mg/kg q12h and simvastatin 10 mg/kg q12h for 72 h. Functional recovery was assessed using motor and spatial learning tasks and neuropathological measurements. Microarray-based transcriptional profiling was used to determine the effect on gene expression at 24 h, 72 h, and 7 days post-CCI. Gene Ontology analysis (GOA) was used to evaluate the effect on relevant biological pathways. Both minocycline and simvastatin improved fine motor function, but not gross motor or cognitive function. Minocycline modestly decreased lesion size with no effect of simvastatin. At 24 h post-CCI, GOA identified a significant effect of minocycline on chemotaxis, blood circulation, immune response, and cell to cell signaling pathways. Inflammatory pathways were affected by minocycline only at the 72 h time point. There was a minimal effect of simvastatin on gene expression 24 h after injury, with increasing effects at 72 h and 7 days. GOA identified a significant effect of simvastatin on inflammatory response at 72 h and 7 days. In conclusion, treatment with minocycline and simvastatin resulted in significant effects on gene expression in the brain reflecting adequate brain penetration without producing significant neurorestorative effects.

FIG. 1.

Concentration-time curves. (A) Minocycline 60 mg/kg oral every 12 h for 72 h. (B) Simvastatin 10 mg/kg orally every 12 h for 72 h. After 12 h, only peak concentrations were obtained. Simvastatin activity was determined as pravastatin equivalents.

FIG. 2.

(A) The locomotor placing task showing movement (+standard error of the mean [SEM]) for days 2, 4, 6, 8, and 10 post-cortical contusion impact (CCI). Overall, both the minocycline (MIN p=0.004) and simvastatin (SIM p=0.019) animals moved more than the vehicle (VEH) animals during the task. (B) The locomotor placing task showing the average fault scores (+SEM) for days 2, 4, 6, 8, and 10 post- CCI. Overall, both the minocycline (p<0.001) and simvastatin (p=0.003) animals performed better than the vehicle animals.

FIG. 3.

(A) The Rotarod test showing the average latency to fall (+standard error of the mean [SEM]) off of the rotating cylinder for days 8–12 post-cortical contusion impact (CCI). No significant differences were found between the treated animals and vehicle (VEH) animals. (B) The MWM reference memory task showing the average latency (+SEM) to reach the platform on days 14–17 post-CCI. No significant differences were found between the treated animals and VEH animals. The MWM working memory task showing the average latency (+SEM) to reach the platform on days 21–23 post-CCI. No significant differences were found between the treated animals and VEH animals. SIM, simvastatin; MIN, minocycline.

FIG. 4.

Lesion analysis. The average (+standard error of the mean) percent reduction score. The minocycline (MIN)-treated group had reduced lesion volumes compared with the vehicle (VEH)-treated group, but the simvastatin (SIM)-treated group showed no improvement. *=p<0.05.

FIG. 5.

TaqMan based RT-PCR validation of the microarray data for the selected genes: Ccr2 (chemokine (C-C) receptor 2), CxCl2 (chemokine [C-X-C motif], ligand 2), Cyp1b1 (cytochrome P450 1b1), Hmox1 (hemeoxygenase 1), Hspb1 (heat shock protein b1), IGF2 (insulin like growth factor 2), Il1b (interleukin 1 beta), Il16 (interleukin 16), Il18 (interleukin 18), IL1rn (interleukin 1 receptor antagonist), Mmp8 (matrix metallopeptidase 8), Mmp9 (matrix metallopeptidase 9), Ptgs2 (prostaglandin-endoperoxide synthase 2), S100a9 (S100 calcium binding protein A9. The RT-PCR data shown in the figure were normalized to the housekeeping gene -actin.