Statins increase neurogenesis in the dentate gyrus, reduce delayed neuronal death in the hippocampal CA3 region, and improve spatial learning in rat after traumatic brain injury

Abstract

Traumatic brain injury (TBI) remains a major public health problem globally. Presently, there is no way to restore cognitive deficits caused by TBI. In this study, we seek to evaluate the effect of statins (simvastatin and atorvastatin) on the spatial learning and neurogenesis in rats subjected to controlled cortical impact. Rats were treated with atorvastatin and simvastatin 1 day after TBI and daily for 14 days. Morris water maze tests were performed during weeks 2 and 5 after TBI. Bromodeoxyuridine (BrdU; 50 mg/kg) was intraperitoneally injected 1 day after TBI and daily for 14 days. Brain tissue was processed for immunohistochemical staining to identify newly generated cells and vessels. Our data show that (1) treatment of TBI with statins improves spatial learning on days 31-35 after onset of TBI; (2) in the non-neurogenic region of the hippocampal CA3 region, statin treatment reduces the neuronal loss after TBI, demonstrating the neuroprotective effect of statins; (3) in the neurogenic region of the dentate gyrus, treatment of TBI with statins enhances neurogenesis; (4) statin treatment augments TBI-induced angiogenesis; and (5) treatment with simvastatin at the same dose provides a therapeutic effect superior to treatment with atorvastatin. These results suggest that statins may be candidates for treatment of TBI.

FIG. 1

The plot shows the spatial learning deficits of rats on days 11–15 after traumatic brain injury (TBI). Rats were trained on the Morris water maze test starting at day 11 after TBI, and daily for 5 days (before sacrifice). Rats in the sham group spent a longer time in the correct quadrant searching for the platform (~55%) than rats from the saline- and atorvastatin-treated groups, with no significant difference between the saline-treated group and atorvastatin-treated group.

FIG. 2

The plot shows the spatial learning deficits of rats on days 31–35 after traumatic brain injury (TBI). Rats were trained on the Morris water maze test on days 31–35 after TBI. Rats in the statin-treated groups showed significant increases in time spent in the correct quadrant on days 33–35 after TBI when compared to the saline-treated group (*p < 0.05). Simvastatin-treated rats showed significant increases of time spent in the correct quadrant at days 34 and 35 when compared to the atorvastatin-treated group (^#p < 0.05).

FIG. 3

Fluorescent staining for MAP-2 (green) counterstained with PI (red) shows the density of neurons in the ipsilateral hippocampal CA3 region in the sham (a), saline-treated (b), atorvastatin-treated (c), and simvastatin-treated (d) animals at 35 days after traumatic brain injury (TBI). Scale bar = 50 μm (d).

FIG. 4

Immunohistochemical staining with diaminobenzidine (DAB) for bromodeoxyuridine (BrdU; counterstained with hematoxylin shows the density of BrdU-labeled cells in the ipsilateral dentate gyrus in the sham (a), saline-treated (b), atorvastatin-treated (c), and simvastatin-treated (d) animals at 35 days after traumatic brain injury (TBI). The lower panel is the inset of the upper panel. Scale bar = 100 μm.

FIG. 5

Double-fluorescent staining for bromodeoxyuridine (BrdU; red) and neuronal nuclei (NeuN; blue) shows the BrdU-labeled (red only) and BrdU/NeuN-colabeled cells (pink) in the ipsilateral dentate gyrus in the sham (a), saline-treated (b), atorvastatin-treated (c), and simvastatin-treated (d) animals at 35 days after traumatic brain injury (TBI). Scale bar = 25μm (d).

FIG. 6

Fluorescein isothiocyanate (FITC)–conjugated dextran perfused vessels were captured by laser confocal microscopy in the ipsilateral dentate gyrus in the sham (a), saline-treated (b), atorvastatin-treated (c), and simvastatin-treated (d) animals at 35 days after traumatic brain injury (TBI). Scale bar = 100 μm (d).

FIG. 7

Bar graph shows that vessels containing bromodeoxyuridine (BrdU)–labeled cells were detected in the saline-treated rats at 15 days after traumatic brain injury (TBI) and the density of vessels increased at 35 days after TBI, demonstrating the presence of TBI-induced angiogenesis in the dentate gyrus, consistent with TBI-induced neurogenesis in the dentate gyrus. Treatment with atorvastatin significantly enhanced TBI-induced angiogenesis at 15 and 35 days after TBI compared with the saline-treated group (*p < 0.05). However, simvastatin is more effective than atorvastatin in the induction of angiogenesis at 35 days after TBI (^#p < 0.05).