Figure 1.
Controlled cortical impact (CCI) injury model in mice.
(A) Impactor tip of an electromagnetic CCI device. (B) Schematic diagram of sites of craniotomy, cortical injury, and EEG electrode placements. (C) Cresyl violet-stained coronal section documenting typical 2 mm CCI injury, which leads to damage through the depth of the neocortex but leaves the underlying hippocampus grossly intact. *cortical injury site.
Figure 2.
The mTORC1 pathway is abnormally activated following TBI.
mTORC1 activation, as reflected by the ratio of P-S6 to total S6 expression, was significantly increased in both hippocampus and neocortex, whereas sham-operated animals showed no such increase. This increase in P-S6 expression started at 3 hr, peaked at 6 hr (A, B) and then decreased within 1 wk, returning to baseline by 2 wk (C, D) after CCI. There is no significant difference between naïve mice (Ctrl) versus sham-operated mice. *p<0.05 vs. Sham at the same time point by two-way repeated measures ANOVA. n = 8 mice per group in A,B and n = 6 mice per group in C,D.
Figure 3.
Rapamycin blocks mTORC1 activation induced by TBI.
(A, B) Rapamycin treatment, initiated one hour after CCI injury and continued daily at 6 mg/kg, inhibited mTORC1 activation at both 6 hr and 3 d following CCI, as reflected by the P-S6/S6 ratio. (C, D) Daily rapamycin treatment for 4 weeks continued to inhibit mTOR activity. (E, F) After rapamycin was stopped, mTOR activity returned to control levels. *p<0.05 vs. Ctrl+Veh; #p<0.05 vs. TBI+Veh at the same time point by two-way repeated measures ANOVA. n = 6 mice per group.
Figure 4.
Rapamycin inhibits increased P-4EBP1, but not P-STAT3, expression induced by TBI.
For comparison with P-S6, the phosphorylation of another downstream mTORC1 target (4EBP1) and a non-mTORC1 mediated phosphorylation pathway (JAK-STAT) was assessed following CCI. (A) P-4EBP1 was elevated following CCI injury and was inhibited by rapamycin. (B) In contrast, P-STAT3 was increased after CCI, but was not inhibited by rapamycin. *p<0.05 vs. Ctrl+Veh; #p<0.05 vs. TBI+Veh at the same time point by two-way repeated measures ANOVA. n = 6 mice per group.
Figure 5.
Rapamycin reduces neuronal degeneration in hippocampus following TBI.
Representative sections of Fluoro-Jade B staining in different regions of hippocampus of control mice (Ctrl, A–C), vehicle-treated TBI mice (TBI+Veh, D–F) and rapamycin-treated TBI mice (TBI+Rap, G–I) three days after CCI are shown. Abundant Fluoro-Jade B positive neurons are seen in vehicle-treated TBI mice in CA1, CA3 and DG, but to a lesser degree in rapamycin-treated TBI mice. Quantitative analysis showed a significant decrease in Fluoro-Jade B positive cells in rapamycin-treated compared to vehicle-treated TBI mice (J–L). *p<0.05 by one-way ANOVA, n = 6 mice per group.
Figure 6.
Rapamycin transiently reduces mossy fiber sprouting following TBI.
(A–D) Timm staining shows mossy fiber sprouting from control mice (Ctrl+Veh, A), and vehicle-treated TBI mice (TBI+Veh, B) and rapamycin-treated TBI mice (TBI+Rap, C) five weeks after CCI. Panels A1, B1 and C1 are higher magnification of boxed regions in panels A, B and C, respectively. Quantitative analysis demonstrates a significant increase in Timm score in vehicle-treated TBI mice compared to control mice and a significant decrease in Timm score in rapamycin-treated compared to vehicle-treated TBI mice (D). (E–H) At sixteen weeks after CCI (12 weeks after rapamycin was stopped), Timm score in rapamycin-treated TBI mice increased back to similar levels of vehicle-treated TBI mice. *p<0.05 by one-way ANOVA, n = 6 mice per group.
Figure 7.
Rapamycin attenuates development of posttraumatic epilepsy in the CCI model.
Representative EEG tracings of seizures (A) and interictal epileptiform abnormalities (B). (C) Rapamycin treatment significantly decreased the development of PTE following TBI (*p<0.05 by Mantel-Cox log-rank test).
Table 1.
Effect of Rapamycin on Posttraumatic Epilepsy (PTE) in the CCI Model.