Imipramine treatment improves cognitive outcome associated with enhanced hippocampal neurogenesis after traumatic brain injury in mice

Abstract

Previous animal and human studies have demonstrated that chronic treatment with several different antidepressants can stimulate neurogenesis, neural remodeling, and synaptic plasticity in the normal hippocampus. Imipramine is a commonly used tricyclic antidepressant (TCA). We employed a controlled cortical impact (CCI) mouse model of traumatic brain injury (TBI) to assess the effect of imipramine on neurogenesis and cognitive and motor function recovery after TBI. Mice were given daily imipramine injections for either 2 or 4 weeks after injury. Bromodeoxyuridine (BrdU) was administered 3-7 days post-brain injury to label the cells that proliferated as a result of the injury. We assessed the effects of imipramine on post-traumatic motor function using a beam-walk test and an assessment of cognitive function: the novel object recognition test (NOR). Histological analyses were performed at 2 and 4 weeks after CCI. Brain-injured mice treated with imipramine showed significantly improved cognitive function compared to a saline-treated group (p<0.001). However, there was no significant difference in motor function recovery between imipramine-treated and saline-treated mice. Histological examination revealed increased preservation of proliferation of Ki-67- and BrdU-positive cells in the hippocampal dentate gyrus (DG) at 2 and 4 weeks after TBI. Immunofluorescence double-labeling with BrdU and neuron-specific markers at 4 weeks after injury showed that most progenitors became neurons in the DG and astrocytes in the hilus. Notably, treatment with imipramine increased preservation of the total number of newly-generated neurons. Our findings provide direct evidence that imipramine treatment contributes to cognitive improvement after TBI, perhaps by enhanced hippocampal neurogenesis.

FIG. 1.

Effects of the antidepressant (AD) imipramine on cognitive and motor function recovery. (A) Motor function was evaluated by beam-walk testing. There was no statistically significant difference observed between the imipramine- and saline-treated groups at any time point. (B) The graph depicts the results of a cognitive function evaluation using a novel object recognition (NOR) test performed in different groups of mice treated with either imipramine or saline. The imipramine-treated mice with controlled cortical impact (CCI) showed a significant improvement in cognitive function compared to the saline-treated CCI group at 3 weeks (*p<0.001; TBI, traumatic brain injury; front, frontlimb; hind, hindlimb).

FIG. 2.

Antidepressant (AD) treatment preserves cell proliferation in the hippocampus following traumatic brain injury (TBI). Images A–D represent 10×and 100×images of the Ki-67-labeled cells in the dentate gyrus. (A) Two-week TBI-vehicle. (B) Two-week TBI-imipramine. (C) Four-week TBI-vehicle. (D) Four-week TBI-imipramine. (E) Summary of the data for chronic imipramine administration after controlled cortical impact (CCI) indicates a significant increase in the number of Ki-67-positive cells in the hippocampus of mice treated with imipramine compared to saline-treated mice at both the 2- and 4-week time points (*p<0.01; **p<0.05; ; scale bars in A–D top row=200 μm; in A and B bottom row=10 μm; in C and D bottom row=20 μm).

FIG. 3.

Imipramine treatment influences the fate and migration of traumatic brain injury (TBI)-induced proliferative cells in the hippocampus. Images A–D represent 10×and 100×images of bromodeoxyuridine (BrdU)-positive cells in the dentate gyrus. (A) Two-week TBI-vehicle. (B) Two-week TBI-imipramine. (C) Four-week TBI-vehicle. (D) Four-week TBI-imipramine. (E) Graph showing the total number of BrdU-positive cells in the dentate gyrus for the different treatment groups at either 2 weeks or 4 weeks after injury (AD, antidepressant; SGZ, subgranular zone; GCL, granule cell layer; scale bars, top row: A=200 μm, B and D=100 μm, C=50 μm; bottom row: A and B=10 μm, C and D=20 μm; (*p<0.01; **p<0.05).

FIG. 4.

Imipramine treatment enhances the maturation of neural progenitor cells in the hippocampus after traumatic brain injury (TBI). (A–D) Low-magnification photomicrographs (20×) showing the morphology and distribution of immature neural progenitor cells expressing doublecortin (DCX) after TBI. (A) Subgranular zone (SGZ) of the ipsilateral dentate gyrus (DG) 2 weeks after TBI. (B) The ipsilateral DG 2 weeks after TBI. (C) Ipsilateral DG taken 4 weeks after TBI, showing reduced numbers of neurons labeled with DCX compared to neurons at 2 weeks. (D) Ipsilateral DG taken 4 weeks after TBI and chronic imipramine treatment, showing reduced numbers of DCX-labeled cells compared to 2 weeks of the same treatment. (A1, B1, C1, and D1) Higher-magnification images of A, B, C, and D, respectively, enabling the visualization of the dendrite morphology of DCX-labeled cells. (E) Graph summarizes the number of DCX-labeled cells in the DG (scale bars in A–D=100 μm, in A1–D1=50 μm; AD, antidepressant; *p<0.05).

FIG. 5.

Progenitor cells differentiate into mature neurons and astrocytes in the hippocampus following traumatic brain injury (TBI). Cells were double-labeled with bromodeoxyuridine (BrdU), and either the neuronal marker NeuN or glial fibrillary acidic protein (GFAP), 4 weeks after TBI. (A–C) Immunofluorescence images show NeuN (green), BrdU (red), and merged cells (orange, arrows) in the granule cell layer (GCL) (A), subgranular zone (SGZ) (B), and hilus (C), indicating that most BrdU-positive cells had migrated to the GCL and SGZ and differentiated into mature neurons. In the hilus (C), most of the BrdU-positive cells were not co-labeled with NeuN, indicating that they did not differentiate into mature neurons. (D–F) These immunofluorescence images show GFAP (green), BrdU (red), and merged cells (orange, arrows) in the SGZ (D) and GCL (E). Few cells have differentiated into astrocytes; however, in the hilus (F), most BrdU-positive cells differentiated into astrocytes, as indicated by co-labeling with BrdU and GFAP. (G) This bar graph summarizes the number of newly-generated cells in the ipsilateral and contralateral DG that differentiated into mature neurons, as indicated by co-labeling with BrdU and NeuN, or astrocytes, as indicated by co-labeling with BrdU and GFAP (AD, antidepressant; CCI, controlled cortical impact; scale bars=100 μm; *p<0.01; **p<0.05).