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.

Introduction

T raumatic brain injury (TBI) is the leading cause of death and a major cause of disability in children and young adults in developed countries (Ariza et al., 2006; Bruns and Hauser, 2003; Cicerone et al., 2005; Colantonio et al., 2009; McCarthy et al., 2005; Tagliaferri et al., 2006). In the U.S., an estimated 1.4 million people sustain some form of TBI annually, and as of the beginning of 2005 approximately 3.17 million Americans were living with TBI-related disabilities (Zaloshnja et al., 2008), not including the nearly 25,000 soldiers now returning from war (Bhattacharjee, 2008). In addition to motor and cognitive deficits, depression is a common and persistent problem following severe TBI, affecting 10–50% of survivors during the course of their recovery (Bowen et al., 1998, 1999; Deb et al., 1999; Jorge et al., 2004; O'Donnell et al., 2004; Rogers and Read, 2007; Seel et al., 2003), especially in the first year after injury (Fann et al., 2004). The increased risk of depression is not limited to those with moderate to severe TBI, but also affects those with mild TBI (Fann et al., 2004; Hoge et al., 2008). In patients with neurological and medical conditions, depression may exacerbate neuropsychological impairment and slow the pace of cognitive recovery (Chen et al., 1996; Jorge et al., 1993; Levin and Kraus, 1994; Malberg et al., 2000; Robinson et al., 1985; Schoenhuber and Gentilini, 1988). As a result, antidepressants are commonly prescribed to treat these patients suffering from depression (Millan, 2006).

Recent studies suggest that the behavioral effects of chronic antidepressant treatment might be mediated by the stimulation of neurogenesis, neural remodeling, and synaptic plasticity in the hippocampus, processes that are induced by tricyclic antidepressants (TCAs) in rodent models of depression (Bessa et al., 2009; Malberg et al., 2000; Santarelli et al., 2003; Wang et al., 2008a) and in non-human primates (Perera et al., 2007). There is also evidence to suggest that upregulation of neurogenesis evoked by antidepressant treatment in the hippocampus can improve outcomes in patients suffering from depression (Czeh et al., 2001). However, no previous studies have examined the effects of antidepressant therapy on neurogenesis following TBI.

Imipramine is the prototype of a class of antidepressants known as the TCAs. TCAs exert their effects by selectively blocking the reuptake of serotonin (5-HT) and norepinephrine (NE) (Kocsis et al., 1988). It has been shown that significant neurogenesis can be induced in the hippocampus in response to chronic imipramine treatment (Santarelli et al., 2003). Although the mechanism of such an effect has not been well established, some studies have shown that imipramine and other antidepressants stimulate neurogenesis through upregulation of brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF2) and their receptors (Bachis et al., 2008; Castren et al., 2007; Rantamaki et al., 2007; Warner-Schmidt and Duman, 2007). In contrast, it has been reported that stress leads to downregulation of BDNF that is reversible with imipramine treatment (Tsankova et al., 2006).

It is commonly accepted that neuronal cell loss and associated neural dysfunction in the cerebral cortex, hippocampus, and thalamus are likely underlying causes of behavioral deficits after TBI (Colicos et al., 1996; Dash et al., 2001). Even though the hippocampus is particularly vulnerable to the consequences of TBI, and pathology or lesions there are frequently associated with cognitive function deficits (Ariza et al., 2006; Bonislawski et al., 2007; Hall et al., 2005; Isoniemi et al., 2006; Smith et al., 1991; Pullela et al., 2006; Tran et al., 2006), it is also a region where endogenous precursors reside that enable neurogenesis throughout the adult life of rodents and primates, including humans (Cameron and McKay, 2001; Eriksson et al., 1998; Kuhn et al., 1996; Kornack and Rakic, 1999; Leuner et al., 2007). Indeed, neuronal cells continuously regenerate from neural progenitor cells in the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. From there they migrate into the granule cell layer and develop into mature granular neurons (Kempermann and Gage, 2000; Ming and Song, 2005; Shapiro and Ribak, 2005; Zhao et al., 2006). In addition, more recent studies suggest that TBI can enhance neurogenesis in general, and specifically may stimulate cell proliferation in the DG of the hippocampus in animals of all ages (Chirumamilla et al., 2002; Chen et al., 2003; Dash et al., 2001; Rice et al., 2003; Sun et al., 2005). Furthermore, these newly-generated granular neurons are capable of integrating into the existing neuronal circuitry, and thereby could contribute to cognitive recovery (Sun et al., 2007). Therefore, reducing TBI-induced damage and enhancing neurogenesis can be a potential treatment strategy to improve the functional recovery of the hippocampus.

It has been shown that various antidepressants, including TCAs such as imipramine and selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, increase adult neurogenesis in the DG and enhance the survival of post-mitotic granule cells (Malberg et al., 2000; Nakagawa et al., 2002; Wang et al., 2008a). Furthermore, antidepressant treatment improved cognitive function, correlating with the development of newly-generated neuronal connections to the surrounding networks in the hippocampus (Wang et al., 2008a). Therefore, in our present study we examined the effect of imipramine, a commonly used TCA, on neurogenesis, and cognitive and motor function recovery after TBI in a mouse model of brain injury.

Methods

Controlled cortical impact brain injury

Male C57BL/6J mice (20–25 g; Jackson Laboratory, Bar Harbor, ME) were used in this study. All protocols involving the use of animals were approved by the University Committee on Animal Resources of the University of Rochester Medical Center, and were in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. A CCI device was used to produce brain injury as previously described (Smith et al., 1995). CCI is widely used in mice to produce TBI resulting in a large cortical contusion centered at the parietotemporal cortex, and hippocampal damage ipsilateral to the injury, in addition to widespread neurodegeneration and axonal damage (Hall et al., 2008; Smith et al., 1995). Briefly, anesthesia was induced in an isoflurane chamber (2.5%) and maintained with a face mask using 2% isoflurane, 33% O₂, and 65.5% N₂O. Body temperature was kept constant at 37.5°C using a feedback-controlled heating pad. After exposing the skull using a midline scalp incision, a 4-mm-diameter rounded craniectomy was performed over the right parietal cortex midway between the lambda and the bregma, keeping the dura mater intact. CCI was performed perpendicular to the brain's surface using the following parameters: diameter of the impact tip, 3 mm; impact velocity, 6.7 m/sec; impact duration, 100 msec; and displacement of the brain, 1 mm. After trauma, the craniotomy was covered with DuraGen^® graft matrix and the scalp was sutured closed immediately. The animals were transferred to an incubator maintained at 37°C until recovery of spontaneous motor activity. For sham surgery groups, a craniotomy was performed without CCI.

Imipramine treatment and BrdU injection

To evaluate the effect of imipramine on brain-injured mice, we subdivided both the CCI and sham-injured mouse cohorts randomly into four groups (n=12 in each group). Briefly, following CCI brain trauma or sham surgery, the animals received either daily IP imipramine injections (20 mg/kg daily) for 2 weeks (n=12) or 4 weeks (n=12), or vehicle (saline) injection for 2 weeks (n=12) or 4 weeks (n=12; Sairanen et al., 2005). The first injection of imipramine was administered 1 h post-injury. All mice received a thymidine analog that labels dividing cells in S-phase, bromodeoxyuridine (BrdU; Takahashi et al., 1992). Daily IP administration of BrdU (200 mg/kg) from the third to the seventh day at 10 am each day after CCI was used to label endogenous cells induced to proliferate by brain trauma (Sun et al., 2009). The marker was also used to determine cell survival and fate 2–4 weeks after post-traumatic imipramine or vehicle treatment.

Cognitive function evaluation

The novel object recognition test (NOR) was used to evaluate cognitive function in all four groups of mice at 1-week intervals after CCI. This method has been described in detail previously (Biegon et al., 2004; Ennaceur and Delacour, 1988; Gaskin et al., 2003). One day before testing, each mouse was individually placed in the testing chamber (a plastic transparent box 60×40 cm in size, covered by gray polyvinyl chloride plastic) for a 1-h habituation period. This was repeated for each animal. On the following day, each mouse was put back into the same chamber with two identical objects. Two object types were used: a Lego pyramid or a 50-mL plastic conical tube affixed to the floor of the chamber using Velcro tape in opposing corners of the chamber. The cumulative time spent by the mouse at each of the objects was recorded during a 5-min period by a video camera positioned over the chamber, and was later scored by an observer blinded to the treatment received. Four hours later, the mouse was reintroduced into the cage, where one of the two objects was replaced by a new one of similar size and complexity. Exploration of an object was defined as the mouse's head being oriented toward the object and within 2 cm of it. Turning around or sitting on the object was not considered as exploratory behavior. The chamber and the objects were carefully cleaned with water between each trial, and cleaned with 70% alcohol at the end of each testing session. The time spent exploring each of the objects was recorded and the total time spent by each animal exploring both old and new objects was calculated; the result is expressed as the percentages of time spent with the new object divided by the total time spent with new and old objects. Normal healthy rodents will spend relatively more time exploring a new object than a familiar one.

Motor function evaluation

Beam-walk testing was used to evaluate differences in fine motor coordination between injured and sham surgery animals as described previously (Faden et al., 1999; Fox et al., 1998). Briefly, the device consists of a narrow wooden beam 9 mm wide and 120 mm in length that is suspended 300 mm above a 60-mm-thick foam rubber pad. The animal was placed on one end of the beam, and the number of foot slippages for the left frontlimb and hindlimb (contralateral to the injured hemisphere) were recorded over 30 steps counted. The percentage of the normal frontlimb and hindlimb steps among all steps was calculated as the final result. The animals were pre-trained to walk across the beam twice before surgery. In all subsequent trials after surgery, performance was assessed 24 h after placement on the beam as a baseline level of competence at this task.

Histological analysis

At 2 weeks or 4 weeks after either CCI or sham surgery, the animals were deeply anesthetized with a mixture of ketamine and xylazine (10:1), and then transcardially perfused with 50 mL phosphate-buffered saline (PBS), followed by 50 mL of 4% paraformaldehyde in PBS. Their brains were dissected, post-fixed in 4% paraformaldehyde overnight, and then cryoprotected in 15% and 30% sucrose solutions. Next, the brains were embedded in embedding medium and sectioned into 20-μm-thick slices using a cryostat throughout the extent of the hippocampus, with all the sections mounted on glass microscope slides, and then stored in a freezer at −80°C. Cell counting was later performed by a blinded observer in every 10th section, and then multiplied by 10 to calculate the final result.

Immunohistochemistry was performed to identify proliferating cells in the hippocampus, as well as the area underlying the contusion. Toward this end, cryostat-cut sections were immunostained for BrdU, as well as the additional proliferation marker Ki-67, and immature neuron marker doublecortin (DCX). In addition, these sections were double-stained for the neuronal marker NeuN, and the glial cell marker glial fibrillary acidic protein (GFAP).

Briefly, for BrdU staining, the brain sections were thawed and air-dried for 30 min, rinsed in PBS (pH 7.4) for 5 min, then incubated at 37°C in 2 M HCl for 40 min to denature the DNA therein. After rinsing in PBS, the sections were incubated in blocking solution (5% rabbit serum and 0.1% Triton X-100) for 60 min, then incubated overnight at 4°C with BrdU rat monoclonal antibody (1:400; Abcam, Cambridge, MA) diluted in 5% normal rabbit serum and 0.1% Triton X-100. The following day, the sections were rinsed in PBS and placed in biotinylated rabbit anti-rat antibody (1:200; Vector Laboratories, Burlingame, CA) for 1 h at room temperature, and then in 3% H₂O₂/PBS for 10 min, followed by application of an avidin-biotin complex (ABC) kit (Vector Laboratories), and visualized with diaminobenzidine (DAB) for quantification. Sections were counterstained with Nissl stain to visualize the cell bodies. For the Ki-67 (1:500 rabbit polyclonal antibody; Abcam) and DCX (1:300 goat polyclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA) labeling, the protocol employed was identical to that used for BrdU minus the DNA denaturating step. The brains underwent double immunofluorescence staining with BrdU (rat, 1:400) and NeuN (1:300, mouse; Chemicon International, Temecula, CA), as well as BrdU with GFAP (1:800; mouse, Sigma-Aldrich, St. Louis, MO). The corresponding secondary antibodies used were Alexa Fluor 488 or 594 rabbit anti-rat (1:1000; Invitrogen, Carlsbad, CA) for BrdU, and Alexa Fluor 488 or 594 goat anti-mouse (1:800, Invitrogen) for NeuN and GFAP. After rinsing in PBS for 5 min, the sections were cover-slipped with Vectashield^® (Vector Laboratories) mounting media and visualized using an Olympus BX- microscope.

Quantification of labeled cells

Ten sections spanning −2.3 to −6.2 mm from the bregma were counted, and the positive cell numbers summarized across all slides for each brain. An observer blinded to the experimental conditions analyzed the BrdU-labeled cells, and Ki-67- and DCX-immunoreactive cells in the hippocampal regions CA1, CA2, and CA3 of the injured and contralateral hemispheres. The cells were visualized with an Olympus BX-51 microscope and digital images were captured at 100×magnification (Olympus DP70 digital camera). The cells were viewed using Olympus software on a large computer screen. For BrdU-, Ki-67- and DCX-labeled cell quantification, all positive cells in the DG, including the SGZ, granule cell layer (GCL), and hilus, were counted on every slide of the ipsilateral and contralateral hippocampus at 100×magnification. Quantification of cells double-labeled with BrdU/NeuN or BrdU/GFAP was done in the same regions (CA1, CA2, and CA3) of the hippocampus. The percentages of double-labeled cells relative to the total number of BrdU-positive cells in the hippocampus were compared among the mice administered different treatments.

Quantification of double-labeled BrdU/NeuN or BrdU/GFAP cells in the hippocampus of the same region of each animal was performed using a confocal laser-scanning microscope (Olympus IX81 FVF with Fluoview FV00 software). The qualities of double-labeled cells in the hippocampus were compared among the different groups.

Statistical analysis

Statistical comparisons of cognitive and motor function data between mouse cohorts were analyzed using separate repeated-measures analysis of variance (ANOVA) tests (group×days). If a significant effect was found, group comparisons adjusting for multiple testing were performed using a one-tailed Dunnett's post-hoc test comparing each group with the CCI-vehicle group. The numbers of labeled cells were compared between groups using the distribution-free Mann-Whitney U statistic. Statistical significance was set at p<0.05. All values were expressed as means±standard error. Data were analyzed with Statistica^® software (StatSoft, Tulsa, OK).

Results

Cognitive but not motor function improved with imipramine treatment after TBI

CCI led to significant motor function deficits in all animals compared to the groups given sham surgery. Particularly, the animals were affected contralateral to their injured hemisphere (their left hindlimbs), where their motor function at 24 h after injury was near zero. Their motor function recovered quickly starting at post-injury day 3, and reached a plateau at 2 weeks. The frontlimbs recovered to near-normal function at 2 weeks, but the left hindlimbs only recovered to approximately 50% of normal function at 4 weeks post-injury. No significant difference in motor function was noted between the antidepressant-treated groups and vehicle-treated groups at any time point (p>0.05; Fig. 1A).

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).

On the other hand, even though the NOR test scores of animals with CCI recovered more slowly than their motor function scores, treatment with imipramine engendered a significant beneficial effect on cognitive recovery. The antidepressant-treated group showed significant improvement in NOR test scores compared to saline-treated animals at 3 weeks after injury (p<0.001; Fig. 1B). Indeed, at week 4 the NOR test scores of the animals treated with imipramine following CCI were higher than those of the sham-surgery groups, even though all of the animals with CCI had lower scores than those given sham surgery during the first 2 weeks. However, this trend did not reach statistical significance (p>0.05).

Cell proliferation, but not survival in the hippocampus, is enhanced by imipramine treatment following TBI

Analysis of the number of BrdU-labeled cells demonstrated that chronic antidepressant administration significantly increased the number of Ki-67-positive cells in the DG relative to vehicle treatment after CCI (Fig. 2). After injured mice were given 2 weeks of imipramine treatment, a significant increase in the number of Ki-67-positive cells was observed in the ipsilateral dentate gyrus (p<0.01), but not in the contralateral side (Fig. 2). After 4 weeks of imipramine treatment, this increase was also noted in the contralateral DG (p<0.05; Fig. 2). The absolute number of Ki-67-positive cells in both groups at 4 weeks was lower than that counted at 2 weeks. The total number of Ki-67-positive cells observed in the sham-surgery group was higher than that seen in the CCI groups at both 2 weeks and 4 weeks following CCI, but the difference was not statistically significant (p>0.05).

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).

To investigate injury-induced cell survival, as well as the cell migration and the fate of newly-proliferated cells, we intraperitoneally injected a marker of DNA replication, BrdU, from the third day to the seventh day post-injury. We found enhanced levels of BrdU-positive cells in the DG of mice with CCI compared to the sham-surgery groups, at both 2 and 4 weeks following brain injury (Fig. 3). Among mice with CCI, those treated with imipramine had more BrdU-positive cells in the hippocampal DG than those given saline (p<0.01 at 2 weeks, p<0.05 at 4 weeks post-injury). The distribution of BrdU-positive cells significantly differed between those with CCI and those given only sham surgery. In the sham-surgery group, most of the BrdU-positive cells were clustered in the SGZ. For the sham-surgery group, there were 78.9% BrdU-positive cells in the imipramine-treated group, and 82.3% in the saline-treated group, at 2 weeks post-surgery. For the CCI group, there were 47% in the imipramine-treated group and 57.3% in the saline-treated group. At 4 weeks post-surgery in the sham group, there were 64.5% BrdU-positive cells in the imipramine-treated group, and 78.5% in the saline-treated group. In the CCI group there were 43.5% in the imipramine-treated group and 46.2% in the saline-treated group.

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).

In contrast, after CCI the BrdU-positive cells were more prevalent in the hilus and GCL in the imipramine-treated group than in the sham-surgery groups. At 2 weeks post-surgery, there were 24% BrdU-positive cells in the imipramine-treated group, and 21.8% in the saline-treated group. In the sham-surgery groups, there were 10.1% BrdU-positive cells in imipramine-treated group, and 8.0% in saline-treated group. At 4 weeks post-surgery, the CCI group had 40% BrdU-positive cells in the imipramine-treated group, and 36% in the saline-treated group. The sham-surgery group had only 20.5% BrdU-positive cells in the imipramine-treated group, and 8.3% in the saline-treated group. Similarly, in the GCL, the BrdU-positive cells were more prevalent in the CCI group than in the sham-surgery group. At 2 weeks post-surgery, there were 29% BrdU-positive cells in the imipramine-treated group, and 20.9% in the saline-treated group. In the sham-surgery groups, there were 10.9% BrdU-positive cells in the imipramine-treated group, and 9.5% in the saline-treated group. At 4 weeks post-surgery, the CCI group had 16% BrdU-positive cells in the imipramine-treated group, and 13.3% in the saline-treated group. The sham-surgery group had only 15% BrdU-positive cells in the imipramine-treated group, and 13.2% in the saline-treated group. Antidepressant treatment seemed to enhance the endogenous proliferative cell migration from the SGZ to the GCL and hilus, likely as part of their development into mature granular neurons and reactive astrocytes.

In order to further characterize the effect of imipramine treatment on neurogenesis and neural protection in the hippocampus of injured animals, cells immunostained with a marker of immature neurons, DCX, were counted. These late neural progenitors are particularly vulnerable to brain injury (Rola et al., 2006; Yu et al., 2008). The numbers of DCX-positive cells were lower in all CCI groups compared to the sham-surgery groups. However, the imipramine-treatment group still had more DCX-positive cells than the saline-treatment group at 4 weeks after injury, but not at 2 weeks post-injury in the bilateral hippocampus. Even though this difference was not statistically significant between the sham-surgery groups given imipramine versus those given saline, it was significant between the CCI groups given imipramine versus those given saline (p<0.05). This indicates that imipramine, and possibly other antidepressants, may have a neural-protective effect after brain trauma. Furthermore, imipramine may have a beneficial effect on maturation as well. DCX-positive cells in the imipramine-treated group had more dendrites (or increased arborization) than those observed in the saline-treated group (Fig. 4). (Wang et al., 2008a)

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).

In both the imipramine- and saline-treated groups, the number of BrdU-positive cells in the SGZ and GCL decreased during the 2–4 weeks following CCI, indicating that antidepressant treatment did not benefit survival. Meanwhile, even though the total number of BrdU-positive cells had decreased over time, there was still an increase in BrdU-positive cells in the imipramine-treated CCI group compared to the sham-vehicle group. The total numbers of BrdU-positive cells decreased 20.4% in the imipramine-treated CCI group, but only 11.5% in the saline-treated group, at 4 weeks post-surgery, and the imipramine-treated group still had more BrdU-immunostained cells than the saline-treated group. Imipramine treatment increased the cellular proliferation and migration in the DG of mice with CCI, as indicated by the presence of more cells in the hilus and GCL at all time points considered, but it did not prolong the survival of these cells. In the contralateral DG, a larger difference was observed between mice with CCI administered imipramine versus those receiving saline, even though the total number of cells was lower than in the ipsilateral DG. Between cohorts of mice with CCI, the sham-surgery group had more BrdU-positive cells in the bilateral DG after imipramine treatment than those given saline. The trend was similar for the contralateral DG between the same groups.

Imipramine treatment induces proliferative cells to differentiate into mature neurons and astrocytes in the hippocampus following TBI

To examine whether imipramine treatment affects the maturation fate of injury-induced proliferative cells in the hippocampus, these cells were double-labeled with BrdU and either NeuN or GFAP (Fig. 5). In the hippocampus, even though most of the BrdU-positive cells were co-labeled with the mature neural marker NeuN, many BrdU-positive cells were also co-labeled with the astrocyte marker GFAP, especially in the areas of the hilus and the molecular layer (ML). In the granular zone (SGZ+GCL) of the ipsilateral hippocampus in mice 4 weeks after CCI, the percentage of newly-generated cells that had differentiated into mature neurons was identified by immunofluorescence co-labeling with BrdU and NeuN. Among the mice given sham surgery, 74±3% of BrdU-positive neurons exhibited this co-labeling of BrdU and NeuN in the saline treatment group, and 78±4% of BrdU-positive neurons were co-labeled in the imipramine-treatment group. In comparison, among mice with CCI, 65%±4% of BrdU-positive neurons in the saline-treatment group and 67±6% of BrdU-positive neurons in the imipramine-treatment group exhibited both markers.

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).

Meanwhile, the percentage of these cells that had differentiated into astrocytes as indicated by co-labeling of BrdU and GFAP was 20±6% of saline-treated mice given sham surgery, 25±4% of imipramine-treated mice given sham surgery, 17±4% of saline-treated mice with CCI, and 18±8% of imipramine-treated mice with CCI (Fig. 5). Both the contralateral and ipsilateral hippocampus exhibited similar trends.

There were no significant differences in the co-labeling of cells with BrdU/NeuN or BrdU/GFAP between the saline and antidepressant treatment groups (p>0.05). Yet, based on the total numbers of BrdU-positive cells in the granular zone (SGZ+GCL), there were more newly-generated neurons in the antidepressant treatment group than in the saline-treatment group. Treatment with the antidepressant seemed to have a beneficial impact on cell proliferation and perhaps neurogenesis in the hippocampus. In contrast to cells in the granular zone, in the hilus most BrdU-positive cells were co-labeled with GFAP, but not with NeuN. Even though the percentages of labeled cells were similar and there were no significant differences among groups, based on the total numbers, more cells in the imipramine-treatment group developed into astrocytes in the hilus.

Discussion

Chronic treatment with a common tricyclic antidepressant, imipramine, improved cognitive function as measured by the NOR test, but not motor function in a mouse model of CCI. These functional benefits seen in mice administered imipramine co-occurred with histological enhancements. Antidepressant treatment has been shown to enhance neurogenesis in the normal hippocampus in a number of previous studies (Encinas et al., 2006;Malberg et al., 2000; Santarelli et al., 2003; Wang et al., 2008a). Our observations support the hypothesis that chronic administration of antidepressants after brain trauma enhances the generation of new neurons that can be incorporated into hippocampal circuits. Notably, our study also provides the first evidence that antidepressants enhance neurogenesis in the DG following TBI. Indeed, the cellular changes induced by imipramine ameliorated neurological functional recovery, as indicated by improved cognitive performance. Meanwhile, gliogenesis also increased in the hippocampus, especially in the hilus.

The role of stress in post-TBI neurogenesis cannot be underestimated. In the study of Wang and associates they used a model of ischemic stroke and ischemic stroke plus chronic mild stress (Wang et al., 2008b). They found that stress worsened outcome in rodents, accompanied by reduced proliferation of progenitor cells, as well as negatively impacting their survival and neurogenic fate (Wang et al., 2008b). Similarly in TBI we know that stress can also have negative effects post-injury. Declining post-TBI neurogenesis and symptoms of stress and depression may be casually connected. It is certainly possible that antidepressants such as imipramine function more to enhance neurogenesis and cell survival; however, they might also contribute to improved outcomes by reducing stress in these animals post-TBI.

One of the important findings of this study is that over time the new neurons seem to diminish in number, yet the behavior continues to improve. It is possible that the neurons that do survive are maturing and refining connections. It is equally possible that at 8 weeks all of the new neurons would be gone. It is likely that re-establishment of neuronal plasticity such as dendritic remodeling and synaptic contacts in the hippocampus, rather than neurogenesis, is the basis for the behavioral improvement that we observed. In fact, one recent study found that the mood-improving actions of antidepressants do not depend on neurogenesis, but are associated with neuronal remodeling (Bessa et al., 2009).

Other studies have shown that TBI-induced cell proliferation peaks approximately 2 days after injury in the ipsilateral SGZ and then decreases to a lower rate, with fewer BrdU-labeled cells than the sham group approximately 2 weeks later (Rice et al., 2003; Rola et al., 2006). In this study, we found that administering an antidepressant increased the number of neurons in the DG following CCI compared to mice given saline, suggesting that antidepressants could stimulate cell proliferation or have a neuroprotective effect that halted cell loss. To distinguish between these two possibilities, the total numbers of neurons in the DG were counted over a 4-week period. To visualize newly-generated neurons following TBI, the marker of DNA replication BrdU was administered from the third to the seventh day post-injury (Sun et al., 2009). We found that numbers of BrdU-positive cells increased immediately after injury, but then decreased 2–4 weeks after CCI. Among mice with CCI, the BrdU-positive cell number in the imipramine-treated group decreased at a faster rate than in the saline-treated groups, although their total number still remained 20% higher than the saline group at 4 weeks. In contrast, in the sham-surgery groups, the imipramine-treated animals had more BrdU-positive cells than the saline groups at all time points, especially at 4 weeks. This finding is consistent with other reports indicating that antidepressant treatment increases proliferation, but not the survival of cells in the hippocampus (Malberg et al., 2000).

However, the timing of labeling associated with proliferation in this study is novel, as the beneficial effects of antidepressants in previously studied normal or depression models were observed only after at least 2 weeks of treatment (Malberg et al., 2000; Wang et al., 2008a). The cells in the current study were labeled within the first week, from the third day after CCI and for 5 consecutive days thereafter. Potentially, antidepressant stimulation of endogenous progenitor proliferation in the DG could start earlier after TBI, or other mechanisms than those previously characterized for antidepressant treatment could change the timing of proliferation observed in this study. There might also be synergism of previously-identified separate mechanisms: TBI-induced neurogenesis and antidepressant-induced neuronal remodeling. Previous studies have documented an increase in brain-derived neurotrophic factor (BDNF) expression in the first week after TBI, particularly in the first 3 days (Hicks et al., 1997; Truettner et al., 1999; Yang et al., 1996), while other studies have shown that antidepressant treatment also stimulates the production of BDNF or other neurotrophic factors, but in a different time window, 3 weeks after treatment initiation (Sairanen et al., 2005). In this study, potentially the combination of the production of the neurotrophic factor following brain injury, juxtaposed with antidepressant treatment, induced a beneficial environment with continued elevations in BDNF that maintained the proliferation of progenitors in the DG. To further characterize the mechanism, future studies should monitor the levels of BDNF and other neurotrophic factors in the hippocampus directly after TBI, and then ongoing along with antidepressant treatment.

The fate of TBI-induced proliferative cells was characterized by immunofluorescence double-labeling with BrdU/NeuN (neurons) and BrdU/GFAP (astrocytes). These experiments in the TBI group showed that antidepressant treatment did not increase the ratio of new neurons in the hippocampus. Still, by increasing the total number of new neurons in the DG, especially in the SGZ and GCL, these newly-generated cells could contribute to functional recovery in the hippocampus. Indeed, new neurons in the GCL have been demonstrated to project axons to the pyramidal cell layer of the CA₃ region of the hippocampal formation (Markakis and Gage, 1999).

The immature neuron marker, DCX, was also detected during this study. Antidepressant treatment stimulated dendritic maturation, as indicated by increased numbers and lengths of branches (data not shown) compared with dendrites in the saline-treated groups, even though most dendritic branches became shorter from 2–4 weeks after injury (data not shown). Long processes from immature neurons (DCX-positive) extended through the GCL to the ML (data not shown), possibly enabling neuronal connections to the surrounding SGZ. This observation is akin to the effects of the selective serotonin reuptake inhibitor (SSRI) fluoxetine on immature neurons, wherein chronic treatment modified the dendritic morphology and accelerated the development of immature neurons (Wang et al., 2008a).

Compared to the animals given sham surgery, fewer DCX-positive cells were found in both the antidepressant and saline TBI groups, especially at 4 weeks post CCI. Since DCX-expressing late neural progenitors are relatively vulnerable to brain injuries (Yu et al., 2008), and antidepressant treatment could accelerate the maturation of these immature cells, the DCX-expressing time window would theoretically be shortened (Wang et al., 2008a), and the number of DCX-positive cells would be relatively lower than in vehicle-treated animals without CCI.

Furthermore, a consideration of the DCX-expressing cells in the TBI groups demonstrated that antidepressant treatment played a protective role with DCX-positive immature neural cells, as more DCX-positive cells were present in the treatment groups than the saline groups post-TBI. Potentially, the observed neuroprotective role of antidepressant treatment could be attributed to the drug's stimulation of neurotrophic factors such as BDNF (Sairanen et al., 2005).

Neural progenitor cells in the DG have been divided into two major subtypes depending on their morphologies, cell markers, and patterns of division (Bull and Bartlett, 2005; Encinas and Enikolopov, 2008). The quiescent neural progenitor (QNPs) subtype has stem cell properties, and only a small fraction (<1%) of these cells can be labeled with BrdU. Another subtype is the QNP's progeny, amplifying neural progenitors (ANPs), which can be labeled by BrdU and differentiate into granular neurons in the DG. A recent research study showed that QNPs are induced by TBI to enter the cell cycle, whereas proliferation of ANPs is not significantly affected; the molecular mechanisms behind this differential response are unknown (Gao et al., 2009). Future studies should investigate the subtypes of neural progenitor cells in the DG targeted by antidepressant treatment following TBI.

It is possible that the key mediator of neuronal fate determination is BDNF (Huang and Reichardt, 2001). Antidepressants stimulate BDNF (Sairanen et al., 2005) and its tyrosine kinase-B (TrkB) receptor (Nibuya et al., 1995), and in doing so stimulate neurogenesis. However, for antidepressant treatment of TBI, the mechanism could be more complex. In the hippocampus, TBI itself can stimulate BDNF expression within 72 h (Hicks et al., 1999; Truettner et al., 1999; Yang et al., 1996), peaking 2–3 h post-injury, and then decreasing very quickly. Meanwhile, BDNF and TrKB expression decrease in the cerebral cortex after TBI (Hicks et al., 1999). Perhaps this is why it is difficult to restore neurogenesis in the cortex of mice with CCI. Temporary increases in BDNF directly after TBI may play more of a neuroprotective role rather than inducing functional recovery and neurogenesis. However, early antidepressant treatment may enable ongoing BDNF expression after TBI, inducing neurogenesis in the DG and enhancing the functional recovery that is observed 3 weeks later.

Other growth factors, such as VEGF and FGF2, can also be induced by chronic treatment with antidepressants in animal models. FGF2 expression increases in both neurons and glial cells in the hippocampus after antidepressant administration (Bachis et al., 2008), and VEGF is a time-dependent key mediator of both the cytogenic and behavioral actions of multiple classes of antidepressants (Warner-Schmidt and Duman, 2007). Therefore, enhancement of neurogenesis in the DG after imipramine treatment of TBI and related cognitive improvements could be a complex phenomenon involving the action of multiple neurotrophic factors at different time points.

In summary, our study demonstrated that chronic administration of imipramine immediately following TBI improved cognitive function, possibly by enhancing cell proliferation and likely via neurogenesis in the hippocampus in a CCI model of brain trauma. In particular, imipramine seemed to increase trauma-induced cell differentiation of neurons in the DG and astrocytes in the hilus. Even though the exact mechanism of action of antidepressant treatment of TBI has not been established, our results suggest that the therapeutic potential of antidepressants might not be limited to the relief of depression and anxiety. Early clinical treatment of TBI (i.e., directly after brain injury) with antidepressants could facilitate neuropsychological functional recovery. Certainly, further studies of the mechanisms of action of imipramine in TBI and the role of different classes of antidepressants in TBI treatment need to be further explored in the future.

Footnotes

Acknowledgments

The authors would like to thank Ms. Beining Cao and Dr. Xiaolin Chen for help with data collection and analysis, and Dr. Ollivier Hyrien for his input on statistical analysis.

This work was supported in part by a University of Rochester SPAC grant (to J.H.H.), and by NIH grant R01-NS-067435 (to J.H.H.).

Author Disclosure Statement

No competing financial interests exist.

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