Temporally specified genetic ablation of neurogenesis impairs cognitive recovery after traumatic brain injury

Abstract

Significant spontaneous recovery occurs after essentially all forms of serious brain injury, although the mechanisms underlying this recovery are unknown. Given that many forms of brain injury such as traumatic brain injury (TBI) induce hippocampal neurogenesis, we investigated whether these newly generated neurons might play a role in recovery. By modeling TBI in transgenic mice, we determined that injury-induced newly generated neurons persisted over time and elaborated extensive dendritic trees that stably incorporated themselves throughout all neuronal layers of the dentate gyrus. When we selectively ablated dividing stem/progenitors at the time of injury with ganciclovir in a nestin-HSV-TK transgenic model, we eliminated injury-induced neurogenesis and subsequently diminished the progenitor pool. Moreover, using hippocampal-specific behavioral tests, we demonstrated that only injured animals with neurogenesis ablated at the time of injury lost the ability to learn spatial memory tasks. These data demonstrate a functional role for adult neurogenesis after brain injury and offer compelling and testable therapeutic options that might enhance recovery.

Figure 1.

Controlled cortical injury induces stable dentate gyrus neurogenesis. A–G, Nestin–CreERT2/stop–YFP–ROSA26 mice given 3 d of tamoxifen show YFP-expressing cells that appear to be neurons (A–F, arrowhead) and type 1 stem/progenitors (D, arrow) in the basal granular layer 2 months after tamoxifen and mock injury. B, E, After injury, YFP-expressing neuronal-appearing cells are found throughout the granular layer (arrowheads) on the injured side. C, F, The uninjured side shows a few neurons outside the basal granular layer (arrowhead), but most YFP-expressing cells are type 1 early progenitors in the subgranular zone (F, arrow). G, Unbiased stereological counts of YFP-expressing cells in the granular zone show a threefold increase only in the injured dentate gyrus. Numbers graphed are total numbers of dentate gyrus cells estimated using unbiased stereology (see Materials and Methods). Results shown are from n = 6 in injured animals and n = 4 in controls. Scale bars: A, 50 μm; D, 200 μm. **p < 0.01 using ANOVA with Bonferroni's post hoc correction.

Figure 2.

YFP-expressing cells in the dentate gyrus express neuronal markers. A–H, Nestin–CreERT2/stop–YFP–ROSA26 mice given 3 d of tamoxifen, injured, and killed 2 months later show YFP-expressing cells that colocalize with the neuronal marker NeuN and the astrocyte/progenitor marker GFAP. Control dentate gyrus shows NeuN/YFP-expressing cells restricted to inner granular layer (A–D, arrows), whereas in injured animals, these cells are more numerous and found in outer granular layer (E–H, arrowheads). Cells expressing both YFP and GFAP remain restricted to the subgranular zone and are not found in reactive astrocytes in the hilus or outside granular layer (C,D, G,H). Scale bar: 50 μm.

Figure 3.

Injury-induced neurogenesis is ablated in ganciclovir-exposed nestin–HSV–TK transgenic mice. A, B, The same injury was performed on nestin–HSV–TK transgenic mice given a single dose of CldU 3 d after injury and treated with ganciclovir for 4 weeks after injury. One month later (2 months after injury), the only mice that demonstrate stable neurogenesis are transgenic mice not given ganciclovir, control mice given ganciclovir, or control mice not given ganciclovir (A). During blinded counting, there is a 90% ablation of neurogenesis only in transgenic animals exposed to ganciclovir (B). C, D, DCX-expressing committed neuroblasts are persistently decreased 2 months after injury and 1 month after ganciclovir treatment in nestin–HSV–TK–GFP transgenic mice. C, In injured transgenic mice given ganciclovir, both the injured and uninjured dentate gyrus have decreased numbers of DCX-expressing cells when unbiased stereological counts are performed 2 months after injury. In control but injured groups, DCX-expressing cells are increased on the uninjured side. D, In uninjured control groups, there are decreased numbers of DCX-expressing cells in the TK mice with ganciclovir. E, F, GFP-expressing early stem/progenitors are persistently decreased 2 months after injury and 1 month after ganciclovir treatment. E, In injured nestin–HSV–TK–GFP transgenic mice, both the injured and uninjured dentate gyrus have equivalent but decreased numbers of GFP-expressing cells when unbiased stereological counts are performed 2 months after injury. In control but injured groups, GFP-expressing progenitors are increased on the uninjured side. F, In uninjured control groups, there is a trend toward decreased numbers of GFP-expressing progenitors in the TK mice with ganciclovir, although this does not reach statistical significance. Numbers graphed are total numbers of dentate gyrus cells estimated using unbiased stereology (see Materials and Methods). *p < 0.05 and **p < 0.01 using t test comparing paired samples in C and E. For B, D, and F, ANOVA with Bonferroni's post hoc analysis was used. n = 5 for Injury+/TK+/gan+, 5 for Injury+/TK+/gan−, 4 for Injury−/TK+/gan+, 4 for Injury−/TK−/gan+, 3 for Control/TK+/gan+, 4 for Control/TK+/gan−, 4 for Control/TK−/gan−, and 3 for Control/TK+/gan−. Scale bar, 50 μm.

Figure 4.

Ganciclovir does not ablate reactive astrocytosis after injury in nestin–HSV–TK transgenic mice. Transgenic nestin–HSV–TK mice were injured and given ganciclovir via implanted osmotic pumps for 30 d and injected with BrdU on days 1–3 after injury. A, E, There was no difference in overall GFAP-expressing astrogliosis in the hippocampus of TK-expressing mice given either vehicle or ganciclovir. A–C, E–G, The number of dividing cells evidenced by BrdU incorporation at 1–3 d after injury was also similar 30 d later (101 ± 13.6 for control, 112 ± 14.4 for Gan-treated; n = 3 for control and 4 for Gan-treated; not significant). D, H, A subset of reactive astrocytes from each group express BrdU, GFAP, and the mature astrocyte marker glutamine synthetase (GS).

Figure 5.

Neither mechanical brain injury nor reduction of neurogenesis affects contextual or cued fear conditioning, but mechanical brain injury results in impaired motor coordination. A, The percentage of time spent freezing during a 5 min test for contextual fear memory (Context) was similar for all mice regardless of injury or treatment. The percentage of cue-dependent freezing (Cue; percentage of time spent freezing during a 3 min presentation of a conditioned auditory stimulus minus the percentage of time spent freezing during an initial 3 min habituation period) during a 6 min test for cued fear memory was also similar for all mice regardless of injury or treatment. For all panels, the data represent means ± SEMs. B, The time for mice to fall off an accelerating rotarod (or turn one full revolution on the rotarod). Trials were conducted across 2 d with four trials per day. Although an initial ANOVA found a significant main effect of both injury and treatment (*p < .05, ***p < .001 for the indicated main effects in a four-way repeated-measures ANOVA), additional analysis found that the two injured groups (Injured + Veh and Injured + Gan) both fell off the rotarod faster than the Sham + Gan group (post hoc Tukey's tests, p < 0.05; no other between-group comparisons were significant). C, In mice lacking the TK transgene, the percentage of time spent freezing during a 5 min test for contextual fear memory (Context) was similar for both vehicle- and ganciclovir-treated mice. The percentage of cue-dependent freezing (Cue), percentage of time spent freezing during a 3 min presentation of a conditioned auditory stimulus minus the percentage of time spent freezing during an initial 3 min habituation period) during a 6 min test for cued fear memory was also similar for vehicle- and ganciclovir-treated mice lacking the TK transgene. D, In mice lacking the TK transgene, the time for mice to fall off an accelerating rotarod (or turn one full revolution on the rotarod) was similar for vehicle- and ganciclovir-treated mice. ***p < .001 for a main effect of trial in a two-way repeated-measures ANOVA.

Figure 6.

Reduction of neurogenesis in mice with traumatic brain injury results in impairments during training in the Morris water maze. A–D, Morris water maze training trials. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated main effects in a three-way repeated-measures ANOVA. A, Latency to reach the hidden platform. The Injured + Gan group took significantly longer to find the hidden platform compared with each of the other three groups (post hoc Tukey's tests, p < 0.05; no other between-group comparisons were significant). B, Average swim speed. The Injured + Gan group exhibited a significantly slower swim speed than the Sham + Veh group (post hoc Tukey's tests, p < 0.01; no other between-group comparisons were significant). C, Total distance traveled. The Injured + Gan group traveled a significantly greater distance while searching for the hidden platform compared with both Sham groups (i.e., Sham + Veh and Sham + Gan; post hoc Tukey's tests, p < 0.01; no other between-group comparisons were significant). D, Percentage of time spent swimming near the walls of the maze (i.e., thigmotaxis). The Injured + Gan group spent significantly more time swimming near the wall of the maze compared with each of the other three groups (post hoc Tukey's tests, p < 0.05; no other between-group comparisons were significant).

Figure 7.

Reduction in neurogenesis results in impaired spatial learning and memory in the Morris water maze and impaired cognitive recovery after traumatic brain injury. A–D, Morris water maze probe trial. *p < 0.05, **p < 0.01 compared with Injured + Gan (post hoc Tukey's tests). ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with target quadrant for A or target platform for B and C (planned comparisons within each group). A, Percentage of time spent swimming in each quadrant of the maze. The target quadrant is the quadrant in which the hidden platform was previously located, and the dotted horizontal line represents chance performance (i.e., 25% of time spent in a quadrant). B, Number of times that mice crossed the former location of the hidden platform (i.e., target platform) and other analogous, identically sized locations in the other three quadrants. C, Average distance (averaged across the 1 min probe trial) from the former location of the hidden platform (i.e., target platform) or from other analogous, identically sized locations in the other three quadrants. D, Average swim speed during the 1 min probe trial.