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Significant spontaneous recovery occurs following essentially all forms of serious brain injury, though 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 following brain injury and offer compelling and testable therapeutic options that might enhance recovery.
The mechanisms underlying spontaneous recovery from a variety of brain injuries remain unclear, though injury-induced neurogenesis may contribute to some of this observed self-recovery (Eriksson et al., 1998; Chirumamilla et al., 2002; Chen et al., 2003; Richardson et al., 2007). The two most well described populations of neural stem and progenitor cells in mammals are the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus (Alvarez-Buylla et al., 2001; Seaberg and van der Kooy, 2002). Normally, the subventricular zone in adults contributes progenitors to the rostral migratory stream to support ongoing olfactory neurogenesis, while the subgranular zone of the dentate gyrus provides new granular neurons throughout life within the hippocampus (Temple and Qian, 1995; Alvarez-Buylla et al., 2001). Following many forms of brain injury, progenitor cells proliferate, though it is still unclear whether this activation results in stable and productive neurogenesis (Richardson et al., 2007; Kernie and Parent, 2010).
Evidence for long-lasting hippocampal neurogenesis after traumatic cortical injury has been accumulating since first described almost a decade ago (Dash et al., 2001; Kernie et al., 2001). The dentate gyrus itself develops mostly during the postnatal period and the reservoir of subgranular zone progenitors persists well into adulthood (Pozniak and Pleasure, 2006). These progenitors develop almost exclusively into granular neurons within the most basal layers of the dentate gyrus and their functions remain largely unknown (Duan et al., 2008). After injury, however, even in adult animals, it is clear that progenitors are activated and increased neurogenesis follows whereby new neurons are found in the outer layers of the dentate gyrus, where normally this would occur only during early development (Dash et al., 2001; Chirumamilla et al., 2002; Chen et al., 2003; Ramaswamy et al., 2005; Richardson et al., 2007; Urrea et al., 2007; Yu et al., 2008). Most of the evidence to support these findings is based on BrdU (5-bromo-2-deoxyuridine) incorporation into dividing cells that when followed over time express markers suggesting mature and stable neurogenesis. Although widely used, there are limitations to BrdU-based approaches, particularly following injury (Gould and Gross, 2002).
There remains tremendous speculation regarding the significance of adult neurogenesis, and there are conflicting data suggesting its function in cognitive processes (Kokovay et al., 2008). The purpose of the present study was to determine whether adult hippocampal neurogenesis plays a functional role in the spontaneous recovery that is observed following brain injury. In order to do this, we utilized a variety of mouse transgenic models to inducibly and persistently label neural stem and progenitor cells, as well as to ablate their proliferation in response to injury. We demonstrate that injury-induced neurogenesis occurred throughout the dentate gyrus and these neurons stably incorporated themselves within the hippcocampus and elaborated sophisticated dendritic arborizations. Moreover, when ablated at the time of injury using a ganciclovir-based system, few newly born neurons were generated, and cognitive recovery was severely impaired.
Experimental animals were housed and cared for in the Animal Resource Center (ARC) at UT Southwestern Medical Center (UTSWMC), which is certified by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All animal experiments were conducted with prior approval of the Institutional Animal Use and Care Committee (IACUC) at UTSWMC in compliance with the highest standard for the humane and compassionate use of animals in biomedical research.
To perform CCI, the standard protocol and a controlled cortical impact device used to generate brain injuries were used as previously described (Kernie et al., 2001). Eight-week old male wild-type or transgenic mice were anesthetized with isoflurane. Mice were placed in a stereotactic frame. A midline incision was made, the soft tissues were reflected, and a 5mm by 5mm craniectomy was made between bregma and lambda and 1mm lateral to the midline. The injury was generated with a 3mm stainless steel tipped impact device with a deformation of 0.6mm and constant speed of 4.4 m/s. After injury, the scalp was fastened with staples and the mice were allowed to recover. Following injury, CldU (100mg/Kg, Sigma) was injected intraperitoneally 3 days after injury. Numbers of animals used for each group are listed in the figure legends.
All mice were deeply anesthetized with ketamine and xylazine mixture as described above before perfusion. The trans-cardiac perfusion was performed with 50ml of 1xPBS, followed by 50ml of 4% paraformaldehyde (PFA) in PBS. Following post-fixation in 4% PFA/1xPBS overnight, whole brains were dissected and embedded in 3% agarose/1xPBS. Serial 50-μm sections were cut with a vibratome (VT1000S, Leica). All sections encompassing the hippocampus were collected sequentially in 12-well plates. Free-floating sections were used for immunohistochemistry. For CldU staining, all sections from a single well were washed with 1xPBS 3 times and rinsed with water. Sections were washed with 0.3% Triton X-100/1xPBS (wash buffer) for 3 times and blocked with 5% normal donkey serum (Sigma-Aldrich) contained wash buffer for 1 hour at room temperature. 1:300 rat-anti CldU (AbD Serotec) antibody was used to label CldU overnight at 4°C. The following day, sections were incubated with mouse anti-NeuN (1:500, Chemicon), washed and then incubated with secondary antibodies (Cy3-conjugated anti-rat antibody and Cy2-conjugated anti-mouse (all 1:200, Jackson Immunoresearch) for 3 hours at room temperature. Additional primary antibodies used in this study were rabbit anti-GFP (1:500, Molecular Biology), goat anti-doublecortin (1:100, Santa Cruz biotech), rat anti-BrdU (1:300, Abcam), mouse anti-glutamine synthetase (1:500, Millipore), and and chicken anti-GFAP (1:500, Aves labs).
Mice were bred to obtain heterozygous Nestin-CreERT2 transgenic and heterozygous stop-YFP-ROSA26 in order to allow for tamoxifen-mediated long-term fate labeling of nestin-expressing progenitors. Twelve double-heterozygous 7 week-old mice were injected with 250μl (6.7mg/ml) of tamoxifen (0.6mg/kg in 9:1 sunflower oil/ethanol) daily for 3 days. Following another 3 days, they underwent CCI injury or mock injury and then allowed to recover for 2 months. Following this, they were perfused and brains processed as outlined above and immunostained for GFP (to detect YFP).
To determine the number of YFP-expressing cells 2 months after injury (Figure 1) and GFP- and DCX-expressing cells in the dentate gyrus after ganciclovir treatment (Figure 2), we used an unbiased stereological approach. A one-in-six series of sections covering the entire hipppocampus in its rostrocaudal extension were cut with a vibrating microtome (Leica) at a 50-μm thickness, immunostained, and visualized under light microscopy with the peroxidase/diaminobenzidine (DAB) method. The cells were counted using a modified optical fractionator and stereological image analysis software (StereoInvestigator, Microbrightfield) operating a computer-driven Olympus microscope regulated in the x, y, and z-axes. Areas to be counted were traced with a 20X objective lens and sample frames (80 μm X 80 μm) were selected at random by the image analysis software. Cells in the counting frame (40 μm X 40 μm) were counted under a 40X objective lens. To avoid oversampling, the uppermost and lowermost focal planes were excluded. The total cell number of cells was estimated by multiplying the resulting counts by 6, since every sixth section had been used. Hence, the numbers obtained in this study are absolute numbers per hippocampus (both sides of the dentate gyrus counted for each) and are independent of the volume of the dentate gyrus.
For cell quantification for double labeling of YFP/NeuN, YFP/GFAP, CldU/NeuN, and GFAP/BrdU-expressing cells, we used a traditional and well-established method to account for oversampling as described in detail elsewhere (Miles and Kernie, 2008; Yu et al., 2008). A Zeiss LSM 510 confocal microscope using Argon 488, He 543 and He 633 lasers and a Zeiss Neofluar 40X/1.3 oil lens was used to determine colocalization of immunohistochemical markers and cell quantification with a pinhole aperture of 3.46 Airy units. Cell quantification was performed from the ipsilateral and contralateral dentate gyrus from injured mice. All brains were embedded in 3% agarose and cut with a vibrating microtome (Leica) at a 50-μm thickness using a sapphire blade in 0.1 M PBS. Free-floating sections through the entire rostral to caudal hippocampal axis were collected in 12-well plates with 5 sections/well. We sampled one well of five sections 50-μm thick, coronally cut, and paraformaldehyde-fixed 600μm apart. Five sections from each well were stained with relevant antibodies as free-floating sections to allow for equal antibody penetration between sections. Sections were mounted using Immunomount with a refractive index of 1.495. Antibody penetration and signal intensity was found to be evenly distributed throughout the entire 50-μm section and was verified on the z-axis in multiple sections from different animals. Each blade of the dentate gyrus was counted separately at 40X under Zeiss immersion oil 518F with 1.5X optical zoom magnification. Nonblinded quantification was performed on cells with fully colocalized antibodies in the granular layer of the denate gyrus. Every double-positive cell was counted. By scanning serially through the z-axis of each cell, a more precise discrimination of the colocalization of relevant cell markers was permissible.
Ganciclovir (200mg/Kg/day, Cytovene-IV, Roche Pharmaceuticals) or vehicle (dH2O) was delivered via osmotic mini pumps that infuse at a constant rate of 0.5 μl per hour for 2 weeks (model 2002, Alzet). The pumps were replaced once after 2 weeks for a total of 4 weeks of treatment. The implantation of pumps was as per the Alzet protocol and is briefly described below: 6-week old mice were anesthetized with isoflurane. A small incision was made in the skin between the scapulae and a small pocket was formed using a hemostat to spread the subcutaneous connective tissue apart. The pumps were inserted into the pocket with the opening of pumps pointing away from the incision. The skin incision was closed with staples.
All behavioral testing was conducted by investigators blind to the experimental treatment conditions. Mice were tested in three cohorts, and the total sample size for each experimental group was as follows: ganciclovir-treated group with CCI injury (“Injured + Gan”), N=12; vehicle-treated group with CCI injury (“Injured + Veh”), N=10; ganciclovir-treated group with sham injury (“Sham + Gan”), N=9; and vehicle-treated group with sham injury (“Sham + Veh”), N=10. All mice were provided with ad lib food and water and kept on a 12 hr light/dark cycle with lights on at 6 am. Behavioral testing was conducted during the light cycle, and behavioral tests occurred in the following order: Morris water maze, visible water maze, fear conditioning, and rotarod.
The Morris water maze task was conducted similar to the method previously described (Powell et al., 2004; Tabuchi et al., 2007; Etherton et al., 2009). A white, circular pool was filled with water that was mixed with gothic white, non-toxic paint to make it opaque, and a circular platform (15 cm in diameter) was submerged 1 cm beneath the surface of the water. The testing room was filled with a number of extra-maze cues. Training in the Morris water maze was conducted over 11 consecutive days, and each training day consisted of 4 individual trials with an inter-trial interval of approximately 1–1.5 minutes. Mice were placed in a different starting location for each of the 4 daily training trials. During training trials, mice were allowed to swim until they found the hidden platform, and if they did not find the platform within 60 seconds, they were guided to it by the experimenter. Mice remained on the platform for 15 seconds before being removed. For each training day, data were averaged across the 4 trials. A probe trial was conducted on day 12; the hidden platform was removed, and mice were placed in the pool and allowed to swim for 60 seconds.
The visible version of the water maze was conducted during a single day, and there were 6 trials with an inter-trial interval of approximately 30 minutes. A visible cue (a black, foam square) was placed on top of the platform. The platform was placed in a new, random location for each trial, and mice were placed in the same starting location for each trial. All data were collected using Ethovision software (Version 2.3.19, Noldus; Leesburg, VA).
For the cued and contextual fear-conditioning task, mice were initially placed in conditioning chambers. They were given a training session that consisted of an initial 2 min habituation period followed by two presentations of a 30 second auditory tone (white noise) that co-terminated with a 2 s, 0.5 mA footshock. There was a 1 min inter-stimulus interval between tone-shock pairings, and mice remained in the conditioning chambers for 1 min before being returned to their home cage. Freezing behavior (motionless except respirations) was measured live by an observer every 5 seconds.
A test session to examine contextual fear memory was conducted 24 hr after the training session. Mice were placed back into the original training context for 5 minutes, and freezing behavior (motionless except respirations) was measured by an observer every 5 seconds. A test session to examine cued fear memory was conducted 3–4 hrs after the contextual fear memory test. Mice were placed in a novel context (visual, olfactory, auditory, and tactile aspects of the general environment were all changed); they experienced an initial 3-minute habituation period followed by a 3-minute presentation of the auditory tone. Freezing behavior (motionless except respirations) was measured by an observer every 5 seconds, and normalized cue-dependent freezing was calculated by subtracting the percent of time spent freezing during the habituation period from the percent of time spent freezing during the tone presentation.
During the rotarod task, mice were placed on a rotating cylindrical rod that accelerated from 4–45 revolutions/min over a period of 5 minutes. Mice were given 4 trials per day over 2 days, and the time to either fall off the rod or turn one full revolution was measured.
All statistical analyses of behavioral results were conducted using Statistica software (version 5.5, StatSoft Inc., Tulsa, OK). Data were initially analyzed using 2-, 3-, or 4-way ANOVAs, as appropriate. Sex was initially included as a between-subjects factor; however, if there was no significant interaction or main effect of Sex, the data were collapsed across Sex. In cases where there was a significant interaction or main effect of Sex, it has been included as a factor in the reported analyses. Other between-subjects factors were Injury (CCI injury vs. sham) and Treatment (vehicle vs. ganciclovir), and within-subjects factors were Day (for Morris water maze), Trial (for visible water maze and rotarod), Quadrant (for Morris water maze), or Platform (for Morris water maze). Further analyses were conducted using post-hoc Tukey HSD tests and planned comparisons (contrast analysis and additional ANOVAs). A thorough description of all statistical results can be found in Supplemental Tables 1 and 2.
Statistical analyses of all non-behavioral results were done using a one-way analysis of variance (ANOVA) for multiple comparisons with a Newman-Keuls multiple comparison test for post hoc analysis. A Student’s t test was performed for paired data. Differences were deemed significant with p<0.05.
We used a previously characterized Nestin-Cre-ERT2/stopYFP-ROSA26 transgenic mouse line that inducibly and specifically labels early stem/progenitors within the mouse brain (Li et al., 2008; Chen et al., 2009). By administering tamoxifen for 3 days to young, adult transgenic (8-week old) mice, we were able to demonstrate that 8 weeks later, there were many YFP-labeled cells that reside predominantly in the most superficial layers of the dentate gyrus (Fig. 1A, D). When mice underwent a unilateral, controlled cortical impact (CCI) injury 3 days after the last tamoxifen injection, there was triple the number of cells generated when compared to the mock-injured controls (Fig. 1, A-G). In addition, the newly generated cells incorporated themselves throughout the dentate gyrus and not just in the most superficial layers (Fig. 1B, E). Notably, these cells expressed long dendritic processes indicative of neurons that have stably integrated themselves within the dentate gyrus (Fig. 1, A-F).
Next, in order to prove that these peripherally incorporated YFP-expressing cells were indeed neurons, we performed triple staining with the mature neuronal marker NeuN as well as the astrocytic and progenitor marker GFAP (Fig. 2). Here, we observed that virtually all YFP-expressing cells in the outer granular layer of the dentate gyrus following injury also expressed NeuN, indicative of stable neurogenesis (Fig 2, E-H). In addition, both in the injured dentate gyrus and in controls, we observed that the YFP-GFAP double expressing cells were almost exclusively limited to the subgranular zone indicative of early GFAP-expressing progenitors (Fig. 2, A-H). We observed 177±1 cells expressing both YFP and NeuN in the ipsilateral injured dentate gyrus compared to 117±10 in mock-injured controls (n=3 each group, p<0.01, unpaired t test). Additionally, we observed 61±11 expressing both YFP and GFAP in injured animals and 51±6 in mock-injured controlleds (n=3, p=0.5, unpaired t test). Notably, we did not observe any reactive astrocytes that also express GFAP within the hilus or molecular layer of the dentate gyrus that also expressed YFP, suggesting that YFP-expressing progenitors became either neurons or remained as progenitors when exposed to injury.
To determine whether these newly-generated neurons had any functional relevance, we developed another transgenic mouse line that expresses a modified version of the herpes simplex virus thymidine kinase (HSV-TK) along with green fluorescent protein (GFP) controlled by the same nestin promoter and allows for ganciclovir-mediated ablation of dividing neural progenitors. We previously demonstrated that 4 weeks of exposure to ganciclovir is sufficient to specifically ablate over 93% of doublecortin-expressing, committed neuroblasts in the adult dentate gyrus (Yu et al., 2008). In order to ablate the injury-induced neurogenesis associated with traumatic brain injury, osmotic mini-pumps with either vehicle or ganciclovir were implanted into the subcutaneous tissue of adult transgenic mice immediately following injury, which exposed the mice to ganciclovir for 4 weeks. In addition, each animal was injected with a single dose of CldU 3 days after injury in order to determine ganciclovir efficacy in ablating neurogenesis. Upon pump removal, each cohort of mice underwent behavioral testing, and then their brains were removed and examined histologically.
By immunostaining for the mature neuronal marker NeuN and CldU, we demonstrated that approximately 90% of injury-induced neurogenesis was ablated with this technique (Fig. 3, A-B). As we previously determined, the administration of ganciclovir only targets dividing progenitors so the relatively quiescent, early stem/progenitor is not susceptible. We therefore also examined the state of Nestin/GFP-expressing early progenitors as well as later doublecortin-expressing committed neuroblasts. Here, we observed a modest reduction in each, suggesting that the progenitor population exhibited some recovery a month after removal of ganciclovir though remained persistently lower than what was seen in mock-injured controls (Fig. 3, C-F).
Reactive gliosis occurs after essentially all forms of brain injury and likely plays roles in how recovery is mediated (Garcia et al., 2004; Myer et al., 2006). In addition, nestin is expressed in other cell types in addition to early progenitors and includes NG2-expressing oligodendrocytes, reactive astrocytes, and endothelial cells (Ridet et al., 1997; Belachew et al., 2003; Aguirre and Gallo, 2004; Yokoyama et al., 2006). Many transgenic animals have been made that express proteins such as GFP under the control of various aspects of nestin regulatory elements including the endogenous nestin promoter with all its enhancer elements as well as a minimal viral promoters with the second intron enhancer (Yamaguchi et al., 2000; Kawaguchi et al., 2001; Lagace et al., 2007). Here, we used the full nestin promoter and the second intron progenitor-specific enhancer in both the nestin-CreERT2 transgenic as well as the nestin-HSV-TK mice. Although endogenous nestin is expressed in reactive astrocytes, we previously demonstrated that genes under the control of the neural progenitor-specific form of the nestin promoter used in the transgenic animals in this study are expressed only in progenitors and not in reactive astrocytes in response to both traumatic and hypoxic-ischemic injuries (Miles and Kernie, 2008; Yu et al., 2008). However, since it remains a formal possibility that this nestin-HSV-TK transgenic could ablate both dividing stem/progenitors as well dividing reactive astrocytes, we performed a detailed investigation.
Nestin-HSV-TK mice that were injured and given either ganciclovir or vehicle for a month following injury were given BrdU during the peak of astrogliosis (once daily, post-injury days 1–3). One month after injury the mice were analyzed for reactive gliosis. We observed that there was a similar amount of overall gliosis in both the ganciclovir and vehicle-treated animals (Fig. 4). Moreover, when quantified, there were similar numbers of proliferative reactive astrocytes in each group as evidenced by co- expression of GFAP and BrdU (101±13.6, control, N=3; 112±14.4, gan-treated, N=4, not significant). Finally, we also observed that the maturation of these reactive astrocytes was similar in each group by performing immunostaining with glutamine synthetase (GS) a marker of mature astrocytes, where we observed that in each group a subset of reactive astrocytes that expressed BrdU, also expressed glutamine synthetase (Fig. 4, D, H).
To determine if the ablation of neurogenesis would modulate the effect of brain injury on non-spatial forms of learning and memory, mice containing the thymidine kinase (TK) were trained in contextual and cued fear conditioning as well as a rotarod task after receiving either CCI or a sham injury and treatment with either ganciclovir (gan) or vehicle (veh). During tests of both cued and contextual fear memory, all mice exhibited comparable levels of freezing (Fig. 5, A; Cued fear conditioning 2-way ANOVA; Injury: P=0.97, Treatment: P=0.27, Injury x Treatment: P=0.85; Contextual fear conditioning 2-way ANOVA; Injury: P=0.09, Treatment: P=0.49, Injury x Treatment: P=0.26; see Supplementary Table 1 for detailed statistical analyses of behavioral tests). These data suggest not only that ablation of neurogenesis did not modulate the effect of brain injury on fear learning and memory but also that neither ablation of neurogenesis alone nor brain injury alone affected this type of learning and memory. Next, motor coordination and learning were assessed using the rotarod task (Fig. 5, B). Although a 4-way repeated measures ANOVA did find significant main effects of Brain Injury and Gancyclovir Treatment (Sex: P<0.02; Injury: P<0.02, Treatment: P<0.04, Trial: P<0.000001; no other significant main effects or interactions), further analysis found that the injured mice with or without ablation of neurogenesis took less time to fall off the rotarod compared to uninjured mice with ablation of neurogenesis (Post-hoc Tukey HSD Tests; “Injured + Gan” vs. “Sham + Gan”: P<0.05, “Injured + Veh” vs. “Sham + Gan”: P<0.002). However, there was no difference between injured mice with and without neurogenesis, suggesting that any mild effect of injury was not exacerbated by the ablation of neurogenesis (Post-hoc Tukey HSD Tests: “Injured + Veh” vs. “Injured + Gan”: P>0.10).
To determine if there were any non-specific effects of ganciclovir on fear conditioning or the rotarod, mice lacking the TK transgene were treated with either vehicle or ganciclovir. Ganciclovir treatment had no effect on cued fear conditioning (Fig. 5, C; 2-way ANOVA; Sex: P<0.006, Treatment: P=0.84, Sex x Treatment: P=0.74), contextual fear conditioning (Fig. 5, C; 1-way ANOVA; Treatment: P=0.69), or the rotarod task (Fig. 5, D; 2-way repeated measures ANOVA; Treatment: P=0.92, Trial: P<0.000001, Treatment x Trial: P=0.95), suggesting that there were no non-specific effects of ganciclovir on these behaviors.
To determine whether ablation of neurogenesis affected injury-induced cognitive deficits, we began behavioral testing one month after ablation of neurogenesis and injury, when injury-induced progenitors have had about four weeks to mature and are most likely to functionally integrate into the hippocampus and participate in memory formation (Ge et al., 2006; Kee et al., 2007). Performance in the Morris water maze was used as the primary cognitive outcome to assess recovery. Mice containing the thymidine kinase (TK) transgene were trained in the Morris water maze after receiving either CCI or a sham injury and treatment with either ganciclovir (gan) or vehicle (veh).
Pharmacogenetic ablation of adult neurogenesis in combination with traumatic brain injury resulted in a modest decrease in initial spatial learning compared to sham controls (Fig. 6, see Supplementary Table 1 for detailed statistical analyses of behavioral tests). Brain injured mice with ablation of neurogenesis (Injured + Gan) exhibited a longer latency to reach the hidden platform compared to all other groups (Figure 6, A; Post-hoc Tukey HSD Tests; “Injured + Gan” vs. “Injured + Veh”: P<0.04, “Injured + Gan” vs. “Sham + Gan”: P<0.006, “Injured + Gan” vs. “Sham + Veh”: P<0.0004 see Supplementary Table 1 for detailed statistical analyses of behavioral tests). Although injured mice without neurogenesis (Injured + Gan) did exhibit a mild decrease in average swim speed (Fig. 6, B; Post-hoc HSD Tukey Tests; “Injured + Gan” vs. “Sham + Veh”: P<0.004), there was no effect of either brain injury or ganciclovir treatment on swim speed during the first day of training (Fig. 6, B; 2-way ANOVA; Injury: P=0.12; Treatment: P=0.86, Injury x Treatment: P=0.56), suggesting that injured mice without neurogenesis (Injured + Gan) did not have any gross motor impairment, but were unable to successfully increase their swim speed as training trials progressed. In another measure of learning that is independent of swim speed, injured mice without neurogenesis (Injured + Gan) travelled longer distances to reach the hidden platform compared to mice that received a sham injury (Fig. 6, C; Post-hoc Tukey HSD Tests; “Injured + Gan” vs. “Sham + Gan”: P<0.0008, “Injured + Gan” vs. “Sham + Veh”: P<0.0002). Although all groups of mice spent a similar percent of time swimming along the walls of the maze (thigmotaxis) during the first day of training (2-way ANOVA; Injury: P=0.17, Treatment: P=0.80, Injury x Treatment: P=0.49), injured mice without neurogenesis (Injured + Gan) spent a greater percent of time swimming along the walls of the maze over the rest of training compared to the other groups of mice (Fig. 6, D; Post-hoc Tukey HSD Tests; “Injured + Gan” vs. “Injured + Veh”: P<0.03, “Injured + Gan” vs. “Sham + Gan”: P<0.0005, “Injured + Gan” vs. “Sham + Veh”: P<0.0003). This suggests that, unlike the other groups, the injured mice without neurogenesis (Injured + Gan) did not progress from the initial use of a thigmotaxic search strategy to the use of an efficient spatial strategy to find the hidden platform, and we interpret this result as a learning deficit (Lipp and Wolfer, 1998). These data suggest that ablation of neurogenesis after brain injury results in mild deficits in learning during the Morris water maze.
During the Morris water maze probe trial, brain-injured mice lacking adult neurogenesis (Injured + Gan) exhibited a complete lack of long-term spatial learning and memory. Using three different behavioral measures, injured mice without neurogenesis (Injured + Gan) did not show any discernable preference for the target quadrant or the previous location of the hidden platform (Fig. 7, A-C, red bars; Planned pairwise comparisons; For Fig. 7A – Target vs. Right: P=0.69, Target vs. Left: P=0.61, Target vs. Opposite: P=0.59; For Fig. 7B – Target vs. Right: P=0.63, Target vs. Left: P=0.44; Target vs. Opposite: P=0.09; For Rig. 7C – Target vs. Right: P=0.71, Target vs. Left: P=0.96, Target vs. Opposite: P=0.82; see paragraph below for similar comparisons within the other groups), suggesting either a lack of spatial memory or greatly impaired spatial learning of the hidden platform’s location. Importantly, the average swim speed during the probe trial was comparable among all groups (Fig. 7, D; 2-way ANOVA; Injury: P=0.67, Treatment: P=0.45, Injury x Treatment: P=0.67), and examination of mice lacking the TK transgene determined that there were no non-specific effects of ganciclovir on behavior (Supplemental Table 2 and Supplemental Figs. 1–3).
Interestingly, a gradient effect of ablating neurogenesis with or without brain injury is also apparent in probe trial measures of spatial memory. Figures 7A and 7B clearly demonstrate that sham-injured mice with intact neurogenesis (Sham + Veh, purple bars) showed a significant preference for the target quadrant (or previous platform location) over all three other quadrants (or analogous zones in the other three quadrants of similar size and relative location as the hidden platform, termed “analogous platform locations”; Planned pairwise comparisons; For Fig. 7A – Target vs. Right: P<0.00004, Target vs. Left: P<0.002, Target vs. Opposite: P<0.00005; For Fig. 7B – Target vs. Right: P<0.00001, Target vs. Left: P<0.002, Target vs. Opposite: P<0.00002). However, mice that received either brain injury alone (Injured + Veh, green bars) or ablation of neurogenesis alone (Sham + Gan, blue bars) significantly preferred the target quadrant (or previous platform location) compared to only one or two of the other quadrants (or analogous platform locations; Planned pairwise comparisons; For Fig. 7A “Injured + Veh” – Target vs. Right: P=0.14, Target vs. Left: P=0.16, Target vs. Opposite: P<0.006; For Fig. 7A “Sham + Gan” – Target vs. Right: P=0.07, Target vs. Left: P<0.04, Target vs. Opposite: P<0.008; For Fig. 7B “Injured + Veh” – Target vs. Right: P<0.04, Target vs. Left: P=0.34, Target vs. Opposite: P<0.02; For Fig. 7B “Sham + Gan” – Target vs. Right: P=0.08, Target vs. Left: P=0.08, Target vs. Opposite: P<0.02). This suggests that both un-injured mice with ablation of neurogenesis (Sham + Gan) and injured mice with intact neurogenesis (Injured + Veh) exhibit an intermediate level of spatial memory that is slightly less robust than that of fully intact mice (Sham + Veh) but more robust than that of injured mice lacking neurogenesis (Injured + Gan). In total, these data suggest that ablation of adult neurogenesis does indeed exacerbate the cognitive effects of traumatic brain injury.
In the visible version of the water maze, all groups of mice were able to reach a visible platform in less than 12 seconds on average (Supplemental Fig. 4), suggesting that neither brain injury nor ablation of neurogenesis caused impairments in vision. However, injured mice without neurogenesis (Injured + Gan) did exhibit some impairments in the visible version of the water maze, suggesting that they had mild impairments in either their ability to learn the basic rules of the water maze task or in their ability to swim directly to the escape platform (Supplemental Fig. 4). Again, these findings suggest that ablation of adult neurogenesis exacerbates the cognitive effects of traumatic brain injury.
Since it is reasonably well established that hippocampal progenitors are activated by injury resulting in increased numbers of new neurons within the dentate gyrus, the primary purpose of this study was to establish the relevance of this phenomenon. The possibilities regarding the significance of injury-induced neurogenesis include the fact that the generation of new neurons might be beneficial and contribute to recovery of learning and memory and possibly other functions impaired by brain injury. However, neurogenesis may contribute to TBI-related morbidity such as epilepsy, which is relatively common following moderate and severe TBI. Finally, this reservoir of progenitors may be nothing more than a developmental remnant that is incapable of providing functionally relevant neurons into the sophisticated hippocampal circuitry. Although we demonstrate here that injury-induced neurogenesis occurs in a long-lasting manner, we also note that the progenitor pool itself may become depleted (Fig. 3). Since there is an apparently permanent depletion of GFP-expressing early progenitors following injury, a number of questions remain open. There may either be damage to the ipsilateral dentate gyrus that impairs its ability to sustain baseline neurogenesis, or the injury itself may accelerate depletion of the overall progenitor pool. Either possibility suggests that recurrent injuries may not result in such a robust neurogenic response and that this ability to self-repair may be limited.
A number of strategies have emerged to test whether hippocampal neurogenesis has physiologic relevance. Methods to inhibit neurogenesis include systemic or local administration of anti-mitotic agents such as Ara-C that are known to non-specifically target all dividing cells including neural progenitors (Doetsch et al., 1999; Lau et al., 2009). Another is to perform cranial irradiation directed to neurogenic zones such as the subventricular zone and dentate gyrus in order to more selectively impair neurogenesis by better controlling what cells are exposed (Hellstrom et al., 2008; Naylor et al., 2008; Clelland et al., 2009; Noonan et al., 2010). Problems with these approaches include both their lack of specificity and potentially toxic side effects that lead to unwanted immune activation that may affect other mediators of recovery or damage not related to neurogenesis. Thus far, the data supporting a role for adult neurogenesis in improving cognitive function in the uninjured state are compelling, but rely almost exclusively on these various imperfect techniques.
Recently, genetically engineered mice have become available that can regulate neurogenesis in a more precise and temporally controlled manner. One strategy is to inducibly express diptheria toxin or its receptor in progenitor cells, which can then be ablated in a temporally controlled manner (Luquet et al., 2005; Durieux et al., 2009). One confounder with this approach is that all progenitors, not just dividing ones, are ablated and therefore the pool becomes depleted and cannot be reactivated. An alternative approach is a transgenic mouse that expresses the herpes simplex virus thymidine kinase (HSV-TK) under the control of the glial fibrillary astrocytic protein (GFAP) promoter. This allows for the inducible ablation of dividing cells that express GFAP, which includes early type 1 hippocampal progenitor cells as well as dividing reactive astrocytes (Sofroniew et al., 1999; Morshead et al., 2003; Saxe et al., 2006). Recently, more specific modified versions of HSV-TK have been shown in nestin-expressing progenitor cells to inducibly ablate early progenitors during normal states as well as after injury (Yu et al., 2008; Clelland et al., 2009).
It remains both controversial and unclear whether adult hippocampal neurogenesis contributes to memory formation during normal adult brain maturation. Some of the conflicting results regarding the impact of neurogenesis on memory have been attributed to the heterogeneous manner in which ablation occurs (Deng et al., 2010). However, even with the most recently developed genetic techniques, there remains evidence for hippocampal-based Morris water maze and contextual fear conditioning deficits in some models of ablated neurogenesis, but not in others (Dupret et al., 2008; Imayoshi et al., 2008; Zhang et al., 2008; Deng et al., 2009). In our present study, we note no differences in fear conditioning in mock-injured animals with or without neurogenesis intact (Figure 5) and only very modest differences in Morris water maze-based spatial memory (Figures 6–7). Although contextual fear conditioning is generally thought to be a hippocampal-dependent behavior, it is possible that other brain regions required for this task, such as the amygdala (Goosens and Maren, 2001; Onishi and Xavier, 2010), were able to compensate and successfully support the contextual fear learning and memory. For example, even if mice were unable to develop an integrated representation of the context, it is possible that they were able to use a combination of discrete cues in the environment to remember specific aspects of the learning context. In addition, the dentate gyrus has been recently linked to pattern separation within the hippocampus (Deng et al., 2010), and neither contextual fear conditioning nor our Morris water maze task specifically tested pattern separation. Our Morris water maze task might not have been sensitive enough to detect the subtle differences that ablation of neurogenesis impart on hippocampal function, and it is possible that we might detect a stronger effect of neurogenesis ablation with more challenging behavior parameters or a spatial learning task that expressly tests pattern separation. Although the most recent studies do suggest functional roles for adult neurogenesis within some aspects of memory formation, they may become most readily apparent when the experimental animals undergo induced induction of neurogenesis with an acquired injury as demonstrated here (Deng et al., 2010).
Although injury-induced neurogenesis clearly occurs in response to a wide variety of injuries, the mechanisms underlying it need more investigation. The injured brain releases numerous extracellular proteins and ions that may play roles in regulating neurogenesis. Two of the best studied of these, potassium chloride and glutamate, have been implicated in enhancement of proliferation in immature cells while at the same time directing toxicity in more mature cell types (Shi et al., 2007; Mattson, 2008). In addition, the injured brain activates both astrocytes and microglia, which are both known to secrete a variety of growth factors as well as immune modulators that may affect progenitor proliferation and survival (Myer et al., 2006; Bessis et al., 2007). Finally, the progenitor cells themselves make physical contact with the vasculature so circulating factors such as cytokines and growth factors that increase after injury may also direct some of these effects (Mignone et al., 2004). Thus, the mechanisms underlying TBI-induced neurogenesis are likely not straightforward nor easily worked out, and therefore remain compelling targets to study, particularly since our data suggest that blockade of such neurogenesis can exacerbate the cognitive deficits results from traumatic brain injury.
Our data show that pharmacogenetic ablation of neurogenesis after brain injury results in impaired learning and memory in the Morris water maze, but ablation of neurogenesis did not modulate the effect of brain injury on motor coordination and learning (as assessed by the rotarod) or fear learning and memory (as assessed by cued and contextual fear conditioning). It is not surprising that the influence of neurogenesis ablation on the behavioral effects of brain injury was limited to spatial learning and memory, given that the most robust neurogenesis within rodents is known to occur within the hippocampus. While further study is needed to determine why neither the ablation of neurogenesis nor brain injury alone affected contextual fear conditioning, which is generally thought to be a hippocampal-dependent behavior, it is possible that there was compensation of other brain regions (as discussed above). There is also evidence that different sub-regions of the hippocampus are required for learning in the contextual fear conditioning and Morris water maze tasks (Richmond et al., 1999), and this could account for the different results in these two tasks. In addition, the modeling demonstrated here affects subventricular zone neurogenesis as well. Although it is less likely that these progenitors contribute to the hippocampal-specific behaviors tested here, it remains formally possible that our observations are confounded by ablation of subventricular zone progenitors.
In multiple measures of learning and memory in the Morris water maze, brain-injured mice lacking neurogenesis (Injured + Gan) exhibited impaired performance compared to all control mice, including mice with either brain injury alone or ablation of neurogenesis alone. As discussed in the Results secion, brain-injured mice lacking neurogenesis (Injured + Gan) also showed increased thigmotaxic behavior during learning in the Morris water maze, suggesting that these mice were unable to progress from the initial use of a thigmotaxic search strategy to the use of an efficient spatial strategy to find the hidden platform (Lipp and Wolfer, 1998; Powell et al., 2004). Although we interpret this as additional evidence for a learning deficit, we acknowledge that we cannot conclusively rule out the possibility that the increased thigmotaxic behavior could be an indication of other possible cognitive deficits, such as increased anxiety-like behavior. Even if that is the case, however, our data still demonstrate that ablation of adult neurogenesis exacerbates the general cognitive deficits resulting from brain injuries and suggest that enhancing hippocampal neurogenesis after injury might be a viable therapeutic strategy.
NIH grant R01 NS048192 (SGK), the Seay Endowed Fund for Research on Brain and Spinal Cord Injuries in Children (SGK), NIMH (CMP), NICHD (CMP), and The Hartwell Foundation (CMP). The authors have no other financial interest to disclose.