Cortical ischemia induces long-term hippocampus-dependent cognitive impairment. We first evaluated how permanent cerebral ischemia affects the formation of enduring memories using contextual fear conditioning (CFC) (Figure 1, A–C). Seven days after surgery, sham-operated and ischemic mice were placed in a chamber (context); after a familiarization phase, several electrical foot shocks were presented, and recent and remote memory (24 hours, 28 days, or 60 days after conditioning) were calculated by measuring the freezing response. Sham-operated and ischemic mice behaved similarly during the training phase, displaying comparable exploratory activity and foot-shock reactivity (Supplemental Figure 1, A and B; P > 0.05; supplemental material available online with this article; https://doi.org/10.1172/JCI120412DS1). In addition, the formation of new memories was not affected by middle cerebral artery occlusion (MCAO), since both sham-operated and MCAO mice displayed similar conditioned response 1 hour after training (Supplemental Figure 1C; P > 0.05).

Figure 1 Cortical stroke impairs long-term memory in mice. (A) Experimental design for panels B and C. Sham-operated and MCAO mice were subjected to CFC (0.6 mA × 3) and tested 28 and 60 days after training. (B and C) Remote memory retention after cerebral ischemia calculated as the percentage of freezing response 28 days (B, P < 0.05 vs. sham operated; sham operated, n = 37; MCAO, n = 63) and 60 days (C, *P < 0.05 vs. sham-operated group; sham operated, n = 8; MCAO, n = 8) after foot shocks. (D) Experimental design for panels E and F. (E) Percentage of freezing in control and MCAO groups after a weak (0.4 mA × 2; left columns; light orange panel; *P < 0.05 vs. sham-operated group; sham operated, n = 7; MCAO, n = 7) or a strong fear-conditioning paradigm (0.8 mA × 5; right columns; dark orange panel; P > 0.05 vs. sham-operated group; sham operated, n = 5; MCAO, n = 5). Retention for both types of conditioning was performed 28 days after training and 60 days after conditioning for the strong one. (F) Memory persistence at 2 months after strong fear-conditioning paradigm. Data are represented as percentage of freezing at 2 months versus that observed at 1 month (*P < 0.05 vs. sham-operated group; sham operated, n = 5; MCAO, n = 5). (G and H) Freezing response after conditioning (0.6 mA × 3) performed 48 hours before surgery (G, *P < 0.05 vs. sham operated; sham operated, n = 9; MCAO, n = 7) or 30 days after MCAO (H, *P < 0.05 vs. sham operated; sham operated, n = 15; MCAO, n = 15), respectively. Data are represented as mean ± SEM. Data were compared using nonparametric 2-tailed Mann-Whitney U test.

During the retention session, although sham-operated and MCAO groups did not show any differences when tested 24 hours after training (Supplemental Figure 1D; P > 0.05), ischemic mice displayed decreased retrieval compared with that of the sham-operated group when remote memory was evaluated 28 days later (Figure 1B; P < 0.05 vs. sham-operated). Importantly, this effect persisted at least 60 days after surgery (Figure 1C; P < 0.05 vs. sham operated).

The degree of memory retention could be modulated by modifying initial memory strength by using a weak or a strong contextual fear training paradigm (Figure 1, D–F): while ischemic mice displayed less freezing behavior than the sham-operated group 28 days after foot-shock presentation when using a weak fear conditioning protocol (Figure 1E; P < 0.05 vs. sham operated), differences between sham-operated and MCAO mice were abolished by a strong fear protocol (Figure 1E; P > 0.05 vs. sham operated). Importantly, even with the latter, ischemic mice presented a low ratio of memory persistence when evaluated 60 days after training (Figure 1F; P < 0.05 vs. sham operated), probably indicating a progressive cognitive decline that mimics that observed in stroke patients (3). We next asked whether the timing of new memory acquisition affects long-term memory retention and whether the long-term memory deficits dissipate with time. Of note, when conditioning was performed either 48 hours before surgery (Figure 1G; P < 0.05 vs. sham operated) or 35 days after MCAO (Figure 1H; P < 0.05 vs. sham operated), similar results were observed, indicating that cerebral ischemia impairs the recall of remote memories independently of the time at which those memories were formed (i.e., before, early after, or even later after stroke).

Memory deficits after stroke are considered to be size and location dependent (for review, see ref. 21). In our model, in which lesions are restricted just to the cortex and the initial and final lesions are directly correlated (Supplemental Figure 2A, P < 0.05), we did not detect any association between damage and degree of cognitive function, determined as freezing response (Supplemental Figure 2B; P > 0.05). Likewise, similar infarct volumes were observed along the anteroposterior axis (Supplemental Figure 2C), with an exclusive cortical affectation, showing that memory deficits were not causally related to infarct size or location.

Enduring contextual fear-conditioning memories have been ascribed to the hippocampus (22). To determine whether cognitive impairment was due to the specific involvement of hippocampus, we examined the ability of retrieval of remote memories after hippocampal inactivation in both sham-operated and ischemic mice (Figure 2A). First, 7 days after the surgery, mice were trained in CFC and tested 28 days after. Twenty-four hours later, mice received a bilateral infusion of vehicle or tetrodotoxin (TTX) in the dorsal hippocampus, and the freezing response was reassessed 3 hours later. TTX administration in both sham-operated and MCAO groups impaired retrieval compared with that of the vehicle-treated group, indicating that memory recall after fear conditioning in both groups was allocated to the hippocampus (Figure 2A; P < 0.05 vs. sham operated).

Figure 2 Long-term memory deficits after MCAO are hippocampus dependent. (A) Design for hippocampus inactivation with TTX before retrieval of fear memory. Sham-operated and MCAO mice were subjected to CFC (0.6 mA × 3) 7 days after surgery and tested 28 days after training (*P < 0.05 vs. sham operated; sham operated, n = 9; MCAO, n = 13). One day later, sham-operated and MCAO mice, allocated in 2 different groups, were bilaterally infused in the dorsal hippocampus with either vehicle or TTX. Three hours later, fear memory was evaluated as percentage of freezing (sham-operated vehicle, n = 4; MCAO vehicle, n = 5; sham-operated TTX, n = 5; MCAO TTX, n = 8; *P < 0.05 vs. sham-operated vehicle; #P < 0.05 vs. MCAO vehicle). (B) Representative images of c-Fos staining (green) in the hippocampus of untrained, sham-operated, and ischemic mice 90 minutes after a 10-minute retrieval session, 28 days after conditioning. Scale bar: 200 μm. (C–F) Barnes maze testing. (C) Experimental design. (D) Representative traces of the paths traveled during retrieval by mice in the Barnes maze obtained by EthoWatcher software. Time spent by sham-operated and MCAO mice around each quadrant (E) or around the target hole (F) in the Barnes maze platform 30 days after training. For E, 2-way ANOVA showed a significant interaction between quadrants and surgery (F (3,116) = 3.50; P = 0.0178) (Bonferroni’s post hoc: *P < 0.05 vs. sham-operated TQ; sham operated, n = 12; MCAO; n = 19). RQ, right quadrant; ATQ, antitarget quadrant; LQ, left quadrant. For F, *P < 0.05 vs. sham-operated group. Data are represented as mean ± SEM. Data were compared by using nonparametric 2-tailed Mann-Whitney U tests (A and F) or nonparametric 2-way ANOVA followed by Bonferroni’s post hoc testing (E).

Accordingly, an altered pattern of hippocampal c-Fos expression was observed in ischemic mice after the retrieval of long-term CFC (Figure 2B). While 90 minutes after the retrieval session, conditioned sham-operated mice displayed an increase in the percentage of c-Fos+ cells in different hippocampal subfields, CA1, CA3, and dentate gyrus (DG), relative to the unconditioned sham-operated group (mice that were exposed to the context during the retrieval session, but did not receive any foot shock 28 days before), the population of hippocampal neurons activated by context reexposure in ischemic mice was smaller in both ipsi- and the contralesional hippocampus compared with that of the sham-operated group (Figure 2B; c-Fos+ cells vs. unconditioned mice: sham operated, 3.79 ± 0.43; MCAO ipsilesional, 1.59 ± 0.29; MCAO contralesional, 1.51 ± 0.36, n = 6–7 mice/group; sham operated vs. MCAO ipsilesional, P = 0.012; sham operated vs. MCAO contralesional, P = 0.033), reinforcing the implication of the hippocampus in MCAO-induced retrieval impairment.

Finally, we explored whether ischemia could also affect other types of hippocampus-dependent memory, such as spatial memory (Figure 2, C–F). Seven days after surgery, mice were trained for 6 consecutive days (3 rounds/d) in the Barnes maze platform, and 28 days later, a probe trial was conducted without the escape box to measure spatial memory retention. Both sham-operated and ischemic mice displayed similar speed and distance travelled and learned similarly to locate the escape hole with the hidden box during the course of the training period, as indicated by a progressive reduction in the time needed to reach the target hole (Supplemental Figure 3, A–D). However, confirming our previous results, ischemic mice displayed a decrease in spatial memory retention 28 days after training compared with the sham-operated group, when estimated either as the time spent in the target quadrant (TQ) (Figure 2E; P < 0.05 vs. sham-operated TQ) or as the time spent around the target hole (Figure 2F; P < 0.05 vs. sham operated). All these data suggest that cortical stroke in mice promotes long-term hippocampus-dependent cognitive impairment.

Stroke-induced neurogenesis in the SGZ positively correlates with memory impairment after cerebral ischemia. Cortical focal ischemia produces a long-term memory impairment that seems to be due to hippocampal affectation. Although no changes were found in overall hippocampal morphology or volume either 1 or 2 months after ischemia using Nissl staining or MRI, respectively (Supplemental Figure 4, A–D; P > 0.05), MCAO mice presented a significant increase in DG granular cell layer (GCL) volume compared with the sham-operated group 35 days after surgery (Figure 3, A and B; P < 0.05 vs. sham operated). Interestingly, a negative relationship between GCL volume and freezing response was found in the ischemic group (Figure 3C; r = –0.4972, P = 0.0016), indicating that animals with higher volumes displayed more severe cognitive deficits at the time studied.

Figure 3 Hippocampal neurogenesis positively correlates with memory impairment. (A and B) Cavalieri estimation of GCL volume in Nissl-stained sections from sham-operated and MCAO mice 35 days after surgery (*P < 0.05 vs. sham operated; sham operated, n = 10; MCAO, n = 40). (B) Representative images. Scale bar: 100 μm. (C) Negative relationship between GCL volume and freezing response in the ischemic group. Linear regression analysis is displayed (MCAO, n = 44; Spearman’s, r = –0.4972, P = 0.0016). (D) Experimental design for cell quantification. (E–H) Quantification of proliferative Ki67+ cells (F) and DCX+ immature neurons (H) in sham-operated and MCAO mice (ipsi- and contralesional DG). For F, a significant interaction between time after surgery and ischemia was found for Ki67+ (F (10,59) = 3.5; P = 0.0010; Bonferroni’s post hoc: *P < 0.05 vs. sham operated; sham operated, n = 4–5; MCAO ipsilesional, n = 4–5; MCAO contralesional, n = 3–5). For H, 2-way ANOVA showed significant differences for ischemia (F (2,57) = 27.90; P < 0.0001) and for time after surgery (F (5,57) = 5.57; P < 0.0001) (Bonferroni’s post hoc: *P < 0.05 vs. sham operated; sham operated, n = 3–5; MCAO ipsilesional, n = 3–5; MCAO contralesional, n = 3–5). (E and G) Representative images of Ki67+ (red) and DCX+ cells (green). Scale bar: 30 μm. (I and J) Spearman’s correlation between ipsilesional DCX+ cells and percentage of freezing 35 days after surgery (I, MCAO, n = 66; Spearman’s, r = –0.651, P < 0.0001) or between DCX+ cells and GCL volume in ischemic mice (MCAO, n = 40; Spearman’s, r = 0.4177, P = 0.0059). Data are represented as mean ± SEM. Data were compared by using nonparametric 2-tailed Mann-Whitney U tests (A) and nonparametric 2-way ANOVA followed by Bonferroni’s post hoc test. (E and G) Correlation analysis was assessed by Spearman’s (C, I, and J).

In the SGZ of the DG, new granular neurons are generated throughout life. These newborn neurons integrate into hippocampal circuits that play a role in memory storage (23–25). Acute brain injury stimulates hippocampal neurogenesis in different experimental models, but this response might have detrimental consequences and promote hippocampal malfunction, as observed in different pathologies, such as epilepsy or schizophrenia (8). Increased SGZ neurogenesis has also been found in different cortical stroke models (26, 27). Since perturbations in neurogenesis may alter GCL/DG size, we reasoned that poststroke neurogenesis could be involved in memory impairment observed after stroke. In our MCAO model (Figure 3D), an almost 2-fold increase in the number of proliferating Ki67+ cells and immature doublecortin+ (DCX+) neurons was found in both ipsi- and contralesional SGZ 14 days after injury, and this persisted at least 21 and 35 days after ischemia, respectively (Figure 3, E–H; P < 0.05 vs. sham operated). Importantly, we found an inverse correlation between the numbers of DCX+ cells and the freezing response, indicating that increased neurogenesis after cerebral ischemia leads to more severe cognitive deficits (Figure 3I; Spearman’s, r = –0.651, P < 0.0001). Accordingly, a positive correlation was found between GCL volume and DCX+ cell number in the SGZ of ischemic mice (Figure 3J; Spearman’s, r = 0.4177, P = 0.0059). We did not detect any differences between sham-operated and MCAO groups in the number of cleaved-caspase-3+ cells (data not shown), but we observed an approximately 1.5-fold increase in the number of new neurons (BrdU+/NeuN+ cells) in both ipsi- and contralesional hippocampus 65 days after ischemia (Supplemental Figure 5, A–D; P < 0.05 vs. sham operated; sham operated, 503.61 ± 33.40 cells; MCAO ipsilesional, 766.52 ± 38.33 cells; MCAO contralesional, 723.34 ± 34.57 cells), indicating that the increase in DCX+ cells and the associated cognitive deficits were not due to a higher death rate of newborn neurons. In addition, the increase in DCX+ cells did not correlate with lesion size (Supplemental Figure 6, A and B; P > 0.05 vs. sham operated) or reductions in the number of postnatal generated neurons as a consequence of ischemic stroke (Supplemental Figure 7).

Enhancement of poststroke neurogenesis exacerbates hippocampal cognitive deficits after ischemia. All these data suggest that levels of neurogenesis and memory deficits after ischemic injury are directly related. To reinforce this correlation, we tested to determine whether interventions directed to further enhance SGZ neurogenesis could exacerbate cognitive deficits in the MCAO group (Figure 4). First, 7 days after surgery, we enhanced neurogenesis in both sham-operated and MCAO mice by allowing them free access to a running wheel, while control animals remain sedentary, with a locked running wheel in their home cages. Twenty-eight days later, running efficiently increased the number of both proliferating Ki67+ cells (Figure 4, A and B; P < 0.05 vs. MCAO sedentary) and DCX+ cells (Figure 4, C and D; P < 0.05 vs. MCAO sedentary) in both sham-operated and MCAO groups compared with sedentary controls (Figure 4, A–D, and Supplemental Figure 8, A and B). To check whether overstimulated neurogenesis after stroke exacerbates cognitive impairment, we evaluated memory retention after CFC (Figure 4E). Consistent with our hypothesis, 28 days after conditioning, running further impaired the recall of remote contextual fear memories relative to that in the sedentary groups (Figure 4E and Supplemental Figure 8C; P < 0.05 vs. sham operated or MCAO sedentary). In addition, MCAO impaired incidental context learning (Supplemental Figure 8D, P < 0.05 vs. sham operated), an effect that was further increased by running (Supplemental Figure 8D, P < 0.05 vs. MCAO sedentary).

Figure 4 Increasing poststroke neurogenesis enhances cognitive impairment. (A–D) Quantification of Ki67+ (A) and DCX+ cells (C) 35 days after surgery in sedentary and runner MCAO mice. (B, D) Representative images. Scale bars: 30 μm. Two-way ANOVA analysis showed significant effect of running in number of Ki67+ (A) (F (1,30) = 50.09; P < 0.0001) and DCX+ cells (C) (F (1,29) = 14.05; P = 0.0008) (Bonferroni’s post hoc: *P < 0.05 vs. MCAO sedentary; MCAO sedentary, n = 8; MCAO runner, n = 9). (E) MCAO mice remained sedentary or ran after CFC and were tested 28 days later (*P < 0.05 vs. MCAO sedentary; MCAO sedentary, n = 13; MCAO runner, n = 8). (F–I) Barnes maze testing. (F) Experimental design. (G) Representative traces. Time spent by MCAO mice around each quadrant (H) or the target hole (I). For H, 2-way ANOVA demonstrated significant interaction between different quadrants and running (F (3,96) = 3.25; P = 0.0251; *P < 0.05 vs. MCAO sedentary TQ; Bonferroni’s post test). For I, *P < 0.05 vs. MCAO sedentary; MCAO sedentary, n = 15; MCAO runner, n = 11. (J–L) MCAO mice treated with MEM showed a reduced freezing response (L) (*P < 0.05 vs. MCAO vehicle; MCAO vehicle, n = 7; MCAO MEM, n = 11) and an increased the number of DCX+ cells (J and K). (J) Two-way ANOVA demonstrated significant effect of MEM in DCX+ cells (F (1,33) = 28.85; P < 0.0001) (Bonferroni’s post hoc: *P < 0.05 vs. MCAO vehicle; MCAO vehicle, n = 7; MCAO MEM, n = 10). Representative images (K). Scale bar: 30 μm. Data are represented as mean ± SEM. Data were compared by using nonparametric 2-tailed Mann-Whitney U tests (E, I, and L) and nonparametric 2-way ANOVA followed by Bonferroni’s post hoc testing (A, C, H and J).

We next used the Barnes maze to determine whether a comparable effect was also found in spatial memory (Figure 4, F–I). Indeed, spatial memory deficits observed in the MCAO sedentary group were exacerbated in runner ischemic mice (Figure 4, H and I), which did not preferentially move toward the TQ (Figure 4H, P < 0.05 vs. MCAO sedentary) or the correct hole (Figure 4I, P < 0.05 vs. MCAO sedentary), showing random behavior with no preference toward any region of the Barnes maze.

To reinforce our results, we next asked whether analogous memory deficits would be observed by using memantine (MEM) (Figure 4, J–L) as a pharmacological approach to increasing neurogenesis after stroke injury (28). Consistent with our previous results, MEM treatment increased poststroke SGZ neurogenesis, estimated as an increase in the number of DCX+ cells (Figure 4, J and K, and Supplemental Figure 9A; P < 0.05 vs. MCAO vehicle) while impairing remote memory recall, as demonstrated by a decreased freezing response (Figure 4L and Supplemental Figure 9B; P < 0.05 vs. MCAO vehicle). All these data support the idea that enhanced hippocampal neurogenesis negatively interferes with the retrieval of memories and could therefore contribute to cognitive deficits after stroke.

Stroke-induced newborn neurons promote differential hippocampal circuitry remodeling. Memory retrieval may result from the reactivation of the same neuronal ensembles at the time of memory encoding (29, 30). Enhanced integration of newborn neurons into hippocampal circuits due to high neurogenesis levels could negatively regulate the ability to recall memories by replacing preexisting synapses and therefore remodeling hippocampal connections where memory is stored, a mechanism that has been demonstrated to mediate forgetting (9). This mechanism might thus explain the inverse correlation found between neurogenesis and hippocampal performance in our cortical ischemia model. However, in our scenario, an alternative and/or additional explanation might be the presence of pathological neurogenesis. Indeed, aberrant morphological alterations have been described for SGZ newborn neurons after MCAO (18, 19). To gain further insight into the mechanisms that contribute to cognitive impairment in our cortical stroke model, we characterized the distribution of the dendritic arborization along granular and molecular layers (ML) in immature neurons (DCX+; Supplemental Figure 10, A–D). Of note, differential patterns were found for ipsi- and contralesional DGs. While a clear increase in the percentage of dendritic arborization in the ML was observed for the contralesional hippocampus, no significant differences were detected for the ipsilesional side (Supplemental Figure 10, A and C; P < 0.05 vs. sham operated and MCAO ipsilesional ML1, respectively). Interestingly, differential results were also found in the number of immature large mossy fiber terminals (LMTs-DCX+) reaching the CA3 region. Whereas a reduction in the number of LMTs in CA3 was detected in the ipsilesional side, in contrast, an increased synaptic rearrangement was found in the contralesional CA3 (Supplemental Figure 10, B, D, and E; P < 0.05 vs. sham operated and MCAO ipsilesional, respectively), further suggesting that differential remodeling processes were taking place in parallel at the ipsi- versus the contralesional hippocampus after ischemia.

To confirm these results, 14 days after surgery, we infused GFP-expressing retrovirus into the DG of sham-operated and ischemic mice in order to evaluate newborn neuron integration and possible circuit remodeling (Figure 5, A and B). Sholl analyses revealed a phenotype comparable to that observed for DCX+ arborization in contralesional DG GFP+ neurons, corroborating an increase in dendritic branching in the distal segment (150–250 μm from the soma; Figure 5, C and D; P < 0.05 vs. sham operated and MCAO ipsilesional, respectively) and in total dendritic length (Figure 5E; P < 0.05 vs. sham operated and MCAO ipsilesional, respectively). Remarkably, an opposite phenotype was found in ipsilesional ischemic GFP+ newborn neurons, with an increased degree of branching in the proximal domain (0–50 μm from the soma; Figure 5, C and D; P < 0.05 vs. sham operated and MCAO contralesional, respectively) and a retraction of the distal domain in the ML. Accordingly, ipsilesional GFP+ neurons also displayed a reduction in total dendritic length (Figure 5E).

Figure 5 Altered features of newborn neurons induced by stroke. (A) Experimental design for GFP retroviral labeling of newborn neurons. (B) Representative images of GFP newborn neurons of each group. Yellow arrow, 112 μm. (C) Sholl analysis of GFP newborn neurons in sham-operated and MCAO groups, 28 days after infection. Two-way ANOVA demonstrates a significant interaction between distance and experimental group in the number of intersections (F (48,3022) = 5.18; P < 0.0001; Bonferroni’s post hoc: *P < 0.05 vs. sham operated; #P < 0.05 vs. MCAO ipsilesional; sham operated, n = 52 neurons/4 mice; MCAO ipsilesional, n = 41 neurons/4 mice; MCAO contralesional, n = 32 neurons/3 mice). (D) Mean average intersections of GFP neurons at different intervals from soma. (*P < 0.05 vs. sham operated; #P < 0.05 vs. MCAO ipsilesional; sham operated, n = 52 neurons/4 mice; MCAO ipsilesional, n = 41 neurons/4 mice; MCAO contralesional, n = 32 neurons/3 mice). (E and F) Quantification of total dendritic length (E, *P < 0.05 vs. sham operated; #P < 0.05 vs. MCAO ipsilesional, respectively; sham operated, n = 18 neurons/4 mice; MCAO ipsilesional, n = 45 neurons/4 mice; MCAO contralesional, n = 29 neurons/3 mice), and apical dendritic length (F, *P < 0.05 vs. sham operated; #P < 0.05 MCAO vs. ipsilesional; sham operated, n = 61 neurons/4 mice; MCAO ipsilesional, n = 60 neurons/4 mice; MCAO contralesional, n = 40 neurons/3 mice) in sham-operated and both ipsi- and contralesional sides of MCAO group. (G) Pie charts display percentage of GFP+ neurons in each group showing apical dendrite lengths of less than 10 μm, 10–40 μm, and more than 40 μm (sham operated, n = 61 neurons/4 mice; MCAO ipsilesional n = 60 neurons/4 mice; MCAO contralesional, n = 40 neurons/3 mice). Data are represented as mean ± SEM. Data were compared by using nonparametric 2-tailed Mann-Whitney U tests (D–F) and nonparametric 2-way ANOVA followed by Bonferroni’s post hoc testing (C).

Mature granule newborn neurons generally display only 1 apical dendrite, which remains almost not ramified until it reaches the ML, where it begins to extend its branches and establishes synapses with afferents of the perforant pathway from the entorhinal cortex (EC). Therefore, apical dendrite growing seems to be a critical factor for the correct integration of newborn neurons (31, 32). Our data also demonstrate a differential pattern of apical dendrite growth in GFP+ neurons of the ipsi- versus the contralesional ischemic DG. Contralesional ischemic GFP+ neurons presented a longer apical dendrite compared with both ipsilesional and sham-operated GFP+ neurons (Figure 5F and Supplemental Figure 11A). In contrast, ipsilesional GFP+ neurons showed a dramatic reduction in apical dendrite length compared with the other groups (Figure 5F; P < 0.05 vs. sham operated and MCAO contralesional, respectively). Interestingly, when we classified GFP+ neurons in each group according to apical dendrite length intervals (Figure 5G and Supplemental Figure 11A), our data demonstrate that apical dendrite growth alteration due to ischemic stroke differentially affected approximately 30% of the newborn neuron population in ipsi- (predominant aberrant phenotype <10 μm) and contralesional sides (predominant aberrant phenotype 40–70 μm), compared with the GFP+ neurons observed in sham-operated mice (predominant phenotype 10–40 μm). We also analyzed to determine whether stroke affected spine density of GFP+ newborn neurons as an index of synaptic integration. Of note, despite their altered dendrite arborization, no differences were observed in the density of dendritic spines of GFP+ newborn granule cells either in the ipsi- or in the contralesional side when compared with the sham-operated group (Supplemental Figure 11, B and C), suggesting that these newborn neurons are integrated into hippocampal circuits receiving inputs from the EC.

As previously demonstrated for poststroke neurogenesis, aberrant features of newborn neurons might also be transient after stroke. For assessing this issue, in a different set of experiments, the GFP-expressing retrovirus was delivered 35 days after stroke (Supplemental Figure 12A), when poststroke neurogenesis had already returned to physiological levels. Interestingly, although the aberrant features of newborn neurons previously detected in Sholl analysis or in mean apical dendrite length were not observed at this time point (Supplemental Figure 12, B and C), 30.5% of the ipsilesional newborn population still displayed apical dendrite growth alterations (aberrant phenotype <10 μm; Supplemental Figure 12, D and E), an effect that might lead to an incorrect integration of axonal projections coming from EC.

All these results indicate that ischemic stroke promotes an increase in neurogenesis that gives rise to the generation of differential populations of newborn neurons with altered morphological features depending on their ipsi- or contralesional location, an effect that remains, at a lower intensity, when the levels of neurogenesis become normalized. The integration of these abnormal neurons could promote aberrant hippocampal circuitry rearrangements and, therefore, contribute to hippocampal cognitive deficits observed after ischemia.

Poststroke memory impairment is reduced by downregulation of ischemia-induced aberrant neurogenesis. Our data support that high levels of poststroke neurogenesis initiate a differential remodeling of hippocampal circuits that contributes to cognitive deficits after stroke. Therefore, we predicted that decreasing aberrant poststroke neurogenesis toward physiological levels might ameliorate ischemia-induced hippocampal deficits. For this, we used both pharmacological and genetic strategies. First, we suppressed poststroke neurogenesis by treating mice with temozolomide (TMZ), a DNA-alkylating agent (Figure 6 and Supplemental Figure 13). TMZ treatment caused a reduction of neurogenesis in MCAO mice, as reﬂected by reduced numbers of proliferating Ki67+ cells and immature neurons expressing DCX (Figure 6, A and B; P < 0.05 vs. MCAO vehicle, and Supplemental Figure 13). Importantly, and confirming our hypothesis, TMZ treatment improved memory retrieval in parallel to a reduction in neurogenesis, as shown by an increased freezing response in mice evaluated 28 days later (Figure 6, C and D; P < 0.05 vs. MCAO vehicle).

Figure 6 TMZ treatment after stroke mitigates hippocampus-dependent memory deficits. (A and B) Number of DCX+ cells in MCAO mice treated with vehicle or TMZ. Representative images of DCX+ cells (B, green) in MCAO mice treated with vehicle or TMZ. Scale bar: 30 μm. For A, 2-way ANOVA analysis showed a significant effect of TMZ in the number of DCX+ cells (F (1,32) = 81.85; P < 0.0001) in both ipsi- and contralesional sides (Bonferroni’s post hoc: *P < 0.05 vs. MCAO vehicle; MCAO vehicle, n = 8; MCAO TMZ, n = 9) at 35 days. (C) Experimental design for poststroke neurogenesis inhibition by TMZ. Seven days after ischemia, mice were subjected to CFC (0.6 mA × 3), and 24 hours later, they were treated i.p. with TMZ (25 mg/kg) for 4 weeks (3 days per week) and tested in the CFC at the end of the treatment. (D) Percentage of freezing response in vehicle- or TMZ-treated ischemic mice 35 days after CFC (*P < 0.05 vs. MCAO vehicle; MCAO vehicle, n = 13; MCAO TMZ, n = 9). Data are represented as mean ± SEM. Data were compared by using nonparametric 2-tailed Mann-Whitney U tests (D) or nonparametric 2-way ANOVA followed by Bonferroni’s post hoc testing (A).

To reinforce our results, we genetically ablated aberrant hippocampus-generated neurons after ischemia by using Nestin-CreERT2/NSE-DTA mice (33–35) (Figure 7A). After tamoxifen administration, a STOP region in the diphtheria toxin fragment A (DTA) cassette gene was deleted in nestin+ neural stem cells (NSCs). These cells die by apoptosis at the beginning of their neuronal differentiation, due to the DTA expression under the neuron-specific enolase 2 (NSE, also known as Eno2) promoter gene. Genetic ablation of newly formed neurons after stroke led to a decrease in DCX+ cells (Figure 7, B and C; P < 0.05 vs. Nestin-CreERT2/NSE-DTA vehicle, and Supplemental Figure 14, A and B) in parallel to a better performance in both contextual fear (Figure 7D, P < 0.05 vs. Nestin-CreERT2/NSE-DTA vehicle, and Supplemental Figure 14C) and the Barnes maze test (Figure 7, E–H, P < 0.05 vs. NestinCreERT2/NSE-DTA vehicle).

Figure 7 Conditional deletion of newborn neurons after stroke decreases cognitive deficits. (A) Genetic strategy used for conditional deletion of newborn neurons after stroke. (B) Number of DCX+ cells in MCAO Nestin-CreERT2/NSE-DTA mice treated with vehicle or tamoxifen. Two-way ANOVA showed a significant effect of tamoxifen in the number of DCX+ cells (F (1,48) = 136.59; P < 0.0001) in both ipsi- and contralesional sides (Bonferroni’s post hoc: *P < 0.05 vs. Nes-CreERT2/NSE-DTA vehicle; vehicle, n = 14; tamoxifen, n = 12). (C) Representative images of DCX+ cells (green) in ischemic Nestin-CreERT2/NSE-DTA mice after vehicle or tamoxifen 35 days after surgery. Scale bar: 30 μm. (D) Percentage of freezing in vehicle- or tamoxifen-Nestin-CreERT2/NSE-DTA treated mice 35 days after CFC (*P < 0.05 vs. Nestin-CreERT2/NSE-DTA vehicle; vehicle, n = 12 and tamoxifen, n = 13). Upper panel: experimental design. Seven days after ischemia, Nestin-CreERT2/NSE-DTA mice were subjected to CFC (0.6 mA × 3), and 24 hours later, they were treated i.p. with vehicle or tamoxifen (150 mg/kg) over 4 consecutive days and tested in the CFC 35 days after surgery. (E–H) Experimental design for assessing spatial memory retention in the Barnes maze (E). Five days after ischemia, Nestin-CreERT2/NSE-DTA mice were trained during 7 days (3 session/d) in the Barnes maze. After training, mice were treated with vehicle or tamoxifen and tested 28 days later. (F) Representative traces during the retention test. (G) Time spent in each quadrant of the Barnes maze platform. Two-way ANOVA demonstrated a significant interaction between quadrants and tamoxifen treatment in ischemic Nestin-CreERT2/NSE-DTA (F (3,68) = 4.41; P = 0.0068; Bonferroni’s post hoc: *P < 0.05 vs. Nestin-CreERT2/NSE-DTA vehicle TQ). (H) Time spent around target hole during testing (*P < 0.05 vs. MCAO Nestin-CreERT2/NSE-DTA vehicle; vehicle, n = 7; tamoxifen, n = 12). Data are represented as mean ± SEM. Data were compared by using nonparametric 2-tailed Mann-Whitney U tests (D and H) and nonparametric 2-way ANOVA followed by Bonferroni’s post hoc testing (B and G).

Together, our data demonstrate that increased levels of poststroke neurogenesis contribute to cognitive hippocampal deficits observed following injury, very likely by producing aberrant remodeling of hippocampal circuits.