Long-term effects of neonatal strokes on behaviors and physical development
We have previously reported behavioral results from post-stroke CD1 mice at P40 (i.e., 4 weeks following the P12 stroke injury). The assays in the previous report were limited to rotorod, open-field, and T-maze spontaneous alternation [21
]. However in the present study, chronic video-monitoring of the 8 ligated mice (n/n=8/11), 1 week per month over a six month period following the stroke was also included, and revealed temporally progressive hyperactivity behaviors that were not noted in the P40 study. Lateralized “mad running” behaviors became apparent in 4 out of the 8 ligation-injured monitored mice (i.e., 50%, 3 clockwise, 1 counterclockwise; 2 males and 2 females) beginning at 2 to 3 months of age. The mice continuously circled the perimeter of their cages for extended periods of time with a running gait. This behavior progressively worsened as evidenced by an increase in episodes and the amount of time spent running around the cage. In 3 of those mice (i.e., 38%, 2 females, 1 male) the activity progressed from running around the periphery of the cage to tighter clockwise rotations at 4 to 5 months of age. This behavior was sometimes associated with “tail chasing” for extended periods of time that was not associated with behavioral features of post-ictal depression like malaise and inactivity. On the contrary there was an overall locomotor hyperactivity. The shams that were monitored for the same periods of time did not show similar behaviors with advancing age. Concomitant weight monitoring over the period of the study, detected considerable lag in weight gain in all the ligation-injured mice (males more than females, ) that was also temporally progressive akin to the increase in hyperactive behaviors. Since the lag in weight gain was not limited to the hyperactive circling mice, the finding cannot be attributed to hyperactivity alone. The differences in weight between ligated and sham-control mice were significant in males (p=0.002) compared to females (p=0.053) at the time when behavioral testing was conducted at 3 months of age (; compare A to B, dotted lines).
Figure 3 Animal weights as a function of time and performance on the Rotarod and spontaneous T-maze alternation tests. A and B. Compared to controls both female (A) and male (B) ligation-injured mice showed impaired weight gain as a function of time over the 7 (more ...)
Motor and cognitive testing
Behavior testing was done on the all the mice in the study (i.e., 11 ligates and 10 sham-controls) at 3 months of age.
Rotorod data was analyzed by a 2-way repeated measures ANOVA with Treatment (sham vs. ligated) and Gender as the between factors and Trial as the repeated measure factor. There was no main effect of Treatment or Trial nor were there any interactions. Only the main effect of Gender was significant (F1,18
= 5.011, p = 0.038). Since the effect of Gender did not impact any other factor it is likely that this main effect was due to the overall shorter fall latencies for all male mice (trial 1: 56.997 ± 7.32; trial 2: 46.1 ± 6.17; trial 3: 53.66 ± 7.79) relative to all female mice (trial 1: 73.89 ± 6.42; trial 2: 62.49 ± 4.99; trial 3: 65.87 ± 7.67). Nevertheless, the rotorod test detected no motor learning deficits for the ligation-injured mice at 3 months after the ligation and thus was similar to previously reported performances at P40 [21
T-maze spontaneous alternation
To measure exploratory behavior as well as memory, we assessed performance on a T-maze spontaneous alternation task. Results from the T-maze spontaneous alternation task can be seen in which shows that the ligation-injured mice alternated at a lower mean rate than sham-control mice that was not significant (p=0.3). There was no difference in the number of trials completed by each groups since. Ligation-injured mice completed on average 12.3±0.9 of the 15 trials compared to the 12.7±0.7 trials by the control group of mice. We also examined the percent of right turns made and found no significant differences between the two groups (43.6±2.8% for sham-control mice and 51.6±4.5% for ligation-injured mice; p=0.15). This indicated that there was no significant lateralized preference for making right turns (i.e., towards the side of the unilateral stroke injury) in the ligation-injured group compared to controls.
Open-field activity and habituation
To examine overall locomotor activity and habituation, spatial distribution and rotational bias, mice were observed in an open-field over 2 consecutive daily sessions. We first examined locomotor activity across the 2 days () and analysis revealed only a main effect of Treatment (F1,20 = 8.322, p = 0.009) reflecting the overall higher levels of activity in the ligates compared to the controls. There was no main effect of Day or a Day × Treatment interaction. Thus, although there was a trend toward less activity in the controls on day 2 relative to day 1 (across session habituation), this difference was not significant. To examine habituation within a session, the total distance traveled during the 30 minute session was analyzed in 5 minute blocks. Analysis of the Day 1 session () revealed significant main effects of Treatment (F1,20 = 7.519, p = 0.013) due to the overall longer distance traveled by the ligated mice, but no effect of Time Block nor a Time Block × Treatment interaction. This was surprising giving the apparent decrement in activity over the course of the session in the sham-control mice, therefore, we performed one-way Anova’s for each treatment group and found a significant effect of Time Block for the sham-controls (F5,20 = 47.317, p < 0.001), indicating that this group habituated, or decreased locomotion over the length of the session. However no such effect was observed in the ligated group. Analysis of the day 2 session () also yielded a significant main effect of Treatment (F1,20 = 10.371, p = 0.004), but in contrast to the day 1 session, the day 2 session analysis also revealed a significant Time Block × Treatment interaction (F5,100 = 2.423, p = 0.041). Posthoc analysis revealed this interaction to be due to a decrease in locomotion over the course of the session in the sham-controls (p<0.001), but not in the ligated mice (p = 0.502). Thus, the ligated mice had higher overall levels of activity, and the only habituation observed was in the sham-control mice within each session.
Figure 4 Open-field test: total open-field activity and habituation. A. Ligation-injured mice showed significantly higher levels of locomotor activity compared to controls in the open-field. B and C. Ligation-injured mice exhibited a complete lack of habituation (more ...)
In addition to habituation, we determined if the spatial distribution of activity differed between the two groups. Therefore, the time mice spent in the four quadrants of the open-field (left front, left rear, right front, and right rear) as well as the time mice spent along the peripheral and the center zones of the field were examined. Analysis of the quadrant data from day 1 yielded no significant main effects or an interaction. In contrast, analysis of the quadrant data from day 2 yielded a significant main effect of Quadrant (F3,60 = 3.095, p = 0.033) due to all mice spending more time in the left rear quadrant overall, and there was a significant Quadrant × Treatment interaction (F3,60 = 3.146, p = 0.032). Follow up analysis revealed that this interaction was most likely due to the ligated mice spending more time in the left rear quadrant relative to the sham mice although the difference was statistically marginal (p = 0.057; data not shown). There were no other significant main effects or interactions. Next we examined the time mice spent in the peripheral and central zones of the field during the two daily sessions and analysis yielded only a significant effect of Zone (Day 1: F1,20 = 401.647, p < 0.001 and Day 2: F1,20 = 415.824, p < 0.001) due to all mice (i.e., ligation-injured and shams) spending more time along the walls of the field than in the center of the field (data not shown).
Finally, we examined the number of clockwise and counter clockwise revolutions made by the mice in the open-field during each of the two daily sessions. Paired comparisons of the data from day 1 did not reveal any significant differences in the numbers of clockwise and counterclockwise rotations for either the shams (19.0 ± 2.08 vs. 40.8 ± 16.69, respectively) or the ligated mice (148.45 ± 61.82 vs. 78.55 ± 53.02 respectively). Similarly comparisons of the data from day 2 did not reveal any significant differences in the numbers of clockwise and counterclockwise rotations for either the shams (17.18 ± 2.37 vs. 18.1 ± 2.52, respectively) or the ligated mice (147.45 ± 62.63 vs. 59.64 ± 34.38 respectively). Analysis of the clockwise rotations across day yielded only a main effect of Treatment (F1,20 = 4.722, p = 0.042; ) due to the higher number of clockwise rotations by the ligates on both days. However analysis of the counter clockwise revolutions across both days did not reveal any significant differences, indicating that the number of counter-clockwise revolutions were similar in the two groups of mice across both days. This open-field test finding was consistent with the clockwise rotational hyperactivity detected in 3 out of 4 ligation-injured mice with lateralized circling hyperactivity of the ligation-injured and video-monitored mice that showed temporal exacerbation.
Unilateral stroke-injury related atrophy in the ipsilateral hemisphere
All the ligated mice introduced in the long-term study (i.e. 100%) showed significant stroke injury in the ipsilateral hemisphere as predicted by their acute seizure scores (, right column compared to the left). Stroke severity was quantified and ranged between 60–80% hemispheric atrophy and 80–90% hippocampal atrophy. Injury-severity in mice randomly assigned to the cage-control and novel-exploration group of ligated mice did not show significant differences (). GCL areas in which counts of BrdU and Arc-positive cells were done were measured. Ipsilateral GCLs in ligation-injured mice were significantly smaller than controls (71602±15749 µm2
and 125127±4775 µm2
respectively for the novel-exploration group, p=0.006 and 53282±4771 µm2
and 160461±12191 µm2
respectively for the cage-control group of mice, p=0.001). Contralateral GCL areas in the ligation-injured mice were not significantly different from sham-controls (p=0.7). Stroke related hippocampal atrophy was severe and not uniform. The dorsal hippocampus was atrophied but relatively spared compared to the ventral hippocampus which in most ligation-injured mice was completely lost to the stroke-injury (, left column) accounting for the overall high percent of atrophy in the ligation-injured mice. As previously reported in the model, CA3 and CA1 neurons are more susceptible to cell death than the DG in the hippocampus [10
]. Therefore, inspite of significant overall hippocampal atrophy, corresponding DGs in the dorsal hippocampi were relatively spared (i.e., GCL areas in ligation- injured mice were 56% and 33% of control in novel-exploration tested and cage-control mice respectively).
At the age of 7 months, BrdU incorporated into dividing cells at P17–21 and detected by green fluorescence (Alexa 488, Chemicon) was distinct in the nuclei of cells in the granule cell layer at low magnifications (10×, ). Total counts of BrdU-positive-cells in the GCL revealed significant differences between ligation-injured and sham-control brains () in the ipsilateral DG. Mean counts of BrdU-positive cells in novel-exploration tested ligation-injured brains were significantly lower compared to the novel-exploration tested shams in the ipsilateral GCLs (30.4±6.4 vs. 72.7±6.3, , black bars, p<0.0001). Total counts of BrdU-positive-cells in the cage-control group of mice also showed a similar trend as would be expected (ligation-injured mice 24.4±4.2 compared to 73.4±4 cells in sham-controls, p <0.0001). In mice that underwent novel-exploration, counts of BrdU-positive-cells in uninjured contralateral hippocampi of ligation- injured brains were 64.6±6.2 compared to 80.3±10.6 cells in sham-controls and therefore marginally but not significantly lower (, gray bars p<0.6). The ipsilateral reduction of endogenous post-stroke neurogenesis had a negative correlation with the corresponding percent atrophy of the injured hippocampi () that did not reach significance (r=−0.55, p=0.1). Counts of BrdU-positive-cells when normalized to their corresponding GCL areas (i.e., cells/mm2 of GCL), were found to be similar to the sham-controls (, compare black to gray bars both for the novel-exploration and cage-control groups of mice), indicating the lower counts of BrdU-positive-cells in the ipsilateral injured hippocampi resulted from the severity of the GCL atrophy associated with the ischemic stroke.
Figure 5 BrdU-positive-cells and Arc-induction in the GCL following novel spatial exploration test conducted 7 months after neonatal-stroke. A1 and 2 show BrdU labeled cells (A1) and basal Arc expression levels (A2) from the same coronal section in a cage-control (more ...)
Novel exploration-dependant Arc-induction in sham and ligation-injured groups of mice at 7 months of age
Every mouse in sham and ligation-injured groups explored all novel objects (i.e. pet toys) spread around the four corners of the novel-exploration field. Neither “mad running” behaviors nor Racine class 1–5 behavioral seizures were noted in the 5 minutes of the novel enriched environment exposure period. Continuous video-monitoring done in the hour following the novel exploration, when the mice were returned to their home cages, did not reveal any behavioral seizures, although “mad running” behaviors were seen in the mice that had shown the behavior in the 6 months preceding testing. Hippocampal circuits activated by exploration have routinely been studied in the dorsal hippocampus. Following novel exploration robust Arc-induction was seen in the control () and ligation-injured mice () after novel-exploration compared to their respective cage-controls (i.e., sham and ligation-injured mice not exposed to the novel-exploration; ) in the dorsal hippocampus. Counts of Arc-positive cells in the ligation-injured mice however were significantly lower than control mice after induction (). This finding indicated that, inspite of significant unilateral stroke-related loss of brain volume, neuronal networks linked to learning and memory, and identified by behavior-dependent induction of the immediate early gene- Arc in GCL neurons, remained functional but were significantly impaired. Total counts of Arc-positive cells in the DG revealed significant differences between ligation-injured and sham-control brains () in both the ipsilateral and contralateral DG. After novel-exploration there were significantly fewer Arc-positive neurons in the ipsilateral DG of ligation-injured brains as compared to the sham-controls (13.4±3.6 vs. 32.8±5.5, p=0.02; , black bars, @). More importantly, counts of Arc-positive cells in uninjured contralateral hippocampi of ligation-injured brains after novel-exploration were also significantly lower than sham-controls (17.2±3 vs. to 32.6±5.6; p=0.02; , gray bars, @). In the cage-control group, counts of Arc-positive cells in ligation-injured mice were lower than the shams, however not significantly (1.5±0.9, vs. 9.8± 4.6, p=0.19 ipsilaterally and 4.1±2 vs. 9.3±4.4, p=0.4 contralaterally). Arc-induction following the novel-exploration test, however was significantly higher in the novel-exploration versus cage-control groups of mice, both in the sham-control (p= 0.01, both ipsi- and contralaterally) and ligation-injured mice (p=0.03 ipsilaterally and p=0.005 contralaterally). The results show the efficacy of the 5 min exposure to the novel environment for robust IEG activation and protein expression when examined 1 h later in the CD1 mouse strain. The impaired induction detected in ligation-injured mice ipsilaterally, is likely directly stroke-injury related. However the contralateral impairment of Arc induction indicated that inter-hemispheric commissural input is essential for bilateral functional hippocampal circuit activation.
Similar to the BrdU-cell counts, Arc-positive cell counts were also normalized to their corresponding GCL areas () and Arc-positive cell densities still maintained significance of induction in the novel-exploration group over the cage-control group in the shams (269.6±50.6 and 62.2±27.7 cells/mm2 respectively ipsilaterally, p=0.015; and 232.6±33.5 and 75.1±34.5 cells/mm2 respectively contralaterally; p= 0.014). In ligation-injured mice the significance between the novel-exploration and cage-control groups was maintained contralaterally (123.7±12.8 and 26.8±14 cells/mm2 respectively; p=0.002) but lost ipsilaterally (417±292.7 and 28.15±17.6 cells/mm2 respectively; p=0.3) due to the large variability associated with stroke-injury related atrophy of the ipsilateral GCLs. After normalization to injury (i.e., corresponding GCL areas in which counts were done) in the group of mice that underwent novel-exploration, Arc-induction remained bilaterally significant in sham-controls and contralaterally in ligation-injured mice, however ipsilaterally this significance was lost (p=0.6). Ipsilaterally, Arc-induction was present but variable and no significant correlation to their corresponding GCL areas was evident (r2=−0.2, p=0.7). This finding may indicate that the variable Arc-induction in the ipsilateral GCLs of ligation-injured mice is dependent on factors other than GCL atrophy, which may include variability of impaired inputs coming in from the injured somatosensory, parietal and visual cortex. In contrast, the density of Arc-positive neurons in the contralateral hippocampus of ligation-injured mice after novel-exploration remained significantly impaired compared to controls after novel exploration. They however showed a significant positive correlation to their corresponding GCL areas (r=0.829, p=0.011) likely due to lack of any ischemic injury related atrophy and thus remained similar to controls. In the cage-control group of mice, baseline Arc activity normalized to injury related GCL atrophy in ligation-injured mice compared to sham-controls, was lower bilaterally, however not significantly [28.2±17.6 vs. 62.2±27.7 ipsilaterally (p=0.4) and 26.8±14 vs. 75.1±34.5 contralaterally (p=0.3); ].
As previously described there was distinct predominance of Arc-positive cells in the upper or dorsal blade of the DG () as compared to the corresponding lower or ventral blade [16
]. This was significantly true for sham-controls that underwent novel-exploration [i.e., 27.3±4.4 and 27.4±4.4 in the upper blade, 5.6±1.5 and 5.1±1.4 in the lower blade, ipsi- (p=0.002) and contralaterally (p=0.001)] respectively] and not statistically significant for sham cage-controls [i.e.7.6±3.9 and 7.4±3.6 in the upper blade, 2.2±0.6 and 1.8±0.9 in the lower blade, ipsi- (p=0.19) and contralaterally (p=0.16) respectively]. Similar observations were made in the ligation-injured group of mice that underwent novel-exploration [i.e., 8.1±2.3 and 12.5±2 in the upper blade, 5.3±1.8 and 4.7±1.2 in the lower blade, ipsi- (p=0.2) and contralaterally (0.002) respectively]. The cage-control group of ligation-injured mice had non-significant trend for a lower baseline of constitutive Arc expression than sham-controls (), and failed to show upper blade predominance of Arc expression due to the lower counts [i.e., 0.9±0.8 and 2.7±1.4 in the upper blade, 0.6±0.4 and 1.4±0.8 in the lower blade of ligation-injured mice, ipsi- (p=0.7) and contralaterally (p=0.4) respectively]. Arc-induction following novel-exploration in the sham-group of mice, when compared to their cage-control littermates, revealed that the significant number of induction related positive cells were in the upper blade bilaterally (* ) as opposed to the lower blade. In the lower blade, the numbers of Arc-positive-cells following novel-exploration in sham-controls were marginally higher however not significantly different from the counts of Arc-positive-cells in the lower blade of their cage-control littermates (p = 0.12 ipsi- and contralaterally). In ligation-injured mice, Arc-induction in the novel-exploration tested mice was also found to be significant in the upper blades of the contralateral DG [p=0.02 for counts, p=0.002 for density (cells/mm2
)] but not in the severely atrophied upper blades of the ipsilateral DGs (p=0.06 for counts and p=0.2 for density; * ). Arc-positive cell densities in ipsilateral upper blades of GCLs were variable relative to the severity of injury in their respective hippocampi and did not show significant correlation to the associated atrophied GCLs (i.e., corresponding GCL areas, r2
= −0.3, p=0.6). Contralateral upper-blade Arc-positive cell densities remained significantly lower than the sham-group that underwent novel exploration (, @). In summary, the present, but reduced, Arc-induction detected the in contralateral uninjured hippocampi was a novel finding for this study. Its persistence at >6 months after neonatal stroke, indicate opportunities for enhancement strategies.
Integration of post-stroke born granule cells into functional circuits
Sub-granular zone neural stem cells proliferating in the 5–9 days after stroke were BrdU-labeled during the S phase of cell division. New-cells that survived the process of differentiation and maturation were then tested after novel-exploration, for their functional integration into hippocampal networks. The number of neurons that were co-labeled with BrdU and Arc represented the newborn cells labeled in the post-stroke period that had integrated into the functional circuits during routine spatial tasks in the cage-control mice and acquisition of novel spatial information in the novel-exploration mice (). As expected, the counts of co-labeled neurons were higher after novel-exploration in the sham-group of mice. Surprisingly, the counts of co-labeled neurons were also higher in the ligation-injured mice () in whom robust Arc-induction had occurred following novel-exploration () compared to their cage-control littermates that was significant in the contralateral DGs (i.e., in the uninjured dorsal hippocampi; p=0.03; ). Following novel exploration, counts of co-labeled cells in the ligation-injured group of mice were 0.65± 0.4 ipsilaterally and 0.25 ± 0.1 contralaterally, therefore were marginally but not significantly lower (p=0.5 ipsilaterally and p=0.1 contralaterally) compared to the shams (1.1±0.5 ipsilaterally and 1.1 ± 0.6 contralaterally). No baseline activity related co-labeled cells were detected in the DG of the cage-control group of ligation-injured mice (i.e., zero both ipsi- and contralaterally). Comparatively in the sham-control mice, a few co-labeled cells were detected (0.12±0.12 ipsilaterally and 0.1 ± 0.1 contralaterally), however the difference was not statistically significant (p=0.4 bilaterally). When normalized to the injury- related atrophy of corresponding GCL areas in which the counts were done () comparisons described above, remained similar. Percentage of new neurons in which Arc-induction occurred in ligation-injured mice compared to sham-controls following novel exploration was 2.4±1.7% vs. 1.5±0.7% ipsi- (p=0.6) and 0.5±0.2% vs. 1.3±0.6% (p=0.2) contralaterally, which again was not significantly different. In the cage-control group of mice, percentage of new neurons in which Arc- induction occurred was also not significantly different between the ligation-injured and sham-group of mice [ 0% vs. 0.16±0.16% ipsi- (p=0.4) and 0% vs. 0.15±0.15% contralaterally (p=0.4) respectively]. These percentages were similar to the percentages of co-labeled new neurons detected when a similar pilot protocol was run on ligation-injured and sham-control CD1 mice at P60 following the P12 unilateral stroke insult. No significant differences were noted between sham-control and ligation-injured mice for new-neuron integration. A pilot study was also conducted at P40 to investigate the earliest post-stroke period at which the new-neurons (i.e. labeled in the week following ligation) could functionally integrate into hippocampal networks involved in novel exploration and no new neurons were found to co-label with Arc (unpublished observations). Therefore new neurons born in early post-stroke period while not yet functionally integrated at P40 were able to integrate into hippocampal functional circuits to a similar degree as new born neurons in the sham-controls at P60 (i.e., 6 weeks after dividing cells were labeled with BrdU) and the trend remained similar in the current study at 6 months after the neonatal stroke.
Figure 6 BrdU-positive-cells (green) expressing Arc protein (red) after exposure to novel spatial environment in ipsilateral DGs of sham and ligation-injured mice contra- and ipsilaterally. A1, B1 and C1 show DGs (10× magnification) for the three groups. (more ...)
Spontaneous behavioral seizure activity
Understanding how post-stroke plasticity can lead to chronic recurrent seizures after a latent period is an important question in epilepsy research. Video-monitoring was done for 25% of the time in the long-term study (i.e., 1 week per month for 6 months). Behavioral seizures graded on the Racine scale [26
] revealed the occurrence of recurrent spontaneous class 2, 3 and 4 behavioral seizures in 5 out of the 8 video-monitored mice (63%; 3 females, 2 males). Only one of the female mice with spontaneous seizures also showed the consistent lateralized circling related hyperactivity. Therefore, while there was overlap between groups, circling behaviors and occurrence of seizures did not occur precisely in the same group of ligation-injured mice. Low frequency seizures (i.e., seizures/day were 0.24 at 1 month, 0.29 at 2 months, 0.19 at 3 months, 0.14 at 4 months, 0.24 at 5 months and 0.25 at 6 months of age) were detected throughout the six-month period in which the mice were behaviorally monitored (). Mean seizure rates were 0.22±0.02 seizures/day for the period of monitoring (i.e., 6 months; min 0.14, max 0.28). No class 5 seizures were detected. Behavioral manifestations of class 2 to 4 seizures ranged from ~10 to 60 seconds in duration. A total of 27 seizures were detected of which 22 were class 3 and 4 seizures and the remaining 5 were class 2 seizures. Of the 27 events, 19 (70%) occurred during the light-cycle, similar to previously reported in rodent models of temporal lobe epilepsy [27
Figure 7 Low frequency spontaneous behavioral seizures (Racine scale, Racine, 1971) in the months following neonatal stroke and absence of mossy fiber sprouting. A. In addition to the temporally progressive increase in overall activity associated with clockwise (more ...)
Experiments exposing neonatal rats to insults that result in infarcts have demonstrated induction of atypical mossy fiber sprouting in the inner molecular layer of the dentate gyrus [28
]. The CD1 neonatal stroke mouse model shows a qualitatively similar type of neuronal cell death in the hippocampus with an affinity for CA1 and CA3 pyramidal neurons [10
]. However, Timm staining done on adjacent series of coronal sections did not show presence of stain product in the inner molecular layer (). Although mossy fiber sprouting has been proposed to be a prerequisite for chronic seizure activity in experimental temporal lobe epilepsy, non-progressive epilepsy is generally not associated with mossy fiber sprouting [29
]. The seizure rates of the post-stroke epilepsy in the CD1 neonatal mouse model remained low for the period monitored in the study and rates did not show a progression as a function of time over the period of monitoring. Therefore, the absence of mossy fiber sprouting was consistent with the absence of clear progressive seizure activity at 7 months following the ischemic insult.
Correlations: neonatal stroke-injury, post-stroke neurogenesis and co-morbidities
In this study, the acute post-stroke seizure scores (i.e., behavioral seizures monitored in the 4h following ligation surgery) did not predict the severity of the post-stroke epilepsy (i.e., counts of spontaneous behavioral seizures video-monitored in the 6 months thereafter; r2=0.22 p=0.3; one tailed). There was a positive correlation between the severity of post-stroke epilepsy (i.e., counts of the spontaneous chronic seizures) in the epileptic mice to the severity of those seizures (i.e., sum of the seizures = grade 3 in severity on the Racine scale; r2=0.97 p=0.0001).
Although no correlation was found between the severity of the post-stroke epilepsy and endogenous post-stroke neurogenesis evaluated by new-neurons labeled by BrdU in the week following the stroke (r2
=0.1, p=0.4) in the ipsilateral injured GCL; contralaterally a significant positive correlation [31
] was found both for mean counts and densities (r2
=0.6, p=0.02 and r2
=0.6, p=0.03 respectively; one tailed; n=11). In contrast, there were negative correlations between the counts of the post-stroke spontaneous behavioral seizures and counts of Arc-positive cells following novel-exploration (n=8) both ipsi- and contralaterally that did not reach statistical significance (r2
=−0.53, p=0.09 ipsi- and r2
=−0.08, p=0.4 contralaterally; one tailed). However, there was a significant negative correlation (r2
=−0.7, p=0.03; one tailed) between the severities of the post-stroke epilepsy and the counts of network integrated new neurons (i.e., BrdU labeled cells that co-labeled with Arc) in the ipsilateral GCL. No similar correlations were noted between acute seizure scores and post-stroke neurogenesis or network activity in new neurons.
With regards to the temporally progressive behavioral co-morbidities detected in the open-field testing, there was a strong positive correlation between total distances covered by the ligation-injured mice (n=11, i.e., with and without novel-exploration) to the counts of total revolutions (r2=0.96, p=0.0001). This finding supported the observation of that the overall temporally progressive hyperactivity was associated lateralized circling behaviors detected with the chronic video-monitoring. Also important for the grouping variability between ligation-injured mice assigned to novel-exploration and cage-control groups was that open-field hyperactivity between the two groups were not different from each other with regards to total activity and lateralized revolutions (p=0.5 and 0.7 respectively). Correlations between the behavioral co-morbidities of hyperactivity and lateralized circling and post-stroke neurogenesis or Arc-induction were consistently negative however none reached significance. Therefore although post-stroke plasticity likely induced the long-latency to onset, progressive hyperactivity behaviors, they did not correlate significantly with the impaired post-stroke neurogenesis or the impaired Arc induction in the injured hippocampi.
Severity of stroke-injury quantified as both hemispheric and hippocampal atrophy had positive correlations with hyperactivity and lateralized circling behaviors quantified with the open-field testing at 3 months of age in the ligation-injured group of mice that only reached significance between hemispheric atrophy and hyperactivity (n=11; p=0.05 and 0.1 for hemispheric atrophy and p= 0.2 and 0.3 for hippocampal atrophy respectively for hyperactivity and counts of lateralized circling behaviors; one tailed). Significant negative correlations between severity of stroke-injury as quantified by hippocampal atrophies (n=11) and counts of BrdU-positive cells in the ipsilateral injured GCLs were noted (r2
=−0.73, p=0.005) as has been reported in prior studies from our group [10
]. Similar negative correlations between hemispheric atrophies and contralateral BrdU-positive cell counts did not reach significance (p=0.1) in this study. Stroke-injury also showed significant negative correlations between severity of hemispheric and hippocampal atrophies and counts of Arc-positive cells in the ipsilateral injured hippocampi (r2
=−0.76, p=0.015 and r2
=−0.73, p=0.021 respectively) that was not significant contralaterally. Therefore severity of the stroke injury predicted the severity of impairment of both post-stroke neurogenesis and exploration induced activation of hippocampal circuits in the injured hippocampi.