Functional integration of new neurons into hippocampal networks and poststroke comorbidities following neonatal stroke in mice

Abstract

Stroke in the developing brain is an important cause of chronic neurological morbidities including neurobehavioral dysfunction and epilepsy. Here, we describe a mouse model of neonatal stroke resulting from unilateral carotid ligation that results in acute seizures, long-term hyperactivity, spontaneous lateralized circling behavior, impaired cognitive function, and epilepsy. Exploration-dependent induction of the immediate early gene Arc (activity-regulated cytoskeleton associated protein) in hippocampal neurons was examined in the general population of neurons versus neurons that were generated approximately 1 week after the ischemic insult and labeled with bromodeoxyuridine. Although Arc was inducible in a network-specific manner after severe neonatal stroke, it was impaired, not only in the ipsilateral injured but also in the contralateral uninjured hippocampi when examined 6 months after the neonatal stroke. Severity of both the stroke injury and the acquired poststroke epilepsy negatively correlated with Arc induction and new neuron integration into functional circuits in the injured hippocampi.

Figure 1

Schematics of experimental protocol for the 7 month study.

Figure 2

Unilateral neonatal stroke injury in the ligation-injured group of mice that had acute seizure scores (A; right panel) compared to control (A; left panel) at 7 months post-ligation (quantified in B and C). Scale bar = 250um. B and C. Stroke-injury related atrophy B. Hippocampal and hemispheric atrophy was not significantly different between the two groups of novel-exploration and cage-control mice within the ligation-injured group of mice. C. In order to control for stroke-injury related atrophy of ipsilateral hippocampi, mean areas of the corresponding GCLs were quantified by high magnification tracings in neuro-investigator (MicroBrightfield Inc.). As expected ipsilateral hippocampi in the ligation-injured group of both novel-exploration and cage-control mice had GCL areas that were significantly smaller (@) than their respective sham-controls, however not significantly different from each other.

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 month duration of the study. The male ligation-injured however showed significantly lower weights than their control litter mates as opposed to females at the time of behavioral testing (dashed line). C. Although ligation-injured mice showed a lower rate of alternation on the T maze, this was not significant compared to their control littermates.

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 within sessions associated with hyperactivity, while control mice showed classic within session habituation both on day 1 (B) and day 2 (C). Neither group showed significant between session habituation. D and E. A clockwise revolution (i.e., lateralized circling) was evident in ligate-injured mice compared to controls by their open-field activity traces (D) that was confirmed with video- monitoring. Quantification of the activity showed significant differences (E, p=0.042)

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 mouse from the sham-group. B1 and 2 show BrdU labeled cells (B1) and Arc-induction 1h after exposure to a 5 min novel spatial exploration test from the sham-group (B2). Note predominantly upper blade induction of Arc in the dorsal hippocampus of the novel-exploration mouse. C1 and 2 show BrdU-labeled cells (C1) and Arc-induction 1h after exposure to a 5 min novel spatial exploration test from the ligation-injured group (C2). D. Mean number of BrdU-positive-cells in the ipsilateral injured hippocampi of ligation-injured mice was significantly lower (@) than controls (black bars; p =0.001) however contralaterally they were similar (gray bars). E. Mean counts of BrdU-positive cells normalized to their respective GCL areas showed mean density counts to be similar between all groups and ipsi- and contralateral DGs. F. Arc-induction following exposure to a novel environment was significantly robust both in the sham and ligation-injured groups of mice as compared to their respective cage-control littermates (*). Mean counts of Arc-positive cells in the ligation-injured group that underwent novel-exploration however was significantly lower (@) than the sham-controls that underwent novel-exploration, both ipsi- and contralaterally. G. Mean Arc-positive cell count densities normalized to their respective GCL areas in ligation-injured mice retained the significance of robust-induction in the contralateral upper blade that was also significantly lower than the sham-control counterparts (@). Ipsilaterally however, densities of Arc-positive cells showed a large variance and on average a higher density of Arc cells /mm² when normalized to their respective GCL areas in the stroke-injured ipsilateral hippocampi. H. Mean counts of Arc-positive cells in the upper vs. lower blades of novel-exploration and cage-control littermates from the sham and ligation-injured mice show higher counts of Arc-positive cells in the upper blades for both groups. However induction related increase in numbers of Arc-positive cells in the novel-exploration group of shams was significantly higher due to activation in upper blade neurons vs. lower blade neurons. I. Mean Arc-positive cell count densities normalized to their respective GCL areas in sham and ligation-injured mice from the upper vs. lower blades of novel-exploration and cage-control littermates also showed similar trends as in H.

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. Bottom panels (A2, B2 and C2) show high magnification view (60×) of co-labeled cells in the three groups with orthogonal views of the stacked z axis. D. On average, counts of BrdU-positive cells that co-labeled with Arc expression were higher in the tested group of sham and ligation-injured mice compared to their cage-control litter-mates both ipsi- and contralaterally. However, in injured mice they were significantly higher (*) only in the contralateral DG of ligation-injured mice (gray bar). E. Total mean density of BrdU/Arc co-labeled cells mirrored total mean counts in the DG. Contralateral density of co-labeled cells in the ligation-injured mice after novel exploration was lower compared to the novel-exploration sham-group but not significantly.

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 running (see Figure 3) progressing to tail-chasing behaviors and a failure to gain weight (see Figure 2A and B) stroke-injured mice were detected to have spontaneous behavioral seizures. These low frequency seizures ranged from grade 2 to 4 on the Racine scale with durations ranging from 5–40 seconds in duration. These low frequency behavioral seizures were not associated with detectable aberrant mossy fiber sprouting (normal mossy fiber shown as brown precipitate (*) in the hilus where the mossy fibers extend to innervate the CA3 neurons) in the inner molecular layer of the DG neither ipsi- nor contralaterally and were therefore similar to controls (compare C and D to B). Scale bar in D = 200 µm applies to B, C and D. H-hilus, GCL-granule cell layer, IML-inner molecular layer