Citalopram enhances neurovascular regeneration and sensorimotor functional recovery after ischemic stroke in mice

Abstract

Recent clinical trials have demonstrated that treatment with selective serotonin reuptake inhibitors after stroke enhances motor functional recovery; however, the underlying mechanisms remain to be further elucidated. We hypothesized that daily administration of the clinical drug citalopram would produce these functional benefits via enhancing neurovascular repair in the ischemic peri-infarct region. To test this hypothesis, focal ischemic stroke was induced in male C57/B6 mice by permanent ligation of distal branches of the middle cerebral artery to the barrel cortex and 7-min occlusion of the bilateral common carotid arteries. Citalopram (10mg/kg, i.p.) was injected 24h after stroke and daily thereafter. To label proliferating cells, bromo-deoxyuridine was injected daily beginning 3 days after stroke. Immunohistochemical and functional assays were performed to elucidate citalopram-mediated cellular and sensorimotor changes after stroke. Citalopram treatment had no significant effect on infarct formation or edema 3 days after stroke; however, citalopram-treated mice had better functional recovery than saline-treated controls 3 and 14 days after stroke in the adhesive removal test. Increased expression of brain-derived neurotrophic factor was detected in the peri-infarct region 7 days after stroke in citalopram-treated animals. The number of proliferating neural progenitor cells and the distance of neuroblast migration from the sub-ventricular zone toward the ischemic cortex were significantly greater in citalopram-treated mice at 7 days after stroke. Immunohistochemical staining and co-localization analysis showed that citalopram-treated animals generated more new neurons and microvessels in the peri-infarct region 21 and 28 days after stroke. Taken together, these results suggest that citalopram promotes post-stroke sensorimotor recovery likely via enhancing neurogenesis, neural cell migration and the microvessel support in the peri-infarct region of the ischemic brain.

Keywords: 2,3,5-triphenyltetrazolium chloride; ANOVA; BDNF; BrdU; DCX; MCA; MCAO; NIHSS; National Institutes of Health stroke scale; NeuN; OCT; PBS; SD; SSRI; SVZ; TTC; TrkB; analysis of variance; angiogenesis; brain-derived neurotrophic factor; bromodeoxyuridine; citalopram; doublecortin; ischemic stroke; middle cerebral artery; middle cerebral artery occlusion; neurogenesis; neuronal nuclei; optimal cutting temperature; phosphate-buffered saline; selective serotonin reuptake inhibitor; standard deviation; sub-ventricular zone; tyrosine kinase receptor.

Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Figures

Figure 1. Citalopram administration does not attenuate infarct volume

Citalopram was administered at 10 mg/kg (i.p.) 30 min after stroke and again at 24 and 48 hrs after stroke. A. Animals were sacrificed at 3 days after stroke. Infarct analysis was performed by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC, red) for live mitochondria in brain cells. The white color area represents dead cells, those negative for TTC staining. B. Indirect volume analyses revealed no significant difference in infarct volume between the saline stroke control and citalopram treatment groups. (n=9 per group; Mean ± SD, p=0.61). C. Students T test revealed no significant differences in infarct volume (mm3) between the saline and citalopram treatment groups. (n=9 per group; Mean ± SD, p=0.73). Water content was measured using the dry-weight method. D. A two-way ANOVA test revealed no significant effect of treatment on cerebral edema after 72 hours. (n=6 per group; Mean ± SD, p=0.10).

Figure 2. Citalopram treatment after stroke significantly enhances sensorimotor functional recovery

Citalopram or saline treatment was administered starting 24h after MCAO and then daily for 14 days. Sensorimotor function was assessed by the forelimb adhesive removal behavioral test. A. Three days after stroke significant deficits in adhesive removal time were detected by one-way ANOVA in saline-treated mice but not in citalopram-treated mice (n= 11 per group, Mean ± SD, # p<0.05 compared to baseline). After 14 days, citalopram-treated animals displayed improved performance of the sensorimotor task, significantly lower than saline-treated animals and not significantly different from baseline vs controls. 3D: 3 days after stroke; 14D: 14 days after stroke. (n=11 per group, Mean ± SD, * p=0.042, Pobserved =95%). B, C, and D. No significant differences in right or left latency or right removal times were detected by two-way Anova between citalopram and saline treated animals. (n=11 per group, Mean ± SD; p=0.075, p=0.22, and p=0.32, respectively.)

Figure 3. Citalopram enhances number and distance of neural precursor migration from the SVZ

Immunohistochemical staining using specific cell markers was performed in brain sections 7 days after stroke. Migration of neural progenitors was visualized using doublecortin (DCX, green) and bromodeoxyuridine (BrdU, red) shows proliferating cells. A and B. Images of SVZ area show BrdU and DCX positive cells. These cells show different distribution (migration) in the migration track towards the ischemic cortex. Arrows point to some double labeled cells. C and D. The migration distance (μm) was measured using Image J. In the summarized bar graph in C, citalopram treatment showed increased neural progenitor migration distance as compared to control (n=5 per group, Mean ± SEM, ** p=0.009). The graph in D shows the number of BrdU and DCX double positive cells quantified using 3-D imaging. E and F. At 7 days post-stroke, citalopram treated animals showed an increase in the number of BrdU-positive neural progenitors compared to saline treated animals. (n=5 per group, Mean ± SEM, ** P=0.002 vs. control). G,H,I. 3-D Z stack image taken in 20× shows BrdU (G,red) and DCX (H, green) co-localization (I).

Figure 4. Citalopram treatment increases new neurons in the peri-infarct region

Neurogenesis was visualized using neuronal marker NeuN (blue) and BrdU (red). A. Astrocytes were labeled with GFAP (green) and B microglia were labeled with Iba1(green) along with NeuN and BrdU to illustrate that the quantified NeuN and BrdU co-labeled cells were not astrocytes or microglia. C. Saline treatment and citalopram treated animals have new neurons 28d after stroke. D. High power image in the peri-infarct cortex shows co-localization of NeuN and BrdU. E. Quantification of the number of co-labeled NeuN and BrdU labeled cells (new neurons) in the peri-infarct region. Both 21d and 28d citalopram treatment groups had significantly more new neurons in the peri-infarct region compared to the saline treated animals. (n=7 per group, Mean ± SEM, ** p<0.01, * p<0.05).

Figure 5. Citalopram treatment enhances vessel representation in the peri-infarct region

Vessels were visualized and counted using vessel basal lamina marker Collagen IV and dividing cells were marked using BrdU. A Immuno-staining for vessels (collagen IV, green) and newly divided cells (BrdU, red) after 28d saline treatment and B citalopram treatment. C. High power image in the peri-infarct shows endothelial cell marker (Glut-1,blue) co-labeled with Collagen IV, and BrdU showing vessel stain specificity. D. Quantification of the number of co-labeled Collagen IV and BrdU vessels signifying proliferating vessels in the peri-infarct region (n=7 per group, Mean ± SEM, p>0.05). D. Vessel density (vessel density/ area of interest) in the peri-infarct. Citalopram treated animals had significantly greater vessel area fraction compared to saline treated groups. (n=7 per group, Mean ± SEM,** p<0.01, * p<0.05).

Figure 6. Citalopram induces BDNF expression in the peri-infarct region

Western blot analysis of BDNF expression in the peri-infarct region (surrounding area 1.2 mm from the edge of the ischemic core). A Western blot gels performed 7 days after stroke. B. Summarized data from assays in A showing significant increase in BDNF protein expression in the peri-infarct region of the citalopram-treated brain (n=5, Mean ± SEM, *p=0.003 vs. control).