- Email: [email protected]
Fluoxetine enhanced neurogenesis is not translated to functional outcome in stroke rats
Neuroscience Letters 603 (2015) 31–36
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research paper
Fluoxetine enhanced neurogenesis is not translated to functional outcome in stroke rats Xiaoyu Sun a,1 , Xuan Sun b,1 , Tingting Liu a , Mei Zhao c , Shanshan Zhao a , Ting Xiao d,e , Jukka Jolkkonen f , Chuansheng Zhao a,∗ a
Neurology, The first hospital of China Medical University, Shenyang, China Interventional Neuroradiology, Beijing Tian Tan Hospital, Capital Medical University, Beijing, China c Cardiology, Shengjing hospital of China Medical University, Shenyang, China d Dermatology, The first hospital of China Medical University, Shenyang, China e Key Laboratory of Immunodermatology, Ministry of Health, Ministry of Education, Shenyang, China f Institute of Clinical Medicine – Neurology, University of Eastern Finland, P. O. Box 1627, 70211 Kuopio, Finland b
h i g h l i g h t s • Fluoxetine encouraged more neuroblasts toward the damaged striatum after stroke. • Fluoxetine increased the dendritic complexity of newborn dentate granule cells. • Fuoxetine treatment did not improve sensorimotor recovery following ischemia.
a r t i c l e
i n f o
Article history: Received 20 March 2015 Received in revised form 2 June 2015 Accepted 28 June 2015 Available online 18 July 2015 Keywords: Stroke Selective serotonin reuptake inhibitors Subventricular zone Neurogenesis Functional recovery
a b s t r a c t Fluoxetine is widely used in clinical practice. It regulates hippocampal neurogenesis, however, the effect of fluoxetine on neurogenesis in the subventricular zone (SVZ) remains controversial. We aimed to study the effect of fluoxetine on neurogenesis in the SVZ and subgranular zone (SGZ) of dentate gyrus (DG) in relation to behavioral recovery after stroke in rats. Adult male Wistar rats were randomly assigned to four groups: sham-operated rats, sham-operated rats treated with fluoxetine, rats subjected to cerebral ischemia, and rats with ischemia treated with fluoxetine. Fluoxetine was orally administrated starting 1 week after ischemia, with a dose of 16 mg/kg/day for 3 weeks. Focal cerebral ischemia was induced by intracranial injection of vasoconstrictive peptide endothelin-1(ET-1). Behavioral recovery was evaluated on post-stroke days 29–31 after which the survival rate and fate of proliferating cells in the SVZ and DG were measured by immunohistochemistry. The production of neuroblasts in both the SVZ and DG was significantly increased after stroke. Chronic post-stroke fluoxetine treatment increased the dendritic complexity of newborn dentate granule cells. However, fluoxetine treatment did not influence the survival or differentiation of newly generated cells. Neither fluoxetine treatment improved sensorimotor recovery following focal cerebral ischemia. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Despite extensive research, current effective treatments for stroke are limited. For most stroke patients, functional impairments are inevitable. However, it is interesting that many stroke patients show some degree of functional recovery even without any treatment. Thus, the brain is considered to be highly plastic after
∗ Corresponding author at: No. 155, North Nanjing Street, Heping District, Shenyang 110001 Liaoning, PR China. Fax: +86 24 83282315. E-mail address: [email protected] (Chuansheng Zhao). 1 Xiaoyu Sun and Xuan Sun contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2015.06.061 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
stroke. Augmenting the self-repair may be an alternative approach to restore the impaired functions after stroke. Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), is commonly prescribed for patients to treat post-stroke depression (PSD). There are studies showing that chronic or subchronic treatment with fluoxetine regulates hippocampal neurogenesis, associated with improved the cognitive functions [15]. However, the effect of fluoxetine on neurogenesis in the subventricular zone (SVZ) of the adult brain remains controversial. Majority of the studies have revealed no significant influences of fluoxetine on neurogenesis in the SVZ [19]. One study showed that chronic administration of fluoxetine for more than 6 weeks even decreased
32
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
neurogenesis in the SVZ of adult mice [13].This study aimed to compare the effects of fluoxetine on the neurogenesis in the SVZ and dentate gyrus (DG) after experimental focal stroke in relation to behavioral recovery. We provided a new finding that fluoxetine had a significant influence on neurogenesis in the SVZ after focal stroke, but enhanced sensorimotor recovery was not seen. 2. Materials and methods 2.1. Animals We used adult male Wistar rats (200–250 g) provided by China Medical University (Shenyang, China). The rats were randomly assigned to four groups: sham-operated rats (n = 8, SHAM), shamoperated rats treated with fluoxetine (n = 8, SHAM + FLUO), rats subjected to cerebral ischemia (n = 8, ISCH), and rats with ischemia treated with fluoxetine (n = 8, ISCH + FLUO). All rats were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle and the rats had free access to food and water. Anesthesia was induced by a mixture of 3% isoflurane in 30% oxygen and 70% nitrous oxide and animals were maintained with 1.5% isoflurane through the surgery. The study protocol was approved by the Institutional Animal Care and Use Committee of China Medical University [permit No.: SCXK (Liao) 2008-0005]. 2.2. Endothelin-1 (ET-1) stroke model
Alexa Fluor 488 (1:500, Invitrogen, USA) for immunofluorescent labeling. Sections were mounted, and then cover slipped (Invitrogen, USA). 2.5. Morphological analyses The immunofluorescence images of BrdU+ /DCX+ cells in the SVZ ipsilateral to the infarct were acquired with a 20× objective (every sixth section between bregma levels +0.96 mm and −0.24 mm, 5 sections per rat). Results were presented as the average areas of BrdU+ /DCX+ cells per section. The number of BrdU+ /NeuN+ cells, BrdU+ / GFAP+ cells and BrdU+ / Iba-1+ cells in the peri-infarct striatum were counted with a 100× objective (every sixth sections between bregma levels +0.96 mm and –0.12 mm, 5 sections per rat). Immunopositive cells were counted in each section by an experimenter blinded to the treatment conditions. DCX-positive cells counted in the DG were obtained using a microscope (Olympus, BS51, Japan) with a 10×objective (every sixth section between bregma levels −3.0 mm and −3.96 mm, 4 sections per rat). Images of DCX-positive cells in the DG were selected on a random basis using a confocal system (Leica SP2, FV-1000, Germany) on a multitrack configuration. Z-series stacks of 1024 × 1024 pixel images were taken using a 40× objective, three-dimensional reconstructions of entire dendritic arbors were made and the total length of the dendrites was traced using NIH Image [5]. A mean cell count was obtained.
To induce focal cerebral ischemia, the vasoconstrictive peptide endothelin-1 (ET-1) (Sigma, USA) was injected at the following coordinates: (1) AP +0.7 mm, ML +2.2 mm, DV −2.0 mm; (2) AP +2.3 mm, ML +2.5 mm, DV −2.3 mm; and (3) AP +0.7 mm, ML +3.8 mm, DV −5.8 mm according to the rat brain atlas by Paxinos and Watson [18]. ET-1 was injected at 0.5 l/min by an infusion pump, and the needle left in situ for 3 min post-injection before being slowly removed to minimize backflow. The volume of each injection was 2 l (0.5 g/l). Sham-operated animals were received the same surgery except saline was injected instead of ET-1.
2.6. Measurement of infarct volume
2.3. Drug treatment and 5-bromo-2-deoxyuridine labeling
2.7. Tapered/ledged beam-walking test
Rats consumed an average of 16 mg/kg/day with drinking water containing 10 mg/ml of fluoxetine for 3 weeks, the dosage was selected based on previous reports [9], which was shown to lead to serum levels of fluoxetine that were equivalent to therapeutic doses used in patients receiving fluoxetine as antidepressant medication. To label newly generated cells, all rats received twice daily intraperitoneal injections of 5-bromo-2-deoxyuridine (BrdU; 100 mg/kg, Sigma–Aldrich) during postoperative days 5-6 (Supplementary Fig. 1A).
Rats were pre-trained for 3 days to traverse the beam before ischemia induction and were then tested at 29 days after ischemia. Performance in the beam walking test was videotaped and later analyzed by calculating the slip ratio of the impaired (contralateral to lesion) forelimb or hindlimb (number of slips/number of total steps). Steps onto the ledge were scored as a full slip and a half slip was given if the limb touched the side of the beam [25].
2.4. Tissue preparation and immunohistochemistry
Rats were placed individually in a clear plexiglas cylinder (20 cm in diameter, 45 cm high) and videotaped from the below for 3 min via an angled mirror. A blind observer viewed the videotapes and counted the contacts by both forelimbs and by either left or right forelimb to the walls of the cylinder. The percentage of impaired (contralateral to lesion) forelimb use was calculated according to the following formula: impaired forelimb contacts/(impaired + unimpaired + both limb contacts) × 100% [24].
Rats were anesthetized and perfused with 4% paraformaldehyde. The brains were removed and postfixed, and embedded in OCT medium. Brain tissue was cut into 40-m-thick sections on a cryotome (Thermo Electron, Waltham, MA, USA). Sections (40 m) were processed for immunohistochemistry as previously described [10]. BrdU staining was preceded by DNA denaturation and incorporated BrdU was detected using sheep anti-BrdU (1:500, Abcam, USA). The following antibodies for phenotyping were applied in combination with anti-BrdU: guinea pig anti-DCX (1:800, Millipore, USA), mouse anti-NeuN Alexa Fluor® 488 conjugated (1:500, Chemicon, USA), rabbit anti-Iba-1 (1:500, Abcam, USA) or rabbit anti-GFAP (1:1000, Abcam, USA). After rinsing, sections were incubated with appropriate secondary antibodies Alexa Fluor 594 or
For assessment of the infarct volume, sections were collected between +4.5 mm and −7.5 mm from the bregma at 1 mm intervals for measurement of the infarct volume with cresyl violet (Sigma, USA). The contralateral and ipsilateral hemisphere areas were measured by a blinded observer using NIH Image J. Infarct volumes were calculated by subtracting the area of the injured hemisphere from the area of the contralateral hemisphere in each section and areas were multiplied by the distance between sections to obtain the respective volumes [14].
2.8. Cylinder test
2.9. Statistics Statistical analyses were conducted using SPSS software (version 19). The data of the behavior tests were analyzed using one-way analysis of variance (ANOVA). Statistical differences between groups were analyzed using the least significant difference
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
33
Fig. 1. Proliferation, migration and survival of neuroblasts in the SVZ and peri-infarct striatum. (A) Representative confocal images for BrdU+ (red)/DCX+ (green) cells in the SVZ. (B) Quantification of BrdU+ /DCX+ cells in the SVZ. (C) Representative confocal images for BrdU+ (red)/NeuN+ (green) cells in the striatum. (D) Quantification of BrdU+ / NeuN+ cells in the striatum. (B:*; P > 0.05 vs SHAM, ; P < 0.01 vs SHAM, #; P < 0.01 vs ISCH, D:*; P > 0.05 vs SHAM, ; P < 0.01 vs SHAM, #; P > 0.05 vs ISCH, n = 6, arrows indicate double-labeled cells, A: scale bar = 100 m, C: scale bar = 40 m).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(LSD) post hoc test. Values presented as mean ± S.E.M. Significance was set at P < 0.05. 3. Results 3.1. Infarct volumes measurement A typical ET-1-induced ischemic infarct comprised extensive cortical and striatal damage. There was no statistically significant difference in infarct volumes between the ISCH group (127.8 ± 9.2 mm3 ) and the ISCH + FLUO group (139.0 ± 12.1 mm3 , F(1,10) = 217.580; P > 0.05). No damages were found in the SHAM group and the SHAM + FLUO group. 3.2. Chronic fluoxetine treatment increased the proliferation, but did not influence the differentiation or survival of the neuroblasts generated in the SVZ In the rat SVZ, the neuroblasts of DCX-positive cells towards the damaged striatum after ischemia were markedly increased compared with the SHAM group (60.7 ± 5.2 vs 32.2 ± 4.5, P < 0.01). After 3 weeks poststroke fluoxetine treatment, the number of DCXpositive was further increased (98.5 ± 4.7, P < 0.01, Fig. 1A).
There was a significant effect in the number of the BrdU+ /DCX+ cells in the peri-infarct striatum (F(3,20) = 41.883; P < 0.001). It showed that the number of BrdU+ /DCX+ cells toward the ipsilateral SVZ after ischemia was significantly increased compared to the SHAM group (10.5 ± 1.8 vs 7 ± 2.0, P < 0.01). After 3 weeks fluoxetine treatment, the number of BrdU+ / DCX+ cells was further increased (38.3 ± 3.2, P < 0.01, Fig. 1B). However, the number of BrdU+ /DCX+ cells did not change in the SHAM + FLUO group compared to the SHAM group (5.2 ± 2.1, P > 0.05). We found that ∼75% of the BrdU positive cells in the striatum were colabeled with neuronal marker (NeuN) and ∼13% were colabeled with astroglia marker (GFAP) and microglia marker (Iba-1). The number of BrdU+ /NeuN+ cells in the peri-infarct striatum was significantly increased after ischemia compared to those of SHAM and SHAM + FLUO groups (19.6 ± 4.4 vs 5.5 ± 2.1; 6.0 ± 3.2, P < 0.01). After 3 weeks of poststroke fluoxetine treatment, the numbers of BrdU+ /NeuN+ cells was unchanged compared to ISCH group (22.2 ± 4.2, P > 0.05, Fig. 1C,D). The integrated densities of GFAP and Iba-1 positive cells in the peri-infarct striatum were increased significantly compared with the SHAM and the SHAM + FLUO groups (GFAP, 91.5 ± 8.7 vs 46 ± 5.0 or 42.7 ± 6.3, P < 0.01; Iba-1, 110.3 ± 8.7 vs 32.5 ± 5.1 or 28.9 ± 6.2, P < 0.01). In comparison with ischemia rats, fluoxetine-treated
34
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
Fig. 2. The proliferation and qualitative assessment of dendritic arbor of DCX-positive cells in the DG. (A) Representative immunofluorescence images for DCX-positive cells in the DG. (B) Quantification of DCX-positive cells in the DG. (C) Representative reconstructed immunofluorescence images of DCX-positive cells in the DG. (D) Quantification of the total dendritic length of DCX-positive cells. (*; P < 0.01 vs SHAM, ; P < 0.01 vs SHAM, #; P < 0.01 vs ISCH, n = 6, A: scale bar = 200 m, C: scale bar = 75 m).
animals showed decreased expression of GFAP and Iba-1(GFAP, 54.3 ± 5.6, P < 0.01, Iba-1, 54.3.4 ± 8.4, P < 0.01, Supplementary Fig.1C,D). 3.3. Chronic fluoxetine treatment increased the proliferation and dendrite complexity of newborn neurons in the DG There was a significant effect in the number of the DCX-positive cells in the DG (F(3,20) = 17.460; P < 0.01). The numbers of DCXpositive cells in the DG of SHAM + FLUO group (52.3. ± 4.8) and ISCH group (58.5 ± 4.3) were markedly increased compared with the SHAM group (36.1 ± 4.8, P < 0.01). Compared with the ISCH group, the number of DCX-positive cells of ISCH + FLUO group (80 ± 3.4, P < 0.01; Fig. 2A,B) was further increased. The total dendritic lengths of the DCX-positive cells in the DG of SHAM + FLUO group (675.2 ± 28.6 m) and ISCH group (602.2 ± 39.1 m) were significantly increased compared with the SHAM group (312.2 ± 29.7 m, P < 0.01). Compared with the ISCH group, the DCX-positive cells of ISCH + FLUO group had significantly longer total dendrite length (961.7 ± 66.4 m, P < 0.01; Fig. 2C,D). 3.4. Chronic fluoxetine treatment did not improve sensorimotor functions In the beam-walking test, there was no statistical difference was found in the slip ratio of the impaired forelimbs and hindlimbs among groups (F(3,20) = 3.172 and F(3,20) = 3.277; P > 0.05; Fig. 3A,B). The slip ratios of the impaired forelimbs (23 ± 4.6% vs 9 ± 2.9%, P < 0.05) and hindlimbs (23.1 ± 5.3% vs 8.8 ± 2.3%, P < 0.05) were increased significantly after ischemia. However, chronic fluoxetine
treatment did not change the slip ratios after ischemia (forelimb, 20.3 ± 4.1%, P > 0.05 and hindlimb, 20 ± 3.8%, P > 0.05). Similarly, no significant overall group effect was found in the cylinder test (F(3,20) = 3.753; P > 0.05; Fig. 3C). The ISCH group increased the number of contralateral contacts compared with the SHAM group (27.5 ± 5.0 vs 49.0 ± 5.9; P < 0.05). But fluoxetine treatment rats showed no statistically significant difference of ipsilateral contacts relative to the ISCH and the SHAM group (31.8 ± 7.8; P > 0.05).
4. Discussion The present study showed that the production of neuroblasts in both the SVZ and DG were significantly increased after stroke. Chronic post-stroke treatment with fluoxetine encouraged more neuroblasts towards the damaged striatum from the SVZ. Fluoxetine also increased the dendritic complexity of newborn dentate granule cells in both sham-operated and ischemic rats. However, fluoxetine treatment did not influence the survival or differentiation of newly generated cells or improve sensorimotor recovery following ischemia. Consistent with previous reports, we showed a high number of DCX-positive cells migrating from the SVZ to the damaged striatum of rats 32 days after stroke [20]. Interestingly, the number of DCXpositive cells was further increased by the post-stroke treatment with fluoxetine. It has been demonstrated that although substantial numbers of neural precursor cells were initially produced after stroke, only small fraction of these cells differentiate and survive in long-term [1,12,20]. It should be noted that the species, strains or age of the experimental animals, different stroke models, BrdU
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
35
Fig. 3. Performance in behavioral tests. (A) Forelimb slip ratio in the beam-walking. (B) Hindlimb slip ratio in the beam-walking. (C) Impaired limb use ratio in the cylinder test. (*; P > 0.05 vs SHAM, ; P < 0.01 vs SHAM, #; P > 0.05 vs ISCH, n = 6).
labeling paradigm including dose, starting or duration of injection are likely to affect the survival rates of newborn neurons generated after stroke. Consistent with the previous study [6], the numbers of BrdU+ /DCX+ , BrdU+ /GFAP+ , BrdU+ /Iba-1 and BrdU+ /NeuN+ cells in the peri-infarct striatum were not influenced by fluoxetine. These results indicated that fluoxetine contributed to the enlarged pool of newborn neurons without affecting the differentiation or survival of newly formed cells. Similar to the SVZ neurogenesis, chronic fluoxetine treatment had a significant effect on cellular proliferation, but did not affect the survival and differentiation of newly formed cells in hippocampus after stroke. However, one study showed that chronic fluoxetine treatment did not affect the proliferation, but enhanced the survival of newborn neurons in the hippocampus of mice after focal stroke [12]. The discrepancy could be explained by differences in experimental design such as the paradigm of BrdU labeling, stroke model, drug administration, drug dosages, and the species used. Consistent with the prior study [22], we found that the dendritic arborization of immature neurons in DG of sham-operated rats treated with fluoxetine was increased. Interestingly, a retroviral cell labeling study demonstrated that newborn neurons in the DG after focal stroke also displayed significant changes in dendritic complexity [11]. In the present study, chronic poststroke fluoxetine
treatment further enhanced the dendritic complexity of immature neurons. Thus, poststroke fluoxetine treatment might have the interactive effects of accelerating the dendritic maturation of newborn dentate granule cells. The mechanisms of enhanced neurogenesis by fluoxetine after focal stroke are not completely understood. Inflammation mediated by activated microglia and astrocyte is regarded as a key component of the cellular response to brain injury [23]. And, fluoxetine is known to have anti-inflammatory effect both in focal and global cerebral ischemia models [11]. In line with this, we showed that fluoxetine suppressed microglia and astroglia activation in the striatum, indicating that it may have a direct anti-inflammatory effect which might be one potential mechanism underling enhanced SVZ neurogenesis. It is well documented that brain derived neurotrophic factor (BDNF) plays important roles of regulating neurogenesis in both physiological and pathological conditions. Fluoxetine has been shown to up-regulate BDNF expression in normal brain, as well as in hippocampal CA1 region following transient global ischemia [17]. A recent study demonstrated that upregulated expression of BDNF was detected in the peri-infarct region after focal stroke in SSRI drug citalopram-treated mice. The production of proliferating neural progenitor cells (NPC) and the distance of neuroblast migrating from the SVZ towards the peri-infarct area were signifi-
36
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
cantly increased after citalopram administration [2]. Therefore, it is reasonable to presume that BDNF-mediated mechanisms might be involved in the enhanced neurogenesis by fluoxetine in ischemic SVZ and DG although the spatial or temporal expressing profiles of BDNF in the brain regulated by fluoxetine after focal stroke remained unexplored. Another possible mechanism of enhanced poststoke neurogenesis by fluoxetine might be related with angiogenesis. It is believed that angiogenesis and neurogenesis are highly linked and coordinated, responsible for restoring brain functions together after stroke [16]. The newly formed blood vessels in the peri-infarct region after stroke appear to secrete several factors such as SDF1␣, MMPs or VEGF to attract and regulate the biological activities of NPCs, including the migration survival and/or differentiation [7,12,16]. Currently, no direct evidences described the potential effects of fluoxetine on the angiogenesis after stroke. However, SSRI drug citalopram was reported to significantly increase the microvessel density in the peri-infarct region [2]. Studies aiming the benefit effects of fluoxetine on poststroke vascular regeneration are required, especially focusing on the combination of fluoxetine and therapeutic angiogenesis to treat ischemic stroke. It remains to be determined whether newly generated neurons contribute to the behavioral recovery after stroke. Direct evidence for stroke-induced neurogenesis which is responsible for functional recovery remains missing and current experimental approaches can show only a causal link between neurogenesis and behavioral recovery [8]. Results from clinical studies suggest that a single dose of fluoxetine or chronic prescription of fluoxetine with physiotherapy enhances the recovery process after stroke [7]. However, the previous studies conducted in animals did not find any significant effect of post-stroke fluoxetine treatment on the recovery of sensorimotor function [4,21]. We neither detect any effects of fluoxetine on the behavioral recovery as assessed by the beam-walking or cylinder tests. It was reported that even up to about 60% of stroke patients will develop PSD, which has an impact on the behavioral recovery after stroke [3]. The stroke patients are more motivated to engage in rehabilitation after fluoxetine treatment. In contrast, a depressed mood is unlikely to exist in the animals subjected with ET-1 induced stroke model [23]. 5. Conclusion In conclusion, our results demonstrated that chronic fluoxetine treatment enhanced neurogenesis in both SVZ and DG, but did not facilitate sensorimotor recovery after focal stroke. Further studies are required to determine how to translate enhanced neuronal plasticity to behavior. Conflicts of interest None. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81372104, No. 30872736), Program for Liaoning Excellent Talents in University (No. LR2013039) and the Research Fund for the Doctoral Program of Higher Education of China (No. 20112104110003). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2015.06. 061
References [1] A. Arvidsson, T. Collin, D. Kirik, Z. Kokaia, O. Lindvall, Neuronal replacement from endogenous precursors in the adult brain after stroke, Nat. Med. 8 (2002) 963–970. [2] A.R. Espinera, M.E. Ogle, X. Gu, L. Wei, Citalopram enhances neurovascular regeneration and sensorimotor functional recovery after ischemic stroke in mice, Neuroscience 247 (2013) 1–11. [3] G. Gainotti, G. Antonucci, C. Marra, S. Paolucci, Relation between depression after stroke, antidepressant therapy, and functional recovery, J. Neurol. Neurosurg. Psychiatry 71 (2001) 258–261. [4] J. Jolkkonen, K. Puurunen, S. Rantakomi, J. Sirvio, A. Haapalinna, J. Sivenius, Effects-of fluoxetine on sensorimotor and spatial learning deficits following focal cerebral ischemia in rats, Restor. Neurol. Neurosci. 17 (2000) 211–216. [5] S.R. Lee, H.Y. Kim, J. Rogowska, B.Q. Zhao, P. Bhide, J.M. Parent, E.H. Lo, Involvement of matrix metalloproteinase in neuroblast cell migration from the subventricular zone after stroke, J. Neurosci. 26 (2006) 3491–3495. [6] J.E. Malberg, A.J. Eisch, E.J. Nestler, R.S. Duman, Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus, J. Neurosci. 20 (2000) 9104–9110. [7] A.B. Marcussen, P. Flagstad, P.E. Kristjansen, F.F. Johansen, U. Englund, Increase in neurogenesis and behavioural benefit after chronic fluoxetine treatment in Wistar rats, Acta Neurol. Scand. 117 (2008) 94–100. [8] J. Minnerup, J.B. Kim, A. Schmidt, K. Diederich, H. Bauer, M. Schilling, J.K. Strecker, E.B. Ringelstein, C. Sommer, H.R. Scholer, W.R. Schabitz, Effects of neural progenitor cells on sensorimotor recovery and endogenous repair mechanisms after photothrombotic stroke, Stroke 42 (2011) 1757–1763. [9] S. Navailles, P.R. Hof, C. Schmauss, Antidepressant drug-induced stimulation of mouse hippocampal neurogenesis is age-dependent and altered by early life stress, J. Comp. Neurol. 509 (2008) 372–381. [10] M. Nibuya, E.J. Nestler, R.S. Duman, Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus, J. Neurosci. 16 (1996) 2365–2372. [11] F. Niv, S. Keiner, Krishna, O.W. Witte, D.C. Lie, C. Redecker, Aberrant neurogenesis after stroke: a retroviral cell labeling study, Stroke 43 (2012) 2468–2475. [12] J.J. Ohab, S. Fleming, A. Blesch, S.T. Carmichael, A neurovascular niche for neurogenesis after stroke, J. Neurosci. 26 (2006) 13007–13016. [13] K. Ohira, T. Miyakawa, Chronic treatment with fluoxetine for more than 6 weeks decreases neurogenesis in the subventricular zone of adult mice, Mol. Brain 4 (2011) 10. [14] A. Popp, N. Jaenisch, O.W. Witte, C. Frahm, Identification of ischemic regions in a rat model of stroke, PLoS One 4 (2009) e4764. [15] F. Possamai, J. dos Santos, T. Walber, J.C. Marcon, T.S. dos Santos, C. Lino de Oliveira, Influence of enrichment on behavioral and neurogenic effects of antidepressants in Wistar rats submitted to repeated forced swim test, Prog. Neuropsychopharmacol. Biol. Psychiatry 58 (2015) 15–21. [16] L. Ruan, B. Wang, Q. ZhuGe, K. Jin, Coupling of neurogenesis and angiogenesis after ischemic stroke, Brain Res. (2015). [17] A.A. Russo-Neustadt, R.C. Beard, Y.M. Huang, C.W. Cotman, Physical activity and antidepressant treatment potentiate the expression of specific brain-derived neurotrophic factor transcripts in the rat hippocampus, Neuroscience 101 (2000) 305–312. [18] S. Soleman, P. Yip, J.L. Leasure, L. Moon, Sustained sensorimotor impairments after endothelin-1 induced focal cerebral ischemia (stroke) in aged rats, Exp. Neurol. 222 (2010) 13–24. [19] A. Surget, M. Saxe, S. Leman, Y. Ibarguen-Vargas, S. Chalon, G. Griebel, R. Hen, C. Belzung, Drug-dependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal, Biol. Psychiatry 64 (2008) 293–301. [20] P. Thored, A. Arvidsson, E. Cacci, H. Ahlenius, T. Kallur, V. Darsalia, C.T. Ekdahl, Z. Kokaia, O. Lindvall, Persistent production of neurons from adult brain stem cells during recovery after stroke, Stem Cells 24 (2006) 739–747. [21] N. Toni, D.A. Laplagne, C. Zhao, G. Lombardi, C.E. Ribak, F.H. Gage, A.F. Schinder, Neurons born in the adult dentate gyrus form functional synapses with target cells, Nat. Neurosci. 11 (2008) 901–907. [22] J.-W. Wang, D.J. David, J.E. Monckton, F. Battaglia, R. Hen, Chronic fluoxetine stimulates maturation and synaptic plasticity of adult-born hippocampal granule cells, J. Neurosci. 28 (2008) 1374–1384. [23] V. Windle, D. Corbett, Fluoxetine and recovery of motor function after focal ischemia in rats, Brain Res. 1044 (2005) 25–32. [24] R. Zhang, Y.Y. Xue, S.D. Lu, Y. Wang, L.M. Zhang, Y.L. Huang, A.P. Signore, J. Chen, F.Y. Sun, Bcl-2 enhances neurogenesis and inhibits apoptosis of newborn neurons in adult rat brain following a transient middle cerebral artery occlusion, Neurobiol. Dis. 24 (2006) 345–356. [25] S. Zhao, M. Zhao, T. Xiao, J. Jolkkonen, C. Zhao, Constraint-induced movement therapy overcomes the intrinsic axonal growth-inhibitory signals in stroke rats, Stroke 44 (2013) 1698–1705.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research paper
Fluoxetine enhanced neurogenesis is not translated to functional outcome in stroke rats Xiaoyu Sun a,1 , Xuan Sun b,1 , Tingting Liu a , Mei Zhao c , Shanshan Zhao a , Ting Xiao d,e , Jukka Jolkkonen f , Chuansheng Zhao a,∗ a
Neurology, The first hospital of China Medical University, Shenyang, China Interventional Neuroradiology, Beijing Tian Tan Hospital, Capital Medical University, Beijing, China c Cardiology, Shengjing hospital of China Medical University, Shenyang, China d Dermatology, The first hospital of China Medical University, Shenyang, China e Key Laboratory of Immunodermatology, Ministry of Health, Ministry of Education, Shenyang, China f Institute of Clinical Medicine – Neurology, University of Eastern Finland, P. O. Box 1627, 70211 Kuopio, Finland b
h i g h l i g h t s • Fluoxetine encouraged more neuroblasts toward the damaged striatum after stroke. • Fluoxetine increased the dendritic complexity of newborn dentate granule cells. • Fuoxetine treatment did not improve sensorimotor recovery following ischemia.
a r t i c l e
i n f o
Article history: Received 20 March 2015 Received in revised form 2 June 2015 Accepted 28 June 2015 Available online 18 July 2015 Keywords: Stroke Selective serotonin reuptake inhibitors Subventricular zone Neurogenesis Functional recovery
a b s t r a c t Fluoxetine is widely used in clinical practice. It regulates hippocampal neurogenesis, however, the effect of fluoxetine on neurogenesis in the subventricular zone (SVZ) remains controversial. We aimed to study the effect of fluoxetine on neurogenesis in the SVZ and subgranular zone (SGZ) of dentate gyrus (DG) in relation to behavioral recovery after stroke in rats. Adult male Wistar rats were randomly assigned to four groups: sham-operated rats, sham-operated rats treated with fluoxetine, rats subjected to cerebral ischemia, and rats with ischemia treated with fluoxetine. Fluoxetine was orally administrated starting 1 week after ischemia, with a dose of 16 mg/kg/day for 3 weeks. Focal cerebral ischemia was induced by intracranial injection of vasoconstrictive peptide endothelin-1(ET-1). Behavioral recovery was evaluated on post-stroke days 29–31 after which the survival rate and fate of proliferating cells in the SVZ and DG were measured by immunohistochemistry. The production of neuroblasts in both the SVZ and DG was significantly increased after stroke. Chronic post-stroke fluoxetine treatment increased the dendritic complexity of newborn dentate granule cells. However, fluoxetine treatment did not influence the survival or differentiation of newly generated cells. Neither fluoxetine treatment improved sensorimotor recovery following focal cerebral ischemia. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Despite extensive research, current effective treatments for stroke are limited. For most stroke patients, functional impairments are inevitable. However, it is interesting that many stroke patients show some degree of functional recovery even without any treatment. Thus, the brain is considered to be highly plastic after
∗ Corresponding author at: No. 155, North Nanjing Street, Heping District, Shenyang 110001 Liaoning, PR China. Fax: +86 24 83282315. E-mail address: [email protected] (Chuansheng Zhao). 1 Xiaoyu Sun and Xuan Sun contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2015.06.061 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
stroke. Augmenting the self-repair may be an alternative approach to restore the impaired functions after stroke. Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), is commonly prescribed for patients to treat post-stroke depression (PSD). There are studies showing that chronic or subchronic treatment with fluoxetine regulates hippocampal neurogenesis, associated with improved the cognitive functions [15]. However, the effect of fluoxetine on neurogenesis in the subventricular zone (SVZ) of the adult brain remains controversial. Majority of the studies have revealed no significant influences of fluoxetine on neurogenesis in the SVZ [19]. One study showed that chronic administration of fluoxetine for more than 6 weeks even decreased
32
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
neurogenesis in the SVZ of adult mice [13].This study aimed to compare the effects of fluoxetine on the neurogenesis in the SVZ and dentate gyrus (DG) after experimental focal stroke in relation to behavioral recovery. We provided a new finding that fluoxetine had a significant influence on neurogenesis in the SVZ after focal stroke, but enhanced sensorimotor recovery was not seen. 2. Materials and methods 2.1. Animals We used adult male Wistar rats (200–250 g) provided by China Medical University (Shenyang, China). The rats were randomly assigned to four groups: sham-operated rats (n = 8, SHAM), shamoperated rats treated with fluoxetine (n = 8, SHAM + FLUO), rats subjected to cerebral ischemia (n = 8, ISCH), and rats with ischemia treated with fluoxetine (n = 8, ISCH + FLUO). All rats were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle and the rats had free access to food and water. Anesthesia was induced by a mixture of 3% isoflurane in 30% oxygen and 70% nitrous oxide and animals were maintained with 1.5% isoflurane through the surgery. The study protocol was approved by the Institutional Animal Care and Use Committee of China Medical University [permit No.: SCXK (Liao) 2008-0005]. 2.2. Endothelin-1 (ET-1) stroke model
Alexa Fluor 488 (1:500, Invitrogen, USA) for immunofluorescent labeling. Sections were mounted, and then cover slipped (Invitrogen, USA). 2.5. Morphological analyses The immunofluorescence images of BrdU+ /DCX+ cells in the SVZ ipsilateral to the infarct were acquired with a 20× objective (every sixth section between bregma levels +0.96 mm and −0.24 mm, 5 sections per rat). Results were presented as the average areas of BrdU+ /DCX+ cells per section. The number of BrdU+ /NeuN+ cells, BrdU+ / GFAP+ cells and BrdU+ / Iba-1+ cells in the peri-infarct striatum were counted with a 100× objective (every sixth sections between bregma levels +0.96 mm and –0.12 mm, 5 sections per rat). Immunopositive cells were counted in each section by an experimenter blinded to the treatment conditions. DCX-positive cells counted in the DG were obtained using a microscope (Olympus, BS51, Japan) with a 10×objective (every sixth section between bregma levels −3.0 mm and −3.96 mm, 4 sections per rat). Images of DCX-positive cells in the DG were selected on a random basis using a confocal system (Leica SP2, FV-1000, Germany) on a multitrack configuration. Z-series stacks of 1024 × 1024 pixel images were taken using a 40× objective, three-dimensional reconstructions of entire dendritic arbors were made and the total length of the dendrites was traced using NIH Image [5]. A mean cell count was obtained.
To induce focal cerebral ischemia, the vasoconstrictive peptide endothelin-1 (ET-1) (Sigma, USA) was injected at the following coordinates: (1) AP +0.7 mm, ML +2.2 mm, DV −2.0 mm; (2) AP +2.3 mm, ML +2.5 mm, DV −2.3 mm; and (3) AP +0.7 mm, ML +3.8 mm, DV −5.8 mm according to the rat brain atlas by Paxinos and Watson [18]. ET-1 was injected at 0.5 l/min by an infusion pump, and the needle left in situ for 3 min post-injection before being slowly removed to minimize backflow. The volume of each injection was 2 l (0.5 g/l). Sham-operated animals were received the same surgery except saline was injected instead of ET-1.
2.6. Measurement of infarct volume
2.3. Drug treatment and 5-bromo-2-deoxyuridine labeling
2.7. Tapered/ledged beam-walking test
Rats consumed an average of 16 mg/kg/day with drinking water containing 10 mg/ml of fluoxetine for 3 weeks, the dosage was selected based on previous reports [9], which was shown to lead to serum levels of fluoxetine that were equivalent to therapeutic doses used in patients receiving fluoxetine as antidepressant medication. To label newly generated cells, all rats received twice daily intraperitoneal injections of 5-bromo-2-deoxyuridine (BrdU; 100 mg/kg, Sigma–Aldrich) during postoperative days 5-6 (Supplementary Fig. 1A).
Rats were pre-trained for 3 days to traverse the beam before ischemia induction and were then tested at 29 days after ischemia. Performance in the beam walking test was videotaped and later analyzed by calculating the slip ratio of the impaired (contralateral to lesion) forelimb or hindlimb (number of slips/number of total steps). Steps onto the ledge were scored as a full slip and a half slip was given if the limb touched the side of the beam [25].
2.4. Tissue preparation and immunohistochemistry
Rats were placed individually in a clear plexiglas cylinder (20 cm in diameter, 45 cm high) and videotaped from the below for 3 min via an angled mirror. A blind observer viewed the videotapes and counted the contacts by both forelimbs and by either left or right forelimb to the walls of the cylinder. The percentage of impaired (contralateral to lesion) forelimb use was calculated according to the following formula: impaired forelimb contacts/(impaired + unimpaired + both limb contacts) × 100% [24].
Rats were anesthetized and perfused with 4% paraformaldehyde. The brains were removed and postfixed, and embedded in OCT medium. Brain tissue was cut into 40-m-thick sections on a cryotome (Thermo Electron, Waltham, MA, USA). Sections (40 m) were processed for immunohistochemistry as previously described [10]. BrdU staining was preceded by DNA denaturation and incorporated BrdU was detected using sheep anti-BrdU (1:500, Abcam, USA). The following antibodies for phenotyping were applied in combination with anti-BrdU: guinea pig anti-DCX (1:800, Millipore, USA), mouse anti-NeuN Alexa Fluor® 488 conjugated (1:500, Chemicon, USA), rabbit anti-Iba-1 (1:500, Abcam, USA) or rabbit anti-GFAP (1:1000, Abcam, USA). After rinsing, sections were incubated with appropriate secondary antibodies Alexa Fluor 594 or
For assessment of the infarct volume, sections were collected between +4.5 mm and −7.5 mm from the bregma at 1 mm intervals for measurement of the infarct volume with cresyl violet (Sigma, USA). The contralateral and ipsilateral hemisphere areas were measured by a blinded observer using NIH Image J. Infarct volumes were calculated by subtracting the area of the injured hemisphere from the area of the contralateral hemisphere in each section and areas were multiplied by the distance between sections to obtain the respective volumes [14].
2.8. Cylinder test
2.9. Statistics Statistical analyses were conducted using SPSS software (version 19). The data of the behavior tests were analyzed using one-way analysis of variance (ANOVA). Statistical differences between groups were analyzed using the least significant difference
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
33
Fig. 1. Proliferation, migration and survival of neuroblasts in the SVZ and peri-infarct striatum. (A) Representative confocal images for BrdU+ (red)/DCX+ (green) cells in the SVZ. (B) Quantification of BrdU+ /DCX+ cells in the SVZ. (C) Representative confocal images for BrdU+ (red)/NeuN+ (green) cells in the striatum. (D) Quantification of BrdU+ / NeuN+ cells in the striatum. (B:*; P > 0.05 vs SHAM, ; P < 0.01 vs SHAM, #; P < 0.01 vs ISCH, D:*; P > 0.05 vs SHAM, ; P < 0.01 vs SHAM, #; P > 0.05 vs ISCH, n = 6, arrows indicate double-labeled cells, A: scale bar = 100 m, C: scale bar = 40 m).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(LSD) post hoc test. Values presented as mean ± S.E.M. Significance was set at P < 0.05. 3. Results 3.1. Infarct volumes measurement A typical ET-1-induced ischemic infarct comprised extensive cortical and striatal damage. There was no statistically significant difference in infarct volumes between the ISCH group (127.8 ± 9.2 mm3 ) and the ISCH + FLUO group (139.0 ± 12.1 mm3 , F(1,10) = 217.580; P > 0.05). No damages were found in the SHAM group and the SHAM + FLUO group. 3.2. Chronic fluoxetine treatment increased the proliferation, but did not influence the differentiation or survival of the neuroblasts generated in the SVZ In the rat SVZ, the neuroblasts of DCX-positive cells towards the damaged striatum after ischemia were markedly increased compared with the SHAM group (60.7 ± 5.2 vs 32.2 ± 4.5, P < 0.01). After 3 weeks poststroke fluoxetine treatment, the number of DCXpositive was further increased (98.5 ± 4.7, P < 0.01, Fig. 1A).
There was a significant effect in the number of the BrdU+ /DCX+ cells in the peri-infarct striatum (F(3,20) = 41.883; P < 0.001). It showed that the number of BrdU+ /DCX+ cells toward the ipsilateral SVZ after ischemia was significantly increased compared to the SHAM group (10.5 ± 1.8 vs 7 ± 2.0, P < 0.01). After 3 weeks fluoxetine treatment, the number of BrdU+ / DCX+ cells was further increased (38.3 ± 3.2, P < 0.01, Fig. 1B). However, the number of BrdU+ /DCX+ cells did not change in the SHAM + FLUO group compared to the SHAM group (5.2 ± 2.1, P > 0.05). We found that ∼75% of the BrdU positive cells in the striatum were colabeled with neuronal marker (NeuN) and ∼13% were colabeled with astroglia marker (GFAP) and microglia marker (Iba-1). The number of BrdU+ /NeuN+ cells in the peri-infarct striatum was significantly increased after ischemia compared to those of SHAM and SHAM + FLUO groups (19.6 ± 4.4 vs 5.5 ± 2.1; 6.0 ± 3.2, P < 0.01). After 3 weeks of poststroke fluoxetine treatment, the numbers of BrdU+ /NeuN+ cells was unchanged compared to ISCH group (22.2 ± 4.2, P > 0.05, Fig. 1C,D). The integrated densities of GFAP and Iba-1 positive cells in the peri-infarct striatum were increased significantly compared with the SHAM and the SHAM + FLUO groups (GFAP, 91.5 ± 8.7 vs 46 ± 5.0 or 42.7 ± 6.3, P < 0.01; Iba-1, 110.3 ± 8.7 vs 32.5 ± 5.1 or 28.9 ± 6.2, P < 0.01). In comparison with ischemia rats, fluoxetine-treated
34
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
Fig. 2. The proliferation and qualitative assessment of dendritic arbor of DCX-positive cells in the DG. (A) Representative immunofluorescence images for DCX-positive cells in the DG. (B) Quantification of DCX-positive cells in the DG. (C) Representative reconstructed immunofluorescence images of DCX-positive cells in the DG. (D) Quantification of the total dendritic length of DCX-positive cells. (*; P < 0.01 vs SHAM, ; P < 0.01 vs SHAM, #; P < 0.01 vs ISCH, n = 6, A: scale bar = 200 m, C: scale bar = 75 m).
animals showed decreased expression of GFAP and Iba-1(GFAP, 54.3 ± 5.6, P < 0.01, Iba-1, 54.3.4 ± 8.4, P < 0.01, Supplementary Fig.1C,D). 3.3. Chronic fluoxetine treatment increased the proliferation and dendrite complexity of newborn neurons in the DG There was a significant effect in the number of the DCX-positive cells in the DG (F(3,20) = 17.460; P < 0.01). The numbers of DCXpositive cells in the DG of SHAM + FLUO group (52.3. ± 4.8) and ISCH group (58.5 ± 4.3) were markedly increased compared with the SHAM group (36.1 ± 4.8, P < 0.01). Compared with the ISCH group, the number of DCX-positive cells of ISCH + FLUO group (80 ± 3.4, P < 0.01; Fig. 2A,B) was further increased. The total dendritic lengths of the DCX-positive cells in the DG of SHAM + FLUO group (675.2 ± 28.6 m) and ISCH group (602.2 ± 39.1 m) were significantly increased compared with the SHAM group (312.2 ± 29.7 m, P < 0.01). Compared with the ISCH group, the DCX-positive cells of ISCH + FLUO group had significantly longer total dendrite length (961.7 ± 66.4 m, P < 0.01; Fig. 2C,D). 3.4. Chronic fluoxetine treatment did not improve sensorimotor functions In the beam-walking test, there was no statistical difference was found in the slip ratio of the impaired forelimbs and hindlimbs among groups (F(3,20) = 3.172 and F(3,20) = 3.277; P > 0.05; Fig. 3A,B). The slip ratios of the impaired forelimbs (23 ± 4.6% vs 9 ± 2.9%, P < 0.05) and hindlimbs (23.1 ± 5.3% vs 8.8 ± 2.3%, P < 0.05) were increased significantly after ischemia. However, chronic fluoxetine
treatment did not change the slip ratios after ischemia (forelimb, 20.3 ± 4.1%, P > 0.05 and hindlimb, 20 ± 3.8%, P > 0.05). Similarly, no significant overall group effect was found in the cylinder test (F(3,20) = 3.753; P > 0.05; Fig. 3C). The ISCH group increased the number of contralateral contacts compared with the SHAM group (27.5 ± 5.0 vs 49.0 ± 5.9; P < 0.05). But fluoxetine treatment rats showed no statistically significant difference of ipsilateral contacts relative to the ISCH and the SHAM group (31.8 ± 7.8; P > 0.05).
4. Discussion The present study showed that the production of neuroblasts in both the SVZ and DG were significantly increased after stroke. Chronic post-stroke treatment with fluoxetine encouraged more neuroblasts towards the damaged striatum from the SVZ. Fluoxetine also increased the dendritic complexity of newborn dentate granule cells in both sham-operated and ischemic rats. However, fluoxetine treatment did not influence the survival or differentiation of newly generated cells or improve sensorimotor recovery following ischemia. Consistent with previous reports, we showed a high number of DCX-positive cells migrating from the SVZ to the damaged striatum of rats 32 days after stroke [20]. Interestingly, the number of DCXpositive cells was further increased by the post-stroke treatment with fluoxetine. It has been demonstrated that although substantial numbers of neural precursor cells were initially produced after stroke, only small fraction of these cells differentiate and survive in long-term [1,12,20]. It should be noted that the species, strains or age of the experimental animals, different stroke models, BrdU
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
35
Fig. 3. Performance in behavioral tests. (A) Forelimb slip ratio in the beam-walking. (B) Hindlimb slip ratio in the beam-walking. (C) Impaired limb use ratio in the cylinder test. (*; P > 0.05 vs SHAM, ; P < 0.01 vs SHAM, #; P > 0.05 vs ISCH, n = 6).
labeling paradigm including dose, starting or duration of injection are likely to affect the survival rates of newborn neurons generated after stroke. Consistent with the previous study [6], the numbers of BrdU+ /DCX+ , BrdU+ /GFAP+ , BrdU+ /Iba-1 and BrdU+ /NeuN+ cells in the peri-infarct striatum were not influenced by fluoxetine. These results indicated that fluoxetine contributed to the enlarged pool of newborn neurons without affecting the differentiation or survival of newly formed cells. Similar to the SVZ neurogenesis, chronic fluoxetine treatment had a significant effect on cellular proliferation, but did not affect the survival and differentiation of newly formed cells in hippocampus after stroke. However, one study showed that chronic fluoxetine treatment did not affect the proliferation, but enhanced the survival of newborn neurons in the hippocampus of mice after focal stroke [12]. The discrepancy could be explained by differences in experimental design such as the paradigm of BrdU labeling, stroke model, drug administration, drug dosages, and the species used. Consistent with the prior study [22], we found that the dendritic arborization of immature neurons in DG of sham-operated rats treated with fluoxetine was increased. Interestingly, a retroviral cell labeling study demonstrated that newborn neurons in the DG after focal stroke also displayed significant changes in dendritic complexity [11]. In the present study, chronic poststroke fluoxetine
treatment further enhanced the dendritic complexity of immature neurons. Thus, poststroke fluoxetine treatment might have the interactive effects of accelerating the dendritic maturation of newborn dentate granule cells. The mechanisms of enhanced neurogenesis by fluoxetine after focal stroke are not completely understood. Inflammation mediated by activated microglia and astrocyte is regarded as a key component of the cellular response to brain injury [23]. And, fluoxetine is known to have anti-inflammatory effect both in focal and global cerebral ischemia models [11]. In line with this, we showed that fluoxetine suppressed microglia and astroglia activation in the striatum, indicating that it may have a direct anti-inflammatory effect which might be one potential mechanism underling enhanced SVZ neurogenesis. It is well documented that brain derived neurotrophic factor (BDNF) plays important roles of regulating neurogenesis in both physiological and pathological conditions. Fluoxetine has been shown to up-regulate BDNF expression in normal brain, as well as in hippocampal CA1 region following transient global ischemia [17]. A recent study demonstrated that upregulated expression of BDNF was detected in the peri-infarct region after focal stroke in SSRI drug citalopram-treated mice. The production of proliferating neural progenitor cells (NPC) and the distance of neuroblast migrating from the SVZ towards the peri-infarct area were signifi-
36
X. Sun et al. / Neuroscience Letters 603 (2015) 31–36
cantly increased after citalopram administration [2]. Therefore, it is reasonable to presume that BDNF-mediated mechanisms might be involved in the enhanced neurogenesis by fluoxetine in ischemic SVZ and DG although the spatial or temporal expressing profiles of BDNF in the brain regulated by fluoxetine after focal stroke remained unexplored. Another possible mechanism of enhanced poststoke neurogenesis by fluoxetine might be related with angiogenesis. It is believed that angiogenesis and neurogenesis are highly linked and coordinated, responsible for restoring brain functions together after stroke [16]. The newly formed blood vessels in the peri-infarct region after stroke appear to secrete several factors such as SDF1␣, MMPs or VEGF to attract and regulate the biological activities of NPCs, including the migration survival and/or differentiation [7,12,16]. Currently, no direct evidences described the potential effects of fluoxetine on the angiogenesis after stroke. However, SSRI drug citalopram was reported to significantly increase the microvessel density in the peri-infarct region [2]. Studies aiming the benefit effects of fluoxetine on poststroke vascular regeneration are required, especially focusing on the combination of fluoxetine and therapeutic angiogenesis to treat ischemic stroke. It remains to be determined whether newly generated neurons contribute to the behavioral recovery after stroke. Direct evidence for stroke-induced neurogenesis which is responsible for functional recovery remains missing and current experimental approaches can show only a causal link between neurogenesis and behavioral recovery [8]. Results from clinical studies suggest that a single dose of fluoxetine or chronic prescription of fluoxetine with physiotherapy enhances the recovery process after stroke [7]. However, the previous studies conducted in animals did not find any significant effect of post-stroke fluoxetine treatment on the recovery of sensorimotor function [4,21]. We neither detect any effects of fluoxetine on the behavioral recovery as assessed by the beam-walking or cylinder tests. It was reported that even up to about 60% of stroke patients will develop PSD, which has an impact on the behavioral recovery after stroke [3]. The stroke patients are more motivated to engage in rehabilitation after fluoxetine treatment. In contrast, a depressed mood is unlikely to exist in the animals subjected with ET-1 induced stroke model [23]. 5. Conclusion In conclusion, our results demonstrated that chronic fluoxetine treatment enhanced neurogenesis in both SVZ and DG, but did not facilitate sensorimotor recovery after focal stroke. Further studies are required to determine how to translate enhanced neuronal plasticity to behavior. Conflicts of interest None. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81372104, No. 30872736), Program for Liaoning Excellent Talents in University (No. LR2013039) and the Research Fund for the Doctoral Program of Higher Education of China (No. 20112104110003). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2015.06. 061
References [1] A. Arvidsson, T. Collin, D. Kirik, Z. Kokaia, O. Lindvall, Neuronal replacement from endogenous precursors in the adult brain after stroke, Nat. Med. 8 (2002) 963–970. [2] A.R. Espinera, M.E. Ogle, X. Gu, L. Wei, Citalopram enhances neurovascular regeneration and sensorimotor functional recovery after ischemic stroke in mice, Neuroscience 247 (2013) 1–11. [3] G. Gainotti, G. Antonucci, C. Marra, S. Paolucci, Relation between depression after stroke, antidepressant therapy, and functional recovery, J. Neurol. Neurosurg. Psychiatry 71 (2001) 258–261. [4] J. Jolkkonen, K. Puurunen, S. Rantakomi, J. Sirvio, A. Haapalinna, J. Sivenius, Effects-of fluoxetine on sensorimotor and spatial learning deficits following focal cerebral ischemia in rats, Restor. Neurol. Neurosci. 17 (2000) 211–216. [5] S.R. Lee, H.Y. Kim, J. Rogowska, B.Q. Zhao, P. Bhide, J.M. Parent, E.H. Lo, Involvement of matrix metalloproteinase in neuroblast cell migration from the subventricular zone after stroke, J. Neurosci. 26 (2006) 3491–3495. [6] J.E. Malberg, A.J. Eisch, E.J. Nestler, R.S. Duman, Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus, J. Neurosci. 20 (2000) 9104–9110. [7] A.B. Marcussen, P. Flagstad, P.E. Kristjansen, F.F. Johansen, U. Englund, Increase in neurogenesis and behavioural benefit after chronic fluoxetine treatment in Wistar rats, Acta Neurol. Scand. 117 (2008) 94–100. [8] J. Minnerup, J.B. Kim, A. Schmidt, K. Diederich, H. Bauer, M. Schilling, J.K. Strecker, E.B. Ringelstein, C. Sommer, H.R. Scholer, W.R. Schabitz, Effects of neural progenitor cells on sensorimotor recovery and endogenous repair mechanisms after photothrombotic stroke, Stroke 42 (2011) 1757–1763. [9] S. Navailles, P.R. Hof, C. Schmauss, Antidepressant drug-induced stimulation of mouse hippocampal neurogenesis is age-dependent and altered by early life stress, J. Comp. Neurol. 509 (2008) 372–381. [10] M. Nibuya, E.J. Nestler, R.S. Duman, Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus, J. Neurosci. 16 (1996) 2365–2372. [11] F. Niv, S. Keiner, Krishna, O.W. Witte, D.C. Lie, C. Redecker, Aberrant neurogenesis after stroke: a retroviral cell labeling study, Stroke 43 (2012) 2468–2475. [12] J.J. Ohab, S. Fleming, A. Blesch, S.T. Carmichael, A neurovascular niche for neurogenesis after stroke, J. Neurosci. 26 (2006) 13007–13016. [13] K. Ohira, T. Miyakawa, Chronic treatment with fluoxetine for more than 6 weeks decreases neurogenesis in the subventricular zone of adult mice, Mol. Brain 4 (2011) 10. [14] A. Popp, N. Jaenisch, O.W. Witte, C. Frahm, Identification of ischemic regions in a rat model of stroke, PLoS One 4 (2009) e4764. [15] F. Possamai, J. dos Santos, T. Walber, J.C. Marcon, T.S. dos Santos, C. Lino de Oliveira, Influence of enrichment on behavioral and neurogenic effects of antidepressants in Wistar rats submitted to repeated forced swim test, Prog. Neuropsychopharmacol. Biol. Psychiatry 58 (2015) 15–21. [16] L. Ruan, B. Wang, Q. ZhuGe, K. Jin, Coupling of neurogenesis and angiogenesis after ischemic stroke, Brain Res. (2015). [17] A.A. Russo-Neustadt, R.C. Beard, Y.M. Huang, C.W. Cotman, Physical activity and antidepressant treatment potentiate the expression of specific brain-derived neurotrophic factor transcripts in the rat hippocampus, Neuroscience 101 (2000) 305–312. [18] S. Soleman, P. Yip, J.L. Leasure, L. Moon, Sustained sensorimotor impairments after endothelin-1 induced focal cerebral ischemia (stroke) in aged rats, Exp. Neurol. 222 (2010) 13–24. [19] A. Surget, M. Saxe, S. Leman, Y. Ibarguen-Vargas, S. Chalon, G. Griebel, R. Hen, C. Belzung, Drug-dependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal, Biol. Psychiatry 64 (2008) 293–301. [20] P. Thored, A. Arvidsson, E. Cacci, H. Ahlenius, T. Kallur, V. Darsalia, C.T. Ekdahl, Z. Kokaia, O. Lindvall, Persistent production of neurons from adult brain stem cells during recovery after stroke, Stem Cells 24 (2006) 739–747. [21] N. Toni, D.A. Laplagne, C. Zhao, G. Lombardi, C.E. Ribak, F.H. Gage, A.F. Schinder, Neurons born in the adult dentate gyrus form functional synapses with target cells, Nat. Neurosci. 11 (2008) 901–907. [22] J.-W. Wang, D.J. David, J.E. Monckton, F. Battaglia, R. Hen, Chronic fluoxetine stimulates maturation and synaptic plasticity of adult-born hippocampal granule cells, J. Neurosci. 28 (2008) 1374–1384. [23] V. Windle, D. Corbett, Fluoxetine and recovery of motor function after focal ischemia in rats, Brain Res. 1044 (2005) 25–32. [24] R. Zhang, Y.Y. Xue, S.D. Lu, Y. Wang, L.M. Zhang, Y.L. Huang, A.P. Signore, J. Chen, F.Y. Sun, Bcl-2 enhances neurogenesis and inhibits apoptosis of newborn neurons in adult rat brain following a transient middle cerebral artery occlusion, Neurobiol. Dis. 24 (2006) 345–356. [25] S. Zhao, M. Zhao, T. Xiao, J. Jolkkonen, C. Zhao, Constraint-induced movement therapy overcomes the intrinsic axonal growth-inhibitory signals in stroke rats, Stroke 44 (2013) 1698–1705.