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Restorative Neurology and Neuroscience 32 (2014) 597–609 DOI 10.3233/RNN-130374 IOS Press

Forced limb-use enhances brain plasticity through the cAMP/PKA/CREB signal transduction pathway after stroke in adult rats Huiling Qua , Mei Zhaob , Shanshan Zhaoa , Ting Xiaoc,d , Xiaoyu Tange , Dongjiao Zhaoe , Jukka Jolkkonenf and Chuansheng Zhaoa,∗ a Neurology,

The First Hospital of China Medical University, Shenyang, China Shengjing Hospital of China Medical University, Shenyang, China c Dermatology, The First Hospital of China Medical University, Shenyang, China d Key Laboratory of Immunodermatology, Ministry of Health, Ministry of Education, Shenyang, China e Biology, Lycoming College, Williamsport, PA, USA f Institute of Clinical Medicine – Neurology, University of Eastern Finland, Kuopio, Finland b Cardiology,

Abstract. Purpose: The mechanism underlying forced limb-use -induced structural plasticity remains to be studied. We examined whether the cyclic adenosine monophosphate (cAMP)–mediated signal transduction pathway was involved in brain plasticity and promoted behavioral recovery induced by forced limb-use after stroke. Methods: Adult rats were divided into a sham group, an ischemia group, an ischemia group with forced limb-use, and an ischemia group with forced limb-use and infusion of N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H89). Forced limb-use began on post-stroke day 7. Biotinylated dextran amine (BDA) was injected into the sensorimotor cortex on post-stroke day 14. Behavioral recovery was evaluated on post-stroke days 29 to 32, and the levels of cAMP, PKA C-␣, phosphorylated CREB (pCREB), synaptophysin, PSD-95, BDA, and BrdU/NeuN were measured. Results: The number of midline-crossing axons and the expression levels of synaptophysin and PSD-95 were increased after forced limb-use. Forced limb-use enhanced the survival of the newborn neurons and increased the levels of cAMP, PKA C-␣ and pCREB. These were significantly suppressed by H89. Behavioral performance improved with forced limb-use and was reversed with H89. Conclusions: Enhanced structural plasticity and the behavioral recovery promoted by post-stroke forced limb-use are suggested to be mediated through the cAMP/PKA/CREB signal transduction pathway. Keywords: Axonal growth, cAMP, forced limb-use, neurogenesis, stroke

1. Introduction Stroke remains a leading cause of adult disability internationally. Despite extensive research, current effective treatments for stroke are limited. Although ∗ Corresponding

author: Chuansheng Zhao, No.155, North Nanjing Street, Heping District, Shenyang 110001, Liaoning, PR China. Tel.: +86 24 83283026; Fax: +86 24 83282315; E-mail: [email protected].

the brain is considered to be highly plastic after stroke, the local microenvironment appears to be unfavorable for the process of self-repair. Constraintinduced movement therapy (CIMT), which has been extensively used for stroke rehabilitation, may induce not only functional reorganization but also structural plasticity (Gauthier et al., 2008; Kononen et al., 2012; Maier et al., 2008; Sterling et al., 2013; Zhao et al., 2013). In our previous study, we found

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that forced limb-use promoted post-stroke synaptic plasticity and axonal growth at least partially by overcoming the intrinsic growth–inhibitory signaling, leading to improved behavioral outcome (Zhao et al., 2013). In addition, forced limb-use after focal experimental stroke has been shown to increase the proliferation of newborn cells labeled by bromodeoxyuridine (BrdU) in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus (Zhao et al., 2009). Furthermore, these studies demonstrated that such enhanced forced limb-useinduced structural plasticity was accompanied by the increased level of stromal cell–derived factor1 (SDF-1) (Zhao et al., 2009, 2013). Interestingly, SDF-1 has been demonstrated to stimulate the cyclic adenosine monophosphate(cAMP)–mediated signaling pathway, which is thought to be associated with processes of neural plasticity such as neurogenesis and axonal growth (Chalasani et al., 2003; Nakagawa et al., 2002; Opatz et al., 2009). However, the direct evidence that the cAMP-mediated signaling pathway is involved in the enhancement of neuroplasticity and behavioral recovery after poststroke forced limb-use remains rare (Ploughman et al., 2007). In the present study, we tested the hypothesis that forced limb-use treatment would enhance structural plasticity through the cAMP–mediated signal transduction pathway after stroke. First, we measured various components in the cAMP cascade in stroke rats with or without forced limb-use treatment. Then we explored whether the enhanced post-stoke structural plasticity induced by forced limbuse was abolished by blockade of the cAMP cascade. Finally, we assessed a possible association between altered structural plasticity and behavioral performances.

2. Materials and methods 2.1. Animals Adult male Wistar rats (200–250 g) were used in the study. The rats were randomly assigned to four groups: sham-operated rats (n = 10, SHAM), rats subjected to cerebral ischemia (n = 12, Ischemia), rats with cerebral ischemia plus forced limb-use (n = 12, Forced limbuse), rats with cerebral ischemia that were treated with forced limb-use and H89 (n = 12, H89). All rats were kept in standardized cages (54.5*39.5*20.0cm3 , 4-5

animals per cage). Each group was raised in a separate cage where the casted rats were separated from the uncasted rats to avoid the plaster cast being taken off. All rats were housed in a room with a 12 h light/dark cycle and the rats had free access to food and water. Anesthesia was induced using a mixture of 3% isoflurane in 30% oxygen and 70% nitrous oxide and animals were maintained with 1.5% isoflurane for the surgeries. All efforts were made to ensure animal welfare and to reduce the number of animals used. 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 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 (Biernaskie and Corbett, 2001; Soleman et al., 2010). ET-1 was injected at 0.5 ␮l/min by an infusion pump, and the needle left in situ for 3 minutes post-injection before being slowly removed. The volume of each injection was 2 ␮l (0.5 ␮g/␮l). All stereotaxic measurements are relative to the bregma and with the depth determined from the brain surface. Sham-operated animals received the same surgery except saline was injected instead of ET-1. 2.3. Forced limb-use Seven days after ischemia, forced limb-use was started by fitting a plaster cast around the unimpaired upper limb of the rats as described previously (Muller et al., 2008). In the present study, the upper torso and the ipsilateral limbs were wrapped in the plaster cast padded with soft cotton gauze, in a naturally retracted position against the animal’s sternum. The plaster applied over the soft cotton gauze may allow considerable mobility of the upper limb in the cast, but not large movements(Ishida et al., 2011). The correct position of the cast was also checked on each animal twice per day. Animals were forced to use their limbs contralateral to the lesion for 3 weeks. All casts were removed before the behavioral test.

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Fig. 1. Study design. The arrows indicate the timing of pre-training, induction of stroke, H89 infusion, BrdU labeling, forced limb-use treatment, behavioral testing and sacrifice.

2.4. Biotinylated dextran amine tracing For BDA injections, the cannulae holes were drilled in the second surgery. Using a Hamilton syringe, 1 ␮l of biotinylated dextran amine (BDA, Molecular Probes, 10.000 MW, 10% wt/vol) in 0.01 M phosphate buffered saline (PBS) was injected stereotactically into the contralateral (right) motor cortex with the following stereotaxic coordinates (Zhao et al., 2013): AP +1.0 mm, ML –2.0 mm; AP 0 mm, ML –1.5 mm; AP –1.0 mm, ML –1.4 mm; and AP +2.0 mm, ML –1.4 mm. The syringe was left in place for 3 min after each injection (Fig. 1). 2.5. H89 injection and 5-bromo-2-deoxyuridine labeling Mini-osmotic pumps (model 2002, flow rate 0.5 ␮l/hour; Alzet, Palo Alto, CA) were implanted in the lateral ventricle (AP –0.8 mm, ML +1.5 mm) on postoperative day 7. The pumps were loaded to deliver the PKA antagonist N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89, Sigma-Aldrich, 1.25 mg/ml in sterile saline) or saline for 14 consecutive days (Lee and Linstedt, 2000; Qiu et al., 2002). All rats received 4 times twice daily intraperitoneal injections of 5-bromo-2-deoxyuridine (BrdU; 100 mg/kg, Sigma-Aldrich) during days 5-6 after ischemia or sham-operation (Inta et al., 2013) (Fig. 1). 2.6. Tapered/ledged beam-walking test Forelimb and hindlimb functions were tested using a tapered/ledged beam (Zhao et al., 2005, 2013). The beam-walking apparatus consisted of a tapered wooden beam with ledges on each side to permit foot faults without falling. The rats were pre-trained for 3 days to traverse the beam before ischemia induction and tested on postoperative day 29. The rats’ performances were

videotaped and later analyzed by calculating the slip ratio of the impaired (contralateral to lesion) forelimb and hindlimb (number of slips/number of total steps) (Fig. 1). 2.7. Tissue preparation After follow-up on day 33, five rats of each group were perfused transcardially with 0.9% sodium chloride followed by perfusion fixation with 4% paraformaldehyde in PBS. The brain and spinal cord were dissected and postfixed for 24 hours at 4◦ C. Samples were then placed in a solution of 30% sucrose for an additional 5 days at 4◦ C. Brain tissue was cut into 30-␮m-thick sections and the C6–C8 spinal cord segments were cut into 50-␮m-thick on a cryotome (Thermo Electron, Waltham, MA, USA). Samples were then processed for immunostaining and placed in antifreeze buffer for storage (–20◦ C) until immunohistochemistry. 2.8. Measurement of tissue loss volume For assessment of tissue loss and ventricular enlargement, serial coronal sections (30 ␮m) were cut from +4.5 to –7.5 mm from the bregma at 1 mm intervals. Sections were mounted on slides, air dried, and stained with cresyl violet (Sigma, USA) (Popp et al., 2009). The contralateral and ipsilateral hemisphere areas were measured using NIH ImageJ. The intact area in the ipsilateral (injured) hemisphere was substracted from the area of the contralateral hemisphere in each section and the areas were multiplied by the distance between sections to obtain the total tissue loss volumes. 2.9. Immunohistochemistry Immunofluorescence staining was performed on free-floating sections as described previously

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(Jessberger et al., 2007). Sections were washed initially in PBS and then blocked in 5% normal goat serum for 90 min and incubated with mouse anti-NeuN Alexa Fluor® 488 conjugated (1:500, Chemicon, USA) antibody overnight at 4◦ C. After rinsing, sections were denatured by incubation in 2 N HCl at 37◦ C for 45 minutes, followed by neutralization in 0.1 mol/l borate buffer (pH 8.5) for 10 min at room temperature. After washing three times in PBS, the sections were blocked in 5% normal donkey serum and incubated with sheep anti-BrdU (1:200, Abcam, USA) antibody overnight at 4◦ C. Sections were rinsed and incubated with Alexa Fluor 594 (1:200, Invitrogen, USA) for 2 hours. For BDA staining, sections were incubated with conjugate streptavidin secondary antibody (1:200, Alexa Fluor 594, Invitrogen) according to the method described previously (Zhou et al., 2003). 2.10. Quantification Axonal growth and sprouting in response to ischemia were evaluated at the caudal cervical enlargement (C6–C8) using a confocal microscope (Olympus FV-1000, Japan) from 6 coronal sections per rat. Three dimensional reconstructions of BDA-labeled fibers were performed from Z-series stacks of confocal images with NIH ImageJ, by using a simple neurite tracer plugin to visualize intact CST fibers and their growth towards the contralateral denervated gray matter. The total length of the crossing CST fibers was traced and analyzed. In order to measure the fractions of BrdU+ cells that expressed the mature neuronal marker NeuN in tissues collected after BrdU injections, cells that exhibited BrdU and NeuN co-expression in the infarcted cortex were identified using a confocal microscope. The percentages of BrdU+ cells that coexpressed NeuN were then estimated on every sixth slice between bregma levels +0.96 mm and –0.12 mm (total of five sections per brain) with a 40× objective. The accuracy of double labeling was verified in a x–y cross-section, as well as in x–z and y–z crosssections and produced by orthogonal reconstructions from a z-series (z-step, 1 ␮m) taken with a 100× objective. 2.11. Measurement of cAMP levels The procedure for measurement of cAMP levels was exactly the same as that reported elsewhere (Cui et al., 2002). In brief, left cortex samples (50 mg) from five

rats of each group around the infarct were collected in 2 ml of 50 mM sodium acetate buffer (pH 4.75). Tissue homogenate was prepared and then treated with dehydrated alcohol (2 ml) for 5 min at room temperature. Tissues samples were prepared following the manufacturer’s protocol. Levels of cAMP were measured using a radioimmunoassay (RIA) with a commercial kit (Furui, Beijing, China). 2.12. Western blot analysis Western blot analysis was performed in brain homogenates obtained from the tissue adjacent to infarct at day 33 after ischemia induction. After decapitation, the heads of five rats of each group were immersed into liquid nitrogen for 10 s, and the left cortex around the infarct was isolated. Nuclear and cytoplasmic protein extracts were prepared from the samples using a NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce, Rockford, IL). Total protein was extracted using the Total Protein Extraction Kit (KeyGEN, Nanjing, China). The protein concentration was determined using the Bradford assay. Proteins were resolved by 10% SDS–PAGE and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, CA, USA). Following transfer, the membranes were blocked with 5% BSA for 1 h at room temperature and incubated at 4◦ C overnight with antibodies directed against anti-PKA C-␣ (1:1000, Cell Signaling), anti-pCREB (1:1000, Cell Signaling), antiCREB (1:1000, Cell Signaling), anti-PSD-95 (1:500, Abcam), anti-synaptophysin (1:500, Millipore), and ␤-actin (Santa Cruz, USA, 1:1000). After incubation with the corresponding secondary antibodies, proteins were visualized using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA). 2.13. Statistics Statistical analyses were performed using SPSS software version 16. The data were analyzed using one-way ANOVA. Post-hoc tests are used at the second stage of the ANOVA if the null hypothesis is rejected. Statistical differences between groups were analyzed using the least significant difference (LSD) post hoc test. All data are presented as mean ± SD, P < 0.05 indicated a significant difference.

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Fig. 2. Effect of ischemia and forced limb-use on cAMP and nuclear PKA C-␣ in the peri-infarct cortex from five animals per group. Quantification of cAMP in the peri-infarct cortex (A) (∗ P < 0.01 vs. SHAM, ∗∗ P < 0.01 vs. ISC). Representative Western immunoblots of nuclear PKA C-␣ in the peri-infarct cortex (B). Quantification of PKA C-␣ in the peri-infarct cortex (C) (∗ P < 0.01 vs. SHAM, ∗∗ P < 0.05 vs. ISC).

3. Results 3.1. Operation From 36 rats injected with ET-1, 5 rats (13%) died before the end of the follow-up. One rat did not show the symptoms of cerebral ischemia and was excluded. All sham-operated rats survived. 3.2. Tissue loss volumes measurement There was no significant difference in the tissue loss volumes between the ischemia group (131.4 ± 13.14 mm3 ), forced limb-use group (129.4 ± 15.37 mm3 ) and the H89 group (145.3 ± 11.85 mm3 ) (P > 0.05). 3.3. Forced limb-use increases cAMP levels cAMP is thought to activate PKA and initiate transcription by CREB in the cell nucleus. The effects of ischemia and forced limb-use on cAMP levels in the cortex were measured by radioimmunoassay. There was a significant overall group effect in the cAMP levels (F(2,12) = 137.923; P < 0.001). A significant decrease in the cAMP level was seen in the cortex after cerebral ischemia (8.68 ± 0.18 pmol/ml vs 7.16 ± 0.12 pmol/ml; P < 0.01) and forced limb-use

Fig. 3. Expression level of nuclear pCREB from five animals per group. Representative Western immunoblots of nuclear pCREB in the peri-infarct cortex (A). Quantification of pCREB in the periinfarct cortex (B) (∗ P < 0.01 vs. SHAM, ∗∗ P < 0.01 vs. ISC, # P < 0.01 vs. Forced limb-use).

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Fig. 4. Expression levels of synaptic markers from five animals per group. Representative Western immunoblots of synaptophysin and PSD-95 in the peri-infarct cortex (A). Quantification of synaptophysin and PSD-95 in the peri-infarct cortex (B) (∗ P < 0.01 vs. SHAM, ∗∗ P < 0.05 vs. ISC, # P < 0.05 vs. Forced limb-use).

reversed this decrease (8.50 ± 0.17 pmol/ml; P < 0.01) (Fig. 2A). 3.4. Forced limb-use enhances PKA C-α expression in nuclear fractions Next we measured PKA C-␣ in the cytosolic and nuclear fractions of the cortex. There was a significant overall group effect in the PKA C-␣ expression (F(2,12) = 151.267; P < 0.001). The immunoreactivity of nuclear PKA C-␣ decreased after cerebral ischemia compared with the sham group, and forced limb-use partially attenuated ischemia-induced decreases in the cortex (Fig. 2B and C). Nonetheless, there were no significant alterations in levels of immunoreactivity of cytosolic PKA C-␣ in these three groups (data not shown). 3.5. Forced limb-use triggers CREB phosphorylation in nuclear fractions Western blot analyses were performed to see whether altered cAMP and PKA levels are reflected

in the temporal profile of the phosphorylation status of CREB. The total CREB levels were not different between the SHAM group, Ischemia group, Forced limb-use group or H89 group. In contrast, there was a significant overall group effect in the pCREB levels (F(3,16) = 324.139; P < 0.001). The pCREB levels decreased during ischemia compared with the SHAM group, but forced limb-use significantly increased the expression of nuclear pCREB in the cortex. The effects of forced limb-use on pCREB expression were completely reversed by the PKA antagonist H89 (Fig. 3A and B).

3.6. Forced limb-use upregulates synaptophysin and PSD-95 levels The expression levels of synaptic markers such as synaptophysin and PSD-95 were examined by Western blot analysis. There were significant overall group effects in the synaptophysin levels (F(3,16) = 71.43; P < 0.001) and PSD-95 levels (F(3,16) = 47.277; P < 0.001).The expression levels of synaptophysin and PSD-95 in the cortex were decreased after cerebral

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A

B

C

Fig. 5. Growth of the intact CST across the midline and into the denervated gray matter from five animals per group. Representative pictures of BDA-immunostaining fibers crossing the midline and growing into the contralateral gray matter (A). Representative pictures of reconstructed BDA labeled CST fibers (B). The sum length of the CST axons that crossed the midline (C). The crossing fibers were counted from C6 to C8 (∗ P < 0.01 vs. SHAM, ∗∗ P < 0.05 vs. ISC, # P < 0.01 vs. Forced limb-use, Scale bar = 100 ␮m).

ischemia compared with the SHAM group. Interestingly, forced limb-use significantly elevated the decreased levels of these synaptic markers, and H89 completely reversed the increase in the cortex (Fig. 4A and B). 3.7. Forced limb-use promotes BDA-labeled fibers to cross the midline Following cerebral ischemia, corticospinal tract (CST) fibers from the intact hemisphere have been shown to sprout collaterals that cross the midline to innervate the denervated ipsilateral CST (Zai et al., 2009). Z-stacks of the scans reconstructed were consistent with previous studies (Fig. 5A and B). There was a

significant overall group effect in the total length of the crossing CST fibers (F(3,16) = 84.649; P < 0.001). In the SHAM group, only a few BDA-labeled axons crossed the midline whereas an increased total length of BDAlabeled CST axons sprouting from the intact CST was observed in the gray matter of the denervated injured hemisphere in the Ischemia group (456.7 ± 76.6 ␮ m vs 995.1 ± 128.9 ␮ m, P < 0.01). After 3 weeks of forced limb-use, there was a significant increase in the length of the fibers within the gray matter of the denervated injured side of the brain compared with the Ischemia group (1646.4 ± 235.4 ␮ m). The increase in the total length of labeled fibers crossing the midline was also ablated by H89 (770.1 ± 120.2 ␮ m) (Fig. 5C).

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A

B

C

Fig. 6. Survival of neuroblasts from five animals per group. Representative confocal images of BrdU/NeuN- positive cells 4 weeks after ischemia (A). Orthogonal reconstructions of double-labeled cells are presented as viewed in the x-z (bottom) and y-z (right) planes (B). Quantification of BrdU+/NeuN+cells (C) (∗ P < 0.01 vs. SHAM, ∗∗ P < 0.01 vs. ISC, # P < 0.01 vs. Forced limb-use, Scale bar = 100 ␮m).

Fig. 7. Performance in behavioral tests from ten animals per group. Forelimb slip ratio in the beam-walking (A) (∗ P < 0.01 vs. SHAM, ∗∗ P < 0.05 vs. ISC, # P < 0.01 vs. Forced limb-use). Hindlimb slip ratio in the beam-walking (B) (∗ P < 0.01 vs. SHAM, ∗∗ P < 0.05 vs. ISC, # P < 0.05 vs. Forced limb-use).

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3.8. Forced limb-use increases the number of BrdU/NeuN double staining cells in the cortex It is known that new neurons become integrated into host neural circuits with difficulty after stroke. One major problem is that the majority of the new neurons die after stroke and very few survive long-term (Thored et al., 2006). To assess whether forced limb-use affected the survival and maturity of these neuroblasts in the peri-infarct cortex, the number of BrdU/NeuN double-labeled cells was estimated at 4 weeks after ischemia. Indeed, there was a significant overall group effect in the number of the BrdU+/NeuN+cells in the peri-infarct cortex (F(3,16) = 385.022; P < 0.001). The number of new adult neurons (BrdU+/NeuN+) in the cortex ipsilateral to the ischemic injury was markedly increased compared with those of the SHAM rats (26.7 ± 3.1 vs 0.3 ± 0.6, P < 0.01). After 3 weeks of forced limb-use, the number of BrdU+/NeuN+ cells in the cortex of rats (47 ± 3) was further increased. This increase was ablated by H89 (26 ± 3.6) (Fig. 6A, B and C). 3.9. Forced limb-use improves behavioral performance In the beam-walking test, there were significant overall group effects in slip ratio with the impaired forelimb (F(3,36) = 219.39; P < 0.001) and hindlimb (F(3,36) = 163.125; P < 0.001). The slip ratios of the impaired forelimb and hindlimb were increased significantly after ischemia compared with the sham group (P < 0.01) and they were decreased after 3 weeks of forced limb-use compared with the ischemia group (P < 0.05). This suggested that motor function was impaired after ischemia and that it can be improved by forced limb-use. This improvement was ablated by H89 compared with the forced limb-use group (P < 0.01 for forelimb, P < 0.05 for hindlimb) (Fig. 7A and B).

4. Discussion The present study showed that forced limb-use enhanced the axonal growth and possible synapse formation from the intact side of the brain to the denervated cervical spinal cord after focal cerebral ischemia in rats. Also, the long-term survival of newborn neurons in the peri-infarct cortex after stroke was

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significantly enhanced after forced limb-use. In addition, forced limb-use increased the level of cAMP and the expression levels of protein kinase A and cAMP-responsive element binding protein in the periinfarct cortex. More importantly, these structural and molecular changes induced by forced limb-use were associated with significant behavioral improvement. Interestingly, the enhanced post-stroke structural plasticity induced by forced limb-use was significantly suppressed by the chronic intraventricular infusion of the PKA antagonist. It is well documented that following unilateral stroke, axons of the CST from the contralateral motor cortex sprout collaterals that cross over into the ipsilateral cervical spinal cord (Chen et al., 2002; Zai et al., 2009; Zhao et al., 2013). The newly formed projections to the ipsilateral spinal cord, which is denervated by the infarcted cortex, may be involved in behavioral recovery (Zai et al., 2009). Consistent with these findings, we showed increases in the number and length of crossing CST fibers of the ischemia group in the present study, which suggested that post-stroke axonal sprouting occurred. Several intrinsic neurite growth-inhibitory molecules such as Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp) were demonstrated to limit the extent of axonal sprouting (Benowitz and Carmichael, 2010). Nogo-A, MAG and OMgp all bind to a common receptor complex and activate downstream target Rhoassociated kinase (ROCK), which ultimately results in inhibition of axon regeneration and growth cone collapse (Pernet and Schwab, 2012). Thus, strategies to modify the inhibitory environment by targeting downstream signaling can enhance rewiring and improve functional outcome in stroke animals (Schwab, 2004). Interestingly, rendering the neurons unresponsive to inhibitory signals at the molecular level is an alternative way to promote axonal regeneration. The secondary messenger, cAMP, and its downstream effectors can make axons unresponsive to inhibitory signals (Spencer and Filbin, 2004). cAMP is thought to activate PKA and initiate transcription by CREB in the nucleus. The cAMP/PKA/CREB signal transduction pathway can increase phosphorylation of RhoA that inactivates Rho Kinase, thus effectively blocking the inhibition of axonal regeneration by MAG and myelin (Cai et al., 1999). which ultimately lead to enhanced axonal growth after injury (Neumann et al., 2002).

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In the present study, we measured the protein expression of the cAMP/PKA/CREB signal transduction pathway on postoperative day 33. We showed that stroke induced by ET-1 significantly decreased the protein levels of the cAMP-mediated cascade, which is in line with previous reports (Tanaka, 2001; Zhu et al., 2004). It is known that the cAMP/PKA/CREB signal transduction pathway is effective in overcoming the intrinsic inhibition of axonal growth after spinal cord injury (Gao et al., 2004; Neumann et al., 2002; Pearse et al., 2004). However, it remains unclear whether manipulation of the cAMP/PKA/CREB signal transduction pathway affects axonal growth after stroke. Our previous study showed that enhanced axonal growth after 3 weeks of forced limb-use treatment is accompanied by decreased expression of Nogo-A/NgR and their downstream targets, RhoA/ROCK, in the peri-infarct cortex on postoperative day 33. This suggests that decreasing Nogo-A/NgR expression even in the late ischemic period might improve axonal regeneration in the ischemic brain (Zhao et al., 2013). In the present study, we showed that there were more CST fibers crossing over the midline and sprouting into the denervated gray matter in the cervical spinal cord in ischemic rats after 3 weeks of forced limb-use treatment. In addition, forced limb-use led to significantly increased expression of synaptophysin and PSD-95, which were used as the marker for axonal growth and reactive synaptogenesis. Interestingly, chronic intraventricular infusion of H89 abolished the enhanced axonal growth in the forced limb-use group. Therefore, the enhanced axonal growth after post-stroke forced limb-use may not only be due to the decrease in the expression of the growth inhibitors but also enhancement of the cAMP/PKA/CREB signal transduction pathway. Many studies reported that ischemic stroke robustly increased adult neurogenesis in both the SVZ and subgranular zone of the dentate gyrus (Ortega and Jolkkonen, 2013; Thored et al., 2006; Yamashita et al., 2006; Zhang et al., 2004). After proliferation, immature neurons migrate towards the lesion through a pathway similar to the rostral migratory stream. This redirected migration occurs over the first 2 weeks, but it can last for several months after stroke (Thored et al., 2006; Yamashita et al., 2006). Consistent with previous reports, we showed a high number of DCX positive neuroblasts migrating from the SVZ to the damaged striatum and peri-infarct cortex of rats by 33 days after stroke. Although substantial numbers of neural pre-

cursor cells were initially produced after stroke and migrated out from the SVZ to the damaged striatum and cortex, only about 10–20% of these cells differentiate and survive in long-term (Ohab et al., 2006; Thored et al., 2006). The local microenvironment appears to be hostile for the long-term survival of newborn neurons generated in the SVZ after stroke. As the cAMP/PKA/CREB signal transduction pathway plays a critical role in multiple aspects of adult neurogenesis (Iguchi et al., 2011; Lepski et al., 2010; Merz et al., 2011), enhanced neurogenesis induced by post-stroke forced limb-use seen in the present study may correlate with the cAMP/PKA/CREB cascade. The following results provide support for this idea. Firstly, the increased cAMP, PKA and phosphorylated CREB by forced limb-use are observed primarily in the peri-infarct cortex where post-stroke neurogenesis occurred. Secondly, activation of the cAMP/PKA/CREB cascade by forced limb-use facilitates the survival of the newborn neurons in the peri-infarct cortex, whereas this effect is blocked by chronic intraventricular infusion of H89. Therefore, post-stroke forced limb-use may make the local microenvironment more favorable for the long-term survival of the newborn neurons by the enhancement of the cAMP/PKA/CREB signal transduction pathway. Consistent with previous studies showing improved motor function after brain injury (Livingston-Thomas et al., 2013; Maclellan et al., 2005; Zhao et al., 2009, 2013), forced limb-use improved behavioral performance in ischemic rats as assessed by the beamwalking test. After stroke, axonal sprouting also occurs from the contralateral motor cortex into the ipsilateral striatum and the contralateral peri-infarct cortex, as well as from the contralateral motor cortex into the red nucleus (Benowitz and Carmichael, 2010). Thus we could not exclude that these new projections other than the newly formed fibers of the CST also mediate functional recovery induced by forced limb-use. Whether newly generated neurons contribute to the behavioral recovery after stroke remains controversial (Ohab et al., 2006; Sugiura et al., 2005). Direct evidence that stroke-induced newborn neurons are responsible for functional recovery remains missing and current experimental approaches can provide only a causal link between neurogenesis and behavioral recovery (Minnerup et al., 2011). Finally, we should note that the intervention used in the present study is an incomplete model of CIMT. CIMT involves intense, functionally oriented

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task practice with the paretic upper extremity along with restraint of the less affected upper extremity (Livingston-Thomas and Tasker, 2013). Rats in our study had a cast through the follow-up and did not have controlled training of impaired forelimb. It is difficult to control over the rehabilitation intensity of animals since the movement is voluntary. Thus, the results from our animal study should be translated to clinical practice carefully because of such limitations. In conclusion, the present study showed that enhanced structural plasticity and the behavioral recovery promoted by post-stroke forced limb-use might be mediated through the cAMP/PKA/CREB signal transduction pathway, which will help us to understand the mechanisms behind forced limb-use and provide a theoretical basis for clinical rehabilitation.

Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos. 81372104, 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).

Conflict of interest The authors declare no conflict of interest.

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