The experiments were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China), and were in accordance with the guidelines of the Institute of Laboratory Animal Resources (Washington, DC, USA). A total of 120 male C57BL/6J wild-type mice (weight, 20–25 g; age, 8–10 weeks old) were purchased from the Tongji Medical College Experimental Animal Centre (Wuhan, China). Animals were housed in a temperature- and humidity-controlled environment with a 12 h light/dark cycle and free access to food and H₂O. Focal stroke was induced by photothrombosis (PT) as described previously (19). Mice were anesthetized using 10% chloral hydrate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) (35 mg/kg) intraperitoneally and placed in a stereotactic apparatus. A midline incision of the skin exposed the skull. A dose of 0.1 ml rose bengal solution (10 mg/ml in normal saline; Sigma-Aldrich; Merck KGaA) was injected intraperitoneally 5 min prior to illumination. For illumination, a cold light source (KL1500 LCD; Zeiss AG, Oberkochen, Germany) with a 4-mm aperture was centred 2 mm lateral from the bregma. The brain was illuminated through the intact skull for 15 min. All mice survived the procedure and exhibited behavioural deficits.

All animals subjected to PT-induced stroke were randomly assigned to receive GUO or vehicle following stroke induction. Researchers were blinded to experimental groups. GUO (0.5 mg/ml in sterile saline; Sigma-Aldrich; Merck KGaA) was administered intraperitoneally (i.p.; 8 mg/kg) beginning 24 h following the stroke and then daily for 7 days. The vehicle group received an equal volume of saline. Bromo-deoxyuridine (BrdU; 10 mg/ml in sterile saline; Sigma-Aldrich; Merck KGaA) was injected i.p. (100 mg/kg) beginning 24 h following the stroke and then twice daily until the animals were sacrificed.

The animals were sacrificed 7 days post-stroke. Anesthetized mice were transcardially perfused with cold PBS followed by 4% paraformaldehyde. Brains were subsequently removed, fixed in fresh 4% formaldehyde solution at 4°C overnight and immersed in 30% sucrose until they sank. Then the brains were frozen at −80°C. The frozen brains were cut into 10-µm coronal sections on a cryostat (CM3050S; Leica Microsystems GmbH, Wetzlar, Germany). For each brain, 15 sequential sections were taken at 100-µm intervals and processed for Nissl staining. Sections were stained with 0.1% crystal violet solution for 10 min at room temperature. Infarct volumes were measured using an image analysis program (ImageJ version 1.46r; National Institutes of Health, Bethesda, MD, USA).

Behavioural tests were carried out prior to the PT procedure and 1, 7, 14 and 28 days following PT using the modified neurological severity scale (mNSS), grid walking test and cylinder test.

mNSS is a composite of motor, sensory, balance and reflex tests. Neurological function is graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18). A total of 1 score point is awarded for the inability to perform the test or for the lack of a tested reflex; therefore, a more severe injury has an increased score (20).

Animals were placed on an elevated wire grid and video-recorded when they walked. The number of contra- and ipsilateral faults for each limb and the total number of steps taken were counted, the ratio between foot faults and total steps was calculated (21).

The animal was placed in a transparent cylinder and video-recorded. Forelimb preference during vertical exploration of the cylinder was evaluated by recording the forelimb contacts. Animals were subjected to one trial on day 1 prior to PT. The asymmetry score for each animal was calculated by the formula previously described (22).

Frozen sections were incubated with a blocking buffer (1X PBS/5% normal goat serum/0.3% Triton X-100, Goodbio, Wuhan, China) for 1 h at room temperature. The sections were subsequently incubated at 4°C overnight with the following primary antibodies: mouse monoclonal anti-BrdU (catalogue no. sc-32323, dilution, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-BrdU (catalogue no. ab19944, dilution, 1:500, Abcam, Cambridge, MA, USA), goat polyclonal anti-doublecortin (DCX; catalogue no. sc-8066, dilution, 1:200; Santa Cruz Biotechnology, Inc.), mouse monoclonal neuronal nuclei (NeuN; Catalogue no. MAB377, dilution, 1:500; EMD Millipore, Billerica, MA, USA) and von Willebrand factor (vWF; catalogue no. ab7356, dilution, 1:1,000; EMD Millipore). Following washing with PBS, the sections were incubated for 1 h at room temperature with two secondary antibodies: Alexa Fluor 488 (catalogue no. CA11001s, dilution, 1:200; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Alexa Fluor 594 (catalogue no. CA11012s, dilution, 1:200; Invitrogen; Thermo Fisher Scientific, Inc.). Incubation was conducted in the dark and then washed with PBS three times. For BrdU immunofluorescence, brain sections were pre-treated with 2 M HCl at 37°C for 30 min and then washed with PBS six times at room temperature before being incubated with blocking solution. The images were captured using a fluorescence microscope (BX51; Olympus Corporation, Tokyo, Japan).

Mice were anesthetized and decapitated at 14 days post-stroke. The ipsilateral peri-infarct cortices and cognate regions from the contralateral hemisphere were sampled. The total protein of each tissue was extracted according to the instructions of the protein reagent kit (Goodbio, Wuhan, China). Protein concentration of each sample was determined using the bicinchoninic acid assay (Sigma-Aldrich; EMD Millipore). Protein samples (20 µg per lane) were subsequently separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Two primary antibodies were incubated with the membranes overnight at 4°C: anti-brain-derived neurotrophic factor (BDNF; catalogue no. AB1779, dilution, 1:200; EMD Millipore), anti-vascular endothelial growth factor (VEGF; catalogue no. sc-152, dilution, 1:200; Santa Cruz Biotechnology, Inc.) and β-actin (catalogue no. 4970, dilution, 1:1,000; Cell Signalling Technology, Danvers, MA, USA). Subsequently, membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (catalogue no. AB501, dilution, 1:1,000; Novoprotein, Shanghai, China) for 2 h at room temperature. was used as a loading control for all experiments. Densitometry analysis was performed using the Image J software with normalization to β-actin.

All data are represented as mean ± standard error of mean and were analysed using SPSS software (version 21.0; IBM SPSS, Armonk, NY, USA). The treatment effects on the behaviour score at different time points were analysed using repeated measures one-way analysis of variance, followed by Tukey-Kramer post hoc tests. When comparing two groups, statistical analysis of data was performed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

There was no statistical difference in infarct volume on day 7 post-stroke between the two groups (1.220±0.110 vs. 1.125±0.120 mm³, n=8 for each group, P>0.05; Fig. 1). In addition, no difference was detected on days 1 and 7 post-stroke, in mNSS score, foot fault or forelimb asymmetry between the vehicle group and GUO group (Fig. 2A-C). However, a significant reduction in the mNSS score and foot fault was observed on days 14 and 28 post-stroke following GUO treatment (P<0.05; Fig. 2A and B). Similarly, treatment with GUO significantly improved the function of the impaired forelimb on day 14, and the effect was maintained up to 28 days post-stroke. The results indicated that delayed administration of GUO was able to promote long-term functional recovery following ischemic stroke (Fig. 2C).

GUO significantly increased the number of BrdU⁺ cells in the ipsilateral SVZ and SGZ when compared with the vehicle group (P<0.05; Fig. 3A-C). These data indicated that administration of GUO enhances cell proliferation following stroke.

In addition, GUO significantly increased the number of BrdU⁺ cells co-labelled with DCX, a marker of neural progenitor cells, in the ipsilateral SVZ and SGZ compared with the vehicle group (P<0.05; Fig. 3D and E) at all time points following stroke induction, suggesting that delayed administration of GUO promotes proliferation of neural progenitor cells in the SVZ and SGZ following stroke.

To further investigate whether the proliferative neural progenitor cells is able to differentiate into functional neurons, double immunostaining was performed with NeuN (a marker of mature neurons) and BrdU. GUO significantly increased the number of BrdU⁺/NeuN⁺ cells in the peri-infarction cortex following stroke, when compared with the vehicle group (P<0.05; Fig. 4A and B). These data indicated that GUO enhances the differentiation of new neural progenitor cells into mature neurons within the peri-infarction region following stroke.

To examine whether GUO influences the formation of new blood vessels in the ischemic brain, all blood vessels in the peri-infarction cortex were counted using vWF immunostaining. vWF is a vascular endothelial cell marker. GUO significantly increased vascular density in the peri-infarction cortex compared with the vehicle group (P<0.05; Fig. 4C and D). Furthermore, these results presented a significant increase in the percentage of BrdU⁺/vWF⁺ cells in mice treated with GUO, when compared with the vehicle group (P<0.05; Fig. 4E). These data suggested that GUO enhances angiogenesis following stroke.

Western blot analysis demonstrated that GUO significantly increased the expression of VEGF and BDNF in the ischemic brain at day 14 post-stroke compared to the vehicle (P<0.05; Fig. 5).