All the procedures were strictly in line with the regulations of the National Institutes for Animal Research. All the animal-related experiments were approved (approval. no. 2022023) by the Animal Care and Management Committee of Beijing Geriatric Hospital (Beijing, China). A total of 90 male C57BL/6 mice (25-30 g, 10 weeks-old) were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental animals were housed in conditions with 12/12-h light/dark cycles, a temperature of 22±3˚C and 60±5% humidity. All the mice had access to a standard rodent diet and tap water.

A MCAO/R mouse model was created as previously described (12,13). The mice were anesthetized with isoflurane (induction dosage 3%; maintenance dosage 1.5%). Through a midline incision on the ventral side of the neck, the common carotid arteries were exposed, the external carotid artery (ECA) and the internal carotid artery (ICA) were carefully isolated. Subsequently, a 6-0 silicone suture was introduced through the ECA and advanced into the ICA to occlude the origin of the middle cerebral artery, ~10-11 mm from the common carotid artery bifurcation. A laser Doppler flow probe was used to confirm the development of MCAO. After 60 min of ischemia, the suture was withdrawn to allow reperfusion. Concurrently, a sham operation was conducted using the same procedure, except for MCA occlusion. After 72 h of the experiment, the mice were euthanized via CO₂ inhalation (50% of the chamber volume per minute), and their brain tissues were collected.

The protocol of the present study, which involves treating with galectin-1 for 3 days, was developed based on previous studies and experimental data of the present study. According to previous data by the authors on zebrafish (14), a 3-day treatment with galectin-1 significantly reduced the generation of oxidative stress in vivo. Additionally, the study by Vallecillo-Zúniga et al (15) observed the anti-ROS effect of galectin-1 in myogenic cells after 2-3 days of treatment (15). Cheng et al (16) found that a 3-day treatment with galectin-1 could promote vascular remodeling, while Arda-Pirincci and Aykol-Celik (17) demonstrated that galectin-1 can reduce the severity of dextran sulfate sodium-induced ulcerative colitis after at least 3 days of treatment. The preliminary data of the present study demonstrated the antioxidative stress effects of galectin-1 in cerebral I/R. Therefore, in the present study, a treatment duration of 3 days was selected.

The dose and administration route for the present study were selected based on previous studies and the preliminary experimental data of the present study. In addition, in the study by Carlos et al (18), rats were treated with recombinant human galectin-1 at a dose of 100 µg per animal, administered intravenously. Similarly, the study by Ye et al (19) involved injecting mice with recombinant human galectin-1 at doses ranging from 250-500 µg per animal, administered intraperitoneally. The preliminary data of the present study indicated that an injection of 500 µg of galectin-1 significantly reduced ROS levels in the mouse brain.

All the male C57BL/6 mice were randomly allocated to three groups (30/group): i) galectin-1 group, the mice received the MCAO/R operation and were treated with recombinant human galectin-1 (500 µg/mouse) (18,19) intraperitoneally starting 1 h after the operation, and this treatment continued for 3 days; ii) MCAO/R group, the mice received the MCAO/R operation and were treated with saline (same amount as galectin-1 intraperitoneally from 1 h after operation, and this treatment continued for 3 days; and iii) sham surgery group, the mice underwent the sham surgery and received saline (same amount as galectin-1) intraperitoneally from 1 h after operation, and this treatment continued for 3 days.

After the brain tissues were collected, 10 mice brain tissues from each group were subjected to examine cerebral infarct volume and microglial cell infiltration, 10 were used for immunohistochemistry (IHC) analysis of allograft inflammatory factor 1 (IBA-1) expression, and the brain tissues of the remaining 10 mice brain tissues from each group were homogenized and stored for further tests (ROS and cytokines levels). Galectin-1 (cat. no. P00388) was purchased from Solarbio Science & Technology Co., Ltd. and dissolved in saline for the experiments based on the previous published study (16).

After the mice were euthanized, the brain tissues were collected and then sliced to a thickness of 2 mm. The slices were stained with 2, 3, 5-triphenyl tetrazolium chloride (2%) for 20 min at 37˚C. Thereafter, the slices were fixed using 4% paraformaldehyde overnight at room temperature. Images of all the stained slices were captured and quantified using ImageJ software (version 1.54; National Institutes of Health). Infarct volumes were calculated using the following formula: Percentage hemisphere lesion volume (% HLV)=[total infarct volume-(volume of intact ipsilateral hemisphere-volume of intact contralateral hemisphere)]/contralateral hemisphere volume x100%.

The neurological deficits of the mice were evaluated using the rotarod test. On 4 consecutive days (before surgery, and 24, 48 and 72 h after surgery), the mice were placed on rotating rods and practiced three times a day. The rods accelerated in speed from 4 to 40 rpm over a 5-min period. The results were expressed as the latency to fall from the rod.

A neurological test was performed to investigate neurological deficit after 72 h of surgery. The degree of neurological impairment was evaluated based on the following features: i) absence of deficits; ii) body bending towards the opposite side; iii) involuntary contralateral circling; iv) tendency to fall to the opposite side; and v) lack of spontaneous movement or a state of unconsciousness (20).

In the present study, the data for the neurological deficit score were presented, exclusively at the 72-h mark, following the precedent set by several other studies in the field (21-25). These studies, focusing on cerebral ischemia in mouse models, typically report neurological deficit scores only at the experiment's endpoint, which is often at 72 h. Consequently, the reporting methodology of the present study was aligned with this standard practice.

ROS generation was estimated using the oxidation-sensitive 2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) dye (cat. no. KGAF018, Nanjing Jiancheng Bioengineering Institute). CM-H2DCFDA becomes fluorescent when oxidized by ROS. In brief, the cerebral cortex tissues of the ischemic hemisphere were collected and gently homogenized in 500 µl of PBS. The concentration of proteins was determined by the BCA method. Thereafter, the brain homogenate was incubated with 25 µl CM-H2DCFDA (10 mM) for 30 min at 37˚C in dark conditions. Subsequently, 500 µl of each sample was loaded into the wells of a 96-well plate. The fluorescence intensities of ROS were measured using a fluorescent microplate reader (excitation: 485 nm, emission: 525 nm). The activity is expressed as U/mg protein (26).

The hemispheres containing the ischemic zones of mice were collected, and then gently homogenized. Tissue homogenates were diluted in the buffer provided by each kit at a concentration of 10 mg tissue/ml. The quantities of cytokines, including interleukin (IL)-6, IL-1 and tumor necrosis factor alpha (TNF-α), and the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidation enzyme (GSH-Px), were measured using ELISA kits (IL-1, cat. no. PI301; IL-6, cat. no. PI326; TNF-α, cat. no. PT513; SOD, cat. no. S0101S; CAT, cat. no. S0051; GSH-Px, cat. no. S0057S) from Beyotime Institute of Biotechnology. Briefly, 96-well plates were first coated with primary antibodies (provided within the kits; ready-to-use) and were filled with 100 µl of either the samples or standards. 100 µl of biotinylated detection antibodies (provided within the kits; ready-to-use) were then added to these wells and left to incubate at room temperature for 1 h. Following this, the wells were emptied, rinsed with PBS, and then filled with 100 µl of the streptavidin complex reagent, for another h of room temperature incubation. A total of 90 µl of 3,3',5,5'-tetramethylbenzidine substrate solution was next added to the wells and left to incubate for 20 min at room temperature. Then 50 µl of stop solution was added to the wells. Absorbance was evaluated using a multimode plate reader at 450 nm. Standard curves were produced using the optical density (OD) values of standard reagents, and these were used to ascertain the cytokine levels. The activity of SOD, CAT and GSH-Px were expressed as U/mg protein.

The lipid peroxidation product MDA is used as an indicator of oxidation. The tissue homogenate was analyzed using an MDA kit following the manufacturers' instructions via the thiobarbituric acid reaction (cat. no. A003-4-1; Nanjing Jiancheng Bioengineering Institute). The samples were measured by using a microplate reader at 532 nm and the values were expressed as nmol/mg of tissue protein.

Brain tissues were collected from euthanized mice. These tissues were first fixed with 10% formalin at room temperature for 24 h, then embedded in paraffin, sliced into 5-µm-thick sections, and arranged on glass slides. The tissue sections were subsequently deparaffinized with xylene at 55˚C and rehydrated in a descending alcohol series (ethanol; 100, 95, 75 and 50%; 3 min each). Thereafter, antigen retrieval was performed by boiling in citrate-EDTA buffer, followed by a 20-min cooling period at room temperature. After a blocking step with 5% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) at room temperature for 1 h, the sections were incubated with primary antibodies diluted against IBA-1 rabbit monoclonal (1:200; cat. no. MA5-36257; Thermo Fisher Scientific, Inc.) and left overnight at 4˚C. Following this, the slides were rinsed with TBS with 0.1% Tween-20, and treated with a horseradish peroxidase-labelled dextran polymer bound with an anti-rabbit antibody (1:1,000; cat. no. A0279; Beyotime Institute of Biotechnology) for 1 h at room temperature. Subsequently, the slides were incubated with DAB solution and inspected under a light microscope (Olympus Corporation). The expression of IBA-1 was assessed under a light microscope at a x40 magnification. The expression level was quantified using a standard IHC staining grade system by measuring positive expression in 5 random fields within the brain tissues, received from three separate non-adjacent sections per mouse.

In addition, a preliminary experiment was conducted to validate the protocol. This included optimizing blocking reagents and antigen retrieval, as well as determining the appropriate concentrations for primary and secondary antibodies. Additionally, the DAB staining step was carefully monitored under a light microscope to prevent overreaction. Furthermore, both positive and negative controls were employed in the staining process. The positive control tissue exhibited strong and specific staining for the target antigen, while no positive staining was observed in the negative control slides.

All the data were analyzed in SPSS 26 (IBM Corp.) for statistical computing and graphics. The quantitative data were expressed as the mean ± standard deviation (SD). Statistical comparisons were performed by one-way ANOVA followed by the Tukey's post hoc test or unpaired Student's t-test as described in figure legends. For data that were not normally distributed in the analyses of neurological scores, The Mann-Whitney U test was used for comparisons between 2 groups. Two-way mixed ANOVA followed by the Bonferroni's post hoc test was used to compare the difference in rotarod test among different groups at 24, 48 and 72 h after surgery. P<0.05 was considered to indicate a statistically significant difference.

In the present study, the results revealed that cerebral ischemia developed in mice after MCAO/R surgery. The neurological outcome was investigated after 3 days of galectin-1 treatment in MCAO/R mice. Compared with the control group that underwent sham surgery, both the neurological deficit and the cerebral infarct area were observed in the MCAO/R mice group (Fig. 1A-D). Moreover, the results demonstrated that treatment with galectin-1 significantly improved the outcome of cerebral ischemia, as evidenced by longer latency on the rotarod, lower neurological deficit scores, and smaller infarct volumes. Data on the neurological deficit score for mice that underwent sham surgery were not included, as no deficits were detected in these mice.

ROS and MDA are both biomarkers for the status of oxidative stress. The data of the present study identified an increased level of both ROS (Fig. 2A) and MDA (Fig. 2B) in the ischemic hemisphere of MCAO/R mice, compared with that in the sham surgery. In addition, treatment with galectin-1 significantly decreased the levels of ROS and MDA in the ischemic hemisphere of MCAO/R mice, compared with that in MCAO/R mice without galectin-1 treatment.

The data of the present study revealed a significantly decreased level of antioxidant defense molecules, including SOD, CAT and GSH-Px, in the ischemic hemisphere of MCAO/R mice, compared with that in the sham surgery. However, compared with the non-galectin-1 treatment group, treatment with galectin-1 significantly increased the levels of SOD, CAT and GSH-Px in the ischemic hemisphere of MCAO/R mice (Fig. 3).

IHC staining was performed to evaluate the activation of microglial cells in the ischemic hemisphere of mouse brain cortex and hippocampus. Galectin-1 treatment significantly decreased the number of IBA-1-positive microglial cells in the ischemic hemisphere of MCAO/R mice, compared with mice without galectin-1 treatment (Fig. 4A and B). In addition, cytokine levels in the ischemic hemisphere tissues of MCAO/R mice were assessed. The results revealed that mice treated with galectin-1 had lower inflammatory cytokines, including IL-6, IL-1 and TNF-α, compared with those in the brains of mice without galectin-1 treatment (Fig. 5A-C).