All the procedures in this study complied with the Guide for the Care and Use of Laboratory Animals. All the experiments were designed to minimize pain and animal numbers, and the study protocol was approved by the Animal Care and Use Committee at Army Medical University. The animals were housed under a 12-h light and 12-h dark cycle and were given free access to food and water.

A total of 150 male Sprague-Dawley (SD) rats, weighing 450–550 g, were provided by the Army Medical University (Chongqing, China). Rats were randomly divided into three groups: the sham-operated group, ICH + vehicle group, and ICH + GLC group. To mimic the clinical condition of ICH, a model was established via injection of autogenous blood, as previously reported (Jiang et al., 2016). Briefly, the animals were anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg). The body temperature of the animals was maintained at 37°C. The animals were positioned in a stereotaxic frame, a cranial burr hole (1 mm diameter) was drilled, and 100 μl autogenous arterial blood (obtained from the right femoral artery) was microinfused using a pump at a constant rate of 10 μl/min into the right caudate nucleus (coordinates: 3.5 mm lateral, 5.5 mm ventral, and 0.2 mm anterior to the bregma) through a 29-G needle. The sham-operated rats were only subjected to needle insertion. All rats survived the ICH induction and no mortality happened.

Glibenclamide (Tocris Bioscience, Ellisville, MO, United States) was administered as previously reported (Jiang et al., 2016). Briefly, dimethyl sulfoxide (DMSO) (50 mg/ml) was used to prepare stock solutions of GLC. The injection solution (200 ng/μl or 1 μg/ml) was made by dilution in 0.9% NaCl, and clarifying the solution using a few microliters of 0.1 N NaOH (final pH ∼ 8.5). At the end of the surgery, GLC was administered in a single loading dose (10 μg/kg intraperitoneal injection) plus continuous subcutaneous infusion (200 ng/h) via a mini-osmotic subcutaneous pump (Alzet, 2001, 1.0 μl/h; Alzet Corp., Cupertino, CA, United States). The vehicle group was treated with vehicle control solutions in the same manner.

The brain water content was examined 72 h following ICH, as previously reported (Li et al., 2015). Briefly, the rats (n = 10/group) were euthanized and decapitated, the brains were quickly removed, and a 4-mm thick section of the coronal brain tissue surrounding the needle entry site was harvested. The brain tissue was divided into four parts: the contralateral cortex, contralateral basal ganglia, ipsilateral cortex, and ipsilateral basal ganglia. The cerebellum served as the internal control. The brain tissue weights were determined immediately after removal and after drying for more than 24 h at 100°C on an electronic analytical balance. The brain water content (%) was calculated using the following formula: (wet weight−dry weight)/wet weight × 100%.

Immunofluorescent labeling was conducted 24 or 72 h following ICH as previously described (Tang et al., 2015). About 18-μm thick brain tissue sections were prepared and stored at −20°C. The specimens were incubated with primary antibodies overnight at 4°C, and then with the appropriate secondary antibodies for 2 h at 37°C. Co-localization was examined by fluorescence microscopy (LSM780; Zeiss, Oberkochen, Germany).

Western blot analysis was conducted 24 h following ICH as previously described (Qi et al., 2019). The perihematomal brain tissues (4-mm-thick) were sampled. The relative densities of the bands were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).

PCR was performed to analyze Sur1 gene expression as previously reported (Jiang et al., 2016). Rats (n = 6/group) were sacrificed by decapitation 6, 12, and 24 h following ICH. The brain tissues were dissected (2 mm posterior and 2 mm anterior to the needle entry site) and immediately flash-frozen with liquid nitrogen. The primers for SUR1 were as follows: forward, 5′-CACAAGAAGCCCATCGACCT-3′; reverse, 5′-ATCGAAGGCCAAGCAGAGTC-3′ (Table 1). A positive standard curve was obtained using a serially diluted complementary DNA sample mixture. Gene expression was normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and quantified with standard samples. The data are expressed as a normalized messenger RNA expression (fold mRNA increase).

We used the corner turn test and forelimb placement test to assess the neurological function of the experimental rats 7 days following ICH as previously described (Hua et al., 2002; Krafft et al., 2014; Tan et al., 2017).

The corner turn test was conducted by two blinded observers. The experimental rats were allowed to proceed to a corner with an angle of 30°. We recorded the direction in which rats turned, and the process was repeated 10 times (60 s between trials); the percentage of right turns was calculated.

The forelimb placement test was conducted by two blinded observers. The rats were held by the torso, and all four limbs were allowed to hang freely in space. A trial was scored if a rat placed its forelimb on the edge of the countertop in response to vibrissae stimulation. Each forelimb was tested 10 times, and the percentage of successful scores was determined.

The Morris water maze test was performed to measure the learning and spatial memory of rats as previously described (Dai et al., 2017). Twenty-three days following ICH, rats (n = 10, per group) were placed in a pool (200 cm in diameter, 50 cm in depth) in which they searched to find a platform (5 cm in diameter, top surface 1.5 cm below the surface of the water) within 120 s. The rats that failed the mission would be picked up and placed on the platform for 15 s to familiar with the situation. The rats were subjected to five consecutive days of trials. The latency time was recorded to assess spatial learning ability. The probe trial was performed on the sixth day by removing the platform, and each rat was allowed to swim freely (120 s). The number of times the target area (previous location of the platform) was crossed, the percent distance and percent time in the target quadrant were analyzed.

The results of this study are expressed as the mean ± SD. Statistical analysis of the data was conducted using one-way analysis ANOVA, followed by Student–Newman–Keuls (SNK). Statistical significance was set as a p-value < 0.05.

The protein expression of Sur1 was significantly upregulated surrounding the hematoma after ICH in aged rats (p < 0.0001, Figures 1A,D), but no significant difference was observed between the rats in the GLC treatment group and the vehicle group (p > 0.05, Figures 1A,D). No difference was observed in KIR6.2 expression among the sham group, GLC treatment group, and vehicle group (p > 0.05, Figure 1C). The Sur1 mRNA expression was examined at 6, 12, and 24 h after ICH. Compared with the sham-treated rats, aged rats with ICH exhibited a remarkable increase in Sur1 mRNA (p < 0.05, p < 0.01, Figure 1B).

The corner turn score and forelimb placing score were used to measure the neurological function of the rats on day 7 after ICH. In these tests, the GLC-treated group had a lower corner turn score (p < 0.05, Figure 2A) and higher forelimb placing score (p < 0.05, Figure 2B) than the vehicle group.

The brain water content of the rats in the vehicle group was significantly increased 72 h after ICH, especially in the ipsilateral basal ganglia (Ipsi-BG: sham vs. vehicle, p < 0.001, Figure 2C). The GLC treatment remarkably decreased the brain water content in the ipsilateral basal ganglia (Ipsi-BG: vehicle vs. GLC, p < 0.05, Figure 2C).

The rats exhibited disadvantageous spatial learning and memory deficits following ICH. The latency of the rats in the GLC group was significantly shorter than that of the rats in the vehicle group beginning on the fourth of five consecutive days of acquisition training (p < 0.05, Figure 3A). The GLC-treated rats spent more time (p < 0.05, Figure 3D) and traveled a greater distance (p < 0.05, Figure 3C) in the target quadrat compared with the vehicle-treated rats. In addition, the rats in the vehicle group crossed the platform fewer times (p < 0.001 and p < 0.05, respectively, vs. sham and GLC, Figure 3B). The results demonstrated that GLC improved spatial learning and memory deficits following ICH.

Immunofluorescence staining suggested a significant increase in brain-derived neurotrophic factor (BDNF) co-localized with neurons in the GLC group compared with that in the vehicle group (Figure 4A). Studied regions were marked with “squares” (Figure 4B). Western blotting examination revealed that the GLC treatment significantly increased the protein expression of BDNF 72 h following ICH (p < 0.01, Figures 4C,D).

We used western blotting and immunofluorescence staining to measure the expression of components of the NF-κB signaling pathway. Immunofluorescence staining demonstrated a remarkable decrease in Ikappa-B kinase (IKKβ) in the GLC group compared with the vehicle group (Figure 5A). The protein expression of NF-κB-p65 was significantly increased 24 h after ICH (sham vs. vehicle, p < 0.001, Figures 5B,C). The GLC treatment significantly decreased the protein expression of NF-κB p65 24 h after ICH (vehicle vs. GLC, p < 0.01, Figures 5B,C).

The expression of inflammatory factors was examined using western blotting analysis. The results revealed that TNF-α, IL-1, and IL-6 expression was significantly increased 24 h after ICH (Figure 6). GLC significantly decreased the expression of TNF-α (p < 0.01, Figure 6A,B). GLC significantly reduced the expression of IL-1 (p < 0.05, Figure 6A,C). GLC decreased the expression of IL-6, but no significant difference was observed (p > 0.05, Figures 6A,D).