The NLRP3 knockout (KO) C57BL/6 mice (NLRP3^−/−; male; age, 6 weeks) were purchased from GemPharmatech Co., Ltd. The age-matched male wild-type (WT) littermates were used as controls. The mice were fed standard chow and water, and were housed under standard experimental conditions (temperature, 20–25°C; humidity, 50–70%) under a 12-h light/dark cycle. The weight range of the mice was 18.22–21.56 g, with a median of 19.34 g. The mice were randomly divided into the following groups: i) Sham-operated group (Sham); ii) ischemia-reperfusion (IR) group; iii) IR + normal glucose group (IR + NG); iv) IR + high glucose group (IR + HG); v) NLRP3^−/− + IR + HG group; and vi) IR + HG + Z-YVAD-FMK (a caspase-1 inhibitor) group. The mice in the NLRP3^−/− + IR + HG group underwent IR and were treated once with a high level of glucose at a rate of 2.5 g/(kg × h) for 2 h (total dose, 5 g/kg). Mice in the IR + HG + Z-YVAD-FMK group were intraperitoneally injected with Z-YVAD-FMK at a dose of 12.5 mg/kg prior to IR. The number of mice in each group was 36. The mice were allowed free access to standard chow and water, and were housed under standard experimental conditions in a specific pathogen-free environment (temperature, 20–25°C; humidity, 50–70%) with a 12-h light/dark cycle.

The entire experimental course consisted of 2 h of ischemia and 24 h of reperfusion. Animals were anesthetized with sodium pentobarbital (30 mg/kg; i.p.) before surgery. The health and behavior of the mice were examined every half hour. If the mice showed signs of awakening during the experiment, sodium pentobarbital (10 mg/kg; i.p.) was supplemented. A total of 22 mice died during the experiment and the rest were euthanized. Respiratory and circulatory failure caused by cerebral edema after cerebral infarction were considered the main cause of death. It has been reported that the mortality rate of mice with middle cerebral artery occlusion (MCAO) was 15% (15), which is consistent with the results (11.96%, 22/184) in the present study.

Euthanasia was performed if mice had difficulty breathing, or became emaciated or dehydrated due to not eating or drinking for 24–48 h. Mice were euthanized by injection with pentobarbital (30 mg/kg; i.p.), followed by cervical dislocation. Heartbeat and breathing were checked to ensure successful euthanasia. All animal experimental procedures including accidental death and euthanasia were approved by The Research Ethics Committee of Guangdong Provincial People's Hospital (Guangzhou, China; approval no. GDRECKY2020-046-01) and were performed following the ARRIVE 2.0 guidelines (16).

Animals were anesthetized with sodium pentobarbital (30 mg/kg intraperitoneal injection) before surgery. As previously described, MCAO was used to establish transient focal cerebral ischemia (15). Briefly, the right middle cerebral artery was blocked with a nylon suture, which was inserted into the internal carotid artery and advanced until the origin of the right middle cerebral artery was closed. After a 2-h occlusion, the suture was removed for reperfusion. The sham mice were subjected to a similar operation, except for the occlusion. During the period of MCAO, high glucose (50% glucose) or normal glucose (5% glucose) was injected through the caudal vein at a rate of 5 ml/(kg × h). The blood glucose levels before, and 2 and 24 h after ischemia were measured using the StatStrip Xpress Glucose Meter (Nova Biomedical Corporation). In addition, 2,3,5,-triphenyl tetrazolium chloride monohydrate (TTC) assessment was used to verify the establishment of the MCAO model. Briefly, brains were removed and cut into 2-mm-thick slices after sacrifice. The slices were immersed in 1% TTC for 30 min at 37°C and fixed with 10% formalin for 6 h at 4°C. Infarcted brain tissue was pale, while normal brain tissue was red. All mice in the IR group developed cerebral infarction (100%), while no mice in the Sham group developed cerebral infarction (0%).

The levels of PbtO₂ were measured according to a previously reported procedure (17). Briefly, a midline incision was performed, a hole was drilled and the dura was punctured. A microsensor was then inserted into the brain tissue. The PbtO₂ was measured at 1, 3, 6, 12 and 24 h after reperfusion using a monitor (Integra CAMO2; Integra LifeSciences Corporation).

The levels of CERO₂ were measured as described previously (17). The right jugular vein was cannulated upstream to collect venous blood from the right side of the brain at 1, 3, 6, 12 and 24 h after reperfusion. Arterial blood samples were also collected from the femoral artery. The blood gas analysis was performed using a blood gas/electrolyte analyzer (Model 5700; Werfen, S.A.). Tests included hemoglobin concentration (Hb), saturation of arterial blood oxygen (SaO₂), saturation of jugular venous blood oxygen, partial pressure of oxygen (PaO₂) and pressure of jugular venous blood oxygen (PjVO₂). The content of jugular venous blood oxygen (CjVO₂), content of arterial blood oxygen (CaO₂) and CERO₂ were calculated using the following formulas:

CaO₂=1.36 × Hb × SaO₂ + 0.0031 × PaO₂

CjVO₂=1.36 × Hb × PjVO₂ + 0.0031 × PjVO₂

CERO₂=(CaO₂-CjVO₂)/CaO₂

The neurological damage to the motor center of the cerebral cortex after IR was examined by rotarod and foot fault tests. The rotarod test was used to assess the coordination and body strength of the mice. The mice were placed on a six-lane acceleration rotary paddle at a rotation rate of 120 rpm and the residence time was recorded. The foot fault test was performed to evaluate limb motor function. Mice were placed on a metal mesh (area, 3 cm²) for 2 min and the number of missing steps on the left foreleg was subsequently recorded. Percentage of foot fault (%)=the number of missing steps/total steps.

Primary microglia were obtained from the brain cortexes of newborn WT or NLRP3^−/− mice (male; age, 1–3 days; weight, 2–3 g), as previously described (17). A total of 40 newborn mice were purchased from GemPharmatech Co., Ltd., and were sacrificed immediately after purchase. Briefly, the mice were anesthetized with sodium pentobarbital (30 mg/kg intraperitoneal injection) and sacrificed by cervical dislocation. Heartbeat and breathing were checked to ensure successful euthanasia. The cortexes were isolated from the entire brain under sterile conditions and the meninges were removed under a dissecting microscope. After which, the cortexes were cut into pieces as large as 1×1 mm and were digested with trypsin for 10 min. The cell suspension was placed in a 15-ml poly-L-lysine-coated flask and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO₂ for 2 weeks. Afterwards, the mixed glial cells were shaken at 1.33 × g at 37°C for 2 h to obtain microglia. The collected cells were seeded in 2-ml poly-L-lysine-coated plates and cultured in DMEM/F12 supplemented with 10% FBS at 37°C. Primary microglia from WT mice were randomly divided into groups: i) Control group (CON); ii) oxygen and glucose deprivation (OGD) group; iii) OGD + NG group (OGD + NG); iv) OGD + HG group (OGD + HG); and v) OGD + HG + Z-YVAD-FMK (an inhibitor of caspase-1) group (OGD + HG + Z-YVAD-FMK). The microglia from the NLRP3^−/− + OGD + HG group originated from NLRP3^−/− mice, and were exposed to OGD and high glucose.

Microglia in the OGD group were cultured in glucose-free DMEM and transferred to a hypoxic chamber with 95% N₂/5% CO₂/0.1% O₂ at 37°C for 6 h. After 6 h of OGD, the cells were returned to high-glucose DMEM with 10% FBS at 37°C in an incubator with 5% CO₂/95% air for 24 h, which was used to reflect IR injury of the ischemic penumbra in cortical areas in vivo. Cells in the OGD + HG group were treated with 25 mM glucose at 37°C for 24 h following 6-h OGD. Meanwhile, the cells in the OGD + HG + Z-YVAD-FMK group were also treated with 10 µM Z-YVAD-FMK at 37°C for 24 h.

The OCR was evaluated using a cellulate OCR Assay Kit (cat. no. BB-48211; Bebo Biotechnology), as previously described (17). Briefly, microglial cells were seeded in 96-well plates. After 6 h of OGD, the cells were restored to the supply of glucose and oxygen at 37°C for 24 h. After which, oxygen fluorescent probes (BBoxiProbe^® R01) and oxygen-mounting media were added to 96-well plates sequentially. The OCR levels were measured every 3 min using a fluorescence microplate reader (Model 9260; LI-COR Biosciences).

The brain tissue from the ischemic penumbra in the cortical area 24 h after ischemia, and primary microglial cells, were lysed on ice with RIPA lysis buffer (cat. no. BB-3101-100T; Bebo Biotechnology) containing protease inhibitors. Notably, there was a region of encephalomalacia following MCAO. The area around the encephalomalacia region was considered the ischemic penumbra. The brain tissue close to the surface of the cerebral hemisphere was extracted from the ischemic penumbra. The BCA protein assay kit (cat. no. 23227; Pierce; Thermo Fisher Scientific, Inc.) was used to measure the total protein concentration. After mixing with 4X loading buffer, the proteins (40 µg per lane) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Then, the membranes were blocked with 5% BSA (cat. no. V900933; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, and were incubated overnight at 4°C with primary antibodies against caspase-1 (1:1,000; cat. no. ab138483; Abcam), GSDMD-FL (1:1,000; cat. no. ab219800; Abcam), GSDMD-N (1:1,000; cat. no. 10137; Cell Signaling Technology), IL-1β (1:1,000; cat. no. ab254360; Abcam), IL-18 (1:1,000; cat. no. ab191860; Abcam) and β-actin (1:1,000; cat. no. ab8226; Abcam). On the following day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000; cat. no. 7074S; Cell Signaling Technology, Inc.) for 2 h at 4°C. Finally, the blots were detected by enhanced chemiluminescence (MilliporeSigma) using an imaging densitometer (ImageQuant LAS 500; GE Healthcare Bio-Sciences).

A total of 24 h after reperfusion, normal saline and 4% paraformaldehyde were injected transcardially under anesthesia, and the brain was immediately collected. The collected brain tissue was fixed with 4% paraformaldehyde for 24 h at 4°C, dehydrated with gradient sucrose, and cut into 4-µm slices. The sections were permeabilized with 0.5% Triton X-100 for 30 min at room temperature and then blocked with and 1% BSA (cat. no. V900933; Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Thereafter, the following appropriate primary antibodies were applied at 4°C overnight: Iba1 (1:200; cat. no. ab283346; Abcam), caspase-1 (1:200; cat. no. 24232; Cell Signaling Technology), GSDMD-N (1:200; cat. no. DF13758; Affinity), IL-1β (1:200; cat. no. ab254360; Abcam) and IL-18 (1:200; cat. no. ab191860; Abcam), followed by secondary antibody incubation for 1 h at room temperature, including Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1:200; cat. no. ab150113; Abcam) and Alexa Fluor^® 594-conjugated donkey anti-rabbit IgG (1:200; cat. no. ab150080; Abcam). Finally, the slices were sealed with mounting medium (cat. no. F6057; MilliporeSigma). The number of cells was decreased in the area of necrosis under a fluorescence microscope (Olympus DP73 Microscope; Olympus). The peripheries of this area were considered the ischemic penumbra. The ischemic penumbra close to the surface of the cerebral hemisphere was observed and imaged. The cells on coverslips were fixed with 4% paraformaldehyde, and the cells underwent permeabilization, blocking and antibody incubation as aforementioned. The average fluorescence (red) density of one single microglia was analyzed using an image analysis system (Image-Pro Plus software; version 7.0; Media Cybernetics, Inc.).

Statistical analysis was performed using SPSS version 22.0 (IBM Corp.). All data are expressed as the mean ± standard deviation. The experiments were repeated six times in vivo and four times in vitro. The Shapiro-Wilk test was performed to determine data distribution, and all data conformed to a normal distribution. An unpaired Student's t-test was used to analyze two-group measurement data. A one-way analysis of variance (ANOVA) was used to analyze the data of four, five or six-group univariate-factor measurements. Multiple comparisons were analyzed by Tukey's test following ANOVA. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of blood glucose levels on neurological function after IR, the blood glucose levels, latency time to fall and percentage of foot fault were assessed. The results demonstrated that there was no difference in blood glucose levels among the Sham, IR, IR + NG, IR + HG and IR + HG + NLRP3^−/− groups at 0 h after ischemia (P>0.05; Fig. 1A). However, the blood glucose levels in the IR + HG and IR + HG + NLRP3^−/− groups were significantly increased compared with the Sham, IR and IR + NG groups (P<0.01), and there was no difference among the Sham, IR and IR + NG groups, as well as between the IR + HG and IR + HG + NLRP3^−/− groups at 2 and 24 h after ischemia (P>0.05; Fig. 1B and C). TTC staining images showed the cerebral infarction in the IR group. The incidence of cerebral infarction was 100% in the IR group, while that of the Sham group was 0% (P<0.01; Fig. 1D); this suggested that the establishment of the MCAO model was successful. The latency to fall in the IR and IR + NG groups were decreased compared with in the Sham group (P<0.01), and it was further decreased in response to high glucose (P<0.01). However, the latency to fall was significantly prolonged in the IR + HG + NLRP3^−/− group compared with the IR + HG group (P<0.01; Fig. 1E). The percentage of foot fault in the IR groups was notably increased compared with in the Sham group (P<0.01) and it was further increased in response to high glucose (P<0.05). However, the percentage was significantly reduced in the IR + HG + NLRP3^−/− group compared with the IR + HG group (P<0.01; Fig. 1F). These results suggested that high glucose can exert an aggravating effect on neurological damage after IR via activating the NLRP3 inflammasome.

The level of CERO₂ in the IR group was notably increased compared with that in the Sham group (1, 3, 6, 12 and 24 h; P<0.01) and it was further increased in response to high glucose (1, 3, 6 and 24 h: P<0.01; 12 h: P<0.05; Fig. 2A). The level of PbtO₂ in the IR group was significantly decreased compared with that in the Sham group (1, 3, 6, 12 and 24 h; P<0.01), and the IR + HG group had the lowest PbtO₂ level as compared with the IR group and Sham group (1 and 24 h, P<0.05; 3, 6 and 12 h, P<0.01; Fig. 2B). The level of OCR in the OGD group was notably increased compared with that in the CON group (5, 15 and 25 min, P<0.05; 10, 20 and 30 min, P<0.01) and it was further increased in response to high glucose (5, 15, 20 and 25 min, P<0.01; 10 and 30 min, P<0.05; Fig. 2C). These results suggested that high glucose treatment aggravated hypoxia by increasing the oxygen extraction rate.

The level of NLRP3 in the KO group was notably decreased compared with that in the WT group (P<0.01), indicating that the NLRP3 KO model was successfully established (Fig. 3A and B). There was an increase in the expression of caspase-1 in the WT + IR group compared with in the WT + Sham group (P<0.01). By contrast, the expression of caspase-1 in the NLRP3^−/− + IR group was notably decreased compared with in the WT + IR group (P<0.01; Fig. 3A and C). There was a decrease in the expression of GSDMD-FL in the WT + IR group compared with in the WT + Sham group (P<0.01). The expression of GSDMD-FL in the NLRP3^−/− + IR group was notably increased compared with in the WT + IR group (P<0.05; Fig. 3A and D). There was an increase in the expression of GSDMD-N in the WT + IR group compared with in the WT + Sham group (P<0.01). The expression of GSDMD-N in the NLRP3^−/− + IR group was notably decreased compared with in the WT + IR group (P<0.01; Fig. 3A and E). There was an increase in the expression of IL-1β in the WT + IR group compared with in the WT + Sham group (P<0.01). The expression of IL-1β in the NLRP3^−/− + IR group was notably decreased compared with in the WT + IR group (P<0.01; Fig. 3A and F). There was an increase in the expression of IL-18 in the WT + IR group compared with in the WT + Sham group (P<0.01). By contrast, the expression of IL-18 in the NLRP3^−/− + IR group was notably decreased compared with in the WT + IR group (P<0.01; Fig. 3A and G). These results suggested that IR can activate the NLRP3 inflammasome, and NLRP3 KO may inhibit activation of the NLRP3 inflammasome and the occurrence of pyroptosis.

The expression levels of caspase-1, GSDMD-FL, GSDMD-N, IL-1β and IL-18 after high glucose treatment were analyzed. The results of western blotting revealed an increase in the expression of caspase-1 in the IR and IR + NG groups compared with in the Sham group (P<0.05); it was further increased in response to high glucose, whereas a decrease in the expression of caspase-1 was detected in the IR + HG + NLRP3^−/− group compared with in the IR + HG group (P<0.01; Fig. 4A and B). The expression of GSDMD-FL in the IR and IR + NG groups was notably decreased compared with in the Sham group (P<0.05), which was further decreased in response to high glucose (P<0.01), but was increased in the IR + HG + NLRP3^−/− group compared with the IR + HG group (P<0.01; Fig. 4A and C). The results revealed an increase in the expression of GSDMD-N in the IR and IR + NG groups compared with in the Sham group (P<0.05), which was further increased in response to high glucose (P<0.01), but decreased in the IR + HG + NLRP3^−/− group compared with the IR + HG group (P<0.01; Fig. 4A and D). Furthermore, the results revealed an increase in the expression of IL-1β in the IR and IR + NG groups compared with in the Sham group (P<0.05); it was further increased in response to high glucose (P<0.01), but a decrease in the expression of IL-1β was detected in the IR + HG + NLRP3^−/− group compared with in the IR + HG group (P<0.01; Fig. 4A and E). The results revealed an increase in the expression of IL-18 in the IR and IR + NG groups compared with in the Sham group (P<0.05), which was further increased in response to high glucose (P<0.01), and a decrease in the expression of IL-18 was observed in the IR + HG + NLRP3^−/− group compared with in the IR + HG group (P<0.01; Fig. 4A and F).

To verify the effect of high glucose on microglial pyroptosis after IR in vivo, the expression levels of caspase-1, GSDMD-N, IL-1β and IL-18 in microglia was detected by double immunofluorescence. It was revealed that IR enhanced the expression of caspase-1 in cerebral microglial cells compared with in the Sham group, and this increase was substantially enhanced in the IR group treated with high glucose; however, the expression of caspase-1 was notably reduced after NLRP3 KO (P<0.01; Fig. 5A and E). IR also enhanced the expression of GSDMD-N in cerebral microglial cells compared with in the Sham group, and this increase was substantially enhanced in the IR group treated with high glucose; however, the expression of GSDMD-N was notably reduced after NLRP3 KO (P<0.01; Fig. 5B and F). IR enhanced the expression of IL-1β in cerebral microglial cells compared with in the Sham group, and this increase was substantially enhanced in the IR group treated with high glucose; however, the expression of IL-1β was notably reduced after NLRP3 KO (P<0.01; Fig. 5C and G). In addition, IR enhanced the expression of IL-18 in cerebral microglial cells compared with in the Sham group, and this increase was substantially enhanced in the IR group treated with high glucose; however, the expression of IL-18 was notably reduced after NLRP3 KO (P<0.01; Fig. 5D and H). These results suggested that NLRP3 KO inhibited high glucose-induced exacerbation of pyroptosis after IR.

To evaluate the effect of Z-YVAD-FMK (a caspase-1 inhibitor) on high glucose-induced pyroptosis in vivo, the expression levels of caspase-1 were analyzed. The results of western blotting revealed that the expression of caspase-1 was markedly reduced after treatment with Z-YVAD-FMK (P<0.01; Fig. 6A and B). To verify the effect of NLRP3 KO and inhibition of caspase-1 on pyroptosis after OGD in vitro, the expression levels of caspase-1, GSDMD-FL, GSDMD-N, IL-1β and IL-18 were analyzed after treatment with high glucose. The results of western blotting revealed an increase in the expression of caspase-1 in the OGD and OGD + NG groups compared with in the CON group (P<0.01), which was further increased in response to high glucose (P<0.01), and a decrease in the expression of caspase-1 was detected in the OGD + HG + NLRP3^−/− group and the OGD + HG + Z-YVAD-FMK group compared with in the OGD + HG group (P<0.01; Fig. 6C and D). The expression of GSDMD-FL in the OGD and OGD + NG groups was notably decreased compared with in the CON group (P<0.01), which was further decreased in response to high glucose (P<0.05), and an increase in the expression of GSDMD-FL was detected in the OGD + HG + Z-YVAD-FMK group compared with the OGD + HG group (P<0.01; Fig. 6E and G). The results also revealed an increase in the expression of GSDMD-N in the OGD and OGD + NG groups compared with in the CON group (P<0.05); it was further increased in response to high glucose (P<0.01), and a decrease in the expression of GSDMD-N was observed in the OGD + HG + Z-YVAD-FMK group compared with in the OGD + HG group (P<0.01; Fig. 6F and G). The results revealed an increase in the expression of IL-1β in the OGD and OGD + NG groups compared with in the CON group (P<0.01), which was further increased in response to high glucose (P<0.01), and a decrease in the expression of IL-1β was detected in the OGD + HG + Z-YVAD-FMK group compared with in the OGD + HG group (P<0.01; Fig. 6G and H). Furthermore, the results revealed an increase in the expression of IL-18 in the OGD and OGD + NG groups compared with in the CON group (P<0.01), which was further increased in response to high glucose (P<0.01), and a decrease in the expression of IL-18 was detected in the OGD + HG + Z-YVAD-FMK group compared with in the OGD + HG group (P<0.01; Fig. 6G and I). These results suggested that NLRP3 KO or inhibition of caspase-1 reversed high glucose-induced exacerbation of pyroptosis after OGD/IR.

To evaluate the effect of NLRP3 KO and inhibition of caspase-1 on high glucose-induced microglial pyroptosis after OGD in vitro, double immunofluorescence was used to examine caspase-1 expression in microglial cells. Enhanced caspase-1 immunofluorescence was observed in the OGD group compared with that in the CON group, and this increase was notably enhanced in the OGD group treated with high glucose; however, the expression of caspase-1 was substantially reduced in the OGD + HG + Z-YVAD-FMK group and the OGD + HG + NLRP3^−/− group (P<0.01; Fig. 7A and E). To verify the effect of inhibition of caspase-1 on microglial pyroptosis after OGD in vitro, double immunofluorescence was used to examine GSDMD-N, IL-1β and IL-18 expression in microglial cells. Enhanced GSDMD-N immunofluorescence was observed in the OGD group compared with that in the CON group, and this increase was notably enhanced in the OGD group treated with high glucose; however, the expression of GSDMD-N was notably reduced in the OGD + HG + Z-YVAD-FMK group (P<0.01; Fig. 7B and F). Enhanced IL-1β immunofluorescence was observed in the OGD group compared with that in the CON group, and this increase was notably enhanced in the OGD group treated with high glucose; however, the expression of IL-1β was notably reduced in the OGD + HG + Z-YVAD-FMK group (P<0.01; Fig. 7C and G). Furthermore, enhanced IL-18 immunofluorescence was observed in the OGD group compared with that in the CON group, and this increase was notably enhanced in the OGD group treated with high glucose; however, the expression of IL-18 was notably reduced in the OGD + HG + Z-YVAD-FMK group (P<0.01; Fig. 7D and H). These results suggested that NLRP3 KO or inhibition of caspase-1 reversed high glucose-induced microglial pyroptosis after OGD.