The current study was approved by the Animal Care Committee of Southwest Medical University (China). A total of 353 male C57BL/6 mice (weighing 18–22 g) were purchased from Chengdu Dossy Experimental Animals Co., Ltd (China). The animals were housed in cages in a temperature-controlled room (22 ± 2 °C) with alternating 12-h light/dark cycle and acclimatized for one week. Before the study, the mice were fasted for 8 h but allowed water ad libitum. All procedures were performed according to the National Institutes of Health guidelines for the use of experimental animals.
2.2. Experimental design
The experimental flow diagram is illustrated in Figure 1.
2.2.1. Experiment 1
To evaluate the time course of endogenous LRP1, TXNIP, NLRP3, and cleaved caspase-1 expression and histopathological changes in the hippocampal tissues of mice after cerebral I/R, 70 mice were randomized into five groups: sham, 6, 12, 24, and 72 h after I/R (n = 14/group). Western blot analysis was performed to assess the protein level of LRP1, TXNIP, NLRP3, and cleaved caspase-1. Then, hematoxylin and eosin (HE) staining was used for histopathological examination of the hippocampal tissues. Additionally, the cellular localization of LRP1 with neurons (Neuronal Nuclei, NeuN), astrocytes (glial fibrillary acidic protein, GFAP), or microglia (calcium-binding adaptor molecule 1, Iba-1) was evaluated by double immunofluorescence staining in the Sham and I/R-72 h group (n = 3/group).
2.2.2. Experiment 2
To determine the beneficial effects of LRP1 activation on neurobehavioral functions, brain water content (BWC), and histopathological changes at 72 h after cerebral I/R, 120 mice were randomized into five groups: sham, I/R + Vehicle, I/R + COG1410 (0.5 mg/kg), I/R + COG1410 (1.0 mg/kg), and I/R + COG1410 (2.0 mg/kg) (n = 24/group). The BWC and histopathological changes were tested at 72 h after reperfusion, while Morris water maze (MWM) was performed for neurobehavior tests on days 3–8 after cerebral I/R.
2.2.3. Experiment 3
To assess the roles of LRP1 activation on microglia, neuroinflammation, and apoptosis at 72 h after cerebral I/R, 36 mice were randomly divided into three groups for western blot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining: sham, I/R + Vehicle, and I/R + COG1410 (n = 12/group).
2.2.4. Experiment 4
In order to investigate the underlying mechanism of TXNIP/NLRP3 pathway in LRP1 mediated microglia polarization modulating, 70 mice were randomized into five groups: sham, I/R + Vehicle, I/R + COG1410, I/R + COG1410 + AAV-NC, I/R + COG1410 + AAV-TXNIP (n = 14/group). The sample for sham, I/R + Vehicle, and I/R + COG1410 groups were shared from experiment 3, and an additional 34 mice were used for the experiment. Moreover, to verify the efficacy of AAV-TXNIP on TXNIP overexpression, 18 mice were randomly divided into three groups: naïve, naïve + AAV-NC, naïve + AAV-TXNIP (n = 6/group). Histopathological changes and the level of oxidative stress were examined, and western blot analysis was performed at 72 h after cerebral I/R.
2.2.5. Experiment 5
To explore the underlying mechanism of TXNIP/NLRP3 pathway in LRP1 mediated long-term neuroprotective effect, 75 mice were randomly divided into five groups: sham, I/R + Vehicle, I/R + COG1410, I/R + COG1410 + AAV-NC, I/R + COG1410 + AAV-TXNIP (n = 15/group). The survival rates of different groups were monitored at 28 days after cerebral I/R, and WMW was conducted on days 28–33 after cerebral I/R.
2.3. Cerebral I/R model
The model of cerebral I/R was established using bilateral common carotid artery occlusion (BCCAO) as described previously . Briefly, mice were anesthetized with pentobarbital sodium (45 mg/kg, intraperitoneally). Then, the trachea was exposed by a 0.5 cm midline neck incision, and the bilateral common carotid arteries (CCAs) were separated from the vagus nerve. Subsequently, the bilateral CCAs were occluded with the microvascular clamp, with the right CCA clamped first. After 30 min of ischemia, both CCAs were reperfused. In the sham operation group mice, the same procedures were performed without CCA occlusion. After infiltration with 0.25% bupivacaine, the wound was closed by sterile sutures. During the surgery, the body temperature of mice was maintained with a heating pad.
2.4. Intraperitoneal administration
COG1410 (acetyl-AS-Aib-LRKL-Aib-KRLL-amide synthesized by GL Biochem, Shanghai, China) was diluted with saline to different concentrations (0.5 mg/kg, 1.0 mg/kg, and 2.0 mg/kg), as described previously (Laskowitz et al., 2007). A volume of 100 μL COG1410 was administered intraperitoneally at 30 min, 24 h, and 48 h after reperfusion. The mice in the sham and I/R + Vehicle groups received equal volumes of saline at the same time point.
2.5. AAV injection
The TXNIP overexpression mouse model was established on day 28 before BCCAO, as described previously . Briefly, the adeno-associated virus vectors (AAV-TXNIP and AAV-NC) were constructed by Syngentech Co., Ltd (Beijing, China). Before exposing the skull, the mice were anesthetized with pentobarbital sodium in a stereotaxic apparatus (RWD, Shenzhen, China). A volume of 1.5 µL of the AAV vectors was injected stereotaxically into the bilateral hippocampal CA1 area (AP-2.0 mm; ML±1.0 mm; DV 2.0 mm) at a rate of 0.5 μL/min, following which the needle was kept in place for 5 min and screwed out slowly. The skin incision was sutured, and the mice were housed in a single cage after they awoke from anesthesia.
2.6. Specimen collection
The mice were sacrificed at 6, 12, 24, or 72 h after reperfusion under deep anesthesia. Some animals were perfused transcardially with ice-cold saline, and then the hippocampal tissues were separated on ice, frozen in liquid nitrogen, and stored at -80 °C for Western blot, ELISA, detection of oxidative stress, and RT-PCR. The other mice were perfused transcardially with 4% paraformaldehyde for HE, TUNEL, and immunofluorescence staining.
2.7. Morris water maze test
The function of spatial learning and memory for mice was assessed using the Morris water maze (MWM) test . The maze (Xinruan, Shanghai, China) consisted of a round tank (height 50 cm, diameter 120 cm), a platform (diameter 10 cm), and a camera analysis system. The tank was divided into four quadrants, filled up to a depth of 30 cm with mixed milk water (22 ± 1 °C). The mice were trained for four trials per day for five consecutive days during the place navigation test. In every trial, mice were placed in a different start quadrant and allowed to find the platform submerged below 1 cm of the water surface within a maximum duration of 60 s; the escape latency was recorded. On day 6, in the probe trial, the platform was withdrawn, and the mice were allowed to navigate freely in water for 60 s. The number of times crosses over the removed platform, duration in the target quarter (%), and swimming speed was recorded during the trial.
2.8. Histopathological examination
Whole-brain tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 3-μm-thick coronal sections. The sections were stained with HE and observed under a light microscope (Olympus, Tokyo, Japan) by two experienced pathologists blinded to the study. The number of normal neurons per square millimeter (cells/mm2) was calculated according to the size of the CA1 subregion in four high-magnification fields of each section, and the average was considered.
2.9. Brain water content
The brain water content (BWC) was estimated by the wet-to-dry brain weight ratio as described previously . Briefly, the whole brains were harvested under deep anesthesia and immediately weighed to obtain the wet weight. Then, the brain tissues were baked at 65 ℃ for 48 h to obtain the dry weight. BWC = (wet weight − dry weight) / wet weight × 100%.
2.10. Assessment of apoptosis
The brains were dehydrated with 30% sucrose and sliced into 8-μm-thick coronal frozen sections using a cryostat (Leica, Germany). The apoptosis of cells in the hippocampus was detected using the TUNEL staining, according to the manufacturer’s protocol (#12156792910, Roche, Basel, Switzerland) . The numbers of TUNEL-positive cells were counted in the CA1 subregion of the hippocampus. Four random high-magnification fields per section over a microscopic field of 400x magnification were averaged. Data were presented as the ratio of TUNEL-positive cells (%).
2.11. Immunofluorescence staining
Immunofluorescence staining was performed as described previously . Briefly, the frozen sections were incubated with primary antibody at 4 °C overnight: mouse anti-Iba1 (1:200, #GT10312, Invitrogen, USA), mouse anti-GFAP (1:200, #3670S, Cell Signaling Technology (CST), USA), mouse anti-NeuN (1:200, #ab104224, Abcam, USA), rabbit anti-LRP1 (1:200, #64099S, CST, USA), rabbit anti-TXNIP (1:200, #14715S, CST, USA), and rabbit anti-NLRP3 (1:200, #15101S, CST, USA). After washing with phosphate-buffered saline (PBS), the sections were incubated with the corresponding secondary antibody (Cy3-labeled goat anti-rabbit IgG, FITC-labeled goat anti-mouse IgG) (1:200, #S0011, #0007, Affinity, USA) at room temperature for 1 h, followed by DAPI (#C0060, Solarbio, China) staining. Finally, the sections were visualized, and images were captured using a fluorescence microscope.
2.12. Detection of inflammatory factors
Hippocampal tissue homogenates were prepared and clarified by centrifugation at 12000 rpm at 4 °C for 15 min. TNF-α, IL-6, IL-1β, and IL-18 levels were measured in the supernatants of hippocampal tissues using the ELISA kits (#MM-0132M1, #MM-0163M1, #MM-0040M1, #MM-0169M1, Meimian Jiangsu, China) by the manufacturer’s instruction as described previously .
2.13. Detection of oxidative stress
Hippocampal tissue homogenates were prepared, and lysates were collected by centrifugation at 3000 rpm at 4 °C for 10 min. The MDA content and SOD activity in the supernatant of hippocampal tissues were detected by commercial biochemical kits (#A003-1-2, #A001-3-2, Nanjing Jiancheng, China) as described previously .
Total RNA was extracted from the hippocampal tissues using an RNA simple total RNA kit (#DP419, Tiangen, Beijing, China) following the standard protocol. This isolated RNA was reverse-transcribed into cDNA using the ReverTra Ace qPCR RT Master Mix (#FSQ-201, Toyobo, Japan) according to the manufacturer’s protocol. Then, the cDNA was amplified using SuperReal PreMix Plus (SYBR Green) (#FP205-02, Tiangen, Beijing, China) on a real-time PCR system (Roche). The amplification parameters were as follows: 95 °C for 15 min, followed by five cycles of 95 °C for 10 s and 60 °C for 32 s. β-actin was used as an internal reference for the quantification of the TXNIP gene expression level. The relative mRNA expression level in the naive group (target mRNA/β-actin value) was set as the normalization to calculate the fold-changes of the mRNA level in other groups. The primer sequences are listed in Table 1.
The PCR primers sequences.
Abbreviation: TXNIP, thioredoxin-interacting protein.
2.15. Western blot analysis
The protein in the hippocampal tissue sample was estimated using an enhanced BCA protein assay kit (#P0012, Beyotime, Shanghai, China). An equivalent of 30 µg protein was separated on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF; Amersham Biosciences, NJ, USA) or a nitrocellulose membrane (Beyotime). Then, the membrane was blocked with 5% non-fat milk at room temperature for 1 h and probed with the primary antibodies at 4 ℃ overnight: anti-LRP1 (1:1000, #64099S, CST), anti-TXNIP (1:1000, #14715S, CST), anti-NLRP3 (1:1000, #15101S, CST), anti-cleaved caspase-1 (1:200, #SC-56036, Santa Cruz, USA), anti-Iba-1 (1:1000, #17198S, CST), anti-iNOS (1:1000, #AF0199, Affinity, USA), anti-Arg-1 (1:1000, #DF6657, Affinity), anti-Bcl-2 (1:1000, #ab59348, Abcam, USA), anti-Bax (1:1000, #50599-2-Ig, Proteintech, China), anti-cleaved caspase-3 (1:1000, #19677-1-AP, Proteintech), and anti-β-actin (1:5000, #6609-1-Ig, Proteintech), followed by incubation with the appropriate horseradish peroxidase (HRP)-labeled secondary antibody (1:5000, #SE131, #SE134, Solarbio, China) at room temperature for 1 h. Then, the membranes were developed using enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, UK). Finally, the immunoreactive bands were analyzed using Image J software (version 1.31; National Institutes of Health (NIH), Bethesda, MD, USA).
2.16. Statistical analysis
The GraphPad Prism 8.3 statistical software (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. All data were expressed as the mean and standard error of the mean (mean ± SEM). One-way analysis of variance (ANOVA) and Tukey’s post hoc test was used for comparisons. For the training phase of the WMW test, the escape latency over time was analyzed by two-way repeated-measures ANOVA. Kaplan-Meier survival curves were analyzed using the log-rank test. P < 0.05 was considered statistically significant.