Specific-pathogen-free male Sprague‒Dawley rats were obtained from Hunan Slake Jingda Co., Ltd. [Changsha, Hunan, China, certificate No. SYXK (Xiang) 2020-0017]. The body weights of the rats ranged from 250 to 280 g. Throughout the experimental period, all the animals were housed at our institution’s animal research center at a constant humidity and temperature with consistent lighting conditions, and they were acclimated to the environment for 1 week before the experiments. During this acclimatization period, the rats were provided unrestricted access to food and water.

A protein electrophoresis system (MINI-PROTEAN3; Bio-Rad, Hercules, CA, USA), Chemiluminescence detection system (1708370; Bio-Rad), Image Lab software (6.0.1; Bio-Rad), a high-speed refrigerated centrifuge (3.16R; Hengnuo, Changsha, Hunan, China), a fluorescence microscope (BA410T; Motic, Xiamen, Fujian, China), a filament (2636-A4; Xinon, Beijing, China), ImageJ software (V.2.0.0; NIH, Bethesda, MD, USA) and Microwave oven (M1-L202B; Midea, Foshan, Guangdong, China) were used in this study.

The main reagents used in this study were as follows. Radioimmunoprecipitation assay (RIPA) buffer (KGB5203; KeyGen, Nanjing, Jiangsu, China), BBG (B0770; Sigma-Aldrich, St. Louis, MO, USA) and 4% paraformaldehyde (G1101; Servicebio, Wuhan, Hubei, China) was used. Primary antibodies against P2X7R (PA5-77665, rabbit anti-rat, Thermo Fisher Scientific, Waltham, MA, USA), NLRP3 (ab263899, rabbit anti-rat, Abcam, Cambridge, MA, USA), Iba-1 (MA5-27726, mouse anti-rat, Thermo Fisher Scientific), caspase-1 (AF5418, rabbit anti-rat, Affinity, Nanjing, Jiangsu, China), IL-18 (DF6252, rabbit anti-rat, Affinity), IL-1$\beta$ (A20527, rabbit anti-rat, ABclonal), and GAPDH (AF7021, rabbit anti-rat, Affinity), as well as goat anti-mouse (SA00001-1) and goat anti-rabbit (SA00001-2) IgG (both from Proteintech, Chicago, IL, USA) were used. 4^′,6-Diamidino-2-phenylindole (DAPI) staining solution (AWC0293a; Abiwell, Changsha, Hunan, China) and IL-6 (E-EL-R0015c), IL-4 (E-EL-R0014c), IL-10 (GER0003) and IL-1$\beta$ (GER0002) kits (all from Servicebio) were also used.

In addition, there are some other auxiliary reagents and consumables, such as: 75% alcohol (67182260463; Annjet, Dezhou, Shandong, China), 0.9% physiological saline (XY4950; QiDuYaoYe, Zibo, Shangdong, China), sodium pentobarbital (BC1040; Protein Biotechnology, Beijing, China), EDTA Buffer (CM12684; Proteintech), nonfat dry milk (GC310001; Servicebio), phosphate buffered saline with tween (PBST, G2157-1L; Servicebio, Wuhan, China), goat serum (50012-8615; Everygreen, Hangzhou, Zhejiang, China), sucrose water (XY4797; JinJianYaoYe, Changsha, Hunan, China), Pure water (G4701-500ML; Servicebio), antifluorescence quencher (G1401-5ML; Servicebio), phosphate buffered saline (PBS, BL302A; Biosharp, Hefei, Anhui, China), Xylene (N1-5L; Taikangyiliao, Wuhan, Hubei, China), Paraffin (C0175A-500g; Beyotime, Shanghai, China), and eppendorf (EP) tube (EP-150-M; Servicebio).

Following a one-week adaptation period, the rats were subjected to right middle cerebral artery occlusion (MCAO). The model rats were subsequently divided into three groups, the stroke (n = 30), stroke + CUMS (n = 20) and stroke + CUMS + intervention (n = 10) groups, utilizing random numbers generated by SPSS 24.0 software (IBM Corp., Chicago, IL, USA). To determine the impacts of CUMS exposure for different durations, the rats in the stroke + CUMS group were further divided into two subgroups and examined at 2 weeks (n = 10) or 4 weeks (n = 10) after MCAO. The stroke-only group was divided into three subgroups and studied at 1 week (n = 10), 2 weeks (n = 10), or 4 weeks (n = 10) after MCAO. Notably, the data collected from rats that had not undergone CUMS at one week after MCAO were used as common baseline data for the stroke + CUMS and stroke-only groups. Notably, two subsets of rats in the stroke + CUMS + intervention group were administered either normal saline (NS; n = 5) or BBG (n = 5) at the beginning of the CUMS protocol (Fig. 1).

Fig. 1.

Diagram of the experimental procedures. BBG, brilliant blue G; NS, normal saline; CUMS, chronic unpredictable mild stress; MCAO, middle cerebral artery occlusion.

A 7 mg/mL solution of BBG was prepared in sterile physiological saline and stored in the dark at 4 °C. This solution was injected into the peritoneal cavity of rats at a standard dose of 50 mg/kg. One week after MCAO, the rats in the stroke + CUMS + BBG group received daily injections of BBG solution beginning 30 minutes after the cessation of stress exposure. Physiological saline served as the control in the stroke + CUMS + NS group. The duration of drug injection was 3 weeks.

As previously described [31], neurological deficit scores were assessed 12 hours after MCAO using the Longa method. A score of 0 represented walking in a straight line, indicating no neurological deficit, whereas a score of 4 represented an inability to walk or impairment of consciousness, indicating very severe neurological impairment and possible death. Therefore, rats with scores ranging from 1 to 3 were deemed successful models of ischemic stroke, whereas those with scores of 0 or 4 were excluded from the analysis.

After ~15% of the MCAO model rats were excluded based on the experimental requirements, a total of sixty rats were included in the statistical analysis.

Prior to testing, all the model rats were allowed to become familiar with and adapt to the testing environment. After each experiment, the equipment was thoroughly cleaned with 75% alcohol to eliminate any residual odor from the preceding animal. Behavioral assessments were consistently conducted at predetermined times to mitigate the potential impact of circadian rhythm variations.

The rats (n = 5 per group) were anesthetized with 1% sodium pentobarbital at 1 week, 2 weeks, and 4 weeks after MCAO. Pentobarbital was administered by intraperitoneal injection into the lower left abdominal quadrant at a dose of 150 mg/kg. The animals were observed for 3–5 minutes. Death was confirmed by loss of consciousness, cessation of respiration and heartbeat, and absence of the pupillary light reflex. The cardiac apex was accessed via a venous indwelling needle, which was then inserted into the left ventricle to the ascending aorta. The right auricle was subsequently cut, and 200 mL of normal saline was rapidly infused. Perfusion with 200 mL of 4% paraformaldehyde was terminated upon observing the stiffness of the tail or extremities. The brains were extracted and immersed in 4% paraformaldehyde overnight in a refrigerator set at 4 °C. Finally, the brain tissue was dehydrated and embedded in paraffin. The embedded tissue was subsequently systematically cut into 5 µm thick slices.

The tissue slices were dewaxed in xylene and subsequently hydrated in graded ethanol solutions. Antigen retrieval was performed by heating the slices in EDTA buffer in a microwave. After deparaffinization with xylene and rehydration in graded ethanol solutions, the slices were subjected to a second round of antigen retrieval with EDTA buffer in a microwave. The slices were subsequently covered completely with 10% goat serum at room temperature to block nonspecific binding. After one hour, the sections were incubated overnight at 4 °C with primary antibodies against P2X7R (1:50), NLRP3 (1:50), or Iba-1 (1:50). After primary antibody incubation, the slices were incubated with 50 µL of secondary antibody (1:100) in an opaque box for 1 hour at 37 °C. Excess antibody was added, and the sections were thoroughly washed with PBS. Finally, the cell nuclei were incubated with DAPI working solution at 37 °C for 15 minutes. An antifluorescence quencher was applied, and the slices were mounted. Staining in the CA1 region was visualized under a fluorescence microscope and imaged in a dark environment. ImageJ software was used to quantify the fluorescence intensity per unit area in the CA1 region. The fluorescence intensities of P2X7R, NLRP3, and Iba-1 were calculated using DAPI as a reference and normalized.

The remaining rats were euthanized with 1% sodium pentobarbital at 1 week, 2 weeks, and 4 weeks after MCAO. Affected hippocampal tissues preserved at –80 °C were fully lysed in RIPA lysis buffer, followed by ultrasonication on ice and centrifugation (12,000 r/min) for 15 minutes at 4 °C. The carefully collected supernatant was mixed with 5$\times$ SDS protein loading buffer and boiled for 5 minutes at 100 °C. After cooling, the sample was stored at –20 °C. Protein concentrations were determined through ultraviolet absorptiometry (A280). The proteins were subsequently separated via SDS‒PAGE at different voltages and then transferred onto a PVDF membrane (BS-PVDF-45; Biosharp) at 4 °C at a constant voltage. The PVDF membrane was blocked with a PBS solution containing 5% nonfat dry milk. Afterward, the PVDF membrane was immersed in an aqueous mixture containing polyclonal anti-caspase-1 (1:1000), polyclonal anti-NLRP3 (1:1000), polyclonal anti-IL-18 (1:1000), polyclonal anti-IL-1$\beta$ (1:1000), polyclonal anti-P2X7R (1:2000), or monoclonal anti-Iba-1 (1:3000) antibodies. After an overnight incubation at 4 °C, PBST was used to wash away the excess primary antibody. The membrane was then immersed in a diluent containing secondary antibody (1:3000) at room temperature for 1 hour. GAPDH (1:10,000) served as a reference protein for quantification, and Image Lab software was used to analyze the gray values of the bands.

Following anesthesia (as described in Section 2.8), blood samples (approximately 1.5 mL) were drawn from the right retroorbital venous plexus of the rats. Samples (n = 5 per group) were collected at 2 and 4 weeks after MCAO and promptly subjected to centrifugation at 4 °C. The resulting plasma was aliquoted into EP tubes and stored at –80 °C. For analysis, the samples were diluted tenfold and processed in accordance with the manufacturer’s instructions. The plasma levels of IL-6, IL-4, IL-10, and IL-1$\beta$ were quantified. All the samples were concurrently analyzed by two experienced experimenters who were blinded to the specific grouping of the samples. Subsequently, standard curves were generated, and the concentration of each cytokine was calculated.

The data were analyzed using SPSS 24.0 software. In this study, all the data were tested with the Shapiro‒Wilk test and met the criteria for a normal distribution. The data collected at 1 week after MCAO were used as the baseline data before chronic stress induction. The data collected at 2 weeks and 4 weeks after MCAO were standardized by calculating the values for 2 w/1 w (%) and 4 w/1 w (%) after MCAO, respectively. Differences in behavioral scores, the relative gray values of the protein bands and immunofluorescence intensity between the stroke group and the stroke + CUMS group were analyzed using two-way ANOVA. When an interaction between CUMS exposure and time was observed, simple effects analysis was performed, with comparisons between groups or between time points adjusted via Bonferroni correction. When no interaction between CUMS exposure and time was observed, main effects analysis with Bonferroni’s post hoc correction was used. Plasma cytokine levels in the stroke group and the stroke + CUMS group were compared via an independent samples t test. Similarly, differences in behavioral scores and the relative gray values of protein bands in the stroke + CUMS + NS group and the stroke + CUMS + BBG group were analyzed via an independent samples t test. Statistical graphs were produced via GraphPad Prism 9.5.0 (Dotmatics, San Diego, CA, USA). A p value 0.05 was considered to indicate statistical significance.

There was no interaction effect between CUMS and time on the expression of Iba-1 [F_{(1, 16)} = 3.716, p = 0.072], caspase-1 [F_{(1, 16)} = 0.007, p = 0.934], IL-18 [F_{(1, 16)} = 1.915, p = 0.185], P2X7R [F_{(1, 16)} = 0.402, p = 0.535] or NLRP3 [F_{(1, 16)} = 0.458, p = 0.508]. Main effect analysis revealed that CUMS had a significant effect on Iba-1 [F_{(1, 16)} = 37.824, p 0.001], caspase-1 [F_{(1, 16)} = 14.115, p = 0.002], and NLRP3 [F_{(1, 16)} = 4.702, p = 0.046] expression. Specifically, the expression of Iba-1 (t = 6.136, p 0.001), caspase-1 (t = 3.758, p = 0.002), and NLRP3 (t = 2.169, p = 0.046) was significantly higher in the stroke + CUMS group than in the stroke group. However, the same results were not found for IL-18 [F_(1,16)= 2.408, p = 0.140] or P2X7R [F_(1,16)= 1.455, p = 0.245] expression (Fig. 3a–g; Supplementary Table 2).

Fig. 3.

Relative expression of Iba-1 and P2X7R/NLRP3 signaling pathway-related proteins in the affected hippocampus in the stroke and stroke + CUMS groups. Representative blots showing the levels of Iba-1 and proteins related to the P2X7R/NLRP3 signaling pathway (a,b). Graphs showing the relative protein expression levels of Iba-1 (c), caspase-1 (d), IL-18 (e), P2X7R (f), NLRP3 (g), and IL-1$\beta$ (h) are presented. The data are presented as the means $\pm$ SDs (n = 5). Unlike for the expression of IL-1$\beta$, there was no interaction effect between CUMS and time on the expression of Iba-1, P2X7R, NLRP3, caspase-1 or IL-18. The stroke + CUMS group vs. the stroke group: *p 0.05, **p 0.01, and ***p 0.001; 4 w/1 w post-MCAO vs. 2 w/1 w post-MCAO: ^#p 0.05, ^#⁢#p 0.01, and ^#⁢#⁢#p 0.001. All comparisons were adjusted via Bonferroni’s correction. P2X7R/NLRP3, Purinergic ligand-gated ion channel 7 receptor/nucleotide-binding oligomerization domain, leucine-rich repeats, and pyrin domain-containing protein 3; IL, interleukin; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Moreover, main effect analysis revealed that time significantly affected the expression of Iba-1 [F_{(1, 16)} = 13.127, p = 0.002], caspase-1 [F_{(1, 16)} = 6.442, p = 0.022], and IL-18 [F_{(1, 16)} = 9.914, p = 0.006] and NLRP3 [F_{(1, 16)} = 5.194, p = 0.037]. Iba-1 (t = 3.610, p = 0.002), caspase-1 (t = 2.539, p = 0.022), IL-18 (t = 3.150, p = 0.006) and NLRP3 (t = 2.279, p = 0.037) expression was increased at 4 weeks after MCAO compared with 2 weeks after MCAO. However, the same results were not obtained for P2X7R [F_{(1, 16)} = 0.754, p = 0.398] (Fig. 3a–g; Supplementary Table 2; The original western blot images for Fig. 3a and Fig. 3b are provided in the Supplementary Figures).

There was an interaction effect between CUMS and time on the expression of IL-1$\beta$ [F_{(1, 16)} = 22.216, p 0.001]. Simple effect analysis revealed that the expression levels of IL-1$\beta$ in the stroke + CUMS group [F_{(1, 16)} = 48.498, p 0.001] were significantly higher at 4 weeks after MCAO than at 2 weeks after MCAO (t = 6.959; p 0.001). However, in the stroke group [F_{(1, 16)} = 0.089, p = 0.769], the difference in IL-1$\beta$ expression between the two time points was not obvious. Simple effect analysis also revealed that at 4 weeks after MCAO [F_{(1, 16)} = 51.700, p 0.001], the expression of IL-1$\beta$ in the stroke + CUMS group was significantly higher than that in the stroke group (t = 7.181, p 0.001). However, this difference was not obvious at 2 weeks after MCAO [F_{(1, 16)} = 0.275, p = 0.607] (Fig. 3h; Supplementary Table 2).

Ischemia can induce an inflammatory response in the hippocampus, and chronic stress can exacerbate structural damage, which is involved in mood disorders [33, 34]. We observed that the levels of some inflammatory proteins increased with prolonged stress, which may have been at least partially related to increased Iba-1 expression or microglial activation. These results suggest that the levels of some inflammatory markers gradually increase over time, as expected, at least in part.

The CA1 region, which is closely associated with the depressive phenotype, has structural and functional importance [35]. Our focus was on the distribution and fluorescence signals of three core proteins within this region. Sections were specifically stained for P2X7R (red fluorescence), NLRP3 (red fluorescence), Iba-1 (green fluorescence) and DAPI (blue fluorescence).

We found no interaction effect between CUMS and time on the fluorescence intensity of Iba-1 [F_{(1, 16)} = 0.789, p = 0.388], NLRP3 [F_{(1, 16)} = 1.440; p = 0.248], or P2X7R [F_{(1, 16)} = 1.967, p = 0.180]. These findings are consistent with the results observed for the whole hippocampus. Main effect analysis revealed that CUMS had a significant effect on the fluorescence intensity of Iba-1 [F_{(1, 16)} = 12.490, p = 0.003], NLRP3 [F_{(1, 16)} = 8.590, p = 0.010] and P2X7R [F_{(1, 16)} = 6.113, p = 0.025]. Specifically, the fluorescence intensities of Iba-1 (t = 3.532, p = 0.003), NLRP3 (t = 2.930, p = 0.010) and P2X7R (t = 2.471, p = 0.025) were significantly higher in the stroke + CUMS group than in the stroke group (Fig. 4; Supplementary Table 3).

Fig. 4.

Comparison of P2X7R, NLRP3 and Iba-1 staining in the affected CA1 region between the stroke and stroke + CUMS groups (400$\mathrm{\times }$). Images of immunofluorescence staining for P2X7R, NLRP3 and Iba-1 in the hippocampal CA1 region on the affected side. Quantitative analysis of the fluorescence intensities of Iba-1 (a,b), NLRP3 (c,d) and P2X7R (e,f) is shown. The data are presented as the means $\pm$ SDs (n = 5). Scale bar = 100 µm. There was no interaction effect between CUMS and time on the fluorescence intensity of Iba-1, P2X7R or NLRP3. The stroke + CUMS group vs. the stroke group: *p 0.05, and **p 0.01. All comparisons were adjusted via Bonferroni’s correction. DAPI, 4^′,6-Diamidino-2-phenylindole.

However, main effect analysis also revealed that time did not significantly affect the fluorescence intensity of Iba-1 [F_{(1, 16)} = 3.332, p = 0.087], NLRP3 [F_{(1, 16)} = 4.312, p = 0.054] or P2X7R [F_{(1, 16)} = 1.616, p = 0.222] (Fig. 4; Supplementary Table 3).

Systemic inflammatory responses occur in somatic lesions caused by chronic stress, indicating that peripheral inflammatory markers are potentially valuable for diagnosing or predicting mood disorders. Since the venous blood of rats could not be obtained or did not meet the requirements for effective analysis at 1 week after MCAO, we compared plasma levels of inflammatory factors between only 2 and 4 weeks after MCAO. Compared with the stroke group, the plasma levels of IL-4 (t = 2.519, p = 0.036) in the stroke + CUMS group was lower 4 weeks after MCAO, but the IL-10 level (t = 2.688, p = 0.028) was increased. However, the plasma levels of IL-6 in the stroke + CUMS group were lower than those in the stroke group at both 2 (t = 7.176, p 0.001) and 4 weeks (t = 5.261, p 0.01) after MCAO. Interestingly, there was no significant difference in plasma IL-1$\beta$ levels between the two groups at 2 or 4 weeks after MCAO, unlike what was observed for hippocampal tissue (Fig. 5a–d).

Fig. 5.

Comparison of peripheral cytokine levels between the stroke and stroke + CUMS groups. (a–d) ELISA results for IL-1$\beta$, IL-4, IL-6, and IL-10, respectively. The data are presented as the means $\pm$ SDs (n = 5). Stroke + CUMS group vs. stroke group: *p 0.05, **p 0.01, and ***p 0.001.

To further investigate the role of the P2X7R/NLRP3 signaling pathway in PSD, we inhibited P2X7R on the cell membrane using BBG, and alterations in downstream inflammatory protein expression were assessed. At 4 weeks after MCAO, compared with the stroke + CUMS + NS group, the stroke + CUMS + BBG group presented increased numbers of rearings (t = 4.706, p = 0.006) and grid crossings (t = 3.243, p = 0.012) in the OFT, an elevated SPI (t = 2.447, p = 0.040), and a shortened latency in the NSFT (t = 3.772, p = 0.005), with no change in food intake in the NSFT (t = 0.175, p = 0.866) (Fig. 6a–d). In addition to reversing the changes in behavioral scores, BBG decreased the expression of IL-1$\beta$ (t = 2.370, p = 0.045), IL-18 (t = 2.548, p = 0.034), NLRP3 (t = 3.492, p = 0.008), and caspase-1 (t = 3.223, p = 0.012) in the affected hippocampus, whereas the expression of Iba-1 remained unaffected (t = 1.215, p = 0.252) (Fig. 6e,f. The original western blot images for Fig. 6e are provided in the Supplementary Figures).

Fig. 6.

BBG alleviates depression-like behaviors and mitigates hippocampal neuroinflammation in rats with stroke. Statistical analysis of the behavioral scores of rats subjected to stroke in the OFT (a), NSFT (b,c), and SPT (d). Western blot bands for inflammatory markers and statistical analysis of the relative gray values of the bands are displayed in (e) and (f), respectively. The data are presented as the means $\pm$ SDs (n = 5). Stroke + CUMS + BBG group vs. stroke + CUMS + NS group: *p 0.05 and **p 0.01.