Specific-pathogen-free healthy adult male Sprague Dawley rats (8 weeks old; 250–280 g; Jinan Pengyue Laboratory Animal Breeding Co., Jinan, Shandong, China; animal license: SCXK (Lu) 20220006) were housed under a 12 h light/dark cycle (22 $\pm$ 1 ℃, 40–60 % humidity). The rats were randomly divided into six groups: one sham group and five CIRI groups, namely CIRI-2 h, 6 h, 12 h, 24 h, and 72 h according to the time after reperfusion. A modified Zea-Longa wire embolization method was used to prepare a middle cerebral artery occlusion (MCAO) model [16]. A wire plug (0.34 $\pm$ 0.02 mm) was inserted into the right common carotid artery (CCA), with the plug head entering the internal carotid artery (ICA) and stopping at a distance of approximately 18.0 mm from the bifurcation of the CCA. Two hours later, the wire was removed for reperfusion. Only the right CCA was isolated in the rats of the sham group. The experimental design of the study is outlined in Fig. 1.

Fig. 1.

Experiment design. Experimental timeline of the sham and CIRI groups, which underwent sham or MCAO operation, and a series tests of MR examination, TEM scans, HE staining, TUNEL staining, IF staining, and WB analysis were performed. CIRI, cerebral ischemia-reperfusion injury; IF, immunofluorescence; HE, hematoxylin-eosin; MCAO, middle cerebral artery occlusion; MR, magnetic resonance; TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WB, western blot.

The neurological examination of CIRI rats was performed at CIRI-2 h according to the Longa scale [16]. Neurological findings were graded on a five-point scale: 0, no neurological deficit; 1, mild focal neurologic deficit with the inability to extend the left forefoot; 2, moderate focal neurologic deficit with circling to the left; 3, severe focal deficit with falling to the left; and 4, inability to walk spontaneously and depressed level of consciousness. Scores of 1–3 were selected for inclusion in the study.

MRI examination was performed using a 7.0 T MRI scanner (BioSpec 70/20 USR; Bruker, Karlsruhe, Germany). Anesthesia was induced by a small animal anesthesia machine (R500, RWD, Shenzhen, Guangdong, China) using a mixture of 5% isoflurane and 95% oxygen, and maintained by a mixture of 2% isoflurane and 98% oxygen. The rats were placed in the center of the magnetic field. The regions of interest (ROIs) were outlined in the cerebral cortex and striatum. All ROI analyses were carried out in the ischemic right hemisphere and in the contralateral left hemisphere using the image analysis software ImageJ (ImageJ 1.6.0, National Institutes of Health, https://imagej.net/ij/index.html, Bethesda, MD, USA).

T2-weighted images were collected using a spin-echo sequence with the following parameters: TR (repetition time) = 3000 ms, TE (echo time) = 34 ms, FOV (field of view) = 3.3 $\times$ 3.1 cm${}^2$, MTX (matrix size) = 160 $\times$ 150, and ST (slice thickness) = 0.5 mm. The midline shift (MLS) quantification method was used to determine the space-occupying effect of the cerebral edema. The largest level of the lateral ventricle was selected. The distance between the outer margin of the cortex and the middle of the lateral ventricle was measured from the ipsilateral (a) and contralateral (b) sides. The calculation formula is as follows: MLS = (a–b) / 2 [17].

The DWI parameters were: TR = 3000 ms, TE = 22 ms, FOV = 3.0 $\times$ 3.0 cm${}^2$, MTX = 128 $\times$ 128, ST = 0.8 mm, and b-value of 0, 1000 s/mm${}^2$. Image processing was performed using small animal MRI workstation image post-processing software to generate apparent diffusion coefficient (ADC) maps. The largest level of the lateral ventricle of the coronal ADC map was selected, and the ischemic side of the cerebral cortex and striatum were manually drawn on the ADC map, as well as the contralateral mirror area as the ROI. ADC values were obtained from each ROI, and its relative mean value was calculated and expressed as the ipsilateral ADC compared with the contralateral ADC values [18].

After MRI scans, all rats were injected with sodium pentobarbital (cat# P3761-5G; 58 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) and subsequently perfused with physiological saline and 4% paraformaldehyde (PFA) through the heart. The brain was removed and fixed in PFA for 24 h. Brain tissue was embedded in paraffin wax. The lateral ventricular level (2.2 mm anterior to 0.26 mm posterior to the bregmatic fontanelle) was localized and sliced using a paraffin slicer (Finesse 325; Thermo Scientific, Waltham, MA, USA) to make 4-µm-thick sections.

The paraffin sections were waxed, hydrated, and stained using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit (MK1014-100; Boster, Wuhan, Hubei, China), according to the manufacturer’s instructions. The sections were then observed and photographed under an upright optical microscope (BX51; Olympus, Tokyo, Japan). TUNEL${}^{+}$ cell counts were recorded as cells/mm${}^2$, and the mean value was adopted.

Paraffin sections of brain tissue were baked at a constant temperature of 60 ℃ for 1 h. The sections were then dewaxed using xylene, hydrated using gradient ethanol, and stained sequentially with hematoxylin-eosin (HE) (G1120; Solarbio, Beijing, China). The sections were then dehydrated in anhydrous ethanol and cleaned in xylene. Finally, the sections were sealed with neutral gum. The morphological changes of cells were observed under an upright optical microscope (BX51; Olympus, Tokyo, Japan).

After anesthesia, rats were perfused with 4% PFA and 2.5% glutaraldehyde buffer (pH 7.4). Brain tissue specimens were collected, and cerebral cortex and striatum on the ischemic side were separated and cut into small pieces (1 $\times$ 1 $\times$ 1 mm${}^3$). Specimens were fixed in 1% osmium tetroxide, dehydrated through gradient ethanol, then embedded in epoxy resin. 40-nm thick sample sections were placed on a 200-mesh copper grid, stained with saturated uranyl acetate and lead citrate, then visualized using transmission electron microscopy (TEM) (HT-7700; Hitachi, Tokyo, Japan).

Immunofluorescence (IF) staining was performed on sections obtained from the sham and CIRI groups. Brain tissue sections were dewaxed and hydrated sequentially, and antigen repair was performed by the water bath thermal repair method. Rabbit anti-AQP4 (16473-1-AP, 1:1000; Proteintech, Wuhan, Hubei, China) and mouse anti-glial fibrillary acidic protein (GFAP) (3670S, 1:300; Cell Signaling Technology, Danvers, MA, USA) primary antibody mixture or rabbit anti-NKCC1 (13884-1-AP, 1:80; Proteintech) and mouse anti-GFAP primary antibody mixture were added as needed, and refrigerated at 4 ℃ overnight. On the next day, goat anti-rabbit IgG (ZF-0511, 1:200; Zhongshan Golden Bridge Biology Company, Beijing, China) and goat anti-mouse IgG (ZF-0513, 1:200; Zhongshan Golden Bridge Biology Company) fluorescent secondary antibody mixes were added, incubated for 1 h and protected from light, and the slices were sealed with fluorescent blocker containing 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI) solution (F6057-20ML; Sigma-Aldrich, St. Louis, MO, USA). The slices were observed and photographed under an upright fluorescent microscope (Axiolab 5; Zeiss, Oberkochen, Germany). The number of GFAP${}^{+}$/AQP4${}^{+}$ and GFAP${}^{+}$/NKCC1${}^{+}$ cells were counted as cells/mm${}^2$, and the mean values were calculated.

Protein concentrations were quantified separately using a bicinchoninic acid protein assay reagent kit (20201ES86, YEASEA, Shanghai, China). After transferring the proteins to the polyvinylidene fluoride membrane, and the membranes were blocked with 5% non-fat milk powder for 2 h. The strips were incubated in rabbit anti-AQP4 (16473-1-AP, 1:5000; Proteintech, Wuhan, Hubei, China) primary antibody, rabbit anti-NKCC1 (13884-1-AP, 1:1000; Proteintech, Wuhan, Hubei, China) primary antibody, and mouse anti $\beta$-tubulin (2128S 1:10,000; Cell Signaling Technology, Danvers, MA, USA) primary antibody and incubated overnight at 4 ℃. The next day, goat anti-rabbit (SA00001-2, 1:10,000; Proteintech, Wuhan, Hubei, China) or goat anti-mouse (SA00001-1, 1:10,000; Proteintech, Wuhan, Hubei, China) secondary antibody was added and incubated at room temperature for 2 h. The proteins were exposed and photographed using a high-performance imaging system, and the relative expression of each protein was analyzed using ImageJ software.

Statistical analyses were performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Shapiro Wilk analysis was used to verify the normality of the measured data, and all normally distributed data are represented as mean $\pm$ standard deviation. MRI data were compared using repeated measures analysis of variance (RMANOVA). Histological data were compared using one way analysis of variance (ANOVA) and further compared using the least significant difference (LSD) test. For correlation analysis, we adopted a method of combining Pearson’s correlation analysis and general linear regression analysis. Statistical significance was set at p 0.05.

T2WI (Fig. 2A) and DWI-MRI (Fig. 2B,C) scans indicated that the brain tissue of the sham group rats had uniform signal and bilateral symmetry, and no abnormal signals were observed. At CIRI-2 h, abnormal signal areas of cerebral edema appeared in the cerebral hemisphere on the ischemic side of the CIRI group, the midline of the brain was shifted to the contralateral side, and the relative apparent diffusion coefficient (rADC) values of the cerebral cortex and striatum decreased compared with the sham group. The midline of the brain was further shifted to the contralateral side at CIRI-6 h and 12 h, and the rADC values were lower than before. At CIRI-24 h, extensive cerebral edema was seen in the cerebral hemisphere on the ischemic side of the CIRI group, the MLS was relatively large, and the rADC value reached a relatively low level. At CIRI-72 h, the MLS was reduced and the rADC value was increased compared to that at CIRI-24 h (Fig. 2D–F, all *p 0.05, n = 6).

Fig. 2.

Dynamic multimodal MRI results. (A) T2WI images of the sham and CIRI groups. (B) DWI images of the sham and CIRI groups. (C) ADC images of the sham and CIRI groups. (D) Schematic diagram of the ROIs of the cortex and striatum and computation of MLS. (E) Analysis of the relative MLS in the sham and CIRI groups. (F) Analysis of the rADC in the sham and CIRI groups. Data are presented as mean $\pm$ standard deviation. *p 0.05 vs. sham group. ADC, apparent diffusion coefficient; DWI, diffusion weighted imaging; MLS, midline shift; rADC, relative apparent diffusion coefficient; ROIs, regions of interest; T2WI, T2-weighted imaging.

HE staining showed that the nerve cells in the cortex and striatum of the sham group were regular in morphology and neatly arranged. At CIRI-24 h, the nerve cells had irregular morphology, disorganized arrangement, and loose structure, and some nuclei were deeply stained (Fig. 3A, n = 6).

Fig. 3.

The results of HE and TUNEL staining in the sham and CIRI-24 h groups. (A) HE images of the sham and CIRI groups. (B) TUNEL images of the sham and CIRI groups. (C) Analysis of the number of TUNEL${}^{+}$ cells. Data are presented as mean $\pm$ standard deviation. *p 0.05 vs. sham group. Scale bar = 20 µm.

TUNEL assay showed that TUNEL${}^{+}$ cells were occasionally observed in the cortex and striatum in the sham group. However, the CIRI-24 h group showed a significantly increased number of TUNEL${}^{+}$ cells in the ischemic cortex and striatum (Fig. 3B,C, *p 0.05, n = 6).

The perivascular astrocytic foot processes (APs) in the cerebral cortex and striatum were relatively thin and flat in the sham group, and the perivascular astrocytic APs were enlarged on the ischemic side of the CIRI-24 h group (Fig. 4, n = 6).

Fig. 4.

TEM images of the sham and CIRI-24 h groups. Enlarged areas indicates an AP. Scale bar = 5 µm in 2000$\times$ figures with rectangular markers, 2 µm in 5000$\times$ figures with no rectangular markers. AP, astrocytic foot process; TEM, transmission electron microscopy.

IF double-label staining showed that the number of GFAP${}^{+}$/NKCC1${}^{+}$ (Fig. 5A) and GFAP${}^{+}$/AQP4${}^{+}$ (Fig. 5B) double-positive cells in the cortex and striatum of the ischemic side in the CIRI groups were significantly different compared with those in the sham group (Fig. 5C–E *p 0.05, n = 6). Moreover, the number of these two double-positive cells in the ischemic side of the cortex in the CIRI-2 h group was significantly higher than that in the sham group (Fig. 5C,D, *p 0.05, n = 6). However, the cell number in the striatum was not significantly different to the sham group. The expression of these two cells on the ischemic side of the cortex in the CIRI group increased at 2 h after reperfusion when compared with the sham group. However, in the striatum, this increased at 6 h after reperfusion. The double-positive cell number peaked at 24 h after reperfusion in the ischemic side of the cortex and at 12 h after reperfusion in the ischemic side of the striatum. The number of double-positive cells was decreased at 72 h after reperfusion compared with 24 h after reperfusion in the ischemic side of the cortex, but were increased in the striatum (Fig. 5C,D, *p 0.05, n = 6)

Fig. 5.

IF staining of GFAP${}^{\mathrm{+}}$/ AQP4${}^{\mathrm{+}}$and GFAP${}^{\mathrm{+}}$/NKCC1${}^{\mathrm{+}}$ in the sham and CIRI groups. (A) GFAP${}^{+}$/AQP4${}^{+}$ IF images of the sham and CIRI groups. (B) GFAP${}^{+}$/NKCC1${}^{+}$ IF images of the sham and CIRI groups. (C) Analysis of the number of GFAP${}^{+}$/AQP4${}^{+}$ cells. (D) Analysis of the number of GFAP${}^{+}$/NKCC1${}^{+}$ cells. (E) Schematic diagram of the ROIs of the cortex and striatum. Data are presented as mean $\pm$ standard deviation.

According to western blot (WB) measurements, the expression of AQP4 and NKCC1 protein in the ischemic cortex (Fig. 6A) and striatum (Fig. 6B) was significantly higher in the CIRI-24 h groups than in the sham group (Fig. 6C,D, *p 0.05, n = 3).

Fig. 6.

WB analysis results of AQP4 and NKCC1 of the sham and CIRI-24 h groups. (A) WB images of AQP4 and NKCC1 in the ischemic cortex of the sham and CIRI-24 h groups. (B) WB images of AQP4 and NKCC1 in the ischemic striatum of the sham and CIRI-24 h groups. (C) Comparison of AQP4 protein expression in the sham and CIRI-24 h groups. (D) Comparison of NKCC1 protein expression in the sham and CIRI-24 h groups. Data are presented as mean $\pm$ standard deviation. *p 0.05 vs. sham group.

Pearson’s correlation analysis and general linear regression showed that at each time point after reperfusion, the number of GFAP${}^{+}$/AQP4${}^{+}$ (Fig. 7A) and GFAP${}^{+}$/NKCC1${}^{+}$ (Fig. 7C) cells on the ischemic side of the cerebral cortex showed a statistically significant negative correlation with rADC. The number of GFAP${}^{+}$/AQP4${}^{+}$ (Fig. 7B) and GFAP${}^{+}$/NKCC1${}^{+}$ (Fig. 7D) cells on the ischemic side of the striatum showed a moderate negative correlation with rADC (Fig. 7, all p 0.01, n = 6).

Fig. 7.

Correlation analysis of the number of GFAP${}^{\mathrm{+}}$/AQP4${}^{\mathrm{+}}$ and GFAP${}^{\mathrm{+}}$/NKCC1${}^{\mathrm{+}}$ cells with rADC value in the sham and CIRI groups at each time point after reperfusion. (A) Correlation analysis of rADC and the number of GFAP${}^{+}$/AQP4${}^{+}$ cells in the cortex. (B) Correlation analysis of rADC and the number of GFAP${}^{+}$/NKCC1${}^{+}$ cells in the cortex. (C) Correlation analysis of rADC and the number of GFAP${}^{+}$/AQP4${}^{+}$ cells in the striatum. (D) Correlation analysis of rADC and the number of GFAP${}^{+}$/NKCC1${}^{+}$ cells in the striatum. Blue and orange colors indicate cortex and striatum data, respectively. AQP4, aquaporin-4; GFAP, glial fibrillary acidic protein; NKCC1, sodium-potassium-chloride cotransport protein 1; rADC, relative apparent diffusion coefficient.