Specific pathogen free (SPF) healthy male Sprague-Dawley (SD) rats (n = 48) with a body weight of 200 $\pm$ 20 g were purchased from the Qinglongshan Animal Experimental Center [SCXK, (Hangzhou, Zhejiang, China), 2019-0002]. Animals were placed in an experimental animal room of the Wannan Medical College and maintained at 24 $\pm$ 2 °C with a light/dark cycle of 12 h/12 h. Free feeding and drinking were allowed, and adaptive feeding was undertaken for one week. Following that, 12 rats were randomly assigned to each of four groups: normal, sham, MCAO, and MCAO+APS. The Laboratory Animal Welfare and Ethics Committee of Wannan Medical College evaluated and approved these animal studies (WNMC-AME-2023134).

The MCAO model was established using the suture-occluded method [10]. Intraperitoneal injection with 1.5% pentobarbital sodium (2 mL/kg, AM00469, Beijing Chemical Reagent Company, Beijing, China) was first used to anesthetize rats. They were then fixed in a supine position on the operating table under a shadowless lamp and the neck skin was disinfected with iodophor. The layers of tissue were separated to expose the right common carotid artery (CCA) and a glass minute hand was used to carefully separate the vagus nerve and CCA. The separation was continued to the bifurcation of the internal carotid artery (ICA) and external carotid artery (ECA), the CCA and distal ECA were ligated, and a spare wire was set up at the proximal end. Ophthalmic scissors were used to make a tiny incision in the ECA so that a threaded plug could be inserted (A2-2432, Beijing Cinonech Co. Ltd, Beijing, China). The ECA was then cut off and the thread was looped into the ICA through the stump of the ECA. The insertion depth was 18.5 $\pm$ 0.5 mm until slight resistance was noted. The tip of the cord was passed through the beginning of the middle cerebral artery and the cord ligated. The incision was then sutured to avoid infection during and after the operation. In the sham group, the blood vessels were separated without being ligated. The room temperature was kept at 22 °C during the operation to allow animals to recover from anesthesia under temperature-controlled conditions. After they awoke from surgery (usually about 2 hours), neurological impairment was evaluated according to the Bederson scoring criteria [11]. Rats with scores of 1–3 were included in the study. Following successful establishment of the model, the MCAO+APS group was given an intraperitoneal injection of 50 mg/kg APS (purity $>$90%, A7970, Solarbio, Beijing, China) once a day for 14 consecutive days, as suggested in the literature [12]. The study designs are displayed in Fig. 1A.

Fig. 1.

Experimental design and behavioral tests. (A) Schematic of the study design. (B) Schematic diagram of statistical area for counting positive cells by Nissl and immunohistochemical staining. (C) Bederson nerve function score; n = 12 per group; ***p 0.001, compared with the sham group; ${}^{\mathrm{\#}}$p 0.05, compared with the MCAO group. (D) Forelimb placement test; n = 12 per group; ***p 0.001, compared with the sham group; ${}^{\mathrm{\#}}$p 0.05, compared with the MCAO group. MACO, middle cerebral artery occlusion; APS, Astragalus polysaccharide.

Neurological impairment in rats was assessed 2 hours after surgery and on days 1, 3, 7, and 14. Neurological function was classified into four levels: 0, no neurological impairment; 1, the forepaw on the opposite side of the tail suspension test could not be fully extended; 2, the resistance of the opposite front paw to thrust decreased; 3, turned to the opposite side after placing.

The forelimb placement test was used to assess the comprehensive ability of visual and proprioceptive perception of rats on days 3, 7, and 14. A square grid (3 cm $\times$ 3 cm) was placed horizontally at a height of 1 meter above ground. On the frame, rats were gently pushed from behind to encourage them to crawl from one side of the grid surface to the other within 2 minutes. During the crawling process, one point was awarded if the forelimbs fell into the grid. The number of times the hemiplegic forelimbs fell into the net during the 2-minute crawl was recorded.

Following treatment, 6 rats in each group were injected intraperitoneally with 1.5% sodium pentobarbital (3 mL/kg) and 4% paraformaldehyde was then infused before brain extraction. Brain tissue was fixed in 4% paraformaldehyde. After dehydration with a sucrose gradient, the tissue was embedded using optimal cutting temperature compound (OTC) in a –20 °C frozen microtome (HM525NX, Thermo Fisher Science, Waltham, MA, USA). It was then cut into 20 µm slices and washed three times with phosphate buffered saline (PBS) for 5 min each before use. Filtered Nissl staining solution was then added dropwise to the tissue slices containing the VPN, and these were placed in a 37 °C incubator for 30 minutes. Excess staining agent was washed off with double distilled water and the sections were dehydrated, made transparent and sealed. Images were observed and recorded at 400$\times$ magnification. The morphology and quantity of Nissl-positive neurons in each visual field was examined using ImageJ software (1.41r, National Institutes of Health, Bethesda, MD, USA).

Frozen sections of brain tissue containing VPN were baked in an incubator in a 60 °C incubator for 2 hours. H${}_2$O${}_2$ (3%) was added to remove endogenous peroxidase and the slices were incubated for 10 minutes at 37 °C. To repair the antigen, the slices were boiled in 10 mM sodium citrate buffer (pH 6.0) for 2 minutes at high temperature and for 14 minutes at low temperature. Non-specific antibody binding was first blocked by incubating with 5% goat serum (AR0004, Boster Biotechnology, Wuhan, Hubei, China) for 30 min. Incubation with rabbit anti-Notch1 (1:200, 10062-AP, Proteintech, Wuhan, Hubei, China), rabbit anti-Hes1 (1:200, DF7569, Affinity Biosciences, Cincinnati, OH, USA), and rabbit anti-p-NF$\kappa$B p65 (1:200, AF2006, Affinity Biosciences, Cincinnati, OH, USA) was conducted at 4 °C. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and 3,3${}^{\prime }$-diaminobenzene (PA 110, TIANGEN Biotech, Beijing, China) were then used for staining and color development. The slices were permeated with xylene and sealed with neutral gum. ImageJ software was used to examine the expression of Notch1, Hes1, and phospho-nuclear factor-$\kappa$B p65 (p-NF$\kappa$B p65) proteins and to obtain a positive cell count.

The remaining 6 rats from each group were anaesthetized by intraperitoneal injection with 1.5% pentobarbital sodium. After decapitation, the brain was removed and placed on an ice box. According to a Stereotaxic map of rat brain described by Paxinos et al. [13], a surgical blade was used on an ice box to cut 2.16 and 3.84 mm behind the anterior fontanel of the brain tissue to collect contains VPN area of the brain tissue, and then the VPN was extracted with microscopic tweezers as described in Fig. 1B. Proteins from the VPN were extracted using radio immunoprecipitation assay (RIPA) lysis buffer, phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitors. The sample protein (40 ug) was run for two hours on an sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis according to the molecular weight of the target protein. Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane using a continuous flow of 300 mA for 1.5 hours. The membranes were blocked for two hours before incubation overnight at 4 °C with primary antibodies against Notch1, Hes1, p-NF$\kappa$B p65, NF$\kappa$B p65 (1:1000, AF5006, Affinity Biosciences, Cincinnati, OH, USA), and GAPDH (1:1000, AF7021, Affinity Biosciences, Cincinnati, OH, USA). Following this, the membranes were incubated with goat anti-rabbit IgG (H+L) secondary antibodies (1:1000, A0208, Beyotime Biotechnology, Shanghai, China) for 1 hour at room temperature, washed, then analyzed with an ECL kit (P0018FS, Beyotime Biotechnology, Shanghai, China). GAPDH was used as the internal reference for Notch1 and Hes1, and NF$\kappa$B p65 was used as the reference for p-NF$\kappa$B p65. The strip gray values were quantified by ImageJ software.

SPSS 26.0 software (IBM Corp., Chicago, IL, USA) was used for statistical analysis of the data, and GraphPadPrism 9.0 (Dotmatics, Boston, MA, USA) for the creation of images. The measurement data were evaluated by a normality test and by a homogeneity test of variance. Data with a normal distribution were reported as the mean $\pm$ standard deviation. The K-W test was used to compare non-normally distributed data, with p 0.05 used as the threshold for statistical significance.

The Bederson nerve function score revealed that rats in the normal group and in the sham operation group had no neurological function defects (Fig. 1C). In contrast, the MCAO group had severe neurological damage and considerably greater neurological impairment scores than those of the sham group at 2 hours after cerebral ischemia and at days 1, 3, 7, and 14 (p 0.001). The neurological function scores of the MCAO+APS group were lower than those of the MCAO group on days 7 and 14 (p 0.05).

The forelimb placement test showed that rats in the normal and sham operation groups would grasp the grid line tightly when crawling, and almost never fall into the grid (Fig. 1D). Rats in the MCAO group showed a significantly higher number of hemiplegic forelimb falls into the grid on days 3, 7 and 14 compared to the sham group (p 0.001). Rats in the MCAO+APS group showed significantly fewer hemiplegic forelimb falls into the grid on days 7 and 14 than those in the MCAO group (p 0.05).

Nissl staining revealed that neurons in the VPN of rats in the normal and sham groups showed a complete morphology, structure, and quantity, with clear outline, centered nuclei, and complete Nissl bodies (Fig. 2A,B). However, in the MCAO group, the volume of neurons in the affected side of the thalamus was smaller, the edge of the cell membrane was blurred, and both necrolysis and nuclear pyknosis were observed. Furthermore, the number of Nissl-positive neurons was significantly lower in the MCAO group than in the sham group (p 0.001). The morphology of neurons in the MCAO+APS group was mostly normal, and the number of Nissl-positive neurons was higher than in the MCAO group (p 0.01).

Fig. 2.

Effect of APS on histopathological changes to ventroposterior thalamic nucleus (VPN). (A) Representative Nissl staining images of each group. (400$\times$, 50 um scale) Red arrows show Nissl-positive neurons and yellow arrows show damaged neurons; n = 6 per group. (B) Statistical analysis of the number of Nissl-positive neurons in each group. ***p 0.001, compared with the sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, compared with the MCAO group.

Immunohistochemistry was used to investigate the expression of Notch1, Hes1, and p-NF$\kappa$B p65 in the right VPN of rats in each group (Fig. 3). Scattered positive cells showed a brown-yellow color. There were no discernible differences in expression between the normal and sham groups (p $>$ 0.05). The MCAO group showed a higher number of positive cells for Notch1, Hes1, and p-NF$\kappa$B p65 than the sham group (p 0.001). However, the MCAO+APS group showed decreased expression of Notch1, Hes1, and p-NF$\kappa$B p65 compared to the MCAO group (p 0.05, p 0.001, and p 0.01, respectively).

Fig. 3.

Immunohistochemistry was used to investigate the effect of APS on the expression of Notch1, Hes1, and p-NF$\kappa$B p65 in the VPN of rats with cerebral ischemia. (A) Representative immunohistochemical staining of Notch1, hairy and Hes1, and p-NF$\kappa$B p65 of rats in each group (400$\times$, 50 um scale); Red arrows show positive neurons. (B) The average positive cell count for Notch1 expression in the MCAO+APS group. ***p 0.001, compared with the sham group; ${}^{\mathrm{\#}}$p 0.05, compared with the MCAO group. (C) The average positive cell count for Hes1 expression in the MCAO+APS group; ***p 0.001, compared with the sham group; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001, compared with the MCAO group. (D) Average positive cell count for p-NF$\kappa$B p65 expression in the MCAO+APS group; ***p 0.001, compared with the sham group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, compared with the MCAO group; n = 6 per group. Notch1, Neurogenic locus notch homolog protein 1; p-NF$\kappa$B p65, phospho-nuclear factor-$\kappa$B p65; Hes1, hairy and enhancer of split 1.

The results obtained with Western blot analysis mirrored those from immunohistochemistry (Fig. 4). The relative expression levels of Notch1 and Hes1 in the VPN were significantly higher in the MCAO group than in the sham group (p 0.01), as was the expression of p-NF$\kappa$B p65/NF$\kappa$B p65 (p 0.05). However, Notch1 and Hes1 expression in the VPN from the MCAO+APS group was significantly lower than that of the MCAO group (p 0.01), while the expression of p-NF$\kappa$B p65/NF$\kappa$B p65 was also lower (p 0.05).

Fig. 4.

Western blotting was used to determine the effect of APS on the expression of Notch1, Hes1, and p-NF$\kappa$B p65 proteins. (A) Representative bands for Notch1, Hes1, p-NF$\kappa$B p65, nuclear factor-$\kappa$B p65 (NF$\kappa$B p65), and GAPDH protein. (B) Quantitative results for Notch1, (C) Hes1, and (D) p-NF$\kappa$B p65 protein; **p 0.01, ***p 0.001 compared with the sham group; ${}^{\mathrm{\#}}$p 0.05, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, compared with the MCAO group; n = 6 per group.