Male Sprague-Dawley rats weighing 280–330 g were procured from the Animal Experimental Center of Shaanxi Provincial People’s Hospital. Animals were maintained in a controlled environment (22 °C, 12 h light/dark cycle) with ad libitum access to food and water. Experimental procedures complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and adhered to the ARRIVE reporting guidelines [15]. The protocol was reviewed and approved by the Laboratory Animal Ethics Committee of Shaanxi Provincial People’s Hospital (Xi’an, China; Approval No. 2021-083). Animals were randomly grouped indiscriminately by means of a table of random numbers. To minimize potential bias, investigators responsible for neurobehavioral assessments, image acquisition and quantification, as well as Western blot analysis were blinded to the group allocation throughout the experiments. Blinding was maintained until completion of data analysis.

A total of 168 male Sprague–Dawley rats were used in this study. Among them, 7 rats died after SAH induction and 11 rats were excluded because of very low SAH grades. Therefore, data from 150 rats were finally included in the analysis and presentation. For all experiments shown in the figures, the sample size was n = 6 animals per group. In the first part of the experiment, rats were randomly assigned to three groups: Sham, SAH, and SAH + WBV. For each group, 6 rats were used for brain water content measurement, 6 rats for immunofluorescence analysis at 24 h, 6 rats for immunofluorescence analysis at 72 h, 6 rats for inflammatory cytokine detection, and 6 rats for neurological function assessment. In the second part of the experiment, rats were divided into four groups: Control, WBV, WBV + 3-TYP, and 3-TYP, with 6 rats per group used for Western blot analysis. In the third part of the experiment, rats were divided into two groups: SAH + WBV and SAH + WBV + 3-TYP. For each group, 6 rats were used for brain water content measurement, 6 rats for immunofluorescence analysis, and 6 rats for inflammatory cytokine detection.

WBV stimulation was administered through a vibration apparatus manufactured by Deca Precision Measuring Instruments (Shenzhen, China). Animals were placed individually on the vibration platform and applied with sinusoidal vibration at 30 Hz, administered twice daily (30 min per session) for a total duration of 30 consecutive days according to a previous published paper [16]. During WBV exposure, animals were monitored to ensure they remained in a natural standing position and to minimize stress or fatigue. The in vivo SAH model was established using a modified endovascular perforation technique, as previously reported [17]. Briefly, animals were anesthetized with an intraperitoneal injection of pentobarbital sodium solution (10 mg/mL) at a dose of 40–50 mg/kg and maintained at a stable depth of anesthesia throughout the procedure. Under appropriate anesthesia and aseptic conditions, a sharpened monofilament was advanced through the internal carotid artery until resistance was encountered, after which the vessel wall was gently perforated to induce SAH. Successful induction of SAH was confirmed by sudden loss of resistance during perforation and subsequent observation of subarachnoid bleeding. Animals in the sham group underwent identical surgical procedures, but without vessel perforation. To inhibit Sirt3 activation in vivo, animals were treated intraperitoneally with 3-TYP, a selective Sirt3 inhibitor (50 mg/kg body weight, suspended in 1% DMSO), once daily for 3 consecutive days prior to SAH induction. This dosing regimen was chosen based on previous studies demonstrating effective inhibition of Sirt3 activity in vivo. The pretreatment paradigm was designed to ensure sufficient suppression of Sirt3 signaling before SAH induction and subsequent WBV intervention. SAH severity was evaluated using a previously established SAH grading system at 24 h after model induction. Briefly, the basal surface of the brain was divided into six segments, and each segment was scored from 0 to 3 according to the amount of subarachnoid blood clot: 0, no blood; 1, minimal blood; 2, moderate blood clot with recognizable arteries; and 3, blood clot covering all arteries within the segment. The total SAH grade was calculated as the sum of the six segment scores, ranging from 0 to 18. Animals with very low SAH grades were considered to have unsuccessful modeling and were excluded from subsequent analyses. SAH grading was performed by an investigator blinded to group allocation.

The selection of experimental time points was based on the temporal characteristics of early brain injury (EBI) following SAH. The 24 h time point was chosen to evaluate acute pathological changes, including brain edema, oxidative stress, neuroinflammation, neuronal apoptosis, and Sirt3-related molecular alterations, as these processes are known to peak during the early phase after SAH. Neurological function was assessed at 48 h post-SAH, a time point at which sensorimotor deficits can be reliably and stably detected. In addition, 72 h was selected for the evaluation of glial activation (Iba-1 and GFAP), as neuroinflammatory responses and glial reactivity may persist or evolve beyond the acute phase. This time-course design allowed us to capture both early injury and subsequent neuroinflammatory changes following SAH.

Brain edema was assessed 24 h after SAH induction using the standard wet–dry weight method, as previously reported [18]. Briefly, rats were deeply anesthetized (Isoflurane, 3–5% in oxygen for induction and 1–2% in oxygen for maintenance) and decapitated, and the brains were rapidly removed and separated into the hemispheres, cerebellum, and brainstem on an ice-cold plate. Each tissue sample was immediately weighed to determine the wet weight (WW). The samples were subsequently dried in a thermostatic oven at 100 °C for 24 h until a constant weight was reached, which was recorded as the dry weight (DW). Brain water content in each region was calculated using the following equation: [(WW – DW) / WW] $\times$ 100%.

Brain tissues embedded in paraffin were sectioned at 4 µm thickness and placed on glass slides. Sections were permeabilized in 0.1% Triton X-100 prepared in PBS for 10 min to facilitate antigen retrieval, and then incubated with 5% bovine serum albumin (BSA) at room temperature for 1 h to minimize nonspecific interactions. Subsequently, sections were incubated overnight at 4 °C with the following primary antibodies: anti-8-OHdG (ab48508, 1:500, Abcam, Shanghai, China), anti-Iba-1 (No.17198, 1:100, Cell Signaling Technology, Shanghai, China), anti-GFAP (No.80788, 1:100, Cell Signaling Technology), and anti-Sirt3 (No.2627, 1:200, Cell Signaling Technology). After three washes with PBS containing 0.05% Tween-20 (PBST, 5 min each), sections were incubated with the appropriate fluorophore-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse IgG, 1:500; Invitrogen, Carlsbad, CA, USA) at 37 °C for 1 h in the dark. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, Roche, Penzberg, Germany) to evaluate neuronal apoptosis. Following this, the nuclei were stained with 4^′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL) for 10 min as a counterstain, and immunofluorescence images were obtained using a Leica SP5 II laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). All imaging parameters (laser intensity, gain, exposure time) were kept constant across experimental groups to allow reliable comparisons. Quantitative analysis of immunofluorescence staining was performed using ImageJ software (1.54j, NIH, Bethesda, MD, USA). For each animal, three coronal brain sections at comparable anatomical levels were selected, and at least three non-overlapping fields within the cortical region were randomly captured per section. The region of interest (ROI) was consistently defined within the ipsilateral cortex to ensure comparability across samples. Fluorescence intensity or the number of positive cells was measured and averaged for each section, and subsequently averaged across sections for each animal. All image acquisition parameters were kept constant across groups. Image analysis was performed by investigators blinded to group allocation using coded images, and the results from each animal were used for statistical analysis.

The concentrations of pro-inflammatory cytokines, including tumor necrosis factor-$\alpha$ (TNF-$\alpha$, ab46087, abcam), interleukin-1$\beta$ (IL-1$\beta$, ab255730, abcam), and interleukin-6 (IL-6, ab234570, abcam), were quantified using commercially available ELISA kits (Anoric-Bio, Tianjin, China), in accordance with the manufacturer’s instructions. Standard curves established with recombinant cytokines were used to determine cytokine levels, which were expressed as pg/mg or pg/mL of protein. All measurements were performed in duplicate to ensure reproducibility, and negative controls were included to exclude nonspecific binding.

Forty-eight hours after SAH, neurological performance was evaluated by the modified Garcia scale (3–18 points) and the beam balance test (1–6 points). The higher score of the modified Garcia scale suggested better sensorimotor function, whereas the lower score of beam balance test indicated better complex movements and coordination. Neurobehavioral assessments were performed by two independent investigators who were blinded to the experimental groups. Each animal was evaluated twice, and the average score was used for statistical analysis to reduce subjective bias.

Protein expression was analyzed by Western blot following established methods. In brief, protein samples (30–50 µg per lane) were mixed with loading buffer and heated at 95 °C for 5 min to ensure complete denaturation. The proteins were then resolved using SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 1 h at room temperature with 5% non-fat milk diluted in Tris-buffered saline containing 0.1% Tween-20 (TBST). After blocking, membranes were incubated overnight at 4 °C with primary antibodies against Sirt3 (1:1000, #2627, Cell Signaling Technology) and GAPDH (1:1000, #5174, Cell Signaling Technology). Following three washes with TBST, HRP-linked secondary antibodies (1:5000; #8889, Cell Signaling Technology) were applied for 1 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) and visualized with a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). For Western blot analysis, band intensities were quantified using ImageJ software by an investigator blinded to the experimental groups. Each sample was analyzed in at least three independent experiments. The relative expression levels of target proteins were normalized to GAPDH and expressed as fold change compared to the control group.

Mitochondrial superoxide production in the cortical region was evaluated using MitoSOX Red staining. Briefly, brain tissues were collected at 24 h after SAH induction, and coronal brain sections at comparable anatomical levels were prepared. After washing with PBS, sections were incubated with MitoSOX Red mitochondrial superoxide indicator working solution (5 µM, Thermo Fisher Scientific, Cat. No. M36008) in the dark at 37 °C for 30 min. Following incubation, the sections were washed three times with PBS to remove excess dye, and nuclei were counterstained with DAPI. Fluorescence images were acquired using a Leica SP5 II laser scanning confocal microscope under identical imaging parameters across all experimental groups.

Statistical analysis was carried out with SPSS software (version 16.0; IBM Corp., Armonk, NY, USA). All results are expressed as the mean $\pm$ standard deviation (SD). The normality of data distribution was first evaluated using the Shapiro–Wilk test, while equality of variances among groups was examined by Levene’s test. Differences between two groups were analyzed using Student’s t-test, with a one-tailed test applied for ratio quantification and a two-tailed test used for the remaining analyses. When more than two groups were compared, statistical differences were assessed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Statistical significance was defined as a p value less than 0.05.

As shown in Fig. 1A, the animals were individually fixed on the vibration table, and a vibration apparatus produced by Deca Precision Measuring Instruments (Shenzhen, China) was used to apply WBV. As shown in Fig. 1B, animals received WBV stimulation at 30 Hz for 30 min per session, twice per day, for 30 days, and were then exposed to SAH. Measurements were collected at the specified time intervals.

Fig. 1.

Equipment and experimental design. (A) The animals were individually fixed on the vibration table, and the vibration parameters were shown in the vibration controller. (B) Schematic illustration of the experimental protocol, including treatments and the timing of different measurements. SAH, subarachnoid hemorrhage.

The influence of WBV on cerebral edema was investigated by measuring brain water content 24 h after SAH induction (Fig. 2A). SAH markedly increased brain water content in the cerebrum, whereas no significant changes were observed in the cerebellum or brainstem. To further assess oxidative DNA damage, immunostaining for 8-OHdG, a well-established biomarker of oxidative nucleic acid modification, was performed (Fig. 2B). SAH resulted in a pronounced increase in 8-OHdG immunoreactivity, which was significantly reduced in animals pretreated with WBV (Fig. 2C). In addition, mitochondrial oxidative stress was evaluated by MitoSOX staining (Fig. 2D). Consistent with the above findings, SAH markedly elevated mitochondrial superoxide levels, while WBV pretreatment significantly suppressed this response (Fig. 2E).

Fig. 2.

WBV reduces neuronal injury following SAH. (A) Evaluation of brain water content in different brain regions, including the cerebrum, cerebellum, and brainstem, at 24 h. (B,C) Representative images of 8-OHdG immunostaining in cortex (B) and quantitative analysis (C). (D,E) Typical images of MitoSOX immunostaining in the cortical region (D) and the related quantification (E). Scale bar = 50 µm. n = 6 rats per group. Data are shown as mean $\pm$ SD. ^#p 0.05 vs. Sham group. ^*p 0.05 vs. SAH group. WBV, whole body vibration.

To assess sensorimotor status, the modified Garcia scale was applied, covering measures of hemiplegia, impaired motor performance, and abnormal postural behavior (Fig. 3A). SAH induction resulted in a significant reduction in Garcia scores, an effect that was effectively abolished by WBV pretreatment. In addition, motor coordination and complex movements were assessed by the beam balance test (Fig. 3B). SAH markedly increased beam balance scores, indicating impaired coordination, and this effect was partially ameliorated by WBV at 48 h post-SAH.

Fig. 3.

WBV improves neurological function following SAH. (A) Modified Garcia scale showing sensorimotor function at 48 h. (B) Beam balance test showing complex movements and coordination at 48 h. Data are shown as mean $\pm$ SD. n = 6 rats per group. Three repeated measurements from each animal were displayed, resulting in 18 plotted data points per group. ^#p 0.05 vs. Sham group. ^*p 0.05 vs. SAH group.

Microglial activation in the cortex was evaluated by Iba-1 immunohistochemistry at two time points after SAH, namely 24 h (Fig. 4A) and 72 h (Fig. 4C). Quantitative analysis revealed that SAH markedly increased Iba-1 expression at both time points (Fig. 4B,D). Notably, WBV pretreatment partially attenuated these SAH-induced increases.

Fig. 4.

WBV treatment decreases cortical Iba-1 expression after SAH. (A,B) Typical images of cortical Iba-1 staining (A) and the related quantitative evaluation (B) at 24 h. (C,D) Representative staining of Iba-1 in the cortex (C) with quantitative analysis (D) at 72 h. Scale bar = 50 µm. n = 6 rats per group. Data are shown as mean $\pm$ SD. ^#p 0.05 vs. Sham group. ^*p 0.05 vs. SAH group.

Cortical astrocyte activation was assessed using GFAP immunostaining at two time points after SAH, namely 24 h (Fig. 5A) and 72 h (Fig. 5C). At 24 h, WBV intervention significantly reduced GFAP expression compared with the SAH group (Fig. 5B). In contrast, GFAP levels at 72 h showed no statistically significant difference between the SAH and SAH + WBV groups (Fig. 5D).

Fig. 5.

WBV reduces the expression of GFAP following SAH. (A,B) Typical images showing GFAP staining in the cortex (A) together with the related quantification (B) at 24 h. (C,D) Cortical GFAP staining images (C) and their quantitative evaluation (D) at 72 h. Scale bar = 50 µm. n = 6 rats per group. Data are shown as mean $\pm$ SD. ^#p 0.05 vs. Sham group. ^*p 0.05 vs. SAH group. DAPI, 4^′,6-diamidino-2-phenylindole; GFAP, Glial fibrillary acidic protein.

Brain homogenates were collected at 24 h following SAH, and SAH markedly increased the levels of TNF-$\alpha$, IL-1$\beta$ and IL-6, which were reduced by WBV (Fig. 6A). We also detected these cytokines in serum, and we found that the SAH-induced increases in these cytokines were attenuated by WBV, although the expression of IL-6 was not statistically decreased (p = 0.13, Fig. 6B).

Fig. 6.

Effects of WBV on inflammatory cytokine responses after SAH. (A) Quantification of inflammatory cytokines in brain tissue at 24 h assessed by ELISA. (B) ELISA-based quantification of serum inflammatory cytokines at 24 h. n = 6 rats per group. Data are shown as mean $\pm$ SD. ^#p 0.05 vs. Sham group. ^*p 0.05 vs. SAH group.

The neuronal apoptosis in the cortex was measured by TUNEL staining (Fig. 7A). Quantitative analysis demonstrated that SAH markedly increased the number of TUNEL-positive cells, and this effect was markedly attenuated by WBV pretreatment (Fig. 7B). Consistently, WBV also reduced the SAH-induced elevation of caspase-1 activity, an established marker of inflammatory apoptosis (Fig. 7C).

Fig. 7.

WBV decreases neuronal death following SAH. (A,B) TUNEL staining in the cortex is presented in representative images (A), with quantitative results shown accordingly (B) at 24 h. (C) Relative caspase-1 activity at 24 h. Scale bar = 100 µm. Data are shown as mean $\pm$ SD. n = 6 rats per group. For Fig. 7B, three repeated measurements from each animal were displayed, resulting in 18 plotted data points per group. ^#p 0.05 vs. Sham group. ^*p 0.05 vs. SAH group.

To better understand the molecular basis of the protective effects associated with WBV, immunohistochemical staining was performed to evaluate Sirt3 expression (Fig. 8A). SAH significantly reduced the Sirt3 protein levels, which was apparently increased by WBV (Fig. 8B). 3-TYP was used to inhibit Sirt3 activity, and we found that WBV-induced Sirt3 expression was reduced by 3-TYP (Fig. 8C). After 3-TYP treatment, the WBV-induced attenuation of 8-OHdG expression following SAH was prevented (Fig. 8D,E). As shown in Fig. 8F,G, a similar result in MitoSOX levels was also observed. In addition, the results of brain water content showed that WBV-induced alleviation of brain water content was significantly reversed by Sirt3 inhibition (Fig. 8H).

Fig. 8.

WBV protects against SAH via activating Sirt3. (A,B) Cortical Sirt3 immunostaining is illustrated in representative images (A), with quantitative evaluation shown in (B). (C) Detection of Sirt3 protein levels by Western blotting. (D,E) Representative cortical images of 8-OHdG staining (D) and their quantitative assessment (E). (F,G) MitoSOX staining in the cortex is presented in representative images (F), with quantitative results shown in (G). (H) Brain edema measurement in cerebrum at 24 h. Scale bar = 50 µm. n = 6 rats per group. Data are shown as mean $\pm$ SD. ^#p 0.05 vs. Sham group. ^*p 0.05 vs. SAH group. ^&p 0.05 vs. WBV group. ^$p 0.05 vs. SAH+WBV group.

Next, we further assayed the role of Sirt3 in WBV-induced effects on neuroinflammation. The results of Iba-1 immunostaining showed that WBV-induced inhibition of Iba-1 expression was ablated by 3-TYP (Fig. 9A,B). 3-TYP treatment also increased GFAP expression in cortex following SAH as compared to that in SAH + WBV group (Fig. 9C,D). We repeated the measurements of ELISA in brain tissues (Fig. 9E) and in serum (Fig. 9F), and the results showed that WBV-induced decreases in inflammatory cytokines expression were nullified by Sirt3 inhibition. As shown in Fig. 9G, a similar result in caspase-1 activity was also observed.

Fig. 9.

WBV regulates neuroinflammation via activating Sirt3. (A,B) Cortical Iba-1 immunostaining is presented in representative images (A), with quantitative results shown in (B). (C,D) Representative images illustrating GFAP staining in the cortex (C) and the corresponding quantification (D). (E,F) ELISA results of inflammatory cytokines levels in brain tissues (E) and in serum (F). (G) Relative caspase-1 activity at 24 h. Scale bar = 50 µm. n = 6 rats per group. Data are shown as mean $\pm$ SD. ^$p 0.05 vs. SAH+WBV group.