All animal procedures were approved by the Ethics Committee of Zhengzhou University (KY2024064) and were conducted following the National Institutes of Health’s “Guide for the Care and Use of Laboratory Animals”. Adult male C57BL/6J mice (18–22 g) were used in this study (Hua FuKang Experimental Animal Company, Beijing, China). Mice were housed under controlled conditions. A total of 164 operated mice were randomly allocated to the following groups:

Part I: Sixty-five male mice were allocated to five groups (n = 13), namely, (1) a sham group and (2) four ICH groups with assessments at different time points (1, 3, 7, and 14 days).

Part II: Ninety-nine male mice were assigned to three groups, namely, (1) a sham group, which received the vehicle consisting of 5% dimethyl sulfoide (DMSO) (D8370, Solarbio, Beijing, China), 30% PEG300 (HY-Y0873, MedChemExpress, Monmouth Junction, NJ, USA), 5% Tween 80 (HY-Y1891, MedChemExpress), and 60% ddH₂O, (2) an ICH + Vehicle group, which also received the vehicle, and (3) an ICH + IDE group, which received IDE (58186-27-9, TargetMol, Boston, MA, USA). Following surgery, the three groups were given vehicle or IDE (100 mg/kg) in a final volume of 100 µL via intraperitoneal injection for three consecutive days.

The ICH model was established based on a previously published protocol [23]. Mice were anesthetized with an intraperitoneal administration of a ketamine-xylazine solution, comprising ketamine (100 mg/kg, H20193336, Jiangsu Hengrui Pharmaceuticals Co., Ltd, Jiangsu, China) and xylazine (10 mg/kg, X0069, TCI Chemical Industry Development Co., Ltd, Shanghai, China). Following this, experimental mice were secured in a stereotaxic apparatus (ZH-BLUESTAR B/S, Anhui Zhenghua Biologic Apparatus Facilities Co., Ltd., Anhui, China) for the surgical procedure. Type VII collagenase (0.075 U; C0773, Sigma-Aldrich, Burlington, MA, USA) was injected into the right striatum using a Hamilton syringe at coordinates 2.0 mm lateral to the bregma and 3.5 mm ventral to the skull base. Experimental animals in the sham group were subjected to the identical surgical protocol without receiving collagenase. Postoperatively, mice were positioned on a warming pad to maintain body temperature until they fully recovered from the anesthesia.

Neurological deficits were assessed using the modified neurological severity score (mNSS) and the corner test on days 1 and 3 post-ICH, as noted in prior reports [24, 25]. The mNSS consists of five components: motor function, sensory function, balance, reflexes, and abnormal movements. Scores range from 0 (normal function) to 18 (maximal neurological impairment), with higher scores indicating greater deficits.

For the corner test, mice were placed between two angled walls (30°), allowing them to move forward and turn. The frequency of left and right turns was documented, and the score was calculated as follows:

$$\text{Corner test score}=\left(\frac{\text{number of right turns}}{\text{total trials}}\right)\times 100\%$$

Higher scores indicate more severe left hemiparesis. All behavioral assessments were conducted by investigators blinded to the group assignments.

The mice were euthanized on day 3 after ICH modeling with carbon dioxide (CO₂) (30% chamber volume displacement/min, followed by decapitation). Brain tissues were collected and dissected into ipsilateral and contralateral hemispheres together with the cerebellum, as previously reported [26]. The wet weight of the sample was measured using an electronic analytical balance, followed by drying at 100 °C for a period of 24 hours, to ascertain the dry weight. The cerebellum served as an internal control. The equation for BWC is shown below:

$$\text{BWC}=\frac{\text{wet weight}-\text{dry weight}}{\text{wet weight}}\times 100\%$$

Brain tissues were collected for protein extraction. Proteins were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (P0013C, Beyotime, Shanghai, China), and the supernatant after centrifugation was collected. Protein concentrations were then quantified using a bicinchoninic acid (BCA) assay (GK10009, GLPBIO, Montclair, CA, USA). Separation of proteins was achieved through Tris-glycine gradient gel electrophoresis, followed by transfer onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with 5% nonfat milk for 2 hours to block nonspecific binding, then incubated overnight with primary antibodies against IL-1$\beta$ (1:1000, ab283818, Abcam, Cambridge, MA, USA), TNF-$\alpha$ (1:1000, ab215188, Abcam), NQO1 (1:1000, R381695, Zen Bioscience, Chengdu, Sichuan, China), $\beta$-actin (1:5000, AP0060, Bioworld, Beijing, China), Keap1 (1:5000, 10503-2-AP, Proteintech, Wuhan, China), Nrf2 (1:5000, 16396-1-AP, Proteintech), matrix metalloproteinase-9 (MMP-9) (1:1000, 10375-2-AP, Proteintech) and zonula occludens-1 (ZO-1) (1:10,000, 21773-1-AP, Proteintech). After washing, the membranes were treated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (1:5000, SA00001-2, Proteintech) for 1 hour. Immunoblots were visualized using an enhanced chemiluminescence (ECL) detection kit (P0018AS, Beyotime, Shanghai, China) with a western blot imaging system (Thermo Fisher Scientific, Waltham, MA, USA). The band densitometry was quantified using ImageJ software (version 1.53; National Institutes of Health, Bethesda, MD, USA). The original images of the Western blot are available in the Supplementary Material.

Mice were euthanized with CO₂ on day 3 post-ICH, and brain tissues were homogenized and centrifuged to collect supernatants. Levels of TNF-$\alpha$ (EK282, MULTI SCIENCES, Zhejiang, China) and IL-10 (SEKM-0010, Solarbio) were quantified according to the manufacturer’s guidelines for ELISA kits.

Brain tissues were collected following cardiac perfusion and then paraffin embedded. The brain tissue that had been fixed in paraffin was then sectioned at a thickness of 5 µm. The sections were then deparaffinized and rehydrated, and then underwent antigen retrieval using citrate buffer. Following 30 minutes of blocking with immunostaining block buffer (P0102, Beyotime), the sections were treated overnight at 4 °C with anti-ionized calcium-binding adaptor molecule 1 (Iba-1) (1:200, 019-19741, Wako, Tokyo, Japan) and anti-myeloperoxidase (MPO) (1:200, ab208670, Abcam) primary antibodies. After rinsing with PBS, the sections were treated with the appropriate secondary antibodies (1:500, 4412, Cell Signaling Technology, Danvers, MA, USA) for 60 minutes in the dark. Following further PBS washes, the sections were mounted using an anti-fluorescence quenching mounting medium containing DAPI (S2110, Solarbio) and imaged using an Olympus microscope (Olympus Corporation, Tokyo, Japan), with positive cell counts were quantified by blinded observers using ImageJ software [27, 28].

The sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After cooling to room temperature, endogenous peroxidase activity was inhibited by treatment with 3% hydrogen peroxide, followed by blocking for 30 minutes. Primary antibodies were applied and incubated overnight at 4 °C. The following primary antibodies were used: Iba-1 (1:2000, ab178846, Abcam) and MPO (1:1000, ab208670, Abcam). Subsequently, HRP-labeled goat anti-rabbit/mouse secondary antibody (PV-8000, ZSBio, Beijing, China) was applied, and the samples were incubated for 20 minutes at ambient temperature. Finally, sections were stained with 3,3^′-Diaminobenzidine solution, and nuclei were counterstained with hematoxylin. Differentiation was performed using ethanol hydrochloride rapid differentiation solution, followed by dehydration and transparency with xylene and ethanol [29].

The BrightGreen apoptosis Detection kit (A112-02, Vazyme, Nanjing, Jiangsu, China) was utilized following the manufacturer’s guidelines. Overnight incubation was conducted with the primary antibody NeuN (1:1000, ab279295, Abcam). The sections were then incubated with the secondary antibody (1:500, 8890, Cell Signaling Technology) for one hour. Images were acquired utilizing an Olympus microscope.

The levels of MDA were measured to assess the degree of lipid peroxidation and oxidative stress. Brain tissues from the perihematomal region were collected three days post-ICH, and MDA levels were measured using a commercially available assay kit (KTB1050, Abbkine, Wuhan, Hubei, China). Absorbance values were determined with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Tissue sections underwent deparaffinization and were subsequently rehydrated using a graded series of ethanol (1030001-04-01, Paini Technology, Zhengzhou, Henan, China) and xylene (1046003-01-01, Paini Technology), followed by staining with H&E (C0105, Beyotime). Randomly selected fields within the hematoma region were evaluated and imaged under optical microscopy.

All experimental data are presented as mean $\pm$ standard deviation (SD). A single-blinded investigator performed the data analysis. Group comparisons were conducted using one-way ANOVA followed by Tukey’s post hoc test. Behavioral test data and BWC were analyzed using two-way ANOVA followed by Tukey’s post hoc test. A p-value 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA).

In order to evaluate alterations in oxidative stress, brain tissue specimens were harvested at 1, 3, 7, and 14 days following ICH. Compared to the sham group, Nrf2 expression in the ICH group increased from day 1, reaching its peak on day 3 (Fig. 1A, p 0.01). Similarly, MDA levels were elevated from day 1, with the highest levels observed on day 3 (Fig. 1B, p 0.01).

Fig. 1.

Time course expression levels of oxidative stress and neuroinflammation after ICH. (A) Western blot analysis of Nrf2 on days 1, 3, 7, and 14 after ICH, with quantification analysis (n = 3). (B) MDA content in the perihematomal tissues was measured using a specific detection kit (n = 6). (C) Immunohistochemical staining for Iba-1 and MPO was illustrated at various time points (scale bar = 50 µm). (D) The quantities of Iba-1+ and MPO+ cells are measured and illustrated in the respective bar graphs (n = 4). Values are expressed as the mean $\pm$ SD, with **p 0.01 for all graphs. ICH, intracerebral hemorrhage; Nrf2, nuclear factor erythroid 2-related factor 2; MDA, Malondialdehyde; Iba-1, ionized calcium-binding adaptor molecule 1; MPO, myeloperoxidase; SD, standard deviation.

To evaluate neuroinflammation following ICH, immunohistochemistry was performed to assess microglial activation and neutrophil infiltration in the perihematomal region (Fig. 1C). The results demonstrated that, in contrast to the sham group, both microglial activation and neutrophil recruitment peaked at day 3 post-ICH (Fig. 1C,D, p 0.01, p 0.01).

In sum, these findings indicated that oxidative stress and neuroinflammation reached their maximum levels three days after ICH. Therefore, day 3 post-ICH was selected for subsequent experiments to evaluate the neuroprotective effects of IDE in subsequent experiments.

The hematoma volumes were quantified using representative brain section images, revealing a significant reduction in hematoma volume in IDE-treated mice compared to those in the ICH + Vehicle group (Fig. 2A, p 0.01). Neurological function was assessed using the mNSS and the corner test before ICH induction and on days 1 and 3 post-ICH. All animals had mNSS scores of 0 before surgery. Following ICH, significant neurological deficits were observed. However, IDE-treated mice displayed significantly lower mNSS scores on days 1 and 3 post-ICH compared to the ICH + Vehicle group (Fig. 2B, p 0.01, p 0.01). Similarly, results from the corner test corroborated these findings, demonstrating a significant reduction in neurological impairment at both time points in the IDE-treated group (Fig. 2C, p 0.05, p 0.01). To further evaluate the neuroprotective effects of IDE, TUNEL/NeuN immunostaining was conducted to measure neuronal death. The proportion of TUNEL/NeuN-positive cells tended to decrease significantly after IDE treatment in comparison with the ICH + vehicle group, indicating that neuronal apoptosis was alleviated (Fig. 2D,E, p 0.01). H&E staining further demonstrated that IDE improved the brain tissue morphology (Fig. 2F). The sham group exhibited uniform staining, well-defined structures, and normal neuronal architecture. However, the ICH + Vehicle group showed neuronal loss, with disrupted architecture, cell fragmentation, and vacuolization. IDE-treated mice showed markedly improved neuronal morphology and structure, with increased cellular organization. These findings indicate that IDE mitigates brain damage and neurological impairments during the acute phase of ICH, supporting its potential as a neuroprotective agent.

Fig. 2.

Effects of IDE on ICH-induced brain injury and neurological deficits in mice. (A) Representative coronal sections of the brain tissue were prepared after formaldehyde fixation to evaluate and quantify hematoma volume (mm³) (scale bar = 5 mm, n = 5). (B,C) Neurological function was assessed using the mNSS test and corner test before ICH and on days 1 and 3 post-ICH (n = 6). (D) The number of TUNEL/NeuN-positive cells was quantified (n = 5). (E) Representative images of TUNEL staining are presented, with DAPI (blue), TUNEL-positive cells (green), NeuN (red) labeling, and white arrows indicating apoptotic neurons (scale bar = 50 µm) for each group are shown, with enlarged views of selected neurons within the white rectangles (scale bar = 5 µm). (F) Representative H&E staining images for each group are shown, with enlarged views of selected cells within the white rectangles. Representative H&E staining images (scale bar = 50 µm) for each group are shown, with enlarged views of selected cells within the white rectangles (scale bar = 10 µm). Values are expressed as the mean $\pm$ standard deviation, with *p 0.05 and **p 0.01 for all graphs. IDE, idebenone; mNSS, modified neurological severity score; TUNEL, TdT-mediated dUTP nick end labeling; DAPI, 4^′,6-diamidino-2-phenylindole; H&E, hematoxylin-eosin.

To assess the anti-oxidative stress effects of IDE, western blotting was used to evaluate the protein levels of Keap1, Nrf2, and its downstream effector NQO1 in perihematomal brain tissues. Compared with the sham group, the ICH + Vehicle group exhibited elevated levels of Nrf2 and NQO1 (Fig. 3A,B, p 0.05, p 0.05), while Keap1 levels were reduced (Fig. 3A,B, p 0.01). Furthermore, IDE treatment significantly decreased Keap1 expression while further increasing Nrf2 and NQO1 levels in comparison with those observed in the ICH + Vehicle group (Fig. 3A,B, p 0.01, p 0.05, p 0.05). To further assess oxidative stress, MDA levels were measured on day 3 following ICH. The results demonstrated that IDE-treated mice had considerably lower levels of MDA compared to the ICH + Vehicle group, indicating reduced lipid peroxidation (Fig. 3C, p 0.05). These findings suggest that IDE alleviates oxidative stress after ICH, potentially by activating the Nrf2 signaling pathway.

Fig. 3.

Effects of IDE on oxidative stress after ICH. (A,B) The expression levels of Nrf2, Keap1 and NQO1 were analyzed using western blotting techniques across all experimental groups, followed by quantitative assessment (n = 3). (C) MDA level was measured in each group (n = 6). Values are expressed as the mean $\pm$ standard deviation, with *p 0.05 and **p 0.01 for all graphs. Keap1, Kelch-like ECH-associated protein 1; NQO1, NAD(P)H quinone oxidoreductase 1.

To evaluate the effects of IDE treatment on neuroinflammation, immunofluorescence staining of Iba-1 (an indicator of microglial activation) and MPO (an indicator of neutrophil infiltration) in the perihematomal region was performed. In the resting state, microglia had an elongated rod-like morphology. However, following ICH, the microglia in the ICH + Vehicle group displayed an amoeboid shape with enlarged cell bodies, indicative of activation. However, IDE-treated mice demonstrated significant reductions in both Iba-1 and MPO-positive cells, suggesting decreased microglial activation and neutrophil recruitment (Fig. 4A, p 0.01, p 0.01).

Fig. 4.

Effects of IDE on microglia, neutrophils, and inflammatory factors release after ICH. (A) Representative immunofluorescence staining for Iba-1 and MPO was performed in the ipsilateral basal ganglia for each group, with magnified views of selected cells shown within the solid rectangles (scale bar = 50 µm, n = 6). (B) Western blot analysis was conducted to evaluate MMP-9, TNF-$\alpha$, and IL-1$\beta$ among each group, with quantification analysis (n = 3). (C) ELISA results show the concentrations of TNF-$\alpha$ and IL-10 in each group. Values are presented as the mean $\pm$ SD, with *p 0.05, **p 0.01 for all graphs. MMP-9, matrix metalloproteinase-9.

Further analysis of the western blotting results indicated reduced expression of MMP-9, IL-1$\beta$, and TNF-$\alpha$ in the ICH + IDE group compared to the ICH + Vehicle group (Fig. 4B, p 0.05, p 0.05, p 0.05). Moreover, ELISA quantification revealed that TNF-$\alpha$ levels were significantly lower in the IDE-treated group, whereas IL-10 levels were significantly increased compared to the ICH + Vehicle group (Fig. 4C, p 0.01, p 0.01). These required results indicate that IDE mitigates neuroinflammation post-ICH by suppressing pro-inflammatory mediators and enhancing the levels of anti-inflammatory cytokines.

These findings imply that IDE inhibits neutrophil recruitment and microglial activation, which modulates the release of inflammatory chemicals to exert anti-neuroinflammatory effects.

To assess the effects of IDE treatment on brain edema following ICH, the BWC was measured in the ipsilateral and contralateral hemispheres as well as in the cerebellum. IDE treatment significantly reduced the BWC in the ipsilateral hemisphere, indicating a protective effect against brain edema in the acute phase of ICH (Fig. 5A, p 0.01). Moreover, the integrity of the BBB was assessed using western blot analysis of ZO-1. The data demonstrated that IDE treatment significantly restored ZO-1 protein expression levels, suggesting that IDE preserves BBB integrity and reduces permeability following ICH (Fig. 5B,C, p 0.01).

Fig. 5.

Effects of IDE on perihematomal edema and BBB dysfunction after ICH. (A) BWC was examined following ICH (n = 6). (B,C) Western blot analysis of ZO-1 expression in each group, with quantification analysis (n = 3). Values are presented as the mean $\pm$ SD, with **p 0.01, ^nsp $>$ 0.05 for all graphs. BWC, brain water content; BBB, blood-brain barrier; ZO-1, zonula occludens-1.