All animal experiments were approved by the Animal Ethics Committee of the Third Affiliated Hospital of Zhengzhou University. Postnatal day 5 (P5) C57BL/6 mice were used in this experiment. Mice were maintained at a constant ambient humidity and temperature under a 12-hour light/dark cycle with ad libitum access to food and water. The construction of the GMH model using collagenase was performed as previously described [17]. P5 mice were anesthetized with 2% isoflurane (R510-22-10, RWD Life Science, Shenzhen, Guangdong, China) on a brain stereotaxic apparatus, and 0.15 U collagenase VII-S (2399, Sigma-Aldrich, St. Louis, MO, USA) dissolved in 1 µL saline was injected into the right subventricular germinal matrix (AP –2 mm, ML –1 mm, and DV +2 mm, using Bregma as reference) using a 28G needle (7803-2, Hamilton, Reno, MA, USA) and a 25 µL Hamilton syringe connected to a syringe pump (KD Scientific Syringe Pump Company, United States). A heating pad (21100-800, Ugo Basile, Gemonio, VA, Italy) was used to keep the body temperature at 37 $\pm$ 0.5 °C throughout the entire surgical procedure. After GMH induction, mice were administered intraperitoneally with RU.521 (5 mg/kg, HY-114180, MedChemExpress, Monmouth Junction, NJ, USA) or vehicle (10% DMSO (HY-Y0320C, MedChemExpress), 40% PEG300 (HY-Y0873, MedChemExpress), 5% Tween-80 (HY-Y1891, MedChemExpress), and 45% saline) at 1 h after GMH [18]. SR-717 (30 mg/kg, HY-131454, MedChemExpress) or vehicle (50% PEG300 and 50% saline) was injected intraperitoneally immediately after GMH [19].

In this study, both male and female pups were randomly assigned to either sham or GMH group. A total of 148 mice were used. At 24 h, 72 h, 7 days or 28 days after GMH, mice were euthanized by over dose of isoflurane and brains were removed and used for quantitative polymerase chain reaction (qPCR), western blot and immunofluorescence.

Experiment 1. To determine the expression and distribution of cGAS and STING after GMH, mice (n = 20, 5/group) were randomly divided into four groups: sham, GMH 24 h, GMH 72 h, and GMH 7 days. The brain tissues were harvested for western blot analysis. For immunohistochemical quantification of cGAS and STING, 18 mice (n = 6/group) from groups of sham, GMH 24 h, and GMH 72 h were used.

Experiment 2. To explore the effects of RU.521 on inflammation, BBB damage, white matter lesions, and neurological deficits, 66 mice were randomly divided into three groups: sham group, GMH + vehicle group, and GMH + RU.521 group. For qPCR analysis of inflammatory cytokines, 15 mice (n = 5/group) were used. Neurological function was tested on 28 days after GMH (n = 30, 10/group). Additionally, immunofluorescence staining (n = 18, 6/group) and western blotting (n = 15, 5/group) were carried out at 24 h and 28 days after GMH.

Experiment 3. To investigate whether the STING agonist SR-717 could reverse the effects of RU.521 on GMH-induced damage, 44 mice were randomly divided into the GMH + RU.521 + vehicle and GMH + RU.521 + SR-717 groups. Neurological function was tested on 28 days after GMH (n = 20, 10/group). Brain samples were collected for western blot analysis (n = 10, 5/group) and immunofluorescence staining (n = 12, 6/group) at 24 h and 28 days after GMH. Another set of mice were used for qPCR analysis (n = 10, 5/group) at 24 h after GMH.

According to the manufacturer’s instructions, total RNA was extracted from brain tissue using the Total RNA Kit (DP419, TIANGEN, Beijing, China). Synthesis of cDNA was conducted using PrimeScrip™ RT Master Mix kit (RR036, TaKaRa, Kyoto, Japan), and qPCR was performed in a LightCycler® 96 gradient machine (Roche, Basel, Switzerland) using PowerUp™ SYBR™ Green premix kit (A25742, Thermo Scientific, Waltham, MA, USA). Relative amount of expression changes in gene levels were calculated using the 2^{-Δ⁢Δ⁢CT} method [20, 21]. Primers used were as follows: GAPDH forward 5^′-AGGTCATCCCAGAGCTGAACG-3^′, reverse 5^′-CACCCTGTTGCTGTAGCCGTAT-3^′, IFN-$\beta$ forward 5^′-GCACTGGGTGGAATGAGACTATTG-3^′, reverse 5^′-TTCTGAGGCATCAACTGACAGGTC -3^′, IL-1$\beta$ forward 5^′-TGAAATGCCACCTTTTGACAG-3^′, reverse 5^′-CCACAGCCACAATGAGTGATAC-3^′, IL-6 forward 5^′-CTGCAAGAGACTTCCATCCAG-3^′, reverse 5^′-AGTGGTATAGACAGGTCTGTTGG-3^′, TNF-$\alpha$ forward 5^′-CCTGTAGCCCACGTCGTAG-3^′, reverse 5^′-GGGAGTAGACAAGGTACAACCC-3^′, iNOS forward 5^′-GGCACAGGGTCATCATCAAA-3^′, reverse 5^′-TCAGGTCACTTTGGTAGGATTT-3^′, CD86 forward 5^′-GTAGAGTCCAGTTGTTCCTGTC-3^′, reverse 5^′-TGGTTCTGTACGAGCACTATTT-3^′, Arginase-1 (Arg-1) forward 5^′-GTCCCTAATGACAGCTCCTTTC-3^′, reverse 5^′-CCACACTGACTCTTCCATTCTT-3^′, CD206 forward 5^′-GTGGTCCTCCTGATTGTGATAG-3^′, reverse 5^′-CACTTGTTCCTG GACTCAGATTA-3^′.

For western blot analysis, brain tissues were dissected from the peri-hematomal area (within 1 mm from the edge of the hematoma) as previously described [22]. RIPA lysis buffer containing protease and phosphatase inhibitors (GRF103, Epizyme, Shanghai, China) was used to extract proteins from brain tissue. Proteins were equally loaded, separated using SDS-polyacrylamide electrophoresis and transferred onto the polyvinylidene fluoride (PVDF) membranes (HVLP06225, Millipore, Darmstadt, Germany). After blocking in 5% skimmed milk for 2 hours, membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies used were: rabbit anti-cGAS (1:1000, 31659), rabbit anti-STING (1:1000, 13647), rabbit anti-TBK1 (1:1000, 3504), rabbit anti-phospho-TBK1 (1:1000, 5483), rabbit anti-IRF3 (1:1000, 4302), rabbit anti-phospho-IRF3 (1:1000, 4947), rabbit anti-$\beta$-actin (1:1000, 4970), rabbit anti-GAPDH (1:1000, 5174, all from Cell Signaling Technology, Boston, MA, USA), rabbit anti-zonula occluden-1 (ZO-1) (1:1000, 61-7300, Invitrogen, Waltham, MA, USA), rabbit anti-occludin (1:1000, ab216327, Abcam, Cambridge, UK), rabbit anti-MBP (1:1000, ab40390, Abcam), goat anti-ionized calcium-binding adapter molecule 1 (Iba1) (1:1000, ab5076, Abcam), mouse anti-nonphosphorylated neurofilament-H (SMI-32, 1:1000, 801701, Biolegend, San Diego, CA, USA). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. Blots were visualized with Image Lab 6.0 software (Bio-Rad Laboratories, Hercules, CA, USA) and quantified using Image J software (1.8.0, NIH, Bethesda, MD, USA).

Cold phosphate-buffered saline (PBS) was used to perfuse mice under deep anesthesia (isoflurane, 2%, R510-22-10, RWD Life Science), followed by 4% paraformaldehyde (PFA, 8187150100, Sigma-Aldrich). Brains were fixed with 4% PFA and then immersed in 20% and 30% sucrose at 4 °C overnight. Coronal sections of 20 µm thickness were blocked with the solution consisting of 1% bovine serum albumin (BSA, V900933, Sigma-Aldrich), consisting 0.3% Triton X-100 (T9284, Sigma-Aldrich) and 3% donkey serum (D9663, Sigma-Aldrich) or goat serum (NS02L, Sigma-Aldrich) and then incubated overnight at 4 °C with primary antibodies: rabbit anti-cGAS (1:200), rabbit anti-STING (1:500, NBP2-24683, NOVUS Biologicals, Centennial, CO, USA), goat anti-Iba1 (1:800, ab5076, Abcam), rabbit anti-Iba1 (1:400, 019-19741, Wako, Osaka, Japan), goat anti-CD31 (1:200, AF3628, R&D Systems, Minneapolis, MN, USA), mouse anti-NeuN (1:1000, MAB377, Millipore), goat anti-GFAP (1:200, ab53554, Abcam), rat anti-CD16 (1:200, 553142, BD Biosciences, Franklin Lakes, NJ, USA), goat anti-CD206 (1:200, AF2535, R&D Systems), rabbit anti-MBP (1:800, ab40390, Abcam), mouse anti-nonphosphorylated neurofilament-H (SMI-32, 1:200, 801701, Biolegend). The second antibodies used were: Alexa Fluor 594-conjugated donkey anti-rabbit IgG (A-21207), Alexa Fluor 488-conjugated donkey anti-goat IgG (A-11055), Alexa Fluor 594-conjugated donkey anti-rat IgG (A-21209), Alexa Fluor 594-conjugated donkey anti-goat IgG (A-11058), Alexa Fluor 488-conjugated donkey anti-mouse IgG (A-21202), Alexa Fluor 488-conjugated goat anti-rat IgG (A-11006), and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (A-21206) (1:1000, all from Invitrogen, diluted in 1% BSA, consisting 0.3% Triton X-100 and 1% donkey or goat serum). Nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI, 1:10,000, 33342, Invitrogen). To measure the leakage of IgG, cerebral sections were incubated with blocking solution (1% BSA, 0.1% Triton X-100, and 5% donkey serum in PBS) for 2 h. Then, the sections were stained with goat anti-CD31 overnight at 4 °C. After washing in PBS, the sections were incubated with Alexa Fluor 594 donkey anti-goat IgG and Alexa Fluor 488-conjugated donkey anti-mouse IgG for 1 h at room temperature. Brain sections were imaged using a fluorescence microscope (Axio Observer 7, ZEISS, Oberkochen, BW, Germany) equipped with a $\times$20 or $\times$40 objective lens. Specific filters were selected based on fluorophore conjugates, and uniform exposure times were set for each channel of all samples to ensure comparability. Then, images were captured with the same pixels and saved in TIFF format to preserve the integrity of the raw data. All images for a given antibody marker were acquired under identical microscope settings. For immunohistochemical quantification, three fields from the brain tissue (Fig. 1A) in three sections were captured under $\times$20 or $\times$40 objectives in each animal. Images were processed using ImageJ software and contrast-enhanced to differentiate positive signals from the background. The fluorescent density of MBP and SMI-32 was measured using the ImageJ plugin “integrated density” analysis tool. The number of cGAS⁺, STING⁺, Iba1⁺, CD16⁺Iba1⁺ and CD206⁺Iba1⁺ cells was examined using the ImageJ “Multi-point” measurement tool and was expressed as cells/mm². To quantify extravascular deposits of IgG, the images were contrast-enhanced to differentiate positive signals from the background. The immunofluorescent density of extravascular IgG was measured using the ImageJ plugin “integrated density” analysis tool.

Fig. 1.

Increased expression of cGAS and STING after GMH. (A) Representative photograph of coronal brain sections showing the location (black boxes) imaged for the cellular protein experiments in immunohistology. Imaging obtained from the similar parts of the brain in sham-operated mice and mice treated with vehicle, RU.521 or RU.521 in combination with SR-717 were used for comparison. (B–E) Representative immunoblots and quantification of cGAS (B,C) and STING (D,E) at 24 h, 72 h, and 7 d after GMH (n = 5). (F–H) Representative images (F) and quantification of cGAS (G) and STING (H) in the peri-hematomal area at 24 h (n = 6). Cell nuclei were stained with DAPI. Scale bar, 50 µm. (I,J) Double immunostaining of cGAS (I) and STING (J) with microglia (Iba1), neuron (NeuN) and astrocytes (GFAP). Cell nuclei were stained with DAPI. Scale bar, 20 µm. Values are mean $\pm$ SD. ***p 0.001, ****p 0.0001. cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; GMH, germinal matrix hemorrhage; DAPI, 4,6-Diamidino-2-phenylindole.

The Y-maze test, as previously described, is employed to examine spatial working memory [23]. The device consists of three arms that diverge at 120 degrees from the central point. Mice were set in the center of the maze and given 5 minutes to move around freely. The experiment was completely videotaped with a camera, and the sequence of arm entries was analyzed by means of ANY-maze 7.4 software (Steolting, Wood Dale, IL USA). The percentage of spontaneous alternation was calculated using the following formula: (number of spontaneous alternation) / (total number of arm entries-2) $\times$ 100 [24].

NOR test was used to assess cognitive memory in mice [25]. This test is divided into two different phases, including the training and testing parts, with a rest period between each phase. In the training phase, mice explored an open-field box with two identical objects for 10 minutes. During the testing stage, mice were placed in the same arena and exposed to two objects, one familiar and one novel object. All experiments were recorded via a video camera and analyzed using ANY-maze 7.4 software. The formula for the calculation of the recognition index is: novel object time/total object time $\times$100 [26].

The rotarod test was used to examine motor coordination of the mice as described previously [27]. Mice were placed on a rotarod cylinder (47650, Ugo Basile, Gemonio, Italy). The speed of rotarod was accelerated from 4 to 40 rpm within 5 minutes. The latency of falling was recorded, and three trials were conducted to calculate the average value. Before surgery, mice were trained for 3 consecutive days.

Data are presented as means $\pm$ standard deviations (SD) and were analyzed by using GraphPad Prism 9 software (Dotmatics, Boston, MA, USA). All data were tested for normality using the Shapiro-Wilk test. Because all data passed the normality test, differences between two groups were performed using unpaired Student’ t test and multiple group comparisons were analyzed by one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test. A value of p 0.05 was considered statistically significant.

The protein levels of cGAS and STING in the hemorrhagic brain hemisphere were assessed at 24 h, 72 h, and 7 d after GMH. A significant increase in cGAS expression was observed at 24 and 72 h and peaked at 24 h post-GMH compared to the sham group (Fig. 1B,C). STING expression was significantly increased at 24 h post-GMH compared to sham (Fig. 1D,E). All original Western blot figures of Fig. 1B,D are available in Supplementary Material 1. Immunostaining revealed a marked increase in the number of cGAS⁺ and STING⁺ cells in the peri-hematomal area at 24 and 72 h after GMH compared to the sham-operated brains (Fig. 1F–H). Double immunostaining of cGAS and STING with Iba1 (microglial marker), NeuN (neuron marker), and GFAP (astrocyte marker) indicated that increased expression of cGAS and STING were colocalized with microglia, whereas only minimal cGAS and STING staining was observed in GFAP⁺ astrocytes and NeuN⁺ neurons (Fig. 1I,J).

To investigate the effects of cGAS following GMH, mice were treated with RU.521, a small molecule that inhibits cGAS by attaching to its catalytic site and preventing its DNA-activated activity [16]. Western blots confirmed a decrease in cGAS expression in the peri-hematomal area in RU.521-treated mice compared to vehicle-treated mice (Supplementary Material 2 Fig. 1A,B). We then found that RU.521 reduced GMH-induced elevation of STING (Supplementary Material 2 Fig. 1C,D). Compared with sham group, microglial number was increased at 24 h after GMH (Fig. 2A,B). Treatment with RU.521 1 h following GMH resulted in a significant reduction in microglial number. Microglia activation leads to the synthesis and secretion of various inflammatory mediators, which contribute to the development of secondary brain damage [28]. We showed that mice treated with RU.521 exhibited significantly lower levels of IL-1$\beta$, IL-6, and TNF$\alpha$ compared with vehicle-treated mice (Fig. 2C–E).

Fig. 2.

Inhibition of cGAS by RU.521 reduces inflammation and drives M2 microglial polarization after GMH. (A,B) Representative images (A) and quantification (B) of Iba1⁺ microglia in the peri-hematomal area at 24 hours after GMH (n = 6). Cell nuclei were stained with DAPI. Scale bar, 50 µm. (C–E) Relative gene expression of IL-1$\beta$ (C), IL-6 (D), and TNF-$\alpha$ (E) (n = 5). (F–I) Relative gene expression of iNOS (F), CD86 (G), Arg-1 (H), and CD206 (I) (n = 5). (J,K) Double immunostaining of CD16 (J) and CD206 (K) with microglia (Iba1). (L,M) Quantification of CD16⁺Iba⁺ pro-inflammatory microglia (L) and CD206⁺Iba1⁺ anti-inflammatory microglia (M) (n = 6). Nuclei were stained with DAPI. Scale bar, 50 µm. Values are mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. Arg-1, Arginase-1; IL-1$\beta$, Interleukin-1$\beta$; IL-6, Interleukin-6; TNF-$\alpha$, Tumor necrosis factor-$\alpha$; iNOS, inducible nitric oxide synthase; CD86, Cluster of differentiation 86.

Neuroinflammation following stroke is primarily attributed to microglial polarization [29, 30]. We found a marked upregulation in the levels of M1 phenotype factors (iNOS, CD86) and a moderate increase in the M2 phenotype factors (Arg-1 and CD206) at 24 h after GMH (Fig. 2F–I). We then observed that RU.521 decreased M1 phenotype-related gene expression while enhancing M2 phenotype-related gene expression. To further test the effects of cGAS inhibition on the M1/M2 polarization, we performed immunofluorescence staining for CD16 and CD206 together with Iba1. The study found that the number of CD16-positive microglia (M1-like microglia) increased after GMH and significantly decreased after RU.521 treatment (Fig. 2J–M). Simultaneously, RU.521 significantly increased the number of CD206-positive microglia (M2-like microglia) after GMH compared with the vehicle group. These results suggest that RU.521 blunted the M1-like polarization and promoted the M2-like polarization of microglia after GMH.

BBB permeability was assessed at 24 h after GMH using IgG and the endothelial cell marker CD31. The findings indicated a notable rise in the perivascular IgG fluorescence density following GMH compared to the sham-operated group (Fig. 3A,B), suggesting disruption of the BBB. RU.521 administration significantly decreased extravascular IgG levels compared to vehicle-treated mice. We next investigated the impact of RU.521 on the degradation of BBB tight junction proteins. We found that RU.521 markedly reduced GMH-induced reduction of the tight junction proteins ZO-1 and occludin in the peri-hematomal regions (Fig. 3C–E). All original Western blot figures of Fig. 3C are available in Supplementary Material 1.

Fig. 3.

The cGAS inhibitor RU.521 reduces blood-brain barrier disruption after GMH. (A,B) Representative images (A) and quantification (B) of IgG perivascular leakage at 24 hours after GMH in sham-operated mice and GMH mice treated with vehicle or RU.521 (n = 6). Microvessels were stained with CD31. Scale bar, 20 µm. (C–E) Representative immunoblots (C) and quantification of tight junction proteins ZO-1 (D) and occludin (E) in the peri-hematomal area (n = 5). Values are mean $\pm$ SD. *p 0.05, ***p 0.001, ****p 0.0001. ZO-1, zonula occluden-1.

At day 28 after GMH, western blot analysis revealed reduced expression of the myelin-associated protein (MBP) and elevated levels of the axonal marker SMI-32 in the corpus callosum compared to sham-operated mice (Fig. 4A–C, Supplementary Material 2 Fig. 2A). All original Western blot figures of Fig. 4A,C are available in Supplementary Material 1. Immunofluorescence quantification revealed that GMH mice exhibited significantly decreased MBP fluorescence intensity and increased SMI-32 staining intensity in the corpus callosum compared to sham-operated mice (Fig. 4D–F, Supplementary Material 2 Fig. 2B). Novel object recognition test, Y-maze test, and rotarod test were used to assess long-term neurological deficits in mice subjected to sham operation and GMH at 28 days after GMH. In the novel object recognition test, GMH mice exhibited a significant decrease in the exploration time for novel objects, as indicated by the recognition index (Fig. 4G). In the Y-maze test, GMH mice exhibited fewer spontaneous alternations, indicating an impairment in working memory (Fig. 4H). The rotarod test showed that locomotor activity was impaired in mice subjected to GMH (Fig. 4I). Treatment with RU.521 enhanced MBP and SMI-32 expression and fluorescence intensity, suggesting decreased demyelination and axonal degeneration. Consistent with these findings, cognitive function and working memory were improved in mice treated with RU.521.

Fig. 4.

RU.521 improves white matter injury and behavioral function after GMH. (A,B) Representative immunoblots (A) and quantification (B) of MBP in the corpus callosum at day 28 after GMH (n = 5). (C) Representative immunoblots of SMI-32 in the corpus callosum (n = 5). (D,E) Representative images (D) and quantification of MBP (E) fluorescence density in the corpus callosum (n = 6). Nuclei were stained with DAPI. Scale bar, 100 µm. (F) Quantification of SMI-32 fluorescence density in the corpus callosum (n = 6). (G–I) RU.521 improved behavioral outcomes in the novel object recognition test (G), Y-maze test (H), and rotarod test (I) (n = 10). Values are mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

The expression of downstream signaling molecules of cGAS was evaluated in the peri-hematomal area by Western blots at 24 h after GMH [31]. We observed robust induction of phospho-TBK1 and phospho-IRF3 (Fig. 5A–C) in GMH mice. All original Western blot figures of Fig. 5A are available in Supplementary Material 1. We then detected a marked increase in the levels of IFN-$\beta$ (Fig. 5D), indicating STING pathway activation [32]. Treatment with the cGAS inhibitor RU.521 reduced GMH-induced activation of TBK1 and IRF3, as well as levels of IFN-$\beta$.

Fig. 5.

Effects of RU.521 and the STING agonist-SR-717 on the expression of pTBK1, pIRF3 and IFN-$\beta$ after GMH. (A) Representative immunoblots of phosphorylated TBK1 (pTBK1) and total TBK1, phosphorylated IRF3 (pIRF3) and total IRF3 in the peri-hematomal area at 24 hours after GMH in sham-operated mice and GMH mice treated with vehicle or RU.521. (B,C) Quantification of pTBK1 (B) and pIRF3 (C) band intensities (n = 5). (D) Relative gene expression of IFN-$\beta$ (n = 5). (E,F) Representative immunoblots (E) and quantification (F) of STING in GMH mice treated with RU.521 or RU.521 in combination with SR-717 (n = 5). (G) Representative immunoblots of pTBK1, total TBK1, pIRF3 and total IRF3. (H) Relative gene expression of IFN-$\beta$ (n = 5). Values are mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

Our subsequent test was to determine whether RU.521’s beneficial impact on GMH-induced damage operates via STING pathways specifically. Western blot analysis indicated that the STING agonist SR-717 reversed RU.521-mediated downregulation of STING (Fig. 5E,F), inactivation of TBK1 and IRF3 (Fig. 5G, Supplementary Material 2 Fig. 3A,B), and efficiently increased the levels of IFN-$\beta$ after RU.521 treatment (Fig. 5H). All original Western blot figures of Fig. 5A,G are available in Supplementary Material 1. Furthermore, treatment with SR-717 dramatically increased the amount of microglia (Fig. 6A,B) and the levels of proinflammatory cytokines in the peri-hematomal area in mice treated with RU.521 (Fig. 6C–E). Previous study demonstrated that administration of SR-717 increased plasma levels of IFN-$\beta$ and IL-6 in naïve mice [33]. To test whether SR-717 alone could influence neuroinflammation, we injected SR-717 intraperitoneally into sham-operated mice. Our results indicated that there was no difference in the expression of Iba1 protein between vehicle-treated and SR-717-treated sham-operated mice (Fig. 6A,B). All original Western blot figures of Fig. 6A are available in Supplementary Material 1. In addition, SR-717 did not significantly change the levels of IL-1$\beta$, IL-6, and TNF-$\alpha$ in sham-operated mice (Fig. 6C–E). These findings are consistent with a report showing that hippocampal injection of cGAMP did not affect neuroinflammation compared with mice treated with vehicle [34].

Fig. 6.

The STING agonist-SR-717 blocked RU. 521-mediated reduction in microglia inflammation and BBB disruption. (A,B) Representative immunoblots (A) and quantification (B) of Iba1 expression in sham-operated mice treated with vehicle or SR-717 (n = 4) and in GMH mice treated with RU.521 or RU.521 in combination with SR-717 (n = 5). (C–E) Relative gene expression of IL-1$\beta$ (C), IL-6 (D), and TNF-$\alpha$ (E) (n = 4–5). (F) Representative immunoblots ZO-1 and occludin (n = 5). (G) Quantification of ZO-1 (n = 5). (H) Quantification of IgG leakage (n = 6). Values are mean $\pm$ SD. ns, not significant, *p 0.05, **p 0.01, ****p 0.0001.

We observed a significant downregulation of the tight junction proteins ZO-1 and occludin in mice treated with RU.521 and SR-717 compared to mice treated with RU.521 alone (Fig. 6F,G, Supplementary Material 2 Fig. 4A). All original Western blot figures of Fig. 6F are available in Supplementary Material 1. Immunofluorescence quantification showed a significant increase in the perivascular IgG fluorescence density treated with both RU.521 and SR-717, compared to those treated solely with RU.521 (Fig. 6H, Supplementary Material 2 Fig. 4B). Western blotting showed that SR-717 reversed RU.521-mediated increase in the expression of MBP and decrease in the expression of SMI-32 in the corpus callosum at day 28 after GMH (Fig. 7A–C). All original Western blot figures of Fig. 7A are available in Supplementary Material 1. Immunofluorescence quantification of MBP and SMI-32 showed that SR-717 blocked RU.521-provided protection against demyelination (Fig. 7D,E) and axon degeneration (Fig. 7D,F). Consistent with these observations, we found that SR-717 exacerbated neurological outcomes in RU.521-treated mice, as shown by novel object recognition test, Y-maze test, and rotarod test (Fig. 7G–I).

Fig. 7.

RU.521-afforded protection on white matter lesions and behavioral outcomes were abolished by the STING agonist-SR-717. (A–C) Representative immunoblots (A) and quantification of SMI-32 (B) and MBP (C) expression in the corpus callosum at day 28 after GMH (n = 5). (D–F) Representative immunostaining (D) and quantification of MBP (E) and SMI-32 (F) fluorescence density in the corpus callosum (n = 6). Nuclei were stained with DAPI. Scale bar, 100 µm. (G–I) Novel object recognition test (G), Y-maze test (H), and rotarod test (I) in mice treated with RU.521 or RU.521 in combination with SR-717 (n = 10). Values are mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.