CXCR4 and CXCL12 Expression Was Upregulated after SAH
As introduction mention demonstrated that CXCR4 was expressed from rodent microglia. The pathway CXCR4/CXCL12 (CXCL12 is the ligand of CXCR4) is the critical factor of the migration and recruitment of microglia. In the SAH rats, we collected the brain tissues from the base of the temporal lobe for western blot to analyze the expression of CXCR4 at 1 h, 6 h, 12 h, 24 h, 48 h, 72 h after SAH. Results showed that the expression of CXCR4 increased gradually after SAH, and the peak elevation occurred at 12 h. Then, it presented a downward trend (**P < 0.01 and ***P < 0.001; Fig. 2b). Meanwhile, we offered another result within 72 h after SAH that compared with the sham group. The expression of CXCL12, the ligand of CXCR4, increased gradually, with the peak at 12 h, then it followed a decreasing trend (**P < 0.01 and ***P < 0.001; Fig. 2c).
The Amount of M1 Microglia Increased and May Peak 24 h after SAH.
Previous studies showed that M1 polarization microglia could express marks (CD86, iNOS, and IL-1β) and release pro-inflammation factors (TNF-α and IL-6) [39, 40]. And CD206 and IL-10 are the special marker and expression factors respectively of M2 polarization microglia [10, 41, 42]. We divided the animals into seven groups according to the specific time point after the SAH model: sham, 1 h, 6 h, 12 h, 24 h, 48 h, 72h. Western blot was performed using the brain tissues to observe the expression levels of CD86, iNOS, and CD206. The result showed the expression of CD86 and iNOS had a rising tendency, and the peak elevation occurred at 24 h. Within 72 h after SAH, the expression level of CD206 of the experiment group had a gradually increasing trend compared with the sham group (*P < 0.05, **P < 0.01 and ***P < 0.001; Fig. 2). Our results suggest that the amount of M1 microglia may peak 24 h after SAH.
CXCR4/CXCL12 Was the Critical Pathway of the Migration of M1 Microglia.
We then performed the transwell experiment in vitro to explore the exact role of the CXCL12/CXCR4 pathway in the migration of M1 microglia. It is known that LPS can activate microglia polarize to M1. Firstly, we used LPS to induce BV2 microglia for 24 h for polarizing cells into the M1 phenotype. The merged image of the immunostaining for Iba-1 and iNOS carried out that LPS induced most of the BV2 cells to M1 (Fig. 3a). Additionally, the protein level of the biomarkers (CD86 and iNOS) of M1 microglia was detected by Western blot assay. The expression of CD86 and iNOS increased after LPS induced (Fig. 3b). The above results validate that the BV-2 microglia acquired M1 polarization characteristics by LPS induced.
To imitate the SAH model, the M1 microglia (LPS-induced BV-2 microglia) was exposed under oxyhemoglobin for 6 h. In this study, AMD3100 was the highly selective antagonist of CXCR4.AMD3100 or vehicle was added into the upper chamber, and CXCL12 or vehicle was added into the lower chamber. Then transwell assay was conducted on the four groups. When both chambers were added vehicle, only a small amount of M1 microglia could be migrated to the opposite surface of the transwell insert, while less M1 microglia migrated to the insert surface when AMD3100 was added to the upper chamber. Conversely, when we added CXCL12 into the lower chamber, more M1 microglia migrate to the surface. In the final group, CXCL12 was added into the lower chamber. Meanwhile, the AMD3100 was added into the upper chamber. We observed the migration of M1 microglia induced by CXCL12 was inhibited by AMD3100 (**P < 0.001; Fig. 3c).
Previous studies found that the expression of CXCL12 increased after the brain injury [43]. The results of the previous assay that CXCL12 had an increasing trend after SAH also confirmed this point. In the CNS, CXCL12 was produced mainly by activated astrocytes [44–46]. Thus, we cultured and seeded the primary astrocytes in the lower chambers, which were induced by oxyhemoglobin to imitate the in vitro SAH environment. The result of the transwell assay showed that in comparison with the control treatment group, the oxyhemoglobin-induced astrocytes could induce the migration of M1 microglia, and the migration phenomenon was significantly inhibited by AMD3100 (**P < 0.001; Fig. 3d).
AMD3100 (an antagonist of CXCR4) Alleviated SAH-induced Inflammation and Improved the Outcome.
Elisa assay was performed to explore the expression level of related inflammation factors after AMD3100 treatment. We divided rats into three groups: sham, SAH, and SAH + AMD3100. Serum samples of each rat were collected for ELISA at 12 h, 24 h, 48 h, and 72 h after SAH modeling. The result showed that AMD3100 did not affect the sham group. Importantly, we found that the expression of inflammatory factor (IL-6, IL-1β, TNF-ɑ), secreted by M1 microglia, had a decreased trend after the AMD3100 treatment compared with the vehicle group. However, we found the expression of the above factors had not significantly different from the vehicle group when SAH rats were treated by AMD3100 later than 24 h (*P < 0.05, ** P < 0.01 and *** P < 0.001; Fig. 4).
To explore the effect of AMD3100 on the prognosis of SAH rats,we performed a series of behavioral experiments. In wire-grip test, we found that the rats of treatment group had a better score than the SAH group (#P < 0.05, ## P < 0.01 and ### P < 0.01; ** P < 0.01 and *** P < 0.001; Fig. 4d). We then performed the Morris water maze test when the muscle strength of the experimental group was not significantly different from that of the sham group. The SAH rats with AMD3100 treated had a shorter latency as the Fig. 4 shows (#P < 0.05 and ## P < 0.01; ** P < 0.01 and *** P < 0.001; Fig. 4e). The above experiment proves that AMD3100 could alleviate the SAH-induced inflammation and improve the outcome.
Inhibition of Inducible NO Synthase/NO• Promoted Ferroptosis in M1 Microglia.
M1 microglia is a major microglia-type in the ischemic boundary zone within the first 72 h post-stroke [47]. We performed Perls’ Prussian blue staining to observe the ferroptosis in the tissues of the temporal-floor brain. The outcomes showed no obvious higher level of non-heme iron in the temporal-floor brain within the first 48 h after the SAH model. Then the level increased significantly at 72 h and showed a gradual upward trend (Fig. 5a). This outcome was in line with that the M1-polarized microglia that were highly resistant to ferroptosis, and M2-polarized microglia were highly sensitive to ferroptosis stimulation.
As we stated before, three programs were included in the cell death program of ferroptosis: iron metabolism, lipid peroxidation, and thiol regulation—the related proteins of these programs like Tfr1, Tf, Fth, Ftl, xCT, GPX4. Moreover, COX2 is a well-recognized biomarker of ferroptosis [48]. BV2 cells were used for the Vitro experiment, and We examined the expression level of the protein associated with ferroptosis by performing the western blot assay. L-NIL and Lip-1 were used to treat, respectively. Our western blot result revealed that compared with the normal group, the expression of Tfr1, Tf, Fth, Ftl, and Cox2 presented an increasing tendency after induced OxyHb while the expression of GPX4 and xCT with a trend toward decrease (#P < 0.05, ##P < 0.01, and ###P < 0.001; Fig. 6). Compared with the vehicle group, protein expression of Tfr1, Tf, Fth, Ftl, and Cox2 showed a rising trend with the L-NIL treated, the GPX4 and xCT expression decreased. The result of Lip-1 treated group showed the opposite trend (*P < 0.05, ** P < 0.01, and *** P < 0.001; Fig. 6).
As mentioned above, lipid peroxidation is associated with ferroptosis. C11-bodipy581 = 591 is very suitable for ratio fluorescence imaging of reactive oxygen species in living cell membranes. Figure 5b showed that OxyHb promoted lipid peroxide formation with the red fluorescence shifted to green fluorescence of the BODIPY, indicating the lipid peroxidation rises after OxyHb induced. We also found that L-NIL facilitated the significant upregulation of lipid peroxidation following OxyHb induced. Significantly, treating L-NIL-treated microglia cells with Lip-1could reduce BODIRY-C11 oxidation following OxyHb induced (Fig. 5b).
The MDA content of M1 microglia increased after being induced by OxyHb. The trend was made more evident after L-NIL treatment, while Lip-1 inhibited the increase in MDA content (#P < 0.05, ##P < 0.01, and ###P < 0.001; *P < 0.05, and **P < 0.01; Fig. 5c).
To sum up, these data suggest that OxyHb resulted in ferroptosis of M1 Microglia while L-NIL promotes this cell death program.
Reduced Quantity of M1 Microglia Alleviated Inflammation after Early Brain Injury.
According to previous studies, the expression levels of Cd86 and iNOS ref lected the M1 polarization of microglia and the count of M1 microglia correspondingly. To validate the effect of AMD3100 and L-NIL on the count of M1 microglia, we performed the following experiments. The rats were categorized into 5 groups: (1) Sham, (2) Vehicle, (3) Vehicle + AMD3100, (4) Vehicle + L-NIL and (5) Vehicle + AMD3100 + L-NIL. Then after 24 h’s successful modeling, rats were euthanized. The brain tissues were used for Western blot. The result showed CD86 and iNOS had a significant rise tendency after SAH. The expression of CD86 and iNOS had a decreasing tendency when AMD3100, L-NIL, or both two drugs were administered (#P < 0.05, ##P < 0.01 and ###P < 0.01; **P < 0.01 and ***P < 0.001; Fig. 7). Hence, these data indicated that AMD3100 and L-NIL treatment could decrease the count of M1 microglia after SAH.
To further investigate the effect of AMD3100 or L-NIL on the amount of M1 microglia in the temporal-floor brain after SAH, we detected M1 marker (iNOS) co-expression with microglia marker Iba-1 at 24 h after SAH. We found the amount of M1 microglia (Merge /Iba-1+) increased significantly at 24 h after SAH. Importantly, the amount of M1 microglia decreased with AMD3100 and/or L-NIL treatment (*P < 0.05, **P < 0.01 and ***P < 0.001; Fig. 7e). The result further confirmed that the AMD3100 and L-NIL treatment could decrease the amount of M1 microglia after SAH.
The inflammation factors in the sera of rats were detected using the specific ELISA kits to confirm the effect of AMD3100 and L-NIL on reducing the inflammation. TNF-ɑ, IL-6, and IL-1β are the classic inflammation factors. Figure 8 showed that the level of inflammation factors of the vehicle group presented a significant upward tendency compared with the sham group. The expression level of inflammation factor compared with vehicle group had a decreasing trend after treatment with AMD3100 and L-NIL (**P < 0.01 and ***P < 0.001; Fig. 8). These results further demonstrated that AMD3100 and L-NIL could alleviate inflammation after SAH.
Both AMD3100 and L-NIL Attenuated Neurological Dysfunctions and Brain Edema after SAH
To evaluate the effect of AMD3100 and L-NIL on neurobehavioral functions and brain edema after SAH. In neurobehavioral functions test, there were no significant differences between each group before SAH. Compared with the sham group, the neurological score of the animals in the vehicle group significantly decreased. We also got the observation that the neurobehavioral was markedly improved when the post-SAH animals were treated with AMD3100 and L-NIL. The result of the neurological score was given in the figure below (*P < 0.05, **P < 0.01; Fig. 9a).
In the brain edema test, both the animals of sham and experiment group were euthanasia 24 h after SAH. We found the brain edema of 24 h post-SAH animals increased significantly by 0.58% in the left hemisphere (##P < 0.01), 0.54% in the right hemisphere (##P < 0.01), and 0.30% (##P < 0.01) in the cerebellum compared with the animals of sham groups, while no change was observed in the brain stem. Our result also showed that the brain water content in the left and right hemisphere decreased after the treatment with AMD3100 or L-NIL (in the left hemisphere, AMD3100, 79.32 ± 0.11 vs. Vehicle, 79.61 ± 0.08, *P < 0.05; L-NIL, 79.42 ± 0.08 vs. Vehicle, 79.61 ± 0.08, *P < 0.05; in the right hemisphere, AMD3100, 79.29 ± 0.11 vs. Vehicle, 79.47 ± 0.06, *P < 0.05; L-NIL, 79.38 ± 0.03 vs. Vehicle, 79.47 ± 0.06, *P < 0.05), the same result was observed in the cerebellum (AMD3100, 79.09 ± 0.05 vs. Vehicle, 79.18 ± 0.04, **P < 0.01; L-NIL, 79.12 ± 0.07 vs. Vehicle, 79.18 ± 0.04, *P < 0.05;). The post-SAH animals which were treated with both AMD3100 and L-NIL had a significantly improved trend in the evaluation of brain edema. The brain water content of treated animals decreased significantly compared with the vehicle group (in the left hemisphere, AMD3100 and L-NIL, 79.20 ± 0.10 vs. Vehicle, 79.61 ± 0.08, **P < 0.01; in the right hemisphere, AMD3100 and L-NIL, 79.09 ± 0.09 vs. Vehicle, 79.47 ± 0.06, **P < 0.01; in the cerebellum, AMD3100 and L-NIL, 79.08 ± 0.07 vs. Vehicle, 79.09 ± 0.05, **P < 0.01; Fig. 9b).