Dexras1 Induces Dysdifferentiation of Oligodendrocytes and Myelin Injury by Inhibiting the cAMP-CREB Pathway after Subarachnoid Hemorrhage

White matter damage (WMD), one of the research hotspots of subarachnoid hemorrhage (SAH), mainly manifests itself as myelin injury and oligodendrocyte differentiation disorder after SAH, although the specific mechanism remains unclear. Dexamethasone-induced Ras-related protein 1(Dexras1) has been reported to be involved in nervous system damage in autoimmune encephalitis and multiple sclerosis. However, whether Dexras1 participates in dysdifferentiation of oligodendrocytes and myelin injury after SAH has yet to be examined, which is the reason for creating the research content of this article. Here, intracerebroventricular lentiviral administration was used to modulate Dexras1 levels in order to determine its functional influence on neurological injury after SAH. Immunofluorescence, transmission electron microscopy, and Western blotting methods, were used to investigate the effects of Dexras1 on demyelination, glial cell activation, and differentiation of oligodendrocyte progenitor cells (OPCs) after SAH. Primary rat brain neurons were treated with oxyhemoglobin to verify the association between Dexras1 and cAMP-CREB. The results showed that Dexras1 levels were significantly increased upon in vivo SAH model, accompanied by OPC differentiation disturbances and myelin injury. Dexras1 overexpression significantly worsened OPC dysdifferentiation and myelin injury after SAH. In contrast, Dexras1 knockdown ameliorated myelin injury, OPC dysdifferentiation, and glial cell activation. Further research of the underlying mechanism discovered that the cAMP-CREB pathway was inhibited after Dexras1 overexpression in the in vitro model of SAH. This study is the first to confirm that Dexras1 induced oligodendrocyte dysdifferentiation and myelin injury after SAH by inhibiting the cAMP-CREB pathway. This present research may reveal novel therapeutic targets for the amelioration of brain injury and neurological dysfunction after SAH.


Introduction
Aneurysmal subarachnoid hemorrhage (aSAH) is a serious neurosurgical disorder with extremely high mortality and disability [1,2]. Despite intensive investigations, pathological mechanisms underlying aSAH have not yet been fully elucidated. Previous research has shown that while neuronal death plays an important part in neurological injury, it is not the only reason for neurological dysfunction after subarachnoid hemorrhage (SAH) [3,4]. In recent years, white matter damage (WMD) after SAH has rapidly become a research hotspot in neuroscience. Yusuke Egashira et.al. [5] found that white matter fiber damage

SAH Model Conducted by Means of Endovascular Perforation
The rat SAH model was established using a previously reported endovascular perforation method [12]. Briefly, the rats were anesthetized via intraperitoneal injection of pentobarbital (50 mg/kg), and the left carotid artery (CA) and its branches were exposed. Then, the distal end of the left external CA was sheared and reflexed to be in line with the left internal CA (ICA). A sharp 4-0 monofilament nylon suture was inserted into the external CA and advanced through the ICA until resistance was felt. The suture was then further advanced for approximately 3 mm to perforate the vessel bifurcation into the anterior and middle cerebral arteries. After puncturing the artery for approximately 10 s, the filament was withdrawn. The sham group underwent identical procedures without the vascular perforation. The severity of SAH was blindly assessed at the time of sacrifice according to the previously described grading system [13]. Rats displaying SAH severity scores between 8-13 were used for further experiments [14]. To study the temporal expression and cellular localization of Dexras1, 35 rats were randomly divided and assigned to seven groups: Sham (n = 5), SAH-6 h (n = 5), SAH-12 h (n = 5), SAH-24 h (n = 5), SAH-48 h (n = 5), SAH-72 h (n = 5), SAH-7 d (n = 5). Western blot analysis was performed to determine the expression changes of Dex-ras1 and other proteins. An additional 12 rats in the Sham (n = 3) and SAH-24 h (n = 3) groups were used for double immunofluorescence staining, electron microscopy. Experiment 2: Seven days after lentivirus injection into the lateral ventricle, to evaluate the effects of Dexras1 on short-term outcome after SAH, 40 rats were randomly divided and assigned to eight groups for Western blot and SAH grade: Sham (n = 5), SAH+LV-scramble (n = 5), SAH+LV-Dexras1 + (2 U, n = 5), SAH+LV-Dexras1 + (4 U, n = 5), and SAH+LV-Dexras1 + (6 U, n = 5), SAH +LV-Dexras1 -(2 U, n = 5), SAH +LV-Dexras1 -(4 U, n = 5), SAH +LV-Dexras1 -(6 U, n = 5). Based on the results of Western blot, Sham (n = 3), SAH+LVscramble (n = 3), SAH+LV-Dexras1 + (6 U, n = 3), and LV-Dexras1 -(6 U, n =3) group rats were used for immunofluorescence staining and electron microscopy to evaluate OPCs differentiation and myelin damage in the ipsilateral hemisphere at 24 h after SAH. To evaluate the effects of Dexras1 on long-term outcome after SAH, Morris water maze of these groups (additional 25 rats, 5 per group) was performed on day 8-13 after SAH. Experiment 3: To explore the effects of Dexras1 on oligodendrocyte differentiation in vitro SAH. Primary cortical neurons transfected with Dexras1 were cocultured with IGF-I induced differentiated OLN-93 cells. Western blot analysis and To study the temporal expression and cellular localization of Dexras1, 35 rats were randomly divided and assigned to seven groups: Sham (n = 5), SAH-6 h (n = 5), SAH-12 h (n = 5), SAH-24 h (n = 5), SAH-48 h (n = 5), SAH-72 h (n = 5), SAH-7 d (n = 5). Western blot analysis was performed to determine the expression changes of Dexras1 and other proteins. An additional 12 rats in the Sham (n = 3) and SAH-24 h (n = 3) groups were used for double immunofluorescence staining, electron microscopy. Experiment 2: Seven days after lentivirus injection into the lateral ventricle, to evaluate the effects of Dexras1 on short-term outcome after SAH, 40 rats were randomly divided and assigned to eight groups for Western blot and SAH grade: Sham (n = 5), SAH+LV-scramble (n = 5), SAH+LV-Dexras1 + (2 U, n = 5), SAH+LV-Dexras1 + (4 U, n = 5), and SAH+LV-Dexras1 + (6 U, n = 5), SAH +LV-Dexras1 − (2 U, n = 5), SAH +LV-Dexras1 − (4 U, n = 5), SAH +LV-Dexras1 − (6 U, n = 5). Based on the results of Western blot, Sham (n = 3), SAH+LV-scramble (n = 3), SAH+LV-Dexras1 + (6 U, n = 3), and LV-Dexras1 − (6 U, n = 3) group rats were used for immunofluorescence staining and electron microscopy to evaluate OPCs differentiation and myelin damage in the ipsilateral hemisphere at 24 h after SAH. To evaluate the effects of Dexras1 on long-term outcome after SAH, Morris water maze of these groups (additional 25 rats, 5 per group) was performed on day 8-13 after SAH. Experiment 3: To explore the effects of Dexras1 on oligodendrocyte differentiation in vitro SAH. Primary cortical neurons transfected with Dexras1 were co-cultured with IGF-I induced differentiated OLN-93 cells. Western blot analysis and immunofluorescence staining was performed to evaluate OPCs differentiation in vitro SAH model. Experiment 4: To explore the mechanism of Dexras1 on oligodendrocyte differentiation in vitro SAH. Primary cortical neurons transfected with Dexras1 were co-cultured with IGF-I-induced differentiated OLN-93 cells, and 8-Bromo-cAMP was added after co-culture for 1 h. Western blot analysis and immunofluorescence staining was performed to evaluate OPCs differentiation in vitro SAH model.  [15]. An RNA duplex with a random sequence was used as the negative control (NC). The transfection efficiency was more than the 90% (0.917 ± 0.005). Puromycin (2 µg/mL) was used to positively identify transfected cells [16] which were then used for the experiments.

Dexras1 Up-and Downregulation in the In Vivo Model of SAH
In this experiment, lentiviral vector-based interventions leading to overexpression and downregulation of Dexras1 were used to verify Dexras1 function in the rat SAH model. Lentivirus-based transfer systems were purchased from Shanghai Genepharma Pharmaceutical Technology Co, Ltd. (the sequences of overexpression and knockdown Dexras1 was showed in Table S1 and S2). The lateral ventricle injection method refers to the procedure introduced by Huang JZ et al. [17] 7 days before the construction of the subarachnoid hemorrhage model. Briefly, anesthetized rats were safely immobilized in a stereotaxic device (Stoelting, Wood Dale, IL, USA). Two to six microliters of the LV-Dexras1 + (109 TU/mL), LV-Dexras1 − (109 TU/mL), or control LV-Scramble (109 TU/mL), were slowly injected into the left lateral ventricle of each rat using a stereotactic frame (1.0 mm posterior to bregma, 1.5 mm lateral to midline, 3.5 mm in depth under the surface of the skull) with a sterile 10-µL Hamilton syringe at a rate of 0.5 µL/min at seven days before SAH induction. A burr hole to the skull was also made in the sham rats but they did not receive intracerebroventricular injections. After the injection and wound closure, the rats were placed on an electric blanket for recovery with free access to food and water.

Evaluation of Short-Term Neurobehavioral Functions and Brain Water Content Detect
The short-term neurobehavioral effects of treatments were assessed by investigators blinded to group ID at 24 h post-SAH according to the updated Garcia scale as previously described [18]. The modified Garcia scoring system includes six subscores. The animals were given an overall score between 0 and 18 after summation of all scores. For brain water content measure as previously described [19], the brains of each group were quickly divided into different portions under deep anesthesia, then weighed immediately (wet weight) and kept in an oven at 105 • C for 72 h (dry weight). The percentage of water content was calculated according to the formula: (wet weight to dry weight)/wet weight × 100%.

Morris Water Maze (MWM)
The MWM test was employed to assess the spatial learning ability and reference memory as previously described [18]. The cued water maze test was conducted one day before SAH, 5 rats per group with no difference were randomly selected for further experiments, and rats showing sensorimotor and/or motivational deficiencies affecting performance during the MWM test were not included in further trials. The spatial water maze test was conducted on days 8 to 13 after SAH. During each acquisition test, rats were allowed 90 s to find concealed platforms. If a rat was unable to find a platform within 90 s, it was directed there and allowed to rest on the platform for 20 s. On the 14th day, the platform was removed and the animals were observed for 90 s with a video recording system. The swimming pattern was assessed by an automatic image capture system to quantify distance, latency, and swimming speed. The number of times the rat crossed the platform and the time spent in the target quadrant were used for statistical analysis.

Cell Culture of Primary Cortical Neurons
Primary cortical neurons were obtained from newborn SD rats as previously described [20]. Briefly, cortical tissues were collected following newborn rat decapitation. The tissues were cut into small pieces and dispersed into cells (5 × 104 cells/mL), which were then grown in poly-D-lysine-coated (PDL-C) plates (Sigma, St. Louis, MO, USA) in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 4 h. Next, the medium was replaced with neurobasal medium (Thermo Fisher Scientific) with 2% B27 reagent, 0.5 mM glutamine (Thermo Fisher Scientific), 100 U/mL penicillin (P), and 100 µg/mL streptomycin (S) at 37 • C with 5% CO 2. The old medium was replaced with fresh medium every 3 days. Primary cortical neurons were used for experiments after culturing for 6 days.

Incubation of Neuronal Cells with Oxyhemoglobin for the Model of SAH In Vitro
To establish in vitro neural cell-based SAH model, primary cortical neurons were treated with 10 µ M oxyhemoglobin (OxyHb, O7109, Sigma, USA) in 1% DMSO for 24 h [21]. Control group cells were treated with 1% DMSO only. Treatment groups (SAH+LV-scramble, SAH +LV-Dexras1 + , and SAH +LV-Dexras1 − ) were prepared with primary cortical cells then exposed to OxyHb for 24 h. In addition, an activator of the cAMP-CREB pathway, 8-Bromo-cAMP sodium salt (MCE, Shanghai, China, HY-12306), was used to verify the effect of Dexras1 on cAMP-CREB pathway in SAH model. After these treatments, each measurement was conducted thrice to enhance the accuracy of the results.

In Vitro Culture of OLN-93 Rat Oligodendroglia Cells
OLN-93 rat oligodendroglial cells were purchased from Shanghai Binsui Biological Technology Co., Ltd. (Shanghai, China). The cells were cultured in DMEM with 10% heat-inactivated FBS, 100 U/mL P, and 100 µg/mL S. Cells were passaged once every 3 days and grown in an incubator at 37 • with 5% CO 2 .

Differentiation of OLN-93 Cells
The differentiation of OLN-93 cells into mature oligodendrocytes was performed as described by Van Meeteren et al. [22]. OLN-93 cells were plated at 24 mm diameter dishes in DMEM with 0.5% FBS. After overnight (O/N) incubation, the medium was replaced with serum-free DMEM and incubated for 3 days, following which the medium was replaced again with a fresh medium with 100 ng/mL IGF-I. Total protein was harvested for Western blot (WB) after 6 days. In addition, IGF-I-induced differentiated OLN-93 cells were seeded on PDL-C-coated 14 mm slides (Nest, China) before immunostaining with fluorescent maturation markers and comparative analysis against non-treated samples.

OLN-93 Cell Differentiation in Co-Culture with Neurons in an In Vitro Model of SAH
Primary neurons were isolated as previously described and were seeded on 24 mm inserts with 0.4 µm pore size (Transwell-Clear Polyester Membrane; Corning Inc., Corning, NY, USA) at a density of 0.5·106 cells with pre-suit corresponding medium, and incubated for 6 days. Then, IGF-I-treated differentiated OLN-93 cells were seeded below the neurons. Samples from each of the treatment groups were collected for protein analysis after coculture for 3 days. For immunofluorescence staining, IGF-I-differentiated cells were also plated on PDL-C-coated 14 mm slides and co-cultured for 3 days with each of the neuron treatment groups.

Western Blot Analysis
WB analysis was performed as previously described [18]. The left cerebral hemispheres of rats were extracted and flash-frozen in liquid nitrogen before storing at −80 • C until further analysis. Cellular proteins were collected by cell scraping. The protein samples were then homogenized in RIPA lysis buffer and centrifuged for 15 min at 12,000× g at 4 • C. Equal quantities of proteins (50 µg) were then separated on 8-15% SDS-PAGE gels and transferred ,anti-NG2 (1:500, Biorbyt, orb382135, Cambridge, UK) and anti-β-actin (1:1000, Cell Signaling, #4970), followed by corresponding secondary antibodies (1:5000, Biorbyt, orb572747/ orb557249) for 1 h at RT, and lastly, protein detection with a chemiluminescent reagent kit (ECL, Engreen Biosystem, Auckland, New Zealand). Non-saturated bands were selected to perform densitometry quantification using Fusion imaging system (Fusion fx 7 Spectra, Paris, France and the results were presented as a percentage, relative to the β-actin levels.

RNA Isolation and Quantitative RT-PCR
Total RNA was isolated from cultured neurons with RNAiso Plus reagent (TaKaRa, 9108, Dalian, China), following the kit operational guidelines. cDNA was prepared from mRNA with the PrimeScript ® RT reagent Kit With gDNA Eraser, following the kit operational guidelines (TaKaRa, RR047A). The relative expression of the genes of interest was determined by quantitative PCR using SYBR ® Premix Ex Taq™ II (TaKaRa, RR820A) and Premix Ex Taq RR390A (TaKaRa, RR390A). qPCR was performed with the BioMark HD Real-Time PCR System (Fluidigm). Thermal cyclic conditions were set to 2 min at 50 • C, 10 min at 95 • C, 30 s at 95 • C (denaturation), 30 s at 58-60 • C (annealing), and 30 s at 72 • C (extension) for 40 cycles. The overall volume per reaction was 10 µL, which consisted of 1 µL diluted cDNA (10 ng/µL), 5 µL Roche SYBR Green Master Mix, 0.5 µL double-distilled water, and 0.5 µL of relevant primers (10-mmol/L concentration).

Enzyme-Linked Immunosorbent Assay (ELISA)
The culture medium was retrieved from all primary neuron cultures and the IL-1β and TNF-α levels measured using an ELISA assay kit (Multisciences, Hangzhou, China), following the manufacturer's instructions.

Transmission Electron Microscopy (TEM)
TEM was performed as described previously [24]. Twenty-four hours after SAH, the deeply anesthetized rats of each group were sacrificed by intracardial perfusion with 0.9% saline followed by 4% paraformaldehyde. Then, the brain tissues were removed and post-fixed with 2% formaldehyde and 2% glutaraldehyde for 30 min. Next, the corpus callosum was minced into 1 mm 3 pieces and maintained O/N at 4 • C in the same fixation mixture, as described earlier. Following dehydration, samples were impregnated with epoxy resin and sectioned, before incubation with uranyl acetate and lead citrate. Finally, the electron micrographs were viewed using the Hitachi-7500 (Hitachi, Tokyo, Japan). Six random visual fields were imaged per section by means of TEM at magnification ×12,000.

Statistical Analysis
Data were expressed as means ± SD and analyzed with GraphPad Prism 8 (GraphPad Prism, San Diego, CA, USA). Unpaired t-tests or one-way ANOVAs were employed to analyze significance among the groups. p < 0.05 was considered significant.

Differentiation Disorders of Oligodendrocyte Precursor Cells and Myelin Damage after Subarachnoid Hemorrhage
First, we successfully established an in vivo animal model of SAH (Figure 2A). In this study, 23 rats died out of 132 SAH rats in total, hence the mortality rate is 17.4% (23/132). The myelin marker protein MBP in the subcortex (corpus callosum) of the left hemisphere was detected by WB, and it was found that MBP expression was significantly reduced after SAH ( Figure 2B,C). The electron microscopy results showed significant loss and dissolution of myelin sheaths and even axonal injury after SAH ( Figure 2F). Next, immunofluorescence and Western blot were used to verify OPC differentiation dysfunction after SAH. The immunofluorescence results showed that the levels of mature oligodendrocyte marker CNPase were decreased and the levels of oligodendrocyte precursor cell marker NG2 were increased after SAH ( Figure 2G). The WB results confirmed the decreased CNPase and increased NG2 levels, showing gradual changes peaking at 24 h ( Figure 2B,D,E).

Dexras1, TNF-α, and IL-1β Brain Expression Was Raised along with Glial Cell Activation after SAH In Vivo
Immunofluorescence staining was applied to determine the histological localization of Dexras1 in neurons, astrocytes, oligodendrocytes, and microglia, after SAH. Based on our data, Dexras1 was ubiquitous in neuronal cytoplasm in the neocortex and hippocampus after SAH, with no conspicuous expression in microglia, astrocytes, and oligodendrocytes ( Figure 3D). Next, the Dexras1, TNF-α, and IL-1β levels, in the subcortex of the left hemisphere at different time intervals after SAH in vivo were examined by WB and ELISA. Compared with the sham group, the expression of Dexras1 after SAH clearly increased, peaking at 24 h ( Figure 3A,B, p < 0.01). Relative to the control values, the expression levels of TNF-α ( Figure 3G p < 0.001) and IL-1β ( Figure 3H p < 0.001) were raised to different degrees. At the same time, the expression of GFAP (astrocyte marker) was also clearly enhanced after SAH ( Figure 3A,C). The analysis of immunofluorescence results showed that the abundance of astrocytes and microglia was clearly increased as compared to the sham, and that astrocyte cell bodies were visibly thicker ( Figure 3E,F).
CNPase and increased NG2 levels, showing gradual changes peaking at 24 h ( Figure  2B,D,E).   After the confirmation of effective sequences used for Dexras1 overexpression and knockdown in vitro ( Figures S1 and S2), lentiviral vectors carrying respective plasmids were injected into the lateral ventricles of SD rats. It was found that six U lentivirus Dexras1 + and Dexras1 − reagents could significantly increase or decrease the expression of Dexras1 after SAH, respectively ( Figure 4A,B). At the same time, the rat myelin marker-myelin basic protein (MBP) expression level was further reduced in the SAH+LV-Dexras1 + rats (p < 0.05, Figure 4A,D), as compared to that in the SAH+LV-Scramble rats. However, after down-regulation by LV-Dexras1 − , relative to the SAH+LV-Scramble rats, the expression of MBP significantly increased (p < 0.05, Figure 4A,D). In the rat brain cortex of the left hemisphere the immunofluorescence staining results showed that MBP expression was further reduced and the texture of the myelin sheath became weaker in the SAH +LV-Dexras1 + group ( Figure 4J). In contrast, MBP expression was markedly increased and the myelin texture was clearer in the SAH +LV-Dexras1 − group relative to the SAH+LV-Scramble rats ( Figure 4J). Structural alterations of myelin sheaths in the corpus callosum were examined by electron microscopy. The results showed that myelin sheaths in the SAH+LV-Dexras1 + group were more severely damaged or even broken, and the axon separation became more evident when compared to alterations in the SAH+LV-Scramble rats ( Figure 4H). However, the myelin sheath swelling and axon separation were clearly alleviated in the SAH +LV-Dexras1 − group ( Figure 4H).
Next, marker proteins for astrocyte (GFAP) and microglial (Iba1) activation were detected by WB. It was found that the expression of GFAP and Iba1 significantly increased after SAH (p < 0.05, Figure 4A,C,E). The expression of GFAP, Iba1, TNF-α, and IL-1β, was elevated in SAH+LV-Dexras1 + rats, as compared to the SAH+LV-Scramble rats, whereas this expression was significantly decreased in SAH +LV-Dexras1 − group (p < 0.05, Figure 4A,C,E,L,M). In addition, the astrocyte and microglial activation marker proteins, GFAP and Iba1, respectively, were detected by immunofluorescence staining in the rat brain cortices. Astrocytic and microglial activation was increased in the SAH+LV-Dexras1 + group, relative to that in the SAH+LV-Scramble rats ( Figure 4J,K). In contrast, the astrocyte and microglial activation was reduced in the SAH +LV-Dexras1 − group relative to the SAH+LV-Scramble rats ( Figure 4J,K).
Then, the changes of CNPase (oligodendrocyte marker) and NG2 (OPC marker) levels after Dexras1 lentivirus-based interventions were investigated by WB and immunofluorescence staining. The results showed that CNPase levels in the SAH +LV-Dexras1 + group were decreased (p < 0.05, Figure 4A,G), while there was a significant increase in NG2 levels (p < 0.05, Figure 4A,F), as compared to the levels in the SAH+LV-Scramble rats. However, there was a recovery of CNPase levels in the SAH +LV-Dexras1 − rats (p < 0.05, Figure 4A,G), while NG2 levels were significantly decreased in comparison to the levels in the SAH+LV-Scramble rats (p < 0.05, Figure 4A,F). The immunofluorescence results also showed that the cerebral expression of CNPase was reduced in the SAH +LV-Dexras1 + rats, accompanied with significantly disordered myelin texture. In addition, NG2 was elevated in the SAH +LV-Dexras1 + rats ( Figure 4I). However, in the SAH +LV-Dexras1 − rats, NG2 expression was significantly reduced relative to the SAH+LV-Scramble rats, whereas CNPase expression was elevated, accompanied with the restoration of myelin structure ( Figure 4I).
The escape latencies (EL) of all five groups of rats that went through acquisition training (5 days) are illustrated in Figure 5G. All rats exhibited sizeable enhancements in the EL after 5 days of training (F (4, 76)6.063, p = 0.002, repeated measures ANCOVA), suggesting that they retained the memory of the escape platform (EP). Repeated measures ANCOVA suggested no relationship between training days and groups (F (4, 76)329.454, p = 0.003). Hence, all the animals had learned the task adequately. Moreover, the EL of the SAH rats was significantly longer, relative to the sham rats (F (4, 76)14.78, p < 0.0001, Figure 5G). In addition, the SAH+ LV-Dexras1 + group showed clearly aggravated learning deficiencies (F (4, 76)2.955, p = 0.0252, Figure 5G) while the SAH +LV-Dexras1 − group did not (F (4, 76)3.732, p = 0.0079, Figure 5G). After the platform was removed, the results indicated that the residence time and the number of platform crossings of the SAH rats were markedly reduced as compared to those of the sham rats (p < 0.01, Figure 5C-F). The SAH+ LV-Dexras1 + rats showed further reductions relative to the SAH+LV-Scramble rats (p < 0.01, Figure 5C-F). However, in the SAH +LV-Dexras1 − group, the residence time, distance, and the number of crossing the platform increased after SAH (p < 0.01, Figure 5C-F).  SAH+LV-Scramble rats; (B) evaluation of water content in brain tissue in each group after SAH, * p < 0.05 sham vs. SAH rats, * p < 0.05 SAH+LV-Dexras1 + vs. SAH+LV-Scramble rats, * p < 0.05 SAH+LV-Dexras1 − vs. SAH+LV-Scramble rats; (C) day 6 tracks of all rats; recording was done for 90 s after training platform removal; (D) representative images of the shortest path of each group to the platform during the acquisition trials in the MWM examination; (E) the number of times each group crossed the platform; recording was done for 90 s after training platform removal, (* p < 0.05, ** p < 0.01); (F) analysis of the percentage of time rats remained in the target quadrant during the spatial exploratory test, (** p < 0.01); (G) 7 days post-SAH, rats were allowed to search for the hidden platform for 90 s; each acquisition trial was part of 4 trials, one per day, for 5 days. Data represent mean ± SD, n = 5, * p < 0.05 sham vs. SAH rats, * p < 0.05 SAH+LV-Dexras1 + vs. SAH+LV-Scramble rats, * p < 0.05 SAH+LV-Dexras1 − vs. SAH+LV-Scramble rats.

Dysdifferentiation of Oligodendrocyte Precursor Cells Occurred in the In Vitro SAH and Was Susceptible to Dextras1 Modulation
In the experiments that followed, we tested the differentiation of oligodendrocyte precursor cells in the model of SAH in vitro and in co-culture with IGF-treated cells. The results showed that the differentiation and maturation of OLN-93 oligodendrocyte precursor cells were inhibited after SAH ( Figure 6A). WB also revealed that NG2 expression was markedly elevated and CNPase was clearly reduced post-SAH in IGF−1-co-cultured cells (p < 0.05, Figure 6B,C). Additionally, the immunofluorescence data revealed that after upregulation of Dexras1, CNPase levels were reduced and NG2 was further increased in the SAH +LV-Dexras1 + rats ( Figure 6D). In contrast, CNPase was elevated, while NG2 was significantly reduced in the SAH +LV-Dexras1 − rats relative to the SAH+LV-Scramble rats ( Figure 6D). Moreover, the WB results also showed that the CNPase levels were further decreased (p < 0.05, Figure 6E,F), while NG2 levels were further significantly increased in the SAH +LV-Dexras1 + rats, relative to the SAH+LV-Scramble rats (p < 0.05, Figure 6E,F). On the contrary, in the SAH +LV-Dexras1 − group, the expression of CNPase increased (p < 0.05, Figure 6E,F), whereas NG2 expression significantly decreased, as compared to SAH+LV-Scramble group levels (p < 0.05, Figure 6E,F).

Dexras1 May Inhibit the Differentiation of Oligodendrocyte Precursor Cells through the cAMP-CREB Pathway
After up-regulation of Dexras1, the expression of IL-1β and TNF-α was elevated in the SAH+LV-Dexras1 + rats relative to the SAH+LV-Scramble rats (p < 0.05, Figure 7A,B,D), accompanied by significant decreases in CREB and pCREB protein levels (p < 0.05, Figure 7A,C). In addition, the expression of IL-1β and TNF-α was significantly lower in the SAH +LV-Dexras1 − rats, relative to the SAH+LV-Scramble rats (p < 0.05, Figure 7A,B,D), accompanied with significantly increased CREB and pCREB protein levels (p < 0.05, Figure 7A,C). Similar to previous experimental results from the in vivo SAH model, it was found that CREB expression was significantly reduced in the SAH in vitro, and IL-1β and TNF-a levels were significantly increased ( Figure 7A-D).
In contrast, CREB and pCREB expression was significantly elevated after administering 8-Bromo-cAMP sodium salt, while the IL-1β and TNF-α levels were drastically decreased (p < 0.05, Figure 8A-C). These results indicate that 8-Bromo-cAMP sodium salt activates the expression and signaling activity of the cAMP-CREB pathway. Next, we found that the levels of CREB and pCREB in the Dexras1 + (overexpression)+8-Bromo-cAMP group were significantly lower than in the SAH+8-Bromo-cAMP group, accompanied with a reduction in CNPase (p < 0.05, Figure 8D,E,F). However, the levels of CREB and pCREB in the Dexras1 − (knockdown)+8-Bromo-cAMP group were significantly higher than in the SAH+8-Bromo-cAMP group, accompanied by an increase in CNPase (p < 0.05, Figure 8D,E,F). Further investigation revealed that TNF-α, IL-1β, and NG2 levels were elevated in the SAH+8-Bromo-cAMP+Dexras1 + group, relative to the SAH+8-Bromo-cAMP-group (p < 0.05, Figure 8D,G). However, TNF-α, IL-1β, and NG2 levels in the cells of SAH+8-Bromo-cAMP+Dexras1 − group were considerably reduced as compared to the levels in SAH+8-Bromo-cAMP-treated cells (p < 0.05, Figure 8D,G).

Discussion
A growing body of research evidence demonstrates the presence of demyelination injury after SAH [25], but there is only a limited number of studies on the underlying mechanism. In this study, we found morphological myelin damage and dysdifferentiation of oligodendrocytes after SAH. Simultaneously, Dexras1 levels increased significantly, and further functional research showed that Dexras1 is related to myelin damage and the impaired oligodendrocyte differentiation. Further research revealed that Dexras1 may cause the impairment of oligodendrocyte differentiation by inhibiting the cAMP-CREB pathway after SAH (Figure 9).
The damage caused by SAH includes neuronal necrosis and apoptosis, delayed cerebral ischemia, and the disruption of the blood-brain barrier. The difference between this present and previously reported research approach is that we focus on white matter damage, which is mainly manifested by myelin damage and oligodendrocyte differentiation disorder. As opposed to gray matter damage, white matter injury is more sensitive to ischemia although being reversible after the brain insult. This reminds us not only to pay attention to neuronal injury, but also to take into account the myelin damage when conducting research on SAH.
Oligodendrocyte precursor cells (OPCs) are the main source of mature oligodendrocytes in CNS, and their impaired differentiation is one of the main causes of demyelination in diseases such as multiple sclerosis and autoimmune encephalitis [8,26]. However, there are few studies dealing with the role of oligodendrocyte differentiation disorders in neurological dysfunction after SAH. This study showed that OPC differentiation disorder took place after SAH, although it could resolve over time. In a previous study, Joseph, M.J [27] showed that the differentiation of OPCs was hindered after intracerebral hemorrhage. We have found that the OPC differentiation disorder occurs acutely, in the early stage after SAH, thus being different from the oligodendrocyte differentiation disorder caused by multiple sclerosis. This may suggest that effective intervention in the early stage of SAH will help to improve the outcome and further prognosis. Moreover, previous studies have found an evident inflammatory response in the early stages after SAH. Our study likewise found that TNF-a and IL-1β brain levels increased significantly at 24 h after SAH, accompanied with the impairment of oligodendrocyte differentiation. Therefore, these results show that inflammatory factors may play a pivotal role in inhibiting the differentiation of OPCs. Hence anti-inflammatory interventions in addition to those based on stem cells, biomaterials, and nanoparticles, may become viable therapeutic options for OPC differentiation disorders [28] and should be studied for the treatment of SAH in the future.

Discussion
A growing body of research evidence demonstrates the presence of demyelination injury after SAH [25], but there is only a limited number of studies on the underlying mechanism. In this study, we found morphological myelin damage and dysdifferentiation of oligodendrocytes after SAH. Simultaneously, Dexras1 levels increased significantly, and further functional research showed that Dexras1 is related to myelin damage and the impaired oligodendrocyte differentiation. Further research revealed that Dexras1 may cause the impairment of oligodendrocyte differentiation by inhibiting the cAMP-CREB pathway after SAH (Figure 9). The damage caused by SAH includes neuronal necrosis and apoptosis, delayed cerebral ischemia, and the disruption of the blood-brain barrier. The difference between this present and previously reported research approach is that we focus on white matter damage, which is mainly manifested by myelin damage and oligodendrocyte differentiation disorder. As opposed to gray matter damage, white matter injury is more sensitive to ischemia although being reversible after the brain insult. This reminds us not only to pay attention to neuronal injury, but also to take into account the myelin damage when conducting research on SAH.
Oligodendrocyte precursor cells (OPCs) are the main source of mature oligodendrocytes in CNS, and their impaired differentiation is one of the main causes of demyelination in diseases such as multiple sclerosis and autoimmune encephalitis [8,26]. However, there are few studies dealing with the role of oligodendrocyte differentiation disorders in neurological dysfunction after SAH. This study showed that OPC differentiation disorder took place after SAH, although it could resolve over time. In a previous study, Joseph, M.J [27] showed that the differentiation of OPCs was hindered after intracerebral hemorrhage. We have found that the OPC differentiation disorder occurs acutely, in the early stage after SAH, thus being different from the oligodendrocyte differentiation disorder caused by multiple sclerosis. This may suggest that effective intervention in the early stage Dexras1 is a small G protein activated by nitric oxide (NO), which is excessively produced in activated microglia/macrophages as well as in neurons [29]. The research done so far on Dexras1 mainly focused on regulating circadian rhythms, tumor cell apoptosis, and calcium ion channels [30,31]. However, Khan et al. [9] found an improvement to myelin damage in experimental optic neuritis after Dexras1 knockdown, which may suggest that Dexras1 modulates myelin damage. We hypothesize that Dexras1 can be also involved in the myelin damage after SAH. Interestingly, in this present study we found that Dexras1 levels significantly increased after subarachnoid bleeding. Further investigations revealed that Dexras1 was involved in WMD after SAH, by mediating the mechanism of OPC differentiation impairment and a subsequent myelin damage. These findings further support the notion that Dexras1 is involved in white matter damage after SAH.
Although in this study we provided a novel insight into the mechanisms of brain injury after SAH, several shortcomings still remain in this regard. For example, we found that there was a positive correlation between the intensity of inflammation and oligodendrocyte differentiation disorder, as well as myelin damage after SAH. However, in order to establish the direct relationship between inflammation, oligodendrocyte differentiation disorder, and myelin damage after SAH, further study is required. In addition, we have investigated the role of Dexras1 only in WMD after SAH. Importantly, the mechanisms of Dexras1mediated modes of cell death after SAH, such as apoptosis, ferroptosis, and pyroptosis, still need to be explored in future studies, which we are planning to pursue.

Conclusions
In summary, in this study we found that Dexras1 may mediate oligodendrocyte differentiation impairment and myelin damage after SAH, and explored the underlying mechanisms. These results may pave the way for further studies of WMD after SAH and provide the molecular basis for novel therapeutic interventions.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.