Loss of monocarboxylate transporter 1 aggravates white matter injury after experimental subarachnoid hemorrhage in rats

Abstract

Monocarboxylic acid transporter 1 (MCT1) maintains axonal function by transferring lactic acid from oligodendrocytes to axons. Subarachnoid hemorrhage (SAH) induces white matter injury, but the involvement of MCT1 is unclear. In this study, the SAH model of adult male Sprague-Dawley rats was used to explore the role of MCT1 in white matter injury after SAH. At 48 h after SAH, oligodendrocyte MCT1 was significantly reduced, and the exogenous overexpression of MCT1 significantly improved white matter integrity and long-term cognitive function. Motor training after SAH significantly increased the number of ITPR2⁺SOX10⁺ oligodendrocytes and upregulated the level of MCT1, which was positively correlated with the behavioral ability of rats. In addition, miR-29b and miR-124 levels were significantly increased in SAH rats compared with non-SAH rats. Further intervention experiments showed that miR-29b and miR-124 could negatively regulate the level of MCT1. This study confirmed that the loss of MCT1 may be one of the mechanisms of white matter damage after SAH and may be caused by the negative regulation of miR-29b and miR-124. MCT1 may be involved in the neurological improvement of rehabilitation training after SAH.

Keywords

microRNAs / monocarboxylate transporter 1 / motor training / subarachnoid hemorrhage / white matter injury

1 Introduction

Subarachnoid hemorrhage (SAH), one of the most common subtypes of hemorrhagic stroke, is a complex cerebrovascular disease with high mortality and disability rates [1]. Although the prevalence of SAH is only 4.4% in all stroke types in China, the mortality rate of SAH patients can reach 50% within 72 h [2,3]. Early brain injury, including white matter injury, may be the main component responsible for the poor prognosis [4,5]. Reijmer et al. detected microstructural white matter abnormalities and cognitive impairment early after aneurysmal SAH [6]. Injury within the white matter tract can damage sensorimotor and cognitive function and lead to severe neurological dysfunction, making the extent of white matter disruption a main indicator of clinical outcomes [7]. White matter accounts for approximately 50% of human brain tissue, and intervention strategies that target white matter can help ameliorate the neurological outcome after experimental SAH [8,9]. However, the underlying mechanism of white matter injury after SAH still needs further research.

A molecular architecture called the “axo-myelinic synapse” (AMS) is present between oligodendrocytes and neurons in the central nervous system (CNS) [10]. Lactate produced by glycolysis can be transferred from oligodendrocytes to axons via the AMS to further decompose and release energy and thus meet the regeneration and maintenance requirements of the axonal myelin sheath [11]. Therefore, AMS protection may promote the remyelination of damaged axons and axon integrity, thus alleviating white matter injury after SAH.

Monocarboxylate transporters (MCTs) comprise a protein family of bidirectional transporters that can transport protons and monocarboxylates, including lactate, pyruvate, and ketone bodies, across the plasma membrane, in which MCT1, MCT2, and MCT4 are localized in the CNS [12]. MCT1 is localized in the oligodendrocytes of the corpus callosum and cerebellar white matter and plays an important role in the energy transport mediated by AMS [13]. This study aims to provide more evidence on the potential role of MCT1 in white matter injury as well as on the changes and associated mechanism of MCT1 expression after experimental SAH.

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a size of 19–24 nucleotides, and they are involved in the regulation of gene expression at the post-transcriptional level. Nearly 90% of human genes are regulated by miRNAs [14]. In both human and mouse, miR-29a, miR-29b, and miR-124 can selectively target the 3′ untranslated regions of MCT1 mRNA [15,16]. Moreover, several reports indicate that the expression levels of miR-29b and miR-124 are significantly increased both in vivo and in vitro after stroke [17,18].

In modern society, physical exercise and its effects on the body and mental health are important. Motor training has a protective effect on white matter in the spinal cord [19,20]. In the CNS, oligodendrocytes envelop axons to form the myelin sheath, which allows rapid action potential conduction and provides energy and metabolic support for neurons. The oligodendrocyte population has been considered as a functionally homogeneous cell group for decades until investigators demonstrated their heterogeneity, and different subtypes of mature oligodendrocytes are enriched in specific regions of the adult brain and can be influenced by motor training [21]. The ITPR2⁺SOX10⁺ oligodendrocytes are distributed in the cortex, hippocampus, and corpus callosum, and they are essential for the rapid myelination for complex motor learning [21,22].

Therefore, the translation inhibition of MCT1 mediated by miR-29b/miR-124 after SAH decreases the protein level of MCT1, thus inhibiting the energy transfer function of AMS, leading to the damage of white matter and its remyelination; motor training can effectively improve the motor dysfunction after SAH, and ITPR2⁺SOX10⁺ oligodendrocytes/MCT1 might be the involved in the underlying mechanism. This study investigated these hypotheses in an experimental SAH model generated by autologous arterial blood injection into the prechiasmatic cisterns of adult male Sprague-Dawley (SD) rats.

2 Materials and methods

2.1 Ethics and animals

Approximately eight-week-old male SD rats weighing 280–320 g were obtained from Zhaoyan New Drug Research Center Co., Ltd. (Suzhou, China). All animals were healthy and had not been given any drugs, tests, or previous procedures. All animals were housed in a quiet room under a regular light/dark schedule and a constant temperature of approximately 23 °C and were allowed to eat and drink freely. All animals were placed under general anesthesia before euthanasia and fixation–perfusion. All institutional and national guidelines for the care and use of laboratory animals were followed. All animal research data were written according to ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines. Sample sizes were determined by power analysis during the animal ethics dossier application.

2.2 Experimental design

2.2.1 Experiment 1: time course and cellular localization of MCT1 and its roles in myelin damage and repair after SAH

Experiment 1 had two parts. In part 1, 60 rats were randomly divided into six groups with 10 rats each, including the sham group and five experimental groups ordered by time points of 3, 6, 12, 24, and 48 h after SAH. At the indicated time point after SAH, rats were euthanized by chloral hydrate and transcardial perfused with phosphate buffered saline (PBS). The first third of the brain tissues were obtained for immunofluorescence analysis, and the corpus callosum separated from the back two thirds were obtained for Western blot. In part 2, 72 rats were randomly assigned to four groups with 18 rats each, including the sham group, SAH group, SAH+ vector group, and SAH+ MCT1 overexpression plasmid (over-MCT1) group. By using a table of random numbers, a technician who did not participate in this study randomly assigned the 18 rats in each group into two subgroups (of six and 12 rats). For six rats, the first third of the brain tissues were obtained for immunofluorescence analysis, and the corpus callosum separated from the back two thirds was obtained for Western blot 48 h after SAH. For the 12 other rats, three behavior tests were conducted until 35 days after SAH, and then the rats were euthanized using chloral hydrate.

2.2.2 Experiment 2: effect of rotarod training on SOX10⁺ITPR2⁺ oligodendrocytes and MCT1

In experiment 2, 80 rats were randomly divided into following four groups with 20 rats each, including the sham group, sham+ rotarod training group, SAH group, and SAH+ rotarod training group. By using a table of random numbers, a technician who did not participate in this study randomly assigned the 20 rats in each group into three subgroups with four, six, and 10 rats. Four rats received two-day rotarod training after SAH, and the first third of their brain tissues were obtained for immunofluorescence analysis. The 16 other rats received seven-day rotarod training after SAH. Six of these rats were euthanized after the last training session, the first third of their brain tissues were obtained for immunofluorescence analysis, and the corpus callosum separated from the back two thirds were obtained for Western blot. The 10 remaining rats were sacrificed after undergoing the rotarod and adhesive removal tests at 35 days after SAH. For the two non-training groups, the rats were euthanized at the expected time point after SAH, and the corresponding brain tissues mentioned above were separated and analyzed together with the training groups.

2.2.3 Experiment 3: roles of miRNA-29b and miRNA-124 in regulating MCT1

In Experiment 3, the CSF and corpus callosum samples from 18 SD rats that had been randomly assigned to three groups of six were first collected to assess the changes in miRNA-29b and miRNA-124 levels over time after SAH. Then, 30 rats were randomly divided into five groups with six rats each, including the sham group, SAH group, SAH+ antagomir-negative control (antagomir-NC) group, SAH+ antagomir-29b-3p group, and SAH+ antagomir-124-3p group. After the indicated treatments, rats were euthanized 48 h after SAH, the first third of their brain tissues were obtained for immunofluorescence analysis, and the corpus callosum separated from the back two thirds were obtained for Western blot, respectively.

Fig. S1 illustrates the experimental designs. The total mortality and exclusion rates of experimental rats are shown in Table S1.

2.3 Establishment of SAH model

The autologous arterial blood was injected into the prechiasmatic cistern to create the experimental SAH model in rats [23]. Briefly, the rats were intraperitoneally injected with 4% chloral hydrate (1 mL/100 g body weight, Aladdin, Shanghai, China) and then fixed in a stereotactic apparatus frame (ZH-Lanxing B type stereotactic frame, Anhui Zhenghua Biological Equipment Co. Ltd., Huaibei, China). After the skull and bregma were exposed, a small hole was drilled 7.5 mm anterior to the bregma and 1–2 mm from the midline. Next, a needle connected to an aseptic syringe with 300 μL of autologous arterial blood collected by cardiac puncture was inserted into the prechiasmatic cistern. The rats in the sham group were injected with 300 μL of physiologic saline solution. Immediately after SAH was induced, rats were injected with 3 mL of physiologic saline solution intraperitoneally to prevent dehydration. Before being returned to their cages, the rats were allowed to recover from the surgery for 60 min, and their heart rate and rectal temperature were monitored, the latter maintained at 37±0.5 °C. Rats were excluded if serious postoperative complications such as hemiplegia, irritability, or dehydration occurred. Subsequently, at a different time point after surgery, the rats were euthanized, and their total brain tissue was removed. The prechiasmatic cistern and arachnoid space of the brain were obviously stained by blood (Fig. S1). Moreover, the SAH grading scores of blood volume and location were performed based on a published grading system [24], and the rats with the grade≤7 within 48 h after SAH were excluded from this study. The brain tissues of the cortex and corpus callosum were obtained and used for Western blot analysis, and the total coronal sections containing the corpus callosum brain tissues were subjected to immunofluorescence staining.

2.4 Plasmid transfection in rats

Plasmid transfection in rats was performed as previously described [25]. Briefly, 10 μL of Entranster in vivo DNA transfection reagent (Engreen Biosystem Co., Ltd., 18688-11-2) was added to 5 μL of MCT1 overexpression plasmid (MCT1 Transcript ID: NM_012716.2). The plasmid sequences are listed in Table S2. Then, the solution was left on ice for 20 min to fully mix. Finally, under the guidance of a stereotaxic apparatus, 15 μL of the solution was injected intracerebroventricularly into the rats after anesthesia. The micro-syringe remained in place for 60 s to prevent reflux. The puncture point of the lateral ventricle was located at 1.5 mm posterior, 1.0 mm lateral, and 3.2 mm below the horizontal plane of the bregma. At 48 h after this process, the SAH model was established. To evaluate transfection efficiency, we used YFP labeled MCT1 overexpressed plasmid. The transfection efficiency of plasmid is shown in Fig. S2.

2.5 Antagomir transfection in rats

Antagomir-29b-3p and antagomir-124-3p (miR30000801-4-5 and miR30000828-4-5, Ribobio Co., Ltd., Guangzhou, China) with a concentration of 10 nmol/L were prepared according to the manufacturer’s instructions. Under the guidance of a stereotaxic apparatus, 20 μL of the solution was injected intracerebroventricularly into the rats after anesthesia. The puncture point of the lateral ventricle was located at 1.5 mm posterior, 1.0 mm lateral, and 3.2 mm below the horizontal plane of the bregma. After 48 h, the SAH model was established. To evaluate transfection efficiency, we used Cy5 labeled antagomir-NC, antagomir-29b-3p, and antagomir-124-3p. The transfection efficiency of antagomir is showed in Fig. S2.

2.6 Western blot analysis

Western blot analysis was performed as preciously described [26]. Briefly, a lysis buffer containing phenylmethylsulfonyl fluoride was used to lyse the brain tissue samples mechanically. The protein concentration was detected through the bicinchoninic acid (BCA) method by using the enhanced BCA protein assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). On a prepared 10% sodium dodecyl sulfate-polyacrylamide gel, the protein samples (40 or 20 μg/lane) and a molecular weight marker (4 μL/lane; Thermo Fisher Scientific, Waltham, MA, USA) were loaded, separated, and transferred to a polydisperse A-vinyl fluoride membrane (Millipore Corporation, Billerica, USA) electrophoretically. Afterward, at room temperature, 5% non-fat milk was used to block the membrane for 1 h. Then, the primary antibodies shown in Tables S3 and S4 were incubated with the membrane overnight at 4 °C. Next, a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was incubated with the membrane for 1 h at room temperature and washed thrice with PBST (PBS+ 0.1% Tween 20). Finally, the membrane was detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA). To analyze the density of the protein bands, we used the ImageJ software (NIH, Bethesda, MA, USA).

2.7 Immunofluorescence staining

As described previously [27], brain tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm-thick sections. The primary antibodies shown in Tables S3 and S4 were used after the antigen was repaired, and the non-specific binding was blocked. The concentration of the same antibody in different experiments or groups was ensured to be consistent. The sections were incubated with these antibodies overnight at 4 °C. Then, the brain sections were incubated with the corresponding secondary antibodies (Alexa Fluor 488 donkey anti-rabbit IgG antibody and Alexa Fluor 555 donkey anti-mouse IgG antibody, Life Technologies, Carlsbad, CA, USA, 1:500 dilution) at 37 °C for 1 h. After covering with anti-fading mounting medium containing 4,6-diamino-2-phenylindole (DAPI, SouthernBiotech, Birmingham, AL, USA), the brain sections were observed under a fluorescence microscope (Olympus BX50/BX-FLA/DP70, Olympus Co., Tokyo, Japan) and analyzed using the Image-Pro Plus program (National Institutes of Health, Beshesda, MD, USA) by a technician who was blinded to the experimental groups. The numbers of β-APP-positive cells and the relative fluorescence intensity of all staining were identified and counted in ipsilateral corpus callosum from three random coronal sections per brain. In each group, the same area was selected for observation, and the final statistical analysis was consistent.

2.8 Behavior tests

2.8.1 Rotarod test

Motor balance and coordination were evaluated using the rotarod test as previously described [28]. Briefly, rats were placed on a rotating rod with an initial speed of 10 rpm accelerating to 40 rpm within 5 min. The total time before falling off the accelerating rod was recorded as the latency to fall by a blinded investigator. Four consecutive trials were conducted for each rat on each testing day with a 5 min intermission time between tests. The average latency time of 2–4 trials was recorded as the result of each testing day on a graph. Rats were trained for 3 days before surgery, and the average latency to fall on the third day was recorded as the baseline value. The testing days were 3, 5, 7, 10, 14, 21, 28, and 35 day after surgery.

2.8.2 Adhesive removal test

To assess sensorimotor function and tactile response, we performed the adhesive removal test as reported previously [29]. Under equal pressure, two adhesive tapes (10 mm × 10 mm) were applied to the animal forepaws, and the positioning of the first adhesive tape (right/left) was switched at each testing sequence. The time for the rats to remove the two adhesive tapes was measured by a blinded observer, and the maximum observation period was set to 120 s. Rats were trained for 3 days before surgery, and the time to remove the adhesive tapes on the last day was recorded as the baseline value. Adhesive removal tests were performed on the same days as the rotarod test.

2.8.3 Morris water maze test

To assess spatial cognitive and memory function, we employed the Morris water maze test as described previously [28]. Briefly, a circular pool (diameter 180 cm, depth 50 cm; type RD1101-MWM-G, Mobile Datum, Shanghai, China) was filled with water to a height of 30 cm and maintained at room temperature. A square platform (12 cm × 12 cm) was placed approximately 1.5 cm under the water surface in one quadrant of the pool. From day 29 to day 33 after SAH, the rat was placed in the quadrant without the platform and allowed to find the hidden platform within 60 s. After the rat stood on the platform for at least 2 s, the time it spent to find the hidden platform was recorded as the escape latency time. The rat was allowed to stand on the platform for 30 s after each trial. Every rat was trained for 3 days before SAH and had three trials on each testing day, and the mean latency was quantified. At day 34 after SAH, the hidden platform was removed to evaluate the memory function. A single 60-s probe trial was conducted for each rat, and the time it spent in the quadrant, where the hidden platform was placed previously, was recorded as the target quadrant time. The swim speed of each rat was recorded using the software provided by the manufacturer.

2.9 Real-time polymerase chain reaction

Total RNA was isolated from CSF and corpus callosum by using the miRNeasy Kit (QIAGEN) according to the manufacturer’s instructions. cDNA was synthesized from 100 ng of total RNA by using the All-in-One qRT-PCR detection kit (QP015, GeneCopoeia, USA). A mixture with a total volume of 25 μL was heated to 37 °C for 60 min, and reactions were stopped by heating to 85 °C for 5 min. qPCR for miRNA was performed using the same kit (QP015, GeneCopoeia, USA). The reactions were carried out in duplicate in total volumes of 20 μL containing 2 μL of first-stand cDNA (1:5 dilution) and 2 μL of All-in-One miRNA qPCR primer (for CSF: rno-miR-29b-3p, RmiRQP0373; rno-miR-124-3p, RmiRQP0074; U6, RmiRQP9003; GeneCopoeia, USA; for corpus callosum: rno-miR-29b-3p, T0703; rno-miR-124-3p, T1111; U6, Lot R0529; Ribobio Co., Ltd., Guangzhou, China) with a final concentration of 200 nmol/L. The parameters for the PCR cycle are as follows: 95 °C/10 min, 40 cycles of 95 °C/10 s, 60 °C/20 s, and 72 °C/15 s, and the melt-curve analysis was performed using StepOne software (ABI, CA, USA) after each experiment to verify the primer specificity. Fold changes in the miRNA expression were determined using the DDCT method [30] by an individual who was blinded to experimental grouping.

2.10 Statistical analysis

All data are presented as the mean±SD. GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Data sets in each group were tested for normality of distribution by using the Kolmogorov–Smirnov test, and all data exhibited a normal distribution. Data were analyzed using two-tailed Student’s t test to compare differences between two groups and using one- or two-way ANOVA to compare multiple groups. Two-tailed Pearson correlation analyses was to determine the correlation between the two groups. A P value less than 0.05 indicated statistically significant difference. The 95% confidence intervals and effect sizes were also considered. Cohen’s D was used to express the magnitudes of the effect of the two-tailed Student’s t test, and η² and partial η² for one- or two-way ANOVA. All statistical analysis methods are summarized in Table S5.

3 Results

3.1 Levels of MCT1 were significantly decreased in brain tissues after a 48 h SAH induction

To investigate the protein levels of MCT1 in rat corpus callosum tissues at different time points after SAH induction, we performed Western blot analysis. The results showed that the expression level of MCT1 was not significantly downregulated until 48 h after SAH induction compared with the sham group (Fig. 1A). After double immunostaining against MCT1 and the oligodendrocyte marker Olig2, MCT1 was localized to the oligodendrocyte in the corpus callosum region, the expression levels of MCT1 48 h after SAH induction was reduced, and the number of oligodendrocyte seemed to be similar (Fig. 1B and 1C). Therefore, 48 h after SAH might serve as the optimal time point to intervene in the expression of MCT1 for further experiments.

3.2 MCT1 overexpression alleviated acute white matter injury after SAH in rats

To determine whether MCT1 is involved in acute white matter injury after SAH, we referred to the overexpression of MCT1 via a recombinant plasmid (Over-MCT1). We detected the efficacy of MCT1 overexpression in brain tissues via Western blot and immunofluorescence analyses. The result of the Western blot assay showed that the levels of MCT1 in the sham and MCT1 overexpression groups were significantly higher than those in the SAH and vector groups, and no significant difference was observed between them (Fig. 2A). The immunofluorescence assay similarly showed that compared with the vector group, the overexpression of MCT1 increases the level of MCT1 localized to the oligodendrocyte in the corpus callosum region (Fig. 2B and 2C). Therefore, the transfection of overexpression plasmid can increase the expression level of MCT1 in brain tissue.

As an established indicator of axonal damage, β-amyloid precursor protein (β-APP) was used to assess white matter injury [31]. The accumulation of β-APP significantly increased along NF200-positive neurofilaments in the corpus callosum at 48 h after SAH in all SAH groups. However, the number of β-APP per mm² was significantly lower in the MCT1-overexpression group than in the vector group, indicating a lower degree of axonal damage (Fig. 2D and 2E). The integrity of myelin and the degree of demyelination were also measured by detecting the changes in myelin basic protein (MBP) and non-phosphorylated neurofilament H (monoclonal clone ID: SMI 32) in the corpus callosum at 48 h after SAH. Following SAH operation, all rats exhibited a decrease in MBP protein level and an increase in SMI 32 protein level compared with the sham group based on Western blot analysis. The MCT1 overexpression group exhibited greater white matter integrity than the vector group, as reflected in the significantly increased MBP protein level and the significantly decreased SMI 32 protein level (Fig. 3A and 3B). Immunofluorescent staining showed that the SMI 32 signal in the corpus callosum was extremely low in the sham group but was readily detectable in the SAH group (Fig. 3C). Then, we calculated the SMI 32/MBP ratio, which measures demyelination [32]. SAH elevated the SMI 32/MBP ratio in the corpus callosum to significantly higher levels in SAH rats than in sham rats, but compared with vector treatment, MCT1 overexpression treatment could significantly reduce this ratio (Fig. 3D).

These findings suggest that the overexpression of MCT1 could attenuate acute white matter injury after SAH at the histological level.

3.3 MCT1 overexpression contributes to the amelioration of SAH-induced long-term neurobehavioral outcomes

We explored whether the overexpression of MCT1 improves or worsens long-term neurobehavioral outcome up to 35 days after SAH or sham operation through three behavioral tests. In comparison with the sham group, all rats in the SAH group exhibited a shorter latency time of falling off the accelerating rotating rod and a longer latency time to remove adhesive tape between 3 and 35 days after SAH operation, indicating SAH-induced sensorimotor impairments. This sensorimotor deficit was recovered more effectively in the MCT1 overexpression group than in the vector group especially, between 21 and 35 days (Fig. 4A and 4B).

The Morris water maze test was performed at 29–34 days after SAH induction to evaluate the long-term spatial cognitive functions. The escape latency time of all rats gradually decreased over time, whereas the rats in the SAH group took longer to find the hidden platform than the rats in the sham group (Fig. 4C). Furthermore, the overexpression of MCT1 could significantly ameliorate SAH-induced spatial cognitive dysfunction, as manifested by the significantly shorter escape latencies and increased time in the target quadrant, indicating an improvement in spatial learning and memory, respectively (Fig. 4D and 4E). No significant differences in swimming speed were observed among the four groups at 34 days after SAH, indicating that the memory test was not affected by major differences in swimming ability (Fig. 4F). These results suggest that the increased protein level of MCT1 could contribute to the amelioration of SAH-induced long-term neurobehavioral outcomes.

3.4 Rotarod training could increase the level of MCT1 in ITPR2⁺SOX10⁺ oligodendrocytes and accelerate long-term neurobehavioral recovery after SAH

The number of ITPR2⁺SOX10⁺ oligodendrocytes in the corpus callosum could increase significantly with two-day motor learning [21]. To confirm and investigate whether the ability of novel motor activity to promote oligodendrocyte differentiation was affected after SAH, we performed rotarod training for 2 days followed by immunofluorescent staining against ITPR2 and SOX10 after sham or SAH operation. The results showed that in both the sham and SAH groups, the training rats exhibited more ITPR2⁺SOX10⁺ cells in the corpus callosum than rats without training. Additionally, no significant difference was found between the sham and SAH groups in terms of the number of ITPR2⁺SOX10⁺ cells, indicating that SAH induction did not affect oligodendrocyte differentiation (Fig. 5A and 5B).

Next, the training period of rats was extended to 7 days after sham or SAH surgery, and then the expression level of MCT1 was assessed. In both the sham and SAH groups, the training rats exhibited an increased protein level of MCT1 (Fig. 5C and 5D) and more MCT1⁺SOX10⁺ cells in the corpus callosum (Fig. 5E and 5F) than the rats without training. Moreover, the number of MCT1⁺SOX10⁺ cells in the corpus callosum was positively correlated with the latency to fall in the rotarod test (Fig. 6A and 6B) and was inversely correlated with the time to remove the adhesive tapes in the adhesive removal test (Fig. 6C and 6D) at 35 days after sham or SAH operation.

Based on these findings, post-SAH motor training could increase the level of MCT1 on ITPR2⁺SOX10⁺ oligodendrocytes and thus accelerate the long-term neurobehavioral recovery after SAH.

3.5 Upregulation of miRNA-29b and miRNA-124 after SAH mediates the acute reduction in MCT1 expression

To explore the underlying regulation mechanisms of MCT1 after SAH, we first detected the levels of miR-29b and miR-124 in the CSF and corpus callosum of SD rats. Both in the CSF and corpus callosum of SAH rats, the levels of miR-29b-3p and miR-124-3p were significantly upregulated at 48 h relative to the sham rats (Figs. 7A, 7B, S3A, and S3B). Then, we examined whether intracerebroventricular injection of antagomir-29b-3p and antagomir-124-3p (neutralizing inhibitors of miR-29b-3p and miR-124-3p, respectively) into experimental rats alleviate the SAH-induced decrease effect of MCT1 at 48 h after SAH, as shown in Fig. 1. As we expected, the rats injected with antagomir-29b-3p and antagomir-124-3p exhibited a higher protein level than the rats injected with antagomir-NC at 48 h after SAH (Fig. 7C). Moreover, the MCT1 signal in the corpus callosum increased after the injection of antagomir-29b-3p and antagomir-124-3p (Fig. 7D and 7E). No significant differences were observed between the antagomir-29b-3p and antagomir-124-3p groups in terms of the level of MCT1. Therefore, the upregulation of miR-29b and miR-124 after SAH may account for the acute reduction in MCT1 expression.

4 Discussion

In the CNS, MCT1 participated extensively in the energy metabolism and exhibited protective effects on neurons and oligodendrocytes. The metabolites including lactate produced by glycolysis of astrocytes can be transmitted to oligodendrocytes by MCTs and connexins; then, oligodendrocytes transport the ingested lactate to axons through MCT1, through which axons obtain energy [33]. Moreover, the specific removal of MCT1 in oligodendrocytes causes axon damage but not oligodendrocyte death [34]. In the current study, we demonstrated the white matter-protective role of MCT1 after experimental SAH based on Western blot analysis, immunofluorescence staining, RT-qPCR, and behavioral criteria. First, the results of Western blot analysis and immunofluorescent staining showed that the expression levels of MCT1 significantly decreased at 48 h after SAH. Then, we further analyzed the effects of MCT1-overexpression via plasmid transfection on acute white matter injury and long-term cognitive and sensorimotor functions. As we expected, the overexpression of MCT1 could remarkably improve the integrity of white matter in the corpus callosum regions in acute injury phases. Moreover, the salutary effects of MCT1 on white matter integrity after SAH might have resulted in the amelioration of long-term cognitive and sensorimotor functions. Therefore, MCT1 participates in the protection of white matter and long-term functional recovery after SAH.

In the present work, we focused on the role of oligodendrocytes MCT1 in axonal injury after SAH. We found that the MCT1 protein level of oligodendrocytes decreased after SAH, and the axonal injury and neurobehavioral disorder can be improved by the specific overexpression of MCT1. Based on Fig. 5, rotarod training itself could promote endogenous MCT1 expression. The rats in each group showed improvement in their behavior during the rotarod test, which may include the contribution of rotarod training itself (Fig. 4). However, each group of rats received the same intensity of rotarod training, and only MCT1 overexpression was a single variable between the SAH+ vector group and SAH+ over-MCT1 group. The results in Fig. 4 can illustrate the influence of MCT1 overexpression on the behavioral ability of SAH mice. After clarifying the function of oligodendrocytes MCT1, we further explored intervention strategies that could promote endogenous MCT1 expression after SAH. Physical training can increase ITPR2⁺SOX10⁺ oligodendrocytes in corpus callosum, and ITPR2⁺SOX10⁺ oligodendrocytes can promote axonal repair [20,21]. In our work, we found that exercise training also could increase the number of ITPR2⁺SOX10⁺ oligodendrocytes in the corpus callosum of SAH rat. Axon repair requires a high energy supply. We hypothesized that exercise training may also promote endogenous MCT1 expression. Exercise training promoted the expression of endogenous MCT1 in oligodendrocytes of SAH rats, and it was positively correlated with the neurobehavioral ability. This result is an important innovation of this work. Physical training affects many other factors besides MCT1 [35].

Physical exercise and its effects on physical and mental health are important. However, the effect of physical activity on the CNS, especially under pathological conditions, is not completely clear. The role of oligodendrocyte MCT1 in axonal injury after SAH in this work may at least partially explain the underlying mechanism of rehabilitation training in SAH patients. Marques et al. found that running for 2 days in a complex wheel-learning paradigm increases the number of ITPR2⁺SOX10⁺ oligodendrocytes by approximately 50% compared with mice of the same age without running training, in the corpus callosum of postnatal 60 days mice [21]. In the present study, eight-week-old SD rats were obtained and allowed to adapt to the environment for 1 week before modeling. The rats were then subjected to running training on a rotating rod. The rats used in this work were approximately the same age as the mice used by Marques et al. and received a similar type of training. Consistent with the report from Marques et al. [21], the number of ITPR2⁺SOX10⁺ oligodendrocytes in the rat corpus callosum significantly increased by running training. In addition, running training for 2 days could also increase the number of ITPR2⁺SOX10⁺ oligodendrocytes in the corpus callosum of SAH model rats. However, we were unable to identify the source of the new ITPR2⁺SOX10⁺ oligodendrocytes. During post-natal development, oligodendrocytes mature is spatiotemporal specific [36,37]. Marques et al. found that approximately 3% of the OPCs in adult mice were in the cell cycle, and that these populations were observed in the corpus callosum of adult mice [21]. Marques et al. also found that ITPR2⁺ cells were the progeny of OPCs via lineage tracing [21]. Therefore, novel motor activity may trigger the rapid differentiation of OPCs into newly formed ITPR2⁺ oligodendrocytes, contributing to myelin formation or repair. This work originally aimed to investigate the potential effects of early motor training on SAH prognosis. Results show that passive training in patients with SAH is more easily performed and has more clinical significance [38]. However, the findings of this study only indicate that early training after SAH is beneficial for the prognosis in rats but cannot be used to determine the training intensity that humans can receive on the day after SAH and the possible effects.

In the present study, the role of MCT1 in SAH was superficially studied. These findings can be confirmed by using MCT1 knockout animals. However, MCT1-deficient mice are embryologically lethal [39]. Currently, MCT1^+/− heterozygous mice are commonly used to observe the chronic process of lipid metabolism [40]. In addition, MCT1 affects the metabolism regulation of neurons [41]. Therefore, we selected corpus callosum injection of MCT1 overexpression plasmid to achieve the transient regulation of MCT1 protein level in oligodendrocytes to study the neuroprotection effect of oligodendrocyte MCT1 in the acute phase of SAH. To further confirm the effect of MCT1 deletion in adult mouse oligodendrocytes on neuronal damage after SAH, we will use MCT1^flox/flox:Olig2-CreERT in the follow-up work to obtain a substantial supplement.

To determine the underlying reasons for the decline of MCT1 after SAH, we examined the levels of miR-29b and miR-124 caused by their reported ability to target the 3′-untranslated regions of MCT1 in both humans and mice [15]. The results of RT-qPCR on SD rats’ CSF showed that SAH induction significantly increased the levels of miR-29b and miR-124. Antagomir, as a commercial miRNA inhibitor, has been applied successfully in many studies of CNS [42,43]. In the present study, the level of MCT1 was increased significantly after intracerebroventricular injection of 10 nmol per rat antagomir-29b or antagomir-124, indicating that miR-29b and miR-124 are partially involved in the downregulation of MCT1 after SAH.

Early brain injury after SAH is a complex pathophysiological process. Both clinical and experimental studies have demonstrated that abnormal white matter signals can be observed with magnetic resonance imaging in the early stage after SAH [6], even in the hyperacute phase (within 4 h) [44], indicating the existence of white matter damage. Nevertheless, although white matter injury can cause severe cognitive impairment after SAH, the potential mechanisms of its occurrence are still poorly understood. Some authors consider that the interruption of the normal connection between astrocytes and oligodendrocytes may cause oligodendrocyte death and subsequent demyelination [33]. Meanwhile, other authors found that the deletion of oligodendrocyte-specific MCT1 results in axon injury rather than oligodendrocyte death [13]. Based on these findings and our results, we speculate a possible mechanism in which, after the onset of SAH, a rapid degeneration and demyelination of white matter occurs accompanied by a decrease in MCT1 in oligodendrocytes in the white matter region, which interferes with the energy supply of oligodendrocytes to axons and aggravates white matter injury. The upregulation of miR-29b and miR-124 may be entirely or partially involved in the decrease of MCT1 after SAH, and motor training might be a promising means of rescue (Fig. 7F).

The in vivo experiments showed that the upregulation of miR-29b and miR-124 after SAH may induce the decrease of MCT1. Based on our results, the specific mechanism of such an increase of miR-29b and miR-124 cannot be determined. The present work focused on the role of oligodendrocytes MCT1 in axonal injury after SAH. We found that the MCT1 protein level of oligodendrocytes decreased after SAH, and the axonal injury and neurobehavioral disorder could be improved by the specific overexpression of MCT1 or upregulation of MCT1 protein level through training. After clarifying the function of oligodendrocytes MCT1, we further explored the factors leading to the decrease of MCT1 after SAH. miR-29b/miR-124 can inhibit the translation of MCT1 mRNA [15,16]. By detecting the level of miR-29b/miR-124 and the intervention of antagomir-29b-3p/124-3p, we found that the increase of miR-29b/miR-124 after SAH was at least partially responsible for the decrease of MCT1 level. However, miR-29b targets IGF-1 and PI3K in skeletal muscle cells [45], and miR-124 targets ferroportin1 in neuron cells [46]. In our model, further studies are needed to determine whether miR-29b/miR-124 specifically targets MCT1. To our best knowledge, in vitro studies have shown that stimulated astrocytes can secrete miR-29b [47]. In addition, miR-124 is widely enriched in neurons and can be transmitted from neurons to microglia through the normal connections between them [14]. The stress activation of astrocytes and the cut-off of the normal connection between neurons and microglia after SAH may increase miR-29b and miR-124. However, further verification is essential, especially in vitro. Finally, we only used male SD rats to simulate human SAH. Whether these results could be replicated in humans remains unknown, and sex differences should also be considered.

In conclusion, this study demonstrated that the decrease of MCT1 that may be mediated by the upregulation of miR-29b, miR-124 was associated with white matter injury after SAH, and exogenous overexpression of MCT1 significantly improved white matter integrity and long-term cognitive function recovery. Additionally, despite the lack of studies on the specific mechanisms, motor training after SAH participated in the upregulation of MCT1, and thus providing a promising direction for the clinical prevention and treatment of white matter injury after SAH.

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