Mitochondrial Transplantation Promotes Remyelination and Long-Term Locomotion Recovery following Cerebral Ischemia

Cerebral ischemia usually leads to axonal degeneration and demyelination in the adjacent white matter. Promoting remyelination still remains a challenging issue in the field. Considering that ischemia deprives energy supply to neural cells and high metabolic activities are required by oligodendrocyte progenitor cells (OPCs) for myelin formation, we assessed the effects of transplanting exogenous healthy mitochondria on the degenerating process of oligodendrocytes following focal cerebral ischemia in the present study. Our results showed that exogenous mitochondria could efficiently restore the overall mitochondrial function and be effectively internalized by OPCs in the ischemic cortex. In comparison with control cortex, there were significantly less apoptotic and more proliferative OPCs in mitochondria-treated cortex. More importantly, higher levels of myelin basic protein (MBP) and more morphologically normal myelin-wrapped axons were observed in mitochondria-treated cortex at 21 days postinjury, as revealed by light and electron microscope. Behavior assay showed better locomotion recovery in mitochondria-treated mice. Further analysis showed that olig2 and lipid synthesis signaling were significantly increased in mitochondria-treated cortex. In together, our data illustrated an antidegenerating and myelination-promoting effect of exogenous mitochondria, indicating mitochondria transplantation as a potentially valuable treatment for ischemic stroke.


Introduction
Stroke is one of the leading causes of disability and dementia around the world [1]. Approximately 80% of stroke is cerebral ischemia, which usually leads to permanent loss of neurons and oligodendrocytes in the injury area. In the past decades, numerous researches focused on and developed various treatments for neuroprotection [2,3]. Relatively, there still lacks effective strategy for preventing the degener-ation of oligodendrocytes and demyelination following ischemia.
One pathological feature of cerebral ischemia is the disruption of energy supply, which usually leads to mitochondria dysfunction [4]. Mitochondria damage is thought as the major cause of cell death and oxidative stress [5]. Restoring or promoting mitochondria function has been regarded as promising for reducing the secondary injury following ischemia [6]. Recently, in vivo transplantation of healthy mitochondria has been proposed as an effective way to boost mitochondria function and exert neuroprotection [7,8]. For example, mitochondrial transplantation exerted remarkable beneficial effects in preventing secondary injury in traumatic brain injury and cognitive decline in aging [9,10]. High metabolic requirement was observed during the myelination process of oligodendrocyte precursor cells (OPCs) [11]. Whether mitochondria transplantation could exert protective effects on oligodendrocyte lineage cells remains interesting to be explored.
In this study, we investigated this issue by evaluating the survival and proliferation of oligodendrocyte progenitor cells (OPCs) and myelination in ischemic cortex following mitochondria transplantation.

Photochemical Ischemia Model.
Adult C57BL/6J male mice (body weight: 20-23 g) with 8-10 weeks of age were adopted. All mice were specific pathogen-free and housed under 12 light/12 dark cycle, controlled temperature (22-24°C) with free access to water and standard rodent chaw. All animal experiments were carried out according to the protocols approved by the Animal Care and Use Committees of Fourth Military Medical University (license number: 20211024). Focal cortical ischemia was induced as described with minor modification [12]. Rose bengal (Sigma) was injected i.v. at 20-25 mg/kg. A skull window was made ranging from 0.4 to 2.4 mm anterior to the Bregma and 0.7 to3.2 mm right to the midline. The brain was illuminated for 15-18 minutes using a cold light source (Zeiss FL2000 LCD).

Mitochondria Preparation and ATP Measurement.
Mitochondria were purified from allogeneic mouse liver as described [10]. Liver tissue was homogenated on ice by mitochondria isolation buffer, and the tissue lysate was centrifuged at 1000 g for 5-8 min. The supernatant was centrifuged at 3500 g for 12 min. The resulting precipitate was resuspended with 15% Percoll and centrifuged at 21000 g to precipitate mitochondria. For mitochondria quantification, mtDNA was isolated and quantified as described [13]. Briefly, plasmids containing ND1 (a mitochondria gene) were diluted into graded concentrations (1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , and 10 8 copies per ml) to make standard curve. qPCR was performed using the following ND1 primers. ND1-F: CCGAGCATCTT ATCCACGCT; ND1-R: ATGGTGGTACTCCCGCTGTA. mtDNA copies were calculated according to the standard curve. Before transplantation, freshly isolated mitochondria were labeled with Mito-Tracker (Mito-Tracker Red CMXRos, M9940, Solarbio) and transplanted immediately after isolation. For mitochondrial transplantation, two bilateral injections with 1 mm distance to lesion center were made. Approximately 2 × 10 7 mitochondria were injected for each site with 50 nl/min at the depth of 0.75 mm and 1.5 mm from dura mater, respectively.
For ATP measurement, brain tissues (including lesion center and brain regions 3 mm within the lesion boarder) were dissected and homogenized. ATP levels were measured by using an ATP assay kit (BC0300, Solarbio Life Sciences). The activity of mitochondrial complex was measured by using assay kits (BC1445, BC3240, BC0945, BC3235, BC0515, and BC0305, Solabio Life Sciences).

Electron Microscopic
Study. At 21 d after mitochondria transplantation, the mice were perfusion fixed with 2% glutaraldehyde. Sections (50 μm in thickness) were prepared and fixed again with 1% osmium tetroxide. Then, sections were subsequently dehydrated with graded ethanol and embedded in Epon 812. Ultrathin sections were cut by using an LKB Nova Ultratome (Bromma). Final counterstaining was performed with uranyl acetate and lead citrate. After that, sections were observed, and images were taken by using a JEM-1230 electron microscope (JEM, Tokyo). The g-ratio was measured as the ratio of the inner to the outer radius of the myelin sheath of the cross section of axons as described [14].

Western
Blotting. Ischemic cortex was dissected and homogenized. SDS-PAGE was conducted and protein transferred to PVDF membrane. Rabbit anti-MBP antibody (1 : 1000, GeneTex) and mouse anti-β-actin antibody (1 : 6000, Proteintech) were incubated with protein-loaded membrane at 4°C overnight. HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1 : 10000; Pierce) were incubated for 1 h at room temperature. Bands were visualized with an ECL kit (Pierce) and images analyzed by Image J.
2.6. Locomotion Assay. Glass sliding test was carried out at 1 d, 3 d, 7 d, and 21 d posttransplantation as described [15]. Mice were videotaped in glass cylinder for 15 min. The numbers of contacts and the numbers of sliding movements of each forelimb at the wall of the cylinder for every spontaneous stand-up were scored. The sliding index was calculated as: the number of sliding/ðnumber of contact + number of slidingÞ × 100.
Rotarod test was conducted at 1 d, 3 d, 7 d, and 21 d posttransplantation. Mice were lowered onto the rotating roller, and the timer was started immediately upon the release 2 Mediators of Inflammation of the tail. The rod was accelerated 5-50 rpm during the course of 5 min. The latency to fall in each trial was recorded. Three trials were performed daily for 3 days. For behavior assays, there were at least 10 mice in each group. The data were presented as means ± S:E: and analyzed by Student's t test or one-way ANOVA, followed by Dunnett post hoc [16]. P values less than 0.05 were considered as statistically significant. SPSS16.0 was adopted to perform statistical analysis.

Internalization of Exogenous Mitochondria by Oligodendrocyte Progenitor Cells (OPCs) in Ischemic Cortex.
Previous studies have demonstrated dysfunction of mitochondria following ischemia [17]. We first explored if 3 Mediators of Inflammation transplanting healthy mitochondria could enhance the oval mitochondria function in the ischemic cortex. As the liver is the organ contains the largest number of mitochondria, mitochondria were isolated from allogeneic liver as reported [10]. 2 × 10 7 mitochondria were injected for each ischemic site. Three days later, the activity of complexes І, II, and III and the levels of ATP were increased significantly in mitochondria-treated cortex, as compared with vehicletreated cortex (Figures 1(a) and 1(b)). Then, we explored whether OPCs could uptake exogenous mitochondria. Internalization of Mito-Tracker labeled mitochondria was evaluated at 24 h and 3 d following transplantation. At 24 h after grafting, approximately 58% NG2-positive cells (OPCs) and 40% CNPase-positive cells (immature oligodendrocytes)   (Figure 1(c)). No Mito-Tracker-labeled MBP-positive cells (mature oligodendrocytes) were detected (data not shown). These data indicated grafted mitochondria could be internalized efficiently by OPCs in ischemic cortex.

Promotion of OPC Survival and Proliferation by
Exogenous Mitochondria. We next examined if exogenous mitochondria could prevent the death of OPCs. Combination of TUNEL and NG2-staining showed that the TUNEUL/ NG2-positive cells in the ischemic region decreased by approximately 45% in mitochondria-grafted cortex at 3 d posttransplantation (Figure 2(a)). In addition, double immunostaining of cleaved caspase-3 (CC-3) with Sox10 (a marker of OPC) showed that CC-3/Sox10-positive cells reduced by approximately 49% at 3 d posttransplantation, as compared with that in vehicle control (Figure 2(b)). These data demonstrated that grafted mitochondria promoted the survival of OPCs in ischemic cortex.
As the development of OPCs is closely associated with their internal metabolic condition, we assessed if mitochondria transplantation could affect the proliferation of OPCs. Double immunostaining of Ki67 (a marker of cellcycle entry) with two OPC markers PDGFRα and Sox10 showed that there were significantly more Ki67/PDGFRαpositive and Ki67/Sox10-positive cells in mitochondriatreated cortex as compared with that of control group (Figures 3(a) and 3(b)).

Improvement of Myelination and Locomotion by
Exogenous Mitochondria. We then evaluated if mitochondria transplantation could attenuate the demyelination in ischemic cortex and the subcortical corpus callosum at 21 d posttransplantation. Both immunostaining and Western blotting demonstrated significantly higher levels of myelin basic protein (MBP) in mitochondria-treated cortex, as compared with vehicle control (Figures 4(a) and 4(b)). In addition, under electron microscope, demyelinated axons with thin and loose myelin could be frequently detected in vehicle-treated cortex, while more axons with thick and intact myelin were observed in mitochondria-treated cortex (Figure 4(c)). The g-ratio of myelin was significantly lower in the mitochondria-treated than that in vehicle-treated cortex (Figure 4(d)). These data indicated that mitochondrial transplantation was beneficial for myelin preservation in ischemic cortex.
In the end, we examined if mitochondria transplantation could affect the locomotion recovery after ischemia. Glass sliding assay which evaluates forelimb activity was conducted. From 3 d posttransplantation, the total activity of affected forelimbs significantly increased in mitochondriatreated group, as compared with that in vehicle-treated group (Figure 4(e)). Accordingly, the sliding activity of affected forelimb reduced significantly in mitochondriatreated group (Figure 4(e)). In addition, Rotarod assay which accesses motor coordination and balance demonstrated that mitochondria-treated mice exhibited significantly longer running time on the rod (Figure 4(f)). These data indicated that exogenous mitochondria was beneficial for promoting functional recovery of ischemic stroke.   (Figure 5(a)). Reportedly, these genes are involved in multiple stages of OPC development [18][19][20][21].
We validated the expression of these genes by real-time RT-PCR. The results showed that the mRNA levels of FABP5, FABP7, FASN, and Olig2 were significantly increased in mitochondria-treated cortex ( Figure 5(b)). As FABP5, FABP7, and FASN play important roles in lipid metabolism, these data indicated that exogenous mitochondria might stimulate remyelination via activating lipid synthesis signaling and Olig2-mediated gene transcription.

Discussion
In this study, we explored the effects of transplantation of exogenous mitochondria on the survival, proliferation, and myelin formation of OPCs. First, we demonstrated the efficient endocytosis of exogenous mitochondria by OPCs. By apoptosis and cell proliferation analysis, we revealed that mitochondria grafting promoted the survival and proliferation of OPCs. More importantly, our data showed that, mitochondria transplantation promoted remyelination in the ischemic cerebral cortex and facilitated long-term functional recovery. As the "energy powerhouse" of every cell which highly needs oxygen, mitochondria were very vulnerable to ischemia. Following cerebral ischemia, astrocytes transfer mitochondria to neurons for emergent energy supplement [22]. This observation inspired mitochondria transplantation as a promising therapy for various ischemic diseases, such as ischemia-infusion-induced heart and kidney injury [23,24]. So far, as we know, only two groups examined the effects of exogenous mitochondria on ischemic stroke by using muscle or mesenchymal stem cell-derived mitochondria and reported reduced infarction size and decreased astrocytic and microglial activation [25,26]. The very short in vitro and extracellular life span of mitochondria may limit the application of mitochondrial transplantation on oligodendrocyte degeneration and remyelination, which persists long after ischemia. However, our data showed exogenous mitochondria could be efficiently endocytosed by OPCs. Internalized mitochondria may either fuse with endogenous mitochondria or function independently. As the life span of mitochondria is about 2-4 weeks in cytoplasm, therefore, it is feasible to use mitochondrial transplantation to rescue energy failure in ischemia-attacked OPCs.
During development, OPCs require active mitochondria for proliferation and subsequent myelin formation ( [11,27]. Our data that NG2-positive and CNPase-positive cells inter-nalize exogenous mitochondria reflected the metabolic requirement of OPCs. The increase of proliferation was consistent with the high energy requirement by OPCs and the boosting of energy supply by exogenous mitochondria. In detail, exogenous mitochondria may restore oxidative phosphorylation, thereby increasing ATP production. In addition, exogenous mitochondria may enhance the buffering of intracellular Ca2+ signaling, which helps to stop the activation of apoptotic signaling cascade promoted cell survival [28].
Interestingly, our data showed that exogenous mitochondria stimulated the expression of multiple lipid synthetic genes. As myelin is extremely lipid rich, and recent studies reported requirement of fatty acid and cholesterol synthesis for oligodendrocyte differentiation and myelination [19,29], it is possible that exogenous mitochondria may steer lipid metabolism towards the direction more favorable for myelin formation [30]. How exogenous mitochondria rebalanced the lipid metabolism is worthy to be further investigated. In the present study, we cannot exclude other cells which internalized mitochondria may also exert beneficial effects. Nevertheless, our data provided a novel strategy for promoting myelination in ischemic stroke and other neurological disorders.

Data Availability
The data used to support the findings of this study are included within the article.