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This figure demonstrates neurogenesis of endogenous neural stem cells. (1) Neurogenesis and proliferation occur in the SVZ and SGZ on the lateral ventricle and hippocampus, respectively. (2) NSPC migration occurs through the rostral migratory stream to the olfactory bulb, where neuroblasts migrate as interneurons through specific cell layers. From the SGZ, NSPCs migrate to the inner granule cell layer. (3) Differentiation occurs once neuroblasts reach glomeruli within the olfactory bulb or the inner granule cell layer. The majority of SVZ-derived neuroblasts become GABAergic granule neurons. After complete differentiation and maturation of neuroblasts from the SGZ, new neurons possess GABAergic and glutamatergic characteristics.
Source publication
Neural stem cells (NSCs) offer a potential therapeutic benefit in the recovery from
ischemic stroke. Understanding the role of endogenous neural stem and progenitor cells under
normal physiological conditions aids in analyzing their effects after ischemic injury, including
their impact on functional recovery and neurogenesis at the site of injury....
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Citations
... Among these, NSCs and MSCs have received the most extensive attention in the literature. 165 Nevertheless, the adverse microenvironment associated with ischemic stroke has been found to significantly diminish the viability and proliferative capacity of stem cells, hence imposing substantial constraints on their therapeutic efficacy. 166 MSCs, whether in their original form or with alterations, have demonstrated significant promise in enhancing the formation of new blood vessels (angiogenesis) in both preclinical studies of ischemic diseases and clinical trials. ...
Angiogenesis, or the formation of new blood vessels, is a natural defensive mechanism that aids in the restoration of oxygen and nutrition delivery to injured brain tissue after an ischemic stroke. Angiogenesis, by increasing vessel development, may maintain brain perfusion, enabling neuronal survival, brain plasticity, and neurologic recovery. Induction of angiogenesis and the formation of new vessels aid in neurorepair processes such as neurogenesis and synaptogenesis. Advanced nano drug delivery systems hold promise for treatment stroke by facilitating efficient transportation across the the blood-brain barrier and maintaining optimal drug concentrations. Nanoparticle has recently been shown to greatly boost angiogenesis and decrease vascular permeability, as well as improve neuroplasticity and neurological recovery after ischemic stroke. We describe current breakthroughs in the development of nanoparticle-based treatments for better angiogenesis therapy for ischemic stroke employing polymeric nanoparticles, liposomes, inorganic nanoparticles, and biomimetic nanoparticles in this study. We outline new nanoparticles in detail, review the hurdles and strategies for conveying nanoparticle to lesions, and demonstrate the most recent advances in nanoparticle in angiogenesis for stroke treatment.
... Cerebral ischemia-reperfusion (I/R) injury in the brain happens during the reperfusion of blocked blood due to the restoration of oxygen-rich blood. The main strategy to mitigate ischemic stroke injury is revascularization, which may lead to I/R injury (Phipps and Cronin 2020;Reis et al. 2017;Zerna et al. 2018). In a recent study, Xie et al. described that mitochondrial transplantation may attenuate cerebral I/R injury. ...
The regeneration capacity after an injury is critical to the survival of living organisms. In animals, regeneration ability can be classified into five primary types: cellular, tissue, organ, structure, and whole-body regeneration. Multiple organelles and signaling pathways are involved in the processes of initiation, progression, and completion of regeneration. Mitochondria, as intracellular signaling platforms of pleiotropic functions in animals, have recently gained attention in animal regeneration. However, most studies to date have focused on cellular and tissue regeneration. A mechanistic understanding of the mitochondrial role in large-scale regeneration is unclear. Here, we reviewed findings related to mitochondrial involvement in animal regeneration. We outlined the evidence of mitochondrial dynamics across different animal models. Moreover, we emphasized the impact of defects and perturbation in mitochondria resulting in regeneration failure. Ultimately, we discussed the regulation of aging by mitochondria in animal regeneration and recommended this for future study. We hope this review will serve as a means to advocate for more mechanistic studies of mitochondria related to animal regeneration on different scales.
... To that end, some researchers have evaluated the administration of stem cells in animal models with a view to regenerating the ischemic brain tissue. NSCs and embryonic stem cells have the potential to differentiate into neural lineages after occlusion of the middle cerebral artery, in light of the evidence that these cells can give rise to neurons at the lesion site [49]. ...
Neurological disorders are a leading cause of morbidity worldwide, giving rise to a growing need to develop treatments to revert their symptoms. This review highlights the great potential of recent advances in cell therapy for the treatment of neurological disorders. Through the administration of pluripotent or stem cells, this novel therapy may promote neuroprotection, neuroplasticity, and neuroregeneration in lesion areas. The review also addresses the administration of these therapeutic molecules by the intranasal route, a promising, non-conventional route that allows for direct access to the central nervous system without crossing the blood–brain barrier, avoiding potential adverse reactions and enabling the administration of large quantities of therapeutic molecules to the brain. Finally, we focus on the need to use biomaterials, which play an important role as nutrient carriers, scaffolds, and immune modulators in the administration of non-autologous cells. Little research has been conducted into the integration of biomaterials alongside intranasally administered cell therapy, a highly promising approach for the treatment of neurological disorders.
... Stem cell therapy is considered to be the most promising therapeutic strategy for ischemic stroke and can greatly extend the therapeutic window [11]. Given the intensive research on the mechanisms involved in stem cell-mediated repair and continuous advances in isolation, culture, induction, monitoring, and transplantation techniques, stem cells have become a hotspot for the treatment of ischemic stroke [12,13]. In particular, neural stem cells (NSCs) can promote the recovery of neurological function in stroke patients by protecting the blood-brain barrier, reducing the inflammatory response, and promoting neurogenesis and angiogenesis. ...
Background
In order to promote the clinical translation of preclinical findings, it is imperative to identify the most optimal therapeutic conditions and adopt them for further animal and human studies. This study aimed to fully
explore the optimal conditions for neural stem cell (NSC)-based ischemic stroke treatment based on animal studies.
Methods
The PubMed, Ovid-Embase, and Web of Science databases were searched in December 2021. The screening of search results, extraction of relevant data, and evaluation of study quality were performed independently by two reviewers.
Results
In total, 52 studies were included for data analysis. Traditional meta-analysis showed that NSCs significantly reduced the modified neurological severity score (mNSS) and volume of cerebral infarct in animal models of ischemic stroke. Network meta-analysis showed that allogeneic embryonic tissue was the best source of NSCs. Further, intracerebral transplantation was the most optimal route of NSC transplantation, and the acute phase was the most suitable stage for intervention. The optimal number of NSCs for transplantation was 1–5×10 ⁵ in mouse models and 1×10 ⁶ or 1.8×10 ⁶ in rat models.
Conclusions
We systematically explored the therapeutic strategy of NSCs in ischemic stroke, but additional research is required to develop optimal therapeutic strategies based on NSCs. Moreover, it is necessary to further improve and standardize the design, implementation, measuring standards, and reporting of animal-based studies to promote the development of better animal experiments and clinical research.
... Existing studies show the feasibility of in vivo magnetic resonance imaging (MRI) tracking of endogenous NSCs in adult rodent brains [45,46]. Pothayee et al. [47] used iron oxide-based MRI to track neuroblasts migration in a naris occlusion model. ...
Ischemic stroke is a very common cerebrovascular accident that occurred in adults and causes higher risk of neural deficits. After ischemic stroke, patients are often left with severe neurological deficits. Therapeutic strategies for ischemic stroke might mitigate neuronal loss due to delayed neural cell death in the penumbra or seek to replace dead neural cells in the ischemic core. Currently, stem cell therapy is the most promising approach for inducing neurogenesis for neural repair after ischemic stroke. Stem cell treatments include transplantation of exogenous stem cells but also stimulating endogenous neural stem cells (NSCs) proliferation and differentiation into neural cells. In this review, we will discuss endogenous NSCs-induced neurogenesis after ischemic stroke and provide perspectives for the therapeutic effects of endogenous NSCs in ischemic stroke. Our review would inform future therapeutic development not only for patients with ischemic stroke but also with other neurological deficits.
... The one that first comes to mind is transplanting exogenous cells aiming to regenerate the injured site or to impose supportive effects on survived cells. The next one is directing or enhancing the functions of endogenous progenitor cells [50][51][52]. The positive effects of this therapeutic method, depending on the type of cell, the timing, and the route of administration include modulating the immune system, replacing the lost neurons, suppling vital neurotrophic factors and ECM components, and subsequently encouraging regeneration [53][54][55]. ...
Spinal cord injury (SCI) is a central nervous system (CNS) devastate event that is commonly caused by traumatic or non-traumatic events. The reinnervation of spinal cord axons is hampered through a myriad of devices counting on the damaged myelin, inflammation, glial scar, and defective inhibitory molecules. Unfortunately, an effective treatment to completely repair SCI and improve functional recovery has not been found. In this regard, strategies such as using cells, biomaterials, biomolecules, and drugs have been reported to be effective for SCI recovery. Furthermore, recent advances in combinatorial treatments, which address various aspects of SCI pathophysiology, provide optimistic outcomes for spinal cord regeneration. According to the global importance of SCI, the goal of this article review is to provide an overview of the pathophysiology of SCI, with an emphasis on the latest modes of intervention and current advanced approaches for the treatment of SCI, in conjunction with an assessment of combinatorial approaches in preclinical and clinical trials. So, this article can give scientists and clinicians' clues to help them better understand how to construct preclinical and clinical studies that could lead to a breakthrough in spinal cord regeneration.
... Various stem cells have been tested in this context, mainly derived from the BM, primarily MSCs and MNCs. When transplantation of these cells is deemed safe for stroke, their efficacy remains elusive, as they exerted or not a functional recovery, depending on the clinical trials reviewed in (Banerjee et al. 2011) and (Reis et al. 2017). Although purified CD34+ stem cells are not being currently preferred for this therapeutic indication, several lines of evidence would favor their use in the future. ...
Although hematopoietic stem cell (HSC) transplantations have been performed daily for more than 50 years, the concept of “regenerative medicine” was only proposed at the beginning of the 21st century, with the goal to structurally and functionally repair damaged tissues and organs using stem cells. Despite the hype and the hope generated by the pluripotency of embryonic or induced pluripotent stem cells, ethical problems and/or risk of teratoma formation and genomic instability hinder their use in the clinic in the foreseeable future. Various types of adult stem cells, mostly isolated from bone marrow (BM), peripheral blood, umbilical cord blood, or adipose tissue, have thus been commonly used to regenerate organs within the past years, with unequal efficacy. CD34⁺ cells emerge now as the more convincing cell type. From their discovery in 1984 until the end of the 1990s, they were considered as being solely HSCs. However, it was progressively demonstrated that endothelial, cardiac muscle, liver, and bone progenitor cells also bear the CD34 marker, giving rise to the transgressive concept of “cell plasticity”, which postulated that HSCs can transdifferentiate into other cell lineages from different germ layers. However, this concept should be definitely rejected because evidence has accumulated that adult BM harbors rare pluripotent stem cells, named very small embryonic-like stem cells (VSELs), which are also CD34⁺. They have been shown to be precursors of hematopoietic, mesenchymal, endothelial, cardiomyocytic, lung epithelial stem cells, and germ cells. To date, no clinical studies have been performed using VSELs. However, the clinical demonstration in recent years of the regeneration potential of CD34⁺ stem cells, which are probably derived from VSELs, could open the way to their greater therapeutic use in the near future.
... When blood flow is blocked, brain tissue in the area of blood supply becomes ischemic and hypoxic, which then leads to neurological dysfunction. Moreover, following blood reperfusion, the damaged brain tissue can be further harmed by restoration of oxygen-rich blood, causing a socalled ischemia-reperfusion (I/R) injury [3,[6][7][8][9]. Major contributors to the pathological process include overproduction of ROS, dramatically increased extracellular glutamate levels, and activation of neuroinflammation responses [6,7,9]. ...
... Among these, the dysfunction of mitochondria in neurons plays a pivotal role [3,9]. Currently, the main strategy to mitigate ischemic stroke injury is revascularization [4,8,10], which may lead to I/R injury. Despite remarkable progress that has been achieved for ischemic stroke, there seem to be no better options for I/R injury. ...
Background. Mitochondrial dysfunctions play a pivotal role in cerebral ischemia-reperfusion (I/R) injury. Although mitochondrial transplantation has been recently explored for the treatment of cerebral I/R injury, the underlying mechanisms and fate of transplanted mitochondria are still poorly understood. Methods. Mitochondrial morphology and function were assessed by fluorescent staining, electron microscopy, JC-1, PCR, mitochondrial stress testing, and metabolomics. Therapeutic effects of mitochondria were evaluated by cell viability, reactive oxygen species (ROS), and apoptosis levels in a cellular hypoxia-reoxygenation model. Rat middle cerebral artery occlusion model was applied to assess the mitochondrial therapy in vivo. Transcriptomics was performed to explore the underlying mechanisms. Mitochondrial fate tracking was implemented by a variety of fluorescent labeling methods. Results. Neuro-2a (N2a) cell-derived mitochondria had higher mitochondrial membrane potential, more active oxidative respiration capacity, and less mitochondrial DNA copy number. Exogenous mitochondrial transplantation increased cellular viability in an oxygen-dependent manner, decreased ROS and apoptosis levels, improved neurobehavioral deficits, and reduced infarct size. Transcriptomic data showed that the differential gene enrichment pathways are associated with metabolism, especially lipid metabolism. Mitochondrial tracking indicated specific parts of the exogenous mitochondria fused with the mitochondria of the host cell, and others were incorporated into lysosomes. This process occurred at the beginning of internalization and its efficiency is related to intercellular connection. Conclusions. Mitochondrial transplantation may attenuate cerebral I/R injury. The mechanism may be related to mitochondrial component separation, altering cellular metabolism, reducing ROS, and apoptosis in an oxygen-dependent manner. The way of isolated mitochondrial transfer into the cell may be related to intercellular connection.
1. Introduction
Stroke is an acute cerebrovascular disease, including ischemic and hemorrhagic stroke, and is considered to be one of the leading causes of human death and disability worldwide [1–4]. Ischemic stroke accounts for over 80% of all strokes and is usually triggered by brain arterial embolism [3, 5]. When blood flow is blocked, brain tissue in the area of blood supply becomes ischemic and hypoxic, which then leads to neurological dysfunction. Moreover, following blood reperfusion, the damaged brain tissue can be further harmed by restoration of oxygen-rich blood, causing a so-called ischemia-reperfusion (I/R) injury [3, 6–9]. Major contributors to the pathological process include overproduction of ROS, dramatically increased extracellular glutamate levels, and activation of neuroinflammation responses [6, 7, 9]. Among these, the dysfunction of mitochondria in neurons plays a pivotal role [3, 9]. Currently, the main strategy to mitigate ischemic stroke injury is revascularization [4, 8, 10], which may lead to I/R injury. Despite remarkable progress that has been achieved for ischemic stroke, there seem to be no better options for I/R injury.
Studies have shown that mitochondria are not only the energy factories of cells but are also closely related to other biological processes, including calcium homeostasis, ROS production, hormone biosynthesis, and cellular differentiation [3, 9, 11]. Mitochondria play an important role in many diseases. Recently, a growing number of studies have begun to apply isolated mitochondria as a therapeutic agent to treat diseases, including kinds of I/R injury [12–20], liver disorders [21, 22], breast cancer [23–25], lung diseases [26, 27], and central nervous system disorders [28–37]. Furthermore, Emani et al. conducted an autologous mitochondrial transplantation clinical study, which showed a promising clinical application [38, 39]. Also, there are studies registered at ClinicalTrials.gov (NCT03639506, NCT02851758, and NCT04998357). Therefore, mitochondrial transplantation holds great therapeutic potential for cerebral I/R injury. One big concern of mitochondrial transplantation is an immune and inflammatory response based on data of mtDNA [40] and damage-associated molecular patterns (DAMPs) [41]. Ramirez-Barbieri et al. demonstrated that there is no direct or indirect, acute or chronic alloreactivity, allorecognition, or DAMP reaction to single or serial injections of allogeneic mitochondria [42].
Recently, several studies have applied isolated mitochondria from various sources as an intervention in many diseases. Four studies focused on cerebral I/R injury have shown benefits of mitochondrial transplantation based on various phenotypes, such as behavioral assessment, infarct size, ROS, and apoptosis. However, the appropriate source of mitochondria, the mechanism of its therapeutic effect, and the fate of isolated mitochondria remain unclear. The clinical application of isolated mitochondria has just begun, and more safety and effectiveness assessments are needed.
In order to answer the above questions, we performed this study. Firstly, we evaluated the source of mitochondria and then assessed the therapeutic effects of mitochondrial transplantation in cellular and animal models. Finally, we mainly focused on the therapeutic mechanisms of mitochondrial transplantation and the fate of transplanted mitochondria.
2. Methods
2.1. Cells
The mouse neural stem cell (mNSC) was obtained and cultured as previously described [43]. Adherent culture of mNSC was performed with Matrigel (Corning, NY, USA, Cat#354277). N2a (Cat#SCSP-5035) and induced pluripotent stem cell (iPSC) (Cat#DYR0100) were purchased from the National Collection of Authenticated Cell Cultures, Shanghai. 293T was provided by Dr. Gao Liu from Zhongshan Hospital, Shanghai Medical College, Fudan University. N2a and 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum. The iPSC was cultured according to the manufacturer’s protocol.
2.2. Animals
Sprague-Dawley rats (7-8 weeks old, 250-300 g) were obtained from Shanghai Super-B&K Laboratory Animal Corp. Ltd. (Shanghai, China). All experimental procedures and animal care were approved by the Animal Welfare and Ethics Group, Laboratory Animal Science Department, Fudan University (ethical approval number 202006013Z) and were carried out according to the Guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health. The rats were divided into three groups: sham, I/R, and I/R+Mito group (sham = sham-operated; I/R = MCAO+reperfusion with saline injection; I/R+Mito = MCAO+reperfusion with mitochondria injection).
2.3. Mitochondrial Isolation
Mitochondria were isolated from N2a and mNSC using the mitochondria isolation kit (ThermoFisher Scientific, USA, Cat#89874) as previously described [32, 44]. Briefly, after cultured cells were orderly digested (trypsin) and centrifuged (300 g, 5 min) and the supernatant was removed, collected cells were resuspended by mitochondrial isolation reagent A (800 μl) in a 2.0 ml microcentrifuge tube and vortexed for 5 s and then incubated for 2 min on ice. Then, the reagent B (10 μl) was further added into the tube and continuously placed in situ for 5 min. Following vortexed at maximal speed for 5 times (each time for 1 min), the reagent C (800 μl) was added into the tube and mixed. Subsequently, the mixed solution was centrifuged (700 g, 10 min, 4°C) and then the supernatant was obtained for further centrifugation (12000 g, 15 min, 4°C). Finally, fresh mitochondria were obtained and used for further experiments. For animal experiments, each rat received mitochondria isolated from cells, and the protein content was about 180 μg-200 μg.
2.4. Transmission Electron Microscopy (TEM)
Cells were fixed with 2.5% glutaraldehyde for 2 h at room temperature and then centrifuged (300×g, 5 min). Subsequently, cells were postfixed with precooled 1% osmic acid (2 h, 4°C) and then centrifuged again (300×g, 5 min). After gradient alcohol dehydration and penetration with a solution of acetone and epoxy resin at different proportions, the cell samples were further embedded into epoxy resin and solidified for 48 h. Subsequently, the embedded samples were sectioned (thickness: 60-100 nm) and then double-stained with 3% uranyl acetate and lead citrate. Finally, the stained sections were observed and imaged by TEM (Tecnai G2 20 TWIN, FEI Company, Oregon, USA).
2.5. Mitochondrial Membrane Potential Analysis
The mitochondrial membrane potential (MMP/ΔΨm) was assessed by JC-1 dye (Beyotime Biotechnology, Shanghai, China, Cat#C2006) and detected by flow cytometry and confocal microscopy, according to previous methods [45, 46]. For flow cytometry, single-cell suspensions of mNSC and N2a were prepared and then coincubated with JC-1 work solution for 20 min at 37°C. Next, sample cells were centrifuged (600 g, 4°C, 5 min) and washed with JC-1 buffer solution 2 times. Subsequently, resuspended cells were subjected to flow cytometry tests. For image, cells were seeded in glass-bottom Petri dishes. 24 hours later, cells were coincubated with JC-1 work solution for 20 min at 37°C, washed with JC-1 buffer, and then examined by confocal microscopy.
2.6. Polymerase Chain Reaction (PCR)
Absolute quantitative PCR was performed as previously described [47–49]. The ratio of mtDNA and nuclear DNA was used to assess relative mtDNA copy number. In this experiment, mt-ND1/β-globin and mt-RNR1/β-actin were used to represent abundance. The sequences of the primers are described in Table S1.
2.7. Mitochondrial Stress Test
A mitochondrial stress test was performed using the Seahorse XF Cell Mito Stress Test Kit according to the manufacturer’s instruction [33, 49, 50]. Oxygen consumption rate (OCR), basic OCR, and maximal OCR were used as the main evaluation indicators. Different levels of cells were tested, including and .
2.8. Hypoxia-Reoxygenation (H/R) Cell Model and Mitochondrial Transplantation
The H/R cell model was induced by 48 h of hypoxia (1% O2) in a tri-gas CO2 incubator and 24 h of routine culture, according to previously described methods [51]. The cultured cells were divided into 3 groups: control group (routine culture (48 h)+replacing medium+routine culture (24 h)), H/R group (hypoxic culture (48 h)+replacing medium+continuing routine culture (24 h)), and H/R+mitochondrial treatment group (hypoxic culture (48 h)+ replacing medium (containing exogenous mitochondria)+continuing routine culture (24 h)). The ratio of mitochondrial donor cell number to the receiver is 5 (e.g., cells need mitochondria isolated from cells).
2.9. Cell Viability Assay
The viability was assessed by Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan, Cat#CK04) according to the manufacturer’s instruction. Briefly, N2a were coincubated with the CCK-8 working solution at 37°C for 3 h in the light-avoided environment. Then, cells were detected at 450 nm by a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
2.10. ROS Measurement by Flow Cytometry
DCFH-DA probes (Beyotime Biotechnology, Shanghai, China, Cat#S0033S) were used to measure the ROS levels in cells according to the manufacturer’s instruction. Fluorescence intensity was detected by flow cytometry and fluorescence plate reader. Briefly, after coincubated with DCFH-DA probes (10 μmol/l, excitation wavelength: 488 nm and emission wavelength: 525 nm) at 37°C for 30 min, cells were detected by a microplate reader (Molecular Devices, Sunnyvale, CA, USA) or collected by centrifugation (300 g, 5 min); after resuspended with PBS, DCFH-DA-labeled cells were further detected by flow cytometry.
2.11. Western Blot
Western blot was performed as previously described [52, 53]. The following primary antibodies were used for WB detection: anti-MFN1 (1 : 500), anti-OPA1 (1 : 1000), and anti-DRP1 antibodies (1 : 1000) were all purchased from Proteintech (Chicago, IL, USA); and anti-Bax (1 : 2000), anti-Bcl-2 (1 : 2000), anti-caspase-3 (1 : 2000), and anti-GAPDH antibodies (1 : 10000) were all purchased from Abcam (Cambridge, Cambs, UK). GAPDH served as internal reference. WB bands were detected with Gel-Pro Analyzer (Media Cybernetics, MD, USA).
2.12. Cell Apoptosis
Cell apoptosis was evaluated using an Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, NJ, USA, Cat#40302) according to the manufacturer’s instruction. Briefly, cells were coincubated with Annexin V-FITC and then propidium iodide for 15 min at RT in a light-avoided environment and then detected by flow cytometry.
2.13. Middle Cerebral Artery Occlusion (MCAO)
Intraluminal filament occlusion was used to induce focal cerebral ischemia injury [54, 55]. Anesthetized by 2% pentobarbital sodium (45 mg/kg), the rats were placed in a prone position. Then, the left common carotid artery, external carotid artery (ECA), and internal carotid artery (ICA) were exposed. Next, a silicon-coated monofilament suture was gradually inserted through the left ECA and was moved up into the left ICA to successfully occlude the left middle cerebral artery (MCA) and remained in situ for 120 min. Subsequently, the suture was carefully removed, the ECA was permanently ligated, and the incision was sutured. Sham group rats were subjected to the same procedure except for the 120 min occlusion of MCA. Experimental animals were then placed into individual cages and provided a standard diet and water. After 120 min occlusion, right before ICA reperfusion, the isolated mitochondria (from cells, the protein content was about 180 μg-200 μg) or saline (10 μl) was injected into the ICA and all incisions were closed.
2.14. Neurobehavioral Evaluation
Neurobehavioral deficits were evaluated 24 h after mitochondrial transplantation using multiple scales, including the Clark general functional deficit score [56, 57], the Clark focal functional deficit score [56, 57], the modified neurological severity score (mNSS) [55, 58], and the rotarod test [55, 59]. Behavioral assessments were conducted by two skillful investigators who were both blinded to the animal groups.
2.15. Cerebral Infarct Area Detection
Triphenyl tetrazolium chloride (TTC) staining was used to display the area of cerebral infarction [60, 61]. Briefly, 24 h after MCAO, the rats were deeply anesthetized and perfused with PBS transcardially, after which the rat brains were obtained and cut into 2 mm thick coronal sections. Subsequently, the brain sections were incubated with a 2% TTC solution at 37°C for 30 min in darkness. Then, stained slices were placed from the frontal to occipital order, and macroscopic images were obtained with a digital camera. Infarct areas were measured by Adobe Photoshop 21.0.0 (Adobe Systems Inc., San Jose, CA, USA).
2.16. Transcriptomic Analysis
RNA sequencing was performed as previously described [62, 63]. Downstream analysis was performed by R (R Foundation for Statistical Computing, Vienna, Austria).
2.17. Mitochondria and Lysosome Labeling
The mitochondrial fluorescent dyes MitoTracker™ Red CMXRos (ThermoFisher Scientific, Waltham, MA, USA), MitoTracker™ Green FM (ThermoFisher Scientific, Waltham, MA, USA), and MitoBright Deep Red (Dojindo Laboratories, Kumamoto, Japan) were used to label mitochondria according to the manufacturer’s instruction. In addition, 293T cells expressing COX8A gene N-terminal signal peptide-mCherry fusion protein were constructed by lentivirus (Inovogen Tech, Chongqi, China, Cat#3512) and the mitochondria were well labeled. The Lyso Dye (Dojindo Laboratories, Kumamoto, Japan, Cat#MD01)was used to label lysosomes according to the manufacturer’s instruction.
2.18. Statistical Analysis
Data that conform to a normal distribution with homogeneous variance are expressed as (SD), and Student’s -test or one-way analysis of variance (ANOVA) was used to compare the differences between two groups or among multiple groups, respectively. Data with a nonnormal distribution are presented as median (25%, 75% quantiles), and Mann-Whitney -test was taken into consideration. Statistical analysis and diagram generation were performed using GraphPad Prism 8.0.1 (GraphPad Software, Inc., San Diego, CA, USA). and were considered to be statistically different.
3. Results
3.1. Characteristics of Mitochondrial Donors
The ideal source of mitochondria is one that is readily available and can be amplified in large numbers. Both stem cells and tumor cells meet this requirement. Therefore, we chose N2a and mNSC as mitochondrial source cells to assess a range of mitochondrial characteristics. To evaluate the ΔΨm, JC-1 dye was used. Representative images showed N2a has more red components than mNSC (Figures 1(a) and 1(b)). Flow cytometry analysis confirmed that N2a had a higher ΔΨm than mNSC (N2a vs. mNSC: vs. , ) (Figure 1(c)). In addition, we observed that the mtDNA abundance of mNSC was higher than N2a based on the mitochondrial-nuclear DNA ratio, mt-ND1/β-globin (N2a vs. mNSC: vs. , ) (Figure 1(d)) and mt-RNR1/β-actin (N2a vs. mNSC: vs. , ) (Figure 1(e)). We subsequently analyzed the oxidative respiration capacity of mitochondria from N2a and mNSC based on the Seahorse XF analysis platform. The OCR-time diagram is shown in Figure 1(f) (N2a vs. mNSC ) and Figure 1(i) (mNSC vs. mNSC ), which implied a huge difference in oxidative respiratory activity between N2a and mNSC. Basal OCR of N2a ( cells) was significantly higher than those of mNSC ( cells) (N2a vs. mNSC: pmol/min vs. pmol/min, ) (Figure 1(g)). Similarly, N2a ( cells) exhibited higher maximal OCR values than those of mNSC ( cells) (N2a vs. mNSC: pmol/min vs. pmol/min, ) (Figure 1(h)). These results suggested that compared to mNSC, mitochondria from N2a exhibited a relatively stronger oxidative respiration capacity. In addition, mitochondrial morphology is presented in Figure S1 and mNSC culture and identification data are presented in Figure S2. Metabolic profiles of N2a and mNSC were quite different (Figure S3). Tumorigenicity evaluation of N2a and mNSC is presented in Figure S4. These results suggested that N2a-derived mitochondria have higher oxidative respiratory activity and lower mtDNA copy number, that the mitochondria from mNSC and N2a have similar morphology, and that they have different metabolomic profiles, and neither is tumorigenic. Therefore, we chose the N2a as a major source of mitochondria for subsequent experiments.
(a)
... Ischemic strokes, stemming from an interruption of blood flow to the brain, make up about 80-85% of all strokes. Despite being the main cause of human cerebral damage, systemic thrombolysis with recombinant tissue plasminogen activator or endovascular treatment remain the only therapeutic options for this disease (Allen and Bayraktutan, 2009;Reis et al., 2017). However, due to short therapeutic window and stringent eligibility criteria, each year less than 1% of stroke patients worldwide receive either therapy (Hacke et al., 2008;Bayraktutan, 2019). ...
Ischaemic stroke continues to be a leading cause of mortality and morbidity in the world. Despite recent advances in the field of stroke medicine, thrombolysis with recombinant tissue plasminogen activator remains as the only pharmacological therapy for stroke patients. However, due to short therapeutic window (4.5 h of stroke onset) and increased risk of haemorrhage beyond this point, each year globally less than 1% of stroke patients receive this therapy which necessitate the discovery of safe and efficacious therapeutics that can be used beyond the acute phase of stroke. Accumulating evidence indicate that endothelial progenitor cells (EPCs), equipped with an inherent capacity to migrate, proliferate and differentiate, may be one such therapeutics. However, the limited availability of EPCs in peripheral blood and early senescence of few isolated cells in culture conditions adversely affect their application as effective therapeutics. Given that much of the EPC-mediated reparative effects on neurovasculature is realised by a wide range of biologically active substances released by these cells, it is possible that EPC-secretome may serve as an important therapeutic after an ischaemic stroke. In light of this assumption, this review paper firstly discusses the main constituents of EPC-secretome that may exert the beneficial effects of EPCs on neurovasculature, then reviews the currently scant literature that focuses on its therapeutic capacity.
... The factors include brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), cytokines like monocyte chemoattractant protein (MCP-1) and macrophage inflammatory protein (MIP-1) [14]. Within a week after the lesion, newly generated neurons appear at the boundary of the damaged area [15], implying that neuroregenerative therapy using endogenous NSCs is highly anticipated as an effective strategy for treating degenerative brain diseases such as ischemic stroke. Nonetheless, this spontaneous regeneration by endogenous NSCs is insufficient for structural or functional restoration of the injured brain, as the majority of these newly formed neurons die before functional maturation, leaving only a limited number of cells which could stably integrate into the neuronal circuitry [16]. ...
Transplantation of neural stem cells (NSCs) has been proposed as an alternative novel therapy to replace damaged neural circuitry after ischemic stroke onset. Nonetheless, albeit the potential of these cells for stroke therapy, many critical challenges are yet to be overcome to reach clinical applications. The major limitation of the NSC-based therapy is its inability to retain most of the donor stem cells after grafting into an ischemic brain area which is lacking of essential oxygen and nutrients for the survival of transplanted cells. Low cell survival rate limits the capacity of NSCs to repair the injured area and this poses a much more difficult challenge to the NSC-based therapy for ischemic stroke. In order to enhance the survival of transplanted cells, several stem cell culture preconditioning strategies have been employed. For ischemic diseases, hypoxic preconditioning is the most commonly applied strategy since the last few decades. Now, the preconditioning strategies have been developed and expanded enormously throughout years of efforts. This review systematically presented studies searched from PubMed, ScienceDirect, Web of Science, Scopus and the Google Scholar database up to 31 March 2020 based on search words containing the following terms: “precondition” or “pretreatment” and “neural stem cell” and “ischemic stroke”. The searched data comprehensively reported seven major NSC preconditioning strategies including hypoxic condition, small drug molecules such as minocycline, doxycycline, interleukin-6, adjudin, sodium butyrate and nicorandil, as well as electrical stimulation using conductive polymer for ischemic stroke treatment. We discussed therapeutic benefits gained from these preconditioned NSC for in vitro and in vivo stroke studies and the detailed insights of the mechanisms underlying these preconditioning approaches. Nonetheless, we noticed that there was a scarcity of evidence on the efficacy of these preconditioned NSCs in human clinical studies, therefore, it is still too early to draw a definitive conclusion on the efficacy and safety of this active compound for patient usage. Thus, we suggest for more in-depth clinical investigations of this cell-based therapy to develop into more conscientious and judicious evidence-based therapy for clinical application in the future.
