The neurovascular unit (NVU), which plays a vital role in neurological disorders, is a multifunctional and morphological entity composed of many cells and materials, including neurons, neurogliocytes, cells of brain vessels (pericytes, endothelial cells, smooth muscle cells), and the extracellular matrix (Potjewyd et al., 2018; Forró et al., 2021). Neurons and neurogliocytes, including oligodendrocytes, astrocytes, and microglia, are key players in maintaining the homeostasis of the microenvironment in the central nervous system (CNS). Under ischemic stroke conditions, hypoxia induces cell death and apoptosis and directly contributes to the release of pro- or anti-inflammatory cytokines and autophagy-related proteins from glia and neurons, leading to neuroprotection or neurotoxicity (Pan et al., 2021; Xin et al., 2021; Hou et al., 2022). Extracellular vesicles (EVs), present in multiple bodily fluids, are cell-derived nanoscale particles enclosed by a protein-rich lipid bilayer (Rodríguez and Vader, 2022). Based on their size, EVs can be classified into three types as follows (Saltarella et al., 2021): 1) apoptotic bodies (1,000–5,000 nm) are derived from apoptotic or necrotic cells that break up into multiple vesicles (Théry et al., 2009; Saltarella et al., 2021); 2) microvesicles (200–1,000 nm) are pinched off from the cell membrane directly (Théry et al., 2009; Saltarella et al., 2021); and 3) exosomes, the smallest EVs (30–150 nm), are produced via an active, energy-dependent, and organized process (Théry et al., 2009; Brinton et al., 2015; Saltarella et al., 2021). Although all classes of EVs and their heterogeneous subsets differ in their biogenesis, diameter, and cargo, owing to their natural composition, EVs have been suggested to exhibit a common feature of good biocompatibility and to transfer homing properties to specific cell types (Möller and Lobb, 2020; Roefs et al., 2020). Therefore, EVs are said to play an important role in intercellular communication as a new paracrine mediator (Möller and Lobb, 2020; Nagelkerke et al., 2021). All the aforementioned types of cells have been found to naturally secrete EVs under normal, physiological, and pathological conditions, owing to the dynamics of the cell membrane (Zhang et al., 2021a; Forró et al., 2021). A great deal of evidence has demonstrated that EVs derived from these cells exert biological functions by modulating specific aspects, such as participation in inflammatory reactions, cell migration, proliferation, apoptosis, and autophagy (Gao et al., 2020; Zhang et al., 2021a; Tallon et al., 2021). However, data on the precise mechanisms underlying such a therapeutic approach are limited. In this review, we summarize the effect of neuron/neurogliocyte-EVs in preclinical studies on treating neurological disorders by regulating different signaling pathways of cellular processes, such as cell apoptosis, inflammation, angiogenesis, and autophagy, and summarize the mutual mechanisms of the interaction between neuron/glial cell-EVs and ischemic stroke conditions.

Despite the identification of well-recognized classes of cell-to-cell communication approaches, EVs have been a hot topic in the past several decades as an essential method for transferring cargo to short or long distances between cells (van Niel et al., 2018). Nevertheless, only a few people are aware of the discovery of EVs that can be traced back to the 19th century using a different nomenclature (Kaddour et al., 2021). In the 1840s, Gulliver’s first studied milky particles in the blood serum, which he called “the molecular base of the chyle,” with very small globules of active Brownian movement and a size ranging from −0.5 to 1 micron; this may have been the first encounter with EVs in the report (Gage and Fish, 1924). All eukaryotes can secrete EVs and exhibit a snapshot of the secreting cells, encapsulating active and specific biomolecules from the donor cell (Marzan and Stewart, 2021). Once EVs are produced in the extracellular space, these nanosized particles can be uptake by recipient cells, and in turn act as messengers and perform biological functions via the delivery of plenty of functional biomolecules including proteins, nucleic acids, lipids, and metabolites, into recipient cells, both near and far from the secreting cell (Palviainen et al., 2019; Marzan et al., 2021). Currently, there are three main subsets of EVs: exosomes, microvesicles, and apoptotic bodies. Exosomes are small EVs with a diameter of 30–150 nm and density of 1.13–1.19 g/ml (Yang et al., 2020). Exosome production is mainly classified into three stages: endocytosis, multivesicular body formation, and release. First, the plasma membranes start endocytosis, which is followed by the fusion of multiple intraluminal vesicles to produce endosomes. Second, the loading of bioactive molecules such as non-coding RNAs (ncRNAs), lipids, and proteins facilitates the formation of multivesicular bodies (van Niel et al., 2018). Finally, multivesicular bodies and plasma membranes are fused to achieve exosome release. Microvesicles with a diameter of 200–1,000 nm are slightly larger EVs generated by budding directly from the cell plasma membrane (Thietart and Rautou, 2020). This process involves lipid rearrangements concerning the asymmetry of the plasma membrane accelerated by membrane translocases, scramblases, and calpain. Conversely, apoptotic bodies, the largest among all types of EVs, are 1,000–5,000 nm in diameter, are secreted by dying cells, and are even more abundant than exosomes or microvesicles under specific conditions (Doyle and Wang, 2019; Battistelli and Falcieri, 2020). Many efforts have been made to identify the emerging role of exosomes and microvesicles in intercellular communication. However, there is little evidence on the value of apoptotic bodies in nanomedicine.

Cell death is the consequence of multiple cellular processes that occur during neurological diseases, including mitochondrial dysfunction, protein aggregation, free radical generation, excitotoxicity, and inflammation (Salucci et al., 2021). Numerous studies have revealed that EVs derived from these cells are involved in cell death in neurological disorders. A previous study assessed the overall effects of exosomes derived from normoxic and hypoxic neurons on the survival and neuritogenesis of rat cortical neurons (Chiang et al., 2021). As presented by Chiang et al. (2021), hypoxic concentrated conditioned media, but not normoxic concentrated conditioned media, significantly reduced neuronal viability. They further pelleted exosomes from both normoxic and hypoxic concentrated conditioned media, and then examined the effects after administration of PBS and 25, 100, or 200 μg/ml exosomes in cultured cortical neurons using the CCK-8 assay. They noted a linear trend in the decrease in viability with increasing hypoxic exosome dose, revealing that hypoxic exosomes impair neuronal survival in a dose-dependent manner. However, low concentrations (25 and 100 μg/ml) of normoxic exosomes did not affect neuronal viability. To further evaluate the effect on axonal outgrowth, dissociated cortical neurons were treated with exosomes and subjected to immunostaining with an anti-Tau antibody. The results showed that exosomes derived from hypoxic neurons, but not EVs obtained from normoxic neurons, impaired both dendritic and axonal outgrowths of cultured cortical neurons (Chiang et al., 2021). Among the various programmed cell death pathways (Datta et al., 2020), apoptosis accounts for a large proportion of cell death through brain injury (Radak et al., 2017), which efficiently removes damaged cells from DNA damage or during development (Fan et al., 2020). Apoptosis plays an essential role in the homeostasis of normal tissues, and scientists have identified that EVs play essential roles in regulating cell apoptosis. Huang et al. (2020) indicated that not only hypoxic neuron-concentrated conditioned media but also EVs derived from hypoxic neurons exacerbate hypoxia-induced injury on transplanted mesenchymal stem cell viability, apoptosis, and oxidative stress in vitro. In addition to neuronal EVs, the effects of EVs derived from glial cells on cell death and apoptosis regulation have been studied more extensively. Bu et al. (2020) performed a series of in vitro experiments and demonstrated that astrocyte-derived exosomes promoted hypoxia-inhibited PC12 cell activity and suppressed cell apoptosis. Likewise, Chun et al. (2021) reported that astrocyte-derived EVs enhanced the survival and electrophysiological function of human cortical neurons. Notably, neuronal apoptosis was significantly increased upon treatment with conditioned medium from necroptotic astrocytes via EVs delivery (Chen et al., 2021). Furthermore, Casella et al. (2020) uncovered oligodendrocyte-derived EVs as an antigen-specific therapy for experimental autoimmune encephalomyelitis. This process was safe and restored immune tolerance by inducing apoptosis of autoreactive CD4⁺ T cells. Concerning the EVs derived from microglia, EV derived from both hypoxic and normoxic microglia can repress neuronal apoptosis and promote neuronal viability in hypoxic cortical neurons (Li et al., 2021a; Zhang et al., 2021a). To date, many studies have assessed the effect of EVs derived from neurons/glial cells in the regulation of apoptosis in CNS diseases (Song et al., 2019a; Xu et al., 2019; Bu et al., 2020; Casella et al., 2020; Datta Chaudhuri et al., 2020; Gao et al., 2020; Nogueras-Ortiz et al., 2020; Zhang et al., 2021a; Chen et al., 2021; Chun et al., 2021; Qi et al., 2021). The characteristics of these studies are summarized in Table 1 .

Autophagy is an evolutionarily conserved cellular mechanism (Wang et al., 2018), which is a program caused by the regulation of the internal conditions of cells (Sun et al., 2018), such as starvation, hypoxic nutrient deficiencies, and infection (Mo et al., 2020), leading to the degradation of toxic proteins, damaged organelles, and invading pathogens via the lysosomal pathway (Liu et al., 2020). On the one hand, autophagy can maintain cellular nerve homeostasis, since it is associated with degraded misfolded or nonfunctional proteins and damaged organelle, suggesting that it plays an essential housekeeping role in the CNS (Peker and Gozuacik, 2020). On the other hand, autophagy is also associated with the promotion of cell death. It is possible that excessive upregulation of autophagy and long-term autophagy eventually result in self-digestion or have harmful effects (Bar-Yosef et al., 2019). Abundant evidence indicates that autophagy and exosomes are inseparable. Autophagy plays a vital role in the synthesis and degradation of extracellular vesicles (EVs). It has been reported that autophagosomes not only have a strong ability to fuse with lysosomes, but also fuse with multivesicular bodies to form amphiphiles. The amphiphiles eventually fuse with lysosomes and dissolve the inner material of the intraluminal vesicles, resulting in a significant reduction in the release of exosomes. Taken together, these results suggest that autophagosome formation plays a key role in EVs secretion and transport (Xu et al., 2018). In addition, much direct evidence has verified that autophagy could be a therapeutic target in neurological treatment by using neuron/neurogliocyte-EVs. For example, in traumatic brain injury, Li et al. (2019) illustrated that neuronal EVs enriched with miR-21-5p can suppress neuronal autophagy induced by scratch injury, directly targeting the Rab11a 3′UTR region to reduce its translation, thus attenuating trauma-induced, autophagy-mediated nerve injury in vitro. Likewise, Liu et al. (2021a) found that M2 type microglia derived EVs could transfer miR-135a-5p into neurons to suppress the expression of thioredoxin-interacting protein, which in turn suppresses the activation of the nod-like receptor protein 3 inflammasome, thereby inhibiting neuronal autophagy induced by ischemia. To date, a vast number of studies have assessed the interaction between neuron/glial cell-EVs and autophagy in CNS diseases (Li et al., 2019; Pei et al., 2019; Guo et al., 2020; Pei et al., 2020; Zang et al., 2020; Liu et al., 2021a). The characteristics of these studies are summarized in Table 2 .

Angiogenesis is the appearance of new microvessels that branch off from pre-existing vessels (Ruan et al., 2015). Hypoxic or insulted tissues can produce vascular endothelial growth factors, and angiogenesis begins along the concentration gradient of vascular endothelial growth factor in neonates (Carmeliet and Tessier-Lavigne, 2005). Angiogenesis plays a vital role in brain injury recovery following an injury because it promotes blood flow and metabolic nutrients to reach the injured regions to promote neural tissue repair by facilitating neurogenesis and synaptic initiation (Hatakeyama et al., 2020; Ma et al., 2021). To investigate whether microglial EVs regulate angiogenesis in vitro, Zhang et al. (2021a) showed that EVs derived from hypoxia-preconditioned microglia labeled with DiI were taken up by bEnd.3 endothelial cells. Further experiments concerning the therapeutic impact of these EVs against hypoxic injury of bEnd.3 cells were performed to evaluate cell viability and cytotoxicity via the MTT and LDH release assays, respectively. The results demonstrated that decreased cell viability and increased cytotoxicity after hypoxia were suppressed by these EVs (Zhang et al., 2021a). Moreover, these EVs can significantly promote bEnd.3 migration during hypoxia according to the scratch migration assay. Meanwhile, EVs derived from preconditioned microglia reversed this effect of impaired tube formation in bEnd.3 cells caused by hypoxia. Following the aforementioned in vitro findings, Zhang et al. (2021a) further indicated that EV administration induces angiogenesis and diminishes cell injury in the ischemic mouse hemispheres. Taken together, the evidence suggests that EVs derived from hypoxic microglia promote cell viability, migration, and angiogenesis in hypoxic injury. As such, the ability of EVs derived from neurons and other glial cells to regulate angiogenesis is unclear, and additional and reliable data are urgently needed.

Neuroinflammation is integral to the neurological pathophysiological process and results in damage to tissue homeostasis (DiSabato et al., 2016; Alawieh et al., 2018), involving acidosis, excitotoxicity, promotion of cytoplasmic Ca²⁺ concentrations, loss of glucose and oxygen, destruction of the blood-brain barrier, and damage to mitochondria (Xu et al., 2021; Chen et al., 2020a; Forrester et al., 2018). However, the inflammatory response is a double-edged sword after injury because it not only intensifies secondary injury to the brain, but also promotes the recovery of neurological function, thereby demonstrating that neuroinflammation is related to the pathogenesis and prognosis of CNS disorders (Zhang et al., 2021a). Several studies have revealed that various neuron/glial cell-EVs are involved in the regulation of inflammation and microglial activation in neurological diseases. Microglia serve as “brain-resident macrophages” that comprise approximately 10% of all the cells in the CNS (Fakhoury, 2018), the activation of microglia represents the first step of an inflammatory response, followed by the activation of other immune cells like neutrophils, T cells, natural killer cells, etc. (Xin et al., 2021; Iadecola and Anrather, 2011; Jin et al., 2010). Thus, the effects of microglial EVs on inflammation regulation have also been studied extensively. Casella et al. (2018) engineered a murine microglia cell line, BV-2 cell, to produce EVs enriched with the endogenous “eat me” signal Lactadherin on the surface to target phagocytes while overexpressing the anti-inflammatory cytokine IL-4. A single injection of these EVs into the cisterna magna upregulated anti-inflammatory markers, such as chitinase 3-like 3 and arginase-1, and significantly suppressed tissue damage in a mouse model of multiple sclerosis and experimental autoimmune encephalomyelitis. Likewise, overexpression of miR-124-3p in EVs derived from microglia following traumatic brain injury can reduce neuronal inflammation and contribute to neurite outgrowth by transferring these EVs into neurons (Huang et al., 2018). Similarly, BV2 cell-secreted EVs enriched with miR-711 could target and suppress Itpkb, thereby suppressing M1 microglial polarization and promoting M2 microglial polarization (Zhang et al., 2020). Besides that, the EVs derived from other glial cells, such as astrocyte and oligodendrocyte, also are involved in the regulation of inflammation (Casella et al., 2020; Li et al., 2020). The additional details are provided in Table 3 . EVs derived from glial cells might, therefore, contribute to suppressing inflammation and microglial activation (Casella et al., 2018; Huang et al., 2018; Zhang et al., 2020), information regarding the modulation of neuronal EVs in inflammation is scarce. An overview of how EVs derived from neurons/glial cells affect neurological recovery is shown in Figure 1 .

A comprehensive literature search of electronic databases, including PubMed, Cochrane Library, EMBASE, Web of Science, and China National Knowledge Infrastructure, was conducted from the inception of these databases until 28 February 2022. We retrieved studies assessing the effect of EV-derived from neurons/glial cells on ischemic stroke adopting the following keywords in accordance with Boolean logic: (“ischemic stroke” OR “middle cerebral artery occlusion” OR “MCAO” OR “ischemia”) and (“exosome” OR “EVs” OR “Extracellular vesicles” OR “microvesicles”) AND (“microglia” OR “neuron” OR “astrocyte” OR “oligodendrocyte”). In addition, all references from the included articles were manually checked to identify potential qualifying studies that were missed in the electronic search results. The process was repeated until no further studies would be obtained. A total of 29 studies were identified in this section (Frühbeis et al., 2013; Fröhlich et al., 2014; Guitart et al., 2016; Xin et al., 2017; Xu et al., 2017; Hira et al., 2018; Yang et al., 2018; Song et al., 2019a; Song et al., 2019b; Pei et al., 2019; Tian et al., 2019; Xu et al., 2019; Chen et al., 2020b; Bu et al., 2020; Frühbeis et al., 2020; Huang et al., 2020; Pei et al., 2020; Wu et al., 2020; Xie et al., 2020; Zang et al., 2020; Liu et al., 2021a; Yang et al., 2021a; Zhang et al., 2021a; Li et al., 2021b; Liu et al., 2021b; Zhang et al., 2021b; Chiang et al., 2021; Du et al., 2021; Raffaele et al., 2021), conducted from 2016 to 2021. The most extensively adopted species and associated stroke models are mice and middle cerebral artery occlusion, respectively. Among these 29 publications, 10 focused on microglia-EVs (Yang et al., 2018; Song et al., 2019a; Tian et al., 2019; Xie et al., 2020; Zang et al., 2020; Liu et al., 2021a; Zhang et al., 2021a; Li et al., 2021b; Zhang et al., 2021b; Raffaele et al., 2021), 11 focused on astrocyte-EVs (Guitart et al., 2016; Xin et al., 2017; Hira et al., 2018; Pei et al., 2019; Xu et al., 2019; Chen et al., 2020b; Bu et al., 2020; Pei et al., 2020; Wu et al., 2020; Liu et al., 2021b; Du et al., 2021), 3 focused on oligodendrocytes (Frühbeis et al., 2013; Fröhlich et al., 2014; Frühbeis et al., 2020) and 5 focused on neuron-EVs (Xu et al., 2017; Song et al., 2019b; Huang et al., 2020; Yang et al., 2021a; Chiang et al., 2021), demonstrating that it is sufficient to suggest that EVs derived from neurons/glial cells play an important role in regulating the progress and prognosis of ischemic stroke.

EVs, ranging in size from 30 to 5,000 nm, secreted by different cell types, are surrounded by cell-segregated membrane complexes with a lipid bilayer and play an important role in the pathological and physiological environments of target cells by transferring a variety of cargo of nucleic acids, lipids, and proteins. Neurons and neurogliocytes, including oligodendrocytes, astrocytes, and microglia, play essential roles in maintaining homeostasis of the microenvironment in the CNS. These cells have been shown to support cell-cell communication via multiple mechanisms, most recently by the release of EVs, which further regulate various mechanisms underlying CNS disorders, including angiogenesis, autophagy, apoptosis, cell death, and inflammation. Ischemic stroke following cerebral artery occlusion is a major cause of chronic disability worldwide. Neuron/neurogliocyte-EVs are involved in the treatment of ischemic stroke under preclinical conditions. Among the mechanisms of these EVs in the treatment of stroke, ncRNAs play a key role, and they can also modulate directly or indirectly various mRNAs signaling pathways such as TGF-β1and NF-κB.