Exosomes were firstly reported to exhibit neuroprotection on CNS injuries in 2012 . Subsequently, many studies have demonstrated that exosomes could provide neuroprotective effects in CNS injuries. The neuroprotection of exosomes was reportedly attributed to their effects on improvement of cognitive function, inhibition of inflammation, suppression of apoptosis, regulation of autophagy, promotion of angiogenesis and protection of BBB (Table 2).
3.1 Cognitive function
In animals, cognitive function is considered to be the ability to learn, retain and recall information. However, in humans, it also represents a complex, multidimensional set of intellectual functions like judgment and evaluation . Thus, in a broader context, cognitive function includes all mental abilities and processes related to knowledge including memory, reasoning, attention, comprehension and language production . Cognitive function was originally thought to be regulated by CNS, but now other systems, for example, the immune system and the intestinal microbiome may also be involved . Cognitive function impairment may occur in CNS injuries and neurodegenerative disease, which is characterized by problems in attention, thinking, memory, language and social communication. Peoples who suffer from cognitive decline experience poor quality of life and demand continuous care from their families and society, thus increasing the burden of family members and social insurance funds .
The effects of exosomes on cognitive function after CNS injuries have been explored. In a rat TBI model, MSCs-derived exosomes significant improved spatial learning as measured by the modified Morris water maze test and recovered sensorimotor function as evidenced by reduced neurological deficits and foot-fault frequency . Furthermore, treatment with exosomes derived from mouse BECs significantly improved neurological and cognitive functional outcome as evaluated by adhesive removal test and odor test in a mouse stroke model . In addition, it has been shown that exosomes from human umbilical cord MSCs attenuated stress-induced hippocampal dysfunctions and improved motor recovery in an acute brain disorder model .
The precise mechanisms underlying how exosomes regulated cognitive function were unclear. It has been revealed that cognitive function impairment involved selective neuronal loss in the hippocampus and cortex . Therefore, exosomes may also improve cognitive function by intervene with these pathological processes.
Inflammation is one of the major determinants of secondary brain damage after CNS injuries . In normal conditions, inflammation is a vital physiological immune response against noxious stimuli (such as injury or infection) and defends the host against pathogenic threats . However, in respond to CNS injuries, excessive inflammation may provoke substantial detrimental effects. This process involves initiating microglia activation and sustaining astrocytic activation. Once activated, these cells can induce a series of events including activation of glial, recruitment of leukocyte, and release of pro-inflammatory cytokines (e.g., IL-1β, IL-2, IL-6, TNF-α, interferon γ (IFN-γ)) and chemokines (e.g., C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 8 (CXCL8)) [65, 66]. These cytokines and chemokines recruit more inflammatory cells to amplify the inflammatory response, leading to BBB breakdown, cerebral edema and cell death .
Numerous studies have proposed that exosomes exerted a central effect in CNS injuries-induced inflammation. The effect of exosomes in CNS injuries-induced inflammation was firstly described by Zhang et al. in 2015 . They found that the density of CD68 + and GFAP + cells was significantly increased in the lesion boundary zone after TBI. MSCs-derived exosomes treatment significantly reduced the CD68 + and GFAP + cells density in the injured cortex compared to the phosphate buffered saline (PBS) treatment, suggesting that MSCs-derived exosomes had anti-inflammatory effects in TBI . Moreover, in a mouse model of SAH, bone marrow MSCs-derived exosomes suppressed the expression and activity of histone deacetylase 3 (HDAC3) and up-regulated the acetylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, thus attenuating neuroinflammation in early brain injury . Furthermore, in ischemic stroke (IS) models, exosomes secreted from the lipopolysaccharide (LPS)-stimulated macrophage promoted microglial polarization from the M1 phenotype to the M2 phenotype and reduced the production of IL-1β, TNF-α in vitro, indicating the anti-inflammatory effect of exosomes . In addition, in has been shown that plasma exosomes could enhance melatonin therapeutic effects against ischemia-induced inflammatory responses and inflammasome-mediated pyroptosis in ischemic stroke .
The underlying mechanisms of exosomes-mediated inflammation are immensely complicated. Studies have indicated that the NF-κB signaling pathway might be the key target and we will discuss the detailed mechanisms in our following sections.
Under physiological conditions, the brain vascular system is stable and contributes to the maintenance and growth of the tissue . When brain vasculature is damage under pathological conditions including injuries, angiogenesis is activated. Angiogenesis is a tightly regulated process through which new blood vessels are formed, it involves the participation of endothelial cells, extracellular matrix and vascular cells to form capillaries. This process requires an orchestrated interplay of many stimulators, inhibitors and matrix components [71, 72]. Angiogenesis facilitates the generation of new vasculature, which further accelerates highly coupled neurorestorative process and promotes tissue perfusion. Angiogenesis is controlled by vascular growth factors such as vascular endothelial growth factor (VEGF) . VEGF owns a mitogenic effect on endothelial cells, thus increasing the vascular permeability and promoting cell migration. Besides its role in angiogenesis, VEGF also shows important effects in the neuronal development and physiological function . VEGF can regulate the ion channels of the neuron membrane and accelerate the development of neurons and neural dendritic .
Since angiogenesis is benefit for CNS injuries-caused secondary injury, exosomes may attenuate brain damage by promoting angiogenesis. Consistent with this hypothesis, Zhang et al. proposed that MSCs-derived exosomes significantly increased the vascular density and angiogenesis as identified by EBA/BrdU + double labeling for newborn endothelial cells in the injured cortex . Furthermore, bone marrow MSCs-derived exosomes increased the number of branch points as proven by tube formation assay in a rat hypoxic-ischemic injury model . In another study, it was shown that miRNA-17-92 cluster-enriched exosomes derived from human bone marrow MSCs increased the formation of blood vessels after TBI, indicating that exosomes could promote angiogenesis. Because angiogenesis is emerging as therapeutic target for CNS injuries, therefore, exosomes-based therapies by targeting angiogenesis might provide opportunities for the development of novel therapeutic strategies for CNS injuries.
Apoptosis is a very tightly programmed cell death (PCD) occurring regularly to eliminate unnecessary and unwanted cells as well as to maintain a homeostatic balance between cell survival and cell death . Apoptosis is critical to animals especially long-lived mammals that must integrate multiple physiological and pathological death signals. Apoptosis therefore represents a physiological and protective response to damage . It has been shown that insufficient apoptosis can trigger cancer or autoimmunity, while excessive activation of apoptosis could be harmful and contribute to abnormal cell death, particularly in pathological conditions such as acute and chronic degenerative diseases, immunodeficiency and trauma . Morphologically, apoptosis renders the cell with shrinkage, which is characterized by DNA fragmentation, chromatin condensation, cytoplasm compacting and plasma membrane blebbing. This is followed by nuclear fragmentation and formation of apoptotic bodies . If apoptosis occurs in CNS injuries, it can cause secondary brain injury, aggravating the damage of brain .
The functions of exosomes in apoptosis have been studied. The results obtained by Song et al. demonstrated that microglia-derived exosomes significantly increased cell survival and decreased neuronal apoptosis in ischemia-reperfusion injury, as demonstrated by neuronal survival, TdT-mediated dUTP Nick-End labeling (TUNEL) staining and the lactate dehydrogenase (LDH) assay . In addition, Ni et al. showed that in a mouse TBI model, bone marrow MSCs-derived exosomes up-regulated the expression of B-cell lymphoma-2 (Bcl-2) while down-regulated the expressions of Bcl-2-associated X protein (Bax), suggesting that bone marrow MSCs-exosomes attenuated cell apoptosis . In another study conducted by Lai et al., they found that MSCs-derived exosomes decreased apoptosis in brain following SAH as shown by increased expression of Bcl-2 and decreased expression of caspase-3 . In conclusion, these data suggested that exosomes could reduce cell apoptosis in models of CNS injury.
Researches so far have only studied the role of exosomes on apoptosis in general. However, apoptosis can be divided into two pathways: the mitochondria-dependent pathway (the intrinsic pathway) and the death receptor-dependent pathway (the extrinsic pathway). The intrinsic pathway involves a chain of intracellular events occurring in the mitochondrion including the release of cytochrome c, formation of the apoptosome with apoptotic protease-activating factor 1 (APAF1), activation of caspase-9 and subsequent caspase-3. The release of cytochrome c is positively regulated by the pro-apoptotic Bcl-2 family members such as Bax, Bcl-2 antagonist killer 1 (Bak), Bid and negatively regulated by the anti-apoptotic Bcl-2 family members such as Bcl-2, B-cell lymphoma-extra large (Bcl-xL). In contrast, the extrinsic pathway is initiated by the binding of TNF ligand to TNF receptor and the binding of Fas ligand to Fas receptor. Upon ligand binding, the death receptors allow the binding of an initiator caspase-8 or -10 to form death inducing signaling complex (DISC) through its death effector domain (DED). The activation of caspase-8 relays the death signal to an execution caspase to bring about apoptosis [85, 86]. Thus, which apoptotic pathway is associated with the effects of exosomes in CNS injuries-induced apoptosis remains unclear and further studies are needed to clarify it.
Autophagy is an evolutionarily conserved lysosomal pathway for the degradation of cytoplasmic components . In conditions of starvation response, cell differentiation and quality control, autophagy is activated and plays an important role in maintaining and regulating cell homeostasis by degrading intracellular components and providing degradation products to cells . Autophagy begins with the formation of a membrane vesicle called the phagophore, which matures into a spherical lipid bilayer vesicle named the autophagosome. The autophagosome then fuses with a lysosome and degrades the contents in autolysosome . Recent studies have revealed that the dysfunction of autophagy was implicated in CNS injuries and extensive activation of autophagy can lead to type II PCD . Up to now, the dual role of autophagy in protective or destructive of CNS injuries remains controversial. Shi et al. found that in cerebral ischemia‑reperfusion rats, inhibiting autophagy by sevoflurane attenuated brain damage, demonstrating a detrimental role of autophagy . Conversely, Ahsan et al. reported that Urolithin A-activated autophagy protected against ischemic neuronal injury by inhibiting endoplasmic reticulum (ER) stress both in vitro and in vivo, suggesting that autophagy played a beneficial role in stroke .
There were also studies showing that exosomes could affect autophagy in CNS injuries. However, the roles of exosomes-regulated autophagy in CNS injuries were also controversial. Li et al. have shown that exosomes from neurons inhibited cell apoptosis and death in TBI by suppression of Rab11a-mediated autophagy, suggesting a detrimental role of autophagy in TBI . Interestingly, in another study conducted by Yuan et al., they found that bone marrow MSCs-derived exosomes decreased ER stress in BV2 cells by induction of disabled homolog 2-interacting protein (DAB2IP)-mediated microglial cell autophagy, suggesting a protective role of exosomes and autophagy in brain injury . The discrepancies may be due to the different source of exosomes and cell types used in these two studies. Taken together, by combination with the previous studies, we though that depending on different CNS injury models, sources of exosomes and cell types, autophagy and cell death may have inhibitory, additive or even synergistic effects.
3.6 BBB function
BBB is a highly specialized, semi-permeable physical barrier that locates at the interface between the CNS and the surrounding environment. It is instrumental in regulating the metabolism of brain, maintaining the microenvironmental homeostasis of CNS and coordinating the functions of peripheral organs . In addition, BBB is a dynamic metabolic interface that can bi-directionally regulate the transport of fluids, solutes and cells . Structurally, BBB is formed by BECs with TJ. Dysfunction of BBB is a common pathological feature in CNS injuries. Several underlying events are involved in BBB destruction, such as disruption of the TJ, breakdown of the BECs and degradation of the extracellular matrix . In an in vitro model of IS, Pan et al. found that MSCs-derived exosomes alleviated BBB disruption in hypoxia/reoxygenation (H/R)-injured endothelial cells by analyzing the Evans blue dye extravasation and brain water content . Moreover, Lai et al. suggested that bone marrow MSCs-derived exosomes attenuated BBB permeability in early brain injury after SAH . Furthermore, another in vivo study confirmed the protective effects of exosomes on BBB in ischemia-reperfusion injury .