Spinal cord injury (SCI) is a severe traumatic disease that often leads to severe and permanent paralysis and carries a heavy burden for individuals, families, and society [1]. SCI occurs in a population of approximately 250,000 to 500,000 worldwide each year. In addition, according to the World Health Organization (WHO), the costs of healthcare, in both time and money, are expected to increase in the coming years ^[2][3][2,3]. When SCI develops, patients’ normal sensory, motor, or autonomic function is greatly affected. They often have numerous multisystem complications, such as motor deficits, gastric dysmotility, cardiac arrest, and bladder dysfunctions ^[4][5][6][4,5,6]. Based on many early primary injuries, progressive secondary injuries can exacerbate the clinical situation [7]. SCI is primarily caused by spinal misalignment and damage, resulting in immediate neuronal cell death, rupture of blood vessels, and damage to the blood–spinal-cord barrier (BSCB). In addition, the wound microenvironment triggers neuronal cell death, inflammatory responses, and vascular changes. Problems like these cause additional nerve damage and dysfunction, further prolonging SCI ^[8][9][8,9]. Due to the complex pathophysiology of SCI, there still remains no effective, definitive treatment or functional recovery strategy for SCI. Exosomes and microRNAs secreted from stem cells can freely pass through the blood–spinal-cord barrier. Once they reach the spinal cord, they may play a role in promoting nervous system repair, including reducing neuronal cell death, promoting vascular remodeling and neurogenesis, reducing neuroinflammation, and promoting axonal remodeling.

After SCI, a large number of neuronal cells are damaged following axonal loss, ischemia, inflammation, and apoptosis ^[10][118]. Neuronal cell death has been reported to occur only in the early stages of SCI, which involves blood vessel rupture, resulting in bleeding and hypoxia ^[3][11][3,119]. Therefore, many studies have shown that using exosomes for treatment in the early stages after SCI can successfully attenuate the apoptosis of neurons ^{[1][5][12][13][14]}[1,5,13,120,121]. There are several types of therapy SCI using exosomes that act by attenuating neuronal apoptosis, promoting neurogenesis, and improving axonal remodeling. For this, in the laboratory, exosomes are isolated using various methods for various purposes and applications. For example, it can be isolated using ultracentrifugation techniques ^[15][16][17][122,123,124], size-based isolation techniques ^[18][19][125,126], polymer precipitation ^[20][21][127,128], and immunoaffinity capture techniques ^[22][23][129,130]. Although various methods have been developed for the isolation and purification of exosomes, a combination of different isolation methods may be better than the isolation effect of a single method. Therefore, in order to obtain ideal exosomes by improving isolation efficiency and enrichment, many research teams have combined different methods of isolating and purifying exosomes to improve yield and purity ^[24][25][131,132].

Apoptosis, which is known as programmed cell death, can be attenuated by intervening with certain factors, such as exosomes, and reducing the ATP-driven process of cell death ^[26][27][133,134]. Apoptotic cell death occurs and contributes to many diseases, such as cancer, restenosis of tissues, stroke, heart failure, neurodegeneration, and AIDS, with cell accumulation and loss ^[27][134]. There are two major pathways for caspase activation: the extrinsic pathway, which is induced by the TNF family of cytokine receptors, such as TNFR1 and Fas, and the intrinsic pathway, which is induced by cytochrome c and elevations in the pro-apoptotic Bcl-2 family proteins such as Bax ^[27][134].

Exosomes and exosomal miRNAs enhance recovery from SCI by attenuating neuronal cell death. Cell apoptosis can be attenuated under the intervention of certain factors such as exosomes ^[26][133]. In preclinical experiments, MSC exosomes have been shown to increase Bcl-2 expression and decrease Bax levels after systemic administration in an SCI mouse or rat model as well as to promote functional improvement and reduce disrupted endothelial cells at the contusion site ^[1][13][1,120]. Another preclinical experiment demonstrated that MSC exosomes could effectively activate the Wnt/β-catenin signaling pathway with anti-apoptotic effects ^[14][121]. Other groups have also reported that MSC exosomes improved the expression of autophagy-related proteins, such as LC3IIB and Beclin-1, and induced autophagosome formation ^[5][12][5,13]. Liu et al. found that bone-MSC-derived exosomes had the potential to reduce lesion size, neuronal apoptosis, and glial scar formation and induced blood vessel density and axonal regeneration in SCI rats by suppressing the activation of astrocytes ^[12][13].

When SCI occurs, the injured spinal column is hypoxic to reduce endogenous neural tissue repair and tissue regeneration with abnormal angiogenesis. Vascular endothelial cells show increased uptake of exosomes to activate protein kinase A (PKA) signaling and promote vascular endothelial growth factor (VEGF) expression as a component of the blood vessel wall ^[28][135]. Cao et al. found that exosomes derived from human urine stem cells stimulated angiogenesis through the PI3K/AKT signaling pathways to enable SCI recovery in the damaged area ^[29][136]. In addition, MSC exosomes have been shown to enhance angiogenesis and axon regeneration by reducing PTEN expression in the damaged spinal cord region and to reduce microglia and astrocyte proliferation in SCI rats ^[30][137]. Exosomes have also been confirmed to promote axon regeneration by regulating the NF-κB p65 signaling pathway in pericytes ^[31][138].

Exosomes are known to contain various proteins including growth factors, such as NGF, which regulates neuronal survival and the release of neurotransmitters and facilitates the plasticity of axons in the adult central and peripheral nervous systems ^[4][32][4,139]. One group reported that exosomes derived from NGF-overexpressed MSCs promoted SCI repair in a mouse model, leading to the regeneration of neuronal axons and differentiation into neurons [4].

miRNAs are 21–23 nucleotides in length and are endogenous nonprotein-coding RNA molecules. Many researchers have predicted that the administration of miRNAs could easily reach a target region that has experienced neuronal death or a damaged organ ^{[3][33][34][35][36][37][38]}[3,16,85,140,141,142,143]. Numerous studies have shown that miRNAs have important roles in some autoimmune diseases and are essential in the development and homeostasis of the immune system ^[39][40][41][144,145,146]. Recently, miRNAs have been identified as potential new targets for SCI treatment ^{[42][43][44][45]}[147,148,149,150]. Accumulating evidence indicates that exosomes with a bilayer membrane structure can be used as valuable carriers for targeting miRNAs at SCI sites. In addition, exosomes can cross the BBB to enhance the therapeutic effect of miRNAs ^[46][151]. MSCs are pre-transfected with specific miRNA plasmids to secrete exosomes containing high levels of specific miRNAs ^[37][142]. Extensive studies have shown that exosomes from MSCs carrying miRNAs exhibit efficient repair effects in SCI.

Lin’s group reported that increased miR-126 could promote angiogenesis, inhibit inflammation, and improve functional recovery after SCI ^[43][148]. However, there remains a limitation regarding the delivery of miRNAs into the damaged tissue. Similar to siRNAs, miRNAs are highly unstable in the local extracellular environment and are delivered to the target tissues by an effective carrier system. In this regard, there are two delivery systems: viral and non-viral systems. The viral system is the most efficient delivery system but has toxic side effects, and the non-viral system contains physical and chemical components that are safe for clinical application ^{[47][48][49][50]}[115,152,153,154]. Therefore, exosome-derived miR-126-treated MSCs were used as a non-viral delivery system and were found to promote angiogenesis and neurogenesis and attenuate apoptosis after SCI in a rat model ^[47][115]. Similarly, there are a few studies regarding exosomes of miRNA-modified MSCs as a therapeutic delivery vehicle to the CNS. Another group found that exosome-derived miR-133b-MSCs contributed to neurite remodeling and improved functional recovery poststroke ^[51][155]. In addition, miR-146b-transferred exosomes were found to have the potential to inhibit tumor growth in brain tumor models ^[52][156]. Zhao et al. demonstrated that exosomes from miR-25-overexpressed-MSCs exerted neuroprotective effects in an ischemic SCI model ^[33][16]. miR-25-enriched exosomes were also shown to inhibit NADPH oxidase 4 expression and increase superoxide dismutase activity and exhibited lower levels of IL-1β and TNF-α.

miR-19b and miR-21 exosomes have been found to enhance neuronal cell viability and inhibit neuronal cell death by inhibiting PTEN/PDCD4 expression. These two miRNAs are found in bone-marrow-derived MSCs, and they inhibit neuronal cell death and promote neuronal differentiation in SCI. When the number of exosomes increases, phosphatase and tensin homolog (PTENC) is inhibited preventing neuronal cell death and promoting neuronal differentiation ^[53][54][157,158]. BMSC-derived exosomes have been shown to have neuroprotective effects in the ischemic SCI. This effect may be due to the pre-transfection of BMSCs to secrete exosomes with high expression of miR-25, thus indicating that miR-25 enhances neuroprotection ^[33][16]. Exosomes secreted from miR-29b-rich MSCs and human neuroepithelial stem cells can exert therapeutic effects on SCI by downregulating PTEN/caspase-3 expression and subsequently inhibiting neuronal apoptosis ^[55][56][159,160]. Injecting exosomes secreted from BMSCs modified with miR-29b into a mouse model showed that these exosomes not only promote nerve regeneration but also accelerate motor function recovery in mice with SCI and reduce pathological damage to spinal cord tissue. This mechanism may also be related to the regulation of the expression of nerve-regeneration-related proteins such as NF200, GAP-43, and GFAP ^[57][161].

There are several studies of BMSC-derived exosomal miR-124-3p, which has been shown to attenuate neurological damage, such as in Parkinson’s disease and spinal cord ischemia-reperfusion injury ^[36][57][141,161]. miR-124-3p and miR-125 are responsible for regulating M2 macrophages. M2 macrophages are key effector cells of the inflammatory response to SCI, and the repair of SCI is based on macrophage activation. miR-125 promotes M2 macrophage polarization and negatively regulates IRF5 to ameliorate SCI. In addition, miR-124-3p promotes M2 macrophage polarization and negatively regulates Ern1 to ameliorate SCI ^[36][38][141,143]. miR-124-3p has also been found to suppress A1 astrocytes by inhibiting the activation of M1 microglia and microglia-induced neuroinflammatory responses through the MYH9/PI3K/AKT/NF-κB signaling pathway ^[58][162]. Exosomes derived from MSCs transformed with miR-126 have been shown to reduce neuronal death and promote functional regeneration after SCI. BMSC-exosome-derived miR-126 promotes angiogenic migration by repressing the expression of SPRED1 and PIK3R2 to promote SCI recovery ^[47][115]. miR-216a-5p has been found to affect the activation of NF-κB signaling by regulating TLR4. Exosomal miR-216a-5p also transforms microglia from an M1 pro-inflammatory phenotype to an M2 anti-inflammatory phenotype, increasing its therapeutic potential by inhibiting TLR4/NF-κB and activating the PI3K/Akt signaling pathway ^[59][60][163,164]. Exosomes secreted from BMSCs containing miR-145-5p improved functional recovery and reduced histopathological damage and inflammation in SCI mice. Exosomes promoted miR-145-5p expression in spinal cord tissue, which specifically targeted TLR4 and inhibited TLR4/NF-κB pathway activation in SCI rats ^[61][165]. MSC-derived miR-26a exosomes increased phosphorylation of PI3K, AKT, and mTOR proteins, promoting neurofilament production and nerve regeneration in neurons. miR-26a exosomes were injected into the tail vein of SCI rats to promote functional recovery in rats and induce neuronal and axonal regeneration by targeting the PTEN and mTOR pathways ^[62][166].

miR-199a-3p/145-5p, which is highly expressed in exosomes secreted from human umbilical cord mesenchymal stem cells, showed an anti-apoptosis effect in vivo. These exosomes could be an effective therapeutic strategy in neuronal injury by affecting TrkA ubiquitination and promoting the NGF/TrkA signaling pathway ^[63][167].

NSC-derived miRNA-124, which is involved in nerve regeneration, ameliorates nerve loss and reduces astrocytes in mice with SCI while increasing neurofilament-200 (NF-200) expression. It has been shown that the expression of nuclear-enriched abundant transcript 1 (Neat1) induces neuron-specific differentiation of neural stem cells by miR-124. It can also activate Wnt/β-catenin signaling to promote neuronal differentiation and migration ability ^[64][168]. NSC-derived MiR-615, which is involved in neuronal survival and axonal regeneration, inhibits LINGO-1 by directly targeting LRR and Ig domain-containing NOGO receptor-interacting protein 1 (LINGO-1), a potent negative regulator of neuronal survival and axonal regeneration, and may contribute to neuronal differentiation through the LINGO-1/RhoA or EGFR signaling pathways. Intrathecal administration of miR-615 to SCI rats suppressed LINGO-1, increased neuronal survival, enhanced axon extension and myelination, and enhanced motor function ^[65][169].

When exosomes encapsulated with miR-133b were injected into SCI rats, STAT3, ERK1/2, and CREB were found to be activated, damaged neurons were shown to be protected, and the recovery of hind limb motor function was demonstrated to be improved. RhoA is a direct target of miR-133b found in exosomes of adipose-derived stem cells. miR-133b protects neurons from apoptosis by downregulating RhoA and regulating Rho-associated kinase (ROCK) to promote ERK phosphorylation. In addition, miR-133b promotes axonal regeneration after SCI by promoting CREB and STAT3 phosphorylation ^[66][170]. Neural stem cell-derived miR-219a-2-3p has also been found to inhibit inflammation by downregulating the YY1 gene and inhibiting NF-κB ^[67][171]. miR-388-5p has been found to downregulate the expression of its target, cannabinoid receptor 1 gene (Cnr1), which increases cAMP accumulation through miR-338-5p, which activates Rap1. In turn, this activates the PI3K/AKT pathway. This pathway inhibits apoptosis and enhances neuronal survival ^[68][172].

Another exosomal miRNA with neuroprotective effects is miR-544. Rat BMSCs were transfected with miR-544 mimics to obtain exosomes highly expressing miR-544, and these exosomes were intravenously injected into a rat model of SCI ^[69][173]. The results of this study showed that miR-544 accelerated the recovery of neuronal function after SCI. In addition, overexpression of miR-544 in BMSC exosomes ameliorated histological defects and neuronal loss due to SCI ^[59][163].

The abovementioned study results demonstrate that exosome-mediated miRNA transport is a new treatment method for SCI because exosomes increase neuron activity through miRNA transport and promote functional recovery by attenuating apoptosis at an early stage during SCI. Table 1 shows the various mechanisms by which miRNAs exert a role after SCI. In addition, Table 2 shows the summary of the function of miRNAs. miRNAs can be observed to modulate processes such as neuroinflammation and apoptosis, can exert neuro-regenerative effects by targeting various molecular mechanisms, and can help recovery from SCI.