EVs are formed when the plasma membrane double invaginates and eventually generates the MVB, which can be degraded in lysosomes or fuse with cell membranes to release inclusions as EVs (Bebelman et al., 2018; Kalluri and LeBleu, 2020). The biogenesis and transport of EVs are associated with a large number of proteins, including ESCRT, Ras-related proteins GTPase Rab, ceramide, and phospholipids (Trajkovic et al., 2008; Mathieu et al., 2019). Exosomes are made of lipid bilayer structures and appear biconcave or cup-shaped when produced by artificially drying during TEM (Yellon and Davidson, 2014; Lobb et al., 2015). Bilayer lipid vesicles in solution are nearly spherical. The method of purification and processing for EM can lead artifactual observation of different shapes (Chernyshev et al., 2015). EVs generated by cells including neurons, glial cells and immune cells can promote cell-to-cell communication and regulate biological processes after nerve injury (López-Leal et al., 2020). In addition, MSCs including bone marrow MSCs, gingival MSCs, adMSCs, and hucMSCs can release EVs to regulate the pathological progression after nerve injury (Bryk et al., 2022; Zhang Y. U. et al., 2022). EVs derived from different cells contain different signaling molecules and surface antigens as well as different cargos including unique proteins, lipids, and genetic material (Toh et al., 2018). This intercellular communication involves two different mechanisms an ESCRT-dependent mechanisms and an ESCRT-independent mechanisms (D'Agnelli et al., 2020). EVs can affect phenotypic and molecular alterations of receptor cells by direct or indirect delivery of signaling molecules to receptor cells through binding with cell surface receptors and receptor-mediated endocytosis or direct membrane fusion (Meldolesi, 2018; Van Niel et al., 2018; Figure 1).

Within the endosomal system, cargos including proteins and nucleic acids are internalized from extracellular space. Cargos are sorted into early endosomes and then mature into MVB through a process that involves interactions with the Golgi complex. MVB can be degraded in lysosomes or fuse with cell membranes to release inclusions as EVs. The biogenesis and transport of EVs are associated with ESCRT and non-ESCRT. ER, endoplasmic reticulum; MVB, multivesicular bodies.

As biomarkers for diagnose and treatments, EVs are at an emerging stage of clinical application. EVs mediating intercellular communication trigger immune responses, inflammatory responses, and many other processes (Harrell et al., 2019; Qiu et al., 2021; Ashrafizadeh et al., 2022). Exosomes presenting in biological fluids and carrying specific membrane proteins are internalized by recipient cell through soluble and juxtacrine signaling events, fusion, and receptor/raft-mediated endocytosis, as well as phagocytosis, and regulate multiple physiological and pathological processes (Hu et al., 2020). The characteristic properties of exosomes during delivery of functional cargos is that they can target specific cells, which also favors their use as therapeutic vehicles (Hessvik and Llorente, 2018). Exosomes affect neuroinflammation and neuropathic pain by delivering proteins, mature miRNAs, and translatable transcripts (Ramanathan and Ajit, 2016). MiR-25-3p, miR-320B, miR-93, miR-126-3p, and RNU48 from serum-derived exosomes in patients with complex regional pain syndrome show significant differential expression (McDonald et al., 2014). Carrying miR-181c-5p containing exosomes repress CCI-induced neuropathic pain through inhibition of neuroinflammation (Zhang Y. U. et al., 2022), which provided the therapeutic rationale for studying EVs to treat neuropathic pain.

Glial cells, also known as neuroglia, modulate neurotransmission at the synaptic level. Now it has been found that glial cells play an important role in neuroinflammation and neuropathic pain (Tsuda and Inoue, 2016; Hirbec et al., 2020). Glial cells are located in DRGs, spinal cord and brain, and show a series of changes such as a shifting phenotype and release of inflammatory mediators in response to a damaging signal within the nervous system to drive central and peripheral sensitization and participate in neuropathic pain evolution (Chen et al., 2018; Matsuda et al., 2019).

Microglia (macrophages residing in the central nervous system) are sensors for the detection of abnormal alterations in response to internal and external insults in the central nervous system (Ji et al., 2016). Studies have found that in SCI models exosomes were delivered to the injury site and significantly enhance axonal regeneration, reduce cell apoptosis, and reduce activation of microglia and astrocytes (Guo et al., 2019). MSC-derived EVs can promote phenotype transformation and function of macrophages/microglia from pro-inflammatory M1 to anti-inflammatory M2 types. The activation of glial cells requires the participation of pro-inflammatory factors which may be related to miRNAs in exosomes, such as miR-34a-5p, miR-21, and miR146a-5p (Domenis et al., 2018). Previous studies have shown that TBI is associated with neuropathic pain (Leung, 2020). Exosomes mediate neuron-glial cell intercellular communication and following TBI, microglia reduce the release of exosomes containing miR-5,121 to inhibit neurite outgrowth and synapse recovery of neurons (Zhao et al., 2021).

The expression of lncGm37494 targeting miR-130b-3p to regulate microglia polarization is increased in exosomes secreted by ADSCs under hypoxia, and has been suggested that exosomes can repair SCI by delivering lncGm37494 (Shao et al., 2020). Intravenous administration of hucMSC-derived exosomes significantly inhibits the transcription levels of astrocytes and microglia, suggesting a reduced inflammatory state from SCI (Kang and Guo, 2022), and single and continuous intrathecal infusion of hucMSC-derived exosomes after SNL can prevent and reverse neuropathic pain. Moreover, it has been found that the analgesic effects of exosomes may involve their actions on neurons and glial cells (Shiue et al., 2019).

As the most abundant glial cell in the peripheral nervous system, Schwann cells undergo phenotypic modulation, proliferate and interact with nociceptive neurons by releasing glial mediators (growth factors and cytokines) after nerve injury. These dramatic alterations in Schwann cells promote nerve regeneration and eventually influence neuropathic pain (Dubový, 2011; Wei et al., 2019). During neuropathic pain, many receptors on Schwann cells are differentially expressed, such as P2X4R, HCAR2 (Boccella et al., 2019; Su et al., 2019). Recent studies have proved the presence of growth factors such as BDNF, NGF, FGF-1, IGF-1, and GDNF in adMSC exosomes. Furthermore, adMSCs-derived exosomes which internalized into the Schwann cells exhibited the ability to promote Schwann cell proliferation in vitro and enhance nerve regeneration following SNI in vivo (Bucan et al., 2019), and interestingly, BDNF and NGF have been identified to correlate with neuropathic pain (Dai et al., 2020; Wu et al., 2021). In addition, exosomal miR-21 was shown to promote the Schwann cell proliferation and the expression of NGF, BDNF, and GDNF after peripheral nerve injury. And exosomal miR-21 secreted by Schwann cells can promote neurite outgrowth and functional repair after SNI (Liu Y. P. et al., 2022; Figure 2).

Lesions of the nervous system from the peripheral nociceptor to the spinal cord and brain may give rise to neuropathic pain. This figure shows the interaction between EVs and glial cells in the central and peripheral nervous system. adMSC, adipose-derived mesenchymal stem cells, hucMSC, human umbilical cord mesenchymal stem cells, ADSC, Adipose tissue-derived mesenchymal stem/stromal cells.

Neuroinflammatory activation of glial cells and immune cells after nerve injury is being increasingly implicated in neuropathic pain (Chambel et al., 2020; Karri et al., 2022). EVs pose tremendous anti-inflammatory capacity following nerve injury and the roles of EVs in neuropathic pain will be a particularly interesting area of future inquiry.

EF-MSCs secrete EVs to repress NLRP3 inflammasome activation and to inhibit the expression of inflammatory cytokines related to neuroinflammation after SCI (Huang et al., 2020). Macrophages change their polarization to anti-inflammatory phenotype which can effectively control the development of inflammation (Ma et al., 2020). HucMSC-derived exosomes can stimulate bone marrow-derived macrophages to transform into M2 type, which can also inhibit inflammation through decreasing inflammatory factors such as TNF-α, MIP-1α, IL-6 and IFN-γ (Sun et al., 2018). MSC derived exosomes can relieve microglia/macrophage and astrocyte-mediated neuroinflammation in rats after TBI (Zhang et al., 2020). Studies suggest a critical role for proteins in the mediation of signaling mechanisms underlying neuropathic pain after SNI. Furthermore, the compliment protein C5a and ICAM-1 were found to be significantly up-regulated in EVs purified from SNI mouse serum (Jean-Toussaint et al., 2020). C5a can help induce the expression of inflammatory factors in microglia and promote the development of their inflammatory status (Liu et al., 2018). Furthermore, C5a and its receptor C5aR can simultaneously participate in the process of neuropathic pain (Quadros and Cunha, 2016).

MSC-derived EVs can facilitate macrophage/microglia polarization from M1 to M2 phenotype. miR-21 and miR-19b in EVs derived from differentiated PC12 cells and MSCs can suppress apoptosis of neurons by decreasing the expression of PTEN which can be applied during post-SCI repair (Xu et al., 2019). We found that miR-216a-5p inside MSC-derived exosomes targeted TLR4 and shifted this M1/M2 polarization to inhibit neuroinflammation after SCI which was mediated by microglial activation (Liu et al., 2020). Exosomes derived from huc-MSC were found to deliver miR-199a-3p/miR-145-5p to neurons causing attenuation of the release of inflammatory factors and promotion of neuronal neurite outgrowth following SCI (Wang et al., 2021). circRNA is a type of noncoding RNA that forms a covalently closed continuous loop. Many of them are enriched in EVs, but whether EV circRNAs regulate neuropathic pain is still a shortage of investigation (Marangon et al., 2023). Circ_003564, a recently discovered circRNA, acted as a critical effector in BMSC-exosome-mediated pyroptosis in the SCI model and these exosomes containing circ_003564 may contribute to treatment efficacy in SCI (Zhao et al., 2022). lncRNA H19 in BMSC derived exosomes can further regulate microglia polarization to release neuroinflammatory signals via sponging miR-29b-3p (Zong et al., 2021). Significantly, the treatment using lncRNA TCTN2-modified exosomes significantly improved functional in vivo recovery in SCI rats and alleviated LPS-induced inflammation in vitro (Liu J. et al., 2022; Figure 3).

This figure illustrates that multiple cells secrete EVs to glial cell and immune cell to mediate neuroinflammation after nerve injury. EF-MSC: epidural fat-mesenchymal stem cells, MSC: mesenchymal stem cells, hucMSC: human umbilical cord mesenchymal stem cells.

Diverse evidence has shown that RNA molecules inside EVs are generally less than 300 nt in length which is smaller than human mRNA, indicating a role for noncoding RNA (Chen et al., 2010). Furthermore, miRNAs represent an important cargo type in EVs can obstruct the expression of target genes by inhibiting target mRNA translation and accelerate mRNA degradation. These miRNA are protected from RNase by the exosomes or cellular membranes even in extreme cases (Koga et al., 2011). The miRNAs enriched in EVs are phagocytosed by receptor cells and can mediate cell-to-cell communication thus participating in the occurrence and maintenance of neuropathic pain (Sosanya et al., 2020).

The expression levels of miR-21 in exosomes derived from blood serum is significantly increased in the pSNL model, and exosomal miR-21 is likely to be a diagnostic biomarker for neuropathic pain (Hori et al., 2016). This confirmed that SNI induced upregulation of miR-21-5p, let7b, miR-124, and miR-134 expression in the lumbar DRG and DRG sensory neurons can release exosomes containing miR-21-5p which were subsequently internalized by macrophages and converting them to the M1 type. The study also indicated that intrathecal injection of the miR-21 antagomir inhibits hyperalgesia and relieves neuropathic pain (Simeoli et al., 2017).

Intravenous and intrathecal administration of MSCs effectively relieves neuropathic pain following CCI or SCI (Yousefifard et al., 2016; Al-Massri et al., 2019) and MSC exosome therapy has shown the same functions as MSCs therapy, such as anti-inflammation, regulation of the immune system, and regulation of cell differentiation (Lee et al., 2021). In CCI rats, miR-181c-5p from MSC-derived exosomes were found to be significantly downregulated in a time-dependent manner. Intrathecal administration of exosomal miR-181c-5p which targets microglia and inhibits the secretion of inflammatory factors alleviated neuropathic pain and the neuroinflammatory response after CCI (Zhang Y. U. et al., 2022).

In the CCI model, downregulated miR-183 in the sciatic nerve targeting transcription factor FoxO1 and tight junction protein claudin-5 promoted mechanical hypersensitivity. Exosomal miR-183 was found decreased from sera of complex regional pain syndrome patients and can regulate neuropathic pain state via regulating microvascular barrier (Reinhold et al., 2022). In conclusion, our study assesses exosomal noncoding RNAs as one modifier of neuropathic pain (Table 1).