Lymphatic vessels (LVs) have conventionally been considered a passive route for transport of fluid and immune cells, playing a critical role in controlling fluid homeostasis, regulating immune responses, absorbing and clearing macromolecular (Oliver et al., 2020). LVs have been largely explored in the central nervous system (CNS), such as meningeal LVs (mLVs) facilitating cerebrospinal fluid drainage and egress of immune cells (Louveau et al., 2015), vertebral LVs (vLVs) connecting to peripheral ganglia and forming metameric vertebral circuits to act as gatekeepers of CNS immunity (Jacob et al., 2019). However, little information is available about the LVs in the peripheral nervous system (PNS). Pioneer studies in 1960s from Sunderland first described the existence of two distinct lymphatic capillary networks inside epineurium and endoneurium of peripheral nerves (Sunderland, 1965), but later works in 2000s from Volpi reported the immunohistochemical identification of LVs in the epineurium of human sural nerves instead of in the endoneurium (Volpi et al., 2006). Interestingly, recent researches showed that LVs were absent in healthy peripheral nerves and functional LVs was required for peripheral nerve repair rather than for the homeostasis of PNS (Hromada et al., 2022; Meng et al., 2020). Therefore, it is still unclear about the existence of pLVs and its specific structural and functional features.

Peripheral nerve injury (PNI) is a major clinical problem causing severe motor and/or sensory dysfunction, even physical disability. Almost 5% of trauma patients (approximately 350,000 people per annum) suffer from PNI and it is challenging to obtain satisfactory functional recovery despite receiving standard treatment, such as microsurgical repair and adjuvant therapies (Singh et al., 2022). So far, pharmacological approaches to accelerate nerve regeneration are sparse mainly due to the difficulty in delivery of therapeutic compounds to the PNS coming from complexities of the peripheral neuroanatomy and the restrictiveness of the blood-nerve barrier (BNB) (Idrisova et al., 2022). To address this fundamental difficulty, several local delivery strategies, such as direct implantation of a bioengineered nerve conduit containing growth factors, administration by injection at epineurium and perineurium with or without disruption of BNB, presynaptic uptake and retrograde axonal transport achieved by viral vectors or bacterial neurotoxins, have been developed to improve the efficiency of targeted delivery to the PNS (Langert and Brey, 2018). However, there are still some obvious drawbacks of these strategies, including limited application scenarios of nerve conduits, collateral neurological damage of epineurium or perineurium injection, immunogenic complications and safety concerns about viral vectors or bacterial neurotoxins. Driven by the slow interstitial fluid flow from the vascular capillaries, LVs offer a channel to transport macromolecules and particles into tissue interstitium (Petrova and Koh, 2020). mLVs have been reported to be a new route for drug delivery to the brain, which can bypass the blood-brain barrier and avoid other issues associated with conventional intravenous administration (Zhao et al., 2020a). Whether pLVs could provide a novel delivery route for promoting peripheral nerve regeneration has yet to be established.

In this study, we successfully characterized the specialized three-dimensional (3D) morphologic features of pLVs by using tissue-clearing methods and light-sheet fluorescent microscopy (LSFM) imaging. It revealed that pLVs existed in both the epineurium and perineurium of healthy peripheral nerves and numbers of newly formed pLVs significantly increased after PNI. Specific phenotypes of lymphatics endothelial cells in peripheral nerves were characterized by single-cell sequencing. Moreover, we used second-window near-infrared (NIR-II) microscopy to in vivo image the trans-lymphatic delivery route toward peripheral nerves in real-time via footpad injection. Exosome's in vivo therapeutic effects on PNI via trans-lymphatic delivery route were also evaluated. Our results demonstrated that pLVs could be a novel delivery route to promote peripheral nerve regeneration.