Electrospun nanofiber sheets incorporating methylcobalamin promote nerve regeneration and functional recovery in a rat sciatic nerve crush injury model
Graphical abstract
Introduction
Peripheral nerve injury is one of common traumas, with an incidence of 2.8% in multi-trauma patients [1]. Although injured peripheral nerves have the capacity to regenerate, axon regeneration proceeds slowly at a rate of only 1–3 mm/day [2]. Therefore, when nerve injury sites are far from their target organs, functional outcomes after peripheral nerve injury are often poor. Regenerative capacity gradually decreases after denervation of target organs, inducing muscle atrophy, chronic pain, and functional disability [3], [4]. Therefore, prompt treatment and acceleration of regeneration are required for functional recovery after peripheral nerve injury. Therapeutic methods and prognoses depend on the nature of the nerve injury [5]. In the presence of nerve defects, placement of an autologous nerve graft is the gold standard for nerve restoration. It has been reported that vein graft and nerve conduit are also useful tools for treating nerve defects [6], [7], [8], [9]. On the other hand, in cases of nerve injury in continuity, i.e., crush injury and entrapment neuropathy, which accounts for 80% of peripheral nerve injury [1], we have no other way than nerve decompression or neurolysis. However, these treatments are often associated with poor outcomes especially in the crush injury. Therefore, increasing curative effects by additional treatments at the operation is required.
Methylcobalamin (MeCbl) is one of the active forms of vitamin B12 homologues, which is delivered favorably to nerve tissues and effective in promoting nerve regeneration and neuronal cell survival [10], [11], [12]. We previously reported that MeCbl promotes neurite outgrowth and neuronal survival in cerebellar granule neurons and dorsal root ganglion (DRG) neurons and improves functional and electrophysiological results in a rat sciatic nerve transection model [12]. High concentrations of MeCbl are likely to be necessary to maximize effectiveness in promoting nerve regeneration. Our previous report showed that effective MeCbl concentrations promoting neurite outgrowth in vitro ranged from 100 nM to 100 μM. Furthermore, we showed that plasma concentrations of MeCbl reached 130 nM through systemic administration (1 mg/kg/day) in a rat model, and this concentration was sufficient to promote functional recovery after peripheral nerve injury [12]. However, systemic prolonged administration tested in animal models is difficult to apply in the clinic. In addition, since the half-life of MeCbl ranges from approximately 20 to 50 min [13], it seems to be difficult to achieve concentrations required for peripheral nerve regeneration even if high amounts of MeCbl are administered in each single dose. In this study, we developed and tested a novel electrospun nanofiber sheet incorporating MeCbl to locally deliver a high concentration of the compound to the peripheral nerve injury site. This approach promotes functional recovery and nerve regeneration in a rat sciatic nerve crush injury model without affecting plasma concentrations of MeCbl.
Section snippets
Isolation of cortical neurons and cell culture in microfluidic chambers
Cortical neurons were cultured as described previously with minor modifications [14]. Cortical neurons were collected easily in large quantities and previously used for evaluating axonal local treatment with compartmentalized culture [15]. Cerebral cortex was collected from Sprague–Dawley rats at embryonic day 18 and put into cold Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The pia mater and blood vessels were removed and residual
Local administration of MeCbl to axon promotes axonal outgrowth in vitro
In a previous report, we demonstrated that MeCbl promoted axonal elongation in cultured cerebellar granule neurons and dorsal root ganglion neurons [12]. However it remained unclear if administration of MeCbl to the axon, rather than soma, influences axonal outgrowth. In the present study, we used cortical neurons, which have less ability to extend neurites in culture than dorsal root ganglion neurons [24], to estimate the axonal outgrowth in the comparatively unfavorable condition and
Discussion
In this study, we fabricated electrospun nanofiber sheets incorporating MeCbl. Local administration of MeCbl at peripheral nerve injury sites promoted nerve regeneration and enhanced functional recovery in a rat sciatic nerve crush injury model. We previously reported that MeCbl promoted neurite outgrowth in neurons [12], differentiation and myelination in Schwann cells [23], and proliferation and migration in myoblasts [31] in vitro. Furthermore, systemic administration of MeCbl with an
Conclusions
A major purpose of this study was to investigate whether local administration of MeCbl using electrospun nanofiber sheets was effective for nerve regeneration and functional recovery in a rat sciatic nerve crush injury model. In a compartmented culture system, local administration of MeCbl to the axon compartment promoted axonal outgrowth equal to axon and soma administration of MeCbl. Furthermore, we fabricated electrospun nanofiber sheets incorporating MeCbl and implanted them at the nerve
Disclosures
The authors have no conflicts of interest to declare.
Acknowledgement
We are grateful to T. Iwahashi and J. Sayanagi for excellent technical assistance. This work was supported by JSPS KAKENHI Grant Number JP15K10405.
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