Age-Dependent Histomorphometrical Values of the Sciatic Nerve Regeneration in Rats after Cutting and Microsurgical Suturing

The effect of ageing on the morphofunctional features of nerves and the process of their posttraumatic regeneration has been studied in details. However, it is not known, whether there are differences of the neuroregeneration potential in the young, adult and mature experimental animals.The aim of the study was to evaluate histomorphometrical parameters of the rat sciatic nerve regeneration after cutting and microsurgical suturing depending on their age.Materials and Methods. The study included 15 white laboratory Wistar rats aged 5–6 (young), 8–10 (adult) and 12 months (mature). The sciatic nerve was transected at the level of the middle third of the thigh with microsurgical scissors and sutured with six epi-perineural stitches in the animals. In 4 months the animals were euthanized. Sections of sciatic nerves distal to the levels of sutures were put into araldite, semi-thin slices (0.5–1.0 mkm) were obtained for histomorphometric analysis. Thirteen intact animals were selected as control. Statistical processing was performed using nonparametric methods.Results. The numerical density of endoneural vessels in nerves of operated rats was higher than that in the intact ones, but in mature animals it was significantly lower than in young and adult ones (p<0.001). The numerical density of endoneural cells nuclei in young experimental animals did not differ from that of the intact ones, but in animals from the adult and mature groups it was more than four times higher than in animals from the young group (p<0.001). The numerical density of regenerating myelinated nerve fibers in animals of the young and adult groups did not differ from that of the intact nerve, in animals of the mature group it increased in 2.5 times (p<0.001). The numerical density of degenerating myelinated fibers in the operated animals of experimental groups was higher than in the intact group (two times, six times and 11 times higher, respectively). The diameter distribution of the regenerating myelinated fibers in animals of all groups differed from that of the intact control animals.Conclusions. The data obtained demonstrate a significant decrease in the regenerative potential of Schwann cells and neurons in adult and mature animals compared with the young ones.

Inverted Human Umbilical Artery as a 3D Scaffold for Sciatic Nerve Regeneration in Rats.

Background: Treatment of peripheral nerve injuries (PNIs) remains a challenge. Interposing a graft delivers better regenerative outcomes. Autografts present major drawbacks which have given rise to the development of alternatives such as artificial scaffolds, some of which are very promising. This study was designed to investigate the potential use of an inverted human umbilical cord artery (iHUA) as a 3D scaffold nerve chamber, for nerve regeneration after transection of the sciatic nerve (SN) in rats.Methods: Rats underwent surgical SN transection in their right hindlimb, followed by suture of the device at the resected stumps. Local tolerance, insert biodegradability and nerve reconstruction over time were thoroughly studied by histopathological and morphometric analysis, completed by functional test assessment of sensitivity and motricity recovery.Results: We have demonstrated that nerve reconstruction in the presence of an iHUA insert is effective. The device is well tolerated and highly biodegraded. Although the regenerated nerve is still immature at the end of our study, signs of sensitivity and partial functional recovery were witnessed, confirming our histological findings. Conclusions: Our results support the potential clinical use of iHUA as a 3D scaffold to bridge nerve discontinuity and guide axonal regrowth in selected cases of PNIs.

In Vitro and In Vivo Effects of Nobiletin on DRG Neurite Elongation and Axon Growth after Sciatic Nerve Injury

Sciatic nerve injury (SNI) leads to sensory and motor dysfunctions. Nobiletin is a major component of polymethoxylated flavonoid extracted from citrus fruits. The role of nobiletin on sciatic nerve regeneration is still unclear. Thus, the purpose of this study was to investigate whether nobiletin increases DRG neurite elongation and regeneration-related protein expression after SNI. Cytotoxicity of nobiletin was measured in a concentration–dependent manner using the MTT assay. For an in vitro primary cell culture, the sciatic nerve on the middle thigh was crushed by holding twice with forceps. Dorsal root ganglion (DRG) and Schwann cells were cultured 3 days after SNI and harvested 36 h later and 3 days later, respectively. In order to evaluate specific regeneration-related markers and axon growth in the injured sciatic nerve, we applied immunofluorescence staining and Western blot techniques. Nobiletin increased cell viability in human neuroblastoma cells and inhibited cytotoxicity induced by exposure to H2O2. Mean neurite length of DRG neurons was significantly increased in the nobiletin group at a dose of 50 and 100 μM compared to those at other concentrations. GAP-43, a specific marker for axonal regeneration, was enhanced in injury preconditioned Schwann cells with nobiletin treatment and nobiletin significantly upregulated it in injured sciatic nerve at only 3 days post crush (dpc). In addition, nobiletin dramatically facilitated axonal regrowth via activation of the BDNF-ERK1/2 and AKT pathways. These results should provide evidence to distinguish more accurately the biochemical mechanisms regarding nobiletin-activated sciatic nerve regeneration.

Development of Multi-channel Electro-conductive Nanofibrous Conduits for Peripheral Nerve Regeneration

Multichannel structures in the design of nerve conduits offer potential advantages for regeneration of damaged nerves due to their bio-mimicking architecture. However, lack of biochemical cues and electrical stimulation could hamper satisfactory nerve regeneration. The aim of this study was to simultaneously evaluate the effects of topographical, biological and electrical cues on sciatic nerve regeneration in a rat model. Accordingly, a series of multichannel nanofibrous nerve conduit was made using longitudinally-aligned laminin-coated electrospun PLGA/CNT nanofibers (NF, mean diameter: 455 ± 362 nm) in the lumen and randomly-oriented PCL NF (mean diameter: 340 ± 200 nm) on the outer surface. In vitro studies revealed that both materials were nontoxic to Schwann cells and able to promote cell attachment and proliferation. To determine the influence of topographical, biological and electrical cues on nerve regeneration, either of hollow PCL conduits, PLGA NF-embedded, PLGA/CNT NF-embedded or laminin-coated PLGA/CNT NF-embedded PCL conduits were implanted in rats. A new surgery method was utilized and results were compared with an autograft. After animal treatments, motor and sensory tests showed significant improvement in the rats treated with NF-embedded PCL conduits. H&E images obtained from cross-sectional and, longitudinal-sections of the regenerated nerves demonstrated the formation of regenerative nerve fibers and also, angiogenesis in laminin-coated PLGA/CNT NF-embedded PCL conduits. Results suggested that these conduits have the potential for clinical application to reconstruct peripheral nerve defects.