In spite of the relatively high success rate of limb replantation, many patients cannot undergo replantation surgery because the preservation time of an amputated limb is only about six hours. In addition, although allotransplantation of composite tissues is being performed more commonly with increasingly greater success rates, the shortage of donors limits the number of patients that can be treated. So the purpose of this study is to examine the feasibility of cryopreservation and replantation of limbs in a rat model.

To preliminarily explore the primary changes in the expression of genes involved in peripheral nerve processes, namely, regeneration, angiogenesis, and the immune response, and to identify important molecular therapeutic targets, 45 Sprague-Dawley (SD) rats were randomly divided into a control group and an injury group. In the injury group, tissue samples were collected at 4 and 7 days after the injury for next-generation sequencing (NGS) analysis combined with gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Venn diagram construction to identify the differentially expressed mRNAs (DEmRNAs) associated with regeneration, angiogenesis, and the immune response of the nerve. The expression of genes in the distal and proximal ends of the injured nerve after injury was analyzed by qRT-PCR. NGS revealed that compared with the control group, the injury group had 4020 DEmRNAs 4 days after injury and 3278 DEmRNAs 7 days after injury. A bioinformatics analysis showed that C-C chemokine receptor type 5 (CCR5), Thy1 cell surface antigen (Thy1), Notch homolog 1 (Notch1), and semaphorin 4A (Sema4A) were all associated with regeneration, angiogenesis, and the immune response of the nerve at both 4 and 7 days after injury, but qPCR revealed no significant difference in the expression of Thy1 (P = 0.29) or Sema4A (P = 0.82) in the proximal end, whereas a significant difference was observed in CCR5 and Notch1 (P < 0.05). The trend in the Notch1 change was basically consistent with the RNA-seq result after injury, which implied its indispensable role during endothelial cell proliferation and migration, macrophage recruitment, and neurotrophic factor secretion.

Peripheral nerve injury (PNI) involving the loss of sensory and movement functions is challenging to repair. Although the gold standard of PNI repair is still the use of autologous nerve grafts, the destruction of the donor side is inevitable. In the present study, peripheral nerve tissueoids (PNTs) composed of a Schwann cell (SC)-based neurotrophin-3 (NT-3) delivery system and a decellularized optic nerve (DON) with naturally oriented channels were engineered to investigate the mechanism of PNTs in nerve regeneration. Proteomic analysis and mRNA sequencing revealed that PNTs have the advantage of promoting nerve regeneration by the three mechanisms of chemotaxis, adhesion and intrinsic mobilisation. The results demonstrated that a local NT-3-enriched pool was constructed by laminin γ3 (LAMC3) in PNTs, creating a niche for the colonization of TrkC-positive SCs, attraction of axons to the defect/graft area, and remyelination. In addition, LAMC3 in PNTs can rapidly promote axon adhesion through integrin aVβ6 and can precisely guide long projecting axons to target tissues. Furthermore, the interactions among the NT-3/TrkC, LAMC3/integrin aVβ6 and the scaffold synergistically activate the PI3K-AKT signalling pathway in damaged neurons, further stimulating the intrinsic regenerative drive within the neurons to ultimately achieve the rapid reinnervation and the improvement of sensory and movement functions in the hindlimb.

An aging-induced decrease in Schwann cell viability can affect regeneration following peripheral nerve injury in mammals. It is therefore necessary to investigate possible age-related changes in gene expression that may affect the biological function of peripheral nerves. Ten 1-week-old and ten 12-month-old healthy male Sprague-Dawley rats were divided into young (1 week old) and adult (12 months old) groups according to their ages. mRNA expression in the sciatic nerve was compared between young and adult rats using next-generation sequencing (NGS) and bioinformatics ( n = 4/group). The 18 groups of differentially expressed mRNA (DEmRNAs) were also tested by quantitative reverse transcription polymerase chain reaction ( n = 6/group). Results revealed that (1) compared with young rats, adult rats had 3608 groups of DEmRNAs. Of these, 2684 were groups of upregulated genes, and 924 were groups of downregulated genes. Their functions mainly involved cell viability, proliferation, differentiation, regeneration, and myelination. (2) The gene with the most obvious increase of all DEmRNAs in adult rats was Thrsp (log ₂ FC = 9.01, P < 0.05), and the gene with the most obvious reduction was Col2a1 (log ₂ FC = –8.89, P < 0.05). (3) Gene Ontology analysis showed that DEmRNAs were mainly concentrated in oligosaccharide binding, nucleotide-binding oligomerization domain containing one signaling pathway, and peptide-transporting ATPase activity. (4) Analysis using the Kyoto Encyclopedia of Genes and Genomes showed that, with increased age, DEmRNAs were mainly enriched in steroid biosynthesis, Staphylococcus aureus infection, and graft-versus-host disease. (5) Spearman's correlation coefficient method for evaluating NGS accuracy showed that the NGS results and quantitative reverse transcription polymerase chain reaction results were positively correlated (r _s = 0.74, P < 0.05). These findings confirm a difference in sciatic nerve gene expression between adult and young rats, suggesting that, in peripheral nerves, cells and the microenvironment change with age, thus influencing the function and repair of peripheral nerves.

Objective: We have demonstrated that angiogenesis in acellular nerve allografts (ANAs) can promote neuroregeneration. The present study aimed to investigate the microvascular regeneration pattern of ANAs in Sprague-Dawley (SD) rats. Methods: Sixty male SD rats were randomly divided into an autologous group and a rat acellular nerve allograft group (rANA), and 10-mm sciatic nerve defects were induced in these rats. On the 7th, 14th and 21st days after surgery, systemic perfusion with Evans Blue (EB) or lead oxide was performed on the rats through carotid intubation. Samples were then collected for gross observation, and the microvessels in the nerves were reconstructed through microscopic CT scans using MIMICS software. The vascular volume fraction (VF, %) and microvessel growth rate (V, mm/d) in both groups were then measured, and 1 month after surgery, NF-200 staining was performed to observe and compare the growth condition of the axons. Results: Early post-operative perfusion with gelatin/EB showed EB permeation around the acellular nerve. Perfusion with gelatin/lead oxide showed that the blood vessels had grown into the allograft from both ends 7 days after the operation. Fourteen days after the operation, the microvessel growth rate of the autologous group was faster than that of the rANA group (0.39 ± 0.17 mm/d vs. 0.26 ± 0.14 mm/d, p < 0.05), and the vascular VF was also higher than that of the rANA group (8.92% ± 1.54% vs. 6.31% ± 1.21%, p < 0.05). Twenty-one days after the operation, the blood vessels at both ends of the allograft had connected to form a microvessel network. The growth rate was not significantly different between the two groups; however, the vascular VF of the autologous group was higher than that of the rANA group (12.18% ± 2.27% vs. 9.92% ± 0.84%, p < 0.05). One month after the operation, the NF-200 fluorescence (IOD) in the autologous group significantly increased compared with that of the rANA group (540,278 ± 17,424 vs. 473,310 ± 14,636, respectively, p < 0.05), suggesting that the results of the repair after nerve injury were significantly better in the autologous group than in the rANA group. Conclusion: Both the autologous nerve and ANAs rely on the permeation of tissue fluids to supply nutrients during the early stage, and microvessel growth mainly starts at both ends of the graft and enters the graft along the long axis. Compared to ANAs, the growth speed of revascularization in autologous nerve grafts was faster, leading to a better outcome in the autologous nerve group.