Gene expression profiling of the rat sciatic nerve in early Wallerian degeneration after injury

Wallerian degeneration is an important area of research in modern neuroscience.A large number of genes are differentially regulated in the various stages of Wallerian degeneration,especially during the early response.In this study,we analyzed gene expression in early Wallerian degeneration of the distal nerve stump at 0,0.5,1,6,12 and 24 hours after rat sciatic nerve injury using gene chip microarrays.We screened for differentially-expressed genes and gene expression patterns.We examined the data for Gene Ontology,and explored the Kyoto Encyclopedia of Genes and Genomes Pathway.This allowed us to identify key regulatory factors and recurrent network motifs.We identified 1 546 differentially-expressed genes and 21 distinct patterns of gene expression in early Wallerian degeneration,and an enrichment of genes associated with the immune response,acute inflammation,apoptosis,cell adhesion,ion transport and the extracellular matrix.Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed components involved in the Jak-STAT,ErbB,transforming growth factor-β,T cell receptor and calcium signaling pathways.Key factors included interleukin-6,interleukin-1,integrin,c-sarcoma,carcinoembryonic antigen-related cell adhesion molecules,chemokine(C-C motif) ligand,matrix metalloproteinase,BH3 interacting domain death agonist,baculoviral IAP repeat-containing 3 and Rac.The data were validated with real-time quantitative PCR.This study provides a global view of gene expression profiles in early Wallerian degeneration of the rat sciatic nerve.Our findings provide insight into the molecular mechanisms underlying early Wallerian degeneration,and the regulation of nerve degeneration and regeneration.     

Gene expression analysis at multiple time‐points identifies key genes for nerve regeneration

Introduction: The purpose of this study was to provide a comprehensive understanding of gene expression during Wallerian degeneration and axon regeneration after peripheral nerve injury. Methods: A microarray was used to detect gene expression in the distal nerve 0, 3, 7, and 14 days after sciatic nerve crush. Bioinformatic analysis was used to predict function of the differentially expressed mRNAs. Microarray results and the key pathways were validated by quantitative real‐time polymerase chain reaction (qRT‐PCR). Results: Differentially expressed mRNAs at different time‐points (3, 7, and 14 days) after injury were identified and compared with a control group (0 day). Nine general trends of changes in gene expression were identified. Key signal pathways and 9 biological processes closely associated with nerve regeneration were identified and verified. Conclusions: Differentially expressed genes and biological processes and pathways associated with axonal regeneration may elucidate the molecular‐biological mechanisms underlying peripheral nerve regeneration. Muscle Nerve 55 : 373–383, 2017     

Critical signaling pathways during Wallerian degeneration of peripheral nerve

Wallerian degeneration is a critical biological process that occurs in distal nerve stumps after nerve injury. To systematically investigate molecular changes underlying Wallerian degeneration, we used a rat sciatic nerve transection model to examine microarray analysis out-comes and investigate significantly involved Kyoto Enrichment of Genes and Genomes (KEGG) pathways in injured distal nerve stumps at 0, 0.5, 1, 6, 12, and 24 hours, 4 days, 1, 2, 3, and 4 weeks after peripheral nerve injury. Bioinformatic analysis showed that only one KEGG pathway (cytokine-cytokine receptor interaction) was significantly enriched at an early time point (1 hour post-sciatic nerve transection). At later time points, the number of enriched KEGG pathways initially increased and then decreased. Three KEGG pathways were studied in further detail: cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and axon guidance. Moreover, temporal expression patterns of representative differentially expressed genes in these KEGG pathways were validated by real time-polymerase chain reaction. Taken together, the above three signaling pathways are important after sciatic nerve injury, and may increase our understanding of the molecular mechanisms underlying Wallerian degeneration.     

The effects of claudin 14 during early Wallerian degeneration after sciatic nerve injury

Claudin 14 has been shown to promote nerve repair and regeneration in the early stages of Wallerian degeneration(0–4 days) in rats with sciatic nerve injury, but the mechanism underlying this process remains poorly understood. This study reported the effects of claudin 14 on nerve degeneration and regeneration during early Wallerian degeneration. Claudin 14 expression was up-regulated in sciatic nerve 4 days after Wallerian degeneration. The altered expression of claudin 14 in Schwann cells resulted in expression changes of cytokines in vitro. Expression of claudin 14 affected c-Jun, but not Akt and ERK1/2 pathways. Further studies revealed that enhanced expression of claudin 14 could promote Schwann cell proliferation and migration. Silencing of claudin 14 expression resulted in Schwann cell apoptosis and reduction in Schwann cell proliferation. Our data revealed the role of claudin 14 in early Wallerian degeneration, which may provide new insights into the molecular mechanisms of Wallerian degeneration.     

Identification of key genes involved in axon regeneration and Wallerian degeneration by weighted gene co-expression network analysis

Peripheral nerve injury repair requires a certain degree of cooperation between axon regeneration and Wallerian degeneration.Therefore,investigating how axon regeneration and degeneration work together to repair peripheral nerve injury may uncover the molecular mechanisms and signal cascades underlying peripheral nerve repair and provide potential strategies for improving the low axon regeneration capacity of the central nervous system.In this study,we applied weighted gene co-expression network analysis to identify differentially expressed genes in proximal and distal sciatic nerve segments from rats with sciatic nerve injury.We identified 31 and 15 co-expression modules from the proximal and distal sciatic nerve segments,respectively.Functional enrichment analysis revealed that the differentially expressed genes in proximal modules promoted regeneration,while the differentially expressed genes in distal modules promoted neurodegeneration.Next,we constructed hub gene networks for selected modules and identified a key hub gene,Kif22,which was up-regulated in both nerve segments.In vitro experiments confirmed that Kif22 knockdown inhibited proliferation and migration of Schwann cells by modulating the activity of the extracellular signal-regulated kinase signaling pathway.Collectively,our findings provide a comparative framework of gene modules that are co-expressed in injured proximal and distal sciatic nerve segments,and identify Kif22 as a potential therapeutic target for promoting peripheral nerve injury repair via Schwann cell proliferation and migration.All animal experiments were approved by the Institutional Animal Ethics Committee of Nantong University,China(approval No.S20210322-008)on March 22,2021.     

Expression changes and bioinformatic analysis of Wallerian degeneration after sciatic nerve injury in rat

Wallerian degeneration (WD) remains an important research topic. Many genes are differentially expressed during the process of WD, but the precise mechanisms responsible for these differentiations are not completely understood. In this study, we used microarrays to analyze the expression changes of the distal nerve stump at 0, 1, 4, 7, 14, 21 and 28 days after sciatic nerve injury in rats. The data revealed 6 076 differentially-expressed genes, with 23 types of expression, specifically enriched in genes associated with nerve development and axonogenesis, cytokine biosynthesis, cell differentiation, cytokine/chemokine production, neuron differentiation, cytokinesis, phosphorylation and axon regeneration. Kyoto Encyclopedia of Genes and Genomes pathway analysis gave findings related mainly to the MAPK signaling pathway, the Jak-STAT signaling pathway, the cell cycle, cytokine-cytokine receptor interaction, the p53 signaling pathway and the Wnt signaling pathway. Some key factors were NGF, MAG, CNTF, CTNNA2, p53, JAK2, PLCB1, STAT3, BDNF, PRKC, collagen II, FGF, THBS4, TNC and c-Src, which were further validated by real-time quantitative PCR, Western blot, and immunohistochemistry. Our findings contribute to a better understanding of the functional analysis of differentially-expressed genes in WD and may shed light on the molecular mechanisms of nerve degeneration and regeneration.     

Nonresident macrophages in peripheral nerve of rat: effect of silica on migration, myelin phagocytosis, and apolipoprotein E expression during Wallerian degeneration

The selective toxicity of silica quartz dust to macrophages was used to assess the role of these cells in Wallerian degeneration and nerve repair. Left sciatic nerves of adult Wistar rats were crushed and one group of animals received repetitive intraperitoneal injections of silica (200 mg two times per week starting 1 day prior to injury), whereas the control group received saline. Unexpectedly, silica treatment did not impair the initial invasion of (hematogenous) macrophages into the degenerating distal nerve stump as revealed by histological and immunocytochemical methods. However, 4 weeks after the lesion three specific events in Wallerian degeneration were significantly inhibited in silica-treated animals: 1) inhibition of phagocytosis and degradation of myelin, 2) delay in disappearance of nonresident macrophages from regenerating nerve, 3) reduction of synthesis and/or secretion of apolipoprotein E in resting macrophages. On the other hand, axonal regrowth and remyelination were not affected by silica. These in situ experiments support and extend previous studies suggesting specific functions for nonresident macrophages in Wallerian degeneration of peripheral nerve.     

Baculoviral inhibitor of apoptosis protein repeat-containing protein 3 delays early Wallerian degeneration after sciatic nerve injury

Wallerian degeneration is a complex biological process that occurs after nerve injury,and involves nerve degeneration and regeneration.Schwann cells play a crucial role in the cellular and molecular events of Wallerian degeneration of the peripheral nervous system.However,Wallerian degeneration regulating nerve injury and repair remains largely unknown,especially the early response.We have previously reported some key regulators of Wallerian degeneration after sciatic nerve injury.Baculoviral inhibitor of apoptosis protein repeat-containing protein 3(BIRC3)is an important factor that regulates apoptosis-inhibiting protein.In this study,we established rat models of right sciatic nerve injury.In vitro Schwann cell models were also established and subjected to gene transfection to inhibit and overexpress BIRC3.The data indicated that BIRC3 expression was significantly up-regulated after sciatic nerve injury.Both BIRC3 upregulation and downregulation affected the migration,proliferation and apoptosis of Schwan cells and affected the expression of related factors through activating c-fos and ERK signal pathway.Inhibition of BIRC3 delayed early Wallerian degeneration through inhibiting the apoptosis of Schwann cells after sciatic nerve injury.These findings suggest that BIRC3 plays an important role in peripheral nerve injury repair and regeneration.The study was approved by the Institutional Animal Care and Use Committee of Nantong University,China(approval No.2019-nsfc004)on March 1,2019.     

Macrophages contribute to the maintenance of stable regenerating neurites following peripheral nerve injury

Normal adult uninjured nerve is unable to support axonal regeneration. We have studied the mechanisms underlying the regeneration of peripheral nerve by culturing adult mouse dorsal root ganglia (DRG) explants on unfixed, longitudinal cryosections of either the uninjured sciatic nerve or the distal segment of the transected sciatic nerve. We found that, initially, DRG grew vigorously on cryosections of both uninjured and postinjury sciatic nerves. However, the neurites began to degenerate shortly after contact with the uninjured nerve, whereas those growing on postinjury nerve substrate remained healthy for up to 9 days in culture. This ability to support stable outgrowth peaked at 8 days, gradually decreased by 10 days, and disappeared by 20 days after injury. Macrophages appeared in the distal segment by 4 days postinjury and had infiltrated its entire length by 8 days. Uninjured nerve cryosections could be rendered supportive of stable outgrowth by preincubation with macrophage-conditioned medium or by brief trypsinization. The activity of the macrophage-conditioned medium was augmented upon activation of macrophages. Together these findings suggest that the environment of the sciatic nerve undergoes a transformation during Wallerian degeneration such that it becomes transiently supportive of the stable outgrowth of neurites. This transformation may be mediated by a proteolytic activity, generated by activated macrophages, that removes a putative "degeneration signal" protein normally present in the adult nerve and thus contributes to the maintenance of stable regenerating neurites.     

Heat shock protein is a key therapeutic target for nerve repair in autoimmune peripheral neuropathy and severe peripheral nerve injury

Guillain-Barré syndrome (GBS) is an autoimmune peripheral neuropathy and a common cause of neuromuscular paralysis. Preceding infection induces the production of anti-ganglioside (GD) antibodies attacking its own peripheral nerves. In severe proximal peripheral nerve injuries that require long-distance axon regeneration, motor functional recovery is virtually nonexistent. Damaged axons fail to regrow and reinnervate target muscles. In mice, regenerating axons must reach the target muscle within 35 days (critical period) to reform functional neuromuscular junctions and regain motor function. Successful functional recovery depends on the rate of axon regeneration and debris removal (Wallerian degeneration) after nerve injury. The innate-immune response of the peripheral nervous system to nerve injury such as timing and magnitude of cytokine production is crucial for Wallerian degeneration. In the current study, forced expression of human heat shock protein (hHsp) 27 completely reversed anti-GD-induced inhibitory effects on nerve repair assessed by animal behavioral assays, electrophysiology and histology studies, and the beneficial effect was validated in a second mouse line of hHsp27. The protective effect of hHsp27 on prolonged muscle denervation was examined by performing repeated sciatic nerve crushes to delay regenerating axons from reaching distal muscle from 37 days up to 55 days. Strikingly, hHsp27 was able to extend the critical period of motor functional recovery for up to 55 days and preserve the integrity of axons and mitochondria in distal nerves. Cytokine array analysis demonstrated that a number of key cytokines which are heavily involved in the early phase of innate-immune response of Wallerian degeneration, were found to be upregulated in the sciatic nerve lysates of hHsp27 Tg mice at 1 day postinjury. However, persistent hyperinflammatory mediator changes were found after chronic denervation in sciatic nerves of littermate mice, but remained unchanged in hHsp27 Tg mice. Taken together, the current study provides insight into the development of therapeutic strategies to enhance muscle receptiveness (reinnervation) by accelerating axon regeneration and Wallerian degeneration.     

Thrombospondin expression in nerve regeneration I. comparison of sciatic nerve crush, transection, and long-term denervation

Patterns of expression of the extracellular matrix molecule thrombospondin (TSP) were examined during peripheral nerve regeneration following sciatic nerve crush or transection. In noninjured nerve, was present in the axoplasm, Schwann cells, endoneurium, and perineurium of the adult mouse sciatic nerve. Following nerve crush or nerve transection, levels of TSP rapidly increased distal to the trauma site. Elevated levels of TSP were present distal to regenerating axons, while expression gradually returned to normal proximal to the regenerating axons. When reinnervation was blocked, TSP levels remained high in the endoneurium in excess of 30 days, but TSP was absent by 60 days. Following reanastomosis of the proximal and distal segments after 60 days of denervation, TSP was re-expressed in the distal nerve stump. These results indicate that TSP, which is involved in neuronal migrations in the embryo and neurite outgrowth in vitro, appears to play a role in axonal regeneration in the adult peripheral nervous system.     

Gene expression profiling and molecular aspects in peripheral nerve regeneration

Regeneration of the peripheral nervous system after injury depends on a complex sequence of histopathological reactions that comprise a highly reproducible sequence of degenerative reactions, termed Wallerian degeneration. During this period a remodelling of the distal nerve stump prepares a microenvironment that permits successful regrowth of nerve fibers from the proximal nerve fragment. This stereotypical sequence of reactions is reflected by a differential and coordinate expression of genes with specific functions in the process of regeneration. This review will summarize cellular and molecular reactions that contribute to peripheral nerve regeneration including data of a pilot study in which membrane based cDNA array expression technology was applied. We examined the expression of 588 annotated genes in response to a crush lesion of rat sciatic nerves. Approximately 40 % of the genes spotted onto the array filters showed expression significantly above background and 55 of these detected genes represented differential expression profiles after nerve lesion. This approach revealed to be suitable for systematic screening of regeneration associated genes.     

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1(TRPV1) provides the sensation of pain(nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517(300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.     

Polyethylene glycol‐fused allografts produce rapid behavioral recovery after ablation of sciatic nerve segments

Restoration of neuronal functions by outgrowths regenerating at ～1 mm/day from the proximal stumps of severed peripheral nerves takes many weeks or months, if it occurs at all, especially after ablation of nerve segments. Distal segments of severed axons typically degenerate in 1–3 days. This study shows that Wallerian degeneration can be prevented or retarded, and lost behavioral function can be restored, following ablation of 0.5–1‐cm segments of rat sciatic nerves in host animals. This is achieved by using 0.8–1.1‐cm microsutured donor allografts treated with bioengineered s olutions varying in ionic and polyethylene glycol (PEG) concentrations (modified PEG‐fusion procedure), being careful not to stretch any portion of donor or host sciatic nerves. The data show that PEG fusion permanently restores axonal continuity within minutes, as initially assessed by action potential conduction and intracellular diffusion of dye. Behavioral functions mediated by the sciatic nerve are largely restored within 2–4 weeks, as measured by the sciatic functional index. Increased restoration of sciatic behavioral functions after ablating 0.5–1‐cm segments is associated with greater numbers of viable myelinated axons within and distal to PEG‐fused allografts. Many such viable myelinated axons are almost certainly spared from Wallerian degeneration by PEG fusion. PEG fusion of donor allografts may produce a paradigm shift in the treatment of peripheral nerve injuries. © 2014 Wiley Periodicals, Inc.     

Axonal autophagy during regeneration of the rat sciatic nerve**★

BACKGROUND: The removal of degenerated axonal debris during Wallerian degeneration is very important for nerve regeneration. However, the mechanism by which debris is removed is not been completely understood. Considerable controversy remains as to the clearance pathway and cells that are involved. OBJECTIVE: To investigate axonal autophagy during removal of degenerated axonal debris by transecting the sciatic nerve in a rat Wallerian degeneration model.DESIGN, TIME AND SETTING: Experimental neuropathological analysis. The experiment was conducted at the Laboratory Animal Service Center of the Southern Medical University between January and June 2005. MATERIALS: Fifty-four adult, Wistar rats of either sex, weighing 180-250 g, were obtained from the Laboratory Animal Service Center of the Southern Medical University. Animals were randomly divided into nine groups of six rats. METHODS: Wallerian degeneration was induced by transecting the rat sciatic nerve, and tissue samples from the distal stump were obtained 0.2, 0.4, 1, 2, 3, 4, 7, 10, and 15 days post-transection. Ultrathin sections were prepared for electron microscopy to study ultrastructure and enzyme cytochemistry staining. MAIN OUTCOME MEASURES: Ultrastructure (axon body, autophagic body, and cystoskeleton) of axons and myelin sheaths observed with electron microscopy; acidic phosphatase activity detected by Gomori staining using electron microscopy. RESULTS: The major changes of degenerating axons after transection were axoplasm swelling and separation of axons from their myelin sheath between five hours and two days post-transection. At four days post-transection, the axoplasm condensed and axons were completely separated from the myelin sheath, forming dissociative axon bodies. Vacuoles of different sizes formed in axons during the early phase after lesion. Larger dissociative axon bodies were formed when the axons were completely separated from the myelin sheath during a late phase. The axolemma surrounding the axon body was derived from the neuronal cell membrane; the condensed axoplasm contained many autophagic vacuoles at all levels. A large number of neurofilaments, microtubules, and microfilaments were arranged in a criss-cross pattern. The autophagic vacuoles exhibited acidic phosphatase activity. Axonal bodies were absorbed after degradation from day 7 onwards, and macrophages were observed rarely in the formative cavity. CONCLUSION: The degenerating axons were cleared mainly by axonal autophagy and Schwann cell phagocytosis during regeneration of the rat sciatic nerve, and macrophages exhibited only an assisting function.