Verapamil inhibits scar formation after peripheral nerve repairin vivo

The calcium channel blocker, verapamil, has been shown to reduce scar formation by inhibiting ifbroblast adhesion and proliferationin vitro. It was not clear whether topical application of verapamil after surgical repair of the nerve in vivo could inhibit the formation of ex-cessive scar tissue. In this study, the right sciatic nerve of adult Sprague-Dawley rats was transected and sutured with No. 10-0 suture. The stoma was wrapped with gelfoam soaked with verapamil solution for 4 weeks. Compared with the control group (stoma wrapped with gelfoam soaked with physiological saline), the verapamil application inhibited the secretion of extracellular matrix from ifbroblasts in vivo, suppressed type I and III collagen secretion and increased the total number of axons and the number of myelinated axons. These ifndings suggest that verapamil could reduce the formation of scar tissue and promote axon growth after peripheral nerve repair.     

Tacrolimus reduces scar formation and promotes sciatic nerve regeneration

A sciatic nerve transection and repair model was established in Sprague-Dawley rats by transecting the tendon of obturator internus muscle in the greater sciatic foramen and suturing with nylon sutures. The models were treated with tacrolimus gavage (4 mg/kg per day) for 0, 2, 4 and 6 weeks. Specimens were harvested at 6 weeks of intragastric administration. Masson staining revealed that the collagen fiber content and scar area in the nerve anastomosis of the sciatic nerve injury rats were significantly reduced after tacrolimus administration. Hematoxylin-eosin staining showed that tacrolimus significantly increased myelinated nerve fiber density, average axon diameter and myelin sheath thickness. Intragastric administration of tacrolimus also led to a significant increase in the recovery rate of gastrocnemius muscle wet weight and the sciatic functional index after sciatic nerve injury. The above indices were most significantly improved at 6 weeks after of tacrolimus gavage. The myelinated nerve fiber density in the nerve anastomosis and the sciatic nerve functions had a significant negative correlation with the scar area, as detected by Spearman’s rank correlation analysis. These findings indicate that tacrolimus can promote peripheral nerve regeneration and accelerate the recovery of neurological function through the reduction of scar formation.     

Balance and coordination training after sciatic nerve injury

Introduction: Numerous therapeutic interventions have been tested to enhance functional recovery after peripheral nerve injuries. Methods: After sciatic nerve crush in rats we tested balance and coordination and motor control training in sensorimotor tests and analyzed nerve and muscle histology. Results: The balance and coordination training group and the sham group had better results than the sedentary and motor control groups in sensorimotor tests. The sham and balance and coordination groups had a significantly larger muscle area than the other groups, and the balance and coordination group showed significantly better values than the sedentary and motor control groups for average myelin sheath thickness and g‐ratio of the distal portion of the nerve. Conclusions: The findings indicate that balance and coordination training improves sciatic nerve regeneration, suggesting that it is possible to revert and/or prevent soleus muscle atrophy and improve performance on sensorimotor tests. Muscle Nerve 44: 55–62, 2011.     

Myelin-associated glycoprotein combined with chitin conduit inhibits painful neuroma formation after sciatic nerve transection

Studies have shown that myelin-associated glycoprotein(MAG)can inhibit axon regeneration after nerve injury.However,the effects of MAG on neuroma formation after peripheral nerve injury remain poorly understood.In this study,local injection of MAG combined with nerve cap made of chitin conduit was used to intervene with the formation of painful neuroma after sciatic nerve transfection in rats.After 8 weeks of combined treatment,the autotomy behaviors were reduced in rats subjected to sciatic nerve transfection,the mRNA expression of nerve growth factor,a pain marker,in the proximal nerve stump was decreased,the density of regenerated axons was decreased,the thickness of the myelin sheath was increased,and the ratio of unmyelinated to myelinated axons was reduced.Moereover,the percentage of collagen fiber area and the percentage of fibrosis marker alpha-smooth muscle actin positive staining area in the proximal nerve stump were decreased.The combined treatment exhibited superior effects in these measures to chitin conduit treatment alone.These findings suggest that MAG combined with chitin conduit synergistically inhibits the formation of painful neuroma after sciatic nerve transection and alleviates neuropathic pain.This study was approved by the Animal Ethics Committee of Peking University People’s Hospital(approval No.2019PHE027)on December 5,2019.     

Spatiotemporal expression of testicular protein kinase 1 after rat sciatic nerve injury

Testicular protein kinase 1 (TESK1), a serine/threonine kinase, has been found expressing in various tissues and cell lines. Previous reports have shown that TESK1 plays an important role in regulating actin reorganization of spreading cell on fibronectin via phosphorylating cofilin. Because of the importance of actin reorganization in radial sorting and remyelination of peripheral nerve regeneration, we investigated the spatiotemporal expression of TESK1 in a rat sciatic nerve crush model. We observed that sciatic nerve crush resulted in a significant upregulation of TESK1 from 5 days to 2 weeks and subsequent return to the control level at 4 weeks. At its peak expression, TESK1 expressed mainly in both Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, upregulation of TESK1 was approximately in parallel with Oct-6, and numerous SCs expressing TESK1 were Oct-6 positive. Experiments with Schwann cell primary cultures revealed that TESK1 accumulated at F-actin-rich lamellipodia of the cell periphery when SCs were plated on fibronectin, whereas it was distributed in the cytoplasm in the case of non-stimulated cells. Thus, these findings suggest that TESK1 plays important roles in promyelinating SCs, potentially through subcellular localization change and participation in integrin-mediated actin reorganization.     

Gene expression profile changes in rat dorsal horn after sciatic nerve injury

Objective: This study aims to investigate gene expression changes in rat dorsal horns after sciatic nerve injury (SNI).

Methods: The GSE18803 microarray data collected from young and adult rats were downloaded from GEO. After preprocessing, differentially expressed genes (DEGs) between SNI and sham-operated groups were selected using Limma package, in young and adult group, respectively, followed by Venn analysis. Then, enrichment analyses were performed for these DEGs using DAVID. The STRING database was used to identify protein–protein interactions (PPIs) among these DEGs, and the module network was further extracted using plugin ClusterONE. Finally, protein domain enrichment analysis of DEGs in each module was performed using InterPro database.

Results: Totally, 210 and 50 DEGs were identified in adult and young group, respectively. Among them, 160 were specific in adult group (e.g. CCL2, NF-κB1 and RAC2); 9 were specific in young group (e.g. ILF3 and LYVE1); and 41 were common in both two groups (e.g. FCER1G, C1QA, C1QB and C1QC). The up-regulated DEGs were mostly enriched in immune response-related biological processes, as well as 15 immune- and inflammation-related pathways. Then, two modules were identified in PPI network. CCL2 and NF-κB1 had high connectivity degrees in module 1, and RAC2, FCER1G and CD68 in module 2.

Conclusion: CCL2, NF-κB1, RAC2, FCER1G and C1Q may contribute to the generation of neuropathic pain after SNI via immune and defense pathways. Among the five genes, the first three are specific in adult population, while the latter two are age-independent. They all might function through involvement of these immune or inflammatory pathways.     

Early cytokine expression in mouse sciatic nerve after chronic constriction nerve injury depends on calpain

Nerve injury initiates Wallerian degeneration with subsequent alterations of cytokine expression contributing to neuropathic pain. To investigate the very early temporal pattern of cytokine regulation we studied 140 mice of C57Bl/6J background after chronic constriction injury (CCI) of the right sciatic nerve and measured the relative mRNA expression of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1beta) and of the anti-inflammatory cytokines IL-4 and IL-10 with quantitative real-time polymerase chain reaction (qRT-PCR). The measurements were performed in ipsi- and contralateral sciatic nerves and dorsal root ganglia (DRG) 1, 3, 6, 9, 12, 24 h, and 3 and 7 days after CCI. We found an ipsilateral upregulation of TNF, IL-1beta and IL-10 mRNA levels as early as one hour after CCI. To investigate upstream regulatory mechanisms, we used inhibitors to the N-methyl-d-aspartate (NMDA) receptor ((+)-MK-801) and to calpain (MDL-28170). MDL-28170, but not (+)-MK-801 inhibited TNF and IL-1beta upregulation one hour after CCI. This leads us to suggest that calpain is one of the earliest mediators of cytokine upregulation in injured peripheral nerves.     

Chemogenetic enhancement of functional recovery after a sciatic nerve injury

Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools used to modulate neuronal excitability. We hypothesized that activation of excitatory (Gq) DREADD by its designer ligand, clozapine‐N‐oxide (CNO), would increase the excitability of neurons whose axons have been transected following peripheral nerve injury, and that this increase will lead to an enhanced functional recovery. The lateral gastrocnemius (LG) muscle of adult female Lewis rats was injected unilaterally with AAV9‐ hsyn‐ hM3Dq‐mCherry (7.6 × 10⁹ viral genomes/μL) to transduce Gq‐DREADD expression in LG neurons. The contralateral LG muscle served as an uninjected control. No significant changes in either spontaneous EMG activity or electrically evoked direct muscle (M) responses were found in either muscle after injection of CNO (1 mg/kg, i.p.). The amplitude of monosynaptic H‐reflexes in LG was increased after CNO treatment exclusively in muscles previously injected with virus, suggesting that Gq‐DREADD activation increased neuronal excitability. After bilateral sciatic nerve transection and repair, additional rats were treated similarly with CNO for up to three days after injury. Electrophysiological data were collected at 2, 4 and 6 weeks after injury. Evoked EMG responses were observed as early as 2 weeks after injury only in Gq‐DREADD expressing virus injected LG muscle. By 4 weeks after injury, both M‐response and H‐reflex amplitudes were significantly greater in muscles previously injected with viral vector than contralateral, uninjected muscles. Increases in the excitability of injured neurons produced by this novel use of Gq‐DREADD were sufficient to promote an enhancement in functional recovery after a sciatic nerve injury.     

Platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury

The effect of platelet-rich plasma on nerve regeneration remains controversial.In this study,we established a rabbit model of sciatic nerve small-gap defects with preserved epineurium and then filled the gaps with platelet-rich plasma.Twenty-eight rabbits were divided into the following groups(7 rabbits/group):model,low-concentrati on PRP(2.5-3.5-fold concentration of whole blood platelets),medium-concentration PRP(4.5-6.5-fold concentration of whole blood platelets),and high-concentration PRP(7...     

Miconazole enhances nerve regeneration and functional recovery after sciatic nerve crush injury

Introduction: Improving axonal outgrowth and remyelination is crucial for peripheral nerve regeneration. Miconazole appears to enhance remyelination in the central nervous system. In this study we assess the effect of miconazole on axonal regeneration using a sciatic nerve crush injury model in rats. Methods: Fifty Sprague‐Dawley rats were divided into control and miconazole groups. Nerve regeneration and myelination were determined using histological and electrophysiological assessment. Evaluation of sensory and motor recovery was performed using the pinprick assay and sciatic functional index. The Cell Counting Kit‐8 assay and Western blotting were used to assess the proliferation and neurotrophic expression of RSC 96 Schwann cells. Results: Miconazole promoted axonal regrowth, increased myelinated nerve fibers, improved sensory recovery and walking behavior, enhanced stimulated amplitude and nerve conduction velocity, and elevated proliferation and neurotrophic expression of RSC 96 Schwann cells. Discussion: Miconazole was beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Muscle Nerve 57 : 821–828, 2018     

Retrograde axonal transport in rat sciatic nerve after nerve crush injury

We investigated the quantitative alterations in retrograde transport of proteins following a nerve crush injury using the 3H N-succinimidyl propionate (3H NSP) method in rat sciatic nerve. After subepineurial injection of 3H NSP into the nerve the amount of radioactively labeled proteins accumulating in the cell bodies of the motor and sensory neurons was determined 1 day or 7 days later in nerves which had been crushed distal to the injection site 1, 3, 5, 7, or 33 days prior to 3H NSP labeling. One day accumulation in the DRG and spinal cord was not altered by nerve crush. Seven day accumulation in the DRG was initially slightly increased, then fell to 73% of control by 7 days, remaining reduced 33 days after crush. Seven day accumulation in the spinal cord was reduced to 25% of control 1 day after crush and remained at that low level except for 5 days post-crush when a normal amount of labeled protein was transported to the spinal cord. The time course of these changes suggests that quantitative alterations in retrograde transport may be involved in the long-term trophic interactions between the cell body and periphery, but are too slow to account for the earliest perikaryal responses to injury. In addition, the difference between the alterations of retrograde transport in motor and sensory neurons may reflect fundamental differences in the composition of retrograde transport in those different systems.     

Matrix metalloproteinase expression and inhibition after sciatic nerve axotomy

Wallerian degeneration is characterized by breakdown of myelin and axons with subsequent macrophage infiltration and removal of the degenerating nerve components. Proteinases of the matrix metalloproteinase (MMP) family seem to play an important role in demyelinating processes, since some of their members have been shown to cleave myelin basic protein. In the present study we investigated the expression of MMP-2 and MMP-9 (gelatinases A and B) during myelin removal after peripheral nerve trauma. After transection of the sciatic nerve an upregulation of MMP-2 and MMP-9 with a first peak 12 h and a second peak 48 h after axotomy was observed by zymography. These peaks correlate with the breakdown of the blood-nerve barrier, the accumulation of granulocytes, and the invasion of macrophages into the damaged nerves, respectively. Furthermore, MMP-2 was found to be upregulated in the contralateral nontransected nerves. Immunocytochemistry for MMP-9 and in situ zymography identified MMP-reactive cells within the distal nerve stump. Chloracetate esterase staining was used to detect granulocytes, which accumulated at the transection site and were colocalized with the in situ zymography signal. Wallerian degeneration of the transected nerve could be delayed either by intraperitoneal injections of hydroxamate (Ro 31-9790), a nonspecific MMP inhibitor, or by local application of an MMP-9-specific antibody. Following these treatment strategies, a decreased number of invading macrophages was seen in the nerves associated with an increased amount of preserved myelin sheaths. These results suggest that the invasion of macrophages into a transected peripheral nerve is accompanied by an increased expression of MMPs, particularly MMP-9. Thus, MMPs may seem to play an important role in the breakdown of the blood-nerve barrier and subsequent cell recruitment from the systemic circulation into the damaged nerve.     

Ursolic acid induces neural regeneration after sciatic nerve injury

In this study,we aimed to explore the role of ursolic acid in the neural regeneration of the injured sciatic nerve.BALB/c mice were used to establish models of sciatic nerve injury through unilateral sciatic nerve complete transection and microscopic anastomosis at 0.5 cm below the ischial tuberosity.The successfully generated model mice were treated with 10,5,or 2.5 mg/kg ursolic acid via intraperitoneal injection.Enzyme-linked immunosorbent assay results showed that serum S100 protein expression level gradually increased at 1-4 weeks after sciatic nerve injury,and significantly decreased at 8 weeks.As such,ursolic acid has the capacity to significantly increase S100 protein expression levels.Real-time quantitative PCR showed that S100 mRNA expression in the L4-6 segments on the injury side was increased after ursolic acid treatment.In addition,the muscular mass index in the soleus muscle was also increased in mice treated with ursolic acid.Toluidine blue staining revealed that the quantity and average diameter of myelinated nerve fibers in the injured sciatic nerve were significantly increased after treatment with ursolic acid.10 and 5 mg/kg of ursolic acid produced stronger effects than 2.5 mg/kg of ursolic acid.Our findings indicate that ursolic acid can dose-dependently increase S100 expression and promote neural regeneration in BALB/c mice following sciatic nerve injury.     

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.     

Exercise training and axonal regeneration after sciatic nerve injury.

In the present study, we aimed to investigate the relationship between exercise training and peripheral nerve regeneration after crush injury. For this purpose, HRP neurohistochemistry and modified Pal-Weigert methods were used to assess the axonal regeneration. In the 2nd and 3rd regeneration week groups, myelin debris was observed, and there was no significant difference between exercise trained and sedentary groups. In the 4th regeneration week group, it was seen that myelin debris was removed, and some myelinated fibers were observed in the exercise trained group. On the other hand, there was no myelinated fiber in the sedentary group, and there was a significant difference between exercise trained and sedentary groups. Consequently, we think that exercise is effective in the 4th regeneration week.