Distal Nerve Stump Research Articles

Identification of peripheral nerve functional fascicles in Sprague-Dawley rats by the carbon quantum dot-Annexin V antibody complex

To explore a method to identify the sensory and motor fascicles of the peripheral nerve to achieve accurate peripheral nerve functional fascicle suture. The peripheral nerve Sunderland V injury model, muscle branch of the femoral nerve and saphenous nerve were established in the bilateral femoral nerves of Sprague-Dawley (SD) rats. The specific samples were grouped as follows: the main trunk of the femoral nerve was exposed bilaterally and cut with microscopic scissors in the main trunk of the femoral nerve to prepare a model of Sunderland V injury in the mixed fascicle of peripheral nerves; the muscle branch of the femoral nerve was exposed bilaterally and cut in the middle section of the muscle branch of the femoral nerve to prepare a model of Sunderland V injury in the motor fascicle of peripheral nerves; the saphenous nerve was exposed bilaterally and cut at 1 cm below the patella to prepare a model of Sunderland V injury to the sensory fascicle of the peripheral nerves. A carbon quantum dot (CD)-annexin V antibody complex was prepared and applied to the distal and proximal nerve stumps of the peripheral nerve Sunderland V injury model groups of SD rats. Under the excitation light source of a 380 nm uv lamp, fluorescence color development was observed under a fluorescence microscope after 5, 10, 15, and 20 min. At 5 min, sections of the bilateral femoral nerve trunk, muscular branches of the femoral nerve, and Sunderland V lesion of the saphenous nerve in SD rats were only dark in color under the microscope, and there was no difference in fluorescence. The intensity of the staining increased significantly for 10–20 min. The sensory fascicles and saphenous nerves of the femoral nerve trunk showed blue fluorescence under the CD-Annexin V antibody complex staining, while the motor fascicles and muscle branches of the femoral nerve trunk showed no fluorescence. Fluorescence intensity gradually decreased after 20 min of staining. There was no significant difference in the staining intensity at 5, 10, 15, and 20 min in each group. Our results suggest that the CD-Annexin antibody complex can be used to identify functional fascicles of peripheral nerves in SD rats.

The use of electrical stimulation to enhance recovery following peripheral nerve injury.

Peripheral nerve injury is common and can have devastating consequences. In severe cases, functional recovery is often poor despite surgery. This is primarily due to the exceedingly slow rate of nerve regeneration at only 1-3 mm/day. The local environment in the distal nerve stump supportive of nerve regrowth deteriorates over time and the target end organs become atrophic. To overcome these challenges, investigations into treatments capable of accelerating nerve regrowth are of great clinical relevance and are an active area of research. One intervention that has shown great promise is perioperative electrical stimulation. Postoperative stimulation helps to expedite the Wallerian degeneration process and reduces delays caused by staggered regeneration at the site of nerve injury. By contrast, preoperative "conditioning" stimulation increases the rate of nerve regrowth along the nerve trunk. Over the past two decades, a rich body of literature has emerged that provides molecular insights into the mechanism by which electrical stimulation impacts nerve regeneration. The end result is upregulation of regeneration-associated genes in the neuronal body and accelerated transport to the axon front for regrowth. The efficacy of brief electrical stimulation on patients with peripheral nerve injuries was demonstrated in a number of randomized controlled trials on compressive, transection and traction injuries. As approved equipment to deliver this treatment is becoming available, it may be feasible to deploy this novel treatment in a wide range of clinical settings.

The Role of Length of Nerve Grafts in Combination with Free Functional Muscle Transplantation for Brachial Plexus Injury: A Single-Center Experience.

Extensive lesions of the brachial plexus, or late cases, require free functional muscle grafts because the expected recovery time exceeds the critical threshold of 1.5 years, beyond which irreversible damage may be expected in the distal nerve stump and in the muscle. The reconstructive concept consists of a two-stage procedure where, in the first step, a nerve transfer is performed (from ipsi- or contralateral donor nerves). In the second step, after successful axonal regeneration within the graft has been confirmed, a free muscle transfer is performed. These grafts often exceed 40 cm in length, particularly for contralateral transfers. The purpose of this study was to assess whether robust motor recovery could be supported by such long nerve grafts. From April 2004 to April 2023, a total of 327 free functional muscle transfers were performed, the nerve graft length ranging from 0 cm (direct coaptation) to 90 cm (serial grafts). Motor recovery was evaluated 1.5 years after surgery according to the MRC scale. A total of 208 patients were available for follow up. Direct coaptation yielded the best results, with 83% of patients reaching an M3 or M4 level of muscle strength. With the application of long (30-60 cm) grafts, 73% of the patients were classified as M3 or M4. The application of serial nerve grafts, however, only resulted in 18% of patients achieving a motor recovery rating of M3. These findings demonstrate that robust motor regeneration is supported by long (30-60 cm) nerve grafts, whereas serial nerve grafting results in a marked reduction in the quality of regeneration.

Dual innervation method to preserve erectile function following prostatectomy

Prostate cancer is the second most prevalent cancer in men. Robot-assisted radical prostatectomy (RARP) has altered the landscape of prostate cancer treatment. Despite the excellent oncological outcomes associated with RARP, the rate of erectile dysfunction (ED) remains high. Primary repair of disrupted cavernous nerves with interpositional nerve grafts has been described; however, the outcomes have been inconsistent. We hypothesize that this is attributed to Schwann cell atrophy and axonal regeneration limitations caused by long nerve grafts. We proposed the use of nerve transfer to support axonal regrowth via an inter-positional graft with additional donor axons. A cadaveric study was performed to evaluate the anatomical feasibility of a vastus lateralis nerve (VLN) transfer to the distal recipient cavernous nerve stump. The VLN is long with multiple branching patterns that allow tension-free coaptation of the cavernous nerve. We postulate that a dual innervation method using VLN nerve transfer together with interpositional nerve graft repair of the transacted cavernous nerves may improve the outcomes of ED post-RARP.

Utilization of the Rat Tibial Nerve Transection Model to Evaluate Cellular and Molecular Mechanisms Underpinning Denervation-Mediated Muscle Injury.

Peripheral nerve injury denervates muscle, resulting in muscle paralysis and atrophy. This is reversible if timely muscle reinnervation occurs. With delayed reinnervation, the muscle's reparative ability declines, and muscle-resident fibro-adipogenic progenitor cells (FAPs) proliferate and differentiate, inducing fibro-fatty muscle degradation and thereby physical disability. The mechanisms by which the peripheral nerve regulates FAPs expansion and differentiation are incompletely understood. Using the rat tibial neve transection model, we demonstrated an increased FAPs content and a changing FAPs phenotype, with an increased capacity for adipocyte and fibroblast differentiation, in gastrocnemius muscle post-denervation. The FAPs response was inhibited by immediate tibial nerve repair with muscle reinnervation via neuromuscular junctions (NMJs) and sensory organs (e.g., muscle spindles) or the sensory protection of muscle (where a pure sensory nerve is sutured to the distal tibial nerve stump) with reinnervation by muscle spindles alone. We found that both procedures reduced denervation-mediated increases in glial-cell-line-derived neurotrophic factor (GDNF) in muscle and that GDNF promoted FAPs adipogenic and fibrogenic differentiation in vitro. These results suggest that the peripheral nerve controls FAPs recruitment and differentiation via the modulation of muscle GDNF expression through NMJs and muscle spindles. GDNF can serve as a therapeutic target in the management of denervation-induced muscle injury.

Brief Electrical Stimulation Promotes Recovery after Surgical Repair of Injured Peripheral Nerves.

Injured peripheral nerves regenerate their axons in contrast to those in the central nervous system. Yet, functional recovery after surgical repair is often disappointing. The basis for poor recovery is progressive deterioration with time and distance of the growth capacity of the neurons that lose their contact with targets (chronic axotomy) and the growth support of the chronically denervated Schwann cells (SC) in the distal nerve stumps. Nonetheless, chronically denervated atrophic muscle retains the capacity for reinnervation. Declining electrical activity of motoneurons accompanies the progressive fall in axotomized neuronal and denervated SC expression of regeneration-associated-genes and declining regenerative success. Reduced motoneuronal activity is due to the withdrawal of synaptic contacts from the soma. Exogenous neurotrophic factors that promote nerve regeneration can replace the endogenous factors whose expression declines with time. But the profuse axonal outgrowth they provoke and the difficulties in their delivery hinder their efficacy. Brief (1 h) low-frequency (20 Hz) electrical stimulation (ES) proximal to the injury site promotes the expression of endogenous growth factors and, in turn, dramatically accelerates axon outgrowth and target reinnervation. The latter ES effect has been demonstrated in both rats and humans. A conditioning ES of intact nerve days prior to nerve injury increases axonal outgrowth and regeneration rate. Thereby, this form of ES is amenable for nerve transfer surgeries and end-to-side neurorrhaphies. However, additional surgery for applying the required electrodes may be a hurdle. ES is applicable in all surgeries with excellent outcomes.

Nerve function restoration following targeted muscle reinnervation after varying delayed periods.

Targeted muscle reinnervation has been proposed for reconstruction of neuromuscular function in amputees. However, it is unknown whether performing delayed targeted muscle reinnervation after nerve injury will affect restoration of function. In this rat nerve injury study, the median and musculocutaneous nerves of the forelimb were transected. The proximal median nerve stump was sutured to the distal musculocutaneous nerve stump immediately and 2 and 4 weeks after surgery to reinnervate the biceps brachii. After targeted muscle reinnervation, intramuscular myoelectric signals from the biceps brachii were recorded. Signal amplitude gradually increased with time. Biceps brachii myoelectric signals and muscle fiber morphology and grooming behavior did not significantly differ among rats subjected to delayed target muscle innervation for different periods. Targeted muscle reinnervation delayed for 4 weeks can acquire the same nerve function restoration effect as that of immediate reinnervation.

Glycolytic System in Axons Supplement Decreased ATP Levels after Axotomy of the Peripheral Nerve.

Wallerian degeneration (WD) occurs in the early stages of numerous neurologic disorders, and clarifying WD pathology is crucial for the advancement of neurologic therapies. ATP is acknowledged as one of the key pathologic substances in WD. The ATP-related pathologic pathways that regulate WD have been defined. The elevation of ATP levels in axon contributes to delay WD and protects axons. However, ATP is necessary for the active processes to proceed WD, given that WD is stringently managed by auto-destruction programs. But little is known about the bioenergetics during WD. In this study, we made sciatic nerve transection models for GO-ATeam2 knock-in rats and mice. We presented the spatiotemporal ATP distribution in the injured axons with in vivo ATP imaging systems, and investigated the metabolic source of ATP in the distal nerve stump. A gradual decrease in ATP levels was observed before the progression of WD. In addition, the glycolytic system and monocarboxylate transporters (MCTs) were activated in Schwann cells following axotomy. Interestingly, in axons, we found the activation of glycolytic system and the inactivation of the tricarboxylic acid (TCA) cycle. Glycolytic inhibitors, 2-deoxyglucose (2-DG) and MCT inhibitors, a-cyano-4-hydroxycinnamic acid (4-CIN) decreased ATP and enhanced WD progression, whereas mitochondrial pyruvate carrier (MPC) inhibitors (MSDC-0160) did not change. Finally, ethyl pyruvate (EP) increased ATP levels and delayed WD. Together, our findings suggest that glycolytic system, both in Schwann cells and axons, is the main source of maintaining ATP levels in the distal nerve stump.

Phrenic nerve neurorrhaphy with a sural nerve graft in unilateral diaphragmatic paralysis

Unilateral diaphragmatic paralysis can present following damage or impairment to a phrenic nerve. In cases where current treatment methods are not feasible, the use of a double phrenic nerve neurorrhaphy with an autologous sural nerve graft should be investigated. In preserved rats, the feasibility of this technique was explored. The sural nerve was excised from the left thigh. Entering into the thoracic cavity bilaterally through the fifth intercostal spaces, the right and left phrenic nerves were identified. The right phrenic nerve was cut to create a distal nerve stump. The sural nerve was grafted onto the distal stump of the right phrenic nerve and the side of the left phrenic nerve. Using six preserved rat cadavers, the end-to-side graft was successfully explored and performed. It was determined that approximately 2.5–3 cm of the sural nerve will be required to be harvested for the graft. The sural nerve was an appropriate size for grafting onto the phrenic nerves. Four 10–0 Nylon sutures were sufficient for each neurorrhaphy. A double phrenic nerve neurorrhaphy with an autologous sural nerve graft is potentially feasible in rat models.

Role of secretomes in cell-free therapeutic strategies in regenerative medicine.

After an injury, peripheral nervous system neurons have the potential to rebuild their axons by generating a complicated activation response. Signals from the damaged axon are required for this genetic transition to occur. Schwann cells (SCs) near a damaged nerve's distal stump also play a role in the local modulation of axonal programs, not only via cell-to-cell contacts but also through secreted signals (the secretome). The secretome is made up of all the proteins that the cell produces, such as cytokines, growth factors, and extracellular vesicles. The released vesicles may carry signaling proteins as well as coding and regulatory RNAs, allowing for multilayer communication. The secretome of SCs is now well understood as being critical for both orchestrating Wallerian degeneration and maintaining axonal regeneration. As a consequence, secretome has emerged as a feasible tissue regeneration alternative to cell therapy. Separate SC secretome components have been used extensively in the lab to promote peripheral nerve regeneration after injury. However, in neurological therapies, the secretome generated by mesenchymal (MSC) or other derived stem cells has been the most often used. In fact, the advantages of cell treatment have been connected to the release of bioactive chemicals and extracellular vesicles, which make up MSCs' secretome.

The best of both worlds: mastering nerve regeneration combining biological and nanotechnological tools.

The best of both worlds: mastering nerve regeneration combining biological and nanotechnological tools.

Trick or treat? Does cancer fool Schwann cells by mimicking axons to promote metastasis into nerves?

Trick or treat? Does cancer fool Schwann cells by mimicking axons to promote metastasis into nerves?

Electrical stimulation accelerates Wallerian degeneration and promotes nerve regeneration after sciatic nerve injury

Following peripheral nerve injury (PNI), Wallerian degeneration (WD) in the distal stump can generate a microenvironment favorable for nerve regeneration. Brief low-frequency electrical stimulation (ES) is an effective treatment for PNI, but the mechanism underlying its effect on WD remains unclear. Therefore, we hypothesized that ES could enhance nerve regeneration by accelerating WD. To verify this hypothesis, we used a rat model of sciatic nerve transection and provided ES at the distal stump of the injured nerve. The injured nerve was then evaluated after 1, 4, 7, 14 and 21 days post injury (dpi). The results showed that ES significantly promoted the degeneration and clearance of axons and myelin, and the dedifferentiation of Schwann cells. It upregulated the expression of BDNF and NGF and increased the number of monocytes and macrophages. Through transcriptome sequencing, we systematically investigated the effect of ES on the molecular processes involved in WD at 4 dpi. Evaluation of nerves bridged using silicone tubing after transection showed that ES accelerated early axonal and vascular regeneration while delaying gastrocnemius atrophy. These results demonstrate that ES promotes nerve regeneration by accelerating WD and upregulating the expression of neurotrophic factors.

Sexual dimorphism of early transcriptional reprogramming in degenerating peripheral nerves.

Sexual dimorphism is a powerful yet understudied factor that influences the timing and efficiency of gene regulation in axonal injury and repair processes in the peripheral nervous system. Here, we identified common and distinct biological processes in female and male degenerating (distal) nerve stumps based on a snapshot of transcriptional reprogramming 24 h after axotomy reflecting the onset of early phase Wallerian degeneration (WD). Females exhibited transcriptional downregulation of a larger number of genes than males. RhoGDI, ERBB, and ERK5 signaling pathways increased activity in both sexes. Males upregulated genes and canonical pathways that exhibited robust baseline expression in females in both axotomized and sham nerves, including signaling pathways controlled by neuregulin and nerve growth factors. Cholesterol biosynthesis, reelin signaling, and synaptogenesis signaling pathways were downregulated in females. Signaling by Rho Family GTPases, cAMP-mediated signaling, and sulfated glycosaminoglycan biosynthesis were downregulated in both sexes. Estrogens potentially influenced sex-dependent injury response due to distinct regulation of estrogen receptor expression. A crosstalk of cytokines and growth hormones could promote sexually dimorphic transcriptional responses. We highlighted prospective regulatory activities due to protein phosphorylation, extracellular proteolysis, sex chromosome-specific expression, major urinary proteins (MUPs), and genes involved in thyroid hormone metabolism. Combined with our earlier findings in the corresponding dorsal root ganglia (DRG) and regenerating (proximal) nerve stumps, sex-specific and universal early phase molecular triggers of WD enrich our knowledge of transcriptional regulation in peripheral nerve injury and repair.

Efficacy of retrograde innervation through the distal stump of the thoracodorsal nerve in single-stage neurovascular latissimus dorsi transfer for facial reanimation: A retrospective analysis of 30 cases

The aim of the study was to analyze the efficacy of retrograde innervation of single-stage free latissimus dorsi (LD) flap for facial reanimation by the distal stump of thoracodorsal nerve (TDN).After the main trunk of TDN was split in half to preserve the function of transverse branch, the proximal stump was coapted with the contralateral facial nerve, and the distal stump was coapted with the ipsilateral masseteric nerve, or vice versa. The length of each nerve stump and the time required for voluntary and spontaneous muscle contraction after surgical treatment were reviewed.Among a total of 30 patients, there was no statistically significant difference in the successful recovery ratio of voluntary smile (antegrade, 86.7%; retrograde, 92.3%; p = 0.323) and of spontaneous smile (antegrade, 61.5%; retrograde, 100.0%; p = 0.079). There was no significant difference in the speed of nerve regeneration of voluntary smile (antegrade, 1.32 cm/month; retrograde, 1.14 cm/month; p = 0.213) and of spontaneous smile (antegrade, 0.91 cm/month; retrograde, 1.09 cm/month, p = 0.120).Within the limitations of the study, it seems that retrograde innervation of neurovascular muscle grafts has the potential to serve as a relevant alternative to other established options of neurovascular muscle transfer, because it provides comparable results in terms of rehabilitation success.

Advancing Our Understanding of the Chronically Denervated Schwann Cell: A Potential Therapeutic Target?

Outcomes for patients following major peripheral nerve injury are extremely poor. Despite advanced microsurgical techniques, the recovery of function is limited by an inherently slow rate of axonal regeneration. In particular, a time-dependent deterioration in the ability of the distal stump to support axonal growth is a major determinant to the failure of reinnervation. Schwann cells (SC) are crucial in the orchestration of nerve regeneration; their plasticity permits the adoption of a repair phenotype following nerve injury. The repair SC modulates the initial immune response, directs myelin clearance, provides neurotrophic support and remodels the distal nerve. These functions are critical for regeneration; yet the repair phenotype is unstable in the setting of chronic denervation. This phenotypic instability accounts for the deteriorating regenerative support offered by the distal nerve stump. Over the past 10 years, our understanding of the cellular machinery behind this repair phenotype, in particular the role of c-Jun, has increased exponentially, creating opportunities for therapeutic intervention. This review will cover the activation of the repair phenotype in SC, the effects of chronic denervation on SC and current strategies to ‘hack’ these cellular pathways toward supporting more prolonged periods of neural regeneration.

Transcription factor SS18L1 regulates the proliferation, migration and differentiation of Schwann cells in peripheral nerve injury.

Transcription factors bind to specific DNA sequences, modulate the transcription of target genes, and regulate various biological processes, including peripheral nerve regeneration. Our previous analysis showed that SS18L1, a gene encoding the transcription factor SS18-like protein 1, was differentially expressed in the distal sciatic nerve stumps after rat sciatic nerve transection injury, but its effect on peripheral nerve injury has not been reported. In the current study, we isolated and cultured primary Schwann cells, and examined the role of SS18L1 for the biological functions of the cells. Depletion of SS18L1 by siRNA in Schwann cells enhanced cell proliferation and inhibited cell migration, as determined by EdU assay and transwell migration assay, respectively. In addition, silencing of SS18L1 inhibited Schwann cell differentiation induced by HRG and cAMP. Bioinformatics analyses revealed an interaction network of SS18L1, including DF2, SMARCD1, SMARCA4, and SMARCE1, which may be implicated in the regulatory functions of SS18L1 on the proliferation, migration and differentiation of Schwann cells. In conclusion, our results revealed a temporal expression profile of SS18L1 in peripheral nerve injury and its potential roles during the process of nerve recovery.

Efficacy of Nerve-Derived Hydrogels to Promote Axon Regeneration Is Influenced by the Method of Tissue Decellularization.

Injuries to large peripheral nerves are often associated with tissue defects and require reconstruction using autologous nerve grafts, which have limited availability and result in donor site morbidity. Peripheral nerve-derived hydrogels could potentially supplement or even replace these grafts. In this study, three decellularization protocols based on the ionic detergents sodium dodecyl sulfate (P1) and sodium deoxycholate (P2), or the organic solvent tri-n-butyl phosphate (P3), were used to prepare hydrogels. All protocols resulted in significantly decreased amounts of genomic DNA, but the P2 hydrogel showed the best preservation of extracellular matrix proteins, cytokines, and chemokines, and reduced levels of sulfated glycosaminoglycans. In vitro P1 and P2 hydrogels supported Schwann cell viability, secretion of VEGF, and neurite outgrowth. Surgical repair of a 10 mm-long rat sciatic nerve gap was performed by implantation of tubular polycaprolactone conduits filled with hydrogels followed by analyses using diffusion tensor imaging and immunostaining for neuronal and glial markers. The results demonstrated that the P2 hydrogel considerably increased the number of axons and the distance of regeneration into the distal nerve stump. In summary, the method used to decellularize nerve tissue affects the efficacy of the resulting hydrogels to support regeneration after nerve injury.

A Therapeutic Strategy for Lower Motor Neuron Disease and Injury Integrating Neural Stem Cell Transplantation and Functional Electrical Stimulation in a Rat Model.

Promising treatments for upper motor neuron disease are emerging in which motor function is restored by brain–computer interfaces and functional electrical stimulation. At present, such technologies and procedures are not applicable to lower motor neuron disease. We propose a novel therapeutic strategy for lower motor neuron disease and injury integrating neural stem cell transplantation with our new functional electrical stimulation control system. In a rat sciatic nerve transection model, we transplanted embryonic spinal neural stem cells into the distal stump of the peripheral nerve to reinnervate denervated muscle, and subsequently demonstrated that highly responsive limb movement similar to that of a healthy limb could be attained with a wirelessly powered two-channel neurostimulator that we developed. This unique technology, which can reinnervate and precisely move previously denervated muscles that were unresponsive to electrical stimulation, contributes to improving the condition of patients suffering from intractable diseases of paralysis and traumatic injury.

Muscle Fiber Composition Changes after Selective Nerve Innervation.

Facial nerve paralysis interferes with mimetic muscle function. To reconstruct natural facial movement, free muscle flaps are transplanted as new muscles. However, it is difficult to maintain resting tonus. A dual innervation technique in which other nerves such as the hypoglossal nerve or contralateral facial nerve are added is often applied. Using 10-week-old rats (n = 10), the masseteric and hypoglossal nerves were cut, and the distal stump of the masseteric nerve and the proximal stump of the hypoglossal nerve were then sutured (suture group). In the other group, the masseteric nerve was cut and cauterized (cut group). Immunohistochemistry and microarray were performed on the extracted masseter muscle. The immunohistochemistry results suggested that the muscles in the suture group obtained oxidative characteristics. The microarray showed the genes involved in mitochondrial function, including Perm1. In summary, our data support the validity of the dualinnervation technique for facial paralysis treatment.