RESUMEN
The use of acellular nerve allografts (ANAs) to reconstruct long nerve gaps (>3 cm) is associated with limited axon regeneration. To understand why ANA length might limit regeneration, we focused on identifying differences in the regenerative and vascular microenvironment that develop within ANAs based on their length. A rat sciatic nerve gap model was repaired with either short (2 cm) or long (4 cm) ANAs, and histomorphometry was used to measure myelinated axon regeneration and blood vessel morphology at various timepoints (2-, 4- and 8-weeks). Both groups demonstrated robust axonal regeneration within the proximal graft region, which continued across the mid-distal graft of short ANAs as time progressed. By 8 weeks, long ANAs had limited regeneration across the ANA and into the distal nerve (98 vs. 7583 axons in short ANAs). Interestingly, blood vessels within the mid-distal graft of long ANAs underwent morphological changes characteristic of an inflammatory pathology by 8 weeks post surgery. Gene expression analysis revealed an increased expression of pro-inflammatory cytokines within the mid-distal graft region of long vs. short ANAs, which coincided with pathological changes in blood vessels. Our data show evidence of limited axonal regeneration and the development of a pro-inflammatory environment within long ANAs.
Asunto(s)
Aloinjertos , Regeneración Nerviosa , Nervio Ciático , Animales , Ratas , Axones/metabolismo , Masculino , Vasos Sanguíneos , Inflamación/patología , Inflamación/metabolismo , Microambiente Celular , Trasplante Homólogo , Citocinas/metabolismo , Ratas Sprague-DawleyRESUMEN
INTRODUCTION/AIMS: Repaired nerve injuries can fail to achieve functional recovery. Therapeutic options beyond surgery, such as systemic tacrolimus (FK506) and electrical stimulation (E-stim), can improve recovery. We tested whether dual administration of FK506 and E-stim enhances regeneration and recovery more than either therapeutic alone. METHODS: Rats were randomized to four groups: E-stim, FK506, FK506 + E-stim, and repair alone. All groups underwent tibial nerve transection and repair. Two sets of animals were created to measure outcomes of early nerve regeneration using nerve histology (n = 36) and functional recovery (n = 42) (21- and 42-day endpoints, respectively). Functional recovery was measured by behavioral analyses (walking track and grid walk) and, at the endpoint, muscle mass and force. RESULTS: Dual E-stim and FK506 administration produced histomorphometric measurements of nerve regeneration no different than either therapeutic alone. All treatments were superior to repair alone (FK506, P < .0001; E-stim, P < .05; FK506 + E-stim, P < .05). The E-stim and FK506 + E-stim groups had improved behavioral recovery compared with repair alone (at 6 weeks: E-stim, P < .05; FK506 + E-stim, P < .01). The FK506 group had improved recovery based on walking-track analysis (at 6 weeks: P < .001) and muscle force and mass (P < .05). The concurrent use of both therapies ensured earlier functional recovery and decreased variability in functional outcomes compared with either therapy alone, suggesting a moderate benefit. DISCUSSION: Dual administration of FK506 and E-stim showed minimal additive effects to further improve regeneration or recovery compared with either therapy alone. The data suggest the combination of FK506 and E-stim appears to combine the relative strengths of each therapeutic.
Asunto(s)
Inmunosupresores , Tacrolimus , Animales , Ratas , Estimulación Eléctrica , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Regeneración Nerviosa/fisiología , Recuperación de la Función/fisiología , Tacrolimus/farmacología , Tacrolimus/uso terapéutico , Nervio Tibial/patología , Distribución AleatoriaRESUMEN
INTRODUCTION/AIMS: Promoting regeneration after segmental nerve injury repair is a challenge, but improving angiogenesis could be beneficial. Macrophages facilitate regeneration after injury by promoting angiogenesis. Our aim in this study was to evaluate the feasibility and effects of transplanting exogenous macrophages to a segmental nerve injury. METHODS: Bone marrow-derived cells were harvested from donor mice and differentiated to macrophages (BMDM), then suspended within fibrin hydrogels to facilitate BMDM transplantation. BMDM survival was characterized in vitro. The effect of this BMDM fibrin hydrogel construct at a nerve injury site was assessed using a mouse sciatic nerve gap injury. Mice were equally distributed to "fibrin+Mφ" (fibrin hydrogels containing culture medium and BMDM) or "fibrin" hydrogel control (fibrin hydrogels containing culture medium alone) groups. Flow cytometry (n = 3/group/endpoint) and immunohistochemical analysis (n = 5/group/endpoint) of the nerve gap region were performed at days 3, 5, and 7 after repair. RESULTS: Incorporating macrophage colony-stimulating factor (M-CSF) improved BMDM survival and expansion. Transplanted BMDM survived for at least 7 days in a nerve gap (~40% retained at day 3 and ~15% retained at day 7). From transplantation, macrophage quantities within the nerve gap were elevated when comparing fibrin+Mφ with fibrin control (~25% vs. 3% at day 3 and ~14% vs. 6% at day 7). Endothelial cells increased by about fivefold within the nerve gap, and axonal extension into the nerve gap increased almost twofold for fibrin+Mφ compared with fibrin control. DISCUSSION: BMDM suspended within fibrin hydrogels at a nerve gap do not impair regeneration.
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Células Endoteliales , Traumatismos de los Nervios Periféricos , Humanos , Estudios de Factibilidad , Fibrina/química , Fibrina/farmacología , Hidrogeles/química , Hidrogeles/farmacología , Macrófagos , Regeneración Nerviosa/fisiología , Nervio Ciático/lesionesRESUMEN
INTRODUCTION: Neuroenhancing therapies are desired because repair of nerve injuries can fail to achieve recovery. We compared two neuroenhancing therapies, electrical stimulation (ES) and systemic tacrolimus (FK506), for their capabilities to enhance regeneration in the context of a rat model. METHODS: Rats were randomized to four groups: ES 0.5 mA, ES 2.0 mA, FK506, and repair alone. All groups underwent tibial nerve transection and repair, and outcomes were assessed by using twice per week walking track analysis, cold allodynia response, relative muscle mass, and nerve histology. RESULTS: Electrical stimulation and FK506 groups demonstrated improved functional recovery and myelinated axon counts distal to the repair compared with repair alone. Electrical stimulation provided improvements in nerve regeneration that were not different from optimized FK506 systemic administration. DISCUSSION: Providing ES after nerve repair improved regeneration and recovery in rats, with minimal differences in therapeutic efficacy to FK506, further demonstrating its clinical potential to improve management of nerve injuries.
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Estimulación Eléctrica/métodos , Inmunosupresores/farmacología , Regeneración Nerviosa/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Tacrolimus/farmacología , Nervio Tibial/lesiones , Animales , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inervación , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Regeneración Nerviosa/fisiología , Procedimientos Neuroquirúrgicos , Traumatismos de los Nervios Periféricos , Ratas , Recuperación de la Función/fisiología , Nervio Tibial/patología , Nervio Tibial/cirugíaRESUMEN
INTRODUCTION: Acellular nerve allografts (ANAs) yield less consistent favorable outcomes compared with autografts for long gap reconstructions. We evaluated whether a hybrid ANA can improve 6-cm gap reconstruction. METHODS: Rat sciatic nerve was transected and repaired with either 6-cm hybrid or control ANAs. Hybrid ANAs were generated using a 1-cm cellular isograft between 2.5-cm ANAs, whereas control ANAs had no isograft. Outcomes were assessed by graft gene and marker expression (n = 4; at 4 weeks) and motor recovery and nerve histology (n = 10; at 20 weeks). RESULTS: Hybrid ANAs modified graft gene and marker expression and promoted modest axon regeneration across the 6-cm defect compared with control ANA (P < 0.05), but yielded no muscle recovery. Control ANAs had no appreciable axon regeneration across the 6-cm defect. DISCUSSION: A hybrid ANA confers minimal motor recovery benefits for regeneration across long gaps. Clinically, the authors will continue to reconstruct long nerve gaps with autografts. Muscle Nerve 57: 260-267, 2018.
Asunto(s)
Regeneración Nerviosa/fisiología , Neuronas/trasplante , Envejecimiento , Aloinjertos , Animales , Axones/fisiología , Biomarcadores/análisis , Expresión Génica , Marcadores Genéticos , Masculino , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/inervación , Ratas , Ratas Endogámicas Lew , Recuperación de la Función , Nervio Ciático/lesiones , Nervio Ciático/cirugía , Estrés FisiológicoRESUMEN
Providing temporally regulated glial cell line-derived neurotrophic factor (GDNF) to injured nerve can promote robust axon regeneration. However, it is poorly understood why providing highly elevated levels of GDNF to nerve can lead to axon entrapment in the zone containing elevated GDNF. This limited understanding represents an obstacle to the translation of GDNF therapies to treat nerve injuries clinically. Here, we investigated how transgenic Schwann cells (SCs) overexpressing GDNF-IRES-DsRed impact nerve regeneration. Cultured primary SCs were transduced with lentiviruses (GDNF-overexpressing transgenic SCs), one of which provides the capability to express high levels of GDNF and regulate temporal GDNF expression. These SC groups were transplanted into acellular nerve allografts (ANAs) bridging a 14 mm rat sciatic nerve defect. GDNF-overexpressing transgenic SCs expressing GDNF for as little as 1 week decreased axon regeneration across ANAs and caused extensive extracellular matrix (ECM) remodeling. To determine whether additional gene expression changes beyond GDNF transgene expression occurred in GDNF-overexpressing transgenic SCs, microarray analysis of GDNF-overexpressing transgenic SCs compared to untreated SCs was performed. Microarray analysis revealed a set of common genes regulated in transgenic SC groups expressing high levels of GDNF compared to untreated SCs. A co-culture model of GDNF-overexpressing transgenic SCs with fibroblasts (FBs) revealed differential FB ECM-related gene expression compared to untreated SCs. These data suggest a component of axon entrapment is independent of GDNF's impact on axons. Biotechnol. Bioeng. 2017;114: 2121-2130. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Proteínas Luminiscentes/metabolismo , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/fisiopatología , Traumatismos de los Nervios Periféricos/terapia , Nervio Ciático/lesiones , Nervio Ciático/trasplante , Aloinjertos , Animales , Sistema Libre de Células , Células Cultivadas , Regeneración Tisular Dirigida/métodos , Sitios Internos de Entrada al Ribosoma/fisiología , Masculino , Ratas , Ratas Endogámicas Lew , Células de Schwann/fisiología , Resultado del TratamientoRESUMEN
BACKGROUND: Although electrical stimulation (ES) can improve nerve regeneration, the impact of nerve block, such as lidocaine (Lido), on the therapeutic benefits of ES remains unclear. We used a rat tibial nerve transection-and-repair model to explore how either preoperative (PreOp) or postoperative (PostOp) nerve block affects ES-related improvement in regeneration. METHODS: Lewis rats were used in 1 of 2 studies. The first evaluated the effects of extraneural Lido on both healthy and injured nerves. In the second study, rats were randomized to 5 experimental groups: No ES (negative control), PreOp Lido, ES + PreOp Lido, PostOp + ES, and ES (positive control). All groups underwent tibial nerve transection and repair. In both studies, nerves were harvested for histological analysis of regeneration distal to the injury site. RESULTS: Application of extraneural Lido did not damage healthy or injured nerve based on qualitative histological observations. In the context of nerve transection and repair, the ES group exhibited improved axon regeneration at 21 days measured by the total number of myelinated fibers compared with No ES. Fiber density and percentage of neural tissue in the ES group were greater than those in both No ES and PreOp Lido + ES groups. ES + PostOp Lido was not different from No ES or ES group. CONCLUSIONS: Extraneural application of Lido did not damage nerves. Electrical stimulation augmented nerve regeneration, but Lido diminished the ES-related improvement in nerve regeneration. Clinical studies on the effects of ES to nerve regeneration may need to consider nerve block as a variable affecting ES outcome.
Asunto(s)
Terapia por Estimulación Eléctrica , Lidocaína , Animales , Ratas , Axones/fisiología , Lidocaína/farmacología , Regeneración Nerviosa/fisiologíaRESUMEN
The immune system has garnered attention for its role in peripheral nerve regeneration, particularly as it pertains to regeneration across segmental injuries. Previous work demonstrated that eosinophils are recruited to regenerating nerve and express interleukin-4, amongst potential cytokines. These results suggest a direct role for eosinophils in promoting nerve regeneration. Therefore, we further considered eosinophils roles in nerve regeneration using a segmental nerve injury and Gata1 knockout (KO) mice, which are severely eosinophil deficient, compared to wild-type BALB/c mice (WT). Mice receiving a sciatic nerve gap injury demonstrated distinct cytokine expression and leukocytes within regenerating nerve. Compared to controls, Gata1 KO regenerated nerves contained decreased expression of type 2 cytokines, including Il-5 and Il-13, and decreased recruitment of eosinophils and macrophages. At this early time point during ongoing regeneration, the macrophages within Gata1 KO nerves also demonstrated significantly less M2 polarization compared to controls. Subsequently, motor and sensory axon regeneration across the gap injury was decreased in Gata1 KO compared to WT during ongoing nerve regeneration. Over longer observation to allow for more complete nerve regeneration, behavioral recovery measured by grid-walk assessment was not different comparing groups but modestly delayed in Gata1 KO compared to WT. The extent of final axon regeneration was not different amongst groups. Our data provide additional evidence suggesting eosinophils contribute to nerve regeneration across a nerve gap injury, but are not essential to regeneration in this context. Our evidence also suggests eosinophils may regulate cytokines that promote distinct macrophage phenotypes and axon regeneration.
Asunto(s)
Traumatismos de los Nervios Periféricos , Neuropatía Ciática , Ratones , Animales , Citocinas/metabolismo , Eosinófilos/metabolismo , Nervios Periféricos/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/metabolismo , Macrófagos/metabolismo , Ratones Noqueados , Neuropatía Ciática/metabolismo , Axones/fisiología , Nervio Ciático/lesionesRESUMEN
Interleukin-4 (IL-4) has garnered interest as a cytokine that mediates regeneration across multiple tissues including peripheral nerve. Within nerve, we previously showed endogenous IL-4 was critical to regeneration across nerve gaps. Here, we determined a generalizable role of IL-4 in nerve injury and regeneration. In wild-type (WT) mice receiving a sciatic nerve crush, IL-4 expressing cells preferentially accumulated within the injured nerve compared to affected sites proximal, such as dorsal root ganglia (DRGs), or distal muscle. Immunohistochemistry and flow cytometry confirmed that eosinophils (CD45+, CD11b+, CD64-, Siglec-F+) were sources of IL-4 expression. Examination of targets for IL-4 within nerve revealed macrophages, as well as subsets of neurons expressed IL-4R, while Schwann cells expressed limited IL-4R. Dorsal root ganglia cultures were exposed to IL-4 and demonstrated an increased proportion of neurons that extended axons compared to cultures without IL-4 (control), as well as longer myelinated axons compared to cultures without IL-4. The role of endogenous IL-4 during nerve injury and regeneration in vivo was assessed following a sciatic nerve crush using IL-4 knockout (KO) mice. Loss of IL-4 affected macrophage accumulation within injured nerve compared to WT mice, as well as shifted macrophage phenotype towards a CD206- phenotype with altered gene expression. Furthermore, this loss of IL-4 delayed initial axon regeneration from the injury crush site and subsequently delayed functional recovery and re-innervation of neuromuscular junctions compared to wild-type mice. Given the role of endogenous IL-4 in nerve regeneration, exogenous IL-4 was administered daily to WT mice following a nerve crush to examine regeneration. Daily IL-4 administration increased early axonal extension and CD206+ macrophage accumulation but did not alter functional recovery compared to untreated mice. Our data demonstrate IL-4 promotes nerve regeneration and recovery after injury.
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Interleucina-4/administración & dosificación , Interleucina-4/biosíntesis , Regeneración Nerviosa/fisiología , Neuropatía Ciática/metabolismo , Animales , Células Cultivadas , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/metabolismo , Ganglios Espinales/inmunología , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica , Inyecciones Intraperitoneales , Interleucina-4/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Compresión Nerviosa/tendencias , Regeneración Nerviosa/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Interleucina-4/biosíntesis , Receptores de Interleucina-4/inmunología , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/inmunologíaRESUMEN
BACKGROUND: Repair of nerve injuries can fail to achieve adequate functional recovery. Electrical stimulation applied at the time of nerve repair can accelerate axon regeneration, which may improve the likelihood of recovery. However, widespread use of electrical stimulation may be limited by treatment protocols that increase operative time and complexity. This study evaluated whether a short-duration electrical stimulation protocol (10 minutes) was efficacious to enhance regeneration following nerve repair using rat models. METHODS: Lewis and Thy1-green fluorescent protein rats were randomized to three groups: 0 minutes of electrical stimulation (no electrical stimulation; control), 10 minutes of electrical stimulation, and 60 minutes of electrical stimulation. All groups underwent tibial nerve transection and repair. In the intervention groups, electrical stimulation was delivered after nerve repair. Outcomes were assessed using immunohistochemistry, histology, and serial walking track analysis. RESULTS: Two weeks after nerve repair, Thy1-green fluorescent protein rats demonstrated increased green fluorescent protein-positive axon outgrowth from the repair site with electrical stimulation compared to no electrical stimulation. Serial measurement of walking tracks after nerve repair revealed recovery was achieved more rapidly in both electrical stimulation groups as compared to no electrical stimulation. Histologic analysis of nerve distal to the repair at 8 weeks revealed robust axon regeneration in all groups. CONCLUSIONS: As little as 10 minutes of intraoperative electrical stimulation therapy increased early axon regeneration and facilitated functional recovery following nerve transection with repair. Also, as early axon outgrowth increased following electrical stimulation with nerve repair, these findings suggest electrical stimulation facilitated recovery because of earlier axon growth across the suture-repaired site into the distal nerve to reach end-organ targets. CLINICAL RELEVANCE STATEMENT: Brief (10-minute) electrical stimulation therapy can provide similar benefits to the 60-minute protocol in an acute sciatic nerve transection/repair rat model and merit further studies, as they represent a translational advantage.
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Axones , Terapia por Estimulación Eléctrica , Animales , Humanos , Ratas , Axones/fisiología , Estimulación Eléctrica/métodos , Regeneración Nerviosa/fisiología , Ratas Endogámicas Lew , Recuperación de la Función/fisiología , Nervio Tibial/lesionesRESUMEN
Background: Therapeutic electrical stimulation (ES) applied to repaired nerve is a promising treatment option to improve regeneration. However, few studies address the impact of ES following nerve graft reconstruction. The purpose of this study was to determine if ES applied to a nerve repair using nerve isograft in a rodent model could improve nerve regeneration and functional recovery. Methods: Adult rats were randomized to 2 groups: "ES" and "Control." Rats received a tibial nerve transection that was repaired using a tibial nerve isograft (1.0 cm length), where ES was applied immediately after repair in the applicable group. Nerve was harvested 2 weeks postrepair for immunohistochemical analysis of axon growth and macrophage accumulation. Independently, rats were assessed using walking track and grid-walk analysis for up to 21 weeks. Results: At 2 weeks, more robust axon regeneration and greater macrophage accumulation was observed within the isografts for the ES compared to Control groups. Both walking track and grid-walk analysis revealed that return of functional recovery was accelerated by ES. The ES group demonstrated improved functional recovery over time, as well as improved recovery compared to the Control group at 21 weeks. Conclusions: ES improved early axon regeneration into a nerve isograft and was associated with increased macrophage and beneficial M2 macrophage accumulation within the isograft. ES ultimately improved functional recovery compared to isograft repair alone. This study supports the clinical potential of ES to improve the management of nerve injuries requiring a nerve graft repair.
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Axones , Regeneración Nerviosa , Animales , Axones/fisiología , Estimulación Eléctrica , Humanos , Isoinjertos , Regeneración Nerviosa/fisiología , Ratas , Recuperación de la Función/fisiologíaRESUMEN
Following traumatic peripheral nerve injury, adequate restoration of function remains an elusive clinical goal. Recent research highlights the complex role that the immune system plays in both nerve injury and regeneration. Pro-regenerative processes in wounded soft tissues appear to be significantly mediated by cytokines of the type 2 immune response, notably interleukin (IL)-4. While IL-4 signaling has been firmly established as a critical element in general tissue regeneration during wound healing, it has also emerged as a critical process in nerve injury and regeneration. In this context of peripheral nerve injury, endogenous IL-4 signaling has recently been confirmed to influence more than leukocytes, but including also neurons, axons, and Schwann cells. Given the role IL-4 plays in nerve injury and regeneration, exogenous IL-4 and/or compounds targeting this signaling pathway have shown encouraging preliminary results to treat nerve injury or other neuropathy in rodent models. In particular, the exogenous stimulation of the IL-4 signaling pathway appears to promote postinjury neuron survival, axonal regeneration, remyelination, and thereby improved functional recovery. These preclinical data strongly suggest that targeting IL-4 signaling pathways is a promising translational therapy to augment treatment approaches of traumatic nerve injury. However, a better understanding of the type 2 immune response and associated signaling networks functioning within the nerve injury microenvironment is still needed to fully develop this promising therapeutic avenue.
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Inflamación , Interleucina-4 , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos , Transducción de Señal/fisiología , Animales , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Regeneración Nerviosa/efectos de los fármacos , Traumatismos de los Nervios Periféricos/tratamiento farmacológico , Traumatismos de los Nervios Periféricos/inmunología , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/fisiopatología , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Understanding the role of macrophages at discrete spatial locations during nerve regeneration after injury is important. But, methodologies that systemically manipulate macrophages can obscure their roles within discrete spatial locations within nerve. NEW METHOD: Liposomes were embedded within fibrin gels to construct a delivery system that facilitated macrophage-specific manipulations at a sole spatial region, as macrophages accumulated within the fibrin. Clodronate liposomes were characterized for their toxicity to specific cells composing nerve in vitro, then tested for macrophage-specific depletion in vivo. This delivery system using clodronate liposomes was used to repair a mouse sciatic nerve gap to evaluate its efficacy and effects. RESULT: Clodronate liposomes showed specific toxicity to macrophages without affecting dorsal root ganglia (DRG)-derived neurons, endothelial cells, or Schwann cells in culture. The delivery system demonstrated sustained release of liposomes for more than 7 days while still retaining liposomes within the fibrin. In vivo, the delivery system demonstrated macrophages were targeted by liposomes, and the use of clodronate liposomes minimized macrophage accumulation within fibrin, while not affecting macrophage accumulation within DRG. Nerve regeneration across the nerve gap repaired using this delivery system was associated with decreased angiogenesis, Schwann cell accumulation, axon growth, and reinnervation of affected muscle. COMPARISON WITH EXISTING METHODS: This delivery system allowed specific perturbation of macrophages locally in nerve. This method could be applicable across species without the need for genetic manipulations or systemic pharmaceuticals. CONCLUSION: Liposomes embedded within fibrin gels locally target macrophages at the site of nerve injury, which enables greater precision in conclusions regarding their roles in nerve.
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Fibrina , Liposomas , Animales , Células Endoteliales , Geles , Macrófagos , RatonesRESUMEN
BACKGROUND: Clinical outcomes following nerve injury repair can be inadequate. Pulsed-current electrical stimulation (ES) is a therapeutic method that facilitates functional recovery by accelerating axon regeneration. However, current clinical ES protocols involve the application of ES for 60 minutes during surgery, which can increase operative complexity and time. Shorter ES protocols could be a strategy to facilitate broader clinical adoption. The purpose of the present study was to determine if a 10-minute ES protocol could improve outcomes. METHODS: C57BL/6J mice were randomized to 3 groups: no ES, 10 minutes of ES, and 60 minutes of ES. In all groups, the sciatic nerve was transected and repaired, and, in the latter 2 groups, ES was applied after repair. Postoperatively, changes to gene expression from dorsal root ganglia were measured after 24 hours. The number of motoneurons regenerating axons was determined by retrograde labeling at 7 days. Histomorphological analyses of the nerve were performed at 14 days. Function was evaluated serially with use of behavioral tests up to 56 days postoperatively, and relative muscle weight was evaluated. RESULTS: Compared with the no-ES group, both ES groups demonstrated increased regeneration-associated gene expression within dorsal root ganglia. The 10-minute and 60-minute ES groups demonstrated accelerated axon regeneration compared with the no-ES group based on increased numbers of labeled motoneurons regenerating axons (mean difference, 202.0 [95% confidence interval (CI), 17.5 to 386.5] and 219.4 [95% CI, 34.9 to 403.9], respectively) and myelinated axon counts (mean difference, 559.3 [95% CI, 241.1 to 877.5] and 339.4 [95% CI, 21.2 to 657.6], respectively). The 10-minute and 60-minute ES groups had improved behavioral recovery, including on grid-walking analysis, compared with the no-ES group (mean difference, 11.9% [95% CI, 3.8% to 20.0%] and 10.9% [95% CI, 2.9% to 19.0%], respectively). There was no difference between the ES groups in measured outcomes. CONCLUSIONS: A 10-minute ES protocol accelerated axon regeneration and facilitated functional recovery. CLINICAL RELEVANCE: The brief (10-minute) ES protocol provided similar benefits to the 60-minute protocol in an acute sciatic nerve transection/repair mice model and merits further studies.
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Axones/fisiología , Estimulación Eléctrica/métodos , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/terapia , Nervio Ciático/fisiopatología , Animales , Masculino , Ratones , Traumatismos de los Nervios Periféricos/fisiopatología , Recuperación de la Función/fisiología , Nervio Ciático/lesionesRESUMEN
Decellularized nerve, or acellular nerve allografts (ANAs), are an increasingly used alternative to nerve autografts to repair nerve gaps to facilitate regeneration. The adaptive immune system, specifically T cells, plays a role in promoting regeneration upon these ANA scaffolds. However, how T cells promote regeneration across ANAs is not clear. Here, we show that T cells accumulate within ANAs repairing nerve gaps resulting in regulation of cytokine expression within the ANA environment. This in turn ultimately leads to robust nerve regeneration and functional recovery. Nerve regeneration across ANAs and functional recovery in Rag1KO mice was limited compared to wild-type (WT) mice. Prior to appreciable nerve regeneration, ANAs from Rag1KO mice contained fewer eosinophils and reduced IL-4 expression compared to ANAs from WT mice. During this period, both T cells and eosinophils regulated IL-4 expression within ANAs. Eosinophils represented the majority of IL-4 expressing cells within ANAs, while T cells regulated IL-4 expression. Finally, an essential role for IL-4 during nerve regeneration across ANAs was confirmed as nerves repaired using ANAs had reduced regeneration in IL-4 KO mice compared to WT mice. Our data demonstrate T cells regulate the expression of IL-4 within the ANA environment via their effects on eosinophils. STATEMENT OF SIGNIFICANCE: The immune system has been emerging as a critical component for tissue regeneration, especially when regeneration is supported upon biomaterials. The role of T cells, and their roles in the regeneration of nerve repaired with biomaterials, is still unclear. We demonstrated that when nerves are repaired with decellularized nerve scaffolds, T cells contribute to regeneration by regulating cytokines. We focused on their regulation of cytokine IL-4. Unexpectedly, T cells do not produce IL-4, but instead regulate IL-4 by recruiting eosinophils, which are major cellular sources of IL-4 within these scaffolds. Thus, our work demonstrated how IL-4 is regulated in a model biomaterial, and has implications for improving the design of biomaterials and understanding immune responses to biomaterials.
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Eosinófilos , Interleucina-4 , Animales , Ratones , Regeneración Nerviosa , Linfocitos T , Trasplante HomólogoRESUMEN
Repair of traumatic nerve injuries can require graft material to bridge the defect. The use of alternatives to bridge the defect, such as acellular nerve allografts (ANAs), is becoming more common and desired. Although ANAs support axon regeneration across short defects (<3â¯cm), axon regeneration across longer defects (>3â¯cm) is limited. It is unclear why alternatives, including ANAs, are functionally limited by length. After repairing Lewis rat nerve defects using short (2â¯cm) or long (4â¯cm) ANAs, we showed that long ANAs have severely reduced axon regeneration across the grafts and contain Schwann cells with a unique phenotype. But additionally, we found that long ANAs have disrupted angiogenesis and altered leukocyte infiltration compared to short ANAs as early as 2â¯weeks after repair. In particular, long ANAs contained fewer T cells compared to short ANAs. These outcomes were accompanied with reduced expression of select cytokines, including IFN-γ and IL-4, within long versus short ANAs. T cells within ANAs did not express elevated levels of IL-4, but expressed elevated levels of IFN-γ. We also directly assessed the contribution of T cells to regeneration across nerve grafts using athymic rats. Interestingly, T cell deficiency had minimal impact on axon regeneration across nerve defects repaired using isografts. Conversely, T cell deficiency reduced axon regeneration across nerve defects repaired using ANAs. Our data demonstrate that T cells contribute to nerve regeneration across ANAs and suggest that reduced T cells accumulation within long ANAs could contribute to limiting axon regeneration across these long ANAs.
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Regeneración Tisular Dirigida/métodos , Regeneración Nerviosa/fisiología , Nervio Ciático/lesiones , Nervio Ciático/trasplante , Linfocitos T/inmunología , Aloinjertos , Animales , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Andamios del TejidoRESUMEN
BACKGROUND: Nerve grafting with an autograft is considered the gold standard. However, the functional outcomes of long (>3 cm) nerve autografting are often poor. The authors hypothesized that a factor contributing to these outcomes is the graft microenvironment, where long compared to short autografts support axon regeneration to different extents. METHODS: A rat sciatic nerve defect model was used to compare regeneration in short (2 cm) and long (6 cm) isografts. Axon regeneration and cell populations within grafts were assessed using histology, retrograde labeling of neurons regenerating axons, immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction, and electron microscopy at 4 and/or 8 weeks. RESULTS: At 8 weeks, for distances of both 1 and 2 cm from the proximal coaptation (equivalent regenerative distance), long isografts had reduced numbers of regenerated fibers compared with short isografts. Similarly, the number of motoneurons regenerating axons was reduced in the presence of long isografts compared with short isografts. Considering the regenerative microenvironments between short and long isografts, cell densities and general populations within both short and long isografts were similar. However, long isografts had significantly greater expression of senescence markers, which included senescence-associated ß-galactosidase, p21, and p16, and distinct chromatin changes within Schwann cells. CONCLUSIONS: This study shows that axon regeneration is reduced in long compared with short isografts, where long isografts contained an environment with an increased accumulation of senescent markers. Although autografts are considered the gold standard for grafting, these results demonstrate that we must continue to strive for improvements in the autograft regenerative environment.
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Regeneración Nerviosa/fisiología , Nervio Ciático/fisiología , Animales , Autoinjertos , Senescencia Celular/fisiología , Masculino , Distribución Aleatoria , Ratas Endogámicas Lew , Nervio Ciático/cirugía , Trasplante Autólogo/métodosRESUMEN
BACKGROUND: The goal of this study was to develop a partial, nonregenerative nerve injury model in a rat that results in permanently reduced motoneuron numbers and function. This model could serve as a platform for the study of therapeutics, such as a reverse end-to-side nerve transfer (i.e., supercharge). The authors hypothesized that transection of one or more of the L4 to L6 nerve roots supplying the sciatic nerve would cause a permanent reduction in muscle force. METHODS: Rats were randomized into five groups that underwent variations of nerve root transections or sham injury. The L4 to L6 nerve roots were selectively transected and capped to prevent regeneration. Tibial and common peroneal nerves were harvested for quantitative histology and retrograde-labeled to assess the number of motoneurons projecting axons. Muscle force and relative muscle mass were assessed as metrics of postinjury motor function. RESULTS: At 6 months, the number of motoneurons projecting axons and myelinated axon counts were reduced in both the tibial and common peroneal nerves after injury in all groups. Transecting both L4 and L5 or both L4 and L6 reduced motoneuron numbers sufficiently below sham numbers to reduce muscle force and mass in major muscles of the hindlimb innervated by both nerves. Transecting L4 reduced muscle force and mass in common peroneal-innervated muscles, whereas transecting L5 reduced muscle force and mass in tibial-innervated muscles. These findings were stable over time. CONCLUSION: Transection of nerve roots produces stable (time-independent) partial nerve injury models with a selective decrease in motor function.
Asunto(s)
Modelos Animales , Traumatismos de los Nervios Periféricos/fisiopatología , Ratas Endogámicas Lew/cirugía , Rizotomía , Raíces Nerviosas Espinales/lesiones , Animales , Axones/patología , Masculino , Neuronas Motoras/patología , Fuerza Muscular , Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , Traumatismos de los Nervios Periféricos/patología , Nervio Peroneo/patología , Distribución Aleatoria , Ratas , Ratas Endogámicas Lew/fisiología , Nervio Ciático , Raíces Nerviosas Espinales/cirugía , Nervio Tibial/patologíaRESUMEN
Acellular nerve allografts (ANAs) and other nerve constructs do not reliably facilitate axonal regeneration across long defects (>3 cm). Causes for this deficiency are poorly understood. In this study, we determined what cells are present within ANAs before axonal growth arrest in nerve constructs and if these cells express markers of cellular stress and senescence. Using the Thy1-GFP rat and serial imaging, we identified the time and location of axonal growth arrest in long (6 cm) ANAs. Axonal growth halted within long ANAs by 4 weeks, while axons successfully regenerated across short (3 cm) ANAs. Cellular populations and markers of senescence were determined using immunohistochemistry, histology, and senescence-associated ß-galactosidase staining. Both short and long ANAs were robustly repopulated with Schwann cells (SCs) and stromal cells by 2 weeks. Schwann cells (S100ß(+)) represented the majority of cells repopulating both ANAs. Overall, both ANAs demonstrated similar cellular populations with the exception of increased stromal cells (fibronectin(+)/S100ß(-)/CD68(-) cells) in long ANAs. Characterization of ANAs for markers of cellular senescence revealed that long ANAs accumulated much greater levels of senescence markers and a greater percentage of Schwann cells expressing the senescence marker p16 compared to short ANAs. To establish the impact of the long ANA environment on axonal regeneration, short ANAs (2 cm) that would normally support axonal regeneration were generated from long ANAs near the time of axonal growth arrest ("stressed" ANAs). These stressed ANAs contained mainly S100ß(+)/p16(+) cells and markedly reduced axonal regeneration. In additional experiments, removal of the distal portion (4 cm) of long ANAs near the time of axonal growth arrest and replacement with long isografts (4 cm) rescued axonal regeneration across the defect. Neuronal culture derived from nerve following axonal growth arrest in long ANAs revealed no deficits in axonal extension. Overall, this evidence demonstrates that long ANAs are repopulated with increased p16(+) Schwann cells and stromal cells compared to short ANAs, suggesting a role for these cells in poor axonal regeneration across nerve constructs.