RESUMO
BACKGROUND: Acellular nerve allografts (ANAs) were developed to replace the autologous nerve grafts (ANGs) to fill the peripheral nerve defects. Poor vascularization relative to ANGs has been a limitation of application of ANAs. METHODS: A total of 60 female Sprague-Dawley rats were assigned 3 groups. The rats in A group received ANGs, the rats in B group received ANAs, and the rats in C group were transplanted with ANA carrying endothelial cells (ANA + ECs). In the 1st, 2nd, 4th, and 12th postoperative weeks, 5 rats were selected from each group for evaluating sciatic function index (SFI), electrophysiology, maximum tetanic force recovery rate, tibialis anterior muscle weights recovery rate, and microvessel density. In the 12th postoperative week, the nerves were harvested and stained with toluidine blue and observed under an electron microscope to compare nerve fibers, myelin width, and G-ratio. RESULTS: All the rats survived. In the first and second postoperative weeks, more microvessels were found in the ANA + EC group. In the 12th postoperative week, the nerve fibers were more numerous, and G-ratio was smaller in the C group compared with the B group. The compound muscle action potential and maximum tetanic force recovery rate in the tibialis anterior muscle in the C group were better than those in the B group in the 12th postoperative week. The A group showed better performances in electrophysiology, maximum tetanic force, muscle wet weight, and nerve regeneration. CONCLUSION: ANA + ECs can promote early angiogenesis, promoting nerve regeneration and neurological function recovery.
Assuntos
Aloenxertos , Células Endoteliais , Regeneração Nervosa , Ratos Sprague-Dawley , Nervo Isquiático , Animais , Feminino , Ratos , Nervo Isquiático/cirurgia , Nervo Isquiático/lesões , Nervo Isquiático/transplante , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/cirurgia , Recuperação de Função Fisiológica , Distribuição AleatóriaRESUMO
BACKGROUND: Autologous nerve grafting, the criterion standard for bridging peripheral nerves, can cause complications at the donor site. We investigated a novel approach to reconstruct the nerve gap with a split cross-sectional unmatched semifascicle autograft, which was harvested from the distal part of the injured nerve. METHODS: A patient diagnosed with left-sided frontal branch facial nerve dissection underwent nerve bridging emergency surgery using a semifascicle nerve graft. A sciatic nerve model was used to validate the feasibility and mechanism of this method. Male Sprague-Dawley rats (n = 36) were randomized into (A) intact fascicle, (B) semifascicle, and (C) semifascicle + conduit groups and further subdivided into 4- and 8-week groups for histological analysis of the neurotissue area, fibers, and Schwann cells. The 8-week groups underwent weekly pain and temperature tests; the wet weight of the gastrocnemius muscle was measured after euthanasia. RESULTS: The frontalis of the patient's injured side exhibited movement at 2 months postsurgery and recovered a symmetrical appearance at 13 months. Group A exhibited more neurotissue areas and fibers than groups B and C at week 4; group B had more neurotissue than group C. Group A had greater neurotissue areas than groups B and C at week 8; groups B and C exhibited no differences. The groups displayed no differences regarding nerve fiber, pain, and temperature analysis at week 8. Muscle wet weight of groups A and B exhibited no differences and was higher than that of group C. CONCLUSION: We demonstrated the clinical translational value of semifascicle nerve grafts; the injured site was both the donor and recipient, thereby avoiding donor site damage and associated complications.
Assuntos
Dor , Nervo Isquiático , Ratos , Animais , Masculino , Humanos , Ratos Sprague-Dawley , Estudos Transversais , Nervo Isquiático/transplante , Autoenxertos , Regeneração Nervosa/fisiologiaRESUMO
In this study, a biological conduit, consisting of an adipocyte-derived mesenchymal stem cell (AdMSCs) sheet and amniotic membrane (AM), was designed for the reconstruction of peripheral nerve defects. To evaluate the effect of the produced conduit on neural regeneration, a 10-mm sciatic nerve defect was created in rats, and experiments were carried out on six groups, i.e., sham control group (SC), negative control group (NC), nerve autograft group (NG), the biological conduit (AdMSCs + AM) group, the commercial PGA tube conduit (PGA) group, and the conduit only consisting of AM (AM) group. The effects of different nerve repair methods on the peripheral nerve and gastrocnemius muscle were evaluated by functional, histological, and immunohistochemical tests. When the number of myelinated axons was compared between the groups of AdMSCs + AM and PGA, it was higher in the AdMSCs + AM group (p < 0.05). The percentage of gastrocnemius collagen bundle area of AdMSCs + AM group was found to be statistically lower than the PGA group (p < 0.05). The muscle fiber diameter of AdMSCs + AM group was lower than that of the NG group, but significantly higher than that of the PGA group and the AM group (p < 0.001). Muscle weight index was significantly higher in the AdMSCs + AM group compared to the PGA group (p < 0.05). It was observed that nerve regeneration was faster in the AdMSCs + AM group, and there was an earlier improvement in pin-prick score and sciatic functional index compared to the PGA group and the AM group. In conclusion, the biological conduit prepared from the AdMSCs sheet and AM is regarded as a new biological conduit that can be used as an alternative treatment method to nerve autograft in clinical applications.
Assuntos
Células-Tronco Mesenquimais , Tecido Nervoso , Humanos , Ratos , Animais , Âmnio , Nervo Isquiático/cirurgia , Nervo Isquiático/transplante , Modelos Animais de Doenças , Regeneração Nervosa/fisiologiaRESUMO
PURPOSE: Various postoperative protocols have been proposed to improve outcomes and accelerate nerve regeneration. Recently, the use of physical exercise in a post-surgical neurorraphy procedure has shown good results when started early. We experimentally investigated the hypothesis that post-operative exercise speeds up results and improves clinical and morphologic parameters. METHODS: Isogenic rats were randomly divided into four groups: 1 SHAM; 2 SHAM submitted to the exercise protocol (EP); 3 Grafting of the sciatic nerve; and 4 Grafting of the sciatic nerve associated with the EP. The EP was based on aerobic activities with a treadmill, with a progressive increase in time and intensity during 6 weeks. The results were evaluated by the sciatic functional index (SFI), morphometric and morphologic analysis of nerve distal to the lesion, and the number of spinal cord motor neurons, positive to the marker Fluoro-Gold (FG), captured retrogradely through neurorraphy. RESULTS: Functional analysis (SFI) did not show a statistical difference between the group grafted with (-50.94) and without exercise (-65.79) after 90 days. The motoneurons count (Spinal cord histology) also showed no diference between these groups (834.5 × 833 respectively). Although functionally there is no difference between these groups, morphometric study showed a greater density (53.62) and larger fibers (7.762) in GRAFT group. When comparing both operated groups with both SHAM groups, all values were much lower. CONCLUSIONS: The experimental model that this aerobic treadmill exercises protocol did not modify nerve regeneration after sciatic nerve injury and repair with nerve graft.
Assuntos
Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Condicionamento Físico Animal , Neuropatia Ciática , Animais , Ratos , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/cirurgia , Traumatismos dos Nervos Periféricos/terapia , Ratos Endogâmicos SHR , Nervo Isquiático/lesões , Nervo Isquiático/transplante , Neuropatia Ciática/cirurgia , Neuropatia Ciática/terapiaRESUMO
BACKGROUND: Increasing evidence demonstrates an interplay between neoangiogenesis and immune cells. We investigated the immune response and revascularization of acellular nerve allografts (ANA) after combined stem cell delivery and surgical angiogenesis in a rat model. METHODS: Unilateral sciatic nerve defects in 60 Lewis rats were repaired with (I) autografts, (II) ANAs, and (III) ANAs wrapped within a pedicled superficial inferior epigastric artery fascial flap to induce surgical angiogenesis, combined with seeding of either (IV) undifferentiated mesenchymal stem cells (uMSCs) or (V) MSCs differentiated into Schwann cell-like cells. Immune cell phenotyping was performed on days 7 and 14. The vascular volume of nerves was measured by microcomputed tomography at 12 and 16 weeks. RESULTS: On day 7, helper T cells (CD4+) were significantly increased in groups IV and V compared to group I. Regulatory T cells (CD4+CD25+) were significantly higher in groups III-IV, and cytotoxic T cells (CD8+) were significantly reduced in groups IV and V compared to group II, respectively. Group II demonstrated the highest levels of natural killer cells (CD161+) compared to groups III-V. On day 14, group IV demonstrated the highest CD4/CD8 ratio. Vascular volume was significantly higher in groups III-V compared to group II at 12 weeks and groups IV and V compared to group II at 16 weeks. The CD4/CD8 ratio demonstrated a positive correlation to vascular volumes at 12 weeks. CONCLUSION: Early favorable immune responses were observed in ANAs treated with surgical angiogenesis with or without stem cell delivery and demonstrated improved vascularity at longer follow-up.
Assuntos
Células-Tronco Mesenquimais , Regeneração Nervosa , Aloenxertos , Animais , Imunidade , Células-Tronco Mesenquimais/fisiologia , Regeneração Nervosa/fisiologia , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático/transplante , Microtomografia por Raio-XRESUMO
Decellularized nerve allograft is an alternative to autologous nerve graft for nerve defects but has shown inferior clinical outcomes. Mesenchymal stem cells can play a key role in improving nerve regeneration of decellularized nerve allografts. The purpose of this study was to compare different mesenchymal stem cell seeding methods and to find the most efficient way to attach cells to nerve grafts for peripheral nerve regeneration. Wharton's jelly mesenchymal stem cells were collected from human umbilical cords and were seeded in the acellular nerve graft in five different ways as follows: PBS injection, fibrin glue drop, Matrigel drop, bioreactor, and Matrigel injection. A 6-mm sciatic nerve defect of Sprague-Dawley rats was bridged using mesenchymal stem cells-laden acellular nerve grafts according to the five seeding methods. Two days after implantation, the nerve tissue was biopsied and analyzed by the immunofluorescence staining of nuclei. The number of Wharton's jelly mesenchymal stem cells (+ h Nuclei) was counted in the inside, outside, and the total area of the graft sections under 200X magnification. The highest efficiency of mesenchymal stem cell attachment inside the graft and the highest total number of attached mesenchymal stem cells was observed in the group using Matrigel injection (p < 0.0001). This study showed mesenchymal stem cells can be more effectively attached to decellularized nerve graft using the injection method with Matrigel than other static or dynamic seeding methods in vivo.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Regeneração Nervosa/fisiologia , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/transplanteRESUMO
Various therapeutic methods have been suggested to enhance nerve regeneration. In this study, we propose a novel approach for enhancement of nerve gap regeneration by applying human epineural conduit (hEC) supported with human mesenchymal stem cells (hMSC), as an alternative to autograft repair. Restoration of 20 mm sciatic nerve defect with hEC created from human sciatic nerve supported with hMSC was tested in 4 experimental groups (n = 6 each) in the athymic nude rat model (Crl:NIH-Foxn1rnu): 1 - No repair control, 2 - Autograft control, 3 - Matched diameter hEC filled with 1 mL saline, 4 - Matched diameter hEC supported with 3 × 106 hMSC. Assessments included: functional tests: toe-spread and pinprick, regeneration assessment by immunofluorescence staining: HLA-1, HLA-DR, NGF, GFAP, Laminin B, S-100, VEGF, vWF and PKH26 labeling; histomorphometric analysis of myelin thickness, axonal density, fiber diameter and myelinated nerve fibers percentage; Gastrocnemius Muscle Index (GMI) and muscle fiber area ratio. Best sensory and motor function recovery, as well as GMI and muscle fiber area ratio, were observed in the autograft group, and were comparable to the hEC with hMSC group (p = 0.038). Significant improvements of myelin thickness (p = 0.003), fiber diameter (p = 0.0296), and percentage of myelinated fibers (p < 0.0001) were detected in hEC group supported with hMSC compared to hEC with saline controls. At 12-weeks after nerve gap repair, hEC combined with hMSC revealed increased expression of neurotrophic and proangiogenic factors, which corresponded with improvement of function comparable with the autograft control. Application of our novel hEC supported with hMSC provides a potential alternative to the autograft nerve repair.
Assuntos
Células-Tronco Mesenquimais , Regeneração Nervosa , Animais , Humanos , Músculo Esquelético , Regeneração Nervosa/fisiologia , Ratos , Nervo Isquiático/transplante , Transplante AutólogoRESUMO
BACKGROUND: Mesenchymal stem cells have the potential to produce neurotrophic growth factors and establish a supportive microenvironment for neural regeneration. The purpose of this study was to determine the effect of undifferentiated and differentiated mesenchymal stem cells dynamically seeded onto decellularized nerve allografts on functional outcomes when used in peripheral nerve repair. METHODS: In 80 Lewis rats, a 10-mm sciatic nerve defect was reconstructed with (1) autograft, (2) decellularized allograft, (3) decellularized allograft seeded with undifferentiated mesenchymal stem cells, or (4) decellularized allograft seeded with mesenchymal stem cells differentiated into Schwann cell-like cells. Nerve regeneration was evaluated over time by cross-sectional tibial muscle ultrasound measurements, and at 12 and 16 weeks by isometric tetanic force measurements, compound muscle action potentials, muscle mass, histology, and immunofluorescence analyses. RESULTS: At 12 weeks, undifferentiated mesenchymal stem cells significantly improved isometric tetanic force measurement and compound muscle action potential outcomes compared to decellularized allograft alone, whereas differentiated mesenchymal stem cells significantly improved compound muscle action potential outcomes. The autografts outperformed both stem cell groups histologically at 12 weeks. At 16 weeks, functional outcomes normalized between groups. At both time points, the effect of undifferentiated versus differentiated mesenchymal stem cells was not significantly different. CONCLUSIONS: Undifferentiated and differentiated mesenchymal stem cells significantly improved functional outcomes of decellularized allografts at 12 weeks and were similar to autograft results in the majority of measurements. At 16 weeks, outcomes normalized as expected. Although differences between both cell types were not statistically significant, undifferentiated mesenchymal stem cells improved functional outcomes of decellularized nerve allografts to a greater extent and had practical benefits for clinical translation by limiting preparation time and costs.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Transferência de Nervo/métodos , Células de Schwann/transplante , Nervo Isquiático/transplante , Neuropatia Ciática/cirurgia , Aloenxertos/fisiologia , Aloenxertos/transplante , Animais , Autoenxertos/fisiologia , Autoenxertos/transplante , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Regeneração Nervosa , Ratos , Células de Schwann/fisiologia , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Transplante Autólogo/métodos , Transplante Homólogo/métodos , Resultado do TratamentoRESUMO
BACKGROUND: The nerve autograft remains the gold standard when reconstructing peripheral nerve defects. However, although autograft repair can result in useful functional recovery, poor outcomes are common, and better treatments are needed. The purpose of this study was to evaluate the effect of purified exosome product on functional motor recovery and nerve-related gene expression in a rat sciatic nerve reverse autograft model. METHODS: Ninety-six Sprague-Dawley rats were divided into three experimental groups. In each group, a unilateral 10-mm sciatic nerve defect was created. The excised nerve was reversed and used to reconstruct the defect. Group I animals received the reversed autograft alone, group II animals received the reversed autograft with fibrin glue, and group III animals received the reversed autograft with purified exosome product suspended in the fibrin glue. The animals were killed at 3 and 7 days and 12 and 16 weeks after surgery. Evaluation included compound muscle action potentials, isometric tetanic force, tibialis anterior muscle wet weight, nerve regeneration-related gene expression, and nerve histomorphometry. RESULTS: At 16 weeks, isometric tetanic force was significantly better in group III (p = 0.03). The average axon diameter of the peroneal nerve was significantly larger in group III at both 12 and 16 weeks (p = 0.015 at 12 weeks; p < 0.01 at 16 weeks). GAP43 and S100b gene expression was significantly up-regulated by purified exosome product. CONCLUSIONS: Local administration of purified exosome product demonstrated improved nerve regeneration profiles in the reverse sciatic nerve autograft rat model. Thus, purified exosome product may have beneficial effects on nerve regeneration, gene profiles, and motor outcomes.
Assuntos
Exossomos , Regeneração Tecidual Guiada/métodos , Traumatismos dos Nervos Periféricos/cirurgia , Nervo Isquiático/transplante , Neuropatia Ciática/cirurgia , Animais , Autoenxertos/fisiologia , Modelos Animais de Doenças , Humanos , Masculino , Regeneração Nervosa , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/lesões , Nervo Isquiático/fisiologiaRESUMO
BACKGROUND: Treatments to manage painful neuroma are needed. An operative strategy that isolates and controls chaotic axonal growth could prevent neuroma. Using long acellular nerve allograft to "cap" damaged nerve could control axonal regeneration and, in turn, regulate upstream gene expression patterns. METHODS: Rat sciatic nerve was transected, and the distal nerve end was reversed and ligated to generate a model end-neuroma. Three groups were used to assess their effects immediately following this nerve injury: no treatment (control), traction neurectomy, or 5-cm acellular nerve allograft cap attached to the proximal nerve. Regeneration of axons from the injured nerve was assessed over 5 months and paired with concurrent measurements of gene expression from upstream affected dorsal root ganglia. RESULTS: Both control and traction neurectomy groups demonstrated uncontrolled axon regeneration revealed using Thy1-GFP rat axon imaging and histomorphometric measures of regenerated axons within the most terminal region of regenerated tissue. The acellular nerve allograft group arrested axons within the acellular nerve allograft, where no axons reached the most terminal region even after 5 months. At 5 months, gene expression associated with regeneration and pain sensitization, including Bdnf, cfos, and Gal, was decreased within dorsal root ganglia obtained from the acellular nerve allograft group compared to control or traction neurectomy group dorsal root ganglia. CONCLUSIONS: Long acellular nerve allografts to cap a severed nerve arrested axon regeneration within the acellular nerve allograft. This growth arrest corresponded with changes in regenerative and pain-related genes upstream. Acellular nerve allografts may be useful for surgical intervention of neuroma.
Assuntos
Axônios/patologia , Regeneração Nervosa/genética , Neuroma/cirurgia , Procedimentos Neurocirúrgicos/métodos , Nervo Isquiático/transplante , Aloenxertos/transplante , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Masculino , Neuroma/genética , Neuroma/patologia , Ratos , Nervo Isquiático/lesões , Transplante Homólogo/métodosRESUMO
In critical nerve gap repair, decellularized nerve allografts are considered a promising tissue engineering strategy that can provide superior regeneration results compared to nerve conduits. Decellularized nerves offer a well-conserved extracellular matrix component that has proven to play an important role in supporting axonal guiding and peripheral nerve regeneration. Up to now, the known decellularized techniques are time and effort consuming. The present study, performed on rat sciatic nerves, aims at investigating a novel nerve decellularization protocol able to combine an effective decellularization in short time with a good preservation of the extracellular matrix component. To do this, a decellularization protocol proven to be efficient for tendons (DN-P1) was compared with a decellularization protocol specifically developed for nerves (DN-P2). The outcomes of both the decellularization protocols were assessed by a series of in vitro evaluations, including qualitative and quantitative histological and immunohistochemical analyses, DNA quantification, SEM and TEM ultrastructural analyses, mechanical testing, and viability assay. The overall results showed that DN-P1 could provide promising results if tested in vivo, as the in vitro characterization demonstrated that DN-P1 conserved a better ultrastructure and ECM components compared to DN-P2. Most importantly, DN-P1 was shown to be highly biocompatible, supporting a greater number of viable metabolically active cells.
Assuntos
Matriz Extracelular/química , Regeneração Nervosa , Nervo Isquiático/fisiologia , Nervo Isquiático/transplante , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Aloenxertos , Animais , Separação Celular , Feminino , Ratos , Ratos Wistar , Nervo Isquiático/citologiaRESUMO
PURPOSE: While many studies have been conducted on peripheral nerve regeneration, few have focused on strengthening the nerve autografts. This study hypothesized that adding autologous stromal vascular fraction (SVF) to a nerve autograft will improve nerve regeneration. The purpose of this study was to compare the results of nerve autograft with and without SVF. METHODS: An adipose tissue sample was excised from the right inguinal region of female Wistar rats, and SVF was separated by centrifugation. The left sciatic nerve was resected at a length of 15 mm and the defect was bridged by a resected nerve autograft. We added SVF with collagen gel around the nerve autograft in the SVF group and added saline in the control group. At 12 weeks after surgery, the wet muscle weight, distal latency, and amplitude of the compound muscle action potential of the tibialis anterior were evaluated by the ratio of left and right sides. Sciatic functional index (SFI) was also evaluated. RESULTS: The wet muscle weight was significantly better in the SVF group than in the control group. The results of distal latency, amplitude, and SFI were not significantly different between the two groups; however, these results tended to be better in the SVF group than in the control group. CONCLUSION: SVF added to artificial nerve grafts has been reported to promote axonal regeneration through secretion of angiogenic, neurotrophic, and anti-apoptotic factors. This study indicates that SVF may also be effective for nerve autografts and improve the clinical result of nerve autograft.
Assuntos
Tecido Adiposo , Regeneração Nervosa , Nervo Isquiático , Tecido Adiposo/citologia , Animais , Autoenxertos , Feminino , Células-Tronco Mesenquimais , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Nervo Isquiático/transplante , Transplante AutólogoRESUMO
BACKGROUND: Current methods to repair ablation-type peripheral nerve injuries (PNIs) using peripheral nerve allografts (PNAs) often result in poor functional recovery due to immunological rejection as well as to slow and inaccurate outgrowth of regenerating axonal sprouts. In contrast, ablation-type PNIs repaired by PNAs, using a multistep protocol in which one step employs the membrane fusogen polyethylene glycol (PEG), permanently restore sciatic-mediated behaviors within weeks. Axons and cells within PEG-fused PNAs remain viable, even though outbred host and donor tissues are neither immunosuppressed nor tissue matched. PEG-fused PNAs exhibit significantly reduced T cell and macrophage infiltration, expression of major histocompatibility complex I/II and consistently low apoptosis. In this study, we analyzed the coding transcriptome of PEG-fused PNAs to examine possible mechanisms underlying immunosuppression. METHODS: Ablation-type sciatic PNIs in adult Sprague-Dawley rats were repaired using PNAs and a PEG-fusion protocol combined with neurorrhaphy. Electrophysiological and behavioral tests confirmed successful PEG-fusion of PNAs. RNA sequencing analyzed differential expression profiles of protein-coding genes between PEG-fused PNAs and negative control PNAs (not treated with PEG) at 14 days PO, along with unoperated control nerves. Sequencing results were validated by quantitative reverse transcription PCR (RT-qPCR), and in some cases, immunohistochemistry. RESULTS: PEG-fused PNAs display significant downregulation of many gene transcripts associated with innate and adaptive allorejection responses. Schwann cell-associated transcripts are often upregulated, and cellular processes such as extracellular matrix remodeling and cell/tissue development are particularly enriched. Transcripts encoding several potentially immunosuppressive proteins (e.g., thrombospondins 1 and 2) also are upregulated in PEG-fused PNAs. CONCLUSIONS: This study is the first to characterize the coding transcriptome of PEG-fused PNAs and to identify possible links between alterations of the extracellular matrix and suppression of the allorejection response. The results establish an initial molecular basis to understand mechanisms underlying PEG-mediated immunosuppression.
Assuntos
Aloenxertos/fisiologia , Perfilação da Expressão Gênica/métodos , Tolerância Imunológica/fisiologia , Polietilenoglicóis/administração & dosagem , Nervo Isquiático/fisiologia , Nervo Isquiático/transplante , Animais , Feminino , Mapas de Interação de Proteínas/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Ablation/segmental loss peripheral nerve injuries (PNIs) exhibit poor functional recovery due to slow and inaccurate outgrowth of regenerating axons. Viable peripheral nerve allografts (PNAs) as growth-guide conduits are immunologically rejected and all anucleated donor/host axonal segments undergo Wallerian degeneration. In contrast, we report that ablation-type sciatic PNIs repaired by neurorrhaphy of viable sciatic PNAs and a polyethylene glycol (PEG)-fusion protocol using PEG immediately restored axonal continuity for many axons, reinnervated/maintained their neuromuscular junctions, and prevented much Wallerian degeneration. PEG-fused PNAs permanently restored many sciatic-mediated behaviors within 2-6 weeks. PEG-fused PNAs were not rejected even though host/donors were neither immunosuppressed nor tissue-matched in outbred female Sprague Dawley rats. Innate and adaptive immune responses to PEG-fused sciatic PNAs were analyzed using electron microscopy, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction for morphological features, T cell and macrophage infiltration, major histocompatibility complex (MHC) expression, apoptosis, expression of cytokines, chemokines, and cytotoxic effectors. PEG-fused PNAs exhibited attenuated innate and adaptive immune responses by 14-21 days postoperatively, as evidenced by (a) many axons and cells remaining viable, (b) significantly reduced infiltration of cytotoxic and total T cells and macrophages, (c) significantly reduced expression of inflammatory cytokines, chemokines, and MHC proteins, (d) consistently low apoptotic response. Morphologically and/or biochemically, PEG-fused sciatic PNAs often resembled sciatic autografts or intact sciatic nerves. In brief, PEG-fused PNAs are an unstudied, perhaps unique, example of immune tolerance of viable allograft tissue in a nonimmune-privileged environment and could greatly improve the clinical outcomes for PNIs relative to current protocols.
Assuntos
Imunidade Adaptativa/fisiologia , Imunidade Inata/fisiologia , Polietilenoglicóis/administração & dosagem , Nervo Isquiático/imunologia , Nervo Isquiático/transplante , Neuropatia Ciática/terapia , Imunidade Adaptativa/efeitos dos fármacos , Aloenxertos/imunologia , Aloenxertos/transplante , Animais , Feminino , Imunidade Inata/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/imunologia , Transplante Homólogo/métodosRESUMO
We developed a sheet of stem cells derived from adipose tissue (ADSC sheet). To improve transplantation, we wrapped decellularized nerves with ADSC sheets and examined the efficacy of this recellularized nerves in nerve regeneration. Decellularized nerves were prepared from sciatic nerves of Sprague-Dawley rats. Wistar rats were subjected to sciatic nerve injury and then randomly assigned to three groups (n = 7 per group), which were transplanted with 15-mm bridge grafts; the first group received a decellularized allogenic nerve implant, the second an ADSC sheet-wrapped decellularized allogenic nerve implant, and the third an autogenous nerves were implant. No significant differences were found in S100-positive and neurofilament-positive areas, axon density, and sciatic functional index (SFI) score between rats transplanted with ADSC sheet-wrapped nerve grafts and those that received autografts. In contrast, these parameters except SFI and the amplitude ratio were significantly larger in rats grafted with ADSC sheet-wrapped nerve than with the decellularized nerve. These results suggest that the number of regenerating axons, as well as their regenerating velocity, and the number of migrating Schwann cells into the implant in rats transplanted with ADSC sheet-wrapped nerves matched those in rats transplanted with autografts. These positive effects are possibly attributable to secretion of growth factors of ADSCs.
Assuntos
Regeneração Tecidual Guiada/métodos , Células-Tronco Mesenquimais , Regeneração Nervosa/fisiologia , Nervo Isquiático/transplante , Animais , Feminino , Ratos , Ratos Sprague-Dawley , Ratos WistarAssuntos
Microambiente Celular , Modelos Animais de Doenças , Neovascularização Fisiológica , Regeneração Nervosa , Traumatismos dos Nervos Periféricos/terapia , Nervo Isquiático/fisiologia , Nervo Isquiático/transplante , Aloenxertos , Animais , Masculino , Traumatismos dos Nervos Periféricos/patologia , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático/lesões , Nervo Isquiático/cirurgiaRESUMO
Acellular nerve allografts (ANAs) are increasingly used to repair nerve gaps following injuries. However, these nerve scaffolds have yet to surpass the regenerative capabilities of cellular nerve autografts; improved understanding of their regenerative mechanisms could improve design. Due to their acellular nature, both angiogenesis and diverse cell recruitment is necessary to repopulate these scaffolds to promote functional regeneration. We determined the contribution of angiogenesis to initial cellular repopulation of ANAs used to repair nerve gaps, as well as the signaling that drives a significant portion of this angiogenesis. Wild-type (WT) mice with nerve gaps repaired using ANAs that were treated with an inhibitor of VEGF receptor signaling severely impaired angiogenesis within ANAs, as well as hampered cell repopulation and axon extension into ANAs. Similarly, systemic depletion of hematogenous-derived macrophages, but not neutrophils, in these mice models severely impeded angiogenesis and subsequent nerve regeneration across ANAs suggesting hematogenous-derived macrophages were major contributors to angiogenesis within ANAs. This finding was reinforced using CCR2 knockout (KO) models. As macrophages represented the majority of CCR2 expressing cells, a CCR2 deficiency impaired angiogenesis and subsequent nerve regeneration across ANAs. Furthermore, an essential role for CCL2 during nerve regeneration across ANAs was identified, as nerves repaired using ANAs had reduced angiogenesis and subsequent nerve regeneration in CCL2 KO vs WT mice. Our data demonstrate the CCL2/CCR2 axis is important for macrophage recruitment, which promotes angiogenesis, cell repopulation, and subsequent nerve regeneration and recovery across ANAs used to repair nerve gaps.
Assuntos
Quimiocina CCL2/metabolismo , Macrófagos/metabolismo , Neovascularização Fisiológica/fisiologia , Regeneração Nervosa/fisiologia , Nervo Isquiático/transplante , Aloenxertos , Animais , Movimento Celular/fisiologia , Regeneração Tecidual Guiada/instrumentação , Regeneração Tecidual Guiada/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR2/metabolismo , Nervo Isquiático/lesões , Alicerces Teciduais , Transplante HomólogoRESUMO
Neurotrophic factors (NTF) secreted by Schwann cells in a sciatic nerve (SN) graft promote retinal ganglion cell (RGC) axon regeneration after either transplantation into the vitreous body of the eye or anastomosis to the distal stump of a transected optic nerve. In this study, we investigated the neuroprotective and growth stimulatory properties of SN grafts in which Schwann cells had been killed (acellular SN grafts, ASN) or remained intact (cellular SN grafts, CSN). We report that both intravitreal (ivit) implanted and optic nerve anastomosed CSN promote RGC survival and when simultaneously placed in both sites, they exert additive RGC neuroprotection. CSN and ASN were rich in myelin-associated glycoprotein (MAG) and axon growth-inhibitory ligand common to both the central nervous system (CNS) and peripheral nervous system (PNS) myelin. The penetration of the few RGC axons regenerating into an ASN at an optic nerve transection (ONT) site is limited into the proximal perilesion area, but is increased >2-fold after ivit CSN implantation and increased 5-fold into a CSN optic nerve graft after ivit CSN implantation, potentiated by growth disinhibition through the regulated intramembranous proteolysis (RIP) of p75NTR (the signalling trans-membrane moiety of the nogo-66 trimeric receptor that binds MAG and associated suppression of RhoGTP). MÏller cells/astrocytes become reactive after all treatments and maximally after simultaneous ivit and optic nerve CSN/ASN grafting. We conclude that simultaneous ivit CSN plus optic nerve CSN support promotes significant RGC survival and axon regeneration into CSN optic nerve grafts, despite being rich in axon growth inhibitory molecules. RGC axon regeneration is probably facilitated through RIP of p75NTR, which blinds axons to myelin-derived axon growth-inhibitory ligands present in optic nerve grafts.
Assuntos
Axônios/patologia , Regeneração Nervosa , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Nervo Isquiático/transplante , Animais , Astrócitos/patologia , Sobrevivência Celular , Células Ependimogliais/patologia , Injeções Intravítreas , Ligantes , Ativação de Macrófagos , Macrófagos/patologia , Masculino , Ratos Endogâmicos F344 , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
In order to repair chronic nerve injuries (injuries repaired after a long delay), the damaged nerve segments are resected and stumps are bridged by grafts. Autografts remain the gold-standard, but outcomes are typically poor, even after long periods of recovery. In a recent study, we described the use of a nerve lengthening device to gradually elongate the proximal stump of a transected nerve towards the distal stump, enabling a tension-free end-to-end repair. This approach showed significantly improved outcomes in comparison to autografts in repairing acutely injured nerves. In this study, we compared the use of nerve lengthening/end-to-end repair (LETER) to isograft repair of chronically transected nerves in a rat model. Structural and functional regenerative outcomes following LETER were comparable to isograft-based repair, with no significant differences found in outcomes involving functional recovery or axon growth. These data demonstrate the feasibility of nerve lengthening as a viable graft-free strategy for repairing chronically injured nerves. Not unexpectedly, outcomes for chronic nerve injuries were less favorable in both groups compared to repair of acutely injured nerves. Nonetheless, the findings provide insight into barriers to restoring function after chronic nerve injury through novel comprehensive characterization of a diverse set of neuromuscular outcomes. This analysis revealed key parameters predicting functional recovery.