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1.
Adv Sci (Weinh) ; 6(22): 1901240, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31763143

RESUMO

Tissue engineering produces constructs with defined functions for the targeted treatment of damaged tissue. A complete spinal cord injury (SCI) model is generated in canines to test whether in vitro constructed neural network (NN) tissues can relay the excitatory signal across the lesion gap to the caudal spinal cord. Established protocols are used to construct neural stem cell (NSC)-derived NN tissue characterized by a predominantly neuronal population with robust trans-synaptic activities and myelination. The NN tissue is implanted into the gap immediately following complete transection SCI of canines at the T10 spinal cord segment. The data show significant motor recovery of paralyzed pelvic limbs, as evaluated by Olby scoring and cortical motor evoked potential (CMEP) detection. The NN tissue survives in the lesion area with neuronal phenotype maintenance, improves descending and ascending nerve fiber regeneration, and synaptic integration with host neural circuits that allow it to serve as a neuronal relay to transmit excitatory electrical signal across the injured area to the caudal spinal cord. These results suggest that tissue-engineered NN grafts can relay the excitatory signal in the completely transected canine spinal cord, providing a promising strategy for SCI treatment in large animals, including humans.

2.
Exp Neurol ; 320: 112965, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31132364

RESUMO

Leucine-rich repeat and immunoglobulin domain-containing protein 1 (LINGO-1) is a transmembrane protein that negatively regulates neural regeneration in the central nervous system. LINGO-1 expression is up-regulated after central nerve injury, and is accompanied by cell death. Both LINGO-1 and cell death in the injury microenvironment are thought to limit neural regeneration, but the relationship between LINGO-1 and cell death has not been characterized. To investigate whether LINGO-1 deletion improves the spinal cord microenvironment after spinal cord injury (SCI) and contributes to cell survival, we generated LINGO-1 knockout (KO) mice. These mice and wild-type control mice were subjected to spinal cord transection. Fourteen days after spinal cord transection, cell apoptosis, inflammation, glial scar, and growth of nerve fibers were evaluated by immunostaining. The results showed that LINGO-1 KO mice demonstrated a profound reduction in expression of caspase-3, transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL), ionized calcium binding adapter molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and chondroitin sulfate proteoglycans (CSPGs) compared to controls. In contrast, expression of neurofilament (NF) at the SCI site in LINGO-1 KO mice was markedly increased compared to that in wild-type mice. These results suggested that LINGO-1 plays a critical role in the injury microenvironment in processes such as cell death, inflammatory response, and glial scar formation. Importantly, LINGO-1 deletion and a positive microenvironment may exert synergistic effects to promote nerve fiber regeneration. Therefore, inhibition of LINGO-1 may be a therapeutic strategy to promote neural regeneration following SCI.


Assuntos
Proteínas de Membrana/deficiência , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/deficiência , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Animais , Apoptose/fisiologia , Feminino , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/patologia
3.
Stem Cell Reports ; 12(2): 274-289, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30661994

RESUMO

The hostile environment of an injured spinal cord makes it challenging to achieve higher viability in a grafted tissue-engineered neural network used to reconstruct the spinal cord circuit. Here, we investigate whether cell survival and synaptic transmission within an NT-3 and TRKC gene-overexpressing neural stem cell-derived neural network scaffold (NN) transplanted into transected spinal cord could be promoted by electroacupuncture (EA) through improving the microenvironment. Our results showed that EA facilitated the cell survival, neuronal differentiation, and synapse formation of a transplanted NN. Pseudorabies virus tracing demonstrated that EA strengthened synaptic integration of the transplanted NN with the host neural circuit. The combination therapy also promoted axonal regeneration, spinal conductivity, and functional recovery. The findings highlight EA as a potential and safe supplementary therapeutic strategy to reinforce the survival and synaptogenesis of a transplanted NN as a neuronal relay to bridge the two severed ends of an injured spinal cord.

4.
Adv Sci (Weinh) ; 5(9): 1800261, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30250785

RESUMO

Tissue engineering-based neural construction holds promise in providing organoids with defined differentiation and therapeutic potentials. Here, a bioengineered transplantable spinal cord-like tissue (SCLT) is assembled in vitro by simulating the white matter and gray matter composition of the spinal cord using neural stem cell-based tissue engineering technique. Whether the organoid would execute targeted repair in injured spinal cord is evaluated. The integrated SCLT, assembled by white matter-like tissue (WMLT) module and gray matter-like tissue (GMLT) module, shares architectural, phenotypic, and functional similarities to the adult rat spinal cord. Organotypic coculturing with the dorsal root ganglion or muscle cells shows that the SCLT embraces spinal cord organogenesis potentials to establish connections with the targets, respectively. Transplantation of the SCLT into the transected spinal cord results in a significant motor function recovery of the paralyzed hind limbs in rats. Additionally, targeted spinal cord tissue repair is achieved by the modular design of SCLT, as evidenced by an increased remyelination in the WMLT area and an enlarged innervation in the GMLT area. More importantly, the pro-regeneration milieu facilitates the formation of a neuronal relay by the donor neurons, allowing the conduction of descending and ascending neural inputs.

5.
Biomaterials ; 181: 15-34, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30071379

RESUMO

We have reported previously that bone marrow mesenchymal stem cell (MSC)-derived neural network scaffold not only survived in the injury/graft site of spinal cord but also served as a "neuronal relay" that was capable of improving the limb motor function in a complete spinal cord injury (SCI) rat model. It remained to be explored whether such a strategy was effective for repairing the large spinal cord tissue loss as well as restoring motor function in larger animals. We have therefore extended in this study to construct a canine MSC-derived neural network tissue in vitro with the aim to evaluate its efficacy in treating adult beagle dog subjected to a complete transection of the spinal cord. The results showed that after co-culturing with neurotropin-3 overexpressing Schwann cells in a gelatin sponge scaffold for 14 days, TrkC overexpressing MSCs differentiated into neuron-like cells. In the latter, some cells appeared to make contacts with each other through synapse-like structures with trans-synaptic electrical activities. Remarkably, the SCI canines receiving the transplantation of the MSC-derived neural network tissue demonstrated a gradual restoration of paralyzed limb motor function, along with improved electrophysiological presentation when compared with the control group. Magnetic resonance imaging and diffusion tensor imaging showed that the canines receiving the MSC-derived neural network tissue exhibited robust nerve tract regeneration in the injury/graft site. Histological analysis showed that some of the MSC-derived neuron-like cells had survived in the injury/graft site up to 6.5 months. Implantation of MSC-derived neural network tissue significantly improved the microenvironment of the injury/graft site. It is noteworthy that a variable number of them had integrated with the regenerating corticospinal tract nerve fibers and 5-HT nerve fibers through formation of synapse-like contacts. The results suggest that the transplanted MSC-derived neural network tissue may serve as a structural and functional "neuronal relay" to restore the paralyzed limb motor function in the canine with complete SCI.


Assuntos
Extremidades/inervação , Células-Tronco Mesenquimais/citologia , Traumatismos da Medula Espinal/terapia , Animais , Células Cultivadas , Imagem de Tensor de Difusão , Cães , Extremidades/fisiologia , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Rede Nervosa , Regeneração Nervosa/fisiologia , Células de Schwann
6.
Exp Neurol ; 307: 37-44, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29852179

RESUMO

CSPGs are components of the extracellular matrix in the nervous system, where they serve as cues for axon guidance during development. After a peripheral nerve injury, CSPGs switch roles and become axon inhibitors and become diffusely distributed at the injury site. To investigate whether the spatial distribution of CSPGs affects their role, we combined in vitro DRG cultures with CSPG stripe or coverage assays to simulate the effect of a patterned substrate or dispersive distribution of CSPGs on growing neurites. We observed neurite steering at linear CSPG interfaces and neurite inhibition when diffused CSPGs covered the distal but not the proximal segment of the neurite. The repellent and inhibitory effects of CSPGs on neurite outgrowth were associated with the disappearance of focal actin filaments on growth cones. The application of an actin polymerization inducer, jasplakinolide, allowed neurites to break through the CSPG boundary and grow on CSPG-coated surfaces. The results of our study collectively reveal a novel mechanism that explains how the spatial distribution of CSPGs determines whether they act as a cue for axon guidance or as an axon-inhibiting factor. Increasing our understanding of this issue may promote the development of novel therapeutic strategies that regulate the spatial distributions of CSPGs to use them as an axon guidance cue.


Assuntos
Citoesqueleto de Actina/fisiologia , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Gânglios Espinais/fisiologia , Regeneração Nervosa/fisiologia , Transdução de Sinais/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Células Cultivadas , Depsipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
7.
J Biomed Mater Res A ; 106(8): 2158-2170, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29577604

RESUMO

Spinal cord injury (SCI) normally results in cell death, scarring, cavitation, inhibitory molecules release, etc., which are regarded as a huge obstacle to reconnect the injured neuronal circuits because of the lack of effective stimulus. In this study, a functional gelatin sponge scaffold was used to inhibit local inflammation, enhance nerve fiber regeneration, and improve neural conduction in the canine. This scaffold had good porosity and modified with neurotrophin-3 (NT-3)/fibroin complex, which showed sustained release in vitro. After the scaffold was transplanted into canine spinal cord hemisection model, hindlimb movement, and neural conduction were improved evidently. Migrating host cells, newly formed neurons with associated synaptic structures together with functional blood vessels with intact endothelium in the regenerating tissue were identified. Taken together, the results demonstrated that using bioactive scaffold could establish effective microenvironment stimuli for endogenous regeneration, providing a potential and practical strategy for treatment of spinal cord injury. © 2018 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2158-2170, 2018.


Assuntos
Inflamação/patologia , Atividade Motora , Fibras Nervosas/fisiologia , Regeneração Nervosa , Neurotrofina 3/farmacologia , Traumatismos da Medula Espinal/fisiopatologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/farmacologia , Movimento Celular/efeitos dos fármacos , Cães , Potencial Evocado Motor/efeitos dos fármacos , Feminino , Fibroínas/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Membro Posterior/fisiopatologia , Atividade Motora/efeitos dos fármacos , Fibras Nervosas/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Próteses e Implantes , Medula Espinal/irrigação sanguínea , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/patologia
8.
Biomaterials ; 160: 37-55, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29353106

RESUMO

The functional multipotency enables mesenchymal stem cells (MSCs) promising translational potentials in treating spinal cord injury (SCI). Yet the fate of MSCs grafted into the injured spinal cord has not been fully elucidated even in preclinical studies, rendering concerns of their safety and genuine efficacy. Here we used a rat spinal cord transection model to evaluate the cell fate of allograft bone marrow derived MSCs. With the application of immunosuppressant, donor cells, delivered by biocompatible scaffold, survived up to 8 weeks post-grafting. Discernible tubes formed by MSCs were observed beginning 2 weeks after transplantation and they dominated the morphological features of implanted MSCs at 8 weeks post-grafting. The results of immunocytochemistry and transmission electron microscopy displayed the formation of perineurium-like sheath by donor cells, which, in a manner comparable to the perineurium in peripheral nerve, enwrapped host myelins and axons. The MSC-derived perineurium-like sheath secreted a group of trophic factors and permissive extracellular matrix, and served as a physical and chemical barrier to insulate the inner nerve fibers from ambient oxidative insults by the secretion of soluble antioxidant, superoxide dismutase-3 (SOD3). As a result, many intact regenerating axons were preserved in the injury/graft site following the forming of perineurium-like sheath. A parallel study utilizing a good manufacturing practice (GMP) grade human umbilical cord-derived MSCs or allogenic MSCs in an acute contusive/compressive SCI model exhibited a similar perineurium-like sheath formed by surviving donor cells in rat spinal cord at 3 weeks post-grafting. The present study for the first time provides an unambiguous morphological evidence of perineurium-like sheath formed by transplanted MSCs and a novel therapeutic mechanism of MSCs in treating SCI.


Assuntos
Células-Tronco Mesenquimais , Nervos Periféricos , Tecidos Suporte , Animais , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Regeneração Nervosa , Neuroproteção , Nervos Periféricos/citologia , Nervos Periféricos/fisiologia , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/terapia , Engenharia Tecidual
9.
Neural Plast ; 2017: 7351238, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28744378

RESUMO

Spinal cord injury (SCI) often results in death of spinal neurons and atrophy of muscles which they govern. Thus, following SCI, reorganizing the lumbar spinal sensorimotor pathways is crucial to alleviate muscle atrophy. Tail nerve electrical stimulation (TANES) has been shown to activate the central pattern generator (CPG) and improve the locomotion recovery of spinal contused rats. Electroacupuncture (EA) is a traditional Chinese medical practice which has been proven to have a neural protective effect. Here, we examined the effects of TANES and EA on lumbar motor neurons and hindlimb muscle in spinal transected rats, respectively. From the third day postsurgery, rats in the TANES group were treated 5 times a week and those in the EA group were treated once every other day. Four weeks later, both TANES and EA showed a significant impact in promoting survival of lumbar motor neurons and expression of choline acetyltransferase (ChAT) and ameliorating atrophy of hindlimb muscle after SCI. Meanwhile, the expression of neurotrophin-3 (NT-3) in the same spinal cord segment was significantly increased. These findings suggest that TANES and EA can augment the expression of NT-3 in the lumbar spinal cord that appears to protect the motor neurons as well as alleviate muscle atrophy.


Assuntos
Neurônios Motores/patologia , Neurônios Motores/fisiologia , Músculo Esquelético/patologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Cauda/inervação , Animais , Células do Corno Anterior/metabolismo , Células do Corno Anterior/patologia , Células do Corno Anterior/fisiologia , Estimulação Elétrica , Eletroacupuntura , Feminino , Neurônios Motores/metabolismo , Atrofia Muscular , Neurotrofina 3/metabolismo , Ratos Sprague-Dawley , Medula Espinal , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapia
10.
Acupunct Med ; 35(2): 122-132, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27841975

RESUMO

OBJECTIVES: In spinal cord demyelination, some oligodendrocyte precursor cells (OPCs) remain in the demyelinated region but have a reduced capacity to differentiate into oligodendrocytes. This study investigated whether 'Governor Vessel' (GV) electroacupuncture (EA) would promote the differentiation of endogenous OPCs into oligodendrocytes by activating the retinoid X receptor γ (RXR-γ)-mediated signalling pathway. METHODS: Adult rats were microinjected with ethidium bromide (EB) into the T10 spinal cord to establish a model of spinal cord demyelination. EB-injected rats remained untreated (EB group, n=26) or received EA treatment (EB+EA group, n=26). A control group (n=26) was also included that underwent dural exposure without EB injection. After euthanasia at 7 days (n=5 per group), 15 days (n=8 per group) or 30 days (n=13 per group), protein expression of RXR-γ in the demyelinated spinal cord was evaluated by immunohistochemistry and Western blotting. In addition, OPCs derived from rat embryonic spinal cord were cultured in vitro, and exogenous 9-cis-RA (retinoic acid) and RXR-γ antagonist HX531 were administered to determine whether RA could activate RXR-γ and promote OPC differentiation. RESULTS: EA was found to increase the numbers of both OPCs and oligodendrocytes expressing RXR-γ and RALDH2, and promote remyelination in the remyelinated spinal cord. Exogenous 9-cis-RA enhanced the differentiation of OPCs into mature oligodendrocytes by activating RXR-γ. CONCLUSIONS: The results suggest that EA may activate RXR signalling to promote the differentiation of OPCs into oligodendrocytes in spinal cord demyelination.


Assuntos
Diferenciação Celular , Doenças Desmielinizantes/terapia , Eletroacupuntura , Oligodendroglia/citologia , Receptores X Retinoide/metabolismo , Animais , Doenças Desmielinizantes/metabolismo , Feminino , Humanos , Oligodendroglia/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Medula Espinal/metabolismo
11.
Neural Regen Res ; 12(12): 2025-2034, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29323042

RESUMO

In our previous study, we found that the edible alcohol extract of the root of the medicinal plant Rhodiola crenulata (RCE) improved spatial cognition in a rat model of Alzheimer's disease. Another study from our laboratory showed that RCE enhanced neural cell proliferation in the dentate gyrus of the hippocampus and prevented damage to hippocampal neurons in a rat model of chronic stress-induced depression. However, the mechanisms underlying the neuroprotective effects of RCE are unclear. In the present study, we investigated the anti-apoptotic effect of RCE and its neuroprotective mechanism of action in a rat model of Alzheimer's disease established by intracerebroventricular injection of streptozotocin. The rats were pre-administered RCE at doses of 1.5, 3.0 or 6.0 g/kg for 21 days before model establishment. ATP and cytochrome c oxidase levels were significantly decreased in rats with Alzheimer's disease. Furthermore, neuronal injury was obvious in the hippocampus, with the presence of a large number of apoptotic neurons. In comparison, in rats given RCE pretreatment, ATP and cytochrome c oxidase levels were markedly increased, the number of apoptotic neurons was reduced, and mitochondrial injury was mitigated. The 3.0 g/kg dose of RCE had the optimal effect. These findings suggest that pretreatment with RCE prevents mitochondrial dysfunction and protects hippocampal neurons from apoptosis in rats with Alzheimer's disease.

12.
Biomaterials ; 109: 40-54, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27665078

RESUMO

Severe spinal cord injury (SCI) causes loss of neural connectivity and permanent functional deficits. Re-establishment of new neuronal relay circuits after SCI is therefore of paramount importance. The present study tested our hypothesis if co-culture of neurotrophin-3 (NT-3) gene-modified Schwann cells (SCs, NT-3-SCs) and TrkC (NT-3 receptor) gene-modified neural stem cells (NSCs, TrkC-NSCs) in a gelatin sponge scaffold could construct a tissue engineering neural network for re-establishing an anatomical neuronal relay after rat spinal cord transection. Eight weeks after transplantation, the neural network created a favorable microenvironment for axonal regeneration and for survival and synaptogenesis of NSC-derived neurons. Biotin conjugates of cholera toxin B subunit (b-CTB, a transneuronal tracer) was injected into the crushed sciatic nerve to label spinal cord neurons. Remarkably, not only ascending and descending nerve fibers, but also propriospinal neurons, made contacts with b-CTB positive NSC-derived neurons. Moreover, b-CTB positive NSC-derived neurons extended their axons making contacts with the motor neurons located in areas caudal to the injury/graft site of spinal cord. Further study showed that NT-3/TrkC interactions activated the PI3K/AKT/mTOR pathway and PI3K/AKT/CREB pathway affecting synaptogenesis of NSC-derived neurons. Together, our findings suggest that NT-3-mediated TrkC signaling plays an essential role in constructing a tissue engineering neural network thus representing a promising avenue for effective exogenous neuronal relay-based treatment for SCI.


Assuntos
Células-Tronco Neurais/transplante , Neurônios/patologia , Células de Schwann/transplante , Traumatismos da Medula Espinal/terapia , Animais , Axônios/patologia , Diferenciação Celular , Sobrevivência Celular , Toxina da Cólera/metabolismo , Técnicas de Cocultura , Fibras Nervosas/metabolismo , Rede Nervosa/patologia , Regeneração Nervosa , Células-Tronco Neurais/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos Sprague-Dawley , Receptor trkC/genética , Receptor trkC/metabolismo , Células de Schwann/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Engenharia Tecidual , Tecidos Suporte
13.
J Biomed Mater Res A ; 104(8): 1902-11, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26991461

RESUMO

Extracellular matrix (ECM) expression is temporally and spatially regulated during the development of stem cells. We reported previously that fibronectin (FN) secreted by bone marrow mesenchymal stem cells (MSCs) was deposited on the surface of gelatin sponge (GS) soon after culture. In this study, we aimed to assess the function of accumulated FN on neuronal differentiating MSCs as induced by Schwann cells (SCs) in three dimensional transwell co-culture system. The expression pattern and amount of FN of differentiating MSCs was examined by immunofluorescence, Western blot and immunoelectron microscopy. The results showed that FN accumulated inside GS scaffold, although its mRNA expression in MSCs was progressively decreased during neural induction. MSC-derived neuron-like cells showed spindle-shaped cell body and long extending processes on FN-decorated scaffold surface. However, after blocking of FN function by application of monoclonal antibodies, neuron-like cells showed flattened cell body with short and thick neurites, together with decreased expression of integrin ß1. In vivo transplantation study revealed that autocrine FN significantly facilitated endogenous nerve fiber regeneration in spinal cord transection model. Taken together, the present results showed that FN secreted by MSCs in the early stage accumulated on the GS scaffold and promoted the neurite elongation of neuronal differentiating MSCs as well as nerve fiber regeneration after spinal cord injury. This suggests that autocrine FN has a dynamic influence on MSCs in a three dimensional culture system and its potential application for treatment of traumatic spinal cord injury. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1902-1911, 2016.


Assuntos
Comunicação Autócrina , Diferenciação Celular , Fibronectinas/farmacologia , Células-Tronco Mesenquimais/citologia , Regeneração Nervosa/efeitos dos fármacos , Neuritos/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Animais , Adesão Celular , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Feminino , Gelatina/química , Imagem Tridimensional , Integrina beta1/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Fibras Nervosas/metabolismo , Neuritos/efeitos dos fármacos , Ligação Proteica , Ratos Sprague-Dawley , Ratos Transgênicos , Traumatismos da Medula Espinal/patologia
14.
Cell Transplant ; 25(8): 1425-38, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26850705

RESUMO

Spinal cord injury (SCI) can cause severe traumatic injury to the central nervous system (CNS). Current therapeutic effects achieved for SCI in clinical medicine show that there is still a long way to go to reach the desired goal of full or significant functional recovery. In basic medical research, however, cell transplantation, gene therapy, application of cytokines, and biomaterial scaffolds have been widely used and investigated as treatments for SCI. All of these strategies when used separately would help rebuild, to some extent, the neural circuits in the lesion area of the spinal cord. In light of this, it is generally accepted that a combined treatment may be a more effective strategy. This review focuses primarily on our recent series of work on transplantation of Schwann cells and adult stem cells, and transplantation of stem cell-derived neural network scaffolds with functional synapses. Arising from this, an artificial neural network (an exogenous neuronal relay) has been designed and fabricated by us-a biomaterial scaffold implanted with Schwann cells modified by the neurotrophin-3 (NT-3) gene and adult stem cells modified with the TrkC (receptor of NT-3) gene. More importantly, experimental evidence suggests that the novel artificial network can integrate with the host tissue and serve as an exogenous neuronal relay for signal transfer and functional improvement of SCI.


Assuntos
Traumatismos da Medula Espinal/terapia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Animais , Transplante de Células , Humanos , Fatores de Crescimento Neural/metabolismo , Rede Nervosa , Regeneração Nervosa/fisiologia , Células de Schwann/citologia , Células de Schwann/fisiologia , Traumatismos da Medula Espinal/metabolismo
15.
Biomaterials ; 83: 233-48, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26774562

RESUMO

Persistent neurotrophic factor delivery is crucial to create a microenvironment for cell survival and nerve regeneration in spinal cord injury (SCI). This study aimed to develop a NT-3/fibroin coated gelatin sponge scaffold (NF-GS) as a novel controlled artificial release therapy for SCI. In vitro, bone marrow-derived mesenchymal stem cells (MSCs) were planted into the NF-GS and release test showed that NF-GS was capable to generate a sustainable NT-3 release up to 28 days. MSCs in NF-GS had high cell activity with excellent cell distribution and phenotype. Then, the NF-GS was transplanted into the injury site of spinal cord of rat and canine in vivo, which exhibited strong biocompatibility during post-transplantation period. Four weeks following transplantation, the concentration of NT-3 was much higher than that in control groups. Cavity areas in the injury/graft site were significantly reduced due to tissue regeneration and axonal extensions associated with myelin sheath through the glial scar into the NF-GS. Additionally, the NF-GS decreased the inflammation by reducing the CD68 positive cells and TNF-α. A striking feature was the occurrence of some cells and myelin-like structure that appeared to traverse the NF-GS. The present results demonstrate that the NF-GS has the property to control the release of NT-3 from the NT-3/fibroin complex thus facilitating regeneration of injured spinal cord.


Assuntos
Axônios/patologia , Gelatina/química , Inflamação/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Neurotrofina 3/uso terapêutico , Poríferos/química , Traumatismos da Medula Espinal/tratamento farmacológico , Tecidos Suporte/química , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Axônios/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Simulação por Computador , Cães , Feminino , Fibroínas/química , Humanos , Inflamação/complicações , Inflamação/patologia , Neuroglia/metabolismo , Neurotrofina 3/farmacologia , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/patologia , Fator de Necrose Tumoral alfa/metabolismo
16.
PLoS One ; 10(12): e0144030, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26640949

RESUMO

Cholera toxin B subunit (CTB) has been extensively used in the past for monosynaptic mapping. For decades, it was thought to lack the ability of transneuronal tracing. In order to investigate whether biotin conjugates of CTB (b-CTB) would pass through transneurons in the rat spinal cord, it was injected into the crushed left sciatic nerve. For experimental control, the first order afferent neuronal projections were defined by retrograde transport of fluorogold (FG, a non-transneuronal labeling marker as an experimental control) injected into the crushed right sciatic nerve in the same rat. Neurons containing b-CTB or FG were observed in the dorsal root ganglia (DRG) at the L4-L6 levels ipsilateral to the tracer injection. In the spinal cord, b-CTB labeled neurons were distributed in all laminae ipsilaterally between C7 and S1 segments, but labeling of neurons at the cervical segment was abolished when the T10 segment was transected completely. The interneurons, distributed in the intermediate gray matter and identified as gamma-aminobutyric acid-ergic (GABAergic), were labeled by b-CTB. In contrast, FG labeling was confined to the ventral horn neurons at L4-L6 spinal segments ipsilateral to the injection. b-CTB immunoreactivity remained to be restricted to the soma of neurons and often appeared as irregular patches detected by light and electron microscopy. Detection of monosialoganglioside (GM1) in b-CTB labeled neurons suggests that GM1 ganglioside may specifically enhance the uptake and transneuronal passage of b-CTB, thus supporting the notion that it may be used as a novel transneuronal tracer.


Assuntos
Toxina da Cólera , Neurônios GABAérgicos/citologia , Gânglios Espinais/citologia , Substância Cinzenta/citologia , Técnicas de Rastreamento Neuroanatômico/métodos , Nervo Isquiático/citologia , Animais , Toxina da Cólera/farmacocinética , Toxina da Cólera/farmacologia , Feminino , Gangliosídeo G(M1)/metabolismo , Neurônios GABAérgicos/metabolismo , Gânglios Espinais/metabolismo , Substância Cinzenta/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismo
17.
Stem Cell Res Ther ; 6: 105, 2015 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-26012641

RESUMO

INTRODUCTION: Severe spinal cord injury often causes temporary or permanent damages in strength, sensation, or autonomic functions below the site of the injury. So far, there is still no effective treatment for spinal cord injury. Mesenchymal stem cells (MSCs) have been used to repair injured spinal cord as an effective strategy. However, the low neural differentiation frequency of MSCs has limited its application. The present study attempted to explore whether the grafted MSC-derived neural-like cells in a gelatin sponge (GS) scaffold could maintain neural features or transdifferentiate into myelin-forming cells in the transected spinal cord. METHODS: We constructed an engineered tissue by co-seeding of MSCs with genetically enhanced expression of neurotrophin-3 (NT-3) and its high-affinity receptor tropomyosin receptor kinase C (TrkC) separately into a three-dimensional GS scaffold to promote the MSCs differentiating into neural-like cells and transplanted it into the gap of a completely transected rat spinal cord. The rats received extensive post-operation care, including cyclosporin A administrated once daily for 2 months. RESULTS: MSCs modified genetically could differentiate into neural-like cells in the MN + MT (NT-3-MSCs + TrKC-MSCs) group 14 days after culture in the GS scaffold. However, after the MSC-derived neural-like cells were transplanted into the injury site of spinal cord, some of them appeared to lose the neural phenotypes and instead transdifferentiated into myelin-forming cells at 8 weeks. In the latter, the MSC-derived myelin-forming cells established myelin sheaths associated with the host regenerating axons. And the injured host neurons were rescued, and axon regeneration was induced by grafted MSCs modified genetically. In addition, the cortical motor evoked potential and hindlimb locomotion were significantly ameliorated in the rat spinal cord transected in the MN + MT group compared with the GS and MSC groups. CONCLUSION: Grafted MSC-derived neural-like cells in the GS scaffold can transdifferentiate into myelin-forming cells in the completely transected rat spinal cord.


Assuntos
Axônios/fisiologia , Células-Tronco Mesenquimais/citologia , Bainha de Mielina/metabolismo , Células-Tronco Neurais/transplante , Traumatismos da Medula Espinal/terapia , Adenoviridae/genética , Animais , Comportamento Animal , Técnicas de Cultura de Células , Transdiferenciação Celular , Células Cultivadas , Feminino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Receptor trkC/genética , Receptor trkC/metabolismo , Recuperação de Função Fisiológica , Regeneração , Tecidos Suporte
18.
J Neurosci ; 35(17): 6801-12, 2015 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-25926456

RESUMO

Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5'-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4(-/-)) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4(-/-) cells. The fission defect was associated with a robust increase of intralysosomal Ca(2+) in Fig4(-/-) cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca(2+) efflux of lysosomes because the endogenous ligand of lysosomal Ca(2+) channel TRPML1 is PI3,5P2 that is deficient in Fig4(-/-) cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca(2+) level and rescued abnormal lysosomal storage in Fig4(-/-) culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca(2+) efflux in Fig4(-/-) culture cells and Fig4(-/-) mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency.


Assuntos
Cálcio/metabolismo , Flavoproteínas/metabolismo , Lisossomos/metabolismo , Células de Schwann/ultraestrutura , Animais , Animais Recém-Nascidos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Flavoproteínas/genética , GTP Fosfo-Hidrolases/metabolismo , Gânglios Espinais/citologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Técnicas In Vitro , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/patologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Fosfatases de Fosfoinositídeos , Células de Schwann/metabolismo , Nervo Isquiático/citologia , Medula Espinal/citologia
19.
Biomaterials ; 53: 184-201, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25890718

RESUMO

Functional deficits following spinal cord injury (SCI) primarily attribute to loss of neural connectivity. We therefore tested if novel tissue engineering approaches could enable neural network repair that facilitates functional recovery after spinal cord transection (SCT). Rat bone marrow-derived mesenchymal stem cells (MSCs), genetically engineered to overexpress TrkC, receptor of neurotrophin-3 (NT-3), were pre-differentiated into cells carrying neuronal features via co-culture with NT-3 overproducing Schwann cells in 3-dimensional gelatin sponge (GS) scaffold for 14 days in vitro. Intra-GS formation of MSC assemblies emulating neural network (MSC-GS) were verified morphologically via electron microscopy (EM) and functionally by whole-cell patch clamp recording of spontaneous post-synaptic currents. The differentiated MSCs still partially maintained prototypic property with the expression of some mesodermal cytokines. MSC-GS or GS was then grafted acutely into a 2 mm-wide transection gap in the T9-T10 spinal cord segments of adult rats. Eight weeks later, hindlimb function of the MSC-GS-treated SCT rats was significantly improved relative to controls receiving the GS or lesion only as indicated by BBB score. The MSC-GS transplantation also significantly recovered cortical motor evoked potential (CMEP). Histologically, MSC-derived neuron-like cells maintained their synapse-like structures in vivo; they additionally formed similar connections with host neurites (i.e., mostly serotonergic fibers plus a few corticospinal axons; validated by double-labeled immuno-EM). Moreover, motor cortex electrical stimulation triggered c-fos expression in the grafted and lumbar spinal cord cells of the treated rats only. Our data suggest that MSC-derived neuron-like cells resulting from NT-3-TrkC-induced differentiation can partially integrate into transected spinal cord and this strategy should be further investigated for reconstructing disrupted neural circuits.


Assuntos
Células-Tronco Mesenquimais/citologia , Rede Nervosa , Neurônios/citologia , Medula Espinal/cirurgia , Animais , Técnicas de Cocultura , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos
20.
Neural Regen Res ; 10(1): 128-35, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25788933

RESUMO

Peripheral nerve injury not only affects the site of the injury, but can also induce neuronal apoptosis at the spinal cord. However, many acupuncture clinicians still focus only on the injury site, selecting acupoints entirely along the injured nerve trunk and neglecting other regions; this may delay onset of treatment efficacy and rehabilitation. Therefore, in the present study, we compared the clinical efficacy of acupuncture at Governor vessel and local meridian acupoints combined (GV/LM group) with acupuncture at local meridian acupoints alone (LM group) in the treatment of patients with peripheral nerve injury. In the GV/LM group (n = 15), in addition to meridian acupoints at the injury site, the following acupoints on the Governor vessel were stimulated: Baihui (GV20), Fengfu (GV16), Dazhui (GV14), and Shenzhu (GV12), selected to treat nerve injury of the upper limb, and Jizhong (GV6), Mingmen (GV4), Yaoyangguan (GV3), and Yaoshu (GV2) to treat nerve injury of the lower limb. In the LM group (n = 15), only meridian acupoints along the injured nerve were selected. Both groups had electroacupuncture treatment for 30 minutes, once a day, 5 times per week, for 6 weeks. Two cases dropped out of the LM group. A good or excellent clinical response was obtained in 80% of the patients in the GV/LM group and 38.5% of the LM group. In a second study, an additional 20 patients underwent acupuncture with the same prescription as the GV/LM group. Electomyographic nerve conduction tests were performed before and after acupuncture to explore the mechanism of action of the treatment. An effective response was observed in 80.0% of the patients, with greater motor nerve conduction velocity and amplitude after treatment, indicating that electroacupuncture on specific Governor vessel acupoints promotes functional motor nerve repair after peripheral nerve injury. In addition, electromyography was performed before, during and after electroacupuncture in one patient with radial nerve injury. After a single session, the patient's motor nerve conduction velocity increased by 23.2%, indicating that electroacupuncture at Governor vessel acupoints has an immediate therapeutic effect on peripheral nerve injury. Our results indicate that Governor vessel and local meridian acupoints used simultaneously promote functional repair after peripheral nerve injury. The mechanism of action may arise from an improvement of the local microenvironment in injured nervous tissue, as well as immediate effects of Governor vessel and local meridian acupoint stimulation to ensure the continuity between the peripheral and central nervous systems.

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