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1.
Nat Commun ; 11(1): 6131, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33257677

RESUMO

After a dorsal root crush injury, centrally-projecting sensory axons fail to regenerate across the dorsal root entry zone (DREZ) to extend into the spinal cord. We find that chemogenetic activation of adult dorsal root ganglion (DRG) neurons improves axon growth on an in vitro model of the inhibitory environment after injury. Moreover, repeated bouts of daily chemogenetic activation of adult DRG neurons for 12 weeks post-crush in vivo enhances axon regeneration across a chondroitinase-digested DREZ into spinal gray matter, where the regenerating axons form functional synapses and mediate behavioral recovery in a sensorimotor task. Neuronal activation-mediated axon extension is dependent upon changes in the status of tubulin post-translational modifications indicative of highly dynamic microtubules (as opposed to stable microtubules) within the distal axon, illuminating a novel mechanism underlying stimulation-mediated axon growth. We have identified an effective combinatory strategy to promote functionally-relevant axon regeneration of adult neurons into the CNS after injury.


Assuntos
Axônios/fisiologia , Lesões por Esmagamento/metabolismo , Microtúbulos/fisiologia , Regeneração Nervosa/fisiologia , Neurônios/fisiologia , Raízes Nervosas Espinhais/fisiologia , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Lesões por Esmagamento/patologia , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/lesões , Gânglios Espinais/fisiologia , Ratos , Ratos Wistar , Medula Espinal , Raízes Nervosas Espinhais/patologia
2.
Plast Reconstr Surg ; 146(6): 1295-1305, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33234960

RESUMO

BACKGROUND: Posttraumatic facial paralysis is a disabling condition. Current surgical management by faciofacial nerve suture provides limited recovery. To improve the outcome, the authors evaluated an add-on strategy based on a syngeneic transplantation of nasal olfactory stem cells in a rat model of facial nerve injury. The main readouts of the study were the recording of whisking function and buccal synkinesis. METHODS: Sixty rats were allocated to three groups. Animals with a 2-mm facial nerve loss were repaired with a femoral vein, filled or not with olfactory stem cells. These two groups were compared to similarly injured rats but with a faciofacial nerve suture. Olfactory stem cells were purified from rat olfactory mucosa. Three months after surgery, facial motor performance was evaluated using video-based motion analysis and electromyography. Synkinesis was assessed by electromyography, using measure of buccal involuntary movements during blink reflex, and double retrograde labeling of regenerating motoneurons. RESULTS: The authors' study reveals that olfactory stem cell transplantation induces functional recovery in comparison to nontransplanted and faciofacial nerve suture groups. They significantly increase (1) maximal amplitude of vibrissae protraction and retraction cycles and (2) angular velocity during protraction of vibrissae. They also reduce buccal synkinesis, according to the two techniques used. However, olfactory stem cell transplantation did not improve axonal regrowth of the facial nerve, 3 months after surgery. CONCLUSIONS: The authors show here that the adjuvant strategy of syngeneic transplantation of olfactory stem cells improves functional recovery. These promising results open the way for a phase I clinical trial based on the autologous engraftment of olfactory stem cells in patients with a facial nerve paralysis.


Assuntos
Traumatismos do Nervo Facial/cirurgia , Paralisia Facial/cirurgia , Transplante de Células-Tronco/métodos , Sincinesia/cirurgia , Enxerto Vascular/métodos , Animais , Técnicas de Observação do Comportamento , Modelos Animais de Doenças , Eletromiografia , Nervo Facial/fisiopatologia , Nervo Facial/cirurgia , Traumatismos do Nervo Facial/complicações , Traumatismos do Nervo Facial/fisiopatologia , Paralisia Facial/diagnóstico , Paralisia Facial/etiologia , Paralisia Facial/fisiopatologia , Feminino , Veia Femoral/transplante , Humanos , Regeneração Nervosa/fisiologia , Mucosa Olfatória/citologia , Ratos , Recuperação de Função Fisiológica , Sincinesia/diagnóstico , Sincinesia/etiologia , Sincinesia/fisiopatologia , Transplante Isogênico/métodos , Vibrissas/inervação , Vibrissas/fisiologia , Gravação em Vídeo
3.
J Vis Exp ; (165)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33191937

RESUMO

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models. We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified. This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.


Assuntos
Axônios/fisiologia , Axotomia , Técnicas de Cocultura/métodos , Modelos Biológicos , Regeneração Nervosa/fisiologia , Neuroglia/citologia , Mucosa Olfatória/citologia , Células Ganglionares da Retina/citologia , Animais , Linhagem Celular , Humanos , Masculino , Ratos Wistar
4.
PLoS One ; 15(10): e0238578, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33001981

RESUMO

The spiral ganglion neurons constitute the primary connection between auditory hair cells and the brain. The spiral ganglion afferent fibers and their synapse with hair cells do not regenerate to any significant degree in adult mammalian ears after damage. We have investigated gene expression changes after kainate-induced disruption of the synapses in a neonatal cochlear explant model in which peripheral fibers and the afferent synapse do regenerate. We compared gene expression early after damage, during regeneration of the fibers and synapses, and after completion of in vitro regeneration. These analyses revealed a total of 2.5% differentially regulated transcripts (588 out of 24,000) based on a threshold of p<0.005. Inflammatory response genes as well as genes involved in regeneration of neural circuits were upregulated in the spiral ganglion neurons and organ of Corti, where the hair cells reside. Prominent genes upregulated at several time points included genes with roles in neurogenesis (Elavl4 and Sox21), neural outgrowth (Ntrk3 and Ppp1r1c), axonal guidance (Rgmb and Sema7a), synaptogenesis (Nlgn2 and Psd2), and synaptic vesicular function (Syt8 and Syn1). Immunohistochemical and in situ hybridization analysis of genes that had not previously been described in the cochlea confirmed their cochlear expression. The time course of expression of these genes suggests that kainate treatment resulted in a two-phase response in spiral ganglion neurons: an acute response consistent with inflammation, followed by an upregulation of neural regeneration genes. Identification of the genes activated during regeneration of these fibers suggests candidates that could be targeted to enhance regeneration in adult ears.


Assuntos
Células Ciliadas Auditivas/fisiologia , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Neurônios Aferentes/fisiologia , Animais , Animais Recém-Nascidos , Expressão Gênica/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Inflamação/genética , Inflamação/fisiopatologia , Ácido Caínico/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Neurogênese/genética , Neurogênese/fisiologia , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/fisiologia , Sinapses/fisiologia , Técnicas de Cultura de Tecidos
5.
J Vis Exp ; (164)2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33104075

RESUMO

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats' paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method's limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.


Assuntos
Análise da Marcha/métodos , Traumatismos dos Nervos Periféricos/fisiopatologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Autoenxertos , Automação , Modelos Animais de Doenças , Nervo Femoral/patologia , Nervo Femoral/fisiopatologia , Marcha/fisiologia , Membro Posterior/fisiopatologia , Abrigo para Animais , Masculino , Regeneração Nervosa/fisiologia , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Neuropatia Ciática/fisiopatologia , Vértebras Torácicas/patologia , Vértebras Torácicas/fisiopatologia
6.
J Neuroimmunol ; 349: 577423, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33074142

RESUMO

Following peripheral nerve injury, macrophages are recruited to the injury site from circulation to clear cellular debris. Injured ß-secretase 1 (BACE1) knockout mice have enhanced macrophage recruitment and debris clearance, which may be due to BACE1 activity in macrophages or the hypomyelination observed in BACE1 knockout mice. To assess if BACE1 expression by macrophages mediates enhanced macrophage recruitment we utilized mice with macrophage specific deletion of BACE1 and saw no increase in macrophage recruitment following injury. This study suggests that expression of BACE1 by macrophages may not be essential for increased recruitment observed previously in global BACE1 KO mice.


Assuntos
Secretases da Proteína Precursora do Amiloide/deficiência , Ácido Aspártico Endopeptidases/deficiência , Macrófagos/metabolismo , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/metabolismo , Animais , Células Cultivadas , Feminino , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismos dos Nervos Periféricos/patologia
7.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 49(4): 500-507, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32985164

RESUMO

Different from neurons in the peripheral nervous system, mature neurons in the mammalian central nervous system often fail to regenerate after injury. Recent studies have found that calcium transduction, injury signaling, mitochondrial transportation, cytoskeletal remodeling and protein synthesis play essential roles in axon regeneration. Firstly, axon injury increases the intracellular concentration of calcium, and initiates the injury signaling pathways including cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and dual leucine kinase (DLK), which are found to promote axon regeneration in multiple animal injury models. The second step for axonal regrowth is to rebuild growth cones. Overexpressing proteins that promote dynamics of microtubules and actin filaments is beneficial for the reassembly of cytoskeletons and initiation of new growth cones. Thirdly, mitochondria, the power factory for cells, also play important roles in growth cone formation and axonal extension. The last but not the least important step is the regulation of gene transcription and protein translation to sustain the regrowth of axons. This review summarizes important findings revealing the functions and mechanisms of these biological progresses.


Assuntos
Axônios , Regeneração Nervosa , Neurologia , Pesquisa , Animais , Axônios/fisiologia , Cones de Crescimento/fisiologia , Modelos Animais , Regeneração Nervosa/fisiologia , Neurologia/tendências , Pesquisa/tendências
8.
J Vis Exp ; (162)2020 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-32925888

RESUMO

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.


Assuntos
Membro Posterior/transplante , Regeneração Nervosa/fisiologia , Alotransplante de Tecidos Compostos Vascularizados/métodos , Animais , Masculino , Modelos Animais , Ratos , Recuperação de Função Fisiológica
9.
J Vis Exp ; (163)2020 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-32955495

RESUMO

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury. The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.


Assuntos
Axônios/fisiologia , Modelos Animais de Doenças , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Animais , Axônios/patologia , Pressão Hidrostática/efeitos adversos , Bainha de Mielina/fisiologia , Compressão Nervosa , Regeneração Nervosa/fisiologia , Traumatismos do Nervo Óptico/etiologia , Ratos , Reprodutibilidade dos Testes
10.
Sci Rep ; 10(1): 15709, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973242

RESUMO

Optic nerve injury induces optic nerve degeneration and retinal ganglion cell (RGC) death that lead to visual disturbance. In this study, we examined if topical ripasudil has therapeutic potential in adult mice after optic nerve crush (ONC). Topical ripasudil suppressed ONC-induced phosphorylation of p38 mitogen-activated protein kinase and ameliorated RGC death. In addition, topical ripasudil significantly suppressed the phosphorylation of collapsin response mediator protein 2 and cofilin, and promoted optic nerve regeneration. These results suggest that topical ripasudil promotes RGC protection and optic nerve regeneration by modulating multiple signaling pathways associated with neural cell death, microtubule assembly and actin polymerization.


Assuntos
Axônios/efeitos dos fármacos , Isoquinolinas/uso terapêutico , Regeneração Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Traumatismos do Nervo Óptico/tratamento farmacológico , Sulfonamidas/uso terapêutico , Animais , Axônios/fisiologia , Isoquinolinas/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Compressão Nervosa , Regeneração Nervosa/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/fisiopatologia , Fosforilação/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Sulfonamidas/administração & dosagem
11.
Plast Reconstr Surg ; 146(2): 250-254, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32740571

RESUMO

The concept of sensate autologous breast reconstruction is not novel, and prior literature has focused mainly on sensate abdominally based breast reconstruction. The goal of this article is to present the authors' results with a novel technique performing sensate implant-based reconstruction. A database was prospectively maintained for patients who underwent implant-based sensate breast reconstruction. The anterior branch of the lateral fourth intercostal is identified and preserved during the mastectomy by the breast surgeon. A processed nerve allograft is used as an interpositional graft connecting the donor nerve to the targeted nipple-areola complex. The sensory recovery process was objectively monitored using a pressure-specified sensory device. Thirteen patients underwent the proposed technique. Eight patients with 15 breasts were monitored for sensory recovery. For sensory measurement, the nipple had a mean threshold of 67.33 ± 34.48 g/nm. The upper inner (29 ± 26.75 g/nm) and upper outer (46.82 ± 32.72 g/nm) nipple-areola complex quadrants demonstrated better scores during the moving test compared with the static test. Mean time between the test and surgery was 4.18 ± 2.3 months, and mean time between the second test and surgery was 10.59 ± 3.57 months. Threshold improvements were documented after the second test for all nipple-areola complex areas evaluated. This is the first study to report on early results obtained after performing sensate implant-based breast reconstruction. More studies are required to determine the long-term outcomes and impact on quality of life and to assess whether patient or breast characteristics impact the success of this procedure.


Assuntos
Implantes de Mama , Mamoplastia/métodos , Regeneração Nervosa/fisiologia , Transferência de Nervo/métodos , Mamilos/cirurgia , Recuperação de Função Fisiológica , Sensação/fisiologia , Adulto , Neoplasias da Mama/cirurgia , Feminino , Seguimentos , Humanos , Mastectomia Subcutânea/métodos , Mamilos/inervação , Estudos Prospectivos , Qualidade de Vida , Fatores de Tempo
12.
J Morphol ; 281(10): 1260-1270, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32770765

RESUMO

The transected lumbar spinal cord of lizards was studied for its ability to recover after paralysis. At 34 days post-lesion about 50% of lizards were capable of walking with a limited coordination, likely due to the regeneration of few connecting axons crossing the transection site of the spinal cord. This region, indicated as "bridge", contains glial cells among which oligodendrocytes and their elongation that are immunolabeled for NOGO-A. A main reactive protein band occurs at 100-110 kDa but a weaker band is also observed around 240 kDa, suggesting fragmentation of the native protein due to extraction or to physiological processing of the original protein. Most of the cytoplasmic immunolabeling observed in oligodendrocytes is associated with vesicles of the endoplasmic reticulum. Also, the nucleus is labeled in some oligodendrocytes that are myelinating sparse axons observed within the bridge at 22-34 days post-transection. This suggests that axonal regeneration is present within the bridge region. Immunolabeling for NOGO-A shows that the protein is also present in numerous reactive neurons, in particular motor-neurons localized in the proximal stump of the transected spinal cord. Ultrastructural immunolocalization suggests that NOGO is synthesized in the ribosomes of these neurons and becomes associated with the cisternae of the endoplasmic reticulum, probably following a secretory pathway addressed toward the axon. The present observations suggest that, like for the regenerating spinal cord of fish and amphibians, also in lizard NOGO-A is present in reactive neurons and appears associated to axonal regeneration and myelination.


Assuntos
Lagartos/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Proteínas Nogo/metabolismo , Medula Espinal/citologia , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Comportamento Animal , Encéfalo/metabolismo , Regeneração Nervosa/fisiologia , Neuroglia/patologia , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Oligodendroglia/metabolismo , Oligodendroglia/ultraestrutura , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia
13.
J Neurosci ; 40(31): 5908-5921, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601248

RESUMO

SORLA is a transmembrane trafficking protein associated with Alzheimer's disease risk. Although SORLA is abundantly expressed in neurons, physiological roles for SORLA remain unclear. Here, we show that cultured transgenic neurons overexpressing SORLA feature longer neurites, and accelerated neurite regeneration with wounding. Enhanced release of a soluble form of SORLA (sSORLA) is observed in transgenic mouse neurons overexpressing human SORLA, while purified sSORLA promotes neurite extension and regeneration. Phosphoproteomic analyses demonstrate enrichment of phosphoproteins related to the epidermal growth factor (EGFR)/ERK pathway in SORLA transgenic mouse hippocampus from both genders. sSORLA coprecipitates with EGFR in vitro, and sSORLA treatment increases EGFR Y1173 phosphorylation, which is involved in ERK activation in cultured neurons. Furthermore, sSORLA triggers ERK activation, whereas pharmacological EGFR or ERK inhibition reverses sSORLA-dependent enhancement of neurite outgrowth. In search for downstream ERK effectors activated by sSORLA, we identified upregulation of Fos expression in hippocampus from male mice overexpressing SORLA by RNAseq analysis. We also found that Fos is upregulated and translocates to the nucleus in an ERK-dependent manner in neurons treated with sSORLA. Together, these results demonstrate that sSORLA is an EGFR-interacting protein that activates EGFR/ERK/Fos signaling to enhance neurite outgrowth and regeneration.SIGNIFICANCE STATEMENT SORLA is a transmembrane trafficking protein previously known to reduce the levels of amyloid-ß, which is critical in the pathogenesis of Alzheimer's disease. In addition, SORLA mutations are a risk factor for Alzheimer's disease. Interestingly, the SORLA ectodomain is cleaved into a soluble form, sSORLA, which has been shown to regulate cytoskeletal signaling pathways and cell motility in cells outside the nervous system. We show here that sSORLA binds and activates the EGF receptor to induce downstream signaling through the ERK serine/threonine kinase and the Fos transcription factor, thereby enhancing neurite outgrowth. These findings reveal a novel role for sSORLA in promoting neurite regeneration through the EGF receptor/ERK/Fos pathway, thereby demonstrating a potential neuroprotective mechanism involving SORLA.


Assuntos
Receptores ErbB/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Regeneração Nervosa/fisiologia , Neuritos/fisiologia , Receptores de LDL/fisiologia , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Genes fos , Hipocampo/fisiologia , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fosforilação , Receptores de LDL/genética
14.
Brain ; 143(7): 2009-2026, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32651949

RESUMO

We only have a rudimentary understanding of the molecular and cellular determinants of nerve regeneration and neuropathic pain in humans. This cohort study uses the most common entrapment neuropathy (carpal tunnel syndrome) as a human model system to prospectively evaluate the cellular and molecular correlates of neural regeneration and its relationship with clinical recovery. In 60 patients undergoing carpal tunnel surgery [36 female, mean age 62.5 (standard deviation 12.2) years], we used quantitative sensory testing and nerve conduction studies to evaluate the function of large and small fibres before and 6 months after surgery. Clinical recovery was assessed with the global rating of change scale and Boston Carpal Tunnel Questionnaire. Twenty healthy participants provided normative data [14 female, mean age 58.0 (standard deviation 12.9) years]. At 6 months post-surgery, we noted significant recovery of median nerve neurophysiological parameters (P < 0.0001) and improvements in quantitative sensory testing measures of both small and large nerve fibre function (P < 0.002). Serial biopsies revealed a partial recovery of intraepidermal nerve fibre density [fibres/mm epidermis pre: 4.20 (2.83), post: 5.35 (3.34), P = 0.001], whose extent correlated with symptom improvement (r = 0.389, P = 0.001). In myelinated afferents, nodal length increased postoperatively [pre: 2.03 (0.82), post: 3.03 (1.23), P < 0.0001] suggesting that this is an adaptive phenomenon. Transcriptional profiling of the skin revealed 31 differentially expressed genes following decompression, with ADCYAP1 (encoding pituitary adenylate cyclase activating peptide, PACAP) being the most strongly upregulated (log2 fold-change 1.87, P = 0.0001) and its expression was associated with recovery of intraepidermal nerve fibres. We found that human induced pluripotent stem cell-derived sensory neurons expressed the receptor for PACAP and that this peptide could significantly enhance axon outgrowth in a dose-dependent manner in vitro [neurite length PACAP 1065.0 µm (285.5), vehicle 570.9 µm (181.8), P = 0.003]. In conclusion, carpal tunnel release is associated with significant cutaneous reinnervation, which correlates with the degree of functional improvement and is associated with a transcriptional programme relating to morphogenesis and inflammatory processes. The most highly dysregulated gene ADCYAP1 (encoding PACAP) was associated with reinnervation and, given that this peptide signals through G-protein coupled receptors, this signalling pathway provides an interesting therapeutic target for human sensory nerve regeneration.


Assuntos
Regeneração Nervosa/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Células Receptoras Sensoriais/metabolismo , Adulto , Idoso , Síndrome do Túnel Carpal , Estudos de Coortes , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade
15.
Acta Cir Bras ; 35(4): e202000405, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555937

RESUMO

PURPOSE: To investigate the effects of Chemically Extracted Acellular Nerves (CEANs) when combined with Adipose-Derived mesenchymal Stem Cell (ADSC) transplantation on the repair of sciatic nerve defects in rabbits. METHODS: A total of 71 six-month-old Japanese rabbit were used in this study. Twenty rabbits served as sciatic nerve donors, while the other 51 rabbits were randomly divided into Autologous Nerve Transplantation Group (ANT, n=17), CEAN group (n=17) and CEAN-ADSCs group (n=17). In all these groups, the rabbit's left sciatic nerves were injured before the experiment, and the uninjured sciatic nerves on their right side were used as the control (CON). Electrophysiological tests were carried out and sciatic nerves were prepared for histomorphology and stretch testing at 24 weeks post-transplant. RESULTS: There were significant differences between ANT and Con groups in amplitude (AMP): P=0.031; motor nerve conduction velocity (MNCV): P=0.029; Maximum stress: P=0.029; and Maximum strain P=0.027. There were also differences between the CEAN and CEAN+ADSCs groups in AMP: P=0.026, MNCV: P=0.024; Maximum stress: P=0.025 and Maximum strain: P=0.030. No significant differences in these parameters were observed when comparing the ANT and CEAN+SACN groups (MNCV: P=0.071) or the CEAN and ANT groups (Maximum stress: P=0.069; Maximum strain P=0.077). CONCLUSION: Addition of ADSCs has a significant impact on the recovery of nerve function, morphology, and tensile mechanical properties following sciatic nerve injury.


Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais , Tecido Nervoso/transplante , Neuropatia Ciática/fisiopatologia , Neuropatia Ciática/cirurgia , Animais , Fenômenos Biomecânicos , Eletromiografia , Masculino , Regeneração Nervosa/fisiologia , Tecido Nervoso/citologia , Coelhos , Valores de Referência , Reprodutibilidade dos Testes , Nervo Isquiático/fisiopatologia , Nervo Isquiático/cirurgia , Resultado do Tratamento
16.
Muscle Nerve ; 62(3): 404-412, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32497302

RESUMO

BACKGROUND: After facial nerve injury and surgical repair in rats, recovery of vibrissal whisking is associated with a high proportion of mono-innervated neuro-muscular junctions (NMJs). Our earlier work with Sprague Dawley (SD)/Royal College of Surgeons (RCS) rats, which are blind and spontaneously restore NMJ-monoinnervation and whisking, showed correlations between functional recovery and increase of fibroblast growth factor-2 (FGF2) and brain-derived neurotrophic factor (BDNF) in denervated vibrissal muscles. METHODS: We used normally sighted rats (Wistar), in which NMJ-polyinnervation is highly correlated with poor whisking recovery, and injected the vibrissal muscle levator labii superioris (LLS) with combinations of BDNF, anti-BDNF, and FGF2 at different postoperative periods after facial nerve injury. RESULTS: Rats receiving anti-BDNF+FGF2 showed low NMJ-polyinnervation and best recovery of whisking amplitude. CONCLUSIONS: Restoration of target reinnervation after peripheral nerve injury requires a complex mixture of trophic factors with a specific time course of availability for each of them.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Fator Neurotrófico Derivado do Encéfalo/imunologia , Traumatismos do Nervo Facial/tratamento farmacológico , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/fisiologia , Vibrissas/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Denervação , Músculos Faciais/efeitos dos fármacos , Músculos Faciais/inervação , Músculos Faciais/fisiopatologia , Traumatismos do Nervo Facial/fisiopatologia , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/efeitos dos fármacos
17.
Proc Natl Acad Sci U S A ; 117(27): 15955-15966, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32554499

RESUMO

Axon regeneration is regulated by a neuron-intrinsic transcriptional program that is suppressed during development but that can be reactivated following peripheral nerve injury. Here we identify Prom1, which encodes the stem cell marker prominin-1, as a regulator of the axon regeneration program. Prom1 expression is developmentally down-regulated, and the genetic deletion of Prom1 in mice inhibits axon regeneration in dorsal root ganglion (DRG) cultures and in the sciatic nerve, revealing the neuronal role of Prom1 in injury-induced regeneration. Elevating prominin-1 levels in cultured DRG neurons or in mice via adeno-associated virus-mediated gene delivery enhances axon regeneration in vitro and in vivo, allowing outgrowth on an inhibitory substrate. Prom1 overexpression induces the consistent down-regulation of cholesterol metabolism-associated genes and a reduction in cellular cholesterol levels in a Smad pathway-dependent manner, which promotes axonal regrowth. We find that prominin-1 interacts with the type I TGF-ß receptor ALK4, and that they synergistically induce phosphorylation of Smad2. These results suggest that Prom1 and cholesterol metabolism pathways are possible therapeutic targets for the promotion of neural recovery after injury.


Assuntos
Antígeno AC133/metabolismo , Axônios/metabolismo , Colesterol/metabolismo , Regeneração Nervosa/fisiologia , Transdução de Sinais , Células-Tronco/metabolismo , Antígeno AC133/genética , Receptores de Ativinas Tipo I , Animais , Axônios/patologia , Colesterol/genética , Regulação para Baixo , Gânglios Espinais/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Nervo Isquiático
18.
PLoS One ; 15(5): e0233531, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453807

RESUMO

Several studies have investigated the use of invasive and non-invasive stimulation methods to enhance nerve regeneration, and varying degrees of effectiveness have been reported. However, due to the use of different parameters in these studies, a fair comparison between the effectiveness of invasive and non-invasive stimulation methods is not possible. The present study compared the effectiveness of invasive and non-invasive stimulation using similar parameters. Eighteen Sprague Dawley rats were classified into three groups: the iES group stimulated with fully implantable device, the tES group stimulated with transcutaneous electrical nerve stimulation (TENS), and the injury group (no stimulation). The iES and tES groups received stimulation for 6 weeks starting immediately after the injury. Motor function was evaluated using the sciatic functional index (SFI) every week. The SFI values increased over time in all groups; faster and superior functional recovery was observed in the iES group than in the tES group. Histological evaluation of the nerve sections and gastrocnemius muscle sections were performed every other week. The axon diameter and muscle fiber area in the iES group were larger, and the g-ratio in the iES group was closer to 0.6 than those in the tES group. To assess the cause of the difference in efficiency, a 3D rat anatomical model was used to simulate the induced electric fields in each group. A significantly higher concentration and intensity around the sciatic nerve was observed in the iES group than in the tES group. Vector field distribution showed that the field was orthogonal to the sciatic nerve spread in the tES group, whereas it was parallel in the iES group; this suggested that the tES group was less effective in nerve stimulation. The results indicated that even though rats in the TENS group showed better recovery than those in the injury group, it cannot replace direct stimulation yet because rats stimulated with the invasive method showed faster recovery and superior outcomes. This was likely attributable to the greater concentration and parallel distribution of electric field with respect to target nerve.


Assuntos
Lesões por Esmagamento/terapia , Regeneração Nervosa/fisiologia , Neuropatia Ciática/terapia , Estimulação Elétrica Nervosa Transcutânea , Animais , Axônios/efeitos da radiação , Lesões por Esmagamento/fisiopatologia , Lesões por Esmagamento/cirurgia , Modelos Animais de Doenças , Humanos , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/efeitos da radiação , Músculo Esquelético/fisiopatologia , Músculo Esquelético/efeitos da radiação , Compressão Nervosa/métodos , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/fisiopatologia , Nervo Isquiático/cirurgia , Neuropatia Ciática/fisiopatologia , Neuropatia Ciática/cirurgia
19.
Nat Commun ; 11(1): 2123, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358558

RESUMO

Mammals differ in their regeneration potential after traumatic injury, which might be caused by species-specific regeneration programs. Here, we compared murine and human Schwann cell (SC) response to injury and developed an ex vivo injury model employing surgery-derived human sural nerves. Transcriptomic and lipid metabolism analysis of murine SCs following injury of sural nerves revealed down-regulation of lipogenic genes and regulator of lipid metabolism, including Pparg (peroxisome proliferator-activated receptor gamma) and S1P (sphingosine-1-phosphate). Human SCs failed to induce similar adaptations following ex vivo nerve injury. Pharmacological PPARg and S1P stimulation in mice resulted in up-regulation of lipid gene expression, suggesting a role in SCs switching towards a myelinating state. Altogether, our results suggest that murine SC switching towards a repair state is accompanied by transcriptome and lipidome adaptations, which are reduced in humans.


Assuntos
Metabolismo dos Lipídeos/fisiologia , Células de Schwann/citologia , Células de Schwann/metabolismo , Animais , Feminino , Humanos , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Bainha de Mielina/metabolismo , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , PPAR gama/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
20.
J Vis Exp ; (158)2020 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-32364547

RESUMO

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species. Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.


Assuntos
Nervo Mediano/fisiologia , Regeneração Nervosa/fisiologia , Fisiologia/métodos , Potenciais de Ação , Animais , Membro Anterior/anatomia & histologia , Membro Anterior/inervação , Força da Mão , Nervo Mediano/anatomia & histologia , Atividade Motora/fisiologia , Músculos/fisiologia , Miografia , Nociceptividade , Ratos Wistar , Recuperação de Função Fisiológica/fisiologia , Temperatura , Termografia , Caminhada
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