RESUMO
This study was designed to see the expression of toll-like receptor 4 (TLR4) and downstream molecules including myeloid differentiation factor 88 (MyD88) and interleukin 1-ß (IL-1ß) in the spinal cord as peripheral nerve injury recovered in mice. We established a model of femoral nerve injury (FNI) in C57BL/6 mice by transection of the motor branch of the femoral nerve, followed by retrograde labeling to show the according motor neurons in the anterior horn of the spinal cord pars lumbar. We observed the motor function recovery of the injured hind limbs using behavioral tests. The expression of TLR4, MyD88, and IL-1ß was examined by immunofluorescent staining and western blot. According to the behavior test, the FNI animals fully recovered within 6-8 weeks. TLR4, MyD88, and IL-1ß were expressed in the ventral horn of the spinal cord both at 72â h till 6 weeks after the femoral nerve transection surgery, and these proteins were mostly co-localized with neurons. IL-1ß also tended to rise in the same surgery groups, but more intimate with microglia surrounding nearby retrograde labeled neurons. And western blot results were consistent with histological findings. The results indicate that peripheral nerve injury may induce innate immune reactions of the central neurons and critical signaling like TLR4/MyD88 in the spinal cord may reflect the recovery of the injury. These findings suggest that peripheral nerve injury triggered the TLR4/MyD88 signal in the soma of spinal neurons may be involved in function and nerve restoration through neuron-glia crosstalk.
Assuntos
Fator 88 de Diferenciação Mieloide , Traumatismos dos Nervos Periféricos , Camundongos , Animais , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismo , Nervo Femoral/metabolismo , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismoAssuntos
Quitosana , Fator Neurotrófico Ciliar , Ratos , Animais , Nervo Óptico/fisiologia , Ratos Sprague-DawleyRESUMO
Alterations in phospholipids have long been associated with spinal cord injury (SCI). However, their specific roles and signaling cascades in mediating cell death and tissue repair remain unclear. Here we investigated whether alterations of cardiolipin (CL), a family of mitochondrion-specific phospholipids, play a crucial role in mitochondrial dysfunction and neuronal death following SCI. Lipidomic analysis was used to determine the profile of CL alteration in the adult rat spinal cord following a moderate contusive SCI at the 10th thoracic (T10) level. Cellular, molecular, and genetic assessments were performed to determine whether CL alterations mediate mitochondrial dysfunction and neuronal death after SCI, and, if so, whether reversing CL alteration leads to neuroprotection after SCI. Using lipidomic analysis, we uncovered CL alterations at an early stage of SCI. Over 50 distinct CL species were identified, of which 50% showed significantly decreased abundance after SCI. The decreased CL species contained mainly polyunsaturated fatty acids that are highly susceptible to peroxidation. In parallel, 4-HNE, a lipid peroxidation marker, significantly increased after SCI. We found that mitochondrial oxidative stress not only induced CL oxidation, but also resulted in CL loss by activating cPLA2 to hydrolyze CL. CL alterations induced mitochondrial dysfunction and neuronal death. Remarkably, pharmacologic inhibition of CL alterations with XJB-5-131, a novel mitochondria-targeted electron and reactive oxygen species scavenger, reduced cell death, tissue damage and ameliorated motor deficits after SCI in adult rats. These findings suggest that CL alteration could be a novel mechanism that mediates injury-induced neuronal death, and a potential therapeutic target for ameliorating secondary SCI.
Assuntos
Cardiolipinas , Traumatismos da Medula Espinal , Ratos , Animais , Cardiolipinas/metabolismo , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Morte Celular , Mitocôndrias/metabolismo , Fosfolipídeos/metabolismo , HomeostaseRESUMO
Our previous studies revealed that etomidate, a non-barbiturate intravenous anesthetic agent, has protective effects on retinal ganglion cells within 7 days after optic nerve transection. Whether this process is related to anti-oxidative stress is not clear. To reveal its mechanism, we established the optic nerve transection injury model by transecting 1 mm behind the left eyeball of adult male Sprague-Dawley rats. The rats received an intraperitoneal injection of etomidate (4 mg/kg) once per day for 7 days. The results showed that etomidate significantly enhanced the number of retinal ganglion cells retrogradely labeled with Fluorogold at 7 days after optic nerve transection. Etomidate also significantly reduced the levels of nitric oxide and malonaldehyde in the retina and increased the level of glutathione at 12 hours after optic nerve transection. Thus, etomidate can protect retinal ganglion cells after optic nerve transection in adult rats by activating an anti-oxidative stress response. The study was approved by the Animal Ethics Committee at Air Force Medical University, China (approval No. 20180305) on March 5, 2018.
RESUMO
Prenatal alcohol exposure (PAE) could lead to developmental disorders of the central nervous system (CNS) and mental retardation. Toll-like receptor (TLR) 4 plays an important role in PAE-induced neurodevelopmental defects. However, how PAE affects TLR4 response in the brain remains controversial. Using a moderate PAE model by feeding pregnant rats with liquid ethanol diet, we investigated the TLR4-mediated response to intraventricular injection of lipopolysaccharide (LPS) in the hippocampus of PEA rats at postnatal day (PND) 30. The results showed that PAE significantly up-regulated the expression of Toll-Interleukin-1 Receptor (TIR)-domain-containing adaptor protein inducing interferon (IFN)-ß (TRIF), TNF-α, and IL-1ß in the rat hippocampus in the absence of LPS, indicated by western blot assay. LPS treatment dramatically up-regulated the expressions of TLR4 and its downstream molecules in the hippocampus of paired-food and control groups. But no such significant changes of those molecules were found in the hippocampus of PAE animals. Moreover, the LPS stimulation even down-regulated the levels of TLR4 and TRIF in the PAE group. These data suggest that the relatively moderate level of PAE may lead to a mild neuroinflammation and a suppression of TLR4-mediated response to LPS in the hippocampus of young rats. As innate immunity plays crucial roles in CNS development, moderate PAE-induced suppression of TLR4-mediated response may serve as a new candidate mechanism of CNS developmental defects.
Assuntos
Etanol/efeitos adversos , Hipocampo/imunologia , Imunidade Inata/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Animais , Células Cultivadas , Regulação para Baixo , Feminino , Injeções Intraventriculares , Interferon beta/biossíntese , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Masculino , Gravidez , Ratos , Fator de Necrose Tumoral alfa/biossíntese , Regulação para Cima/efeitos dos fármacosRESUMO
PURPOSE: This study's aim was to investigate the beneficial effects of branched-chain amino acids (BCAAs) on the neuronal survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) transection. METHOD: The experimental rats received daily BCAA injections through the caudal vein after left intra-orbital ON transection. Neuroprotection was evaluated by counting Fluorogold-labeled RGCs. The role of mammalian target of rapamycin (mTOR) pathway activation in promoting RGC survival was studied after rapamycin administration. Moreover, a peripheral nerve (PN) graft was transplanted onto the transected ON to study the effects of BCAAs on axon regeneration of injured RGCs. RESULTS: Our results showed that BCAAs alleviated the death of RGCs 7 and 14 days after ON transection, accompanied by an activation of mTOR pathway in RGCs. Blocking mTOR pathway with rapamycin eliminated such neuroprotective effects of BCAAs. Moreover, BCAAs also promoted axon regeneration of injured RGCs into a PN graft. CONCLUSION: Our results suggest a neuroprotection of BCAAs through the activation of mTOR pathway. BCAAs also have a beneficial effect on axon regeneration of injured RGCs. Therefore, BCAAs could be considered for the clinical treatment of ON injury.
Assuntos
Aminoácidos de Cadeia Ramificada/uso terapêutico , Regeneração Nervosa/efeitos dos fármacos , Traumatismos do Nervo Óptico/tratamento farmacológico , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologiaRESUMO
Cell therapy has been shown to be a key clinical therapeutic option for central nervous system diseases or damage. Standardization of clinical cell therapy procedures is an important task for professional associations devoted to cell therapy. The Chinese Branch of the International Association of Neurorestoratology (IANR) completed the first set of guidelines governing the clinical application of neurorestoration in 2011. The IANR and the Chinese Association of Neurorestoratology (CANR) collaborated to propose the current version "Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)". The IANR council board members and CANR committee members approved this proposal on September 1, 2016, and recommend it to clinical practitioners of cellular therapy. These guidelines include items of cell type nomenclature, cell quality control, minimal suggested cell doses, patient-informed consent, indications for undergoing cell therapy, contraindications for undergoing cell therapy, documentation of procedure and therapy, safety evaluation, efficacy evaluation, policy of repeated treatments, do not charge patients for unproven therapies, basic principles of cell therapy, and publishing responsibility.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Humanos , Regeneração Nervosa/fisiologia , Controle de QualidadeRESUMO
Sphingosine-1-phosphate and its structural analog FTY720 (fingolimod) are important in the inhibition of osteoclast differentiation and bone resorption, however, it remains unknown whether they enhance osteogenic differentiation of the bone marrow mesenchymal stem cells (BMMSCs). The present study investigated the effect of FTY720 on the osteogenic differentiation of BMMSCs from the femurs of the ovariectomized (OVX) rats. Three different concentrations (1, 10 and 100 nM) of FTY720 were demonstrated to markedly upregulate mRNA expression levels of Runtrelated transcription factor 2 (Runx2) and Sp7 transcription factor (Sp7) at 2 weeks, and alkaline phosphatase (ALP) at 3 weeks. The osteocalcin (OCN) expression was similar at weeks 2 and 3. The protein expression levels of Runx2, Sp7, OCN and ALP induced by three different concentrations of FTY720 were higher than those in the control groups at 3 weeks in the OVX and sham groups. The findings of the current study suggested a beneficial effect of FTY720 on bone formation in OVX rats, and provided a potential therapeutic method of FTY720 to prevent alveolar bone resorption in patients with postmenopausal osteoporosis.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Biomarcadores , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Imunofenotipagem , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Osteoporose/diagnóstico por imagem , Osteoporose/etiologia , Osteoporose/metabolismo , Osteoporose/patologia , Ovariectomia , RatosRESUMO
Peripheral nerve (PN) grafts can be used to bridge tissue defects in the CNS. Using a PN-to-optic nerve (ON) graft model, we combined gene therapy with pharmacotherapy to promote the long-distance regeneration of injured adult retinal ganglion cells (RGCs). Autologous sciatic nerve was sutured onto the transected ON and the distal end immediately inserted into contralateral superior colliculus (SC). Control rats received intraocular injections of saline or adeno-associated virus (AAV) encoding GFP. In experimental groups, three bi-cistronic AAV vectors encoding ciliary neurotrophic factor (CNTF) were injected into different regions of the grafted eye. Each vector encoded a different fluorescent reporter to assess retinotopic order in the regenerate projection. To encourage sprouting/synaptogenesis, after 6 weeks some AAV-CNTF injected rats received an intravitreal injection of recombinant brain-derived neurotrophic factor (rBDNF) or AAV-BDNF. Four months after surgery, cholera toxin B was used to visualize regenerate RGC axons. RGC viability and axonal regrowth into SC were significantly greater in AAV-CNTF groups. In some cases, near the insertion site, regenerate axonal density resembled retinal terminal densities seen in normal SC. Complex arbors were seen in superficial but not deep SC layers and many terminals were immunopositive for presynaptic proteins vGlut2 and SV2. There was improvement in visual function via the grafted eye with significantly greater pupillary constriction in both AAV-CNTF+BDNF groups. In both control and AAV-CNTF+rBDNF groups the extent of light avoidance correlated with the maximal distance of axonal penetration into superficial SC. Despite the robust regrowth of RGC axons back into the SC, axons originating from different parts of the retina were intermixed at the PN graft/host SC interface, indicating that there remained a lack of order in this extensive regenerate projection.
Assuntos
Comportamento Animal , Encéfalo/anatomia & histologia , Terapia Genética , Nervos Periféricos/transplante , Retina/anatomia & histologia , Vias Visuais/anatomia & histologia , Animais , Axônios/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fator Neurotrófico Ciliar/metabolismo , Feminino , Neurogênese/efeitos dos fármacos , Nistagmo Optocinético/efeitos dos fármacos , Nervo Óptico/transplante , Ratos , Reflexo Pupilar/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Nervo Isquiático/transplante , Visão Ocular/efeitos dos fármacosRESUMO
To mimic multilevel nerve root compression and intervertebral foramina stenosis in human, we established a new animal model of the chronic compression of unilateral multiple lumbar DRGs (mCCD) in the rat. A higher occurrence of signs of spontaneous pain behaviors, such as wet-dog shaking and spontaneous hind paw shrinking behaviors, was firstly observed from day 1 onward. In the meantime, the unilateral mCCD rat exhibited significant bilateral hind paw mechanical and cold allodynia and hyperalgesia, as well as a thermal preference to 30°C plate between 30 and 35°C. The expression of activating transcription factor 3 (ATF3) was significantly increased in the ipsilateral and contralateral all-sized DRG neurons after the mCCD. And the expression of CGRP was significantly increased in the ipsilateral and contralateral large- and medium-sized DRG neurons. ATF3 and CGRP expressions correlated to evoked pain hypersensitivities such as mechanical and cold allodynia on postoperative day 1. The results suggested that bilateral neuropathy of primary sensory neurons might contribute to bilateral hypersensitivity in the mCCD rat.
Assuntos
Gânglios Espinais/fisiopatologia , Hiperalgesia/fisiopatologia , Síndromes de Compressão Nervosa/fisiopatologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Hiperalgesia/etiologia , Masculino , Síndromes de Compressão Nervosa/complicações , Medição da Dor , Doenças do Sistema Nervoso Periférico/etiologia , Ratos , Ratos Sprague-DawleyRESUMO
Neuroprotection of lithium for axotomized retinal ganglion cells (RGCs) is attributed to upregulated intraretinal Bcl-2. As lithium also upregulates brain-derived neurotrophic factor (BDNF) which can rescue axotomized RGCs, it is hypothesized that lithium could protect RGCs through BDNF. This study investigated this hypothesis and a possible relationship between the dose and protection of lithium. All adult experimental rats received daily intraperitoneal injections of lithium chloride (LiCl) at 30, 60 or 85 mg/kg·bw until they were euthanized 2, 7 or 14 days after left intraorbital optic nerve (ON) transection. Our results revealed that RGC densities promoted and declined with increased dose of LiCl and the highest RGC densities were always in the 60 mg/kg·bw LiCl group at both 7 and 14 day points. Similar promotion and decline in the mRNA and protein levels of intraretinal BDNF were also found at the 14 day point, while such BDNF levels increased in the 30 mg/kg·bw LiCl group but peaked in the 60 and 85 mg/kg·bw LiCl groups at the 7 day point. These findings suggested that lithium can delay the death of axotomized RGCs in a dose-dependent manner within a certain period after ON injury and such beneficial effect is interrelated with an upregulated level of intraretinal BDNF.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cloreto de Lítio/farmacologia , Substâncias Protetoras/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Relação Dose-Resposta a Droga , Feminino , Imuno-Histoquímica , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Células Ganglionares da Retina/metabolismo , Fatores de TempoRESUMO
We aimed to investigate whether peripheral low-dose lipopolysaccharide (LPS) induces the breakdown of the blood-brain barrier (BBB) and/or the activation of toll-like receptor 4 (TLR4) in the neonatal rat brain. Neonatal rats received intraperitoneal injections of low-dose LPS (0.3 mg/kgâbw), and the BBB compromise was detected by Evans Blue extravasation and electron microscopy. Meanwhile, TLR4, adaptin myeloid differentiation factor 88 (MyD88), nuclear transcription factor kappa-B (NF-κB) p50 and tumor necrosis factor alpha (TNFα) in the neonatal rat brain were determined by quantitative real-time polymerase chain reaction (PCR) and Western Blot. Immunohistochemistry was used to determine the distribution and activation of microglia in the brain after LPS administration. It was demonstrated that Evans Blue extravasation was not observed in the brain parenchyma, and that tight junctions of cerebral endothelial cells remained intact after systemic injections of LPS in neonatal rats. Although intracerebroventricular injections of LPS activated microglia and up-regulated the expression of TLR4, MyD88, NF-κB p50 and TNFα in the neonatal rat brain, systemic LPS did not induce these responses. These findings indicate that while the neonatal rat brain responds to the direct intra-cerebral administration of LPS through robust TLR4 activation, systemic low-dose LPS does not induce the innate immune reaction or compromise the BBB in neonatal rats.
Assuntos
Barreira Hematoencefálica/ultraestrutura , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Ratos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Animais Recém-Nascidos , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/microbiologia , Feminino , Injeções , Masculino , Microglia/imunologia , Microglia/microbiologia , Ratos/microbiologia , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Recent studies demonstrated that mature atrocytes have the capacity for de-differentiating into neural stem/progenitor cells (NSPCs) in vitro and in vivo. However, it is still unknown what signals endow astroglial cells with a de-differentiation potential. Furthermore, the signaling molecules and underlying mechanism that confer astrocytes with the competence of NSPC phenotypes have not been completely elucidated. Here, we found that sonic hedgehog (Shh) production in astrocytes following mechanical injury was significantly elevated, and that incubation of astrocyes with the injured astrocyte conditioned medium (ACM) causes astrocytes to gradually lose their immunophenotypical profiles, and acquire NSPC characteristics, as demonstrated by down-regulation of typical astrocytic markers (GFAP and S100) and up-regulation of markers that are generally expressed in NSCs, (nestin, Sox2, and CD133). ACM treated astrocytes exhibit self-renewal capacity and multipotency similar to NSPCs. Concomitantly, in addition to Ptc, there was a significant up-regulation of the Shh downstream signal components Gli2 and Cyclin D1 which are involved in cell proliferation, dramatic changes in cell morphology, and the disruption of cell-cycle G1 arrest. Conversely, the depletion of Shh by administration of its neutralizing antibody (Shh n-Ab) effectively inhibited the de-differentiation process. Strikingly, Shh alone had little effect on astrocyte de-differentiation to NSPCs. These data above suggest that Shh is a key instructive molecule while other molecules secreted from insulted astrocytes may synergistically promote the de-differentiation event.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Desdiferenciação Celular , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Animais , Astrócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Ciclina D1/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Receptores Patched , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Estresse Mecânico , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteína Gli2 com Dedos de ZincoRESUMO
It has long been promulgated that microglial cells serve beneficial roles in the central nervous system (CNS). The beneficial role of microglial cells is considered to be linked with microglial activation and consequent up-regulation of various trophic factors. However, what triggers microglial activation and consequent elevated level of trophic factors, especially brain-derived neurotrophic factor (BDNF), following traumatic CNS injury has become a crucial but elusive issue. Furthermore, an effort still remains in understanding of the cellular and molecular mechanisms underlying the endogenous neuroprotection of activated microglial cells. In this study, we demonstrated that mechanically-injured astrocyte conditioned medium (ACM) could provoke beneficial activation of microglial cells and thus promote the transcription, synthesis and release of BDNF in cultured microglial cells. The microglia-derived BDNF can exerted a demonstrable biological role in promoting neurite outgrowth and intimate terminal contacts of dorsal root ganglion (DRG) neurons co-cultured with microglial cells. Moreover, ACM induced remarkable p38MAPK phosphorylation in cultured microglial cells that preceded the burst of BDNF. Activating p38-MAPK by anisomycin resulted in salutary effects similar to those seen with ACM, whereas specific inhibition of the p38MAPK by SB203580 abrogated all the positive effects of ACM, including BDNF promotion and subsequent neurite outgrowth of DRG neurite outgrowth of DRG neurons and their intimate terminal contacts with microglial cells. Together, our results indicated that the neuroprotection of the microglial source is mainly caused by micro-environmental soluble molecules released from injured astrocytes, and ACM-induced BDNF production and release from microglial cells may be mediated through p38-MAPK signaling pathway. Therefore, these findings may lay a foundation to further investigations on the microglial beneficial activation role in the repair of traumatic CNS injury and neurodegenerative diseases.
Assuntos
Astrócitos/fisiologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Ativação de Macrófagos/fisiologia , Microglia/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Western Blotting , Células Cultivadas , Imunofluorescência , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/fisiologia , Microglia/fisiologia , Neuritos/fisiologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Medula Espinal/citologia , Estresse Mecânico , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
PURPOSE: To compare neuroprotection and therapeutic time windows of two diazepam regimens on retinal ganglion cells (RGCs) after rat optic nerve transection (ONT). METHODS: Adult rats received initial intraperitoneal diazepam injections 30 minutes before left ONT, followed by daily diazepam (regimen-A) or every 8 hours for 3 days (regimen-B) until they were killed at day 7 or 14. Initial diazepam in regimen-A and regimen-B was delayed to 3, 6, 7, 9, 10, 12 and 6, 7, 8, 9, 10, 12 hours after ONT and these animals survived for 7 days. The effect of daily combinational uses of diazepam and bicuculline was assayed at 7 days. RESULTS: Regimen-A induced higher RGC densities than those in control and regimen-B groups at day 7, but lower density than regimen-B did at day 14. When initial diazepam was delayed beyond 6 or 8 hours after ONT with regimen-A and regimen-B, the promoting effects of diazepam on RGC densities disappeared. Bicuculline completely inhibited the protection of diazepam. CONCLUSIONS: Prolonged neuroprotection on RGCs at day 14 and extended therapeutic time window for 8 hours can be achieved by regimen-B, while regimen-A induces a stronger neuroprotection at day 7. Diazepam neuroprotection is mediated through GABAA receptor.
Assuntos
Diazepam/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Traumatismos do Nervo Óptico/tratamento farmacológico , Células Ganglionares da Retina/efeitos dos fármacos , Envelhecimento , Animais , Axotomia/efeitos adversos , Contagem de Células/métodos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Injeções Intraperitoneais/métodos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine (L-NA) on the survival of retinal ganglion cells (RGCs) at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 (1.0, 3.0 and 4.5 mg/kg) and L-NA (1.5, 3.0, 4.5 and 6.0 mg/kg) increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.
RESUMO
Radial glial cells play a significant role in the repair of spinal cord injuries as they exert critical role in the neurogenesis and act as a scaffold for neuronal migration. Our previous study showed that mature astrocytes of spinal cord can undergo a de-differentiation process and further transform into pluripotential neural precursors; the occurrence of these complex events arise directly from the induction of diffusible factors released from scratch-insulted astrocytes. However, it is unclear whether astrocytes can also undergo rejuvenation to revert to a radial glial progenitor phenotype after the induction of scratch-insulted astrocytes conditioned medium (ACM). Furthermore, the mechanism of astrocyte de-differentiation to the progenitor cells is still unclear. Here we demonstrate that upon treating mature astrocytes with ACM for 10 days, the astrocytes exhibit progressive morphological and functional conversion to radial glial cells. These changes include the appearance of radial glial progenitor cells, changes in the immunophenotypical profiles, characterized by the co-expression of nestin, paired homeobox protein (Pax6) and RC2 as well as enhanced capability of multipotential differentiation. Concomitantly, ErbB2 protein level was progressively up-regulated. Thereby these results provide a potential mechanism by which ACM could induce mature astrocytes to regain the profile of radial glial progenitors due to activating the ErbB2 signaling pathways.
Assuntos
Astrócitos/citologia , Diferenciação Celular , Receptor ErbB-2/metabolismo , Animais , Sequência de Bases , Western Blotting , Meios de Cultivo Condicionados , Primers do DNA , Imuno-Histoquímica , Neuroglia/citologia , Neuroglia/metabolismo , Reação em Cadeia da Polimerase , RatosRESUMO
In addition to a fast activating and immediately inactivating inward sodium current, many types of excitable cells possess a noninactivating or slowly inactivating component: the persistent sodium current (I(NaP)). The I(NaP) is found in normal primary sensory neurons where it is mediated by tetrodotoxin-sensitive sodium channels. The dorsal root ganglion (DRG) is the gateway for ectopic impulses that originate in pathological pain signals from the periphery. However, the role of I(NaP) in DRG neurons remains unclear, particularly in neuropathic pain states. Using in vivo recordings from single medium- and large-diameter fibers isolated from the compressed DRG in Sprague-Dawley rats, we show that local application of riluzole, which blocks the I(NaP), also inhibits the spontaneous activity of A-type DRG neurons in a dose-dependent manner. Significantly, riluzole also abolished subthreshold membrane potential oscillations (SMPOs), although DRG neurons still responded to intracellular current injection with a single full-sized spike. In addition, the I(NaP) was enhanced in medium- and large-sized neurons of the compressed DRG, while bath-applied riluzole significantly inhibited the I(NaP) without affecting the transient sodium current (I(NaT)). Taken together, these results demonstrate for the first time that the I(NaP) blocker riluzole selectively inhibits I(NaP) and thereby blocks SMPOs and the ectopic spontaneous activity of injured A-type DRG neurons. This suggests that the I(NaP) of DRG neurons is a potential target for treating neuropathic pain at the peripheral level.
Assuntos
Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Ativação do Canal Iônico/efeitos dos fármacos , Neurônios/patologia , Riluzol/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Animais , Gânglios Espinais/fisiopatologia , Hiperalgesia/complicações , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Potenciais da Membrana/efeitos dos fármacos , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/patologia , Neurônios/efeitos dos fármacos , Radiculopatia/complicações , Radiculopatia/patologia , Radiculopatia/fisiopatologia , Ratos , Ratos Sprague-DawleyRESUMO
In adult mammals, restoration of function after peripheral nerve injury is often poor and effective therapies are not available. Previously we have shown in mice that a peptide which functionally mimics the human natural killer cell (HNK)-1 trisaccharide epitope significantly improves the outcome of femoral nerve injury. Here we evaluated the translational potential of this treatment using primates. We applied a linear HNK-1 mimetic or a functionally inactive control peptide in silicone cuffs used to reconstruct the cut femoral nerves of adult cynomolgus monkeys (Macaca fascicularis). Functional recovery was evaluated using video-based gait analysis over a 160-day observation period. The final outcome was further assessed using force measurements, H-reflex recordings, nerve histology, and ELISA to assess immunoreactivity to HNK-1 in the treated monkeys. Gait deficits were significantly reduced in HNK-1 mimetic-treated compared with control peptide-treated animals between 60 and 160 days after injury. Better outcome at 160 days after surgery in treated versus control animals was also confirmed by improved quadriceps muscle force, enhanced H-reflex amplitude, decreased H-reflex latency, and larger diameters of regenerated axons. No adverse reactions to the mimetic, in particular immune responses resulting in antibodies against the HNK-1 mimetic or immune cell infiltration into the damaged nerve, were observed. These results indicate the potential of the HNK-1 mimetic as an efficient, feasible, and safe adjunct treatment for nerve injuries requiring surgical repair in clinical settings.
Assuntos
Neuropatia Femoral/tratamento farmacológico , Mimetismo Molecular/fisiologia , Polissacarídeos/uso terapêutico , Receptores Semelhantes a Lectina de Células NK/uso terapêutico , Trissacarídeos/uso terapêutico , Animais , Modelos Animais de Doenças , Estudos de Viabilidade , Neuropatia Femoral/fisiopatologia , Macaca fascicularis , Masculino , Peptídeos Cíclicos/fisiologia , Peptídeos Cíclicos/uso terapêutico , Polissacarídeos/agonistas , Polissacarídeos/fisiologia , Receptores Semelhantes a Lectina de Células NK/agonistas , Receptores Semelhantes a Lectina de Células NK/fisiologia , Recuperação de Função Fisiológica , Trissacarídeos/agonistas , Trissacarídeos/fisiologiaRESUMO
Transplantation of olfactory ensheathing cells (OECs) becomes one of the promising strategies in restoring lost functions of injured central nervous system. Elevated level of expressed brain-derived neurotrophic factor (BDNF) was revealed in the previous studies to be related to the protective effects of OECs on injured cortical and brain stem neurons as well as retinal ganglion cells (RGCs), but no evidence has been obtained to demonstrate whether transplanted OECs protect injured central neurons directly by their secreted BDNF. In the present study, the effects of BDNF neutralization on the neuroprotection of adult OEC-conditioned medium (OEC-CM) on scratch-insulted RGCs were examined. The results showed that OEC-CM protected cultured RGCs from scratch insult, and neutralization of BDNF by BDNF neutralizing antibody attenuated such neuroprotection of the medium. It is thus concluded that neurotrophic factors including BDNF secreted by OECs can protect injured OECs in vitro and BDNF plays a major role in such a protection of OECs.