RESUMO
A variety of organic compounds in human exhaled breath were measured online by mass spectrometry using the fifth (206 nm) and fourth (257 nm) harmonic emissions of a femtosecond ytterbium (Yb) laser as the ionization source. Molecular ions were enhanced significantly by means of resonance-enhanced, two-color, two-photon ionization, which was useful for discrimination of analytes against the background. The limit of detection was 0.15 ppm for acetone in air. The concentration of acetone in exhaled breath was determined for three subjects to average 0.31 ppm, which lies within the range of normal healthy subjects and is appreciably lower than the range for patients with diabetes mellitus. Many other constituents, which could be assigned to acetaldehyde, ethanol, isoprene, phenol, octane, ethyl butanoate, indole, octanol, etc., were observed in the exhaled air. Therefore, the present approach shows potential for use in the online analysis of diabetes mellitus and also for the diagnosis of various diseases, such as COVID-19 and cancers.
Assuntos
Testes Respiratórios , Lasers , Espectrometria de Massas , Humanos , Testes Respiratórios/métodos , Espectrometria de Massas/métodos , COVID-19/diagnóstico , Expiração , Acetona/análise , Compostos Orgânicos Voláteis/análise , Diabetes Mellitus/diagnóstico , SARS-CoV-2/isolamento & purificação , Limite de DetecçãoRESUMO
A sample mixture of fatty acid methyl esters (FAMEs) was measured by femtosecond laser ionization mass spectrometry (fsLIMS) using the fifth (206 nm) and fourth (257 nm) harmonic emissions of an ytterbium (Yb) laser (1030 nm). Molecular ions were observed as the major signals in this technique, providing valuable information concerning the molecular weight and the number of double bonds in the molecule. The mass spectral data were then used as explanatory variables in machine learning based on artificial intelligence (AI) to correlate with objective variables such as the cetane number, kinematic viscosity, specific gravity, a higher heating value, an iodine value, flash point, oxidative stability index, and a cloud point measured for reference biofuel samples containing various FAMEs. The properties of biofuels, i.e., the objective variables, were evaluated from the mass spectral data obtained for unknown samples. The errors in the evaluation were a few percent when the distribution of the FAMEs in the unknown biofuel sample was similar to those of the biofuels used for machine learning. As demonstrated herein, the present approach, involving a combination of fsLIMS and AI, has the potential for use in evaluating the properties of a biofuel and then in solving of environmental issues associated with global warming.
RESUMO
A standard sample mixture containing thirty-seven fatty acid methyl esters (FAMEs) was measured by femtosecond laser ionization mass spectrometry. FAME molecules with double bonds were efficiently ionized via resonance-enhanced two-photon ionization by absorbing the first photon at 206 nm at the edge of the absorption band of the πâπ* transition and subsequently ionized by absorbing the second photon at 257 nm. The intensity of the molecular radical ion was enhanced significantly using this two-color ionization scheme, which minimizes the excess energy in the ionized state, when compared with electron ionization mass spectrometry and vacuum-ultraviolet photoionization mass spectrometry. This approach was then used for the reliable identification of FAMEs contained in an actual sample of biofuel.
RESUMO
Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are suspected to be highly carcinogenic and mutagenic compounds that are present in the environment. Gas chromatography combined with mass spectrometry (GC-MS) is the most frequently used technique for trace analysis. The electron ionization techniques that are currently used in MS, however, typically do not result in the formation of a molecular ion, thus making the determination of these compounds more difficult. In this study, we report on the use of a compact highly-repetitive (low-pulse-energy) ultraviolet (UV) femtosecond laser as the ionization source in combination with a miniature time-of-flight mass analyzer and a time-correlated ion counting system. The UV laser pulses emitted at 343, 257, and 206 nm were produced by harmonic generations of a femtosecond Yb laser emitting at 1030 nm and were utilized for single-color multiphoton ionization. A combination of the 343-nm and 257-nm pulses was further employed to achieve two-color two-photon ionization. This technique was found to be more useful for sensitive detection and also resulted in the formation of a molecular ion. A pump-and-probe technique using these pulses was examined in a proof-of-concept study to measure the femtosecond lifetimes of the nitro-PAHs separated by GC, providing additional information for use in the characterization of the analyte. The developed technique was applied in the analysis of an authentic sample, an organic solvent extract from diesel exhaust particulates. The nitro-PAHs contained in a standard reference material (SRM1975) were determined on a two-dimensional GC-MS display, suggesting that this technique would be useful for the practical trace analysis of nitro-PAHs in environmental samples.
RESUMO
In most cases, a molecular ion is observed in femtosecond laser ionization mass spectrometry, which provides information concerning the molecular weight of the analyte. However, the Ti:sapphire laser currently used as the ionization source is costly and involves special skills for operation and maintenance, which prevents its practical use in many applications. In this study, we report on the development of a miniature time-of-flight mass analyzer with a flight tube length of 65 mm for use in combination with a compact highly-repetitive (120-560 kHz) femtosecond Yb laser and a time-correlated single ion counting system. The fundamental beam (1030 nm) was converted into ultraviolet beams emitting at 343, 257, and 206 nm, which was utilized as an efficient two-photon ionization source. A mass resolution of 670 was achieved for the molecular ion of chlorobenzene, the minimum time for measuring a mass spectrum being 0.1 s. This mass spectrometer was used in the on-site real-time monitoring of products appeared by the combustion of plastic, some nerve agent analogs, and an explosive in the air. The interference arising from nitrogen and oxygen in the air was suppressed, since they require nonresonant four- and three-photon ionizations, respectively. The mass spectrometer was combined with a gas chromatograph and used for the comprehensive analysis of polycyclic aromatic hydrocarbons, suggesting its potential advantage for use in the practical trace analysis of organic compounds in the environmental and forensic sciences.
Assuntos
Lasers , Hidrocarbonetos Policíclicos Aromáticos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Espectrometria de Massas/métodos , Fótons , Hidrocarbonetos Policíclicos Aromáticos/análiseRESUMO
An analyte molecule was ionized using a femtosecond laser as the ionization source and was measured by a twin-type time-of-flight mass spectrometer with long (42 cm) and short (6.4 cm) flight tubes. The signal was measured using an analog signal digitizer and a time-correlated single ion counting system, and performance was evaluated by comparing data obtained from both instruments. The short mass spectrometer had a mass resolution of 450 and was used in the trace analysis of allergenic substances in a fragrance.
RESUMO
INTRODUCTION: The diagnosis and treatment of deep vein thrombosis/pulmonary embolism (DVT/PE) are important issues not only in acute-care hospitals, but also in rehabilitation hospitals. To test the hypothesis that DVT/PE occurring at rehabilitation hospitals is carried over from acute-care hospitals, we evaluated a method of DVT screening on admission that combined D-dimer (D-D) measurement and compression ultrasound (CUS). MATERIAL AND METHODS: This prospective single-center observational study included 1043 patients who were admitted to our rehabilitation hospital between August 1, 2007, and August 1, 2011, after excluding those meeting the exclusion criteria. We screened patients on admission and observed the occurrence of DVT/PE until discharge. RESULTS: Of the 1043 patients, 152 (14.6%) had a D-D level of ≥ 3.0 µg/mL on admission. CUS was performed for these patients and indicated the presence of DVT in 15 patients (1.4%), who were subsequently treated. Of these 15 patients, six (40%) had no DVT symptoms, and five of these six patients had spinal cord injury. Of 137 patients who were CUS negative, two developed DVT/PE within 8 days of hospitalization, and recovery was achieved by treatment. No subsequent occurrence was observed. CONCLUSIONS: These results indicated all cases were carried over from acute-care hospitals. Six out of 15 patients had no symptoms of DVT/PE. Thus, this method of DVT screening on admission to a rehabilitation hospital is useful for risk management.
Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Embolia Pulmonar/diagnóstico , Trombose Venosa/diagnóstico , Adulto , Idoso , Hospitalização , Humanos , Programas de Rastreamento , Pessoa de Meia-Idade , Estudos Prospectivos , Embolia Pulmonar/sangue , Embolia Pulmonar/diagnóstico por imagem , Ultrassonografia , Trombose Venosa/sangue , Trombose Venosa/diagnóstico por imagemRESUMO
The aim of this study was to evaluate whether transplantation of human bone marrow stromal cell-derived Schwann cells (hBMSC-SC) promotes functional recovery after contusive spinal cord injury of adult rats. Human bone marrow stromal cells (hBMSC) were cultured from bone marrow of adult human patients and induced into Schwann cells (hBMSC-SC) in vitro. Schwann cell phenotype was confirmed by immunocytochemistry. Growth factors secreted from hBMSC-SC were detected using cytokine antibody array. Immunosuppressed rats were laminectomized and their spinal cords were contused using NYU impactor (10 g, 25 mm). Nine days after injury, a mixture of Matrigel and hBMSC-SC (hBMSC-SC group) was injected into the lesioned site. Five weeks after transplantation, cresyl-violet staining revealed that the area of cystic cavity was smaller in the hBMSC-SC group than that in the control group. Immunohistochemistry revealed that the number of anti-growth-associated protein-43-positive nerve fibers was significantly larger in the hBMSC-SC group than that in the control group. At the same time, the number of tyrosine hydroxylase- or serotonin-positive fibers was significantly larger at the lesion epicenter and caudal level in the hBMSC-SC group than that in the control group. In electron microscopy, formation of peripheral-type myelin was recognized near the lesion epicenter in the hBMSC-SC group. Hind limb function recovered significantly in the hBMSC-SC group compared with the control group. In conclusion, the functions of hBMSC-SC are comparable to original Schwann cells in rat spinal cord injury models, and are thus potentially useful treatments for patients with spinal cord injury.
Assuntos
Recuperação de Função Fisiológica , Células de Schwann/transplante , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/cirurgia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Diferenciação Celular , Cistos/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Regeneração Nervosa/fisiologia , Ratos , Ratos Wistar , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura , Traumatismos da Medula Espinal/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Adulto JovemRESUMO
Macrophage migration inhibitory factor (MIF) is a multipotential protein that acts as a proinflammatory cytokine, a pituitary hormone, and a cell proliferation and migration factor. The objective of this study was to elucidate the role of MIF in spinal cord injury (SCI) using female MIF knockout (KO) mice. Mouse spinal cord compression injury was produced by application of a static load (T8 level, 20 g, 5 min). We analyzed the motor function of the hind limbs and performed histological examinations. Hind-limb function recovered significantly in the KO mice starting from three weeks after injury. Cresyl-violet staining revealed that the number of surviving neurons in the KO mice was significantly larger than that of WT mice six weeks after injury. Immunohistochemical analysis revealed that the number of NeuN/caspase-3-active, double-positive, apoptotic neurons in the KO mice was significantly smaller than that of the WT mice 24 and 72 h after SCI. These results were related to in-vitro studies showing increased resistance of cerebellar granular neurons from MIF-KO animals to glutamate neurotoxicity. These results suggest that MIF existence hinders neuronal survival after SCI. Suppression of MIF may attenuate detrimental secondary molecular responses of the injured spinal cord.
Assuntos
Morte Celular , Locomoção , Fatores Inibidores da Migração de Macrófagos/deficiência , Neurônios/metabolismo , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal , Análise de Variância , Animais , Caspase 3/metabolismo , Células Cultivadas , Cerebelo/patologia , Proteínas de Ligação a DNA , Extremidades/fisiopatologia , Feminino , Ácido Glutâmico/toxicidade , Imuno-Histoquímica , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Proteínas Nucleares/metabolismo , Compressão da Medula Espinal/complicações , Traumatismos da Medula Espinal/etiologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
OBJECT: The authors previously reported that Schwann cells (SCs) could be derived from bone marrow stromal cells (BMSCs) in vitro and that they promoted axonal regeneration of completely transected rat spinal cords in vivo. The aim of the present study is to evaluate the efficacy of transplanted BMSC-derived SCs (BMSC-SCs) in a rat model of spinal cord contusion, which is relevant to clinical spinal cord injury. METHODS: Bone marrow stromal cells were cultured as plastic-adherent cells from the bone marrow of GFPtransgenic rats. The BMSC-SCs were derived from BMSCs in vitro with sequential treatment using beta-mercaptoethanol, all-trans-retinoic acid, forskolin, basic fibroblast growth factor, platelet derived-growth factor, and heregulin. Schwann cells were cultured from the sciatic nerve of neonatal, GFP-transgenic rats. Immunocytochemical analysis and the reverse transcriptase-polymerase chain reaction were performed to characterize the BMSC-SCs. For transplantation, contusions with the New York University impactor were delivered at T-9 in 10- to 11-week-old male Wistar rats. Four groups of rats received injections at the injury site 7 days postinjury: the first received BMSCSCs and matrigel, a second received peripheral SCs and matrigel, a third group received BMSCs and matrigel, and a fourth group received matrigel alone. Histological and immunohistochemical studies, electron microscopy, and functional assessments were performed to evaluate the therapeutic effects of BMSC-SC transplantation. RESULTS: Immunohistochemical analysis and reverse transcriptase-polymerase chain reaction revealed that BMSC-SCs have characteristics similar to SCs not only in their morphological characteristics but also in their immunocytochemical phenotype and genotype. Histological examination revealed that the area of the cystic cavity was significantly reduced in the BMSC-SC and SC groups compared with the control rats. Immunohistochemical analysis showed that transplanted BMSCs, BMSC-SCs, and SCs all maintained their original phenotypes. The BMSC-SC and SC groups had a larger number of tyrosine hydroxilase-positive fibers than the control group, and the BMSC-SC group had more serotonin-positive fibers than the BMSC or control group. The BMSC-SC group showed significantly better hindlimb functional recovery than in the BMSC and control group. Electron microscopy revealed that transplanted BMSC-SCs existed in association with the host axons. CONCLUSIONS: Based on their findings, the authors concluded that BMSC-SC transplantation reduces the size of the cystic cavity, promotes axonal regeneration and sparing, results in hindlimb functional recovery, and can be a useful tool for spinal cord injury as a substitute for SCs.
Assuntos
Axônios/fisiologia , Transplante de Medula Óssea/métodos , Regeneração Nervosa , Células de Schwann , Traumatismos da Medula Espinal/terapia , Células Estromais/citologia , Animais , Diferenciação Celular , Masculino , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Vértebras TorácicasRESUMO
The aim of this study was to evaluate the efficacy in adult rat completely transected spinal cord of adenovirus vector-mediated brain-derived neurotrophic factor (BDNF) ex vivo gene transfer to bone marrow stromal cells (BMSC). BMSC were infected with adenovirus vectors carrying beta-galactosidase (AxCALacZ) or BDNF (AxCABDNF) genes. The T8 segment of spinal cord was removed and replaced by graft containing Matrigel alone (MG group) or Matrigel and BMSC infected by AxCALacZ (BMSC-LacZ group) or AxCABDNF (BMSC-BDNF group). Axons in the graft were evaluated by immunohistochemistry and functional recovery was assessed with BBB locomotor scale. In the BMSC-BDNF group, the number of fibers positive for growth associated protein-43, tyrosine hydroxylase, and calcitonin gene-related peptide was significantly larger than numbers found for the MG and BMSC-LacZ groups. Rats from BMSC-BDNF and BMSC-LacZ groups showed significant recovery of hind limb function compared with MG rats; however, there was no significant difference between groups in degree of functional recovery. These findings demonstrate that adenovirus vector-mediated ex vivo gene transfer of BDNF enhances the capacity of BMSC to promote axonal regeneration in this completely transected spinal cord model; however, BDNF failed to enhance hind limb functional recovery. Further investigation is needed to establish an optimal combination of cell therapy and neurotrophin gene transfer for cases of spinal cord injury.
Assuntos
Transplante de Medula Óssea/métodos , Fator Neurotrófico Derivado do Encéfalo/genética , Técnicas de Transferência de Genes , Traumatismos da Medula Espinal/terapia , Células Estromais/transplante , Adenoviridae/genética , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Células Cultivadas , Modelos Animais de Doenças , Vetores Genéticos/genética , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Masculino , Regeneração Nervosa/genética , Plasticidade Neuronal/genética , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Células Estromais/metabolismo , Células Estromais/virologia , Resultado do TratamentoRESUMO
The aim of the present study was to elucidate the effects of granulocyte colony-stimulating factor (G-CSF)-mediated mobilization of bone marrow-derived stem cells on the injured spinal cord. Bone marrow cells of green fluorescent protein (GFP) transgenic mice were transplanted into lethally irradiated C57BL/6 mice. Four weeks after bone marrow transplantation, spinal cord injury was produced by a static load (20 g, 5 min) at T8 level. G-CSF (200 microg/kg/day) was injected subcutaneously for 5 days. Immunohistochemistry for GFP and cell lineage markers was performed to evaluate G-CSF-mediated mobilization of bone marrow-derived cells into injured spinal cord. Hind limb locomotor recovery was assessed for 6 weeks. Immunohistochemistry revealed that G-CSF increased the number of GFP-positive cells in injured spinal cord, indicating that bone marrow-derived cells were mobilized and migrated into injured spinal cord. The numbers of double positive cells for GFP and glial markers were larger in the G-CSF treated mice than in the control mice. Luxol Fast Blue staining revealed that G-CSF promoted white matter sparing. G-CSF treated mice showed significant recovery of hind limb function compared to that of the control mice. In conclusion, G-CSF showed efficacy for spinal cord injury treatment through mobilization of bone marrow-derived cells.
Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Movimento Celular/fisiologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Compressão da Medula Espinal/terapiaRESUMO
OBJECT: The use of human umbilical cord blood (HUCB) cells has been reported to improve functional recovery in cases of central nervous system injuries such as stroke, traumatic brain injury, and spinal cord injury (SCI). The authors investigated the effects of hemopoietic stem cells that were derived from HUCB and transplanted into the injured spinal cords of rats. METHODS: One week after injury, an HUCB fraction enriched in CD34-positive cells was transplanted into the experimental group. In control animals, vehicle (Matrigel) was transplanted. Recovery of motor functions was assessed using the Basso-Beattie-Bresnahan Locomotor Scale, and immunohistochemical examinations were performed. Cells from HUCB that were CD34 positive improved functional recovery, reduced the area of the cystic cavity at the site of injury, increased the volume of residual white matter, and promoted the regeneration or sparing of axons in the injured spinal cord. Immunohistochemical examination revealed that transplanted CD34-positive cells survived in the host spinal cord for at least 3 weeks after transplantation but had disappeared by 5 weeks. The transplanted cells were not positive for neural markers, but they were positive for hemopoietic markers. There was no evidence of an immune reaction at the site of injury in either group. CONCLUSIONS: These results suggest that transplantation of a CD34-positive fraction from HUCB may have therapeutic effects for SCI. The results of this study provide important preclinical data regarding HUCB stem cell-based therapy for SCI.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Transplante de Células-Tronco Hematopoéticas , Traumatismos da Medula Espinal/terapia , Transplante Heterólogo , Animais , Antígenos CD34/fisiologia , Modelos Animais de Doenças , Membro Posterior/fisiopatologia , Humanos , Recém-Nascido , Masculino , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
We compared the effects of hematopoietic stem cell and marrow stromal cell transplantation for spinal cord injury in mice. From green fluorescent protein transgenic mouse bone marrow, lineage-negative, c-kit- and Sca-1-positive cells were sorted as hematopoietic stem cells and plastic-adherent cells were cultured as marrow stromal cells. One week after injury, hematopoietic stem cells or marrow stromal cells were injected into the lesioned site. Functional recovery was assessed and immunohistochemistry was performed. In the hematopoietic stem cell group, a portion of green fluorescent protein-positive cells expressed glial marker. In the marrow stem cell group, a number of green fluorescent protein and fibronectin-double positive cells were observed. No significant difference was observed in the recovery between both groups. Both hematopoietic stem cells and marrow stromal cells have the potential to restore the injured spinal cord and to promote functional recovery.
Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/fisiologia , Traumatismos da Medula Espinal/cirurgia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Fibronectinas/metabolismo , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de TempoRESUMO
The aim of this study was to evaluate whether transplantation of Schwann cells derived from bone marrow stromal cells (BMSC-SCs) promotes axonal regeneration and functional recovery in completely transected spinal cord in adult rats. Bone marrow stromal cells (BMSCs) were induced to differentiate into Schwann cells in vitro. A 4-mm segment of rat spinal cord was removed completely at the T7 level. An ultra-filtration membrane tube, filled with a mixture of Matrigel (MG) and BMSC-SCs (BMSC-SC group) or Matrigel alone (MG group), was grafted into the gap. In the BMSC-SC group, the number of neurofilament- and tyrosine hydroxylase-immunoreactive nerve fibers was significantly higher compared to the MG group, although 5-hydroxytryptamine- or calcitonin gene-related peptide-immunoreactive fibers were rarely detectable in both groups. In the BMSC-SC group, significant recovery of the hindlimb function was recognized, which was abolished by retransection of the graft 6 weeks after transplantation. These results demonstrate that transplantation of BMSC-SCs promotes axonal regeneration of lesioned spinal cord, resulting in recovery of hindlimb function in rats. Transplantation of BMSC-SCs is a potentially useful treatment for spinal cord injury.
Assuntos
Axônios/transplante , Transplante de Medula Óssea/métodos , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/fisiologia , Células de Schwann/transplante , Traumatismos da Medula Espinal/fisiopatologia , Animais , Imuno-Histoquímica , Masculino , Atividade Motora/fisiologia , Ratos , Ratos Wistar , Células de Schwann/citologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Células Estromais/citologia , Células Estromais/transplante , Vértebras Torácicas/inervação , Vértebras Torácicas/cirurgia , Fatores de TempoRESUMO
The purpose of this study was to use a cDNA microarray to identify new genes involved in healing of spinal cord injury. C57BL/6 mice (7-8 weeks, male) were subjected to spinal cord compression injury (SCI) at the T7/8 level (20 g, 5 min; SCI group). For the control group, mice underwent only laminectomy. Mice were killed at 1, 3 and 7 days. cDNA transcribed from mRNA was hybridized to NIA mice 15K microarrays at each time point. We found 84 genes showing significant expressional changes, including higher and lower expression levels in the SCI groups than in the control [more than 1.0 or less than -1.0 using log ratio (base 2)]. Five genes were selected for further quantitative gene expression analysis by real-time reverse transcription (RT)-PCR. For histological examination, we applied in situ hybridization and fluorescence immunohistochemistry. Cathepsin D, metallothionein-1 (MT-1), metallothionein-2 (MT-2), osteopontin (OPN), and tenascin-C were selected for quantitative and histological analysis. Microarray analysis revealed that SCI led to the up-regulation of OPN and cathepsin D expression at 7 days and also of MT-1, MT-2, and tenascin-C expression at 1 day. Tenascin-C was re-up-regulated at 7 days. These values agreed with those of real-time RT-PCR analysis. By double labeling with in situ hybridization and fluorescence immunohistochemistry, MT-1, MT-2 and tenascin-C expression was observed in neurons and glial cells at 1 day, whereas at 7 days the main MT-2 and tenascin-C expression was found in fibronectin-positive fibroblasts. The main cathepsin D and OPN expression was observed in activated macrophages/microglia at 3 and 7 days. The five genes picked up by microarray gene expression profiling were shown to exhibit temporal and spatial changes of expression after SCI. This system is potentially useful for identifying genes that are involved in the response to SCI.
Assuntos
Catepsina D/metabolismo , Metalotioneína/metabolismo , Sialoglicoproteínas/metabolismo , Traumatismos da Medula Espinal/genética , Tenascina/metabolismo , Animais , Comportamento Animal , Catepsina D/genética , Análise por Conglomerados , Imunofluorescência/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/fisiologia , Hibridização In Situ/métodos , Masculino , Metalotioneína/genética , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Osteopontina , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sialoglicoproteínas/genética , Medula Espinal/citologia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Tenascina/genética , Fatores de TempoRESUMO
Neurotrophins have been shown to promote axonal regeneration, but the techniques available for delivering neurotrophins have limited effectiveness. The aim of this study was to evaluate the effect of adenovirus vector mediated gene transfer of brain-derived neurotrophic factor (BDNF) on axonal regeneration after spinal cord injury. We prepared adenovirus vectors encoding either beta-galactosidase (AxCALacZ) or BDNF (AxCABDNF). AxCALacZ was used to assess infection levels of the adenovirus BDNF produced by AxCABDNF was detected by Western blotting and its bioactivity was confirmed by bioassay. As a model of spinal cord injury, the rat spinal cord was completely transected at the T8 level. Immediately after transection, the vectors were injected into both stumps of the spinal cord. Axonal regeneration after transection was assessed by retrograde and anterograde tracing. In AxCALacZ-injected rats, adenovirus-infected cells were observed not only at the injected site but also in brainstem nuclei, as shown by LacZ expression. After the injection of the retrograde tracer fluorogold (FG) distal portion to the transection, AxCABDNF-injected rats showed FG-labeled neurons in the red nucleus. The anterograde tracer biotinylated dextran amine (BDA) injected into the red nucleus was also found in regenerating rubrospinal fibers distal to the transection. These tracing experiments demonstrated the regeneration of descending axons. In addition, rats of the AxCABDNF group showed significant locomotor recovery of hindlimb function, which was completely abolished by re-transection. These results indicate that the recovery was caused by regeneration of rubrospinal axons, not by simple enhancement of the central pattern generator.
Assuntos
Axônios/fisiologia , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Técnicas de Transferência de Genes , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/terapia , Adenoviridae , Animais , Vetores Genéticos/uso terapêutico , Óperon Lac/fisiologia , Masculino , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Macrophage migration inhibitory factor (MIF) is a multipotential protein that acts as a pro-inflammatory cytokine, pituitary hormone, immunoregulator, and mitogen. To elucidate function of MIF in spinal cord injury, we examined expression of MIF after compression-induced spinal cord injury using Northern blot analysis, in situ hybridization and immunohistochemistry. The MIF mRNA was up-regulated in injured spinal cord, peaking 3 days after injury shown by Northern blot analysis. In situ hybridization revealed up-regulation of MIF in microglia accumulating in the lesion epicenter 3 days after injury and astrocytes around the cystic cavity 1 week after injury. Double staining showed co-localization of MIF and tomato lectin in the lesioned site, indicating that microglia accumulating to the lesion epicenter express MIF. The time course of MIF expression is different from that of previous reports about cytokine expression peaking at earlier time points; thus, it is unlikely that MIF acts as a pro-inflammatory factor in the present study. The MIF may contribute to proliferation of astrocytes around the lesioned site in spinal cord injury because of its cell proliferation-promoting property.
Assuntos
Regulação da Expressão Gênica/fisiologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Compressão da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Regulação para Cima/fisiologia , Animais , Northern Blotting/métodos , Contagem de Células/métodos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Microglia/metabolismo , Lectinas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Compressão da Medula Espinal/complicações , Traumatismos da Medula Espinal/etiologia , Fatores de TempoRESUMO
Semaphorin 3A (Sema3A) is a secreted repulsive axon guidance protein. It appears to play important roles in axon fasciculation, branching, neuronal migration, and tissue differentiation during embryonic development. In adults, Sema3A is expressed in spinal motoneurons and in some neurons in the brain. Here, we demonstrate changes in Sema3A expression in the spinal cord after complete transection and in the brain after spinal cord hemisection at the Th8 level in laboratory rats. Semi-quantitative reverse transcriptase-PCR analysis showed that the expression of Sema3A mRNA, which was present in the normal spinal cord, rapidly decreased after transection, reaching its lowest level 1 day after injury. Thereafter, Sema3A expression levels recovered and reached four-fifths of the normal level at 28 days. Double staining by in situ hybridization and fluorescence immunohistochemistry showed that Sema3A was expressed in NeuN-positive neurons, but not in glia in the spinal cord. Sema3A expression was up-regulated in the contralateral cerebral cortex and in the ipsilateral spinal trigeminal nucleus 1-3 days after spinal cord hemisection. It is likely that the up-regulation occurred in neurons whose descending fibers were transected. These results suggest that Sema3A is regulated differently in spinal motoneurons and brain neurons following axonal injury.
Assuntos
Córtex Cerebral/citologia , Regulação da Expressão Gênica , Neurônios/metabolismo , Semaforina-3A/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/citologia , Análise de Variância , Animais , Contagem de Células/métodos , Modelos Animais de Doenças , Lateralidade Funcional/fisiologia , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Laminectomia/métodos , Masculino , Oligorribonucleotídeos Antissenso/metabolismo , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Semaforina-3A/genética , Fatores de TempoRESUMO
Recovery in central nervous system disorders is hindered by the limited ability of the vertebrate central nervous system to regenerate lost cells, replace damaged myelin, and re-establish functional neural connections. Cell transplantation to repair central nervous system disorders is an active area of research, with the goal of reducing functional deficits. Recent animal studies showed that cells of the hematopoietic stem cell (HSC) fraction of bone marrow transdifferentiated into various nonhematopoietic cell lineages. We employed a mouse model of spinal cord injury and directly transplanted HSCs into the spinal cord 1 week after injury. We evaluated functional recovery using the hindlimb motor function score weekly for 5 weeks after transplantation. The data demonstrated a significant improvement in the functional outcome of mice transplanted with hematopoietic stem cells compared with control mice in which only medium was injected. Fluorescent in situ hybridization for the Y chromosome and double immunohistochemistry showed that transplanted cells survived 5 weeks after transplantation and expressed specific markers for astrocytes, oligodendrocytes, and neural precursors, but not for neurons. These results suggest that transplantation of HSCs from bone marrow is an effective strategy for the treatment of spinal cord injury.