RESUMO
BACKGROUND: Mesenchymal stem cell therapy in renal failure is rarely used because of low rates of cell engraftment after systemic delivery. Repeated intra-arterial cell administration may improve results; however, no current delivery method permits repeated intra-arterial infusions in a rat model. In this study, we developed an intra-arterial delivery system for repeated stem cell infusion via the aorta, catheterizing the left femoral artery to the suprarenal aorta under fluoroscopic guidance in rats with adenosine-induced renal failure. METHODS: First, we compared our intra-arterial catheter system (C group, n = 3) with tail vein injection (V group, n = 3) for engraftment efficacy, using mesenchymal stem cells from luciferase transgenic rats. Rats were infused with the cells and euthanized the following day; we performed cell-tracking experiments using a bioluminescence imaging system to assess the distribution of the infused cells. Second, we assessed the safety of the system over a 30-day period in a second group of six rats receiving infusions every 7 days. RESULTS: Cells infused through our delivery system efficiently engrafted into the kidney, compared with peripheral venous infusion. In five of the six rats in the safety study, the delivery system remained patent for at least 9 days (range, 9-24 days). Complications became evident only after 10 days. CONCLUSION: Our intra-arterial catheter system was effective in delivering cells to the kidney and permitted repeated injection of cells.
Assuntos
Transplante de Células-Tronco Mesenquimais/instrumentação , Insuficiência Renal/terapia , Animais , Cateteres de Demora , Modelos Animais de Doenças , Ratos Endogâmicos LewRESUMO
PURPOSE: To investigate the use of diffusion-tensor imaging (DTI) to detect denervation of the nigrostriatal pathway in a nonhuman primate model of Parkinson disease (PD) after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MATERIALS AND METHODS: This study was approved by the institutional committee for animal experiments. DTI was performed in marmosets (n = 6) by using a 7-T magnetic resonance (MR) imager before and 10 weeks after administration of MPTP. Fixed brains of a normal marmoset and a marmoset model of PD (n = 1) were analyzed by using microscopic tractography. Tyrosine-hydroxylase staining of dopaminergic neurons and three-dimensional histologic analysis also were performed in normal marmosets (n = 2) and a PD marmoset model (n = 2) to validate the course of the nigrostriatal pathway revealed at tractography. Statistical analysis of voxel-based and post hoc region-of-interest analyses of DTI maps was performed by using a paired t test. RESULTS: At voxel-based analysis of DTI before and after treatment, MPTP-treated marmoset brains showed significantly increased axial and radial diffusivity in the bilateral nigrostriatal pathway (P < .05, false discovery rate corrected). The largest area of significantly increased diffusivity was an area of axial diffusivity in the right hemispere (177 mm(3)) that corresponded to the location of dopaminergic neurodegeneration at histologic evaluation. Region-of-interest analysis revealed a 27% increase in axial diffusivity in the right hemisphere (1.198 mm(2)/sec ± 0.111 to 1.522 mm(2)/sec ± 0.118; P = .002). Three-dimensional histologic analysis with tyrosine-hydroxylase staining showed the course of the nigrostriatal pathway and degeneration in the PD marmoset model as the absence of a tyrosine-hydroxylase stained region. Microscopic tractography showed that the connection of the substantia nigra to the striatum followed the same course as the nigrostriatal pathway and fewer fiber tracts in the PD marmoset model. CONCLUSION: DTI and microscopic tractography showed the loss of fiber structures of the nigrostriatal pathway in the marmoset model of PD. The results of this study provide a potential basis for the use of DTI in the clinical diagnosis of PD.
Assuntos
Corpo Estriado/patologia , Imagem de Difusão por Ressonância Magnética , Vias Neurais/patologia , Doença de Parkinson/diagnóstico , Substância Negra/patologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , Animais , Callithrix , Modelos Animais de Doenças , MasculinoRESUMO
This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.
Assuntos
Alginatos , Colágeno , Dependovirus , Microesferas , Dependovirus/genética , Alginatos/química , Animais , Colágeno/química , Camundongos , Técnicas de Transferência de Genes , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Hidrogéis/química , Géis/químicaRESUMO
We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture.
Assuntos
Doença de Fabry/patologia , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Vírus Sendai/genética , Diferenciação Celular , Linhagem Celular , Doença de Fabry/genética , Doença de Fabry/metabolismo , Fibroblastos/metabolismo , Vetores Genéticos , Humanos , Fator 4 Semelhante a Kruppel , Neurônios/metabolismo , Neurônios/patologia , Pele/metabolismo , Pele/patologia , Transdução GenéticaRESUMO
Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.
Assuntos
Cromonas/uso terapêutico , Regeneração Nervosa/efeitos dos fármacos , Semaforina-3A/antagonistas & inibidores , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Xantonas/uso terapêutico , Animais , Células COS , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Células de Schwann/efeitos dos fármacos , Semaforina-3A/genética , Semaforina-3A/metabolismo , Medula Espinal/fisiologia , Traumatismos da Medula Espinal/reabilitação , alfa-Defensinas/metabolismoRESUMO
During rodent experiments, the caudal ventral artery (CVA) is useful for blood pressure (BP) measurement. However, CVA measurements may not reflect the true BP. This study was performed to verify the site-specific accuracy of invasive arterial BP monitoring during surgery in rats. Invasive arterial BP was simultaneously measured in rats via the CVA and the common carotid artery (CCA). The BP values were analysed while the rats were subjected to cooling of the head or tail. Additionally, the rats underwent digital subtraction angiography and histological examination of these arteries. The pressure difference was more significant in the tail cooling group than in the head cooling group. Digital subtraction angiography revealed that angiospasms occurred more frequently in the CVA than in the CCA upon cooling. This phenomenon was supported by histological analysis, which showed that the tunica media area was significantly larger in the CVA than in the CCA. CVA pressure is susceptible to environmental changes and may not accurately reflect the true BP without a strictly controlled laboratory environment. Therefore, understanding the pitfalls of this method is necessary to avoid cooling of the tail during BP measurement.
Assuntos
Determinação da Pressão Arterial/métodos , Pressão Sanguínea , Artérias Carótidas/fisiologia , Cauda/irrigação sanguínea , Animais , Determinação da Pressão Arterial/normas , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
Hundreds of inbred mouse strains are established for use in a broad spectrum of basic research fields, including genetics, neuroscience, immunology, and cancer. Inbred mice exhibit identical intra-strain genetics and divergent inter-strain phenotypes. The cognitive and behavioral divergences must be controlled by the variances of structure and function of their brains; however, the underlying morphological features of strain-to-strain difference remain obscure. Here, in vivo microscopic magnetic resonance imaging was optimized to image the mouse brains by using an isotropic resolution of 80 µm. Next, in vivo templates were created from the data from four major inbred mouse strains (C57Bl/6, BALB/cBy, C3H/He, and DBA/2). A strain-mixed brain template was also created, and the template was then employed to establish automatic voxel-based morphometry (VBM) for the mouse brain. The VBM assessment revealed strain-specific brain morphologies concerning the gray matter volume of the four strains, with a smaller volume in the primary visual cortex for the C3H/He strain, and a smaller volume in the primary auditory cortex and field CA1 of the hippocampus for the DBA/2 strain. These findings would contribute to the basis of for understanding morphological phenotype of the inbred mouse strain and may indicate a relationship between brain morphology and strain-specific cognition and behavior.
Assuntos
Encéfalo/anatomia & histologia , Microscopia Intravital/métodos , Camundongos Endogâmicos BALB C/anatomia & histologia , Camundongos Endogâmicos C3H/anatomia & histologia , Camundongos Endogâmicos C57BL/anatomia & histologia , Camundongos Endogâmicos DBA/anatomia & histologia , Animais , Córtex Auditivo/anatomia & histologia , Substância Cinzenta/anatomia & histologia , Processamento de Imagem Assistida por Computador , Microscopia Intravital/veterinária , Imageamento por Ressonância Magnética/veterinária , Masculino , Camundongos , Especificidade da Espécie , Córtex Visual/anatomia & histologiaRESUMO
Allodynia, a form of neuropathic pain, is defined as pain in response to a non-nociceptive stimulus. The brain regions responsible for pain, which are not normally activated, can be activated in allodynic mice by providing a suitable stimulus to Aß-fibers, which transmit signals from tactile sensory fibers. Functional MRI (fMRI) can be used to objectively observe abnormal brain activation. In the present study, fMRI was conducted to investigate allodynia in mice; allodynia was generated by surgical injury at the L4 spinal nerve root, thus selectively stimulating sensory nerve fibers. In intact mice, only the primary somatosensory cortex (S1) was activated by stimulation of Aß-fibers. Meanwhile, allodynic mice showed significantly higher BOLD signals in the anterior cingulate area (ACA) and thalamus. Using resting state fMRI, both degree and eigenvector centrality were significantly decreased in the contralateral S1, clustering coefficient and local efficiency were significantly increased in the ACA, and betweenness centrality was significantly higher in the ventral posterolateral nucleus of the thalamus. These results suggest that the observed abnormal BOLD activation is associated with defects in Aß-fibers when Aß-fibers in allodynic mice are selectively stimulated. The objective approach enabled by fMRI can improve our understanding of pathophysiological mechanisms and therapeutic efficacy.
Assuntos
Encéfalo/fisiologia , Neuroimagem Funcional/métodos , Hiperalgesia/diagnóstico , Fibras Nervosas/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dor , Estimulação Física , Nervos Espinhais/cirurgia , TatoRESUMO
Embryonic stem cell research has facilitated the generation of many cell types for the production of tissues and organs for both humans and companion animals. Because ≥30% of pet cats suffer from chronic kidney disease (CKD), xenotransplantation between pigs and cats has been studied. For a successful pig to cat xenotransplant, the immune reaction must be overcome, especially hyperacute rejection. In this study, we isolated the gene for feline decay-accelerating factor (fDAF), an inhibitor of complement proteins, and transfected a swine endothelial cell line with fDAF to "felinize" the pig cells. These fDAF-expressing cells were resistant to feline serum containing anti-pig antibodies, suggesting that felinized pig cells were resistant to hyperacute rejection. Our results suggest that a "felinized" pig kidney can be generated for the treatment of CKD in cats in the future.
Assuntos
Antígenos CD55/genética , Células Endoteliais/metabolismo , Transplante Heterólogo , Sequência de Aminoácidos , Animais , Antígenos CD55/química , Doenças do Gato/cirurgia , Gatos , Linhagem Celular , Células Clonais/metabolismo , Clonagem Molecular , Expressão Gênica , Rejeição de Enxerto , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Dados de Sequência Molecular , Insuficiência Renal Crônica/veterinária , Suínos , Transfecção , Transplante Heterólogo/efeitos adversosRESUMO
Using histological and electrophysiological methods, we identified the neuroanatomical properties of the common marmoset corticospinal tract (CST), which underlies hand/arm motor control. Biotinylated dextran amine (BDA) was injected into the primary motor cortex to anterogradely label CST axons in the cervical segments, revealing that most CST axons descend in the contralateral dorsolateral funiculus (DLF; 85.0%), and some in the ipsilateral DLF (10.7%). Terminal buttons were mainly found in the contralateral lamina VII of the gray matter, but projection to lamina IX, where forelimb motoneurons are located, was rare. Bilateral projections were more abundant than found in the rat CST, resembling the CST organization of other primates. Intracellular recordings were made from 57 forelimb motoneurons on the contralateral side to stimulation, which revealed no monosynaptic excitatory postsynaptic potentials (EPSPs), but di- or polysynaptic EPSPs and inhibitory synaptic potentials were commonly found. Local field potentials showed monosynaptic excitation mainly in laminae VII, where abundant BDA-labeled CST terminals were observed. These results suggest that direct corticomotoneuronal projection is absent in common marmosets but di- or oligosynaptic effects would be mediated by spinal interneurons.
Assuntos
Membro Anterior/inervação , Neurônios Motores/fisiologia , Músculo Esquelético/inervação , Tratos Piramidais/anatomia & histologia , Tratos Piramidais/fisiologia , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Callithrix , Fenômenos Eletrofisiológicos , Potenciais Pós-Sinápticos Excitadores , Substância Cinzenta/fisiologia , Substância Cinzenta/ultraestrutura , Potenciais da Membrana , Vias Neurais , Substância Branca/fisiologia , Substância Branca/ultraestruturaRESUMO
Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung's disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret(-/-) mouse, an animal model of Hirschsprung's disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret(-/-) mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung's disease capable of colonizing the affected bowel and giving rise to neurons.
RESUMO
BACKGROUND: Pallido-ponto-nigral degeneration (PPND), a major subtype of frontotemporal dementia with parkinsonism related to chromosome 17 (FTDP-17), is a progressive and terminal neurodegenerative disease caused by c.837 T > G mutation in the MAPT gene encoding microtubule-associated protein tau (rs63750756; N279K). This MAPT mutation induces alternative splicing of exon 10, resulting in a modification of microtubule-binding region of tau. Although mutations in the MAPT gene have been linked to multiple tauopathies including Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy, knowledge regarding how tau N279K mutation causes PPND/FTDP-17 is limited. RESULTS: We investigated the underlying disease mechanism associated with the N279K tau mutation using PPND/FTDP-17 patient-derived induced pluripotent stem cells (iPSCs) and autopsy brains. In iPSC-derived neural stem cells (NSCs), the N279K tau mutation induced an increased ratio of 4-repeat to 3-repeat tau and accumulation of stress granules indicating elevated cellular stress. More significant, NSCs derived from patients with the N279K tau mutation displayed impaired endocytic trafficking as evidenced by accumulation of endosomes and exosomes, and a reduction of lysosomes. Since there were no significant differences in cellular stress and distribution of subcellular organelles between control and N279K skin fibroblasts, N279K-related vesicle trafficking defects are likely specific to the neuronal lineage. Consistently, the levels of intracellular/luminal vesicle and exosome marker flotillin-1 were significantly increased in frontal and temporal cortices of PPND/FTDP-17 patients with the N279K tau mutation, events that were not seen in the occipital cortex which is the most spared cortical region in the patients. CONCLUSION: Together, our results demonstrate that alterations of intracellular vesicle trafficking in NSCs/neurons likely contribute to neurodegeneration as an important disease mechanism underlying the N279K tau mutation in PPND/FTDP-17.
Assuntos
Demência Frontotemporal/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação de Sentido Incorreto , Mutação Puntual , Transporte Proteico , Vesículas Transportadoras/fisiologia , Proteínas tau/genética , Sítios de Ligação/genética , Células Cultivadas , Corpos Embrioides , Endossomos/fisiologia , Éxons/genética , Exossomos/fisiologia , Feminino , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lisossomos/fisiologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Lobo Occipital/metabolismo , Lobo Occipital/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Lobo Temporal/metabolismo , Lobo Temporal/patologiaRESUMO
Sox21, a high-mobility group box transcription factor, is expressed throughout the immature neural stem/progenitor population in ventricular zone but not in the cortical plate in embryonic mouse brain and its expression is restricted to the subventricular zone in the adult brain. In undifferentiated PC12 cells, endogenous Sox21 expression is detected but its expression ceases during the nerve growth factor (NGF)-induced neuronal differentiation. Overexpression of Sox21 results in a substantial repression of NGF-induced neurite outgrowth in PC12 cells. Further, we biochemically identified a Sox21-associating protein, Y-box binding protein 1 which not only binds to Sox21 but also partially restores NGF-induced neurite outgrowth of PC12 cells inhibited by Sox21. These results suggest that Sox21 is a repressor of neuronal differentiation in the developing nervous system.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Neoplasias/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/farmacologia , Córtex Cerebral/citologia , Feto , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/genética , Camundongos , Fatores de Transcrição NFI , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/citologia , Células PC12 , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXE , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Proteína 1 de Ligação a Y-BoxRESUMO
An RNA-binding protein, hnRNP K, has been studied extensively because of its involvement in neural development through the post-transcriptional regulation of its downstream target genes; however, its binding mode remains unclear. According to structural features of the binding sites, we have presumed the existence of possible unique structures 'j-motifs' that are similar to known i-motifs, the difference being that the initial cluster comprises successive U nucleic acids instead of C. It was suspected that the motifs could be recognized by hnRNP K to regulate the translation levels of target proteins, however, there were virtually no methods to verify their existence except computational methods: regular expression searches and theoretical molecular orbital (MO) calculations. Here, we first show a list of 16 genes having j-motif-like sequences we discovered under refined search conditions. The list was highly related to neural development from both subjective and objective aspects. Additionally, MO calculations revealed the similarity of non-canonical base pairs found in i- and j-motifs qualitatively, leading to a proposal of the possible existence of the j-motifs. When taken into consideration, it was indicated that the j-motifs could be formed and play some role in the neural development.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Motivos de Nucleotídeos , RNA/química , Animais , Pareamento de Bases , Inibidor de Quinase Dependente de Ciclina p21/genética , Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Humanos , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Mensageiro/químicaRESUMO
Spinal cord injury (SCI) is a devastating neurological injury. At present, pharmacological, regenerative, and rehabilitative approaches are widely studied as therapeutic interventions for motor recovery after SCI. Preclinical research has been performed on model animals with experimental SCI, and those studies often evaluate hand and arm motor function using various indices, such as the success rate of the single pellet reaching test and the grip force. However, compensatory movement strategies, involuntary muscle contraction, and the subject's motivation could affect the scores, resulting in failure to assess direct recovery from impairment. Identifying appropriate assessments of motor impairment is thus important for understanding the mechanisms of motor recovery. In this study, we developed a motion capture system capable of reconstructing three-dimensional hand positions with millimeter and millisecond accuracy and evaluated hand kinematics during food retrieval movement in both healthy and hemispinalized common marmosets. As a result, the endpoint jerk, representing the accuracy of hand motor control, was asserted to be an appropriate index of upper limb motor impairment by eliminating the influence of the subject's motivation, involuntary muscle contraction, and compensatory strategies. The result also suggested that the kinematics of the limb more consistently reflects motor restoration from deficit due to spinal cord injury than the performance in the single pellet reaching test. Because of recent attention devoted to the common marmoset as a nonhuman primate model for human diseases, the present study, which clarified arm-reaching movements in spinalized marmosets, provides fundamental knowledge for future therapeutic studies.
Assuntos
Lateralidade Funcional , Movimento/fisiologia , Amplitude de Movimento Articular/fisiologia , Traumatismos da Medula Espinal/diagnóstico , Traumatismos da Medula Espinal/fisiopatologia , Extremidade Superior/fisiopatologia , Animais , Fenômenos Biomecânicos , Callithrix , Modelos Animais de Doenças , Feminino , Masculino , Movimento (Física) , Desempenho Psicomotor/fisiologia , Gravação em VídeoRESUMO
Naked mole-rats have a variety of distinctive features such as the organization of a hierarchical society (known as eusociality), extraordinary longevity, and cancer resistance; thus, it would be worthwhile investigating these animals in detail. One important task is the preparation of a brain atlas database that provide comprehensive information containing multidimensional data with various image contrasts, which can be achievable using a magnetic resonance imaging (MRI). Advanced MRI techniques such as diffusion tensor imaging (DTI), which generates high contrast images of fiber structures, can characterize unique morphological properties in addition to conventional MRI. To obtain high spatial resolution images, MR histology, DTI, and X-ray computed tomography were performed on the fixed adult brain. Skull and brain structures were segmented as well as reconstructed in stereotaxic coordinates. Data were also acquired for the neonatal brain to allow developmental changes to be observed. Moreover, in vivo imaging of naked mole-rats was established as an evaluation tool of live animals. The data obtained comprised three-dimensional (3D) images with high tissue contrast as well as stereotaxic coordinates. Developmental differences in the visual system were highlighted in particular by DTI. Although it was difficult to delineate optic nerves in the mature adult brain, parts of them could be distinguished in the immature neonatal brain. From observation of cortical thickness, possibility of high somatosensory system development replaced to the visual system was indicated. 3D visualization of brain structures in the atlas as well as the establishment of in vivo imaging would promote neuroimaging researches towards detection of novel characteristics of eusocial naked mole-rats.
RESUMO
BACKGROUND: Despite numerous articles on the use of artificial nerve conduits, autologous nerve transplants remain the most effective for nerve repair. To improve this technique, the authors examined conduits containing gelatin hydrogel as a carrier enabling the sustained release of basic fibroblast growth factor (bFGF). METHODS: To confirm sustained bFGF release in vivo, nerve-guide tubes containing iodine-125-labeled bFGF with or without gelatin hydrogel were implanted under the skin of mice, and the remaining radioactivity was measured. Next, a 15-mm segment of the sciatic nerve was resected and repaired with autologous nerve (group 1), a tube with gelatin hydrogel and bFGF (group 2), a tube with bFGF alone (group 3), or a tube only (group 4). Histologic and functional analyses were performed for 16 weeks after surgery. RESULTS: The radioactivity from iodine-125-labeled bFGF incorporated into gelatin hydrogel decreased more slowly than iodine-125-labeled bFGF alone. Four weeks after surgery, significantly more regenerating axons were detected in group 2 than in groups 3 and 4, but the axonal density in group 2 was lower than in group 1. Similarly, the animals in group 2 showed significantly better motor performance than those in groups 3 and 4, but worse than those in group 1. The animals in groups 1 and 2 showed significantly better sensory recovery than those in groups 3 and 4. CONCLUSIONS: The nerve-guide tube containing gelatin hydrogel and bFGF promoted axonal regeneration after peripheral nerve injury, but not as well as autologous transplants. Understanding the limitations of this technique will facilitate its improvement for clinical applications.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Transferência de Nervo/métodos , Nervo Isquiático/cirurgia , Análise de Variância , Animais , Comportamento Animal , Preparações de Ação Retardada/farmacologia , Modelos Animais de Doenças , Feminino , Gelatina , Hidrogel de Polietilenoglicol-Dimetacrilato , Imuno-Histoquímica , Contração Muscular/fisiologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/fisiologia , Cloreto de Tolônio , Transplante Autólogo , Resultado do TratamentoRESUMO
The paraneoplastic neurologic disorders target several families of neuron-specific RNA binding proteins (RNABPs), revealing that there are unique aspects of gene expression regulation in the mammalian brain. Here, we used HITS-CLIP to determine robust binding sites targeted by the neuronal Elav-like (nElavl) RNABPs. Surprisingly, nElav protein binds preferentially to GU-rich sequences in vivo and in vitro, with secondary binding to AU-rich sequences. nElavl null mice were used to validate the consequence of these binding events in the brain, demonstrating that they bind intronic sequences in a position dependent manner to regulate alternative splicing and to 3'UTR sequences to regulate mRNA levels. These controls converge on the glutamate synthesis pathway in neurons; nElavl proteins are required to maintain neurotransmitter glutamate levels, and the lack of nElavl leads to spontaneous epileptic seizure activity. The genome-wide analysis of nElavl targets reveals that one function of neuron-specific RNABPs is to control excitation-inhibition balance in the brain.
Assuntos
Encéfalo/citologia , Proteínas ELAV/metabolismo , Regulação da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Neurônios/fisiologia , Splicing de RNA/genética , Regiões 3' não Traduzidas/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Biologia Computacional , Modelos Animais de Doenças , Proteínas ELAV/deficiência , Eletroencefalografia , Epilepsia/genética , Epilepsia/fisiopatologia , Regulação da Expressão Gênica/genética , Glutaminase/genética , Glutaminase/metabolismo , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Multipotent somatic stem cells have been identified in various adult tissues. However, the stem/progenitor cells of the peripheral nerves have been isolated only from fetal tissues. Here, we isolated Schwann-cell precursors/immature Schwann cells from the injured peripheral nerves of adult mice using a floating culture technique that we call "Schwann-spheres." The Schwann-spheres were derived from de-differentiated mature Schwann cells harvested 24 hours to 6 weeks after peripheral nerve injury. They had extensive self-renewal and differentiation capabilities. They strongly expressed the immature-Schwann-cell marker p75, and differentiated only into the Schwann-cell lineage. The spheres showed enhanced myelin formation and neurite growth compared to mature Schwann cells in vitro. Mature Schwann cells have been considered a promising candidate for cell-transplantation therapies to repair the damaged nervous system, whereas these "Schwann-spheres" would provide a more potential autologous cell source for such transplantation.
Assuntos
Envelhecimento/patologia , Células de Schwann/patologia , Células de Schwann/transplante , Nervo Isquiático/lesões , Esferoides Celulares/patologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Neuritos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/patologia , Esferoides Celulares/metabolismo , Células-Tronco/metabolismoRESUMO
Previous work from our laboratory demonstrated that the RNA-binding protein HuD binds to and stabilizes the GAP-43 mRNA. In this study, we characterized the expression of HuD and GAP-43 mRNA in the hippocampus during two forms of neuronal plasticity. During post-natal development, maximal expression of both molecules was found at P5 and their levels steadily decreased thereafter. At P5, HuD was also present in the subventricular zone, where it co-localized with doublecortin. In the adult hippocampus, the basal levels of HuD and GAP-43 were lower than during development but were significantly increased in the dentate gyrus after seizures. The function of HuD in GAP-43 gene expression was confirmed using HuD-KO mice, in which the GAP-43 mRNA was significantly less stable than in wild type mice. Altogether, these results demonstrate that HuD plays a role in the post-transcriptional control of GAP-43 mRNA in dentate granule cells during developmental and adult plasticity.