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1.
PLoS One ; 11(7): e0158571, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27385031

RESUMO

Cochlear and deep brain implants are prominent examples for neuronal prostheses with clinical relevance. Current research focuses on the improvement of the long-term functionality and the size reduction of neural interface electrodes. A promising approach is the application of carbon nanotubes (CNTs), either as pure electrodes but especially as coating material for electrodes. The interaction of CNTs with neuronal cells has shown promising results in various studies, but these appear to depend on the specific type of neurons as well as on the kind of nanotubes. To evaluate a potential application of carbon nanotube coatings for cochlear electrodes, it is necessary to investigate the cytocompatibility of carbon nanotube coatings on platinum for the specific type of neuron in the inner ear, namely spiral ganglion neurons. In this study we have combined the chemical processing of as-delivered CNTs, the fabrication of coatings on platinum, and the characterization of the electrical properties of the coatings as well as a general cytocompatibility testing and the first cell culture investigations of CNTs with spiral ganglion neurons. By applying a modification process to three different as-received CNTs via a reflux treatment with nitric acid, long-term stable aqueous CNT dispersions free of dispersing agents were obtained. These were used to coat platinum substrates by an automated spray-coating process. These coatings enhance the electrical properties of platinum electrodes, decreasing the impedance values and raising the capacitances. Cell culture investigations of the different CNT coatings on platinum with NIH3T3 fibroblasts attest an overall good cytocompatibility of these coatings. For spiral ganglion neurons, this can also be observed but a desired positive effect of the CNTs on the neurons is absent. Furthermore, we found that the well-established DAPI staining assay does not function on the coatings prepared from single-wall nanotubes.


Assuntos
Eletrodos , Nanotubos de Carbono/química , Platina/química , Gânglio Espiral da Cóclea/fisiologia , Animais , Animais Recém-Nascidos , Materiais Revestidos Biocompatíveis/química , Proteínas de Fluorescência Verde/metabolismo , Teste de Materiais , Camundongos , Células NIH 3T3 , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes
2.
PLoS One ; 9(11): e112142, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25392995

RESUMO

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.


Assuntos
Morte Celular/fisiologia , Degeneração Retiniana/fisiopatologia , Animais , Animais Geneticamente Modificados , Calpaína/fisiologia , Morte Celular/genética , GMP Cíclico/fisiologia , Modelos Animais de Doenças , Histona Desacetilases/fisiologia , Transdução de Sinal Luminoso/genética , Camundongos , Mutação , Poli Adenosina Difosfato Ribose/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Ratos , Degeneração Retiniana/genética
3.
PLoS One ; 9(3): e92157, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24642701

RESUMO

Sensorineural deafness is caused by damage of hair cells followed by degeneration of the spiral ganglion neurons and can be moderated by cochlear implants. However, the benefit of the cochlear implant depends on the excitability of the spiral ganglion neurons. Therefore, current research focuses on the identification of agents that will preserve their degeneration. In this project we investigated the neuroprotective effect of Rolipram as a promising agent to improve the viability of the auditory neurons. It is a pharmaceutical agent that acts by selective inhibition of the phosphodiesterase 4 leading to an increase in cyclic AMP. Different studies reported a neuroprotective effect of Rolipram. However, its significance for the survival of SGN has not been reported so far. Thus, we isolated spiral ganglion cells of neonatal rats for cultivation with different Rolipram concentrations and determined the neuronal survival rate. Furthermore, we examined immunocytologically distinct proteins that might be involved in the neuroprotective signalling pathway of Rolipram and determined endogenous BDNF by ELISA. When applied at a concentration of 0.1 nM, Rolipram improved the survival of SGN in vitro. According to previous studies, our immunocytological data showed that Rolipram application induces the phosphorylation and thereby activation of the transcription factor CREB. This activation can be mediated by the cAMP-PKA-signalling pathway as well as via ERK as a part of the MAP-kinase pathway. However, only in cultures pre-treated with BDNF, an endogenous increase of BDNF was detected. We conclude that Rolipram has the potential to improve the vitality of neonatal auditory nerve cells in vitro. Further investigations are necessary to prove the effect of Rolipram in vivo in the adult organism after lesion of the hair cells and insertion of cochlear implants.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Neurônios/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Rolipram/farmacologia , Gânglio Espiral da Cóclea/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Neurônios/citologia , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Ratos , Transdução de Sinais , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/metabolismo
4.
PLoS One ; 8(2): e57163, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23468924

RESUMO

Retinitis pigmentosa (RP) relates to a group of hereditary neurodegenerative diseases of the retina. On the cellular level, RP results in the primary death of rod photoreceptors, caused by rod-specific mutations, followed by a secondary degeneration of genetically normal cones. Different mechanisms may influence the spread of cell death from one photoreceptor type to the other. As one of these mechanisms a gap junction-mediated bystander effect was proposed, i.e., toxic molecules generated in dying rods and propagating through gap junctions induce the death of healthy cone photoreceptors. We investigated whether disruption of rod-cone coupling can prevent secondary cone death and reduce the spread of degeneration. We tested this hypothesis in two different mouse models for retinal degeneration (rhodopsin knockout and rd1) by crossbreeding them with connexin36-deficient mice as connexin36 represents the gap junction protein on the cone side and lack thereof most likely disrupts rod-cone coupling. Using immunohistochemistry, we compared the progress of cone degeneration between connexin36-deficient mouse mutants and their connexin36-expressing littermates at different ages and assessed the accompanied morphological changes during the onset (rhodopsin knockout) and later stages of secondary cone death (rd1 mutants). Connexin36-deficient mouse mutants showed the same time course of cone degeneration and the same morphological changes in second order neurons as their connexin36-expressing littermates. Thus, our results indicate that disruption of connexin36-mediated rod-cone coupling does not stop, delay or spatially restrict secondary cone degeneration and suggest that the gap junction-mediated bystander effect does not contribute to the progression of RP.


Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Retinose Pigmentar/metabolismo , Animais , Morte Celular , Conexinas/genética , Modelos Animais de Doenças , Progressão da Doença , Deleção de Genes , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Retinose Pigmentar/genética , Proteínas rho de Ligação ao GTP/deficiência , Proteína delta-2 de Junções Comunicantes
5.
J Comp Neurol ; 521(5): 1119-35, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22965528

RESUMO

Pannexin1 (Panx1) belongs to a class of vertebrate proteins that exhibits sequence homology to innexins, the invertebrate gap junction proteins, and which also shares topological similarities with vertebrate gap junction proteins, the connexins. Unlike gap junctional channels, Panx1 forms single-membrane channels, whose functional role in neuronal circuits is still unsettled. We therefore investigated the subcellular distribution of Panx1 in the mouse retina of wildtype and Panx1-null mice by reverse-transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and electron microscopy. Use of Panx1-deficient mice served as a model to assess the physiological role of Panx1 by electroretinographic recordings and also to ensure the specificity of the anti-Panx1 antibody labeling. Expression of Panx1 was found in type 3a OFF bipolar cells and in dendrites and axonal processes of horizontal cells. Panx1 was also found in horizontal cell dendrites representing the lateral elements of the triad synapse at cone and rod terminals. In vivo electroretinography of Panx1 knockout mice indicated an increased a- and b-wave compared to Panx1 wildtype mice under scotopic conditions. The effect on the b-wave was confirmed by in vitro electroretinograms from the inner retina. These results suggest that Panx1 channels serve as sinks for extracellular current flow making them possible candidates for the mediation of feedback from horizontal cells to photoreceptors.


Assuntos
Conexinas/deficiência , Proteínas do Tecido Nervoso/deficiência , Neurônios/fisiologia , Retina/anatomia & histologia , Vias Visuais/fisiologia , Animais , Conexinas/genética , Eletrorretinografia , Potenciais Evocados Visuais/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Glicosídeo Hidrolases/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/classificação , Neurônios/ultraestrutura , Células Bipolares da Retina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Tirosina 3-Mono-Oxigenase/metabolismo
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