Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Fernandes, Artur José de Brum; Mendes, Niele Dias; Almeida, Gláucia Maria; Nogueira, Giovanna Orlovski; Machado, Carla de Moraes; Horta-Júnior, J. A. C.; Assirati, João Alberto; García-Cairasco, Norberto; Neder, Luciano; Sebollela, Adriano

doi:10.3791/59845

Cited by 11 publications

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“…Briefly, a fragment of human frontal cortex was surgically collected, sliced (200μm thick) and cultured for 2 days, as described in detail in (20,21).…”

Section: Ex-vivo Human Brain Slicesmentioning

confidence: 99%

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity

et al. 2021

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Tau is a microtubule-associated protein responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. Here, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and humans. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots of the same extracts were stored for at least 2 weeks at either -20°C or -80°C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ~25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry analysis in excised bands revealed this ~25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80°C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.

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“…Briefly, a fragment of human frontal cortex was surgically collected, sliced (200μm thick) and cultured for 2 days, as described in detail in (20,21).…”

Section: Ex-vivo Human Brain Slicesmentioning

confidence: 99%

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity

et al. 2021

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“…The procedures carried out using ex-vivo human brain slices (20) were approved by the Ribeirão Preto Medical School Ethics Committee, under the protocol HCRP #17578/15. Briefly, a fragment of human frontal cortex was surgically collected, sliced (200μm thick) and cultured for 2 days, as described in detail in (20,21).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, a fragment of human frontal cortex was surgically collected, sliced (200μm thick) and cultured for 2 days, as described in detail in (20,21).…”

Section: Ex-vivo Human Brain Slicesmentioning

confidence: 99%

A freeze-and-thaw induced-fragment of the microtubule-associated protein Tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity

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et al. 2018

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Tau is a neuronal, microtubule-associated protein responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation, but in some neurological diseases it becomes aberrantly hyperphosphorylated, thus contributing to the pathogenesis of several neurological diseases such as Alzheimer's disease. Western Blot (WB) has widely been employed in the assessment of Tau levels and fragmentation in neurological disease models. However, quantification of Tau levels/cleavage by WB should be interpreted with care, as this approach has been recognized as a complex, multi-step technique prone to produce artifactual results if not properly performed. Here, we evaluated the influence of freeze-and-thaw, a common storage procedure preceding WB, to the integrity of Tau in rat brain extracts. We observed that samples submitted to freeze-and-thaw presented a ~25 kDa fragment shown to be derived from Tau. Based on this observation, we strongly recommend that molecular analysis of Tau levels in rodent brain samples in future investigations be performed using fresh extracts. Finally, we discuss the impact of our findings to the translational power of studies involving Tau-induced neurodegeneration.

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“…O modelo de fatias cerebrais humanas já foi padronizado por nosso grupo em trabalhos anteriores, nos quais mostramos que estas se encontram viáveis e funcionais por até 5DIV, com a preservação da citoarquitetura do tecido neural, e com a presença dos principais tipos celulares: neurônios, micróglia e astrócitos (Fig. 2) (Mendes et al, 2018;Fernandes et al, 2019), características importantes para que o modelo possa ser utilizado em estudos de neuropatogênese viral.…”

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“…Barra de escala 100 µm. Dados retirados dos artigos publicados por nosso grupo(Mendes et al 2018;Fernandes et al, 2019).…”

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Estudo da infecção do sistema nervoso central pelo vírus Oropouche em um modelo de fatias de cérebro humano adulto

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Almeida, Glaucia Maria. Study of central nervous system infection by the Oropouche vírus in adult human brain slice cultures. Ribeirão Preto, 2020. The Oropouche virus (OROV), of the family Peribunyaviridae, genus Orthobunyavirus, is a causative agent of arbovirus called "Oropouche fever". This virus has the significant spreading capacity, and in recent years has been found in nonendemic region, especially in South American countries, including Brazil. Although some clinical evidences in alterations in central nervous system, as meningitis, has been associated with OROV infection, few studies involving OROV neuropathogenesis have been studied, and all knowledge obtained were showed from neonatal murine models. This difficulty in studying OROV neuropathogenesis in the human central nervous system is related to the lack of models that maintain the complexity of human brain. Therefore, in this study, we evaluated the OROV neuroinfection using adult human brain slices cultures. We showed for the first time that OROV infects human neural cells and caused tissue damage within 48 h post infection, such as loss of cell viability and TNF-α release, a proinflammatory cytokine. We showed, by immunofluorescence, microglia cells were the cell most susceptible to OROV infection, but that it retains the ability to infect neurons. This is the first study to report OROV infection in adult human brain model and should become useful in neuropathogenesis studies related to this emerging pathogen.

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Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Cited by 11 publications

References 0 publications

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity

A freeze-and-thaw induced-fragment of the microtubule-associated protein Tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity

Estudo da infecção do sistema nervoso central pelo vírus Oropouche em um modelo de fatias de cérebro humano adulto

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