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1.
Nat Commun ; 9(1): 226, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29335532

RESUMO

Quantifying heterogeneities within cell populations is important for many fields including cancer research and neurobiology; however, techniques to isolate individual cells are limited. Here, we describe a high-throughput, non-disruptive, and cost-effective isolation method that is capable of capturing individually targeted cells using widely available techniques. Using high-resolution microscopy, laser microcapture microscopy, image analysis, and machine learning, our technology enables scalable molecular genetic analysis of single cells, targetable by morphology or location within the sample.


Assuntos
Separação Celular/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Análise de Célula Única/métodos , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Aprendizado de Máquina , Células Piramidais/citologia , Células Piramidais/metabolismo , Reprodutibilidade dos Testes
2.
Acta Neuropathol Commun ; 4(1): 78, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27487831

RESUMO

Functional and molecular changes associated with pathophysiological conditions are relatively easily detected based on tissue samples collected from patients. Population specific cellular responses to disease might remain undiscovered in samples taken from organs formed by a multitude of cell types. This is particularly apparent in the human cerebral cortex composed of a yet undefined number of neuron types with a potentially different involvement in disease processes. We combined cellular electrophysiology, anatomy and single cell digital PCR in human neurons identified in situ for the first time to assess mRNA expression and corresponding functional changes in response to edema and increased intracranial pressure. In single pyramidal cells, mRNA copy numbers of AQP1, AQP3, HMOX1, KCNN4, SCN3B and SOD2 increased, while CACNA1B, CRH decreased in edema. In addition, single pyramidal cells increased the copy number of AQP1, HTR5A and KCNS1 mRNAs in response to increased intracranial pressure. In contrast to pyramidal cells, AQP1, HMOX1and KCNN4 remained unchanged in single cell digital PCR performed on fast spiking cells in edema. Corroborating single cell digital PCR results, pharmacological and immunohistochemical results also suggested the presence of KCNN4 encoding the α-subunit of KCa3.1 channels in edema on pyramidal cells, but not on interneurons. We measured the frequency of spontaneous EPSPs on pyramidal cells in both pathophysiological conditions and on fast spiking interneurons in edema and found a significant decrease in each case, which was accompanied by an increase in input resistances on both cell types and by a drop in dendritic spine density on pyramidal cells consistent with a loss of excitatory synapses. Our results identify anatomical and/or physiological changes in human pyramidal and fast spiking cells in edema and increased intracranial pressure revealing cell type specific quantitative changes in gene expression. Some of the edema/increased intracranial pressure modulated and single human pyramidal cell verified gene products identified here might be considered as novel pharmacological targets in cell type specific neuroprotection.


Assuntos
Edema Encefálico/metabolismo , Hipertensão Intracraniana/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Adulto , Edema Encefálico/patologia , Edema Encefálico/cirurgia , Feminino , Regulação da Expressão Gênica , Substância Cinzenta/metabolismo , Substância Cinzenta/patologia , Substância Cinzenta/cirurgia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Hipertensão Intracraniana/patologia , Hipertensão Intracraniana/cirurgia , Pressão Intracraniana/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Neocórtex/patologia , Neocórtex/cirurgia , Neurônios/patologia , RNA Mensageiro/metabolismo , Técnicas de Cultura de Tecidos
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