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Neuroscience
Yetişkin İnsan Beyninden Kısa Süreli Serbest Kayan Dilim Kültürleri
Yetişkin İnsan Beyninden Kısa Süreli Serbest Kayan Dilim Kültürleri
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Yetişkin İnsan Beyninden Kısa Süreli Serbest Kayan Dilim Kültürleri

Full Text
9,770 Views
09:14 min
November 5, 2019

DOI: 10.3791/59845-v

Artur Fernandes1,2, Niele Dias Mendes1,3, Glaucia Maria Almeida1, Giovanna Orlovski Nogueira1, Carla de Moraes Machado4, Jose de Anchieta de Castro Horta-Junior4, João Alberto Assirati Junior5, Norberto Garcia-Cairasco2, Luciano Neder3, Adriano Sebollela1

1Department of Biochemistry and Immunology, Ribeirão Preto Medical School,University of São Paulo, 2Department of Physiology, Ribeirão Preto Medical School,University of São Paulo, 3Department of Pathology and Forensic Medicine, Ribeirão Preto Medical School,University of São Paulo, 4Department of Anatomy, Institute of Biosciences,São Paulo State University, 5Clinical Hospital at the Ribeirão Preto Medical School,University of São Paulo

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Overview

This study presents a protocol for preparing free-floating slice cultures from adult human brain tissue, enhancing the understanding of neurodegeneration in age-associated brain diseases. The method is a simplified and cost-effective alternative to traditional slice culture techniques, suitable for biochemical and immunohistochemical assays.

Key Study Components

Area of Science

  • Neuroscience
  • Cell Biology
  • Neurodegeneration

Background

  • Existing slice culture methods can be complex and costly.
  • Neurodegenerative diseases present significant research challenges.
  • Advancements in culture techniques can improve biological insights.
  • This research involves collaboration among graduate students and researchers.

Purpose of Study

  • To simplify the preparation of slice cultures from human brain tissue.
  • To facilitate short-term assays investigating neurodegeneration mechanisms.
  • To provide a reliable culture system for neurobiological research.

Methods Used

  • Free-floating slice cultures using human brain tissue.
  • Protocol involves the excision of meninges and precision slicing using a vibratome.
  • No multiomics work is described in the text.
  • Slicing produces 200 micrometer thick sections, followed by culture in BDNF-supplemented medium.
  • Histological assessment is conducted post-culture.

Main Results

  • The study indicates that slice preservation allows for normal cellular morphology and neurophysiological responses post-culture.
  • Neuronal cell types and glial cells present typical architecture even after surgical procedures.
  • Short-term neurophysiological assays confirm neuron responsiveness.

Conclusions

  • This protocol simplifies access to human brain tissue for neurobiological studies.
  • Contributes to the understanding of neurodegeneration mechanisms in aged populations.
  • Implications for enhanced research methodologies in studying age-associated neurological disorders.

Frequently Asked Questions

What are the advantages of this slice culture method?
This method is simpler and more cost-effective than traditional slice culture protocols, making it more accessible for researchers.
How is the main biological model implemented in this study?
Human brain tissue from living donors is used, allowing for the study of neurodegeneration mechanisms directly relevant to human health.
What types of data can be obtained from using these slice cultures?
The methodology facilitates biochemical assays, immunohistochemistry, and assessment of neuronal and glial morphology.
Can this method be adapted for other types of neural tissue?
While primarily designed for human brain tissue, the protocol could be customized for various neural tissue types with appropriate adjustments.
What are the key limitations or considerations in this study?
The complexity of some procedural steps may require visual demonstration to ensure accurate execution.
How does this study contribute to understanding neurodegeneration?
By elucidating cellular responses and morphology under cultured conditions, it advances knowledge of neurodegenerative processes associated with aging.
What is the expected cell morphology in cultured slices?
Cultured slices exhibit typical neuronal and glial architecture, demonstrating the method’s efficacy in preserving cell types.

Yetişkin insan beyninden serbest yüzen dilim kültürleri hazırlamak için bir protokol sunulmaktadır. Protokol membran ekler kullanarak yaygın olarak kullanılan dilim kültür yönteminin bir varyasyonudur. Bu basit, maliyet-etkin, ve yaşilişkili beyin hastalıkları arkasında nörodejenerasyon mekanizmaları çözmek için amaçlayan kısa vadeli tahliller çalıştırmak için önerilir.

Rezeksiyon beyin cerrahisine gönderilen canlı insan donörlerinden toplanan beyin dokusundan serbest çesitli dilim kültürleri hazırlamak için bir protokol sayılmiyoruz. Bu kültür biyokimyasal ve hücre biyolojisi tahlilleri veya immünohistokimya yapmak için münakat. Bu ilişkili beyin hastalıklarında nörodejenerasyon altında yatan mekanizmanın açıklanmasına katkıda bulunması bekleniyor.

Bu tekniğin en büyük avantajı membran kesici uçlar kullanarak klasik dilim kültürü protokolüne daha basit ve uygun maliyetli bir alternatif olmasıdır. Genel protokol karmaşık olmasa da, menenjlerin çıkarılması, dokunun vibratom numune diskine yapıştırMa ve immünohistokimya rezeksiyonu gibi bazı adımlar görsel olarak gösterildiğinde en iyi şekilde anlaşılmaktadır. Prosedürü göstermek için yardımcı Niele Mendes ve Glaucia Almedia yüksek lisans öğrencileri, ve Giovanna Nogueira, başka bir yüksek lisans öğrencisi olacaktır.

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