Isolation and characterization of intact mitochondria from neonatal rat brain

Nishadi Rajapakse, Katsuyoshi Shimizu, Mark Payne, David Busija

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58 Scopus citations

Abstract

Poor outcome after neonatal brain injury may be associated with alterations in mitochondrial function. Thus, isolated mitochondria have been a useful tool in understanding the underlying mechanisms of mitochondrial dysfunction. However, isolation and characterization of mitochondria from neonatal rat brain are not fully described. Thus, the aim of this study was to develop a rapid method for the isolation and characterization of functional mitochondria from neonatal rat brain. Mitochondria were isolated from 7-day-old rat brain weighing ∼500 mg using a discontinuous Percoll density gradient. Brains were homogenized in 12% Percoll/sucrose buffer and layered onto a 26% Percoll/40% Percoll gradient followed by centrifugation. Four methods were used for assessing mitochondrial integrity and function: (1) electron microscopy to assess the morphology of the mitochondria and to determine the relative purity of the preparation; (2) fluorescence of chloromethyl-X-rosamine (Mito Tracker Red) in mitochondria as an indicator of mitochondrial membrane potential (Δψm); (3) state 3 and 4 respiration; and (4) protein import into mitochondria using an in vitro-synthesized mitochondrial malate dehydrogenase (mMDH). These studies demonstrated that the morphology of mitochondria is maintained with intact outer membranes and well-developed cristae, and Δψm is preserved. Respiration measurements revealed tightly coupled mitochondria with a respiration control ratio (RCR) of 4.1±0.18 (n=6). Import of precursor mMDH into mitochondria increased in a time-dependent manner maximizing at 15 min. The results indicate that neonatal brain mitochondria isolated using this method are well coupled, morphologically intact and are capable of protein import across the outer and inner mitochondrial membranes.

Original languageEnglish (US)
Pages (from-to)176-183
Number of pages8
JournalBrain Research Protocols
Volume8
Issue number3
DOIs
StatePublished - Dec 31 2001

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Keywords

  • CMX-rosamine
  • Electron microscopy
  • Energy metabolism
  • Mitochondria

ASJC Scopus subject areas

  • Neuroscience(all)

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