title: Isolation and characterization of intact mitochondria from neonatal rat brain.
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 approximately 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 (Delta psi(m)); (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 Delta psi(m) 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.
authors.affiliation: Department Of Physiology/Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston Salem, NC 27157, USA. crajapak@wfubmc.edu
authors.forename: N
authors.initials: N
authors.lastname: Rajapakse
authors.forename: K
authors.initials: K
authors.lastname: Shimizu
authors.forename: M
authors.initials: M
authors.lastname: Payne
authors.forename: D
authors.initials: D
authors.lastname: Busija
mesh_terms: D000818:Animals; D000831:Animals, Newborn / Q000502:physiology*; D001921:Brain / Q000648:ultrastructure*; D001923:Brain Chemistry; D004396:Coloring Agents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008291:Malate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry / Q000648:ultrastructure*; D009930:Organic Chemicals; D010101:Oxygen Consumption / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D004396:Coloring Agents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009930:Organic Chemicals; D011506:Proteins; C107472:red dye CMXRos; D008291:Malate Dehydrogenase