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Vol. 30, No. 5, 2008
Issue release date: September 2008
Free Access
Dev Neurosci 2008;30:319–324
(DOI:10.1159/000121416)

An Isolation Method for Assessment of Brain Mitochondria Function in Neonatal Mice with Hypoxic-Ischemic Brain Injury

Caspersen C.S.a · Sosunov A.b · Utkina-Sosunova I.a · Ratner V.I.a · Starkov A.A.c · Ten V.S.a
Departments of aPediatrics and bNeurological Surgery, Columbia University, and cDepartment of Neurology, Weill-Cornell University, New York, N.Y., USA
email Corresponding Author

Abstract

This work was undertaken to develop a method for the isolation of mitochondria from a single cerebral hemisphere in neonatal mice. Mitochondria from the normal mouse brain hemisphere isolated by the proposed method exhibited a good respiratory control ratio of 6.39 ± 0.53 during glutamate-malate-induced phosphorylating respiration. Electron microscopy showed intact mitochondria. The applicability of this method was tested on mitochondria isolated from naïve mice and their littermates subjected to hypoxic-ischemic insult. Hypoxic-ischemic insult prior to reperfusion resulted in a significant (p < 0.01) inhibition of phosphorylating respiration compared to naïve littermates. This was associated with a profound depletion of the ATP content in the ischemic hemisphere. The expression for Mn superoxide dismutase and cytochrome C (markers for the integrity of the mitochondrial matrix and outer membrane) was determined by Western blot to control for mitochondrial integrity and quantity in the compared samples. Thus, we have developed a method for the isolation of the cerebral mitochondria from a single hemisphere adapted to neonatal mice. This method may serve as a valuable tool to study mitochondrial function in a mouse model of immature brain injury. In addition, the suggested method enables us to examine the mitochondrial functional phenotype in immature mice with a targeted genetic alteration.


 goto top of outline Key Words

  • Mitochondria
  • Respiration
  • Neonatal mouse
  • Hypoxia-ischemia

 goto top of outline Abstract

This work was undertaken to develop a method for the isolation of mitochondria from a single cerebral hemisphere in neonatal mice. Mitochondria from the normal mouse brain hemisphere isolated by the proposed method exhibited a good respiratory control ratio of 6.39 ± 0.53 during glutamate-malate-induced phosphorylating respiration. Electron microscopy showed intact mitochondria. The applicability of this method was tested on mitochondria isolated from naïve mice and their littermates subjected to hypoxic-ischemic insult. Hypoxic-ischemic insult prior to reperfusion resulted in a significant (p < 0.01) inhibition of phosphorylating respiration compared to naïve littermates. This was associated with a profound depletion of the ATP content in the ischemic hemisphere. The expression for Mn superoxide dismutase and cytochrome C (markers for the integrity of the mitochondrial matrix and outer membrane) was determined by Western blot to control for mitochondrial integrity and quantity in the compared samples. Thus, we have developed a method for the isolation of the cerebral mitochondria from a single hemisphere adapted to neonatal mice. This method may serve as a valuable tool to study mitochondrial function in a mouse model of immature brain injury. In addition, the suggested method enables us to examine the mitochondrial functional phenotype in immature mice with a targeted genetic alteration.

Copyright © 2008 S. Karger AG, Basel


 goto top of outline References
  1. Gilland E, Puka-Sundvall M, Hillered L, Hagberg H: Mitochondrial function and energy metabolism after hypoxia-ischemia in the immature rat brain: involvement of NMDA-receptors. J Cereb Blood Flow Metab 1998;18:297–304.
  2. Puka-Sundvall M, Wallin C, Gilland E, Hallin U, Wang X, Sandberg M, Karlsson J, Blomgren K, Hagberg H: Impairment of mitochondrial respiration after cerebral hypoxia-ischemia in immature rats: relationship to activation of caspase-3 and neuronal injury. Brain Res Dev Brain Res 2000;125:43–50.
  3. Liu XH, Kwon D, Schielke GP, Yang GY, Silverstein FS, Barks JD: Mice deficient in interleukin-1 converting enzyme are resistant to neonatal hypoxic-ischemic brain damage. J Cereb Blood Flow Metab 1999;19:1099–1108.
  4. Ten VS, Bradley-Moore M, Gingrich JA, Stark RI, Pinsky DJ: Brain injury and neurofunctional deficit in neonatal mice with hypoxic-ischemic encephalopathy. Behav Brain Res 2003;145:209–219.
  5. Lafemina MJ, Sheldon RA, Ferriero DM: Acute hypoxia-ischemia results in hydrogen peroxide accumulation in neonatal but not adult mouse brain. Pediatr Res 2006;59:680–683.
  6. Polster BM, Robertson CL, Bucci CJ, Suzuki M, Fiskum G: Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release. Cell Death Differ 2003;10:365–370.
  7. Erecinska M, Cherian S, Silver IA: Energy metabolism in mammalian brain during development. Prog Neurobiol 2004;73:397–445.
  8. Sims N: Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 1990;55:698–707.
  9. Anderson MF, Sims NR: Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples. Brain Res Brain Res Protoc 2000;5:95–101.
  10. Rosenthal RE, Hamud F, Fiskum G, Varghese PJ, Sharpe S: Cerebral ischemia and reperfusion: prevention of brain mitochondrial injury by lidoflazine. J Cereb Blood Flow Metab 1987;7:752–758.
  11. Slinko S, Caspersen C, Ratner V, Kim JJ, Alexandrov P, Polin R, Ten VS: Systemic hyperthermia induces ischemic brain injury in neonatal mice with ligated carotid artery and jugular vein. Pediatr Res 2007;62:65–70.
  12. Clarkson A, Clarkson J, Jackson D, Sammut I: Mitochondrial involvement in transhemispheric diaschisis following hypoxia-ischemia: clomethiazole-mediated amelioration. Neuroscience 2007;144:547–561.
  13. Paila YD, Pucadyil TJ, Chattopadhyay A: The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin 1A receptor. Mol Membr Biol 2005;22:241–249.
  14. Brown MR, Sullivan PG, Dorenbos KA, Modafferi EA, Geddes JW, Steward O: Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria. J Neurosci Methods 2004;137:299– 303.
  15. Brustovetsky N, Jemmerson R, Dubinsky JM: Calcium-induced cytochrome c release from rat brain mitochondria is altered by digitonin. Neurosci Lett 2002;332:91–94.
  16. Tretter L, Mayer-Takacs D, Adam-Vizi V: The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria. Neurochem Int 2007;50:139–147.
  17. Herculano-Houzel S, Mota B, Lent R: Cellular scaling rules for rodent brains. Proc Natl Acad Sci USA 2006;103:12138–12143.

 goto top of outline Author Contacts

Vadim S. Ten, MD, PhD
Department of Pediatrics, Columbia University
3959 Broadway, CHN 1201
New York, NY 10032 (USA)
Tel. +1 212 342 0503, Fax +1 212 305 8796, E-Mail vt82@columbia.edu


 goto top of outline Article Information

Received: September 6, 2007
Accepted after revision: November 28, 2007
Published online: March 19, 2008
Number of Print Pages : 6
Number of Figures : 2, Number of Tables : 0, Number of References : 17


 goto top of outline Publication Details

Developmental Neuroscience

Vol. 30, No. 5, Year 2008 (Cover Date: September 2008)

Journal Editor: Campagnoni A.T. (Los Angeles, Calif.)
ISSN: 0378–5866 (Print), eISSN: 1421–9859 (Online)

For additional information: http://www.karger.com/DNE


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

This work was undertaken to develop a method for the isolation of mitochondria from a single cerebral hemisphere in neonatal mice. Mitochondria from the normal mouse brain hemisphere isolated by the proposed method exhibited a good respiratory control ratio of 6.39 ± 0.53 during glutamate-malate-induced phosphorylating respiration. Electron microscopy showed intact mitochondria. The applicability of this method was tested on mitochondria isolated from naïve mice and their littermates subjected to hypoxic-ischemic insult. Hypoxic-ischemic insult prior to reperfusion resulted in a significant (p < 0.01) inhibition of phosphorylating respiration compared to naïve littermates. This was associated with a profound depletion of the ATP content in the ischemic hemisphere. The expression for Mn superoxide dismutase and cytochrome C (markers for the integrity of the mitochondrial matrix and outer membrane) was determined by Western blot to control for mitochondrial integrity and quantity in the compared samples. Thus, we have developed a method for the isolation of the cerebral mitochondria from a single hemisphere adapted to neonatal mice. This method may serve as a valuable tool to study mitochondrial function in a mouse model of immature brain injury. In addition, the suggested method enables us to examine the mitochondrial functional phenotype in immature mice with a targeted genetic alteration.



 goto top of outline Author Contacts

Vadim S. Ten, MD, PhD
Department of Pediatrics, Columbia University
3959 Broadway, CHN 1201
New York, NY 10032 (USA)
Tel. +1 212 342 0503, Fax +1 212 305 8796, E-Mail vt82@columbia.edu


 goto top of outline Article Information

Received: September 6, 2007
Accepted after revision: November 28, 2007
Published online: March 19, 2008
Number of Print Pages : 6
Number of Figures : 2, Number of Tables : 0, Number of References : 17


 goto top of outline Publication Details

Developmental Neuroscience

Vol. 30, No. 5, Year 2008 (Cover Date: September 2008)

Journal Editor: Campagnoni A.T. (Los Angeles, Calif.)
ISSN: 0378–5866 (Print), eISSN: 1421–9859 (Online)

For additional information: http://www.karger.com/DNE


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Gilland E, Puka-Sundvall M, Hillered L, Hagberg H: Mitochondrial function and energy metabolism after hypoxia-ischemia in the immature rat brain: involvement of NMDA-receptors. J Cereb Blood Flow Metab 1998;18:297–304.
  2. Puka-Sundvall M, Wallin C, Gilland E, Hallin U, Wang X, Sandberg M, Karlsson J, Blomgren K, Hagberg H: Impairment of mitochondrial respiration after cerebral hypoxia-ischemia in immature rats: relationship to activation of caspase-3 and neuronal injury. Brain Res Dev Brain Res 2000;125:43–50.
  3. Liu XH, Kwon D, Schielke GP, Yang GY, Silverstein FS, Barks JD: Mice deficient in interleukin-1 converting enzyme are resistant to neonatal hypoxic-ischemic brain damage. J Cereb Blood Flow Metab 1999;19:1099–1108.
  4. Ten VS, Bradley-Moore M, Gingrich JA, Stark RI, Pinsky DJ: Brain injury and neurofunctional deficit in neonatal mice with hypoxic-ischemic encephalopathy. Behav Brain Res 2003;145:209–219.
  5. Lafemina MJ, Sheldon RA, Ferriero DM: Acute hypoxia-ischemia results in hydrogen peroxide accumulation in neonatal but not adult mouse brain. Pediatr Res 2006;59:680–683.
  6. Polster BM, Robertson CL, Bucci CJ, Suzuki M, Fiskum G: Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release. Cell Death Differ 2003;10:365–370.
  7. Erecinska M, Cherian S, Silver IA: Energy metabolism in mammalian brain during development. Prog Neurobiol 2004;73:397–445.
  8. Sims N: Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 1990;55:698–707.
  9. Anderson MF, Sims NR: Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples. Brain Res Brain Res Protoc 2000;5:95–101.
  10. Rosenthal RE, Hamud F, Fiskum G, Varghese PJ, Sharpe S: Cerebral ischemia and reperfusion: prevention of brain mitochondrial injury by lidoflazine. J Cereb Blood Flow Metab 1987;7:752–758.
  11. Slinko S, Caspersen C, Ratner V, Kim JJ, Alexandrov P, Polin R, Ten VS: Systemic hyperthermia induces ischemic brain injury in neonatal mice with ligated carotid artery and jugular vein. Pediatr Res 2007;62:65–70.
  12. Clarkson A, Clarkson J, Jackson D, Sammut I: Mitochondrial involvement in transhemispheric diaschisis following hypoxia-ischemia: clomethiazole-mediated amelioration. Neuroscience 2007;144:547–561.
  13. Paila YD, Pucadyil TJ, Chattopadhyay A: The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin 1A receptor. Mol Membr Biol 2005;22:241–249.
  14. Brown MR, Sullivan PG, Dorenbos KA, Modafferi EA, Geddes JW, Steward O: Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria. J Neurosci Methods 2004;137:299– 303.
  15. Brustovetsky N, Jemmerson R, Dubinsky JM: Calcium-induced cytochrome c release from rat brain mitochondria is altered by digitonin. Neurosci Lett 2002;332:91–94.
  16. Tretter L, Mayer-Takacs D, Adam-Vizi V: The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria. Neurochem Int 2007;50:139–147.
  17. Herculano-Houzel S, Mota B, Lent R: Cellular scaling rules for rodent brains. Proc Natl Acad Sci USA 2006;103:12138–12143.