J Korean Med Sci.  2013 Jul;28(7):1071-1076. 10.3346/jkms.2013.28.7.1071.

Quantitative in Vivo Detection of Brain Cell Death after Hypoxia Ischemia Using the Lipid Peak at 1.3 ppm of Proton Magnetic Resonance Spectroscopy in Neonatal Rats

Affiliations
  • 1Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. wonspark@skku.edu
  • 2Department of Pediatrics, Ajou University College of Medicine, Suwon, Korea.
  • 3Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea.
  • 4Department of Pediatrics, Haeundae Paik Hospital, College of Medicine, Inje University, Busan, Korea.
  • 5Department of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
  • 6Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.

Abstract

This study was performed to determine the accuracy of proton magnetic spectroscopy (1H-MRS) lipid peak as a noninvasive tool for quantitative in vivo detection of brain cell death. Seven day-old Sprague Dawley rats were subjected to 8% oxygen following a unilateral carotid artery ligation. For treatment, cycloheximide was given immediately after hypoxic ischemia (HI). Lipid peak was measured using 1H-MRS at 24 hr after HI, and then brains were harvested for fluorocytometric analyses with annexin V/propidium iodide (PI) and fluorescent probe JC-1, and for adenosine-5'-triphosphate (ATP) and lactate. Increased lipid peak at 1.3 ppm measured with 1H-MRS, apoptotic and necrotic cells, and loss of mitochondrial membrane potential (DeltaPsi) at 24 hr after HI were significantly improved with cycloheximide treatment. Significantly reduced brain ATP and increased lactate levels observed at 24 hr after HI showed a tendency to improve without statistical significance with cycloheximide treatment. Lipid peak at 1.3 ppm showed significant positive correlation with both apoptotic and necrotic cells and loss of DeltaPsi, and negative correlation with normal live cells. Lipid peak at 1.3 ppm measured by 1H-MRS might be a sensitive and reliable diagnostic tool for quantitative in vivo detection of brain cell death after HI.

Keyword

Hypoxia-Ischemia, Brain; Animals; Newborn; Magnetic Resonance Spectroscopy; Flow Cytometry

MeSH Terms

Adenosine Triphosphate/*analysis
Animals
Animals, Newborn
*Apoptosis
Brain/metabolism/pathology
Cycloheximide/pharmacology
Hypoxia-Ischemia, Brain/*metabolism/*pathology
Lactic Acid/*analysis
Lipids/*analysis
Magnetic Resonance Spectroscopy
Membrane Potential, Mitochondrial
Rats
Rats, Sprague-Dawley
Adenosine Triphosphate
Cycloheximide
Lactic Acid
Lipids

Figure

  • Fig. 1 Representative MRI, 1H-MRS and flow cytometry data for the ipsilateral cerebral cortex after hypoxia-ischemia injury in each group. Flow cytometry data fall into four areas: Q1 (annexin V-/PI+), Q2 (annexin V+/PI+), Q3 (annexin V-/PI-), and Q4 (annexin V+/PI-). Positive annexin V denotes apoptosis and positive PI denotes necrosis. (A) Magnetic resonance image. (B) Proton magnetic resonance spectroscopy (MRS) with TE 30 msec. (C) MRS with TE 135 msec. (D) Flow cytometry with annexin V & PI. (E) Flow cytometry with JC-1. NC, normoxic control; HI, hypoxic ischemia; HI-CHX, hypoxic ischemia with cycloheximide; FC, flow cytometry.

  • Fig. 2 Correlation of the lipid/NAA ratio with the flow cytometry results. Q2 (annexin V+/PI+) cells (A), Q3 (annexin V-/PI-) cells (B), and JC-1 (green+/red-) damaged cells (C).


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