carbocyanines and Brain-Infarction

We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) with a fluorescent probe, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolo-carbocyanine iodide), for the quantitative evaluation of neonatal hypoxic-ischemic brain injury. Seven-day-old rat pups were subjected to 2h of 8% oxygen following unilateral carotid artery ligation. Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI), and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI), terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c. Flow cytograms of JC-1 showed two distinct sub-populations with different ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V(+)/PI(+) necrotic cells, TUNEL-positive cells, TTC infarct area and western blot of cytosolic cytochrome c, and negative correlation with annexin V(-)/PI(-) live cells. In summary, low ΔΨ measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI. Therefore, fluorocytometric analysis of ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.

Topics: Animals; Animals, Newborn; Annexin A5; Benzimidazoles; Brain; Brain Infarction; Carbocyanines; Cell Death; Cytochromes c; Disease Models, Animal; Flow Cytometry; Fluorescent Dyes; Hypoxia-Ischemia, Brain; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Neurons; Propidium; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Time Factors

Recent studies have shown that neuroblasts migrate from the subventricular zone (SVZ) into the injured area after ischemic brain insults. However, it is not well understood which mechanism mediates this ectopic migration and which types of cells migrate into the damaged region from the SVZ. The present study was designed to investigate the characteristics of the migration of nestin-positive neural stem cells toward the region of ischemic injury after focal cortical ischemia. Nestin-eGFP transgenic mice were used to effectively model the migration of SVZ cells. Photothrombotic ischemia was induced by injection of rose bengal (30 mg/kg) and exposure to cold light. Migration of nestin-positive cells was examined using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and bromodeoxyuridine (BrdU) labeling. The number of nestin-positive cells was increased significantly in the peri-infarct area at 5 and 7 days after photothrombosis. A subset of nestin-positive cells was co-labeled with DiI or BrdU. Some of the nestin-positive cells co-expressed doublecortin (DCX) and only a few nestin-positive cells co-labeled with anti-epidermal growth factor receptor (EGFr) antibody. However, no nestin-positive cells were immunoreactive for glial fibrillary acidic protein (GFAP). The inhibition of matrix metalloproteinases (MMPs) using the MMP inhibitor, FN-439, decreased nestin-positive cells in the peri-infarct region at 7 days after photothrombosis. Although MMP-9 was not co-expressed in the nestin-positive cells in the peri-infarct cortex, MMP-9 did co-localize with GFAP-positive astrocytes. These results suggest that nestin-positive neural progenitor cells migrate into the peri-infarct cortex after photothrombotic ischemia and that MMP-9 is involved in the migration.

Topics: Animals; Brain Infarction; Brain Ischemia; Bromodeoxyuridine; Carbocyanines; Cell Movement; Doublecortin Domain Proteins; Doublecortin Protein; ErbB Receptors; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Hydroxamic Acids; Immunohistochemistry; Intermediate Filament Proteins; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Transgenic; Microinjections; Microtubule-Associated Proteins; Nerve Tissue Proteins; Nestin; Neuroglia; Neurons; Neuropeptides; Oligopeptides; Rose Bengal; Stem Cells