carbocyanines and triphenyltetrazolium

The blood-brain barrier (BBB) disruption following cerebral ischemia (stroke) contributes to the development of life-threatening brain edema. Recent studies suggested that the ischemic BBB disruption is not uniform throughout the affected brain region. The aim of this study was to establish in vivo optical imaging methods to assess the size selectivity and spatial distribution of the BBB disruption after a focal cerebral ischemia. The BBB permeability was assessed in mice subjected to a 60-min middle cerebral artery occlusion and 24 h of reperfusion using in vivo time domain near-infrared optical imaging after contrast enhancement with two tracers of different molecular size, Cy5.5 (1 kDa) and Cy5.5 conjugated with bovine serum albumin (BSA) (67 kDa). Volumetric reconstruction of contrast-enhanced brain areas in vivo and ex vivo indicated that the BSA-Cy5.5-enhancement is identical to the volume of infarct determined by TTC staining, whereas the volume of enhancement with Cy5.5 was 40% greater. The volume differential between areas of BBB disruption for small and large-size molecules could be useful for determining the size of peri-infarct tissues (penumbra) that can respond to neuroprotective therapies.

Topics: Animals; Blood-Brain Barrier; Brain; Carbocyanines; Cattle; Cone-Beam Computed Tomography; Contrast Media; Disease Models, Animal; Fluorescence; Histocytochemistry; Humans; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Mice; Mice, Inbred Strains; Microtomy; Molecular Imaging; Reperfusion Injury; Serum Albumin; Tetrazolium Salts

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