carbocyanines and Hypoxia-Ischemia--Brain

The early antecedents of cerebral palsy (CP) are unknown but are suspected to be due to hypoxia-ischemia (H-I). In our rabbit model of CP, the MRI biomarker, apparent diffusion coefficient (ADC) on diffusion-weighted imaging, predicted which fetuses will develop postnatal hypertonia. Surviving H-I fetuses experience reperfusion-reoxygenation but a subpopulation manifested a continued decline of ADC during early reperfusion-reoxygenation, which possibly represented greater brain injury (RepReOx). We hypothesized that oxidative stress in reperfusion-reoxygenation is a critical trigger for postnatal hypertonia. We investigated whether RepReOx predicted postnatal neurobehavior, indicated oxidative stress, and whether targeting antioxidants at RepReOx ameliorated motor deficits, which included testing of a new superoxide dismutase mimic (MnTnHex-2-PyP). Rabbit dams, 79% gestation (E25), were subjected to 40 min uterine ischemia. Fetal brain ADC was followed during H-I, immediate reperfusion-reoxygenation, and 4-72 h after H-I. Endpoints were postnatal neurological outcome at E32, ADC at end of H-I, ADC nadir during H-I and reperfusion-reoxygenation, and area under ADC curve during the first 20 min of reperfusion-reoxygenation. Antioxidants targeting RepReOx were administered before and/or after uterine ischemia. The new MRI-ADC biomarker for RepReOx improved prediction of postnatal hypertonia. Greater superoxide production, mitochondrial injury, and oligodendroglial loss occurred in fetal brains exhibiting RepReOx than in those without. The antioxidants, MnTnHex-2-PyP and Ascorbate and Trolox combination, significantly decreased postnatal motor deficits and extent of RepReOx. The etiological link between early injury and later motor deficits can thus be investigated by MRI, and allows us to distinguish between critical oxidative stress that causes motor deficits and noncritical oxidative stress that does not.

Topics: Age Factors; Animals; Animals, Newborn; Antioxidants; Ascorbic Acid; Benzimidazoles; Blood Flow Velocity; Brain; Brain Mapping; Carbocyanines; Chromans; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Embryo, Mammalian; Female; Flow Cytometry; Hypoxia-Ischemia, Brain; Ionophores; Laser-Doppler Flowmetry; Membrane Potential, Mitochondrial; Metalloporphyrins; Microvessels; Mitochondria; Movement Disorders; Muscle Hypertonia; O Antigens; Pregnancy; Rabbits; Reperfusion Injury; Superoxides; Time Factors; Valinomycin

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

Accumulating evidence indicates that the polyphenol resveratrol (trans-3, 5, 4"-trihydroxystibene, RVT) potently protects against cerebral ischemia neuronal damage due to its oxygen free radicals scavenging and antioxidant properties. However, it is unknown whether RVT can attenuate ischemia-induced early impairment of neuronal excitability. To address this question, we simulated ischemic conditions by applying oxygen-glucose deprivation (OGD) to acute rat hippocampal slices and examined the effect of RVT on OGD-induced pyramidal neuron excitability impairment using whole-cell patch clamp recording. 100 microM RVT largely inhibited the 15 min OGD-induced progressive membrane potential (Vm) depolarization and the reduction in evoked action potential frequency and amplitude in pyramidal neurons. In a parallel neuronal viability study using TO-PRO-3 iodide staining, 20 min OGD induced irreversible CA1 pyramidal neuronal death which was significantly reduced by 100 microM RVT. No similar effects were found with PQQ treatment, an antioxidant also showing potent neuroprotection in the rat rMCAO ischemia model. This suggests that antioxidant action per se, is unlikely accounting for the observed early effects of RVT. RVT also markedly reduced the frequency and amplitude of AMPA mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons, which is also an early consequence of OGD. RVT effects on neuronal excitability were inhibited by the large-conductance potassium channel (BK channel) inhibitor paxilline. Together, these studies demonstrate that RVT attenuates OGD-induced neuronal impairment occurring early in the simulated ischemia slice model by enhancing the activation of BK channel and reducing the OGD-enhanced AMPA/NMDA receptor mediated neuronal EPSCs.

Topics: Action Potentials; Animals; Antioxidants; Carbocyanines; Cell Death; Hippocampus; Hypoxia-Ischemia, Brain; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Nerve Degeneration; Organ Culture Techniques; Potassium Channel Blockers; Pyramidal Cells; Rats; Resveratrol; Stilbenes; Time Factors

Progesterone modulates gamma-aminobutyric acid and excitatory amino acid neurotransmitter systems and has neuroprotective properties in models of hypoxia-ischemia. This study examined the in vitro effects of allopregnanolone, the active progesterone metabolite, in models of N-methyl-D-aspartate (NMDA)-induced necrosis and apoptosis. Cultured NT2 neurons were exposed to 1 mM NMDA. Lactate dehydrogenase (LDH) release was measured 24 h later. NMDA at a concentration of 1 mM produced a 39 +/- 19% release of total LDH. Exposure to 10 microM allopregnanolone prior to NMDA exposure reduced LDH release by 51% (P = 0.0028). NMDA stimulated apoptotic cell changes defined by terminal dUTP nick-end labeling (TUNEL) and 5,5', 6,6'-tetrachloro-1,1,3,3'-tetra ethlybenzimidazolycarbocyanide iodide staining were reduced to baseline values by both 10 microM allopregnanolone and 100 microM MK-801. Pretreatment with allopregnanolone (0-10 microM) reduced the percentage of TUNEL-positive cells in a dose-dependent manner (EC(50) = 2.7 +/- 0.1 nM). Physiologic concentrations of allopregnanolone provided protection against both necrotic and apoptotic injury induced by NMDA excitotoxicity.

Topics: Apoptosis; Asphyxia Neonatorum; Benzimidazoles; Carbocyanines; Cell Count; Cell Survival; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Fluorescent Dyes; Humans; Hypoxia-Ischemia, Brain; In Situ Nick-End Labeling; Infant, Newborn; L-Lactate Dehydrogenase; Membrane Potentials; Mitochondria; N-Methylaspartate; Neurons; Neuroprotective Agents; Neurotoxins; Pregnancy; Pregnanolone; Progesterone; Tumor Cells, Cultured