fg-9041 and Reperfusion-Injury

We have developed an approach to directly probe neuronal excitability during the period beginning with induction of global ischemia and extending after reperfusion using transgenic mice expressing channelrhodopsin-2 (ChR2) to activate deep layer cortical neurons independent of synaptic or sensory stimulation. Spontaneous, ChR2, or forepaw stimulation-evoked electroencephalogram (EEG) or local field potential (LFP) records were collected from the somatosensory cortex. Within 20 s of ischemia, a >90% depression of spontaneous 0.3-3 Hz EEG and LFP power was detected. Ischemic depolarization followed EEG depression with a ∼2 min delay. Surprisingly, neuronal excitability, as assessed by the ChR2-mediated EEG response, was intact during the period of strong spontaneous EEG suppression and actually increased before ischemic depolarization. In contrast, a decrease in the somatosensory-evoked potential (forepaw-evoked potential, reflecting cortical synaptic transmission) was coincident with the EEG suppression. After 5 min of ischemia, the animal was reperfused, and the ChR2-mediated response mostly recovered within 30 min (>80% of preischemia value). However, the recovery of the somatosensory-evoked potential was significantly delayed compared with the ChR2-mediated response (<40% of preischemia value at 60 min). By assessing intrinsic optical signals in combination with EEG, we found that neuronal excitability approached minimal values when the spreading ischemic depolarization wave propagated to the ChR2-stimulated cortex. Our results indicate that the ChR2-mediated EEG/LFP response recovers much faster than sensory-evoked EEG/LFP activity in vivo following ischemia and reperfusion, defining a period where excitable but synaptically silent neurons are present.

Topics: Anesthetics, Local; Animals; Bacterial Proteins; Carrier Proteins; Channelrhodopsins; Disease Models, Animal; Electroencephalography; Evoked Potentials; Excitatory Amino Acid Antagonists; Forelimb; Hyperalgesia; In Vitro Techniques; Ischemia; Luminescent Proteins; Membrane Potentials; Mice; Mice, Transgenic; Neurons; Optogenetics; Physical Stimulation; Quinoxalines; Reperfusion Injury; Tetrodotoxin; Valine

To investigate the effects of different antagonists on the alteration of I?B kinase (IKK) activity in rat hippocampus following global brain ischemia.. Using 4-vessel occlusion (4-VO) as brain ischemia model, IKK protein expression was examined by immunoblotting and immunoprecipitation, and IKK activity was assayed by in vitro kinase assay.. There was no alteration of IKK protein expression following ischemia or ischemia/reperfusion different time points, but IKK activity reached its peak level at ischemia 30 min. Pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, non-NMDA receptor antagonist DNQX, or NF-kappaB inhibitor PDTC decreased the IKK activity following brain ischemia 30 min. The increase in substrate myelin basic protein (MBP) phosphorylation by IKK is associated with an increase in autophosphorylation of IKK, which can also be antagonized by ketamine, DNQX, and PDTC.. NMDA receptor and non-NMDA receptor mediate the increase of IKK activity following global brain ischemia in rat hippocampus, which contributes to the alterations of expression and activity of downstream factor NF-kappaB.

Topics: Animals; Brain Ischemia; Excitatory Amino Acid Antagonists; Hippocampus; I-kappa B Kinase; Ketamine; Male; NF-kappa B; Phosphorylation; Proline; Protein Serine-Threonine Kinases; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Thiocarbamates

Recent studies suggest that nuclear factor NF-kappaB may be involved in excitotoxin-induced cell apopotosis. To analyze the variation of NF-kappaB, levels of NF-kappaB were measured after the rats were subjected to 30 min of four-vessel occlusion and sacrificed in selected reperfusion time points. Induction of NF-kappaB consisting mainly of p65 and p50 subunits was detected by Western blot with anti p65, p50 antibodies, respectively. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our studies indicate that ischemia-induced NF-kappaB nuclear translocation is time-dependent. Inductions or binding activity of NF-kappaB in nucleus increased about 10-fold from 6 to 12 h as compared with that of the control group, then gradually declined in the following 24, 72 h. To further analyze the regulation by ionotropic glutamate receptor and L-type voltage-gated Ca(2+) channel (L-VGCC) in vivo, N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione and L-VGCC antagonist nifedipine were given 20 min prior to 30 min of ischemia. The NF-kappaB nuclear translocation was completely blocked by these three antagonists in a dose-dependent manner after ischemia/reperfusion 6 h. Increased phosphorylation of the NF-kappaB regulatory unit IkappaB-alpha was detected by Western blot. Decrement of IkappaB-alpha was found after 3 h reperfusion in the cytoplasm following global ischemia, which was also blocked by such three antagonists. These results illustrate that glutamate-gated ionotropic NMDA or non-NMDA receptors and voltage-gated Ca(2+) channels are important routes to mediate NF-kappaB activation during brain ischemic injury. Active NF-kappaB may attend the excitotoxin-induced cell death in turn. Our studies also suggest that IkappaB-alpha is an important regulatory unit that controls the activation of NF-kappaB after its phosphorylation and degradation and resynthesis in rat hippocampus following global ischemia.

Topics: Animals; Brain Ischemia; Calcium Channel Blockers; Calcium Channels, L-Type; Cytoplasm; Excitatory Amino Acid Antagonists; Hippocampus; I-kappa B Proteins; Ketamine; Male; NF-kappa B; Nifedipine; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Amino Acid; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Time Factors

The effects of three drugs including ketamine(KT), a noncompetitive antagonist of NMDA receptor (NR), nifedipine(ND), a voltage gated calcium channel (VGCC) antagonist and 6, 7dinitroquinoxaline -2, 3-dione(DNQX), a non-NMDA receptor antagonist on the contents of phospho-tyrosine proteins (p-tyr-pr) in the synaptosomal(P2), the crude membrane (P3), and the cytosolic (S3) fractions of hippocampus in forebrain ischemia of mongolian gerbils were studied.. (1) the contents of p-tyr-pr in all three fractions (P2, P3, S3) decreased 15 min after ischemia, but the contents of p-tyr-pr in S3 fraction decreased more obviously than the others did, With the increase of reperfusion time, the contents of p-tyr-pr in all of these fractions recovered gradually, but the p-tyr-pr in S3 fraction increased more rapidly among them, in the P2 fraction, the contents of p-tyr-pr increased slowly, but significantly and sustained longer during reperfusion when compared with that of P3 did. (2) The increase in p-tyr-pr contents induced by cerebral ischemia/reperfusion was partially antagonisted by KT and ND administration prior to cerebral ischemia, under these conditions, DNQX has no effect on it.. the increase of p-tyr-Pr contents induced by cerebral ischemia/reperfusion is related to NR channel and L-type VGCC, but not to non-NR channel.

Topics: Animals; Brain Ischemia; Calcium Channel Blockers; Calcium Channels; Excitatory Amino Acid Antagonists; Gerbillinae; Hippocampus; Ketamine; Nifedipine; Phosphorylation; Phosphotyrosine; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury

The effects of ischemia/reperfusion on the levels of protein tyrosine phosphorylation in the synaptosome of gerbil hippocampus and the effects of three drugs, ketamine (KT), a noncompetitive antagonist of NMDA receptor, nifedipine (ND), a voltage gated calcium channel (VGCC) antagonist and 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, on the phosphorylation were studied. The results showed that (1) 15 min of transient forebrain ischemia caused a marked decrease in the level of tyrosine phosphorylation of many protein bands, but, if followed by 15 min to 48 h of reperfusion, many protein bands including the 180 kD protein appeared to be increased; (2) the degree of tyrosine phosphorylation of the protein bands was higher than that of the sham-operated control, e.g. that of 180 kD protein was 1.8 fold of control; (3) administration of KT and ND before ischemia attenuated the increase of 180 kD protein tyrosine phosphorylation, while DNQX had no effect; and (4) immunoprecipitation and Western blot confirmed that the NR2B subunits of the NMDA receptors were among the phosphorylated 180 kD protein and ischemia /reperfusion did not affect the level of protein expression of NR2B. The above results suggest that the increase of tyrosine phosphorylation of NR2B induced by ischemia/reperfusion may further activate NR channels and aggravate neuronal injury, and that NR channels and other protein can be regulated by tyrosine phosphorylation not only through NR channels themselves but also via L-type VGCCs. Consequently, antagonists of both NR channels and L-type VGCCs may play a certain role in prevention and cure of ischemic brain injury.

Topics: Animals; Brain Ischemia; Calcium Channels, L-Type; Gerbillinae; Hippocampus; Ketamine; Nifedipine; Phosphorylation; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Synaptosomes; Tyrosine