tetrodotoxin and Reperfusion-Injury

Hypoxia typically accompanies acute inflammatory responses in patients and animal models. However, a limited number of studies have examined the effect of hypoxia in combination with inflammation (Hypo-Inf) on neural function. We previously reported that neuronal excitability in hippocampal CA1 neurons decreased during hypoxia and greatly rebounded upon reoxygenation. We attributed this altered excitability mainly to the dynamic regulation of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels and input resistance. However, the molecular mechanisms underlying input resistance changes by Hypo-Inf and reperfusion remained unclear. In the present study, we found that a change in the density of the delayed rectifier potassium current (I

Topics: Action Potentials; Animals; CA1 Region, Hippocampal; Cell Hypoxia; Culture Media; Delayed Rectifier Potassium Channels; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; In Vitro Techniques; Inflammation; Kinetics; Membrane Potentials; Neuroprotective Agents; Patch-Clamp Techniques; Rats; Reperfusion; Reperfusion Injury; Tetrodotoxin

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

Intracellular Na(+) concentration ([Na(+)](i)) rises in the heart during ischemia, and on reperfusion, there is a transient rise followed by a return toward control. These changes in [Na(+)](i) contribute to ischemic and reperfusion damage through their effects on Ca(2+) overload. Part of the rise of [Na(+)](i) during ischemia may be caused by increased activity of the cardiac Na(+)/H(+) exchanger (NHE1), activated by the ischemic rise in [H(+)](i). In support of this view, NHE1 inhibitors reduce the [Na(+)](i) rise during ischemia. Another possibility is that the rise of [Na(+)](i) during ischemia is caused by Na(+) influx through channels. We have reexamined these issues by use of two different NHE1 inhibitors, amiloride, and zoniporide, in addition to tetrodotoxin (TTX), which blocks voltage-sensitive Na(+) channels. All three drugs produced cardioprotection after ischemia, but amiloride (100 microM) and TTX (300 nM) prevented the rise in [Na(+)](i) during ischemia, whereas zoniporide (100 nM) did not. Both amiloride and zoniporide prevented the rise of [Na(+)](i) on reperfusion, whereas TTX was without effect. In an attempt to explain these differences, we measured the ability of the three drugs to block Na(+) currents. At the concentrations used, TTX reduced the transient Na(+) current (I (Na)) by 11 +/- 2% while amiloride and zoniporide were without effect. In contrast, TTX largely eliminated the persistent Na(+) current (I (Na,P)) and amiloride was equally effective, whereas zoniporide had a substantially smaller effect reducing I (Na,P) to 41 +/- 8%. These results suggest that part of the effect of NHE1 inhibitors on the [Na(+)](i) during ischemia is by blockade of I (Na,P). The fact that a low concentration of TTX eliminated the rise of [Na(+)](i) during ischemia suggests that I (Na,P) is a major source of Na(+) influx in this model of ischemia.

Topics: Amiloride; Anesthetics, Local; Animals; Cardiotonic Agents; Cell Separation; Female; Guanidines; In Vitro Techniques; Intracellular Space; Myocardium; Patch-Clamp Techniques; Pyrazoles; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium; Sodium Channel Blockers; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Tetrodotoxin

Ischemic incubation significantly increased amino acid release from rat striatal slices. Reoxygenation (REO) of the ischemic slices, however, enhanced only taurine and citrulline levels in the medium. Ischemia-induced increases in glutamate, taurine and GABA outputs were accompanied with a similar amount of decline in their tissue levels. Tissue final aspartic acid level, however, was doubled by ischemia. Lactate dehydrogenase (LDH) leakage was not altered by ischemia, but enhanced during REO. Presence of tetrodotoxine (TTX) during ischemic period caused significant decline in ischemia-induced glutamate output, but not altered REO-induced LDH leakage. Although omission of extracellular calcium ions from the medium during ischemic period protected the slices against REO-induced LDH leakage, this treatment failed to alter ischemia-induced glutamate and GABA outputs. The release of other amino acids, however, declined 50% in calcium-free medium. Blockade of the glutamate uptake transporter by L-trans-PDC, on the other hand, doubled ischemia induced glutamate and aspartic acid outputs. These results indicate that more than one mechanisms probably support the ischemia-evoked accumulation of glutamate and other amino acids in the extracellular space. Although LDH leakage enhanced during REO, processes involved in this increment were found to be dependent on extracellular calcium ions during ischemia but not REO period.

Topics: Amino Acids; Animals; Aspartic Acid; Brain; Calcium; Citrulline; Corpus Striatum; Female; gamma-Aminobutyric Acid; Glutamic Acid; Hypoxia; Ions; Ischemia; L-Lactate Dehydrogenase; Male; Oxygen; Rats; Rats, Wistar; Reperfusion Injury; Taurine; Tetrodotoxin; Time Factors

Glutamate extracellular accumulation is an early event in brain ischemia triggering excitotoxic neuron damage. We have investigated how to control the glutamate efflux from human cerebrocortical slices superfused in conditions simulating an acute ischemic insult (oxygen and glucose deprivation). The efflux of previously accumulated [3H]D-aspartate or endogenous glutamate increased starting 18 min after exposure to ischemia and returned almost to basal values in 6 min reperfusion with standard medium. Superfusion with Ca2+-free, EGTA (0.5 mM)-containing medium or with medium containing tetrodotoxin (TTX; 0.5 microM) inhibited the ischemia (24 min)-evoked [3H]D-aspartate efflux by about 50% and 65%, respectively. The ischemia (24 or 36 min)-evoked efflux of [3H]D-aspartate or endogenous glutamate was reduced at least 40% by the adenosine A(2A) receptor antagonist SCH 58261 (1 microM); the compound was effective when added up to 15 min after exposure to ischemia. No effect of SCH 58261 on the ischemia-evoked [3H]D-aspartate was found in Ca2+-free, EGTA-containing medium. To conclude, a significant component of the ischemia-evoked glutamate efflux in human cerebrocortical slices seems to occur by a vesicular-like mechanism. Endogenously released adenosine is likely to activate A(2A) receptors that enhance vesicular-like glutamate release during ischemia; A(2A) receptor antagonists would deserve consideration for their neuroprotective potential.

Topics: Adenosine A2 Receptor Antagonists; Adult; Aged; Anesthetics, Local; Aspartic Acid; Brain Ischemia; Calcium; Cerebral Cortex; Female; Glucose; Glutamic Acid; Humans; In Vitro Techniques; Male; Middle Aged; Neuroprotective Agents; Pyrimidines; Reperfusion Injury; Synaptic Vesicles; Tetrodotoxin; Triazoles

The aim of this study was to examine whether the antioxidant alpha-lipoic acid protects retinal neurons from ischemia-reperfusion injury. Rats were injected intraperitoneally with either vehicle or alpha-lipoic acid (100 mg/kg) once daily for 11 days. On the third day, ischemia was delivered to the rat retina by raising the intraocular pressure above systolic blood pressure for 45 min. The electroretinogram was measured prior to ischemia and 5 days after reperfusion. Rats were killed 5 or 8 days after reperfusion and the retinas were processed for immunohistochemistry and for determination of mRNA levels by RT-PCR. Ischemia-reperfusion caused a significant reduction of the a- and b-wave amplitudes of the electroretinogram, a decrease in nitric oxide synthase and Thy-1 immunoreactivities, a decrease of retinal ganglion cell-specific mRNAs and an increase in bFGF and CNTF mRNA levels. All of these changes were clearly counteracted by alpha-lipoic acid. Moreover, in mixed rat retinal cultures, alpha-lipoic acid partially counteracted the loss of GABA-immunoreactive neurons induced by anoxia. The results of the study demonstrate that alpha-lipoic acid provides protection to the retina as a whole, and to ganglion cells in particular, from ischemia-reperfusion injuries. alpha-Lipoic acid also displayed negligible affinity for voltage-dependent sodium and calcium channels.

Topics: Anesthetics, Local; Animals; Antioxidants; Binding, Competitive; Brain-Derived Neurotrophic Factor; Calcium; Calcium Channel Blockers; Cells, Cultured; Ciliary Neurotrophic Factor; Diltiazem; Dizocilpine Maleate; DNA Primers; Dose-Response Relationship, Drug; Drug Interactions; Electroretinography; Fibroblast Growth Factors; Glial Fibrillary Acidic Protein; Glyceraldehyde-3-Phosphate Dehydrogenases; N-Methylaspartate; Nifedipine; Rats; Reperfusion Injury; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Sodium; Tetrodotoxin; Thioctic Acid; Thy-1 Antigens; Veratridine

Prior induction of heat shock protein 70 (HSP70) protects against ischemia-reperfusion (I/R) mucosal injury, but the ability of HSP70 to affect I/R-induced alterations in epithelial cell function is unknown. Rats subjected to whole body hyperthermia (41.5-42 degrees C for 6 min) increased HSP70 and heat shock factor 1 mRNA expression, reaching a maximum 2 h after heat stress and declining thereafter. HSP70 production was maximally elevated at 4 h after heat stress and remained elevated until after 12 h. Heat stress alone had no effect on mucosal function except to enhance secretion in response to ACh. Heat stress provided complete morphological protection against I/R-induced mucosal injury but did not confer a similar protection against I/R-induced decreases in mucosal resistance, sodium-linked glucose absorption, or tachykinin-mediated chloride secretion. Heat stress, however, attenuated the I/R-induced suppression of ACh response, and this effect was dependent on enteric nerves. Thus induction of heat shock protein 70 is associated with the preservation of mucosal architecture and attenuation of some specific functional alterations induced by I/R.

Topics: Absorption; Acetylcholine; Animals; Chlorides; Enteric Nervous System; Glucose; Hot Temperature; HSP70 Heat-Shock Proteins; Hyperthermia, Induced; Intestinal Mucosa; Ischemia; Male; Permeability; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Stress, Physiological; Tetrodotoxin

Using middle cerebral artery occlusion (MCAO) and in vivo microdialysis, we have evaluated the changes in extracellular concentrations of the excitatory amino acids (EAA) glutamate and aspartate during varying periods of MCAO (0, 30, 60 min) in the striatum of spontaneously hypertensive rats (SHR). A positive correlation between occlusion time-dependent elevations in EAAs and the resulting ischemic injury was observed. This is the first demonstration of the temporal profile of EAA efflux during transient focal ischemia in SHRs. Possible sources and mechanisms of ischemia-induced EAA efflux were examined during 60 min of MCAO. Removal of Ca(2+) from the microdialysis infusion media significantly attenuated ischemia-induced increases in both glutamate (from ischemic peak of 4892 +/- 1298 to 1144 +/- 666% of preischemic values) and aspartate (from 2703 +/- 682 to 2090 +/- 599% of preischemic values). Similarly, infusion of the voltage dependent Na(+) channel blocker tetrodotoxin (TTX; 10 microM) significantly attenuated MCAO-induced increases in glutamate (to 1313 +/- 648%) and aspartate (to 359 +/- 114%). Infusion of the GLT-1 selective nontransportable inhibitor, dihydrokainate (DHK; 1 mM) also significantly attenuated the ischemia-induced increases in both EAAs (1285 +/- 508 and 1366 +/- 741% of the preischemic levels, respectively). These results indicate that during transient focal ischemia the increase in extracellular EAAs originates from both the neuronal pool, via conventional exocytotic release, and glial sources via the reversal of the GLT-1 transporter.

Topics: Amino Acid Transport System X-AG; Animals; Aspartic Acid; ATP-Binding Cassette Transporters; Brain; Brain Ischemia; Extracellular Space; Glutamic Acid; Infarction, Middle Cerebral Artery; Kainic Acid; Male; Rats; Rats, Inbred SHR; Reperfusion Injury; Tetrodotoxin; Time Factors

There is ample evidence that ischaemia is associated with partial denervation of the detrusor muscle and that this is responsible for much of its abnormal contractile behaviour, resulting in bladder dysfunction (instability). In guinea-pig nerves are very susceptible to the ischaemic damage as compared to the muscle cells. The purpose of this study was to assess the neuroprotection afforded by taurine on guinea-pig detrusor under ischaemic-like conditions. Guinea-pig detrusor strips were subjected for 60 min to ischaemic-like conditions, followed by 150 min reperfusion. Intrinsic nerves underwent every 30 min electrical field stimulation (EFS) by 5-s trains of square voltage pulses of 0.05 ms duration (15 Hz, 50 V). Detrusor strips were perfused with 0.1, 1, 3 or 10 mM taurine during the ischaemia-like exposure and the first 30 min of reperfusion. Taurine (1 and 3 mM) significantly improved the response of the strips to EFS both at the end of ischaemia and reperfusion. On the contrary, neither 0.1 nor 10 mM taurine had significant effects. It is concluded that taurine can partially counteract the ischaemia-reperfusion injury in the guinea-pig urinary bladder.

Topics: Aminoethylphosphonic Acid; Animals; Atropine; Electric Stimulation; Evoked Potentials; Glucose; Guinea Pigs; Hypoxia; Ischemia; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Neuroprotective Agents; Oxygen; Purinergic P2 Receptor Antagonists; Reperfusion Injury; Suramin; Taurine; Tetrodotoxin; Urinary Bladder

Ischemia reperfusion injury (IRI) contributes significantly to posttransplant graft dysfunction. An emphasis, therefore, has been directed toward the identification of novel renoprotective agents. In this study, the renoprotective effect of tetrodotoxin (TTX) alone, or in combination with a thromboxane synthetase inhibitor (OKY-046), was investigated in a 60-min warm ischemia, 72-h reperfusion, IRI rodent model. Unilateral nephrectomized rats were treated with the test vehicle alone, 1, 2, or 4 microgram/kg of TTX or 2 mg/kg of OKY-046 intravenously, either 15 min pre- or postischemia, or 2 microgram/kg TTX administered simultaneously with OKY-046 (2 mg/kg), following the ischemic interval. Baseline, 24, and 72 h mean plasma creatinine (Cr) and urea nitrogen (BUN) were compared. Maximal renoprotection was demonstrated by significantly improved 72-h Cr and BUN levels with the 2 microgram/kg of TTX or with 2 mg/kg of OKY-046, each administered after ischemia (ischemic control Cr = 8. 01 +/- 1.07 mg/dl vs TTX = 3.84 +/- 0.80 mg/dl, P = 0.008; vs OKY-046 = 4.0 +/- 1.5, P + 0.008; ischemic control BUN = 241.3 mg/dl +/- 32.8 vs TTX = 85.7 mg/dl +/- 18.7, P < 0.008; vs OKY-046 = 52.6 +/- 22.5, P = 0.008). The combination therapy utilizing TTX with OKY-046 resulted in reduced animal survival, demonstrating no renoprotection as measured with the biochemical parameters. These results support the renoprotective effects of TTX in a severe, rodent IRI model. The exact mechanism of action, as well as the therapeutic potential of TTX in preservation/transplantation, warrants further study.

Topics: Animals; Blood Urea Nitrogen; Creatinine; Drug Therapy, Combination; Enzyme Inhibitors; Kidney; Male; Methacrylates; Organ Size; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Survival Rate; Tetrodotoxin; Thromboxane-A Synthase; Time Factors

To clarify functional changes of dopaminergic neurons and dopamine (DA) reuptake during and after ischemia, extracellular DA levels in striatum were determined using in vivo brain microdialysis in a 4-vessel occlusion model of male Wistar rats with and without pharmacological interventions. Without interventions, the extracellular DA levels markedly increased during ischemia, but upon reperfusion, rapidly returned to control level. Infusion of tetrodotoxin, a blocker of voltage-dependent Na+ channels, was without effect on the DA surge during ischemia, but decreased the DA levels after reperfusion to the same extent as in control rats. Pretreatment with nomifensine, an inhibitor of DA reuptake, was also without effect on the surge, but reduced the rate of DA decline after reperfusion to one-fifth of the rate without the pretreatment. When nomifensine was administered 40 min after reperfusion, extracellular DA levels increased to the same extent as in control rats. Infusion of high K+ 1 h after reperfusion induced a smaller increase in extracellular DA levels than that in control rats. It took 96 h for this reduced response to high K+ stimulation to recover after reperfusion. These results suggest that the DA surge during ischemia is mainly derived from action potential-independent DA release (means dysfunction of dopaminergic neurons), although activity of DA reuptake is completely inhibited. After reperfusion, the basal function of dopaminergic neurons and activity of DA reuptake rapidly recover, but the neurons are functionally disturbed to release less DA in response to a given stimulus for several days.

Topics: Animals; Chromatography, High Pressure Liquid; Corpus Striatum; Dialysis; Dopamine; Ischemic Attack, Transient; Male; Neurons; Nomifensine; Potassium; Prosencephalon; Rats; Rats, Inbred Strains; Reperfusion Injury; Tetrodotoxin