tetrodotoxin and Ischemia

Ischaemia is known to cause massive neuronal depolarization, termed anoxic depolarization (AD), due to energy failure and loss of membrane ion gradients. The neuromodulator adenosine accumulates extracellularly during ischaemia and activates four metabotropic receptors: A. We performed patch-clamp recordings of MSNs in rat striatal slices subjected to oxygen and glucose deprivation (OGD) to investigate the effects of A. Our data indicate that the A. Adenosine, released during striatal OGD, activates A

Topics: Adenosine; Animals; Glucose; Glutamic Acid; Ion Channels; Ischemia; Ligands; Neurons; Oxygen; Rats; Rats, Wistar; Receptor, Adenosine A2A; 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

Resveratrol has been demonstrated to be protective in the cardiovascular system. The aim of this study was to assess the effects of resveratrol on hydrogen peroxide (H(2)O(2))-induced increase in late sodium current (I(Na.L)) which augmented the reverse Na(+)-Ca(2+) exchanger current (I(NCX)), and the diastolic intracellular Ca(2+) concentration in ventricular myocytes.. I(Na.L), I(NCX,) L-type Ca(2+) current (I(Ca.L)) and intracellular Ca(2+) properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes.. Resveratrol (10, 20, 40 and 80 µM) decreased I(Na.L) in myocytes both in the absence and presence of H(2)O(2) (300 µM) in a concentration dependent manner. Ranolazine (3-9 µM) and tetrodotoxin (TTX, 4 µM), I(Na.L) inhibitors, decreased I(Na.L) in cardiomyocytes in the presence of 300 µM H(2)O(2). H(2)O(2) (300 µM) increased the reverse I(NCX) and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H(2)O(2) of the diastolic intracellular Ca(2+) fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca(2+) transient rise and decay. As expected, 2 µM TTX had no effect on I(Ca.L).. Resveratrol protects the cardiomyocytes by inhibiting the H(2)O(2)-induced augmentation of I(Na.L.)and may contribute to the reduction of ischemia-induced lethal arrhythmias.

Topics: Acetanilides; Animals; Antioxidants; Arrhythmias, Cardiac; Calcium; Diastole; Dose-Response Relationship, Drug; Electrophysiology; Female; Heart Ventricles; Hydrogen Peroxide; Ischemia; Male; Muscle Cells; Patch-Clamp Techniques; Piperazines; Rabbits; Ranolazine; Resveratrol; Sodium; Stilbenes; Temperature; Tetrodotoxin

Recent studies have identified the important contribution of glial cells to the plasticity of neuronal circuits. Resting microglia, the primary immune effector cells in the brain, dynamically extend and retract their processes as if actively surveying the microenvironment. However, just what is being sampled by these resting microglial processes has not been demonstrated in vivo, and the nature and function of any interactions between microglia and neuronal circuits is incompletely understood. Using in vivo two-photon imaging of fluorescent-labeled neurons and microglia, we demonstrate that the resting microglial processes make brief (approximately 5 min) and direct contacts with neuronal synapses at a frequency of about once per hour. These contacts are activity-dependent, being reduced in frequency by reductions in neuronal activity. After transient cerebral ischemia, the duration of these microglia-synapse contacts are markedly prolonged (approximately 1 h) and are frequently followed by the disappearance of the presynaptic bouton. Our results demonstrate that at least part of the dynamic motility of resting microglial processes in vivo is directed toward synapses and propose that microglia vigilantly monitor and respond to the functional status of synapses. Furthermore, the striking finding that some synapses in the ischemic areas disappear after prolonged microglial contact suggests microglia contribute to the subsequent increased turnover of synaptic connections. Further understanding of the mechanisms involved in the microglial detection of the functional state of synapses, and of their role in remodeling neuronal circuits disrupted by ischemia, may lead to novel therapies for treating brain injury that target microglia.

Topics: Action Potentials; Anesthetics, Local; Animals; Calcium; Calcium-Binding Proteins; Cell Movement; Cerebral Cortex; Electron Microscope Tomography; Green Fluorescent Proteins; Ischemia; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Microglia; Nerve Net; Neurons; Presynaptic Terminals; Synapses; Temperature; Tetrodotoxin; Time Factors; Visual Pathways

The aim of the present study was to explore whether endogenous activation of different purine receptors by ATP and adenosine contributes to or inhibits excess glutamate release evoked by ischemic-like conditions in rat hippocampal slices. Combined oxygen-glucose deprivation (OGD) elicited a substantial, [Ca(2+)](o)-independent release of [(3)H]glutamate, which was tetrodotoxin (1 microM)-sensitive and temperature-dependent. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 0.1-10 microM), and the selective P2X(7) receptor antagonist Brilliant Blue G (1-100 nM), decreased OGD-evoked [(3)H]glutamate efflux indicating that endogenous ATP facilitates ischemia-evoked glutamate release. The selective A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1-250 nM) and the selective A(2A) receptor antagonists 4-(2-[7-amino-2-)2-furyl(triazolo-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385, 0.1-20 nM) and 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261, 2-100 nM) decreased OGD-evoked [(3)H]glutamate efflux, indicating that endogenous adenosine also facilitates glutamate release under these conditions. The effect of DPCPX and ZM241385 was reversed, whereas the action of P2 receptor antagonists was potentiated by the selective ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL67156, 50 microM). The binding characteristic of the A(2A) ligand [(3)H]CGS21680 to hippocampal membranes did not change significantly in response to OGD. Taken together these data suggest that while A(1) receptors might became desensitized, A(2A) and P2X receptor-mediated facilitation of glutamate release by endogenous ATP and its breakdown product adenosine remains operational under long-term OGD. Therefore the inhibition of P2X/A(2A) receptors rather than the stimulation of A(1) adenosine receptors could be an effective approach to attenuate glutamatergic excitotoxicity and thereby counteract ischemia-induced neurodegeneration.

Topics: Adenosine; Adenosine Triphosphate; Analgesics; Animals; Dose-Response Relationship, Drug; Glucose; Glutamic Acid; Hippocampus; Hypoxia; In Vitro Techniques; Ischemia; Male; Phenethylamines; Purinergic Agonists; Purinergic Antagonists; Pyrimidines; Rats; Rats, Wistar; Receptors, Purinergic; Sodium Channel Blockers; Tetrodotoxin; Triazines; Triazoles; Xanthines

The contribution of intracellular stores to axonal Ca2+ overload during chemical ischemia in vitro was examined by confocal microscopy. Ca2+ accumulation was measured by fluo-4 dextran (low-affinity dye, KD approximately 4 microM) or by Oregon Green 488 BAPTA-1 dextran (highaffinity dye, KD approximately 450 nM). Axonal Na+ was measured using CoroNa Green. Ischemia in CSF containing 2 mM Ca2+ caused an approximately 3.5-fold increase in fluo-4 emission after 30 min, indicating a large axonal Ca2+ rise well into the micromolar range. Axonal Na+ accumulation was enhanced by veratridine and reduced, but not abolished, by TTX. Ischemia in Ca2+-free (plus BAPTA) perfusate resulted in a smaller but consistent Ca2+ increase monitored by Oregon Green 488 BAPTA-1, indicating release from intracellular sources. This release was eliminated in large part when Na+ influx was reduced by replacement with N-methyl-D-glucamine (NMDG+; even in depolarizing high K+ perfusate), Li+, or by the application of TTX and significantly increased by veratridine. Intracellular release also was reduced significantly by neomycin or 1-(6-[(17beta-methoxyestra-1,3,5 [10]-trien-17-yl) amino] hexyl)-1H-pyrrole-2,5-dione (U73122 [GenBank]) (phospholipase C inhibitors), heparin [inositol trisphosphate (IP3) receptor blocker], or 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; mitochondrial Na+/Ca2+ exchange inhibitor) as well as ryanodine. Combining CGP37157 with U73122 [GenBank] or heparin decreased the response more than either agent alone and significantly improved electrophysiological recovery. Our conclusion is that intra-axonal Ca2+ release during ischemia in rat optic nerve is mainly dependent on Na+ influx. This Na+ accumulation stimulates three distinct intra-axonal sources of Ca2+: (1) the mitochondrial Na+/Ca2+ exchanger driven in the Na+ import/Ca2+ export mode, (2) positive modulation of ryanodine receptors, and (3) promotion of IP3 generation by phospholipase C.

Topics: Anesthetics, Local; Animals; Axons; Calcium; Chelating Agents; Drug Interactions; Egtazic Acid; Enzyme Inhibitors; Fluorescent Dyes; Glucose; In Vitro Techniques; Ischemia; Male; Microscopy, Confocal; Nitrogen; Optic Nerve; Rats; Rats, Long-Evans; Sodium; Tetrodotoxin; Time Factors; Veratridine

Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Anesthetics, Local; Animals; Bicuculline; Caffeine; Calcium; Cell Count; Cells, Cultured; Cerebral Cortex; Deoxyadenosines; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fura-2; GABA Antagonists; gamma-Aminobutyric Acid; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Vitro Techniques; Ischemia; Membrane Potentials; Microscopy, Confocal; Microtubule-Associated Proteins; Neurons; Patch-Clamp Techniques; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrodotoxin; Time Factors; Triazines; Tritium; Xanthines

The mammalian CNS contains an abundance of chelatable zinc that is sequestered in the vesicles of glutamatergic presynaptic terminals and co-released with glutamate. Considerable Zn(2+) is also released during cerebral ischemia and reperfusion (I/R) although the mechanism of this release has not been elucidated. We report here the real time observation of increase of the concentration of extracellular Zn(2+) ([Zn(2+)](o)), accompanied by a rapid increase of intracellular free Zn(2+)concentration, in the areas of dentate gyrus (DG), CA1 and CA3 in acute rat hippocampus slices during ischemia simulated by deprivation of oxygen and glucose (OGD) followed by reperfusion with normal artificial cerebrospinal fluid. A brief period of OGD caused a sustained increase of [Zn(2+)](o). Subsequent reperfusion with oxygenated medium containing glucose resulted in a further increase of [Zn(2+)](o). Longer periods of OGD caused greater increases of [Zn(2+)](o,) and subsequent reperfusion caused still further increases of [Zn(2+)](o,) regardless of OGD duration. The Zn(2+) chelator CaEDTA (10 mM) significantly reduced the increase of [Zn(2+)] induced by OGD and reperfusion. Significant regional differences of [Zn(2+)](o) over the areas of the DG, CA1 and CA3 were not observed during I/R. Neither sodium channel blockade by tetrodotoxin (2 microM), perfusion with nominally calcium-free medium nor anatomical disassociation of the DG, CA1 and CA3 regions from one another by lesioning affected the increase of [Zn(2+)](o). The non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine methyl ester (1 mM), however, blocked the increase of [Zn(2+)](o) during ischemia and reperfusion. The data indicate the important role of NO in causing the release of Zn(2+) during I/R and suggest that NOS inhibitors may be used to reduce Zn(2+)-induced neuronal injury.

Topics: Anesthetics, Local; Animals; Edetic Acid; Enzyme Inhibitors; Extracellular Fluid; Hippocampus; Ischemia; Male; Nitric Oxide Synthase; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Reperfusion; Tetrodotoxin; Time Factors; Zinc

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

An ischemia-induced change in glutamatergic transmission was investigated in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by use of the whole cell patch-clamp technique; the ischemia was simulated by superfusing an oxygen- and glucose-free medium (ISM). Following ISM superfusion, 21 of 37 SG neurons tested produced an outward current (23 +/- 4 pA at a holding potential of -70 mV), which was followed by a slow and subsequent rapid inward current; the remaining neurons had only inward currents. During such a change in holding currents, spontaneous excitatory postsynaptic currents (EPSCs) were remarkably decreased in a frequency with time (half-decay time of the frequency: about 65 s). The frequency of spontaneous EPSCs was reduced to 28 +/- 13% (n = 37) of the control level during the generation of the slow inward current (about 4 min after the beginning of ISM superfusion) without a change in the amplitude of spontaneous EPSCs. When ISM was superfused together with either bicuculline (10 microM) or CGP35348 (20 microM; GABA(A) and GABA(B) receptor antagonists, respectively), spontaneous EPSC frequency reduced by ISM recovered to the control level and then the frequency markedly increased [by 325 +/- 120% (n = 22) and 326 +/- 91% (n = 17), respectively, 4 min after ISM superfusion]; this alteration in the frequency was not accompanied by a change in spontaneous EPSC amplitude. Superfusing TTX (1 microM)-containing ISM resulted in a similar recovery of spontaneous EPSC frequency and following increase (by 328 +/- 26%, n = 12) in the frequency; strychnine (1 microM) did not affect ISM-induced changes in spontaneous EPSC frequency (n = 5). It is concluded that the ischemic simulation inhibits excitatory transmission to SG neurons, whose action is in part mediated by the activation of presynaptic GABA(A) and GABA(B) receptors, probably due to GABA released from interneurons as a result of an ischemia-induced increase in neuronal activities. This action might protect SG neurons from an excessive excitation mediated by L-glutamate during ischemia.

Topics: Age Factors; Anesthetics, Local; Animals; Bicuculline; Excitatory Postsynaptic Potentials; GABA Antagonists; gamma-Aminobutyric Acid; Glutamic Acid; Glycine; Glycine Agents; Ischemia; Male; Neural Inhibition; Organ Culture Techniques; Patch-Clamp Techniques; Rats; Strychnine; Substantia Gelatinosa; Tetrodotoxin

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

In this study, the properties of ischemic condition-induced and veratridine-evoked [3H]noradrenaline ([3H]NA) release from rat spinal cord slices were compared. It was expected that ischemia mimicked by oxygen and glucose deprivation results in the impairment of Na+/K+ -ATPase with a consequent elevation of the intracellular Na+ -level which reverses the NA carrier and promotes excessive NA release, and veratridine, by the activation of Na+ channels, releases NA both carrier-mediated and Ca2+ -dependent, i.e. vesicular manner. In our experiments, veratridine (1-100 microM) dose-dependently increased the resting [3H]NA release, and its effect was only partially blocked by low temperature or the lack of external calcium, whereas the sodium channel inhibitor tetrodotoxin (TTX, 1 microM) completely prevented it, indicating that veratridine induces NA release via axonal depolarization and reversing the transporters by eliciting Na+ -influx. In contrast to TTX, the local anesthetic lidocaine (100 microM) only partially blocked the veratridine-induced [3H]NA release due to its inhibitory action on K+ channels. The ischemia-induced [3H]NA release was abolished at 12 degrees C, a temperature known to block only the transporter-mediated release of transmitters. However, lidocaine was also partially effective to reverse the action of ischemia on the NA release, indicating that lidocaine is not a useful compound in the treatment of spinal cord-injured patients against the excessive excytotoxic NA release.

Topics: Animals; Cell Hypoxia; Cold Temperature; Glucose; In Vitro Techniques; Ischemia; Male; Norepinephrine; Rats; Spinal Cord; Tetrodotoxin; Tritium; Veratridine

Transmitters and cotransmitters of the sympathetic nervous system are involved in the regulation of a variety of immune cell functions. However, it is not entirely clear what stimuli lead to the release of these molecules in immune organs. In this study, we investigated whether local ischemia can cause the parallel release of norepinephrine and its cotransmitter, ATP, in the spleen. Ischemic-like conditions, simulated by transient (15 min) O(2) and glucose deprivation, elicited a reversible increase in the release of both norepinephrine and purines from superfused spleen strips preloaded with [3H]norepinephrine or [3H]adenosine. HPLC analysis of the released tritium label revealed a net increase in the amount of ATP, ADP, AMP, adenosine, inosine, hypoxanthine and xanthine in response to ischemic-like condition. Selective O(2) or glucose deprivation, and Ca(2+)-free conditions differentially affected the outflow of [3H]norepinephrine and [3H]purines, indicating that they derived from different sources. The ABC transporter inhibitors glibenclamide (100 microM) and verapamil (100 microM) as well as low-temperature inhibited [3H]purine release evoked by ischemic-like conditions. Surgical denervation of the spleen reduced endogenous catecholamine content and [3H]norepinephrine uptake of the spleen, but not that of [3H]adenosine. In summary, these results demonstrate the release of norepinephrine and purines in response to an ischemic-like condition in an immune organ. Although both could provide an important source of extracellular catecholamines and purines involved at various levels of immunomodulation, the source and mechanism of norepinephrine and purine efflux seem different.

Topics: Adenine; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Calcium; Hypoxanthine; Inosine; Ischemia; Male; Norepinephrine; Rats; Rats, Wistar; Spleen; Sympathectomy; Sympathetic Nervous System; Sympathomimetics; Tetrodotoxin; Tritium; Vasodilator Agents; Xanthine

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

The aim of the study was to address discrepant findings in the literature regarding coupling between decreased functional demand during disuse and reduced capillarity. We previously reported [K. Tyml, O. Mathieu-Costello, and E. Noble. Microvasc. Res. 49: 17-32, 1995] that severe disuse of rat extensor digitorum longus (EDL) muscle caused by a 2-wk application of tetrodotoxin (TTX) on the sciatic nerve is not accompanied by capillary loss. Using the same animal model, the present study examined whether this absence of coupling could be explained in terms of 1) too short a duration of disuse and 2) muscle-specific response to disuse. Fischer 344 rats were exposed to either no treatment (control) or to 2- or 8-wk TTX applications. Fiber size, capillary density per fiber cross-sectional area, and capillary-to-fiber (C/F) ratio were determined by morphometry in the EDL muscle (control, 2- and 8-wk groups) and in the superficial portion of medial gastrocnemius (Gas) muscle (control, 2 wk). In both muscles, microvascular blood flow was evaluated by intravital microscopy [red blood cell velocity in capillaries (V(RBC))] and by laser Doppler flowmetry (LDF). Regardless of duration of TTX application or muscle type, TTX-induced disuse resulted in a significant reduction of fiber area (44-71%). However, capillary density increased in EDL muscle (both at 2 and 8 wk) but not in Gas muscle. C/F ratio decreased in EDL muscle at 8 wk (18%) and in Gas muscle (39%). This indicates that the effect on capillarity depended on duration of disuse and on muscle type. V(RBC) and LDF signal were significantly larger in EDL than in Gas muscle. Analysis of change in capillarity vs. V(RBC) suggested that the outcome of disuse may be modulated by blood flow. We conclude that the duration of skeletal muscle disuse per se does not dictate capillary loss, and we hypothesize that discrepant findings of coupling between functional demand and capillarity could be due to the presence/absence of flow-related angiogenesis superimposed on the capillary removal process during disuse.

Topics: Animals; Blood Volume; Body Weight; Capillaries; Hemodynamics; Hindlimb; Ischemia; Male; Microcirculation; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Inbred F344; Sciatic Nerve; Tetrodotoxin; Time Factors

A steep inwardly directed Na+ gradient is essential for glial functions such as glutamate reuptake and regulation of intracellular ion concentrations. We investigated the effects of glucose deprivation, chemical hypoxia, and simulated ischemia on intracellular Na+ concentration ([Na+]i) in cultured spinal cord astrocytes using fluorescence ratio imaging with sodium-binding benzofuran isophthalate (SBFI) AM. Glucose removal or chemical hypoxia (induced by 10 mM NaN3) for 60 min increased [Na+]i from a baseline of 8.3 to 11 mM. Combined glycolytic and respiratory blockage by NaN3 and 0 glucose saline caused [Na+]i to increase by 20 mM, similar to the [Na+]i increases elicited by blocking the Na+/K+-ATPase with ouabain. Recovery from large [Na+]i increases (>15 mM) induced by the glutamatergic agonist kainate was attenuated during glucose deprivation or NaN3 application and was blocked in NaN3 and 0 glucose. To mimic in vivo ischemia, we exposed astrocytes to NaN3 and 0 glucose saline containing L-lactate and glutamate with increased [K+] and decreased [Na+], [Ca2+], and pH. This induced an [Na+]i decrease followed by an [Na+]i rise and a further [Na+]i increase after reperfusion with standard saline. Similar multiphasic [Na+]i changes were observed after NaN3 and 0 glucose saline with only reduced [Na+]e. Our results suggest that the ability to maintain a low [Na+]i enables spinal cord astrocytes to continue uptake of K+ and/or glutamate at the onset of energy failure. With prolonged energy failure, however, astrocytic [Na+]i rises; with loss of their steep transmembrane Na+ gradient, astrocytes may aggravate metabolic insults by carrier reversal and release of acid, K+, and/or glutamate into the extracellular space.

Topics: Animals; Animals, Newborn; Antimetabolites; Astrocytes; Benzofurans; Cell Hypoxia; Deoxyglucose; Energy Metabolism; Enzyme Inhibitors; Ethers, Cyclic; Excitatory Amino Acid Agonists; Fluorescent Dyes; Fluorides, Topical; Glucose; Glycolysis; Homeostasis; Ischemia; Kainic Acid; Neurotoxins; Ouabain; Rats; Rats, Sprague-Dawley; Sodium; Sodium Azide; Sodium Fluoride; Sodium-Potassium-Exchanging ATPase; Spinal Cord; Tetrodotoxin

The principal finding of the present study with rat spinal cord slices was the novel demonstration of the [Ca2+]o-independent effect of ischemia on norepinephrine release and its antagonism by tetrodotoxin and low temperature (10 degrees C). Our finding that tetrodotoxin antagonized the effects of glucose deprivation on norepinephrine release in a [Ca2+]o-independent way suggests that Na+ channel block alone, i.e., the prevention of Na+ accumulation, may account for the protective action. Low temperature completely prevented the effect of ischemia on norepinephrine release but did not change the release associated with axonal activity. This finding is in good agreement with the observation that small changes in brain temperature critically determine the extent of neuronal injury from ischemia and suggests that both [Ca2+]o-independent release and cell injury are associated with the norepinephrine membrane carrier. It is suggested, therefore, that drugs able to attenuate the increase in [Na+]i during ischemia may be useful agents to protect against ischemic damage if given before the insult.

Topics: 4-Aminopyridine; Anesthetics, Local; Animals; Calcium; Calcium Channel Blockers; Cold Temperature; Hypoglycemia; Hypoxia; Ischemia; Lidocaine; Male; Norepinephrine; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Spinal Cord; Tetrodotoxin

It has previously been demonstrated that muscle atrophy associated with aging and disuse is accompanied by changes in microvascular function including absolute loss of capillaries, increased mean red blood cell velocity (VRBC), and absence of reactive hyperemia. The purpose of the present study was to determine whether disuse could account for these changes. The right extensor digitorum longus muscle in male Fisher 344 rats was subjected to 15 days of disuse through the neural application of tetrodotoxin (TTX). Microvascular function, as assessed using intravital microscopy, was compared for muscles from control (n = 8) and TTX-treated (n = 5) animals. The TTX-induced disuse was associated with a 40.5% decrease in muscle weight, a 51.6% decrease in fiber cross-sectional area, a 62% decrease in mitochondrial volume density, and increased capillary damage (TTX, 11% control, 1.1%). Although capillary density in the disused muscle increased (by 139%), when corrected for muscle atrophy, the absolute number of capillaries was maintained. With TTX disuse, VRBC heterogeneity was not different from that in the control rats while the mean velocity increased 3.18x. TTX disuse did not alter the pattern of reactive hyperemia following 30 min of complete ischemia. These results suggest that short-term TTX-induced atrophy affects both microvascular structure and resting state blood flow in rat skeletal muscle, but it does not affect the vascular responsiveness following a metabolic challenge.

Topics: Animals; Blood Flow Velocity; Endothelium, Vascular; Ischemia; Male; Microcirculation; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Inbred F344; Tetrodotoxin

Tetrodotoxin has been reported to cause prolonged systemic hypotension without resultant ischemic damage. We tested its ability to protect the kidney during 60 minutes of warm ischemia in uninephrectomized rats. Protection was observed when tetrodotoxin was given intravenously at two microgram./kg. and four microgram./kg. as assessed by serial plasma blood urea nitrogen and creatinine measurements over two weeks. Tetrodotoxin was protective when given immediately before or immediately after the ischemic period. The renal protection of tetrodotoxin was not due to its effects on renal nerves as renal denervation did not protect the kidney from the ischemic damage. The renal protective effects of four microgram. tetrodotoxin/kg. were similar to those of four mg. captopril/kg. but the combination of the two was paradoxically without effect. We tested whether tetrodotoxin and captopril chemically antagonized each other, but in the presence of tetrodotoxin, captopril was still a potent inhibitor of the conversion of angiotensin I to angiotensin II. These results indicate that tetrodotoxin could be useful in elucidating the sequence of events associated with ischemic-reperfusion renal injury and in identifying ways of preserving renal function during renal surgery.

Topics: Acute Kidney Injury; Animals; Blood Pressure; Blood Urea Nitrogen; Captopril; Creatinine; Ischemia; Kidney; Male; Peptidyl-Dipeptidase A; Rats; Rats, Inbred Strains; Tetrodotoxin

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Chickens; Dizocilpine Maleate; Edema; Electron Transport; Glycolysis; Hypoglycemia; Hypoxia; Iodoacetates; Iodoacetic Acid; Ischemia; N-Methylaspartate; Potassium Cyanide; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Retina; Tetrodotoxin

Myotonic discharges in rats given 20, 25-diazacholesterol hydrochloride and fibrillation discharges in denervated rat muscle both were silenced by procaine hydrochloride, tetrodotoxin or ischemia, or potassium chloride (after initial activation). They both were activated by succinylcholine, but only the fibrillations were silenced by alpha-bungarotoxin or atropine sulfate. It is hypothesized that fibrillations and diazacholesterol-induced myotonia are mediated through mechanisms involving ionic channels, that both can be produced by activation of the junctional/nonjunctional acetylcholine receptors (or some mechanism coupled to the receptors), but that an unfettered alpha-bungarotoxin-binding portion of the acetylcholine-receptor molecule and an unblocked atropine-binding site are obligatory only for production of fibrillations.

Topics: Acetylcholine; Animals; Atropine; Azacosterol; Bungarotoxins; Evoked Potentials; Ischemia; Male; Muscle Denervation; Muscle Spasticity; Muscles; Myotonia; Potassium Chloride; Procaine; Pyridostigmine Bromide; Rats; Receptors, Cholinergic; Succinylcholine; Tetrodotoxin; Tubocurarine

Topics: Animals; Blood Pressure; Cardiac Output; Cardiology; Coronary Circulation; Coronary Vessels; Heart; Heart Arrest, Induced; Heart Rate; Injections; Ischemia; Pulse; Rats; Tetrodotoxin; Time Factors

Topics: Animals; Atropine; Dogs; Electric Conductivity; Electric Stimulation; Electrolytes; Ganglia; Gastrointestinal Motility; Guanidines; Hexamethonium Compounds; In Vitro Techniques; Ischemia; Jejunum; Methacholine Compounds; Morphine; Muscle, Smooth; Nicotine; Piperazines; Spectrophotometry; Sulfates; Sympathomimetics; Tartrates; Tetrodotoxin