Effects of ion channel blockade on the distribution of Na, K, Ca and other elements in oxygen-glucose deprived CA1 hippocampal neurons.

The pathophysiology of brain ischemia and reperfusion injury involves perturbation of intraneuronal ion homeostasis. To identify relevant routes of ion flux, rat hippocampal slices were perfused with selective voltage- or ligand-gated ion channel blockers during experimental oxygen-glucose deprivation and subsequent reperfusion. Electron probe X-ray microanalysis was used to quantitate water content and concentrations of Na, K, Ca and other elements in morphological compartments (cytoplasm, mitochondria and nuclei) of individual CA1 pyramidal cell bodies. Blockade of voltage-gated channel-mediated Na+ entry with tetrodotoxin (1 microM) or lidocaine (200 microM) significantly reduced excess intraneuronal Na and Ca accumulation in all compartments and decreased respective K loss. Voltage-gated Ca2+ channel blockade with the L-type antagonist nitrendipine (10 microM) decreased Ca entry and modestly preserved CA1 cell elemental composition and water content. However, a lower concentration of nitrendipine (1 microM) and the N-, P-subtype Ca2+ channel blocker omega-conotoxin MVIIC (3 microM) were ineffective. Glutamate receptor blockade with the N-methyl-D-aspartate (NMDA) receptor-subtype antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP; 100 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptor subtype blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM/100 microM glycine) completely prevented Na and Ca accumulation and partially preserved intraneuronal K concentrations. Finally, the increase in neuronal water content normally associated with oxygen-glucose deprivation/reperfusion was prevented by Na+ channel or glutamate receptor blockade. Results of the present study demonstrate that antagonism of either postsynaptic NMDA or AMPA glutaminergic receptor subtypes provided nearly complete protection against ion and water deregulation in nerve cells subjected to experimental ischemia followed by reperfusion. This suggests activation of ionophoric glutaminergic receptors is involved in loss of neuronal osmoregulation and ion homeostasis. Na+ channel blockade also effectively diminished neuronal ion and water derangement during oxygen-glucose deprivation and reperfusion. Prevention of elevated Nai+ levels is likely to provide neuroprotection by decreasing presynaptic glutamate release and by improving cellular osmoregulation, adenosine triphosphate utilization and Ca2+ clearance. Thus, we suggest that voltage-gated tetrodotoxin-sensitive Na+ channels and glutamate-gated ionotropic NMDA or AMPA receptors are important routes of ion flux during nerve cell injury induced by oxygen-glucose deprivation/reperfusion.

Experimental spinal cord injury: spatiotemporal characterization of elemental concentrations and water contents in axons and neuroglia.

To examine the role of axonal ion deregulation in acute spinal cord injury (SCI), white matter strips from guinea pig spinal cord were incubated in vitro and were subjected to graded focal compression injury. At several postinjury times, spinal segments were removed from incubation and rapidly frozen. X-ray microanalysis was used to measure percent water and dry weight elemental concentrations (mmol/kg) of Na, P, Cl, K, Ca, and Mg in selected morphological compartments of myelinated axons and neuroglia from spinal cord cryosections. As an index of axon function, compound action potentials (CAP) were measured before compression and at several times thereafter. Axons and mitochondria in epicenter of severely compressed spinal segments exhibited early (5 min) increases in mean Na and decreases in K and Mg concentrations. These elemental changes were correlated to a significant reduction in CAP amplitude. At later postcompression times (15 and 60 min), elemental changes progressed and were accompanied by alterations in compartmental water content and increases in mean Ca. Swollen axons were evident at all postinjury times and were characterized by marked element and water deregulation. Neuroglia and myelin in severely injured epicenter also exhibited significant disruptions. In shoulder areas (adjacent to epicenter) of severely injured spinal strips, axons and mitochondria exhibited modest increases in mean Na in conjunction with decreases in K, Mg, and water content. Following moderate compression injury to spinal strips, epicenter axons exhibited early (10 min postinjury) element and water deregulation that eventually recovered to near control values (60 min postinjury). Na(+) channel blockade by tetrodotoxin (TTX, 1 microM) perfusion initiated 5 min after severe crush diminished both K loss and the accumulation of Na, Cl, and Ca in epicenter axons and neuroglia, whereas in shoulder regions TTX perfusion completely prevented subcellular elemental deregulation. TTX perfusion also reduced Na entry in swollen axons but did not affect K loss or Ca gain. Thus graded compression injury of spinal cord produced subcellular elemental deregulation in axons and neuroglia that correlated with the onset of impaired electrophysiological function and neuropathological alterations. This suggests that the mechanism of acute SCI-induced structural and functional deficits are mediated by disruption of subcellular ion distribution. The ability of TTX to reduce elemental deregulation in compression-injured axons and neuroglia implicates a significant pathophysiological role for Na(+) influx in SCI and suggests Na(+) channel blockade as a pharmacotherapeutic strategy.

Oxygen/glucose deprivation in hippocampal slices: altered intraneuronal elemental composition predicts structural and functional damage.

Effects of oxygen/glucose deprivation (OGD) on subcellular elemental composition and water content were determined in nerve cell bodies from CA1 areas of rat hippocampal slices. Electron probe x-ray microanalysis was used to measure percentage water and concentrations of Na, P, K, Cl, Mg, and Ca in cytoplasm, nucleus, and mitochondria of cells exposed to normal and oxygen/glucose deficient medium. As an early (2 min) consequence of OGD, evoked synaptic potentials were lost, and K, Cl, P, and Mg concentrations decreased significantly in all morphological compartments. As exposure to in vitro OGD continued, a negative DC shift in interstitial voltage occurred ( approximately 5 min), whereas general elemental disruption worsened in cytoplasm and nucleus (5-42 min). Similar elemental changes were noted in mitochondria, except that Ca levels increased during the first 5 min of OGD and then decreased over the remaining experimental period (12-42 min). Compartmental water content decreased early (2 min), returned to control after 12 min of OGD, and then exceeded control levels at 42 min. After OGD (12 min), perfusion of hippocampal slices with control oxygenated solutions (reoxygenation) for 30 min did not restore synaptic function or improve disrupted elemental composition. Notably, reoxygenated CA1 cell compartments exhibited significantly elevated Ca levels relative to those associated with 42 min of OGD. When slices were incubated at 31 degreesC (hypothermia) during OGD/reoxygenation, neuronal dysfunction and elemental deregulation were minimal. Results show that in vitro OGD causes loss of transmembrane Na, K, and Ca gradients in CA1 neurons of hippocampal slices and that hypothermia can obtund this damaging process and preserve neuronal function.

Electron probe x-ray microanalysis: a quantitative electron microscopy technique for measurement of elements and water in nervous tissue cells.

Although structure and function of the major cells comprising nervous tissue have been studied extensively, very little detailed information exists concerning subcellular distributions of water and such elements as Na, K, Cl, and Ca. This information gap limits our understanding of cell physiology since transmembrane gradients of corresponding ionic species are critically involved in modulating the metabolic and signaling behavior of neuronal and glial cells (1). Moreover, substantial evidence indicates that neuropathic conditions induced by a variety of injury events (e.g., xenobiotic intoxication, disease processes, trauma) involve shifts in subcellular ion composition and volume regulation (2-4). Several techniques (e.g., atomic absorption spectrophotometry, ion selective microelectrodes, ion-sensitive fluorescent dyes) have been used to measure tissue or cellular levels of elements (ions) in normal and injured nervous tissue. In our laboratory, we have used electron probe X-ray microanalysis (EPMA) to investigate the role of ion and water deregulation in different central and peripheral neuropathies (5-7). EPMA is a quantitative electron microscope technique that measures both water content (percentage water) and total (free plus bound) concentrations of biologically relevant elements (e.g., Na, K, S, P, Cl, Ca, and Mg in mmol/kg dry or wet weight) in cellular morphological compartments. Unlike other methods of ion/element measurements, EPMA permits simultaneous determinations of multiple elements and allows optical differentiation of nervous tissue cell types and their processes (e.g., nerve and glial cell bodies, dendrites, axons) with subsequent analyses of submembrane regions or organelles (e.g., axoplasm, mitochondria).

Rubidium uptake and accumulation in peripheral myelinated internodal axons and Schwann cells.

To study mechanisms of K+ transport in peripheral nerve, uptake of rubidium (Rb+), a K+ tracer, was characterized in rat tibial nerve myelinated axons and glia. Isolated nerve segments were perfused with zero-K+ Ringer's solutions containing Rb+ (1-20 mM) and x-ray microanalysis was used to measure water content and concentrations of Rb, Na, K, and Cl in internodal axoplasm, mitochondria, and Schwann cell cytoplasm and myelin. Both axons and Schwann cells were capable of removing extracellular Rb+ (Rb+(o)) and exchanging it for internal K+. Uptake into axoplasm, Schwann cytoplasm, and myelin was a saturable process over the 1-10 mM Rb+(o) concentration range, although corresponding axoplasmic uptake rates were higher than respective glial velocities. Mitochondrial accumulation was a linear function of axoplasmic Rb+ concentrations, which suggests involvement of a nonenzymatic process. At 20 mM Rb+(o), a differential stimulatory response was observed; i.e., axoplasmic Rb+ uptake velocities increased more than fivefold relative to the 10 mM rate, and glial cytoplasmic uptake rose almost threefold. Finally, Rb+(o) uptake rate into axons and glia was completely inhibited by ouabain (2-4 mM) exposure or incubation at 4 degrees C. These results suggest that Rb+ uptake into peripheral nerve internodal axons and Schwann cells is mediated by Na+,K+-ATPase activity and implicate the presence of axonal- and glial-specific Na+ pump isozymes.

Acrylamide intoxication modifies in vitro responses of peripheral nerve axons to anoxia.

Decreased axolemmal Na+/K(+)-ATPase activity has been considered as a possible mechanism for peripheral nerve axon damage induced by acrylamide (ACR) or 2,5-hexanedione (HD). Reduced activity of this enzyme is also presumed to be the basis of peripheral nerve resistance to ischemia or hypoxia associated with other neuropathies (e.g., diabetes). In the present study, we tested the hypothesis that peripheral nerve of ACR (50 mg/kg/d x 10 d) or HD (400 mg/kg/d x 20 d) exposed rats are resistant to oxygen-limiting conditions as a result of reduced axonal Na+/K(+)-ATPase activity. As an index of resistance, effects of in vitro anoxia on subaxonal concentrations of Na, K and Ca were assessed in isolated segments of tibial nerve from control and neurotoxicant-treated animals. Results show axons from HD rats were not resistant to anoxic challenge; i.e., axons exhibited disrupted elemental composition comparable to anoxic control changes. In contrast, ACR-exposed axons displayed anoxic resistance. Ouabain-exposed tibial axons subjected to anoxic conditions were also resistant, but the corresponding elemental pattern did not resemble that associated with ACR axons. Moreover, ACR axons were capable of maintaining elemental gradients during normoxic exposure which should not be possible if Na+ pump activity is depressed. Considered together, these data are not consistent with a role for diminished Na+/K(+)-ATPase activity in neurotoxicant-induced peripheral axonopathy. We also assessed the ability of ACR- and HD-exposed tibial nerve axons to recover from anoxia. Unlike control fibers which can fully restore normal elemental composition, neurotoxicant-exposed axons were incapable of such restoration. These data suggest the axonal machinery responsible for post-anoxia recovery (e.g., energy metabolism, ion translocation, Ca2+ and free radical buffering) is compromised by ACR or HD intoxication.