Anoxic and ischemic injury of myelinated axons in CNS white matter: from mechanistic concepts to therapeutics

White matter of the brain and spinal cord is susceptible to anoxia and ischemia. Irreversible injury to this tissue can have serious consequences for the overall function of the CNS through disruption of signal transmission. Myelinated axons of the CNS are critically dependent on a continuous supply of energy largely generated through oxidative phosphorylation. Anoxia and ischemia cause rapid energy depletion, failure of the Na(+)-K(+)-ATPase, and accumulation of axoplasmic Na+ through noninactivating Na+ channels, with concentrations approaching 100 mmol/L after 60 minutes of anoxia. Coupled with severe K+ depletion that results in large membrane depolarization, high [Na+]i stimulates reverse Na(+)-Ca2+ exchange and axonal Ca2+ overload. A component of Ca2+ entry occurs directly through Na+ channels. The excessive accumulation of Ca2+ in turn activates various Ca(2+)-dependent enzymes, such as calpain, phospholipases, and protein kinase C, resulting in irreversible injury. The latter enzyme may be involved in "autoprotection," triggered by release of endogenous gamma-aminobutyric acid and adenosine, by modulation of certain elements responsible for deregulation of ion homeostasis. Glycolytic block, in contrast to anoxia alone, appears to preferentially mobilize internal Ca2+ stores; as control of internal Ca2+ pools is lost, excessive release from this compartment may itself contribute to axonal damage. Reoxygenation paradoxically accelerates injury in many axons, possibly as a result of severe mitochondrial Ca2+ overload leading to a secondary failure of respiration. Although glia are relatively resistant to anoxia, oligodendrocytes and the myelin sheath may be damaged by glutamate released by reverse Na(+)-glutamate transport. Use-dependent Na+ channel blockers, particularly charged compounds such as QX-314, are highly neuroprotective in vitro, but only agents that exist partially in a neutral form, such as mexiletine and tocainide, are effective after systemic administration, because charged species cannot penetrate the blood-brain barrier easily. These concepts may also apply to other white matter disorders, such as spinal cord injury or diffuse axonal injury in brain trauma. Moreover, whereas many events are unique to white matter injury, a number of steps are common to both gray and white matter anoxia and ischemia. Optimal protection of the CNS as a whole will therefore require combination therapy aimed at unique steps in gray and white matter regions, or intervention at common points in the injury cascades.     

Transient prolongation of ventricular action potential duration after metabolic inhibition

Transient prolongation of the action potential duration was observed in canine ventricular muscle during the reoxygenation period following metabolic inhibition. We investigated the effects of verapamil, lanthanum (La3+), and hexamethyleneamiloride (HMA) on the recovery time course of the action potential and its rebound prolongation. The time course of the intracellular resistivity was estimated from the conduction velocity and electrograms. The action potentials of canine left ventricular trabeculae were recorded by the conventional microelectrode technique. After a control tracing was obtained, the preparation was perfused with a hypoxic, acidic solution for 20 min and then reoxygenated with regular Tyrode's solution. After reoxygenation, action potential prolongation exceeding the control value by 21.0 +/- 7.3% was observed depending on the degree of metabolic inhibition. Verapamil depressed the rebound prolongation when it was added before the start of metabolic inhibition, but not when added after reoxygenation was started. La3+ and HMA depressed the rebound phenomenon. Intracellular resistivity was increased during metabolic inhibition, but showed no significant changes during the period of action potential prolongation. It was concluded that the rebound action potential prolongation was related to the accumulation of intracellular Ca2+ during metabolic inhibition. Other ions, such as Na+ and H+ may also contribute to the phenomenon by modulating outward currents.     

Ion channel redistribution and function during development of the myelinated axon

The development of myelinated axons represents one of the most complex interactions among different cell types in the nervous system. Striking changes occur in both morphology and function in the early postnatal period. Myelination effectively isolates electrically most of the axolemma and dramatically alters the pathways for current flow that are required for rapid, reliable, and efficient conduction. Correspondingly, ion channels must be directed to and stabilized at their required sites. In the case of Na+ channels, this requires a 25-fold increase in density within nodes of Ranvier, and, in mammalian fibers, a virtually complete spatial separation from voltage-dependent K+ channels. Nodes must also be properly spaced to ensure a high conduction velocity and energy efficiency without compromising the safety factor for reliable propagation. In this review, we consider the events responsible for axon development, emphasizing the involvement of ion channels. We discuss the current state of research in this area, including some controversies regarding mechanisms of neuron-glial communication.     

Potassium current kinetics in Myxicola axons. Effects of conditioning prepulses

In Myxicola giant axons the time constants for activation of the potassium conductance (GK) after prepulses less depolarized than a test pulse are comparable to the time constants for turn off of GK after prepulses more depolarized than the same test pulse. The absolute magnitude of the steady-state level of GK is also independent of prepulse amplitude in Myxicola. The results are contrasted with recent observations on voltage-clamped frog nodes.     

Effect of membrane surface potential on the uptake and the inhibition of cationic compounds in rat intestinal brush-border membrane vesicles and liposomes

The effect of membrane surface potential on the uptake of tryptamine, an organic cation, by rat intestinal brush-border membrane vesicles was investigated. In the presence of an inside-negative K(+)-diffusion potential, the manner of initial uptake of tryptamine appeared to be pH-dependent and the uptake in the acidic medium was lower than that in the neutral medium. Changes in surface potential of brush-border membrane vesicles were monitored using 8-anilino-1-naphthalenesulfonic acid (ANS) and the results suggested that the membrane surface potential (negative charge on the membrane surface) decreased in the acidic medium. A good correlation was observed between the K(+)-diffusion potential-dependent uptake of tryptamine and membrane surface potential monitored by ANS at various pH levels. The uptake of tryptamine by liposomes (large unilamellar vesicles), which contained various amounts of dipalmitoylphosphatidylserine (DPPS), was also examined. The uptake of tryptamine decreased with a decrease of DPPS content in the liposomes, and was correlated with the membrane surface potential monitored by ANS. Moreover, the effect of organic cations on the uptake of tryptamine by intestinal brush-border membrane vesicles was examined. The uptake of tryptamine was inhibited by tetracaine and imipramine. The inhibitory effect of these cations was well correlated with changes in the membrane surface potential in the presence of tetracaine or imipramine. These results suggest that the K(+)-diffusion potential-dependent uptake of tryptamine by intestinal brush-border membrane vesicles is affected by membrane surface potential, and the inhibition of tryptamine uptake originates in changes in the membrane surface potential caused by the organic cations.     

Multiple layers of basement membrane around myelinated and unmyelinated fibers in cellular blue nevus

Concentric multiple layers of basement membranes around Schwann cells in cellular blue nevus are reported.     

Thoracic epidural analgesia in aortocoronary bypass surgery. II: Effects on the endocrine metabolic response

Thoracic epidural analgesia (TEA) may offer haemodynamic benefits for patients with coronary heart disease going through major surgery. This may-in part-be secondary to an effect on the endocrine and metabolic response to surgery. We therefore investigated the effect of TEA on the endocrine metabolic response to aortocoronary bypass surgery (ACBS). Thirty male patients (age < 65 years, ejection fraction > 0.5) were randomized into 3 groups; the HF group receiving a high dose fentanyl (55 micrograms.kg-1) anaesthesia, the HF+TEA group with the same fentanyl dose+TEA with 10 ml bupivacain 5 mg.ml-1, followed by 4 ml every hour, and the LF+TEA group receiving fentanyl 15 micrograms.kg-1 + TEA. Adrenalin, noradrenalin, systemic vascular resistance (SVR), glucose, cortisol, lactate and free fatty acids were followed during the operation and for 20 h postoperatively. A significant increase in adrenalin, noradrenalin and SVR was found in the HF group whereas this increase was blocked in both epidural groups. An increase in glucose and cortisol was noticed in all groups, but the increase was delayed in the epidural groups. Our results suggest that a more effective blockade of the stress response during ACBS is obtained when TEA is added to general anaesthesia than with high dose fentanyl anaesthesia alone.     

UV-B-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Impairment of donor and acceptor side components

Inhibition of photosystem II electron transport by UV-B radiation has been studied in isolated spinach photosystem II membrane particles using low-temperature EPR spectroscopy and chlorophyll fluorescence measurements. UV-B irradiation results in the rapid inhibition of oxygen evolution and the decline of variable chlorophyll fluorescence. These effects are accompanied by the loss of the multiline EPR signal arising from the S2 state of the water-oxidizing complex and the induction of Signal IIfast originating from stabilized Try-Z+. The EPR signals from the QA-Fe2+ acceptor complex, Tyr-D+, and the oxidized non-heme iron (Fe3+) are also decreased during the course of UV-B irradiation, but at a significantly slower rate than oxygen evolution and the multiline signal. The decrease of the Fe3+ signal at high g values (g = 8.06, g = 5.6) is accompanied by the induction of another EPR signal at g = 4.26 that arises most likely from the same Fe3+ ion in a modified ligand environment. UV-B irradiation also affects cytochrome b-559. The g = 2.94 EPR signal that arises from the dark- oxidized form is enhanced, whereas the light inducible g = 3.04 signal that arises from the photo-oxidizable population of cytochrome b-559 is diminished. UV-B irradiation also induces the degradation of the D1 reaction center protein. The rate of the D1 protein loss is slower than the inhibition of oxygen evolution and of the multiline signal but follows closely the loss of Signal IIslow, the QA-Fe2+ and the Fe3+ EPR signals, as well as the release of protein-bound manganese. It is concluded from the results that UV-B radiation affects photosystem II redox components at both the donor and acceptor side. The primary damage occurs at the water-oxidizing complex. Modification and/or inactivation of tyrosine-D, cytochrome b-559, and the QAFe2+ acceptor complex are subsequent events that coincide more closely with the UV-B-induced damage to the protein structure of the photosystem II reaction center.     

Baseline consideration of liposomal contrast agent. CNS transport by macrophages in experimental allergic encephalomyelitis

The purpose of this study was to investigate a specialized liposomal contrast agent for magnetic resonance imaging (MRI), as part of a program to examine infiltrating immune cells in lesions of experimental allergic encephalomyelitis (EAE). A potent investigational liposomal contrast agent, phosphatidylethanolamine-DTPA-gadolinium, was chosen which has been shown to remain tightly liposomal-associated, with long persistence in vivo. Europium (Eu3+), a fluorescent paramagnetic metal, was also utilized in these experiments in place of gadolinium. This material is avidly taken up by monocytes in vivo. Thirty-four animals received some form of liposomal material either before or during the opening of the blood-brain barrier (BBB). Twenty-seven Hartley guinea pigs were inoculated for EAE with homogenized brain and Complete Freunds Adjuvant (CFA) and seven control animals received CFA alone. Eighty-two percent of the experimental animals exhibited degeneration of the BBB with inflammation and edema in the brain, while all control animals had normal brain scans. T1-weighted MRI, performed to detect the presence of liposomal contrast material in experimental animals, was not different from untreated animals. Fluorescent microscopy revealed no characteristic changes associated with Eu3+ presence in the brains of treated or control animals. Therefore, it would seem that insufficient material crosses the disrupted BBB, either in free form or subsequent to macrophage ingestion, to be detected by MRI or fluorescent microscopic examination.     

Degeneration and regeneration in rabbit peripheral nerve with long-term nerve cuff electrode implant: a stereological study of myelinated and unmyelinated axons

Spatio-temporal patterns of neuronal activity after the induction of long-term potentiation (LTP) in mouse hippocampal slices were studied with the use of a real-time high-resolution optical recording system. The slices were stained with voltage-sensitive dye and then high-frequency pulses (tetanus) were delivered to Schaffer collaterals of CA3 at the stratum radiatum of CA1. Optical signals as well as field potential in response to test pulses were potentiated after tetanus. The area of response measured by optical recording was slightly but significantly enlarged after tetanus, suggesting that the propagation of optical responses from CA1 towards the subiculum was enlarged. It was suggested that a great increase in neuronal activity was elicited at CA1 and the subiculum after LTP. These changes of spatio-temporal patterns of neuronal activity may contribute to learning and memory. The effect of trichloroethylene (TCE) on LTP was studied with the use of both electrical and optical and recording. The hippocampus from mice injected with 300 mg/kg or 1000 mg/kg TCE was sliced 24 hours after TCE-administration. Test pulses were delivered to Schaffer collaterals every 30 sec and the field potential from the stratum pyramidale of CA1 was recorded. At 40 min after the application of tetanus, population spikes (PS) were potentiated in all groups, but the post/pre ratio of PS was smaller in TCE groups than in the control. Optical recording was also carried out in 1000 mg/kg TCE-injected mice. At 40 min after tetanus, the optical signal response to the test pulse was potentiated in both TCE and control groups, but the post/pre ratio of the optical signals was smaller in TCE than in the control. No significant difference was detected in the increase in the neuronal response area between TCE and the control. It was suggested that TCE depressed the LTP, whereas the increase in the area after tetanus meant that a good amount of neuronal activity was still potentiated.     

Effects of television on metabolic rate: potential implications for childhood obesity

The effects of television viewing on resting energy expenditure (metabolic rate) in obese and normal-weight children were studied in a laboratory setting. Subjects were 15 obese children and 16 normal-weight children whose ages ranged from 8 to 12 years. All subjects had two measured of resting energy expenditure obtained while at rest and one measurement of energy expenditure taken while viewing television. Results indicated that metabolic rate during television viewing was significantly lower (mean decrease of 211 kcal extrapolated to a day) than during rest. Obese children tended to have a larger decrease, although this difference was not statistically significant (262 kcal/d vs 167 kcal/d, respectively). It was concluded that television viewing has a fairly profound lowering effect of metabolic rate and may be a mechanism for the relationship between obesity and amount of television viewing.     

Purinergic inhibition of glucose transport in cardiomyocytes

ATP is known to act as an extracellular signal in many organs. In the heart, extracellular ATP modulates ionic processes and contractile function. This study describes a novel, metabolic effect of exogenous ATP in isolated rat cardiomyocytes. In these quiescent (i.e. noncontracting) cells, micromolar concentrations of ATP depressed the rate of basal, catecholamine-stimulated, or insulin-stimulated glucose transport by up to 60% (IC50 for inhibition of insulin-dependent glucose transport, 4 microM). ATP decreased the amount of glucose transporters (GLUT1 and GLUT4) in the plasma membrane, with a concomitant increase in intracellular microsomal membranes. A similar glucose transport inhibition was produced by P2 purinergic agonists with the following rank of potencies: ATP approximately ATPgammaS approximately 2-methylthio-ATP (P2Y-selective) > ADP > alpha,betameATP (P2X-selective), whereas the P1 purinoceptor agonist adenosine was ineffective. The effect of ATP was suppressed by the poorly subtype-selective P2 antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid but, surprisingly, not by the nonselective antagonist suramin nor by the P2Y-specific Reactive Blue 2. Glucose transport inhibition by ATP was not affected by a drastic reduction of the extracellular concentrations of calcium (down to 10(-9) M) or sodium (down to 0 mM), and it was not mimicked by a potassium-induced depolarization, indicating that purinoceptors of the P2X family (which are nonselective cation channels whose activation leads to a depolarizing sodium and calcium influx) are not involved. Inhibition was specific for the transmembrane transport of glucose because ATP did not inhibit (i) the rate of glycolysis under conditions where the transport step is no longer rate-limiting nor (ii) the rate of [1-14C]pyruvate decarboxylation. In conclusion, extracellular ATP markedly inhibits glucose transport in rat cardiomyocytes by promoting a redistribution of glucose transporters from the cell surface to an intracellular compartment. This effect of ATP is mediated by P2 purinoceptors, possibly by a yet unknown subtype of the P2Y purinoceptor family.     

Adaptation of intestinal nutrient transport in health and disease. Part II

The first part of this review dealt with the physiology of glucose transport with specific emphasis on transporters of the brush border membrane (BBM) and the basolateral membrane (BLM). On the BBM, the sodium (Na)/glucose transporters (SGLT1 and SGLT2), the Na-independent transporter (GLUT5) and on the BLM the hexose transporter (GLUT2) are discussed. The molecular biology of these transporters is also reviewed. In the second part of the review, we discuss the manner in which intestinal adaptation may be modified by alterations in the diet, especially the lipid constituents, and two important examples of intestinal adaptation will be given: diabetes mellitus and inflammatory bowel disease.     

Effect of metabolic inhibitors on K+ transport across the lamprey (Lampetra fluviatilis) erythrocyte membrane

In order to assess the contribution of oxidative metabolism to K+(86Rb+) transport across the lamprey red cell membrane, the effects of various metabolic inhibitors were examined. The influx of K+ was reduced markedly in the presence of 20 mumol/l 2,4-dinitrophenol (2,4-DNP) or rotenone, and to a lesser extent by 1 mmol/l cyanide. Rotenone produced complete inhibition of the K+ active transport and a partial blockade of K+ channels by 28% on the average. Addition of 2,4-DNP to incubation media resulted in a significant reduction of both active transport of K+ (by 47%) and of K+ movement via channels (by 57%). The inhibitory effect of 2,4-DNP on total K+ influx was independent on decreasing extracellular pHe from 7.4 to 6.5. The blocking action of 1 mmol/l Ba2+ on K+ channels was abolished in the red cells incubated at pHe 6.5. Treatment of the red cells with 1 mmol/l cyanide diminished active transport of K+ to about 34% of control values but did not affect K+ channels. The obtained data indicate that in the lamprey red blood cells at least a half of energy needed for the active transport of K+ is supplied with ATP produced by oxidative phosphorylation. It may be suggested that NADH dehydrogenase is the key enzyme required for active transport of K+ in the cells, as rotenone, a selective blocker of this enzyme, causes a complete blockade of the Na+, K(+)-pump.     

Effects of oxidants and glutamate receptor activation on mitochondrial membrane potential in rat forebrain neurons

Both glutamate and reactive oxygen species have been implicated in excitotoxic neuronal injury, and mitochondria may play a key role in the mediation of this process. In this study, we examined whether glutamate-receptor stimulation and oxidative stress interact to affect the mitochondrial membrane potential (delta psi). We measured delta psi in rat forebrain neurons with the ratiometric fluorescent dye JC-1 by using laser scanning confocal imaging. Intracellular oxidant levels were measured by using the oxidation-sensitive dyes 2',7'-dichlorodihydrofluorescein (DCFH2) and dihydroethidium (DHE). Application of hydrogen peroxide (0.3-3 mM) or 1 mM xanthine/0.06 U/ml xanthine oxidase decreased delta psi in a way that was independent of the presence of extracellular Ca2+ and was not affected by the addition of cyclosporin A, suggesting the presence of either a cyclosporin A-insensitive form of permeability transition, or a separate mechanism. tert-Butylhydroperoxide (730 microM) had less of an effect on delta psi than hydrogen peroxide despite similar effects on intracellular DCFH2 or DHE oxidation. Hydrogen peroxide-, tert-butylhydroperoxide-, and superoxide-enhanced glutamate, but not kainate, induced decreases in delta psi. The combined effect of peroxide or superoxide plus glutamate was Ca2+ dependent and was partially inhibited by cyclosporin A. These results suggest that oxidants and glutamate depolarize mitochondria by different mechanisms, and that oxidative stress may enhance glutamate-mediated mitochondrial depolarization.