Ian A. Silver

Effects of Hypothermia on Energy Metabolism in Mammalian Central Nervous System

This review analyzes, in some depth, results of studies on the effect of lowered temperatures on cerebral energy metabolism in animals under normal conditions and in some selected pathologic situations. In sedated and paralyzed mammals, acute uncomplicated 0.5- to 3-h hypothermia decreases the global cerebral metabolic rate for glucose (CMRglc) and oxygen (CMRO2) but maintains a slightly better energy level, which indicates that ATP breakdown is reduced more than its synthesis. Intracellular alkalinization stimulates glycolysis and independently enhances energy generation. Lowering of temperature during hypoxia–ischemia slows the rate of glucose, phosphocreatine, and ATP breakdown and lactate and inorganic phosphate formation, and improves recovery of energetic parameters during reperfusion. Mild hypothermia of 12 to 24-h duration after normothermic hypoxic–ischemic insults seems to prevent or ameliorate secondary failures in energy parameters. The authors conclude that lowered head temperatures help to protect and maintain normal CNS function by preserving brain ATP supply and level. Hypothermia may thus prove a promising avenue in the treatment of stroke and trauma and, in particular, of perinatal brain injury.

Interactions of bioactive glasses with osteoblasts in vitro : effects of 45S5 Bioglass®, and 58S and 77S bioactive glasses on metabolism, intracellular ion concentrations and cell viability

In a cell culture model of murine osteoblasts three particulate bioactive glasses were evaluated and compared to glass (either borosilicate or soda-lime-silica) particles with respect to their effect on metabolic activity, cell viability, changes in intracellular ion concentrations, proliferation and differentiation. 45S5 Bioglass caused extra- and intracellular alkalinization, a rise in [Ca2+]i and [K+]i, a small plasma membrane hyperpolarization, and an increase in lactate production. Glycolytic activity was also stimulated when cells were not in direct contact with 45S5 Bioglass particles but communicated with them only through the medium. Similarly, raising the pH of culture medium enhanced lactate synthesis. 45S5 Bioglass had no effect on osteoblast viability and, under most conditions, did not affect either proliferation or differentiation. Bioactive glasses 58S and 77S altered neither the ion levels nor enhanced metabolic activity. It is concluded that: (1) some bioactive glasses exhibit well-defined effects in osteoblasts in culture which are accessible to experimentation; (2) 45S5 Bioglass causes marked external and internal alkalinization which is, most likely, responsible for enhanced glycolysis and, hence, cellular ATP production; (3) changes in [H+] could contribute to alternations in concentrations of other intracellular ions; and (4) the rise in [Ca2+]i may influence activities of a number of intracellular enzymes and pathways. It is postulated that the beneficial effect of 45S5 on in vivo bone growth and repair may be due to some extent to alkalinization, which in turn increases collagen synthesis and crosslinking, and hydroxyapatite formation.

Ion Homeostasis in Rat Brain in vivo: Intra- and Extracellular [Ca2+] and [H+] in the Hippocampus during Recovery from Short-Term, Transient Ischemia

Changes in intra- and extracellular [Ca2+] and [H+], together with alterations in tissue Po2 and local blood flow, were measured in areas CA1 and CA3 of the hippocampus during recovery (up to 8 h) after an 8-min period of low-flow ischemia. Restoration of blood supply was followed by an immediate rise in flow and tissue Po2 above normal, with large fluctuations in both persisting for up to 4 h. In area CA1, [Ca2+]i decreased rapidly from an ischemic mean value of 30 μM to a control mean level of 73.1 nM in 20–30 min, whereas normalization of [Ca2+]e took ∼1 h. Recovery of [Ca2+]i was accelerated by preischemic administration of a calcium antagonist, nifedipine, and a free radical scavenger, N-tert-butyl-α-phenylnitrone (PBN), but not by MK-801, a blocker of N-methyl-d-aspartate receptors. There was a secondary rise in [Ca2+]i in many cells beginning ∼2 h after reperfusion. This was attenuated somewhat by PBN but not clearly influenced by either nifedipine or MK-801. Changes of [Ca2+]i in area CA3 were much smaller and slightly slower than in area CA1 and were not affected by the drugs mentioned above. In both areas CA1 and CA3, pHe and pHi fell during ischemia to an average value of 6.2, from which there was a rapid initial recovery in the first 5–10 min when blood flow was restored. Thereafter tissue pH rose slowly and did not reach control levels for ∼1 h, and in some microareas not at all. It is concluded that (a) effective mechanisms for restoring normal [Ca2+]i remain intact after 8 min of low-flow ischemia; (b) in neurons of area CA1, some insidious change in the homeostasis of calcium triggers a secondary rise in its free cytosolic concentration, which may be causally related to activation of irreversible cell damage; and (c) the changes in [Ca2+]i and [Ca2+]e during and following 8 min of ischemia can be adequately accounted for by movements of a fixed pool of Ca between intra- and extracellular compartments, and possible mechanisms are discussed.

Ion homeostasis in brain cells: differences in intracellular ion responses to energy limitation between cultured neurons and glial cells.

Intracellular concentrations of sodium, potassium and calcium together with membrane potentials were measured in cultured murine cortical neurons and glial cells under conditions which mimicked in vivo hypoxia, ischemia and hypoglycemia. These included; glucose omission with and without added pyruvate, addition of rotenone in the presence and absence of glucose and substitution of 2-deoxyglucose for glucose with and without rotenone. Cellular energy levels ([ATP], [ADP], [phosphocreatine], [creatine]) were measured in suspensions of C6 cells incubated in parallel under identical conditions. [Na+]i and [Ca2+]i rose while [K+]i fell and plasma membrane depolarized when energy production was limited. Intracellular acidification was observed when glycolysis was the sole source for ATP synthesis. There was a positive correlation between the extent of energy depletion in glial cells and the magnitude and velocity of alterations in ion levels. Neither glycolysis alone nor oxidative phosphorylation alone were able to ensure unaltered ion gradients. Since oxidative phosphorylation is much more efficient in generating ATP than glycolysis, this finding suggests a specific requirement of the Na pump for ATP generated by glycolysis. Changes in [Na+]i and [K+]i observed during energy depletion were gradual and progressive whereas those in [Ca2+]i were initially slow and moderate with large elevations occurring only as a late event. Increases in [Na+]i were usually smaller than reductions in [K+]i, particularly in the glia, suggestive of cellular swelling. Glia were less sensitive to identical insults than were neurons under all conditions. Results presented in this study lead to the conclusion that the response to energy deprivation of the two main types of brain cells, neurons and astrocytes, is a complex function of their capacity to produce ATP and the activities of various pathways which are involved in ion homeostasis.

Optical measurements of intracellular oxygen concentration of rat heart in vitro.

Abstract The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a3 at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a3 at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O2 (P50) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O2 titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a3 as intracellular O2 indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a3, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O2 affinities between myoglobin and cytochrome a + a3. The results indicate that there may be an O2 gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a3 can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a3 is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.

Head cooling with mild systemic hypothermia in anesthetized piglets is neuroprotective

Hypothermia is potentially therapeutic in the management of neonatal hypoxic‐ischemic brain injury. However, not all studies have shown a neuroprotective effect. It is suggested that the stress of unsedated hypothermia may interfere with neuroprotection. We propose that selective head cooling (SHC) combined with mild total‐body hypothermia during anesthesia enhances local neuroprotection while minimizing the occurrence of systemic side effects and stress associated with unsedated whole‐body cooling. Our objective was to determine whether SHC combined with mild total‐body hypothermia while anesthetized for a period of 24 hours reduces cerebral damage in our piglet survival model of global hypoxia‐ischemia. Eighteen anesthetized piglets received a 45‐minute global hypoxic‐ischemic insult. The pigs were randomized either to remain normothermic or to receive SHC. We found that the severity of the hypoxic‐ischemic insult was similar in the SHC versus the normothermic group, and that the mean neurology scores at 30 and 48 hours and neuropathology scores were significantly better in the SHC group versus the normothermic group. We conclude that selective head cooling combined with mild systemic hypothermia and anesthesia is neuroprotective when started immediately after the insult in our piglet model of hypoxic‐ischemic encephalopathy. Ann Neurol 2003;53:000–000

Breast cancer cells have a high capacity to acidify extracellular milieu by a dual mechanism.

The extracellular pH in malignant tumors is known to be lower than in normal tissues and may therefore facilitate extracellular activation of secreted lysosomal cathepsins. We have tested the capability of human mammary cells (continuous cell lines and primary culture) to acidify their extracellular environment, using two techniques. By measuring pH changes through alterations of phenolsulfone phthaleine absorbance, we found that the more aggressive MDA-MB-231 human breast cancer cells were more active in acidifying a non-buffered balanced salt solution than the estrogen receptor positive MCF7 and ZR75 cell lines and than normal mammary epithelial cells in primary culture. Metastatic breast cancer cells from pleural effusions were up to 200-fold more active in acidifying their extracellular milieu than non-malignant mammary cells cultured in the same conditions, strongly suggesting that this difference also occurs in vivo. The use of inhibitors in the presence or absence of glucose showed that both lactate and an ATP-driven proton pump sharing some characteristics of the vacuolar H+ pump were involved. Bafilomycin A1, a specific inhibitor of the vacuolar (V-type) ATP-H+ pump inhibited part of the acidification by MCF7 cells, but not by MDA-MB-231 cells. We also used microelectrodes to measure extracellular pH, in close contact to the MCF7 breast cancer cells. The pH at the free surface of MCF7 cells was lower by 0.33±0.14 unit than that of the surrounding medium, while insertion of the microelectrode tip beneath the attached surface of the cells showed a greater lowering of pH from 0.3 to 1.7 pH unit as long as cell attachment on the substrate prevented H+ diffusion. We conclude that breast carcinoma cells have a higher capacity for acidifying their extracellular milieu than normal mammary cells, and that both a plasma membrane H+-ATPase, and lactic acid production are involved in this acidification. It is therefore possible that the aspartyl and cysteinyl pro-cathepsins secreted in excess by tumor cells may be activated extracellularly in vivo close to the basement membrane.

The Distribution of Types I and III Collagen and Fibronectin in the Healing Equine Tendon

During tissue response to injury the glycoproteins fibronectin and Type III collagen are synthesized in increased amounts. We have studied the distribution of these molecules in the healing tendon at various times after injury by comparison with that of the major constituent of normal tendon, Type I collagen. Immunofluorescent localization demonstrated the presence of fibronectin throughout the tendon within one week after injury. Staining was found in the matrix, both around capillaries and around fibroblast-like cells. Fibronectin was still apparent in the healing tendon at one month after injury, but after a further two months was no longer detectable. Type III collagen was present both in pericellular and matrix locations until three months after injury, and matrix staining was apparent during the entire fourteen-month period under study. Type III collagen was also found throughout the matrix of the contralateral superficial flexor tendon during this period.

Oxygen and Ion Concentrations in Normoxic and Hypoxic Brain Cells

The goal of the present contribution is to discuss the relationships among brain oxygen tension, energy (ATP) level, and ion gradients and movements. The function of the CNS, the generation and transmission of impulses, is determined to a large extent by the movements of ions. Hence elucidation of these relationships is necessary to the understanding of how brain works. Moreover, such knowledge is indispensable for the design of rational therapies for treatment of a large group of pathological states caused by lack of oxygen. This paper is partly a review and partly an original contribution although the former involves to a considerable extent, results obtained in our laboratories. It is divided into 3 parts: a) a very brief general introduction which reminds the reader some well-known facts; b) presentation and discussion of data; and c) conclusions and/or predictions.

Energetic demands of the Na+/K+ ATPase in mammalian astrocytes

Cultured astrocytes and cell lines derived therefrom maintain a high energy level ([ATP]/[ADP]) through operation of oxidative phosphorylation and glycolysis. The contribution from the latter to total ATP production is 25–32%. A powerful Na+/K+ pump maintains potassium, sodium, and calcium gradients out of equilibrium. [Na+]i is about 20 mM, [K+]i is 130 mM and [Ca2+]i is less than 100 nM. Under non‐stimulated conditions, the Na+/K+ ATPase consumes 20% of astrocytic ATP production. Inhibition of the pump by ouabain decreases energy expenditure, raises [creatine phosphate]/[creatine], and leads to a leakage of sodium, potassium, and calcium ions. Decrease in the pump function via a fall in [ATP] also collapses ion gradients; the rate and extent of the fall correlates positively with cellular energy state. Under “normal” conditions (i.e., when ATP production pathways are not inhibited), there appears to be no preferential utilization of energy produced by either glycolysis or oxidative phosphorylation for the support of pump function. GLIA 21:35–45, 1997.