Esther Yoles

Brain mitochondrial redox state, tissue haemodynamic and extracellular ion responses to four-vessel occlusion and spreading depression in the rat

Fibre-optic surface fiuorometer reflectrometry was used to monitor the NADH (nicotine adenine dinucleotide) redox state from rat brain during three- or four-vessel occlusion. To compare the completeness of the electrocauterization of the vertebral arteries and the effectiveness of the anterior cerebral arteries, two light guides were implanted above the cerebral hemispheres. The NADH level was measured and correlated with the changes in the intensity of the reflected light at the excitation wavelength (366 nm) and to the ECoG (electrocorticogram). In the present study, we used ten rats in which unilateral and bilateral carotid occlusion were performed. In a second group of rats we tested the effects of four-vessel occlusion on the metabolic and extracellular K+ and Ca2+ activities as compared with those recorded under spreading depression conditions. These experiments were done by using the multiprobe assembly (MPA) approach. The results could be summarized as follows: (1) in the four-vessel occlusion model, the level of cerebral ischaemia could be estimated quantitatively, in real-time, by monitoring the NADH redox state; (2) unilateral carotid occlusion (after vertebral coagulation) led to a variable level of ipsilateral ischaemia, depending upon the blood flow compensation between the two hemispheres; (3) fibre-optic fluorometry enabled the correlation of NADH redox state with other physiological parameters as well as during after-brain ischaemia; (4) using the MPA in rats exposed to four-vessel occlusion as well as spreading depression, we identified the differences between the two pathological states, although there were some similarities in the ion homeostasis responses.

The effect of hyperbaric hyperoxia on brain function in the newborn dog in vivo.

Abstract Age is a natural factor that has been found to significantly affect sensitivity to hyperbaric hyperoxia (HBO). Exposure to HBO may lead to damages in the energy metabolism of the brain cells. The aim of this study was to test the effect of HBO on the metabolic, hemodynamic and electrical activities in the newborn dog. The study was performed using one-day- to 70-day-old puppies. The puppies were placed in a pressure chamber. The pressure of pure 02 in the chamber was raised by 5 atmospheres (ATA, 75 psi= 6 ATA) within 10 min. The first biochemical change to take place during HBO was oxidation of mitochondrial NADH. The age of the puppy was found to affect the time to the initiation of seizures. In the puppies under the age of 24 days, the average time was 35.1 ±5.9 min. In the puppies of 24 days old and older, the average time was 5.1 ±0.8 min. In the younger puppies, there was a later occurrence of blood vessel contractions and a longer life span compared to the older puppies. The comparison between the puppies of different ages during exposure to HBO showed differences in the metabolic response, hemodynamic changes and electrical activity. These differences can partially explain the higher resistance in the younger puppies to HBO. [Neurol Res 2000; 22: 404-408]

Some electrophysiological properties of developing rat skeletal myotubes grown in serum-free, chemically defined medium

Serum‐free, chemically defined media have been reported to provide suitable conditions for growth and proliferation of mammalian skeletal muscle, but there is no information regarding the ability of myotubes to develop normal electrophysiological properties in these media. We have recorded transmembrane resting and action potentials from rat skeletal myotubes grown in both serum‐containing (GM) and serum‐free chemically defined (CDM) growth media. Muscle cells in CDM do not develop as high resting Em as their counterparts in conventional growth media. CDM myotubes also had a lower incidence and frequency of spontaneously occurring action potentials. Treatment with ouabain or decrease in temperature of the recording medium reduced resting Em of both GM and CDM cells to the same level. We found that the sensitivity of CDM cells to ouabain was about 10‐fold higher than that of GM cells. An increase in temperature of the recording medium increased Em of GM myotubes but not of CDM myotubes. The change in resting Em in response to a 10‐fold change in extracellular K+‐ion concentration was the same for both groups of cells thus indicating that there was no difference in membrane permeability to K+‐ion. We conclude that the difference in Em can be accounted for largely, if not entirely, by differences in activity or amount of electrogenic Na+‐K+ ATPase.

Brain vascular ionic and metabolic responses to ischemia in the Mongolian gerbil.

Since the pioneering work of Levine and Payan / l / more than 20 years ago, the Mongolian gerbil /2,3/ became one of the most popular animal models for ischemia studies /4-6/. The incompleteness of the Circle of Willis in this animal exhibits two unique consequences when a unilateral or a bilateral carotid artery occlusion is performed. As in all mammals, blood is supplied to the brain by two main sources, namely, the two common carotid arteries and the two vertebral arteries merging into one basilar artery entering the hind brain area /7 / . In most gerbils the connection between the carotid and basilar arteries system is missing; therefore, by occlusion of the two carotid arteries, a complete ischemia could be achieved. The connection between the two anterior cerebral arteries is varied between various gerbils ranging from no connection at all to a well developed connection similar to that found in the rat /7/ . As a result of this brain vasculature anomaly, it is possible to create two levels of ischemia in the same animal /8 / . In order to evaluate the level of ischemia achieved, it is necessary to

EFFECTS OF AGE ON THE METABOLIC, IONIC AND ELECTRICAL RESPONSES TO ANOXIA IN THE NEWBORN DOG BRAIN IN VIVO

The interrelation between brain energy metabolism, electrical activity and ion homeostasis developing under experimental anoxia in animals of different ages is of significant value in the understanding of brain damage occurring under similar conditions of clinical neuropathology. The purpose of the present study was to compare brain energy states and extracellular ion homeostasis during anoxia in newborn puppies of various ages. We have developed and used a multiparametric monitoring device by which various functions of the brain can be recorded in a real-time mode from a 5 mm diameter area on the surface of the cortex. Intracellular oxygen balance was evaluated in newborn puppies of various ages by monitoring the intramitochondrial NADH redox state using a fluorescence technique. The electrical activity was measured by recording the spontaneous ECoG (electrocorticogram) and DC (direct current) steady potential. Ion homeostasis was evaluated using surface potassium and calcium mini-electrodes. Newborn puppies were anesthetized, the dura mater was removed and the multiprobe assembly was placed on the brain and cemented to the skull. Five groups of puppies (0-1, 2-7, 8-14, 15-21 days and 3-24 weeks) were exposed to 5 minutes of complete O2 deprivation (100% nitrogen exposure) and were monitored during the recovery period until all parameters returned to baseline values. The results may be summarized as follows: 1. Resting baseline levels of extracellular K+ were in the same range as described for other young and adult mammals (2.9 +/- 0.05 mM). 2. Extracellular Ca2+ levels were higher than those published for other mammals (1.6 +/- 0.07 mM). 3. During 5 minutes of anoxia, a significant increase in K+ levels was recorded. This increase was not accompanied by measurable changes in extracellular Ca2+. 4. The effect of age on the length of time to the elevation of the extracellular K+ concentration and on the rate of K+ accumulation from the onset of the anoxic condition was significant, i.e., the younger the animal the longer the time and the lower the rate. 5. The rate of energy depletion was age dependent as indicated by the rate of NADH accumulation during anoxia. However, no significant effect of age on the basal aerobic metabolism was found as measured by the maximum percent increase of NADH during anoxia.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolic, ionic and electrical responses to oxygen deficiency in the newborn dog in vivo.

The interrelation between energy metabolism and the functional state of the newborn brain is a critical factor in understanding cerebrovascular disturbances in newborn babies after birth. Changes in the cerebral blood flow after birth may lead to brain injury due to a decrease in oxygen supply and to an imbalance between demand and supply. Neonatal asphyxia which ended up in hypoxic encephalopathy had been reported in 1% of the total deliveries (1).

Metabolic and ionic responses to global brain ischemia in the newborn dog in vivo: II. Post-natal age aspects

Abstract The main difference between newborn and adult brains is expressed in the relative resistance of the newborn brain to oxygen deprivation. The aim of the present study was to examine the effect of global ischemia in canine puppies of three different ages on the metabolic, ionic and electrical activity of the brain and to study the basic mechanisms underlying the relative resistance of the newborn brain in ischemic episode. The puppies were divided into three age groups. The young group included 0-6-day-old puppies (n= 16), the intermediate group included 7-19-day-old puppies (n = 2l), and the ‘adult’ group included puppies aged 20 days or more (n = 7 7). Statistical analysis of the results led to the following conclusions: The younger the puppy, the longer is the time until the occurrence of the secondary reflectance increase SRI (13.0±l.9 min vs. 5.3 ± 0.5 min). The younger the puppy, the longer the time until onset of potassium leakage from the cells (0.9± 0.7 min vs. 0.35 ± 0.05 min) and the lower the amount of potassium leakage (9.6±2.8mM vs. 21.7±4.8 mM). The rate of pumping of the potassium ions into the cells during the recovery stage was higher in the oldest group (1,2± 0.2 mM min–1 vs. 0.38 ±0.1 mMmin–1J. It was possible to speculate that in the young puppies there is uncoupling of the oxidative phosphorylation from respiration and as a result, there is a lower, if any, rate of ATP synthesis. It seems that the newborn brain is able to cope with a decrease in available energy for a longer period of time. This is apparently due to differences in membrane characteristics and an improved ability to retain ionic equilibrium across both sides of the membrane. [Neurol Res 2000; 22: 623–629]

Brain metabolic and ionic responses to systemic hypoxia in the newborn dog in vivo

Newborns are less sensitive than adults to hypoxic/ischemic injury. However, research into the mechanism of the newborns relative resistance to reduced brain oxygen levels is relatively scarce, and the time-scale for the disappearance of resistance is not known. The multiprobe assembly (MPA) has enabled us to examine the resistance of puppies at various ages to hypoxia via continuous, simultaneous, on-line measurement of various ionic, metabolic and electrical parameters from the cerebral cortex. The parameters measured included electrocorticogram (ECoG), direct current (DC) steady state potential, extracellular potassium (Ke+) and calcium ion concentrations and intra-mitochondrial Nicotine amide adenine dinucleotide NADH redox levels. These parameters were measured under various degrees of hypoxia (fraction of inspiration oxygen was between 0-10%) in 6-h-old to 24-week-old puppies (n = 44). Sensitivity to hypoxia increased with age, being expressed in the leakage of potassium ions out of the cells (0.3 +/- 0.07 mM in the younger puppies and 3.0 +/- 1.3 mM in the older puppies) following an increase in intra-mitochondrial NADH redox levels. Potassium ion (Ke+) leakage was apparently due to depleted energy stores resulting from an impairment in the balance between oxygen supply and demand. Although the overall effect was similar, the kinetics of these changes were much faster in the older puppies. The time to initial increase of extracellular K+ was 2.5 +/- 0.1 min in the younger puppies and 0.9 +/- 0.1 min in the older puppies. The time to maximum increase of NADH was 3.2 +/- 0.2 min in the younger puppies and 1.4 +/- 0.1 min in the older puppies. Our results indicate that the older puppies utilize the existing oxygen faster than the younger puppies. It is concluded that the increased resistance of newborn puppies to hypoxia is due to intrinsic properties of the brain itself, like the ability of the membrane to maintain ionic homeostasis.

Brain metabolic and ionic responses to global brain ischemia in the newborn dog in vivo: 1. Methodological aspects.

Abstract A variety of methods has been used in order to obtain a state of acute cerebral ischemia. Most of these methods suffered from drawbacks such as irreversible ischemia, difficult to obtain total ischemia and heart injury. The aim of this study was to develop a new method for induction of global or partial cerebral ischemia in the newborn dog at various post-natal ages. A multi-parameter monitoring system (MPA) measures the metabolic (mitochondria NADH oxidation/reduction state), hemodynamic (reflectance), ionic (extracellular potassium and calcium) and electrical changes (ECoG) continuously and simultaneously In the puppy’s brain in vivo. A hole was made in the chest cavity, the two large arteries supplying blood to the brain, the brachiocephalic and the subclavian arteries (B + S) were isolated and occluded during the monitoring. In most of the animals, occlusion of these two arteries alone resulted in partial ischemia. For obtaining 1 00% ischemia, we occluded both the B + S arteries as well as the aortic arch. Immediately at the onset of ischemia, an increase (reduction) of NADH begins. During complete ischemia the average time until maximal increase was 4 min, compared to ischemia of up to 50% of the maximal reduction of the NADH where the average time was 1 min. After reperfusion of the brain, mitochondria recovery was very rapid and the average time until return of this parameter to its pre-ischemic level was 1.4±0.2 min. The ionic changes which occurred immediately upon the onset of ischemia were the accumulation of extracellular potassium ions was recorded. The rate of potassium ion accumulation was dependent on the severity of the ischemia (range 0.19±0.08-2.2±0.4 mM min-1). The increase in the extracellular potassium ion concentration occurs in two stages, an initial slow stage and a second rapid stage (13.0±1.8 mM). The results presented in this paper suggest and prove the usefulness of a new approach for global and partial ischemia in the newborn dogs. In addition, our results assess the brain metabolic, ionic, hemodynamic and electrical responses to brain ischemia in the puppies. [Neurol Res 2000; 22: 505-511]

Evidence for a functional role of acetylcholinesterase in cultured chick myotubes

This study was undertaken in order to assess the functional role of acetylcholinesterase (AChE) in cultures of chick skeletal muscle cells. Cultures of skeletal myotubes were prepared by mechanical dissociation of limb muscle removed from 11-day-old chick embryos and plating at a concentration of 0.8 X 10(6) cells/ml. Cultures incubated for 4-10 days were used for electrophysiological studies with intracellular microelectrodes. Individual myotubes differed with respect to the time course of repolarization following depolarization by acetylcholine (ACh), some cells repolarizing within 2-3 min and others only after 8-10 min. Physostigmine (10(-8)-10(-6) M) prolonged or sometimes completely prevented repolarization following ACh-induced depolarization. These results demonstrate that hydrolysis of ACh by AChE in cultured chick skeletal myotubes plays an important role in the repolarization of these cells following ACh-induced depolarization.