Differential Fall in ATP Accounts for Effects of Temperature on Hypoxic Damage in Rat Hippocampal Slices

J. Wang,1 G. Chambers,1 J. E. Cottrell,1 and I. S. Kass1,2

 1Department of Anesthesiology and  2Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Wang, J., G. Chambers, J. E. Cottrell, and I. S. Kass. Differential Fall in ATP Accounts for Effects of Temperature on Hypoxic Damage in Rat Hippocampal Slices. J. Neurophysiol. 83: 3462-3472, 2000. Intracellular recordings, ATP and cytosolic calcium measurements from CA1 pyramidal cells in rat hippocampal slices were used to examine the mechanisms by which temperature alters hypoxic damage. Hypothermia (34°C) preserved ATP (1.7 vs. 0.8 nM/mg) and improved electrophysiologic recovery of the CA1 neurons after hypoxia; 58% of the neurons subjected to 10 min of hypoxia (34°C) recovered their resting and action potentials, while none of the neurons at 37°C recovered. Increasing the glucose concentration from 4 to 6 mM during normothermic hypoxia improved ATP (1.3 vs. 0.8 nM/mg) and mimicked the effects of hypothermia; 67% of the neurons recovered their resting and action potentials. Hypothermia attenuated the membrane potential changes and the increase in intracellular Ca2+ (212 vs. 384 nM) induced by hypoxia. Changing the glucose concentration in the artificial cerebrospinal fluid primarily affects ATP levels during hypoxia. Decreasing the glucose concentration from 4 to 2 mM during hypothermic hypoxia worsened ATP, cytosolic Ca2+, and electrophysiologic recovery. Ten percent of the neurons subjected to 4 min of hypoxia at 40°C recovered their resting and action potentials; this compared with 60% of the neurons subjected to 4 min of normothermic hypoxia. None of the neurons subjected to 10 min of hypoxia at 40°C recovered their resting and action potentials. Hyperthermia (40°C) worsens the electrophysiologic changes and induced a greater increase in intracellular Ca2+ (538 vs. 384 nM) during hypoxia. Increasing the glucose concentration from 4 to 8 mM during 10 min of hyperthermic hypoxia improved ATP (1.4 vs. 0.6 nM/mg), Ca2+ (267 vs. 538 nM), and electrophysiologic recovery (90 vs. 0%). Our results indicate that the changes in electrophysiologic recovery with temperature are primarily due to changes in ATP and that the changes in depolarization and Ca2+ are secondary to these ATP changes. Both primary and secondary changes are important for explaining the improved electrophysiologic recovery with hypothermia.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Oxygen deprivation, even for relatively short periods of time, can cause irreversible damage to the mammalian brain. Neuroprotective strategies include the use of glutamate receptor antagonists, calcium channel blockers, free-radical scavengers, and induced hypothermia (Fay 1959; Newberg et al. 1986; Rader and Lanthorn 1989; Shadid et al. 1998). Mild and moderate hypothermia may not only protect against neuronal injury (Minamisawa et al. 1990) but also improve clinical outcome after traumatic brain injury (Wass and Lanier 1996). Mild hyperthermia worsens ischemia-induced neuronal injury (Dietrich et al. 1990).

Experimental observations have led to hypotheses that ischemia-induced neuronal damage results from glutamate excitotoxicity, an increase in intracellular calcium, and/or oxidative stress. Reductions in ATP concentrations and increases in sodium influx are thought to increase cytosolic calcium concentrations that trigger damaging cascades such as the overactivation of proteases, kinases, phospholipases, and endonucleases and an increase in free radical formation (Lipton 1999). Small variations in brain temperature can influence synaptic glutamate accumulation and thereby excitotoxicity (Busto et al. 1989a,b). Hypothermia and hyperthermia may modulate free radical production by decreasing or increasing, respectively, cytosolic calcium, hydrogen, and sodium, and mitochondrial free radical formation (Choi 1988; Kil et al. 1996; Wolbarsht and Fridovich 1989). Glutamate is normally transported into neurons and glia against a large concentration gradient by transporters that utilize the Na+ and K+ gradients as an energy source (Bouvier et al. 1992). ATP depletion produced by ischemia inhibits the Na/K pump and results in ion gradient reductions that can reverse the glutamate transporters (Erecinska and Dagani 1990). Hypothermia preserves intracellular ATP and potassium concentrations and attenuates the increase in sodium during hypoxia; hyperthermia exacerbates these changes in ATP, potassium, and sodium (Amorim et al. 1999).

Hippocampal CA1 pyramidal neurons are known to be extremely vulnerable to hypoxia and ischemia and demonstrate a stereotyped response characterized by an initial hyperpolarization followed by a depolarization. When the membrane completely depolarizes, the damage becomes irreversible, even if oxygen is reintroduced (Rader and Lanthorn 1989). The aim of the present study is to investigate the effects of temperature on irreversible membrane dysfunction induced by hypoxia using intracellular recordings, and ATP and cytosolic calcium measurements. The current studies complement previously published work that examined the effect of hypothermia and hyperthermia on hypoxic changes in ATP, Na+, and K+ concentrations and the recovery of extracellular potentials (Amorim et al. 1999). Together these studies examine important triggers for long-term damage and help explain why hypothermia ameliorates and hyperthermia exacerbates hypoxic damage in the slice preparation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slice preparation

All procedures involving animals were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Sprague-Dawley rats (100-120 days old) were anesthetized with 2% isoflurane and decapitated, and their brain was quickly removed and placed into chilled (2-4°C) artificial cerebrospinal fluid (ACSF) (Zhu et al. 1997). Hippocampal slices of 400 µm thickness for intracellular electrophysiologic recording and 300 µm for calcium imaging were sectioned in chilled ACSF (4-6°C) using a vibratome (Wang and Kass 1997). The thinner sections improved visualization for the imaging experiments. The slices were stored in a beaker containing ACSF saturated with 95% O2-5% CO2 and remained there for approximately 2 h at room temperature. The composition of the ACSF was (in mmol/l) 126 NaCl, 3 KCl, 1.4 KH2PO4, 26 NaHCO3, 4 glucose, 1.3 MgSO4, 1.4 CaCl2, at pH 7.4, and was equilibrated with 95% O2-5% CO2. Slices were transferred to a tissue chamber and maintained at the protocol's temperature in this chamber. Hypoxia was generated by switching the gas to 95% N2-5% CO2.

Electrophysiology

A Fine Science Tools recording chamber was used for the electrophysiologic experiments. The hippocampal slice was submerged in the recording chamber and perfused with ACSF at a rate of 3.0 ml/min. The temperature in the recording chamber was maintained at either 34, 37, or 40°C according to the experimental protocol.

A bipolar stimulating electrode was placed in the Schaffer collateral pathway, and then a CA1 pyramidal neuron was impaled with a glass micropipette filled with 4 mol/l KAc (70-120 MOmega ). A hyperpolarizing current was applied through the recording electrode to facilitate the recovery of the neuron from the injury of the impalement. The hyperpolarizing current was gradually reduced to zero before starting the experiment. Only neurons with stable resting potentials of at least -60 mV for 15 min with high-amplitude, short-duration action potentials that showed spike frequency accommodation and were activated by short-latency Schaffer collateral stimulation were examined. Electrophysiologic activity was recorded with an AxoClamp 2B amplifier (Axon Instruments) and displayed on an oscilloscope and a chart recorder. The data were also stored for later analysis on IBM-compatible computers using Pclamp 6 and Axotape 2.0 software from Axon Instruments.

The electrophysiologic parameters were measured as previously described by Tanaka et al. (1997). The latency of the rapid depolarization was measured from the onset of anoxia to onset of the rapid depolarization, which was estimated by extrapolating the slope of the rapid depolarization to the slope of the slow depolarization (Fig. 1). The onset of the rapid depolarization was measured at the membrane potential crossing the extrapolated slope of the slow depolarization and the rapid depolarization. The peak potential was measured as the membrane potential deflection from the rapid depolarization to 0 mV. The amplitude of the rapid depolarization was measured as the difference between the peak potential and the onset potential of the rapid depolarization.



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Fig. 1. Measured parameters of the rapid depolarization. The latency of the rapid depolarization was measured from the onset of hypoxia to onset of the rapid depolarization. The onset of the rapid depolarization was measured at the membrane potential crossing the extrapolated slope of the slow depolarization and the rapid depolarization (1). The peak potential was measured as the membrane potential deflection from the rapid depolarization to 0 mV (2 to 3). The amplitude of the rapid depolarization was measured as the difference between the peak potential and the onset potential of the rapid depolarization (4). These are the same measurements that were used by Tanaka et al. (1997).

ATP measurement

ATP was measured in the CA1 region of individual slices (Amorim et al. 1999). Slices were incubated in beakers under conditions similar to those described for the electrophysiologic experiments. For these experiments a period of 5 or 10 min of hypoxia was examined. After hypoxia, or an equivalent time period, the slices were removed from the beaker, rapidly frozen in liquid nitrogen, lyophilized and the CA1 regions dissected and weighed. The ATP was then extracted by homogenizing the tissue in 3N ice-cold perchloric acid and measured, after neutralization, using the firefly luciferin-luciferase assay (Kass et al. 1992).

Calcium imaging

Two slices were placed in a small beaker containing 6 ml ACSF, 9 µmol/l fura-2 AM (Molecular Probes, Eugene, OR), 0.01% Pluronic acid, and 50 µl of DMSO for 45 min. They were then washed in fresh ACSF and incubated for an additional 45 min to remove extracellular fura-2 and allow the AM moiety of intracellular fura-2 AM to hydrolyze. The slices were maintained at 33°C from the initial incubation until they were placed in a tissue chamber on the microscope stage; maintenance at this temperature improved dye loading. The slices were maintained at either 34, 37, or 40°C in the tissue chamber on the microscope stage throughout the experiment. An Incyt Im2 dual wave length imaging system and its associated software was used for measuring Ca2+ (Intracellular Imaging, Cincinnati, OH). A long working distance Nikon Plan Fluor ×20 UV objective (n.a. 0.5) and a Nikon TMS inverted microscope were attached to a filter changer (340 and 380 nm filter), a 300 w Xenon light source, and a low light level charge-coupled device (CCD) camera for image acquisition.

We used Ca buffers in solution for calibration. We recognize that they yield only a relative measure of intracellular calcium concentrations. This standardization allows for the comparison of results across laboratories using different imaging systems and over time in the same laboratory as imaging equipment components age. All values represent Ca2+ concentrations corrected for background fluorescence in time-matched, unlabeled (no fura-2) slices subjected to hypoxia (Fig. 2). The absolute light intensity of the unlabeled tissue was subtracted from the light intensity of the fura-2-labeled tissue at each wavelength for each time point and the 340/380 ratio was calculated from these corrected values. This was done to account for the increase in background fluorescence due to the increase in NADH levels during hypoxia (Brooke et al. 1996).



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Fig. 2. Raw fluorescence data after 340 and 380 nm excitation from a fura-2-loaded slice and an unloaded slice. A: changes in fluorescence in a fura-2-loaded slice. B: changes in fluorescence in an unloaded slice.

Either chi 2, ANOVA followed by Dunnett's multiple comparison test, or Student's t-test were used, as appropriate, to test significance (Prism, GraphPad Software, San Diego, CA). Data are expressed as means ± SD; P < 0.05 was considered significant. Percentage values are used in the text to enhance readability and facilitate comparison; however, all statistics are done on the absolute numbers, which are shown in the figures and tables. Any stated difference in the results section is a statistically significant difference.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrophysiologic recovery

Intracellular recordings were made from a total of 106 CA1 pyramidal neurons in rat hippocampal slices before, during, and after hypoxia. The mean resting potentials before hypoxia were -63.8 ± 3.2 mV at 34°C, -63.5 ± 3.4 mV at 37°C, and -64.2 ± 4.0 mV at 40°C. Hypoxia caused an initial hyperpolarization, followed by a slow depolarization and finally a rapid and complete depolarization (Figs. 3 and 4). This final depolarization was concomitant with a complete loss of excitability.



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Fig. 3. Effect of temperature on electrophysiologic responses in CA1 pyramidal cells before, during, and after 10 min of hypoxia. Continuous recordings from the neurons at 34, 37, and 40°C are displayed. The slices are unstimulated; action potentials on the traces are due to spontaneous activity. Hypoxia is indicated by the solid bar below the traces. The resting potential remained at 0 mV after hypoxia at 37 and 40°C even though the slices were reoxygenated. The resting potential recovered after hypoxia at 34°C. Scale: 20 mV, 1 min.



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Fig. 4. Effect of temperature on the membrane potential measured in CA1 pyramidal cells before, during, and after hypoxia. Hypothermia enhanced the initial hyperpolarization (P < 0.01), delayed the rapid depolarization (P < 0.01), and reduced the final depolarization (P < 0.01) during hypoxia. Hyperthermia decreased the initial hyperpolarization (P < 0.05) and accelerated the rapid depolarization (P < 0.01). The resting potential recovered in hypothermic (P < 0.01 vs. normothermic) but not normothermic or hyperthermic slices after hypoxia. Values are means ± SD; n = 12 for each group.

Hippocampal slices were subjected to 10 min of hypoxia and then reperfused with oxygenated ACSF for 60 min. None of the neurons subjected to hypoxia at 37°C recovered their resting potentials. Hypothermia significantly improved the recovery of the resting potential after hypoxia. In the 34°C group, 7 of 12 neurons (58%) subjected to 10 min of hypoxia recovered their resting potential after reoxygenation. There is a significant difference between the hypothermic and normothermic groups (P < 0.05, chi 2 test). All neurons that recovered their resting potential also responded to intracellular current injection and Schaffer collateral stimulation after hypoxia by generating action potentials.

Hippocampal slices subjected to 4 min of hypoxia under hypothermic conditions demonstrated significantly improved recovery of resting and action potentials compared with normothermic slices. All of the neurons in the 34°C group recovered their resting and action potentials, this compares with 6 of 10 neurons in the 37°C group (P < 0.05, chi 2 test). Only 1 of 10 neurons subjected to 4 min of hypoxia at 40°C recovered its resting and action potential; this was significantly worse than normothermia (P < 0.05, chi 2 test; Fig. 5).



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Fig. 5. Effect of temperature on the recovery of resting and action potentials after 4 min of hypoxia. Hippocampal slices subjected to 4 min of hypoxia under hypothermic conditions demonstrated significantly improved recovery of resting and action potentials compared with normothermic slices. Hyperthermia (40°C) significantly reduced the recovery after hypoxia compared with normothermia. chi 2 test; P < 0.05.

Effects of altered glucose concentrations on ATP levels and electrophysiologic recovery

In slices maintained at 37°C, the ATP fell to 22 and 14% of its normoxic concentration after 5 and 10 min of hypoxia, respectively. None of the neurons recovered their resting and action potentials on reoxygenation. Hypothermia significantly attenuated the fall in ATP concentration during hypoxia. At 5 and 10 min of hypoxia, the ATP concentration fell to only 50 and 26%, respectively; 58% of the neurons in the hypothermic group recovered their resting and action potentials (Table 1).


                              
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Table 1. Effects of temperature and glucose concentrations on intracellular ATP concentrations (nmol/mg dry weight) and the functional recovery

Increasing the glucose concentration of the ACSF from 4 to 6 mM under normothermic conditions increased the ATP level during hypoxia and improved the recovery of resting and action potentials. At 5 and 10 min of hypoxia the ATP concentrations fell to only 42 and 23%, respectively; 67% of these neurons recovered their resting and action potentials. Thus like hypothermia, increased glucose preserved ATP and improved the recovery (Table 1).

Decreasing the glucose concentration from 4 to 2 mM during hypothermia accelerated the fall in ATP during hypoxia and decreased electrophysiologic recovery on reoxygenation. At 5 and 10 min of hypoxia the ATP concentrations fell to 34 and 15%, respectively; none of the neurons in the hypothermic-2 mM glucose group recovered their resting and action potentials on reoxygenation (Table 1).

Hyperthermia enhanced damage; the ATP concentration fell to 32 and 13% of its normoxic concentration at 5 and 10 min of hypoxia. Increasing the glucose concentration of the ACSF from 4 to 8 mM under hyperthermic conditions preserved the ATP level during hypoxia and improved the recovery of resting and action potentials. At 5 and 10 min of hypoxia, the ATP concentrations fell to only 56 and 29%, respectively; 90% of the neurons recovered their resting and action potentials. This mimicked the protection found with hypothermia (Table 1).

Initial hyperpolarization

Hypothermia significantly increased the amplitude (51%) and prolonged the duration (28%) of the initial hyperpolarization compared with normothermia (Table 2). Hyperthermia significantly decreased the amplitude (36%) and shortened the duration (31%) of the initial hyperpolarization compared with normothermia (Table 2).


                              
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Table 2. Effects of temperature on the initial hyperpolarization and the slow depolarization

Reducing glucose from 4 to 2 mM during normothermic hypoxia shortened the duration and reduced the amplitude of the initial hyperpolarization; these changes mimicked those induced by hyperthermia. Increasing the glucose concentration to 6 mM significantly prolonged the duration and increased the amplitude of the initial hyperpolarization (Fig. 6A); this mimicked the changes induced by hypothermia.



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Fig. 6. Effect of altered glucose concentrations on membrane potential changes induced by hypoxia at 3 temperatures. A: reducing glucose from 4 to 2 mM during normothermic hypoxia shortened the duration (P < 0.01) and reduced the amplitude of the initial hyperpolarization (P < 0.01), shortened the duration (P < 0.01) and increased the slope of the slow depolarization (P < 0.01), and shortened the latency (P < 0.01) and increased the amplitude of the rapid depolarization (P < 0.01). These changes mimicked those induced by hyperthermia. Increasing the glucose concentration to 6 mM significantly prolonged the duration (P < 0.01) and increased the amplitude of the initial hyperpolarization (P < 0.01), prolonged the duration (P < 0.01) and decreased the slope of the slow depolarization (P < 0.01), and prolonged the latency (P < 0.01) and decreased the amplitude of the rapid depolarization (P < 0.01); this mimicked the changes induced by hypothermia. B: reducing glucose from 4 to 2 mM during hypothermic hypoxia shortened the duration (P < 0.01) and decreased the amplitude of the initial hyperpolarization (P < 0.01), shortened the duration (P < 0.01) and increased the slope of the slow depolarization (P < 0.01), and shortened the latency (P < 0.01) and increased the amplitude of the rapid depolarization (P < 0.01). C: increasing glucose to 8 mM with hyperthermic hypoxia prolonged the duration (P < 0.01) and increased the amplitude of the initial hyperpolarization (P < 0.01), prolonged the duration (P < 0.01) and decreased the slope of the slow depolarization (P < 0.01), and prolonged the latency (P < 0.01) and decreased the amplitude of the rapid depolarization (P < 0.01).

Reducing glucose from 4 to 2 mM during hypothermic hypoxia shortened the duration and decreased the amplitude of the initial hyperpolarization (Fig. 6B). Increasing glucose to 8 mM with hyperthermic hypoxia prolonged the duration and increased the amplitude of the initial hyperpolarization (Fig. 6C). Thus the effects of temperature on the initial hyperpolarization could be prevented by altering glucose.

Slow depolarization

Hypothermia significantly increased the amplitude (54%), prolonged the duration (83%), and decreased the slope of the slow depolarization (17%) compared with normothermia (Table 2). Hyperthermia significantly decreased the amplitude (42%) and shortened the duration of the slow depolarization (50%) compared with normothermia. The slope of the slow depolarization showed a trend to increase with hyperthermia (11%) compared with normothermia; however, this difference was not significant (Table 2).

Reducing glucose from 4 to 2 mM during normothermic hypoxia mimicked the changes induced by hyperthermia. The duration of the slow depolarization was shortened, and the slope of the slow depolarization was increased. Increasing glucose to 6 mM during normothermic hypoxia significantly prolonged the duration and decreased the slope of the slow depolarization (Fig. 6A); this mimicked the changes induced by hypothermia.

Reducing glucose to 2 mM during hypothermic hypoxia shortened the duration and increased the slope of the slow depolarization compared with 4 mM glucose hypothermic hypoxia (Fig. 6B). Increasing glucose to 8 mM during hyperthermic hypoxia prolonged the duration and decreased the slope of the slow depolarization compared with 4 mM glucose hyperthermic hypoxia (Fig. 6C).

Rapid depolarization

Hypothermia significantly prolonged the latency (43%), and reduced the maximal slope (32%), the amplitude (29%), and the peak potential (45%) of the rapid depolarization compared with normothermia (Table 3). Hyperthermia significantly shortened the latency (34%), and increased the maximal slope (26%), the amplitude (25%), and the peak potential (33%) of the rapid depolarization compared with normothermia.


                              
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Table 3. Effects of temperature on the rapid depolarization

Reducing the glucose concentration from 4 to 2 mM during normothermic hypoxia mimicked the changes induced by hyperthermia. The latency of the rapid depolarization was shortened, and the amplitude of the rapid depolarization was increased. Increasing the glucose concentration to 6 mM significantly prolonged the latency and decreased the amplitude of the rapid depolarization (Fig. 6A); this mimicked the changes induced by hypothermia.

Reducing the glucose concentration from 4 to 2 mM during hypothermic hypoxia shortened the latency and increased the amplitude of the rapid depolarization (Fig. 6B). Increasing the glucose concentration to 8 mM during hyperthermic hypoxia prolonged the latency and decreased the amplitude of the rapid depolarization (Fig. 6C).

Intracellular Ca2+ concentration

The intracellular Ca2+ concentration before hypoxia at 34°C was significantly lower than at 37 and 40°C (P < 0.05). During hypoxia at all temperatures a slow increase in intracellular Ca2+ concentrations was followed by a rapid increase. Hypothermia significantly attenuated the increase in intracellular Ca2+ during hypoxia (146 vs. 197%, P < 0.01) and delayed the onset of the rapid increase (7.5 ± 0.8 min vs. 4.9 ± 0.4 min, P < 0.01). Hypoxia induced a significantly greater increase in intracellular Ca2+ at 40°C than at 37°C (255 vs. 197%, P < 0.01), and the onset of the rapid increase was significantly faster (3.1 ± 0.3 min vs. 4.9 ± 0.4 min, P < 0.01; Fig. 7, A and B).



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Fig. 7. Effect of temperature on intracellular calcium concentrations induced by hypoxia. A: ratio of light output after 340 and 380 nm excitation in slices loaded with fura-2. Hypothermia attenuates and hyperthermia exacerbates the change in ratio during hypoxia. Each trace is the output from 1 slice. B: the intracellular Ca2+ concentration before hypoxia at 34°C was significantly lower than those at 37 and 40°C (P < 0.05). Hypothermia attenuated the increase in intracellular Ca2+ during hypoxia (P < 0.01 at 10 min of hypoxia) and delayed the onset of its rapid increase (P < 0.01). Hyperthermia induced a more rapid (P < 0.01) and greater (P < 0.01) increase in intracellular Ca2+ during hypoxia. Values are means ± SD; n = 10 each group.

Decreasing the glucose concentration of the ACSF from 4 to 2 mM during hypothermic hypoxia increased the intracellular Ca2+ concentration induced by hypoxia (289 vs. 146%, P < 0.01); this mimicked the Ca2+ change induced by normothermia (4 mM glucose; Fig. 8A). Increasing the glucose concentration of the ACSF from 4 to 8 mM during hyperthermic hypoxia significantly attenuated the increase in intracellular Ca2+ induced by hypoxia (141 vs. 255%, P < 0.01), which mimicked the Ca2+ change induced by hypothermia (Fig. 8B). Thus using glucose to manipulate ATP levels during normothermic hypoxia duplicated the effects of hypothermic and hyperthermic hypoxia. Reducing the ATP preservation during hypothermic hypoxia by reducing glucose blocked the effects of hypothermia.



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Fig. 8. Effect of altered glucose concentrations on intracellular Ca2+ changes induced by hypoxia during hypothermia and hyperthermia. A: reducing the glucose concentration from 4 to 2 mM during hypothermic hypoxia leads to a more rapid (P < 0.05) and greater increase (P < 0.01) in intracellular Ca2+ during hypoxia. B: increasing the glucose concentration from 4 to 8 mM during hyperthermic hypoxia attenuated the increase in intracellular Ca2+ during hypoxia (P < 0.01) and delayed the onset of its rapid increase (P < 0.01). Values are the means ± SD; n = 6 each group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study (Amorim et al. 1999) we found that hypothermia (34 vs. 37°C) improved the recovery of the evoked population spike and attenuated the fall in ATP, the rise in sodium, and the fall in potassium during 7 min of hypoxia. Hyperthermia (40 vs. 37°C) reduced the recovery after 3 min of hypoxia and exacerbated the fall in ATP and potassium (Amorim et al. 1999). The current study extends these results to examine intracellular electrophysiologic recordings and Ca2+ concentrations and tests the importance of ATP on temperature induced hypoxic alterations.

Hippocampal CA1 pyramidal neurons are very sensitive to the effects of hypoxia. Ten minutes of normothermic hypoxia induced an initial hyperpolarization, followed by a slow depolarization and then a rapid depolarization. When the membrane depolarizes further and approaches 0 mV, the change in membrane potential becomes irreversible, and there is no recovery when oxygen is reintroduced (Rader and Lanthorn 1989). Hypothermia improves the recovery of resting and action potentials after hypoxia, while hyperthermia decreases the recovery of these potentials. These results agree with those from other animal models of hypoxia and ischemia (Busto et al. 1987, 1989a; Onitsuka et al. 1998a; Rosen and Morris 1994) and with clinical studies, which indicate that mild hypothermia improves and hyperthermia degrades the neurological outcome in patients with ischemic brain injury (Wass and Lanier 1996).

Initial hyperpolarization

The hypoxia-induced hyperpolarization is most likely mediated by an increase in K+ conductance (Fujiwara et al. 1987; Hansen et al. 1982; Leblond and Krnjevic 1989). It has been suggested that the initial hyperpolarization is due to activation of a Ca2+-dependent K+ conductance (Belousov et al. 1995; Katchman and Hershkowitz 1993; Leblond and Krnjevic 1989), and/or activation of KATP channels by depletion of [ATP]i (Fujimura et al. 1997; Godfraind and Krnjevic 1993; Grigg and Anderson 1989). Hypoxia induced a deeper hyperpolarization with mild hypothermia than with normothermia; this hyperpolarization was attenuated during hyperthermic hypoxia. A recent study from our laboratory showed that there was only a small decrease in [ATP]i and [K+]i during the initial 3 min of hypoxia at 34°C; however, there was a greater decrease in [ATP]i and [K+]i during hypoxia at 37 or 40°C (Amorim et al. 1999). Increasing the glucose concentration preserves ATP and mimics the effects of hypothermia on the initial hyperpolarization. We make the assumption that the main effect of changing the glucose concentration is to alter the ATP level during hypoxia. Preliminary data indicate that the changes in glucose concentrations we used do not substantially alter pH during hypoxia in this in vitro preparation (Kass, unpublished observations). This is probably due to the large chamber volume, high flow rate, and fixed CO2 concentration in our studies. Our results suggest a possible explanation for the deeper hypoxic hyperpolarization induced by hypothermia; the higher ATP concentration preserves the activity of Na+-K+ pump, maintains the [K+]i concentration, and thereby the hyperpolarizing equilibrium potential of potassium. The changes in the initial hyperpolarization during hypothermic hypoxia could also be due to a decrease in the slow depolarization.

Slow depolarization

Hypothermia prolonged the duration and decreased the slope of the slow depolarization while hyperthermia shortened its duration. The slow depolarization during hypoxia is probably due to the depression of Na+-K+ pump activity caused by reduced ATP levels and the resultant slow increase in [K+]o (Ben-Ari 1990; Fujiwara et al. 1987; Martin et al. 1994). This will lead to a gradual shift of the potassium equilibrium potential causing depolarization. When the slow depolarization passes a threshold, there is a rapid depolarization and a large flux of sodium and calcium into the neuron and potassium out of the neuron. This leads to an increase of [K+]o and [Na+]i and a decrease of [K+]i and [Na+]o (Amorim et al. 1999; Fried et al. 1995; Hansen et al. 1982; Jiang and Haddad 1991, 1992; Kass et al. 1992; Kass and Lipton 1982). Hypothermia inhibited and hyperthermia exacerbated the changes in [K+]i and [Na+]i induced by hypoxia, presumably by attenuating or exacerbating, respectively, the reduction in ATP during hypoxia (Amorim et al. 1999). Increasing the glucose concentration preserved ATP and significantly prolonged the duration of slow depolarization in both normothermia and hyperthermia; reducing the glucose concentration exacerbated the fall in ATP and shortened the duration of slow depolarization even in hypothermic hypoxia. It is likely that the enhanced ATP levels with high glucose preserved Na-K pump function, better maintained the ionic gradients, and thereby attenuated the slow depolarization.

The Ca2+-dependent release of glutamate from nerve terminals is increased by the elevation of [K+]o (Choi 1995; Dawson and Snyder 1994), and the plasma membrane glutamate transporter can be reversed when [K+]o and [Na+]i increase (Nicholls and Attwell 1990). Both mechanisms of glutamate release are temperature dependent (Busto et al. 1989b; Madl and Allen 1995; Mitani and Kataoka 1991; Takagi et al. 1994). Thus the hypothermia-induced delay of the slow depolarization may involve an inhibition of these processes. Hyperthermia induced an increase of glutamate release during anoxia (Takagi et al. 1994), which would trigger Na+ entry through alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor channels and might explain the earlier onset of both the slow and rapid depolarization during hyperthermic hypoxia.

Rapid depolarization

Our results agree with those of Onitsuka et al. (1998a) and demonstrate that hypothermia significantly prolonged the latency, reduced the maximal slope, and decreased the peak amplitude and the peak potential of the rapid depolarization. We also demonstrated that hyperthermia significantly shortened the latency, increased the maximal slope, and increased the peak amplitude and the peak potential of the rapid depolarization.

The threshold voltage for initiating the rapid depolarization was increased with hypothermia. This could be due to the decreased slope of the slow depolarization with hypothermia, causing accomodation of the neurons thereby increasing their threshold for initiating the rapid depolarization. Thus decreasing the slope of the slow depolarization would increase the latency of the rapid depolarization by both increasing the time until a fixed threshold in reached and increasing the threshold voltage. The threshold voltage for initiating the rapid depolarization was also increased with a higher concentration of glucose in both normothermic and hyperthermic conditions.

Calcium concentration

Normothermic hypoxia induced a slow increase in [Ca2+]i followed by a large rapid increase. Hypothermia attenuated the increase in [Ca2+]i during hypoxia and delayed the onset of this rapid increase; hyperthermia had the opposite effect. The onset of the rapid increase in [Ca2+]i induced by hypoxia was well matched with the onset of the rapid depolarization at all three temperatures. These results suggest that the marked increase in [Ca2+]i is associated with the rapid depolarization. Some studies have suggested that the rapid depolarization during hypoxia may be due to a nonselective increase in permeability to many ions including Na+, K+, Cl-, and Ca2+ (Hansen 1985; Kudo et al. 1989; Rothman 1985; Tanaka et al. 1994, 1997). Recent studies from our laboratory showed that 5 min of hypoxia induced a significant decrease in [K+]i and an increase in [Na+]i (Amorim et al. 1999; Fried et al. 1995); the current study demonstrates that the increase in [Ca2+]i corresponds to the rapid depolarization. Thus our results support the suggestion that the rapid depolarization may be due to a nonselective increase in permeability to many ions. We have extended these results to demonstrate that hypothermia attenuated and hyperthermia enhanced the increase of this nonselective permeability during hypoxia.

Recovery after hypoxia

Hypothermia significantly protects the CA1 neurons against the membrane dysfunction induced by hypoxia, while hyperthermia enhances this dysfunction. Hypothermia may protect against hypoxic brain damage by reducing energy demand (Michenfelder and Theye 1968), attenuating ATP depletion (Kramer et al. 1968) and intracellular acidosis (Chopp et al. 1989), reducing glutamate release (Busto et al. 1989b), and inhibiting protein kinases (Cardell et al. 1991; Churn et al. 1990). The present study shows that reducing the glucose concentration to 2 mM in normothermic slices mimics the damage in hyperthermic slices with normal glucose (4 mM). Increasing the glucose concentration to 6 mM in normothermic slices mimics the protection by hypothermia during hypoxia with normal glucose. During hyperthermia, increasing the glucose concentration to 8 mM maintained a higher ATP level during hypoxia and improved electrophysiologic recovery. Reducing the glucose concentration during hypothermia exacerbated the fall in ATP during hypoxia and reduced electrophysiologic recovery. These results demonstrate that preserving ATP is an important mechanism by which hypothermia protects neurons against hypoxia. Our results show that the attenuation of Ca2+ accumulation induced by hypoxia may be another important mechanism by which hypothermia protects against ischemic brain damage. Ischemia increased extracellular glutamate accumulation and triggered Ca2+ influx via the NMDA channels (Ben-Ari 1990; Grigg and Anderson 1989; Tanaka et al. 1997; Zhang and Lipton 1999). Hypothermia depressed the release of glutamate induced by ischemia (Matsumoto et al. 1993) and attenuated the glutamate-evoked [Ca2+]i increase (Tymianski et al. 1998). Decreasing the glucose concentration of the ACSF from 4 to 2 mM during hypothermic hypoxia increased the intracellular Ca2+ concentration induced by hypoxia; this mimicked the Ca2+ change induced by normothermia (4 mM glucose). Increasing the glucose concentration of the ACSF from 4 to 8 mM during hyperthermic hypoxia significantly attenuated the increase in intracellular Ca2+ induced by hypoxia; this mimicked the Ca2+ change induced by hypothermia. Using glucose to manipulate ATP levels during hypoxia duplicated the effects of hypothermic and hyperthermic hypoxia on cytosolic Ca2+. Reducing the ATP preservation during hypothermic hypoxia by reducing the glucose concentration blocked the effects of hypothermia on cytosolic Ca2+. Thus the changes in glucose concentrations primarily alter ATP concentrations during hypoxia; this may explain the changes in recovery and calcium concentrations with both hypothermia and hyperthermia. Calcium has been implicated as an important trigger of the secondary processes that lead to neuronal damage, and we have clearly demonstrated that hypothermia attenuates and hyperthermia exacerbates the increase in calcium during hypoxia.

The increase in [Ca2+]i induced by ischemia activates a variety of Ca2+-dependent enzymes, such as protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaM kinase II), calpain phospholipase A2, phospholipase C, and NO synthase (Choi and Rothman 1990; Katsura et al. 1993; Meldrum and Garthwaite 1990; Mody and Macdonald 1995; Onitsuka et al. 1998b), which may lead to long-term neuronal damage. Since hypothermia decreases and hyperthermia increases the calcium elevation induced by hypoxia, this will alter these Ca2+-dependent biochemical processes and may contribute to the temperature effects on neuronal damage. Hypothermia prevented the decrease in PKC and CaM kinase II activity induced by ischemia, whereas mild hyperthermia reduced the activity of these enzymes (Busto et al. 1994; Churn et al. 1990; Tohyama et al. 1998). Mild hyperthermia markedly enhanced calpain activation during ischemia, and this may contribute to the enhanced ischemic brain damage (Morimoto et al. 1997).

We conclude that ATP depletion during hypoxia leads to depolarization and a cascade of ionic events, including an increase in cytosolic Ca2+, that in turn, triggers the irrevesible damage. Hypothermia protects by attenuating the ATP depletion, delaying the depolarization and reducing the subsequent ionic changes; this blocks the triggering of biochemical cascades that lead to irreversible neuronal damage. Hyperthermia exacerbates the reduction in ATP and worsens the damage by a similar sequence of events.


    ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid from the Heritage Affiliate of the American Heart Association and a grant from the National Institute of General Medical Sciences to I. S. Kass.


    FOOTNOTES

Address for reprint requests: I. S. Kass, Dept. of Anesthesiology, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Ave., Box 6, Brooklyn, NY 11203.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 March 1999; accepted in final form 1 March 2000.


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ABSTRACT
INTRODUCTION
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