1Department of Anesthesiology and 2Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 M). 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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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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Either 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.
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RESULTS |
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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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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, 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, 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,
2 test; Fig.
5).
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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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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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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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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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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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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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DISCUSSION |
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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
-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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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