1Department of Physiology and Neuroscience Program, Tulane University School of Medicine, New Orleans, Louisiana 70112-2699; and 2Departments of Neurology and Anatomy, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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Kreisman, Norman R. and Joseph C. LaManna. Rapid and Slow Swelling During Hypoxia in the CA1 Region of Rat Hippocampal Slices. J. Neurophysiol. 82: 320-329, 1999. The role of swelling in hypoxic/ischemic neuronal injury is incompletely understood. We investigated the extent and time course of cell swelling during hypoxia, and recovery of cell volume during reoxygenation, in the CA1 region of rat hippocampal slices in vitro. Cell swelling was measured optically and compared with simultaneous measurements of the extracellular DC potential, extracellular [K+], and synaptic transmission in the presence and absence of hypoxic depolarization. Hypoxia-induced swelling consisted of rapid and/or slow components. Rapid swelling was observed frequently and always occurred simultaneously with hypoxic depolarization. Additionally, rapid swelling was followed by a prolonged phase of swelling that was ~15 times slower. Less frequently, slow swelling occurred independently, without either hypoxic depolarization or a preceding rapid swelling. For slices initially swelling rapidly, recovery of both cell volume and the slope of field excitatory postsynaptic potentials were best correlated with the duration of hypoxia (r = 0.77 and 0.87, respectively). This was also the case for slices initially swelling slowly (r = 0.70 and 0.58, respectively). In contrast, the degree of recovery of cell volume was the same at 30 or 60 min of reoxygenation, indicating that prolonging the duration of reoxygenation within these limits was ineffective in improving recovery. Spectral measurements indicated that the hypoxia-induced changes in light transmittance were related to changes in cell volume and not changes in the oxidation state of mitochondrial cytochromes. The persistent impairment of synaptic transmission in slices swelling slowly (i.e., without hypoxic depolarization) indicates that swelling may play a role in this injury and that hypoxic depolarization is not required. Additionally, the correlation between the degree of recovery of cell volume and the degree of recovery of synaptic transmission during reoxygenation supports a role for swelling in hypoxic neuronal injury.
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INTRODUCTION |
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Selected regions of the brain, including cerebral
cortex, cerebellum, striatum, and hippocampus, can be damaged by
episodes of ischemia lasting only several minutes (Pulsinelli et
al. 1982; Schmidt-Kastner and Freund 1991
). A
critical factor in the triggering of ischemic brain injury is
hypoxic depolarization (Vysko
il et al.
1972
), which consists of a profound increase in membrane permeability, dissipation of transmembrane ion gradients, and depolarization of cell membranes (Hansen 1985
;
Siesjö 1992
; Somjen et al. 1990
).
The combination of hypoxia, the increased membrane permeability, and
activation of the Na-K pump all contribute to depletion of ATP
(Kass and Lipton 1982
; Lipton and Whittingham 1982
; Siesjö 1992
). Additionally, brain
cells swell because of an associated osmotic influx of water
(Hansen and Olsen 1980
; Hossmann 1971
;
Korf et al. 1988
; Nemoto 1982
). Prolonged
hypoxic depolarization leads to irreversible neuronal damage because of the associated rise in intracellular [Ca2+]
(Choi 1988
; Kass and Lipton 1982
;
Roberts and Sick 1988
; Siesjö 1992
;
Somjen 1990
).
Whereas hypoxic depolarization and elevations in intracellular
[Ca2+] are accepted widely as playing a role in
neuronal injury, the role of cell swelling is more controversial. Some
investigators believe that swelling contributes to brain damage by
compressing brain tissue and blood vessels in the bony cavity of the
skull (see Lutz and Nillson 1994). However, swelling may
play a role in neuronal injury in the absence of elevated intracranial
pressure. Diffusion-weighted magnetic resonance imaging, which measures changes in diffusion of water molecules as an index of cytotoxic edema,
has shown that focal swelling is an important marker of both the extent
of injury and recovery of function after restoration of blood flow and
oxygenation in animal models of global ischemia and reperfusion
(Busza et al. 1992
; Hossmann et al. 1994
;
Minematsu et al. 1992
). Additionally, focal swelling can
serve as a marker of impaired function because both
electrophysiological and metabolic recovery are related spatially and
temporally to recovery of normal volume (Hossmann et al.
1994
). Moreover results of investigations in brain slices,
where intracranial compression is not a factor, suggest that swelling
can contribute directly to irreversible damage (Balestrino
1995
).
Hypoxic cell swelling has been confirmed in brain slices by
measurements of extracellular volume (Hansen and Olsen
1980; Pérez-Pinzón et al. 1995
;
Rice and Nicholson 1991
; Syková et al.
1994
), extracellular resistance (Chebabo et al.
1995
; Jing et al. 1994
), optical properties of
brain tissue (Kreisman et al. 1995a
; Turner
et al. 1995
), and intracellular volume (Melzian et al.
1996
). Increasing the osmolarity of solutions bathing
hippocampal slices with agents such as mannitol delays the onset of
hypoxic depolarization (Balestrino 1995
) and enhances
posthypoxic recovery of orthodromic responses in the CA1 region of the
hippocampus (Huang et al. 1996
). Conversely, prior
hypotonic cell swelling exacerbates the hypoxic injury to synaptic
transmission (Payne et al. 1996
). In the present
experiments, we characterized the extent and time course of cell
swelling during hypoxia and re-oxygenation in the hippocampal slice
preparation. Our rationale for using hypoxia in this preparation is
that neurons most vulnerable to ischemia in vivo are also the first to
be affected by hypoxia in vitro (Dong et al. 1988
;
Somjen et al. 1990
). Swelling and extracellular
potential were compared in the presence and absence of hypoxic
depolarization. Additionally, we investigated the relationship between
recovery of synaptic transmission and recovery from swelling during
reoxygenation. Some of the results have been presented in abstracts
(Kreisman et al. 1994
, 1995b
).
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METHODS |
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Preparation of hippocampal slices
Male Sprague-Dawley rats from Charles River (200-500 g) were anesthetized deeply with ether and perfused through the heart with 60 ml of bathing medium (4°C). The rats were decapitated, and the brain was removed and placed immediately in iced bathing medium with the following composition (in mM): 129 NaCl, 3.5 KCl, 2 MgSO4, 1 NaH2PO4, 2.7 CaCl2, 26 NaHCO3, and 10 glucose (osmolarity was 295-300 mosM/l). The hippocampi were removed from the brain, and 400-µm-thick slices were cut transversely with a tissue chopper (Stoelting, Wood Dale, IL). Slices were incubated in an interface-style holding chamber for a minimum of 90 min at room temperature (23-24°C). The slices then were placed on the nylon mesh of an interface-style recording chamber or, on a few occasions, submersed below another mesh. The bathing medium was maintained at 32-34°C and was pumped through the chamber at 0.6 ml/min. Warmed, humidified 95% O2-5% CO2 flowed over the surface of the bathing fluid at a rate of 480 ml/min.
Electrical stimulation and recording
CA1 pyramidal cells were activated orthodromically by applying
constant current pulses (400 µA; 0.2 ms; 20- to 60-s intervals) to
bipolar stimulating electrodes placed on the Schaffer collaterals. Extracellular field potentials were recorded from either the stratum pyramidale or stratum radiatum with glass micropipettes filled with 150 mM NaCl (tip resistance 5-20 M). Only stable recordings of
population spikes with a minimum amplitude of 3 mV were acceptable. Thereafter, recording electrodes usually were relocated to the CA1
stratum radiatum for recording extracellular DC potentials and
excitatory postsynaptic potentials (fEPSPs). fEPSPs were triggered by
stimulating the Schaffer collaterals with constant current pulses
(10-100 µA; 1 ms) to produce a response that was 50-75% of maximum
amplitude. In some experiments, extracellular resistance was estimated
by measuring the extracellular voltage response (Ve) to a 1-ms constant current
stimulus. Extracellular DC levels and optical signals were recorded
continuously on a stripchart recorder, and evoked field potentials were
recorded on either magnetic tape or computer disk via a MacLab digital
data recorder.
Measurement of extracellular [K+]
Extracellular [K+] was measured from
double-barreled glass pipettes with tip diameters of 2-4 µm
(modified from Kreisman and Smith 1993). The
K+-sensing barrel was filled with 5-10%
tri-n-butylchlorosilane (Pfaltz and Bauer, Waterbury, CT) in
CCl4 and the electrode baked at 180°C for
2 h. Thereafter, the tip was filled with potassium liquid ion
exchanger, IE190 (World Precision Instruments, Sarasota, FL) to a
height of 1-2 mm. The sensing barrel was back-filled with 150 mM KCl,
and the reference barrel was filled with 150 mM NaCl. Extracellular
[K+] and DC potential were measured with an
Axon Instruments Axoprobe 1-A differential amplifier. Calibrations were
conducted in the recording chamber before and after experiments.
Acceptable calibration slopes were 50-58 mV for the 10-fold change in
[K+] between 3 and 30 mM.
Measurement and imaging of light transmittance
White light from a quartz-halogen source was delivered to the
bottom of the recording chamber via a 3.2-mm-diam fiberoptic bundle.
Transmitted light was detected by a silicon photodiode (Ealing, model
78-7821), coupled to one ocular of a Nikon binocular dissecting
microscope via a 6.4-mm-diam fiberoptic bundle. The optical field was
~0.4 × 1.2 mm at ×80 magnification, which included the CA1
stratum radiatum and stratum pyramidale. To calibrate the optical
signal, basal light transmittance (T) was set to 0 V at the beginning
of the experiment, using the offset of a DC amplifier. The dark value
was then determined by shutting off the light source. Variations in
light intensity (T) were calculated as
T/T in percent.
To image light transmittance, the photodiode was replaced with an 8-bit
digital video camera (Dage, model CCD72, Dage MTI, Michigan City, IN).
Black level and dynamic range were set manually, based on a histogram
of light transmittance values obtained from each slice during normoxia.
As with the photodiode measurements, relative changes in light
transmittance (T) were expressed as a percent of the
basal transmittance (T). Images were captured by a
framegrabber every 1-3 s, depending on the experiment, using National
Institutes of Health Image or custom imaging software (Synetic,
Montreal, Canada). Stored images were analyzed off-line. Areas of
interest were demarcated on an image of the slice and sequential values
of
T/T were plotted as a function of time for each area.
Spectroscopy
Spectral measurements were performed by collecting light from the ocular of the Nikon dissecting microscope, via a 3.2-mm fiber optic bundle, to a rapid-scanning spectrophotometer (World Precision Instruments, Sarasota, FL). Wavelength precision was checked by placing filters with narrow optical band-pass in the light path. The optical field for these measurements was a spot ~1.5 mm in diameter. Each reported spectrum is the average of seven raw spectra, each integrated over a 10-s period.
Control and measurement of bath oxygenation
Hypoxia was induced by switching the gas mixture superfusing the
slice from 95% O2-5% CO2
to 95% N2-5% CO2. Graded
levels of hypoxia were produced by proportionally mixing the gases with flowmeters. In several experiments, PO2 in the
bathing medium was measured polarographically using a 250-µm-diam
platinum electrode exposed only at the tip. The electrode was polarized
to 0.7V (relative to an Ag/AgCl wire connected to ground), where its
current output was a linear function of PO2.
Calibrations were conducted at room air and 100%
N2. Mean PO2 at 1 mm below
the surface of the bath was 443 ± 19 (mean ± SE) mmHg
during "normoxia," 98 ± 6 mmHg during "moderate
hypoxia," and 20 ± 3 mmHg during "severe hypoxia"
(n = 9). All hypoxic episodes reported in
RESULTS should be considered as severe hypoxia unless noted
otherwise. Also all data were obtained from interface slices unless
noted otherwise.
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RESULTS |
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Hippocampal swelling occurs at two different rates during hypoxia
Responses to hypoxia and reoxygenation were assessed in the CA1
region of rat hippocampal slices by simultaneously recording the
extracellular potential (DC), light transmittance
(T/T), and occasionally the extracellular
potassium ion concentration ([K+]o). Hypoxia-induced
swelling proceeded at two different rates in hippocampal slices,
hereafter designated as rapid and slow swelling (Fig.
1). Rapid swelling was associated
consistently with hypoxic depolarization, as indicated by the
synchronous occurrence of sharp decrease in
T/T, a rapid negative shift in the
extracellular DC potential, and an abrupt increase in extracellular
[K+], which peaked at levels exceeding 20 mM
(Fig. 1A). In contrast, slow swelling occurred only in the
absence of classic hypoxic depolarization; the DC shift was either
small or absent and increases in extracellular
[K+] were limited in rate and magnitude (Fig.
1B). In five hypoxic slices showing slow swelling,
[K+]o rose from a
normoxic baseline value of 4.3 ± 0.2 mM to a plateau of only
6.9 ± 0.7 mM (P < 0.05 by a one-tailed
Student's t-test) while the DC potential shifted
1.2 ± 1.6 mV. In 10 submerged slices, hypoxia produced only slow swelling
and classic hypoxic depolarization never occurred.
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Rapid swelling occurred in 29 of 44 slices (66%) and was observed commonly in response to either severe or moderate hypoxia. Slow swelling occurred in 15 of 44 slices (34%) and was observed most often in response to moderate hypoxia. These percentages likely underestimate the true probability of rapid swelling and overestimate the true probability of slow swelling because we purposely attempted to enhance the occurrence of slow swelling in many experiments by exposing slices to moderate hypoxia and/or slowing the rate of onset of hypoxia. However, we did not investigate the role of these factors systematically because these manipulations did not guarantee that slow swelling would occur. It should be noted, for comparison, that durations of hypoxia tended to be longer for slices swelling slowly (mean 16.5 ± 2.3 min; n = 14) compared with those swelling rapidly (mean 9.2 ± 1.1 min; n = 22). Hypoxia was imposed usually for longer periods in slices responding with slow swelling because it was difficult to discern whether slow swelling had in fact occurred with shorter durations of hypoxia.
Slices in the rapidly swelling group actually displayed two rates of
swelling, as indicated by curve fitting with a double exponential
function (Fig. 2). The initial rate,
which only lasted 20-60 s, had a time constant (Tau 1) that was ~17
times faster than the second time constant (Tau 2) (Table
1). The peak T/T reached during the initial rate was nearly 12%, whereas the maximum
T/T attained by the end of hypoxia was about
double this value. In contrast, slices in the slow-swelling group
displayed only a single time constant of swelling that was virtually
identical to the second, slower time constant of swelling in the
initially fast swelling slices (Table 1).
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An example of a slice responding with rapid swelling is shown in Fig.
1A. A 6-min episode of severe hypoxia initially induced a
small, slow increase in T/T of 1.6%
(indicating modest cell shrinkage), which was associated with both a
slow rise in [K+]o and a
slow negative shift in the DC potential from baseline. Synaptic
transmission diminished and failed during this early period (arrow a)
then recovered partially (arrow b) but failed again at the onset of
hypoxic depolarization. Rapid swelling accompanied hypoxic
depolarization, indicated by a sharp decrease in
T/T to 27.3% below baseline within 2 min.
Simultaneously, [K+]o
rose to a peak value of 28 mM, and the DC potential shifted
10 mV.
Reoxygenation promptly reversed the direction of
T/T, indicating amelioration of swelling.
After 23 min of reoxygenation, [K+]o already had
recovered to baseline, whereas
T/T returned
only to 12.5% below baseline (i.e., a 54.2% recovery), indicating
that hippocampal cells remained somewhat swollen. Population spikes did
not yet show any recovery from hypoxic blockade.
A typical example of slow hippocampal swelling, induced in this case by
severe hypoxia, is shown in Fig. 1B. Hypoxia initially caused a brief increase in T/T by 2.2%
(indicating modest cell shrinkage) but
T/T
then decreased slowly for the duration of hypoxia, to a nadir of 34%
below baseline at 20 min of hypoxia (indicating cell swelling). As in
the previous example, reoxygenation abruptly suspended swelling. Arrow
a marks the time of synaptic blockade. Synaptic blockade persisted
during hypoxia in this slice, as in all slices responding to hypoxia
with slow swelling. There was no transient recovery as in many slices
showing rapid swelling. Additionally, extracellular DC potential
decreased by 5 mV while [K+]o increased slowly
and moderately. Despite maintained hypoxia, the DC potential recovered
to baseline while [K+]o
remained elevated. Twenty minutes of reoxygenation produced only a
small (32.4%) recovery of
T/T, to a plateau
at 23% below the original baseline. This indicates persistent swelling
after reoxygenation.
In addition to using T/T as an index of cell
swelling, the extracellular voltage response
(Ve) to a 1-ms constant current stimulus was measured in 13 slices. An increase of
Ve (i.e., a decrease in the
volume of the extracellular space) should accompany cell swelling and
be correlated with
T/T.
Ve increased linearly as a function of
T/T (Fig. 3)
with correlation coefficients ranging from 0.69 to 0.95.
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Recovery of hippocampal cell volume during reoxygenation
During reoxygenation, both T/T and
population spike amplitudes often failed to recover fully to normoxic
values. As expected, the degree of recovery of cell volume was
inversely related to the duration of hypoxia. Measurements of changes
in [K+]o, extracellular
DC potential, and light transmittance (
T/T) during two sequential hypoxia-reoxygenation episodes from the same
slice help illustrate this point (Fig.
4). The first episode of hypoxia lasted 3 min, and the second episode lasted 4 min. Depolarization occurred
within 2 min after onset of hypoxia as indicated by the rapid increase
in [K+]o and the
simultaneous negative DC shift. There was a concomitant decrease in
T/T, indicative of cell swelling, which
followed a time course similar to that of the DC trace. Reoxygenation
after 80 s of hypoxic depolarization resulted in recovery and
overshoot of
T/T, indicating a rebound
decrease in cell volume. The second episode of hypoxic depolarization,
which lasted 140 s, produced a larger decrease in
T/T (i.e., greater swelling) than with the previous episode. Little recovery of
T/T
accompanied reoxygenation, indicating persistent swelling of cells in
the slice. Extracellular [K+] also failed to
recover fully in this case.
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Factors contributing to the reoxygenation-induced recovery from rapid
and slow swelling were evaluated by making scatter plots and
calculating first-order regression coefficients (Fig.
5). The degree of recovery of cell volume
from rapid swelling was related inversely to the duration of hypoxia
(Fig. 5A) (r = 0.732; P < 0.001; n = 22), the duration of hypoxic depolarization
(r = 0.640; P < 0.005), and maximal
T/T (r = 0.678;
P < 0.001). Recovery from rapid swelling tended to be
good if the duration of severe hypoxia was <5 min but recovery was
poor if the duration of severe hypoxia exceeded 10 min (Fig. 5,
A and C). Increasing the duration of
reoxygenation failed to enhance recovery (Fig. 5B and Table
2). Likewise, the degree of recovery from
slow swelling was related inversely to both the duration of hypoxia
(Fig. 5C; r = 0.698; P = 0.017; n = 11) and the maximal
T/T attained during hypoxia (r = 0.670; P = 0.020). As with rapid swelling, the degree of recovery from slow swelling was not enhanced by increasing the
duration of reoxygenation (Fig. 5D and Table
3).
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Comparisons were made of the magnitude of hypoxia-induced swelling and
reoxygenation-induced recovery of cell volume in rapidly and slowly
swelling slices that were exposed to equal durations of hypoxia (Table
4). Peak T/T
during hypoxia was significantly greater, and the percent recovery of
T/T during reoxygenation was significantly
lower, in rapidly swelling slices. This comparison should be made with
caution, however, because we often purposely attenuated both the degree
of hypoxia and its rate of onset to enhance the probability of slices
responding with slow swelling.
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Recovery of fEPSPs and population spikes during reoxygenation
Initial slopes of fEPSPs from the CA1 stratum radiatum were
measured in 13 of 22 slices responding to hypoxia with rapid swelling. In all slices, fEPSPs were blocked within minutes of onset of hypoxia.
Recovery of fEPSP slope during reoxygenation was related to recovery of
T/T (Figs. 6 and
7B, and Table 2). The degree of recovery of the initial fEPSP slope was correlated roughly with
recovery of
T/T at 30 min of reoxygenation but
the correlation improved considerably at 60 min of reoxygenation (Table
2). Despite the differences in correlation coefficients, mean
recoveries of fEPSP slope and
T/T were similar
at both 30 and 60 min of reoxygenation (Table 2). Likewise, recovery of
population spike amplitudes and
T/T were
similar to each other both at 30 and 60 min of reoxygenation in five
slices that swelled slowly during hypoxia (Fig.
8 and Table 3). The degree of recovery of
population spike amplitude was related generally to the degree of
recovery of
T/T (compare Fig. 8, A
and B). However, the degree of recovery was less in slowly
swelling slices than in rapidly swelling slices (compare data in Tables
2 and 3), likely because slowly swelling slices were exposed to longer
durations of hypoxia.
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Hypoxic swelling in the various strata of CA1
The largest hypoxia-induced changes in
T/T occurred consistently in the stratum
radiatum, with lesser changes in the strata oriens and pyramidale,
respectively (e.g., Fig. 9, C
and D). This order of swelling was similar to that observed
in response to treatment of the slices with hypoosmotic media (Fig.
9A). Two phases of
T/T often were
evoked by hypoxia in both rapidly and slowly swelling slices: a small
increase in
T/T (shrinkage) followed by a
larger decrease in
T/T (swelling). The extent
of reoxygenation-induced recovery of
T/T in
each layer of CA1 varied in different experiments (e.g., compare
responses in Fig. 9, B-D).
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Spectral transmittance during hypoxia
Transmission spectra were measured from two hippocampal slices during normoxia, hypoxia, isosmotic, and hypoosmotic conditions (Fig. 10). Transmission spectra during two separate episodes of normoxia taken 10 min apart were indistinguishable, indicating stable baseline transmittance over such time periods (Fig. 10A). In the same slice, spectral transmittance at 10 min of hypoxia decreased by as much as 10% compared with the immediately preceding period of normoxia, particularly at wavelengths >525 nm (Fig. 10B). This was nearly identical to the shift in the transmission spectra during hypoosmotic swelling during normoxia (Fig. 10C), produced by decreasing osmolarity of the medium from 295 to 250 mosM/l. There was no evidence of absorbance changes at specific wavelengths characteristic of mitochondrial cytochromes.
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DISCUSSION |
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Rapid versus slow swelling
One of the most important findings of this investigation is that
there are two rates of swelling in the CA1 region of hippocampal slices
during hypoxia. This was confirmed by the best fit of two exponential
functions to the T/T traces in response to
hypoxia (Fig. 2 and Table 1). Fast swelling, as indicated by a profound and rapid change in
T/T, was associated
consistently with hypoxic depolarization, as indicated by a
simultaneous negative shift of the extracellular potential and a sharp
increase in extracellular [K+]. In contrast,
slow swelling occurred either in the absence of hypoxic depolarization
(1/3 of the slices) or subsequent to hypoxic depolarization (2/3 of the
slices). Hypoxic depolarization is triggered by a nonspecific
increase in membrane permeability and the associated efflux of
K+ and influx of Na+,
Ca2+, and Cl
(Hansen and Zeuthen 1981
; Vysko
il et al.
1972
), a permeability change identical to that seen in
spreading depression (Kraig and Nicholson 1978
). Rapid
swelling would be expected to accompany hypoxic depolarization and
spreading depression because the influx of Na+,
Ca2+, and Cl
should
osmotically obligate the influx of water (Hansen 1985
). In conjunction with these findings, the optical responses indicative of
rapid hypoxic swelling observed here are remarkably similar to those
reported during spreading depression in hippocampal slices (Snow
et al. 1983
).
Cell swelling during hypoxia has been inferred from repeated
observations of increases in tissue impedance (Hossmann
1971; Jing et al. 1994
; Korf et al.
1988
), which are consistent with decreases in interstitial
volume. Similar increases in impedance have been measured during severe
hypoosmotic swelling in hippocampal slices (Chebabo et al.
1995
) and during spreading depressions and hypoxic
depolarizations (Jing et al. 1994
). Other investigators measured hypoxia-induced decreases in the extracellular volume fraction
(Hansen and Olsen 1980
; Lundbæk and Hansen
1992
; Pérez-Pinzón et al. 1995
;
Syková et al. 1994
), which were derived from
changes in diffusion of an extracellular space marker (Rice and
Nicholson 1991
). Additionally, tissue swelling during hypoxia
was confirmed recently by measuring changes in the intracellular
concentration of a fluorescent dye (Melzian et al.
1996
). Finally, brain tissue swelling during ischemia has been
measured in situ by magnetic resonance imaging of the apparent
diffusion coefficient of water (Busza et al. 1992
;
Hossmann et al. 1994
; Minematsu et al.
1992
). None of these investigations, however, differentiated
fast versus slow swelling, probably because the time resolution of
their measurements was set to measure only peak changes.
Inferences about a slow process, likely involving slow depolarization
and swelling, can be made from existing reports of responses of neural
tissues to hypoxia. Some tissues, such as spinal cord and peripheral
nerves, respond to hypoxia with slow depolarization and seldom, if
ever, respond with rapid depolarization (Collewijn and Van
Harreveld 1966; Wright 1947
). Fujiwara et
al. (1987)
reported slow transmembrane depolarization of 1-2
mV/min in 50% of the hippocampal neurons they tested during hypoxia.
Membrane potentials at 20 min of hypoxia were depolarized ~25 mV from
the resting level, which would be expected to inactivate voltage-gated
conductances. Additionally, slow depolarization was observed in both
hypoglossal and neocortical neurons during hypoxia (O'Reilly et
al. 1995
). Slow depolarization would be expected to be
accompanied by slow swelling in response to qualitatively the same
ionic fluxes described in the preceding text. This is supported in our
experiments by the temporal association of moderate increases in
extracellular [K+] and a small negative DC
shift with slow changes in both
T/T and
extracellular resistance. A virtually identical correlation between
shrinkage of the extracellular space and a gradual decay of
K+ homeostasis was reported during hypoxia in
submerged striatal slices (Rice and Nicholson 1991
).
Finally, Croning and Haddad (1998)
observed hypoxic
changes in extracellular [K+] in submerged
hippocampal slices that look identical to those recorded here during
slow swelling. Proof of a causal relationship between slow
depolarization and slow swelling would require recording of
transmembrane potentials in conjuntion with optical measurements. The
factors that determine whether fast or slow depolarization and swelling
will occur during hypoxia are unknown but likely involve a variety of
ion channels, and possibly transport mechanisms, in the cell membrane.
Recovery of cell volume and synaptic transmission after rapid versus slow hypoxic swelling
Our results emphasize that incomplete recovery of synaptic
transmission can occur both in slices responding to hypoxia with rapid
depolarization and swelling or slow swelling (and presumably slow
depolarization). Heretofore it generally had been accepted that the
likelihood of permanent block of synaptic transmission after
reoxygenation is related directly to the duration of rapid hypoxic
depolarization and not the duration of hypoxia per se (Balestrino and Somjen 1986). However, these and other
investigators also emphasize that the duration of hypoxic
depolarization is not the only factor involved (Balestrino et
al. 1988
; Rader and Lanthorn 1989
). Our findings
indicate that both duration of hypoxia and duration of rapid hypoxic
depolarization are important factors in failure of synaptic
transmission to recover during reoxygenation. Our results show also
that recovery of synaptic transmission is impaired after slow hypoxic
swelling (and presumably slow depolarization). The correlation between
duration of hypoxia and the degree of impairment on reoxygenation is
similar to that observed for slices swelling rapidly. In support of our
observations, others also have reported persistently impaired synaptic
transmission in the absence of conventional, fast hypoxic
depolarization (Chen et al. 1996
; Croning and
Haddad 1998
; Schiff and Somjen 1987
).
Additionally, neuroprotective agents can work independently of their
effect to delay hypoxic depolarization (Chen et al.
1996
), supporting a role for other factors in impaired
posthypoxic synaptic transmission. Chen et al. (1996)
also suggest that the persistent synaptic blockade during reoxygenation
after slow hypoxic depolarization is more characteristic of the
ischemic penumbra than the ischemic core because slow depolarization
appears most often to be associated with moderate hypoxia.
Relationship of rapid and slow swelling to severity of hypoxia
The tendency of slow swelling to occur most often during more
moderate degrees and slopes of hypoxia suggests that the rate of
swelling is related to the severity of hypoxia. However, the relationship is not a simple one because sometimes severe hypoxia also
led to slow swelling. Additionally, rapid swelling occurred during
either severe or moderate hypoxia. Unfortunately, the unpredictability and relatively infrequent appearance of slow swelling made it difficult
to investigate this relationship systematically in our experiments. In
contrast with our results in interface slices, the few submerged slices
we investigated always responded to hypoxia with slow swelling.
Additionally, classic hypoxic depolarization was never observed in our
submerged slices, in agreement with results of others (Croning
and Haddad 1998). Spot checks of bath PO2
in our submerged slices revealed lower PO2 levels
in normoxia and higher PO2 levels in hypoxia than
in our interface slices. These preliminary observations support the
suggestion that slow swelling is related to milder hypoxia but do not
rule out the possibility that other factors play a role.
Are recoveries of cell volume and synaptic transmission causally related?
Although the correlation between recovery of synaptic transmission
and recovery from swelling observed here suggests a causal relationship, our data neither provide proof nor suggest an underlying mechanism. We speculate that swelling might contribute to posthypoxic depolarization, e.g., through dilution of intracellular
[K+], which would inactivate voltage-gated ion
conductances and depress excitability. In fact, intraneuronal
[K+] is lower than normal after reoxygenation
(Jiang and Haddad 1991; Kass and Lipton
1982
), but some investigators argue that the low intracellular
[K+] during reoxygenation is insufficient to
account for the persistent blockade of excitability (Kass and
Lipton 1982
). Another qualification is that hypoosmotic
swelling alone does not depolarize either hippocampal or neocortical
neurons (Ballyk et al. 1991
; Rosen and Andrew
1990
; Saly and Andrew 1993
), but hypoxic
swelling may have different consequences. Most importantly,
measurements of membrane potentials from neurons exposed to hypoxia of
several min duration show persistent posthypoxic depolarization despite prolonged reoxygenation (Fujiwara et al. 1987
;
O'Reilly et al. 1995
; Rader and Lanthorn
1989
). At minimum, resolution of this issue will require
additional experiments in which simultaneous measurements are made of
all the relevant variables.
Methodological considerations
One potential criticism of the use of light transmittance changes
as an index of hypoxia-induced changes in cell volume is that other
factors might affect light transmittance during hypoxia. The most
likely interference is from changes in the oxidation-reduction state of
mitochondrial cytochromes (Sick and LaManna 1995). There was no evidence of absorbance changes at specific wavelengths characteristic of mitochondrial cytochromes in spectra from our slices.
If these changes occur, they are orders of magnitude smaller than the
volume-related optical signal.
In conclusion, the persistent impairment of synaptic transmission in slices swelling slowly (i.e., without hypoxic depolarization) indicates that swelling may play a role in this injury and that hypoxic depolarization is not required. Additionally, the correlation between the degree of recovery of cell volume and the degree of recovery of synaptic transmission during reoxygenation supports a role for swelling in hypoxic neuronal injury.
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ACKNOWLEDGMENTS |
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We thank F. Kriedt, S.-C. Liao, C.-W. Lin, M. Patel, S. Soliman, and E. Yeh for technical assistance in various phases of the project and Drs. K. Elmslie and G. Schofield for assistance with exponential curve fitting.
This work was supported by National Institutes of Health Grants NS-22077 and HL-42215 to J. C. LaManna and by the American Heart Association-La. Affiliate to N. R. Kreisman.
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FOOTNOTES |
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Address for reprint requests: N. R. Kreisman, Dept. of Physiology (SL39), Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112-2699.
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 13 July 1998; accepted in final form 16 March 1999.
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REFERENCES |
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