1Department of Cell Biology, 2Department of Neurobiology, 3Department of Physics, and 4Department of Surgery (Neurosurgery), Duke University; and 5Durham Veterans Affairs Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Bahar, S.,
D. Fayuk,
G. G. Somjen,
P. G. Aitken, and
D. A. Turner.
Mitochondrial and Intrinsic Optical Signals Imaged During
Hypoxia and Spreading Depression in Rat Hippocampal Slices.
J. Neurophysiol. 84: 311-324, 2000.
During hypoxia
in the CA1 region of the rat hippocampus, spreading-depression-like
depolarization (hypoxic spreading depression or HSD) is accompanied by
both a negative shift of the extracellular DC potential
(Vo), and a sharp decrease in light
transmittance (intrinsic optical signal or IOS). To investigate
alterations in mitochondrial function during HSD and normoxic spreading
depression (SD), we simultaneously imaged mitochondrial depolarization,
using rhodamine-123 (R123) fluorescence, and IOS while monitoring
extracellular voltage. Three major phases of the R123 signal were
observed during hypoxia: a gradual, diffuse fluorescence increase, a
sharp increase in fluorescence coincident with the HSD-related
Vo, primarily in the CA1 region, and a
plateau-like phase if reoxygenation is delayed after HSD onset,
persisting until reoxygenation occurs. Two phases occurred following
re-oxygenation: an abrupt and then slow decrease in fluorescence to
near baseline and a slow secondary increase to slightly above baseline
and a late recovery. Parallel phases of the IOS response during hypoxia
were also observed though delayed compared with the R123 responses: an
initial increase, a large decrease coincident with the HSD-related
Vo, and a trough following HSD. After
reoxygenation, there occurred a delayed increase in transmittance and
then a slow decrease, returning to near baseline. When Ca2+
was removed from the external medium, resulting in complete synaptic blockade, the mitochondrial response to hypoxia did not significantly differ from control (normal Ca2+) conditions. In slices
maintained in low-chloride (2.4 mM) medium, a dramatic reversal in the
direction of the IOS signal associated with HSD occurred, and the R123
signal during HSD was severely attenuated. Normoxic SD induced by
micro-injection of KCl was also associated with a decrease in light
transmittance and a sharp increase in R123 fluorescence but both
responses were less pronounced than during HSD. Our results show two
mitochondrial responses to hypoxia: an initial depolarization that
appears to be caused by depressed electron transport due to lack of
oxygen and a later, sudden, sharp depolarization linked to HSD. The
depression of the second, sharp depolarization and the inversion of the
IOS in low-chloride media suggest a role of Cl
-dependent
mitochondrial swelling. Lack of effect of Ca2+-free medium
on the R123 and IOS responses suggests that the protection against
hypoxic damage by low Ca2+ is not due to the
prevention of mitochondrial depolarization.
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INTRODUCTION |
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In many areas of the CNS, including the
CA1 and fascia dentata regions of the hippocampus, neurons and glial
cells undergo a severe and sudden depolarization when oxygen is
withdrawn (Hansen 1985; Somjen et al.
1993
). Because of its phenomenological similarity to classical
normoxic spreading depression (SD) (Leão 1944
,
1947
), this response to hypoxia has been termed hypoxic SD-like
depolarization or hypoxic spreading depression (HSD). Both SD and HSD
are marked by cessation of neural activity, sudden negative shift in
extracellular potential
Vo, severe
depolarization of neurons and glia, and drastic redistribution of ions
across the membrane. If oxygen is restored in a timely manner, the
tissue can recover with no apparent ill effects, but even after a few
minutes permanent damage can occur, particularly in hypoxia-sensitive
regions such as the CA1 region of the hippocampus. The neural
response to hypoxia is complex and poorly understood, but we do know
that the duration of HSD and, in particular, Ca2+
entry into neurons during HSD, are both important factors in determining later neuronal survival following hypoxia (Somjen et
al. 1993
).
Both SD and HSD are accompanied by changes in the optical properties of
brain tissue slices, usually referred to as intrinsic optical signals
(IOS). The initial response includes a light transmittance increase and
a reflectance decrease, indicating reduced scattering of light. Reduced
scattering is thought to be linked to cell swelling (Aitken et
al. 1999; Kreisman and LaManna 1999
;
Kreisman et al. 1994
; Müller and Somjen
1999
). This is followed by a reversal of the IOS, noted as a
propagating wave of either a sharp drop in light transmittance or
increase of reflectance. This increased scattering moves as a
wave through the CA1 region of hippocampal slices
(Aitken at al 1998
; Müller and Somjen
1999
; Snow et al. 1983
). The onset of the
Vo at any point in the tissue
coincides with the arrival of the leading edge of the wave of increased light scattering. The increased scattering is chloride-dependent but,
unlike the initial decrease in scattering, is not directly related to
cell swelling (Müller and Somjen 1999
).
Müller and Somjen (1999)
have suggested that
this second phase may be linked to swelling of mitochondria.
A role for intracellular calcium
([Ca2+]i) in the neural
response to ischemia/hypoxia is not surprising given its normally tight regulation and its function as a regulator of many intracellular processes. Interstitial calcium
([Ca2+]o) decreases
drastically during SD and HSD, indicating massive uptake into cells
(Hansen 1985; Nicholson and Kraig 1981
).
Withdrawing Ca2+ from brain slices can prevent
lasting damage of the tissue by periods of hypoxia that would be lethal
in preparations maintained in the presence of normal
[Ca2+]o
(Balestrino and Somjen 1986
; Roberts and Sick
1988
). These and similar observations have lead to the
conclusion that Ca2+ entry during HSD and the
resulting cascade of intracellular reactions are critical early events
in acute hypoxic damage (Siesjö 1981
; Somjen et al. 1990
). Other processes are clearly
involved, as hypoxic neurons will eventually die even with
Ca2+ removed from the extracellular medium, but
the influx of Ca2+ during HSD appears to greatly
accelerate neuron damage. The early stages of the response to
hypoxia/ischemia are important to understand to assist with development
of therapeutic interventions.
As the organelles of oxidative metabolism, mitochondria are expected to
be critically involved in the response to hypoxia. Recent reports have
revived the idea that mitochondria buffer cytosolic
Ca2+ elevations (David et al.
1998). Excess Ca2+ uptake into
mitochondria could, however, lead to their injury and therefore
eventually to the demise of the host cell (Crompton 1999
; Gunter et al. 1994
). It has also been
reported that elevated Ca2+ can trigger the
mitochondrial permeability transition (mPT), and it is suggested that
this transition can initiate apoptotic or necrotic cell injury
(Crompton 1999
; Lemasters et al. 1997
). Finally, Ca2+ released from mitochondria has been
blamed for delayed elevation of
[Ca2+]i and consequent
injury to the host cell (Grøndahl and Langmoen 1996
;
Zhang and Lipton 1999
). Given that a rise in
[Ca2+]i is an important
part of the neural response to hypoxia and that mitochondrial function
is affected by [Ca2+]i,
it is possible that the effects of Ca2+ entry
during HSD are mediated, at least partly, via effects on mitochondria.
In this paper, we investigate the temporal and spatial relationships
among mitochondrial depolarization, hypoxia and HSD and SD in
hippocampal slices. Mitochondrial depolarization was measured by
changes in rhodamine-123 (R123) fluorescence, which is highly specific
in tissue culture and isolated mitochondrial studies (Bindokas
et al. 1998; Dubinsky and Levi 1998
;
Duchen 1992
; Grouselle et al. 1990
;
Johnson et al. 1981
). The occurrence of either SD or HSD
was recorded as the sudden negative shift in extracellular potential,
Vo, and was also mapped as changes
in the IOS. We find that an initial slow, diffuse mitochondrial
depolarization is accompanied by a light transmittance increase through
most regions of the hippocampus after the imposition of hypoxia. This pre-HSD phase is then followed by the propagation of a sharp, wavelike
increase in fluorescence associated with the onset of HSD and
subsequent spread into some regions. In stratum radiatum of CA1, the
rapid, wavelike R123 signal is found to coincide with a sharp decrease
in light transmittance and with
Vo.
In some experiments, the mitochondrial depolarization extended into
regions that did not undergo a light transmittance decrease.
Additionally, there was little effect of removing
Ca2+ from the bath on the hypoxic R123
fluorescence response as well as on IOS. Normoxic SD, induced by KCl
injection, also led to an increased R123 fluorescence concurrent with
the IOS but less pronounced than that associated with hypoxia.
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METHODS |
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Slice preparation
Hippocampal tissue slices were prepared from male Sprague-Dawley
rats (60-250 g body wt). The rats were anesthetized with diethyl ether
or fluothane (halothane) and decapitated. The brain was rapidly (1-2
min) removed from the skull and placed in chilled artificial
cerebrospinal fluid (ACSF, composition defined in the following text)
for 1-2 min. One hippocampus was isolated, and transverse 400-µm
slices were cut with a tissue chopper. These experiments were fully
approved by the Institutional Animal Care Committee at Duke University.
After preparation, slices were incubated for 15-20 min in 5 µg/ml
R123 and then transferred to an "Oslo-style" interface chamber in
one of two physiology and imaging workstations. The chambers were
similar in design, were maintained at 35 ± 1oC, were continuously aerated with 95%
O2-5% CO2 (400 ml/min), and were perfused with oxygenated ACSF flowing at a rate of 1.5 ml/min.
After placement in the interface chamber the slices were allowed to
recover for 60 min before viability was assessed by measurement of
evoked potentials in the s. radiatum and s. pyramidale. Hypoxia was
induced by replacing the 95% O2-5%
CO2 atmosphere with 95%
N2-5% CO2, at the same rate.
Solutions
The ACSF contained (in mM) 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 1.2 CaCl2, 1.2 MgSO4, and 10 dextrose (pH 7.4). Calcium-free medium was identical to ACSF except that no CaCl2
was added, the concentration of MgSO4 was 4.8 mM,
and 1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was added to
chelate any residual Ca2+ remaining in the
extracellular space. In low-chloride medium, KCl and NaCl were replaced
with KCH3SO4 and
NaCH3SO4.
R123 staining
For dye staining, slices were incubated in continuously
oxygenated ACSF containing 5 µg/ml rhodamine-123 (Molecular Probes) at room temperature. R123 was diluted 1:2000 from a 10 mg/ml stock in
95% EtOH. Slices were left in the staining solution for 15-20 min and
briefly washed in dye-free ACSF before being placed in the interface
chamber. R123 enters cells and partitions across the mitochondrial
membranes according to the membrane potential. Under normal conditions,
most of the dye is inside mitochondria and, due to self-quenching, the
overall fluorescence is low. When the mitochondria depolarize, dye
leaves the mitochondria and enters the cytoplasm, and reduced quenching
results in greater overall fluorescence. To verify that R123
fluorescence was in fact due to mitochondrial depolarization, we
recorded R123 fluorescence in the presence and absence of carbonyl
cyanide p(trifluoromethoxy)phenyl-hydrazone (FCCP), a highly
effective uncoupler of mitochondrial oxidative phosphorylation
(Heytler 1980). FCCP was administered either through the
bath (1 µM FCCP in ACSF) or by injecting it (100 µM) into the s.
radiatum region of CA1 either in separate slices or after another
manipulation. As also reported by Bindokas et al.
(1998)
, we verified that R123 fluorescence is significantly
enhanced during FCCP treatment. The maximal R123 fluorescence under
these conditions was comparable to, though no larger than, that
observed during prolonged hypoxic conditions. Since the penetration of
both the R123 and FCCP was variable between slices, the absolute values of the fluorescence induced by all of the conditions described here as
well as FCCP differed, so the magnitude of the fluorescence could not
be directly compared. However, the FCCP fluorescent response was
equivalent to that observed during the most severe condition in our
experiments, prolonged hypoxia (for
10 min).
KCl micro-injections
Normoxic SD was triggered with a brief high-pressure (20-60
lb/in2; 20-100 ms) micro-injection of 1 M KCl
into CA1 s. radiatum via a glass micropipette (tip diameter 5 µm)
connected to a Picospritzer (General Valve). In each experiment, the
pulse pressure and duration were adjusted to find the minimal values
capable of evoking reproducible SD in one of the slices for a given
micropipette. The same pulse through the same pipette was then used to
inject KCl into other slices. For data analysis, we used only the first
normoxic SD induced in each slice, with the requirement that full
recovery of the excitatory postsynaptic potential (EPSP) amplitude was obtained during the first 15 min after SD. The recording electrode was
placed in the s. radiatum of CA1 between the KCl injection pipette and
CA3 region at ~500 µm from the injection site.
Voltage recording
Extracellular measurements of evoked and DC potentials were made
using thin-walled, single-barreled borosilicate glass microelectrodes (World Precision Instruments) with a tip resistance of ~5 M. The
electrodes were filled with 150 mM NaCl.
Imaging
Slices in the interface chambers were imaged through either a
Nikon upright microscope (UM-2) with a compound lens (×4) or a Nikon
dissecting microscope (SMZ-U), using a linear, cooled 12-bit
charge-coupled device (CCD) camera. Because of the two different
configurations, epifluorescence was used in one and transmission
fluorescence was used in the other. These two separate imaging
workstations, though similar in most respects, did show minor
differences between some of the imaging and fluorescence results, but
in all significant respects the results were highly consistent. Two
12-bit cameras were used: a Princeton Instruments PentaMAX system, with
1,024 × 512 frame transfer CCD (at 35°C) or a Cooke
Instruments Sensicam, with 640 × 480 interline CCD (at
15°C).
Images were taken once every 1-2 s (50-500 ms exposure for each
image). The images were usually acquired as 2 × 2 binned images,
reducing the effective spatial resolution to either 256 × 256 or
320 × 240 pixels. The direct digital images for both cameras were
transferred to the host computer and stored as 12-bit files. Each
binned pixel corresponded to a slice region of 6-16 µm2 depending on the magnification used.
For imaging of R123 fluorescence, slices were either epi- or trans-illuminated, using excitation light consisting of wavelengths centered at either 485 nm (with a long-pass emission filter 520 nm), or 535 nm (using a long-pass 580-nm emission filter). Both (the longer and shorter wavelength) pairs of excitation/emission filters gave a large fluorescence signal due to the wide bandwidth of R123 excitation. Emitted light was also passed through an infrared filter (cutoff at 700 nm) to eliminate extraneous near infrared light. To assess whether there was intrinsic slice fluorescence (using the preceding wavelengths), unstained slices were imaged in the same manner using hypoxia; no intrinsic slice fluorescence could be detected at the frequencies used for the R123 fluorescence with the intensity of light normally used for excitation. Likewise using both epifluorescence and transmission fluorescence, we assessed whether there was any bleed-through of excitation light past the blocking filters, and this was <1% of our measured signals, using representative light intensities.
For imaging of the IOS, the chamber was illuminated from below using a stabilized xenon arc lamp. The transmission light source was filtered to a narrowband-pass centered at 650 nm to minimize any direct R123 excitation. This combination of light sources allowed alternating images using the same filter system and camera by alternating only the excitation source since the direct transmitted light for the intrinsic optical signal (IOS) determination passed directly through the emission filters for the fluorescence. The optimal transmitted light intensity was ~10-15 times less than the intensity of light used for R123 excitation, again minimizing any unexpected R123 fluorescence contribution to the intrinsic optical signal. Thus it should be emphasized that the alternate measurements of IOS were independent of the R123 fluorescence except that the concurrent intrinsic optical changes in the tissue also influence the direct transmission of the R123 excitation and emission light through the tissue. However, the intrinsic optical signal and R123 fluorescence usually responded in opposite directions (i.e., a decrease in IOS occurring together with an increase in R123 fluorescence), leading to only a small likelihood of significant interaction between the two signals.
Data analysis
Each series of images was analyzed using regions of interest,
generating a series of 12-bit intensity numbers for plotting. Difference images were also calculated for each series, using a control
image as a baseline for each further image in the series: I/I = (Imagen - Imagecontrol)/Imagecontrol.
In these difference images, no difference was scaled as a value of 128, while a decrease showed smaller values (to black) and an increase showed larger values (to white). Data were analyzed for statistical significance using t-tests and ANOVA where needed. Data are
shown as means ± SD.
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RESULTS |
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Short-term hypoxia
PHASES OF RESPONSE TO HYPOXIA.
Overall, three major phases of the R123 signal were observed in
response to hypoxia: a gradual, diffuse fluorescence increase over much
of the slice following the removal of oxygen, lasting several minutes
(pre-HSD phase), a sharp increase in fluorescence coincident with the
HSD-related Vo, occurring primarily
in the CA1 region, but also occasionally in the dentate gyrus (HSD
phase), and a plateau-like elevation that persists for the duration of hypoxia, if reoxygenation is delayed following the onset of HSD (hypoxic plateau phase). Following reoxygenation there were two additional phases: an abrupt initial decrease in fluorescence followed
by a more gradual decrease of R123 signal to near baseline (initial
reoxygenation phase) and a late reoxygenation phase dependent on the
duration of hypoxia, consisting of a slow delayed increase to above
baseline and then a slow recovery toward the rest level. The IOS
response to hypoxia was parallel to the R123 signal response but showed
some differences in time course: an initial increase, before onset of
HSD, coincident with the pre-HSD R123 phase; a large decrease
coincident with the HSD-related
Vo
and R123 HSD phase; and a trough following HSD, with a delayed,
persistent decrease. On reoxygenation a delayed and often overshooting
increase in transmittance occurred, after the R123 signal has returned to baseline, followed by a further late decrease with an eventual return to near baseline, occurring during and after the late
reoxygenation R123 phase. The R123 signal more closely followed the
reoxygenation of the slice tissue while the intrinsic optical signal
responded with a more complex set of phases and more delay following reoxygenation.
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PRE-HSD PHASE. During normal oxygenation and just after onset of hypoxia, no visible changes in either R123 fluorescence or IOS were observed compared with the control images. This baseline is indicated by the uniform gray color throughout the slice in both second images (at 0 and 1 s) and by the baseline values in the plots in Fig. 2. The initial pre-HSD phase of the R123 response began shortly (10-30 s) after the withdrawal of oxygen. This phase consisted of a slow ramp of fluorescence increase occurring widely over the slice (Fig. 1, left, at 180 and 186 s), accompanied by a gradual brightening of the transmitted light signal (right, at 181 and 187 s). In contrast to the diffuse R123 fluorescence, the translucence increase was mostly restricted to the CA1 region (Fig. 1, 181 and 187 s). The average duration of this pre-HSD phase was 162 ± 52 s (n = 26), measured in one of the two interface chambers. Once started, this gradual increase of the R123 signal and translucence continued until either HSD onset or reoxygenation if this was instituted prior to HSD onset (data not shown).
The time course of both the R123 signal and the IOS varied considerably depending on the location of measurement, with CA3 and dentate gyrus (DG) showing considerably different responses from those in the CA1 region, as shown in Fig. 2 for different regions. Figure 2B shows the R123 signal in CA1 s. radiatum by itself, and the arrows point to inflection points for the start of the pre-HSD phase and the onset of the HSD (discussed in the following text). For example, the slope of R123 fluorescence increase during this pre-HSD phase was slightly higher in the CA3 region than in other regions, particularly s. pyramidale in the CA1 region (the slopes are shown in more detail in Table 1, and given as percentage change per second). However, the translucence values were opposite: a lesser rate was noted in the CA3 region compared with that observed in st. pyramidale of CA1. There did not appear to be a correlation between the duration of the pre-HSD phase and the total extent of the increase of either R123 fluorescence or intrinsic optical signal.
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HSD PHASE.
By images 202/203 of Fig. 1, the HSD has started in the CA1 region,
with a marked decrease in the transmission IOS and an increase in the
same region in the R123 signal. The sharp increase in R123 fluorescence
moved as a wave through CA1, in the same direction and generally
overlapping the progressive regions involved with the IOS, as noted in
images 218/219. The increased light scattering indicated by the
decreased transmittance during HSD (on the order of 15%) would be
expected to depress the R123 signal because both the excitation light
and the emitted fluorescence are hindered in their passage through
tissue. In spite of this expected decrease due to the interaction
between the signals, the R123 signal increased significantly in
amplitude (ranging from 32.8 ± 5.7% for CA1 s. radiatum to
22.0 ± 7.7% for CA3 s. radiatum, n = 29 slices),
suggesting that the signal would be even larger if there were no
interference from the intrinsic optical signal and passive transmission
characteristics of the tissue during the peak of the HSD. The peak
values for R123 maximal increase during HSD varied both between slices
(due to variable loading) and between regions, but CA1 s. radiatum
usually showed the most intense response as depicted in Fig. 1. There
was also some difference in the absolute maximal change noted between
the two chambers used for this study, beyond slice loading of R123 and
regional variation. These differences can likely be interpreted as
resulting primarily from the differences in illumination intensity in
the different experimental setups.
INITIAL REOXYGENATION PHASE. Termination of hypoxia led to an abrupt but partial fall of R123 fluorescence in all regions of hippocampal slice, irrespective of the total duration of hypoxia. The example shown in Figs. 1 and 2 indicates the rapid changes occurring in both R123 and IOS immediately following reoxygenation, which occurred directly after the HSD onset. During the first 15 s of reoxygenation, R123 fluorescence decreased more than twofold, indicating a rapid mitochondrial repolarization to the renewed presence of oxygen. At the same time, the IOS continued to decrease slowly in the regions experiencing HSD (CA1) with no direct change in time course. But in those regions in which HSD did not appear (CA3 and DG in Fig. 2), the IOS, which was slightly enhanced during hypoxia, began to recover slowly. The initial rapid reduction of the R123 signal was followed by a slower decrease of fluorescence that lasted roughly until the beginning of IOS recovery, which consisted of a slow return to baseline. At about this time, the DC extracellular potential also started to recover quickly (Fig. 2A). The recovery of IOS in the regions experiencing HSD started with some delay (~30-40 s) after termination of hypoxia (here we describe recovery process after a short-term HSD episode with rapid reoxygenation). Since the R123 signal is reliably linked to restoring oxygen to the tissue, the delay in the IOS likely reflects a recovery process that is ongoing after restoration of oxygen. At the end of this phase, both the R123 and IOS signals were close to baseline values.
LATE REOXYGENATION PHASE. Further recovery included a final, late phase of R123 and IOS responses following hypoxia and then reoxygenation. First, there followed a period of concurrent further decrease in the R123 signal toward baseline, and a rapid increase of the IOS, with an overshoot above baseline noted particularly in the CA1 s. radiatum region (Fig. 2A). Next, there occurred a period of a secondary increase of R123 fluorescence above the baseline level, and secondary slow decrease of the IOS, lasting ~3-4 min. This secondary increase is minor in Fig. 2A and more apparent in Fig. 2C, in the CA1 s. pyramidale region, and was partially dependent on the duration of the hypoxic conditions. In this particular example, there was rapid reoxygenation so the secondary late increase in the R123 signal was minor compared with longer durations of hypoxia. Eventually there was a final recovery to near the rest level of both the R123 and IOS signals, which often required 20-30 min (data not shown). This time for recovery coincided with recovery of synaptic field potentials (not shown).
Variations in short-term hypoxic response
In 5 of 47 experiments we observed a somewhat different pattern of response, which included a large R123 signal in regions that did not show a drop in IOS, particularly in the CA3 region. Images for this less common outcome are shown as a montage in Fig. 3, and the time course of ROIs for the CA1 s. radiatum and CA3 s. radiatum for one such typical experiment are shown in Fig. 4. Both the images and the plots reveal a robust biphasic (gradual pre-HSD increase followed by rapid HSD) R123 signal in both CA1 and CA3, though the expected IOS decrease was not observed in CA3. In the more typical short-term hypoxia responses demonstrated in Figs. 1 and 2, the large, rapid R123 signal present at the start of HSD onset was consistently colocalized with a large, decreasing IOS.
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As discussed in the preceding text, the presence of the IOS decrease (a lessened transmittance through the tissue) likely led to a decline in the overall observed R123 response, but the interaction between the two independent signals could be more complex than just a linear summation. In contrast, the example shown in Figs. 3 and 4 demonstrates a minimal concurrent IOS change together with a large, rapid R123 signal, indicating that the R123 signal clearly does occur in the absence of a large, negative IOS response. Thus the summation in the more typical experiments may approach a linear combination of the two effects, reducing the effective, imaged R123 fluorescence signal to less than the actual generated signal.
Multiple short hypoxic episodes
In some cases, short-term hypoxic treatments (with rapid
reoxygenation after HSD occurrence) were repeatedly applied to the same
slice, though allowing complete recovery of the evoked, field synaptic
potentials between episodes. Recovery was indicated by return to the
control amplitude of the field EPSP, usually occurring 30 min after
onset of the previous HSD episode, associated with full recovery of the
intrinsic optical signal. For such successive hypoxia treatment, the
R123 and IOS responses were very similar (data not shown) across all
trials. We have administered up to six successive short-term hypoxic
treatments in the same slice, but on each trial, there was a slight but
progressive reduction of the duration of the pre-HSD phase without
significant slope changes of either the pre-HSD or HSD phases (data not
shown). For example, comparison of the R123 fluorescence slopes
(pre-HSD and HSD slopes) in s. radiatum of CA1 from pairs of successive hypoxic trials (next/preceding) revealed no significant difference, averaging 1.14 ± 0.32 and 1.00 ± 0.12 for pre-HSD and HSD
phases, respectively (mean ± SD, 21 comparisons in 16 slices;
compare to Table 1). This observed consistency in subsequent short-term episodes is critical when performing multiple hypoxia treatments under
different conditions in the same slice, to be described in the
following text. Typical protocols for such experiments consisted of
alternation of hypoxic trials in control and experimental ACSF
conditions (see following text), and then the pairs of successive responses at different conditions were analyzed. These results confirm
that analysis of successive short-term hypoxic episodes is a valid
scheme to assess treatment effects.
Prolonged hypoxia
The short-term hypoxia conditions described in the preceding text
focused on reoxygenation within 20 s following onset of HSD as
determined by the extracellular recording of
Vo in CA1 s. radiatum. This
paradigm reliably resulted in complete recovery of the field synaptic
potential within 30-60 min. However, after long-term (up to ~10 min)
hypoxia, slices fail to recover electrical function as determined by
the absence of field synaptic potentials even after 1 h of
recovery following HSD occurrence (data not shown). Moreover recovery
of IOS was poor following such prolonged hypoxia. However, R123
fluorescence recovers rapidly after reoxygenation, irrespective of
hypoxia duration
10 min. Plots of typical R123 and IOS responses in
s. radiatum of CA1 during successive short- and long-term hypoxic
treatments applied in the same slices are presented in Fig.
5. With extension of hypoxia beyond the
immediate HSD phase, R123 fluorescence tended to reach a plateau of
increased fluorescence after ~1-3 min, which is designated the
hypoxic plateau phase. This plateau-like level occurred in slice
regions even if there was no direct involvement in HSD propagation.
However, the maximal relative increase and plateau levels of R123
fluorescence were substantially larger in regions that experienced HSD.
This hypoxic plateau level was not necessarily completely stable and could either slowly increase or decrease, depending on the specific region of the slice. The maximal levels for the hypoxic plateau were
slightly higher than those noted for the experiments in which rapid
reoxygenation was performed, in the range of 30-40%, but most of the
R123 response occurred within the first 12-20 s after onset of HSD.
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As shown in Fig. 5, the time course for the rapid decrease in
R123 fluorescence immediately following reoxygenation (initial reoxygenation phase) did not differ between the short and prolonged hypoxic episodes. The presence of the hypoxic plateau of the R123 signal and its maintenance for 10 min of hypoxia without significant decline strongly suggests that there is little bleaching of the R123
dye or loss of the dye from the cells in the hippocampal slice. Also
that the mitochondrial depolarization can rapidly reverse and normalize
almost immediately with reoxygenation indicates the likely sustained
viability of the mitochondria giving rise to the fluorescent signal.
The rapid resumption of mitochondrial function suggests that the
observed mitochondrial depolarization is reversible in spite of
profound mitochondrial depolarization after
10 min of hypoxia and
argues against any form of irreversible mitochondrial alteration. The
rapid normalization of the R123 fluorescence also appears to imply a
real recovery of mitochondrial potential as shown by the ability of a
subsequent (3rd) hypoxia to again cause a large increase in R123
fluorescence of amplitude similar to previous episodes (Fig.
5B). Since the absolute maximum R123 fluorescence does not
appear to wane significantly with subsequent episodes of hypoxia, there
may be minimal actual cell leakage of the dye or bleaching of the dye
with prolonged fluorescence excitation. The rapid reversibility of the
mitochondrial signal in spite of the loss of the field potential with
prolonged hypoxia also suggests a dissociation between conditions
suitable for mitochondrial survival and repolarization versus those
necessary for neuronal survival and recovery of electrical activity.
In contrast to the rapidly reversible R123 response with prolonged
hypoxia, both the extracellular field potential and IOS showed much
less reversibility and recovery (Fig. 5). There was a large, abrupt
extracellular shift (25-mV peak) associated with the first two HSD
events shown in Fig. 5. However, after the second HSD, there was no
recovery of the field potential, and the peak extracellular shift on
the third HSD was only
8 mV and exhibited a much slower time course.
This lack of recovery was mirrored in the intrinsic signal as well
since the third HSD began on a much more negative level than the
previous two events.
Also, with the longer episodes of hypoxia duration, there appeared a much more pronounced secondary increase of R123 fluorescence during the late reoxygenation phase (Fig. 5). As shown in Fig. 5B, there is only a minimal late secondary increase of the R123 signal with the short hypoxia (to the left), whereas with the prolonged hypoxia there is much larger secondary increase (middle response). This late increase in R123 fluorescence may be related to hyperoxidation occurring with mitochondrial overactivity during the recovery phase, following reoxygenation. The late increase of R123 fluorescence was also accompanied by a further decrease in IOS, suggesting profound tissue changes following this severe, prolonged hypoxia. However, in this series of experiments, we focused on the short-term changes with hypoxia, and further experiments will be required to determine whether or not a clear relationship exists between hypoxia duration and the amplitude of the late R123 elevation during the late reoxygenation phase. This relationship may clarify the time course of late mitochondrial and neuronal damage following reoxygenation.
Hypoxia in Ca2+-free medium
Hippocampal slices have been previously found to recover from
prolonged (9 min) hypoxia (and HSD) experienced in
Ca2+-free media, whereas slices in medium with
normal Ca2+ concentration failed to recover after
a similar prolonged hypoxia (Balestrino and Somjen
1986). It has also been demonstrated that high concentration of
free cytosolic Ca2+ can induce mitochondrial
depolarization in neurons (Dubinsky and Levi 1998
;
Loew et al. 1994
). These findings suggest that the
likely large influx of extracellular Ca2+
associated with hypoxia and HSD might be in part responsible for the
induction of the observed profound mitochondrial depolarization. To
prevent the large Ca2+ influx, hypoxia
experiments were performed as described in the preceding text, with
imaging of R123 fluorescence and IOS but in
Ca2+-free medium (no Ca2+
and 1 mM EGTA added). To assess whether the media had the expected physiological effect, we routinely monitored extracellular synaptic responses in the CA1 region; the Ca2+-free medium
reliably resulted in complete loss of the synaptic signal within 5-10 min.
We compared R123 responses during successive HSD episodes induced with hypoxia first in normal and then in Ca2+-free ACSF in the same slices (n = 3 experiments). The time to onset of HSD did not change significantly in the Ca2+-free media, and the actual HSD appeared similar electrophysiologically. In addition, Ca2+ withdrawal did not lead to any significant change of the R123 signals, including maxima or slope of the initial and HSD phases (see Fig. 6). Since Ca2+ influx is markedly reduced in these conditions, these results imply that most of the mitochondrial depolarization observed is not directly dependent on an elevation of Ca2+ via Ca2+ influx. However, release of Ca2+ from internal Ca2+ stores could potentially account for at least a partial Ca2+ induction of the mitochondrial depolarization. Additionally the lack of oxygen for mitochondrial respiration may be the key factor leading to the mitochondrial depolarization during the peak of the HSD rather than necessarily Ca2+ influx.
|
Hypoxia in low-chloride medium
It has recently been shown (Müller and Somjen
1999) that the sharp increase in light scattering associated
with hypoxic spreading depression (the IOS described here) is dependent
on [Cl
]o. Mechanisms
other than cell swelling may account for the complex series of IOS
changes described with hypoxia, particularly the anomalous signal with
HSD. Mitochondrial (or other organelle) swelling is another possible
mechanism. Since mitochondrial swelling can be associated with
depolarization of the mitochondrial inner membrane, we have studied the
R123 response during HSD induced in low Cl
medium. Figure 7 shows plots of R123 and
IOS response as measured in s. radiatum of CA1 successively under
control (A) and low-chloride (B) conditions in
the same slice. The slope of the sharp increase in R123 fluorescence
(at HSD onset) was significantly reduced when hypoxia was applied to
the same slice, but in medium containing only 2.4 mM
Cl
(as CaCl2) (Fig.
7B). The data from paired (normal/low
Cl
) hypoxic trials revealed that the R123 HSD
slope was 3.14 ± 1.78 (n = 8 trials in
n = 5 slices) times steeper in normal compare to low
Cl
ASCF (P < 0.01), but the
same calculation for pre-SD slopes
1.05 ± 0.65 did not realize
significance between the two conditions. In agreement with the finding
of Müller and Somjen (1999)
, the sharp decrease in
light transmittance with HSD onset was abolished in the low-chloride
condition, instead demonstrating a large and rapid increase in
transmittance. However, the pre-HSD phase of the IOS persisted
unchanged, indicating that this phase (and possibly also the early and
late reoxygenation phases) was unaffected by the
low-Cl
media, in contrast to the HSD phase
reversal. The significant diminution of the R123 HSD response and
reversal of the IOS during the HSD phase strongly suggests that the two
signals are closely linked to Cl
flux, whereas
the other phases are less affected and may be relatively independent of
Cl
flux, either across the cell membrane or the
mitochondrial membrane. Alternatively, the low
Cl
condition may in some manner protect
mitochondria, leading to the attenuated slope and reduced maxima in
this situation.
|
R123 fluorescence during normoxic spreading depression
Intrinsic optical changes have been observed in tissue
experiencing normoxic SD as well as hypoxia and HSD (Aitken et
al. 1998, 1999
; Basarsky and Feighan 1999
;
Basarsky et al. 1998
; Obeidat and Andrew
1998
; Snow et al. 1983
). Also, normoxic SD and
HSD are similar in electric and ionic responses. To assess the relative degree of mitochondrial stress, we imaged R123 fluorescence and light
transmittance during normoxic SD induced by micro-injection of KCl. A
typical experiment is shown in Fig. 8 as
subtracted images and in Fig. 9 as plots
of percentage fluorescence change in various ROIs. Micro-injection of 1 M KCl into s. radiatum of CA1 region evoked a propagating wave of SD
accompanied by intense IOS and R123 changes, heralded by the sudden
negative shift in extracellular potential
(
Vo) (n = 12). The
R123 maximal response averaged 10.2 ± 3.1% in CA1 s. radiatum,
significantly less than the maximal response noted during HSD. The SD
typically propagated through the most of CA1 region but failed to
spread beyond CA1, stopping abruptly at both the CA3 and subicular
border (Aitken et al. 1998
). As analyzed near the
recording electrode location, both optical signals changed
simultaneously with
Vo (see Fig. 9A). R123 fluorescence showed a rapid increase in intensity,
following first a brief increase in the IOS signal and coincident with
a rapid decrease in IOS. In 11 of 12 experiments, the decrease in IOS
was preceded by a brief initial (2-4 s) increase, also reported in
earlier works (Aitken et al. 1999
; Basarsky et
al. 1998
). Because optical data were limited by the accuracy
permitted by a 2-s inter-image interval for the same parameter (IOS or
R123 fluorescence), we cannot reliably determine whether the initial
IOS increase truly anticipates the slightly delayed increase in R123
fluorescence. Interesting, the maximal increase in R123 fluorescence,
decrease in IOS and decrease of Vo
occurred almost simultaneously. The discrepancy between peak times for
the various signals averaged 1.1 ± 3.6, 0.8 ± 5.1, and
1.9 ± 3.9 s for IOS and Vo,
R123 fluorescence and Vo, and R123 and
IOS, respectively (n = 12; CA1 s. radiatum, near the
recording electrode location) and 0.2 ± 4.2,
0.6 ± 6.4, and
1.7 ± 3.0 s for IOS and R123 fluorescence, as measured
in s. radiatum, s. pyramidale, and s. oriens, respectively.
|
|
Detailed analysis of IOS and R123 fluorescence changes revealed that these parameters did not spread uniformly in all subregions of CA1 (Fig. 9). During SD phase, the reduction in IOS (calculated from the amplitude of the initial increase) and the rise in R123 fluorescence were maximal in s. radiatum and s. oriens but minimal in s. pyramidale. The extent of the IOS decrease and R123 fluorescence increase in s. pyramidale varied significantly from slice to slice, and we found that this discrepancy was likely due to the orientation of the cell body layer with respect to the axis of imaging (that is, perpendicular to the slice surface). Because the width of s. pyramidale is much less than the overall slice thickness (~40 µm compared with 400 µm), the optical signal from this layer cannot be completely isolated from the signals of adjacent tissue, particularly if the s. pyramidale is not perpendicular to the slice orientation. If the s. pyramidale is slanted with respect to the slice cut, then imaging along the optical axis will contaminate the s. pyramidale signal with that from adjacent layers. However, the example shown in Figs. 8 and 9 does illustrate the situation where the axis of the cell body layer is parallel to the imaging axis. Here the initial rise in translucence in s. pyramidale was not followed by a decrease as appeared in s. radiatum and s. oriens but instead was maintained at a high level during all SD phases. The recovery phase also differed for the response in CA1 st. pyramidale with a much slower return to baseline than for the dendritic layers. In most other examples, the s. pyramidale response is mixed with that of adjacent layers due to the slant of the layer through the tissue slice.
Recovery began almost simultaneously for optical and electrical signals
but proceeded in a more delayed and more complex fashion for IOS and
R123 fluorescence than for the extracellular potential. Although
Vo was completely restored within
~30-50 s following the peak of the SD, final restoration of IOS and
R123 fluorescence was not observed until 5-10 min later. According
to the time course and character of changes (similar to hypoxia
experiments) several phases of the IOS and R123 fluorescence response
with SD can be distinguished, including the SD phase, similar to the HSD phase described above for hypoxia. During the SD phase, there is a
rapid increase in the IOS, followed by a rapid decrease to below the
control level, as described in the preceding text. The initial recovery
phase after the peak of the SD can be characterized as a secondary fast
IOS increase to about the same level as was reached during initial SD
peak, concurrent with partial recovery of R123 fluorescence to the
level of ~30% of the maximal response during the SD phase (best
observed in Fig. 9, B and D). Duration of this
stage did not exceed 1 min. During the next recovery phase, the IOS
decreased to values lower than the rest level. The R123 signal often
showed a gradual late increase at this point. The extent of this late
R123 fluorescence increase strongly correlated with the degree of
simultaneous IOS decrease and was most pronounced in s. pyramidale.
This phase lasted ~1.5-2 min. Final restoration occurred when both
IOS and R123 fluorescence recovered to the rest levels, which often
extended for 10-12 min, coincident with recovery of the synaptic field
potential (not shown). It is important to note that complete recovery
of optical and electrical signals occurred after normoxic SD episodes
in all experiments (data not shown).
During SD, less mitochondrial depolarization is observed in the cell body layer than in the dendritic layer. However, R123 fluorescence does rise in the post-SD period. The correlation between R123 fluorescence increase and IOS decrease in different regions suggests a definite relationship between the IOS decrease and mitochondrial depolarization during SD. Note, however, that the presence of SD alone generates a mitochondrial depolarization that is small in contrast to that induced by the metabolic stress of hypoxia.
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DISCUSSION |
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We have demonstrated that mitochondrial membrane potential, measured via R123 fluorescence, and the intrinsic optical signal exhibit multiple phases when hippocampal slices are temporarily made hypoxic. The pre-HSD phase represents a progressive loss of mitochondrial function, leading to depolarization, in response to hypoxia. The HSD phase is similar to that observed during normoxic SD. The R123 fluorescence signal is minimally affected by a lack of external Ca2+, whereas decreased external chloride concentration leads to a profound blunting of the R123 response and a reversal of the intrinsic optical signal. Even with extended hypoxia from which slices do not recover electrophysiologically, the R123 fluorescence rapidly recovers on reoxygenation, though with an enhanced later response, suggestive of reoxygenation stress. These various aspects will be discussed in further detail.
Phases of R123 fluorescence and intrinsic optical signal response to hypoxia
Hypoxia of brain tissue in vitro proceeds in three epochs.
1) First the membrane potential of many but not all neurons
hyperpolarizes due to increased membrane potassium conductance, and
synaptic transmission is depressed because voltage-gated
Ca2+ channels fail to open in presynaptic
terminals (Hansen 1985; Young and Somjen
1992
). 2) This initial epoch is followed by
SD-like depolarization, or HSD. Initially, this state, however
drastically outside normal physiological limits, is still completely
reversible (Hansen 1985
; Somjen et al.
1993
). 3) If hypoxia continues, neurons become
irreversibly injured. Cells can be irreversibly damaged without
undergoing HSD, but the demise of cerebral neurons is greatly
accelerated by the massive entry of Ca2+ ions
during HSD. This is shown by the fact that withholding
Ca2+ from the bathing fluid can greatly extend
the period of revocability (Balestrino and Somjen 1986
;
Roberts and Sick 1988
) even though HSD is not prevented.
Yet the influx of Ca2+ does not kill neurons
instantly; there seems to be a critical minimal period during which
high [Ca2+]o must act
before it endangers neuron survival.
The data presented in this paper speak to the pathophysiological
processes occurring during the first two epochs of hypoxia, which
correspond to the five phases of R123 fluorescence and intrinsic optical signals we have described. During the first phase (and epoch 1, prior to HSD onset), we have observed a diffuse, slow initial
brightening of the R123 signal over wide areas of the tissue slice,
indicating gradual depolarization of mitochondria. This was followed by
a sudden increase in R123 fluorescence that spreads as a wave over the
tissue, the onset of which at different sites coincided with the onset
of a decrease in light transmittance and of the SD-like
Vo (the HSD epoch or phase 2).
Heterogeneity of IOS responses during SD in different regions of
hippocampal slice has also been described in earlier works
(Aitken at al. 1998
; Basarsky et al.
1998
; Kreisman and LaManna 1999
; Obeidat and Andrew 1998
).
During normoxic, KCl-injection-induced SD, we have observed a similar
sudden, wave-like increase in R123 fluorescence, concomitant with
Vo, and a decrease in light
transmittance but without the preceding slow fluorescence increase and
quantitatively exhibiting a lower level of R123 fluorescence. This
suggests that the initial R123 brightening early in hypoxia represents
mitochondrial depolarization that is specific to diffuse hypoxic
stress, while the sharp, wave-like increase of the R123 signal is
directly related to both normoxic and hypoxic SD and the resulting
profound metabolic demand imposed on mitochondria for ion pumping
needs. This stress would be expected to be much greater with HSD since
the tissue has already experienced a profound depletion of oxygen prior
to the onset of HSD. The association of these two signals is further
underscored by the suppression of both the decrease in light
transmittance and the sharp increase of the R123 fluorescence in
Cl
-free medium.
We observed a much closer correspondence of the R123 and IOS signals during normoxic SD than in hypoxic SD. This finding is not surprising given the difference between the two conditions. In normoxic SD, the mitochondria are likely responding to a single stress, namely the redistribution of ions that is caused by SD. Since the IOS changes are also caused, directly or indirectly, by this ionic flux, one would expect a good temporal correlation between the two measures. In hypoxia, however, things are much more complex. Mitochondria are responding not only to ionic fluxes but also to oxygen deprivation, redox state changes, and other changes that are a part of hypoxic but not of normoxic SD. Therefore lack of a close temporal correlation between the IOS and R123 signals is to be expected.
In early reports on mitochondrial volume changes, it has been asserted
that isotonic mitochondrial swelling can take place if the medium
contains "permeant anions" (e.g., Azzone et al. 1976a,b
, and previous work quoted by these authors). It
is not clear how the substitution of methylsulfate for chloride affects the resting anion composition of the cytosol, but it undoubtedly prevents the massive influx of Cl
during HSD.
For this reason, the chloride dependence of the light scattering
increase during HSD seemed to suggest that it may be due directly to
the swelling of mitochondria (Müller and Somjen 1999
). Clearly, mitochondrial swelling can be only part of the mechanism of IOS changes, as evidenced by the frequent discrepancies between the IOS and R123 signals in our experiments. It is important to
keep in mind the fact that R123 measures mitochondrial membrane potential, not swelling, and while swelling and depolarization are
often closely correlated, they are not the same thing. Mitochondrial depolarization is frequently, though not invariably, associated with
mitochondrial swelling. Thus the chloride dependence of the sharp,
wave-like increase of the R123 signal (phase 2) agrees with the idea
that it, as well as the transmittance decrease of the IOS, is in some
way related to mitochondrial swelling. This conclusion is further
supported by the distribution of the IOS and R123 signals, both of
which are much more pronounced in regions rich in mitochondria, such as
the neuropil, than in mitochondria-poor layers, as s. pyramidale
(Bindokas et al. 1998
; Nafstad and Blackstad 1966
). Yet the sharp increase of R123 fluorescence sometimes
could invade areas that did not demonstrate an IOS, and which are
relative resistant to HSD (shown in Figs. 3 and 4) (Balestrino
et al. 1989
; Basarsky et al. 1998
;
Kawasaki et al. 1990
). It seems that, while SD-like
depolarization invariably leads to severe depolarization of
mitochondria, mitochondrial depolarization can occur without the
SD-like change and, inferentially, without mitochondrial swelling. A
problem with this train of thought is the well-known fact that in
suspensions of isolated mitochondria, swelling of these organelles is
accompanied by decreased light scattering (increased transmittance) (Raaflaub 1953
). Our suggestion rests on the assumption
that within cells, the refractive index of the mitochondria may differ
from that of the cytosol, in which case an expansion of their surface would increase scattering. Clearly this issue is not settled. Among
other reasons for changes in tissue scattering and light transmittance
may be changes in the aggregation of macromolecules. Thus while there
are several possible mechanisms for the discrepancy between the
increased light transmittance noted during the early phase of hypoxia
and the profound decreased transmittance observed during the peak of
HSD and normoxic SD, the swelling of intracellular organelles may be an
important aspect of these SD-related signals.
There is ample evidence for a correlation between the duration of HSD
and hypoxic neuron injury (Adachi et al. 1998;
Balestrino 1995
; Balestrino and Somjen
1986
; Balestrino et al. 1999
; Lee and
Lowenkopf 1993
; Tombaugh 1994
; Watson and
Lanthorn 1995
). The uptake of Ca2+ into
neurons is a critical event in the injury (Balestrino and Somjen
1986
; Roberts and Sick 1988
; Siesjö
and Bengtsson 1989
). Since elevated
[Ca2+]i triggers the
mitochondrial permeability transition (mPT) and this transition
depolarizes mitochondria and impairs their function (Crompton
1999
; Gunter et al. 1994
; Lemasters et
al. 1997
; Loew et al. 1994
), we have asked
whether the influx of Ca2+ is required for the
sharp increase of R123 fluorescence. The answer to this question was
negative: withdrawing Ca2+ from the preparation
did not prevent the sudden mitochondrial depolarization. While our data
do not address the question of neuronal survival, it seems that the
injury by excessive intracellular Ca2+ is not due
directly to mitochondrial depolarization but rather to the catalysis of
some other biochemical process leading to cell autolysis
(Siesjö 1981
; Siesjö and Bengtsson
1989
).
However, there is a discrepancy between prolonged, irreversible hypoxia
(in terms of slice recovery) and the rapid repolarization of
mitochondria with reoxygenation, suggesting that mitochondria may
remain viable far beyond neuronal survival. In addition, the late
increase in R123 fluorescence (late reoxygenation phase) may represent
a form of reoxygenation damage to mitochondria (Leyssens et al.
1996). This type of mitochondrial stress evident on
reoxygenation has also been attributed to hyperoxidation since the
rapid return of oxygen may in itself damage electron transport in
mitochondria through overactivation (Pérez-Pinzón et
al. 1998
). However, the appearance of a small secondary
increase in R123 fluorescence even after KCl-injection-induced SD
suggests that there may be other explanations as well. Qualitatively,
the degree of the late reoxygenation elevation in R123 fluorescence
appeared to be correlated with the duration of hypoxia, particularly
following HSD, and further studies may be helpful in assessing this
potential correlation.
Does the large R123 change represent a mitochondrial permeability transition?
It is not clear whether the sharp increase of R123 fluorescence
represents mPT or some other mechanism of mitochondrial depolarization. The opening of the permeability pores can be a prelude to irreversible injury (Gunter et al. 1994; Lemasters et al.
1997
), yet the hippocampal slices do recover
electrophysiological function after short periods of HSD and the
associated large change in R123 fluorescence. Recently it has become
clear, however, that transient, low-conductance mPT can also occur
during normal function (Ichas et al. 1997
). Likewise
even after prolonged hypoxia, there is still a rapid decrease in R123
fluorescence with reoxygenation, indicating that mitochondria can still
sequester R123 during and/or after repolarization and arguing against
any form of irreversible mitochondrial injury. Moreover,
Müller and Somjen (1999)
found that the
administration of cyclosporin-A did not prevent the SD-like IOS
response (though there may be some doubt about permeation of the
cyclosporin into slices). None of these observations are completely
decisive, but, for now, it seems more probable that the SD-related
sharp depolarization of mitochondria is due to some mechanism other
than the opening of the permeability transition pore. For example, loss
of oxygen as a critical mitochondrial substrate may in itself serve to
cause the observed depolarization, and this explanation would be
consistent with the observed rapid recovery of the R123 fluorescence
signal with reoxygenation.
Usefulness of R123 fluorescence in slices
Although slices exhibited variable loading of the R123 dye, the qualitative response and phases we have described were consistent between slices. Clearly loading of mitochondria in slices represents both those from both glial and neuronal elements, and single-cell studies will be required to assess the relative contribution of individual cell types to these phases of hypoxia-induced signals. The fluorescence was robust and was detected using both epifluorescence and transmission fluorescence techniques, and with a range of excitation/emission filter sets (though still appropriate to the dye wavelengths). Surprisingly, there appeared to be neither significant bleaching or fade with prolonged signal elevations and there was minimal leaching from cells with mitochondrial depolarization. The R123 signal was appreciably more rapid in response than the IOS, which usually was delayed following a sudden tissue change, such as with SD occurrence. The fluorescence responded appropriately to critical control conditions, particularly FCCP, so mitochondrial localization similar to that of other in vitro preparations, such as tissue culture, appears an appropriate assumption. Thus R123 shows a rapid response and a high degree of fluorescence and appears to be specific to mitochondrial depolarization, from critical previous studies of mitochondrial function. However, the linkage between R123 fluorescence and mitochondrial membrane states, such as the permeability transition, remain unclear, and further types of mitochondrial-specific dyes may be needed to more fully delineate the presence or absence of such a large transition with HSD and SD occurrence.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Michael Müller for suggestions and a critical reading of the manuscript.
This work was supported by National Institutes of Health Grants NS-18670 and AG-13165 and a Veterans Affairs Merit Review Award.
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FOOTNOTES |
---|
Address for reprint requests: D. A. Turner, Box 3807, Neurosurgery, Duke University Medical Center, Durham, NC 27710 (E-mail: dennis.turner{at}duke.edu)
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 15 November 1999; accepted in final form 30 March 2000.
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REFERENCES |
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