Intracellular calcium and survival of tadpole forebrain cells in anoxia
1 Department of Anesthesia, University of California, San Francisco, CA
94143-0542, USA
2 Department of Biological Sciences, California State University, Hayward,
Hayward, CA 94542-3083, USA
* Author for correspondence at address 2 (e-mail: mhedrick{at}csuhayward.edu)
Accepted 7 December 2004
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Summary |
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Key words: cell calcium, amphibian, Rana catesbeiana, neuroprotection, anoxia, Fura 2-FF, BTC, Fura-AM, propidium iodide
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Introduction |
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In contrast to the situation in mammals, some species, such as the
freshwater turtle (Trachemys scripta and Chrysemys picta),
are able to tolerate extended periods of anoxia
(Hochachka and Lutz, 2001;
Bickler and Donohoe, 2002
;
Bickler, 2004
;
Lutz and Nilsson, 2004
). One
adaptation of turtle neurons to survive anoxia is the ability to maintain
ionic homeostasis and limit increases in [Ca2+]i during
prolonged anoxia (Bickler,
1998
). Turtles subjected to anoxia for several weeks exhibit
moderate and sustained increases in neuronal [Ca2+]i
(Bickler, 1998
), and this
appears to be linked to anoxia-induced suppression of NMDA receptors
(Bickler et al., 2000
). This
pattern is also seen in hypoxia-tolerant neonatal mammalian neurons
(Bickler and Hansen, 1998
).
Thus, studies in mammals and turtles suggest there may be common mechanisms of
neuroprotection in vertebrate neurons that involve moderate increases in cell
calcium.
There is much less known about the cellular mechanisms that allow other
vertebrates to survive periods of severe hypoxia and anoxia. A number of
studies indicate that frogs are intermediate in anoxia tolerance relative to
anoxia-intolerant mammals and anoxia-tolerant turtles. For example, Ranid
frogs survive only a few days of anoxia at low temperatures
(Hutchison and Dady, 1964;
Christiansen and Penney, 1973
;
Lillo, 1980
;
Stewart et al., 2004
) and
about 3 h of anoxia at room temperature
(Lutz and Reiners, 1997
;
Knickerbocker and Lutz, 2001
).
During anoxia at room temperature, brain ATP levels slowly decrease and when
ATP falls to about 35% of normoxic levels, ionic homeostasis is no longer
maintained and there is a slow increase in extracellular K+
([K+]o)
(Knickerbocker and Lutz,
2001
). If anoxia is continued, this slow increase of
[K+]o is followed by a sharp increase in
[K+]o and release of the amino acid neurotransmitters
GABA and glutamate. Thus, the `slow death' of the anoxic frog brain exhibits
all the hallmarks of acute energy failure, similar to that seen in mammals,
but on a significantly extended time scale
(Wegner and Krause, 1993
;
Lutz and Reiners, 1997
;
Knickerbocker and Lutz, 2001
;
Lutz and Nilsson, 2004
).
Although it is not known whether large increases in cell calcium are
correlated with the limits of survival in the anoxic frog brain, total plasma
calcium concentration triples after 4 days of anoxic submergence in the frogs
Rana catesbeiana and Rana pipiens
(Stewart et al., 2004
). An
additional observation on the hypoxia tolerance of amphibians is that tadpoles
have a greater hypoxia tolerance than do adult frogs
(Bradford, 1983
;
Crowder et al., 1998
). This is
consistent with the general observation that developing animals are more
hypoxia tolerant than their adult counterparts (e.g.
Duffy et al., 1975
).
There is virtually nothing known about the cellular mechanisms underlying the increased hypoxia tolerance of larval amphibians, nor have any studies examined the effects of hypoxia on [Ca2+]i in amphibian neurons. Given that survival of anoxic neurons appears to be enhanced with moderate increases of [Ca2+]i, we sought to examine the relationship between [Ca2+]i and survival of tadpole forebrain cells during anoxia. Specifically, we wished to test the hypothesis that moderately large increases in [Ca2+]i may be associated with prolonged, but survivable, hypoxia in tadpole brain cells. To test this hypothesis, we measured cell survival and [Ca2+]i during anoxic exposure (up to 18 h) on a mixed population of cells (neurons and glia) acutely dissociated from tadpole forebrain tissue.
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Materials and methods |
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Isolation of tadpole forebrain cells
In each experiment, the forebrain tissue from 2-3 tadpoles was used to
yield a sufficient number of cells for study. Isolation of forebrain cells
followed a modified procedure described previously for isolation of
sympathetic ganglion neurons in adult frogs
(Selyanko et al., 1990).
Briefly, tadpoles were anesthetized in ice-cold tapwater (5-10°C) that had
been bubbled with 2-2.5% isoflurane (balance O2) for 15-20 min (cf.
Firestone et al., 1993
).
Tadpoles were placed in the cold tapwater until all movements ceased (ca. 10
min). The animal was removed and decapitated. The forebrain was exposed by
opening a hole in the skull with a pair of sharp iris scissors, removed and
placed in cold artificial cerebrospinal fluid (aCSF). The composition of aCSF
was (in mmol l-1): NaCl, 104.0; KCl 4.0; MgCl2, 1.4;
NaHCO3, 25.0; CaCl2, 2.4; glucose, 10.0
(Winmill and Hedrick, 2003
).
Removal of the forebrain usually required less than 2 min to complete.
Forebrain tissue was minced finely with sharp scissors in cold aCSF and then
placed in trypsin (1 mg ml-1) for enzymatic digestion at room
temperature for 1 h. Following this, brain tissue was triturated 2-3 times
with a fire-polished glass pipette in a neuroprotective external solution
containing no Mg2+ or Ca2+
(Selyanko et al., 1990
).
Tissue was centrifuged for 1 min at approximately 3000 g and
the tissue pellet resuspended in aCSF. Aliquot samples of the triturated
tissue suspension (150-200 µl) in aCSF were placed onto 10 mm coverslips
pre-coated with Cell-Tak (Collaborative Research, Bedford, MA, USA) and left
undisturbed for 1 h at room temperature to allow cells to adhere to the
substrate.
Assessment of cell death
Cell viability was assessed by propidium iodide (PI; Molecular Probes,
Eugene, OR, USA) fluorescence. Propidium iodide, a highly polar fluorescent
molecule, penetrates damaged plasma membranes and binds irreversibly to DNA.
The bound PI fluoresces while the unbound PI does not fluoresce. Excitation
light was 490 nm and emission was at 590 nm. Prior to viability
determinations, cells were incubated with 1-5 µmol l-1 PI for
15-30 min. Determination of the percentage of living and dead cells was made
by counting the total number of cells exhibiting PI fluorescence. For cell
viability measurements with PI, the percentage of live cells relative to the
total number of cells was calculated.
In one experiment with forebrain tissue from 2 tadpoles, we used immunohistochemistry for a neuron-specific nuclear protein (neuN) to determine the relative number of neurons in the acutely dissociated cell population. We found that greater than 90% of the cells that adhered to coverslips were immunopositive for neuN, indicating that the vast majority of cells studied were neurons rather than glia.
Measurements of intracellular calcium levels
We estimated intracellular calcium concentrations in isolated tadpole
forebrain cells using three different calcium-sensitive indicators. Several
different indicators were employed because we anticipated that anoxia-induced
[Ca2+]i changes could be quite large, and no single
indicator is appropriate for estimating [Ca2+]i under
both basal conditions and after hypoxic stress
(Hyrc et al., 2000).
Therefore, we chose indicators with low calcium affinity (fura-2FF and
benzothiazole coumarin; BTC) and higher calcium affinity (Fura-2), in order to
encompass the possible range of calcium concentrations that might occur in
cells. Further, with calcium indicators such as fura-2 and fura-2FF, the
excitation [Ca2+]i and emission wavelengths of the dyes
overlap with fluorescent compounds such as NADH, which are labile during
hypoxic stress, making interpretation difficult. The indicator BTC involves
the use of longer wavelengths and avoids these pitfalls. Isolated cells were
incubated with 5-10 µmol l-1 of the indicators for 60 min before
measurements.
Calibration of calcium estimates were done as follows. First, the
dissociation constant (KD) of all three dyes was
determined with calcium buffer calibration kit supplied by Molecular Probes
using the same light source, optical path and filters on the microscope stage.
Measured KD values were 0.26 µmol l-1
(fura-2), 8.1 µmol l-1 (fura 2-FF) and 9.1 µmol
l-1 (BTC). Measurements and calibration of
[Ca2+]i were made in dissociated cells using a dual
excitation fluorescence spectrometer (Photon Technology International, South
Brunswick, NJ, USA) and a Nikon Diaphot inverted microscope. Intracellular
calcium concentration in studies involving fura-2 and fura-2FF was calculated
from the 340/380 nm fluorescence intensity at an emission wavelength of 510
nm, using the equations of Buck and Bickler
(1995). With BTC, excitation
wavelengths were 400 nm and 480 nm and emission intensity was measured at 540
nm.
Experimental protocol
Following dissociation and isolation of forebrain cells, coverslips
containing forebrain cells suspended in aCSF were placed in a 2 l
Billups-Rothenberg chamber (Del Mar, CA, USA). Cells were exposed to an anoxic
atmosphere by allowing 100% N2 to flow through the chamber (ca. 1 l
min-1) for 15-20 min with the outflow tube placed in a beaker of
water to create a slight positive pressure in the chamber. A moist paper towel
was placed inside the chamber to create a humidified atmosphere and prevent
desiccation of the cells. After 15-20 min, the flow was stopped and the
chamber sealed until a coverslip was removed for measurement of
[Ca2+]i or PI. Each time a coverslip was removed, the
remaining cells were briefly exposed to room air for approximately 30 s. The
chamber was resealed and N2 gas was allowed to flow through again
as above and the chamber sealed after 15-20 min of N2 exposure. In
most experiments, coverslips were removed at 1, 2, 3, 4, 6 and 18 h after the
start of anoxia for measurement of [Ca2+]i or PI.
Individual coverslips containing normoxic control cells were placed into a
24-well plate on the bench beside the chamber containing anoxic cells. The
control coverslips with forebrain cells in aCSF were exposed to the same
temperature as the anoxic cells and to room air for the same duration of time
as the anoxic cells.
Statistical analysis
Data were analyzed using a one-way analysis of variance (ANOVA) followed by
the Neuman-Keuls post hoc test after determining homogeneity of
variances using Bartlett's test (Zar,
1974). If significant differences were found among variances, a
non-parametric analysis by ranks (Kruskal-Wallis test) was used instead of
ANOVA. Percentage data were arcsine transformed prior to statistical analysis
(Zar, 1974
). Statistical
analyses were done using commercially available software (GraphPad Prism, v.
4.0, San Diego, CA, USA or StatistiXL v.1.3).
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Results |
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Anoxia and [Ca2+]i
We used three ratiometric calcium indicators with varying calcium
affinities to determine [Ca2+]i since we were unsure to
what extent calcium levels might change during anoxic exposure. Fura-2, which
has a relatively high calcium affinity, gave a resting (normoxic)
[Ca2+]i of 47±3 nmol l-1
(Table 1;
Fig. 2). Fura-2 is a more
appropriate indicator for measurement of resting calcium levels than either
BTC or fura 2-FF, both of which have relatively low calcium affinities. Owing
to the low calcium affinity of fura 2-FF and BTC, these indicators would
overestimate resting cell calcium, as indicated by normoxic calcium values of
0.8 µmol l-1 for fura 2-FF and 3.1 µmol l-1 for
BTC (Table 1).
|
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Regardless of the type of calcium indicator used, [Ca2+]i increased significantly after 3-6 h anoxia, but the maximal levels of [Ca2+]i varied by nearly 30-fold, owing to the different calcium affinities of the indicators. For example, there was no significant increase in [Ca2+]i measured by fura-2 until 6 h anoxia (Fig. 2). Intracellular calcium levels measured by this method produced a maximum [Ca2+]i of 0.83±0.56 µmol l-1 (Table 1). By contrast, [Ca2+]i, determined with fura 2-FF, increased significantly after 3 h anoxia (P<0.05) and increased to a maximal value of 9.2±5.5 µmol l-1 (P<0.01) after 4 h anoxia (Table 1; Fig. 3). Measurement of [Ca2+]i with BTC produced similar results, with a significant increase in [Ca2+]i after 4 h anoxia (P<0.01) and the highest [Ca2+]i measured of 31.8 ± 8.0 µmol l-1 (Table 1; Fig. 4). Using the combination of fura-2 for estimating resting calcium levels and fura 2-FF or BTC for estimating maximal calcium levels during anoxia, the overall change in [Ca2+]i during a 4-6 h exposure to anoxia produced an approximate 200 to 600-fold increase in [Ca2+]i.
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Discussion |
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Calcium and cell survival during anoxia
The major results from this study demonstrate that tadpole forebrain cells
survive and recover from prolonged anoxia while experiencing relatively large
increases in [Ca2+]i. Compared to mammalian neurons,
which show evidence of increased [Ca2+]i and cell death
rapidly (within minutes to hours) with brief (<10 min) hypoxia, tadpole
forebrain cells endured prolonged increases in [Ca2+]i
associated with anoxia without evidence of membrane damage. This finding is
remarkable because it has been widely assumed that increases in
[Ca2+]i during anoxia are a central cause of cellular
injury and death during severe hypoxia
(Kristian and Siesjö,
1998). Indeed, preventing increases in
[Ca2+]i has been a common goal of therapies to treat
brain ischemia or hypoxia (Choi,
1995
). Survivable anoxia associated with large increases in
[Ca2+]i is concordant with data showing that
anoxia-tolerant neurons from freshwater turtles (Chrysemys) undergo a
2-3-fold increase of [Ca2+]i during prolonged and
survivable hypoxia (Bickler,
1998
; Bickler and Buck,
1998
). Note that [Ca2+]i estimates made by
Buck and Bickler were all performed with fura-2 and thus true
[Ca2+]i may have been considerably higher (see below).
Therefore, relatively large increases in [Ca2+]i are not
necessarily associated with cellular injury.
A number of studies involving mammalian neurons demonstrate that increases
in [Ca2+]i are associated with neuroprotective
consequences. For example, some forms of the phenomenon of ischemic
preconditioning (wherein a mild stress induces tolerance of a later severe
ischemic insult) depend on increases in [Ca2+]i produced
by activation of NMDA receptors, and can be blocked by calcium chelators,
calcium channel blockers or NMDA receptor blockers
(Kato et al., 1992).
Furthermore, there is good evidence that survival mechanisms associated with
ischemic preconditioning involve calcium-dependent processes such as
activation of protein kinase C (Raval et
al., 2003
), increased expression of MAP kinase cascades, nitric
oxide (Nandagopal et al.,
2001
) and the survival factors bax and bcl-2 activated by Akt
(protein kinase B; Mattson,
1997
). In mammalian hippocampal neurons, moderate increases in
[Ca2+]i produced by calcium-selective ionophores protect
by activating calcium-dependent signal cascades
(Bickler and Fahlman, 2004
).
Furthermore, it is clear that too little calcium at critical times induces
apoptosis in neurons and other cells (Lee
et al., 1999
). Altogether, the available information suggests that
moderate increases in [Ca2+]i may actually be crucial to
surviving hypoxic stress.
Intracellular calcium estimates during anoxia
We used several different calcium indicator compounds to show that
survivable anoxia is associated with relatively large increases in
[Ca2+]i in tadpole forebrain cells. This approach
greatly increases the probability that we are correct in the assertion that
the calcium increases were substantial. Fura-2 has a KD of
approximately 300 nmol l-1 and is therefore an appropriate dye for
estimating calcium levels in the low nanomolar range. The estimate of resting
[Ca2+]i of 50 nmol l-1 using fura-2 is in the
same range as estimates of resting [Ca2+]i in snail
neurons made using Ca2+ microelectrodes
(Kennedy and Thomas, 1996) and
in bullfrog sympathetic neurons using the ratiometric indicators fura-2
(Nohmi et al., 1988
) and
fluo-3 (Yu et al., 1994
).
Estimates of resting calcium in turtle neurons, measured using fura-2, are
higher (100-180 nmol l-1) than we have measured in tadpole cells
(Bickler, 1998
;
Buck and Bickler, 1995
;
Bickler et al., 2000
). In
addition, our estimate of [Ca2+]i in anoxic forebrain
cells of about 0.83 µmol l-1
(Table 1) measured with fura-2
is similar to maximal values (1 µmol l-1) measured with fura-2
in bullfrog sympathetic neurons permeabilized with digitonin
(Tokimasa et al., 1997
).
During hypoxia maximal calcium concentrations estimated with fura-2 are
limited because of at least two factors. First, significant labile background
fluorescence occurs at the same wavelengths employed with fura-2, particularly
that from NADH. Second, the KD of fura-2 makes detection
of [Ca2+]i changes problematic when
[Ca2+]i exceeds 600 nmol l-1
(Hyrc et al., 1997). BTC
avoids the first problem because the excitation and emission wavelengths are
greater than those of fura dyes. Fura-2FF, with a KD of
approximately 8 µmol l-1, avoids the second problem. However,
both low-affinity calcium indicators are poor for estimating resting calcium
levels, evidenced by the very high resting values obtained with these
indicators (Table 1).
Regardless of the type of calcium indicator used, all results agree with the
conclusion that survivable anoxia in tadpole forebrain cells is associated
with increases in [Ca2+]i on the order of hundreds of
nmol l-1.
The finding that large increases in [Ca2+]i are not
associated with increased cell death is also consistent with recent studies in
adult frogs showing that energy loss and neuronal death are not tightly
linked. For example, exposure of Rana pipiens to anoxia causes a
large (80%) fall in brain ATP in about 1 h, but the frog survives an
additional 1-2 h before the release of excitotoxic neurotransmitters
(Lutz and Reiners, 1997).
Anoxia tolerance in the adult frog is also extended by maintaining ion
homeostasis during anoxia (Knickerbocker
and Lutz, 2001
). An emerging view from recent studies with
amphibians is that anoxia results in a `slow death' of neurons with the same
sequence of degenerative events as seen in mammals, but on a longer time scale
(Milton et al., 2003
;
Lutz and Nilsson, 2004
).
Natural history observations suggest that early-stage Rana
tadpoles are more tolerant to extended periods of hypoxia or anoxia than
late-stage tadpoles or adults (Bradford,
1983; Crowder et al.,
1998
), and perhaps part of the increased hypoxia tolerance relates
to tolerance of large increases in [Ca2+]i during
cellular anoxia. By contrast, adult amphibians are relatively intolerant of
anoxia or severe hypoxia for longer than a few days at 3°C
(Hutchison and Dady, 1964
;
Christiansen and Penney, 1973
;
Lillo, 1980
;
Stewart et al., 2004
) or a few
hours at room temperature. These survival rates are consistent with studies at
the cellular level (Lutz and Reiners,
1997
; Knickerbocker and Lutz,
2001
).
The increases in [Ca2+]i observed in tadpole
forebrain cells are similar in magnitude, but different in time scale, to
those seen in more anoxia-tolerant neurons from Western painted turtles
Chrysemys picta. Painted turtles survive several months of anoxia
during winter dormancy (Ultsch and
Jackson, 1982). In laboratory-induced dormancy,
[Ca2+]i in the cerebrocortex of this species increases
to about twice normal over the course of several hours
(Bickler et al., 2000
) and
remains elevated for weeks to months
(Bickler, 1998
). It should be
noted, however, that previous estimates of [Ca2+]i in
turtle neurons were made exclusively with fura-2, perhaps underestimating the
maximal increases in [Ca2+]i that might occur during
anoxia. In light of the results obtained using ratiometric indicators with low
calcium affinities (fura-2FF and BTC), it might be useful to reexamine the
degree to which [Ca2+]i increases in anoxia-tolerant
species such as turtles. Thus, even though the increase in
[Ca2+]i during the first several hours of hypoxia is
rather similar in tadpole and turtle neurons, the long-term survival is much
more pronounced in turtle neurons. It is of interest to know more about how
moderate increases in [Ca2+]i relate to neuroprotective
signaling.
In conclusion, we have demonstrated that anoxia tolerance in tadpole forebrain cells is associated with moderate to large increases in [Ca2+]i, but is not associated with increased cell death. The large increases in [Ca2+]i were measurable only by the use of ratiometric indicators with varying affinities for calcium. The large increase in [Ca2+]i may play a neuroprotective role for tadpole forebrain cells in anoxia.
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Acknowledgments |
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