From the Center for the Study of Neurological Disease, The Queen's
Medical Center, Honolulu, Hawaii.
Correspondence to Tibor Kristián, PhD, Center for the Study of Neurological Disease, The Queen's Medical Center, University Tower 8th Floor, 1356 Lusitana St, Honolulu, HI 96813. E-mail tibor{at}www.cns.queens.org
Summary of Review—The original concepts encompass the hypothesis
that cell damage in ischemia-reperfusion is due to enhanced
activity of phospholipases and proteases, leading to release of free
fatty acids and their breakdown products and to degradation of
cytoskeletal proteins. It is equally clear that a coupling exists
between influx of calcium into cells and their production of
reactive oxygen species, such as ·O2-,
H2O2, and ·OH. Recent results have
underscored the role of calcium in ischemic cell death. A
coupling has been demonstrated among glutamate release, calcium influx,
and enhanced production of reactive metabolites such as
·O2-, ·OH, and nitric oxide. It
has become equally clear that the combination of
·O2- and nitric oxide can yield
peroxynitrate, a metabolite with potentially devastating effects. The
mitochondria have again come into the focus of interest. This is
because certain conditions, notably mitochondrial calcium accumulation
and oxidative stress, can trigger the assembly (opening) of a
high-conductance pore in the inner mitochondrial membrane. The
mitochondrial permeability transition (MPT) pore leads to a collapse of
the electrochemical potential for H+, thereby arresting ATP
production and triggering production of reactive oxygen
species. The occurrence of an MPT in vivo is suggested by the dramatic
anti-ischemic effect of cyclosporin A, a virtually specific
blocker of the MPT in vitro in transient forebrain ischemia.
However, cyclosporin A has limited effect on the cell damage incurred
as a result of 2 hours of focal cerebral ischemia, suggesting
that factors other than MPT play a role. It is discussed whether this
could reflect the operation of phospholipase A2 activity
and degradation of the lipid skeleton of the inner mitochondrial
membrane.
Conclusions—Calcium is one of the triggers involved in
ischemic cell death, whatever the mechanism.
In 1977, Nicholson et al8 showed that anoxia
triggers rapid translocation of Ca2+ from
extracellular to intracellular spaces of cerebellar tissues. This, as
well as other findings, led to the hypothesis of calcium-mediated
neuronal death in ischemia/hypoxia, hypoglycemia, and
status epilepticus.9 As applied to brain tissues,
the hypothesis of calcium-mediated cell death has some special
features. The most important of these is that since the BBB has a very
low permeability to
Ca2+,10 11 12 the calcium
translocated into cells is, at least in the short perspective, that
contained in the cerebral extracellular fluids. It was tempting to
speculate, therefore, that certain neurons in the brain are selectively
vulnerable to ischemic, hypoglycemic, and epileptic insults
because they possess a high density of calcium channels in their plasma
membranes.9 At that time, many neurons were known
to have an innate proclivity to fire synchronously in an epileptic
fashion, the epileptiform activity being driven by calcium spikes, ie,
by calcium influx through VSCC, localized to the dendritic fields of
these neurons.13 Accordingly, it could be assumed
that selectively vulnerable neurons are those that show such
calcium-related, "epileptogenic" spikes, neurons that thus could be
assumed to accumulate the brunt of the calcium load under adverse
conditions.
An important step forward was taken when it could be demonstrated in
cultured neurons14 and brain
slices15 that glutamate and related EAAs trigger
neuronal cell death. Such excitotoxic cell injury, which typically
affects dendrites and neuronal somata, was originally assumed to be
osmolytic, reflecting influx into cells of Na+
and Cl-, with osmotically obliged
water.16 However, it was soon demonstrated by
Choi17 that whereas the early cell swelling was
usually reversible, cells exposed to glutamate showed a type of delayed
cell death that was calcium mediated.18 19 This
means that the excitotoxic hypothesis is a variant of the calcium
hypothesis of cell death, but with agonist-operated channels playing
the major role of translocating calcium into cells.
With this as a background, we will probe into the role of calcium in
ischemic cell death in the brain. The first paragraphs will be
devoted to calcium fluxes between extracellular and intracellular
fluids and between blood and brain tissues. On the basis of the results
quoted, we will discuss the role of calcium in rapidly occurring or
delayed cell death. The notion will be considered that a coupling
exists among calcium influx, generation of free radicals, and
mitochondrial dysfunction, with mitochondria also emerging as important
generators of ROS. It seems justified to begin, though, with a brief
summary of cell calcium metabolism in normal cells. For
further discussion of the topics discussed, the reader is referred to
recent articles from the laboratory.20 21 22 23
Presynaptic and postsynaptic calcium channels ("conductances") at
an excitatory synapse in which signal transduction is mediated by
glutamate encompass N, P, L, and T types of VSCCs. The first two may be
mainly localized to presynaptic endings, while the L and T types abound
at postsynaptic membranes (see, for example, References 26 through 2926 27 28 29 ).
Two ionotropic glutamate receptors exist, one being selectively
activated by AMPA and the other by NMDA.
Under normal circumstances, signal transduction begins with release of
glutamate from the presynaptic ending and with activation of AMPA and
NMDA receptors. Since the AMPA receptor–gated channel is permeable to
monovalent cations, Na+ will enter the cell along
its electrochemical gradient, depolarizing the postsynaptic membrane.
The depolarization has two effects: it relieves the
Mg2+ block of the
Ca2+-permeable channel gated by the NMDA
receptor, and it opens VSCCs. Calcium thus enters the cell by multiple
pathways. At least in some cells additional VSCCs exist, and it is
debated at present whether changes in the subunit composition of
the AMPA receptor can render the channel it gates permeable to
calcium.30 31 32 Additional channels may be opened
under adverse conditions, eg, those comprising unspecific cation
channels and those activated by ROS, notably
H2O2 (F. Mendez and R.
Penner, personal communication, 1997). It is of interest that the
latter inactivate very slowly, if at all. Under some
circumstances, such as depolarization and intracellular
Na+ accumulation, the
3Na+-Ca2+ exchanger can
operate in the reversed mode, causing calcium to accumulate in the cell
(see below).
Since physiological signals usually have a short
duration, mechanisms must exist for terminating the glutamate (and
calcium) transients. This occurs by several mechanisms. The major
mechanism involves reuptake of glutamate through an electrogenic
Na+/glutamate symporter, which derives its energy
from the electrochemical gradient for
Na+.33 Although some
glutamate may be taken up by presynaptic and postsynaptic neuronal
elements, glial cells represent major sinks for glutamate. The
glial cells convert glutamate to glutamine or lactate, which are then
exported to neurons for resynthesis of glutamate or energy
production.34 35 Other mechanisms
encompass activation of Ca2+- or ATP-dependent
K+ channels or of
Fig 1
A third determinant of intracellular calcium movements, and thereby of
[Ca2+]i, is the
mitochondrion. It is now widely accepted that the balance between
influx and efflux of Ca2+ across the inner
membrane regulates mitochondrial dehydrogenases, which are
rate-limiting for citric acid cycle
metabolism.46 In summary, when cell
activity leads to a substantial or excessive rise in
[Ca2+]i, the mitochondria
may accumulate large amounts of calcium.47 This
is because the uniporter, carrying calcium into the mitochondrion along
the electrochemical gradient, has a much higher total activity than the
export pathways, which encompass
2Na+-Ca2+ exchange. In
other words, if the net influx of Ca2+ exceeds
the capacity of the extrusion pathway through
2Na+-Ca2+ exchange,
intramitochondrial Ca2+ concentration
([Ca2+]m) increases, and
Ca2+ will be sequestered within the mitochondria.
Under special circumstances, which will be discussed below, a large
conductance pore will be opened. This, which is called the MPT pore,
allows Ca2+ to leave the mitochondria.
Given this background, we will discuss calcium fluxes during and after
ischemia. Before we do that, though, we wish to bring up two
issues of conceptual importance. These relate to the total tissue and
total cell calcium content and to the type of ischemia
encountered.
As remarked, intracellular and extracellular ("free") calcium
concentrations are normally approximately 0.1 and 1000 µmol/L,
respectively. However, since the total cell calcium content (excluding
the extracellular spaces) is approximately 1000 µmol/L (ie,
1 mmol/L · kg-1), it follows that
more than 99% of the total cell calcium content is bound to proteins
or phospholipids, or sequestered into ER, to so-called calciosomes and
mitochondria. Clearly, although the source of a rise in
[Ca2+]i is often the
extracellular calcium content, the cell contains enough (bound or
sequestered) calcium to markedly increase
[Ca2+]i. This could occur
if the bound calcium is displaced from its binding sites or if the
calcium sequestered in ER or in mitochondria is released to the
cytosol. An uncontrolled release of calcium from the ER (or related
intracellular stores) is now believed to predispose to
Ca2+-related cell
damage.45 48 49 Furthermore, calcium
metabolism of the mitochondria has become the focus of
interest (see below).
As discussed elsewhere,22 50 51 it appears
justified that a distinction is made between ischemia of the
"cardiac arrest" type and that of the "stroke type." The
former, which is usually of short duration (5 to 15 minutes), can be
studied as such, ie, in models of cardiac arrest (eg, Reference 5252 );
however, most workers in the field prefer to use forebrain
ischemia in gerbils and rats.53 This is
because these models allow some remaining flow to brain stem centers
during ischemia, obviating the use of intensive care measures
during reperfusion. The second type of ischemia ("stroke")
is a focal one, which is usually of much longer duration, if not
permanent. It is commonly induced by permanent or transient occlusion
of one MCA.
The most important differences between the two types of
ischemia relate to the duration of the ischemia (and
its density). In global/forebrain ischemia of brief duration,
the tissue damage is usually confined to the neuronal population, and
cell death is characteristically delayed by hours or
days.54 55 56 Cells outside the selectively
vulnerable areas are usually not affected, nor are glial cells or
vascular endothelium. Furthermore, any inflammatory
component is of moderate degree. In contrast, focal ischemia of
long duration leads to pannecrosis, ie, to the death of all types of
cells (infarction).57 58 This implies that
vascular damage is a prominent feature of the lesions and that a strong
inflammatory response is elicited.59 60 61
Figure 2
During ischemia,
[Ca2+]e decreases to
values less than 10% of control, ie, to approximately 0.1 mmol/L.
In addition, the extracellular fluid space decreases to approximately
50% of control. This means that almost all extracellular calcium is
translocated into cells. With an extracellular fluid space of 20% of
tissue volume and a
[Ca2+]e of approximately
1.3 mmol/L, the calcium load to which cells are exposed is
approximately 250 µmol · kg-1 of
tissue. Since it is not likely that much calcium uptake occurs into
glial cells, the calcium load of neurons could well be twice that
figure, meaning that the total neuronal cell calcium content may
increase to 150% of control or more. Clearly, if some neurons have
very high calcium conductances, they could be exposed to an even higher
load.
In the hippocampus, CA1 cells have a very high density of NMDA
receptors. It is of interest, therefore, that Silver and
Erecinska63 64 demonstrated that ischemia
causes [Ca2+]i to
increase from approximately 0.1 µm to values of 30 to 60
µm. Clearly, these are nonphysiological increases
in [Ca2+]i.
Additional information on ischemic calcium transients has been
collected in experiments in which
[Ca2+]e and DC potential
shifts were measured in transient ischemia of the forebrain
type. This type of ischemia has the drawback of giving a less
distinct "onset" of ischemia but the advantage of readily
allowing reperfusion events to be studied. Figure 3
These experiments challenge the postulate that any damage incurred by
hypoxic cells is proportional to the duration of the calcium transient
and to the rise in Ca2+i, ie, to
the time integral under the
Ca2+i curve. Although it is
noteworthy that hyperglycemic animals have a worse outcome after a
10-minute period of ischemia, whether one uses a neurological
or histopathological end point, the difference in duration of calcium
uptake and DC potential shift between normoglycemic and hyperglycemic
animals is relatively trivial. However, it becomes more obvious if the
nominal ischemic period is only 5 minutes (Fig 4
The relationship between calcium influx and neuronal injury is even
more complicated than indicated in Fig 3
Calcium and Delayed Neuronal Death
The question arises of how the perturbation of cell calcium
metabolism during and immediately after a transient period
of ischemia influences the cascade of events that leads to
delayed cell injury. There are many possibilities since
Ca2+ activates phospholipases,
endonucleases, and proteases, since it affects protein
phosphorylation by altering the activity of protein
kinases and phosphatases, and since it activates enzymes that
give rise to the production of ROS and
NO.9 28 76 77
Three schemes have been elaborated that purport to explain delayed
neuronal death. One puts the emphasis on a sustained perturbation of
the signal transduction pathway, ie, the sequence of events that starts
with the activation of receptors for EAAs and neurotrophins, continues
with the activation or deactivation of protein kinases and
phosphatases, and ends with an altered activity of major response
elements, mainly those regulating gene expression and
protein.78 79 80 One feels intuitively that nuclear
events, particularly if they involve fragmentation of DNA, could be
devastating for cell survival. Furthermore, a sustained suppression of
protein synthesis could be equally harmful since it bereaves the cells
of molecules required for survival, such as antioxidative enzymes and
trophic factors.
The second hypothesis is one in which cell death is assumed to be due
to a sustained perturbation of cell calcium metabolism,
leading to a slow, gradual rise in
[Ca2+]i and to eventual
mitochondrial calcium overload.21 81 82 We will
describe the origin of that hypothesis, attempt to integrate it with
data suggesting a failure of the signal transduction pathway, and
discuss data that give a novel perspective on postischemic
mitochondrial dysfunction.
In the beginning of the 1980s, it was known that ischemia is
accompanied by translocation of Ca2+ from
extracellular to intracellular fluids8 83 84 and
also that reperfusion restored
[Ca2+]e to normal within
15 to 20 minutes.85 However, it was not known
whether any changes occurred in total tissue Ca2+
content, nor was there information on calcium metabolism in
the period of recirculation preceding the delayed neuronal death. In
1984, Dienel86 reported results on transient
forebrain ischemia of 20 minutes' duration. His data revealed
an increased incorporation of
45 Ca2+ into the subiculum
of the CA1 sector and into the lateral caudoputamen after
24 hours of reperfusion; however, this increased incorporation seemed
to occur without an increase in the total cell calcium content. On the
basis of these results, our laboratory explored the time course of
changes in the total tissue calcium concentration of the dorsal
hippocampus, correlating it with light microscopic signs of cell
death.81 The results showed that the total tissue
calcium content during reperfusion did not increase until late (between
24 and 48 hours) and that an increase in
[Ca2+]i seemed to precede
morphological signs of cell death. The results, which are illustrated
in Fig 5
This hypothesis requires (1) that mitochondria accumulate
Ca2+ before cell death becomes manifest and (2)
that [Ca2+]i rises
gradually during reperfusion. Both of these requirements seem to be
fulfilled. Thus, Dux et al90 showed that a second
wave of Ca2+ precipitates in the mitochondria of
CA1 neurons after 24 hours of recirculation following 5 minutes of
transient ischemia in the gerbil. Furthermore, Zaidan and
Sims,91 studying forebrain ischemia of 20
minutes' duration in rats and employing fractionation of tissue by
centrifugation, directly showed a two-phase
accumulation of calcium by the mitochondria, one occurring immediately
after ischemia and the other many hours later. Finally, the
results reported by Silver and Erecinska64
demonstrated that although reperfusion in rats subjected to forebrain
ischemia normalized
[Ca2+]i within 10 to 20
minutes, continued recirculation seemed to give rise to a secondary,
gradual rise in
[Ca2+]i.
The third hypothesis is that published by Abe et
al.92 Like the second of the other two hypotheses
discussed, it predicts that the ultimate cell damage is due to
mitochondrial failure. However, the mechanisms proposed are different.
The background is that the respiratory complexes, ie, the enzymes that
shuttle electrons along the respiratory chain and extrude
H+, are encoded for by both mitochondrial and
nuclear DNA. For mitochondria to gain genetic material for synthesis of
all relevant proteins, the mitochondria must be transported to the
nucleus by being propelled along cytoskeletal elements by transport
proteins such as dynorphin and kinesin. Abe et
al92 submit that this process is halted when the
cytoskeleton is broken down by calcium-activated proteases and
by calcium-dependent disassembly of microtubuli (see Reference 9393 ). The
long-term result of this would be reduced activities of respiratory
enzymes, such as complex I or complex IV, with devastating effects on
mitochondrial generation of ATP.
Calcium Accumulation and Mitochondrial Dysfunction
According to the chemiosmotic theory of
Mitchell,100 electron transport in the
respiratory chain of mitochondria causes the extrusion of
H+, creating a large electrochemical potential
difference across the inner mitochondrial membrane. This potential
(
Mitochondrial ATP production thus depends on the regulated
entry of H+ across the inner mitochondrial
membrane. However, the literature on mitochondrial function in vitro
contains many reports demonstrating that exposure of mitochondria to
calcium causes them to swell and to release intramitochondrial
components into the medium.94 95 It is now
realized that this sequence of events reflects the assembly of an MPT
pore in the inner mitochondrial membrane. This pore allows the release
of Ca2+ and Mg2+ as well as
of various low- and high-molecular-weight compounds. In this process
the mitochondria show osmotic swelling. Furthermore, the assembly of
the pore leads to the collapse of
The seminal work of Crompton and
collaborators98 101 identified the major factors
triggering the assembly of an MPT pore in isolated mitochondria. These
were a decrease in the ATP and increase in the Pi
concentration, oxidative stress, and calcium accumulation. The last two
factors have emerged as major determinants in other experimental
paradigms.102 103 Furthermore, the coupling among
a decrease in mitochondrial membrane potential, the assembly of an MPT
pore, and enhanced mitochondrial production of ROS has been
established in thymocytes that have been committed to die by an
apoptotic mechanism, after exposure to
dexamethasone.104 105 In these and
other rapidly proliferating cells, the first event in the sequence
leading to (apoptotic) cell death is a decrease in
The conclusions that can be drawn from these experiments are that
oxidative stress and mitochondrial calcium accumulation predispose to
an MPT and to the consequences of such an event, eg, the
depolarization-coupled production of ROS. This is where the
action of CsA comes in. In all experimental paradigms studied in vitro,
whether on cells of nonneuronal or neuronal origin, CsA proved to be an
almost specific inhibitor of the
MPT.96 This is presumably because CsA, which
combines with a series of cyclophilin proteins, blocks the MPT pore by
competing with the effect of Ca2+-cyclophilin for
occupancy on the transition pore proteins.
Two years ago, our laboratory obtained results showing that CsA
dramatically ameliorates the CA1 damage, provided that it can pass the
BBB. The results were obtained in experiments in which growth
factor–producing cells were injected into the CA1 sector of one
hemisphere, CsA being given to suppress the immune response (Uchino et
al106 ). Analyses of the primary
experiments and additional experiments revealed that the combination of
systematically injected CsA and a unilateral needle lesion almost
eliminated the CA1 damage after 7 or 10 minutes of forebrain
ischemia (Fig 6
Calcium-Mediated Mechanisms of Delayed Neuronal Death: A
Speculative Synthesis
Release of excitatory transmitters, depolarization, and an increase in
[Ca2+]i trigger enhanced
production of the traditional ROS (
·O2-,
H2O2, and ·OH) as
well as of NO.110 111 In general, activation of
PLA2 leads to the production of ROS
because arachidonic acid, generated by
PLA2 activity, is metabolized by
cyclooxygenase (and lipoxygenase)
to yield a variety of degradation products and, in that process,
·O2- is
formed.112 Another source of ROS is activation of
the Ca2+-calmodulin–dependent NOS
pathway, which produces NO from arginine. As proposed by Beckman et
al,113 114 NO can then react with
·O2- to yield
peroxynitrate (ONOO), the latter decomposing with the
production of ·OH, a highly toxic free radical. The
reaction sequences yield NO, peroxynitrate, and ·OH, three
reactive metabolites of potentially destructive nature. Interestingly,
both NO and peroxynitrate predispose to an MPT and could thus act as
triggers of a pore opening in partially calcium-loaded
mitochondria.115 116 117
It has been shown beyond doubt that transient forebrain
ischemia is accompanied by an enhanced production of
ROS, which is maximal during the first 10- to 60-minute period of
recirculation.118 119 120 However, it has been more
difficult to demonstrate an anti-ischemic effect of free
radical scavengers. For example, although the spin trap PBN ameliorates
neurological deficit and ischemic cell death in the
gerbil,118 121 it lacks an effect in rats
subjected to forebrain ischemia.122
However, a fairly robust effect was obtained with a very high dose of
the ·OH scavenger dimethylthiourea in rats
subjected to 15 minutes of forebrain
ischemia.123 It seems likely, therefore,
that the postischemic production of ROS contributes
to delayed neuronal death in both gerbils and rats.
Fig 7
It should be emphasized that mitochondrial dysfunction and bioenergetic
failure are delayed phenomena. Thus, in the early recirculation period
a normal bioenergetic status is quickly
achieved,126 and respiratory functions of
isolated mitochondria are resumed.127–129 The
mitochondria also have a normal capacity to respond to
metabolic challenges. Thus, although the "resting"
metabolic rate of brain tissues is reduced, challenges such
as spreading depression (Reference 130130 and T. Kristián, G.
Gidö, and B.K. Siesjö, unpublished data, 1996) and
epileptic seizures131 trigger the expected
responses, allowing maintenance of bioenergetic and ionic
homeostasis. Clearly, it appears unlikely that the initial oxidative
burst during recirculation causes direct damage to mitochondria. A more
likely scenario is that slow calcium accumulation ultimately triggers
an MPT, production of free radicals, and mitochondrial damage
(see above) or that breakdown of the cytoskeleton with damage to the
motor proteins arrests shuttling of mitochondria between the periphery
and the nucleus, affecting proteins encoded for by nuclear
DNA.92
Recovery of Mitochondrial Function
The reason why mitochondrial respiratory functions are not resumed
after long ischemic periods, particularly in hyperglycemic
subjects, is not known. It has been generally held that mitochondria
are either damaged by PLA2-mediated breakdown of
the lipid backbone of the inner mitochondrial membrane or by oxidation
of protein components mediating electron transport,
H+ extrusion, or ATP production. However,
no agreement has been reached on the targets. Thus, some studies
implicate the pyruvate dehydrogenase complex, others one or more
respiratory complexes (I through V), and still others the
adenylate translocase.135 136 137 In fact,
the pyruvate dehydrogenase complex has also been incriminated in
ischemia of briefer duration in starved
animals.91 138 Since PLA2
is a calcium-dependent enzyme and since calcium activates
several enzymes producing ROS, the mitochondrial failure, whether acute
or delayed, can be traced back to a perturbed calcium
metabolism. However, the molecular defect remains
unclarified.
In this context, recent results obtained in experiments on focal
ischemia of 2 hours' duration are intriguing. In core and
penumbral tissues, this period of MCA occlusion was accompanied by a
decrease in tissue ATP content to approximately 10% and 25%,
respectively, and by increases in lactate content to approximately
15 mmol/L ·
kg-1.139 After 1 hour of
reperfusion, ATP increased to 50% to 70% of control. Since the
adenine nucleotide pool (
Subsequent results showed that the time course of changes in cellular
bioenergetic state was paralleled by corresponding changes in ADP-
and uncoupler-stimulated respiration of tissue homogenates
in focal and penumbral tissues.140 These results
suggest that the partial recovery of ATP concentrations and the failure
of recovery of normal lactate concentrations reflect sustained
mitochondrial dysfunction. It could further be shown that the free
radical spin trap PBN and the immunosuppressant FK-506, both of which
ameliorate tissue damage due to transient
ischemia,141 142 143 also prevent the
secondary deterioration of mitochondrial respiratory
capacity.144 145
In view of the results obtained with brief to intermediate periods of
ischemia, it is perhaps not surprising that mitochondria fail
to resume normal respiratory functions after 2 hours of MCA occlusion.
However, it is more surprising that the activity of respiratory
complexes (I, II-III, IV) and of two associated enzymes (citrate
synthase and glutamate dehydrogenase) did not
decrease.146
These results suggest that failure of mitochondria to resume normal
respiratory functions, in vivo or in vitro, may encompass mechanisms
other than those discussed for global or forebrain ischemia. In
vivo, and perhaps also when homogenates are used to study
mitochondrial respiratory functions in vitro, increased FFA
concentrations may uncouple mitochondria or inhibit their
respiration.147 148 149 150 An additional possibility is
that the mitochondria are partially calcium-loaded and prone to
assemble a permeability transition pore.151 As
remarked, this would promote mitochondrial production of free
radicals and accelerate breakdown of mitochondrial phospholipids. Very
likely, conditions during reperfusion favor the opening of an MPT pore,
particularly if NO and peroxynitrate are formed.
Calcium Metabolism
Although the results are clear-cut, they raise a series of important
questions. First, is there an influx of calcium from blood to tissue
because Ca2+e is reduced,
creating a suitable transport gradient? Second, is there a rise in
total tissue calcium content because cells fail to extrude
Ca2+ against whatever gradient may be existing?
Third, if cells continuously accumulate Ca2+, is
this "extra" calcium sequestered by the mitochondria?
Recent results obtained in an experimental setting of 2 hours of MCA
occlusion, with reperfusion for 1 to 8 hours, provide some answers to
these questions.156 156A First, in the focus of the lesion,
reperfusion leads to rephosphorylation of ADP to ATP
(see above) and to normalization of
K+e, but
Ca2+e is only restored to
approximately 50% of control, leaving a transport gradient for
translocation of Ca2+ from plasma to tissue (T.K.
and B.K.S, unpublished data, 1997). Second, the 2-hour period of
ischemia leads to a 20% increase in the total tissue calcium
content, reflecting the influx of plasma calcium during the
ischemic period. During the first 3 to 4 hours of
recirculation, the calcium content remains the same or increases
slowly, but after 6 hours (or 24 hours) there is a substantial increase
in tissue calcium content (T.K. and B.K.S, unpublished data, 1997).
Changes in a neocortical penumbral area were qualitatively similar, and
massive calcium "loading" occurred after 6 (and 24) hours of
recovery (T.K. and B.K.S, unpublished data, 1997).
These results lead to a number of questions. A major question can be
posed as follows: Why is Ca2+e
in focal areas not normalized during recirculation? Provided that one
can assume that calcium influx occurs along a transport gradient
created by the low Ca2+e, where
does the calcium accumulate? Does it accumulate in mitochondria?
However, if mitochondria accumulate calcium, how can such accumulation
be reconciled with the notion of an MPT, which should be accompanied by
release of calcium to the cytosol? This leads to a general question:
Where is the accumulated calcium localized? Furthermore, how is this
localization/sequestration related to cell death? Does it cause cell
death, or does it occur because dying cells accumulate calcium? To
provide answers to some of these questions, we will examine in vitro
experiments.
Exposure of cultured cells to EAAs in vitro has been claimed to be a
useful paradigm for ischemic disease in vivo. Two facts suggest
that the extrapolation from in vitro conditions is fraught with
difficulties. First, while in vitro results demonstrate that 2 to 3
minutes of glutamate exposure leads to extensive neuronal necrosis in
vitro,159 this duration of ischemia does
not lead to cell damage in any region other than the CA1 sector of the
hippocampus.58 69 In other words, cells are more
vulnerable in vitro to anoxic/excitotoxic transients. Second, it has
been demonstrated that acidosis in vitro protects cells against anoxic
and excitotoxic insults160 161 162 ; however, it has
been demonstrated beyond doubt that acidosis in vivo exaggerates
ischemic brain damage (for reviews, see References 67 and
16367 163 ).
It seems likely that these differences can be explained by the fact
that, in vitro, cells are exposed to an unlimited source of calcium,
allowing translocation of large amounts of Ca2+
into cells.164 This conclusion is supported by
results obtained in vitro, demonstrating that the total calcium content
during a 5- to 10-minute exposure to glutamate can increase
severalfold.165 Such a massive influx does not
occur in vivo, since the amount acutely accumulated is restricted by
the calcium content of the extracellular fluid (see above). By analogy,
and recalling that acidosis reduces the rate of calcium influx through
NMDA- and voltage-operated channels,166 we can
deduce that, in vitro, acidosis protects cells by limiting
ischemia-induced calcium influx into cells. In vivo, acidosis
can retard the rate of calcium influx in ischemia, but since
the calcium source is restricted, factors other than the total calcium
load determine the final outcome.23
Despite these reservations, it must be concluded that experiments on
neurons in vitro represent a useful paradigm for studies of
ischemia in vivo. The following results, obtained on cells in
vitro, are particularly relevant.
(1) A transient insult leads to a delayed increase in
Ca2+i after the initial
increase. It has been documented in numerous publications that a
glutamate transient leads to a marked rise in
Ca2+i. A few studies have
documented that if the excitotoxic insult is sufficiently severe or
sufficiently prolonged, recovery of
Ca2+i occurs after the initial
insult but is followed by a secondary rise in
Ca2+i, obviously preceding
deregulation of Ca2+ homeostasis and cell
death.158 167 168 In fact, if the excitotoxic
insult is sufficiently severe,
Ca2+i will not recover when the
stimulus is removed.158 169 We recognize that
these results mimic those obtained in vivo after brief and sustained
ischemic periods.
(2) The cell damage incurred in EAA transients is not related to
changes in Ca2+i but to changes
in total calcium influx. Results obtained from several
laboratories158 170 171 suggest that the
(initial) rise in Ca2+i is a
poor indicator of the outcome. A variable that correlates better
with the outcome of an excitotoxic insult is the total amount of
calcium translocated into the cells165 172 173
(for review, see Reference 165165 ). There are several possible
implications of the findings, supporting this notion. One is that
activation of NMDA receptors may be detrimental because the
receptor-gated channel translocates more Ca2+
into the cell than other agonist-operated channels or voltage-dependent
channels. Another implication is that the vulnerability of neurons in
vitro is exaggerated because much more calcium can be translocated into
intracellular fluids in vitro because of the unlimited source of
Ca2+ available (see above).
(3) The initial calcium load, at least in part, is sequestered by the
mitochondria. Data obtained more than one decade ago suggested that the
mitochondria served as a "sink" for calcium loads associated with
intense physiological activity or pathological
transients.47 Subsequent work performed on
neuronal cultures in vitro gave substance to this assumption and
provided information on pathological calcium transients (Thayer and
Miller174 [1990], Werth and
Thayer175 [1994], White and
Reynolds176 [1995],Wang et
al177 [1994], White and
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from these studies is that when substantial amounts of
Ca2+ enter the cell, a large part is sequestered
in the mitochondria. This process occurs by uptake of
Ca2+ through the electrogenic uniporter, while
egress of Ca2+ probably occurs by
Ca2+-2Na+ exchange. The
absolute amounts of Ca2+ taken up are not known,
however, ie, we do not know how much of a given tissue calcium
"overload" can be attributed to calcium sequestration.
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production of ROS. As mentioned previously, ischemia
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ROS.118 119 120 Evidence now exists that a
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(5) Cell death is preceded by mitochondrial membrane depolarization and
is inhibited by CsA. Recently the EAA stimulation of surface receptors,
increase in Ca2+i, sequestration
of Ca2+ by mitochondria, and mitochondrial
generation of ROS have been further investigated. Thus, results have
been published which show that the effect of EAA on cells encompasses a
decrease in the mitochondrial
Received October 1, 1997;
accepted December 9, 1997.
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© 1998 American Heart Association, Inc.
Basic Science Review
Calcium in Ischemic Cell Death
Abstract
Top
Abstract
Introduction
Cell Calcium Homeostasis
Forebrain or Global Ischemia...
Global or Focal Ischemia...
Excitotoxicity, Calcium Influx,...
References
Background—This review article deals
with the role of calcium in ischemic cell death. A
calcium-related mechanism was proposed more than two decades ago to
explain cell necrosis incurred in cardiac ischemia and muscular
dystrophy. In fact, an excitotoxic hypothesis was advanced to explain
the acetylcholine-related death of muscle end plates. A similar
hypothesis was proposed to explain selective neuronal damage in the
brain in ischemia, hypoglycemic coma, and status
epilepticus.
Key Words: calcium • cerebral ischemia • free radicals • mitochondria
Introduction
Top
Abstract
Introduction
Cell Calcium Homeostasis
Forebrain or Global Ischemia...
Global or Focal Ischemia...
Excitotoxicity, Calcium Influx,...
References
The calcium
hypothesis of ischemic cell death was originally launched to
explain the relationship between excessive calcium influx and the cell
damage that is incurred in myocardial
ischemia1 as well as in muscle
dystrophy.2 In fact, studies conducted at that
time led to the formulation of an excitotoxic hypothesis, predicting
that excessive release of acetylcholine at the motor end plate was what
caused damage to skeletal muscle.3 4 The work
performed at that time on ischemic muscle damage was almost
visionary since it forestalled the pivotal role of release of
transmitters in enhancing the influx of Ca2+ at
postsynaptic sites and also predicted that a
nonphysiological rise in
[Ca2+]i could exert its
adverse effects by overactivating cellular proteases and
lipases.2 3 A link to mitochondrial dysfunction
was also suggested since free fatty acids, liberated as a result of
PLA2 activity,5 6 were
supposed to increase the permeability of mitochondrial membranes and to
uncouple respiration and oxidative phosphorylation in
isolated mitochondria.7
Cell Calcium Homeostasis
Top
Abstract
Introduction
Cell Calcium Homeostasis
Forebrain or Global Ischemia...
Global or Focal Ischemia...
Excitotoxicity, Calcium Influx,...
References
There is normally a very large electrochemical driving force
tending to translocate calcium into cells. This force has two
components: the difference in calcium concentration (extracellular
fluids having a 10 000-fold higher concentration than intracellular
ones) and the electrical potential across plasma membranes (the inside
being 60 to 90 mV negative to the outside). The electrochemical
potential is upheld because calcium fluxes associated with signal
transduction are usually small and tightly regulated and because a rise
in [Ca2+]i triggers the
extrusion of calcium by
3Na+-Ca2+ exchange and by
an ATP-driven Ca2+-2H+
exchanger.24 25 The pump-leak relationships thus
upheld normally maintain
[Ca2+]i at values of 100
to 200 nmol/L, but the concentration rises transiently during cell
activation. 
-aminobutyric
acid–activated Cl-
channels.36 37 Activation of such channels would
tend to repolarize or hyperpolarize membranes. Yet another mechanism,
of theoretical and practical interest, can be deduced from the known
dependence of NMDA-activated ion currents on pH. Since
Ca2+ ion conductance decreases steeply when
extracellular pH is reduced below 7.0,38 39 40 one
can envisage that strong excitatory stimuli (which reduce intracellular
and extracellular pH) are subjected to feedback inhibition by this
mechanism. However, since the pKa for the
effect of pH on NMDA channel currents is approximately 6.7, one can
also deduce that alkalosis (ie, an increase in
pHe above normal) increases the tendency to
calcium-mediated cell firing, perhaps acting as a trigger for
epileptogenic discharges.41 
summarizes cell calcium
metabolism in a wider perspective, taking into account not
only pathways of Ca2+ entry but also mechanisms
for extrusion, binding, and sequestration. The upper part of the figure
illustrates what has already been discussed, ie, the pump-leak
relationship for Ca2+ across the plasma membrane.
Undoubtedly, this relationship will set, or at least modulate, the
value of [Ca2+]i. Another
determinant is the corresponding calcium traffic across the membranes
of the ER, which is supposed to contain fluids having a free cytosolic
calcium concentration close to that of the extracellular
fluid.42 The calcium sequestered by the ER can be
released by the operation of a sequence of events that starts with
activation of surface receptors coupled to phospholipase C, continues
with the formation of IP3 and its activation of
IP3 receptors on ER membranes, and ends with the
release of Ca2+ from the ER. This is undoubtedly
an important source of calcium, which could contribute to a rise in
[Ca2+]i.42 43 44 45
Furthermore, since resequestration of Ca2+ into
the ER requires ATP, the traffic of Ca2+ across
the ER membranes represents another aspect of the pump-leak
relationship for Ca2+.
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Figure 1. Calcium handling by cells. The calcium enters the
cell through voltage- and agonist-operated channels and also by more
unspecific channels, such as those opened by ROS, while
Ca2+ is extruded from the cell by an ATP-driven pump or by
Ca2+-Na+ exchange. Cytosolic calcium
(Ca2+i) is sequestered by mitochondria and ER
at the expense of respiratory energy or ATP, and it is bound to
calcium-binding molecules (A). Release of Ca2+ from ER
occurs in response to agonist activation of receptors coupled to
phospholipase C (PLC) and the formation of IP3 from
phosphatidyl inositol bisphophate (PIP2). A rise in
Ca2+i can cause translocation of protein kinase
C (PKC) to membranes where the kinase may be activated by
diglycerides (DG) formed during PIP2 hydrolysis.
Accumulation of Ca2+ in mitochondria can lead to the
activation of an MPT pore, leading to the release of Ca2+
and other molecules with molecular weight up to 1.5 kD from
intramitochondrial compartments. Quis indicates quisqualate; APCD,
1-aminocyclopentane-1S,3R-dicarboxylic acid; PDH, pyruvate
dehydrogenase; ICDH, isocitrate dehydrogenase; and OGDH,
oxoglu-tarate dehydrogenase. Modified from Reference 82.
Forebrain or Global Ischemia of Brief to Intermediate
Duration
Top
Abstract
Introduction
Cell Calcium Homeostasis
Forebrain or Global Ischemia...
Global or Focal Ischemia...
Excitotoxicity, Calcium Influx,...
References
Calcium Fluxes and Changes in Extracellular and Intracellular
Calcium Concentration
Under this heading, we will discuss events occurring after brief
periods of forebrain ischemia, under optional reflow
conditions. However, we will also consider results obtained with
sudden, complete ischemia since they give unequivocal
information on rates of calcium influx (for reviews, see References 22,
37, and 6222 37 62 ). illustrates calcium influx into
cells of neocortical tissue after complete ischemia, as this
influx is reflected in
[Ca2+]e. In normoglycemic
animals the time to ischemic depolarization is approximately 50
seconds, and calcium influx is rapid. The raised plasma (and tissue)
glucose concentrations in hyperglycemic subjects lead to delayed
depolarization and to a two-phase influx of Ca2+,
the second one being very slow. Clearly, hyperglycemia delays
depolarization, probably by providing additional substrate for
(anaerobic) ATP production, and it reduces the rate
of calcium influx. The latter effect is obviously due to the
exaggerated tissue acidosis since excessive hypercapnia, induced in
normoglycemic animals, duplicates the effects of
preischemic hyperglycemia. Finally, since the NMDA
antagonist MK-801 has effects similar to those of
hyperglycemia and of hypercapnia, the results must largely reflect the
blocking effect of acidosis on NMDA receptor–gated
Ca2+ influx.
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Figure 2. Recordings of extracellular calcium
concentration (Ca2+e) in complete
ischemia during control (normoglycemic and normocapnic)
conditions, in conditions with accentuated tissue acidosis
(hyperglycemia and hypercapnia), and in animals given dizocilpine
maleate (MK-801). Data from Reference 166. illustrates the
[Ca2+]e transients
accompanying ischemia of 15 minutes' duration in hypoglycemic,
normoglycemic, and hyperglycemic animals.65 66
The results illustrate that hyperglycemic animals show a delay before
depolarization occurs (see above) compared with normoglycemic animals
but also that they repolarize earlier than their normoglycemic controls
(see also Fig 4 for 5- and 2.5-minute ischemia). This means
that the duration of the ischemic calcium transient is shorter
in hyperglycemic than in normoglycemic animals (for review, see
Reference 6767 ); however, the ischemic damage is exaggerated in
hyperglycemic animals.67 68 69
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Figure 3. Typical recordings of extracellular
calcium concentration (Ca2+e)
transients during 15 minutes ischemia in hyperglycemic (Hyper),
normoglycemic (Normo), and hypoglycemic (Hypo) animals. Downward arrows
indicate the onset of ischemia, upward arrows the termination
of ischemia. Data from Reference 183.
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Figure 4. Anoxic depolarization time (latency from onset of
ischemic to massive cell depolarization) (A) in the cortex and
hippocampus during 2.5 minutes (top) and 5 minutes (bottom) of
ischemia; total depolarization periods (B) during 2.5 minutes
(top) and 5 minutes (bottom) of ischemia in the cortex and
hippocampus. ***P<.001, **P<.01,
two-factor ANOVA followed by Scheffé's test.
P<.01, Mann-Whitney U test. Open
circles indicate normoglycemic rats; solid circles, hyperglycemic
animals; lined circles, animals that did not show any depolarization
during 2.5 minutes of ischemia. Data from Reference 69.
, data from Reference 6969 ). With this
period of ischemia, the duration of the
Ca2+e/DC potential transient in
hyperglycemic subjects is only 50% of that observed in normoglycemic
animals. Despite that, the former incurred at least as much neuronal
damage and, in contrast to normoglycemic subjects, they showed a
tendency to develop postischemic seizures. Clearly,
although it is likely that Ca2+ constitutes a
major mediator of ischemic cell death, it must act in concert
with other mediators, such as exaggerated intracellular acidosis.
Predictably, hyperthermia is an additional aggravating
factor.70 . Thus, recent results
demonstrate that hyperglycemic rats subjected to only 2.5 minutes of
ischemia show 15% to 20% damage to the CA1 sector of the
hippocampus even though the cells never depolarize during
ischemia (Fig 4) and even though the
[Ca2+]e remains unchanged
or increases somewhat.69 Very probably,
depolarization does not occur because the increased glucose content
provides additional ATP from anaerobic
glycolysis.71 Clearly, any rise in the
[Ca2+]i must have been
small, and other factors must have contributed to the delayed neuronal
death. Such factors could encompass acidosis caused by the
anaerobic glycolysis and/or the redox change that
accompanies ischemia/reperfusion.
In this type of ischemia, the neuronal injury is truly
delayed since cells repolarize and resume
physiological and metabolic functions
before they suffer secondary damage and die. Signs of ongoing adverse
processes are present, however. These encompass a sustained
depression of metabolic rate72 and of
overall protein synthesis.73 Other signs of
metabolic perturbation are the expression of mRNAs for
immediate early genes and for neurotrophins and the synthesis of
"stress" or "heat shock" proteins (for extensive discussions of
these issues, see recent volumes edited by Moskowitz and
Caplan74 and by Siesjö and
Wieloch75 ). , were subsequently confirmed by
measurements with proton-intensified x-ray emission (PIXE), allowing
analyses of the different layers of the CA1 and CA3
sectors.87 Based on these results and on the
theory proposed by Alkon and Rasmussen,88 we
advanced the hypothesis of delayed calcium-related cell death. This
hypothesis predicts that the initial ischemic transient gives
rise to a sustained perturbation of plasma membrane handling of
Ca2+, resulting in a gradual rise in
[Ca2+]i. When the latter
exceeds the "set point" for calcium sequestration in the
mitochondria, these begin accumulating Ca2+ until
they are "overloaded" and become dysfunctional. It should be
emphasized that the plasma membrane may not be the only type of
membranes that are perturbed by the ischemic transient. Thus,
evidence for an involvement of ER membranes in the delayed cell death
was reported by Tsubokawa et al,48 89 who induced
ischemia of 5 minutes in gerbils, allowed reperfusion for 2.5
to 3.5 days, and prepared hippocampal slices for patch clamping of CA1
cells. Tetanic stimulation of the input to these cells caused
irreversible depolarization, as did injection of
IP3 via the patch pipette. The depolarization
could be prevented by antibodies to IP3 or to the
kinase that converts IP3 to
IP4. This suggests that release of calcium from
the ER could contribute to the rise in
Ca2+i and that a perturbed
signal transduction affecting the
IP3-IP4 system could be
part of the pathogenetic defect leading to dysregulation of calcium
metabolism (see also Reference 4545 ).
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Figure 5. Changes in total tissue calcium content in dorsal
hippocampus in control animals (C) and at 6, 24, 48, and 72 hours of
reperfusion after 15 minutes of forebrain ischemia. There was a
significant increase in tissue Ca2+ after 48 and 72 hours
of reperfusion. **P<0.01 ANOVA, post hoc Dunnett's test.
Data from Reference 81.
The postulate that mitochondrial dysfunction contributes to
delayed neuronal death has recently received support from results
obtained with the immunosuppressant drug CsA. The background is as
follows. It has been known for decades that massive calcium
accumulation triggers mitochondrial damage.47
This was originally thought to reflect activation of mitochondrial
PLA2 which, by breaking down the lipid backbone
of the inner mitochondrial membrane, gives rise to a nonspecific
increase in mitochondrial membrane
permeability.94 However, a different mechanism
giving rise to such an increase in permeability involves the formation
("assembly") of a proteinaceous pore, the MPT pore, which has such
a high conductance that it allows the passage of ions and molecules
with a molecular mass less than 1500 D.94 95 96 97 98 99 
H+) consists of an
H+ concentration gradient and an electrical
potential difference (m). The membrane is
normally impermeable to H+ and other ions, and it
only allows passage of ions (or substrates) for which specific
transport systems exist. This means that H+ can
normally only reenter the mitochondria through the F1F0-ATPase. In that
process, ATP is formed. µH+ and
thereby to cessation of ATP production. As will be discussed
below, an additional consequence is a burst of production of
ROS. m. What then follows is a burst of free
radical production, cell shrinkage, and cell death. ). We interpreted
the results to show that the needle lesion enhanced the BBB
permeability of CsA and that CsA acted by preventing the assembly of an
MPT pore in calcium-loaded mitochondria. At present, this is a
tentative interpretation since CsA has effects other than that of
blocking the MPT pore. These may be related to its immunosuppressant
effects and its ability to combine with cyclophilin, a modulator of the
phosphatase calcineurin. This enzyme affects several
metabolic events, including NO production by
NOS.107 However, FK 506, which is a stronger
immunosuppressant than CsA and which, like CsA, inhibits calcineurin,
is less efficacious than CsA in forebrain
ischemia.108 109 Since FK 506 does not
act as blocker of the MPT, the results suggest that CsA works by
preventing a Ca2+-triggered MPT during
reperfusion.
View larger version (16K):
[in a new window]
Figure 6. Percentage of necrotic cells in the CA1 sector of
the hippocampus after 7 minutes of forebrain ischemia, as
assessed after 7 days of recirculation. Animals treated with CsA and
needle insertion (BBB opened) showed a dramatic decrease in CA1 damage.
***P<.0001, ANOVA, followed by Scheffé's test.
Data from Reference 106.
The hypotheses discussed encompass events that are possibly
linked. For example, a perturbation of signal transduction may alter
the pump-leak relationship for calcium across plasma and intracellular
membranes so that the result is a gradual rise in
Ca2+i and eventual mitochondrial
calcium overload. Since the hypothesis of Abe et
al92 requires further support, we will discuss
the hypothesis of delayed calcium-mediated cell death. In its simplest
form, this hypothesis predicts that the initial ischemic
transient gives rise to a sustained perturbation of membrane handling
of calcium. This then sets the stage for a gradual, secondary rise in
[Ca2+]i, which eventually
causes mitochondrial calcium overload. If this hypothesis is viewed
against the background of our present knowledge of the MPT,
affecting mitochondria that accumulate calcium, we can envisage a
coupling among plasma membrane perturbation, gradual cell calcium
accumulation, mitochondrial dysfunction, and delayed ischemic
cell death. illustrates how these findings can
be incorporated into the general hypothesis discussed. Realizing that
ischemia leads to the influx of Ca2+,
causing [Ca2+]i to rise,
and that reperfusion triggers the extrusion of the calcium accumulated
with normalization of
[Ca2+]i, we must probe
into the mechanisms that are triggered during and immediately after the
ischemic transient, when ROS are formed. As proposed in a
recent article by Siesjö et al,82 ROS could
directly modulate Ca2+ entry pathways or
Ca2+ extrusion mechanisms by oxidation of
proteins or lipids, thereby resetting the pump-leak relationship for
calcium. An alternative possibility is that the effect of ROS is
indirect, reflecting activation of protein kinases and phosphatases or
of endonucleases, which indirectly affect membrane handling of calcium.
For example, ischemia with reperfusion translocates protein
kinase C to membranes, downregulates protein kinase A, alters the
activity of Ca2+-calmodulin–protein
kinase II, reduces or arrests protein synthesis, and alters gene
expression.80 124 125 Any of these events could
modulate, over long periods, membrane handling of
Ca2+. We recognize that a rise in
[Ca2+]i per se
could trigger many of these effects. As discussed above, this does not
necessarily imply that it is the plasma membrane function that is
altered since a correspondingly altered pump-leak relationship for
calcium could exist at the level of ER membranes. The important feature
of the hypothesis is that ischemia and reperfusion perturb
membrane handling of calcium in such a way that
[Ca2+]i is gradually
increased, triggering sequestration of calcium in mitochondria. Cell
death could then be the direct result of a calcium-triggered MPT, since
the latter induces a burst of free radical generation and leads to the
collapse of µH+.
View larger version (17K):
[in a new window]
Figure 7. Coupling between calcium-induced signal
transduction and calcium-triggered production of free radicals.
Enhanced influx of calcium through leak pathways leads to a rise in
Ca2+i that activates protein kinases,
thereby influencing membrane conductance to ions including
Ca2+, mitochondrial respiration, gene expression, and
protein synthesis. Free radicals produced by mitochondria and/or
calcium-activated enzymes may influence leak pathways and
calcium pumps in such a way that the rise in
Ca2+i is sustained. In addition, free radicals
can (by protein oxidation) lead to a downregulation of protein kinases,
thus disturbing the signal transduction that is triggered by calcium
influx. PKC indicates protein kinase C; CAMK-II,
Ca2+-calmodulin–protein kinase II. Modified
from Reference 82.
Global or Focal Ischemia of Long Duration
Top
Abstract
Introduction
Cell Calcium Homeostasis
Forebrain or Global Ischemia...
Global or Focal Ischemia...
Excitotoxicity, Calcium Influx,...
References
When global or forebrain ischemia is prolonged, the free
interval between the insult and the final damage is shortened and, if
the ischemia is of very long duration, recirculation may fail
to restore mitochondrial function and cellular bioenergetic state.
Experiments with extended periods of ischemia also reveal a
rapidly developing, massive calcium accumulation in the tissue. We will
consider in turn recovery of mitochondrial function and changes in
calcium metabolism.
Under adverse conditions, recirculation may fail to be accompanied
by resumption of oxidative phosphorylation of isolated
mitochondria or lead to partial recovery for a limited period only.
Such conditions encompass ischemia of long duration and
ischemia with superimposed hyperglycemia. For example,
incomplete forebrain ischemia of 30 minutes' duration in fed
rats (which became hyperglycemic) was followed by additional
deterioration of mitochondrial respiratory rates during
recirculation.132 133 Furthermore, rapid
maturation of mitochondrial damage has been found in hyperglycemic dogs
subjected to anoxia-ischemia,129 in rats
subjected to long ischemic
periods,134 135 and in gerbils subjected to 30
minutes of forebrain ischemia.136 
-ATP+ADP+AMP) had decreased
accordingly, this degree of recovery must reflect a virtually complete
rephosphorylation of the ADP available to ATP. However,
recirculation for 2 hours did not further increase ATP concentration or
the size of the adenine nucleotide pool and, after 4 hours,
signs of a secondary decrease in ATP concentration were apparent. Since
tissue lactate concentrations showed little tendency to decrease at 1
and 2 hours and increased further at 4 hours, the results suggest
partial recovery of cell energy metabolism and
mitochondrial functions at 1 and 2 hours and secondary failure at 4
hours.
After brief to intermediate periods of ischemia, massive
calcium accumulation, reflecting net transfer of
Ca2+ from blood to tissue, is observed many hours
(or days) after the initial insult (see above). After long periods of
ischemia, the "free" interval is shortened, and substantial
amounts of calcium may accumulate during the first 2 to 3 hours of
reperfusion.78 152 A similarly accelerated influx
of calcium into the brain occurs in permanent or transient focal
ischemia. In some published studies the calcium content was
measured by atomic absorption spectrophotometry, giving quantitative
values for calcium content and the rate of calcium flux from blood to
tissue.11 153 In others,
45Ca autoradiography was
used.154 155 This technique gives good spatial
resolution, but the data cannot be used for quantitative estimates
since the tissue activity must depend on both calcium content and
45Ca transfer rates across the BBB. Despite this
reservation, however, published data are consistent in showing
a rapidly developing true increase in tissue calcium content, whether
the ischemia is permanent or sustained for 1 to 3 hours.
Excitotoxicity, Calcium Influx, and Cell Death In Vitro
Top
Abstract
Introduction
Cell Calcium Homeostasis
Forebrain or Global Ischemia...
Global or Focal Ischemia...
Excitotoxicity, Calcium Influx,...
References
There is now extensive literature on pathological calcium
transients in cultured neurons. A major part of this work was inspired
by the discovery that glutamate and related EAAs are neurotoxic and
that the toxicity is related to Ca2+ influx into
cells14 17 (for early reviews, see References 16
and 7716 77 ). During the last 8 to 10 years, a large body of evidence has
been accumulated that expands the central postulate, ie, that damage is
prone to develop when cells are exposed to sufficiently high
concentrations of EAAs for a sufficiently long period of time and that
the damage depends heavily on the Ca2+ influx
that occurs in response to the EAA
exposure.157 158 ; furthermore, CsA was found to not
only reduce the incidence (and degree) of depolarization but also to
reduce the incidence of cell death.176 182 There
are thus reasons to believe that EAAs act by accelerating
Ca2+ influx into cells, with an ensuing rise in
Ca2+i, that calcium is
sequestered in mitochondria, and that this sequestration gives rise to
the assembly of an MPT pore as well as to production of ROS by
the mitochondria. Clearly, this sequence of events could explain the
delayed cell death after brief periods of ischemia, the rapidly
maturing cell death after long periods of ischemia, or cell
death complicated by preischemic hyperglycemia.
Selected Abbreviations and Acronyms
AMPA
=
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate
BBB
=
blood-brain barrier
CsA
=
cyclosporin A
EAA
=
excitatory amino acid
ER
=
endoplasmic reticulum
IP3, IP4
=
inositol 1,4,5-trisphosphate, inositol
1,3,4,5-tetrakisphosphate
MCA
=
middle cerebral artery
MPT
=
mitochondrial permeability transition
NMDA
=
N-methyl-D-aspartate
NO
=
nitric oxide
NOS
=
nitric oxide synthase
PBN
=
-phenyl-N-tert-butyl nitrone
PLA2
phospholipase A2
ROS
=
reactive oxygen species
VSCC
=
voltage-sensitive calcium channels
Acknowledgments
This study was supported by the Swedish Medical Research Council
(14X-263), the US Public Health Service through the National Institutes
of Health (5 R01-NS-07838), and the Queen's Medical Center,
Honolulu, Hawaii.
References
Top
Abstract
Introduction
Cell Calcium Homeostasis
Forebrain or Global Ischemia...
Global or Focal Ischemia...
Excitotoxicity, Calcium Influx,...
References
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R. Schmid-Elsaesser, E. Hungerhuber, S. Zausinger, A. Baethmann, H.-J. Reulen, and J. A. Zivin Combination Drug Therapy and Mild Hypothermia : A Promising Treatment Strategy for Reversible, Focal Cerebral Ischemia • Editorial Comment: A Promising Treatment Strategy for Reversible, Focal Cerebral Ischemia Stroke, September 1, 1999; 30 (9): e1891 - 1899. [Abstract] [Full Text] [PDF]
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G. P. Schielke, N. C. Kupina, P. A. Boxer, C. F. Bigge, D. F. Welty, and C. Iadecola The Neuroprotective Effect of the Novel AMPA Receptor Antagonist PD152247 (PNQX) in Temporary Focal Ischemia in the Rat • Editorial Comment Stroke, July 1, 1999; 30(7): 1472 - 1477. [Abstract] [Full Text] [PDF]
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 R. C Tasker Pharmacological advance in the treatment of acute brain injury Arch. Dis. Child., July 1, 1999; 81(1): 90 - 95. [Full Text]
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 N. Jovanovic and A. TERZIC Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca2+ homeostasis under metabolic stress FASEB J, May 1, 1999; 13(8): 923 - 929. [Abstract] [Full Text]
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 S. P. Yu, C. Yeh, U. Strasser, M. Tian, and D. W. Choi NMDA Receptor-Mediated K+ Efflux and Neuronal Apoptosis Science, April 9, 1999; 284(5412): 336 - 339. [Abstract] [Full Text]
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L. Kiss and S. J. Korn Modulation of N-Type Ca2+ Channels by Intracellular pH in Chick Sensory Neurons J Neurophysiol, April 1, 1999; 81(4): 1839 - 1847. [Abstract] [Full Text] [PDF]
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C. O. Wambi-Kiesse and Z. S. Katusic Inhibition of copper/zinc superoxide dismutase impairs NO ·-mediated endothelium-dependent relaxations Am J Physiol Heart Circ Physiol, March 1, 1999; 276(3): H1043 - H1048. [Abstract] [Full Text] [PDF]
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 C. Iadecola, C. A. Salkowski, F. Zhang, T. Aber, M. Nagayama, S. N. Vogel, and M. Elizabeth Ross The Transcription Factor Interferon Regulatory Factor 1 Is Expressed after Cerebral Ischemia and Contributes to Ischemic Brain Injury J. Exp. Med., February 15, 1999; 189(4): 719 - 727. [Abstract] [Full Text] [PDF]
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M. Shimizu-Sasamata, T. Kano, J. Rogowska, G. L. Wolf, M. A. Moskowitz, E. H. Lo, and C. Iadecola YM872, a Highly Water-Soluble AMPA Receptor Antagonist, Preserves the Hemodynamic Penumbra and Reduces Brain Injury After Permanent Focal Ischemia in Rats • Editorial Comment Stroke, October 1, 1998; 29(10): 2141 - 2148. [Abstract] [Full Text] [PDF]
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K.-z. Zhang, P. J. Lindsberg, T. Tatlisumak, M. Kaste, H. S. Olsen, and L. C. Andersson Stanniocalcin: A molecular guard of neurons during cerebral ischemia PNAS, March 28, 2000; 97(7): 3637 - 3642. [Abstract] [Full Text] [PDF]
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