Clinical perspectives: neuroprotection lessons from hypoxia-tolerant organisms
Department of Anesthesia, University of California, San Francisco, CA 94143-0542, USA
e-mail: bicklerp{at}anesthesia.ucsf.edu
Accepted 12 March 2004
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Summary |
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Key words: brain, ischemia, hypoxia, neuron, adaptation, hypothermia
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Introduction |
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The vast majority of the current research directed to finding treatments for ischemic or hypoxic brain injuries have been based on identifying, in molecular detail, what goes wrong when the brain is deprived of oxygen and nutrients. These efforts have identified hundreds of events in a number of complex and interactive cascades, which involve, very broadly, the following processes: (1) failure of energy balance (loss of ATP), (2) excitotoxicity (runaway excitatory neurotransmitter release), (3) free-radical damage, (4) inflammation and immune system over-activation, and (5) delayed cell death. Identifying the best target for therapy is thus not easy.
Developing a broad phylogenetic and evolutionary approach to treating brain hypoxia
The hypoxic/ischemic brain presents a myriad of possible targets for
intervention. In addition, neurons are capable of mounting an elaborate series
of defenses against hypoxic injury. A fundamental question is thus raised:
when studying brain hypoxia or ischemia, how can damaging events be separated
from useful defense mechanisms? In hypoxia-sensitive neurons the answer to
this question is often not obvious. Hypoxia-tolerant neurons can provide
critical insights into this problem. One may reasonably argue that, because
studying how cells die has not yielded a treatment for hypoxic brain damage,
it is time to study cells that survive oxygen lack. A main theme of this paper
is that understanding the adaptive responses of neurons to hypoxia can be a
very valuable tool for the pursuit of new neuroprotective strategies. Pursuing
this theme, however, leads to a second and equally important question: how can
such information be translated into new therapies? While it may be foolish to
think that concepts can be directly transferred from the comparative
physiology laboratory to the clinic, why shouldn't breakthroughs in our
understanding of how cells adapt to hypoxic conditions eventually lead to
treatments? Models of such transfer of information to clinical medicine
include the alphastat concept of acidbase balance during
cardiopulmonary bypass (derived from ectothermic vertebrates) and the use of
hypothermia as a treatment for heart attack and trauma (based on hibernation
and winter dormancy).
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Adaptive capacities of neurons to hypoxia: fertile ground for clinical lessons |
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Immediate adaptation to anoxia |
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There are several examples of how turtle neurons have provided new ideas or
insights for neuroprotection concepts that may be useful clinically. One
example concerns the role of Ca2+ in the death of hypoxic or
ischemic neurons. Increases in intracellular calcium
([Ca2+]i) are considered central to ischemic injury in
mammalian neurons, and have provided a target for numerous stroke therapy
trials. Strikingly, moderate increases in [Ca2+]i
(50300 nmol l1) are associated with long-term hypoxic
survival of hypoxia-tolerant neurons from freshwater turtles
(Bickler, 1998), Rana
tadpoles (M. S. Hedrick and P. E. Bickler, personal observation), garden
snails (P. H. Donohoe and P. E. Bickler, personal observation), and even
hippocampal neurons from mammalian neonates
(Bickler and Hansen, 1998
). The
proposition that moderate increases in calcium can be protective has some
foundation in mammalian cells as well. Neuroprotective effects of moderate
increases in [Ca2+]i produced by depolarization or
N-methyl-D-aspartate (NMDA) receptor activation have been
observed in cultured mammalian neurons
(Franklin and Johnson, 1994
).
Moderate increases in Ca2+ are essential for the activation and
full expression of several critical survival pathways, including the MAPK
p42/44 pathway (Fahlman et al.,
2002
) and the Akt pathways
(Cheng et al., 2003
). These
pathways are best known for their neuroprotective effects mediated by
activated growth factor receptors. In addition, hypoxia-induced changes in
gene expression mediated by hypoxia inducible factor (HIF-1
) are
modulated by Ca2+ via calmodulin and MAPKs
(Mottet et al., 2003
). A
[Ca2+]i of 50100 nmol l1 is
typical of healthy neurons and an increase in [Ca2+]i of
100200 nmol l1 is a signal associated with growth and
development (Berridge et al.,
2000
). Conversely, excessively low [Ca2+]i
can promote apoptotic neuron death (Lampe
et al., 1995
). It is thus reasonable to conjecture that moderate
[Ca2+]i increases before, during, or after brain
ischemia may be substantially more conducive to neuronal survival than low
[Ca2+]i during the same periods
(Lee et al., 1999
). This
controversial hypothesis predicts that preventing all but large increases in
[Ca2+]i in neurons during or following brain ischemia
may be detrimental.
Transferring concepts from turtles to mammalian neurons: the role of calcium
Because increases in calcium are associated with surviving anoxia in
hypoxia-tolerant neurons, we have re-examined the role of calcium in cell
death in mammalian neurons, testing the hypothesis that moderate increases in
calcium are protective. We used the rat hippocampal slice culture (HSC) model
(Stoppini et al., 1991) to explore this idea because it is particularly suited
to pharmacologic manipulations and the examination of delayed cell death. We
find that by simply treating the HSCs with ionophores to increase
[Ca2+]i produces impressive resistance to subsequent
oxygen and glucose deprivation. The mechanisms of this protective effect
involve the activation of several recognized neuroprotective signaling
cascades (MAPK ERK1/2 and Akt or protein kinase B). Blocking ERK or Akt or
applying ionophores in calcium-depleted medium reverses the usual protection
(Bickler and Fahlman, 2004).
Further, these studies have has led us to appreciate that other
pre-conditioning strategies may involve increases in
[Ca2+]i as a centrally important event. It is probable
that Ca2+ is directly required as a potentiating cofactor for
activation of critical signaling cascades. These are illustrated in
Fig. 2. Transferred to the
clinic, this concept has the potential of avoiding the deleterious effects of
over-zealous calcium blockade. Of note, the list of failed stroke treatments
involving blocking calcium increases is particularly long. Could this
situation have been at least partially avoided by a more comprehensive
understanding of calcium homeostasis in hypoxic neurons?
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Other findings from turtle neurons are also relevant to clinical
considerations. Studies of anoxia-tolerant neurons from freshwater turtle
(C. scripta) cerebrocortex show that NMDA receptor function is
decreased, but definitely not eliminated, during anoxia
(Bickler, 1998;
Bickler et al., 2000
), and that
glutamate release and re-uptake are modulated during anoxia
(Milton et al., 2002
). This
again is a challenge to a long-held belief that NMDA receptors should be
blocked to stop stroke damage. Finally, turtle neurons can inform us about the
significance of complex signaling events during or following anoxia. The
pro-apoptotic cofactor Bax is upregulated during re-oxygenation in anoxic
turtle cerebrocortex (J. J. Haddad, unpublished data), possibly indicating
that Bax is not always associated with death signaling, or at least that its
role is more complex than currently appreciated.
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Developmental adaptation to hypoxia |
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It has been appreciated for centuries that the neonate is more tolerant of
hypoxia than the adult (Boyle,
1725). Low in utero oxygen tension correlates with the
significantly greater hypoxia tolerance of embryonic or neonatal rats and
their neurons (Bickler and Hansen,
1998
; Friedman and Haddad,
1993
). One notable feature of neonatal neurons is that they have
the capacity to avoid excessive calcium influx mediated by glutamate receptors
and other mechanisms (Bickler and Hansen,
1998
; Friedman and Haddad,
1993
). During hypoxia, neonatal neurons avoid death even though
the concentration of glutamate in the brain tissues may be sufficient to
saturate glutamate receptors (Bickler and
Hansen, 1998
; Cherici et al.,
1991
; Marks et al.,
1996
; Puka-Sundvall et al.,
1997
). Glutamate excitotoxicity is a significant threat to
immature neurons when oxygen is present
(Ikonomidou et al., 1989
). It
is therefore striking that the NMDA receptors that predominate in the neonatal
brain generate large calcium currents
(Burgard and Hablitz, 1993
;
Hestrin, 1992
), which,
although critical for strengthening of synapses
(Durand et al., 1996
;
Tovar and Westbrook, 1999
),
would seem to increase the severity of glutamate excitotoxicity during hypoxic
stress.
The observation that hypoxia silences NMDARs in turtle neurons has produced
interesting new perspectives on the role of hypoxia during development and the
innate hypoxia-tolerance of neonatal tissues. We hypothesized that hypoxia
inhibits NMDA currents in neurons from the mammalian neonate just as it does
in turtles. This proposition must take into account developmental changes in
oxygen tension (in utero brain tissue oxygen tension is about 10 mmHg
and increases to 30 mmHg at birth) as well as the significant changes in NMDAR
composition and properties during the perinatal period. During the first
23 weeks of life, the NMDA receptor subunits NR2A and NR2C are added to
or replace NR2B and NR2D subunits in functional receptors
(Dunah et al., 1996;
Wang et al., 1995
;
Wenzel et al., 1996
). The
increase in NR2A and NR2C subunits in cortex, hippocampus and cerebellum over
the first few weeks of life almost exactly parallels the decrease in the
hypoxia-tolerance of neonatal rats (Adolph,
1948
) and their neurons
(Bickler and Hansen, 1998
;
Friedman and Haddad,
1993
).
It was significant, therefore, that we found that NR1/NR2D receptor
currents were decreased by hypoxia and that NR1/NR2C receptor currents were
increased by hypoxia (Fig. 3;
Bickler et al., 2003). These
studies were done using a Xenopus oocyte expression system, because
NR2D expression in hippocampus peaks between birth and 1 week of age
(Dunah et al., 1996
;
Kirsen et al., 1999
;
Wenzel et al., 1996
). This
effect may explain the inhibition of NMDAR responses during hypoxia in
immature hippocampal neurons. In contrast, hypoxia activated receptors
containing the NR2C subunit. Whereas NR2C subunits are scarce in the neonatal
brain, they are expressed in the cerebellum and other areas after several
weeks of postnatal life (Zhong et al.,
1995
). The pattern of appearance of NR2C receptors in the
cerebellum fits the behavioral effects of hypoxia in rats: in neonates,
hypoxia rapidly induces motor retardation, but in animals older than 2 weeks
hypoxia at least transiently causes increases in motor activity. The
hypoxia-tolerance of rat pups is greatly reduced after 2 weeks of life
(Duffy et al., 1975
).
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An additional and novel insight from this work concerns the increase in oxygen tension at birth; oxygen is probably a signal for synaptic development because it promotes greater currents through NMDARs. The transition from the in utero level of ca. 10 mmHg to 30 mmHg is sufficient to do this. It is of interest that while normal oxygen levels are predicted to promote synapse formation, hypoxia would have the opposite effect and possibly retard normal synaptic development at a crucial period.
There are many fundamental unanswered questions concerning the neonate. One
is how the neonatal brain avoids ATP loss during hypoxia. This response may be
absolutely fundamental, and shared by all hypoxia tolerant cells
(Hochachka, 1986;
Hochachka et al., 1996
). Are
neonatal mitochondria more efficient producers of ATP? Neonatal neurons appear
to `shut-down' and adopt a stasis-like hypometabolic condition that is common
to other anoxia-tolerant organisms
(Hochachka and Guppy, 1987
).
Understanding this posture could pinpoint new approaches to helping vulnerable
cells adapt to hypoxic conditions.
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Slow adaptation |
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In contrast, intermittent hypoxia destroys neurons and provides a valuable
contrast for adaptation to chronic hypoxia. For example, rats tolerate
intermittent hypoxia very poorly, with even mild hypoxia (10% oxygen) coming
in 90 s episodes during the night, causing death of hippocampal neurons and
induction of a number of `stress genes'
(Gozal et al., 2002).
Clinically, individuals with the intermittent hypoxia of sleep apnea suffer
memory loss. Experimental models of chronic versus acute hypoxia
could help identify injurious versus adaptive events.
Risks of engineering hypoxia tolerance: oncogenesis?
Oxygen is also a signal important to oncogenesis, to the growth of tumors
and the growth of blood vessels within them. There is now preliminary evidence
that hypoxia-tolerance mechanisms may confer survival advantage to tumors, or
may themselves be oncogenic. For example, KCNK9 (TASK 3) potassium channels
are upregulated in breast cancer, and when these channels are transfected into
other cells they make the cells hypoxia tolerant, probably because they
hyperpolarize the cell during impending energy failure
(Pei et al., 2003). We have
also demonstrated this tolerance in cultured hippocampal slices transfected
using a Sindbis virus vector (C. S. Yost and P. E. Bickler, unpublished data).
Not all potassium channels are protective, for unclear reasons. The enthusiasm
for potassium channels as a strategy for stroke treatment may be dampened by
these considerations.
Another case of pre-adaptation: hypothermia-associated hypoxia tolerance
Hypothermia may be one of our most valuable clinical tools in the search
for treatments for brain ischemia. All known hypoxia-tolerant neurons tolerate
and benefit substantially from hypothermia. Euthermic mammalian tissues do not
tolerate hypothermia for prolonged periods; problems include cellular edema,
energy loss, activation of stress responses and disorders of intermediary
metabolism, blood coagulation, autonomic regulation, electrical excitability
and stability of the heart, to name a few. Additional clinical issues include
altered drug clearance and increases in metabolism during rewarming (shivering
can lead to myocardial ischemia in some individuals a stress test).
Increasing the hypothermia tolerance of tissues could possibly allow hypoxia
tolerance to be achieved at the same time
(Hochachka, 1986).
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Conclusions and clinical lessons |
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Acknowledgments |
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