Roundtable,4 the authors stress the need of earlier trials to be

scrutinized, emphasizing that new strategies must be used in

the future. In their article, Savitz and Fisher1 put strong emphasis on the improvement of structural stroke readouts, postulating that better magnetic resonance imaging tools may
enable study success. The authors also suggest that combinations of neuroprotectants may be more promising in patients
than single drugs.
The lack of drug efficacy is a problem not only in the
stroke field. Drug failure also worries researchers in other areas of clinical neurology, such as neurodegenerative diseases
and brain cancer, where therapeutic progress was also poor
recently. In view of drug failure being a more general issue,
we doubt that better study end points alone will lead stroke
studies to new breakthroughs.
The translation from animals to humans is a particularly
crucial step in drug development. At this stage, patient groups
and time windows are defined, and drug doses are selected.
Drug biodistribution is a highly complex process with numerous denominators.5 It differs considerably between species and
is profoundly altered in various pathophysiological states.5 As a
consequence, drug concentrations in humans can hardly be
predicted without empirical information.
With this letter, we plead for more precise biodistribution
studies to be made in preparation for drug efficacy trials.
Until now, it has not been possible to measure drug concentrations directly in human brains because of the lack of noninvasive techniques. This has recently changed with the possibility of quantitative drug measurements using positron
emission tomography.5 We predict that more careful bioaccumulation data should lead to more reliable dose selections,
which should greatly increase the overall success of pharmacological studies.
Whether combinations of drugs may improve the success
of future trials, as Savitz and Fisher1 suggested, requires careful scrutiny. Drug combinations confer additional uncertainties to drug therapies, brought about by drug interactions or
alterations in drug biodistribution. Doubts remain whether
the benefits of drug combinations outweigh their unfavorable
consequences.
Department of Neurology, University Hospital Zurich, Zurich,
Switzerland

Reply
Sean Savitz, MD1 and Marc Fisher, MD2
We thank Drs Hermann and Bassetti for their comments in
response to our article on neuroprotection for acute stroke.1
Our colleagues make two important points in their letter.
First, they argue that biodistribution studies of putative neuroprotective agents are needed before advancing to efficacy
trials. They point out that positron emission tomography can
be used to measure drug concentrations in human brain. We
agree that positron emission tomography imaging would be a
useful addition to the evaluation package of neuroprotective
agents to provide topographic and quantitative information.
Second, they raise doubts about combining drugs to improve
the chance of finding an effective therapy. In our article, we
point out that use of single agents targeting multiple pathways of the ischemic cascade is preferable to using multiple
agents, each with only one mechanism of action. However,
there is precedent that combination therapy has potential to
improve outcome, at least in animal models, as witnessed by
recent studies on caffeinol, a combination of ethanol and caffeine2 that has been shown to be safe in patients.3
1

Department of Neurology, University of Texas Houston

Medical School, Houston, TX, and 2Department of Neurology,
University of Massachusetts Memorial Health Care, Worcester,
MA

References
1. Savitz SI, Fisher M. Future of neuroprotection for acute stroke:
in the aftermath of the SAINT trials. Ann Neurol 2007;61:
396 402.
2. Aronowski J, Strong R, Shirzadi A, Grotta JC. Ethanol plus caffeine (caffeinol) for treatment of ischemic stroke: preclinical experience. Stroke 2003;34:1246 1251.
3. Piriyawat P, Labiche LA, Burgin WS, et al. Pilot dose-escalation
study of caffeine plus ethanol (caffeinol) in acute ischemic stroke.
Stroke 2003;34:12421245.

DOI: 10.1002/ana.21237

Female Gonadal Hormones, Migraine, and

Spreading Depression
Ali Gorji, MD

References
1. Savitz SI, Fisher M. Future of neuroprotection in the aftermath
of the SAINT trials. Ann Neurol 2007;61:396 402.
2. Shuaib A, Lees KR, Lyden P, Grotta J, Davalos A, Davis SM,
Diener HC, Ashwood T, Wasiewski WW, Emeribe U. SAINT
II Trial Investigators. NXY-059 for the treatment of acute ischemic stroke. N Engl J Med 2007;357:562571.
3. Lees KR, Zivin JA, Ashwood T, et al, Stroke-Acute Ischemic
NXY Treatment (SAINT I) Trial Investigators. NXY-059 for
acute ischemic stroke. N Engl J Med 2006;354:588 600.
4. Stroke Therapy Academic Industry Roundtable. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 1999;30:27522758.
5. Hermann DM, Bassetti CL. Implications of ATP-binding cassette transporters for brain pharmacotherapies. Trends Pharmacol
Sci 2007;28:128 134.

DOI: 10.1002/ana.21210

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Although sex differences in the incidence of migraine appear

to be related to high circulating levels of female gonadal hormones, the underlying mechanisms are not yet fully understood.1 One possibility is that gonadal hormones may influence cortical spreading depression (CSD), a phenomenon
that was demonstrated as human aura equivalent and suggested to correlate of the neurological symptoms in migraine.2 Brennan and colleagues3 assumed that an increased
cortical excitability could be related to the greater prevalence
of migraine in women as they observed a reduced threshold
for CSD occurrence in female compared with male mice.
Furthermore, although the estrous cycle in female animals
was not monitored, they suggested that female mice likely
were sampled from different estrous phases and concluded
that the increased cortical excitability of the female brain is
not temporally correlated with cyclical hormonal variation.

In my view, the last conclusion is not convincing. The sample rate of their female mice was too small to see any significant differences in CSD threshold among female mice based
on their possible estrous phases. All female mice were in
some extent under the effect of female gonadal hormones
independent of the exact estrous phase. Sachs and colleagues4
investigated the effect of a wide range of estrogen and progesterone concentrations on CSD induced by superfusion of
hypotonic artificial cerebrospinal fluid or KCl injection in rat
neocortical slices. This study indicates a dose-dependent enhancement of the neocortical susceptibility to CSD occurrence in the presence of ovarian hormones. CSD occurred
more frequently and with larger amplitude even at low concentrations (0.1mol/L) of both -estradiol and
progesterone-treated neocortical tissues. Therefore, it would
not be surprising if all female mice at any estrous phase demonstrated a lower threshold for CSD.
Brennan and colleagues3 also did not explain the background mechanism of increased cortical excitability that led
to a greater CSD occurrence. Sachs and colleagues4 reported
a facilitatory effect of female hormones on the somatosensory
neocortical synaptic transmission. Several other experiments
also pointed to the excitatory effect of ovarian hormones on
different neuronal activities. Estrogen increases the number
of N-methyl-D-aspartate (NMDA) binding sites and enhances NMDA receptormediated currents in neuronal tissues.5 From these results, it can be speculated that treatment
with ovarian hormones enhance NMDA receptor activity,
providing the grounds for a facilitated potentiation of synaptic strength. Activation of NMDA receptors is critical for
induction of spreading depression in different neuronal tissues. Therefore, both neuronal and synaptic facilitation induced by these hormones may reduce the CSD threshold
and may be responsible for increased migraine attacks in
women.
Institut fur Physiologie I, Westfalische Wilhelms-Universitat
Munster, Munster, Germany

References
1. MacGregor EA. Oestrogen and attacks of migraine with and
without aura. Lancet Neurol 2004;3:354 361.
2. Gorji A. Spreading depression: a review of the clinical relevance.
Brain Res Rev 2001;38:33 60.
3. Brennan KC, Reyes MR, Lopez Valdes HE, et al. Reduced
threshold for cortical spreading depression in female mice. Ann
Neurol 2007;61:603 606.
4. Sachs M, Pape HC, Speckmann EJ, Gorji A. The effect of estrogen and progesterone on spreading depression in rat neocortical tissues. Neurobiol Dis 2007;25:2734.
5. Foy MR, Xu J, Xie X, et al. 17beta-estradiol enhances NMDA
receptor-mediated EPSPs and long-term potentiation. J Neurophysiol 1999;81:925929.

DOI: 10.1002/ana.21184

Reply
K. C. Brennan, MD and Andrew Charles, MD
We appreciate Dr Gorjis comments on our article and the
opportunity to respond to them. Our results show a sex dif-

ference in the threshold for spreading depression in mice

sampled randomly without estrous cycle monitoring.1 We
conclude from these results that female mice have a reduced
threshold for induction of cortical spreading depression
(CSD) that is unlikely to be explained entirely by increased
excitability correlated with a specific individual phase of the
estrous cycle. Importantly, we do not suggest that cyclical
variations in CSD thresholds associated with the estrous cycle do not occur, or that gonadal hormones do not play a
key role in altering CSD threshold. Rather, we simply conclude that female mice have a lowered threshold for CSD
that is not necessarily temporally synchronized with the estrous cycle. This increased susceptibility to CSD could involve changes in cortical excitability that are mediated by
sustained exposure to gonadal hormones over days to
months, developmental effects of hormones, chromosomal
effects that are independent of hormones, or any combination of these events.
Dr Gorjis groups study offers potential mechanistic insights into our results. Sachs and colleagues2 find that acute
exposure of rat brain cortical slices to estradiol or progesterone increases the amplitude of the electrophysiological
changes of CSD and increases the number of CSD events
evoked by a sustained stimulus. Although Sachs and colleagues2 report involves a substantially different experimental
model than the one we used, their findings that both estradiol and progesterone change CSD properties are indeed
consistent with a sex difference in CSD thresholds that could
occur at multiple phases of the estrous cycle, or in noncycling female mice.
We agree with Dr Gorji that sustained low levels of either
estradiol or progesterone, greater than what are found in
male mice, could contribute to the difference in cortical excitability we see in female mice. However, we question his
assertion that the 0.1M (27ng/ml) concentration of estradiol used in his studies represents a physiological level of the
hormone. Circulating estradiol concentrations in female rodents3,4 and humans5 range from 10 to 400pg/ml, orders of
magnitude lower than the concentration at which Sachs and
colleagues2 found an effect on CSD. Thus, it is unclear
whether the results of Sachs and colleagues2 are directly applicable to our results in vivo. It is also difficult to assess the
significance of the effects of acute progesterone exposure on
CSD thresholds, given that acute effects of progesterone on
neuronal preparations are believed to be primarily mediated
by its metabolite 5-pregnan-3-ol-20-one, or allopregnanolone, via activity at the GABAA receptor.6
Having demonstrated a fundamental sex difference in
CSD thresholds, characterizing the mechanisms underlying
this difference will involve a standard neuroendocrinological
approach.7 First, characterization of CSD in ovariectomized
mice with and without exogenous administration of different
gonadal hormones, and in mice at different phases of the
estrous cycle, will allow determination of the role of the
acute or activational effects of individual sex hormones. More
permanent organizational effects of sex hormones can be investigated during different stages of development. Finally,
novel transgenic mouse models can elucidate sex differences
that are independent of gonadal hormones and are thought

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