Magnesium Research 2013; 26 (2): 67-73
ORIGINAL ARTICLE
doi:10.1684/mrh.2013.0340
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 31/07/2017.
FAST-Mag protocol with or without
mild hypothermia (35◦C) does not
improve outcome after permanent
MCAO in rats
Bruno P. Meloni, Jane L. Cross, Laura M. Brookes, Vincent W. Clark,
Kym Campbell, Neville W. Knuckey
Centre for Neuromuscular and Neurological Disorders/University of Western Australia, Australian Neuro-Muscular Research Institute and Department of Neurosurgery, Sir Charles
Gairdner Hospital, Nedlands, Western Australia, Australia
Correspondence: B P. Meloni. Australian Neuro-Muscular Research Institute. A Block, 4th floor, QEII Medical
Centre, Nedlands, Western Australia, 6009, Australia
<[email protected]>
Abstract. The current study assessed the neuroprotective efficacy of magnesium using a FAST-Mag trial treatment protocol alone, and in combination
with mild hypothermia, in Sprague Dawley rats subjected to permanent, middle
cerebral artery occlusion (MCAO). Treatment with magnesium (MgSO4 .7H2 O)
consisted of an intravenous loading dose (LD: 360 ␮mol/kg) and a 24 hour infusion (120 ␮mol/kg/h), while mild hypothermia at 35◦ C was maintained for 24
hours. Treatment groups consisted of animals receiving: i) saline; ii) magnesium
LD/infusion at 1.5 h/2.5 h post-MCAO; iii) magnesium LD/infusion at 1.5 h/2.5 h
post-MCAO and hypothermia commencing at 2.5 h post-MCAO; iv) magnesium
LD and hypothermia at 1.5 h and magnesium infusion at 2.5 h post-MCAO;
v) hypothermia commencing at 1.5 h post-MCAO and magnesium LD/infusion
at 2.5 h post-MCAO; and vi/vii) hypothermia commencing at 1.5 h or 2.5 h
post-MCAO. No treatment significantly reduced infarct volumes or improved
adhesive tape removal time when measured 48 hours after MCAO. These findings indicate that FAST-Mag treatment alone or with mild hypothermia may
not provide benefit after ischemic stroke, associated with permanent cerebral
artery occlusion.
Key words: stroke, FAST-Mag, magnesium, mild hypothermia, Sprague Dawley
rats, MCAO
Magnesium is currently under investigation in
a phase 3 clinical trial, which aims to determine whether therapy commencing within two
hours of stroke onset improves outcomes [1]. While
some laboratories have demonstrated neuroprotective outcomes, we have observed that when
given as a sole treatment, magnesium is usually not neuroprotective in either focal or global
cerebral ischemia rat models [2-6]. This finding
is consistent with the largely negative magnesium IMAGES trial [7]. One of the reasons
provided for the negative IMAGES findings was
the late recruitment of patients (>95% treated
after 3 h). Consequently, a second magnesium trial
called FAST-Mag was designed, which consisted
of treatment with a magnesium loading dose in
the field by ambulance paramedics within two
hours of stroke symptoms, and following hospital
arrival, followed by a 24-hour magnesium infusion. Patient recruitment in the FAST-Mag trial
was completed early 2013, with results expected
to be published later in the year.
In a number of experimental studies we
have shown that when magnesium treatment is
67
To cite this article: Meloni BP, Cross JL, Brookes LM, Clark VW, Campbell K, Knuckey NW. FAST-Mag protocol with or
without mild hypothermia (35◦ C) does not improve outcome after permanent MCAO in rats. Magnes Res 2013; 26(2): 67-73
doi:10.1684/mrh.2013.0340
B.P. MELONI, ET AL.
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combined with mild hypothermia (35◦ C/24 h), it
can enhance the reduction of brain injury [5, 6].
Furthermore, due to the potential application of
mild hypothermia in awake stroke patients [2] and
its use in a large European phase 3 clinical stroke
trial [8], we chose 35◦ C as the target temperature in the present experiments. Therefore, in this
study, we aimed to simulate FAST-Mag trial treatment conditions in a permanent MCAO rat stroke
model, and to combine this with mild hypothermia
treatment (35◦ C/24 h), either starting at the time
of the magnesium loading dose (1.5 h post-MCAO)
or the magnesium infusion (2.5 h post-MCAO). We
also assessed mild hypothermia alone, with treatment starting at 1.5 or 2.5 hours after stroke onset.
Methods and materials
Experimental groups and treatments
All treatments were randomized and administered in a blinded manner (figure 1). Randomization was performed using a web-based computer
random number generator [9]. Infusion of magnesium or vehicle treatment solution was performed
at 1.5 or 2.5 hours post-MCAO. The dose of magnesium used was based on our previous studies [4-6],
and which results in a near doubling of serum
magnesium levels [10]. The magnesium solution
comprised MgSO4 .7H2 O in 0.9% NaCl, to provide
an intravenous loading dose of 360 ␮mol/kg and a
24 hour intravenous infusion at 120 ␮mol/kg/h. By
volume, animals received an initial loading dose of
300 ␮L given over 10 minutes (100 ␮L heparinised
0.9% NaCl in priming line followed by 200 ␮L
MgSO4 .7H2 O solution), then an infusion rate of
1 ␮L/minute over 24 hours, the total volume being
1740 ␮L. Vehicle infusion comprised 0.9% NaCl at
the same rates as for the magnesium infusion.
Rat permanent focal cerebral ischemia
model
This study was approved by the Animal Ethics
Committee of the University of Western Australia.
Male Sprague Dawley rats weighing 270-350 g
were kept under controlled housing conditions
with a 12-hour light-dark cycle, and with free
access to food and water. Experimental animals
were fasted overnight and subjected to permanent
middle cerebral artery occlusion (MCAO) as follows.
Anaesthesia was induced with 4% isoflurane
and a 2:1 mix of N2 O and O2 via a mask. Anaesthesia was maintained with 1.7-2% isoflurane.
Cerebral blood flow (CBF) was monitored continuously using laser Doppler flowmetry (Blood
FlowMeter, AD Instruments, Sydney, Australia).
The probe was located 1 mm caudal and 4 mm lateral (right) to the bregma. A cannula was inserted
into the right femoral artery to monitor blood pressure continuously and to provide samples for blood
glucose and blood gas readings. Blood glucose was
measured using a glucometer (MediSense Products, Abbott Laboratories, Bedford, MA, USA)
and blood gases were measured using a blood gas
analyser (ABL5, Radiometer, Copenhagen, Denmark). Blood pressure was maintained at 80100 mmHg. During surgery, rectal temperature
was maintained at 37 ± 0.5◦ C, and warming when
Outline of treatment schedules used in trial
pMCAO
1.5 h 2.5 h
≈ 26-27 h
Mg or saline infusion + normothermia or hypothermia (35º)
Saline
Saline
Mg LD
Mg infusion
Mg LD
Mg infusion + Hypo
Mg LD + Hypo
Mg infusion + Hypo
Hypo
Mg LD + Mg infusion + Hypo
Saline
Saline + Hypo
Saline + Hypo
Saline + Hypo
48 h
Body temperature self-regulated
All animals housed at 25ºC, until
euthanasia at 48 h post-MCAO
Figure 1. Timeline of experimental procedures and treatment interventions used in trial (not to scale).
LD = loading dose, Hypo = hypothermia. Saline-control animal received a saline bolus and infusion at
1.5 and 2.5 hours post-MCAO respectively.
68
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FAST-Mag and hypothermia after stroke
necessary with a fan heater. For the intravenous
infusions, a length of PVC line primed with heparinised saline was tied in place in the right
jugular vein, and then externalised through a
dorsal, mid-scapular incision to a tether/swivel
system (Instech Laboratories, Philadelphia, USA)
designed to permit free movement.
The right common carotid artery (CCA) was
exposed via a ventral neck incision. The external
carotid artery (ECA) was isolated after cauterisation of the superior thyroid and occipital arteries.
The isolated section of the ECA was ligated and
cauterised to create a stump. The carotid body was
removed and the pterygopalatine artery was ligated. A 4-0 nylon monofilament with a 0.39 mm
diameter silicone tip (Doccol, Redlands, CA, USA)
was inserted through the ECA stump into the CCA
and advanced rostrally into the internal carotid
artery (ICA) until the laser Doppler flowmetry
recorded a >30% decrease from baseline of cerebral blood flow. The monofilament was secured in
two places (at the base of the ECA stump and
on the ICA) for the remainder of the experiment.
Animals were given post-operative analgesia consisting of pethidine (3 mg/kg intramuscular) and
bupivacaine (1.5 mg/kg subcutaneously) at head
and leg incision sites.
Post-surgical temperature monitoring
A radio-transmitting temperature probe was
inserted into the abdominal cavity during the
surgical procedure for the MCAO to enable
post-operative telemetric thermoregulation (LabVIEW 2010 version 10.0, National Instruments,
Australia) as described previously [5, 6]. The
thermistor probe sends core body temperature data at ≈30 second intervals to a radio
receiver, and computer-controlled software activates a cooling/heating fan and a water mister to
maintain the desired animal body temperature.
Post-surgery animals were allowed to recover
in a climate-controlled chamber and their core
body temperature maintained at normothermia
(37 ± 0.2◦ C) using a cooling/heating fan when
required. At 1.5 or 2.5 hours post-MCAO, mild
hypothermia (35◦ C) was induced and maintained
using the telemetric thermoregulation system.
The cooling period was 20 minutes; 35◦ C ± 0.2◦ C
target temperature was maintained for 24 hours
before gradual rewarming to 37◦ C over 1 hour.
Normothermic animals continued to have their
body temperature maintained at 37 ± 0.2◦ C.
Tissue processing and infarct volume
measurement
Animals were sacrificed 48 hours post-MCAO with
intra-peritoneal injections of sodium pentobarbitone (900 mg/kg). After euthanasia, the brain
was removed and placed in a sterile container
of 0.9% NaCl and then placed in a freezer at
-80◦ C for seven minutes. The brain was then
sliced coronally from the junction of the cerebellum and cerebrum to 12 mm rostral to this point in
2 mm thick slices. Slices were immediately stained
with 1% 2,3,5 triphenyltetrazolium chloride (TTC,
Sigma, St Louis, MO, USA) at 37◦ C for 20 minutes, followed by fixation in 4% formalin at room
temperature for at least 18-24 hours before infarct
volume measurements. Slices were scanned and
images were analysed by an operator blind to
treatment status using ImageJ 3rd edition (NIH,
USA). The total infarct volume was determined by
adding the areas of infarcted tissue on both sides
of the 2 mm sections. These measured areas were
multiplied by half-slice thickness (1 mm), and
corrected for cerebral oedema by multiplying the
ratio of affected to normal hemisphere areas [5].
Adhesive tape removal test
This test measured the detection of (sensory
parameter) and reaction to (motor component)
small pieces of adhesive tape placed on the forelimbs [11]. Animals had adhesive removal tests
performed prior to surgery and at 48 hours postMCAO. Animals were placed in a transparent
enclosure for two minutes prior to starting the
tests in order to adapt to placement in a new
enclosure (habituation). Adhesive tape (Diversified Biotech, Dedham, MA, USA) was placed on
the palmar surface of the paw and the time from
first contact (detection) of the adhesive tape to the
time of removal of the tape was measured and
recorded for each forelimb.
Statistical analysis
For infarct volume measurements, each treatment group was compared to its respective vehicle
control group by analysis of variance (ANOVA)
followed by post-hoc Fisher’s PLSD test. ANOVA
was employed to compare physiological parameters between groups. For the adhesive tape
test, data were log transformed prior to ANOVA
with the statistical package R (version 2.11.1). A
69
B.P. MELONI, ET AL.
value of P<0.05 was considered significant for
all data sets. Data in figures are presented as
mean ± standard deviation. Statistical power calculations showed that a reduction in mean infarct
volume of 40% would be required to show significance with the present sample sizes (note for
groups with numbers ≥9) with 80% power.
Experimental groups and treatments
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Infusion of magnesium or vehicle treatment solution was performed at 1.5 or 2.5 hours post-MCAO
(figure 1). The magnesium solution comprised
MgSO4 .7H2 O in 0.9% NaCl, to provide an intravenous loading dose of 360 ␮mol/kg and a 24-hour
intravenous infusion at 120 ␮mol/kg/h. Vehicle
infusion consisted of 0.9% NaCl at the same rates
as for the magnesium infusion. At 1.5 or 2.5
hours post-MCAO, mild hypothermia (35◦ C) was
induced and maintained using a thermoregulation
system.
There were two animal mortalities during the
trial: one in the magnesium 1.5 h/hypothermia
2.5 h treatment group (≈ 43 h post-MCAO), and
one in the normothermia/saline treatment group
(≈ 45 h post-MCAO). One animal in the magnesium 1.5 h/hypothermia 2.5 h treatment group
was euthanised after suffering a seizure (≈ 45 h
post-MCAO). Note: infarct volume measurements
were obtained for all unplanned animal deaths
(denoted by † in figure 2).
Infarct volume measurements
The mean total infarct volumes for each treatment
group were determined 48 hours after the MCAO
and are presented in figure 2. No treatment significantly reduced infarct volume compared to the
normothermic vehicle-treated rats.
Functional assessment
Adhesive tape removal was highly variable within
groups and no significant improvement was
observed between control and treatment groups
for either the left or right paw (figure 3).
Results
Physiological measurements, body
temperature control and animal deaths
The physiological parameters for the control
and treatment groups assessed pre-ischemia, and
mean core body temperatures during the temperature control period for each experimental group
are presented in table 1. There were no statistically significant differences between the groups.
Discussion
In 2004, the IMAGES trial returned a negative
outcome, however analysis of 79 patients treated
with magnesium within three hours of stroke
onset showed a positive treatment trend [7]. With
Table 1. Physiological variables (mean ± SD) for trial (immediately before occlusion).
Control
Saline
(N = 10)
Mg LD: 1.5 h Mg LD: 1.5 h Mg LD: 1.5 h Hypo: 1.5 h Hypo: 1.5 h
Mg INF: 2.5 h Mg INF: 2.5 h Hypo: 1.5 h Mg LD: 2.5 h
(N = 6)
(N = 9)
Hypo: 2.5 h Mg INF: 2.5 h Mg INF: 2.5 h
(N = 8)
(N = 9)
(N = 11)
PO2 (mmHg)
Before MCAO
117.4 ± 27.75 126.88 ± 24.21 133.88 ± 25.84 117.11 ± 28.05 134.45 ± 25.43
PCO2 (mmHg)
Before MCAO
43.3 ± 8.86
42.0 ± 6.71
42.25 ± 9.05
42.67 ± 8.25
42.73 ± 8.73
pH
Before MCAO
7.34 ± 0.05
7.35 ± 0.04
7.38 ± 0.05
7.36 ± 0.05
7.35 ± 0.05
Glucose
(mmol/L)
Before MCAO
6.15 ± 1.61
6.94 ± 1.93
5.83 ± 0.68
5.84 ± 1.53
6.39 ± 1.7
MAP (mmHg)
Before/During
80.28 ± 11.06 82.31 ± 9.62
82.98 ± 9.14
78.46 ± 6.30
74.99 ± 8.5
MCAO
Temperature
(◦ C)
During 24 h
37.08 ± 0.33 36.99 ± 0.28
35.08 ± 0.35
35.10 ± 0.36
35.09 ± 0.36
maintenance
period
LD = loading dose, INF = 24 h IV infusion, Hypo = 35◦ C for 24 h.
70
Hypo: 2.5 h
(N = 5)
128.67 ± 20.5 117.2 ± 29.85
46.83 ± 7.78
40.4 ± 9.76
7.30 ± 0.05
7.35 ± 0.04
6.88 ± 2.11
5.82 ± 0.84
78.55 ± 6.29
77.2 ± 10.5
35.08 ± 0.39
35.08 ± 0.35
FAST-Mag and hypothermia after stroke
500
†s
3
Total Infarct Volume (mm )
450
400
350
†
300
250
200
150
100
50
†
0
Mg LD: 1.5h Mg LD: 1.5h Mg LD: 1.5h Hypo: 1.5h Hypo: 1.5h
Mg INF: 2.5h Mg INF: 2.5h Hypo: 1.5 h Mg LD: 2.5h
Hypo: 2.5 h Mg INF: 2.5h Mg INF: 2.5h
Hypo: 2.5h
Treatment Groups
Figure 2. Infarct volume measurements following different magnesium (IV loading dose:
360␮mol/kg/infusion: 120 ␮mol/kg/h/24 h) and/or
hypothermia (35◦ C/24 h) treatment combinations starting at different times after permanent
MCAO. LD = loading dose, INF = infusion,
HYPO = hypothermia. Means ± SD. † Denotes animals that died 43-45 hours post-MCAO. †s Denotes
an animal that was euthanised after suffering a
seizure 45 hours post-MCAO.
respect to positive treatments, hypothermia is
considered the gold standard for neuroprotection [12], following favourable outcomes in animal
cerebral ischaemia studies [13] and cardiac arrest
3
3
Time to detect tape
Time log10 (sec)
Time log10 (sec)
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Control
(saline)
clinical trials [14, 15]. Consequently, moderate
hypothermia (33◦ C) is used clinically following
cardiac arrest, and mild hypothermia (34-35◦ C)
is the subject of a phase 3 clinical stroke trial
[8]. Importantly, we have previously demonstrated
that magnesium can improve the neuroprotective efficacy of mild hypothermia (35◦ C) in both
global cerebral ischaemia (i.e. ischaemia that
occurs after cardiac arrest) and stroke models
[5]. However, the present study using a permanent MCAO stroke model, and administration of
a magnesium loading dose at 1.5 or 2.5 hours
after occlusion, followed by a 24-hour magnesium infusion, either alone or combined with mild
hypothermia, did not significantly reduce infarct
volume.
These findings are contrary to a previous study
from our laboratory that demonstrated a neuroprotective effect of combined magnesium/mild
hypothermia therapy when begun up to four hours
after permanent MCAO [5]. However, in line with
our previous study, treatment with magnesium
or mild hypothermia alone were ineffective when
administered two hours after permanent MCAO.
We do not know why the current combined magnesium/hypothermia findings are contradictory, but
they could be related to: i) magnesium loading
and infusion treatments being performed one hour
apart, instead of sequentially; ii) different animal operators performing the trial; iii) use of the
2
1
0
Time to remove tape
2
1
0
Right pawprior
Left pawprior
Right pawpost
Treatment Groups
Left pawpost
Right pawprior
Left pawprior
Right pawpost
Left pawpost
Treatment Groups
Figure 3. Functional assessment using the adhesive tape removal test in Sprague Dawley rats prior to
and post-stroke. Treatment groups from left to right: i) saline; ii) magnesium LD/infusion at 1.5 h/2.5 h
post-MCAO; iii) magnesium LD/infusion at 1.5 h/2.5 h post-MCAO and hypothermia commencing at
2.5 h post-MCAO; iv) magnesium LD and hypothermia at 1.5 h and magnesium infusion at 2.5 h
post-MCAO; v) hypothermia commencing at 1.5 h post-MCAO and magnesium LD/infusion at 2.5 h
post-MCAO; vi) hypothermia commencing at 1.5 h post-MCAO; and vii) hypothermia commencing at
2.5 h post-MCAO. Post-stroke assessment was performed immediately prior to euthanasia (48 h after
MCAO). No treatment significantly improved adhesive tape detection or removal times for the left or
right paw. Values are mean (± SD) times in seconds (sec; log transformed), maximum time 120 seconds.
71
B.P. MELONI, ET AL.
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thermoregulation system to maintain normothermia from immediately after surgical recovery
instead of only during the normothermia or
hypothermia maintenance phase; and iv) the differential response of different batches of Sprague
Dawley rats to MCAO. Regardless of whether one
or more of these factors has influenced our results,
what is critical is that it appears that treatment
with magnesium and/or mild hypothermia may
not provide robust neuroprotection. Furthermore,
based on these negative findings we predict that
the FAST-Mag trial will return a similar outcome,
at least in stroke patients suffering a permanent
cerebral artery occlusion.
It is possible that by starting magnesium and
hypothermia treatment earlier (e.g. 0.5 h versus 1.5 h post-MCAO), we would have observed
a beneficial effect. We chose a late FAST-Mag
trial treatment starting point for magnesium
in our study to determine if a wider therapeutic window was effective, especially when
combined with mild hypothermia. It should also be
mentioned that in stroke associated with revascularisation, spontaneous or induced (thrombolysis
therapy or mechanical intervention), treatment
with magnesium and hypothermia at 1.5-2.5
hours post-stroke or immediately after reperfusion at even later time points (e.g. 3-6 h
post-stroke) may be effective in improving outcomes. To this end, studies in our laboratory have
shown that following transient MCAO (90min),
treatment with magnesium and mild hypothermia is effective when started immediately or 30
minutes post-reperfusion, but not at 90 minutes
post-reperfusion [16].
A potential limitation of the study that may
have contributed to the negative outcome was
the level of statistical power based on animal numbers used to detect a treatment effect.
Animal numbers in the control, magnesium
only, magnesium 1.5 h/hypothermia 1.5 h, and
hypothermia 1.5 h/magnesium 2.5 h treatment
groups ranged from nine to 11, while magnesium
1.5 h/hypothermia 2.5 h, and hypothermia only
treatment groups ranged from five to eight animals. While increasing animal numbers would
have increased the statistical power, we feel that
considering that there was no observable trend
in mean infarct volume reductions for treatment
groups with nine to 11 animals, our current
findings are unlikely to represent false negative
results, at least for groups with ≥ nine animals.
72
Conclusions
If FAST-Mag results turn out to be positive, magnesium will be the first clinical stroke treatment
considered to have a direct neuroprotective action.
If however, they are negative, magnesium it will
join a long list of agents that have failed at clinical trial. However, magnesium is an important
electrolyte that if not maintained within normal
limits in the serum and CNS, is likely to worsen
ischemic brain injury [17]. Therefore, maintaining
magnesium homeostasis should still be considered to be an important intervention following
stroke, and whether it can be combined with other
therapies to enhance any neuroprotective effects
requires additional investigation. Similarly, for
mild hypothermia (35◦ C) to be beneficial after
stroke, it may require adjunct pharmacotherapy
to improve efficacy. Finally, the present findings
mainly relate to stroke arising from a permanent occlusion in a major cerebral artery, and
hence better outcomes are likely to be achievable
with magnesium/mild hypothermia therapy associated with cerebral revascularization and/or mild
stroke.
Disclosure
Financial support: This study was supported by
the National Stroke Foundation (Australia) and
by an equipment grant from the Rebecca Copper Research Foundation (temperature system
upgrade). Conflict of interest: none.
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