J Appl Physiol 112: 2068 –2076, 2012.
First published April 5, 2012; doi:10.1152/japplphysiol.00092.2012.
Prothrombolytic action of normobaric oxygen given alone or in combination
with recombinant tissue-plasminogen activator in a rat model of
thromboembolic stroke
H. N. David,1 B. Haelewyn,2 M. Degoulet,2 D. G. Colomb Jr.,3 J. J. Risso,4 and J. H. Abraini1,2
1
Centre de Recherche, Centre Hospitalier Affilié Universitaire Hôtel-Dieu de Lévis, Lévis, Quebec, Canada; 2Université de
Caen-Basse Normandie, Caen, France; 3Navy Experimental Diving Unit, Panama City, Florida; and 4Institut de Recherche
Biomédicale des Armées, ERRSO, Toulon, France
Submitted 19 January 2012; accepted in final form 3 April 2012
acute ischemic stroke; normobaric oxygen; thrombolysis; tissue-plasminogen activator
is a major cause of mortality and
long-term neurologic morbidity in the adult population. The
primary cause of acute ischemic stroke is a disruption of
cerebral blood flow through thromboembolism that leads to an
oxygen and glucose deprivation for the cell, and subsequent
secondary excitotoxic mechanisms (8, 27). Limitation of the
primary vascular insult by early reperfusion still remains the
only approved therapy of acute ischemic stroke to date, since
blockade of the excitotoxic cascade has failed to be efficient in
humans. Although new techniques and devices for mechanical
removal of the thrombus are being developed (13), the only
approved therapy of acute ischemic stroke to date is thrombolysis by recombinant human tissue plasminogen activator
(rtPA). Although adverse hemorrhagic effects have been reported, early reperfusion by rtPA has benefited ischemic stroke
patients if given within 3– 6 h of symptoms onset (14, 25, 42).
ACUTE ISCHEMIC STROKE
Address for reprint requests and other correspondence: J. H. Abraini,
Centre Hospitalier Affilié Universitaire Hôtel-Dieu de Lévis, Lévis, Quebec, Canada and Université de Caen Basse, Normandie, Caen, France
(e-mail: [email protected]).
2068
Unfortunately, although single centers have reported higher
rates of rtPA administration, US national and population-based
estimates of the use of rtPA therapy have been estimated and
recently confirmed to be substantially less than 5% of acute
ischemic stroke patients mainly due to some contraindications
to treatment and most importantly to the narrow therapeutic
time window of rtPA (23, 34).
Alternatively, as tissue hypoxia plays a critical role in the
primary and secondary events that lead to ischemia-induced
neuronal death (46), tissue oxygenation with oxygen-enriched
medical air or 100 vol% normobaric oxygen (NBO) is generally thought to be a logical therapeutic stroke strategy. Accordingly, experimental studies have demonstrated beneficial effects of NBO if started early after cerebral ischemia (9, 19, 22,
37, 38). However, in contrast, clinical studies performed with
oxygen-enriched medical air or NBO have led to more puzzling conclusions. While one half of these studies has led to
negative results (33, 34), the other half has concluded that
NBO may benefit the ischemic patient (4, 36) possibly by
increasing reperfusion (36). In line with this latter study, recent
magnetic resonance imaging (MRI) data from a mechanical
model of acute ischemic stroke have shown that NBO increases
cerebral blood flow (CBF) in the ischemic penumbra (2).
However, the only two studies performed in rats subjected to
thromboembolic middle cerebral artery occlusion (MCAO)
have failed to show significant NBO-induced changes in CBF
(10, 20). However, it is noteworthy that these latter studies
were performed in rats that were shifted to NBO immediately
after nitrous oxide, a condition that could have biased results
since nitrous oxide has been shown to inhibit potently thrombolysis (17).
To advance the critical question of the use of NBO in acute
ischemic stroke, we studied the effects of NBO administered
alone or in combination with rtPA on CBF and ischemic brain
damage in rats exposed to thromboembolic MCAO-induced
brain ischemia, a clinically relevant model of acute ischemic
stroke in rodents.
MATERIALS AND METHODS
Animals. All animal-use procedures were examined by a local ethic
committee (Cyceron, Caen, France) in accordance with the European
Communities Council Directive of 24 November 1986 for the use of
animals in biomedical experimentation (86/609/EEC, also known as
the Declaration of Helsinki), and approved with permit number
14 –27. Before surgery, all animals were housed at 21 ⫾ 0.5°C with
lights on from 8:00 pm to 8:00 am in a group of four in Perspex home
cages with free access to food and water. After surgery, all animals
were housed individually in similar environmental conditions.
http://www.jappl.org
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David HN, Haelewyn B, Degoulet M, Colomb DG Jr, Risso JJ,
Abraini JH. Prothrombolytic action of normobaric oxygen given alone or in
combination with recombinant tissue-plasminogen activator in a rat model of
thromboembolic stroke. J Appl Physiol 112: 2068–2076, 2012. First published April 5, 2012; doi:10.1152/japplphysiol.00092.2012.—The potential
benefit of 100 vol% normobaric oxygen (NBO) for the treatment of
acute ischemic stroke patients is still a matter of debate. To advance
this critical question, we studied the effects of intraischemic normobaric oxygen alone or in combination with recombinant tissue-plasminogen activator (rtPA) on cerebral blood flow and ischemic brain
damage and swelling in a clinically relevant rat model of thromboembolic stroke. We show that NBO provides neuroprotection by
achieving cerebral blood flow restoration equivalent to 0.9 mg/kg
rtPA through probable direct interaction and facilitation of the fibrinolytic properties of endogenous tPA. In contrast, combined NBO and
rtPA has no neuroprotective effect on ischemic brain damage despite
producing cerebral blood flow restoration. These results 1) by providing a new mechanism of action of NBO highlight together with
previous findings the way by which intraischemic NBO shows beneficial action; 2) suggest that NBO could be an efficient primary care
therapeutic intervention for patients eligible for rtPA therapy; 3) indicate
that NBO could be an interesting alternative for patients not eligible for
rtPA therapy; and 4) caution the use of NBO in combination with rtPA
in acute stroke patients.
Prothrombolytic Effect of Oxygen
David HN et al.
2069
Assessment of brain damage, brain hemorrhages, and disruption of
the blood-brain barrier. Rats were killed by decapitation under
isoflurane anesthesia 24 h after the onset of ischemia or injection of
NMDA or NMDA plus rtPA. Studies with excitotoxic, transient, or
permanent ischemic models of brain injury have shown using magnetic resonance imaging, triphenyltetrazolium chloride, thionin, and
neuronal nuclei immunochemical staining techniques that assessment
of infarct size at 24 h is a time condition sufficient to obtain
consolidated neuronal death, i.e., whose assessment would be similar
if performed one or several days later (11, 15, 21). Before being
killed, rats were given Evans blue intravenously (4% in 1 ml saline
solution). Then the brain was removed and frozen in isopentane.
Coronal brain sections (20 ␮m) were cryostat-cut, mounted on slides,
stained with thionin, and digitized on a computer. The lesion areas
were delineated by the pallor of staining in the necrotic tissue
compared with the surrounding healthy tissue; then volumes of brain
damage were estimated by integration over the whole brain of the
infarct surfaces calculated with the ImageJ software analyzer (Scion,
MD), corrected for tissue edema when needed (ischemia) by calculating and dividing volume by the ipsilateral/contralateral brain hemispheres ratio, and expressed in cubic millimeters. While cutting the
unstained brain mounted on the cryostat, macrophotographs were
taken, digitized on a computer, and analyzed using the ImageJ
software analyzer to assess brain hemorrhages and disruption of the
blood-brain barrier as detailed previously (6). Evans blue extravasation was delineated, and multiplied by the extravasation’s optical
density. Brain hemorrhages were delineated as blood evident at the
macroscopic level, and expressed in square millimeters. The experiments were performed in a blinded manner according to the Stroke
Therapy Academy Industry Roundtable (STAIR) recommendations as
follows: the investigators who performed the surgery and treatment
gave each group of rats a secret code that remained unknown to the
experimenters in charge of assessing brain tissue damage, brain
hemorrhages, and disruption of the blood-brain barrier until the end of
the study.
rtPA and reteplase catalytic efficiency assay. The effects of oxygen
at 0, 25, 50, 75, and 100 vol% on the catalytic efficiency of tPA
(alteplase) and reteplase, a tPA-derived drug that lacks the fibronectin
type I, EGF-like, and kringle 1 domains and thereby only possesses
the kringle 2 and catalytic domains, were assessed as detailed previously (6). Briefly, the recombinant form of human tPA in the form of
Actilyse, reteplase in the form of Rapilysin (Actavis, Le PlessisRobinson, France), and their specific chromogenic substrate methylsulfonyl-D-phenyl-glycil-arginine-7-amino-4-methylcoumarin acetate
(spectrozyme XF, ref. 444; American Diagnostica, Stamford, CT)
were separately diluted in 1 ml of distilled water in 1.5-ml sterile
tubes. Each tube containing 0.4 ␮M of tPA, 0.09 unit of reteplase, or
10 ␮M of the tPA substrate was saturated for 20 min with oxygen of
0 to 100 vol% (with the remainder being nitrogen when necessary).
The catalytic efficiency of human tPA and reteplase (for each, n ⫽ 12
per oxygen concentration) was assessed using the initial rate method
by incubating 50 ␮l of tPA with 50 ␮l of substrate at 37°C in a
spectrofluorometer microplate reader.
In vitro thrombolysis experiments. In vitro thrombolysis experiments were performed as detailed previously (6). Briefly, whole blood
samples of 500 ␮l volume drawn from male mature Sprague-Dawley
rats weighing 600 – 650 g (n ⫽ 4) were transferred in preweighed
tubes of 1.5 ml and incubated at 37°C for 3 h. After clot formation and
total serum removal, each tube was weighed to determine the clot
weight. Blood clots were selected in the same weight range (0.296 ⫾
0.009 g) to reduce variability. Each tube was filled with saline solution
containing 1 ␮g/ml of tPA in the form of Actilyse saturated with 100
vol% oxygen (n ⫽ 13) or air (n ⫽ 12), and incubated at 37°C for a
90-min period. Then the fluid was removed, and the tubes were
weighed again to assess the percentage of clot lysis induced by rtPA
in the presence of medical air or 100 vol% oxygen.
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Middle cerebral artery occlusion-induced ischemia. Male SpragueDawley rats weighing 250 –275 g were used to assess the effects of
NBO given alone or in combination with rtPA on CBF and MCAOinduced brain damage. Rats were subjected to MCAO-induced ischemia by administration of an autologous blood clot as described in
detail previously (6). Briefly, 24 h before the animals were subjected
to ischemia a whole caudal blood sample of 200 ␮l was withdrawn
and allowed to clot at 37°C for 2 h. Then, the clot was extruded from
the catheter into a saline-filled petri dish and stored at 4°C for 22 h
before being used the day after. On the day of surgery, the rats were
anesthetized with 2 vol% isoflurane in medical air composed of 25
vol% oxygen and 75 vol% nitrogen, intubated, and ventilated artificially. Catheters were inserted into the femoral vein to allow injection
of rtPA or saline solution, and in the femoral artery for continuous
monitoring of heart rate, diastolic, systolic, and mean arterial pressures, and for the periodic analysis of blood gases and pH. Rats were
placed prone in a stereotaxic frame, and a laser-Doppler flowmetry
probe was positioned onto the right parietal bone, previously thinned
with a saline-cooled dental drill (coordinates 1.7 mm posterior, 5.5
mm lateral from the bregma), to assess successful induction of
cerebral ischemia and to monitor changes in CBF continuously.
Changes in CBF were expressed as a percentage from CBF control
values recorded during a 20-min preocclusion period. A single clot
measuring 40 mm in length was injected in a volume of 50 ␮l saline
solution through microtubing directed into the internal carotid artery
up to 2 mm after the pterygopalatin-internal carotid artery bifurcation.
Following a 45-min period of occlusion during which all rats were
given medical air, microtubing was removed from the internal carotid
artery to the external carotid artery and the rats were given rtPA ⫹
medical air, NBO ⫹ saline, or NBO ⫹ rtPA. rtPA alone or in
combination with NBO was given over a 45-min period at the dose of
0.9 mg/kg in 1 ml saline solution (10% bolus and 90% perfusion), a
dose shown to be more clinically relevant particularly in gas pharmacology studies than that of 10 mg/kg often used in rodents studies
(18). NBO alone was also given for 45 min. Then all rats were given
medical air again. Rats were maintained normothermic throughout the
surgery and ischemia-reperfusion protocol. Then the rats were returned back to their home cage and allowed to move freely with food
and water ad libitum. To mimic clinical recommendations (31), rats
showing less than 50% (controls, n ⫽ 1; rtPA, n ⫽ 1; NBO, n ⫽ 2;
NBO ⫹ rtPA, n ⫽ 2) or more than 85% reduction (rtPA, n ⫽ 2; NBO,
n ⫽ 1) in CBF as calculated over a 15-min period before treatment
were excluded from the study design. Also, rats that died during
MCAO-induced ischemia (n ⫽ 7) and those that died before being
used for histologic analysis (n ⫽ 3) were excluded from statistical
data. The final number of animals taken into account for statistical
analysis was n ⫽ 5 per group. Brain damage was assessed as
described below.
NMDA-induced neuronal death in vivo. On the day of surgery, rats
were anesthetized with 1.5% halothane in medical air and mounted on
a stereotaxic apparatus with the incisor bar set at 3.9 mm below the
horizontal zero. During surgery, body temperature was kept at 37 ⫾
0.5°C. A burr hole was drilled and a micropipette (⬃10 ␮m at the tip)
was lowered into the right striatum (anterior, 0.6 mm; lateral, 3.0 mm;
ventral, 5.8 mm, from bregma) to allow injection of 50 nmol of
N-methyl-D-aspartate (NMDA) alone or in combination with 3 ␮g
rtPA in the form of Actilyse in 1 ␮l of PBS (pH 7.4) over a 2-min
period. After an additional 5-min period, the micropipette was removed and the animals were returned to their home cage with free
access to food and water. Sixty minutes after administration of
NMDA or NMDA plus rtPA, rats were treated for 3 h with medical air
or 100 vol% oxygen as described above. The number of animals was
as follows: NMDA ⫹ air, n ⫽ 12; NMDA ⫹ rtPA ⫹ air, n ⫽ 8;
NMDA ⫹ rtPA ⫹ NBO, n ⫽ 8. No rat was excluded from statistical
analysis. Brain damage was assessed as described below.
•
2070
Prothrombolytic Effect of Oxygen
•
David HN et al.
Table 1. Values of PaCO2, arterial pH, and temperature
PaCO2, mmHg
Air
NBO
pH
Temp, °C
Ctrl
Isch
Reperf
Ctrl
Isch
Reperf
Ctrl
Isch
Reperf
40 ⫾ 1
38 ⫾ 2
38 ⫾ 1
38 ⫾ 2
39 ⫾ 1
39 ⫾ 1
7.45 ⫾ 0.01
7.49 ⫾ 0.01
7.43 ⫾ 0.01
7.47 ⫾ 0.01
7.39 ⫾ 0.01
7.45 ⫾ 0.01
37.4 ⫾ 0.2
37.8 ⫾ 0.1
37.5 ⫾ 0.1
37.6 ⫾ 0.1
37.4 ⫾ 0.2
37.5 ⫾ 0.1
Data are expressed as means ⫾ SE. PaCO2, arterial carbon dioxide partial pressure; Temp, temperature; Ctrl, control values recorded during a 20-min period
of control before the onset of ischemia; Isch, mean values recorded during the 45-min period of ischemia; Reperf, mean values recorded during the 45-min period
of treatment; NBO, normobaric oxygen at 100 vol%. *P ⬍ 0.02 vs. medical air.
fact that NBO increased the catalytic activity of tPA by approximately
the same amplitude as 37.5 vol% xenon did decrease it (7): mean ⫾
SD for rtPA ⫽ 163 ⫾ 59 mm3; mean ⫾ SD for xenon at 37.5 vol% ⫽
317 ⫾ 62 mm3; ␣ error level (confidence level) ⫽ 0.05; ␤ error level
(or statistical power, 1 ⫺ ␤) ⫽ 0.2 (or 0.8); minimum number of
animals required, n ⫽ 3.
RESULTS
NBO has dual effects depending on if given alone or in
combination with rtPA. First, we studied the effects of NBO
given alone or in combination with rtPA in male adult SpragueDawley rats subjected to thromboembolic brain ischemia.
Upon clot injection, CBF dropped to about 30% of preischemic
baseline, and remained stable throughout the 2-h duration of
the study in control animals but not in rats treated with rtPA
and/or NBO. Physiological parameters remained within normal
range in all groups (Table 1). Before NBO, arterial PO2 levels
were between 75 and 85 mmHg in all rats. NBO given alone or
in combination with rtPA rapidly and markedly elevated arterial PO2 levels to above 400 mmHg (Table 2).
Control rats treated with saline solution and medical air
showed no CBF restoration (Fig. 1, A and B), had ischemic
brain damage and swelling, respectively, of 395 mm3 (25–75
percentiles: 370 – 402 mm3; Fig. 1C) and 21.9% (16.7–23.7%;
Fig. 1D), and further exhibited brain hemorrhages and disruption of the blood-brain barrier (Fig. 1, E and F), respectively,
in the form of discrete hematomas and dark and discrete areas
of Evans blue extravasation (Fig. 1, G and H).
As expected, rats treated with rtPA (and medical air) had
sustained CBF restoration (Fig. 1, A and B), and reduced
ischemic brain damage and swelling, respectively, of 167 mm3
(25–75 percentiles: 160 –209 mm3; P ⬍ 0.01; Fig. 1C) and
10.1% (25–75 percentiles: 7.3–12%; P ⬍ 0.01; Fig. 1D)
compared with control animals. However, despite these beneficial effects and in line with the well-known adverse properties
of rtPA therapy, rtPA-treated rats showed no reduction of brain
hemorrhages (Fig. 1, E, G, and H) and disruption of the
blood-brain barrier (Fig. 1, F–H) compared with control rats.
Likewise, rats treated with NBO (and saline solution) showed
sustained CBF restoration (Fig. 1, A and B), and exhibited reduced
ischemic brain damage and swelling, respectively, of 93 mm3
Table 2. Values of PaO2, SaO2, and MAP
PaO2, mmHg
Air
NBO
SaO2
MAP, mmHg
Ctrl
Isch
Reperf
Ctrl
Isch
Reperf
Ctrl
Isch
Reperf
88 ⫾ 12
84 ⫾ 3
85 ⫾ 14
76 ⫾ 3
93 ⫾ 2
506 ⫾ 28*
96 ⫾ 1
97 ⫾ 1
96 ⫾ 2
96 ⫾ 1
97 ⫾ 1
100 ⫾ 0*
90 ⫾ 11
96 ⫾ 3
85 ⫾ 11
96 ⫾ 3
77 ⫾ 10
105 ⫾ 2*
Data are expressed as means ⫾ SE. PaO2, arterial oxygen partial pressure; SaO2, arterial hemoglobin saturation of oxygen; MAP, mean arterial pressure
*P ⬍ 0.02 vs. medical air.
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NMDA-induced Ca2⫹ influx in neuronal cell cultures. Cerebral
cortexes from 14- to 15-day-old embryos were dissected in Dulbecco’s
modified Eagle medium containing 5% fetal bovine serum (FBS), 5%
horse serum, and 2 mM glutamine (all from Sigma-Aldrich, Lyon,
France). Cortical cells were dissociated for trituration, plated in glass
petri dishes coated with 0.1 mg/ml poly-D-lysine (Sigma-Aldrich,
Lyon, France) and 0.02 mg/ml laminin (Invitrogen, Villebon-surYvette, France), and cultured in Dubelcco’s modified Eagle medium
changed twice a week in a humidified atmosphere containing 5% CO2.
Treatments were performed after 14 days in vitro. Intracellular free
Ca2⫹ in neurons was measured using fura-2 fluorescence videomicroscopy on the stage of a Nikon Eclipse inverted microscope
equipped with a 100-W Xenon lamp and oil immersion objective
(Nikon 40⫻, NA 1.3). Cultures on glass bottom dish were loaded for
45 min with 10 ␮M fura-2/acetoxymethyl ester (AM; F-1201, Invitrogen, Villebon-sur-Yvette, France) and 0.2% pluronic acid and
incubated for an additional 15 min at room temperature in a HEPESbuffered saline solution. Fura-2 (ex: 340 ⫾ 5 nm/380 ⫾ 6.5 nm; em:
510 nm) ratio images were acquired and digitized through a CCD
camera in a computer at a resolution time of 2 s per successive
measurement using Metafluor 6.3 software (Universal Imaging, Chester, PA). Changes in intraneuronal free Ca2⫹ were induced by a
stimulation of 30-s duration induced by 25 ␮M NMDA given alone or
after exposure of cortical neurons to 20 ␮g/ml rtPA for a 15-min
period in the presence or absence of NBO. During the experiments,
the cells were continuously perfused at a rate of 2 ml/min using a
peristaltic pump with a HEPES-buffered saline solution previously
saturated with medical air or NBO. Size of samples was n ⫽ 105–120
per condition.
Gas pharmacology. Oxygen and nitrogen of medical grade were
bought to Air Liquide Santé (Paris, France). Gas mixtures containing
oxygen at 0 to 100 vol%, with the remainder being nitrogen when
necessary, were obtained using computer-driven flowmeters and gas
analyzers.
Statistical analysis. Data were analyzed using the Statview software (SAS Institute, Cary, NC). In vitro data, given as means ⫾
standard error of the mean (SE), were analyzed using parametric
ANOVA and Student’s t-test. In vivo data, given as median value ⫾
25th–75th percentiles, were analyzed using the Kruskal-Wallis nonparametric ANOVA and Mann-Whitney nonparametric unpaired Utest. The level of significance was set at P ⬍ 0.05. The number of
animals per group for the in vivo ischemic studies was assessed by
performing power analysis through an online software (www.dssresearch.com/toolkit/sscalc/size_a2.asp) with the following variables
taken from a previous study with xenon at 37.5 vol%, based on the
Prothrombolytic Effect of Oxygen
David HN et al.
2071
NBO reduces NMDA plus rtPA-induced neuronal death and
intraneuronal Ca2⫹ influx. Next, because administration of
rtPA has been reported in addition to its beneficial thrombolytic action to increase excitotoxic neuronal death and ischemia-induced brain damage through NMDA-mediated mechanisms (32, 43, 44), we hypothesized that the adverse effects
produced by combined NBO and rtPA in rats subjected to
ischemia could have resulted from a facilitating effect of NBO
on the rtPA-mediated potentiation of NMDA-induced excitotoxic processes. To answer this question, we first investigated
NMDA-induced brain damage in rats treated with NMDA and
medical air, NMDA plus rtPA and medical air, and NMDA
plus rtPA and NBO. Administration of NMDA in the striatum
led to a volume of excitotoxic neuronal death of 18.6 mm3
(25–75 percentiles: 16.7–19.8 mm3). As expected from previous studies, rats injected with NMDA plus rtPA showed
greater brain damage than those injected with NMDA alone,
with a volume of neuronal death of 33.2 mm3 (25–75 percentiles: 29.6 –36.8 mm3; P ⬍ 0.001). NBO decreased neuronal
death produced by coinjection of NMDA plus rtPA, so that rats
treated with NBO and NMDA plus rtPA had a lower volume of
25.9 mm3 (25–75 percentiles: 22.7–27.9 mm3; P ⬍ 0.01; Fig. 3A).
Next, we investigated in the effects of NBO on NMDA- and
NMDA plus rtPA-induced intraneuronal Ca2⫹ influx, a process
well known to play a key role in excitotoxic neuronal death.
rtPA increased NMDA-induced intraneuronal Ca2⫹ influxes.
Consistent with our in vivo findings, we found that NBO
reduced NMDA plus rtPA-induced intraneuronal Ca2⫹ influxes
in cultured neurons (P ⬍ 0.001; Fig. 3B) in a manner similar
to its in vivo effects in rats.
DISCUSSION
Neuroprotective effects of NBO administered alone. We showed
that NBO achieved clot lysis similar to 0.9 mg/kg rtPA, and
thereby reduced ischemia-induced brain damage, swelling, and
hemorrhages. Because we showed that NBO facilitates the catalytic and thrombolytic efficiency of rtPA through direct interaction with the fibronectin type I, EGF-like, and/or kringle 1 tPA
domains, we assume that the in vivo prothrombolytic effects of
NBO occur through facilitation of the fibrinolytic properties of
endogenous tPA, which activity in contrast with what is generally
thought is significantly increased within the ischemic brain (44).
Also, in line with previous data that have reported that NBO given
before rtPA did reduce rtPA-induced brain hemorrhages (42), we
found that NBO-treated rats had reduced brain hemorrhages
compared with control rats. In light of our findings that NBO has
a facilitating action on the catalytic and thrombolytic efficiency of
tPA, it is likely that the reduction of brain hemorrhages in this
latter study could result from the fact that NBO yet has produced
recanalization or partial recanalization through activation of endogenous tPA before rtPA injection. Taken together, these results
provide experimental evidence for a small clinical pilot study in
patients ineligible for rtPA therapy that has first hypothesized that
NBO could reduce ischemic brain damage by increasing reperfusion (36), as well as basic mechanisms for previous data that have
shown in animal models that NBO allows delaying the transition
of ischemia to infarction (“buying time”; 22). Taken together with
previous findings that have shown that NBO decreases ischemiainduced acidosis and disruption of energy metabolism (40), in-
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(25–75 percentiles: 89 –114 mm3; P ⬍ 0.02; Fig. 1C) and 6.3%
(25–75 percentiles: 0 –12.3%; P ⬍ 0.02; Fig. 1D) compared with
control animals. Also, NBO-treated rats had reduced brain hemorrhages in the form of petechial hemorrhages rather than discrete
hematomas compared with control animals (P ⬍ 0.05; Fig. 1, E,
G, and H), and further showed reduced disruption of the bloodbrain barrier compared with both control and rtPA-treated rats
(P ⬍ 0.02– 0.05; Fig. 1, F–H).
Rats treated with NBO and rtPA in combination had sustained CBF restoration similar to that of rtPA- and NBOtreated rats (Fig. 1A). Although the lack of synergistic effect of
combined NBO and rtPA on CBF restoration could appear
surprising at first view, this result is in excellent agreement
with previous data that have shown that CBF restoration
produced by 10 mg/kg rtPA is only twice that produced by the
clinical dose of 0.9 mg/kg rtPA (18) used in the present study
(R ⫽ 0.9074, ddl ⫽ 23, P ⬍ 0.001; Fig. 1B). Despite showing
CBF restoration, rats treated with combined NBO and rtPA had
ischemic brain damage and swelling, respectively, of 435 mm3
(25–75 percentiles: 393– 466 mm3; Fig. 1C) and 26.7% (25–75
percentiles: 21.4 –32.6%; Fig. 1D) that were not different from
those of control animals and greater than those of rtPA and
NBO-treated rats (P ⬍ 0.01– 0.05; Fig. 1, C and D). Also, rats
treated with combined NBO and rtPA showed a trend toward
increase of brain hemorrhages that reached statistical difference compared with NBO-treated rats (P ⬍ 0.02; Fig. 1, E, G,
and H), and further exhibited lower (P ⬍ 0.05) and higher (P ⬍
0.02) disruption of the blood-brain barrier, in the form of light
and diffuse Evans blue extravasation that often goes beyond
the ischemic brain damage areas, compared with rtPA- and
NBO-treated rats, respectively (Fig. 1, F–H).
NBO facilitates the catalytic and thrombolytic efficiency of
rtPA in vitro. Next, to examine whether NBO could have
actually promoted thrombolysis in vivo by interacting directly
with endogenous tPA, we characterized in vitro by the initial
rate method the effects of various concentrations of oxygen on
the catalytic efficiency of rtPA and reteplase, a tPA-derived
drug that lacks the fibronectin type I, EGF-like, and kringle 1
domains and only possesses the kringle 2 and catalytic domains. We found that the addition of oxygen into the medium
increases the catalytic efficiency of rtPA in a concentrationdependent manner (P ⬍ 0.001). Interestingly, the absence of
oxygen into the medium led to a decrease of the catalytic
activity of rtPA compared with control values obtained with
medical air, a result that indicates as a novel concept that the
lack of oxygen in an ischemic brain area would play a key role
in the thrombosis process. Contrasting with the strong facilitating effect of oxygen on the catalytic efficiency of rtPA, we
found that oxygen at the higher concentrations of 75 vol% and
100 vol% induced a modest decrease of the catalytic efficiency
of reteplase (P ⬍ 0.05), indicating that NBO would produce
thrombolysis in vivo by interacting with endogenous tPA
through the fibronectin type I, EGF-like, and/or kringle 1
domains (Fig. 2A). To demonstrate this possibility, we studied
the effect of NBO on the thrombolytic efficiency of rtPA and
reteplase in vitro in whole blood samples drawn from male mature
rats. NBO increased rtPA-induced thrombolysis by about 40%
compared with whole blood samples treated with rtPA and medical air (P ⬍ 0.005) but failed to alter the thrombolytic efficiency
of the tPA-derived drug reteplase (Fig. 2B).
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creases tissue oxygenation (26), and reduces NMDA-mediated
excitotoxicity (16), our results highlight the mechanisms by which
intraischemic NBO has beneficial action on brain damage, swelling, and hemorrhages.
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David HN et al.
Lack of neuroprotective action of combined NBO and rtPA.
In contrast with the beneficial effects of NBO (and rtPA)
administered alone, we found that rats treated with combined
NBO and rtPA despite showing CBF restoration had ischemic
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Prothrombolytic Effect of Oxygen
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David HN et al.
2073
brain damage and swelling equivalent to those of control
animals, and greater than those of rtPA- and NBO-treated rats.
Because NBO alone induces a facilitation of the catalytic
activity of tPA, we hypothesized that the adverse effects of
combined NBO and rtPA on brain damage and swelling could
have resulted from a NBO-induced potentiation of the wellknown rtPA-induced facilitation of ischemia- and NMDAmediated excitotoxic processes (32, 43, 44). However, such a
mechanism is unlikely to be true since as shown in this study
NBO reduces the facilitating effects of rtPA on NMDAinduced intraneuronal calcium influxes and neuronal death, a
result in agreement with previous data that have reported redox
modulation of the NMDA receptor with reduction producing a
potentiation and oxidation an inhibition of the NMDA receptor
response and glutamate-induced neuronal death (25, 39). These
contrasting effects of combined NBO and rtPA on ischemiaand NMDA-induced brain damage clearly support that the
basic mechanisms involved in the lack of neuroprotective
action of combined NBO and rtPA are likely to occur at the
neurovascular rather than the parenchymal side of the central
nervous system. Strong support for this and our findings are
recent data that have shown on one hand that rtPA and NBO,
respectively, increases and decreases serum concentrations of
matrix metalloproteinase-9, whose expression is known to be
critically associated with ischemia-induced disruption of the
blood-brain barrier, and on the other hand that combining
oxygen and rtPA suppresses the beneficial effects of NBO at
decreasing matrix metalloproteinase-9 (30). Based on these
findings, the authors suggest as found in the present report that
combining NBO and rtPA could not be a safe strategy (30).
Comparison with previous data. Our findings that NBO
restores CBF oppose previous investigations that have reported
no effect of NBO alone on CBF and ischemic brain damage in
rats subjected to a thromboembolic model of acute ischemic
stroke (10, 20) or showed that NBO could be coadministered
safely with rtPA (20). Differences in the duration of NBO
treatment—less than 1 h until reperfusion in the present study
vs. 3 h or 3.5 h from before to after reperfusion in these latter
studies— could account for these discrepancies since postischemic NBO is known to exacerbate brain tissue damage and
deteriorate neurologic outcome (16, 31). Also, such discrepancies could mainly result from the fact that the above mentioned
studies were performed in rats anesthetized with 70 vol%
nitrous oxide that were shifted to NBO immediately after
Fig. 1. A: time-course effects of recombinant tissue-plasminogen activator (rtPA) and normobaric oxygen (NBO) given alone or in combination on cerebral blood
flow (CBF) in a rat model of thromboembolic stroke. Rats treated with rtPA, NBO, or coadministration of rtPA and NBO showed similar CBF restoration.
Untreated control rats exhibited no reperfusion. B: individual data of cumulative CBF during treatment from T to T= in control rats (black), and rats treated with
NBO (yellow), tPA (blue), or combined NBO-tPA (green). To plot data, because NBO and rtPA at 0.9 mg/kg had similar effects on CBF restoration, we assume
that NBO may be considered equivalent to 0.9 mg/kg rtPA, and the combination of NBO plus rtPA equivalent to 1.8 mg/kg. Values with 10 mg/kg rtPA (red)
were obtained from a previous study (17). Data analysis shows a high and significant correlation coefficient (R ⫽ 0.9074, ddl ⫽ 23, P ⬍ 0.001) that indicates
that the lack of significant effect of combined NBO and rtPA at increasing cerebral reperfusion compared with NBO or rtPA alone is in good agreement with
the fact that CBF restoration produced by 10 mg/kg rtPA is only twice that produced by 0.9 mg/kg rtPA. C–F: scores of brain damage (C), brain swelling (D),
brain hemorrhages (E), and Evans blue extravasation (F) in rats treated with rtPA, NBO, or coadministration of rtPA and NBO. Top part and bottom part of the
histograms in C represent subcortical and cortical brain damage, respectively. Rats treated with rtPA and NBO alone, but not in combination, had decreased brain
damage, swelling, and hemorrhages compared with middle cerebral artery occlusion (MCAO) control rats. No significant difference was found between rtPAand NBO-treated rats. G: typical examples of brain from rats treated with rtPA and NBO alone or in combination. It is noteworthy that, uniquely among other
groups, rats treated with combined NBO and rtPA had brain hemorrhages in the upper layers of the cortex as shown by arrows. H: typical examples of brain
slices from control rats and rats treated with rtPA and NBO alone or in combination. Left: Evans blue extravasation; right: Evans blue extravasation and
comparison with neuronal death. Data are expressed as the median value and 25th–75th percentiles. The number of animals is n ⫽ 5 per group. **P ⬍ 0.01,
*P ⬍ 0.05 vs. MCAO control rats; $P ⬍ 0.01, ‡P ⬍ 0.05 vs. rtPA-treated rats; #P ⬍ 0.05 vs. NBO-treated rats.
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Fig. 2. A: concentration-dependent effects of oxygen of 0 to 100 vol% on the catalytic efficiency of human rtPA (black square) and the tPA-derived drug reteplase
(RPA) that lacks the fibronectin type I, EGF-like, and/or kringle 1 tPA domain (open square) compared with their own controls performed in medical air
composed of 25 vol% oxygen and 75 vol% nitrogen taken as a 100% value (gray square). NBO at concentration of 50 –100 vol% increases the catalytic efficiency
of tPA. In contrast, the absence of oxygen reduces the catalytic efficiency of tPA, thereby suggesting as a novel concept that the lack of oxygen in an ischemic
brain area would play a key role in the thrombosis process. Size of samples is N ⫽ 3, n ⫽ 12 per oxygen concentration. *P ⬍ 0.001 vs. rtPA own controls; ‡P ⬍
0.05 vs. RPA own controls. B: effects of NBO (100 vol% oxygen) on the thrombolytic efficiency of rtPA and retelase (RPA) in vitro on blood clots obtained
from whole blood samples. NBO increases the thrombolytic activity of rtPA but not of reteplase. Size of samples is N ⫽ 6, n ⫽ 12 per condition. *P ⬍ 0.005
vs. rtPA own controls. Data are expressed as means ⫾ SE. Taken together, these data indicate that NBO decreases the catalytic and thrombolytic activity of rtPA
through direct interaction with the fibronectin type I, EGF-like, and/or kringle 1 tPA domain.
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Prothrombolytic Effect of Oxygen
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David HN et al.
Fig. 3. A: effects of NBO on rtPA-induced potentiation of NMDA-induced neuronal death. Administration of NBO 1 h after coinjection of NMDA plus rtPA
decreases rtPA-induced potentiation of NMDA-induced neuronal death. The number of animals is n ⫽ 8 –12 per group. Data are expressed as the median value
and 25th–75th percentiles. *P ⬍ 0.005 vs. control rats treated with NMDA; ‡P ⬍ 0.01 vs. rats treated with NMDA plus rtPA. B: effects of NBO on rtPA-induced
potentiation of NMDA-evoked Ca2⫹ influx in cortical neuronal cultures. A 30-s exposure to NMDA (25 ␮M) produced a rapid increase in intracellular free Ca2⫹,
which recovered over the following minutes. Preincubation of cortical neurons with rtPA (20 ␮g/ml) for a 15-min period increases NMDA-evoked Ca2⫹ influx.
NBO reduces rtPA-induced potentiation of NMDA-evoked Ca2⫹ influx. Size of samples is N ⫽ 3, n ⫽ 90 –120 per condition. Data are expressed as means ⫾
SEM. *P ⬍ 0.001 vs. control neurons treated with NMDA; ‡P ⬍ 0.001 vs. neurons treated with NMDA plus rtPA.
interactions in the above mentioned studies (10, 20). Also, it is
possible that the dose of 10 mg/kg rtPA used in these studies
could have masked the effect of NBO on rtPA, since the use of
such a high dose of rtPA could have allowed the presence of
rtPA molecules free of oxygen (18) in contrast with the dose
of 0.9 mg/kg used in the present report and in previous studies
on inert gas modulation of rtPA thrombolytic and proteolytic
properties (6, 17).
Clinical perspectives. From a clinical perspective, our results provide experimental evidence that the use of NBO alone
may be an efficient alternative therapeutic strategy to rtPA,
particularly for the vast majority of patients who cannot be
treated with rtPA, and/or a primary care therapeutic intervention for those eligible for rtPA therapy. If such, since
delayed NBO has no beneficial effect and can even deteriorate outcome, it is likely that the use of NBO should be
restricted to the ischemic period to promote reperfusion and
then stopped once reperfusion has been assessed using CBF
monitoring. Also and in contrast with the beneficial effect of
NBO alone, our results further suggest that NBO should not
be administered concomitantly with rtPA therapy in acute
ischemic stroke patients.
We believe that our findings of 1) prothrombolytic action of
NBO should be tested and compared with rtPA in carefully
designed clinical trials in acute ischemic stroke patients, and
2) adverse interactions between NBO and rtPA should be used
as a principle of caution to avoid combining NBO and rtPA
until our results will or will not be confirmed in acute ischemic
stroke patients.
ACKNOWLEDGMENTS
We thank Dr. Olivier Nicole for technical support in calcium imaging
studies.
GRANTS
This work was supported by funding from the University of Caen-Basse
Normandie, the Centre de Recherche Centre Hospitalier Affilié Universitaire Hôtel-Dieu de Lévis, the Direction Générale de l’Armement of the
French Ministry of Defence, and NNOXe Pharmaceuticals (Quebec City,
QC, Canada).
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nitrous oxide (10, 20), a condition that could have biased
results and altered NBO-induced facilitation of tPA catalytic
and thrombolytic activity since nitrous oxide has been recently
shown to inhibit potently the catalytic and thrombolytic activity of tPA (17). Indeed, inert gases and oxygen bind and
compete for binding to proteins within hydrophobic cavities as
a function of multiple factors among which affinity, as defined
by the Hildebrand coefficient and the Meyer-Overton rule
(with xenon ⬎ nitrous oxide ⬎ krypton ⬎ argon ⬎ oxygen ⬎
nitrogen ⬎ helium), play a critical role (5, 28, 45). Accordingly, once bound, inert gases with actually higher affinity than
oxygen, such as xenon and nitrous oxide, do not remove out
from the brain as soon as it is generally thought when administration is stopped. For instance, clinical trials have shown that
the time to obtain full recovery from 8-h moderate sedation
with xenon at 35 vol% [that is half the minimum anesthetic
concentration (MAC) of xenon] is 35 min (3). Given the MAC
value of nitrous oxide that is 1.5-fold higher that of xenon (7;
which indicates that nitrous oxide given at equal concentration
as xenon is less potent at producing narcosis/anesthesia) and
the blood/gas partition coefficient of nitrous oxide that is 0.46
vs. 0.12 for xenon (12; which indicates that nitrous oxide has
a longer wash-out than xenon), it can be estimated that the time
to obtain full recovery from a similar nitrous oxide sedation
would be ⬃90 min. Support for such long-lasting effect of
nitrous oxide and our findings that NBO increases CBF are
recent MRI data that have demonstrated in rats subjected to
mechanical acute ischemic stroke that oxygen, given ⬃90 min
after nitrous oxide and MCAO procedure (with medical air in
between), increases CBF in the ischemic penumbra in a concentration-dependent manner (2). Other examples for longlasting effects of nitrous oxide are also available, for instance
in pain research. In that way, particularly, and in line with our
findings that nitrous oxide inhibits thrombolysis (17), clinical
studies in patients anesthetized with or without nitrous oxide
for noncardiac surgery have shown that the use of nitrous oxide
is associated with an increased risk of postoperative myocardial ischemia (1). Therefore, it can be concluded with little
doubt that administration of nitrous oxide at 70 vol% before
NBO treatment may have actually altered NBO-induced facilitation of tPA thrombolytic activity and thereby NBO-rtPA
Prothrombolytic Effect of Oxygen
DISCLAIMER
Funders had no role in study design, data collection, and analysis, decision
to publish, or preparation of the manuscript.
Lt. D. G. Colomb, Jr., is a military service member and this work was
prepared as part of his official duties. The views expressed in this article are
those of the author and do not reflect the official policy or position of the
Department of the Navy, Department of Defense, or the U.S. Government.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: H.N.D., B.H., D.G.C., J.-J.R., and J.H.A. conception
and design of research; H.N.D., B.H., and M.D. performed experiments;
H.N.D., B.H., M.D., D.G.C., and J.H.A. analyzed data; H.N.D., D.G.C.,
J.-J.R., and J.H.A. interpreted results of experiments; H.N.D. and J.H.A.
prepared figures; H.N.D., D.G.C., J.-J.R., and J.H.A. drafted manuscript;
H.N.D., B.H., M.D., D.G.C., J.-J.R., and J.H.A. approved final version of
manuscript; J.H.A. edited and revised manuscript.
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