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RESEARCH ARTICLE
Hyperbaric oxygen and hyperbaric air treatment result
in comparable neuronal death reduction and improved behavioral
outcome after transient forebrain ischemia in the gerbil
Michal Malek •Malgorzata Duszczyk •
Marcin Zyszkowski •Apolonia Ziembowicz •
Elzbieta Salinska
Received: 23 July 2012 / Accepted: 19 September 2012 / Published online: 2 October 2012
ÓThe Author(s) 2012. This article is published with open access at Springerlink.com
Abstract Anoxic brain injury resulting from cardiac arrest
is responsible for approximately two-thirds of deaths. Recent
evidence suggests that increased oxygen delivered to the
brain after cardiac arrest may be an important factor in pre-
venting neuronal damage, resulting in an interest in hyper-
baric oxygen (HBO) therapy. Interestingly, increased
oxygen supply may be also reached by application of nor-
mobaric oxygen (NBO) or hyperbaric air (HBA). However,
previous research also showed that the beneficial effect of
hyperbaric treatment may not directly result from increased
oxygen supply, leading to the conclusion that the mechanism
of hyperbaric prevention of brain damage is not well
understood. The aim of our study was to compare the effects
of HBO, HBA and NBO treatment on gerbil brain condition
after transient forebrain ischemia, serving as a model of
cardiac arrest. Thereby, we investigated the effects of
repetitive HBO, HBA and NBO treatment on hippocampal
CA1 neuronal survival, brain temperature and gerbils
behavior (the nest building), depending on the time of initi-
ation of the therapy (1, 3 and 6 h after ischemia). HBO and
HBA applied 1, 3 and 6 h after ischemia significantly
increased neuronal survival and behavioral performance and
abolished the ischemia-evoked brain temperature increase.
NBO treatment was most effective when applied 1 h after
ischemia; later application had a weak or no protective
effect. The results show that HBO and HBA applied between
1 and 6 h after ischemia prevent ischemia-evoked neuronal
damage, which may be due to the inhibition of brain tem-
perature increase, as a result of the applied rise in ambient
pressure, and just not due to the oxygen per se. This per-
spective is supported by the finding that NBO treatment was
less effective than HBO or HBA therapy. The results pre-
sented in this paper may pave the way for future experiment al
studies dealing with pressure and temperature regulation.
Keywords Ischemia Hyperbaric oxygen therapy
Hyperbaric air Neuronal damage Brain temperature
Gerbil
Introduction
Sudden cardiac arrest is a common manifestation of car-
diovascular disease affecting a large number of people, and
despite advances in resuscitation techniques, the percent-
age of surviving patients is still very low (Nolan et al.
2008). The mortality in patients who achieve return of
spontaneous circulation (ROSC) is due largely to post-
cardiac arrest syndrome which comprises anoxic brain
injury, post-cardiac arrest myocardial dysfunction, ische-
mia/reperfusion response and persistent precipitating
pathology (Nolan et al. 2008). Among these anoxic brain
injury is a major cause of mortality, responsible for
approximately two-thirds of deaths. The mechanisms of
brain tissue damage triggered by cardiac arrest and resus-
citation include energy deficit, anoxic depolarization,
excitotoxicity, disturbed calcium homeostasis, formation of
free radicals and activation of cell death signaling path-
ways (Busl and Greer 2010). Currently, hypothermia is the
only proven treatment for anoxic brain injury after cardiac
arrest, and although some drugs appear to have short-term
M. Malek M. Duszczyk A. Ziembowicz E. Salinska (&)
Department of Neurochemistry, Mossakowski Medical Research
Centre, Polish Academy of Sciences, 5 Pawinskiego Str.,
02-106 Warsaw, Poland
e-mail: elas@cmdik.pan.pl
M. Zyszkowski
Department of Anesthesiology and Intensive Care,
Military Institute of Medicine, Warsaw, Poland
123
Exp Brain Res (2013) 224:1–14
DOI 10.1007/s00221-012-3283-5
benefits, no specific drug therapy has been confirmed to
improve long-term survival in randomized controlled trials
(Morley 2011; Deakin et al. 2010).
There is a lot of recent evidence that hyperbaric oxygen
(HBO) prevents neuronal damage and improves neuro-
logical outcome after cardiac arrest or stroke (for review
see Harch and Neubauer 2009), and there is the opinion
that amount of oxygen delivered to the brain in a short time
after cardiac arrest or after stroke may be an important
factor in preventing neuronal damage resulted from brain
ischemia (Neumar 2011; Rosenthal et al. 2003; Van Meter
et al. 2008). The neurological outcome after cardiac arrest
may be greatly affected by the oxygen inhaled immediately
after resuscitation, and there is a growing body of evidence
suggesting that delivering too much oxygen too quickly
may increase injury associated with postischemic reperfu-
sion. There are reports that postresuscitation hyperoxemia
(PaO
2
C300 mmHg) exposure in the first 60 min after
ROSC increased the mortality in patients and was associ-
ated with lower independent functional status among
patients who survived (Kilgannon et al. 2011).
Despite many reports on beneficial results of HBO in
global cerebral ischemia/anoxia in animal models and
human clinical studies (for review see Harch and Neubauer
2009), this therapy did not find the common use in neu-
rological diseases treatment. Pressure-related complica-
tions of HBO and growing concern that HBO treatment
may generate toxic free radicals, resulted in skepticism
about the safety and efficacy of HBO in cerebral ischemia
(Kot and Mathieu 2011). Nevertheless, HBO still remains
the subject of interest of many scientists: moreover, the
critical analysis of negative results and data from new
animal studies renewed the interest in HBO therapy
(Michalski et al. 2011). Recent publications have provided
essential information about the important factors influenc-
ing HBO efficacy such as the therapeutic time window and
the mechanism of action (Rockswold et al. 2010; Lou et al.
2004). It is now known that HBO not only significantly
increases oxygen pressure and concentration in the arteries,
resulting in better oxygen supply to the ischemic areas, but
also increases cerebral blood flow (CBF) into the injured
brain, the so-called inverse steal phenomenon mediated by
nitric oxide (NO) (Lassen and Palvo
¨lgyi 1968; Atochin
et al. 2003; Dean et al. 2003). HBO also ameliorates blood
flow velocity due to increased deformability of red blood
cells (van Hulst et al. 2003).
There is now increasing experimental evidence that
HBO can afford neuroprotection after experimental global
cerebral ischemia induced by vascular occlusion. It has
been shown that HBO treatment results in the reduction in
neuronal death and accelerated neurological recovery in
different animal models of global ischemia (Iwatsuki et al.
1994; Kapp et al. 1982; Krakovsky et al. 1998; Takahashi
et al. 1992) and also in an experimental cardiac arrest
model in dogs (Rosenthal et al. 2003). In addition, HBO
improves also the rate of return of spontaneous circulation
after prolonged cardiopulmonary arrest (Van Meter et al.
2008), decreases lactate concentration in cerebrospinal
fluid, improves mitochondrial function after focal cerebral
ischemia (Rockswold et al. 2001; Daugherty et al. 2004),
triggers multiple neuroprotective effects such as reduction
in concentration of cyclooxygenase-2 (COX-2) (Yin et al.
2002) and intracellular adhesion molecule-1 (ICAM-1)
(Buras et al. 2000), and decreases the expression of proa-
poptotic genes including hypoxia inducible factor-1a(HIF-
1a), p53, caspase-9 and caspase-3 (Li et al. 2005). It was
also shown that HBO may cause hypothermia due to
increased oxygen partial pressure (Fenton et al. 1993).
Regardless of these beneficial effects of HBO on brain
tissue, the consequences of forced oxygen application on
other organs have to be considered. Lung toxicity is a
known phenomenon following exposure to a high oxygen
concentration. Prolonged exposure to hyperoxia resulting
from HBO and from normobaric oxygen (NBO) can
damage pulmonary epithelial cells, and concomitantly
occurring chronic pulmonary diseases are additional con-
traindications in the use of oxygen in cerebral ischemia (Li
et al. 2007; Sinclair et al. 2004).
The application of increased oxygen to ischemic areas
dates from the mid-1900s and in the early experiments
compressed air was used in hyperbaric medicine (for
review see Singhal 2006), and later on Smith et al. (1961)
and Illingworth (1962) showed the beneficial effect of
HBO on cerebral ischemia. To date there is little data
showing the effect of hyperbaric air (HBA) on postische-
mic survival. HBA is not used even as a control to compare
the effects of HBO. The few papers containing data about
the effects of HBA treatment on brain tissue concern the
hypoxic tolerance induced by hyperbaric preconditioning
or the impact on cell viability of hypoxic brain slices (Peng
et al. 2008;Gu
¨nther et al. 2004). Recent publications
showing that the beneficial effect of HBO may not be the
only result of increased oxygen supply (Rosenthal et al.
2003; Miljkovic-Lolic et al. 2003) suggest that the mech-
anisms of hyperbaric prevention of brain damage still
remain unclear.
The aim of our study was to compare the effects of
HBO, HBA and NBO treatment on gerbil brain condition
after transient forebrain ischemia as a model of cardiac
arrest, by testing neuronal survival in the hippocampus,
changes in the brain temperature and also basic behavior in
the nest-building test. Additionally, possible differences
dependent on the time of initiation of the hyperbaric
therapy were investigated.
2 Exp Brain Res (2013) 224:1–14
123
Experimental procedures
Animals
All the experiments used male Mongolian gerbils (Meri-
ones unguiculatus), bred in the Animal Colony of the
Medical Research Centre, Polish Academy of Sciences in
Warsaw, aged 12–13 weeks and weighing about 60 g. The
animals were kept at the room temperature (24–25 °C), fed
ad libitum and randomly assigned into experimental groups
(group 1—sham operated, group 2—non treated ischemia,
group 3—ischemia ?HBO, group 4—ischemia ?HBA
and group 5—ischemia ?NBO). The animals from groups
2—5 were submitted to a 3-min forebrain ischemia, fol-
lowed by either hyperbaric therapy or normobaric oxygen
application, started at three different times after ischemia
(Fig. 1). Sham-operated animals served as controls in
evaluation of neuronal cell loss and in behavioral experi-
ments. Animals used in nest-building test experiments
served also for neuronal loss evaluation. The number of
animals per experimental group ranged from n=5to
n=9 (exact number given in figure legends). The total
number of animals used in this study n=120. All animal
experiments were carried out according to the Polish and
European Community Council regulations concerning
experiments on animals.
The animal experiments were approved by the Local
Ethical Committee in Warsaw, Poland, and performed in
accordance with Polish governmental regulations
(Dz.U.97.111.724) and the European Community Council
Directive of 24 November 1986 (86/609/EEC). All efforts
were made to minimize animal suffering and the number of
animals used.
Forebrain ischemia
The brain ischemia was performed as described earlier
(Chandler et al. 1985; Duszczyk et al. 2006b). Briefly, the
animals were anesthetized with 4 % halothane in a gas
mixture containing 30 % O
2
and 70 % N
2
O, and 2 min
before the operation, the concentration of halothane was
reduced to 2 % and kept at this concentration during
ischemia. The carotid arteries were isolated through an
anterior middle cervical incision, made after the injection
of local anesthetics. Cerebral ischemia was induced by the
occlusion of both common carotid arteries with miniature
aneurismal clips for 3 min. Sham-operated animals were
exposed to similar surgery without carotid artery occlusion.
During the surgery, animals were kept on the heating pad at
37 °C. After wound closure animals were put into separate
cages and moved to the room designed for behavioral test
or brain temperature measurements accordingly. Both
groups of animals were then submitted to hyperbaric
therapy or normobaric oxygen. In this study, ischemic
protocol resulted in less than 3 % mortality and there was
no mortality that could be associated with hyperbaric or
normobaric oxygen treatment.
Hyperbaric treatment
Animals subjected to ischemia were divided into three
groups. Animals from group 3 were treated with HBO
therapy (compression in 100 % oxygen), group 4 was
compressed in atmospheric air (HBA) and group 5 con-
sisted of animals treated with normobaric oxygen (100 %
oxygen at 1 ATA). As a control served sham-operated
animals treated with HBO. Animals were placed into the
hyperbaric chambers (for HBO—Model B-11 Animal
Research Chamber, Reimers Systems, INC., USA; for
HBA—Hyperbaric Chamber, Technika Podwodna, Poland)
for 60 min 1, 3 or 6 h after ischemia and compressed to 2.5
ATA (rate of compression and decompression 1 ATA/
min). Animals from the NBO group were placed in an
unpressurized hyperbaric chamber with a constant 100 %
oxygen flow through the chamber at a rate of 0.5 l/min.
The treatment was repeated for 3 following days. The
temperature in the hyperbaric chambers was monitored
during the whole session and was maintained at 24–25 °C.
Measurement of brain temperature
For continuous recording and analysis of the brain tem-
perature in conscious and freely moving gerbils, a tele-
metric system to measure the brain temperature was
utilized (Mini Mitter VitalView hardware and software
system, Mini Mitter Co. Inc. Oregon, USA). The temper-
ature was measured in gerbils submitted to 3-min forebrain
ischemia and treated with HBO, NBO and HBA. The
procedure for the brain temperature probe (probe type Mini
Mitter XM-FH-BP) implantation has previously been
described in detail (Duszczyk et al. 2006a). Briefly, the
ischemia
0
0
0
0
1
3
6
25
27
30
49
51
54
Hours after ischemia
a
b
c
d
untreated ischemia
treatment initiated
1 h after ischemia
treatment initiated
3 h after ischemia
treatment initiated
6 h after ischemia
Fig. 1 Scheme of the procedure of experiments including untreated
3-min ischemia (a) and HBO, HBA or NBO treatment initiated at
different times after ischemia (b,c,d). Each time point indicates
initiation of the treatment lasting 60 min; treatments were repeated in
24-h intervals (for details of the protocol see section ‘‘Experimental
procedures’’ )
Exp Brain Res (2013) 224:1–14 3
123
holders of the brain temperature probes were implanted
unilaterally in gerbils submitted to halothane anesthesia
and the tips into the striatum, approximately to the same
depth as the hippocampus. Two days later, the probes were
inserted into the probe holders and the gerbils were placed
in plastic cages resting on the telemetry receiver. Tem-
perature measurements began 3 h before an ischemic epi-
sode and continued for 72 h. The temperature signals from
the probe were sampled every 30 s, to ensure strict control
of the temperature. The mean temperatures were calculated
for individual time points (usually every 1 h). Because of
technical limitations, brain temperature was not measured
during hyperbaric sessions (around 60 min) and the mea-
surements were continued immediately after returning the
animals from hyperbaric chamber into the cages. It was
shown earlier that insertion of small temperature probe into
brain caused only minor injury that did not affect the main
results (Duszczyk et al. 2006a; Nurse and Corbett 1994;
Zhang et al. 1997).
Histochemistry
One week (for TUNEL staining) or 2 weeks (for cresyl
violet staining) after ischemia, animals were killed for
brain examination (3–5 from each experimental group).
This time was chosen according to earlier observations that
after transient forebrain ischemia, most of the apoptotic
processes in gerbil brain is finished, whereas one week
after ischemia apoptosis is still observed (Ko et al. 2009;
Goda et al. 2012). Animals were anesthetized with halo-
thane and subjected to intracranial perfusion fixation with
4 % neutralized formalin (Sigma-Aldrich, St. Louis, Mis-
souri, USA). The brains were removed and immersed in
4 % formalin for 1 week, then transferred to absolute
ethanol and embedded in paraffin. Ten-lm cross sections
from the dorsal part of the hippocampus (between 2.2 and
3.5 mm posterior to bregma) were used to evaluate the
brain damage size and hippocampal apoptotic neurons.
Sections were stained with cresyl violet (Sigma, St. Louis,
Missouri, USA) or terminal deoxynucleotidyl transferase-
mediated dUTP-nick end labeling (TUNEL, In Situ Cell
Death Detection Kit, Fluorescein; Roche, Switzerland). For
each animal, at least 5 sections of the central part of CA1
region in both hippocampi were analyzed for neuron den-
sity or TUNEL stained cells (number of animals per group
n=3–5). The number of neurons was counted in central
CA1 area of 0.5 mm length using AxioVison imaging
program (Carl Zeiss, Aalen, Germany). A mean number of
neurons stained with cresyl violet were expressed as a
percentage of mean number of neurons of sham-operated
gerbils. The mean number of pyramidal neurons in CA1
region of sham-operated gerbils amounts to 155 per
0.5 mm.
Nest-building test
Nest-building behavior was evaluated in gerbils submitted
to 3-min forebrain ischemia (group 2) and those treated
after ischemia with HBO, HBA and NBO (group 3, 4 and
5, respectively) starting at different times after operation
(1, 3 and 6 h). Additional groups consisted of sham-oper-
ated gerbils (group 1) exposed to HBO, HBA and NBO at
the same times as the ischemic groups.
After surgery, each animal was placed into a cage
covered with a paper towel and the nest building was
assessed each day for 7 days following the ischemic epi-
sode. Paper shredding was scored on a 4-point scale
adapted from Babcock et al. (1993): 0 =none; 1 =pieces
[4cm
2
;2=pieces between 2 and 4 cm
2
;3=pieces
\2cm
2
(Duszczyk et al. 2006a). Some of these animals
were subsequently used for evaluation of neuronal
damage.
Statistical analysis
Apart from nest-building scores, the results are expressed
as mean ±SEM for each experimental group. Statistical
analysis of that data was performed by one way ANOVA,
with further analysis using the post hoc least significance
test for significant differences between groups (GraphPad
Prism, version 5.01; GraphPad Software Inc., La Jolla,
California, USA). Differences were considered significant
with p value less than 0.05.
The nest-building scores are presented as medians,
estimated by calculation of the interquartile range (IQR)
and tested by two-way analysis of variance (ANOVA).
Results
HBO and HBA prevent neurodegeneration
The counting of pyramidal neurons in CA1 area of hippo-
campus showed that 3-min ischemia resulted in a significant,
82 % neuronal loss in comparison with control, sham-
operated animals (F
1,15
=135, p\0.001) (Fig. 2). Ische-
mia-initiated apoptotic processes and one week after ische-
mic insult 45 TUNEL-positive cells in examined CA1 area
(0.5 mm length) were observed (Fig. 3). The application of
HBO treatment 1 h after ischemia increased the number of
neurons observed in this region to 54 % of control (signifi-
cant difference from ischemia, F
1,26
=21.19, p\0.001)
(Fig. 2). Application of HBO 3 or 6 h after ischemia resulted
in significant increase in number of neurons to 41 % in both
cases (F
1,14
=92.2, p\0.001 and F
1,14
=60.38,
p\0.001, respectively). HBO also significantly reduced the
number of TUNEL-positive cells observed in CA1 by 80, 62
4 Exp Brain Res (2013) 224:1–14
123
and 49 %, respectively, to the time of treatment initiation
(F
3,13
=25.1, p\0.001) (Fig. 3) The use of HBA also
significantly increased the number of neurons (Fig. 2).
Application of HBA 1 h after ischemia resulted in an
increase in viable neurons to 49 % (F
1.12
=29.24,
p\0.001), and HBA therapy applied 3 or 6 h after ischemia
in 43 and 41 % of control, respectively (F
1,12
=45,
p\0.001 and F
1,15
=71.1, p\0.001). In comparison with
untreated ischemia, HBA treatment decreased the number of
TUNEL-positive cells by 67 % when initiated 1 h after
ischemia and by 58 and 40 % when initiated 3 and 6 h after
ischemia, respectively (F
3,10
=13.2, p\0.001). Statistical
analysis of results comparing HBO and HBA therapy did not
reveal significant differences between their effectiveness.
The application of NBO 1 and 3 h after ischemia resulted in
only a slight increase in the number of surviving neurons (29
and 26 % in both times, respectively, compared to 18 % of
surviving neurons in non-treated ischemia; F
1,14
=7.63,
p\0.05 and F
1,27
=9.28, p\0.01, respectively), which
was significantly lower compared to HBO and HBA
1h
3h
6h
HBO HBA NBO
100 µm
sham ischemia
Quantity of neurons
(% of control)
0
20
40
60
80
100
1 h 3 h 6 h
ischemia HBO
HBA NBO
****
*
**
*
**
Fig. 2 The effect of HBO, HBA and NBO treatment initiated at
different times after ischemia (1, 3 and 6 h) on number of neurons in
hippocampal CA1 region of the gerbil brain, stained with cresyl
violet. The hyperbaric and NBO treatment was applied 3 times in
24-h intervals. Brain tissue was examined 14 days after ischemia. Top
right corner graph represents results expressed as percentage of
surviving neurons compared to the mean control level of 290 cell/mm
in central CA1 region in sham-operated animals. Number of analyzed
animals per group n=5. Results on the graph are mean val-
ues ±SEM; *p\0.05, **p\0.001
Exp Brain Res (2013) 224:1–14 5
123
(F
2,34
=5.9, p\0.01 and F
2, 25
=11.6, p\0.001,
respectively). NBO applied 1 h after ischemia reduced the
TUNEL-positive cells number by 70 % and by 42 % when
initiated 3 h after ischemic insult (F
1,8
=39.9, p\0.001
and F
1,8
=11.6, p\0.01, respectively). The application of
NBO at 6 h resulted neither in significant increase in number
of neurons observed in CA1 region of hippocampus nor in
decrease in TUNEL-positive cells (Figs. 2,3).
1 h
3 h
6 h
HBO HBA NBO
sham ischemia
TUNEL-positive cells (0.5 mm)
0
10
20
30
40
50
1 h 3 h 6 h
ischemia HBO
HBA NBO *
#
Fig. 3 TUNEL-positive cells detected in CA1 region in HBO, HBA
and NBO treated animals. Treatment was initiated at different times
after ischemia (1, 3 and 6 h). The hyperbaric and NBO treatment was
applied 3 times in 24-h intervals. Brain tissue was examined 7 days
after ischemia. Top right corner graph represents results expressed as
number of TUNEL-positive cells in the central CA1 area of 0.5 mm
length. Number of analyzed animals per group n=3–4. Results on
the graph are mean values ±SEM. *—significantly different from
HBO and HBA groups, p\0.001;
#
—significantly different from
HBO group, p\0.01
6 Exp Brain Res (2013) 224:1–14
123
The effect of HBO and HBA on gerbil brain
temperature
Brain temperature measurements showed that the mean
temperature of gerbil brain after implantation of the probe
varied between 36.5 and 37 °C. A significant increase, up
to 37.8 °C, in brain temperature was observed up to 4 h
after ischemia which remained through the whole mea-
surement period. The application of HBO therapy 1 or 3 h
after ischemic incident resulted in a significant decrease in
brain temperature (F
1,14
=5.03, p\0.04 and
F
1,20
=17.9, p\0.001, respectively) and was observed
up to 10 h after HBO treatment; subsequently, the tem-
perature remained in the range registered before ischemia
(Fig. 4a, b). HBO applied 6 h after ischemia also prevented
brain temperature increase (F
1,20
=22.16, p\0.001)
(Fig. 4c). The application of HBA to the ischemic gerbils 1
or 3 h after ischemia also resulted in a significant decrease
in the brain temperature (F
1,20
=19.5, p\0.001 and
F
1,18
=7.56, p\0.05, respectively) and was observed for
up to 10 h after the first HBA treatment (Fig. 4a, b); con-
versely, the application of HBA 6 h after ischemic incident
resulted in only a slight, insignificant decrease in brain
temperature and was only observed up to 3 h after treat-
ment (Fig. 4c). Hyperbaric sessions themselves had an
influence on the temperature of the brain, and after each
session an additional, significant transient drop of tem-
perature was observed.
NBO treatment to ischemic gerbils resulted in a signif-
icant decrease in brain temperature only when NBO was
applied 1 h after ischemia and measured temperatures were
significantly different from the ischemic control only up to
3 h after NBO (p\0.01). Application of NBO 3 and 6 h
after ischemia produced a short transient decrease which
disappeared when NBO treatment was terminated (Fig. 4).
The effect of HBO and HBA on nest-building behavior
in ischemic gerbils
The nest-building behavior of gerbils was monitored for
7 days following the ischemic insult. Our current and
previous experiments showed that naı
¨ve and sham-operated
animals start nest building immediately after placing them
into the experimental cage (Duszczyk et al. 2006a), and
after 3-min ischemia, the gerbils exhibited a 2-day delay in
the initiation of nest building and a paper shredding score
of 3 was reached at the end of the observation period (day 6
or 7) (Fig. 5). In the present experiment, sham-operated
animals were treated with HBO and HBA, which did not
significantly influence the nest-building behavior. In all
experimental groups, the nest-building process improved
over time but significant differences in the time required to
reach the highest score were observed.
The HBO therapy in gerbils submitted to 3-min ische-
mia resulted in significant improvement in the nest-build-
ing scores. The animals which received the HBO therapy 1
or 3 h after the insult started to build the nest within 24 h
after ischemia, and the nest-building process was signifi-
cantly improved compared to non-treated animals
(F=22.28, p\0.001 for HBO and F=22.02,
p\0.001) (Fig. 4a, b). The delay of nest building in ani-
mals treated with HBO 6 h after ischemia was only 1 day,
but the difference in behavior of treated and non-treated
animals was significant (F=18.65, p\0.001) (Fig. 5c).
Animals treated with HBA also showed significant
improvement in nest-building behavior compared to non-
treated (F=18.43, p\0.001 for HBA treatment started
1 h after insult, F=21.57, p\0.001 and F=21.32,
p\0.001 for treatment initiated 3 and 6 h after ischemia,
respectively), and this was not significantly different from
that observed after HBO. NBO treatment initiated 1 and
6 h after ischemia also resulted in quicker nest building
(F=27.40, p\0.001 and F=33.07, p\0.001, respec-
tively) (Fig. 5a, c), although the NBO treatment applied
3 h after ischemia did not.
Discussion
The results presented in this paper demonstrate that both
HBO and HBA, applied up to 6 h after transient forebrain
ischemia in gerbils, induce morphological protection of the
CA1 pyramidal neurons and also reduce behavioral deficits
in the nest-building task to a higher degree than the
application of NBO.
The CA1 pyramidal neurons both in experimental ani-
mals and in humans undergo selective cell death after
transient forebrain ischemia (Bonnekoh et al. 1990). This
phenomenon is termed ‘‘delayed neuronal death’’ because
pyramidal neurons of CA1 mostly remain unchanged at the
early stage after the event but begin to die in a time win-
dow of usually 3–4 days after ischemia due to apoptotic
processes (Nitatori et al. 1995). As known from experi-
mental research, the CA1 pyramidal neurons of the gerbil
hippocampus degenerate within 2–4 days after 5-min
transient global ischemia and it was reported that only
5.8 % of the neurons survive 3 weeks later (Bonnekoh
et al. 1990). Our results show that one week after an
ischemic insult, lasting 3-min apoptotic processes were still
in progress, and after 2 weeks, only 18 % of CA1 pyra-
midal neurons were intact. HBO therapy significantly
reduced the number of dying neurons, and protection was
most effective when HBO was initiated 1 h after ischemia;
however, the therapeutic window for HBO was still open
6 h after ischemia. These results are in agreement with
previously published data reporting a therapeutic window
Exp Brain Res (2013) 224:1–14 7
123
for HBO (from 1.5 to 3 ATA) using different animal
models of both global and focal ischemia, where neuronal
protection effect was observed even when this therapy was
applied up to 12 h after ischemic insult (Li et al. 2005; Lou
et al. 2004; Niklas et al. 2004; Wang et al. 2008; Zhang
et al. 2005).
There are reports that normobaric hyperoxia adminis-
tered early in the injury and for a prolonged period of time
prevents delayed post-ischemic neuronal death (Kumaria
and Tolias 2009). NBO applied during ischemia had been
shown to significantly reduce neuronal cell death, and brain
infarct volume both in vivo and in vitro inhibits the
upregulation of matrix metalloproteinase-2 and attenuate
nitric oxide production (Yuan et al. 2010; Kim et al. 2005;
Gu
¨nther et al. 2004). Studies using NBO and HBO after
experimental cerebral ischemia provide evidence for
blood–brain barrier stabilization, although the effect of
NBO was rather weak (Veltkamp et al. 2005). Our results
show that NBO applied after ischemia resulted in only a
slight reduction in apoptosis and increase in the number of
surviving neurons.
The results presented in this paper indicate a more
potent protective effect of HBO than NBO and are in
agreement with data published by Beynon et al. (2007)
35
35,5
36
36,5
37
37,5
38
38,5
0246810 12 14 16 18 20 22 24 26
ischemia HBO
HBA NBO
Brain temperature (
°C)
*
****
#
#
#
##
35
36
37
38
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
35
35,5
36
36,5
37
37,5
38
38,5
0246810 12 14 16 18 20 22 24 26
ischemia HBO
HBA NBO
Brain temperature (
°C)
*
****
#
#
####
35
36
37
38
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
35
35,5
36
36,5
37
37,5
38
38,5
0246810 12 14 16 18 20 22 24 26
Time after brain temperature probe implantation (h)
ischemia HBO
HBA NBO
Brain temperature (
°C)
**
***
#
35
36
37
38
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
A1 h
B 3 h
C6 h
Fig. 4 Effect of HBO, HBA
and NBO treatment started 1 h
(a), 3 h (b) and 6 h (c) after
ischemia on gerbil brain
temperature. The hyperbaric
and NBO treatment was applied
3 times in 24-h intervals. Main
graphs show temperature
measurement during first 27 h.
Brain temperature was
measured for 74 h; complex
results are presented on small
graphs. Three-min ischemia was
induced at 4th hour after probe
implantation (arrow). Results
are mean values ±SEM;
n=4–5 per each group.
*—HBO group significantly
different from untreated
ischemia, p\0.01;
#
—HBA
group significantly different
from untreated ischemia,
p\0.01
8 Exp Brain Res (2013) 224:1–14
123
showing that even delayed HBO treatment is more effec-
tive than early prolonged NBO. Surprisingly, in our
experiments, HBA therapy appeared to be more effective
than NBO. HBA applied up to 6 h after ischemia resulted
in decrease in apoptotic neurons and significant increase in
the number of surviving neurons in the CA1 region of the
gerbil hippocampus. Only few studies describe the effect of
HBA on postischemic survival, and published data show
only a slight, if any, protective effect of HBA (Peng et al.
2008;Gu
¨nther et al. 2004). This makes the results pre-
sented in this paper more interesting, since the beneficial
effect of HBA was not only limited to a reduced number of
dying neurons but also manifested itself in the animals’
behavior. These results may also resolve doubts concerning
the effectiveness of oxygen administration by aviators
mask in air pressurized chambers (Harch and Neubauer
2009).
Although the neuronal death after ischemic episode is
delayed, during the initial postischemic period preceding
the death of CA1 neurons, gerbils demonstrate a behavioral
deficit, which seems to be linked to hippocampal dys-
function (Duszczyk et al. 2006a). The control gerbils
exhibit stereotypical nest-building behavior, characterized
by a species-specific shredding of the nest material, in this
case a soft paper towel. It is well documented that global
brain ischemia in gerbils results in disturbances in nest-
building behavior, and this correlates directly with the
extent of ischemic morphological damage (Antonawich
et al. 1997; Baldwin et al. 1993; Duszczyk et al. 2006a). A
typical male gerbil builds a nest within 12–24 h. In our
studies, sham-operated animals serving as controls started
nest building immediately after placing them in the
experimental cage. Animals submitted to 3-min ischemia
showed a 2-day delay in the initiation of nest building. It
A 1 h
0
1
2
3
4
1234567
Day after ischemia
ischemia
ischemia + HBO
sham + HBO
ischemia + NBO
Nest building score
0
1
2
3
4
12345 67
Day after ischemia
ischemia
ischemia + HBA
sham + HBA
B 3 h
0
1
2
3
4
12345 67
Day after ischemia
ischemia
ischemia + HBO
sham + HBO
ischemia + NBO
Nest building score
0
1
2
3
4
1234567
Day after ischemia
ischemia
ischemia + HBA
sham + HBA
C 6 h
0
1
2
3
4
1234567
Day after ischemia
ischemia
ischemia + HBO
sham + HBO
ischemia + NBO
Nest building score
0
1
2
3
4
1234567
Day after ischemia
ischemia
ischemia + HBA
sham + HBA
Fig. 5 Nest-building behavior
in gerbils after HBO, HBA and
NBO treatment started at 1 h
(a), 3 h (b) and 6 h (c) after
ischemia. The hyperbaric and
NBO treatment was applied 3
times in 24-h intervals. Nest
building was assessed each day
for 7 days following ischemic
episode. Values are median
nest-building score, IQR for
each data point B1. Group size:
sham n=5; ischemia n=9;
HBO, HBA and NBO each time
group n=5
Exp Brain Res (2013) 224:1–14 9
123
has been suggested that disruption of nest building is not a
secondary effect of increased motor activity, which usually
accompanies transient global ischemia, but results from
delays in habituation and spatial mapping resulting from
hippocampal damage (Antonawich et al. 1997; Babcock
et al. 1993). Administration of HBO treatment after
ischemia significantly improved nest building, which may
reflect a protective effect of hyperoxygenation on neurons.
Animals which received NBO and HBA treatment after
ischemia also showed an improvement in nest-building
behavior, which was not significantly different from that
observed after HBO. Generally, delivery of oxygen under
increased partial pressure within 6 h after ischemia sig-
nificantly improved animal’s nest-building behavior. The
beneficial effect of HBO on neurological deficits graded on
the Garcia scale (Garcia et al. 1995) was reported earlier in
the rat model of focal cerebral ischemia (Miljkovic-Lolic
et al. 2003; Beynon et al. 2007); however, no beneficial
effect of NBO was observed (Beynon et al. 2007) and, to
our knowledge, there are no reports regarding the effect of
HBA on animal behavior after ischemia. Thus, results
presented in this paper for the first time show that HBA
may not only protect CA1 neurons from ischemia-induced
damage but also improve nest-building behavior in gerbils.
These results indicate that the increased survival of neurons
observed in our experiments correlates with an improved
neurological outcome.
There are many mechanisms proposed to explain the
beneficial effects of HBO treatments, including increased
oxygenation of ischemic and penumbra area in the brain,
reduction in blood–brain barrier damage, elevation of
autophagic activity, attenuation of nitric oxide production
and inhibition of apoptotic protein expression (Ostrowski
et al. 2005; Yuan et al. 2010; Veltkamp et al. 2005; Yan
et al. 2011). There are reports that supplemental oxygen-
ation applied during reperfusion may result in intensifica-
tion of injury (Rink et al. 2010). However, increased
oxygenation evoked by HBO reduces neuronal death and
improves neurological outcome, which was shown not only
in our experiments but also after canine cardiac arrest
(Rosenthal et al. 2003), circulatory occlusion in cats (Kapp
et al. 1982) and cardiopulmonary arrest in pigs (Van Meter
et al. 2008). It was recently shown that the improvement in
neurological function and reduced neuronal cell death
observed after cardiac arrest and reperfusion does not result
from increased cerebral oxygen delivery or oxygen con-
sumption (Rosenthal et al. 2003). It is possible that
hyperbaric oxygen or hyperbaric treatment itself may
trigger more protective mechanisms and the maintenance
of brain temperature might be one of them.
Previous studies show that hyperthermia is frequently
observed in the first 72 h after resuscitation from cardiac
arrest and were associated with poor outcome (Takasu
et al. 2001). Similarly, ischemic stroke is usually followed
by hyperthermia, resulting from both a stroke-induced
inflammatory reaction and disturbances in cell metabolism
(Zaremba 2004). Mild therapeutic hypothermia is currently
the only therapy that improved survival and brain function
after initial resuscitation from cardiac arrest (Janata and
Holzer 2009; Sugerman and Abella 2009). It protects the
brain after ischemia by decreasing metabolism, inhibiting
excitatory amino acid release, and also by attenuation of
reactive oxygen species formation and the immune
response during reperfusion (Janata and Holzer 2009).
Mongolian gerbils are particularly susceptible to bilat-
eral carotid occlusion, which results in global forebrain
ischemia, due to the incomplete circle of Willis (Du et al.
2011). In this model of ischemia, brain temperature often
significantly decreases during occlusion but then quickly
returns to normal after the onset of recirculation (Zhang
et al. 1997; Colbourne et al. 1993). However, at 10–20 min
after the start of recirculation, brain temperature increases
by 0.7–1 °C and this postischemic hyperthermia is
observed for 45–90 min.; thereafter, temperature returns to
normal. Brain temperature measurements presented in this
paper are mostly in agreement with previous observations.
However, in our experiments, the increase in brain tem-
perature lasted for almost 6 h after reperfusion and
remained slightly increased to the end of measurements.
Previously, we also observed a prolonged (3–4 h) period of
post-ischemic hyperthermia in gerbils submitted to 3-min
global ischemia (Duszczyk et al. 2005,2006a). The basis of
postischemic temperature increase in the gerbil remains
uncertain; however, the hyperthermia described in certain
focal ischemia models in rats has been suggested to be
caused by altered blood flow in the hypothalamus (He et al.
1999; Zhao et al. 1994), although damage of the blood–
brain barrier and development of inflammatory processes
may also be an explanation (Veltkamp et al. 2005).
Wood and Gonzales (1996) showed that hyperthermia
increases the imbalance between energy supply and
demand following ischemia. Temperature-dependent
changes in functional neurological outcome, histopathol-
ogy, intraneuronal calcium accumulation and the levels of
enzymes mediating calcium effects, including neuronal
excitability, synaptic modulation and release of excitatory
neurotransmitters, were demonstrated in number of animal
models of cardiac arrest and stroke (Dietrich et al. 1996;
Ginsberg and Busto 1998; Wass et al. 1995; Coimbra et al.
1996). These changes may cause further development of
postischemic injury of neurons, leading to irreversible
lesions. Hyperthermia may also induce additional dys-
function of the blood–brain barrier, facilitating the regional
influx of leukocytes as a result of the ischemia-evoked
10 Exp Brain Res (2013) 224:1–14
123
inflammatory reaction (Zaremba 2004). Thus, the preven-
tion of hyperthermia within the first hours after resuscita-
tion from cardiac arrest or stroke is important for
preventing further damage to the brain.
The results presented in this paper show that both HBO
and HBA treatment applied up to 3 h after ischemia pre-
vents ischemia-evoked increase in brain temperature,
especially that observed up to 10 h after reperfusion.
Within 72 h, brain temperature of animals treated with
hyperbaric therapy remained slightly lower or the same
level as before ischemia. Hyperbaric treatment applied 6 h
after ischemic insult also prevented the increase in brain
temperature, although HBA was less effective than HBO.
NBO treatment effectively prevented brain temperature
increase only when applied 1 h after ischemia. The results
presented in this paper show the correlation between
morphological protection, behavioral improvement and
attenuation of postischemic hyperthermia. This suggests
the causal connection between presented data and indicates
a possible key role of brain temperature decrease in ben-
eficial effects of hyperbaric treatments.
It is known from the literature that increased partial
pressure of oxygen causes hypothermia. It has been shown
in rats that HBO is associated with a significant decrease in
body temperature and that this effect is evoked mostly by
an increase in the partial pressure of oxygen, and only in
small part by heat loss due to pressure alone (Fenton et al.
1993). In our experiments, a decrease in brain temperature
during both HBO and HBA was also observed, while only
a slight temperature drop was observed in gerbils subjected
to NBO. This observation is difficult to explain because the
oxygen partial pressure during NBO is two times higher
than during HBA (data not shown); however, perhaps the
effect of pressurization itself should not be totally exclu-
ded. In fact, Fenton et al. (1993) observed a decrease in
body temperature in pressure control experiments, where
the partial pressure of oxygen at 4 ATA was the same as
the partial pressure in air at 1 ATA. Recently, Tsai et al.
(2005) found beneficial effects of 8 % oxygen pressurized
to 253 kPa (2.5 ATA) in resuscitating rats with experi-
mental heatstroke. The mechanism of this hyperbaric
treatment induced hypothermia is still not clear, although
the initially proposed involvement of 5-HT
1A
receptors was
excluded (Fenton et al. 1993). These results together with
results presented in this paper suggest that the pressuriza-
tion itself (occurring during HBO and HBA) might be the
causal reason for the observed beneficial effects of HBO
and HBA.
Concerning the potential mechanisms of pressure-rela-
ted hypothermia, the suggestion that decrease in CBF
caused by high pressure may induce hypothermia and also
does not seem to be good explanation because pressure
used in presented in this paper experiments (2.5 ATA) only
slightly changes CBF (Demechenko et al. 2005). Further,
one of the explanations of observed hypothermia may be
the increased oxygen supply delivered to the ischemic and
penumbra regions of the brain and in consequence
improvement in aerobic metabolism of injured regions, at
the time window which may guarantee at least partial
prevention of neuronal damage (Rockswold et al. 2001).
However, Rosenthal et al. (2003) using the animal cardiac
arrest model showed no increase in cerebral oxygen
delivery and consumption during hyperbaric treatment. It
was suggested that probably there is no ongoing ischemia
during postischemic hypoperfusion and that energy
metabolism may not be limited by oxygen delivery but
rather by the activity of aerobic metabolic enzymes
(McKinley et al. 1996; Rosenthal et al. 2003).
Hyperbaric reduction in blood–brain barrier damage
preventing inflammatory processes and inhibition of
neutrophil adhesion to the endothelium are also factors
which may contribute in neuronal protection (Buras and
Reenstra 2007; Veltkamp et al. 2005). There is now
agreement that the best time window for hyperbaric
therapy is within the first 6 h after ischemic insult. Our
results show that this time window is effective both for
HBO and HBA. The best time for NBO application is up
to 3 h after ischemia, and during this time, NBO may
serve as a useful adjunct therapy widening the time
window for reperfusion by as much as 2 h (Kim et al.
2005). However, clinical practice shows that oxygen
applied less than 1 h after resuscitation from cardiac
arrest results in a dose–dependent association between
supranormal oxygen tension and risk of in-hospital death
(Kilgannon et al. 2011).
In conclusion, the results presented here show for the
first time that not only HBO but also HBA applied between
1 and 6 h after transient forebrain ischemia may prevent
ischemia-induced neuronal damage in the CA1 region of
the gerbil brain and that this protection is protection is
mostly likely due to a pressure-related inhibition of brain
temperature increase which typically originates from
ischemia. Although early applied NBO also resulted in
neuronal protection, this treatment was less effective than
delayed HBO or HBA therapy.
Acknowledgments This study was supported by Polish Ministry of
Sciences and Higher Education grant no. N N401 003935. We would
like to thank Technika Podwodna for making accessible the hyper-
baric chamber used in HBA experiments. We are also grateful to Prof.
Jerzy Lazarewicz from Medical Research Centre and Dr Jacek Kot
from National Center for Hyperbaric Medicine in Gdynia for reading
the manuscript and useful comments.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
Exp Brain Res (2013) 224:1–14 11
123
References
Antonawich FJ, Melton CS, Wu P, Davis JN (1997) Nesting and
shredding behavior as an indicator of hippocampal ischemic
damage. Brain Res 764:249–252
Atochin DN, Demechenko IT, Astern J, Boso AE, Piantadosi CA,
Huang PL (2003) Contributions of endothelial and neuronal
nitric oxide synthase to cerebrovascular responses to hyperoxia.
J Cereb Blood Flow Metab 23:1219–1226
Babcock A, Baker D, Lovec R (1993) Locomotor activity in the
ischemic gerbil. Brain Res 625:351–354
Baldwin HA, Jones JA, Cross AJ, Green AR (1993) Histological,
biochemical and behavioural evidence for the neuroprotective
action of chlormetazole following prolonged carotid occlusion.
Neurodegeneration 2:139–146
Beynon C, Sun L, Marti HH, Heiland S, Veltkamp R (2007) Delayed
hyperbaric oxygenation is more effective than prolonged
normobaric hyperoxia in experimental focal cerebral ischemia.
Neurosci Lett 425:141–145
Bonnekoh P, Barbier A, Oschlies U, Hossmann K-A (1990) Selective
vulnerability in the gerbil hippocampus: morphological changes
after 5-min ischemia and long survival times. Acta Neuropathol
80:18–25
Buras JA, Reenstra WR (2007) Endothelial-neutrophil interactions
during ischemia and reperfusion injury: basic mechanisms of
hyperbaric oxygen. Neurol Res 29:127–131
Buras JA, Stahl GL, Svoboda KK, Reenstra WR (2000) Hyperbaric
oxygen downregulates ICAM-1 expression induced by hypoxia
and hypoglycemia: the role of NOS. Am J Physiol Cell Physiol
278:C292–C302
Busl KM, Greer DM (2010) Hypoxic-ischemic brain injury: patho-
physiology, neuropathology and mechanisms. NeuroRehabilita-
tion 26:5–13
Chandler MJ, DeLeo JA, Carney JM (1985) An unanesthetized gerbil
model of cerebral ischemia-induced behavioral changes. J Phar-
macol Methods 14:137–146
Coimbra C, Drake M, Moller FB, Wieloch T (1996) Long-lasting
neuroprotective effect of postischemic hypothermia and treat-
ment with an anti-inflammatory/antipyretic drug. Stroke
27:1578–1585
Colbourne F, Nurse SM, Corbett D (1993) Temperature changes
associated with forebrain ischemia in the gerbil. Brain Res
602:264–267
Daugherty WP, Levasseur JE, Sun D, Rockswold GL, Bullock MR
(2004) Effects of hyperbaric oxygen therapy on cerebral
oxygenation and mitochondrial function following moderate
lateral fluid-percussion injury in rats. J Neurosurg 101:499–504
Deakin CD, Morrison LJ, Morley PT, Callaway CW, Kerber RE,
Kronick SL, Lavonas EJ, Link MS, Neumar RW, Otto CW, Parr
M, Shuster M, Sunde K, Peberdy MA, Tang W, Hoek TL, Bo
¨ttiger
BW, Drajer S, Lim SH, Nolan JP, Advanced Life Support Chapter
Collaborators (2010) Part 8: advanced life support: 2010 interna-
tional consensus on cardiopulmonary resuscitation and emergency
cardiovascular care science with treatment recommendations.
Resuscitation 81(Suppl 1):e93–e174
Dean JB, Mulkey DK, Garcia AJ 3rd, Putnam RW, Jenderson RA 3rd
(2003) Neuronal sensitivity to hyperoxia, hypercapnia, and inert
gases at hyperbaric pressures. J Appl Physiol 95:883–909
Demechenko IT, Luchakov YI, Moskvin AN, Gutsaeva DR, Allen
BW, Thalmann ED, Piantadosi CA (2005) Cerebral blood flow
and brain oxygenation in rats breathing oxygen under pressure.
J Cereb Blood Flow Metab 25:1288–1300
Dietrich WD, Busto R, Globus MY, Ginsberg MD (1996) Brain
damage and temperature: cellular and molecular mechanisms.
Adv Neurol 71:177–194
Du XY, Zhu XD, Dong G, Lu J, Wang Y, Zeng L, Zhao TY, Ye HH,
Li RS, Bai JY, Chen ZW (2011) Characteristics of circle of
Willis variations in the Mongolian gerbil and newly established
ischemia-prone gerbil group. ILAR e-J 52:e1–e7
Duszczyk M, Gadamski R, Ziembowicz A, Danysz W, Lazarewicz
JW (2005) NMDA receptor antagonism does not inhibit
induction of ischemic tolerance in gerbil brain in vivo. Neurotox
Res 7(4):283–292
Duszczyk M, Ziembowicz A, Gadamski R, Lazarewicz JW (2006a)
Behavioral evaluation of ischemic damage to CA1 hippocampal
neurons: effects of preconditioning. Acta Neurobiol Exp
66:311–319
Duszczyk M, Gadamski R, Ziembowicz A, Lazarewicz JW (2006b)
Antagonists of group I metabotropic glutamate receptors do not
inhibit induction of ischemic tolerance in gerbil hippocampus.
Neurochem Int 48:478–484
Fenton LH, Beck G, Djali S, Robinson MB (1993) Hypothermia
induced by hyperbaric oxygen is not blocked by serotonin
antagonists. Pharmacol Biochem Behav 44:357–364
Garcia JH, Wagner S, Liu K-F, Hu X-J (1995) Neurological deficit
and extent of neural necrosis attributable to middle cerebral
artery occlusion in rats. Stroke 26:627–635
Ginsberg MD, Busto R (1998) Combating hyperthermia in acute
stroke: a significant clinical concern. Stroke 29:529–534
Goda W, Satoh K, Nakashima M, Hara A, Niwa M (2012) PBN fails
to suppress in delayed neuronal death of hippocampal CA1
injury following transient forebrain ischemia in gerbils. Neurosci
Lett 517:47–51
Gu
¨nther A, Manaenk A, Franke H, Wagner A, Schneider D,
Berrouschot J, Reinhardt R (2004) Hyperbaric and normobaric
reoxygenation of hypoxic rat brain slices—impact on purine
nucleotide and cell viability. Neurochem Int 45:1125–1132
Harch PG, Neubauer RA (2009) HBO therapy in global cerebral
ischemia/anoxia and coma. In: Jain KK (ed) Textbook of
hyperbaric medicine, 5th edn. Hogrefe & Huber Publishers,
Go
¨ttingen, pp 235–274
He Z, Yamawaki T, Yang S, Day AL, Simpkins JW, Naritomi H
(1999) Experimental model of small deep infarcts involving the
hypothalamus in rats: changes in body temperature and postural
reflex. Stroke 30:2743–2751
Illingworth C (1962) Treatment of arterial occlusion under oxygen at
two atmospheres pressure. Br Med J 5315:1271–1275
Iwatsuki N, Takahashi M, Ono K, Tajima T (1994) Hyperbaric
oxygen combined with nicardipine administration accelerates
neurologic recovery after cerebral ischemia in canine model. Crit
Care Med 22:858–863
Janata A, Holzer M (2009) Hypothermia after cardiac arrest. Prog
Cardiovasc Dis 52:168–179
Kapp JP, Phillips M, Markov A, Smith RR (1982) Hyperbaric oxygen
after circulatory arrest: modification of postischemic encepha-
lopathy. Neurosurgery 11:496–499
Kilgannon JH, Jones AE, Parrillo JE, Dellinger RP, Milcarek B,
Hunter K, Shapiro NI, Trzeciak S, Emergency Medicine Shock
Research Network (EMShockNet) Investigators, (2011) Rela-
tionship between supranormal oxygen tension and outcome
after resuscitation from cardiac arrest. Circulation 123:2717–
2722
Kim HY, Singhal AB, Lo EH (2005) Normobaric hyperoxia extends
the reperfusion window in focal cerebral ischemia. Ann Neurol
57:571–575
Ko IG, Shin MS, Kim BK, Kim SE, Sung YH, Kim TS, Shin MC,
Cho HJ, Kim SC, Kim SH, Kim KH, Shin DH, Kim CJ (2009)
Tadalafil improves short-term memory by suppressing ischemia-
induced apoptosis of hippocampal neuronal cells in gerbils.
Pharmacol Biochem Behav 91:629–635
12 Exp Brain Res (2013) 224:1–14
123
Kot J, Mathieu D (2011) Controversial issues in hyperbaric oxy-
gen therapy: a European Committee for Hyperbaric Medicine
Workshop. Diving Hyperb Med 41:101–104
Krakovsky M, Rogatsky G, Zarchin N, Mayevsky A (1998) Effect of
hyperbaric oxygen therapy on survival after global cerebral
ischemia in rats. Surg Neurol 49:412–416
Kumaria A, Tolias CM (2009) Normobaric hyperoxia therapy for
traumatic brain injury and stroke: a review. Br J Neurosurg
23:576–584
Lassen NA, Palvo
¨lgyi R (1968) Cerebral steal during hypercapnia and
the inverse reaction during hypocapnia observed by the 133
xenon technique in man. Scand J Clin Lab Invest Suppl 102:XIII
Li Y, Zhou C, Calvert JW, Colohan ART, Zhang JH (2005) Multiple
effects of hyperbaric oxygen on the expression of HIF-1aand
apoptotic genes in global ischemia-hypotension rat model. Exp
Neurol 191:198–210
Li LF, Liao SK, Ko YS, Lee CH, Quinn DA (2007) Hyperoxia
increases ventilation-induced lung injury via mitogen-activated
protein kinases: a prospective, controlled animal experiment.
Crit Care 11(1):R25
Lou M, Eschenfelder CC, Herdegen T, Brecht S, Deuschl G (2004)
Therapeutic window for use of hyperbaric oxygenation in focal
transient ischemia in rats. Stroke 35:578–583
McKinley BA, Morris WP, Parmley CL, Butler BD (1996) Brain
parenchyma P
O2
,P
CO2
and pH during and after hypoxic,
ischemic brain insult in dogs. Crit Care Med 24:1858–1868
Michalski D, Ha
¨rtig W, Schneider D, Hobohm C (2011) Use of
normobaric and hyperbaric oxygen in acute focal cerebral
ischemia—a preclinical and clinical review. Acta Neurol Scand
123:85–97
Miljkovic-Lolic M, Silbergleit R, Fiskum G, Rosenthal RE (2003)
Neuroprotective effects of hyperbaric oxygen treatment in
experimental focal cerebral ischemia are associated with reduced
brain leukocyte myeloperoxidase activity. Brain Res 971:90–94
Morley PT (2011) Drugs during cardiopulmonary resuscitation. Curr
Opin Crit Care 17:214–218
Neumar RW (2011) Optimal oxygenation during and after cardio-
pulmonary resuscitation. Curr Opin Crit Care 17:236–240
Niklas A, Brock D, Schober R, Schultz A, Schneider D (2004)
Continuous measurement of cerebral tissue oxygen pressure
during hyperbaric oxygenation—HBO effects on brain edema
and necrosis after severe brain trauma in rabbits. Neurol Sci
219:77–82
Nitatori T, Sato N, Waguri S, Karasawa Y, Araki H, Shibanai K,
Kominami E, Uchiyama Y (1995) Delayed neuronal death in the
CA1 pyramidal cell layer of the gerbil hippocampus following
transient ischemia is apoptosis. J Neurosci 15(2):1001–1011
Nolan JP, Neumar RW, Adrie C, Aibiki M, Berg RA, Bo
¨ttinger BW,
Callaway C, Clark RSB, Geocadin RG, Jauch EC, Kern KB,
Laurent I, Longstreth WT, Merchant RM, Morley P, Morrison
LJ, Nadkarni V, Peberdy MA, Rivers EP, Rodriguez-Nunez A,
Sellke FW, Spaulding C, Sunde K, Hoek TV (2008) Post-cardiac
arrest syndrome: epidemiology, pathophysiology, treatment, and
prognostication. A Scientific Statement from the International
Liaison Committee on Resuscitation; the American Heart
Association Emergency Cardiovascular Care Committee; the
Council on Cardiovascular Surgery and Anesthesia; the Council
on Cardiopulmonary, Perioperative, and Critical Care; the
Council on Clinical Cardiology; the Council on Stroke. Resus-
citation 79:350–379
Nurse S, Corbett D (1994) Direct measurement of brain temperature
during and after intraischemic hypothermia: correlation with
behavioral, physiological, and histological endpoints. J Neurosci
14(12):7726–7734
Ostrowski RP, Colohan ART, Zhang JH (2005) Mechanisms of
hyperbaric oxygen-induced neuroprotection in rat model of
subarachnoid hemorrhage. J Cereb Blood Flow Metab
25:554–571
Peng Z, Ren P, Kang Z, Du J, Lian Q, Liu Y, Zhang JH, Sun X (2008)
Up-regulated HIF-1ais involved in hypoxic tolerance induced
by hyperbaric oxygen preconditioning. Brain Res 1212:71–78
Rink C, Roy S, Khan M, Ananth P, Kuppusamy P, Sen CK, Khanna S
(2010) Oxygen-sensitive outcomes and gene expression in acute
ischemic stroke. J Cereb Blood Flow Metab 30:1275–1287
Rockswold SB, Rockswold GL, Vargo JM, Erickson CA, Sutton RL,
Bergman TA, Biros MH (2001) Effects of hyperbaric oxygen-
ation therapy on cerebral metabolism and intracranial pressure in
severely brain injured patients. J Neurosurg 94:403–411
Rockswold SB, Rockswold GL, Zaun DA, Zhang X, Cerra CE,
Bergman TA, Liu J (2010) A prospective, randomized clinical
trial on compare the effect of hyperbaric to normobaric
hyperoxia on cerebral metabolism, intracranial pressure, and
oxygen toxicity in severe traumatic brain injury. J Neurosurg
112:1080–1094
Rosenthal RE, Silbergleit R, Hof PR, Haywood Y, Fiskum G (2003)
Hyperbaric oxygen reduces neuronal death and improves neuro-
logical outcome after canine cardiac arrest. Stroke 34:1311–1316
Sinclair SE, Altemeier WA, Matute-Bello G, Chi EY (2004)
Augmented lung injury due to interaction between hyperoxia
and mechanical ventilation. Crit Care Med 32:2496–2501
Singhal AB (2006) Oxygen therapy in stroke: past, present, and
future. Int J Stroke 1:101–200
Smith G, Lawson DD, Renfrew S, Ledingham IM, Sharp GR (1961)
Preservation of cortical activity by breathing oxygen at two
atmospheres of pressure during cerebral ischemia. Surg Gynecol
Obstet 113:289–292
Sugerman NT, Abella BS (2009) Hospital-based use of therapeutic
hypothermia after cardiac arrest in adults. J Neurotrauma
26:371–376
Takahashi M, Iwatsuku N, Ono K, Tajima T, Akama M, Koga Y
(1992) Hyperbaric oxygen therapy accelerates neurologic recov-
ery after 15-minute complete global cerebral ischemia in dogs.
Crit Care Med 20:1588–1594
Takasu A, Saitoh D, Kaneko N, Sakamoto T, Okada Y (2001)
Hyperthermia: is it an ominous sign after cardiac arrest?
Resuscitation 49:273–277
Tsai HM, Gao CJ, Li WX, Lin MT, Niu KC (2005) Resuscitation
from experimental heatstroke by hyperbaric oxygen therapy. Crit
Care Med 33:813–818
Van Hulst RA, Haitsma JJ, Klein J, Lachmann B (2003) Oxygen
tension under hyperbaric conditions in healthy pig brain. Clin
Physiol Funst Imaging 23:143–148
Van Meter K, Sheps S, Jriedt F, Moises J, Barratt D, Murphy-Lavoie
H, Harch PG, Bazan N (2008) Hyperbaric oxygen improves rate
of return of spontaneous circulation after prolonged normother-
mic porcine cardiopulmonary arrest. Resuscitation 78:200–214
Veltkamp R, Siebing DA, Sun L, Heiland S, Biber K, Marti HH,
Nagel S, Schwab S, Schwaninger M (2005) Hyperbaric oxygen
reduces blood-brain barrier damage and edema after transient
focal cerebral ischemia. Stroke 36:1679–1683
Wang X-L, Zhao Y, Yang Y-J, Xie M, Yu X-H (2008) Therapeutic
window of hyperbaric oxygen therapy for hypoxic-ischemic
brain damage in newborn rats. Brain Res 1222:87–94
Wass CT, Lanier W, Hofer RE, Sheithauer BW, Adnrews AG (1995)
Temperature changes of C1 degree alter functional neurologic
outcome and histopathology in canine model of complete
cerebral ischemia. Anesthesiology 83:325–335
Wood SC, Gonzales R (1996) Hypothermia in hypoxic animal:
mechanisms, mediators, and functional significance. Comp
Biochem Physiol Part B Biochem Mol Biol 113:37–43
Yan W, Zhang H, Bai X, Lu Y, Dong H, Xiong L (2011) Autophagy
activation is involved in neuroprotection induced by hyperbaric
Exp Brain Res (2013) 224:1–14 13
123
oxygen preconditioning against focal cerebral ischemia in rats.
Brain Res 1402:109–121
Yin W, Badr AE, Mychaskiw G, Zhang JH (2002) Down regulation of
COX-2 is involved in hyperbaric oxygen treatment in rat
transient focal cerebral ischemia model. Brain Res 926:165–171
Yuan Z, Liu W, Liu B, Schnell A, Liu KJ (2010) Normobaric
hyperoxia delays and attenuates early nitric oxide production in
focal cerebral ischemic rats. Brain Res 1352:248–254
Zaremba J (2004) Hyperthermia in ischemic stroke. Med Sci Monit
10:RA148–RA153
Zhang L, Mitani A, Yanase H, Kataoka K (1997) Continuous
monitoring and regulation of brain temperature in the conscious
and freely moving ischemic gerbil: effect of MK-801 and delayed
neuronal death in hippocampal CA1. J Neurosci Res 47:440–448
Zhang JH, Lo T, Mychaskiw G, Colohan A (2005) Mechanisms of
hyperbaric oxygen and neuroprotection in stroke. Pathophysiol-
ogy 12:65–80
Zhao Q, Memezawa H, Smith ML, Siesjo BK (1994) Hyperthermia
complicates middle cerebral artery occlusion induced by intra-
luminal filament. Brain Res 649:253–259
14 Exp Brain Res (2013) 224:1–14
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