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Prolonged and intermittent normobaric hyperoxia induce different degrees of ischemic tolerance in rat brain tissue
BR A I N R ES E A RC H 1 1 5 2 ( 2 00 7 ) 2 2 8 –23 3
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Research Report
Prolonged and intermittent normobaric hyperoxia induce different degrees of ischemic tolerance in rat brain tissue Mohammad Reza Bigdeli a,b , Sohrab Hajizadeh a,⁎, Mehdi Froozandeh b , Bahram Rasulian c , Ali Heidarianpour a , Ali Khoshbaten d a
Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran Department of Medical Biotechnology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran c Trauma Research Center, Baqiyatallah Medical Sciences University, Tehran, Iran d Research Center for Chemical Injuries, Baqiyatallah Medical Sciences University, Tehran, Iran b
A R T I C LE I N FO
AB S T R A C T
Article history:
Prior prolonged oxygen exposure is associated with some protection against ischemia–
Accepted 13 March 2007
reperfusion (IR) injury to rat brain tissue, but also with toxic effects. We sought to compare
Available online 28 March 2007
the magnitude of protection offered by prolonged and intermittent oxygen pretreatments against IR injury to the rat brain. Rats were divided into four experimental groups, each of
Keywords:
21 animals. The first two were exposed to 95% inspired (normobaric hyperoxia, NBHO) for
Prolonged hyperoxia
4 h/day for 6 consecutive days (intermittent NBHO) or for 24 continuous hours (prolonged
Intermittent hyperoxia
NBHO). The second two groups acted as controls were exposed to 21% oxygen. After 24 h,
Brain ischemic tolerance
they were subjected to 60 min of right middle cerebral artery occlusion (MCAO) followed by
Ischemic preconditioning
24 h of reperfusion. The animals were sacrificed for assessment of infarct volume, brain
Neuroprotection
edema, and blood–brain barrier (BBB) permeability, respectively. Prolonged and
Stroke
intermittent NBHO pretreatment reduced infarct volume by 63.3% and 73.7%, respectively, when compared to the respective NBNO groups. Intermittent NBHO (when compared to intermittent NBNO) also reduced the post-ischemic increment of brain water content significantly (81.53 ± 0.8%, vs. 80.12 ± 0.79%) and Evans Blue extravasation (7.49 ± 2.89 ± g/g tissue vs. 3.9 ± 0.79 μg/g tissue, P < 0.001), while prolonged NBHO had no significant effect on brain water content (81.69 ± 1.16% vs. 80.74 ± 0.94%) and EB extravasations (6.48 ± 2.42 μg/g tissue vs. 4.31 ± 1.07 μg/g tissue). Intermittent hyperoxia had relatively more significant effects on brain edema and BBB protection. Although preconditioning with both prolonged and intermittent oxygen exposure protects rat brain tissue against IR injury, the intermittent hyperoxia could have relatively more protective effects in this regard. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic preconditioning (IPC) is an endogenous phenomenon that can induce ischemic tolerance (IT) in a variety of
organs such as the heart and kidney, but also the brain. Kitagawa et al. (1990) found that gerbils subjected to sublethal transient global cerebral ischemia exhibited a reduction in hippocampal CA1 neural death after 5 min of global cerebral
⁎ Corresponding author. Fax: +98 21 88006544. E-mail address: [email protected] (S. Hajizadeh). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.03.068
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ischemia. IPC-like phenomena have similarly been also demonstrated in brain slices (Raval et al., 2003) and in neural cell cultures (Romera et al., 2004), and can be induced by diverse stimuli including seizures (Plamondon et al., 1999), anoxia (Perez-Pinzon et al., 1996), heat (Chopp et al., 1989; Kitagawa et al., 1991), oxidative stress(Ohtsuki et al., 1992), polyunsaturated fatty acid (PUFA) pretreatment (Patel et al., 1998; Maingret et al., 2000), and inhibition of oxidative phosphorylation (Riepe et al., 1997). Most such stimuli, however, lack potential for clinical translation due to associated toxicity. For this reason, safe non-pharmacological stimuli have been sought. One such is hyperbaric hyperoxia (HBHO), shown to induce neuroprotection against ischemic injury in gerbil hippocampus, rabbit spinal cord and in mouse and rat brain (Wada et al., 1996, 2002; Prass et al., 2000; Xiong et al., 2000). More recently, similar protection has been shown to be conferred by normobaric hyperoxia (NBHO) (Dong et al., 2002; Zhang et al., 2004), perhaps (as in other situations (Ravati et al., 2001; Rauca et al., 2000)) though the generation of oxygen free radicals (OFR) (Zhang et al., 2004; Wada et al., 2002, 1996) and hydroxyl radicals (OH*). Continuous prolonged exposure to hyperoxia is however associated with toxicity, which may partly offset the benefits of IT induction. Little, however, is known of the benefits of (possibly less toxic) intermittent hyperoxic exposure. We thus sought to compare the impact of prolonged and intermittent normobaric oxygen pretreatment on brain infarct volume, neurobehavioral deficits, blood–brain barrier (BBB) permeability and brain edema generation after exposure to focal cerebral ischemia.
2.
Result
2.1.
Experimental conditions parameters
Fig. 1 shows oxygen content (%) in the container in hyperoxia and normoxia conditions. Arterial blood gas analysis confirmed clinical hyperoxia in the treated groups (Table 1).
Table 1 – ABG tests at the end of pretreatment Experimental groups Intermittent NBNO Intermittent NBHO Prolonged NBNO Prolonged NBHO
PO2 (mmHg)
Respiratory rate (Hz)
pH
PCO2 (mmHg)
7.4 ± 0.03
41.16 ± 1.4
7.3 ± 0.02
39.2 ± 1.9
7.37 ± 0.052
40.2 ± 1.3
93.2 ± 5.2
1.60 ± 0.11
7.35 ± 0.027
37.8 ± 2.38
363 ± 14.3 ***
1.24 ± 0.12
92.8 ± 3.3 351.1 ± 17.6 ***
1.62 ± 0.09 1.37 ± 0.12
*** P < 0.001.
2.2. Effects of intermittent and prolonged NBHO on neurologic deficit scores Median neurologic deficit scores (NDS) were reduced by hyperoxic exposure, being 0 (range: 0–3), 1 (range: 0–5) and 2 (range: 0–5) in the intermittent NBHO, prolonged NBHO and NBNO groups, respectively, but these differences did not reach statistical significance (Table 2). Those with no deficit all showed EB extravasation, confirming the fact that focal cerebral ischemia had been induced. The putative beneficial effects of NBHO were confirmed by a reduction in infarct volume not seen in NBNO (Fig. 2). The neuroprotection exerted by NBHO was mainly seen in the penumbra (cortex).
2.3. Effects of intermittent and prolonged NBHO on infarct volume Preconditioning with both prolonged and intermittent NBHO 24 h before MCAO resulted in a reduction of infarct volume. Intermittent and prolonged NBNO had no effects on infarct volume (Fig. 2). The neuroprotection exerted by NBHO was mainly seen in the penumbra (cortex), while was slightly observed in the infarct core.
2.4. Effects of intermittent and prolonged NBHO on the brain water content Focal cerebral ischemia significantly increased the brain water content in the ischemic hemisphere in prolonged (P = 0.001) and intermittent NBNO groups (P = 0.002), while the brain water content was not different in right and left brain hemispheres of sham-operated rats (Fig. 3). Intermittent NBHO reduced the post-ischemic brain water content increment (P = 0.017), while prolonged NBHO did not significantly decrease post-ischemic water content compare to prolonged NBHO. Prolonged NBHO was less effective than intermittent NBHO (Fig. 3).
2.5. Effects of intermittent and prolonged NBHO on the blood–brain barrier
Fig. 1 – Oxygen concentration in the hyperoxia container during normobaric hyperoxia (NBHO) and normobaric normoxia (NBNO).
Brain edema formation was associated with increase in BBB permeability at 24 h. The EB concentration in ischemic cerebral tissue was 6.48 ± 2.42 μg/g tissue in prolonged NBNO and 7.49 ± 2.89 μg/g tissue in intermittent NBNO, while the EB level in non-ischemic cerebral tissue was 3.80 ± 1.05 μg/g tissue in
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Table 2 – The distribution of neurologic deficit score in each groups No.
1 2 3 4 5
Experimental groups
Intermittent NBNO Intermittent NBHO Prolonged NBNO Prolonged NBHO Total
NDS in each groups(N) 0
1
2
3
4
5
1 11 2 7 21
4 3 3 7 17
8 5 5 2 20
3 1 6 3 13
3 0 2 0 5
2 0 2 1 5
Total (N)
Median
Statistical results (P value)
21 20 20 20 81
2 0 2 1 –
1:2 2:4 4:3 1:4 2:3
sig. (0.000) nonsig. (0.314) sig. (0.029) sig. (0.006) sig. (0.000)
NDS: neurologic deficit score; N: the number of cases in each groups; sig.: significant; nonsig.: nonsignificant.
left hemisphere of prolonged NBNO group, 3.60 ± 0.59 μg/g tissue in left hemisphere of intermittent NBNO group, 3.31 ± 0.5 μg/g tissue and 3.36 ± 0.43 μg/g tissue in right and left hemisphere of prolonged sham-operated NBNO group, and 3.19 ± 0.4 μg/g tissue and 3.1 ± 0.35 μg/g tissue in right and left hemispheres of intermittent sham-operated NBNO group, respectively. Intermittent NBHO was associated with a reduction in EB extravasation in the ischemic brain, not seen with prolonged NBHO. BBB permeability in the contralateral hemisphere was not significantly affected in the brain pretreated with intermittent NBHO (Fig. 4). EB extravasations in other groups were unchanged (Fig. 4). In NBHO groups, EB entered mainly into the core but not into the penumbra intercellular environment, while in NBNO groups it entered into both penumbra and core. Therefore, the BBB protection caused by NBHO was mainly in the penumbra (cortex), not in the core.
3.1.
3.
These findings indicate that hyperoxia may influence the brain water content and brain water homeostasis by increasing BBB integrity (Helms et al., 2005). Therefore, the increase of BBB integrity modulates the cell volume of neurons and astrocytes directly. The means through which hyperoxia improves BBB integrity are not clear, but may hinge upon the production of ROS and TNF-α in the brain tissue (Saini et al., 2004; Pradillo et al., 2006).
Discussion
Our data suggest that intermittent normobaric hyperoxia may reduce infarct volume, brain edema, BBB permeability, and neurobehavioral deficit scores more effectively than prolonged NBHO in a reliable and reproducible animal model of stroke followed by reperfusion (Longa et al., 1989). These data are supported by other experimental studies showing that hyperoxia may induce cerebral ischemic tolerance (Dong et al., 2002; Zhang et al., 2004).
Fig. 2 – The effects of normobaric hyperoxia in various doses on infarct volume.
NBHO produces ischemic tolerance in the brain
The amount and duration of oxygen concentration change the rate of ischemic tolerance in the experimental brain tissues. Therefore, the qualities of hyperoxia administration are important in ischemic tolerance induction, side effects and toxicities of hyperoxia on the body. Our results show that prolonged hyperoxia in some cases produce lung pulmonary insufficiency. Subarachnoid hemorrhage sometimes results in mortality in the various experimental groups. Because, subarachnoid hemorrhage and hypothalamic infection as a cause of hyperthermia has been identified recently as a complication of intraluminal filament model of middle cerebral artery occlusion (MCAO) (Dittmar et al., 2003).
3.2. NBHO decreases the formation of brain edema and BBB integrity
3.3. Which one is more effective and less toxic in IT induction: intermittent or prolonged NBHO? Past studies have suggested that, to be effective, prolonged NBHO should involve at least 24 h of exposure (Zhang et al., 2004). However, such durations are associated with toxicity (Al-Motabagani, 2005): exposure to 95% O2 for 24 h has been suggested to result in severe pulmonary congestion with extravasations of red blood cell, edema, and alteration in the alveolar structure; recovery in room air for 2 weeks did not result in repair of distorted alveolar structure. Further, the benefits of prolonged hypoxic preconditioning may be conferred by the reoxygenation rather than by hypoxia per se (Milano et al., 2002). Our study is consistent with such an observation, with intermittent repetitive hyperoxia conferring a possible greater benefit (Figs. 3 and 4). Further, intermittent hyperoxia is less toxic to the lung than prolonged hyperoxic exposure (Hendricks et al., 1977). In general, with consideration of evidence, the intermittent NBHO is more effective and safe than prolonged hyperoxia.
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Fig. 3 – Brain water content in various experimental groups including intermittent sham-operated NBNO – right (1) and left (2) hemisphere; prolonged sham-operated NBNO – right (3) and left (4) hemisphere; intermittent NBNO – right (5) and left (6) hemisphere; intermittent NBHO – right (7) and left (8) hemisphere; prolonged NBHO – right (9) and left (10) hemisphere; and prolonged NBNO – right (11) and left (12) hemisphere.
3.4. Other mechanisms of hyperoxia-mediated neuroprotection Although our data suggest that hyperoxia-mediated neuroprotection is due to the reduction of postischemic infarct volume and brain edema, other mechanisms may be at work. Hyperoxia can produce angiogenesis and increase vessel density in the brain (Helms et al., 2005), and can inhibit intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil accumulation (Zhang et al., 1995; Bowes et al., 1994). Wada et al. described increase in expression of FRO as well as Bcl-2, an inhibitor of apoptosis, after repeated hyperbaric oxygen exposure in gerbils, which correlated with increased neural survival (Wada et al., 2000). On the other hand, the increase of FRO and superoxide dismutase were associated with decreased expression of hypoxia-induced factor-1α, leading to improved BBB function via decreased vascular growth factor (Ostrowski et al., 2005). In addition, ROS may increase TNF-α in the blood and produce ischemic tolerance via TNF-α receptors (Pradillo et al., 2006). Accordingly, the effects of NBHO on brain may reduce glutamate and superoxide anion-mediated toxicity. As a result, NBHO can decrease excitotoxicity via ROS and TNF-α. In conclusion, our data suggest that NBHO may induce ischemic tolerance in the rat brain. Further work is required to extend these observations. Ultimately, it is hoped that novel cerebroprotective strategies may be developed for those at risk of stroke, or in whom cerebral perfusion is electively reduced perhaps at the time of surgery.
4.
Experimental procedures
4.1.
Animals and group assignment
Male Sprague-Dawley (250–380 g) rats were divided randomly into 4 groups of 21 animals. Two of these groups were placed
231
in an environmental chamber and exposed to a hyperoxic atmosphere (90% oxygen: normobaric hyperoxic groups, or NBHO) either intermittently (for 4 continuous hours of each day for each of 6 consecutive days, yielding a total hyperoxic exposure of 24 h) or for 24 h continuously. The two other groups were similarly placed in the environmental chamber and exposed to room-air equivalent (21% oxygen: normobaric normoxic groups, NBNO) for similar time periods: the full 6 days (‘intermittent’ NBNO) or for just 24 h (‘prolonged’ NBNO). Animals were then placed in ordinary room air for a further 24 h, after which all were subjected to 60 min of middle cerebral artery occlusion (MCAO). 24 h later, neurobehavioural studies were performed before the animals from each group were split into three subgroups of seven, each of which was sacrificed and study made of infarct volume, brain edema, and blood–brain barrier [BBB] permeability, respectively. In addition, two other groups (each of 14 animals) were managed according to the prolonged and intermittent NBNO protocols, respectively, but underwent surgery without MCAO. When sacrificed, these sham-operated animal groups were divided into subgroups (n = 7 in each) for evaluation of brain edema, and BBB permeability, respectively. In a subset of animals, arterial blood gas analysis was performed just prior to removal from the environmental chamber.
4.2.
Environmental chamber
All rats underwent adaptation for 1 week in the animal room. The environmental chamber comprised an airtight box (650 × 350 × 450 mm3) with a gas inlet and outlet port. Internal pressure was continuously monitored by a manometer. Oxygen (90%) or room air (by an aquarium pump) was delivered at a rate of < 5 l/min through the inlet port. The oxygen concentration inside the container was continuously monitored (Lutron-Do5510 oxygen sensor, Taiwan), and
Fig. 4 – EB extravasations in various experimental groups including intermittent sham-operated NBNO – right (1) and left (2) hemisphere; prolonged sham-operated NBNO – right (3) and left (4) hemisphere; intermittent NBNO – right (5) and left (6) hemisphere; intermittent NBHO – right (7) and left (8) hemisphere; prolonged NBHO – right (9) and left (10) hemisphere; and prolonged NBNO – right (11) and left (12) hemisphere.
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carbon dioxide cleared using soda lime (BDH Limited, Poole, England) at the bottom of the container. Oxygen concentration was maintained at 90% or 21% according to the experimental protocol.
4.3.
Focal cerebral ischemia
The rats were weighed and intraperitoneally (i.p.) anesthetized with chloral hydrate (Merck, Germany) (400 mg/kg). MCAO was performed as described by Longa et al. (1989). Briefly, using a surgical microscope, the right common carotid artery (CCA) and external carotid artery (ECA) were exposed, and carefully separated from vagus nerve and surrounding tissues. The right internal carotid artery (ICA) was then dissected to the level of pterygopalatine artery (PA). The PA was then ligated using a silk suture placed loosely around the ECA stump, and the CCA and ICA temporarily occluded using a microvascular clip. A small incision was then made in the ECA, through which a blunt 3-0 nylon monofilament was passed. With the ECA thread tensioned to prevent bleeding, the ICA microvascular clip was removed, and the nylon thread carefully and slowly advanced for 20–22 mm from the CCA bifurcation (depending on animal weight) to position the tip of nylon thread at the beginning of anterior cerebral artery, when a mild resistance was felt. In this manner, the origin of anterior and middle cerebral arteries was occluded. Reperfusion was started by withdrawing the suture after 60 min of ischemia. Rectal temperature was monitored (Citizen-513w) and maintained at 37.0 °C by surface heating and cooling during surgery. The body temperature, blood gases, cardiovascular rate were maintained within physiologic range throughout.
4.4.
Neurobehavioral evaluation
After the suture was withdrawn, the rats were returned to their separate cages. 24 h later, the rats were assessed neurologically by an observer who was blind to the animal groups. The neurobehavioral scoring was performed using a six-point scale as was previously described by Longa et al. (1989): normal motor function = 0; flexion of contralateral forelimb upon suspended vertically by tail or failure to extend forepaw = 1; circling to the contralateral side but have normal posture at rest = 2; loss of righting reflex = 3; no spontaneous motor activity = 4. Death was considered as score 5 only when a large infarct volume was present in the absence of subarachnoid hemorrhage. If the rats died due to subarachnoid hemorrhage or pulmonary insufficiency and asphyxia, they were eliminated from the study.
4.5.
Infarct volume assessment
After sacrifice with chloral hydrate (800 mg/kg), the animals were decapitated and the brains rapidly removed and cooled in 4 °C saline for 15 minutes. Eight 2-mm-thick coronal sections were cut (Brain Matrix, Iran) through the brain, beginning at the olfactory bulb. The slices were immersed in 2% 2,3,5-triphenyltetrazolium chloride solution (Merck, Germany), and kept at 37 °C in a water bath for 15 min. The slices were then photographed by the digital camera (Nokia 6630, Finland) connected to a computer. Unstained areas were defined as
infarct, and were measured using image analysis software (Image Tools, NIH). The infarct volume was calculated by measuring the unstained and stained area in each hemisphere slice, and multiplied by slice thickness (2 mm), and then summating all of the eight slices according to the method of Swanson et al. (1990): corrected infarct volume = left hemisphere volume − (right hemisphere volume − infarct volume).
4.6.
Brain water content measurement
After decapitation, the brain was removed. The cerebellum, pons, and olfactory bulb were separated and their wet weights (WW) measured (Vakili et al., 2005). Dry weights (DW) were assessed after 24 h at 120 °C. Brain water content (BWC) was calculated as [(WW − DW)/WW] × 100.
4.7.
Blood–brain barrier permeability
The integrity of BBB was evaluated by using Evans Blue (EB, Sigma Chemicals, USA) dye extravasations, as described by Kaya et al. (2003). Briefly, the rats received 4 ml/kg of 2% EB solution in saline by tail vein injection 30 min after MCAO. 24 h after reperfusion, the thoracic cavity was opened under anesthesia. The rats were perfused with 250 ml saline transcardially to wash out intravascular EB until colorless perfusion fluid was obtained from the atrium. After decapitation, the brains were removed and the hemispheres separated and weighed. The right and left hemispheres were separately homogenized in 2.5 ml phosphate-buffered saline to extract the EB, and to precipitate protein 2.5 ml of 60% trichloroacetic acid was added and mixed by vortex for 3 min. The samples were then placed at 4 °C for 30 min and centrifuged for 30 min at 1000×g. The amount of EB in the supernatants was measured at 610 nm using spectrophotometer (Spectronic 20D MILTON ROY). EB levels were expressed as μg/g of brain tissue against a standard curve.
4.8.
Statistical analysis
Infarct volumes, brain water content, EB extravasations, and arterial blood gases (ABG) were compared using one-way ANOVA test and t-test. The neurologic deficit scores were analyzed using the Mann–Whitney U test. Data were expressed as means±SD. P< 0.05 was considered significant.
Acknowledgments This study was supported by a grant from Iran national sciences foundation (INSF). We thank Professor Hugh Montgomery for critical review on the manuscript.
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Research Report
Prolonged and intermittent normobaric hyperoxia induce different degrees of ischemic tolerance in rat brain tissue Mohammad Reza Bigdeli a,b , Sohrab Hajizadeh a,⁎, Mehdi Froozandeh b , Bahram Rasulian c , Ali Heidarianpour a , Ali Khoshbaten d a
Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran Department of Medical Biotechnology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran c Trauma Research Center, Baqiyatallah Medical Sciences University, Tehran, Iran d Research Center for Chemical Injuries, Baqiyatallah Medical Sciences University, Tehran, Iran b
A R T I C LE I N FO
AB S T R A C T
Article history:
Prior prolonged oxygen exposure is associated with some protection against ischemia–
Accepted 13 March 2007
reperfusion (IR) injury to rat brain tissue, but also with toxic effects. We sought to compare
Available online 28 March 2007
the magnitude of protection offered by prolonged and intermittent oxygen pretreatments against IR injury to the rat brain. Rats were divided into four experimental groups, each of
Keywords:
21 animals. The first two were exposed to 95% inspired (normobaric hyperoxia, NBHO) for
Prolonged hyperoxia
4 h/day for 6 consecutive days (intermittent NBHO) or for 24 continuous hours (prolonged
Intermittent hyperoxia
NBHO). The second two groups acted as controls were exposed to 21% oxygen. After 24 h,
Brain ischemic tolerance
they were subjected to 60 min of right middle cerebral artery occlusion (MCAO) followed by
Ischemic preconditioning
24 h of reperfusion. The animals were sacrificed for assessment of infarct volume, brain
Neuroprotection
edema, and blood–brain barrier (BBB) permeability, respectively. Prolonged and
Stroke
intermittent NBHO pretreatment reduced infarct volume by 63.3% and 73.7%, respectively, when compared to the respective NBNO groups. Intermittent NBHO (when compared to intermittent NBNO) also reduced the post-ischemic increment of brain water content significantly (81.53 ± 0.8%, vs. 80.12 ± 0.79%) and Evans Blue extravasation (7.49 ± 2.89 ± g/g tissue vs. 3.9 ± 0.79 μg/g tissue, P < 0.001), while prolonged NBHO had no significant effect on brain water content (81.69 ± 1.16% vs. 80.74 ± 0.94%) and EB extravasations (6.48 ± 2.42 μg/g tissue vs. 4.31 ± 1.07 μg/g tissue). Intermittent hyperoxia had relatively more significant effects on brain edema and BBB protection. Although preconditioning with both prolonged and intermittent oxygen exposure protects rat brain tissue against IR injury, the intermittent hyperoxia could have relatively more protective effects in this regard. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic preconditioning (IPC) is an endogenous phenomenon that can induce ischemic tolerance (IT) in a variety of
organs such as the heart and kidney, but also the brain. Kitagawa et al. (1990) found that gerbils subjected to sublethal transient global cerebral ischemia exhibited a reduction in hippocampal CA1 neural death after 5 min of global cerebral
⁎ Corresponding author. Fax: +98 21 88006544. E-mail address: [email protected] (S. Hajizadeh). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.03.068
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ischemia. IPC-like phenomena have similarly been also demonstrated in brain slices (Raval et al., 2003) and in neural cell cultures (Romera et al., 2004), and can be induced by diverse stimuli including seizures (Plamondon et al., 1999), anoxia (Perez-Pinzon et al., 1996), heat (Chopp et al., 1989; Kitagawa et al., 1991), oxidative stress(Ohtsuki et al., 1992), polyunsaturated fatty acid (PUFA) pretreatment (Patel et al., 1998; Maingret et al., 2000), and inhibition of oxidative phosphorylation (Riepe et al., 1997). Most such stimuli, however, lack potential for clinical translation due to associated toxicity. For this reason, safe non-pharmacological stimuli have been sought. One such is hyperbaric hyperoxia (HBHO), shown to induce neuroprotection against ischemic injury in gerbil hippocampus, rabbit spinal cord and in mouse and rat brain (Wada et al., 1996, 2002; Prass et al., 2000; Xiong et al., 2000). More recently, similar protection has been shown to be conferred by normobaric hyperoxia (NBHO) (Dong et al., 2002; Zhang et al., 2004), perhaps (as in other situations (Ravati et al., 2001; Rauca et al., 2000)) though the generation of oxygen free radicals (OFR) (Zhang et al., 2004; Wada et al., 2002, 1996) and hydroxyl radicals (OH*). Continuous prolonged exposure to hyperoxia is however associated with toxicity, which may partly offset the benefits of IT induction. Little, however, is known of the benefits of (possibly less toxic) intermittent hyperoxic exposure. We thus sought to compare the impact of prolonged and intermittent normobaric oxygen pretreatment on brain infarct volume, neurobehavioral deficits, blood–brain barrier (BBB) permeability and brain edema generation after exposure to focal cerebral ischemia.
2.
Result
2.1.
Experimental conditions parameters
Fig. 1 shows oxygen content (%) in the container in hyperoxia and normoxia conditions. Arterial blood gas analysis confirmed clinical hyperoxia in the treated groups (Table 1).
Table 1 – ABG tests at the end of pretreatment Experimental groups Intermittent NBNO Intermittent NBHO Prolonged NBNO Prolonged NBHO
PO2 (mmHg)
Respiratory rate (Hz)
pH
PCO2 (mmHg)
7.4 ± 0.03
41.16 ± 1.4
7.3 ± 0.02
39.2 ± 1.9
7.37 ± 0.052
40.2 ± 1.3
93.2 ± 5.2
1.60 ± 0.11
7.35 ± 0.027
37.8 ± 2.38
363 ± 14.3 ***
1.24 ± 0.12
92.8 ± 3.3 351.1 ± 17.6 ***
1.62 ± 0.09 1.37 ± 0.12
*** P < 0.001.
2.2. Effects of intermittent and prolonged NBHO on neurologic deficit scores Median neurologic deficit scores (NDS) were reduced by hyperoxic exposure, being 0 (range: 0–3), 1 (range: 0–5) and 2 (range: 0–5) in the intermittent NBHO, prolonged NBHO and NBNO groups, respectively, but these differences did not reach statistical significance (Table 2). Those with no deficit all showed EB extravasation, confirming the fact that focal cerebral ischemia had been induced. The putative beneficial effects of NBHO were confirmed by a reduction in infarct volume not seen in NBNO (Fig. 2). The neuroprotection exerted by NBHO was mainly seen in the penumbra (cortex).
2.3. Effects of intermittent and prolonged NBHO on infarct volume Preconditioning with both prolonged and intermittent NBHO 24 h before MCAO resulted in a reduction of infarct volume. Intermittent and prolonged NBNO had no effects on infarct volume (Fig. 2). The neuroprotection exerted by NBHO was mainly seen in the penumbra (cortex), while was slightly observed in the infarct core.
2.4. Effects of intermittent and prolonged NBHO on the brain water content Focal cerebral ischemia significantly increased the brain water content in the ischemic hemisphere in prolonged (P = 0.001) and intermittent NBNO groups (P = 0.002), while the brain water content was not different in right and left brain hemispheres of sham-operated rats (Fig. 3). Intermittent NBHO reduced the post-ischemic brain water content increment (P = 0.017), while prolonged NBHO did not significantly decrease post-ischemic water content compare to prolonged NBHO. Prolonged NBHO was less effective than intermittent NBHO (Fig. 3).
2.5. Effects of intermittent and prolonged NBHO on the blood–brain barrier
Fig. 1 – Oxygen concentration in the hyperoxia container during normobaric hyperoxia (NBHO) and normobaric normoxia (NBNO).
Brain edema formation was associated with increase in BBB permeability at 24 h. The EB concentration in ischemic cerebral tissue was 6.48 ± 2.42 μg/g tissue in prolonged NBNO and 7.49 ± 2.89 μg/g tissue in intermittent NBNO, while the EB level in non-ischemic cerebral tissue was 3.80 ± 1.05 μg/g tissue in
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Table 2 – The distribution of neurologic deficit score in each groups No.
1 2 3 4 5
Experimental groups
Intermittent NBNO Intermittent NBHO Prolonged NBNO Prolonged NBHO Total
NDS in each groups(N) 0
1
2
3
4
5
1 11 2 7 21
4 3 3 7 17
8 5 5 2 20
3 1 6 3 13
3 0 2 0 5
2 0 2 1 5
Total (N)
Median
Statistical results (P value)
21 20 20 20 81
2 0 2 1 –
1:2 2:4 4:3 1:4 2:3
sig. (0.000) nonsig. (0.314) sig. (0.029) sig. (0.006) sig. (0.000)
NDS: neurologic deficit score; N: the number of cases in each groups; sig.: significant; nonsig.: nonsignificant.
left hemisphere of prolonged NBNO group, 3.60 ± 0.59 μg/g tissue in left hemisphere of intermittent NBNO group, 3.31 ± 0.5 μg/g tissue and 3.36 ± 0.43 μg/g tissue in right and left hemisphere of prolonged sham-operated NBNO group, and 3.19 ± 0.4 μg/g tissue and 3.1 ± 0.35 μg/g tissue in right and left hemispheres of intermittent sham-operated NBNO group, respectively. Intermittent NBHO was associated with a reduction in EB extravasation in the ischemic brain, not seen with prolonged NBHO. BBB permeability in the contralateral hemisphere was not significantly affected in the brain pretreated with intermittent NBHO (Fig. 4). EB extravasations in other groups were unchanged (Fig. 4). In NBHO groups, EB entered mainly into the core but not into the penumbra intercellular environment, while in NBNO groups it entered into both penumbra and core. Therefore, the BBB protection caused by NBHO was mainly in the penumbra (cortex), not in the core.
3.1.
3.
These findings indicate that hyperoxia may influence the brain water content and brain water homeostasis by increasing BBB integrity (Helms et al., 2005). Therefore, the increase of BBB integrity modulates the cell volume of neurons and astrocytes directly. The means through which hyperoxia improves BBB integrity are not clear, but may hinge upon the production of ROS and TNF-α in the brain tissue (Saini et al., 2004; Pradillo et al., 2006).
Discussion
Our data suggest that intermittent normobaric hyperoxia may reduce infarct volume, brain edema, BBB permeability, and neurobehavioral deficit scores more effectively than prolonged NBHO in a reliable and reproducible animal model of stroke followed by reperfusion (Longa et al., 1989). These data are supported by other experimental studies showing that hyperoxia may induce cerebral ischemic tolerance (Dong et al., 2002; Zhang et al., 2004).
Fig. 2 – The effects of normobaric hyperoxia in various doses on infarct volume.
NBHO produces ischemic tolerance in the brain
The amount and duration of oxygen concentration change the rate of ischemic tolerance in the experimental brain tissues. Therefore, the qualities of hyperoxia administration are important in ischemic tolerance induction, side effects and toxicities of hyperoxia on the body. Our results show that prolonged hyperoxia in some cases produce lung pulmonary insufficiency. Subarachnoid hemorrhage sometimes results in mortality in the various experimental groups. Because, subarachnoid hemorrhage and hypothalamic infection as a cause of hyperthermia has been identified recently as a complication of intraluminal filament model of middle cerebral artery occlusion (MCAO) (Dittmar et al., 2003).
3.2. NBHO decreases the formation of brain edema and BBB integrity
3.3. Which one is more effective and less toxic in IT induction: intermittent or prolonged NBHO? Past studies have suggested that, to be effective, prolonged NBHO should involve at least 24 h of exposure (Zhang et al., 2004). However, such durations are associated with toxicity (Al-Motabagani, 2005): exposure to 95% O2 for 24 h has been suggested to result in severe pulmonary congestion with extravasations of red blood cell, edema, and alteration in the alveolar structure; recovery in room air for 2 weeks did not result in repair of distorted alveolar structure. Further, the benefits of prolonged hypoxic preconditioning may be conferred by the reoxygenation rather than by hypoxia per se (Milano et al., 2002). Our study is consistent with such an observation, with intermittent repetitive hyperoxia conferring a possible greater benefit (Figs. 3 and 4). Further, intermittent hyperoxia is less toxic to the lung than prolonged hyperoxic exposure (Hendricks et al., 1977). In general, with consideration of evidence, the intermittent NBHO is more effective and safe than prolonged hyperoxia.
BR A I N R ES E A RC H 1 1 5 2 ( 2 00 7 ) 2 2 8 –2 33
Fig. 3 – Brain water content in various experimental groups including intermittent sham-operated NBNO – right (1) and left (2) hemisphere; prolonged sham-operated NBNO – right (3) and left (4) hemisphere; intermittent NBNO – right (5) and left (6) hemisphere; intermittent NBHO – right (7) and left (8) hemisphere; prolonged NBHO – right (9) and left (10) hemisphere; and prolonged NBNO – right (11) and left (12) hemisphere.
3.4. Other mechanisms of hyperoxia-mediated neuroprotection Although our data suggest that hyperoxia-mediated neuroprotection is due to the reduction of postischemic infarct volume and brain edema, other mechanisms may be at work. Hyperoxia can produce angiogenesis and increase vessel density in the brain (Helms et al., 2005), and can inhibit intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil accumulation (Zhang et al., 1995; Bowes et al., 1994). Wada et al. described increase in expression of FRO as well as Bcl-2, an inhibitor of apoptosis, after repeated hyperbaric oxygen exposure in gerbils, which correlated with increased neural survival (Wada et al., 2000). On the other hand, the increase of FRO and superoxide dismutase were associated with decreased expression of hypoxia-induced factor-1α, leading to improved BBB function via decreased vascular growth factor (Ostrowski et al., 2005). In addition, ROS may increase TNF-α in the blood and produce ischemic tolerance via TNF-α receptors (Pradillo et al., 2006). Accordingly, the effects of NBHO on brain may reduce glutamate and superoxide anion-mediated toxicity. As a result, NBHO can decrease excitotoxicity via ROS and TNF-α. In conclusion, our data suggest that NBHO may induce ischemic tolerance in the rat brain. Further work is required to extend these observations. Ultimately, it is hoped that novel cerebroprotective strategies may be developed for those at risk of stroke, or in whom cerebral perfusion is electively reduced perhaps at the time of surgery.
4.
Experimental procedures
4.1.
Animals and group assignment
Male Sprague-Dawley (250–380 g) rats were divided randomly into 4 groups of 21 animals. Two of these groups were placed
231
in an environmental chamber and exposed to a hyperoxic atmosphere (90% oxygen: normobaric hyperoxic groups, or NBHO) either intermittently (for 4 continuous hours of each day for each of 6 consecutive days, yielding a total hyperoxic exposure of 24 h) or for 24 h continuously. The two other groups were similarly placed in the environmental chamber and exposed to room-air equivalent (21% oxygen: normobaric normoxic groups, NBNO) for similar time periods: the full 6 days (‘intermittent’ NBNO) or for just 24 h (‘prolonged’ NBNO). Animals were then placed in ordinary room air for a further 24 h, after which all were subjected to 60 min of middle cerebral artery occlusion (MCAO). 24 h later, neurobehavioural studies were performed before the animals from each group were split into three subgroups of seven, each of which was sacrificed and study made of infarct volume, brain edema, and blood–brain barrier [BBB] permeability, respectively. In addition, two other groups (each of 14 animals) were managed according to the prolonged and intermittent NBNO protocols, respectively, but underwent surgery without MCAO. When sacrificed, these sham-operated animal groups were divided into subgroups (n = 7 in each) for evaluation of brain edema, and BBB permeability, respectively. In a subset of animals, arterial blood gas analysis was performed just prior to removal from the environmental chamber.
4.2.
Environmental chamber
All rats underwent adaptation for 1 week in the animal room. The environmental chamber comprised an airtight box (650 × 350 × 450 mm3) with a gas inlet and outlet port. Internal pressure was continuously monitored by a manometer. Oxygen (90%) or room air (by an aquarium pump) was delivered at a rate of < 5 l/min through the inlet port. The oxygen concentration inside the container was continuously monitored (Lutron-Do5510 oxygen sensor, Taiwan), and
Fig. 4 – EB extravasations in various experimental groups including intermittent sham-operated NBNO – right (1) and left (2) hemisphere; prolonged sham-operated NBNO – right (3) and left (4) hemisphere; intermittent NBNO – right (5) and left (6) hemisphere; intermittent NBHO – right (7) and left (8) hemisphere; prolonged NBHO – right (9) and left (10) hemisphere; and prolonged NBNO – right (11) and left (12) hemisphere.
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carbon dioxide cleared using soda lime (BDH Limited, Poole, England) at the bottom of the container. Oxygen concentration was maintained at 90% or 21% according to the experimental protocol.
4.3.
Focal cerebral ischemia
The rats were weighed and intraperitoneally (i.p.) anesthetized with chloral hydrate (Merck, Germany) (400 mg/kg). MCAO was performed as described by Longa et al. (1989). Briefly, using a surgical microscope, the right common carotid artery (CCA) and external carotid artery (ECA) were exposed, and carefully separated from vagus nerve and surrounding tissues. The right internal carotid artery (ICA) was then dissected to the level of pterygopalatine artery (PA). The PA was then ligated using a silk suture placed loosely around the ECA stump, and the CCA and ICA temporarily occluded using a microvascular clip. A small incision was then made in the ECA, through which a blunt 3-0 nylon monofilament was passed. With the ECA thread tensioned to prevent bleeding, the ICA microvascular clip was removed, and the nylon thread carefully and slowly advanced for 20–22 mm from the CCA bifurcation (depending on animal weight) to position the tip of nylon thread at the beginning of anterior cerebral artery, when a mild resistance was felt. In this manner, the origin of anterior and middle cerebral arteries was occluded. Reperfusion was started by withdrawing the suture after 60 min of ischemia. Rectal temperature was monitored (Citizen-513w) and maintained at 37.0 °C by surface heating and cooling during surgery. The body temperature, blood gases, cardiovascular rate were maintained within physiologic range throughout.
4.4.
Neurobehavioral evaluation
After the suture was withdrawn, the rats were returned to their separate cages. 24 h later, the rats were assessed neurologically by an observer who was blind to the animal groups. The neurobehavioral scoring was performed using a six-point scale as was previously described by Longa et al. (1989): normal motor function = 0; flexion of contralateral forelimb upon suspended vertically by tail or failure to extend forepaw = 1; circling to the contralateral side but have normal posture at rest = 2; loss of righting reflex = 3; no spontaneous motor activity = 4. Death was considered as score 5 only when a large infarct volume was present in the absence of subarachnoid hemorrhage. If the rats died due to subarachnoid hemorrhage or pulmonary insufficiency and asphyxia, they were eliminated from the study.
4.5.
Infarct volume assessment
After sacrifice with chloral hydrate (800 mg/kg), the animals were decapitated and the brains rapidly removed and cooled in 4 °C saline for 15 minutes. Eight 2-mm-thick coronal sections were cut (Brain Matrix, Iran) through the brain, beginning at the olfactory bulb. The slices were immersed in 2% 2,3,5-triphenyltetrazolium chloride solution (Merck, Germany), and kept at 37 °C in a water bath for 15 min. The slices were then photographed by the digital camera (Nokia 6630, Finland) connected to a computer. Unstained areas were defined as
infarct, and were measured using image analysis software (Image Tools, NIH). The infarct volume was calculated by measuring the unstained and stained area in each hemisphere slice, and multiplied by slice thickness (2 mm), and then summating all of the eight slices according to the method of Swanson et al. (1990): corrected infarct volume = left hemisphere volume − (right hemisphere volume − infarct volume).
4.6.
Brain water content measurement
After decapitation, the brain was removed. The cerebellum, pons, and olfactory bulb were separated and their wet weights (WW) measured (Vakili et al., 2005). Dry weights (DW) were assessed after 24 h at 120 °C. Brain water content (BWC) was calculated as [(WW − DW)/WW] × 100.
4.7.
Blood–brain barrier permeability
The integrity of BBB was evaluated by using Evans Blue (EB, Sigma Chemicals, USA) dye extravasations, as described by Kaya et al. (2003). Briefly, the rats received 4 ml/kg of 2% EB solution in saline by tail vein injection 30 min after MCAO. 24 h after reperfusion, the thoracic cavity was opened under anesthesia. The rats were perfused with 250 ml saline transcardially to wash out intravascular EB until colorless perfusion fluid was obtained from the atrium. After decapitation, the brains were removed and the hemispheres separated and weighed. The right and left hemispheres were separately homogenized in 2.5 ml phosphate-buffered saline to extract the EB, and to precipitate protein 2.5 ml of 60% trichloroacetic acid was added and mixed by vortex for 3 min. The samples were then placed at 4 °C for 30 min and centrifuged for 30 min at 1000×g. The amount of EB in the supernatants was measured at 610 nm using spectrophotometer (Spectronic 20D MILTON ROY). EB levels were expressed as μg/g of brain tissue against a standard curve.
4.8.
Statistical analysis
Infarct volumes, brain water content, EB extravasations, and arterial blood gases (ABG) were compared using one-way ANOVA test and t-test. The neurologic deficit scores were analyzed using the Mann–Whitney U test. Data were expressed as means±SD. P< 0.05 was considered significant.
Acknowledgments This study was supported by a grant from Iran national sciences foundation (INSF). We thank Professor Hugh Montgomery for critical review on the manuscript.
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