- Email: [email protected]
Hyperbaric oxygen induces rapid protection against focal cerebral ischemia
Brain Research 1037 (2005) 134 – 138 www.elsevier.com/locate/brainres
Research report
Hyperbaric oxygen induces rapid protection against focal cerebral ischemia Roland Veltkampa,*, Dirk A. Siebinga, Sabine Heilanda, Philip Schoenffeldt-Varasa, Claudia Veltkampb, Markus Schwaningera, Stefan Schwaba a
Department of Neurology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany b Department of Internal Medicine IV, Ruprecht-Karls-University Heidelberg, 69120 Heidelberg, Germany Accepted 1 January 2005
Abstract Background and purpose. The timing and mechanisms of protection by hyperbaric oxygen (HBO) in cerebral ischemia have only been partially elucidated. We monitored the early in vivo effects of HBO after 2 h transient focal ischemia using repetitive MRI. Methods. Wistar rats underwent filament occlusion of the middle cerebral artery (MCAO). 40 min after MCAO, rats were placed in a HBO chamber and breathed either 100% O2 at 3.0 atmospheres absolute (ata; n = 24) or at 1.0 ata (control; n = 24) for 1 h. Diffusion, perfusion and T2-weighted MR-images were obtained after 15 min and 3, 6 and 24 h of reperfusion. In 6 axial MR slices, volume of abnormal diffusion and T2w signals were measured in the ischemic hemisphere. Furthermore, hemispheric mean apparent diffusion coefficient- (ADC) and T2 values were calculated for statistical analysis. Results. HBO significantly reduced volume of abnormal DWI signal beginning immediately after reperfusion (control: 92 F 28 mm3; HBO: 64 F 17) and lesion size on T2w (control: 375 F 91 mm3; HBO: 225 F 39) after 24 h. Correspondingly, mean ADC levels were lower and T2 values higher in the ischemic hemisphere in the control group. HBO reduced histological infarct size at 24 h. Conclusion. High-dose intraischemic HBO therapy has an immediate protective on the brain which is superior to normobaric oxygen. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Cerebrovascular disorders; Neuroprotection; MRI; Hypoxia
1. Introduction Progression of tissue damage during the first hours of cerebral ischemia has remained a major challenge in acute stroke management [4,10] Because cerebral hypoxia is an important component of immediate and early secondary cell damage, improving oxygen delivery to the ischemic penumbra has been a logical concept for tissue protection. Several experimental studies have shown that normobaric
T Corresponding author. Fax: +49 6221 561740. E-mail address: [email protected] (R. Veltkamp). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.01.006
hyperoxia can protect the brain against focal cerebral ischemia [6,14,19,20]. Administering oxygen at increased ambient pressure–i.e. hyperbaric oxygen treatment (HBO)– can further increase the pO2 in blood plasma and in normal brain tissue. The effectiveness of HBO in cerebral ischemia, however, has remained controversial for many years [15]. While many of the older experimental studies had methodological shortcomings (reviewed in [25]), more recent work confirmed a protective effect of HBO at least under certain circumstances [11,18,21,25]. Clinical pilot studies [1,16,17] have failed to show a benefit from HBO, perhaps because a long time window between symptom-onset and initiation of therapy was allowed [30]. Indeed, appropriate judgment of the therapeutic potential of HBO is hampered because the
R. Veltkamp et al. / Brain Research 1037 (2005) 134–138
timing of its protective effect and the corresponding pathophysiological targets are largely unknown. In the present study, we used repetitive in vivo magnetic resonance imaging (MRI) to gain an insight into the timing of the protective effects of HBO on ischemic tissue and thus narrow the search for the involved mechanisms.
2. Materials and methods 2.1. Surgical preparation Experiments were performed on male Wistar rats (Charles River, Germany) weighing 280–320 g. The procedures were approved by the governmental animal care authorities. Focal cerebral ischemia was induced by filament occlusion of the middle cerebral artery (MCAO) for 120 min according to Zea-Longa et al. [29] with some modifications [18]. After MCAO, animals were placed into the MRI scanner for perfusion weighted imaging (PWI). Then, anesthesia was discontinued and animals breathed 100% O2-enriched air. Forty minutes later, rats were transferred into a rodent HBO chamber. Animals were randomly assigned to either the control group, which breathed 100% O2 at atmospheric pressure (1 atmosphere absolute (ata)), or the HBO group which received 100% O 2 at 3.0 ata. In the HBO group, compression was started 40 min after filament introduction and was performed over 5 min. Decompression was begun 105 min after filament introduction at the same rate. Rats were reanesthetized and the filament was removed 120 min after introduction. A second MRI was performed 15 min after reperfusion. Animals were allowed to awaken in an O2-enriched environment and transferred to their cages. Additional MRI scans were performed 3, 6 and 24 h after reperfusion.
135
2.2. Magnetic resonance imaging The animals were examined in a 2.35-T scanner (Biospec 24/40, BRUKER Medizintechnik, Ettlingen, Germany) using a previously described configuration [8]. The MR-protocol consisted of a diffusion-weighted spinecho echo-planar imaging (SE-EPI) sequence (b values = 200, 300, 400, 500, 600 and 700 s/mm2) and a multi-spinecho sequence (12 echoes with echo times of 8, 16, 24, ... 96 ms). For PWI, we used a gradient-echo echo-planar imaging (GE-EPI) sequence (repetition time = 1 s, echo-time = 15 ms, 20 repetitions with a time resolution of 1 s/image data set) for monitoring the bolus passage of 1 mmol/kg of a paramagnetic contrast agent (Omniscan, Nycomed Amersham, Oslo, Norway). 2.3. Data analysis During MR imaging, a six-slice data set was obtained covering the MCA territory, the first axial slice containing the olfactory bulb. DWI and T2w data were analyzed by an operator-driven and a largely operator-independent mode. In the first method, areas of abnormal signal intensity were encircled for each MR section using a side to side comparison on the screen. Volume of lesion on DWI and T2w was calculated by multiplying the total area with section thickness. The second method was performed to circumvent the problem of adequate threshold determination. For each slice, each hemisphere was separately encircled as the region of interest. For each hemisphere, we calculated the ADC from the diffusion-weighted data and T2 from the T2w sequence as described previously [8]. In a second step, the ischemic and the nonischemic hemisphere were manually segmented for all slices. Then, ADC and T2 were calculated within these regions on a pixel-by-pixel basis. From these ADC and T2 maps, a
Fig. 1. Schematic drawing showing method of bobjectiveQ MRI data analysis. From 6 adjacent 2 mm thick slices, ADC and T2 data were calculated. For each hemisphere, data were transferred into frequency histograms. Histogram shows distribution at 6 h for ADC in the ischemic hemisphere for control and HBO treated group.
136
R. Veltkamp et al. / Brain Research 1037 (2005) 134–138
frequency histogram was created showing the number of pixels with T2 or ADC in certain ranges (Fig. 1). The ADC histogram was determined in steps of 50d 10 6 mm2/s, the T2 histogram in steps of 5 ms. For analysis of PWI, the relative cerebral blood volume (rCBV) was calculated as a ratio of values obtained from two predefined corresponding regions of interest (parietal cortex, striatum) in the ischemic and nonischemic hemisphere from the signal-time-curve determined from the PWI data as described in [7]. 2.4. Determination of infarct size Postmortem, 20 Am coronal cryosections was cut at 400 Am intervals, stained with the high-contrast silver infarct method as described [26] and analyzed using the public domain Scion Image program (release beta 4.0.2). To account for edema formation, infarct size was determined according to Swanson et al. [22] with modifications as described [9]. 2.5. Statistical analysis All values are expressed as mean F standard deviation (SD). For comparison of physiologic values, MRI data and infarct volumes between groups, ANOVA and t tests were used, respectively. A P value b 0.05 was regarded as statistically significant.
Fig. 2. Volume of tissue with abnormal hyperintense signal in the ischemic hemisphere on diffusion-weighted imaging (after 15 min, 3 h and 6 h of reperfusion) and T2w images (after 24 h of reperfusion) (*P b 0.05).
(375 F 91 mm3) than in HBO treated rats (225 F 39; P b 0.05). Objective analysis showed significantly longer mean T2 values at 24 h in the control (105.9 F 6.1 ms) than in the HBO group (101.0 F 6.9). In the nonischemic hemispheres of both groups, mean T2 was 93 F 3.0 ms. Finally, volume of histological infarct (24 h) before correction for edema was 262 F 140 mm3 in the control and 151 F 77 mm3 in the HBO group. After correction for edema, infarct volumes were 171 F 82 mm3 (control) vs. 100 F 58 mm3 (HBO; P b 0.05).
4. Discussion 3. Results Physiologic parameters before MCAO and 30 min after reperfusion were not significantly different between groups. Perfusion deficit during ischemia and reperfusion after filament removal did not differ between groups (ischemia: cortical rCBV was 0.50 F 0.32 (control) vs. 0.43 F 0.27 (HBO); reperfusion: cortical rCBV was 0.96 F 0.19 (control) vs. 0.87 F 0.20 (HBO)). Abnormally hyperintense signal on DWI was detected already 15 min after reperfusion and became more extensive on subsequent scans at 3 and 6 h of reperfusion (Fig. 2). HBO significantly reduced the volume of diffusion abnormality beginning immediately after reperfusion (Fig. 2). Mean abnormal diffusion volumes (mm3) at the various time points were at 15 min of reperfusion: 92 F 28 (control) vs. 64 F 17 (HBO); at 3 h: 180 F 51 (control) vs. 122 F 26 (HBO); at 6 h: 217 F 66 (control) vs. 144 F 32 (HBO; all P b 0.05). Correspondingly, objective analysis yielded significantly lower mean ADC values in the ischemic hemisphere of control animals compared to HBO. Mean ADC values were at 15 min: 0.78 F 0.05 (control) vs. 0.81 F 0.05; at 6 h: 0.72 F 0.03 vs. 0.77 F 0.03 (HBO). Mean ADC values ranged between 0.83 and 0.82 F 0.05 in the nonischemic hemisphere. T2w imaging at 24 h demonstrated a larger volume of hyperintense signal in control
Cerebral ischemia triggers a multiphasic sequence of cellular and molecular cascades resulting in brain damage [4]. Consequently, therapies directed against specific pathophysiological targets have a defined window of opportunity [4,10]. To date, it has remained largely speculative in which phase of ischemia HBO is effective—a matter which determines the timing of its administration and is relevant for the identification of its protective mechanisms. The major new finding of our study is that high-dose HBO treatment during transient focal ischemia has an immediate protective effect on the brain. Protection by HBO was superior to the inhalation of 100% O2 at atmospheric pressure. Diffusion-weighted MRI showed a reduced volume of signal abnormality immediately after HBO therapy. Thus, HBO counteracts early cascades of ischemic cell damage in tissue at risk. Improvement of oxygen-dependent energy metabolism in the ischemic brain parenchyma is a candidate mechanism based on this timing and because reduction of the ADC in DWI probably results from hypoxic failure of the ATP-dependent Na+–K+ pump. Sunami et al. [21] calculated enhanced oxygen delivery to the ischemic area, but reversal of tissue hypoxia and improvement of aerobic cell energy metabolism by HBO have not been demonstrated directly to date. Timing of HBO-induced protection is also compatible with counteracting the early neurotoxic
R. Veltkamp et al. / Brain Research 1037 (2005) 134–138
effects of excitatory neurotransmitters [4]. Interestingly, reduced glutamate concentration has been measured in striatal microdialysates after HBO therapy [3]. In contrast, previously reported effects of HBO on leucocyte adhesion and infiltration [2,12,23,24], cyclo-oxygenase-2 [28] or apoptosis [5,27] cannot account for the immediate protection in our study because the contribution of these pathophysiological pathways to the demise of ischemic tissue is delayed for several hours or even days [4]. Several studies have shown a protective effect of normobaric hyperoxia in focal ischemia [6,14,19,20]. Administration of HBO to stroke patients imposes a considerable logistical challenge which could be justified only if HBO-induced protection was superior to normobaric hyperoxia. Compared to normobaric oxygen, HBO reduced the parenchymal lesion size by 30 to 40% depending on the measured parameter and time point. This corresponds to our previous long-term outcome study where histological infarct sizes were reduced by 35% after intraischemic high-dose HBO compared to 100% O2 or low-dose HBO [25]. Similarly, Lou and colleagues [11] demonstrated a better protection by HBO than normobaric hyperoxia when administered during the first 6 h of reperfusion. Thus, there is a moderate additional benefit of high-dose HBO in transient focal ischemia. Protection by HBO in permanent ischemia was superior to room air in a recent MRI monitoring study [18]. In contrast, Lou et al. [11] found no difference between HBO and normobaric hyperoxia in this setting. In conclusion, the present findings demonstrate that early HBO therapy can immediately preserve tissue at risk in transient ischemia. Although we caution against uncritical translation of our data into the clinical setting, such bbridgingQ until reperfusion may have implications for the treatment of acute stroke because it could increase the volume of salvageable tissue for reperfusion therapies.
Acknowledgment This study was supported by a grant from the Deutsche Forschungsgemeinschaft (VE 196/2-1).
References [1] D.C. Anderson, A.G. Bottini, W.M. Jagiella, B. Westphal, S. Ford, G.L. Rockswold, R.B. Loewenson, A pilot study of hyperbaric oxygen in the treatment of human stroke, Stroke 22 (1991) 1137 – 1142. [2] D.N. Atochin, D. Fisher, I.T. Demchenko, S.R. Thom, Neutrophil sequestration and the effect of hyperbaric oxygen in a rat model of temporary middle cerebral artery occlusion, Undersea Hyperbaric Med. 27 (2000) 185 – 190. [3] A.E. Badr, W. Yin, G. Mychaskiw, J.H. Zhang, Effect of hyperbaric oxygen on striatal metabolites: a microdialysis study in awake freely moving rats after MCA occlusion, Brain Res. 916 (2001) 85 – 90. [4] U. Dirnagl, C. Iadecola, M.A. Moskowitz, Pathobiology of ischaemic stroke: an integrated view, Trends Neurosci. 22 (1999) 391 – 397.
137
[5] C.C. Eschenfelder, M. Lou, T. Herdegen, G. Deuschl, Mechanisms of hyperbaric oxygenation (HBO)-induced neuroprotection in transient focal ischemia in rats, Cerebrovasc. Dis. 17 (Suppl. 5) (2004) 13, (abstract). [6] E.P. Flynn, R.N. Auer, Eubaric hyperoxemia and experimental cerebral infarction, Ann. Neurol. 52 (2002) 566 – 572. [7] S. Heiland, W. Reith, M. Forsting, K. Sartor, Perfusion weighted magnetic resonance imaging using a new gadolinium complex as contrast agent in a rat model of focal cerebral ischemia, J. Magn. Reson. Imaging 7 (1997) 1109 – 1115. [8] S. Heiland, K. Sartor, E. Martin, H.J. Bardenheuer, K. Plaschke, In vivo monitoring of age-related changes in rat brain using quantitative diffusion magnetic resonance imaging and magnetic resonance relaxometry, Neurosci. Lett. 334 (2002) 157 – 160. [9] O. Herrmann, V. Tarabin, S. Suzuki, N. Attigah, I. Coserea, A. Schneider, J. Vogel, S. Prinz, S. Schwab, H. Monyer, F. Brombacher, M. Schwaninger, J. Cereb. Blood Flow Metab. 23 (2003) 406 – 415. [10] K.A. Hossmann, Viability thresholds and the penumbra of focal ischemia, Ann. Neurol. 36 (1994) 557 – 564. [11] M. Lou, C.C. Eschenfelder, T. Herdegen, S. Brecht, G. Deuschl, Therapeutic window for use of hyperbaric oxygenation in focal transient ischemia in rats, Stroke 35 (2004) 578 – 583. [12] M. Miljkovic-Lolic, R. Silbergleit, G. Fiskum, R.E. Rosenthal, Neuroprotective effects of hyperbaric oxygen treatment in experimental focal cerebral ischemia are associated with reduced brain leukocyte myeloperoxidase activity, Brain Res. 971 (2003) 90 – 94. [13] O. Miyamoto, R. Auer, Hypoxia, hyperoxia, ischemia and brain necrosis, Neurology 54 (2000) 362 – 367. [14] N. Nighoghossian, P. Trouillas, Hyperbaric oxygen in the treatment of acute ischemic stroke: an unsettled issue, J. Neurol. Sci. 150 (1997) 27 – 31. [15] N. Nighoghossian, P. Trouillas, P. Adeleine, F. Saford, Hyperbaric oxygen in the treatment of acute ischemic stroke: a double-blind pilot study, Stroke 26 (1995) 1369 – 1372. [16] D.E. Rusyniak, M.A. Kirk, J. May, Kao Louise, E.J. Brizendine, J.L. Welch, W.H. Cordell, R.J. Alonso, Hyperbaric oxygen therapy in acute ischemic stroke: results of the hyperbaric oxygen in acute ischemic stroke trial pilot study, Stroke 34 (2003) 571 – 574. [17] W.R. Schabitz, H. Schade, S. Heiland, R. Kollmar, J. Bardutzky, N. Henninger, H. Mller, U. Carl, S. Toyokuni, C. Sommer, S. Schwab, Neuroprotection by hyperbaric oxygenation after experimental focal cerebral ischemia monitored by MR imaging, Stroke 35 (2004) 1175 – 1179. [18] A.B. Singhal, R.M. Dijkhuizen, B.R. Rosen, E.H. Lo, Normobaric hyperoxia reduces MRI diffusion abnormalities and infarct size in experimental stroke, Neurology 26 (2002) 945 – 952. [19] A.B. Singhal, X. Wang, T. Sumii, T. Mori, E.H. Lo, Effects of normobaric hyperoxia in a rat model of focal cerebral ischemiareperfusion, J. Cereb. Blood Flow Metab. 22 (2002) 861 – 868. [20] K. Sunami, Y. Takeda, M. Hashimoto, M. Hirakawa, Hyperbaric oxygen reduces infarct volume in rats by increasing oxygen supply to the ischemic periphery, Crit. Care Med. 28 (2000) 2831 – 2836. [21] R.A. Swanson, M.T. Morton, G. Tsao-Wu, R.A. Savalos, C. Davidson, F.R. Sharp, A semiautomated method for measuring brain infarct volume, J. Cereb. Blood Flow Metab. 10 (1990) 290 – 293. [22] S.R. Thom, Functional inhibition of leucocyte B2 integrins by hyperbaric oxygen in carbon monoxide-mediated brain injury in rats, Toxicol. Appl. Pharmacol. 123 (1993) 248 – 256. [23] S.R. Thom, I. Mendiguren, K. Hardy, T. Bolotin, D. Fisher, M. Nebolon, L. Kilpatrick, Inhibition of human neutrophil B2-integrindependent adherence by hyperbaric O2, Am. J. Physiol. 272 (1997) C770 – C777. [24] R. Veltkamp, D.S. Warner, F. Domoki, A.D. Brinkhous, J.F. Toole, D.W. Busija, Hyperbaric oxygen decreases infarct size and behavioral deficit after transient focal cerebral ischemia in rats, Brain Res. 853 (2000) 68 – 73. [25] J. Vogel, C. Mfbius, W. Kuschinsky, Early delineation of ischemic
138
R. Veltkamp et al. / Brain Research 1037 (2005) 134–138
tissue in rat brain cryosections by high-contrast silver staining, Stroke 30 (1999) 1134 – 1141. [26] W. Yin, A.E. Badr, G. Mychaskiw, J.H. Zhang, Down regulation of COX-2 is involved in hyperbaric oxygen treatment in a rat transient focal cerebral ischemia model, Brain Res. 926 (2002) 165 – 171. [27] D. Yin, C. Zhou, I. Kusaka, J.W. Calvert, A.D. Parent, A. Nanda, J. Zhang, Inhibition of apoptosis by hyperbaric oxygen in a rat focal
cerebral ischemic model, J. Cereb. Blood Flow Metab. 23 (2003) 855 – 864. [28] E. Zea-Longa, P.R. Weinstein, S. Carlson, R. Cummins, Reversible middle cerebral artery occlusion without craniotomy in rats, Stroke 20 (1989) 84 – 91. [29] J.H. Zhang, A.B. Singhal, J.F. Toole, Oxygen therapy in ischemic stroke, Stroke 34 (2003) e152, (letter).
Research report
Hyperbaric oxygen induces rapid protection against focal cerebral ischemia Roland Veltkampa,*, Dirk A. Siebinga, Sabine Heilanda, Philip Schoenffeldt-Varasa, Claudia Veltkampb, Markus Schwaningera, Stefan Schwaba a
Department of Neurology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany b Department of Internal Medicine IV, Ruprecht-Karls-University Heidelberg, 69120 Heidelberg, Germany Accepted 1 January 2005
Abstract Background and purpose. The timing and mechanisms of protection by hyperbaric oxygen (HBO) in cerebral ischemia have only been partially elucidated. We monitored the early in vivo effects of HBO after 2 h transient focal ischemia using repetitive MRI. Methods. Wistar rats underwent filament occlusion of the middle cerebral artery (MCAO). 40 min after MCAO, rats were placed in a HBO chamber and breathed either 100% O2 at 3.0 atmospheres absolute (ata; n = 24) or at 1.0 ata (control; n = 24) for 1 h. Diffusion, perfusion and T2-weighted MR-images were obtained after 15 min and 3, 6 and 24 h of reperfusion. In 6 axial MR slices, volume of abnormal diffusion and T2w signals were measured in the ischemic hemisphere. Furthermore, hemispheric mean apparent diffusion coefficient- (ADC) and T2 values were calculated for statistical analysis. Results. HBO significantly reduced volume of abnormal DWI signal beginning immediately after reperfusion (control: 92 F 28 mm3; HBO: 64 F 17) and lesion size on T2w (control: 375 F 91 mm3; HBO: 225 F 39) after 24 h. Correspondingly, mean ADC levels were lower and T2 values higher in the ischemic hemisphere in the control group. HBO reduced histological infarct size at 24 h. Conclusion. High-dose intraischemic HBO therapy has an immediate protective on the brain which is superior to normobaric oxygen. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Cerebrovascular disorders; Neuroprotection; MRI; Hypoxia
1. Introduction Progression of tissue damage during the first hours of cerebral ischemia has remained a major challenge in acute stroke management [4,10] Because cerebral hypoxia is an important component of immediate and early secondary cell damage, improving oxygen delivery to the ischemic penumbra has been a logical concept for tissue protection. Several experimental studies have shown that normobaric
T Corresponding author. Fax: +49 6221 561740. E-mail address: [email protected] (R. Veltkamp). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.01.006
hyperoxia can protect the brain against focal cerebral ischemia [6,14,19,20]. Administering oxygen at increased ambient pressure–i.e. hyperbaric oxygen treatment (HBO)– can further increase the pO2 in blood plasma and in normal brain tissue. The effectiveness of HBO in cerebral ischemia, however, has remained controversial for many years [15]. While many of the older experimental studies had methodological shortcomings (reviewed in [25]), more recent work confirmed a protective effect of HBO at least under certain circumstances [11,18,21,25]. Clinical pilot studies [1,16,17] have failed to show a benefit from HBO, perhaps because a long time window between symptom-onset and initiation of therapy was allowed [30]. Indeed, appropriate judgment of the therapeutic potential of HBO is hampered because the
R. Veltkamp et al. / Brain Research 1037 (2005) 134–138
timing of its protective effect and the corresponding pathophysiological targets are largely unknown. In the present study, we used repetitive in vivo magnetic resonance imaging (MRI) to gain an insight into the timing of the protective effects of HBO on ischemic tissue and thus narrow the search for the involved mechanisms.
2. Materials and methods 2.1. Surgical preparation Experiments were performed on male Wistar rats (Charles River, Germany) weighing 280–320 g. The procedures were approved by the governmental animal care authorities. Focal cerebral ischemia was induced by filament occlusion of the middle cerebral artery (MCAO) for 120 min according to Zea-Longa et al. [29] with some modifications [18]. After MCAO, animals were placed into the MRI scanner for perfusion weighted imaging (PWI). Then, anesthesia was discontinued and animals breathed 100% O2-enriched air. Forty minutes later, rats were transferred into a rodent HBO chamber. Animals were randomly assigned to either the control group, which breathed 100% O2 at atmospheric pressure (1 atmosphere absolute (ata)), or the HBO group which received 100% O 2 at 3.0 ata. In the HBO group, compression was started 40 min after filament introduction and was performed over 5 min. Decompression was begun 105 min after filament introduction at the same rate. Rats were reanesthetized and the filament was removed 120 min after introduction. A second MRI was performed 15 min after reperfusion. Animals were allowed to awaken in an O2-enriched environment and transferred to their cages. Additional MRI scans were performed 3, 6 and 24 h after reperfusion.
135
2.2. Magnetic resonance imaging The animals were examined in a 2.35-T scanner (Biospec 24/40, BRUKER Medizintechnik, Ettlingen, Germany) using a previously described configuration [8]. The MR-protocol consisted of a diffusion-weighted spinecho echo-planar imaging (SE-EPI) sequence (b values = 200, 300, 400, 500, 600 and 700 s/mm2) and a multi-spinecho sequence (12 echoes with echo times of 8, 16, 24, ... 96 ms). For PWI, we used a gradient-echo echo-planar imaging (GE-EPI) sequence (repetition time = 1 s, echo-time = 15 ms, 20 repetitions with a time resolution of 1 s/image data set) for monitoring the bolus passage of 1 mmol/kg of a paramagnetic contrast agent (Omniscan, Nycomed Amersham, Oslo, Norway). 2.3. Data analysis During MR imaging, a six-slice data set was obtained covering the MCA territory, the first axial slice containing the olfactory bulb. DWI and T2w data were analyzed by an operator-driven and a largely operator-independent mode. In the first method, areas of abnormal signal intensity were encircled for each MR section using a side to side comparison on the screen. Volume of lesion on DWI and T2w was calculated by multiplying the total area with section thickness. The second method was performed to circumvent the problem of adequate threshold determination. For each slice, each hemisphere was separately encircled as the region of interest. For each hemisphere, we calculated the ADC from the diffusion-weighted data and T2 from the T2w sequence as described previously [8]. In a second step, the ischemic and the nonischemic hemisphere were manually segmented for all slices. Then, ADC and T2 were calculated within these regions on a pixel-by-pixel basis. From these ADC and T2 maps, a
Fig. 1. Schematic drawing showing method of bobjectiveQ MRI data analysis. From 6 adjacent 2 mm thick slices, ADC and T2 data were calculated. For each hemisphere, data were transferred into frequency histograms. Histogram shows distribution at 6 h for ADC in the ischemic hemisphere for control and HBO treated group.
136
R. Veltkamp et al. / Brain Research 1037 (2005) 134–138
frequency histogram was created showing the number of pixels with T2 or ADC in certain ranges (Fig. 1). The ADC histogram was determined in steps of 50d 10 6 mm2/s, the T2 histogram in steps of 5 ms. For analysis of PWI, the relative cerebral blood volume (rCBV) was calculated as a ratio of values obtained from two predefined corresponding regions of interest (parietal cortex, striatum) in the ischemic and nonischemic hemisphere from the signal-time-curve determined from the PWI data as described in [7]. 2.4. Determination of infarct size Postmortem, 20 Am coronal cryosections was cut at 400 Am intervals, stained with the high-contrast silver infarct method as described [26] and analyzed using the public domain Scion Image program (release beta 4.0.2). To account for edema formation, infarct size was determined according to Swanson et al. [22] with modifications as described [9]. 2.5. Statistical analysis All values are expressed as mean F standard deviation (SD). For comparison of physiologic values, MRI data and infarct volumes between groups, ANOVA and t tests were used, respectively. A P value b 0.05 was regarded as statistically significant.
Fig. 2. Volume of tissue with abnormal hyperintense signal in the ischemic hemisphere on diffusion-weighted imaging (after 15 min, 3 h and 6 h of reperfusion) and T2w images (after 24 h of reperfusion) (*P b 0.05).
(375 F 91 mm3) than in HBO treated rats (225 F 39; P b 0.05). Objective analysis showed significantly longer mean T2 values at 24 h in the control (105.9 F 6.1 ms) than in the HBO group (101.0 F 6.9). In the nonischemic hemispheres of both groups, mean T2 was 93 F 3.0 ms. Finally, volume of histological infarct (24 h) before correction for edema was 262 F 140 mm3 in the control and 151 F 77 mm3 in the HBO group. After correction for edema, infarct volumes were 171 F 82 mm3 (control) vs. 100 F 58 mm3 (HBO; P b 0.05).
4. Discussion 3. Results Physiologic parameters before MCAO and 30 min after reperfusion were not significantly different between groups. Perfusion deficit during ischemia and reperfusion after filament removal did not differ between groups (ischemia: cortical rCBV was 0.50 F 0.32 (control) vs. 0.43 F 0.27 (HBO); reperfusion: cortical rCBV was 0.96 F 0.19 (control) vs. 0.87 F 0.20 (HBO)). Abnormally hyperintense signal on DWI was detected already 15 min after reperfusion and became more extensive on subsequent scans at 3 and 6 h of reperfusion (Fig. 2). HBO significantly reduced the volume of diffusion abnormality beginning immediately after reperfusion (Fig. 2). Mean abnormal diffusion volumes (mm3) at the various time points were at 15 min of reperfusion: 92 F 28 (control) vs. 64 F 17 (HBO); at 3 h: 180 F 51 (control) vs. 122 F 26 (HBO); at 6 h: 217 F 66 (control) vs. 144 F 32 (HBO; all P b 0.05). Correspondingly, objective analysis yielded significantly lower mean ADC values in the ischemic hemisphere of control animals compared to HBO. Mean ADC values were at 15 min: 0.78 F 0.05 (control) vs. 0.81 F 0.05; at 6 h: 0.72 F 0.03 vs. 0.77 F 0.03 (HBO). Mean ADC values ranged between 0.83 and 0.82 F 0.05 in the nonischemic hemisphere. T2w imaging at 24 h demonstrated a larger volume of hyperintense signal in control
Cerebral ischemia triggers a multiphasic sequence of cellular and molecular cascades resulting in brain damage [4]. Consequently, therapies directed against specific pathophysiological targets have a defined window of opportunity [4,10]. To date, it has remained largely speculative in which phase of ischemia HBO is effective—a matter which determines the timing of its administration and is relevant for the identification of its protective mechanisms. The major new finding of our study is that high-dose HBO treatment during transient focal ischemia has an immediate protective effect on the brain. Protection by HBO was superior to the inhalation of 100% O2 at atmospheric pressure. Diffusion-weighted MRI showed a reduced volume of signal abnormality immediately after HBO therapy. Thus, HBO counteracts early cascades of ischemic cell damage in tissue at risk. Improvement of oxygen-dependent energy metabolism in the ischemic brain parenchyma is a candidate mechanism based on this timing and because reduction of the ADC in DWI probably results from hypoxic failure of the ATP-dependent Na+–K+ pump. Sunami et al. [21] calculated enhanced oxygen delivery to the ischemic area, but reversal of tissue hypoxia and improvement of aerobic cell energy metabolism by HBO have not been demonstrated directly to date. Timing of HBO-induced protection is also compatible with counteracting the early neurotoxic
R. Veltkamp et al. / Brain Research 1037 (2005) 134–138
effects of excitatory neurotransmitters [4]. Interestingly, reduced glutamate concentration has been measured in striatal microdialysates after HBO therapy [3]. In contrast, previously reported effects of HBO on leucocyte adhesion and infiltration [2,12,23,24], cyclo-oxygenase-2 [28] or apoptosis [5,27] cannot account for the immediate protection in our study because the contribution of these pathophysiological pathways to the demise of ischemic tissue is delayed for several hours or even days [4]. Several studies have shown a protective effect of normobaric hyperoxia in focal ischemia [6,14,19,20]. Administration of HBO to stroke patients imposes a considerable logistical challenge which could be justified only if HBO-induced protection was superior to normobaric hyperoxia. Compared to normobaric oxygen, HBO reduced the parenchymal lesion size by 30 to 40% depending on the measured parameter and time point. This corresponds to our previous long-term outcome study where histological infarct sizes were reduced by 35% after intraischemic high-dose HBO compared to 100% O2 or low-dose HBO [25]. Similarly, Lou and colleagues [11] demonstrated a better protection by HBO than normobaric hyperoxia when administered during the first 6 h of reperfusion. Thus, there is a moderate additional benefit of high-dose HBO in transient focal ischemia. Protection by HBO in permanent ischemia was superior to room air in a recent MRI monitoring study [18]. In contrast, Lou et al. [11] found no difference between HBO and normobaric hyperoxia in this setting. In conclusion, the present findings demonstrate that early HBO therapy can immediately preserve tissue at risk in transient ischemia. Although we caution against uncritical translation of our data into the clinical setting, such bbridgingQ until reperfusion may have implications for the treatment of acute stroke because it could increase the volume of salvageable tissue for reperfusion therapies.
Acknowledgment This study was supported by a grant from the Deutsche Forschungsgemeinschaft (VE 196/2-1).
References [1] D.C. Anderson, A.G. Bottini, W.M. Jagiella, B. Westphal, S. Ford, G.L. Rockswold, R.B. Loewenson, A pilot study of hyperbaric oxygen in the treatment of human stroke, Stroke 22 (1991) 1137 – 1142. [2] D.N. Atochin, D. Fisher, I.T. Demchenko, S.R. Thom, Neutrophil sequestration and the effect of hyperbaric oxygen in a rat model of temporary middle cerebral artery occlusion, Undersea Hyperbaric Med. 27 (2000) 185 – 190. [3] A.E. Badr, W. Yin, G. Mychaskiw, J.H. Zhang, Effect of hyperbaric oxygen on striatal metabolites: a microdialysis study in awake freely moving rats after MCA occlusion, Brain Res. 916 (2001) 85 – 90. [4] U. Dirnagl, C. Iadecola, M.A. Moskowitz, Pathobiology of ischaemic stroke: an integrated view, Trends Neurosci. 22 (1999) 391 – 397.
137
[5] C.C. Eschenfelder, M. Lou, T. Herdegen, G. Deuschl, Mechanisms of hyperbaric oxygenation (HBO)-induced neuroprotection in transient focal ischemia in rats, Cerebrovasc. Dis. 17 (Suppl. 5) (2004) 13, (abstract). [6] E.P. Flynn, R.N. Auer, Eubaric hyperoxemia and experimental cerebral infarction, Ann. Neurol. 52 (2002) 566 – 572. [7] S. Heiland, W. Reith, M. Forsting, K. Sartor, Perfusion weighted magnetic resonance imaging using a new gadolinium complex as contrast agent in a rat model of focal cerebral ischemia, J. Magn. Reson. Imaging 7 (1997) 1109 – 1115. [8] S. Heiland, K. Sartor, E. Martin, H.J. Bardenheuer, K. Plaschke, In vivo monitoring of age-related changes in rat brain using quantitative diffusion magnetic resonance imaging and magnetic resonance relaxometry, Neurosci. Lett. 334 (2002) 157 – 160. [9] O. Herrmann, V. Tarabin, S. Suzuki, N. Attigah, I. Coserea, A. Schneider, J. Vogel, S. Prinz, S. Schwab, H. Monyer, F. Brombacher, M. Schwaninger, J. Cereb. Blood Flow Metab. 23 (2003) 406 – 415. [10] K.A. Hossmann, Viability thresholds and the penumbra of focal ischemia, Ann. Neurol. 36 (1994) 557 – 564. [11] M. Lou, C.C. Eschenfelder, T. Herdegen, S. Brecht, G. Deuschl, Therapeutic window for use of hyperbaric oxygenation in focal transient ischemia in rats, Stroke 35 (2004) 578 – 583. [12] M. Miljkovic-Lolic, R. Silbergleit, G. Fiskum, R.E. Rosenthal, Neuroprotective effects of hyperbaric oxygen treatment in experimental focal cerebral ischemia are associated with reduced brain leukocyte myeloperoxidase activity, Brain Res. 971 (2003) 90 – 94. [13] O. Miyamoto, R. Auer, Hypoxia, hyperoxia, ischemia and brain necrosis, Neurology 54 (2000) 362 – 367. [14] N. Nighoghossian, P. Trouillas, Hyperbaric oxygen in the treatment of acute ischemic stroke: an unsettled issue, J. Neurol. Sci. 150 (1997) 27 – 31. [15] N. Nighoghossian, P. Trouillas, P. Adeleine, F. Saford, Hyperbaric oxygen in the treatment of acute ischemic stroke: a double-blind pilot study, Stroke 26 (1995) 1369 – 1372. [16] D.E. Rusyniak, M.A. Kirk, J. May, Kao Louise, E.J. Brizendine, J.L. Welch, W.H. Cordell, R.J. Alonso, Hyperbaric oxygen therapy in acute ischemic stroke: results of the hyperbaric oxygen in acute ischemic stroke trial pilot study, Stroke 34 (2003) 571 – 574. [17] W.R. Schabitz, H. Schade, S. Heiland, R. Kollmar, J. Bardutzky, N. Henninger, H. Mller, U. Carl, S. Toyokuni, C. Sommer, S. Schwab, Neuroprotection by hyperbaric oxygenation after experimental focal cerebral ischemia monitored by MR imaging, Stroke 35 (2004) 1175 – 1179. [18] A.B. Singhal, R.M. Dijkhuizen, B.R. Rosen, E.H. Lo, Normobaric hyperoxia reduces MRI diffusion abnormalities and infarct size in experimental stroke, Neurology 26 (2002) 945 – 952. [19] A.B. Singhal, X. Wang, T. Sumii, T. Mori, E.H. Lo, Effects of normobaric hyperoxia in a rat model of focal cerebral ischemiareperfusion, J. Cereb. Blood Flow Metab. 22 (2002) 861 – 868. [20] K. Sunami, Y. Takeda, M. Hashimoto, M. Hirakawa, Hyperbaric oxygen reduces infarct volume in rats by increasing oxygen supply to the ischemic periphery, Crit. Care Med. 28 (2000) 2831 – 2836. [21] R.A. Swanson, M.T. Morton, G. Tsao-Wu, R.A. Savalos, C. Davidson, F.R. Sharp, A semiautomated method for measuring brain infarct volume, J. Cereb. Blood Flow Metab. 10 (1990) 290 – 293. [22] S.R. Thom, Functional inhibition of leucocyte B2 integrins by hyperbaric oxygen in carbon monoxide-mediated brain injury in rats, Toxicol. Appl. Pharmacol. 123 (1993) 248 – 256. [23] S.R. Thom, I. Mendiguren, K. Hardy, T. Bolotin, D. Fisher, M. Nebolon, L. Kilpatrick, Inhibition of human neutrophil B2-integrindependent adherence by hyperbaric O2, Am. J. Physiol. 272 (1997) C770 – C777. [24] R. Veltkamp, D.S. Warner, F. Domoki, A.D. Brinkhous, J.F. Toole, D.W. Busija, Hyperbaric oxygen decreases infarct size and behavioral deficit after transient focal cerebral ischemia in rats, Brain Res. 853 (2000) 68 – 73. [25] J. Vogel, C. Mfbius, W. Kuschinsky, Early delineation of ischemic
138
R. Veltkamp et al. / Brain Research 1037 (2005) 134–138
tissue in rat brain cryosections by high-contrast silver staining, Stroke 30 (1999) 1134 – 1141. [26] W. Yin, A.E. Badr, G. Mychaskiw, J.H. Zhang, Down regulation of COX-2 is involved in hyperbaric oxygen treatment in a rat transient focal cerebral ischemia model, Brain Res. 926 (2002) 165 – 171. [27] D. Yin, C. Zhou, I. Kusaka, J.W. Calvert, A.D. Parent, A. Nanda, J. Zhang, Inhibition of apoptosis by hyperbaric oxygen in a rat focal
cerebral ischemic model, J. Cereb. Blood Flow Metab. 23 (2003) 855 – 864. [28] E. Zea-Longa, P.R. Weinstein, S. Carlson, R. Cummins, Reversible middle cerebral artery occlusion without craniotomy in rats, Stroke 20 (1989) 84 – 91. [29] J.H. Zhang, A.B. Singhal, J.F. Toole, Oxygen therapy in ischemic stroke, Stroke 34 (2003) e152, (letter).