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Max-Planck-Institute for Neurological Research, Department of Experimental Neurology, Cologne, Germany;
* Department of Cardiology and Pneumology, Free University of Berlin; and
Institute of Pharmacology and Toxicology, Medical Faculty (Charité) of the Humboldt University, Germany
2Correspondence: University Hospital Benjamin Franklin, Department of Cardiology and Pneumology, Free University of Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail: thomas.walther{at}ukbf.fu-berlin.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: A II • focal cerebral ischemia • transgenic mice • penumbra • collateral flow
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. In contrast, inhibition of ACE or long-term blockade of the angiotensin receptor subtype AT1 (AT1) in rat brain improves the neurological outcome and reduces the infarct volume after experimental focal cerebral ischemia (3
4
5
6)
and in stroke patients (7)
. These findings define A II as a significant contributor to the pathophysiology of ischemic stroke. Several findings, however, indicate a benefit from A II in brain ischemia. In the Captopril Prevention Project, a higher risk in fatal and nonfatal strokes was detected in hypertensive patients treated with the ACE inhibitor captopril vs. conventional therapy (8)
. Moreover, infusion of A II or AT2 receptor stimulation has been shown to decrease the mortality rate in gerbils with unilateral carotid ligation (9
, 10)
. The promotion of angiogenesis and recruitment of preexisting collateral circulation by A II in case of acute ischemia have been suggested as a possible mechanism of this benefit (11)
.
The major goal of the present study was to further clarify the role of A II and the AT1 receptor in brain ischemia. The severity of ischemic injury evaluated 1 and 24 h after middle cerebral artery occlusion (MCAO) was investigated either in the presence of a chronic increase in A II supply using transgenic mice expressing a rat angiotensinogen transgene (TGM123) (12)
or in response to a defect in the transmission of the A II signal via its main receptor in knockout mice lacking the AT1A receptor (13)
.
It has to be considered that in the brain, AT1 and AT2 receptors are expressed in cerebral microvessels and cerebral arteries (14
, 15)
as well as in neurons (reviewed in refs16
, 17
). In in vivo studies, therefore, it is not possible to clearly differentiate whether the effects of A II are mediated by vascular or neuronal receptors. To clearly define the involvement of neuronal AT1 receptors in the severity of ischemic neuronal injury, we included in vitro experiments using primary neuronal cultures obtained from embryos (E14) of AT1-deficient mice and their wild-type littermates. Oxygen-glucose deprivation (OGD) served as a model of ischemic injury.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 13)
were obtained from breeding stocks of T.W. at the UKBF in Berlin. The animals were housed in groups of two to three at 22 ± 1°C in a 12 h/12 h light/dark cycle with food and beverage available ad libitum.
In situ hybridization
In situ hybridization was performed as described elsewhere (18)
using a digoxigenin-labeled probe matching the mouse AT1 receptor mRNA. The probe was synthesized from a T vector system (Promega, Madison, WI) derivative harboring a 352 bp fragment homologous to the AT1 receptor mRNA using the DIG-RNA labeling kit (Roche Biochemicals, Mannheim, Germany).
Animal preparation
Animals were anesthetized with 1.5% halothane and maintained at 1% halothane in 70% N20 and 30% O2. For measurement of arterial blood gases and blood pressure, the left femoral artery was cannulated with PE-50 polythene tubing. In all animals, the mean arterial blood pressure was recorded continuously and the arterial blood gas status was determined before and 10 min after permanent MCAO. Focal cerebral ischemia was induced by occluding the left middle cerebral artery (MCA) using a modified intrafilament technique (19
, 20)
. Animals selected for 24 h survival were returned to their cages after MCAO and received an intraperitoneal (i.p.) injection of saline with glucose (5%) to maintain proper water and glucose balance (0.8 ml, twice a day).
Autoradiographic measurements and tissue processing
Forty-five minutes and 2 min before the end of the experiment, animals were administered L-[4,5-3H]-leucine (300 mCi/animal, specific activity 151 Ci/mmol; Amersham, Braunschweig, Germany) and 4-iodo-N-methyl-[14C]-antipyrine (20 mCi/animal, specific activity 40 to 60 mCi/mmol; Biotrend Chemicals, Cologne, Germany) i.p. to evaluate cerebral protein synthesis (CPS) and cerebral blood flow (CBF) rates, respectively (18
, 20)
. After the predetermined ischemic periods, experiments were terminated by in situ freezing with liquid nitrogen. Brains and hearts were removed in a cold temperature cabinet (-20°C) and brains were cut into 20 µm-thick coronal cryostat sections at -20°C. Sections were mounted on coverslips for ATP bioluminescence, on object holders for CPS and CBF autoradiography.
Laser Doppler flow
Laser Doppler flow was recorded with a probe attached to the skull above the territory of the MCA (2 mm caudal to bregma and 6 mm lateral to midline), and was monitored from the beginning of surgery until 15 min after reperfusion. Only those animals were included in the study in which the laser Doppler flow signal intensity dropped below 25% of the control value after MCAO.
Regional measurement of ATP, cerebral protein synthesis, and blood flow
Pictorial measurements of ATP were carried out using the ATP-specific bioluminescence method and of CPS and CBF by a [14C]-[3H] double tracer technique. For CBF autoradiography, slices were exposed for 12 days to Hyperfilm (Kodak, Rochester, NY) together with calibrated [14C] standards. For CPS measurement, the same slices were incubated in 10% trichloroacetic acid to remove labeled free leucine and metabolites other than labeled proteins. Subsequently, slices were exposed for 14 days with [3H] standards to a tritium sensitive film (Hyperfilm [3H]; Amersham) as described previously (21)
. For quantitative CBF measurement, the blood was sampled from the frozen heart to obtain the final arterial [14C] radioactivity and CBF rates were calculated, as described elsewhere (22)
.
Morphometric analysis of ischemia-induced metabolic disturbances
Bioluminescence and autoradiographic images were digitized with a CCD camera system and analyzed using a modified version (ImageMG) of the public domain software NIH Image (W. Rasband, National Institutes of Health, Bethesda, MD). ATP depletion was defined as a decline to less than 30% of the mean value of the contralateral side. The threshold for CPS inhibition was set to the lowest CPS value of the nonischemic hemisphere, excluding fiber tracts. The areas of ATP depletion and CPS inhibition were measured at the level of caudate-putamen and expressed as percentage of the contralateral hemisphere. For flow threshold pixel analysis, the CBF and CPS autoradiogram, and the corresponding ATP image were aligned. The ipsilateral cortex was outlined, ATP and CPS pixels were registered in flow classes of 10 ml/100 g/min, and the percentage of pixels with metabolic disturbance was calculated. The perfusion threshold value was then defined as the flow value at which 50% of pixels exhibited metabolic failure (22)
.
Primary neuronal cell culture, oxygen-glucose deprivation, and cell death assay
Primary neuronal cultures of cerebral cortex were obtained from embryos (E14-E15) of wild-type and AT1 receptor knockout mice. Cultures were prepared from mice brains according to the method introduced for rat brains by Brewer (23)
modified as described previously (24)
. Cultures were kept at 36.5°C and 5% CO2, and were fed beginning on the fourth day in vitro (DIV) with serum-free cultivating medium by replacing half of the medium twice a week.
On days 8 to 10, medium was removed from the cultures and preserved. Cultures were rinsed twice with PBS, then subjected to OGD for 120 min in a balanced salt solution at pO2 < 2 mm Hg, followed by replacement of the preserved medium as described previously (24
, 25)
. For the pharmacological blockade of AT1 receptors, cortical cultures of wild-type mice were pretreated with losartan (dissolved in water) 1 h before OGD at a final concentration of 1 or 10 µM.
Neuronal injury was quantitatively assessed by the measurement of lactate dehydrogenase (LDH) in the medium of the cortical cultures at 24 h after OGD (26)
. The enzyme standard for kinetic LDH test was obtained from Sigma Chemie GmbH (Deisenhofen, Germany).
Statistical analysis
All values are expressed as means ± SD. Differences in physiological values, metabolic parameters, and regional blood flow at both time points (1 and 24 h after MCAO) were compared between the transgenic subgroups and the corresponding wild-type littermates using ANOVA, followed by the Scheffé test. Differences in physiological values before and 10 min after MCAO were analyzed applying a paired t test. Differences in LDH release in culture were analyzed by one-way ANOVA, followed by Tukey’s test. P < 0.05 was considered to indicate statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The corresponding wild-type littermates exhibiting normal A II levels served as controls. CBF was determined in the infarct core and the penumbra using a modified autoradiograhic 14C-iodoantipyrine technique (21)
.
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Although TGM123 animals exhibited significantly higher mean arterial blood pressure (Table 1
a), the ischemic lesion size as defined by ATP depletion did not differ from that in the wild-type subgroup after 1 and 24 h MCAO (Fig. 2
). After 1 h permanent MCAO, however, the penumbra was significantly smaller in transgenic mice (2.2±0.8% of contralateral hemisphere) than in the wild-type littermates (12.5±4.6%; P<0.01). The size of the penumbra became negligible at 24 h of permanent MCAO in both the angiotensinogen-overexpressing (1.2±1.7%) and wild-type subgroup (0.3±0.7%). Since A II is known to exhibit vasoconstrictor properties, we wanted to assess hemodynamic differences between the experimental groups. As summarized in Fig. 2
, regional CBF was significantly lower in the transgenic subgroup than in corresponding wild-type mice in the ischemic core at 1 h (TGM123: 3.8±1.7 ml/100 g/min vs. wild-type: 7.6±2.7 ml/100 g/min; P<0.05), and in the penumbra region (TGM123: 23.0±5.3 ml/100 g/min vs. wild-type: 36.2±5.2 ml/100 g/min; P<0.01) and the lesion area at 24 h after permanent MCAO (TGM123: 2.9±2.7 ml/100 g/min vs. wild-type: 7.1±2.9 ml/100 g/min; P<0.02), respectively. To exclude that the deterioration of the ischemic injury was due to a significant up- or down-regulation of the AT1 receptor in TGM123 mice induced by elevated brain A II concentrations, the AT1 receptor mRNA concentrations have been characterized by in situ hybridization showing no obvious changes between transgenic and wild-type mice (Fig. 3
).
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Importance of AT1 receptor in focal ischemia
To evaluate the importance of AT1 receptors in mediating the detrimental effect of A II after MCAO, the sizes of the infarct core and penumbral area were measured in transgenic animals deficient for the AT1 receptor and compared with respective wild-type littermates. In contrast to TGM123 mice, the mean arterial blood pressure was lower in AT1 knockout mice (Table 1b)
, but ischemic lesion size (the ATP-depleted area) was smaller at 1 h permanent MCAO than in the wild-type animals (AT1 knockout: 53.9±7.3% of contralateral hemisphere vs. wild-type: 64.9±4.6%; P<0.05). Moreover, the size of the penumbra was significantly larger in the absence of the AT1 subtype (14.7±4.0%) than in corresponding wild-type species (7.7±3.5%; P<0.05), although the area of inhibited CPS did not differ significantly (Fig. 4
). Since the penumbra evolved into the core 24 h later, a similarly small penumbra size was observed at this time in both subgroups (AT1 knockout: 1.7±2.9; wild-type: 0.5±0.4%). Average CBF was higher in the core (AT1 knockout: 11.7±5.1 ml/100 g/min, wild-type: 4.6±1.1 ml/100 g/min; P<0.03) and penumbra areas (AT1 knockout: 31.0±3.0 ml/100 g/min, wild-type: 24.6±3.1 ml/100 g/min; P<0.03) in AT1-deficient animals after 1 h MCAO than in the controls. After 24 h permanent MCAO, regional CBF in the lesion area was comparable (AT1 knockout: 6.0±2.6 ml/100 g/min vs. wild-type: 6.7±1.5 ml/100 g/min). As in TGM123 mice, the arterial blood gas status did not differ significantly between mutant and wild-type mice before or shortly after MCAO irrespective of the ischemic duration (Table 1b)
.
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Ischemia-induced metabolic disturbances
Using a unique pixel analysis approach that comprises cerebral blood flow and protein synthesis double tracer autoradiography combined with bioluminescent ATP imaging, it is possible to determine a flow rate below which cortical energy failure and/or protein synthesis inhibition occurs after MCAO (22
, 27)
. In the present study, this approach was used with transgenic animals for the first time to characterize possible differences in the ischemic sensitivity of brain tissue after focal cerebral ischemia. At 1 h MCAO, a significant rise of the perfusion threshold for ATP depletion was determined in angiotensinogen-overexpressing mice (29±1 ml/100 g/min) vs. their wild-type littermates (20±6 ml/100 g/min; P<0.05), which suggests an elevated ischemic sensitivity of cortical tissue for energy failure in transgenic mutants. The flow threshold for the inhibition of overall protein synthesis in angiotensinogen-overexpressing mice, on the other hand, remained unchanged (Fig. 5
a). In AT1 receptor-deficient mice, however, perfusion threshold analyses revealed a significantly lesser sensitivity of cortex for metabolic disturbances after onset of focal cerebral ischemia. Although there was a trend toward lower threshold flow rates for energy failure in AT1 knockout mice (AT1 knockout: 18±2 ml/100 g/min vs. wild-type: 23±7 ml/100 g/min; n.s.), the significantly lower perfusion threshold values for protein synthesis inhibition in AT1-deficient mutants compared with wild-type littermates (AT1 knockout: 34±6 ml/100 g/min vs. wild-type: 45±4 ml/100 g/min; P<0.05) clearly indicated that cortical tissue of AT1 knockout mice in vivo was more resistant to an ischemic impact than that of wild-type controls (Fig. 5b
).
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Involvement of neuronal AT1 receptors in ischemic injury in vitro
Exposure of primary cultures of cortical neurons derived from AT1 wild-type mice to OGD for 120 min resulted in a considerable increase in the release of LDH activity indicating neuronal cell damage (Fig. 6
A). The release of LDH in response to the same degree of ischemic injury was significantly reduced in cortical neurons derived from AT1 knockout mice. A comparable protection of the neurons against OGD was achieved when the corresponding wild-type neurons were pretreated with the AT1 receptor antagonist losartan (1 and 10 µM; Fig. 6B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, who showed better collateral circulation and smaller lesion size in angiotensinogen knockout mice than in controls after 1 h MCAO, indicating a beneficial effect of lower A II levels before the onset of ischemia. Comparable evidence has been obtained by pharmacological blockade of A II synthesis or the AT1 receptor (3
4
5
6
7)
.
The detrimental effects of A II could be addressed to its vasoconstrictor effect, but also to the development of hypertrophy of cerebral vessels, which may influence the focal vascular reactivity of the collateral vessels. Growing evidence indicates that the AT1 receptor is responsible for the majority of the classical and known biological effects of A II, including hypertension, vasoconstriction, cardiomyocyte hypertrophy, proliferation of the vascular smooth muscle cells, and adventitial thickening (29
30
31)
. Several mechanisms have been discussed as being responsible for the beneficial effect of AT1 receptor blockade in brain ischemia. Long-term blockade of central AT1 receptors has been shown to reduce the expression of c-Fos and c-Jun in response to cerebral ischemia (5)
. A dose-dependent expression of various immediate early gene-encoded transcription factors in various brain regions has been found after intracerebroventricular injection of A II in conscious rats (32
, 33)
. In spontaneously hypertensive rats, the protective effect of AT1 receptor antagonism in cerebral ischemia has been related to the normalization of cerebrovascular autoregulation in the marginal ischemic zone, resulting in decreased neuronal injury (6)
. In the present study, long-term hypertension induced by continuous overexpression of A II may have further contributed to the vascular hypertrophy and shift in the blood pressure CBF autoregulation curve toward higher blood pressure values (34)
. This results in an earlier and more severe decline of CBF at a less severe decrease in perfusion pressure, and thus may prevent maintenance of the CBF above the threshold of breakdown of energy metabolism. The association of a higher blood flow with a smaller ATP-depleted lesion area and a larger penumbra in the AT1 knockout mice, and/or the lower blood flow with the larger ATP-depleted region and smaller penumbra in the angiotensinogen-overexpressing mice, supports this notion.
The in vitro data clearly indicate that the deleterious effects of A II in ischemic injury, however, do not depend exclusively on vasoconstriction and proliferation of vascular tissue. In primary neuronal cultures, which are free of vascular components and completely independent of hemodynamic parameters, neurons were resistant to a transient loss of oxygen and glucose supply when the function of the AT1 receptor was ruled out either by AT1 knockout or pharmacologically by receptor blockade with losartan. The involvement of AT1 stimulation in apoptosis caused by A II has been shown in myocytes (35)
and endothelial cells in vitro (36)
and in rat blood vessels in vivo (37)
. A participation of bax, caspase-3, or activation of protein kinase C in the pathway of apoptosis triggered by AT1 stimulation has been discussed (35
, 36)
. In the present study, we prove for the first time that AT1 activation also initiates proapoptotic stimuli in neuronal cells.
In conclusion, we provide clear evidence that neuroprotection against ischemia/hypoxia can be achieved by a drug- as well as a transgene-induced suppression of AT1 receptor function in vivo and in vitro. These findings may have clinical implications and disclose the utility of AT1 blockade as a promising neuroprotective strategy in stroke. Our data may also provide an experimental basis by which to understand the recent clinical finding that ACE gene polymorphism or pharmacological interactions of the renin-angiotensin system appear to significantly alter the neurological outcome and survival of stroke patients (1
, 2
, 7)
. However, it has to be considered that most of the beneficial effects described in our in vivo experiments are not maintained throughout the 24 h period of permanent MCAO. Moreover, the already developed vascular hypertrophy and disturbed autoregulation in risk patients with hypertension and secondary heart failure cannot be influenced immediately by pharmacological AT1 blockade in the acute phase of ischemic stroke. Nevertheless, the detrimental effects of A II in brain ischemia as demonstrated in the present study should be recognized and controlled in time. Although the detrimental effect of A II is clearly prominent, with timely treatment the above-mentioned therapeutic interventions at the AT1 receptor level might have a significant influence on the evolution of infarct size after onset of an ischemic stroke.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 20, 2001. Accepted for publication October 18, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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