Selective Blockade of Endothelin-B Receptors Exacerbates Ischemic Brain Damage in the Rat
Background and Purpose— Endothelins act through 2 receptors, namely, ETA and ETB. In the cerebral circulation, ETA mediates marked and prolonged vasoconstriction, and its blockade increases cerebral blood flow (CBF) and reduces ischemic brain damage. However, the role of ETB receptors remains unclear. In this study we examined, in rats, the kinetics of expression of ETB and the effects of ETB blockade on changes in CBF and brain damage after focal cerebral ischemia and N-methyl-d-aspartate (NMDA)–induced excitotoxic injury.
Methods— Rats were subjected to transient (60 minutes) focal cerebral ischemia or cortical injection of NMDA. The selective ETB antagonist BQ-788 was injected intracerebroventricularly 30 minutes before and 30 minutes after the onset of ischemia. Cortical perfusion was monitored by laser-Doppler flowmetry. The volume of infarction or NMDA-induced cortical lesion was assessed at 24 hours after the insult. The reverse transcription–polymerase chain reaction technique was used to assess ETB expression.
Results— Cerebral ischemia failed to alter the expression of ETB mRNA in both acute and chronic stages. Administration of BQ-788 resulted in an increase in infarction volume (178%; P<0.05) accompanied by a decrease in residual CBF (−26.7% versus control; P<0.01). In these animals we found a positive correlation between the volume of infarction and the severity of the decrease in CBF. NMDA-induced cortical lesions were not affected by the administration of BQ-788.
Conclusions— Our results suggest that the ETB antagonist BQ-788 induces deleterious effects that are mediated by the reduction of residual blood flow after ischemia and argue that the optimal therapeutic strategy in stroke would be to target the use of selective ETA antagonists and not mixed ETA/ETB antagonists.
Endothelins are a family of 3 isopeptides, termed endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3), recognized for their potent vasomotor activity.1,2⇓ Two distinct types of endothelin receptors have been identified: ETA and ETB. The ETA receptor is characterized by a higher affinity toward ET-1 than ET-2 and ET-3, while the ETB receptor displays a similar affinity toward all 3 endothelins.3,4⇓ Overall, the elements of the endothelin system (ET-1, ET-3, ETA, ETB, and endothelin-converting enzymes), but not ET-2, are expressed in both vascular and nonvascular compartments of the central nervous system.5,6⇓ In the cerebral circulation, ETA receptors mediate a potent and long-lasting vasoconstriction;7 however, the function of ETB receptors is more controversial. As shown in many studies, the selective activation of ETB receptors induces vasodilatation mediated by the generation of the endothelium-derived relaxing factor (nitric oxide and/or prostacyclin).8–10⇓⇓ Under some conditions ETB can also induce vasoconstriction.10–12⇓⇓ This dual role of ETB receptor, which is well described in some peripheral vessels in terms of pharmacological approaches, has led to a proposed subdivision of ETB receptors into ETB1 receptors (on endothelial cells) that mediate vasodilatation and ETB2 receptors (on smooth muscle cells) that mediate vasoconstriction.4,13,14⇓⇓ However, no molecular correlates of these ETB receptor subtypes have been reported.15 The demonstration of constrictive ETA along with dilatory and/or constrictive ETB receptors has important implications for the development of endothelin receptor antagonists with an optimal pharmacological profile (selective ETA or mixed ETA/ETB) for the treatment of cerebrovascular diseases such as cerebral ischemia.16
Three lines of evidence point to a major role of ET-1 in the pathophysiology of cerebral ischemia: (1) the ability of exogenous ET-1 to reduce cerebral blood flow (CBF) to levels that induce infarction17; (2) the increase in plasma, cerebrospinal fluid, and cerebral tissue ET-1 levels after cerebral ischemia18,19⇓; and (3) the efficacy of selective ETA or mixed ETA/ETB antagonists in some, but not all, studies to reduce brain damage after experimental stroke.20–22⇓⇓ The precise mechanisms of ET-1 actions in cerebral ischemia have not yet been fully elucidated. The majority of studies that have intervened with endothelin receptor antagonists after experimental stroke have focused on the role of ETA receptors as mediators of brain damage. In contrast, the role of ETB receptors in ischemic pathology is still to be elucidated.
In the present study we focused on ETB receptors and addressed 3 questions: (1) the kinetics of ETB mRNA expression after cerebral ischemia; (2) the effects of selective ETB blockade on CBF changes and brain damage after ischemia; and (3) the effects of ETB blockade on N-methyl-d-aspartate (NMDA)–induced excitotoxic brain damage.
Materials and Methods
Anesthesia and General Preparation
The experiments were performed on adult male Sprague-Dawley rats weighing 347±9 g (R. Janvier Breeding Center) according to the appropriate European Directives and French National Legislation. Anesthesia was induced with halothane (5%) and maintained during surgery (0.7% to 1.3%) in an O2/N2O mixture (30%/70%). The animals were orally intubated and mechanically ventilated (Harvard Apparatus 683). Rectal temperature was recorded and kept close to 37.5°C with a heating pad (Thermalert). The caudal artery was cannulated with polyethylene tubing for continuous arterial pressure monitoring (Stoelting) as well as for gas analyses and pH (Ciba Corning).
Rats were assigned to the following experimental groups: (1) kinetics of expression of ETB mRNA after cerebral ischemia; (2) effects of BQ-788 on changes in CBF and brain damage after focal cerebral ischemia; and (3) effects of BQ-788 on cortical lesions induced by NMDA administration.
Middle Cerebral Artery Occlusion
Temporary focal cerebral ischemia was induced by occlusion of the right middle cerebral artery (MCAO) with the use of the intraluminal filament technique.23 Briefly, a nylon thread (0.18 mm in diameter) with a distal cylinder (3 mm in length and 0.38 mm in diameter) was inserted into the lumen of the external carotid artery and advanced to the origin of the MCA. The nylon thread was removed 60 minutes later to allow reperfusion. For sham-operated animals, the nylon thread was advanced to the origin of the MCA and immediately removed. The animals were then allowed to recover from anesthesia and were killed 24 hours after MCAO for histological analysis or at different time points for reverse transcription–polymerase chain reaction (RT-PCR) analyses.
Measurement of CBF
To examine the effects of the ETB antagonist (BQ-788) on cortical brain perfusion, rats were placed prone in a stereotaxic frame (Kopf Instruments). A laser-Doppler flowmetry (LDF) probe (0.7 mm in diameter, FloLab Moor Instruments) was positioned on the right parietal bone (coordinates 1.7±0.1 mm posterior, 5.5±0.1 mm lateral to the bregma)24 thinned with a saline-cooled dental drill. CBF data were collected continuously before the occlusion up to 15 minutes after reperfusion and expressed as percentage of the mean of 10-minute preocclusion values.
NMDA-Induced Excitotoxic Damage
In halothane-anesthetized rats, excitotoxic lesions were induced by a microinjection (2 μL) into the right parietal cortex (coordinates 5 mm lateral, 3.4 mm ventral to the bregma)24 of NMDA (50 nmol) at a rate of 1 μL/min. The lesion volume was quantified 24 hours after the injection of NMDA.
Drug Characteristics and Administration
BQ-788 is a potent selective and competitive ETB receptor antagonist with an approximately 1000-fold relative selectivity for the ETB receptor.25 BQ-788 was purchased as a pure powder and dissolved in sterile dimethyl sulfoxide (DMSO) following the recommendations of the supplier (France-Biochem). The peptide nature of BQ-788 precluded its intravenous administration. BQ-788 or its vehicle was infused over 5 minutes into the right lateral ventricle (3 μL) (coordinates 0.8 mm posterior, 1.5 mm lateral, 4.3 mm ventral to the bregma)24 30 minutes before and 30 minutes after the onset of MCAO or cortical administration of NMDA. NMDA was purchased as pure powder (Sigma-Aldrich) and dissolved in PBS buffer.
Reverse Transcription–Polymerase Chain Reaction
Brain tissue was collected 3 hours, 6 hours, 24 hours, or 3 days after transient MCAO. Total RNAs were isolated from the ipsilateral or contralateral hemispheres through the use of the RNaxel kit (Eurobio). Two micrograms of total RNAs was reverse transcripted into cDNA with the use of poly(dT) oligonucleotides. ETB receptor oligonucleotide probes (sense: 5′-TTCACCTCAGCAGGATTCTG-3′; antisense: 5′-AGGTGTGGAAAGTTAGAACG-3′) were synthesized from the published probe sequence of Wang and colleagues,26 who verified their specificity by sequencing the PCR product. The probes of β-actin (as housekeeping gene) (sense: 5′-GTGGGCCGCTCTAGGCACAA-3′; antisense: 5′-CTCTTTGATGTCACGCACGATTTC-3′) were synthesized according to the published sequence of Ali and colleagues.27 Amplification conditions with the use of a thermocycler (Eppendorf 53.32) were the following: 95°C (40 seconds), 55°C (40 seconds), and 72°C (1 minute). The number of cycles was 27 for β-actin and 35 for the ETB receptor. Amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide.
Measurement of Infarct Volume
Under deep anesthesia, 24 hours after the insult, rats were killed, and the brains were removed rapidly and frozen in cooled isopentane at −65°C for 15 seconds. Whole brains were cut in 20-μm-thick sections with a cryostat (Leica CM3050), and 1 section in every 40 was collected on a glass slide and stained by thionin. The infarcted area was quantified by image analysis (Scion Image, See Scan). Infarction volume, corrected for edema as described previously,28 was calculated by the integration of infarct areas over 16 equidistant brain slices that encompassed the whole lesion.
Data and Statistical Analysis
The results are presented as mean±SEM. Statistical analyses were performed with ANOVA followed by the Bonferroni-Dunn test as indicated. P<0.05 was accepted as significant.
ETB mRNA Expression After MCAO
The expression of ETB mRNA receptors was examined through the use of the RT-PCR technique at 3 hours, 6 hours, 24 hours, and 3 days after 1-hour temporary ischemia (3 rats at each time point were used). We verified the presence of ischemia-induced brain damage using the neurological test of Bederson and coworkers29 in rats examined at 3 and 6 hours and using histology for rats examined at 24 hours and 3 days. For each experiment we obtained a single PCR product of the expected size (475 bp for ETB and 539 bp for β-actin). The expression of ETB receptor mRNA in the ipsilateral hemisphere, at the time points analyzed, showed no change after MCAO compared with that observed in the respective contralateral hemisphere and with sham-operated animals. The housekeeping gene β-actin also showed no change over time (Figure 1). Similarly, the lesion induced by NMDA injection did not result in any changes in ETB mRNA expression as examined at 1, 12, and 24 hours after the injection (data not shown).
Effects of BQ-788 on CBF and Brain Damage
In pilot experiments, we tested the effect of 2 doses of BQ-788 (4 and 40 μg) administered into the lateral ventricle, 30 minutes before and 30 minutes after MCAO, on brain damage. The volumes of hemispheric infarction were 78.5±13.8, 141.4±28.3, and 156.6±20.3 mm3 in rats treated with vehicle (n=8), BQ-788 at a dose of 4 μg (n=9), and BQ-788 at a dose of 40 μg (n=9), respectively. The ETB antagonist elicited a statistically significant increase in infarction volume (P=0.029) only when a dose of 40 μg was used (P=0.076 for the dose of 4 μg). We subsequently used the dose 40 μg in other groups of animals in which physiological parameters as well as CBF were monitored.
Physiological parameters (mean arterial pressure, Paco2, Pao2, pH, and body temperature) were not different in BQ-788–treated animals (n=8) compared with those treated with vehicle (n=7) (Table). In these animals and as found in the pilot experiments, BQ-788 injected into the lateral ventricle increased dramatically the volume of infarction, essentially in the cortex, compared with vehicle (178%; P=0.027) (Figure 2A and 2B). BQ-788 also increased the volume of brain edema, although statistical significance was not reached (P=0.14) (Figure 2C). BQ-788 per se administered into the ventricle failed to induce any discernible lesion in nonischemic animals.
The analysis of LDF data showed that BQ-788 did not affect the basal CBF during the 30-minute period before MCAO (P=0.2) (Figure 3A). MCAO induced a similar initial decrease in CBF in both BQ-788– and vehicle-treated rats (Figure 3A). The CBF decrease remained stable during the whole period of ischemia in vehicle-treated animals. However, a further decrease in CBF in animals treated with BQ-788 was observed in the second 30-minute period of MCAO (Figure 3A). The comparison of CBF values measured during the second 30-minute period of ischemia showed a statistically significant difference between BQ-788– and vehicle-treated animals (−26.7%; P=0.006, ANOVA followed by Bonferroni-Dunn test) (Figure 3B). After reperfusion, hyperemia was observed, and CBF returned to preocclusion values after 15 minutes in a similar manner in both groups (P=0.15) with, however, high interindividual variability (Figure 3B). In addition, there was a significant correlation (P<0.001) between the volume of infarction and the change in residual CBF during MCAO (Figure 4).
Effects of BQ-788 on NMDA-Induced Excitotoxic Brain Damage
The glutamatergic agonist NMDA (50 nmol) produced a well-defined cortical lesion 24 hours after injection (Figure 5B). Administration of BQ-788 (40 μg) into the lateral ventricle 30 minutes before and 30 minutes after NMDA injection failed to change the volume of cortical damage (39.7±7.5 mm3 for NMDA+vehicle [n=6] and 37.3±7.6 mm3 for NMDA+BQ-788 [n=6]) (Figure 5A). Physiological parameters (mean arterial pressure, Paco2, Pao2, pH, body temperature, and heart rate) were not different in BQ-788–treated animals compared with those treated with vehicle (Table).
Three novel findings arise from the present study. First, cerebral ischemia failed to modify the expression of ETB receptor mRNA examined in both the acute and chronic stages of ischemia. Second, selective ETB receptor blockade dramatically exacerbated brain damage and reduced cortical perfusion after ischemia. Third, NMDA-induced excitotoxic damage was not affected by ETB receptor blockade.
Because of the potent vasoconstrictive actions of ET-1 and its upregulation after stroke, this peptide has been implicated in the pathogenesis of ischemic brain lesions.10,16–21⇓⇓⇓⇓⇓⇓ The definitive mechanisms of action of endothelin, however, are not yet fully established. Selective ETA or mixed ETA/ETB antagonists have been shown in some, but not all, studies to ameliorate CBF and neurological outcome and to reduce infarction volume in models of global and focal cerebral ischemia in rats and cats.20–22⇓⇓ One of the key issues is whether selective ETA or mixed ETA/ETB antagonists are likely to be effective in treating stroke.30
In the cerebral circulation, it is well established that ETA mediates a potent and long-lasting constriction17,31⇓; however, the role of ETB receptor, in both physiological and pathophysiological conditions, remains not fully elucidated. Although the blockade of ETB receptors, under physiological conditions, does not change the diameter of cerebral arterioles,10 the majority of studies have shown that selective activation of ETB receptors mediates vasodilatation.8–10,32⇓⇓⇓ Nonetheless, in some reports, cerebrovascular ETB receptor has been linked to the contractile properties of endothelins because those are more widely observed in the systemic circulation.33–37⇓⇓⇓⇓ In the present work we focused specifically on the roles of ETB receptors in focal cerebral ischemia in the rat.
The analysis of the kinetics of expression of ETB receptors mRNA in the ipsilateral hemisphere showed no change through the acute and chronic stages of ischemia. In each rat, the presence of ischemia-induced lesion was verified by neurological tests29 for acute time points (3 and 6 hours) and by histological analysis for chronic time points (24 hours and 3 days). The absence of changes in expression observed through the acute and chronic stages may be due to the heterogeneity of brain tissue used for this analysis, which probably encompasses regions with different degrees of ischemia (ie, severely ischemic tissue, penumbral tissue, and healthy tissue). However, we have previously observed upregulation of some elements of the endothelin system, such as ET-1 and ETA receptors, in the same rats using the same technique (ie, RT-PCR) as in the present study.38 In global cerebral ischemia, Yamashita and coworkers39 reported an increase in ETB receptor binding in microglia in the hippocampal pyramidal cell field 7 days after global ischemia. The authors suggested that ET-1 released from astrocytes, in response to ischemia, would activate microglia with ETB receptors to participate in neuronal death in the hippocampus. Moreover, an increase in ETB receptor agonist binding sites and ETB mRNA receptors after experimental subarachnoid hemorrhage has been shown.40,41⇓ Our findings and the aforementioned results show that ETB receptors are present in vascular and nonvascular brain tissue after cerebrovascular insults. However, the functionality and the significance of these receptors cannot be ascertained from these descriptive data.
The present study provides the first definitive evidence that blockade of ETB receptor enhances ischemic brain damage. Our data demonstrate that intracerebroventricular administration of the well-characterized ETB antagonist BQ-788 remarkably increased the volume of infarction (178%) as well as the volume of edema (200%) and significantly decreased the residual cortical CBF (−26.7%). BQ-788 per se is not toxic in the normal brain.20
Two potential mechanisms may explain the ETB blockade–induced exacerbation of ischemic brain lesion. First, given the direct vasomotor actions mediated by ETB receptor, one would assume that BQ-788 counteracts the relaxing actions of ETB during ischemia. In this case, the overall effects of postischemic endogenous ET-1 would be the result of a balance between ETA-mediated vasoconstriction and ETB-mediated vasodilatation. Moreover, it has been reported in many peripheral tissues, eg, heart and lung, that ETB receptor can act as a clearance receptor for ET-1.42–44⇓⇓ If this occurs in the brain, where ETB receptors are widely expressed, mainly in astrocytes, treatment with BQ-788 would increase the availability of the postischemic released ET-1 for ETA receptors. This would also result in enhancement of vasoconstriction.
Our data support these ETB-mediated vascular effects since the blockade of this receptor resulted in an exacerbation of MCAO-induced hypoperfusion. In addition, there was a good correlation between the volume of infarction and changes in residual CBF. The available data indicate that the fate of the penumbral tissue, characterized by a relatively mild hypoperfusion, could be determined by subtle changes in CBF.45 Moreover, the failure of BQ-788 to modify the lesion size in NMDA-induced brain injury, a model in which excitotoxicity is the main factor that induces cell death and in which vascular mechanisms are not predominant,46 reinforces the idea that ETB antagonism likely enhances ischemic damage through its action on the cerebral circulation.
Second, BQ-788 could act on nonvascular tissue to enhance deleterious events in ischemic tissue. The mammalian brain predominantly contains the ETB receptor subtype expressed mainly on endothelial and glial cells.47–49⇓⇓ This widespread nonvascular distribution of ETB receptors suggests a role for ET receptors in nonvascular functions. It has been suggested that endothelins act, through ETB, as growth factors for astrocytes, regulating biological processes such as proliferation during brain development or injury.6,50–52⇓⇓⇓ Ho and coworkers53 demonstrated that astrocytes without ET-1 are more vulnerable to hypoxic/ischemic injuries and that upregulation of astrocytic ET-1 is essential for the survival of astrocytes. The roles of ETB in nonvascular brain tissue after experimental ischemia would be best addressed in brain cellular cultures devoid of vasculature.
Whatever the mechanisms underlying BQ-788–mediated effects in cerebral ischemia, our data suggest that the optimal therapeutic strategy in stroke targeting endothelins would be the use of selective ETA antagonists and not mixed ETA/ETB antagonists.
This work was supported by the CNRS, University of Caen, and the Conseil Régional de Basse-Normandie.
- Received March 27, 2002.
- Revision received July 3, 2002.
- Accepted July 17, 2002.
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- ↵Seo B, Oemar BS, Siebenmann R, von Segesser L, Luscher TF. Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels. Circulation. 1994; 89: 1203–1208.
- ↵Chuquet J, Ali C, Vivien D, MacKenzie ET, Roussel S, Touzani O. Expression of ET-1, ET-3, ECE-1a, ET-A, and ET-B mRNA after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab. 2001; 21: S435.Abstract.
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