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β-Adrenoreceptor Antagonists Attenuate Brain Injury After Transient Focal Ischemia in Rats

Goyagi, Toru, MD; Kimura, Tetsu, MD; Nishikawa, Toshiaki, MD; Tobe, Yoshitsugu; Masaki, Yoko, PhD

Section Editor(s): Gelb, Adrian W.

doi: 10.1213/01.ane.0000228859.95126.69
Neurosurgical Anesthesia: Research Report
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β-adrenoreceptor antagonists experimentally reduce cardiac and renal injury after ischemia and are also clinically useful for myocardial infarction and severe burns. In addition, β-adrenoreceptor antagonists provide neuroprotective effects after focal cerebral ischemia in experimental settings. We conducted the present study to compare the neuroprotective effects of several β-adrenoreceptor antagonists in rat transient focal cerebral ischemia. Halothane-anesthetized normothermic adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion using the intraluminal suture technique confirmed by laser Doppler flowmetry. Rats received an IV infusion of saline 0.5 mL/h, propranolol 100 μg · kg−1 · min−1, carvedilol 4 μg · kg−1 · min−1, esmolol 200 μg · kg−1 · min−1, or landiolol 50 μg · kg−1 · min−1 (n = 6 in each group). Infusion was initiated 30 min before middle cerebral artery occlusion and continued for 24 h. Additional rats received esmolol 50 μg · kg−1 · min−1 or landiolol 10 μg · kg−1 · min−1 intrathecally (IT) via the cisterna magna (n = 5 in each group), according to the same experimental protocol. The neurological deficit score was evaluated at 22 h after reperfusion, and the brains were removed and stained with triphenyltetrazolium chloride for evaluation of infarct volume. Additional rats that received saline, esmolol, and landiolol IV (n = 6 in each group) were allowed to survive for 7 days followed by measurement of infarct size. Neurological deficit scores were smaller in rats treated with propranolol-IV, carvedilol-IV, esmolol-IV, landiolol-IV, esmolol-IT, and landiolol-IT compared with saline-treated rats (P < 0.05). Cortical and striatum infarct volumes were less in the rats receiving β-adrenoreceptor antagonists via either IV or IT than in saline-treated rats (P < 0.05). We conclude that β-adrenoreceptor antagonists improve neurological and histological outcomes after transient focal cerebral ischemia in rats independent of administration route.

IMPLICATIONS: We evaluated the neuroprotective effect of four different β-adrenoreceptor antagonists in the rat after transient focal cerebral ischemia. Both IV and intrathecal administration of β-adrenoreceptor antagonists improved neurological and histological outcomes.

From the Department of Anesthesia and Intensive Care Medicine, Akita University School of Medicine, Akita, Japan.

Accepted for publication May 11, 2006.

Presented, in part, at the 52nd Annual Meeting of Japanese Society of Anesthesiologists, Kobe, Japan, June 2, 2005 and 22nd International Symposium on Cerebral Blood Flow, Metabolism, and Function, June 8, Amsterdam, Netherlands.

Supported, in part, by Grant-in-Aid for Young Scientists (B) project number 15790809.

Address correspondence and reprint requests to Toru Goyagi, MD, Akita University School of Medicine, 1-1-1 Hondo, Akita-city, Akita, 010-8543, Japan. Address e-mail to tgoyagi@doc.med.akita-u.ac.jp.

Although the mechanisms of ischemic brain injury have gradually been elucidated, clinically proven pharmacologic interventions have not yet emerged. β-adrenoreceptor antagonists have shown the efficiency for cardiac and renal ischemia in experimental models (1–4), as well as cardiac protection in clinical settings (5,6). Also, d-propranolol and l-propranolol have been shown to have neuroprotective effects in experimental models (7–10). Carvedilol, an α and β-adrenoreceptor antagonist, provides neuroprotection against focal and global cerebral ischemia by antiapoptotic effects (11,12) and prevents apoptosis in myocardial ischemia (13).

On the other hand, clenbuterol, a β2-adrenoreceptor agonist, has been shown to have neuroprotective activity after transient forebrain ischemia through increasing nerve growth factor (NGF) expression (14–16). In addition, delivery of NGF prevents neuronal damage resulting from apoptosis in vitro (17,18) as well as in vivo (19). Thus, the stimulation of the β2-adrenoreceptor seems to play an important role in activating NGF against neural damage.

Esmolol and landiolol, which are selective β1-adrenoreceptor antagonists, have a short elimination half-life, are metabolized by red blood cell esterase, are hydrophilic, and are available clinically. Esmolol showed the protective effect of ischemic precondi tioning in the rabbit heart (20). In vitro, several β1-adrenoreceptor antagonists reduced Na+ influx into cortical synaptosomes by direct interaction with sodium channels (21). However, it is not clear whether β1-adrenoreceptor antagonists affect ischemic brain injury.

Therefore, we investigated whether esmolol and landiolol would have neuroprotective effect against focal cerebral ischemia, along with comparing the effects of other β-adrenoreceptor antagonists, and whether the neuroprotection by the IV and intrathecal (IT) administration of esmolol and landiolol would be equivalently effective.

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METHODS

The Animal Subjects Committee of Akita University School of Medicine approved this study. Male Sprague-Dawley rats weighing 280–340 g were used. Rats had free access to food and water before the experiment. After anesthesia was induced with 4% halothane and 60% nitrous oxide in oxygen, anesthesia was maintained with 0.5%–1.5% halothane and 60% nitrous oxide in oxygen delivered by mask. The right femoral artery was cannulated to monitor arterial blood pressure and arterial blood gases. After the femoral vein was cannulated to infuse drugs, the catheter was tunneled subcutaneously and exteriorized through a swivel sutured over the dorsal mid-thorax that allowed the rat to move freely in the cage after emergence from anesthesia. Rectal and temporalis muscle temperatures were maintained with a heating pad and lamp throughout the experiment. The perfusion in the cortical area of the middle cerebral artery (MCA) was measured with laser Doppler flowmetry (LDF) (Advance Laser Flowmeter ALF 21D; Advance, Tokyo, Japan). Transient focal cerebral ischemia was produced by modifying a previously described technique for intraluminal MCA occlusion (MCAO) (22,23). Briefly, rats were laid in the supine position with spontaneous ventilation. The right common carotid artery was exposed and separated carefully from the vagus nerve and ligated at the more proximal side through a slight right paramedian incision. The external carotid artery (ECA) was ligated at the more distal side and cut at the proximal side. The occipital artery of the ECA was coagulated. The internal carotid artery was carefully exposed and the pterygopalatine artery was ligated with a 5-0 silk suture. Ischemia was produced by advancing a 4-0 monofilament nylon suture, with its distal tip rounded by heat, into the internal carotid artery through the ECA until the LDF signal was significantly decreased. After placement, the intraluminal suture was secured with a 5-0 silk suture tied around the ECA. Reperfusion was produced by withdrawal of the intraluminal suture. Rats were excluded from the study when the LDF signal did not decrease below 40% of baseline or if restoration of the LDF signal did not occur at reperfusion. LDF measurements were averaged over 5-min periods at 5, 15, 30, 60, 90, and 120 min of MCAO and at 15 min of reperfusion.

Rats received one of the following drugs IV 30 min before MCAO: saline 0.5 mL/h, propranolol 100 μg · kg−1 · min−1 (propranolol-IV), carvedilol 4 μg · kg−1 · min−1 (carvedilol-IV), esmolol 200 μg · kg−1 · min−1 (esmolol-IV), or landiolol 50 μg · kg−1 · min−1 (landiolol-IV) (n = 6 in each group). Additional rats received esmolol 50 μg · kg−1 · min−1 (esmolol-IT), or landiolol 10 μg · kg−1 · min−1 (landiolol-IT) via the cisterna magna (n = 5 in each group) at a rate of 0.1 mL/h, according to the same experimental protocol. The catheter (PE 90) was inserted through the hole that was drilled on the posterior end of the interparietal bone to a depth of 6 mm and fixed by an adhesive, Alone Alpha® (Toa Gosei, Tokyo, Japan). Consequently, the tip of the catheter was placed in the cisterna magna. All drugs were administered continuously until 22 h after reperfusion.

Rats were neurologically evaluated using a neurological deficit score (Table 1) (24,25) at 22 h after reperfusion, and then decapitated under deep halothane anesthesia. Additional rats that received saline, esmolol, and landiolol IV (n = 6 in each group) were allowed to survive for 7 days. After neurologic evaluation each brain was harvested and sliced into 7 2-mm-thick coronal sections for staining with 1% triphenyltetrazolium chloride (TTC) in saline at 37°C for 30 min, as previously described (22,23). Infarct volume was measured by a blinded observer using digital imaging (C-4040, Olympus, Tokyo, Japan) and image analysis software (SigmaScan Pro, Systat Software, Point Richmond, CA). The infarcted area was numerically integrated across each section and over the entire ipsilateral hemisphere. Infarct volumes were measured separately in cerebral cortex and striatum as previously described (22,23).

Table 1

Table 1

Data are presented as mean ± sem. Infarction volume and physiological data were compared among treatment groups by Scheffé’s F test as a post hoc test following one-way analysis of variance. For analysis, neurological deficit scores among the groups were compared by the Kruskal-Wallis analysis followed by the Bonferroni test. A P value of <0.05 value was considered significant.

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RESULTS

No significant differences among the experimental groups were detected in physiological data (arterial blood pressure, heart rate, Paco2, Pao2, pH, temporalis muscle temperature, rectal temperature, blood glucose, and hemoglobin) at baseline, during MCAO, and at reperfusion (Table 2).

Table 2

Table 2

Neurological deficit scores were significantly lower in all treatment rats (propranolol-IV; 15.3 ± 3.7, carvedilol-IV; 13.6 ± 3.3, esmolol-IV; 12.2 ± 1.4, landiolol-IV; 10.5 ± 1.2, esmolol-IT; 3.6 ± 4.3, landiolol-IT; 10.6 ± 1.3) than in the saline-treated rats (28.0 ± 3.6) at 24 h after ischemia, whereas there were no significant differences in neurological deficit scores in all treatment rats. At 7 days after ischemia, neurological deficit scores in saline-treated rats (7.2 ± 2.1) were significantly greater versus rats treated with esmolol-IV (0 ± 0) or landiolol-IV (1.6 ± 0.7). There were no significant differences in weight change 1 day and 7 days after ischemia among groups. LDF signals showed similar changes among the treatment groups as compared with saline-treated rats (Fig. 1).

Figure 1.

Figure 1.

TTC-determined infarct volumes in the cortex at 22 h of reperfusion were significantly less in rats treated with propranolol-IV (72 ± 33 mm3), carvedilol-IV (64 ± 25 mm3), esmolol-IV (65 ± 18 mm3), landiolol-IV (44 ± 18 mm3), esmolol-IT (17 ± 6 mm3), and landiolol-IT (39 ± 30 mm3) when compared with saline-treated rats (205 ± 28 mm3; P < 0.05) (Fig. 2). Similarly, infarct volumes in the striatum were significantly smaller in rats treated with propranolol-IV (44 ± 15 mm3), carvedilol-IV (26 ± 8 mm3), esmolol-IV (22 ± 7 mm3), landiolol-IV (12 ± 6 mm3), esmolol-IT (17 ± 7 mm3), and landiolol-IT (8 ± 4 mm3) compared with saline-treated rats (82 ± 2 mm3; P < 0.05) (Fig. 2). However, there were no significant differences in infarct volume of both cortex and striatum between rats receiving IV and IT administration of esmolol and landiolol (Fig. 2). Moreover, TTC-determined infarct volumes in the cortex and the striatum at 7 days after reperfusion, were significantly smaller in rats treated with esmolol- and landiolol-IV compared with saline-treated rats (Fig. 3), whereas there were no significant differences in infarct volume between the esmolol- and landiolol-treated rats.

Figure 2.

Figure 2.

Figure 3.

Figure 3.

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DISCUSSION

In this study, we demonstrated that four different β-adrenoreceptor antagonists decreased infarct volumes and impaired neurological deficit scores after transient focal cerebral ischemia in rats. In addition, attenuation of infarct volume by the selective β1-adrenoreceptor antagonists, esmolol and landiolol, was independent of administration route. These results suggest that β-adrenoreceptor antagonists of even short duration have a neuroprotective effect after transient focal cerebral ischemia in rats.

Propranolol has been shown to provide neuroprotection in rats or cats in focal cerebral ischemia (7–10). According to previous reports, propranolol decreases oxygen consumption with minimal effect on cerebral blood flow in normocapnia (26) and inhibits platelet aggregation and serotonin release induced by adenosine diphosphate, epinephrine, collagen, and thrombin (27). Propranolol also interferes with calcium influx by reducing the population of receptor-operated channels and abolishing cyclic AMP-dependent protein kinase-mediated phosphorylation of slow inward current channels (28,29) and reduces calcium-dependent phospholipase A2 activity in platelets (30). Furthermore, carvedilol provided neuroprotection in rat transient focal cerebral ischemia by inhibiting apoptosis and attenuating the expression of tumor necrosis factor-α and interleukin-1β (12). The neuroprotective effect of propranolol and carvedilol demonstrated in the present study is consistent with those previous results, although the methods of drug administration were different. We used continuous IV administration of the treatment drugs because the two short-acting β-blockers, esmolol and landiolol, require continuous infusion to maintain effective plasma concentrations.

On the other hand, the neuroprotection by clenbuterol, a β2-adrenoreceptor agonist, has been shown in vivo and in vitro (17–19). Clenbuterol enhances NGF expression in rat brain (31,32). NGF exerts neurotrophic actions on cholinergic neurons in the basal forebrain (33,34) and protects them against axotomy-induced neurodegeneration and age-related atrophy (35). In addition, NGF ameliorates neuronal degeneration in rat cerebral cortex and hippocampus after ischemic insults (36,37) and fibroblasts producing NGF protect hippocampal neurons after ischemia (38). Therefore, stimulation of the β2-adrenoreceptor seems to play an important role in inducing the neurotrophic factor.

Propranolol is a nonselective β-adrenoreceptor antagonist, whereas carvedilol is an α- and nonselective β-adrenoreceptor antagonist that blocks β1-, β2-, and α1-receptors. In contrast, esmolol and landiolol are selective β1-adrenoreceptor antagonists. Therefore, it is speculated that the mechanisms of neuroprotection induced by the administration of propranolol and carvedilol may be attributed to factors other than NGF because propranolol was reported to inhibit the induction of NGF synthesis caused by clenbuterol. The neuroprotective effect of propranolol is thus likely to involve a decrease in oxygen consumption and inhibition of platelet aggregation and calcium influx. Likewise, several studies have reported that the neuroprotective action of carvedilol is likely to be caused by its antioxidant and antiapoptotic effects and antiinflammatory response (11,12).

In contrast, selective β1-adrenoreceptor antagonists seem to be different from other β-adrenoreceptor antagonists with respect to the mechanism of neuroprotection. Autoradiographic analysis of the distribution of β1- and β2-adrenoreceptors in the rat brain showed high levels of β1-receptors in the cingulated cortex, layers I and II of the cerebral cortex, the hippocampus, the Islands of Calleja, and the gelatinosus, mediodorsal, and ventral nuclei of the thalamus (39). High levels of β2-receptors were found in the molecular layer of the cerebellum, over pia mater, and in the central, paraventricular, and caudal lateral posterior thalamic nuclei. Approximately equal levels of β1- and β2-receptors were noted in the substantia nigra, the olfactory tubercle, layer IV of the cerebral cortex, the medial preoptic nucleus, and all nuclei of the medulla. Moreover, most of the β-adrenergic receptors in the brain are located on astrocytes (40). The larger proportion of β1-receptors than β2-receptors in the area injured by MCAO may help explain the neuroprotective effect of selective β1-adrenoreceptor antagonists. NGF induced by esmolol and landiolol may also contribute to neuroprotection. In addition, there is a report showing that β1- adrenoreceptor antagonists reduce Na+ influx into cortical synaptosomes by direct interaction with Na+ channels (21).

Both esmolol and landiolol are hydrophilic compounds, have short elimination half-lives, and are metabolized by red blood cell esterases. In the present study, therefore, we chose continuous infusion to achieve the effect concentration. Because it is difficult for hydrophilic compounds to cross the blood-brain barrier (41), we compared the effects of IT versus IV esmolol and landiolol administration. Surprisingly, the infarct volumes in the cortex and the striatum were similarly reduced by both IT and IV administration of esmolol and landiolol in this study. Because the blood-brain barrier might collapse at the early phase of ischemia and reperfusion injury (41), hydrophilic β-adrenergic antagonists might have crossed the barrier at an early phase of ischemic injury, possibly providing the neuroprotective effects.

Because we have selected and studied only one dose of each drug according to previous reports (10,12,20), the optimal dose of each drug could not be determined from this study. An equivalent effectiveness of β-adrenoreceptor antagonists was observed in this study. As described above, esmolol and landiolol are rapidly hydrolyzed by blood esterases. Hydrolysis of esmolol in human and dog blood is mediated by an esterase present in the cytosol of red blood cells (42). Because blood esmolol esterase activity is higher in rats than humans (43), the blood concentration in rats may be different from that in humans. In the present study we did not measure the blood concentration of esmolol and landiolol. Further study is needed to determine the optimal dose.

In conclusion, we demonstrated neuroprotective effects of four different β-adrenoreceptor antagonists after focal cerebral ischemia in rats. All the β-adrenoreceptor antagonists—propranolol, carvedilol, esmolol, and landiolol—decreased the infarct volumes of cortex and striatum and improved neurological deficit scores. The effects of hydrophilic β-adrenoreceptor antagonists, esmolol and landiolol, were similar regardless of route of administration. Further study is needed to clarify the neuroprotective mechanism of esmolol and landiolol.

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