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.
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).
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.
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).
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).
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.
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.
1. Pinelli A, Trivulzio S, Tomasoni L, et al. Isoproterenol-induced myocardial infarction in rabbits. Protection by propranolol or labetalol: a proposed non-invasive procedure. Eur J Pharm Sci 2004;23:277–85.
2. Solez K, Freshwater MF, Su CT. The effect of propranolol on postischemic acute renal failure in the rat. Transplantation 1977;24:148–51.
3. Stowe N, Emma J, Magnusson M, et al. Protective effect of propranolol in the treatment of ischemically damaged canine kidneys prior to transplantation. Surgery 1978;84:265–70.
4. Yao H, Sadoshima S, Shiokawa O, et al. Renal blood flow in acute cerebral ischemia in spontaneously hypertensive rats: effects of alpha- and beta-adrenergic blockade. Stroke 1987;18:629–33.
5. Kloner RA, Rezkalla SH. Cardiac protection during acute myocardial infarction: where do we stand in 2004? J Am Coll Cardiol 2004;44:276–86.
6. Stevens RD, Burri H, Tramer MR. Pharmacologic myocardial protection in patients undergoing noncardiac surgery: a quantitative systematic review. Anesth Analg 2003;97:623–33.
7. Standefer M, Little JR. Improved neurological outcome in experimental focal cerebral ischemia treated with propranolol. Neurosurgery 1986;18:136–40.
8. Latchaw JP, Little JR, Slugg RM, et al. Treatment of acute focal cerebral ischemia and recirculation with d-propranolol. Neurosurgery 1985;16:18–22.
9. Capraro JA, Reedy DP, Latchaw JP, et al. Treatment of acute focal cerebral ischemia with propranolol. Stroke 1984;15:486–91.
10. Little JR, Latchaw JP Jr, Slugg RM, et al. Treatment of acute focal cerebral ischemia with propranolol. Stroke 1982;13:302–7.
11. Rabasseda X. Carvedilol: an effective antihypertensive drug with antiischemic/antioxidant cardioprotective properties. Drugs Today 1998;34:905–26.
12. Savitz SI, Erhardt JA, Anthony JV, et al. The novel beta-blocker, carvedilol, provides neuroprotection in transient focal stroke. J Cereb Blood Flow Metab 2000;20:1197–204.
13. Oliveira PJ, Goncalves L, Monteiro P, et al. Are the antioxidant properties of carvedilol important for the protection of cardiac mitochondria? Curr Vasc Pharmacol 2005;3:147–58.
14. Semkova I, Krieglstein J. Neuroprotection mediated via neurotrophic factors and induction of neurotrophic factors. Brain Res Rev 1999;30:176–88.
15. Culmsee C, Junker V, Kremers W, et al. Combination therapy in ischemic stroke: synergistic neuroprotective effects of memantine and clenbuterol. Stroke 2004;35:1197–202.
16. Junker V, Becker A, Huhne R, et al. Stimulation of beta- adrenoceptors activates astrocytes and provides neuroprotection. Eur J Pharmacol 2002;446:25–36.
17. Ordway GA, Gambarana C, Frazer A. Quantitative autoradiography of central beta adrenoceptor subtypes: comparison of the effects of chronic treatment with desipramine or centrally administered l-isoproterenol. J Pharmacol Exp Ther 1988; 247:379–89.
18. Semkova I, Schilling M, Henrich-Noack P, et al. Clenbuterol protects mouse cerebral cortex and rat hippocampus from ischemic damage and attenuates glutamate neurotoxicity in cultured hippocampal neurons by induction of NGF. Brain Res 1996;717:44–54.
19. Tamura A, Graham DJ, McCulloch J, Teasdale GM. Focal cerebral ischemia in the rat: regional cerebral blood flow determined by [14
C]iodoantipyrine autoradiography following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1981;1:61–8.
20. Iliodromitis EK, Tasouli A, Andreadou I, et al. Intravenous atenolol and esmolol maintain the protective effect of ischemic preconditioning in vivo
. Eur J Pharmacol 2004;499:163–9.
21. Chidlow G, Melena J, Osborne NN. Betaxolol, a β1
-adrenoceptor antagonist, reduces Na(+) influx into cortical synaptosomes by direct interaction with Na(+) channels: comparison with other β-adrenoceptor antagonists. Br J Pharmacol 2000;130:759–66.
22. Goyagi T, Goto S, Bhardwaj A, et al. Neuroprotective effect of sigma(1)-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) is linked to reduced neuronal nitric oxide production. Stroke 2001;32:1613–20.
23. Goyagi T, Toung TJ, Kirsch JR, et al. Neuroprotective kappa-opioid receptor agonist BRL 52537 attenuates ischemia-evoked nitric oxide production in vivo
in rats. Stroke 2003;34:1533–8.
24. Maier CM, Sun GH, Kunis D, et al. Delayed induction and long-term effects of mild hypothermia in a focal model of transient cerebral ischemia. J Neurosurg 2001;94:90–6.
25. Baker AJ, Zornow MH, Grafe MR, et al. Hypothermia prevents ischemia-induced increases in hippocampal glycine concentrations in rabbits. Stroke 1991;22:666–73.
26. Mackenzie ET, McCulloch J, Harper AM. Influence of endogenous norepinephrine on cerebral blood flow and metabolism. Am J Physiol 1976;231:483–8.
27. Weksler BB, Gillick M, Pink J. Effects of propranolol on platelet function. Blood 1977;49:185–96.
28. Braunwald E. Mechanism of action of calcium channel blocking agents. N Engl J Med 1982;307:1618–27.
29. Lindemann JP, Bailev JC, Watanabe AM. Potential biochemical mechanisms for regulation of the slow inward current: Theoretical basis for drug action. Am Heart J 1982;103:746–56.
30. Van der hoek JY, Feinstein MB. Local anesthetics, chlorpromazine and propranolol inhibit stimulus activation of phospholipase A2
in human platelets. Mol Pharmacol 1978;16:171–80.
31. Follesa P, Mocchetti I. Regulation of basic fibroblast growth factor and nerve growth factor mRNA by β-adrenergic receptor activation and adrenal steroids in rat central nervous system. Mol Pharmacol 1992;43:132–8.
32. Hayes VY, Isackson PJ, Fabrazzo M, et al. Induction of nerve growth factor and basic fibroblast growth factor mRNA following clenbuterol: contrasting anatomical and cellular localization. Exp Neurol 1995;132:33–41.
33. Hartikka J, Hefti F. Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes. J Neurosci 1988;8:2967–85.
34. Whittemore SR, Seiger A. The expression, localization and functional significance of β-nerve growth factor in the central nervous system. Brain Res Rev 1987;12:439–46.
35. Hefti F, Dravid A, Hartikka J. Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions. Brain Res 1984;293:305–11.
36. Buchan AM, Williams L, Bruederlin B. Nerve growth factor: pretreatment ameliorates ischemic hippocampal neuronal injury. Stroke 1990;21:177–182.
37. Shigeno T, Mima T, Takaura K, et al. Amelioration of delayed neuronal death in the hippocampus by nerve growth factor. J Neurosci 1991;11:2914–9.
38. Pechan PA, Yoshida T, Panahian N, et al. Genetically modified fibroblasts producing NGF protect hippocampal neurons after ischemia in the rat. NeuroReport 1995;6:669–72.
39. Rainbow TC, Parsons B, Wolfe BB. Quantitative autoradiography of b1
- and b2
- adrenergic receptors in rat brain. Proc Natl Acad Sci U S A 1984;81:1585–9.
40. Shao Y, Sutin J. Noradrenergic facilitation of motor neurons: localization of adrenergic receptors in neurons and nonneuronal cells in the trigeminal motor nucleus. Exp Neurol 1991;114:216–27.
41. Gasche Y, Fujimura M, Morita-Fujimura Y, et al. Early appearance of activated matrix metalloproteinase-9 after focal cerebral ischemia in mice: a possible role in blood-brain barrier dysfunction. J Cereb Blood Flow Metab 1999;19:1020–8.
42. Quon CY, Stampfli HF. Biochemical properties of blood esmolol esterase. Drug Metab Dispos 1985;13:420–4.
© 2006 International Anesthesia Research Society
43. Quon CY, Mai K, Patil G, Stampfli HF. Species differences in the stereoselective hydrolysis of esmolol by blood esterases. Drug Metab Dispos 1988;16:425–8.