Propranolol is a nonselective β-adrenoreceptor antagonist that shows neuroprotection against focal cerebral ischemia.1–4 Goyagi et al.5 showed that esmolol and landiolol, which are selective β1-adrenoreceptor antagonists, also have this property in rats. β2-adrenoreceptor agonism plays an important role in neuroprotection through activation of neuron growth factor,6,7 and the results for esmolol and landiolol show that β1-adrenoreceptor blockade is also associated with neuroprotection against focal cerebral ischemia. Therefore, blockade of β1-adrenoreceptors, rather than β2-adrenoreceptors, seems to be required for neuroprotection against focal cerebral ischemia.
The effects of neuroprotective drugs differ in different types of experimental brain ischemia, probably because the pathophysiological responses to focal stroke and global ischemia are inherently different.8–10 However, the effects of β-adrenoreceptor antagonists on experimental global cerebral ischemia are unknown; that is, the efficacy of these antagonists in protection of vulnerable brain regions after ischemic insult remains to be determined. Therefore, this study was conducted to investigate whether IV administration of propranolol, esmolol, and landiolol has a neuroprotective effect against forebrain ischemia in rats, and to determine the dependence of this effect on the time of administration with respect to the onset of ischemia. Thus, the effects of these β-antagonists on outcome after cerebral ischemia were assessed using administration before ischemia or after cerebral reperfusion.
All experimental protocols were approved by the Animal Care and Use Committee of Nara Medical University. Male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan) weighing 270 to 330 g were fasted for 12 hours. Anesthesia was induced with 5% isoflurane with oxygen, and the trachea was intubated. The isoflurane concentration was then reduced to 1.5% to 2.0%. Lungs were ventilated mechanically with a mixture of oxygen and air at a fraction of inspired oxygen of 0.3. A needle thermometer was inserted between the temporal muscle and the skull, and the pericranial temperature was maintained at 37.5°C ± 0.5°C by surface heating or cooling. Needle electroencephalographic electrodes were inserted in a biparietal configuration, and the electroencephalogram was monitored continuously (EEG-4217, Nihon Kohden, Tokyo, Japan). A cannula was inserted into the tail artery using PE-50 tubing, and the mean arterial blood pressure (MAP) was monitored continuously. A second cannula for withdrawal of blood was inserted into the extrajugular vein using PE-60 tubing.
Implantation of a Microinfusion Pump
Test drugs were diluted in physiological saline and administered systemically through the right femoral vein using an osmotic pump (Alzet model 2ML1, Durect Corp., Cupertino, CA). Each osmotic pump was preincubated in physiological saline at 37°C according to the instructions for use of the Alzet pump. After the femoral vein was cannulated for infusion of drugs, the catheter was tunneled subcutaneously in the back and the connecting tube and osmotic pump were placed subcutaneously in the dorsal area. The infusion rate was set at 10 μL · h−1 for each dose, with physiological saline infused as a control. Because the procedure took <20 minutes, implantation of the microinfusion pump was performed before or after ischemia according to the experimental protocols (see below). Continuous infusion of the drugs was used because this procedure is required to maintain effective concentrations of the 2 short-acting β-blockers, esmolol and landiolol.
Induction of Forebrain Ischemia Using Bilateral Carotid Artery Occlusion
With the animal in the supine position, the anesthetic concentration was changed to 1.5% isoflurane and a period of 10 to 15 minutes was allowed for equilibration of the anesthetic level. Blood samples were obtained for determination of preischemic values of hematocrit (Hct), glucose, pH, PaCO2, and PaO2. Ventilation was adjusted to achieve PCO2 of 35 to 40 mm Hg and PaO2 of ≥100 mm Hg. MAP and heart rate were monitored throughout the experiment. Heparin (100 U · kg−1) was administered IV, and 5 minutes later, hypotension was induced by withdrawal of blood from the superior vena cava catheter into a prewarmed syringe. Once the MAP decreased to 35 mm Hg, both carotid arteries were occluded with vascular clamps for a period of 8 minutes. Ischemia was confirmed by observation of an isoelectric electroencephalogram. During the occlusion period, MAP was maintained at 35 mm Hg by withdrawal or reinfusion of blood. After the period of ischemia, reperfusion of the brain was established by removal of the vascular clamps and reinfusion of the withdrawn blood. The vascular catheters were then removed, and the wounds were closed. Isoflurane was discontinued when the wound closure was completed. The effect of heparin was reversed with intraperitoneal administration of protamine (0.3 mg). Ventilation was continued with 100% O2, and the animals were allowed to recover from anesthesia. A rectal probe was used for intermittent temperature monitoring in the first 3 hours postoperatively, and the rectal temperature was recorded every hour and maintained at 37.5 °C ± 1.0°C by surface heating or cooling, or management of the temperature of the recovery chamber. Once spontaneous ventilation resumed, which usually occurred immediately after discontinuation of isoflurane, the animal was transferred to a prewarmed oxygen-rich humidified recovery chamber. The trachea was extubated after observation of spontaneous movement.
Motor function tests were performed on the fifth postoperative day.11 Briefly, the rats were placed on a 30 × 30 cm screen (grid size 1.0 × 1.0 cm) that could be rotated from 0° (horizontal) to 90° (vertical). The animal was placed on the horizontally positioned screen, and the screen was then rotated into the vertical plane. The duration of time for which the animal was able to hold onto the vertical screen was recorded up to a maximum of 15 seconds (a total score of 3 points). Next, the animal was placed at the center of a horizontal wooden rod (2.5-cm diameter) and the time that the animal was able to remain balanced on the rod was recorded up to a maximum of 30 seconds (a total score of 3 points). Finally, a prehensile traction test was administered, in which the time that the animal was able to cling to a horizontal rope was recorded up to a maximum of 5 seconds (cannot = 1, failed midway = 2, and complete = 3). The total motor score (up to 9 points) was determined from these 3 tests.
Harvesting and Analysis of Brain Tissue
After evaluation of weight loss compared with the preischemic state, the animal was reanesthetized using 2.5%to 3% isoflurane through a mask, and sodium thiopental (2–3 mg · kg−1) was then injected intraperitoneally. For histological experiments, after confirmation of loss of consciousness and the absence of a pain response, transcardial perfusion was performed with 150 mL saline at a rate of 40 mL · min−1, followed by 200 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 20 mL · min−1. The brain was removed and stored in 4% paraformaldehyde in 0.1 M phosphate buffer for later dissection. The brains were prepared for histological analysis by dehydration in graded concentrations of ethanol and butanol, followed by embedding in paraffin. Coronal sections (5 μm) were prepared and stained with hematoxylin and eosin. An investigator blinded to the group assignment evaluated the CA1 of the hippocampus in a coronal plane 3300 μm posterior to the bregma. These regions were determined on each slice by referring to the rat brain atlas of Palkovits and Brownstein.12 Viable and nonviable neurons were counted manually, and the percentage of nonviable neurons was calculated for quantification of neuronal damage in a counting frame (0.5 × 0.5 mm at a magnification ×200). To standardize placement of the counting frame across animals, the middle zone of each field of the hippocampus was targeted for assessment of the neuronal damage. Nonviable neurons were identified by cytoplasmic eosinophilia with loss of Nissl substance and by the presence of pyknotic homogeneous nuclei. By convention, values from the hemisphere with the worst damage were used for the final analysis.
Rats received saline 10 μL · h−1, propranolol 100 μg · kg−1 · min−1, esmolol 200 μg · kg−1 · min−1, or landiolol 50 μg · kg−1 · min−1 IV 30 minutes before bilateral carotid artery occlusion (BCAO) (experiment 1) or 60 minutes after BCAO (experiment 2). All drugs were administered continuously until 5 days after reperfusion. Each group in each experiment included 13 to 14 rats.
Physiological data and cell counts were compared among groups by analysis of variance (ANOVA) or ANOVA for repeated measures. If the ANOVA identified significant differences, an unpaired t test with a Bonferroni correction was used for intergroup comparison. Comparisons of motor activity among groups were performed using the Kruskal-Wallis test. Mortality was compared using a χ2 test or Fisher exact test. All data except for the motor activity score and mortality rate are shown as means ± SD.
Several rats died during the 5-day recovery period. Some animals died in the early recovery period because of apparent upper airway obstruction or seizure. The causes of other deaths were not identified because these deaths were not witnessed.
Of 55 animals used in experiment 1, 22 died after ischemia. The number of deaths and the mortality rate in each group in experiment 1 are shown in Table 1. The mortality in the propranolol group was 64%, but the rates did not differ significantly among the groups. Physiological values for PaCO2, PaO2, and pericranial and rectal temperatures were maintained around the target values described in the Methods section, and preischemic physiological variables including Hct, glucose, MAP, and heart rate were also similar among the groups (Table 2). Changes in MAP and heart rate (Fig. 1) did not differ among the groups, weight loss after the recovery period of 5 days was insignificant in all groups, and rectal temperature before death was similar among the groups (data not shown). The percentages of injured neurons in the CA1 sector after ischemia identified histologically are shown in Figure 2. These data also showed no significant differences among the groups. For motor activity, the propranolol group tended to have a worse score, but again, there was no significant difference between this score and those for other groups (Fig. 3).
Of 53 animals used in experiment 2, 16 died after ischemia (Table 3). The mortality rate in the propranolol group decreased from 64% (in experiment 1) to 46%, and the rates did not differ among the groups. PaCO2, PaO2, and pericranial and rectal temperatures were maintained around the target values given in the Methods section, and preischemic Hct, glucose, MAP, and heart rate were also similar among the groups (Table 4). Changes in MAP and heart rate (Fig. 4) were also similar among the groups, weight loss after ischemia was negligible in all groups, and rectal temperature before death was similar among the groups (data not shown). The percentage of injured neurons in the CA1 sector after ischemia (Fig. 5) was significantly decreased by esmolol and landiolol compared with saline, but was not decreased by propranolol. However, there were no differences in motor activity in rats treated postischemically with a β-adrenoreceptor antagonist compared with those that received saline (Fig. 6).
It has been shown that preischemic treatment with β-adrenoreceptor antagonists induces neuroprotection against focal cerebral ischemia.1–5 In contrast, preischemic treatment with propranolol, esmolol, or landiolol in this study did not provide neuroprotection of the hippocampus against forebrain ischemia induced by 8-minute BCAO. However, interestingly, postischemic treatment with esmolol and landiolol reduced neuronal injury after forebrain ischemia. Focal ischemic models have normally been used to assess neuroprotection by β-adrenoreceptor antagonists,1–5 but several reports have shown different effects of neuroprotective drugs in different experimental brain ischemia models, probably because of inherent differences in pathophysiological responses to focal stroke and forebrain ischemia.8–10 Therefore, the difference in experimental design (focal versus forebrain ischemia) may have contributed to the failure to find protection of the hippocampus in preischemic treatment in this study. Focal ischemia typically gives rise to localized brain infarction,8 whereas forebrain ischemia results in selective neuronal injury within vulnerable brain regions (e.g., CA1).8 This suggests that blockade of β-adrenoreceptors may not be effective in reducing neuronal injury in ischemia-sensitive regions.
In contrast to preischemic treatment, we found that postischemic administration of esmolol or landiolol reduced neuronal injury in vulnerable brain regions. Therefore, β-adrenoreceptor blockade is apparently effective in reducing neuronal injury in ischemia-sensitive regions. In this study, BCAO with hypotension induced by hemorrhagic shock was used to create forebrain ischemia. Luchette et al.13 reported that β-blockade was associated with a significant increase in plasma lactate and epinephrine in response to hemorrhagic shock, and Le Tulzo et al.14 showed that propranolol administration was associated with increased messenger RNA levels for the inflammatory cytokines interleukin-1β, tumor necrosis factor-α, and transforming growth factor β1 in the lungs after hemorrhage. Raddatz et al.15 also demonstrated that pretreatment with a β1-adrenoreceptor agonist, dobutamine, improved liver function after hemorrhagic shock through induction of hemeoxygenase-1, and that esmolol blocked the protective effect of dobutamine. Collectively, these results indicate that concomitant β-adrenoreceptor blockade with hemorrhagic shock can worsen systemic conditions, and this might have contributed to the histological outcome in this study. Perfusion of ischemic brain tissue might also have differed from controls in animals treated with adrenoceptor antagonists, although the MAP and heart rate were similar across groups. However, this is speculative because we did not measure cerebral perfusion. It is of note that the mortality in some β-blocker groups tended to be higher than that in the saline group for preischemic drug administration, and the motor activity score (especially with propranolol) tended to be worse than in the saline group. Exacerbation of systemic conditions by β- adrenoreceptor blockade during hemorrhagic shock might mask the neuroprotective effect, and simultaneous β1- and β2-blockade during hemorrhagic shock might have a more significant influence on outcome compared with selective β1-blockade. A less severe model that does not require manipulation of systemic hemodynamics, such as 4-vessel occlusion, might produce different results, and a further study is required to address this issue.
Postischemia treatment with esmolol and landiolol (selective β-adrenoreceptor 1 antagonists) but not with propranolol (nonselective β-adrenoreceptor blocker) significantly reduced neuronal injury after BCAO with hypotension. The detailed neuroprotective mechanisms of β-blockers are unclear; however, possible mechanisms have been proposed. β- Adrenoreceptor blockade decreases oxygen consumption with a minimal effect on cerebral blood flow in normocapnia16 and inhibits platelet aggregation and serotonin release induced by adenosine diphosphate, epinephrine, collagen, and thrombin.17 β-Adrenoreceptor blockade also interferes with calcium influx by reducing the population of receptor-operated channels and abolishing cyclic adenosine monophosphate– dependent protein kinase-mediated phosphorylation of slow inward current channels,18,19 and reduces calcium-dependent phospholipase A2 activity in platelets.20 In addition, Goyagi et al.5 proposed that selective β1-blockade may be especially effective against focal cerebral ischemia because of the larger proportion of β1-receptors than β2-receptors in the area injured by middle cerebral artery occlusion (MCAO). In contrast, β2-adrenoreceptor stimulation produces neuroprotection through neuron growth factor expression.6,7 Thus, specific types of β-adrenoreceptor blockade might be neuroprotective in certain situations. We speculate that postischemic treatment with propranolol failed to show neuroprotection after BCAO with hemorrhagic shock because of, at least in part, β2-adrenoreceptor blockade. Esmolol and landiolol are very weak β2-antagonists, and this may explain the neuroprotective effect on ischemia-sensitive regions after forebrain ischemia with hemorrhagic shock based on drug administration started in the recovery period from shock. Use of 2 different β1-selective drugs could provide strong evidence that postischemic β1-adrenoreceptor blockade is important to reduce neuronal damage in forebrain ischemia with hemorrhagic shock. Postischemic administration of β-blockers in focal ischemia models is of interest, and the results of this study show that this is effective against focal cerebral ischemia. However, it remains to be determined whether preischemic or postischemic treatment is the better approach.
Several drugs have been reported to be neuroprotective in animal models of transient global ischemia, which are representative of clinical cerebral ischemia in cardiac arrest. However, almost all of these drugs must be administered before ischemia to prevent neuronal death. Because most patients do not receive medical care until hours after the ischemic insult, the clinical application of such drugs in humans is limited. Therefore, neuroprotective drugs that reduce neuronal damage when administered after ischemia are very attractive, and our results suggest that selective β1-adrenoreceptor antagonists might have an important role in postischemic treatment paradigms. However, β-blockers may coincidentally be used preischemically for cerebral ischemia due to perioperative cardiac events, because Mangano et al.21 reported that perioperative β-blockade significantly reduced mortality and the incidence of cardiac events in patients with coronary artery disease undergoing noncardiac surgery. However, prophylactic perioperative β-blockade for prevention of cardiac events has been questioned after reevaluation of this approach in the Metoprolol after Vascular Surgery study, Diabetic Postoperative Mortality and Morbidity trial, and POISE (PeriOperative Ischemic Evaluation) trial.22–24 In particular, the POISE trial showed that perioperative β-blockade increased mortality and stroke, although it did decrease myocardial infarction.24 In addition, the trial showed that mortality and stroke were associated with hypotension induced by metoprolol. As mentioned above, concomitant β-blockade in critical situations might work as a systemic depressant, rather than a neuroprotectant, and result in exacerbation of cerebral ischemia.
Several limitations of the study should be considered in interpreting the results. First, we studied only 1 dose of each drug, and the doses used might not have been appropriate for preischemic treatment in our experimental protocol. However, the doses were chosen based on the MCAO study performed by Goyagi et al.,5 in which the same preischemic treatment regimens of β-blockers significantly reduced infarct volume produced by MCAO in rats. Furthermore, the same doses of β-adrenoreceptor antagonists used postischemically were effective. Considering the higher mortality rates when administration was started before ischemia, it is reasonable to assume that larger doses of β-blockers would not have produced improved neuroprotection in this model of forebrain ischemia. Regarding the effect of smaller doses, further studies are required to address this question. Another concern is that preischemic treatment with esmolol tended to have a higher mortality rate compared with preischemic landiolol, even though these drugs showed equivalent efficacy in postischemic administration. Esmolol is metabolized by red blood cell esterases,25 whereas landiolol is metabolized by plasma cholinesterases.26 Because concomitant β1-adrenoreceptor blockade with hemorrhagic shock may alter systemic conditions,15 the activity of these enzymes might be modified during hemorrhagic shock, and this might affect the β1-adrenorecepror antagonist activities of the drugs. Regarding the discrepancy between functional and morphological outcomes, the moderate neuroprotection in this study (reduction of neuronal damage from 80% to 60%) might be related to the failure to detect functional improvement. In particular, greater histological improvement in hippocampal areas might have improved learning and memory. A final concern is that β-adrenoceptor antagonists may affect body temperature27 when administered after ischemia over several days, and this might have influenced the results. We only measured body temperature during ischemia and in the early reperfusion period. However, we also found similar rectal temperatures across the groups on the fifth day after ischemia, which suggests that the β-adrenoceptor antagonists had no significant effect on body temperature.
In summary, posttreatment but not pretreatment with esmolol and landiolol (selective β-adrenoreceptor 1 antagonists) provided neuroprotection in the hippocampus in rats after transient forebrain ischemia. However, propranolol (a nonselective β-adrenoreceptor antagonist) failed to give neuroprotection. It is likely that the discrepancy in the neuroprotective efficacy of β-blockade between our results using a BCAO model and results obtained in MCAO models is a function of differences in the experimental models. In particular, concomitant β-blockade and shock might work as a systemic depressant, rather than a neuroprotectant, and exacerbate cerebral ischemia.
Landiolol and esmolol were donated by ONO Pharmaceutical Co., Ltd. and Maruishi Pharmaceutical Co., Ltd., respectively.
1. Standefer M, Little JR. Improved neurological outcome in experimental focal cerebral ischemia treated with propranolol. Neurosurgery 1986;18:136–40
2. Latchaw JP, Little JR, Slugg RM, Lesser RP, Stowe N. Treatment of acute focal cerebral ischemia and recirculation with d-propranolol. Neurosurgery 1985;16:18–22
3. Capraro JA, Reedy DP, Latchaw JP, Slugg RM, Stowe NT, Lesser RP, Little JR. Treatment of acute focal cerebral ischemia with propranolol. Stroke 1984;15:486–91
4. Little JR, Latchaw JP Jr, Slugg RM, Lesser RP, Stowe NT. Treatment of acute focal cerebral ischemia with propranolol. Stroke 1982;13:302–7
5. Goyagi T, Kimura T, Nishikawa T, Tobe Y, Masaki Y. Beta-adrenoreceptor antagonists attenuate brain injury after transient focal ischemia in rats. Anesth Analg 2006;103:658–63
6. Semkova I, Schilling M, Henrich-Noack P, Rami A, Krieglstein J. 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
7. Semkova I, Krieglstein J. Neuroprotection mediated via neurotrophic factors and induction of neurotrophic factors. Brain Res Rev 1999;30:176–88
8. Ginsberg MD, Busto R. Rodent models of cerebral ischemia. Stroke 1989;20:1627–42
9. Li H, Colbourne F, Sun P, Zhao Z, Buchan AM, Iadecola C. Caspase inhibitors reduce neuronal injury after focal but not global cerebral ischemia in rats. Stroke 2000;31:176–82
10. Bond A, Ragumoorthy N, Monn JA, Hicks CA, Ward MA, Lodge D, O'Neill MJ. LY379268, a potent and selective group II metabotropic glutamate receptor agonist, is neuroprotective in gerbil global, but not focal, cerebral ischaemia. Neurosci Lett 1999;273:191–4
11. Miura Y, Grocott HP, Bart RD, Pearlstein RD, Dexter F, Warner DS. Differential effects of anesthetic agents on outcome from near-complete but not incomplete global ischemia in the rat. Anesthesiology 1998;89:391–400
12. Palkovits M, Brownstein MJ. Maps and Guide to Microdissection of the Rat Brain. New York, NY: Elsevier, 1980
13. Luchette FA, Robinson BR, Friend LA, McCarter F, Frame SB, James JH. Adrenergic antagonists reduce lactic acidosis in response to hemorrhagic shock. J Trauma 1999;46:873–80
14. Le Tulzo Y, Shenkar R, Kaneko D, Moine P, Fantuzzi G, Dinarello CA, Abraham E. Hemorrhage increases cytokine expression in lung mononuclear cells in mice: involvement of catecholamines in nuclear factor-kappaB regulation and cytokine expression. J Clin Invest 1997;99:1516–24
15. Raddatz A, Kubulus D, Winning J, Bauer I, Pradarutti S, Wolf B, Kreuer S, Rensing H. Dobutamine improves liver function after hemorrhagic shock through induction of heme oxygenase-1. Am J Respir Crit Care Med 2006;174:198–207
16. Mackenzie ET, McCulloch J, Harper AM. Influence of endogenous norepinephrine on cerebral blood flow and metabolism. Am J Physiol 1976;231:483–8
17. Weksler BB, Gillick M, Pink J. Effects of propranolol on platelet function. Blood 1977;49:185–96
18. Braunwald E. Mechanism of action of calcium channel blocking agents. N Engl J Med 1982;307:1618–27
19. 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
20. 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
21. Mangano DT, Layug EL, Wallace A, Tateo I. Effect of atenolol on mortality and cardiovascular morbidity after noncardiac surgery. Multicenter Study of Perioperative Ischemia Research group. N Engl J Med 1996;335:1713–20
22. Yang H, Raymer K, Butler R, Parlow J, Roberts R. The effects of perioperative beta-blockade: results of the Metoprolol after Vascular Surgery (MaVS) study, a randomized controlled trial. Am Heart J 2006;152:983–90
23. Juul AB, Wetterslev J, Kofoed-Enevoldsen A, Callesen T, Jensen G, Gluud C. Diabetic Postoperative Mortality and Morbidity group. The Diabetic Postoperative Mortality and Morbidity (DIPOM) trial: rationale and design of a multicenter, randomized, placebo-controlled, clinical trial of metoprolol for patients with diabetes mellitus who are undergoing major noncardiac surgery. Am Heart J 2004;147:677–83
24. Devereaux PJ, Yang H, Yusuf S, Guyatt G, Leslie K, Villar JC, Xavier D, Chrolavicius S, Greenspan L, Pogue J, Pais P, Liu L, Xu S, Málaga G, Avezum A, Chan M, Montori VM, Jacka M, Choi P. Effects of extended-release metoprolol succinate in patients undergoing non-cardiac surgery (POISE trial): a randomised controlled trial. Lancet 2008;371:1839–47
25. Quon CY, Stampfli HF. Biochemical properties of blood esmolol esterase. Drug Metab Dispos 1985;13:420–4
26. Iguchi S, Iwamura H, Nishizaki M, Hayashi A, Senokuchi K, Kobayashi K, Sakaki K, Hachiya K, Ichioka Y, Kawamura M. Development of a highly cardioselective ultra short-acting β-blocker, ONO-1101. Chem Pharm Bull 1992;40:1462–9
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27. Soszynski D, Kozak W, Conn CA, Rudolph K, Kluger MJ. Beta-adrenoceptor antagonists suppress elevation in body temperature and increase in plasma IL-6 in rats exposed to open field. Neuroendocrinology 1996;63:459–67