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Anesthesia & Analgesia:
doi: 10.1213/ANE.0b013e318280e26d
Neuroscience in Anesthesiology and Perioperative Medicine

Treatment with a Highly Selective β1 Antagonist Causes Dose-Dependent Impairment of Cerebral Perfusion After Hemodilution in Rats

Hu, Tina MSc*; Beattie, W. Scott MD, PhD; Mazer, C. David MD*; Leong-Poi, Howard MD; Fujii, Hiroko MD; Wilson, David F. PhD§‖; Tsui, Albert K. Y. PhD*; Liu, Elaine MD*; Muhammad, Maimoona BSc*; Baker, Andrew J. MD*; Hare, Gregory M. T. MD, PhD*

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Author Information

From the *Department of Anesthesia, and Division of Cardiology, Department of Medicine, Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto; Department of Anesthesia, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Ontario, Canada; and Departments of §Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania.

Accepted for publication November 27, 2012.

This work was funded through an investigator-initiated trial grant from the Forest Research Institute (subsidiary of Forest Laboratories), the Departments of Anesthesia (St. Michael’s Hospital and Toronto General Hospital, University of Toronto), the International Anesthesia Research Society–Society of Cardiovascular Anesthesiologists, The Physician’s Services Incorporated Foundation, the R. Fraser Elliott Endowment, the Ministry of Training, Colleges, and Universities, and the Institute of Medical Science at the University of Toronto. Drs. Beattie, Mazer, Baker, and Hare have received Merit Awards from the Department of Anesthesia, University of Toronto.

See Disclosures at end of article for Author Conflicts of Interest. This report was previously presented, in part, at the Canadian Anesthesiologists’ Society Meeting, Canadian Cardiovascular Congress Meeting, and the Society of Cardiovascular Anesthesiologists Meeting.

Address correspondence and reprint requests to Gregory M. T. Hare, MD, PhD, Department of Anesthesia, Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, 30 Bond St., Toronto, ON M5B 1W8, Canada. Address e-mail to hareg@smh.ca.

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Abstract

BACKGROUND: Acute β-blockade has been associated with a dose-dependent increase in adverse outcomes, including stroke and mortality. Acute blood loss contributes to the incidence of these adverse events. In an attempt to link the risks of acute blood loss and β-blockade, animal studies have demonstrated that acute β-blockade impairs cerebral perfusion after hemodilution. We expanded on these findings by testing the hypothesis that acute β-blockade with a highly β1-specific antagonist (nebivolol) causes dose-dependent cerebral hypoxia during hemodilution.

METHODS: Anesthetized rats and mice were randomized to receive vehicle or nebivolol (1.25 or 2.5 mg/kg) IV before hemodilution to a hemoglobin concentration near 60 g/L. Drug levels, heart rate (HR), cardiac output (CO), regional cerebral blood flow (rCBF, laser Doppler), and microvascular brain PO2 (PBrO2, G2 Oxyphor) were measured before and after hemodilution. Endothelial nitric oxide synthase (NOS), neuronal NOS (nNOS), inducible NOS, and hypoxia inducible factor (HIF)-1α were assessed by Western blot. HIF-α expression was also assessed using an HIF-(ODD)-luciferase mouse model. Data were analyzed using analysis of variance with significance assigned at P < 0.05, and corrected P values are reported for all post hoc analyses.

RESULTS: Nebivolol treatment resulted in dose-specific plasma drug levels. In vehicle-treated rats, hemodilution increased CO and rCBF (P < 0.010) whereas PBrO2 decreased to 45.8 ± 18.7 mm Hg (corrected P < 0.001; 95% CI 29.4–69.7). Both nebivolol doses comparably reduced HR and attenuated the CO response to hemodilution (P < 0.012). Low-dose nebivolol did not impair rCBF or further reduce PBrO2 after hemodilution. High-dose nebivolol attenuated the rCBF response to hemodilution and caused a further reduction in PBrO2 to 28.4 ± 9.6 mm Hg (corrected P = 0.019; 95% CI 17.4–42.7). Both nebivolol doses increased brain endothelial NOS protein levels. Brain HIF-1α, inducible NOS, and nNOS protein levels and brain HIF-luciferase activity were increased in the high-dose nebivolol group after hemodilution (P < 0.032).

CONCLUSIONS: Our data demonstrate that nebivolol resulted in a dose-dependent decrease in cerebral oxygen delivery after hemodilution as reflected by a decrease in brain tissue PO2 and an increase in hypoxic protein responses (HIF-1α and nNOS). Low-dose nebivolol treatment did not result in worsened tissue hypoxia after hemodilution, despite comparable effects on HR and CO. These data support the hypothesis that acute β-blockade with a highly β1-specific antagonist causes a dose-dependent impairment in cerebral perfusion during hemodilution.

Cardioselective β-adrenergic antagonists have been broadly prescribed to treat coronary artery disease1 based on their efficacy in reducing mortality and reinfarction after an acute myocardial infarction.2,3 Two early clinical trials supported the use of β-adrenergic antagonists to confer cardiac protection and reduce mortality in surgical patients with cardiovascular risk factors,2,3 leading to their application in perioperative medicine.4

Despite their efficacy at reducing myocardial events, subsequent clinical trials failed to demonstrate improved survival; paradoxically, an increased incidence of adverse clinical outcomes including bradycardia, hypotension, stroke, and increased mortality were observed.5–9 The largest of these trials (POISE) demonstrated an increased risk of ischemic stroke and mortality in patients who were randomized to receive metoprolol.10 Subsequent studies have proposed that metoprolol, a relatively poor β1-selective antagonist, may be associated with a pronounced increase in adverse clinical outcomes, relative to other more highly β1-selective antagonists.6–8,11 These adverse outcomes have been observed in acute care settings, in which acute β-blocker administration, blood loss, and anemia occur. Each of these factors may independently contribute to the observed increased incidence of vital organ ischemia and mortality.6,7,9,10,12–14 The finding that acute blood loss and anemia are associated with an increase in major adverse cardiac events provides further incentive to explore the relationships among β-antagonism, anemia, and vital organ perfusion.9,15

In an experimental model of acute hemodilution, metoprolol was shown to significantly impair cerebral perfusion in vivo16 by attenuating the cardiac output (CO) response to acute anemia, a predominantly β1-mediated effect. An additional study in isolated cerebral arteries demonstrated that metoprolol can also inhibit β2-mediated cerebral vasodilation in vitro.17 The current experimental study was designed to test the hypothesis that acute β-blockade with a highly β1-specific antagonist (nebivolol) causes dose-dependent cerebral tissue hypoxia during hemodilution.

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METHODS

Animal Model of Acute Hemodilution

All animal procedures were approved by the Animal Care Committee at St. Michael’s Hospital (Toronto, ON, Canada) and the Toronto Centre for Phenogenomics (Toronto, ON, Canada) in accordance with the standards set by the Canadian Council on Animal Care. A summary of all experimental protocols and sampling times for outcome measures is provided in Figure 1. Male Sprague-Dawley rats (400–500 g) were purchased from Charles River Laboratories (Montreal, QC, Canada) and hypoxia inducible factor (HIF)-(oxygen degradation domain [ODD])-luciferase mice (25–30 g) were purchased from Jackson’s Laboratory (Bar Harbor, ME). All animals were housed under standard conditions with food and water ad libitum.

Figure 1
Figure 1
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All rats were anesthetized with isoflurane (2%–3%; Abbott Laboratories, Saint-Laurent, QC, Canada) in 50% oxygen, tracheotomized, and ventilated to achieve normocapnia and normoxia. All incision sites were infiltrated with 2% Xylocaine. The tail artery was cannulated to measure mean arterial blood pressure (MAP), arterial blood gases, and hemoglobin (Hb) concentration by cooximetry (Radiometer ALB 500 and OSM 3; London Scientific, London, ON, Canada). The tail vein was cannulated to administer vehicle or nebivolol IV and to perform acute hemodilution. Heart rate (HR) was measured by using electrocardiogram electrodes, and rectal temperature was maintained near 37°C with a warming pad. All physiologic data were acquired digitally (Power Lab 16/30; ADInstruments, Colorado Springs, CO). A total of 115 rats, 12 C57BL/6J black mice, and 11 HIF-(ODD)-luciferase mice were used as outlined in 5 different experimental protocols.

After establishing a stable baseline, rats were randomized to receive either the selective β1 antagonist (nebivolol at the 1.25 or 2.5 mg/kg dose; Forest Laboratories, New York, NY) or vehicle (50%:50% cyclodextrin and saline) IV, targeting a 20% reduction in HR and based on previous experimental studies in rodents (Bertek Pharmaceuticals Inc., report R67555). Thirty minutes after drug or vehicle administration, different groups of rats were either observed and monitored (time control, no hemodilution) or hemodiluted by simultaneously exchanging 30 mL/kg arterial blood (50% estimated blood volume) with an equivalent volume of warmed 10% pentastarch (Pentaspan, osmotic and oncotic pressure of 326 mOsm and 40 mm Hg, respectively; Bristol-Myers Squibb, Montreal, QC, Canada) at a fixed rate over 10 minutes by using a push-pull infusion pump (PHD 2000; Harvard Apparatus, Saint-Laurent, QB, Canada) to achieve a target Hb concentration of 60 g/L. Data were recorded electronically for 1 hour after hemodilution in all protocols. Arterial blood gas and cooximetry samples were collected before baseline, 30 minutes after nebivolol or vehicle administration, 30 minutes after hemodilution, and 1 hour after hemodilution in all protocols (Fig. 1).

C57BL/6J black mice were subjected to vehicle or nebivolol treatment (1.25 or 2.5 mg/kg) to assess the effect of nebivolol doses on HR. HIF-(ODD)-luciferase mice were subjected to vehicle or nebivolol treatment (1.25 or 2.5 mg/kg) and hemodiluted to an Hb concentration of approximately 60 g/L as previously described.18 Brain tissue was extracted 3 hours after hemodilution. Protein concentration was determined using a protein assay (BCA Protein Assay; ThermoScientific, Rockford, IL) and HIF-luciferase activity was assessed in vitro using the FLUOstar OPTIMA (Fisher Scientific, Nepean, ON, Canada).18

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Statistical Analyses

Sample size calculations are presented for each experimental protocol assuming a power of 0.8 and an α of 0.05. Data were analyzed using Sigma Plot version 11.0 (Systat Software Inc., San Jose, CA). All physiologic data were assessed to be normally distributed with homogeneity of variance as assessed by Shapiro-Wilk and Levene tests (all P > 0.11), respectively. All physiologic and arterial blood gas data were assessed independently using 2-way repeated-measures analysis of variance (ANOVA) for time, group, and interaction effects. Plasma drug levels were assessed by 1-way ANOVA whereas protein levels (Western blot) and HIF-luciferase data were assessed by 1-way ANOVA on ranks using a Dunn test for post hoc analysis. All data are presented as mean ± SD with significance assigned at P < 0.05. Post hoc Tukey and Dunn tests were used to compare means when an adequate F ratio was achieved and comparisons are presented as corrected P values.

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Protocol 1: Effect of Nebivolol and Hemodilution on Plasma Drug Levels, HR, MAP, and Regional Cerebral Blood Flow

Previously obtained pharmacokinetic data (Bertek Pharmaceuticals Inc., report R67555) demonstrated a mean plasma drug level of 72 ± 13 nM at 2 hours after an IV dose of nebivolol (1.25 mg/kg). Based on these numbers and an expected doubling of drug level with a higher dose (2.5 mg/kg), we estimated that a sample size of n = 4 per group was needed to assess drug levels (vehicle, 1.25, and 2.5 mg/kg nebivolol). Regional cerebral blood flow (rCBF) measurements were performed in this group of experimental animals to maximize the number of secondary outcomes obtained.

Vehicle (50%:50% cyclodextrin and saline) or nebivolol (1.25 or 2.5 mg/kg) was administered IV to anesthetized rats, and HR, MAP, and rCBF (laser Doppler) were measured. Drug-level measurements were determined at 1 hour after drug injection without hemodilution to establish the expected nebivolol plasma levels at the time of starting hemodilution. In an additional group of rats, drug levels were also determined at 1 hour after hemodilution (2 hours after drug injection) (n = 4–6 per group). These animals were instrumented for rCBF measurements. To collect plasma for drug-level measurements, blood samples were collected from the descending aorta and centrifuged at 3000 rpm for 30 minutes. Plasma drug levels were measured in a blinded manner using liquid chromatography tandem mass spectrometry as previously described.19 These drug doses were chosen based on previously available pharmacokinetic data with a dose of 1.25 mg/kg expected to produce an estimated 20% reduction in HR.

To assess rCBF, rats were placed in a stereotaxic frame with a 2-mm bur hole trephined between the lambda and bregma on the right side of the sagittal sinus, and a laser Doppler flow probe (OxyFlo; Oxford Optronix, Oxford, UK) was placed over the cerebral cortex. Rats were randomized to receive IV nebivolol (dose of 1.25 or 2.5 mg/kg) or vehicle. In the first group of rats, HR, MAP, and rCBF were measured over 60 minutes without hemodilution in rats treated with vehicle or nebivolol (n = 6 per group). In the second group, HR, MAP, and rCBF were assessed in rats treated with vehicle or nebivolol before and after hemodilution (n = 6 per group).

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Protocol 2: Effect of Nebivolol and Hemodilution on CO

Based on previous measurements of CO16 at baseline (138 ± 28 mL/min), after hemodilution (208 ± 19 mL/min), and after hemodilution and β-blockade (142 ± 20 mL/min), we estimated a sample size of n = 5 per group to enable the detection of a mean difference in CO of 66 ± 28 mL/min between vehicle- and nebivolol-treated anemic animals.

CO was assessed by echocardiography using a Sonos 5500 echocardiographic system (Philips Medical Systems Canada, Markham, ON, Canada) equipped with a high-frequency (5- to 12-MHz broad-bandwidth) phased-array transducer (S12, Philips Ultrasound) as previously reported.16 CO measurements were taken before and after hemodilution in rats treated with vehicle or nebivolol (n = 5 per group). Two-dimensional and M-mode echocardiographic imaging along with spectral Doppler acquisition was performed by using standard parasternal and apical windows. Left ventricular end-diastolic and end-systolic dimensions were obtained while fractional shortening ([left ventricular end-diastolic diameter – left ventricular end-systolic diameter]/left ventricular end-diastolic diameter) and stroke volume (SV) (left ventricular outflow tract area × left ventricular outflow tract velocity time) were calculated. CO was determined by multiplying SV and HR. All echocardiographic data were assessed in a blinded manner.

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Protocol 3: Effect of Nebivolol and Hemodilution on Microvascular Cerebral Cortical Oxygen Tension

Based on previous measurements of microvascular brain PO2 (PBrO2)16 at baseline (73 ± 13 mm Hg), after hemodilution (54 ± 5 mm Hg), and after hemodilution and β-blockade (40 ± 9 mm Hg), we estimated a sample size of n = 10 per group to enable the detection of a mean difference in PBrO2 of 14 ± 9 mm Hg between vehicle- and nebivolol-treated anemic animals. Because the primary interest was to differentiate between the brain PO2 values after the 2 different drug doses, randomization was performed in a 1:2:2 distribution (vehicle, 1.25, and 2.5 mg/kg nebivolol, respectively) to minimize the number of animals used. Randomization was performed on the day of the experiment using a computer-generated randomized schedule (www.randomization.com) in blocks of 5 and conducted in a blinded manner by an independent study coordinator who was not directly involved with the experimental protocol. Drug treatment group was concealed from the study investigators.

After blinded randomization and drug-dose concealment, rats received vehicle or nebivolol (dose of 1.25 or 2.5 mg/kg) before undergoing hemodilution (n = 5, 10, and 11 per group, respectively). Two bilateral bur holes were trephined as previously described and 4-mm-diameter fiberoptic excitation and detection light guides were positioned such that the light path crossed through the cerebral cortex and deeper brain structures. Rats were injected with Oxyphor G2-phosphorescent dye (Pd-meso-tetra-[4-carboxyphenyl]-tetrabenzoporphyrin, 1 mg in 0.2 mL saline), allowing measurements of phosphorescence quenching by oxygen through the PMOD 5000 probes (Oxygen Enterprises, Philadelphia, PA), as previously described.16 Data were recorded continuously for 1 hour after hemodilution.

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Protocol 4: Effect of Nebivolol and Hemodilution on Brain HIF-α and Nitric Oxide Synthase Protein Levels

Based on previous measurements of normalized cerebral cortical HIF-α protein levels16 after hemodilution (1.00 ± 0.35), after β-blockade alone (1.16 ± 0.36), and after hemodilution and β-blockade (2.00 ± 0.20), we estimated a sample size of n = 5 per group to enable the detection of a mean difference in HIF-α protein levels of 0.86 ± 0.36 between vehicle- and nebivolol-treated anemic animals.

Brain protein responses were assessed by Western blot analysis (n > 5 per group) and immunohistologic analysis (n = 3 per group) as previously described.18,20 Rats underwent hemodilution after being given vehicle or nebivolol (1.25 or 2.5 mg/kg). Western blot analysis was performed on cerebral cortex tissue removed from euthanized rats at the completion of protocol 3. Total protein was extracted from homogenates of the cerebral cortex, and total protein levels were quantified (BCA Protein Assay; ThermoScientific, Rockford, IL) before running samples on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The protein was then transferred to a nitrocellulose membrane and specific protein bands were identified using the appropriate primary polyclonal antibodies for endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) nitric oxide synthases, HIF-1α, and S-nitrosoglutathione reductase (GSNOR) (SC654, Santa Cruz Biotechnology, Santa Cruz, CA; 610431, BD Biosciences, Mississauga, ON, Canada; 610309, BD Biosciences; AF1935, R&D Systems, Toronto, ON, Canada; 11051-1-AP, Proteintech Group, Chicago, IL, respectively) and labeled secondary antibodies conjugated with horseradish peroxidase. Band densities were quantified using densitometry and normalized to α-tubulin (T6199; Sigma-Aldrich Canada, Oakville, ON, Canada). For immunohistochemical and fluorescent staining, 10-μm fixed tissue sections (4% paraformaldehyde) were incubated overnight at 4°C with the diluents of the primary polyclonal antibody for HIF-1α (AF1935, R&D Systems; or NB 100-131, Novus Biologicals, Cedar Lane Labs, Burlington, ON, Canada) and nNOS (610309; BD Biosciences). TO-PRO®-3 iodine was used for nuclear staining (T3605; Life Technologies, Grand Island, NY) and specific binding was determined with a labeled secondary antibody. Microscopy was performed using fluorescent and confocal microscopes (Nikon ECLIPSE 90i and Bio-Rad Radiance 2100; St. Michael’s Hospital Keenan Research Centre Medical Imaging Facility, Toronto, ON, Canada).

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Protocol 5: Effect of Hemodilution and Nebivolol on Brain HIF-α Luciferase Activity in HIF-(ODD)-Luciferase Mice

Based on previous measurements of cerebral cortical HIF-α luciferase activity18 in control (8.05 ± 1.58 LU/μg protein) and anemic mice (16.22 ± 4.01 LU/μg protein), we estimated a sample size of n = 4 per group to enable the detection of a mean difference in HIF-α luciferase activity of 8.17 ± 2.97 between vehicle- and nebivolol-treated anemic animals.

As previously described,18 a transgenic mouse model [HIF-(ODD)-luciferase mice] was used to assess the impact of acute hemodilution on real-time brain HIF-α luciferase activity after vehicle, low-, or high-dose nebivolol treatment. Mice were treated with vehicle or nebivolol and hemodiluted to a target Hb concentration of 60 g/L while spontaneously breathing room air under isoflurane anesthesia (2%). The mice were euthanized under anesthesia 3 hours after hemodilution and cerebral cortex tissue was extracted and prepared for assessment of tissue luciferase activity.18

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RESULTS

Effect of Acute Hemodilution on Arterial Blood Gas and Cooximetry Values

For all experimental protocols, pH, PaCO2, and PaO2 remained unchanged without significant differences between vehicle-treated control rats and rats treated with nebivolol (Table 1). The baseline Hb concentration across all 3 treatment groups was approximately 145 g/L and a target Hb concentration near 60 g/L was reached in all groups after hemodilution. Blood oxygen content and Hb decreased comparably in all treatment groups after hemodilution, relative to baseline (Table 1, time effect, P < 0.001 for all).

Table 1
Table 1
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Protocol 1: Effect of Nebivolol and Hemodilution on Plasma Drug Levels, HR, MAP, and rCBF

No drug was detected in vehicle-treated control rats. In nonhemodiluted rats, plasma drug levels reached 109 ± 35 nM and 202 ± 22 nM 1 hour after receiving low- or high-dose nebivolol, respectively (Fig. 2A). In a different group of rats, plasma drug levels were measured 1 hour after hemodilution and 2 hours after vehicle or drug administration (Fig. 2B). Drug levels reached 56 ± 21 nM and 122 ± 32 nM after receiving low- or high-dose nebivolol and after hemodilution, respectively (Fig. 2, treatment group effect, P < 0.001). There was a significant difference between low- and high-dose nebivolol treatment 2 hours after drug administration (Fig. 2, corrected P = 0.011).

Figure 2
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Both doses of nebivolol reduced HR by approximately 20% relative to baseline in nonhemodiluted rats (Fig. 3A, treatment group, time, and interaction effect, P < 0.002 for all). There was a significant reduction in HR in both drug groups versus vehicle-treated control rats (corrected P < 0.001 for both), but no significant difference in HR between the 2 drug doses (corrected P = 0.984). There was a small but significant decrease in MAP with both doses of nebivolol relative to baseline (Fig. 3B, treatment group, time, and interaction effect, P < 0.020 for all), but no difference between drug treatment groups (corrected P = 0.753). No changes in basal rCBF were observed in any treatment group over time after vehicle or drug administration (Fig. 3C).

Figure 3
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In a different group of rats that underwent vehicle or nebivolol treatment and hemodilution, similar postdrug effects on HR and MAP were observed (Fig. 4, A and B). Administration of both low and high doses of nebivolol decreased HR (~20%) relative to baseline (Fig. 4A, treatment group, time, and interaction effect, P < 0.01 for all). There was a significant reduction in HR in both drug groups versus vehicle-treated controls (corrected P < 0.006 for both), but no significant difference in HR between the 2 drug doses (corrected P = 0.887). Both nebivolol doses caused a small but significant decrease in MAP relative to baseline (Fig. 4B, treatment group, time, and interaction effect, P < 0.001 for all), but there was no significant difference in MAP between the 2 drug doses (Fig. 4B, corrected P = 0.226). After acute hemodilution, HR and MAP were maintained in vehicle-treated controls, but were reduced by both nebivolol doses (Fig. 4, A and B; corrected P < 0.001 for both). For rCBF measurements, there were significant treatment group, time, and interaction effects (Fig. 4C, P < 0.012 for all). After acute hemodilution, there was a significant increase in rCBF in both the vehicle-treated control and low-dose nebivolol groups relative to baseline and compared with the high-dose nebivolol group (Fig. 4C, corrected P < 0.001 for all), with mean posthemodilution values of 1.9 ± 0.5 (95% confidence interval [CI] 1.2–2.6) and 1.7 ± 0.4 (95% CI 1.3–2.6), respectively. By contrast, there was no significant increase in rCBF in the high-dose nebivolol treatment group relative to baseline (Fig. 4C, corrected P = 0.974, 1.1 ± 0.1, 95% CI 1.0–1.3).

Figure 4
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Protocol 2: Effect of Nebivolol and Hemodilution on CO

Vehicle-treated control rats demonstrated a characteristic increase in CO after hemodilution (190 ± 8; 95% CI 179–196 mL/min) relative to baseline (122 ± 10; 95% CI 106–132 mL/min), but this was severely attenuated in both low- and high-dose nebivolol treatment groups after hemodilution (150 ± 18; 95% CI 140–174 and 144 ± 16; 95% CI 117–161 mL/min, respectively) (Table 2, treatment group, time, and interaction effect, P < 0.018 for all). After hemodilution, there was a significant difference in CO in both low- and high-dose nebivolol groups, relative to vehicle-treated controls (Table 2, corrected P < 0.014 for both), but no difference between drug treatment groups (Table 2, corrected P = 0.486). In both drug-treated groups, nebivolol resulted in a comparable reduction in HR (~20%); however, the increase in SV was preserved (Table 2). Left ventricular end-diastolic diameter, left ventricular end-diastolic area, left ventricular outflow tract, left ventricular outflow tract velocity time integral, and fractional area change increased after hemodilution in all 3 groups, relative to baseline (Table 2, corrected P < 0.025 for all). No differences in cardiac variables were observed between low- and high-dose nebivolol treatment groups.

Table 2
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Protocol 3: Effect of Nebivolol and Hemodilution on Microvascular Cerebral Cortical Oxygen Tension

In this group of rats, the HR and MAP responses were comparable to previous protocols with both drug doses causing significant and comparable reductions in HR and MAP relative to vehicle-treated controls (Fig. 5, A and B, treatment group, time, and interaction effects, P < 0.05 for all). In all hemodiluted groups, there was a significant reduction in brain microvascular PBrO2 as previously reported16 (Fig. 5C, time effect, P < 0.001) with a significant time and treatment group interaction (P = 0.024). In vehicle-treated controls, the PBrO2 was reduced to 45.8 ± 18.7 mm Hg after hemodilution (Fig. 5, corrected P < 0.001, 95% CI 29.4–69.7), consistent with previously reported values.16,18 After hemodilution, low-dose nebivolol did not result in a further reduction in brain microvascular PBrO2, relative to vehicle-treated control rats (Fig. 5C, corrected P = 0.432, 36.0 ± 4.9 mm Hg, 95% CI 28.8–43.1). In contrast, high-dose nebivolol resulted in a significant further reduction in brain microvascular PBrO2 relative to vehicle-treated control rats after hemodilution (Fig. 5C, corrected P = 0.019, 28.4 ± 9.6 mm Hg, 95% CI 17.4–42.7).

Figure 5
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Protocol 4: Effect of Nebivolol and Hemodilution on Brain HIF-α and NOS Protein Levels

Treatment with high-dose nebivolol resulted in an increase in all 3 cerebral cortical NOS isoform protein levels, relative to vehicle-treated controls at 1 hour after hemodilution. Vehicle-treated controls versus high-dose nebivolol had the following NOS values (median [25%–75%]): eNOS (0.020 [0.015–0.041] vs 0.176 [0.156–0.183], P = 0.004; iNOS (0.339 [0.292–0.387] vs 0.523 [0.461–1.173], P = 0.006); nNOS (0.311 [0.278–0.378] vs 0.518 [0.378–0.716], P = 0.027) (Fig. 6, corrected P < 0.05 for all). Protein levels were not significantly elevated with low-dose nebivolol treatment versus controls for any NOS isoform.

Figure 6
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Similarly, compared with vehicle-treated controls, cerebral cortical expression of HIF-1α was increased in the high-dose nebivolol treatment group after hemodilution (0.275 [0.146–0.294] vs 0.427 [0.334–0.584], P = 0.032, respectively) (Fig. 7, corrected P < 0.05). By contrast, expression of GSNOR was reduced in the high-dose nebivolol treatment group compared with vehicle-treated controls after hemodilution (0.464 [0.401–0.513] vs 0.305 [0.209–0.340], P = 0.013) (Fig. 7, corrected P < 0.05). Immunostaining for HIF-1α and nNOS demonstrated increased cellular and perivascular HIF-1α staining, most predominantly after hemodilution and high-dose nebivolol treatment (Figs. 8 and 9).

Figure 7
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Figure 8
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Figure 9
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Protocol 5: Effect of Hemodilution and Nebivolol on Brain HIF-α Luciferase Activity in HIF-(ODD)-Luciferase Mice

Treatment with low- and high-dose nebivolol resulted in a similar ~20% reduction in HR in mice (Fig. 10, corrected P = 0.006 for both relative to baseline). After hemodilution to an Hb target of 60 g/L, there was a significant increase in the cerebral cortical HIF-luciferase signal in the high-dose nebivolol treatment group, relative to vehicle-treated controls (2.486 [2.062–2.535] vs 0.977 [0.559–1.441], P = 0.006) (Fig. 11). There was also a significant increase in the liver HIF-luciferase signal in the high-dose nebivolol treatment group after hemodilution, relative to vehicle-treated controls (1.718 [1.516–1.933] vs 1.075 [0.826–1.174], P = 0.033) (Fig. 11). There was no difference in the HIF-luciferase signal in the heart or kidney after hemodilution (Fig. 11, P = 0.994 and 0.731, respectively).

Figure 10
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Figure 11
Figure 11
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CONCLUSIONS

These data demonstrate that acute high-dose β-antagonism impaired cerebral perfusion after hemodilution. A clear dose response was observed regarding the attenuation in the rCBF response, a reduction in microvascular PBrO2, and an increase in the expression of hypoxic cellular responses in the brain. All of these treatment effects were significantly accentuated by high-dose nebivolol treatment, relative to both low-dose and vehicle-treated groups. These findings support the hypothesis that treatment with a highly β1-selective antagonist causes a dose-dependent impairment in cerebral oxygen delivery during acute hemodilution.

In a blinded protocol with concealed drug dosing, we observed that treatment with a higher dose of nebivolol resulted in a significant reduction in microvascular PBrO2, relative to vehicle-treated controls, in acutely anemic rats. These noninvasive and quantitative measurements of PBrO2 are comparable to previously published values for PBrO2 at baseline21,22 and after hemodilution.16,18 The lower PBrO2 observed in the high-dose nebivolol group corresponded to an increased expression of hypoxic proteins (HIF-1α, iNOS, and nNOS), suggesting that the measured reduction in PBrO2 was sensed at the cellular level and was therefore biologically relevant. In addition, we have confirmed this dose-dependent effect of high-dose nebivolol on real-time cerebral HIF-α expression in a previously established transgenic mouse model of acute hemodilution [HIF-(ODD)-luciferase mice].18 These mice demonstrated an increase in HIF-α expression (firefly luciferase activity) in the anemic high-dose nebivolol treatment group. To demonstrate that not all gene expression was increased in our model, we measured GSNOR protein levels,18 which decreased during anemia and β-blockade. This finding supports the conclusion that the observed increase in hypoxic molecules (HIF-1α, nNOS, and iNOS) was specific to the measured degree of tissue hypoxia.

HIF-1α and nNOS were measured in this study because they are established markers of reduced brain tissue PO2 during both hypoxia23,24 and anemia.20,25–28 Although the observed increases in HIF-1α and nNOS protein levels during anemia could be either adaptive or maladaptive, we favor the likelihood that they are adaptive physiologic changes directed at maintaining oxygen homeostasis and cell survival during anemia based on the following reasoning. Mice lacking nNOS have been shown to be susceptible to anemia-induced mortality and die earlier and at higher Hb concentrations, relative to wild-type nNOS-replete mice.18 In addition, nNOS-deficient mice lack the appropriate cardiovascular (CO) and hypoxic cellular responses (HIF-α) to anemia, providing a potential mechanistic explanation for the increase in mortality.18 The timeframe of the nNOS and HIF response is rapid, consistent with the need to adapt suddenly to changes in oxygen delivery during acute anemia. Both HIF and nNOS responses can occur within 30 to 60 minutes of exposure to reductions in brain tissue PO2.29–33 These rapid increases are consistent with the need for dynamic cell responses to anemia-induced tissue hypoxia. However, in the context of acute β-blockade and surgical blood loss in patients with cardiovascular morbidity, the increased expression of hypoxic cellular mediators may also reflect inadequate cerebral perfusion and contribute to the mechanism of increased stroke and mortality observed in patients.10 Further study is required to determine whether modifying the dose or type of β-blocker used can limit morbidity and mortality in high-risk surgical populations.

Although we have previously demonstrated upregulation of HIF-dependent proteins responsible for increased glucose transport and glycolytic flux in the brain during anemia,18 we have not directly measured changes in metabolism associated with anemia and β-blockade in the current study. Recently, noninvasive measurements of tissue PO2 using phosphorescence quenching or endogenous nicotine adenine dinucleotide, reduced (NADH) fluorescence have been used to facilitate oxygen imaging using 2-photon microscopy.21,22,34 The collective data from these studies demonstrate that the brain microvascular PO2 is in the range of ~80 mm Hg in the arteriole decreasing rapidly to ~60 mm Hg within the capillary at points near red blood cells, with a further decrease to ~30 mm Hg in the plasma more distant from the red blood cells, and to ~20–25 mm Hg in brain tissue. Reduction in oxygen delivery by reducing the fraction of inspired oxygen or inhibiting ventilation results in rapid decreases in tissue PO2.21,22 In addition, NADH florescence measurements have demonstrated that the increase in intrinsic NADH fluorescence that occurs at a tissue threshold near 10 mm Hg is an indicator of metabolically limiting tissue hypoxia. Although we have not directly measured PO2 at the capillary-tissue level, the reduction of our noninvasive microvascular tissue PO2 values from ~70 to ~20 mm Hg are within the range of metabolically limiting tissue hypoxia.34

Nebivolol exerted a comparable negative chronotropic effect in both drug-dose treatment groups, likely by blocking β1-adrenoreceptors at the cardiac myocyte.35 As a result, both drug doses had a comparable effect on attenuating the CO response to anemia. This suggests that plasma drug levels in both low- and high-dose treatment groups (109 ± 35 nM and 202 ± 22 nM, respectively) were adequate to bind with high affinity to the β1-adrenoreceptors (binding affinity ~0.7 nM).36 By contrast, only the high-dose treatment group achieved drug levels (202 ± 22 nM) that approached the affinity of nebivolol for the vascular β2-adrenoreceptors (225 nM).36 Interaction of nebivolol with cerebral vascular β2-adrenoreceptors in the high-dose group provides a potential explanation for the observed differential effects on cerebral perfusion in our model of acute hemodilution. By inference, the drug concentration achieved with the high nebivolol dose may inhibit β2-mediated responses, including cerebral vasodilation.17,37 Further mechanistic studies are required to establish this potential mechanism. Regardless of the mechanism, our data support that low-dose nebivolol limited cardiac oxygen demand (attenuating HR and CO) while maintaining adequate cerebral oxygen delivery in the setting of acute hemodilutional anemia. Of interest, neither dose of nebivolol inhibited the increase in SV, which contributes to maintaining the increase in CO during acute hemodilution.38 This is in contrast with a previous study in which metoprolol attenuated both the HR and SV responses to acute hemodilution.16 This preservation of SV may be advantageous and provides some indirect evidence that nebivolol may have vasodilatory capacity (increased venous return) in this setting. The mechanism by which nebivolol mediates vasodilation may include increased eNOS expression36 as supported by its increased expression in the current study.

There are several limitations to our study. We used a fraction of inspired oxygen of 50% and maintained animals near 37°C to model conditions in the human operating room.16 These conditions may not perfectly model the conditions of human anesthesia and surgery. Rats have a much higher baseline HR compared with humans and thus may be more profoundly affected by β-blockade. We attempted to use a clinically relevant dose of nebivolol39 that reduced HR by a clinically relevant target of ~20%.38,40 We did not directly assess the effect of high-dose nebivolol on β2-adrenoreceptors. Future studies could examine the effect of the 2 doses of nebivolol on resistance artery function.17 The method of measuring rCBF using laser Doppler was focal and semiquantitative and is associated with lower sensitivity and spatial/temporal resolution,22,41 but these measurements did show significant differences in relative CBF during our study. We did not assess different regions of the brain, which may limit the interpretation of results because of the known heterogeneity in metabolic demands in different brain regions.22 Similarly, quantitative, noninvasive measurements of microvascular tissue PO2 lack spatial resolution, although they are independent of changes in other inert gases and are not influenced by optical properties of the brain tissue.22,41 Finally, all of our experiments used young and healthy rats without associated comorbidities and we did not study the effect of the 2 doses of nebivolol using an experimental stroke model, which would provide additional insight into the impact of β-adrenergic blockade on ischemic stroke.

In summary, we have provided novel data demonstrating that treatment with a highly β1-selective antagonist caused a dose-dependent decrease in brain perfusion during hemodilution. Two nebivolol doses comparably reduced HR and MAP and attenuated the CO response to hemodilution (primarily a β1 effect); however, only the higher dose of nebivolol limited brain oxygen delivery as supported by quantitative PBrO2 measurements and expression of hypoxic proteins in vivo (HIF-1α, iNOS, and nNOS). Because only the higher dose of nebivolol resulted in plasma drug levels that approached the affinity of nebivolol for the β2-adrenoreceptor, these data support the derivation of a new hypothesis: that cerebral perfusion was maintained during acute hemodilution after treatment with low-dose nebivolol by minimizing the effect of β2-mediated antagonism of cerebral vasodilation.

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DISCLOSURES

Name: Tina Hu, MSc.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Tina Hu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: W. Scott Beattie, MD, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: W. Scott Beattie has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: C. David Mazer, MD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: C. David Mazer has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Howard Leong-Poi, MD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Howard Leong-Poi has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Hiroko Fujii, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Hiroko Fujii has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: David F. Wilson, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: David F. Wilson has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Albert K. Y. Tsui, PhD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Albert K. Y. Tsui has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Elaine Liu, MD.

Contribution: This author helped conduct the study.

Attestation: Elaine Liu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Maimoona Muhammad, BSc.

Contribution: This author helped conduct the study.

Attestation: Maimoona Muhammad has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Andrew J. Baker, MD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Andrew J. Baker has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Gregory M. T. Hare, MD, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Gregory M. T. Hare has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Conflicts of Interest: Gregory M. T. Hare received research funding from Forest Research Institute. This work was funded through an investigator-initiated trial grant from Forest Research Institute (subsidiary of Forest Laboratories).

This manuscript was handled by: Gregory J. Crosby, MD.

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ACKNOWLEDGMENTS

The authors acknowledge Dr. Henry Li and Dr. Julie Wangsa (Forest Research Institute, New York, NY) for assistance in determining nebivolol plasma levels, Mr. Sanjay Yagnik (Research Coordinator, Department of Anesthesia, St. Michael’s Hospital, Toronto, ON, Canada) for his help in blinding the experiments, and Ms. Judy Trogardis (Chief Technologist, Bio-Imaging Facility, Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON, Canada) for assistance in obtaining immunofluorescence images.

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