Artificial Cells, Nanomedicine, and Biotechnology, 2015; 43: 87–92 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.916716
Hemoglobin-based oxygen carrier attenuates cerebral damage by improving tissue oxygen preload in a dog model of cardiopulmonary bypass Qian Li1*, Shen Li2*, Qian Yang3, Tao Li1, Jin Liu1 & Chengmin Yang2 1Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu,
Sichuan, P. R. China, 2Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, P. R. China, and 3Department of Pharmacy, Chengdu Medical College, Chengdu, P. R. China
Hemoglobin-based oxygen carrier (HBOC) was initially developed as blood substitutes for surgical and trauma patients with hemorrhagic shock, especially in emergency and military settings (Chang 2005, Varnado et al. 2013). It possesses inherent advantages compared to stored erythrocytes, such as higher oxygen (O2) affinity, lower viscosity, and smaller mean diameter, which may be helpful in providing sufficient microcirculation perfusion and thereby alleviating organ I/R injury (Wu et al. 2011). Glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) is a particularly promising HBOC developed in China (Li et al. 2006). Our previous studies have demonstrated that it could provide protections on various organs against I/R injury, including heart (Li et al. 2009a, 2009b, 2010a, 2010b, 2011), lung (Li et al. 2013), kidney (Li et al. 2012b) and liver (You et al. 2013). The underlying molecular mechanisms are implicated in attenuation of apoptosis, quenching of oxidative stress, and restoration of nitroso-redox balance (Li et al. 2009a, 2009b, 2010b). Here, we tested the hypothesis that pretreatment with PolyPHb could attenuate CPB-induced cerebral damage.
Abstract In order to investigate whether hemoglobin-based oxygen carrier (HBOC) attenuates cardiopulmonary bypass (CPB)-induced cerebral damage. Male adult Beagle dogs were randomly divided into sham, control, and HBOC groups. After establishment of CPB model, hearts were arrested for 2 h and reperfused for 2 h. HBOC improved intracerebral O2 tensions and reduced the releases of biomarkers for cerebral damage, including neuron-specific enolase and S100b in both cerebrospinal fluid and serum. Moreover, HBOC attenuated the releases of tumor necrosis factor-a and interleukin-1b after CPB. Therefore, our findings suggest that HBOC could reduce cerebral damage after CPB, which was probably associated with improvement of tissue O2 preload. Keywords: brain, cardiopulmonary bypass, hemoglobin-based oxygen carrier, inflammation, ischemia/reperfusion injury
Introduction During cardiac surgery with cardiopulmonary bypass (CPB), normal circulation may be temporarily halted to ensure a clean, bloodless operation field. As a consequence, many organs may be suffered from ischemia/ reperfusion (I/R) injury, which is the primary cause of death and poor prognosis in patients undergoing cardiac surgery (de Vries et al. 2013). The brain is the organ most vulnerable to ischemic injury during this period, possibly leading to postoperative neurocognitive decline (POCD) (Taylor 1998). As the population ages, this problem is likely to be worse given that elderly brain are more susceptible to I/R injury (de Tournay-Jetté et al. 2011). Thus, strategies that provide cerebral protection in CPB are urgently required.
Materials and methods All of the experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University, and all of the animals received human care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult male beagle dogs, approximately 6–8 months old and weighing 8–10 kg, were housed at a constant temperature (22 3°C) in a 12-h light/dark cycle and were given free access to food and water.
*Qian Li and Shen Li contributed equally to this study. Correspondence: Tao Li, PhD, Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu 610041, P. R. China. Tel: (6828)85164040. Fax: (6828)-85423593. E-mail: [email protected] (Received 24 March 2014; accepted 16 April 2014)
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HBOC The HBOC used in this study was PolyPHb (8–10 gHb/dL, methemoglobin 3%, tetrameric hemoglobin 1%), which was prepared as reported previously (Li et al. 2006). Briefly, purified and viral inactivated fresh human placenta hemoglobin (Tianjin Union Stem Cell Genetic Engineering Ltd, Tianjin, China) was modified with bis(3,5-dibromosalicyl) fumarate to achieve optimal O2 affinity. After cross-linkage with glutaraldehyde, the mixture was subject to ultrafiltration and molecular sieve chromatography. The final product had a molecular weight of 64–600 kDa.
Dog CPB model Dog CPB model was established as described previously (Li et al. 2012a, 2014). After induction (4 mg/kg propofol, 0.1 mg/kg midazolam, and 5 μg/kg fentanyl) and muscle relaxation (1 mg/kg scoline), all the dogs were intubated with an Fr. 7.5 endotracheal tube and mechanically ventilated with tidal volume of 10 mL/kg (Datex-Ohmeda Excel 210, Soma Technology, Cheshire, Connecticut, USA). Each group received a continuous infusion of fentanyl at 0.3 μg/kg/min and vecuronium bromide at 0.2 mg/kg/h during surgery. Anesthesia was maintained with 150 μg/kg/min propofol. After heart exposure through a mid-sternal incision and heparinization (3 mg/kg), the ascending aorta and the right atrial appendage were cannulated. The CPB circuit was composed of a rolling pump (StÖckert II, Munich, Germany), a membrane oxygenator (1500 mL/min, Kewei Medical Ltd., Guangdong, China), and an arterial filter (Kewei Medical Ltd., Guangdong, China). The CPB was primed with Lactate Ringer’s solution containing 5% sodium bicarbonate (10 mL/L), 20% mannitol (2.5 mL/L), furosemide (0.5–1.0 mg/L), dexamethasone (5 mg/L), heparin (10 mg/L), and 10% potassium chloride (5 mL/L). Also, a 10% calcium gluconate (2–4 mL) was added every 30 min for four times.
Thomas’ solution (STS), and then reperfused for 2 h using aortic declamping. The dogs without cardiac arrest and reperfusion were allocated into the sham group.
Hemodynamic monitoring A Swan–Ganz float catheter (No. 7, Edwards Laboratories, Irvine, CA, USA) was inserted via femoral vein and advanced to pulmonary artery, the blood temperature and hemodynamics parameters, including heart rate, pulmonary artery wedge pressure (PAWP), pulmonary arterial pressure (PAP), central venous pressure (CVP) were collected using a PowerLab data-acquisition system (ADInstruments Pty, Bella Vista, NSW, Australia).
Measurement of blood gas values Arterial and venous blood samples were collected in polyethylene catheter placed in the left femoral artery or the Swan–Ganz float catheter. The blood gas parameters, including pH, arterial O2 partial pressure (PaO2), venous O2 partial pressure (PvO2) were measured immediately by a blood gas analyzer (ABL800 FLEX, Radiometer Medical A/S, Copenhagen, Denmark).
Intracerebral O2 tension and blood flow measurements A scalp incision was made to expose the right parietal bone. After drilling a 5-mm burr hole, the tip of the composite tissue PO2 laser Doppler flow probe with thermocouple (Oxford Optronix, Ltd, Oxford, UK) was inserted into the parasagittal parietal lobe to a depth of approximately 5 mm below the dura mater. Then, the probe was fixed to the skull with tissue glue, and supported with a custom-made probe holder. The intracerebral O2 tensions (tPO2) and cerebral blood flow (CBF) were continuously collected using a PowerLab data-acquisition system.
Experimental protocol
Measurement of neurological markers and inflammatory cytokines
The experimental protocol is schematically illustrated in Figure 1. Eighteen adult male beagle dogs were randomly divided into three groups (n 6): sham, control, and HBOC groups. Before heart exposure, animals were pretreated with intravenous injection of 0.1 gHb/kg PolyPHb (HBOC group) or equal amount of physiological saline (control group). After establishment of CPB and equilibration, hearts were arrested for 2 h using intra-aortic infusion of 40 mL/kg St.
Cerebrospinal fluid (CSF) and serum samples were collected before CPB and after 2 h of reperfusion and stored at 80°C until used. The levels of neuron-specific enolase (NSE) and S100β were analyzed in duplicate with commercially available ELISA test kits (Nanjing Jiancheng, Nanjing, China). The releases of tumor necrosis factor (TNF)-α and Interleukin (IL)-1β in serum were also determined (R&D, Minneapolis, MN, USA).
Figure 1. A schematic representation of the experimental protocol.
HBOC and CPB-induced cerebral damage
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Statistical analysis All values are presented as mean SD. An unpaired Student’s t test was used to detect significant differences when two groups were compared. One-way or two-way ANOVA was used to compare the differences among three groups followed by Bonferroni’s multiple comparison tests as applicable (SPSS 16.0 software). In addition, a simple linear regression analysis was performed to estimate the correlation between cortical tPO2 and neurological markers. P values less than 0.05 were considered statistically significant. Figure 2. HBOC improved cortical tPO2. Values are presented as the means SD (n 3). tPO2: tissue oxygen partial pressure.
Results HBOC elevates intracerebral O2 supply As shown in the Figure 2, infusion of HBOC was associated with an increase of tPO2 in parietal lobe cortex, from 11.75 1.28 to a peak of 28.79 3.98 torr at 16 min after infusion (P 0.001, n 3). Even though the tPO2 decreased gradually to 23.73 7.69 torr at 30 min after infusion, it still greatly higher than that of the animal received physiological saline (11.48 2.44 torr, P 0.01). The CBF values were not altered after HBOC infusion (data not shown).
was companied by elevations of PAP and PAWP. We confirmed that in the control group, both PAP and PAWP greatly increased as compared to that of the sham group (P 0.001 and P 0.001, respectively). This elevation was significantly reduced by HBOC pretreatment (P 0.01 and P 0.01 vs. the control group, respectively). Besides, no significant differences of CVP, pH, and blood temperature were found among groups.
HBOC improves hemodynamic and blood gas parameters after CPB
HBOC inhibits the releases of NSE and S100b
As presented in the Table I, HBOC increased the PaO2 and reduced the PvO2 during reperfusion, suggesting the circulatory O2 supply was well preserved by HBOC after CPB-induced I/R injury. Usually, circulatory dysfunction
As markers of neurological damage, the releases of NSE and S100β were measured. There were no significant differences of NSE and S100β releases before CPB. At 2 h after CPB, the NSE levels were significantly increased in the control group, in both serum and CSF (P 0.001 and P 0.001
Table I. The hemodynamic and blood gas parameters at baseline and during reperfusion. Reperfusion Baseline T (oC) Sham Control HBOC HR (bpm) Sham Control HBOC PaO2 (mmHg) Sham Control HBOC PvO2 (mmHg) Sham Control HBOC CVP (mmHg) Sham Control HBOC PAP (mmHg) Sham Control HBOC PAWP (mmHg) Sham Control HBOC
30 min
60 min
90 min
120 min
38.95 0.07 38.77 1.21 38.8 0.14
34.45 1.06 33.97 1.31 33.45 1.34
34.3 0.57 33.97 1.11 33.45 0.92
34.4 0.56 34.03 0.95 33.55 0.64
34.85 0.92 34.2 0.8 33.8 0.57
139 11 142 12 140 11
139 10 138 13 136 13
145 11 135 12 137 10
149 11 140 13 139 11
142 12 138 10 136 11
352.83 73.91 256.00 63.37 292 47.94*
418.83 61.81 265.83 78.98 294.50 69.63*
414.33 56.11 372.17 69.84 339.50 74.81 413.00 59.28 302.50 62.11 252.50 58.89 409.50 61.83 329.50 60.62* 288.17 70.04* 34.50 9.05 34.50 12.01 36.83 10.34
31.83 10.50 70.33 23.43 42.67 12.94*
38.00 11.51 63.67 38.69 36.00 13.16*
35.00 16.14 54.50 20.57 32.33 16.31**
37.17 12.81 58.83 23.71 35.83 19.26*
8.83 0.80 9.17 0.82 9.00 0.98
9.00 0.98 9.75 0.98 9.75 1.19
8.67 0.91 9.10 0.96 9.30 1.13
8.33 0.87 9.33 0.92 9.00 1.05
8.67 0.79 8.83 0.86 9.05 0.93
17.00 1.12 17.17 1.42 16.83 1.35
17.00 1.99 22.50 2.37 18.67 2.40**
17.07 2.08 23.50 2.54 18.50 2.52**
16.83 1.72 22.83 1.87 18.50 1.91**
16.67 1.85 23.67 1.82 19.00 1.93**
11.33 1.33 11.50 1.51 12.17 1.46
11.50 1.15 13.59 1.17 11.67 1.16**
11.17 1.31 14.50 1.27 12.33 1.25**
11.00 1.33 15.43 1.37 12.83 1.31**
11.03 1.37 15.33 1.45 12.33 1.41**
T, temperature; HR, heart rate; PaO2, arterial O2 partial pressure; PvO2, venous O2 partial pressure, CVP, central venous pressure; PAP, pulmonary arterial pressure; PAWP, pulmonary artery wedge pressure. Values were reported as Mean SD (n 6), *P 0.05; **P 0.01 versus the control group.
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Figure 3. HBOC reduced the releases of NSE and S100β in both serum (A and B) and CSF (C and B). Values were expressed as the means SD (n 6). *P 0.05 and ***P 0.001 vs. the control group. CSF: cerebrospinal fluid; NSE: neuron-specific enolase.
vs. the sham group). In contrast, animals received HBOC appeared a great reduction in NSE level (P 0.05 and P 0.001 vs. the control group; Figure 3A and B). Consistently, in the control group, the S100β release in serum and CSF was highly elevated (P 0.001 and P 0.001 vs. the sham group), while in the HBOC group, this increase
was greatly attenuated (P 0.05 and P 0.001 vs. the control group; Figure 3C and D). It is worth pointing out that this inhibitory effect was more prominent on neurological biomarkers in CSF. Moreover, linear regression analysis indicated a strong correlation between cortical tPO2 and releases of NSE and S100β after CPB (Figure 4).
Figure 4. The linear regression analysis of the relationship between cortical tPO2 and releases of NSE (A and C) and S100β (B and D) (n 6). CSF: cerebrospinal fluid; NSE: neuron-specific enolase; tPO2: tissue oxygen partial pressure.
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Figure 5. The levels of TNF-α (A) and IL-1β (B) in serum before and after CPB. Values are presented as mean SD (n 6). *P 0.05 and **P 0.01 versus the control group. CPB: cardiopulmonary bypass; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β.
HBOC reduces the releases of TNF-a and IL-1b after CPB The levels of both TNF-α and IL-1β in the control group were increased after CPB (P 0.001 and P 0.001 vs. the sham group, respectively; Figure 5). HBOC treatment significantly inhibited the releases of these inflammatory cytokines (P 0.05 and P 0.01 vs. the control group, respectively), suggesting the inflammation induced by CPB was greatly depressed.
Discussion In theory, HBOC allows delivery of more O2 to hypoxic tissues due to its higher O2 affinity, lower viscosity, and smaller mean diameter than human erythrocytes. These features help to provide sufficient perfusion of the microcirculation and thereby alleviate I/R injury. In the current study, we confirmed that the cerebral tissue O2 supply can be improved by HBOC as evidenced by increased cortical tPO2. This increased O2 preload for brain would be beneficial to against following I/R injury. For the early diagnosis of ischemic brain injury due to CPB, brain biomarkers, such as NSE and S100β, are commonly used. These biomarkers have been well-documented to present into blood and CSF during CPB and may reflect both cerebral injury and increased permeability of the blood–brain barrier (Mercier et al. 2013, Mondello et al. 2011). Consistent with increased cerebral O2 preload, our study indicated that HBOC greatly reduced the levels of both NSE and S100β after CPB, suggesting a significant protection on brain. As we know, except for I/R injury, CPB may cause systemic inflammatory response syndrome (SIRS). The mechanism is likely an abnormal regulation of various immune-modulating agents triggered by trauma of the blood cells or ischemia injury of the organs. As a consequence, cytokines are released, and the aggregate of these responses could lead to microcirculatory dysfunction and ultimately to renal dysfunction and/or pulmonary edema, which would jointly cause cerebral damage (Pahari et al. 2013). Therefore, it is reasonable that the elevated tissue O2 preload is also capable of reducing possible SIRS and brain damage during CPB. Our data clearly supported this notion that both the inflammation and cerebral damage were attenuated by high O2 supply from HBOC. Even though the present data cannot figure out the exact molecular signaling pathway underlying the cerebral
protective effect of HBOC, we believe improvement of O2 supply in brain plays an important role in this effect. As reported previously, the O2 releasing capacity of solution with HBOC is nearly 1.5-fold higher than that of the control solution, and the inhibited release of lactic acid from tissue provided additional evidence that the O2 supply could be enhanced by HBOC (Li et al. 2010b). In our study, by use of tissue PO2 and laser Doppler flow detection system, we continuously measured the cortical tPO2 before and 30 min after HBOC infusion. The data clearly indicated that the O2 supply in the cortex was remarkably increased as compared to the control group, even though the blood flow was no changed. We think this increased O2 supply should be attributed to the excellent O2 releasing capacity of HBOC. Oxygen consumption is regarded to be strongly correlated with organ performance (Gutterman and Cowley 2006). The present study confirmed this fact and further proved that the cerebral tPO2 was significantly correlated with the level of cerebral damage, suggesting tPO2 in brain is predictive of cerebral damage in surgery with CPB. The possible clinical application of this finding is that we should pay more attention to the patients whose cerebral tPO2 decreases sharply during surgery, and we could have taken some positive measures to improve it before it comes apparent, such as use of HBOC or just by inhalation of high O2.
Conclusion In conclusion, with a dog CPB model, we demonstrated that HBOC pretreatment exerts a profound protective effect to against possible cerebral I/R injury, and the proposed mechanism for this protection is associated with the improved tissue O2 preload.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by grants from the National Nature Science Foundation of China (81300110 and 81100180), the 2013 Research Fund for Outstanding Young Scholars of Sichuan University, and the Specialized Research Fund for the Doctoral Program of Higher Education (20100181120090).
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References Chang TMS. 2005. Therapeutic applications of polymeric artificial cells. Nat Rev Drug Discov. 4:221–235. de Tournay-Jetté E, Dupuis G, Bherer L, Deschamps A , Cartier R, Denault A . 2011. The relationship between cerebral oxygen saturation changes and postoperative cognitive dysfunction in elderly patients after coronary artery bypass graft surgery. J Cardiothorac Vasc Anesth. 25:95–104. de Vries DK , Kortekaas KA , Tsikas D, Wijermars LG, van Noorden CJ, Suchy M-T, et al. 2013. Oxidative damage in clinical ischemia/ reperfusion injury: a reappraisal. Antioxid Redox Signal. 19: 535–545. Gutterman DD, Cowley AW. 2006. Relating cardiac performance with oxygen consumption: historical observations continue to spawn scientific discovery. Am J Physiol Heart Circ Physiol. 291: H2555–H2556. Li T, Li J, Liu J, Zhang P, Wu W, Zhou R, et al. 2009a. Polymerized placenta hemoglobin attenuates ischemia/reperfusion injury and restores the nitroso-redox balance in isolated rat heart. Free Radic Biol Med. 46:397–405. Li T, Liu J, Yang C. 2010a. Pretreatment with hemoglobin-based oxygen carriers protect isolated rat heart from myocardial infarction. Artif Cells Blood Substit Immobil Biotechnol. 38: 115–118. Li T, Liu J, Yang Q, Wu W, Zhang P, Yang J, et al. 2009b. Polymerized placenta hemoglobin improves cardiac functional recovery and reduces infarction size of isolated rat heart. Artif Cells Blood Substit Immobil Biotechnol. 37:48–52. Li T, Wu W, You Z, Zhou R, Li Q, Zhu D, et al. 2012a. Alternative use of isoflurane and propofol confers superior cardioprotection than using one of them alone in a dog model of cardiopulmonary bypass. Eur J Pharmacol. 677:138–146. Li T, Yu R, Zhang HH, Wang H, Liang WG, Yang XM, Yang CM. 2006. A method for purification and viral inactivation of human placenta hemoglobin. Artif Cells Blood Substit Immobil Biotechnol. 34: 175–188. Li T, Zhang P, Liu J, Zhou R, Li Q, You Z, Dian K . 2010b. Protective effects of hemoglobin-based oxygen carrier given to isolated heart during ischemia via attenuation of mitochondrial oxidative damage. Free Radic Biol Med. 48:1079–1089.
Notice of correction The version of this article published online ahead of print on 28 May 2014 contained multiple labeling errors in Table 1. The errors have been corrected for this version.
Li T, Zhang Z, Liao D, Chen Y, Yang C, Xu X, Liu J. 2012b. The effect of polymerized placenta hemoglobin on renal ischemia/ reperfusion injury. Artif Cells Blood Substit Immobil Biotechnol. 40:396–399. Li T, Zhang Z, Wu W, Liao D, Chen Y, Li S, et al. 2013. Resuscitation with polymerized human placenta hemoglobin attenuated hemorrhagic shock-induced lung injury. Artif Cells Nanomed Biotechnol. 41:27–31. Li T, Zhou R, Xiang X, Zhu D, Li Y, Liu J, et al. 2011. Polymerized human placenta hemoglobin given before ischemia protects rat heart from ischemia reperfusion injury. Artif Cells Blood Substit Immobil Biotechnol. 39:392–397. Li T, Zhou R, Yao Y, Yang Q, Zhou C, Wu W, et al. 2014. Angiotensinconverting enzyme inhibitor captopril reverses the adverse cardiovascular effects of polymerized hemoglobin. Antioxid Redox Signal. [Epub ahead of print]. Mercier E, Boutin A , Lauzier F, Fergusson DA , Simard J-F, Zarychanski R, et al. 2013. Predictive value of S-100β protein for prognosis in patients with moderate and severe traumatic brain injury: systematic review and meta-analysis. BMJ. 346:f1757. Mondello S, Muller U, Jeromin A , Streeter J, Hayes RL, Wang KK . 2011. Blood-based diagnostics of traumatic brain injuries. Expert Rev Mol Diagn. 11:65–78. Pahari DR, Gu YJ, van Oeveren W, El‐Essawi A , Harringer W, Brouwer RM. 2013. Effect of minimized perfusion circuit on brain injury markers carnosinase and brain‐type fatty binding protein in coronary artery bypass grafting patients. Artif Organs. 37:128–135. Taylor KM. 1998. Brain damage during cardiopulmonary bypass. Ann Thorac Surg. 65: S20–S28. Varnado CL, Mollan TL, Birukou I, Smith BJ, Henderson DP, Olson JS. 2013. Development of recombinant hemoglobin-based oxygen carriers. Antioxid Redox Signal. 18:2314–2328. Wu W, Li T, Liu J, Yang C. 2011. Pretreatment before ischemia induction with polymerized human placenta hemoglobin (PolyPHb) attenuates ischemia/reperfusion injury-induced myocardial apoptosis. Artif Cells Blood Substit Immobil Biotechnol. 39:3–6. You Z, Li Q, Li B, Yang C, Liu J, Li T. 2013. Isovolemic hemodilution with glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) attenuated rat liver ischemia/reperfusion injury. Artif Cells Nanomed Biotechnol. 42:83–87.
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Hemoglobin-based oxygen carrier attenuates cerebral damage by improving tissue oxygen preload in a dog model of cardiopulmonary bypass Qian Li1*, Shen Li2*, Qian Yang3, Tao Li1, Jin Liu1 & Chengmin Yang2 1Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu,
Sichuan, P. R. China, 2Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, P. R. China, and 3Department of Pharmacy, Chengdu Medical College, Chengdu, P. R. China
Hemoglobin-based oxygen carrier (HBOC) was initially developed as blood substitutes for surgical and trauma patients with hemorrhagic shock, especially in emergency and military settings (Chang 2005, Varnado et al. 2013). It possesses inherent advantages compared to stored erythrocytes, such as higher oxygen (O2) affinity, lower viscosity, and smaller mean diameter, which may be helpful in providing sufficient microcirculation perfusion and thereby alleviating organ I/R injury (Wu et al. 2011). Glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) is a particularly promising HBOC developed in China (Li et al. 2006). Our previous studies have demonstrated that it could provide protections on various organs against I/R injury, including heart (Li et al. 2009a, 2009b, 2010a, 2010b, 2011), lung (Li et al. 2013), kidney (Li et al. 2012b) and liver (You et al. 2013). The underlying molecular mechanisms are implicated in attenuation of apoptosis, quenching of oxidative stress, and restoration of nitroso-redox balance (Li et al. 2009a, 2009b, 2010b). Here, we tested the hypothesis that pretreatment with PolyPHb could attenuate CPB-induced cerebral damage.
Abstract In order to investigate whether hemoglobin-based oxygen carrier (HBOC) attenuates cardiopulmonary bypass (CPB)-induced cerebral damage. Male adult Beagle dogs were randomly divided into sham, control, and HBOC groups. After establishment of CPB model, hearts were arrested for 2 h and reperfused for 2 h. HBOC improved intracerebral O2 tensions and reduced the releases of biomarkers for cerebral damage, including neuron-specific enolase and S100b in both cerebrospinal fluid and serum. Moreover, HBOC attenuated the releases of tumor necrosis factor-a and interleukin-1b after CPB. Therefore, our findings suggest that HBOC could reduce cerebral damage after CPB, which was probably associated with improvement of tissue O2 preload. Keywords: brain, cardiopulmonary bypass, hemoglobin-based oxygen carrier, inflammation, ischemia/reperfusion injury
Introduction During cardiac surgery with cardiopulmonary bypass (CPB), normal circulation may be temporarily halted to ensure a clean, bloodless operation field. As a consequence, many organs may be suffered from ischemia/ reperfusion (I/R) injury, which is the primary cause of death and poor prognosis in patients undergoing cardiac surgery (de Vries et al. 2013). The brain is the organ most vulnerable to ischemic injury during this period, possibly leading to postoperative neurocognitive decline (POCD) (Taylor 1998). As the population ages, this problem is likely to be worse given that elderly brain are more susceptible to I/R injury (de Tournay-Jetté et al. 2011). Thus, strategies that provide cerebral protection in CPB are urgently required.
Materials and methods All of the experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University, and all of the animals received human care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult male beagle dogs, approximately 6–8 months old and weighing 8–10 kg, were housed at a constant temperature (22 3°C) in a 12-h light/dark cycle and were given free access to food and water.
*Qian Li and Shen Li contributed equally to this study. Correspondence: Tao Li, PhD, Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu 610041, P. R. China. Tel: (6828)85164040. Fax: (6828)-85423593. E-mail: [email protected] (Received 24 March 2014; accepted 16 April 2014)
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HBOC The HBOC used in this study was PolyPHb (8–10 gHb/dL, methemoglobin 3%, tetrameric hemoglobin 1%), which was prepared as reported previously (Li et al. 2006). Briefly, purified and viral inactivated fresh human placenta hemoglobin (Tianjin Union Stem Cell Genetic Engineering Ltd, Tianjin, China) was modified with bis(3,5-dibromosalicyl) fumarate to achieve optimal O2 affinity. After cross-linkage with glutaraldehyde, the mixture was subject to ultrafiltration and molecular sieve chromatography. The final product had a molecular weight of 64–600 kDa.
Dog CPB model Dog CPB model was established as described previously (Li et al. 2012a, 2014). After induction (4 mg/kg propofol, 0.1 mg/kg midazolam, and 5 μg/kg fentanyl) and muscle relaxation (1 mg/kg scoline), all the dogs were intubated with an Fr. 7.5 endotracheal tube and mechanically ventilated with tidal volume of 10 mL/kg (Datex-Ohmeda Excel 210, Soma Technology, Cheshire, Connecticut, USA). Each group received a continuous infusion of fentanyl at 0.3 μg/kg/min and vecuronium bromide at 0.2 mg/kg/h during surgery. Anesthesia was maintained with 150 μg/kg/min propofol. After heart exposure through a mid-sternal incision and heparinization (3 mg/kg), the ascending aorta and the right atrial appendage were cannulated. The CPB circuit was composed of a rolling pump (StÖckert II, Munich, Germany), a membrane oxygenator (1500 mL/min, Kewei Medical Ltd., Guangdong, China), and an arterial filter (Kewei Medical Ltd., Guangdong, China). The CPB was primed with Lactate Ringer’s solution containing 5% sodium bicarbonate (10 mL/L), 20% mannitol (2.5 mL/L), furosemide (0.5–1.0 mg/L), dexamethasone (5 mg/L), heparin (10 mg/L), and 10% potassium chloride (5 mL/L). Also, a 10% calcium gluconate (2–4 mL) was added every 30 min for four times.
Thomas’ solution (STS), and then reperfused for 2 h using aortic declamping. The dogs without cardiac arrest and reperfusion were allocated into the sham group.
Hemodynamic monitoring A Swan–Ganz float catheter (No. 7, Edwards Laboratories, Irvine, CA, USA) was inserted via femoral vein and advanced to pulmonary artery, the blood temperature and hemodynamics parameters, including heart rate, pulmonary artery wedge pressure (PAWP), pulmonary arterial pressure (PAP), central venous pressure (CVP) were collected using a PowerLab data-acquisition system (ADInstruments Pty, Bella Vista, NSW, Australia).
Measurement of blood gas values Arterial and venous blood samples were collected in polyethylene catheter placed in the left femoral artery or the Swan–Ganz float catheter. The blood gas parameters, including pH, arterial O2 partial pressure (PaO2), venous O2 partial pressure (PvO2) were measured immediately by a blood gas analyzer (ABL800 FLEX, Radiometer Medical A/S, Copenhagen, Denmark).
Intracerebral O2 tension and blood flow measurements A scalp incision was made to expose the right parietal bone. After drilling a 5-mm burr hole, the tip of the composite tissue PO2 laser Doppler flow probe with thermocouple (Oxford Optronix, Ltd, Oxford, UK) was inserted into the parasagittal parietal lobe to a depth of approximately 5 mm below the dura mater. Then, the probe was fixed to the skull with tissue glue, and supported with a custom-made probe holder. The intracerebral O2 tensions (tPO2) and cerebral blood flow (CBF) were continuously collected using a PowerLab data-acquisition system.
Experimental protocol
Measurement of neurological markers and inflammatory cytokines
The experimental protocol is schematically illustrated in Figure 1. Eighteen adult male beagle dogs were randomly divided into three groups (n 6): sham, control, and HBOC groups. Before heart exposure, animals were pretreated with intravenous injection of 0.1 gHb/kg PolyPHb (HBOC group) or equal amount of physiological saline (control group). After establishment of CPB and equilibration, hearts were arrested for 2 h using intra-aortic infusion of 40 mL/kg St.
Cerebrospinal fluid (CSF) and serum samples were collected before CPB and after 2 h of reperfusion and stored at 80°C until used. The levels of neuron-specific enolase (NSE) and S100β were analyzed in duplicate with commercially available ELISA test kits (Nanjing Jiancheng, Nanjing, China). The releases of tumor necrosis factor (TNF)-α and Interleukin (IL)-1β in serum were also determined (R&D, Minneapolis, MN, USA).
Figure 1. A schematic representation of the experimental protocol.
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Statistical analysis All values are presented as mean SD. An unpaired Student’s t test was used to detect significant differences when two groups were compared. One-way or two-way ANOVA was used to compare the differences among three groups followed by Bonferroni’s multiple comparison tests as applicable (SPSS 16.0 software). In addition, a simple linear regression analysis was performed to estimate the correlation between cortical tPO2 and neurological markers. P values less than 0.05 were considered statistically significant. Figure 2. HBOC improved cortical tPO2. Values are presented as the means SD (n 3). tPO2: tissue oxygen partial pressure.
Results HBOC elevates intracerebral O2 supply As shown in the Figure 2, infusion of HBOC was associated with an increase of tPO2 in parietal lobe cortex, from 11.75 1.28 to a peak of 28.79 3.98 torr at 16 min after infusion (P 0.001, n 3). Even though the tPO2 decreased gradually to 23.73 7.69 torr at 30 min after infusion, it still greatly higher than that of the animal received physiological saline (11.48 2.44 torr, P 0.01). The CBF values were not altered after HBOC infusion (data not shown).
was companied by elevations of PAP and PAWP. We confirmed that in the control group, both PAP and PAWP greatly increased as compared to that of the sham group (P 0.001 and P 0.001, respectively). This elevation was significantly reduced by HBOC pretreatment (P 0.01 and P 0.01 vs. the control group, respectively). Besides, no significant differences of CVP, pH, and blood temperature were found among groups.
HBOC improves hemodynamic and blood gas parameters after CPB
HBOC inhibits the releases of NSE and S100b
As presented in the Table I, HBOC increased the PaO2 and reduced the PvO2 during reperfusion, suggesting the circulatory O2 supply was well preserved by HBOC after CPB-induced I/R injury. Usually, circulatory dysfunction
As markers of neurological damage, the releases of NSE and S100β were measured. There were no significant differences of NSE and S100β releases before CPB. At 2 h after CPB, the NSE levels were significantly increased in the control group, in both serum and CSF (P 0.001 and P 0.001
Table I. The hemodynamic and blood gas parameters at baseline and during reperfusion. Reperfusion Baseline T (oC) Sham Control HBOC HR (bpm) Sham Control HBOC PaO2 (mmHg) Sham Control HBOC PvO2 (mmHg) Sham Control HBOC CVP (mmHg) Sham Control HBOC PAP (mmHg) Sham Control HBOC PAWP (mmHg) Sham Control HBOC
30 min
60 min
90 min
120 min
38.95 0.07 38.77 1.21 38.8 0.14
34.45 1.06 33.97 1.31 33.45 1.34
34.3 0.57 33.97 1.11 33.45 0.92
34.4 0.56 34.03 0.95 33.55 0.64
34.85 0.92 34.2 0.8 33.8 0.57
139 11 142 12 140 11
139 10 138 13 136 13
145 11 135 12 137 10
149 11 140 13 139 11
142 12 138 10 136 11
352.83 73.91 256.00 63.37 292 47.94*
418.83 61.81 265.83 78.98 294.50 69.63*
414.33 56.11 372.17 69.84 339.50 74.81 413.00 59.28 302.50 62.11 252.50 58.89 409.50 61.83 329.50 60.62* 288.17 70.04* 34.50 9.05 34.50 12.01 36.83 10.34
31.83 10.50 70.33 23.43 42.67 12.94*
38.00 11.51 63.67 38.69 36.00 13.16*
35.00 16.14 54.50 20.57 32.33 16.31**
37.17 12.81 58.83 23.71 35.83 19.26*
8.83 0.80 9.17 0.82 9.00 0.98
9.00 0.98 9.75 0.98 9.75 1.19
8.67 0.91 9.10 0.96 9.30 1.13
8.33 0.87 9.33 0.92 9.00 1.05
8.67 0.79 8.83 0.86 9.05 0.93
17.00 1.12 17.17 1.42 16.83 1.35
17.00 1.99 22.50 2.37 18.67 2.40**
17.07 2.08 23.50 2.54 18.50 2.52**
16.83 1.72 22.83 1.87 18.50 1.91**
16.67 1.85 23.67 1.82 19.00 1.93**
11.33 1.33 11.50 1.51 12.17 1.46
11.50 1.15 13.59 1.17 11.67 1.16**
11.17 1.31 14.50 1.27 12.33 1.25**
11.00 1.33 15.43 1.37 12.83 1.31**
11.03 1.37 15.33 1.45 12.33 1.41**
T, temperature; HR, heart rate; PaO2, arterial O2 partial pressure; PvO2, venous O2 partial pressure, CVP, central venous pressure; PAP, pulmonary arterial pressure; PAWP, pulmonary artery wedge pressure. Values were reported as Mean SD (n 6), *P 0.05; **P 0.01 versus the control group.
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Figure 3. HBOC reduced the releases of NSE and S100β in both serum (A and B) and CSF (C and B). Values were expressed as the means SD (n 6). *P 0.05 and ***P 0.001 vs. the control group. CSF: cerebrospinal fluid; NSE: neuron-specific enolase.
vs. the sham group). In contrast, animals received HBOC appeared a great reduction in NSE level (P 0.05 and P 0.001 vs. the control group; Figure 3A and B). Consistently, in the control group, the S100β release in serum and CSF was highly elevated (P 0.001 and P 0.001 vs. the sham group), while in the HBOC group, this increase
was greatly attenuated (P 0.05 and P 0.001 vs. the control group; Figure 3C and D). It is worth pointing out that this inhibitory effect was more prominent on neurological biomarkers in CSF. Moreover, linear regression analysis indicated a strong correlation between cortical tPO2 and releases of NSE and S100β after CPB (Figure 4).
Figure 4. The linear regression analysis of the relationship between cortical tPO2 and releases of NSE (A and C) and S100β (B and D) (n 6). CSF: cerebrospinal fluid; NSE: neuron-specific enolase; tPO2: tissue oxygen partial pressure.
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Figure 5. The levels of TNF-α (A) and IL-1β (B) in serum before and after CPB. Values are presented as mean SD (n 6). *P 0.05 and **P 0.01 versus the control group. CPB: cardiopulmonary bypass; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β.
HBOC reduces the releases of TNF-a and IL-1b after CPB The levels of both TNF-α and IL-1β in the control group were increased after CPB (P 0.001 and P 0.001 vs. the sham group, respectively; Figure 5). HBOC treatment significantly inhibited the releases of these inflammatory cytokines (P 0.05 and P 0.01 vs. the control group, respectively), suggesting the inflammation induced by CPB was greatly depressed.
Discussion In theory, HBOC allows delivery of more O2 to hypoxic tissues due to its higher O2 affinity, lower viscosity, and smaller mean diameter than human erythrocytes. These features help to provide sufficient perfusion of the microcirculation and thereby alleviate I/R injury. In the current study, we confirmed that the cerebral tissue O2 supply can be improved by HBOC as evidenced by increased cortical tPO2. This increased O2 preload for brain would be beneficial to against following I/R injury. For the early diagnosis of ischemic brain injury due to CPB, brain biomarkers, such as NSE and S100β, are commonly used. These biomarkers have been well-documented to present into blood and CSF during CPB and may reflect both cerebral injury and increased permeability of the blood–brain barrier (Mercier et al. 2013, Mondello et al. 2011). Consistent with increased cerebral O2 preload, our study indicated that HBOC greatly reduced the levels of both NSE and S100β after CPB, suggesting a significant protection on brain. As we know, except for I/R injury, CPB may cause systemic inflammatory response syndrome (SIRS). The mechanism is likely an abnormal regulation of various immune-modulating agents triggered by trauma of the blood cells or ischemia injury of the organs. As a consequence, cytokines are released, and the aggregate of these responses could lead to microcirculatory dysfunction and ultimately to renal dysfunction and/or pulmonary edema, which would jointly cause cerebral damage (Pahari et al. 2013). Therefore, it is reasonable that the elevated tissue O2 preload is also capable of reducing possible SIRS and brain damage during CPB. Our data clearly supported this notion that both the inflammation and cerebral damage were attenuated by high O2 supply from HBOC. Even though the present data cannot figure out the exact molecular signaling pathway underlying the cerebral
protective effect of HBOC, we believe improvement of O2 supply in brain plays an important role in this effect. As reported previously, the O2 releasing capacity of solution with HBOC is nearly 1.5-fold higher than that of the control solution, and the inhibited release of lactic acid from tissue provided additional evidence that the O2 supply could be enhanced by HBOC (Li et al. 2010b). In our study, by use of tissue PO2 and laser Doppler flow detection system, we continuously measured the cortical tPO2 before and 30 min after HBOC infusion. The data clearly indicated that the O2 supply in the cortex was remarkably increased as compared to the control group, even though the blood flow was no changed. We think this increased O2 supply should be attributed to the excellent O2 releasing capacity of HBOC. Oxygen consumption is regarded to be strongly correlated with organ performance (Gutterman and Cowley 2006). The present study confirmed this fact and further proved that the cerebral tPO2 was significantly correlated with the level of cerebral damage, suggesting tPO2 in brain is predictive of cerebral damage in surgery with CPB. The possible clinical application of this finding is that we should pay more attention to the patients whose cerebral tPO2 decreases sharply during surgery, and we could have taken some positive measures to improve it before it comes apparent, such as use of HBOC or just by inhalation of high O2.
Conclusion In conclusion, with a dog CPB model, we demonstrated that HBOC pretreatment exerts a profound protective effect to against possible cerebral I/R injury, and the proposed mechanism for this protection is associated with the improved tissue O2 preload.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by grants from the National Nature Science Foundation of China (81300110 and 81100180), the 2013 Research Fund for Outstanding Young Scholars of Sichuan University, and the Specialized Research Fund for the Doctoral Program of Higher Education (20100181120090).
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References Chang TMS. 2005. Therapeutic applications of polymeric artificial cells. Nat Rev Drug Discov. 4:221–235. de Tournay-Jetté E, Dupuis G, Bherer L, Deschamps A , Cartier R, Denault A . 2011. The relationship between cerebral oxygen saturation changes and postoperative cognitive dysfunction in elderly patients after coronary artery bypass graft surgery. J Cardiothorac Vasc Anesth. 25:95–104. de Vries DK , Kortekaas KA , Tsikas D, Wijermars LG, van Noorden CJ, Suchy M-T, et al. 2013. Oxidative damage in clinical ischemia/ reperfusion injury: a reappraisal. Antioxid Redox Signal. 19: 535–545. Gutterman DD, Cowley AW. 2006. Relating cardiac performance with oxygen consumption: historical observations continue to spawn scientific discovery. Am J Physiol Heart Circ Physiol. 291: H2555–H2556. Li T, Li J, Liu J, Zhang P, Wu W, Zhou R, et al. 2009a. Polymerized placenta hemoglobin attenuates ischemia/reperfusion injury and restores the nitroso-redox balance in isolated rat heart. Free Radic Biol Med. 46:397–405. Li T, Liu J, Yang C. 2010a. Pretreatment with hemoglobin-based oxygen carriers protect isolated rat heart from myocardial infarction. Artif Cells Blood Substit Immobil Biotechnol. 38: 115–118. Li T, Liu J, Yang Q, Wu W, Zhang P, Yang J, et al. 2009b. Polymerized placenta hemoglobin improves cardiac functional recovery and reduces infarction size of isolated rat heart. Artif Cells Blood Substit Immobil Biotechnol. 37:48–52. Li T, Wu W, You Z, Zhou R, Li Q, Zhu D, et al. 2012a. Alternative use of isoflurane and propofol confers superior cardioprotection than using one of them alone in a dog model of cardiopulmonary bypass. Eur J Pharmacol. 677:138–146. Li T, Yu R, Zhang HH, Wang H, Liang WG, Yang XM, Yang CM. 2006. A method for purification and viral inactivation of human placenta hemoglobin. Artif Cells Blood Substit Immobil Biotechnol. 34: 175–188. Li T, Zhang P, Liu J, Zhou R, Li Q, You Z, Dian K . 2010b. Protective effects of hemoglobin-based oxygen carrier given to isolated heart during ischemia via attenuation of mitochondrial oxidative damage. Free Radic Biol Med. 48:1079–1089.
Notice of correction The version of this article published online ahead of print on 28 May 2014 contained multiple labeling errors in Table 1. The errors have been corrected for this version.
Li T, Zhang Z, Liao D, Chen Y, Yang C, Xu X, Liu J. 2012b. The effect of polymerized placenta hemoglobin on renal ischemia/ reperfusion injury. Artif Cells Blood Substit Immobil Biotechnol. 40:396–399. Li T, Zhang Z, Wu W, Liao D, Chen Y, Li S, et al. 2013. Resuscitation with polymerized human placenta hemoglobin attenuated hemorrhagic shock-induced lung injury. Artif Cells Nanomed Biotechnol. 41:27–31. Li T, Zhou R, Xiang X, Zhu D, Li Y, Liu J, et al. 2011. Polymerized human placenta hemoglobin given before ischemia protects rat heart from ischemia reperfusion injury. Artif Cells Blood Substit Immobil Biotechnol. 39:392–397. Li T, Zhou R, Yao Y, Yang Q, Zhou C, Wu W, et al. 2014. Angiotensinconverting enzyme inhibitor captopril reverses the adverse cardiovascular effects of polymerized hemoglobin. Antioxid Redox Signal. [Epub ahead of print]. Mercier E, Boutin A , Lauzier F, Fergusson DA , Simard J-F, Zarychanski R, et al. 2013. Predictive value of S-100β protein for prognosis in patients with moderate and severe traumatic brain injury: systematic review and meta-analysis. BMJ. 346:f1757. Mondello S, Muller U, Jeromin A , Streeter J, Hayes RL, Wang KK . 2011. Blood-based diagnostics of traumatic brain injuries. Expert Rev Mol Diagn. 11:65–78. Pahari DR, Gu YJ, van Oeveren W, El‐Essawi A , Harringer W, Brouwer RM. 2013. Effect of minimized perfusion circuit on brain injury markers carnosinase and brain‐type fatty binding protein in coronary artery bypass grafting patients. Artif Organs. 37:128–135. Taylor KM. 1998. Brain damage during cardiopulmonary bypass. Ann Thorac Surg. 65: S20–S28. Varnado CL, Mollan TL, Birukou I, Smith BJ, Henderson DP, Olson JS. 2013. Development of recombinant hemoglobin-based oxygen carriers. Antioxid Redox Signal. 18:2314–2328. Wu W, Li T, Liu J, Yang C. 2011. Pretreatment before ischemia induction with polymerized human placenta hemoglobin (PolyPHb) attenuates ischemia/reperfusion injury-induced myocardial apoptosis. Artif Cells Blood Substit Immobil Biotechnol. 39:3–6. You Z, Li Q, Li B, Yang C, Liu J, Li T. 2013. Isovolemic hemodilution with glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) attenuated rat liver ischemia/reperfusion injury. Artif Cells Nanomed Biotechnol. 42:83–87.
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