Neuroglobin Expression And Its Protective Effect Against Ischemic Brain Injury During Deep Hypothermic Circulatory Arrest

Various techniques for the heart, brain, lung, liver, and kidney protection have been developed and with increasing cooperation between researchers from different fields, deep hypothermic circulatory arrest (DHCA) has gained more extensive clinical application. Currently, DHCA has become a very important means for surgical management of some congenital cardiac defects in infants as well as of complicated major blood vessel abnormalities in adults. Among the vital organs of the body, the brain is especially vulnerable to anoxia and ischemia. Hypothermia allows chances for the organs to evade from disastrous damages in the context of circulatory arrest by significantly lower the metabolic rate, but even so, prolonged DHCA still results in nerve system impairment of various degrees. The mechanism of DHCA-induced brain injury is complex, and most researches on brain protection following circulatory arrest give their primary attention to the means of lowering brain metabolism, which, however, does not seem to provide a solution for all the problems. A typical solution for brain protection may lie in the exploit of the mechanism of brain oxygen metabolism, so as to develop methods to promote brain oxygen preservation, transport and economic consumption. Basic studies of brain oxygen metabolismhave made great progress in recent years.In 2000, Burmester et al discovered an oxygen-carrying globulin specific to the brain of human and mice, and gave it the name neuroglobin (Ngb), which shed light on a new scope of cerebral anoxia research. As a highly conservative single-copy gene in human, Ngb gene is located on chromosome 14q24 with a full cDNA length of 1909 bp that encodes 151 amino acids. Ngb is mainly expressed in the neuronal cytoplasm of vertebrates with high affinity to oxygen and functionally resembles hemoglobin in that it provides oxygen supply specifically to the brain and modulates the oxygen supply of the brain at the molecular level. As an important endogenous neuroprotective factor, the expression level of Ngb is positively associated with the tolerance of anoxia of the brain tissue. Ngb probably performs the functions of (1) oxygen preservation, in that it binds to oxygen in physiological conditions and release oxygen in anoxia, (2) oxygen transport, it carries oxygen through the blood-brain barrier and accelerates the diffusion of the oxygen into the mitochondria in the neurons, (3) anoxia sensors, which protects from downstream anoxic damages, and (4) clearing toxic substances such as NO.For a long time Hemin has been used for treatment of anemia but recently, its protective role in other systems is given increasing attention. Experimental evidences have proved that Hemin can significantly enhance Ngb expression in vitro in a dose- and time-dependent manner. As a substrate and inducer of heme oxygenase (HO), Hemin can induce the expression of Ngb, resist free radical injury and reduce the toxicity of excitatory amino acids, thus provides important protection of the heart, brain, and guts against ischemic and anoxic injuries.As techniques of molecular biology are widely applied in medical science research, increasing understanding of the pathological basis of brain injury has been obtained, and the roles of free radicals, NO, cytokines and adhesion molecules, and protein biochemical markers in extracorporeal circulation and DHCA have attracted increasing attention.S-100β, a protein found in bovine brain by Moore in 1965 with relative molecular mass of 21×103, exists concentration-dependently in the central nervous system as an acidic calcium-binding protein. S-100βis overexpressed in the brain tissue and rapidly released into the circulation following brain injury, but due to its very short half-life, the level of this protein in the blood soon declines. Delayed functional impairment or progressive death of the glial cells following brain injury leads to S-100βleakage, and secondary brain injury causes further damage of the blood-brain barrier. As the condition worsens, secondary elevation of S-100βor persistent presence of high-level S-100βmay occur, which suggests progressive secondary brain injury.Myelin basic protein (MBP) is one of the major components of the myelin sheath that insulates axons and binds tightly with myelin to maintain the integrity and functional stability of the neural sheath. In the event of neural sheath destruction, MBP can be released in the nervous system and blood circulation, and finally degraded and cleared through urination. Current data suggest that MBP is associated with acute or chronic cerebrovascular diseases, experimental allergic encephalomyelitis, multiple sclerosis and many other neurological diseases, and considered a specific biomedical indicator of organic damage of the nervous system, particularly demyelination. MBP also serves the purpose of therapeutic effect evaluation and prognostic assessment.Tumor necrosis factorα(TNFα) is a extensively studied cytokine secreted by various types of cells. Studies suggest that this cytokine plays a double-faced role in ischemic brain injury in that TNFαworsens neuronal damage following ischemic brain injury but may also execute neuroprotective functions. But due to the insufficiency of systemic research data demonstrating the beneficial effect of TNFαin ischemic brain injury, most researchers inclined to believe that TNFαparticipates in the pathological process of brain injury as an inflammatory agent. This seems to provide new insight into therapy of ischemic brain injury.Vascular cell adhesion molecule-1 (VCAM-1), a member of the adhesion molecule immunoglobulin superfamily, is located on the vascular endothelial cell membrane. Stimulation with inflammatory agents results in increased expression of VCAM-1 which participates in lymphocyte activation and migration and the growth and development of hemopoietic cells and is also involved in such pathological processes as inflammation and tumor metastasis. Researches have identified important roles of VCAM-1 in the inflammatory response in relation to cerebral ischemia and its progression. VCAM-1 expression can alleviate the symptoms of brain ischemia and protect against ischemic brain injury.In this study, real-time fluorescence quantitative PCR was employed to analyze Ngb mRNA expression during extracorporeal circulation and DHCA in relation to the expressions of S-100β, MBP, TNFαand VCAM-1. The value of Ngb and other indices related to brain injury was explored in the diagnosis, therapy and prognostic assessment of brain injury due to DHCA.Objective:Ngb expression and plasma levels of S-100β, MBP, TNFαand VCAM-1 were detected at different time points during DHCA in dogs with or without Hemin treatment. Ultrastructural changes of the cerebral cortex was also observed to reveal the role and mechanism of Ngb and the other factors in protection against early-stage brain injury due to DHCA, thereby finding effective means for prevention and therapy of brain injury due to DHCA.Methods:Ten healthy hybrid dogs were randomized into control group and Hemin treatment group, and in the latter, the dogs were given 50 mg/kg Hemin 24 hours prior to the experiment. The dogs were anesthetized with 3% sodium pentobarbital followed by tracheal intubation with artificial ventilation. Cannulation of the left jugular vein was performed to collect cerebral venous blood sample, and the left carotid artery was also cannulated to measure the mean arterial blood pressure (MBP). Craniotomy was performed on the right side of the skull to obtain cerebral cortex sample. Cannulation of the aorta and the inferior and superior vena cava was performed to establish extracorporeal circulation, which was terminated till the nasopharyngeal temperature dropped to 18℃. Ninety minutes later, the circulation was restarted and terminated till a nasopharyngeal temperature of 37℃. One hour after extracorporeal circulation termination, the dogs were killed by blood depletion. Venous blood samples were obtained from the left jugular vein immediately after extracorporeal circulation establishment, 0 and 60 minutes after extracorporeal circulation termination, and 45 minutes of reperfusion, respectively, for measurement of S-100β, MBP, TNFα, and VCAM-1 levels. Cortical samples were also obtained at these time points and frozen for real-time fluorescence quantitative PCR for Ngb mRNA measurement. At 60 minutes of reperfusion, 4% paraformaldehyde was perfused through the carotid artery and cerebral cortex samples were harvested for transmission electron microscopic observation of the ultrastructures.The data of the measurement were statistically analyzed with SPSS11. 5 software by repeated measure analysis of variance and one-way ANOVA. Bonfferoni test was performed for paired comparison. A P value no greater than 0.05 was considered to indicate significant difference.RESULTS1,The establishment of extracorporeal circulation resulted in increased Ngb mRNA expression, which was especially obvious after body temperature reduction, and at 60 min after DHCA its expression reached the peak level((?)±S=565680681.56±223521679.77Copy Number/ug), followed by slight decrease after body temperature recovery. The concentration of Ngb mRNA showed significant differences between different time points (F=16.745, P＜0.001), but were comparable between the Hemin and control groups (F=5.153, P=0.392). There is no crossover effect of the concentration of Ngb mRNA between groups and different time points (F=0.249, P=0.862). At each time points of measurement, Ngb mRNA concentration exhibited different patterns of alteration, specifically, it increased after body temperature reduction till circulation arrest, reaching the peak level at 60 minutes of DHCA and a slight reduction was noted at 45 minutes of reperfusion when Ngb mRNA level was still higher than that measured at the initiation of DHCA. Ngb mRNA level at 60 minutes of DHCA and at 45 minutes of reperfusion was significantly higher than that measured before extracorporeal circulation establishment and at the start of DHCA (P＜0.048).2,Following extracorporeal circulation establishment plasma S-100βprotein level showed significant increment, which was most obvious within the period between extracorporeal circulation initiation and DHCA. As DHCA was prolonged, S-100βprotein level kept increasing even after reperfusion, but the increment showed a gradually lowered magnitude. S-100βprotein level showed significant differences between the time points of measurement (F=1114.099, P＜0.001). But between Hemin and control groups, S-100βprotein levels showed no significant difference (F=0.599, P=0.461). There is no crossover effect of S-100βprotein level between groups and different time points (F=0.249, P=0.862). Most obvious changes of S-100βprotein level was noted in the period between extracorporeal circulation initiation and DHCA (P＜0.001). The highest S-100βprotein level occurred at 45 minutes of reperfusion((?)±S=17.59±0.35 ng/ml), but this high level showed no significant difference from that measured at 60 minutes of DHCA (P=0.289).3,Plasma MBP level increased gradually following extracorporeal circulation initiation till 60 minutes of DHCA, and at reperfusion, plasma BMA reached its peak level ((?)±S=4.17±0.88 ng/ml), but at 45 minutes of reperfusion, MBP level underwent a significant reduction even to a level below that before extracorporeal circulation initiation. MBP level showed significant differences between the time points of measurement (F=44.999, P＜0.001). But between Hemin and control groups, MBP level showed no significant difference (F=1.988, P=0.196). There is no crossover effect of MBP level between groups and different time points (F=1.081, P=0.376). Between extracorporeal circulation initiation and 60 minutes of DHCA, plasma DHCA level did not undergo significant changes until the termination of DHCA and initiation of reperfusion (P＜0.001), but at 45 minutes of reperfusion, BMP level recovered and became even lower than the level before extracorporeal circulation, showing significant difference from the level at 60 minutes of DHCA (P=0.001).4,Plasma TNFαlevel was slowly reduced after extracorporeal circulation initiation till 60 minutes of DHCA, and at the time of reperfusion, TNFαlevel reached its lowest ((?)±S=316.17±3.07 ng/ml), followed by slow increase at 45 minutes of reperfusion but failed to recover its former level. Its level showed significant differences between the time points (F=4.258, P=0.015), but these differences were not supported statistically after multiple comparison (P＞0.152). TNFαlevel was not significantly different between the control and Hemin groups (F=1.108, P=0.323). There is no crossover effect of TNFαlevel between groups and different time points (F=0.614, P=0.613).5,Plasma VCAM-1 level increased gradually following extracorporeal circulation initiation and at 45 minutes of reperfusion, plasma VCAM-1 level reached its peak level ((?)±S=35.14±3.26ng/ml). Plasma VCAM-1 level showed significant differences between the time points of measurement (F=15.838, P=0.001). But between Hemin and control groups, Plasma VCAM-1 level showed no significant difference (F=0.525, P=0.489). There is no crossover effect of Plasma VCAM-1 level between groups and different time points (F=0.077, P=0.972). In the former 3 stages of the experiment, VCAM-1 level did not undergo significant alterations, but from 60 minutes of DHCA to 45 minutes of reperfusion, VCAM-1 level showed obvious increase (P=0.013). In other words, VCAM-1 level began to increase obviously following reperfusion after brain temperature recovery.6,Correlation analyses between Ngb mRNA expression and the brain injury indices described above revealed that Ngb mRNA expression was in close positive correlation with plasma S-100βlevel during DHCA (with coefficient of partial correlation of 0.4603, P=0.004).7,After DHCA and reperfusion, obvious changes were observed in the ultrastructure of the cortical neurons, including swelling of the mitochondria and reduction of the mitochondrial crest. The cell membrane remained continuous and intact with clear bilayer structure. Lysosome proliferation was obvious, and phagocytosed mitochondria could be seen. These changes were not significantly different between the control and Hemin groups.CONCLUSIONHemin pretreatment may enhance Ngb mRNA expression at different phases of DHCA, but due to the large magnitude of the mRNA copies, no statistically significant differences are produced (F=5.153, P=0.053). Hemin failed to show obvious effect in improving brain injury during DHCA in comparison with the control group. Hemin and anoxia induce Ngb expression through different mechanisms, and the former seems to induce Ngb expression at a much lower pace than the latter.As currently no effective means are available to sufficiently stimulate Ngb expression, the protective effect of Ngb against brain injury is not obvious during DHCA, and the mechanism needs further exploration.Plasma S-100βprotein, MBP, and VCAM-1 can be sensitive indicators of early brain injury during DHCA, but S-100βprotein and VCAM-1 serve this purpose better than MBP. TNFαdoes not seem to suit for this purpose, and its double effects in brain injury in relation to DHCA needs further investigation.Ngb has the potential as a novel sensitive indicator for brain injury during DHCA, but its value in the diagnosis, prognostic evaluation and protective effect in brain injury awaits more extensive laboratory and clinical research.Analysis of the time-concentration curve of the nervous system-related proteins in the blood following brain injury and comprehensive evaluation of the dynamic changes of multiple markers, other than single such proteins, can be more valuable for brain injury severity assessment, prognostic evaluation and treatment planning.