Significance of neuroglobin in serum of acute atherosclerotic cerebral infarction patients*★
Shoucai Zhao, Zhaohu Chu, Lingsong Ma, Yinong Chen, Lei Wang, Benxiao Wang, Zili Huang, Jun Zhang

Department of Neurology, Yijishan Hospital, Wannan Medical College, Wuhu 241001, Anhui Province, China

Abstract
This study sought to examine neuroglobin (NGB) in the serum of acute cerebral infarction patients with double-antibody sandwich enzyme-linked immunosorbent assay to identify all risk factors, calculate infarct size, assess neurological impairment, and analyze the relation between NGB and each of these factors. The double-antibody sandwich assay indicated that levels of NGB in serum were unaltered within 6 hours following acute cerebral infarction compared with normal levels. NGB levels then underwent a distinct change, peaking at 24 hours then returning to normal levels in 72 hours. The results suggest that the level of NGB might be related to infarct size and low-density lipoprotein at 24 hours after acute cerebral infarction. There were no significant differences in neurological impairment scores and infarct size at different periods following infarction. The findings indicated that the level of NGB in serum of acute cerebral infarction patients was correlated with infarct time.
Key Words: neuroglobin; acute cerebral infarction; onset time; morbidity; infarct size; neurological impairment score 

INTRODUCTION
   
Neuroglobin (NGB) exists in the brains of vertebrates, including humans, and belongs to a branch of the globin family[1-2], which can reversibly bind oxygen with an affinity that increases as pH decreases[3-5]. Previous studies have reported that NGB acts as an oxygen reservoir, and combats reactive oxygen species[2, 6-9], which are increased by neuronal hypoxia in vitro and focal cerebral ischemia in vivo. As such, NGB is useful for reducing infarction volume[10-14]. Zhou et al [15] suggested that transduction of NGB may provide a way of treating cerebrovascular and neurodegenerative diseases.
NGB is widely distributed in neurons in cerebrum tissue, including the cerebral cortex, hippocampus, cerebellum, suprachiasmatic nucleus and astrocytes[16-23]. According to Jin et al [24], NGB exists highly in areas of peripheral ischemia in brain tissue biopsy of acute cerebral infarction (ACI) patients; Shang et al [25] concludes in rat experiments that the level of NGB is ascending in serum after ischemia- reperfusion. However, few investigations have examined the change of NGB in human body fluid[26-27]. Up to now, there are few relevant reports concerning the variation of NGB in serum of ACI patients. Therefore, the current study sought to provide a detailed analysis of the dynamic change of NGB, and elucidate the relationship between NGB and each risk factor in the serum of ACI patients using an enzyme-linked immunosorbent assay (ELISA).

RESULTS

Quantitative analysis and general data of involved patients
From December 2008 to December 2009, a total of 108 cases were included in this study and divided into two groups, an ACI group (n = 74) and a control group (n = 34). All of these cases were included in the final analysis. There were no significant differences regarding the type and degree of education, sex, diabetes mellitus, smoking, history of stroke, concentrations of low-density lipoprotein (LDL), total cholesterol and serum glucose between two groups. Importantly, we compared certain risk factors including age and blood pressure between ACI patients and the control group (Table 1), finding significant differences in each of these risk factors between groups.
Multiple regression analysis between each risk factor and NGB in the ACI group
Each risk factor, including diabetes mellitus, history of hypertensive disease, smoking,  
stroke, and drinking, was regarded as a dependent variable, while NGB and other risk factors such as infarct size and the National Institutes of Health Stroke Scale (NIHSS) score on admission were regarded as independent variables. Logistic regression analysis was then conducted. We did not find statistically significant differences between NGB and each risk factor. A multiple linear regression was performed, taking the infarct size and NIHSS on admission as independent variables, while NGB as dependent variable. The results revealed a linear correlation between NGB and infarct size (B = 2.553, SE = 0.492, t = 5.191, P = 0.000) and no statistically significant difference between NGB and NIHSS on admission (Beta In=-1.67(a), t = -1.394, P = 0.171; Table 2).


Comparison among the risk factors of different infarct sizes in the ACI group (Table 3)

There were 28 cases in the large infarct size subgroup and 46 cases in the small infarct subgroup. The inclusion criteria for each group were based on the cranial CT and/or MRI scanning results. Based on these criteria, infarcts exceeding 3.0 cm2 and involving at least two regions of cranial anatomy can be regarded as a large infarct, while those between 1.5-3.0 cm2 and occupying one region of cranial anatomy was referred to as a small infarct. Each risk factor in the ACI patients was determined within 6 hours, at 24 hours, and at 72 hours after onset respectively. The results revealed a significant difference between infarct size and LDL in the 24-hour subgroup (P < 0.05), while no significant differences in any risk factors were observed in other subgroups, even if the time and infarct size varied     (P > 0.05; Table 3).
Relationship between infarct size, NIHSS score and the level of NGB in the ACI group
One-way analysis of variance test revealed that the level of NGB in serum at 24 hours in the ACI subgroup was significantly higher than that in the control group, and at  6, and 72 hours in ACI subgroups (P < 0.01). We found no significant differences in NIHSS or infarct size between the various subgroups (P > 0.05; Table 4).

Relationship between the level of NGB in each subgroup and infarct size
The independent sample t-test revealed a clear relationship between NGB levels and various infarct sizes in the 72-hour subgroup. The results demonstrated that the NGB levels for larger infracts were significantly higher than those for small infarcts (P = 0.01). In the    ≤ 6-hour and 24-hour subgroups, the level of NGB in various infarct size exhibited no significant differences  (P > 0.05; Table 5).

DISCUSSION

Although the level of NGB has been previously examined in serum after ischemia-reperfusion in rats and in cerebrospinal fluid of chronic headache patients[25-27], this study is the first investigation of NGB levels in the serum in ACI patients. The experimental results were in accordance with previous studies, confirming that the overall level of NGB was quite low in most cases, and appeared to be correlated with age. NGB levels were shown to rise in the early stages of ACI patients, there were significant differences in NGB levels at different time points. These results indicated that the level of NGB in the serum of ACI patients was ascending.
A number of previous studies in rats have reported that NGB is over-expressed in neurons in areas of peripheral ischemia following ischemia/reperfusion injury[11-12, 28-29]. Shang et al [30] reported that the expression of NGB in the ischemic penumbra was dependent on time following forebrain ischemia in rats, finding that the level ascended gradually in early ischemia, peaked at 24 hours, then descended over time[30]. In addition, the level of NGB in serum was reported to ascend at 8 hours and peak at  48 hours following the ischemic event. The current results were in accordance with these previous findings, showing that the amount of NGB increased at 1-6 hours after the onset in serum of ACI patients, peaked at    24 hours, then gradually descended to normal levels at 72 hours. This evidence supports the notion that NGB, as an oxygen-carrying protein, increases its expression in the early period following cerebral ischemia.
The current results also revealed significant differences between the level of NGB in serum of ACI patients and infarct size in multiple linear regression analysis, but no significant differences between the levels of NGB in various infarct sizes in the ≤ 6-hour and 24-hour subgroups in the ACI group. However, there was a significant difference in the 72-hour subgroup. Although the level of NGB in serum of ACI patients decreased to the normal level at 72 hours, the levels of NGB were significantly higher in the large infract group than the small infarct group. There was no significant difference in neurological impairment score and the risk factors between two subgroups. Khan et al [13] examined NGB-overexpressing transgenic mice compared with wild-type littermates, finding that the volume of cerebral infarcts after occlusion of the middle cerebral artery was reduced by approximately 30%. Shang et al [30] reported a significant correlation between NGB levels in serum and the severity of neuronal damage in the gerbil brain. Our study was limited to testing a single-centre patient population, possibly explaining the discrepancy between the current findings and the results of Shang et al [30].
Several limitations of the current study must also be taken into account. First, ACI patients were observed over the period between onset to admission, without blinding, while the control group were non-cerebrovascular disease patients. As such, there may have been differences in risk factors between the groups, potentially biasing the results. In addition, the results revealed significant differences between NGB levels with varying infarct sizes at 72 hours, but no difference was found at ≤ 6 hours and 24 hours. This discrepancy may be related to the small sample size in the current study. Further studies are needed to validate the results using multi-center large-sample clinical trials.
In conclusion, the current results indicated that NGB increased in the early stages following ACI onset. In addition, our findings indicated that the level of NGB might be related to onset time, but not related to infarct size and neurological impairment score. NGB may at least partially reflect the general conditions of neurological impairment and the state of corresponding recovery.

SUBJECTS AND METHODS

Design
A non-randomized controlled case study.
Time and setting
Patients were recruited from Department of Neurology, Yijishan Hospital, Wannan Medical College, China between December 2008 and December 2009. NGB in serum was tested in March 2010, at the Experimentation Center, Yijishan Hospital, Wannan Medical College, China.
Subjects
Patient group
A total of 267 ACI patients were hospitalized in the Department of Neurology, Yijishan Hospital, Wannan Medical College, China from December 2008 to December 2009. According to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification[31], 148 cases were diagnosed as atherosclerotic ACI. Among these, 56 cases were excluded from the study because of the exclusion criteria, and 18 cases disagreed with the informed consent. In total, 74 cases gave informed consent and were included in this study. This sample included 42 males and 32 females, aged 38-79 years. All patients were first onset, and there was no family heredity history for any patients.
Inclusion criteria: Patients were diagnosed according to clinical symptoms and physical signs using cranial CT and/or MRI scanning. Cases of hemorrhage-related stroke, such as cerebral hemorrhage and subarachnoid hemorrhage, were not included. All included patients suffered from atherosclerotic ACI, according to the TOAST classification.
Exclusion criteria: Patients with severe dysfunction of the liver, kidney and heart, thyroid hypofunction, arthrolithiasis, autoimmune disease, tumor, severe systemic infection, surgery and trauma within 4 weeks, or taking drugs containing methotrexate, estrogen, contraceptive drug, decoagulant, inflammation suppressive agent or anti-epileptic drugs such as diphenylhydantoin sodium and carbamazepine were all excluded.
Control group
As the control group, we recruited 34 control subjects, including 16 males and 18 females, aged 38-79 years. All patients suffered from non-cerebrovascular disease, and were in the same hospital at the same period.
The study was conducted according to the 33rd rule of Hospital Administration Regulation of China[32]. All patients and/or their dependents were informed of the methods of examination, and informed consent was obtained from all members of the patient and control groups before the procedure.
Methods
Blood sample collection  
All patients in the ACI group received 8 mL venous blood collection upon admission. Blood samples were placed into a test tube containing natrium citricum for anticoagulation, well mixed, then centrifuged at
3 000 r/min for 20 minutes. The plasma was collected and stored below -80°C in a refrigerator. The venous blood of control subjects was collected in the early morning, and the other experimental steps were identical to those for ACI patients.
Experimental grouping
According the onset time of ACI and the period of the hospitalization, the group of ACI patients were classified into ≤ 6-hour, 24-hour and 72-hour subgroups. Patients were divided into a large infarct subgroup (n = 28) and a small infarct subgroup (n = 46) based on the Adams classification scheme[31]. The assignment depended on the results of cranial CT imaging and/or MRI: infarcts exceeding 3.0 cm2 and involving two regions of cranial anatomy were considered large infarcts; while those between 1.5-3.0 cm2 and occupying one region of cranial anatomy were considered small infarcts. Infarct size was diagnosed and measured by a neurologist who had been engaged in neurology for at least five years.
Neurological impairment scores
The neurological impairment of ACI patients upon admission was assessed using the NIHSS[33] score. A score of < 4 represented low-degree impairment, between 4 and 15 represented medium-degree impairment, and a score of > 15 represented severe-degree impairment. Two neurologists independently scored the participants using the NIHSS. If the difference between the neurologists’ scores did not exceed 3, the average of the two scores was recorded; if the difference was above 3, the score was confirmed by another chief physician of neurology.
Density of NGB in plasma using double-antibody sandwich ELISA method
Double-antibody measurement was adopted to test the contents of NGB according to the kit instructions (Shanghai Runweida Industry Corporation, China).
Ten standard wells on the plates were coated with ELISA. 100 μL of standard solution was added to the first and the second wells, then 50 μL standard dilution was added and mixed well. 100 μL from the first and the second wells were supplemented to the third and the forth wells separately, which were then added with 50 μL standard dilution and mixed well. 50 μL from the third and the forth wells were added to the fifth and the sixth wells, which were then added with 50 μL standard dilution and mixed well. Another 50 μL from the fifth and the sixth wells were added to the seventh and the eighth wells, which were then added with 50 μL standard dilution and mixed well. In addition, 50 μL from the seventh and the eighth wells were added to the ninth and tenth wells, which was added with 50 μL standard dilution and mixed well. 50 μL from the ninth and tenth wells was discarded, then each well was added with   50 μL samples after diluting (density: 30 μg/L, 20 μg/L, 10 μg/L, 5 μg/L, 2.5 μg/L). Blank and testing sample wells were set up. First, 40 μL sample dilution was added to testing sample well, then 10 μL testing sample was put into the well (the sample had been diluted five times). The sample was gently mixed, avoiding touching the well wall. After the culture plate was enclosed with closure membrane, samples were incubated for 30 minutes at 37°C. Then, 20-fold wash solution was diluted with distilled water 20 times and reserved for later use. The closure membrane was uncovered to pour out the liquid, the plate was dried, washing buffer was applied to each well, maintained for 30 seconds then drained out. All experimental steps were repeated 5 times, and the plate was dried again. Horseradish peroxidase-conjugate reagent 50 μL was added to each well, except the blank ones. Samples were re-incubated for 30 minutes at 37°C. Re-washing was then performed, as above. Chromogen solution A (50 μL) and Chromogen solution B (50 μL) were then added to each well, mixed gently, and kept at 37°C in the dark for 15 minutes to develop. Stop solution (50 μL) was added to each well to terminate the reaction (the blue color turned to yellow). Taking the blank wells as zero, absorbance value at 450 nm were recorded within 15 minutes.
Statistical analysis
All data were analyzed with SPSS 13.0 software (SPSS, Chicago, IL, USA), numeration data were analyzed with the crosstabs chi-square test, and expressed as mean ± SD, while measurement data were compared using dual-sample t-tests for homogeneity of variance and expressed as percentages. The average data of multiplicity samples were calculated with logistic regression analysis, multiple linear regression and one-way analysis of variance. The mean among groups was measured using SNK-q tests. A significant difference was defined at the 0.05 level.

Author contributions: Zhaohu Chu was responsible for experimental design and implementation, monitoring patient enrollment, validating and checking the manuscript. Shoucai Zhao headed the experiment, proposed and designed the study, and conducted the experimental procedures. He was responsible for case screening, data analysis, writing the manuscript and statistical analysis. Lingsong Ma participated in implementing the experiment and screening cases. Yinong Chen, Lei Wang, Benxiao Wang, Zili Huang were responsible for the collection of cases, NIHSS scoring, and specimen collection. Jun Zhang provided technical support and examined laboratory samples using ELISA.
Conflicts of interest: None declared.
Ethical approval: The experiment complied with the Ethic Committee of Yijishan Hospital of Wannan Medical College, China.
Funding: This study was financially sponsored by the Science and Technology Plan of Anhui Province, No. 08020304111.
Supplementary information: Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 27, 2011 after selecting the “NRR Current Issue” button on the page.

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(Edited by Guo ZJ, Sai XY/Yang Y/Wang L)