by Yetty Ramli, MD, SpN(K); Fadhlan Rusdi, MD, SpN; Mohammad Kurniawan, MD, SpN(K); Mohamad Sadikin, MD, DSc; and Florencia Evelyn, MD

Drs. Ramli, Rusdi, Kurniawan, and Evelyn are with the Department of Neurology, Faculty of Medicine, Universitas Indonesia in Jakarta, Indonesia. Dr. Sadikin is with the Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia in Jakarta, Indonesia.

Funding: This research was funded by Universitas Indonesia Research Grant, International Indexed Publication program 2023.

Disclosures: The authors have no conflicts of interest relevant to the content of this article.

Innov Clin Neurosci. 2024;21(10–12):38–43.

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Abstract

Background: Prognostic markers can optimize the management of acute ischemic stroke (AIS). Neuroglobin (Ngb), which plays a role in intraneuronal oxygen transport and hypoxia resistance, is a potential prognostic marker in AIS.

Methods: A cohort study was conducted on patients with AIS treated at Dr. Cipto Mangunkusumo National Referral Hospital from March to April 2023. Serum samples for Ngb examination were collected three days after the onset of the stroke, while a modified Rankin Scale (mRS) was obtained after seven days and again after six months. National Institutes of Health Stroke Scale (NIHSS), Barthel Index (BI), and Montreal Cognitive Assessment (MoCA-Ina) scores were obtained on the seventh day. Significance analysis and receiver operating characteristic (ROC) curve were used to determine the relationship between Ngb and AIS outcomes.

Results: A total of 42 subjects underwent analysis. Serum Ngb levels were higher in subjects with mRS score of 3 to 6, compared to those with scores of 0 to 2 (median [range]: 12.42ng/mL [3.57–50.43] vs. 4.79ng/mL [2.25–37.32], p=0.005). The association with mRS persisted until six months post-AIS (p=0.004). The area under the ROC curve (AUC) was 0.75. Ngb levels were also higher in groups with higher NIHSS at discharge (p=0.03), lower BI (p=0.01), and lower MoCA-Ina scores (p=0.002). Clinical assessments (BI and NIHSS), along with evaluations of cognitive function and Ngb markers, can be employed to monitor patient progress and predict stroke outcomes up to six-months post-AIS.

Conclusion: Higher serum Ngb levels in AIS are associated with poorer functional outcomes. Further research is needed before clinical application.

Keywords: Acute ischemic stroke, neuroglobin, prognostic marker

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Acute ischemic stroke (AIS) is the result of cerebral ischemia, which causes high mortality and morbidity globally and in Indonesia. Global incidence of stroke reached 12.2 million per year in 2019, which shows an increase of 70 percent in the last 20 years.¹ In Indonesia, the prevalence of stroke in people aged over 15 years was 1.09 percent in 2018, an increase from 2013 when the prevalence was 0.7 percent.² Stroke is the second-leading cause of death worldwide (11.6%) and the third-leading cause of combined death and disability (5.7% of the total disability-adjusted life years [DALYs]).¹

Affordable and easily performed prognostic markers, such as biological markers detectable in blood, might be able to supplement the modalities available to clinicians for managing stroke. One of the biological markers that has the potential to fulfill this role is neuroglobin (Ngb). Ngb is a protein that was discovered in 2000 by Burmester et al.³ Since then, there have been many publications regarding its chemical properties and role in disease management. Like other globin proteins, it is predicted that Ngb has a role in oxygen transport. Several in vitro and in vivo studies have also shown that Ngb protects neurons from hypoxic and ischemic injury.^4–6 

The choice of Ngb as a marker for stroke outcomes stems from its potential to enhance the precision of outcome assessments beyond clinical evaluations alone. While clinical assessments provide valuable insights into a patient’s condition, incorporating Ngb as a marker offers a more nuanced understanding of the underlying molecular and cellular changes associated with stroke recovery. Ngb is found in the brain and has been implicated in neuroprotection and tissue oxygenation. Monitoring its levels can contribute to a comprehensive evaluation, allowing clinicians to assess not only the overt clinical symptoms but also the intricate biochemical responses and neural recovery processes. By integrating Ngb assessments into the overall evaluation framework, a more holistic and nuanced perspective on stroke outcomes can be attained, potentially leading to more tailored and effective therapeutic interventions.

Since its discovery, Ngb expression patterns and functions have been studied in both animals and humans. The protein Ngb is mainly expressed in the central and peripheral nervous system, including the retina. In addition, Ngb has also been found in the adrenal glands.⁴

Most of the processes that trigger an increase in Ngb are related to direct (hypoxia) or indirect (reactive oxygen species [ROS]) disturbances of oxygen metabolism. Hypoxia increases levels of hypoxia-inducible factor (HIF)-1α by reducing its hydroxylation, ubiquitination, and proteasomal degradation. Next, HIF-1α activates the transcription of hypoxia-responsive genes and through intermediate proteins promotes Ngb gene transcription.⁷ In addition to functioning in intraneuronal oxygen binding and transport, Ngb is also thought to play a role in protecting neurons from oxidative injury, promoting neuronal survival, and resisting apoptosis. In the acute phase, Ngb plays a crucial role in indicating the severity of the ischemic process in the brain.^5,8,9

Given its localization and regulation expression, Ngb has the potential to be a biomarker that is specific for AIS. The neuronal lysis and increased permeability of the blood-brain barrier (BBB) resulting from the ischemic cascade and release of cytokines in AIS¹⁰ allow elevated levels of Ngb to be detected in the circulation. This study aims to establish the relationship between serum Ngb levels in AIS and the patient’s functional outcome at discharge.

Methods

From March to April 2023, we conducted a cross-sectional study on patients with AIS at Dr. Cipto Mangunkusumo National Referral Hospital in Jakarta, Indonesia. Patients who were at least 18 years of age and arrived within 96 hours after stroke onset were included. Subjects with a history of epilepsy, use of hormonal contraception, renal impairment requiring routine hemodialysis, severe systemic infection, and malignancy were excluded. Serum samples were collected from 44 subjects; however, only 42 were included in the analysis due to the identification of exceptionally high Ngb values in two subjects. Basic characteristics, including age, sex, and other clinical data, were documented during the patients’ emergency room (ER) admissions. The study was conducted in accordance with the Declaration of Helsinki and approved by the Health Research Ethics Committee of the Faculty of Medicine at Universitas Indonesia – Cipto Mangunkusumo Hospital. Informed consent to participate was obtained from either the patient or their guardian.

At 72 to 96 hours after stroke onset, 3cc of blood was collected without additives. The samples were then centrifuged and stored at –200°C. All samples underwent simultaneous examination using an enzyme-linked immunosorbent assay (ELISA) kit provided by Elabscience® (Houston, Texas, US). Microplates precoated with a monoclonal antibody specific to Ngb were utilized. Standard and serum samples were pipetted into the wells, allowing Ngb to bind to the fixed antibody. Subsequently, an enzyme-linked monoclonal antibody specific to Ngb was added, followed by washing to remove any remaining antibody-enzyme reagent. The substrate solution was then introduced, causing a color change proportional to the amount of Ngb bound in the previous step. The addition of a stop solution halted the color change in each well. Optical density was measured using a microplate reader, and Ngb concentration values were determined using standard formulas/curves.

Outcome assessments were conducted on the seventh day or the day of discharge, whichever occurred first. The primary clinical outcome parameter was the modified Rankin Scale (mRS), an ordinal scale ranging from 0 (indicating complete independence without symptoms) to 6 (indicating death). For the significance test, the mRS scores were further categorized into favorable (0–2, reflecting at least the ability to carry out self-care independently) and unfavorable outcomes (3–6). Additionally, functional independence was evaluated using the Barthel Index (BI), which uses a scale from 0 to 100, with a BI score of 60 or more considered a favorable outcome. Functional outcomes were re-evaluated by administering the mRS by phone after six months.¹¹

Neurologic deficits were assessed using the National Institutes of Health Stroke Scale (NIHSS), with a higher score (≥6) indicating a poorer outcome.¹² Cognitive function was assessed using the Bahasa Indonesia version of the Montreal Cognitive Assessment (MoCA-Ina). The cutoff point used to determine a favorable outcome was 22.¹³

Data collection was done manually using existing research forms. Data processing was carried out using SPSS version 23. Data on the distribution of subject characteristics in categorical form were presented in the form of proportions (n, %). For data in numerical form, a normality test was performed first. Data that were normally distributed were presented using means, while medians were used for data with a normal distribution. To find statistical significance, bivariate analyses were performed. For the primary outcome, the analyzed variables were categorical and numerical, and the data distribution was abnormal, so the test used was the Mann–Whitney U test.

Results

Clinical characteristics. Initially, there were 51 participants who met the inclusion criteria, but seven patients were excluded based on the exclusion criteria. Subsequently, samples were taken and measurements were conducted on the seventh day. Further Ngb examinations were carried out in the sixth month. However, due to the presence of two subjects with extreme values, the total number of participants included this study was 42.

The mean±standard deviation age of the subjects was 58.30±13.58 years. Of the 42 patients included, 61.4 percent were male. The most common risk factor for ischemic stroke was hypertension, which was present in 81.8 percent of patients. The majority of subjects had 2 to 3 risk factors. The most common stroke subtype based on the Bamford classification was lacunar infarction (LACI), found in 21 patients (50%; Table 1).

Ngb levels. Out of 44 patients, there were two with extreme concentrations of Ngb (143.7ng/mL and 205.6ng/mL). For the remaining 42 subjects, the Shapiro–Wilk test showed an abnormal distribution (p<0.001), with a median Ngb level of 8.97ng/mL (2.25–50.43ng/mL). Ngb levels did not differ significantly based on age, sex, or the number of risk factors (p-values were 0.554, 0.072, and 0.088, respectively). Ngb levels also did not differ between groups of patients with different admission NIHSS scores (p=0.091). Ngb levels were significantly higher in anterior circulation stroke (total anterior circulation infarct and partial anterior circulation infarct) than posterior circulation infarct and LACI (median: 13.4ng/mL vs. 4.8ng/mL, p=0.005).

Ngb and stroke types. In this study, we also aimed to compare lacunar and nonlacunar strokes. The results revealed a significant difference between the two groups (p=0.005), with the nonlacunar stroke group showing higher levels of Ngb compared to the lacunar stroke group. The difference in stroke types (lacunar and nonlacunar) was also found to be significant in relation to NIHSS scores (p=0.013; Table 2). This can occur because, in lacunar strokes, there is typically less extensive damage, resulting in a lower degree of hypoxia. Consequently, the levels of Ngb, which are often elevated in response to hypoxic conditions, remain lower in lacunar strokes compared to nonlacunar strokes.

Ngb and AIS outcomes. A comparative test using the Mann–Whitney U test showed higher serum Ngb levels in subjects with poorer outcomes (median [range]: 12.42ng/mL [3.57–50.43] vs. 4.79ng/mL [2.25–37.32], p=0.005; Figure 1). Six months after the ischemic event, all patients were followed up and completed the mRS by phone, and the association was still detected (p=0.004; Table 3).

Receiver operating characteristic (ROC) curve (Figure 2) and area under the curve (AUC) were used to assess the ability of serum Ngb levels to predict poor outcomes. The AUC was 0.751, which means that Ngb was fairly accurate in predicting the outcome of AIS (standard error: 0.078, p=0.005).

Secondary outcome parameters in this study were neurological deficits assessed by the NIHSS, functional independence assessed by the BI, and cognitive function assessed by the MoCA-Ina. At seven days poststroke, 27 patients had relatively good NIHSS (<6) and 15 had poor NIHSS (≥6). Serum Ngb levels were found to be higher in the group with worse NIHSS (Table 4). Meanwhile, 19 patients had a BI score of 60 or more, and 23 patients had a score below 60. Ngb levels were significantly higher in the group with a lower BI score (Table 4). Assessment of cognitive function using MoCA-Ina was performed on 32 patients (76.2%); MoCA-Ina score could not be assessed in 10 patients due to loss of consciousness or aphasia. MoCA-Ina scores of 22 or higher were found in 20 patients, while the rest scored below 22. There was a significant tendency for MoCA-Ina scores to be lower in patients with higher Ngb levels (Table 4).

Discussion

The clinical characteristics of the subjects of this study are not different compared to various stroke studies in Asia, particularly in Indonesia. A previous study by Harris et al,¹⁴ involving 5,411 patients with stroke in Indonesia, found that the proportion of male patients was greater than that of female patients (59.33%). In addition, hypertension was found in 79.2 percent of patients, and the most common stroke subtype was also LACI (45.07%).

The mRS score is the most commonly used functional independence assessment in various stroke studies. Besides being simple and having good reliability, mRS also correlates well with infarct size. In addition, mRS also has fairly good predictive validity, characterized by short-term mRS that correlates with long-term stroke disability. In general, an mRS score of 0 to 2 can be considered good, because it indicates that the patient can carry out self-care and daily activities that are quite complex, although not necessarily able to carry out outdoor activities.¹⁵

In this study, we found that there was a relationship between serum Ngb levels and mRS assessed at seven days and six months after the onset of ischemic stroke. An autopsy study by Jin et al¹⁶ showed that increased Ngb could be detected in the peri-infarct tissue of the brain after ischemic stroke. In another study conducted by Shang et al¹⁷ in 2006, it was found that serum Ngb concentrations in mice increased starting eight hours after occlusion of the carotid artery and reached a peak at 48 hours. A significant correlation exists between serum Ngb levels and the severity of brain tissue damage, as evidenced by higher Ngb levels in cases of delayed reperfusion.

Despite considerable preclinical research, clinical evidence regarding the association of Ngb and AIS outcomes has not been extensively investigated. A 2017 study by Xue et al¹⁸ on 40 patients with ischemic stroke found that higher serum Ngb levels in the first 24 to 96 hours from the onset of ischemic stroke were associated with poorer outcomes after three months. Patients with poor outcomes (mRS score of 3–6) had higher levels of acute phase serum Ngb (31.01±3.23 vs. 20.05±2.02μg/L; p=0.004), compared to patients with good outcomes (mRS score of 0–2 ).¹⁸ With increasing evidence, further research on this subject is quite promising. The higher Ngb level compared to this study (25.76±3.15μg/L) was probably due to the difference in the ELISA kit and the exclusion of lacunar strokes in the previous study.

There have been no other studies that exclusively investigated the relationship between Ngb and ischemic stroke outcomes. Studies on aneurysmal subarachnoid hemorrhage (aSAH) showed that serum Ngb was also associated with mortality and outcomes at six months as measured by mRS.^19,20 This finding is most likely due to hypoxic conditions that can occur in aSAH through a vasospasm mechanism that occurs due to stimulation by blood and its degradation products in the subarachnoid space.¹⁹

Based on our ROC curve analysis, an AUC value of 0.751 was obtained, which indicates that Ngb levels can fairly predict poor outcomes in AIS. Ngb should not be used alone, but together with other clinical, radiological, and laboratory examinations. To determine the predictive role of Ngb along with other factors, further research with a larger number of samples is needed.

Higher Ngb levels also appear to be associated with a poorer BI score. Despite arriving at the same conclusion, there was a slight difference between the mRS and BI in classifying patients into favorable or unfavorable outcomes. This discrepancy arose because the threshold value of 60 on the BI is not equivalent to a score of 3 on the mRS. A study by Liu et al²¹ found that the threshold for BI that is equivalent to mRS score of 3 is about 75. Furthermore, another study revealed an overlap in the distribution of BI scores on the mRS, especially in the acute phase (first 10 days).²²

The increase in Ngb in the acute phase of ischemic stroke is consistent with the notion that Ngb is a responsive compound whose expression increases during brain ischemia and hypoxia. However, these findings cannot confirm whether this increase is an adaptive response or just a transfer of Ngb from intracellular space to systemic circulation. In addition to increasing intracellular Ngb expression, the pathophysiological process of ischemic stroke can cause neuronal apoptosis and increased permeability of the blood-brain barrier, resulting in increased levels of Ngb in serum.¹⁰ Examination of upstream molecules that regulate Ngb expression, such as Ngb messenger ribonucleic acid (mRNA) or HIF-1α, should be able to confirm this.

We also found that serum Ngb levels 3 to 4 days after ischemic stroke were associated with higher NIHSS scores on Day 7. This is logical because the NIHSS itself has been known to correlate well with infarct size.¹⁵ Larger infarct size indicates a wider ischemic hypoxic area, thus involving more neurons. Data on this were not available before. The study by Xue et al¹⁸ only examined the relationship between Ngb and NIHSS scores at admission.

Immediately after the acute phase, up to 40 percent of patients with ischemic stroke experience cognitive impairment. In this study, the MoCA-Ina was selected as an outcome parameter for cognitive function. This choice was based on its applicability, stronger assessment of executive function and visual construction tasks compared to the Mini-Mental State Examination (MMSE), and its ability to predict the development of chronic poststroke cognitive impairment.²³ The lower normal limit value (≥22) was chosen because it is more representative in the acute phase of stroke.¹³ In this study, the MOCA-Ina examination could be performed seven days after the onset of stroke in 32 (76.2%) patients. This is similar to previous findings, where MoCA could be applied to 73 to 82.5 percent of patients with acute stroke.^23,24 Decreased consciousness, high NIHSS, and lesions in the left hemisphere (language disorders) were barriers to implementing MoCA.

Ngb levels also appear to be related to cognitive function outcomes in patients with AIS. Ngb levels were statistically higher in the group with the lower MoCA-Ina scores. This strengthens the theory regarding the processes and factors that influence the expression of Ngb and its release into the systemic circulation, as described above. This clinical evidence in ischemic stroke is a novelty.

Meanwhile, in patients with hemorrhagic stroke, Gao et al²⁵ found that lower Ngb was associated with a higher incidence of poststroke cognitive impairment (4.7±0.9ng/mL vs. 7.5±1.1ng/mL, p≤0.05). These contradictory results might be related to differences in the pathophysiology of ischemic and hemorrhagic strokes. The process that dominates the pathophysiology of hemorrhagic stroke is not an ischemic cascade but a mass effect. Ischemic/hypoxic processes contribute to a smaller portion, specifically in secondary injury when there is continued expansion of the hematoma.²⁶ Therefore, the expression of Ngb in hemorrhagic stroke is relatively lower and does not represent the most dominant process.

Another study by Ding et al¹⁹ on patients with aSAH showed consistent results with this study, where Ngb levels in the first seven days were higher in patients with poorer functional and cognitive outcomes after 12 months. This can be explained by the pathophysiology of vasospasm common in aSAH. This vasospasm results in hypoxia, which can increase Ngb through a similar mechanism as AIS.

Out of 44 patients, there were two patients with very high Ngb levels. Both patients had worse neurological deficits, functional independence, and cognitive function. Although this finding does not contradict the general trend, it is necessary to investigate the cause of this very high increase in Ngb levels that might influence interpretation. The differentiating factor between these two subjects was the presence of nonconvulsive status epilepticus (NCSE), as evidenced by electroencephalography. Prolonged seizures are associated with physiological changes in the body, including alterations in heart rate, respiratory function, blood pressure, blood glucose, electrolyte concentrations, and body temperature. During the initial 20 to 40 minutes, homeostatic mechanisms can compensate for the metabolic demands of the convulsing brain and contracting muscles. However, the failure of these compensatory mechanisms typically begins approximately 20 to 40 minutes after onset. Although most of the pathophysiological studies on SE come from animal models, similar processes are likely to occur in humans.²⁷ The severity of cerebral hypoxia during convulsive SE is aggravated in the presence of other conditions, such as hyperthermia, hypotension, hypoglycemia, and acidosis.

There is evidence that nonconvulsive seizures also cause some degree of brain injury. When prolonged seizures are induced in paralyzed and mechanically ventilated primates, brain injury occurs, albeit less severely. Epileptic activity itself can lead to neuronal damage and death, primarily through activation of glutamate receptors and neuronal influx of Ca²⁺.²⁷ Therefore, after ruling out other possibilities, we concluded that NCSE contributed to the marked increase in serum Ngb in these two subjects.

Limitations. The relatively small sample size of our study was a limitation. With only 42 participants, the statistical power of our findings is limited, and the results might not be fully generalizable to the broader population of patients with AIS. Our study did not specifically investigate whether patients with pre-existing dementia or neurodegenerative diseases exhibit higher Ngb levels. Amyloid beta 42 oligomers trigger Ngb expression via oxidative stress, suggesting a protective response mechanism in neurons.²⁸ Given the neuroprotective role of Ngb, it would be valuable to explore this potential association in future research. 

We acknowledge that our method of collecting mRS score via phone after six months has its limitations. Variations in the type and intensity of rehabilitation received by patients during this period could significantly impact their recovery outcomes. Recognizing these factors is crucial for interpreting the study results accurately and planning future research to control for these variables more rigorously.

Conclusion

This study showed that serum Ngb levels in acute-phase ischemic stroke were higher in patients with unfavorable functional outcomes. Ngb might be useful as a supporting test to predict the prognosis of patients with AIS, but its clinical application still requires further research. It is necessary to consider serial examinations to better understand the pattern of Ngb expression in the acute phase of stroke. Further studies also need to consider examining other biomarkers in Ngb expression, such as Ngb mRNA or HIF-1α, to ensure the occurrence of an adaptive response to hypoxia. Establishing a predictive model that incorporates multiple prognostic markers, with a larger sample size and better subject selection criteria, can improve accuracy and increase the utility of the Ngb examination.

Acknowledgment 

We would like to thank Universitas Indonesia Research Grant, International Indexed Publication program 2023. We extend our gratitude to the Neurology Department of the Faculty of Medicine at Universitas Indonesia and Cipto Mangunkusumo General Hospital for facilitating this research. 

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. 

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