Exploring the Association Between Serum Neurogranin, Nardilysin, and Ischemic Stroke: A Case-Control Study Conducted in the Emergency Department

Introduction

Ischemic stroke (IS) is a leading cause of long-term mortality and disability globally, with a growing incidence due to an aging population [1,2]. The prompt identification and management of IS in the emergency department (ED) is crucial, as early intervention can significantly improve patient outcomes [3]. However, the complex pathophysiology of IS, which involves endothelial dysfunction, reduced nitric oxide bioavailability, platelet activation, and neuronal injury, presents challenges in effectively diagnosing and predicting the prognosis of affected individuals [4]. In this context, the identification of novel biomarkers that improve the biochemical diagnosis and prognostic assessment of IS has significant clinical relevance.

Neurogranin (Ng) is another protein, consisting of 78 amino acids, predominantly found in pyramidal cells with a granule-like appearance in the hippocampus and cortex [5,6]. Its name reflects its association with these specific cells, and it is recognized for its role in neurosynaptic plasticity, regeneration, and long-term potentiation [7,8]. Due to its involvement in postsynaptic activity, recent research has focused on exploring the connection between Ng and various neurological conditions [5,9]. Ng has been used as a biochemical test for diagnosing and predicting the prognosis of numerous neurological and psychiatric disorders, including neurodegenerative diseases, Alzheimer disease, Parkinson disease, IS, and schizophrenia [5,10]. Additionally, studies have suggested that serum and cerebrospinal fluid (CSF) Ng levels vary in response to diffuse axonal injury, highlighting its potential role as a biomarker in traumatic brain injury models [10]. Moreover, biomarkers like Ng can be analyzed more conveniently than CSF, further emphasizing their significance in studying this group of diseases [5].

N-arginine dibasic convertase, also known as Nardilysin (NRDC), is a metallopeptidase belonging to the M16 family [11,12]. It facilitates the ectodomain shedding of various membrane proteins, including tumor necrosis factor-α and heparin-binding epidermal growth factor, in the extracellular zone [12,13]. Additionally, it regulates transcriptional functions in the nucleus by providing communication between the nucleus and cytoplasm [12,14]. NRDC plays a critical role as a positive regulator in axonal maturation and myelination. Its impact on myelin thickness has been demonstrated in a dose-dependent manner in vivo, showing its potential as a pharmacological target for demyelinating conditions such as multiple sclerosis. Furthermore, NRDC contributes to axonal repair following injury [15]. Evidence from in vivo mouse models also suggests that NRDC helps prevent amyloid plaque formation in Alzheimer disease [16]. These experimental findings underscore NRDC’s involvement in a diverse array of neurological disorders [15].

Considering that NRDC and Ng have been investigated in various neurological diseases and found to have significant associations, we hypothesized that these biomarkers might also have a clinical correlation in IS cases, which involve severe neuronal damage. Given their previously established relevance in neurodegenerative and ischemic processes, we propose that NRDC and Ng levels may exhibit meaningful changes in acute IS cases due to neuronal injury. This hypothesis forms the basis of our study, aiming to determine whether these biomarkers could serve as valuable indicators of ischemic cerebrovascular disease.

In this study, we aimed to explore the diagnostic and prognostic potential of Ng and NRDC in IS in the ED. By examining their serum levels in a case-control framework, we assessed their clinical utility in identifying IS and predicting patient outcomes. Understanding the relationship between these biomarkers and IS pathophysiology could provide valuable insights into neuronal damage mechanisms and contribute to the development of novel biochemical tools for improving early diagnosis and risk stratification in acute stroke management.

Material and Methods

ETHICAL FRAMEWORK, PATIENT CONSENT, AND RESEARCH CENTER CHARACTERISTICS:

Approval for this study was obtained from the 2^nd Ethics Committee of the hospital, where the cases were collected, with approval number E2-23-4178, on May 26, 2023. The researchers provided detailed information about the study to the patients or their legal guardians who were eligible to participate, and informed written consent was obtained from those who agreed. The study was conducted in a tertiary-care, high-volume hospital that serves as a referral center for both medical and surgical emergencies. Patients were admitted through direct emergency visits or transfers via land and air ambulances from surrounding hospitals. This center is fully equipped for stroke management, including advanced imaging modalities and interventional radiology for embolectomy procedures.

STUDY DESIGN, PATIENT SELECTION, AND DIAGNOSIS:

This study was designed as a prospective case-control study. The patient and control groups were recruited between June 1 and October 1, 2023. The patient group consisted of individuals diagnosed with IS in the ED, while the control group comprised healthy individuals matched for age and sex.

Patients included in the IS group were those presenting to the ED with an acute neurological deficit. Diagnosis was confirmed through cranial computed tomography (CT) and diffusion-weighted magnetic resonance imaging (MRI), demonstrating an ischemic lesion corresponding to the clinical findings. The control group consisted of individuals without any symptoms suggestive of cerebrovascular disease, as confirmed by a detailed neurological examination and the absence of ischemic lesions on neuroimaging.

Participants included in the study were required to be at least 18 years old, provide informed consent, and have no history of chronic neurological or psychiatric disorders. Patients with active infection, malignancy, recent trauma, or pre-existing neurodegenerative diseases that could influence biomarker levels were excluded.

BIOCHEMICAL ASSESSMENT OF NG AND NRDC:

For biochemical analysis, 5-mL venous blood samples were collected from all participants into red-capped tubes at the time of admission. The venous blood samples were centrifuged at 1300×g for 10 minutes to separate the serum. The separated serum was aliquoted into Eppendorf tubes and stored at -80°C until further analysis.

Ng and NRDC levels were measured using commercially available ELISA kits in accordance with the manufacturer’s protocols. Ng was quantified using an ELISA kit (Fine Test, Wuhan, China; catalog no: EH2396; lot no. H2396H085 E), while NRDC was measured using another ELISA kit from the same manufacturer (catalog no. EH10653; lot no. H10653H085 E). Before analysis, serum samples were diluted 1: 2 with the recommended dilution buffer, as per the manufacturer’s instructions.

The 96-well ELISA plates were precoated with anti-Ng and anti-NRDC antibodies. Standard solutions and patient samples were added to the wells, followed by an incubation period. Unbound conjugates were washed away using a buffer, and a biotinylated detection antibody was added to bind with the specific antigen-antibody complexes. After another washing step, horseradish peroxidase (HRP)-streptavidin solution was applied, followed by 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate to visualize the enzymatic reaction. The reaction was stopped, and optical density (OD) readings were measured at 450 nm using a microplate reader.

The concentration of Ng and NRDC was determined by comparing OD values to a standard curve, with the detection ranges and assay precision as follows:

To ensure unbiased data collection, the laboratory technician performing ELISA was blinded to the patients’ clinical data, imaging results, and group assignments. Similarly, the ED physician responsible for diagnosing patients and interpreting neuroimaging studies was blinded to the biomarker measurements.

DIAGNOSTIC AND PROGNOSTIC ENDPOINTS:

The primary outcome measure was the assessment of Ng and NRDC levels as potential diagnostic and prognostic biomarkers in IS patients. Serum Ng and NRDC levels were evaluated for their ability to differentiate between IS cases and healthy controls. Secondary outcomes included their association with disease severity and the requirement for intensive care unit (ICU) admission.

Additionally, biochemical parameters, such as urea, creatinine, and hemoglobin levels, were analyzed. The study also aimed to assess the diagnostic accuracy of Ng and NRDC through ROC analysis.

STATISTICAL ANALYSES:

All statistical analyses were conducted using SPSS version 18.0 (IBM, Inc.) and Analyse-it (Analyse-it Software, Ltd). The normality of data distribution was assessed using the Kolmogorov-Smirnov test. Comparisons between the IS and control groups were performed using the t test or Mann-Whitney U test, as appropriate, while categorical variables were analyzed using Pearson’s chi-squared test.

ROC analysis was performed to evaluate the diagnostic performance of Ng and NRDC, with area under the curve (AUC) values and optimal cut-off points determined using the highest likelihood ratio. A P value <0.05 was considered statistically significant.

To ensure the statistical robustness of the findings, post hoc power analysis was performed using the G-Power program. With 44 cases in the IS group and 44 in the control group, at an alpha error of 0.05, the calculated effect size was 0.7069, yielding a study power of 95%.

Results

BASELINE CHARACTERISTICS:

A total of 44 patients with IS and 44 age- and sex-matched healthy controls were included in the study. Sex distribution was similar between the 2 groups, with no statistically significant difference observed (P=0.83). The mean age of IS patients was 71.48±14.19 years, slightly older than that of the control group (66.52±8.93 years), although the difference did not reach statistical significance (P=0.1). The mean body mass index (BMI) was significantly higher in IS patients (26.25±4.35) compared to controls (24.11±2.12; P=0.005), suggesting a potential association between higher BMI and IS risk (Table 1).

NEUROLOGICAL SYMPTOMS IN IS PATIENTS:

The analysis of clinical symptoms in IS patients revealed that dysarthria was the most frequently reported neurological concern, observed in 34% of patients. This was followed by right lateral deficits (31%) and facial asymmetry (22%). Additional symptoms included left lateral deficits (20%), confusion (18%), and visual impairment (9%). These findings indicate the multifactorial neurological involvement in IS, demonstrating the diversity of clinical manifestations (Table 2).

COMPARISON OF BIOCHEMICAL AND HEMATOLOGICAL PARAMETERS:

Ng levels were significantly higher in IS patients (281.12±32.12 pg/mL) compared to controls (265.71±24.54 pg/mL; P=0.01). In contrast, NRDC levels showed no significant difference between groups (6.35±4.13 ng/mL in IS patients vs 6.99±4.16 ng/mL in controls; P=0.47) (Table 3).

Among other biochemical markers, IS patients exhibited significantly higher levels of aspartate aminotransferase (36.75±34.03 U/L; P=0.04), urea (47±21.61 mg/dL; P=0.003), and creatinine (1.01±0.41 mg/dL; P=0.02) compared to controls. Hemoglobin levels were lower in IS patients (12.11±1.79 g/dL) than in controls (12.99±1.69 g/dL; P=0.02). Additionally, lymphocyte counts were significantly lower in IS patients (1.801±0.798×109/L) compared to the control group (2.181±0.829×109/L; P=0.03) (Table 3).

In the IS patient group, Ng levels were significantly higher in individuals admitted to the ICU (311.50±46.13 pg/mL) compared to those hospitalized in other services (277.23±28.40 pg/mL; P=0.023). This finding suggests a potential relationship between elevated Ng levels and IS severity, although its prognostic significance remains to be fully clarified. In contrast, NRDC levels did not show a significant difference between ICU admissions (7.46±7.69 ng/mL) and other services (6.22±3.60 ng/mL; P=0.226). These results emphasize the potential role of Ng in identifying patients at higher risk of requiring intensive care interventions (Table 4).

DIAGNOSTIC UTILITY OF NG:

ROC analysis demonstrated that the Ng levels yielded an AUC of 0.624 (95% CI: 0.508–0.741) for diagnosing IS, reflecting moderate diagnostic accuracy (Figure 1).

Discussion

LIMITATIONS AND FUTURE STUDIES:

Certain limitations, however, should be acknowledged. One major limitation is that our study relied solely on serum biomarker analysis using ELISA, without validation through other methods such as tissue protein or mRNA expression. Due to ethical and practical constraints, we were unable to collect tissue samples from patients, which precluded the use of these techniques. This restricted our ability to confirm that Ng and NRDC are highly specific biomarkers for IS. Additionally, the study did not include CSF analyses, which could have provided additional insights, as prior research indicates stronger biomarker correlations in CSF compared to plasma. Furthermore, infarct-specific data were not included, limiting our ability to comprehensively evaluate the relationship between Ng levels and the severity of IS. These omissions represent areas for further exploration.

Future research should validate these findings in larger, multi-center cohorts and address these limitations. Incorporating complementary validation methods such as tissue protein or mRNA expression analyses, when ethically and practically feasible, would strengthen biomarker specificity. Additionally, including CSF analyses alongside plasma Ng and NRDC measurements and exploring additional biomarkers may enhance diagnostic and prognostic utility. Expanding the scope to include infarct-specific data and investigating dynamic biomarker changes over time could provide a more comprehensive understanding of the clinical relevance of Ng and NRDC in IS management.

Conclusions

This study identified distinct roles of Ng and NRDC as biomarkers in IS. Plasma Ng levels had a moderate association with its clinical relevance, whereas NRDC did not appear to have significant utility. Additionally, higher Ng levels show a moderate correlation with the need for intensive care, suggesting a potential link with stroke severity. While Ng may have a meaningful role in clinical assessment, the utility of NRDC remains limited. Further research with larger patient groups and different methodologies is needed to validate Ng’s role and explore its potential integration into stroke management strategies.