Review and Monograph: Recent Advances in Prognostic Biomarkers after Cerebral Infarction: Mechanisms and Clinical Applications

Jingtang Nong¹,²,³, Chengmin Yang², Shenglong Mo², Zhicheng Lu¹,², Lina Tang¹,², Chongdong Jian²,⁴, Jingwei Shang²,⁴

¹Graduate School of Youjiang Medical College for Nationalities, Baise 533000, China
²Department of Neurology, Affiliated Hospital of Youjiang Medical College for Nationalities, Baise 533000, China
³Key Laboratory of Metabolic Diseases in Baise City, Baise 533000, China

*Corresponding authors: Chongdong Jian, Professor; E-mail: jianchongdong@163.com; Jingwei Shang, Associate Researcher; E-mail: jw_shang@aliyun.com

Nong Jingtang and Yang Chengmin are co-first authors

Abstract

Ischemic stroke (IS) is one of the leading causes of disability and death worldwide. Various biomarkers closely associated with IS prognosis have been identified in recent years, including cytokines, microRNAs (miRNAs), and extracellular vesicles. This paper reviews the mechanisms of action of blood cell analysis related indicators, omega-3 polyunsaturated fatty acids, IL-6, TNF-α, miRNAs, and extracellular vesicles in ischemic stroke and their research progress as predictive biomarkers. The concentrations of blood cell analysis related indicators, omega-3 polyunsaturated fatty acids, IL-6 and TNF-α are closely related to the severity and prognosis of IS. miR-21 improves neuronal functional recovery by inhibiting apoptosis and promoting neuronal survival, while miR-155 exacerbates brain injury by regulating inflammatory responses, with its expression level predicting IS recurrence. Moreover, miR-126 plays a crucial role in angiogenesis and neuroprotection. Extracellular vesicles, carrying anti-inflammatory factors, neurotrophic factors, antioxidant enzymes, and heat shock proteins, mitigate inflammation and damage caused by ischemia-reperfusion, significantly influencing IS prognosis. Despite the tremendous potential of these biomarkers in IS prognosis assessment, their clinical application faces several challenges, including interindividual differences, inadequate research on long-term effects and safety, and issues with technical standardization. Future research should focus on elucidating the mechanisms of action of these biomarkers, developing standardized detection methods, and conducting large-scale clinical validation, to apply them in clinical practice to improve the prognosis and quality of life of IS patients.

Keywords: Brain Infarction; Prognosis; Biomarkers; Cytokines; MicroRNAs; Extracellular vesicles; Review

Introduction

Stroke, also known as cerebrovascular accident, is classified into ischemic and hemorrhagic types, both of which can cause various neurological deficits that severely impair patients' language, motor, and other daily living functions¹. Ischemic stroke, also called cerebral infarction, accounts for approximately 85% of all stroke cases². According to the 2019 Global Burden of Disease (GBD) report, there were 12.2 million incident cases of stroke globally, 101 million prevalent cases, and 6.55 million deaths³. In China, the situation is particularly severe, with 3.94 million incident cases, 28.76 million prevalent cases, and 2.19 million deaths⁴. Stroke has become the second leading cause of death and the third leading cause of disability worldwide, representing a major public health challenge that urgently needs to be addressed³. It is estimated that 33–42% of stroke patients still require assistance with daily living activities six years after onset, while 36% remain disabled five years post-stroke⁵, imposing a heavy burden on patients' families and society.

Current treatments for ischemic stroke mainly include intravenous thrombolysis, mechanical thrombectomy, and antiplatelet therapy. Intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) is limited by a narrow time window of 4.5 hours post-onset and carries a risk of hemorrhage. Although mechanical thrombectomy can extend the treatment window to 6–24 hours, it is only suitable for patients with large vessel occlusion and carries risks of vascular injury and re-thrombosis. Antiplatelet therapy is primarily used to prevent stroke recurrence but has limited efficacy in acute phase treatment. Despite significant progress in secondary prevention and treatment of ischemic stroke over recent decades, the disability and mortality rates remain high due to treatment time window limitations and risks of ischemia-reperfusion injury. Therefore, early diagnosis and timely treatment are crucial for improving ischemic stroke prognosis.

Good prognosis can alleviate patients' physical and psychological suffering, improve their quality of life, and reduce the economic burden on families and society. In recent years, the development and application of prognostic biomarkers for ischemic stroke have become a research focus for scholars worldwide. These markers have important clinical reference value in predicting ischemic stroke progression and prognosis. Blood cell analysis, as a commonly used clinical test, offers rapid and convenient detection. Inflammatory responses play an indispensable role in the occurrence and development of ischemic stroke, with inflammatory factors being key mediators. Additionally, recent studies have found that omega-3 polyunsaturated fatty acids (ω-3 PUFA), microRNAs (miRNA), and exosomes are closely associated with ischemic stroke prognosis.

Given the broad scope of biomarkers, this review focuses on recent domestic and international research progress regarding blood cell analysis indicators [such as red cell volume distribution width (RDW)], ω-3 PUFA, miRNA, and exosomes as prognostic markers for ischemic stroke. We selected these biomarkers because they play important roles in the pathogenesis and prognosis assessment of ischemic stroke and have been extensively reported in the literature. In our discussion, we first introduce biomarkers with strong evidence that have been applied clinically or validated in large cohort studies, followed by exploratory findings from smaller studies, aiming to provide new insights and approaches for prognosis assessment and clinical treatment of ischemic stroke.

Literature Search Strategy: We conducted computerized searches of CNKI, VIP, Wanfang Data, Web of Science, and PubMed for studies on prognostic biomarkers after cerebral infarction from database inception to March 2024, using a combination of MeSH terms and free-text keywords. Chinese search terms included: "cerebral infarction AND blood cell analysis," "cerebral infarction AND inflammatory factors," "cerebral infarction AND polyunsaturated fatty acids," "cerebral infarction AND microRNA," and "cerebral infarction AND exosomes." English search terms included: "(cerebral infarction OR ischemic stroke) AND red cell volume distribution width," "(cerebral infarction OR ischemic stroke) AND neutrophil to lymphocyte ratio," "(cerebral infarction OR ischemic stroke) AND platelet to lymphocyte ratio," "(cerebral infarction OR ischemic stroke) AND inflammatory factors," "(cerebral infarction OR ischemic stroke) AND PUFA," "(cerebral infarction OR ischemic stroke) AND miRNA," and "(cerebral infarction OR ischemic stroke) AND exosomes." Inclusion criteria were: (1) Chinese or English language only; (2) studies focusing on prognostic biomarkers after cerebral infarction; and (3) research including mechanisms and clinical applications. Studies without full-text availability were excluded.

1. Blood Cell Analysis Indicators and PUFA in Cerebral Infarction

The potential mechanisms of blood cell analysis indicators and PUFA in predicting disease progression and outcomes during cerebral infarction are illustrated in Figure 1 [FIGURE:1].

1.1 Red Cell Volume Distribution Width (RDW)

RDW is an indicator measuring the variability in red blood cell volume in peripheral blood, with increased values indicating greater heterogeneity in red cell size⁶. Elevated pro-inflammatory cytokine levels in plasma may inhibit erythropoietin (EPO)-induced maturation and proliferation of red blood cells and reduce erythropoietin receptor expression, thereby increasing RDW⁶⁻⁷. Traditionally, RDW has been used for classification and differential diagnosis of anemia⁸, but recent studies have revealed its important role in stroke prognosis assessment⁷. Although the exact mechanism remains unclear, it may be related to hemodynamic changes. Elevated RDW leads to anisocytosis, affecting peripheral blood circulation function and potentially serving as an independent or synergistic factor for increased circulation resistance and vascular occlusion⁹.

Xue et al.¹⁰ conducted a retrospective analysis of 629 patients with acute ischemic stroke and found through multivariate analysis that higher RDW was significantly associated with moderate to severe stroke (OR=2.21, 95%CI=1.30–3.75, P=0.003), modified Rankin Scale scores of 3–6 at 3 months (OR=1.86, 95%CI=1.02–3.41, P=0.044), and Barthel Index scores <85 (OR=2.27, 95%CI=1.25–4.12, P=0.007). Multiple logistic regression analysis further confirmed the correlation between RDW and stroke severity and poor functional outcomes. Li et al.¹¹ performed a retrospective analysis of 606 acute ischemic stroke patients aged ≥80 years and found that higher RDW was significantly positively correlated with in-hospital mortality (HR=3.31, 95%CI=2.47–4.45, P<0.001). Additional studies have reported that higher baseline RDW is not only associated with increased risk of recurrent ischemic stroke but also negatively correlated with time to stroke recurrence¹²⁻¹³. Therefore, RDW shows promise as a reliable and economical indicator for assessing prognosis and recurrence risk in ischemic stroke patients, providing important evidence for clinical diagnosis and prognosis evaluation.

1.2 Neutrophil-to-Lymphocyte Ratio (NLR)

Neutrophils, derived from granulocyte-monocyte progenitors in bone marrow, are terminally differentiated, short-lived phagocytic cells that play crucial roles in various diseases including trauma, infectious diseases, metabolic disorders, and autoimmune conditions¹⁴. Inflammatory responses are key factors causing cerebral ischemia injury¹⁵, with neutrophils directly damaging neurons through release of proteolytic enzymes (such as neutrophil elastase) and causing microvascular mechanical obstruction through aggregation in blood vessels¹⁶. Cai et al.¹⁷ conducted a case-control study of 225 Chinese Han patients with acute ischemic stroke and found that in patients with infarct area >1.5 cm, both neutrophil count (R²=0.07, P=0.0208) and NLR (R²=0.07, P=0.0447) were positively correlated with infarct area, revealing that elevated peripheral blood neutrophil levels in the early stage of stroke predict poor prognosis. As a composite parameter, NLR can more accurately reflect immune cell activity and offers higher stability, sensitivity, and specificity¹⁸. NLR has been validated as a prognostic marker for various diseases including cardiovascular disease, immune disorders, cancer, and infection¹⁹⁻²⁰.

Kocaturk et al.²¹ found that in acute ischemic stroke, NLR was significantly correlated with infarct volume in anterior circulation stroke (ACS) and was an independent predictor of 3-month mortality. Infarct volume in ACS patients showed significant correlation with NLR (r=0.482, P≤0.001). Multivariate analysis revealed that NLR was the only independent predictor of 3-month mortality (OR=1.186, 95%CI=1.032–1.363, P=0.016), with specificity and sensitivity of 76.5% and 63.6%, respectively, when NLR ≥4.7. Another case-control study showed that NLR was an independent predictor of in-hospital gastrointestinal bleeding in acute ischemic stroke patients receiving dual antiplatelet therapy, with predictive sensitivity of 87.8% and specificity of 85.7%²². Similarly, Xu et al.²³ conducted a prospective study of 341 ischemic stroke patients and demonstrated that NLR was closely associated with stroke progression (SP) and poor outcomes, with an area under the ROC curve (AUC) of 0.6117 (95%CI=0.5341–0.6893, P=0.0032) for predicting poor functional outcomes, an optimal cutoff value of 4.2139, and predictive sensitivity and specificity of 52.7% and 72.0%, respectively. As a low-cost, easily accessible biomarker, NLR holds significant importance in stroke diagnosis and prognosis evaluation.

1.3 Platelet-to-Lymphocyte Ratio (PLR)

Platelets, derived from mature megakaryocytes in bone marrow, are essential for maintaining vascular integrity and hemostasis. In cerebral ischemia, inflammatory responses damage endothelial cells, subsequently activating and aggregating platelets to form thrombi that obstruct vessels and cause cerebral tissue ischemia and infarction²⁴. PLR, as a novel inflammatory indicator, can simultaneously reflect thrombotic and inflammatory responses and is closely associated with the occurrence, severity, and prognosis of acute ischemic stroke.

Tsalta-Mladenov et al.²⁵ conducted a prospective study of 141 acute ischemic stroke patients and found that PLR was significantly associated with poor prognosis (P<0.001) and could predict the possibility of hemorrhagic transformation (HT) in young acute ischemic stroke patients. ROC curve analysis showed that when the optimal cutoff value of PLR for predicting 3-month poor prognosis was 122.6, sensitivity was 77.8% and specificity was 61.5% (AUC=0.613, P=0.031). Another prospective observational study indicated that PLR was associated with stroke severity and might serve as a prognostic marker for ischemic stroke, helping to prevent potential complications²⁶. Wen et al.²⁷ performed a retrospective study of 157 young patients with first-episode acute ischemic stroke and found that PLR was associated with independent risk factors for hemorrhagic transformation. ROC analysis showed that when the cutoff value of PLR for predicting hemorrhagic transformation was 109.073, sensitivity and specificity were 80.6% and 67.4%, respectively. Li et al.²⁸ conducted a retrospective study of 158 acute ischemic stroke patients and found that PLR was significantly associated with recurrent ischemic stroke (P<0.001). ROC analysis showed that PLR>115.9 provided optimal predictive effect for recurrent stroke, with sensitivity of 78.9% and specificity of 57.2% (AUC=0.72, P=0.021). Additionally, Sun et al.²⁹ found that PLR at 24 hours after rtPA intravenous thrombolysis was significantly increased in patients with poor prognosis and death, independently associated with increased risk of poor outcomes and mortality. Therefore, PLR may serve as a simple yet effective indicator for ischemic stroke prognosis, offering high predictive value for assessing disease outcomes.

2. Inflammatory Factors and Their Correlation with Ischemic Stroke Prognosis

Inflammatory responses are crucial in the course of ischemic stroke³⁰, with microglia (MG) playing a key role. The M1 phenotype of MG can produce various pro-inflammatory and cytotoxic factors, including but not limited to interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-23 (IL-23), and tumor necrosis factor α (TNF-α)³². Polarization toward the M1 phenotype exacerbates brain injury by releasing multiple inflammatory factors, reactive oxygen species (ROS), and proteases, upregulating C-reactive protein (CRP) expression, inducing neuronal excitotoxicity, and damaging the blood-brain barrier (BBB)³³. Additionally, matrix metalloproteinases (MMPs) further affect BBB integrity by degrading extracellular matrix (ECM) and tight junction proteins (TJs)³⁴. Given the importance of inflammatory responses in the pathological process of ischemic stroke, inflammatory factor concentrations may be key determinants of disease progression and thus have potential as prognostic biomarkers for ischemic stroke. The key mechanisms of inflammatory factors in predicting disease outcomes after cerebral ischemia are illustrated in Figure 2 [FIGURE:2].

2.1 C-Reactive Protein (CRP)

CRP is a non-glycosylated protein synthesized by hepatocytes that significantly increases during various inflammatory processes and serves as a sensitive marker for assessing inflammatory responses³⁵. However, due to limited sensitivity of CRP in clinical practice, detection of high-sensitivity C-reactive protein (hs-CRP) has become particularly important as a key tool for predicting disease progression and prognosis. Pu et al.³⁶ measured pre-admission serum hs-CRP levels in 119 acute ischemic stroke patients and found that patients with good prognosis had significantly lower median hs-CRP and NIHSS scores than those with poor prognosis (P<0.001), indicating that serum hs-CRP levels are closely associated with prognosis in acute ischemic stroke patients, with higher levels predicting worse outcomes. When the hs-CRP cutoff value was set at 11.835 mg/L, sensitivity for predicting 90-day outcomes in acute ischemic stroke patients was as high as 95% and specificity reached 92.5% (AUC=0.986), demonstrating the efficacy of hs-CRP in predicting acute ischemic stroke prognosis. Chen et al.³⁷ further confirmed that admission hs-CRP levels were significantly positively correlated with mortality, recurrence risk, and poor prognosis in ischemic stroke patients. High hs-CRP levels indicate poor prognosis and may exacerbate stroke recurrence and mortality rates. Cheng et al.³⁸ conducted a retrospective study of 212 acute ischemic stroke patients and found through logistic regression analysis that hs-CRP>1.60 mg/L was negatively correlated with good thrombolytic response rate (OR=0.496, 95%CI=0.266–0.927, P=0.028). Therefore, hs-CRP >1.6 mg/L can serve as a predictor of poor prognosis in acute ischemic stroke patients receiving intravenous thrombolysis, providing important reference for evaluating thrombolytic efficacy and predicting prognosis. In summary, hs-CRP, as a sensitive inflammatory marker, demonstrates significant application value in predicting prognosis of acute ischemic stroke patients and is expected to become an important tool for prognosis assessment and clinical decision-making.

2.2 Matrix Metalloproteinase-9 (MMP-9)

MMPs are key regulatory factors of vascular injury after stroke, mainly derived from activated macrophages, astrocytes, and extravasated neutrophils, with the ability to decompose and degrade various protein components including collagen, elastin, and fibronectin in the extracellular matrix³⁴. Among them, MMP-2, MMP-9, MMP-3, and MMP-12 have been confirmed to be associated with BBB damage after cerebral ischemia³⁹. Regarding the role of MMPs, particularly MMP-9, in predicting ischemic stroke prognosis, Yuan et al.⁴⁰ conducted a case-control study including 168 ischemic stroke patients and 40 healthy controls, using enzyme-linked immunosorbent assay (ELISA) to measure plasma MMP-9 concentrations and brain computed tomography (CT) or magnetic resonance imaging 3–14 days after stroke onset to diagnose spontaneous hemorrhagic transformation (sHT). Results showed that when MMP-9 concentration >181.7 ng/mL, it demonstrated high sensitivity (82.9%) and specificity (81.3%) for predicting hemorrhagic transformation, with positive predictive value of 48% and negative predictive value of 95.8%. Additionally, other studies have indicated a significant positive correlation between MMP-9 expression levels and stroke severity⁴¹. Li et al.⁴² further found that MMP-9 and brain-derived neurotrophic factor (BDNF) showed time-dependent association in prognosis prediction for acute ischemic stroke patients. Specifically, gradual decrease in MMP-9 levels and concurrent increase in BDNF levels often predicted better prognosis. Notably, dynamic changes in MMP-9 and BDNF showed greater advantage than baseline measurements in predicting acute ischemic stroke outcomes. In summary, MMP-9 demonstrates significant potential in ischemic stroke prognosis prediction, with its level changes being associated not only with post-stroke BBB damage and hemorrhagic transformation risk but also closely related to stroke severity, making it a promising biomarker for acute ischemic stroke prognosis assessment.

2.3 Interleukin-6 (IL-6)

IL-6 is an inflammatory cytokine with immunomodulatory and chemotactic functions that plays an important role in post-stroke inflammatory responses⁴³. Elevated serum IL-6 concentration after stroke leads to neuronal death and BBB damage, exacerbating brain injury⁴⁴. Multiple studies have demonstrated a close association between IL-6 concentration and stroke severity and prognosis. Shaafi et al.⁴⁵ conducted a cross-sectional study of 45 acute ischemic stroke patients, evaluating NIHSS and modified Rankin Scale scores on days 1, 5, 90, and 365, and measuring serum IL-6 levels on days 1 and 5 by ELISA. Results showed that IL-6 was significantly positively correlated with NIHSS and modified Rankin Scale scores, with higher IL-6 levels in deceased patients, indicating that higher IL-6 levels predict worse stroke prognosis. Li et al.⁴⁶ conducted a prospective study of 180 patients with first-episode acute ischemic stroke and found that IL-6 levels were significantly elevated in the poor prognosis group, establishing IL-6 as an independent risk factor for functional outcomes, with higher levels indicating worse prognosis. Furthermore, IL-6 can serve as an indicator of futile reperfusion in acute ischemic stroke patients undergoing mechanical thrombectomy. Mechtouff et al.⁴⁷ collected peripheral blood samples from 164 acute ischemic stroke patients treated with mechanical thrombectomy (MT) before pre-hospital intravenous (IV) thrombolysis, at 6 h, 24 h, 48 h, and 3 months after admission, and measured IL-6 using ELISA kits. CT scans were performed on day 1 and follow-up MRI on day 6. Multivariate analysis showed that high IL-6 levels within 24 hours after admission (OR=6.15, 95%CI=1.71–22.10) were associated with futile reperfusion. In summary, IL-6 has important value in predicting ischemic stroke prognosis, and serum IL-6 levels can be used to predict stroke severity, functional outcomes, and occurrence of futile reperfusion after MT.

2.4 Other Inflammatory Factors

TNF-α plays a key role in neurotoxic substance generation and local inflammatory responses and is considered an important indicator for predicting stroke prognosis⁴⁸. Studies have shown that TNF-α levels within 24 hours of ischemic stroke onset are closely related to infarct size⁴⁹. In patients with lacunar infarction, blood TNF-α levels are independently associated with poor prognosis at 3 months⁵⁰. Additionally, lipoprotein-associated phospholipase A2 (Lp-PLA2), as a vascular-specific inflammatory marker, has been identified as an independent predictor of ischemic stroke⁵¹. Research shows that elevated Lp-PLA2 levels are significantly associated with poor functional outcomes at 3 months and 1 year after ischemic stroke⁵². Li et al.⁵³ further demonstrated that elevated serum Lp-PLA2 levels are significantly correlated with incidence, severity, and recurrence rate of acute ischemic stroke. In summary, TNF-α and Lp-PLA2 have important value in predicting ischemic stroke prognosis and are expected to serve as key biomarkers for clinical diagnosis and assessment, providing new perspectives and evidence for prognosis prediction and clinical treatment of ischemic stroke patients.

3. Correlation Between PUFA and Ischemic Stroke Prognosis

Fatty acids can be divided into saturated and unsaturated fatty acids, with the latter further subdivided into monounsaturated and polyunsaturated fatty acids (PUFA), including ω-3 and ω-6 types. ω-3 PUFA includes α-linolenic acid (ALA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). Studies have indicated that ω-3 PUFA plays an important role in reducing neural damage caused by ischemic stroke, exerting neuroprotective effects⁵⁴ and effectively reducing the risk of ischemic stroke⁵⁵. Lin et al.⁵⁶ revealed that ω-3 PUFA inhibits microglial release of TNF-α by suppressing a disintegrin and metalloproteinase 17 (ADAM17) activity and promotes microglial release of exosomal nerve growth factor while activating the neuroprotective TNF-α/nuclear factor κB (NF-κB) pathway. These two mechanisms work synergistically to achieve neuronal protection.

Recent research advances have further confirmed the important value of ω-3 PUFA in predicting and improving prognosis of ischemic stroke patients. Suda et al.⁵⁷ conducted a retrospective study of 281 Japanese patients diagnosed with acute ischemic stroke within 24 hours of onset and found that early neurological deterioration (END) was negatively correlated with EPA/AA ratio (OR=0.18, P=0.003), DHA/AA ratio (OR=0.045, P=0.001), and (EPA+DHA)/AA ratio (OR=0.45, P=0.002), revealing that low serum ω-3 PUFA/ω-6 PUFA ratio at admission may predict neurological deterioration in acute ischemic stroke. Song et al.⁵⁸ performed a prospective study showing that ω-3 PUFA was negatively correlated with admission severity and 3-month poor outcomes in ischemic stroke, suggesting that ω-3 PUFA can serve as a potential blood biomarker for prognosis in acute non-cardiogenic ischemic stroke patients. Additionally, another cross-sectional study indicated that EPA was negatively correlated with ischemic stroke prevalence⁵⁹. Shojima et al.⁶⁰ further confirmed that low EPA/AA ratio at admission was associated with poor long-term prognosis and increased mortality in ischemic stroke patients. In summary, ω-3 PUFA, as a potential blood biomarker for ischemic stroke prognosis, plays an important role in prognosis assessment and treatment of ischemic stroke, with its concentration and ratio having significant clinical implications.

4. Correlation Between miRNA and Ischemic Stroke Prognosis

miRNAs are non-coding RNAs that play critical roles in gene expression regulation and demonstrate important regulatory functions in multiple aspects of acute ischemic stroke pathology, including energy failure, inflammatory responses, and apoptosis⁶¹. Consequently, miRNAs are expected to become reliable blood biomarkers for acute ischemic stroke risk prediction, precise diagnosis, and prognosis assessment. Numerous studies have confirmed that many miRNAs are closely related to the occurrence, development, and clinical course of acute ischemic stroke⁶².

4.1 miR-21

miR-21 has been clearly identified as a key factor that effectively inhibits apoptosis and strongly promotes neuronal survival during the initiation and progression of ischemic stroke⁶³. Zhou et al.⁶⁴ revealed in an oxygen-glucose deprivation (OGD) model study that miR-21 could significantly inhibit apoptosis in mouse neuroblastoma (N2A) cells after oxygen-glucose deprivation/reperfusion (OGD/R) treatment. Additionally, Yan et al.⁶⁵ showed in a middle cerebral artery occlusion (MCAO) mouse model that downregulating miR-21 expression significantly upregulated p53 and Bax expression while downregulating Bcl-2 expression, thereby exacerbating ischemic neuronal damage, demonstrating the core role of the miR-21/p53/Bcl-2/Bax signaling pathway in ischemic injury regulation. Notably, miR-21 expression levels are closely related to prognosis in ischemic stroke patients. Specifically, high miR-21 expression often predicts better prognosis, likely due to its ability to inhibit apoptosis, reduce brain damage, and promote neural tissue repair and regeneration, thereby effectively improving patient outcomes⁶⁶. Yuan et al.⁶⁷ further noted that post-stroke cognitive impairment (PSCI) patients had significantly higher miR-21 expression levels than cognitively normal (PSCN) patients, suggesting that higher serum miR-21 levels at admission in ischemic stroke patients correspond to higher risk of post-stroke cognitive impairment. Therefore, miR-21 alone or combined with specific indicators (such as FA values) may serve as important diagnostic biomarkers for distinguishing PSCI from PSCN.

4.2 miR-155

miR-155 is an immune regulatory microRNA that plays a significant role in ischemic brain injury by precisely regulating downstream signaling pathways⁶⁸. Studies have shown that upregulated miR-155 expression can activate inflammatory signaling pathways in macrophages and T lymphocytes (TC), promoting release of inflammatory factors such as TNF-α and IL-1β and exacerbating inflammatory responses and brain tissue damage⁶²,⁶⁹. Additionally, miR-155 can regulate NF-κB and MAPK signaling pathway activity, profoundly affecting inflammatory mediator release and regulation⁷⁰,⁷¹. Notably, knocking down miR-155 expression significantly alleviates inflammatory responses and brain damage after cerebral circulation ischemia⁶⁸. Therefore, miR-155 plays an important role in regulating inflammatory responses and ischemic brain injury and holds promise as a potential biomarker for ischemic stroke prognosis assessment and a novel therapeutic target.

Yang et al.⁷² found in a transient middle cerebral artery occlusion (tMCAO) mouse model that miR-155-5p expression in choroid plexus epithelium (CPE) significantly increased after OGD/R treatment, while inhibition of miR-155-5p markedly reduced autophagy levels in mouse brain tissue. Zhang et al.⁷³ conducted a case-control study including 93 ischemic stroke patients and 70 non-stroke controls and revealed that plasma endothelial microparticles (EMV) and EMVs-miR-155 levels were significantly elevated in acute and subacute phases of ischemic stroke and positively correlated with infarct volume and NIHSS scores. Multivariate logistic regression analysis further indicated that plasma EMVs and EMVs-miR-155 were important independent risk factors for ischemic stroke. The study also suggested that EMVs-miR-155 could serve as a predictive factor for ischemic stroke occurrence in non-stroke individuals. Chen et al.⁷⁴ found that in acute ischemic stroke patients, miR-155 expression was higher after treatment, while ROC curve analysis showed that when miR-155>2.665, sensitivity and specificity for predicting acute ischemic stroke occurrence were 77.05% and 65.75%, respectively. Follow-up results showed that recurrent patients had higher miR-155 expression than non-recurrent patients, and when miR-155>2.630, sensitivity and specificity for predicting ischemic stroke recurrence were 88.89% and 69.77%, respectively. Therefore, serum miR-155 has good predictive function for both onset and prognosis of acute ischemic stroke.

4.3 miR-126

miR-126 is an endothelial cell-specific miRNA involved in regulating angiogenesis. Under cerebral hypoxic conditions, miR-126 effectively inhibits ischemia-hypoxia-induced oxidative stress and inflammatory responses by regulating endothelial cell function⁶¹,⁶³. miR-126 alleviates BBB damage by inhibiting MMPs expression and preventing degradation of junction proteins (such as ZO-1, claudin-5, and occludin)⁷⁵. Additionally, upregulating miR-126 expression can stimulate vascular endothelial growth factor (VEGF) and PI3K/Akt signaling pathways, thereby promoting angiogenesis and neural repair⁷⁵,⁷⁶.

miR-126 expression levels in ischemic stroke are closely related to infarct size, disease severity, and prognosis. Specifically, upregulated miR-126 expression can promote neurovascular repair and regeneration and is therefore considered a positive prognostic factor for ischemic stroke. Pan et al.⁷⁷ established a cerebral ischemia-reperfusion (MCAO) mouse model and confirmed that miR-126-3p/miR-126-5p could significantly reduce ischemic stroke volume, alleviate cerebral edema, reduce degradation of tight junction (TJ) proteins and IgG leakage, and significantly improve prognosis in mice with ischemic stroke. Furthermore, upregulated expression of miR-126-3p and miR-126-5p can inhibit expression of pro-inflammatory cytokines such as IL-1β, TNF-α, and adhesion molecules⁷⁴. These results suggest that miR-126 may play roles in angiogenesis and neuroprotection, thereby reducing brain damage and improving prognosis. Other studies have shown that plasma miR-126 levels in acute ischemic stroke patients were lower than in controls and negatively correlated with NIHSS scores and TNF-α, IL-1β, and IL-6 levels, suggesting that higher plasma miR-126 levels correspond to lower disease risk, lower severity, and better prognosis⁷⁸. Additionally, Qi et al.⁷⁹ found that miR-126 expression levels were associated with neurological function, self-care ability, and prognosis in ACI patients, holding important value for predicting patient outcomes.

miRNAs play key regulatory roles in cell differentiation, biological development, and disease processes. Currently, more than 2,000 human miRNAs have been registered in annotation databases⁸⁰. In addition to miR-21, miR-155, and miR-126, many other miRNAs are closely associated with ischemic stroke prognosis⁶⁰,⁶²,⁶⁶. For example, the neuroprotective role of miR-124 in cerebral ischemia injury has been extensively studied and recognized. Studies have shown that miR-124 can effectively promote neuronal survival and regeneration by regulating inflammatory responses and apoptosis, playing important neuroprotective roles after stroke. Additionally, research has found that miR-223 has significant effects in regulating inflammatory responses and reducing brain damage, with its high expression levels closely associated with better functional outcomes. Furthermore, miR-181 family members play important roles in cell death and inflammatory responses after cerebral ischemia. Specifically, inhibiting miR-181a expression significantly reduces brain damage and improves neurological function, demonstrating its great potential in post-stroke neuroprotection. Similarly, miR-30d exerts important protective effects after cerebral ischemia by regulating autophagy-related gene expression, with its high expression levels closely associated with better prognosis. In summary, multiple miRNAs play crucial roles in the pathophysiological process of ischemic stroke and are closely related to patient prognosis. In-depth investigation of the mechanisms and regulatory networks of these miRNAs is expected to provide novel biomarkers and therapeutic targets for precise diagnosis, effective treatment, and prognosis assessment of stroke.

5. Correlation Between Exosomes and Ischemic Stroke Prognosis

Exosomes are membrane-bound vesicles 30–150 nm in diameter secreted by various cells through exocytosis, carrying bioactive molecules including proteins, lipids, RNA, and DNA. Exosomes enter target cells by binding to receptors on the target cell membrane or through endocytosis, thereby playing central roles in intercellular signal transduction and material exchange, profoundly affecting numerous physiological and pathological processes including immune regulation, apoptosis, angiogenesis, and neural repair⁸¹. In the pathological process of ischemic stroke, exosomes demonstrate multiple mechanisms: carrying anti-inflammatory factors to alleviate inflammation induced by ischemic stroke, promoting neuroprotection and repair in damaged areas; transporting neurotrophic factors (such as BDNF and nerve growth factor) to assist neuronal survival and axonal regeneration; delivering antioxidant enzymes and heat shock proteins to effectively mitigate oxidative stress and apoptosis triggered by ischemia-reperfusion; and strengthening BBB integrity to reduce cerebral edema and secondary damage after ischemic stroke⁸²⁻⁸³.

In recent years, research on exosomes in ischemic stroke prognosis assessment has attracted increasing attention⁸⁴. Multiple studies have revealed that specific miRNAs in exosomes (such as miR-124 and miR-21) are closely associated with prognosis in ischemic stroke patients⁸⁵. These miRNAs significantly affect neurological function recovery by finely regulating inflammatory responses and apoptosis. Specifically, miR-21 improves post-stroke neurological function rehabilitation by inhibiting apoptosis and promoting neuronal survival⁶³, while miR-124 plays a key role in neuroprotection and regeneration, helping to reduce tissue damage caused by ischemic stroke⁸⁶. Additionally, protein markers carried by exosomes, such as heat shock protein 70 (HSP70) and tissue plasminogen activator (tPA), are also considered potential biomarkers for predicting ischemic stroke prognosis⁸⁷. These proteins play important roles in neuroprotection and repair. Studies have found that HSP70 in exosomes reduces cell damage caused by ischemia-reperfusion through anti-inflammatory and antioxidant mechanisms, while tPA effectively alleviates ischemic stroke severity by dissolving thrombi and restoring blood flow⁸⁸. The mechanisms of miRNA and exosomes in predicting cerebral infarction prognosis are illustrated in Figure 3 [FIGURE:3].

Despite the tremendous potential of exosomes as prognostic biomarkers for ischemic stroke, their clinical translation faces several challenges. The primary issue is significant interindividual variability in exosome composition and function, which challenges their reliability as universal biomarkers. Secondly, the long-term effects and safety of exosomes in humans remain insufficiently understood, limiting their clinical feasibility and efficacy. Finally, standardized techniques for exosome extraction, purification, and detection are lacking, hindering their widespread application in clinical practice. Therefore, future research should focus on developing standardized technologies, exploring mechanisms of action in depth, and conducting large-scale clinical validation to effectively apply this novel biomarker in ischemic stroke prognosis assessment and treatment strategies, ultimately improving patient outcomes and quality of life.

Discussion

This review summarizes the importance and potential mechanisms of various biomarkers in ischemic stroke prognosis assessment, covering hematological indicators, inflammatory factors (such as IL-6, TNF-α, and Lp-PLA2), ω-3 PUFA, miRNAs (such as miR-21, miR-155, and miR-126), and exosomes. Among these, blood cell analysis, as a routine clinical test, provides critical information for ischemic stroke prognosis judgment due to its rapid and convenient characteristics. Inflammatory factors IL-6, TNF-α, and Lp-PLA2 significantly affect ischemic stroke prognosis by regulating inflammatory processes. Notably, blood routine indicators such as RDW are also associated with ischemic stroke prognosis, with higher baseline RDW correlating with increased risk of recurrent ischemic stroke and negatively correlating with time to stroke recurrence. Additionally, ω-3 PUFA demonstrates important value in predicting and improving prognosis of ischemic stroke patients, while specific miRNAs play crucial roles by regulating apoptosis, inflammatory responses, and angiogenesis. Exosomes, as a research hotspot in recent years, show great application potential in stroke prognosis assessment through their carried miRNAs and proteins.

Although existing research has preliminarily revealed the application prospects of these biomarkers, their clinical promotion still faces numerous challenges. For each marker, this article elaborates on its clinical advantages and limitations, and points out that future research directions should focus on the following aspects: First, standardized extraction and analysis techniques need to be developed to ensure consistency and reliability of experimental results. Second, the specific roles of exosomes and miRNAs in stroke pathological processes and repair mechanisms should be thoroughly investigated. Third, large-scale, rigorously designed preclinical and clinical studies (including cross-sectional studies, case-control studies, cohort studies, and randomized controlled trials) should be conducted, with clear specification of sample types, sample sizes, ethnic distributions, and sample collection methods to validate the reliability and effectiveness of these biomarkers across different patient populations. Finally, systematic evaluation of their long-term effects and safety in vivo should be performed to provide solid scientific evidence for clinical application. Through further in-depth research and clinical validation, these biomarkers are expected to play more precise roles in stroke prognosis assessment and treatment, promoting the development of personalized diagnosis and treatment strategies. In particular, if these biomarkers are established, specific application strategies in disease assessment and drug target selection should be strengthened to significantly improve stroke patient prognosis, enhance their quality of life, and reduce the economic burden on families and society.

Author Contributions: Jingtang Nong conducted the research, wrote and revised the manuscript; Shenglong Mo, Zhicheng Lu, and Lina Tang performed literature retrieval and screening; Chengmin Yang extracted and analyzed data; Chongdong Jian proposed the research idea, provided funding, and guided the research; Jingwei Shang provided technical and material support and revised the manuscript.

Conflict of Interest: The authors declare no conflict of interest.

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Received: 2024-09-10; Revised: 2024-11-20
Edited by: Mao Yamin