This prospective observational cohort study enrolled patients with spontaneous aneurysmal SAH admitted to the Second Affiliated Hospital of Zhejiang University and Zhejiang Provincial People’s Hospital between October 2021 and October 2023 (ClinicalTrials.gov NCT06009016). Eligible patients were required to be ≥18 years old, diagnosed with aneurysmal SAH, and have a modified Fisher score (mFS) of 2-4 by Computed Tomography Angiography (CTA) within 24 h of symptom onset. Exclusion criteria included: (1) non-aneurysmal SAH (e.g., trauma, arteriovenous malformations, angiogram-negative SAH); (2) previous central nervous system (CNS) disorders (stroke, traumatic brain injury, CNS infections); (3) severe comorbidities within 6 months prior to SAH (e.g., malignancies, drug-resistant cardiovascular diseases, coagulation disorders, or organ dysfunction). Patients with normal pressure hydrocephalus (NPH) aged ≥18 years were enrolled as the control group, presenting at least two symptoms of the Hakim-Adams triad (gait disturbance, urinary incontinence, and cognitive decline), CSF pressure <200 mm H₂O, normal CSF content, and exclusion of other medical causes [9]. Ethical approval was granted by the Institutional Review Board of the Second Affiliated Hospital of Zhejiang University (Approval No. 2023-059). Informed consent was obtained from participants or their family members, or waived by the board.

Digital subtraction angiography (DSA) was performed within 24 h of admission to identify the responsible aneurysm, followed by embolization or clipping by experienced neurointerventional or neurosurgical teams. LD was routinely performed for patients with an mFS of 3-4, except when acute hydrocephalus necessitated EVD. Postoperative brain CT scans were initially performed, with follow-up scans every 1–3 days, as clinically indicated, until discharge. Nimodipine was administered to prevent cerebral vasospasm, and intravenous rehydration was used to maintain normal blood volume.

Sample size estimation was conducted by G*Power (version 3.1) to determine the number of patients of SAH (one-sample case), with an α error probability of 0.05, effect size [d] of 0.4, study power (1-β) of 0.9. The result was 55 aSAH patients. We totally enrolled 63 patients to ensure the study’s statistical power.

Functional outcomes assessed by the mRS were collected via telephone calls or hospital and clinic visits 3 months after discharge. mRS scores of 0–2 were considered good outcomes, while scores of 3–6 indicated poor outcomes. Assessments were conducted by independent, trained investigators blinded to study details to minimize bias.

Baseline characteristics (age, gender, BMI, medical history, and social history) were assessed by the treating physician. Clinical data upon admission, including the Glasgow Coma Scale (GCS), Hunt and Hess (HH) grade, and World Federation of Neurosurgeons Scale (WFNS), were recorded. Treatment-related data, such as surgical approaches, ICU length of stay, and duration of mechanical ventilation, were also documented. SAH-related complications, including seizures and delayed cerebral ischemia (DCI), were monitored. DCI was defined as focal neurological deficits or a ≥2-point decline in GCS, caused by cerebral vasospasm or infarction (new infarcts on CT or MRI, excluding those occurring within 48 h post-surgery). Functional outcomes were evaluated at 3 months post-discharge using the mRS, with good prognosis defined as mRS scores of 0–3 and poor prognosis as scores of 4–6. Radiological data collected from head CT scans included mFS, Subarachnoid Hemorrhage Early Brain Edema Score (SEBES), intraventricular hemorrhage (IVH), and the highest Hounsfield unit (HU) in the hemorrhagic area. CSF samples were collected via LD or EVD within 72 h postoperatively. Following collection, samples were centrifuged at 3,000 rpm for 10 min at 4 °C to remove erythrocytes and immune cells, and stored at −80 °C until ELISA analysis.

CSF ApoC3 concentrations were determined using the Human Apolipoprotein CIII ELISA Kit (ab154131, Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol. The intra-assay coefficient of variation (CV) is 6% and the inter-assay CV is 11%. All samples were tested in duplicate, and mean values were calculated for further analysis. Samples were analyzed on two plates within the same day to minimize variability between assays. A standard curve was generated using a four-parameter logistic regression model, and ApoC3 concentrations were calculated by interpolation. All experiments and analyses were conducted by separate investigators, blinded to patients’ clinical information and 3-month outcome status. Sample identifiers were anonymized prior to analysis.

Descriptive statistics are presented as counts (n) and percentages (%) for categorical variables, and mean ± standard deviation (SD) for continuous variables. Differences in patient characteristics were evaluated using unpaired Student’s t-tests or non-parametric Mann-Whitney U tests for continuous variables, and Pearson chi-square or Fisher’s exact tests for categorical variables. Normality of the data was assessed using the Shapiro-Wilk test. The predictive value of ApoC3 for mRS outcomes was assessed by ROC curve analysis, with optimal cutoff points determined by the Youden index. Statistical analyses were conducted using GraphPad Prism 8.2.1 (GraphPad Software, San Diego, CA, USA) and SPSS 23.0 (IBM, Armonk, NY, USA). A p-value <0.05 was considered statistically significant.

A total of 63 patients with aSAH were enrolled between June 2021 and March 2023. Of these, 54% were female, with a mean age of 59.7 ± 11.53 years. The median hospital length of stay (LOS) was 12 days (IQR: 10–22 days), and the median neurosurgical intensive care unit (NSICU) stay was 6 days (IQR: 2–18 days). At admission, 61.9% of patients had a Glasgow Coma Scale (GCS) score of 13–15, whereas 20.6% had scores of 3–5. Regarding severity scales, 46% of patients had WFNS grades 3–5, and 44.4% presented with Hunt & Hess grades 3-4. Smoking and hypertension were common comorbidities, observed in 33.3% and 57.1% of patients, respectively. CSF examination showed a median nucleated cell count of 442 × 10⁶/L (IQR: 169–1,487) and a median protein level of 128.3 mg/dL (IQR: 69–157.6). The median CSF ApoC3 concentration was 3,149.5 ng/mL (IQR: 1,174.77–7,768.51). Aneurysms most frequently involved the anterior cerebral artery (49.2%), with sizes predominantly between 5.1 and 10 mm (52.4%). Regarding treatment, 66.7% underwent aneurysm clipping, and 33.3% received endovascular coiling. Post-treatment complications included clinical vasospasm in 36.5% and delayed infarction in 42.9% of patients. At 3-month follow-up, 47.6% of patients had poor outcomes (mRS scores 3–6). Detailed demographic and clinical data are presented in Table 1. The demographic characteristics of NPH patients are shown in Supplementary Table 1.

Univariate analysis showed that several clinical factors significantly correlated with poor outcomes (Table 2). Patients with unfavorable outcomes had significantly longer hospital (p = 0.002) and NSICU (p < 0.001) stays. Poor outcomes were also associated with advanced age (p = 0.022) and higher admission systolic blood pressure (p = 0.037). Patients with lower admission GCS scores (3–5) had significantly worse outcomes (p < 0.001). Higher WFNS grades (p < 0.001) and Hunt & Hess grades (p < 0.001) were also strongly linked to poor outcomes. Among CSF parameters, elevated ApoC3 levels (p < 0.001), increased protein levels (p = 0.017), and abnormal chloride (Cl-) concentrations (p = 0.001) were significantly associated with unfavorable outcomes. Severity scores, including mFS, SEBES, and mGS, were strongly predictive of poor prognosis (all p < 0.001). Complications such as clinical vasospasm (p = 0.002), delayed infarction (p < 0.001), and hydrocephalus (p < 0.001) were significantly more frequent among patients with poor outcomes. The Shapiro-Wilk test p-values for key continuous variables are present in Supplementary Table 2.

CSF ApoC3 concentrations were compared between 63 aSAH patients and 17 NPH controls (Figure 1A). ApoC3 levels were significantly higher in aSAH patients compared to controls (p < 0.0001). Univariate linear regression analysis indicated significant linear correlations between ApoC3 concentrations and various clinical scores, including WFNS (p = 0.0152, Figure 1B), mFS (p < 0.0001, Figure 1D), and SEBES (p = 0.006, Figure 1E). Higher CSF ApoC3 concentrations correlated significantly with worse GCS (p < 0.01, Figure 1F) and more severe mFS groups (p < 0.05, Figure 1H). Although several clinical scores aren’t correlated with ApoC3 concentration (Figure 1C,G,I). To control for confounding factors and assess ApoC3 as an independent predictor of prognosis, multivariable logistic regression was performed, including ApoC3, age, WFNS score, Fisher score, and hydrocephalus (Table 3). The results indicated that elevated CSF ApoC3 levels were independently correlated with poor 3-month outcomes.

ROC curve analysis was performed to evaluate the predictive ability of CSF ApoC3 concentrations for 3-month outcomes (Figure 2). The results demonstrated that ApoC3 levels effectively predicted poor outcomes (AUC = 0.7899, 95% CI: 0.6739–0.9059). The optimal cutoff value, determined by the Youden index, was 4,463 ng/mL, with a sensitivity of 0.6667 (95% CI: 0.4878–0.8077) and specificity of 0.8788 (95% CI: 0.7267–0.9518).

Subsequently, the optimal cutoff (4,463 ng/mL) was used to classify 63 aSAH patients into high ApoC3 and low ApoC3 groups. Univariate analysis of clinical factors between these groups is presented in Table 4.

Patients in the high ApoC3 group exhibited significantly worse clinical scores upon admission. Specifically, they showed lower GCS scores (p = 0.001), higher WFNS grades (p = 0.004), higher mFS scores (p = 0.006), and higher mGS scores (p = 0.006). Moreover, patients with elevated ApoC3 experienced significantly longer NSICU stays (p = 0.018) and longer durations of mechanical ventilation (p = 0.021). Regarding surgical interventions, no significant differences existed between the two groups in the rates of coiling, clipping, or LD/EVD drainage. However, patients undergoing decompression were significantly more likely to have elevated ApoC3 (p = 0.015). CSF analysis revealed significantly increased glucose, protein, and Cl⁻ levels in the high ApoC3 group. Notably, the high ApoC3 group exhibited significantly worse 3-month functional outcomes (p < 0.001). Additionally, complications such as delayed infarction (p = 0.001) and hydrocephalus (p = 0.012) occurred significantly more often in the high ApoC3 group.

Three months post-discharge, the proportion of patients with poor outcomes (mRS 3–6) decreased from 96.9% at discharge to 47.6% at 3 months (Figure 3A). However, patients in the high ApoC3 group (CSF ApoC3 >4,463 ng/mL) had consistently poor mRS scores at discharge, with 100% scoring 3–6 (Figure 3B). Even after 3 months of rehabilitation, poor outcomes remained prevalent (83.3%) in the high ApoC3 group, while decreasing to 25.6% in the low ApoC3 group (Figure 3C).

The negative correlation observed between elevated CSF ApoC3 levels and poor prognosis may be related to inflammation, oxidative stress, and dysregulated lipid ratios. Previous studies have demonstrated that ApoC3 mediates NLRP3 inflammasome activation in human monocytes and induces reactive oxygen species production via caspase-8 activation and Toll-like receptor (TLR) 2 and 4 dimerization, processes that impede endothelial regeneration and promote kidney injury [15], and previous study has shown that elevated inflammatory level in the CSF are associated with the development of complication following SAH [16]. Thus, elevated CSF ApoC3 may exacerbate inflammatory responses and oxidative stress, thereby aggravating brain injury in aSAH patients. Additionally, ApoC3 accumulates in circulating blood and performs an essential function in TRL metabolism. Elevated CSF ApoC3 may suggest increased penetration of peripheral substances into the brain, contributing to more severe pathology. Changes in lipid ratios associated with elevated CSF ApoC3 levels may also contribute to secondary injury following SAH [10].

Moreover, it is recognized that lipid metabolites in CSF are associated with the risk, complications, and outcomes in patients with aSAH. Levels of lipid peroxides are positively correlated with an increased incidence of symptomatic vasospasm [17]. Total cholesterol levels negatively correlate with the risk of delayed infarction and mortality in aSAH patients [18]. Elevated CSF ApoC3 may increase the prevalence of complications (particularly delayed infarction and hydrocephalus) in aSAH patients by altering lipid metabolism, leading to vasospasm, neuroinflammation, blood-brain barrier (BBB) disruption, and endothelial fragility [18–21]. Collectively, these mechanisms may explain how elevated CSF ApoC3 worsens clinical outcomes in aSAH patients.

Increased ApoC3 may originate from circulating blood, with ApoC3 levels rising in parallel with total protein or albumin quotients, reflecting the extent of BBB disruption. Regardless of whether ApoC3 is synthesized in the brain or derived from peripheral circulation, the clear correlation between ApoC3 levels and the prognosis of aSAH patients supports its utility as a valuable marker for clinical decision-making. In addition, considering that hydrocephalus and cerebral edema are crucial indicators for evaluating aSAH patients [16], we selected NPH patients with hydrocephalus and normal CSF composition as the control group. It should be noted that NPH patients might exhibit abnormal CSF dynamics. However, due to ethical constraints, obtaining CSF from healthy individuals is not feasible; thus, CSF form NPH patients is currently the most suitable control in clinical practice without increasing patient burden. And CSF sample from NPH patients is chosen as the standard control group in aSAH research [22].