Higher Serum Levels of Lactate Dehydrogenase Before Microsurgery Predict Poor Outcome of Aneurysmal Subarachnoid Hemorrhage

Introduction: We explored whether higher preoperative serum levels of lactate dehydrogenase (LDH) predicted outcome 3 months after surgery in patients with aneurysmal subarachnoid hemorrhage (aSAH) treated using microsurgical clipping in our institution. Methods: Patients with aSAH treated at our institution between 2010 and 2018 were enrolled. The following parameters were recorded: age, sex, smoking and drinking history, medical history, Hunt–Hess and Fisher grades, aneurysm location, aneurysm size, surgical treatment, delayed cerebral ischemia (DCI), intracranial infection, hydrocephalus, pneumonia, and preoperative serum LDH levels within 24 h of aSAH. We investigated whether preoperative serum LDH levels were associated with Hunt–Hess grade, Fisher grade, and functional neurological outcome. Results: In total, 2,054 patients with aSAH were enrolled, 874 of whom were treated using microsurgical clipping. The average serum LDH level (U/L) was significantly lower in the good outcome group (180.096 ± 50.237) than in the poor outcome group (227.554 ± 83.002; p < 0.001). After propensity score matching, the average serum LDH level (U/L) was still lower in the good outcome group (205.356 ± 76.785) than in the poor outcome group (227.119 ± 86.469; p = 0.029). The area under the receiver operating characteristic (ROC) curve was 0.702 (95% confidence interval [CI]: 0.650–0.754; p < 0.001). Based on the ROC curve, the optimal cutoff value for serum LDH levels as a predictor of poor 3-month outcome (modified Rankin Scale score > 2) was 201.5 U/L. The results revealed that Hunt–Hess grade, Fisher grade, DCI, pneumonia, and serum LDH (>201.5 U/L) were significantly associated with poor outcome. After propensity score matching, serum LDH levels > 201.5 U/L were still considered an independent risk factor for poor outcome (odds ratio: 2.426, 95% CI = 1.378–4.271, p = 0.002). Serum LDH levels were associated with Hunt–Hess and Fisher grades and were correlated with functional neurological outcomes (p < 0.001). Conclusions: Our findings showed that higher preoperative serum levels of LDH correlated with Hunt–Hess grade, Fisher grade, and neurological functional outcome, and predicted the outcome of aSAH treated by microsurgical clipping at 3 months, which was involved in the related mechanisms of early brain injury and showed its potential clinical significance in patients with aSAH.


INTRODUCTION
Lactate dehydrogenase (LDH) is a glycolytic enzyme that occurs in all important human organs, including the liver, heart, skeletal muscle, kidney, lung, and brain (1,2). It catalyzes the dehydrogenation of lactic acid to pyruvic acid, promotes anaerobic glycolysis, and prevents lactic acid accumulation; the latter are associated with unfavorable clinical outcomes of traumatic brain injury (3). When cytolysis occurs or the cell membrane is destroyed, LDH is released into the blood, resulting in an increase in serum LDH (4). LDH activity can be detected in malignant tumor tissues and leukemic cells (5), and serum LDH levels are correlated with the prognosis of adult T-cell leukemialymphoma (6), prostate cancer (7), acute myeloid leukemia (8), melanoma (9), neuroblastoma (10), glioblastoma multiforme (11), acute encephalopathy (12), and Mycoplasma pneumoniae pneumonia (13). Serum LDH levels reflect the degree of brain tissue injury, and Yu et al. demonstrated that serum LDH activity is associated with middle cerebral artery occlusion in a dosedependent manner (14). Several reports have shown that LDH quantification predicts neuronal injury (15,16) and may predict poor prognosis of traumatic brain injury (17) and neonatal intracranial hemorrhage (18).
Several risk factors contribute to the poor prognosis in aneurysmal subarachnoid hemorrhage (aSAH), such as hypertension, poor Hunt-Hess grade, higher Fisher grade, hydrocephalus, pneumonia, and treatment modalities (19)(20)(21). However, few reports have explored the clinical significance of serum LDH levels in patients with aSAH, and the role of LDH in aSAH has not been fully established. At least two sources may contribute to higher serum LDH levels in patients with aSAH: (1) apoptotic/necrotic/damaged neurons or glial cells, (2) lytic red blood cells (RBCs) after release into the cerebrospinal fluid (CSF). Lu et al. reported that the number of apoptotic/necrotic/damaged cells was positively correlated with clinical condition in patients with aSAH, as well as with their Hunt-Hess grade (22). Similarly, the number of RBCs in the cerebra cisterna, sulcus, and/or ventricle was correlated with Fisher grade. Frontera found that early brain ischemia injury was associated with worse Hunt-Hess grade, which indicates poor acute neurological status and is correlated with worse functional outcomes after SAH (23). Claassen et al. showed that SAH completely filling the cistern or fissure, as well as intraventricular hemorrhage (IVH) on computed tomography (CT), were risk factors for delayed cerebral ischemia (DCI) (24), which is correlated with poor outcomes after SAH. However, few reports have explored the relationship between serum LDH levels and the extent of cerebral tissue injury in patients with aSAH. In the present study, we explored the clinical significance of serum LDH in patients with aSAH treated using microsurgical clipping in our institution. We hypothesized that higher preoperative serum levels of LDH, which may be correlated with Hunt-Hess grade and Fisher grade, predict 3-month outcome in patients with aSAH treated using microsurgical clipping.

Participants
Patients were enrolled in the study based on the following criteria: (1) diagnosis of SAH confirmed by CT; (2) presence of intracranial aneurysms confirmed using CT angiography (CTA) or digital subtraction angiography (DSA); (3) all aneurysms treated using microsurgical clipping; (4) CTA and/or DSA performed postoperatively. (5) patients were admitted 24 h after the onset of SAH. The exclusion criteria were as follows: (1) aSAH detected > 24 h after occurrence; (2) other cerebrovascular diseases (such as cerebral arteriovenous malformations, intracranial arteriovenous fistula, or moyamoya syndrome/disease) or intracranial tumors; (3) history of myocardial infarction, hepatitis, malignant tumor, pulmonary infarction, leukemia, hemolytic anemia, kidney disease, or progressive muscular atrophy. The data of patients with aSAH at our institution between 2010 and 2018 were collected. The following parameters were recorded: age, sex, smoking and drinking history, medical history (hypertension, diabetes, coronary heart disease, cerebral stroke), Hunt-Hess and Fisher grades, aneurysm location, aneurysm size, surgical treatment (conventional or decompressive craniotomy), delayed cerebral ischemia (DCI), intracranial infection, hydrocephalus, pneumonia, and preoperative serum LDH levels within 24 h of aSAH.

Treatment
After confirmation, ruptured intracranial aneurysms were treated using microsurgical clipping within 72 h of aSAH onset. After surgery, patients were treated according to current aSAH guidelines (25), including prevention of cerebral arterial narrowing, improvement of cerebral blood flow, neurotrophic treatment, stress ulcer prevention, and nutritional support.

Follow-Up Visit and Definition of Outcome
Postoperative complications were evaluated using CT scanning within 24 h of surgery. The neurological outcome was assessed at the 3-month follow-up and classified according to the modified Rankin Scale (mRS) score. Good clinical outcome was defined as an mRS score of 0-2, while poor outcome was assigned to patients with an mRS score of 3-6. Functional outcome was divided into four levels according to mRS: no symptoms (mRS = 0), no significant to slight disability (mRS = 1-2), moderate to serious disability (mRS = 3-4), and severe disability to death (mRS = 5-6). To define the relationship between serum LDH levels and clinical outcome in patients with aSAH, we investigated whether preoperative serum LDH levels were associated with Hunt-Hess grade, Fisher grade, or the upper four functional outcomes.

Statistical Analysis
All statistical analyses were performed using SPSS for Windows (version 25.0; IBM Corp., Armonk, NY, USA). The Kolmogorov-Smirnov test was used to check whether the data had a normal distribution. One-way analysis of variance or the Student's ttest were used to determine the significance of differences in continuous data. The χ 2 test or Fisher's exact test was used to determine the significance of differences in qualitative data. Multivariable analysis was carried out using a backward stepwise logistic regression model that included all variables with a p-value of <0.10 in the univariate analysis. In the multivariable analysis model, age was divided into "≤ 60 years" and "> 60 years" (26), Hunt-Hess grade into "low grade" (I-III) and "high grade" (IV-V), Fisher grade into "low grade" (1-3) and "high grade" (4), and serum LDH levels into "≤ optimal cutoff value" and "> optimal cutoff value." Statistical significance was set at a p-value of <0.05. The receiver operating characteristics (ROC) curve (MedCalc for Windows version 15.2.2; Mariakerke, Belgium) was generated to analyze the specificity and sensitivity of serum LDH levels for mRS. Propensity-score matching (PSM) was performed to remove imbalances in basic clinical characteristics between the good outcome and poor outcome groups, as well as between the pneumonia and non-pneumonia groups. Conditional probability was estimated using a logistic regression model. The good outcome and poor outcome groups were matched at a ratio of 1:1 using the nearest neighboring matching algorithm.

RESULTS
A total of 2,054 patients with aSAH were treated in our institution between 2010 and 2018, and 874 patients treated using microsurgical clipping were enrolled based on the above criteria (Figure 1). The incidence of poor outcomes following aSAH was   in patients with aSAH at the 3-month follow-up is shown in Figure 2. The area under the ROC curve (AUC) was 0.702 (95% confidence interval [CI]: 0.650-0.754; p < 0.001). The optimal cutoff value for serum LDH levels as a predictor of poor 3-month outcome (mRS > 2) was determined to be 201.5 U/L based on the ROC curve. At this level, the sensitivity was 59.5% and the specificity 76.4%.
Interestingly, serum LDH levels were associated with Hunt-Hess and Fisher grade, with levels of 163.880 ± 35.571 U/L in the Hunt-Hess grade I group, lower than those in the grade II (174.981 ± 49.616), III (188.306 ± 50.702), IV (225.609 ± 69.509), and V groups (252.851 ± 93.302). There were significant differences among the groups in this regard (p < 0.001), and there was a marked trend whereby serum LDH levels increased  alongside Hunt-Hess grade (Figure 3). Serum LDH levels were 169.492 ± 41.621 in the Fisher grade 1 group, lower than in the grade 2 (177.097 ± 42.621), grade 3 (198.709 ± 72.553), and grade 4 groups (210.811 ± 68.962). There were statistically significant differences between grades 4 and 3, grades 4 and 2, grades 4 and 1, grades 3 and 2, grades 3 and 1 (p < 0.001 in all cases). There was a marked trend whereby serum LDH levels increased alongside Fisher grade (Figure 4).
Serum LDH levels were also correlated with functional neurological outcome at the 3-month follow-up. The serum LDH levels were 179.247 ± 46.761 in the mRS 0 group, lower than in the no significant to slight disability (mRS 1-2; 193.977 ± 69.399), moderate to serious disability (mRS 3-4; 205.918 ± 59.203), and severe disability to death groups (mRS 5-6; 234.188 ± 108.336). There were significant differences among the groups in this regard (p < 0.001). There was a marked trend whereby serum LDH levels increased as neurological function deteriorated (Figure 5).

DISCUSSION
Our findings showed a marked trend whereby serum LDH levels increased alongside Hunt-Hess and Fisher grades. In addition, Hunt-Hess grade, Fisher grade, DCI, pneumonia, and higher serum LDH levels predicted and contributed to poor outcome in patients with aSAH at 3 months. The optimal cutoff value for serum LDH levels as a predictor of 3-month poor outcome (mRS > 2) was 201.5 U/L. Moreover, serum LDH levels were correlated with functional neurological outcomes. There was a marked trend whereby serum LDH levels increased as neurological function deteriorated. After PSM, serum LDH (>201.5 U/L) was still considered an independent risk factor of poor outcome.
Serum LDH levels are correlated with the prognosis of adult T-cell leukemia-lymphoma (6), prostate cancer (7), acute myeloid leukemia (8), melanoma (9), neuroblastoma (10), glioblastoma multiforme (11), acute encephalopathy (12), and Mycoplasma pneumoniae pneumonia (13). However, few studies have investigated the relationship between LDH and aSAH. Regional cerebral blood flow and arteriovenous difference of oxygen are reduced due to primary aSAH injury (27), and cerebral ischemia causes an anaerobic shift of metabolism, leading to lactic acidosis and upregulation of serum LDH levels (28). There is a significant correlation between serum LDH and lactic acid levels, and both reflect the degree of tissue damage (27,29).
Serum LDH levels can also reflect the severity of brain tissue injury. Neuronal apoptosis and necrosis have been observed 24 h after SAH (30,31) and can result in cytolysis and cell membrane destruction. Subsequently, LDH is released into the blood from the damaged or dead cells, resulting in increased serum LDH (4). In the study by Yu et al. (14), serum LDH activity was associated with infarct volume and degree of middle cerebral artery occlusion in a dose-dependent manner. Several studies have shown that LDH can be quantified to predict neuronal damage (15,16), and inhibition of LDH release may reduce neuronal apoptosis (14). Rao et al. reported that a significant increase in serum LDH levels was a predictor of severe brain damage and poor prognosis of traumatic brain injury (17), while Engelke et al. indicated that LDH was significantly correlated with subsequent seizures, hydrocephalus, and adverse long-term outcomes of neonatal intracranial hemorrhage (18).
In the present study, serum LDH levels increased alongside Hunt-Hess grade. We deduced that serum LDH levels were correlated with Hunt-Hess grade, and that they reflected the degree of early brain injury and the clinical condition of patients with aSAH. Furthermore, there was a marked trend whereby serum LDH levels increased as neurological function at the 3-month follow-up deteriorated. Subarachnoid clots in sulci/fissures induce spreading depolarizations and acute cerebral infarction of the adjacent cortex after cerebral aneurysm rupture (30). This is a mechanism of early brain injury after SAH (32) and contributes to the clinical condition of patients with aSAH. In a study by Frontera et al. (23), early ischemic brain injury was related to worse Hunt-Hess grade, higher rates of death, and severe disability/death (mRS 4-6) at the 3-month followup. Increased ischemic lesion volume has been associated higher Hunt-Hess grade and 3-month mRS (33), corroborating our own findings.
Our findings also showed a marked trend whereby serum LDH levels increased alongside Fisher grade. Fisher grade is higher when there is more blood in the subarachnoid space, and higher Fisher grade correlates with poor aSAH outcome (34,35). After cerebral aneurysm rupture, the blood brain barrier is destroyed and RBCs are released into the subarachnoid space from the artery. These RBCs break down in the cerebrospinal fluid (CSF) (35) and LDH from the lysed RBCs is absorbed into the blood after being released into the CSF (17), increasing serum LDH levels. Therefore, higher serum LDH levels are associated with higher Fisher grade, which is closely related to poor outcome in patients with aSAH (34,36,37). However, our study had some limitations. Firstly, LDH occurs in all important human organs and lacks specificity to the central nervous system. LDH was not collected and measured in the CSF in our patients, and serum LDH levels do not directly reflect the true levels in brain tissue. Secondly, imaging data were not available to confirm the relationship between serum LDH levels and the degree of brain tissue damage. Thirdly, serum LDH levels were within the normal range in some patients with poor outcomes, so they do not fully explain these patients' prognosis; the detailed mechanism needs further exploration.Fourthly, LDH, like C-reactive protein, neutrophil to lymphocyte ratio and white blood cell count, are related to the prognosis of SAH. They are biomarkers of the prognosis of SAH. However, the specific mechanism of LDH is still unclear. And there is still no effective treatment to reduce the mortality and disability rate of aSAH patients.

CONCLUSIONS
Our findings showed that higher preoperative serum levels of LDH correlated with Hunt-Hess grade, Fisher grade, and functional neurological outcome, and that they predicted the 3-month outcome in patients with aSAH, which is associated with mechanisms of early brain injury and may have clinical significance in patients with aSAH.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Fujian Medical University. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.