Higher leukocyte count predicts 3-month poor outcome of ruptured cerebral aneurysms

It is not fully established whether leukocyte can predict the poor outcome for ruptured cerebral aneurysms (CA) or not. Here, we retrospectively analyzed the clinical data of 428 patients with ruptured CA between 2010 and 2015. Patients’ demographic data, including gender, age, history of smoking, alcohol, hypertension, diabetes and hypercholesterolemia, Hunt-Hess and Fisher grade, occurrence of hydrocephalus, aneurysm location, time to surgery, delayed ischemic neurological deficit (DIND) and peak leukocyte of blood test from day 1 to 3 after aneurysmal rupture were recorded and analyzed. In the multivariable analysis model, gender, Fisher grade, time to surgery and hydrocephalus were not relevant to poor outcome. However, Hunt-Hess grade, DIND and preoperative leukocyte count (>13.84 × 109/L) were significantly associated with adverse outcome. The respective increased risks were 5.2- (OR 5.24, 95% CI 1.67–16.50, p = 0.005), 6.2-(OR 6.24, 95% CI 3.55–10.99, p < 0.001) and 10.9-fold (OR 10.93, 95% CI 5.98–19.97, p < 0.001). The study revealed that Hunt-Hess grade, DIND and preoperative leukocyte count (>13.84 × 109/L) were independent risk factors for poor outcome of ruptured CA at 3 months. Higher leukocyte count is a convenient and useful marker to predict 3-month poor outcome for ruptured CA.

Statistical Analysis. All the statistical analyses were performed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). The significance of differences in continuous data was determined with one-way variance (ANOVA) and Student's t test. Qualitative data were compared using a chi-squared test (χ 2 test) or Fisher's exact test. Multivariable analysis logistic regression analyses included all variables significance level at p < 0.15 in univariate analysis. For inclusion in the multivariable analysis model, age was dichotomized as "less than 60 years" and "more than 60 years" 15 , Hunt-Hess grade as "low grade (Grade I-III)" and "high grade (Grade IV-V)", Fisher grade as "low grade (Grade 1, 2, 3)" and "high grade (Grade 4)", surgical time as "less than 3 days" and "more than 3 days", leukocyte counts as "≤ optimal cutoff value" and "> optimal cutoff value". P < 0.05 was deemed statistically significant. A MedCalc 15.2.2 (MedCalc Software, Mariakerke, Belgium) was used to generate the receiver operating curve (ROC) and analyze the specificity, sensitivity, negative predictive values of leukocytes and positive predictive values of leukocytes for mRS.

Results
Four hundred and twenty eight patients were included in the retrospective study according to the upper inclusion and exclusion criteria. The clinical characteristics of these patients with ruptured CA are shown in Table 1. The mean age of 307 (72%) patients in the good outcome group was55.8 ± 10.6 years, and 121 (28%) patients with a poor outcome was 56.6 ± 10.8 years (p > 0.05). The univariate analysis indicated there were significant differences were detected in gender, Fisher grade, hydrocephalus, time to surgery, DIND, leukocyte count between favorable and poor groups (p < 0.05). One hundred and ninety-four patients (45%) were male, 234 (55%) were female, the difference of the gender distribution between the two groups was statistically significant (p < 0.05). There were no significant statistical differences in the number of the patients with history of smoking, alcohol, hypertension, diabetes, hypercholesterolemia between the two groups (p > 0.05). The aneurysm location between the two groups was not significantly statistical difference (p > 0.05). There was no significant difference of preoperative rebleeding between the two groups (6 patients in the favorable and 4 in the poor outcome group) (p > 0.05).
The median peak leukocyte count (×10 9 /L) in the good outcome group (10.34 ± 3.23) was lower than that in the poor outcome group (15.24 ± 5.47). The receiver operating characteristic (ROC) curve is shown in Fig. 1. The optimal cutoff value for leukocyte as a predictor for 3-month status was determined as 13.84 (×10 9 /L) in the ROC curve (sensitivity was 60.3%, and the specificity was 88.3%). In addition, the mean of leukocyte count in patients with Hunt-Hess grade IV-V (16.98 ± 6.54) was higher than that in Hunt-Hess grade I-III (11.33 ± 4.12), the difference was statistically significant (p < 0.001).

Discussion
The incidence of poor outcome reported in previous studies ranged from 20 to 40% 16,17 . In this 6-year review of 428 consecutive patients with ruptured CA, the risk factors including gender, Fisher grade on admission, time of surgery, hydrocephalus and preoperative higher leukocyte count between the two groups are statistically significant, which may be associated with 3-month poor outcome. Previous studies indicated that hydrocephalus and time to surgery were associated with poor outcome of ruptured CA 18,19 . However, in the logistic regression models, it was found that only three variables (Hunt-Hess grade, DIND and preoperative higher leukocyte count) Scientific RepoRts | (2018) 8:5799 | DOI:10.1038/s41598-018-23934-x were the independent risk factors for predicting 3-month poor outcome, while female gender, higher Fisher grade, hydrocephalus and time to surgery not.
Neuronal and axonal damage may occur in the pathophysiological process after aneurysmal rupture 20 , and a deterioration of the clinical neurological condition was observed. It is no doubt that poor Hunt-Hess grade on admission strongly predicted the poor outcome in aneurysmal subarachnoid hemorrhage, and our finding is consistent with prior report 21 . We found that the leukocyte count in the patients with Hunt-Hess grade I-III on admission was lower than that in Hunt-Hess grade IV-V within 3 days after aneurysm rupture. Thus, leukocyte count in peripheral blood may be associated with the severity of the acute primary injury of the brain tissue. Furthermore, our findings suggested that Hunt-Hess grade IV-V and a serum leukocyte count greater than 13.84 × 10 9 /L in this study were associated with developing DIND, which was related to poor outcome [22][23][24] .
As we know, the increased leukocyte count may be the result of acute phase response and reflects the severity of disease and tissue inflammatory response. In 1974, Neil-Dwyer and Cruichshank 14 firstly revealed that higher leukocyte count was associated with the unfavorable outcome of ruptured CA. On the contrary, Spallone 12 found no relationship between the admission higher leukocyte count and ischemic complications. Therefore, studies assessing the role of leukocyte in the adverse outcome of ruptured CA have yielded conflicting conclusions 12,13 .
Interestingly, the main finding of our study revealed that higher leukocyte count before microsurgical treatment was an independent risk factor for poor outcome of ruptured CA at 3 months. Previous results showed that the inflammatory reaction occured after cerebral aneurysm rupture. Leukocytes in peripheral blood would be recruited within the first several hours because of the blood released into the subarachnoid space 25 , which would increase intracranial pressure and lead to early brain injury (EBI). Accompanying with releasing chemotactic factor into the central nervous system and an increase in the number of leukocytes in peripheral blood, the leukocyte will be recruited in the brain microvasculature 26 , which is associated with blood-brain barrier   disruption and basal lamina degradation 27 . As a result, brain edema might occur or be aggravated together with neuroinflammation-promoted apoptosis in sympathetic neurons, which would further aggravate secondary brain damage 28 . It is noteworthy that several molecular mechanisms including Angiotensin II (Ang II) 29 , myeloperxidase 30 , NF-κB 31 , cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) 32 , were associated with the adverse outcome after aneurysm rupture, but the detailed mechanisms of leukocyte in cerebral aneurysm remain obscure. A growing body of evidence suggested that leukocyte infiltration in the aneurysmal wall contributed to the formation, growth and rupture of cerebral aneurysm, which is increasingly recognized as an inflammatory process 33 . Furthermore, histopathological findings of aneurysmal wall indicated that the more leukocytes were observed, the more fragile aneurysms became 34 .
Several inflammatory mediators synthesized and/or expressed by leukocyte, such as IL-1β, IL-6, TNF-α, TLR4, and matrix metalloproteinases, will further aggravate the local inflammatory response which is closely related to an adverse outcome 35 . Maiuri, et al. 36 and McGirt, et al. 11 came to a common conclusion that leukocyte   counts had a high prognostic value in predicting cerebral vasospasm which was associated with neurological deterioration and poor outcome. Inhibiting the inflammatory response and reducing leukocyte activity was highly beneficial in limiting secondary brain injury and increasing survival after aneurysm rupture 37 . Limitation. In the retrospective study, the other biomarkers, such as Erythrocyte Sedimentation Rate (ESR), C Reactive Protein (CRP), complement C3 and C9, were not included in the study. Further other biomarkers examination should be performed.

Conclusion
Hunt-Hess grade, DIND and preoperative higher leukocyte count (>13.84 × 10 9 /L) before microsurgical treatment were independent risk factors for poor outcome of ruptured CA at 3 months. Higher leukocyte count before microsurgical treatment is a convenient and useful marker to predict poor outcome of ruptured cerebral aneurysm at 3 months.