The Quantitative and Functional Changes of Postoperative Peripheral Blood Immune Cell Subsets Relate to Prognosis of Patients with Subarachnoid Hemorrhage: A Preliminary Study

doi:10.1016/j.wneu.2017.08.091

World Neurosurgery

Volume 108, December 2017, Pages 206-215

https://doi.org/10.1016/j.wneu.2017.08.091 Get rights and content

Objective

It has been suggested that the preoperative (PRE) and postoperative (POST) immune system alteration triggered by aneurysmal subarachnoid hemorrhage (SAH) and surgical treatment itself may affect patients' prognosis and contribute to POST complications. The mechanisms may be attributed to immune suppression–triggered infection or immune overreaction–triggered aseptic inflammation. In this study, we investigated the dynamic changes in peripheral immune cell subsets as well as the alterations of inflammatory cytokines in patients with aneurysmal SAH who received craniotomy and clipping surgery. In addition, we studied the association of those changes with POST complications and clinical prognosis.

Methods

We investigated 27 patients who received craniotomy and clipping surgery for aneurysmal SAH. The operations were all performed within 24 hours after the occurrence of aneurysm rupture. Detailed immune monitoring (peripheral blood leukocytes and lymphocyte subsets and inflammatory cytokines) was performed on PRE (on admission), day 1, day 3, and day 6 after operation.

Results

Our data showed that the percentage of CD3+, CD8+, natural killer T (NKT), CD4+, and regulatory T (Treg) cells significantly decreased and the level of interleukin 4 (IL-4), interferon γ, and IL-2 significantly increased 1 day after surgery compared with the data in PRE. On the contrary, natural killer (NK), NK group 2 (NKG2D), and B cells increased and the level of IL-10 in plasma decreased. In study of the relationship between POST fever and the change in immune cell subgroups, the fever group had a lower percentage of CD3+, CD4+, NKT, Tregs, and B cells on day 1, day 3, and day 6 after surgery compared with the patients who did not have fever, whereas the CD8+, NK, and NKG2D subsets showed the opposite trend. Furthermore, we analyzed the association between immune profile changes and the prognosis of those patients. The patients were divided into those with an unfavorable prognosis (n = 6) and those with a favorable prognosis (n = 21) according to Glasgow Outcome Scale score and postoperation (POST) coma. Our results showed that except for B cells, patients with a favorable prognosis had a relatively higher percentage of CD3+, CD4+, CD8+, NK, NKT, NKG2D, and Treg cells compared with the unfavorable prognosis group from PRE to day 6 POST.

Conclusions

Our results indicated that patients with aneurysmal SAH undergoing craniotomy and clipping surgery had a profound transient deterioration in immune function. In addition, the changes in immune cell subgroups had a strong association with POST fever. The changes in immune cell subgroups were also directly associated with clinical prognosis of the patients. These association findings might be attributable to a better biomarker to predict patient diagnosis.

Introduction

Subarachnoid hemorrhage (SAH) is a complex and potentially devastating disorder, with only 25% of patients likely to live independently.1, 2 The clinical and socioeconomic burden of SAH is unacceptably high. SAH caused by intracranial aneurysm rupture, namely aneurysmal SAH (aSAH), are most common. Surgical treatment, both endovascular coiling and neurosurgical clipping, is the only therapy for aSAH, but high fever and other bad complications are still risk factors that contribute to patients' unfavorable postoperative (POST) outcome.3, 4

POST infection, which is a leading cause of major disability and death, together with cerebral vasospasm play the rule of second strike. In neurosurgery intensive care units, infection is a common problem. The overall POST infection rate ranges from 6.5% to 11.2% in most hospitals. Among those patients staying longer than 48 hours in a neurosurgery intensive care unit, the infection rate is higher than 30%.5, 6

Previous studies have shown that the occurrence of systemic infections after SAH may be a symptom of impaired immune competence.7, 8 Shortly after the onset of SAH, the blood components and irritated brain tissue expose antigens that are recognized by components of the innate immune system, which leads to its activation and acute inflammatory response.9, 10 The activated primary effector cells are phagocytes (polymorphonuclear neutrophils, macrophages, and monocytes)11, 12 and innate lymphocytes (natural killer [NK] and NKT).¹³ These cells attach to the endothelium of proinflammatory vessels in the brain by binding with various adhesion factors. They produce cytokines14, 15 (interleukin 2 [IL-2], IL-6, interferon γ [IFN-γ], and IL-10) that induce and regulate inflammation, ingest and destroy microbes, and clear the damaged cells. Subsequently, innate immune responses generate molecules that present signals, in addition to antigens, to activate naive T and B lymphocytes.10, 16

Immune activation after SAH has been proved to play an important role in host defense against infection. Besides neutrophils and monocytes, the activated lymphocytes also release proinflammatory cytokines, directly eliminating damaged cells, killing the infected cells, or neutralizing microbes.¹⁷ There is increasing evidence that, after acute central nervous system injury, a temporary impairment of cellular immune function acts as an important risk factor in the occurrence of infections. For this reason, the occurrence of systemic infections with patients POST aSAH needs to be further explored. In this pilot study, we assessed the preoperative (PRE) and POST immune profile changes (peripheral immune subset count) of patients with aSAH undergoing clipping surgery. In addition, we studied the association of those changes with POST complication (fever), as well as the relationship between immune profile changes and clinical outcome of patients.

Section snippets

Patients

Patients were enrolled from May 2015 to July 2015 in the Neurosurgery Department of the Second Xiangya Hospital of Central South University, Changsha, Hunan, China. We investigated 27 adult patients who received clipping surgery for an intracerebral aneurysm after SAH. The inclusion criteria were: 1) SAH confirmed by cranial computed tomography; 2) cerebral angiogram showing intracranial aneurysm (the location of aneurysms of these 27 patients was the anterior communicating artery, posterior

Adaptive Immune System Was Inhibited in POST Periods

For all patients, we performed detailed immune monitoring from PRE to day 6 POST. As shown in Figure 1, the percentage of CD3⁺, CD8⁺, NKT, CD4⁺, and Tregs significantly decreased on day 1 POST, compared with data in PRE. On the contrary, NK, NK group 2, member D (NKG2D), and B cells increased on day 1 POST. These findings may indicate that the secondary surgical damage may lead to inhibition of the adaptive immune system and activation of the innate immune system. NK cells returned to

Discussion

Brain damage (ischemic or hemorrhagic) leads to systemic inflammatory changes and immune system disorders,21, 22 including the fluctuation of immune cell subsets and inflammatory factor level.8, 15 These changes may lead to immune suppression or overreaction, which critically contributes to the patient's prognosis. The immune suppression may make the human body unable to resist the infection of bacteria or virus, whereas immune system overreaction also causes tissue damage and increases the

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An analysis of the in-hospital outcome using the GOS revealed that lymphopenia and increased PL-R were associated with poor prognosis after IA rupture. Zhou et al., in a group of 27 SAH patients, found that favorable prognosis assessed with the GOS was associated with a higher percentage of CD3+CD4+CD8+ cells in peripheral blood.31 In particular, immunodepression with lower CD4+CD8+ cell counts was observed in aSAH patients with symptomatic DCI4 and in patients who died in the hospital.32
The rupture of an intracranial aneurysm (IA) causes a systemic response that involves an immune/inflammatory reaction. We sought to characterize the systemic response to IA rupture.
We included 19 patients in the acute phase of IA rupture and 20 control subjects. Flow cytometry was used to analyze alterations in the level of mononuclear leukocytes. Cell-related parameters, including the neutrophil-to-lymphocyte ratio (NL-R), lymphocyte-to-monocyte ratio (LM-R), platelet-to-lymphocyte ratio (PL-R), and systemic immune-inflammation index (SII), were calculated, and the relationship between the analyzed hematological parameters and clinical status was investigated.
Patients with ruptured IAs presented with significantly higher white blood cells (WBC) and neutrophil counts but lower lymphocyte counts than control subjects. NL-R and SII values were higher and the LM-R was lower in the acute phase after IA rupture. Analyzing the severity of clinical status and the outcome of patients with subarachnoid hemorrhage, we found that patients with poor clinical status, as measured by the Glasgow Coma Scale (GCS) and the Hunt and Hess scale, had significantly lower lymphocyte counts and higher NL-R, PL-R and SII values than those with good clinical status. Additionally, patients with lower GCS scores presented a lower proportion of CD3+CD4-CD8- cells. Worse outcomes assessed at discharge were associated with lower lymphocyte counts but higher PL-R values.
The current study pointed to the significance of systemic immune and inflammatory responses after IA rupture and the potential clinical utility of hematological parameters, which can be easily calculated. In particular, the role of DN T cells and the significance of the SII as a marker related to clinical status should be further investigated.
Peripheral Monocytosis at Admission to Predict Cerebral Infarct and Poor Functional Outcomes in Subarachnoid Hemorrhage Patients
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Hence NLR might not always be transposable or useful to predict outcomes in nonsmokers or former smoker patients. Recently, Jiang et al24 showed that natural killer cells in peripheral blood are associated with poor outcomes in SAH patients, which adds more evidence of the complexity of the inflammatory process preceding brain damage in SAH. Finally, is remarkable that any of the previous studies have shown a high sensitivity and specificity of a single marker to predict complications or outcomes in SAH; therefore future prospective studies should address the potential utility as prognosis markers of inflammatory cells at different time points.
Increasing evidence points monocytes' role to be larger than thought in developing cerebral infarction (CI) after subarachnoid hemorrhage (SAH). However, there is no clinical evidence of the relationship between peripheral monocytes and CI and clinical outcomes. Therefore we determine whether an increase in monocytes in the acute phase is useful to predict CI and functional outcomes in SAH patients.
We included 204 patients with an SAH diagnosis. We collected patient-related factors, comorbidities, Hunt-Hess grade, modified Fisher grade, treatment, delayed cerebral ischemia, CI, aneurysm characteristics, and peripheral monocytes from vein blood at admission. Poor outcomes were defined as modified Rankin Scale score ≥3.
Fifty (24.5%) patients had CI before discharge. In a multivariate model, increased monocytes at admission were significantly associated with CI after adjusting for IV-V Hunt-Hess grade and delayed cerebral ischemia (odds ratio: 3.193, 95% confidence interval: 1.069–9.532, P = 0.037). In receiver operating characteristic curve analysis, a monocyte count of 0.60 was identified as the best cutoff value to discriminate the development of CI (area under the curve = 0.622, P = 0.010; CI for monocytes <0.60 17.4% vs. CI for monocytes ≥0.60 29.1% P = 0.046). Admission monocytes ≥0.60 predicted poor functional outcomes at discharge (monocytes <0.60 52% vs. monocytes ≥0.60 64.7%) and at 12 months (monocytes <0.60 29.4% vs. monocytes ≥0.60 70.6%).
Increased peripheral monocytes at admission is a risk factor for developing CI after SAH. Moreover, short- and long-term poor clinical outcomes were associated with higher monocyte count. Therefore monocytes could be a convenient biomarker for prognosis unfavorable outcomes and a possible target for new therapeutic strategies.
Compartmentalisation of the inflammatory response following aneurysmal subarachnoid haemorrhage
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Other factors, particularly relating to clinical aSAH grade and necrotic load, will also contribute to the severity of this SIRS (e.g. sepsis and organ failure). Moreover, after acute CNS injury, a temporary impairment of cellular immune function is believed to be a risk factor for systemic infections [24]. In those who will develop/have developed DIND, this response is more pronounced (probably proportional to the concentration/quantity of the original causative agent(s) within the initial haemorrhagic blood load).
There is some evidence to suggest that a systemic and central nervous system (CNS) inflammatory response occurs following aneurysmal subarachnoid haemorrhage (aSAH) which may be related to the pathophysiology of early brain injury and delayed ischaemic neurological deficit (DIND). The aim of this study was to measure inflammatory mediator levels in plasma and cerebrospinal fluid (CSF) in the days following aSAH and to determine their association with aSAH, DIND and clinical outcome.
Plasma and CSF samples were obtained prospectively from patients with aSAH on days 1–3, 5, 7 and 9 and profiled for interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-15, IL-17, IL-18, macrophage chemotactic protein (MCP)-1, vascular endothelial growth factor (VEGF) and tumour necrosis factor (TNF)-α. Plasma and CSF samples from non-aSAH patients undergoing spinal anaesthesia were used as controls.
The CSF levels of all cytokines investigated except for IL-1α were significantly higher in aSAH compared to controls in the first seven days of ictus. CSF levels of IL-1α (p = 0.014), IL-18 (p = 0.016), IL-6 (p = 0.0006) and IL-8 (p = 0.006) showed significant increases in the days following aSAH. Conversely IL-17 demonstrated a decrease. In particular, IL-4 was higher in the CSF of patients who had DIND at all time-points (p = 0.032). Plasma IL-6 and IL-8 levels were higher, and IL-1α levels lower, than controls at most time-points. All mediators demonstrated persistent elevation in the CSF compared to plasma apart from IL-1α and IL-18 which followed the opposite trend. Day 3 plasma IL-6 levels predicted poor outcome at six months (Exp(B) 1.12 1.03-1.22, P = 0.012), although this association was lost in the second analysis incorporating Fisher grade, WFNS grade and age.
The post aSAH inflammatory response peaks on days 5–7 post ictus and remains largely compartmentalised within the CNS. IL-4 may have a particular association with DIND although its precise role in the pathophysiology of the disorder remains unclear. IL-6 predicted poor outcome but not independently of clinical grade, suggesting that it may be a surrogate marker of early brain injury.
Plasma Levels of IL-6, IL-8, IL-10, ICAM-1, VCAM-1, IFNγ, and TNFα are not Associated with Delayed Cerebral Ischemia, Cerebral Vasospasm, or Clinical Outcome in Patients with Subarachnoid Hemorrhage
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In peripheral blood, plasma levels of interleukin-6 (IL-6) have been reported to correlate with outcome13,14 and with post-SAH complications,15 although an association with outcome could not be found in a later study.16 A recent study reported that plasma interleukin-10 (IL-10) decreased in the weeks after SAH,17 but no studies have investigated the systemic IL-10 concentration in relation to vasospasm, DCI, or outcome. Likewise, no study has investigated systemic TNFα levels during the vasospasm phase, although TNFα measured early after SAH was not associated with radiologic or clinical parameters.18
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In 90 patients with SAH, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, high sensitivity C-reactive protein (HsCRP), interleukin-8, interleukin-10, interferon gamma, and tumor necrosis factor alpha were measured in peripheral blood day 3 and day 8 after SAH. Any occurrence of DCI or infection was recorded, and computed tomography angiography was performed on day 8. Clinical outcome was assessed after 3 months.
HsCRP on day 3 was higher in patients with angiographic vasospasm (P = 0.003), and HsCRP on day 8 was higher in patients with poor outcome (P = 0.014). No association with DCI, vasospasm, or outcome was found for any of the remaining analyzed substances.
High plasma levels of HsCRP were significantly associated with angiographic vasospasm and clinical outcome. Plasma levels of interleukin-6, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, interleukin-8, interleukin-10, interferon gamma, and tumor necrosis factor alpha were not associated with DCI, angiographic vasospasm, or clinical outcome at 3 months.
Sterile Inflammation, Potential Target in Aneurysmal Subarachnoid Hemorrhage
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However, the therapeutic potential of DAMPs after SAH has not yet been explored in detail. Increasing evidence reveals upregulation of inflammatory cascades characterized by elevated DAMPs levels and activation of immune cells and release of cytokines.10,21-30 Because DAMPs are the initiators and sustainers of sterile inflammation, they represent important therapeutic targets to contain and limit inflammation during post-SAH complications.
Interleukin-4 Modulates Neuroinflammation by Inducing Phenotypic Transformation of Microglia Following Subarachnoid Hemorrhage
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: Conflict of interest statement: The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Original ArticleThe Quantitative and Functional Changes of Postoperative Peripheral Blood Immune Cell Subsets Relate to Prognosis of Patients with Subarachnoid Hemorrhage: A Preliminary Study

Objective

Methods

Results

Conclusions

Introduction

Section snippets

Patients

Adaptive Immune System Was Inhibited in POST Periods

Discussion

Neuroscience

J Neuroimmunol

Lancet

Prog Mol Biol Transl Sci

Biochem Biophys Res Commun

Brain Res Bull

Metamorphosis of subarachnoid hemorrhage research: from delayed vasospasm to early brain injury

Mol Neurobiol

Risk classification after aneurysmal subarachnoid hemorrhage

Surg Neurol

Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group

Stroke

Intravascular inflammation triggers intracerebral activated microglia and contributes to secondary brain injury after experimental subarachnoid hemorrhage (eSAH)

Transl Stroke Res

Endovascular therapy for vasospasm after aneurysmatic subarachnoid hemorrhage

Br J Neurosurg

Innate inflammatory responses in stroke: mechanisms and potential therapeutic targets

Curr Med Chem

Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation

J Exp Med

Temporal and spatial dynamics of cerebral immune cell accumulation in stroke

Stroke

Immune cell infiltration in malignant middle cerebral artery infarction: comparison with transient cerebral ischemia

J Cereb Blood Flow Metab

Original Article
The Quantitative and Functional Changes of Postoperative Peripheral Blood Immune Cell Subsets Relate to Prognosis of Patients with Subarachnoid Hemorrhage: A Preliminary Study