Methods
Patients and control subjects
This study included ten patients who underwent surgical obliteration of cerebral aneurysms at Aichi Medical University Hospital within 1 day of the onset of SAH. These patients, three men and seven women, ranged from 41 to 75 years of age (mean age of 57 years). Their baseline clinical characteristics are shown in Table
1. After their aneurysms were clipped, cisternal catheters were placed into their chiasmatic or prepontine cisterns, and postoperative CSF samples were collected on each of the following 14 days. One patient developed delayed ischemic neurological deficits. All the other patients had normal postoperative courses. Glasgow outcome scale was evaluated 3 months after the onset of SAH. No patients suffered from infection. CSF samples were obtained from another five patients (mean age of 62 years) who had undergone clipping for unruptured cerebral aneurysms and were used as controls. All samples were immediately centrifuged upon collection, and the supernatants were stored at −80 °C until analysis. The Ethics Committee of Aichi Medical University approved this clinical experiment.
Table 1
Clinical data pertaining to ten patients with subarachnoid hemorrhage
1 | 45, F | ICPC | 2 | 3 | − | 14 | GR |
2 | 71, F | A. com | 2 | 3 | − | 14 | MD |
3 | 53, M | MCA | 3 | 4 | + | 14 | VD |
4 | 65, F | MCA | 2 | 3 | − | 14 | GR |
5 | 49, M | A. com | 4 | 3 | − | 14 | GR |
6 | 75, F | A. com | 2 | 4 | − | 14 | MD |
7 | 41, M | A. com | 2 | 3 | − | 7 | MD |
8 | 46, F | ICPC | 2 | 3 | − | 10 | GR |
9 | 60, F | A. com | 3 | 4 | − | 10 | GR |
10 | 64, F | A. com | 4 | 3 | − | 10 | MD |
CSF analysis
The concentrations of IL-6, MCP-1, IP-10, and MIG were measured using enzyme immunoassays (EIA; R&D Systems, Inc., Minneapolis, MN, USA). The detection limits of the assays were 0.70 pg/ml for IL-6, 1.7 pg/ml for MCP-1, 1.67 pg/ml for IP-10, and 3.84 pg/ml for MIG.
Statistical analysis
Data are expressed as the mean ± SEM. Significant differences between groups were assessed using one-way ANOVA, followed by Fisher’s PLSD for multiple comparisons. Correlations between cytokine and chemokine levels were assessed by Pearson’s correlation coefficient. Significant differences between groups were assessed by the Mann-Whitney U test. Significance was set at p < 0.05.
Discussion
In this study, we demonstrated that the concentration of IL-6 increased immediately after the onset of SAH, followed by an increase in MCP-1, whose level peaked on day 3, and increases in IP-10 and MIG, whose levels peaked on day 5. There were strong correlations between the maximum levels of IL-6 and MCP-1 and IP-10 and MIG on day 5.
We have previously shown that long-lasting cerebral vasospasm is induced by intracisternal injections of IL-6 in canines [
9]. We have also confirmed that IL-6 induces phosphorylation of JAK1 and STAT3 in the rat basilar artery after SAH, which transduces signals into adjacent cell nuclei, resulting in transcription of acute-phase genes [
10]. IL-6 is a reliable early indicator of increased vasospasm risk after SAH [
11], which is in agreement with our data indicating that poor outcome patients exhibited higher IL-6 levels. IL-6 plays a role in amplifying leukocyte accumulation at sites of inflammation by activating STAT3. This is achieved in part by augmenting local production of MCP-1 and ICAM-1 [
12]. MCP-1 belongs to the β or CC family of chemokines and stimulates migration of monocytes to inflammatory tissues. Previous studies have shown that activated mononuclear leukocytes can be found surrounding the major cerebral arteries in the subarachnoid space after SAH [
13]. In addition, MCP-1 has been found to be significantly expressed in the major cerebral arteries in the setting of cerebral vasospasm in rats [
14]. Given these findings and our data showing that a strong correlation exists between the maximum levels of IL-6 and MCP-1, immediate expression of IL-6 may play an important role in inducing MCP-1 expression via STAT3 after SAH.
IP-10 belongs to the α or CXC family of chemokines and was originally identified as an interferon-γ-inducible protein. IP-10 stimulates migration of monocytes and T cells to inflammatory tissues. However, it does not induce a chemotactic activity in neutrophils. In experimental autoimmune encephalomyelitis (EAE), astrocytes were found to secrete MCP-1 and IP-10 and to play a significant role in intrathecal inflammation [
15]. MCP-1 and IP-10 are also expressed in the CSF of patients with viral meningitis and induce chemotaxis of peripheral blood mononuclear cells (PBMCs) [
16]. MIG is also a member of the CXC chemokine family and activates lymphocytes. MIG and IP-10 were found to be highly expressed in the synovial tissues of patients with rheumatoid arthritis (RA) [
17], which suggests that these chemokines may play an important role in the pathophysiology of RA. In the case of EAE, MIG was expressed predominantly by macrophages/microglia, while IP-10 was produced by astrocytes surrounding inflammatory lesions [
18]. IP-10 and MIG have different spatial and cellular localizations in the CNS, which suggests that these chemokines have specialized functions. We found that the concentrations of IP-10 and MIG increased significantly during the first 5 days after SAH and that there was a significant correlation between the concentrations of IP-10 and MIG on day 5, which suggests that these chemokines may play a role in the development of delayed ischemic neurological deficits through simultaneous activation of monocytes and lymphocytes. More studies are needed to explore the CNS expression sites of these chemokines after SAH.
In wound healing, recruitment of various leukocyte subtypes to wound sites is essential. Using a skin wound model, macrophage migration was induced by MCP-1 expression from day 2 onward. The migration of lymphocytes was initiated by the expression of MCP-1 and was accompanied by the subsequent expression of IP-10 and MIG after 4 days [
19]. This time course of chemokine expression is in agreement with our data, suggesting that the dynamic expression of chemokines and their subsequent recruitment of leukocyte subtypes play important roles in inflammation in the CNS after SAH.
In this study, we explored serial changes in the levels of inflammatory cytokines and chemokines after SAH. There were several limitations to this study. We enrolled only a small number of patients of widely varying ages and with widely varying outcomes. It is possible that these factors affected the cytokine concentrations in the CSF of these patients. Moreover, our results are purely observational, as we performed no animal experiments to corroborate our findings. Only one patient in this study suffered from symptomatic vasospasm; therefore, it is unclear whether the abovementioned cytokines play roles in the pathogenesis of delayed ischemic neurologic deficits after SAH. The sample size was not large enough to allow patient subgroup analyses. Our preliminary data confirmed only that strong correlations exist between the maximum levels of IL-6 and MCP-1 and IP-10 and MIG on day 5 after SAH. Additional studies involving larger numbers of patients are necessary to determine whether these factors are associated with the development of delayed brain injury after SAH.
Acknowledgements
The authors thank Kenta Murotani for his assistance with the statistical analysis, Shin-ichi Satoh for critical reading of this manuscript, and Hisae Inui for providing technical assistance.