Elevated levels of several chemokines in the cerebrospinal fluid of patients with subarachnoid hemorrhage are associated with worse clinical outcome

Abstract

Background: Chemokines are small cytokines that exert chemotactic actions on immune cells and are involved in many inflammatory processes. The present study aims to provide insight in the role of this relatively unexplored family of proteins in the inflammatory pathophysiology of subarachnoid hemorrhage (SAH).

Materials and methods: Cerebrospinal fluid of 29 patients (17 female; mean age 57 years) was collected at days 1, 4 and 10 after SAH, centrifuged and frozen at -70°C. Analysis of 92 inflammation-related proteins was performed using Target 96 Inflammation ® assay (Olink Proteomics, Uppsala, Sweden) based on Proximity Extension Assay technology. The panel included 20 chemokines (CCL2 (or MCP-1), CCL3, CCL4, CCL7 (or MCP-3), CCL8 (or MCP-2), CCL11 (or Eotaxin), CCL13 (or MCP-4), CCL19, CCL20, CCL23, CCL25, CCL28, CXCL1, CXCL5, CXCL6, CXCL8 (or IL-8), CXCL9, CXCL10, CXCL11 and CX3CL1 (or Fractalkine)) that were analyzed for their temporal patterns of expression and compared in dichotomized clinical groups based on World Federation of Neurosurgical Societies (WFNS) admission score and amount of blood on admission CT based on Fisher scale; presence of delayed cerebral ischemia(DCI)/delayed ischemic neurological deficit (DIND); and clinical outcome based on Glasgow Outcome Scale. Protein expression levels were provided in output unit Normalized Protein Expression (NPX). ANOVA models were used for statistical analyses.

Results: Four temporal patterns of expression were observed (i.e., early, middle, late peak and no peak). Significantly higher day 10 mean NPX values were observed in patients with poor outcome (GOS 1-3) for chemokines CCL2, CCL4, CCL7, CCL11, CCL13, CCL19, CCL20, CXCL1, CXCL5, CXCL6 and CXCL8. In the WFNS 4-5 group, CCL11 showed significantly higher day 4 and day 10 mean NPX values and CCL25 significantly higher day 4 values. In patients with SAH Fisher 4, CCL11 showed significantly higher mean NPX values on days 1, 4 and 10. Finally, patients with DCI/DIND had significantly higher day 4 mean NPX values of CXCL5.

Conclusion: Higher levels of multiple chemokines at the late stage of SAH seemed to correlate with worse clinical outcome. A few chemokines correlated with WFNS score, Fisher score and occurrence of DCI/DIND. Chemokines may be useful as biomarkers for describing the pathophysiology and prognosis of SAH. Further studies are needed to better understand their exact mechanism of action in the inflammatory cascade.

Fig 1. Representative graphs of temporal pattern of expression for each group of chemokines: Four different temporal patterns of expression were noticed, and the chemokines were grouped accordingly.

Representative graphs for each chemokine group are illustrated with their means and 0.95 CI for each day. More specifically, CCL4 is shown for Group A with its early peak and decreasing trend; CCL28 was the only chemokine of Group B with a significant middle peak on day 4; CXCL6 with its increasing trend and late peak is shown for Group C; and finally, CCL2 is representing Group D with stable values throughout the observation period. Note that NPX values of CCL4 (representing Group A), CXCL6 (Group C) and CCL2 (Group D) are shown on the left Y-axis (L) whereas values of CCL28 (representing Group B) are shown on the right Y-axis (R).

Fig 2

Comparisons of CCL2 NPX values in patient groups: Mean NPX values and 0.95 CI for each day in dichotomized patient groups based on a) WFNS score on admission (top left), b) Fisher scale of admission CT (top right), c) occurrence of DCI/DIND (bottom left) and d) clinical outcome (bottom right). The asterisk on the bottom right image marks significantly higher day 10 mean NPX values for patients with unfavorable outcome.

Fig 3

Comparisons of CXCL8 NPX values in patient groups: Mean NPX values and 0.95 CI for each day in dichotomized patient groups based on a) WFNS score on admission (top left), b) Fisher scale of admission CT (top right), c) occurrence of DCI/DIND (bottom left) and d) clinical outcome (bottom right). The asterisk on the bottom right image marks significantly higher day 10 mean NPX values for patients with unfavorable outcome.

Fig 4

Comparisons of CCL11 NPX values in patient groups: Mean NPX values and 0.95 CI for each day in dichotomized patient groups based on a) WFNS score on admission (top left), b) Fisher scale of admission CT (top right), c) occurrence of DCI/DIND (bottom left) and d) clinical outcome (bottom right). Asterisks on the top left and right images as well as the bottom right image mark statistically significant differences in mean NPX values for the respective day in each group.