Elevated peripheral neutrophils and matrix metalloproteinase 9 as biomarkers of functional outcome following subarachnoid hemorrhage

Abstract

There is growing evidence supporting the role of inflammation in early brain injury and cerebral vasospasm following subarachnoid hemorrhage (SAH). Matrix metalloproteinases (MMPs) are released by inflammatory cells and can mediate early brain injury via disruption of the extracellular matrix and mediate vasospasm by cleaving endothelin-1 into vasoactive fragments. We hypothesize that inflammation marked by neutrophil elevation and MMP-9 release in human SAH is associated with vasospasm and with poor clinical outcome. We enrolled consecutive SAH subjects (N = 55), banked serial blood and cerebrospinal fluid (CSF) samples, and evaluated their 3-month modified Rankin scores (mRS). Vasospasm was defined as >50% vessel caliber reduction on angiography 6-8 days post-SAH. A poor outcome was defined as mRS > 2. We compared blood leukocyte and neutrophil counts during post-SAH days 0-14 with respect to vasospasm and 3-month outcome. In a subset of SAH subjects (N = 35), we compared blood and CSF MMP-9 by enzyme-linked immunosorbent assay (ELISA) on post-SAH days 0-1, 2-3, 4-5, 6-8, and 10-14 with respect to vasospasm and to 3-month outcome. Persistent elevation of blood leukocyte (p = 0.0003) and neutrophil (p = 0.0002) counts during post-SAH days 0-14 are independently associated with vasospasm after adjustment for major confounders. In the same time period, blood neutrophil count (post-SAH days 2-3, p = 0.018), blood MMP-9 (post-SAH days 4-5, p = 0.045), and CSF MMP-9 (post-SAH days 2-3, p = 0.05) are associated with poor 3-month SAH clinical outcome. Neutrophil count correlates with blood MMP-9 (post-SAH days 6-8, R = 0.39; p = 0.055; post-SAH days 10-14, R = 0.79; p < 0.0001), and blood MMP-9 correlates with CSF MMP-9 (post-SAH days 4-5, R = 0.72; p = 0.0002). Elevation of CSF MMP-9 during post-SAH days 0-14 is associated with poor 3-month outcome (p = 0.0078). Neither CSF nor blood MMP-9 correlates with vasospasm. Early rise in blood neutrophil count and blood and CSF MMP-9 are associated with poor 3-month SAH clinical outcome. In blood, neutrophil count correlates with MMP-9 levels, suggesting that neutrophils may be an important source of blood MMP-9 early in SAH. Similarly, CSF and blood MMP-9 correlate positively early in the course of SAH, suggesting that blood may be an important source of CSF MMP-9. Blood and CSF MMP-9 are associated with clinical outcome but not with vasospasm, suggesting that MMP-9 may mediate brain injury independent of vasospasm in SAH. Future in vitro studies are needed to investigate the role of MMP-9 in SAH-related brain injury. Larger clinical studies are needed to validate blood and CSF MMP-9 as potential biomarkers for SAH outcome.

Fig. 1

Peripheral white blood cell count is elevated in SAH subjects with angiographic vasospasm (VSP+; n = 22) compared with those without Vasospasm (VSP-; n = 33) during post-SAH days 0–14 (p = 0.0003). * indicates between-group difference on that specific post-SAH day is significant at a level of p < 0.05

Fig. 2

Peripheral neutrophil count is elevated in SAH subjects with angiographic vasospasm (VSP+; n = 15) compared with those without vasospasm (VSP-; n = 23) during post-SAH days 0–14 (p = 0.0027). * indicates between-group difference on that specific post-SAH day is significant at a level of p < 0.05

Fig. 3

Peripheral neutrophil count is elevated during post-SAH days 0–14 in SAH subjects with poor (mRS > 2; n = 12) 3-month outcome compared with those with good outcome (mRS ≤ 2; n = 25; p = 0.018). * indicates between-group difference on that specific post-SAH day is significant at a level of p < 0.05

Fig. 4

a Elevation of blood MMP-9 levels during post-SAH days 0–14 in SAH patients showed a trend of association with poor (mRS > 2; n = 10) 3-month outcome versus good (mRS ≤ 2; n = 21) outcome (p = 0.09). b Elevation of CSF MMP-9 levels during post-SAH days 0–14 in SAH patients is associated with poor (mRS > 2; n = 9) 3-month outcome versus good (mRS ≤ 2; n = 22; p = 0.0078). * indicates between-group difference on that specific post-SAH day is significant at a level of p < 0.05)

Fig. 5

Peripheral neutrophil counts correlate with blood MMP-9 levels a on post-SAH days 6–8 (n = 24; R = 0.39; p = 0.055) and b on post-SAH days 10–14 (n = 23; R = 0.79; p < 0.0001)

Fig. 6

Blood MMP-9 correlates with CSF MMP9 on post-SAH days 4–5 (n = 22; R = 0.72; p = 0.0002)

Fig. 7

a Blood MMP-9 levels during post-SAH days 0–14 in SAH patients showed no association with presence (VSP+; n = 17) or absence of angiographic vasospasm (VSP-; n = 16; p = 0.42). b CSF MMP-9 levels during post-SAH days 0–14 in SAH patients showed no association with presence (VSP+; n = 17) or absence of angiographic vasospasm (VSP-; n = 15; p = 0.32)