Neutrophils mediate early cerebral cortical hypoperfusion in a murine model of subarachnoid haemorrhage

Abstract

Cerebral hypoperfusion in the first hours after subarachnoid haemorrhage (SAH) is a major determinant of poor neurological outcome. However, the underlying pathophysiology is only partly understood. Here we induced neutropenia in C57BL/6N mice by anti-Ly6G antibody injection, induced SAH by endovascular filament perforation, and analysed cerebral cortical perfusion with laser SPECKLE contrast imaging to investigate the role of neutrophils in mediating cerebral hypoperfusion during the first 24 h post-SAH. SAH induction significantly increased the intracranial pressure (ICP), and significantly reduced the cerebral perfusion pressure (CPP). At 3 h after SAH, ICP had returned to baseline and CPP was similar between SAH and sham mice. However, in SAH mice with normal neutrophil counts cortical hypoperfusion persisted. Conversely, despite similar CPP, cortical perfusion was significantly higher at 3 h after SAH in mice with neutropenia. The levels of 8-iso-prostaglandin-F2α in the subarachnoid haematoma increased significantly at 3 h after SAH in animals with normal neutrophil counts indicating oxidative stress, which was not the case in neutropenic SAH animals. These results suggest that neutrophils are important mediators of cortical hypoperfusion and oxidative stress early after SAH. Targeting neutrophil function and neutrophil-induced oxidative stress could be a promising new approach to mitigate cerebral hypoperfusion early after SAH.

Figure 1

Overview of animal randomisation and mortality.

Figure 2

Murine model of subarachnoid haemorrhage. (A,B) Time courses of CPP and ICP changes after induction of SAH or sham surgery. Note that ICP increases minutes after SAH induction but returns to near baseline after 3 h, leading to a similar CPP as that before induction. (*p < 0.05; error bars indicate SEM; group sizes: SAH-neutropenia/cerebral perfusion group 2, n = 7; SAH-control/cerebral perfusion group 4, n = 6; sham-neutropenia/cerebral perfusion group 1, n = 4; sham-control/cerebral perfusion group 3, n = 5). (C) Representative images of cerebral cortical perfusion before and after induction of SAH in mice receiving vehicle or anti-Ly6G antibody to induce neutropenia. (D) Panels 1, 2, and 3 show representative images 15 min, 3 h, and 24 h after induction of SAH.

Figure 3

Gene expression of inflammation markers 24 h after SAH. Gene expression of IL-1β (A), iNOS (B), TNFα (C) 24 hours after induction of SAH (biochemistry groups 3 (n = 5) and 6 (n = 5), *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4

Enhanced cerebral cortical perfusion after SAH by induction of neutropenia. (A,B) Relative cerebral cortical perfusion after induction of SAH or sham surgery. (*p < 0.05; error bars indicate SEM; group sizes: SAH-neutropenia/cerebral perfusion group 2, n = 7; SAH-control/cerebral perfusion group 4, n = 6; sham-neutropenia/cerebral perfusion group 1, n = 4; sham-control/cerebral perfusion group 3, n = 5).

Figure 5

Reduced oxidative stress marker (8-isoprostane) accumulation in the subarachnoid haematoma of mice with induced neutropenia. (A) Isolation of the basal subarachnoid haematoma (drawing by Stefan Kindel, Mainz, Germany). (B) 8-isoprostane levels in the subarachnoid haematoma after induction of neutropenia or vehicle treatment. Note that the significant increase in the 8-isoprostane levels 3 h after SAH in the vehicle group was markedly attenuated in the neutropenia group. (*p < 0.05; error bars indicate SEM; group sizes: SAH-neutropenia (biochemistry groups 1–3): 15 min n = 6, 3 h n = 5, 24 h n = 5 ; SAH-control (biochemistry groups 4–6): 15 min n = 6; 3 h n = 4; 24 h n = 5).