Neutrophil Enzyme Myeloperoxidase Modulates Neuronal Response in a Model of Subarachnoid Hemorrhage by Venous Injury

Abstract

Background and Purpose: Aneurysmal subarachnoid hemorrhage (SAH) is associated with the development of delayed cognitive deficits. Neutrophil infiltration into the central nervous system is linked to the development of these deficits after SAH. It is however unclear how neutrophil activity influences central nervous system function in SAH. The present project aims to elucidate which neutrophil factors mediate central nervous system injury and cognitive deficits after SAH. Methods: Using a murine model of SAH and mice deficient in neutrophil effector functions, we determined which neutrophil effector function is critical to the development of deficits after SAH. In vivo and in vitro techniques were used to investigate possible pathways of neutrophils effect after SAH. Results: Our results show that mice lacking functional MPO (myeloperoxidase), a neutrophil enzyme, lack both the meningeal neutrophil infiltration (wild type, sham 872 cells/meninges versus SAH 3047, P =0.023; myeloperoxidase knockout [MPOKO], sham 1677 versus SAH 1636, P =NS) and erase the cognitive deficits on Barnes maze associated with SAH (MPOKO sham versus SAH, P =NS). The reintroduction of biologically active MPO, and its substrate hydrogen peroxide (H 2 O 2 ), to the cerebrospinal fluid of MPOKO mice at the time of hemorrhage restores the spatial memory deficit observed after SAH (time to goal box MPOKO sham versus MPOKO+MPO/H 2 O 2 , P =0.001). We find evidence of changes in neurons, astrocytes, and microglia with MPO/H 2 O 2 suggesting the effect of MPO may have complex interactions with many cell types. Neurons exposed to MPO/H 2 O 2 show decreased calcium activity at baseline and after stimulation with potassium chloride. Although astrocytes and microglia are affected, changes seen in astrocytes are most consistent with inflammatory changes that likely affect neurons. Conclusions: These results implicate MPO as a mediator of neuronal dysfunction in SAH through its effect on both neurons and glia. These results show that, in SAH, the activity of innate immune cells in the meninges modulates the activity and function of the underlying brain tissue.

Modulating neutrophils leads to changes in CNS output

“…To maintain the neutrophil depletion throughout the experiment, mice were injected every 2 days with anti-Ly6G antibodies intraperitoneally. Barnes maze was based on previous protocol used in our laboratory 14 . The protocol was expanded to test reversal learning and strength of memory.…”

“…Flow cytometry was performed on the meninges as previously described 14 . To summarize, a single cell suspension was generated by digesting dissected meninges using 1 mg/ml DNAse and 1.4 U/ml collagenase in Hanks Balanced Salt Solution with magnesium and calcium.…”

“…Our laboratory has shown that subarachnoid hemorrhage leads to a significant increase of mature neutrophils in the meninges 14 . Surprisingly, our data show that the injury has no effect on the number of immature neutrophils in the meninges (Figure 4B).…”

“…SAH or sham surgeries were performed as previously reported 14 . Briefly, mice were anesthetized, placed in a prone position, and a 3mm incision made on the back of the neck.…”

“…Neutrophils are the most abundant myeloid cell and their activation has been linked to immune modulation, tissue damage and healing after injury [11][12][13] . Our laboratory has shown that neutrophil function in the meninges is critical for the development of cognitive deficits after subarachnoid hemorrhage (SAH) 14 . As expected, most neutrophils infiltrating the meninges after SAH are blood derived, although we found the presence of neutrophils in the meninges prior to the injury suggesting a meningeal neutrophil population.…”