The neutrophil enzyme myeloperoxidase modulates neuronal response after subarachnoid hemorrhage, a sterile injury model

Background: Aneurysmal subarachnoid hemorrhage (SAH) is associated with the development of delayed cognitive deficits. Neutrophil infiltration into the central nervous system (CNS) is linked to the development of these deficits after SAH. It is however unclear how neutrophil activity influences CNS function in SAH. As such, the present project aims to elucidate neutrophil factors and mechanisms mediating CNS 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. Also, in vitro techniques were used to elucidate how neutrophils affect cellular function of neurons and glia after SAH. Results: Our results show that following SAH, neutrophils infiltrate the meninges, and not the brain parenchyma. Mice lacking functional myeloperoxidase (MPO KO), a neutrophil enzyme, lack both the meningeal neutrophil infiltration and the cognitive deficits associated with SAH. The re-introduction of biologically active MPO, and its substrate hydrogen peroxide, to the cerebrospinal fluid of MPO KO mice at the time of hemorrhage restores the spatial memory deficit observed after SAH. Furthermore, in culture, MPO affects the function of both primary neurons and astrocytes, though not microglia. Neurons exposed to MPO and its substrate show decreased calcium activity at baseline and after stimulation with potassium chloride. In addition, MPO and its substrate lead to significant astrocyte loss in culture, phenocopying a result observed in the brain after SAH. Conclusions: These results implicate MPO as a mediator of neuronal dysfunction in SAH through their effect on both neurons and astrocytes. Finally, these results show that, in SAH, the activity of innate immune cells in the meninges can modulate the activity and function of the underlying brain tissue. Further analysis showed no effect on the number of at this point (C). As expected, the injury led to a significant increase and in not


Introduction
Neutrophils are pathological in a multitude of neurological disorders. For example, neutrophil infiltration into the substantia nigra leads to neuronal loss in mice (1). Neutrophil adherence to cerebral capillary beds is linked to cognitive decline in a mouse model of Alzheimer's disease(2), and Brain images of sections labelled with anti-iba1 antibody, were converted to 8-bit files. The iba1 fluorescence threshold of the images was set at 90% intensity, and a 10 x 10 grid was placed on each image. To randomize data acquisition and minimize bias, the fifth row of squares was cropped out and analyzed. Cells were only included in the analysis if fully located within the selected row. The 'Scholl analysis' application was then used to quantify the number of processes observed on selected cells using the protocol previously described (19).

Vasospasm
Previous studies from our lab show the presence of vascular spasm in the middle cerebral artery (MCA) of SAH mice early (at day 1) and, more importantly, in a delayed manner (6 days after the hemorrhage) corresponding to DCI in mice(5). Since the peak neutrophil infiltration occurs in our model 3 days post-hemorrhage and delayed vasospasm is correlated with DCI, we focused our vasospasm analysis on the delayed vasospasm occurring on the 6 th day after SAH.
The arterial tree of each mouse was labeled using the vascular dye Microfil (Flow Tech, Carver, MA), and the cross-sectional MCA diameter was measured. Briefly, animals were anesthetized using sodium pentobarbital, transcardially perfused with 20 ml of cold PBS followed by 20 ml of cold 1% paraformaldehyde in PBS, then injected with 10 ml of Microfil dye. Brains were dissected, rinsed in PBS, then cleared using methyl salicylate (Sigma-Aldrich, St Louis MO US). The ventral surface of each brain was then imaged using a Leica DM 2000 LED light microscope. Because vasospasm leads to non-uniform constriction of arteries, there are affected and unaffected areas. The percent constriction was calculated as a ratio of the smallest diameter to the largest cross-sectional diameter of the MCA within a 2 mm segment distal to the posterior wall of the internal carotid using ImageJ and converted to a percentage.

Behavior analysis
Prior to surgery, mice were randomly assigned to treatment group using a random number generator.
In addition, treatment conditions were blinded to the person administering the behavioral test.

Barnes Maze
Previous studies show that mice with SAH develop deficits in performance on the Barnes maze task 6-8 days after the hemorrhage(5). Therefore, in the present study, this task was used to assess the presence of cognitive deficits after SAH. Briefly, mice were habituated to the maze for 2 days before the surgery. Habituation was performed by placing mice in the middle of the maze and leaving them to explore for 5 min. Testing started on day three post-surgery and continued for 7 consecutive days thereafter. During testing, mice were placed in the middle of the maze and given a total of 300 seconds to find the escape hole. Each trial ended when the mouse entered the escape hole, stayed in the entry zone of the escape hole (defined by a circle of 5 cm diameter around the escape hole) for 5 consecutive seconds, or the 300 second maximum had elapsed. All trials were recorded and analyzed using the tracking software Ethovision 13 (Noldus, Leesburg, VA, US).

Rotarod
To determine whether the naïve MPO KO and WT mice showed different motor coordination and learning, mice motor performance was analyzed using the rotarod test. During this test, only the latency to fall was analyzed. In brief, mice were placed on the rod at low speed and left to acclimate for 60 seconds. Mice were then tested with a continuous increased speed on the rod for 5 consecutive minutes. Experiments ended when mice fell of the rod or the 5 min have elapsed. Each mouse was given 3 unique trials separated by a 30 minutes rest period in the home cage.
Briefly, cortices of P0 neonates were dissociated, in minimal essential medium (MEM) with 10% heat inactivated FBS and penicillin/streptomycin, using a 1000ul micropipette. Cells were filtered using a 70um cell filter and seeded on sterile 12 well plates coated with poly-D-lysine (0.1 mg/ml). Two hours after seeding, media was changed to neurobasal A media with B27, glutamax and penicillin/streptomycin. 25% of cultures media was subsequently exchanged every two days. Seven days in vitro (DIV) cultures were incubated with either 0.8 ug of MPO with or without H 2 O 2 (0.0012%) or H 2 O 2 alone. All cultures conditioned were imaged using a widefield Leica DM6000B microscope outfitted with a CCD camera. Before stimulation, a 5-minute time-lapse baseline video (at a rate of 2 frames per second) was obtained for each culture. Following the 5 min baseline recoding, neurons were stimulated with either MPO with or without H 2 O 2 or H 2 O 2 alone, followed by a 10minutes timelapse recording. Cultures were left to acclimate to the stimulus for 2.5 hours. Cells were then stimulated with 10mM potassium chloride (KCl) solution. The first 10 min of the KCl stimulation were also recorded. Prior to analysis, all time-lapse recordings were blinded. Using Imaris (Bitplane, Concord, USA), 8-10 cells were chosen at random and a maximum fluorescent intensity value was generated for each cell at each frame acquired in the time lapse video. The values were plotted on excel and firing rate (peaks) were counted before and after stimulation in each experimental group (control, MPO, MPO+H2O2, and H2O2). Percent baseline values were then determined by dividing the firing rate of each cell after the stimulation to the firing rate before the stimulation. Values were averaged across each cell to generate a single value for that time lapse recording.

Primary astrocyte culture
Primary astrocyte cultures were established as previously described(22). In brief, brain cortex was collected from P0 pups, dissociated in 2.5% Trypsin in HBSS solution, and vigorously pipetted using a 10ml serological pipette to generate a single cell suspension. Cells were then spun and resuspended in astrocyte media (DMEM, high glucose, 10% heat inactivated FBS, and penicillin/streptomycin). Cells were then plated on T75 culture flask and incubated at 37 degrees. 2 day after plating, media was changed and every 3 days thereafter. On the 7 th day DIV, culture flask was shaken and rinsed to remove microglia and oligodendrocyte progenitor cells. The generated pure astrocyte culture was further incubated for 12-14 days, after which the culture was split. 2 days after the split, cultures were detached, rinsed and transferred to 15mm culture dish for testing. As with the primary neuronal culture, astrocytes cultures were imaged, exposed to 3 experimental conditions, MPO, MPO with H 2 O 2 , or H 2 O 2 alone, then incubated for 4 hours. At the end of the incubation period, cultures were imaged.
Cells were counted in the before and after images to determine the toxic/morphological effect of each experimental condition on astrocytes.
Of note, for both neuron and astrocyte cultures, each mouse brain was plated on 12 culture dishes (or 1 12-well culture plate). Triplicates of each experimental condition (control, MPO, MPO+H2O2, and H2O2) were performed for each mouse. For analysis, data was collected from each triplicate, and averaged to generate a single data point for a given mouse brain.

2-photon imaging of neuronal calcium activity
All imaging experiments were performed on an Olympus FVMPE-RS system equipped with sensitive GaAsP detectors and a resonant scanner. A cranial window was prepared on Thy1-GCaMP3 mice as previously described(23). In brief, mice were anaesthetized, mounted on the stereotactic instrument, and the skin on the scalp removed to expose the skull. A 3mm diameter circle was made in the parietal bone with a microdrill 2 mm lateral and 2 mm caudal to the bregma. The edge of the circle was further thinned by drilling until the circled bone cap was able to be flipped and removed with fine-tipped forceps. A few drops of 0.9% NaCl saline solution was applied on top of the exposed dura mater. A small area of dura was carefully cut with a fine needle and a solution of drug (MPO or saline) applied directly to the cortex. The cranial window was covered with a glass coverslip, sealed at the edges with super glue. To facilitate the injection of KCl into the cortex, a 1mm diameter burr hole was prepared 3 mm caudal and lateral to the cranial window. A flattened nail (used as a handle to orient mouse head during imaging) was horizontally glued the contralateral occipital bone.
The cranial window implanted mice were kept under anesthesia for the duration of the imaging session. 3 unique regions were imaged within the cranial window separated by a 10 minutes interval.
Images were acquired using an Olympus 25X NA 1.05 objective lens with an 920 nm infrared laser.
The line averaging on the resonant scanner was set to 6 to obtain images with 512X512 resolution and 2.5 Hz acquisition speed. A target field of view was selected at a depth between 100 nm and 200 nm and continuous 5 minutes-long T-series imaging session was recorded.
To induce KCl-triggered spreading depolarization, a 20um diameter pulled micropipette was filled with 1 M KCl, mounted on a nanoliter injector (WPI) and inserted into the burr hole. For imaging, a 1minute baseline was first recorded. Followed by cortical spreading depression generated by the application of 100 nL of 1 M KCl to the cortex though the burr hole, and 4 additional minutes of recording.
Each generated file was blinded and then transferred to the Imaris software (Bitplane, Concord, USA).
Within each image, 10 individual neurons were selected at random. Maximum fluorescent intensity was collected for each cell at every frame during the recording. Values were averaged across all cells and the 3 unique field of view for each mouse to generate a single data point for each given mouse.
Finally, increase fluorescence from baseline (peak and depression) were calculated across groups to determine whether the addition of the MPO, saline or SAH affected the neuronal response in cortical spreading depression.

Statistics
Graphpad Prism 8.1 (Graphpad, La Jolla,CA, US) software was used to analyze all of the data obtained.
Student's t-test or analysis of variance (ANOVA) were used to determine whether differences between treatment groups were statistically significant. Significance was attributed to p values less than 0.05.

Results
The neutrophil enzyme myeloperoxidase is critical to the development of cognitive deficits after subarachnoid hemorrhage (SAH).
In our model of sterile brain injury, peripheral depletion of neutrophils impedes the development of spatial memory deficits after SAH(4). To determine whether neutrophil effector function plays a role in these deficits, mice deficient in the neutrophil effector proteins myeloperoxidase, elastase and NADPH oxidase (that have been previously implicated in neuronal damage after ischemic stroke)(8,10,25), were tested ( Fig. 1). Both elastase and NADPH oxidase knockout (KO) mice developed delayed spatial memory deficits after SAH ( Fig. 1A and 1B). On the other hand, the MPO KO mouse was protected against the development of delayed spatial memory deficits (Fig. 1C), suggesting that neutrophil derived MPO is critical to the development of this syndrome.
To determine whether the loss of MPO activity leads to improved cognitive function, biologically active MPO was added to the CSF of MPO KO mice at the time of the hemorrhage. The addition of MPO and its substrate, hydrogen peroxide (H 2 O 2 ), to the MPO KO mouse recapitulates the spatial memory deficits previously present in our model ( Fig. 1G and 1H). The addition of MPO alone (Fig. 1E) or hydrogen peroxide alone (Fig. 1F) was not sufficient to cause deficits suggesting that the peroxidase activity of the MPO enzyme is important for late cognitive deficits associated with SAH.
Finally, an identifying hallmark of both murine and human SAH is the presence of delayed vasospasm in large cerebral vessels(26). In the MPO KO mouse, the diameter of the middle cerebral artery showed no spasm 6 days after SAH (supplemental figure 1) Neutrophils infiltrate the meningeal parenchyma but not the brain after SAH.
In order to locate the site of action of neutrophils in the CNS, the meninges and brain parenchyma were analyzed for neutrophil infiltration after SAH. Neutrophils were readily found in the meninges ( Fig. 2) and not the brain parenchyma (supplemental fig.2, confirming our previous finding(4)) of the SAH and sham mouse. In the meninges, neutrophils are found in the venous sinuses (supplemental figure 3A) as well as the meningeal parenchyma ( Fig. 2A). Although no significant changes were observed in the number of neutrophils found within the sinuses of the sham and SAH mice (supplemental figure 3B), a significantly higher number of neutrophils were observed in the meningeal parenchyma of the SAH mouse 3 days after hemorrhage compared to sham (Fig. 2B).
Further characterization, using intravenous CD45 injection, showed that this increase is due to active infiltration of neutrophils into the meninges after SAH (Fig. 2C).
On the other hand, MPO KO mice showed few to no neutrophils in the meninges after SAH ( Fig. 2A and   2B). The persistent presence of neutrophils in the meningeal venous sinuses of the MPO KO mice (Supplemental figure 3.) suggests that extravasation, rather than recruitment, may be impaired in the MPO KO. This is consistent with previous reports that show that the loss of functional MPO affects both neutrophil migration and production of key cytokines by neutrophils(27,28). Furthermore, characterization of the MPO KO mouse with biologically active MPO and H 2 O 2 showed few neutrophils in the meninges (data not shown), suggesting that the recapitulation of cognitive behavior in this experimental group is due to MPO activity and not increase recruitment of neutrophils into the meningeal parenchyma.

The loss of functional MPO in neutrophils skews the innate immune landscape of the CNS at baseline
The lack of functional MPO in neutrophils affects neutrophil secretion of cytokines, including IL-6, the lungs during infection(28). Recent evidence shows immune cells populate the meninges under normal conditions(29). To determine whether the congenital loss of functional MPO affects the baseline immune makeup of the brain and meninges, flow cytometry was performed on both CNS compartments, brain and meninges, of the naïve MPO KO and WT mice (supplemental figure 4 gating strategy).
The MPO KO mouse has more microglia and non-microglia CD45 + cells in the brain than the WT mouse (supplemental figure 5A). As a proportion of non-microglia innate immune cells (CD45 + CD11b + ), MPO KO and WT mice had a comparable number of neutrophils in the vasculature of the brain. Analysis of monocyte populations was divided into circulating inflammatory (ly6c hi ) and patrolling/alternative (Ly6c lo ) subsets(30,31). Overall, the MPO KO mouse had more monocytes in the brain than the WT mouse (supplemental figure 5A). This increase was attributed to the high number of Ly6c lo monocytes. Although the identity of these cells is unknown, we postulate that the Ly6c lo population is comprised most likely of perivascular macrophages(32).
In the meninges, a comparable number of CD45 + immune cells were found in the MPO KO and WT mice. No difference was observed in the number of neutrophils between the two mice, suggesting that the changes we observed in the SAH model are due to a failure to recruit peripheral neutrophils into the meninges in the MPO KO mouse. Similar to the brain, more Ly6c lo monocytes populate the meninges of the MPO KO mouse at baseline.
Finally, to make sure that the baseline behavior does not differ between the WT and MPO KO mice, Barnes maze and rotarod test were performed. Although both mice have similar performance on the rotarod test, the naïve MPO KO mouse takes significantly longer to find the goal box in the Barnes maze tasks than the WT mouse contrary to the improved function seen after SAH (supplemental figure 5D). Further analyses are needed to determine if the failure to perform in the Barnes maze is due to distress (fear/anxiety) or a lack of motivation to escape.

MPO modulates neuronal response to stimulation
In our model, MPO appears to play a role in the development of the behavioral deficits associated with SAH. A possible mechanism for the development of these deficits is that MPO enters the brain to act on neurons after injury. Therefore, to determine whether MPO can act on neurons, primary neuronal cultures were generated using the Thy1GCaMP3 mice. Using the calcium signal as an indicator of activity, we characterized neuronal activity after MPO, H 2 O 2 , or MPO+H 2 O 2 administration. The calcium traces show that the addition of MPO with H 2 O 2 significantly dampens neuronal activity (Fig.   3A) and results in minimal neuronal loss (Fig. 3B). The addition of MPO without its substrate has little to no effect on neuronal activity (Fig 3A) Because the in vitro system does not fully mimic in vivo conditions, 2-photon microscopy and KClinduced cortical spreading depolarizations (CSD) were tested to determine if MPO alters neuronal response to stimuli in the live animal. Using calcium signaling as a measure of neuronal activity, we determined the peak calcium signal during CSD and the subsequent signal depression in the presence of MPO. We compared this change to the CSD induced signal and depression in the SAH mouse. Of note, because the highest level of neutrophil infiltration occurs 3 days after SAH in our model, our analysis of CSD was focused to this time point in the SAH mouse.
As demonstrated in the traces generated by calcium signaling within the neurons, under all treatment conditions, MPO or SAH, neuronal depolarization (i.e. increase in calcium signal) and depression (decrease in calcium signal) was detected within minutes of KCl administration to the cortex (Fig. 3C).
Analysis of the maximum signal intensity (baseline -peak) showed no difference between groups, suggesting that MPO and SAH do not affect the depolarization potential of neurons. Analysis of depression (baseline -minimal signal) showed that both MPO administration and SAH leads to less depression in the neurons compared to controls (Fig. 3D). We interpret these changes to suggest that there are resting state abnormalities in neurons due to SAH and MPO that are consistent with our previous findings in hippocampal neurons (4).
To our knowledge, these experiments are the first to demonstrate that MPO has a neuromodulatory effect in sterile brain injury. However, we have been unable to locate MPO protein within the brain at any timepoint after SAH. This raises the question of contributions of intermediate cells, between the neutrophils in the meninges and the neurons in the brain parenchyma.

Astrocytes but not microglia are affected by meningeal derived MPO
It is unclear how neutrophils in the meninges affect neurons within the parenchyma of the brain. We investigated the possibility that MPO's effect on neurons in the hippocampus may also act through a cell intermediary. To determine whether MPO acts on inflammatory glial cells in the brain, we characterized the effect of SAH on microglia and astrocytes in the hippocampus.
Microglia morphology was characterized using Iba1 expression in the hippocampus with the understanding that infiltrating macrophages will also express Iba1 (supplemental figure 6A).
Interestingly, no changes were observed in the number of Iba1 + cells or their fluorescent intensity in the CA1 region of the WT and MPO KO mouse at either day 3 or 6 after SAH (data not shown). To determine whether the activation phenotype of these cells was affected after SAH, the number of cell processes was quantified using a modified Scholl analysis. In the WT mouse hippocampus, no changes are observed in microglia ramification after SAH at day 3 (supplemental figure 6B). 6 days after injury, the number of processes detectable on the injured mouse decreased significantly (supplemental figure 6B). In the MPO KO mouse, at day 3 after injury, microglia lost the ramified morphology observed in the sham mouse. This change reverted to control levels 6 days after SAH (supplemental figure 6B). These results suggest the changes in the microglia population are not due to MPO but a function of the injured/inflamed state of the CNS.
Astrocytes were characterized in the CA1 region of the hippocampus by their expression of GFAP and vimentin (Fig. 4). At day 3, in WT mice although no changes were observed in the number of GFAP + astrocytes after SAH (Fig. 4C), GFAP intensity was significantly decreased in the CA1 region (Fig. 4B).
Furthermore, more astrocytes colocalized GFAP and Vimentin 3 days after SAH than control (Fig. 4D), suggesting increased activation of these cells(33). At day 6, both GFAP + cell number and intensity were significantly decreased after SAH (Figs. 4E-G) with no change in the colocalization of GFAP and vimentin (Fig. 4H). This suggests that in SAH, there is astrocyte loss (suggested by low GFAP at day 3 and decreased astrocytes at day 6) with increased concomitant vimentin/GFAP colocalized astrocytes at day 3. No early decrease in GFAP or astrocyte loss were observed in the MPO KO mouse after SAH at either timepoint (Fig. 4A-E), nor was there an increase in colocalization of vimentin and GFAP compared to control suggesting that astrocytes are affected by SAH and that this is recovered in the MPO KO mouse.

MPO activity leads to astrocyte death in culture
The data above suggests MPO can affect neuron and astrocyte function within the brain. Because MPO is not detectable in the parenchyma of the brain, the proximity of astrocytes to the meninges make these cells a likely intermediary. To test this hypothesis, primary astrocyte cultures were stimulated with MPO, H 2 O 2 , or MPO+H 2 O 2 for 4 hours. After which, cell morphology and survival were assessed.
Consistent with the in vivo experiment that shows that SAH leads to astrocyte death, exposure of cultured astrocytes to MPO+H 2 O 2 led to significant cell death in culture as well. MPO alone had very little effect on astrocyte morphology and survival suggesting the presence of its substrate is important H 2 O 2 alone led to significant cell death in culture as well (supplemental figure 7), suggesting that astrocytes are especially vulnerable to the oxidative stress caused by MPO+H 2 O 2 and peroxide . These results further support the loss of astrocytes evident in the mouse brain after SAH (Fig. 4).
To determine whether there are MPO-induced astrocyte secreted molecules can affect neuronal function (independent of cell death), primary neuronal culture activity was analyzed after exposure to astrocyte conditioned media. Both H 2 O 2 and MPO with H 2 O 2 led to astrocyte death making conditioned media unreliable as a source of secreted molecules. The MPO conditioned media had no discernable effect on neuronal activity (data not shown). suggesting that MPO effects on the brain are likely due to MPO-mediated astrocyte death, leading to the dysregulation of neuronal support.

Summary
The results of the present project provide clear evidence that immune activity in the meningeal compartment affects functions of the underlying brain. Using subarachnoid hemorrhage (SAH) as a sterile injury model, we show that through the action of its enzyme myeloperoxidase (MPO), neutrophils affect neuronal function. Our 2-photon and in vitro experiments demonstrate that MPO can affect neuronal activity similar to SAH. We also find evidence that MPO modulates astrocyte survival in vivo and in vitro. Finally, the removal of functional MPO in non-injured mice, leads to significant change in the immune landscape within and around the brain. This suggests that neutrophils and myeloperoxidase likely play an important role in how immune cells populate the meninges.
The effect of MPO on behavior offers an opportunity to investigate the physiology that underlies delayed neuronal dysfunction after SAH. There are two likely routes by which MPO could mediate neuronal function: effect on neurons, and/or through an intermediate cell type such as astrocytes or microglia. Histological evidence in our studies suggests against microglial activation as a mediator of MPO based neuronal dysfunction. The data in neurons and astrocytes is more promising.

Myeloperoxidase directly modulates neuronal activity
Previous findings from our laboratory demonstrate that SAH leads to delayed cognitive deficits in mice(5). These deficits are attributed to the loss of late long-term potentiation (L-LTP) in the hippocampus(4). Changes in L-LTP and NMDAR subunits after SAH suggest an effect of the injury on neuronal physiology. Here, we find that MPO null mice do not develop the behavioral changes associated with SAH. We also find that MPO and MPO+ H 2 O 2 significantly decrease neuronal activity in vitro (although it is important to note that the toxic effect of H 2 O 2 alone could suggest an alternate interpretation that MPO catalysis is removing free H 2 O 2 , thereby mitigating its toxic potential). Furthermore, our in vivo experiments using 2-photon microscopy, show that MPO's activity, like SAH, decreases neuronal depression after cortical spreading depression.
These experiments show a modulatory effect of MPO on neuronal activity but raise an important question about how MPO gets from the meninges to hippocampal neurons. Immunohistochemistry in the brain after SAH does not show significant quantities of MPO in the brain which suggests that other mechanisms exist. These are areas of interest in our group.
In addition, the mechanism(s) through which MPO's activity affects neurons is not clear. Because MPO has no identified, dedicated cell surface receptor, it is unclear which moieties on the neuronal membrane are being acted upon. However, we do know that MPO's main enzymatic activity is the production of reactive oxygen species (ROS), specifically the chlorinating agent, HOCl(34). ROS have been implicated in neuronal dysfunction and degeneration in a multitude of models(35). These data most support a mechanism of MPO modulating neuronal membrane properties i.e. receptors and channels, on the surface evidenced by changes in calcium imaging. Alternately, another possible mechanism is that MPO is internalized through a non-cell surface protein mechanism and lead to subsequent intracellular activity which could lead to calcium changes (36) .

Enzymatic activity of myeloperoxidase leads to impaired astrocyte function
Another possible pathway for neuronal dysfunction is the action of MPO on supporting brain cells that affect neuronal dysfunction. SAH in WT mice shows a significant loss of astrocytes in the hippocampus six days after SAH. This decrease is preceded by a significant decrease in GFAP coverage, which is indicative of astrocyte loss in the hippocampus after SAH. Furthermore, the GFAP decrease coincides with an increase in vimentin expression. Vimentin/GFAP co-expression in astrocytes occurs in precursor/immature astrocytes(37). As such, the increase vimentin coexpression could be indicative of an increase proliferation of astrocyte progenitor cells. These changes are absent in the MPO KO mouse, suggesting that neutrophil activity in the meninges affects astrocytes in the cortex. Astrocytes are critical to the maintenance of homeostatic balance within the nervous system. Their ability to support neurons, both structurally and through the provision of neurotransmitters, lactate, etc., is critical to the maintenance of normal neuronal function in the CNS(38). Astrocytes also maintain the integrity of the CNS milieu by acting as a barrier, the bloodbrain and blood-CSF barriers, between the circulatory system and the CNS. These barriers are especially critical during inflammation (39,40). For example, the loss of GFAP positive astrocytes leads to increased leukocyte infiltration in the brain after stab injury(40) and in EAE(39). Although our model is devoid of CNS immune infiltration, the loss and/or impaired function of astrocytes may hinder their ability to 'provide' the necessary support to neurons after SAH and may represent a complimentary mechanism leading to neuronal dysfunction.

Conclusions
This paper provides evidence that activity of neutrophils outside the brain can modulate neuronal function during injury. We show that MPO is critical to the behavioral changes observed in SAH mice.
Its effects include modulation of both astrocytic and neuronal activity within the parenchyma (Fig. 5).
One possible interpretation of the data suggests that after SAH, neutrophils degranulate in the subarachnoid space, leading to activation of astrocytes that form the glia limitans in the subpial space. Reactive oxygen species from MPO's enzymatic activity in this space leads to the down regulation of GFAP and cellular death at this critical interface. The astrocyte cell death can then affect brain function. Furthermore, MPO dampens neuronal response to stimuli, thereby dysregulating whole circuit activity. Although other mechanisms are likely at play, we propose that this dysregulation is likely a critical factor in the development of cognitive deficits associated with SAH.    Figure 1 Myeloperoxidase is critical to the development of delayed cognitive deficits after SAH.
Barnes maze memory task were used to determine the mechanism of action of neutrophils   The enzymatic activity of MPO modulates neuronal activity. Primary neuronal cultures, obtained from Thy1-GCaMP3 mice, were incubated with MPO and/or H2O2. Time-lapsed images were taken from cultures before and after the addition of the MPO and/or H2O2.
After 2.5 hours of incubation, cells were further stimulated with 10mM KCl (A).
Representative traces of neuronal activity, as denoted by cellular calcium signal, showed the effect of MPO and/or H2O2 on baseline firing rate. MPO alone had no effect on neuronal activity, while H2O2 decreases activity by more than 75%. On the other hand, addition of MPO together with H2O2 decreased neuronal activity by 50%. Furthermore, the addition of   Neutrophil derived myeloperoxidase directly affects brain parenchymal cells after sterile injury subarachnoid hemorrhage. The results presented in this study clearly demonstrate that myeloperoxidase, MPO, can directly modulate CNS output by either affecting neuronal firing and response to stimulation or dysregulating astrocyte function. WE postulate that 3 days after SAH, neutrophils infiltrate the meninges. Therein, they release MPO, a granule protein, into the subarachnoid space. MPO's activity within this space leads to dysregulation and death of astrocytes at the subpial brain interface. Further leading to dysregulation of neuronal support and ultimately CNS output.

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