Following SAH, activated macrophages release pro-inflammatory cytokines/chemokines for the recruitment of circulating macrophages to aid in RBC clearance (19). Phagocytosis of RBCs and subsequent death of the infiltrated leukocytes (occurring 2–4 days post-ictus) in the cerebrospinal fluid leads to release of a plethora of toxic molecules (35) including endothelins, oxygen free radicals, and hemoglobin/heme (and byproducts), thereby potentiating pro-inflammatory pathways and endothelial cellmediated thrombosis. The latter event induces a positive feedforward loop, potentiating thromboinflammation by continuing to recruit systemic immune cells for debris clean-up, causing cell adhesion molecules to continuedly activate platelets, leading to microthrombi formation (36) (**Figure 1**).

Production/release of reactive oxygen species and nitric oxide adds to endothelial damage (37), stimulating endothelial expression of adhesion molecules (e.g*.*, ICAM-1, P-selectin, VCAM, collagen, fibrinogen, etc.) (37, 38), and release of tissue factor and vWF, thereby activating platelets. This process is controlled by ADAMTS13 (a disintegrin-like metalloprotease with thrombospondin type 1 repeats-13), which acts to control clot formation by cleaving hyperactive ultralarge vWF to prevent platelet adherence (39). Interestingly, aSAH patients who go on to develop DCI are more hypercoagulable than aSAH patients who will not develop DCI (40, 41). Auxiliary to greater/sustained microthrombi formation, continued activation of coagulation by endothelial cells and platelets further promote inflammation *via* activation of circulating leukocytes through protease activated receptor-1 and toll-like receptor 4 signaling (36).

Finally, emerging evidence further displays the delicate balance that inflammation plays in modulating coagulation (36). In the non-diseased state, systemic leukocytes aid in preventing coagulation by expressing several anticoagulant factors (42–44). Yet, when pro-inflammatory signaling is initiated, leukocytes change phenotypes, thereby aiding in coagulation through the release of procoagulant factors (45, 46) and reduced expression and degradation of anti-coagulation factors (47–49). While leukocytes play a critical role in inducing and propagating inflammation, leukocytes also promote a hypercoagulable state after SAH (36) and may play a crucial role in DCI *via* thromboinflammation.

#### Clinical Evidence

To date, the exact role thromboinflammation plays in patients suffering from aSAH is not known. Yet, the following clinical studies suggest that thromboinflammation may be a key contributor to DCI after aSAH, and thus should be considered as a therapeutic target.

First, microthrombi have been reported as an associative cause of DCI and worse neurological outcome (50, 51). In human autopsy studies, microthrombi, reported to be throughout the brain, are associated with regions of infarct (50, 51), and also develop with a timing similar to DCI (6). Following aSAH, platelet activation is over-stimulated, resulting in microthrombi within small arterioles (51), peaking within 2 days and then again from 1 to 2 weeks post-ictus (50). Platelet-derived thromboxane B2 is higher and platelet count is lower in DCI patients compared to non-DCI patients (52, 53), suggesting platelet aggregation as a potential cause of microthrombi. In addition, aSAH patients are hypercoagulable with changes in platelet-activating factor (54), vWF (40, 54), and tissue plasminogen activator (55, 56) correlating with the incidence of DCI (54, 55) and poor patient outcome (28). Specific polymorphisms of plasminogen activator inhibitor (PAI) have higher PAI activity (57) leading to a higher incidence of DCI (41). Finally, in addition to an over active coagulation cascade, fibrinolysis after aSAH is impaired (40, 56). ADAMTS13, proposed as a critical link between inflammation and thrombosis (39), has been observed to have low activity after aSAH, and is associated with an increased risk of ischemic stroke (58) and may potentiate microthrombi formation. The inherent roles of leukocytes and platelets in inflammation and thrombosis, as well as the involvement of ADAMTS13, argue for thromboinflammation being considered as a therapeutic target after SAH.

Not only is the coagulation cascade not functioning properly, so too is inflammation continually hyperactive. Several proinflammatory cytokines have been linked with worse clinical outcome after aSAH (25–27, 59, 60). Increases in tumor necrosis factor α, interleukin-1α, interleukin-1β, interleukin-6, and

interleukin-8 have all been found in the cerebrospinal fluid and blood (59, 61–68), with several factors also being associated with poor outcome and DCI (59, 61). Additionally, elevated adhesion molecules in the blood have also been observed after aSAH in patients, remaining elevated for 6–8 days, and are associated with DCI (69–71). Blood P-selectin is elevated in patients with DCI, suggesting that inflammation and platelet activation adhesion are associated with DCI (72, 73). Indeed, adhesion molecules are well documented to be at the crux of thromboinflammation (39, 74–76). These findings, which suggest thromboinflammation is active, are also supported by increased leukocyte infiltration into the brain following SAH (77–80) and also correlate with ischemia after SAH (81). Thus, the so-called "leukocyte–endothelial cell interaction" (78) may play a role in SAH pathophysiology (i.e., thromboinflammation).

A recent study on 106 aSAH patients showed that platelet activation and inflammation (*via* C-reactive protein measurement) occurred simultaneously and were associated with worse early brain injury and 3-month outcome (82). Similar to others (40, 54), Frontera et al. found that platelet activation and C-reactive protein levels were associated with DCI, but even more interesting, the authors reported that higher platelet activation and C-reactive protein levels at 72 h post-SAH correlate with DCI (82). The findings by Frontera et al. further support the potential role of thromboinflammation in DCI after aSAH, suggesting thromboinflammation may be an unexplored therapeutic target for improving aSAH outcome.

#### Evidence from Experimental Models

Thromboinflammation has also not been specifically studied in experimental models of SAH, nor has it been examined for correlation with microthrombi, ischemia, or DCI in animals. While DCI is unmeasurable in animals, several pathological measures are used as correlates for DCI (e.g., vasospasm, microthrombi formation, ischemia/infarct/cell death).

Several groups have reported the existence of microthrombi within the vasculature both locally, as well as distally from the SAH insult in experimental SAH models (83–86). In rodent models of SAH using autologous blood infusion, microthrombi and occluded blood vessels have been reported to occur throughout the brain at 2 days post-SAH (84) and can be observed for up to 7 days post-ictus (86). While microthrombi have been observed in puncture models of SAH (87, 88), it is the blood infusion models which offer the most insight into the potential role of thromboinflammation in clot formation after experimental SAH. Indeed, thrombi may be thrown downstream from the vessel rupture in endovascular perforation models as the mechanism by which microthrombi occur in the vasculature; however, no such vessel rupture is present in the autologous blood infusion models. In the blood injection SAH models, the likely cause of microthrombi formation and deposition within the cerebral vasculature distal from the SAH insult is inflammation.

Furthermore, following experimental SAH, endothelial damage resulting from leukocyte infiltration may lead to the downward spiral that is thromboinflammation. Indeed, post-SAH inflammation is devastating (19) and includes cytokine/ chemokine upregulation (89, 90), increased expression of adhesion molecules (91, 92), and leukocyte infiltration (93–95). In fact, either depleting neutrophils or reducing neutrophil activity decreases microvascular injury following SAH in rats (93).

Similar to clinical findings, P-selectin has been observed to increase after experimental SAH, leading to activation of platelets and thrombi formation (84). Interestingly, platelet aggregates have been observed to extravasate into the brain parenchyma in the rodent SAH autologous blood injection model which likely propagates pro-inflammatory signaling (22), further showing that there is cross talk between inflammation and thrombosis after SAH.

Evidence from experimental models of SAH, similar to clinical findings, suggest that significant overlap between inflammation and thrombosis occurs post-SAH and may be involved in secondary injury and neurological deterioration. Future studies are warranted to investigate the exact role(s) thromboinflammation plays in SAH and its potential as a therapeutic target.

### THROMBOINFLAMMATION AS A THERAPEUTIC TARGET TO PREVENT DCI

The detrimental roles thromboinflammation play in brain pathologies has led to thromboinflammation to be suggested as a therapeutic target (32, 96), but not for SAH. Based on the review presented above, thromboinflammation should also be considered a therapeutic target for patients of SAH (54). Potential targets of thromboinflammation may include expression of adhesion molecules, release of factors activating leukocytes and platelets simultaneously (e.g., selectins), disrupting platelet-leukocyte formations, and targeting ADAMTS13. To date, no clinical trials have studied these factors as specific therapeutic targets. Below we review some of the clinical trials which have targeted either coagulation or inflammation, and show that targeting these physiological events separately does not seem to hold the answer for SAH treatment.

#### Previous Trials

#### Targeting Coagulation and Clot Clearance

Several early clinical trials investigated therapeutics directed at targeting coagulation following SAH, many of which used antiplatelet therapies. However, meta-analysis of antiplatelet therapy indicates that antiplatelet therapy has a trend for improving outcome in aSAH patients, with tendencies to decrease secondary ischemia (except for acetylsalicylic acid which shows no benefit) (97). Some of the antiplatelet therapies thus far investigated are acetylsalicylic acid, ticlopidine (adenosine diphosphate receptor inhibitor), dipyridamole (phosphodiesterase inhibitor), OKY-046/Calaclot (thromboxane synthetase inhibitor), a thromboxane A2 antagonist, and E5880 (inhibitor of platelet-activating factor receptor) (97). Of these agents, Calaclot and E5880 seemed to improve functional outcome, but additional studies need to be undertaken to determine the therapeutic benefit associated with antiplatelet therapies.

Studies have also examined the benefit of targeting clot clearance. In this regard, recombinant tissue plasminogen activator (administered intraventricularly) has been investigated in several clinical trials which have shown that although recombinant tissue plasminogen activator can improve clot clearance, it fails to attenuate cerebral vasospasm or DCI (98), reduce mortality (99), or improve functional outcome (100). These findings indicate that clot clearance alone may not be enough for aSAH.

#### Targeting Inflammation

Trials targeting inflammation have had varied success. Erythropoietin-β, despite having mechanisms of action against vasospasm and inflammation (101), had no effect on cerebral vasospasm, but did reduce DCI and improve functional outcome (102). This was a small study and is at high risk for bias (103), so additional trials are needed. Another anti-inflammatory agent, methylprednisolone, was found to improve functional outcome at 1 year despite having no effect on cerebral vasospasm or hypodensity on CT scans (104). It should be noted that methylprednisolone is an anti-inflammatory and thus may have no effect on vasospasm, although the lack of change in CT hypodensity is puzzling. Regardless, these trials suggest that inflammation indeed plays a major role in SAH outcome. Since there is considerable feedforward/back mechanisms between thrombosis and inflammation, anti-inflammatory agents may indirectly reduce thromboinflammation. While previous trials have shown promise, additional studies are needed to determine any benefit to patient outcome for anti-inflammatory agents as well as the relationship to thromboinflammation.

#### Current Trials

The lack of promising data for therapeutics targeting either cerebral vasospasm (100, 103) or clot clearance suggest that SAH prognosis may lie within inflammation, platelet activation, or at the intersection of the two (i.e., thromboinflammation). To date, the only FDA approved treatment after aSAH is nimodipine. While the initial mechanism of action was purported to be *via* anti-vasospasm (105, 106), its effects seem to be more than just preventing vasospasm. Indeed, nimodipine can improve patient outcome irrespective of vasospasm attenuation (107). Although the exact mechanism of nimodipine is unknown, it may reduce microthrombosis, be neuroprotective, and inhibit cortical spreading ischemia (14, 108). Furthermore, nimodipine has been implicated as being anti-inflammatory (109), and it is important to note that calcium channel blockers may reduce leukocyte infiltration (110), and therefore reduce thromboinflammation. Mechanistic studies for the role(s) of nimodipine are needed to clarify the mechanisms by which nimodipine improves patient outcome after SAH. These studies may also shed light onto the therapeutic targets for thromboinflammation.

Fasudil is currently used in Asia instead of nimodipine, because the latter is not commercially available. Fasudil is a rho-kinase inhibitor which is reported to reduce hemodynamic dysfunction (i.e., vasoconstriction, endothelial injury) and inflammation through downstream signaling pathways *via* inhibition of rhokinase (111). Initially, fasudil was used to target cerebral vasospasm, but fasudil was found to also reduce cerebral infarction, and improve functional outcome after aSAH (112). Thus, fasudil also seems to provide therapeutic benefit for aSAH patients, but still requires validation in large randomized controlled clinical trials (113). Being a rho-kinase inhibitor, fasudil may prevent a number of downstream signaling pathways involved in SAH pathophysiology [smooth muscle cell contraction and endothelial dysfunction (95, 114), inflammation, and leukocyte activation (115)], arguing that drugs with pleiotropic effects may be an answer. Similar to nimodipine, the exact mechanisms by which fasudil improves outcome following SAH remain a mystery, and translational studies for SAH will benefit for additional studies on the mechanism(s) of fasudil.

The recent study by Wessell et al. found that infusion of lowdose heparin administered with nimodipine is associated with increased odds of patient discharge (116), and, in a preliminary trial, heparin reduced cerebral vasospasm and infarct (117). These studies support the notion that combining therapies which reduce distinct injury mechanisms can provide increased effectiveness. Furthermore, these studies indicate that there may be therapeutic potential in anti-coagulation and anti-inflammation (103). Currently, heparin is currently being explored in the ASTROH trial (NCT02501434) which will examine the efficacy of heparin for reducing vasospasm and delayed neurological deficits in SAH patients.

Specific information regarding the role of thromboinflammation and its potential as a therapeutic target may be elucidated through the SoSTART trial (NCT03153150) and the etanercept (TNFα receptor antagonist) trial (NCT01865630). The SoSTART trial is set to investigate seven anti-coagulants for therapeutic benefit in patients of cerebral hemorrhage, including aSAH. These proposed drugs are known to be reduce coagulability and prevent inflammation (118, 119), which may reduce the overall thromboinflammation.

#### Challenges in Targeting Thromboinflammation

Several challenges exist when considering thromboinflammation as a therapeutic target for preventing DCI. First and foremost, special care needs to be taken when identifying potential therapies for thromboinflammation since the ideal candidate will only target newly formed microthrombi and not have any significant effect on the clot formed at the site of aneurysm rupture. Once the aneurysm has been secured with clip or coil, the aneurysm is of

#### REFERENCES

less concern, but potential complications such as post-operative bleeding or bleeding along the ventriculostomy remain a risk.

In most pathologies, including SAH, both pro- and antiinflammatory pathways are activated to promote debris/toxin clearance and repair of injured tissue, respectively. Since proinflammatory cytokines and downstream signaling is linked with thromboinflammation (34), it is critical to pursue agents which mitigate pro-inflammatory cytokines. So far research has largely focused on this, however, anti-inflammatory signaling aids in repair and healing, thus this arm of inflammation should remain active (or upregulated) after reducing thromboinflammation.

Finally, although experimental studies are required to understand pathophysiology, uncover signaling mechanisms, and investigate novel drugs and potential therapeutic targets, animals (including non-human primates) have differences when it comes to coagulation (120, 121), as well as inflammation (122, 123). Therefore, it follows that animals may have distinct differences when it comes to thromboinflammation and its treatment. Future studies should examine these differences to aid in therapeutic development against thromboinflammation.

#### CONCLUSION

Thromboinflammation, the cross talk between the thrombotic and inflammatory pathways, likely plays a critical role in the development of DCI after SAH, thereby having significant impact on overall patient outcome. The current understanding of thromboinflammation and its role in SAH pathophysiology is only beginning to emerge, but evidence from experimental and clinical studies suggest that preventing thromboinflammation has the potential for benefiting patients of aSAH.

#### AUTHOR CONTRIBUTIONS

All authors contributed to the conception and drafting of this article.

## FUNDING

This work was supported by a NIH grant (JZ, NS081740) and by a seed grant provided by The Vivian L. Smith Department of Neurosurgery at The University of Texas Health Science Center at Houston (DM).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 McBride, Blackburn, Peeyush, Matsumura and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Hypothesis on the role of Cryptochromes in inflammation and subarachnoid Hemorrhage outcome

*Adriano Barreto Nogueira1,2\*, Ariel Barreto Nogueira3 , José Carlos Esteves Veiga2 and Manoel Jacobsen Teixeira1,4*

*1Division of Neurosurgery Clinic, Hospital das Clinicas, Faculty of Medicine, University of Sao Paulo, Sao Paulo, Brazil, 2Discipline of Neurosurgery, Santa Casa Faculty of Medical Sciences, Department of Surgery, Sao Paulo, Brazil, 3 Faculty of Medicine, Department of Radiology, Hospital das Clinicas, University of Sao Paulo, Sao Paulo, Brazil, 4 Faculty of Medicine, Department of Neurology, University of Sao Paulo, Sao Paulo, Brazil*

#### *Edited by:*

*Fernando Testai, University of Illinois at Chicago, United States*

#### *Reviewed by:*

*Christoph Stretz, Yale University, United States Minjee Kim, Northwestern University, United States Benjamin Aaron Emanuel, Keck School of Medicine of USC, United States*

*\*Correspondence:*

*Adriano Barreto Nogueira<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neurocritical and Neurohospitalist Care, a section of the journal Frontiers in Neurology*

*Received: 21 July 2017 Accepted: 14 November 2017 Published: 28 November 2017*

#### *Citation:*

*Nogueira AB, Nogueira AB, Veiga JCE and Teixeira MJ (2017) Hypothesis on the Role of Cryptochromes in Inflammation and Subarachnoid Hemorrhage Outcome. Front. Neurol. 8:637. doi: 10.3389/fneur.2017.00637*

We have recently found that the temperature variability (TV) in the day–night cycle may predict the mean intracranial pressure in the following 24 h (ICP24) in subarachnoid hemorrhage (SAH) patients under multimodality monitoring, sedation, and hypothermia (<35°C). Specifically, we found that ICP24 = 6 (4 − TV) mmHg. TV is the ratio between the coefficient of variation of temperature during the nocturnal and the preceding diurnal periods. This result suggests that the circadian clock reflects brain plasticity mechanisms and its malfunctioning leads to deterioration of the neurologic status. The sleep–wake cycle is absent in these patients and their circadian clock can function properly only by environment light-independent mechanisms. One mechanism involves the circadian clock proteins named cryptochromes (CRYs). CRYs are highly preserved and widespread in the evolutionary tree, are expressed in different cell types in humans [type II CRYs, in two forms: human cryptochrome 1 and 2 (hCRY1 and hCRY2)], and in certain species, respond to blue light and play role in magnetoreception. Interestingly, SAH outcome seems to correlate with inflammation, and CRYs decrease inflammatory activity. Our hypothesis derived from these observations is that CRYs modulate the circadian oscillation of temperature even during therapeutic hypothermia and improve outcome in SAH through decrease in inflammation. A strategy to test this hypothesis is to measure periodically during the acute phase of high-grade SAH the level of CRYs in cerebrospinal fluid (CSF) and circulating white blood cells, and to correlate these levels with outcome, TV, ICP24, and pro- and anti-inflammatory markers in CSF and blood. If this hypothesis is true, the development of therapies targeting inflammation in SAH could take advantage of cryptochrome properties. It has been shown that blue light phototherapy increases the expression of CRYs in blood mononuclear cells in jaundiced neonates. Likewise, visual stimulus with flashing light improves Alzheimer's disease features in experimental model and there is a prominent expression of CRYs in the retina. Remarkably, recent evidence showed that hCRY2 responds to electromagnetic fields, which could be one elusive mechanism of action of transcranial magnetic stimulation and a reason for its use in SAH.

Keywords: cryptochromes, subarachnoid hemorrhage, inflammation, neurogenesis, circadian rhythm, intracranial pressure prediction, therapeutic hypothermia, targeted temperature management

## INTRODUCTION

Our previous work indicates that circadian rhythms are a primary factor to predict brain injury (1–6). First, we showed that the anterior hypothalamus displays a prominent expression of neurogenesis-related markers in adult humans (5, 6). The functions of the anterior hypothalamus include circadian rhythm control and thermoregulation. Because of these features, we suspected that the monitoring of circadian oscillation of temperature could measure putative brain regeneration and plasticity mechanisms and, therefore, anticipate the intensity of brain injury. Following this rationale, we discovered that the ratio between the mean temperature during the sleep and the preceding wake period correlates with the likelihood of seizure occurrence in the following 24 h in epileptic patients (1, 3). We have recently showed that this concept works even with high-grade subarachnoid hemorrhage (SAH) patients under sedation and therapeutic hypothermia induced by intravascular catheter (2). In this case, the ratio between the coefficient of variation of temperature during the nocturnal and the preceding diurnal periods correlates with the mean intracranial pressure in the following 24 h (ICP24) (2). Collectively, these findings open the possibility to reduce brain injury through circadian rhythm modulation.

Circadian rhythms may be modulated by interfering on the level of the clock proteins cryptochromes (CRYs). CRYs are evolutionarily old flavoproteins expressed in two types, in species ranging from plants to humans (7). Mammals display ubiquitous expression of type II CRYs (8, 9). In humans, there are two forms of type II CRYs, named human CRY1 (hCRY1) and CRY2 (hCRY2) (8, 9). CRYs are highly expressed in the suprachiasmatic nucleus (10). The suprachiasmatic nucleus is the hypothalamus structure that orchestrates the circadian rhythms by the central nervous system. CRYs also interfere in the other mechanism of circadian rhythm control (peripheral mechanism), which includes immune system cells (7). Importantly, hCRY levels can be potentially modulated by blue light (11) and electromagnetic field (8), which are currently used in clinical practice.

## HYPOTHESIS

We hypothesize that the ratio between the level of expression of hCRYs during the nocturnal and the preceding diurnal periods (CRYn/d) correlates with inflammation and predicts neurologic signs in the following day–night cycle and long-term outcome in SAH.

## RATIONALE OF THE HYPOTHESIS

#### Adult Human Neurogenesis

The discovery of adult mammalian neurogenesis opened a novel perspective on brain plasticity and neuroregeneration therapies (4–6). The current prevailing concept that the adult mammalian brain harbors two primary neurogenic niches in the subgranular zone of the dentate gyrus and in the subventricular zone replaced the long-lasting dogma of no-new neurons after birth (4–6). Adult human neurogenesis is controversial, but we have previously shown that the adult human brain harbors what we referred to as potential neurogenic system (4–6). The potential neurogenic system was revealed by the detection of expression of neural stem cell markers in the circumventricular organs (4–6). The circumventricular organs are the brain structures located principally in the hypothalamus that display no blood–brain barrier. Moreover, brain structures adjacent to the circumventricular organs, which form part of the hypothalamic and limbic system circuits, express the immature or newly formed neuron marker doublecortin (DCX) (5, 6). We interpreted these results as a potential constitutive mechanism of neurogenesis that could participate in the maintenance of certain brain circuits and in their regeneration after injury. Next, we pursued a method to measure the effects of this potential plasticity mechanism in clinical practice.

## Brain Injury Prediction through Circadian Rhythm Monitoring

#### Presence of Sleep–Wake Cycle

Because the hypothalamus displays robust expression of neurogenesis-related markers and modulates circadian rhythms and temperature, we suspected that these functions parallel endogenous brain plasticity. Indeed, alterations in the hypothalamus-related functions anticipate worsening in neurologic status, maybe partially due to alterations in endogenous neurogenesis mechanisms. In practice, we found that the ratio between the mean skin temperature during sleep and the preceding wake period (Ts/w) correlates with the likelihood of seizure occurrence in the following 24 h in epileptic patients (1, 3). This method is non-invasive and involves the use of wristband with sensors of vital signs (1, 3). Importantly, skin temperature has been shown to be a reliable circadian rhythm marker (1, 3).

#### Absence of Sleep–Wake Cycle

A stronger indication that the circadian rhythm is a primary factor to predict brain injury is being unveiled by our ongoing research showing that the pattern of oscillation of core body temperature in the day–night cycle in high-grade SAH patients correlates with further intracranial pressure (2). The patients analyzed in this study underwent sedation and targeted temperature management (33–33.9°C) using intravascular catheter. Circadian rhythm correlated with further neurologic signs even in this situation of decreased level of consciousness, lack of sleep–wake cycle, and strict temperature control. Specifically, the ratio between the coefficient of variation of the nocturnal (starting at 18:00) and the preceding diurnal periods (starting at 6:00) [temperature variability (TV)] correlated with ICP24 (*p* < 0.001). The formula derived from regression analysis is ICP24 = 6 (4 − TV) mmHg (2).

The findings of our line of research indicate the existence of a light-independent mechanism of circadian rhythm that participates in brain plasticity (9, 12). The discovery of a factor involved in this mechanism could lead to the development of a circadian rhythm-guided therapy to prevent brain injury.

### Cryptochrome As Potential Prediction Factor in SAH

Cryptochromes display circadian expression in the suprachiasmatic nucleus (10) and in peripheral tissues (7) such as white blood cells (11) through light-independent (7, 9) and lightdependent mechanisms (8, 10, 12).

The expression of hCRYs decreases during the day and increases during the night (13). Insufficient increase in hCRYs during the night correlates with higher levels of inflammation in the following morning, as has already been demonstrated in rheumatoid arthritis patients (13). In SAH patients, inflammation seems to lead to poor outcome (4).

The circadian pattern of temperature oscillation correlates with further neurologic signs (i.e., seizure in epileptic patients and intracranial hypertension in high-grade SAH) (1–3), and an elusive mechanism of this finding is that a normal circadian rhythm reflects normal hypothalamic neurogenesis (**Figure 1**) (1–6). Intriguingly, another potential marker of circadian rhythm, which could theoretically form a loop involving hCRYs and neurogenesis is blue light emission by the body. Endogenous blue light emission has been shown from human body (14). This emission displays circadian pattern and correlates with metabolism and reactive oxygen species (ROS) formation (14). Blue light stimulates CRY expression (11), and both—CRY (15) and ROS (16)—stimulate neurogenesis. Conversely, neurogenesis could close the loop by decreasing ROS formation, blue light emission, and CRY stimulation.

These observations raise the possibility that the circadian pattern of CRY expression correlates with circadian patterns of markers of inflammation and neurogenesis, neurologic signs in the following day–night cycle, and outcome in high-grade SAH.

#### HYPOTHESIS TESTING

The primary analysis to test our hypothesis is the correlation of the ratio between the level of hCRYs in the cerebrospinal fluid (CSF) during the nocturnal and the preceding diurnal periods (CRYn/d) of acute phase of high-grade SAH patients

with outcome after 1 year. This level can be determined by immune-enzymatic assay (ELISA). In addition, bearing in mind the relationship between CRYs and inflammation, the ratio can be calculated also regarding the expression of hCRYs in white blood cells (WBCCn/d) (and their different types, e.g., mononuclear cells).

Due to potential therapeutic implications of the hypothesis (as detailed below), a crucial analysis is the correlation between CRYn/d and TV and ICP24. A caveat of this analysis is that TV has been shown to correlate with ICP24 only under strict control of temperature, with daily mean <35°C (2).

The complete study to test the hypothesis proposed here includes our published protocol to assess multimodality monitoring, inflammation, and neuroregeneration markers in CSF and blood of highgrade SAH patients aiming at revealing a prognostic biomarker (4). Because of our previous work, we deem worthwhile including in our original protocol principally the analysis (from blood and CSF samples of SAH patients) of markers related to circadian rhythms (melatonin), thermoregulation (RANK) (17), and hypothalamusor hypophysis-produced hormones (growth hormone-releasing hormone, growth hormone, gonadotropin-releasing hormone, and oxytocin) (**Table 1**). Accordingly, the pineal gland and neurohypophysis are circumventricular organs, and the hormones mentioned above stimulate neurogenesis (18, 19).

Also bearing in mind its intriguing presence in the circumventricular organs [pineal gland (20)/habenular zone (21), choroid plexus (22), hypothalamus parenchyma (22)], it would be interesting to quantify through cell sorting mast cells in the CSF of the patients enrolled in this type of protocol. The potential neurogenic system arises from zones without blood–brain barrier, whose permeability is dynamic across the sleep–wake cycle (23). An indication that mast cells may contribute to dynamic features of blood–brain barrier is that they contain granules of histamine, which increases locally the permeability of blood– brain barrier (21, 22). Histamine also decreases inflammation *via* activation of H2 receptor and is involved in neuroprotection associated with preconditioning (24). Furthermore, histamine increases neural stem cell proliferation and favors cell fate toward neuronal differentiation *via* activation of neural stem cell H2 and H1 receptors, respectively (24). The presence of mast cells in circumventricular organs, action of histamine to increase blood–brain barrier permeability, decrease inflammation, protect neurons from secondary injury, and stimulate neurogenesis suggest that mast cells may play a role in endogenous mechanisms of neuroprotection and neuroregeneration.

#### EXPECTED RESULTS

In decreasing order of importance, the expected results for the study protocol summarized above are:

Some remarks should be mentioned regarding hypothesis testing and expected results (**Table 1**).

#### TABLE 1 | Parameters for correlation analysis with CRYn/d.

*CSF collection at 6:00 and 18:00 at minimum every 3 days for CRYn/d determination when external ventricular drain is placed, until 14 days after bleeding. Minimum, maximum, and mean CRYn/d for each patient should be compared with any of the remaining parameters to search for correlation. WBCCn/d and mononuclear WBC CRY levels can be used as an alternative parameter for circadian CRY level. Main parameters are in bold. The remaining parameters are options that can be selected according to the goal of the protocol or resources of the service.*

Sickness Impact Profile Clinical assessment Health-related QoL Discharge, 1, 3, 6, and 12mo Short-form 36 Clinical assessment Health-related QoL Discharge, 1, 3, 6, and 12mo

*ADP, adenosine diphosphate; ATP, adenosine triphosphate; CRP, C-reactive protein; CRY, cryptochrome; CRYn/d, ratio between CRY level at night (18:00) and the preceding day (6:00) in CSF; CSF, cerebrospinal fluid; DCI, delayed cerebral ischemia; EEG, electroencephalogram; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescent-activated cell sorting; GH, growth hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; GOS, Glasgow Outcome Scale; ICP, intracranial pressure; ICP24, mean ICP in the day following TV monitoring; ICU, intensive care unit; IL, interleukin; MCAV, middle cerebral artery velocity; MMSE, mini-mental status exam; MCP, monocyte chemoattractant protein; MoCA, Montreal cognitive assessment; mRS, modified Rankin scale; PtiO2, brain tissue oxygen pressure; RANK, receptor-activator of nuclear factor* κ *B; qRT-PCR, real-time polymerase chain reaction; QoL, quality of life; SDF, stromal cell-derived factor; T, temperature; Th, T helper; TNF, tumor necrosis factor; TV, temperature variability; WBCCn/d, ratio between CRY expression in white blood cells at night (18:00) and the preceding day (6:00).*

Cerebrospinal fluid collection for CRY determination will be carried out at 6:00 and 18:00, because they are the times of extreme core body temperature values. This procedure will be performed when an external ventricular drain is placed, at least every 3 days, and until 14 days after bleeding. For each patient, we will test three manners to calculate CRYn/d: highest ratio, lowest ratio, and mean ratio of all daily CRYn/d values. CRY level will be calculated as hCRY1, hCRY2, and hCRY1 plus hCRY2 levels.

The role of inflammation in stroke and particularly in SAH pathophysiology is not totally established (25). For example, tumor necrosis factor alpha correlates with neuron damage or protection depending on the membrane receptor on which it acts (4). Interleukin-6 (IL-6) shows a similar dual effect depending on microenvironment conditions (4). Likewise, microglia participates in inflammation in the acute phase of stroke model, but in a later phase contributes to migration and differentiation of newly formed immature neurons (i.e., neuroblasts) (4).

The multifactorial nature of SAH pathophysiology hampers the determination of correlation between outcome and inflammation (25). SAH and brain injury trigger clinical features that lead to systemic inflammation (26), which on its turn may cause brain damage through seizures, for example (26). However, some studies showed that serum level of inflammation markers such as C-reactive protein (CRP) (27) and IL-6 (28, 29) correlates with SAH outcome.

Cerebrospinal fluid composition could in theory reflect a more straightforward level of brain inflammation, but factors such as amount of bleeding and use of external ventricular drain may interfere on results. A stronger correlation in comparison with serum inflammation markers was demonstrated between CSF level of the inflammation-related marker high mobility group box-1 protein and SAH outcome after 3 months (30). This marker and CRP (27) also correlated with clinical (Hunt and Hess, World Federation of Neurological Societies, and Glasgow Coma scales) and radiological (Fisher) scales known to be prognostic factors in SAH.

The use of the ratio between CRY level during day and night is necessary to assess the role of circadian rhythm in SAH and is an approach that may contribute to overcome certain caveats mentioned above.

First, a higher CRYn/d would result from more remarkable sequential alterations between increase and decrease CRY level collected, respectively, during night and day, supposedly reflecting a closer to normal circadian rhythm. The use of this proportion instead of absolute values may diminish the influence of amount of bleeding on results.

Second, because CRY expression is ubiquitous, the cell type that serves as source of CRY in the CSF cannot be determined in this study protocol. A low CRYn/d may reflect low increase in CRY expression in white blood cells during night and, therefore, a lower anti-inflammatory activity. Additionally, a low CRYn/d may reflect a lower number of hypothalamus neurons in the same way that the level of the hypothalamus-produced hormones oxytocin and vasopressin is lower in the CSF of SAH cases with poor outcome (31). An indication whether the level of CRY in the CSF is altered due to less intense increase of CRY expression in white blood cells or due to brain damage may be revealed from the analysis of WBCCn/d (or alternatively of CRY expression in CSF leukocytes).

This study protocol intends to be a pilot study and the primary endpoint will be 1-year outcome defined according to modified Rankin scale as good (0–3) or poor (4–6). This option may yield a preliminary conclusion using Mann–Whitney *U*-test.

Complementary, we will analyze other functional scales, namely, Glasgow Outcome Scale and Barthel Index. Because half of the survivors after SAH displays good functional outcome measured by modified Rankin scale, but half of these patients shows neuropsychological alterations (32), we will explore this issue through the Mini-Mental Status Exam (33) and Montreal Cognitive Assessment (MoCA) (32, 34, 35). Moreover, SAH outcome studies have been taking into account analyses of healthrelated quality of life, which in our study will be evaluated through Sickness Impact Profile and Short-Form 36 (36, 37). Regarding timing, all outcome tests will be performed at discharge, and 1, 3, 6, and 12 months after bleeding.

#### POTENTIAL IMPLICATIONS OF THE HYPOTHESIS

The confirmation of the expected results would represent one more piece of evidence that circadian rhythms parallel endogenous mechanisms of brain plasticity and can be used to predict neurologic status (**Figure 1**). Regarding the study protocol we propose here, neurologic status corresponds to intracranial hypertension during the acute phase of SAH and to functional outcome in the long term.

This confirmation may underpin therapies guided by circadian rhythm modulation. A recent study showed that hCRY2 senses magnetic field (8). Magnetic field can be manipulated in clinical practice through transcranial magnetic stimulation, which interestingly increases neurogenesis (38). Perhaps transcranial magnetic stimulation improves neurologic outcome in a series of neuropsychiatric diseases through stimulation of hCRY expression and consequent neurogenesis.

Blue light stimulation is another clinical measurement that may modulate circadian rhythm. In this regard, flashing light improves Alzheimer's disease in experimental model (39). The retina is a major structure where hCRYs are expressed (10). Blue light also acts on hCRY expression in peripheral tissues. For example, jaundice improvement in children with blue light phototherapy correlates with increase in hCRY expression in mononuclear white cells (11). Therefore, blue light interferes in the level of hCRYs through central and peripheral mechanisms, and this is the reason for which it is interesting to assess hCRY expression in CSF and white blood cells in high-grade SAH patients and the correlation of this expression with TV and ICP24. In clinical practice, monochromatic phototherapy could follow the protocol described by Chen et al. who demonstrated CRY increase during treatment of jaundice in neonates (11). For example, in the first day of treatment, 20 W cool fluorescent bulbs could be used from 18:00 to 6:00 of the next day to reach 500 µW/cm2 measured by a illuminance meter. This procedure is expected to increase CRY during night (which could be monitored by further CSF collections). In the following days, light exposure could be tailored aiming at reaching a CRYn/d that further studies occasionally reveal to be associated with good outcome.

If CRYn/d or WBCCn/d correlates with TV and ICP24 and transcranial magnetic stimulation and blue light interfere in the level of hCRYs, then the endpoint of these therapies in high-grade SAH could also be the value of CRYn/d or WBCCn/d that leads to a TV higher than 0.666 and consequently to a predicted ICP24 lower than 20 mmHg (**Figure 2**). Nonetheless, it is important to emphasize that these remarks are highly speculative and the practical use of these concepts depends on the confirmation of

FIGURE 2 | Key temperature variability (TV) values and corresponding predicted mean intracranial pressure in the following 24 h (ICP24). This figure is a schematic representation of key TV values and their corresponding predicted ICP24 [for detailed explanation, please see Ref. (2)]. Numbers between parentheses correspond to 80% prediction interval. These values could guide a further circadian rhythm modulation by stimulation of hCRY expression through transcranial magnetic and blue light stimulations.

from which (A–C) were adapted; (D–F) have not been published previously.

the hypothesis explained here, the replication of the study on ICP prediction by TV, and in the improvement of the TV analysis in such a way that it includes mean daily temperature values higher than 34.9°C (**Figure 3**). In our series, days with mean daily temperature higher than 34.9°C displayed higher temperature range. In these cases, the night–day ratio of coefficient of variation (SD/ mean) was significantly different from the night–day ratio of SD, because mean night temperature over mean day temperature was not equal to one (contrarywise to what happens during hypothermia) (**Figure 3**). We are currently testing whether ICP24 can be predicted by TV during non-hypothermia periods using variables derived from the mean coefficient of variation of harmonics of 12 h obtained during day or night, similar to the formula to predict ICP24 already described (**Figure 3**).

### CONCLUSION

We have previously shown that the adult human brain harbors a potential neurogenic system whose integrity could be monitored by the analysis of circadian rhythms such as temperature oscillation. The pattern of circadian rhythms correlates with further brain injury, even under conditions in which the sleep–wake

## REFERENCES

cycle is absent and the temperature is strictly controlled. This finding led us to postulate that light-independent mechanisms of circadian rhythm control may be involved in endogenous brain plasticity.

The circadian proteins CRYs display light-independent mechanism of circadian rhythm control, can be assessed in acute severe neurologic conditions, and their expression could theoretically be modulated by blue light and transcranial magnetic stimulation.

## AUTHOR CONTRIBUTIONS

AN (first author): developed the hypothesis and wrote the paper. AN (second author): participated in previous work of the group on the paper subject, provided ideas, and revised the manuscript. JV and MT: participated in previous work of the group on this subject, provided ideas, and revised the manuscript.

## FUNDING

National Council for Scientific and Technological Development (*Conselho Nacional para o Desenvolvimento Científico e*  Tecnológico (CNPq)), grant #401002/2013-6.

*Information Across Wake and Sleep Periods*. World Intellectual Property Organization (WIPO) WO2017/007808, United States patent pending PCT/ US2016/041085 (2016).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Nogueira, Nogueira, Veiga and Teixeira. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Time courses of inflammatory Markers after aneurysmal subarachnoid hemorrhage and Their Possible relevance for Future studies

*Anke Höllig1 \*, Birgit Stoffel-Wagner <sup>2</sup> , Hans Clusmann1 , Michael Veldeman1 , Gerrit A. Schubert1 and Mark Coburn3*

*1Department of Neurosurgery, University Hospital RWTH Aachen, Aachen, Germany, 2Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany, 3Department of Anesthesiology, University Hospital RWTH Aachen, Aachen, Germany*

#### *Edited by:*

*J. Marc Simard, University of Maryland, Baltimore, United States*

#### *Reviewed by:*

*Minjee Kim, Northwestern University, United States Robert F. James, University of Louisville, United States*

*\*Correspondence: Anke Höllig<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neurocritical and Neurohospitalist Care, a section of the journal Frontiers in Neurology*

*Received: 20 July 2017 Accepted: 04 December 2017 Published: 22 December 2017*

#### *Citation:*

*Höllig A, Stoffel-Wagner B, Clusmann H, Veldeman M, Schubert GA and Coburn M (2017) Time Courses of Inflammatory Markers after Aneurysmal Subarachnoid Hemorrhage and Their Possible Relevance for Future Studies. Front. Neurol. 8:694. doi: 10.3389/fneur.2017.00694*

Object: Aneurysmal subarachnoid hemorrhage triggers an intense inflammatory response, which is suspected to increase the risk for secondary complications such as delayed cerebral ischemia (DCI). However, to date, the monitoring of the inflammatory response to detect secondary complications such as DCI has not become part of the clinical routine diagnostic. Here, we aim to illustrate the time courses of inflammatory parameters after aneurysmal subarachnoid hemorrhage (aSAH) and discuss the problems of inflammatory parameters as biomarkers but also their possible relevance for deeper understanding of the pathophysiology after aSAH and sophisticated planning of future studies.

Materials and methods: In this prospective cohort study, 109 patients with aSAH were initially included, *n* = 28 patients had to be excluded. Serum and—if possible—cerebral spinal fluid samples (*n* = 48) were retrieved at days 1, 4, 7, 10, and 14 after aSAH. Samples were analyzed for leukocyte count and C-reactive protein (CRP) (serum samples only) as well as matrix metallopeptidase 9 (MMP9), intercellular adhesion molecule 1 (ICAM1), and leukemia inhibitory factor (LIF) [both serum and cerebrospinal fluid (CSF) samples]. Time courses of the inflammatory parameters were displayed and related to the occurrence of DCI.

results: We illustrate the time courses of leukocyte count, CRP, MMP9, ICAM1, and LIF in patients' serum samples from the first until the 14th day after aSAH. Time courses of MMP9, ICAM1, and LIF in CSF samples are demonstrated. Furthermore, no significant difference was shown relating the time courses to the occurrence of DCI.

conclusion: We estimate that the wide range of the measured values hampers their interpretation and usage as a biomarker. However, understanding the inflammatory response after aSAH and generating a multicenter database may facilitate further studies: realistic sample size calculations on the basis of a multicenter database will increase the quality and clinical relevance of the acquired results.

Keywords: inflammatory response, subarachnoid hemorrhage, early brain injury, delayed cerebral ischemia, observational research

## INTRODUCTION

Early brain injury (EBI) after aneurysmal subarachnoid hemorrhage (aSAH) repeatedly has been associated with a rise of inflammatory parameters measured in serum and cerebrospinal fluid (CSF). However, results are ambiguous concerning the prognostic value of inflammatory serum or CSF parameters. Several studies have proposed single inflammatory markers to be predictive for delayed cerebral ischemia (DCI) or outcome (1–8). Thus far, due to the wide individual and interindividual range, inflammatory parameters are limited serving as biomarkers to detect complications as DCI or to predict outcome on an individual basis. However, current data are heterogeneous and partly conflicting as there is no consensus, e.g., on the compartment for sample acquisition (serum or CSF or extracellular fluid—ECF—acquired *via* microdialysis) or the time point for sampling. Neuroinflammation is a major aspect of EBI. However, it remains a challenge to differentiate beneficial from devastating inflammatory components. Furthermore, the inflammatory response to the initial injury consists of various components such as cellular reactions as in terms of microglia activation (9), induction of cytokines and chemokines (1, 8, 10), leukocyte–endothelial cell interactions (11), and modulation of receptor expression (12). A complex pattern of immunoreaction is initiated after aSAH. Besides the inflammatory activation, there are various other pathophysiological reactions, which are initiated after SAH; such as microthrombosis, cortical spreading depolarization, microvasospasm, and blood–brain barrier breakdown, altogether resulting in dysfunction of the cerebral microcirculation (13). In turn, microcirculatory dysfunction may aggravate the inflammatory activation. Thus, the interactions of the initiated reactions hamper the interpretation of a single parameter's dynamics. There is a vast amount of data on inflammatory parameters after aSAH usually in search for biomarkers predicting DCI. However, none of the examined parameters has found its way into clinical routine. This is most likely due to the abovementioned enormous individual and interindividual range and the low specificity of inflammatory parameters in general. Here, we present our data from a prospective observational study to illustrate the courses of various inflammatory parameters after aSAH. We hypothesize that monitoring of inflammatory parameters during the acute phase after aSAH does not allow an individual risk estimation (e.g., regarding the occurrence of DCI). However, detailed documentation of a vast amount of parameters may increase the understanding of the pathophysiological reaction after aSAH. Sophisticated knowledge of the pro-inflammatory reaction after aSAH and its interactions may possibly allow identification of specific anti-inflammatory therapeutic approaches.

Taken together, we aim to demonstrate that individual risk estimation for secondary complications after aSAH using inflammatory biomarkers is not promising. Instead, we vote for a multicenter database clarifying the courses and interactions of inflammatory parameters after aSAH to provide deeper insight of the pathophysiology of EBI.

## MATERIALS AND METHODS

The prospective cohort study was approved by the local ethics committee (Ethikkommission an der Medizinischen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn, Germany; EK 199/08), written informed consent according to the Declaration of Helsinki was obtained from patients or legal representatives. Data from this study have already been published elsewhere (8, 14, 15). A total of 109 consecutive patients with aSAH were screened for eligibility within a 21-month period. Patients were not eligible if they were younger than 18 years, enrolled in other clinical trials, admitted more than 12 h after onset or if informed consent could not be obtained. Due to these criteria, 28 patients were excluded (*n* = 15 declined participation or data were lost, *n* = 9 were delayed admitted, *n* = 4 participated in another trial). We documented demographic, clinical, laboratory, and radiological data within an anonymized file history. Inflammatory parameters in serum and CSF were assessed at days 1, 4, 7, 10, and 14 after aSAH. CSF was tested in patients with necessity for CSF drainage mostly due to hydrocephalus (*n* = 48). Sample acquisition regularly took place in line with routine diagnostic (between 5:00 and 6:00 a.m.).

#### Sample Processing

Blood and CSF samples were centrifuged for 10 min at 2,000 × *g* before processing. Samples were analyzed at the Department of Clinical Chemistry and Clinical Pharmacology, University of Bonn, either as part of routine diagnostics [leukocyte count (G/L), determination of C-reactive protein (CRP, mg/L), and interleukin 6 (IL6, pg/mL)] or for study purposes, by means of enzyme-linked immunofluorescence assays for determination matrix metallopeptidase 9 (MMP9, ng/mL), intercellular adhesion molecule 1 (ICAM1, ng/mL), and leukemia inhibitory factor (LIF, pg/mL) in serum samples (all assays purchased by IBL International GmbH, Hamburg, Germany).

#### Analysis

Courses of serum parameters (all patients included), additionally CSF parameters and the corresponding serum parameters of the patients with CSF drainage were plotted dependent on time of sample acquisition. Graphs were acquired using GraphPad Prism® 6. Furthermore, courses were analyzed using the Mann–Whitney *U* test. Occurrence of DCI was used as grouping variable to illustrate the usual approach in search of a biomarker indicating the occurrence of DCI. Therefore, DCI was defined as secondary neurologic worsening (increase in modified National Institutes of Health Stroke Scale >2 points). Furthermore, clinical improvement after induced hypertension and new cerebral ischemia or perfusion deficit confirmed by cranial computed tomography or magnetic resonance imaging (in case of consciousness or sedation or persistent neurologic deficit) were defined as indicators of DCI (if not explained by other causes such as embolism during angiography). Other relevant known causes such as infection, seizure, and metabolic, or electrolyte disturbances were excluded before determination of DCI. Tests were carried out using SPSS® 21.

## RESULTS

Baseline characteristics of the patients included is presented as supplemental data table (see Table S1 in Supplementary Material). Infectious complications occurred regularly with *n* = 8 (9.9%) at day 1, *n* = 27 (33.3%) at day 4, *n* = 38 (46.9%) at day 7, *n* = 36 (44.4%) at day 10, and *n* = 26 (32.1%) at day 14.

### Interleukin 6

Serum course of IL6 differs from the pattern observed in CSF samples (**Figures 1B,C**). The peak of the measured serum level represents values obtained at day 1 after aSAH, whereas course of IL6 in CSF peaks somewhat later (at day 4) (**Figure 1A**). CSF values are far higher than serum levels (up to 10-fold). Of note, confidence intervals especially for serum values are huge due to the enormous variability of individual values.

#### Matrix Metallopeptidase 9

Matrix metallopeptidase 9 peaks earlier in CSF samples (at day 4) than in serum samples (at day 7) (**Figure 2**). Contrary to IL6, MMP9 is found in patients' serum in far higher concentrations then in CSF.

#### Intercellular Adhesion Molecule 1

Intercellular adhesion molecule 1 is detectable in all of the acquired serum samples. In CSF samples, lower limit of detection (6.3 ng/mL) was not reached in 45.8% (*n* = 22) of the samples. Highest value of CSF ICAM1 expression was 56.8 ng/mL. Of note, the peak of the course is observed at day 4 after aSAH (**Figure 3**). However, variability especially at day 4 is extraordinary high.

## Leukemia Inhibitory Factor

Leukemia inhibitory factor was detected reliably in patients' CSF samples (LOD: 3.13 pg/mL). The course of LIF measured in CSF differs a lot from the one measured in serum samples (**Figure 4**): in CSF, a very early peak is observed followed by a rapid drop (**Figure 4A**). The assessed CSF levels are quite homogeneous demonstrated by low SDs. Serum levels of LIF are far lower (up to 10-fold) (**Figures 4B,C**).

### CRP and Leukocyte Count

The levels of CRP and leukocyte count were only assessed in serum samples. Of note, courses of CRP and leukocyte count act almost inversely. At day 4 after aSAH CRP levels peaks (**Figure 5**), whereas leukocyte count shows its nadir (**Figure 6**).

## Courses of Inflammatory Parameters Related to DCI

In 18 patients (22.2%), occurrence of DCI was detected (day 3: *n* = 1; day 4: *n* = 5; day 6: *n* = 2; day 7: *n* = 8; day 9: *n* = 1; day 10: *n* = 1). Comparing the courses of the assessed parameters related

Figure 1 | Time courses of interleukin 6 (IL6) in cerebrospinal fluid (CSF) samples [(A); IL6csf], serum samples of the patients with CSF samples [(B); IL6serum], and all serum samples available [(C); IL6serum all] and their 95% confidence interval are displayed. Differences between nadir and peak values assessed *via* Wilcoxon Rank Test: IL6serum: day 14 vs. day 1: *p* = 0.029; IL6csf: day 1 vs. day 4: *p* = 0.184.

Figure 2 | Time courses of matrix metallopeptidase 9 (MMP9) in cerebrospinal fluid (CSF) samples [(A); MMP9csf], serum samples of the patients with CSF samples [(B); MMP9serum], and all serum samples available [(C); MMP9serum all] and their 95% confidence interval are displayed. Differences between nadir and peak values assessed *via* Wilcoxon Rank Test: MMP9serum: day 14 vs. day 7: *p* = 0.103; MMP9csf: day 10 vs. day 4: *p* = 0.001.

Figure 4 | Time courses of leukemia inhibitory factor (LIF) in cerebrospinal fluid (CSF) samples [(A); LIFcsf], serum samples of the patients with CSF samples [(B); LIFserum], and all serum samples available [(C); LIFserum all] and their 95% confidence interval are displayed. Differences between nadir and peak values assessed *via* Wilcoxon Rank Test: LIFserum: day 14 vs. day 4: *p* = 0.081; IL6csf: day 4 vs. day 1: *p* < 0.0001.

to occurrence of DCI, no significant differences were detected (Mann–Whitney *U* test). Marginal significance was only seen for leukocyte count at day 4 (*p* = 0.054). Courses are provided in **Figure 7**.

### Courses of Inflammatory Parameters Related to Fisher Scale Score

Courses of inflammatory parameters additionally were compared related to Fisher scale score. Grading according to Fisher scale score

Figure 6 | Time courses of leukocyte count in serum samples of the patients with cerebrospinal fluid samples [(A); leukocyte countserum] and all serum samples available [(B); leukocyte countserum all] and their 95% confidence interval are displayed. Differences between nadir and peak values assessed *via* Wilcoxon Rank Test: leukocyte count: day 4 vs. day 10: *p* < 0.0001.

was available for *n* = 81 patients [Fisher 1: *n* = 1 (1.2%); Fisher 2: *n*= 13 (16%); Fisher 3: *n*= 27 (33.3%); and Fisher 4: *n*= 40 (49.4%)]. For statistical purposes, Fisher grading was dichotomized according to severity of aSAH (Fisher A: Fisher 1 and 2; Fisher B: Fisher 3 and 4). Especially course of CRP was related to Fisher grading: the measured values significantly differed related to dichotomized Fisher scale score at day 1 (*p* = 0.019), day 4 (*p* = 0.001), and day 7 (*p* = 0.033; Mann–Whitney *U* test). Significant difference was also seen for serum IL6 at day 1 (*p* = 0.006) and day 4 (*p* = 0.002), further for serum LIF at day 7 (*p* = 0.038; Mann–Whitney *U* test). All courses are displayed in **Figure 8**.

### Courses of Inflammatory Parameters Related to WFNS Scale Score

Courses of inflammatory parameters also have been related to the impact of hemorrhage expressed according to WFNS scale score, which may reflect the EBI. The score was available for *n* = 81 patients [WFNS 1: *n* = 24 (29.6%); WFNS 2: *n* = 15 (18.5%); WFNS 3: *n* = 5 (6.2%); WFNS 4: *n* = 14 (7.3%); WFNS 5: *n* = 23 (28.5%)]. Again, grading scale scores were dichotomized for statistical purposes (WFNS A: WFNS 1–3; WFNS B: WFNS 4 and 5). Particularly courses of IL6 levels were related to the impact of hemorrhage expressed according to WFNS scale score: significant differences of the values for the WFNS A vs. WFNS B group were found at day 1 (*p* = 0.001); day 7 (*p* = 0.007); day 10 (*p* = 0.010), and day 14 (*p* = 0.035; Mann–Whitney *U* test). From day 1 to 10, mean IL6 values in the WFNS A group were lower than in the WFNS B group, but at day 14 it changed to higher mean values in the WFNS A group compared with WFNS B.

Levels of CRP measured early after the impact of aSAH also were found to be related to WFNS scale score: at days 1 and 4 significantly higher values were seen in the WFNS B group

compared with the WFNS A group (day 1: *p* = 0.039; day 4: *p* = 0.034; Mann–Whitney *U* test).

No significant differences related to dichotomized WFNS scale score were found for leukocyte count, MMP9, LIF, and ICAM.

All courses are displayed in **Figure 9**.

#### DISCUSSION

Here, we illustrate the courses of several inflammatory parameters (IL6, MMP9, ICAM1, and LIF) in CSF and serum samples after aSAH, additionally courses of CRP and leukocyte count in serum. The levels of IL6 and LIF are far higher in CSF samples than in serum samples during the entire observation period, whereas MMP9 and ICAM1 were detected in higher concentrations in serum samples. The courses of CRP and leukocyte count nearly act inversely.

Our evaluation of the measured values related to the occurrence of DCI illustrated the usual problem that the wide range of the measured values hampers their interpretation and usage as a biomarker. However, understanding the inflammatory response after aSAH and generating a database may facilitate further studies: realistic sample size calculations on the basis of a multicenter database will increase the quality and clinical relevance of the acquired results.

Interleukin 6 is a pleiotropic cytokine. Its secretion is induced by various stimuli (such as infectious disease, trauma, and other causes of tissue damage, but also during chronic conditions, e.g., depression or chronic pain). There are plenty of publications dealing with the influence of IL6 secretion after aSAH on outcome and occurrence of DCI (1, 2, 8, 10, 14, 16–19). Time courses of cytokine levels in CSF provided by us are similar to those illustrated by Niwa and colleagues (10), However, others

demonstrate a later peak (1, 20). In total, schedules for sampling acquisition differ a lot among the previously mentioned studies; thus, comparison of results or courses is hampered. Anyway, wide ranges of IL6 levels are seen in all of the studies. Of note, related to WFNS scale score IL6 levels were found to be indicators for injury's severity in our study. Nevertheless, the usage of IL6 as a biomarker is hampered by its non-specificity and its ambiguous effect showing both pro-inflammatory and destructive as well as regenerative aspects.

Higher levels of MMP9 have been associated with unfavorable outcome (21). Similar to our data, SDs (i.e., variance of measured values) are pronounced, and serum levels are more than 10-fold higher than the CSF levels.

The interpretation of ICAM1 in CSF samples is hampered by the LOD of the used assay, as the LOD was not reached in 45.8% of the samples. The ICAM1 CSF levels in our cohort are somewhat lower than those presented by Kaynar and colleagues (22), serum levels are similar. Nevertheless, comparison of time course is not possible due to different schedule for sample acquisition. Mack and colleagues also have presented ICAM1 serum levels over time after aSAH (23). Time courses seem to be similar; however, serum levels measured are slightly lower.

There are no previous data on LIF after aSAH. We demonstrate an early peak in CSF samples with far higher overall levels than in serum samples.

Leukocyte count and CRP levels have been associated with prognosis as well as vulnerability for DCI (5, 21, 24–27). Both parameters are unspecific and therefore not useful as biomarkers. Anyway, pronounced leukocytosis seems to reflect severity of disease and consecutively prognosis.

We present time courses of CSF and serum inflammatory parameters after aSAH. The results of the study are limited by the fact that time courses are displayed only descriptively. Furthermore, sample size of the presented data is quite low.

However, we estimate that mere description is useful to understand the complex inflammatory response after aSAH.

Due to their marginal specificity and wide interindividual range, inflammatory parameters hardly serve as biomarkers. Nevertheless, there is a distinct inflammatory response after aSAH, which includes a vast spectrum of parameters interacting with each other. Beneficial as well as damaging stimuli may result from the inflammatory reaction. The interactions of this reaction are poorly understood. As far as our experience goes, we hold that further illustration of courses and possible interactions of inflammatory reaction after aSAH is essential before associations with outcome or occurrence of DCI in search for a biomarker are promising. Generally, setting up multicenter databases to further illustrate and hopefully understand inflammatory response after aSAH has more prospect of success than searching for a single biomarker resulting in controversial, clinically irrelevant results. Furthermore, these data may lay the groundwork for further studies' realistic sample size calculations to create clinically relevant and reliable results. Thus, we encourage everyone involved in research of inflammation after aSAH to cooperate and build up networks to share acquired data.

#### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of "Ethikkommission an der Medizinischen Fakultät der Rheinischen Friedrich-Wilhelms Universität Bonn name of guidelines, name of committee" with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the local ethics committee (Ethikkommission an der Medizinischen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn, Germany; EK 199/08).

#### AUTHOR CONTRIBUTIONS

AH conceived the study, analyzed the data, and wrote the preliminary draft. BS-W organized and supervised analysis of serum and CSF samples; assisted in interpretation of data. MV and HC participated in the analyses and helped to draft the manuscript. GS helped with data analyses and assisted the revision of the manuscript. MC participated in the design of the study and coordination and helped to draft the manuscript. All the authors read, revised, and approved the final manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fneur.2017.00694/ full#supplementary-material.

Table S1 | Baseline characteristics of patients included.

#### REFERENCES

*J Neurosurg Anesthesiol* (2006) 18(1):68–72. doi:10.1097/01.ana.0000181693. 30750.af

an observational study. *Eur J Anaesthesiol* (2016) 33(9):662–9. doi:10.1097/ EJA.0000000000000494

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Höllig, Stoffel-Wagner, Clusmann, Veldeman, Schubert and Coburn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Heparin: The Silver Bullet of Aneurysmal Subarachnoid Hemorrhage?

*Nicolas K. Khattar and Robert F. James\**

*Department of Neurological Surgery, University of Louisville School of Medicine, Louisville, KY, United States*

Various neurological diseases have recently been associated with neuroinflammation and worsening outcomes. Subarachnoid hemorrhage has been shown to generate a potent neuroinflammatory response. Heparin is a potential effective anti-inflammatory agent to prevent initial injury as well as delayed neurological decline. Different mechanisms of action for heparin have been proposed including, but not limited to the binding and neutralization of oxyhemoglobin, decreased transcription and signal transduction of endothelin-1, inhibition of binding to vessel wall selectins and vascular leakage into the subarachnoid space as well as direct binding and neutralization of inflammatory molecules. With a reasonably safe side-effect profile, heparin has shown significant promise in small series in human studies of aneurysmal subarachnoid hemorrhage in decreasing both initial and delayed neurological injury. Further studies are needed to validate various neuroprotective features of heparin in subarachnoid hemorrhage as well as other disease states.

#### *Edited by:*

*J. Marc Simard, University of Maryland, Baltimore, United States*

#### *Reviewed by:*

*Jeanne Teitelbaum, McGill University, Canada Benjamin Aaron Emanuel, University of Southern California, United States*

#### *\*Correspondence:*

*Robert F. James<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neurocritical and Neurohospitalist Care, a section of the journal Frontiers in Neurology*

*Received: 09 September 2017 Accepted: 12 February 2018 Published: 27 March 2018*

#### *Citation:*

*Khattar NK and James RF (2018) Heparin: The Silver Bullet of Aneurysmal Subarachnoid Hemorrhage? Front. Neurol. 9:97. doi: 10.3389/fneur.2018.00097*

Keywords: unfractionated heparin, neuroprotection, neuroinflammation, subarachnoid hemorrhage, delayed neurological injury, vasospasm

## INTRODUCTION

Neuroinflammation has been recently associated with worse functional outcomes in patients afflicted by a plethora of neurological diseases, but especially in aneurysmal subarachnoid hemorrhage (aSAH) (1). Inflammation has been designated as a primary cytotoxic event affecting neurons in both acute and chronic neurological diseases. This has led to specific targeting of the inflammatory cascade (2). Unfractionated heparin (UFH) is a safe and commonly used anticoagulant in the treatment of deep venous thrombosis, pulmonary emboli, and other hypercoagulable conditions. In addition to its anticoagulant properties, UFH has been shown to have very promising significant neuroprotective anti-inflammatory properties, especially in association with subarachnoid hemorrhage (3–5). Studies have shown that heparin is prevalent in various invertebrates with no hematological system, indicating that its primary physiological role is not anticoagulation (6). Even though extrapolation to other species is not evident, the role of heparin is worth investigating. In this short review, we focus on the ever-expanding connection between heparin and neuroprotection in the setting of aSHA.

### PROFILE: CHARACTERISTICS AND MECHANISM OF ACTION

Unfractionated heparin is a highly sulfated glycosaminoglycan polymer with the most negative charge of any biological molecule (7). Inflammatory mediators are generally positively charged and binding to the heparin molecule disturbs the biochemical and electrostatic microenvironment, effectively neutralizing the actions of these mediators (8). Particularly in hemorrhagic stroke, heparin is able

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to bind to oxyhemoglobin and neutralize the toxic effects of free hemoglobin on other structures in the brain (9). Given the large size and uniform distribution of the negative charge along the protein, heparin can stoichiometrically bind four molecules of oxyhemoglobin, thereby quickly neutralizing the noxious effects of the oxygen free radicals on the endothelium as well as the brain parenchyma (9).

On the molecular level, heparin was found to have various neuroprotective interactions. It is able to decrease the transcription of endothelin-1 (ET-1) and the ET-1 promoter. In addition, heparin decreased the expression of the erythroid transcription factor family (GATA)-binding capacity, which is essential for ET-1 function in the endothelial cells (10, 11). ET-1 displays significant vasoconstrictive effects mediated in vascular smooth muscle cells through the epidermal growth factor receptor (EGFR) (12). Heparin-binding epidermal growth factor, a ligand of EGFR, modulates its transactivation (12). Binding of heparin to the ligand prevents EGFR receptor transactivation and was found to dampen any peri-hemorrhagic vasoconstriction and further cerebral injury in both murine and human *in vitro* models (12, 13).

Heparin and its low-molecular weight derivatives are potent inhibitors of the adhesion molecules P- and L-selectin, which mediate leukocyte rolling, the initial event governing leukocyte transmigration from vessel walls into areas of inflammation (14, 15). These mechanisms mirror effects observed in cancer metastasis, where UFH (and not other anticoagulants) was able to decrease the speed of oncologic spread by inhibiting the selectins (15). This particular effect has been further explored in cancer research and has been shown to be potent with any type of adhesion molecules (galectins, integrins, etc.), in addition to being shown to be completely unrelated to the anticoagulant properties of heparin (16). All effects were preserved with modified 2- or 6-O desulfated, N-acetylated heparins in both the oncologic and neurovascular models (16, 17). Heparin was also found to bind all the pro-inflammatory molecules such as cytokines, chemokines, as well as mediators of inflammation such as elastase and major basic protein (18–20).

Translocation of inflammatory leukocytes into an injured area through vascular leakage is a hallmark of the initiation of the inflammatory response. Presence of leukocytes in the subarachnoid space is a specific marker of inflammation (21). The accepted mechanism of action of leukocyte extravasation is through interaction of cell glycoproteins with the selectin family of proteins, generally expressed on endothelial cells (22). Heparin has been shown to specifically decrease the number of leukocytes participating in the inflammatory response to any insult to the central nervous system (23, 24).

Prior studies have also shown that microthromboembolisms may have contributed to delayed neurological deficits, therefore possibly implicating the anticoagulation effect of heparin in the neuroprotective effects observed (25). This hypothesis has been demonstrated in experimental animal models of subarachnoid hemorrhage. Mice in the experimental group showed an increase in the number of microthrombi when compared with the sham group (26). On the other hand, the advent of modifications of heparin lacking the anticoagulation domains and showing anti-inflammatory neuroprotection provides some evidence to suggest microthromboemolisms do not represent the entire pathological picture of aSHA (27). A head-to-head comparison of both forms of heparin will need to be conducted to elucidate the roles of the various domains of heparin. The various proposed mechanisms of action of UFH are summarized in **Table 1**.

### NEUROPROTECTIVE EFFECTS OF HEPARIN IN SUBARACHNOID HEMORRHAGE

Some authors hypothesize that a combination of the hemorrhage volume, vasoparalysis, and decreased cerebrospinal fluid resorption associated with aSHA triggers an increase in intracranial pressure leading to significant transient ischemia. This has been considered by various authors to be the cause of the initial neurological injury following the ictus, especially when associated with loss of consciousness at presentation (28, 29). Heparin and its nonanticoagulant derivatives have been shown to effectively counter the initial injury in various rat models of ischemia/reperfusion mimicking the initial insult following aneurysmal rupture (27, 30). The caspase pathway is triggered in the initial phase of the injury of subarachnoid hemorrhage, especially given that activation of caspase 1 was first observed in ischemic stroke (31). Countering the early activation of caspases 1, 3, 8, 9, and 11 in global cerebral ischemia can prevent neurological devastation. Animal studies demonstrated a significant decrease in cleaved caspase 3 in samples obtained from subarachnoid hemorrhage animals that were

Table 1 | Mechanisms of action of heparin to prevent delayed neurological injury associated with subarachnoid hemorrhage.

*Direct chelation of hemoglobin in the subarachnoid space*

– Heparin binds oxidized hemoglobin that is released from damaged erythrocytes. Oxyhemoglobin is believed to have a major role in the induction of vasospasm

*Decreased free-radical release*

– Heparin is able to directly bind to specific molecules and inhibit the formation of free radicals through the inhibition of various pro-inflammatory molecules that contribute to their formation

*Inhibition of endothelin-1 (ET-1)*

*Prevention of K+ channel down-regulation induced by oxyhemoglobin release*

– Downregulation of potassium channels causes a depolarization of vascular smooth muscle cells, increased incidence of calcium influxes and increased activation causing increased vasoconstriction and neurological decline

*Suppression of vascular smooth cell hyperplasia*

– Smooth muscle and myofibroblast proliferation, associated with cell necrosis, lead to increased vasoconstriction, ischemia, and further neurological decline. Pathological proliferation of smooth muscle cells and neovascularization may prevent further progression of neurological injury

*Inhibition of neuroinflammatory pathways*

given heparin pharmacotherapy. The downregulation of apoptotic effectors leads to decreased neuroinflammation, demyelination, and decreased burden of injury (32).

The evidence supporting heparin's anti-inflammatory role is further supported with evidence from traumatic brain injury (TBI) and chronic neurodegeneration research. Nagata et al. has shown that early administration of heparin is associated with a significant decrease in post-TBI inflammation and preservation of cognitive outcomes. These results were further supported by prior studies investigating the role of low-molecular weight fractionated heparin, enoxaparin (2, 23, 33–35). Human studies investigated the role of enoxaparin (low-molecular weight fractionated heparin) in aneurysmal SAH with mixed results (36, 37). There was a statistically significant reduction in delayed ischemia and vasospasm in the enoxaparin group in one trial but no obvious benefit in others (36, 37). A retrospective cohort study showed significant benefits of UFH in Fisher grade 3 subarachnoid hemorrhage patients (4). The patients in the heparin group had significantly less clinical and radiographic vasospasm, as well as a decrease in vasospasmrelated infarction. In addition, there were a significantly higher proportion of patients who were discharged home from the hospital, instead of having to be discharged to a rehabilitation facility (4). Human studies highlighting the neuroprotective effects of heparin in various disease states are summarized in **Table 2**.

In addition to the transient ischemia state associated with SAH, significant vasogenic edema and blood–brain barrier dysfunction plague this patient population and represent a dismal prognostic factor (39). Heparin has been shown to counter cerebral edema in general in various pathological states including but not limited to TBI, meningitis, ischemia, and intracerebral hemorrhages (23, 40–43). Various interactions in animal models have been reported between heparin and molecules specifically associated with cerebral edema (VEGF, bradykinin, etc.) but no proven connection linking the action of heparin on these molecules was proven.

These neuroprotective effects have been shown to correlate with a decrease in clinical vasospasm and a specific decrease in the delayed neurological injury, namely long-term cognitive decline following subarachnoid hemorrhage (32, 44, 45). Despite the fact that the number of patients included in these series remains limited; there is a positive trend and a budding interest in a closer

#### REFERENCES

Table 2 | Human studies showing neuroprotection of heparin in various neurological injuries.

*Prevention of delayed neurological injury following aneurysmal subarachnoid hemorrhage*

*Prevention of neurological sequelae following traumatic brain injury (TBI)*

correlation of heparin with improved cognitive outcomes. One such effort is the Aneurysmal Subarachnoid hemorrhage Trial RandOmizing Heparin, a Phase II multi-center randomized trial, studying the effects of low-dose intravenous heparin infusion on 90-day cognitive outcomes using the Montreal Cognitive Assessment test. Enrollment is expected to be complete in December 2018 (NCT02501434).

#### CONCLUSION AND FUTURE DIRECTIONS

Heparin is today a venerable, ever-young drug that has not ceased to bewilder and amaze. With its seemingly ubiquitous properties, heparin seems to show promise in various neuropathologies, with a predictable and manageable side-effect profile. Further research is needed to establish heparin as a completely safe and effective intervention in patients with subarachnoid hemorrhage. Together, with its success in other experimental neurological insults such as TBI, stroke, meningitis, cancer, etc., heparin seems to be emerging as a potential silver bullet for mitigating the delayed neurological injury commonly seen after aSHA.

#### AUTHOR CONTRIBUTIONS

RJ designed the concept of the review. NK conducted the review, compiled the information, and drafted the initial manuscript. Both authors critically reviewed and approved the final version of the manuscript.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Khattar and James. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Neuroinflammation as a Target for Intervention in Subarachnoid Hemorrhage

#### *Airton Leonardo de Oliveira Manoel1,2\* and R. Loch Macdonald3*

*1Adult Critical Care Unit, Hospital Paulistano – United Health Group, São Paulo, Brazil, 2Keenan Research Center for Biomedical Science, Department of Surgery, Li Ka Shing Knowledge Institute, University of Toronto, Toronto, ON, Canada, 3Division of Neurosurgery, St. Michael's Hospital, Labatt Family Centre of Excellence in Brain Injury and Trauma Research, Keenan Research Centre for Biomedical Science, Department of Surgery, Li Ka Shing Knowledge Institute, University of Toronto, Toronto, ON, Canada*

Aneurysmal subarachnoid hemorrhage (SAH) is a sub-type of hemorrhagic stroke associated with the highest rates of mortality and long-term neurological disabilities. Despite the improvement in the management of SAH patients and the reduction in case fatality in the last decades, disability and mortality remain high in this population. Brain injury can occur immediately and in the first days after SAH. This early brain injury can be due to physical effects on the brain such as increased intracranial pressure, herniations, intracerebral, intraventricular hemorrhage, and hydrocephalus. After the first 3 days, angiographic cerebral vasospasm (ACV) is a common neurological complication that in severe cases can lead to delayed cerebral ischemia and cerebral infarction. Consequently, the prevention and treatment of ACV continue to be a major goal. However, most treatments for ACV are vasodilators since ACV is due to arterial vasoconstriction. Other targets also have included those directed at the underlying biochemical mechanisms of brain injury such as inflammation and either independently or as a consequence, cerebral microthrombosis, cortical spreading ischemia, blood–brain barrier breakdown, and cerebral ischemia. Unfortunately, no pharmacologic treatment directed at these processes has yet shown efficacy in SAH. Enteral nimodipine and the endovascular treatment of the culprit aneurysm, remain the only treatment options supported by evidence from randomized clinical trials to improve patients' outcome. Currently, there is no intervention directly developed and approved to target neuroinflammation after SAH. The goal of this review is to provide an overview on anti-inflammatory drugs tested after aneurysmal SAH.

Keywords: neuroinflammation, subarachnoid hemorrhage, early brain injury, secondary brain injury, vasospasm, delayed cerebral ischemia

## INTRODUCTION

Aneurysmal subarachnoid hemorrhage (SAH) is a complex cerebrovascular disease with profound systemic complications (1–3). Despite the advances achieved in the management of those patients, high disability and mortality rates continue to devastate this patients' population (4).

Aneurysm rebleeding is the most threatening early neurological complication and usually occurs in the first 24 h after the initial hemorrhage (5, 6). However, delayed cerebral ischemia (DCI) remains the major cause of morbidity and mortality among patients who survive after repair of the ruptured aneurysm (7, 8).

#### *Edited by:*

*Fernando Testai, University of Illinois at Chicago, United States*

#### *Reviewed by:*

*Adel Helmy, University of Cambridge, United Kingdom Elisa R. Zanier, Istituto Di Ricerche Farmacologiche Mario Negri, Italy*

#### *\*Correspondence:*

*Airton Leonardo de Oliveira Manoel<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neurocritical and Neurohospitalist Care, a section of the journal Frontiers in Neurology*

*Received: 15 February 2018 Accepted: 16 April 2018 Published: 02 May 2018*

#### *Citation:*

*de Oliveira Manoel AL and Macdonald RL (2018) Neuroinflammation as a Target for Intervention in Subarachnoid Hemorrhage. Front. Neurol. 9:292. doi: 10.3389/fneur.2018.00292*

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For decades, angiographic cerebral vasospasm (ACV), a common neuroradiological finding after SAH, has been proposed as the primary cause of DCI and DCI-related cerebral infarction (9). In turn, severe angiographic vasospasm can cause DCI and DCI-related cerebral infarction, which made the prevention and treatment of ACV a major target in the management of SAH. Crowley et al. analyzed 381 patients who were part of a randomized clinical trial and all of whom underwent serial CT scanning and angiography (10). Angiographic vasospasm was graded as none/mild (209, 55%), moderate (118, 31%), or severe (54, 14%). Delayed cerebral infarctions occurred in 3% of those with no/mild, 10% with moderate, and 46% with severe vasospasm. The data also show that a minority of patients can develop DCI without ACV (10). Angiographic vasospasm occurs in approximately 70% of patients during the first 2 weeks after SAH, but the incidence of DCI is only around 30% (7). Some patients with severe ACV do not develop ischemia or infarction for the same reasons that a patient with a carotid artery occlusion may not, which includes (a) the recruitment of collateral blood flow through the anterior and posterior communicating arteries (i.e., the primary collateral pathway), or through the ophthalmic artery and leptomeningeal vessels (i.e., the secondary collateral pathway) (11, 12) and (b) the cerebral vasoreactivity status (i.e., intact versus impaired) (13). In addition, patients may develop cerebral infarction in vascular territories that are not affected by ACV (14, 15). Moreover, enteral nimodipine is the only pharmacological treatment shown to improve functional outcome, despite the fact that it does not lead to marked cerebral vasodilation (16, 17).

Also, a meta-analysis that included 14 randomized controlled trials in SAH, involving 4,235 patients, showed that the tested drugs significantly reduced angiographic vasospasm, but clinical outcomes remained unaffected (18).

Because of the failure of several pharmacological trials and the dissociation between vasospasm-related morbidity and functional outcomes, other possible mechanisms, such as early brain injury, cortical spreading ischemia, microthrombosis, cerebral autoregulation impairment, and capillary transit time heterogeneity, are thought to play a role in the pathophysiology of DCI and DCI-related cerebral infarction (1, 3, 19, 20). These additional mechanisms tend to be more difficult to assess clinically and the dissociation mentioned above could also be due to drug toxicity and off-target effects, effects of rescue therapy, inadequate trial sample size and other trial design problems, and testing of only one dose and dose regimen.

Another common biochemical mechanism after brain injury is neuroinflammation, which may also contribute to many of the causes of brain damage after SAH. Subarachnoid blood and subsequent hemoglobin degradation can in turn trigger the inflammatory cascade, which also develops in the brain probably secondary to ischemia, blood–brain barrier breakdown, and such. There is an increasing interest in the understanding of the role of neuroinflammation in the pathophysiology of DCI and other severe complications after SAH.

The main goal of this review article is to summarize the concept of neuroinflammation following SAH, and its potential contribution to ACV, DCI, and systemic complications. We also address the possible benefits of targeting the inflammatory cascade after aneurysmal SAH. Finally, we review studies of drugs directed at neuroinflammation after SAH (**Table 1**).

#### SEARCH STRATEGY

A PubMed search was performed from inception to November 2017 for articles published in English by using the terms "Subarachnoid Hemorrhage" [Mesh] AND ("Neuroinflammation" [Title/Abstract] OR "inflammation" [Title/Abstract]). A total of 272 articles were found, including 155 articles on human subjects. The authors' own databases additionally were used as a source for this review article. This review was limited to studies in humans, because the literature on experimental animal models is vast, and it is beyond the scope of this article.

#### DCI and Angiographic Vasospasm

The majority of patients (approximately 70%) develop some radiological degree of vasospasm between 3 and 14 days after a single SAH (9). However, only 30% of those will progress to develop DCI and even fewer to delayed cerebral infarction (15, 34). While ACV is an important contributor to DCI, there is evidence that other processes are involved (1, 3, 35–38).

Two major neurological consequences of aneurysmal SAH are currently described, early brain injury and ACV/DCI. Early brain injury refers to the acute effects of aneurysm rupture. Arterialized blood flows into the subarachnoid space, which suddenly increases the intracranial pressure. Consequently, the cerebral perfusion pressure is acutely reduced leading to a reduction in cerebral blood flow that produces global cerebral ischemia. Clinically, the phenomenon is characterized by transient loss of consciousness that can progress to intracranial circulatory arrest in severe cases (39, 40). There also are probably effects from the subarachnoid blood as well as brain injury from other physical and biochemical processes. Ischemia with its associated cell death causes influx of inflammatory cells, activation of microglia and neuroinflammation. Patients who survive the initial hemorrhage are at risk of DCI that is the main determinant of unfavorable outcome after SAH (41). DCI is a clinical syndrome characterized by "the occurrence of focal neurological impairment (such as hemiparesis, aphasia, apraxia, hemianopia, or neglect) and/or a decrease of at least two points on the Glasgow coma scale (either on the total score or on one of its individual components, such as eye, motor on either side, or verbal) (7). This should last for at least 1 h, is not apparent immediately after aneurysm occlusion, and cannot be attributed to other causes by means of clinical assessment, computed tomographic, or magnetic resonance imaging of the brain and appropriate laboratory studies". The pathophysiology of DCI is multifactorial and remains to be completely elucidated, however, it is hypothesized that ACV is one if not the primary contributor, along with cortical spreading ischemia, impaired cerebral autoregulation and microcirculation constriction, microthrombosis, capillary transit time heterogeneity, and neuroinflammation (1, 3, 35–38, 42). The risk of DCI is increased by the amount of subarachnoid blood (i.e., the volume, density, and persistence of blood in the subarachnoid space) (43–45);

Frontiers in Neurology

by the initial level of consciousness (i.e., early brain injury) (46, 47); and by factors that alter the relationship between the brain oxygen/glucose supply and demand (1).

### Inflammation as an Additional Contributor to DCI

There is recent evidence suggesting that neuroinflammation may play an important role in the damage of cerebral cells after SAH (42, 48). The products of erythrocyte degradation in the subarachnoid space lead to the accumulation of hemoglobin and its products (i.e., methemoglobin, heme, and hemin), which activate toll-like receptor 4, initiating the inflammatory cascade. Microglia, the resident inflammatory cells in the central nervous system, are activated, which triggers the upregulation of a large number of endothelial adhesion molecules, allowing the inflammatory cells to reach the subarachnoid space (49). Macrophages and neutrophils, once in the subarachnoid space, start the process of phagocytosis of the product of degraded red cells, with the attempt of removing the extravascular hemoglobin, the process of which may promote neuronal healing (50). The clearance of hemoglobin from subarachnoid space depends on and is accelerated by the ligation of hemoglobin to haptoglobin. The complex hemoglobin/haptoglobin is than phagocyted by immune cells (51).

Macrophages and neutrophils are indispensable for the clearance of subarachnoid blood, however, they may become imprisoned within the subarachnoid space after the eventual reestablishment of the blood–brain barrier, and also because of the changes occurring in the cerebral spinal fluid (CSF) flow. The main problem with the entrapment of inflammatory cell in the subarachnoid space is that they suffer degranulation, releasing several inflammatory and vasoactive factors, such as endothelins. Also, these inflammatory and vasoactive factors are not restricted to the subarachnoid space, but generalized throughout the central nervous system, inducing cerebritis, aseptic meningitis, and cerebral vasoconstriction (50).

### Monitoring Inflammation After SAH

Several methods exist to monitor systemic and neurological inflammation in the patients with SAH. They include classic systemic signs of inflammation such as fever, leukocytosis, and systemic inflammatory response (SIRS), as well as the evaluation of pro- and anti-inflammatory cytokines in the peripheral blood, CSF, and cerebral extracellular fluid (52).

Increased levels of inflammatory cytokines are found in the CSF, cerebral extracellular fluid, and blood of patients with SAH (53). Two sets of authors reviewed published data reporting biomarkers for assessing outcome after SAH (54, 55). Neuron and astrocyte proteins, cell adhesion proteins, and extracellular matrix molecules, vascular, coagulation, and cardiac proteins have all been measured in various studies of CSF and blood after SAH. Inflammatory substances in blood that have been shown to be elevated after SAH included C-reactive protein (CRP), tumor necrosis factor α (TNF-α), and interleukin-1 receptor antagonist (IL-1Ra) and IL-6 (IL-6). Elevated levels of TNF-α, IL-1Ra, IL-6, and IL-8 were also described in the CSF (56–58). IL-1Ra concentrations were measured in blood and were significantly higher after SAH than in controls, in poor-grade SAH patients compared to good grades and in patients who had unfavorable as compared to favorable outcome (56). DCI further increased the concentration of IL-1Ra.

In one study, CSF TNF-α concentrations increased between 4 and 10 days after SAH in patients with unfavorable outcome whereas they did not if there was favorable outcome (56). The same rough correlation of increased CSF TNF-α and unfavorable outcome was reported by other investigators (59). Similarly, CSF levels of IL-6 and IL-8 were shown to be significantly elevated in SAH patients compared to controls, and this elevation was associated with the development of symptomatic vasospasm between day 5 and 7 (58).

Subarachnoid increases in IL-1, IL-6, and TNF-α may precede the hemodynamic abnormalities detected by transcranial Doppler and to be associated with the development of severe ACV diagnosed by transcranial Doppler ultrasound.

Elevated IL-6 levels also may be an early marker of DCI and unfavorable outcome. Muroi et al. showed in a cohort of 138 SAH patients that early elevation of IL-6 (days 3–7) was significantly associated with the occurrence of DCI and unfavorable outcome, after the adjustment for confounding factors such as infection and therapeutic hypothermia (60). In addition, elevated IL-6 levels detected in the cerebral extracellular fluid by cerebral microdialysis in patients of all grades predicted the occurrence of DCI in one study (61).

Complement system is another important component of inflammatory response after SAH. Complement activation and the formation of membrane attack complex contribute to the development of cerebral edema and the disruption in the blood– brain barrier. Interestingly, the process of complement activation and membrane attack complex formation is also implicated in the inflammation and degradation of the cerebral aneurysm wall, with consequent aneurysm rupture (62).

The complement activation in association with the formation of membrane attack complex may contribute to the development of angiographic vasospasm (63). In one study investigated the role of sequential measurement of serum complements (i.e., CH50, C3, C4) in the first 3 weeks of hemorrhage. Hunt and Hess grade was associated with C4 levels, and C4 levels were also markedly reduced in patients with severe angiographic vasospasm with neurological deficits (64).

Although, the measurement of cytokines and their receptors in the CSF, cerebral extracellular fluid, and blood seem to be feasible, their use remains experimental. Also, inflammation is the fundamental response of tissues to injury and is generally required for tissue healing. There are situations where inflammation is detrimental. Therefore, it is going to be very complex to determine which aspect of cerebral inflammation is beneficial or harmful (52). More adequate, well controlled and thus basically prospective multicenter studies would be needed before we could draw conclusions on the clinical utility of inflammatory biomarkers (53).

### Inflammation, Brain Injury, and Systemic Complications

There is systemic immune response activation after SAH and it is commonly manifested by high levels of circulating cytokines, such as IL-1, IL-6, and TNF-α (65). These are key mediators of systemic inflammation. Clinically, this process is usually manifested by fever, leukocytosis, tachycardia, and tachypnea. These are components of SIRS, which has been defined by the presence of two or more of the following: temperature <36 or >38°C, heart rate >90 bpm, respiratory rate of >20 breaths/ min, and white blood cell count of <4,000 or >12,000/mm3 (66, 67). In 2 reports that included 276 and 413 patients with SAH, respectively, SIRS was present on admission in over half and in 63–85% within 4 days (68–70). Its occurrence was associated with poorer neurological grade, larger amount of subarachnoid blood on computed tomography. In one of the two studies, it was an independent predictor of angiographic vasospasm, systemic complications, unfavorable outcome, and death.

Systemic inflammatory response is a relatively nonspecific clinical syndrome that reflects a complex interaction among inflammation, coagulation, sympathoadrenal activation, and endothelial cell activation and dysfunction (71). This complex process generates and perpetuates tissue hypoperfusion, ultimately culminating with microthrombosis and compromised microcirculation blood flow, which is manifested by the multiorgan dysfunction syndrome (71, 72). In addition, catecholamines released into the systemic circulation after SAH may induce cardiac and pulmonary dysfunction (i.e., myocardial stunning and neurogenic pulmonary edema) (73, 74). Sympathoadrenal activation, represented by high circulating catecholamine levels, has also been shown to be highly associated with coagulopathy, endotheliopathy, and functional outcome in patients with isolated traumatic brain injury (75, 76).

Other indirect surrogate markers of systemic inflammatory activity after SAH include hyperthermia, elevated white blood cell count, hyperglycemia, high erythrocyte sedimentation rate, high CRP, transthyretin, and negative nitrogen balance (60, 64, 66, 72–82). Those systemic inflammatory parameters are associated with angiographic vasospasm, DCI, and unfavorable outcome (66, 68).

### Platelet Activation and Inflammation After SAH

Inflammation is intimately related to platelet adhesion, activation of coagulation cascade, and consequently the formation of microthrombi (82). Injury to a blood vessel leads to endothelial platelet adhesion, which triggers the coagulation cascade. Thrombin, von Willebrand factor, and collagen are capable of activating the platelets, which release a large number of procoagulant factors, responsible for the development of a hemostatic clot (82–84).

In SAH patients, microthrombosis was described in an autopsy study of seven patients who died within 3 days of SAH (85). The density of thrombi (microclot burden) was associated with clinical and radiological evidence of delayed ischemia. Also, microclot burden was significantly associated with histological evidence of ischemia.

Additional evidence for microthromboembolism includes a prospective study of SAH patients monitored with transcranial Doppler (86). Microembolic signals were detected in 16 of 23 patients (70%), and 44 of 138 arteries (32%) monitored. Microembolic signals were more common in patients with compared with patients without clinical vasospasm (83 versus 54%). These data at least show an association between inflammation, activation of coagulation cascade and formation of microthrombi after SAH and may be an additional component of DCI (83).

Inflammation and platelet activation have been associated with both early brain injury and DCI after SAH (82, 84). Frontera et al. (82), in a prospective study enrolled 106 consecutive SAH patients, comparing them with 26 control subjects. Compared to controls, SAH patients demonstrated higher levels o inflammatory biomarkers (i.e. higher levels of C-reactive protein), and higher levels of platelets activation by thromboelastography. Platelet activation and inflammation were related to the severity of early brain injury, and it was also associated with the development of DCI and unfavorable functional outcome (82).

Interestingly, nimodipine has been shown to increase endogenous fibrinolysis, which may partially explain the benefit of nimodipine use by the reduction in microthrombosis after SAH (87). However, the exact mechanism by which nimodipine exerts its beneficial effects remains to be determined, but its probably multifactorial, including fibrinolysis, inhibition of cortical spreading ischemia, and the blockage of calcium influx to neurons after ischemia (1, 20).

### Antiplatelet Drugs After SAH

Cyclooxygenase (COX) enzymes are fundamental for the balance of vascular homeostasis (88). Thromboxane A2 (TxA2) is released by platelets as a result of COX-1 activity, and prostacyclin is synthetized mainly in vascular endothelium by COX-2 (**Figure 1**). TxA2 induces platelet aggregation, vasoconstriction, and smooth muscle proliferation, while prostacyclin antagonizes TxA2 effects in the macrovascular endothelium through smooth muscle relaxation and vasodilator effects. Prostacyclin also inhibits platelet aggregation through prostacyclin receptors.

Acetylsalicylic acid [aspirin, a non-steroidal anti-inflammatory drug (NSAID)] inhibits cyclic prostanoid synthesis (TxA2, prostacyclin and other prostaglandins, **Figure 2**). The mechanism of its antithrombotic effect is by irreversible acetylation of COX-1, an enzyme constitutively expressed platelets and most other cells. This inhibits platelet aggregation. Its anti-inflammatory effects are due to inhibition of COX-2, which is induced by inflammation and generates inflammatory prostaglandins.

Dorhout Mees et al. conducted a meta-analysis of seven randomized clinical trials (1,385 patients) that evaluated the effect of antiplatelet drugs on outcome, case fatality, secondary ischemia, hemorrhagic intracranial complications, and aneurysm rebleeding in patients with SAH (28). The studies tested different drugs and regimens, including suppositories or oral aspirin, oral ticlopidine, oral or intravenous dipyridamole, continuous infusion of OKY-046, and continuous infusion of cataclot (90–94). There was no effect of the antiplatelet drugs on any of the outcome measures. Ticlopidine was associated with a significant reduction

constitutively expressed in many cells and COX-2 that is induced by inflammatory stimuli.

in poor outcome but this was based on one randomized trial of 135 patients (95).

#### Steroids for SAH

Corticosteroids are anti-inflammatory. They inhibit the formation of adhesion molecules, arachidonic acid metabolites, cytokines, and chemokines. The mechanism of action that accounts for their anti-inflammatory effects is direct binding of the glucocorticoid to the glucocorticoid receptor, forming a glucocorticoid/glucocorticoid receptor complex. This is transported in to the nucleus where it binds to glucocorticoid responsive elements in the promoter region of genes, or interacts with other transcription factors, in particular activating protein-1 or nuclear factor-κB (96).

Several different types doses and dose regimens of corticosteroids have been studied in patients with SAH. Overall, the studies show mixed results and sample sizes are too small to draw conclusions among other shortcomings of the studies (23–25). The largest study was a retrospective cohort that included 309 patients, 101 (33%) who received dexamethasone. Dexamethasone significantly reduced in the odds of unfavorable outcome although it had no effect on DCI or the rate of infections (26). Glucocorticoids are not recommended for routine use in patients with SAH.

Important to mention is that a large randomized clinical trial, including more than 10,000 patients with moderate-to-severe traumatic brain injury, compared the effect of 48 h infusion of methylprednisolone with placebo. The risk of death from all causes was increased in the intervention group (97, 98). The exact cause of worse outcome with the use of steroids after traumatic brain injury is not well established.

#### Immune-Suppressive Treatment in SAH

There are at least two studies that tested the use of immunesuppressive treatment in SAH. The first study was a randomized clinical trial, which included 25 patients (22). Nine received the intervention (cyclosporine A orally 6–9 mg/kg/day to maintain level of cyclosporine in the blood at 100–400 ng/ml). Patients treated with early clipping (up to 72 h after SAH) plus cyclosporine A had significantly better neurological outcome. The second study treated nine SAH patients with Fisher Grade 3 SAH with cyclosporine A (21). Despite the small number of patients, cyclosporine A was considered to be safe but to not prevent ACV or DCI.

Many SAH patients admitted to the critical care unit are exposed to invasive procedures and catheter placement, such as mechanical ventilation, external ventricular drain, invasive intracranial monitors, and vascular catheters. These invasive tools are fundamental for the management of poor-grade SAH patients (1), however, they may increase the risk of hospital-acquired infections (99). Therefore, the use of immunosuppressive treatment, including the use of glucocorticoids, might increase the risk of infection in this patient population, which increases the hospital morbidity and mortality.

#### NSAIDs for SAH

Non-steroidal anti-inflammatory drugs inhibit COX (88). Traditionally they inhibited COX-1 and COX-2 although there are some specific COX-2 inhibitors. Thus, they inhibit inflammation mediated by COX-2. While aspirin is the classic NSAID, the effects differ between the newer NSAIDs and from aspirin probably due to the relative potencies against COX-1 and COX-2 and their effects on other targets. For example, ibuprofen reduces the expression of adhesion molecules by the endothelium, which in turn decreases the inflammation in the subarachnoid space. In a propensity score-matched study, Nassiri et al. studied 178 patients with SAH who were matched by propensity scoring into 89 who received NSAIDS and 89 who did not (29). Use of NSAIDs was associated with lower in-hospital mortality and shorter intensive care and hospital stay. There was, however, no significant difference in functional outcome, in the development of delayed ischemic neurological deficits, angiographic vasospasm or need for rescue therapy. Muroi and colleagues found that NSAID use was associated with lower systemic IL-6 and CRP concentrations and with better outcome in 138 patients with SAH (100). There is at least one randomized clinical trial of NSAIDs in 81 patients with SAH (101). Meloxicam had no significant effect on ACV or clinical outcome compared to placebo. Currently, the treatment of SAH patients with NSAIDs cannot be recommended.

#### Other Potentially Anti-Inflammatory Drugs

Several other medications with anti-inflammatory activity have been tested in patients with SAH (**Table 1**). The statins are the best studied and have been completely ineffective at least in the doses and dose regimens studied (102). Albumin has multiple systemic and cerebral effects, including antioxidant and scavenger properties; the capacity to modulate cellular apoptosis; enhance of microcirculatory and organ blood flow; and an antiinflammatory effect through the decrease in leukocyte rolling and adherence. Albumin has been studied in a dose-escalation, Phase I, pilot study (33). Doses up to 1.25 g/kg/day × 7 days were well tolerated, with a trend toward better 3-month functional outcome. A Phase III placebo control trial will be carried out in the near future.

### CONCLUSION

There is an increasing interest in the understanding of the role of neuroinflammation in the pathophysiology of early brain injury and DCI after SAH. Inflammatory biomarkers are associated with the occurrence of ACV, DCI, and unfavorable outcome. However, the use of anti-inflammatory agents was studied in only a small numbers of subjects. Acetylsalicylic acid, other NSAIDs, thromboxane synthase inhibitors, steroids, nitric oxide donors, and immunosuppressant therapies have not shown beneficial clinical effects. On the other hand, none have been studied in enough detail or in adequate, well-controlled clinical trials to reach a definitive conclusion about safety and efficacy. Currently, there is no intervention directly developed and approved to target neuroinflammation after SAH, therefore anti-inflammatory treatments are not suggested in SAH, at least in the doses and dose regimens studied.

### AUTHOR CONTRIBUTIONS

AM wrote the first version of the manuscript. RM made crucial extensive revisions.

#### FUNDING

RM receives grant support from the Brain Aneurysm Foundation, Canadian Institutes for Health Research, Genome Canada, and the Ontario Genomics.

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**Conflict of Interest Statement:** RM receives grant support from the Brain Aneurysm Foundation, Canadian Institutes for Health Research, Genome Canada, and the Ontario Genomics Institute; and he is Chief Scientific Officer of Edge Therapeutics, Inc. The other author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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