Introduction
Subarachnoid hemorrhage (SAH) is associated with a disproportionately elevated morbidity and mortality. The overall case fatality is estimated in 40 to 50% with almost 10 to 15% of the patients dying before reaching medical attention [
1,
2]. Of the survivors, 46% may have long-term cognitive impairment and 30% require lifelong supportive care [
3,
4]. This condition often affects young adults and may be responsible for almost a quarter of all the years lost because of stroke [
5]. Despite intense research, progress toward the development of effective treatments for SAH has been disappointingly slow. Nimodipine remains the only pharmacological treatment for this condition. The beneficial effect of nimodipine, however, is limited and its use is often restricted by the occurrence of hypotension [
6]. New and effective treatments for SAH are largely needed. Studies performed over the past decade have yielded a preponderance of evidence supporting a link between inflammation and stroke outcome [
7-
10]. Inflammation plays an important role in brain damage by contributing to neural injury, diminished vascular reactivity, microthrombosis, and enhanced blood brain barrier (BBB) permeability [
7,
8,
11,
12]. These observations support immunomodulation as a potential target for intervention following SAH. Fingolimod (FTY720) is an immunomodulatory agent has been approved by the Food and Drugs Administration (FDA) for the treatment of multiple sclerosis. Fingolimod is a sphingosine-1-phosphate (S1P) analog that crosses the BBB and regulates critical cellular processes including proliferation, apoptosis, endothelial barrier permeability, and inflammation [
13,
14]. Studies performed in association with cerebral ischemia and intraparenchymal hemorrhage have shown that FTY720 reduces stroke-related neuroinflammation, brain edema, and neuronal death and improves neurological outcome [
15-
17]. Thus, we hypothesized that fingolimod would be efficacious in the treatment of SAH. To that end, we investigated the effect of FTY720 in a rat model of SAH. In particular we addressed leukocyte adhesion to pial venules, microvascular (pial arteriolar) dilating reactivity, and neurological outcome.
Discussion
In this study we demonstrated that the use of fingolimod was associated with a restricted leukocyte adhesion to pial vessels, preserved arteriolar vasodilation responses, and improved neurological function after SAH.
FTY720 has been used with success in models of cerebral ischemia and parenchymal hemorrhage [
15-
17]. This, however, is the first study addressing the protective effect of this drug in animals subjected to SAH. The dose of FTY720 is in agreement with studies done in non-SAH models [
15,
17,
23,
24]. SAH is characterized by acute headache resulting in patients seeking early medical attention. Therefore, the 3 hours post-SAH treatment time point constitutes a therapeutic window similar to that encountered in clinical practice.
FTY720 is an agonist of S1P receptors type 1, 3, 4 and 5 which are ubiquitously distributed in cells of different lineages. Fingolimod induces the internalization and subsequent ubiquitination and proteosomal degradation of S1P-receptor type 1 on lymphocytes, which are retained in lymph organs [
25]. In our study, FTY720 decreased circulating lymphocytes with virtually no effect on neutrophils, an observation that is consistent with the known mechanism of action of this drug [
25,
26]. FTY720 has a high volume of distribution and its half-life is of approximately 9 to 10 days [
14]. Thus, it was not unexpected that a single dose of this drug led to an immunosuppressive state that persisted for at least 48 hours. In addition, the treatment with FTY720 restricted the adhesion of leukocytes to pial vessels and improved neurological outcome after SAH. The role of leukocytes in SAH is not well known. Immediately after SAH, there is a rapid increase in the ICP leading to transient global hypoperfusion with subsequent restoration of the cerebral blood flow [
27]. In this context, SAH has some similarities with transient cerebral ischemia. Studies performed in ischemic models illustrate that tissue hypoxia upregulates the expression of adhesion molecules that facilitate the interaction of circulating leukocytes with vascular elements. These release vasoactive and pro-inflammatory mediators that contribute to brain damage [
10,
28,
29]. In SAH, the initial insult induces an inflammatory state characterized by early recruitment of leukocytes and increased production of cytokines and chemokines in both plasma and CSF [
9,
12,
30]. This immune response correlates with SAH severity and has been linked to neural cell injury, BBB dysfunction and delayed cerebral ischemia [
31-
33]. Accordingly, immunomodulation has emerged as a potential treatment strategy that could ameliorate brain injury and improve outcome after SAH. Our previous findings indicate that blocking leukocyte adhesion and transmigration into the brain parenchyma via LJP-1586, a selective vascular adhesion protein-1 (VAP-1) inhibitor, improves neurological outcome in SAH [
34]. LJP-1586 is nonspecific and inhibits the adhesion of neutrophils, monocytes and lymphocytes. In comparison, FTY720 has a selective effect on lymphocytes. Studies performed in ischemic models confirm the contribution of both innate and adaptive immunity to stroke outcome. Understanding the role of lymphocytes in cerebral ischemia is confounded by the existence of specific subpopulations that are either protective, deleterious, or both [
29]. Despite the limited information regarding the specific contribution of lymphocytes to SAH outcome, the selective effect of FTY720 supports the key role of this cell type on SAH-associated brain injury. Animal models, however, do not necessarily recapitulate the complex pathogenic mechanisms that take place in humans. From the immunological standpoint, the predominant circulating leukocytes in humans are neutrophils (50 to 70%) and in rodents lymphocytes (60 to 80%) [
35]. In addition, immune-related molecules are differentially expressed in different species [
35]. Therefore, the results obtained in experimental models may not necessarily apply to humans. Both similarities and differences have been described in the immune responses triggered by SAH in rodents and humans [
31]. Significantly, it was observed that methylprednisolone, a drug that preferentially targets lymphocytes, is beneficial in rodents and patients with SAH [
36-
38]. The studies were too small to draw solid conclusions but they support the active role of lymphocytes in SAH-associated brain injury.
In our study we also observed that the use of FTY720 preserved microvascular dilating function which was tested using different vasodilating agents. Hypercapnia and Ach induce cerebral arteriolar dilation via nitric oxide (NO) synthase/cyclooxygenase and endothelium-dependent mechanisms, respectively. In comparison, ADO tests G-coupled receptor-dependent vascular smooth muscle responses and SNAP interrogates vascular smooth muscle NO reactivity. Time-dependent alterations in the response of small arteries to vasodilators occur within minutes of the SAH and peak at 48 hours [
22]. The upstream processes governing vascular tone on SAH are not completely understood. An association between inflammation and abnormal vasomotor responses in SAH has been established in large conduit arteries [
39,
40]. Lymphocytes, in particular, have been associated with the emergence of late vasospasm [
41]. Pharmacological blockage of VAP-1 and neutrophil inhibition, in addition, ameliorate microvascular dysfunction in animal models suggesting that immunological factors regulate vasoreactivity in both small and large intracranial vessels [
39]. Interestingly, there is evidence supporting the notion that microvascular dysfunction is a major contributor to microthrombi formation, tissue hypoxia, and outcome after SAH [
42-
44]. The improvement in neurological outcome, in parallel with decreased neuroinflammation and preserved arteriolar responses observed in association with FTY720, is in agreement with this paradigm. It should be pointed out, however, that this drug has a direct stabilizing effect on the endothelium and has been shown to enhance the endothelial synthesis of NO in non-neural tissues [
14,
45]. In our study the arteriolar responses in the sham-vehicle and sham-FTY720 were comparable (
P > 0.05) indicating that, under the conditions used in this study, FTY720 does not have a direct vasodilating effect. FTY720 has a direct epigenetic effect via histone deacetylases regulation and also targets different components of the neurovascular unit. In particular, it inhibits astrogliosis, increases neuronal survival in cerebral ischemia, and enhances endothelial-barrier function [
25,
46-
48]. Therefore, the preserved arteriolar function and neurological improvement observed in our study cannot be conclusively ascribed to the inhibition of lymphocytes.
Studies done in ischemia models demonstrate that FTY720 decreases brain edema, reduces neuronal cell death, and improves long-term neurobehavioral outcome [
15,
17,
49] Furthermore, the early treatment of intracerebral hemorrhage patients with FTY720 reduces hematoma-associated edema and improves neurological outcome at 3 months [
50]. Since SAH is associated with unique pathogenic mechanisms, the extrapolation of findings gathered in other stroke models should be done with caution. Current data, however, demonstrate that FTY720 has pleiotropic effects and can influence key elements of SAH, including neuronal survival, BBB permeability, neuroinflammation, and microvascular dysfunction [
15,
17,
49].
Similar to other brain-injury models, a profound immunodepression develops in SAH patients [
51]. This temporary state lasts for at least 3 days after the bleed and is associated with a high rate of infections, particularly pneumonia. Thus, the potential translation of our findings to clinical practice may be limited by the immunomodulatory nature of fingolimod. Fingolimod is known to retain naïve T cells and central memory T cells in lymph nodes. However, this drug has a partial effect on peripheral effector memory T cells, which are important against infections [
14]. There is limited information regarding the effect of fingolimod on infection rates on stroke. However, studies done in the rodent model of cerebral ischemia have shown that treatment with fingolimod reduces circulating leukocytes and improves outcome without increasing the rate of bacterial lung infections [
52]. In addition, two recent small studies investigating the effect of fingolimod on patients with ischemic stroke and intracerebral hemorrhage showed similar rates of infection in both the treatment and placebo groups [
50,
53].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HLX and FDT were involved in the design of the study, carried out the experiments, and participated in the data analysis and manuscript preparation. DAP and CP participated in the data analysis and manuscript preparation. All authors read and approved the final manuscript.