Introduction
Sudden cardiac arrest (CA) is a leading cause of global mortality [
1]. Despite many advances in optimizing the techniques of cardiopulmonary resuscitation (CPR), the overall prognosis is still unsatisfactory after out-of-hospital CA with successful resuscitation, mainly due to post-CA syndrome [
2,
3]. Brain injury represents an essential hallmark of the pathophysiology of post-CA syndrome [
4], profoundly impairing neurological function and leading to lifelong disability and even death among CA survivors. Nevertheless, no clinically effective pharmacological intervention is available to reduce neurologic deficiency in patients with CA/CPR at present [
5].
Currently, most neuroprotective methods after CA/CPR have focused on protecting neurons. However, endothelial dysfunction, an inevitable pathologic process of ischemia/reperfusion during CA plays a governing role in the progression of post-CA brain injury and evokes further neuronal damage [
6]. Blood–brain barrier (BBB) integrity is compromised by CA/CPR, leading to intractable cerebral edema. More significantly, cerebral edema exacerbates clinical outcomes and is a strong prognostic factor of outcomes after CA [
7‐
9]. Microglia/macrophages are spectacularly plastic and obtain multiple subtypes, such as pro-inflammatory and anti-inflammatory subtypes, to fulfill different activities in health and disease. Altered microglia/macrophages functional subtypes and consequent neuroinflammation have an intimate relationship with the integrity of the BBB. The unwanted entry of serum proteins into the brain after the collapse of the BBB is a pivotal cause of neuroinflammation [
10], facilitating the transition of microglia/macrophages to a pro-inflammatory status. The pro-inflammatory phenotype of microglia, in turn, can simultaneously exaggerate BBB insult [
11]. In this way, a vicious cycle between BBB breakdown and pro-inflammatory microglia/macrophage status is created to continuously escalate post-CA/CPR brain injury. Therefore, only drugs that modulate multiple targets, such as BBB destruction, neuroinflammation and neuronal injury, are likely to achieve clinical translation. However, the molecular mechanisms responsible for controlling the restrictive feature of the BBB and microglia/macrophage polarization in post-CA/CPR brain injury remain largely elusive.
Transient receptor potential M4 (TRPM4), a nonselective monovalent cation channel activated by elevated intracellular Ca
2+, has been shown to be critical in regulating BBB function [
12]. The findings of studies conducted by us and others indicated that pharmacological inhibition of the subunits of sulfonylurea receptor 1-transient receptor potential M4 (SUR1-TRPM4) or gene deletion of
Trpm4 could function in preserving BBB integrity in animal models of status epilepticus [
13], acute ischemic stroke [
14], spinal cord injury [
15], intracerebral hemorrhage [
16], and subarachnoid hemorrhage [
17]. Indeed, we have reported that glibenclamide, a selective inhibitor of SUR1, effectively improved survival and neurologic outcome in rodent models of CA/CPR [
18‐
20], but its protective effect on the BBB after CA/CPR and whether it works by blocking the SUR1-TRPM4 channel remain unclear. Furthermore, the SUR1-TRPM4 channel was shown to be expressed in microglia and participated in regulating pro-inflammatory gene expression [
21]. However, whether the TRPM4 channel can affect microglia polarization has not yet been understood. We hypothesized that interfering with the TRPM4 channel may therapeutically manipulate BBB function and the phenotypic shift of microglia/macrophages, which could serve as a promising opportunity for minimizing brain injury resulting from CA/CPR.
Flufenamic acid (FFA) is a non-steroidal anti-inflammatory drug that has been applied for analgesia for pain related to rheumatic disorders. Since the TRPM4 channel was found to be highly sensitive to FFA and can be inhibited by low concentrations of FFA, FFA has attracted extensive attention as a convenient and relatively selective TRPM4 inhibitor to study the physiological effects of TRPM4 [
22,
23]. Emerging studies have suggested that FFA confers neuroprotection against several neurological diseases, such as spinal cord injury [
24], Alzheimer’s disease [
25], and epilepsy [
26]. FFA inhibited capillary fragmentation and secondary hemorrhage by blocking TRPM4 after spinal cord injury [
24]. However, the protective effects of FFA on brain injury secondary to CA/CPR and the underlying mechanisms have not been addressed. These studies prompted us to logically postulate that FFA could elicit its neuroprotective effects after CA/CPR by ameliorating BBB breakdown and improving neurologic outcome by inhibiting TRPM4.
Here, we investigated whether FFA could improve neurologic outcome in a mouse model of CA/CPR. Moreover, we aimed to explore whether gene deletion of Trpm4 (Trpm4−/−) could exert an effect similar to that of FFA and whether TRPM4 blockage represents part of the mechanism accounting for FFA-mediated neuroprotection in CA/CPR.
Discussion
Hypoxic ischemic brain injury after CA is the primary cause of death of CA survivors [
35], thus it is imperative to develop an effective strategy to combat brain injury for CA victims with successful resuscitation. In this study, we investigated the neuroprotective effects of FFA and the underlying mechanisms after experimental CA/CPR in mice. We presented that FFA treatment remarkably improved 7-day survival and neurologic outcome, lessened neuropathological impairment, reduced brain edema and mitigated BBB breakdown. Additionally, pro-inflammatory microglia/macrophage polarization was suppressed, while anti-inflammatory microglia/macrophage polarization was promoted in CA/CPR mice administered with FFA. Lastly, we demonstrated that
Trpm4−/− mice exhibited comparable effects to FFA-treated mice, and
Trpm4−/− mice treated with FFA showed no benefit of superposition after CA/CPR. Taken together, our results supported that the neuroprotective effects of FFA against brain injury resulting from CA/CPR, at least partially depended on the inhibition of the overactivated TRPM4 channel in the neurovascular unit (Fig.
10). Our study highlighted the significance of TRPM4 in the development of post-CA brain injury and comprehensively evaluated the functions of FFA in post-CA brain injury.
Numerous studies have revealed that FFA can be an ion channel modulator [
22,
23,
36,
37]. Since then, interest in the “off targets” of FFA (i.e., beyond its well-known effect of COX inhibition) protection has been rekindled, and the pleiotropic effects of FFA have been reevaluated. These intriguing findings gave rise to several studies indicating that FFA afforded neuroprotection in central nervous system (CNS) diseases in terms of dampening the inflammatory response, facilitating angiogenesis, preserving myelin and motor neurons, inhibiting glial activation and so on [
24,
25]. Here, we stepped forward to elucidate the role of FFA in the model of CA/CPR. Our exciting findings in this study were that FFA treatment showed benefits in improving survival and neurological function and lessening neuropathological injuries following CA/CPR, which further supported the use of FFA as a lifesaving neuroprotectant to address CA/CPR-induced brain injury.
BBB disintegration is a threatening event in hypoxic ischemic encephalopathy after CA, causing fatal brain edema closely associated with poor prognosis. Osmotherapy is the conventional treatment for cerebral edema, by employing hypertonic saline or mannitol. However, under BBB dysfunction conditions, hypertonic saline or mannitol may accumulate within the brain, therefore potentiating edema formation. In fact, previous studies have indicated that hypertonic treatment fails to improve the outcome when administered after CA/CPR [
38,
39]. Besides, osmotherapy is unlikely to alleviate, but may aggravate, the pro-inflammatory response of extravasated toxic substances [
40]. From a clinical point of view, osmotherapy after CA/CPR remains an understudied topic due to these counterproductive effects. In this respect, preventing edema by enhancing BBB integrity is preferable to osmotherapy for treating the already swollen brain. Accumulating studies have demonstrated that targeting TRPM4 could be a new perspective for maintaining BBB integrity [
14,
16,
41]. FFA was reported to ameliorate capillary fragmentation and secondary hemorrhage following spinal cord injury by blocking TRPM4 [
42]. In the current study, we further provide encouraging evidence that FFA ameliorates BBB disruption and consequent edema formation following CA/CPR, supporting FFA as a promising agent in the rescue of BBB breakdown, which has important clinical implications given the considerable incidence rate of cerebral edema in CA/CPR patients.
Neuroinflammation plays a cardinal role in hypoxic ischemic encephalopathy resulting from CA/CPR [
33]. Microglia may become activated for many weeks and develop a macrophage-like capacity to initiate inflammatory cascades of the CNS after CA/CPR. Classically activated microglia/macrophages secrete adverse cytokines that worsen brain injury and impede damaged tissue remodeling, whereas alternatively activated microglia/macrophages release anti-inflammatory mediators that hasten brain repair and potentiate the phagocytosis of dying cells [
43]. Endowing activated microglia with a neuroprotective phenotype was found to improve outcomes in a model of CA/CPR [
44,
45]. Moreover, a previous study demonstrated that microglia depletion by intrahippocampal injection of liposome-encapsulated clodronate was not sufficient to salvage neuronal degeneration after CA/CPR [
46], which further supports that the balance between the numbers of reparative versus deleterious microglia/macrophages phenotypes rather than indiscriminate suppression of microglia activation may be instrumental in optimal brain repair and neurologic recovery. A recent study found that the administration of FFA inhibited microglia activation [
24]. Herein, we add to the current knowledge that FFA transforms microglia/macrophages from a pro-inflammatory functional status into an anti-inflammatory status after CA/CPR. Strikingly, CA/CPR not only triggered an elevation in the percentage of pro-inflammatory microglia/macrophages, but also increased the percentage of anti-inflammatory microglia/macrophages, suggesting self-protection of the brain in response to CA/CPR. Previous studies also found that anti-inflammatory cytokines, such as TGF-β and IL-10 increased in the brain following CA/CPR [
44,
47].
Sustained confrontation of dying cells is a major stimulus for harmful inflammatory reactions [
48]. After CA/CPR, the selectively vulnerable regions with extensive cellular debris and cell corpses act as reservoirs for various cytotoxicities and pro-inflammatory cytokines, conducing to overwhelming neuroinflammation and the enlargement of secondary injury. In addition to triggering inflammation, necrotic tissue hampers neural reorganization and repair. Considering that interfering with apoptosis and necrosis is still difficult to achieve in the clinical real-world setting, the promotion of the reparative microglia/macrophages phenotype associated with augmented clearance function toward damaged cells and cellular debris may be indispensable for timely eliminating the source of inflammation and ultimately enabling effective functional recovery. We illustrated that the phagocytosis of microglia/macrophages was strengthened after FFA administration, which could be a reasonable explanation for the reduced accumulation of damaged cells in the brain. Besides, the adhesion molecule ICAM-1 is largely exposed due to glycocalyx degradation during CA/CPR, which promotes neuroinflammation by mediating leukocyte adhesion to the BBB and eventually transmigration to brain tissue [
49]. We found that FFA treatment lowered the expression of ICAM-1, which contributed to the arrested amplification of neuroinflammation after CA/CPR. Altogether, our data reveal that FFA treatment after CA/CPR significantly optimizes the tissue-reparative function of microglia/macrophages to allow for better brain cleanup and a stronger capacity for neuroinflammation resolution, thereby averting further neuronal injury and favoring neurologic recovery.
The TRPM4 channel is a nonselective monovalent cation that is upregulated in a variety of CNS diseases [
14,
17,
42] and leads to excessive influx of extracellular sodium, causing oncotic cell death and BBB breakdown, while gene deletion of this channel has been shown to afford neuroprotection in several models of neurological disease [
13,
14]. Beyond its predominant effect of attenuating edema and preserving BBB function, increasing attention has been given to the study of the TRPM4 channel as a novel target for abating neuroinflammatory burden in several CNS diseases [
50,
51]. Kurland et al. [
21] indicated that in microglia in vivo and in vitro, gene silencing of
Trpm4 decreased pro-inflammatory gene expression following TLR4 activation in microglia, hinting at a delicate connection between TRPM4 and microglia polarization. In particular, the activation of TRPM4 requires calcium overload, which virtually occurs under conditions of CA/CPR [
4]. In line with previous findings in the model of CA/CPR [
19,
52], the expression of TRPM4 was upregulated and colocalized with the neurovascular unit. In the present study, we found more favorable neurologic outcome, milder histological injury, less IgG leakage, and more anti-inflammatory microglia/macrophage polarization in
Trpm4−/− mice after CA/CRR, which were comparable to the effects of FFA. Since FFA shows high potency in blocking the TRPM4 channel [
22,
23], these results together further confirm the vital role of TRPM4 in the pathogenesis of post-CA brain injury. TRPM4, which can lead to catastrophic BBB disruption and a persistent neuroinflammatory response, could represent a promising and clinically relevant target for the future treatment of post-CA induced brain injury. As a step forward, we observed that FFA treatment significantly inhibited the upregulation of TRPM4, and FFA treatment combined with
Trpm4 deficiency showed no additive protective effect, which indicated that the suppression of TRPM4 in the neurovascular unit by FFA may be the main reason for better outcome following CA/CPR.
Although there is no doubt about the fundamental role of the endothelium in forming a barrier to restrict BBB permeability, the interaction of the endothelium with the components of the neurovascular unit should not be overlooked. In contrast to pericytes and astrocytes, the role of microglia in the regulation of BBB function is only beginning to be defined. Following ischemia, microglia form perivascular clusters, which display the intricate interplay between microglia and brain vessels. The communication of aggregated microglia with the endothelium exerts both beneficial and detrimental effects on BBB function depending on the microenvironment [
53]. After BBB disruption, peripheral immune cells and toxic substances are recruited into the brain and whereby amplify neuroinflammation, including classically activated microglia/macrophage polarization [
54]. These classically activated pro-inflammatory microglia may hinder BBB remodeling and result in vascular leakage, while anti-inflammatory microglia assist in the recovery of BBB damage [
11]. Besides, neuroinflammation accompanying CNS diseases has been shown to accelerate BBB breakdown [
55]. On this basis, the cross-talk between BBB dysfunction and microglia/macrophage polarization plays a key role in the vicious circle contributing to the pathogenesis of post-CA induced brain injury, which necessitates a conceptual shift from the widely recognized emphasis on protecting neurons to a novel treatment paradigm of targeting the entire neurovascular unit, especially microglia/macrophages. We propose that the effect of FFA on maintaining BBB integrity may not only be through directly suppressing TRPM4 on the endothelium, but also be derived from regulating the functional status of microglia/macrophages, namely that FFA treatment is an effective strategy to break the vicious cycle. Additionally, the pathophysiology of post-CA induced brain injury encompasses a heterogeneous cascade. Thus, using agents that affect multifaceted targets simultaneously may be more efficacious than modulating a single point to alleviate secondary brain injury after CA/CPR. We are convinced that FFA is promised to stand out as an attractive candidate drug for a polyvalent approach to stabilize the BBB following CA/CPR.
Apart from blocking the TRPM4 channel, FFA also affects the activity of other nonselective cationic channels [
37]. In fact, specific pharmacological inhibition of TRPM4 is not practicable at present, probably due to structural similarities of TRPM4 to other channels. To date, pharmacological inhibition of TRPM4 by FFA has been pursued in several models [
22‐
24]. Besides, TRPM4 has a relatively higher affinity for FFA than other ion channels [
37]. It is suggested that low concentrations of FFA (~ 10 μM) may be suitable to determine the physiological effect of TRPM4 in situ, because it has little to no influence on other ion channels whose FFA sensitivity is much lower [
37]. Accordingly, TRPM4 modulation may commonly account for the effects of FFA given its upregulation in the CA/CPR model and high sensitivity to FFA. Of note, plasma concentrations of 4–12 μM, measured in conditions of FFA clinical use, are sufficient to potently inhibit TRPM4 [
56], which further support FFA as a TRPM4 inhibitor that could be translated into clinical use. To our knowledge, other channels potentially modulated by FFA are not expected to alleviate microvascular dysfunction, since this protective effect is specific for the TRPM4 channel. More importantly, as gene deletion of
Trpm4 mimicked the effect of FFA and no additional benefit was found in FFA-treated
Trpm4−/− mice, the participation of other ion channels is unlikely. Moreover, despite its primary characterization as a COX inhibitor, FFA showed lower effectiveness than other non-steroidal anti-inflammatory drugs, and it has been reported that FFA provides neuroprotection by inhibiting excitotoxicity and limiting neuroinflammation, which is independent of COX inhibition [
25,
57]. Collectively, our findings corroborated that TRPM4 is the most likely regulator of the neuroprotective impact of FFA on CA/CPR-induced brain injury.
This study has some limitations. First, we did not study the effects of different doses of FFA on brain injury after CA/CPR. However, the FFA dose used in this study was derived from two previous well-designed studies that showed that FFA could inhibit TRPM4 and reduce spinal cord injury [
24,
42]. Second, the lack of an additional protective effect of FFA in
Trpm4−/− mice may be due to the floor effect caused by FFA alone or Trpm4 knockout itself. Therefore, a direct observation of FFA inhibiting TRPM4 current in subsequent studies would provide more evidence. Nevertheless, we found that FFA did inhibit the upregulated expression of TRPM4, suggesting that the protective effect of FFA is at least partially mediated by inhibiting the TRPM4 channel.
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