4 DISCUSSION
Hypoxic ischemic brain injury after CA is the primary cause of death of CA survivors [36], 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 as well as mitigated BBB breakdown. Additionally, pro-inflammatory microglia/macrophages polarization was suppressed while anti-inflammatory microglia/macrophages polarization was promoted in CA/CPR mice administered with FFA. Lastly, we demonstrated that Trpm4−/− mice exhibited comparable effect to FFA and Trpm4−/−mice treated with FFA showed no benefit of superposition after CA/CPR. Taken together, our results supported the neuroprotection of FFA against brain injury resulting from CA/CPR, probably depended on inhibition of the overactivated TRPM4 channel in the neurovascular unit. Our study highlights the significance of TRPM4 in the development of post-CA brain injury and comprehensively evaluates the functions of FFA in post-CA brain injury.
Numerous studies have revealed that FFA can be an ion channel modulator [22-23, 37-38]. Since then, interest in FFA’s “off targets” (ie, beyond its well-known effect of COX inhibition) protection has been rekindled and pleiotropic effects of FFA have been reevaluated. These intriguing findings gave rise to several studies indicating that FFA afforded neuroprotection in the central nervous system (CNS) diseases, in term of dampening inflammatory response, facilitating angiogenesis, preserving myelin and motor neurons, inhibiting glial activation and so on [24-26]. 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, neurological function and lessening neuropathological injuries following CA/CPR, which further supported 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, dehydrates also accumulate within the brain, therefore potentiating edema formation. Virtually, previous studies have indicated that hypertonic treatment fails to improve the outcome when administered after CA/CPR [39, 40]. Besides, osmotherapy is unlikely to alleviate, but may aggravate, the pro-inflammatory response of the extravasated toxic substances [41]. 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, 42]. FFA was reported to ameliorate capillary fragmentation and secondary hemorrhage following spinal cord injury through blocking TRPM4 [43]. In the current study, we further offer encouraging evidence that FFA ameliorates BBB disruption and consequent edema formation following CA/CPR, supporting FFA as a viable agent in the rescue of BBB breakdown, which has important clinical implications given considerable incidence rate of cerebral edema in CA/CPR patients.
Neuroinflammation serves as a cardinal role in hypoxic ischemic encephalopathy resulting from CA/CPR [34]. Microglia may get activated for many weeks and develop 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 phagocytosis of dying cells [44]. Endowing activated microglia with a neuroprotective phenotype improved outcome in the model of CA/CPR [45, 46]. 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 [47], 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 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 one 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 [45, 48].
Sustained confrontation of dying cells is a major stimulus for harmful inflammatory reaction [49]. After CA/CPR, the selectively vulnerable regions with extensive cellular debris and cell corpses act as a reservoir for various cytotoxicity and pro-inflammatory cytokines, conducing to overwhelming neuroinflammation and enlargement of secondary injury. In addition to triggering inflammation, the 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, promotion of 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 efferocytosis of microglia/macrophages was strengthened after FFA administration, which could be a reasonable explanation for less damaged cells accumulated in the brain. Besides, the adhesion molecule ICAM-1 is largely exposed due to glycocalyx degradation during CA/CPR, which is critical for mediation of neuroinflammation [50]. We found that FFA treatment lowered expression of ICAM-1, which contributed to arrest 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 stronger capacity of neuroinflammation resolution, thereby averting further neuronal injury and favoring neurologic recovery.
TRPM4 channel is a nonselective monovalent cation that upregulates in a variety of CNS diseases [14, 17, 43] and leads to excessive influx of extracellular sodium, causing oncotic cell death and BBB breakdown, while gene deletion of this channel affords neuroprotection in several models of neurological disease [13, 14]. Beyond its predominant effect of attenuating edema and preserving BBB function, increasing attention has been attracted to study TRPM4 channel as a novel target for abating neuroinflammatory burden in several CNS diseases [51, 52]. 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 a delicate connection between TRPM4 and microglia polarization. In particular, activation of TRPM4 requires calcium overload, which is virtually occurs under conditions of CA/CPR [4]. In line with the previous findings in the model of CA/CPR [19, 53], the expression of TRPM4 was unregulated and co-localized with the neurovascular unit. In the present study, we found more favorable neurological outcome, lighter histological injury, less IgG leakage, and more anti-inflammatory microglia/macrophages 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 persistent neuroinflammatory response, could represent a promising and clinical 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 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 endothelium in forming a barrier to restrict BBB permeability, the interaction of endothelium with the components of neurovascular unit should not be overlooked. In contrast to pericytes and astrocytes, the role of microglia in 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 vessel. The communication of aggregated microglia with endothelium exerts both beneficial and detrimental effects in BBB function depending on the microenvironment [54]. After BBB disruption, peripheral immune cells and toxic substances are recruited into the brain and whereby amplify neuroinflammation, including classically activated microglia/macrophages polarization [55]. 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 [56]. On this basis, the cross-talk between BBB dysfunction and microglia/macrophages polarization plays a key role in the vicious circle contributing to the pathogenesis of post-CA induced brain jury, which necessitates a significant 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 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 circle. 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 blockage of the TRPM4 channel, FFA also affects the activity of other nonselective cationic channels [38]. In fact, specific pharmacological inhibition of TRPM4 is not practicable at present, probably due to structural similarities of TRPM4 to other channels. So far, pharmacological inhibition of TRPM4 in several models by FFA has been pursued [22-25]. Besides, TRPM4 has relatively higher affinity to FFA than other ion channels [38]. 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 [38]. Accordingly, TRPM4 modulation may commonly account for the effects of FFA given its upregulation in 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 [57], which further supports FFA to be a TRPM4 inhibitor in clinical translation. To our knowledge, other channels potentially modulated by FFA are not be 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-treatedTrpm4−/− mice, the participation of other ion channels is unlikely. Moreover, despite its primary characterization as COX inhibitors, FFA showed lower effectiveness than other non-steroidal anti-inflammatory drugs and it is reported that FFA provided neuroprotection through inhibiting excitotoxicity and limiting neuroinflammation, which was independent of COX inhibition [26, 58]. Collectively, our findings corroborated that TRPM4 is the most likely regulator of the neuroprotective impact of FFA on CA/CPR-induced brain injury.