1 INTRODUCTION
Sudden cardiac arrest (CA) is a leading cause of global mortality [1]. Despite much 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 the post-CA syndrome [2, 3]. Brain injury represents an essential hallmark of the pathophysiology of the post-CA syndrome [4], profoundly impairs the neurological function and conduces to lifelong disability among CA survivors and even death. However, 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 damages [6]. The blood-brain barrier (BBB) integrity is compromised by CA/CPR, leading to intractable cerebral edema. More significantly, cerebral edema exacerbates clinical outcomes and becomes a potent prognostication after CA [7-9]. Microglia/macrophages are spectacularly plastic and obtain multiple subtypes, such as pro-inflammatory and anti-inflammatory status, to fulfill different activities in health and disease. Altered microglia/macrophages functional subtypes and consequent neuroinflammation have an intimate relationship with the integrity of BBB. The unwanted entry of serum proteins into brain after the collapse of 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 circle between BBB breakdown and pro-inflammatory microglia/macrophages status is created to escalate post-CA/CPR brain injury continuously. Therefore, only drugs that modulate multiple targets such as BBB destruction, neuroinflammation and neuronal injury are likely to achieve clinical translation.
Although the molecular mechanisms responsible for controlling the restrictive feature of BBB remain largely elusive, transient receptor potential M4 (TRPM4), a nonselective monovalent cation channel activated by elevated intracellular Ca2+, has been shown to be critical in regulating the BBB function [12]. 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 ofTrpm4 could function in preserving the 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 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], implying that TRPM4 channel may affect microglia polarization. Therefore, the therapeutic manipulation of the BBB function and phenotypic shift of microglia/macrophages by interfering with the TRPM4 channel may 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 against 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 suggested that FFA conferred neuroprotection against several neurological diseases, such as spinal cord injury [24, 25], Alzheimer’s disease [26], and epilepsy [27]. FFA inhibited capillary fragmentation and secondary hemorrhage via blocking TRPM4 after spinal cord injury [24, 25]. These studies prompted us to logically postulate that FFA could elicit its neuroprotective effects through 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−/− ) exerts similar effect to FFA and whether blockage of TRPM4 represents part of the mechanism accounting for FFA-mediated neuroprotection in CA/CPR.