Therapeutic Strategies of Ischemic Stroke Based on Proton-Activated Chloride Channel

Introduction
Ischemic stroke is a multifactorial disease with high rate of morbidity, mortality and disability rate worldwide [1]. When acute ischemia occurs, it leads to excessive oxidative stress, causing irreversible damage to nervous tissue [2,3]. However, current treatment strategies of ischemic stroke are relatively limited, actually, a large number of neuroprotective agents that have proved ineffective in clinical trials [4]. PAC, also known as ASOR (acid-sensitive outwardly rectifying anion channel) [5] or PAORAC (proton-activated outwardly rectifying anion channel) [6], which was first observed in rat Sertoli cells in 2003[7], has shown encouraging results in providing partial neuroprotection against tissue acidosis caused by ischemic stroke.

Pac Channel and its Therapeutic Potential

Under normal physiological condition, the pH of extracellular fluid, including the blood plasma, is normally tightly regulated between 7.32 and 7.42 by the chemical buffers, the respiratory system, and the renal system [8]. However, in cerebral acidosis caused by ischemic stroke, the pH of the ischemic core may be as low as 6.0 [9]. Ischemic tissue acidosis is a sensitive metabolic indicator for the progression of cerebral ischemic injury. PAC was reported to be activated by extracellular acidity as well as its sensitivity to temperature and pH [5]. The threshold pH for PAC activation is relatively low, around pH 5.5 at room temperature and around pH 6.0 at 37℃ [10]. As a highly conserved channel ubiquitously expressed in mammals, the core protein of PAC (TMEM206) has the highest molecular expression in the cerebral cortex of human [11]. By cooperating with other ion transporters and channels such as V-ATPase, acid-sensing ion channel 1a (ASIC1a), and Na+/ H+ exchanger (NHE), PAC is activated by extracellular acidity and mediates Cl- influx into the cell, cytotoxic edema and cell necrosis ensue [12-14]. The simultaneous increase in intracellular Ca2+ promotes cell death cascade associated with intracellular Ca2+ accumulation [15].
Several studies have reported that inhibition of PAC provides partial neuroprotection against tissue acidosis after ischemic stroke:
1. Neurons undergoing massive necrosis 1h after exposure to acidic solution were not only protected by PAC channel blockers but also by cooling down to 25 ℃ [10],
2. Knocking down the core component of PAC (TMEM206) almost abolished proton activated Cl- current in rat neurons and reduced neuronal cell death caused by acid treatment [16],
3. Application of the chloride channel blocker 4,4′-Diisothiocyano- 2,2′-stilbenedisulfonic acid (DIDS) in vivo attenuated cell injury induced by ischemia-reperfusion in hippocampal CA1 neurons [17],
4. Knockout of mouse PAC abolished proton-activated Cl- current in neurons and attenuated brain damage after ischemic stroke [11]. Based on above lines of evidence, PAC is a promising therapeutic target to protect neurons in cerebral ischemia.

Therapeutic Strategies of Ischemic Stroke Targeting PAC Channels

Metabolic acidosis occurring in ischemic stroke is a sensitive metabolic indicator capable of targeting drugs to the salvageable ischemic penumbra with high specificity. Targeting ischemic brain tissue using pH sensitive marker polymers or nanoparticles has enabled medical imaging to accurately distinguish ischemic tissues from normal ones [18,19]. The development of bio-responsive materials that undergo conformational or solubility changes under acidic environments offers great promise for the development of smart targeted drug delivery nano-systems [20], such as the hydrophobically modified chitosan nanoparticles (Chit NPs) with C6-side chains were released the Ca2+ channel blocker nimodipine (NIMO) drug at the pH of ischemic tissue (=6.0), while at normal pH (=7.4) the drug molecules was remained closed in polymer shell [21]. pH directing effect and bio-responsive nanomaterials have been applied in solid tumor therapy and inspired the application of nanotechnology in the treatment of ischemic stroke [22,23]. Selective delivery of PAC specific blockers to the ischemic penumbra via a pH responsive smart nanosystem holds promise for the treatment of injury sites without adverse nontargeted side effects. However, currently there are only several universal drugs available to target chloride channels, such as DIDS, niflumic acid (NFA), and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), specific PAC blocker has not yet been reported [11,12].
Therapeutic hypothermia has been regarded as one of the most effective neuroprotective strategies since 1987 [24,25]. Over the past few decades, the neuroprotective effects of hypothermia in cardiac arrest [26] and resuscitation from neonatal hypoxicischemic encephalopathy [27] have been confirmed by multiple clinical trials. Numerous preclinical studies based on vascular recanalization models have shown that hypothermia exerts neuroprotective effects mainly by reducing the cerebral metabolic rate [28], but also involves cell death, inflammation and white matter integrity [29], and the temperature sensitivity of PAC also provides potential mechanisms [16]. Intra-arterial selective cooling infusion (IA-SCI), as a novel therapeutic method of hypothermia, utilizes the characteristics of high blood flow to the brain to directly perfuse hypothermic fluid into the cerebral arteries for rapid and selective cooling [30]. IA-SCI can precisely achieve regional cooling of brain tissue with a much faster cooling compared with traditional superficial cooling and systemic transvenous cooling and can be combined with mechanical thrombectomy to avoid the side effects brought by systemic cooling [31,32], which has been clinically validated in rodent models [33], large animal models [34] and ischemic stroke patients [35]. Precise cooling of the acidotic brain tissue in ischemic stroke by IA-SCI can elevate opening threshold of PAC, thereby shutting the channel, thereby reducing the influx of Cl-, improving cytotoxic edema, and achieving neuroprotection [10,12].

Conclusion
Targeted delivery of PAC blockers by smart nano-systems is highly specific and efficient and enables the gradual release of drugs to extend the time of drug exposure, reducing the risk of adverse side effects or off target toxicity. Compared with the simultaneous closure / blockade of multiple cationic channels associated with cellular edema (such as ASICs and NHEs, etc.), the blockade of a single and unique anion channel (PAC) by hypothermia has higher applicability and feasibility experimentally or clinically. Although there is currently a lack of sufficient experiments to validate the above two therapeutic strategies, smart drug targeting nanosystems and hypothermia therapy that inhibit PAC also pose an promising opportunity for translational trials to protect brain tissue. 

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