Neuroprotective effects of amiodarone in a mouse model of ischemic stroke

Ion channels play a crucial role in the development of ischemic brain injury [1]. After ischemic insult, Na⁺-K⁺- adenylpyrophosphatase (Na⁺-K⁺-ATPase) becomes inactivated due to the depletion of cellular ATP. Intracellular Na⁺ accumulates and the brain cell membrane becomes depolarized. Excessive glutamate release and subsequent activation of voltage-gated Na⁺ and Ca²⁺ channels occur, resulting in neuronal hyperexcitability with further accumulation of intracellular Na⁺ and Ca²⁺. As a result, cellular necrosis due to osmotic swelling occurs [1]. Recently, many studies have reported that the blockade of the Na⁺ and Ca²⁺ channels improves outcomes in experimental models of ischemic stroke [1‐5].

Amiodarone, one of the most effective drugs for the treatment of life-threatening arrhythmia, works as a multiple ion channel blocker and its characteristics cover all four Vaughan-Williams classes [6]. Besides its inhibitory effects on K⁺ channels, amiodarone also has inhibitory effects on the Na⁺ and Ca²⁺ channels [6]. Considering amiodarone’s pharmacological profile as a multiple ion channel blocker, there is a possibility that amiodarone has neuroprotective effects on the ischemic brain. It is known that amiodarone indirectly contributes to the prevention of ischemic stroke by maintaining sinus rhythm in patients with atrial fibrillation [7]. However, the direct neuroprotective effect of amiodarone has not been clarified.

With the above in mind, the purpose of this study was to evaluate the neuroprotective effect of amiodarone on ischemic brain injury. We tested whether pre-treatment or post-treatment with amiodarone effectively attenuates ischemic brain injury because it can be used both before and after ischemic insult caused by life-threatening or refractory arrhythmia in clinical situations. Our hypothesis was that pre-treatment and post-treatment with amiodarone attenuate ischemic brain injury. We examined the following outcomes with amiodarone treatment: infarct volume, neurological function, hemodynamics, and arrhythmias.

Experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. The experimental protocol was reviewed and approved by the University of Yamanashi Animal Care Committee.

In the present study, we found that amiodarone administered before ischemic brain insult lessened the infarct volume and improved neurological function.

Systemically administered amiodarone passes the blood–brain barrier [13, 14] and can exert its pharmacological effects on the central nervous system [15]. The pharmacological profile of amiodarone is complex. It works as a multiple ion channel blocker and has inhibitory effects on Na⁺, Ca²⁺, and K⁺ channels, Na⁺-K⁺-ATPase, and Na⁺/Ca²⁺ exchanger. [6, 16, 17] Previous studies have suggested the neuroprotective effect of various types of ion channel blockers including Na⁺ [2, 5], Ca²⁺ [18], and K⁺ blockers [3, 4]. Blockade of Na⁺ or Ca²⁺ channels prevents hyperexcitability and accumulation of Na⁺ and Ca²⁺ after ischemic injury by stabilizing the cellular membrane [1, 2]. Recent studies also revealed beneficial effects of K⁺ channel blockade in the ischemic brain [3, 4]. Amiodarone blocks the delayed rectifier potassium current and ATP-sensitive and Ach-sensitive potassium currents, and it prolongs the cellular action potential duration. Because mitochondrial K_ATP channels play a crucial role in pharmacological preconditioning effects against ischemic insult [19, 20], blockade of the K⁺ channel can be detrimental. However, amiodarone blocks K_ATP channels but has no effect on mitochondrial K_ATP channels [21]. Interestingly, some studies have revealed that inhibition of K_ATP channels creates a neuroprotective effect through the effect on microglia [4]. Effects of Na⁺-K⁺-ATPase and Na⁺/Ca²⁺ exchanger blockade on the ischemic brain are unclear and controversial [22, 23]. Depending on the cellular electrophysiological condition, these effects can be both harmful and neuroprotective. With a shortage of ATP, inactivation of Na⁺-K⁺-ATPase helps to preserve cellular ATP. If the ATP shortage is critical, however, inactivation of Na⁺-K⁺-ATPase may result in the further accumulation of cellular Na⁺ and Ca²⁺ and exacerbate neuronal death [1]. Na⁺/Ca²⁺ exchanger, an antiporter membrane protein, transports Ca²⁺ out as Na⁺ enters the cell [24]. In ischemic states with accumulated intracellular Na⁺, Na⁺/Ca²⁺ exchanger promotes in the reverse mode, which produces an opposite transporting direction (Ca²⁺ influx and Na + efflux) [25]. The mode in which the Na⁺/Ca²⁺ exchanger operates depends on the Na⁺ and Ca²⁺ transmembrane gradient. These characteristics and the complexity of Na⁺/Ca²⁺ exchanger may explain differing results, indicating both neuroprotective and harmful effects of Na⁺/Ca²⁺ exchanger blockers on ischemic brain injury [22, 23].

Non-electrophysiological effects of amiodarone include inhibitory effects on beta-adrenergic receptors [26]. Blockade of beta-adrenergic receptors has been reported to exert neuroprotective effects possibly by decreasing oxygen consumption, platelet aggregation, and Ca²⁺ influx [27]. In addition to its inhibitory effect on beta-adrenergic receptors, amiodarone also prevents excessive pro-inflammatory cytokine production [28‐31] and suppresses reactive oxidative stress [30‐32], which can also lead to neuroprotection against ischemic brain injury. Due to its complex pharmacological profile, it is difficult to attribute the neuroprotective effect of amiodarone observed in this study to one simple mechanism. However, the present results suggest that systemically administered amiodarone prior to ischemic brain injury has neuroprotective effects, at least in part, via blocking the sodium channels. We also used isoproterenol to offset the inhibitory effect of amiodarone on beta-adrenergic receptors. However, isoproterenol did not inhibit the neuroprotective effect of amiodarone, indicating that blockade of beta-adrenergic receptors and decrease in oxygen consumption were not likely the mechanisms underlying the neuroprotective effect of amiodarone observed in the present study.

We could not identify a neuroprotective effect of amiodarone post-treatment. It is reported that post-treatment with Na⁺ [2], Ca²⁺ [18], and K⁺ blockers [3, 4], beta-blockers [27], anti-inflammatory drugs [33, 34], and anti-oxidants [35] provides neuroprotection in experimental models of ischemic stroke. One possible reason for this discrepancy is that amiodarone could not reach the ischemic site due to the permanent coagulation of the MCA that did not produce reperfusion. Different results might be obtained by studies using brain ischemia/reperfusion models.

The clinical significance of our study is that systemically administered amiodarone prior to ischemic brain insult has the potential to improve neurological functional outcomes. Amiodarone is one of the most commonly prescribed drugs for life-threatening and refractory arrhythmias, and it is currently a second-line drug for the treatment of ventricular fibrillation during cardiopulmonary resuscitation [36]. Cardiac arrest leads to brain ischemia, and neurological function and recovery after resuscitation are of the greatest concern. We used 50 mg/kg of amiodarone, which is estimated to be equivalent to a cardiac arrest dosage for an adult human (300 mg) based on body surface area [37].

This study had several limitations. Since amiodarone has a complex pharmacological profile, there remains a possibility that other mechanisms are involved in the neuroprotective effect of amiodarone observed in this study. Furthermore, amiodarone could have diverse roles and exert different effects based on various factors or the severity of brain injury. Although there was no statistical difference in blood pressure among the groups, there was a trend toward lower blood pressure in the pre-treatment and post-treatment groups. Hypotension is a major side effect of single-bolus amiodarone [38] that can largely affect brain injury severity. We used relatively young male mice that had no degenerative vascular or cellular changes. Future studies should test these findings in aged mice and female mice with different menopausal states, as sex and menopausal state can significantly affect stroke outcomes [39]. In this study, we conducted infarct volume analysis 48 h after MCAO. However, considering its long elimination half-life and various side effects, the long-term neurological outcomes and effects of amiodarone on ischemic stroke need to be determined.

Amiodarone pre-treatment, but not post-treatment, attenuates ischemic brain injury and improves functional outcomes without affecting heart rhythm and blood pressure. The neuroprotective properties of amiodarone demonstrated in the present study may be explained by its complex pharmacological profile as a multiple ion channel blocker. The results of the present study may indicate that systemically administered amiodarone prior to ischemic brain insult has the potential to improve neurological outcomes. Further studies are needed to elucidate the underlying mechanisms and therapeutic time windows of amiodarone for ischemic brain injury.