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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: J Mol Neurosci. 2013 Mar 27;51(1):92–98. doi: 10.1007/s12031-013-0005-9

Sexually dimorphic response of TRPM2 inhibition following cardiac arrest-induced global cerebral ischemia in mice

S Nakayama 1, R Vest 2, RJ Traystman 2,3, PS Herson 2,3
PMCID: PMC3728170  NIHMSID: NIHMS460792  PMID: 23532768

Abstract

Transient global cerebral ischemia due to cardiac arrest followed by resuscitation (CA/CPR) causes significant neurological damage in vulnerable neuron populations within the brain, such as hippocampal CA1 neurons. In recent years, we have implicated the transient receptor potential M2 (TRPM2) channel as a mediator of ischemic injury to neurons. We previously demonstrated that genetic and pharmacological strategies that reduce TRPM2 function preferentially protect male neurons in vitro and reduce infarct volume following experimental stroke. Due to the narrow therapeutic window for intervention following ischemic stroke, it is important to assess the role of TRPM2 in other models of cerebral ischemia. Therefore, this study utilized a modified mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR) to mimic more accurately the clinical condition by maintaining body and head temperatures near the physiological range throughout. Here we report that inhibition of TRPM2 activity with clotrimazole (CTZ) reduces hippocampal CA1 neuronal injury when administered 30 minutes after resuscitation from cardiac arrest. Consistent with our previous observations, neuroprotection was observed in male mice and no effect on injury was observed in the female. These findings provide further evidence for TRPM2 as a target for protection against cerebral ischemia in the male brain.

Keywords: Brain, ischemia, TRPM2, cardiac arrest

Introduction

Sudden cardiac arrest (CA) is fatal unless witnessed and followed quickly by cardiopulmonary resuscitation (CPR). Even with CPR, mortality rates are high post-resuscitation and many CA/CPR survivors experience serious cognitive and motor dysfunction due to ischemic neurological damage (Allen et al. 2012; Lim et al. 2004; Madl et al. 2004; Roger et al. 2011; Roine et al. 1993; Rosamond et al. 2008; Saxon 2005). In the brain, transient global ischemia causes delayed neuronal cell death in vulnerable neuronal populations such as the CA1 region of the hippocampus (Allen et al. 2012; Garcia 1988; Garcia et al. 1989). Ischemic neuronal cell death is mediated through the excitotoxic release of the neurotransmitter glutamate, overstimulating glutamate receptors (Kostandy 2012; Lau et al. 2010; Szydlowska et al. 2010). Activation of these glutamatergic receptors allows extracellular sodium and calcium into the cell, resulting in cell death through sodium/calcium overload, mitochondrial dysfunction, oxidative stress, energy depletion, and the activation of death-inducing signaling cascades (Kostandy 2012; Lau et al. 2010; Szydlowska et al. 2010). In an effort to improve functional outcome and neuronal survival for patients suffering the effects of CA/CPR, researchers have focused on inhibiting the initial excitotoxic activation of glutamatergic receptors. Unfortunately, these receptors are also critical for normal neurological function and learning-memory formation (Riedel et al. 2003). Thus, while pharmacological inhibition of ionotropic glutamatergic receptors is indeed neuroprotective in many experimental models of ischemia, pharmacological inhibition of glutamatergic receptors produces unacceptable side effects that have caused the inhibitors to fail in clinical trials (Ginsberg 2008). Therefore, it remains important to identify effective treatments for CA/CPR without intolerable side effects to prevent neuronal cell death and improve neurological outcome. An ideal treatment in CA/CPR would be one that can interrupt the death-inducing cell signaling event triggered by ischemia without also interrupting normal, healthy neurological function. Ideally, this treatment could be applied later in the signaling process to interrupt delayed cell death, thus extending the therapeutic window for treatment. One candidate for further study is the Transient Receptor Potential M2 (TRPM2) ion channel (Jiang et al. 2010; Sumoza-Toledo et al. 2011).

TRPM2 is a sodium, calcium, and potassium permeable ion channel activated in a calcium-dependent manner (McHugh et al. 2003; Olah et al. 2009; Starkus et al. 2007) by adenine dinucleotide phosphate ribose (ADP-ribose) (Inamura et al. 2003; Kolisek et al. 2005; Kraft et al. 2004; Kühn et al. 2005; Kühn et al. 2004; Perraud et al. 2005), and under some conditions by 2′-O-acetyl-ADP-ribose (OAADPR) (Grubisha et al. 2006; Tong et al. 2010). Little is known about the physiological role of the TRPM2 channel, but evidence indicates that TRPM2 is a significant contributor to pathological neuronal Ca2+ overload and cell death caused by H2O2-induced oxidative stress (Aarts et al. 2005; Kühn et al. 2005; Miller 2006; Miller et al. 2011). Oxidative stress is a major factor in neurological damage in ischemia (Saito et al. 2005; Sugawara et al. 2004), thus many have hypothesized an important role for TRPM2 in ischemia (Aarts et al. 2005; Jiang et al. 2010; Miller 2006; Miller et al. 2011; Takahashi et al. 2011). We recently tested this hypothesis in vitro and in vivo and to our surprise, TRPM2 inhibition or knockdown was neuroprotective in the male neurons or brain specifically, but have no effect on outcome in females (Jia et al. 2011; Verma et al. 2012). This gender specific effect of TRPM2 inhibition in vitro and focal cerebral ischemia is of particular interest considering difference in vulnerability to cerebral ischemia between male and females in the human population (Kim et al. 2001; Vukmir 2003; Wigginton et al. 2002).

While our previous work indicates that TRPM2 plays an important role in OGD and focal cerebral ischemia in male but not female neurons or mice (Jia et al. 2011; Verma et al. 2012), we do not know whether this sex-difference translates to transient global ischemia. Thus, in this study we test the hypothesis that TRPM2 plays an important role in global cerebral ischemia in the male but not the female mouse brain. Here we demonstrate that TRPM2 inhibition is neuroprotective in male but not female mice in a normothermia CA/CPR model of global cerebral ischemia. Coupled with our previous publications, these results suggest that TRPM2 inhibition may hold therapeutic potential as a neuroprotectant against ischemic neurological damage in men.

Results

Asystolic CA was induced and followed by resuscitation in male and female C57Bl/6 wild type mice (22–25g) to determine whether TRPM2 inhibition is neuroprotective in a gender specific manner in mice following global cerebral ischemia. The experimental procedures were performed as previously described (Allen et al. 2011; Nakano et al. 2010), with modifications to temperature control to maintain temperatures near physiological level. These experiments were carried out in a randomized manner by a single investigator blinded to treatment. Animals were anesthetized with isoflurane, intubated, and mechanically ventilated during surgery and at time of resuscitation. Temperature probes were placed into the left temporalis muscle and the rectum. The right jugular vein was cannulated for drug administration. Electrocardiogram was monitored throughout the experimental procedures. CA was induced by intravenous injection of 50µL cold 0.5 mol/l KCL and was confirmed by appearance of asystole on the electrocardiography monitor and no spontaneous breathing. Animals were resuscitated 8 minutes after CA with chest compressions (300/min), slow administration of epinephrine (0.5ml, 16µg/ml), and ventilation with 100% oxygen. As soon as return of spontaneous circulation (ROSC) was achieved, defined as electrocardiographic activity with visible cardiac contractions, chest compression was stopped. Vehicle control or the TRPM2 inhibitor clotrimazole (CTZ) were administered by subcutaneous injection 30 minutes after resuscitation. 30 minutes was chosen as it represents a reasonable therapeutic window for drug administration following CA/CPR. CPR duration, epinephrine dose, and survival rates were not different between vehicle and CTZ treatment groups (Table 1). However, survival rates were significantly higher in the female vs. male mice (Table 1). In contrast to our previous CA/CPR model which consisted of elevated head temperatures in combination with peripheral hypothermia (Allen et al. ; Kofler et al. 2004; Nakano et al. 2010)), CA/CPR was performed under normothermic conditions (Figure 1). Rectal body temperature was allowed to decrease during the arrest to a minimum of 35°C and then restored to physiological temperatures following resuscitation. Pericranial temperature was maintained at 37.5°C throughout the procedure. Pericranial temperature was regulated by a circulating heated-water coil placed around the head. Figure 1 illustrates the tight temperature control obtained and the lack of difference observed in treatment group or gender. One female mouse treated with vehicle, two male mice treated with vehicle, and three male mice treated with clotrimazole were excluded from the reported data due to surgical complications.

Table 1. CA/CPR related parameters in male and female mice.

Survival rates were significantly higher in females compared to males (P<0.05). Otherwise, no significant difference was observed across conditions. N indicates number of mice included in each experimental group. BW indicates body weight. Neuroscore is a general health assessment including awareness and motor control/strength and is presented as median with 25% and 75% quartiles. All other data indicates mean average � standard error of the mean (SEM).

Male mice Female mice
Vehicle Clotrimazole Vehicle Clotrimazole
N 11 11 9 8
Body weight (g) 25.4 ± 0.6 26.2 ± 0.6 23.3 ± 0.3 23.1 ± 0.5
Day 1 21.6 ± 0.5 22.2 ± 0.5 19.9 ± 0.4 19.9 ± 0.5
Day 2 21.0 ± 0.5 21.8 ± 0.4 19.0 ± 0.5 18.5 ± 0.4
Day 3 21.5 ± 0.6 22.3 ± 0.4 19.8 ± 0.8 20.0 ± 0.6
Total weight loss (%) −15.0 ± 1.8 −14.6 ± 2.1 −15.2 ± 3.0 −13.4 ± 2.6
CPR duration (sec) 73.3 ± 6.3 74.5 ± 5.9 69.8 ± 6.6 76.8 ± 3.1
Epinephrine (µg) 8.1 ± 0.1 8.4 ± 0.2 8.2 ± 0.2 8.2 ± 0.2
Epinephrine (µg/g BW) 0.3 ± 0.01 0.3 ± 0.01 0.4 ± 0.01 0.4 ± 0.02
Neuroscore
Day 1 3 (3–6.5) 4 (3–5) 4 (3–10) 4 (3–4)
Day 2 3 (1–5.5) 2 (1–2.5) 3 (2–6.0) 3 (2–3)
Day 3 2 (1–3.5) 2 (1–2.5) 2 (1–4.0) 2 (1.8–2.0)
Surviving animals (%) 62 (13/21) 70 (14/20) 91 (10/11) 89 (8/9)
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Figure 1. CA/CPR was performed under normothermic conditions in male and female mice.

Figure 1

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Rectal body temperature was monitored in male (A) and female (C) mice. Rectal body temperature decreased during CA, but was to physiological temperatures following resuscitation. Pericranial temperature was monitored in male (B) and female (D) mice and was maintained at 37.5°C throughout the procedure except for a brief decrease in temperature during resuscitation.

General health condition was assessed daily during light phase by an investigator who was blinded to the experimental group following CA/CPR (Table 1). The general health condition score was determined by adding up evaluation scores under each category as previously described (Nakano et al. 2010; Allen et al., 2011) and latency to move was evaluated by the time (sec) required for the mouse to move to the outside of a 12-cm diameter circle after being placed in the center. General health condition and latency to move analysis did not reveal differences among treatment groups or gender. Three days (72 hours) after CA/CPR, mice were deeply anesthetized with 5% isoflurane and transcardially perfused and fixed with 10% formalin. Brains were removed, embedded in paraffin and 6µm coronal sections were serially cut. The CA1 region of the hippocampus was analyzed, three levels (100 µm apart) from −1.5 mm bregma. Sections were stained with hematoxylin and eosin (H&E) for analysis of damaged neurons, determined by the presence of pink eosinophilic cytoplasm and dark pyknotic nucleus. All viable and nonviable neurons were counted for each microscopic field, and the percentage of nonviable neurons was calculated for the entire CA1 region. CA/CPR induced delayed neuronal cell death in the CA1 region of the hippocampus in both male and female mice. Consistent with previous reports, the degree of neuronal injury following ischemia in females (27.3±11.1%) was less than in males (46.4±11.0, P<0.05). There was no statistically significant difference between vehicle and clotrimazole treated female mice (Figure 2, CTZ: 23.2 ± 6.6%, Vehicle: 27.3 ± 11.1%). In contrast, CTZ reduced CA/CPR induced neuron death in the CA1 region of the hippocampus in male mice (Figure 3, CTZ: 18.4 ± 5.2%, Vehicle: 46.4 ± 11.0%, p<0.05). Combined with previously reported results, these results indicate that TRPM2 inhibition is neuroprotective in the male but not female mouse following CA/CPR, lending support to the hypothesis that TRPM2 inhibition has therapeutic potential for the treatment of cerebral ischemia in men.

Figure 2. TRPM2 inhibition has no effect on CA/CPR induced delayed neuronal cell death in the CA1 region of the hippocampus in female mice.

Figure 2

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There was no significant difference in neuronal cell death in the hippocampus between vehicle and clotrimazole treated female mice (Figure 2, CTZ: 23.2 ± 6.6%, Vehicle: 27.3 ± 11.1%). Bottom panels (B, C) show representative hematoxylin and eosin stained hippocampal sections from vehicle or clotrimazole treated mice. Sections were analyzed for damaged neurons and quantified in (A). Damaged neurons were determined by the presence of pink eosinophilic cytoplasm and dark pyknotic nucleus. Representative damaged neurons indicated by red arrows and injured neurons by red arrows.

Figure 3. TRPM2 inhibition reduces CA/CPR induced delayed neuronal cell death in the CA1 region of the hippocampus in male mice.

Figure 3

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Cell death was higher in vehicle treated males than in females (compare with figure 2). (A) Clotrimazole significantly reduced neuronal cell death in male hippocampus (CTZ: 18.4 ± 5.2%, Vehicle: 46.4 ± 11.0%, p<0.05). Bottom panels (B, C) show representative hematoxylin and eosin stained hippocampal sections. Representative damaged neurons indicated by red arrows and injured neurons by red arrows

Discussion

The results presented here demonstrate that clotrimazole, a TRPM2 inhibitor, is neuroprotective in a normothermic CA/CPR model of global cerebral ischemia in the male but not the female mouse. These results are consistent with our previous results showing a male-specific response to TRPM2 inhibition or shRNA-mediated knockdown in OGD in vitro, and with our recent studies demonstrating male-specific neuroprotection following TRPM2 inhibition and knockdown in an in vivo model of experimental stroke (Jia et al. 2011; Verma et al. 2012). In our CA/CPR model, we observed decreased neuronal damage in females vs. males, consistent with previous studies indicating that the female mouse is less vulnerable to ischemic brain damage (Herson et al. 2009), likely due to the neuroprotective effects of estrogen (Herson et al. 2009; Lang et al. 2008). This observation raises the possibility that clotrimazole was not protective in females because endogenous female hormones had already provided the maximum possible neuroprotection against CA/CPR. However, we and others have demonstrated neuroprotection in females with various compounds (Canal Castro et al. 2012; Kelley et al. 2011; Lebesgue et al. 2009), and thus we believe the lack of protection by clotrimazole is due to a male-specific role of TRPM2 in ischemia.

It is unclear why the TRPM2 channel induces neurotoxicity in the male but not the female following cerebral ischemia in mice. Several possibilities present themselves. TRPM2 is activated by intracellular ADP-ribose (Fonfria et al. 2004; Kolisek et al. 2005; Kraft et al. 2004; Perraud et al. 2005). It is possible that cerebral ischemia results in the higher production of ADP-ribose in males vs. females, or that oxidative stress is greater in males vs. females. Indeed, studies have shown that the male is particularly vulnerable to the over-activation of poly (ADP-ribose) polymerase-1 (PARP-1) (Lang et al. 2008; Vagnerova et al. 2010). PARP-1 generates branching chains of ADP-ribose, which is then hydrolyzed to monomeric ADP-ribose by the enzyme poly (ADP-ribose) glycohydrolase (PARG) (Blenn et al. 2011). However, evidence for sexually dimorphic activation of PARP-1 following cerebral ischemia remains sparse. Several other enzymes can also produce the ADP-ribose necessary to activate the TRPM2 channel. These enzymes include those that metabolize NAD+ such as CD38, mono-ADP-ribosyl transferase, and ADP-ribose cyclases (Ma et al. 2012). TRPM2 is also activated by OAADPR in a puromycin injury model of cell death in HEK293 cells (Grubisha et al. 2006). OAADPR is product of SIRT2 activity (Tong et al. 2010). SIRT2 is a histone deacetylase enzyme implicated in cell cycle control and neurite outgrowth (Harting et al. 2010; Sauve et al. 2012). Little is known about the role of SIRT2 in ischemia, but recent evidence suggests that SIRT2 may be involved in oxidative stress in a model of Parkinson’s disease (Liu et al. 2012), a disease that affects more men than women (Van Den Eeden et al. 2003). Thus, it is possible that SIRT2 contributes to TRPM2 mediated cell death in a gender specific manner.

A limiting factor in progress regarding physiological and pathophysiological roles of TRPM2 is the lack of selective inhibitors. Several compounds have been demonstrated to inhibit TRPM2 in various experimental settings, including 2-aminoethoxy diphenyl borate (2-APB), N-(p-amylcinnamoyl)anthrancilic acid (ACA), flufenamic acid and clotrimazole (Harteneck 2005). Our previous in vitro studies utilized all four of these compounds to confirm the role of TRPM2 in male-specific ischemic injury (Jia et al. 2011; Verma et al. 2012). The fact that each compound has a different spectrum of non-TRPM2 effects makes it reasonable to conclude that our data demonstrating that each of these inhibitors protects male neurons from OGD without effecting female neurons implicates TRPM2 in male injury. Furthermore, our recent work with clotrimazole in experimental stroke makes it the most promising pharmacological tool to assess the role of TRPM2 in CA/CPR-induced neuronal injury. A possible limitation of this study is the ability of clotrimazole to interact with various other ion channels, including Ca2+-activated K+ channels (Jensen et al. 1998), the ATP-gated K+ channels (Jager et al. 2004) and the L-type voltage gated Ca2+ channels (Fearon et al. 2000; Thomas et al.). However, we consider it a remote possibility that CTZ neuroprotection is via one of these off-target ion channels as none of them have been shown to have the sex-specific effect observed here. More importantly, our previous work with CTZ in culture and in vivo models of hypoxia and ischemia have shown that the protective effects of CTZ in the male mouse neuron is most likely due to TRPM2 inhibition (Jia et al. 2011; Verma et al. 2012). Thus, while it is possible that the protective effects of clotrimazole are due to non-TRPM2 targets, it is probable that neuroprotection in males is due to TRPM2 inhibition in this model of global cerebral ischemia. Development of more selective pharmacological inhibitors of TRPM2 would provide a powerful new tool for researchers interested in the role of this ion channel in disease. These results together with our previously reported studies provide evidence that TRPM2 activation mediates neuronal cell death in ischemia in the male mouse.

These results are particularly interesting in light of observations that indicate that the male brain is more susceptible to ischemic insult. Additionally, TRPM2 is activated under pathological conditions following glutamate receptor activation, downstream of oxidative stress (Aarts et al. 2005; McNulty et al. 2005; Miller 2006; Miller et al. 2011; Takahashi et al. 2011). The lack of identifiable physiological role for TRPM2 and the relatively delayed activation of TRPM2 following injury may result in fewer side-effects and a longer therapeutic window for the treatment of ischemia, making TRPM2 an attractive target for therapeutic intervention for cerebral ischemia in males.

Acknowledgements

This work supported in part by R01NS058792 and the Walter S. and Lucienne Driskill Foundation Award.

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