Inhibition of cytosolic phospholipase A2 alpha protects against focal ischemic brain damage in mice

brain research 1471 (2012) 129–137

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Research Report

Inhibition of cytosolic phospholipase A2 alpha protects against focal ischemic brain damage in mice Jian Zhang, Noah Barasch, Rung-Chi Li, Adam Sapirsteinn Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, 720 Rutland Ave, Ross 347, Baltimore, MD 21205, USA

art i cle i nfo

ab st rac t

Article history:

It is postulated that inhibition of cytosolic phospholipase A2 alpha (cPLA2a) can reduce

Accepted 25 June 2012

severity of stroke injury. This is supported by the finding that cPLA2a-deficient mice are

Available online 20 July 2012

partially protected from transient, focal cerebral ischemia. The object of this study was to

Keywords:

determine the effect of cPLA2a inhibition with arachidonyl trifluoromethyl ketone (ATK) on

Arachidonic acid

stroke injury in mice. Male C57BL/6 mice were subjected to 1 h of focal cerebral ischemia

Cyclooxygenase-2

followed by 24 or 72 h of reperfusion. Mice were treated with ATK or vehicle by intermittent

Stroke

intraperitoneal injection or continuous infusion via an implanted infusion pump. ATK

Inflammation

injections 1 h before and then 1 and 6 h after the start of reperfusion significantly reduced infarction volumes in striatum and hemisphere after 24 h of reperfusion. ATK did not reduce injury if it was not administered before onset of ischemia or was not administered after 6 h of reperfusion. Intermittent doses of ATK failed to reduce infarct volume after 72 h of reperfusion. Continuous infusion with ATK throughout 72 h of reperfusion significantly reduced cortical and whole hemispheric infarct volume compared to vehicle treatment. Following ischemia and reperfusion, ATK treatment significantly reduced brain PLA2 activity. These results are the first to demonstrate a therapeutic effect of cPLA2a inhibition on ischemia and reperfusion injury and define a therapeutic time window. cPLA2a activity augments injury in the acute and delayed phases of cerebral ischemia and reperfusion injury. We conclude that cPLA2a inhibition may be clinically useful if started before initiation of cerebral ischemia. & 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Brain injury after ischemia and reperfusion is a complex process that is the result of acute necrosis and delayed injury mechanisms such as excitotoxicity, inflammation, and apoptosis (Moskowitz et al., 2010). Metabolism of arachidonic acid and the resulting eicosanoid products have been implicated in these injury mechanisms (Ward et al., 2011). In large part, cellular levels of arachidonic acid are controlled by the phospholipase A2 (PLA2) enzymes. PLA2s comprise an enzyme family that hydrolyzes the ester bond at the second (sn-2) n

Corresponding author. Fax: þ1 410 955 8978. E-mail address: [email protected] (A. Sapirstein).

0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.06.031

position of glycerolphopholipids to liberate free fatty acids. Cerebral ischemia has long been associated with PLA2 activity, and this PLA2 activity has been correlated with neuronal injury (Arai et al., 2001; Bonventre et al., 1997; Brady et al., 2006; Ward et al., 2011). The PLA2s include a large number of proteins that are classified according to their structures, cellular location, enzymatic mechanisms, and calcium dependence. The cytosolic PLA2 class contains several members that have a common cytosolic intracellular location. The cPLA2 alpha (cPLA2a) form has unique characteristics that include its

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brain research 1471 (2012) 129–137

preference for arachidonic acid at the sn-2 position of phospholipid substrate molecules. Therefore it has been postulated that cPLA2a may play a significant role in generating neurological injury following ischemic brain injury. Direct evidence for the role of cPLA2a in ischemic brain injury has been shown in cPLA2a-knockout (cPLA2a/) mice, which are partially protected from transient focal cerebral ischemia (Bonventre et al., 1997). Subsequent experiments in rats demonstrated that cPLA2a expression is increased 1 day after ischemia and reperfusion and that cPLA2a activity correlates with brain injury (Nito et al., 2008). In mice subjected to 2 h of regional ischemia, cPLA2a expression was correlated with increased oxidative stress and cellular changes even before reperfusion (Kishimoto et al., 2010). Studies of the cyclooxygenase-2 enzyme (COX-2) have also indicated that arachidonic acid metabolism can have a profound impact on the propagation of experimental and clinical stroke. Inhibition of COX-2 has been shown to reduce experimental stroke (Candelario-Jalil et al., 2007; Dore´ et al., 2003; Iadecola et al., 2001; Nogawa et al., 1997; Sugimoto and Iadecola, 2003). Unfortunately, clinical use of specific COX-2 inhibitors increases the overall cardiac risk profile of patients, and these drugs are not recommended for prophylaxis against stroke injury (van Staa et al., 2008). Substantial experimental evidence indicates that cPLA2a is a key component along the pathway of stroke injury (Bonventre et al., 1997; Brady et al., 2006; Kishimoto et al., 2010; Shen et al., 2007). This evidence has been obtained, in large part, through experiments that have used cPLA2-deficient mice. In the present studies, we evaluated whether the use of a chemical inhibitor of cPLA2a could reduce stroke injury in a mouse model of unilateral cerebral ischemia and reperfusion. In addition, we characterized the therapeutic time window of cPLA2a inhibition in relation to the timing of the ischemic event.

2.

Results

We evaluated the effectiveness of a mixed cPLA2a/GVIA inhibitor, arachidonyl trifluoromethyl ketone (ATK), on mouse brain injury following transient, focal ischemia and reperfusion because it has been shown to be effective in vivo in other

models of central nervous system (CNS) injury (Kalyvas and David, 2004). In the first experiments, we modified a protocol that had been used previously to demonstrate that inhibition of COX-2 effectively reduces the injury from cerebral ischemia and reperfusion in mice (Nagayama et al., 1999). Male C57BL/6 mice were given intraperitoneal (IP) injections of ATK (either 1 or 10 mg/kg) or an equivalent volume of vehicle 1 h before 1 h of unilateral ischemia. At 1 and 6 h after reperfusion, ATK was administered at the same dosage as the initial dose. The COX2-specific inhibitor NS398 (10 mg/kg) was administered to a separate group of mice according to the same protocol as a positive control for injury prevention. In a separate group of mice we initiated laser Doppler flowmetry immediately preceding treatment with ATK 10 mg/kg or vehicle and continued it until 15 min after reperfusion. There was no difference in relative blood flow attributed to ATK at any time (Fig. 1). Mice were sacrificed after 24 h of reperfusion, and the relative amount of tissue injury was measured by triphenyltetrazolium chloride (TTC) staining. ATK 1 mg/kg treatment had no effect on infarction when compared to vehicle treated mice (Supplementary Fig. S1). ATK 10 mg/kg effectively decreased the cerebral injury caused by ischemia and reperfusion with a 43% reduction in infarct volume of the ischemic hemisphere when compared to the vehicle-treated mice (Fig. 2A, Po0.05, n¼ 10). This difference in injury was largely due to protection conferred in the striatum, which suffered significantly less infarction than did the same region in the vehicle-treated mice (57% relative reduction in injury; Po0.05). There was also a trend toward decreased injury in the cortex of mice treated with ATK (ATK, 22.276.4% vs. vehicle, 36.475.3%). When compared to mice that were treated with NS398 (Fig. 2B), ATK provided similar injury reduction in cortex and hemisphere (ATK, 22.276.4%, 17.273.9% vs. NS398, 17.276.4%, 15.174.1%; P¼0.59, 0.62). We next conducted an experiment to determine if delayed inhibition of cPLA2a protects mice from ischemia and reperfusion injury. We administered ATK 10 mg/kg or vehicle 1 and 6 h after reperfusion but did not treat the mice before ischemia. There was no difference in injury after 24 h of reperfusion between ATK and vehicle in this post-ischemia treatment experiment (data not shown).

Fig. 1 – ATK treatment does not alter relative cerebral blood flow. Laser Doppler flow measurement of cerebral blood flow was performed (as described in Methods) immediately before 10 mg/kg ATK or vehicle treatment. Relative blood flow was measured continuously throughout 60 min of ischemia and first 15 min of reperfusion.

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Fig. 3 – Acute inhibition of cPLA2a with ATK does not provide protection from focal cerebral ischemia after 72 h reperfusion. C57BL/6 mice were treated with vehicle or 10 mg/kg ATK 1 h before ischemia and at 1 and 6 h after initiation of reperfusion. Infarct volumes (7SD) in striatum, cortex, and hemisphere were measured after 72 h of reperfusion (n ¼9/group).

Fig. 2 – ATK or NS398 treatment protects mice from focal cerebral ischemia and 24 h of reperfusion. (A) C57BL/6 mice were injected intraperitoneally with 10 mg/kg ATK or an equal volume of vehicle 1 h before ischemia and again 1 and 6 h after initiation of reperfusion. Striatal and hemispheric infarct volumes were significantly less in the ATK-treated mice (n¼ 7) than in the vehicle-treated mice (n ¼10) at 24 h of reperfusion. (B) Mice were injected intraperitoneally with 10 mg/kg NS398 or an equal volume of vehicle 1 h before ischemia and again 1 and 6 h after initiation of reperfusion. Cortical, striatal, and hemispheric infarct volumes were significantly less in the NS398-treated mice (n ¼10) than in the vehicle-treated mice (n ¼10) at 24 h of reperfusion. Po0.05.

We further examined whether inhibition of cPLA2a for periods of treatment less than 7 h after injury could also protect mice from stroke injury. We treated the mice with one dose of ATK 10 mg/kg or vehicle 1 h before ischemia or with ATK doses 1 h before ischemia and 1 h after reperfusion. We again examined infarct volume 24 h after reperfusion. In each case, infarct volume did not differ between mice treated with one or two doses of ATK and those treated with vehicle (data not shown). The absence of neurologic protection with only two doses of ATK suggested that cPLA2a activity contributes to mechanisms of injury that occur in later phases of stroke progression. To test this possibility, we repeated treatment of the mice with ATK at 1 h before and 1 and 6 h after reperfusion initiation and measured the cerebral infarct volume at 72 h of reperfusion. As expected, the injury had evolved by 72 h of reperfusion as demonstrated by increased infarct volumes (Fig. 3). In contrast to the results at 24 h of reperfusion, the three-dose treatment regimen with ATK conferred no protection at 72 h of reperfusion when compared to vehicle-treated mice (Fig. 3). ATK was originally described as a suicide inhibitor of cPLA2a (Street et al., 1993). However, ATK is metabolized within cells

Fig. 4 – Continuous infusion with ATK reduces ischemiainduced brain infarct volume after 72 h of reperfusion. Mice were treated with 10 mg/kg ATK (n¼ 10) or vehicle (n ¼ 10) 1 h before ischemia and at 1 and 6 h after initiation of reperfusion. In addition, ATK (0.4 lmol per day) or vehicle was continuously administered by an osmotic pump implanted in the peritoneum at the time of operation for ischemia. Infarct volumes (7SD) in striatum, cortex, and hemisphere were measured after 72 h of reperfusion. Cortical and hemispheric infarct volumes were significantly smaller in ATK-treated mice than in vehicle-treated mice. Po0.05.

to an inactive metabolite, a fact that may limit the duration of its activity in our in vivo model of cerebral ischemia and reperfusion. To determine if continuous cPLA2a inhibition during reperfusion could decrease cerebral injury, we delivered ATK by continuous IP infusion with an implanted osmotic pump. Mice were treated with a continuous infusion of ATK (0.4 mmol/day) or the equivalent volume of vehicle for a period of 72 h starting 1 h before initiation of ischemia. Continuous ATK treatment significantly reduced the infarct volume in the ischemic hemisphere (Fig. 4; ATK, 11.273.1% vs. vehicle, 31.176.4%, Po0.05, n¼ 10/group). In contrast to 24 h of reperfusion, at 72 h of reperfusion, ATK significantly protected the cortex but not the striatum from injury (ATK cortex, 12.575.5% vs. vehicle cortex, 38.278.1%, Po0.05).

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Functional neurologic deficits were measured in mice treated with intermittent and continuous drug delivery at 24 and 72 h after reperfusion. After 24 h of reperfusion, there was a small trend toward reduction in deficits in the drugtreated groups that did not reach significance (Fig. 5). Interestingly, the neurologic deficit scores (NDS) in the intermittent ATK, intermittent NS398, and continuous ATK treatment groups were essentially identical after 24 h of reperfusion. In contrast to the findings after 24 h reperfusion, after 72 h there was statistically significant reduction in the NDS of continuous ATK infusion mice compared to the continuous vehicletreated mice (Fig. 5). Because ATK inhibits both cPLA2a and GVIA PLA2 we measured these activities in brain homogenates from mice that had been treated with the 3 doses of either vehicle or ATK. We were unable to detect any differences in either cPLA2a or GVIA PLA2 activity following ATK treatment in mice that had not been subjected to cerebral ischemia and reperfusion (data not shown). However, following 1 h of

Fig. 6 – ATK treatment inhibits cPLA2a and GVIA PLA2 activity in brain homogenates collected following ischemia and reperfusion. C57BL/6 mice were injected intraperitoneally with ATK 10 mg/kg or an equal volume of vehicle 1 h before ischemia and again 1 and 6 h after initiation of reperfusion. PLA2 activity was measured in homogenates of the ischemic (ipsilateral) and non-ischemic (contralateral) hemispheres using in vitro assays optimized for (A) cPLA2a and (B) GVIA PLA2. PLA2 activity was reduced by ATK when compared to vehicle treatment for each condition. Data are shown as means7SD; n¼ 3/group. Po0.05; Po0.01; Po0.001.

Fig. 5 – Continuous ATK treatment improves the neurologic deficit score (NDS) after 72 h of reperfusion. (A) NDS after 24 h of reperfusion in mice subjected to 1 h cerebral ischemia and injected IP with vehicle (n ¼ 10), 10 mg/kg NS398 (n ¼10), or 10 mg/kg ATK (n ¼7). (B) NDS after 24 and 72 h of reperfusion in mice subjected to 1 h cerebral ischemia and continuous infusion with vehicle or ATK (n¼ 10/group; Po0.05 by Mann–Whitney Rank Sum test).

ischemia and 6 h of reperfusion ATK administration significantly inhibited both cPLA2a (Fig. 6A) and GVIA PLA2 activity (Fig. 6B) in both the ipsilateral and contralateral hemispheres. It has been reported that at high concentrations, ATK inhibits COX-2 in vitro. We next determined if the doses of ATK used in this study could inhibit COX-2 in vivo. Because the liver has significantly higher levels of COX-2 than brain, we performed COX activity assays on liver obtained from mice immediately after 72 h administration of either vehicle or ATK (Yang et al., 2004). The COX activities in the livers of ATK- and vehicle-treated mice were identical (ATK, 12.5971.16 nmol/min/mg vs. vehicle, 13.2771.19 nmol/min/ mg, P40.05, n¼ 3), indicating that ATK did not inhibit COX in the protocol that was used in this study. Because ATK inhibits both cPLA2a and GVIA PLA2 it is possible that its protective effect is due in part to GVIA PLA2 inhibition. To test this we treated cPLA2a/ mice with 3 doses of either ATK 10 mg/kg or vehicle using the same cerebral ischemia and reperfusion protocol. There was no difference in the brain injury measured after 24 h of reperfusion between the treatments (Fig. 7). This leads us to

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Fig. 7 – ATK treatment of cPLA2a/ mice does not reduce brain injury from cerebral ischemia and reperfusion. cPLA2a/ mice were injected intraperitoneally with 10 mg/kg ATK (n ¼8) or an equal volume of vehicle (n¼ 7) 1 h before ischemia and again 1 and 6 h after initiation of reperfusion. Infarct volumes were measured 24 h after reperfusion.

conclude that the protection of ATK treatment is the result of cPLA2a inhibition. Inhibition of any PLA2 activity and in particular cPLA2a has the potential to alter the levels of arachidonic acid and its metabolites. The generation of prostaglandin E2 through the COX-2 enzyme is an important step in the signaling cascade for fever in the mouse (Ushikubi et al., 1998). Therefore, it is possible that inhibition of cPLA2a by ATK could disrupt temperature regulation after ischemia and reperfusion. To determine if ATK altered the temperature regulation of mice and if a lowered core temperature was an unanticipated mechanism for the neuroprotection of ATK, we measured mouse core body temperature throughout the 72 h experimental window. We implanted radiofrequency temperature probes in the contralateral side of the neck of the mice during the surgery to perform filament placement in the middle cerebral artery. This allowed remote temperature measurement of the mice and prevented stress induced hyperthermic responses (Bouwknecht et al., 2007). The temperatures of individual mice were measured remotely every 15 min during the ischemic period and then every hour for the first 6 h of reperfusion. In the subsequent period, the temperature was recorded every day. As shown in Fig. 8, temperatures did not differ between the ATK- and vehicle-treated mice. There were also no differences in the temperatures when compared as a function of the time from ischemia onset.

3.

Discussion

This is the first study to demonstrate that acute inhibition of cPLA2a effectively reduces brain injury after cerebral ischemia and reperfusion in mice. Furthermore this set of experiments describes a therapeutic time window for the efficacy of ATK in stroke injury amelioration. Unfortunately this study indicates that even a small delay in the administration of the cPLA2a inhibitor causes failure to protect from injury. In addition, these experiments also indicate that it is necessary

Fig. 8 – Core temperature of mice is not altered by ATK treatment after transient cerebral ischemia. C57BL/6 mice were treated with 10 mg/kg ATK or vehicle 1 h before onset of ischemia and at 1 and 6 h after initiation of reperfusion. Temperature was monitored remotely by radiofrequency probe. Data are shown as means7SD; n ¼9/group.

to maintain inhibition throughout the reperfusion period in order to limit stroke progression. These results have noteworthy implications for the potential use of cPLA2a inhibitors in the clinical setting. It has been recognized for over 10 years that the cPLA2a/ mouse is partially protected from brain injury after transient ischemia and reperfusion (Bonventre et al., 1997). Subsequent work on the role of cPLA2a in stroke also utilized this mouse model in which cPLA2a expression has been genetically removed from all cells (Kishimoto et al., 2010; Shen et al., 2007). The possibilities that compensation for the congenital absence of cPLA2a or that nonenzymatic properties of cPLA2a could impact the results of studies with the cPLA2a/ mouse have not been previously tested. Indeed, we and others have demonstrated that basal and stimulated central nervous system (CNS) levels of COX-2 are reduced in the cPLA2a/ mouse (Bosetti and Weerasinghe, 2003; Sapirstein et al., 2005).

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Therefore, it is possible that neuroprotection by genetic inactivation of cPLA2a in a variety of neuronal injury models is due to chronic, secondary effects of cPLA2a deletion such as decreased COX-2 expression or changes in expression of other forms of PLA2. The fact that inhibition of cPLA2a is protective in the transient, focal ischemia model indicates that its activity contributes to the injury cascade following reperfusion. This finding is consistent with previous work in which ATK prevented both the increased Ca2þ transient and broadening of the action potential in stimulated CA1 hippocampal neurons that were exposed to NMDA (Shen et al., 2007). In other work, we demonstrated that cPLA2a/ mice are also protected from the early morphological changes and oxidative stress that follow ischemia and reperfusion (Kishimoto et al., 2010). The effect of cPLA2a on ischemia and reperfusion injury extends beyond the phase of acute injury. If cPLA2a played a role only in early phase injury mechanisms then we would have expected the three-dose ATK protocol to have demonstrated protection 72 h after reperfusion. Instead we found that 72 h after reperfusion, the injury was equivalent in ATKand vehicle-treated groups. This finding implies that a secondary injury mechanism is also cPLA2-dependent. Such mechanisms have been suggested previously because blood–brain barrier disruption after filament occlusion of the middle cerebral artery in rats is associated with increased cPLA2a activity at 24 h of reperfusion (Nito et al., 2008). Interestingly, levels of cPLA2a and phosphorylated cPLA2a proteins peak 3 day after reperfusion in the rat model (Nito et al., 2008). Our current work also supports a role for cPLA2a in secondary phases of the injury cascade, as continuous infusion with ATK reduced the injury at 72 h of reperfusion relative to the vehicle-treated mice. The apparent reduction in the infarct volumes following 72 h of reperfusion of mice that were treated with a continuous infusion of vehicle compared to mice that were treated with 3 injections of vehicle may be the result of the abdominal surgery (compare Fig. 2A with Fig. 4). The intra-abdominal implantation of the osmotic pump is a major difference between this experiment and intermittent injection and it could have had an attenuating effect on the severity of the cerebral injury through a variety of mechanisms. The NDS is not as sensitive to focal ischemic injuries as histological analysis. However, the finding that NDS is reduced after 72 h in the mice that were continuously infused with ATK (Fig. 5) does support the theory that cPLA2a is contributing to functional injury. We cannot speculate as to the effects of continued inhibition of cPLA2a beyond 72 h of reperfusion. It is possible that this could have harmful or beneficial effects on stroke injury and remodeling. Future studies on the effects of cPLA2a on long term outcomes will be needed to assess these possibilities. We used ATK to inhibit cPLA2a because it had been used previously in a model of CNS injury and had demonstrated effects in the CNS (Kalyvas and David, 2004). ATK inhibits both cPLA2a and GVIA (calcium-independent) PLA2, both of which exist in the brain (Yang et al., 1999b). Indeed, the relative enzymatic activity of GVIA PLA2 in the brain far exceeds that of cPLA2a(Yang et al., 1999b). In our experiments

it appears that ATK inhibited CNS PLA2 activity only after ischemia and reperfusion injury. This result suggests that blood brain barrier injury may be required for this drug to reach CNS sites. Furthermore, we demonstrated that it significantly inhibited not only cPLA2a but also the GVIA PLA2 activity (Fig. 6). For this reason, the current series of experiments does not eliminate the possibility that the observed neuroprotection may result from inhibition of GVIA PLA2. However, we do not think that inhibition of GVIA PLA2 is the protective mechanism in our experiments because (1) ATK treatment did not further reduce injury in cPLA2a/ mice (Fig. 7), (2) previous data indicate that the required PLA2 activity in excitotoxic injury is Ca2þ-dependent (Shen et al., 2007), and (3) ATK treatment confers a level of neuroprotection to ischemia and reperfusion injury similar to that seen in cPLA2a/ mice (Bonventre et al., 1997). Future experiments that measure dose response and use more selective cPLA2a inhibitors will be needed to verify whether cPLA2a inhibition is the mechanism for neuroprotection by ATK. In these experiments, the cPLA2a inhibitor was administered systemically rather than within the CNS. This method of administration has been demonstrated previously to be effective in vivo (Kalyvas and David, 2004). In pilot experiments, we administered ATK by intracerebral injection. This method of administration caused cerebral injury and confounded evaluation of the drug effect versus the injection effect (data not shown). The effect of IP ATK injection on neuroprotection could result from both systemic and central cPLA2a inhibition. For example, cPLA2a is required for normal leukocyte, macrophage, and platelet function (Adler et al., 2008; Bonventre et al., 1997; Wong et al., 2002), and since these circulating cells may adhere to cerebral microvessels and infiltrate the CNS, they can exacerbate ischemia and amplify neuroinflammation (Barone et al., 1995; Ren et al., 2011; Yilmaz and Granger, 2010). Systemic inhibition of cPLA2a could also modulate COX-2 levels in both circulating and parenchymal cells in a number of organs. Importantly, in vitro measurement of COX activity in the liver indicated that ATK did not inhibit COX-2 and by extension did not reduce its level of expression. Because our previous work showed that cPLA2a deficiency and ATK administration decrease the excitotoxic effects of NMDA treatment in hippocampal slices, we believe that the early effects of ATK on ischemia and reperfusion injury are most likely the result of CNS inhibition (Shen et al., 2007). Additional experiments will be needed to distinguish between the systemic and central effects of cPLA2a inhibition on neuroinflammation following ischemia and reperfusion. The original description of neuroprotection in the cPLA2a/ mouse after transient cerebral ischemia and reperfusion boosted hopes that a chemical cPLA2a inhibitor might one day be used clinically as a stroke therapy. The results of this study now suggest that such a drug is unlikely to be of benefit if started after the ischemic insult. This result will reduce enthusiasm for the use of cPLA2a inhibition as an acute stroke therapy. However it is still possible that prophylactic cPLA2a inhibition can reduce brain injury in clinical settings where patients are known to be at risk of ischemia and reperfusion. This possibility should motivate further exploration of this potential therapy.

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4.

Experimental procedures

4.1.

Materials

Model 3000D Alzet osmotic infusion pumps were obtained from DURECT Corporation (Cupertino, CA). IPTT-200 implantable temperature transponders and the DAS-5002 notebook scanner were from Bio Medic Data Systems (Seaford, DE). The cPLA2a inhibitor ATK and the COX-2 inhibitor NS398 were purchased from Cayman Chemical Co. (Ann Arbor, MI) and dissolved in 150 mM NaCl (pH 12) for all experiments. Unless otherwise stated, all other compounds were purchased from Sigma-Aldrich Company (St. Louis, MO).

4.2.

Animal care

All experiments were conducted in accordance with the guidelines of the National Institutes of Health and approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Male C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice deficient in cPLA2a (cPLA2a/) were the gift of Joseph V. Bonventre (Brigham and Women’s Hospital and Harvard Medical School; Boston, MA.). All mice were housed in a facility with a 12 h diurnal light cycle and had free access to food and water.

4.3.

Focal cerebral ischemia

Anesthesia was induced and maintained in mice by spontaneous ventilation of isoflurane in 30% O2. A thermostatically controlled warming pad and infrared light were used to maintain the rectal temperature at 37.570.5 1C during all phases of the surgery. In order to monitor the regional blood flow in the ischemic core of the right hemisphere a laser-Doppler flow probe was secured on the skull. The flow probe was affixed with glue 2 mm posterior and 3 mm lateral to the bregma 1 mm caudal to the coronal suture on exposed temporal bone of the ipsilateral hemisphere. This location corresponds to the territory of the middle cerebral artery that becomes deeply ischemic upon occlusion (Huang et al., 1994). Transient focal ischemia was induced by intraluminal placement of a silicone coated 7.0 nylon filament in the middle cerebral artery (MCA) of male C57BL/6 and cPLA2a/ mice that were between 12 and 16 weeks of age. Filament placement in the right middle cerebral artery and sham surgery were carried out through a vertical cervical incision. The right common carotid artery was proximally ligated and the external carotid artery was divided to leave a proximal arterial stump. An arteriotomy was made in the external carotid artery stump through which the filament was directed under operative microscopy into the internal carotid artery and advanced into the MCA. Occlusion of flow was documented by decrease of laser Doppler flow by greater than 70% of baseline. One hour after filament placement, the mice were re-anesthetized, the filament was removed, and the mice were placed in a temperaturecontrolled environment. In these experiments there was an 8.75% mortality which was associated with intracerebral bleeding that was most likely due to arterial puncture with the suture. Mice that died during or immediately after the surgery (within the first hour) were not included in the analysis. During these

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experiments there was no delayed mortality. In preliminary experiments we compared body temperature measurements made with an IPTT-200 implantable temperature transponder that was placed either in the contralateral side of the neck through the surgical incision or intraabdominally to temperature measurements made with a rectally placed temperature probe. We found that temperature measurements from the neck were systematically though not significantly lower than intra-abdominal measurements (Supplementary Fig. S2). Placement of the probe through the cervical incision allowed us to reliably obtain the temperature of the unrestrained mice remotely at indicated time points with the DAS-5002 scanner. Functional stroke injury was evaluated by NDS immediately after reperfusion and before euthanasia. An observer who was blinded to treatment groups assigned an NDS between 0 and 4 in which 0¼ no deficit; 1¼ forelimb weakness; 2¼ circling to affected side; 3¼unable to bear weight on affected side; 4¼no spontaneous motor activity (Longa et al., 1989). Stroke injury was measured after either 24 or 72 h of reperfusion by using laboratory standard volumetric analysis of anterior and posterior views of five coronal slabs stained with TTC and corrected for swelling. The effect of systemic treatment with the cPLA2a inhibitor ATK or the COX-2 inhibitor NS398 on stroke injury was compared to treatment with vehicle. In some experiments ATK, NS398, or vehicle was delivered intermittently by IP injection using various modifications to a three-dose schedule in which 10 mg/kg was given 1 h before onset of ischemia, 1 h after reperfusion, and 6 h after reperfusion. In one experiment, mice that underwent 72 h of reperfusion and were treated with the three-dose regimen were supplemented by continuous IP drug delivery through an osmotic infusion pump that was implanted into the peritoneal cavity at the beginning of ischemia.

4.4.

PLA2 assays

Mouse hemispheres were rapidly harvested after perfusion with 4 1C PBS and immediately homogenized by polytron in iced buffer (10 mM HEPES (pH 7.4), 1 mM EDTA, 10 mM PMSF, 10% v/v glycerol). The homogenate was centrifuged at 10,000g at 4 1C for 15 min, the supernatant was removed and protein concentration was measured using a modified Bradford assay. Modified, group specific, mixed micelle PLA2 assays were performed in duplicate using 30 mg of supernatant protein by slight modification to previously described assays (Sapirstein et al., 1996; Yang et al., 1999a). In brief, PLA2 substrate was prepared with 40 nCi of 1-Palmitoyl-2-[1-14C]arachidonyl-phosphatidylcholine (100,000 CPM/assay) and incubated with protein in assay buffer for 30 min at 37 1C. The assay buffer for cPLA2a was 75 mM Tris (pH 7.4), 5 mM CaCl2, and 2 mM DTT. The assay buffer for GVIA PLA2 was 100 mM HEPES (pH 7.4), 5 mM EDTA, 1 mM ATP, and 2 mM DTT. Free aracahidonic acid was extracted by phase separation and radioactivity was measured by liquid scintillation counting.

4.5.

COX-2 assay

Liver tissue was removed from mice that underwent 72 h of reperfusion and were administered vehicle or ATK. Frozen

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tissue was weighed and homogenized by a dounce homogenizer in 100 mL/10 mg ice-cold lysis buffer (0.1 M Tris–HCl [pH 7.8], 1 mM EDTA), followed by brief sonication. Homogenate was centrifuged at 10,000g for 15 min at 4 1C and supernatant collected for assay. Protein concentration was determined by modified Bradford assay. COX activity was measured by using a commercially available kit (Cayman Chemical Co., Ann Arbor, MI) according to the manufacturer’s instructions. Equal amounts of protein were assayed in 100 mL sample volumes, and COX activity was determined by absorbance at 595 nm.

4.6.

Statistical analysis

Group means were compared by unpaired t-test or ANOVA followed by post-hoc t-tests. Comparison of neurologic deficit scores was made with the Mann–Whitney Rank Sum test. P values o0.05 were considered to be statistically significant. All data are presented as means7SD.

Acknowledgments This work was supported by the Department of Anesthesiology and Critical Care Medicine of Johns Hopkins School of Medicine and Grants from the NIH-NINDS (R01NS48978 to AS) and the American Heart Association (09GRNT2261454 to AS). The authors would like to thank Claire Levine for editing the manuscript and Ray Koehler for scientific review and advice.

Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.brainres.2012.06.031.

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