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Food and Chemical Toxicology 100 (2017) 175e182

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate/ foodchemtox

UPEI-400, a conjugate of lipoic acid and scopoletin, mediatesneuroprotection in a rat model of ischemia/reperfusion

Barry J. Connell a, Monique C. Saleh a, Desikan Rajagopal c, Tarek M. Saleh a, b, *

a Department of Biomedical Science, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canadab Department of Biomedical Science, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canadac Department of Chemistry & Pharmaceutical Chemistry, School of Advanced Sciences, VIT University, Vellore, India

a r t i c l e i n f o

Article history:Received 21 October 2016Received in revised form29 November 2016Accepted 20 December 2016Available online 23 December 2016

Keywords:StrokeAntioxidantCo-drugReperfusion-injuryAnimal model

* Corresponding author. Department of BiomedicaCollege, University of Guelph, Guelph, Ontario, Canad

E-mail address: tsaleh@uoguelph.ca (T.M. Saleh).

http://dx.doi.org/10.1016/j.fct.2016.12.0260278-6915/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

Previously, our laboratory provided evidence that lipoic acid (LA) covalently bonded to various antiox-idants, resulted in enhanced neuroprotection compared to LA on its own. The naturally occurringcompound scopoletin, a coumarin derivative, has been shown in various in vitro studies to have bothantioxidant and anti-inflammatory mechanism of actions. The present investigation was designed todetermine if scopoletin on its own, or a co-drug consisting of LA and scopoletin covalently bondedtogether, named UPEI-400, would be capable of demonstrating a similar neuroprotective efficacy. Using arat stroke model, male rats were anesthetized (Inactin®; 100 mg/kg, iv), the middle cerebral artery waspermanently occluded for 6 h (pMCAO), or in separate animals, occluded for 30 min followed by 5.5 h ofreperfusion (ischemia/reperfusion; I/R). Pre-administration of either scopoletin or UPEI-400 significantlydecreased infarct volume in the I/R model (p < 0.05), but not in the pMCAO model of stroke. UPEI-400was ~1000 times more potent compared to scopoletin alone. Since UPEI-400 was only effective in amodel of I/R, it is possible that it may act to enhance neuronal antioxidant capacity and/or upregulateanti-inflammatory pathways to prevent the neuronal cell death.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

According to the most recent published data in 2016, death anddisability due to stroke remains a global medical issue and thera-peutic options remain limited (Mozaffarian et al., 2016). Ischemicstroke, resulting from the occlusion of a cerebral artery, accounts forup to 80% of all strokes (Ginsberg, 2008). A transient or permanentreduction of cerebral blood flow results in hypoxia, hypoglycemia,the failure of ATP-dependent pumps and a disruption of ionic equi-librium, leading to the generation of reactive oxygen species (ROS)and free radicals, and the upregulation of cytokine inflammatorypathways, ultimately ending in neuronal damage and death(Ginsberg, 2008; Margaill et al., 2005). Research into antioxidant andanti-inflammatory therapy to prevent and/or scavenge ROS and freeradical generation has provided hope that the damage due toreperfusion injury may be mitigated, and that the window of op-portunity for therapeutic treatment, such as tissue-plasminogen-activator (TPA), may be extended beyond the currently accepted

l Science, Ontario Veterinarya.

time frame of 3e4 h following the onset of stroke.Scopoletin (6-methoxy-7-hydroxycoumarin) is a coumarin de-

rivative found in various plants and has a long history of use for itsmedicinal properties in traditional Chinese medicine (Kang et al.,1999). Scopoletin is isolated from Canarium patentinervium Miq(Burseraceae Kunth), a rare plant from the family of Burseraceae andgenus Canarium found in the Asia-Pacific region and has been used inwound healing by the indigenous people of Malaysia (Mogana et al.,2011). In particular, evidence has been provided to suggest that sco-poletin has both antioxidant and anti-inflammatory properties andhas been used to ameliorate the symptoms of various inflammatory,rheumatoid and digestive disorders (Kang et al.,1999). In vitro studieshave demonstrated that scopoletin has antioxidant properties, is aneffective inhibitor of the activation of 5-lipoxygenase and has anti-acetylcholinesterase activity (Mogana et al., 2013). To date, scopole-tin has not been tested in any in vivo model of stroke (ischemia orischemia/reperfusion), but other coumarin derivatives have beentested with some success (Moa et al., 2011; Sun et al., 2003).

Clinically, single drug therapies following stroke have not yieldedthe positive clinical trial results expected when compared to thelaboratory testing of these compounds. Due to the multifactorial na-ture of ischemia and reperfusion injury, it is likely that a particulardrug will be unable to affect several pathways simultaneously. Thus,

B.J. Connell et al. / Food and Chemical Toxicology 100 (2017) 175e182176

there has been a recent interest in the use of co-drugs as a therapeuticapproach in various pathologies (Bansal and Sliakari, 2014;Minnerupand Schabitz, 2009). The development and administration of a co-drug, containing lipoic acid (LA) covalently linked to another com-pound, have been demonstrated to have a greater efficacy/potencycompared totheadministrationofamixtureof the twodrugs (Connellet al., 2012a,2012b,2014;Daset al., 2010;Salehetal., 2014;Sozioet al.,2010). The antioxidant LA, is a known free radical scavenger (Bilskaand Wlodek, 2005; Biewengo et al., 1997), and many researchershave shown that in several different stroke models administration oflipoic acid on its own can be neuroprotective but only at relativelyhighdoses (Connell et al., 2011;Richardet al., 2011;Clarkeet al., 2001;Panigrahi et al., 1996; Wolz and Krieglstein, 1996; Cao and Phillis,1995). Further, combining lipoic acid with other drugs has beenshown to produce an additive or synergistic protective effect inseveral different animal models of pathology (Mufherjee et al., 2011;Bano and Bhatt, 2010; Babu et al., 2011; Garcia-Estrada et al., 2009;Sola et al., 2005; Shotton et al., 2004; Gonzalez-Perez et al., 2002),when compared to the effect of either drug alone. Our laboratory hasdemonstrated that LA covalently bonded to various naturally occur-ring antioxidant compounds has provided superior neuroprotectionfollowing I/R injury compared to either drug alone or a mixture of 2compounds administered simultaneously (Connell et al., 2012a,2012b, 2014; Saleh et al., 2014, 2015). Based on the above observa-tionsdemonstrating thebeneficialeffectsofdevelopingaco-drug,ourlaboratory has developed a new synthetic co-drug that is a covalentconjugate between LA and scopoletin, named UPEI-400.

In the present study, using a model of acute stroke and reper-fusion injury (I/R) in male rats developed in our laboratory (Connelland Saleh, 2010), we determined whether the administration ofscopoletin alone is neuroprotective, and if the administration ofUPEI-400 would provide a further improvement of the neuro-protection observed with scopoletin alone. Further, a neuro-protective dose of UPEI-400 was then tested at various time pointsfollowing reperfusion to examine the neuroprotective capability ofUPEI-400 in a more clinically relevant paradigm.

2. MATERIALS and METHODS

2.1. Ethics statement

All experiments were carried out in accordance with theguidelines of the Canadian Council on Animal Care and wereapproved by the University of Prince Edward Island Animal CareCommittee (protocol #11-045 and 13-036).

2.2. General surgical procedures for in vivo stroke surgery

All experiments were conducted on male Sprague-Dawley rats(300e350 g; Charles Rivers; Montreal, PQ, CAN). For all animals, foodand tap water were available ad libitum. Rats were anesthetized withthe long-lasting barbiturate anesthetic, sodium thiobutabarbital

(Inactin®; Sigma-Aldridge; St. Louis, MO, USA; 100 mg/kg; ip). Supple-ments were given if a withdrawal reflex was elicited following cornealor toe pinch every 10min. All animalswere tracheotomized to facilitatebreathing and then placed on a heating blanket to maintain bodytemperature at 37 ± 1 �C (Physitemp Instruments; Clifton, NJ, USA).

2.3. Transient and permanent middle cerebral artery occlusion(tMCAO and pMCAO)

We have previously published the detailed methodology forocclusion of the middle cerebral artery (Connell et al., 2012a;Connell and Saleh, 2010; Saleh et al., 2014, 2015) in both perma-nent (pMCAO) and transient (tMCAO) models. Briefly, the middlecerebral artery (MCA) was exposed via a craniotomy, and suturethread placed under 3 separate branches of the MCA. The sutureswere either occluded for 6 h (pMCAO) or occluded for 30 min,followed by removal of the sutures and the MCA allowed toreperfuse for an additional 5.5 h (tMCAO).

2.4. UPEI-400 synthesis

Scopoletin and lipoic acid were chemically linked via an esterbond using a simple synthetic route. Accordingly, scopoletin(0.012 g) was placed in a dry 100 ml flask followed by lipoic acid(0.025M in 0.02 ml of dimethylaminopyridine; DMAP) in 100 mlanhydrous dichloromethane (CH2Cl2). Dicyclo-hexylcarbodiimide(DCC) (0.025M) was added in small quantities over a period of1 h under nitrogen atmosphere. The reaction was stirred overnightand the compound was purified using silica column chromatog-raphy after an aqueous work up. Proton (1H) nuclear magneticresonance spectroscopy and mass spectrometry was used to char-acterized the pure compound, UPEI-400.

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2.5. Drug injection protocol in tMCAO model

Scopoletin, UPEI-400 or vehicle were administered (1 ml/kg;i.v.) 30 min prior to the onset of MCAO (�60 on schematic). Thesutures were left in place for 30 min (duration of ischemia; MCAOon schematic), and then removed to allow the return of blood flowand reperfusion which lasted for 5.5 h (0e330 min on the sche-matic). Post-MCAO administration of UPEI-400 was investigated byadministering the lowest dose of UPEI-400 which was previouslyshown to produce significant neuroprotection at the following timepoints during the ischemia/reperfusion protocol: 15 min prior tothe onset of reperfusion (�15), and 30, 90, 150 or 210min followingthe start of reperfusion.

2.6. Effect of scopoletin on ischemia-reperfusion (tMCAO model)

In the first set of animals, scopoletin (1.0 (n ¼ 6), 0.1 (n ¼ 6) or0.01 (n¼ 6)mg/kg) was administered in a volume of 1ml/kg (i.v.) orvehicle (30% DMSO; 1 ml/kg; i.v.; n¼ 8) 30 min prior to the onset ofMCAO (�30 min). Following this 30 min occlusion, sutures wereremoved and the region allowed to reperfuse for an additional5.5 h.

2.7. Effect of UPEI-400 on ischemia-reperfusion (tMCAO model)

In the second set of experiments, UPEI-400 (0.1 (n ¼ 6), 0.01(n¼ 6), 0.001 (n ¼ 6) or 0.0001 (n ¼ 6) mg/kg) was administered ina volume of 1 ml/kg (i.v.) or vehicle (0.05% DMSO; 1 ml/kg; i.v.;n ¼ 6) 30 min prior to the onset of MCAO (�30 min). The sutureswere left in place for 30 min, followed by 5.5 h of reperfusion.

2.8. Effect of UPEI-400 on infarct volume when administered eitherduring the occlusion or reperfusion

To determine if UPEI-400 was neuroprotective when adminis-tered during the 30 min period of occlusion, the optimal dose ofUPEI-400, which based on the dose-response-curve describedabove was 0.001 mg/kg, was administered 15 min into the 30 minocclusion period. The sutures were then removed for an additional5.5 h of reperfusion.

To examine the effect of UPEI-400 on reperfusion injury alone,injections of the optimal dose of UPEI-400 (0.001 mg/kg; 1 ml/kg;iv; n ¼ 5e6 per group) were made 30, 90, 150 or 210 min followingsuture removal (start of reperfusion). Experiments were thenterminated at the end of the 5.5 h of reperfusion. In all cases, thecomparison group far statistical analysis was UPEI-400 vehicle(0.05% DMSO; n ¼ 6).

2.9. Effect of UPEI-400 on ischemia alone (permanent occlusion;pMCAO)

To determine if administration of UPEI-400 (0.001 mg/kg; iv;

1 ml/kg; n ¼ 5) was neuroprotective on ischemia-induced celldeath only, injections of UPEI-400 were made 30 min prior topMCAO. Following 6 h of occlusion, the experiment was termi-nated. Similar to the studies on tMCAO described above, the vehiclefor UPEI-400 tested in the pMCAO model was also 0.05% DMSO(1 ml/kg; iv; n ¼ 5).

2.10. Effect of UPEI-400 on baseline blood pressure and heart rate

To determine the hemodynamic effect of UPEI-400 on baseline(resting) blood pressure and heart rate, continuous recordings ofblood pressure and heart rate were taken prior to and for 2 hfollowing UPEI-400 (0.001 mg/kg; 1 ml/kg; iv; n ¼ 4) or vehicle(0,05% DMSO, 1 ml/kg; iv) administration. Cardiovascular data

acquisition and analysis was done as described in our previousstudies (Connell and Saleh, 2010). Measurements of the bloodpressure and heart rate values were made 5 min immediately priorto drug administration and at 5, 10, 15, 30, 45, 60, 90 and 120 minfollowing drug administration.

2.11. Histological procedures

At the end of each experiment where infarct volume wasmeasured, animals were overdosed with Inactin (100 mg/kg), theirdiaphragm transected and left ventricle of the heart perfused withphosphate buffered saline (PBS 0.1 M; 200 ml) via an 18-gaugeneedle. The brains were removed and infarct volumes measuredas previously described (Connell et al., 2012a; Connell and Saleh,2010; Saleh et al., 2014, 2015). Digital images were coded prior tobeing sent for analysis of infarct volume to allow the analyst to beblinded to treatment groups.

2.12. Statistical analysis

All data was analyzed using SigmaStat and SigmaPlot (JandelScientific, Tujunga, CA). Data are presented as a mean ± standarderror of the mean (S.E.M). Using analysis of variance (ANOVA) fol-lowed by a Bonferroni posthoc analysis, differences were consid-ered significant if p � 0.05. If only two groups were beingcompared, Student's t-test was used.

3. Results

3.1. The effect of pre-administration of scopoletin on ischemia-reperfusion (tMCAO)

Pre-administration of scopoletin resulted in a dose-dependentneuroprotection with 1.0 mg/kg resulting in a significant decreasein infarct volume compared to the administration of vehicle(p � 0.05; Fig. 1A and B).

(A) Representative photomicrographs of TTC-stained, 1 mmthick coronal slices illustrating the extent of the infarct within the

Fig. 1. Dose-dependent neuroprotection observed with scopoletin.Fig. 2. Dose-dependent neuroprotection observed with UPEI-400 following tMCAO,but not pMCAO.

B.J. Connell et al. / Food and Chemical Toxicology 100 (2017) 175e182178

prefrontal cortex following pretreatment (30 min prior to MCAO;i.v.) with either vehicle (30% DMSO) or scopoletin (1.0 mg/kg)following ischemia-reperfusion (tMCAO). (B) Bar graph summari-zing the dose-response relationship between increasing doses ofscopoletin and infarct volume (mm3) calculated from TTC-stained,1 mm thick coronal sections following tMCAO. Each bar representsthe mean ± S.E.M. (n ¼ 6 or 8/group) and * indicates significance(p � 0.05) from the vehicle-treated control group.

3.2. The effect of pre-administration of UPEI-400 on ischemia-reperfusion (tMCAO)

Pre-administration of UPEI-400 resulted in a dose-dependentneuroprotection with 0.001, 0.01 and 0.1 mg/kg resulting in a sig-nificant decrease in infarct volume compared to the administrationof vehicle (p � 0.05; Fig. 2A1 and 2B1).

(A) Representative photomicrographs of TTC-stained, 1 mmthick coronal slices illustrating the extent of the infarct within theprefrontal cortex following pretreatment (30 min prior to MCAO;i.v.) with either vehicle (0.05% DMSO) or UPEI-400 (0.001 mg/kg)following ischemia-reperfusion (tMCAO) or permanent ischemia

(pMCAO). (B) Bar graph summarizing the dose-response relation-ship between increasing doses of UPEI-400 and infarct volume(mm3) calculated from TTC-stained, 1 mm thick coronal sectionsfollowing tMCAO. Also, a bar graph illustrating the effect of UPEI-400 (0.001 mg/kg; n ¼ 5) injected 30 min prior to 6 h of perma-nent middle cerebral artery occlusion (pMCAO) on infarct volume(mm3) calculated from TTC-stained, 1 mm thick coronal sections.(vehicle ¼ 0.05% DMSO; n ¼ 5). Each bar represents themean ± S.E.M. (n ¼ 6/group) and * indicates significance (p � 0.05)from the vehicle-treated control group.

3.3. The effect of pre-administration of UPEI-400 on permanentischemia (pMCAO)

To examine the effect of UPEI-400 on ischemia-induced celldeath only, administration of the optimal dose of UPEI-400 iden-tified in the dose-response curve in Fig. 2A2 (0.001 mg/kg) orvehicle were made 30 min prior to 6 h of pMCAO (sutures left inplace for 6 h). UPEI-400 pre-administration did not produce sig-nificant neuroprotection when infarct volume was measured 6 hfollowing the start of pMCAO (p � 0.05; Fig. 2B2).

Fig. 3. Time-course of UPEI-400 induced neuroprotection.

B.J. Connell et al. / Food and Chemical Toxicology 100 (2017) 175e182 179

3.4. The effect of UPEI-400 administration on infarct volume whenadministered either during ischemia or after the start of reperfusion

Administration of UPEI-400 (0.001 mg/kg) 15 min into the30 min period of MCAO (15 min prior to the start ofreperfusion; �15 min on schematic) produced significant neuro-protection compared to vehicle when infarct volumewasmeasuredfollowing 5.5 h of reperfusion (p � 0.05; Fig. 3A).

(A) Representative photomicrographs of TTC-stained, 1 mmthick coronal slices illustrating the extent of the infarct within theprefrontal cortex following treatment with either vehicle (0.05%DMSO) or UPEI-400 (0.001 mg/kg) 15 min prior to the beginning ofreperfusion (�15), immediately prior to suture removal (0), or 30,90, 150, or 210 min (n ¼ 5 or 6/group). (B) Effect of UPEI-400(0.001 mg/kg) on infarct volume (mm3) following injection15 min prior to the beginning of reperfusion (�15), immediatelyprior to suture removal (0), or 30, 90, 150, or 210 min (n ¼ 5 or 6/group) following the start of reperfusion. Each bar represents themean ± S.E.M. and * indicates significance (p � 0.05) from thevehicle control group (0.05% DMSO; n ¼ 5) injected 30 min prior to

tMCAO.We determined the effect of UPEI-400 on reperfusion injury

only by measuring the infarct volume when the drug was onlyadministered at 30, 90, 150 or 210min after the start of reperfusion.Administration of UPEI-400 at all time points up to 150 minfollowing the start of reperfusion resulted in significantly smallerinfarct volumes compared to vehicle administration (p � 0.05 ateach time point; Fig. 3B).

3.5. The effect of UPEI-400 administration on blood pressure andheart rate

The following experiment was designed to determine the he-modynamic effect of administration of UPEI-400 (0.001 mg/kg) onmean arterial blood pressure and heart rate for a period of 2 hfollowing administration. Baseline mean arterial pressure andmean heart rate prior to drug administration was 110 ± 9 mm/Hgand 385 ± 16 bpm for the vehicle (0.05% DMSO) treated group, and,112 ± 5 mm/Hg and 390 ± 15 bpm for the UPEI-400 (0.001 mg/kg)treated group. Administration of either UPEI-400 or vehicle did not

B.J. Connell et al. / Food and Chemical Toxicology 100 (2017) 175e182180

significantly alter either arterial blood pressure or heart rate(p � 0.05) at any time point compared to respective pre-administration (baseline) values. Further, drug administrationvalues for blood pressure and heart rate were not significantlydifferent from those measured in vehicle-treated animals at anytime point (p � 0.05 at each time point).

4. Discussion

In this study, we determined that UPEI-400, a chemical combi-nation of two naturally occurring compounds, LA, and scopoletin,produced dose-dependent neuroprotection against neuronal celldeath as observed in a previously validated, novel model ofischemia-reperfusion (I/R) (Connell and Saleh, 2010). The resultsdemonstrated that UPEI-400 produced neuroprotection following5.5 h of reperfusion in a model of focal ischemia, which is restrictedto the prefrontal cerebral cortex. Further, the dose of UPEI-400required to produce significant neuroprotection (0.001 mg/kg)was 1000 fold less compared to the dose required for scopoletinalone (1.0 mg/kg), and 5000 fold less compared to the optimalneuroprotective dose of LA alone, observed previously in our lab(Connell et al., 2011). Also, the optimal dose of UPEI-400 producedsignificant neuroprotection when administered 15 min prior to thestart of reperfusion, and when administered 30, 90, and 150 minfollowing the onset of reperfusion. Clinically, the paradigm ofadministering UPEI-400 during, and/or following the occlusionwasto mimic the clinical situation in which therapy would be admin-istered at the time a patient presents to a hospital following theonset of a stroke, or following administration of thrombolytictherapy (to prevent reperfusion injury). Interestingly, UPEI-400 didnot produce neuroprotection when administered prior to a 6-hpermanent occlusive stroke (no reperfusion; pMCAO). This sug-gests that UPEI-400 may act to attenuate the oxidative stressobserved following transient occlusion (I/R), whereas UPEI-400 isineffective against the necrotic cell death primarily observed inpermanent ischemia. These results also suggest that UPEI-400provides neuroprotection against reperfusion injury alone, likelydue to the scavenging of free radicals, by the attenuation of ROSproduction, or the inhibition of upregulated inflammationpathways.

Many studies have demonstrated anti-inflammatory and anti-oxidant properties of scopoletin, but to the best of our knowledge,the neuroprotective capacity of scopoletin has not been tested in anin vivo stroke model. The utility of drug therapy on reactive oxygenspecies and inflammation-induced growth of ischemic injury usingmodels of reperfusion injury have been established by many labs(Ginsberg, 2008; Margaill et al., 2005; Wang et al., 2007). In vitrostudies have demonstrated that scopoletin suppressed the pro-duction of pro-inflammatory cytokines in RAW 246.7 and HMC-1 cell lines (Moon et al., 2007; Kim et al., 2004). Scopoletin has beenshown to inhibit the enzymatic activity of 5-lipoxygenase andacetylcholinesterase (Mogana et al., 2013; Khunnawutmanothamet al., 2016) and selectively inhibit MAO-b and increase dopaminelevels in rodent brain tissue in vitro (Basu et al., 2016). Scopoletinhas also been shown to have antioxidant properties when tested invarious antioxidant assays in vitro (Shaw et al., 2003; Malik et al.,2011) and is protective against glutamate-induced neurotoxicityas well as anti-cholinesterase activity in an HT22 cell line (Lee et al.,2015). To date, most of the investigations on the therapeutic effi-cacy of scopoletin in vivo have focused on systemic disorders. In vivostudies have demonstrated that scopoletin is able to ameliorate thesymptoms of various inflammatory, rheumatoid and digestivedisorders (TN1Deng et al., 2012; X1Ding et al., 2012).

Other coumarin derivatives have been shown to have antioxi-dant properties, which translated to neuroprotection. The

coumarin derivative esculetin has been shown to reduce oxidativestress in several in vitro models (Lee et al., 2011; Kim et al., 2008)and demonstrate anti-inflammatory properties in vivo (Kwon et al.,2011; Witaicenis et al., 2010). Esculetin has been shown to beneuroprotective in a mouse model of I/R following transient oc-clusion of the middle cerebral artery (Wang et al., 2012). Theseauthors also demonstrated that esculetin administration decreasedcleaved caspase-3 levels, upregulated levels of Bcl-2 and down-regulated levels of Bax. In addition, delayed administration ofesculetin up to 4 h following the start of reperfusion resulted in adecreased infarct size after 24 h of reperfusion. The coumarin de-rivative, osthole, displayed dose-dependent neuroprotection whencultured cortical neurons were exposed to 4 h of oxygen-glucosedeprivation followed by 24 h of reperfusion (Chen et al., 2011).These authors demonstrated that osthole prolonged the activationof extracellular signal-regulated kinase 1/2 (ERK1/2) and preventedto activation of c-Jun N-terminal kinase (JNK), two members of thefamily of mitogen-activated protein kinases (MAPK). Osthole hasalso been tested an in vivo rodent stroke model. Osthole adminis-tration at the beginning of reperfusion reduced infarct volumefollowing stroke induced by 2 h of monofilament suture placementat the base of the MCA (Moa et al., 2011). Osthole has also beenreported to be protective against myocardial, renal and intestinalischemia/reperfusion (Dong et al., 2013; XY1Dong et al., 2013;Zheng et al., 2013).

In the present study, UPEI-400 was not able to protect againstneuronal death measured following permanent MCAO. Permanentischemia is characterized by glutamate-induced neuronal toxicityultimately leading to necrosis (Ginsberg, 2008). Generation ofoxidative radicals and the upregulation of inflammatory pathwaysdo not appear to play an important role in ischemic damage andsubsequent cell death in an environment where there is a lack ofblood flow. Therefore, anti-oxidant and anti-inflammatory agentssuch as scopoletin or lipoic acid, would not be effective incombating the permanent ischemia and necrotic cell death asdemonstrated in the pMCAO model used in the current study.

Our results indicate that the chemical combination of scopoletinand lipoic acid produced a 1000-fold increase in I/R-induced neu-roprotection compared to scopoletin alone. Similar results havealso been presented from our laboratory. For example, using thesame stroke model, the synthetic combination of lipoic acid withresveratrol (UPEI-200; (Saleh et al., 2014)) produced a 200 foldincrease in neuroprotection compared to resveratrol alone, a 100fold increase in neuroprotection was found when lipoic acid wascombinedwith apocynin (UPEI-100; (Connell et al., 2012a)), and a 5fold increase in neuroprotection was found when lipoic acid wascombined with edaravone (UPEI-300; (Connell et al., 2014)). Otherlaboratories have also demonstrated the benefits of using chemicalcombinations of LA with other compounds to provide neuro-protective effects greater than either of the two compoundsadministered on their own. Covalent linkage of LA with ibuprofenhas been demonstrated to be neuroprotective in rodent models ofAlzheimer's disease where administration of the co-drug decreasedthe oxidative damage due to the infusion of Ab (1-40) (Minnerupand Schabitz, 2009). Also, a co-drug produced by chemically link-ing LA with L-dopa or dopamine decreased neuronal oxidativedamage associated with the administration of L-dopa or dopamineon their own (Di Stefano et al., 2006).

Many authors have described the multiplicity of mechanismsinvolved in ischemia- and reperfusion-induced neuronal damagefollowing an occlusive stroke and the difficulty in providing treat-ment in a clinical setting (Ginsberg, 2008; Green, 2008). Therefore,drugs targeting multiple pathways might overcome this dilemma.The synthesis and development of co-drugs using biologicallyrelevant molecules provide the ability to simultaneously target

B.J. Connell et al. / Food and Chemical Toxicology 100 (2017) 175e182 181

multiple pathways involved in the pathogenesis of neurologicaldiseases, specifically, pathways involved in the initiation of oxida-tive stress and inflammation following reperfusion. The resultspresented above support the idea that the combination of antiox-idant and/or anti-inflammatory compounds at subthreshold dosescan produce equal or enhanced neuroprotection. The advantage ofusing lower doses to achieve comparable or improved therapeuticeffects is to minimize side effects on other physiological systems asis seen in many current therapies. Our findings support theadvantage of combination therapy in stroke treatment both pro-phylactically and even during ischemia-reperfusion. UPEI-400 is apotential therapeutic candidate to protect against the negativeoutcomes associated with reperfusion injury.

Author contributions

TMS, BJC and MCS conceived and designed the experiments; BCand MCS performed the experiments; TMS and BJC analyzed thedata; DR contributed the UPEI-400 material; TMS wrote the paper.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The authors wish to thank the Atlantic Canada OpportunityAgency's Atlantic Innovation fund (file # 199294) awarded to TMSfor funding this project.

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.fct.2016.12.026.

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