edaravone offers neuroprotection in a diabetic stroke model via inhibition of endoplasmic reticulum...

Edaravone Offers Neuroprotection in a Diabetic StrokeModel via Inhibition of Endoplasmic Reticulum Stress

Krishnamoorthy Srinivasan and Shyam S. Sharma

Molecular Neuropharmacology Laboratory, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education &Research (NIPER), Punjab, India

(Received 19 May 2011; Accepted 3 July 2011)

Abstract: Recent investigations have postulated a link between oxidative stress and endoplasmic reticulum (ER) dysfunctionin cerebral ischaemic ⁄ reperfusion (I ⁄ R) injury. Diabetes is common amongst elderly patients with stroke and has been postu-lated to aggravate brain I ⁄ R damage by triggering oxidative as well as ER stress. We investigated whether treatment with eda-ravone (1–10 mg ⁄ kg), a potent free radical scavenger protects against cerebral I ⁄ R injury in rats associated with comorbidtype 2 diabetes. Diabetic rats exposed to 2-hr middle cerebral artery occlusion (MCAO) and 22 hr of reperfusion significantlyhad increased infarct, oedema volume and functional neurological deficits as compared to sham-operated rats. Also, the mas-sive DNA fragmentation accompanied by significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nickend labelling (TUNEL) positive cells was noticed in the ipsilateral penumbral brain region of diabetic I ⁄ R rats. The effects ofI ⁄ R injury were associated with significant up-regulation of 78 kDa-glucose-regulated protein (GRP78), CCAAT ⁄enhancerbinding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP ⁄ GADD153) and activationof caspase-12, markers of ER stress ⁄ apoptosis. Treatment with edaravone (3 and 10 mg ⁄ kg) significantly diminished thecerebral infarct, oedema volume and improved functional recovery of neurological deficits. In addition, edaravone treatmentameliorated the DNA fragmentation concomitantly with a significant decrease in induction of GRP78, CHOP ⁄ GADD153immunoreactivity ⁄ expression and activation of caspase-12 in the ischaemic brain hemispheres. Overall, the present dataindicate that edaravone offers good neuroprotection against diabetic stroke by interrupting the ER stress-mediated apoptoticpathways involving CHOP ⁄ GADD153 and caspase-12.

Endoplasmic reticulum (ER) stress plays an important rolein the pathogenesis of cerebral ischaemic ⁄ reperfusion (I ⁄ R)injury and diabetes. Diabetes is a proven risk factor forstroke, with a two to threefold increased risk for diabeticpatients compared with non-diabetic patients [1]. Further,diabetes exacerbates brain ischaemic damage which has beenrecently shown to be mediated via augmenting ER stress andapoptosis [2]. The ER is a eukaryotic organelle involved inprotein synthesis, folding and trafficking, calcium homoeo-stasis and lipid and steroid synthesis. Perturbations of ERhomoeostasis because of certain stress stimuli such as ischae-mia, nutrient deprivation, oxidative stress and ER Ca2+

depletion lead to accumulation of unfolded or misfoldedproteins within the lumen of ER, a condition known as ERstress [3]. To alleviate this stress, ER triggers an evolution-arily conserved adaptive signalling cascade termed as theunfolded protein response (UPR). The UPR is essentiallycarried out by three ER-transmembrane effectors proteins:the RNA-activated protein kinase-like ER-resident kinase(PERK), inositol-requiring enzyme (IRE1) and activatingtranscription factor 6 (ATF6). In the unstressed state,PERK, IRE1 and ATF6 activity is suppressed by binding of

the ER chaperone glucose-regulated protein 78 (GRP78) tothe ER-transmembrane effectors. In response to ER stress,GRP78 dissociates and binds to the unfolded protein tofacilitate refolding; this allows the activation of PERK,IRE1 and ATF6. The UPR includes translational attenua-tion, induction of molecular chaperones (GRP78) and ER-associated degradation. Overall, UPR activation can helpthe cell to cope with the ER stress, but if it is severe or unre-solved, the inflicted cells may undergo programmed celldeath [4].

The wealth of evidence suggests that reactive oxygen spe-cies (ROS) or oxidative stress plays a detrimental role in thepathophysiology of cerebral I ⁄ R injury [5,6]. ROS robustlygenerated during cerebral I ⁄ R are reported to induce ERstress and apoptotic cell death [7]. Further, the studies withtransgenic animals over expressing superoxide dismutase-1(SOD-1) revealed the reduced oxidative damage to ER andischaemic neuronal cell death [8,9]. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) (EDR), a potent novel free radicalscavenger, has been approved only in Japan for the treatmentof acute ischaemic stroke and still under clinical investigationin some countries [10]. EDR has been reported to reducebrain infarction and oedema after I ⁄ R injury in animal mod-els [11–13] as well as in stroke patients [10,14]. Experimentalevidence suggests that the possible mechanism of action ofEDR mainly encompasses decreasing oxidative stress ⁄ lipidperoxidation, pro-inflammatory response and protecting

Author for correspondence: Shyam S. Sharma, Department ofPharmacology and Toxicology, National Institute of PharmaceuticalEducation and Research (NIPER), Sector 67, S.A.S. Nagar, 160 062Punjab, India (fax 91-172-2214692, e-mail [email protected]).

Basic & Clinical Pharmacology & Toxicology, 110, 133–140 Doi: 10.1111/j.1742-7843.2011.00763.x

� 2011 The AuthorsBasic & Clinical Pharmacology & Toxicology � 2011 Nordic Pharmacological Society

neurovascular tissues after ischaemic stress [12,15]. Interest-ingly, EDR has been shown to elicit neuroprotection byinhibiting ER dysfunction in a mouse model of hypoxic-ischaemia under normoglycaemic conditions [16]. In additionto these anti-stroke effects, EDR has also been tested againstoxidative damage to various extracerebral organs. Of note,EDR exhibited protective effect against multiple low doses ofSTZ-induced diabetes [17] and certain microvascular diabeticcomplications [18].

Although the neuroprotective potential of EDR has beeninvestigated earlier, to our knowledge, there is no report thatdescribes neuroprotective effect of EDR in a rat model offocal cerebral I ⁄ R injury associated with comorbid type 2diabetes. In the present investigation, we tested whetherEDR would also be effective against diabetic stroke by sup-pressing the augmented ER stress ⁄ apoptotic cell death. Thisstudy also bears significance in view of updated stroke ther-apy academic industry roundtable (STAIR, 2009) recommen-dation reinforcing that experiments shall also be performedin animal models with comorbid conditions for more clinicalrelevance and better translation of efficacy of test com-pounds from pre-clinical models to clinical trials [19].

Materials and Methods

Animals. Male Sprague-Dawley rats (120–140 g) were procured fromthe central animal facility of the institute. The rats were fed regularpellet feed (Ashirwad Industries, Chandigarh) and potable water adlibitum. The experiments were duly permitted by the institutionalanimal ethics committee (IAEC), NIPER and performed in accor-dance with guidelines of the committee for the purpose of controland supervision of experiments on animals (CPCSEA), Governmentof India.

Induction of type 2 diabetes in rats. The induction of type 2 diabeteswas carried out in rats by combination of high-fat diet (HFD) feed-ing and low dose of streptozotocin (STZ; Sigma, St. Louis, MO,USA) treatment as previously described [20]. Briefly, the rats werefed with HFD for 2 weeks and then rendered diabetic by a singlelow dose of STZ (35 mg ⁄ kg, i.p.) treatment. Blood samples were col-lected initially and at the end of 4 weeks. The various biochemicalparameters such as plasma glucose, triglycerides and total cholesterollevels were measured using commercially available colorimetric kits(Accurex India Pvt Ltd, Mumbai, India). Plasma insulin was deter-mined using an ELISA kit (Linco Research, St. Charles, MO, USA).Only those rats with plasma glucose level of >300 mg ⁄ dl at the endof 4 week were considered diabetic and included in the study.

Induction of focal cerebral ischaemia in diabetic rats. The rats wererandomly assigned to the following experimental groups, namely 1)diabetic sham 2) diabetic I ⁄ R + Vehicle 3) diabetic I ⁄ R + EDR (1,3 and 10 mg ⁄ kg) treated. Focal cerebral ischaemia was induced indiabetic rats as per the middle cerebral artery occlusion (MCAO)method as previously described [21]. Briefly, these type 2 diabetic ratswere priorly administered with atropine sulphate (0.5 mg ⁄ kg, i.p.)before induction of anaesthesia with 4% halothane in a mixture of70% nitrous oxide and 30% oxygen. Anaesthesia was maintained byadministering 1% halothane using an anaesthesia system (HarvardApparatus Ltd, Eden Bridge, Kent, UK). Occlusion was performedusing a 3 ⁄ 0 nylon monofilament coated with 0.01% poly-L-lysine(Sigma, St. Louis, MO, USA) which was advanced up to 21 mm asthe distance from bifurcation or till the resistance was felt throughthe lumen of the external carotid artery into the internal carotidartery to block the origin of the right middle cerebral artery. Occlu-

sion was performed for a period of 2 hr followed by 22 hr of reperfu-sion by complete withdrawal of filament. After 22 hr of reperfusion,the rats were evaluated for certain neurological functional deficits,cerebral infarct and oedema volume. In the sham-operated group,only the filament was introduced into the external carotid artery butnot advanced. Rectal temperature was continuously monitored andmaintained at 37.0 € 0.5�C using a feedback-controlled homoeother-mic blanket system (Harvard Apparatus, Eden Bridge, Kent, UK).The anaesthetic gaseous mixtures were fine-adjusted to reach thevital physiological parameters such as pO2 (110–145 mm Hg), pCO2(30–45 mmHg) and pH (7.3–7.5) close to physiological limits andmaintained before, during MCAO and reperfusion [22].

Drug preparation and treatment. EDR (Tocris Bioscience, Ellisville,MO, USA) (1, 3 and 10 mg ⁄ kg), a potent free radical scavenger, wasdissolved in 1 N NaOH solution and neutralized with 1 N HCl toadjust the pH to 7.4 at the dose volume of 2 ml ⁄ kg. EDR wasadministered intraperitoneally (i.p.) immediately (within 2 min.) afterthe MCAO. The doses of EDR were selected based on a literaturereport [16]. The neuroprotective potential of EDR was evaluatedfrom both histological and functional neurological studies after22 hr of reperfusion as described below as compared to vehicle treat-ment.

Assessment of functional neurological deficits. The neurological defi-cits were evaluated after 22 hr of reperfusion as reported earlier [21].Neurological findings were scored on a five-point scale. No neurolog-ical deficit = 0, failure to extend right paw fully = 1, circling if pulledby tail = 2, spontaneous circling = 3 did not walk spontaneouslyand had depressed level of consciousness = 4.

Estimation of brain infarct and oedema volume. The rats were killed22 hr after reperfusion for the estimation of brain infarct andoedema volume. The brains were sliced into 2 mm thick coronal sec-tions and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC)solution, and the area of infarction and oedema volume was mea-sured using image analysis software (Leica Qwin, Wetzlar, Germany)as reported earlier [23]. Briefly, the infarct areas of all brain sectionswere cumulated to reach total infarct area that was multiplied by thethickness of brain sections to reach the volume of infarction.Oedema correction of infarct volume was performed using theformula, volume correction = (infarct volume · contralateralvolume) ⁄ ipsilateral volume. The volumes of both hemispheres werecalculated from which the oedema volume was derived by subtract-ing the contralateral volume from the ipsilateral volume.

Estimation of DNA fragmentation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) assay wascarried out to identify the extent of DNA fragmentation in paraffin-embedded brain section as described previously [23]. The 3¢ end ofthe fragmented DNA was labelled using the DNA fragmentationdetection kit – TdT-FragEL according to the manufacturer’s instruc-tions (Merck, USA). The TUNEL positive cells were counted fromthe penumbral region of the brain sections and expressed as percent-age of TUNEL positive cells compared with total cells.

Immunohistochemistry. Immunohistochemistry (IHC) analysis forthe in situ expression of various ER stress proteins such as GRP78and CHOP ⁄ GADD153 was performed as previously described usingVecta stain ABC kit (Vector Labs, Burlingame, CA, USA) [24]. Thespecific labelling was detected using diaminobenzidine as a substrate.The sections were counterstained with haematoxylin and observedunder light microscope (Leica) over ipsilateral penumbral region,and images were acquired with a CCD camera (Leica). As GRP78 isa constitutive protein, the immunoreactivity was measured based onimmunohistochemical scoring pattern. IHC scoring was carried outbased on the intensity of staining as follows: 1 – slight or no colour;2 – very low staining; 3 – moderate staining; and 4 – very intensestaining. However, CHOP ⁄ GADD153 is an inducible protein that is

134 KRISHNAMOORTHY SRINIVASAN AND SHYAM S. SHARMA

� 2011 The AuthorsBasic & Clinical Pharmacology & Toxicology � 2011 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 110, 133–140

quantitatively represented as per cent dark brown stained CHOP ⁄GADD153 positive cells as compared to total cells.

Western blotting. Western blotting was performed as described previ-ously [25]. For western blotting analysis, aliquots containing equalamount of protein were loaded in each well and subjected to 10–12%SDS-PAGE. The separated proteins were transferred onto nitrocellu-lose membrane and blocked with 3% bovine serum albumin for 2 hr.The membranes were then probed for GRP78 or caspase-12 proteinby incubating with the primary antibodies such as GRP78 (1:500,Santa Cruz Biotech. Inc., Santa Cruz, USA) or caspase-12 (1:1000;Cell Signaling, Davers, MA, USA) followed by incubation with alka-line phosphatase (AP)-conjugated secondary antibody (1: 5000,Sigma, USA) for 2 hr at room temperature. Blots were visualized byenzymatic reaction by incubating with a mixture of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium atroom temperature. Equal protein loading was confirmed by measur-ing b- actin. The densitometric analysis was performed using NIHImageJ analysis software. The values were normalized with b- actin.

Statistical analysis. Statistical analyses were performed using thestatistical analysis software Sigma Stat 2.0, USA. Data are presentedas mean € SEM unless otherwise stated. All the parameters except

neurological score were analysed using one-way analysis of variance(ANOVA) followed by Tukey’s multiple comparison test. Neurologicalscore is expressed as median and was analysed using Kruskal–Wallisone-way analysis of variance on ranks test followed by post hocDunn’s multiple comparison test. Differences were considered to besignificant if p < 0.05.

Results

Experiment 1: Effect of HFD ⁄ STZ on body weight andbiochemical parameters in rats.The feeding of HFD and STZ injection in rats produced typ-ical characteristics of type 2 diabetic conditions associatedwith significant increase in plasma glucose, triglyceride andtotal cholesterol levels. The effects were seen without signifi-cant reduction in plasma insulin levels as well as body weightcompared with normal control rats at the end of 4 weeks(table 1).

Experiment 2: Effect of EDR on histological and functionaloutcome measures.After I ⁄ R injury, the diabetic rats exhibited significantlylarger brain infarct and oedema volume when compared withthe sham-operated diabetic rats (fig. 1). The single-dosetreatment with EDR (3 and 10 mg ⁄ kg) produced a signifi-cant reduction in brain infarct and oedema volume. How-ever, low dose of EDR (1 mg ⁄ kg) did not significantly alterneurological damage (fig. 1). In addition, monitoring of thevital physiological parameters revealed that EDR treatmentelicited no significant change in body temperature as well asblood glucose level as compared to vehicle treatment (datanot shown).

Further, based upon functional assessment, the diabeticrats subjected to I ⁄ R manifested significant impairment inneurological score. Treatment with EDR (3 and 10 mg ⁄ kg)significantly improved functional recovery of neurologicaldeficits, as reflected from reduction in neurological score

Table 1.Body weight and biochemical profiles (plasma glucose, triglycerides,total cholesterol and insulin levels) of normal and diabetic rats.

Parameters Normal Diabetic

Body weight (g) 256.3 € 6.1 265.2 € 4.2Plasma glucose (mg ⁄ dl) 109.3 € 3.2 401.3 € 20.8***Plasma triglycerides (mg ⁄ dl) 42.6 € 13.2 181.4 € 20.1***Plasma total cholesterol (mg ⁄ dl) 53.3 € 4.3 174.3 € 17.2***Plasma insulin (ng ⁄ ml) 1.0 € 0.1 0.8 € 0.1

The combination of high-fat diet (HFD) and low-dose streptozoto-cin (STZ) treatment produced characteristic features of type 2diabetes marked by hyperglycaemia, hypertriglyceridaemia andhypercholesterolaemia in the presence of almost normal circulatinginsulin concentration (relative insulin deficiency) at the end of4 weeks of dietary manipulation as compared to normal control rats.The values are expressed as mean € SEM. n = 5–7. ***p < 0.001versus normal control rat group.

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Fig. 1. Effect of EDR on cerebral infarct and oedema volume in focal cerebral I ⁄ R injury associated with diabetes. The left panel indicates therepresentative TTC stained brain coronal sections of diabetic sham-, vehicle- and EDR- (1, 3, 10 mg ⁄ kg, i.p.) treated diabetic I ⁄ R rat groups(A). The vehicle-treated diabetic I ⁄ R rats had increased brain infarct (panel B) and oedema volume (panel C) that were significantly inhibitedby EDR (3 and 10 mg ⁄ kg, i.p.) treatment. The values are expressed as mean € SEM, n = 5–7. ***p < 0.001 versus diabetic sham; ###p < 0.001versus vehicle-treated diabetic I ⁄ R group. EDR-edaravone.

EDARAVONE OFFERS NEUROPROTECTION IN A DIABETIC STROKE MODEL 135


(fig. 2). However, there was no significant improvementfound with the low dose of EDR (1 mg ⁄ kg).

Experiment 3: Effect of EDR on DNA fragmentation.The ischaemic injury caused extensive DNA fragmentationas evidenced from marked increase in TUNEL positive cells

in the penumbral ipsilateral brain region of the diabetic ratsas compared to the sham-operated group. Treatment withEDR (10 mg ⁄ kg) significantly attenuated DNA fragmenta-tion in ipsilateral penumbral region (fig. 3). However, werarely detected any TUNEL positive cells in the contralateralregion of the brain (data not shown).

Experiment 4: Effect of EDR on GRP78 andCHOP ⁄ GADD153 immunoreactivity.The results from the immunohistochemistry experimentsrevealed a significant increase in the immunoreactivity of ERstress ⁄ apoptotic proteins viz., GRP78 and CHOP ⁄ GADD153in the ischaemic penumbral region of the diabetic I ⁄ R ratscompared with the sham-operated rats. Nevertheless, thetreatment with EDR (10 mg ⁄ kg) resulted in a significantreduction in GRP78 and CHOP ⁄ GADD153 immunoreactiv-ity as reflected from decreased IHC score and percentCHOP ⁄ GADD153 positive cells, respectively, compared withvehicle treatment (fig. 4).

Experiment 5: Effect of EDR on GRP78 and capsase-12expression.Consistent with IHC results, western blot analysis also sub-stantiated the similar pattern of GRP78 protein expressionin the various groups (fig. 5). In addition, the diabetic ratsexposed to I ⁄ R showed significant activation of caspase-12associated with pronounced reduction in the level in theischaemic, ipsilateral brain hemispheres after 22 hr of rep-erfusion. On the other hand, EDR treatment (10 mg ⁄ kg)significantly replenished caspase-12 level, possibly by inhib-iting its activation as compared to vehicle treatment(fig. 5).

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Fig. 2. Effect of EDR on functional neurological deficits in focalcerebral I ⁄ R injury associated with diabetes. There was significantimpairment in neurological score in vehicle-treated diabetic I ⁄ R ascompared to sham-operated rats. However, EDR (3 and 10 mg ⁄ kg,i.p.) significantly improved functional recovery of neurological defi-cits. The values are expressed as median, n = 5–7. *p < 0.05 versusdiabetic sham; #p < 0.05 versus vehicle-treated diabetic I ⁄ R group.EDR-edaravone.

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Fig. 3. Effect of EDR on DNA fragmentation in focal cerebral I ⁄ R injury associated with diabetes. The left panel indicates representative brainimages showing TUNEL positive cells (A, C and E) against corresponding DAPI-stained total cell population (B, D and F) of diabetic sham-operated, vehicle- and EDR- (10 mg ⁄ kg) treated diabetic I ⁄ R groups, respectively. The vehicle-treated group of rats was found to have moreTUNEL positive cells (indices of DNA fragmentation) in the ipsilateral penumbral brain region following 22 hr of reperfusion as compared tothe sham group. EDR (10 mg ⁄ kg) significantly decreased TUNEL positive cells (panel G). The values are expressed as mean € SEM, n = 3–4.***p < 0.001, **p < 0.01 versus diabetic sham; ###p < 0.001 versus vehicle-treated diabetic I ⁄ R group. The images were taken at 40X and themicron bar = 50 lm. EDR-edaravone; DAPI (nuclear stain)-4¢-6-diamidino-2-phenylindole.



Discussion

In the present study, we have demonstrated that EDR offersneuroprotection in experimental diabetic stroke throughreducing ER stress and apoptotic DNA fragmentation.Growing evidence suggests the involvement of ER stress invarious neurodegenerative (cerebral ischaemia) and meta-bolic disorders (obesity, diabetes and atherosclerosis) and

further targeting ER function could provide future therapeu-tic opportunities [26,27]. However, the availability of agentsspecifically targeting ER stress pathways is very limited forlaboratory investigation. Nevertheless, two therapeutic strate-gies have been mainly relied to target ER stress. Firstly, ther-apeutic interventions could be targeted against suppressingthe pathological process leading to functional ER impair-ment and secondly facilitating the folding of unfolded

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Fig. 4. Effect of EDR on GRP78 and CHOP ⁄ GADD153 immunoreactivity in focal cerebral I ⁄ R injury associated with diabetes. The represen-tative photomicrographs showing GRP78 (A, B and C) and CHOP ⁄ GADD153 (D, E and F) immunoreactivity of diabetic sham, vehicle- andEDR- (10 mg ⁄ kg) treated diabetic I ⁄ R group, respectively. The diabetic rats treated with vehicle exhibited significant increase in GRP78 IHCscore (panel G) and CHOP ⁄ GADD153 positive cells (panel H) in peri-infarct brain region, markers of ER stress ⁄ apoptosis as compared to thediabetic sham group. EDR (10 mg ⁄ kg) significantly reduced the immunoreactivity of both GPR78 and CHOP ⁄ GADD153. The values areexpressed as mean € SEM, n = 3–4. ***p < 0.001, **p < 0.01 versus diabetic sham; ###p < 0.001 versus the vehicle-treated diabetic I ⁄ R group.The representative dark brown stained cells indicated with arrows are CHOP ⁄ GADD153 positive cells. The photographs were taken at 40X andthe micron bar = 50 lm. EDR-edaravone.

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Fig. 5. Effect of EDR on the GRP78 and caspase-12 expression in focal cerebral I ⁄ R injury associated with diabetes. Western blot analysis alsorevealed a consistent increase in GRP78 expression and also detected significant activation of caspase-12 accompanied with its reduced levelsfollowing I ⁄ R in vehicle-treated rats compared with the diabetic sham group. EDR (10 mg ⁄ kg) markedly decreased the induction of GRP78and reduced the activation of caspase-12. The values are expressed as mean € SEM. Each value is average of three or four independent experi-ments. ***p < 0.001, *p < 0.05 versus diabetic sham group; ###p < 0.001, #p < 0.05 versus vehicle- treated diabetic I ⁄ R group. EDR-edaravone.



proteins so that it restores ER function and protects cellsfrom irreversible cell damage [27]. The major pathologicalprocess resulting in ER stress in various acute disordersincluding cerebral ischaemia is oxidative disorder caused bya rise in ROS to levels exceeding antioxidant defence. Oxy-gen free radicals produced inside and outside the ER lumenperturb ER function and result in the accumulation ofunfolded proteins and the induction of apoptosis as investi-gated in ischaemic brain [9]. The study also demonstratedthat the rats over expressing SOD1, ischaemia-induced ERdysfunction were indeed markedly suppressed [9]. Thus, itappears that drugs having antioxidant activity could be usedto decrease or block ROS-induced impairment of ER func-tioning. Therefore, we decided to use and evaluate the neuro-protective potential of EDR, potent free radical scavengeragainst focal cerebral ischaemia associated with comorbidtype 2 diabetic conditions, an animal model that mimicshuman stroke. The HFD ⁄ STZ type 2 diabetic rat model usedin the study replicates the natural history and the character-istic features of human type 2 diabetes viz. hyperglycaemia,hyperlipidaemia and hypercholesterolaemia under normal orslightly elevated absolute circulating insulin concentration(relative insulin deficiency) as compared to normal controlrats which is compatible with our earlier results [20].

Upon exposure to I ⁄ R, the diabetic rats developed greaterneurological damage and impairment in neurological func-tions which is consistent with earlier reports [2,28,29]. On theother hand, treatment with EDR not only produced signifi-cant inhibition of cerebral infarct and oedema volume butalso improved functional recovery of neurological deficits sig-nifying its potent neuroprotective activity in diabetic stroke aswell. This result is in agreement with previous reports ofEDR on cerebral ischaemia under normoglycaemic condi-tions [16]. The neuroprotective effect of EDR was further evi-denced from reduction in TUNEL positive cells in theipsilateral penumbral region of the brain as compared to thevehicle-treated rats. TUNEL positive cells are indices ofDNA fragmentation ⁄ apoptosis that is predominantly respon-sible for neurological damage in the ischaemic penumbralregion [30]. We next examined whether neuroprotective effectsof EDR were because of amelioration of ER stress by analy-sing respective molecular biomarkers viz. GRP78, CHOP ⁄GADD153 and caspase-12, after 22 hr of reperfusion in thebrains of the various groups. Up-regulation of the ER-resi-dent chaperone, GRP78 is a hallmark of adaptive UPR andhas been commonly used as a marker of ER stress [31]. Theexpression of GRP78 remained significantly elevated evenafter 22 hr of reperfusion indicating the involvement of pro-longed ER stress as compared to the sham-operated rats. Thisfinding is in agreement with brain ischaemia under normogly-caemic conditions [32]. Despite the increase in protectiveGRP78 chaperone levels, marked apoptotic DNA fragmenta-tion was strikingly evident at 22 hr after reperfusion. As thestress was too severe and prolonged, the compensatory UPRso noticed with up-regulation of GRP78 might have appearedto be relatively insufficient and unsuccessful to cope with aug-mented ER stress and protect the cells dying from apoptosis

in the vehicle-treated I ⁄ R rats [2]. The latter ER stress-induced apoptosis has been shown to involve induction ofCHOP ⁄ GADD153 as well as activation of caspase-12-specificmediators of ER stress-associated cell death. We observed anincrease in expression or immunoreactivity of proapoptotictranscription factor CHOP ⁄ GADD153 and marked activa-tion of caspase-12 which is in agreement with earlier reportson normoglycaemic as well as diabetic stroke models[2,33,34].

The cells under unstressed state express only very low levelof CHOP ⁄ GADD153 which becomes markedly elevated inresponse to ER stress through transcriptional induction.Although a precise mechanism by which CHOP ⁄ GADD153mediates apoptosis is unknown, CHOP ⁄ GADD153 isreported to activate transcription of numerous cell deathgenes including Gadd34, Ero1, Bim and Trb3 and suppressionof anti-apototic Bcl-2 protein. Further, CHOP ⁄ GADD153 - ⁄ -cells and mice have been shown to be protected from ERstress-induced apoptosis, whereas CHOP ⁄ GADD153 overexpression resulted in increased apoptosis [4]. Likewise, cas-pase-12-deficient mice are also found to be resistant to ERstress-induced apoptosis [35]. Caspase-12 is an ER-residentpro-caspase that is proteolysed or activated in response to ERstress conditions, releasing active cleaved fragments which inturn are responsible for downstream intrinsic apoptosis sig-nalling eventually resulting in DNA fragmentation [36]. Onthe other hand, treatment with EDR significantly inhibitedthe induction of CHOP ⁄ GADD153 as well as activation ofcaspase-12 in parallel with reduced apoptosis. These findingssuggest that EDR treatment might probably share a commonmechanism in ameliorating ER stress in this diabetic strokemodel as observed with deletion of CHOP ⁄ GADD153 ordeficiency of caspase-12.

It has been postulated that ROS generated during I ⁄ Rcould accelerate protein misfolding in ER lumen by oxidizingamino acids in folding proteins or modifying endogenouschaperones functions or damaging ER-resident proteins bothdirectly or indirectly through formation of peroxidated lipidor peroxynitrite [8,37]. The ability of the antioxidative stressresponse to combat ROS accumulation and protein misfold-ing may be especially important for function and survival ofcells [38]. We thus speculate that EDR, being a potent freeradical scavenger, might have significantly quenched ROSproduction and subsequently reduced the formation andaccumulation of ROS-induced misfolded proteins within ERlumen following I ⁄ R. These alleviative effects of EDRagainst protein misfolding might have finally reduced theactivation of downstream apoptotic signalling pathwaysinvolving CHOP ⁄ GADD153 and caspase-12. Of furthernote, the neuroprotective potential of EDR in this diabeticstroke model was found independent of its effect on bloodsugar level and body temperature, thereby excluding the fac-tors of hypothermia or hypoglycaemia underlying itsneuroprotection. However, it remains to be determinedwhether the inhibition of ER stress response associated withantioxidant treatment is specific to EDR only or a commonmechanism to all other antioxidants.



Conclusions

Taken together, these experimental findings demonstrate thepotent neuroprotective potential of EDR in a rat model ofdiabetic stroke. The effect is likely to result from theinhibition of ER stress and apoptotic DNA fragmentationinvolving CHOP ⁄ GADD153 and caspase-12. Further, thedata obtained from the present study as well as fromprevious studies may pave the way for future clinicalinvestigations aimed at exploring therapeutic benefits ofEDR not only against cerebral I ⁄ R damage but alsoconcomitantly against underlying diabetes-induced oxidativetissue damage ⁄ complication in diabetic stroke subjects.

AcknowledgementsKrishnamoorthy Srinivasan acknowledges the financial

assistance from the Department of Science and Technology(DST), New Delhi, Government of India for this researchwork via their SERC FAST track scheme (LS-134 ⁄ 2008). Wealso thank Mr. Jang Bhadhur and Mr. Yavinder for theirassistance in the preparation of HFD.

Disclosure StatementThe authors declare that there are no conflicts of interest.

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edaravone offers neuroprotection in a diabetic stroke model via inhibition of endoplasmic reticulum...

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