Neuroprotective effect of lercanidipine in middle cerebral artery occlusion model of stroke in rats

    Neuroprotective effect of lercanidipine in middle cerebral artery occlusion model of stroke in rats Sangeetha Gupta, Uma Sharma, Naranamangalam R Jagannathan, Yogendra Kumar Gupta PII: DOI: Reference:

S0014-4886(16)30345-4 doi: 10.1016/j.expneurol.2016.10.014 YEXNR 12423

To appear in:

Experimental Neurology

Received date: Revised date: Accepted date:

25 June 2016 24 September 2016 24 October 2016

Please cite this article as: Gupta, Sangeetha, Sharma, Uma, Jagannathan, Naranamangalam R, Gupta, Yogendra Kumar, Neuroprotective effect of lercanidipine in middle cerebral artery occlusion model of stroke in rats, Experimental Neurology (2016), doi: 10.1016/j.expneurol.2016.10.014

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ACCEPTED MANUSCRIPT Title:

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Neuroprotective Effect of Lercanidipine in Middle Cerebral Artery

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Occlusion Model of Stroke in Rats

Sangeetha Gupta1, Uma Sharma2, Naranamangalam R Jagannathan2, Department of Pharmacology and 2Department of NMR & MRI Facility, All India Institute of Medical Sciences, New Delhi-110029, India

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*Corresponding author:

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Yogendra Kumar Gupta1*

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Dr. Y. K. Gupta Professor & Head, Department of Pharmacology, All India Institute of Medical Sciences, New Delhi – 110 029, India Tel: 91-11-26593282 Fax: 91-11-26588641, 26588663 Email: [email protected]

ACCEPTED MANUSCRIPT Abstract Oxidative stress, inflammation and apoptotic neuronal cell death are cardinal mechanisms

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involved in the cascade of acute ischemic stroke. Lercanidipine apart from calcium channel blocking activity possesses anti-oxidant, anti-inflammatory and anti-apoptotic properties. In the

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present study, we investigated neuroprotective efficacy and therapeutic time window of lercanidipine in a 2 h middle cerebral artery occlusion (MCAo) model in male Wistar rats. The

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study design included: acute (pre-treatment and post-treatment) and sub-acute studies. In acute studies (pre-treatment) lercanidipine (0.25, 0.5 and 1 mg/kg, i.p.) was administered 60 min prior

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MCAo. The rats were assessed 24 h post-MCAo for neurological deficit score (NDS), motor deficit paradigms (grip test and rota rod) and cerebral infarction via 2,3,5-triphenyltetrazolium

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chloride (TTC) staining. The most effective dose was found to be at 0.5 mg/kg, i.p., which was considered for further studies. Regional cerebral blood flow (rCBF) was monitored till 120 min

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post-reperfusion to assess vasodilatory property of lercanidipine (0.5 mg/kg, i.p.) administered at two different time points: 60 min post-MCAo and 15 min post-reperfusion. In acute studies

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(post-treatment) lercanidipine (0.5 mg/kg, i.p.) was administered 15 min, 120 min and 240 min

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post-reperfusion. Based on NDS and cerebral infarction via TTC staining assessed 24 h postMCAo, effectiveness was evident upto 120 min. For sub-acute studies same dose/vehicle was repeated for next 3 days and magnetic resonance imaging (MRI) was performed 96 h after the last dose. Biochemical markers estimated in rat brain cortex 24 h post-MCAo were oxidative stress (malondialdehyde, reduced glutathione, nitric oxide, superoxide dismutase), blood brain barrier damage (matrix metalloproteinases-2 and-9) and apoptotic (caspase-3 and -9). Lercanidipine significantly reduced NDS, motor deficits and cerebral infarction volume as compared to the control group. Lercanidipine (60 min post-MCAo) significantly increased rCBF (86%) as compared to vehicle treated MCAo group (64%) 120 min post-reperfusion, but failed to show vasodilatation with 15 min post-reperfusion group. Lercanidipine (13.78 ± 2.78%) significantly attenuated percentage infarct volume as evident from diffusion-weighted (DWI) and T2-weighted images as compared to vehicle treated MCAo group (25.90 ± 2.44%) investigated 96 h post-MCAo. The apparent diffusion coefficient (ADC) was also significantly improved in lercanidipine group as compared to control group. Biochemical alterations were significantly ameliorated by lercanidipine till 120 min post-reperfusion group and MMP-9 inhibition observed

ACCEPTED MANUSCRIPT even with 240 min group. Thus, lercanidipine revealed significant neuroprotective effect

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mediated through attenuation of oxidative stress, inflammation and apoptosis.

Keywords: Neuroprotection, Lercanidipine, Stroke, MCAo, Anti-oxidant, Anti-inflammatory,

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Anti-apoptotic, Calcium channel blocker

Highlights:

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 Lercanidipine was investigated in MCAo model of stroke in rats.  Lercanidipine reduced neurological deficit score, motor deficits, and cerebral infarction.

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 Biochemical alterations and cerebral blood flow was improved.  Neuroprotective effect was mediated through anti-oxidant, anti-inflammatory, vasodilatory

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Abbreviations:

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and anti-apoptotic mechanisms.

MCAo, middle cerebral artery occlusion; MRI, magnetic resonance imaging; DWI, diffusionweighted imaging; ADC, apparent diffusion coefficient; FDA, Food and drug administration; MMP, matrix metalloproteinases; TTC, 2,3,5-triphenyltetrazolium chloride; HED, Human equivalent dose; i.p., intraperitoneally; LDF, Laser doppler flowmetry; rCBF, regional cerebral blood flow; rt-PA, recombinant tissue plasminogen activatior; MDA, malondialdehyde; GSH, reduced glutathione; NO, nitric oxide; SOD, superoxide dismutase; CCB, calcium channel blocker; MABP, mean arterial blood pressure

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1. Introduction

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Stroke is the second leading cause of death accounting 11.13% worldwide with an incidence of 7,95,000 patients every year (AHA 2015). Current treatment of acute stroke is restricted to

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recombinant tissue-plasminogen activator (rt-PA) which is the only FDA approved thrombolytic drug. The acceptability of rt-PA is limited due to its accessibility to less than 10% of patients,

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time-window of 4.5 h after stroke onset and hemorrhagic transformation risk. Further, risk factors and recurrence associated with stroke further necessitates exploration of prophylactic

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approach apart from acute treatment strategies. A cascade of events is initiated such as overload of intracellular calcium, excitotoxicity, cerebral edema, activation of proteolytic enzymes and

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inflammatory mediators, free radical generation, apoptosis and necrosis after onset of ischemic

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stroke (Dirnagl et al., 1999). Numerous neuroprotective agents have been evaluated, yet none has been found to be efficacious despite significant research effort (Lutsep and Clark, 2001).

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Previous clinical trials for calcium channel blockers (CCBs) such as nimodipine, isradipine have failed to show efficacy, as calcium channel blocking property was the sole action. It has been well documented that 1,4-dihydropyridine (DHPs) binding sites are present in rat cerebral tissues (Middlemiss and Spedding, 1985) and possess an ability to cross blood brain barrier (BBB) (Larkin et al., 1992). Lercanidipine is a highly lipophilic, vasoselective, third generation DHP L-type CCB. It has a longer duration of action with a plasma life of 8-10 h attaining peak plasma levels within 1.5-3 h. It induces less peripheral edema and reflex tachycardia as compared with other DHPs (Borghi, 2005). Apart from CCB blocking activity lercanidipine also possesses anti-oxidant, anti-inflammatory, vasodilatory and anti-apoptotic property (Martinez et al., 2008a; Martinez et al., 2008b; Menne et al., 2006; Wu et al., 2009). Anti-oxidant and anti-inflammatory effect of lercanidipine is well evident from lipid peroxidation and matrix metalloproteinases (MMPs) activity reduction in alloxan-induced diabetic and two-kidney, one-clip (2K-1C) hypertensive rats (Martinez et al., 2008a; Martinez et al., 2008b). The anti-proliferative effect of lercanidipine is illustrated in rat vascular smooth muscle cell cultures and balloon injury rat carotid artery model mediated through inhibition of

ACCEPTED MANUSCRIPT reactive oxygen species (ROS) , Ras-MEK1/2-ERK1/2, and PI3K-Akt pathways (Wu et al., 2009). Lercanidipine was found to be superior from nicardipine, lisinopril, valsartan, and hydralazine in occlusion-induced delayed neuronal death in stroke-prone spontaneously

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hypertensive rats. The hippocampal cells were protected in dementia model associated with

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essential hypertension (Sakurai-Yamashita et al., 2011).

Lercanidipine dose-dependently suppressed ST depression in vasopressin and methacholine-

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induced angina model. The antianginal activity of lercanidipine was found to be superior to nifedipine, amlodipine and benidipine attributed to its antispasmolytic coronary vasodilating

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activity (Sasaki et al., 2008). Lercanidipine (2.5 mg/kg/day for 3 weeks) improved renal function in a double-transgenic rat model overexpressing human renin and angiotensinogen genes.

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Angiotensin I-mediated PKC-α and PKC-δ activation was inhibited by lercanidipine attributed to intracellular calcium flux reduction (Menne et al., 2006).

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The aim of the present study was to investigate the neuroprotective effect of lercanidipine in a transient middle cerebral artery occlusion (MCAo) model of focal cerebral ischemia in rats.

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Acute (pre-treatment and post-treatment), subacute studies and the possible mechanisms were

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explored mediated through anti-oxidant, anti-inflammatory and anti-apoptotic action.

2. Materials and methods

2.1. Animals

Albino male Wistar rats weighing 240–260 g were procured from the Central Animal Facility of All India Institute of Medical Sciences, New Delhi and group housed in polypropylene cages (38 x 23 x 10 cm) with not more than 4 animals per cage. The animals were maintained under standard laboratory conditions with natural dark-light cycle (14 ± 60 min light; 10 ± 60 min dark). They were allowed free access to standard dry rat diet (Aashirwad Industries, Chandigarh) and tap water ad libitum. All experimental procedures were reviewed and approved by the Institutional Animal Ethics Committee (536/IAEC/13).

2.2. Drugs and chemicals

ACCEPTED MANUSCRIPT Lercanidipine was procured as a gift sample from Glenmark Pharmaceuticals Ltd., Mumbai. The chemicals purchased accordingly as chloral hydrate (Fluka Chemicals, USA), nylon filaments 3-0 and absorbable sutures 6-0 (ethicon, Johnson & Johnson, India), 2,3,5-

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triphenyltetrazolium chloride and formaldehyde (Sigma Chemical Co., USA), Quick start bradford protein assay kit (cat. no. K5000201; Biorad, USA), SensoLyte® Plus 520 MMP-9 (cat.

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no. AS-72017) and MMP-2 assay kit (cat. no. AS-72224; AnaSpec, San Jose, CA, USA),

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caspase-3/CPP32 (cat. no. K105-100) and caspase-9 (cat. no. K118-100; BioVision Inc. CA, USA)) fluorometric assay kit. All other reagents were of analytical grade and obtained from

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Central Drug House (P) Ltd. and Sisco Research Laboratories Pvt. Ltd., India.

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2.3. Dose selection of lercanidipine for stroke experiments The usual human dose of lercanidipine for essential hypertension is 10 mg once daily orally.

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Lercanidipine dose considered = 10 mg for 60 kg; hence per kg = 0.17 mg/kg. According to

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formula; animal dose (mg/kg) = HED (mg/kg) x (human Km/animal Km) = 0.17 x (37/6) = 1.03 mg/kg (FDA, CDER, 2005). Thus, selected doses were 1, 0.5 and 0.25 mg/kg, intraperitoneally

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(i.p.), once daily. Lercanidipine was prepared in 20% ethanol, 20% DMSO and 60% normal saline and was administered with a volume not exceeding 1 ml/1000 g. 2.4. Experimental design

Protocol 1 (Acute studies)

Protocol 1a. Acute studies (Pre-treatment): The rats were randomly divided into 5 groups: group 1 was sham-operated received only vehicle, 60 min prior MCAo and no MCAo was performed (n = 4). Group 2 was MCAo control group (n = 6) in which vehicle was administered 60 min prior MCAo. Groups 3, 4 and 5 received lercanidipine in doses of 0.25 (n = 5), 0.5 (n = 6) and 1 mg/kg (n = 6), i.p. respectively, 60 min prior MCAo (Fig. 1A). Groups 3 to 5 were used to decide most effective dose of lercanidipine for further studies assessed 24 h after MCAo through parameters: neurological deficit score (NDS), motor deficit paradigms (grip test score and rota rod) and cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

ACCEPTED MANUSCRIPT Protocol 1b. Acute studies (rCBF): Group 1 was MCAo control group (n = 4) received vehicle 60 min post-MCAo, group 2 was lercanidipine administered 0.5 mg/kg, i.p., 60 min post-MCAo (n = 5) and in group 3 was lercanidipine (0.5 mg/kg, i.p.) administered 15 min post-reperfusion

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(n = 6). The regional cerebral blood flow (rCBF) was assessed during MCAo and till 2 h postreperfusion with a baseline prior to MCAo. The NDS and cerebral infarction using TTC staining

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was assessed 24 h after MCAo (Fig. 1A).

Protocol 1c. Acute studies (Post-treatment): Rats were randomly divided into following 3 groups

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in which MCAo was performed and received lercanidipine (0.5 mg/kg, i.p.) at three time-points (Fig. 1A): group 1 received 15 min post-reperfusion (n = 6); group 2 received 120 min post-

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reperfusion (n = 6) and group 3 received 240 min post-reperfusion (n = 5). The rats were assessed 24 h after MCAo for NDS and cerebral infarction volume (TTC staining).

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Protocol 1d. Acute studies (Biochemical estimations): Rats were randomly divided into 6 groups

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treated with either vehicle/lercanidipine (0.5 mg/kg, i.p.) and assessed 24 h after MCAo: group 1 (n = 4) sham operated and group 2 (n = 4) MCAo control, both was treated with vehicle; group 3

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(n = 6), lercanidipine was administered 60 min prior MCAo. Groups 4, 5 and 6 received lercanidipine 15 min (n = 4), 120 min (n = 6) and 240 min (n = 6) post-reperfusion respectively (Fig. 1A).

Protocol 2 (Sub-acute studies)

Rats were randomly divided into 3 groups: group 1 (n = 6), MCAo was performed followed by magnetic resonance imaging (MRI) within 30-60 min post-reperfusion, considered as baseline; group 2 (n = 4), vehicle-treated MCAo control and group 3 (n = 6), lercanidipine (0.5 mg/kg, i.p.) treated (Fig. 1B). Based on protocol 1c, in group 2 or group 3, lercanidipine or vehicle respectively was administered 120 min post-reperfusion and further once daily for 3 days. NDS was performed on day 1 and day 4 followed by MRI and TTC staining on 4th day.

2.5. Middle cerebral artery occlusion (MCAo) model of focal cerebral ischemia

ACCEPTED MANUSCRIPT Overnight fasted rats were anesthetized with chloral hydrate (400 mg/kg, i.p.). A midline incision was made and the right common carotid artery, external carotid artery and internal carotid arteries were exposed. A 3-0 monofilament nylon thread (Ethicon, Johnson and Johnson)

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with its tip rounded by heating quickly with a flame was used to occlude the MCA. The filament was advanced from the external carotid artery into the lumen of the internal carotid artery upto

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17-20 mm until a resistance was felt which ensured the occlusion at the origin of MCA. The

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nylon filament was allowed to remain in the place for 2 h and then gently retracted so as to allow the reperfusion. In sham operated rats, the filament was introduced into the external carotid

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artery but was not advanced into the internal carotid artery (Longa et al., 1989). The rCBF

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reduction approximately to 20 to 30% of baseline was used to confirm the occlusion.

2.6. Measurement of physiological parameters

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Rectal temperature was maintained at 37°C using thermostatically regulated heating pad

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throughout the surgical procedure and till recovery using blower. Mean arterial blood pressure (MABP) was measured using non-invasive small animal tail blood pressure systems, NIBP200A

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amplifier equipped with tail cuff sensor [RXTCUFSENSOR9.5 (9.5mm)]; Biopac Systems, Inc. USA. The readings were monitored using AcqKnowledge 4.0 software in normal rats with a baseline reading 30 min prior and at 30, 60, 120 and 180 min after lercanidipine (1 mg/kg, i.p.) administration. Formula used for MABP was (2 x diastolic blood pressure + systolic blood pressure) / 3.

2.7. Regional cerebral blood flow by laser doppler flowmetry Regional cerebral blood flow (rCBF) was determined by laser doppler flowmetry (Module LDF100C Biopac Systems, Inc., USA). A small incision was made in the skin overlying the temporalis muscle to measure rCBF using a flexible 0.5 mm fiber-optic extension to needle probe (Model No. TSD144). An accelerator was used to fix the probe on the ipsilateral side of the superior portion of temporal bone over the ischemic cortex at a dimension (2 mm posterior and 6 mm lateral from the bregma). The sampling depth was around 0.5-1 mm with a sampling volume of 0.3-0.5 mm3. Baseline rCBF was determined 30 min prior MCAo and then every 30 min after MCAo till 120 min post-reperfusion for vehicle-treated MCAo and lercanidipine (0.5

ACCEPTED MANUSCRIPT mg/kg, i.p.) treated rats. Laser doppler signals were measured as microvacular blood perfusion units (BPU) using AcqKnowledge 4.0 software, which is a relative unit scale defined using a motility standard latex spheres undergoing Brownian motion. BPU = number of moving red

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blood cells in volume sampled x mean velocity of red blood cells. The rCBF data is expressed as

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percentage of the preocclusion value (Dirnagl et al., 1989; Hara et al., 1996).

2.8. Neurological deficit score

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Neurological deficit score (NDS) in vehicle and lercanidipine treated rats was evaluated 24 h after MCAo in a blinded fashion using a standard 6–point scale as: 0 = no neurological deficits, 1

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= failure to extend left forepaw fully, 2 = circling to the left, 3 = paresis to the left, 4 = no spontaneous walking and 5 = death (Tatlisumak et al., 1998; Gupta et al., 2005).

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2.9.1. Grip test score

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2.9. Motor function test

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Grip test score was performed according to the protocol reported earlier (Moran et al., 1995; Gupta et al., 2005). The apparatus consisted of a string of 50 cm length which was pulled taut between two vertical supports at a height of 40 cm from a flat surface. After placing rat on the midway between on the string between supports rats were evaluated according to scoring system: 0 = fall off, 1 = hangs onto string by two forepaws, 2 = as for 1 but attempts to climb on string, 3 = hangs onto string by two forepaws plus one or both hind paws, 4 = hangs onto string by all fore paws plus tail wrapped around string and 5 = escape. 2.9.2. Rota rod test Rota rod (Ugo Basile, Italy) test is widely used to assess the muscle co-ordination. A training session for conditioning to the rota rod at a constant speed of 8 rpm was performed one day before and prior to the MCAo rats. The rats which achieved the criterion of remaining on the rotating spindle for 60 s were selected for further studies. Each rat then received a single baseline test trial on the accelerating rota rod at a speed of 4–40 rpm with a cut off period of 5 min. Test trial was again performed 24 h post-MCAo (Rogers et al., 1997; Gupta et al., 2005).

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2.10. Assessment of cerebral infarction

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2.10.1. 2,3,5-triphenyltetrazolium chloride staining Cerebral infarction volume was assessed using 2,3,5-triphenyltetrazolium chloride (TTC)

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staining method. Rat brains were isolated after cervical dislocation in anaesthetized rats followed by decapitation for TTC staining and were kept at -80°C for 15 min. Acrylic Rat Brain Slicer

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Matrix 2.0 mm (Zivic labs, USA) was used to obtain consecutive 2 mm thick coronal brain slices. Brain slices were stained with TTC (0.1%) solution prepared in 37°C phosphate buffer

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(0.2 M Na2HPO4 and 0.2 M NaH2PO4, pH 7.4-7.6) and kept at 37°C in water bath for 10 min. For consistent staining slices were flipped after 5 min anteriorly and posteriorly. Images were

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captured within 3 days after fixing in 10% formalin. Image-J (Ver 1.45) software (http://rsb.info.nih.gov/nih-image) was used for infarct volume analysis. Infarct volume was calculated as (S1+S2+S3+S4+S5) x 2 mm3, where S1 to S5 represented slice area from 1 to 5 in

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mm2 each with a slice thickness of 2 mm. Corrected infarct volume was calculated by formula =

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(infarct volume x contralateral volume)/ipsilateral volume (Bederson et al., 1986b; Rupadevi et

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al., 2011; Swanson et al., 1990).

2.10.2. Magnetic resonance imaging 2.10.2.1. Magnetic resonance imaging protocol Magnetic resonance imaging (MRI) studies were carried out using an animal MRI 7.0 T scanner (Bruker, Biospin MRI GmbH Biospec 70/20 US). The instrument was equipped with 72 mm transmit only 1H circularly polarized coil with transmit only capability (112/72 mm, outer/inner diameter) in combination with rat brain surface coil, 1H circularly polarized coil with receive only capabilities. Cerebral infarct was identified by diffusion weighted imaging (DWI) within 30-60 min post-reperfusion and on 4th day. The ischemic region was identified by the acquisition of multislice T2-weighted pilot images using rapid acquisition with rapid enhancement (RARE) sequence using parameters: repetition time (TR) = 2500 ms, echo time (TE) = 33 ms, slice thickness = 1 mm, slices = 14, averages = 2, matrix size = 256 x 256 mm, field of view (FOV) = 35 x 35 mm. Diffusion-weighted images were then acquired using

ACCEPTED MANUSCRIPT parameters: TR/TE = 3000/32.50 ms, slice thickness = 1 mm, slices = 14, matrix size = 96 x 96, averages = 2, diffusion gradient scaling/maximum gradient strength per channel = 22.25, 31.47,

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200, 400, 1000, 1500 and 1800 s/mm2 (Chauhan et al., 2012).

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49.76, 60.94, 66.76 % and 100.19, 141.69, 224.04, 274.39, 300.59 mT/m and six b values = 0,

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2.10.2.2. Calculation for apparent diffusion coefficient (ADC), percentage infarct volume and signal intensity

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Paravision 6.0 software was used for image analysis, and contrast of DWI and T2 images were adjusted using the grey scale. Hand-drawn region of interest (ROIs) were traced to obtain

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infarcted region and complete areas of each slice. Each value was multiplied by slice thickness to get the slice volume (mm3). Infarct volume was calculated by summation of infarcted slices and then percentage infarction was obtained from total brain volume. Signal intensity and ADC were

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calculated by drawing ROIs on each infarcted slice both ipsilaterally and contralaterally. ADC

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ratio calculated as ADC value of ipsilateral (right)/ ADC value of contralateral (left) hemisphere (R/L). Data was analysed by an observer who was blinded to treatment groups (Chauhan et al.,

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2012; Gupta et al., 2005).

2.11. Biochemical estimations

Rats were decapitated under ether anesthesia 24 h after MCAo. The brain except cerebellum was quickly removed and cleaned with chilled saline. The ipsilateral (ischemic) hemisphere of brain was dissected to separate out cortex. For the estimation of malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO) and superoxide dismutase (SOD) cortex was homogenized with 10 times (w/v) ice-cold 0.1 M phosphate buffer (pH 7.4). For estimation of metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2) activity cortex

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homogenized in the assay buffer provided in the kit containing 0.1% Triton-X 100, and then centrifuged for 15 min at 10,000x g at 4◦C. Supernatant was collected and stored at -70◦C until use. Brain samples for caspase estimation, were homogenized in ice-cold cell lysis buffer and centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was collected and stored at -70◦C for

ACCEPTED MANUSCRIPT use. Protein concentration was measured using the Quick Start Bradford Protein Assay Kit, Biorad (cat. no. K5000201).

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2.11.1. Estimation of oxidative stress markers

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2.11.1.1. Malondialdehyde levels

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The lipid peroxidation indicator MDA was measured as described by Ohkawa et al., 1979. In 0.1 ml of processed tissue sample, acetic acid 1.5 ml (20%), pH 3.5, 1.5 ml thiobarbituric acid

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(0.8%) and 0.2 ml sodium dodecyl sulfate (8.1%), were added and gently vortexed. The mixture was then heated at 100ºC for 60 min in a water bath. The mixture was cooled with tap water and

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5 ml of n-butanol-pyridine (15:1) and 1 ml of distilled water was added. After centrifugation at 4000 rpm for 10 min, the organic layer was withdrawn and absorbance was measured at 532 nm. Standard curve was prepared by using 1,1,3,3 tetraethoxypropane (TEP) as MDA standard. The

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MDA concentration is expressed in nM/mg of protein.

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2.11.1.2. Reduced glutathione levels

The GSH levels were measured according to the method of Ellman et al., 1959. Equal quantity of homogenate was mixed with 10% trichloroacetic acid and centrifuged to separate the proteins. To 0.1 ml of this supernatant, 2 ml of 0.3 M phosphate buffer (pH 8.4), 0.5 ml of 5,5′-dithiobis (2-nitrobenzoic acid) and 0.4 ml of double distilled water were added. The mixture was vortexed and the absorbance was read at 412 nm within 15 min. The GSH concentration is expressed in µg/mg of protein.

2.11.1.3. Nitric oxide levels Nitrite (NO2¯) concentration is used as an index of NO production was quantified using Griess colorimetric assay. Briefly, 50 μl of brain supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-napthylethylenediamine dihydrochloride). Standard curve was prepared using sodium nitrite. The optical density was read after 10 min at 550 nm and expressed in uM/mg of protein (Griess, 1879).

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2.11.1.4. Superoxide dismutase activity

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Superoxide dismutase (SOD) activity was estimated by pyrogallol oxidation inhibition

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method. Pyrogallol auto-oxidizes to a yellow color compound pyrogallin which is augmented in presence of superoxide anions. The reaction mixture consisted of 950 µl Tris-HCl buffer (Tris-

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HCl 50 mM and EDTA 1 mM, pH 8.2), 50 µl of 4 mM pyrogallol and 50 µl sample. The rate of auto-oxidation was measured at 420 nm every 30 s for 3 min. One SOD unit is defined as the

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amount of enzyme required to inhibit the auto-catalyses of pyrogallol by 50%. The result is expressed as percentage of pyrogallol inhibition (Marklund and Marklund, 1974).

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2.11.2. Neuro-inflammatory markers as indicators of BBB damage: MMP-9 and MMP-2 activity The specific enzymatic activity of active MMP-9 and MMP-2 was measured with SensoLyte®

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Plus 520 MMP-9 (cat. no. AS-72017) and MMP-2 assay kit (cat. no. AS-72224; AnaSpec, San Jose, CA, USA). Active MMP-9 and MMP-2 was captured by immobilized monoclonal antihuman-MMP-9 or MMP-2 antibodies, and proteolytic activity was then quantified using a 5FAM/QXL™520 fluorescence resonance energy transfer (FRET) peptide. Fluorescence of 5FAM (fluorophore) was quenched by QXL™520 (quencher) in the intact FRET peptide. Upon 5FAM/QXL™520 cleavage by MMP-9/MMP-2, the fluorescence of free 5-FAM was monitored at excitation/emission wavelength = 490 ± 20 nm/520 ± 20 nm with cut off at 515 nm. The samples (100 µg) were placed in a 96-well plate containing 50 µL of assay buffer quantified using a fluorescence microplate reader (SpectraMax® M5/M5e Multimode Plate Reader, Molecular Devices, USA). The zymogen of MMPs existing as pro-MMPs was activated by 4aminophenylmercuric acetate. The reading from all wells was subtracted with the reading from blank control, which contains FRET substrate but no MMPs. The proteolytic resonance energy transfer peptide was expressed as a fold-change in fluorescence intensity as compared to sham group.

ACCEPTED MANUSCRIPT 2.11.3. Apoptotic markers: caspase-3 and caspase-9 activity The apoptotic markers were measured with caspase-3/CPP32 (cat. no. K105-100) and caspase9 (cat. no. K118-100) fluorometric assay kit (BioVision Inc. CA, USA). A total of 100 μg of

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homogenate was incubated with 2 × reaction buffer (50 μl) in a 96-well plate. The DEVD-AFC or LEHD-AFC substrate upon cleavage by caspase-3 or caspase-9 respectively emits blue light

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(λmax = 400 nm). The free AFC emits a yellow-green fluorescence (λmax = 505 nm), quantified

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using a fluorescence microplate reader (SpectraMax® M5/M5e Multimode Plate Reader,

intensity as compared to the sham group.

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2.12. Statistical analysis

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Molecular Devices). The caspase activity was expressed as a fold-change in fluorescence

Statistical analysis was performed using Graph pad prism 5.0 version (GraphPad Software

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Inc., San Diego, CA, USA). Non-parametric Kruskal-Wallis test was applied to compare groups

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for NDS and grip test scores followed by multiple comparisons using Mann-Whitney U test. Data is represented as grouped median (minimum-maximum). The rCBF data was analyzed by

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two-way Analysis of Variance (ANOVA) to detect differences between groups and over time. Independent Student’s t-test and One-way ANOVA followed by Bonferroni’s t-test was applied for other parameters. Data represented as mean ± SEM and p < 0.05 represents a level of significance. 3. Results

3.1. General observations A total of 132 rats were included in the present study out of which 102 rats were analysed for various parameters, 19 rats died and 11 rats were excluded. Exclusion criteria included operation time more than 20 min, excessive bleeding while performing surgery, rCBF more than 30% after occlusion, subarachnoid haemorrhage and no infarction. No mortality was observed in sham group. The survival rate of MCAo rats after exclusion included: MCAo control group, 73.9% (19/14); lercanidipine (0.25 mg/kg) pretreatment, 83.3% (6/5), lercanidipine (0.5 mg/kg) pretreatment, 82.85% (12/10); lercanidipine (1 mg/kg) pretreatment, 85% (7/6); lercanidipine (0.5 mg/kg) 60 min post-MCAo, 100% (5/5); lercanidipine (0.5 mg/kg) 15 min post-reperfusion,

ACCEPTED MANUSCRIPT 100% (16/16); lercanidipine (0.5 mg/kg) 120 min post-reperfusion, 85.7% (14/12); lercanidipine (0.5 mg/kg) 240 min post-reperfusion, 73.2% (15/11). In MCAo control rats subjected to MRI the survival on 4th day was only 43.9% (7/3), while with lercanidipine was 85.7% (7/6). Rats

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which showed dropdown of rCBF below 30% were included in the study. The LDF signals of rats subjected to MCAo were reduced from 1264.09 ± 23.17 BPU of baseline (prior to MCAo) to

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225.80 ± 10.07 BPU (18%) after MCAo and recovered to 709.69 ± 17.52 BPU (56%) post-

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reperfusion.

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3.2. Effect of lercanidipine on MABP

The highest dose of lercanidipine, 1 mg/kg, i.p. chosen for the study was found to be non-

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hypotensive as there was no significant change in the MABP from baseline monitored till 3 h. The MABP (mmHg) values were baseline (112.5 ± 4.80), 30 min (112.3 ± 2.71), 60 min (110.9

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± 1.75), 120 min (118.7 ± 3.54) and 180 min (113.5 ± 4.14) after lercanidipine administration.

3.3. Effect of lercanidipine on NDS

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There was no evidence of neurological deficits in vehicle-treated sham-operated rats scored as 0. Significant increase in NDS was evident [4 (3-4), n = 13] representing [grouped median (minimum-maximum)], in vehicle-treated MCAo control rats (p = 0.0095) as compared to sham rats (n = 4). A Kruskal-Wallis H test followed by multiple comparisons using Mann-Whitney U test showed that there was a statistically significant difference in NDS among the groups, Kruskal Wallis statistic, KW (11,75) = 46.94, p < 0.0001, n = 4-13 (Fig. 2A). A significant reduction in the NDS was observed with lercanidipine 0.5 mg/kg, pretreatment [3 (2-3), n = 5, p = 0.0096]; 0.5 mg/kg, 60 min post-MCAo [2.5 (1-3), n = 6, p = 0.0049]; 0.5 mg/kg postreperfusion at 15 min [2 (1-3), n = 12, p = 0.0002] and 120 min [2 (1-3), n = 11, p = 0.0001]. Lercanidipine pre-treatment doses 0.25 mg/kg, [4 (3-4), n = 5]; 1 mg/kg, [3 (2-4), n = 6] and 0.5 mg/kg, 240 min post-reperfusion [3 (2-4), n = 5] did not produce significant reduction in NDS (Fig. 2A). The NDS was also significantly reduced in 96 h lercanidipine treated group [1 (1-3), n = 5, p = 0.0498] as compared to 96 h vehicle-treated rats [4 (3-4)].

3.4. Effect of lercanidipine on motor function test

ACCEPTED MANUSCRIPT There was a significant reduction in grip test score in vehicle-treated MCAo control rats [1.5 (1-3), (p = 0.0048)] representing grouped median (minimum-maximum), as compared to sham rats [4 (4-5)]. There was a statistically significant difference in grip test score among the groups

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when Kruskal-Wallis H test followed by multiple comparisons using Mann-Whitney U test was applied, Kruskal Wallis statistic, KW (5, 27) = 15.15, p = 0.0044 (Fig. 2B). A significant

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reduction in the NDS was observed with lercanidipine 1 mg/kg, pretreatment [3 (2-4), p =

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0.0281]. Lercanidipine at lower pretreatment doses 0.25 mg/kg, [2 (1-2)] and 0.5 mg/kg, [3 (14)] did not produce significant reduction in grip test score.

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A significant difference in the motor coordination of rats was evident among all the groups [F (4, 26) = 10.631, p < 0.0001; n = 4-6] as assessed by rota rod test. There was a significant

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reduction in the time spent on rotating spindle in MCAo control rats (56.33 ± 6.91 s, p = 0.0001) as compared to the sham rats (159.25 ± 17.29 s). Lercanidipine pretreatment at 1 mg/kg only showed significant improvement in motor performance was observed (124.17 ± 18.00 s, p =

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0.007; Fig. 2C). Lercanidipine pre-treatment doses i.e. 0.25 and 0.5 mg/kg did not showed

Fig. 2A, B, C

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significant improvement in the motor coordination.

3.5. Effect of lercanidipine on cerebral infarction volume 3.5.1. Assessed by TTC staining

There was a significant difference in cerebral infarction volume as analyzed by one-way ANOVA among all the groups [F (10, 66) = 15.16, p < 0.0001; n = 4-12]. The vehicle-treated MCAo demarcated significant infarction in ipsilateral hemisphere as compared to the sham group (p = 0.0001). The percentage inhibition for cerebral infarct volume as compared to MCAo control group decreased dose-dependently from 20.12 ± 7.42%, 42.04 ± 6.02% and 43.94 ± 7.49% in 0.25, 0.5 and 1.0 mg/kg lercanidipine pretreated rats, respectively. As the significant decrease observed with lercanidipine at 0.5 and 1.0 mg/kg didn’t differed significantly, thus for further studies 0.5 mg/kg was considered. Percentage inhibition included with 60 min postMCAo (51.52 ± 7.11%, p = 0.0001); 15 min post-reperfusion (79.90 ± 3.32%, p = 0.0001) and 120 min post-reperfusion (61.84 ± 2.71%, p = 0.0001). Thus, a significant decrease was observed with lercanidipine administered till 120 min post-reperfusion which then declined in 240 min

ACCEPTED MANUSCRIPT post-reperfusion group (25.32 ± 3.79%; Fig. 3A, B). The infarct volume was also significantly reduced in 96 h lercanidipine treated group (50.49 ± 9.59%, p < 0.05) as compared to 96 h

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vehicle-treated rats.

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Fig. 3A, B

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3.5.2. Assessed by MRI

The ipsilateral hemisphere ADC value (0.37 ± 0.02) × 10˗3 mm2/s was significantly; t (9) =

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4.32, p < 0.01 lower than the normal contralateral hemisphere (0.81 ± 0.09) × 10˗3 mm2/s in 30 min post-reperfusion baseline group. Similarly, in 96 h vehicle-treated MCAo group the ADC

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value of ipsilateral hemisphere (0.48 ± 0.05) × 10˗3 mm2/s was significantly; t (4) = 3.28, p < 0.05 lower than the normal contralateral hemisphere (0.64 ± 0.02) × 10˗3 mm2/s. This implicates restricted cellular movement of water and progression of tissue injury in the MCA region. There

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was no significant difference in the ADC values in either hemisphere (0.51 ± 0.03) × 10˗3 or

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(Fig. 4A; Table 1).

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(0.61 ± 0.02) × 10˗3 mm2/s; respectively, t (7) = 2.40, p < 0.05 in 96 h lercanidipine treated group

Significant difference in the ADC (R/L) ratio was obtained among groups as assessed by one way ANOVA [F (2,12) = 8.86, p < 0.05; n = 4-6]. The ADC ratio was significantly improved in 96 h lercanidipine treated group as compared to baseline 30 min post-reperfusion group and fall near to sham ADC ratio considered as 1. The ADC ratio of 96 h control group was not significantly different from baseline. There was a significant difference in % cerebral infarct volume among groups as assessed by one-way ANOVA [F(2,14)=8.696, P<0.05; n=4-6]. Significant increase in % infarct volume in 96 h MCAo group was observed as compared with 30 min post-reperfusion group evaluated by DWI. In lercanidipine-treated group, the % infarct volume was significantly less than that of 96 h vehicle-treated MCAo group and the infarction was near to baseline indicating arrest of cellular injury (Fig. 4A; Table 1). The signal intensity increased significantly t (10) = 9.546, p < 0.001; t (4) = ˗4.003, p < 0.05 in ipsilateral hemisphere as compared to contralateral hemisphere in 30 min post-reperfusion and

ACCEPTED MANUSCRIPT 96 h MCAo group respectively (Fig. 4A, Table 1). There was a decrease in signal intensity of 96 h lercanidipine treated group as compared to vehicle treated MCAo group in ipsilateral

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hemisphere, but was not significant.

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Fig. 4A, B; Table 1

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3.6. Effect of lercanidipine on rCBF

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The relative changes in rCBF as assessed by LDF before MCAo and till 120 min of postreperfusion is depicted in Fig. 4B. There was a significant reduction in rCBF immediately after

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MCAo in vehicle-treated, lercanidipine 0.5 mg/kg, administered 60 min post-MCAo group and lercanidipine 0.5 mg/kg, administered 15 min post-reperfusion group to approximately 12 to 25% of the baseline value. The rCBF after reperfusion was significantly higher in lercanidipine

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60 min post-MCAo group as compared to vehicle group, but no significant increment in rCBF

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was observed when lercanidipine was administered 15 min post-reperfusion. Two-way ANOVA showed significant difference among three groups w.r.t. time F (9, 120) = 226.5, p < 0.0001 and

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treatment F (2, 120) = 64.34, p < 0.0001. Multiple comparisons using Sidak-Bonferroni method showed a significant difference (p < 0.0001) between vehicle and lercanidipine 60 min postMCAo group at time points from 120 min, t = 5.58; 150 min, t = 5.78; 180 min, t = 6.49; 210 min, t = 5.10; 240 min, t = 4.28. The rCBF at 240 min recovered approximately to 86% in lercanidipine group, while in MCAo it was only 64% of baseline. On the other hand in lercanidipine 15 min post-reperfusion group, the rCBF was not significantly different from MCAo control group accounting 63%.

Fig. 4 A, B

3.7. Effect of lercanidipine on biochemical markers 3.7.1. Oxidative stress markers 3.7.1.1 Malondialdehyde and reduced glutathione levels

ACCEPTED MANUSCRIPT There was a significant difference in the brain MDA levels [F (5, 27) = 20.911, p < 0.0001] and GSH [F (5, 27) = 5.383, p = 0.002] among all the groups. Post hoc analysis showed a significant increase in MDA levels (p < 0.0001) and a significant decrease in GSH levels (p =

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0.0035) in the vehicle-treated MCAo control group as compared to sham group. Lercanidipine significantly reduced MDA levels when administered 60 min prior MCAo (p = 0.0038); 15 min

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(p < 0.0001); 120 min (p = 0.0007) and 240 min post-reperfusion (p = 0.0053) as compared to

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vehicle-treated MCAo group (Table 2). Also, post hoc analysis showed that GSH levels increased significantly when lercanidipine administered 15 min (p = 0.0189) and 120 min post-

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reperfusion (p = 0.0153), as compared to vehicle-treated MCAo group (Table 2). No significant differences were observed in GSH levels in lercanidipine 60 min pretreatment and 240 min post-

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reperfusion groups.

3.7.1.2. Nitric oxide and superoxide dismutase levels

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There were significant differences among the groups in the brain NO levels [F (5, 27) = 16.80,

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p < 0.0001] and SOD levels [F (5, 27) = 8.798, p = 0.0001]. Post hoc analysis showed a significant increase in NO levels (p < 0.0001) and a significant decrease in SOD levels (p <

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0.0001) in the vehicle-treated MCAo control group as compared to sham group. Lercanidipine (0.5 mg/kg, i.p.) administered 60 min prior MCAo (p = 0.0036); 15 min (p < 0.0001) and 120 min post-reperfusion (p = 0.0069) significantly reduced the NO levels as compared to vehicletreated MCAo group as illustrated in Table 2. Similarly, lercanidipine showed significant increase in SOD levels when administered 60 min prior MCAo (p = 0.0106); 15 min (p = 0.0007) and 120 min post-reperfusion (p = 0.0165) as compared to vehicle-treated MCAo group (Table 2). No significant differences were observed in NO and SOD levels in lercanidipine 240 min post-reperfusion group. Table 2

3.7.2. MMP-9 and MMP-2 activity Significant difference in MMP-2 and MMP-9 activity was observed between the groups [F (5, 27) = 7.239, p = 0.0004] and [F (5, 27) = 29.350, p < 0.0001] respectively. Total MMP-9

ACCEPTED MANUSCRIPT activities including both pro- and active forms of MMP-2 and MMP-9 in ipsilateral cortex increased significantly (p < 0.001), 2.94 ± 0.64 and 2.66 ± 0.15 fold respectively in vehicletreated MCAo rats as compared to sham group. MMP-9 activity declined significant in all

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lercanidipine treated groups till 240 min post-reperfusion, while MMP-2 activity was inhibited

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only till 120 min post-reperfusion (Fig. 5A,B).

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3.7.3. Caspase-3 and caspase-9 activity

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Caspase-3 and caspase-9 activity in ipsilateral cortex of vehicle-treated MCAo rats was increased 3.10 ± 0.23 and 2.38 ± 0.18 fold significantly (p < 0.001) as compared to sham group. There was a significant difference in caspase-3 and caspase-9 activity between the groups [F (5,

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27) = 10.930, p < 0.0001] and [F (5, 27) = 6.482, p = 0.0008] respectively. Caspase-3 activity decreased significantly in lercanidipine 15 min (p = 0.0003) and 120 min (p = 0.0229) post-

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reperfusion groups, while lercanidipine 60 min pretreatment and 240 min post-reperfusion

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groups showed insignificant reduction in caspase-3 activity. Significant reduction in caspase-9

4. Discussion

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activity with all groups except at 240 min post-reperfusion group (Fig. 5C,D). Fig. 5A,B,C,D

4.1. Protection mediated via NDS, motor function and cerebral infarction In the present study lercanidipine produced a significant neuroprotection as evident from reduction in NDS, motor deficits and cerebral infarction volume in MCAo model of stroke in rats. We studied non-hypotensive dose-dependent (0.25, 0.5 and 1 mg/kg, i.p.) effect of lercanidipine in pretreatment acute studies administered 60 min prior MCAo. The protective effect was evident with 1 and 0.5 mg/kg, i.p. with insignificant difference among doses. Thus, most effective dose 0.5 mg/kg, i.p. was investigated for other studies. Further, the therapeutic time window in post-treatment study was carried at 15, 120 and 240 min post-reperfusion. The significant neuroprotective effect was evident with 15 and 120 min post-reperfusion, with a declination at 240 min assessed via NDS and cerebral infarction.

ACCEPTED MANUSCRIPT Early detection of cerebral injury was evident from DWI within 30 min post-reperfusion which is not possible with T2 images are in concordance with Moseley et al., 1990. In the present study infarcted area (ipsilateral hemisphere) was observed as hyperintense region on T2

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weighted as well as DW images. The ADC value in ipsilateral hemisphere significantly reduced as compared to contralateral hemisphere especially in the frontoparietal cortex and lateral

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caudoputamen region is in corroboration with previous lab studies (Chauhan et al., 2012; Gupta

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et al., 2005; Wang et al., 2002). This may be attributed to an imbalance in water and ion homeostasis, failure of Na+/K+ pump and further aggravation due to reperfusion. The ADC value

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of contralateral hemisphere also declined on 4th day as compared to that observed after 30 min post-reperfusion. Reduction in CBF and localized vasospasm may be the ascribing factor for

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regional differences in ADC (Wang et al., 2002). Lercanidipine significantly reduced cerebral infarction (14%) as evident from DWI and T2 images as compared to the 96 h MCAo control rats (25%). The cerebral infarction in 30 min post-reperfusion (10%) was not further aggravated

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after lercanidipine treatment administered 120 min post-reperfusion and followed further for 3

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days. The regional differences between ipsilateral and contralateral ADC value and signal intensity was evident with 30 min post-reperfusion and 96 h MCAo control group, which was not

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observed with lercanidipine treated rats. Moreover, ADC ratio was improved in lercanidipine treated rats as compared to 30 min post-reperfusion rats, which was not observed with 96 h MCAo control rats.

4.2. Lercanidipine-induced vasodilatation In the current investigation vasodilatory property of lercanidipine was evident by LDF method. Compared with other methods LDF is non-invasive and possess an ability of continuous measurement, though lacking the ability of monitoring of absolute blood flow and sensitivity to physiological parameters such as altered haematocrit, blood gases etc. (Kramer et al., 1996). The rCBF reached 80-90% with lercanidipine when administered during ischemia, but failed to show vasodilatation when administered post-reperfusion. The rCBF of MCAo control and lercanidipine post-reperfusion group reached 50-65% of baseline value 30-120 min postreperfusion. This further strengthens the pathophysiological mechanism of stroke elucidating central mechanism for cellular damage during ischemia is elevated cytosolic calcium levels due to sudden loss of high energy adenosine triphosphate, depolarization, glutamate release,

ACCEPTED MANUSCRIPT anaerobic glucolysis and acidosis (White et al., 2000; Siesjo et al., 2008). On the contrary, key events after reperfusion includes excessive free radical generation such as superoxide, NO and peroxynitrite, lipid peroxidation, inflammation and caspase activation (White et al., 2000). Thus,

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in the present study, the neuroprotective effect mediated by lercanidipine pretreatment predominantly involves vasodilatory mechanisms, while for post-reperfusion involves reduction

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of oxidative stress, inflammatory and apoptotic markers. SB 201823-A, a neuronal CCB

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inhibited calcium influx in central neurons (hippocampal and cerebellar) in vitro and in vivo model of focal cerebral ischemia (Barone et al., 1995). Inositol trisphosphate receptor-type 2

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knockout mice protected astrocytes primarily involved in calcium signaling in a photothrombosis-induced stroke model (Li et al., 2015). Our study demonstrates that with a

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decline in rCBF the NDS and cerebral infarct volume increases, is in agreement with previous finding (Hedna et al., 2015). Various mechanisms such as autoregulation, pressure gradient, carbon dioxide pressure changes, and hyperperfusion have been illustrated for increased NDS

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with a drop in rCBF in ischemic zone (Alexandrov et al., 2007; Hedna et al., 2015). During

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occlusion due to paradoxically hypercapnia induced vasodilation in normal area, there is shifting of blood flow more towards non-ischemic area as compared to ischemic area termed as

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“Reversed Robin Hood syndrome or cerebral steal” (Alexandrov et al., 2007). The classical phenomenon resulting in redistribution of blood to an ischemic area as compared to nonischemic area is known as “Robin Hood Phenomenon” or “inverse-steal” (Alexandrov et al., 2007; Hedna et al., 2015). Thus, in the present study as lercanidipine increased rCBF in ischemic area, Robin Hood Phenomenon may be one of the contributing mechanisms in providing protection and decreased NDS. Other mechanism regulating rCBF includes autoregulation executed via endothelial NOS-NO pathway and neurovascular coupling executed via neuronal NOS-NO pathway (Garry et al., 2015).

4.3. Anti-oxidant effects of lercanidipine Oxidative stress and inflammation mainly contribute to the neuronal cell death following ischemic stroke (Andersen et al., 2004; Wang et al., 2007). Our study demonstrated that the levels of MDA and NO were significantly lowered; while the levels of GSH and SOD were increased in lercanidipine treated rats at a dose of 0.5 mg/kg,i.p. as compared to MCAo control

ACCEPTED MANUSCRIPT rats. Lercanidipine treated rats showed significant amelioration of above altered biochemical markers most effectively even till 120 min after post-reperfusion. During oxidative stress, an imbalance between ROS production and anti-oxidant enzyme

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levels leads to cerebral infarction. Oxidative stress induced ROS is involved in various destructive mechanisms including glial cell activation, mitochondrial dysfunction, protein

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misfolding, proteasome malfunction and programmed cell death (Andersen et al., 2004).

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Calcium is another vital mediator for ischemic-reperfusion injury. Intracellular calcium overload during ischemia due to translocation of extracellular calcium into cells activate lipases, proteases,

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kinases, phosphatases, and endonucleases and enhances the production of ROS (Siesjo et al., 1995). ROS induced polyunsaturated fatty acids derived lipid peroxides products are unstable

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and decompose forming complex compounds such as MDA (Chen et al., 2011). ROS also leads to disruption of BBB and extracellular matrix ultimately imposing to cerebral infarction due to release of various cytotoxic components such as MMPs, cytokines, chemokines, integrins etc

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(Andersen et al., 2004; Wang et al., 2007). Glutathione (L-c-glutamyl-L-cysteinylglycine; GSH),

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an intracellular thiol tripeptide has been shown to reduce cerebral infarction in vivo in ischemia model and in vitro in murine brain endothelial cells (Song et al., 2015).

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During cerebral ischemia superoxide radicals (O2 ) generated are detoxified to hydrogen peroxide (H2O2) by SOD enzyme, which is in turn is converted to water (H2O2) by catalases or glutathione peroxidases (GSHPx) (Chen et al., 2011). Substantial evidences suggest beneficial as well as detrimental role of NO during cerebral ischemic-reperfusion injury (del Zoppo et al., 2000). In acute stroke patient’s poor neurological status was associated with elevated serum MDA levels alongwith decreased SOD, GSH-Px and catalase activities (Milanlioglu et al., 2015). Lercanidipine drug in hypertensive patients reduced oxidative stress via suppression of free radical generation (Incandela et al., 2001).

4.4. Lercanidipine inhibitory effect on neuroinflammation MMPs are primarily involved during stroke leading to BBB degradation, cerebral hemorrhage, and edema with a pro-inflammatory nature (Cuadrado et al., 2008). In the present study, MMP-2 and MMP-9 activity was increased as compared to MCAo control rat evident 24 h post-MCAo. Thus, mechanisms elucidated above may contribute for lercanidipine MMP-2

ACCEPTED MANUSCRIPT inhibitory activity which was evident 120 min post-reperfusion. The MMP-9 inhibition was observed even at 240 min post-reperfusion, may be implicated in combination with delayed t-PA treatment. Anti-inflammatory effect of lercanidipine has been elucidated in rat vascular smooth

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muscle cells exposed to lipopolysaccharide and interferon-γ via suppression of NO, ROS and TNF-α through down-regulation of iNOS, MMP-2/MMP-9, and HMGB1 (Yeh et al., 2013).

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MMP-2, gelatinase A, EC 3.4.24.24 is secreted mainly by vascular endothelium and smooth

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muscle cells. MMP-2 mediated endothelin-1[1–21] generation and platelet aggregation is crucial in various inflammatory and cardiovascular conditions including stroke (Fernandez-Patron et al.,

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1999; Sawicki et al., 1997).

It has been well elucidated that extracellular proteolysis system required for neurite and axon

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extension is mainly executed through MMPs and t-PA. Numerous mechanisms have been elucidated for risk of hemorrhagic transformation associated with delayed t-PA treatment. These include interaction of t-PA with non-fibrin substrates such as enhancement of N-methyl-D-

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aspartate receptor mediated signaling and MMPs particularly MMP-9 dysregulation in the

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neurovascular unit apart from plasmin (Nicole et al., 2001; Lo EH et al., 2004). In a study FeTMPyP, a peroxynitrite decomposition catalyst decreased ischemic MMP-9 levels upregulated

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after t-PA treatment in MCAo rat model (Chen et al., 2015). In stroke patients elevated plasma MMP-9 levels associated with poor neurological outcome has been illustrated (Castellanos et al., 2003). Thus, targeting t-PA-MMP signaling pathway may be one of the fruitful strategies to increase safety and efficacy of t-PA in stroke.

4.5. Anti-apoptotic effects of lercanidipine Two major pathways of cerebral ischemia induced cell death include: irreversible necrosis in core region, while reversible apoptosis in ischemic penumbra. Thus, cells in the ischemic penumbra provide an opportunity for salvage and vision for neuroprotective agents. Findings of our study revealed significant inhibition of caspase-3 and caspase-9 activity (3.0 and 2.5 fold respectively) in MCAo control group as compared with the sham group. This indicates that caspase-3 and caspase-9 plays a crucial role in the pathogenesis of stroke. Lercanidipine significantly attenuated caspase-3 and caspase-9 activity till 120 min post-reperfusion, but not at 240 min.

ACCEPTED MANUSCRIPT Apoptosis is executed through extrinsic and intrinsic pathway via caspases which is a family of cysteine-dependent aspartate-directed proteases. The extrinsic apoptosis pathway activates caspase-8 which either activates executioner caspases such as caspase-3, or intrinsic apoptotic

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pathway. In intrinsic pathway, loss of transmembrane potential leads to mitochondrial respiration failure, enhanced free radicals generation and loss of energy production. There is cytochrome C

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release from mitochondria into the cytoplasm complexing with procaspase-9 and other proteins

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(such as Apaf1) forming apoptosome, thereby activating caspase-9. Once downstream caspases are activated such as caspase-3, DNA breaking enzymes (e.g., endonucleases) and repair

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enzymes (Poly ADP-ribose polymerase) activate leading to cell death (Broughton et al., 2009; Rami et al., 2008). Intranasal delivery of caspase-9 inhibitor in MCAo model reduced cerebral

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injury and improved neurological functions mediated through inhibition of caspase-6 dependent axon degeneration (Akpan et al., 2011). In a cerebral ischemic-reperfusion injury rat model aloe polysaccharides reduced elevated neuronal caspase-3 protein and mRNA expression (Lu et al.,

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4.6. Conclusions

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2012).

The present study is of clinical relevance as lercanidipine has been found be neuroprotective in focal cerebral ischemic-reperfusion injury model. The prê-treatment, post-treatment efficacy till 120 min and sub-acute studies imposes lercanidipine as a treatment option in acute stroke patient population associated with risk factors and recurrence. The neuroprotective effect may be attributed to anti-oxidant, anti-inflammatory and anti-apoptotic property, along with calcium channel blocking activity. The limitation of delayed treatment associated hemorrhagic transformation with thrombolytic agents can be overcome with lercanidipine due to its MMPs inhibitory activity will be an added advantage.

5. Limitations of the present study

The major lacunae of the present study are deficiency of chronic study and exploration of combination effects with rt-PA as lercanidipine was found to have MMPs inhibitory activity.

ACCEPTED MANUSCRIPT Acknowledgment The authors are thankful to Department of Science & Technology, New Delhi, India, for

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financially assisting in the form of Women Scientist Fellow (File No. SR/WOS-A/LS-342/2013). Lercanidipine was provided as a kind gift sample from Glenmark Pharmaceuticals Ltd., Mumbai,

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India is duly acknowledged.

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Fig. 1. Figure depicts schematic representation of experimental protocol followed for the

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lercanidipine study. (A) Protocol 1. Acute studies. (B) Protocol 2. Sub-acute studies.

Fig. 2. Neuroprotective effect of lercanidipine (Protocol 1) on neurological deficit score (NDS;

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Fig. 2A), grip test score (Fig. 2B) and rota rod test (Fig. 2C) assessed 24 h after MCAo in a 2 h MCAo model of focal cerebral ischemia in rats. Protocol 1a: vehicle or lercanidipine (0.25, 0.5

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and 1 mg/kg, i.p.) administered 60 min prior MCAo; Protocol 1b: vehicle/lercanidipine (0.5 mg/kg, i.p.) administered 60 min post-MCAo and Protocol 1c: lercanidipine (0.5 mg/kg, i.p.)

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administered 15 min, 120 min and 240 min post-reperfusion. NDS and grip test score is represented as grouped median (minimum-maximum) in a scatter vertical plot. Middle horizontal line represents median value, while line above median represents 25th percentile and line below

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individual rat. Kruskal-Wallis test followed by Mann-Whitney U test was applied for NDS and grip test score. Rota rod test represents time spent (s) on rotating spindle. One-way ANOVA

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followed by Bonferroni’s post hoc test was applied for rota rod test. **p < 0.01,

***

p < 0.001 vs.

sham group; #p < 0.05, # #p < 0.01, # # #p < 0.001 vs. MCAo control group.

Fig. 3. Neuroprotective effect of lercanidipine (Protocol 1 and 2) on cerebral infarction volume assessed 24 h after MCAo by TTC staining in a 2 h MCAo model of focal cerebral ischemia in rats. (A) Representative TTC stained coronal rat brain sections of lercanidipine treated rats: a. Vehicle + sham, b. Vehicle + MCAo, c. Lercanidipine 0.25 mg/kg, i.p., 60 min prior + MCAo, d. Lercanidipine 0.5 mg/kg, i.p., 60 min prior + MCAo, e. Lercanidipine 1 mg/kg, i.p., 60 min prior + MCAo, f. MCAo + Lercanidipine 0.5 mg/kg, i.p., 60 min post-MCAo, g. MCAo + Lercanidipine 0.5 mg/kg, i.p., 15 min post-reperfusion, h. MCAo + Lercanidipine 0.5 mg/kg, i.p., 120 min post-reperfusion, i. MCAo + Lercanidipine 0.5 mg/kg, i.p., 240 min post-reperfusion, j. MCAo + Vehicle 96 h and k. MCAo + Lercanidipine 0.5 mg/kg, i.p., 96 h . Red color region indicates non-infarct area, while white color region indicates infarct area of coronal brain sections. (B) Effect of lercanidipine on cerebral infarction volume (mm3) assessed 24 h and 96 h after MCAo. Bullets of different shapes represent value of an individual rat. One-way ANOVA

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Fig. 4. (A) Representative T2-weighted (Panel 1) and diffusion-weighted (Panel 2) MRI images of rat brain slices: a) 30 min post-reperfusion (baseline), b) 96 h vehicle-treated MCAo and c) 96

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h lercanidipine (0.5 mg/kg ,i.p.) treated rats. Focal cerebral ischemia was evident as hyperintense

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region in the ipsilateral right hemisphere. Lercanidipine treatment ameliorated the lesions evident as less hyperintense region. Neuroprotective effect of lercanidipine on regional cerebral blood

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flow (rCBF) and cerebral infarction using magnetic resonance imaging (MRI). (B) The rCBF was monitored using LDF in the MCA region over a period of 2 h during MCAo and and further

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for 2 h post-reperfusion at an interval of 30 min, with a baseline 30 min prior to MCAo. The graph represents three groups MCAo + vehicle control group, lercanidipine (0.5 mg/kg, i.p., 60 min post-MCAo) and lercanidipine (0.5 mg/kg, i.p., 15 min post-reperfusion). Each value for

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control group.

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ANOVA applied w.r.t. treatment and time.*p < 0.05, **p < 0.01, ***p < 0.001 vs. MCAo + vehicle

Fig. 5. Effect of lercanidipine on matrix metalloproteinases (A) MMP-2, (B) MMP-9, (C) caspase-3 and (D) caspase-9 activity estimated in rat brain cortex 24 h after MCAo in a 2 h MCAo model of focal cerebral ischemia in rats. Horizontal line represents median value and bullet represents value of an individual rat in scatter vertical plots, n = 4-6.*p < 0.05, ***p < 0.001 vs. sham group; # # #p < 0.001, # #p < 0.01, #p < 0.05 vs. MCAo control group.

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Table 1 Neuroprotective effect of lercanidipine (0.5 mg/kg, i.p.) on signal intensity, apparent diffusion

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coefficient (ADC), ADC (R/L) ratio and percentage (%) infarct volume in MCAo model of focal cerebral ischemia in rats. Data presented as mean ± SEM, n = 4-6. @p < 0.05,

@ @

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control group.

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Table 2

Effect of lercanidipine on malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide

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(NO) and superoxide dismutase (SOD) levels estimated in rat brain cortex 24 h after MCAo in a 2 h MCAo model of focal cerebral ischemia in rats. Data represented as mean ± SEM, n = 4-6.*p ***

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Table 1 96 h MCAo control Ipsilateral Contralateral

Signal Intensity (arbitrary units)

178.82 ± 7.23@ 81.80 ± 7.13

170.83 ± 15.22 @

107.47 ± 4.42

ADC values (10-3 mm2/s)

0.36 ± 0.02@@

0.48 ± 0.05 @

0.64 ± 0.02

0.51 ± 0.02

% infarct volume

10.16 ± 2.07

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157.23 ± 7.73

0.51 ± 0.03

99.62 ± 10.79

0.61 ± 0.02

0.75 ± 0.10

0.82 ± 0.05 *

25.90 ± 2.44 *

13.78 ± 2.78#

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ADC (R/L) ratio

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96 h Lercanidipine Ipsilateral Contralateral

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Parameters

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NO levels (µM/mg of protein)

Vehicle + Sham

34.4 ± 5.8

132.2 ± 14.9

29.6 ± 2.6

77.5 ± 4.3

Vehicle + MCAo

173.1 ± 7.3 ***

62.8 ± 6.9**

136.4 ± 13.1***

31.7 ± 4.6***

Ler 0.5 mg/kg, i.p., 60 min prior + MCAo

111.5 ± 13.8 ##

89.8 ± 5.5

73.1 ± 15.1 ##

MCAo + Ler 0.5 mg/kg, i.p., 15 min post-reperfusion

63.1 ± 7.3 ###

120.3 ± 9.4#

MCAo + Ler 0.5 mg/kg, i.p., 120 min post-reperfusion

106.4 ± 10.3 ##

116.9 ± 13.1#

83.5 ± 5.6##

57.4 ± 3.4#

MCAo + Ler 0.5 mg/kg, i.p., 240 min post-reperfusion

119.7 ± 9.6

93.6 ± 6.3

121.6 ± 8.9

50.5 ± 6.6

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SOD levels (% inhibition of control)

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MDA levels (nM/mg of protein)

Groups

61.8 ± 7.7# 70.7 ± 6.3###