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NEUROPROTECTIVE AND ANTI-APOPTOTIC EFFECTS OF LIRAGLUTIDE IN THE RAT BRAIN FOLLOWING FOCAL CEREBRAL ISCHEMIA S. BRIYAL, a S. SHAH b AND A. GULATI a*

hemisphere compared to sham-operated group. The number of TUNEL-positive cells in vehicle group was 73.5 ± 3.3 and 85.5 ± 5.2/750 lm2 in non-diabetic and diabetic vehicle-treated MCAO rats, respectively. Following liraglutide treatment the number of TUNEL-positive cells was remarkably attenuated to 25.5 ± 2.8 and 41.5 ± 4.1/750 lm2 (p < 0.001) in non-diabetic and diabetic rats, respectively. The results demonstrate that glucagon-like peptide 1 (GLP-1) agonist, liraglutide, is a neuroprotective agent and attenuates the neuronal damage following cerebral ischemia in rats by preventing apoptosis and decreasing oxidative stress. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Department of Pharmaceutical Sciences, Chicago College of Pharmacy, Midwestern University, Downers Grove, IL 60515, USA b Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, IL 60515, USA

Abstract—Stroke is a leading cause of death and serious, long-term disability worldwide. We report that rats receiving liraglutide show markedly attenuated infarct volumes and neurological deficit following ischemic insult. We have also investigated the effect of liraglutide on apoptosis and oxidative stress pathways after ischemic injury in diabetic and non-diabetic rats. Male Sprague-Dawley rats weighing 300– 350 g were used. Diabetes was induced by streptozotocin. Rats were pretreated with either vehicle or liraglutide (50 lg/kg, s.c.) for 14 days and thereafter subjected to middle cerebral artery occlusion (MCAO). Twenty-four hours after occlusion, rats were assessed for neurological deficit, motor function and subsequently sacrificed for estimation of infarct volume, oxidative stress and apoptotic markers. Vehicle-treated non-diabetic and diabetic rats showed significant (p < 0.001) neurological deficit following cerebral ischemia. Liraglutide pretreatment resulted in significantly (p < 0.001) less neurological deficit compared to vehicletreated MCAO rats. Cerebral ischemia produced significant (p < 0.0001) infarction in vehicle-treated rats; however, the infarct volume was significantly (p < 0.001) less in liraglutide-pretreated rats. Oxidative stress markers were increased following ischemia but were attenuated in liraglutide-treated rats. Anti-apoptotic protein Bcl-2 expression was decreased and pro-apoptotic protein Bax expression was increased in vehicle-treated MCAO rats compared to sham (p < 0.0001). On the other hand liraglutide pretreatment showed significantly (p < 0.01) increased expression of Bcl-2 and decreased expression of Bax in MCAO rats. In vehicle-treated group, the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells significantly (p < 0.0001) increased in the ischemic

Key words: cerebral ischemia, GLP-1 receptor, liraglutide, oxidative stress, apoptosis.

INTRODUCTION Stroke is the leading cause of adult disability and represents a major health and economical threat (Donnan et al., 2008). Despite the severity of this condition, the only currently available FDA-approved pharmacological treatment for ischemic stroke is recombinant tissue plasminogen activator (rtPA), which has complications of a relatively short window of time between infarct and treatment (3–4 h) and an increased risk of subarachnoid hemorrhage (Micieli et al., 2009). A large number of other agents, broadly classified as neuroprotective and aiming to slow or stop the secondary damage associated with the ischemic cascade following stroke, have shown promise in the initial stages of research but have thus far failed to demonstrate efficacy in clinical trials (Ly et al., 2006; Stankowski and Gupta, 2011). Unfortunately, to this point, with the notable exception of rtPA, no pharmaceutical interventions have proven efficacious in human clinical trials. A new approach is therefore needed to investigate these conditions and the pathways and mechanisms which lead to them in an attempt to fully understand the etiology of the disease and where it may be curtailed by human intervention. Type 2 diabetes mellitus (T2DM) is a major risk factor for cardiovascular events, including stroke (Luitse et al., 2012). In addition, patients with T2DM have two- to sixfold increased risk for severe strokes and have worse outcome than patients without T2DM (Megherbi et al., 2003; Reeves et al., 2010). Moreover, it increases the risks of morbidity and mortality after stroke. Oxidative

*Corresponding author. Address: Chicago College of Pharmacy, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. Tel: +1-630-971-6417; fax: +1-630-971-6097. E-mail address: [email protected] (A. Gulati). Abbreviations: AD, Alzheimer’s disease; EDTA, ethylenediaminetetraacetic acid; Ex-4, exendin-4; GLP-1, glucagon-like peptide 1; GLP-1R, GLP-1 receptor; GSH, glutathione; MCAO, middle cerebral artery occlusion; MDA, malondialdehyde; NGF, nerve growth factor; PFA, paraformaldehyde; ROS, reactive oxygen species; RPM, rotations per minute; rtPA, recombinant tissue plasminogen activator; SOD, superoxide dismutase; STZ, streptozotocin; T2DM, type 2 diabetes mellitus; TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. http://dx.doi.org/10.1016/j.neuroscience.2014.09.064 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 269

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stress plays an essential role in the pathogenesis of cerebral ischemic injury (Vinik and Flemmer, 2002; Niizuma et al., 2009), which causes apoptosis and delayed death of cells through oxidative damage to lipids, proteins, and DNA in the ischemic penumbral region (Warner et al., 2004; Nakka et al., 2008; Niizuma et al., 2010). Assessing new stroke therapies for patients with diabetes mellitus is essential, because diabetes mellitus is an important risk factor for stroke. Glucagon-like peptide 1 (GLP-1) is an incretin hormone that is released into the blood stream postprandially from the gut and binds to the GLP-1 receptor (GLP-1R) (Bell et al., 1983). Currently, the GLP-1 receptor agonists exendin-4 (Ex-4), liraglutide and lixisenatide are approved for treatment of T2DM (Lovshin and Drucker, 2009; Wohlfart et al., 2013). A selective inhibitor of dipeptidylpeptidase-4 (DPP-4), functioning as a long-acting agonist of GLP-1, is also in clinical use worldwide for patients with T2DM (Drucker and Nauck, 2006; Darsalia et al., 2013; Yang et al., 2013). The beneficial effects of GLP-1 are not limited to the treatment of diabetes but also produce significant neuroprotection in animal models of cerebral ischemia and Alzheimer’s disease (AD) (During et al., 2003; Lee et al., 2011; Briyal et al., 2012; Sato et al., 2013; McClean and Holscher, 2014). GLP-1 acts as a growth factor in the brain (Buteau et al., 1999) and has been shown to protect against oxidative injury (Perry et al., 2007). Furthermore, the distribution of GLP-1R in the brain suggests they play a central role in the regulation of neuronal activity and protect the brain tissue (Banks et al., 2004; Sharma et al., 2014). GLP1-Rs have become well accepted as having anti-apoptotic properties. Both GLP-1 and Ex-4 augment cellular integrity and overall survival following exposure to a range of pro-apoptotic agents such as peroxides, cytokines and fatty acids (Hui et al., 2003; Li et al., 2003; Harkavyi and Whitton, 2010). In addition GLP-1 appears to increase expression of anti-apoptotic genes Bcl-2 and Bcl-xl (Buteau et al., 2004). Studies have also shown that Ex-4 enhances nerve growth factor (NGF)-induced neuronal differentiation and attenuates neural degeneration following NGF withdrawal indicating a potential neuroprotective role of GLP-1Rs (Drucker, 2001; Perry et al., 2002a,b). Liraglutide is an analog of GLP-1 that acts through GLP-1R. It has been shown that liraglutide improves cognitive function and reduces amyloid plaque deposition in a mouse model of AD, and it is now being tested in clinical trials in AD patients (McClean and Holscher, 2014). Liraglutide crosses the blood–brain barrier and has neuroprotective effects in rats (Hunter and Holscher, 2012). Studies in our laboratory using the middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia in rats demonstrated that chronic administration of Ex-4 significantly improved infarct volume, neurological deficit and oxidative stress parameters in ischemic rats (Briyal et al., 2012). In another study, it was found that liraglutide, administered intraperitoneally after induction of stroke, reduced infarct volume, oxidative stress parameters and increased cortical vascular endothelial growth factor (Sato et al., 2013).

Patients with T2DM may be receiving liraglutide as part of their treatment. It was thought worthwhile to investigate whether liraglutide treatment will offer any protection to the CNS damage following cerebral ischemia. In an effort to more closely model the clinical situation, we studied the effect of liraglutide given subcutaneously 2 weeks prior to the induction of ischemia stroke in diabetic and non-diabetic rats. The effects of GLP-1 receptor agonist liraglutide on infarct area, neurological and motor deficit, oxidative stress, and apoptotic markers were determined in a rat model of MCAO.

EXPERIMENTAL PROCEDURES Animals Male Sprague-Dawley rats (300–350 g) obtained from Harlan, Indianapolis, IN were allowed to acclimate for at least 4 days before use. Rats were housed in a room with controlled temperature (23 ± 1 °C), humidity (50 ± 10%), and light (6:00 A.M. to 6:00 P.M.). Food and water were available continuously. Care and use of rats along with experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Midwestern University.

Drugs Ketamine (Butler Animal Health Supply, Dublin, OH, USA) and xylazine (Lloyd Laboratories, Shenandoah, IA, USA) were administered intraperitoneally (i.p.) in doses of 100 mg/kg and 10 mg/kg, respectively. Streptozotocin (Enzo Life Sciences, Inc., Farmingdale, NY, USA) was freshly dissolved in 0.01 M sodium citrate buffer (pH 4.3) and administered i.p. in the dose of 45 mg/kg. Liraglutide (Novo Nordisk, Inc., Princeton, New Jersey, USA) was dissolved in saline and administered at the dose of 50 lg/kg, subcutaneously (s.c.) for 14 days prior to MCAO.

Experimental protocol Rats were randomly divided into five groups of six animals each. Group 1 animals were subjected to a sham operation. Rats in groups 2–6 underwent MCAO and were treated as follows – Group 2: Vehicle + MCAO (ND); Group 3: Liraglutide + MCAO (ND); Group 4: Vehicle + MCAO (D); Group 5: Liraglutide + MCAO (D); Group 6: Insulin + MCAO (D); (ND: Non-diabetic and D: Diabetic). T2DM was induced in rats belonging to the diabetic group by administering streptozotocin (STZ). Rats were fed with high-fat diet (Research Diets, Inc., New Brunswick, NJ, USA) for 2 weeks prior to STZ administration. On day 3, blood glucose was obtained from the rat-tail and tested for hyperglycemia using the SureStep Complete Blood Glucose monitor kit. Rats with blood glucose value >300 mg/dL were considered diabetic.

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MCAO to induce focal cerebral ischemia Induction of focal cerebral ischemia via MCAO was performed according to the method of Koizumi et al. (1986). Rats were anesthetized with ketamine and xylazine, a rectal core temperature of 37 ± 1 °C (measured with a probe using Cole Palmer Animal Monitoring Thermometer (Vernon Hills, IL, USA)) was maintained using the thermo-controlled base of the operating table. Animal secured in supine position, a midline incision was made and the right common carotid artery, external carotid artery, and internal carotid artery were exposed. A 4.0 monofilament nylon thread (CP Medical, Portland, OR, USA) with rounded tip by briefly heating the end was used to occlude the middle cerebral artery. The filament was advanced from the external carotid artery into the lumen of the internal carotid artery until a resistance was felt (20 mm), indicating occlusion of the middle cerebral artery. In order to create a permanent model of focal cerebral ischemia the filament remained in place and was not removed. In sham-operated animals, the common carotid artery and external carotid artery were exposed and without touching the internal carotid artery the incision was sutured using 3.0 silk surgical sutures (Ethicon, Inc., Blue Ash, Ohio, USA). Neurological evaluation Neurological evaluation was performed prior to and 24 h following MCAO. The neurological evaluation was based on a six-point scale as described by Tatlisumak et al. (1998). The scoring was as follows: 0 = no deficits, 1 = failure to fully extend left forepaw, 2 = circling to the left, 3 = paresis to the left, 4 = no spontaneous walking and 5 = death. Motor performance tests Motor activity and coordination of the animals was assessed prior to and 24 h post MCAO using a grip test, foot fault test, rota rod and spontaneous locomotor activity. Grip test. The grip test was performed using a string 50 cm in length, pulled taut between two vertical supports and elevated 40 cm above a flat surface. The animal was placed on the string midway between the supports and evaluated according to a six-point scale (Moran et al., 1995). The scoring was a follows: 0 = falls off, 1 = hangs on by two forepaws, 2 = hangs on by two forepaws and attempts to climb on, 3 = hangs on by 3 + paws, 4 = hangs on by all paws plus tail, and 5 = escapes. Foot fault test. Animals were placed on an elevated grid floor with a mesh size of 30 mm for 1 min to acclimate. They were then observed for 1 min and evaluated for foot fault errors (i.e. a misplaced limb falling through the grid) compared with paired steps as follows (Markgraf et al., 1992): % foot fault error = (number of faults/number of paired steps)  100 Rota rod. Animals were acclimated to the rotating spindle of the rota rod apparatus (Rota-Rod 47700, Ugo Basile, Italy) prior to MCAO. Rats were placed on the

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rotating spindle, set to a constant eight rotations per minute (RPM), until they demonstrated the ability to remain on the spindle for 60 s. Rats were then subjected to a baseline test trial on the accelerating spindle (4–40 RPM) over 5 min. The acceleration trial was repeated at 24 h after MCAO, and the time at which they fell off was recorded in seconds (Rogers et al., 1997). Spontaneous locomotor activity. Spontaneous locomotor activity was assessed using an animal activity meter (Opto-Varimex-4 Auto-Track System, Columbus Instruments, Columbus, OH, USA) prior to and at 24 h following MCAO. Each animal was observed for a period of 10 min in a square enclosed area equipped with infrared photocells along the X, Y, and Z axes to quantitatively measure spontaneous horizontal and vertical motion. Assessment of cerebral infarct volume After completion of neurological and motor function testing 24 h post MCAO, rats were euthanized by decapitation, and the brains were quickly removed and chilled in saline at 4 °C for 5 min to determine infarct volume. They were then cut into 2-mm-thick coronal slices using a Brain Matrix (Harvard Apparatus, Holliston, Massachusetts, USA). Sections were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, MO) dissolved in saline for 15 min at 37 °C. The stained sections were stored in 10% formalin and refrigerated at 4 °C for further analysis (Li et al., 1997). Infarct volumes were calculated by sampling each side of the coronal sections with a digital camera (Nikon). The infarct area, outlined in white, was measured using image analysis software (Adobe Photoshop CS4). Infarct size is expressed as infarction volume in mm3 as the sum of infarct areas in each slice, corrected for edema. Estimation of oxidative stress markers Brain levels of malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were estimated 24 h post MCAO. The animals were decapitated, and the brains were quickly removed and washed in chilled saline, then stored at 80 °C. The biochemical analyses were performed within 48 h. Measurement of lipid peroxidation. MDA was estimated according to the method of Ohkawa et al. (1979). Brains were homogenized with 10 times (w/v) in 0.1 M sodium phosphate buffer (pH 7.4). 1.5-ml acetic acid (20%, pH 3.5), 1.5-ml thiobarbituric acid (0.8%), and 0.2-ml sodium dodecyl sulfate (8.1%) were added to 0.1 ml of processed tissue sample. The mixture was heated at 100 °C for 60 min and then 5 ml of n-butanol:pyridine (15:1%, v/v) and 1 ml of distilled water were added to the cooled mixture, shaking vigorously. After centrifugation at 4000 RPM for 10 min, the organic layer was withdrawn and absorbance was measured at 532 nm using a spectrophotometer. Measurement of reduced GSH. Reduced GSH was measured according to the method of Ellman (1959) with

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minor modifications. Brains were homogenized with 10 times (w/v) in 0.1 M sodium phosphate buffer (pH 7.4) and then centrifuged with 5% trichloroacetic acid to separate out proteins. Next, 2 ml of phosphate buffer (pH 8.4), 0.5 ml of 50 5 dithiobis (2-nitrobenzoic acid), and 0.4 ml of double distilled water were added to 0.1 ml of homogenate. The mixture was vortexed and the absorbance was read within 15 min at 412 nm using a spectrophotometer. Measurement of SOD. SOD was measured according to the method of Kakkar et al. (1984). Brains were homogenized with 10 times (w/v) sodium phosphate buffer (pH 7.4). To 0.1-ml homogenized tissue, 1.2-ml sodium pyrophosphate buffer (0.052 M, pH 8.3), 0.1-ml phenazine methosulfate (186 lM), 0.3-ml nitro blue tetrazolium (300 lM), and 0.2 ml NADH (780 lM) were added. The mixture was incubated at 30 °C for 90 min after that 4 ml of n-butanol and 1 ml of acetic acid were added, and the mixture was shaken vigorously. Subsequently centrifugation was carried out at 4000 RPM for 10 min, the organic layer was removed, and absorbance was measured at 560 nm using a spectrophotometer. Estimation of apoptotic markers Brain tissues were washed in chilled saline and homogenized in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris–HCl pH 7.5, 120 mM NaCl, 1.0% Triton X100, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 10% glycerol, 1 mM EDTA and 1 protease inhibitor, Roche, San Francisco, California, USA). Proteins were isolated in solubilized form and concentrations was determined using Folin–Ciocalteu’s Reagent (Lowry et al., 1951). Solubilized protein (60 lg) was denatured in Laemmli sample buffer (Bio-Rad, Hercules, California, USA), resolved in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred on nitrocellulose membrane (Sigma–Aldrich, St. Louis, MO, USA). The membrane was then blocked with 5% bovine serum albumin (BSA) (w/v) in TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween 20) for 30 min at room temperature. The membranes were incubated with rabbit polyclonal anti-Bax and anti-Bcl-2 antibodies (1:1000) (Sigma– Aldrich, St. Louis, MO, USA) at 4 °C overnight, followed by incubation with goat anti-rabbit immunoglobulin G (IgG), horseradish peroxidase-conjugated (HRP) secondary antibody (1:1000) (Santa Cruz Biotech., Santa Cruz, CA, USA) for 2 h at room temperature. b-actin (1:10,000; Sigma–Aldrich, St. Louis, MO, USA) was used as a loading control. The labeled proteins were visualized with SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific, Schaumburg, Illinois, USA) using the Kodak Gel Logic 1500 Imaging System (Carestream Health Inc., New Haven, CT, USA). Protein expression was analyzed using ImageJ (NIH) software. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Twenty-four hours following MCAO, animals underwent transcardial perfusion to fix the brains. Briefly, the

animals were anesthetized with urethane (0.15 g/kg, IP), and the chest opened to expose the heart. A feeding needle, connected to an infusion pump by a tube, was inserted into the left ventricle up to the aorta. Two ventricles were blocked with hemostatic forceps, and the right atrium was cut open to allow the perfusate to drain. A perfusion of normal saline (200–300 ml) was infused to displace the blood in the vessels, until the draining perfusate was clear. An infusion of 4% paraformaldehyde (300 ml) then displaced the saline to fix the tissue. The animals were then decapitated and the brains removed. The brains were post-fixed in 50 ml of 4% paraformaldehyde (PFA) in NaPO4 buffer solution for 2 h, and then placed in 20% sucrose/4% PFA, pH 7.4, 50 ml/ brain at 4 °C for 48 h. The brains were then sliced into 10-lm-thick slices for immunofluorescent analyses at 20 °C using a cryostat (Microtome cryostat HM 505E; Walldorf, Germany). Coronal cryostat sections were processed according to the manufacturer’s instructions for TUNEL assay using Click-iT TUNEL Alexa Fluor Imaging Assay kit (Molecular Probes, Invitrogen, NY, U.SA). Cells exhibiting DNA fragmentation (TUNEL positive) were counted in the tissues by the use of a 60 objective of inverted fluorescent microscope (Nikon eclipse Ti series) in a blinded fashion (Husse et al., 2003; Yu et al., 2004). Statistical analysis Data are presented as mean ± S.E.M. A one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc comparison test was used for intergroup comparison in behavioral data, evaluating apoptotic and oxidative stress markers. A p value of less than 0.05 was considered to be significant. The statistical analysis was processed with GraphPad Prism 6.00 (GraphPad, San Diego, CA, USA).

RESULTS Effect on blood glucose levels following STZ injection The mean blood glucose levels (473.0 ± 10.0, 482.0 ± 6.0, and 475.0 ± 23.0 mg/dl, respectively) after STZ injection in diabetic vehicle, diabetic liraglutide, and diabetic insulin-treated rats were significantly (p < 0.0001) higher compared to levels before STZ injection (120.0 ± 2.0, 120.0 ± 1.0, and 120.0 ± 1.0 mg/dl, respectively). This indicates increased blood glucose levels 3 days following STZ injection. Effect on neurological deficit in middle cerebral artery occluded rats MCAO was performed on the right side of the brain and 24 h later left hind paresis was observed in rats. The mean neurological score (3.62 ± 0.18 and 4.25 ± 0.16, respectively) in non-diabetic and diabetic vehicle-treated MCAO rats was significantly (p < 0.0001) higher compared to sham-operated rats, indicating neurological deficit following cerebral ischemia. Diabetic rats showed significantly (p < 0.05) greater neurological deficit

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compared to non-diabetic rats. Liraglutide pretreated nondiabetic and diabetic rats resulted in significantly (p < 0.001) less neurological deficit (1.62 ± 0.18 and 2.0 ± 0.19, respectively) compared to vehicle-treated MCAO rats. Liraglutide pretreatment had similar effects on both diabetic and non-diabetic rats. Neurological deficit was greater in diabetic insulin-treated MCAO rats compared to sham. Neurological deficit in diabetic insulin-treated was similar to vehicle-treated MCAO rats (Table 1). Effect on motor coordination in middle cerebral artery occluded rats Effect on muscular strength using grip test. The mean grip test score in the non-diabetic and diabetic vehicletreated MCAO rats (1.5 ± 0.18 and 0.62 ± 0.2, respectively) was significantly (p < 0.0001) decreased compared to sham-operated rats (4.62 ± 0.18). This indicates reduced muscular strength following MCAO. Diabetic rats showed significantly (p < 0.05) less muscular strength compared to non-diabetic rats. Liraglutide-pretreated non-diabetic and diabetic rats demonstrated significantly (p < 0.001) greater retention of muscular strength (2.62 ± 0.32 and 2.37 ± 0.26, respectively) compared to vehicle-treated MCAO rats. Liraglutide pretreatment had similar effects on both diabetic and non-diabetic rats. Grip test score was decreased in diabetic insulin-treated MCAO rats compared to sham and was similar to vehicle-treated MCAO rats (Table 1). Effect on motor coordination using foot fault test. The percent foot fault errors were significantly (p < 0.0001) higher in non-diabetic and diabetic vehicle-treated MCAO rats (81.62 ± 2.29% and 93.0 ± 2.01%, respectively) compared to sham group (7.25 ± 0.31%), indicating motor incoordination. Diabetic rats showed significantly (p < 0.05) more foot fault errors compared to nondiabetic rats. Non-diabetic and diabetic rats pretreated with liraglutide produced significantly (p < 0.001) fewer foot fault errors (38.5 ± 1.5% and 41.37 ± 1.88%, respectively) compared to vehicle-treated MCAO rats. Liraglutide pretreatment had similar effects on both diabetic and non-diabetic rats. Foot fault error was

greater in diabetic insulin-treated MCAO rats compared to sham. Foot fault error in diabetic insulin-treated was similar to vehicle-treated MCAO rats (Table 1). Effect on rota rod duration. The mean number of seconds spent on the rota rod were significantly (p < 0.0001) decreased in non-diabetic and diabetic vehicle-treated MCAO rats (44.87 ± 3.66 s and 33.62 ± 1.46 s, respectively) compared to sham group (185.8 ± 5.68 s), indicating motor incoordination. Diabetic rats showed significantly (p < 0.05) greater motor incoordination compared to non-diabetic rats. Non-diabetic and diabetic rats pretreated with liraglutide spent significantly (p < 0.001) more time on the rotating spindle (92.75 ± 3.13 s and 83.25 ± 5.2 s, respectively) compared to vehicle-treated MCAO rats. Liraglutide pretreatment had similar effects on both diabetic and non-diabetic rats. Time spent on rota rod was less in diabetic insulin-treated MCAO rats compared to sham and was similar to vehicle-treated MCAO rats (Table 1). Effect on spontaneous locomotor activity. Spontaneous locomotor activity was observed over a period of 10 min for each rat. Vehicle-treated rats showed a decrease in spontaneous locomotor activity as measured by distance traveled. Animals in the liraglutide pretreatment group, on the other hand, demonstrated a greater amount of spontaneous locomotor activity (p < 0.001) as compared to vehicle group. Spontaneous locomotor activity was decreased in diabetic insulin-treated MCAO rats compared to sham. Spontaneous locomotor activity in diabetic insulintreated was similar to vehicle-treated MCAO rats (Table 1). Effect of liraglutide on infarct volume Cerebral ischemia was evident in all MCAO rats. Cerebral ischemia by MCAO produced significant (p < 0.0001) infarction in non-diabetic and diabetic vehicle-treated rats with infarct volumes 169.0 ± 5.0 and 196.0 ± 4.0 mm3, respectively. The infarct volume was significantly smaller in liraglutide-pretreated non-diabetic and diabetic rats (57.0 ± 5.0 and 60.0 ± 4.0 mm3, respectively) compared to non-diabetic and diabetic vehicle-treated rats. Liraglutide pretreatment had similar

Table 1. Effect of liraglutide on neurological deficit and motor performance in MCAO rats Treatment groups

Neurological evaluation (6 point scale)

Grip test (6 point scale)

Foot fault error (%)

Rota rod duration (s)

Distance traveled (cm)

Sham Vehicle + MCAO (ND) Vehicle + MCAO (D) Liraglutide + MCAO (ND) Liraglutide + MCAO (D) Insulin + MCAO (D)

0

4.75 ± 0.16 1.37 ± 0.18* # 0.5 ± 0.19* @ 2.75 ± 0.25 2.5 ± 0.19@ 1.37 ± 0.18*

8.12 ± 0.44 83.37 ± 2.2* # 92.4 ± 1.7* 39.37 ± 1.40@ 44.12 ± 2.11@ 90.62 ± 1.42*

179.87 ± 3.91 51.9 ± 2.4* # 35 ± 1.9* @ 124.37 ± 4.9 111.12 ± 5.04@ 47.5 ± 2.92*

4880 ± 361.3 569 ± 95.4* 367 ± 97.5* 2553 ± 349.9@ 1996 ± 425.1@ 535 ± 69.3*

3.5 ± 0.18* # 4.37 ± 0.18* @ 1.75 ± 0.16 1.87 ± 0.22@ 3.87 ± 0.22*

Values expressed as Mean ± S.E.M. * p < 0.0001 compared to sham. # p < 0.05 compared to ND-vehicle. @ p < 0.001 compared to ND and D-vehicle (ND: Non-diabetic and D: Diabetic).

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effects on both diabetic and non-diabetic rats. Although the infarct volume (178.0 ± 5.0 mm3) in diabetic insulintreated MCAO rats was significantly (p < 0.0001) higher compared to sham-operated rats, no attenuation was seen and volumes were similar to vehicle-treated MCAO rats (Fig. 1). Effect on oxidative stress markers in middle cerebral artery occluded rats

Effect on brain MDA levels. The levels of MDA in the brain were significantly (p < 0.0001) increased following cerebral ischemia in non-diabetic and diabetic vehicletreated groups (578.0 ± 16.0 and 676.0 ± 14.0 nmol/g wet tissue, respectively) compared to sham-operated

rats (114.0 ± 2.0 nmol/g wet tissue). Liraglutide pretreatment in non-diabetic and diabetic rats produced significant (p < 0.001) protection as evident by attenuation of the raised MDA levels (312.0 ± 11.0 and 342.0 ± 21.0 nmol/g wet tissue, respectively) seen after MCAO compared to non-diabetic and diabetic vehicle-treated groups. Although the MDA level (615.0 ± 21.0 nmol/g wet tissue) in diabetic insulintreated MCAO rats was significantly (p < 0.0001) higher compared to sham-operated rats, no improvements were seen and levels were similar to vehicle-treated MCAO rats (Fig. 2A). Effect on brain GSH levels. In vehicle-treated nondiabetic and diabetic MCAO rats, there was a significant (p < 0.0001) decrease in levels of GSH in the brain

Fig. 1. (A) 2 mm coronal sections of brains stained with TTC to visualize the infarct area 24 h post middle cerebral artery occlusion (red indicates normal tissue and white indicates infarct tissue). (B) Effect of GLP-1 receptor agonist, liraglutide, on infarct volume in middle cerebral artery occluded rats. Values are expressed as mean ± SEM. ⁄p < 0.001 vs. sham. #p < 0.05 vs. vehicle + MCAO (ND); @p < 0.05 vs. ND and D-vehicle + MCAO (ND: Non-diabetic and D: Diabetic). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Effect of GLP-1 receptor agonist, liraglutide, on oxidative stress parameters following middle cerebral artery occlusion in rats. (A) Malondialdehyde levels in the occluded rat brain. (B) Reduced glutathione levels in the occluded rat brain. (C) Superoxide dismutase levels in the occluded rat brain. Liraglutide (50 lg/kg, s.c.) or isotonic saline (1 ml/kg, s.c.) was injected 2 weeks prior to MCAO. Values are expressed as mean ± SEM. ⁄p < 0.001 vs. sham. #p < 0.05 vs. vehicle + MCAO (ND); @p < 0.05 vs. ND and D-vehicle + MCAO (ND: Non-diabetic and D: Diabetic).

(94.0 ± 4.0 and 62.0 ± 3.0 lg/g wet tissue, respectively) compared to sham-operated rats (241.0 ± 12.0 lg/g wet tissue). Liraglutide pretreatment in non-diabetic and diabetic rats produced significant (p < 0.001) protection as evident by higher GSH levels (172.0 ± 5.0 and 186.0 ± 6.0 lg/g wet tissue, respectively) seen after MCAO compared to non-diabetic and diabetic vehicletreated groups. Although the GSH level (90.0 ± 5.0 lg/g wet tissue) in diabetic insulin-treated MCAO rats was significantly (p < 0.0001) lower compared to shamoperated rats, no improvements were seen and levels were similar to vehicle-treated MCAO rats (Fig. 2B). Effect on brain SOD levels. The levels of SOD in the brain were significantly (p < 0.0001) decreased following cerebral ischemia in non-diabetic and diabetic vehicle-treated groups (15.0 ± 1.0 and 8.0 ± 1.0 U/mg protein, respectively) compared to sham-operated rats (37.0 ± 2.0 U/mg protein). Liraglutide pretreatment in non-diabetic and diabetic rats produced significant (p < 0.001) protection as evident by higher in SOD levels (23.0 ± 1.0 and 25.0 ± 1.0 U/mg protein, respectively) seen after MCAO compared to nondiabetic and diabetic vehicle-treated groups. Although the SOD level (12.0 ± 1.0 U/mg protein) in diabetic insulin-treated MCAO rats was significantly (p < 0.0001)

lower compared to sham-operated rats, no improvements were seen and levels were similar to vehicle-treated MCAO rats (Fig. 2C). Effects on the expression of Bcl-2 and Bax The protein expression of Bcl-2 in ischemic hemisphere was decreased in the vehicle-treated rat (Fig. 3). In rats receiving pretreatment with liraglutide, the protein expression of Bcl-2 was increased in the ischemic hemisphere (Fig. 3, Lane 6) compared to vehicle (p < 0.001). On the other hand, protein expression of Bax in ischemic hemisphere was increased in the vehicle-treated rat brain after cerebral ischemia (Fig. 4). In rats pretreated with liraglutide, the protein expression of Bax was decreased in the ischemic hemisphere (Fig. 4, Lane 6) compared to vehicle (p < 0.001). Effects on TUNEL staining DNA fragments reach a peak between 24 and 48 h after cerebral ischemia. 24 h after MCAO, TUNEL staining was employed to study the effect of liraglutide on the level of apoptosis. The cells were scored as apoptotic when they were TUNEL-positive staining (Markgraf et al.). In the vehicle-treated group, the number of the TUNEL-positive cells significantly (p < 0.0001) increased

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Fig. 3. Expression of Bcl-2 receptor protein levels with b-actin as a loading control. Lane 1 – Sham (LH); Lane 2 – Sham (Buteau et al.); Lane 3 – Vehicle + MCAO (LH); Lane 4 – Vehicle + MCAO (Buteau et al.); Lane 5 – Liraglutide + MCAO (LH); Lane 6 – Liraglutide + MCAO (Buteau et al.). The blot is representative of five different experiments with similar results and bar graph showing relative folds change in the expression of Bcl-2 receptor in brain 24 h following middle cerebral artery occlusion in non-diabetic (A) and diabetic (B) rats. LH = Left hemisphere; RH = Right hemisphere. Values are expressed as mean ± S.E.M. ⁄p < 0.0001 compared to sham, #p < 0.001 compared to Vehicle (LH) and @p < 0.0001 compared to vehicle.

Fig. 4. Expression of Bax receptor protein levels with b-actin as a loading control. Lane 1 – Sham (LH); Lane 2 – Sham (Buteau et al.); Lane 3 – Vehicle + MCAO (LH); Lane 4 – Vehicle + MCAO (Buteau et al.); Lane 5 – Liraglutide + MCAO (LH); Lane 6 – Liraglutide + MCAO (Buteau et al.). The blot is representative of five different experiments with similar results and bar graph showing relative folds change in the expression of Bax receptor in brain 24 h following middle cerebral artery occlusion in non-diabetic (A) and diabetic (B) rats. LH = Left hemisphere; RH = Right hemisphere. Values are expressed as mean ± S.E.M. ⁄p < 0.0001 compared to sham, #p < 0.001 compared to Vehicle (LH) and @p < 0.0001 compared to vehicle.

in ischemic hemisphere as compared with the shamoperated group, where TUNEL staining was barely evident. The number of TUNEL-positive cells in the vehicle-group was 73.5 ± 3.3 and 85.5 ± 5.2 per 750 lm2 in non-diabetic and diabetic vehicle-treated MCAO rats, respectively. Pretreatment with liraglutide in non-diabetic and diabetic rats resulted in a remarkably fewer TUNEL-positive cells (25.5 ± 2.8 and 41.5 ± 4.1

per 750 lm2, respectively) when compared to vehicle group (Fig. 5).

DISCUSSION The aim of this study was to assess whether liraglutide pretreatment had any protective effect on cerebral ischemia in diabetic and non-diabetic rats. Neurological

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Fig. 5. TUNEL-positive cells/750 lm2 in the ischemic region was detected by TUNEL staining 24 h after MCAO of sham-operated rats, vehicletreated rats (non-diabetic and diabetic), and liraglutide-treated rats (non-diabetic and diabetic). Values are expressed as mean ± S.E.M. ⁄ p < 0.0001 compared to sham; #p < 0.001 compared to Vehicle + MCAO; @p < 0.05 compared to liraglutide + MCAO (ND); (ND: Non-diabetic and D: Diabetic).

deficit, infarct volume and oxidative stress and apoptotic biomarkers were determined following MCAO in diabetic and non-diabetic rats receiving liraglutide. Experimental cerebral ischemia results in a marked deficit in neurological and motor functions, as well as an increase in infarct size, oxidative stress and apoptotic markers, 24 h after MCAO. Liraglutide pretreatment resulted in a smaller infarct size as well as fewer neurological and motor function deficits. These beneficial effects were associated with inhibition of oxidative stress or neuronal apoptosis-related pathways, such as reduction of the MDA content, elevation of GSH and SOD activities, when administration commenced 2 weeks prior to MCAO. Liraglutide increased the expression of Bcl-2 and decreased the expression of Bax in cerebral

ischemia. Liraglutide pretreatment had similar effects on both diabetic and non-diabetic rats. These results suggest that liraglutide treatment may attenuate cerebral injury in diabetic and non-diabetic rats. Several studies have demonstrated the therapeutic effect of GLP-1 analog on animal models of AD and stroke (Isacson et al., 2011; Briyal et al., 2012; Darsalia et al., 2012; Parthsarathy and Holscher, 2013; Sato et al., 2013; McClean and Holscher, 2014; Holscher, 2014b). GLP-1 analog has been shown to reduce endogenous levels of b-amyloid in the brain (Perry et al., 2003; McClean et al., 2011) and prevents the impairment in learning new spatial tasks, synaptic loss and reduce plaque load (Wang et al., 2010; Gengler et al., 2012; McClean and Holscher, 2014). Moreover, Perry et al.

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observed that GLP-1 stimulated neurite outgrowth in rat PC12 cultured cells in a manner similar to nerve growth factor indicating GLP-1Rs could be stimulants for neuronal growth and suggesting a potential neuroprotective role of GLP-1Rs (Perry et al., 2002b; Harkavyi and Whitton, 2010). Studies in our laboratory using the MCAO model of focal cerebral ischemia in rats demonstrated that repeated administration of Ex-4 significantly improved infarct volume, neurological deficit and oxidative stress parameters in ischemic rats (Briyal et al., 2012). It has also been shown that Ex-4 protects against ischemiainduced neuronal death possibly by increasing GLP-1R expression against transient cerebral ischemic damage (Lee et al., 2011). In another study, it was found that liraglutide reduced infarct volume and oxidative stress parameters as well as increasing cortical vascular endothelial growth factor (Sato et al., 2013). These findings led to the suggestion that stimulation of GLP-1Rs could be of value in neurological disorders such as stroke. In the present study, diabetic and non-diabetic rats undergoing MCAO received 2 weeks pretreatment with either vehicle (isotonic saline) or GLP-1 receptor agonist liraglutide before MCAO. Twenty-four hours after the occlusion, animals were evaluated for neurological and motor deficit, and their brains were removed for analysis of infarct area, oxidative stress and apoptotic markers. Occlusion of the middle cerebral artery resulted in severe neurological and motor deficits, with animals demonstrating weakened or paralyzed limbs. Pretreatment with liraglutide resulted in significantly less neurological deficit and motor impairment as measured by the grip test, foot fault, rota rod and spontaneous locomotor activity when compared with the vehicletreated group. Loss of muscle strength and coordination following cerebral ischemia was significantly less in liraglutide-treated rats. The observed functional deficits coincided with the results of the TTC staining for infarct volume; the liraglutide group presented with significantly smaller infarct volumes than those of the vehicle-treated groups in both diabetic and non-diabetic rats. Ischemic stroke remains one of the leading causes of death and disability worldwide, and the poor prognosis for stroke is largely due to a lack of effective therapies (Mehta et al., 2007; Barone, 2009). Recent insight into the basic mechanism involved in ischemic stroke indicates that oxidative stress along with apoptosis and neuroinflammation represent key elements in the occurence and development of ischemic brain damage that results in cell damage and death (Gursoy-Ozdemir et al., 2004; Gong et al., 2014). It is well known that oxidative stress plays an important role in brain injury associated with stroke (Chan, 1996). It has been indicated that reactive oxygen species (ROS) overproduction during ischemia leads to oxidative stress and attacks lipids, proteins and DNA in ischemic brain tissue (Taylor and Crack, 2004; Manzanero et al., 2013). ROS produce MDA, an end product of lipid peroxidation, and are scavenged by endogenous antioxidant enzymes, such as SOD and GSH that could prevent the deleterious stroke-induced ROS generation (Chan, 2001; Dinkova-Kostova and Talalay, 2008). Therefore, measuring the levels of such

enzymes enables the amount of oxidative stress to be estimated. In the present study the levels of MDA, GSH and SOD were measured to estimate the extent of oxidative stress. Our results showed that cerebral ischemia increased the lipid peroxidation and decreased the activity of endogenous antioxidant enzymes. Pretreatment with liraglutide resulted in significantly lower levels of MDA and greater activities of GSH and SOD in the brain tissue of MCAO rats. These results indicate that GLP-1 agonist, liraglutide, plays the fundamental roles as an antioxidant and a neuroprotector against cerebral ischemic injury through the amelioration of oxidative stress. Abundant evidence suggests that apoptosis plays a significant role in regulating cell death after cerebral ischemic injury (Choi, 1996; Yao et al., 2001; Broughton et al., 2009). Apoptosis is one of the main forms of neuronal death in the brain during the progression of ischemic stroke. Interactions between the pro-apoptotic Bax and anti-apoptotic Bcl-2 family proteins on the mitochondria are believed to play an important role in cell survival (Vela et al., 2013). GLP1-Rs have become well accepted as having anti-apoptotic properties. Both GLP-1 and Ex-4 augment cellular integrity and overall survival following exposure to a range of pro-apoptotic agents such as peroxides, cytokines and fatty acids (Hui et al., 2003; Li et al., 2003; Harkavyi and Whitton, 2010). In addition GLP-1 appears to increase expression of anti-apoptotic genes Bcl-2 and Bcl-xl (Buteau et al., 2004). Oxidative stress, ionic imbalance and excitotoxicity result in nerve cell apoptosis. The Bcl-2 family has been considered to be the most important regulator of apoptosis. The anti-apoptotic protein Bcl-2 is capable of preventing cytochrome c release, which is an activator of apoptosis, and promotes cell survival, while the pro-apoptotic protein (Bax) promotes cell death (Nakka et al., 2008; Martinou and Youle, 2011). By regulating the Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) balance, the Bcl-2 family maintains mitochondrial stabilization (Hu et al., 2004). Our results showed that pretreatment with liraglutide significantly attenuated the decreased expression of Bcl-2 and increased expression of Bax that was observed in vehicle-treated MCAO group, suggesting that liraglutide may mediate the protective effect against cerebral ischemia by inhibiting apoptosis. In addition, we also used TUNEL staining to detect DNA fragmentation as a marker of apoptosis. DNA fragmentation is a characteristic manifestation of apoptosis and a marker for neuronal cell death in cerebral ischemia. Our study showed that liraglutide significantly inhibited apoptosis of neurons caused by cerebral ischemia, which was proved by DNA fragmentation. Liraglutide treatment significantly reduced the number of TUNEL-positive cells in the brain 24 h after ischemia. Liraglutide could effectively attenuate apoptosis through inhibiting DNA fragmentation and Bax expression and by up-regulating Bcl-2 expression. These factors may explain the mechanism of the neuroprotective effects of liraglutide on ischemia stroke. The attenuation of physiological deficit, infarct area, and oxidative stress and apoptotic markers after liraglutide administration in the current study indicates that the

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GLP-1 receptor may be a new therapeutic target for neuroprotection following ischemia stroke in both diabetic and non-diabetic conditions. It is well known that diabetes aggravates brain damage in experimental and clinical stroke subjects. Diabetes accelerates maturation of neuronal damage, increases infarct volume and exaggerates ischemic brain damage (Li et al., 1998; Gisselsson et al., 1999). Hyperglycemia is an exacerbating factor in ischemic stroke in patients with T2DM. In T2DM, insulin signaling is impaired, often caused by a desensitization of the insulin response (Steculorum et al., 2014). Therefore, in the present study we also determined the effect of insulin treatment on MCAO rats compared to liraglutide. While both treatments reduce hyperglycemia, only liraglutide had positive effects. This would suggest that the mere reduction of hyperglycemia is not the main mechanism of neuroprotection. Studies have shown that insulin is neuroprotective in neurodegenerative disorders, however, they have administered insulin intranasal or directly in the CNS with the aim of stimulating CNS insulin receptors ((Bake et al., 2014; Holscher, 2014a). The route of administration and experimental approach could account for the difference observed in the present study. Liraglutide pretreatment resulted in significantly lower neurological deficit and smaller infarct volume even in the diabetic rats. In the clinical situation, however, it is likely that T2DM patients who suffer an ischemic event are already receiving treatment for their diabetes; GLP-1 treatment may lead to a better outcome or recovery. However, further studies will be conducted using GLP-1R antagonist to verify that the effects of liraglutide are specific to activation of GLP-1Rs. Finally, recent studies have demonstrated that GLP-1 and analogs of GLP-1 induce cell proliferation in vivo and in vitro (Perry et al., 2002b; Li et al., 2010; Parthsarathy and Holscher, 2013) and may be associated with the neurogenic and angiogenic pathways (Kang et al., 2013; Lennox et al., 2013). GLP-1 receptors are expressed throughout the brain especially on neurons in the hippocampus and cortex, where they enhance cell survival (Hamilton and Holscher, 2009). Whether stimulation of GLP-1Rs may enhance this process following cerebral ischemia is as yet unknown. Future studies are planned which hope to address these questions in addition to gaining a more complete picture of the mechanism of action by which GLP-1Rs protect the brain following ischemia.

CONCLUSION The results of this study demonstrate that GLP-1 analog, liraglutide, is a neuroprotective agent in rats and prevents some of the neuronal damage associated with cerebral ischemia. Our previous and current reports indicate that GLP-1 analogs promote neuronal survival and attenuate apoptosis and oxidative stress in the brain. Both the reduction of oxidative stress and anti-apoptotic effects of GLP-1 analog, liraglutide, appear to play a role in neurological recovery following cerebral ischemia as evidenced by the results of the present study. Our findings suggest that liraglutide in both diabetic and non-diabetic rats provides significant neuroprotection

following a cerebral ischemic event. Liraglutide acts on GLP-1 receptors and stimulating these receptors may be making CNS less prone to damage due to cerebral ischemia.

Acknowledgment—Funding for this project was provided by Novo Nordisk, Inc. and Midwestern University.

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(Accepted 29 September 2014) (Available online 6 October 2014)

Neuroprotective and anti-apoptotic effects of liraglutide in the rat brain following focal cerebral ischemia.

Stroke is a leading cause of death and serious, long-term disability worldwide. We report that rats receiving liraglutide show markedly attenuated inf...
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