neuroprotective effects of neuropeptide y-y2 and y5 receptor agonists in vitro and in vivo

Neuropeptides 43 (2009) 235–249

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Neuropeptides

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Neuroprotective effects of neuropeptide Y-Y2 and Y5 receptor agonistsin vitro and in vivo

Maria Smiałowska a,*, Helena Domin a, Barbara Zieba a, Ewa Kozniewska b, Radosław Michalik b,c,Piotr Piotrowski d, Małgorzata Kajta e

a Department of Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Kraków, Polandb Department of Neurosurgery, M. Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Polandc Department of Neurooncology, The Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Polandd Department of Experimental and Clinical Neuropathology, M. Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Polande Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Kraków, Poland

a r t i c l e i n f o

Article history:Received 3 September 2008Accepted 13 February 2009Available online 24 March 2009

Keywords:NPYNPY receptorsKainic acidPrimary neuronal culturesHippocampus excitotoxicityApoptosisMiddle cerebral artery occlusionNeuroprotection

0143-4179/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.npep.2009.02.002

* Corresponding author. Tel.: +48 12 66 23 270; faxE-mail address: [email protected] (M. Smiało

a b s t r a c t

It is generally assumed that neurodegeneration is connected with glutamatergic hyperactivity, and thatneuropeptide Y (NPY) inhibits glutamate release. Some earlier studies indicated that NPY may have neu-roprotective effect; however, the results obtained so far are still divergent, and the role of different Yreceptors remains unclear. Therefore in the presented study we investigated the neuroprotective poten-tial of NPY and its Y2, Y5 or Y1 receptor (R) ligands against the kainate (KA)-induced excitotoxicity inneuronal cultures in vitro, as well as in vivo after intrahippocampal KA injection and also in an ischemicmiddle cerebral artery occlusion model after intraventricular injection of Y2R agonist. NPY compoundswere applicated 30 min, 1, 3 or 6 h after the start of the exposure to KA, or 30 min after the onset of ische-mia. Our results indicate the neuroprotective activity of NPY and its Y2R and Y5R ligands against the kai-nate-induced excitotoxicity in primary cortical and hippocampal cultures. Importantly, NPY was effectivewhen given as late as 6 h, while Y2R or Y5R agonists 3 h, after starting the exposure to KA. In in vitro stud-ies those protective effects were inhibited by the respective receptor antagonists. Neuroprotection wasalso observed in vivo after intrahippocampal injection of Y2R and Y5R agonists 30 min or 1 h after KA.No protection was found either in vitro or in vivo after the Y1R agonist. The Y2R agonist also showed neu-roprotective activity in the ischemic model. The obtained results indicate that neuropeptide Y producesneuroprotective effect via Y2 and Y5 receptors, and that the compounds may be effective after delayedapplication.

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1. Introduction

Neuropeptide Y (NPY) is a 36 amino acid peptide, widely dis-tributed in the nervous system where it plays the role of a neuro-transmitter and neuromodulator (Chronwall et al., 1985; Gray andMorley, 1986). In the mammalian forebrain, NPY is present inmany inhibitory interneurons and it modulates – mainly inhibits– the release of other neurotransmitters (Kohler et al., 1986; Col-mers, 1990; Greber et al., 1994). NPY plays an important role inthe regulation of neuronal activity: it reduces epileptiform activityin the hippocampus both in vitro and in vivo (Colmers and Bleak-man, 1994; Woldbye et al., 1997), and inhibits glutamate release(Colmers and Bleakman, 1994; Schwarzer et al., 1998; Greber

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et al., 1994; Vezzani et al., 1999; Silva et al., 2005b). The suppres-sion of epileptiform activity by NPY was also observed in the fron-tal cortex (Bijak, 1999). Injection of NPY into the lateral ventriclereduced kainic acid (KA)-induced seizures in the rat (Baraban,2002). Moreover, increases in the synthesis and content of NPYin limbic structures were found after seizures of different origin(Marksteiner et al., 1990; Bellmann et al., 1991; Bendotti et al.,1991; Schwarzer et al., 1996; Smiałowska et al., 2003; Silva et al.,2005b).

All these findings suggest an important, probably inhibitory,role of NPY in neuronal excitability. It is generally assumed thatglutamatergic overactivation may lead to excitotoxic cell death,and that inhibition of the toxic glutamatergic hyperactivity mayproduce neuroprotection. Therefore the inhibitory action of NPYmay result in its protective effect on neurons. In fact, our earlierstudies showed the neuroprotective effects of NPY against KA-in-duced neurotoxicity both in vivo in rat hippocampus (Smiałowskaet al., 2003) and in vitro in hippocampal and cortical neuronal

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236 M. Smiałowska et al. / Neuropeptides 43 (2009) 235–249

cultures (Domin et al., 2006). Moreover, the protective effects ofNPY were observed after delayed treatment with the peptide30 min to even 6 h after KA. On the other hand, the results of otherauthors were controversial as they showed both protective andtoxic effects of NPY (Cheung and Cechetto, 2000; Chen and Cheung,2002). The cause of such a discrepancy seems to lie in the fact thatNPY activates different Y receptors (YR).

Six types of YR have been described, all belonging to the super-family of G-protein-coupled, heptahelical receptors (Wahlestedtet al., 1985; Wahlestedt and Reis, 1993; Michel et al., 1998). AmongNPY receptors, Y1, Y2 and Y5-R are believed to play the most impor-tant role in the regulation of neurotoxicity and neuroprotection.Specific agonists and antagonists of those receptors have been syn-thesized (Fuhlendorff et al., 1990; Aguirre et al., 1990; Rudolf et al.,1994; Wieland et al., 1998; Doods et al., 1999; Cabrele et al., 2000;Dumont et al., 2000), which makes it possible to study the role ofparticular receptors in neuroprotection; however the results arestill controversial. Activation of Y1 receptors has been found to in-duce both neuroprotective (Silva et al., 2003) and neurotoxic effects(Gariboldi et al., 1998) in different models of hippocampal excito-toxicity. The neuroprotective effects of agonists of Y2 and Y5 recep-tors were observed in the hippocampus after kainate (KA)excitotoxicity (Silva et al., 2003), but these results are still incom-plete. Moreover, in the majority of studies, NPY and its ligands wereapplied before or simultaneously with a neurotoxic treatment,which is a model substantially different from the situation facedin clinical practice. Therefore in the present study we investigatedthe role of Y1, Y2 and Y5 receptors in neuroprotection; however,our attention was focused chiefly on the effectiveness of a delayedtreatment (even a few hour delay), which seemed to resemble moreclosely the situation of patients, who were usually treated sometime after an injury.

As a model of neurodegeneration we used the kainic acid-in-duced excitotoxicity. This model was described as a good and val-idate simulation of normally occurring excitotoxicity, connectedwith the secondary release of endogenous glutamate (Wanget al., 2005; Coyle, 1983; Ferkany and Coyle, 1983; Malva et al.,1998). The possibility of neuroprotective action of studied com-pounds, especially after delayed treatment, was investigated bothin vitro in neuronal cultures and in vivo in rat hippocampus. Addi-tionally, we used transient middle cerebral artery occlusion(MCAO) model of ischemia.

2. Materials and methods

2.1. In vitro studies: cortical and hippocampal primary neuronalcultures

2.1.1. MaterialsThe experiments were performed on primary cultures of mouse

cortical and hippocampal neurons. Neuronal tissues were takenfrom Swiss mouse embryos on days 15/16 (for cortical cultures)and 17/18 (for hippocampal cultures) of gestation, and were culti-vated essentially as described previously (Brewer, 1995; Kajtaet al., 2004). Pregnant females were anesthetized with CO2 vapor,killed by cervical dislocation and subjected to cesarean section inorder to remove fetal brains. The dissected cortical and hippocam-pal tissues were minced, then gently digested with trypsin (0.1%;for 15 min at room temperature; [RT], Sigma, USA), triturated inthe presence of 10% fetal calf serum (Gibco, USA) and DNAse I(170 Kunitz units per ml, Sigma, USA), and finally centrifuged for5 min at 100g. The cells were then suspended in phenol red-freeNeurobasal medium (Gibco, USA) supplemented with 5% fetal calfserum and plated at a density of 1.5 � 105 cells per cm2 onto poly-ornithine (0.01 mg per ml, Sigma, USA) coated multi-well plates

(TPP). After two days, the culture medium was exchanged to neu-robasal medium supplemented with B27 (200 ll/100 ml; Gibco,USA). This procedure typically yields cultures that contain about90% of neurons and 10% of astrocytes. The cultures were main-tained at 37 �C in a humidified atmosphere containing 5% CO2

and were cultivated for 8 days prior to the experiment.

2.1.2. Treatment with drugsIn order to evoke toxic effects, primary neuronal cultures were

exposed to 150 lM kainic acid (KA; Tocris, USA), dissolved in redis-tilled water, for 24 (hippocampal cultures) or 48 h (cortical cul-tures). The concentration of KA used in our experiments waschosen on the basis of our earlier studies (Kajta et al., 1999; Kajtaand Lason, 2000; Domin et al., 2006) and also other authors re-search (Shih et al., 2002, 2004). The time-course of kainate effectson caspase-3 activity and LDH release on primary neuronal cul-tures was worked out and described in our earlier paper (Dominet al., 2006). In our present experiments into neuroprotection,NPY and NPY receptor specific ligands were used. NeuropeptideY (NPY) (Tocris, USA), which activates all Y receptors, [Leu31,Pro34]-NPY (Tocris, USA) a Y1R agonist, NPY13-36 (Tocris, USA) aY2R agonist, or [cPP1-17, NPY19-23, Ala31, Aib32, Gln34]-hPP (Tocris,USA) a Y5R agonist, at concentrations of 0.01, 0.1, 0.5 and 1 lMwere added 30 min, 1, 3 or 6 h after starting the exposure to KA.NPY concentrations were chosen on the basis of some earlier stud-ies performed by us and other authors (Bijak and Smiałowska,1995; Klapstein and Colmers, 1997; Domin et al., 2006). The con-centrations of Y1, Y2 or Y5 receptor agonists were chosen on thebasis of experiments performed by Silva et al. (2003). Additionally,specific antagonists of Y receptors were added 10 min before theappropriate agonists: (R)-N-[[4-aminocarbonylaminomethyl) phe-nyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate(BIBO3304; [Boehringer-Ingleheim, Biberach, Germany], a Y1Rantagonist), (N-[(1S)-4-[(Aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide (BIIE0246;[Tocris, USA], a Y2R antagonist), N-[[trans-4-[[(4-Amino-2-quinaz-olinyl)amino]methyl]cyclohexyl]methyl]-1-naphthalenesulfona-mide hydrochloride (CGP 71683 [Tocris, USA], a Y5R antagonist).The concentrations of BIBO3304 were chosen on the basis of ourpilot experiments, while those of BIIE0246 were chosen based onour pilot experiments and a study by Silva et al. (2003). The con-centrations of CGP 71683 were chosen on the basis of a study byNanobashvili et al. (2004). NPY; [Leu31, Pro34]-NPY; NPY13-36;[cPP1-17, NPY19-23, Ala31, Aib32, Gln34]-hPP; BIBO3304; BIIE0246were dissolved in redistilled water, and CGP 71683 in DMSO. Thecontrol cultures were supplemented with the same amount of anappropriate vehicle.

2.1.3. Evaluation of cell death and protection2.1.3.1. Measurement of lactate dehydrogenase (LDH) activity. In or-der to quantify cell death, lactate dehydrogenase (LDH) releasedfrom damaged cells into the cell culture media was measured24 h (hippocampal cultures) and 48 h (cortical cultures) after start-ing the treatment with kainate. A colorimetric assay was used,according to which the amount of formazan salt, formed after con-version of lactate to pyruvate and then by reduction of tetrazoliumsalt, was proportional to LDH activity in the sample. Cell-free cul-ture supernatants were collected from each well and incubatedwith the appropriate reagent mixture according to the supplier’sinstructions (Cytotoxicity Detection Kit, Roche) at RT for 60 min.The intensity of red color formed in the assay and measured at awavelength of 490 nm was proportional to LDH activity and tothe number of damaged cells. The data were normalized to theactivity of LDH released from vehicle-treated cells (100%) and ex-

M. Smiałowska et al. / Neuropeptides 43 (2009) 235–249 237

pressed as a percent of the control ± SEM established from n P 6wells per one experiment from three separate experiments. Absor-bance of blanks, determined as no-enzyme control, has been sub-tracted from each value.

2.1.3.2. Measurement of caspase-3 activity. For evaluation of apopto-sis, caspase-3 activation was measured. The method was per-formed according to Nicholson et al. (1995) and Kajta et al.(2005, 2007) in samples treated for 6 h with the neurotoxic agentKA, alone or in combination with ligands studied, applied 30 minafter KA. After replacing the media with Caspase Assay Buffer(50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA,10% glycerol, and 10 mM dithiothreitol), the cell lysates (25 lgper sample) were incubated at 35 �C with a colorimetric substratepreferentially cleaved by caspase-3-Ac-DEVD-pNA (N-acetyl-asp-glu-val-asp p-nitro-anilide; 40 lM Sigma, USA). The amounts ofp-nitro-anilide were continuously monitored over 60 min with aplate reader (Multiscan, Labsystems). Absorption was measuredat 405 nm and only the data within the linear slope of the reactioncurve provided consistent measure of caspase-3 activity. To con-firm the correlation between signal detection and caspase activity,we used Ac-DEVD-CHO (aldehyde substrate; Molecular Probes,USA), which is a specific caspase-3 protease inhibitor. The datawere normalized to the absorbance in vehicle-treated cells and ex-pressed as a percent of control ± SEM established from n P 6 wellsper one experiment from two separate experiments. Absorbance ofblanks, determined as no-enzyme control, has been subtractedfrom each value.

2.1.3.3. Identification of apoptotic cells. Apoptotic cells were visual-ized by fluorescent staining using Hoechst 33342 (MolecularProbes, USA). Cells for those studies were cultured, as describedabove, on round cover-glasses (Menzel-Glasser, Germany), placedin 24-holes culture plates. The cultures were treated with KA alone,or with KA and the NPY, Y2R or Y5R agonists. At 24 h (hippocampalcultures) or 48 h (cortical ones) after the start of incubation withKA (or without KA in control groups), the cultures were washedwith PBS, fixed for 20 min with 4% paraformaldehyde, washed sev-eral times in PBS, and then exposed to Hoechst 33342 (0.6 lg/ml inPBS) for 10 min at room temperature. Hoechst 33342 stains con-densed DNA fragments characteristic for apoptotic cells. Bright-blue fluorescence of the condensed chromatine was observed un-der a fluorescence microscope (Nicon Optiphot 2) using a wavelength of 330–380 nm. Microphotographs were made using SPOT32 camera (Diagnostic Instruments Inc.).

2.1.4. Data analysisThe data after normalization as a percentage of control ± SEM

were analyzed using GraphPad Prism 4.0 software. One-way anal-ysis of variance (ANOVA) was used to determine overall signifi-cance. Differences between control and experimental groupswere assessed with post hoc Tukey test. The level of significancewas determined as P < 0.05.

2.2. In vivo studies: kainic acid-induced excitotoxicity in rathippocampus

2.2.1. AnimalsMale Wistar rats weighing about 250–300 g were used for the

experiments. The rats were age-matched and were housed six toa cage on a 12:12 light–dark cycle, with free access to food andtap water. The rats after cannulae implantation were housed sin-gly. During the experiment, all efforts were made to minimize ani-mal suffering and to reduce the number of animals used, inaccordance with the Local Bioethical Commission Guide for theCare and Use of Laboratory Animals.

2.2.2. Cannulae implantationThe rats were anaesthetized with equithesin and were stereo-

taxically, bilaterally implanted with chronic quide cannulae aimedat the dorsal hippocampus CA1 region. The guide cannulae(23-gauge stainless steel tubing), secured by dental cement, wereanchored to the skull by three stainless steel screws. In order toprevent clogging, stainless steel stylets were placed in the guidecannulae and left until the animals were microinjected.

2.2.3. Drug treatmentsSeven days after cannulae implantation, the rats were unilater-

ally microinjected with kainic acid (KA) into the right dorsal hippo-campus CA1 region (coordinates: A + 5.7, L ± 2.1, H + 7.2 mm fromthe interaural line, according to the Paxinos and Watson stereo-taxic atlas (Paxinos and Watson, 1986). KA was freshly dissolvedin 0.1 M phosphate buffer, pH 7.4 and was microinjected in a doseof 2.5 nmol/1 ll. Y1R, Y2R or Y5R agonists were injected in a doseof 470 pmol/1 ll into the same CA1 region of the right hippocam-pus. [Leu31, Pro34]-NPY (Y1R agonist) was administered 30 minafter KA; NPY 13-36 (Y2R agonist) or [cPP1-7, NPY19-23, Ala31,Aib32, Gln34]-hPancreatic Polypeptide (Y5R agonist) were given30 min, 1 or 3 h after KA. The contralateral hippocampus of eachrat was microinjected with a phosphate buffer and used as a con-trol side. The doses of KA and NPY were chosen on the basis of ourearlier study (Smiałowska et al., 2003). The doses of Y1, Y2 or Y5receptor agonists were chosen on the grounds of our pilot studies.

2.2.4. Evaluation of damage and protection in CA region ofhippocampus2.2.4.1. Tissue preparation and histology. Seven days later, the ratswere killed by an overdose of pentobarbital, their brains were re-moved, fixed in a cold 4% paraformaldehyde for 7 days, and werethen immersed in a buffered 20% sucrose solution for at least5 days at 4 �C. The brains were then frozen on dry ice, and 30 lmcoronal sections were cut at levels containing the dorsal hippocam-pus (between bregma �2.12 to �4.30 mm, according to the Paxi-nos and Watson atlas (Paxinos and Watson, 1986). The sectionswere mounted on glass slides, dried, stained with Cresyl Violet,cover-slipped with Permount, and were used for verification ofthe injection site and for a histological analysis of the lesion.

2.2.4.2. Stereology. The total number of neurons in the pyramidallayer of the CA of the dorsal hippocampus was evaluated by stereol-ogical counting. The procedures were performed using a microscope(Leica, DMLB; Leica, Denmark) equipped with a projecting cameraand a microscope stage connected to an xyz stepper (PRIOR ProScan)controlled by a computer using the Olympus Denmark CAST2 soft-ware, as described previously (Ossowska et al., 2005, 2006).

Systemic uniform random sampling was used to choose the sec-tions. The first sampling item was randomly taken from the frontalpart of the dorsal hippocampus, and all the following samplingitems were taken at a fixed distance from the previous one. At least10–12 sections through the entire length of the dorsal hippocam-pus were sampled.

The total number of cells (N) in the pyramidal layer of the hip-pocampal CA region was estimated by measuring the referencevolume (Vref; the area that contains the population of the cells)and the numerical density (Nv) of the cells within the Vref:

N ¼ Vref � Nv

The pyramidal layer of the dorsal hippocampus CA region wasoutlined at a lower magnification (5�). CAST2 software providestemplates of points in various arrays used in point counting for ref-erence volume estimation. The Vref value was determined by usingpoint counting methods and applying Cavalieri’s principle (Gun-dersen and Jensen, 1987) according to the formula:


Vref ¼X

pi� AðpiÞ � t

whereP

pi is the sum of the number of points (pi) counted, A(pi) isthe area associated with each point, and t is the known distance be-tween sections. The area of the counting frame wasA(fr) = 3382 lm2.

For determination of the density of cells in the hippocampal CAregion, the computer software generated a random selection ofsites within the outlined area, from which the density was deter-mined under higher magnification (63�). Cell density (Nv) wasestimated by using the optical dissector method according to theformula:

Nv ¼X

Q=X

P � vðdisÞ

whereP

Q is the sum of cells counted from all the dissector frames,PP is the total number of all the dissector points, and v(dis) is the

total volume of the dissector.

2.2.5. Data analysisStatistical analysis was carried out using GraphPad Prism 4.00

software. Differences between the control (contralateral) and KA-lesioned hippokampi (ipsilateral) were compared by a pairedtwo-tailed t-test. Differences between KA-lesioned and KA + Y ago-nist-treated hippocampi were compared by an unpaired two-tailedt-test. P-value less than 0.05 was considered statistically significant.

2.3. In vivo studies: transient focal cerebral ischemia in rats

2.3.1. AnimalsThirteen male Wistar rats weighing 270–320 g were used for

this part of the study. The animals had free access to food (conven-tional chew pallets) and water until the experiment.

2.3.2. Transient middle cerebral artery occlusion (transient MCAO)For the experiment, rats were anesthetized with chloral hydrate

(36 mg/100 g b.wt) and placed on the thermostatic heating blanket(Homeothermic Blanket System, Harvard Apparatus, England).Rectal temperature was kept between 37 and 38 �C throughoutthe experiment and until the animals were awaken thereafter.

Transient focal cerebral ischemia was induced for 120 minusing a modification of Longa’s intraluminal suture occlusionmethod (Longa et al., 1989). Briefly, anesthetized animals wereplaced in supine position and a midline incision was made on theirneck to expose the right common carotid artery. After careful prep-aration of the area around the carotid sinus, the internal and exter-nal carotid arteries were identified under a surgical microscope.Then, the pterygopalatine artery was identified, carefully cleanedof the surrounding tissue and ligated. The external carotid arterywas freed likewise from the extravascular tissue; small branchingvessels were coagulated and the vessel was cannulated in a retro-grade fashion with PE-50 polyethylene tubing. A 3-0 surgical nylonsuture with the attached silicone-coated filament (model4037Pk10; Doccol Co., Redlands, CA, USA) was inserted throughthe tubing to the level just above the knot on the pterygopalatinebranching. Afterwards, the animal was carefully placed in a proneposition and its head was fixed in a head holder. The skin over thescalp was cut along the saggital sinus and the cranium was de-nuded. Two burr holes, each of 1.5 mm in diameter, were drilledsymmetrically on both sites 8 mm lateral and 1 mm posterior tothe bregma using a saline-cooled dental drill. That procedure al-lowed thinning the bone sufficiently to see vessels on the surfaceof the brain. In those holes, laser-Doppler probes were later placedto measure microflow. The third burr hole of 1 mm in diameterwas made on the right side at 1.5 mm lateral and 0.5 mm posteriorto the bregma. Through that burr hole, the cannula for vehicle or

NPY13-36 infusion was introduced into the right lateral ventricle.LDF probes were placed in the respective burr holes with a micro-manipulator avoiding large blood vessels. The holes were coveredwith transparent gel. Microcirculation in the brain cortex was con-tinuously monitored using a laser-Doppler flowmeter (DRT4,Moore Instruments Ltd., England).

Occlusion of the MCA was performed after at least 15 min ofstable LDF recording by advancing the suture further 8–10 mm un-til the LDF signal decreased unilaterally to ischemic levels. Bilateraldecrease of LDF indicated subarachnoid hemorrhage and the ter-mination of the experiment. The suture was left in place for120 min.

2.3.3. Drug administrationThirty minutes after MCA occlusion, NPY13-36 (10 lg/6 ll) dis-

solved in freshly prepared and filtered artificial cerebrospinal fluidwas administered to the right lateral ventricle of six animals for30 s through the 23G cannula connected to the Hamilton microsy-ringe. The cannula was left in place for 2 min and was then with-drawn. In seven control animals the same volume of artificialcerebrospinal fluid was administered in the identical manner,30 min after MCA occlusion. After two hours of occlusion, the fila-ment was withdrawn to allow reperfusion. Then, the wounds weresutured and infiltrated with lidocaine. The rats were kept normo-thermic with the help of the heating blanket until they awoke.

2.3.4. Evaluation of brain damageSeventy two hours after reperfusion, the animals were anaes-

thetized with 5% halothane in N2O/O2 (70%:30%) and decapitated.Their brains were quickly removed and placed in a dish with ice-cold physiological saline for 2 min. Then they were placed in ratbrain matrices, cut into 2-mm thick coronal sections and immersedin 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, USA) inphosphate-buffered saline for 10 min at 37 �C in darkness (Beder-son et al., 1986). TTC reacts with intact mitochondrial respiratoryenzymes to generate a bright red color to contrast with the palecolor of the infarct. Stained sections were fixed in phosphate-buf-fered 10% formalin for at least 24 h before a morphometric analy-sis. The morphometric analysis was done using a computed basedimage analysis system (GIMP 2). The infarct volume was calculatedin relation to the volume of the contralateral hemisphere and wascorrected for the edema component (Swanson et al., 1990).

2.3.5. Data analysisStatistical analysis of LDF changes was performed using a one-

way ANOVA and a post hoc Tukey test. The infarction volume datawere analyzed by a Student t-test for unpaired variables. The dif-ferences were considered to be statistically significant at P < 0.05.

3. Results

3.1. In vitro studies in primary cultures

3.1.1. The effects of NPYApplication of NPY attenuated the kainate-induced LDH release

in both cortical (Fig. 1A) and hippocampal (Fig. 1C) cultures. The ef-fects were dose- and time-dependent. When NPY was added30 min or 1 h after KA, significant diminution of LDH release (about20% in cortical and 38% in hippocampal cultures) was observed at avery low NPY concentration of 0.1 lM. The diminution was morepronounced at higher NPY concentrations of 0.5 and 1 lM (about40% in cortical and 50% in hippocampal cultures). When NPY wasapplied 3 h or 6 h after the start of KA intoxication, the effectiveconcentrations were 0.5 and 1 lM. The diminution of LDH releaseaveraged 28% in cortical cultures and 38% in hippocampal ones

Fig. 1. (A and C) The effects of NPY on kainate (KA; 150 lM)-induced LDH release in the primary cultures of mouse cortical (A) and hippocampal (C) neurons. LDH wasmeasured 48 h (cortical) or 24 h (hippocampal cultures) after KA administration. NPY was added to the culture medium 30 min, 1 h, 3 h or 6 h after KA. (B, D) – the effects ofNPY on KA-induced increase in caspase-3 activity in mouse primary cortical (B) and hippocampal cultures (D). Caspase-3 was measured 6 h after starting KA intoxication. NPYwas added to cultures 30 min after KA. Each bar represents the mean of n P 6 platings ± SEM from 3 to 4 independent experiments. ***P < 0.001 versus control cultures;#P < 0.05, ##P < 0.01, and ###P < 0.001 versus the cultures exposed to KA.


when NPY was applied 3 h after KA, and 25–32% when it was given6 h after KA. NPY did not significantly change the kainate-inducedLDH release in either cortical or hippocampal cultures when it wasused at a concentration of 0.01 lM (Fig. 1A and C). NPY alone, ap-plied into untreated cultures, did not influence LDH activity at anydose studied. The measurement of caspase-3 activity showed po-tent activation of that apoptotic enzyme after 6 h of KA intoxica-tion, reaching ca. 170% of the control value. NPY atconcentrations of 0.1, 0.5 and 1 lM significantly diminished theactivation of caspase-3 in both cortical and hippocampal cultures(Fig. 1B and D). The level of caspase-3 activity after addition ofNPY at the highest concentration almost reached the control value.The highest dilution of NPY (0.01 lM) had no significant effect(only a tendency towards a decrease). NPY alone added to the cul-tures, did not influence caspase-3 activity.

3.1.2. The effects of Y2 receptor agonistApplication of NPY13-36, a specific Y2R agonist, significantly

decreased the KA-induced LDH release in both cortical and hippo-campal cultures. The effects were slightly stronger in the latter cul-tures. In cortical cultures, significant effects were observed whenNPY13-36 was added at a concentration of 0.5 or 1 lM at 30 min(a 45–48% decrease) or 1 h (a 35–37% decrease) after KA. A higherdilution of the agonist, as well as its application at 3 or 6 h after KAwere not effective (Fig. 2A). In hippocampal cultures, attenuationof the KA-induced LDH release by the Y2R agonist was observednot only at its concentration of 0.5 or 1 lM (a 63% or 66% decrease),

but also at 0.1 lM (a 53% decrease) when the agonist was applied30 min after KA. When it was added 1 h after KA, LDH release wasdiminished by about 46–55%, and only its concentrations of 0.5and 1 lM were effective. Moreover, the peptide was also effectiveafter a more delayed application (3 h after KA) (a 28–32 decrease)(Fig. 2C). No effect on LDH was found in either cultures, when theY2R agonist was added 6 h after KA.

The strong decrease in the KA-induced LDH release, evoked bythe Y2R agonist at a concentration of 1 lM, 30 min after KA, wascompletely abolished by the specific Y2R antagonist BIIE0246, ata concentration of 3 lM, applied 10 min before NPY13-36(Fig. 3A and C). BIIE0246 alone, added in the cultures at the samedilution, had no effect on LDH level. Similarly, NPY13-36 alone gi-ven to untreated cultures did not influence LDH.

Measurement of caspase-3 activity showed that the strong acti-vation of the enzyme by KA (about 170% of the control value) wasconsiderably diminished by the Y2R agonist applied at concentra-tions of 0.5 or 1 lM to cortical cultures and 0.1, 0.5 or 1 lM to hip-pocampal cultures 30 min after KA (Figs. 4A and 5A). Its effect oncaspase-3 was prevented by the Y2R antagonist BIIE0246 given10 min before NPY13-36 (Figs. 4B and 5B). Neither NPY13-36 norBIIE0246 alone, given to non-treated cultures influenced caspase-3 activity.

3.1.3. The effects of Y5 receptor agonistThe specific Y5R agonist, [cPP1-17, NPY19-23, Ala31, Aib32, Gln34]-

hPP, significantly diminished the kainate-induced LDH release

Fig. 2. The effects of Y2R agonist (NPY13-36) (A, C) and Y5R agonist ([cPP1-7, NPY19-23, Ala31, Aib32, Gln34]-hPP) (B, D) on the KA (150 lM)-induced LDH release in primarycultures of mouse cortical (A and B) and hippocampal (C and D) neurons. LDH was measured 48 h (cortical) or 24 h (hippocampal cultures) after KA administration. The Y2 orY5R agonists were added to a culture medium 30 min, 1 h, 3 h or 6 h after KA. Each bar represents the mean of n P 6 platings ± SEM 3–4 independent experiments. ***P < 0.001versus control cultures; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the cultures exposed to KA.


when it was added into cortical cultures at 30 min after KA (a 29–51% decrease, depending on the agonist concentration) or 1 h afterKA (26–29%), as well as when it was administered into hippocam-pal cultures at 30 min (a 56–63% decrease), 1 h (a 54–70% de-crease) or 3 h (a 38–55% decrease) after KA. When given 30 minafter KA, the Y5R agonist was effective even at its lowest concen-tration (0.01 lM) in both those cultures (a 29% decrease in corticalcultures and a 56% fall in hippocampal ones); however, in the caseof a more delayed treatment, only its concentrations of 0.1, 0.5 or1 lM (after 1 h), and 0.5 or 1 lM (after 3 h in hippocampal cul-tures) attenuated the KA-induced LDH release. The results areshown in Fig. 2B and D. The Y5R agonist was not effective when gi-ven 6 h after KA. Application of the Y5R specific antagonist CGP71683 at a concentration of 10 lM completely counteracted Y5Ragonist effect on LDH release (Fig. 3B and D).

When given 30 min after KA, the Y5R agonist significantly pre-vented the kainate-induced activation of caspase-3 in both thosecultures ( Figs. 4C and 5C). The effects were stronger after the1 lM concentration in the hippocampal cultures, reaching almostthe control level. The protective effect of Y5R agonist was pre-vented by Y5R antagonist (Figs. 4D and 5D).

3.1.4. The effects of Y1 receptor agonistThe specific Y1R agonist [Leu31, Pro34]-NPY did not reduce the

kainate-induced LDH release in either the cortical or the hippo-campal cultures at any concentration studied (Fig. 6A and C). Onthe other hand, LDH release was slightly, but significantly, attenu-ated by the Y1R antagonist BIBO3304 at concentrations of 0.01, 0.1and 1 lM (Fig. 6B and D).

3.1.5. Morphological identification of apoptotic cells by Hoechst 33342staining

Kainate applicated into the cultures at a concentration of150 lM induced the appearance of apoptotic bodies after 24 h inhippocampal cultures, or 48 h in cortical ones. The apoptoticbodies were visibly less numerous when NPY, Y2R or Y5R agonistswere added into cultures 30 min after KA. Fig. 7 presents the re-sults obtained in hippocampal cultures after KA, KA + NPY andKA + Y2R agonist. The effect of Y5R agonist in not shown, but itlooks similarly as Y2R one.

3.2. The effects of Y2, Y5 or Y1 receptor agonists in in vivo studies

Kainate injected unilaterally in a dose of 2.5 nmol into the CA1region of the dorsal hippocampus induced extensive degenerationof CA pyramidal neurons (Fig. 8A, left photo). Stereological count-ing showed a strong, ca. 50% reduction in the number of neurons inthe pyramidal layer of the ipsilateral dorsal hippocampus in com-parison to the contralateral side (Fig. 8).

Injection of the Y2R agonist NPY13-36, in a dose of 470 nmol,30 min or 1 h after KA, visibly attenuated the KA-induced lesion.The number of living neurons in the CA pyramidal layer signifi-cantly increased after post-treatment with Y2R agonist (Fig. 8B).The extent of the lesion decreased by ca. 60–20% in comparisonto the lesion after KA alone. No protection was seen when theY2R agonist was given 3 h after KA.

Neuroprotective effects were also observed after the Y5R ago-nist (in a similar dose as Y2R) given 30 min or 1 h after KA admin-istration. The results of stereological counting showed a significant

Fig. 3. The effects of Y2R antagonist (BIIE0246) (A and C), or Y5R antagonist (CGP 71683) (B and D) on changes in LDH release induced by KA and the respective YR agonists.LDH was measured 48 h (cortical) or 24 h (hippocampal cultures) after KA administration. The agonists of Y2 or Y5 receptors were added to the culture medium 30 min afterthe KA; the Y2 or Y5R antagonists were applied 10 min before the respective agonists. Each bar represents the mean of n P 6 platings ± SEM from 3 to 4 independentexperiments. ***P < 0.001 versus control cultures; ###P < 0.001 versus the cultures exposed to KA; $$$P < 0.001 versus the cultures exposed to KA+NPY13-36, orKA+[cPP1–7,NPY19–23,Ala31,Aib32,Gln34]-hPP.


diminution of KA lesion by ca. 45–37% in comparison to KA alone(Fig. 8C).

Microinjection of the Y1R agonist in a dose of 470 nmol at30 min after KA did not induce any protection; in contrast atendency towards an increase of the lesion was observed(Fig. 8D).

3.3. The effects of Y2 receptor agonist (NPY13-36) in transient MCAO

Intracerebroventricular injection of NPY13-36 diminished thebrain damage after MCAO. The total volume of infarction was by

58% smaller (P < 0.0005) in the group treated with NPY13-36 com-pared to the control one (Fig. 9A and B), which suggests a neuro-protective effect of the peptide. In the control group, 26.7 ± 1.2%of the hemisphere was infarcted. In the group treated withNPY13-36, the infarction volume was only 11.2 ± 2.3% of that ofthe noninjured hemisphere.

NPY13-36 did not significantly influence brain blood flow. Thedecrease in LDF upon MCA occlusion was similar in either group(control and treated) (Fig. 9C). No statistically significant differ-ences in LDF were observed, either, throughout 2 h of occlusionand upon reperfusion.

Fig. 4. The effects of Y2R (A, B) and Y5R (C, D) agonists and antagonists on the KA-induced increase in caspase-3 activity measured in mouse cortical cultures. Caspase-3 wasmeasured 6 h after starting the incubation with KA. The agonists of Y receptors were added to the cultures 30 min after KA, and the antagonists 10 min before the respectiveagonists. Each bar represents the mean of n P 6 platings ± SEM from 3 to 4 independent experiments. ***P < 0.001 versus control cultures; #P < 0.05, ##P < 0.01, and###P < 0.001 versus the cultures exposed to KA; $P < 0.05 versus the cultures exposed to KA + NPY13-36, or KA + [cPP1-7, NPY19-23, Ala31, Aib32, Gln34]-hPP.


4. Discussion

Our studies were carried out with three models: (1) in vitro, inprimary cultures of mouse cortical and hippocampal neurons; (2)in vivo in adult rat hippocampi, as these structures are very sensi-tive to excitotoxicity, and (3) in vivo, using an ischemic MCAOmodel. In the first and the second model, excitotoxic degenerationwas induced by kainic acid (KA) applied into the culture medium(in vitro studies), or microinjected into the hippocampus (in vivostudies).

The obtained results indicated a neuroprotective effects of NPY,as well as of specific Y2 and Y5 receptor agonists against the KA-in-duced neurotoxicity, since the peptides inhibited LDH increase and

prevented apoptosis. The protective activity of NPY, which is anagonist of all YR types, is in agreement with our earlier resultsshowing such effects against KA intoxication both in vivo, afterintrahippocampal injection (Smiałowska et al., 2003), and in vitro(Domin et al., 2006). In our present experiment we found that inneuronal cultures NPY was protective not only at concentrationsof 0.5 and 1 lM (as described previously), but also at 0.1 lM (butnot 0.01 lM). It is noteworthy that in neuronal cultures NPY waseffective against kainate excitotoxicity when it was given as lateas 6 h after KA, although the effects were much weaker: a 22–26% decrease occurred in cortical cultures and a 28–34% fall in hip-pocampal ones compared to a 25–45% and a 38–52% decrease,respectively, when NPY was applied 30 min after KA. The neuro-

Fig. 5. The effects of Y2R (A and B) and Y5R (C and D) agonists and antagonists on the KA-induced increase in caspase-3 activity measured in mouse hippocampal cultures.Other explanations as in Fig. 4.


protective effects of NPY were also observed in vivo by Thiriet et al.(2005) after intracerebroventricular injection into mice, but theseauthors did not study delayed effects as the peptide was injected1 h before the toxin. Effectiveness of delayed NPY application,found in cultures in our previous (Domin et al., 2006) and presentstudies, is in agreement with the in vivo results of Wu and Li (2005)who found neuroprotection in mouse hippocampus after intracer-ebroventricular NPY injection at 2 and 8 h after KA.

The results obtained by other authors concerning the neuropro-tective effects of NPY are divergent and controversial, hence we at-tempted to determine the role of different Y receptors (YR) usingspecific ligands. Our results showed the neuroprotective activityof Y2R and Y5R agonists against the KA-induced toxicity in bothmodels studied. The effects were significant even after delayedapplication: a few hours after starting KA intoxication. Our

in vitro findings agree with the data revealed by other authors. Sil-va et al. (2003) showed the protective properties of Y2R and Y5Ragonists in organotypic hippocampal cultures; however, theyadded those agonists 24 h before and simultaneously with a toxicsubstance (AMPA or KA). A protective effects after delayed applica-tion of the Y2R agonist was found by Xapelli et al. (2007) in thesame model of a hippocampal organotypic culture. The authors ob-served neuroprotection when NPY13-36 was added 30, but not60 min, after KA. In our studies, significant protection was inducedby that peptide applied not only 30 min, but also 1 h (cortical cul-tures) or 3 h (hippocampal cultures) after KA. The discrepancy be-tween our and Xapelli’s results may have arisen from differentculture models, doses of peptide, and different methods of evalua-tion of degeneration. Our in vitro study also showed neuroprotec-tive effects of [cPP1-7, NPY19-23, Ala31, Aib32, Gln34]-hPancreatic

Fig. 6. The effects of Y1R agonist ([Leu31, Pro34]-Neuropeptyd Y) and Y1R antagonists (BIBO3304) on the KA (150 lM)-induced LDH release in primary cultures of mousecortical (A and B) and hippocampal (C and D) cultures. The Y1R receptor agonist or antagonist was added to the culture medium 30 min after the KA. Each bar represents themean of n P 6 platings ± SEM from 3 to 4 independent experiments. ***P < 0.001 versus control cultures; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the cultures exposed toKA.


Polypeptide, which activates mainly Y5R. This finding may speakfor the neuroprotective effect of Y5R activation. That effect oc-curred at similar doses and time points as the effect of the Y2R ago-nist; also hippocampal cultures were better protected than corticalones. To date, only few papers have described the neuroprotectivepotential of Y5 receptors (Silva et al., 2003, 2005a).

The effectiveness of a delayed application of NPY and YR ago-nists in KA-excitotoxicity models, observed in our present study,seems to be very important to future clinical investigations.Although the diminution of KA-induced LDH release was weakerwhen the peptides were given 3 or 6 h after KA (compared to a30-min or a 1-h delay), the obtained results seem to suggest a pos-sibility of neuroprotection after delayed treatment. The mecha-nism of these effects may be connected with the fact that thesecondary release of endogenous glutamate plays a significant rolein the KA-induced degeneration (Coyle, 1983; Ferkany and Coyle,1983; Malva et al., 1998).

We also found the antiapoptotic action of peptide NPY, Y2 andY5R agonists, as those substances decreased the KA-induced cas-pase-3 activation and the number of apoptotic bodies visible inHoechst 33342 staining. It is known that KA disturbs the homeo-stasis of calcium ions in cells, cytochrome c release and caspase-3 activation, which leads to cell death (Wang et al., 2005). It istherefore proposed that the antiexcitatory and antiapoptotic prop-erties of the peptides studied are connected with a decrease in theinput of calcium ions into neurons and a decrease in glutamate re-lease. Also Thiriet et al. (2005) suggested that one of the possiblemechanism of the neuroprotective action of NPY may be inhibitionof calcium channels. Summing up our discussion of the delayed ef-fects, it is proposed that the peptides studied may inhibit the excit-atory cascade that develops gradually after KA application.

In the present study we attempted to determine whether theneuroprotective effects of Y2R and Y5R agonists are specifically re-lated to specific receptors. In fact, the effects of those peptides on

Fig. 7. Selected microphotographs illustrating the fluorescence staining with Hoechst 33342 (a marker of apoptosis) in hippocampal cultures. Cells with bright fragmentednuclei (apoptotic bodies, arrows) showing condensation of chromatin were identified as dying in apoptotic mode. (A) A control culture with few apoptotic bodies; (B) aculture after KA exposure (150 lm; 24 h). Fewer healthy cells and more apoptotic bodies can be seen; (C) a culture exposed to KA and NPY (1 lM; 30 min after KA); (D) aculture exposed to KA and Y2R agonist (NPY13-36, 1 lM; 30 min after KA). Some neuroprotection can be seen (in C and D) as a decrease in the number of apoptotic bodies incomparison with KA (B).


both LDH and caspase-3 activity were prevented by the respectivereceptor antagonists BIIE0246 and CGP 71683. Our results are inline with the findings of other authors who observed preventionof the neuroprotective effects of the Y2R agonist NPY13-36 bythe specific receptor antagonist BIIE0246 in organotypic hippo-campal cultures (Silva et al., 2003; Xapelli et al., 2007, 2008). Nostudies with the Y5R antagonist concerning neuroprotection havebeen conducted so far by other authors, but the role of Y2 andY5R in the protection of neurons is suggested by electrophysiolog-ical results obtained both in vitro (in the hippocampal slices) andin vivo. They show the suppression of epileptiform bursting or sei-zures by Y2R and/or Y5R agonists and the inhibition of these effectsby specific receptors antagonists (Woldbye et al., 1997; Bijak,1999; Marsh et al., 1999; Vezzani et al., 1999; Baraban, 2002;Nanobashvili et al., 2004; El Bahh et al., 2005).

Interesting finding of our in vitro experiments was that NPY, thewhole molecule, provided better neuroprotection than did peptideanalogs specific to the respective Y2 or Y5 receptors. It may suggestthat simultaneous activation of both these receptors (as occursafter NPY) induces stronger protection than activation of onereceptor type only.

In the present study we found that Y2R and Y5R agonists wereneuroprotective not only in vitro, but also in vivo after their injec-tion into rat hippocampus. Those agonists were effective even afterdelayed treatment. Significant diminution of degeneration was ob-served when the peptides were injected 30 or 60 min, but not 3 h,after KA. These results are in line with the findings of our previousin vitro studies, but are not so promising as those obtained by Wuand Li (2005). The latter authors observed inhibition of the KA-in-

duced apoptosis in mouse hippocampus by NPY or Y2 and Y5receptor agonists given 8 h after KA injection. The discrepancy be-tween our and Wu and Li’s results may arise from differences inthe methodology: in our experiments we used rats and injectedKA and the peptides locally into the hippocampus, while theabove-quoted authors used mice and injected KA intraperitonealyand the peptides intraventricularly.

We have found that, in contrast to the Y2 and Y5R agonists, theY1R agonist did not exhibit any neuroprotective effects: instead,even a tendency to increase the KA-induced damage was observedboth in vitro and in vivo. Moreover, the Y1R antagonist BIBO3304,added 30 min after KA, revealed neuroprotective properties in cor-tical and hippocampal cultures. The results of other authors con-cerning the role of Y1 receptors are divergent. Neuroprotectionwas described by Silva et al. (2003) and Xapelli et al. (2007), whoused a model of rat organotypic hippocampal slice cultures. Thefindings of some other authors are in line with our results. Gari-boldi et al. (1998) found that Y1R activation increased, while itsinhibition decreased the KA-induced seizures. In the model of atransient middle cerebral artery occlusion, intracerebroventricularmicroinjection of the Y1R agonist increased the infarct volume,while its antagonist reduced that volume (Chen and Cheung,2003). Also in oxygen- and glucose-deprived neuronal cultures,the Y1R activation worsened, whereas its blockade improved cellviability (Chen and Cheung, 2004). The neurotoxic effects of Y1Rstimulation observed in some experiments may be due to the factthat these receptors are situated mainly postsynaptically, and theiractivation induces calcium influx and inhibition of potassiumchannels, which increases neuronal excitability (Dumont et al.,

Fig. 8. Microphotographs of coronal sections of rat brain hippocampi stained with cresyl violet, and the respective results of the stereological counting of neurons. Arrowsindicate a CA pyramidal layer where the neurons were counted. (A) loss of neurons and extensive gliosis can be seen in the hippocampus after KA microinjection (2.5 nmol/1 ll) (left photo) in comparison with the non-degenerated contralateral side (right photo). (B) Neuroprotective effect of the Y2R agonist injected 30 min after KA. The lesion ismuch smaller, as seen in the microphoto on the left side. The results of stereological counting are shown on the right side – neurons are more numerous in comparison withkainate-treated rats. (C) a similar neuroprotective effect was observed after Y5R agonist microinjection 30 min after KA. (D) Microinjection of the Y1R agonist has noprotective effect against KA. In contrast, a tendency towards an increase of the lesion can be seen. Each bar represents the mean ± SEM of n = 6 per group. ***P < 0.001contralateral versus KA (ipsilateral); #P < 0.05 KA lesioned (ipsilateral) versus KA + YR agonists (ipsilateral) hippocampi. Calibration bars, 200 lm.


1992; Silva et al., 2002; Gobbi et al., 1996). Stimulation of calciumrelease from the endoplasmic reticulum (Aakerlund et al., 1990;Michel et al., 1998) and enhancement of nitric oxide production(Bitran et al., 1999; Chen et al., 2002), has also been proposed as

possible mechanisms of neurotoxicity after Y1R activation, is alsopostulated. On the other hand it has been found in the brain thatsome Y1R are located presynaptically (Kopp et al., 2002) andmay thus have inhibitory effects by diminishing of glutamate re-

Fig. 9. (A and B) Effect of Y2R agonist NPY13-36 (10 lg/6 ll) on the infarct volumeproduced by a middle cerebral artery occlusion (MCAO) in rats. NPY13-36 (10 lg/6 ll) was given intracerebroventricularly 30 min after starting MCAO. (A) Repre-sentative examples of TTC staining of coronal slices of rat brains, taken 72 h afterfinishing MCAO, showing the viable brain tissue in red (in the photo in dark) andinfarcted brain tissue in white. Slices from the MCAO brain treated with vehicle(upper panel) and treated with NPY13-36 (lower panel) illustrate a diminution ofinfarction after the peptide treatment. (B) The results of a morphometric analysis ofthe infarct volume after MCAO or MCAO + Y2R agonist treatment. Each barrepresents the mean ± SEM of n = 6 per group. ###P < 0.001 compared withvehicle-treated group (MCAO). (C) graph presenting results of microflow measure-ments of a blood flow (LDF) during the occlusion and the onset of reperfusion. Nosignificant differences are seen between the control MCAO group andMCAO + NPY13-36 treated group.


lease (McQuiston et al., 1996; Silva et al., 2001). Hence divergentresults of different authors – neuroprotective or neurotoxic effectsof Y1R agonists – may stem from the fact that those authors stud-ied different brain structures, using different models.

Additionally, we attempted to find out whether the neuropro-tective effects of YR activation occurred not only in the case ofKA excitotoxicity, but also in an ischemic model. For that experi-ment we chose the Y2R agonist NPY13-36 as the most promisingligand, on the basis of our earlier results with kainate models. Inthe transient MCAO model we showed strong and significant pro-tective action of the peptide given 30 min after the onset of ische-mia. To our knowledge, no studies into the neuroprotective effectsof Y2R agonists in MCAO have been conducted so far. Moreover, anincrease in infarct volume was observed after NPY or Y1R agonistmicroinjection into rat lateral ventricle during or after ischemia(Chen and Cheung, 2002; Cheung and Cechetto, 2000). Theabove-cited authors attributed those effects to the hemodynamicaction of NPY, which worsened reperfusion. In our study, the mea-surement of blood flow did not show any significant influence ofNPY13-36 on LDF changes observed in the transient MCAO, hencethat peptide did not seem to induce vasoconstriction. That obser-vation is in line with some earlier results of other authors whofound that vasoconstriction after NPY was mediated by Y1R, whichprevailed in cerebral arteries (Abounader et al., 1995, 1999). Incontrast, no such effects were found after Y2R activation (Lewiset al., 1999; Edvinsson, 2006; Zukowska et al., 2003; Chronwalland Zukowska, 2004). Moreover Y2R activation may be beneficialfor ischemic revascularization, as it stimulates angiogenesis (Abeet al., 2007; Kuo et al., 2007).

Summing up, the present data obtained using kainate andischemic models point to the neuroprotective effects of NPY withengagement of Y2 and Y5 receptors. No protection has been foundafter the Y1R agonist. The specific activation of Y2R seems partic-ularly promising, as it is also effective in an ischemic model. It isof particular importance that the studied peptides reveal their pro-tective activity even after delayed treatment, 30 min or even a fewhours after starting the damaging action. The effectiveness of sucha late application indicates a potential therapeutic use of similarcompounds in patients to whom a neuroprotective treatment canbe introduced a few hours after damage.

Acknowledgments

This study was supported by Grant No. 2P05A 114 28 from theMinistry of Science and Higher Education, Poland.

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neuroprotective effects of neuropeptide y-y2 and y5 receptor agonists in vitro and in vivo

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