Anticonvulsant effect of neural regeneration peptide 2945 onpentylenetetrazol-induced seizures in ratsAzadeh Sajadian a1 Sana Esteghamat a1 Fariba Karimzadeh b1 Arezou Eshaghabadi a1Frank Sieg c Erwin-Josef Speckmann e Sven Meuth d Thomas Seidenbecher eThomas Budde e Ali Gorji adefga Shefa Neuroscience Research Center Tehran Iranb Departments of Neuroscience School of Advanced Technology of Medical Tehran Medical University Tehran Iranc CuroNZ Ltd 29 Nugent Street Grafton Auckland New Zealandd Department of Neurology Westfaumllische Wilhelms-Universitaumlt Muumlnster Muumlnster Germanye Institute of Physiology I Westfaumllische Wilhelms-Universitaumlt Muumlnster Muumlnster Germanyf Epilepsy Research Center Westfaumllische Wilhelms-Universitaumlt Muumlnster Muumlnster Germanyg Klinik und Poliklinik fuumlr Neurochirurgie Westfaumllische Wilhelms-Universitaumlt Muumlnster Muumlnster Germany

A R T I C L E I N F O

Article historyReceived 5 June 2014Accepted 19 November 2014Available online

KeywordsNeuroinflammationEpilepsyNeuropharmacologyBrainIn vivo

A B S T R A C T

Neuron regeneration peptides (NRPs) are small synthetic peptides that stimulate neural proliferationmigration and differentiation with no apparent toxicity and high target specificity in CNS The aim ofthis study was to investigate the effect of NRP2945 on seizure activity induced by pentylenetetrazol (PTZ)in rats Using behavioural assessment and electrocorticographical recordings the effects of different dosesof NRP2945 (5ndash20 μgkg) were tested on seizure attacks induced by PTZ injection In addition the effectof NRP2945 was evaluated on the production of dark neurons and expression of GABAA receptor α andβ subunits and GAD-65 in the hippocampus and somatosensory cortex of the rat brain Intraperitonealinjection of NRP2945 at 20 μgkg prevented seizure attacks after PTZ injection NRP2945 at doses of 5and 10 μgkg significantly decreased the total duration of seizure attacks and reduced the amplitudeduration and latency of epileptiform burst discharges induced by PTZ In addition the peptide signifi-cantly inhibited the production of dark neurons in the hippocampus and somatosensory cortex of epilepticrats NRP2945 also significantly increased the expression of GABAA receptor α and β subunits and GAD-65 in the hippocampus and somatosensory cortex compared with PTZ treated rats This study indicatesthat NRP2945 is able to prevent the seizure attacks and neuronal injuries induced by PTZ likely by stimu-lating GABAA and GAD-65 protein expression andor protecting these components of GABAergic signallingfrom PTZ-induced alteration Further studies are needed to elucidate the potential role of NRP2945 asan antiepileptic drug

copy 2014 Elsevier Ltd All rights reserved

1 Introduction

Neural regeneration peptides (NRPs) are small synthetic pep-tides that promote proliferation migration differentiation andsurvival of neural precursors and neural stem cells under patho-logical conditions affecting the central nervous system (Gorba et al2006 Singh et al 2010) The discovery of NRPs dates back to ex-perimentation with a newly developed in vitro model of traumatic

brain injury (Sieg et al 1999) Administration of biochemically frac-tionated cell culture supernatant derived from hippocampalorganotypic tissue cultures (OTCs) led to a neuronal bridge forma-tion in thalamocortical OTC co-cultures within 3ndash4 days (Landgraf2005) The first completely identified gene that encodes for bioactivepeptides displaying potency for neuronal survival-promoting ac-tivity was experimentally shown to be encoded on chromosome 12in mice and is coding for a 135 amino acid long protein (Gorba et al2006) NRP2945 derives from the human NRP gene located at po-sition 7q3135 also known as calcium- dependent activator of proteinsecretion 2 (CAPS2 Sadakata et al 2004) and represents amino acidposition 40ndash50 within the CAPS2 sequence (Speidel et al 2003)NRPs have both neuroregenerative and anti-inflammatory effects(Gorba et al 2006 Landgraf 2005 Sieg and Antonic 2007) Severalstudies using human primary brain cells human embryonic neuralstem cells and animal cells subsequently confirmed that NRPs at

Abbreviations DAB 3-3prime-diaminobenzidine NRPs neuron regeneration pep-tides OTCs organotypic tissue cultures PFA paraformaldehyde PTZpentylenetetrazol PBS phosphate buffered saline

Corresponding author Epilepsy Research Center Muumlnster University Robert-Koch-Straszlige 27a D-48149 Muumlnster Germany

E-mail address gorjialuni-muensterde (A Gorji)1 The first four authors contributed equally to this manuscript

httpdxdoiorg101016jnpep2014110020143-4179copy 2014 Elsevier Ltd All rights reserved

Neuropeptides (2014) ndash

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Contents lists available at ScienceDirect

Neuropeptides

journal homepage wwwelseviercom locate npep

sub-nanomolar concentrations have both neuroprotective and anti-inflammatory effects suitable for the treatment of neuroinflammatorydiseases (Gorba et al 2006)

Over the past two decades an increasing amount of evidenceindicates that activation of inflammatory processes occurs duringepileptogenesis which contribute in concert to the adverse out-comes (Vezzani and Ruumlegg 2011) Increased levels of inflammatorymolecules and upregulation of their receptors in glial and neuro-nal cells as well as microvascular endothelial cells have beendemonstrated in human brain tissues of medically refractory epi-leptic patients suggesting that proinflammatory pathways areactivated in seizure foci (Aronica et al 2012 Choi et al 2009Vezzani et al 2013) It is proposed that some patients withchronic epilepsy might benefit from anti-inflammatory orimmunomodulatory therapies Neuroprotective and anti-inflammatory drugs significantly decreased expression ofinflammatory molecules and cell loss in the brain and can slowepileptogenesis (Dedeurwaerdere et al 2012) Therefore prevent-ing the activation of inflammatory cascades represents a promisingstrategy to improve treatment of epilepsy

Several screening models are currently used to investigatecompounds for anticonvulsant activity Seizure induction by pen-tylenetetrazol (PTZ) application as a model for acute generalisedclonic type seizures (absence or myoclonic Ferrendelli et al 1989Meldrum 2002) is commonly employed in anticonvulsant screen-ing structure-activity research drug design and other approachesfor antiepileptic drug development (Kupferberg 2001) Pre-treatment with well-established anticonvulsants such asclonazepam carbamazepine valproate or ethosuximide (15ndash30 min before PTZ injection) significantly increased the seizurethreshold in rats (Loumlscher et al 1991) This study was designed toevaluate the effect of NRP2945 on behavioural and histopathologi-cal consequences of PTZ-induced convulsions in adult rats

2 Materials and methods

Adult male Wistar rats (200ndash250 g n = 40) were housed undercontrolled conditions (ambient temperature = 22 degC humidi-ty = 40 12-h lightdark cycle) with food and water ad libitumAnimals were age- and weight-matched and randomly divided intothree groups (i) Control group animals underwent all of the sur-gical procedures and treated with saline intraperitoneally (ip) butdid not receive PTZ (Santa Cruz Heidelberg Germany) or NRP2945(n = 8) (ii) PTZ group rats received saline 30 min before ip injec-tion of PTZ (50 mgkg dissolved in 09 saline PTZ + saline n = 8)(iii) NRP2945 group animals received ip injection of NRP2945 (510 or 20 μgkg) 30 min before ip injection of PTZ (PTZ + NRP2945n = 24 eight rats for each concentration) NRP2945 was gener-ously donated by CuroNZ Ltd Auckland New Zealand Allexperiments were carried out according to the protocol approvedby the Animal Ethics Committee of Shefa Neuroscience CenterTehran Iran

21 Behavioural tests

After each PTZ-injection the convulsive behaviour was as-sessed for 30 min and resultant seizures were scored as follows0 = normal behaviour 1 = immobility 2 = rigid posture 3 = repet-itive scratching circling or head bobbing 4 = forelimb clonus rearingand falling 5 = repeated occurrence of level four behaviour and6 = severe tonic-clonic behaviour or status epilepticus (Karimzadehet al 2013 Morrison et al 1996) Latency of seizure onset wasdefined as the average length of time in seconds between PTZ ad-ministration and initially detectable seizure A generalised seizurewas characterised by symmetric forelimb and hind limb tonus fol-lowed by hind limb clonus and subsequent jumping activity Since

animals occasionally had more than one seizure the seizure dura-tion was assessed as the sum of durations of these multiple seizures

22 Surgical procedure and electrocorticogram (ECoG) recording

Animals were anaesthetised with chloral hydrate 35 (dis-solved in normal 09 saline 1 mL100 g ip Sigma-Aldrich) andthe head of each rat was placed in a stereotaxical instrument(Stoelting Instruments Wood Dale IL USA) Epidural recording elec-trodes were stereotaxically implanted above the left and rightsomatosensory cortices (30 mm posteriorly to Bregma and 30 mmlaterally from the midline) under continuous chloral hydrate an-aesthesia The reference electrode is located at the nasal bone ECoGwas recorded via monopolar silver electrodes connected to an am-plifier (EXT-02 F NPI Tamm Germany with band-pass filters at 05ndash30 kHz sampling rate 10 kHz) and stored by a digital oscilloscopeRats were allowed to recover 1 week after surgery ECoG record-ings were performed for 30 min after receiving saline or NRP2945(5 10 20 μgkg) and 60 min after the injection of PTZ Latency du-ration amplitude and frequency of epileptic spikes activity werecalculated using AxoScope 10 software (Axon Instruments FosterCity CA USA)

23 Stereological methods and physical dissector

The volume-weighted mean volume of normal neurons was cal-culated directly by point-sampled intercept on 10 uniformsystematically and randomly sampled microscopic fields of the py-ramidal layers of hippocampal areas CA1 and CA3 as well as thesomatosensory cortex A lattice of test points on lines was super-imposed randomly onto the traced nuclear profiles in each particularfield Nuclei of neurons were marked and two isotropic lines fromrandomly selected directions were centred on this neuron and su-perimposed The intersection of each line with the outer surface ofthe respective neuronrsquos soma was marked These lines producedpoint-sampled intercepts the lengths of which were measured andcubed The mean was multiplied by π3 and finally all interceptswere averaged to give an estimate of the volume-weighted meanneuronal volume For quantitative analysis of dark neurons the phys-ical dissector method was used Ten pairs of sections with 8-mmdistance were collected from each brain The first section of eachpair was designated as the reference and the second one was usedfor comparison On each pair of sections at least 10 microscopicfields were selected by uniform systematic-random sampling in everyarea of interest Using the unbiased frame and physical dissectorcounting rule the counting of dark neurons in each field was carriedout (Jafarian et al 2010 Sadeghian et al 2012)

24 Histological studies

Rats were sacrificed after 90 min of PTZ injection Animals weredeeply anaesthetised with chloral hydrate (35 Sigma-Aldrich) andperfused transcardially with 200 mL of saline followed by 200 mLof a solution containing 1 paraformaldehyde (PFA) After perfu-sion all rats were decapitated and the brains were removed Thebrains were kept in 1 PFA for at least 10 days and were then pro-cessed for histological studies as follows The brain was cut intotransverse sections of 8 μm thickness Sections were stained withtoluidine blue The sections were selected by uniform systematicrandom sampling in every area of interest Different areas (hippo-campal CA1 and CA3 areas and somatosensory neocortex) werestudied under a light microscope (BX51 Olympus Tokyo Japan)equipped with a digital camera Digital photographs were taken usinga 40 times oil immersion objective lens (Olympus) The magnificationwas calculated using an objective micrometre

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25 Immunohistochemistry

Animals were deeply anaesthetised with chloralhydrate and per-fused transcardially with 200 mL of saline followed by 600 mL of4 PFA solution The brains were extracted and kept in 4 PFA forat least 4 days at 4 degC and were then processed for immunohisto-chemistry studies The serial (8 μm) coronal sections were preparedTen paraffin-embedded sections of different brain regions (the so-matosensory cortex and the hippocampal CA1 and CA3 areas) in minus18to minus33 mm to the bregma (medialndashlateral to the midline 7 mm depthof 7 mm) were cleared and rehydrated through a series of xylol andalcohol and washed with phosphate buffered saline (PBS) three timesand incubated in blockage solution (3 H2O2methanol for 5 min)After washing in PBS slides were boiled in sodium citrate buffer at95 degC for 10 min After cooling to room temperature for 20 min slideswere washed with PBS three times and incubated overnight at 4 degCwith commercial rabbit polyclonal anti-rat antibody (Abcam Cam-bridge UK) against GABAA α GABAA β or GAD-65 overnight Antibodyconcentration for GABAA α was 1100 for GABAA β was 150 andfor Glutamic acid decarboxylase-65 (GAD-65) was 1200 in a so-lution containing 1ndash5 NGS in 03 Triton X-100 and 01 M PBS atpH 74 The sections were then rinsed three times in PBS (10 mineach) and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Abcam) diluted at 1400 in PBS with03 Triton X-100 and 5 NGS at 22 degC for 1 h After several wash-ings with PBS the slides were incubated and developed in 3-3prime-diaminobenzidine (DAB Roche 05 μL DAB and 15 μL peroxidebuffer) for 5ndash10 min Sections were counterstained withhaematoxylin Control for the specificity of immunostaining was per-formed by omission of the primary antibody Images for analysiswere acquired with a digital camera attached to the microscope(BX51WI Olympus) The number of cells labelled for GABAA αGABAA β or GAD-65 was measured in different sections from controlPTZ + saline and PTZ + NRP2945 (20 μgkg) rats Three areas in-cluding the somatosensory cortex hippocampal CA1 area and CA3regions from each section were analysed All images analysis wascarried out in blind

Data were expressed as mean plusmn SEM Statistical analysis wascarried out by using one-way analysis of variance (ANOVA) fol-lowed by Tukeyrsquos post-hoc test The criterion for statisticalsignificance was P lt 005 Correlation between the number of darkneurons and the volume of neurons was analyzed by Pearsonrsquos test

3 Results

31 The effect of NRP2945 on PTZ-induced seizures

All animals of the control group did not exhibit any seizurebehaviour In the present study PTZ was used to induce epilepticseizures in rats Rats of the PTZ group developed typical seizurebehaviour about 2ndash3 min following PTZ injection PTZ-induced sei-zures were completely prevented by administration of NRP2945 ata dose of 20 μgkg At lower doses (5 and 10 μgkg) NRP2945reduced the total duration of seizure attacks after PTZ injection com-pared with PTZ animals (P lt 005 Fig 1A Table 1) but was notaffecting the latency and severity of seizure attacks (Fig 1A)

32 The effect of NRP2945 on PTZ-induced epileptiformECoG activity

ECoG recording in anesthetised rats were performed to vali-date the behavioural observations Recordings in control rats didnot show any epileptiform field potentials ECoG recording in PTZgroup consisted of a series of surface positive flat topped deflec-tions These waves had durations of 20ndash200 ms and a frequency of03ndash4 Hz Following injection of PTZ (50 mgkg) the first epilepti-form spike occurred within 20ndash185 (45 plusmn 11) s of the injection Thespikes steadily increased in size and reached amplitudes of14 plusmn 01 mV durations of 180 plusmn 72 s and an occurrence of 15 plusmn 12min within 5ndash10 min The epileptiform burst discharges then remainsconstant for another 30 min (PTZ group) Application of NRP2945at a dose of 20 μgkg 30 min before PTZ injection completely pre-vented development of epileptiform field potentials (Fig 1B)NRP2945 at doses of 5 and 10 μgkg significantly increased thelatency time to the onset of the first spike waves and decreased theamplitude duration and repetition rate of the epileptiform field po-tentials compared with PTZ group (P le 0001 Fig 1B and C)

33 The effect of NRP2945 on production of dark neurons

The appearance of neurons with condensed darkly stained nucleiand bright cytoplasm was used to assess neuronal degeneration fol-lowing PTZ treatment (Garman 2011) Dark neurons were identifiedby neuronal shrinkage cytoplasmic eosinophillia nuclear pykno-sis and surrounding spongiosis (Jafarian et al 2010 Sadeghian et al

Table 1Data display the effect of NRP2945 at doses of 5 10 and 20 μgkg on behaviour (PTZ-induced seizure attacks) epileptiform burst discharges recorded by electrocorticography(ECoG) the mean number of dark neurons and the mean volume of normal neurons after injection of PTZ NRP2945 at different doses was administered 30 min before PTZinjection and Indicate P lt 005 and P lt 0001 respectively

Control + saline PTZ + saline PTZ + NRP (5 μgkg) PTZ + NRP (10 μgkg) PTZ + NRP (20 μgkg)

Behavioural testLatency (s) ndash 1442 plusmn 118 2302 plusmn 106 1502 plusmn 74 -Duration (s) ndash 605 plusmn 05 3583 plusmn 59 3767 plusmn 73 -Severity ndash 4 plusmn 0 367 plusmn 021 367 plusmn 021 -

ECoGLatency (s) ndash 27 plusmn 7 326 plusmn 24 417 plusmn 37 -Duration (s) ndash 180 plusmn 72 60 plusmn 12 60 plusmn 21 -Amplitude (mV) ndash 14 plusmn 01 11 plusmn 004 084 plusmn 01 -Frequency (per min) ndash 15 plusmn 12 47 plusmn 03 667 plusmn 09 -

Dark neuronsCA1 131 plusmn 7 256 plusmn 23 153 plusmn 13 88 plusmn 4 137 plusmn 9CA3 119 plusmn 7 270 plusmn 14 131 plusmn 17 80 plusmn 7 126 plusmn 11Cortex 127 plusmn 7 363 plusmn 25 142 plusmn 12 78 plusmn 9 130 plusmn 8

Mean volume of normal neuronsCA1 4663 plusmn 69 3849 plusmn 56 3809 plusmn 71 4131 plusmn 44 4519 plusmn 55CA3 4766 plusmn 66 3909 plusmn 31 3879 plusmn 32 4386 plusmn 56 4741 plusmn 51Cortex 3924 plusmn 35 3419 plusmn 36 3408 plusmn 31 3957 plusmn 61 3879 plusmn 75

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2012) The mean number of dark neurons was significantly higherin all evaluated area in PTZ group compared with control rats(P le 0001 Fig 2A and B) Density of dark neurons in the hippo-campal CA1 and CA3 areas and the somatosensory cortex wassignificantly decreased after administration of NRP2945 com-pared with PTZ rats The mean number of dark neurons in thehippocampal CA1 and CA3 areas as well as the cortex in controlgroup was 131 plusmn 7 119 plusmn 7 and 127 plusmn 7 respectively The meannumber of dark neurons in the hippocampal CA1 area decreasedfrom 256 plusmn 23 in PTZ rats to 153 plusmn 13 88 plusmn 4 and 137 plusmn 9 after ap-plication of 5 10 and 20 μgkg respectively (P le 0001 Fig 2A andB Table 1) The mean number of dark cells in the hippocampal CA3area also significantly reduced from 270 plusmn 14 in control rats to131 plusmn 17 80 plusmn 7 and 126 plusmn 11 after application of 5 10 and 20 μgkg respectively (P le 0001 Fig 2 Table 1) NRP2945 also reducedproduction of dark neurons by PTZ injection in the neocortex Themean number of dark cells in the somatosensory cortex in PTZ groupwas 363 plusmn 25 Administration of NRP2945 at 5 10 and 20 μgkgbefore injection of PTZ reduced the number of these neurons to

142 plusmn 12 78 plusmn 9 130 plusmn 8 respectively (P le 0001 Fig 2A and BTable 1)

34 The effect of NRP2945 on the mean volume of normal neurons

Administration of PTZ significantly decreased the volume-weighted mean volume of normal neurons compared with controlrats (P le 0001 Fig 3A) The mean volume of normal neuronsin the hippocampal CA1 and CA3 areas as well as the neocortexin control group was 4663 plusmn 69 4766 plusmn 67 and 3924 plusmn 35 μm3respectively The mean volume of normal neurons in the hip-pocampal CA1 and CA3 areas as well as the neocortex in PTZgroup decreased to 3849 plusmn 56 3909 plusmn 31 and 3419 plusmn 36 μm3 re-spectively (P le 0001 Fig 3A) In addition there was a significantdifference in the mean volume of normal neurons observed in thehippocampus and in the somatosensory cortex after applicationof different doses of NRP2945 compared with PTZ rats (Fig 3ATable 1) NRP2945 at 5 μgkg did not affect the mean volume ofnormal neurons after PTZ-induced seizures However the

Fig 1 The effect of NRP2945 on seizure activities as well as epileptic burst discharges induced by injection of pentylenetetrazol (PTZ) in rats (A) The effect of NRP2945 atdoses of 5 and 10 μgkg on the latency duration and severity of seizure attacks induced by PTZ injection assessed by behavioural observation The duration of seizure attackswas significantly shorter in rate received NRP2945 at doses of 5 or 10 μgkg compared with PTZ group NRP2945 at a dose of 20 μgkg prevented PTZ-induced convulsion(B) Representative examples of epileptiform burst discharges recorded by electrocorticography (ECoG) in the neocortices of control rats PTZ rats as well as animals treatedwith different doses of NRP2945 (C) Effect of NRP2945 at doses of 5 and 10 μgkg on the latency frequency amplitude and duration of epileptiform burst discharges inducedby PTZ injection assessed by ECoG recordings The amplitude of spikes was significantly smaller and the duration of bursts was significantly shorter in rats treated with 5and 10 μgkg before PTZ injection compared with PTZ + saline animals The frequency of burst discharges was significantly lower than control rats and Indicate P lt 005and P lt 0001 respectively

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Fig 2 The inhibitory effect of NRP2945 administration on production of dark neurons (A) Light-microscopic appearance of toluidine blue stained dark neurons in 8-μmsections of the hippocampal CA1 and CA3 areas as well as the neocortex in rats (B) NRP2945 at doses of 5 10 and 20 μgkg inhibited production of dark neurons in thehippocampal CA1 and CA3 areas as well as the somatosensory cortex in rats after induction of seizures by intraperitoneal pentylenetetrazol (PTZ) injection and x IndicateP lt 001 and P lt 0001 respectively

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hippocampal CA1 and CA3 areas as well as the somatosensory cortexhad a larger mean volume of normal neurons after application ofNRP2945 at 10 and 20 μgkg compared with PTZ group (P le 0001Fig 3A Table 1)

Correlation analysis between the volume-weighted mean volumeof normal neurons and the number of dark neurons revealed thatan increase in the volume-weighted mean volume of normal neuronswas accompanied by a decrease in the number of dark neurons inthe hippocampal CA1 (r = 0084 P le 0001) and CA3 (r = 0147P le 00001) areas as well as in the somatosensory cortex (r = 0302P le 0001) after application of NRP2945 (Fig 3B)

35 The effect of NRP2945 on GABAA receptor α and β subunitsand GAD-65

To evaluate a possible mechanism of the anticonvulsant effectof NRP2945 the expression of GABAA receptor α and β subunits andGAD-65 after PTZ injection was assessed by immunohistochemi-cal staining The expression of GABAA receptor α and β subunits andGAD-65 was significantly reduced after ip injection of PTZ in thehippocampal CA1 area compared with control group (P le 0001Fig 4AndashF) PTZ injection significantly decreased expression of GABAA

receptor α subunit and GAD-65 in the neocortex (P le 0001Fig 4AndashF) A significant reduction of GAD-65 was also observed inCA3 area after PTZ injection (P le 0001 Fig 4C and F) A significantenhancement in the expression of GABAA receptor α subunit in thesomatosensory cortex and hippocampal CA1 area following treat-ment with NRP2945 (20 μgkg) was observed when compared withPTZ rats (P le 0001 Fig 4A and D) Application of NRP2945 at 20 μgkg only enhanced the expression of GABAA receptor β subunit in theCA1 hippocampal region compared with PTZ rats (P le 0001 Fig 4B

and E) We also examined the effects of NRP2945 on GAD-65immunoreactivity in the hippocampus and neocortex The immu-nohistochemical labelling for GAD-65 in the somatosensory cortexas well as in the CA1 and CA3 hippocampal areas revealed a sig-nificant increase in the expression in of GAD-65 immunoreactiveneurons in these brain regions after application of NRP2945 (20 μgkg) compared with PTZ rats (P le 0001 Fig 4C and F)

4 Discussion

The present study points to anticonvulsant effect of NRP2945 onthe PTZ-induced seizure behaviours and epileptiform burst dis-charges In addition NRP2945 prevents damage to cell structureproduced by seizure activities after PTZ injection NRP2945 reducedthe number of dark neurons and increased the mean volume ofnormal neurons in different brain regions A correlation betweenprevention of cell injury and enhancement of the mean volume ofnormal neurons by NRP2945 was observed Furthermore NRP2945enhanced expression of GABAA receptor α and β subunits and GAD-65 in the hippocampus and somatosensory cortex if applied beforeinjection of PTZ in rats

Administration of NRP2945 (at 20 μgkg) 30 min before PTZ in-jection prevented seizure attack and at lower doses reduced theduration of seizure attacks NRP2495 at all doses reduced the latencyfrequency amplitude and duration of PTZ-induced epileptiform fieldpotentials This suggests possible anticonvulsive effects of NRP2945for prevention of seizure attacks PTZ model is suggested to be pre-dictive of anticonvulsant drug activity against non-convulsive seizureattacks (Krall et al 1978) However several anticonvulsive medi-caments that protect against non-convulsive seizures in epilepticpatients failed in the PTZ test therefore testing with other models

Fig 3 The effects of different doses of NRP2945 administration on volume-weighted mean volume of normal neurons in the hippocampal CA1 and CA3 areas and the so-matosensory cortex after PTZ-induced seizures in rats (A) Administration of PTZ significantly decreased the mean volume of neurons compared with control rats The meanvolume of normal neurons in rats treated with NRP2945 at doses of 10 and 20 μgkg before PTZ injection was higher in different brain regions compared with PTZ + salinerats (B) Correlation between the volume-weighted mean volume of normal neurons and the number of dark neurons revealed that an increase in the volume-weightedmean volume of normal neurons was accompanied by a decrease in the number of dark neurons in hippocampal CA1 and CA3 areas as well as in the somatosensory cortexafter application of NRP2945 Indicates P lt 0001

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Fig 4 The expression of GABAA α and β receptors as well as GAD-65 in the hippocampal CA1 and CA3 regions and in the somatosensory cortex after application of NRP2945(20 μgkg) 30 min before PTZ injection (AndashC) Photomicrographs of immunohistochemistry of GABAA α and β receptors as well as GAD-65 expression in the neocortex andhippocampal CA1 and CA3 areas in control PTZ + saline and PTZ + NRP2945 rats (DndashF) The bar graphs show the quantitative results (mean plusmn SEM) of GABAA α and β re-ceptors as well as GAD-65 expression in the neocortex and hippocampal CA1 and CA3 areas The expression of GABAA α and β receptors and GAD-65 was significantly decreasedafter PTZ injection compared with control rats Treatment with NRP2945 before PTZ injection resulted in a higher expression of GABAA α subunit in the neocortex and CA1hippocampal region and a higher GABAA β subunit expression in CA1 area GAD-65 expression was significantly higher in both the neocortex and the hippocampal CA1 andCA3 regions after treatment with NRP2945 compared with PTZ + saline rats Indicate P lt 0001

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of non-convulsive seizures (such as genetic rat mutant model of con-vulsion) is needed for correct prediction of NRP2495 efficacy againstnon-convulsive seizures (Loumlscher 2011) PTZ is thought to inter-fere with GABA-mediated inhibition in particular a reduction ofGABAA receptor-mediated inhibitory postsynaptic potentials (Lewekeet al 1990) A single PTZ-induced convulsion as well as chronic ap-plication of PTZ was reported to be associated with rapid changesin expression of GABAA receptors These changes involved tran-sient decreases of GABAA receptor α and β subunitsrsquo mRNAs and adecrease in the coupling between the GABA and benzodiazepine rec-ognition sites in the receptor (Follesa et al 1999 Walsh et al 1999)Application of PTZ (50 mgkg ip) reduced GABAA-mediated recur-rent inhibition (by 19ndash25) in the CA1 hippocampal area anddecreased the density of GABAA receptors in the hippocampus (by19) and cortex (by 14) of mice (Psarropoulou et al 1994) PTZmodified the GABA benzodiazepine receptor complex and sup-pressed GABA-stimulated Cl- influx into the neurons (Rocha et al1996) Reduction of GABAA receptor expression had a functional sig-nificance on seizure threshold (El Idrissi et al 2003) and theactivation of GABA receptors exhibited a marked anticonvulsive effecton clonicndashtonic seizures in PTZ-induced seizures in rats (Becker et al1994 Rundfeldt et al 1995 Tirassa et al 2005) Intraperitonealadministration of GABA prevented development of behavioural con-vulsions and decreased the intensity of bioelectrical seizure activityinduced by PTZ (Smiałowski 1980) Antiepileptic substances withmain effect on GABA system such as vigabatrin and tiagabine arealso reported to prevent seizure behaviour and EEG changes inducedby PTZ injection in mice and rats (for review see Loumlscher 2011)

Our study revealed increased immunohistochemical labelling ofGABAA receptor α and β subunits and GAD-65 after application ofNRP2945 in both cortical and subcortical brain regions comparedwith PTZ-treated rats While reduction of hippocampal GAD im-munoreactivity was accompanied by a decreased hippocampal GABAexpression after PTZ treatment in rats (Tirassa et al 2005) admin-istration of antisense oligodeoxynucleotides to GAD-67 inhibited theexpression of GAD-67 mRNA and the concentration of GABA in thehippocampal tissue and increased the severity and the frequencyof PTZ-induced seizures (He et al 2002) NRP2945 may thereforeexert its anticonvulsive effect via increasing inhibitory tone in cor-tical and subcortical brain regions

It has been shown that PTZ-induced seizures produced neuro-nal damage in the supragranular region (Holmes et al 1999) andin the CA3 hippocampal area (Huang et al 2002) Repeated briefseizures elicited by PTZ treatment are capable of inducing progres-sive and selective neuronal loss and cell injury (ie pyknosis) in thehippocampus and other limbic structures (Fang and Lei 2010Mortazavi et al 2005) Neuronal damage and cell loss in the hip-pocampus was correlated with behavioural alterations such asdecreased exploratory locomotion as well as the severity of con-vulsions in rats treated with PTZ (Franke and Kittner 2001) Oneof the main factors mediating the neuronal damaging effects of PTZin the brain is oxidative stress (Rauca et al 1999) A significant de-crease in cysteine and mixedsymmetric disulfides as well as anincrease in lipid peroxidation protein oxidation levels and free hy-droxyl radicals after PTZ-induced seizure was observed (Patsoukiset al 2004 Rauca et al 1999) GABAergic neurons in different brainregions such as the amygdala hippocampus and temporal cortexare particularly susceptible to oxidative stress and convulsion(Tuunanen et al 1996) NRP2945 reduced the mean number of darkneurons after PTZ-induced convulsion Dark cells as a measure ofdegenerating neurons have been shown in mechanical neuronaltrauma ischaemia hypoglycaemia exposure to excitatory aminoacids and after induction of spreading depression in addition to ep-ilepsy (Ishida et al 2004 Sadeghian et al 2012) PTZ-induced seizurewas correlated with increased mean number of dark neurons in thehippocampus and neocortex (Karimzadeh et al 2013) The mouse

24mer NRPs exhibited a significant neuroprotective effect againstneuronal death and oxidative stress induced by a mixed injury par-adigm using the glutamate and the succinate dehydrogenase activityinhibitor in hippocampal cultures (Gorba et al 2006) NRPs havebeen shown to reduce caspase-3 expression in neonatal rats fol-lowing acute brain insults (Svedin et al 2007) NRP2945 may protectneurons against PTZ-caused seizures and neuronal damage by en-hancement of GABAA- and GAD65-derived inhibitory tone Activationof GABAA receptors plays a role in the inhibition of neuronal damageinduced by brain injury (Ito et al 1999)

Our data indicate that reduction of GABAA receptor α and β sub-units expression after PTZ injection and enhancement of theirexpression after application of NRP2495 in the hippocampus wasrestricted to CA1 area Regional differences in the changes in GABAA

receptor mRNA levels in the hippocampus after PTZ-induced seizureactivities were reported in rat brain (Walsh et al 1999) It has beenshown that GABA levels were changed differentially in CA1 and CA3areas of the hippocampus after induction of epileptiform burst dis-charges The extracellular GABA values were increased with differenttime courses in CA3 and CA1 regions and changes in GABA immu-noreactivity in mossy fibers in CA3 area usually occurred with alonger latency compared with the hippocampal CA1 area (Ding et al1998) Regional differences in the expression of GABAA receptor afterPTZ-induced seizure activity may be determined at least in part byregional differences in GABAA receptor subunit composition andor neuronal function (Walsh et al 1999)

The dosendashresponse of NRP2495 on production of dark neuronsdisplayed a bell shape ie less dark cells were observed by appli-cation of 10 μgkg compared with 5 and 20 μgkg in all tested areasThe survival promoting actions of several substances such as mela-tonin erythropoietin and bucladesine followed a bell-shaped dosendashresponse curve in in vivo and in vitro experimental models (Beniet al 2004 Vakilzadeh et al 2014 Weishaupt et al 2004) Al-though the exact mechanism of this dose-dependent biphasicresponse is not clear it has been suggested that this response maybe due to an overstimulation of the relevant receptors leading toa desensitisation of the response at a distinct concentration(Bronnikov et al 1999 Kim et al 2008)

The data emerging from this study indicate that NRP2945 ex-hibits neuroprotective and anticonvulsant effects in the PTZ seizuremodel likely by stimulation andor recovery of the PTZ-induced al-terations of GABAA and GAD65 expression Further investigationsare needed to elucidate the potential role of NRP2945 as anantiepileptic and putative neuroprotective drug

Acknowledgements

This work was funded by BMBF (01DR12096 to TB) and the ShefaNeuroscience Center grant related to Dr-Thesis 23657 (to AG andSE)

References

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ARTICLE IN PRESS

Please cite this article in press as Azadeh Sajadian et al Anticonvulsant effect of neural regeneration peptide 2945 on pentylenetetrazol-induced seizures in rats Neuropeptides(2014) doi 101016jnpep201411002

8 A Sajadian et alNeuropeptides (2014) ndash

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Kim D Kwon YK Cho KH 2008 The biphasic behavior of incoherent feed-forwardloops in biomolecular regulatory networks Bioessays 30 1204ndash1211

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Singh AT Keelan JA Sieg F 2010 Regulation of trophoblast migration and survivalby a novel neural regeneration peptide Reprod Biomed Online 21 237ndash244

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Vezzani A Ruumlegg S 2011 The pivotal role of immunity and inflammatory processesin epilepsy is increasingly recognized introduction Epilepsia 52 S1ndashS4

Vezzani A Aronica E Mazarati A Pittman QJ 2013 Epilepsy and braininflammation Exp Neurol 244 11ndash21

Walsh LA Li M Zhao TJ Chiu TH Rosenberg HC 1999 Acute pentylenetetrazolinjection reduces rat GABAA receptor mRNA levels and GABA stimulation ofbenzodiazepine binding with No effect on benzodiazepine binding site densityJ Pharmacol Exp Ther 289 1626ndash1633

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ARTICLE IN PRESS

Please cite this article in press as Azadeh Sajadian et al Anticonvulsant effect of neural regeneration peptide 2945 on pentylenetetrazol-induced seizures in rats Neuropeptides(2014) doi 101016jnpep201411002

9A Sajadian et alNeuropeptides (2014) ndash