pokusne životinje u znanstvenim istraživanjima ... · drugi simpozij hrvatskog društva za...

KNJIGA SAŽETAKA / BOOK OF ABSTRACTS

Drugi simpozijHrvatskog društva za znanost o laboratorijskim životinjamas međunarodnim sudjelovanjem

Pokusne životinjeu znanstvenimistraživanjimaZagreb, 10. listopad, 2014.

2nd Symposiumof Croatian Laboratory Animal Science Associationwith international participation

Experimental animalsin scientificresearchZagreb, October 10, 2014.

Drugi simpozij Hrvatskog društva za znanost o laboratorijskim životinjama s međunarodnim sudjelovanjem

“Pokusne životinje u znanstvenim istraživanjima”

Zagreb, 10. listopad, 2014.

2nd Symposium of Croatian Laboratory Animal Science Association with international participation

“Experimental animals in scientific research”

Zagreb, October 10, 2014.

Knjiga sažetaKa / BooK of aBstracts

SPONZORI / SPONSORS:

Biološki odsjek Prirodoslovno-matematičkog fakulteta Sveučilišta u Zagrebu

Department of Biology, Faculty of Science, University of Zagreb

Gradski ured za zdravstvo Grada Zagreba City Office for Health, The city of Zagreb

ORGANIZATORI / ORGANISERS

Hrvatsko društvo za znanost o laboratorijskim životinjama (CroLASA)

Croatian Laboratory Animal Science Association (CroLASA)

Veterinarski fakultet Sveučilišta u Zagrebu Faculty of Veterinary Medicine, University of Zagreb

Biološki odsjek Prirodoslovno-matematičkog fakulteta Sveučilišta u Zagrebu

Department of Biology, Faculty of Science, University of Zagreb

Medicinski fakultet Sveučilišta u Zagrebu School of Medicine, University of Zagreb

Institut Ruđer Bošković, Zagreb Rudjer Boskovic Institute, Zagreb

POKROVITELJSTVO / AUSPICE

Grad Zagreb/ The city of Zagreb

Genetically altered models-transgenic animals

Animalni modeli bolesti • Animal models of human disease

Patološka, patohistološka i toksikološka istraživanja u • animalnim modelima Pathology, histopathology and toxicology in animal models

Ponašanje životinja, dobrobit životinja i alternativne metode • Animal behavior, animal welfare and alternative methods

Okrugli stol-Direktiva EU 2010/63-kritički osvrt • Round Table-Directive EU 2010/63-critical review

REGISTRACIJA / REGISTRATION

Registracija putem web stranice simpozija www.hdzlz.hr i za vrijeme trajanja kongresa ispred dvorane u prizemlju glavne zgrade Veterinarskog fakulteta Sveučilišta u Zagrebu, Heinzelova ulica 55, 10000 Zagreb Registration via symposium website www.hdzlz.hr and during the symposium in the front of the hall in the ground floor of the main building of the Faculty of Veterinary Medicine, University of Zagreb, Heinzelova ulica 55, 10000 Zagreb, Croatia

ORGANIZACIJSKI ODBOR / ORGANIZING COMMITTEE

Jadranka Bubić Špoljar, Julija Erhardt, Maja Lang Balija, Blanka Smolić, Dubravka Švob Štrac

ZNANSTVENI ODBOR / SCIENTIFIC COMMITTEE

Tomislav Dobranić, Željko Grabarević, Mirko Hadžija, Marija Heffer, Nataša Jovanov-Milošević, Marijan Klarica, Nada Oršolić, Bojan Polić, Ranko Stojković

KONTAKT / CONTACT

putem web stranice simpozija www.hdzlz.hr ili e-maila: [email protected] via website www.hdzlz.hr or e-mail: [email protected]

OPĆE INFORMACIJE / GENERAL INFORMATION

MJEsTO OdRžAvANJA sIMPOzIJA / syMPOsIuM vENuE

Veterinarski fakultet Sveučilišta u Zagrebu/ Faculty of Veterinary Medicine, University of Zagreb

Heinzelova ulica 55, 10000 Zagreb, Croatia

GLAvNE TEME sIMPOzIJA / syMPOsIuM kEy TOPICsGenetski izmijenjeni modeli-transgenične životinje •

sLužBENI JEzICI sIMPOzIJA / syMPOsIuM OFFICIAL LANGUAGES

Hrvatski i engleski jezik / Croatian and English

POSTERI / POSTERS

Posteri se donose osobno i postavljaju između 8.00 i 9.00 sati na dan Simpozija. Materijal za postavljanje postera biti će osiguran u organizaciji Simpozija. Od autora se očekuje prisutnost uz poster tijekom vremena predviđenog za izlaganje postera i raspravu. Stručno Povjerenstvo dodijelit će nagrade najboljim posterima na svečanoj dodijeli nagrada prije zatvaranja Simpozija.

Posters should be brought in person and mounted between 8:00 and 9:00 a.m. on the day of the Symposium. Material needed for putting up the posters will be provided in the organization of the Symposium. The authors are expected to be present by their posters during the poster presentation and discussion period. Awards for the best posters selected by the Expert Committee will be presented at the awards ceremony before the closing of the Symposium.

VREDNOVANJE I POTVRDA O SUDJELOVANJU / CERTIFICATE OF ATTENDANCE

Hrvatska veterinarska komora će doktorima veterinarske medicine sudionicima navedenog stručnog skupa, vrednovati stručno usavršavanje sa 2 boda. Povjerenstvo za medicinsku izobrazbu liječnika Hrvatske Liječničke komore kategoriziralo je i vrednovalo stručni skup sa 15 bodova za aktivno

KOTIZACIJA / REGISTRATION FEE

KOTIZACIJA/ REGISTRATION FEE

RANA REGISTRACIJA/ EARLy REGISTRATION(do 15. srpnja 2014/ until July 15th 2014)

KASNA REGISTRACIJA/ LATE REGISTRATION(od 15. srpnja 2014/ from July 15th 2014)

Članovi CroLASA-e CroLASA members

150 kuna 200 kuna

Ne-članovi Non-members

200 kuna 250 kuna

Studenti Students

50 kuna 100 kuna

kOTIzACIJA ukLJuČuJE / REGIsTRATION FEE INCLudEsAktivno/pasivno sudjelovanje u aktivnostima i potvrdnicu o • sudjelovanju (na zahtjev) Attendance to all scientific sessions and “Certificate of attendance” (on request)

Kongresni materijal (identifikacijska značka, knjiga sažetaka • i program kongresa) Official symposium documentation (Congress badge, Book of abstracts, Programme)

Domjenak • Lunch

Osvježenja u stankama programa • Coffee and Refreshments

Dinko Mitrečić Medicinski fakultet Sveučilišta u Zagrebu/ School of Medicine, University of Zagreb

Bojan Polić Medicinski fakultet Sveučilišta u Rijeci/ School of Medicine, University of Rijeka

Melita Šalković – Petrišić Medicinski fakultet Sveučilišta u Zagrebu/ School of Medicine, University of Zagreb

Maja Šrut Prirodoslovno-matematički fakultet Sveučilišta u Zagrebu/ Faculty of Science, University of Zagreb

Guglielmo Vismara Tecniplast, Italy

Anna Wilkinson University of Lincoln, UK

sudjelovanje i 10 bodova za pasivno sudjelovanje.

Potvrda o sudjelovanju na Simpoziju izdati će se sudionicima na zahtjev.

Certificate of Attendance will be issued to participants on request.

POzvANI PREdAvAČI / INvITEd sPEAkERs

Lidija Bach-Rojecky Farmaceutsko-biokemijski fakultet Sveučilišta u Zagrebu/ Faculty of Pharmacy and Biochemistry, University of Zagreb

Tihomir Balog Institut Ruđer Bošković, Zagreb/ Ruđer Bošković Institute, Zagreb

Željko Grabarević Veterinarski fakultet Sveučilišta u Zagrebu/ Faculty of Veterinary Medicine, University of Zagreb

Andrea Gudan Kurilj Veterinarski fakultet Sveučilišta u Zagrebu/ Faculty of Veterinary Medicine, University of Zagreb

Dubravka Hranilović Prirodoslovno-matematički fakultet Sveučilišta u Zagrebu/ Faculty of Science, University of Zagreb

PREdvIĐENO VRIJEME / TIME

TEMA / TOPIC PREdAvAČ / LECTURER

8:00 - 9:00 REGISTRACIJA I POSTAVLJANJE POSTERA Registration and Mounting of Posters

9:00 - 9:30 POZDRAVNA RIJEČ I OTVARANJE OPENING CEREMONY

J. Bubić Špoljar

seKcija animalni modeli section animal models9:30 - 10:00 Proizvodnja i upotreba genetski

modificiranih miševa u eksperimentalnoj medicini Production and use of genetically modified mice in experimental medicine

B. Polić

10:00 - 10:15 Eksperimentalni model sporadične Alzheimerove bolesti The experimental model of sporadic Alzheimer’s disease

M. Šalković – Petrišić

10:15 - 10:30 Uloga hiperserotoninemije u neurorazvojnim poremećajima:istaživanja na štakoru s perinatalno poremećenim metabolizmom serotonina The role of hyperserotonemia in neurodevelopmental disorders: studies on rats with perinatally altered serotonin metabolism

D. Hranilović

10:30 - 10:45 Mišji model za moždani udar u regenerativnoj neuroznanosti Mouse model for stroke in regenerative neuroscience

D. Mitrečić

PROGRAM/ PROGRAMME 10:45 - 11:00 RASPRAVA DISCUSSION

11:00 - 11:30 PAUZA ZA KAVU COFFEE BREAK

seKcija PatološKa, PatohistološKa i toKsiKološKa istraživanja u animalnim modelimasection Pathology, histoPathology and toxicology in animal models11:30 - 12:00 Komparativna onkologija -

sličnosti i razlike najčešćih tumora ljudi i pasa Comparative oncology – similarities and differences between most common human and canine tumors

Ž. Grabarević

12:00 - 12:15 Mikro PET kamera za male životinje, utjecaj hiperoksije na distribuciju F18-FDG u mozgu miševa Micro PET camera in animal research, different F18-FDG uptake in mouse brain after normobaric hyperoxia treatment

T. Balog

12:15 - 12:30 Najčešća patološka stanja u laboratorijskih miševa, štakora i kunića The most common pathologic conditions in laboratory mices, rats and rabbits

A. Gudan Kurilj

12:30 - 12:45 Zebrice (Danio rerio) kao modelni organizmi – genomske promjene kao posljedica genotoksičnog djelovanja Zebrafish (Danio rerio) as a model organism - genome changes as a consequence of genotoxicity

M. Šrut

12:45 - 13:00 RASPRAVA DISCUSSION

15“exPerimental animals in scientific research”

13:00 - 14:00 RUČAK LUNCH

14:00 - 15:30 POSTER SEKCIJA POSTER SECTION

seKcija Ponašanje životinja, doBroBit životinja i alternativne metode section animal Behavior, animal welfare and alternative methods15:30 - 16:00 Cold – Blooded Cognition: The

Missing ClassA. Wilkinson

16:00 - 16:15 Eksperimentalni in vivo modeli u istraživanju boli – postoje li alternative? Eksperimental in vivo models in the study of pain – are there any alternatives?

L. Bach Rojecky

16:15 - 16:45 Development of Configuration and standards of the Laboratory Animal Equipment

G. Vismara

16:45 - 17:00 RASPRAVA DISCUSSION

17:00 - 17:15 PAUZA ZA KAVU COFFEE BREAK

17:15 - 19:30 OKRUGLI STOL Direktiva EU 2010/63 – kritički osvrt ROUND TABLE Directive EU 2010/63 – critical review

19:30 - 20:00 ZATVARANJE SIMPOZIJA Podjela nagrada najboljim posterima CLOSENING CEREMONY Prize giving ceremony for the best posters

POPIS POSTERA / LIST OF POSTERS

Broj Postera Poster numBer

a seKcija - animalni modelia section – animal models

01. Ivo Dumić-Čule, Jadranka Bubić-Špoljar, Lovorka Grgurević; Slobodan Vukičević

NOVEL RAT MODEL FOR OSTEOPOROSIS RESEARCH

02. Vedrana Ivić, Marta Balog, Senka Blažetić, Irena Labak, Marija Heffer

ACUTE AND CHRONIC STRESS MODEL IN SPRAGUE-DAWLEY RATS

03. Maja Kesić, Gordana Mokrović, Lipa Čičin-Šain

BEHAVIORAL RESPONSE IN WISTAR-ZAGREB 5HT MODEL OF RATS WITH CONSTITUTIONALLY ALTERED SEROTONIN TRANSPORTER

04. Marijan Klarica, Miroslav Vukić, Milan Radoš , Ivana Jurjević , Gorislav Erceg, Darko Orešković

DEVELOPMENT OF NEW MODELS ON LARGE EXPERIMENTAL ANIMALS AND ITS INFLUENCE ON CRANIO-SPINAL HYDRODYNAMICS RESEARCH

05. Maja Lazarus, Tatjana Orct, Jasna Jurasović, Maja Blanuša

SUCKLING RATS AS EXPERIMENTAL MODEL FOR TOXIC METAL EXPOSURE AND SELENIUM SUPPLEMENTATION IN THE EARLY PERIOD OF LIFE

06. Andrija Lončar, Ana Knezović, Melita Šalković-Petrišić

ACUTE CHANGES OF ACETHYLCHOLINESTERASE ACTIVITY AND TAU PROTEIN PHOSPHORYLATION IN MOUSE MODEL OF SPORADIC ALZHEIMER’S DISEASE

16 “PoKusne životinje u znanstvenim istraživanjima” 17“exPerimental animals in scientific research”

09. Marina Tišljar, Nataša Jovanov Milošević, Željko Grabarević, Marija Mišić, Vladimir Savić, Branka Artuković, Petar Džaja, Tajana Amšel Zelenika, Sven Seiwerth, Krešimir Severin, Susan Semple-Rowland

POULTRY AND OTHER BIRDS SUFFICIENTLY REPRESENTED AS ANIMAL MODELS IN BIOMEDICAL RESERACH OF SOME HUMAN DISEASES IN CROATIA?

10. Josipa Vlainić, Dubravka Švob Štrac, Maja Jazvinšćak Jembrek

THE ASSESMENT OF ANTICONVULSANT PROPERTIES OF THE DRUG – PENTYLENETETRAZOLE SEIZURE MODEL

Broj PosteraPoster numBer

B seKcija - PatološKa, PatohistološKa i toKsiKološKa istraživanja u animalnim modelimaB section - Pathology, histoPathology and toxicology in animal models

11. Davorka Breljak, Vedran Micek, Ivana Vrhovac, Dean Karaica, Giuliano Ciarimboli, Herman Koepsell, Ivan Sabolic

CELLULAR LOCALIZATION AND SEX-RELATED PROTEIN EXPRESSION OF ORGANIC CATION TRANSPORTERS OCT1 AND OCT2 IN MOUSE KIDNEYS AND LIVER

12. Dean Karaica, Davorka Breljak, Jovica Lončar, Ivan Mihaljević, Marija Ljubojević, Carol M. Herak-Kramberger,Vedran Micek, Ivana Vrhovac, Jana Ivković, Birgitta Burckhardt, Gerhard Burckhardt, Tvrtko Smital, Ivan Sabolić

IMMUNOLOCALIZATION OF CHLORIDE-FORMATE EXCHANGER (Slc26a6) IN VARIOUS RAT TISSUES; SEX-DEPENDENT EXPRESSION IN KIDNEYS

13. Marina Korolija, Mirko Hadžija, Marijana Popović Hadžija

NEURAL TUBE DEFECTS IN DIABETIC EMBRYOPATHY

06-A Vedrana Mužić, Gordana Jurić-Lekić, Marta Himelreich, Željka Majić, Nino Sinčić; Ana Katušić, Maja Vlahović, Ljiljana Šerman, Jelena Lončarević, Floriana Bulić-Jakuš

demethylating agent 5-azacytidine negatively affects Proliferation of mammalian limB Bud cells in an organ-culture system

07. Nada Oršolić, Eleonora Goluža, Domagoj Đikić, Duje Lisičić, Željko Jeleč, Maja Vihnanek Lazarus, Tatjana Orct

ROLE OF FLAVONOIDS ON OXIDATIVE STRESS AND MINERAL CONTENTS IN THE RETINOIC ACID-INDUCED BONE LOSS MODEL OF RAT

07-A Nino Sinčić, Anastas Gospodinov, Maja Vlahović Maja, Gordana Jurić-Lekić, Floriana Bulić-Jakuš

IMPACT OF RNA INTERFERENCE WITH STEMNESS GENE EXPRESSION IN EXPERIMENTAL MOUSE TERATOMA GROWN IN VITRO

07-B Nino Sinčić, Maja Vlahović, Zdenko Herceg, Frane Paić, Ljiljana Šerman, Ana Katušić, Floriana Bulić Jakuš

Impact of 5-azacytidine on Oct4 and Nanog DNA methylation/expression in experimental mouse teratocarcinoma

08. Una Smailović, Ana Knezović, Silvia Mandel, Moussa Youdim, Melita Šalković-Petrišić

THERAPEUTIC POTENTIAL OF MULTIFUNCTIONAL IRON-CHELATING AGENT M30 IN A RAT MODEL OF SPORADIC ALZHEIMER’S DISEASE

08-A Nikola Sobočan, Nino Sinčić, Željka Majić, Ana Katušić, Maja Vlahović, Ljiljana Šerman, Robert Beuc, Gordana Jurić-Lekić, Floriana Bulić-Jakuš

N-TERT-BUTYL-Α-PHENYLNITRONE IMPROVES EX VIVO GROWTH OF THE RAT EMBRYO AT THE AIR-LIQUID INTERFACE IN A CHEMICALLY DEFINED MEDIUM


Broj PosteraPoster numBer

c seKcija - Ponašanje životinja, doBroBit životinja i alternativne metodec section - animal Behavior, animal welfare and alternative methods

21. Vladimir Farkaš, Robert Bagarić, Ivo Dumić-Čule, Alfred Švarc

RAT CARDIAC IMAGING - COREGISTERED 18FDG-PET AND GOLD NANOPARTICLES CT

22. Vladimir Farkaš, Robert Bagarić, Alfred Švarc

LABORATORY ANIMALS PET IMAGING THROUGH THE EYES OF A 3R’S PRINCIPLE

23. Maja Lang Balija, Maja Šantak, Maja Markušić, Dubravko Forčić

THE EVALUATION OF NEUROVIRULENCE OF MUMPS VIRUS STRAINS WITH ALTERNATIVE NEWBORN RAT-BASED SAFETY TEST

24. Maja Lang Balija, Tihana Kurtović, Marija Brgles, Beata Halassy

THE ROAD TOWARDS IN VITRO ALTERNATIVE TO IN VIVO SNAKE VENOM TOXICITY AND ANTIVENOM POTENCY ASSAYS

25. Željka Lončarić, Branimir K. Hackenberger

OPTIMIZATION OF THE NUMBER OF EXPERIMENTAL ANIMALS USING NEW COMPUTING TECHNIQUES

26. Darko Modun, Lidija Bach-Rojecky

FREE TEACHING RESOURCE: E-HANDBOOK TO ACCOMPANY MICROLABS FOR PHARMACOLOGISTS

27. Janko Samardžić, Dubravka Švob Štrac, Miloš Đurić, Dragan I Obradović

BICUCCULINE ANTAGONIZES THE EFFECTS OF DEHYDRO- EPIANDROSTERONE ON RAT BEHAVIOR

14. Anja Mikolić, Antonija Sulimanec Grgec, Martina Piasek

ORAL CADMIUM EXSPOSURE DURING PREGNANCY: ASSESSMENT OF MICROELEMENT DISTRIBUTION IN MOTHER RAT AND FOETUS AT TERM

15. Nada Oršolić, Nikola Car, Duje Lisičić, Vesna Benković, Anica Horvat Knežević, Domagoj Đikić, József Petrik

SYNERGISM BETWEEN PROPOLIS AND HYPERTHERMAL INTRAPERITONEAL CHEMOTHERAPY WITH CISPLATIN ON EHRLICH ASCITES TUMOR IN MICE

16. Nada Oršolić, Jadranka Skurić, Domagoj Đikić, Gabrijela Stanić, Darko Kolarić

INHIBITORY EFFECT OF A PROPOLIS ON DI-N-PROPYL DISULFIDE OR N-HEXYL SALICILATE-INDUCED SKIN IRRITATION, OXIDATIVE STRESS AND INFLAMMATORY RESPONSES IN MICE

17. Damir Sirovina, Nada Oršolić, Marijana Zovko Končić, Goran Kovačević, Vesna Benković, Gordana Gregorović

QUERCETIN VS CHRYSIN: EFFECT ON LIVER HISTOPATHOLOGY IN DIABETIC MICE

18. Damir Sirovina, Nada Oršolić, Ivan Ivić, Sanja Novak, Goran Gajski, Vera Garaj-Vrhovac, Marijana Zovko Končić

ANTIOXIDATIVE AND ANTIDIABETIC EFFECTS OF NARINGIN AND CURCUMIN IN VITRO AND IN VIVO

19. Sandra Sobočanec, Ana Šarić, Željka Mačak Šafranko, Marijana Popović Hadžija, Gorana Aralica, Marina Korolija, Borka Kušić, Tihomir Balog

THE ROLE OF ESTROGEN IN HYPEROXIA-INDUCED OXIDATIVE STRESS IN LIVER OF CBA/H MICE

20. Ivana Vrhovac, Davorka Breljak, Dean Karaica, Hermann Koepsell, Ivan Sabolic

EXPRESSION OF SODIUM-D-GLUCOSE COTRANSPORTER 1 IN MURINE TISSUES; SEX-DEPENDENT EXPRESSION IN KIDNEYS


28. Daša Ševeljevic-Jaran

ESLAV AWARNESS PRESENTATION29. Šlipogor Vedrana, Millesi, Eva; Bugnyar Thomas

SALIVARY CORTISOL AS INDICATOR OF PERSONALITY TYPES IN COMMON MARMOSETS?

30. Dubravka Švob Štrac, Josipa Vlainić Josipa, Željka Krsnik, Janko Samardžić, Julija Erhardt

EFFECTS OF CHRONIC DHEAS ADMINISTRATION ON SEIZURE THRESHOLD, LOCOMOTOR ACTIVITY, MOTOR COORDINATION AND BODY WEIGHT IN MALE AND FEMALE MICE

sAžECI / ABsTRACTs


PREDAVANJA / LECTURES


EXPERIMENTAL IN vIvO MOdELs IN THE sTudy OF PAIN – ARE THERE ANy ALTERNATIvEs?

Bach-rojecky lidija1; drinovac višnja1

1Department of Pharmacology, University of Zagreb School of Pharmacy and Biochemistry, Zagreb, Croatia

E-mail: [email protected]

Animals have been employed in pain research to explore basic physiological mechanisms of pain and to predict analgesic efficacy leading to clinical drug development. By far, most research on pain and nociception has been performed in small rodents. Based on aetiology, pain assays can be described as: acute, inflammatory and neuropathic. Acute assays involve applying a noxious stimulus (thermal, mechanical, electrical and chemical) to a convenient rodent body part (hind paw, tail, muscle, abdomen), which causes nocifensive withdrawal or other simple behaviours that can be easily scored. For studying inflammatory pain, longer-lasting inflammatory assays (lasting from hours to days) which include injection of various algogenic substances, such as formalin, capsaicin, carrageenan and immune system-activating substances, were developed. Long-lasting models of peripheral neuropathic pain commonly include partial damage to a specific nerve (constriction, partial transection, freezing), while polyneuropathy can be induced by certain injection protocols with toxic substances/drugs; e.g. streptozotocin to induce diabetic neuropathy, paclitaxel and vincristine to induce chemotherapeutic-induced neuropathy, antiretrovirals to induce AIDS therapy-induced neuropathy, cyclophosphamide to induce cystitis. The rodent behaviours typically measured are spinal reflexes (withdrawal), spino-bulbal reflexes (jumping)

or simple behaviours (vocalization, licking, guarding, biting). However, the majority of these evoked withdrawal responses measure hyperalgesia and allodynia, not spontaneous pain itself. A drawback of current rodent model systems is that they are labour intensive, very expensive and time-consuming and, due to ethical issues, only small groups of animals can be used. Recently the first papers have been published using adult as well as larvae zebrafish as a model to study nociception. Fish possess the physiological and neuro-anatomical structures required for nociceptive responses similar to mammals. Most studies in fish use injection of diluted acetic acid as noxious stimulus and the resulting behaviours are fully described. The range of genetic and behavioural tools available and the possibility to do large-scale screens with larvae model will help to understand the mechanisms involved in nociception and will hopefully lead to the discovery of new analgesics, which can then be further validated using higher vertebrate models. However, in the meantime, despite ethical considerations, limitations, cost and limited success in predicting analgesic efficacy and side-effects of novel therapeutics, rodent models of pain continue to be extensively used as an important tool in advancing our understanding of pain and searching for novel analgesics.

Keywords: pain research; animal models; rodents; zebrafish


DIFFERENT F18-FDG UPTAKE IN MOUSE BRAIN AFTER NORMOBARIC HyPEROXIA TREATMENT

šarić ana1; sobočanec sandra1; mačak šafranko željka1; Bagarić robert2; farkaš vladimir2, švarc alfred2; Balog tihomir1

1Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia2Division of Experimental Physics, Ruđer Bošković Institute, Zagreb, Croatia


The free radical theory of aging first described by Denham Harman strongly correlate aging with level of macromolecules damage caused by reactive oxygen species (ROS). The model of oxidative stress induced by hyperoxia using pure oxygen (92-95%) is well known. Reactive oxygen species (ROS), superoxide anion and hydrogen peroxide can be produced from inhaled oxygen in mitochondria and cell cytoplasm. The response to oxidative stress is sex-related, with female animals being more resistant to oxidative damage than males. In addition females have lower levels of ROS in cell, higher activity of antioxidant enzymes and higher mitochondrial respiratory chain capacity, which results with lower oxidative damage of macromolecules. The inverse relationship between glucose consumption and oxidative stress in brain tissue was already described by C. Borras et al. In addition there is little or no data of different response to hyperoxia between sex and age in relation to glucose consumption. This work will described the effect of hyperoxia on brain glucose consumption as a potential parameter of oxidative stress. Our previous data already shown that after hyperoxia exposure female mice have higher survival rate in

comparison to males. Ovariectomy diminished female resistance to hyperoxia oxidative damage, while estrogen implants in males increased their resistance to hyperoxia. Results of F18 FDG uptake demonstrated that older female mice have lower glucose consumption in the back brain in relation to young females. The same is not observed with males. In addition the effect of hyperoxia with pure oxygen (92-95%) on glucose consumption in beck brain is gender related with lower glucose consumption in female in relation to male animals. This data will additionally explain differences between age and sex in response to hyperoxia using brain glucose consumption status as marker of oxidative stress after hyperoxia treatment.

Keywords: oxidative stress; hyperoxia; sex and age differences; F18-FDG


COMPARATIvE ONCOLOGy – sIMILARITIEs ANd DIFFERENCES OF THE MOST FREQUENT TUMOURS IN MEN AND DOGS

grabarević željko1

1Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia


There are two well known facts in comparative pathology considering tumours in men and animals. First one is that animals develop tumours in various organs and tissues which are pathohistologically very similar or even identical to its human counterparts, but their biological behaviour or malignancy grade is very different. Second one is that predominant trend in pathology is finding spontaneous animal tumours which are the most similar to the same tumours in men (histologically, etiologically and biologically) which could be used in preclinical tumour investigations. Although these investigations are very necessary and they were proven to be very helpful in experimental oncology of the human tumours, the first fact is unfairly neglected. The knowledge of the reasons for different tumour grades of the spontaneous tumours in animals could significantly contribute to the targeted tumour therapy of tumours in men and animals as well. There are numerous examples for this. Testicular tumours in dogs with almost identical histology as in men are biologically benign, while in males they are predominantly malignant. In humans hepatocellular carcinomas affects severely damaged liver (hepatitis, cirrhosis, etc.) while in dogs they appear in unchanged liver tissue. Mammary tumours in bitches are much more frequent than breast tumours in women, while in cats

they are less frequent but almost in all cases malignant. Canine lymphomas are equally frequent as in humans, but they are in the group of non-Hodgkin lymphomas, because Hodgkin tumours in dogs are extremely rare. This work presents comparative tumour analysis and the results of the investigations conducted at the Department of Veterinary Pathology of the Veterinary Faculty Zagreb.

Keywords: comparative tumour analysis; HCC; testicular tumours; mammary carcinoma; lymphoma; men; dogs


THE MOST COMMON PATHOLOGIC CONDITIONS IN LABORATORy MICEs, RATs ANd RABBITs

gudan Kurilj andrea1

1Department of Veterinary Pathology, Faculty of Veterinary Medicine University of Zagreb, Zagreb, Croatia


Despite nowadays alternative methods, laboratory rabbits, rats and mice in particular, are an important part of biomedical research. Since these laboratory animals are kept in a “clean” facilities and are regularly monitored in quarantine and health controls, the infectious agents are not as likely to cause substantial morbidity or mortality that raises concern and leads to further investigation. However, as there are multiple strains of laboratory animals, some strains are more susceptible to infections, and also it should bear in mind the possibility of inapparent infections that can modulate the immune system and may interfere with diverse research areas. Therefore, here will be presented the most frequent spontaneous infectious diseases that primarily affect the enterohepatic, respiratory, immune system and skin or are multisystemic. Besides, a very important part of the health status of laboratory animals are noninfectious diseases and conditions whose causes are dependent on the sex and age of animals, genetics and immune status, microbial status, diet and environmental factors. According to this, here will be summarized the most common noninfectious conditions that affect skin, heart diseases, amyloidosis, nephropathy and frequent noninfectious conditions in the nervous and reproductive system as well as the most common tumors.

Keywords: diseases; infectious; noninfectious; mouse; rat; rabbit


THE ROLE OF HyPERsEROTONEMIA IN NEURODEVELOPMENTAL DISORDERS: STUDIES ON RATs WITH PERINATALLy ALTEREd sEROTONIN METABOLISM

hranilović dubravka1

1Department of Animal Physiology, Division of Biology, Faculty of Science, University of Zagreb, Croatia


In mammals, serotonin (5HT) is present both in the brain (central 5HT compartment) and peripheral tissues (peripheral 5HT compartment). In the developing brain, serotonin acts as a key regulator of serotonergic outgrowth and synaptogenesis. Therefore, perinatally altered 5HT homeostasis could lead to deviations from optimal 5HT concentrations, affecting so the development of the brain serotonergic system and later leading to the related behavioral deficits. Although central 5HT disturbances are strongly indicated in several neurodevelopmental disorders, the role of peripheral 5HT dysregulation, and its relationship to the central 5HT malfunctioning are less clear. In order to study the consequences of perinatal disturbances in serotonin metabolism, we exposed developing Wistar rats to treatments with the immediate 5HT precursor 5-hydroxytryptophan (5HTP, 25mg/kg), or the monoamine oxidase (MAO) inhibitor tranylcypromine (TCP, 2mg/kg) from gestational day 12 until postnatal day 21. Treatment with 5HTP significantly raised peripheral but not central 5HT concentrations, while treatment with TCP induced significant 5HT elevations in both compartments, allowing us to compare the extent of 5HT-related disturbances induced by alteration in only peripheral vs. peripheral and central 5HT

homeostasis. Developmental consequences were observed in both groups of animals (compared to the saline-treated controls, pups had decreased viability, significantly lower body mass throughout the entire postnatal period, altered formation of the barrel cortex and increased separation anxiety) but were more prominent in TCP-treated rats. In 5HTP-treated rats, peripheral 5HT homeostasis was re-established while modest decrease in 5HT concentration in frontal cortex remained at adult age. In TCP-treated rats, imbalances in 5HT homeostasis remained at adult age, both peripherally (as hyperserotonemia) and centrally (as altered 5HT metabolism with markedly decreased 5HT concentrations). The obtained results indicate that exposure of developing brain to the increased 5HT concentrations may lead to structural and behavioral abnormalities indicative of developmental delay. Developmental disturbances induced by perinatal alterations in both 5HT compartments were much more pronounced than those induced by perinatal alteration only in the peripheral 5HT-compartment indicating that hyperserotonemia alone might not be sufficient to cause 5HT-related disturbances in neurodevelopmental syndromes. Still, transient fetal/neonatal (along with maternal) hyperserotonemia in 5HTP-treated animals was sufficient to induce measurable 5HT-related changes, suggesting a caution in the use of this 5HT enhancer by pregnant women. Finally, permanent alterations in the central 5HT homeostasis induced by developmental disturbances in 5HT synthesis or degradation suggest that genes which regulate 5HT metabolism might be considered as potential candidates in 5HT-related behavioral disorders.

Keywords: serotonin; hyperserotonemia; neurodevelopmental disorders; 5-hydroxytryptophan; tranylcypromine


MOUSE MODEL FOR STROKE IN REGENERATIVE NEUROSCIENCE

mitrečić dinko1

1Laboratory for Stem Cells, Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Zagreb, Croatia


Ischemic brain injury is the most common form of stroke and represents one of the most significant medical problems of human society. Costs of medical care of patients suffering from stroke in European Union reached 800 billion euros per year, which is, for comparison, three times larger than treatment costs of all malignant diseases together. The high incidence of stroke and a regular occurrence of long or life-long impairment and absence of any specific therapy urge for a constant quest for new approaches in the treatment of stroke. The most widely accepted mouse stroke model is induced by the occlusion of the medial cerebral artery (MCAO): while the mouse is under inhalation anesthesia, a thin filament is inserted into the internal carotid artery and subsequently to the medial cerebral artery which is occluded for 60 minutes. This results in an ischemic lesion of the striatum and part of the cortex. In our Laboratory we additionally improved this method by application of Doppler detector which measures blood flow in temporarily occluded region. In this way we achieve the maximal level of comparability among all operated animals. To estimate the expected stroke damage, every operated mouse is checked for signs of neurologic deficits: unilateral weakness of limbs and asymmetrical walk. Subsequent CT scans and histological analyses allow to precisely measure volume of the tissue affected by stroke. The main application of mouse stroke

model in our Laboratory is analyses of potential therapeutic effects of transplantation of stem cells. One day after operation we transplant neural stem cells using stereotaxic instrument in various brain regions: cortex, corticostriatal border and/or hippocampus. Bioluminescence and immunohistochemistry are then applied to trace migration of cells, their differentiation and survival in the periods up to three months. This mouse stroke model allowed to detect the best routes for stem cell delivery, rates of differentiation and survival of stem cells and subsequent acceleration of tissue regeneration. Obtained results are constantly translated into clinical trials, which makes this mouse model irreplaceable and invaluable tool for regenerative neuroscience.

Keywords: stroke; MCAO; mouse model; neural stem cells


PROduCTION ANd usE OF GENETICALLy MOdIFIEd MICE IN EXPERIMENTAL MEDICINE

Polić Bojan1

1Department of Histology & Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia


Interventions in the mouse genome have been extensively used for research of various gene functions in vivo and for modelling of human diseases. Implementation of the classical transgenic technology in the early 70thies, which comprises construction and microinjection of a transgene into the zygote pronucleus, gave huge impulse to the investigation of gene functions, usually in excess of gene expression. However, limitations of this technology, like random integration of the transgene, use of non-physiological promoters, ectopic and excess expression of transgene, became a bottleneck for a more refine research of gene functions. This was overridden in 80thies by introduction of the gene targeting technology which gave an opportunity to do very precise manipulation at desired site of the mouse genome. The mutations were introduced in the genetic locus of interest of mouse embryonic stem (ES) cells by homologous recombination of a targeting vector. Very precise mutations (to a single base) of the desired gene locus enabled production of mouse models for precise studying of gene functions and their impact on the physiological and pathophysiological processes in vivo. However, inactivation of developmentally essential genes by this technology leaded to the early embryonic death, which hampered investigation of particular gene functions in adult organism. Therefore, in 90thies conditional gene targeting

was developed which employed Cre/LoxP or Flp/FRT systems. It enabled inactivation of any gene of interest in a cell specific and time dependent manner, which gave an opportunity for precise definition of various gene functions in a very complex multicellular organism. Thus, conventional and conditional gene targeting became commonly used technologies for specific investigation of gene functions in vivo as well as for modelling of various human diseases.

Keywords: transgenic mice; gene targeting; targeting vectors; ES cells; blastocyst microinjection; embryo-transfer; mouse models


THE EXPERIMENTAL MODEL OF SPORADIC ALZHEIMER’S DISEASE

šalković-Petrišić melita1

1Department of Pharmacology, University of Zagreb School of Medicine, Zagreb, Croatia


Alzheimer’s disease (AD) is the most common progressive neurodegenerative disorder in the elderly which is responsible for 60-80% of all dementia cases. The vast majority of AD cases are of a sporadic origin (sAD), unknown cause and ineffective treatment which all indicate a need for in-depth research of sAD ethiopathogenesis and pathophysiology. Development of animal model which would mimic human sAD condition as much as possible seems to be the first step in this research since widely exploited transgenic mice AD models represent a rare, genetically caused familial AD form. Considering post-mortem findings of the insulin resistant brain state (IRBS) in sAD patients, induction of IRBS in animals by intracerebroventricular (icv) administration of streptozotocin (STZ, a compound selectively toxic to insulin producing/secreting and insulin receptor expressing cells), has been recognized as promising experimental approach. STZ-icv rat (as well as mouse and monkey) model shares a numerous behavioural (cognitive deficits), neurochemical (cholinergic deficit, IRBS, tau hyperphosphorylation, oxidative stress), metabolic (brain glucose hypometabolism) and structural (neuroinflammation, pathological amyloid accumulation) similarities with sAD patients. Further characterization and validation of STZ-icv animal model which is still in progress, will hopefully facilitate animal-to-human data translational

process and contribute to design of preclinical and clinical drug trials, finally leading to more effective disease modifying therapy of sAD.

Keywords: sporadic Alzheimer’s disease; animal model; streptozotocin; intracerebroventricular


ZEBRAFISH (DANIO RERIO) AS A MODEL ORGANISM - GENOME CHANGES AS A CONSEQUENCE OF GENOTOXICITy

šrut maja1, štambuk anamaria1, Bourdineaud jean-Paul2, Klobučar i.v.goran1

1Department of Zoology, Faculty of Science, University of Zagreb, Zagreb, Croatia2Arcachon Marine Station, CNRS, UMR EPOC 5805, University of Bordeaux, Arcachon, France


Zebrafish (Danio rerio) became a prominent model vertebrate in a variety of biological disciplines including genetics, development, pharmacology and cancerogenesis. The large amount of data collected from the developmental and genetic research, along with the sequenced genome makes zebrafish an attractive and powerful model also in toxicological research. Recently, zebrafish larvae have been described as particularly sensitive models in environmental toxicity testing which can replace the use of adult specimens in such studies and thus fit well into the 3R principles. However, there is a lack of data concerning the larval genotoxic responses in comparison to adult life stages. Therefore, to explore window of sensitivity of zebrafish larvae toward genotoxic stress we assessed primary DNA damage and existence of potentially persistent DNA alterations in the zebrafish genome using larval and adult zebrafish life stages upon exposure to model genotoxic agents. Genotoxicity assessed using comet assay served as an initial screening for evaluation of general impact on DNA integrity under genotoxic stress. Quantitative RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment

length polymorphism) were applied to test for genomic effects upon genotoxic stress. Furthermore, expression of a suite of genes involved in DNA repair, oxidative stress response and xenobiotic metabolism was monitored in order to get better overview of zebrafish genome responses to genotoxic stressors. Additionally, AFLP method was applied on adult specimens one year after their exposure to genotoxicants at the larval stage in order to further evaluate longevity of observed DNA alterations. Zebrafish larvae proved to be extremely sensitive zebrafish model for revealing genotoxic effects, which was evidenced by the high incidence of DNA alterations and the lack of significant DNA repair at the early stages of larval development. Some of the genomic alterations observed in larvae were still detectable in the adulthood, indicating the formation of persistent genomic modifications. These results thus underline zebrafish larvae as particularly sensitive model in genotoxicity testing and prove their applicability as a good replacement for the use of adult fish in such surveys.

Keywords: zebrafish larvae; genotoxicity model; 3R


DEVELOPMENT OF CONFIGURATION AND sTANdARds OF THE LABORATORy ANIMAL EQUIPMENT

vismara guglielmo1

1Tecniplast Spa, Italy


The history of caging rodents for biomedical research is a constant path of innovation. Since the first cage in plastic until the last model of Individually Ventilated Cage (IVC), able to offer a standardized and enriched environment as well as a efficient tool to handle by researchers. The development of housing devices must follow the international guidelines for Lab Animal Research, and provide tools that allow the users to apply for certifications like AAALAC. The quality of the air, the ventilation parameters and the electronic management of the sensors are capable to provide high animal welfare and microbiological conditions. The design of the housing systems drives to the accomplishment of the “3R” rule, since it is a key point for the standardizations of the experiments.

Keywords: IVC; SPF mice; housing; HEPA; standardization; 3R

COLD-BLOODED COGNITION: THE MISSING CLASS

wilkinson anna1

1School of Life Sciences, College of Science, University of Lincoln, Lincoln, United Kingdom


Very little is known about the cognitive abilities of reptiles. They have traditionally been considered to be “sluggish and unintelligent creatures” (Yerkes 1901, p 520) and have largely been ignored in the study of animal cognition. However, to gain an understanding of the evolution of cognition in amniotes, it is necessary to carry out direct experimental investigations of the learning and memory abilities of reptiles that parallel the extensive work already available in mammals and birds. Chelonia (turtles, tortoises and terrapins) are of particular interest because they are considered to be at the base of the archosorian branch. Therefore, examination of the cognitive mechanisms underlying the behaviour of these animals can provide crucial information about the evolution of the brain. This talk will present some recent research on the learning and memory abilities of reptiles, particularly tortoises, and compare them to what is known about these processes in other animals.

Keywords: reptiles; learning and memory; behaviour


POSTERI / POSTERS


CELLULAR LOCALIZATION AND SEX-RELATED PROTEIN EXPRESSION OF ORGANIC CATION TRANsPORTERs OCT1 ANd OCT2 IN MOusE kIdNEys AND LIVER

Breljak davorka1; micek vedran1; vrhovac ivana1; Karaica dean1; ciarimboli giuliano2; Koepsell herman3; sabolić ivan1

1Molecular Toxicology, Institute for Medical Research and Occupational Health, Zagreb, Croatia2Department of Internal Medicine D, Experimental Nephrology, University Hospital Münster, Münster, Germany3Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany


Organic cation transporters (OCT; family SLC22) mediate (re)absorption of various endogenous and exogenous organic cations. In rodents, Oct1/Slc22a1 and Oct2/Slc22a2 are expressed in major excretory organs including kidneys and liver. Due to absence of specific antibodies and corresponding knockout (KO) mice, cellular localization of these transporters and their sex-related protein expression in this species have not been characterized. Here we used adult wild-type (WT) and double Oct1/Oct2 KO mice of both sexes to study a) localization of Oct1 and Oct2 by immunocytochemistry in cryosections of the liver and kidneys, b) expression of these transporters by Western blotting of isolated total cell membranes (TCM), and c) sex-dependency in localization and expression of both transporters. Using specific antibodies, in WT mice the renal Oct1 and Oct2 were localized in the basolateral membrane (BLM) of proximal tubules (PT) exhibiting sex differences (males>females) in the

staining intensity. However, Oct1 was detected in the PT S1/S2 segments, whereas Oct2 was expressed in the PT S2/S3 segments. In the liver, Oct1 was stained in the sinusoidal membrane of hepatocytes, whereas Oct2 was undetected. In TCM of WT mice, the ~75-kDa Oct1 protein band was labeled in both organs, whereas the ~75-kDa Oct2 protein band was labeled exclusively in kidneys. Both transporters were undetected in Oct1/Oct2 KO mice. Collectively, in the mouse kidneys, the Oct1 and Oct2 proteins are located in the BLM of specific PT segments with male-dominant expression, whereas in the mouse liver, only the sex-independent Oct1 expression was found in the hepatocyte sinusoidal membrane.

Keywords: immunolocalization; knockout mice; organic cations; membrane transporters; sex differences; Western blotting


NOVEL RAT MODEL FOR OSTEOPOROSIS RESEARCH

dumić-Čule ivo1; Bubić-špoljar jadranka1; grgurević lovorka1; vukičević slobodan1

1Laboratory for mineralized tissues, Center for Translational and Clinical Research, Medical School, University of Zagreb, Croatia


Osteoporosis is one of the most common metabolic diseases of bones. The field of research on osteoporosis has grown in recent years and numerous potential drugs were tested. For better understanding of drug efficacy and molecular mechanism on bone metabolism there is a great need for establishing appropriate animal model without influence of hormones that affect bone remodeling. Experiments were carried out in male Sprague-Dawley rats, weighting approximately 350-380g. Removal of the thyroid and parathyroid glands (thyroparathyroidectomy - TPTx) was done surgically by a ventral approach under general anesthesia. TPTx resulted in a hypocalcaemia with significantly lower serum calcium levels as compared to sham operated control rats. TPTx further resulted in a decreased C-telopeptide and osteocalcin serum level as compared to intact rats. TPTx animals had decreased thyroid hormone and PTH serum level. 1,25-D(3) was at day 7 decreased about 10 times while TSH was increased 4 to 5 times due to the lack of T3/T4. The effect of TPTx on the trabecular bone was assessed by microCT and biomechanical tests, showing a significantly decreased bone volume and loss of biomechanical strength. We established TPTx rat model and developed conditions for testing the direct influence of several drugs on bone in the absence of calciotropic hormones. After one week rats had a significant trabecular bone

loss and they were ready for experimental research.

Keywords: osteoporosis; bone research; thyroparathyroidectomy


RAT CARDIAC IMAGING - COREGISTERED 18FDG-PET AND GOLD NANOPARTICLES CT

farkaš vladimir1; Bagarić robert1; dumić-Čule ivo2; švarc alfred1

1Division of Experimental Physics, Ruđer Bošković Institute, Zagreb, Croatia 2 Laboratory for mineralized tissues, Center for Translational and Clinical Research, Medical School, University of Zagreb, Croatia


Positron emission tomography (PET) is a method of nuclear medicine that allows to determine spatial and temporal distribution of radiolabelled molecules - radiopharmaceuticals. The most commonly used radiopharmaceutical in the PET imaging is a glucose analogue 2-fluoro-2-deoxy-glucose (18FDG). 18FGD-PET imaging enables visualization of glucose metabolism and it is considered as a functional imaging. On the other hand, computed tomography (CT) provides insight in tissue morphology and it represents typical anatomical imaging. Native CT is not a method of choice for displaying the rat heart anatomy, but CT with gold nanoparticles (GNP) as an contrast agent becomes very useful cardiac imaging method. When GNP is applied intravenously, because of the high absorption of x-rays and low interaction with the tissues, it becomes an excellent contrast agent for x-ray, including CT. The presence of GNP in the bloodstream provides a detailed display of the blood vessels with high contrast. The combination of functional and anatomical imaging methods is often used in research and diagnostics. The overlapping of images from different imaging methods with anatomical accuracy is called coregistration. Coregistered PET and CT images together provide much more

information than PET or CT separately. Applied in cardiology, 18FDG-PET displays the glucose uptake in the myocardium while GNP-CT provides a very detailed view of the blood vessels at the base of the heart and cardiac chambers, especially the ventricles. Successfully coregistered 18FDG-PET and GNP-CT images represent an excellent method in cardiology preclinical research.

Keywords: positron emission tomography; cardiac 18FDG-PET; rat; GNP contrast CT


LABORATORy ANIMALs PET IMAGING THROuGH THE EyEs OF A 3R’s PRINCIPLE

farkaš vladimir1; Bagarić robert1; švarc alfred1

1Division of Experimental Physics, Ruđer Bošković Institute, Zagreb, Croatia


Positron emission tomography (PET) has evolved rapidly through the last decade and it now enables excellent imaging possibilities in wide range of preclinical studies. In 1959, Russel and Burch have proposed the 3R’s concept (replacement, refinement and reduction) as a leading ethical principle in conducting animal experiments. At first not that important, but as global awareness of animal welfare grows, implementing of 3R’s principle in animal experiments becomes inevitable. PET represents the cutting-edge technology in medical imaging and when applied in experiments including laboratory animals it meets the 3R’s principle in many ways. In PET imaging, every animal can be scanned „before“ as a control animal, and „after“ as a experimental animal. Basically, it means that every animal can be a control for itself. This is crucial for reduction because in this case it is not necessary to have two groups of animals. The use of inhalation anesthesia, which also provides analgesia, becomes a standard protocol in PET imaging. That protocol enhances welfare of animals and affects refinement in a positive way. After experiment, PET images remain saved and they can be analyzed retrospectively. This retrospective studies can bring out a whole new experiment with new objectives that are not connected with the original experiment. For results obtained in that manner it can be said that no animals were used and count it

as the replacement method. The importance of implementing the 3R’s principle lies not just in improvement of the animal welfare but also in obtaining accurate results.

Keywords: positron emission tomography; laboratory animal welfare; 3R’s principle


ACUTE AND CHRONIC STRESS MODEL IN SPRAGUE-dAWLEy RATs

ivić vedrana1; Balog marta1; Blažetić senka2; labak irena2; heffer marija1

1University of Osijek, Faculty of Medicine, Osijek, Croatia2University of Osijek, Department of Biology, Osijek, Croatia


Various acute and chronic stressors challenge homeostasis maintenance. They cause different effects: acute stress causes “fight-or-flight” response, while chronic stress eventually leads to metabolic syndrome and more serious changes of physiology. One of important issues in the stress research field is the lack of standard protocol so the results from different studies are not comparable. The aim of this study was to create a standard stress protocol and to measure the parameters that prove development of metabolic syndrome caused by stress. Study included 72 four-months-old Sprague-Dawley rats divided in males, non-ovariectomized (NON-OVX) and ovariectomized females (OVX) group. These groups were subdivided in acute, chronic and control group. Acute stress was provoked by cold restraint, and chronic stress used the combination of cold exposure with additional stressors. Chronic stress was repeated 3 times in 10-day stress sessions with 3 weeks of no stressors in-between. Serum glucose and cholesterol concentrations were analyzed as biochemical markers of metabolic syndrome. Glucose tolerance was measured in chronic stress group before any stress, after acute and after each stress session. Body weights were monitored in control and chronic stress group. Acute and chronic stress exposure caused decrease in plasma glucose and cholesterol.

Males significantly lost weight, OVX gained and there were no changes in body weight of NON-OVX females. GTT profile significantly changed in males and NON-OVX, but not in OVX. Measured parameters confirmed that described stress protocol induced stress response in rats and it can be suggested as a standard acute and chronic stress protocol.

Keywords: acute stress; chronic stress; protocol; metabolic syndrome


IMMUNOLOCALIZATION OF CHLORIDE-FORMATE EXCHANGER (Slc26a6) IN VARIOUS RAT TISSUES; sEX-dEPENdENT EXPREssION IN kIdNEys

Karaica dean1; Breljak davorka1; lončar jovica2; mihaljević ivan2; ljubojević marija1; herak-Kramberger carol m1; micek vedran1; vrhovac ivana1; ivković jana1; Burckhardt Birgitta3; Burckhardt gerhard3; smital tvrtko2; sabolić ivan1

1Molecular Toxicology, Institute for Medical Research and Occupational Health, Zagreb, Croatia2Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia3Institute of Systemic Physiology and Pathophysiology, Center of Physiology and Pathophysiology, University Medical Center Göttingen, Göttingen, Germany


Chloride-formate exchanger (CFEX), a member of the Solute carrier family 26 (Slc26a6), in various mammalian organs mediates transport of chloride, bicarbonate, oxalate, formate and hydroxyl ions. Its cellular localization in rat organs/tissues is poorly documented. Here we used a commercial polyclonal anti-CFEX antibody (CFEX-Ab) to investigate the CFEX protein expression in the kidneys, intestine, liver and pancreas of Wistar rats by immunofluorescence cytochemistry (IFC) in tissue cryosections and by Western blotting (WB) of total cell membranes (TCM) isolated from the respective organs. To determine sex-dependent renal expression, the CFEX protein abundance was studied in the kidneys of prepubertal, adult intact and gonadectomized rats of both sexes, and of sex hormone-treated castrates. By IFC, the CFEX-Ab stained

the brush-border membrane (BBM) of proximal tubules with heterogeneous intensity (S1~S2>S3), other nephron segments being CFEX-negative. The renal CFEX protein expression was a) male-dominant, b) downregulated by castration, c) not affected by ovariectomy, d) upregulated by androgen treatment, and e) low and sex-independent in prepubertal animals. In the intestine, CFEX was localized in the BBM of duodenal and jejunal enterocytes (duodenum>jejunum); no staining was seen in the ileum, caecum and colon. By WB of TCM, the CFEX-Ab labeled a single protein band of ~120 kDa whose density matched the IFC findings. The protein was also immunolocalized to the hepatocyte canalicular membrane and apical domain of pancreatic ducts. Therefore, the CFEX protein is expressed in various rat organs/tissues, whereas in the kidneys, its expression is male-dominant due to post-pubertal androgen stimulation.

Keywords: androgens; brush-border membrane; gastrointestinal tract; liver; pancreas; proximal tubule


BEHAVIORAL RESPONSE IN WISTAR-ZAGREB 5HT MOdEL OF RATs WITH CONsTITuTIONALLy ALTEREd SEROTONIN TRANSPORTER

Kesić maja; mokrović gordana; Čičin-šain lipa

Laboratory of Neurochemistry and Molecular Neurobiology, Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia


Serotonin (5HT), a monoamine neurotransmitter plays an important modulatory role in a variety of behaviors and behavioral disorders including anxiety and depression, as well in cognitive performances. In order to investigate the relationship between constitutional differences in serotonergic activity and emotive/cognitive functions, we use Wistar-Zagreb 5HT rats, an animal model with constitutionally altered serotonin homeostasis, developed in our laboratory by selective breeding toward extremes of peripheral serotonin transporter (5HTT). Selective breeding led also to the alterations in the brain serotonergic homeostasis between high-5HT and low-5HT sublines of this model. Different forms of behaviour were tested in rats from 5HT-sublines of Wistar-Zagreb 5HT rats as follows: anxiety-related behaviour, depression-related behaviour and memory and learning. In the elevated plus maze test animals from high-5HT subline had lower score in time spent in open arms, showing a higher level of anxiety-related behaviour. In the Morris water maze test, high-5HT rats learned platform position faster than low-5HT rats, but there were no differences in memory score between 5HT-sublines. In the forced swim test, low-5HT animals showed increased immobility, being an indication for their increased depression-like phenotype. Differences in multiple

types of behaviour between animals from 5HT-sublines, make this animal model a valuable tool for further investigation of serotonergic modulation of various aspects of emotional behaviours and cognitive functions.

Keywords: serotonin; Wistar-Zagreb 5HT rats; anxiety; depression; memory; learning


DEVELOPMENT OF NEW MODELS ON LARGE EXPERIMENTAL ANIMALS AND ITS INFLUENCE ON CRANIO-sPINAL HydROdyNAMICs REsEARCH

Klarica marijan1; vukić miroslav2; radoš milan1; jurjević ivana1; erceg gorislav1; orešković darko3

1University of Zagreb, School of Medicine, Department of Pharmacology and Croatian Institute for Brain Research, Zagreb, Croatia2Department of Neurosurgery, School of Medicine University of Zagreb, Zagreb3Ruđer Bošković Institute, Department of Molecular Biology, Zagreb, Croatia

Increased cerebrospinal fluid (CSF) pressure inside the cranium (intracranial hypertension) and hydrocephalus are still, despite many years of research, clinically challenging and difficult to treat. This is probably because we still don’t know enough about basic CSF physiology, as well as the mechanisms which lead to an increase of CSF pressure. A generally accepted hypothesis of CSF physiology and pathophysiology (on which current therapy is based) is set according to the results obtained mostly from small experimental animals (for example rats, mice), i.e. according to the distribution of dye and test substances in small CSF samples obtained mostly from a single CSF compartment. Contrary to that, in some new, but also some earlier studies performed on larger experimental animals (rabbits, cats, dogs) in which it was physically possible to obstruct individual parts of CSF system and obtain samples from every other part of it, it was observed that CSF and other intracranial fluids have a different faith. In our laboratory, over the last 30 years we have developed a substantial number of new experimental models (acute and subchronic

aqueduct blockade, acute and subchronic stenosis of cervical subarachnoid space, application with change of microvolume, ventriculo-aqueductal perfusion, simultaneous recording of CSF pressure in multiple sites of measurement, etc.), by which a new concept of CSF physiology and pathophysiology, as well as intracranial pressure was created. Results of our research open up a need for revision of currently existing hypotheses about the occurrence of pathological CSF-related disorders (intracranial hypertension, hydrocephalus), as well as for further persuit of new and more efficient approaches to treatment. It seems that in investigation of certain medical disorders it is extremely important to choose an appropriate experimental animal species, and also to subsequently confirm the obtained results by further investigation on humans.

Keywords: cerebrospinal fluid; intracranial pressure; large experimental animals


-20,39%/1 mg/kg and -18,34%/3 mg/kg). Results suggest long-term cognitive deficits which tend to correlate with observed cholinergic deficit at the highest STZ dose regimen, varying from the acute changes, followed by normalization and finally progressive decompensation effects.

Keywords: sporadic Alzheimer’s disease, streptozotocin, cognitive deficit, cholinergic transmission

CHOLINERGIC AND COGNITIVE DEFICITS IN STREPTOZOTOCIN-INDUCED RAT MODEL OF SPORADIC ALZHEIMER’S DISEASE

Knezović ana1; šalković-Petrišić melita1

1Department of Pharmacology and Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Croatia


Rats treated intracerebroventricularly with streptozotocin (STZ-icv) have been proposed as a model for sporadic Alzheimer’s disease (sAD). We aimed to characterize the STZ-icv dose- and post-treatment time-dependency of cognitive impairment and cholinergic deficit in the brain of the STZ-icv rat model. Male Wistar rats were given STZ (0.3, 1 and 3 mg/kg dose) or vehicle (controls) icv and sacrificed one, three, six or nine months afterwards. Cognitive functions were tested by Passive Avoidance Test. Acetylcholinesterase (AChE) activity was measured in hippocampus (HPC) and cortex (CTX) by Ellman’s method. Protein expression of muscarinic cholinergic receptor M1 in HPC and CTX was measured by Western blot analysis. Data were analysed by Kruskal-Wallis and Mann-Whitney U test (p<0.05). STZ-icv rats exhibit significant cognitive decline, emphasized with higher doses (-45% to -90%). AChE activity in the STZ-icv (3 mg/kg) treated rats was significantly elevated in HPC after one (+20%) and nine (+32%) months. One and 3 mg STZ dose significantly altered the expression of muscarinic M1 receptors three months after the injection, found increased in CTX (+82,89% and +67,83%) and decreased in HPC (-18,06% and 15,01%). After 9 months, the expression of M1 receptor in CTX was decreased with all three STZ doses (-22,5%/0.3 mg/kg,


NEuRAL TuBE dEFECTs IN dIABETIC EMBRyOPATHy

Korolija marina1; hadžija mirko1; Popović hadžija marijana1

1Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia


Maternal diabetes increases the rate of congenital malformations in embryos, causing diabetic embryopathy which mostly affects hart and neural tube. In this work, we have employed inbred non-obese diabetic (NOD) mouse strain maintained at the Ruđer Bošković Institute animal facility, in order to assess the incidence of neural tube defects (NTDs) in diabetic condition. NTDs are the consequence of the failed neural tube closure which can occur at different levels along rostrocaudal body axis, producing distinct morphological features with affected brain (exencephaly), spine (spina bifida aperta) or entire neural tube (craniorachischisis). We analyzed embryos from 15 non-diabetic and 15 alloxan-induced diabetic pregnancies at 10.5 days post coitum (time-point at which the neurulation in mice is completed). Both groups yielded nearly the same total number of implants. Embryos were categorized according to external morphology as normal, malformed, affected by NTD or resorbed. There was no significant difference between groups in crown-rump length and somite number of morphologically normal embryos. No significant difference between groups was observed in any category except NTDs, which occurred exclusively in diabetic pregnancies. Out of 15 analyzed diabetic pregnant females, 11 of them had embryos affected by at least one form of non-syndromic open NTD: overall 17 exencephalic embryos were found, 2 embryos had spina bifida aperta and 4 embryos had

craniorachischisis. Obtained data suggest high specificity and sensitivity of the NOD model of diabetic embryopathy, which makes it particularly suitable and advantageous over other strains for studying the influence of maternal hyperglycemia on neurulation.

Keywords: neural tube defects; diabetic embryopathy; non-obese diabetic mice


THE ROAD TOWARDS IN VITRO ALTERNATIVE TO IN vIvO sNAkE vENOM TOXICITy ANd ANTIvENOM POTENCy AssAys

lang Balija maja1ǂ; Kurtović tihana2; Brgles marija2; halassy Beata2

1Institute of Immunology, Inc., Zagreb, Croatia2University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Zagreb, Croatia

ǂEequally contributing authors


Continuous quality control of venoms and antivenoms is necessary for a successful production of the only specific treatment of victims of venomous snake bites in Europe. Control is based on two in vivo tests: (a) the venom lethal toxicity assay (determination of median lethal dose of venom or LD50) and (b) test for determining the neutralization potency (effectiveness) of produced antivenoms (determination of mean effective antivenom dose or ED50). Both these tests, which cause suffering, pain and death of the experimental animals and also require a large number of animals, were identified by ECVAM (European Centre for Validation of Alternative Methods) as assays that are necessary to be replaced with alternative methods. Snake venoms are complex mixtures of more than a hundred mostly proteins and peptides, and non-protein compounds with different biochemical and pharmacological effects. Such complex mixtures are used as an antigen for animal immunization in antivenom production. For the development of in vitro tests, it is necessary to identify the components that contribute to the venom’s overall toxicity or specificity of antibodies that effectively neutralize

them. So far several molecules, mainly from the family of metalloproteinases (haemorrhagins, the dominant cause of the human envenomation pathology) and phospholipase A2 (ammodytoxins, the most toxic molecules in the venom known to date) have been isolated and characterized from the European vipers’ venom. However, there were no literature data for their immunogenicity. Our study showed that ammodytoxin content, determined by two developed biochemical methods, HPLC and the “sandwich” ELISA, highly correlated with the lethal toxicity of the whole viper venom. Further we examined the role of ammodytoxin (Atx)- and haemorrhagin (H)-specific antibodies in the venom lethal toxicity neutralization. The results showed that functional anti-Atx antibodies were only partially involved in the neutralization of the venom toxicity. On the other hand, functional anti-H antibodies did not provide protection at all. Developed methods for anti-Atx or anti-H determination, due to only limited involvement of the mentioned antibodies in venom toxicity neutralization, cannot be a substitute for the in vivo assays. Deeper insight into snake venom composition at the molecular level and the participation of the particular components in the venom toxicity has yet to be done before solving this task. On the other hand, Atx content determination in different venom batches could be a good screening method in the selection of the best antigen for immunisation.

Keywords: quality control; snake venom; LD50; snake antivenom; ED50; in vitro alternative


THE EVALUATION OF NEUROVIRULENCE OF MUMPS VIRUS STRAINS WITH ALTERNATIVE NEWBORN RAT-BAsEd sAFETy TEsT

lang Balija maja1; šantak maja2; markušić maja2; forčić dubravko2

1Institute of Immunology, Inc., Zagreb, Croatia2University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Zagreb, Croatia


Because of neurotropic and neurovirulent properties of mumps virus, neurovirulence testing of live attenuated vaccine is required by most national regulatory organisations. Such testing is preformed in monkeys (Ph.Eu. 01/2008:20618-Test for neurovirulence of live virus vaccines). But results obtained from these tests do not necessarily distinguish among the neurovirulent strain from those that are not. As a part of an international collaborative study we investigated the neurovirulence of three vaccine strains (JL5, Urabe AM9 and L-Zagreb) and two wild–type mumps viruses isolated in Croatia (9218/Zg98 and MuVi/Zagreb.HRV/28.12) by the neurovirulence assay in newborn rats. The results obtained by alternative assay on two vaccine strains (JL5 and Urabe AM9) correspond to the results obtained by the National Institute for Biological Standards (NIBSC) in the United Kingdom and the Food and Drug Administration in the United Station. Strain L-Zagreb also showed reduced neurovirulent properties as expected because it is a vaccine strain. Wild-type mumps virus isolates showed high neurovirulence. These results indicate as that the test in newborn rats is suitable for assessing the neurovirulence of mumps viruses. The test is robust, reproducible and follows the 3R principles.

Keywords: rat-based neurovirulence safety test; mumps virus


SUCKLING RATS AS EXPERIMENTAL MODEL FOR TOXIC METAL EXPOSURE AND SELENIUM suPPLEMENTATION IN THE EARLy PERIOd OF LIFE

lazarus maja1; orct tatjana1; jurasović jasna1; Blanuša maja1

1Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia


Oral cadmium and mercury exposure during early life has been recognized as critical due to physiological specificities of the developing organism including increased gastrointestinal uptake and metal retention in the brain. Selenium as essential element may reduce retention of both toxic metals in adult animals, but its effects in newborns are not entirely elucidated. Only a low percentage of mothers exclusively breast-feed their children beyond 6 months and intake from milk accounts for very low portion of the total metal amount ingested. Our rat model has simulated newborn oral exposure from sources other than breast-feeding (additional nutrition, oral exploration, hand-to-mouth activity) using original methods for artificial feeding in suckling rats. Water solutions of cadmium or mercury (8 or 6 µmol/kg body weight a day in form of chloride) were administered two times a day (5 or 4 days) and in between all rats (Wistar) were allowed to suckle their own mother rats. Selenium solution (in form of sodium selenite) was given before and during cadmium (4+5 days) or mercury (3+4 days) exposure in equimolar dose to respective toxic metal. On postnatal day 15, suckling rats were anesthetised (Narketan/Xylapan) and urine, faeces, blood, and organs (after exsanguination from the abdominal aorta) were sampled to determine cadmium and mercury retentions.

Selenium supplementation reduced cadmium levels in blood, brain, liver, and kidney and mercury levels in plasma, brain, and kidney. In conclusion, selenium can decrease retention of cadmium and mercury in the tissues of suckling rats and thus possibly diminish toxic metal effects.

Keywords: suckling rat; cadmium; mercury; selenium supplementation; toxic metal retention


ACuTE CHANGEs OF ACETHyLCHOLINEsTERAsE ACTIvITy ANd TAu PROTEIN PHOsPHORyLATION IN MOUSE MODEL OF SPORADIC ALZHEIMER’S DISEASE

lončar andrija1; Knezović ana2; šalković-Petrišić melita2

1Department of Zagreb County Emergency Medicine, Croatian Institute of Emergency Medicine, Vrbovec, Croatia2Department of Pharmacology and Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Zagreb, Croatia


Intracerebroventricular (icv) administration of streptozotocin (STZ) generates a mouse model of sporadic Alzheimer’s disease. Long term effects of STZ-icv treatment have been well documented but the acute neurochemical impairment have not been investigated thoroughly enough. We aimed to explore acute (within 24 hours post icv injection) cholinergic and tau protein changes in STZ-icv mouse model. Mice (strain C57Bl/6) were injected icv with STZ (1 and 1,5 mg/kg) or vehicle-citrate buffer (controls). The animals were sacrificed 15 min, 1, 6 and 24 hours following the STZ-icv treatment. Acetylcholinesterase (AChE) activity in hippocampus (HPC) and parietal cortex (PC) was measured spectrophotometrically by Ellman’s method. Protein expression of phosphorylated tau protein (PHF13 and AT8) in HPC and PC was measured by SDS-PAGE electrophoresis, followed by Western blot analysis. Data were analysed by Mann-Whitney U test (p<0.05). One hour after STZ-icv injection only the expression of PHF13 was found significantly increased in HPC (+26%) and PC (+37%), while the expression of AT8 was found significantly increased (+96%) in PC only. The AChE activity in PC was significantly increased 6 (+10%) and 24 hours

(+15.5%) following the STZ-icv injection. Results indicate that changes in tau phosphorylation are occurring earlier than those of AChE activity. Additionally, both changes occur earlier in PC than in the HPC, which indicates that STZ-icv administration triggers different acute post-treatment time- and brain-region dependent changes in the mouse brain.

Keywords: streptozotocin; tau protein; acetylcholinesterase; sporadic Alzheimer’s disease


various statistical methods of designing and determining sample sizes for individual endpoints measured in laboratory mice, rats, zebra fish and earthworms are used. The results showed that the use of these techniques for experimental designs the number of animals can be significantly reduced with the same parameters of statistical inference.

Keywords: sample size, design of experiments, resampling, statistical techniques, Bayes

OPTIMIZATION OF THE NUMBER OF EXPERIMENTAL ANIMALS USING NEW COMPUTING TECHNIQUES

lončarić željka1; hackenberger K. Branimir1

1Subdepartment of Quantitative Ecology, Department of Biology, Josip Juraj Strossmayer University, Osijek, Croatia


Modern research involving animal testing are required to reduce the number of animals that will be used. In addition to the usual reasons such as cost reduction, ethical reasons are becoming more prominent. Therefore, the requirements for reducing the number of animals in the experiments are growing. Besides the importance of minimizing the number of animals used, there is also a need for obtaining accurate and reliable results. Therefore, one of the crucial tasks of experimental design which includes use of animals is optimization of sample size. Classical statistical methods can fairly accurate determine the sample size required for appropriate decisions making regarding acceptance or rejection of the hypothesis. However, due to the nature of endpoints that are measured during the experiment, this number is often relatively high. Modern statistical techniques combined with modern computers and calculation techniques, are enabling a substantial reduction of animals in experiments. Although in such approaches additional requirements are set for researchers in terms of the use of existing biological knowledge and understanding of statistical methods, they allow much better assay performance, in economical and ethical sense. Resampling methods in combination with the use of Bayesian statistics and parallel computing form the basis of optimal design of experiments using animals. In this paper application of


ORAL CAdMIuM EXsPOsuRE duRING PREGNANCy: ASSESSMENT OF MICROELEMENT DISTRIBUTION IN MOTHER RAT AND FOETUS AT TERM

mikolić anja1; sulimanec grgec antonija1; Piasek martina1

1Analytical Toxicology and Mineral Metabolism Unit, Institute of Medical Research and Occupational Health, Zagreb, Croatia


Foodstuffs are the main source of metal exposure for the general population. Cadmium is a highly toxic metal with ability to accumulate within a mammalian body during lifetime. Most of its adverse health effects result from interaction with essential elements. During pregnancy occur numerous physiological changes in the body of expectant mother to fulfil increased demands for maintenance of foetal nutrition and viability. We evaluated the effects of oral cadmium exposure on microelement distribution in Wistar rats orally exposed to 50 mg Cd/L of demineralised water (prepared from CdCl2xH2O) during 20 days of pregnancy. Controls received demineralised water. Non-pregnant rats were treated under the same experimental conditions. The rats were supplied standard pelleted feed (Mucedola, Italy). All rats were then euthanized in general anaesthesia and samples of blood, liver, kidney, placenta, and foetus were dissected and prepared for element analyses (by AAS). Cadmium body retention was higher in pregnant than in non- pregnant exposed rats as evidenced by higher cadmium blood levels and organ cadmium contents. Liver zinc increased in all of the exposed rats. Iron levels decreased in liver and kidney of exposed pregnant rats. Placental zinc and foetal iron decreased. In all of the pregnant vs. non-pregnant rats, copper

levels were lower in liver and higher in kidney. In conclusion, cadmium accumulation and tissue perturbations of essential microelements were more pronounced in exposed pregnant than exposed non-pregnant rats. Maternally-mediated oral cadmium exposure disrupts transplacental passage of iron and zinc and thus may compromise perinatal growth and development.

Keywords: cadmium; oral exposure; pregnancy; placenta; foetus; micronutrients


FREE TEACHING RESOURCE: E-HANDBOOK TO ACCOMPANy MICROLABs FOR PHARMACOLOGIsTs

modun darko1; Bach-rojecky lidija2

1Department of Pharmacology, University of Split School of Medicine, Split, Croatia2Department of Pharmacology, University of Zagreb School of Pharmacy and Biochemistry, Zagreb, Croatia


Microlabs for Pharmacologists is a free PC-based resource for teaching Pharmacology. It consists of several modules about different topics in Pharmacology, and it includes Tutorials, Simulations, Databases, Videos and Case studies. The author of Microlabs, retired Professor of Pharmacology, Hendrik van Wilgenburg, gave his permission to the authors to produce an e-Handbook to accompany Microlabs. The e-Handbook is primarily focused on Simulations modules of Microlabs, virtual experiments in Pharmacodynamics (Isolated ileum) and Pharmacokinetics (Kinetic). Regarding teaching Pharmacodynamics (PD), the students are guided to draw Concentration-Response curve(s), Lineweaver–Burk and Schild plots and to determine different PD parameters, like EC50, Emax and pA2, for different agonists and antagonists, by using the provided raw data. Regarding teaching Pharmacokinetics (PK), the students are guided to draw Time-Concentration curve(s), and to estimate different PK parameters, like t1/2, Cl, Vd and AUC, for different drugs and clinical cases. For the more advanced students, or young scientists, there is a tutorial how to calculate different PK parameters, by using the provided raw data and non-compartmental analysis approach. E-Handbook

about virtual experiments in Microlabs for Pharmacologists is available as a free PDF to download.

Keywords: education; virtual experiments; pharmacology


dEMETHyLATING AGENT 5-AzACyTIdINE NEGATIvELy AFFECTs PROLIFERATION OF MAMMALIAN LIMB BUD CELLS IN AN ORGAN-CuLTuRE sysTEM

mužić vedrana1,2; jurić-lekić gordana3; himelreich marta3; majić željka1; sinčić nino1; Katušić ana1; vlahović maja1; šerman ljiljana1; lončarević jelena3; Bulić-jakuš floriana1

1Department of Biology, School of Medicine, University of Zagreb, Zagreb, Croatia2Department of Rehabilitation and Orthopaedic Devices; University Hospital Centre Zagreb, Croatia3Department of Histology and Embryology School of Medicine University of Zagreb, Croatia


The aim of this investigation was to assess proliferative capacity at the single cell level in ex vivo cultivated rat limb buds under the influence of the epigenetic drug 5-azacytidine (5-azaC). 13-days-old embryos were isolated from pregnant Fisher rat females and limb buds were microsurgically isolated under the stereomicroscope. They were cultivated in an organ-culture system at the air-liquid interface in MEM and 50% rat serum. 5-azacytidine (30µmol) was added to the culture medium. During the second week, explants were processed for immunohistochemistry on Anti-Proliferating Cell Nuclear Antigen (PCNA). PCNA positive cells were stereologically evaluated using numerical density (Nv) and results were statistically compared with Student’s t-test. In both fore-limb and hind-limb explants, either treated or controls, differentiation proceeded in comparison to 13-days-old limb buds. PCNA was

expressed in some cells of the cartilage, stratified squamous epithelium and mesenchyme. Nv values for PCNA were significantly lower (p<0.001) in both fore- and hind- limbs treated with 5-azaC. It can be concluded that teratogenicity of this epigenetic drug is, at least, partially due to impaired capacity for proliferation.

Keywords: 5-azacytidine; proliferative capacity; cultivated rat limb buds


syNERGIsM BETWEEN PROPOLIs ANd HyPERTHERMAL INTRAPERITONEAL CHEMOTHERAPy WITH CIsPLATIN ON EHRLICH ASCITES TUMOR IN MICE

oršolić nada1; car nikola2; lisičić duje1; Benković vesna1; horvat Knežević anica1; Đikić domagoj1; Petrik józsef3

1Department of Animal Physiology, Faculty of Science, University of Zagreb, Zagreb, Croatia2Pliva Croatia Ltd., Zagreb, Croatia3Department of Medical Biochemistry and Haematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

E-mail: [email protected]; [email protected]

We investigated antitumor, genotoxic, chemopreventive and immunostimulative effects of local chemoimmunotherapy and hyperthermal intraperitoneal chemotherapy (HIPEC) in a mouse bearing Ehrlich ascites tumor. Mice were treated with water-soluble derivative of propolis (WSDP) at dose of 50 mg kg-1) 7 and 3 days before implantation of EAT cells, while cisplatin (5 or 10 mg kg-1) was injected 3 days after implantation of EAT cells at 37°C and 43°C. The following variables were analyzed: the total number of cells, differential count of the cells present in the peritoneal cavity, functional activity of macrophages, comet assay and micronucleus assay. Combination of WSDP + CIS at 37°C resulted in tumor growth inhibition and increased the survival of mice by additional 115.25% (CIS5). WSDP with HIPEC increased survival of mice by additional 160.3% as compared with HIPEC. WSDP reduce cisplatin toxic and genotoxic effect to normal cells without effecting cisplatin cytotoxicity on EAT cells. In addition, WSDP with HIPEC

increase the cytotoxic actions of macrophages to tumor cells. WSDP increases macrophage activity and sensitivity of tumor cells to HIPEC and reduces cisplatin toxicity to normal cells.

Keywords: hyperthermia; chemotherapy; immunomodulation; propolis; cisplatin; Ehrlich ascites tumor


ROLE OF FLAVONOIDS ON OXIDATIVE STRESS AND MINERAL CONTENTS IN THE RETINOIC ACID-INDUCED BONE LOSS MODEL OF RAT

oršolić nada1; goluža eleonora2; Đikić domagoj1; lisičić duje1; jeleč željko3; vihnanek lazarus maja4; orct tatjana4

1Department of Animal Physiology, Faculty of Science, University of Zagreb, Zagreb, Croatia2Department of Anaesthesiology, Reanimatology and Intensive Medicine, University Hospital Centre- Zagreb, Zagreb, Croatia3Department of Orthopaedic Surgery, General Hospital “Dr. Ivo Pedišić”, Sisak, Croatia4Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia


Reactive oxygen species (ROS) play a role in a number of degenerative conditions including osteoporosis. Flavonoids as phytoestrogens exert physiological effects against oxidative stress diseases. We developed a retinoic acid-induced bone loss model (RBL) of rats to assess whether flavonoids and alendronate as positive control have role against oxidative stress and mineral contents in osteoporosis in vivo. Three month-old female rats of the Y59 strain were given quercetin, chrysin, naringenin (100 mg kg-1) or alendronate (40 mg kg-1, a positive control) immediately before retinoic acid treatment (80 mg kg-1) once daily for 14 days by a single intragastric (i.g.) application. In the second part of the study, we assessed the effect of those flavonoids on the skeletal system of healthy rats using single i.g. application on the respective flavonoids during 14 days. Twenty four hours after the treatment, we analyzed bone mineral density

(BMD) and the total content of bone calcium and phosphorus in the femur, the geometric and physical characteristics of thigh bones and lipid peroxidation and glutathione levels of liver and kidney cells. All flavonoids improved the decrease in bone weight coefficient, the length and the diameter of the bone, the content of bone ash and calcium and phosphorus content induced by retinoic acid. Chrysin and quercetin showed promise as preventive agents. Flavonoids were superior to alendronate according to some criteria. These results suggest that the dietary flavonoids could reduce retinoic acid-induced oxidative stress and bone loss and that flavonoids may be useful therapeutics for prevention of skeletal diseases.

Keywords: flavonoids; retinoic Acid; bone loss prevention; oxidative stress; rat


INHIBITORy EFFECT OF A PROPOLIs ON dI-N-PROPyL dIsuLFIdE OR N-HEXyL sALICILATE-INduCEd skIN IRRITATION, OXIdATIvE sTREss ANd INFLAMMATORy REsPONsEs IN MICE

oršolić nada1; skurić jadranka2; Đikić domagoj1; stanić gabrijela3; Kolarić darko4

1Department of Animal Physiology, Faculty of Science, University of Zagreb, Zagreb, Croatia2Clinic of Anaesthesia and Intensive Care Unit, Sveti Duh General Hospital, Zagreb, Croatia3Department of Pathology, Sveti Duh General Hospital, Zagreb, Croatia4Ruđer Bošković Institute, Centre for Informatics and Computing, Zagreb, Croatia


Thermal imaging has been utilized, both preclinically and clinically, as a tool for assessing inflammation. Psoriasis is a chronic inflammatory skin disease characterized by hyperkeratosis, dermal inflammatory infiltrate and increased angiogenesis. The aim of the present study was to assess usefulness of thermography in psoriatic lesion regression after topically treatment with bee propolis, recognized as potent antioxidants and anti-inflammatory agents. We monitored the inflammation process induced by irritants such as n-Hexyl salicilate (HXS) or Di-n-Propyl Disulfide (PPD by hystopatological assessment of skin, thermographic scanning, total number of inflammatory cells in peritoneal cavity, differential analysis of cells in peritoneal cavity, macrophage spreading index, haematological and biochemical parameters, frequencies of micronucleated

reticulocytes, lipid peroxidation and glutathione assay in skin. Topically applied ethanolic extract of propolis (EEP) with HXS or PPD reduced the lipid peroxidation in skin and total number of inflammatory cells in skin and peritoneal cavity, functional activity of macrophages, the number of micronuclei in mouse peripheral blood reticulocytes and enzymatic activity of ALP and AST. These results demonstrate that topical application of EEP may improve psoriatic-like skin lesions by suppressing functional activity of macrophages and ROS production. Taken together, it is suggested that EEP can safely be utilized in the prevention of psoriasis-related inflammatory changes without causing any toxic effect.

Keywords: mouse models of psoriasis; skin lesions; propolis; lipid peroxidation; glutathione level; thermography


BICUCCULINE ANTAGONIZES THE EFFECTS OF dEHydROEPIANdROsTERONE ON RAT BEHAvIOR

samardžić janko1; švob štrac dubravka2; Đurić miloš1; obradović i dragan1

1Institute of Pharmacology, Clinical Pharmacology and Toxicology, Medical Faculty, University of Belgrade, Belgrade, Serbia

2laboratory for molecular neuropharmacology, division of molecular medicine, ruđer Bošković institute, zagreb, croatia


Neurosteroid dehydroepiandrosterone (DHEA) has been associated with various functions in the central nervous system including memory and behavior; however its mechanisms of action are not fully understood. The behavioral profile of DHEA was investigated in rats in the forced swim test (FST) and active avoidance (AA) paradigm, as well as its impact on the locomotor activity. Male Wistar rats received DHEA (2, 10, and 50 mg/kg) or vehicle intraperitoneally, 30 min prior testing. The capability of bicuculline (0.5, 1, and 2 mg/kg) to antagonize effects of DHEA was checked. 10 mg/kg of DHEA significantly decreased the duration of immobility in FST, suggesting antidepressant-like effects and facilitated the retrieval of avoidance responses in AA testing. At higher doses (50 mg/kg) DHEA performance in the retention session was attenuated. These effects were antagonized by bicuculline (2 mg/kg), a specific GABAA receptor antagonist. DHEA did not show significant effects on struggling behavior, locomotor activity, habituation crossings and intertrial crossings. Hence,

DHEA in a manner resembling an inverted U shape, facilitated retrieval of memory task imposed to rats in AA, suggesting enhanced formation of implicit, procedural and hippocampal-independent memory task. In addition, data suggest that DHEA might have triggered antidepressant-like effects in rats. The observed actions of DHEA were not confounded by change in motor function, but were abolished with bicuculline, confirming partially GABA-ergic mediation of the effects. However, the molecular and neuronal substrates linking the actions of DHEA to specific GABAA receptors remain to be further elucidated and linked to human neuropsychiatric disorders.

Keywords: dehydroepiandrosterone; Wistar rats; forced swim test; active avoidance; motor activity; bicuculline


IMPACT OF RNA INTERFERENCE WITH STEMNESS GENE EXPRESSION IN EXPERIMENTAL MOUSE TERATOMA GROWN IN VITRO

sinčić nino1; gospodinov anastas2; vlahović maja1; jurić-lekić gordana3; Bulić-jakuš floriana1

1Department of Medical Biology; School of Medicine, University of Zagreb, Croatia2Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria3Department of Histology and Embryology, School of Medicine, University of Zagreb, Croatia


RNA interference provides an epigenetic mechanism that has been used in targeted silencing of gene expression at the mRNA level for therapy of human diseases (e.g. wet macular degeneration with bevasiranib- siRNA against VEGF). This investigation has been dedicated to discover impact on developmental parameters of the experimental mouse teratoma by silencing expression of stemness (Oct3/4, Nanog) and Trrap genes. E7,5 C3H mouse embryos were isolated under the dissecting microscope and grown at the air-liquid interface in a serum-supplemented medium for seven days. Their axes were measured each day from the beginning of the 7-day-culture period by an ocular micrometer to assess overall growth of the tumor. EsiRNAs were constructed according to following sequences: Oct3/4 (NM_013633.1), Nanog (XM_132755.3), Trrap (NM_133901.2), GFP (132-591) as a negative control. They were applied to the medium by lipofectamine 2000. In teratomas, derivatives of all three germ layers were discovered:

epidermis, neural tissue, vegetative ganglionic cells, smooth muscle, myotubes, cylindrical epithelium and gastric epithelium with cells typical for the fundic region (parietal and chief cells). In comparison to esiGFP (control), esiOct3/4 and especially esiNanog have significantly diminished overall growth of teratomas. EsiTrrap did not significantly diminish overall growth, but it seems to have induced major apoptotic activity. It is possible that silencing of the stemness genes has had impact on the cell proliferation. On the other hand in esiTrapp treated teratomas, where stemness genes were not silenced, they probably could compensate for overall growth through cell proliferation despite of the seemingly pronounced apoptotic activity.

Keywords: RNA interference; experimental mouse teratoma; stemness gene expression; in vitro


IMPACT OF 5-AzACyTIdINE ON OCT4 ANd NANOG dNA METHyLATION/EXPREssION IN EXPERIMENTAL MOUSE TERATOCARCINOMA

sinčić nino1; vlahović maja1; herceg zdenko2; Paić frane1; šerman ljiljana1; Katušić ana1, Bulić jakuš floriana1

1Department of Biology, School of Medicine, University of Zagreb, Zagreb, Croatia2Epigenetics Group, International Agency for Research on Cancer, Lyon, France


DNA methylation is an epigenetic mechanism regulating normal embryonal development and development of cancer. Experimental mouse teratocarcinoma connects embryogenesis and cancerogenesis. Gastrulating mouse embryo transplanted at an ectopic site gives rise to teratocarcinoma. Beside differentiated tissue it contains embryonal carcinoma cells (EC). Self-renewal and pluripotency of EC cells seems to be determined by Oct4 and Nanog, members of core transcriptional regulatory circuitry. 5-azacytidine (5azaC), as an epidrug, induces DNA hypomethylation and alters gene expression. We are reporting changes in the DNA methylation of Oct4 and Nanog induced by 5azaC in experimental mouse teratocarcinoma and consequent modification in gene expression. 7,5–days-old C3H embryos were transplanted under the kidney capsule of syngeneic adults. First group of animals was killed after 4 weeks. Other two groups were treated with 5azaC or saline (control) twice a week for 4 weeks. Cancer samples were isolated and weighted. DNA was isolated. After bisulfite conversion and PCR amplification, DNA methylation of Oct4 and Nanog was analyzed by pyrosequencing.

RNA was isolated using commercial kits. Standard protocol for qPCR was performed. Gene expression was determined in comparison to housekeeping gene Hprt1. Lowest Oct4 and Nanog DNA methylation status was found in embryos. In 4-week-old tumors DNA methylation was significantly higher and Oct4 and Nanog expression was detected. In 8-week-old tumors methylation was slightly higher compared to 4-week-old tumors, but surprisingly, the expression was significantly higher. In treated 8-week-old tumors Oct4 and Nanog methylation was slightly lower than in control as well as expression (more than 50%). Highest tumor growth was observed during second 4 weeks and was considerably reduced under 5azaC treatment. The highest DNA methylation level in 4-week-old tumors reflects the differentiation process in teratocarcinoma during its transition from embryo. Expression of stemnes genes corresponds to the high intensity of growth during second 4 weeks which is in concordance to their role in maintaining pluripotency and self-renewal. Epidrug 5azaC significantly reduced Oct4 and Nanog expression leading to growth arrest although without change in their DNA methylation status. This leads to conclusion that Oct4 and Nanog expression may be moderated by a slight DNA demethylation or some other mechanism.

Keywords: DNA methylation; gene expression; mouse teratocarcinoma; Oct4; Nanog; 5-azacytidine


QuERCETIN vs CHRysIN: EFFECT ON LIvER HIsTOPATHOLOGy IN dIABETIC MICE

sirovina damir1; oršolić nada2; zovko Končić marijana3; Kovačević goran1; Benković vesna2; gregorović gordana1

1Department of Biology, Department of Zoology, Faculty of Science, University of Zagreb, Zagreb, Croatia2Department of Biology, Department of Animal Physiology, Faculty of Science, University of Zagreb, Zagreb, Croatia3Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia


The antioxidant and reducing ability of quercetin and chrysin and their ability to chelate Fe2+ ions in vitro were studied and compared with their effects on lipid peroxidation and histopathological changes in liver of diabetic mice. Diabetes was induced in Swiss albino mice with a single intravenous injection of alloxan (75 mg kg-1). Two days after alloxan injection, flavonoids preparations (50 mg kg-1 per day) were given intraperitoneally for 7 days in diabetic mice. Antioxidant activity was analyzed as DPPH free radical scavenging activity, reducing power and the capability to chelate iron (II) ions. In vitro chrysin demonstrated notable antiradical activity albeit much lower than quercetin which was an extremely effective radical scavenger. The reducing power of both flavonoids was notable but lower than the activity of ascorbic acid. Both flavonoids demonstrated notable chelating ability towards ferrous ion, chrysin being more active of the two. The lipid peroxidation was evaluated by measuring the MDA production using the 2-thiobarbituric acid TBA test. Administration of quercetin and chrysin to diabetic

mice resulted in a significant decrease of lipid peroxidation level in liver tissue. Treatment of diabetic mice with flavonoids solutions results in decreased number of vacuolated cells and degree of vacuolization of the liver tissue. Quercetin and chrysin have beneficial effects on liver histopathology in diabetic mice, according to their antioxidant capacity in vitro. The protective role of flavonoids against the ROS induced damages in diabetic mice gives a hope that they may exert similar protective action in humans.

Keywords: diabetes; quercetin; chrysin; lipid peroxidation; histopathology


ANTIOXIDATIVE AND ANTIDIABETIC EFFECTS OF NARINGIN AND CURCUMIN IN VITRO AND IN VIVO

sirovina damir1ǂ; oršolić nada2ǂ; ivić ivan3; novak sanja4; gajski goran5; garaj-vrhovac vera5; zovko Končić marijana6

1Department of Biology, Department of Zoology, Faculty of Science, University of Zagreb, Zagreb, Croatia2Department of Biology, Department of Animal Physiology, Faculty of Science, University of Zagreb, Zagreb, Croatia3University of Pécs, Medical School, Department of Pathophysiology and Gerontology, Pécs, Hungary4Department of Physiology and Immunology, Medical faculty Osijek, J.J. Strossmayer University Osijek, Osijek, Croatia5Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia6Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

ǂEqual contributors


The in vitro antioxidant effects of polyphenols naringin and curcumin and their effects on DNA damage caused by alloxan-induced diabetes in mice as well as the overall state of health are compared. Diabetes was induced in Swiss albino mice with a single intravenous injection of alloxan at dose of 75 mg kg-1 body weight. Two days after alloxan injection, naringin or curcumin preparations (50 mg kg-1) were given intraperitoneally for 7 days. Antioxidant activity was analyzed as DPPH free radical scavenging activity, reducing power and the capability to chelate iron (II) ions. In vitro curcumin showed appreciable scavenging properties, high reducing power and good chelating

activity. Naringin showed lower but notable scavenging and chelating activity and negligible reducing power. Comet assay showed decreased DNA damage in lymphocytes and increased level of DNA damage in liver and kidney cells of mice treated with polyphenols compared to the untreated diabetic animals. The same pattern was demonstrated by the micronucleus test. However, beneficial antidiabetic effects of naringin or curcumin in vivo are confirmed by a significant increase in the body weight and 100% survival of mice. It seems that antioxidant capacity of curcumin and naringin was crucial in preventing further oxidative damage, particularly double DNA breaks and repair of single DNA breaks while untreated alloxan-induced diabetes leads to the rapid loss of the kidneys and liver cells. This protective role of investigated polyphenols in diabetic mice gives a hope that they may exert protective action in humans but further research is needed.

Keywords: diabetes; naringin; curcumin; antioxidant activity; DNA damage


THERAPEUTIC POTENTIAL OF MULTIFUNCTIONAL IRON-CHELATING AGENT M30 IN A RAT MOdEL OF SPORADIC ALZHEIMER’S DISEASE

smailović una1; Knezović ana1; mandel silvia2; youdim moussa2; šalković-Petrišić melita1

1Department of Pharmacology and Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Croatia2Eve Topf Center for Neurodegenerative Diseases Research and Department of Molecular Pharmacology, Faculty of Medicine, Technion, Haifa, Israel


Central administration of streptozotocin (STZ-icv) is shown to induce Alzheimer-like changes due to the STZ-icv rat model has been proposed as an animal model of sporadic Alzheimer’s disease (sAD). We aimed to assess the therapeutic potential of a novel multifunctional iron-chelating compound M30 in a STZ-icv rat model. Adult male Wistar rats were injected icv with STZ (3 mg/kg) or vehicle (control). STZ-icv injected rats were treated orally with water (STZ) or M30 (10 mg/kg 3x a week) starting 8 days after icv treatment (STZ+M30) and sacrificed 2 weeks after icv treatment. Protein expression of phospho(p)-tau protein PHF13 and AT8, phospho and total glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5) in hippocampus was measured by SDS-PAGE. Brain iron levels were detected by Prussian blue iron staining. Data were analyzed by Kruskal-Wallis and Mann-Whitney U test (p<0.05). M30 treatment decreased STZ-icv-induced increment in p-tau PHF13 expression (p<0.05), compared to STZ-icv treatment alone. AT8 expression and GSK3β activity remained unchanged

in all groups. CDK5 expression was found significantly decreased both in STZ and STZ+M30 compared to the control (p<0.05). M30 treatment reduced positive signal of pathological iron accumulation in the STZ-icv rat brain. M30 treatment demonstrates therapeutic potential in STZ-icv model of sAD and further supports the neuroprotective role of multifunctional iron-chelator M30 providing evidence on the molecular mechanisms of the therapeutic potential of M30 in STZ-icv model of sAD.

Keywords: sporadic Alzheimer’s disease; streptozotocin; iron-chelator M30


N-TERT-BuTyL-Α-PHENyLNITRONE IMPROvEs EX vIvO GROWTH OF THE RAT EMBRyO AT THE AIR-LIQuId INTERFACE IN A CHEMICALLy dEFINEd MEDIUM

sobočan nikola1,2; sinčić nino1; majić željka1; Katušić ana1; vlahović maja1; šerman ljiljana1; Beuc robert3; jurić-lekić gordana4; Bulić-jakuš floriana1

1Department of Medical Biology, School of Medicine, University of Zagreb, Croatia2University Clinic Merkur, Zagreb, Croatia3Institute of Physics, Zagreb, Croatia4Department of Histology and Embryology, School of Medicine, University of Zagreb, Croatia


In our unpublished results, N-tert-Butyl-α-phenylnitrone (PBN), the spin-trapping agent with an antioxidant activity, has been shown to improve growth of fetuses treated with the teratogen 5-azacytidine. To investigate PBN’s direct impact upon the embryo itself, we have now used an original ex vivo model, a protein-free, chemically defined medium model of postimplantation rat embryo development where biological activities of various substances were more pronounced than in the serum-supplemented medium (Bulić-Jakuš et al. 1999). Rat Fischer strain females were sacrificed in ether and microsurgically isolated. 9,5-days-old embryos (embryonic shields without extraembyonic membranes) were cultivated for two weeks at the air-liquid surface in Eagle’s MEM with 5-azacytidine (5μM), and/or PBN (22.6 µM) and controls in MEM or in MEM with 50% rat serum. Explant diameters were measured by an

ocular micrometer at the beginning of culture and every other day. Growth areas were determined in arbitrary units and data normalized to those obtained in MEM. Explant growth was highest in the serum-supplemented medium and lowest with a DNA demethylating epigenetic drug and a teratogen. PBN ameliorated growth of 5-azaC treated explants for approximately 25%. Moreover, PBN improved growth for 25% in comparison to control grown only in MEM. According to our results with gastrulating-embryo culture it seems that PBN is valuable for reaching “the ultimate goal-to eliminate serum and develop effective serum-free or even protein-free or chemically defined media“ for regenerative medicine purposes.

Keywords: 5-azacytidine; N-tert-Butyl-α-phenylnitrone; ex vivo model; rat embryo; growth


THE ROLE OF EsTROGEN IN HyPEROXIA-INduCEd OXIDATIVE STRESS IN LIVER OF CBA/H MICE

sobočanec sandra1; šarić ana1; mačak šafranko željka1; Popović-hadžija marijana1; aralica gorana2; Korolija marina1; Kušić Borka1; Balog tihomir1

1Department of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia2Department of Pathology Medical School University of Zagreb and University Hospital Dubrava, Zagreb, Croatia


Estrogen has well established cardioprotective, antioxidant and neuroprotective role and exerts vast range of biological effects in both males and females. In this study we examined estrogen effect on stress resistance, oxidative damage markers (LPO and DNA damage), mediators of oxidative stress response (Sirt1, ppar-γ, eNOS) and antioxidant parameters (MnSOD) using hyperoxia as a model of acute oxidative stress load in liver of ovariectomized/estrogen implemented CBA/H mice of both sexes. We have found that hyperoxia induced oxidative damage only in males, followed by their decreased survival. Histopathological examination revealed that differences in survival were not the consequence of acute lung injury induced by hyperoxia. Ovariectomy diminished female resistance to oxidative damage, while the E2 treatment markedly increased resistance to hyperoxia in males as well as in ovariectomized females. Moreover, antioxidant parameters were upregulated upon estrogen addition in hyperoxia-treated animals, which may imply protective effect of estrogen under oxidative stress conditions. This study clarified the role of E2 in sex-related

differences to oxidative stress resistance, including the role of sirt1 as a mediator of oxidative stress response, thus contributing to understanding about the appropriate strategies for treatment of various age-related diseases.

Keywords: estrogen, hyperoxia, sex-related, Sirt1, ovariectomy, ROS


ESLAV AWARNESS PRESENTATION

ševeljevic-jaran daša1

1ESLAV Board ordinary member, Nat Reps coordinator - on behalf of ESLAV Board


The European Society of Laboratory Animal Veterinarians (ESLAV) was created at the 6th FELASA Symposium held in June 1996 in Basel, Switzerland. The Society is registered as a non-profit organization in France. ESLAV gives veterinarians a forum to discuss issues which concern them in the field of laboratory animal science (LAS) and by doing so addresses the important issues that are the humane care and use of laboratory animals for scientific purposes which is in an equally important domain of general public interest. The society’s objectives are to promote and disseminate expert veterinary knowledge within the field of LAS, accomplished through:

Organization of annual scientific meetings always in • conjunction with a local LAS organization. Also the organization of lectures, discussions and publications (semi-annual Society’s magazine ‘’Briefing’’)

ESLAV sets the right environment and support for the activity • of the European College of Laboratory Animal Medicine (ECLAM). The College represents the academic component in our field of laboratory animal medicine.

The advancement of veterinary knowledge and skills in •

subjects connected with the breeding, health, welfare and use of laboratory animals

Collaboration and exchange of information with other • Societies (LAVA, FELASA, AAALAC, FVE-EVERI, NC3Rs, AALAS, ACLAM, etc) and allied scientific disciplines

Active encouragement of its Members to provide training for • veterinarians practicing or wishing to practice in the field of LAS, both at the under- and postgraduate level

Representation of the veterinary “voice” at regulatory and • governmental bodies, by representation at Expert Working Group meetings and National Contact Point meetings regarding 2010/63/EU

Keywords: ESLAV; laboratory animal veterinarians;, laboratory animal science


sALIvARy CORTIsOL As INdICATOR OF PERsONALITy TyPEs IN COMMON MARMOsETs?

šlipogor vedrana1, millesi eva2; Bugnyar thomas1

1Department of Cognitive Biology, Faculty of Life Sciences, University of Vienna, Vienna, Austria2Department of Behavioural Biology, Faculty of Life Sciences, University of Vienna, Vienna, Austria


Individual differences or personalities are a correlated suite of behavioural traits, consistent across time and/or contexts. They are often causally linked to physiological traits that may provide a proximate explanation to the evolutionary maintenance of the variation in these behavioural correlations, however this notion received surprisingly little interest among non-human primate studies so far. In the present study we combined behavioural and physiological parameters to study consistent inter-individual differences in common marmosets (Callithrix jacchus). We assayed salivary cortisol levels and their relation to the personality trait aggressiveness. Individually separated marmosets (N = 15) were tested in two blocks of behavioural tests. In the Mirror Test, a mirror was placed in front of the experimental enclosure; in the Video Test, the mirror was replaced by a computer screen on which an image of an unfamiliar but same sex individual was presented. Saliva samples were taken before, immediately after and 10 minutes after the tests, while all the behavioural responses during the tests were recorded with two video cameras from different angles. In both tests, we predicted that seeing an “unfamiliar conspecific“ would evoke a (primarily) aggressive response in this highly territorial species, yet that

this response may vary consistently across individuals. Whether inter-individual differences in hormonal levels are linked to the personality trait aggressiveness, i.e. whether more aggressive individuals show higher levels of salivary cortisol after seeing an unfamilar conspecific than less aggressive individuals will be discussed.

Keywords: animal personality; individual differences; salivary cortisol; hormones; common marmosets


EFFECTS OF CHRONIC DHEAS ADMINISTRATION ON sEIzuRE THREsHOLd, LOCOMOTOR ACTIvITy, MOTOR COORdINATION ANd BOdy WEIGHT IN MALE AND FEMALE MICE

švob štrac dubravka1; vlainić josipa1; Krsnik željka2; samardžić janko3; erhardt julija4

1Laboratory for Molecular Neuropharmacology, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia2Croatian Institute for Brain Research, Department of Neuroscience, School of Medicine, University of Zagreb, Croatia 3Institute of Pharmacology, Clinical Pharmacology and Toxicology, Medical Faculty, University of Belgrade, Serbia4Department of Animal Physiology, Faculty of Science, University of Zagreb, Croatia


Dehydroepiandrosterone sulphate (DHEAS) is a neurosteroid associated to various important brain functions, such as neuronal plasticity, cognition and emotions. It appears that the effects of DHEAS in the central nervous system are primarily mediated through its action on several neurotransmitter systems, including GABAergic and glutamatergic neurotransmission, which are also involved in regulating the balance between excitation and inhibition in the brain. It is possible that enhancement of brain excitability and seizures might limit DHEAS potential therapeutic applications. In order to investigate the effects of chronic neurosteroid treatment, DHEAS has been administered intraperitoneally to female and male mice once daily for 4 weeks in a dose of 10 mg/kg. The effects of long-term DHEAS administration on the picrotoxin-, pentylentetrazole- and NMDA-

induced seizures, locomotor activity, motor coordination and body weight of the animals of both sexes have been studied. Chronic DHEAS has not modified the locomotor activity, motor coordination and body weight of both male and female mice. The results also failed to demonstrate significant effects of long-term DHEAS treatment on the doses of intravenously administered picrotoxin, pentylentetrazole and NMDA, needed to produce convulsant signs in male and in female mice. However, sex differences in the susceptibility to seizures became more prominent following chronic DHEAS administration to mice. Although our findings suggest that long-term DHEAS treatment might be safe for various potential therapeutic applications, they also support reported interaction of DHEAS with male and female hormonal status, which may underline observed sex differences in the relationship between DHEAS and various health outcomes.

Keywords: dehydroepiandrosterone sulphate; mice; sex differences; seizure threshold; motor activity; body weight


ARE POuLTRy ANd OTHER BIRds suFFICIENTLy REPRESENTED AS ANIMAL MODELS IN BIOMEDICAL REsERACH OF sOME HuMAN dIsEAsEs IN CROATIA?

tišljar marina1; jovanov milošević nataša3; grabarević željko2; mišić marija5; savić vladimir1, artuković Branka2; džaja Petar4; amšel zelenika tajana1; seiwerth sven5; severin Krešimir4; semple-rowland susan6

1Poultry Centre, Croatian Veterinary Institute, Zagreb, Croatia2Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Zagreb, Croatia3Croatian Institute for Brain Research, Department of Neuroscience, School of Medicine, University of Zagreb, Croatia4Department of Forensic Veterinary Medicine, Faculty of Veterinary Medicine, University of Zagreb, Croatia5Department of Pathology, School of Medicine, University of Zagreb, Croatia6Department of Neuroscience, University of Florida, Florida, USA


Numerous studies on poultry as an important animal models in the investigation of non-infectious diseases in humans have been successfully carried out and published in numerous scientific journals around the world. This report should not be taken as definitive, rather as a short review of some successfully completed investigations on poultry as animal models in Croatia (cardiovascular diseases in chickens). It is also questioning the justification of the current obstacles to the continuation and completion of research of e.g. degenerative retinopathy in chickens (very similar to retinitis pigmentosa in humans), acute heart diseases (in broiler turkey as a model), and also the

neglection of other species of birds as animal models (Parrot Proventricular Dilatation Disease as the model in the study of Guillain-Barré syndrome in humans).

Keywords: poultry; birds; animal models; non-infectious diseases in humans; Croatia


THE ASSESMENT OF ANTICONVULSANT PROPERTIES OF THE dRuG – PENTyLENETETRAzOLE sEIzuRE MODEL

vlainić josipa1; švob štrac dubravka1; jazvinšćak jembrek maja1

1Laboratory for Molecular Neuropharmacology, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia


The need for new antiepileptic drugs and the appropriate model for their identification are always present. Different approaches can be used and one of them also used in our laboratory is systemic administration of the chemoconvulsant pentylenetetrazol (PTZ), a non-competitive GABA antagonist. Namely, minimal doses of PTZ needed to induce different stages of seizure are recorded as a seizure threshold. During experiments the threshold for different types of seizures (myoclonic twitch, generalized clonus with loss of righting reflexes, and tonic backward extension of forelimbs, and death) are assessed using i.v. application of PTZ, while the traditional seizure test with PTZ s.c. injection accounts for generalized clonic seizures in mice. The most important factors which influence an estimation of the drug anticonvulsive potency in PTZ seizure model are bishaped dose-response curve (decline in anticonvulsant dose-response at high doses), the route of PTZ administration (i.v. vs. s.c.), species and strain differences in drug metabolism, differences in drug potencies (administered doses compared to ‘active’ drug concentrations in plasma), endpoints used for PTZ test, etc. Namely, i.v. PTZ seizure threshold may be useful in assessing the anticonvulsant effect of the drug at different stages of convulsions although the important role of technical, biological and pharmacological

factors in the interpretation of the results should be taken into account. In conclusion, PTZ seizure model is effective in predicting the efficacy of a substance against the myoclonic petit mal seizures in humans, though one can not predict the drug efficacy against absence seizures.

Keywords: epilepsy; anticonvulsant drugs; pentylenetetrazol; experimental seizures


EXPRESSION OF SODIUM-D-GLUCOSE COTRANSPORTER 1 IN MURINE TISSUES; SEX-dEPENdENT EXPREssION IN kIdNEys

vrhovac ivana1; Breljak davorka1; Karaica dean1; Koepsell hermann2; sabolic ivan1

1Molecular Toxicology Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia2Institute of Anatomy & Cell Biology, University of Würzburg, Würzburg, Germany


Sodium-D-glucose cotransporter SGLT1 (SLC5A1) in the brush-border membrane (BBM) of small intestinal enterocytes and renal proximal tubule (PT) epithelium is responsible for glucose absorption and reabsorption, respectively. Detailed localization of SGLT1 in other mammalian organs is poorly known. Since mice are frequently used experimental animals in preclinical testing, it is important to determine the expression of mouse Sglt1 (mSglt1) in their organs/tissues. To investigate the mSglt1 protein expression in different organs/tissues by immunocytochemistry (IC) in tissue cryosections and by Western blotting (WB) in isolated cell membranes, we used the adult wild-type (WT) and Sglt1 knock-out (KO) mice of both sexes. Specificity of the polyclonal mSglt1-antibody was confirmed in Sglt1 KO mice; in WT mice, by WB in the jejunal and renal BBM the antibody labeled a single protein band of ~75-kDa, whereas in the Sglt1 KO mice membranes, these protein bands were absent. The IC studies in the kidneys of WT mice revealed localization of the protein in PT BBM, exhibiting segmental (S2>S3), zonal (cortex>outer stripe) and sex (males>females) differences in

staining intensity, and in the apical membrane of thick ascending limb of Henle and macula densa. However, contrary to the male-dominant protein expression, the renal Sglt1 mRNA expression, analyzed by qPCR, was female-dominant. The mSglt1-antibody further stained the luminal domain of bile and pancreatic ducts, whereas the spleen, cerebrum, cerebellum, fat and skeletal muscle, remained unstained. The observed distribution of mSglt1 protein in various mouse organs will enable further studies of its role in patho/physiological, pharmacological and toxicological conditions.

Keywords: intestine; glucose transporters; mouse organs; proximal tubules; sex differences, SGLT1


BILJEŠKE / NOTES


POPIS AUTORA / INDEX OF AUTHORS


AAmšel Zelenika Tajana 110Aralica Gorana 102Artuković Branka 110

BBach-Rojecky Lidija 24, 78Bagarić Robert 26, 50, 52Balog Marta 54Balog Tihomir 26, 102Benković Vesna 82, 94Beuc Robert 100Blanuša Maja 68Blažetić Senka 54Bourdineaud Jean-Paul 40Breljak Davorka 46, 56, 114Brgles Marija 64Bubić-Špoljar Jadranka 48Bugnyar Thomas 106Bulić-Jakuš Floriana 80, 90, 92, 100Burckhardt Birgitta 56Burckhardt Gerhard 56

CCar Nikola 82Ciarimboli Giuliano 46

Č, ĆČičin-Šain Lipa 58

DDrinovac Višnja 24Dumić-Čule Ivo 48, 50

DžDžaja Petar 110

ĐĐikić Domagoj 82, 84, 86Đurić Miloš 88

EErceg Gorislav 60Erhardt Julija 108

FFarkaš Vladimir 26, 50, 52Forčić Dubravko 66

GGajski Goran 96Garaj-Vrhovac Vera 96Goluža Eleonora 84Gospodinov Anastas 90Grabarević Željko 28, 110Gregorović Gordana 94Grgurević Lovorka 48Gudan Kurilj Andrea 30

HHackenberger K. Branimir 74Hadžija Mirko 62Halassy Beata 64


Heffer Marija 54Herak-Kramberger M Carol 56Herceg Zdenko 92Himelreich Marta 80Horvat Knežević Anica 82Hranilović Dubravka 32

IIvić Ivan 96Ivić Vedrana 54Ivković Jana 56

JJazvinšćak Jembrek Maja 112Jeleč Željko 84Jovanov Milošević Nataša 110Jurasović Jasna 70Jurić-Lekić Gordana 80, 90, 100Jurjević Ivana 60

KKaraica Dean 46, 56, 114Katušić Ana 80, 92, 100Kesić Maja 58Klarica Marijan 60Klobučar I.V. Goran 40Knezović Ana 62, 72, 98Koepsell Herman 46, 114Kolarić Darko 86Korolija Marina 64, 102Kovačević Goran 94

Krsnik Željka 108Kurtović Tihana 66Kušić Borka 102

LLabak Irena 54Lang Balija Maja 66, 68Lazarus Maja 70Lisičić Duje 82, 84Lončar Andrija 72Lončar Jovica 56Lončarević Jelena 80Lončarić Željka 74

LJLjubojević Marija 56

MMačak Šafranko Željka 26, 102Majić Željka 80, 100Mandel Silvia 98Markušić Maja 68Micek Vedran 46, 56Mihaljević Ivan 56Mikolić Anja 76Millesi Eva 106Mišić Marija 110Mitrečić Dinko 34Modun Darko 78Mokrović Gordana 58Mužić Vedrana 80


NNovak Sanja 96

OObradović I Dragan 88Orct Tatjana 70, 84Orešković Darko 60Oršolić Nada 82, 84, 86, 94, 116

PPaić Frane 92Petrik József 82Piasek Martina 72Polić Bojan 36Popović-Hadžija Marijana 64, 102

RRadoš Milan 60

SSabolić Ivan 46, 56, 114Samardžić Janko 88, 108Savić Vladimir 110Seiwerth Sven 110Semple-Rowland Susan 110Severin Krešimir 110Sinčić Nino 80, 90, 92, 100Sirovina Damir 94, 96Skurić Jadranka 86Smailović Una 98Smital Tvrtko 56Sobočan Nikola 100

Sobočanec Sandra 26, 102Stanić Gabrijela 86Sulimanec Grgec Antonija 76

ŠŠalković-Petrišić Melita 38, 62, 72, 98Šantak Maja 68Šarić Ana 36, 102Šerman Ljiljana 80, 92, 100Ševeljevic-Jaran Daša 104Šlipogor Vedrana 104Šrut Maja 40Štambuk Anamaria 40Švarc Alfred 26, 50, 52Švob Štrac Dubravka 88, 108, 112

TTišljar Marina 110

VVihnanek Lazarus Maja 84Vismara Guglielmo 42Vlahović Maja 80, 90, 92, 100Vlainić Josipa 108, 112Vrhovac Ivana 46, 56, 114Vukičević Slobodan 48Vukić Miroslav 60

WWilkinson Anna 43YYoudim Moussa 98

132 “PoKusne životinje u znanstvenim istraživanjima”

ZZovko Končić Marijana 94, 96

POKROVITELJSTVO / AUSPICE

Grad Zagreb/ The city of Zagreb

SPONZORI / SPONSORS:

Biološki odsjek Prirodoslovno-matematičkog fakulteta Sveučilišta u ZagrebuDepartment of Biology, Faculty of Science, University of Zagreb

Gradski ured za zdravstvo Grada ZagrebaCity Office for Health, The city of Zagreb

pokusne životinje u znanstvenim istraživanjima ... · drugi simpozij hrvatskog društva za...

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