Pokusne životinje u znanstvenim istraživanjima Experimental

Transcription

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
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
ORGANIZATORI / ORGANISERS
SPONZORI / SPONSORS:
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
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
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:
CroLASA14@gmail.com
via website www.hdzlz.hr or e-mail: CroLASA14@gmail.com
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 TOPICS
•Genetski izmijenjeni modeli-transgenične životinje
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
KOTIZACIJA / REGISTRATION FEE
KOTIZACIJA/
REGISTRATION FEE
Članovi CroLASA-e
CroLASA members
Ne-članovi
Non-members
Studenti
Students
SLUŽBENI JEZICI SIMPOZIJA / SYMPOSIUM OFFICIAL
LANGUAGES
RANA
KASNA
REGISTRACIJA/
REGISTRACIJA/
EARLY
LATE REGISTRATION
REGISTRATION
(od 15. srpnja 2014/
(do 15. srpnja 2014/ from July 15th 2014)
until July 15th 2014)
150 kuna
200 kuna
200 kuna
250 kuna
50 kuna
100 kuna
KOTIZACIJA UKLJUČUJE / REGISTRATION FEE INCLUDES
•Aktivno/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
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
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
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
PROGRAM/ PROGRAMME
PREDVIĐENO
VRIJEME /
TIME
8:00 - 9:00
TEMA / TOPIC
REGISTRACIJA I
POSTAVLJANJE POSTERA
Registration and Mounting of
Posters
9:00 - 9:30
POZDRAVNA RIJEČ I
OTVARANJE
OPENING CEREMONY
SEKCIJA ANIMALNI MODELI
SECTION ANIMAL MODELS
9:30 - 10:00
Proizvodnja i upotreba genetski
modificiranih miševa u
eksperimentalnoj medicini
Production and use of genetically
modified mice in experimental
medicine
10:00 - 10:15
Eksperimentalni model sporadične
Alzheimerove bolesti
The experimental model of
sporadic Alzheimer’s disease
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
10:30 - 10:45
Mišji model za moždani udar u
regenerativnoj neuroznanosti
Mouse model for stroke in
regenerative neuroscience
10:45 - 11:00
PREDAVAČ /
LECTURER
J. Bubić Špoljar
B. Polić
M. Šalković –
Petrišić
D. Hranilović
D. Mitrečić
RASPRAVA
DISCUSSION
11:00 - 11:30
PAUZA ZA KAVU
COFFEE BREAK
SEKCIJA PATOLOŠKA, PATOHISTOLOŠKA I TOKSIKOLOŠKA
ISTRAŽIVANJA U ANIMALNIM MODELIMA
SECTION PATHOLOGY, HISTOPATHOLOGY AND TOXICOLOGY IN
ANIMAL MODELS
11:30 - 12:00
Komparativna onkologija Ž. Grabarević
sličnosti i razlike najčešćih tumora
ljudi i pasa
Comparative oncology –
similarities and differences
between most common human and
canine tumors
T. Balog
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
12:15 - 12:30
Najčešća patološka stanja u
A. Gudan Kurilj
laboratorijskih miševa, štakora i
kunića
The most common pathologic
conditions in laboratory mices, rats
and rabbits
12:30 - 12:45
Zebrice (Danio rerio) kao modelni M. Šrut
organizmi – genomske promjene
kao posljedica genotoksičnog
djelovanja
Zebrafish (Danio rerio) as a model
organism - genome changes as a
consequence of genotoxicity
12:45 - 13:00
RASPRAVA
DISCUSSION
“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
METHODS
15:30 - 16:00
Cold – Blooded Cognition: The
A. Wilkinson
Missing Class
L. Bach Rojecky
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?
16:15 - 16:45
Development of Configuration and G. Vismara
standards of the Laboratory Animal
Equipment
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
01.
02.
03.
04.
A SEKCIJA - ANIMALNI MODELI
A SECTION – ANIMAL MODELS
Ivo Dumić-Čule, Jadranka Bubić-Špoljar, Lovorka
Grgurević; Slobodan Vukičević
NOVEL RAT MODEL FOR OSTEOPOROSIS
RESEARCH
Vedrana Ivić, Marta Balog, Senka Blažetić, Irena Labak,
Marija Heffer
ACUTE AND CHRONIC STRESS MODEL IN
SPRAGUE-DAWLEY RATS
Maja Kesić, Gordana Mokrović, Lipa Čičin-Šain
BEHAVIORAL RESPONSE IN WISTAR-ZAGREB
5HT MODEL OF RATS WITH CONSTITUTIONALLY
ALTERED SEROTONIN TRANSPORTER
Marijan Klarica, Miroslav Vukić, Milan Radoš , Ivana
Jurjević , Gorislav Erceg, Darko Orešković
05.
DEVELOPMENT OF NEW MODELS ON LARGE
EXPERIMENTAL ANIMALS AND ITS INFLUENCE ON
CRANIO-SPINAL HYDRODYNAMICS RESEARCH
Maja Lazarus, Tatjana Orct, Jasna Jurasović, Maja Blanuša
06.
SUCKLING RATS AS EXPERIMENTAL MODEL
FOR TOXIC METAL EXPOSURE AND SELENIUM
SUPPLEMENTATION IN THE EARLY PERIOD OF LIFE
Andrija Lončar, Ana Knezović, Melita Šalković-Petrišić
ACUTE CHANGES OF ACETHYLCHOLINESTERASE
ACTIVITY AND TAU PROTEIN PHOSPHORYLATION
IN MOUSE MODEL OF SPORADIC ALZHEIMER’S
DISEASE
15
16
“POKUSNE ŽIVOTINJE U ZNANSTVENIM ISTRAŽIVANJIMA”
06-A
07.
07-A
07-B
08.
08-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
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 ACIDINDUCED BONE LOSS MODEL OF RAT
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
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
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
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 AIRLIQUID INTERFACE IN A CHEMICALLY DEFINED
MEDIUM
09.
10.
BROJ
POSTERA
POSTER
NUMBER
11.
12.
13.
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?
Josipa Vlainić, Dubravka Švob Štrac, Maja Jazvinšćak
Jembrek
THE ASSESMENT OF ANTICONVULSANT
PROPERTIES OF THE DRUG –
PENTYLENETETRAZOLE SEIZURE MODEL
B SEKCIJA - PATOLOŠKA, PATOHISTOLOŠKA I
TOKSIKOLOŠKA ISTRAŽIVANJA U ANIMALNIM
MODELIMA
B SECTION - PATHOLOGY, HISTOPATHOLOGY AND
TOXICOLOGY IN ANIMAL MODELS
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
Dean Karaica, Davorka Breljak, Jovica Lončar, Ivan
Mihaljević, Marija Ljubojević, Carol M. HerakKramberger,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
Marina Korolija, Mirko Hadžija, Marijana Popović Hadžija
NEURAL TUBE DEFECTS IN DIABETIC
EMBRYOPATHY
17
18
14.
15.
16.
17.
18.
19.
20.
Anja Mikolić, Antonija Sulimanec Grgec, Martina Piasek
ORAL CADMIUM EXSPOSURE DURING
PREGNANCY: ASSESSMENT OF MICROELEMENT
DISTRIBUTION IN MOTHER RAT AND FOETUS AT
TERM
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
Nada Oršolić, Jadranka Skurić, Domagoj Đikić, Gabrijela
Stanić, Darko Kolarić
INHIBITORY EFFECT OF A PROPOLIS ON DI-NPROPYL DISULFIDE OR N-HEXYL SALICILATEINDUCED SKIN IRRITATION, OXIDATIVE STRESS
AND INFLAMMATORY RESPONSES IN MICE
Damir Sirovina, Nada Oršolić, Marijana Zovko Končić,
Goran Kovačević, Vesna Benković, Gordana Gregorović
BROJ
POSTERA
POSTER
NUMBER
21.
22.
23.
24.
C SEKCIJA - PONAŠANJE ŽIVOTINJA, DOBROBIT
ŽIVOTINJA I ALTERNATIVNE METODE
C SECTION - ANIMAL BEHAVIOR, ANIMAL WELFARE
AND ALTERNATIVE METHODS
Vladimir Farkaš, Robert Bagarić, Ivo Dumić-Čule, Alfred
Švarc
RAT CARDIAC IMAGING - COREGISTERED 18FDGPET AND GOLD NANOPARTICLES CT
Vladimir Farkaš, Robert Bagarić, Alfred Švarc
LABORATORY ANIMALS PET IMAGING THROUGH
THE EYES OF A 3R’S PRINCIPLE
Maja Lang Balija, Maja Šantak, Maja Markušić, Dubravko
Forčić
THE EVALUATION OF NEUROVIRULENCE OF
MUMPS VIRUS STRAINS WITH ALTERNATIVE
NEWBORN RAT-BASED SAFETY TEST
Maja Lang Balija, Tihana Kurtović, Marija Brgles, Beata
Halassy
25.
THE ROAD TOWARDS IN VITRO ALTERNATIVE
TO IN VIVO SNAKE VENOM TOXICITY AND
ANTIVENOM POTENCY ASSAYS
Željka Lončarić, Branimir K. Hackenberger
ANTIOXIDATIVE AND ANTIDIABETIC EFFECTS OF
NARINGIN AND CURCUMIN IN VITRO AND IN VIVO
Sandra Sobočanec, Ana Šarić, Željka Mačak Šafranko,
Marijana Popović Hadžija, Gorana Aralica, Marina
Korolija, Borka Kušić, Tihomir Balog
26.
OPTIMIZATION OF THE NUMBER OF
EXPERIMENTAL ANIMALS USING NEW
COMPUTING TECHNIQUES
Darko Modun, Lidija Bach-Rojecky
THE ROLE OF ESTROGEN IN HYPEROXIA-INDUCED
OXIDATIVE STRESS IN LIVER OF CBA/H MICE
Ivana Vrhovac, Davorka Breljak, Dean Karaica, Hermann
Koepsell, Ivan Sabolic
27.
QUERCETIN VS CHRYSIN: EFFECT ON LIVER
HISTOPATHOLOGY IN DIABETIC MICE
Damir Sirovina, Nada Oršolić, Ivan Ivić, Sanja Novak,
Goran Gajski, Vera Garaj-Vrhovac, Marijana Zovko Končić
EXPRESSION OF SODIUM-D-GLUCOSE
COTRANSPORTER 1 IN MURINE TISSUES; SEXDEPENDENT EXPRESSION IN KIDNEYS
FREE TEACHING RESOURCE: E-HANDBOOK
TO ACCOMPANY MICROLABS FOR
PHARMACOLOGISTS
Janko Samardžić, Dubravka Švob Štrac, Miloš Đurić,
Dragan I Obradović
BICUCCULINE ANTAGONIZES THE EFFECTS OF
DEHYDRO- EPIANDROSTERONE ON RAT BEHAVIOR
19
20
28.
Daša Ševeljevic-Jaran
29.
ESLAV AWARNESS PRESENTATION
Šlipogor Vedrana, Millesi, Eva; Bugnyar Thomas
30.
SALIVARY CORTISOL AS INDICATOR OF
PERSONALITY TYPES IN COMMON MARMOSETS?
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
21
22
PREDAVANJA / LECTURES
23
24
EXPERIMENTAL IN VIVO MODELS IN THE STUDY OF
PAIN – ARE THERE ANY ALTERNATIVES?
Bach-Rojecky Lidija1; Drinovac Višnja1
Department of Pharmacology, University of Zagreb School of
Pharmacy and Biochemistry, Zagreb, Croatia
1
E-mail: lbach@pharma.hr
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 systemactivating 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 sideeffects 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
25
26
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
Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb,
Croatia
2
Division of Experimental Physics, Ruđer Bošković Institute, Zagreb,
Croatia
1
E-mail: balog@irb.hr
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 (9295%) 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; F18FDG
27
28
1
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.
E-mail: zeljko.grabarevic@vef.hr
Keywords: comparative tumour analysis; HCC; testicular tumours;
mammary carcinoma; lymphoma; men; dogs
COMPARATIVE ONCOLOGY – SIMILARITIES AND
DIFFERENCES OF THE MOST FREQUENT TUMOURS
IN MEN AND DOGS
Grabarević Željko1
Department 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
29
30
THE MOST COMMON PATHOLOGIC CONDITIONS IN
LABORATORY MICES, RATS AND RABBITS
Gudan Kurilj Andrea1
Medicine University of Zagreb, Zagreb, Croatia
1
E-mail: agudan@vef.hr
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
31
32
THE ROLE OF HYPERSEROTONEMIA IN
NEURODEVELOPMENTAL DISORDERS: STUDIES
ON RATS WITH PERINATALLY ALTERED SEROTONIN
METABOLISM
Hranilović Dubravka1
Department of Animal Physiology, Division of Biology, Faculty of
Science, University of Zagreb, Croatia
1
E-mail: dubravka@biol.pmf.hr
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 5HTPtreated 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
33
34
MOUSE MODEL FOR STROKE IN REGENERATIVE
NEUROSCIENCE
Mitrečić Dinko1
Laboratory for Stem Cells, Croatian Institute for Brain Research,
School of Medicine, University of Zagreb, Zagreb, Croatia
1
E-mail: dominic@mef.hr
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
35
36
PRODUCTION AND USE OF GENETICALLY MODIFIED
MICE IN EXPERIMENTAL MEDICINE
Polić Bojan1
Department of Histology & Embryology, Faculty of Medicine,
University of Rijeka, Rijeka, Croatia
1
E-mail: bojan.polic@medri.uniri.hr
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
37
38
THE EXPERIMENTAL MODEL OF SPORADIC
ALZHEIMER’S DISEASE
Šalković-Petrišić Melita1
Medicine, Zagreb, Croatia
1
E-mail: melitas@mef.hr
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
39
40
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
Department of Zoology, Faculty of Science, University of Zagreb,
Zagreb, Croatia
2
Arcachon Marine Station, CNRS, UMR EPOC 5805, University of
Bordeaux, Arcachon, France
1
E-mail: maja.srut@biol.pmf.hr
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
41
42
DEVELOPMENT OF CONFIGURATION AND
STANDARDS OF THE LABORATORY ANIMAL
EQUIPMENT
Vismara Guglielmo1
1
Tecniplast Spa, Italy
E-mail: gvismara@tecniplast.it
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
School of Life Sciences, College of Science, University of Lincoln,
Lincoln, United Kingdom
1
E-mail: awilkinson@lincoln.ac.uk
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
43
44
POSTERI / POSTERS
45
46
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
Molecular Toxicology, Institute for Medical Research and
Occupational Health, Zagreb, Croatia
2
Department of Internal Medicine D, Experimental Nephrology,
University Hospital Münster, Münster, Germany
3
Institute of Anatomy and Cell Biology, University of Würzburg,
Würzburg, Germany
1
E-mail: dbreljak@imi.hr
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) sexdependency 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
47
48
NOVEL RAT MODEL FOR OSTEOPOROSIS RESEARCH
loss and they were ready for experimental research.
Dumić-Čule Ivo1; Bubić-Špoljar Jadranka1; Grgurević Lovorka1;
Vukičević Slobodan1
Keywords: osteoporosis; bone research; thyroparathyroidectomy
Laboratory for mineralized tissues, Center for Translational and
Clinical Research, Medical School, University of Zagreb, Croatia
1
E-mail: ivodc1@gmail.com
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 SpragueDawley 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
49
50
RAT CARDIAC IMAGING - COREGISTERED 18FDG-PET
AND GOLD NANOPARTICLES CT
Farkaš Vladimir1; Bagarić Robert1; Dumić-Čule Ivo2; Švarc Alfred1
Croatia
2
Laboratory for mineralized tissues, Center for Translational and
Clinical Research, Medical School, University of Zagreb, Croatia
1
E-mail: vfarkas@irb.hr
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
51
52
LABORATORY ANIMALS PET IMAGING THROUGH
THE EYES OF A 3R’S PRINCIPLE
Farkaš Vladimir1; Bagarić Robert1; Švarc Alfred1
Croatia
1
E-mail: vfarkas@irb.hr
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
53
54
ACUTE AND CHRONIC STRESS MODEL IN SPRAGUEDAWLEY RATS
Ivić Vedrana1; Balog Marta1; Blažetić Senka2; Labak Irena2; Heffer
Marija1
1
University of Osijek, Faculty of Medicine, Osijek, Croatia
University of Osijek, Department of Biology, Osijek, Croatia
2
E-mail: vedrana.ivic@mefos.hr
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, nonovariectomized (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 10day 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
55
56
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
Molecular Toxicology, Institute for Medical Research and
2
Laboratory for Molecular Ecotoxicology, Division for Marine and
Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia
3
Institute of Systemic Physiology and Pathophysiology, Center
of Physiology and Pathophysiology, University Medical Center
Göttingen, Göttingen, Germany
1
E-mail: dkaraica@imi.hr
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 CFEXAb 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
57
58
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
E-mail: mkesic@irb.hr
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: anxietyrelated 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
59
60
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
University of Zagreb, School of Medicine, Department of
Pharmacology and Croatian Institute for Brain Research, Zagreb,
Croatia
2
Department of Neurosurgery, School of Medicine University of
Zagreb, Zagreb
3
Ruđer Bošković Institute, Department of Molecular Biology, Zagreb,
Croatia
1
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
61
62
NEURAL TUBE DEFECTS IN DIABETIC EMBRYOPATHY
Korolija Marina1; Hadžija Mirko1; Popović Hadžija Marijana1
Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb,
Croatia
1
E-mail: mkorolija@irb.hr
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 nonsyndromic 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
63
64
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
Institute of Immunology, Inc., Zagreb, Croatia
University of Zagreb, Centre for Research and Knowledge Transfer in
Biotechnology, Zagreb, Croatia
1
2
Eequally contributing authors
ǂ
E-mail: mbalija@imz.hr
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
65
66
THE EVALUATION OF NEUROVIRULENCE OF MUMPS
VIRUS STRAINS WITH ALTERNATIVE NEWBORN RATBASED SAFETY TEST
Lang Balija Maja1; Šantak Maja2; Markušić Maja2; Forčić Dubravko2
Institute of Immunology, Inc., Zagreb, Croatia
University of Zagreb, Centre for Research and Knowledge Transfer in
Biotechnology, Zagreb, Croatia
1
2
E-mail: mbalija@imz.hr
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
67
68
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
Analytical Toxicology and Mineral Metabolism Unit, Institute for
Medical Research and Occupational Health, Zagreb, Croatia
1
E-mail: mlazarus@imi.hr
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 breastfeeding (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
69
70
ACUTE CHANGES OF ACETHYLCHOLINESTERASE
ACTIVITY AND TAU PROTEIN PHOSPHORYLATION IN
MOUSE MODEL OF SPORADIC ALZHEIMER’S DISEASE
Lončar Andrija1; Knezović Ana2; Šalković-Petrišić Melita2
Department of Zagreb County Emergency Medicine, Croatian
Institute of Emergency Medicine, Vrbovec, Croatia
2
Department of Pharmacology and Croatian Institute for Brain
Research, School of Medicine, University of Zagreb, Zagreb, Croatia
(+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.
1
E-mail: andrija.loncar.dr@gmail.com
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 MannWhitney 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
Keywords: streptozotocin; tau protein; acetylcholinesterase;
sporadic Alzheimer’s disease
71
72
ORAL CADMIUM EXSPOSURE DURING PREGNANCY:
ASSESSMENT OF MICROELEMENT DISTRIBUTION IN
MOTHER RAT AND FOETUS AT TERM
Mikolić Anja1; Sulimanec Grgec Antonija1; Piasek Martina1
Analytical Toxicology and Mineral Metabolism Unit, Institute of
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.
1
E-mail: akatic@imi.hr
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
Keywords: cadmium; oral exposure; pregnancy; placenta; foetus;
micronutrients
73
74
FREE TEACHING RESOURCE: E-HANDBOOK TO
ACCOMPANY MICROLABS FOR PHARMACOLOGISTS
about virtual experiments in Microlabs for Pharmacologists is
available as a free PDF to download.
Modun Darko1; Bach-Rojecky Lidija2
Keywords: education; virtual experiments; pharmacology
Department of Pharmacology, University of Split School of
Medicine, Split, Croatia
2
Pharmacy and Biochemistry, Zagreb, Croatia
1
E-mail: darko.modun@mefst.hr
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
75
76
DEMETHYLATING AGENT 5-AZACYTIDINE
NEGATIVELY AFFECTS PROLIFERATION OF
MAMMALIAN LIMB BUD CELLS IN AN ORGANCULTURE SYSTEM
Mužić Vedrana1,2; Jurić-Lekić Gordana3; Himelreich Marta3; Majić
Željka1; Sinčić Nino1; Katušić Ana1; Vlahović Maja1; Šerman Ljiljana1;
Lončarević Jelena3; Bulić-Jakuš Floriana1
Department of Biology, School of Medicine, University of Zagreb,
Zagreb, Croatia
2
Department of Rehabilitation and Orthopaedic Devices; University
Hospital Centre Zagreb, Croatia
3
Department of Histology and Embryology School of Medicine
University of Zagreb, Croatia
1
E-mail: floriana.bulic@mef.hr
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 organculture 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
77
78
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
Department of Animal Physiology, Faculty of Science, University of
Zagreb, Zagreb, Croatia
2
Pliva Croatia Ltd., Zagreb, Croatia
3
Department of Medical Biochemistry and Haematology, Faculty of
Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
1
E-mail: nada.orsolic@biol.pmf.hr; norsolic@yahoo.com
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 watersoluble 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
79
80
ROLE OF FLAVONOIDS ON OXIDATIVE STRESS
AND MINERAL CONTENTS IN THE RETINOIC ACIDINDUCED BONE LOSS MODEL OF RAT
Oršolić Nada1; Goluža Eleonora2; Đikić Domagoj1; Lisičić Duje1; Jeleč
Željko3; Vihnanek Lazarus Maja4; Orct Tatjana4
2
Department of Anaesthesiology, Reanimatology and Intensive
Medicine, University Hospital Centre- Zagreb, Zagreb, Croatia
3
Department of Orthopaedic Surgery, General Hospital “Dr. Ivo
Pedišić”, Sisak, Croatia
4
Analytical Toxicology and Mineral Metabolism Unit, Institute for
1
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
81
82
INHIBITORY EFFECT OF A PROPOLIS ON DI-NPROPYL DISULFIDE OR N-HEXYL SALICILATEINDUCED SKIN IRRITATION, OXIDATIVE STRESS AND
INFLAMMATORY RESPONSES IN MICE
Oršolić Nada1; Skurić Jadranka2; Đikić Domagoj1; Stanić Gabrijela3;
Kolarić Darko4
2
Clinic of Anaesthesia and Intensive Care Unit, Sveti Duh General
Hospital, Zagreb, Croatia
3
Department of Pathology, Sveti Duh General Hospital, Zagreb,
Croatia
4
Ruđer Bošković Institute, Centre for Informatics and Computing,
Zagreb, Croatia
1
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
83
84
BICUCCULINE ANTAGONIZES THE EFFECTS OF
DEHYDROEPIANDROSTERONE ON RAT BEHAVIOR
Samardžić Janko1; Švob Štrac Dubravka2; Đurić Miloš1; Obradović I
Dragan1
Institute of Pharmacology, Clinical Pharmacology and Toxicology,
Medical Faculty, University of Belgrade, Belgrade, Serbia
1
2
Laboratory for Molecular Neuropharmacology, Division of
Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia
E-mail: jankomedico@yahoo.es
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 hippocampalindependent 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
85
86
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
Department of Medical Biology; School of Medicine, University of
Zagreb, Croatia
2
Institute of Molecular Biology, Bulgarian Academy of Sciences,
Sofia, Bulgaria
3
Department of Histology and Embryology, School of Medicine,
1
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
87
88
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
Department of Biology, School of Medicine, University of Zagreb,
Zagreb, Croatia
2
Epigenetics Group, International Agency for Research on Cancer,
Lyon, France
1
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 selfrenewal. 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
89
90
QUERCETIN VS CHRYSIN: EFFECT ON LIVER
HISTOPATHOLOGY IN DIABETIC MICE
Sirovina Damir1; Oršolić Nada2; Zovko Končić Marijana3; Kovačević
Goran1; Benković Vesna2; Gregorović Gordana1
Department of Biology, Department of Zoology, Faculty of Science,
University of Zagreb, Zagreb, Croatia
2
Department of Biology, Department of Animal Physiology, Faculty of
Science, University of Zagreb, Zagreb, Croatia
3
Faculty of Pharmacy and Biochemistry, University of Zagreb,
Zagreb, Croatia
1
E-mail: sirovina@biol.pmf.com
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
91
92
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
Department of Biology, Department of Zoology, Faculty of Science,
University of Zagreb, Zagreb, Croatia
2
Department of Biology, Department of Animal Physiology, Faculty of
Science, University of Zagreb, Zagreb, Croatia
3
University of Pécs, Medical School, Department of Pathophysiology
and Gerontology, Pécs, Hungary
4
Department of Physiology and Immunology, Medical faculty Osijek,
J.J. Strossmayer University Osijek, Osijek, Croatia
5
Mutagenesis Unit, Institute for Medical Research and Occupational
Health, Zagreb, Croatia
6
Faculty of Pharmacy and Biochemistry, University of Zagreb,
Zagreb, Croatia
1
Equal contributors
ǂ
E-mail: sirovina@biol.pmf.hr
The in vitro antioxidant effects of polyphenols naringin and
curcumin and their effects on DNA damage caused by alloxaninduced 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
93
94
THERAPEUTIC POTENTIAL OF MULTIFUNCTIONAL
IRON-CHELATING AGENT M30 IN A RAT MODEL OF
Smailović Una1; Knezović Ana1; Mandel Silvia2; Youdim Moussa2;
Šalković-Petrišić Melita1
Research, School of Medicine, University of Zagreb, Croatia
2
Eve Topf Center for Neurodegenerative Diseases Research and
Department of Molecular Pharmacology, Faculty of Medicine,
Technion, Haifa, Israel
1
E-mail: Unna88@gmail.com
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 STZicv 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 ironchelator 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; ironchelator M30
95
96
N-TERT-BUTYL-Α-PHENYLNITRONE IMPROVES EX
VIVO GROWTH OF THE RAT EMBRYO AT THE AIRLIQUID INTERFACE IN A CHEMICALLY DEFINED
MEDIUM
Sobočan Nikola1,2; Sinčić Nino1; Majić Željka1; Katušić Ana1; Vlahović
Maja1; Šerman Ljiljana1; Beuc Robert3; Jurić-Lekić Gordana4; BulićJakuš Floriana1
Department of Medical Biology, School of Medicine, University of
Zagreb, Croatia
2
University Clinic Merkur, Zagreb, Croatia
3
Institute of Physics, Zagreb, Croatia
4
Department of Histology and Embryology, School of Medicine,
1
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
97
98
THE ROLE OF ESTROGEN IN HYPEROXIA-INDUCED
OXIDATIVE STRESS IN LIVER OF CBA/H MICE
Sobočanec Sandra1; Šarić Ana1; Mačak Šafranko Željka1; PopovićHadžija Marijana1; Aralica Gorana2; Korolija Marina1; Kušić Borka1;
Balog Tihomir1
Department of Molecular Medicine, Ruđer Bošković Institute,
Zagreb, Croatia
2
Department of Pathology Medical School University of Zagreb and
University Hospital Dubrava, Zagreb, Croatia
1
E-mail: ssoboc@irb.hr
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
99
100
ESLAV AWARNESS PRESENTATION
subjects connected with the breeding, health, welfare and use
of laboratory animals
Ševeljevic-Jaran Daša1
ESLAV Board ordinary member, Nat Reps coordinator - on behalf of
ESLAV Board
1
E-mail: dseveljevicjaran@gmail.com
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 (semiannual 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
•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
101
102
SALIVARY CORTISOL AS INDICATOR OF PERSONALITY
TYPES IN COMMON MARMOSETS?
Šlipogor Vedrana1, Millesi Eva2; Bugnyar Thomas1
Department of Cognitive Biology, Faculty of Life Sciences, University
of Vienna, Vienna, Austria
2
Department of Behavioural Biology, Faculty of Life Sciences,
University of Vienna, Vienna, Austria
1
E-mail: vedrana.slipogor@univie.ac.at
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
103
104
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
2
Croatian Institute for Brain Research, Department of Neuroscience,
School of Medicine, University of Zagreb, Croatia
3
Institute of Pharmacology, Clinical Pharmacology and Toxicology,
Medical Faculty, University of Belgrade, Serbia
4
Zagreb, Croatia
1
E-mail: dsvob@irb.hr
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
105
106
ARE POULTRY AND OTHER BIRDS SUFFICIENTLY
REPRESENTED AS ANIMAL MODELS IN BIOMEDICAL
RESERACH OF SOME HUMAN DISEASES IN CROATIA?
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).
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
Keywords: poultry; birds; animal models; non-infectious diseases in
humans; Croatia
Poultry Centre, Croatian Veterinary Institute, Zagreb, Croatia
Medicine, University of Zagreb, Croatia
3
Croatian Institute for Brain Research, Department of Neuroscience,
School of Medicine, University of Zagreb, Croatia
4
Department of Forensic Veterinary Medicine, Faculty of Veterinary
Medicine, University of Zagreb, Croatia
5
Department of Pathology, School of Medicine, University of Zagreb,
Croatia
6
Department of Neuroscience, University of Florida, Florida, USA
1
2
E-mail: marina.tisljar@gmail.com
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
107
108
THE ASSESMENT OF ANTICONVULSANT PROPERTIES
OF THE DRUG – PENTYLENETETRAZOLE SEIZURE
MODEL
Vlainić Josipa1; Švob Štrac Dubravka1; Jazvinšćak Jembrek Maja1
1
E-mail: josipa.vlainic@irb.hr
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
109
110
EXPRESSION OF SODIUM-D-GLUCOSE
COTRANSPORTER 1 IN MURINE TISSUES; SEXDEPENDENT EXPRESSION IN KIDNEYS
Vrhovac Ivana1; Breljak Davorka1; Karaica Dean1; Koepsell Hermann2;
Sabolic Ivan1
Molecular Toxicology Unit, Institute for Medical Research and
2
Institute of Anatomy & Cell Biology, University of Würzburg,
Würzburg, Germany
1
E-mail: ivrhovac@imi.hr
Sodium-D-glucose cotransporter SGLT1 (SLC5A1) in the brushborder 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 maledominant 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
111
112
CHOLINERGIC AND COGNITIVE DEFICITS IN
STREPTOZOTOCIN-INDUCED RAT MODEL OF
Knezović Ana1; Šalković-Petrišić Melita1
Research, School of Medicine, University of Zagreb, Croatia
1
E-mail: anaknezovic@yahoo.com
Rats treated intracerebroventricularly with streptozotocin (STZicv) have been proposed as a model for sporadic Alzheimer’s
disease (sAD). We aimed to characterize the STZ-icv doseand 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,
-20,39%/1 mg/kg and -18,34%/3 mg/kg). Results suggest longterm 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
113
114
OPTIMIZATION OF THE NUMBER OF EXPERIMENTAL
ANIMALS USING NEW COMPUTING TECHNIQUES
Lončarić Željka1; Hackenberger K. Branimir1
Subdepartment of Quantitative Ecology, Department of Biology,
Josip Juraj Strossmayer University, Osijek, Croatia
1
E-mail: hack@biologija.unios.hr
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
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
115
116
BILJEŠKE / NOTES
117
118
119
120
121
122
123
124
POPIS AUTORA / INDEX OF AUTHORS
125
126
A
Amšel Zelenika Tajana
Aralica Gorana
Artuković Branka
106
98
106
B
Bach-Rojecky Lidija
Bagarić Robert
Balog Marta
Balog Tihomir
Benković Vesna
Beuc Robert
Blanuša Maja
Blažetić Senka
Bourdineaud Jean-Paul
Breljak Davorka
Brgles Marija
Bubić-Špoljar Jadranka
Bugnyar Thomas
Bulić-Jakuš Floriana
Burckhardt Birgitta
Burckhardt Gerhard
24, 74
26, 50, 52
54
26, 98
78, 90
96
68
54
40
46, 56, 110
64
48
102
76, 86, 88, 96
56
56
C
Car Nikola
Ciarimboli Giuliano
78
46
Č, Ć
Čičin-Šain Lipa
58
D
Drinovac Višnja
Dumić-Čule Ivo
24
48, 50
Dž
Džaja Petar
106
Đ
Đikić Domagoj
Đurić Miloš
78, 80, 82
84
E
Erceg Gorislav
Erhardt Julija
60
104
F
Farkaš Vladimir
Forčić Dubravko
26, 50, 52
66
G
Gajski Goran
Garaj-Vrhovac Vera
Goluža Eleonora
Gospodinov Anastas
Grabarević Željko
Gregorović Gordana
Grgurević Lovorka
Gudan Kurilj Andrea
92
92
80
86
28, 106
90
48
30
H
Hackenberger K. Branimir
Hadžija Mirko
Halassy Beata
114
62
64
127
128
Heffer Marija
Herak-Kramberger M Carol
Herceg Zdenko
Himelreich Marta
Horvat Knežević Anica
Hranilović Dubravka
54
56
88
76
78
32
I
Ivić Ivan
Ivić Vedrana
Ivković Jana
92
54
56
J
Jazvinšćak Jembrek Maja
Jeleč Željko
Jovanov Milošević Nataša
Jurasović Jasna
Jurić-Lekić Gordana
Jurjević Ivana
108
80
106
68
76, 86, 96
60
K
Karaica Dean
Katušić Ana
Kesić Maja
Klarica Marijan
Klobučar I.V. Goran
Knezović Ana
Koepsell Herman
Kolarić Darko
Korolija Marina
Kovačević Goran
46, 56, 110
76, 88, 96
58
60
40
70, 94, 112
46, 110
82
62, 98
90
Krsnik Željka
Kurtović Tihana
Kušić Borka
104
64
98
L
Labak Irena
Lang Balija Maja
Lazarus Maja
Lisičić Duje
Lončar Andrija
Lončar Jovica
Lončarević Jelena
Lončarić Željka
54
64, 66
68
78, 80
70
56
76
114
LJ
Ljubojević Marija
56
M
Mačak Šafranko Željka
Majić Željka
Mandel Silvia
Markušić Maja
Micek Vedran
Mihaljević Ivan
Mikolić Anja
Millesi Eva
Mišić Marija
Mitrečić Dinko
Modun Darko
Mokrović Gordana
Mužić Vedrana
26, 98
76, 96
94
66
46, 56
56
72
102
106
34
74
58
76
129
130
Sobočanec Sandra
Stanić Gabrijela
Sulimanec Grgec Antonija
26, 98
82
72
Š
Šalković-Petrišić Melita
Šantak Maja
Šarić Ana
Šerman Ljiljana
Ševeljevic-Jaran Daša
Šlipogor Vedrana
Šrut Maja
Štambuk Anamaria
Švarc Alfred
Švob Štrac Dubravka
38, 70, 94, 112
66
36, 98
76, 88, 96
100
100
40
40
26, 50, 52
84, 104, 108
60
T
Tišljar Marina
106
46, 56, 110
84, 104
106
106
106
106
76, 86, 88, 96
90, 92
82
94
56
96
V
Vihnanek Lazarus Maja
Vismara Guglielmo
Vlahović Maja
Vlainić Josipa
Vrhovac Ivana
Vukičević Slobodan
Vukić Miroslav
80
42
76, 86, 88, 96
104, 108
46, 56, 110
48
60
N
Novak Sanja
92
O
Obradović I Dragan
Orct Tatjana
Orešković Darko
Oršolić Nada
84
68, 80
60
78, 80, 82, 90, 112
P
Paić Frane
Petrik József
Piasek Martina
Polić Bojan
Popović-Hadžija Marijana
88
78
72
36
62, 98
R
Radoš Milan
S
Sabolić Ivan
Samardžić Janko
Savić Vladimir
Seiwerth Sven
Semple-Rowland Susan
Severin Krešimir
Sinčić Nino
Sirovina Damir
Skurić Jadranka
Smailović Una
Smital Tvrtko
Sobočan Nikola
W
Wilkinson Anna
Y
Youdim Moussa
53
94
131
132
Z
Zovko Končić Marijana
90, 92
POKROVITELJSTVO / AUSPICE
Grad Zagreb/ The city of Zagreb
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

Pokusne životinje u znanstvenim istraživanjima Experimental

Transcription

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