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VOLUME 159, SUPPLEMENT 1

PALACKÝ UNIVERSITY PRESS, OLOMOUC

2015

OF THE FACULTY OF MEDICINE AND DENTISTRY

OF PALACKÝ UNIVERSITY, OLOMOUC

CZECH REPUBLIC

BIOMEDICAL PAPERS

Volume 159, Supplement 1

Published quarterlyMK ČR E 12793

Published and printed by Palacký University Press, OlomoucKřížkovského 8, 771 47 Olomouc, IČO 61989592

Olomouc 2015

ISSN 1213-8118eISSN 1804-7521

http://dx.doi.org/10.5507/bp.2015.042

Czech Anatomical SocietyCzech Society for Histo- and Cytochemistry

Palacký University in OlomoucFaculty of Medicine and Dentistry in Olomouc

MORPHOLOGY 201549th International Congress of the Czech Anatomical Society

52nd Lojda Symposium on Histochemistry

Under the Auspices of

prof. Mgr. Jaroslav Miller, M.A., Ph.D.Rector of the Palacký University in Olomouc

Prof. MUDr. Milan Kolář, Ph.D.Dean of the Faculty of Medicine and Dentistry in Olomouc

September 6–8, 2015Olomouc, Czech Republic

Sponsors

Agel, a.s.

Bamed, s.r.o.

CASA DEL CAFFÉ, s.r.o.

Fagron, a.s.

Hanácká kyselka, s.r.o.

I.T.A. – Intertact, s.r.o.

Melites, s.r.o.

Město Olomouc

Nikon, s.r.o.

Olympus Czech Group, s.r.o.

Scintila, s.r.o.

Sigma – Aldrich, s.r.o.

Spinchem, s.r.o.

YBUX, s.r.o.

ZENA-R, s.r.o.

Pivovar ZUBR, a.s.

Scientifi c Committee

Prof. MUDr. Jiří Ehrmann, Ph.D.Prof. MUDr. Karel Smetana, DrSc.Doc. MUDr. Ondřej Naňka, Ph.D.Prof. MUDr. Jaroslav Mokrý, Ph.D.

Prof. RNDr. Petr Dubový, CSc.Doc. MUDr. Vojtěch Kamarád, DrSc.Doc. MUDr. Stanislav Laichman, CSc.

Doc. RNDr. Petr Mlejnek, Ph.D.

Organization Committee

Doc. MUDr. Vojtěch Kamarád, DrSc.Prof. MUDr. Jiří Ehrmann, Ph.D.Doc. RNDr. Petr Mlejnek, Ph.D.

Doc. MUDr. Stanislav Laichman, CSc.

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GENERAL INFORMATION

VenueBuilding of the Theoretical Institutes of the Faculty of Medicine and DentistryHněvotínská 3, Olomouc

Registration and Information DeskBuilding of the Theoretical Institutes of the Faculty of Medicine and Dentistry

Offi ce Hours:Sunday, September 6, 2015 15:00–17:00Monday, September 7, 2015 08:00–17:00Tuesday, September 8, 2015 08:00–14:00

Languages of the conferenceEnglish, Czech, Slovak

Topics of the conference• Stem Cells• Morphogenesis• Neurosciences• Clinical Anatomy• History• Anatomy and Histology Teaching• Various Topics

PresentationsPlenary lectures - 20 min, lectures – 10 min, discussion – 5 min

PostersThe size of a poster panel is 100 cm by 100 cm

SOCIAL PROGRAM

Welcome Party, free of chargeSunday, September 6, at 7 p.m.

Get Together Party will take place at Restaurant FONTANA, Smetanovy Sady 603, Olomouc, on Monday, September 7, at 7.00 p.m., price 500 Kč

Czech Anatomical Society – member of the Council of Scientifi c Societies of the Czech RepublicSecretary offi ce: Charles University, First Faculty of MedicineU nemocnice 3, 128 00 Prague 2, Czech Republicphone: +420224965770, Fax: +420224965770 http://cas.lf1.cuni.cz

Czech Society for Histo-and Cytochemistry – member of the Council of Scientifi c Societies of the Czech RepublicSecretary offi ce: Simkova 870, 500 38 Hradec Kralové phone +420495816294 Fax +420495816376 http://www.cshc.cz

Authors are responsible for English.

Czech Anatomical SocietyCzech Society for Histo- and Cytochemistry

Palacký University in OlomoucFaculty of Medicine and Dentistry in Olomouc

Programme

MORPHOLOGY 201549th International Congress of the Czech Anatomical Society

52nd Lojda Symposium on Histochemistry

September 6–8, 2015Olomouc, Czech Republic

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SUNDAY, SEPTEMBER 6, 2015

15.00–17.00 Registration17.00–19.00 Opening CeremonyWords of WelcomeCultural program

Prof. Obručník memory dedicated lectureSedmera DElucidating cardiac conduction system formation with phylogenic approaches

Winning lecture of the 2014 Czech Anatomical Society & Olympus AwardOralová V, Matalová E, Janečková E, Drobná Krejčí E, Knopfová L, Šnajdr P, Tucker AS, Veselá I, Šmarda J, Buchtová MRole of c-Myb in chondrogenesis

Winning lecture of the 2014 Czech Society for Histochemistry and Cytochemistry & Nikon AwardKodet O, Lacina L, Krejčí E, Vlček Č, Šáchová J, Dvořánková B, Grim M, Štork J, Kodetová D, Kolář M,Strnad H, Smetana KMelanoma cells infl uence the diff erentiation pattern of human epidermal keratinocytes

19.00–20.30 Welcome party

20.00 Meetings of the national committees of ČAS and ČSHC

MONDAY, SEPTEMBER 7, 2015

9.00–9.40Plenary lecturesChairs: Naňka O, Mlejnek P

9.00–9.20Holibka VAnatomy teaching at the medico-surgical school in Olomouc, 1782–1875

9.20–9.40Druga RThe claustrum – history research

9.45–10.45Stem CellsChairs: Smetana K, Ehrmann J

9.45–10.00Smetana K Jr., Lacina L, Dvořánková BWound repair, ageing and cancer: challenge for new therapeutic modalities

10.00–10.15Kvasilová A, Dvořánková B, Drobná Krejčí E, Smetana K, Grim MExpression of some markers of stem cells and tissue-specifi c progenitors in human postnatal skin

10.15–10.30Čížková D, Vávrová J, Mičuda S, Doleželová E, Brůčková L, Filip S, Mokrý JRole of transplanted bone marrow cells in the skeletal muscle regeneration

10.30–10.45Měšťanová V, Varga I, Bencat M, Adamkov MMyoid cells of thymus in question of origin and function

10.45 –11.15 Coff ee Break 11.15 –12.00 Plenary session of ČAS 12.00 –12.10 Congress Photo 12.10 –13.00 Lunch

13.00–14.15Sessions in Section ANeurosciences Chairs: Mechírová E, Mazurová Y

13.00–13.15Mazurová Y, Bezrouk ATransgenic rat model of Huntington’s disease – a useful tool for study of the development of progressive neuronal degeneration

13.15–13.30Dubový P, Klusáková I, Korimová A, Joukal M, Hradilová-Svíženská IUpregulation of Toll-like receptors after nerve injury and infl ammatory signaling from Wallerian degeneration

13.30–13.45Joukal M, Kuklová A, Solár P, Klusáková I, Dubový PKolmer cells and their immunophenotyping in the rat choroid plexus after peripheral nerve injury

13.45–14.00Salaj M, Barinka F, Kubová H, Druga RDiff erences in expression of calcium binding proteins in the perirhinal and retrosplenial cortex of the rat

14.00–14.15Bálentová S, Hajtmanová E, Filová B, Borbelyová V, Lehotský J, Adamkov MCognitive and histopathological changes in the rat brain following fractionated irradiation

14.15–14.45 Coff ee Break

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14.45–16.00Poster Sessions Chairs: Smetana K, Dubový P

16.10–16.40 Plenary session of CSHC

13.00–16.00Sessions in Section BTeaching MorphologyChairs: Ehrmann J, Kachlík D, Krajčí D

13.00–13.20Ehrmann J, Lužná P, Čížková KRole of histology in the era of molecular biology

13.20–13.35Kachlík D, Štěpánková T, Musil V, Báča VAnatomical eponyms

13.35–14.35 Round Table Kachlík D, Slížová D, Lovásová KRole of the anatomy practical teaching in current curriculum

14.35–16.00E-Learning in Teaching Histology

14.35–14.50Lichnovská R, Krajčí D (Mrs.), Erdosová B, Krajčí DTeaching practical histology with digital (virtual) slides

14.50–15.05Krajčí D (Mrs.), Lichnovská R, Edosová B, Krajčí D Application of electronic testing in histology and anatomy practicals

15.05–15.25Krajčí D, Kopečný T, Novotný R A new system of electronic evaluation of results of PC-delivered tests

15.25–15.45Krajčí D, Kylar P GAMETIX – a new software for managing MCQ databases and creation of examination sheets

19.00 Dinner

TUESDAY, SEPTEMBER 8, 2015

9.00–10.15MorphogenesisChairs: Stingl J, Rybárová S

9.00–9.15Naňka O, Vicente-Steijn R, Blom NA, Jongbloed MRM, Sedmera DRemodelling of atrioventricular canal myocardium determines atrioventricular conduction patterns

9.15–9.30Čížková K, Rajdová A, Ehrmann JCYP epoxygenases and their relevance for human prenatal development

9.30–9.45 Mlejnek PABC transporters and their role in the drug resistance

9.45–10.00Pisal RCloning of synthetic intron into coding sequence of red fl uorescent gene for confi rmatory expression of micro RNA cloned within the intron

10.00–10.15Šimůnková P, Djakow J, Cinek O, Vajner L, Uhlík JUltrastructural and videomicroscopic fi ndings in patients with genetically proven primary ciliary dyskinesia

10.15–10.45 Coff ee Break

10.45–11.30Cardiovascular SystemChairs: Mokrý J, Kučera T

10.45–11.00Stingl J, Pirk J, Straka Z, Setina M, Kachlík D, Stach J, Musil VMorphology of vasa vasorum in the explanted bypassed saphenous veins

11.00–11.15Kučera T, Smorodinova N, Rozsívalová K, Přidal J, Ďuřišová M, Pirk J, Bláha M, Melenovský V, Kautzner J Infl ammatory cells in human atrial myocardium from patients undergoing open heart surgery

11.15–11.30Tonar Z, Kochová P, Cimrman R, Perktold J, Witter K Segmental diff erences in the orientation of smooth muscle cells in the tunica media of porcine aortae

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11.30–12.15AnatomyChairs: Slížová D, Druga R

11.30–11.45 Kachlík D., Hajek P, Konařík M, Báča VArteria brachioradialis – overview

11.45–12.00Kachlík D, Blanková A, Báča V Supernumerary carpal bones

12.00–12.15Výborná E, Dorko F, Holibka V, Tokarčík J, Chylíková J Historical overview of morphological studies of palatine tonsil

11.00–11.30Histology practical room in the new Theoretical Departments building, fl oor 2, room n. 2.540

Erdosová B, Krajčí (Mrs.), Lichnovská R, Krajčí DA live demonstration of histology practical with digital slides

12.10–13.30Various TopicsChairs: Laichman S, Kamarád V

12.15–12.30Bonczek O, Šerý O, Hloušková A, Lochman J, Izakovičová-Hollá L, Šoukalová J, Štembírek J, Míšek I, Černochová P, Krejčí P, Vaněk JSequencing analysis of PAX9 and MSX1 genes in the Czech population

12.30–12.45 Blažková Z, Oborná I, Filipčíková R, Bezdíčková M, Hubáček P, Mlčoch M, Dobiáš M, Pastucha DPregnant woman as participants of traffi c accident in anatomical-clinical relation- research intent and preliminary study

12.45–13.00Horváthová F, Danielisová v, Kuchárová B, Solár P, Rybárová S, Hodorová I, Mihalik JThe eff ect of deprenyl administration on antioxidant status of rat testis

13.00–13.15Ostrovska L, Vostejnova L, Dzugan J, Slama P, Kubina T, Ukraintsev E, Kraličkova M, Hubalek Kabalcova MThe biological evaluation of ultra-fi ne titanium with improved mechanical strength for tissue engineering

13.15–13.30Überall IIconology of disease in Ranaisence art

13.30Closing SessionAuthors of the best poster award

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POSTERS

1. Beznoska P, Hrebíková H, Chvátalová J, Pisal R, Čížková D, Diaz D, Mokrý J Analysis of decellularized skeletal muscle scaff olds and their settlement by myogenic cells 2. Bludovská M, Křížková V, Kotyzová D, Kubíková T Pleuran (beta-glucan from Pleurotus ostreatus ) potentiates free radial induced hepatotoxic eff ects of hexavalent

chromium in rats 3. Boleková A, Tomášová L, Hvizdošová N, Bona M, Lovásová K, Matéff y S, Kluchová D Postnatal development of nitrergic hippocampal neurons after prenatal infl uence of retinoic acid 4. Celá P, Shylo N, Weatherbee SD, Buchtová M Transmembrane protein 107 is a critical factor for craniofacial development 5. Danková M, Mechírová E, Domoráková I, Janitorová Poliačíková M, Stebnický M

Pharmacological preconditioning: chance for formation of ischemic tolerance in rabbit’s spinal cord 6. Domoráková I, Mechírová E, Danková M, Janitorová Poliačiková M, Stebnický M Glial reaction in the spinal cord ischemia after noradrenaline preconditioning 7. Dorko F, Výborná E, Holibka V, Tokarčík J, Chylíková J, Horňáková L Autonomic innervation of renal haemolymph node in rats 8. Dosedělová H, Veselá I, Krejčí P, Kunová M, Buchtová M Morphological analysis of mouse skeleton following AZD4547 treatment 9. Gregová K, Tóth S, Bilecová-Rabajdová M, Kudláčková M, Kovalčíková S, Jonecová Z The eff ect of quercetin on the expression of infl ammatory mediators10. Hodorová I, Fiala P, Mihalik J, Horváthová F, Rybárová S Immunohistochemistry of colorectal carcinoma: expression of p53 and MDM211. Holanová V, Zahradníček O Continuosly regeneration dentition in anoline lizards: the model of postnatal teeth development leading to

changes in tooth shapes and numbers12. Hrebíková H, Chvátalová J, Díaz D, Beznoska P, Mokrý J Naturally derived scaff old for skeletal muscle regeneration13. Hvizdošová N, Tomášová L, Bona M, Matéff y S, Kluchová D Retinoic acid in the development of the NADPH-d positive neurons in rat brain14. Chvátalová J, Mokrý J, Hrebíková H, Pisal R Colonization of muscle scaff olds by diff erent cell types 15. Chyliková J, Kamarád V, Výborná E, Dvořáčková J, Holéczy P M1/M2 Macrophages polarization in white aditose tissue of morbid obese patients16. Jirkovská M, Zuska K The infl uence of maternal diabetes on proliferative potential of human term placenta17. Jonecová Z, Tóth S, Maretta M, Gregová K, Varga J Modifi cation of apoptotic cell rate in the intestinal graft mucosa during cold preservation18. Kikalová K, Kopecký M, Charamza J, Tomanová J, Zemanek P Blood pressure of girls aged 10 to 18 with respect to intra –abdominal fat tissue19. Killinger M, Veselá I, Buchtová M Eff ect of WNT5a on chondrogenesis and limb development20. Klepáček I, Shbat A, Zedníková-Malá P Abbot Seka’s cranial morfology and 2D reconstruction of his face with individual landmarks21. Kolesová H, Čapek M, Janáček J, Sedmera D Comparison of diff erent tissue clearing methods for 3D imaging techniques in connexin40: GFP – expressing

embryonic and adult mice22. Kolinko Y, Cendelín J, Tonat Z, Králíčková M Stereological assessment of cerebellum microcirculation in the Lurcher mice 23. Krajčí D (Mrs.), Lichnovská R, Erdösová B, Kopečný T, Novotný R, Krajčí D Evaluation of results of MCQ tests applied in electronic format24. Kuklová A, Solár P, Joukal M, Klusáková I, Dubový P Study of changes in the rat choroid plexus after peripheral nerve injury using fl uorescent conjugated dextran25. Langová P, Balková S, Buchtová M Microarray analysis of mandible regionalization during mouse development

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26. Lichnovská R, Krajčí D (Mrs.), Erdösová B, Krajčí D Impact of E-learning format using virtual microscopy in practical histology sessions27. Makovický P, Tůmová E, Makovický P, Rimárová K The eff ect of food restriction on the liver morphology: experimental study on the chicken model28. Matéff y S, Vecanová J, Tomášová L, Hvizdošová N, Rozprávková M, Pribulová J, Matuševská M Diff erence in description of topographic position of communicating branches in diff erent medical literatures29. Matuševská M, Pribulová J, Rozprávková M, Redaj L, Ohlasová J, Matéff y S, Lovásová K, Kluchová D Anatomy and pathological anatomy of periodontium depending to the state of oral health30. Mechírová E, Gdovinová Z, Skorvanek M, Domoráková I, Danková M Immunohistochemical visualization of α-synuclein in colonic lamina propria mucosae31. Miklošová M, Dankovčík R, Herich R Application of double staining method for fetal specimens32. Oralová V, Matalová E, Knopfová L, Švandová E, Šmarda J, Buchtová M A role of c-Myb in ossifi cation33. Pribulová J, Matuševská M, Rozprávková M, Redaj L, Ohlasová J, Matéff y S, Lovasová K, Kluchová D The variation in morphology of fi rst upper premolar34. Putnová B, Buchtová M, Jekl V, Hodan R, Machoň V, Štembírek J Healing of soft and hard tissues of rabbit temporomandibular joint following the application of stem cells35. Rejtarová O, Kuchař M, Eliáš P Anatomical variations of popliteal artery – a possible case of lower limb ischaemia36. Riedlová J, Paulová M, Vignerová J, Soumar J Children of obese mothers in their fi rst 18 months37. Rozprávková M, Redaj L, Ohlasová J, Lovasová K, Matuševská M, Pribulová J, Matéff y S, Kluchová D Anatomical changes of temporomandibular joint in the aging process38. Rybárová S, Hodorová I, Mihalik J, Vecanová J Expression of carbonic anhydrase IX and its correlation with clinicopathological characteristics in lung cancer

cells39. Salaj M, Barinka F, Kubová H, Druga R Diff erences in expression of calcium binding proteins in the perihinal and retrosplenial cortex of the rat40. Sedláčková M, Anger M, Matiješčuková N Intercellular junctions of corona radiata cells and oocyte in preovulatory period 41. Tokarčík J, Dorko F, Výborná E, Chyliková J, Bražina D Interactive 3D Models42. Tomašová L, Hvizdošová N, Šmajda B, Bona M, Matéff y S, Kluchová D Various noxious factors and their eff ect on the developing nervous system43. Tóth S, Maretta M, Gregová K, Šoltés J, Švaňa M, Pribula M, Kušnier M, Jonecová Z Regulatory action of quercetin on cyclooxygenase -2 in the model of ischemia/reperfusion injury of small

intestine44. Výbohová D, Mellová Y, Adamicová K Blood and lymphatic microvascular changes in relation to VEGF-A and VEGF-C expression in psoriatic lesions45. Wurst Z, Zach P, Ibrahim I, Bartoš A, Musil V, Patzelt M, Mrzílková J Tractography of corpus callosum and gyrus cinguli in patients with Alzheimer’s s Disease

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1).

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Editorial

Dear colleagues,It is our great pleasure to invite you to read in the ab-

stracts of MORPHOLOGY 2015, the 49th International Congress of the Czech Anatomical Society and the 52nd Lojda Symposium on Histochemistry. Within a wide-ranging programme, we are highlighting the themes of Stem Cells, Morphogenesis, Neurosciences, and Clinical Anatomy. We organize as well the roundtable about Anatomy and Histology Teaching. These interdisciplin-ary topics spanning both basic and clinically oriented re-search showcase the broad range of topics studied by the

morphologists in the Czech Republic. We are happy to notice many fruitful collaborations with colleagues from abroad. We find also hopeful that the morphological dis-ciplines continue to attract attention of young colleagues.

We cordially welcome you in Olomouc, hoping that this congress will be both scientifically and socially suc-cessful meeting.

Kind regards,

On behalf of the Organizing SocietiesOndřej Naňka and Jaroslav Mokrý


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Cognitive and histopathological changes in the rat brain following fractionated irradiation

Sona Balentovaa, Eva Hajtmanovab, Barbora Filovac, Veronika Borbelyovad, Jan Lehotskye, Marian Adamkova

aInstitute of Histology and Embryology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovak RepublicbDepartment of Radiotherapy and Oncology, Martin University Hospital, Martin, Slovak RepubliccInstitute of Medical Physics, Biophysics, Informatics and Telemedicine, Faculty of Medicine, Comenius University in Bratislava, Slovak RepublicdInstitute of Molecular Biomedicine, Faculty of Medicine, Comenius University in Bratislava, Slovak RepubliceInstitute of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovak Republic

Aims. In the present study we investigated whether expected radiation-induced histopathological changes in the rat brain will be associated with appropriate neuro-cognitive dysfunction.

Methods. Adult male Wistar rats randomized into sham (0 Gy) and 2 experimental groups (survived 30 and 100 days after treatment) received fractionated whole-brain irradiation (one 5 Gy fraction /week for 4 weeks) with a total dose of 20 Gy of gamma rays. Morris water maze cognitive testing, histochemistry, immuno-histochemistry and confocal microscopy were used to determine whether the cognitive changes are associated with the alteration of neurogenesis, astrocytic response and activation of microglia in two neurogenic regions: the hippocampal dentate gyrus (DG) and the subventricular zone-olfactory bulb axis (SVZ-OB axis) represented by the anterior SVZ, vertical arm, elbow and horizontal arm.

Results. Irradiation negatively influenced the spatial memory in Morris water maze 100 days after radiation treatment, however the reference memory remained in-tact. Neurodegenerative changes were characterized by a significant increase of Fluoro-Jade-positive cells 30 days after irradiation in both of investigated brain regions. The SVZ-OB axis displayed a steep decrease of neuroblasts and less obvious decline of stem/precursor cells until 100 days after treatment. Strong decline or re-expression of astrocytes was seen in the SVZ-OB axis until 100 days after irradiation, however the hippocampal DG did not showed any significant changes. A decrease and subse-quent re-expression of the activated microglia was seen during the experiment.

Conclusion. Results showed, that fractionated irradia-tion led to cognitive impairment closely associated with the initiation of neuronal cell death, inhibition of neuro-genesis, activation of astrocytes and microglia indicated an early delayed radiation-induced changes.

ACKNOWLEDGEMENTS

The study was supported by a projects "Center of translational medicine"/"Creating a new diagnostic algo-rithm for selected cancer diseases", ITMS: 26220220021 and ITMS: 26220220022 co-financed from EU sources and European Regional Development Fund.

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Analysis of decellularized skeletal muscle scaffolds and their settlement by myogenic cells

Pavel Beznoska, Hana Hrebikova, Jana Chvatalova, Rishikaysh Pisal, Dana Cizkova, Daniel Diaz, Jaroslav Mokry

Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic

Aims. Tissue engineering represents a progressing branch of research with the aim to develop biologi-cally equivalent replacements of tissues and organs. Construction of the carriers is based on the knowledge of nanotechnology, or tissue decellularization. Suitable bio-compatible and biodegradable carriers are then colonized with the desired cell populations. The decellularized non-toxic scaffold with 3D structure cultured in vitro provides a temporary mechanical support for the implanted cells and moreover, it forms suitable environment for adhesion, growth and proliferation of the cells, and stimulates them to production of extracellular matrix.

Methods. In this work we decellularized the intact and cardiotoxin-injured mouse skeletal muscles and analyzed the formed scaffolds for presence of components of the extracellular matrix. Then, the scaffolds were cultivated with C2C12 mouse myoblasts to compare penetration of the cells under physiological and pathophysiological con-ditions. The muscle injury was provoked in C57BL6 /J mice by injecting 75 μL of cardiotoxin (0.06 μg/μL) along the longitudinal axis of the tibialis anterior muscle. The intact and cardiotoxin-injured skeletal muscles (4 days after injection) were decellularized using immersion in the detergent solution 1% SDS (sodium dodecyl sulfate) and simultaneous mechanical agitation for 48 h. Then, the tissue was rinsed in sterile PBS buffer with antibi-otics (penicillin, streptomycin, amphotericin B) for 24 h and DNase was subsequently used to remove residual cellular DNA. After washing in PBS, the scaffolds were sterilized by UV radiation for 30 min. Then, the scaffolds were cultivated in vitro and seeded with C2C12 mouse myoblasts. After 8 and 14 days of cultivation the scaffold with cells were fixed in 10% formalin and embedded into paraffin. The sections were stained by hematoxylin-eosin, green trichrome, Weigert resorcin-fuchsin and Sirius Red. Immunohistochemical detections were performed by in-direct three-step (LSAB) methods.

Results. The decellularized skeletal muscles kept 3D structure, retained components of the extracellular matrix,


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i.e. collagen and elastic fibers and served as a scaffold for myoblasts, which in various degrees adhered to its surface and penetrated into it.

Conclusion. According to the preliminary results of the digital image analysis, the myoblasts penetrated in greater percentage into the scaffold prepared from the toxin-injured skeletal muscle, which highlights a role of the bioscaffold microenvironment for a successful recel-lularization and opens new approaches for modification of bioconstructs in tissue engineering.

ACKNOWLEDGEMENTS

This study was supported by Grant Agency of Charles University in Prague No. SVV-2015-260179, PRVOUK P37/06 and with European Social Fund No. CZ.1.07/2.3.00/30.0061.

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Pregnant women as participants of traffic accident in anatomical-clinical relation – research

intent and preliminary studyZdeňka Blazkovaa, Ivana Obornab, Radka Filipcikovaa, Marcela Bezdickovaa, Petr Hubacekc, Michal Mlcochd,

Martin Dobiase, Dalibor Pastuchaf

aDepartment of Anatomy, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic bDepartment of Obstetrics and Gynecology, University Hospital, Olomouc, Czech Republic cDepartment of Emergency, University Hospital, Olomouc, Czech Republic dDepartment of Obstetrics and Gynecology, Regional Hospital of Tomas Bata, Zlin, Czech Republic eDepartment of Forensic Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic fDepartment of Rehabilitation, Faculty of Medicine, University of Ostrava, Czech Republic

Aims. With increasing traffic density pregnant woman participate daily as the driver, co-driver, cyclist, a pas-senger in public transport or walker. These facts cause increase of probability of a collision. The consequences of traffic collisions can be crucial not only for the woman and her fetus, but can also have significant health and eco-nomic impact on the whole society. The aim of our work is based on anatomical and clinical research which will lead to greater social interest in this issue. Next purpose is to achieve enclosure of the results of our work into the preclinical and clinical teaching of medical students and disciplines allied to medicine.

Methods. We only know statistics data (from UZIS Company) about the total abortion respectively the num-ber of abortions, and we have data on total maternal mortality too. However, unlike in other countries, more detailed information on the relationship between abor-tion and maternal mortality incidental to road accidents

is not available. Considering that we discuss about mar-ginal issue at present, we want to get a greater amount of detailed information from more workplaces on the extent and nature of the trauma, but other injuries to pregnant women and her fetus too. Using case reports analysis we want to focus on strength vectors interaction during traf-fic accident and anatomical-clinical variations in pregnant woman and her fetus.

Results. The outputs will be used not only for teaching anatomy of forensic medicine, emergency medicine and obstetrics, but they are also directly applicable to clini-cal practice. Depending on the stage of pregnancy and the severity of the trauma of pregnant women there is an increased risk of injury or death of the fetus. Moreover, even relatively small wounds of mothers might have fatal consequences for the fetus.

Conclusion. Traffic accidents consequences can occur at both organisms of pregnant and fetus immediately, but they might be captured from a distance or, especially psy-chological, over the long-term horizons. Although there are not many recorded cases, this issue is very topical, because the threats are always on two lives. It is therefore crucial underpinning of the incidence in order to obtain valid data from medical facilities throughout the Czech Republic and determine the true prevalence of traffic ac-cidents of pregnant women and their health effects.

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Postnatal development of nitrergic hippocampal neurons after prenatal influence of retinoic acidAdriana Bolekova, Lenka Tomasova, Natalia Hvizdosova,

Martin Bona, Kvetuse Lovasova, Stanislav Mateffy, Darina Kluchova

Department of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic

Aims. Retinoic acid (RA) is a biologically active de-rivate of vitamin A that plays important role during the development. The aim of the study was to investigate the influence of prenatal application of RA on postnatal de-velopment of hippocampal nitrergic neurons in the rat. Toxicity of RA could be a growing concern both in devel-oping (where large therapeutic doses are taken to control vitamin A deficiency) and developed countries (where intake of vitamin A often exceeds recommended dietary allowances).

Methods. 24 female Wistar rats were used in the ex-periment. 18 pregnant animals were administrated by RA intraperitoneally in dose 1 mg/kg during three consecutive gestational days: 14th, 15th and 16th (RA group); 6 preg-nant rats were without experimental influence (control – K group). The offspring of both groups were divided into 5 groups and underwent experimental procedures in different postnatal days: in the 1st, 7th, 14th and 21st and in the adulthood (in the age of 3 month). Animals in each group were anaesthetized and perfused transcardially,


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brains were removed and processed for NADPH-d histo-chemical method. Nitrergic neurons of the hippocampus were investigated on the coronal sections of each brain sample.

Results. During physiological conditions without re-ceiving RA by pregnant rats (K groups) the NADPH-d positive interneurons were appeared in the all hippocam-pal layers since the 7th postnatal day. The number and density of nitrergic neurons increased until the 14th post-natal day, than decreased and stabilized on the 21th day to the pattern of the adult hippocampus. These neurons were morphologically developed on 14th postnatal day already and were seen to be similar to those in the adulthood. Numerous NADPH-d positive interneurons were found in the hilus of dentate gyrus. Single layers of hippocampus proprius and dentate gyrus significantly differed in their NADPH-d staining. In the groups after RA administra-tion (RA groups), the number of nitrergic neurons in hip-pocampal formation markedly decreased in all postnatal periods compared to control animals. Neurons seem to be damaged and delayed in their development. The number and intensity of positive interneurons was decreased in deep layers of hippocampus and hilus of dentate gyrus. The number and density of neurons increased very gently until 21th day, they supposed to be finally developed this time. In the adult animals prenatally influenced by RA, the pattern of NADPH-d staining of hippocampal regions was identical to the control group.

Conclusion. Changes of nitrergic interneurons con-firmed that prenatal administration of RA can modify NADPH-diaphorase activity in the rat hippocampus. The morphology and intensity of NADPH-d positive hippo-campal neurons has changed during the postnatal devel-opment. It can be resumed that prenatal application of RA could play the specific role in different regions of hippocampal formation during postnatal development of nitrergic neurons in the rat.

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Sequencing analysis of PAX9 and MSX1 genes in the Czech population

Ondrej Bonczeka,b, Omar Serya,b, Alena Hlouskovaa, Jan Lochmana, Lydie Izakovicova-Hollac, Jana Soukalovac,

Jan Stembirekb,d, Ivan Misekb, Pavlina Cernochovac, Premysl Krejcie, Jiri Vanekc

aLaboratory of DNA Diagnostic, Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech RepublicbLaboratory of Animal Embryology, Institute of Animal Physiology and Genetics, v.v.i., The Academy of Sciences of the Czech Republic, Brno, Czech RepubliccClinic of Stomatology, Faculty of Medicine, Masaryk University, Brno and St. Anne's University Hospital in Brno, Czech RepublicdDepartment of Oral and Maxillofacial Surgery, University Hospital Ostrava, Czech RepubliceDepartment of Dentistry, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

Aims. Tooth agenesis is one of the most common de-velopmental anomalies in humans. Tooth development (odontogenesis) is a very complicated and complex pro-cess that involves interplay between oral ectoderm and mesenchyme. These interactions are mediated by more than 350 signaling proteins. Synergistic and antagonistic interactions between these proteins may lead to local ac-tivation or inhibition of the expression. The aim of our study was to investigate the relationship between PAX9 and MSX1 genes variants and tooth agenesis in the Czech population.

Methods. The selected regions of PAX9 and MSX1 genes were analyzed by direct sequencing and compared with the reference sequence from the GenBank on-line database (NCBI).

Results. We found several novel variants in the PAX9 and MSX1 genes. The newly identified causative g.9527G>T mutation is located in intron 2, in the area recognized as the splicing site between exon 2 and intron 2 of the PAX9 gene. The other mutations in the PAX9 gene e.g. insertion g.5100_5101insC (rs11373281) with simulta-neous substitution g.5272C>G (rs4904155) in exon 1, and mutation g.10934C>T (Gly203Gly, rs61754301) in exon 3 seem to be polymorphisms in the Czech population. We also observed polymorphisms g.10276A>G (rs12882923) and g.10289A>G (rs12883049) in IVS2 (intervening se-quence 2) previously related to tooth agenesis in Polish studies.

In the MSX1 gene we identified novel heterozy-gous mutation that may cause amino acid substitutions Ala40Gly. This mutation was found both in the group of patients suffering from hypodontia and in the control group. Furthermore, intron and exon mutations without influence on amino acid change have been identified.

Conclusion. We identified a novel g.9527G>T muta-tion associated with oligodontia in a family comprising nine members. All the other described PAX9 and MSX1 genetic variants were present both in patients with tooth


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agenesis and controls. We hypothesize that tooth agenesis in our cohort of patients was caused by mutations in re-gions different from the PAX9 and MSX1 exons analyzed in this study or in different genes.

ACKNOWLEDGMENT

This project was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic IGA MZ ČR No. NT/11420-6.

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Pleuran (beta-glucan from Pleurotus ostreatus) potentiates free radical induced hepatotoxic

effects of hexavalent chromium in ratsMonika Bludovskaa, Vera Krizkovab, Dana Kotyzovaa,

Tereza Kubikovab,c

aDepartment of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech RepublicbDepartment of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech RepubliccBiomedical Centre, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic

Aims. β-Glucans are widely used immunomodulatory nutritional supplements. Many studies have also proved their antioxidant and hepatoprotective properties. The study was designed to evaluate the effects of pleuran, β-(1.3/1.6) glucan from Pleurotus ostreatus, on the toxic and oxidative effects and histological changes caused by an acute Cr(VI) intoxication in rats.

Methods. Wistar albino male rats were divided into three groups: control, Cr, and Cr + pleuran. Pleuran (250 mg/kg/day) was administered by oral use for 10 (half of the animals for 11) consecutive days. K2Cr2O7 (40 mg/kg/day) was administered i.p. as a single dose on the 10th day of the experiment. Half of animals in each group were sacrificed 24 h and the rest 48 h after the administra-tion of Cr. Lipid peroxidation (LP), reduced glutathione (GSH) and activities of glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) and the content of Cr were estimated in the liver, the activities of ALT, AST and GLDH were determined in serum. Liver tissue samples were collected for histological and imuno-histochemical examination.

Results. Administration of Cr(VI) significantly de-creased liver content of GSH and significantly increased LP. The activities of ALT, AST and GLDH were signifi-cantly increased in serum. Treatment with pleuran signifi-cantly increased the content of Cr in the liver, increased the effect of chromium on LP (24 and 48 h post Cr dose), on the activities of ALT, AST and GLDH (24 hours post Cr dose) and on chromium-induced histopathologi-cal changes. The control group of animals was without

prominent changes in liver parenchyme and connective tissue. This finding was in contrast to the Cr animal group and especially to Cr + pleuran group of animals. In the mentioned group, some striking histological changes were observed: swollen hepatocytes near to central vein, dilated sinusoids and vacuolated hepatocytes in portal area in addition to connective tissue changes.

Conclusion. The only antioxidant effect of pleuran administration was an increase in the content of GSH in the liver, which was decreased by Cr administration. From histology point of view was the most affected Cr + pleuran group of animals with the most conspicuous tis-sue changes.

ACKNOWLEDGEMENT

We wish to thank Ms. M. Šlajerová for the excellent technical assistance. The work was supported by the SVV project No. 260 047 and by the project ED2.1.00/03.0076 from European Regional Development Fund.

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Transmembrane protein 107 is a critical factor for craniofacial development

Petra Celaa,b, Natalia Shyloc, Scott D. Weatherbeec, Marcela Buchtovaa,b

aInstitute of Animal Physiology and Genetics, The Academy of Sciences of the Czech Republic, Brno, Czech Republic bDepartment of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno, Czech RepubliccDepartment of Genetics, Yale University, New Haven, CT, USA

Aims. Primary cilium is a microtubule-based exten-sion of the cell surrounded by a membrane, which plays a striking role in many developmental patterning events, including establishment of left-right asymmetry or skeletal formation. Moreover, primary cilia have been implicated in the regulation of several key developmental signaling pathways. Functional cilia are required for activation of SHH pathway, which is essential for morphogenesis of variety tissues and organs. Recently, Transmembrane protein 107 (TMEM107) was found to be essential for ciliogenesis during embryonic development (Christopher et al., 2012). Tmem107 homozygous mutant embryos were described as embryonic lethal and displaying SHH-related defects such as polydactyly, which is also a hallmark of defective cilia.

Methods. First, we analyzed the external craniofacial phenotype of Tmem107 mutant mice during period of palate formation (E12.5 – E15.5). Wild type, heterozy-gous and homozygous mice were compared. Transversal histological sections stained with H&E were used for mi-croscopical analysis of palatal fusion.

Results. Tmem107 homozygous null embryos exhib-ited abnormalities in head shape with microphthalmia or


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anophthalmia and exencephaly. Furthermore, shorten-ing of the snout and cleft lip was observed. Histological analysis revealed defects in patterning and size of nasal cavity, narrow or shorter nasal septum and smaller cupola-shaped tongue. The nasal cartilages were deformed and nasal septum was curved when unilateral cleft lip was visible. At E12.5, Tmem-/- mice had deficiency of mid-line structures, both nasal cavities were connected and exhibited simple shallow morphology. At E15.5, cleft of secondary palate occurred or palatal shelves were fused in midline; however, they were not attached to the short nasal septum.

Conclusion. In summary, we found large spectrum of craniofacial defects in Tmem107 null homozygous mice, while heterozygous animals displayed normal morphol-ogy. Thus, Tmem107 seems to be essential for proper for-mation of lip and palate during embryonic development and further determination of its role in palatogenesis will advance our understanding of causations of ciliopathy disorders.

ACKNOWLEDGEMENTS

This study was supported by the Grant Agency of the Czech Republic (14-37368G) and institutional support (RVO:67985904).

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Role of transplanted bone marrow cells in the skeletal muscle regeneration

Dana Cízkovaa, Jiřina Vavrovab, Stanislav Micudac, Eva Dolezelovac, Lenka Bruckovaa, Stanislav Filipd,

Jaroslav Mokrya

aDepartment of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech RepublicbDepartment of Radiobiology, Faculty of Military Health Sciences in Hradec Kralove, University of Defence, Hradec Kralove, Czech Republic cDepartment of Pharmacology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic dDepartment of Oncology and Radiotherapy, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic

Aims. Recently discovered ability of the adult bone marrow cells (BMCs), incl. hematopoietic and mesenchy-mal stem cells, to contribute to injury–induced skeletal muscle regeneration has raised new possibilities in treat-ment of skeletal muscle diseases. However, mechanisms by which BMCs participate in regenerative myogenesis have still remained to be fully elucidated.

Methods. To extend our knowledge of the role of exog-enous adult BMCs in the skeletal muscle regeneration, we intravenously transplanted mouse lacZ+ o r GFP+ freshly

isolated BMCs into whole-body lethally irradiated immu-nocompetent mice 7 hours after or 4 weeks before the cardiotoxin-induced injury of the recipients’ tibialis ante-rior muscles. Seven to 33 days after the toxin injection, the injured and intact contralateral skeletal muscles were excised and fixed in 4% paraformaldehyde, processed for X-gal histochemistry to detect lacZ+ cells, embedded into GMA resin (to obtain 1 μm thin sections) or paraffin or fixed in 2% paraformaldehyde, frozen in methylbutane, chilled in liquid nitrogen, cryosectioned and examined for GFP fluorescence. The presence of lacZ gene in injured muscles was determined by qPCR. For transmission elec-tron microscopy, the excised injured and intact contralat-eral skeletal muscles were fixed in 3% paraformaldehyde and 0,05% glutaraldehyde in 0.1 M Sörensen buffer and embedded into LR White resin. Immunohistochemical detections were performed by indirect three-step (LSAB) methods using primary antibodies: monoclonal rabbit anti-desmin, clone Y66 (GeneTex) and polyclonal rabbit anti-nestin (Sigma).

Results. The skeletal muscles of recipients injured 7 h before the transplantation did not regenerate, never-theless, X-gal positivity was predominantly identified in desmin- and nestin- multinucleated cells resembling for-eign body giant cells located in the injured areas, 14 and 33 days after grafting. On the contrary, the recipients’ muscles injured 4 weeks after the transplantation fully regenerated. X-gal or GFP positivity was observed in the regenerating muscles excised 7 days after the injury in numerous inflammatory cells, in some newly formed myo-blasts and myotubes and in some cells of the endomysium and in the regenerated muscles examined 28 days after the toxin injection in some endomysial cells and rarely in newly formed myofibers. qPCR verified presence of transplanted lacZ+ BMCs in injured recipients’ muscles.

Conclusion. Our results confirmed ability of intra-venously transplanted exogenous BMCs to settle in the injured skeletal muscle and participate in the skeletal muscle regeneration. They generated blood cells that infil-trated endomysium and took part in the cleaning reaction, the first step of the regeneration process, and moreover, they contributed to new myofibers formation.

ACKNOWLEDGEMENTS

This work was supported by the grant projects: No. 15-09161S from the Grant Agency of the Czech Republic and PRVOUK P37/06.


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9

CYP epoxygenases and their relevance for human prenatal development

Katerina Cizkovaa, Aneta Rajdovaa, Jiri Ehrmanna,b

aDepartment of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepublicbDepartment of Clinical and Molecular Pathology and Laboratory of Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

Aims. Cytochrome P450 (CYPs) are involved in metabolism of various endogenous and exogenous com-pounds, including clinically used drugs. There is growing evidence that some of the CYP enzymes ale play role in development of organism. In this regard, CYP epoxygen-ases (CYP2C and CYP2J subfamilies) are very interest-ing. CYP epoxygenases convert arachidonic acid (AA) to four regioisomeric epoxyeicosatrienoic acids (EETs). EETs have relativelly short half-life in vivo. They are me-tabolized to less active dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). Because of EETs regulate a variety of processes potentially relevant to development, such as cell proliferation and differentia-tion, and cell signalling, we decided to estimate expression pattern of EETs-producing and EETs-degrading proteins in human embryonic/foetal intestines, liver and kidney samples from 7th week to 20th week of intrauterine devel-opment (IUD) to consider possible importance of EETs for human prenatal development.

Methods. We compared expression patterns of CYP epoxygenases (CYP2C8, CYP2C9, CYP2C19, CYP2J2) and sEH in human embryonic/foetal intestines, liver and kidney samples from 7th week to 20th week of IUD. The expression patterns in these organs were obtained by two-step immunohistochemistry method.

Results. We confirmed expression of CYP epoxygen-ases and sEH in tested human embryonic/foetal intes-tines, liver and kidney. In intestines, distribution of CYP epoxygenases and sEH expression could lead to diverse concentration of EETs along crypt-villus axis. In crypt area, where new cells of intestinal epithelia are generated, is strong expression of CYP2C8 and CYP2C9 together with low expression of sEH. The main product of these epoxygenases is 14,15-EET which has the strongest pro-liferation potential from all EETs regioisomers. On the other hand, in apex of villi, where are fully differentiated cells, expression of CYP2C8 and CYP2C9 is lower and expression of sEH is higher than in crypt area. In kid-ney samples, CYP epoxygenases as well as sEH are ex-pressed in renal corpuscles and tubular system. On the other hand, nephrogenic zone, where new nephrons are developed, is possitive for CYP epoxygenases but nega-tive for sEH.

Conclusion. Distribution of CYP epoxygenases and sEH in intestinal epithelium and nephrogenic zone of the kidney suggests an influence of EETs on cell prolifera-tion and differentiation and consequently development

of these organs. If EETs really play role in development, alterations in the strict spatio-temporal pattern of expres-sion of CYP epoxygenases and / or sEH during human prenatal development by xenobiotics could have adverse consequences for developing organism.

ACKNOWLEDGEMENTS

We thank to Ivana Travnickova and Jitka Stastna for excellent technical support. This study was supported by LF_2015_008.

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Pharmacological preconditioning: chance for formation of ischemic tolerance in rabbit’s

spinal cordMarianna Dankovaa, Eva Mechirovaa, Iveta Domorakovaa,

Miroslava Janitorova Poliacikovaa, Milan Stebnickyb

aDepartment of Histology and Embryology, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republicb2nd Depatrment of Surgery, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic

Aims. The present study was designed to evaluate the noradrenaline and bradykinin preconditioning on spinal cord ischemic injury using a transient lumbar spinal cord ischemia model in rabbits. Noradrenaline is known to be responsible for the body's reaction to different types of stress. Bradykinin is a biologically active nonapeptide that mediates inflammatory response and increases vasodila-tion. The main purpose of this study is to summarize the results dealing with acquisition of ischemic tolerance and utilization of preconditioning for protection of the vulner-able neurons in the rabbit’s spinal cord against ischemia and reperfusion. Pharmacological preconditioning is a form of organ and tissue protection induced by a sublethal dose of various chemical agents against lethal ischemic insult. Preconditioning involves the induction of cellular protective mechanisms.

Methods. The New Zealand white rabbits were divided into 4 groups: 1. control group (n – 7), 2. ischemic group – 20 min of ischemia/48 h of reperfusion (n – 7), 3. ex-perimental group (n – 11) – preconditioned with bolus of bradykinin 48 h prior to 20 min of ischemia and 48 h of reperfusion and 4. experimental group (n – 9) – precon-ditioned with bolus of noradrenalin 48 h prior to 20 min of ischemia and 48 h of reperfusion.

We demonstrated the distribution of proteins NeuN and ubiquitin after pharmacological preconditioning prior to ischemic insult. Tarlov scoring system was used for as-sessment of neurological function of hind limbs.

Results. In the groups 2, 3, and 4 of experimental animals 20 min of ischemia caused different intensity of ubiquitin positivity in the motor neurons of lamina IX and interneurons of intermediate zone of spinal cord. Dark


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brown aggregates of ubiquitin were visible in the cyto-plasm of ischemic motor neurons of 2. ischemic group. Accumulation of stress proteins leads the neurons to the necrosis. Ubiquitin positive structures were also discerned in the surrounding neuropil. Ubiquitin aggregates are signs of damaged cells that lost the ability of intracellular hydrolytic degradation of altered proteins. We assume that neurons, unable to synthesize new ubiquitin, appear to be those that succumb to the degenerative process induced by ischemia.

Significant decrease of NeuN positive neurons (P<0.001) was observed after bradykinin and noradrena-lin preconditioning in both groups of animals after 20 min of ischemia followed by 48 h of reperfusion in com-parison with the control group. Despite of this significant decrease in number of NeuN positive neurons, it seems there were enough viable neurons that are able to guaran-tee active movement of the hind limbs.

Conclusion. The results indicated that degree of spinal cord damage after ischemia reperfusion is associated with number of ubiquitin and NeuN positive neurons. The as-sessment of neurological function of hind limbs showed that pharmacological preconditioning can partially im-prove these results.

ACKNOWLEDGEMENTS

Supported by VEGA grant 1/0815/14 and 9/GSD/2012.

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Glial reaction in the spinal cord ischemia after noradrenaline preconditioning

Iveta Domorakovaa, Eva Mechirovaa, Marianna Dankovaa,Miroslava Janitorova Poliacikovaa, Milan Stebnickyb

aDepartment of Histology and Embryology, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republicb2nd Department of Surgery, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic

Aims. Protein homeostasis is essential for proper func-tion of neurons and oligodendrocytes. Missfolded proteins are degraded by ubiquitin proteasome system. Astrocytes have neuroprotective effect, while small changes in pro-tein homeostasis in oligodendrocytes have detrimental effect to neurons. Our study was focused to ubiquitin reac-tion in oligodendrocytes and comparison between astro-cytic GFAP and ubiquitin reactions in the rabbit spinal cord after pharmacologic preconditioning followed by ischemia/ reperfusion.

Methods. New Zealand male rabbits were precondi-tioned by noradrenaline 2 days prior to 20 min of spinal cord ischemia induced by occlusion of abdominal aorta. Animals were divided into 4 groups (n=5). Group (1) – sham operated; (2) – 20 min of ischemia/ reperfusion

2 days; (3) – noradrenaline preconditioning/ 20 min of ischemia/ 24 h of reperfusion; (4) – noradrenaline pre-conditioning/ 20 min of ischemia/ 48 h of reperfusion. Monoclonal mouse anti-GFAP (glial fibrillary acidic pro-tein) and polyclonal anti-ubiquitin were used for demon-stration of ubiquitin reaction (UR) in oligodendrocytes and GFAP and UR in the astrocytes.

Results. Our previous study showed that noradrenaline preconditioning followed by 20 min of ischemia followed by 24 h of reperfusion (3), induces ischemic tolerance in motor neurons. Moreover, number of surviving neurons significantly increases and hind limb function was im-proved in comparison to ischemic group (2). Acquisition of ischemic tolerance slows down deleterious effect of 20 min of ischemia. However, longer period of reperfusion in the group (4) showed reduction of surviving neurons vs. group (3). In ischemic group (2) reactive GFAP posi-tive astrocytes were present in the white matter, their nu-clei and processes showed strong UR. Astrocytes were grouped around the damaged areas of the grey matter. Efferent axons radiated from anterior horns were swollen and surrounded by oligodendrocytes with strong UR in the nuclei and cytoplasm. In preconditioned group (3) astrocytes have thin processes both in the grey and white matter. In the grey matter moderate UR was observed in the rounded nuclei of astrocytes, however in the white matter UR was found also in the astrocytic processes and also in the nuclei of oligodendrocytes. In preconditioned group (4) reactive astrocyte GFAP positive and strong UR was found mainly in the white matter. Efferent axons extended from anterior horns were swollen, varicosed and surrounded by number of ubiquitin positive glial nuclei.

Conclusion. Ischemic tolerance induced by precon-ditioning is the way how to protect vulnerable neurons exposed to ischemia/ reperfusion. Further research should found optimal “time window” for application of ischemic postconditioning which is more relevant for clinical prac-tice.

ACKNOWLEDGEMENTS

Supported by VEGA grant 1/0815/14 and 9/GSD/2012.


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12

Autonomic innervation of renal haemolymph node in rats

František Dorkoa, Eva Vybornaa, Vladimir Holibkaa, Jan Tokarcika, Jaroslava Chylikovab, Lubica Hornakovac

aDepartment of Anatomy, Faculty of Medicine, University of Ostrava, Czech Republic bDepartment of Histology and Embryology, Faculty of Medicine, University of Ostrava, Czech RepubliccSmall Animal Clinic, University of Veterinary Medicine and Pharmacy in Kosice, Slovak Republic

Aims. Haemolymph nodes are part of the lymphatic system and its lymphatic sinuses contain erythrocytes at various stages of development, associated with phenome-na haemosiderosis, which causes brownish-red macroscop-ic appearance of these nodes. Renal haemolymph node (RHLU) in rats is located near the a. renalis. Significant dominance of medullary sinuses and strands that contact directly with the fibrous capsule or the subcapsular sinus is seen in RHLU. The intermediate and medullary sinuses contain many erythrocytes as a rosette clusters with mac-rophages with considerable accumulation of hemosiderin in there. Haemolymph renal gland cortex has irregular areas of lymphoid tissue, which form irregular nodules of lymphocytes without the typical bright germinal centres. We studied RHLU autonomic innervation in rats.

Methods. For studying RHLU innervation we used 25 rats of both sexes, Wistar strain, aged 6 months. Animals were anesthetised with pentobarbital, 20 mg/kg, i.p. Removed RHLU were fixed in 4% formaldehyde for 2 to 4 h. To prove ACHE – positive nerve fibres we used di-rect tiocholin method modified by El-Badawi and Schenk. Adrenergic neural components were depicted using gly-oxic acid fluorescence histochemical method modified by Švalev and Zučkova.

Results. Specifically fluorescing adrenergic nerve fi-bres enter the RHLU in common bundle with arteries, through the nodes hilus, but sporadically at other loca-tions of the nodes surface. We recorded rich periarterial nerve plexus running in medullary strands. Toward the nodes surface, as the arteriolar branching is gradually reduced, the number of penetratingly fluorescent accom-panying nerve fibres. The largest relative density of adr-energic nerve fibres is detected around the arterioles and the parenchyma of the cortex and medulla sections where the amount of erythrocytes and lymphoid cells were seen. These cellular components come into intimate contact with adjacent nerve fibres. In areas that are free cells poor-er and do not contain the rosette clusters of macrophages, adrenergic innervation is limited to a small amount of fluorescent periarterial profiles. ACHE – positive nerve fibres in the hilar lymph nodes enter the medulla through the medullary columns and in the capsule and subcapsu-lar plexuses. In the medulla ACHE – positive nerve fibres creating perivascular tangles, but we've also noticed a free ACHE – positive nerves, which was not accompanied

vessels. Fibres from these perivascular tangle penetrated through the cortical border into cortex and paracortical area. In paracortical area we observed abundant perivas-cular tangle. In the cortex ACHE – positive nerve fibres was located parafolliculary.

Conclusion. In renal haemolymph node adrenergic and ACHE positive nerves innervate the same sections as in the other lymph nodes. Their total number is larger in this structure, mainly in areas with maximum erytrophages and siderophages concentration. Nerve fibres enter the nodes in the common bundle of the arteries through the hilus. Renal haemolymph node is an organ that eliminates the red cells from the lymph as well as controls immune responses.

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Morphological analysis of mouse skeleton following AZD4547 treatment

Hana Dosedelovaa,b, Iva Veselaa,b, Pavel Krejcic, Michaela Kunovac, Marcela Buchtovaa,b

aDepartment of Anatomy, Embryology and Histology, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic bLaboratory of Animal Embryology, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech RepubliccDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Aims. FGF-receptor tyrosine kinases (FGFR1-4) rep-resent attractive therapy targets, as activating mutations, gene amplifications or chromosomal translocations cre-ating fusion genes involving FGFR genes was associated with several cancers. Many FGFR inhibitors have been developed in the past years, including recently discovered compound AZD4547. As FGFR inhibitors may be useful for achondroplasia therapy, we aim to analyze the effect of AZD4547 on cartilage growth at in vivo experimental conditions.

Methods. Newborn mice were injected intraperito-neally by AZD4547 at concentration 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 250 μM, 1000 μM or 2000 μM in amount of 50 μL. Control group of animals was injected by DMSO in appropriate concentration. Injection was repeated every week for 4 weeks and animals were eu-thanized by cervical dislocation at day 28 after the first injection. Skin was removed from animals and they were fixed in 100% ethanol, stained with Alizarin red/Alcian blue solution, and cleared in KOH/Glycerol. The body weight was measured at the time of injection (every week). The length of the body (from snout to tip of the tail) and tail (from radix to tip of the tail) was measured at the end of experiment.

Results. No changes in weigh and length of animals were observed by 0.5 μM, 1 μM, 5 μM and 10 μM of AZD4547. Small increase in weight and length was found in animals injected by 50 μM concentration. On the other hand, animal weight decreased with higher doses


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of AZD4547. At 1000 μM and 2000 μM concentration, AZD4547 led to death in 32% and 43% of mice, respec-tively. The organs mostly affected by 1000 μM AZD4547 were liver (necrotic areas, lymphocytes accumulation), lungs (alveolar hemorrhage), and kidneys (reduction of renal tubules). The lethality occurred in 2-3 days after the first injection. Next, we analyzed the effect of AZD4547 on long bones and skull morphology. We found no signifi-cant changes of skeletal growth with low concentrations of AZD4547. Only dose of 50 μM AZD4547 caused the improvement of bone growth but difference in compari-son to DMSO control was not statistically significant. In contrast, higher concentrations than 250 μM resulted in shortening of long bones and skull. General bone mor-phology was similar to controls in all analyzed bones. Bones of chondrocranium as well as desmocranium were affected by treatment.

Conclusion. High doses of AZD4547 caused signifi-cant changes of skeletal growth where the inhibition of bone growth was accompanied by severe toxicity in the mouse. The face exhibited shorter morphology resembling syndromic craniosynostoses such as Apert syndrome, which are also caused by disruptions of FGF signaling. Therefore, only 50 μM concentration demonstrated posi-tive effect on animal growth and elongation of long bones during postnatal stages.

ACKNOWLEDGEMENTS

This research was supported by the Grant Agency of the Czech Republic (14-31540S) and institutional support (RVO: 67985904).

14

The claustrum – history of researchRastislav Druga

Institute of Anatomy, 1st and 2nd Faculty of Medicine, Charles University in Prague, Czech RepublicInstitute of Physiology, The Academy of Sciences of the Czech Republic, Prague, Czech Republic

Although the first descriptions of the claustrum ap-peared in 19 century

(Burdach 1812) majority of exact data were pub-lished in postwar period. Claustrum has been identified in all eutherian and metartherian mammals. Differences in morphology and relationships to cortex allow us to distinguish its two parts – dorsal (insular) and ventral (piriform) claustrum. An increasing body of evidence has been collected about its transcription factors expressed during development. The dorsal claustrum was classified as being a derivative of the lateral pallium, whereas the ventral claustrum was considered to be a derivative of the ventral pallium and thus the claustrum is regarded as a telencephalic, pallial subcortical mass. With using of basic staining in all mammals cells of various sizes and shapes

were found but most common are medium-sized neurons with triangular, multipolar, oval and fusiform perikarya. Golgi impregnation studies reveal two major classes of neurons: polymorphous projection neurons with spiny dendrites and local interneurons characterized by oval perikarya and smooth aspiny dendrites. In electron mi-croscopic studies from three to five classes of neurons were identified.

Most of the neurons in the claustrum are glutama-tergic (excitatory) projection neurons. The proportion of GABA-ergic interneurons was estimated at between 7–12%. The population of neurons containing calcium-binding proteins (CBPs) has been estimated at 12 – 15% of all claustral neurons. The dorsal as well as the ven-tral claustrum contain several neuropeptides and nitric oxide synthase which frequently colocalize with CBPs. Claustrum specific proteins (cadherins, netrin G2, la-texin, Gng2) were recently demonstrated.

Majority of afferent connections of the claustrum orig-inate in the neocortex, weaker projection originate in the limbic cortical regions. Cortico-claustral projections are topographically organized and significantly overlapped. Subcortical projections originate in the thalamus, amyg-dala and several brainstem structures. The main output of the claustrum is the return pathway to the cortex. The ipsilateral projection is much stronger than the contra-lateral one. The fact that the claustrum has bidirectional connections with all sensory, motor, associative and lim-bic areas suggests participation of this nucleus in more general and more complex functions.

The convergence of information from different corti-cal areas and subcortical structures indicate that the claus-trum may be the site at which modality specific signals are bound into a multimodal representation. The complex connectivity of the claustrum represents a backround of hypotheses suggesting that this telencephalic structure is part of the system involved in salience detection and that the claustrum has importat role in cognitive processing.

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Upregulation of Toll-like receptors after nerve injury and inflammatory signaling from Wallerian

degenerationPetr Dubovya,b, Ilona Klusakovaa,b, Andrea Korimovaa,b,

Marek Joukala,b, Ivana Hradilova-Svizenskaa,b

aDepartment of Anatomy, Division of Neuroanatomy, Faculty of Medicine, Masaryk University, Kamenice 3, 62500 Brno, Czech RepublicbCentral European Institute of Technology (CETEC), Masaryk University, Kamenice 3, 62500 Brno, Czech Republic

Aims. Wallerian degeneration (WD) distal to nerve injury is considered to be aseptic inflammation includ-ing fragmentation of axons and their mitochondria, up-regulation of ECM molecules and their cleavage. All the products of WD named as damage associated molecular


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patterns (DAMPs) can be released into blood circulation and reach to remote structures without blood barriers. Here, DAMPs are detected by Toll-like receptors (TLRs) that mediate inflammatory reaction. We hypothesize that upregulation of TLRs is responsible for inflammatory re-actions in the dorsal root ganglia (DRG) both associated and non-associated with injured nerve.

Methods. The right sciatic nerve of adult rats was crushed (n=15), ligated (n=15) or transected (n=15) and animals were left to survive for 1, 3, 7, 14 and 21 days. The sciatic nerve and DRG of naïve rats (n=6) and sham-operated rats (n=6) were used as controls. TLR2, 3, 4, and 9 were detected by standard indirect immunofluorescence in the cryostat sections through distal and proximal nerve stumps as well as contralateral sciatic nerve. In addition, bilateral DRG of L4-L5 and C7-C8 segments were used for TLRs detection. Activated Schwann or satellite glial cells were identified by co-localization with monoclonal S100 antibody. In addition, we tested upregulation of TLR9 in Schwannoma cells (RT4) by N-formyl-methionyl-leucyl-phenylalanine (fMLF), the prototypical N-formylated peptide released by fragmented mitochondria.

Results. Sciatic nerve injuries induced upregulation of investigated TLRs in Schwann cells distal and proximal as well as in neurons and satellite glial cells of DRG from ip-silateral and contralateral sides. Schwann cells cultivated in medium supplied with fMLP displayed an increased expression of TLR9 in endosomes and patches of plasma membrane particularly in cytoplasmic processes or small protrusions. Medium containing fMLP also induced in-crease of NF-kappa B, TNF alpha and IL1beta.

Conclusion. Our results shown that TLRs are upregu-lated in Schwann cells activated by nerve injury and in DRG not only associated, but also non-associated with injured nerve. The increased expression of TLRs can be regulated by DAMPs that are responsible for dissemi-nation of inflammatory reactions into remote nervous structures.

ACKNOWLEDGEMENTS

We would like to thank Ms. Marta Lněníčková, Ms. Jitka Mikulášková and Ms. Dana Kutějová for their skillful technical assistance. The work was supported by the Project “CEITEC – Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund.

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Porcine liver vascular corrosion casts – our up to now experience

Lada Eberlovaa,b, Vaclav Liskab,c, Hynek Mirkab,d, Tomas Gregore, Zbynek Tonarb,f,g, Richard Palekb,c,

Martin Skalab,c, Michael Emingerb,c, Ondrej Troupb,c, Jachym Rosendorfb,c, Milena Kralickovab,g,

Alois Lametschwandtnerh

aDepartment of Anatomy, Faculty of Medicine in Pilsen, Charles University in Prague,Karlovarska 48, 301 66 Pilsen, Czech RepublicbBiomedical Centre, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic cDepartment of Surgery, Faculty of Medicine in Pilsen, Charles University in Prague and University Hospital in Pilsen, Alej Svobody 80, 304 60 Pilsen, Czech RepublicdDepartment of Imaging Methods, Faculty of Medicine in Pilsen, Charles University in Prague and University Hospital in Pilsen, Alej Svobody 80, 304 60 Pilsen, Czech RepubliceNew Technology and Materials Centre – CENTEM, University of West Bohemia, Univerzitni 8, 306 14 Pilsen, Czech RepublicfEuropean Centre of Excellence, University of West Bohemia, Univerzitni 8, 306 14 Pilsen, Czech RepublicgDepartment of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Karlovarska 48, 301 66 Pilsen, Czech RepublichDepartment of Organismic Biology, University Salzburg, Hellbrunner Str. 34, A-5020 Salzburg, Austria

Aims. In experimental medicine, pig is frequently used as a surrogate animal model. The aim of our study was to assess porcine liver corrosion casts we made with the use of Biodur E20 (Biodur products, Heidelberg, Germany).

Material and Methods. 4 porcine livers were filled via the portal vein with Biodur E 20, the volume injected ranged around 700 mL. Corrosion casts were examined by using: 1. multi-slice human CT (Somatom Sensation 64, Siemens, Forchheim, Germany), slice thickness 0.6 mm, voxel size 0.4 x 0.4 x 0.6 mm, 2. micro-CT (Xradia XCT 400, Pleasanton, CA, USA), the pixel size used for imaging was 17 μm, 9.5 μm and 4.5 μm, 3. scanning electrone microscopy (SEM): specimens were sputtered with gold for 60 s and examined in Stereoscan 250 SEM (Cambridge, U.K.) at an accelerating voltage of 10 kV.

Results. Biodur E20 filled liver sinusoids and in some places continued into the central veins. Interlobular arter-ies as well as peribiliary plexuses were misssing. Tortuous, globular structures on the course of sinusoids appeared to be resin extravasations. Biodur E20 is alcohol resistant, the quality of vascular replica was satisfactory. For the detailed morphological description, micro-CT needs to be completed by SEM examination.

Conclusion. Biodur E20 is suitable for high volume corrosion casting. Micro-CT images allow stereological assessment of the microvessels, in combination with SEM corrosion casting enhances the possibility of morphomet-rical analysis of vascular network in health and disease.


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ACKNOWLEDGEMENTS

This work was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic under Project No. IGA MZ ČR 13326, by the proj-ect CZ.1.05/2.1.00/03.0076 from European Regional Development Fund and SVV No 260 170, by CENTEM project reg. No. CZ.1.05/2.1.00/03.0088, and partly sup-ported also by the European Regional Development Fund under Project “NTIS – New Technologies for Information Society”, European Centre of Excellence, CZ.1.05/1.1.00/02.0090.

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Role of histology in the era of molecular biologyJiři Ehrmann, Katerina Cizkova, Pavla Luzna,

Anna Konieczna

Department of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

Aims. The implementation of molecular biology methods in histology/embryology had important conse-quences, as the new discoveries may significantly extend the current knowledge about cells and tissues. The aim of this overview is to give some examples how „molecular histology“ may serve as good model for clinical medicine, in this particular case in the area of study of multidrug re-sistance and study of precancerous lesions of oesophagus.

Methods. Broad spectrum of molecular biology ap-proaches were applied with emphasis to use formalin fixed, paraffin embedded samples which are still gold standard for histology assessment.

Results. Based on molecular profiling of histologic samples, identification of new biomarkers for prediction of development of oesophageal adenocarcinoma and new therapeutic perspectives for multidrug resistance overcom-ing were suggested.

Conclusion. The engagement of new molecular tech-niques into the current morphological concept will open new horizons in histology/embryology research. However, relevant changes in the system of histology/embryology education are necessary.

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The effect of quercetin on the expression of inflammatory mediatorsKristina Gregovaa, Stefan Totha,

Miroslava Bilecova-Rabajdovab, Michaela Kudlackovaa, Sona Kovalcikovaa, Zuzana Jonecovaa

aDepartment of Histology and Embryology, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicbDepartment of Medical and Clinical Biochemistry and LABMED, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic

Aims. The aim of this work was to observe the changes in expression of inflammatory mediators after ischemia-reperfusion injury (IRI) of small intestine in rats after quercetin administration. Quercetin is a natural flavonoid, able to neutralized free radicals. Quercetin acts as an an-tihistamine and an anti-inflammatory mediator. Systemic inflammatory reaction is a result of IRI of the small in-testine. Experiment was focused on mRNA expression analyses of pro-inflammatory cytokine TNFα and anti-inflammatory cytokine IL-10 after IRI under quercetin regulatory action.

Methods. Adult male Wistar rat SPF–Charles River (n=28) were randomly assigned to the control group (E) or experimental groups (Q). Quercetin dissolved in etha-nol or ethanol vehiculum waere applied intraperitoneally 30 min before the complete ischemia of a. mesenterica cra-nialis. Ischemia was performed by microvascular clamp, lasted 1 h and was followed by 1 h (E1, Q1) and 4 h (E4, Q4) of reperfusion period. After the reperfusion period, bioptic samples were harvested. Total mRNA was isolated from the complete jejunal wall; subsequently RT-PCR was used followed by electrophoresis and semiquantitative data evaluation.

Results. We studied quercetin effect on mRNA lev-els of pro-inflammatory cytokine TNFα and anti-inflam-matory cytokine IL-10 referring to reperfusion periods. The protective effect of quercetin was most dominant after 4 h of reperfusion, when we observed decrease in mRNA levels in both pro-inflammatory cytokine TNFα P≤0,001 (227±7%) and anti-inflammatory IL10 P≤0,001 (82.8±31%). Histological analyses provided similar ten-dencies and supported the theory of quercetin protective character in the inflammation process.

Conclusion. The obtained results suggest a great pos-sible therapeutic effect of antioxidant quercetin on the expression of inflammatory mediators and therefore its use in preconditioning might be considered.

ACKNOWLEDGEMENT

This study was supported by VEGA 1/0478/2014 grant.


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Immunohistochemistry of colorectal carcinoma: expression of p53 and MDM2

Ingrid Hodorovaa, Patrik Fialab, Jozef Mihalika, Frantiska Horvathovaa, Silvia Rybarovaa

aDepartment of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicbDepartment of Pathological Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic

Aims. Investigation of p53 and MDM2 immunoreac-tivity, as a potential marker of p53 function, in formalin-fixed paraffin-embedded tissues of colorectal carcinoma. Colorectal carcinoma is diagnosed in approximately one million people annually, and it has one of the highest cancer-related mortalities, with 694 000 deaths reported globally in 2012. About 40% of patients have a metastatic disease at diagnosis. Mechanisms of colorectal carcino-genesis involve multiple tumour supressor genes (APC, TP53, TGF-b) and oncogenic genes (RAS, BRAF, PTEN and PIK3CA). Inactivation of TP53 pathway by TP53 mu-tation is present in nearly half of colorectal tumours and is one of the key genetic steps in colorectal carcinogenesis.

Methods. For detection of p53 (mouse anti-p53, DO7, Biogenex) and MDM2 (mouse anti MDM2, clone SMP14, Santa Cruz Biotechnology, inc.) we have used indirect immunohistochemical method. Study population consists of 32 patients of colorectal carcinoma (17 males and 15 females) and median age at diagnosis was 72.5 years (range 57-88 years).

Results. Our study revealed undetectable reaction of p53 in the majority (87.5%) of colorectal carcinomas. Positive immunoreaction was observed only in four cases (12.5%) of colorectal carcinomas. None tissue samples shown MDM2 expression.

Conclusion. Our findings are not consistent with pre-vious study indicating that p53 mutations occur in half of colorectal carcinoma, but detection of p53 may help predict a tendency toward malignancy in patients with this type of cancer.

ACKNOWLEDGEMENT

Thank Mgr. Ivana Čigášová Perunská and Mgr. Diana Gagyiová for your kind cooperation.

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The effect of deprenyl administration on antioxidant status of rat testis

Frantiska Horvathovaa, Viera Danielisovab, Barbora Kucharovac, Peter Solarc, Silvia Rybarovaa,

Ingrid Hodorovaa, Jozef Mihalika

aDepartment of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicbInstitute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak RepubliccDepartment of Cell Biology, Faculty of Science, P. J. Safarik University in Kosice, Slovak Republic

Aims. Despite the fact that a certain level of reactive oxygen species (ROS) is needed for sperm capacitation and acrosome reaction, ROS is considered as one of the main factors causing male infertility. We focused on cho-sen antioxidant enzymes (CuZnSOD, MnSOD, and CAT) in rat testis, sperm count and sperm damage after depre-nyl administration.

Methods. 18 male Wistar rats (390 g, aged 8-9 weeks) were randomly divided into 3 groups and daily intraperito-neally injected for 30 days with either saline, low-dose of deprenyl (LDD, 0.0025 mg/kg per day, Sigma M003) or high-dose of deprenyl (HDD, 0.25 mg/kg per day). After the last drug administration animals were killed by lethal dose of thiopental. The activities of antioxidant enzymes were measured by spectrophotometric method and their localiza-tion was identified by immunohistochemistry. The sperm damage was determined employing flow cytometry, when TMRE and Annexin V/FITC analyses were performed.

Results and Conclusions.CuZnSOD and MnSOD ac-tivity were elevated in both experimental groups. Also an increase of approximately 20% in sperm count was found in the LDD group. As by SOD enzymes, CAT levels were elevated, too. Average data of TMRE and Annexin V/PI cells staining indicates, that spermatozoa were not af-fected the deprenyl administration. Histological evaluation performed on testicular sections of all groups showed no alterations compared to control. Immunohistochemical evaluation of CuZnSOD sections showed positive signals in spermatids and Leydig cells, in HDD also in spermato-gonia. MnSOD detection was positive in spermatogonia, spermatocytes, spermatids and Leydig cells. CAT positive signals were recorded in Leydig cells, germ cells and sper-matids. We can conclude, that deprenyl administration did not alter seminiferous epithelium and sperm vitality. Moreover, the low dose had positive influence on sperm count and both deprenyl doses caused elevation of anti-oxidant enzymes activities in rat testis.

ACKNOWLEDGEMENTS

This work was supported by Scientific Agency of the Slovak Ministry of Education and Slovak Academy of Sciences (VEGA) under projects 1/0928/11, 1/0394/15, and 1/0224/12.


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Naturally derived scaffold for skeletal muscle regeneration

Hana Hrebikova, Jana Chvatalova, Daniel Diaz, Pavel Beznoska, Jaroslav Mokry

Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic

Aims. Tissue engineering is closely associated with production of biomaterials. One of these strategies is orientated to decellularization which is able to produce scaffolds with preserved architecture, vasculature and innervation pathways for subsequent regeneration and reconstruction of the tissue. Decellularization provides extracellular matrix (ECM) with its attachment sites and adequate environment for cells colonizing this scaffold, reconstituting the decellularized organ.

Methods. The protocol is based on the immersion of tissues in detergent solution in combination with agitation and consists of the following steps: 1. hypertonic buffer 5M NaCl2. hypotonic buffer 10 mM TrisHCl 3. detergent 1% SDS 4. 0.1% peracetic acid 5. PBS buffer 6. DNase I7. PBS buffer

Decellularization efficiency was determined by his-tological staining (haematoxylin-eosin, Alcian blue, Sirius Red). The preservation of the ECM components was assessed by immunohistochemistry (collagen IV). Preservation of collagen as a main component of ECM was assessed by biochemical method: hydroxyprolin as-say. Removal of cell nuclei was determined by quantifica-tion of DNA. Cytocompatibility was tested by seeding the muscle scaffolds with murine myoblasts for 12 days. Decelularized scaffold was transplanted into mouse and in terms from 1 to 4 weeks scaffolds were removed from mice and histologically analysed (haematoxylin-eosin).

Results. Whole-organ decellularization was achieved with the described protocol. Histological sections were stained with standard haematoxylin-eosin, Alcian blue and by Sirius Red. These stainings revealed absent nuclei or cy-toplasmic components compared to physiological skeletal muscle, presence of glycosaminoglycans and preservation of collagen fibers which are major components of ECM. Preservation of basal lamina was determined with immu-nostaining of collagen IV which showed that this structure retained solid. Quantification of this protein was analysed with hydroxyproline assay which proved preservation of collagen. DNA content was significantly reduced under 50 ng of DNA per mg of tissue. Adhesion and viability of the scaffold was confirmed by re-seeding with C2C12 murine myoblasts and cell morphology was examined in paraffin-embedded sections at 12 days after cultivation. Histological examination of scaffolds implanted into the

murine tibial anterior muscle confirmed its good integra-tion with the muscle and rich colonization by recipient cells.

Conclusion. We succeeded in producing decellular-ized skeletal muscle tissue with well-preserved major components (basal lamina, collagen fibers and glycos-aminoglycans) that fulfiled requirement for successful decellularization (elimination of cell and nuclear com-ponents, reduction of DNA content and preservation of 3D architecture of ECM). Cytocompatibility of scaffold was proved with a successful in vitro recellularization technique and implantation of decelularized scaffold into the mouse muscle, which resulted in rich scaffold recel-lularization.

ACKNOWLEDGEMENTS

This study was supported by Grant Agency of Charles University in Prague No. SVV-2015-260179, GAČR No. 15-09161S and with European Social Fund No. CZ.1.07/2.3.00/30.0061

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Retinoic acid in the development of the NADPH-d positive neurons in rat brain

Natalia Hvizdosova, Lenka Tomasova, Martin Bona, Stanislav Mateffy, Darina Kluchova


Aims. This study describes the presence of nitrergic neurons in selected area of the rat brain during postna-tal development, under physiological condition and after exposure retinoic acid. We have focused on prefrontal cortex of rats.

Methods. Wistar rats were used in the experiment. Retinoic acid was prenatally administered between 14-16 days of pregnancy. Dosage was 1 mg RA/kg body weight. Prefrontal cortex was investigated at postnatal days (P) 7, 14 and 21. The section of brain tissues were processed for NADPH-d histochemical method.

Results. Nitrergic neurons were differentiated during a relatively short period of time and reach their struc-tural maturity by the end of the second week of postnatal development. At P7 and P21 control animals the image was comparable with animals exposed to retinoic acid. We observed the change in nitrergic neurons of exposure animals at P14, these neurons had shorter processes in compared to control group (P<0.001).

Conclusion. The results suggest inhibitory effect of retinoic acid, supplementation during 14-16 days of preg-nancy, on length of processes of neurons at age 14 days but 7 and 21 postnatal days were without changes in mor-phological characteristics of the prefrontal cortex.


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Colonization of muscle scaffolds by different cell types

Jana Chvatalova, Jaroslav Mokry, Hana Hrebikova, Rishikaysh Pisal

aDepartment of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic

Aims. To extend our knowledge of processes associ-ated with skeletal muscle repair we utilized a model based on application of several of muscle cell types into scaf-folds. In our case we used bioscaffolds derived from the skeletal muscle decellularization. We examined adher-ence, growth and development of different types of cells in scaffolds after 10 and 21 days from recellularization.

Methods. We studied implantation of 8 types of cells – muscle derived stem cells (MDSC) in three stages of isolation (passage 3 – PP3, passage 5 – PP5 and passage 6 – PP6); mouse embryonic stem cells (mES); muscle murine fibroblasts; myoblasts (C2C12 cell line); satel-lite cells isolated from muscle fibers and stem cells iso-lated from the bone marrow of mouse using separation Lineage Cell Depletion Kit and positive separation with antibody Sca1 (lin-Sca1+). The scaffolds were obtained from C5Bl/6J mice (precisely from tibialis anterior mus-cle) washing in 5 M NaCl in distilled water, hypotonic 10 mM TrisHCl, incubation with 1% SDS and steriliza-tion in 0.1% peracetic acid solution. Cell nuclei residues were removed with DNase. We implanted 10 μL of cell suspension of each cell line into two scaffolds. We used volume of cells in previously known concentration. The cell lineages were cultured in common cultivation medium (DMEM, glutamine, 10% fetal bovine serum, penicillin and streptomycin). Cultivation medium was replaced with fresh one every two days. The scaffolds were removed in specified period of time after implantation (10th and 21st day), fixed in formalin, processed for paraffin-embedded sections and stained with standard methods or indirect immunohistochemistry.

Results. The scaffolds and their cells were evaluated by various histological and histochemical methods. After 10 days, of 8 cell types only 4 were observed in recellularized scaffolds: C2C12 myoblasts, mES, lin-Sca1+ and murine muscle fibroblasts. The structure of cells is very close. They cells elongated, begin to fuse and able to survive and proliferate. Only few cells migrated to the middle of scaffold, most of them were left on the surface of scaffold. Ten days in vitro the cells migrating over the outer surface. After 21 days in vitro, cells penetrated deep areas into scaf-fold where they accumulated although some other deep areas of scaffolds were left unoccupied.

Conclusion. Our experiments provided evidence the decellularized scaffolds preserved well components of extracellular matrix and their microarchitecture. The acellular scaffolds can be colonized by cells; the best

recellularization was achieved with C2C12 myoblasts, mouse embryonic stem cells, lin-Sca1+ and murine muscle fibroblasts.

ACKNOWLEDGEMENTS

Supported by SVV-2015-260179, GAČR No. 15-09161S and PRVOUK P37/06.

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The influence of maternal diabetes on proliferative potential of human term placenta

Marie Jirkovska, Krystof Zuska

Institute of Histology and Embryology, 1st Faculty of Medicine, Charles University in Prague, Czech Republic

Aims. Placenta is a temporary organ finishing its func-tion at parturition. This study compares the proliferatiove potential of normal term placentas and placentas from pregnancies complicated by either maternal gestational diabetes (GDM) or diabetes type 1 (DM1).

Methods. Histological sections of placental specimens taken by systematic uniform random sampling were used for immunocytochemical detection of Ki-67 antigen. The proliferative potential was expressed as the numbers of labelled nuclei per square milimeter of villous section counted in cytotrophoblast, stroma and vascular wall in stem, intermediate (IV) and terminal villi (TV). Placental groups were compared by the Mann-Whitney test, results were considered to be significant at P ≤ 0.05.

Results. Histological analysis revealed variable amount of pathological villi (hypovascularized and hypervascular-ized) in DM1 placentas, and only infrequent occurrence of hypovascularized villi in GDM placenta. The fact that both pathological forms of villi displayed nearly no Ki-67 labelled nuclei reflects itself in results of measurement. In the villous cytotrophoblast of terminal villi, the frequency of Ki-67 labelled nuclei per square milimeter was signifi-cantly lower in both DM1 and GDM placentas (19.87 ± 12.25 and 18.66 ± 9.88 respectively) than that in the control group (32.21 ± 14.70). In the capillary wall, the frequency was sigificantly lower in DM1 group as com-pared to the control group (5.23 ± 4.50 vs 10.11 ± 4.65). In intermediate villi, significant differences were found between DM1 and control group in the villous cytotro-phoblast (9.46 ± 6.12 vs 18.26 ± 7.74) and between DM1 and GDM group in the villous stroma (5.43 ± 6.16 vs 1.53 ± 1.83). No differences were found among stem villi of all studied groups.

Conclusion. The lower proliferative potential of cyto-trophoblast in TV of both diabetic groups as well as the decreased one in capillaries of TV imply a lower ability to sustain the growth of placental components ensuring ma-ternofetal transport. It may adversely influence the fetal well-being in terminal phases of intrauterine development


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and during perinatal period in pregnancies complicated by both types of maternal diabetes.

ACKNOWLEDGEMENTS

The work was supported by PRVOUK P25/LF1/2.

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Modification of apoptotic cell rate in the intestinal graft mucosa during cold preservation

Zuzana Jonecovaa, Stefan Totha, Milan Marettab, Kristina Gregovaa, Jan Vargac

aDepartment of Histology and Embryology, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicbDepartment of Neurology, Louis Pasteur University Hospital, Kosice, Slovak RepubliccDepartment of Gynaecology and Obstetrics, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic

Aims. The aim of present study was to investigate whether duration and frequency of ischemic precondi-tioning (IPC) procedure affects cold preservation injury in the small intestine grafts. The epithelial apoptosis and extracellular matrix protein laminin expression were stud-ied because of their importance in failure or maintaining the barrier function of the graft intestinal mucosa.

Methods. Three groups of Sprague-Dawley rats were recruited (n=11 each) as follows: the short IPC (SIPC) performed through 4 cycles of mesenteric ischemia of 4 min each followed by 10 min of reperfusion, the long IPC (LIPC) obtained by 2 ischemic cycles of 12 min each fol-lowed by 10 min of reperfusion, and the control group (C) without IPC. Grafts were then stored in cold histidine-tryptophan-ketoglutarate solution and samples were taken at 0, 3, 6 and 9 hours lasting preservation. Apoptosis was detected by using the terminal deoxynucleotidyl-transfer-ase-mediated deoxyuridine triphosphate in situ nick end-labeling (TUNEL). Apoptotic cells (AC) were quantified per 1 intestinal villus. Anti-laminin immunohistochemical method for the laminin evaluation was used. The laminin positive structures in basement membrane (BM) of the epithelial lining were expressed as a mean per 1 intestinal villus according to the semiquantitative immunohisto-chemical scale 0-3.

Results. BM laminin expression in the jejunal graft mucosa at the beginning of preservation was similar in all groups (C=0.80±0.17; SIPC=0.98±0.14; LIPC=1.12±0.07). A parallel significant increase in laminin expression with the histopathological injury rate after 6 hours of preservation was observed in all grafts (C=1.82±0.06, P<0.001; SIPC=2.08±0.06, P<0.001; LIPC=1.90±0.16, P<0.01) in comparison with the level at time 0. The similar results after 9 hours of preservation were found (C=1.86±0.21; SIPC=2.10±0.09; LIPC=1.32±0.11, all of them in comparison with the starting time P<0.001). When compared to the C group, in both IPC groups AC

rate was significantly reduced at the beginning of cold preservation (C=1.46±0.34; SIPC=0.60±0.09, P<0.05; LIPC=0.62±0.05, P<0.05). In the LIPC group the AC rate increased during initial 3 hours and its level remained fairly constant during the further 6 hours of preserva-tion (0.88±0.09 at the end of preservation), thus probably preventing necrosis and improving graft viability. In SIPC group the decrease in AC rate during 9 hours of preserva-tion was evident and only 0.26±0.14 positive cells were found at the end of preservation.

Conclusion. A higher graft protection was observed when fewer, but longer periods of ischemia were used in IPC, thanks to the improved morphology of the intestinal mucosa and sustentative level of the cell apoptosis rate during the cold preservation. Better preserved graft ben-efits to graft survival during following retransplantation.

AKNOWLEDGEMENTS

This study was supported by the grant VEGA 1/0478/2014 and VVGS-2013-111.

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Kolmer cells and their immunophenotyping in the rat choroid plexus after peripheral nerve injury

Marek Joukala, Adela Kuklovaa, Peter Solara, Ilona Klusakovaa,b, Petr Dubovya,b

aDepartment of Anatomy, Faculty of Medicine, Masaryk University, Brno, Czech RepublicbCEITEC, Masaryk University, Brno, Czech Republic

Aims. The unilateral chronical constriction injury (CCI) used as a neuropathic pain model causes the eleva-tion of proinflammatory cytokines levels not only in the structures directly related with damaged nerve but also in remote structures of central nervous system (CNS). The way how the neuroinflammatory molecules affect CNS is not completely clear. We purpose that one of the possible pathways for spread of neuroinflammation could be over the blood-cerebrospinal fluid barrier localized in choroid plexus (CP). CP is formed by highly vascularized stromal core with fenestrated capillaries covered by cuboidal cells on the ventricular side responsible for cerebrospinal fluid secretion. Epiplexal Kolmer cells (KC) are attached on the surface of the cuboidal cells. They are known as the scavenger cells processing the signals from blood to ce-rebrospinal fluid. Therefore, the aim of our study was to investigate changes of KC and CP after peripheral nerve injury.

Methods. Twelve Wistar male rats were used in our experiments. Rats were anesthetized with a mixture of ket-amine and xylazine administered intraperitoneally. CCI of the left sciatic nerve was performed by three ligatures that reduced the nerve diameter by approximately one-third. The animals were exposed to CCI for 3 (n=5) and


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21 (n=5) days, two rats were naive. After time of survival, animals were sacrificed in CO2, perfused transcardially by Zamboni’s fixative; the brain was removed and fixed for three days. Immunohistochemical detection for resident (ED2) and activated (ED1) macrophages and dendritic cells (OX-42) was performed on coronal cryostat sections through the brain.

Results. Immunostaining for ED1 showed activated macrophages in the stroma of CP and in epiplexal posi-tion. The number of ED1+ cells in CP increased with du-ration of nerve compression whereas the most ED1+ cells were found in epiplexal position after 21 days of nerve compression. Immunohistochemical detection for OX42+ showed the presentation of cells only in epiplexal position not in a stroma. Their number gradually increased respect-ing the time of CCI. KC positive for ED2 were found in CCI operated animals but not in naïve rats.

Conclusion. We demonstrated that population of KC is formed by cells with different immunophenotypes namely ED1, ED2 and OX42. We can conclude that the number of KC increased after peripheral nerve injury. KC are blood derived therefore the changes of their number could be caused by alteration of tight junctions that are found between the cuboidal cells. These changes could result in a spread of neuroinflammatory reaction from peripheral nervous system to the CNS structures.

ACKNOWLEDGEMENTS

We would like to thank Ms. Marta Lněníčková, Ms. Jitka Mikulášková, Ms. Dana Kutějová and Mr. Lumír Trenčanský for their skillful technical assistance. The work was supported by MUNI/A/1215/2014.

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Anatomical eponymsDavid Kachlika,b, Tereza Stepankovaa, Vladimir Musilc,d,

Vaclav Bacaa,b

aDepartment of Anatomy, Third Faculty of Medicine, Charles University in Prague, Czech RepublicbDepartment of Health Care Studies, College of Polytechnics Jihlava, Czech RepubliccCentre of Scientific Information, Third Faculty of Medicine, Charles University in Prague, Czech RepublicdInstitute of Information Studies and Librarianship, Faculty of Arts, Charles University in Prague, Czech Republic

Eponyms in anatomy are a constant part of the ter-minology. Due to its shortness and relative uniqueness they have gained an irreplaceable position. Although they were excluded from the Latin anatomical nomenclature in Parisiensia Nomina Anatomica in 1955, they have re-mained an integral part of the anatomical terminology both, in the oral communication and even in the scientific literature. The last revisions of the Latin nomenclatures (Terminologia Anatomica, Terminologia Histologica, Terminologia Embryologica) are completed with lists of eponyms as an appendix.

This discrepancy between the official approach and the general state needs to be approached. The authors suggest a three-leveled classification of eponyms: Level A for every physician and student, Level B for special-ists in one individual medical branch and Level C for superfluous terms (which should be omitted from future communication).

AKNOWLEDGEMENTS

Supported by Charles University Grant 236028/IRP/2013, 236055/IPUK/2014, 236070/IPUK/2015.

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Arteria brachioradialis – overviewDavid Kachlika,b, Petr Hajekc, Marek Konarik, Vaclav Bacaa,b

aDepartment of Anatomy, Third Faculty of Medicine, Charles University in Prague, Czech RepublicbDepartment of Health Care Studies, College of Polytechnics Jihlava, Czech RepubliccDepartment of Anatomy, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic

Arteria brachioradialis is the most common varia-tion of the arterial trunks of the upper limb. The over-all incidence is reported in 14% of cases on average. By long-time theory, it is derived from its embryological precedent termed the arteria brachialis superficialis. The development of the upper limb arteries is not a simple process, closely related to the bone development. Arteria brachioradialis is the radial artery featuring a „high origin“ (proximal to the cubital fossa). It is typically located more ventrally and laterally to the proper brachial artery and crosses ventrally the median nerve. It can be classified as “mere” arteria brachioradialis if shows typical course of radial artery in the forearm or as arteria brachioradialis superficialis when crossing over the tendons delimiting the snuff box. Secondly, it can be classified into four types ac-cording to its origin, as it usually branches in the proximal third of the arm from the arteria brachialis (66%) but not exceptionally from the axillary artery as well. Finally, it can be classified according to the pattern of branches present “on the way” along the arm and forearm. The anatomical knowledge of arteria brachioradialis existence and course is essential for radiodiagnostic, surgical and traumatologic procedures and the superficially coursing artery brings a higher risk of heavy bleeding in unexpect-ed situations. The presentation gives an overview of the anatomy and variability of the arteria brachioradialis and the implications in the clinical medicine, mainly for the interventional cardiologists and reconstructive surgeons.

AKNOWLEDGEMENTS

Supported by Charles University, Project PRVOUK # 33.


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Supernumerary carpal bonesDavid Kachlika,b, Alzbeta Blankovaa, Vaclav Bacaa,b

aDepartment of Anatomy, Third Faculty of Medicine, Charles University in Prague, Czech RepublicbDepartment of Health Care Studies, College of Polytechnics Jihlava, Czech Republic

The usual number of carpal bones is fairly constant. In spite of this fact, variations have been reported, based on the presence of the accessory elements, of the coalition or even of the absence of one carpal bone. A particular review of reference literature showed a log row of different variants with incoherent and inconsistent nomenclature. A need of the classification of carpal bones variations for anatomical research, education and clinical practice was the main hint of this contribution. Embryologically, more than eight ossification centers appear in the wrist, some of them later disappear, other ones persist or fuse to form definitive carpal bones. A disturbance in the development can result in presence of a separated bony element (a supernumerary bone) or a process of a constant carpal bone. These variations can stem from pathologic states as well: ossification of synovial folds or posttraumatic os-sification.

Variations of carpal bones are classified into four groups: accessory carpal elements, multipartite carpal bones, absent carpal bones and collation of carpal bones. The clinical relevance comprises problems with diagno-sis in the X-ray image to reveal the origin of the patient problems (developmental or traumatic) to choose the ap-propriate treatment.

Supported by Charles University, Project PRVOUK # 33.

AKNOWLEDGEMENTS

Supported by Charles University Grant 236028/IRP/2013, 236055/IPUK/2014, 236070/IPUK/2015.

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Effect of WNT5a on chondrogenesis and limb development

Michael Killingera,b, Iva Veselaa, Marcela Buchtovaa,b

aLaboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of Czech Republic, Brno, Czech RepublicbDepartment of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

Aims. WNT family includes many critical mediators of cell-cell signaling events during embryogenesis. WNTs members are cystein-rich secreted extracellular ligands es-sential for physiological, pathological and developmental

processes such as patterning of the body axis, limbs and identifying planar cell polarity. WNTs are associated with receptors Frizzled and low density lipoprotein receptor-related protein family (LRP5/6). Members of WNT can signal through β-catenin (canonical) or β-catenin indepen-dent (non-canonical) pathway. WNT5a belongs among β-catenin independent ligands. Loss-of-function experi-ment inhibited the formation of columnar growth plate architecture during chondrogenesis. Our aim was to un-cover a possible role of WNT5a in chondrogenesis while using gain-of-function approach.

Methods. Mouse tibias were isolated from E18 embry-os and were cultured in media supplemented by WNT5a (40 ng/mL) or FGF2 (50 ng/mL) or mixture of both pro-teins. Control tibias were cultured in media and four bio-logical replicates were used for each treatment. The length of tibias was measured at the first day and at the end of ex-periment to analyze growth differences. Chicken embryos were selected as a model organism for in vivo experiment because of their easy access in the shell. Fertilized eggs were incubated for 3.5 days (38 ˚C) and bead soaked with WNT5a (20 μg/mL and 40 μg/mL) was implanted into wing bud. Embryos were collected into 100% ethanol fol-lowing 11 days of incubation. Wings were stained with 3% Alcian Blue for cartilage visualization and 0.1% Alizarin Red to analyze bone morphology. Samples were cleared in 4% KOH and photographed in 50% glycerol.

Results. We observed statistically significant differenc-es in degree of tibias growth based on treatments. FGF2 caused shortening of tibias while WNT5a by itself did not effect cartilage growth. However, WNT5a rescued FGF2 phenotype when both proteins were added into the media. To uncover effects of WNT5a on early cartilage develop-ment, beads soaked with WNT5a were implanted into wing bud. We observed similar phenotypes when using two different concentrations but the frequency of external abnormalities differed (10% abnormal wings at concentra-tion 20 μg/mL and 36% of defected wings at concentra-tion 40 μg/mL). Wings treated with WNT5a were longer and thinner than control wings. We also observed mor-phological differences in zeugopod and autopod areas such as curved zeugopod and abnormal sizes of fingers.

Conclusion. Here, we found that WNT5a protein af-fected chondrogenesis in vivo and could rescue FGF2-induced inhibition of tibias growth. Next, we aim to further uncover cross-talk of FGF and WNT signaling pathways during limb development and determine the role of WNT5a in causing the malformations of posterior structures (ulna/fibula) during limb development.

ACKNOWLEDGEMENTS

This research was supported by the Grant Agency of the Czech Republic (14-31540S) and institutional support (RVO: 67985904).


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Abbot Seka’s cranial morphology and 2D reconstruction of his face with individual

landmarksIvo Klepaceka, Andrej Shbata, Pavla Zednikova-Malab

aInstitute of Anatomy, First Faculty of Medicine, Charles Universit in Prague, Czech Republic bDepartment of Anthropology and Biology, Institute of Criminalistics, Prague, Czech Republic

Aims. To make an anthropological reconstruction of an abbot Ferdinand Seka’s face. He was the important personality of the premonstrate order in the first half of 19th century. No detailed realistic portrait is known.

Methods. Due to well-preserved facial skeleton with incomplete dentition was at the disposal, a modified two-dimensional (2D) method (Gerasimov 1955, Rynn et al. 2010) was applied. The cranium in question was recog-nized as dinar type and examined by CT.

Results. No malformations or traumatic changes were found. Malar surfaces of facial bones were free of cracks, sutures were clearly defined. A slight depression was de-tected on the left suborbital area. Thus, the following an-thropometric points: supraglabella, glabella, nasion, mid nasal, rhinion, mid-philtrum, prosthion, supramentale, pogonion, gnathion, mid mandibular, menton, zygion and gonion were identified to be critical making en face and profile contours. A slight lateral depression of pro-truded nose and ptosis of the right eye were created. Our reconstruction assumes probable facial asymmetry with relatively massive and curved nose in comparison with later found contemporary abbot’s effigy.

Conclusion. It is accepted that the shape and size of the individual parts of the soft tissues in face as well as shape of the bone structures lying underneath them have measurable relationship. The detailed relief of the Seka’s face was constructed using combination of predictive rules (supported statistically: DeGreef et al. 2006, George 1987, 1993, Stephan and Simpson 2008, Prokopec and Uberlaker 2002, Rynn et al. 2010, Wilkinson 2004, 2010). Due to that, it was possible to estimate the size and shape even of individual elements (eyes, nose, mouth), because abbot’s skull type was recognized. It must be emphasized that even in the ideal case (where the cranium is fully complete), the anthropologic methods have limits and the final result is still partially influenced by the subjective approach of the author's reconstruction.

ACKNOWLEDGEMENTS

Partially supported by GACR P101/12/1306 and UNCE 20401

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Comparison of different tissue clearing methods for 3D imaging techniques in GFP-expressing

embryonic and adult miceHana Kolesovaa, Martin Capekb, Jiri Janacekb,

David Sedmeraa,b

aInstitute of Anatomy, First Faculty of Medicine, Charles University in Prague, Czech RepublicbInstitute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic

Aims. To find a suitable tissue clearing protocol for whole mount imaging of embryonic and adult hearts of transgenic mice preserving GFP fluorescence.

Methods. We compared various published organic solvent and water-based clearing protocols claiming to preserve GFP fluorescence in central nervous system: tetrahydrofurane dehydration and dibenzyl ether (DBE), SCALE, CLARITY, and CUBIC. Tissue transparency and preservation of GFP fluorescence was evaluated in hearts of Cx40:GFP knock in mice at stages: ED10.5 – 18.5 ED, P1-P10, and adults. Imaging was performed on a dissecting microscope equipped with epifluorescence and an upright single photon confocal microscope (Olympus) with 4x, 10x, and 25x objectives after mounting the speci-mens into cavity slides or custom imaging chambers.

Results. Despite careful control of all critical pa-rameters, DBE clearing protocol did not preserve GFP fluorescence; in addition, it caused considerable tissue shrinking and worse deformation than golden standard BABB protocol used for whole mount immunohistochem-istry. The SCALE clearing resulted in good tissue trans-parency up to ED12.5; at later stages and in the adults the useful depth of imaging was limited by tissue light scattering. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The CUBIC method resulted in very well cleared specimens even at the adult stages, and GFP fluorescence was also preserved. In addition, it de-colorized the blood and myocardium by removing iron in the tissues. High-resolution images were thus obtained even from deep tissue layers with ScaleView immersion 25x objective. Simultaneously, intact specimens could be imaged by optical projection tomography.

Conclusion. From the tested methods, the CUBIC protocol turned out to be the best for whole mount GFP cardiac specimens. Scale technique is also suitable for younger embryos. These protocols will be used for three dimensional reconstructions of normal and abnormal specimens to visualize the developing cardiac conduction system and coronary vasculature.


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ACKNOWLEDGEMENTS

Supported by 13-12412S from the Grant Agency of the Czech Republic, Ministry of Education PRVOUK P35/LF1/5, and Charles University UNCE

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Stereological assessment of cerebellum microcirculation in the Lurcher mice

Yaroslav Kolinkoa,c, Jan Cendelinb,c, Zbynek Tonara,c, Milena Kralickovaa,c

aDepartment of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Karlovarska 48, 301 66 Pilsen, Czech Republic bDepartment of Pathophysiology, Faculty of Medicine in Pilsen, Charles University in Prague, Lidicka 1, 301 66 Pilsen, Czech RepubliccBiomedical Centre, Faculty of Medicine in Pilsen, Charles University in Prague, Alej Svobody 76, 301 00 Pilsen, Czech Republic

Aims. Lurcher mouse is a natural model of the olivo-cerebellar degeneration caused by a semi-dominant mu-tation (Grid2Lc) in the δ2 glutamate receptor encoding gene. We aimed to characterize the microvascular network of cerebellum in the three months old Lurcher mouse.

Methods: Paraformaldehyde-fixed brains of B6CBA (n=5) and Lurchr (n=5) mice were processed into 18-μm thick serial sections (n=1973) with random orientation. Microvessels were visualized using polyclonal rabbit anti-laminin antibody. In total 4,226 stacks comprising three 5-μm thick optical sections were recorded using system-atic uniform random sampling. Numerical density of mi-crovessels was estimated by counting the branching nodes using the optical disector. Total lengths were assessed using the optical fractionator technique. For statistical analysis a Mann–Whitney U-test was used.

Results: The total number of microvessels in the control group was 232,332±31,843 (mean±SD) with a total length of 11,193.3±1,205.54 mm (within a volume of 32.3±3.16 mm3). In the Lurcher cerebellum (volume 9.2±0.96 mm3 P=0.012) the microvessel number was 108,181±14,677 (P=0.022) and the length was 3,927±2,507 mm (P=0.012). The volumes of the individual layers of the Lurcher cerebellum except there nuclei was also sig-nificantly lower (P=0.012): molecular layer and Purkinje cells by 81.6% to 2.43±0.25 mm3, granular layer by 76.9% to 3.12±0.36 mm3, and white matter layer by 47.8% to 2.77±0.23 mm3. Both number and length were quanti-fied in separate layers of the cerebellum. We observed a significant reduction (P=0.012) in the number and length of vessels in molecular (by 61.2% to 29,649±6,026; by 68.6% to 1,262.6±148.23 mm) and granular (by 62.4% to 45,420±4,304; by 72.3% to 1,522.3±94.4 mm) layers, as well as reducing of total vessels length in white matter by 48% to 756.6±107.1 mm (P=0.012). In the cerebellar

nuclei numerical density of vessels and their lengths re-mained unchanged.

Conclusion: We have proved the quantitative changes of microvascular network in individual layers of Lurcher mice cerebellum, which are most expressed in its cortex.

ACKNOWLEDGEMENTS

Supported by the project ED2.1.00/03.0076 from European Regional Development Fund and the Charles University Research Fund (project number P36).

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GaMeTix – a software for administration of MCQ databases and creation of examination papers

Dimitrolos Krajcia, Pavel Kylarb

aDepartment of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic bPrivate IT Programmer, Krelov, Czech Republic

Aims. Examination of student’s knowledge by ap-plication of written or electronic tests is widely used in modern pedagogy. Large collections of examination ques-tions must be managed systematically and securely using dedicated software that allows user to add new questions progressively and to edit these questions written in various MCQ formats. Consecutive creation of question sheets and their publication for printed or electronic delivery is another required feature of such a software. We have coded and practically tested a stand-alone portable appli-cation called GaMeTix. In this report we analyze specific features of this application and demonstrate its functional-ity in practical testing in Histology for medical students.

Methods. The program is created under the .NET framework in the C# language. Database of the test ques-tions is stored in a file encrypted using the symmetric cryptography. Questions and generated tests are stored in UTF-8 format and can be imported or exported from/into various formats. In order to generate tests, one or more databases simultaneously can be used. Access to the program is protected by a username and password. Several user accounts can be created.

Results. The program is composed of five units that cover Database Management, Generation of Tests, Test Printing, Test Export and Database Backup. A Database Management pane provides the key function of this soft-ware to add new questions to the database and to catego-rize them by different criteria (ID number, topic, theme, semester, difficulty level, date of the last usage, frequency of use). A search filter available in this pane is to sort questions according to these criteria. The question for-mats are MCQ, MRQ, TF, and they can be supplemented with pictures in bmp, jpg and gif formats. In the Test Generator, some filters can be set to specify the range and the number of questions that should appear in the


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test. The generator checks whether the selected databases contain sufficient number of the questions for the speci-fied filters and then – using a random number generator – it generates the tests. Tests of the same topic can be generated in several versions, each version contains dif-ferent, randomly selected and differently sorted questions to prevent possible cheating.

The generated tests can be exported into the text file format or directly printed. Before printing, the header of the test can be edited. Printing of the test on the printer or into a PDF file is followed by printing of the key to the correct answers for easy evaluation of test results. The saved tests can be also exported into txt and xlsx files for on-paper editing or importing sets of questions into elec-tronic test creators like Articulate Quizmaker 13.

Conclusion. The GaMeTix is a dedicated stand-alone application to manage several databases of MCQs in a secure and portable manner. It provides educators with a simple tool to create sets of examination question sheets with random selection of questions on predefined topics in various MCQ formats.

ACKNOWLEDGEMENTS

Development of this software and application of electronic testing was supported by ESF-OPVK grant n.CZ.1.07/2.2.00/28.0089 in years 2012-2014.

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A new system of electronic evaluation of results of PC-delivered tests

Dimitrolos Krajcia, Tomas Kopecnyb, Radko Novotnya

aDepartment of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic bDean`s Administration Office, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

Aims. Electronic application of summative tests pro-vides an immediate information about test results to stu-dents and examiners. When the test results are processed electronically the answers of individual students can be collected and statistically evaluated. This provides a valu-able feedback to test creators about the difficulty of ap-plied test and quality of questions. Electronic evaluation of test results can be provided by some LMS systems and also by payed Cloud-based services of software-producing companies. In order to extend the practicality of our own electronic testing system we have developed our own stor-age database of test results located on university servers with protected web connection to computers in histology practical room.

Methods. For creation of electronic tests we use a soft-ware Articulate Quizmaker 13. This software provides im-mediate on-screen summative scoring of students’ results, but fails in analysis of responses to single questions and

in further statistical analysis of question difficulty. The software is able to register all data about each test ques-tion in html-formatted results table and, after enabling the Print Results function, the result table can be printed. Our modification of this examination system required reprogramming of the Quizmaker’s 13 report.html file in order to be able to send result data directly to a web server installed on the teacher’s PC. On this PC all result reports are stored in html and xml formats for backup and, furthermore, the html-formatted reports are sent to the MySQL database on the database server for statisti-cal analysis.

Results. After password-protected logging into the Database of Test Results the graphical user interface shows indexes to four panels with functions – Overview of Test Reports, Statistical Analysis of Tests, Import of Tests and a Setup panel. The Overview panel displays information about all deposited test results sorted by se-mester, date of examination, title of examination, class group and name of the PC used by a student. All these criteria can be used to filter the displayed groups of test results. From this panel test results of each student can be open, correct and incorrect answers can be evaluated and the final percentage score can be read out. The statistical evaluation panel provides a summary information about each group of tests selected by a date of examination, name of the examination or a class group of students. The statistical information contains data about number of students in the group, numbers of passing and failing stu-dents, number of questions in the test, a mean difficulty rating of the test, a mean time spent for answering each question and a mean time spent to complete the test. All questions in a particular test are displayed with informa-tion about numbers of students passing and failing these answers, mean time spent to answer each question and a difficulty value of each question expressed in percentage values and color coded. A histogram of difficulty indices is also displayed in this panel.

Conclusion. Electronic evaluation of PC-delivered tests is an essential tool in teacher’s hand to manage and evalu-ate results of large groups of students. It provides detailed information about students’ performance in each test that can be analyzed and discussed with a student without giv-ing out any printed copy of the test. Statistical evaluation of test results provides an additional input about the dif-ficulty of tests and question items related to performance of students.

ACKNOWLEDGEMENTS

Development of the database and application of electronic testing was supported by ESF-OPVK grant n.CZ.1.07/2.2.00/28.0089 in years 2012-2014.


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Application of electronic testing in histology and anatomy practical

Drahomira Krajcia, Radka Lichnovskaa, Bela Erdosovaa, Dimitrolos Krajcia, Libor Machalekb

aDepartment of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepublicbDepartment of Anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

Aims. Our aim was to find a system, which guarantees a straightforward and quick use of question packages, im-mediate and objective evaluation and reporting of results, limitation of lateral student’s communication and cross-talk, and a good prevention of leaking questions outside of the examination room.

Methods. We use a special software ARTICULATE Quizmaker ’13 which has proved to be simple and easy to use by teachers having no programming skills. The user interface of this software is similar to the standard envi-ronment of MS Office applications. Since this software has a selective option to shuffle sequences of questions and also to shuffle all distracters in the quiz content ran-domly on monitors of examined students, only one ver-sion of histology or anatomy test was enough to prepare for one examination session. A time limit for display of each question and a total time allowed for a complete test were settable during test creation. Quizmaker’13 offers the possibility to use a mix various formats of questions like – MCQ (one correct answer), MRQ (one or more of correct answers), and Drag-&-Drop format (dragging and aligning several correct options to the same number of statements). For identification of structures in displayed micrographs a Hot-Spot format of question (clicking with a mouse on a structure in question) proved to be very use-ful in histology and anatomy testing.

Results. Prior to exporting tests for PC application the teacher can decide about information that will be visible to student during examination time. These are: the test title, countdown display of the total examination time, point value of each question and a current student's score as achieved during testing. On completing the test, the software automatically summarizes and displays value of correct answers in percentages and shows a minimum passing limit for that exam. When completely authored, the test can be exported into different application formats for web (for use on a website or a PC-equipped lab), for CD or DVD distribution, for LMS-managed classrooms, and it can be also exported into a simple MS Word docu-ment for archival documentation.

Conclusion. PC-based testing of students’ knowledge may be used not only as regular in-course assessments in practical sessions, but also as in-group summative exami-nation tool at the end of the teaching semester. Evaluation results show that computerized in-course examination of theory and practical knowledge has been well accepted by

students of our histology and anatomy classes and teach-ers alike.

ACKNOWLEDGEMENTS

Development and application of electronic testing was supported by ESF-OPVK grant n. CZ.1.07/2.2.00/28.0089 in years 2012-2014.

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Evaluation of results of MCQ tests applied in electronic format

Drahomira Krajcia, Radka Lichnovskaa, Bela Erdosovaa, Tomas Kopecnyb, Radko Novotnya, Dimitrolos Krajcia

aDepartment of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepublicbDean’s Administration office of the Faculty of Medicine, Palacky University Olomouc, Czech Republic

Aims. Three years ago we have introduced electronic testing in our system of teaching practical histology using a dedicated software Articulate Quizmaker 13. This soft-ware provides immediate on-screen summative scoring of students’ results, but fails in analysis of responses to single questions and in further statistical analysis of question difficulty. Here we report our innovations in collection of test results locally and their difficulty analysis up to the level of a single question.

Methods. In order to examine theoretical as well as practical knowledge of histology we use four formats of questions in our tests, namely Multiple Choice, Multiple Response, Drag & Drop and Hot Spot questions. The final test-scoring page informs students about their re-sults. The software is able to register all data about each test question in html-formatted results table and, after enabling the Print Results function, the result table can be printed. In its original configuration, the Quizmaker 13 failed to collect and sent these data through the network to any hardware storage for further analysis. Our modi-fication of this examination system required reprogram-ming of the Quizmaker’s 13 report.html file to be able to send result data directly to a web server installed on the teacher’s PC. This web server processes the result data into a modified results table, saves the table on teachers’ PC for immediate access, and simultaneously it sends the table to a dedicated email address “histexam” located on university servers.

Results. On the “histexam” location, the examination results are categorized by student’s name, date of exam, contents of the exam etc. The downloaded result tables of every student are further manually converted to standard xlsx spreadsheet table and further analyzed to calculate values of summary points, net points, easiness index and difficulty index in percentage values. Based on these data, we can evaluate performance of each student. We also evaluate each question in the test to classify it as easy,


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moderate and difficult one. We presented the evaluation data in the form of columnar graphs where difficulty in-dices are color coded according to our own classification scale. This evaluation of tests gives us important data on the quality of tests applied but also on the overall pre-paredness of students for the particular topic. It can also indicate possible cheating activities of students.

Conclusion. Application of electronic tests is a use-ful tool for teachers and students alike. We consider the regular evaluation of electronic tests as very important follow-up feature of the examination procedure. It reveals improperly formulated questions, incorrect sets of dis-tracters and overall difficulty of examinations. This activ-ity also provides a summative information about classes of students and stimulates students in their preparation for examinations.

ACKNOWLEDGEMENTS

This communication was supported by ESF-OPVK grant n. CZ.1.07/2.2.00/28.0089 in years 2012 – 2014.

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Inflammatory cells in human atrial myocardium from patients undergoing open heart surgery

Tomas Kuceraa, Natalia Smorodinovaa, Karolína Rozsivalovaa, Jaromir Pridala, Maria Durisovaa,

Jan Pirkc, Martin Blahab, Vojtech Melenovskyb, Josef Kautznerb

aInstitute of Histology and Embryology, The First Faculty of Medicine, Charles University in Prague, Czech RepublicbDepartment of Cardiology, Institute for Clinical and Experimental Medicine – IKEM, Prague, Czech RepubliccDepartment of Cardiovascular Surgery, Institute for Clinical and Experimental Medicine – IKEM, Prague, Czech Republic

Aims. In this study we focused on morphological and functional changes in endomysium of atrial myocardium with special attention to CD45+- cells, representing cells of bone marrow origin. In addition, we characterize differ-ent types of resident or infiltrating immune cells.

Methods. We analyzed atrial biopsies obtained from patients undergoing bypass or mitral valve surgery. The patients had a regular sinus rhythm or were suffering from atrial fibrillation. The atrial samples were fixed with 4% paraformaldehyde and embedded into paraffin. Sections from atrium were histologically examined using routine hematoxylin-eosin staining. For detection of different types of immune cells the following markers were de-tected immunohistochemically: CD45 as a pan-leukocyte marker, CD3 for T-lymphocytes, CD68 for monocyte/macrophages, mast cell tryptase for mast cells and DC-SIGN for immature dendritic cells. Three-step immuno-peroxidase detection was performed on paraffin sections after antigen retrieval and images were taken using Leica

DMLB microscope equipped with DC300 camera. For double labeling we used immunofluorescence approach and images were taken using a confocal laser scanning microscope (FV1000, Olympus).

Results. In all atrial samples from both groups we de-tected CD45+-cells in atrial myocardium as well as in en-docardium and epicardium. The frequency of these cells was variable. Atria from patients with atrial fibrillation had tendency for a higher infiltration with CD45+ cells. Apparently, CD45+ cells formed a heterogeneous group of cells. We also detected CD68+cells, CD3+cells, mast cells and DC-SIGN-positive dendritic cells. The most abundant subpopulation from all CD45+ cells was CD68-positive cells. In the left atrial myocardium there was significantly higher frequency of these cells in patients with atrial fibril-lation compared to those in sinus rhythm. Similar finding was in case of CD3+cells, but the numbers of these cells were several times lower.

Conclusion. Our results document that CD45+ cells are a heterogeneous cell population in atrial myocardium from patients undergoing open heart surgery and these cells can be detected regardless of the heart rhythm. The finding of higher frequency of CD68+ and CD3+ cells in AF samples from left atria indicates increased susceptibil-ity of this part of the heart to inflammatory processes re-lated to atrial fibrillation. Colocalization between CD68+ and DC-SIGN demonstrated that the most frequent im-mune cells in the atrial myocardium are dendritic cells.

ACKNOWLEDGEMENTS

This work was supported by the Research Program of Charles University – PRVOUK -P25/LF1/2.

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Study of changes in the rat choroid plexus after peripheral nerve injury using fluorescent

conjugated dextranAdela Kuklovaa, Peter Solara, Marek Joukala,

Ilona Klusakovaa,b, Petr Dubovya,b

aDepartment of Anatomy, Faculty of Medicine, Masaryk University, Brno, Czech RepublicbCEITEC, Masaryk University, Brno, Czech Republic

Aims. Peripheral nerve injury can lead to cellular and molecular changes in the peripheral and central nerv-ous system structures which are not directly related with damaged nerve. We suppose that these structures can be influenced by damage associated molecular patterns (DAMPs) produced by Wallerian degeneration and dif-fused via blood-cerebrospinal fluid barrier (B-CSF-B). This barrier is present in the choroid plexus (CP) of brain ventricles. CP consists of highly vascularized stromal core with fenestrated capillaries and cuboidal cells covering the


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ventricular side. Epiplexal Kolmer cells (KC) attached on the surface of the cuboidal cells are involved in neu-roimmunological regulations. The aim of our study was to investigate the changes of CP after peripheral nerve injury. For this purpose we used intravenously injected fluorescent conjugated dextran FluoroEmerald (FE) with different time of circulation.

Methods. Twenty Wistar rats (male, 250-300 g) were anaesthetized with a mixture of ketamine and xylazine and unilateral chronic constriction injury (CCI) of the sciatic nerve was performed. CCI operated rats were left to survive for 3 days (3D) (n=8) and 21 days (21D) (n=8). Four naive rats were used as control. After time of CCI survival the FE was intravenously applied after anesthesia to the left external jugular vein. Time of FE circulation was 5 h (n=4) and 18 h (n=4) in each period of CCI survival. Then, the rats were deeply anaesthetized, perfused transcardially by Zamboni’s fixative and the brains were removed and fixed for three days. Coronal cryostat sections (20 μm) were cut through the lateral and third ventricles. Distribution of FE and simultaneous immunohistochemical detection of resident (ED2) and activated (ED1) macrophages, antigen presenting cells (APC; MHC-II), T-cells (OX-52) and dendritic cells (OX-42) were analyzed using fluorescence microscope.

Results. FE particles were found inside the cuboidal cells, the immune stromal cells and the KC. Statistically significant increase of number of FE+ cells was found in CP after 3 and 21 days of CCI and FE circulation for 18, but not for 5hours. Most of FE+ cells were found in CP 21 days from CCI and circulation for 18 h. FE parti-cles were detected inside stromal cells of the CP which displayed immunophenotypes of activated macrophages (ED1+) and antigen presenting cells (MHC-II+). KC with-out FE particles were immunostained for ED1, MHC-II and OX42. Number of OX42+ cells in CP increased with duration of nerve compression. FE positive KC were si-multaneously positive for ED2 and OX52, respectively.

Conclusion. CP responds to the chronic peripheral nerve damage with presence of FE+ cells in the stroma. Therefore, we assume that CP is a possible way how pro-inflammatory molecules from peripheral nerve injury could spread into the central nervous system. These CP changes occur with prolonged peripheral nerve damage by contrast to dorsal root ganglia. We also demonstrated increased penetration of immune cells through B-CSF-B induced by peripheral nerve injury. These changes could be caused by alteration in tight junctions.

ACKNOWLEDGEMENTS

We thank Ms. Dana Kutějová, Ms. Marta Lněníčková, Ms. Jitka Mikulášková and Ms. Jana Vachová for their skilful technical assistance. This work was supported by MUNI/A/1215/2014.

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Expression of some markers of stem cells and tissue-specific progenitors in human postnatal

skinAlena Kvasilova, Barbora Dvorankova, Eliska Drobna Krejci,

Karel Smetana, Milos Grim

Institute of Anatomy, First Faculty of Medicine, Charles University in Prague, Czech Republic

Introduction. This study is a part of a project focused on cellular and molecular characteristics of neonatal human skin with the respect to its property of scarless wound healing after reconstructive surgery of the lip cleft. We hypothesize that early postnatal skin has a greater potential to regenerate because it is not yet fully differ-entiated. We therefore decided to asses whether its cells express markers of various tissue-specific progenitors, and adult stem cells.

Aims. The aim of this study is to determine the expres-sion of Sox2, Nanog, Oct4 and nestin in human postnatal skin of different ages.

Methods. Histological sections of the residual tissue removed during the lip cleft surgery were processed for immunohistochemical detection of Sox2, Oct4, Nanog and nestin. Merkel cells were detected by anti-Keratin 7/8. Samples of adult human skin and mouse skin from neonatal and adult animals were used for comparison of findings.

Results. Sox2 is expressed in the cells of epidermis, in the dermal papilla of hair follicles, in the perifollicular cells and in scattered cells of the dermis. Some perifoli-cular cells expres Oct4 and filaments of Nestin suggesting continuing process of follicle differentiation. Sox2 and Nanog positivity was observed in all Merkel cells. This finding is difficult to interpret because Merkel cells are differentiated cells and are considered to be postmitotic.

Conclusion. Transcription factors Sox2, Nanog and Oct4 involved in maintaining stem cell phenotype and pluripotency in the embryonic and adult progenitors were demonstrated in the cells of human postnatal skin. It is likely that this expression is an important factor for scar-less skin healing.


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Microarray analysis of mandible regionalization during mouse development

Petra Langovaa,b, Simona Balkovaa, Marcela Buchtovaa,c

aInstitute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic bDepartment of Stomatology, Faculty of Medicine, Masaryk University, Brno, Czech RepubliccDepartment of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

Aims. The mammalian teeth function to capture and chew food and they have become specialized for these purposes producing a complex and distinctive dentition consisting of unicuspid and multicuspid teeth. To achieve this specialization, tooth development is characterized by a complex series of reciprocal interactions that occur between the epithelium and the underlying cranial neural crest-derived mesenchyme that regulates tooth morpho-genesis and differentiation at molecular level. Up to now several homeobox genes are known to be expressed in specific pattern of mouse mandible that specifies the inci-sor and molar area. However, there are still many unsolved questions how complex signaling between the epithelium and mesenchyme is regulated during initial development of different classes of teeth. To reveal these complex molecular bases of tooth development, a polygenetic ap-proach is needed. Here, we aim to uncover signaling cas-cades between the oral epithelium and mesenchyme along the jaw that underlay heterodont dentition in mammals.

Methods. Tissues were collected from the rostral and caudal areas of the lower jaw at E11.5 and E12.5. Total RNA was send to Institute of Molecular Genetics of the ASCR, where hybridization and scanning of array chips was performed. GeneChips Mouse Genome 430A 2.0 Arrays (Affymetrix) were used for hybridization (3 bio-logical replicates for each time point and each area of in-terest). The evaluation and statistic analysis were made in program Gene Spring GX (Agilent Technologies). Three levels of selection were used during investigation: 1) if there is present or marginal expression level in at least two samples; 2) one way ANOVA (P=0.05) followed by post-hoc testing; 3) if there is at least 1.5 fold change versus control. These methods allowed us to generate a high-confidence list of genes upregulated or downregu-lated in early dentinogenous tissue during initiation of odontogenesis.

Results. The rostral area of the lower jaw was charac-terized by high expression of Alx3, Bmp4, Msx1, Msx2, Pax3 and Satb2. In the caudal area, Fgf8, Ina, Neurod1 and Neurog1 were detected. Moreover, several genes such as Ankrd15, Asb4, Dlg2, Dpep1, Rerg1, Tmem16A have not been linked to mandibular prominence or teeth develop-ment yet.

Conclusion. We applied microarray technology to decipher differences in molecular regulations during ini-tiation of tooth in the incisiform and molariform areas

of early mouse embryos. Using microarray essays, novel genes expressed in restricted patterns in dental epithe-lium and mesenchyme of incisor and molar areas were determined in mouse and also the known genes, which have not been connected to odontogenesis yet. Next, new candidate genes will be analyzed by whole mount in situ hybridization to uncover their exact expression pattern during odontogenesis.

ACKNOWLEDGEMENTS

This work was supported by the Grant Agency of the Czech Republic (14-37368G) and institutional support (RVO:67985904).

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Teaching practical histology with digital (virtual) slides

Radka Lichnovska, Drahomira Krajci, Bela Erdosova, Dimitrolos Krajci


Aims. We created new format of practical teaching of histology using digital scans of classical glass histol-ogy slides. These virtual slides enable students to study structures of tissues and organs on high quality, variably magnified images simulating on their personal computers microscope-like examination of structures. The core of this e-learning system is our new database of histology practical.

Methods. The database of histology practical is based on the format of MS Excel document. It is available in two identical language versions, Czech and English. After logging into a welcome desktop screen, students are hy-perlinked into the content page of the histology practical database. Each topic of practical session (24 themes in total) systematically deals with General Histology and Microscopic Anatomy of various body systems including notes on development of various organs. Each practical session contains a set of virtual slides with slide proper-ties, keywords, file size information and overview pictures of virtual slides. Additional supporting documents in pdf and ppsx format are also available for each of the histol-ogy topic.

Results. A guide to the practical session includes de-scription and labelled micrographic visualization of all virtual slides available in the session. This information is supplemented with didactic tools like control questions, schematic pictures with legends and references to external bibliographic and internet resources or quizzes. Teacher’s presentation for pre-lab session contains micrographs and schematic drawings of slides that are introduced to students at the beginning of the practical session with teacher’s explanation and diagnostic notes. A document


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– presentation of electron micrographs – briefs students with basic information about ultrastructure features of specific types of cells in body systems. A folder of mov-ies, animations and embryology notes guides students through the difficult visualization of 3D shapes of devel-oping organs (ossification process, tooth eruption, muscle contraction etc.).

During their own study of virtual slides, students can copy to clipboard selected areas of virtual slides viewed in the Olyvia, (OLYMPUS) viewer, and paste them directly into their own self-prepared pptx presentation for later revisions.

Conclusion. The e-learning format of histology prac-tical based on virtual slides proves to be a modern and didactically efficient method of teaching histology to medical students. It standardizes the set of histology slides and gives all students in a large teaching group equal op-portunity to see the same high quality slides in practical sessions. Majority of students evaluated positively the use of virtual slides, as they allowed them to study and also to discuss various details of cells and tissues clearly at various magnifications. A live demonstration of this system will be available during the Congress hours at a scheduled time.

ACKNOWLEDGEMENTS

This project was supported by ESF-OPVK grant n. CZ.1.07/2.2.00/28.0089 in years 2012-2014.

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Impact of E-learning format using virtual microscopy in practical histology sessions

Radka Lichnovska, Drahomira Krajci, Bela Erdosova, Dimitrolos Krajci


Aims. In order to use virtual slides successfully in histology sessions, we have developed our own system of application of virtual slides in a classroom equipped with PC technology. We have evaluated the impact of this method on our pedagogic effort from teacher’s and stu-dent’s points of view.

Methods. Each practical session contains a set of virtual slides including information on slide proper-ties, keywords, file size, and overview of virtual slides. Additional supporting documents are also available for each of the topic. During study, students can screen-co-py selected areas of virtual slides viewed in the Olyvia viewer (Olympus), and paste them into their own pptx presentation for later revisions. Simultaneously students watch projection of virtual slides on a wide screen ac-companied by teacher’s explanation. For electronic testing of student’s knowledge we have prepared quizzes using

Articulate Quizmaker 13 software. This software has a selective option to shuffle sequences of questions and also to shuffle all distracters randomly on monitors of student’s PCs.

Results. The long-term application of E-learning for-mat brought several benefits to our teaching experience. The uniform set of virtual slides of the same quality used for all students prevented loss of this valuable material. Supporting documents were suitable materials for later revisions and self-study. New model of teaching histology supports active student’s approach and their engagement in group activities on digital slides. This helps with ever-in-creasing numbers of students at our Faculty of Medicine. New teaching format also fulfils student’s expectations to use innovative technologies (PC, laptops and tablets) during their studies. Access to images anywhere and at any time was provided through the external login to the database of virtual slides which correspond to those in practical sessions. This open image database contains short annotations of typical structures that are important for identification of slides. Objective evaluation and quick procedure of examination of formative and summative in-course quizzes are important benefits during practi-cal testing. Drawback noticed during our practice is a tendency of some students to passively follow the slide demonstration. They also preferred to observe slides in their digital form rather than to use classical microscopes that were also available.

Conclusion. The new e-learning format proved to be a didactically efficient method of medical teaching histol-ogy. Students readily accepted to use of PC for observa-tion of virtual slides. Teachers benefited from a uniform quality of presented slides and also from a straightfor-ward and easy personal communication with students in the class when personal guidance and explanation was needed. PC-based class also provides an easy environment for computerized testing of student’s practical knowledge. Most of the mentioned drawbacks can be prevented by proper organization of practical sessions.

ACKNOWLEDGEMENTS

This communication was supported by ESF-OPVK grant n. CZ.1.07/2.2.00/28.0089 in years 2012 – 2014.


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The effect of food restriction on the liver morphology: experimental study on the chicken

modelPeter Makovickya, Eva Tumovab, Pavol Makovickyc,

Kvetoslava Rimarovad

aCzech Centre for Phenogenomics (BIOCEV), Institute of Molecular Genetics, Department of Transgenic Models of Diseases, ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic bDepartment of Animal Husbandry, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences in Prague, Kamycka 129, 165 21 Prague 6, Czech Republic cDepartment of Biology, Pedagogical Faculty, Selye Janos University, Bratislavska 3322, 945 01 Komarno, Slovak Republic dDepartment of Public Health and Hygiene, Faculty of Medicine, P. J. Safarik University in Kosice, Trieda SNP 1, 040 11 Kosice, Slovak Republic

Aims. Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome, characterized by simple steatosis with or without inflammation, steatohepatitis with or without fibrosis or cirrhosis. Also classified as an NAFLD is hepatocellular carcinoma. Factors that are involved in mechanisms of NAFLD development are the subject of several studies. The objective of our study was to describe the proportion of interstitial tissue to the liver parenchyma and to measure hepatocyte diameter depend-ing on the stage of food restriction using an experimental chicken model.

Methods. This experiment was performed on 48 chick-ens, which were divided into three groups. The control group (C) was fed ad libitum until the end of the experi-ment. The second group (ADL1) was fed 80% ADL in the 2nd-week and the last group (ADL2) was fed 65% ADL in the 2nd-week of the experiment and after this one week were all chickens fed ad libitum until the end of the experiment. The experiment lasted five weeks and at the end, 16 chickens were selected from each group for histopathological study. The liver samples were fixed in 10% formalin solution, standardly processed and embed-ded in paraffin blocks. Serial sections were stained with hematoxylin-eosin and with Sirius red kit.

Results. Mild liver steatosis and liver inflammation was visible in C group. The weight of the liver was in C group (46.98 g), ADL1 group (45.24 g) and ADL2 group (46.83 g). The average hepatocyte diameter was measured at 49.93 μm (C-group), 47.86 μm (ADL1 group) and 46.31 μm (ADL2 group), (P < 0.001). The proportional content of interstitial tissue in relation to the liver paren-chyma was measured at 8.20% (C group), 6.60% (ADL1 group) and 6.29% (ADL2 group), (P < 0.001).

Conclusion. Results shows that food restriction cor-relates with changes in liver morphology. An increase in the steatosis histologic grade is associated with inflam-matory changes, with hepatocyte diameter and with a proportion of interstitial tissue to the liver parenchyma. Early restriction is not associated with the development of

fibrosis of the liver tissue and can have a protective effect on NAFLD development.

ACKNOWLEGDEMENTS

The work was institutionally supported by RVO 68378050, LM2011032 (MEYS) and the project BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109; European Regional Development Fund) to R.S., and by NAZV QJ 15100192.

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Difference in description of topographic position of communicating branches in different medical

literaturesStanislav Mateffya, Janka Vecanovaa, Lenka Tomasovaa,

Natalia Hvizdosovaa, Mirela Rozpravkovab, Jana Pribulovac, Maria Matusevskad

aDepartment of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicbDepartment of Orthodontics, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepubliccPrivat Dental Clinic, Presov, Slovak RepublicdPrivat Dental Clinic, Lubotice, Slovak Republic

Aims. It is generally known that the communicating branches (CBs) connect the sympathetic trunk with the peripheral nervous system, and the sympathetic neurons send their axons via CBs. The topographic position of these structures is very important for a neurosurgical treatment of specific diseases connected with hyperfunc-tion of the sympathetic innervation, how is hyperhidro-sis, Raynaud's phenomenon, and the others.

Methods. 3 human donor’s cadavers were used for the study of the topographic position of the sympathetic trunk and the CBs.

Results. The difference in description of topographic position of the CBs was found in anatomical and clinical literatures used in Eastern block countries in compari-son to those used in Western countries. In English and German anatomical books, the position of the white CB, known as a pleural branch, is described more distally than the grey CB. The pleural branch carrying preganglionic fibres is visible through the parietal pleura and is easily ac-cessible for a surgical operation, and the grey CB contain-ing postganglionic fibres is located behind the ganglion of sympathetic trunk. On the other hand, in the anatomical books used in Slovak and Czech Republics, and in many other former socialistic countries, where was dominant the influence of soviet anatomists, there is the just op-posite description of topographic position of the same grey and white CBs. The same difference was found not only between the anatomical books, but between clinical books and scientific journals both parts of world as well.


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Conclusion. Proper knowledge of position of the CBs is very important for good diagnostic and therapeutic processes in a clinical practice. It can be resumed, that unification of those information in the anatomical and consequently clinical literatures would demand more at-tention and observations in several theoretical and clinical disciplines in future.

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Anatomy and pathological anatomy of periodontium depending to the state of oral

healthMaria Matusevskaa, Jana Pribulovab, Mirela Rozpravkovac,

Lubomír Redajd, Janka Ohlasovae, Stanislav Mateffyf, Kvetuse Lovasovaf, Darina Kluchovaf

aPrivat Dental Clinic HYG – DENT, Presov Slovak RepublicbPrivat Dental Clinic, DENT-PREV, Presov, Slovak RepubliccDepartment of Orthodontics, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepublicbPrivate Dental Clinic, Studio Smile, Kosice, Slovak RepublicePrivat Dental Clinic, Kosice, Slovak RepublicfDepartment of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic

Aims. A very few of dentists are interested in the treat-ment of periodontitis because the lack of information about the periodontium in the Slovak Republic. The basis is the comparison of physiological anatomy and patho-logical anatomy of the periodontium pointing out the ap-propriate and inappropriate approaches to the process of treatment. The main aim is to investigate and describe the physiological anatomy of periodontium in four groups: healthy tooth, the tooth with infection in the periapical area, the healthy tooth with periodontitis and the infected tooth with periodontitis.

Methods. Recognition of changes in the anatomy of periodontium accordingly upon the state of oral health. The extracted teeth based on their condition are appoint-ed to the corresponding group according to periodontal ligament spaces performed under X – ray examination. The state of cementum, periodontal ligaments, dentine tubules and dental pulp by histological examination will be examined. The main reason is to compare changes in the anatomy of root cementum of teeth, periodontal ligaments, cemento-enamel junction and the area of the root apex.

Results. Assuming that anatomy of tooth with peri-odontal infection in the periapical area will influence only the periapical area. In the third group, expected changes are in the area of periodontal ligament spaces, in the al-veolar bone and in the cementum of the tooth root. In the last group, assumed changes are in the area of periodontal ligament spaces, in the alveolar bone and in the cemen-tum of tooth root, also including changes in the area of dentine and dental pulp too.

Conclusion. This research could help to explain changes in structure of periodontal tissue depending to the pathological state. This knowledge can positively in-fluence the therapy of periodontitis. It should prevent the occurrence of periodontal tissue damage leaving out excessive or undesirable procedures for the patient.

ACKNOWLEDGEMENTS

I want to cordially thank of Department of Anatomy, Faculty of Medicine, University of Pavol Jozef Safarik in Kosice and all my colleagues for cooperation on this design.

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Transgenic rat model of Huntington’s disease – a useful tool for study of the development of

progressive neuronal degenerationYvona Mazurovaa, Ales Bezroukb

aDepartment of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech RepublicaDepartment of Medical Biophysics, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic

Aims. Following up on our previous findings in rats transgenic for Huntington’s disease (tgHD51 CAG rats), we focused on the most significant change in the course of the disease, i.e. the degeneration of striatal neurons.

Methods. Homozygous tgHD51 CAG rats and their age-matched wild-type (wt) littermates 2-3, 6, 12, 18 and 22-24 months old were compared. Immunohistochemical and immunofluorescent detections using the array of primary antibodies (primarily anti-NeuN, anti-beta-III-tubulin, anti-MAP2, anti-Iba1, anti-polyQ-huntingtin) were made. Quantitative analysis of the median diameter of neuronal nuclei labelled with NeuN antibody (50 in-dependent measurements) was made on serial paraffin sections in a sequence of 3 slices, each 7 μm thick, in the distance of 15 sections (i.e. approximately 105 μm) and followed by statistical analysis.

Results. Neurodegenerative process (NDP) of HD phenotype primarily manifests by gradual decrease in size of neuronal bodies and nuclei (with maintenance of nucleo-cytoplasmic rate) and finally, although slowly, results in the death of affected neurons, ultimately scav-enged by microglia. Our morphological findings were supplemented by quantitative analysis. Also the propor-tion of age-related changes in this process was assessed. The quantitative analysis proved that the rate of neuronal degeneration reaches maximum at the end of the first year of animal’s age, and then, in following 12 months, it proceeds rather slowly. It is potentiated with age-relat-ed changes particularly in the oldest animals. However,


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neuronal degeneration in wt rats can be attributed only to the debit of the aging process. Indeed, the reduction in a volume of neuropil and alterations in a character of syn-apses are at least of the same importance. Additionally, detection of polyglutamine deposits using polyQ-hunting-tin provided interesting findings which complete histo-pathological picture of the progression of NDP of HD phenotype.

Conclusion. The results of quantitative analysis proved that the end of the first year represents the turn in the development of morphological changes related to the progression of NDP in tgHD51 rats. These findings cor-respond to the course of HD in human brain, in which also behavioral and motor changes precede the loss of striatal neurons.

ACKNOWLEDGEMENTS

Supported by the Charles University research program PRVOUK P37/06 and P37/09.

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Immunohistochemical visualization of α-synuclein in colonic lamina propria mucosae

Eva Mechirovaa, Zuzana Gdovinovab, Matej Skorvanekb, Iveta Domorakovaa, Marianna Dankovaa

aDepartment of Histology and Embryology, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicbDepartment of Neurology, Faculty of Medicine, P. J. Safarik University in Kosice andL. Pasteur University Hospital Kosice, Slovak Republic

Aims. There is an urgent need for biomarkers of Parkinson’s disease (PD). Routine colonic biopsies allow the demonstration of α-synuclein pathology in colonic mu-cosa and submucosa in living PD patients. Localization of α-synuclein aggregation is typical not only in the enteric nervous system (ENS), but also in other cells frequently seen in the digestive tract (macrophages, plasma cells).

Methods. Four healthy control, 4 PD patients and 9 healthy patients with positive screening for PD risk factors underwent routine colonoscopy and the bioptic material from ascendent and sigmoid colon was analysed by immu-nohistochemistry using antibodies against α-synuclein and phosphorylated α-synuclein. For visualization of CD68 positive cell population we used anti human macrophage (CD68) monoclonal antibody and secondary anti-mouse fluorescent antibody Alexa Fluor 488.

Results. α-synuclein positive aggregates were detect-ed in nerve fibres of lamina propria mucosae of all PD patients and 6 out of 9 patients with positive screening. Strong α- synuclein positive monocyte-macrophage cell population was detected in PD and positive screening patients. Phosphorylated α-synuclein was present in PD, positive screening and also healthy control patients. In the

fluorescent microscope CD68 positive cells were present in both PD and positive screening patients.

Conclusion. Our results showing presence of α-synuclein aggregates in colonic nerve fibres and mono-cyte-macrophage cell population in healthy patients at risk for development of PD, support the theory of colonic α-synuclein biopsies as potential premotor biomarker of PD and can serve as a model for testing this hypothesis.

ACKNOWLEDGEMENTS

Supported by VEGA grant 1/0024/14 and APVV-14-0415.

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Myoid cells of thymus in question of origin and function

Veronika Mestanovaa, Ivan Vargab, Marian Bencatc, Marian Adamkova

aDepartment of Histology and Embryology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovak RepublicbDepartment of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Slovak RepubliccAlpha Medical, Patologia, s.r.o., Martin, Slovak Republic

Aims. Complex developing process of pro-T-lympho-cytes requires unique cellular and excessive antigenic de-mands accomplished in thymus. This central lymphoid organ comprises of heterogenous cell populations that reflect thymus complicated embryological development, thus endo-ectodermal origin with direct participation of pluripotent cells migrating from transient embryonic neu-ral crest. Present research was focused on a rare popula-tion of thymic myoid cells (MC) which, despite their strict evolutionary conservation, have not revealed their neither origin nor the physiological role.

Methods. Using the 49 formalin-fixed paraffin-embed-ded thymic tissues obtained from corrective cardiovascu-lar surgery, immunohistochemical analysis was performed on monoclonal antibodies palette of actin, desmin and MyoD1 specific for striated muscle antigens expressed by myoid cells as well as bcl2, p53 and survivin – proteins regulating the apoptotic process.

Results. Semiquantitative analysis proved the predomi-nant location of myoid cells in the medullary compart-ment. Concerning the embryological origin, we detected decrease in number of MC in tissue from congenital heart defects which etiopathogenesis is closely related with dis-rupted migration of neural crest cells. Considering the physiological function, we demonstrated inverse propor-tion in number of MC in correlation with the apoptotic markers.

Conclusion. To summarize our results, myoid cells rep-resent a scarce population localised in thymic medulla


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and in the proximity of cortico-medullary junction, with the probable physiological counteraction on regulation of programmed cell death during selection processes under-gone by immunocompetence gaining T- lymphocytes. In a case of corrupted development of myoid cells population and subsequent defective selection processes regulation, the clinical manifestations may arise in form of immuno-deficiency disorders or autoimmune diseases.

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Application of double staining method for fetal specimens

Maria Miklosova , Robert Dankovcikb, Robert Herichc

aDepartment of Anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic b2nd Department of Gynecology and Obstetrics, Faculty of Medicine, P. J. Safarik University Kosice and L. Pasteur University Hospital Kosice, Slovak RepubliccInstitute of Pathological Anatomy, University of Veterinary Medicine and Pharmacy, Kosice, Slovak Republic

Aims. The improved double-staining technique to dem-onstrate cartilage and bone can now be used in teratologi-cal and toxicological studies as well as in morphological studies of large specimens. The principle used here is the affinity of Alizarin red S to bind with calcium of bones, while Alcian blue reacts with acid mucopolysacharides in cartilage. The end product of this technique is a specimen in which the ossified parts of bone are stained red and cartilages are stained blue. This staining allows to observe primary ossification centers, transition cartilage to bone, and the related abnormalities.

Methods. We examined large fetuses and neonates. Fresh, eviscerated specimens are incubated with Alcian Blue 8 GS and Alizarin Red S. The cartilage stains blue in color and bones are red, both embedded in the trans-parent (cleared) muscle. Before examination or storage, the double stained specimens must be cleared in graded concentration arrays of glycerin. The specimens can be stored in pure glycerin.

Results and Conclusion. Although fragile not ideal for mounting, this type of specimen is excellent for de-tailed scientific skeletal studies in 3D, and also may be used in multiple settings in university teaching curricula. Distinguishing of colors is very good. Fresh, formalde-hyde-fixed or alcohol-fixed specimens from various spe-cies worked well.

ACKNOWLEDGEMENTS

Supported by the grant VEGA 1/0678/15.

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Remodelling of the atrioventricular canal myocardium determines atrioventricular

conduction patternsOndrej Nankaa, Rebecca Vicente-Steijnb, Nico A Blomc,

Monique RM Jongbloedb, David Sedmeraa,d

aInstitute of Anatomy, 1st Faculty of Medicine, Charles University in Prague, Czech RepublicbDepartment of Anatomy and Embryology, Leiden University Medical Center, Leiden, The NetherlandscDepartment of Pediatric Cardiology, Leiden University Medical Center, Leiden, The Netherlands dInstitute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic

Aims. Based on our previous study showing acceler-ated conversion of ventricular activation sequence during hypoxic incubation correlating with increased apoptosis in the atrioventricular canal (AVC), we hypothesized that this slight increase in apoptosis could lead to an acceler-ated pruning of myocardial atrioventricular connections, which would manifest as earlier appearance of mature apex-to-base activation patterns.

Methods. To test this hypothesis, apoptosis was blocked with a caspase inhibitor (zVAD-fmk) by intraperi-cardial injection in chick embryos at embryonic day (ED) 4. The epicardial contribution to AVC remodeling was studied by performing epicardial outgrowth inhibitions – microsurgical epicardial ablation atED3. Ventricular activation was studied by optical mapping and apopto-sis rates were quantified by Lysotracker Red and Active Caspase-3 immunofluorescence staining.

Results. Apoptosis measurements and quantification in zVAD treated hearts shows decreased apoptosis in AVC myocardium atED7. zVAD-treated and epicardial ablat-ed hearts showed increased frequency of immature ven-tricular activation patterns. In 5 of the 21 zVAD-treated hearts we observed activation originating from the right AVC. This pattern was not observed in neither control nor epicardial ablation hearts. The epicardial ablation hearts showed activation originating from the left AVC. Ventricular activation time in Base-to-Apex activation pat-terns in the zVAD-treated hearts was significantly longer than in controls.

Conclusion. AVC maturation requires apoptosis in the AVC myocardium and epicardium-derived ingrowth. Disruption of proper AVC morphogenesis can lead to persistence of accessory atrioventricular connections, which form a morphological substrate for ventricular pre-excitation.

ACKNOWLEDGEMENTS

Supported by Charles University PRVOUK 1105-280002 and GACR 13-12412S.


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Amygdalar volume in narcolepsy type 1 and type 2 and in controls

Veronika Nemcovaa, Jan Krasenskyb, Pavel Petrovickya, Manuela Vaneckovab, Zdenek Seidlb, Jitka Buskovac,

Karel Sonkac

a Institute of Anatomy, 1st Faculty of Medicine, Charles University in Prague, Czech RepublicbDepartment of Radiology, 1st Faculty of Medicine, Charles University in Prague, Czech RepubliccDepartment of Neurology, 1st Faculty of Medicine, Charles University in Prague, Czech Republic

Aims. Amygdala has a key role in detection and pro-cessing of emotional signals. Emotions trigger cataplexy in narcolepsy type 1 (N1). Abnormal amygdalar reactiv-ity on humorous stimuli on fMRI was described in N1. Recently several microstructural changes were noted in amygdala in N1 patients but not in narcolepsy type 2 (N2) subjects. Our preliminary results showed a reduced volume of amygdala in N1 patients, thus we decided to compare amygdalar volume in N1 and N2 patients and healthy subjects using more sophisticated methods.

Methods. Twenty one patients with N1, 45 patients with N2 and 37 healthy controls participated in the study. They were examined in the same MR scanner 1.5 T, the measurement was carried out as T1W 3D image with a slice thickness of 1.0/0 mm.The amygdalar volume was as-sessed manually in ScanView.cz program (man.) and au-tomatically by brain segmentation using Freesurfer (FS).

Results. The average left amygdala volume was man. 1.44 cm3 (FS: 1.46 cm3) in N1, man. 1.45 cm3 (FS: 1.55 cm3) in N2 and man. 1.45 cm3 (FS: 1.53 cm3) in controls. The average right amygdala volume was man. 1.41 cm3 (FS: 1.54 cm3) in N1, man. 1.44 cm3 (FS: 1.62 cm3) in N2 and man. 1.4 cm3 (FS: 1.68cm3) in controls. No dif-ferences were found in amygdalar absolute volumes or in amygdalar volumes related to whole brain volumes.

Conclusion. Automatic nor manual amygdalar volu-mometry did not found any differences betweenN1 and N2 patients and controls.

ACKNOWLEDGEMENTS

Supported by the grant IGA NT 13238-4/2012.

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A role of c-Myb in ossificationVeronika Oralovaa,b, Eva Matalovaa,c, Lucie Knopfovab,

Eva Svandovaa, Jan Smardab, Marcela Buchtovaa,b

aInstitute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech RepublicbDepartment of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech RepubliccDepartment of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

Aims. Bone formation can proceed by intramem-branous (direct differentiation of mesenchymal cells into osteoblasts) or endochondral ossification (gradual replacement of cartilage by bone during development). Developmental processes are influenced by several mol-ecules pathways including FGF, HH, BMP and WNT as well as series of transcription factors. c-Myb is one of tran-scription factors involved in proliferation, differentiation, cell death and survival (Oh et Reddy, 1999). Previously, c-Myb was linked with undifferentiated state but recent experiments suggested its role in differentiation of chon-drocytes and odontoblats (Matalova et al., 2011; Bhattarai et al., 2013; Lungova et al. 2012). Here, we aim to examine a role of c-Myb in osteogenic developmental processes.

Methods. Mesenchymal cells were isolated from fore-limbs of CD1 mouse embryos at E12.0 and spotted in high density to obtain primary micromass cultures. Cells were transfected with the c-myb coding vector (pcDNA3-mcMYB) or control vector (pcDNA-3). Gene expression changes were analyzed by PCR Array designed for osteo-genic markers.

Results. Overexpression of c-myb increased expres-sion of several cartilage and bone markers such as Bgn, Bmpr1a, Smad1, Msx1, Col1a2, Fgfr2, Tgfb3 following 24h after treatment. In addition, a strong increase of NF-κb1, Icam1, Vcam1 and Vegfa expression was evident at this early time point. NF-κb is known to control the dif-ferentiation and activity of the major skeletal cell types – osteoclasts, osteoblasts, osteocytes and chondrocytes (Novack, 2011). Icam1 and Vcam1, belongs to cell ad-hesion molecules, both have also been found to be ex-pressed in osteoblasts and involved in osteoclastogenesis (Tanaka et al., 1995). Moreover, Vegf and its receptors are expressed by osteoblastic cells in vitro (Maes et al., 2002). To further reveal the role of the c-Myb transcription factor in regulation of skeletogenesis, we analyzed the effect of c-myb overexpression in long-term cultivated micromasses. Cultures were stained by Alkaline phosphatase, Alizarin red and Osteocalcin following 14 days of cultivation. c-myb overexpression caused the increase of mineralization and positivity for all three labeling in areas surrounding individual cartilage nodules confirming enhanced osteo-genic activity of cultures.

Conclusion. We found that overexpression of c-myb in micromass cultures accelerated the expression of wide range of osteogenic genes including transcription factors,


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growth factors and their receptors. Next, we intend to ex-amine the effect of c-myb modulation on cell line MC3T3, which was established from newborn mouse calvaria to confirm our findings.

ACKNOWLEDGEMENTS

This study was supported by the Centre of Excellence of the Grant Agency of the Czech Republic (14-37368G) and institutional support (RVO: 67985904).

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The biological evaluation of ultra-fine titanium with improved mechanical strength for tissue

engineeringLucie Ostrovskaa, Lucie Vistejnovaa, Jan Dzuganb, Peter Slamab, Tomas Kubinab, Egor Ukraintsevc,

Dana Kubiesd, Milena Kralickovaa, Marie Hubalek Kalbacovaa,e

aBiomedical Center, Medical Faculty in Pilsen, Charles University in Prague, Pilsen, Czech Republic bComtes FHT a.s., Dobrany, Czech RepubliccInstitute of Physics, The Academy of Sciences of the Czech Republic, Prague, Czech RepublicdInstitute of Macromolecular Chemistry, The Academy of Sciences of the Czech Republic, Prague, Czech RepubliceInstitute of Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University in Prague, Czech Republic

Aims. Titanium, the gold standard for implant materi-als, is distinguished by its exceptional biocompatibility; however, from a long-term perspective, titanium still lacks sufficient loading strength. The aim of our work was to evaluate the cytocompatibility of a commercially pure ti-tanium grade 2 (Ti material) with the ultra-fine grain size and mechanically treated surface which provides it with an enhanced mechanical strength.

Methods. Original Ti material (marked as S) was pro-cessed by the ECAP-Conform technique and two novel types of Ti material (marked as M, Z) with enhanced mechanical resistance were obtained. These ultra-fine grained Ti samples with grain sizes ≤ 1 μm (Z), 4.6 μm (M) and 30 μm (S) and mechanically treated surfaces (grinding, polishing) were characterized using atomic force microscopy, and their surface wettability was as-sessed. Osteoblast cell line (SAOS-2) and human primary mesenchymal stem cells (hMSC) were used as an appro-priate in vitro model. SAOS-2 and hMSC were seeded on Ti samples and polystyrene culture plastic (PS control) in concentrations 15 000 cells/cm2 and 10 000 cells/cm2, respectively. After 2 and 48 h of incubation cells were fixed and visualized by immunofluorescent staining (nuclei-DAPI, actin-phalloidin-Alexa 488 and vinculin-primary antibody against vinculin-secundary antibody Alexa 568). The number of cells on cm2 was estimated

by a program Cell profiler (open-source) from the images of nuclei. Cell area in μm² was defined from the images of actin cytoskeleton also using Cell profiler. Metabolic activity was measured after 48 h of incubation by Cell Titer 96® AQueous One kit (Promega) for MTS assay.

Results. Our study revealed that primary adhesion (2 h) of both cells types to Ti material was increased. Additionally, elevated proliferation and metabolic activity of SAOS-2 cells after 48 h indicated that both original Ti material S and mechanical improved Ti materials M and Z enhanced the growth of osteoblasts. Although prolif-eration of hMSC after 48 h was slightly decreased they remained metabolically active. Interestingly, cell area for both cell types was lower in comparison to PS control at both time points. Different cell morphology and ar-rangement and shape of focal adhesions confirmed that surface treatment has an influence on cell attachment and spreading.

Conclusion. Our results revealed that the novel Ti ma-terials M and Z were equivalent to the original Ti material S, thus confirming their suitability for osteoblast and mes-enchymal stem cell growth and proliferation. The present study proved the cytocompatibility of the novel forms of Ti with superior mechanical properties and revealed their potential for designing improved biomedical implants.

ACKNOWLEDGEMENTS

This work was suppor ted by the projects CZ.1.05/2.1.00/03.0076, CZ.1.05/2.1.00/03 and CZ.1.05/1.1.00/02.0109 of the European Regional Development Fund, project PRVOUK-P24/LF1/3 of First Faculty of Medicine and project PRVOUK P36 of Faculty of Medicine in Pilsen and the program of support and co-operation of companies and universities in the Pilsen region called “Innovation Vouchers”.

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Cloning of synthetic intron into coding sequence of red fluorescent gene for confirmatory

expression of micro RNA cloned within the intronRishikaysh Pisal, Hana Hrebikova, Jana Chvatalova,

Daniel Diaz Garcia, David Kunke, Tomas Soukup, Jaroslav Mokry

Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic

Aims. To clone a synthetic intron into the gene encod-ing red fluorescent protein derived from Discosoma sp. This system will act as an easy tool to confirm the expres-sion and processing of micro RNA (miR) cloned within the intron by simple visualization of cells using fluores-cence microscopy after transduction or transfection of the target cells with the construct. To determine the course


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and efficiency of reprogramming by cloning mir 302/367 cluster into the intronic region of the construct.

Method. Sequencing of the construct was performed to check any deviations in the sequence. Proper process-ing and expression of individual miR was confirmed by us-ing Taqman probes specific to a particular miR’s. Human fibroblasts were reprogrammed using mir 302/367 cluster cloned into the construct. Pluripotency was confirmed by detecting expression of pluripotency markers Oct4, Sox2, Alkaline phosphatase, TRA 1-81, SSEA.

Results. Sequencing results confirmed successful clon-ing of intron within the red fluorescent gene. Three to four percent of the cells expressing the red fluorescence successfully dedifferentiated to induced pluripotent stem cells (iPSC’s).

Conclusion. This system of miR expression assures the expression of the miR with the advantage of tracking the outcome in this case the reprogramming by compar-ing fluorescence to the morphological change in the fi-broblasts as they dedifferentiate into induced pluripotent stem cells.

ACKNOWLEDGEMENT

This study was supported by Grant Agency of Charles University in Prague (GAUK grant no. 1854214), No. SVV-2015-260179, and with European Social Fund No. CZ.1.07/2.3.00/30.0061.

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The variation in morphology of first upper premolar

Jana Pribulovaa, Maria Matusevskab, Mirela Rozpravkovac, Lubomir Redajd, Jana Ohlasovae, Stanislav Mateffyf,

Kvetuse Lovasovaf, Darina Kluchovaf

aPrivat Dental Clinic, DENT-PREV, Presov, Slovak RepublicbPrivat Dental Clinic, HYG-DENT,Presov, Slovak RepubliccDepartement of Orthodontics, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepublicbPrivate Dental Clinic, Studio Smile, Kosice, Slovak RepublicePrivat Dental Clinic, Kosice, Slovak RepublicfDepartment of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic

Aims. Perform endodontic treatment precisely and efficiently is based primarily on a perfect knowledge of the anatomy of the tooth and its variations. If we don’t know anatomy we may cause irreversible damage to the entire canal system of the tooth, which can no longer be maintained. The first upper premolar conceals more anatomical and morphological traits. The number of roots varies and roots are usually fragile, which in the case of curvature substantially compromises its endodontic treat-ment. The amount of root channels varies from 1-3 with different anatomical complexity, not excluding the api-cal variations, and accessory channels. The main task of

endodontic treatment is a hermetic sealing of the entire channel system. Treatment success depends on varying degrees of anatomical complexity. Therefore, knowledge of anatomical and morphological diversity plays a major role in the success of therapy.

Methods. The first upper premolars, extracted essen-tially because orthodontics treatment, was dipped in 3% hydrogen peroxide solution for 72 hours and after that were flushed with clean water. The teeth were bundled into Lucasteric bags and sterilized in an autoclave. On these premade teeth we will further study the external and internal anatomy of the tooth.

Results. We assume that about 70% of the extracted first upper premolars will have two roots with two root canals. 25% will have one root with two root canals. The remaining percentage will consist of different morphologi-cal traits like premolars with three roots and with three roots canals. We do not expect a high percentage of first upper jaw premolars with only one root canal.

Conclusion. Success in endodontic therapy at the first upper premolar is mainly dependent on the complexity of the internal anatomy of the tooth in the form and amount of curvature of root canals.

ACKNOWLEDGEMENTS

Especially I want to thank all my colleagues to col-laborate on the implementation of this project, especially the Department of Anatomy in Kosice.

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Healing of soft and hard tissues of rabbit temporomandibular joint following the

application of stem cellsBarbora Putnovaa, Marcela Buchtovaa,b, Vladimir Jeklc,

Radek Hodand, Vladimir Machone, Jan Stembireka,d

aInstitute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech RepublicbDepartment of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech RepubliccClinic of Small Animals, University of Veterinary and Pharmaceutical Sciences Brno, Czech RepublicdDepartment of Oral and Maxillofacial Surgery, University Hospital Ostrava, Czech RepubliceDepartment of Oral and Maxillofacial Surgery, 1st Faculty of Medicine, Charles University in Prague, Czech Republic

Aims. Temporomandibular joint (TMJ) as a pair connection between processus condylaris mandibulae and temporal bone (fossa mandibularis) is characteris-tic for mammals (Bermejo et al., 1993). TMJ is one of the areas drawing a lot of attention in human medicine lately as the incidence of temporomandibular disorders (TMD) increased along with improvement of diagnos-tic success. It was proposed that the rising incidence of TMD is connected with increased occurrence of stress in


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our population, which plays an important role in TMD progress. It was estimated that 50% to 70% of human population suffers from some kind of TMD symptoms at least once during the life. Pain in the facial area, earache, toothache, joint stiffness, various noises from TMJ, jaw dislocation and, finally, limitation of movement are the most common symptoms of TMJ disorders. At present, the therapy is only symptomatic as current medicine lacks the resources for either cartilage regeneration or surgical replacement of the affected joint. The main aim of our study was to evaluate the influence of stem cells on heal-ing process of soft and hard tissues of TMJ. This study is unique in using fresh heterologous stem cells, which were not previously cultivated with any factors interfering with their differentiation.

Methods. Rabbit was selected as a model animal be-cause of easy experimental access to joint cavity in con-trast to mouse and pig. We used 17 adult New Zeland white rabbits in this study. Nine animals were used as an experimental group, eight animals as a control group. Cartilaginous damage was induced surgically in the left joint under general anaesthesia using open approach to the joint. The bone defect was created using a tool with round head. Following that, human mesenchymal stem cells isolated from adipose tissue were applied into the wound for the experimental group while only saline solu-tion was used in the control group. The collection of the stem cells was performed using a special separator. The main advantage of using adipose stem cells is their easy collection causing much less distress to the patient than that of bone marrow stem cells, which are usually used. Rabbits were euthanized at two time points – 11 days (6 animals) and 1 month (11 animals) after the treatment.

Inflammatory changes and tissue response to stem cells were assessed based on macroscopic changes found during the autopsy as well as on results of histopathologi-cal analysis of serial sections stained with hematoxylin and eosin. For evaluation of proliferation, immunohisto-chemical methods were used.

Results. In the experimental group, we found a higher tendency to form granulation of tissue. Due to the fact that the rabbits were medically immunosuppressed prior to the surgery, we could not properly evaluate immune re-sponse of human stem cells to foreign antigens. However, we have not observed any difference in immune response between experimental and control groups based on hema-toxylin and eosin staining.

Conclusion. Our aim was to examine the mechanism of stem cell influence on the structures in TMJ and es-pecially to confirm an expected positive effect on bone healing. We were also interested in finding possible side effects such as uncontrolled proliferation during healing process. Next, we will focus on a more detailed analysis of inflammatory markers in soft and hard tissues of treated TMJ.

ACKNOWLEDGEMENTS

This study was supported by the Grant Agency of the Czech Republic (14-37368G, 14-29273P) and institutional support (RVO: 67985904).

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Anatomical variations of popliteal artery – a possible case of lower limb ischaemia

Olga Rejtarovaa, Michal Kuchara, Pavel Eliasb

aDepartment of Anatomy, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech RepublicbDepartment of Radiology University Hospital in Hradec Kralove, Czech Republic

Aims. Our aim is to present bilateral abnormal branch-ing of the anterior tibial artery (ATA) arising proximally to the popliteal muscle and passing between the muscle and tibial condyle to the upper shin.

Methods. Nine cadavers and four solitary lower limbs used in 2014/2015 anatomy practical classes were ob-served for possible popliteal variations. In cooperation with the Department of Radiology University Hospital in Hradec Kralove a recent cases of anatomical claudications were searched for.

Results. On dissected cadavers we found a rare varia-tion of ATA and an atypical lateral course of popliteal artery in close contact with plantaris muscle belly. CT scans presented examples of claudication of the lower limb caused by compression of PA with supernumerary belly of medial gastrocnemius muscle.

Conclusion. Risk of ATA damage in knee joint surgery is high in the case of atypical course of this vessel. The potential danger of entrapment must be considered as well, although it is more common for PA – be it caused by muscle abnormalities or its irregular position inside the popliteal fossa. All of these examples are presented in our paper.

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Children of obese mothers in their first 18 monthsJitka Riedlovaa, Marketa Paulovab, Jana Vignerovac,

Jan Soumard

aDepartment of Anatomy, Third Faculty of Medicine, Charles University in Prague, Czech RepublicbThe National Institute of Public Health, Prague, Czech RepubliccNational Lactation Center, Prague, Czech RepublicdNational Museum, Prague, Czech Republic

Aims. Obesity and overweight are becoming a big problem of our society. They adversely affect not only the mother, but they have an impact on health and develop-ment of the child, in which eg. risk of developing obesity, hypertension and dyslipidemia increases, birth weight is


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more often over 90th percentile and the probability of full breastfeeding at the time of maternity hospital discharge is lower. Non-breastfed children have a higher risk of de-veloping overweight than children fully breastfed for 4 – 6 months. Family environment contributes to the develop-ment of obesity, too.

The object of the study was to evaluate basic anthropo-metric parameters of children of obese mothers until 18 months of age and compare them with percentile growth charts used for growth assessment of Czech children population.

Methods. Data of 1765 children collected in 2009 – 2010 were used for the study of obese mothers` children. The children were divided according to BMI categories of mothers. Software RustCZ based on the current growth charts was used for finding the percentile values of length for age, weight for age, weight for length and BMI for age for all measurements of every child at all monitored ages (0, 3, 6, 12 and 18 months). Reference data are results of the 5th and 6th Nationwide Anthropological Survey (1991 and 2001).

Results. The group included 132 obese mothers (7.6 % of dataset), 9 of whom with a BMI even above 40 kg/m2. Among the obese mothers there was a highest number of apprenticed and lowest number of women with university education, only 55% non-smokers and the highest number of divorced women. 76% of obese mothers` children were fully breastfed at the maternity hospital discharge, where-as in case of mothers with a normal BMI the percentage was 90%. Obese mothers breastfed their babies exclusively a month less than other women and the total average dura-tion of breastfeeding was 6.1 months only, while others were breastfed for an average of 8.5 months. Children of obese mothers had the highest weight, BMI and weight for length during all the monitoring period and they also were the longest ones at the age of 18 months. Length of breastfeeding had no proved impact on the weight of obese mothers` children at the age of 18 months.

Conclusion. With an increasing BMI of mothers, dura-tion of breastfeeding shortens and weight of the children increases. However, we also found lean children among those of obese mothers.

ACKNOWLEDGEMENTS

Supported by the grant IGA MZ ČR NS9974-4/2008 and Prvouk P02.

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Anatomical changes of temporomandibular joint in the aging process

Mirela Rozpravkovaa, Lubomir Redajb, Jana Ohlasovac, Kvetuse Lovasovad, Maria Matusevskae, Jana Pribulovaf,

Stanislav Mateffyd, Darina Kluchovad

aDepartement of Orthodontics, Faculty of Medicine of Dentistry, Palacky University Olomouc, Czech RepublicbPrivate Dental Clinic, Studio Smile, Kosice, Slovak RepubliccPrivate Dental Clinic, Ohlasdent, Kosice, Slovak RepublicdDepartement of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicePrivate Dental Clinic, Lubotice, Slovak RepublicfPrivate Dental Clinic, Presov, Slovak Republic

Aims. Temporomandibular joint (TMJ) and adjacent orofacial region (pterygomandibular space) is a key point localization of important anatomical structures and pos-sible occurrence of pathological processes. The aim of our research is to define morphological changes of TMJ in the aging process. In the natural process of aging TMJ undergoes degenerative and proliferative changes. Rate compensation of incongruences of articular surfaces is determined by biconcave articular disc.

Methods. In our anatomical study we used 8 human adult male and female cadaveric heads (sagittal sections) aged 30 to 80 years with different dental status. The ca-daveric material from Department of Anatomy, Faculty of Medicine, Pavol Jozef Safarik University in Kosice was fixed in 10% formalin.

Results. This study focused on discus changes in the natural aging process. It provides the review of psy-chophysiological concept of TMJ disorders with a pre-dominance of stress factor. Especially highlights the importance of morphometric parameters of the lower jaw in Orthodontics. Orofacial Anatomy knowledge necessary for interpretation of cephalometric X-ray in Orthodontics specifies the treatment plan of patients with fixed orth-odontic appliances. Our study presents topographic par-ticularities of the mandibular canal in toothless mandible – atrofic changes of alveolar process, localisation of man-dibular canal, as well as relationship of external morphol-ogy of the mandible and the course of mandibular canal.

Conclusion. The exact determination of the anatomi-cal joint ratios and structures in Infratemporal Fossa is essential for the successful implementation of diagnostic, therapeutic or surgical procedures.

ACKNOWLEDGEMENTS

We would like to thank for support and for valuable advice during realization of this study, especially our sci-entific supervisor, doc. MVDr. Kvetuse Lovasova, PhD, as well as Head of the Department of Anatomy, prof. MUDr. Darina Kluchova, PhD.


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61

Expression of carbonic anhydrase IX and its correlation with clinicopathological characteristics

in lung cancer cellsSilvia Rybarova, Ingrid Hodorova, Jozef Mihalik,

Janka Vecanova


Aims. The aim of our study was to reveal the expres-sion of protein carbonic anhydrase (CAIX) in lung cancer cells and correlate its levels with basic clinicopathological parameters (type of tumor, stage and grade). Hypoxia in cancers is associated with poor prognosis. Protein CAIX belongs to the most strongly induced proteins in response to hypoxia. Tumor hypoxia is clinically very important phenomenon that affects response to therapy, cancer progression and survival of cancer patients. Therefore, detection of hypoxic regions within tumors is important for stratification of patients for suitable treatment regi-mens. CAIX is interesting for its low expression in normal tissue, but on the other side is strongly associated with tumor cells and is expressed abnormally in a broad palette of human tumors.

Patients and Methods. Indirect immunohistochemical (IHC) method was used to detect the expression of CAIX in 135 samples of non-small cell lung cancers (NSCLC). The samples of NSCLC were divided according the types into adenocarcinomas (N=56), squamocellular carcinoma (ca) (N=58), large cell ca (N= 12) and other types (N=9). Divission according to the stage was into 1A+1B, 2A+2B, 3A+3B and 4. Finally, we have evaluated the CAIX ex-presssion according to the grade (G)1, G2, G3 and G4. The IHC expression was scored as follow: 0 positive cells = – (negative sample), 1-10% of positive cells = 1+ (nega-tive sample), 11-90% = 2+ (positive sample), 91-100% = 3+ (positive sample). The patients gave their informed consent before their inclusion into the study.

Results. The highest degree of positive samples reached the squamocellular samples in number 39/67% and 19/33% of samples were CAIX negative. Large cell ca samples were positive in 7/58%, the rest 5/42% were nega-tive. Adenocarcinoma samples were CAIX immunoreac-tive in 27/48% and 29/52% were negative. Stage 1A+1B achieved the highest positivity, it was 52/58%, 2A+2B = 9/47%, 3A+3B = 11/52% and stage 4 = 2/40%. In case of 4 grades, G3 reached the most CAIX positive samples, 41/63%. In G4 it was 6/55%, G2 25/52% and in G1 2/18%. We evaluated our results also by statistical methods. For statistical evaluation we have used the Kruskal –Wallis test and chi-square test. We did not find any correlation between expression of CAIX and clinicopathological pa-rameters.

Conclusion. CAIX have attracted considerable at-tention, not only as a functional component of tumor physiology, but also as a tumor biomarker and potential

therapeutic targets. Nevertheless, we did not find any cor-relation between expression of CAIX and clinicopatho-logical parameters.

ACKNOWLEDGEMENTS

We thank Mgr. Ivana Čigašová-Perunská and Mgr. Diana Gagyiová for their skillful technical assistance. The authors declare that they have no competing interests.

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Differences in expression of calcium binding proteins in the perirhinal and retrosplenial cortex

of the ratMartin Salaja, Filip Barinkad, Hana Kubovac,

Rastislav Drugaa,b

aDepartment of Anatomy, 2nd Faculty of Medicine, Charles University in Prague, Czech RepublicbInstitute of Anatomy, 1nd Faculty of Medicine, Charles University in Prague, Czech RepubliccInstitute of Physiology, The Academy of Sciences of the Czech Republic, Prague, Czech RepublicdKlinik und Poliklinik für Neurologie, Universität Regensburg, Regensburg, Germany

Aims. The main goal was to describe interneuronal population expressing calcium binding proteins calretinin (CR) and parvalbumin (PV) in the perirhinal (PRC) and retrosplenial (RSC) cortex of the rat. These two cortical areas differ strikingly in their connectivity and function, which could be caused also by different composition of the interneuronal populations. Having a precise knowl-edge of the cellular composition of any cerebral area forms one of the basic input parameters and tenets for computational modeling of neuronal networks and for understanding some pathological conditions, like generat-ing and spreading of epileptic activity.

Methods. The brains of 8 male 3-month-old Wistar rats were used in this study. The brains were cut into 50 μm thick coronal section and these were stained for NISSL (cresyl violet), CR and PV. We perfomed qualitative and quantitative analysis on these sections, using stereologi-cal and densitometric approach. All data aquired were statistically evaluated.

Results. PRC possesses higher absulute and relative densities of CR+ and PV+ neurons than RSC, but the CR: PV ratio is higher in the RSC, which is similar to the neocortex. The bipolar/bitufted neurons are most com-mon type of CR+ population, while the majority of PV+ neurons show multipolar morphology.

Conclusion. In our study we described the pattern of CaBP immunoreactivity, the density of CR+ and PV+ neu-rons as well as the overall neuronal density in rat perirhi-nal and retrosplenial cortex. Current results indicate that main difference between analysed areas is in density of


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CR+ neurons, which was significantly higher in the PRC. Our results coupled with works of other authors show that there are significant differences in the interneuronal com-position and distribution of heretofore seemingly similar cortical areas. This results may contribute to the better understanding of the mechanism of function of this corti-cal region in normal and diseased states.

ACKNOWLEDGEMENTS

We would like to thank to Mrs. B. Čejková for excel-lent technical assistance.

This work was supported by Grant Agency of Charles University, grant no. 35407.

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Intercellular junctions of corona radiata cells and oocyte in preovulatory period

Miroslava Sedlackova, Martin Anger, Natalie Matijescukova

Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Aims. Oocyte maturation takes place under natural conditions in close contact with corona radiata cells. Thin cytoplasmic processes of corona radiata cells penetrate the zona pellucida and form the intercellular junctions with the oocyte. Factors produced by the corona radiata cells maintain the arrest of meiosis and prevent the re-sumption of the first meiotic division of the oocyte. When the follicles are punctured to obtain oocytes for in vitro fertilization, whole oocyte-cumulus oophorus complexes are aspirated, but also some cumulus-free oocytes cov-ered by zona pellucida only are obtained. The aim of the presented work was to determine on a mouse model the structure of the perivitelline space of oocytes surrounded by cumulus and cumulus-free oocytes and to compare the morphology of both types of oocytes.

Methods. 6 oocyte-cumulus oophorus complexes (group 1) and 6 cumulus-free oocytes (group 2) obtained by puncture of antral ovarian follicles of mice unstimu-lated ovaries were processed standardly for electron mi-croscopy.

Results. Group 1 oocytes: long thin processes of co-rona radiata cells after penetration through zona pellucida were connected with the oocyte by small desmosomes or gap junctions. Oocytes exhibited a structure typical for oocyte with germinal vesicle.

Group 2 oocytes: the zona pellucida seemed denser in comparison with the group 1, processes of the corona ra-diata cells persisted in the zona and in perivitelline space, intercellular junctions were mostly preserved. Some oo-cytes did not show any morphological alteration on ultra-structural level, the only exception was the higher number of myelin structures. The other oocytes (with normal ap-pearance on the light-microscopic level), showed more

significant changes, which can be considered as signs of incipient degeneration. Especially secondary lysosomes in the form of autophagic vacuoles were numerous.

Conclusion. The oocytes with precocious loss of co-rona radiata cells, are likely to be in the initial stage of extinction, even though some of them seem intact and do not show any degenerative changes. Fertilization of these oocytes may be successful, but their developmental potential is probably limited.

ACKNOWLEDGEMENTS

This study is supported by the Grant Agency of the Czech Republic (15-04844S).

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Elucidating cardiac conduction system formation with phylogenetic approaches

David Sedmeraa,b

aInstitute of Anatomy, First Faculty of Medicine, Charles University in Prague, Czech RepublicbInstitute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic

Aims. To complement our developmental studies in birds and mammals, we study remodeling of the atrioven-tricular canal and development of the ventricular conduc-tion system also from the phylogenetic perspective. After finding a functional, but not morphological, equivalent of His-Purkinje system in fish and amphibians, we focused our attention on reptilian models.

Methods. We performed optical mapping and histolog-ical examination of partially septated reptilian hearts of monitor lizard Varanus acanthurus and completely septat-ed crocodilian hearts (Crocodylus siamensis). Conduction system markers were detected on serial paraffin sections using in situ hybridization and immunohistochemistry.

Results. Optical mapping revealed first ventricular epi-cardial breakthroughs in the area of the forming interven-tricular septum on the left side in the later stage Varanus hearts. Post-septation crocodilian hearts showed an apex-to-base activation pattern with dual breakthrough near the ventricular apices. Conduction system markers Irx1 and 2, Tbx5 and Myh6 were expressed in the interventricular septum in the Varanus heart. Crocodilian hearts showed distinct positivity in the pacemaker area as well as in the interventricular septum for the HNK1 epitope, similar to situation in the rat or chick hearts.

Conclusion. Our preliminary results show that nascent conduction system is present in advanced reptilian hearts. In the absence of fossil evidence, this is the closest we can get to understand the evolution of cardiac conduction system and its relationship to ventricular septation and homeiothermy.


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ACKNOWLEDGEMENTS

Supported by Ministry of Education PRVOUK P35/LF1/5 and institutional grant RVO:67985823 (Academy of Sciences).

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Ultrastructural and videomicroscopic findings in patients with genetically proven primary ciliary

dyskinesiaPetra Simunkovaa, Jana Djakowa, Ondrej Cineka,

Ludek Vajnerb, Jiri Uhlikb

aDepartment of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital in Motol, Prague, Czech RepublicbDepartment of Histology and Embryology, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic

Aims. Primary ciliary dyskinesia (PCD) is a multi-genic autosomal recessive disorder affecting respiratory tract and other organs where ciliary motility is required. Its clinical manifestations are recurrent or chronic respi-ratory infections consequent from stagnation of mucus, subfertility or infertility and laterality defects. The classic triad of chronic sinusitis, bronchiectasis and situs vis-cerum inversus is known as the Kartagener syndrome. The diagnosis of PCD is based on clinical presentation, high-speed videomicroscopy (HSVM) and transmission electron microscopy (TEM). It can be proven by a genetic examination; although only in 60 – 65% of all cases a genetic defect could be found due to the enormous hetero-geneity of PCD. We decided to review our TEM, HSVM and genetic results in patients with PCD to evaluate the validity of both morphological examinations.

Methods. Total of 27 patients with genetically proven PCD were selected from the cohort of 105 patients exam-ined by TEM in last 20 years. All patients recruited from the whole Czech Republic were indicated for HSVM and TEM after the thorough clinical examination. The result of HSVM was not known to TEM investigator and vice versa. The DNA analysis was performed at the end of the diagnostic algorithm. Our TEM and HSVM findings were compared with expected findings described in the literature and corresponding with particular genotypes.

Results. Mutations of 6 PCD genes were detected in our patients. All the genes are known as causative with specific TEM and HSVM findings. Our TEM results were consistent with expected findings in 20/27 cases, HSVM corresponded to expectations in 24/25 patients. Most un-expected TEM findings concerned the erroneous assess-ments of inner dynein arms (IDA), which are very subtle and can be easily misinterpreted. TEM of one patient was primarily described as a normal ultrastructure. This result was later re-assessed to the defect of outer dynein arms (ODA) using an electron microscope allowing higher

resolution. No HSVM records of our genetically proven PCD patients were interpreted as “normal”.

Conclusion. The results of our retrospective study con-firmed the validity of both morphological examinations in diagnostic protocol of PCD.

ACKNOWLEDGEMENTS

The work was supported by the Ministry of Health of the Czech Republic (grant No. NT11469-5) and by the Conceptual Development Project of Research Organization, University Hospital Motol, Prague, the Czech Republic (00064203).

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Wound repair, ageing and cancer: challenge for new therpaeutic modalities

Karel Smetana, Jr., Lukas Lacina, Barbora Dvorankova

Institute of Anatomy, 1st Faculty of Medicine, Charles University in Prague, Czech Republic

Aims. Incidence of different types of cancer has an in-creasing tendency in developed countries worldwide along with increasing average life expectancy, also described as "the grey tsunami". This unfavourable situation in perspec-tive of demographic situation and cancer cell biology was analysed.

Methods. This lecture reviews our own results from previous decade and published data of other authors.

Results. In animals with strong regenerative potential, e.g. flatworms, their relatively limited lifespan is accom-panied by low incidence of tumors. On the other hand, the regenerative potential of humans is low and human life expectancy is rapidly increasing. Notably, incidence of malignant diseases is also increasing. This situation can be elucidated by the hypothesis that the risk of tumor formation is dependent on the number of tissue stem cell divisions, which is naturally also increased with aging. Reduced efficacy of gene repair mechanisms in elderly supports this theory. The risk of "the grey tsunami" ac-companied by epidemy-like incidence of malignant tumors urgently requires development of new generations of an-ticancer drugs that will target not only cancer cells but also the cancer microenvironment as a niche for cancer stem cell.

Conclusion. The unfavourable demographic situation in developed countries and increasing incidence of tu-mors tasks to develop the new therapeutic modalities. The therapeutic manipulation of stroma can be a good target for anticancer therapy.


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ACKNOWLEDGEMENTS

This study was supported by Grant Agency of the Czech Republic No. 304/12/1333.

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Morphology of vasa vasorum in the explanted bypassed saphenous veins

Josef Stingla, Jan Pirkb, Zbynek Strakac, Marek Setinad, David Kachlika, Josef Sache, Vladimir Musilf,g

aDepartment of Anatomy, Third Faculty of Medicine, Charles University in Prague, Czech RepublicbClinic of Cardiovascular Surgery, Institute of Clinical and Experimental Medicine, Prague, Czech RepubliccDepartment of Cardiac Surgery, Third Faculty of Medicine, Charles University in Prague and University Hospital Kralovske Vinohrady, Prague, Czech RepublicdDepartment of Cardiosurgery, Second Faculty of Medicine, Charles University in Prague, and University Hospital Motol, Prague, Czech RepubliceDepartment of Pathology, Third Faculty of Medicine, Charles University in Prague and University Hospital Kralovske Vinohrady, Prague, Czech RepublicfCentre of Scientific Information, Third Faculty of Medicine, Charles University in Prague, Czech RepublicgInstitute of Information Studies and Librarianship, Faculty of Arts, Charles University in Prague, Czech Republic

Aims. The aim of the study was to describe the pres-ence and participation of the vasa vasorum (VV) on the supply of the failed great saphenous vein (GSV) grafts in different time intervals after their implantation as aorto-coronary bypasses (ACB).

Methods. The study was done on the segments of GSV-ACB bypasses, explanted either post mortem or per-operatively from 39 patients. The specimens obtained were divided into 5 groups after the duration of bypasses functioning: A – up to 1 month (7 cases), B – 1-6 months (3 cases), C – 7-24 months (2 cases), D – opened, but functionally insufficient bypasses (6 cases), and E – above 24 months (21 cases). The specimens were investigated qualitatively by light microscopy using 4 different stain-ings.

Results. Group A: the GSV wall without an apparent arterialisation, with some thromboses of different extent, VV without evident changes. Group B: beginning arte-rialisation of the venous wall joint by intensive intimal hyperplasia; VV without evident proliferation. Group C: complete arterialisation of the media and beginning ath-erosclerosis of the intima, apparent proliferation of VV into the media. Group D: complete arterialisation of the media, with intensive intimal atherosclerosis, an intensive proliferation of the VV into the whole media. Group E: bypasses with nearly completely occluded lumen, caused by extensive intimal hyperplasia; VV invade the whole bypass wall.

From the results obtained it is evident that the walls of grafts, used as ACB, undergo completely different morphological changes, e.i. arterialisation of media and intimal hyperplasia, and according to their extents joined with the proliferation of the VV.

Conclusion. An apparent proliferation of the VV in the walls of the ACB grafts is present in bypasses at in-tervals longer than 6 months after their implantation, and depends, above all, on the extent of the intimal hy-perplasia. Therefore, the proliferation of the VV has to be considered as a secondary process, reacting on the previous pathological changes of media and intima of the failed grafts.

ACKNOWLEDGEMENT

The study was supported by project of Charles University PRVOUK P38.

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Interactive 3D ModelsJan Tokarcika, Frantisek Dorkoa, Eva Vybornaa,

Jaroslava Chylikovab, David Brazinac

aDepartment of Anatomy, Faculty of Medicine, University of Ostrava, Czech RepublicbDepartment of Histology and Embryology, Faculty of Medicine, University of Ostrava, Czech RepubliccDepartment of Informatics and Computers, Faculty of Science, University of Ostrava, Czech Republic

Aims. Our goal is to simplify teaching and test stu-dents’ knowledge of the acquired knowledge. To better understand the different topographical relationships of anatomical structures of the human body, we created 3D models, using modelling and visualization techniques.

Methods. To construct the models of the human body, resources from the Department of Anatomy Medical School were used. The models of the human body were processed in Eon Player Experience. Body Parts were con-verted from 2D video sources into 3D object structures. The resulting structures were subsequently adjusted in EON Studio and MAYA respectively – EON Creator. The output images were supported by the software solution EON Experience Player, which performs highly interac-tive visualization. Animations are presented with the aid of a projector, controlled by a computer.

Results. The biggest pros include easy handling of 3D objects. A fully usable functional site with a database of the cardiovascular system, heart, skeletal system, respira-tory system, and digestive system in a usable graphical interface. The acquired knowledge can be improved stu-dent and then checked in the “Quiz”.The test consists of open and closed questions. For open questions the correct answer must be entered for closed questions, the system offers four options, from which the student may choose


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one correct answer. The system automatically evaluates the accuracy of the answers after each question.

In the case of incorrect answers, the student continues the test. In conclusion, the system evaluates the overall test, including the number of correct and incorrect re-sponses and the overall evaluation of the test.

Conclusion. Teaching anatomy is currently not pos-sible without accompanying image processing, lectures, and practical exercises. Teaching has one goal, and that is to increase the quality of the educational process. This approach places greater demands on the teacher, who has to have a thorough knowledge of computers and how to most effectively use new technologies, not only in the education itself, but also in the development of teaching materials. The future of teaching anatomy can be seen in conjunction with conventional methods of interconnec-tion of imaging in clinical practice. Interest in creating 3D models was expressed mainly by students of medicine and bachelor’s candidates for the continuing validation of knowledge in the study, and in the repetition of the test anatomy.

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Various noxious factors and their effect on the developing nervous system

Lenka Tomasovaa, Natalia Hvizdosovaa, Benadik Smajdab, Martin Bonaa, Stanislav Mateffya, Darina Kluchovaa

aDepartment of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicbDepartment of Animal Physiology, Institute of Biology and Ecology, Faculty of Science, P. J. Safarik University in Kosice, Slovak Republic

Aims. The developing central nervous system (CNS) is very sensitive to the action of various biological, chemi-cal and physical exogenous factors, which can lead to the various types of damages. The aim of our study was to compare influence of two different noxious factors (physi-cal and chemical) on the morphology and function of the brain.

Methods. The study was performed on adult Wistar rats, the progeny of mothers exposed to ionizing radiation (IR) (1 Gy of gamma rays on the 17. day of gestation) or to retinoic acid (RA) (1 mg/kg body weight on the 14. – 16. day of embryonic development). We tested emotional behaviour of rats in the plus maze test and after the behav-ioural test we determined the presence and morphology of nitrergic neurons in the amygdala.

Results. The results obtained from the plus maze test showed increased level of anxiety and fear in animals pre-natally exposed to IR or RA. Reducing the time, which animals spent in the open arms of apparatus, manifested it. Statistically significant changes of emotional behav-iour were observed after exposure to ionizing radiation. Histological observation revealed that nitrergic neurons of control animals, animals exposed to IR and animals

exposed to RA had the same morphological structure. The cells had oval or elongated body with long dendrites and the body size and length of dendrites were compa-rable between all groups of animals. Also number of nitrergic neurons of amygdala did not differ between ex-perimental groups.

Conclusion. These findings confirm the fact that de-veloping nervous system is very sensitive to the action of external factors. Our results also indicate that the level and type of damages of CNS need not specifically depend on the type of acting factor; different types of noxious fac-tors acting in the same phase of embryonic development can have the same effect on the function and morphology of the central nervous system.

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Segmental differences in the orientation of smooth muscle cells in the tunica media of porcine aortae

Zbynek Tonara, Petra Kochovab, Robert Cimrmanc, Josef Perktoldd, Kirsti Wittere

aDepartment of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech RepublicbNew Technologies – Research Centre, University of West Bohemia, Pilsen, Czech RepubliccOutremont QC H3T1E9, Quebec, Canada dInstitute of Anatomy, Histology and Embryology, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria

Aims. The orientation of vascular smooth muscle cells of porcine aortae was assessed to test the widely accepted assumption that these smooth muscle cells are arranged in two helices.

Methods. We used tangential histological sections of 82 samples of five anatomical segments of thoracic and abdominal porcine aortae and three age groups in animals ranging in age from 5-210 days. The distribution of the orientation of smooth muscle cell nuclei in five proxi-modistal segments of the porcine aortae was determined using an algorithm that fitted a mixture of one to five von Mises probability distributions of the data retrieved from histological micrographs. Automated tracking of the nuclei was confirmed by and consistent with manual histological analysis.

Results. The orientation of the vascular smooth muscle cells was successfully fitted using two von Mises distributions in most of the samples with different ages, wall thicknesses, and anatomical positions, which cor-responds to two populations of vascular smooth muscle cells. A minor fraction of samples also required a tertiary von Mises distribution to describe the orientation of the smooth muscle cell nuclei. The distribution of vascular smooth muscle cells in five aortic segments ranging from the thoracic ascending aorta to the abdominal intrare-nal aorta exhibited similar main directions but different shapes.


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Conclusion. The results are consistent with the widely used model of two muscular helices intermingling in the arterial wall. Furthermore, we calculated the central an-gles of symmetry and the mean value of angles between the two assumed smooth muscle directions. We also suc-cessfully approximated the orientation of the smooth muscle cells using a mixture of von Mises distributions and our open source software named dist_mixtures. This method is openly available to researchers who are inter-ested in mathematically assessing the orientation of cell nuclei in various tissues.

ACNOWLEDGEMENTS

The study was supported by the ERDF projects No. ED.1.05/2.1.00/03.0076, CZ.1.05/1.1.00/02.0090, and the Prvouk P36 Project of the Charles University in Prague.

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Regulatory action of quercetin on cyclooxygenase-2 in the model of ischemia/

reperfusion injury of small intestineStefan Totha, Milan Marettab , Kristina Gregovaa, Jan Soltesa,

Martin Svanaa, Martin Pribulaa, Matus Kusniera, Zuzana Jonecovaa

aDepartment of Histology and Embryology, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak RepublicbDepartment of Neurology, Louis Pasteur University Hospital, Kosice, Slovak Republic

Aims. Quercetin, a polyphenolic bioflavonoid, pos-sesses multiple pharmacological actions including anti-inflammatory and anti-tumor properties. The aim of present work was to investigate the effect of quercetin on cyclooxygenase-2 (COX-2), an important mediator in inflammation promotion, during intestinal ischemia/re-perfusion injury (IRI) of the small intestine in rats.

Methods. Adult male Wistar rat SPF–Charles River (n=28) were randomly assigned to the control group (E) or experimental groups (Q). Quercetin dissolved in etha-nol or ethanol vehiculum were applied intraperitoneally 30 min before the complete ischemia of a. mesenterica cra-nialis. Ischemia was performed by microvascular clamp, lasted 1 h and was followed by 1 h (E1, Q1) and 4 h (E4, Q4) of reperfusion period. After the reperfusion period, bioptic samples were harvested and histological sections were stained by H&E staining method and COX-2 immu-nohistochemical method. The numbers of COX-2 positive cells in the epithelium were calculated per 1 mm of the epithelium lining.

Results. Quercetin had no effect on the number of COX-2 positive cells in the epithelium of the samples tak-en after 1 h of reperfusion, in both E1 and Q1 groups the results were similar (20.48±1.07 and 20.59±2.28). After 4 h of reperfusion in group E4 non-significant increase in it

was found (26.46±1.76); but in the group Q4 decrease in COX-2 positive cell number was observed (12.37±0.91). The decrease was significant in comparison with both Q1 (P<0.01) and E4 (P<0.001) groups.

Conclusion. COX-2 is an immediate-early response gene that is induced by a variety of mitogenic and inflam-matory stimuli. Our results confirmed quercetin inhibito-ry effect of COX-2 family in the model of rat IRI, since the number of COX-2 positive cells in the epithelium of the jejunum after 4 h of reperfusion was significantly reduced.

AKNOWLEDGEMENTS

This study was supported by the grant VEGA 1/0478/2014.

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Iconology of diseases in Renaissance artIvo Uberalla, Ales Filipb

aDepartment of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepublicbDepartment of Musiciology, Faculty of Arts, Masaryk University Brno, Czech Republic

Aims. The aim of this paper will be to highlight the selected examples of symptoms in Renaissance art.

Methods. From the point of view of methodology iconographic-iconological approach will be applied. Some of the pathological diagnoses in Renaissance art will be introduced to and will be set in the historical-social frame-work.

Results. Renaissance is a historical period, which is apart from anything else characterized by concern of man about himself. In combination with the development of anatomy as a science, first appearance of pathologies in pictures began to appear. Leaving aside the appear-ance of malformations and pathologies that are secular and symbolic nature (eg. St. Roch as the patron saint of plague patients), secular themes remain among the few, that offer an insight into the health status of the popula-tion. Besides conventional medical books in which we meet with numerous diseases (De recondita abscessuum natura, 1643 or Selecta praxis medico-chirurgicae, 1652) we can rarely observe the iconographical representation of different pathologies in artistic artifacts. Lets name some of them for example The old man and his grandson (Domenico Ghirlandaio, 1490) or Person with syphilis (Albrecht Dürrer, 1496). A special chapter consists of scenes that use pathological changes to amplify the effect of a caricature. Examples of art work combining secu-lar and medical theme are for example A representation of the plague infected Temptation of St. Anthony (Mathis Grünewald, 1510-1515), goitre at Assumpta from Desna (unknown author, 1450) or symptoms of leprosy in the work Plate with three apostles (unknown author, 1510) .A


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special place in the Renaissance iconology disease plays Michelangelo's depiction of incipient cancer of the statue "Night" in the Medici Chapel in the cathedral in Florence (1521) or distorted breast by cancer on a fresco of the Sistine Chapel (1508-1541) by the same author.

Conslusion. The presence of signs of disease in the arts and their reproducibility testifies about the observation capability and suggests that Renaissance artists used live models. The frequency of display of various pathologies gives some idea about the population health status.

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Blood and lymphatic microvascular changes in relation to VEGF–A and VEGF–C expression in

psoriatic lesionsDesanka Vybohovaa, Yvetta Mellovaa, Katarina Adamicovab

aInstitute of Anatomy, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovak RepublicbInstitute of Pathological Anatomy, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovak Republic

Aims. Present study focuses on the evaluation of quan-titative changes of blood and lymphatic microcirculatory bed, VEGF–A and VEGF–C expression and their mutual relations within the psoriatic lesions.

Methods. Skin samples of psoriatic lesions located at the abdominal and back regions were taken from untreat-ed patients (in age range 32-55 years) without any system-ic disorder and compared with control healthy skin from identical regions. Immunohistochemical analysis with an-tibodies to CD34, podoplanin (D2 – 40), VEGF–A and VEGF–C was used to determine the blood and lymphatic vessels area (BVA and LVA) in standard fields of the pap-illary dermis and VEGF–A and VEGF–C positive area in epidermis and dermis. All mentioned parameters were evaluated by using exact morphometric analysis software.

Results. Measurements revealed that both lesional and healthy skin samples showed significantly higher BVA than LVA, and higher VEGF–A than VEGF–C expres-sion. BVA values in psoriatic lesions showed almost 3 – fold increase. Althought LVA values were significantly lower than BVA, they showed 2 – fold increase in com-parison to control healthy skin. VEGF–A and VEGF–C were strongly overexpressed in both lesional epidermis and dermis. VEGF–A expression in psoriatic lesions was significantly higher in epidermis (27 – fold increase) than in epidermis (20 – fold increase). Similarly, VEGF–C ex-pression achieved 22-fold increase in psoriatic epidermis and 14 – fold increase in psoriatic dermis. Obtained re-sults confirmed significant positive correlation between VEGF–A expression in epidermis and BVA (r=0.659, P=0.027) in papillary dermis of psoriatic lesions. However, correlation between VEGF–A expression in der-mis and BVA was only mild and statistically insignificant (r=0.309, P=0.224). Mild but statistically insignificant cor-relation was demonstrated between VEGF–C expression

in epidermis and LVA as well as in dermis and LVA in papillary dermis of psoriatic lesions (r=0.311, P=0.351; r=0.455, P=0.159 respectively).

Conclusion. These data support the hypothesis that angiogenesis and lymphangiogenesis in psoriasis are to a greater extent driven by VEGF–A and VEGF–C expres-sion in epidermis.

It probably results from epidermal changes, mainly hyperplasia, which are prominent in pathogenesis of pso-riasis.

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Historical overview of morphological studies of palatine tonsil

Eva Vybornaa, Frantisek Dorkoa, Vladimir Holibkaa, Jan Tokarcika, Jaroslava Chylikovab

aDepartment of Anatomy, Faculty of Medicine, University of Ostrava, Czech RepublicbDepartment of Histology and Embryology, Faculty of Medicine, University of Ostrava, Czech Republic

The human palatine tonsil, as part of the Waldayer lymphatic ring, represents the first point of contact with antigens from inhaled air and food. As an organ suffer-ing from frequent inflammations, it has been studied not only by morphologists but also ENT specialists, imunolo-gists and specialists from other medical fields. In their contrinution, authors summarize findings about tonsil-lar lymphoid tissue, tonsillar structure and ultrastructure (lymphoreticular parenchyma, surface and crypt epithe-lium, microcirculation, etc.) as well as its function, as they were found in the literature and obtained by self-study.

The research of the human palatine tonsil over time has been closely related to the evolution of mi-croscopy techniques and methodology. Using a light microscope, the structure of the human palatine ton-sil was firstly established by Stöhr in 1891; followed by Lenz who in 1972 further described its cryptic epithe-lium (“Ultrakanalsystem“). The definition of the human palatine tonsil in anatomical terms dates back to 1961 (Fioretti). The boom of the scanning electron microscopy in 70ies and 80ies of the 20th century significantly en-hanced the means of study and resulted in a number of new findings. Perry (1988, 1994, 1998) confirmed the conclusions of Lenz by studying in TEM and SEM the lymphoreticular stroma of the tonsil. Oláh pioneered to describe the microfold cells (“M-cells“). Together with researchers from Japan (Kodama, Hoshino 1977, Maeda, Mogi 1982), those authors significantly contributed to the research of the lymphatic tissue by describing, among oth-ers, the ultrastructure of the follicular areas with dendritic cells, the reticular epithelium as well as the Langerhans cells containing so called Birbeck granules. They also studied the microcirculation of tonsils resulting in the discovery of the High Endothelial Venules (HEV).

In Czechoslovakia and then the Czech Republic, Slípka devoted most of his research to the study of the


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lymphatic tissue, for example, by comparing the develop-ment of thymus and tonsil. Holibka (1992) described in detail the tonsil‘s ultrastructure and, based on microcir-culation studies, post-capillary venules and fenestrated capillaries. Holibková (1989) dealt with the relationship between glandular and lymphatic tissue and Kutal (1998) described the tonsil of insectivores. Stemming overwhelm-ingly from tonsillectomies, the human material used for the research implies a pathological modification of the tissues. Therefore, some authors (Holibka 1992, 1994, Výborná 1997) devoted their study to ultrastructure of the human palatine tonsil in the prenatal period, i.e. without the presumed antigenic stimulation. Using the method of metallized histological sections and based on a sample of 76 tonsils from 16th to 40th prenatal week, the au-thors studied in particular the tonsil’s lymphoreticular parenchyma, surface epithelium and crypts. The research revealed the presence of reticular epithelium, including fenestrated capillaries, as of the 30th prenatal week and concluded that the tonsil is ready to function already be-fore the birth. Recently, the research of the human pala-tine tonsil continues focusing on the Mucosa Associated Lymphatic Tissues („MALT“) of both human (NALT – Perry, 1988, 1994, 1998) and animal organs (rabbits, sheep, chicken – Oláh, 1975, 1988, 2003; neat – Palmer, 2009; and dromedary – Zidan, 2009).

The contribution summarizes knowledge on the struc-ture and ultrastructure of the tonsil (epithelium surface and crypt epithelium, its reticularization, intraepithelial pathways and migration of lymphocytes as well as special-ized vascular structures) and also mentions other com-ponents of MALT. The findings were obtained by using of light, transmission and scanning electron microscopy.

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Tractography of corpus callosum and gyrus cinguli in patients with Alzheimer’s Disease

Zdenek Wursta, Petr Zacha, Ibrahim Ibrahimb, Ales Bartosc,d, Vladimir Musile, Matej Patzelta, Jana Mrzilkovaa

aDepartment of Anatomy, Third Faculty of Medicine, Charles University in Prague, Czech RepublicbMR Unit, Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, Prague, Czech RepubliccAD Centre, National Institute of Mental Health, Klecany, Czech Republic dDepartment of Neurology, Third Faculty of Medicine, Charles University in Prague and University Hospital Kralovske Vinohrady, Prague, Czech RepubliceCentre of Scientific Information, Third Faculty of Medicine, Charles University in Prague, Czech Republic

Aims. The most frequent cause of dementia in middle-age and elderly is currently Alzheimer's Disease (AD). It is a chronic progressive nervous system disease mani-fested by neurodegenerative process, accompanied by characteristic histopathological changes (pathological

deposition of amyloid β protein and protein τ). AD dis-rupts brain function and causes a decline in cognitive function (thinking, memory, judgment). The aim is to verify the hypothesis, based on volumetric changes of the corpus callosum (CC) and its specific parts in the group of AD patients and controls using tractographical displaying. Examined parameters are the number, volume and length of the fibres in defined areas of the corpus callosum, the corresponding functional involvement. The same parameters are also measured in the gyrus cinguli, another structure involved in memory consolidation, are connected in the limbic circuit.

Methods. We have used 3T DTI MRI scans of the pa-tients diagnosed as probably AD and control persons from the Department of Radiodiagnostic and Interventional Radiology IKEM. These scans were analysed by the com-puter program DSI Studio. Using the region of interest maps we have displayed the fibres of the front and rear part of the gyrus cinguli and five functional parts of the corpus callosum.

Tractography is a method for neuronal fibres of the brain displaying. The axonal membrane, myelin, and the other structures significantly affect Brown’s movement of the water molecules. The resulting vector is largest in the longitudinal direction of the fibre. Tractography is calculated separately for each voxel wherein the fibre direction depends on the direction of the resultant vec-tor of diffusional tensor. Composition of neighbouring vectors according to set parameters (angle of deviation, fractional anisotropy) direction and reconstruct the result-ing waveform fibres. This allows all the pathways the areas in which they were ranked numbers of nerve fibres, their volume and average length. Visual control of fibres and their participation is made possible 3D reconstruction MRI sequences. Statistically we were compared between patients and controls coherent age.

Results. There is statistically significant decrease in the volume and number of fibres in the front and rear part of the CC. Decrease in the number of fibres has also been important in CC medial anterior and CC centralis. The fibre length did not change in any of the areas measured by us. We have also measured neuronal damage param-eters: fractional anisotropy (FA) expresses the neuronal arrangement, apparent diffusional coefficient (ADC) presents diffusivity in structural volume, axial diffusivity (AX) – characterize microtubules and microfilaments, radial diffusivity (RD) – expresses myelin damage.

Conclusion. Although the gyrus cinguli contributes to memory consolidation, the measured parameters in AD groups to shift there. Most of the monitored parts of corpus callosum in the AD patients were changed. The exception are the functional connections of fibres parietal and temporal lobe, which are between AD and controls unchanged and is probably related to the natural age loss of neurons. All studied structures in our study indicate statistically significant decrease of neuronal damage pa-rameters.


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ACKNOWLEDGEMENTS

The research was supported by projects of Charles University PRVOUK P34, P38 and 260168/SVV/2015, NUDZ project ED 2.1.00/03.00/0078, and project MZCR00023001IKEM.

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Blood pressure of girls aged 10 to 18 with respect to intra-abdominal fat tissue

Katerina Kikalovaa, Miroslav Kopeckyb, Jiri Charamzab, Jitka Tomanovac, Petr Zemanekc

aDepartment of Anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepublicbDepartment of Specialised Subjects and Practical Skills, Faculty of Health Sciences, Palacky University Olomouc, Czech RepubliccDepartment of Anthropology and Health Education, Faculty of Education, Palacky University Olomouc, Czech Republic

Aims. This paper focused (i) to determine basic an-thropometric parameters (body height, body weight, BMI), (ii) to determine the values of intra-abdominal fat, (iii) to measure the systolic and diastolic blood pressure, (iv) to classify subjects into BMI zones, in order to assess the impact of intra-abdominal fat tissue on the blood pres-sure in girls aged 10-18.

Methods. The research group consisted of 1075 girls aged 10-18. Their blood pressure was measured with a digital tonometer A&D Medical UA 787 Plus using an appropriate cuff. The blood pressure was measured in a sitting position, on the right arm, after subjects have been five minutes in rest. Oscillometric method was chosen on the basis of anthropometric research arrangement, due to a significant noise in the room where the measurement was conducted. Body height was determined by standard anthropometric method. The values of intra-abdominal fat were obtained using bioimpedance device InBody 230. The classification of subjects into BMI categories was performed using a 7-zone distribution according to WHO. We used the Statistica 12 software for the evaluation and the correlation of particular parameters.

Results. The mean values of body height in the age groups of girls aged 10 to 18 were following: 144.10, 150.84, 157.86, 160.68, 163.86, 164.35, 166.05, 164.85, 165.22 cm; similarly, the mean body weight values were: 37.35, 42.40, 48.42, 51.56, 55.69, 57.55, 60.96, 58.85, 61.34 kg; and the mean values of intra-abdominal fat were: 51.37, 54.81, 55.51, 58.97, 63.31, 69.01, 79.54, 73.55, 77.60 cm2. Values of intra-abdominal fat up to 100 cm2 were considered normal. Importantly, such value or higher was found in 12.74% of girls. The classification into BMI zones for the entire set of girls followed normal distribu-tion. Only the subgroup of girls in the zone of obesity was larger than normal (8.19% vs. 3% in normal population). Systolic blood pressure was stable up to 15 years of age and later started to rise slowly. Diastolic blood pressure exhibited a steady rise, from 71.87 Torr in 10 years to 80.49 Torr at the age of 18. Systolic and diastolic blood

pressure positively correlated with body height, body weight, and BMI values as well as with intraabdominal fat in the whole group, irrespective of age. The highest dependency rate was present between blood pressure and body weight.

Conclusion. Our results correspond to the secular trend of increasing body weight. The blood pressure in-creased with the volume of intra-abdominal fat. For a more precise correlation of parameters the use of multi-variate analysis would be required.

ACKNOWLEDGEMENTS

Research was suppor ted by the project PL.3.22/2.3.00/11.02576, The obesity epidemic – a shared problem: the transfer of knowledge, education, prevention, from Operational Programme Cross-border Cooperation Czech Republic – Poland 2007-2013.

77

Continuously regenerating dentition in anoline lizards: the model of postnatal teeth development leading to changes in tooth shapes and numbers

Veronika Holanovaa, Oldrich Zahradnicekb

aDepartment of Zoology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague, Czech RepublicbDepartment of Teratology, Institute of Experimental Medicine, The Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic

Aims. The deciduous and permanent dentitions in human and other diphyodont mammals mostly dif-fer in teeth size, external and internal morphology and teeth number. The necessary condition for development of such differences between deciduous and permanent dentition is formation of successive teeth generations. Dental characters correspond to feeding preferences and phylogenetical relationships of taxons. The differences between first functional teeth generation and successive generation(s) were not observed only in mammals but also in other toothed vertebrate clades. The increasing number of teeth and teeth size are the most common char-acteristics which change between new teeth generations throughout life. Species of some non-mammalian lineages could significantly change morphology of teeth between teeth generations of juveniles and adults. Here we focus on small group of four anoline lizard species from Cuba, the Cuban false chameleons, which are uniquely adapted to crushing snail shells. Their morphology of dentitions significantly changes between juvenile and adult teeth generations. Such dentitions evolved from the dentition adapted to insectivory and saurivory. Except for Cuban false chameleons we used in this study giant green anole Anolis baracoae with generalized type of anoline dentition to construct possible story of evolution of molluscivory dentition among these specialists. Species of Cuban false


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chameleons could differ in the grade of mallacophagous specialization.

Methods. The lower jaws of specimens were separated from the upper jaws and then were cleaned mechanically and chemically by bleaching solution from soft tissue. The coating by a thin layer of gold was made after bones dehy-dratation in acetone and dry owen. The SEM microscope JEOL SEM 6380 LV operating at 25 kV was used for the dentition documentation.

Results. We showed that each examined species dif-fers in morphology of juvenile and adult dentition, while in adults were differences more expressed. The two main different types of dentition evolved in Cuban false cha-meleon lizards. One group of Cuban false chameleon lizards noticeably increases teeth number throughout life and resemble situation in anoles with generalized type of dentition, while second group of these lizards does not increase teeth number at all or just minimally what we interpreted as one of dentition adaptations for the mol-luscivory. The shapes of teeth in juveniles of Cuban false chameleon lizards resemble insectivorous and saurivorous teeth of anoles, while teeth of adults in the caudal area of jaws significantly differ from anoles by greater robustness, loosing or minimalizing side cusps and by forming more rounded tip of the tooth crown.

Conclusion. We hypothesize that the shape differences correspond to feeding preferences and that adults of A. guamuhaya are possibly insectivorous and saurivorous as well as molluscivorous. Adults of other species of Cuban false chameleon lizards seem to be mainly molluscivorous specialists.

ACKNOWLEDGEMENTS

We thank for the financial support to GACR 14-37368G.

78

M1/M2 Macrophages polarization in white adipose tissue of morbid obese patients

Jaroslava Chylikovaa, Vojtech Kamaradb, Eva Vybornac , Jana Dvorackovad, Pavol Holeczye

aDepartment of Histology, Faculty of Medicine, University of Ostrava, Ostrava – Zabreh, Czech RepublicbDepartment of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech RepubliccDepartment of Anatomy, Faculty of Medicine, University of Ostrava, Ostrava – Zabreh, Czech RepublicdDepartment of Pathology, Faculty of Medicine, University of Ostrava, Ostrava – Zabreh, Czech RepubliceDepartment of Surgery, Vitkovice Hospital a.s., Ostrava – Vitkovice, Czech Republic

Aims. An important factor in pro-inf lammatory conditions associated with obesity is polarization of macrophages, which accumulates in adipose tissue.

Macrophages are heterogeneous cell populations with functional variability dependent on the polarization state. Adipose tissue macrophages may be present in different activation status, known as M1 and M2. M1 and M2 mac-rophages perform different functions, among other things, producing the pro- or anti-inflammatory factors. The aim of our study was to characterize the activation status (phe-notype) of adipose tissue macrophages in morbidly obese using immunohistochemistry.

Methods. In our work, we studied the presence of CD68 and CD204 positive macrophages in white adi-pose tissue. The material was obtained during intraop-erative biopsy in patients undergoing bariatric surgery for morbid obesity. Our group includes 42 men aged 21-66 years with a BMI between 34-58, and 64 women, aged between 21-60 years with a BMI between 30-54. We in-vestigated both subcutaneous and visceral white adipose tissues. The formalin fixed samples were embedded in paraffin. Paraffin sections were stained with hematox-ylin-eosin. Two-steps immunohistochemical indirect method has been used. In first step we used EnVision detection system (Dako), visualization was done using DAB chromogen. Macrophages were detected using an antibody CD68 (Mouse Monoclonal Anti-Human CD68, DakoCytomation). Anti-MSR1 antibody was produced in rabbit (Sigma).

Results. CD68 positive macrophages were found in the interstitium – isolated in the thin septa between two adi-pocytes, and in groups surrounding a small blood vessels or at the point of contact between more fat cells. We also found the so-called „crown-like“ structures consisting of macrophages fully surrounding dead adipocyte, but they were less frequent.

Especially in obese with diabetes the number of CD68 + macrophages was greater in visceral adipose tissue and in the vicinity of hypertrophic adipocytes. CD 204+ macrophages we found in the same localizations as the CD68+. In general, their number was smaller in both sub-cutaneous and visceral adipose tissue. They are present also in the “crown-like” structures, however, they form only a small part from their macrophages. In the obese without diabetes is the presence of CD204+ macrophages comparable with CD68+.

Conclusion. CD68+ macrophages and CD204+ are present in both subcutaneous and visceral adipose tissue. In the morbidly obese with diabetes is the presence of CD68+ macrophages higher especially in visceral adipose tissue. In patients without diabetes, anti-inflammatory oriented M2 (CD204+) macrophages are found more frequently.


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79

ABC transporters and their role in the drug resistancePetr Mlejnek

Department of Anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

Background. In the last decades a significant attention has been dedicated to the role of ABC (ATP binding cas-sette) transporters, namely ABCB1 (P-glycoprotein (P-gp)), ABCG2 (breast cancer resistance protein (BCRP)), and ABCC1 (the multidrug resistance protein (MRP1)). This was mainly due to the convincing laboratory results. Unfortunately, efforts to translate this discovery to the clinic have failed. Indeed, the outcome of clinical trials utilising drug transporter inhibitors to reverse resistance mediated by P-gp was mostly disappointing. A clear cor-relation between drug transporter expression and resis-tance was also difficult to establish in clinical samples. However, the overexpression of ABC transporters, includ-ing ABCB1, ABCG2, and ABCC1 were associated with

poor outcome. These facts open the question whether ABC transporters could mediate drug resistance in tu-mors. The discrepancy between laboratory research and clinical study were mostly attributed to both the complex-ity of the phenomenon of drug resistance at organismal level and to the methodical problems.

Results. However, we think that many disappointing results are due to oversimplifying approach to this com-plex issue. We believe that more rigorous design of the in vitro experiments may provide novel results which will help to better understand the phenomenon of drug resis-tance and shed a light on contradictory results that exist. For example, we observed that the resistance to antican-cer drugs mediated by main ABC transporters, ABCB1 and ABCG2, strongly depends on their expressions at protein levels.

Conclusion. Expression level of a particular drug trans-porter is relevant to the understanding its clinical role.

ACKNOWLEDGEMENT

This work was supported by grant IGA_LF 2015_029 (Internal grant of Palacky University).


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Adamicova K. 73Adamkov M. 1, 49Anger M. 63Baca V. 27, 28, 29Balentova S. 1 Balkova S. 41Barinka F. 62Bartos A. 75Bencat M. 49Bezdickova M. 3Beznoska P. 2, 21Bezrouk A. 47Bilecova-Rabajdova M. 18Blaha M. 38Blankova A. 29Blazkova Z. 3Blom A. N. 51Bludovska M. 6Bolekova A. 4Bona M. 4, 22, 69Bonczek O. 5Borbelyova V. 1Brazyna D. 68Bruckova L. 8Buchtova M. 7, 13, 30, 41, 53, 57Buskova J. 52Capek M. 32Cela P. 7Cendelin J. 33Cernochova P. 5Charamza J. 76Chvatalova J. 2, 21, 23, 55Chylikova J. 12, 68, 74, 78Cimrman R. 70Cinek O. 65Cizkova D. 2, 8Cizkova K. 9, 17Danielisova V. 20Dankova M. 10, 11, 48Dankovcik R. 50Diaz D. 2, 21, 55Djakow J. 65Dobias M. 3Dolezelova E. 8Domorakova I. 10, 11, 48Dorko F. 12, 68, 74Dosedelova H. 13Drobna Krejci E. 40Druga R. 14, 62Dubovy P. 15, 26, 39Durisova M. 38Dvorackova J. 78Dvorankova B. 40, 66Dzugan J. 54Eberlova L. 16Ehrmann J. 9, 17

Elias P. 58Eminger M. 16Erdosova B. 36, 37, 42, 43Fiala P. 19Filip A. 72Filip S. 8Filipcikova R. 3Filova B. 1Gdovinova Z. 48Gregor T. 16Gregorova K. 18, 25, 71Grim M. 40Hajek P. 28Hajtmanova E. 1Herich R. 50Hlouzkova A. 5Hodan R. 57Hodorova I. 19, 20, 61Holanova V. 77Holeczy P. 78Holibka V. 12, 74Hornakova L. 12Horvathova F. 20Hradilova-Svizenska I. 15Hrebikova H. 2, 21, 23, 55Hubacek P. 3Hubalek Kalbacova M. 54Hvizdosova N. 4, 22, 45, 69Ibrahim I. 75Izakovicova-Holla L. 5Janacek J. 32Janitorova Poliacikova M. 10, 11Jekl V. 57Jirkovska M. 24Jonecova Z. 18, 25, 71Jongbloed M. R. M. 51Joukal M. 15, 26, 39Kachlik D. 27, 28, 29, 67Kamarad V. 78Kautzner J. 38Kikalova K. 76Killinger M. 30Klepacek I. 31Kluchova D. 4, 22, 46, 56, 60, 69Klusakova I. 15, 26, 39Knopfova L. 53Kochova P. 70Kolesova H. 32Kolinko Y. 33Konarik M. 28Konieczna A. 17Kopecky M. 76Kopecny T. 36, 37Korimova A. 15Kotyzova D. 6Kovalcikova S. 18

AUTHOR INDEX

Krajci D. 34, 35, 36, 37, 42, 43Krajci D. (Mrs.) 36, 37, 42, 43Kralickova M. 16, 33, 54Krasensky J. 52Krejci P. 5, 13Krizkova V. 6Kubies D. 54Kubikova T. 6Kubina T. 54Kubova H. 62Kucera T. 38Kuchar M. 58Kucharova B. 20Kudlackova M. 18Kuklova A. 26, 39Kunke D. 55Kunova M. 13Kusnier M. 71Kvasilova A. 40Kylar P. 34Lacina L. 66Lametschwandtner A. 16Langova P. 41Lehotsky J. 1Lichnovska R. 36, 37, 42, 43Liska V. 16Lochman J. 5Lovasova K. 4, 46, 56, 60Luzna P. 17Machalek L. 36Machon V. 57Makovicky Pa. 44Makovicky Pe. 44Maretta M. 25, 71Matalova E. 53Mateffy S. 4, 22, 45, 46, 56, 60, 69Matijescukova N. 63Matusevska M. 45, 46, 56, 60Mazurova Y. 47Mechirova E. 10, 11, 48Melenovsky V. 38Mellova Y. 73Mestanova V. 49Micuda S. 8Mihalik J. 19, 20, 61Miklosova M. 50Mirka H. 16 Misek I. 5Mlcoch M. 3Mlejnek P. 79Mokry J. 2, 8, 21, 23, 55Mrzilkova J. 75Musil V. 27, 67, 75Nanka O. 51Nemcova V. 52Novotny R. 35, 37


S47

Oborna I. 3Ohlasova J. 46, 56, 60Oralova V. 53Ostrovska L. 54Palek R. 16Pastucha D. 3Patzelt M. 75Paulova M. 59Perktold J. 70Petrovicky P. 52Pirk J. 38, 67Pisal R. 2, 23, 55Pribula M. 71Pribulova J. 45, 46, 56, 60Pridal J. 38Putnova B. 57Rajdova A. 9Redaj L. 46, 56, 60Rejtarova O. 58Riedlova J. 59Rimarova K. 44Rosendorf J. 16Rozpravkova M. 45, 46, 56, 60Rozsivalova K. 38Rybarova S. 19, 20, 61Sach J. 67Salaj M. 62Sedlackova M. 63Sedmera D. 32, 51, 64

Seidl Z. 52Sery O. 5Setina M. 67Shbat A. 31Shylo N. 7 Simunkova P. 65Skala M. 16Skorvanek M. 48Slama P. 54Smajda B. 69Smarda J. 53Smetana K., Jr. 40, 66Smorodinova N. 38Solar P. 20, 26, 39Soltes J. 71Sonka K. 52Soukalova J. 5Soukup T. 55Soumar J. 59Stebnicky M. 10, 11Stembirek J. 5, 57Stepankova T. 27Stingl J. 67 Straka Z. 67Svana M. 71Svandova E. 53Tokarcik J. 12, 68, 74Tomanova J. 76Tomasova L. 4, 22, 69

Tonar Z. 16, 33, 70Toth S. 18, 25, 71Troup O. 16Tumova E. 44Uberall I. 72Uhlik J. 65Ukraintsev E. 54Vajner L. 65Vaneckova M. 52Vanek J. 5Varga I. 49Varga J. 25Vavrova J. 8Vecanova J. 45, 61Vesela I. 13, 30Vicente-Steijn R. 51Vignerova J. 59Vistejnova L. 54Vybohova D. 73Vyborna E. 12, 68, 74, 78Weatherbee D. S. 7Witter K. 70Wurst Z. 75Zach P. 75Zahradnicek O. 77Zednikova-Mala P. 31Zemanek P. 76Zuska K. 24

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