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Home , Human skin, Lymphangiogenesis, Palatine tonsil, Secondary palate, Terminologia Histologica, Vascular smooth muscle

iŒ’ˆ‡Œ†„ê w„“ˆ•–

OF THE FACULTY OF AND DENTISTRY OF PALACKÝ UNIVERSITY, OLOMOUC CZECH REPUBLIC

VOLUME 159, SUPPLEMENT 1

PALACKÝ UNIVERSITY PRESS, OLOMOUC 2015 BIOMEDICAL PAPERS

Volume 159, Supplement 1

Published quarterly MK ČR E 12793

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

Olomouc 2015

ISSN 1213-8118 eISSN 1804-7521 http://dx.doi.org/10.5507/bp.2015.042 Czech Anatomical Society Czech Society for Histo- and Cytochemistry

Palacký University in Olomouc Faculty of Medicine and Dentistry in Olomouc

MORPHOLOGY 2015 49th 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, 2015 Olomouc, 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. GENERAL INFORMATION

Venue Building of the Theoretical Institutes of the Faculty of Medicine and Dentistry Hněvotínská 3, Olomouc

Registration and Information Desk Building of the Theoretical Institutes of the Faculty of Medicine and Dentistry

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

Languages of the conference English, Czech, Slovak

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

Presentations Plenary lectures - 20 min, lectures – 10 min, discussion – 5 min

Posters The size of a poster panel is 100 cm by 100 cm

SOCIAL PROGRAM

Welcome Party, free of charge Sunday, 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 Republic Secretary offi ce: Charles University, First Faculty of Medicine U nemocnice 3, 128 00 Prague 2, Czech Republic phone: +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 Republic Secretary offi ce: Simkova 870, 500 38 Hradec Kralové phone +420495816294 Fax +420495816376 http://www.cshc.cz

Authors are responsible for English.

vi Czech Anatomical Society Czech Society for Histo- and Cytochemistry Palacký University in Olomouc Faculty of Medicine and Dentistry in Olomouc

Programme

MORPHOLOGY 2015 49th International Congress of the Czech Anatomical Society 52nd Lojda Symposium on Histochemistry

September 6–8, 2015 Olomouc, Czech Republic SUNDAY, SEPTEMBER 6, 2015 10.00–10.15 Kvasilová A, Dvořánková B, Drobná Krejčí E, 15.00–17.00 Registration Smetana K, Grim M 17.00–19.00 Opening Ceremony Expression of some markers of stem cells and - Words of Welcome specifi c progenitors in human postnatal Cultural program 10.15–10.30 Prof. Obručník memory dedicated lecture Čížková D, Vávrová J, Mičuda S, Doleželová E, Sedmera D Brůčková L, Filip S, Mokrý J Elucidating cardiac conduction system formation with Role of transplanted marrow cells in the skeletal phylogenic approaches muscle regeneration

Winning lecture of the 2014 Czech Anatomical Society & 10.30–10.45 Olympus Award Měšťanová V, Varga I, Bencat M, Adamkov M Oralová V, Matalová E, Janečková E, Drobná Krejčí E, Myoid cells of in question of origin and Knopfová L, Šnajdr P, Tucker AS, Veselá I, Šmarda J, function Buchtová M Role of c-Myb in chondrogenesis 10.45 –11.15 Coff ee Break 11.15 –12.00 Plenary session of ČAS Winning lecture of the 2014 Czech Society for 12.00 –12.10 Congress Photo Histochemistry and Cytochemistry & Nikon Award 12.10 –13.00 Lunch Kodet O, Lacina L, Krejčí E, Vlček Č, Šáchová J, Dvořánková B, Grim M, Štork J, Kodetová D, Kolář M, 13.00–14.15 Strnad H, Smetana K Sessions in Section A cells infl uence the diff erentiation pattern of Neurosciences human epidermal Chairs: Mechírová E, Mazurová Y

19.00–20.30 Welcome party 13.00–13.15 Mazurová Y, Bezrouk A 20.00 Meetings of the national committees of ČAS and Transgenic rat model of Huntington’s disease ČSHC – a useful tool for study of the development of progressive neuronal degeneration

MONDAY, SEPTEMBER 7, 2015 13.15–13.30 Dubový P, Klusáková I, Korimová A, Joukal M, 9.00–9.40 Hradilová-Svíženská I Plenary lectures Upregulation of Toll-like receptors after Chairs: Naňka O, Mlejnek P injury and infl ammatory signaling from Wallerian degeneration 9.00–9.20 Holibka V 13.30–13.45 Anatomy teaching at the medico-surgical school in Joukal M, Kuklová A, Solár P, Klusáková I, Dubový P Olomouc, 1782–1875 Kolmer cells and their immunophenotyping in the rat plexus after peripheral nerve injury 9.20–9.40 Druga R 13.45–14.00 The claustrum – history research Salaj M, Barinka F, Kubová H, Druga R Diff erences in expression of binding in the perirhinal and retrosplenial of the rat 9.45–10.45 Stem Cells 14.00–14.15 Chairs: Smetana K, Ehrmann J Bálentová S, Hajtmanová E, Filová B, Borbelyová V, Lehotský J, Adamkov M 9.45–10.00 Cognitive and histopathological changes in the rat brain Smetana K Jr., Lacina L, Dvořánková B following fractionated irradiation Wound repair, ageing and : challenge for new therapeutic modalities 14.15–14.45 Coff ee Break

viii 14.45–16.00 TUESDAY, SEPTEMBER 8, 2015 Poster Sessions Chairs: Smetana K, Dubový P 9.00–10.15 Morphogenesis Chairs: Stingl J, Rybárová S 16.10–16.40 Plenary session of CSHC 9.00–9.15 Naňka O, Vicente-Steijn R, Blom NA, Jongbloed MRM, 13.00–16.00 Sedmera D Sessions in Section B Remodelling of atrioventricular canal myocardium Teaching Morphology determines atrioventricular conduction patterns Chairs: Ehrmann J, Kachlík D, Krajčí D 9.15–9.30 13.00–13.20 Čížková K, Rajdová A, Ehrmann J Ehrmann J, Lužná P, Čížková K CYP epoxygenases and their relevance for human Role of histology in the era of molecular prenatal development

13.20–13.35 9.30–9.45 Kachlík D, Štěpánková T, Musil V, Báča V Mlejnek P Anatomical eponyms ABC transporters and their role in the resistance

9.45–10.00 13.35–14.35 Pisal R Round Table Cloning of synthetic intron into coding sequence of red Kachlík D, Slížová D, Lovásová K fl uorescent for confi rmatory expression of micro Role of the anatomy practical teaching in current RNA cloned within the intron curriculum 10.00–10.15 Šimůnková P, Djakow J, Cinek O, Vajner L, Uhlík J 14.35–16.00 Ultrastructural and videomicroscopic fi ndings in E-Learning in Teaching Histology patients with genetically proven primary ciliary dyskinesia 14.35–14.50 Lichnovská R, Krajčí D (Mrs.), Erdosová B, Krajčí D 10.15–10.45 Coff ee Break Teaching practical histology with digital (virtual) slides 10.45–11.30 14.50–15.05 Cardiovascular System Krajčí D (Mrs.), Lichnovská R, Edosová B, Krajčí D Chairs: Mokrý J, Kučera T Application of electronic testing in histology and anatomy practicals 10.45–11.00 Stingl J, Pirk J, Straka Z, Setina M, Kachlík D, Stach J, 15.05–15.25 Musil V Krajčí D, Kopečný T, Novotný R Morphology of in the explanted bypassed A new system of electronic evaluation of results of PC- saphenous delivered tests 11.00–11.15 15.25–15.45 Kučera T, Smorodinova N, Rozsívalová K, Přidal J, Krajčí D, Kylar P Ďuřišová M, Pirk J, Bláha M, Melenovský V, Kautzner J GAMETIX – a new software for managing MCQ Infl ammatory cells in human atrial myocardium from databases and creation of examination sheets patients undergoing open surgery

11.15–11.30 19.00 Dinner Tonar Z, Kochová P, Cimrman R, Perktold J, Witter K Segmental diff erences in the orientation of cells in the of porcine aortae

ix 11.30–12.15 12.10–13.30 Anatomy Various Topics Chairs: Slížová D, Druga R Chairs: Laichman S, Kamarád V

11.30–11.45 12.15–12.30 Kachlík D., Hajek P, Konařík M, Báča V Bonczek O, Šerý O, Hloušková A, Lochman J, Arteria brachioradialis – overview Izakovičová-Hollá L, Šoukalová J, Štembírek J, Míšek I, Černochová P, Krejčí P, Vaněk J 11.45–12.00 Sequencing analysis of PAX9 and MSX1 in the Kachlík D, Blanková A, Báča V Czech population Supernumerary carpal 12.30–12.45 12.00–12.15 Blažková Z, Oborná I, Filipčíková R, Bezdíčková M, Výborná E, Dorko F, Holibka V, Tokarčík J, Chylíková J Hubáček P, Mlčoch M, Dobiáš M, Pastucha D Historical overview of morphological studies of palatine Pregnant as participants of traffi c accident in anatomical-clinical relation- research intent and preliminary study

11.00–11.30 12.45–13.00 Histology practical room in the new Theoretical Horváthová F, Danielisová v, Kuchárová B, Solár P, Departments building, fl oor 2, room n. 2.540 Rybárová S, Hodorová I, Mihalik J The eff ect of deprenyl administration on Erdosová B, Krajčí (Mrs.), Lichnovská R, Krajčí D status of rat testis A live demonstration of histology practical with digital slides 13.00–13.15 Ostrovska L, Vostejnova L, Dzugan J, Slama P, Kubina T, Ukraintsev E, Kraličkova M, Hubalek Kabalcova M The biological evaluation of ultra-fi ne titanium with improved mechanical strength for tissue engineering

13.15–13.30 Überall I Iconology of disease in Ranaisence art

13.30 Closing Session Authors of the best poster award

x POSTERS

1. Beznoska P, Hrebíková H, Chvátalová J, Pisal R, Čížková D, Diaz D, Mokrý J Analysis of decellularized 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 after prenatal infl uence of retinoic acid 4. Celá P, Shylo N, Weatherbee SD, Buchtová M Transmembrane 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 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 mediators 10. Hodorová I, Fiala P, Mihalik J, Horváthová F, Rybárová S Immunohistochemistry of colorectal carcinoma: expression of p53 and MDM2 11. Holanová V, Zahradníček O Continuosly regeneration dentition in anoline lizards: the model of postnatal teeth development leading to changes in tooth shapes and numbers 12. Hrebíková H, Chvátalová J, Díaz D, Beznoska P, Mokrý J Naturally derived scaff old for skeletal muscle regeneration 13. Hvizdošová N, Tomášová L, Bona M, Matéff y S, Kluchová D Retinoic acid in the development of the NADPH-d positive neurons in rat brain 14. Chvátalová J, Mokrý J, Hrebíková H, Pisal R Colonization of muscle scaff olds by diff erent types 15. Chyliková J, Kamarád V, Výborná E, Dvořáčková J, Holéczy P M1/M2 polarization in white aditose tissue of morbid obese patients 16. Jirkovská M, Zuska K The infl uence of maternal diabetes on proliferative potential of human term 17. Jonecová Z, Tóth S, Maretta M, Gregová K, Varga J Modifi cation of apoptotic cell rate in the intestinal graft mucosa during cold preservation 18. Kikalová K, Kopecký M, Charamza J, Tomanová J, Zemanek P of girls aged 10 to 18 with respect to intra –abdominal fat tissue 19. Killinger M, Veselá I, Buchtová M Eff ect of WNT5a on chondrogenesis and limb development 20. Klepáček I, Shbat A, Zedníková-Malá P Abbot Seka’s cranial morfology and 2D reconstruction of his with individual landmarks 21. 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 mice 22. Kolinko Y, Cendelín J, Tonat Z, Králíčková M Stereological assessment of cerebellum 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 format 24. 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 dextran 25. Langová P, Balková S, Buchtová M Microarray analysis of mandible regionalization during mouse development

xi 26. Lichnovská R, Krajčí D (Mrs.), Erdösová B, Krajčí D Impact of E-learning format using virtual microscopy in practical histology sessions 27. Makovický P, Tůmová E, Makovický P, Rimárová K The eff ect of food restriction on the morphology: experimental study on the chicken model 28. 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 literatures 29. Matuševská M, Pribulová J, Rozprávková M, Redaj L, Ohlasová J, Matéff y S, Lovásová K, Kluchová D Anatomy and pathological anatomy of depending to the state of oral health 30. Mechírová E, Gdovinová Z, Skorvanek M, Domoráková I, Danková M Immunohistochemical visualization of α-synuclein in colonic lamina propria mucosae 31. Miklošová M, Dankovčík R, Herich R Application of double staining method for fetal specimens 32. Oralová V, Matalová E, Knopfová L, Švandová E, Šmarda J, Buchtová M A role of c-Myb in ossifi cation 33. 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 premolar 34. Putnová B, Buchtová M, Jekl V, Hodan R, Machoň V, Štembírek J Healing of soft and hard tissues of rabbit temporomandibular following the application of stem cells 35. Rejtarová O, Kuchař M, Eliáš P Anatomical variations of popliteal – a possible case of lower limb ischaemia 36. Riedlová J, Paulová M, Vignerová J, Soumar J Children of obese mothers in their fi rst 18 months 37. 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 process 38. Rybárová S, Hodorová I, Mihalik J, Vecanová J Expression of carbonic anhydrase IX and its correlation with clinicopathological characteristics in cancer cells 39. Salaj M, Barinka F, Kubová H, Druga R Diff erences in expression of calcium binding proteins in the perihinal and retrosplenial cortex of the rat 40. 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 Models 42. Tomašová L, Hvizdošová N, Šmajda B, Bona M, Matéff y S, Kluchová D Various noxious factors and their eff ect on the developing 43. 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 44. Výbohová D, Mellová Y, Adamicová K Blood and lymphatic microvascular changes in relation to VEGF-A and VEGF-C expression in psoriatic lesions 45. 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

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

Dear colleagues, morphologists in the Czech Republic. We are happy to It is our great pleasure to invite you to read in the ab- notice many fruitful collaborations with colleagues from stracts of MORPHOLOGY 2015, the 49th International abroad. We find also hopeful that the morphological dis- Congress of the Czech Anatomical Society and the 52nd ciplines continue to attract attention of young colleagues. Lojda Symposium on Histochemistry. Within a wide- We cordially welcome you in Olomouc, hoping that ranging programme, we are highlighting the themes of this congress will be both scientifically and socially suc- Stem Cells, Morphogenesis, Neurosciences, and Clinical cessful meeting. Anatomy. We organize as well the roundtable about Kind regards, Anatomy and Histology Teaching. These interdisciplin- ary topics spanning both basic and clinically oriented re- On behalf of the Organizing Societies search showcase the broad range of topics studied by the Ondřej Naňka and Jaroslav Mokrý

xiii

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

1 ACKNOWLEDGEMENTS Cognitive and histopathological changes in the rat The study was supported by a projects "Center of brain following fractionated irradiation translational medicine"/"Creating a new diagnostic algo- rithm for selected cancer diseases", ITMS: 26220220021 Sona Balentovaa, Eva Hajtmanovab, Barbora Filovac, and ITMS: 26220220022 co-financed from EU sources Veronika Borbelyovad, Jan Lehotskye, Marian Adamkova and European Regional Development Fund. aInstitute of Histology and , Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, 2 Slovak Republic bDepartment of Radiotherapy and Oncology, Martin University Analysis of decellularized skeletal muscle Hospital, Martin, Slovak Republic scaffolds and their settlement by myogenic cells cInstitute of Medical Physics, Biophysics, Informatics and Telemedicine, Faculty of Medicine, Comenius University in Pavel Beznoska, Hana Hrebikova, Jana Chvatalova, Bratislava, Slovak Republic Rishikaysh Pisal, Dana Cizkova, Daniel Diaz, Jaroslav Mokry dInstitute of Molecular Biomedicine, Faculty of Medicine, Comenius University in Bratislava, Slovak Republic Department of Histology and Embryology, Faculty of Medicine eInstitute of Medical Biochemistry, Jessenius Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, in Martin, Comenius University in Bratislava, Martin, Slovak Czech Republic Republic Aims. In the present study we investigated whether Aims. Tissue engineering represents a progressing expected radiation-induced histopathological changes in branch of research with the aim to develop biologi- the rat brain will be associated with appropriate neuro- cally equivalent replacements of tissues and organs. cognitive dysfunction. Construction of the carriers is based on the knowledge of Methods. Adult male Wistar rats randomized into nanotechnology, or tissue decellularization. Suitable bio- sham (0 Gy) and 2 experimental groups (survived 30 compatible and biodegradable carriers are then colonized and 100 days after treatment) received fractionated with the desired cell populations. The decellularized non- whole-brain irradiation (one 5 Gy fraction /week for 4 toxic scaffold with 3D structure cultured in vitro provides weeks) with a total dose of 20 Gy of gamma rays. Morris a temporary mechanical support for the implanted cells water maze cognitive testing, histochemistry, immuno- and moreover, it forms suitable environment for adhesion, histochemistry and confocal microscopy were used to growth and proliferation of the cells, and stimulates them determine whether the cognitive changes are associated to production of . with the alteration of neurogenesis, astrocytic response Methods. In this work we decellularized the intact and and activation of in two neurogenic regions: the cardiotoxin-injured mouse skeletal muscles and analyzed hippocampal dentate gyrus (DG) and the subventricular the formed scaffolds for presence of components of the zone-olfactory bulb axis (SVZ-OB axis) represented by extracellular matrix. Then, the scaffolds were cultivated the anterior SVZ, vertical arm, elbow and horizontal arm. with C2C12 mouse myoblasts to compare penetration of Results. Irradiation negatively influenced the spatial the cells under physiological and pathophysiological con- memory in Morris water maze 100 days after radiation ditions. The muscle injury was provoked in C57BL6 /J treatment, however the reference memory remained in- mice by injecting 75 μL of cardiotoxin (0.06 μg/μL) along tact. Neurodegenerative changes were characterized by a the longitudinal axis of the tibialis anterior muscle. The significant increase of Fluoro-Jade-positive cells 30 days intact and cardiotoxin-injured skeletal muscles (4 days after irradiation in both of investigated brain regions. The after ) were decellularized using immersion in SVZ-OB axis displayed a steep decrease of neuroblasts the detergent 1% SDS (sodium dodecyl sulfate) and less obvious decline of stem/precursor cells until 100 and simultaneous mechanical agitation for 48 h. Then, days after treatment. Strong decline or re-expression of the tissue was rinsed in sterile PBS buffer with antibi- was seen in the SVZ-OB axis until 100 days otics (penicillin, streptomycin, amphotericin B) for 24 after irradiation, however the hippocampal DG did not h and DNase was subsequently used to remove residual showed any significant changes. A decrease and subse- cellular DNA. After washing in PBS, the scaffolds were quent re-expression of the activated microglia was seen sterilized by UV radiation for 30 min. Then, the scaffolds during the experiment. were cultivated in vitro and seeded with C2C12 mouse Conclusion. Results showed, that fractionated irradia- myoblasts. After 8 and 14 days of cultivation the scaffold tion led to cognitive impairment closely associated with with cells were fixed in 10% formalin and embedded into the initiation of neuronal cell death, inhibition of neuro- paraffin. The sections were stained by hematoxylin-eosin, genesis, activation of astrocytes and microglia indicated green trichrome, Weigert resorcin-fuchsin and Sirius Red. an early delayed radiation-induced changes. 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,

S1 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). i.e. and elastic fibers and served as a scaffold for is not available. Considering that we discuss about mar- myoblasts, which in various degrees adhered to its surface ginal issue at present, we want to get a greater amount of and penetrated into it. detailed information from more workplaces on the extent Conclusion. According to the preliminary results of and nature of the trauma, but other injuries to pregnant the digital image analysis, the myoblasts penetrated in women and her fetus too. Using case reports analysis we greater percentage into the scaffold prepared from the want to focus on strength vectors interaction during traf- toxin-injured skeletal muscle, which highlights a role of fic accident and anatomical-clinical variations in pregnant the bioscaffold microenvironment for a successful recel- woman and her fetus. lularization and opens new approaches for modification Results. The outputs will be used not only for teaching of bioconstructs in tissue engineering. anatomy of forensic medicine, emergency medicine and obstetrics, but they are also directly applicable to clini- cal practice. Depending on the stage of and ACKNOWLEDGEMENTS the severity of the trauma of pregnant women there is an increased risk of injury or death of the fetus. Moreover, This study was supported by Grant Agency of even relatively small wounds of mothers might have fatal Charles University in Prague No. SVV-2015-260179, consequences for the fetus. PRVOUK P37/06 and with European Social Fund No. Conclusion. Traffic accidents consequences can occur CZ.1.07/2.3.00/30.0061. 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 3 are not many recorded cases, this issue is very topical, Pregnant women as participants of traffic because the threats are always on two lives. It is therefore crucial underpinning of the incidence in order to obtain accident in anatomical-clinical relation – research valid data from medical facilities throughout the Czech intent and preliminary study Republic and determine the true prevalence of traffic ac- cidents of pregnant women and their health effects. Zdeňka Blazkovaa, Ivana Obornab, Radka Filipcikovaa, Marcela Bezdickovaa, Petr Hubacekc, Michal Mlcochd, Martin Dobiase, Dalibor Pastuchaf 4 Postnatal development of nitrergic hippocampal aDepartment of Anatomy, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic neurons after prenatal influence of retinoic acid bDepartment of Obstetrics and Gynecology, University Hospital, Olomouc, Czech Republic Adriana Bolekova, Lenka Tomasova, Natalia Hvizdosova, cDepartment of Emergency, University Hospital, Olomouc, Czech Martin Bona, Kvetuse Lovasova, Stanislav Mateffy, Republic dDepartment of Obstetrics and Gynecology, Regional Hospital of Darina Kluchova Tomas Bata, Zlin, Czech Republic Department of Anatomy, Faculty of Medicine, P. J. Safarik e Department of Forensic Medicine, Faculty of Medicine and University in Kosice, Slovak Republic Dentistry, Palacky University, Olomouc, Czech Republic f Department of Rehabilitation, Faculty of Medicine, University of Aims. Retinoic acid (RA) is a biologically active de- Ostrava, Czech Republic rivate of that plays important role during the Aims. With increasing traffic density pregnant woman development. The aim of the study was to investigate the participate daily as the driver, co-driver, cyclist, a pas- influence of prenatal application of RA on postnatal de- senger in public transport or walker. These facts cause velopment of hippocampal nitrergic neurons in the rat. increase of probability of a collision. The consequences Toxicity of RA could be a growing concern both in devel- of traffic collisions can be crucial not only for the woman oping (where large therapeutic doses are taken to control and her fetus, but can also have significant health and eco- vitamin A deficiency) and developed countries (where nomic impact on the whole society. The aim of our work intake of vitamin A often exceeds recommended dietary is based on anatomical and clinical research which will allowances). lead to greater social interest in this issue. Next purpose Methods. 24 female Wistar rats were used in the ex- is to achieve enclosure of the results of our work into the periment. 18 pregnant animals were administrated by RA preclinical and clinical teaching of medical students and intraperitoneally in dose 1 mg/kg during three consecutive disciplines allied to medicine. gestational days: 14th, 15th and 16th (RA group); 6 preg- Methods. We only know statistics data (from UZIS nant rats were without experimental influence (control Company) about the total abortion respectively the num- – K group). The offspring of both groups were divided ber of abortions, and we have data on total maternal into 5 groups and underwent experimental procedures in mortality too. However, unlike in other countries, more different postnatal days: in the 1st, 7th, 14th and 21st and detailed information on the relationship between abor- in the adulthood (in the age of 3 month). Animals in each tion and maternal mortality incidental to road accidents group were anaesthetized and perfused transcardially,

S2 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). brains were removed and processed for NADPH-d histo- 5 chemical method. Nitrergic neurons of the hippocampus were investigated on the coronal sections of each brain Sequencing analysis of PAX9 and MSX1 genes sample. in the Czech population Results. During physiological conditions without re- ceiving RA by pregnant rats (K groups) the NADPH-d Ondrej Bonczeka,b, Omar Serya,b, Alena Hlouskovaa, positive were appeared in the all hippocam- Jan Lochmana, Lydie Izakovicova-Hollac, Jana Soukalovac, pal layers since the 7th postnatal day. The number and Jan Stembirekb,d, Ivan Misekb, Pavlina Cernochovac, density of nitrergic neurons increased until the 14th post- Premysl Krejcie, Jiri Vanekc natal day, than decreased and stabilized on the 21th day to the pattern of the adult hippocampus. These neurons were aLaboratory of DNA Diagnostic, Department of Biochemistry, morphologically developed on 14th postnatal day already Faculty of Science, Masaryk University, Brno, Czech Republic and were seen to be similar to those in the adulthood. bLaboratory of Animal Embryology, Institute of Animal Numerous NADPH-d positive interneurons were found in and Genetics, v.v.i., The Academy of Sciences of the Czech the hilus of dentate gyrus. Single layers of hippocampus Republic, Brno, Czech Republic c proprius and dentate gyrus significantly differed in their Clinic of Stomatology, Faculty of Medicine, Masaryk University, NADPH-d staining. In the groups after RA administra- Brno and St. Anne's University Hospital in Brno, Czech Republic dDepartment of Oral and Maxillofacial Surgery, University tion (RA groups), the number of nitrergic neurons in hip- Hospital Ostrava, Czech Republic pocampal formation markedly decreased in all postnatal eDepartment of Dentistry, Faculty of Medicine and Dentistry, periods compared to control animals. Neurons seem to be Palacky University Olomouc, Czech Republic damaged and delayed in their development. The number and intensity of positive interneurons was decreased in Aims. Tooth agenesis is one of the most common de- deep layers of hippocampus and hilus of dentate gyrus. velopmental anomalies in humans. Tooth development The number and density of neurons increased very gently (odontogenesis) is a very complicated and complex pro- until 21th day, they supposed to be finally developed this cess that involves interplay between oral and time. In the adult animals prenatally influenced by RA, . These interactions are mediated by more the pattern of NADPH-d staining of hippocampal regions than 350 signaling proteins. Synergistic and antagonistic was identical to the control group. interactions between these proteins may lead to local ac- Conclusion. Changes of nitrergic interneurons con- tivation or inhibition of the expression. The aim of our firmed that prenatal administration of RA can modify study was to investigate the relationship between PAX9 NADPH-diaphorase activity in the rat hippocampus. The and MSX1 genes variants and tooth agenesis in the Czech morphology and intensity of NADPH-d positive hippo- population. campal neurons has changed during the postnatal devel- Methods. The selected regions of PAX9 and MSX1 opment. It can be resumed that prenatal application of genes were analyzed by direct sequencing and compared RA could play the specific role in different regions of with the reference sequence from the GenBank on-line hippocampal formation during postnatal development of database (NCBI). nitrergic neurons in the rat. 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

S3 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). agenesis and controls. We hypothesize that tooth agenesis prominent changes in liver parenchyme and connective in our cohort of patients was caused by mutations in re- tissue. This finding was in contrast to the Cr animal group gions different from the PAX9 and MSX1 exons analyzed and especially to Cr + pleuran group of animals. In the in this study or in different genes. mentioned group, some striking histological changes were observed: swollen near to central , dilated sinusoids and vacuolated hepatocytes in portal area in ACKNOWLEDGMENT addition to changes. Conclusion. The only antioxidant effect of pleuran This project was supported by the Internal Grant administration was an increase in the content of GSH Agency of the Ministry of Health of the Czech Republic in the liver, which was decreased by Cr administration. IGA MZ ČR No. NT/11420-6. From histology point of view was the most affected Cr + pleuran group of animals with the most conspicuous tis- 6 sue changes. Pleuran (beta-glucan from Pleurotus ostreatus) potentiates free radical induced hepatotoxic ACKNOWLEDGEMENT effects of hexavalent chromium in rats We wish to thank Ms. M. Šlajerová for the excellent

a b a technical assistance. The work was supported by the SVV Monika Bludovska , Vera Krizkova , Dana Kotyzova , project No. 260 047 and by the project ED2.1.00/03.0076 Tereza Kubikovab,c from European Regional Development Fund. aDepartment of and Toxicology, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech 7 Republic bDepartment of Histology and Embryology, Faculty of Medicine in Transmembrane protein 107 is a critical factor Pilsen, Charles University in Prague, Pilsen, Czech Republic cBiomedical Centre, Faculty of Medicine in Pilsen, Charles for craniofacial development University in Prague, Pilsen, Czech Republic Petra Celaa,b, Natalia Shyloc, Scott D. Weatherbeec, Marcela Buchtovaa,b Aims. β-Glucans are widely used immunomodulatory nutritional supplements. Many studies have also proved aInstitute of Animal Physiology and Genetics, The Academy of their antioxidant and hepatoprotective properties. The Sciences of the Czech Republic, Brno, Czech Republic study was designed to evaluate the effects of pleuran, bDepartment of Animal Physiology and Immunology, Institute of β-(1.3/1.6) glucan from Pleurotus ostreatus, on the toxic Experimental Biology, Masaryk University, Brno, Czech Republic c and oxidative effects and histological changes caused by Department of Genetics, Yale University, New Haven, CT, USA an acute Cr(VI) intoxication in rats. Methods. Wistar albino male rats were divided into Aims. Primary is a -based exten- three groups: control, Cr, and Cr + pleuran. Pleuran sion of the cell surrounded by a membrane, which plays (250 mg/kg/day) was administered by oral use for 10 (half a striking role in many developmental patterning events, of the animals for 11) consecutive days. K2Cr2O7 (40 mg/ including establishment of left-right asymmetry or skeletal kg/day) was administered i.p. as a single dose on the 10th formation. Moreover, primary cilia have been implicated day of the experiment. Half of animals in each group were in the regulation of several key developmental signaling sacrificed 24 h and the rest 48 h after the administra- pathways. Functional cilia are required for activation of tion of Cr. Lipid peroxidation (LP), reduced glutathione SHH pathway, which is essential for morphogenesis of (GSH) and activities of glutathione peroxidase (GPx), variety tissues and organs. Recently, Transmembrane glutathione reductase (GR) and catalase (CAT) and the protein 107 (TMEM107) was found to be essential for content of Cr were estimated in the liver, the activities of during embryonic development (Christopher ALT, AST and GLDH were determined in serum. Liver et al., 2012). Tmem107 homozygous mutant embryos were tissue samples were collected for histological and imuno- described as embryonic lethal and displaying SHH-related histochemical examination. defects such as polydactyly, which is also a hallmark of Results. Administration of Cr(VI) significantly de- defective cilia. creased liver content of GSH and significantly increased Methods. First, we analyzed the external craniofacial LP. The activities of ALT, AST and GLDH were signifi- phenotype of Tmem107 mutant mice during period of cantly increased in serum. Treatment with pleuran signifi- formation (E12.5 – E15.5). Wild type, heterozy- cantly increased the content of Cr in the liver, increased gous and homozygous mice were compared. Transversal the effect of chromium on LP (24 and 48 h post Cr dose), histological sections stained with H&E were used for mi- on the activities of ALT, AST and GLDH (24 hours post croscopical analysis of palatal fusion. Cr dose) and on chromium-induced histopathologi- Results. Tmem107 homozygous null embryos exhib- cal changes. The control group of animals was without ited abnormalities in head shape with microphthalmia or

S4 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). anophthalmia and exencephaly. Furthermore, shorten- isolated BMCs into whole-body lethally irradiated immu- ing of the snout and cleft was observed. Histological nocompetent mice 7 hours after or 4 weeks before the analysis revealed defects in patterning and size of nasal cardiotoxin-induced injury of the recipients’ tibialis ante- cavity, narrow or shorter nasal septum and smaller cupola- rior muscles. Seven to 33 days after the toxin injection, shaped . The nasal were deformed and the injured and intact contralateral skeletal muscles were nasal septum was curved when unilateral cleft lip was excised and fixed in 4% paraformaldehyde, processed for visible. At E12.5, Tmem-/- mice had deficiency of mid- X-gal histochemistry to detect lacZ+ cells, embedded into line structures, both nasal cavities were connected and GMA resin (to obtain 1 μm thin sections) or paraffin or exhibited simple shallow morphology. At E15.5, cleft of fixed in 2% paraformaldehyde, frozen in methylbutane, secondary palate occurred or palatal shelves were fused chilled in liquid nitrogen, cryosectioned and examined for in midline; however, they were not attached to the short GFP . The presence of lacZ gene in injured nasal septum. muscles was determined by qPCR. For transmission elec- Conclusion. In summary, we found large spectrum of tron microscopy, the excised injured and intact contralat- craniofacial defects in Tmem107 null homozygous mice, eral skeletal muscles were fixed in 3% paraformaldehyde while heterozygous animals displayed normal morphol- and 0,05% glutaraldehyde in 0.1 M Sörensen buffer and ogy. Thus, Tmem107 seems to be essential for proper for- embedded into LR White resin. Immunohistochemical mation of lip and palate during embryonic development detections were performed by indirect three-step (LSAB) and further determination of its role in palatogenesis will methods using primary : monoclonal rabbit advance our understanding of causations of ciliopathy anti-, clone Y66 (GeneTex) and polyclonal rabbit disorders. anti-nestin (Sigma). Results. The skeletal muscles of recipients injured 7 h before the transplantation did not regenerate, never- ACKNOWLEDGEMENTS theless, X-gal positivity was predominantly identified in desmin- and nestin- multinucleated cells resembling for- This study was supported by the Grant Agency of the eign body giant cells located in the injured areas, 14 and Czech Republic (14-37368G) and institutional support 33 days after grafting. On the contrary, the recipients’ (RVO:67985904). 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 8 numerous inflammatory cells, in some newly formed myo- Role of transplanted cells in the blasts and myotubes and in some cells of the and in the regenerated muscles examined 28 days after skeletal muscle regeneration the toxin injection in some endomysial cells and rarely Dana Cízkovaa, Jiřina Vavrovab, Stanislav Micudac, in newly formed myofibers. qPCR verified presence of + Eva Dolezelovac, Lenka Bruckovaa, Stanislav Filipd, transplanted lacZ BMCs in injured recipients’ muscles. a Conclusion. Our results confirmed ability of intra- Jaroslav Mokry venously transplanted exogenous BMCs to settle in the aDepartment of Histology and Embryology, Faculty of Medicine injured skeletal muscle and participate in the skeletal in Hradec Kralove, Charles University in Prague, Hradec Kralove, muscle regeneration. They generated blood cells that infil- Czech Republic trated endomysium and took part in the cleaning reaction, bDepartment of Radiobiology, Faculty of Military Health Sciences the first step of the regeneration process, and moreover, in Hradec Kralove, University of Defence, Hradec Kralove, Czech they contributed to new myofibers formation. Republic cDepartment of Pharmacology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech ACKNOWLEDGEMENTS Republic dDepartment of Oncology and Radiotherapy, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, This work was supported by the grant projects: No. Czech Republic 15-09161S from the Grant Agency of the Czech Republic and PRVOUK P37/06. 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 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

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9 of these organs. If EETs really play role in development, alterations in the strict spatio-temporal pattern of expres- CYP epoxygenases and their relevance for human sion of CYP epoxygenases and / or sEH during human prenatal development prenatal development by xenobiotics could have adverse consequences for developing organism. Katerina Cizkovaa, Aneta Rajdovaa, Jiri Ehrmanna,b aDepartment of Histology and Embryology, Faculty of Medicine ACKNOWLEDGEMENTS and Dentistry, Palacky University Olomouc, Czech Republic bDepartment of Clinical and Molecular Pathology and Laboratory of Molecular Pathology, Faculty of Medicine and Dentistry, We thank to Ivana Travnickova and Jitka Stastna for Palacky University Olomouc, Czech Republic excellent technical support. This study was supported by LF_2015_008. Aims. Cytochrome P450 (CYPs) are involved in of various endogenous and exogenous com- pounds, including clinically used . There is growing 10 evidence that some of the CYP ale play role in Pharmacological preconditioning: chance development of organism. In this regard, CYP epoxygen- ases (CYP2C and CYP2J subfamilies) are very interest- for formation of ischemic tolerance in rabbit’s ing. CYP epoxygenases convert arachidonic acid (AA) spinal cord to four regioisomeric epoxyeicosatrienoic acids (EETs). a a a EETs have relativelly short half-life in vivo. They are me- Marianna Dankova , Eva Mechirova , Iveta Domorakova , tabolized to less active dihydroxyeicosatrienoic acids Miroslava Janitorova Poliacikovaa, Milan Stebnickyb (DHETs) by soluble epoxide hydrolase (sEH). Because aDepartment of Histology and Embryology, Faculty of Medicine, P. of EETs regulate a variety of processes potentially relevant J. Safarik University in Kosice, Slovak Republic to development, such as cell proliferation and differentia- b2nd Depatrment of Surgery, Faculty of Medicine, P. J. Safarik tion, and cell signalling, we decided to estimate expression University in Kosice, Slovak Republic pattern of EETs-producing and EETs-degrading proteins in human embryonic/foetal intestines, liver and Aims. The present study was designed to evaluate the samples from 7th week to 20th week of intrauterine devel- noradrenaline and bradykinin preconditioning on spinal opment (IUD) to consider possible importance of EETs cord ischemic injury using a transient lumbar spinal cord for human prenatal development. ischemia model in rabbits. Noradrenaline is known to be Methods. We compared expression patterns of CYP responsible for the body's reaction to different types of epoxygenases (CYP2C8, CYP2C9, CYP2C19, CYP2J2) stress. Bradykinin is a biologically active nonapeptide that and sEH in human embryonic/foetal intestines, liver and mediates inflammatory response and increases vasodila- kidney samples from 7th week to 20th week of IUD. The tion. The main purpose of this study is to summarize the expression patterns in these organs were obtained by two- results dealing with acquisition of ischemic tolerance and step immunohistochemistry method. utilization of preconditioning for protection of the vulner- Results. We confirmed expression of CYP epoxygen- able neurons in the rabbit’s spinal cord against ischemia ases and sEH in tested human embryonic/foetal intes- and reperfusion. Pharmacological preconditioning is a tines, liver and kidney. In intestines, distribution of CYP form of and tissue protection induced by a sublethal epoxygenases and sEH expression could lead to diverse dose of various chemical agents against lethal ischemic concentration of EETs along crypt-villus axis. In crypt insult. Preconditioning involves the induction of cellular area, where new cells of intestinal epithelia are generated, protective mechanisms. is strong expression of CYP2C8 and CYP2C9 together Methods. The New Zealand white rabbits were divided with low expression of sEH. The main product of these into 4 groups: 1. control group (n – 7), 2. ischemic group epoxygenases is 14,15-EET which has the strongest pro- – 20 min of ischemia/48 h of reperfusion (n – 7), 3. ex- liferation potential from all EETs regioisomers. On the perimental group (n – 11) – preconditioned with of other hand, in apex of villi, where are fully differentiated bradykinin 48 h prior to 20 min of ischemia and 48 h of cells, expression of CYP2C8 and CYP2C9 is lower and reperfusion and 4. experimental group (n – 9) – precon- expression of sEH is higher than in crypt area. In kid- ditioned with bolus of noradrenalin 48 h prior to 20 min ney samples, CYP epoxygenases as well as sEH are ex- of ischemia and 48 h of reperfusion. pressed in renal corpuscles and tubular system. On the We demonstrated the distribution of proteins NeuN other hand, nephrogenic zone, where new are and ubiquitin after pharmacological preconditioning prior developed, is possitive for CYP epoxygenases but nega- to ischemic insult. Tarlov scoring system was used for as- tive for sEH. sessment of neurological function of hind limbs. Conclusion. Distribution of CYP epoxygenases and Results. In the groups 2, 3, and 4 of experimental sEH in intestinal and nephrogenic zone of the animals 20 min of ischemia caused different intensity of kidney suggests an influence of EETs on cell prolifera- ubiquitin positivity in the motor neurons of lamina IX and tion and differentiation and consequently development interneurons of intermediate zone of spinal cord. Dark

S6 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). brown aggregates of ubiquitin were visible in the cyto- 2 days; (3) – noradrenaline preconditioning/ 20 min of plasm of ischemic motor neurons of 2. ischemic group. ischemia/ 24 h of reperfusion; (4) – noradrenaline pre- Accumulation of stress proteins leads the neurons to the conditioning/ 20 min of ischemia/ 48 h of reperfusion. necrosis. Ubiquitin positive structures were also discerned Monoclonal mouse anti-GFAP (glial fibrillary acidic pro- in the surrounding . Ubiquitin aggregates are tein) and polyclonal anti-ubiquitin were used for demon- signs of damaged cells that lost the ability of intracellular stration of ubiquitin reaction (UR) in hydrolytic degradation of altered proteins. We assume that and GFAP and UR in the astrocytes. neurons, unable to synthesize new ubiquitin, appear to be Results. Our previous study showed that noradrenaline those that succumb to the degenerative process induced preconditioning followed by 20 min of ischemia followed by ischemia. by 24 h of reperfusion (3), induces ischemic tolerance in Significant decrease of NeuN positive neurons motor neurons. Moreover, number of surviving neurons (P<0.001) was observed after bradykinin and noradrena- significantly increases and hind limb function was im- lin preconditioning in both groups of animals after 20 proved in comparison to ischemic group (2). Acquisition min of ischemia followed by 48 h of reperfusion in com- of ischemic tolerance slows down deleterious effect of 20 parison with the control group. Despite of this significant min of ischemia. However, longer period of reperfusion decrease in number of NeuN positive neurons, it seems in the group (4) showed reduction of surviving neurons there were enough viable neurons that are able to guaran- vs. group (3). In ischemic group (2) reactive GFAP posi- tee active movement of the hind limbs. tive astrocytes were present in the , their nu- Conclusion. The results indicated that degree of spinal clei and processes showed strong UR. Astrocytes were cord damage after ischemia reperfusion is associated with grouped around the damaged areas of the . number of ubiquitin and NeuN positive neurons. The as- Efferent radiated from anterior horns were swollen sessment of neurological function of hind limbs showed and surrounded by oligodendrocytes with strong UR in that pharmacological preconditioning can partially im- the nuclei and . In preconditioned group (3) prove these results. 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 ACKNOWLEDGEMENTS matter UR was found also in the astrocytic processes and also in the nuclei of oligodendrocytes. In preconditioned Supported by VEGA grant 1/0815/14 and 9/ group (4) reactive GFAP positive and strong GSD/2012. 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. 11 Conclusion. Ischemic tolerance induced by precon- Glial reaction in the spinal cord ischemia after ditioning is the way how to protect vulnerable neurons exposed to ischemia/ reperfusion. Further research should noradrenaline preconditioning found optimal “time window” for application of ischemic Iveta Domorakovaa, Eva Mechirovaa, Marianna Dankovaa, postconditioning which is more relevant for clinical prac- Miroslava Janitorova Poliacikovaa, Milan Stebnickyb tice. aDepartment of Histology and Embryology, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic ACKNOWLEDGEMENTS b2nd Department of Surgery, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic Supported by VEGA grant 1/0815/14 and 9/ GSD/2012. Aims. Protein is essential for proper func- tion of neurons and oligodendrocytes. Missfolded proteins are degraded by ubiquitin 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 . Animals were divided into 4 groups (n=5). Group (1) – sham operated; (2) – 20 min of ischemia/ reperfusion

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12 vessels. Fibres from these perivascular tangle penetrated through the cortical border into cortex and paracortical Autonomic innervation of renal haemolymph area. In paracortical area we observed abundant perivas- node in rats cular tangle. In the cortex ACHE – positive nerve fibres was located parafolliculary. František Dorkoa, Eva Vybornaa, Vladimir Holibkaa, Conclusion. In renal haemolymph node adrenergic and Jan Tokarcika, Jaroslava Chylikovab, Lubica Hornakovac ACHE positive innervate the same sections as in the other nodes. Their total number is larger in this aDepartment of Anatomy, Faculty of Medicine, University structure, mainly in areas with maximum erytrophages b of Ostrava, Czech Republic Department of Histology and and siderophages concentration. Nerve fibres enter the Embryology, Faculty of Medicine, University of Ostrava, Czech nodes in the common bundle of the through the Republic cSmall Animal Clinic, University of Veterinary Medicine and hilus. Renal haemolymph node is an organ that eliminates Pharmacy in Kosice, Slovak Republic the red cells from the lymph as well as controls immune responses. Aims. Haemolymph nodes are part of the and its lymphatic sinuses contain erythrocytes at various stages of development, associated with phenome- 13 na haemosiderosis, which causes brownish-red macroscop- Morphological analysis of mouse skeleton ic appearance of these nodes. Renal haemolymph node (RHLU) in rats is located near the a. renalis. Significant following AZD4547 treatment dominance of medullary sinuses and strands that contact Hana Dosedelovaa,b, Iva Veselaa,b, Pavel Krejcic, directly with the fibrous capsule or the subcapsular sinus Michaela Kunovac, Marcela Buchtovaa,b is seen in RHLU. The intermediate and medullary sinuses contain many erythrocytes as a rosette clusters with mac- aDepartment of Anatomy, Embryology and Histology, University rophages with considerable accumulation of hemosiderin of Veterinary and Pharmaceutical Sciences Brno, Czech Republic in there. Haemolymph renal cortex has irregular bLaboratory of Animal Embryology, Institute of Animal Physiology areas of lymphoid tissue, which form irregular nodules of and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic without the typical bright germinal centres. c We studied RHLU autonomic innervation in rats. Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Methods. For studying RHLU innervation we used 25 rats of both sexes, Wistar strain, aged 6 months. Animals Aims. FGF-receptor tyrosine kinases (FGFR1-4) rep- were anesthetised with pentobarbital, 20 mg/kg, i.p. resent attractive therapy targets, as activating mutations, Removed RHLU were fixed in 4% formaldehyde for 2 to gene amplifications or chromosomal translocations cre- 4 h. To prove ACHE – positive nerve fibres we used di- ating fusion genes involving FGFR genes was associated rect tiocholin method modified by El-Badawi and Schenk. with several . Many FGFR inhibitors have been Adrenergic neural components were depicted using gly- developed in the past years, including recently discovered oxic acid fluorescence histochemical method modified by compound AZD4547. As FGFR inhibitors may be useful Švalev and Zučkova. for achondroplasia therapy, we aim to analyze the effect Results. Specifically fluorescing adrenergic nerve fi- of AZD4547 on growth at in vivo experimental bres enter the RHLU in common bundle with arteries, conditions. through the nodes hilus, but sporadically at other loca- Methods. Newborn mice were injected intraperito- tions of the nodes surface. We recorded rich periarterial neally by AZD4547 at concentration 0.5 μM, 1 μM, 5 nerve plexus running in medullary strands. Toward the μM, 10 μM, 50 μM, 250 μM, 1000 μM or 2000 μM in nodes surface, as the arteriolar branching is gradually amount of 50 μL. Control group of animals was injected reduced, the number of penetratingly fluorescent accom- by DMSO in appropriate concentration. Injection was panying nerve fibres. The largest relative density of adr- repeated every week for 4 weeks and animals were eu- energic nerve fibres is detected around the and thanized by cervical dislocation at day 28 after the first the parenchyma of the cortex and sections where injection. Skin was removed from animals and they were the amount of erythrocytes and lymphoid cells were seen. fixed in 100% ethanol, stained with Alizarin red/Alcian These cellular components come into intimate contact blue solution, and cleared in KOH/Glycerol. The body with adjacent nerve fibres. In areas that are free cells poor- weight was measured at the time of injection (every week). er and do not contain the rosette clusters of macrophages, The length of the body (from snout to tip of the tail) and adrenergic innervation is limited to a small amount of tail (from radix to tip of the tail) was measured at the end fluorescent periarterial profiles. ACHE – positive nerve of experiment. fibres in the hilar lymph nodes enter the medulla through Results. No changes in weigh and length of animals the medullary columns and in the capsule and subcapsu- were observed by 0.5 μM, 1 μM, 5 μM and 10 μM of lar plexuses. In the medulla ACHE – positive nerve fibres AZD4547. Small increase in weight and length was found creating perivascular tangles, but we've also noticed a free in animals injected by 50 μM concentration. On the ACHE – positive nerves, which was not accompanied other hand, animal weight decreased with higher doses

S8 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). of AZD4547. At 1000 μM and 2000 μM concentration, were found but most common are medium-sized neurons AZD4547 led to death in 32% and 43% of mice, respec- with triangular, multipolar, oval and fusiform perikarya. tively. The organs mostly affected by 1000 μM AZD4547 Golgi impregnation studies reveal two major classes of were liver (necrotic areas, lymphocytes accumulation), neurons: polymorphous projection neurons with spiny (alveolar hemorrhage), and kidneys (reduction of and local interneurons characterized by oval renal tubules). The lethality occurred in 2-3 days after the perikarya and smooth aspiny dendrites. In electron mi- first injection. Next, we analyzed the effect of AZD4547 croscopic studies from three to five classes of neurons on long bones and morphology. We found no signifi- were identified. cant changes of skeletal growth with low concentrations Most of the neurons in the claustrum are glutama- of AZD4547. Only dose of 50 μM AZD4547 caused the tergic (excitatory) projection neurons. The proportion improvement of bone growth but difference in compari- of GABA-ergic interneurons was estimated at between son to DMSO control was not statistically significant. In 7–12%. The population of neurons containing calcium- contrast, higher concentrations than 250 μM resulted in binding proteins (CBPs) has been estimated at 12 – 15% shortening of long bones and skull. General bone mor- of all claustral neurons. The dorsal as well as the ven- phology was similar to controls in all analyzed bones. tral claustrum contain several neuropeptides and nitric Bones of chondrocranium as well as desmocranium were oxide synthase which frequently colocalize with CBPs. affected by treatment. Claustrum specific proteins (, netrin G2, la- Conclusion. High doses of AZD4547 caused signifi- texin, Gng2) were recently demonstrated. cant changes of skeletal growth where the inhibition of Majority of afferent connections of the claustrum orig- bone growth was accompanied by severe toxicity in the inate in the neocortex, weaker projection originate in the mouse. The face exhibited shorter morphology resembling limbic cortical regions. Cortico-claustral projections are syndromic craniosynostoses such as Apert syndrome, topographically organized and significantly overlapped. which are also caused by disruptions of FGF signaling. Subcortical projections originate in the thalamus, amyg- Therefore, only 50 μM concentration demonstrated posi- dala and several brainstem structures. The main output tive effect on animal growth and elongation of long bones of the claustrum is the return pathway to the cortex. The during postnatal stages. 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- ACKNOWLEDGEMENTS bic areas suggests participation of this nucleus in more general and more complex functions. This research was supported by the Grant Agency of The convergence of information from different corti- the Czech Republic (14-31540S) and institutional support cal areas and subcortical structures indicate that the claus- (RVO: 67985904). 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 14 hypotheses suggesting that this telencephalic structure is The claustrum – history of research part of the system involved in salience detection and that the claustrum has importat role in cognitive processing. Rastislav Druga Institute of Anatomy, 1st and 2nd Faculty of Medicine, Charles 15 University in Prague, Czech Republic Institute of Physiology, The Academy of Sciences of the Czech Upregulation of Toll-like receptors after nerve Republic, Prague, Czech Republic injury and inflammatory signaling from Wallerian Although the first descriptions of the claustrum ap- degeneration peared in 19 century a,b a,b a,b (Burdach 1812) majority of exact data were pub- Petr Dubovy , Ilona Klusakova , Andrea Korimova , lished in postwar period. Claustrum has been identified Marek Joukala,b, Ivana Hradilova-Svizenskaa,b in all eutherian and metartherian . Differences aDepartment of Anatomy, Division of Neuroanatomy, Faculty of in morphology and relationships to cortex allow us to Medicine, Masaryk University, Kamenice 3, 62500 Brno, Czech distinguish its two parts – dorsal (insular) and ventral Republic (piriform) claustrum. An increasing body of evidence has bCentral European Institute of Technology (CETEC), Masaryk been collected about its transcription factors expressed University, Kamenice 3, 62500 Brno, Czech Republic during development. The dorsal claustrum was classified as being a derivative of the lateral pallium, whereas the Aims. Wallerian degeneration (WD) distal to nerve ventral claustrum was considered to be a derivative of the injury is considered to be aseptic includ- ventral pallium and thus the claustrum is regarded as a ing fragmentation of axons and their mitochondria, up- telencephalic, pallial subcortical mass. With using of basic regulation of ECM molecules and their cleavage. All the staining in all mammals cells of various sizes and shapes products of WD named as damage associated molecular

S9 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). patterns (DAMPs) can be released into blood circulation 16 and reach to remote structures without blood barriers. Here, DAMPs are detected by Toll-like receptors (TLRs) Porcine liver vascular corrosion casts – our up that mediate inflammatory reaction. We hypothesize that to now experience upregulation of TLRs is responsible for inflammatory re- actions in the dorsal root ganglia (DRG) both associated Lada Eberlovaa,b, Vaclav Liskab,c, Hynek Mirkab,d, and non-associated with injured nerve. Tomas Gregore, Zbynek Tonarb,f,g, Richard Palekb,c, Methods. The right sciatic nerve of adult rats was Martin Skalab,c, Michael Emingerb,c, Ondrej Troupb,c, crushed (n=15), ligated (n=15) or transected (n=15) and Jachym Rosendorfb,c, Milena Kralickovab,g, animals were left to survive for 1, 3, 7, 14 and 21 days. h The sciatic nerve and DRG of naïve rats (n=6) and sham- Alois Lametschwandtner operated rats (n=6) were used as controls. TLR2, 3, 4, and aDepartment of Anatomy, Faculty of Medicine in Pilsen, Charles 9 were detected by standard indirect immunofluorescence University in Prague, in the cryostat sections through distal and proximal nerve Karlovarska 48, 301 66 Pilsen, Czech Republic stumps as well as contralateral sciatic nerve. In addition, bBiomedical Centre, Faculty of Medicine in Pilsen, Charles bilateral DRG of L4-L5 and C7-C8 segments were used for University in Prague, Pilsen, Czech Republic TLRs detection. Activated Schwann or satellite glial cells cDepartment of Surgery, Faculty of Medicine in Pilsen, Charles were identified by co-localization with monoclonal S100 University in Prague and University Hospital in Pilsen, Alej Svobody 80, 304 60 Pilsen, Czech Republic . In addition, we tested upregulation of TLR9 in d Schwannoma cells (RT4) by N-formyl-methionyl-leucyl- Department of Imaging Methods, Faculty of Medicine in Pilsen, Charles University in Prague and University Hospital in Pilsen, Alej phenylalanine (fMLF), the prototypical N-formylated Svobody 80, 304 60 Pilsen, Czech Republic peptide released by fragmented mitochondria. eNew Technology and Materials Centre – CENTEM, University of Results. Sciatic nerve injuries induced upregulation of West Bohemia, Univerzitni 8, 306 14 Pilsen, Czech Republic investigated TLRs in Schwann cells distal and proximal as fEuropean Centre of Excellence, University of West Bohemia, well as in neurons and satellite glial cells of DRG from ip- Univerzitni 8, 306 14 Pilsen, Czech Republic silateral and contralateral sides. Schwann cells cultivated gDepartment of Histology and Embryology, Faculty of Medicine in in medium supplied with fMLP displayed an increased Pilsen, Charles University in Prague, Karlovarska 48, 301 66 Pilsen, Czech Republic expression of TLR9 in and patches of plasma h membrane particularly in cytoplasmic processes or small Department of Organismic Biology, University Salzburg, Hellbrunner Str. 34, A-5020 Salzburg, Austria protrusions. Medium containing fMLP also induced in- crease of NF-kappa B, TNF alpha and IL1beta. Aims. In experimental medicine, is frequently used Conclusion. Our results shown that TLRs are upregu- as a surrogate animal model. The aim of our study was to lated in Schwann cells activated by nerve injury and in assess porcine liver corrosion casts we made with the use DRG not only associated, but also non-associated with of Biodur E20 (Biodur products, Heidelberg, Germany). injured nerve. The increased expression of TLRs can be Material and Methods. 4 porcine were filled via regulated by DAMPs that are responsible for dissemi- the portal vein with Biodur E 20, the volume injected nation of inflammatory reactions into remote nervous ranged around 700 mL. Corrosion casts were examined structures. 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 ACKNOWLEDGEMENTS XCT 400, Pleasanton, CA, USA), the pixel size used for imaging was 17 μm, 9.5 μm and 4.5 μm, 3. scanning We would like to thank Ms. Marta Lněníčková, Ms. electrone microscopy (SEM): specimens were sputtered Jitka Mikulášková and Ms. Dana Kutějová for their with gold for 60 s and examined in Stereoscan 250 SEM skillful technical assistance. The work was supported (Cambridge, U.K.) at an accelerating voltage of 10 kV. by the Project “CEITEC – Central European Institute Results. Biodur E20 filled liver sinusoids and in some of Technology” (CZ.1.05/1.1.00/02.0068) from the places continued into the central veins. Interlobular arter- European Regional Development Fund. 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 18 This work was supported by the Internal Grant The effect of quercetin on the expression Agency of the Ministry of Health of the Czech Republic of inflammatory mediators under Project No. IGA MZ ČR 13326, by the proj- ect CZ.1.05/2.1.00/03.0076 from European Regional Kristina Gregovaa, Stefan Totha, Development Fund and SVV No 260 170, by CENTEM Miroslava Bilecova-Rabajdovab, Michaela Kudlackovaa, project reg. No. CZ.1.05/2.1.00/03.0088, and partly sup- Sona Kovalcikovaa, Zuzana Jonecovaa ported also by the European Regional Development Fund under Project “NTIS – New Technologies for aDepartment of Histology and Embryology, Faculty of Medicine, P. Information Society”, European Centre of Excellence, J. Safarik University in Kosice, Slovak Republic CZ.1.05/1.1.00/02.0090. bDepartment of Medical and Clinical Biochemistry and LABMED, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic 17 Role of histology in the era of molecular biology Aims. The aim of this work was to observe the changes in expression of inflammatory mediators after ischemia- Jiři Ehrmann, Katerina Cizkova, Pavla Luzna, reperfusion injury (IRI) of small intestine in rats after Anna Konieczna quercetin administration. Quercetin is a natural flavonoid, able to neutralized free radicals. Quercetin acts as an an- Department of Histology and Embryology, Faculty of Medicine tihistamine and an anti-inflammatory mediator. Systemic and Dentistry, Palacky University Olomouc, Czech Republic inflammatory reaction is a result of IRI of the small in- Aims. The implementation of molecular biology testine. Experiment was focused on mRNA expression methods in histology/embryology had important conse- analyses of pro-inflammatory TNFα and anti- quences, as the new discoveries may significantly extend inflammatory cytokine IL-10 after IRI under quercetin the current knowledge about cells and tissues. The aim regulatory action. of this overview is to give some examples how „molecular Methods. Adult male Wistar rat SPF–Charles River histology“ may serve as good model for clinical medicine, (n=28) were randomly assigned to the control group (E) in this particular case in the area of study of multidrug re- or experimental groups (Q). Quercetin dissolved in etha- sistance and study of precancerous lesions of oesophagus. nol or ethanol vehiculum waere applied intraperitoneally Methods. Broad spectrum of molecular biology ap- 30 min before the complete ischemia of a. mesenterica cra- proaches were applied with emphasis to use formalin nialis. Ischemia was performed by microvascular clamp, fixed, paraffin embedded samples which are still gold lasted 1 h and was followed by 1 h (E1, Q1) and 4 h (E4, standard for histology assessment. Q4) of reperfusion period. After the reperfusion period, Results. Based on molecular profiling of histologic bioptic samples were harvested. Total mRNA was isolated samples, identification of new biomarkers for prediction from the complete jejunal wall; subsequently RT-PCR was of development of oesophageal adenocarcinoma and new used followed by electrophoresis and semiquantitative therapeutic perspectives for multidrug resistance overcom- data evaluation. ing were suggested. Results. We studied quercetin effect on mRNA lev- Conclusion. The engagement of new molecular tech- els of pro-inflammatory cytokine TNFα and anti-inflam- niques into the current morphological concept will open matory cytokine IL-10 referring to reperfusion periods. new horizons in histology/embryology research. However, The protective effect of quercetin was most dominant relevant changes in the system of histology/embryology after 4 h of reperfusion, when we observed decrease in education are necessary. 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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19 20 Immunohistochemistry of colorectal carcinoma: The effect of deprenyl administration expression of p53 and MDM2 on antioxidant status of rat testis Ingrid Hodorovaa, Patrik Fialab, Jozef Mihalika, Frantiska Horvathovaa, Viera Danielisovab, Frantiska Horvathovaa, Silvia Rybarovaa Barbora Kucharovac, Peter Solarc, Silvia Rybarovaa, a a aDepartment of Anatomy, Faculty of Medicine, P. J. Safarik Ingrid Hodorova , Jozef Mihalik University in Kosice, Slovak Republic aDepartment of Anatomy, Faculty of Medicine, P. J. Safarik b Department of Pathological Anatomy, Faculty of Medicine, P. J. University in Kosice, Slovak Republic Safarik University in Kosice, Slovak Republic bInstitute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak Republic Aims. Investigation of p53 and MDM2 immunoreac- c tivity, as a potential marker of p53 function, in formalin- Department of , Faculty of Science, P. J. Safarik University in Kosice, Slovak Republic fixed paraffin-embedded tissues of colorectal carcinoma. Colorectal carcinoma is diagnosed in approximately one Aims. Despite the fact that a certain level of reactive million people annually, and it has one of the highest oxygen species (ROS) is needed for sperm capacitation cancer-related mortalities, with 694 000 deaths reported and reaction, ROS is considered as one of the globally in 2012. About 40% of patients have a metastatic main factors causing male infertility. We focused on cho- disease at diagnosis. Mechanisms of colorectal carcino- sen antioxidant enzymes (CuZnSOD, MnSOD, and CAT) genesis involve multiple tumour supressor genes (APC, in rat testis, sperm count and sperm damage after depre- TP53, TGF-b) and oncogenic genes (RAS, BRAF, PTEN nyl administration. and PIK3CA). Inactivation of TP53 pathway by TP53 mu- Methods. 18 male Wistar rats (390 g, aged 8-9 weeks) tation is present in nearly half of colorectal tumours and is were randomly divided into 3 groups and daily intraperito- one of the key genetic steps in colorectal carcinogenesis. neally injected for 30 days with either saline, low-dose of Methods. For detection of p53 (mouse anti-p53, deprenyl (LDD, 0.0025 mg/kg per day, Sigma M003) or DO7, Biogenex) and MDM2 (mouse anti MDM2, clone high-dose of deprenyl (HDD, 0.25 mg/kg per day). After the SMP14, Santa Cruz Biotechnology, inc.) we have used last drug administration animals were killed by lethal dose indirect immunohistochemical method. Study population of thiopental. The activities of antioxidant enzymes were consists of 32 patients of colorectal carcinoma (17 males measured by spectrophotometric method and their localiza- and 15 females) and median age at diagnosis was 72.5 tion was identified by immunohistochemistry. The sperm years (range 57-88 years). damage was determined employing flow cytometry, when Results. Our study revealed undetectable reaction of TMRE and Annexin V/FITC analyses were performed. p53 in the majority (87.5%) of colorectal carcinomas. Results and Conclusions.CuZnSOD and MnSOD ac- Positive immunoreaction was observed only in four cases tivity were elevated in both experimental groups. Also an (12.5%) of colorectal carcinomas. None tissue samples increase of approximately 20% in sperm count was found shown MDM2 expression. in the LDD group. As by SOD enzymes, CAT levels were Conclusion. Our findings are not consistent with pre- elevated, too. Average data of TMRE and Annexin V/ vious study indicating that p53 mutations occur in half PI cells staining indicates, that spermatozoa were not af- of colorectal carcinoma, but detection of p53 may help fected the deprenyl administration. Histological evaluation predict a tendency toward in patients with performed on testicular sections of all groups showed no this type of cancer. alterations compared to control. Immunohistochemical evaluation of CuZnSOD sections showed positive signals in and Leydig cells, in HDD also in spermato- ACKNOWLEDGEMENT gonia. MnSOD detection was positive in spermatogonia, , spermatids and Leydig cells. CAT positive Thank Mgr. Ivana Čigášová Perunská and Mgr. Diana signals were recorded in Leydig cells, germ cells and sper- Gagyiová for your kind cooperation. 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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21 murine tibial anterior muscle confirmed its good integra- tion with the muscle and rich colonization by recipient Naturally derived scaffold for skeletal muscle cells. regeneration Conclusion. We succeeded in producing decellular- ized skeletal with well-preserved major Hana Hrebikova, Jana Chvatalova, Daniel Diaz, components (, collagen fibers and glycos- Pavel Beznoska, Jaroslav Mokry aminoglycans) that fulfiled requirement for successful decellularization (elimination of cell and nuclear com- Department of Histology and Embryology, Faculty of Medicine ponents, reduction of DNA content and preservation of in Hradec Kralove, Charles University in Prague, Hradec Kralove, 3D architecture of ECM). Cytocompatibility of scaffold Czech Republic was proved with a successful in vitro recellularization Aims. Tissue engineering is closely associated with technique and implantation of decelularized scaffold into production of biomaterials. One of these strategies is the mouse muscle, which resulted in rich scaffold recel- orientated to decellularization which is able to produce lularization. scaffolds with preserved architecture, vasculature and innervation pathways for subsequent regeneration and reconstruction of the tissue. Decellularization provides ACKNOWLEDGEMENTS extracellular matrix (ECM) with its attachment sites and adequate environment for cells colonizing this scaffold, This study was supported by Grant Agency of Charles reconstituting the decellularized organ. University in Prague No. SVV-2015-260179, GAČR Methods. The protocol is based on the immersion of No. 15-09161S and with European Social Fund No. tissues in detergent solution in combination with agitation CZ.1.07/2.3.00/30.0061 and consists of the following steps: 1. hypertonic buffer 5M NaCl 2. hypotonic buffer 10 mM TrisHCl 22 3. detergent 1% SDS Retinoic acid in the development of the NADPH-d 4. 0.1% peracetic acid 5. PBS buffer positive neurons in rat brain 6. DNase I Natalia Hvizdosova, Lenka Tomasova, Martin Bona, 7. PBS buffer Stanislav Mateffy, Darina Kluchova Decellularization efficiency was determined by his- tological staining (haematoxylin-eosin, Alcian blue, Department of Anatomy, Faculty of Medicine, P. J. Safarik Sirius Red). The preservation of the ECM components University in Kosice, Slovak Republic was assessed by immunohistochemistry (collagen IV). Preservation of collagen as a main component of ECM Aims. This study describes the presence of nitrergic was assessed by biochemical method: hydroxyprolin as- neurons in selected area of the rat brain during postna- say. Removal of cell nuclei was determined by quantifica- tal development, under physiological condition and after tion of DNA. Cytocompatibility was tested by seeding exposure retinoic acid. We have focused on prefrontal the muscle scaffolds with murine myoblasts for 12 days. cortex of rats. Decelularized scaffold was transplanted into mouse and Methods. Wistar rats were used in the experiment. in terms from 1 to 4 weeks scaffolds were removed from Retinoic acid was prenatally administered between 14-16 mice and histologically analysed (haematoxylin-eosin). days of pregnancy. Dosage was 1 mg RA/kg body weight. Results. Whole-organ decellularization was achieved Prefrontal cortex was investigated at postnatal days (P) with the described protocol. Histological sections were 7, 14 and 21. The section of brain tissues were processed stained with standard haematoxylin-eosin, Alcian blue and for NADPH-d histochemical method. by Sirius Red. These stainings revealed absent nuclei or cy- Results. Nitrergic neurons were differentiated during toplasmic components compared to physiological skeletal a relatively short period of time and reach their struc- muscle, presence of glycosaminoglycans and preservation tural maturity by the end of the second week of postnatal of collagen fibers which are major components of ECM. development. At P7 and P21 control animals the image Preservation of basal lamina was determined with immu- was comparable with animals exposed to retinoic acid. nostaining of collagen IV which showed that this structure We observed the change in nitrergic neurons of exposure retained solid. Quantification of this protein was analysed animals at P14, these neurons had shorter processes in with hydroxyproline assay which proved preservation of compared to control group (P<0.001). collagen. DNA content was significantly reduced under Conclusion. The results suggest inhibitory effect of 50 ng of DNA per mg of tissue. Adhesion and viability retinoic acid, supplementation during 14-16 days of preg- of the scaffold was confirmed by re-seeding with C2C12 nancy, on length of processes of neurons at age 14 days murine myoblasts and cell morphology was examined in but 7 and 21 postnatal days were without changes in mor- paraffin-embedded sections at 12 days after cultivation. phological characteristics of the prefrontal cortex. Histological examination of scaffolds implanted into the

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23 recellularization was achieved with C2C12 myoblasts, mouse embryonic stem cells, lin-Sca1+ and murine muscle Colonization of muscle scaffolds by different cell . types

Jana Chvatalova, Jaroslav Mokry, Hana Hrebikova, ACKNOWLEDGEMENTS Rishikaysh Pisal Supported by SVV-2015-260179, GAČR No. 15-09161S aDepartment of Histology and Embryology, Faculty of Medicine and PRVOUK P37/06. in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic 24 Aims. To extend our knowledge of processes associ- The influence of maternal diabetes on ated with skeletal muscle repair we utilized a model based on application of several of types into scaf- proliferative potential of human term placenta folds. In our case we used bioscaffolds derived from the Marie Jirkovska, Krystof Zuska skeletal muscle decellularization. We examined adher- ence, growth and development of different types of cells Institute of Histology and Embryology, 1st Faculty of Medicine, in scaffolds after 10 and 21 days from recellularization. Charles University in Prague, Czech Republic Methods. We studied implantation of 8 types of cells – muscle derived stem cells (MDSC) in three stages of Aims. Placenta is a temporary organ finishing its func- isolation (passage 3 – PP3, passage 5 – PP5 and passage tion at parturition. This study compares the proliferatiove 6 – PP6); mouse embryonic stem cells (mES); muscle potential of normal term and placentas from murine fibroblasts; myoblasts (C2C12 cell line); satel- complicated by either maternal gestational lite cells isolated from muscle fibers and stem cells iso- diabetes (GDM) or diabetes type 1 (DM1). lated from the bone marrow of mouse using separation Methods. Histological sections of placental specimens Lineage Cell Depletion Kit and positive separation with taken by systematic uniform random sampling were used antibody Sca1 (lin-Sca1+). The scaffolds were obtained for immunocytochemical detection of Ki-67 . The from C5Bl/6J mice (precisely from tibialis anterior mus- proliferative potential was expressed as the numbers of cle) washing in 5 M NaCl in distilled water, hypotonic labelled nuclei per square milimeter of villous section 10 mM TrisHCl, incubation with 1% SDS and steriliza- counted in cytotrophoblast, and vascular wall in tion in 0.1% peracetic acid solution. Cell nuclei residues stem, intermediate (IV) and terminal villi (TV). Placental were removed with DNase. We implanted 10 μL of cell groups were compared by the Mann-Whitney test, results suspension of each cell line into two scaffolds. We used were considered to be significant at P ≤ 0.05. volume of cells in previously known concentration. The Results. Histological analysis revealed variable amount cell lineages were cultured in common cultivation medium of pathological villi (hypovascularized and hypervascular- (DMEM, glutamine, 10% fetal bovine serum, penicillin ized) in DM1 placentas, and only infrequent occurrence and streptomycin). Cultivation medium was replaced with of hypovascularized villi in GDM placenta. The fact that fresh one every two days. The scaffolds were removed in both pathological forms of villi displayed nearly no Ki-67 specified period of time after implantation (10th and 21st labelled nuclei reflects itself in results of measurement. In day), fixed in formalin, processed for paraffin-embedded the villous cytotrophoblast of terminal villi, the frequency sections and stained with standard methods or indirect of Ki-67 labelled nuclei per square milimeter was signifi- immunohistochemistry. cantly lower in both DM1 and GDM placentas (19.87 Results. The scaffolds and their cells were evaluated by ± 12.25 and 18.66 ± 9.88 respectively) than that in the various histological and histochemical methods. After 10 control group (32.21 ± 14.70). In the wall, the days, of 8 cell types only 4 were observed in recellularized frequency was sigificantly lower in DM1 group as com- scaffolds: C2C12 myoblasts, mES, lin-Sca1+ and murine pared to the control group (5.23 ± 4.50 vs 10.11 ± 4.65). muscle fibroblasts. The structure of cells is very close. In intermediate villi, significant differences were found They cells elongated, begin to fuse and able to survive between DM1 and control group in the villous cytotro- and proliferate. Only few cells migrated to the middle of phoblast (9.46 ± 6.12 vs 18.26 ± 7.74) and between DM1 scaffold, most of them were left on the surface of scaffold. and GDM group in the villous stroma (5.43 ± 6.16 vs 1.53 Ten days in vitro the cells migrating over the outer surface. ± 1.83). No differences were found among stem villi of After 21 days in vitro, cells penetrated deep areas into scaf- all studied groups. fold where they accumulated although some other deep Conclusion. The lower proliferative potential of cyto- areas of scaffolds were left unoccupied. trophoblast in TV of both diabetic groups as well as the Conclusion. Our experiments provided evidence the decreased one in of TV imply a lower ability to decellularized scaffolds preserved well components of sustain the growth of placental components ensuring ma- extracellular matrix and their microarchitecture. The ternofetal transport. It may adversely influence the fetal acellular scaffolds can be colonized by cells; the best well-being in terminal phases of intrauterine development

S14 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). and during perinatal period in pregnancies complicated rate was significantly reduced at the beginning of cold by both types of maternal diabetes. 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 ACKNOWLEDGEMENTS fairly constant during the further 6 hours of preserva- tion (0.88±0.09 at the end of preservation), thus probably The work was supported by PRVOUK P25/LF1/2. 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 25 found at the end of preservation. Modification of apoptotic cell rate in the intestinal Conclusion. A higher graft protection was observed when fewer, but longer periods of ischemia were used in graft mucosa during cold preservation IPC, thanks to the improved morphology of the intestinal Zuzana Jonecovaa, Stefan Totha, Milan Marettab, mucosa and sustentative level of the cell rate Kristina Gregovaa, Jan Vargac during the cold preservation. Better preserved graft ben- efits to graft survival during following retransplantation. aDepartment of Histology and Embryology, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic bDepartment of Neurology, Louis Pasteur University Hospital, AKNOWLEDGEMENTS Kosice, Slovak Republic c Department of Gynaecology and Obstetrics, Faculty of Medicine, This study was supported by the grant VEGA P. J. Safarik University in Kosice, Slovak Republic 1/0478/2014 and VVGS-2013-111. Aims. The aim of present study was to investigate whether duration and frequency of ischemic precondi- tioning (IPC) procedure affects cold preservation injury 26 in the small intestine grafts. The epithelial apoptosis and Kolmer cells and their immunophenotyping in the extracellular matrix protein laminin expression were stud- ied because of their importance in failure or maintaining rat choroid plexus after peripheral nerve injury the barrier function of the graft intestinal mucosa. Marek Joukala, Adela Kuklovaa, Peter Solara, Methods. Three groups of Sprague-Dawley rats were Ilona Klusakovaa,b, Petr Dubovya,b recruited (n=11 each) as follows: the short IPC (SIPC) performed through 4 cycles of mesenteric ischemia of 4 aDepartment of Anatomy, Faculty of Medicine, Masaryk min each followed by 10 min of reperfusion, the long IPC University, Brno, Czech Republic (LIPC) obtained by 2 ischemic cycles of 12 min each fol- bCEITEC, Masaryk University, Brno, Czech Republic lowed by 10 min of reperfusion, and the control group (C) without IPC. Grafts were then stored in cold histidine- Aims. The unilateral chronical constriction injury tryptophan-ketoglutarate solution and samples were taken (CCI) used as a neuropathic model causes the eleva- at 0, 3, 6 and 9 hours lasting preservation. Apoptosis was tion of proinflammatory levels not only in the detected by using the terminal deoxynucleotidyl-transfer- structures directly related with damaged nerve but also in ase-mediated deoxyuridine triphosphate in situ nick end- remote structures of (CNS). The labeling (TUNEL). Apoptotic cells (AC) were quantified way how the neuroinflammatory molecules affect CNS is per 1 . Anti-laminin immunohistochemical not completely clear. We purpose that one of the possible method for the laminin evaluation was used. The laminin pathways for spread of neuroinflammation could be over positive structures in (BM) of the the blood-cerebrospinal fluid barrier localized in choroid epithelial lining were expressed as a mean per 1 intestinal plexus (CP). CP is formed by highly vascularized stromal villus according to the semiquantitative immunohisto- core with fenestrated capillaries covered by cuboidal cells chemical scale 0-3. on the ventricular side responsible for cerebrospinal fluid Results. BM laminin expression in the jejunal graft secretion. Epiplexal Kolmer cells (KC) are attached on mucosa at the beginning of preservation was similar in all the surface of the cuboidal cells. They are known as the groups (C=0.80±0.17; SIPC=0.98±0.14; LIPC=1.12±0.07). scavenger cells processing the signals from blood to ce- A parallel significant increase in laminin expression rebrospinal fluid. Therefore, the aim of our study was to with the histopathological injury rate after 6 hours of investigate changes of KC and CP after peripheral nerve preservation was observed in all grafts (C=1.82±0.06, injury. P<0.001; SIPC=2.08±0.06, P<0.001; LIPC=1.90±0.16, Methods. Twelve Wistar male rats were used in our P<0.01) in comparison with the level at time 0. The experiments. Rats were anesthetized with a mixture of ket- similar results after 9 hours of preservation were found amine and xylazine administered intraperitoneally. CCI (C=1.86±0.21; SIPC=2.10±0.09; LIPC=1.32±0.11, all of of the left sciatic nerve was performed by three ligatures them in comparison with the starting time P<0.001). that reduced the nerve diameter by approximately one- When compared to the C group, in both IPC groups AC 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, This discrepancy between the official approach and animals were sacrificed in CO2, perfused transcardially by the general state needs to be approached. The authors Zamboni’s fixative; the brain was removed and fixed for suggest a three-leveled classification of eponyms: Level three days. Immunohistochemical detection for resident A for every physician and student, Level B for special- (ED2) and activated (ED1) macrophages and dendritic ists in one individual medical branch and Level C for cells (OX-42) was performed on coronal cryostat sections superfluous terms (which should be omitted from future through the brain. communication). 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- AKNOWLEDGEMENTS ration of nerve compression whereas the most ED1+ cells were found in epiplexal position after 21 days of nerve Supported by Charles University Grant 236028/ compression. Immunohistochemical detection for OX42+ IRP/2013, 236055/IPUK/2014, 236070/IPUK/2015. 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 28 CCI operated animals but not in naïve rats. Arteria brachioradialis – overview Conclusion. We demonstrated that population of KC is formed by cells with different immunophenotypes David Kachlika,b, Petr Hajekc, Marek Konarik, Vaclav Bacaa,b namely ED1, ED2 and OX42. We can conclude that the aDepartment of Anatomy, Third Faculty of Medicine, Charles number of KC increased after peripheral nerve injury. KC University in Prague, Czech Republic are blood derived therefore the changes of their number bDepartment of Health Care Studies, College of Polytechnics could be caused by alteration of tight junctions that are Jihlava, Czech Republic found between the cuboidal cells. These changes could cDepartment of Anatomy, Faculty of Medicine in Hradec Kralove, result in a spread of neuroinflammatory reaction from Charles University in Prague, Hradec Kralove, Czech Republic peripheral nervous system to the CNS structures. Arteria brachioradialis is the most common varia- tion of the arterial trunks of the upper limb. The over- ACKNOWLEDGEMENTS all incidence is reported in 14% of cases on average. By long-time theory, it is derived from its embryological We would like to thank Ms. Marta Lněníčková, Ms. precedent termed the arteria brachialis superficialis. The Jitka Mikulášková, Ms. Dana Kutějová and Mr. Lumír development of the upper limb arteries is not a simple Trenčanský for their skillful technical assistance. The process, closely related to the bone development. Arteria work was supported by MUNI/A/1215/2014. 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 27 crosses ventrally the median nerve. It can be classified as Anatomical eponyms “mere” arteria brachioradialis if shows typical course of radial artery in the forearm or as arteria brachioradialis David Kachlika,b, Tereza Stepankovaa, Vladimir Musilc,d, superficialis when crossing over the delimiting the Vaclav Bacaa,b snuff box. Secondly, it can be classified into four types ac- cording to its origin, as it usually branches in the proximal a Department of Anatomy, Third Faculty of Medicine, Charles third of the arm from the arteria brachialis (66%) but not University in Prague, Czech Republic exceptionally from the axillary artery as well. Finally, it bDepartment of Health Care Studies, College of Polytechnics Jihlava, Czech Republic can be classified according to the pattern of branches cCentre of Scientific Information, Third Faculty of Medicine, present “on the way” along the arm and forearm. The Charles University in Prague, Czech Republic anatomical knowledge of arteria brachioradialis existence dInstitute of Information Studies and Librarianship, Faculty of and course is essential for radiodiagnostic, surgical and Arts, Charles University in Prague, Czech Republic traumatologic procedures and the superficially coursing artery brings a higher risk of heavy in unexpect- Eponyms in anatomy are a constant part of the ter- ed situations. The presentation gives an overview of the minology. Due to its shortness and relative uniqueness anatomy and variability of the arteria brachioradialis and they have gained an irreplaceable position. Although they the implications in the clinical medicine, mainly for the were excluded from the Latin anatomical nomenclature interventional cardiologists and reconstructive surgeons. in Parisiensia Nomina Anatomica in 1955, they have re- mained an integral part of the both, in the oral communication and even in the scientific AKNOWLEDGEMENTS literature. The last revisions of the Latin nomenclatures (, , Supported by Charles University, Project PRVOUK Terminologia Embryologica) are completed with lists of # 33. eponyms as an . S16 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1).

29 processes such as patterning of the body axis, limbs and identifying planar cell polarity. WNTs are associated with Supernumerary carpal bones receptors Frizzled and low density lipoprotein receptor- David Kachlika,b, Alzbeta Blankovaa, Vaclav Bacaa,b related protein family (LRP5/6). Members of WNT can signal through β-catenin (canonical) or β-catenin indepen- aDepartment of Anatomy, Third Faculty of Medicine, Charles dent (non-canonical) pathway. WNT5a belongs among University in Prague, Czech Republic β-catenin independent ligands. Loss-of-function experi- bDepartment of Health Care Studies, College of Polytechnics ment inhibited the formation of columnar growth plate Jihlava, Czech Republic architecture during chondrogenesis. Our aim was to un- cover a possible role of WNT5a in chondrogenesis while The usual number of carpal bones is fairly constant. In using gain-of-function approach. spite of this fact, variations have been reported, based on Methods. Mouse tibias were isolated from E18 embry- the presence of the accessory elements, of the coalition os and were cultured in media supplemented by WNT5a or even of the absence of one carpal bone. A particular (40 ng/mL) or FGF2 (50 ng/mL) or mixture of both pro- review of reference literature showed a log row of different teins. Control tibias were cultured in media and four bio- variants with incoherent and inconsistent nomenclature. logical replicates were used for each treatment. The length A need of the classification of carpal bones variations for of tibias was measured at the first day and at the end of ex- anatomical research, education and clinical practice was periment to analyze growth differences. Chicken embryos the main hint of this contribution. Embryologically, more were selected as a model organism for in vivo experiment than eight centers appear in the wrist, some because of their easy access in the shell. Fertilized eggs of them later disappear, other ones persist or fuse to form were incubated for 3.5 days (38 ˚C) and bead soaked with definitive carpal bones. A disturbance in the development WNT5a (20 μg/mL and 40 μg/mL) was implanted into can result in presence of a separated bony element (a wing bud. Embryos were collected into 100% ethanol fol- supernumerary bone) or a process of a constant carpal lowing 11 days of incubation. Wings were stained with 3% bone. These variations can stem from pathologic states Alcian Blue for cartilage visualization and 0.1% Alizarin as well: ossification of synovial folds or posttraumatic os- Red to analyze bone morphology. Samples were cleared sification. in 4% KOH and photographed in 50% glycerol. Variations of carpal bones are classified into four Results. We observed statistically significant differenc- groups: accessory carpal elements, multipartite carpal es in degree of tibias growth based on treatments. FGF2 bones, absent carpal bones and collation of carpal bones. caused shortening of tibias while WNT5a by itself did not The clinical relevance comprises problems with diagno- effect cartilage growth. However, WNT5a rescued FGF2 sis in the X-ray image to reveal the origin of the patient phenotype when both proteins were added into the media. problems (developmental or traumatic) to choose the ap- To uncover effects of WNT5a on early cartilage develop- propriate treatment. ment, beads soaked with WNT5a were implanted into Supported by Charles University, Project PRVOUK wing bud. We observed similar phenotypes when using # 33. 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- AKNOWLEDGEMENTS tion 40 μg/mL). Wings treated with WNT5a were longer and thinner than control wings. We also observed mor- Supported by Charles University Grant 236028/ phological differences in zeugopod and autopod areas IRP/2013, 236055/IPUK/2014, 236070/IPUK/2015. such as curved zeugopod and abnormal sizes of . Conclusion. Here, we found that WNT5a protein af- fected chondrogenesis in vivo and could rescue FGF2- 30 induced inhibition of tibias growth. Next, we aim to Effect of WNT5a on chondrogenesis and limb further uncover cross-talk of FGF and WNT signaling pathways during limb development and determine the development role of WNT5a in causing the malformations of posterior Michael Killingera,b, Iva Veselaa, Marcela Buchtovaa,b structures (ulna/fibula) during limb development. aLaboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of Czech ACKNOWLEDGEMENTS Republic, Brno, Czech Republic b Department of Experimental Biology, Faculty of Science, This research was supported by the Grant Agency of Masaryk University, Brno, Czech Republic the Czech Republic (14-31540S) and institutional support Aims. WNT family includes many critical mediators (RVO: 67985904). of cell-cell signaling events during embryogenesis. WNTs members are cystein-rich secreted extracellular ligands es- sential for physiological, pathological and developmental

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31 32 Abbot Seka’s cranial morphology and 2D Comparison of different tissue clearing methods reconstruction of his face with individual for 3D imaging techniques in GFP-expressing landmarks embryonic and adult mice Ivo Klepaceka, Andrej Shbata, Pavla Zednikova-Malab Hana Kolesovaa, Martin Capekb, Jiri Janacekb, a,b aInstitute of Anatomy, First Faculty of Medicine, Charles Universit David Sedmera b in Prague, Czech Republic Department of Anthropology and aInstitute of Anatomy, First Faculty of Medicine, Charles University Biology, Institute of Criminalistics, Prague, Czech Republic in Prague, Czech Republic bInstitute of Physiology, The Czech Academy of Sciences, Prague, Aims. To make an anthropological reconstruction of Czech Republic an abbot Ferdinand Seka’s face. He was the important personality of the premonstrate order in the first half of Aims. To find a suitable tissue clearing protocol for 19th century. No detailed realistic portrait is known. whole mount imaging of embryonic and adult of Methods. Due to well-preserved facial skeleton with transgenic mice preserving GFP fluorescence. incomplete dentition was at the disposal, a modified two- Methods. We compared various published organic dimensional (2D) method (Gerasimov 1955, Rynn et al. solvent and water-based clearing protocols claiming to 2010) was applied. The cranium in question was recog- preserve GFP fluorescence in central nervous system: nized as dinar type and examined by CT. tetrahydrofurane dehydration and dibenzyl ether (DBE), Results. No malformations or traumatic changes were SCALE, CLARITY, and CUBIC. Tissue transparency found. Malar surfaces of facial bones were free of cracks, and preservation of GFP fluorescence was evaluated in sutures were clearly defined. A slight depression was de- hearts of Cx40:GFP knock in mice at stages: ED10.5 – tected on the left suborbital area. Thus, the following an- 18.5 ED, P1-P10, and adults. Imaging was performed on a thropometric points: supraglabella, glabella, nasion, mid dissecting microscope equipped with epifluorescence and nasal, rhinion, mid-, prosthion, supramentale, an upright single photon confocal microscope (Olympus) pogonion, gnathion, mid mandibular, menton, zygion with 4x, 10x, and 25x objectives after mounting the speci- and gonion were identified to be critical making en face mens into cavity slides or custom imaging chambers. and profile contours. A slight lateral depression of pro- Results. Despite careful control of all critical pa- truded nose and ptosis of the right eye were created. Our rameters, DBE clearing protocol did not preserve GFP reconstruction assumes probable facial asymmetry with fluorescence; in addition, it caused considerable tissue relatively massive and curved nose in comparison with shrinking and worse deformation than golden standard later found contemporary abbot’s effigy. BABB protocol used for whole mount immunohistochem- Conclusion. It is accepted that the shape and size of istry. The SCALE clearing resulted in good tissue trans- the individual parts of the soft tissues in face as well as parency up to ED12.5; at later stages and in the adults shape of the bone structures lying underneath them have the useful depth of imaging was limited by tissue light measurable relationship. The detailed relief of the Seka’s scattering. The CLARITY method considerably improved face was constructed using combination of predictive tissue transparency at later stages, but also decreased GFP rules (supported statistically: DeGreef et al. 2006, George fluorescence intensity. The CUBIC method resulted in 1987, 1993, Stephan and Simpson 2008, Prokopec and very well cleared specimens even at the adult stages, and Uberlaker 2002, Rynn et al. 2010, Wilkinson 2004, 2010). GFP fluorescence was also preserved. In addition, it de- Due to that, it was possible to estimate the size and shape colorized the blood and myocardium by removing iron even of individual elements (eyes, nose, mouth), because in the tissues. High-resolution images were thus obtained abbot’s skull type was recognized. It must be emphasized even from deep tissue layers with ScaleView immersion that even in the ideal case (where the cranium is fully 25x objective. Simultaneously, intact specimens could be complete), the anthropologic methods have limits and the imaged by optical projection tomography. final result is still partially influenced by the subjective Conclusion. From the tested methods, the CUBIC approach of the author's reconstruction. 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 ACKNOWLEDGEMENTS dimensional reconstructions of normal and abnormal specimens to visualize the developing cardiac conduction Partially supported by GACR P101/12/1306 and system and coronary vasculature. UNCE 20401

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ACKNOWLEDGEMENTS nuclei numerical density of vessels and their lengths re- mained unchanged. Supported by 13-12412S from the Grant Agency of the Conclusion: We have proved the quantitative changes Czech Republic, Ministry of Education PRVOUK P35/ of microvascular network in individual layers of Lurcher LF1/5, and Charles University UNCE mice cerebellum, which are most expressed in its cortex.

33 ACKNOWLEDGEMENTS Stereological assessment of cerebellum Supported by the project ED2.1.00/03.0076 from microcirculation in the Lurcher mice European Regional Development Fund and the Charles Yaroslav Kolinkoa,c, Jan Cendelinb,c, Zbynek Tonara,c, University Research Fund (project number P36). Milena Kralickovaa,c 34 aDepartment of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Karlovarska 48, 301 66 Pilsen, GaMeTix – a software for administration of MCQ Czech Republic bDepartment of Pathophysiology, Faculty of Medicine in Pilsen, databases and creation of examination papers Charles University in Prague, Lidicka 1, 301 66 Pilsen, Czech a b Republic Dimitrolos Krajci , Pavel Kylar cBiomedical Centre, Faculty of Medicine in Pilsen, Charles aDepartment of Histology and Embryology, Faculty of Medicine University in Prague, Alej Svobody 76, 301 00 Pilsen, Czech and Dentistry, Palacky University Olomouc, Czech Republic Republic bPrivate IT Programmer, Krelov, Czech Republic Aims. Lurcher mouse is a natural model of the olivo- cerebellar degeneration caused by a semi-dominant mu- Aims. Examination of student’s knowledge by ap- tation (Grid2Lc) in the δ2 glutamate receptor encoding plication of written or electronic tests is widely used in gene. We aimed to characterize the microvascular network modern pedagogy. Large collections of examination ques- of cerebellum in the three months old Lurcher mouse. tions must be managed systematically and securely using Methods: Paraformaldehyde-fixed brains of B6CBA dedicated software that allows user to add new questions (n=5) and Lurchr (n=5) mice were processed into 18-μm progressively and to edit these questions written in various thick serial sections (n=1973) with random orientation. MCQ formats. Consecutive creation of question sheets Microvessels were visualized using polyclonal rabbit anti- and their publication for printed or electronic delivery laminin antibody. In total 4,226 stacks comprising three is another required feature of such a software. We have 5-μm thick optical sections were recorded using system- coded and practically tested a stand-alone portable appli- atic uniform random sampling. Numerical density of mi- cation called GaMeTix. In this report we analyze specific crovessels was estimated by counting the branching nodes features of this application and demonstrate its functional- using the optical disector. Total lengths were assessed ity in practical testing in Histology for medical students. using the optical fractionator technique. For statistical Methods. The program is created under the .NET analysis a Mann–Whitney U-test was used. framework in the C# language. Database of the test ques- Results: The total number of microvessels in the tions is stored in a file encrypted using the symmetric control group was 232,332±31,843 (mean±SD) with a cryptography. Questions and generated tests are stored total length of 11,193.3±1,205.54 mm (within a volume in UTF-8 format and can be imported or exported from/ of 32.3±3.16 mm3). In the Lurcher cerebellum (volume into various formats. In order to generate tests, one or 9.2±0.96 mm3 P=0.012) the microvessel number was more databases simultaneously can be used. Access to 108,181±14,677 (P=0.022) and the length was 3,927±2,507 the program is protected by a username and password. mm (P=0.012). The volumes of the individual layers of Several user accounts can be created. the Lurcher cerebellum except there nuclei was also sig- Results. The program is composed of five units that nificantly lower (P=0.012): molecular layer and Purkinje cover Database Management, Generation of Tests, Test cells by 81.6% to 2.43±0.25 mm3, granular layer by 76.9% Printing, Test Export and Database Backup. A Database to 3.12±0.36 mm3, and white matter layer by 47.8% to Management pane provides the key function of this soft- 2.77±0.23 mm3. Both number and length were quanti- ware to add new questions to the database and to catego- fied in separate layers of the cerebellum. We observed a rize them by different criteria (ID number, topic, theme, significant reduction (P=0.012) in the number and length semester, difficulty level, date of the last usage, frequency of vessels in molecular (by 61.2% to 29,649±6,026; by of use). A search filter available in this pane is to sort 68.6% to 1,262.6±148.23 mm) and granular (by 62.4% questions according to these criteria. The question for- to 45,420±4,304; by 72.3% to 1,522.3±94.4 mm) layers, mats are MCQ, MRQ, TF, and they can be supplemented as well as reducing of total vessels length in white matter with pictures in bmp, jpg and gif formats. In the Test by 48% to 756.6±107.1 mm (P=0.012). In the cerebellar Generator, some filters can be set to specify the range and the number of questions that should appear in the

S19 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). test. The generator checks whether the selected databases in further statistical analysis of question difficulty. The contain sufficient number of the questions for the speci- software is able to register all data about each test ques- fied filters and then – using a random number generator tion in html-formatted results table and, after enabling – it generates the tests. Tests of the same topic can be the Print Results function, the result table can be printed. generated in several versions, each version contains dif- Our modification of this examination system required ferent, randomly selected and differently sorted questions reprogramming of the Quizmaker’s 13 report.html file to prevent possible cheating. in order to be able to send result data directly to a web The generated tests can be exported into the text file server installed on the teacher’s PC. On this PC all result format or directly printed. Before printing, the header of reports are stored in html and xml formats for backup the test can be edited. Printing of the test on the printer and, furthermore, the html-formatted reports are sent to or into a PDF file is followed by printing of the key to the the MySQL database on the database server for statisti- correct answers for easy evaluation of test results. The cal analysis. saved tests can be also exported into txt and xlsx files for Results. After password-protected logging into the on-paper editing or importing sets of questions into elec- Database of Test Results the graphical user interface tronic test creators like Articulate Quizmaker 13. shows indexes to four panels with functions – Overview Conclusion. The GaMeTix is a dedicated stand-alone of Test Reports, Statistical Analysis of Tests, Import of application to manage several databases of MCQs in a Tests and a Setup panel. The Overview panel displays secure and portable manner. It provides educators with a information about all deposited test results sorted by se- simple tool to create sets of examination question sheets mester, date of examination, title of examination, class with random selection of questions on predefined topics group and name of the PC used by a student. All these in various MCQ formats. 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 ACKNOWLEDGEMENTS the final percentage score can be read out. The statistical evaluation panel provides a summary information about Development of this software and application of each group of tests selected by a date of examination, electronic testing was supported by ESF-OPVK grant name of the examination or a class group of students. n.CZ.1.07/2.2.00/28.0089 in years 2012-2014. 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 35 rating of the test, a mean time spent for answering each A new system of electronic evaluation of results of question and a mean time spent to complete the test. All questions in a particular test are displayed with informa- PC-delivered tests tion about numbers of students passing and failing these Dimitrolos Krajcia, Tomas Kopecnyb, Radko Novotnya answers, mean time spent to answer each question and a difficulty value of each question expressed in percentage aDepartment of Histology and Embryology, Faculty of Medicine values and color coded. A histogram of difficulty indices and Dentistry, Palacky University Olomouc, Czech Republic is also displayed in this panel. b Dean`s Administration Office, Faculty of Medicine and Dentistry, Conclusion. Electronic evaluation of PC-delivered tests Palacky University Olomouc, Czech Republic is an essential tool in teacher’s hand to manage and evalu- ate results of large groups of students. It provides detailed Aims. Electronic application of summative tests pro- information about students’ performance in each test that vides an immediate information about test results to stu- can be analyzed and discussed with a student without giv- dents and examiners. When the test results are processed ing out any printed copy of the test. Statistical evaluation electronically the answers of individual students can be of test results provides an additional input about the dif- collected and statistically evaluated. This provides a valu- ficulty of tests and question items related to performance able feedback to test creators about the difficulty of ap- of students. 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 ACKNOWLEDGEMENTS companies. In order to extend the practicality of our own electronic testing system we have developed our own stor- Development of the database and application of age database of test results located on university servers electronic testing was supported by ESF-OPVK grant with protected web connection to computers in histology n.CZ.1.07/2.2.00/28.0089 in years 2012-2014. 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

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36 students of our histology and anatomy classes and teach- ers alike. Application of electronic testing in histology and anatomy practical ACKNOWLEDGEMENTS Drahomira Krajcia, Radka Lichnovskaa, Bela Erdosovaa, Dimitrolos Krajcia, Libor Machalekb Development and application of electronic testing was supported by ESF-OPVK grant n. CZ.1.07/2.2.00/28.0089 aDepartment of Histology and Embryology, Faculty of Medicine in years 2012-2014. and Dentistry, Palacky University Olomouc, Czech Republic bDepartment of Anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic 37 Evaluation of results of MCQ tests applied in Aims. Our aim was to find a system, which guarantees a straightforward and quick use of question packages, im- electronic format mediate and objective evaluation and reporting of results, Drahomira Krajcia, Radka Lichnovskaa, Bela Erdosovaa, limitation of lateral student’s communication and cross- Tomas Kopecnyb, Radko Novotnya, Dimitrolos Krajcia talk, and a good prevention of leaking questions outside of the examination room. aDepartment of Histology and Embryology, Faculty of Medicine Methods. We use a special software ARTICULATE and Dentistry, Palacky University Olomouc, Czech Republic Quizmaker ’13 which has proved to be simple and easy to bDean’s Administration office of the Faculty of Medicine, Palacky use by teachers having no programming skills. The user University Olomouc, Czech Republic interface of this software is similar to the standard envi- Aims. Three years ago we have introduced electronic ronment of MS Office applications. Since this software testing in our system of teaching practical histology using has a selective option to shuffle sequences of questions a dedicated software Articulate Quizmaker 13. This soft- and also to shuffle all distracters in the quiz content ran- ware provides immediate on-screen summative scoring of domly on monitors of examined students, only one ver- students’ results, but fails in analysis of responses to single sion of histology or anatomy test was enough to prepare questions and in further statistical analysis of question for one examination session. A time limit for display of difficulty. Here we report our innovations in collection of each question and a total time allowed for a complete test test results locally and their difficulty analysis up to the were settable during test creation. Quizmaker’13 offers level of a single question. the possibility to use a mix various formats of questions Methods. In order to examine theoretical as well as like – MCQ (one correct answer), MRQ (one or more of practical knowledge of histology we use four formats of correct answers), and Drag-&-Drop format (dragging and questions in our tests, namely Multiple Choice, Multiple aligning several correct options to the same number of Response, Drag & Drop and Hot Spot questions. The statements). For identification of structures in displayed final test-scoring page informs students about their re- micrographs a Hot-Spot format of question (clicking with sults. The software is able to register all data about each a mouse on a structure in question) proved to be very use- test question in html-formatted results table and, after ful in histology and anatomy testing. enabling the Print Results function, the result table can Results. Prior to exporting tests for PC application the be printed. In its original configuration, the Quizmaker 13 teacher can decide about information that will be visible failed to collect and sent these data through the network to student during examination time. These are: the test to any hardware storage for further analysis. Our modi- title, countdown display of the total examination time, fication of this examination system required reprogram- point value of each question and a current student's score ming of the Quizmaker’s 13 report.html file to be able to as achieved during testing. On completing the test, the send result data directly to a web server installed on the software automatically summarizes and displays value of teacher’s PC. This web server processes the result data correct answers in percentages and shows a minimum into a modified results table, saves the table on teachers’ passing limit for that exam. When completely authored, PC for immediate access, and simultaneously it sends the the test can be exported into different application formats table to a dedicated email address “histexam” located on for web (for use on a website or a PC-equipped lab), for university servers. CD or DVD distribution, for LMS-managed classrooms, Results. On the “histexam” location, the examination and it can be also exported into a simple MS Word docu- results are categorized by student’s name, date of exam, ment for archival documentation. contents of the exam etc. The downloaded result tables of Conclusion. PC-based testing of students’ knowledge every student are further manually converted to standard may be used not only as regular in-course assessments in xlsx spreadsheet table and further analyzed to calculate practical sessions, but also as in-group summative exami- values of summary points, net points, easiness index and nation tool at the end of the teaching semester. Evaluation difficulty index in percentage values. Based on these data, results show that computerized in-course examination of we can evaluate performance of each student. We also theory and practical knowledge has been well accepted by evaluate each question in the test to classify it as easy,

S21 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). moderate and difficult one. We presented the evaluation DMLB microscope equipped with DC300 camera. For data in the form of columnar graphs where difficulty in- double labeling we used immunofluorescence approach dices are color coded according to our own classification and images were taken using a confocal laser scanning scale. This evaluation of tests gives us important data on microscope (FV1000, Olympus). the quality of tests applied but also on the overall pre- Results. In all atrial samples from both groups we de- paredness of students for the particular topic. It can also tected CD45+-cells in atrial myocardium as well as in en- indicate possible cheating activities of students. docardium and epicardium. The frequency of these cells Conclusion. Application of electronic tests is a use- was variable. Atria from patients with atrial fibrillation ful tool for teachers and students alike. We consider the had tendency for a higher infiltration with CD45+ cells. regular evaluation of electronic tests as very important Apparently, CD45+ cells formed a heterogeneous group of follow-up feature of the examination procedure. It reveals cells. We also detected CD68+cells, CD3+cells, mast cells improperly formulated questions, incorrect sets of dis- and DC-SIGN-positive dendritic cells. The most abundant tracters and overall difficulty of examinations. This activ- subpopulation from all CD45+ cells was CD68-positive ity also provides a summative information about classes cells. In the left atrial myocardium there was significantly of students and stimulates students in their preparation higher frequency of these cells in patients with atrial fibril- for examinations. 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. ACKNOWLEDGEMENTS Conclusion. Our results document that CD45+ cells are a heterogeneous cell population in atrial myocardium This communication was supported by ESF-OPVK from patients undergoing open heart surgery and these grant n. CZ.1.07/2.2.00/28.0089 in years 2012 – 2014. 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- 38 ity of this part of the heart to inflammatory processes re- Inflammatory cells in human atrial myocardium lated to atrial fibrillation. Colocalization between CD68+ and DC-SIGN demonstrated that the most frequent im- from patients undergoing open heart surgery mune cells in the atrial myocardium are dendritic cells. Tomas Kuceraa, Natalia Smorodinovaa, Karolína Rozsivalovaa, Jaromir Pridala, Maria Durisovaa, ACKNOWLEDGEMENTS Jan Pirkc, Martin Blahab, Vojtech Melenovskyb, Josef Kautznerb This work was supported by the Research Program of Charles University – PRVOUK -P25/LF1/2. aInstitute of Histology and Embryology, The First Faculty of Medicine, Charles University in Prague, Czech Republic bDepartment of Cardiology, Institute for Clinical and 39 Experimental Medicine – IKEM, Prague, Czech Republic cDepartment of Cardiovascular Surgery, Institute for Clinical and Study of changes in the rat choroid plexus Experimental Medicine – IKEM, Prague, Czech Republic after peripheral nerve injury using fluorescent Aims. In this study we focused on morphological and conjugated dextran functional changes in endomysium of atrial myocardium a a a with special attention to CD45+- cells, representing cells Adela Kuklova , Peter Solar , Marek Joukal , of bone marrow origin. In addition, we characterize differ- Ilona Klusakovaa,b, Petr Dubovya,b ent types of resident or infiltrating immune cells. aDepartment of Anatomy, Faculty of Medicine, Masaryk Methods. We analyzed atrial biopsies obtained from University, Brno, Czech Republic patients undergoing bypass or surgery. The bCEITEC, Masaryk University, Brno, Czech Republic 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 Aims. Peripheral nerve injury can lead to cellular and from were histologically examined using routine molecular changes in the peripheral and central nerv- hematoxylin-eosin staining. For detection of different ous system structures which are not directly related with types of immune cells the following markers were de- damaged nerve. We suppose that these structures can tected immunohistochemically: CD45 as a pan-leukocyte be influenced by damage associated molecular patterns marker, CD3 for T-lymphocytes, CD68 for / (DAMPs) produced by Wallerian degeneration and dif- macrophages, tryptase for mast cells and DC- fused via blood-cerebrospinal fluid barrier (B-CSF-B). SIGN for immature dendritic cells. Three-step immuno- This barrier is present in the choroid plexus (CP) of brain peroxidase detection was performed on paraffin sections ventricles. CP consists of highly vascularized stromal core after antigen retrieval and images were taken using Leica with fenestrated capillaries and cuboidal cells covering the

S22 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). ventricular side. Epiplexal Kolmer cells (KC) attached 40 on the surface of the cuboidal cells are involved in neu- roimmunological regulations. The aim of our study was Expression of some markers of stem cells and to investigate the changes of CP after peripheral nerve tissue-specific progenitors in human postnatal injury. For this purpose we used intravenously injected fluorescent conjugated dextran FluoroEmerald (FE) with skin different time of circulation. Alena Kvasilova, Barbora Dvorankova, Eliska Drobna Krejci, Methods. Twenty Wistar rats (male, 250-300 g) were Karel Smetana, Milos Grim anaesthetized with a mixture of ketamine and xylazine and unilateral chronic constriction injury (CCI) of the Institute of Anatomy, First Faculty of Medicine, Charles University sciatic nerve was performed. CCI operated rats were in Prague, Czech Republic left to survive for 3 days (3D) (n=8) and 21 days (21D) (n=8). Four naive rats were used as control. After time Introduction. This study is a part of a project focused of CCI survival the FE was intravenously applied after on cellular and molecular characteristics of neonatal anesthesia to the left external jugular vein. Time of FE with the respect to its property of scarless circulation was 5 h (n=4) and 18 h (n=4) in each period after reconstructive surgery of the lip cleft. of CCI survival. Then, the rats were deeply anaesthetized, We hypothesize that early postnatal skin has a greater perfused transcardially by Zamboni’s fixative and the potential to regenerate because it is not yet fully differ- brains were removed and fixed for three days. Coronal entiated. We therefore decided to asses whether its cells cryostat sections (20 μm) were cut through the lateral express markers of various tissue-specific progenitors, and and third ventricles. Distribution of FE and simultaneous adult stem cells. immunohistochemical detection of resident (ED2) and Aims. The aim of this study is to determine the expres- activated (ED1) macrophages, antigen presenting cells sion of Sox2, Nanog, Oct4 and nestin in human postnatal (APC; MHC-II), T-cells (OX-52) and dendritic cells (OX- skin of different ages. 42) were analyzed using . Methods. Histological sections of the residual tissue Results. FE particles were found inside the cuboidal removed during the lip cleft surgery were processed for cells, the immune stromal cells and the KC. Statistically immunohistochemical detection of Sox2, Oct4, Nanog significant increase of number of FE+ cells was found and nestin. Merkel cells were detected by anti- in CP after 3 and 21 days of CCI and FE circulation for 7/8. Samples of adult human skin and mouse skin from 18, but not for 5hours. Most of FE+ cells were found in neonatal and adult animals were used for comparison of CP 21 days from CCI and circulation for 18 h. FE parti- findings. cles were detected inside stromal cells of the CP which Results. Sox2 is expressed in the cells of , displayed immunophenotypes of activated macrophages in the dermal papilla of follicles, in the perifollicular (ED1+) and antigen presenting cells (MHC-II+). KC with- cells and in scattered cells of the . Some perifoli- out FE particles were immunostained for ED1, MHC-II cular cells expres Oct4 and filaments of Nestin suggesting and OX42. Number of OX42+ cells in CP increased with continuing process of follicle differentiation. Sox2 and duration of nerve compression. FE positive KC were si- Nanog positivity was observed in all Merkel cells. This multaneously positive for ED2 and OX52, respectively. finding is difficult to interpret because Merkel cells are Conclusion. CP responds to the chronic peripheral differentiated cells and are considered to be postmitotic. nerve damage with presence of FE+ cells in the stroma. Conclusion. Transcription factors Sox2, Nanog and Therefore, we assume that CP is a possible way how pro- Oct4 involved in maintaining phenotype and inflammatory molecules from peripheral nerve injury pluripotency in the embryonic and adult progenitors were could spread into the central nervous system. These CP demonstrated in the cells of human postnatal skin. It is changes occur with prolonged peripheral nerve damage likely that this expression is an important factor for - by contrast to dorsal root ganglia. We also demonstrated less skin healing. 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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41 of early mouse embryos. Using microarray essays, novel genes expressed in restricted patterns in dental epithe- Microarray analysis of mandible regionalization lium and mesenchyme of incisor and molar areas were during mouse development determined in mouse and also the known genes, which have not been connected to odontogenesis yet. Next, new Petra Langovaa,b, Simona Balkovaa, Marcela Buchtovaa,c candidate genes will be analyzed by whole mount in situ a hybridization to uncover their exact expression pattern Institute of Animal Physiology and Genetics, v.v.i., Academy of during odontogenesis. Sciences of the Czech Republic, Brno, Czech Republic bDepartment of Stomatology, Faculty of Medicine, Masaryk University, Brno, Czech Republic cDepartment of Experimental Biology, Faculty of Science, Masaryk ACKNOWLEDGEMENTS University, Brno, Czech Republic This work was supported by the Grant Agency of the Aims. The mammalian teeth function to capture and Czech Republic (14-37368G) and institutional support chew food and they have become specialized for these (RVO:67985904). purposes producing a complex and distinctive dentition consisting of unicuspid and multicuspid teeth. To achieve this specialization, tooth development is characterized 42 by a complex series of reciprocal interactions that occur Teaching practical histology with digital (virtual) between the epithelium and the underlying cranial neural crest-derived mesenchyme that regulates tooth morpho- slides genesis and differentiation at molecular level. Up to now Radka Lichnovska, Drahomira Krajci, Bela Erdosova, several homeobox genes are known to be expressed in Dimitrolos Krajci specific pattern of mouse mandible that specifies the inci- sor and molar area. However, there are still many unsolved Department of Histology and Embryology, Faculty of Medicine questions how complex signaling between the epithelium and Dentistry, Palacky University Olomouc, Czech Republic and mesenchyme is regulated during initial development of different classes of teeth. To reveal these complex Aims. We created new format of practical teaching molecular bases of tooth development, a polygenetic ap- of histology using digital scans of classical glass histol- proach is needed. Here, we aim to uncover signaling cas- ogy slides. These virtual slides enable students to study cades between the oral epithelium and mesenchyme along structures of tissues and organs on high quality, variably the jaw that underlay heterodont dentition in mammals. magnified images simulating on their personal computers Methods. Tissues were collected from the rostral and microscope-like examination of structures. The core of caudal areas of the lower jaw at E11.5 and E12.5. Total this e-learning system is our new database of histology RNA was send to Institute of Molecular Genetics of the practical. ASCR, where hybridization and scanning of array chips Methods. The database of histology practical is based was performed. GeneChips Mouse Genome 430A 2.0 on the format of MS Excel document. It is available in Arrays (Affymetrix) were used for hybridization (3 bio- two identical language versions, Czech and English. After logical replicates for each time point and each area of in- logging into a welcome desktop screen, students are hy- terest). The evaluation and statistic analysis were made in perlinked into the content page of the histology practical program Gene Spring GX (Agilent Technologies). Three database. Each topic of practical session (24 themes in levels of selection were used during investigation: 1) if total) systematically deals with General Histology and there is present or marginal expression level in at least Microscopic Anatomy of various body systems including two samples; 2) one way ANOVA (P=0.05) followed by notes on development of various organs. Each practical post-hoc testing; 3) if there is at least 1.5 fold change session contains a set of virtual slides with slide proper- versus control. These methods allowed us to generate a ties, keywords, file size information and overview pictures high-confidence list of genes upregulated or downregu- of virtual slides. Additional supporting documents in pdf lated in early dentinogenous tissue during initiation of and ppsx format are also available for each of the histol- odontogenesis. ogy topic. Results. The rostral area of the lower jaw was charac- Results. A guide to the practical session includes de- terized by high expression of Alx3, Bmp4, Msx1, Msx2, scription and labelled micrographic visualization of all Pax3 and Satb2. In the caudal area, Fgf8, Ina, Neurod1 virtual slides available in the session. This information is and Neurog1 were detected. Moreover, several genes such supplemented with didactic tools like control questions, as Ankrd15, Asb4, Dlg2, Dpep1, Rerg1, Tmem16A have not schematic pictures with legends and references to external been linked to or teeth develop- bibliographic and internet resources or quizzes. Teacher’s ment yet. presentation for pre-lab session contains micrographs Conclusion. We applied microarray technology to and schematic drawings of slides that are introduced to decipher differences in molecular regulations during ini- students at the beginning of the practical session with tiation of tooth in the incisiform and molariform areas teacher’s explanation and diagnostic notes. A document

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– presentation of electron micrographs – briefs students Articulate Quizmaker 13 software. This software has a with basic information about ultrastructure features of selective option to shuffle sequences of questions and specific types of cells in body systems. A folder of mov- also to shuffle all distracters randomly on monitors of ies, animations and embryology notes guides students student’s PCs. through the difficult visualization of 3D shapes of devel- Results. The long-term application of E-learning for- oping organs (ossification process, tooth eruption, muscle mat brought several benefits to our teaching experience. contraction etc.). The uniform set of virtual slides of the same quality used During their own study of virtual slides, students can for all students prevented loss of this valuable material. copy to clipboard selected areas of virtual slides viewed in Supporting documents were suitable materials for later the Olyvia, (OLYMPUS) viewer, and paste them directly revisions and self-study. New model of teaching histology into their own self-prepared pptx presentation for later supports active student’s approach and their engagement revisions. in group activities on digital slides. This helps with ever-in- Conclusion. The e-learning format of histology prac- creasing numbers of students at our Faculty of Medicine. tical based on virtual slides proves to be a modern and New teaching format also fulfils student’s expectations didactically efficient method of teaching histology to to use innovative technologies (PC, laptops and tablets) medical students. It standardizes the set of histology slides during their studies. Access to images anywhere and at and gives all students in a large teaching group equal op- any time was provided through the external login to the portunity to see the same high quality slides in practical database of virtual slides which correspond to those in sessions. Majority of students evaluated positively the practical sessions. This open image database contains use of virtual slides, as they allowed them to study and short annotations of typical structures that are important also to discuss various details of cells and tissues clearly for identification of slides. Objective evaluation and quick at various magnifications. A live demonstration of this procedure of examination of formative and summative system will be available during the Congress hours at a in-course quizzes are important benefits during practi- scheduled time. 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 ACKNOWLEDGEMENTS their digital form rather than to use classical microscopes that were also available. This project was supported by ESF-OPVK grant n. Conclusion. The new e-learning format proved to be a CZ.1.07/2.2.00/28.0089 in years 2012-2014. 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 43 quality of presented slides and also from a straightfor- Impact of E-learning format using virtual ward and easy personal communication with students in the class when personal guidance and explanation was microscopy in practical histology sessions needed. PC-based class also provides an easy environment Radka Lichnovska, Drahomira Krajci, Bela Erdosova, for computerized testing of student’s practical knowledge. Dimitrolos Krajci Most of the mentioned drawbacks can be prevented by proper organization of practical sessions. Department of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic ACKNOWLEDGEMENTS Aims. In order to use virtual slides successfully in histology sessions, we have developed our own system This communication was supported by ESF-OPVK of application of virtual slides in a classroom equipped grant n. CZ.1.07/2.2.00/28.0089 in years 2012 – 2014. 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

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44 of the liver tissue and can have a protective effect on NAFLD development. The effect of food restriction on the liver morphology: experimental study on the chicken model ACKNOWLEGDEMENTS Peter Makovickya, Eva Tumovab, Pavol Makovickyc, The work was institutionally supported by RVO Kvetoslava Rimarovad 68378050, LM2011032 (MEYS) and the project BIOCEV – Biotechnology and Biomedicine Centre aCzech Centre for Phenogenomics (BIOCEV), Institute of Molecular of the Academy of Sciences and Charles University Genetics, Department of Transgenic Models of Diseases, ASCR, (CZ.1.05/1.1.00/02.0109; European Regional v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic Development Fund) to R.S., and by NAZV QJ 15100192. 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 45 cDepartment of Biology, Pedagogical Faculty, Selye Janos University, Bratislavska 3322, 945 01 Komarno, Slovak Republic Difference in description of topographic position dDepartment of Public Health and Hygiene, Faculty of Medicine, P. of communicating branches in different medical J. Safarik University in Kosice, Trieda SNP 1, 040 11 Kosice, Slovak Republic literatures a a a Aims. Non-alcoholic fatty liver disease (NAFLD) is Stanislav Mateffy , Janka Vecanova , Lenka Tomasova , a clinicopathological syndrome, characterized by simple Natalia Hvizdosovaa, Mirela Rozpravkovab, Jana Pribulovac, steatosis with or without inflammation, steatohepatitis Maria Matusevskad with or without fibrosis or cirrhosis. Also classified as a an NAFLD is hepatocellular carcinoma. Factors that are Department of Anatomy, Faculty of Medicine, P. J. Safarik involved in mechanisms of NAFLD development are the University in Kosice, Slovak Republic bDepartment of Orthodontics, Faculty of Medicine and Dentistry, subject of several studies. The objective of our study was Palacky University Olomouc, Czech Republic to describe the proportion of interstitial tissue to the liver cPrivat Dental Clinic, Presov, Slovak Republic parenchyma and to measure diameter depend- dPrivat Dental Clinic, Lubotice, Slovak Republic ing on the stage of food restriction using an experimental chicken model. Aims. It is generally known that the communicating Methods. This experiment was performed on 48 chick- branches (CBs) connect the sympathetic trunk with the ens, which were divided into three groups. The control peripheral nervous system, and the sympathetic neurons group (C) was fed ad libitum until the end of the experi- send their axons via CBs. The topographic position of ment. The second group (ADL1) was fed 80% ADL in these structures is very important for a neurosurgical the 2nd-week and the last group (ADL2) was fed 65% treatment of specific diseases connected with hyperfunc- ADL in the 2nd-week of the experiment and after this tion of the sympathetic innervation, how is hyperhidro- one week were all chickens fed ad libitum until the end sis, Raynaud's phenomenon, and the others. of the experiment. The experiment lasted five weeks and Methods. 3 human donor’s cadavers were used for at the end, 16 chickens were selected from each group for the study of the topographic position of the sympathetic histopathological study. The liver samples were fixed in trunk and the CBs. 10% formalin solution, standardly processed and embed- Results. The difference in description of topographic ded in paraffin blocks. Serial sections were stained with position of the CBs was found in anatomical and clinical hematoxylin-eosin and with Sirius red kit. literatures used in Eastern block countries in compari- Results. Mild liver steatosis and liver inflammation son to those used in Western countries. In English and was visible in C group. The weight of the liver was in C German anatomical books, the position of the white CB, group (46.98 g), ADL1 group (45.24 g) and ADL2 group known as a pleural branch, is described more distally than (46.83 g). The average hepatocyte diameter was measured the grey CB. The pleural branch carrying preganglionic at 49.93 μm (C-group), 47.86 μm (ADL1 group) and fibres is visible through the parietal pleura and is easily ac- 46.31 μm (ADL2 group), (P < 0.001). The proportional cessible for a surgical operation, and the grey CB contain- content of interstitial tissue in relation to the liver paren- ing postganglionic fibres is located behind the ganglion of chyma was measured at 8.20% (C group), 6.60% (ADL1 sympathetic trunk. On the other hand, in the anatomical group) and 6.29% (ADL2 group), (P < 0.001). books used in Slovak and Czech Republics, and in many Conclusion. Results shows that food restriction cor- other former socialistic countries, where was dominant relates with changes in liver morphology. An increase in the influence of soviet anatomists, there is the just op- the steatosis histologic grade is associated with inflam- posite description of topographic position of the same matory changes, with hepatocyte diameter and with a grey and white CBs. The same difference was found not proportion of interstitial tissue to the liver parenchyma. only between the anatomical books, but between clinical Early restriction is not associated with the development of books and scientific journals both parts of world as well.

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Conclusion. Proper knowledge of position of the CBs Conclusion. This research could help to explain is very important for good diagnostic and therapeutic changes in structure of periodontal tissue depending to processes in a clinical practice. It can be resumed, that the pathological state. This knowledge can positively in- unification of those information in the anatomical and fluence the therapy of periodontitis. It should prevent consequently clinical literatures would demand more at- the occurrence of periodontal tissue damage leaving out tention and observations in several theoretical and clinical excessive or undesirable procedures for the patient. disciplines in future.

46 ACKNOWLEDGEMENTS Anatomy and pathological anatomy of I want to cordially thank of Department of Anatomy, Faculty of Medicine, University of Pavol Jozef Safarik periodontium depending to the state of oral in Kosice and all my colleagues for cooperation on this health design. Maria Matusevskaa, Jana Pribulovab, Mirela Rozpravkovac, Lubomír Redajd, Janka Ohlasovae, Stanislav Mateffyf, 47 Kvetuse Lovasovaf, Darina Kluchovaf Transgenic rat model of Huntington’s disease aPrivat Dental Clinic HYG – DENT, Presov Slovak Republic – a useful tool for study of the development of bPrivat Dental Clinic, DENT-PREV, Presov, Slovak Republic progressive neuronal degeneration cDepartment of Orthodontics, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic Yvona Mazurovaa, Ales Bezroukb bPrivate Dental Clinic, Studio Smile, Kosice, Slovak Republic ePrivat Dental Clinic, Kosice, Slovak Republic aDepartment of Histology and Embryology, Faculty of Medicine fDepartment of Anatomy, Faculty of Medicine, P. J. Safarik in Hradec Kralove, Charles University in Prague, Hradec Kralove, University in Kosice, Slovak Republic Czech Republic aDepartment of Medical Biophysics, Faculty of Medicine in Hradec Aims. A very few of dentists are interested in the treat- Kralove, Charles University in Prague, Hradec Kralove, Czech ment of periodontitis because the lack of information Republic about the periodontium in the Slovak Republic. The basis is the comparison of physiological anatomy and patho- Aims. Following up on our previous findings in rats logical anatomy of the periodontium pointing out the ap- transgenic for Huntington’s disease (tgHD51 CAG rats), propriate and inappropriate approaches to the process of we focused on the most significant change in the course treatment. The main aim is to investigate and describe the of the disease, i.e. the degeneration of striatal neurons. physiological anatomy of periodontium in four groups: Methods. Homozygous tgHD51 CAG rats and their healthy tooth, the tooth with in the periapical age-matched wild-type (wt) littermates 2-3, 6, 12, 18 and area, the healthy tooth with periodontitis and the infected 22-24 months old were compared. Immunohistochemical tooth with periodontitis. and immunofluorescent detections using the array of Methods. Recognition of changes in the anatomy of primary antibodies (primarily anti-NeuN, anti-beta-III- periodontium accordingly upon the state of oral health. tubulin, anti-MAP2, anti-Iba1, anti-polyQ-huntingtin) The extracted teeth based on their condition are appoint- were made. Quantitative analysis of the median diameter ed to the corresponding group according to periodontal of neuronal nuclei labelled with NeuN antibody (50 in- spaces performed under X – ray examination. dependent measurements) was made on serial paraffin The state of , periodontal , dentine sections in a sequence of 3 slices, each 7 μm thick, in the tubules and dental pulp by histological examination will distance of 15 sections (i.e. approximately 105 μm) and be examined. The main reason is to compare changes followed by statistical analysis. in the anatomy of root cementum of teeth, periodontal Results. Neurodegenerative process (NDP) of HD ligaments, cemento-enamel junction and the area of the phenotype primarily manifests by gradual decrease in root apex. size of neuronal bodies and nuclei (with maintenance Results. Assuming that anatomy of tooth with peri- of nucleo-cytoplasmic rate) and finally, although slowly, odontal infection in the periapical area will influence only results in the death of affected neurons, ultimately scav- the periapical area. In the third group, expected changes enged by microglia. Our morphological findings were are in the area of periodontal ligament spaces, in the al- supplemented by quantitative analysis. Also the propor- veolar bone and in the cementum of the tooth root. In the tion of age-related changes in this process was assessed. last group, assumed changes are in the area of periodontal The quantitative analysis proved that the rate of neuronal ligament spaces, in the alveolar bone and in the cemen- degeneration reaches maximum at the end of the first tum of tooth root, also including changes in the area of year of animal’s age, and then, in following 12 months, dentine and dental pulp too. it proceeds rather slowly. It is potentiated with age-relat- ed changes particularly in the oldest animals. However,

S27 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). neuronal degeneration in wt rats can be attributed only to fluorescent microscope CD68 positive cells were present the debit of the aging process. Indeed, the reduction in a in both PD and positive screening patients. volume of neuropil and alterations in a character of syn- Conclusion. Our results showing presence of apses are at least of the same importance. Additionally, α-synuclein aggregates in colonic nerve fibres and mono- detection of polyglutamine deposits using polyQ-hunting- cyte- cell population in healthy patients at tin provided interesting findings which complete histo- risk for development of PD, support the theory of colonic pathological picture of the progression of NDP of HD α-synuclein biopsies as potential premotor biomarker of phenotype. PD and can serve as a model for testing this hypothesis. 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 ACKNOWLEDGEMENTS progression of NDP in tgHD51 rats. These findings cor- respond to the course of HD in , in which Supported by VEGA grant 1/0024/14 and APVV-14- also behavioral and motor changes precede the loss of 0415. striatal neurons. 49 ACKNOWLEDGEMENTS Myoid cells of thymus in question of origin and

Supported by the Charles University research program function PRVOUK P37/06 and P37/09. Veronika Mestanovaa, Ivan Vargab, Marian Bencatc, Marian Adamkova 48 aDepartment of Histology and Embryology, Jessenius Faculty of Immunohistochemical visualization of α-synuclein Medicine in Martin, Comenius University in Bratislava, Slovak Republic in colonic lamina propria mucosae bDepartment of Histology and Embryology, Faculty of Medicine, a b b Comenius University in Bratislava, Slovak Republic Eva Mechirova , Zuzana Gdovinova , Matej Skorvanek , cAlpha Medical, Patologia, s.r.o., Martin, Slovak Republic Iveta Domorakovaa, Marianna Dankovaa aDepartment of Histology and Embryology, Faculty of Medicine, P. Aims. Complex developing process of pro-T-lympho- J. Safarik University in Kosice, Slovak Republic cytes requires unique cellular and excessive antigenic de- bDepartment of Neurology, Faculty of Medicine, P. J. Safarik mands accomplished in thymus. This central lymphoid University in Kosice and organ comprises of heterogenous cell populations that L. Pasteur University Hospital Kosice, Slovak Republic reflect thymus complicated embryological development, thus endo-ectodermal origin with direct participation of Aims. There is an urgent need for biomarkers of pluripotent cells migrating from transient embryonic neu- Parkinson’s disease (PD). Routine colonic biopsies allow ral crest. Present research was focused on a rare popula- the demonstration of α-synuclein pathology in colonic mu- tion of thymic myoid cells (MC) which, despite their strict cosa and in living PD patients. Localization of evolutionary conservation, have not revealed their neither α-synuclein aggregation is typical not only in the enteric origin nor the physiological role. nervous system (ENS), but also in other cells frequently Methods. Using the 49 formalin-fixed paraffin-embed- seen in the digestive tract (macrophages, plasma cells). ded thymic tissues obtained from corrective cardiovascu- Methods. Four healthy control, 4 PD patients and 9 lar surgery, immunohistochemical analysis was performed healthy patients with positive screening for PD risk factors on monoclonal antibodies palette of , desmin and underwent routine colonoscopy and the bioptic material MyoD1 specific for striated muscle expressed by from ascendent and was analysed by immu- myoid cells as well as bcl2, p53 and survivin – proteins nohistochemistry using antibodies against α-synuclein and regulating the apoptotic process. phosphorylated α-synuclein. For visualization of CD68 Results. Semiquantitative analysis proved the predomi- positive cell population we used anti human macrophage nant location of myoid cells in the medullary compart- (CD68) monoclonal antibody and secondary anti-mouse ment. Concerning the embryological origin, we detected fluorescent antibody Alexa Fluor 488. decrease in number of MC in tissue from congenital heart Results. α-synuclein positive aggregates were detect- defects which etiopathogenesis is closely related with dis- ed in nerve fibres of lamina propria mucosae of all PD rupted migration of neural crest cells. Considering the patients and 6 out of 9 patients with positive screening. physiological function, we demonstrated inverse propor- Strong α- synuclein positive monocyte-macrophage cell tion in number of MC in correlation with the apoptotic population was detected in PD and positive screening markers. patients. Phosphorylated α-synuclein was present in PD, Conclusion. To summarize our results, myoid cells rep- positive screening and also healthy control patients. In the resent a scarce population localised in thymic medulla

S28 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). and in the proximity of cortico-medullary junction, with 51 the probable physiological counteraction on regulation of programmed cell death during selection processes under- Remodelling of the atrioventricular canal gone by immunocompetence gaining T- lymphocytes. In a myocardium determines atrioventricular case of corrupted development of myoid cells population and subsequent defective selection processes regulation, conduction patterns the clinical manifestations may arise in form of immuno- Ondrej Nankaa, Rebecca Vicente-Steijnb, Nico A Blomc, deficiency disorders or autoimmune diseases. Monique RM Jongbloedb, David Sedmeraa,d 50 aInstitute of Anatomy, 1st Faculty of Medicine, Charles University in Prague, Czech Republic Application of double staining method for fetal bDepartment of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands specimens cDepartment of Pediatric Cardiology, Leiden University Medical a b c Center, Leiden, The Netherlands Maria Miklosov , Robert Dankovcik , Robert Herich dInstitute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic aDepartment of Anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic Aims. Based on our previous study showing acceler- b nd 2 Department of Gynecology and Obstetrics, Faculty of ated conversion of ventricular activation sequence during Medicine, P. J. Safarik University Kosice and L. Pasteur University hypoxic incubation correlating with increased apoptosis Hospital Kosice, Slovak Republic cInstitute of Pathological Anatomy, University of Veterinary in the atrioventricular canal (AVC), we hypothesized that Medicine and Pharmacy, Kosice, Slovak Republic this slight increase in apoptosis could lead to an acceler- ated pruning of myocardial atrioventricular connections, Aims. The improved double-staining technique to dem- which would manifest as earlier appearance of mature onstrate cartilage and bone can now be used in teratologi- apex-to-base activation patterns. cal and toxicological studies as well as in morphological Methods. To test this hypothesis, apoptosis was studies of large specimens. The principle used here is the blocked with a caspase inhibitor (zVAD-fmk) by intraperi- affinity of Alizarin red S to bind with calcium of bones, cardial injection in chick embryos at embryonic day (ED) while Alcian blue reacts with acid mucopolysacharides in 4. The epicardial contribution to AVC remodeling was cartilage. The end product of this technique is a specimen studied by performing epicardial outgrowth inhibitions in which the ossified parts of bone are stained red and – microsurgical epicardial ablation atED3. Ventricular cartilages are stained blue. This staining allows to observe activation was studied by optical mapping and apopto- primary ossification centers, transition cartilage to bone, sis rates were quantified by Lysotracker Red and Active and the related abnormalities. Caspase-3 immunofluorescence staining. Methods. We examined large fetuses and neonates. Results. Apoptosis measurements and quantification Fresh, eviscerated specimens are incubated with Alcian in zVAD treated hearts shows decreased apoptosis in AVC Blue 8 GS and Alizarin Red S. The cartilage stains blue myocardium atED7. zVAD-treated and epicardial ablat- in color and bones are red, both embedded in the trans- ed hearts showed increased frequency of immature ven- parent (cleared) muscle. Before examination or storage, tricular activation patterns. In 5 of the 21 zVAD-treated the double stained specimens must be cleared in graded hearts we observed activation originating from the right concentration arrays of glycerin. The specimens can be AVC. This pattern was not observed in neither control stored in pure glycerin. nor epicardial ablation hearts. The epicardial ablation Results and Conclusion. Although fragile not ideal hearts showed activation originating from the left AVC. for mounting, this type of specimen is excellent for de- Ventricular activation time in Base-to-Apex activation pat- tailed scientific skeletal studies in 3D, and also may be terns in the zVAD-treated hearts was significantly longer used in multiple settings in university teaching curricula. than in controls. Distinguishing of colors is very good. Fresh, formalde- Conclusion. AVC maturation requires apoptosis in hyde-fixed or alcohol-fixed specimens from various spe- the AVC myocardium and epicardium-derived ingrowth. cies worked well. Disruption of proper AVC morphogenesis can lead to persistence of accessory atrioventricular connections, which form a morphological substrate for ventricular ACKNOWLEDGEMENTS pre-excitation.

Supported by the grant VEGA 1/0678/15. ACKNOWLEDGEMENTS

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

S29 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1).

52 53 Amygdalar volume in narcolepsy type 1 and type 2 A role of c-Myb in ossification and in controls Veronika Oralovaa,b, Eva Matalovaa,c, Lucie Knopfovab, Veronika Nemcovaa, Jan Krasenskyb, Pavel Petrovickya, Eva Svandovaa, Jan Smardab, Marcela Buchtovaa,b b b c Manuela Vaneckova , Zdenek Seidl , Jitka Buskova , aInstitute of Animal Physiology and Genetics, v.v.i., Academy of Karel Sonkac Sciences of the Czech Republic, Brno, Czech Republic bDepartment of Experimental Biology, Faculty of Science, a Institute of Anatomy, 1st Faculty of Medicine, Charles University Masaryk University, Brno, Czech Republic in Prague, Czech Republic cDepartment of Physiology, University of Veterinary and bDepartment of Radiology, 1st Faculty of Medicine, Charles Pharmaceutical Sciences, Brno, Czech Republic University in Prague, Czech Republic c st Department of Neurology, 1 Faculty of Medicine, Charles Aims. Bone formation can proceed by intramem- University in Prague, Czech Republic branous (direct differentiation of mesenchymal cells into ) or endochondral ossification (gradual Aims. Amygdala has a key role in detection and pro- replacement of cartilage by bone during development). cessing of emotional signals. Emotions trigger cataplexy Developmental processes are influenced by several mol- in narcolepsy type 1 (N1). Abnormal amygdalar reactiv- ecules pathways including FGF, HH, BMP and WNT as ity on humorous stimuli on fMRI was described in N1. well as series of transcription factors. c-Myb is one of tran- Recently several microstructural changes were noted in scription factors involved in proliferation, differentiation, amygdala in N1 patients but not in narcolepsy type 2 cell death and survival (Oh et Reddy, 1999). Previously, (N2) subjects. Our preliminary results showed a reduced c-Myb was linked with undifferentiated state but recent volume of amygdala in N1 patients, thus we decided to experiments suggested its role in differentiation of chon- compare amygdalar volume in N1 and N2 patients and drocytes and odontoblats (Matalova et al., 2011; Bhattarai healthy subjects using more sophisticated methods. et al., 2013; Lungova et al. 2012). Here, we aim to examine Methods. Twenty one patients with N1, 45 patients a role of c-Myb in osteogenic developmental processes. with N2 and 37 healthy controls participated in the study. Methods. Mesenchymal cells were isolated from fore- They were examined in the same MR scanner 1.5 T, the limbs of CD1 mouse embryos at E12.0 and spotted in measurement was carried out as T1W 3D image with a high density to obtain primary micromass cultures. Cells slice thickness of 1.0/0 mm.The amygdalar volume was as- were transfected with the c-myb coding vector (pcDNA3- sessed manually in ScanView.cz program (man.) and au- mcMYB) or control vector (pcDNA-3). Gene expression tomatically by brain segmentation using Freesurfer (FS). changes were analyzed by PCR Array designed for osteo- Results. The average left amygdala volume was man. genic markers. 1.44 cm3 (FS: 1.46 cm3) in N1, man. 1.45 cm3 (FS: 1.55 Results. Overexpression of c-myb increased expres- cm3) in N2 and man. 1.45 cm3 (FS: 1.53 cm3) in controls. sion of several cartilage and bone markers such as Bgn, The average right amygdala volume was man. 1.41 cm3 Bmpr1a, Smad1, Msx1, Col1a2, Fgfr2, Tgfb3 following (FS: 1.54 cm3) in N1, man. 1.44 cm3 (FS: 1.62 cm3) in 24h after treatment. In addition, a strong increase of NF- N2 and man. 1.4 cm3 (FS: 1.68cm3) in controls. No dif- κb1, Icam1, Vcam1 and Vegfa expression was evident at ferences were found in amygdalar absolute volumes or in this early time point. NF-κb is known to control the dif- amygdalar volumes related to whole brain volumes. ferentiation and activity of the major skeletal cell types Conclusion. Automatic nor manual amygdalar volu- – , osteoblasts, and mometry did not found any differences betweenN1 and (Novack, 2011). Icam1 and Vcam1, belongs to cell ad- N2 patients and controls. 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 ACKNOWLEDGEMENTS expressed by osteoblastic cells in vitro (Maes et al., 2002). To further reveal the role of the c-Myb transcription factor Supported by the grant IGA NT 13238-4/2012. 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,

S30 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). growth factors and their receptors. Next, we intend to ex- by a program Cell profiler (open-source) from the images amine the effect of c-myb modulation on cell line MC3T3, of nuclei. Cell area in μm² was defined from the images which was established from newborn mouse calvaria to of actin also using Cell profiler. Metabolic confirm our findings. 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 ACKNOWLEDGEMENTS (2 h) of both cells types to Ti material was increased. Additionally, elevated proliferation and metabolic activity This study was supported by the Centre of Excellence of SAOS-2 cells after 48 h indicated that both original Ti of the Grant Agency of the Czech Republic (14-37368G) material S and mechanical improved Ti materials M and and institutional support (RVO: 67985904). 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 54 both cell types was lower in comparison to PS control The biological evaluation of ultra-fine titanium at both time points. Different cell morphology and ar- rangement and shape of focal adhesions confirmed that with improved mechanical strength for tissue surface treatment has an influence on cell attachment engineering and spreading.

a a b Conclusion. Our results revealed that the novel Ti ma- Lucie Ostrovska , Lucie Vistejnova , Jan Dzugan , terials M and Z were equivalent to the original Ti material Peter Slamab, Tomas Kubinab, Egor Ukraintsevc, S, thus confirming their suitability for and mes- Dana Kubiesd, Milena Kralickovaa, enchymal stem cell growth and proliferation. The present Marie Hubalek Kalbacovaa,e study proved the cytocompatibility of the novel forms of Ti with superior mechanical properties and revealed their aBiomedical Center, Medical Faculty in Pilsen, Charles University potential for designing improved biomedical implants. in Prague, Pilsen, Czech Republic bComtes FHT a.s., Dobrany, Czech Republic cInstitute of Physics, The Academy of Sciences of the Czech ACKNOWLEDGEMENTS Republic, Prague, Czech Republic dInstitute of Macromolecular Chemistry, The Academy of Sciences of the Czech Republic, Prague, Czech Republic This work was supported by the projects eInstitute of Inherited Metabolic Disorders, 1st Faculty of Medicine, CZ.1.05/2.1.00/03.0076, CZ.1.05/2.1.00/03 and Charles University in Prague, Czech Republic CZ.1.05/1.1.00/02.0109 of the European Regional Development Fund, project PRVOUK-P24/LF1/3 of First Aims. Titanium, the gold standard for implant materi- Faculty of Medicine and project PRVOUK P36 of Faculty als, is distinguished by its exceptional biocompatibility; of Medicine in Pilsen and the program of support and however, from a long-term perspective, titanium still lacks co-operation of companies and universities in the Pilsen sufficient loading strength. The aim of our work was to region called “Innovation Vouchers”. 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 55 an enhanced mechanical strength. Cloning of synthetic intron into coding sequence Methods. Original Ti material (marked as S) was pro- cessed by the ECAP-Conform technique and two novel of red fluorescent gene for confirmatory types of Ti material (marked as M, Z) with enhanced expression of micro RNA cloned within the intron mechanical resistance were obtained. These ultra-fine grained Ti samples with grain sizes ≤ 1 μm (Z), 4.6 μm Rishikaysh Pisal, Hana Hrebikova, Jana Chvatalova, (M) and 30 μm (S) and mechanically treated surfaces Daniel Diaz Garcia, David Kunke, Tomas Soukup, (grinding, polishing) were characterized using atomic Jaroslav Mokry force microscopy, and their surface wettability was as- sessed. Osteoblast cell line (SAOS-2) and human primary Department of Histology and Embryology, Faculty of Medicine in mesenchymal stem cells (hMSC) were used as an appro- Hradec Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic priate in vitro model. SAOS-2 and hMSC were seeded on Ti samples and polystyrene culture plastic (PS control) Aims. To clone a synthetic intron into the gene encod- in concentrations 15 000 cells/cm2 and 10 000 cells/ ing red fluorescent protein derived from Discosoma sp. cm2, respectively. After 2 and 48 h of incubation cells This system will act as an easy tool to confirm the expres- were fixed and visualized by immunofluorescent staining sion and processing of micro RNA (miR) cloned within (nuclei-DAPI, actin-phalloidin-Alexa 488 and vinculin- the intron by simple visualization of cells using fluores- primary antibody against vinculin-secundary antibody cence microscopy after transduction or transfection of the Alexa 568). The number of cells on cm2 was estimated target cells with the construct. To determine the course

S31 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). and efficiency of reprogramming by cloning mir 302/367 endodontic treatment is a hermetic sealing of the entire cluster into the intronic region of the construct. channel system. Treatment success depends on varying Method. Sequencing of the construct was performed degrees of anatomical complexity. Therefore, knowledge to check any deviations in the sequence. Proper process- of anatomical and morphological diversity plays a major ing and expression of individual miR was confirmed by us- role in the success of therapy. ing Taqman probes specific to a particular miR’s. Human Methods. The first upper premolars, extracted essen- fibroblasts were reprogrammed using mir 302/367 cluster tially because orthodontics treatment, was dipped in 3% cloned into the construct. Pluripotency was confirmed by hydrogen peroxide solution for 72 hours and after that detecting expression of pluripotency markers Oct4, Sox2, were flushed with clean water. The teeth were bundled Alkaline phosphatase, TRA 1-81, SSEA. into Lucasteric bags and sterilized in an autoclave. On Results. Sequencing results confirmed successful clon- these premade teeth we will further study the external and ing of intron within the red fluorescent gene. Three to internal anatomy of the tooth. four percent of the cells expressing the red fluorescence Results. We assume that about 70% of the extracted successfully dedifferentiated to induced pluripotent stem first upper premolars will have two roots with two root cells (iPSC’s). canals. 25% will have one root with two root canals. The Conclusion. This system of miR expression assures remaining percentage will consist of different morphologi- the expression of the miR with the advantage of tracking cal traits like premolars with three roots and with three the outcome in this case the reprogramming by compar- roots canals. We do not expect a high percentage of first ing fluorescence to the morphological change in the fi- upper jaw premolars with only one root canal. broblasts as they dedifferentiate into induced pluripotent Conclusion. Success in endodontic therapy at the first stem cells. 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. ACKNOWLEDGEMENT

This study was supported by Grant Agency of Charles ACKNOWLEDGEMENTS University in Prague (GAUK grant no. 1854214), No. SVV-2015-260179, and with European Social Fund No. Especially I want to thank all my colleagues to col- CZ.1.07/2.3.00/30.0061. laborate on the implementation of this project, especially the Department of Anatomy in Kosice. 56 57 The variation in morphology of first upper premolar Healing of soft and hard tissues of rabbit temporomandibular joint following the Jana Pribulovaa, Maria Matusevskab, Mirela Rozpravkovac, Lubomir Redajd, Jana Ohlasovae, Stanislav Mateffyf, application of stem cells Kvetuse Lovasovaf, Darina Kluchovaf Barbora Putnovaa, Marcela Buchtovaa,b, Vladimir Jeklc, d e a,d aPrivat Dental Clinic, DENT-PREV, Presov, Slovak Republic Radek Hodan , Vladimir Machon , Jan Stembirek b Privat Dental Clinic, HYG-DENT,Presov, Slovak Republic aInstitute of Animal Physiology and Genetics, v.v.i., Academy of c Departement of Orthodontics, Faculty of Medicine and Dentistry, Sciences of the Czech Republic, Brno, Czech Republic

Palacky University Olomouc, CzechRepublic bDepartment of Experimental Biology, Faculty of Science, b Private Dental Clinic, Studio Smile, Kosice, Slovak Republic Masaryk University, Brno, Czech Republic e Privat Dental Clinic, Kosice, Slovak Republic cClinic of Small Animals, University of Veterinary and f Department of Anatomy, Faculty of Medicine, P. J. Safarik Pharmaceutical Sciences Brno, Czech Republic University in Kosice, Slovak Republic dDepartment of Oral and Maxillofacial Surgery, University Aims. Perform endodontic treatment precisely and Hospital Ostrava, Czech Republic e st efficiently is based primarily on a perfect knowledge of Department of Oral and Maxillofacial Surgery, 1 Faculty of the anatomy of the tooth and its variations. If we don’t Medicine, Charles University in Prague, Czech Republic know anatomy we may cause irreversible damage to the Aims. Temporomandibular joint (TMJ) as a pair entire canal system of the tooth, which can no longer connection between processus condylaris mandibulae be maintained. The first upper premolar conceals more and temporal bone (fossa mandibularis) is characteris- anatomical and morphological traits. The number of roots tic for mammals (Bermejo et al., 1993). TMJ is one of varies and roots are usually fragile, which in the case of the areas drawing a lot of attention in human medicine curvature substantially compromises its endodontic treat- lately as the incidence of temporomandibular disorders ment. The amount of root channels varies from 1-3 with (TMD) increased along with improvement of diagnos- different anatomical complexity, not excluding the api- tic success. It was proposed that the rising incidence of cal variations, and accessory channels. The main task of TMD is connected with increased occurrence of stress in

S32 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). our population, which plays an important role in TMD ACKNOWLEDGEMENTS progress. It was estimated that 50% to 70% of human population suffers from some kind of TMD symptoms at This study was supported by the Grant Agency of the least once during the life. Pain in the facial area, earache, Czech Republic (14-37368G, 14-29273P) and institutional toothache, joint stiffness, various noises from TMJ, jaw support (RVO: 67985904). 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 58 the resources for either cartilage regeneration or surgical Anatomical variations of popliteal artery replacement of the affected joint. The main aim of our study was to evaluate the influence of stem cells on heal- – a possible case of lower limb ischaemia ing process of soft and hard tissues of TMJ. This study is Olga Rejtarovaa, Michal Kuchara, Pavel Eliasb unique in using fresh heterologous stem cells, which were not previously cultivated with any factors interfering with aDepartment of Anatomy, Faculty of Medicine in Hradec Kralove, their differentiation. Charles University in Prague, Hradec Kralove, Czech Republic b Methods. Rabbit was selected as a model animal be- Department of Radiology University Hospital in Hradec Kralove, cause of easy experimental access to joint cavity in con- Czech Republic trast to mouse and pig. We used 17 adult New Zeland white rabbits in this study. Nine animals were used as Aims. Our aim is to present bilateral abnormal branch- an experimental group, eight animals as a control group. ing of the anterior tibial artery (ATA) arising proximally Cartilaginous damage was induced surgically in the left to the popliteal muscle and passing between the muscle joint under general anaesthesia using open approach to and tibial to the upper shin. the joint. The bone defect was created using a tool with Methods. Nine cadavers and four solitary lower limbs round head. Following that, human mesenchymal stem used in 2014/2015 anatomy practical classes were ob- cells isolated from were applied into the served for possible popliteal variations. In cooperation wound for the experimental group while only saline solu- with the Department of Radiology University Hospital in tion was used in the control group. The collection of the Hradec Kralove a recent cases of anatomical claudications stem cells was performed using a special separator. The were searched for. main advantage of using adipose stem cells is their easy Results. On dissected cadavers we found a rare varia- collection causing much less distress to the patient than tion of ATA and an atypical lateral course of popliteal that of bone marrow stem cells, which are usually used. artery in close contact with plantaris muscle belly. CT Rabbits were euthanized at two time points – 11 days (6 scans presented examples of claudication of the lower animals) and 1 month (11 animals) after the treatment. limb caused by compression of PA with supernumerary Inflammatory changes and tissue response to stem belly of medial gastrocnemius muscle. cells were assessed based on macroscopic changes found Conclusion. Risk of ATA damage in knee joint surgery during the autopsy as well as on results of histopathologi- is high in the case of atypical course of this vessel. The cal analysis of serial sections stained with hematoxylin potential danger of entrapment must be considered as and eosin. For evaluation of proliferation, immunohisto- well, although it is more common for PA – be it caused chemical methods were used. by muscle abnormalities or its irregular position inside Results. In the experimental group, we found a higher the . All of these examples are presented tendency to form granulation of tissue. Due to the fact in our paper. 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, 59 we have not observed any difference in immune response Children of obese mothers in their first 18 months between experimental and control groups based on hema- toxylin and eosin staining. Jitka Riedlovaa, Marketa Paulovab, Jana Vignerovac, Conclusion. Our aim was to examine the mechanism Jan Soumard of stem cell influence on the structures in TMJ and es- a pecially to confirm an expected positive effect on bone Department of Anatomy, Third Faculty of Medicine, Charles healing. We were also interested in finding possible side University in Prague, Czech Republic bThe National Institute of Public Health, Prague, Czech Republic effects such as uncontrolled proliferation during healing cNational Center, Prague, Czech Republic process. Next, we will focus on a more detailed analysis of dNational Museum, Prague, Czech Republic inflammatory markers in soft and hard tissues of treated TMJ. Aims. 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, and dyslipidemia increases, birth weight is

S33 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). more often over 90th percentile and the probability of full 60 breastfeeding at the time of maternity hospital discharge is lower. Non-breastfed children have a higher risk of de- Anatomical changes of temporomandibular joint veloping overweight than children fully breastfed for 4 – 6 in the aging process months. Family environment contributes to the develop- ment of obesity, too. Mirela Rozpravkovaa, Lubomir Redajb, Jana Ohlasovac, The object of the study was to evaluate basic anthropo- Kvetuse Lovasovad, Maria Matusevskae, Jana Pribulovaf, metric parameters of children of obese mothers until 18 Stanislav Mateffyd, Darina Kluchovad months of age and compare them with percentile growth charts used for growth assessment of Czech children aDepartement of Orthodontics, Faculty of Medicine of Dentistry, population. Palacky University Olomouc, Czech Republic Methods. Data of 1765 children collected in 2009 – bPrivate Dental Clinic, Studio Smile, Kosice, Slovak Republic cPrivate Dental Clinic, Ohlasdent, Kosice, Slovak Republic 2010 were used for the study of obese mothers` children. d The children were divided according to BMI categories of Departement of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic mothers. Software RustCZ based on the current growth ePrivate Dental Clinic, Lubotice, Slovak Republic charts was used for finding the percentile values of length fPrivate Dental Clinic, Presov, Slovak Republic for age, weight for age, weight for length and BMI for age for all measurements of every child at all monitored ages Aims. Temporomandibular joint (TMJ) and adjacent (0, 3, 6, 12 and 18 months). Reference data are results of orofacial region () is a key point the 5th and 6th Nationwide Anthropological Survey (1991 localization of important anatomical structures and pos- and 2001). sible occurrence of pathological processes. The aim of Results. The group included 132 obese mothers (7.6 % our research is to define morphological changes of TMJ of dataset), 9 of whom with a BMI even above 40 kg/m2. in the aging process. In the natural process of aging TMJ Among the obese mothers there was a highest number of undergoes degenerative and proliferative changes. Rate apprenticed and lowest number of women with university compensation of incongruences of articular surfaces is education, only 55% non-smokers and the highest number determined by biconcave articular disc. of divorced women. 76% of obese mothers` children were Methods. In our anatomical study we used 8 human fully breastfed at the maternity hospital discharge, where- adult male and female cadaveric heads (sagittal sections) as in case of mothers with a normal BMI the percentage aged 30 to 80 years with different dental status. The ca- was 90%. Obese mothers breastfed their babies exclusively daveric material from Department of Anatomy, Faculty a month less than other women and the total average dura- of Medicine, Pavol Jozef Safarik University in Kosice was tion of breastfeeding was 6.1 months only, while others fixed in 10% formalin. were breastfed for an average of 8.5 months. Children of Results. This study focused on discus changes in obese mothers had the highest weight, BMI and weight the natural aging process. It provides the review of psy- for length during all the monitoring period and they also chophysiological concept of TMJ disorders with a pre- were the longest ones at the age of 18 months. Length dominance of stress factor. Especially highlights the of breastfeeding had no proved impact on the weight of importance of morphometric parameters of the lower jaw obese mothers` children at the age of 18 months. in Orthodontics. Orofacial Anatomy knowledge necessary Conclusion. With an increasing BMI of mothers, dura- for interpretation of cephalometric X-ray in Orthodontics tion of breastfeeding shortens and weight of the children specifies the treatment plan of patients with fixed orth- increases. However, we also found lean children among odontic appliances. Our study presents topographic par- those of obese mothers. 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- ACKNOWLEDGEMENTS ogy of the mandible and the course of mandibular canal. Conclusion. The exact determination of the anatomi- Supported by the grant IGA MZ ČR NS9974-4/2008 cal joint ratios and structures in Infratemporal Fossa is and Prvouk P02. 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 therapeutic targets. Nevertheless, we did not find any cor- relation between expression of CAIX and clinicopatho- Expression of carbonic anhydrase IX and its logical parameters. correlation with clinicopathological characteristics

in lung cancer cells ACKNOWLEDGEMENTS Silvia Rybarova, Ingrid Hodorova, Jozef Mihalik, Janka Vecanova We thank Mgr. Ivana Čigašová-Perunská and Mgr. Diana Gagyiová for their skillful technical assistance. The Department of Anatomy, Faculty of Medicine, P. J. Safarik authors declare that they have no competing interests. University in Kosice, Slovak Republic

Aims. The aim of our study was to reveal the expres- 62 sion of protein carbonic anhydrase (CAIX) in lung cancer Differences in expression of calcium binding cells and correlate its levels with basic clinicopathological parameters (type of tumor, stage and grade). Hypoxia in proteins in the perirhinal and retrosplenial cortex cancers is associated with poor prognosis. Protein CAIX of the rat belongs to the most strongly induced proteins in response a d c to hypoxia. Tumor hypoxia is clinically very important Martin Salaj , Filip Barinka , Hana Kubova , phenomenon that affects response to therapy, cancer Rastislav Drugaa,b progression and survival of cancer patients. Therefore, aDepartment of Anatomy, 2nd Faculty of Medicine, Charles detection of hypoxic regions within tumors is important University in Prague, Czech Republic for stratification of patients for suitable treatment regi- bInstitute of Anatomy, 1nd Faculty of Medicine, Charles University mens. CAIX is interesting for its low expression in normal in Prague, Czech Republic tissue, but on the other side is strongly associated with cInstitute of Physiology, The Academy of Sciences of the Czech tumor cells and is expressed abnormally in a broad palette Republic, Prague, Czech Republic of human tumors. dKlinik und Poliklinik für Neurologie, Universität Regensburg, Patients and Methods. Indirect immunohistochemical Regensburg, Germany (IHC) method was used to detect the expression of CAIX in 135 samples of non-small cell lung cancers (NSCLC). Aims. The main goal was to describe interneuronal The samples of NSCLC were divided according the types population expressing calcium binding proteins calretinin into adenocarcinomas (N=56), squamocellular carcinoma (CR) and parvalbumin (PV) in the perirhinal (PRC) and (ca) (N=58), large cell ca (N= 12) and other types (N=9). retrosplenial (RSC) cortex of the rat. These two cortical Divission according to the stage was into 1A+1B, 2A+2B, areas differ strikingly in their connectivity and function, 3A+3B and 4. Finally, we have evaluated the CAIX ex- which could be caused also by different composition of presssion according to the grade (G)1, G2, G3 and G4. the interneuronal populations. Having a precise knowl- The IHC expression was scored as follow: 0 positive cells edge of the cellular composition of any cerebral area = – (negative sample), 1-10% of positive cells = 1+ (nega- forms one of the basic input parameters and tenets for tive sample), 11-90% = 2+ (positive sample), 91-100% = computational modeling of neuronal networks and for 3+ (positive sample). The patients gave their informed understanding some pathological conditions, like generat- consent before their into the study. ing and spreading of epileptic activity. Results. The highest degree of positive samples Methods. The brains of 8 male 3-month-old Wistar rats reached the squamocellular samples in number 39/67% were used in this study. The brains were cut into 50 μm and 19/33% of samples were CAIX negative. Large cell ca thick coronal section and these were stained for NISSL samples were positive in 7/58%, the rest 5/42% were nega- (cresyl violet), CR and PV. We perfomed qualitative and tive. Adenocarcinoma samples were CAIX immunoreac- quantitative analysis on these sections, using stereologi- tive in 27/48% and 29/52% were negative. Stage 1A+1B cal and densitometric approach. All data aquired were achieved the highest positivity, it was 52/58%, 2A+2B = statistically evaluated. 9/47%, 3A+3B = 11/52% and stage 4 = 2/40%. In case of Results. PRC possesses higher absulute and relative 4 grades, G3 reached the most CAIX positive samples, densities of CR+ and PV+ neurons than RSC, but the 41/63%. In G4 it was 6/55%, G2 25/52% and in G1 2/18%. CR: PV ratio is higher in the RSC, which is similar to the We evaluated our results also by statistical methods. For neocortex. The bipolar/bitufted neurons are most com- statistical evaluation we have used the Kruskal –Wallis mon type of CR+ population, while the majority of PV+ test and chi-square test. We did not find any correlation neurons show multipolar morphology. between expression of CAIX and clinicopathological pa- Conclusion. In our study we described the pattern of rameters. CaBP immunoreactivity, the density of CR+ and PV+ neu- Conclusion. CAIX have attracted considerable at- rons as well as the overall neuronal density in rat perirhi- tention, not only as a functional component of tumor nal and retrosplenial cortex. Current results indicate that physiology, but also as a tumor biomarker and potential main difference between analysed areas is in density of

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CR+ neurons, which was significantly higher in the PRC. significant changes, which can be considered as signs of Our results coupled with works of other authors show that incipient degeneration. Especially secondary in there are significant differences in the interneuronal com- the form of autophagic were numerous. position and distribution of heretofore seemingly similar Conclusion. The oocytes with precocious loss of co- cortical areas. This results may contribute to the better rona radiata cells, are likely to be in the initial stage of understanding of the mechanism of function of this corti- extinction, even though some of them seem intact and cal region in normal and diseased states. do not show any degenerative changes. Fertilization of these oocytes may be successful, but their developmental potential is probably limited. ACKNOWLEDGEMENTS

We would like to thank to Mrs. B. Čejková for excel- ACKNOWLEDGEMENTS lent technical assistance. This work was supported by Grant Agency of Charles This study is supported by the Grant Agency of the University, grant no. 35407. Czech Republic (15-04844S).

63 64 Intercellular junctions of corona radiata cells and Elucidating cardiac conduction system formation oocyte in preovulatory period with phylogenetic approaches Miroslava Sedlackova, Martin Anger, Natalie Matijescukova David Sedmeraa,b

Department of Histology and Embryology, Faculty of Medicine, aInstitute of Anatomy, First Faculty of Medicine, Charles University Masaryk University, Brno, Czech Republic in Prague, Czech Republic bInstitute of Physiology, The Czech Academy of Sciences, Prague, Aims. Oocyte maturation takes place under natural Czech Republic conditions in close contact with corona radiata cells. Thin Aims. To complement our developmental studies in cytoplasmic processes of corona radiata cells penetrate birds and mammals, we study remodeling of the atrioven- the and form the intercellular junctions tricular canal and development of the ventricular conduc- with the oocyte. Factors produced by the corona radiata tion system also from the phylogenetic perspective. After cells maintain the arrest of meiosis and prevent the re- finding a functional, but not morphological, equivalent of sumption of the first meiotic division of the oocyte. When His-Purkinje system in fish and amphibians, we focused the follicles are punctured to obtain oocytes for in vitro our attention on reptilian models. fertilization, whole oocyte- complexes Methods. We performed optical mapping and histolog- are aspirated, but also some cumulus-free oocytes cov- ical examination of partially septated reptilian hearts of ered by zona pellucida only are obtained. The aim of the monitor lizard Varanus acanthurus and completely septat- presented work was to determine on a mouse model the ed crocodilian hearts (Crocodylus siamensis). Conduction structure of the of oocytes surrounded system markers were detected on serial paraffin sections by cumulus and cumulus-free oocytes and to compare the using in situ hybridization and immunohistochemistry. morphology of both types of oocytes. Results. Optical mapping revealed first ventricular epi- Methods. 6 oocyte-cumulus oophorus complexes cardial breakthroughs in the area of the forming interven- (group 1) and 6 cumulus-free oocytes (group 2) obtained tricular septum on the left side in the later stage Varanus by puncture of antral ovarian follicles of mice unstimu- hearts. Post-septation crocodilian hearts showed an apex- lated were processed standardly for electron mi- to-base activation pattern with dual breakthrough near the croscopy. ventricular apices. Conduction system markers Irx1 and Results. Group 1 oocytes: long thin processes of co- 2, Tbx5 and Myh6 were expressed in the interventricular rona radiata cells after penetration through zona pellucida septum in the Varanus heart. Crocodilian hearts showed were connected with the oocyte by small or distinct positivity in the pacemaker area as well as in the gap junctions. Oocytes exhibited a structure typical for for the HNK1 epitope, similar to oocyte with germinal vesicle. situation in the rat or chick hearts. Group 2 oocytes: the zona pellucida seemed denser in Conclusion. Our preliminary results show that nascent comparison with the group 1, processes of the corona ra- conduction system is present in advanced reptilian hearts. diata cells persisted in the zona and in perivitelline space, In the absence of fossil evidence, this is the closest we intercellular junctions were mostly preserved. Some oo- can get to understand the evolution of cardiac conduction cytes did not show any morphological alteration on ultra- system and its relationship to ventricular septation and structural level, the only exception was the higher number homeiothermy. of structures. The other oocytes (with normal ap- pearance on the light-microscopic level), showed more

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ACKNOWLEDGEMENTS resolution. No HSVM records of our genetically proven PCD patients were interpreted as “normal”. Supported by Ministry of Education PRVOUK P35/ Conclusion. The results of our retrospective study con- LF1/5 and institutional grant RVO:67985823 (Academy firmed the validity of both morphological examinations of Sciences). in diagnostic protocol of PCD.

65 ACKNOWLEDGEMENTS Ultrastructural and videomicroscopic findings in The work was supported by the Ministry of Health patients with genetically proven primary ciliary of the Czech Republic (grant No. NT11469-5) and dyskinesia by the Conceptual Development Project of Research

a a a Organization, University Hospital Motol, Prague, the Petra Simunkova , Jana Djakow , Ondrej Cinek , Czech Republic (00064203). Ludek Vajnerb, Jiri Uhlikb aDepartment of Paediatrics, 2nd Faculty of Medicine, Charles 66 University in Prague and University Hospital in Motol, Prague, Czech Republic Wound repair, ageing and cancer: challenge for bDepartment of Histology and Embryology, 2nd Faculty of new therpaeutic modalities Medicine, Charles University in Prague, Czech Republic Karel Smetana, Jr., Lukas Lacina, Barbora Dvorankova Aims. Primary ciliary dyskinesia (PCD) is a multi- genic autosomal recessive disorder affecting respiratory Institute of Anatomy, 1st Faculty of Medicine, Charles University in tract and other organs where ciliary motility is required. Prague, Czech Republic Its clinical manifestations are recurrent or chronic respi- ratory consequent from stagnation of mucus, Aims. Incidence of different types of cancer has an in- subfertility or infertility and laterality defects. The classic creasing tendency in developed countries worldwide along triad of chronic sinusitis, bronchiectasis and situs vis- with increasing average life expectancy, also described as cerum inversus is known as the Kartagener syndrome. "the grey tsunami". This unfavourable situation in perspec- The diagnosis of PCD is based on clinical presentation, tive of demographic situation and cancer cell biology was high-speed videomicroscopy (HSVM) and transmission analysed. electron microscopy (TEM). It can be proven by a genetic Methods. This lecture reviews our own results from examination; although only in 60 – 65% of all cases a previous decade and published data of other authors. genetic defect could be found due to the enormous hetero- Results. In animals with strong regenerative potential, geneity of PCD. We decided to review our TEM, HSVM e.g. flatworms, their relatively limited lifespan is accom- and genetic results in patients with PCD to evaluate the panied by low incidence of tumors. On the other hand, validity of both morphological examinations. the regenerative potential of humans is low and human Methods. Total of 27 patients with genetically proven life expectancy is rapidly increasing. Notably, incidence PCD were selected from the cohort of 105 patients exam- of malignant diseases is also increasing. This situation ined by TEM in last 20 years. All patients recruited from can be elucidated by the hypothesis that the risk of tumor the whole Czech Republic were indicated for HSVM and formation is dependent on the number of tissue stem cell TEM after the thorough clinical examination. The result divisions, which is naturally also increased with aging. of HSVM was not known to TEM investigator and vice Reduced efficacy of gene repair mechanisms in elderly versa. The DNA analysis was performed at the end of supports this theory. The risk of "the grey tsunami" ac- the diagnostic algorithm. Our TEM and HSVM findings companied by epidemy-like incidence of malignant tumors were compared with expected findings described in the urgently requires development of new generations of an- literature and corresponding with particular genotypes. ticancer drugs that will target not only cancer cells but Results. Mutations of 6 PCD genes were detected in also the cancer microenvironment as a niche for cancer our patients. All the genes are known as causative with stem cell. specific TEM and HSVM findings. Our TEM results were Conclusion. The unfavourable demographic situation consistent with expected findings in 20/27 cases, HSVM in developed countries and increasing incidence of tu- corresponded to expectations in 24/25 patients. Most un- mors tasks to develop the new therapeutic modalities. The expected TEM findings concerned the erroneous assess- therapeutic manipulation of stroma can be a good target ments of inner dynein arms (IDA), which are very subtle for anticancer therapy. 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

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ACKNOWLEDGEMENTS From the results obtained it is evident that the walls of grafts, used as ACB, undergo completely different This study was supported by Grant Agency of the morphological changes, e.i. arterialisation of media and Czech Republic No. 304/12/1333. intimal hyperplasia, and according to their extents joined with the proliferation of the VV. Conclusion. An apparent proliferation of the VV in 67 the walls of the ACB grafts is present in bypasses at in- Morphology of vasa vasorum in the explanted tervals longer than 6 months after their implantation, and depends, above all, on the extent of the intimal hy- bypassed saphenous veins perplasia. Therefore, the proliferation of the VV has to Josef Stingla, Jan Pirkb, Zbynek Strakac, Marek Setinad, be considered as a secondary process, reacting on the David Kachlika, Josef Sache, Vladimir Musilf,g previous pathological changes of media and intima of the failed grafts. aDepartment of Anatomy, Third Faculty of Medicine, Charles University in Prague, Czech Republic bClinic of Cardiovascular Surgery, Institute of Clinical and ACKNOWLEDGEMENT Experimental Medicine, Prague, Czech Republic c Department of Cardiac Surgery, Third Faculty of Medicine, The study was supported by project of Charles Charles University in Prague and University Hospital Kralovske University PRVOUK P38. Vinohrady, Prague, Czech Republic dDepartment of Cardiosurgery, Second Faculty of Medicine, Charles University in Prague, and University Hospital Motol, 68 Prague, Czech Republic eDepartment of Pathology, Third Faculty of Medicine, Charles Interactive 3D Models University in Prague and University Hospital Kralovske Vinohrady, Prague, Czech Republic Jan Tokarcika, Frantisek Dorkoa, Eva Vybornaa, fCentre of Scientific Information, Third Faculty of Medicine, Jaroslava Chylikovab, David Brazinac Charles University in Prague, Czech Republic gInstitute of Information Studies and Librarianship, Faculty of aDepartment of Anatomy, Faculty of Medicine, University of Arts, Charles University in Prague, Czech Republic Ostrava, Czech Republic bDepartment of Histology and Embryology, Faculty of Medicine, University of Ostrava, Czech Republic Aims. The aim of the study was to describe the pres- cDepartment of Informatics and Computers, Faculty of Science, ence and participation of the vasa vasorum (VV) on the University of Ostrava, Czech Republic supply of the failed great saphenous vein (GSV) grafts in different time intervals after their implantation as aorto- coronary bypasses (ACB). Aims. Our goal is to simplify teaching and test stu- Methods. The study was done on the segments of dents’ knowledge of the acquired knowledge. To better GSV-ACB bypasses, explanted either post mortem or per- understand the different topographical relationships of operatively from 39 patients. The specimens obtained anatomical structures of the , we created 3D were divided into 5 groups after the duration of bypasses models, using modelling and visualization techniques. functioning: A – up to 1 month (7 cases), B – 1-6 months Methods. To construct the models of the human body, (3 cases), C – 7-24 months (2 cases), D – opened, but resources from the Department of Anatomy Medical functionally insufficient bypasses (6 cases), and E – above School were used. The models of the human body were 24 months (21 cases). The specimens were investigated processed in Eon Player Experience. Body Parts were con- qualitatively by light microscopy using 4 different stain- verted from 2D video sources into 3D object structures. ings. The resulting structures were subsequently adjusted in Results. Group A: the GSV wall without an apparent EON Studio and MAYA respectively – EON Creator. The arterialisation, with some thromboses of different extent, output images were supported by the software solution VV without evident changes. Group B: beginning arte- EON Experience Player, which performs highly interac- rialisation of the venous wall joint by intensive intimal tive visualization. Animations are presented with the aid hyperplasia; VV without evident proliferation. Group C: of a projector, controlled by a computer. complete arterialisation of the media and beginning ath- Results. The biggest pros include easy handling of 3D erosclerosis of the intima, apparent proliferation of VV objects. A fully usable functional site with a database of into the media. Group D: complete arterialisation of the the cardiovascular system, heart, skeletal system, respira- media, with intensive intimal , an intensive tory system, and digestive system in a usable graphical proliferation of the VV into the whole media. Group E: interface. The acquired knowledge can be improved stu- bypasses with nearly completely occluded lumen, caused dent and then checked in the “Quiz”.The test consists of by extensive intimal hyperplasia; VV invade the whole open and closed questions. For open questions the correct bypass wall. answer must be entered for closed questions, the system offers four options, from which the student may choose

S38 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). one correct answer. The system automatically evaluates exposed to RA had the same morphological structure. the accuracy of the answers after each question. The cells had oval or elongated body with long dendrites In the case of incorrect answers, the student continues and the body size and length of dendrites were compa- the test. In conclusion, the system evaluates the overall rable between all groups of animals. Also number of test, including the number of correct and incorrect re- nitrergic neurons of amygdala did not differ between ex- sponses and the overall evaluation of the test. perimental groups. Conclusion. Teaching anatomy is currently not pos- Conclusion. These findings confirm the fact that de- sible without accompanying image processing, lectures, veloping nervous system is very sensitive to the action of and practical . Teaching has one goal, and that external factors. Our results also indicate that the level is to increase the quality of the educational process. This and type of damages of CNS need not specifically depend approach places greater demands on the teacher, who on the type of acting factor; different types of noxious fac- has to have a thorough knowledge of computers and how tors acting in the same phase of embryonic development to most effectively use new technologies, not only in the can have the same effect on the function and morphology education itself, but also in the development of teaching of the central nervous system. 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 70 3D models was expressed mainly by students of medicine and bachelor’s candidates for the continuing validation Segmental differences in the orientation of smooth of knowledge in the study, and in the repetition of the muscle cells in the tunica media of porcine aortae test anatomy. Zbynek Tonara, Petra Kochovab, Robert Cimrmanc, 69 Josef Perktoldd, Kirsti Wittere Various noxious factors and their effect on the aDepartment of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, Czech Republic developing nervous system bNew Technologies – Research Centre, University of West Bohemia, Pilsen, Czech Republic a a b Lenka Tomasova , Natalia Hvizdosova , Benadik Smajda , cOutremont QC H3T1E9, Quebec, Canada Martin Bonaa, Stanislav Mateffya, Darina Kluchovaa dInstitute of Anatomy, Histology and Embryology, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria aDepartment of Anatomy, Faculty of Medicine, P. J. Safarik University in Kosice, Slovak Republic Aims. The orientation of cells bDepartment of Animal Physiology, Institute of Biology and of porcine aortae was assessed to test the widely accepted Ecology, Faculty of Science, P. J. Safarik University in Kosice, assumption that these smooth muscle cells are arranged Slovak Republic in two helices. Methods. We used tangential histological sections of Aims. The developing central nervous system (CNS) 82 samples of five anatomical segments of thoracic and is very sensitive to the action of various biological, chemi- abdominal porcine aortae and three age groups in animals cal and physical exogenous factors, which can lead to the ranging in age from 5-210 days. The distribution of the various types of damages. The aim of our study was to orientation of smooth muscle cell nuclei in five proxi- compare influence of two different noxious factors (physi- modistal segments of the porcine aortae was determined cal and chemical) on the morphology and function of using an algorithm that fitted a mixture of one to five the brain. von Mises probability distributions of the data retrieved Methods. The study was performed on adult Wistar from histological micrographs. Automated tracking of rats, the progeny of mothers exposed to ionizing radiation the nuclei was confirmed by and consistent with manual (IR) (1 Gy of gamma rays on the 17. day of gestation) or histological analysis. to retinoic acid (RA) (1 mg/kg body weight on the 14. – Results. The orientation of the vascular smooth 16. day of embryonic development). We tested emotional muscle cells was successfully fitted using two von Mises behaviour of rats in the plus maze test and after the behav- distributions in most of the samples with different ages, ioural test we determined the presence and morphology wall thicknesses, and anatomical positions, which cor- of nitrergic neurons in the amygdala. responds to two populations of vascular smooth muscle Results. The results obtained from the plus maze test cells. A minor fraction of samples also required a tertiary showed increased level of anxiety and fear in animals pre- von Mises distribution to describe the orientation of the natally exposed to IR or RA. Reducing the time, which smooth muscle cell nuclei. The distribution of vascular animals spent in the open arms of apparatus, manifested smooth muscle cells in five aortic segments ranging from it. Statistically significant changes of emotional behav- the thoracic ascending aorta to the abdominal intrare- iour were observed after exposure to ionizing radiation. nal aorta exhibited similar main directions but different Histological observation revealed that nitrergic neurons shapes. of control animals, animals exposed to IR and animals

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Conclusion. The results are consistent with the widely was found (26.46±1.76); but in the group Q4 decrease in used model of two muscular helices intermingling in the COX-2 positive cell number was observed (12.37±0.91). arterial wall. Furthermore, we calculated the central an- The decrease was significant in comparison with both Q1 gles of symmetry and the mean value of angles between (P<0.01) and E4 (P<0.001) groups. the two assumed smooth muscle directions. We also suc- Conclusion. COX-2 is an immediate-early response cessfully approximated the orientation of the smooth gene that is induced by a variety of mitogenic and inflam- muscle cells using a mixture of von Mises distributions matory stimuli. Our results confirmed quercetin inhibito- and our open source software named dist_mixtures. This ry effect of COX-2 family in the model of rat IRI, since the method is openly available to researchers who are inter- number of COX-2 positive cells in the epithelium of the ested in mathematically assessing the orientation of cell after 4 h of reperfusion was significantly reduced. nuclei in various tissues.

AKNOWLEDGEMENTS ACNOWLEDGEMENTS This study was supported by the grant VEGA The study was supported by the ERDF projects No. 1/0478/2014. 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. 72 71 Iconology of diseases in Renaissance art Regulatory action of quercetin on Ivo Uberalla, Ales Filipb cyclooxygenase-2 in the model of ischemia/ aDepartment of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech reperfusion injury of small intestine Republic b a b a a Department of Musiciology, Faculty of Arts, Masaryk University Stefan Toth , Milan Maretta , Kristina Gregova , Jan Soltes , Brno, Czech Republic Martin Svanaa, Martin Pribulaa, Matus Kusniera, Zuzana Jonecovaa Aims. The aim of this paper will be to highlight the selected examples of symptoms in Renaissance art. a Department of Histology and Embryology, Faculty of Medicine, P. Methods. From the point of view of methodology J. Safarik University in Kosice, Slovak Republic iconographic-iconological approach will be applied. Some bDepartment of Neurology, Louis Pasteur University Hospital, Kosice, Slovak Republic of the pathological diagnoses in Renaissance art will be introduced to and will be set in the historical-social frame- Aims. Quercetin, a polyphenolic bioflavonoid, pos- work. sesses multiple pharmacological actions including anti- Results. Renaissance is a historical period, which is inflammatory and anti-tumor properties. The aim of apart from anything else characterized by concern of man present work was to investigate the effect of quercetin about himself. In combination with the development of on cyclooxygenase-2 (COX-2), an important mediator in anatomy as a science, first appearance of pathologies inflammation promotion, during intestinal ischemia/re- in pictures began to appear. Leaving aside the appear- perfusion injury (IRI) of the small intestine in rats. ance of malformations and pathologies that are secular Methods. Adult male Wistar rat SPF–Charles River and symbolic nature (eg. St. Roch as the patron saint of (n=28) were randomly assigned to the control group (E) plague patients), secular themes remain among the few, or experimental groups (Q). Quercetin dissolved in etha- that offer an insight into the health status of the popula- nol or ethanol vehiculum were applied intraperitoneally tion. Besides conventional medical books in which we 30 min before the complete ischemia of a. mesenterica cra- meet with numerous diseases (De recondita abscessuum nialis. Ischemia was performed by microvascular clamp, natura, 1643 or Selecta praxis medico-chirurgicae, 1652) lasted 1 h and was followed by 1 h (E1, Q1) and 4 h (E4, we can rarely observe the iconographical representation Q4) of reperfusion period. After the reperfusion period, of different pathologies in artistic artifacts. Lets name bioptic samples were harvested and histological sections some of them for example The old man and his grandson were stained by H&E staining method and COX-2 immu- (Domenico Ghirlandaio, 1490) or Person with syphilis nohistochemical method. The numbers of COX-2 positive (Albrecht Dürrer, 1496). A special chapter consists of cells in the epithelium were calculated per 1 mm of the scenes that use pathological changes to amplify the effect epithelium lining. of a caricature. Examples of art work combining secu- Results. Quercetin had no effect on the number of lar and medical theme are for example A representation COX-2 positive cells in the epithelium of the samples tak- of the plague infected Temptation of St. Anthony (Mathis en after 1 h of reperfusion, in both E1 and Q1 groups the Grünewald, 1510-1515), goitre at Assumpta from Desna results were similar (20.48±1.07 and 20.59±2.28). After 4 (unknown author, 1450) or symptoms of leprosy in the h of reperfusion in group E4 non-significant increase in it work Plate with three apostles (unknown author, 1510) .A

S40 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). special place in the Renaissance iconology disease plays in epidermis and LVA as well as in dermis and LVA in Michelangelo's depiction of incipient cancer of the statue papillary dermis of psoriatic lesions (r=0.311, P=0.351; "Night" in the Medici Chapel in the cathedral in Florence r=0.455, P=0.159 respectively). (1521) or distorted breast by cancer on a fresco of the Conclusion. These data support the hypothesis that Sistine Chapel (1508-1541) by the same author. and lymphangiogenesis in are to a Conslusion. The presence of signs of disease in the arts greater extent driven by VEGF–A and VEGF–C expres- and their reproducibility testifies about the observation sion in epidermis. capability and suggests that Renaissance artists used live It probably results from epidermal changes, mainly models. The frequency of display of various pathologies hyperplasia, which are prominent in pathogenesis of pso- gives some idea about the population health status. riasis.

73 74 Blood and lymphatic microvascular changes in Historical overview of morphological studies of relation to VEGF–A and VEGF–C expression in psoriatic lesions Eva Vybornaa, Frantisek Dorkoa, Vladimir Holibkaa, Desanka Vybohovaa, Yvetta Mellovaa, Katarina Adamicovab Jan Tokarcika, Jaroslava Chylikovab aInstitute of Anatomy, Jessenius Faculty of Medicine in Martin, aDepartment of Anatomy, Faculty of Medicine, University of Comenius University in Bratislava, Martin, Slovak Republic Ostrava, Czech Republic bInstitute of Pathological Anatomy, Jessenius Faculty of Medicine bDepartment of Histology and Embryology, Faculty of Medicine, in Martin, Comenius University in Bratislava, Martin, Slovak University of Ostrava, Czech Republic Republic The human palatine tonsil, as part of the Waldayer Aims. Present study focuses on the evaluation of quan- lymphatic ring, represents the first point of contact with titative changes of blood and lymphatic microcirculatory antigens from inhaled air and food. As an organ suffer- bed, VEGF–A and VEGF–C expression and their mutual ing from frequent , it has been studied not relations within the psoriatic lesions. only by morphologists but also ENT specialists, imunolo- Methods. Skin samples of psoriatic lesions located at gists and specialists from other medical fields. In their the abdominal and back regions were taken from untreat- contrinution, authors summarize findings about tonsil- ed patients (in age range 32-55 years) without any system- lar lymphoid tissue, tonsillar structure and ultrastructure ic disorder and compared with control healthy skin from (lymphoreticular parenchyma, surface and crypt epithe- identical regions. Immunohistochemical analysis with an- lium, microcirculation, etc.) as well as its function, as they tibodies to CD34, podoplanin (D2 – 40), VEGF–A and were found in the literature and obtained by self-study. VEGF–C was used to determine the blood and lymphatic The research of the human palatine tonsil over vessels area (BVA and LVA) in standard fields of the pap- time has been closely related to the evolution of mi- illary dermis and VEGF–A and VEGF–C positive area croscopy techniques and methodology. Using a light in epidermis and dermis. All mentioned parameters were microscope, the structure of the human palatine ton- evaluated by using exact morphometric analysis software. sil was firstly established by Stöhr in 1891; followed by Results. Measurements revealed that both lesional and Lenz who in 1972 further described its cryptic epithe- healthy skin samples showed significantly higher BVA lium (“Ultrakanalsystem“). The definition of the human than LVA, and higher VEGF–A than VEGF–C expres- palatine tonsil in anatomical terms dates back to 1961 sion. BVA values in psoriatic lesions showed almost 3 (Fioretti). The boom of the scanning electron microscopy – fold increase. Althought LVA values were significantly in 70ies and 80ies of the 20th century significantly en- lower than BVA, they showed 2 – fold increase in com- hanced the means of study and resulted in a number of parison to control healthy skin. VEGF–A and VEGF–C new findings. Perry (1988, 1994, 1998) confirmed the were strongly overexpressed in both lesional epidermis conclusions of Lenz by studying in TEM and SEM the and dermis. VEGF–A expression in psoriatic lesions was lymphoreticular stroma of the tonsil. Oláh pioneered to significantly higher in epidermis (27 – fold increase) than describe the microfold cells (“M-cells“). Together with in epidermis (20 – fold increase). Similarly, VEGF–C ex- researchers from Japan (Kodama, Hoshino 1977, Maeda, pression achieved 22-fold increase in psoriatic epidermis Mogi 1982), those authors significantly contributed to the and 14 – fold increase in psoriatic dermis. Obtained re- research of the lymphatic tissue by describing, among oth- sults confirmed significant positive correlation between ers, the ultrastructure of the follicular areas with dendritic VEGF–A expression in epidermis and BVA (r=0.659, cells, the reticular epithelium as well as the Langerhans P=0.027) in papillary dermis of psoriatic lesions. cells containing so called Birbeck granules. They also However, correlation between VEGF–A expression in der- studied the microcirculation of resulting in the mis and BVA was only mild and statistically insignificant discovery of the High Endothelial (HEV). (r=0.309, P=0.224). Mild but statistically insignificant cor- In Czechoslovakia and then the Czech Republic, relation was demonstrated between VEGF–C expression Slípka devoted most of his research to the study of the

S41 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). lymphatic tissue, for example, by comparing the develop- deposition of β protein and protein τ). AD dis- ment of thymus and tonsil. Holibka (1992) described in rupts brain function and causes a decline in cognitive detail the tonsil‘s ultrastructure and, based on microcir- function (thinking, memory, judgment). The aim is to culation studies, post-capillary venules and fenestrated verify the hypothesis, based on volumetric changes of capillaries. Holibková (1989) dealt with the relationship the corpus callosum (CC) and its specific parts in the between glandular and lymphatic tissue and Kutal (1998) group of AD patients and controls using tractographical described the tonsil of insectivores. Stemming overwhelm- displaying. Examined parameters are the number, volume ingly from , the human material used for and length of the fibres in defined areas of the corpus the research implies a pathological modification of the callosum, the corresponding functional involvement. The tissues. Therefore, some authors (Holibka 1992, 1994, same parameters are also measured in the gyrus cinguli, Výborná 1997) devoted their study to ultrastructure of the another structure involved in memory consolidation, are human palatine tonsil in the prenatal period, i.e. without connected in the limbic circuit. the presumed antigenic stimulation. Using the method of Methods. We have used 3T DTI MRI scans of the pa- metallized histological sections and based on a sample tients diagnosed as probably AD and control persons from of 76 tonsils from 16th to 40th prenatal week, the au- the Department of Radiodiagnostic and Interventional thors studied in particular the tonsil’s lymphoreticular Radiology IKEM. These scans were analysed by the com- parenchyma, surface epithelium and crypts. The research puter program DSI Studio. Using the region of interest revealed the presence of reticular epithelium, including maps we have displayed the fibres of the front and rear fenestrated capillaries, as of the 30th prenatal week and part of the gyrus cinguli and five functional parts of the concluded that the tonsil is ready to function already be- corpus callosum. fore the birth. Recently, the research of the human pala- Tractography is a method for neuronal fibres of the tine tonsil continues focusing on the Mucosa Associated brain displaying. The axonal membrane, myelin, and the Lymphatic Tissues („MALT“) of both human (NALT other structures significantly affect Brown’s movement – Perry, 1988, 1994, 1998) and animal organs (rabbits, of the water molecules. The resulting vector is largest sheep, chicken – Oláh, 1975, 1988, 2003; neat – Palmer, in the longitudinal direction of the fibre. Tractography 2009; and dromedary – Zidan, 2009). is calculated separately for each voxel wherein the fibre The contribution summarizes knowledge on the struc- direction depends on the direction of the resultant vec- ture and ultrastructure of the tonsil (epithelium surface tor of diffusional tensor. Composition of neighbouring and crypt epithelium, its reticularization, intraepithelial vectors according to set parameters (angle of deviation, pathways and migration of lymphocytes as well as special- fractional anisotropy) direction and reconstruct the result- ized vascular structures) and also mentions other com- ing waveform fibres. This allows all the pathways the areas ponents of MALT. The findings were obtained by using in which they were ranked numbers of nerve fibres, their of light, transmission and scanning electron microscopy. volume and average length. Visual control of fibres and their participation is made possible 3D reconstruction MRI sequences. Statistically we were compared between 75 patients and controls coherent age. Tractography of corpus callosum and gyrus cinguli Results. There is statistically significant decrease in the volume and number of fibres in the front and rear part in patients with Alzheimer’s Disease of the CC. Decrease in the number of fibres has also been Zdenek Wursta, Petr Zacha, Ibrahim Ibrahimb, Ales Bartosc,d, important in CC medial anterior and CC centralis. The Vladimir Musile, Matej Patzelta, Jana Mrzilkovaa fibre length did not change in any of the areas measured by us. We have also measured neuronal damage param- aDepartment of Anatomy, Third Faculty of Medicine, Charles eters: fractional anisotropy (FA) expresses the neuronal University in Prague, Czech Republic arrangement, apparent diffusional coefficient (ADC) bMR Unit, Department of Diagnostic and Interventional presents diffusivity in structural volume, axial diffusivity Radiology, Institute for Clinical and Experimental Medicine, (AX) – characterize and , Prague, Czech Republic c radial diffusivity (RD) – expresses myelin damage. AD Centre, National Institute of Mental Health, Klecany, Czech Conclusion. Although the gyrus cinguli contributes Republic dDepartment of Neurology, Third Faculty of Medicine, Charles to memory consolidation, the measured parameters in University in Prague and University Hospital Kralovske Vinohrady, AD groups to shift there. Most of the monitored parts of Prague, Czech Republic corpus callosum in the AD patients were changed. The eCentre of Scientific Information, Third Faculty of Medicine, exception are the functional connections of fibres parietal Charles University in Prague, Czech Republic and temporal , which are between AD and controls unchanged and is probably related to the natural age loss Aims. The most frequent cause of dementia in middle- of neurons. All studied structures in our study indicate age and elderly is currently Alzheimer's Disease (AD). statistically significant decrease of neuronal damage pa- It is a chronic progressive nervous system disease mani- rameters. fested by neurodegenerative process, accompanied by characteristic histopathological changes (pathological

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ACKNOWLEDGEMENTS pressure positively correlated with body height, body weight, and BMI values as well as with intraabdominal The research was supported by projects of Charles fat in the whole group, irrespective of age. The highest University PRVOUK P34, P38 and 260168/SVV/2015, dependency rate was present between blood pressure and NUDZ project ED 2.1.00/03.00/0078, and project body weight. MZCR00023001IKEM. 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 76 more precise correlation of parameters the use of multi- Blood pressure of girls aged 10 to 18 with respect variate analysis would be required. to intra-abdominal fat tissue Katerina Kikalovaa, Miroslav Kopeckyb, Jiri Charamzab, ACKNOWLEDGEMENTS Jitka Tomanovac, Petr Zemanekc Research was supported by the project aDepartment of Anatomy, Faculty of Medicine and Dentistry, PL.3.22/2.3.00/11.02576, The obesity epidemic – a shared Palacky University Olomouc, Czech Republic problem: the transfer of knowledge, education, prevention, bDepartment of Specialised Subjects and Practical Skills, Faculty from Operational Programme Cross-border Cooperation of Health Sciences, Palacky University Olomouc, Czech Republic Czech Republic – Poland 2007-2013. cDepartment of Anthropology and Health Education, Faculty of Education, Palacky University Olomouc, Czech Republic Aims. This paper focused (i) to determine basic an- 77 thropometric parameters (body height, body weight, Continuously regenerating dentition in anoline BMI), (ii) to determine the values of intra-abdominal fat, (iii) to measure the systolic and diastolic blood pressure, lizards: the model of postnatal teeth development (iv) to classify subjects into BMI zones, in order to assess leading to changes in tooth shapes and numbers the impact of intra-abdominal fat tissue on the blood pres- a b sure in girls aged 10-18. Veronika Holanova , Oldrich Zahradnicek Methods. The research group consisted of 1075 girls aDepartment of Zoology, Faculty of Science, Charles University in aged 10-18. Their blood pressure was measured with a Prague, Vinicna 7, 128 44 Prague, Czech Republic digital tonometer A&D Medical UA 787 Plus using an bDepartment of Teratology, Institute of Experimental Medicine, appropriate cuff. The blood pressure was measured in a The Academy of Sciences of the Czech Republic, Videnska 1083, sitting position, on the right arm, after subjects have been 142 20 Prague, Czech Republic five minutes in rest. Oscillometric method was chosen on the basis of anthropometric research arrangement, due to Aims. The deciduous and permanent dentitions a significant noise in the room where the measurement in human and other diphyodont mammals mostly dif- was conducted. Body height was determined by standard fer in teeth size, external and internal morphology and anthropometric method. The values of intra-abdominal teeth number. The necessary condition for development fat were obtained using bioimpedance device InBody 230. of such differences between deciduous and permanent The classification of subjects into BMI categories was dentition is formation of successive teeth generations. performed using a 7-zone distribution according to WHO. Dental characters correspond to feeding preferences and We used the Statistica 12 software for the evaluation and phylogenetical relationships of taxons. The differences the correlation of particular parameters. between first functional teeth generation and successive Results. The mean values of body height in the age generation(s) were not observed only in mammals but groups of girls aged 10 to 18 were following: 144.10, also in other toothed vertebrate clades. The increasing 150.84, 157.86, 160.68, 163.86, 164.35, 166.05, 164.85, number of teeth and teeth size are the most common char- 165.22 cm; similarly, the mean body weight values were: acteristics which change between new teeth generations 37.35, 42.40, 48.42, 51.56, 55.69, 57.55, 60.96, 58.85, throughout life. Species of some non-mammalian lineages 61.34 kg; and the mean values of intra-abdominal fat were: could significantly change morphology of teeth between 51.37, 54.81, 55.51, 58.97, 63.31, 69.01, 79.54, 73.55, 77.60 teeth generations of juveniles and adults. Here we focus cm2. Values of intra-abdominal fat up to 100 cm2 were on small group of four anoline lizard species from Cuba, considered normal. Importantly, such value or higher the Cuban false chameleons, which are uniquely adapted was found in 12.74% of girls. The classification into BMI to crushing snail shells. Their morphology of dentitions zones for the entire set of girls followed normal distribu- significantly changes between juvenile and adult teeth tion. Only the subgroup of girls in the zone of obesity was generations. Such dentitions evolved from the dentition larger than normal (8.19% vs. 3% in normal population). adapted to insectivory and saurivory. Except for Cuban Systolic blood pressure was stable up to 15 years of age false chameleons we used in this study giant green anole and later started to rise slowly. Diastolic blood pressure Anolis baracoae with generalized type of anoline dentition exhibited a steady rise, from 71.87 Torr in 10 years to to construct possible story of evolution of molluscivory 80.49 Torr at the age of 18. Systolic and diastolic blood dentition among these specialists. Species of Cuban false

S43 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Sep; 159 (Supplement 1). chameleons could differ in the grade of mallacophagous Macrophages are heterogeneous cell populations with specialization. functional variability dependent on the polarization state. Methods. The lower jaws of specimens were separated Adipose tissue macrophages may be present in different from the upper jaws and then were cleaned mechanically activation status, known as M1 and M2. M1 and M2 mac- and chemically by bleaching solution from . The rophages perform different functions, among other things, coating by a thin layer of gold was made after bones dehy- producing the pro- or anti-inflammatory factors. The aim dratation in acetone and dry owen. The SEM microscope of our study was to characterize the activation status (phe- JEOL SEM 6380 LV operating at 25 kV was used for the notype) of adipose tissue macrophages in morbidly obese dentition documentation. using immunohistochemistry. Results. We showed that each examined species dif- Methods. In our work, we studied the presence of fers in morphology of juvenile and adult dentition, while CD68 and CD204 positive macrophages in white adi- in adults were differences more expressed. The two main pose tissue. The material was obtained during intraop- different types of dentition evolved in Cuban false cha- erative biopsy in patients undergoing bariatric surgery for meleon lizards. One group of Cuban false chameleon morbid obesity. Our group includes 42 men aged 21-66 lizards noticeably increases teeth number throughout life years with a BMI between 34-58, and 64 women, aged and resemble situation in anoles with generalized type of between 21-60 years with a BMI between 30-54. We in- dentition, while second group of these lizards does not vestigated both subcutaneous and visceral white adipose increase teeth number at all or just minimally what we tissues. The formalin fixed samples were embedded in interpreted as one of dentition adaptations for the mol- paraffin. Paraffin sections were stained with hematox- luscivory. The shapes of teeth in juveniles of Cuban false ylin-eosin. Two-steps immunohistochemical indirect chameleon lizards resemble insectivorous and saurivorous method has been used. In first step we used EnVision teeth of anoles, while teeth of adults in the caudal area of detection system (Dako), visualization was done using jaws significantly differ from anoles by greater robustness, DAB chromogen. Macrophages were detected using an loosing or minimalizing side cusps and by forming more antibody CD68 (Mouse Monoclonal Anti-Human CD68, rounded tip of the tooth crown. DakoCytomation). Anti-MSR1 antibody was produced in Conclusion. We hypothesize that the shape differences rabbit (Sigma). correspond to feeding preferences and that adults of A. Results. CD68 positive macrophages were found in the guamuhaya are possibly insectivorous and saurivorous as interstitium – isolated in the thin septa between two adi- well as molluscivorous. Adults of other species of Cuban pocytes, and in groups surrounding a small blood vessels false chameleon lizards seem to be mainly molluscivorous or at the point of contact between more fat cells. We also specialists. found the so-called „crown-like“ structures consisting of macrophages fully surrounding dead , but they were less frequent. ACKNOWLEDGEMENTS Especially in obese with diabetes the number of CD68 + macrophages was greater in visceral adipose tissue and We thank for the financial support to GACR 14- in the vicinity of hypertrophic . CD 204+ 37368G. 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 78 also in the “crown-like” structures, however, they form M1/M2 Macrophages polarization in white only a small part from their macrophages. In the obese without diabetes is the presence of CD204+ macrophages adipose tissue of morbid obese patients comparable with CD68+. Jaroslava Chylikovaa, Vojtech Kamaradb, Conclusion. CD68+ macrophages and CD204+ are Eva Vybornac , Jana Dvorackovad, Pavol Holeczye present in both subcutaneous and visceral adipose tissue. In the morbidly obese with diabetes is the presence of aDepartment of Histology, Faculty of Medicine, University of CD68+ macrophages higher especially in visceral adipose Ostrava, Ostrava – Zabreh, Czech Republic tissue. In patients without diabetes, anti-inflammatory bDepartment of Histology and Embryology, Faculty of Medicine oriented M2 (CD204+) macrophages are found more and Dentistry, Palacky University Olomouc, Czech Republic frequently. cDepartment of Anatomy, Faculty of Medicine, University of Ostrava, Ostrava – Zabreh, Czech Republic dDepartment of Pathology, Faculty of Medicine, University of Ostrava, Ostrava – Zabreh, Czech Republic eDepartment of Surgery, Vitkovice Hospital a.s., Ostrava – Vitkovice, Czech Republic

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

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79 poor outcome. These facts open the question whether ABC transporters could mediate drug resistance in tu- ABC transporters and their role in the drug mors. The discrepancy between laboratory research and resistance clinical study were mostly attributed to both the complex- ity of the phenomenon of drug resistance at organismal Petr Mlejnek level and to the methodical problems. Results. However, we think that many disappointing Department of Anatomy, Faculty of Medicine and Dentistry, results are due to oversimplifying approach to this com- Palacky University Olomouc, Czech Republic plex issue. We believe that more rigorous design of the in vitro experiments may provide novel results which will Background. In the last decades a significant attention help to better understand the phenomenon of drug resis- has been dedicated to the role of ABC (ATP binding cas- tance and shed a light on contradictory results that exist. sette) transporters, namely ABCB1 (P-glycoprotein (P- For example, we observed that the resistance to antican- gp)), ABCG2 (breast cancer resistance protein (BCRP)), cer drugs mediated by main ABC transporters, ABCB1 and ABCC1 (the multidrug resistance protein (MRP1)). and ABCG2, strongly depends on their expressions at This was mainly due to the convincing laboratory results. protein levels. Unfortunately, efforts to translate this discovery to the Conclusion. Expression level of a particular drug trans- clinic have failed. Indeed, the outcome of clinical trials porter is relevant to the understanding its clinical role. utilising drug transporter inhibitors to reverse resistance mediated by P-gp was mostly disappointing. A clear cor- relation between drug transporter expression and resis- ACKNOWLEDGEMENT tance was also difficult to establish in clinical samples. However, the overexpression of ABC transporters, includ- This work was supported by grant IGA_LF 2015_029 ing ABCB1, ABCG2, and ABCC1 were associated with (Internal grant of Palacky University).

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AUTHOR INDEX

Adamicova K. 73 Elias P. 58 Krajci D. 34, 35, 36, 37, 42, 43 Adamkov M. 1, 49 Eminger M. 16 Krajci D. (Mrs.) 36, 37, 42, 43 Anger M. 63 Erdosova B. 36, 37, 42, 43 Kralickova M. 16, 33, 54 Baca V. 27, 28, 29 Fiala P. 19 Krasensky J. 52 Balentova S. 1 Filip A. 72 Krejci P. 5, 13 Balkova S. 41 Filip S. 8 Krizkova V. 6 Barinka F. 62 Filipcikova R. 3 Kubies D. 54 Bartos A. 75 Filova B. 1 Kubikova T. 6 Bencat M. 49 Gdovinova Z. 48 Kubina T. 54 Bezdickova M. 3 Gregor T. 16 Kubova H. 62 Beznoska P. 2, 21 Gregorova K. 18, 25, 71 Kucera T. 38 Bezrouk A. 47 Grim M. 40 Kuchar M. 58 Bilecova-Rabajdova M. 18 Hajek P. 28 Kucharova B. 20 Blaha M. 38 Hajtmanova E. 1 Kudlackova M. 18 Blankova A. 29 Herich R. 50 Kuklova A. 26, 39 Blazkova Z. 3 Hlouzkova A. 5 Kunke D. 55 Blom A. N. 51 Hodan R. 57 Kunova M. 13 Bludovska M. 6 Hodorova I. 19, 20, 61 Kusnier M. 71 Bolekova A. 4 Holanova V. 77 Kvasilova A. 40 Bona M. 4, 22, 69 Holeczy P. 78 Kylar P. 34 Bonczek O. 5 Holibka V. 12, 74 Lacina L. 66 Borbelyova V. 1 Hornakova L. 12 Lametschwandtner A. 16 Brazyna D. 68 Horvathova F. 20 Langova P. 41 Bruckova L. 8 Hradilova-Svizenska I. 15 Lehotsky J. 1 Buchtova M. 7, 13, 30, 41, 53, 57 Hrebikova H. 2, 21, 23, 55 Lichnovska R. 36, 37, 42, 43 Buskova J. 52 Hubacek P. 3 Liska V. 16 Capek M. 32 Hubalek Kalbacova M. 54 Lochman J. 5 Cela P. 7 Hvizdosova N. 4, 22, 45, 69 Lovasova K. 4, 46, 56, 60 Cendelin J. 33 Ibrahim I. 75 Luzna P. 17 Cernochova P. 5 Izakovicova-Holla L. 5 Machalek L. 36 Charamza J. 76 Janacek J. 32 Machon V. 57 Chvatalova J. 2, 21, 23, 55 Janitorova Poliacikova M. 10, 11 Makovicky Pa. 44 Chylikova J. 12, 68, 74, 78 Jekl V. 57 Makovicky Pe. 44 Cimrman R. 70 Jirkovska M. 24 Maretta M. 25, 71 Cinek O. 65 Jonecova Z. 18, 25, 71 Matalova E. 53 Cizkova D. 2, 8 Jongbloed M. R. M. 51 Mateffy S. 4, 22, 45, 46, 56, 60, 69 Cizkova K. 9, 17 Joukal M. 15, 26, 39 Matijescukova N. 63 Danielisova V. 20 Kachlik D. 27, 28, 29, 67 Matusevska M. 45, 46, 56, 60 Dankova M. 10, 11, 48 Kamarad V. 78 Mazurova Y. 47 Dankovcik R. 50 Kautzner J. 38 Mechirova E. 10, 11, 48 Diaz D. 2, 21, 55 Kikalova K. 76 Melenovsky V. 38 Djakow J. 65 Killinger M. 30 Mellova Y. 73 Dobias M. 3 Klepacek I. 31 Mestanova V. 49 Dolezelova E. 8 Kluchova D. 4, 22, 46, 56, 60, 69 Micuda S. 8 Domorakova I. 10, 11, 48 Klusakova I. 15, 26, 39 Mihalik J. 19, 20, 61 Dorko F. 12, 68, 74 Knopfova L. 53 Miklosova M. 50 Dosedelova H. 13 Kochova P. 70 Mirka H. 16 Drobna Krejci E. 40 Kolesova H. 32 Misek I. 5 Druga R. 14, 62 Kolinko Y. 33 Mlcoch M. 3 Dubovy P. 15, 26, 39 Konarik M. 28 Mlejnek P. 79 Durisova M. 38 Konieczna A. 17 Mokry J. 2, 8, 21, 23, 55 Dvorackova J. 78 Kopecky M. 76 Mrzilkova J. 75 Dvorankova B. 40, 66 Kopecny T. 36, 37 Musil V. 27, 67, 75 Dzugan J. 54 Korimova A. 15 Nanka O. 51 Eberlova L. 16 Kotyzova D. 6 Nemcova V. 52 Ehrmann J. 9, 17 Kovalcikova S. 18 Novotny R. 35, 37

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Oborna I. 3 Seidl Z. 52 Tonar Z. 16, 33, 70 Ohlasova J. 46, 56, 60 Sery O. 5 Toth S. 18, 25, 71 Oralova V. 53 Setina M. 67 Troup O. 16 Ostrovska L. 54 Shbat A. 31 Tumova E. 44 Palek R. 16 Shylo N. 7 Uberall I. 72 Pastucha D. 3 Simunkova P. 65 Uhlik J. 65 Patzelt M. 75 Skala M. 16 Ukraintsev E. 54 Paulova M. 59 Skorvanek M. 48 Vajner L. 65 Perktold J. 70 Slama P. 54 Vaneckova M. 52 Petrovicky P. 52 Smajda B. 69 Vanek J. 5 Pirk J. 38, 67 Smarda J. 53 Varga I. 49 Pisal R. 2, 23, 55 Smetana K., Jr. 40, 66 Varga J. 25 Pribula M. 71 Smorodinova N. 38 Vavrova J. 8 Pribulova J. 45, 46, 56, 60 Solar P. 20, 26, 39 Vecanova J. 45, 61 Pridal J. 38 Soltes J. 71 Vesela I. 13, 30 Putnova B. 57 Sonka K. 52 Vicente-Steijn R. 51 Rajdova A. 9 Soukalova J. 5 Vignerova J. 59 Redaj L. 46, 56, 60 Soukup T. 55 Vistejnova L. 54 Rejtarova O. 58 Soumar J. 59 Vybohova D. 73 Riedlova J. 59 Stebnicky M. 10, 11 Vyborna E. 12, 68, 74, 78 Rimarova K. 44 Stembirek J. 5, 57 Weatherbee D. S. 7 Rosendorf J. 16 Stepankova T. 27 Witter K. 70 Rozpravkova M. 45, 46, 56, 60 Stingl J. 67 Wurst Z. 75 Rozsivalova K. 38 Straka Z. 67 Zach P. 75 Rybarova S. 19, 20, 61 Svana M. 71 Zahradnicek O. 77 Sach J. 67 Svandova E. 53 Zednikova-Mala P. 31 Salaj M. 62 Tokarcik J. 12, 68, 74 Zemanek P. 76 Sedlackova M. 63 Tomanova J. 76 Zuska K. 24 Sedmera D. 32, 51, 64 Tomasova L. 4, 22, 69

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Book The titles of journals should be abbreviated in accordance with 3. Winer BJ. Statistical principles in experimental design. New Index Medicus. York: McGraw-Hill; 1971. 4. Gilstrap LC, Cunningham FG, VanDorsten JP, editors. Footnotes Operative obstetrics. 2nd ed. New York: McGraw Hill; 2002. Symbols indicating author affiliation should be superscript letters:a,b,c. Chapter in a book 5. Shatkin AJ. Colorimetric reactions for DNA, RNA, and protein determinations. In: Habel K, Salzman NP, editors. FEE NOTIFICATION Fundamental techniques in virology. New York: Academic Press; 1969. p. 231-237. The fee of 10,000 Czech crowns (or an equivalent sum in Euro) will apply to all manuscripts accepted for publication. Accepted Conference paper manuscripts will not proceed further without receipt of payment. 6. Wefers H, Sies H. Generation of photoemissive species during quinone redox cycling. In: Alexander P, editor. Bioreduction in the Activation of Drugs. Proceedings of the Second Biochemical REJECTION OF MANUSCRIPTS Pharmacology Symposium; 1985 25-26 July; Oxford, UK. Oxford: Pergamon Press; 1986. p. 26-34. Manuscripts dealing with topics that have been well-studied in the literature, and that do not resolve questions raised by previous Journal article on the internet studies, or manuscripts that are statistically underpowered, are 7. Abood S. Quality improvement initiative in nursing homes: likely to be rejected without peer review. the ANA acts in an advisory role. Am J Nurs [serial on the Internet]. 2002 Jun [cited 2002 Aug 12]; 102(6):[about 3 p.]. Available from: http://www.nursingworld.org/AJN/2002/june/ Wawatch.htm