20.500.12556/DKUM-75885 Razvoj metode tkivne rezine in vpliv farmakoloških agensov ter pulzirajočega elektromagnetnega polja na delovanje gladkomišičnih celic sečnega mehurja miši Development of the Acute Tissue Preparation Technique and Influence of Pharmacologic Agents and Pulsed Electromagnetic Field on Murine Detrusor Smooth Muscle Cells Function Fiziologija detruzorja je zelo zapletena, zaradi česar predstavlja preučevanje njegove fiziologije in odzivov na različne dražljaje velik izziv. Do danes je bilo v literaturi opisanih več in vitro metod preučevanja fiziologije sečnega mehurja, vsaka s svojimi omejitvami. Po drugi strani se uroginekologi v svoji klinični praksi srečujejo z naraščanjem incidence prekomerne aktivnosti sečnega mehurja (PASM). Vse to nakazuje potrebo po razvoju novih in učinkovitih metod preučevanja detruzorja, ki bi se potencialno lahko uporabile tudi pri preučevanju različnih terapevtskih možnosti zdravljenja PASM. Namen naše raziskave je bil razviti novo metodo za preučevanje fiziološke aktivnosti GMC detruzorja, ki bi zajemala akutno pripravo tkivnega preparata in uporabo konfokalne mikroskopije. Dodatno smo želeli to metodo uporabiti še za opredelitev učinka pulzirajočega elektromagnetnega polja (PEMP), ki je ena izmed možnih metod zdravljenja PASM, na GMC detruzorja, saj po naših podatkih le-ta v literaturi še ni bil opredeljen. Za pripravo tkivnega preparata smo uporabili izolirane delce detruzorja predhodno žrtvovane odrasle miši NMRI. S svetlobno in transmisijsko elektronsko mikroskopijo smo preučili morfologijo GMC znotraj tkiva. Nato smo s pomočjo konfokalne mikroskopije preučevali kalcijevo dinamiko posameznih GMC med stimulacijo z naraščajočimi koncentracijami karbamilholina (CCh). GMC smo identificirali na podlagi njihove morfologije in kalcijevih odzivov. Odzive GMC smo opredelili z naslednjimi spremenljivkami: zamik do odziva, rekrutacija in relativna aktivnosti celic ter kontrakcija tkiva. Za opredelitev vpliva PEMP na GMC detruzorja smo dva preparata detruzorja iste živali dvakrat zapored dražili s 25 µM CCh. En delec smo med obema stimulacijama izpostavili PEMP, drugi pa je služil kot kontrola. Nato smo opredelili naslednje spremenljivke odziva GMC pred in po stimulaciji s PEMP v primerjavi s kontrolno skupino: število aktivnih GMC, kontrakcija tkiva, čas do kontrakcije tkiva, maksimalni premik tkiva in površina kontrahiranega tkiva. S pomočjo stimulacije tkiva z naraščajočimi koncentracijami CCh smo opredelili tri različne fenotipe odzivov GMC: spontano aktivne GMC z in brez odziva na stimulacijo s CCh ter GMC brez spontane aktivnosti, ki so se odzvale na stimulacijo s CCh. Rekrutacija GMC v aktivno stanje je potekala znotraj ozkega koncentracijskega območja (1–25 µM), ki je povzročilo aktivacijo praktično vseh GMC. Maksimalna kalcijeva aktivnost GMC je bila pri koncentraciji 25 µM, kar je sovpadalo z vidno kontrakcijo tkiva. Dodatno smo pri višjih koncentracijah CCh opažali krajše zamike do pričetka odzivov celic. Ugotovili smo tudi, da bi PEMP lahko imelo neposreden učinek na GMC detruzorja, saj je bil po stimulaciji s PEMP v primerjavi s kontrolno skupino prisoten manjši maksimalni premik tkiva med drugo stimulacijo s CCh kot med prvo stimulacijo s CCh. Dodatno smo ugotavljali tudi statistično značilno manjše razmerje premika tkiva med obema stimulacijama s CCh v primerjavi s kontrolno skupino in večjo razliko v površini kontrahiranega dela tkiva med obema stimulacijama s CCh v primerjavi s kontrolno skupino. Zaključimo lahko, da se je naša nova in situ metoda preučevanja morfologije in fiziologije detruzorja izkazala za učinkovito in reproducibilno. Odzivi GMC na stimulacijo s CCh so bili znotraj fiziološkega območja in primerljivi z rezultati drugih raziskav s tega področja. Ker sama metoda omogoča zadostno prostorsko in časovno ločljivost za preučevanje odzivov več GMC hkrati, verjamemo, da bi lahko imela velik doprinos k preučevanju fiziologije detruzorja. Dodatno smo uspeli dokazati možen učinek PEMP na GMC detruzorja, kar predstavlja pomembno izhodišče za nadaljnje raziskave. Te bodo v prihodnosti skušale raziskati terapevtski učinek na večjem vzorcu in za večjo translacijsko relevantnost modela tudi na človeškem tkivu, pridobljenem pri operacijah sečnega mehurja. Physiology of detrusor muscle is very complex, which makes the investigation of detrusor physiology and its responses to different stimuli very challenging. Several different in vitro techniques for investigating urinary bladder physiology have been described in the literature, each with inherent drawbacks. On the other hand, urogynecologists are faced with increasing incidence of overactive bladder (OAB) syndrome. All of this emphasizes a need to develop new and efficient approaches to investigating detrusor physiology that could potentially also be used in evaluation of new OAB therapeutic modalities. The aim of our study was to develop a novel approach to investigating mouse detrusor SMCs physiological activity by utilizing an acute tissue dissection technique and confocal calcium imaging. Then, we aimed to use this novel approach to evaluate the effect of pulsed electromagnetic field (PEMF) stimulation, which is one of the possible OAB therapeutic options, on mouse detrusor SMCs, as it has not been previously investigated in the literature. For acute tissue preparation, the bladder of a sacrificed adult female NMRI mouse was dissected. We used light and transmission electron microscopy to assess morphology of SMCs within the tissue. Calcium imaging in individual SMCs was performed using confocal microscopy during stimulation with increasing concentrations of carbamylcholine (CCh). SMCs were identified according to their morphology and calcium activity. We determined several parameters describing the SMC responses: delays to response, recruitment, relative activity, and contraction of the tissue. For investigation of PEMF effect on detrusor SMCs, two pieces of detrusor muscle of the same animal were stimulated with 25 µM CCh two times consecutively. One piece was exposed to PEMP in between the two stimulations, while the other served as a control. Then, we determined the following parameters describing the SMC responses before and after PEMP compared to the control group: number of active SMCs, tissue contraction, time to tissue contraction, maximal tissue displacement, and the area of contracted tissue. CCh stimulation revealed three different SMC phenotypes: spontaneously active SMCs with and without CCh-enhanced activity and SMCs with CCh-induced activity only. SMCs were recruited into an active state in response to CCh-stimulation within a narrow range (1–25 µM); causing activation of virtually all SMCs. Maximum calcium activity of SMCs was at about 25 µM, which coincided with a visible tissue contraction. Finally, we observed shorter time lags before response onsets with higher CCh concentrations. Moreover, we discovered that PEMF might exhibit a direct effect on detrusor SMCs responses, as maximal tissue displacement during second stimulation with CCh was smaller than during the first one in preparations, exposed to PEMF, but not in the control group. Additionally, we observed lower ratio of maximal tissue displacement between both stimulations with CCh and larger difference in the area of contracted tissue between both stimulations with CCh in preparations, exposed to PEMF, compared to controls. In conclusion, our novel in situ approach proved to be a robust and reproducible method to study detrusor SMC morphology and physiology. SMC responses to CCh stimulation were within physiologic range and comparable to results of other studies in this field. We believe that our method could add an important insight to detrusor physiology investigation, as it allows sufficient spatial and temporal resolution to investigate responses of several SMCs simultaneously. Furthermore, we managed to demonstrate a possible effect of PEMF on detrusor SMCs, which represents an important foundation for future studies. These shall clarify the abovementioned therapeutic potential on a larger sample, and for a better translational relevance on human tissue obtained during bladder operations. sečni mehur detruzor gladkomišična celica konfokalna mikroskopija miš fiziologija disekcija tkiva prekomerna aktivnost sečnega mehurja pulzirajoče elektromagnetno polje magnetna stimulacija bladder detrusor smooth muscle cell calcium imaging mouse physiology tissue dissection overactive bladder pulsed electromagnetic field magnetic stimulation true true false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2020-02-25 23:00:23 2020-07-17 07:37:48 2022-08-09 11:52:22 2020-07-18 03:11:04 2020 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 22824707 URN:SI:UM:DK:JEKD9HTW DOK_Serdinsek_Tamara_2020.pdf DOK_Serdinsek_Tamara_2020.pdf 1 ED69B109FEC8EE9DF1E86D89BB2C71A2 386be55af4efe5fe547d2d7a827d22df85169f2929264ad6c42659b5010df0f3 64b91b77-7c11-11eb-bb7a-00155d0001ca 20.500.12556/dkum/a487ac23-6115-4c88-8242-0e9f5fc4b7c9 https://dk.um.si/Dokument.php?lang=slv&id=143415 Medicinska fakulteta 0 0 0