Title Kutana inervacija miša s isključenim genom St8sia1
Title (english) Cutaneous innervation of St8sia1 knockout mice
Author Renata Brčić
Mentor Natalija Filipović (mentor)
Committee member Snježana Mardešić Brakus (predsjednik povjerenstva)
Committee member Ivana Pavlinac Dodig (član povjerenstva)
Committee member Marino Vilović (član povjerenstva)
Granter University of Split School of Medicine Split
Defense date and country 2021-07-14, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Basic Medical Sciences Cytology, Histology and Embryology
Abstract Ciljevi: Cilj ovog rada bio je istražiti da li će isključivanje gena St8sia1 utjecati na perifernu inervaciju kože uške miša i na gustoću različitih tipova živčanih vlakana s obzirom na to da se mutacije gena St8sia1 povezuju s neurodegenerativnim procesima.
Materijali i metode: U istraživanju je korišteno trinaest miševa u dobi od šest mjeseci, od kojih je osam miševa s isključenim genom St8sia1, a pet je kontrolnih miševa divljeg tipa (C57BL/6 soj). Nakon eutanazije, uzeto je tkivo uške miša koje je zatim uklopljeno u parafinske blokove. Dobiveni histološki preparati bojani su imunohistokemijski po standardnom postupku. Primarna protutijela koja su se koristila su Anti–NF52, Anti–CGRP, Anti–TH i Anti–ChAT. Nakon inkubacije s primarnim protutijelima, uzorci su ispirani s PBS-om te su nakon ispiranja inkubirani sat vremena sa sekundarnim protutijelima. Korištena sekundarna protutijela su: Donkey Anti-Rabbit IgG, Alexa Fluor® 488 i Donkey Anti–Sheep IgG H+L Alexa Fluor® 488. Preparati su pregledani BX51 Olympus mikroskopom opremljenim hlađenom digitalnom kamerom te su uzete fotografije za daljnju analizu. Mikrofotografije su dalje analizirane u Adobe Photoshop CS2 programu, a područje od interesa bili su koža i potkožno tkivo uške miša. Rezultati su statistički obrađeni u GraphPad Prism 8.0.1. programu, a za statistički značajnu razliku postavljena je vrijednost P < 0,05.
Rezultati: Analizom postotaka površine N52 imunoreaktivnih živčanih vlakana pronađena je statistički značajna razlika u smanjenu gustoće inervacije kože miša uspoređujući WT i KO skupine (P = 0,049). Isto tako, gustoća ChAT-imunoreaktivnih živčanih vlakana bila je smanjena u miševa s isključenim genom St8sia1 (P = 0,035). Za razliku od prethodnog, nije pronađena značajna razlika u gustoći simpatičke inervacije kože (TH marker) i potkožnog tkiva između miševa divljeg tipa i miševa s isključenim genom St8sia1 (P = 0,122). Nadalje, gustoća inervacije peptidergičkim, CGRP imunoreaktivnim, živčanim vlaknima nije se razlikovala između dvaju istraživanih sojeva (P = 0,349).
Zaključci: Isključivanje gena St8sia1 u miševa rezultira poremećajem periferne inervacije, koji se očituje smanjenjem gustoće perifernih živčanih vlakana u koži. Poremećaj periferne inervacije ima selektivan učinak samo na mijelinizirana živčana vlakna, dok nemijelinizirani neuroni nisu bili zahvaćeni.
Abstract (english) Diploma thesis title: Cutaneous innervation of St8sia1 knockout mice
Objectives: The aim of this study was to investigate whether the knock out of the St8sia1 gene will affect peripheral innervation of mouse ear skin and the density of different types of nerve fibers since mutations in the St8sia1 gene are associated with neurodegenerative processes.
Materials and methods: Thirteen six-month-old mice were used in the study, eight of them were St8sia1-knock out mice and five of them were control-type wild-type mice (C57BL / 6 strain). After euthanasia, mouse ear tissue was taken and then embedded in paraffin blocks. Obtained histological preparations were stained immunohistochemically according to standard procedures. The primary antibodies used were Anti-NF52, Anti-CGRP, Anti-TH and Anti-ChAT. After incubation with the primary antibodies, the samples were washed with PBS and after being washed out the samples were incubated for one hour with the secondary antibodies. Secondary antibodies that were used are Donkey Anti-Rabbit IgG, Alexa Fluor® 488 and Donkey Anti-Sheep IgG H + L Alexa Fluor® 488. Histological preparations were examined with a BX51 Olympus microscope equipped with a cooled digital camera and photographs were taken for further analysis. The microphotographs were further analyzed in Adobe Photoshop CS2 and the areas of interest were the skin and subcutaneous tissue of the mouse ear. The results were statistically processed in GraphPad Prism 8.0.1. program and for a statistically significant difference was set the value of P <0,05.
Results: Analysis of the percentage of N52 surface area of immunoreactive nerve fibers has shown a statistically significant difference in the reduced innervation density of mouse skin comparing WT and KO groups (P = 0,049). Likewise, the density of ChAT-immunoreactive nerve fibers was reduced in the St8sia1 knock out mice (P = 0,035). Unlike the previous one, no significant difference was found in the density of sympathetic innervation of the skin (TH marker) and subcutaneous tissue between wild-type mice and St8sia1 knock out mice (P = 0,122). Furthermore, the density of innervation by peptidergic CGRP immunoreactive nerve fibers did not differ between the two studied strains (P = 0,349).
Conclusions: Knock-out of the St8sia1 gene in mice results in a disturbance of peripheral innervation, which is manifested by a decrease in the density of peripheral nerve fibers in the skin. Peripheral innervation disorder has a selective effect only on myelinated nerve fibers, while unmyelinated neurons were not affected.
Keywords
gen St8sia1
GD3 sintaza
gangliozidi
TH
ChAT
N52
CGRP
Keywords (english)
St8sia1 gene
GD3 synthase
gangliosides
TH
ChAT
N52
CGRP
Language croatian
URN:NBN urn:nbn:hr:171:534960
Study programme Title: Medicine Study programme type: university Study level: integrated undergraduate and graduate Academic / professional title: doktor/doktorica medicine (doktor/doktorica medicine)
Type of resource Text
File origin Born digital
Access conditions Open access
Terms of use
Created on 2021-07-19 14:09:23