Canine muscle cell culture and consecutive patch-clamp measurements - a new approach to characterize muscular diseases in dogsReport as inadecuate




Canine muscle cell culture and consecutive patch-clamp measurements - a new approach to characterize muscular diseases in dogs - Download this document for free, or read online. Document in PDF available to download.

BMC Veterinary Research

, 8:227

Cardiovascular, gastrointestinal, urological medicine

Abstract

BackgroundThe recognition of functional muscular disorders, e.g. channelopathies like Myotonia is rising in veterinary neurology. Morphologic e.g. histology and even genetic based studies in these diseases are not able to elucidate the functional pathomechanism. As there is a deficit of knowledge and skills considering this special task, the aim of the current pilot study was to develop a canine muscle cell culture system derived from muscle biopsies of healthy client-owned dogs, which allows sampling of the biopsies under working conditions in the daily veterinary practise.

ResultsMuscular biopsies from 16 dogs of different age and breed were taken during standard surgical procedures and were stored for one to three days at 4°C in a transport medium in order to simulate shipping conditions. Afterwards biopsies were professionally processed, including harvesting of satellite cells, inducing their proliferation, differentiating them into myotubes and recultivating myotubes after long-term storage in liquid nitrogen. Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation. Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.

ConclusionWe have developed a canine muscle cell culture system, which allows sampling of biopsies from young and old dogs of different breeds under practical conditions. Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

KeywordsMyotubes Voltage gated ion channels Functional Dog Animal models AbbreviationsAqua destDistilled water

°CDegree Celsius

CaClCalcium Chloride

Cat. No.Category number

CO2Carbondioxide, cm, Cubic-centimeter

DMSODimethyl sulfoxide

idIdentification of biopsy donor

DAB3,3′-diaminobenzidine-tetrahydrochloride

DABCO1, 4-Diazabicyclo 2.2.2 octane

DAPI4′, 6-diamidino-2- 327 phenylindole, dihydrochloride

DMEMDulbecco’s modified Eagle Medium

FBSFoetal Bovine Serum

gGramm

HEPES3.6 g N- 2-hydroxyethyle piperazine-N-2- ethansulphonic acid

H2O2Hydrogenperoxide

KClPotassium Chloride

KOHPotassium hydroxide

mmCubic-millimetres

MgCl2Magnesium Chloride

MΩMega Ohm

mLMillilitre

mMMillimolar

mosmLMilliosmol

mVMillivolt

μmMicrometers

min.Minutes

NaClSodium Chloride

NaOHSodium hydroxide

PSGPorcine Skin Gelatine, PFA, Paraformaldehyde

PBSPhosphate Buffered Saline, RT, Room Temperature

TBSTRIS-buffered saline.

Electronic supplementary materialThe online version of this article doi:10.1186-1746-6148-8-227 contains supplementary material, which is available to authorized users.

Download fulltext PDF



Author: Henning Christian Schenk - Klaus Krampfl - Wolfgang Baumgärtner - Andrea Tipold

Source: https://link.springer.com/







Related documents