title: TTC staining of damaged brain areas after MCA occlusion in the rat does not constrict quantitative gene and protein analyses.
abstract: In models of ischemic stroke, TTC (2,3,5-triphenyltetrazolium chloride) staining is commonly applied for the fast and reliable visualization of hypoxic brain tissue and for defining the size of cerebral infarction and penumbra. Deciphering molecular processes of pathogenesis within the penumbra is of particular interest for the development of therapeutic strategies. The aim of this study was to assess whether TTC-stained tissues can easily and in a reliable quantitative manner be processed for further molecular and biochemical analyses. We applied phenol-based RNA isolation, protein lysis by conventional RIPA buffer, and combined RNA/protein isolation with NucleoSpinRNA/Protein-Kit. Gene and protein expression analyses were performed by RT-rtPCR and Western-blotting. Middle cerebral arteria occlusion (MCAO) in rats was performed following a standardized experimental procedure. After MCAO, TTC staining revealed massive cell death in cortical and sub-cortical areas. TTC processing did not affect the quality of tissue RNA and protein. The expression of housekeeping and regulatory genes and proteins revealed no difference between control and TTC-stained groups. The expression of known stroke-regulated genes such as TNFalpha and IL1beta revealed similar induction profiles after TTC staining as described in the literature. TTC staining allows the precise delineation of lesioned and primarily non-lesioned brain areas for subsequent dissection of selected tissue pieces for molecular analysis. Our study demonstrates that TTC-stained tissues in stroke animal models can be used for quantitative gene and protein expression analyses without constriction. Pathomechanisms of ongoing tissue damage within the penumbra region can now be investigated in detail.
authors.affiliation: Faculty of Medicine, Institute of Neuroanatomy, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Kramer
authors.forename: Jon
authors.initials: J
authors.lastname: Dang
authors.forename: Fabian
authors.initials: F
authors.lastname: Baertling
authors.forename: Bernd
authors.initials: B
authors.lastname: Denecke
authors.forename: Tim
authors.initials: T
authors.lastname: Clarner
authors.forename: Christoph
authors.initials: C
authors.lastname: Kirsch
authors.forename: Cordian
authors.initials: C
authors.lastname: Beyer
authors.forename: Markus
authors.initials: M
authors.lastname: Kipp
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D004396:Coloring Agents; D004195:Disease Models, Animal; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006652:Histological Techniques; D020244:Infarction, Middle Cerebral Artery / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D008297:Male; D015336:Molecular Probe Techniques; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020521:Stroke / Q000378:metabolism / Q000473:pathology; D013778:Tetrazolium Salts; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D053583:Interleukin-1beta; D011506:Proteins; D013778:Tetrazolium Salts; D014409:Tumor Necrosis Factor-alpha; D012313:RNA; C009591:triphenyltetrazolium