In vivo perfusion staining of atherosclerotic lesions and a novel quantification method for lesion size in sequential aortic root sectioning in murine models

ObjectivesTodevelopasimple,accurateandreproduciblemethod,whichcombinesmacroandhistopathologicaltechniquesfordeterminingthedegreeoflipiddepositioningeneticallymodifiedmice.MethodTheentireaortasfromC57BL/6,ldlr-/-andapoE-/-micewerestainedwithSudanⅣusingeitherinvivoperfusionortraditionalinvitroenfacestainingtechniques.Histologicalsectionsofaorticrootandheartswereembeddedintissuefreezingmediumandcutwithacryostat,thenstainedwithOilRedO.Thecalculatedaorticrootareabasedontheaorticrootcircumferencewasusedtoreducemeasurementerrors.ResultsTheinvitroenfacestainingcanstainallfat,whichincludetheadventitialtissuearoundaorta.Howevertheinvivoperfusionstainingcanspecificallystainthefattydepositioninsideofaorta.Bothentireaortaandaorticrootsectionstainingshowedthattherewasahighlysignificantincreaseinfattydepositionintheaortasofthegeneticmodifiedmice.Althoughallmicegeneticbackgroundwassame,theapoE-/-micehadlargeratheroscleroticlesionsthanldlr-/-mice.ConclusionsThenewinvivoperfusionmethodismoreaccuratethantheinvitroenfacemethod.Thecombinationofthesemacroandmicroscopictechniquesovercomestheshortcomingsoftheearlierpublishedmethodswhicharegenerallylimitedtothemeasurementoffattyredstainingareasonly,neglectingnon-specificadventitialfatstainingaroundaortaandaorticrootsectiontissuedistortion.