Aims & Methods Flashcards
(16 cards)
What were your two hypotheses?
1) there is an association between VSMC senescence & cSVD in individuals aged 80+
2) delayed VSMC senescence may act as a resilience factor against vascular pathology in individuals aged 80+
What were your aims?
-To determine whether HISTOPATHOLOGICAL measures of VSMC senescence in individuals AGED 80+ are associated wirh POST MORTEM cSVD DIAGNOSIS
Give a one senetence summary of your methods
- Immunohistochemistry to label for a nuclear marker of senescence (H3K9me3) on sections of brain from 27 individuals aged 80+
- Analysis was done blind to clinical data
What were the inclusion criteria for your cohort?
- aged 80+
- minimal AD pathology
- no hereditary CSVD
- clinical info for: age, sex, HTN status, PMI, cSVD score
Tell me about the brain sections
- 6um formalin fixed paraffin embedded sections
- in 10% formalin for at least 1month before wax embedding
- subcortical frontal matter (relevant to cSVD, Lammie 2002)
How was the neuropathological score calculated?
- adjecent slides were sent to a specialist neuropathologist
- H&E and luxol fast blue slides
- PARENCHYMAL PATHOLOGY giving 0-6 score
- 2x sections given 0-3 score
Score based on:
- pallor/loss of mylein staining
- loosening of parenchymal tissue
- enlargement of perivascular spaces
What was the primary antibody used?
- anti-H3K9me3
- unconjugated
- rabbit
- polyclonal
What was the seconary antibody used?
- anti-IgG
- conjugated to HRP
- polymer
What was the positive control antibody?
- anti-hsMM
- unconjugated
- mouse
- monoclonal
What is the difference between monocloncal & polyclonal antibodies?
Monoclonal:
- from one Ab producing B cells
- binds one epitpope
- more specific
- but vulnerable to loss of that epitope
Polyclonal:
- from mutliple B cells
- binds >1 epitope
- higher affinity
- detects low conc or slightly denatured antigens
- risk of some background staining
What was the negative control?
-No primary antibody
Summarise the immunohistochemistry protocol
- dewax
- endogenous peroxidase block
- HIAR
- BSA block
- primary antibody overnight 4degrees (1:10,000)
- washes in PBT
- secondary antibody for 1hr
- washes
- diaminobenzidine
- copper sulfate
- light green SF
- transfer to xylene
- cover slip
Describe the microscopy
- upright light microscope
- photographed vessels 20-160um in diameter on 63x lens
- blind to clinical data
What were your measures of senescnece & how did you generate these?
RATIO:
- manual nuclear counts
- senescent/appearing over total
AF:
- deconvultion into red, green, brown, on brown image
- select stained areas 1-500 sq pixels i.e. stippled nuclei not large uniformly dark
- draw around outer circumference of vessel & create area fraction of the stippled within this
How did you determine a senescent appearing nucleus?
- Identificaiton of SAHF via staining (H3K9me3 at core)
- confident identification of stipples across the whole nucleus
- excluded nuclei with homogenous dark staining or staining just around the nuclear membrane
-also exlcuded endothelial & cells within lumen
Describe the statistics you used & why?
- CASE MEANS (from 6-13 vessels per slide)
- Descriptive statistics including anderson darling normality (normal p>0.05) to determine further statistical analysis (non parametric I chose)
- Mann Whitney U Test (equlaity of two populations - binary data e.g. senescence vs sex, HTN, Y/N dx)
- Pearson correlation (degree of linear relationship - continuous variables e.g. ratio, AF, age)
- Spearman’s rank order correlation (correlation of ranked variables - ordinal variables e.g. cSVD score)
-All on software minitab
