Fig 1.
Dosing and sacrifice schedule.
Illustration of the dosing schedule exhibiting the treatment of the 3 groups of mice kept.
Fig 2.
Thioflavin-S positive plaque quantification.
Tissue sections were incubated with an an antibody against amyloid-β, SC-28365 (i) or 6E10 (ii) (-red- excitation λ: 555 emission λ:580). The sections were then co-stained with Thioflavin-S (-green- excitation λ: 430 emission λ:550). Areas of co-localisation were considered as fibrillar (insoluble) amyloid deposits.
Fig 3.
Immunoblot analysis of the CD88 marker revealing increased expression of the receptor in 5XFAD animals treated with EP67, both at 3 months and 6 months compared to both the wild type and untreated 5XFAD animals. (Immunoblot images indicate representative sample runs and were cropped as indicated by the dotted white line).
Fig 4.
(a) Representative sagittal brain sections from 3 and 6 month old wild type mice (ai–aiii, x–xii respectively), 5XFAD (iv–vi, xiii–xv respectively) mice and 5XFAD mice treated with EP67 (vii–ix, xvi–xviii respectively). These sections were co-stained with Thioflavin-S (-green- excitation λ: 430 emission λ:550) and an anti-amyloid-β antibody (-red- excitation λ: 555 emission λ:580). Representative images of the cortex, thalamus and HPC are shown. Scale bar: 300μm. Serial sections were double stained with Thioflavin-S and an anti β-Amyloid antibody to quantify amyloid plaque deposition in the hippocampus (HPC) (b) and cortex (c). n = 6/group/age. Mean ± 1SD. (d&e) Whole hemisphere brain homogenates from wild type, 5XFAD and 5XFAD mice treated with EP67 at ages 3 and 6 months were used to measure the amount Aβ 40 (d) and Aβ 42 (e), n = 6/group/age.
Fig 5.
(a) Wild type, 5XFAD and 5XFAD EP67 treated mice of 3 and 6 months of age were given the spontaneous alternation behavioural test using a Y-maze. (b) The number of arm entries for each group was recorded and exhibited no significant difference among any group of animals. n = 6/group/age. Mean ± 1SD.
Fig 6.
Immunoblots against synaptophysin were prepared. n = 6/group/age. Mean ± 1SD. (Immunoblot images indicate representative sample runs and were cropped as indicated by the dotted white line).
Fig 7.
Representative sagittal cortex sections from 3 and 6 month old wild type (a, a’ & d, d’), 5XFAD (b, b’ & e, e’) and 5XFAD EP67 treated (c, c’ & f, f’) mice sections were co-stained with an antibody against Synaptophysin (-red- excitation λ: 358 emission λ:461) and DAPI nuclear staining (-blue- excitation λ: 555 emission λ:580). Scale bars: a-c & d-f 150 μm, a’- c’ 75 μm and d’-f’ 48 μm.
Fig 8.
Representative sagittal cortex sections from 3 and 6 month old wild type (a, a’ & d, d’), 5XFAD (b, b’ & e, e’) and 5XFAD EP67 treated (c, c’ & f, f’) mice sections were co-stained with an antibody against NeuN (-red- excitation λ: 358 emission λ:461) and DAPI nuclear staining (-blue- excitation λ: 555 emission λ:580). Scale bars: a-c & d-f 150 μm, a’- c’ 75 μm and d’-f’ 48 μm.
Fig 9.
(a) Immunoblots against GFAP were prepared. n = 6/group/age. Mean ± 1SD. (b) Immunoblots against F4/80 were also carried out. n = 6/group/age. Mean ± 1SD. (d) (Immunoblot images indicate representative sample runs and were cropped as indicated by the dotted white line).
Fig 10.
Representative sagittal cortex sections from 6 month old wild type (a & a’), 5XFAD (b & b’) and 5XFAD EP67 treated (c & c’) mice sections were co-stained with an antibody against GFAP (-red- excitation λ: 358 emission λ:461). Scale bars: a-c 150 μm, a’- c’ 75 μm.
Fig 11.
Representative sagittal cortex sections from 3 and 6 month old wild type (a, a’ & d, d’), 5XFAD (b, b’ & e, e’) and 5XFAD EP67 treated (c, c’ & f, f’) mice sections were co-stained with an antibody against F4/80 (-red- excitation λ: 358 emission λ:461) and DAPI nuclear staining (-blue- excitation λ: 555 emission λ:580). Scale bars: a-c & d-f 150 μm, a’- c’ 75 μm and d’-f’ 48 μm.
Fig 12.
Phagocytes gene expression levels in the brain.
The levels of gene transcripts were measured through RT-QPCR. (a) MCP-1 a monocyte chemoattractant was found to be significantly increased in animals receiving the EP67 treatment, MIP- 2a (b) the leukocyte chemoattractant appears to be massively overexpressed following treatment with EP67. The expression of the microglia specific marker TMEM119 (c) remains unchanged amongst all groups. CCR2 (d) and LY6C (e), both found to be expressed in monocytes also appear to be significantly increased in animals receiving the EP67 treatment. Comparative CT Method (ΔΔCt) against relevant wild type samples, n = 5/group/age.
Fig 13.
Representative sagittal cortex sections from 6 month old 5XFAD (i-iii) and 5XFAD EP67 treated (iv-vi) mice were co-stained with Thioflavin-S (-green- excitation λ: 430 emission λ: 550), an antibody against the neutrophil marker Neutrophil Elastase (ELANE) (-red- excitation λ: 555 emission λ:580) and DAPI (-blue- excitation λ: 350 emission λ:470). Immunoblots against another neutrophil marker LY6G were also prepared. n = 6/group/age. Mean ± 1SD. Scale bar: 150 μm. (Immunoblot images indicate representative sample runs and were cropped as indicated by the dotted white line).