Fig 2: TRPV4 activation enhances the expression of GFAP. A Immunofluorescence images show GFAP protein expression profiles in rat retinal vertical slices acquired from sham-operated retinas (saline-injected; control) (A1), 1 µM GSK101-injected retinas (A2), and 10 µM GSK101-injected retinas (A3). Retinas that received no GFAP antibody served as negative controls (A4). Double immunofluorescence staining showing GFAP expression when the GFAP antibody was pre-adsorbed with its blocking peptide (BP) (A5). Scale bar: 20 µm. B Representative immunoblots showing changes in GFAP protein levels in control and 10 µM GSK101-injected retinas. C Bar chart summarizing mean expression levels of GFAP under different conditions. n = 6. *p < 0.05 vs control

Fig 3: Cellular composition of rat brains with and without orthotopic tumours.Cell populations were determined using flow cytometry in the healthy brain and the tumour-bearing brain, after 7 and 8 days of tumour growth following the implantation of 250,000 RG2-GFP cells (N=6 biological replicates). (a) Pie charts of cellular content from fluorescence-activated cell sorting analysis of healthy and tumour-bearing brains. The other counts include cells, but also debris that could not be gated out successfully. The amount of neurons appeared constant between the different brains. The amount of microglia cells was slightly increased in the presence of the tumour, likely due to the recruitment of TAM. In the tumour-bearing brain, a small amount of tumour cells was positive for GFAP labelling, in accordance with the astrocytic origin of RG2 tumours (green). Representative confocal images of microglia (white) (b–d), astrocytes (white) (e–g) and neurons (white) (h–j) in the tumour bulk, around the periphery of the tumour and on the edge of the ipsilateral hemisphere. (c,f) High density of microglia and astrocytes border the tumour periphery and recruitment of activated microglia/tumour-associated macrophages is notable in the tumour bulk (b). For each cell type and area the image is representative of three slides from one animal. All images were stained with DAPI (blue) for nuclear visualization. (Scale bar, 50 µm).

Fig 4: Chemical constructions of the three compounds, NSCs culture and AD cell model establishment. Chemical constructions of the three compounds. (A) 3,6'-disinapoyl sucrose (DISS); (B) onjisaponin B (OB); (C) tenuifolin (TEN). Hippocampal cells were isolated from newborn C57BL/6 mice; and cultured in proliferation medium for 7 days to obtain neurospheres; and then dissociated the neurospheres into single cells cultured in differentiation medium for 7 days to test the multi-directional differentiation ability. To establish AD cell model; the neurospheres were transfected with APP gene; and RT-PCR and Western blot analysis were used to determine the transfection. (D) The NSCs were double positive for Sox-2 (green) and Nestin (red); Scale bar = 100 µm; (E) The neurons; astrocytes and oligodendrocytes were marketed by NF-M; GFAP and NG2; respectively. Scale bar = 25 µm. (F) The 293 T cells and NSCs transfected with APP-GFP and GFP plasmids expressed green fluorescent protein. Scale bar = 100 µm; (G) The Aß protein and mRNA levels in GFP-NSCs and APP-NSCs were measured by RT-PCR and Western blot assay; respectively; (H-I) Semi-quantification of Aß protein (H) and mRNA (I) as in G. **P < 0.01 vs. GFP-NSCs group. Data are represented as mean ± sem. Data were analyzed by t-test

Fig 5: Confocal images illustrating cellular tropism of NPs 4 and 24 h in the healthy brain.(a–d) Brain slices stained for astrocytes (GFAP in white), showing that PLA–HPG–CHO NPs (red) produced an up-regulation of GFAP protein, characteristic of reactive astrocytes (d), while PLA, PLA–PEG (red) and PLA–HPG NPs (red) did not (a,b,c) at 4 h. (e–h; GFAP in white) After 24 h, all particles except PLA–HPG NPs (red) (g) induced an up-regulation of GFAP proteins. (i–l) Brain slices stained for microglia (Iba-1 in white), showing that PLA (red) and PLA–HPG–CHO NPs (red) activated microglia (i,l, microglia present mostly in amoeboid shape), while PLA–PEG (red) and PLA–HPG NPs (red) did not (j and k, microglia retain their ramified state) at 4 h. (m–p; iba-1 in white) After 24 h, all particles except PLA–HPG NPs (red) (o) activated microglia, with PLA–HPG–CHO NPs (red) showing the largest amount of particles in the perinuclear space (p, Supplementary Fig. 8). For each particle type and staining, the image is representative of three slides from one animal. All images were stained with DAPI (blue) for nuclear visualization. (Scale bar, 20 µm for zoomed in images and 50 µm for larger images).