Fig 2: AQP4 depolarization was worsened in midbrain of A53T mice following blocking of meningeal lymphatics. a Immunofluorescence showed that AQP4 was restrictively expressed at astrocyte endfeet (arrowheads) surrounding CD31 positive microvessels in SN of WT-sham mice, but AQP4 immunoreactive signals were abnormally present at the neuropil domains (stars) of the other three groups, especially in A53T mice that had Ldclns. b Percentage of AQP4 positive area significantly increased in both WT-LDclns mice and A53T-LDclns mice (genotype, F(1,12) = 32.82, p < 0.0001; ligament, F(1,12) = 46.37, p < 0.0001; interaction, F(1,12) = 0.1126, p = 0.7430). c AQP4 polarization, a ratio of perivascular AQP4 immunointensity to neutrophil AQP4 immunointensity, was lower in A53T-LDclns mice than WT-LDclns mice and A53T-sham controls respectively (genotype, F(1,12) = 23.4, p = 0.0004; ligament, F(1,12) = 26.22, p = 0.0003; interaction, F(1,12) = 0.1357, p = 0.7191). d-e Representative immunoblotting bands and densitometry analysis of AQP4 expression from ventral midbrain samples. Levels of AQP4 were increased in both A53T mice and WT mice after LDclns (genotype, F(1,12) = 15.69, p = 0.0019; ligament, F(1,12) = 27.04, p = 0.0002; interaction, F(1,12) = 0.2051, p = 0.6587). f Representative images showed the distribution pattern of AQP4 and a-syn. Notably, dense AQP4 immunoreactivity was closely surrounding a-syn positive cells. Data represent mean ± SEM from 4 mice per group; data in e are from two independent results

Fig 3: (a) Fluorescence staining images and (b) quantified fluorescence intensity of CD31 in ECs on the EC/SMC co-culture scaffold and ECs-only culture scaffold. Scale bars are 10 µm. The CD31 and nucleus of ECs and SMCs were stained with mouse anti-CD31 antibody and DAPI solution, respectively. (c) Real-time PCR histogram of a-SMA and OPN of SMCs on the EC/SMC co-culture scaffold (a-SMA: 20.92 ± 0.91%; OPN: 1.09 ± 0.29%) and SMCs-only culture scaffold (a-SMA: 9.61 ± 0.73%; OPN: 2.86 ± 0.51%). The data are presented as mean ± standard error of the mean. Student's two-tailed t-test was performed to calculate p. Samples were deemed significantly different at a p-value < 0.05 (n = 3).

Fig 4: Immunohistochemistry of NTN4 expression within NB. Representative light microscopy images of a neuroblastoma sample from a female patient (age > 18M); tumor with retroperotineal location and in disseminated stage. (a) Hematoxylin and eosin staining reveals presence of ganglionar-differentiated cells (yellow arrowhead) and primitive neuroblasts (yellow asterisk). (b) Immunohistochemistry of NTN4 with its corresponding negative control (inset). (c) PCNA staining. (d) NTN4 is expressed preferentially in the endothelium (yellow arrows), as confirmed by CD31 staining (inset). (e) Detail of ganglionar-differentiated cells with strong NTN4 cytoplasmic expression in a characteristic punctuated pattern (yellow arrowhead). (f) Close up image of (b) as indicated, highlighting NTN4 expression within the stroma (yellow asterisk). A, B, C; Scale bar = 40 µm. D, E, F; Scale bar = 10 µm; inset = 10 µm.

Fig 5: Bioprinted tissue-on-chip with vascular-like channel. Schematic of the tissue-on-chip design. Confocal images of hiPSC-NPCs encapsulated within 3 wt% ELP-RGD bioprinted on top of a channel seeded with HUVECs and cultured for 5 days. hiPSC-NPCs were visualized with Sox2 immunostaining, HUVECs were visualized with CD31 immunostaining, and all cells were visualized by nuclear staining with DAPI. Scale bars represent 50 µm.