Fig 2: CCN2 localization in normal mouse liver. CCN2/EGFP reporter mice were fed control diet for 3 weeks, after which the mice were sacrificed and liver sections subjected to dual IHC staining as follows: (A) EGFP and HNF4α; (B) CCN2 and CK19; (C) EGFP and LYVE1; (D) CCN2 and desmin; (E) CCN2 and αSMA; (F) EGFP and collagen I; (G) EGFP and F4/80; and (H) EGFP and CD45. White arrowheads show representative colocalization of each pair of signals to the same cell. Scale bars: 20 µm, except for 10 µm for lower panel of (A) and 50 µm for upper panel of (C).

Fig 3: Evaluation of hydrocephalus, neurological function and CSF drainage in Cx3cr1-/- mice and control mice after IVH. Cx3cr1-/- mice were generated using CRISPR/Cas9. (A) Schematic diagram depicting the process used to generate Cx3cr1-/- mice. (B) Mouse genotyping by PCR. F\R1 indicates for knockout sequence, and F\R2 indicates the wild-type sequence. (C) Representative WB images of CX3CR1 in the meninges of mice in the Sham, IVH (WT) and IVH (Cx3cr1-/-) groups 24 h after IVH. α-Tubulin was used as an internal reference protein. (D) Quantification of CX3CR1 expression (n = 6, one-way ANOVA). (E, G) Representative T2-weighted images and three-dimensional reconstructions of the lateral ventricles three and seven days after IVH in the Sham group, IVH (WT) group, and IVH (Cx3cr1-/-) group. (F, H) Quantification of the volume of lateral ventricles in each group (n = 6, one-way ANOVA). (I) Representative movement trajectories of mice in the different groups in the open field test 7 days after IVH. (J) The summary data show the distance traveled in the open field experiment (n = 6, one-way ANOVA). (K) Average speed of the different groups in the open field test (n = 6, one-way ANOVA). (L) Representative photomicrographs of LYVE-1-stained dCLNs in Sham, IVH (WT) and IVH (Cx3cr1-/-) group mice were captured 2 h after i.c.m. injection of OVA647. (M) The mean fluorescence intensity of OVA647 in dCLNs was quantified (n = 6, one-way ANOVA). *P < 0.05, **p < 0.01. The data are means ± SEMs.

Fig 4: Neutrophils, NETs, and fibrin were present in mLVs after IVH. Twenty-four h after IVH, the meninges and CLNs of mice were collected for observation. (A) Representative fluorescence photomicrographs showing the immunolabeling of CitH3 in mLVs following IVH. (B) Representative images showing NETs in the TS. (C) Representative fluorescence photomicrographs showing the immunolabeling of Ly6g in mLVs after IVH. (D) Representative fluorescence images showing the immunolabeling of fibrin in mLVs after IVH. (E) Representative fluorescence images showing the immunolabeling of NETs in dCLNs after IVH. (F) Images showing CFSE-labeled neutrophils in Lyve1+ mLVs and dCLNs. (G) TEM revealed the ultrastructural composition of the TS, including sinus ECs, LECs, lymphocytes, neutrophils, and leukocyte interactions after 24 h of IVH. (H) SEM revealed the ultrastructural composition of the TS, including the sinus, mLVs, and entrainment of erythrocytes, polymorphonuclear neutrophils, and fibrin within mLVs following IVH. (I) Representative cases of ICH-IVH. CT imaging showing the presence of hyperdense blood accumulation in the right basal ganglia and ventricular system. (J) Overview of patient characteristics. (K) Assessment of double-stranded DNA (dsDNA) concentrations in CSF within a three-day timeframe in both control individuals and patients diagnosed with ICH-IVH (n =9, non-parametric tests). The data are means ± SEMs (SD), *** p < 0.001.

Fig 5: Evaluation of hydrocephalus, neurological function and CSF drainage with or without CX3CR1 knockdown by AAV after IVH. AAV-shRNAcx3cr1 was engineered to knock down the expression of CX3CR1 in the mLVs of mice by i.c.v. injection. AAV-shRNAnc was injected as a control. Autologous blood IVH mice were generated 28 days after viral injection. (A) Representative photomicrographs of CX3CR1 and LYVE-1 staining in the TS of Sham, AAV-shRNAnc+IVH or AAV-shRNAcx3cr1+IHV groups 24 h after IVH. (B) Assessment of the number of CX3CR1+ LECs in the TS of the meninges (n = 5, one-way ANOVA). (C) Representative WB images of CX3CR1 in the meninges of mice in the Sham, AAV-shRNAnc+IVH and AAV-shRNAcx3cr1+IVH groups 24 h after IVH. ɑ-Tubulin was used as an internal reference protein. (D) Quantification of CX3CR1 expression (n = 6, one-way ANOVA). (E, G) T2-weighted images and three-dimensional reconstructions of the lateral ventricles were obtained three and seven days after IVH in the Sham group, AAV-shRNAnc+IVH group, and AAV-shRNAcx3cr1+IVH group and served as representative examples. (F, H) Quantification of the volume of lateral ventricles in each group (n = 6, 5, one-way ANOVA). (I) Representative open field movement trajectories of mice in the different groups were assessed 7 days after IVH. (J) The summary data show the distance traveled in the open field experiment (n = 6, one-way ANOVA). (K) Average speed of the different groups in the open field test (n = 6, one-way ANOVA). (M) Representative photomicrographs of LYVE-1-stained dCLNs in Sham, AAV-shRNAnc+IVH and AAV-shRNAcx3cr1+IVH mice were captured 2 h after i.c.m. injection of OVA647 (n = 6). (N) The mean fluorescence intensity of OVA647 in dCLNs was quantified (n = 6, one-way ANOVA). (L) mNSSs of the different groups (n = 6, non-parametric tests). ns: P > 0.05, *P < 0.05, **p < 0.01. The data are means ± SEMs.