Fig 2: Inhibition of NKCC1 in DRGs. (a): The mRNA expression level of NKCC1 gene was detected by qRT-PCR. (b): The protein level of NKCC1 was detected by WB analysis. (c): NKCC1 protein concentration in the three groups using the BCA assay. (d): DAPI staining of DRGs section with outlined spinal nerve dorsal root area and a squared inset of observed ganglion. (e-f): Quantification of NKCC1 immunoreactivity (green) with representative immunofluorescence images of DRGs sections. DAPI, blue; scale bar, 50 μm.

Fig 3: Schematic illustration of ischemic stroke-induced changes of the ChP blood-CSF barrier integrity. Ischemic stroke triggers the damage of blood-CSF barrier and results in permeability increase which lasts at least 7 days post-stroke, which reflected by uptake of the albumin by CPECs, the breakdown of TJs and infiltration of circulating immune cells, etc. Ischemic stroke also causes upregulation of proinflammatory Lcn2 gene and LCN2 protein expression especially in the early acute post-stroke stage, accompanied with sustained reduction of choroidal SPAK-NKCC1 protein expression but activation of SPAK as well as NKCC1 phosphorylation. Ischemic stroke also induced infiltration of CD45+/CD11b+ myeloid cells into the ChP. Taken together, stroke-mediated damages of the ChP may lead to inflammation and dysregulation of CSF.

Fig 4: Dynamic changes of SPAK-NKCC1 complex phosphorylation in LVCP after ischemic stroke. (A) Representative confocal immunofluorescence staining images of phosphorylated SPAK and NKCC1 proteins in LVCP (IL: ipsilateral) at 5-day post-stroke or sham surgery. Arrow: high expression. Arrowhead: low expression. (B) Quantification of pSPAK and pNKCC1 fluorescence intensity. Data are mean ± SEM, n = 3. **p < 0.01; ***p < 0.001.

Fig 5: Inhibition of NKCC1 in spinal cords. (a): The mRNA expression level of NKCC1 gene was detected by qRT-PCR. (b): The protein level of NKCC1 was detected by WB analysis. (c): NKCC1 protein concentration in the three groups using the BCA assay. (d): DAPI staining of spinal cord section with outlined gray matter area and a squared inset of observed ventral horn. (e): Immunostaining of NKCC1 (green) in the T10 region of spinal cords in different groups. DAPI, blue; sale bar, 50 μm. (f): Quantification of NKCC1 immunoreactivity. n.s.: Not significant between the three groups.