Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: BBB maturation stages in the postnatal cerebral cortex based on glial configuration. (A) 3D images of AQP4, Iba1, lectin, and DAPI staining in the rat cerebral cortex at P1–30 are shown. Scale bar indicates 30 μm. AQP4 gradually accumulated in the blood vessels and localized to the blood vessels after P15. The number of Iba1+ microglia significantly increased after P10 and peaked at P15, changing their shape from ameboid to ramified. (B) Temporal changes in biotin permeability are corrected to a maximum value of 100%. Typical images of P4 and P15 rat brain cortical regions stained with streptavidin are shown. A solid line was drawn perpendicular to the blood vessel to quantify the fluorescence, which is indicated by the dashed line, and the red fluorescence value was measured, as shown in the upper right graph. The traces show the average of 70–76 transverse vessel lines for the respective postnatal ages. The maximum fluorescence value was set at 100, and the sum of the fluorescence values 20 μm to the left and to the right was calculated. The peak became sharper as the age increased. Astrocyte and microglial coverage rates were corrected to a P30 value of 100%. BBB formation and maturation in rats are classified into three phases based on biotin leakage and astrocyte and microglial contact with blood vessels . The phases are as follows: the “immature stage” when biotin permeability is still high; the “organic formation stage” when glial structures around blood vessels are formed; and the “completion stage” when the structures around blood vessels become stable and the morphology of microglia changes to a ramified type. Abbreviations: AQP4: aquaporin 4; BBB: blood–brain barrier; DAPI: 4′,6-diamidino-2-phenylindole; Iba1: ionized calcium binding adapter protein 1; P: postnatal day.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Staining, Permeability, Fluorescence, Binding Assay

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Expression of blood vessel developmental markers in the postnatal cerebral cortex. (A) Images of CD31, CD34, CD146, agrin, and Tie2 co-stained with lectin and DAPI in the rat cerebral cortex at P1–30 are shown. Scale bar indicates 50 μm. CD31 and Tie2 signals gradually increased in blood vessels, whereas CD34, CD146, and agrin signals gradually decreased. (B) The graphs show temporal changes in the percentage of marker+ area relative to the total vessel area. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 Images were obtained from the cerebral cortical regions of three rat pups of each age with a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01. vs. P1 value, while # p < 0.05, ## p < 0.01 vs. P10 value. (C) Graph summarizing results for B and B. Changes in biotin permeability, CD34, CD146, and agrin were corrected to a P1 value of 100%, and the astrocyte coverage rate, microglial coverage rate, CD31, and Tie2 were corrected to a P30 value of 100%. The grey zone represents the organic formation term (P4P15). Abbreviations: CD31, cluster of differentiation 31 or platelet endothelial cell adhesion molecule 1; CD146, melanoma cell adhesion molecule; DAPI: 4′,6-diamidino-2-phenylindole; P, postnatal day; Tie2, TEK receptor tyrosine kinase; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy, Permeability

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Analysis of vascular TJ protein expression in the postnatal cerebral cortex. (A) Images of claudin-5, occludin, and ZO-1 co-stained with lectin and DAPI in the rat cerebral cortex at P1–30. Scale bar indicates 100 μm. Claudin-5 and occludin signals increased in blood vessels during development, whereas ZO-1 signals remained unchanged. (B) The graphs show changes in the percentage of marker+ area of the total vessel area at P4, P15, and P30. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 images were obtained from the cerebral cortical regions of three rat pups from each age group using a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01 vs. P4 value, and # p < 0.05 vs. P15 value. DAPI: 4′,6-diamidino-2-phenylindole; P, postnatal day; TJ, tight junction; ZO-1: zonula occludens-1; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy

Journal: Frontiers in Neuroanatomy

Article Title: Search for marker proteins to assess blood–brain barrier development

doi: 10.3389/fnana.2026.1717532

Figure Lengend Snippet: Analysis of vascular expression of transporters and receptor proteins in the postnatal cerebral cortex. (A) Images of P-gp, BCRP, Glut1, and TfR co-stained with lectin and DAPI in the rat cerebral cortex at P1–30. Scale bar indicates 100 μm. The arrowhead shows the co-localization areas of the marker and lectin. P-gp and BCRP signals gradually increased in the blood vessels during development, whereas Glut1 and TfR signals were high in the blood vessels at P1 and remained unchanged. (B) The graphs show changes in the percentage of marker+ area of the total vessel area at P4, P15, and P30. The colocalized signals between the markers and blood vessels were calculated, and the areas of the colocalized signals were normalized to the total blood vessel area in each image. Approximately 9–15 images were obtained from the cerebral cortical regions of three rat pups from each age group using a Nikon A1R-A1 confocal microscope. Data are shown as averaged value ± SEM. Data were analyzed using an ANOVA followed by a Tukey’s multiple range test. * p < 0.05, ** p < 0.01 vs. P4 value. Abbreviations: BCRP: breast cancer resistance protein; DAPI: 4′,6-diamidino-2-phenylindole; Glut1: glucose transporter type 1; P: postnatal day; P-gp: P-glycoprotein; TfR: transferrin receptor; ANOVA, analysis of variance.

Article Snippet: After rinsing, the sections were stained with lectin (1:200; DL1177; Vector Labs, Newark, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; 342-07431, Dojindo, Kumamoto, Japan).

Techniques: Expressing, Staining, Marker, Microscopy