Intracerebroventricular (i.c.v.) injection of thrombin (20 U/animal, i.c.v.) causes reductions in brain microvascular endothelial cell (BMVEC) immunoreactivity after 1 day, and subsequent BMVEC proliferation around the rat brain vessels in lacunosum moleculare layer (LMol) of the hippocampus after 7 days and 14 days. Panels A-C show rats following sham operations labeled for BrdU, bromodeoxyuridine (A), RECA-1, rat endothelial antigen-1 (B) and the overlay or Merged image (C). RECA-1⁺ cells demonstrate the tube-shape of brain capillaries (arrows in panel C). Panels D to F show BrdU (D), RECA-1 (E) and the Merged image (F) at 1 day after thrombin injections. Compared with the sham group, RECA-1⁺ cells tend to lose their tube-shape at 1 day following thrombin injections (arrows in panel F) and there were no BrdU⁺ cells co-labeled with RECA-1 at one day (panel F). Panels G-I show the staining for BrdU (G), RECA-1 (H) and the Merged image (I) 1 day after thrombin plus PP2 injections. PP2 administration at day 0, immediately after thrombin injection, blocks the thrombin-induced loss of tube-shape of RECA-1⁺ cells. (Legend for first portion of Figure 2, Panels A-I).
Panels J-L show the staining of BrdU (J), RECA-1 (K) and the Merged image (L) 7 days after thrombin injection. Compared to 1 day, BrdU⁺ cells are increased 7 days after thrombin injection. Some of these BrdU⁺ cells are co-labeled with RECA-1 (arrows in panel L and M). A few brain capillaries regained their tube-shape, though not completely. Panel M shows a higher power image of Panel L (area within dashed lines). RECA-1 stained BMVEC are red. The BrdU⁺/RECA-1⁺ double-labeled new born BMVEC nuclei are yellow. Panels N to P show the staining for BrdU (M), RECA-1 (N) and the Merged image (P) 14 days after thrombin injection. Compared to 7 days, BrdU⁺ cells are decreased, but some BrdU⁺ cells (N) remain co-labeled with RECA-1 (arrow in panel P), and more and more brain capillaries regained the tube-shape 14 days after the thrombin injection. Scale bars: A-P, 50 μm. (Legend for second portion of Figure 2, Panels J-P).
Panel Q shows the density of BrdU⁺ RECA-1⁺ double-labeled cells counted in LMol layer of hippocampus in each experimental group. Each column and vertical bar represents the mean ± standard error of the mean. ** p<0.01 vs. Cont (one-way ANOVA followed by Tukey's post hoc test). Cont = control. PP2 = non-specific src family kinase inhibitor. Throm = thrombin. RECA-1 = rat endothelial cell antigen-1. BrdU = bromodeoxyuridine, a marker of cell proliferation. (Legend for third portion of Figure 2, Panel Q).

Brain sodium fluorescein (NF, panel A) and Evans blue (EB, panel B) extravasation increased 1 day after thrombin (Throm) injections (20U, i.c.v.), and decreased at 7 and 14 days. The thrombin inhibitor hirudin (Hir, 20U) blocked thrombin-induced NF/EB extravasation at 1 day after co-injection into the cerebral ventricles. PP2 (src family kinase inhibitor) administered with thrombin (day 0) blocked the NF/EB extravasation at 1 day after thrombin injection, whereas delayed PP2 administration (days 2-6) postponed alleviation of NF/EB extravasation at 7 days post-thrombin injection. Each column and vertical bar represents the mean ± standard error of the mean. ** p<0.01 vs. Cont; ^#p<0.05, ^##p<0.01 vs. Throm/1day, ^‡‡ p<0.01 vs. Throm/7days (one-way ANOVA followed by Tukey's post hoc test).

The Lacunosum moleculare layer (LMol) is located between CA1 pyramidal neurons and the molecular layer of the dentate gyrus (MoDG) in the hippocampus. The LMol is characterized by a series of large (10 - 50 μm diameter) blood vessels (marked with stars) that were perpendicular to the plane of these coronal sections in every animal in this study. Scale bar: 500 μm.

Intracerebroventricular injection of thrombin (20 U/animal, i.c.v.) causes reductions in astrocyte GFAP immunoreactivity after 1 day, and subsequent astrocyte proliferation around the rat brain vessels in lacunosum moleculare layer (LMol) of the hippocampus after 7 days and 14 days. Panels A-C show rats with sham operation labeled for BrdU (A), GFAP (B), and Merged image (C). GFAP⁺ cells envelop most all of the brain vessel (arrows in panel B). Panels D-F show BrdU (D), GFAP (E) and the Merged image (F) at 1 day after thrombin injection. Compared with the sham group, there is decreased GFAP immunoreactivity around brain vessels. There are a few BrdU⁺/GFAP^- cells located close to the vessel (arrows in panel F). G-I show the staining for BrdU (G), RECA-1 (H) and the Merged image (I) 1 day after thrombin plus PP2 injections. PP2 administration at day 0, immediately after thrombin injection, blocks the thrombin-induced reductions in GFAP immunoreactivity. (Legend for first portion of Figure 3, Panels A-I).
J-L show the staining for BrdU (J), GFAP (K) and the Merged image (L) 7 days after thrombin injection. Compared to 1 day, BrdU⁺ cells are increased 7 days after thrombin injection (J, arrows). Many of these BrdU⁺ cells are co-labeled with GFAP (arrows in panel L). Panel M shows a higher power image of Panel L (area within dashed lines). GFAP stained astrocytes are red. The BrdU⁺ GFAP⁺ double-labeled new born astrocytic nuclei are yellow (arrows, Panel M). Panels N-P show the staining for BrdU (N), GFAP (O) and the Merged image (P) 14 days after thrombin injection. Compared to 7 days, BrdU⁺ cells are decreased 14 days after thrombin injection. Some BrdU⁺ cells (N, arrow) remain co-labeled with GFAP (arrow in panel P) 14 days after the thrombin injection. Scale bars: A-P, 50 μm. (Legend for second portion of Figure 3, panels J-P).
Panel Q shows density of BrdU⁺ GFAP⁺ double-labeled cells counted in the LMol of hippocampus in different conditions. Each column and vertical bar represents the mean ± standard error of the mean. ** p<0.01 vs. Cont (one-way ANOVA followed by Tukey's post hoc test). Abbreviations are the same as Figure 2. GFAP = astrocyte marker. Cont=control. Throm = thrombin. PP2 = non-specific src family kinase inhibitor. (Legend for third portion of Figure 3, Panel Q).

Brain edema (water content) increased at 1 day after thrombin (Throm) injections (20U, i.c.v.), and decreased by 7 and 14 days. The thrombin inhibitor hirudin (Hir, 20U, i.c.v.) blocked elevation thrombin-induced brain water content at 1 day after co-injection into the cerebral ventricle. Administration of PP2 (src family kinase inhibitor) at day 0 blocked the increase in brain water content observed at 1 day after thrombin injection, whereas delayed PP2 administration (days 2-6) prevented the resolution of brain water content at 7 days post-thrombin injection. Each column and vertical bar represents the mean ± standard error of the mean. ** p<0.01 vs. Cont; ^#p<0.05, ^##p<0.01 vs. Throm/1day, ^‡ p<0.05 vs. Throm/7days (one-way ANOVA followed by Tukey's post hoc test).