Histidine provides long-term neuroprotection after cerebral ischemia through promoting astrocyte migration

Abstract

The formation of glial scar impedes the neurogenesis and neural functional recovery following cerebral ischemia. Histamine showed neuroprotection at early stage after cerebral ischemia, however, its long-term effect, especially on glial scar formation, hasn't been characterized. With various administration regimens constructed for histidine, a precursor of histamine, we found that histidine treatment at a high dose at early stage and a low dose at late stage demonstrated the most remarkable long-term neuroprotection with decreased infarct volume and improved neurological function. Notably, this treatment regimen also robustly reduced the glial scar area and facilitated the astrocyte migration towards the infarct core. In wound-healing assay and transwell test, histamine significantly promoted astrocyte migration. H2 receptor antagonists reversed the promotion of astrocyte migration and the neuroprotection provided by histidine. Moreover, histamine upregulated the GTP-bound small GTPase Rac1, while a Rac1 inhibitor, NSC23766, abrogated the neuroprotection of histidine and its promotion of astrocyte migration. Our data indicated that a dose/stage-dependent histidine treatment, mediated by H2 receptor, promoted astrocyte migration towards the infarct core, which benefited long-term post-cerebral ischemia neurological recovery. Therefore, targeting histaminergic system may be an effective therapeutic strategy for long-term cerebral ischemia injury through its actions on astrocytes.

Figure 1. Histidine provides neuroprotection on the neurological function and cognitive abilities after focal cerebral ischemia.

Neurological scores were evaluated on 1 d, 3 d, 7 d after ischemia under the treatment of histamine (A, 200, 500 or 1000 mg/kg), and evaluated on 14 d, 28 d, 42 d, 56 d after ischemia under the treatment of histidine treatment (B, 1000 mg/kg for the first week but 0, 200, 500, or 1000 mg/kg for later weeks, named as His 1000–0, His 1000–200, His 1000–500 or His 1000–1000). Cognitive abilities were examined by Morris water maze (D: Day 22–24; F: Day 50–52) and contextual and cued fear conditioning test (E: Day 27, G: Day 55) after ischemia. The representative swim paths on Day 24 in (D) were shown in (C), and the gray circle indicates the location of the platform. n = 13–15 for A,B (Day 14 and Day 28), D and E; n = 7–9 for B (Day 42 and Day 56), F and G. *P < 0.05, **P < 0.01, ***P < 0.001, compared with sham group within each test day or each test, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with tMCAO group within each test day or each test.

Figure 2. Histidine reduces infarct area and glial scar area after focal cerebral ischemia.

Under the different regimens of histidine treatment, the infarct area was estimated with TB staining on 28 d (C) and 56 d (E) after tMCAO, with the representative layer shown in (A). The glial scar area was also estimated with GFAP staining on 28 d (D) and 56 d (F) after tMCAO, with the representative photo shown in (B) (B1 shows the area quantified; B2-B5 shows the enlarged glial scar edge). n = 10–12 for C and D; n = 6–7 for E and F. B1: bar = 500 μm; B2–5: bar = 100 μm. *P < 0.05, ***P < 0.001, compared with tMCAO group.

Figure 3. Histidine promotes reactive astrocytes migration towards the infarct core.

GFAP immunostaining was performed on 7 d and 14 d after tMCAO (A), and the infarct area surrounded by reactive astrocytes was quantified (B). The morphology of astrocytes from glial scar edge indicated by arrows in A were shown in C1–3, with enlarged images in C4–6 (GFAP: green; DAPI: blue). Arrows in C4–6 indicate the polarized astrocytes. The length (D), the width (E), the ratio of length to width of protrusions (F) and the percentage of polarized astrocyte (G) were quantified at glial scar edge on 14 d after tMCAO. n = 6–8. A: bar = 1 mm; C1–3: bar = 100 μm; C4–6: bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, compared with tMCAO group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with His 1000–0 group.

Figure 4. Histamine promotes astrocyte migration in wound-healing assay and transwell migration assay in vitro.

Phase-contrast micrographs show astrocyte migration under the treatment of histamine (HA) 10⁻⁷ mol/L at 0 h, 24 h and 48 h after the scratch (A, white lines indicate wound edges). The migration distance was quantified under the different concentrations of histamine at 24 h after scratch (B). The effect of histamine on astrocyte migration was also analyzed in transwell migration assay (F), with representative images of migrated astrocytes under the treatment of histamine 10⁻⁷ mol/L (D). Cells with lamellipodia at wound boundary were counted at 2 h or 4 h after scratch under the treatment of 10⁻⁷ mol/L histamine (G), with representative images stained with rhodamine-phalloidin to show F-actin (C, arrows indicate cells with lamellipodia). At 24 h after scratch, the cells under the treatment of histamine 10⁻⁷ mol/L were stained with GFAP in green and DAPI in blue. The morphology of astrocytes at the wound boundary was analyzed, including the length (H), the width (I), the ratio of length to width of protrusions (J) and the percentage polarized astrocyte (K), with the representative images shown in (E). The percentage of astrocytes adhering to poly-L-lysine and laminin 30 min after plating was quantified in L. Values are from 3 to 4 independent experiments. C: bar = 50 μm; D, E: bar = 100 μm. *P < 0.05, **P < 0.01,***P < 0.001, compared with control (CON) group.

Figure 5. Histidine promotes astrocyte migration and provides neuroprotection through H2 receptor.

In wound-healing assay, cimetidine (Cime) at 10⁻⁷ mol/L, famotidine (Famo) at 10⁻⁷ mol/L, or Rp-cAMP at 10⁻⁵ mol/L was administrated after the scratch, while 10⁻⁷ mol/L histamine (HA) or 10⁻⁸ mol/L amthamine (Amth) was added 30 min later. Their effects on astrocyte migration were shown in (A). Cimetidine treatments were also divided into two phases (first week and later week), during which cimetidine was not given or given at the dose of 20 or 100 mg/kg at 30 min before each injection of histidine, including Cime 20–20, Cime 100–100, Cime 0–100 and Cime 100–0 regimens. The morphology of astrocytes from the glial scar edge 14 days after tMCAO was shown in B (GFAP: green; DAPI: blue). The length (C), the width (D), the ratio of length to width of protrusions (E) and the percentage polarizing astrocyte (F) were quantified. The infarct area surrounded by reactive astrocytes was quantified in (G). The neurological deficit score was evaluated on 1 d, 3 d, 7 d and 14 d after tMCAO (H), while the infarct area was also estimated with TB staining (I). A: values are from 3 to 4 independent experiments; B–I: n = 10–12. Bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group (A) or tMCAO group (C–I) within each test day or each test; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with histamine or His 1000–500 group within each test day or each test.

Figure 6. Inhibition of small GTPase Rac1 impedes astrocyte migration and neurological recovery provided by histidine.

In wound-healing assay, cimetidine (Cime, 10⁻⁷ mol/L) or Rp-cAMP (10⁻⁵ mol/L), Rac1 inhibitor NSC 23766 at indicated concentration were administrated after the scratch, while 10⁻⁷ mol/L histamine (HA) was added 30 min later. The GTP-bound small GTPase Rac1 was examined by Western blotting analysis at 24 h after scratch (A). The quantification of migration distance at 24 h after scratch was shown in B. After tMCAO, 50 μg NSC23766 was delivered into cerebral ventricle at 30 min before each injection of histidine from 7 d to 14 d after tMCAO. Its effects on the infarct area surrounded by reactive astrocytes and on the morphology of astrocytes at the glial scar edge 14 days after tMCAO were shown in (C,D) with GFAP immunostaining (GFAP: green; DAPI: blue). The length (E), the width (F), the ratio of length to width of protrusions (G) and the percentage polarized astrocyte (H) were quantified. The neurological deficit score (I) was evaluated on 1 d, 3 d, 7 d, 14 d and 28 d after tMCAO, while the cognitive abilities were evaluated by contextual and cued fear conditioning test (Day 27, J) and Morris water maze (Day 22–24, K) with representative swim paths (Day 24, the gray circle indicates the location of the platform). The glial scar area (L) and infarct area (M) from TB staining were determined on 28 d after tMCAO. A and B: values are from 3 to 4 independent experiments; C–L: n = 10–12. D: bar = 50 μm; L: bar = 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group (A,B) or sham group (J,K) within each test day or each test; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with histamine treatment group A and B) or tMCAO group (C–M); &P < 0.05, &&P < 0.01, &&&P < 0.001, compared with His 1000–500 group within each test day or each test.