Angiopoietin-Like 4 Attenuates Brain Edema and Neurological Deficits in a Mouse Model of Experimental Intracerebral Hemorrhage

Figure 1. All animals exhibited similar and marked neurological impairments at day 1 following ICH. (A) Rotarod test. Rotarod score was significantly lower for mice in the control group compared with the ANGPTL4 group from day 3 following ICH. (B) mNSS test. Significant behavioral improvement was observed in ANGPTL4-treated mice compared with the control group from day 7 following ICH. (C) Corner turn test. Significant differences were observed between groups from day 3 to day 14; however, no significant difference was observed at days 1, 21, and 28. Data are presented as the mean±standard deviation, n=8 mice/group/test, * P<0.05. and # P<0.01 vs. ANGPTL4. ANGPTL4 – angiopoietin-like 4; ICH – intracerebral hemorrhage; mNSS – modified neurological severity score.

Figure 2. ANGPTL4 decreased brain damage and edema formation induced by ICH. (A) Hematoxylin and eosin staining revealed tissue damage in the contralateral and ipsilateral striata of control and ANGPTL4-treated mice following intrastriatal collagenase injection. No tissue damage was observed in the contralateral striata. (B) Quantification of hematoma volume normalized to the striatum volume in control and ANGPTL4-treated mice. (C) Brain water content of contralateral and ipsilateral hemispheres and the cerebellum in both groups at 3 days following ICH. (D) Brain water content of the ipsilateral hemispheres of control and ANGPTL4-treated mice at 1, 3, and 7 days following ICH. (E) The difference in brain water content between ipsilateral and contralateral hemispheres in the control group at 1, 3, and 7 days following ICH. (F) The difference in brain water content between ipsilateral and contralateral hemispheres in group at 1, 3, and 7 days following ICH. Data are presented as the mean ± standard deviation. (Hematoxylin-eosin, n=6 mice/group; Brain water content, n=8 mice/group/time-point). * P<0.05 and # P<0.01. ANGPTL4 – angiopoietin-like 4; ICH – intracerebral hemorrhage.

Figure 3. ANGPTL4 specifically protected interendothelial junctions in ICH. (A) Immunostaining of VE-cadherin and claudin-5 in contralateral and ipsilateral striata of control and ANGPTL4-treated mice. Quantification of (B) VE-cadherin- and (C) claudin-5-positive areas normalized to the total EC surface area (Isolectin B4 staining) in contralateral and ipsilateral striata of control and ANGPTL4-treated mice. Data are presented as mean ± standard deviation. Magnification: ×400, Scale bar=20 μm. n=6 mice/group, # P<0.01. ANGPTL4 – angiopoietin-like 4; ICH – intracerebral hemorrhage; VE – vascular endothelial; EC – endothelial cells.

Figure 4. Electron microscopy images of the striata from C57BL/6J mice at 24 and 72 h following ICH. (A, B) Representative images derived from the perihematoma tissue of sham mice revealed a normal blood-brain barrier structure. (C) Markedly swollen astrocytic processes and mitochondria with disorganized cristae were observed in the cytoplasm of all cells around the capillaries in control mice at 1 day following ICH. (D) The structure of basal laminae, astrocytic foot processes, and mitochondria were indistinct, even collapsed, in control mice at day 3 following ICH. (E, F) Mitochondria with moderate swollen cristae were observed in endothelial cells in ANGPTL4-treated mice at days 1 and 3 following ICH. Magnification: ×7800. n=3 mice/group/time-point. ANGPTL4 – angiopoietin-like 4; ICH – intracerebral hemorrhage.

Figure 5. ANGPTL4 specifically protected against ICH-induced VE-cadherin and claudin-5 disruption following stroke. (A) Representative Western blot of protein expressions in the perihematoma regions 24 h following ICH. Relative density was normalized with β-actin and quantified using Image J software. A significant increase in (B) VE-cadherin and (C) claudin-5 levels was observed in the ipsilateral striata of ANGPTL4-treated mice. (D) ANGPTL4 significantly attenuated the level of pSrc compared with the control mice. Data are presented as the mean ± standard deviation. n=6 mice/group, # P<0.01. ANGPTL4 – angiopoietin-like 4; ICH – intracerebral hemorrhage; VE – vascular endothelial; p – phosphorylated.