Aquaporin-4 activation facilitates glymphatic system function and hematoma clearance post-intracerebral hemorrhage

Abstract

Efficient clearance of hematomas is crucial for improving clinical outcomes in patients with intracerebral hemorrhage (ICH). The glymphatic system, facilitated by aquaporin-4 (AQP4), plays a crucial role in cerebrospinal fluid (CSF) entry and metabolic waste clearance. This study examined the role of the glymphatic system in ICH pathology, with a focus on AQP4. Collagenase-induced ICH models were established, with AQP4 expression regulated through mifepristone as an agonist, TGN-020 as an inhibitor, and Aqp4 gene knockout. Fluorescence tracing and multimodal magnetic resonance imaging (MRI) were employed to observe glymphatic system functionality, hematoma, and edema volumes. Neurological deficit scoring was performed using the modified Garcia Scale. AQP4 expression was quantified using RT-qPCR and Western blotting, and cellular localization was explored using immunofluorescence. The brain tissue sections were examined for neuronal morphology, degenerative changes, and iron deposition. Three days post-ICH, the AQP4 agonist group showed increased AQP4 protein expression and perivascular polarization, decreased hemoglobin levels, and reduced iron deposition. Conversely, the inhibition group exhibited contrasting trends. AQP4 activation improved glymphatic system function, leading to a wider distribution, improved neurological function, and reduced hematoma. Pharmacological inhibition and genetic knockout of AQP4 have opposing effects. The glymphatic system, facilitated by AQP4, plays a crucial role in hematoma clearance following cerebral hemorrhage. Upregulation of AQP4 improves glymphatic system function, facilitates hematoma clearance, and promotes brain tissue recovery.

Keywords: AQP4; glymphatic system; hematoma clearance; intracerebral hemorrhage.

FIGURE 1

Impact of AQP4 on neurological damage, BBB permeability, hemoglobin content, and brain water content after ICH. (a) Experimental schematic. The brain tissue samples and coronal sections of brain tissue with hemorrhagic lesions were obtained. (b) Coronal section image of hematoma in mice brain after ICH. Black scale bar: 2 mm (c) Neurobehavioral assessments of mice at various time points in different groups. (n = 5). (d) Quantification of AQP4 mRNA and protein expression levels in each mouse group 3 days post‐ICH, along with representative Western blot (WB) images(n = 6). Comparison of blood–brain barrier permeability (e), hemoglobin levels (f), and brain water content (g) among different groups. (n = 6). *p < 0.05/****p < 0.0001 versus Sham group at the same time point. ^# p < 0.05/ ^## p < 0.01/ ^### p < 0.001/^#### p < 0.0001 versus ICH group at the same time point. ^&&&& p < 0.0001 versus the same group at day 1 and day 7. AQP4, aquaporin‐4; ICH, intracerebral hemorrhage.

FIGURE 2

Influence of AQP4 on neuronal dynamics and iron deposition post‐ICH. (a)Representative images and (b) quantification of Nissl staining in brain tissue at 3 days post‐intracerebral hemorrhage (n = 6). The red arrows indicate normal neurons. Black scale bar:50 μm. (c) Representative images of FJC staining on 3 days after ICH. (d) Quantification of FJC staining around the hematoma (n = 4). Yellow scale bar:50 μm. (e) Representative brain sections stained with Perl's at day 7 post‐ICH in mice. Black scale bar:50 μm. (f) Quantification of iron deposition area at the hematoma core (n = 4). The blue arrows indicate iron deposition. ns: p > 0.05 versus Sham group. *p < 0.05/****p < 0.0001 versus Sham group. ^# p < 0.05/^### p < 0.001 /^#### p < 0.0001 versus ICH group. AQP4, aquaporin‐4; ICH, intracerebral hemorrhage.

FIGURE 3

Investigating the impact of AQP4 on hematoma clearance following ICH through fluorescent tracing methodology. (a) Experimental schematic of the temporal distribution of fluorescent tracers in the mouse brain. (b) Fluorescent tracers flow through the vascular structures in the brain. (t = 60 min). Black arrows show tracer‐filled vessels; white arrows show untraced ones. (c) Representative images demonstrate the distribution of tracer in coronal brain sections and deep cervical lymph node sections over time. (d) Quantification reveals the permeated tracer area (n = 3). ****p < 0.0001 versus Time post‐injection 60 min group in brain slices. ^&&& p < 0.001/^&&&& p < 0.0001 versus time post‐injection 60 min group in lymph node slices. (e) Distribution of fluorescent tracers at different time points in various groups Fluorescent image of FITC‐d4000 distribution in brain and magnified view of the white box in Panel. Clearly visible hematoma and injection site in the image. (f) Quantification of fluorescent area proportion in coronal sections of brain tissue (n = 3). *p < 0.05/**p < 0.01/***p < 0.001/****p < 0.0001 versus ICH group at the same time point. ns: no significant versus ICH group at the same time point. (g) Fluorescent tracer distribution and quantification (h) in deep cervical lymph nodes (n = 3). *p < 0.05/**p < 0.01/***p < 0.001/****p < 0.0001 versus ICH group at the same time point. ns: No significant versus ICH group at the same time point. White scale bar:2 mm. AQP4, aquaporin‐4; ICH, intracerebral hemorrhage.

FIGURE 4

Investigating the role of AQP4 in hematoma clearance after ICH through MRI scanning. (a) Experimental schematic. (b) Diffusion distribution of contrast agent (GD‐DTPA) under continuous scanning, with labeled images a‐f representing consecutive coronal sections. Yellow, blue, and red arrows indicate contrast agents distribution in ventricles, brain parenchyma, and cervical vessels/lymph nodes, respectively. (c) The distribution of contrast agent Gd‐DTPA was assessed using MRI at 1, 3, and 7 days post‐cerebral hemorrhage (T1WI sequences). Quantification of contrast agent diffusion area in brain (n = 3). (d) The volume of cerebral hemorrhage was evaluated using MRI at 1, 3, and 7 days post‐ICH (SWI sequences). Quantification of hematoma volume in brain.(n = 3) (e) The volume of edema was evaluated using MRI at 1, 3, and 7 days post‐ICH (T2WI sequences). Quantification of edema volume in brain. (n = 3). *p < 0.05/ **p < 0.01/***p < 0.001/****p < 0.0001 versus ICH group at the same time point. ns: No significant versus ICH group at the same time point. AQP4, aquaporin‐4; ICH, intracerebral hemorrhage.

FIGURE 5

The cellular localization of AQP4. (a) Immunofluorescent double‐label images of AQP4 and GFAP for each group, along with co‐localization analysis. AQP4 is marked with red fluorescence, GFAP with green fluorescence and “Merge” represents the overlay of the first three images. “Magnified” shows locally enlarged images. (b) Polarized distribution of AQP4 observed by calculating co‐localization with CD31. AQP4 is marked with red fluorescence, CD31 with green fluorescence. CD31 is an adhesion molecule for platelets and endothelial cells, serving as a marker for blood vessels. Yellow scale bar:50 μm. AQP4, aquaporin‐4; ICH, intracerebral hemorrhage.