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. 2024 Apr 12;10(8):e29483.
doi: 10.1016/j.heliyon.2024.e29483. eCollection 2024 Apr 30.

The protective effects of methylene blue on astrocytic swelling after cerebral ischemia-reperfusion injuries are mediated by Aquaporin-4 and metabotropic glutamate receptor 5 activation

Yu Lai  1 , Jie Han  1 , Dongxian Qiu  2 , Xinyan Liu  3 , Kan Sun  1 , Yuzhu Fan  4 , Chunliang Wang  1 , Song Zhang  1
Affiliations

The protective effects of methylene blue on astrocytic swelling after cerebral ischemia-reperfusion injuries are mediated by Aquaporin-4 and metabotropic glutamate receptor 5 activation

Yu Lai et al. Heliyon. .

Abstract

Methylene blue (MB) was found to exert neuroprotective effect on different brain diseases, such as ischemic stroke. This study assessed the MB effects on ischemia induced brain edema and its role in the inhibition of aquaporin 4 (AQP4) and metabotropic glutamate receptor 5 (mGluR5) expression. Rats were exposed 1 h transient middle cerebral artery occlusion (tMCAO), and MB was injected intravenously following reperfusion (3 mg/kg). Magnetic resonance imaging (MRI) and 2,3,5-triphenyltetrazolium chloride (TTC) staining was performed 48 h after the onset of tMCAO to evaluate the brain infarction and edema. Brain tissues injuries as well as the glial fibrillary acidic protein (GFAP), AQP4 and mGluR5 expressions were detected. Oxygen and glucose deprivation/reoxygenation (OGD/R) was performed on primary astrocytes (ASTs) to induce cell swelling. MB was administered at the beginning of reoxygenation, and the perimeter of ASTs was measured by GFAP immunofluorescent staining. 3,5-dihydroxyphenylglycine (DHPG) and fenobam were given at 24 h before OGD to examine their effects on MB functions on AST swelling and AQP4 expression. MB remarkably decreased the volumes of T2WI and ADC lesions, as well as the cerebral swelling. Consistently, MB treatment significantly decreased GFAP, mGluR5 and AQP4 expression at 48 h after stroke. In the cultivated primary ASTs, OGD/R and DHPG significantly increased ASTs volume as well as AQP4 expression, which was reversed by MB and fenobam treatment. The obtained results highlight that MB decreases the post-ischemic brain swelling by regulating the activation of AQP4 and mGluR5, suggesting potential applications of MB on clinical ischemic stroke treatment.

Keywords: AQP 4; Ischemic stroke; Methylene blue; brain edema; mGluR5.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Experimental design schematic diagram.
Fig. 2
Fig. 2
MRI testing was performed prior to MB treatment (before reperfusion) to determine the lesion volume. (A). ADC images of rats in tMCAO + vehicle and tMCAO + MB groups prior to MB treatment (before reperfusion) to ensure the lesion volumes. (B). Quantitative analyses on the ADC lesions in vehicle and MB treatment groups before reperfusion.
Fig. 3
Fig. 3
Effect of MB on brain edema resulting from focal transient brain ischemia. (A). ADC and T2WI images for the MB- and vehicle-treated rats at 48 h following tMCAO. (B). ADC evolution defines the volumes of ischemia lesions in MB and vehicle treatment groups at 48 h. (C). Quantitative analyses of the corrected lesion volume on T2WI in the MB and vehicle treatment groups at 48 h following tMCAO. (D). Quantitative analyses of brain edema predicted with T2WI images at 48 h following tMCAO. All values are shown as the mean ± SD, **p < 0.01, ***p < 0.001 vs. tMCAO + vehicle group.
Fig. 4
Fig. 4
Effect of MB on transient brain ischemia induced brain infarction and edema was evaluated using TTC staining. (A). Typical TTC staining for cerebral sections in MB and vehicle treatment groups at 48 h after tMCAO. (B) Quantitative analyses of the lesion volumes defined by TTC in MB- and vehicle-treated rats. (C) Quantitative analyses of TTC staining in MB- and vehicle-treated rats. All values are shown as the mean ± SD, ***p < 0.001 vs. tMCAO + vehicle group.
Fig. 5
Fig. 5
Effect of MB on the GFAP and AQP4 expression in tMCAO rats. (A). HE staining images showing neuropathological changes in each group. (B). IHC staining and quantitative analysis showing AQP4 and GFAP expression at the ischemic penumbra of cortical regions. AQP4 expression around blood vessels was indicated by white arrows. Scale bars show 20 μm. Three fields of the IHC images in the ischemic penumbra of cortical regions from 5 rats in each group were randomly selected to calculate the mean optical density. All values are shown as the mean ± SD, ###p < 0.001 vs. tMCAO group, ***p < 0.001 vs. sham group.
Fig. 6
Fig. 6
Effect of MB on astrocyte swelling induced by OGD/R. Astrocytes were injured by OGD/R and treated with different concentrations of MB (0.01–10 μM). Immunofluorescence staining for GFAP in cultured astrocytes. Scale bars show 50 μm. Quantitative analyses of the image data obtained based on the panels within (A) revealed the astrocyte perimeters in each group. Ten cells in each field under high magnification (200 × ) were randomly selected. Three fields in three wells were measured in every group, and the values of nine fields were calculated as the cell perimeter of each group. Values are displayed as the mean ± SD. ###p < 0.001 vs. OGD/R group, ***p < 0.001 vs. Control.
Fig. 7
Fig. 7
Effect of MB (10 μM) treatment on AQP4 expression in cultured astrocytes. (A). immunofluorescence staining for AQP4 in cultured astrocytes. Scale bars show 50 μm. (B). Protein expression of AQP4 determined by Western blot and quantitative data. All results are obtained on the basis of 3 repeated independent samples which were repeated for 3 times. Values are displayed as the mean ± SD. ###p < 0.001 vs. OGD/R group, ***p < 0.001 vs. Control.
Fig. 8
Fig. 8
Co-expression of AQP4 and mGluR5 in astrocytes. (A) Immunofluorescence double staining of AQP4 and mGluR5 in cultured astrocytes. (B) GFAP and AQP4 double immunofluorescence staining in the brain tissues of each group. Scale bars show 20 μm.
Fig. 9
Fig. 9
Effect of MB on the expression of mGluR5 and AQP4 in tMCAO brain tissue. (A). The expression of AQP4 and mGluR5 in cultured astrocytes by Western blotting and its subsequent analysis using ImageJ software (B). Double immunofluorescence staining showing the expression of mGluR5 and AQP4 in pericapillary areas in the normal brain and cell membranes of astrocytes after tMCAO, and MB treatment. Scale bars show 20 μm. All results are obtained on the basis of 3 repeated independent samples which were repeated for 3 times. Values are shown as the mean ± SD, ***p < 0.001 vs. control; ##p < 0.01, ###p < 0.001 vs. OGD/R group.
Fig. 10
Fig. 10
The effect of MB on astrocyte swelling and AQP4 (regulation of mGluR5). (A). immunofluorescence staining for GFAP in cultured astrocytes. Scale bars show 50 μm. (B). Astrocyte perimeters in each group measured according to Panel (A). Ten cells in each field under high magnification (200 × ) were randomly selected. Three fields in three wells were measured in every group, and the values of nine fields were calculated as the cell perimeter of each group. (C). AQP4 protein expression in each group determined through Western blotting. (D). Quantitative analysis of AQP4 protein expression in each group. All results are obtained on the basis of 3 repeated independent samples which were repeated for 3 times. All data are shown as the mean ± SD, *p < 0.05 vs. control.
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References

    1. Yao Y., Zhang Y., Liao X., Yang R., Lei Y., Luo J. Potential therapies for cerebral edema after ischemic stroke: a mini review. Front. Aging Neurosci. 2021;12 - PMC - PubMed
    1. Assefa B.T., Gebre A.K., Altaye B.M. Reactive astrocytes as drug target in Alzheimer's disease. BioMed Res. Int. 2018;2018 - PMC - PubMed
    1. Mamtilahun M., Tang G., Zhang Z., Wang Y., Tang Y., Yang G.-Y. Targeting water in the brain: role of aquaporin-4 in ischemic brain edema. Curr. Drug Targets. 2019;20:748–755. - PubMed
    1. Chu H., Huang C., Ding H., Dong J., Gao Z., Yang X., Tang Y., Dong Q. Aquaporin-4 and cerebrovascular diseases. Int. J. Mol. Sci. 2016;17:1249. - PMC - PubMed
    1. Salman M.M., Kitchen P., Halsey A., Wang M.X., Törnroth-Horsefield S., Conner A.C., Badaut J., Iliff J.J., Bill R.M. Emerging roles for dynamic aquaporin-4 subcellular relocalization in CNS water homeostasis. Brain. 2021;145:64–75. - PMC - PubMed

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