Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes

doi:10.12122/j.issn.1673-4254.2024.06.08

Abstract

Abstract:

Objective To investigate the protective effect of exogenous leptin against focal cerebral ischemia-reperfusion (I/R) injury in mice and explore the underlying mechanism. Methods A total of 100 C57BL/6 mice were randomly divided into 5 groups, including a sham-operated group, cerebral I/R model group, and 3 leptin treatment groups with intraperitoneal injections of 0.5, 1.0 or 2.0 leptin immediately after occlusion of the internal carotid artery. At 24 h after reperfusion, neurological function scores of the mice were assessed, and TTC staining was used to determine the area of cerebral infarction. The pathological changes in the cortical brain tissue of the mice were observed using HE staining, and degenerative damage of the cortical neurons were assessed with Fluoro-Jade C staining. The expression of glial fibrillary acidic protein in cortical brain tissues was detected using immunohistochemistry and Western blotting. In another 45 C57BL/6 mice with sham operation, I/R modeling, or leptin (1 mg/kg) treatment, glutamic acid in the cortical brain tissue was detected using glutamate assay, and cortical glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) protein expressions were detected using immunohistochemistry. Results Compared with the I/R model mice, the leptin-treated mice had significantly lower neurological deficit scores, smaller cerebral infarct area, milder pathologies in the cortical brain tissue, and lessened cortical neuronal damage with normal morphology and less excessive proliferation of the astrocytes. Leptin treatment significantly up-regulated the expressions of GLT-1 and GLAST and lowered the content of glutamic acid in the brain tissue of the I/R mice. Conclusion Exogenous leptin has obvious neuroprotective effect against cerebral I/R injury in mice, mediated probably by controlling excessive astrocyte proliferation and up-regulating cortical GLT-1 and GLAST expressions to reduce glutamate-mediated excitotoxic injury of the astrocytes.

Key words: leptin, cerebral ischemia-reperfusion injury, astrocytes, glutamate transporter-1, glutamate-aspartate transporter, excitotoxicity

Jie CHEN, Chenxu LIU, Chun WANG, Li LI, Weiting TAO, Jingru XUN, Honghui TANG, Li HUANG. Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes[J]. Journal of Southern Medical University, 2024, 44(6): 1079-1087.

Figures/Tables 8

Fig.1 Microscopic observation of mouse right common carotid artery (CCA) and its branches.

Fig.2 Posture of the mice following brain I/R modeling (A) and neurological deficit scores of the mice in different groups (B), n=9. ****P<0.0001 vs Sham group; ####P<0.0001 vs I/R group.

Fig.3 TTC staining for determining the size of cerebral infarct area of the mice. A: TTC staining of the brain tissue sections. B: Quantitative analysis of brain infarct area (n=3). ****P<0.0001 vs Sham group; ###P<0.001, ####P<0.0001 vs I/R group.

Fig.4 HE staining for examining histopathological changes in the cortical brain tissues of the mice (scale bar=20 μm).

Fig.5 FJC staining for assessing cortical neuronal degenerative damage. A: FJC staining iamges (scale bar=20 μm). B: Positive rate of the degenerated neurons. ****P<0.0001 vs Sham group; ####P<0.0001 vs I/R group.

Fig.6 Expression of GFAP protein in mouse cortical brain tissue. A: Expression of GFAP detected by immunohistochemistry (Original magnification: ×40). B: Expression of GFAP detected by Western blotting (n=3). C: Quantitative analysis of GFAP expression. ***P<0.001 vs Sham group; ##P<0.01 vs I/R group.

Fig.7 Expression of GLT-1 and GLAST proteins in mouse cortical brain tissue (×40).

Fig.8 Changes in the expression of GLT-1 and GLAST proteins and Glu content in mouse cortical brain tissue. A: Expression of GLT-1 and GLAST detected by Western blotting (n=3). B: Quantitative analysis of GLT-1 expression. C: Quantitative analysis of GLAST expression; D: Content of Glu (n=3). ***P<0.001, ****P<0.0001 vs Sham group; #P<0.01, ###P<0.001 vs I/R group.

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