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. 2020 May 6;23(5):101136. doi: 10.1016/j.isci.2020.101136

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© 2020 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Insulin Decreases Astrocyte and Neuron Death and Protects the Brain Damage during I/R Injury by Enhancing Astrocytic GP Activity

(A) Glycogen level in cultured astrocytes at 12 h after OGD/R (n = 8, one-way ANOVA with the LSD multiple comparisons test). Insulin (1 μM) was added to the medium immediately after reoxygenation. VEH represents the vehicle group, and IS represents the insulin group. Sg represents astrocytes infected with scrambled sgRNA lentiviruses. Sg-Pygb represents GP-knockdown astrocytes.

(B) Relative cell viability of the cultured astrocytes at 24 h after reoxygenation (n = 8, one-way ANOVA with the LSD multiple comparisons test). The control condition means that the astrocytes received culture medium changes (containing glucose) without OGD/R stress.

(C) The numbers of apoptotic cultured astrocytes were analyzed by TUNEL staining at 24 h after reoxygenation (n = 7, one-way ANOVA with the LSD multiple comparisons test). Scale bars represent 100 μm.

(D) Viability of neurons treated at 24 h after reoxygenation in the astrocyte-neuron coculture system (n = 8, one-way ANOVA with the LSD multiple comparisons test). The control condition means that the cocultured neurons received culture medium changes (containing glucose) without OGD/R stress.

(E) The numbers of apoptotic neurons in coculture were analyzed by TUNEL staining at 24 h after reoxygenation (n = 7, one-way ANOVA with the LSD multiple comparisons test). Scale bars represent 100 μm.

(F) LDH release in the coculture medium was analyzed (n = 8, one-way ANOVA with the Dunnett T3 multiple comparisons test).

(G) Glycogen levels in the ischemic penumbra at 12 h after MCAO/R in mice (n = 7, one-way ANOVA with the LSD multiple comparisons test). Insulin (10 μM) was injected into the lateral ventricle immediately after reperfusion to achieve a final concentration of approximately 1 μM in the cerebrospinal fluid. Sh represents mice infected with scrambled shRNA AAVs. Sh-Pygb represents PYGB knockdown mice.

(H) The infarct volumes at 24 h after MCAO/R in mice were analyzed by TTC staining (n = 9, one-way ANOVA with the LSD multiple comparisons test). Scale bars represent 1 mm.

(I) A corner test was performed to analyze the numbers of right turns in 10 trials before (Pre) and after reperfusion in the mouse model of MCAO (n = 7, repeated measures analysis).

(J) A grid-walking test was performed to assess the total steps (left panel) and foot fault ratios (right panel) before (Pre) and after reperfusion in the mouse model of MCAO (n = 7, repeated measures analysis).

(K) Nissl staining was used to assess infarct volumes at day 14 after MCAO/R in mice (n = 7, one-way ANOVA with the LSD multiple comparisons test). Scale bar represents 2 mm.

The data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figures S11 and S12.