Experimental protocol

As shown in Figure 1, this study was divided into two separate sections.

Diagram of timeline of experimental procedures.

In section 1, we mainly evaluated the effect of different doses and durations of DSCG on MCT expression to formulate the subsequent experimental protocol. Two independent experiments were performed in this section.

In experiment 1, mice (PND 7) from each litter (4 L, 6 mice pups in each litter, 24 mice pups in total), regardless of gender, were randomly allocated into one of four treatment protocols: sham controls (C), GA-treated (A), GA + DSCG treated group 1 (AD-1), GA + DSCG treated group 2 (AD-2).

In experiment 2, neonatal mice with the same condition were randomly allocated into another one of four treatment protocols: sham controls (C), GA-treated (A), GA + DSCG treated group 3 (AD-3), GA + DSCG treated group 4 (AD-4).

Mice in C group did not receive any intervention; mice in A group received only isoflurane exposure on PND 7; mice in AD-1 group received isoflurane exposure on PND 7 and DSCG intraperitoneally injection during PND 7–21 (25 mg/kg/day, 2 Weeks); mice in AD-2 group received isoflurane exposure on PND 7 and DSCG intraperitoneally injection during PND 7–21 (50 mg/kg/day, 2 Weeks); mice in AD-3 group received isoflurane exposure on PND 7 and DSCG intraperitoneally injection during PND 7–28 (25 mg/kg/day, 3 Weeks); mice in AD-4 group received isoflurane exposure on PND 7 and DSCG intraperitoneally injection during PND 7–28 (50 mg/kg/day, 3 Weeks). Twenty-four hours after the DSCG treatment phase, brain tissues were harvested from each group to detect the expression levels of MCT (PND 22 and PND 29, respectively).

In section 2, we mainly verified the pharmacological effect of DSCG. Mice (PND 7) from each litter (40 L, 5–8 mice pups in each litter, 224 mice pups in total), regardless of gender, were randomly allocated into one of four treatment protocols: sham controls (C), sham + DSCG treated (CD), GA-treated (A), GA + DSCG treated (AD).

Mice in C group did not receive any intervention; mice in CD group received only DSCG intraperitoneally injection during PND 7–21 (25 mg/kg/day, 2 Weeks); mice in A group received only isoflurane exposure on PND 7; mice in AD group received isoflurane exposure on PND 7 and DSCG intraperitoneally injection during PND 7–21 (25 mg/kg/day, 2 Weeks). After that, four independent experiments were performed in this section.

In experiment 1, behavioral testing was performed during PND 22–36 (4 Weeks) in each group.

In experiment 2, behavioral testing was performed during PND 50–64 (8 Weeks) in each group.

In experiment 3, mice were sacrificed at PND 22 (4 Weeks). Brain was harvested to measure MCs by toluidine blue staining, determine the expression of MCT, chondroitin sulfate proteoglycan 4 (CSPG4), oligodendrocyte transcription factor 2 (OLIG2), myelin basic protein (MBP), synaptosomal associated protein 25 kDa (SNAP25), postsynaptic density protein 95 kDa (PSD95) and brain-derived neurotrophic factor (BDNF) by Western blotting, and detect glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule 1 (IBA1) by Immunofluorescent staining.

In experiment 4, mice were sacrificed at PND 50 (8 Weeks). Brain was harvested to determine the expression of MCT, CSPG4, OLIG2, MBP, SNAP25, PSD95, and BDNF by Western blotting, and detect GFAP and IBA1 by Immunofluorescent staining.

Mice did not receive any intervention during PND 22–49 in experiment 2 and 4.