Fig 2: Experimental timeline. The mice were administered Netrin-1 (45 μg/kg) or vehicle control (sterile PBS) at 1 h after surgery (Day 0). The mice in Cohort 1 were used to detect behavior 6 h after surgery. Once the behavior test was completed, they were sacrificed immediately for immunofluorescence and ELISA. The mice in Cohort 2 were used to detect behavior at 9 h after surgery. Once the behavior test was completed, they were sacrificed immediately for WB. The mice in Cohort 3 were used to detect behavior 24 h after surgery. Twenty mice in Cohort 4 were injected intravenously with 100 μl 10-kDa dextran at 6 h after surgery. Fifteen minutes after injection, each mouse was prepared for immunofluorescence. Another 20 mice in Cohort 4 were injected intravenously with 100 μl 10-kDa dextran at 24 h after surgery. Fifteen minutes after injection, each mouse was prepared for spectrophotometric quantification of 10-kDa dextran.

Fig 3: Expression of Netrin-1 and Neogenin in human melanoma cells. (A) Western blot results show different levels of Netrin-1 and Neogenin expression in human melanoma cell lines. The human neuroblastoma cell line SH-SY5Y and the normal human epidermal keratinocyte cell line HEKn were used as positive-expressing and poorly expressing controls for cellular expression of Netrin-1, respectively. (B) Results from immunofluorescence (IF) staining shows more intense expression of Neogenin in the aggressive melanoma cell lines C8161 and Sk-Mel28 compared to the poorly aggressive melanoma cell line UACC1273. SH-SY5Y was used as positive control for the IF staining for Neogenin. (Original magnification 20X). (C) The histogram shows the ratio of Neogenin to Netrin-1 expression determined from densitometric analysis of the respective WB bands and represented as a fold difference relative to the Neogenin-to-Netrin-1 ratio calculated for SH-SY5Y, which was used as control. These results clearly show that Neogenin expression relative to Netrin-1 is significantly greater in aggressive compared to poorly aggressive melanoma cells (* p < 0.05, n = 3).

Fig 4: Knockdown MEF2A weakened the therapeutic effect of dexmedetomidine combined with Netrin-1 after MCAO and OGD injuries. (A,B) Immunofluorescence staining exhibited downregulation in MEF2A expression in the hippocampus at 14 days after AAV9-MEF2A RNAi microinjection. (C,D) Western blot analysis shows downregulation in MEF2A expression in the hippocampus at 14 days after AAV9-MEF2A RNAi microinjection. (E) The mNSS result after MEF2A knockdown. (F) The step-through latency result after MEF2A knockdown. (G,H) The OPS recovery rate after MEF2A knockdown. (I–K) Western blot analysis of ERS-related proteins CHOP and GRP78 in the hippocampus at 14 days after AAV9-MEF2A RNAi microinjection, β-actin was used as an internal control. (L–N) Western blot analysis of ERS-related proteins ERK5 and p-ERK5 in the hippocampus at 14 days after AAV9-MEF2A RNAi microinjection, β-actin was used as an internal control. ∗∗P < 0.01 and ∗∗∗P < 0.001 using one-way ANOVA followed by Tukey’s post hoc tests.

Fig 5: Combination of dexmedetomidine and Netrin-1 reduced the neuronal injury after MCAO injury. (A) TTC staining after MCAO injury, the red area represents normal tissue and the white area represents infarcted tissue. (B) Representative images of TUNEL-positive cells (green) in the hippocampal CA1 region. The nuclei were stained with DAPI. Scale bar = 200 μm. (C) Nissl staining of the surviving cells in the hippocampal CA1 region. Scale bar = 200 μm. (D) Percentage of infarct volume after TTC staining. (E) Quantitative analysis of TUNEL-positive neurons. (F) Quantitative analysis of Nissl-positive neurons. Data are shown as the mean ± SD. ∗∗P < 0.01 and ∗∗∗P < 0.001 using one-way ANOVA followed by Tukey’s post hoc tests.