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Environmental enrichment combined with fasudil promotes motor function recovery and axonal regeneration after stroke - PubMed

Environmental enrichment combined with fasudil promotes motor function recovery and axonal regeneration after stroke

Yi-Tong Zhu et al. Neural Regen Res. 2021 Dec.

Abstract

Fasudil, a Rho-associated protein kinase (ROCK) inhibitor, has a protective effect on the central nervous system. In addition, environmental enrichment is a promising technique for inducing the recovery of motor impairments in ischemic stroke models. The present study aimed to explore whether environmental enrichment combined with fasudil can facilitate motor function recovery and induce cortical axonal regeneration after stroke. First, a mouse model of ischemic cerebral stroke was established by photochemical embolization of the left sensorimotor cortex. Fasudil solution (10 mg/kg per day) was injected intraperitoneally for 21 days after the photothrombotic stroke. An environmental enrichment intervention was performed on days 7-21 after the photothrombotic stroke. The results revealed that environmental enrichment combined with fasudil improved motor function, increased growth-associated protein 43 expression in the infarcted cerebral cortex, promoted axonal regeneration on the contralateral side, and downregulated ROCK, p-LIM domain kinase (LIMK)1, and p-cofilin expression. The combined intervention was superior to monotherapy. These findings suggest that environmental enrichment combined with fasudil treatment promotes motor recovery after stroke, at least partly by stimulating axonal regeneration. The underlying mechanism might involve ROCK/LIMK1/cofilin pathway regulation. This study was approved by the Institutional Animal Care and Use Committee of Fudan University, China (approval No. 20160858A232) on February 24, 2016.

Keywords: Nissl bodies; Rho/ROCK pathway; axon regeneration; biotinylated dextran amines; environmental enrichment; fasudil; growth-associated protein 43; ischemic stroke; motor recovery.

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Conflict of interest statement

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Figures

Figure 1
Figure 1

Time course of interventions (left) and the EE cage (right). BDA: Biotinylated dextran amines; EE: enriched environment; PT: photothrombotic stroke.

Figure 2
Figure 2

EE combined with fasudil promotes motor recovery after PT. (A) Latency to fall off the rod in the accelerating rotarod test. (B, C) Histogram of the latency to fall off the rod on post-PT days 14 and 21 in the accelerating rotarod test. (D) Laterality index in the cylinder test. (E, F) Histogram of the laterality index in each group on post-PT days 14 and 21 in the cylinder test. (G) Forelimb slip rate in the rung walking test. (H, I) Histogram of the forelimb slip rate in each group on post-PT days 14 and 21 in the rung walking test. EE combined with fasudil promoted the performance of model mice in the rotarod, cylinder, and forelimb slip tests. Data are expressed as the mean ± SEM (n = 15). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way analysis of variance followed by Tukey's post-hoc test). EE: Enriched environment; PT: photothrombotic stroke. Sham group: mice received saline; PT group: mice received photochemical embolization and saline; PT + EE group (PE): mice received photochemical embolization, EE intervention, and saline; PT + fasudil group (PF): mice received photochemical embolization and fasudil treatment at 10 mg/kg per day for 3 weeks; and PT + EE + fasudil group (PEF): mice received photochemical embolization, EE intervention, and fasudil treatment at 10 mg/kg per day for 3 weeks.

Figure 3
Figure 3

EE combined with fasudil relieves the damage in cortical neurons following PT (Nissl staining). Representative micrographs of Nissl staining and quantification of Nissl-stained neurons. Surviving cells showing well-stained Nissl bodies are marked with red arrows, while damaged cells forming dense necrotic dendritic fragments are marked with black arrows. The neurons in the sham group were arranged regularly, and there were almost no swollen or necrotic cells. The Nissl bodies in the cell bodies and dendrites were large and numerous, and were dyed dark blue or purple. In the PT group, the arrangement of neurons was disordered, and there were many necrotic cells. There were many darkly stained cell fragments in the cell body and dendrites, and Nissl bodies were almost invisible. The numbers of surviving neurons in the PT + EE, PT + fasudil, and PT + EE + fasudil groups were significantly higher than those in the PT group. Scale bars: 50 μm, original magnification 40×. Data are expressed as the mean ± SEM (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey's multiple comparisons). EE: Enriched environment; PT: photothrombotic stroke. Sham group: mice received saline; PT group: mice received photochemical embolization and saline; PT + EE group (PE): mice received photochemical embolization, EE intervention, and saline; PT + fasudil group (PF): mice received photochemical embolization and fasudil treatment at 10 mg/kg per day for 3 weeks; and PT + EE + fasudil group (PEF): mice received photochemical embolization, EE intervention, and fasudil treatment at 10 mg/kg per day for 3 weeks.

Figure 4
Figure 4

EE combined with fasudil increases GAP43 immunopositivity in the peri-infarct cortex following PT (immunohistochemistry staining). Representative micrographs of GAP43 staining and quantification of the GAP43-positive area. Compared with the PT group, GAP43 immunopositivity was significantly higher in the PT + EE + fasudil group at 22 days after surgery. Scale bars: 100 μm, original magnification 10× (upper); 50 μm, original magnification 20× (lower). Data are expressed as the mean ± SEM (n = 3). ***P < 0.001 (one-way ANOVA followed by Tukey's multiple comparisons). EE: enriched environment; PT: photothrombotic stroke. Sham group: mice received saline; PT group: mice received photochemical embolization and saline; PT + EE group (PE): mice received photochemical embolization, EE intervention, and saline; PT + fasudil group (PF): mice received photochemical embolization and fasudil treatment at 10 mg/kg per day for 3 weeks; and PT + EE + fasudil group (PEF): mice received photochemical embolization, EE intervention, and fasudil treatment at 10 mg/kg per day for 3 weeks.

Figure 5
Figure 5

EE combined with fasudil increases the BDA-labeled neurons and axons in the bilateral cortex following PT (immunohistochemical staining). (A) Representative micrographs of BDA-labeled neurons and axons in the bilateral cortex. (B) Quantification of the BDA-reactive area in the bilateral cortex. BDA-labeled neurons and axon fibers were observed in the contralesional cortex, whereas only BDA-labeled axon fibers were observed in the ipsilesional cortex. The PT + fasudil and PT + EE + fasudil groups had greater areas of labeled neurons and sprouting axons compared with the PT group in both the contralesional and ipsilesional cortices. Scale bars: 50 μm, and 20 μm in the enlarged part. Data are expressed as the mean ± SEM (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey's multiple comparisons). BDA: Biotinylated dextran amines; EE: enriched environment; PT: photothrombotic stroke. Sham group: mice received saline; PT group: mice received photochemical embolization and saline; PT + EE group (PE): mice received photochemical embolization, EE intervention, and saline; PT + fasudil group (PF): mice received photochemical embolization and fasudil treatment at 10 mg/kg per day for 3 weeks; and PT + EE + fasudil group (PEF): mice received photochemical embolization, EE intervention, and fasudil treatment at 10 mg/kg per day for 3 weeks.

Figure 6
Figure 6

Effects of EE and fasudil on protein expression in the ROCK/LIMK1/cofilin pathway in the ischemic cortex. (A–C) Quantitative results of ROCK (A), LIMK1, p-LIMK1 (B), cofilin, and p-cofilin (C). GAPDH was used as an internal reference. Data are expressed as the mean ± SEM (n = 9). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey's multiple comparisons). GAPDH: Glyceraldehyde phosphate dehydrogenase. Sham group: mice received saline; PT group: mice received photochemical embolization and saline; PT + EE group (PE): mice received photochemical embolization, EE intervention, and saline; PT + fasudil group (PF): mice received photochemical embolization and fasudil treatment at 10 mg/kg PER day for 3 weeks; and PT + EE + fasudil group (PEF): mice received photochemical embolization, EE intervention, and fasudil treatment at 10 mg/kg PER day for 3 weeks.

Figure 7
Figure 7

Correlation of motor function with the relative expression levels of p-cofilin/cofilin and p-LIMK1/LIMK1. (A, B) Correlation of rotarod results with the relative levels of p-cofilin/cofilin and p-LIMK1/LIMK1 (r = −0.5101, P = 0.0003; r = −0.3174, P = 0.037). (C, D) Correlation of forelimb slip rate with the relative levels of p-cofilin/cofilin and p-LIMK1/LIMK1 (r = 0.6407, P < 0.0001; r = 0.3639, P = 0.0140). Correlations were analyzed using Pearson's correlation coefficient method. r: Pearson's correlation coefficient

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