Enriched environment boosts the post-stroke recovery of neurological function by promoting autophagy

Abstract

Autophagy is crucial for maintaining cellular homeostasis, and can be activated after ischemic stroke. It also participates in nerve injury and repair. The purpose of this study was to investigate whether an enriched environment has neuroprotective effects through affecting autophagy. A Sprague-Dawley rat model of transient ischemic stroke was prepared by occlusion of the middle cerebral artery followed by reperfusion. One week after surgery, these rats were raised in either a standard environment or an enriched environment for 4 successive weeks. The enriched environment increased Beclin-1 expression and the LC3-II/LC3-I ratio in the autophagy/lysosomal pathway in the penumbra of middle cerebral artery-occluded rats. Enriched environment-induced elevations in autophagic activity were mainly observed in neurons. Enriched environment treatment also promoted the fusion of autophagosomes with lysosomes, enhanced the lysosomal activities of lysosomal-associated membrane protein 1, cathepsin B, and cathepsin D, and reduced the expression of ubiquitin and p62. After 4 weeks of enriched environment treatment, neurological deficits and neuronal death caused by middle cerebral artery occlusion/reperfusion were significantly alleviated, and infarct volume was significantly reduced. These findings suggest that neuronal autophagy is likely the neuroprotective mechanism by which an enriched environment promotes recovery from ischemic stroke. This study was approved by the Animal Ethics Committee of the Kunming University of Science and Technology, China (approval No. 5301002013855) on March 1, 2019.

Keywords: autophagy; brain; central nervous system; factor; injury; pathways; protection; regeneration; repair; stroke.

Figure 1

Rehabilitation apparatus of the enriched environment (EE) and experimental timeline. (A, B) Superior (A) and lateral (B) view of an EE cage. (C) To take food, rats had to climb up to the highest platform of the EE and grab the food with two forelimbs; thus, the ischemic stroke-impaired limbs were repeatedly stimulated during EE treatment. (D) Timeline of the experimental procedure.

Figure 2

Effects of EE treatment on autophagic activity in the penumbra of ischemic stroke rats as detected by western blot assay. MCAO + EE group: MCAO animals were housed in EE cages; MCAO group: MCAO rats were housed in standard cages; sham group: sham surgery rats were housed in EE cages. (A) Protein bands. (B, C) Quantitative results of Beclin-1 protein expression and ratio of LC3-II/LC3-I. Data are expressed as the mean ± SEM of the ratio to the sham group (n = 6). #P < 0.05, ##P < 0.01 (one-way analysis of variance followed by Dunnett’s test). EE: Enriched environment; LC3: light chain 3; MCAO: middle cerebral artery occlusion; n.s.: no significance.

Figure 3

Efficacy of EE treatment to promote autophagic activity in the penumbra of ischemic stroke rats was verified by double immunofluorescence to investigate cellular localization. MCAO + EE group: MCAO animals were housed in EE cages; MCAO group: MCAO rats were housed in standard cages; sham group: the sham surgery rats were housed in EE cages. (A–C) The percentage of LC3-/NeuN-positive cells was significantly elevated by 4 weeks of EE treatment in the MCAO + EE group compared with the MCAO group. However, the ratios of LC3-/GFAP- and LC3-/Iba-1-positive cells in the MCAO + EE group were similar to those in the MCAO group. Green (stained by Alexa Fluor-488) indicates LC3 and GFAP; red (stained by Alexa Fluor-594) indicates LC3, NeuN, and Iba-1; blue indicates total cells; and yellow indicates LC3-/NeuN-, LC3-/GFAP-, and LC3-/Iba-1-positive cells. Scale bars: 50 µm. High-magnification images of the boxed areas are shown in the inserts. (D–F) Quantitative results of LC3-/NeuN- (neuron), LC3-/GFAP- (astrocyte) and LC3-/Iba-1-positive (microglia) cells. (G) The black square in G represents the selected penumbra area that was used to count positively stained cells. Data are expressed as the mean ± SEM of the ratio to the sham group (n = 6). #P < 0.05, ##P < 0.01 (one-way analysis of variance followed by Dunnett’s test). EE: Enriched environment; GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium-binding adaptor molecule 1; LC3: light chain 3; MCAO: middle cerebral artery occlusion; n.s.: no significance.

Figure 4

Proteins in the autophagic/lysosomal pathway are detected in the penumbra of ischemic stroke rats to evaluate the efficacy of EE treatment on autophagic flux. MCAO + EE group: MCAO animals were housed in EE cages; MCAO group: MCAO rats were housed in standard cages; sham group: the sham surgery rats were housed in EE cages. (A) Protein bands. (B–G) Quantitative results of proteins in the autophagic/lysosomal pathway. Data are expressed as the mean ± SEM of the ratio to the sham group (n = 6). #P < 0.05, ##P < 0.01, ###P < 0.001 (one-way analysis of variance followed by Dunnett’s test). EE: Enriched environment; LAMP-1: lysosomal-associated membrane protein 1; LC3: light chain 3; MCAO: middle cerebral artery occlusion; n.s.: no significance; SQSTM1: sequestosome 1.

Figure 5

Effects of EE treatments on autolysosomal function in the penumbra of ischemic stroke rats as evaluated by double immunofluorescence. MCAO + EE group: MCAO animals were housed in EE cages; MCAO group: MCAO rats were housed in standard cages; sham group: the sham surgery rats were housed in EE cages. (A, B) The ratios of LC3-II-/LAMP-1- and p62-/cathepsin B-positive cells were markedly increased in the MCAO + EE group compared with the MCAO group. This implied that EE treatment was not only able to boost the fusion of autophagosomes with lysosomes, but could also enhance autophagic degradation. Green (stained by Alexa Fluor-488) indicates LAMP-1 and p62; red (stained by Alexa Fluor-594) indicates LC3 and cathepsin B; blue indicates total cells; and yellow indicates LC3-II-/LAMP-1- and p62-/cathepsin B-positive cells. Scale bars: 50 µm. (C, D) Quantitative results of LC3-II-LAMP-1- and p62-cathepsin B-positive cells. Data are expressed as the mean ± SEM of the ratio to the sham group (n = 6). ##P < 0.01 (one-way analysis of variance followed by Dunnett’s test). DAPI: 4′,6-Diamidino-2-phenylindole; EE: enriched environment; LAMP1: lysosomal-associated membrane protein 1; LC3: light chain 3; MCAO: middle cerebral artery occlusion; n.s.: no significance.

Figure 6

Effect of EE treatment on neurological deficit in ischemic stroke rats. The mNSS in the MC6AO + EE group was significantly reduced compared with the MCAO group. Data are expressed as the mean ± SEM of the ratio to the sham group (n = 6). ###P < 0.001 (one-way analysis of variance followed by Dunnett’s test). EE: Enriched environment; MCAO: middle cerebral artery occlusion; mNSS: modified neurological severity score.

Figure 7

Effects of EE treatment on infarct volume in ischemic stroke rats. MCAO + EE group: MCAO animals were housed in EE cages; MCAO group: MCAO rats were housed in standard cages; sham group: the sham surgery rats were housed in EE cages. (A) With TTC staining, normal brain tissue is red and infarct tissue is pale. Infarct volume was attenuated by 4 weeks of EE treatment compared with the MCAO group. (B) Quantitative results of the percentage of infarct volume. Data are expressed as the mean ± SEM of the ratio to the sham group (n = 6). ###P < 0.001 (one-way analysis of variance followed by Dunnett’s test). EE: Enriched environment; MCAO: middle cerebral artery occlusion; TTC: triphenyltetrazolium chloride.

Figure 8

Effects of EE treatment on neuronal survival in the penumbra of ischemic stroke rats as assessed by immunofluorescence. MCAO + EE group: MCAO animals were housed in EE cages; MCAO group: MCAO rats were housed in standard cages; sham group: the sham surgery rats were housed in EE cages. (A) The percentage of NeuN-positive cells in the MCAO + EE group was significantly higher than in the MCAO group. Green (stained by Alexa Fluor-488) indicates NeuN-positive cells; blue indicates total cells. Scale bars: 50 µm. (B) Quantitative results of the percentage of NeuN-positive cells. Data are expressed as the mean ± SEM of the ratio to the sham group (n = 6). ##P < 0.01 (one-way analysis of variance followed by Dunnett’s test). DAPI: 4′,6-Diamidino-2-phenylindole; EE: enriched environment; MCAO: middle cerebral artery occlusion.