An enriched environment reduces hippocampal inflammatory response and improves cognitive function in a mouse model of stroke

Abstract

An enriched environment is used as a behavioral intervention therapy that applies sensory, motor, and social stimulation, and has been used in basic and clinical research of various neurological diseases. In this study, we established mouse models of photothrombotic stroke and, 24 hours later, raised them in a standard, enriched, or isolated environment for 4 weeks. Compared with the mice raised in a standard environment, the cognitive function of mice raised in an enriched environment was better and the pathological damage in the hippocampal CA1 region was remarkably alleviated. Furthermore, protein expression levels of tumor necrosis factor receptor-associated factor 6, nuclear factor κB p65, interleukin-6, and tumor necrosis factor α, and the mRNA expression level of tumor necrosis factor receptor-associated factor 6 were greatly lower, while the expression level of miR-146a-5p was higher. Compared with the mice raised in a standard environment, changes in these indices in mice raised in an isolated environment were opposite to mice raised in an enriched environment. These findings suggest that different living environments affect the hippocampal inflammatory response and cognitive function in a mouse model of stroke. An enriched environment can improve cognitive function following stroke through up-regulation of miR-146a-5p expression and a reduction in the inflammatory response.

Keywords: cognitive function; enriched environment; isolated environment; miR-146a-5p; neuroinflammation; nuclear factor κB p65; photothrombotic model; stroke; tumor necrosis factor receptor-associated factor 6.

Figure 1

Flow chart of this study. SE group: Stroke + standard environment group; EE group: enriched environment group; IE group: stroke + isolated environment group; HE: hematoxylin-eosin; TRAF6: factor receptor-associated factor 6; TNF-α: tumor necrosis factor-α; IL-6: interleukin 6; NF-κBp65: nuclear factor kappa-Bp65; TTC: triphenyl tetrazolium chloride; RT-qPCR: real time quantitative polymerase chain reaction.

Figure 2

The different cage environments. The figure shows enriched environment (A and B), standard environment (C and D), and isolated environment (E and F) cages.

Figure 3

Triphenyl tetrazolium chloride staining of the brain in control mice (A) and photothrombotic stroke model mice (B). The non-ischemic necrosis area was red, and the avascular necrosis area was white. The brain tissue of mice in the sham mice was normal with red color (A), while the left cortex and subcortex in mice with photothrombotic stroke were white (B) under triphenyl tetrazolium chloride staining, which suggests ischemic infarction.

Figure 4

The enriched environment improved learning and memory of a mouse model of stroke in the Morris water maze. (A) Average escape latency on days 1–5. (B) The number of platform zone crossings on day 6. Data are expressed as the mean ± SD (n = 8). *P < 0.05, vs. sham group; #P < 0.05, vs. SE group; †P < 0.05, vs. SE group (one-way analysis of variance followed by least significant difference test). EE group: stroke + enriched environment group; IE group: stroke + isolated environment group; SE group: stroke + standard environment group.

Figure 5

Effects of the enriched environment on the swimming trajectory of a mouse model of stroke on day 5 of the Morris water maze test. (A) (A) Sham group. (B) SE group. (C) EE group. (D) IE group. After stroke, the swimming trajectory of the mice looking for the platform was extended. The enriched environment shortened the swimming trajectory of mice, and the isolated environment further extended the swimming trajectory of mice. The blue dot indicates the point at which mice were placed into the water; the red dot indicates the end of the swimming track; the big red circle indicates the edge of the pool; the small red circle indicates the platform. EE group: stroke + enriched environment group; IE group: stroke + isolated environment group; SE group: stroke + standard environment group.

Figure 6

Effects of the enriched environment on the histopathology in the hippocampal CA1 region of a mouse model of stroke. (A–D) Hematoxylin-eosin (A1, B1, C1, D1) and Nissl (A2, B2, C2, D2) staining in the hippocampal CA1 region in the sham (A), SE (B), EE (C), and IE (D) groups (original magnification 400×). Red arrows indicate nerve cells that were obviously irregular, with nuclear pyknosis and vacuoles; and green arrows indicate normal nerve cells. The morphology of hippocampal CA1 cells in the sham group was normal, hippocampal CA1 cells in the SE group were damaged, the morphology of CA1 cells in the EE group was improved compared to SE, and hippocampal CA1 cells in the IE group were more damaged compared to SE. (E) Number of Nissl bodies in the hippocampal CA1 region (per 400-fold field). Data are expressed as the mean ± SD (n = 6). *P < 0.05, vs. sham group; #P < 0.05, vs. SE group (one-way analysis of variance followed by least significant difference test). EE group: stroke + enriched environment group; IE group: stroke + isolated environment group; SE group: stroke + standard environment group.

Figure 7

Effects of the enriched environment on the protein expression levels of TRAF6, NF-κBp65 (nucleus), TNF-α, and IL-6 in the hippocampus of a mouse model of stroke using Western blot assay. (A) Bands of TRAF6, TNF-α, and IL-6 protein. (B) Bands of NF-κBp65 (nucleus) protein. (C–F) Quantitative results of TRAF6, TNF-α, IL-6, and NF-κBp65 (nucleus) protein expression. The expression of TRAF6, TNF-α, and IL-6 protein was normalized against GAPDH, and the expression of NF-κBp65 (nucleus) protein was normalized against Histone H3. Data are expressed as the mean ± SD (n = 6). *P < 0.05, vs. sham group; #P < 0.05, vs. SE group (one-way analysis of variance followed by least significant difference test). EE group: stroke + enriched environment group; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IE group: stroke + isolated environment group; IL-6: interleukin 6; NF-κBp65: nuclear factor kappa-Bp65; SE group: stroke + standard environment group; TNF-α: tumor necrosis factor-α; TRAF6: factor receptor-associated factor 6.

Figure 8

Effects of the enriched environment on the expression levels of miR-146a-5p (A) and TRAF6 mRNA (B) in the hippocampus of a mouse model of stroke using quantitative reverse transcription-polymerase chain reaction. Data are expressed as the mean ± SD (n = 6), and were analyzed using nonparametric tests. *P < 0.05, vs. sham group; #P < 0.05, vs. SE group (Kruskal-Wallis test followed by Mann-Whitney U test). EE group: stroke + enriched environment group; IE group: stroke + isolated environment group; SE group: stroke + standard environment group; TRAF6: tumor necrosis factor receptor-associated factor 6.

Figure 9

TRAF6 as a target gene of miR-146a-5p according to the database prediction comparison analysis. There were three potential binding sites of the TRAF6 mRNA 3′ untranslated region to miR-146a-5p according to the principle of base complementary pairing (A=T, C=G). TRAF6: Tumor necrosis factor receptor-associated factor 6.