Sage Journals: Discover world-class research

Abstract

Several studies have demonstrated the neuroprotective effect of enriched environment (EE) and its positive effect on cognitive performance in pathological conditions, such as neurodegenerative diseases, epilepsy, and traumatic brain injury. However, the immunomodulatory effect of EE in normal rodents is not well characterized. To assess the immunomodulatory effect of EE, we randomly assigned normal mice to EE housing or standard environmental (SE) housing for 3 weeks. Behavioral alterations were evaluated by open field, fear conditioning, and Morris water maze tests. Immunohistochemical staining was performed to assess the expression of behavioral-related proteins, and enzyme-linked immunosorbent assay (ELISA) for brain-derived neurotrophic factor (BDNF), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) was also performed. We also measured the levels of RIP1 and RIP3 proteins using western blotting. EE significantly improved the cognitive performance which was associated with the increased expressions of BDNF, ionized calcium-binding adapter molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP); EE did not influence any morphological changes in the brain tissue in adult mice; however, increased resistance to inflammation induced by TNF-α was observed. These findings indicate that EE can positively influence cognitive and behavioral performance in healthy adult mice by exerting environ-immuno effect on neural function.

Keywords

behavior enriched environment inflammation mice TNF-α

Introduction

Numerous studies have demonstrated enriched environment (EE)-induced neuroplastic changes and/or behavioral improvements in normal animals as well as in animal models of various central nervous system (CNS) diseases.^1–3 EE can affect the inflammation and outcome not only in the disease animal models but also in the health animal models.^4–6

Homeostasis of neuroimmune signaling pathways in the brain is maintained by circulating immune cells and proteins,⁶ which may influence cognition, behavior, and mood.⁷ Previous studies have revealed that the beneficial effect of EE was attributed not only to alter neurobiological and behavioral response, but, possibly, also through other immune markers, such as glial cells,⁸ cytokines, and chemokines⁷ in rodents placed in the presence of an EE.

In a recent study, EE restored stress-induced impairment in learning, memory, and behavioral depression through reversal of adult neurogenesis.⁹ Environmental enrichment (EE) refers to a housing condition that improves physical and psychological well-being by providing various stimuli meeting the special needs of inhabitants.^1–4,10 EE has been shown to modulate plasticity within the hippocampus and other cortical regions in rodent brains¹¹ and to slow down the process of neuronal aging.¹² Moreover, EE was also shown to increase levels of neurotrophins, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which play an integral role in neuronal signaling.¹³

Interestingly, EE-housed rats showed an attenuated response of interleukin-1β (IL-1β) and members of the tumor necrosis factor (TNF) family within the hippocampus.⁸ The finding that peripheral TNF-α and interferon gamma (INF-γ) influences the CNS either at a neuroimmune level or at the behavioral level¹⁴ led to a speculation that EE plays a crucial role in the healthy and injured brains via regulating the relationship between the immune system and brain functioning. In this study, we sought to examine the potential immunomodulatory effect of EE on behavior and on the brain microenvironment in healthy mice and to assess the potential role of EE in the prevention of behavioral and cognitive diseases.

Results

Experimental protocol

The complete experimental protocol is illustrated in Figure 1. The animals were randomly divided into two groups (EE group and standard environment (SE) group, n = 20). After spending 3 weeks in either EE or SE housing conditions, half the mice were randomly assigned to the protocol A and the other half to the protocol B. The details of protocols are as follows. The protocol A includes major five steps. At first, experimental animals were put into EE or SE housing conditions for 3 weeks. And then, we performed the behavioral tests, including three3 steps, such as open field test (OFT) for 2 days, fear condition for 1 day, and MWM for 5 days. The last step is to sacrifice animals for other studies. The protocol B includes three major steps. The first step is the same as the protocol A. And then, animals were injected with TNF-α. Finally, after injection for 5 h, we sacrificed the animals for further studies.

Figure 1.

Schematic illustration of the experimental protocol. Mice were reared either in EE housing or SE housing for 3 weeks (n = 20).

Housing conditions

As shown in Figure 2, all the animals were reared either in EE housing (Figure 2(a)) or in SE housing (Figure 2(b)). The details of protocols are as follows. EE housing includes many interesting tools such as that are beneficial for the experimental animals. And there are more animals in the EE housing which are helpful for social activities among animals. However, there are no tools for animals and too few animals in SE housing.

Figure 2.

Housing conditions: (a) enriched environment (EE) housing, eight mice per cage and (b) standard environment (SE) housing, three mice per cage.

Behavioral tests

Behavioral performance was assessed at 22 days after treatment (Figure 3). Locomotor activity assessed using OFT showed a significant difference between two groups (SE vs EE: 3180 ± 206 cm vs 1750 ± 115 cm, Figure 3(a); *P < 0.05). The effect of EE on learning and memory was assessed by the contextual fear conditioning test. Data showed that EE mice had higher freezing levels during the context and tone-dependent memory test compared to SE mice (SE vs EE: 37% ± 3% vs 62% ± 5%, SE vs EE: 60% ± 6% vs 91% ± 8%, respectively; *P < 0.05, Figure 3(b) and (c)). In order to determine the effect of spatial learning acquisition, we performed the Morris water maze (MWM) test and measured the following four indices: escape latency and distance to locate the removed platform, the time spent in the platform quadrant, and the times crossing over the platform. Figure 3(d) and (e) shows that the mean escape latency and escape distance in the EE group were shorter than those in the SE group (SE vs EE: 24.15 ± 6.52 vs 11.43 ± 3.4, SE vs EE: 591.54 ± 78.17 vs 264.3 ± 50.1; Figure 3(d) and (e), respectively, *P < 0.05). The time spent in the platform quadrant showed significant between-group difference (SE vs EE: 29 ± 4 vs 53 ± 6; Figure 3(f), *P < 0.05). Similarly, times crossing over the platform was also found to be significantly different between the two groups (SE vs EE: 2.1 ± 0.6 vs 4.3 ± 1.2, Figure 3(g), *P < 0.05).

Figure 3.

Behavioral performance tests: (a) total distance traveled in EE-reared and SE-reared mice (*P < 0.05), (b) contextual fear conditioning (*P < 0.05), (c) EE enhanced delay tone-dependent fear conditioning (*P < 0.05), (d) escape latency in the removed platform trial (*P < 0.05), (e) escape distance in the removed platform trial (*P < 0.05), (f) time spent in the platform quadrant in the removed platform trial (*P < 0.05), and (g) times crossing the platform in the removed platform trial (*P < 0.05).

Upregulation of glial fibrillary acidic protein and Iba1 expression in EE group

EE-reared mice had a greater expression of glial fibrillary acidic protein (GFAP) and Iba1 as compared to that in SE-reared mice. (Figure 4(a) and (c) shows hippocampal tissues from the SE-reared mice, while Figure 4(b) and (d) shows the hippocampal tissues from the EE-reared mice.)

Figure 4.

The related protein expression measured with immunohistochemistry staining following 3 weeks of EE (200×). (a and b) The expression of GFAP both in the SE and EE mice. (c and d) The expression of Iba1 both in the SE and EE mice.

EE decreased the expression of RIP1 and RIP3 proteins

To determine whether EE impacts necroptosis signaling after brain injury, we analyzed the expression of RIP1 and RIP3, the key markers of necroptosis. There was a marked decrease in RIP1 and RIP3 expression in the EE mice compared to the SE mice group (Figure 5).

Figure 5.

Expression of proteins related to necroptosis from SE and EE mice. Figure 5 represents the data of Western blotting analysis.

EE mice had increased expression of BDNF, but the expression of TNF-α and IL-1β remained unchanged

To assess the potential effect of EE on the brain microenvironment, we measured the classical inflammtatory cytokines (TNF-α and IL-1β) as well as the growth factor (BDNF) within the hippocampus of both groups of mice. The expression of BDNF was significantly greater in the EE group (SE vs EE: 291 ± 27 vs 503 ± 51, *P < 0.05, Figure 6(a)), while the levels of TNF-α and IL-1β were not significantly different in EE-reared mice when compared to the SE group (SE vs EE: 22 ± 4 vs 17 ± 3; SE vs EE: 19 ± 3 vs 15 ± 2, respectively, Figure 6(b)).

Figure 6.

Expression of inflammatory cytokines and growth factors within the hippocampus measured by ELISA. (a) The expression of BDNF protein from hippocampus was higher in the EE group mice than in the SE group mice (*P < 0.05) and (b) there was a trend of decrease in the expressions of TNF-α in the EE and SE groups, as well as IL-1β, but no significant difference was noted.

EE-reared mice attenuated expression of the inflammatory marker, CD68, after TNF-α injection

Following TNF-α injection, we assayed the expression of the inflammatory marker, CD68, which indicates activated microglia/macrophages. As shown in Figure 7(a)–(c), the number of CD68+ cells in the hippocampus in EE-reared mice was significantly lower than that in the SE-reared mice (SE vs EE: 67 ± 7 vs 38 ± 4, *P < 0.05).

Figure 7.

Analysis of the inflammatory marker, CD68 (activated microglia/macrophages). CD68 protein expression in (a) SE mice and (b) EE mice (b) (200×). (c) Number of CD68+ cells per field in SE- and EE mice (*P < 0.05).

EE-reared mice showed increased resistance to inflammation induced by TNF-α

We used TNF-α to induce an inflammatory response and assayed the expression of the critical inflammatory cytokines, TNF-α and IL-1β, in the hippocampus, 5 h after TNF-α or phosphate-buffered saline (PBS) injection. As shown in Figure 8, both the pro-inflammatory cytokines TNF-α and IL-1β were increased within the hippocampus after TNF-α injection; however, this increase was significantly attenuated in EE-reared mice compared to SE-reared mice (TNF-α: SE-PBS vs EE-PBS: 24 ± 4 vs 18 ± 3 ng/g, SE-TNF-α vs EE-TNF-α: 731 ± 120 vs. 438 ± 75 ng/g; IL-1β: SE-PBS vs EE-PBS: 23 ± 3 vs 17 ± 2 ng/g, SE-TNF-α vs EE-TNF-α: 529 ± 71 vs 278 ± 56 ng/g; *P < 0.05).

Figure 8.

Results of ELISA for expression of inflammatory cytokines in the hippocampal tissue (*P < 0.05, **P < 0.01).

Discussion

EE is defined as an experimental form of rehabilitation in which laboratory animals are reared in an environment of cognitive, motor and sensory stimulation at levels much greater than that in standard housing conditions.^{1–6,9,10,15,16} Although EE has been shown to benefit physical and cognitive ability, as well as social interaction, the preventive role of EE in the healthy adult mice and the effect of EE on brain microenvironment have remained unclear.

We subjected normal healthy adult mice to EE and then measured the behavioral performance. The MWM test for learning and memory is dependent on the hippocampus.¹⁷ We found that EE-reared mice had better cognitive performance than SE-reared mice and that during the 60-s probe trial, the EE-reared mice showed a stronger preference for the platform quadrant than the SE-reared mice, which is consistent with earlier studies.¹⁸ After 3 weeks of EE-rearing, there was a marked increase in the locomotor activity and freezing responses in the OFT and fear conditioning test, respectively. Contextual fear learning has traditionally been attributed to both the hippocampus and the amygdala.^19–21 Hence, our data suggest that EE enhanced not only the long-term memory and spatial navigation but also exploration and nonspatial learning ability, such as depression-like behavior.

In contrast, EE-reared mice showed increased protein expressions of both Iba1 (marker of microglia) and GFAP (marker of astrocytes) in the hippocampus, which is consistent with findings of previous studies which showed increased generation of glia under EE conditions⁸ and as well as after wheel running exercises.²² EE has been shown to upregulate neurotrophins, such as BDNF, which play an essential role in these enrichment-induced alterations, both at the behavioral and cellular level.^23,24 In this study, EE was associated with a markedly increased expression of BDNF. Therefore, our data support the view that BDNF can contribute to the alterations following EE, including learning enhancement²⁵ and freezing response for fear conditioning.²⁶

Microglia, resident brain parenchymal macrophages, play either a beneficial or deleterious role depending on their differential polarization²⁷ and regulate cytokines and inflammatory response in brain and in major depression behaviors.²⁸ Astrocytes and microglia cells of the CNS secrete TNF-α in reaction to inflammatory or infectious stimuli.²⁹ This discovery of significant and complex relationship between immune system, EE, and brain function has led to a speculation that EE may exert an inhibitory effect on inflammatory diseases.

IL-1β is a well-known pro-inflammatory molecule that is important for normal brain function.^30,31 Moreover, physiological levels of IL-1β are essential for normal learning and memory function.³² Both too low or too high levels have a harmful effect.³³ TNF-α is a pleiotropic cytokine that is recognized as a central not exclusive mediator in the CNS under physiological and pathological conditions.³⁴ Our study shows that exposure to an EE significantly reduced the expression of TNF-α and IL-1β in the hippocampus after injection of TNF-α in the healthy mice. Before TNF-α injection, EE decreased the expression of TNF-α and IL-1β; however, there was no statistical difference. We also found that the expression of RIP1 and RIP3 was decreased in EE-reared mice after injection of TNF-α in healthy mice. Our study shed light on the underlying mechanism of the neuroprotective effect of EE and suggested that the improved behavioral performance under EE conditions is mediated via the regulation of microenvironment in healthy adult mice and prevention of the inflammatory response.

Although our study showed that EE improved cognitive and behavioral performance and attenuated inflammatory response after injection of TNF-α, there are still many limitations that need to further studied. What changes did TNF and EE treatments bring to the construction of brain tissue and how did these changes happen and the relationship between the changes of brain tissue and behavioral performance are the issues that need to be studied in depth.

These findings provided evidence that EE can positively influence cognitive and behavioral performance in healthy adult mice by exerting environ-immuno effect on neural function and that EE attenuates inflammatory response after injection of TNF-α in healthy adult mouse.

Experimental procedures

Animals and experimental design

In all, 40 adult male C57BL/6 10-week-old mice were purchased from the Shanghai Laboratory Animal Center (SLAC), Chinese Academy of Sciences. Animals were randomly divided into two groups and reared in two different housing conditions for 3 weeks. All the animals in this study were allowed free access to water and food and reared in accredited room with 12-h light/dark cycle. After 3 weeks, we carried out behavioral tests on mice. Following these operation and analysis, mice were sacrificed and brain samples used for subsequent study. The study protocol was approved by the Institutional Animal Care and Use Committee of Fudan University, Shanghai, China (20160966A284, 27 February 2016).

Housing condition

EE

Eight mice were housed in one EE cage consisting of a large plastic cage (56 × 40 × 22 cm). The EE cage had various items for the mice to play and manipulate. The stimuli were as follows: plastic tunnels, wooden climbing frame, platforms, hiding shelters, house, exercise wheel, chew toys, and other novel objects designed specifically for small animals. In order to maintain novelty, the items in the cage were rearranged every other day and were replaced completely when the cage was cleaned twice a week. After 3 weeks, half of the mice were subjected to a series of behavioral tests and then sacrificed for further study, and the other half were administered intravenous injection of recombinant murine TNF-α (30 μg/kg body weight; R&D Systems, Minneapolis, MN, USA) or vehicle (sterile PBS, control group) and then sacrificed for further analysis.

SE

One SE cage contained three mice. SE cage was a standard plastic rodent cage without special equipment (31 × 21 × 13 cm). We observed the state of mice every day and cages were cleaned completely once a week.

Behavioral tests

EE-reared and SE-reared animals (n = 20) were subjected to a series of behavioral tests. All experimental animals were subjected to OFT and fear condition test on 22, 24, and 25 days after being kept in EE or SE environment; the water maze test was conducted between days 27 and 32. The workers involved in conducting behavioral tests and data analysis were blinded to the group identity of the animals.

OFT

An OFT was used to evaluate a series of behaviors, such as exploratory, anxiety, and spontaneous locomotor activity as described previously³⁵ with slight modifications. The mouse was placed in an open field apparatus having a camera attached at the center, which was linked to a computer. The mouse was allowed to freely move around for 15 min after a 2-min habituation period. Total distance traveled in the apparatus was determined. The apparatus was cleaned with 75% ethanol after each test. Total distance traveled in the apparatus was determined.

Contextual fear conditioning test

The methodology used was as described previously,²⁴ but with slight modifications. The apparatus for fear conditioning included a conditioning chamber equipped with an automated shock generator and a computer software system. During the conditioning session, mice were placed in the conditioning chamber for 150 s and were then given a 30 s tone (10 kHz, 75 dB sound pressure level (SPL)) and a foot shock (2 s, 0.7 mA, constant current). Freezing responses were monitored for 30 s after tone and foot shock, and then, the mice were returned to their home cages. Mice were put back into the conditioning chamber 24 h after the conditioning training, and freezing responses were recorded for 5 min by two researchers who were blinded to the experimental conditions.

MWM test

The MWM test was conducted to measure spatial learning and memory of mice for six consecutive days as described previously by our team.³⁶ Water maze test was started after 3 weeks of rearing in EE or SE housing. In brief, a cylindrical water pool was filled with water (22°C–25°C) and then rendered as opaque by adding edible white pigment in order to cover the platform. An escape platform was placed in the water pool 1 cm below the surface of the water and fixed at a stationary location. The experiment was divided into two sections. The first section served as the training section for five consecutive days; the second one was the test section. On days 1–5, mice were trained to find the escape platform. The trial was not stopped until mouse reached the platform or lasted a maximum of 60 s. On day 6, the platform was removed. The time spent in passing through and the time spent in the platform quadrant as well as escape latency and distance to cross over the platform were recorded using a computer software linked with a camera (Nikon Eclipse Ti-S; Nikon, Tokyo, Japan).

Histopathological examination and immunofluorescence staining

Following behavioral test, animals were anesthetized with 10% chloral hydrate and then perfused with PBS. After sacrifice, brains were removed and fixed in 4% paraformaldehyde solution at 4°C overnight. Subsequently, the brains were dehydrated in 30% sucrose solution and 25-μm-thick sections prepared. Sections were rinsed with PBS and blocked with PBS/5% milk for 1 h and probed with primary antibodies in 3% bovine serum albumin (BSA) solution overnight at 4°C. On the second day, the sections were incubated with secondary antibodies tagged with fluorescent dye (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at room temperature. Finally, the slides were mounted with ProLong Gold antifade reagent (Life Technologies, Grand Island, NY, USA) and the images captured using a fluorescence microscope (Nikon Eclipse Ti-S). The following primary antibodies were used: goat polyclonal anti-GFAP and Iba1 (1:300; Santa Cruz Biotechnology, Inc.).

For the immunohistochemical examination, we performed 3,3′-diaminobenzidine (DAB) staining according to the protocol described elsewhere³⁷ with slight modifications. Briefly, brains were removed, paraffin-embedded, and 25-μm-thick sections prepared. These were incubated in 0.3% H₂O₂ in PBS for 30 min and then incubated in PBS containing 0.3% Triton X-100 for 15 min at 25°C. Following a 30-min preincubation in 10% (wt./vol.) BSA (in PBS), the sections were incubated overnight at 4°C with primary antibody (CD68, 1:300). The sections were then washed with PBS and treated with appropriate secondary antibodies (Vector Laboratories, Burlingame, CA, USA). After rinsing in PBS, immunoreactivity was visualized by the avidin–biotin complex (ABC) method (Vectastain ABC kit; Vector Laboratories) and developed using diaminobenzidine (DAB) staining. Several fields were photographed in each section, and at least five serial sections spaced 200 μm were counted for each mouse. In every third section, CD68-positive cells were counted per field by a researcher blinded to the feature of the photographs.

Western blotting analysis

After 3 weeks of injection, mice were deeply anesthetized with 10% chloral hydrate, and then, hippocampal regions of the brains were dissected and rinsed with homogenization buffer. Total protein extracts and Western blotting analysis were performed as previously described.³⁶ Tissues were lysed in a homogenizer containing radioimmunoprecipitation assay (RIPA) lysis buffer keeping in 0°C–4°C (Beyotime Biotechnology, Shanghai, China). Each protein sample of 40 μg was dispensed to the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS/PAGE) gel well and separated by 10%–15% SDS/PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA). After that, membranes were blocked with 5% milk solution and immunoblotted with primary and secondary antibodies. We measured the levels of RIP1 and RIP3 (1:1000; Santa Cruz Biotechnology, Inc.), respectively.

Enzyme-linked immunosorbent assay

Concentrations of TNF-α, IL-1β, and BDNF proteins from the hippocampal homogenates were quantified using enzyme-linked immunosorbent assay (ELISA) kits (Mouse TNF-α ELISA Kit, Mouse IL-1β ELISA Kit, Mouse BDNF ELISA Kit; RayBiotech, Norcross, GA, USA) in accordance with the manufacturer’s protocol. Data from each sample were normalized for the protein concentration.

Statistical analyses

Values were reported as mean ± standard deviation (SD). Between-group differences were assessed by two-tailed t test. P < 0.05 was considered indicative of statistically significant difference. All graphs were constructed using GraphPad Prism (version 5, GraphPad Software, San Diego, CA, USA).

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Funding

This study was supported by the Natural Science Foundation of China (NSFC,nos. 81472150),the Key Projects of Shanghai Science and Technology on Biomedicine (nos. 13411951000),and the Key Construction Projects of Shanghai Health and Family Planning on Weak Discipline (nos. 2015ZB0401).

References

Doulames

Lee

Shea

(2013) Environmental enrichment and social interaction improve cognitive function and decrease reactive oxidative species in normal adult mice. International Journal of Neuroscience 124(5): 369–376.

Cracchiolo

Mori

Nazian

et al . (2007) Enhanced cognitive activity-over and above social or physical activity-is required to protect Alzheimer’s mice against cognitive impairment, reduce Ab deposition, and increase synaptic immunoreactivity. Neurobiology of Learning and Memory 88: 277–294.

Jadavji

Kolb

Metz

(2006) Enriched environment improves motor function in intact and unilateral dopamine depleted rats. Neuroscience 140: 1127–1138.

Smith

Lyons

Correa

et al . (2016) Behavioral and physiological consequences of enrichment loss in rats. Psychoneuroendocrinology 77: 37–46.

Hüttenrauch

Salinas

Wirths

(2016) Effects of long-term environmental enrichment on anxiety, memory, hippocampal plasticity and overall brain gene expression in C57BL6 mice. Frontiers in Molecular Neuroscience 9: 62.

Ron-Harel

Cardon

Schwartz

(2011) Brain homeostasis is maintained by “danger” signals stimulating a supportive immune response within the brain’s borders. Brain, Behavior, and Immunity 25: 1036–1043.

Singhal

Jaehne

Corrigan

et al . (2014) Cellular and molecular mechanisms of immunomodulation in the brain through environmental enrichment. Frontiers in Cellular Neuroscience 8: 97.

Williamson

Chao

Bilbo

(2012) Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus. Brain, Behavior, and Immunity 26: 500–510.

Veena

Srikumar

Raju

et al . (2009) Exposure to enriched environment restores the survival and differentiation of new born cells in the hippocampus and ameliorates depressive symptoms in chronically stressed rats. Neuroscience Letters 455(3): 178–182.

10.

Zheng

Zhang

et al . (2014) Oxytocin mediates early experience-dependent cross-modal plasticity in the sensory cortices. Nature Neuroscience 17: 391–399.

11.

Baamonde

Martinez-Cue

Florez

et al . (2011) G-protein-associated signal transduction processes are restored after postweaning environmental enrichment in Ts65Dn, a down syndrome mouse model. Developmental Neuroscience 33(5): 442–450.

12.

Young

Lawlor

Leone

et al . (1999) Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective. Nature Medicine 5: 448–453.

13.

Filiano

Tustison

et al . (2016) Unexpected role of interferon-γ in regulating neuronal connectivity and social behaviour. Nature 535(7612): 425–429.

14.

Zhang

Baloch

(2016) Pathogenetic and therapeutic applications of tumor necrosis factor-α (TNF-α) in major depressive disorder: A systematic review. International Journal of Molecular Sciences 17(5): E733.

15.

Livingston-Thomas

Nelson

Karthikeyan

et al . (2016) Exercise and environmental enrichment as enablers of task-specific neuroplasticity and stroke recovery. Neurotherapeutics 13(2): 395–402.

16.

Morris

Garrud

Rawlins

et al . (1982) Place navigation is impaired in rats with hippocampal lesions. Nature 297(5868): 681–683.

17.

Zhang

Wang

et al . (2016) Increased protein expression levels of pCREB, BDNF and SDF-1/CXCR4 in the hippocampus may be associated with enhanced neurogenesis induced by environmental enrichment. Molecular Medicine Reports 14: 2231–2237.

18.

Phillips

LeDoux

(1992) Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behavioral Neuroscience 106: 274–285.

19.

Maren

(2001) Neurobiology of Pavlovian fear conditioning. Annual Review of Neuroscience 24: 897–931.

20.

Goosens

Maren

(2001) Contextual and auditory fear conditioning are mediated by the lateral, basal, and central amygdaloid nuclei in rats. Learning & Memory 8: 148–155.

21.

Ehninger

Kempermann

(2003) Regional effects of wheel running and environmental enrichment on cell genesis and microglia proliferation in the adult murine neocortex. Cerebral Cortex 13: 845–851.

22.

Rossi

Angelucci

Costantin

et al . (2006) Brain-derived neurotrophic factor (BDNF) is required for the enhancement of hippocampal neurogenesis following environmental enrichment. European Journal of Neuroscience 24: 1850–1856.

23.

Falkenberg

Mohammed

Henriksson

et al . (1992) Increased expression of brain-derived neurotrophic factor mRNA in rat hippocampus is associated with improved spatial memory and enriched environment. Neuroscience Letters 138: 153–156.

24.

Piazza

Segabinazi

Centenaro

et al . (2014) Enriched environment induces beneficial effects on memory deficits and microglial activation in the hippocampus of type 1 diabetic rats. Metabolic Brain Disease 29: 93–104.

25.

Liu

Lyons

Mamounas

et al . (2004) Brain-derived neurotrophic factor plays a critical role in contextual fear conditioning. Journal of Neuroscience 24(36): 7958–7963.

26.

Wang

et al . The biphasic function of microglia in ischemic stroke. Progress in Neurobiology. Epub ahead of print 1 February 2016. DOI: 10.1016/j.pneurobio.2016.01.005.

27.

Kim

Myint

et al . (2016) The role of pro-inflammatory cytokines in neuroinflammation, neurogenesis and the neuroendocrine system in major depression. Progress in Neuro-Psychopharmacology & Biological Psychiatry 64: 277–284.

28.

Kronfol

Remick

(2000) Cytokines and the brain: Implications for clinical psychiatry. American Journal of Psychiatry 157: 683–694.

29.

Yirmiya

Goshen

(2011) Immune modulation of learning, memory, neural plasticity and neurogenesis. Brain, Behavior, and Immunity 25: 181–213.

30.

Tanaka

Kondo

Kanda

et al . (2011) Involvement of interleukin-1 in lipopolysaccharide-induced microglial activation and learning and memory deficits. Journal of Neuroscience Research 89: 506–514.

31.

Cibelli

Fidalgo

Terrando

et al . (2010) Role of interleukin-1beta in postoperative cognitive dysfunction. Annals of Neurology 68: 360–368.

32.

Goshen

Avital

Kreisel

et al . (2009) Environmental enrichment restores memory functioning in mice with impaired IL-1 signaling via reinstatement of long-term potentiation and spine size enlargement. Journal of Neuroscience 29: 3395–3403.

33.

Williamson

Sholar

Mistry

et al . (2011) Microglia and memory: Modulation by early-life infection. Journal of Neuroscience 31: 15511–15521.

34.

Clark

Alleva

Vissel

(2010) The roles of TNF in brain dysfunction and disease. Pharmacology & Therapeutics 128(3): 519–548.

35.

Miki

Ishibashi

Sun

et al . (2009) Intensity of chronic cerebral hypoperfusion determines white/gray matter injury and cognitive/motor dysfunction in mice. Journal of Neuroscience Research 87(5): 1270–1281.

36.

Zhang

Wang

et al . (2016) Necrostatin-1 attenuates inflammatory response and improves cognitive function in chronic ischemic stroke mice. Medicines 3(3): 16.

37.

Huang

Tang

et al . (2013) CXCR4 antagonist AMD3100 protects blood-brain barrier integrity and reduces inflammatory response after focal ischemia in mice. Stroke 44: 190–197.

Enriched environment improves behavioral performance and attenuates inflammatory response induced by TNF-α in healthy adult mice

Abstract

Keywords

Introduction

Results

Experimental protocol

Housing conditions

Behavioral tests

Upregulation of glial fibrillary acidic protein and Iba1 expression in EE group

EE decreased the expression of RIP1 and RIP3 proteins

EE mice had increased expression of BDNF, but the expression of TNF-α and IL-1β remained unchanged

EE-reared mice attenuated expression of the inflammatory marker, CD68, after TNF-α injection

EE-reared mice showed increased resistance to inflammation induced by TNF-α

Discussion

Experimental procedures

Animals and experimental design

Housing condition

EE

SE

Behavioral tests

OFT

Contextual fear conditioning test

MWM test

Histopathological examination and immunofluorescence staining

Western blotting analysis

Enzyme-linked immunosorbent assay

Statistical analyses

Footnotes

Declaration of conflicting interests

Funding

References