Enriched Environment Causes Epigenetic Alterations in the Hippocampus and Improve Long-term Cognitive Function Sepsis-Induced

Background: Sepsis is dened as the presence of life-threatening organ dysfunction. The presence of oxidative stress and inammatory mediators in sepsis leads to dysregulated gene expression, leading to a hyperinammatory response. Environmental conditions play an important role in various pathologies depending on the stimulus it presents. A standard environment may offer reduced sensory and cognitive stimulation, but an enriched environment improves spatial learning, prevents cognitive decits induced by disease stress, and is an important modulator of epigenetic enzymes. The study evaluated the epigenetic alterations and the effects of the environmental enrichment (EE) protocol in the brain of animals submitted to sepsis by cecal ligation and perforation (CLP). Methods: Male Wistar rats were divided into sham and CLP at 24 hours, 72 hours, 10 days and 30 days after sepsis. Other male Wistar rats were distributed in a standard environment or in EE for forty-ve days. Behavioral tests, analysis of epigenetic enzymes:histone acetylase (HAT), histone deacetylase (HDAC) and DNA methyltransferase (DNMT), biochemical and synaptic plasticity analyzes were performed. Results: An increase in HDAC and DNMT activities was observed at 72 hours, 10 days and 30 days. There was a positive correlation between epigenetic enzymes DNMT and HDAC 24 hours, 10 days and 30 days. After EE, HDAC and DNMT enzyme activity decreased, cognitive impairment was reversed, IL1-β levels decreased and there was an increase in PSD-95 levels in the hippocampus. Conclusion: Interventions in environmental conditions can modulate the outcomes of cognitive consequences supporting the idea of the EE. (1:1000). Secondary The using ECL. Densitometry analysis of the lms was performed using the ImageJ v.1.34 software. All results were as a relative between anti-PSD-95 and GAPDH.


Background
Sepsis and septic shock are potentially life-threatening conditions that are associated with high morbidity and mortality [1,2]. A relevant characteristic of sepsis is the presence of encephalopathy followed by microglial activation [3] and the consequent expression of in ammatory mediators (cytokines and chemokines) and oxidative stress [4][5][6][7][8]. Recent studies show how oxidative stress insults and in ammatory events can lead to the vulnerability of the expression of some genes and epigenetic alterations [9][10][11][12].
Changes in gene expression during sepsis initiation and progression are related [13][14][15]. Epigenetic mechanisms are considered exible parameters and can be altered by various stimuli, such as stress and changes in the environment. Failures in the establishment or maintenance of epigenetic marks can result in improper activation or inhibition of several genes and alter normal cell physiology leading to the appearance of pathologies [16,17]. The main epigenetic regulations are related to the dynamic states of chromatin, such as changes in histones and in the DNA methylation pattern. Histones are small proteins that, by complexing with DNA, form the nucleosome core. These proteins can be in one of two different forms, acetylated or deacetylated, which are regulated by the enzymes histone acetylases (HATs) and histone deacetylases (HDACs). Histone acetylation increases genic transcription while histone deacetylation represses it [18]. In addition, epigenetic modi cations of DNA also regulate genic transcription, such as DNA methylation. Methylation of DNA involves covalent binding of a methyl group to cytosines by enzymes called DNA methyltransferases (DNMTs), which lead to promoter methylation and gene transcription is suppressed [19] (Figure 1).
Environmental conditions play a fundamental role in the cognitive performance of several neuropsychiatric diseases, affecting in a positive or negative way, depending on the stimulus that this environment offers and are considered factors that can modulate epigenetic mechanisms [20,21]. The environment considered standard is one that offers reduced sensory and cognitive stimulation, whereas the enriched environment (EE) is de ned as an environment that presents physical and social stimuli [22,23]. The negatively altered behavioral and neuroendocrine response is seen in a standard setting [21,24]. Evidence shows that patients admitted to the ICU are subject to this environment, which may have negative effects on cognitive performance [25,26]. However, an EE can improve spatial learning, decrease mortality and prevent cognitive de cits induced by stress from a certain pathology [22,27].
In order to defend these hypotheses, current animal studies show that these neurocognitive alternations were accompanied by signi cant changes in the biomarkers of the immune response and synaptic plasticity of the hippocampus [28][29][30]. Thus, it is understood that the effects of environmental conditions on cognitive function are reversible, with an improvement in cognitive performance when animals are governed in an EE [28].
Epigenetic changes can provide mechanistic information about the occurrence and treatment of diseases, but little is known about these mechanisms during the development of sepsis in the brain. Here, epigenetic changes in the hippocampus of animals submitted to the sepsis model have been reported and we suggest that interventions in environmental conditions may modulate the results of the long-term cognitive consequences associated with sepsis, supporting the idea of the potential bene ts of an EE.

Ethics
The experimental procedures involving animals were carried out in accordance with the ARRIVE guidelines and the Brazilian legislation on animal welfare, with the approval of the Ethics Committee on the Use of Animals of the University of the Extreme South of Santa Catarina -Unesc (protocol number: 008-2017 / 2).

Animals
The study was divided into two experiments. Adult male Wistar rats, 60 days old, weighing between 200-300g were used. The rats were kept in light-dark cycles of ± 12 hours (7:00 am to 7:00 pm) at a temperature between 18 and 22 ° C, relative humidity between 55 and 65%. The animals had free access to water and food. At the end of the experiments, the animals were euthanized by the decapitation method. The choice of not using females for this study is because that sex has hormonal changes that lead to an increase in mortality.
After 48 hours, the animals were distributed in a standard environment or EE and kept in these housing conditions for forty-ve days. After this period, the animals were subjected to behavioral tests and, nally, euthanasia was performed using the decapitation method and the hippocampus was removed for later neurochemical and immunological analyzes ( Figure 3).

Sepsis induction -cecal ligation and perforation (CLP) model
Male Wistar rats were subjected to CLP as previously described [31]. Brie y, animals were anesthetized using a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg), given intraperitoneally. Under aseptic conditions, a 3 cm midline laparotomy was performed to expose the cecum and adjoining intestine. The cecum was ligated with a 3.0 silk suture at its base, below the ileocecal valve, and was perforated once with a 14-gauge needle. The cecum was then squeezed gently to extrude a small amount of feces through the perforation site. The cecum was then returned to the peritoneal cavity, and the laparotomy was closed with 4.0 silk sutures. Animals were resuscitated with saline (NaCl 0.09%, 50 mL/kg) subcutaneously (s.c.) immediately after and 12 h after CLP. All animals received antibiotics (ceftriaxone at 30 mg/kg) and dipyrone (80mg/kg) every 6 h s.c. for 3 days. The same investigator always performed the CLP procedure to minimize variability between different experiments. The mortality of this model is around 40% that is consistent with severe sepsis.

Environmental conditions
After recovery from sepsis-inducing surgery (48 hours), the animals were randomly assigned to two different environmental conditions: standard environment or EE and kept in these housing conditions for forty-ve days. The standard environment consisted of common cage housing (41 cm × 34 cm × 18 cm), while the EE consisted of larger cages (80 cm × 45 cm × 22 cm) containing a small house and a racing wheel for voluntary exercise. and toys that were changed three times a week with new toys of different shapes and colors. For the standard environment, the animals were grouped in ve per box [28] ( Figure 4).

HAT, HDAC, DNMT activity
At determined times (24h, 72h, 10 and 30 days), the animals were killed by decapitation, and the hippocampus was dissected (n=5). The obtained samples were ash-frozen and stored at −80 °C until nuclear proteins could be extracted. The hippocampus was subjected to a nuclear extraction protocol using a commercial Nuclear Extraction kit (Chemicon, USA). Nuclear extraction was used to evaluate the levels of HAT, HDAC, and DNMT activity.
In order to evaluate the activity of HAT, HDAC and DNMT, the nuclear extracts were submitted to an assay for the evaluation of HAT, HDAC, and DNMT activity using assay kit (colorimetric detection) according to the manufacturer's instructions (Epigentek cod. HAT P-4003-96; HDAC P-4002-96; DNMT P-3001-2). A standard curve was performed with serial dilutions of subtracts from each kit and positive and negative controls were added to the plate. Colorimetric reading was performed on an ELISA plate reader. The calculation of enzymes activity was performed based on the standard curve, and the values were presented in nM/μg of protein. Protein dosages were determined by the method of Lowry [31] and bovine serum albumin was used as a standard.

Behavioral tasks
After enriched environment protocol, the animals were subjected to habituation to the open-eld task and the object recognition test. After the behavioral tests, the animals were sacri ced by decapitation, the hippocampus was immediately isolated on dry ice and stored at -80°C for other analysis.

Open-eld test
Behavior was assessed in an open-eld apparatus to evaluate both locomotor and exploratory activity. The apparatus was a 40 × 60 cm open eld surrounded by 50-cm-high dark grey walls and a glass front wall. Black lines divided the oor of the open eld into nine rectangles. Each animal was gently placed in the center of the open eld and was left to explore the arena for 5 min (training session). The number of crossings (i.e., the number of times that each animal crossed the black lines, an assessment of locomotor activity) and rearing movements (i.e., the exploratory behavior observed in rats subjected to a new environment) were measured. Immediately after this procedure, the animals were taken back to their home cage. Twenty-four hours later, they were subjected to a second open-eld session (test session). In both sessions, the number of crossings and rearings was counted during a 5-min period. A reduction in the number of crossings and rearings between the two sessions was considered as a measure of the retention of memory. The same experimenter, who was blind to group allocation, performed all the behavioral testing and manual scoring [33]. The experimental box was thoroughly cleaned with 70% ethanol between testing sessions.

Object recognition
For object recognition animals were allowed to explore an open eld. Training was conducted by placing rats in the eld in which two identical cubes (objects A1 and A2) were positioned. Twenty-four hours posttraining animals were allowed to explore the eld in the presence of the familiar object A but a novel object C (a sphere with a square-shaped base). A recognition index was calculated and reported as the ratio TB/ (TA + TB) (TA = time spent exploring the familiar object A; TB = time spent exploring the novel object B) [34].

Assessment of the concentration of IL-1β
Brie y, the hippocampus was homogenized in PBS buffer (100 mg of tissue per 1 mL). The levels of IL-1β were determined using commercially available enzyme-linked immunosorbent assays (ELISA), following the instructions supplied by the manufacturer (DuoSet kits, R&D Systems, Minneapolis, MN, USA). The results are expressed as pg/mL.

Synaptic Protein Levels
Levels of synaptophysin (a speci c synaptic vesicle marker that plays an important role in the release of neurotransmitters) were determined in the hippocampus using the commercial enzyme-linked immunosorbent assay kit (ELISA) [35]. To perform immunoblotting of PSD-95 (postsynaptic density protein 95), tissue samples of hippocampus was homogenized in Laemmli buffer (62.5 mM TrisHCl, pH 6.8, 1% (w/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol) and equal amounts of protein (30 µg/well) were fractionated by polyacrylamide gel electrophoresis-sodium dodecyl sulfate (SDS-PAGE) and electro transferred to nitrocellulose membranes. The e ciency of the electro transfer was veri ed by Ponceau staining, and the membrane was then blocked in Tris-Tween buffer saline (TTBS: 100 mM Tris HCl, pH 7.5, containing 0.9% NaCl and 0.1% Tween 20) with 5% albumin. The membranes were incubated overnight at 4 o C with rabbit monoclonal anti-PSD-95 (1:1000). Secondary anti-rabbit IgG was incubated with the membrane for 2h (1:10000). The immunoreactivity was detected by chemiluminescence using ECL. Densitometry analysis of the lms was performed using the ImageJ v.1.34 software. All results were expressed as a relative ratio between anti-PSD-95 and GAPDH.

Statistics
The data from the behavioral test of habituation in the open eld were evaluated through the mean and the standard error of the mean (EPM), being EPM the statistical difference calculated through the Student T test for independent samples. For the object recognition test, the results were expressed as median and interquartile range and the differences between training and test were evaluated by the Mann-Whitney U test followed by the Wilcoxon test.
For biochemical and immunological analyzes, data were expressed as mean ± standard deviation and compared with one-way analysis of variance (ANOVA) followed by the Tukey test. For the correlation of epigenetic enzymes, data were expressed using Pearson's correlation coe cient, considering the "r" value. Western blotting was quanti ed by the ImageJ ® program and presented as a ratio between protein and GAPDH. All tests were analyzed using SPSS version 20 and / or GraphPad Prism 5.0. In all analyzes, a level of p <0.05 was adopted for statistical signi cance.

Results
Time-dependent alterations on HAT, HDAC and DNMT activities in hippocampus of animals submitted to sepsis.
HDAC and HAT are enzymes associated with histone acetylation and deacetylation respectively. In HDAC activity, there was no difference at 24 hours after sepsis (sham 24 hours= 2,2±0,2; CLP 24

Object recognition
To further explore the potential protective effect of the environmental enrichment protocol, the object recognition task was used to assess long-term memory. The CLP + Standard Environment group showed a memory de cit, as they did not present greater latency and remained exploring the new object (CLP + Standard Environment: training= 0,440±0,200; test= 0,450±0,060). However, the CLP + EE group showed a signi cant difference in the object recognition index between training and testing (CLP + EE: training= 0,280±0,100; test= 0,710±0,070), manifesting a long-term memory and con rming its ability to reverse the changes generated by sepsis (Figure 9). IL-1β levels in hippocampus after enriched environment protocol in animals submitted to sepsis IL-1β levels were analyzed in the hippocampus after the environmental enrichment protocol in animals submitted to sepsis. The CLP + Standard Environment group (3,00±0,75) demonstrated high levels of IL-1β compared to the Sham + Standard Environment group (1,70±0,15) and the Sham + EE group (1,70±0,25). However, septic animals that were submitted to the environmental enrichment protocol (1,53±0,40) had a signi cant decrease in IL-1β levels ( Figure 10).
To determine the effect of the EE protocol on synaptic plasticity, a highly stable synapse structural protein, also known as PSD-95, was evaluated. The CLP + Standard Environment group (0,15±0,16) showed a decrease in PSD-95 levels compared to the Sham + Standard Environment group (0,48±0,20).

Discussion
This study reported the presence of epigenetic changes in an animal model of sepsis, rst at different times and later showed that exposure to an EE protocol can reduce these changes and decrease the de cits generated by the pathophysiology of sepsis.
HAT and HDAC are enzymes related to histone modi cation, more speci cally acetylation and deacetylation of these proteins. Numerous enzymes act together to add or remove covalent modi cations in histones, interacting with each other and with other mechanisms to maintain chromatin conformation and transcript control [36,37]. DNMTs are enzymes responsible for DNA methylation, they replace the cytosine H5 of DNA by a methyl group. Methylation of the gene leads to inhibition of transcription and it is related to gene silencing, in addition to leading to chromosomal instability [38]. When hypomethylation or hypermethylation occurs then an imbalance in gene expression occurs, leading to important consequences such apoptosis and exacerbated in ammation. In this study, there was no difference between groups in HAT activity. However, changes in HDAC and DNMT activities were notable. A signi cant increase in HDAC and DNMT activities was reported up to 30 days. These results suggest alterations in the genetic transcription in an animal model of sepsis, mainly related to silencing of gene expression up to 30 days, and this is related to cognitive impairment that we demonstrated previously [6,7].
In vitro research suggests that epigenetic changes are essential for establishing the tolerance generated to the pathogen. In autoimmune diseases, cancer and neurological disorders, several epigenetic changes have been reported, which have been associated with the onset or worsening of diseases [39]. In addition, recent studies have demonstrated the involvement of epigenetic changes in targeting the macrophage phenotype, as well as in the function of dendritic cells during sepsis [40][41][42].
Epigenetic regulation occurs through several mechanisms, but in general it is characterized as a regulated organization of gene loci in transcriptionally active or silent states [43]. Recent studies have shown that in postmortem analyzes of the brains of septic patients, the expression of the HDAC enzyme was high [44]. It is known that under normal conditions, the activity of the enzymes HAT and HDAC remains in balance. However, this pattern is modi ed at the beginning of the pathophysiology of sepsis, as there is an intense expression of pro-in ammatory mediators, making it necessary for the HAT enzyme to open the chromatin structure to transcript in ammatory genes, such as IL-1β, which is involved in the central nervous effects of sepsis [45,46], as we showed here. This process ends up by HDAC enzymes, which can be activated or induced by bacterial compounds [47], leading to the reconstitution of chromatin closely linked to the silencing of the gene [48], which can be associated with immunosuppression [49,50].
However, it is possible to correlate the activity of the enzyme HDAC and DNMT during the pathophysiology of sepsis, as these two epigenetic enzymes showed a positive correlation at the times of 24 hours, 10 days and 30 days after sepsis. The interactions between DNA methylation and chromatin modi cation have been known and described for many years [9]. There are studies that already prove that methylated DNA can recruit HDAC enzymes via MeCP2 (methyl-CpG-binding protein 2), leading to histone deacetylation and chromatin condensation, consequently disfavoring transcriptional initiation [44]. Another study showed that the use of tricostatin A, an HDAC inhibitor, attenuated the DNA repression induced by its own methylation, pointing to a cross effect between these two epigenetic changes [38].
Here we con rm that exposing Wistar rats to an EE was able regulated enzymes activities HDAC and DNMT in hippocampus. According to Lambert et al. [51] EE can produce changes in the hippocampus related to learning and memory. EE is associated with neurochemical and physiological changes in the brain. The increase in dendridic rami cations, including in the pyramidal cortical II/III and V, synapses, neuronal size, neurogenesis, neurotropin expression and neurotransmitter release have been related to EE [52,53].
Current studies demonstrate that HDACs are elevated in the brain of septic animals, showing their relationship in neurodegeneration and in the cognitive functions generated by sepsis [17]. Because of this, it is understood that epigenetic modulation is involved in the septic brain and the inhibition of HDACs can serve as a potential therapeutic approach for the treatment of sepsis-associated encephalopathy [50].
The induction of sepsis in animals generates de cits in memory, learning and often exhibits symptoms of a depressive type [3,54]. It is known that when there is exposure to an environment characterized as a standard, these factors do not improve. The data from the present study show that septic animals submitted to a standard environment showed cognitive impairment and exhibited a long-term memory de cit when compared between their training and the test. It is known that the neurocognitive consequences generated by the pathophysiology of sepsis can perpetuate in a standard environment, but when there is exposure to an enriched environment, studies show that these consequences generated by the disease show improvements [28,55]. These ndings are in agreement with the results evidenced in this study, since the septic animals submitted to the AE protocol obtained the ability to reverse the changes generated by sepsis, as they had a long-term memory. Notably, the role of environmental conditions on cognitive function is reversible, showing an improvement in cognitive performance when subjected to an AE protocol, which is characterized by leading to an increase in spatial learning and preventing cognitive de cits generated by sepsis [25,26,30].
Evidence shows that DNA methylation is dynamically modulated by events dependent on neuronal activity [56,57]. DNMTs enzymes not only play a role in gene printing and transcription regulation in the early stages of CNS development, but are also indispensable in learning, memory and cognition, and there are recent reports that show the participation of DNMTs in consolidating the memory of long-term [58-60].
It is known that IL-1β is considered an initial marker of sepsis, as it expresses pro-in ammatory effects that contribute to an intense cellular response to the pathogen, in addition to having the ability to release secondary mediators [61,62]. In the present study, IL-1β levels remained elevated in the hippocampus, forty-ve days after sepsis. However, animals submitted to the AE protocol showed signi cantly decreased levels in brain structure. It is then understood that brain in ammation persists late in septic animals, but that AE has the ability to reverse these damages generated by the pathophysiology of sepsis.
Synapses are vulnerable in neurodegenerative conditions, so sepsis can destroy synaptic proteins and contribute to cognitive de cit [63]. PSD-95 is a highly stable synapse structural protein that binds to NMDA receptors and stabilizes them in postsynaptic neurons, participates in the maturation of synaptic buttons, and favors excitatory synapse [64,65]. Another signaling-dependent protein mentioned above is synaptophysine, a membrane protein present in synaptic vesicles, which can be used as a speci c marker of synaptic vesicles present in the synaptic terminals of presynaptic neuron that plays an important role for neurotransmitter release [35,66]. The present study showed that sepsis decreases PSD-95 levels in the brain, with the hippocampus being the most affected. However, these levels increase in septic animals submitted to the EE protocol. Above all, the synaphysophysin presynaptic protein was not affected in septic animals. A study by Moraes et al.
[67] exposed that synaptophysin did not show changes in its levels, however the PSD-95 protein demonstrated signi cantly reduced levels in the hippocampus of mice 24 hours and 3 days after sepsis induction, however 30 days after sepsis the co-location of PSD -95 was established at normal levels.
There is a correlation between the loss of synapses and the cognitive impairment generated in sepsis.
Therefore, interventions such as the EE protocol can be bene cial in the synaptic plasticity of this pathology. It is important to emphasize that the limitation of this study is the choice of not using females because of that sex they present hormonal changes that lead to an increase in mortality.

Conclusion
In conclusion, the physiological stress that is generated and transmitted to the immune system during severe sepsis, can have signi cant effects on gene expression in the brain, speci cally in the hippocampus tissue, both in the short and long term. Because of this, EE may have the ability to delay and improve cognitive de cits induced by the disease and be a strategy with great potential for modulation of epigenetic enzymes in an animal model of sepsis.

Declarations
Ethics approval and consent to participate Animal studies were approved by the ethics commitment of our institutional ethics committee (protocol number: 008-2017/2).

Availability of data and materials
Material will available are request

Competing interests
The authors declare that they have no competing interests.  Histone acetylation and deacetylation through HAT and HDAC enzymes respectively.

Figure 2
Timeline of the rst experiment, dates of sepsis induction, animal euthanasia and structure removed for analysis.

Figure 3
Timeline of the second experiment, date of sepsis induction, behavioral tests and structure removed for analysis.    Behavioral test of exploratory and locomotor activity after the environmental enrichment protocol in animals submitted to sepsis. The bars represent mean ± SEM, the statistical difference being calculated Page 22/24 using the Student T test for independent samples. nº = 10 each group. * p <0.05 when comparing the test versus training of your own group. EE: environmental enrichment; SE: standard environment.

Figure 8
Object recognition task after the environmental enrichment protocol in animals submitted to sepsis. The recognition index is presented as median and interquartile ranges. Differences between training and testing were assessed using the Mann-Whitney U test followed by the Wilcoxon test. nº = 10 each group. * p <0.05 when comparing the test versus training of your own group. EE: environmental enrichment; SE: standard environment.   PSD-95 levels in the hippocampus after the environmental enrichment protocol in animals submitted to sepsis. PSD-95 levels were measured at the end of the environmental enrichment protocol in the hippocampus of animals subjected to sepsis. The data were expressed as mean ± SD and compared with one-way analysis of variance (ANOVA) followed by the Tukey test. nº = 6 each group. * p <0.05 different from sham + SE; # different from CLP + SE. EE: environmental enrichment; SE: standard environment.