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 hours= 2,2±0,22). However, from 72h to 30 days (sham 72 hours= 2,08±0,39; CLP 72 hours= 5,8±0.41; sham 10 days= 2,0±0,45; CLP 10 days= 6,0±0,58; sham 30 days= 2,1±0,24; CLP 30 days= 5,5±0,51) after sepsis an increase in the activity of this enzyme was noted when compared septic to sham group (Figure 5a). It was not observed changes in the activity of HAT enzyme in any time-point studied (sham 24 hours= 0,057±0,013; CLP 24 hours= 0,042±0,007; sham 72 hours= 0,028±0,006; CLP 72 hours= 0,038±0,006; sham 10 days= 0.05±0,008; CLP 10 days= 0,053±0,009; sham 30 days= 0,05±0,01; CLP 30 days= 0,042±0,009) (Figure 5b). DNMT is associated with DNA methylation. There was no difference in DNMT activity 24 hours after sepsis (sham 24 hours= 0,05±0,006; CLP 24 hours= 0,042±0,009). After 72h, 10 and 30 days of sepsis induction there was an increase in the activity of DNMT (sham 72 hours= 0,022±0,004; CLP 72 hours= 0,057±0,022; sham 10 days= 0,21±0,006; CLP 10 days= 0,067±0,006; sham 30 days= 0,02±0,004; CLP 30 days= 0,077±0,009) (Figure 5c).
Correlation of DNMT and HDAC Epigenetic Enzymes
The correlation of the activity of epigenetic enzymes DNMT and HDAC was evaluated in the hippocampus of animals submitted to sepsis. It is understood that the DNMT enzyme is related to DNA methylation and the HDAC enzyme with changes in chromatin, and these factors interact with each other. It was noticed that in the times of 24 hours (r = 0,767) (Figure 6a), 10 days (r = 0,378) (Figure 6c), and 30 days (r = 0,567) (Figure 6d), the activity of the two enzymes showed a significantly positive correlation. However, the 72-hour time (r = 0,224) (Figure 6b) showed a correlation, however, not significantly.
Enriched environment effects on HAT, HDAC and DNMT activities in hippocampus of animals submitted to sepsis.
It was not observed changes in the activity of HAT enzyme after EE protocol (sham + SE= 0,074125±0,00317; sham + EE= 0,074100±0,002130; CLP + SE= 0,074000±0,006825; CLP + EE= 0,73100±0,003512) (Figure 7a). In HDAC (sham + SE= 1,750000±0,099247; sham + EE= 1,586000±0,173136; CLP + SE= 5,032000±0,373148; CLP + EE= 2,044420±0,178453) and DNMT (sham + SE= 0,054830±0,004216; sham + EE= 0,059132±0,003548; CLP + SE= 0,086234±0,003587; CLP + EE= 0,042636±0,003278) enzymes activities there were an increase in CLP + Standard Environment group and in both enzyme activities, EE protocol reverse these alterations (Figure 7b and 7c).
Open-field test
In order to assess habituation memory and impairment of animals, the habituation test in the open field was performed. The CLP + Standard Environment group showed cognitive impairment, as there was no significant difference between training and testing in exploratory (rearing) and locomotor (crossing) activity (CLP + Standard Environment: rearing training= 16,88±1,172; rearing test= 13,63±1,603; crossing training= 42,50±3,655; crossing test= 38,50±3,495). However, the CLP + EE group showed a significant difference between training and testing, (CLP + EE: rearing training= 20,91±1,771; rearing test= 11,18±1,174; crossing training= 41,45±4,196; crossing test= 26,36±2,684). demonstrating an improvement in habituation memory, confirming its ability to reverse the changes generated by sepsis (Figure 8).
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 deficit, 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 significant 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 confirming 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 significant decrease in IL-1β levels (Figure 10).
Synaptic Protein Levels
Synaptophysin is considered a specific marker of synaptic vesicles, which plays an important role in the release of neurotransmitters. There were no significant changes in synaptophysin levels in any of the groups analyzed after the EE protocol in animals submitted to sepsis (sham + SE= 0,73±0,11; sham + EE= 0,68±0,14; CLP + SE= 0,68±0,09; CLP + EE= 0,64±0,08) (Figure 11a).
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). However, the EE protocol (0,90±0,30) was able to increase PSD-95 levels in the hippocampus of animals subjected to sepsis (Figure 11b).