Neurofilament degradation is involved in laparotomy-induced cognitive dysfunction in aged rats
An excessive neuroinflammatory response involved in the pathogenesis of postoperative cognitive dysfunction (POCD), which increases morbidity and mortality. However, the precise mechanism remains unclear. Trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to be anti-inflammatory. Therefore, we aimed to explore whether TSA can inhibit the surgery-induced neuroinflammation and improve POCD and further reveal the complex neuropathogenesis underlying POCD. Methods To explore the
... olecular mechanisms by which surgery-induced POCD in aged rats, TSA (1 mg/kg) was intraperitoneally injected, and hippocampal microglial activation and neuroinflammation were observed. We investigated changes in the protein profile of the hippocampus using a proteomics approach [isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano liquid chromatography-mass spectrometry] at the peak of surgery-induced neuroinflammation, and significant alterations of proteins were verified using western blotting and immunofluorescence. Then, proteins associated with signaling pathways in the surgery + TSA and surgery groups were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Results After laparotomy, aged rats had prolonged escape latencies on days 4 and 5 postsurgery, spent less time in the target quadrant than control rats (p < 0.05), and exhibited excessive hippocampal microglia activation and IL-1β and TNF-α release. iTRAQ and bioinformatics analyses at 6 h after surgery showed that neurofilaments (NFs), including the NEFH, NEFM and NEFL proteins, were significantly upregulated, and TSA pretreatment could mitigate these changes. Subsequently, KEGG analysis revealed that nine pathways were enriched in the surgery + TSA group vs. the surgery group (p < 0.05). Among them, two signaling pathways, "focal adhesion" and "ECM-receptor interaction", were associated with significant upregulation of collagen and downregulation of NF 4 proteins, indicating these as possibly important pathways involved in NF degradation in the hippocampus of aged brains after surgery-induced POCD. Conclusion Surgery-induced neuroinflammation upregulated NFs, resulting NF degradation and aggregation in the hippocampus of aged rats, which might lead to hippocampusindependent learning and memory impairment, contributing to POCD. Additionally, TSA diminished surgery-induced neuroinflammatory responses and modulated the NFassociated changes in cognitive dysfunction in aged brains, which might be related to activation of the "focal adhesion" and "ECM-receptor interaction" pathways. Postoperative cognitive dysfunction (POCD) commonly occurs in older adults after surgery and is frequently under-recognized. Development of POCD has been associated with worse outcomes, longer hospital stays, decreased quality of life, increased mortality and risk of dementia. POCD has a high incidence (10%-54%) and is currently more often recognized clinically because of an increased awareness [1, 2]. In recent years, many studies have suggested that the pathogenesis of POCD is a synergistic effect of various factors, including tau phosphorylation, amyloid β accumulation , blood brain barrier (BBB) disruption  and immune system disorders [5, 6]. However, the precise pathophysiology of POCD remains unclear. The hippocampus is an important component of the limbic system and is associated with the regulation of learning, memory and behaviors. Our previous studies revealed that laparotomy could increase the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) in the hippocampus and cause cognitive dysfunction to persist for several weeks in aged rats . However, the precise mechanism of this neuroinflammatory reaction, which has been confirmed to be a key factor mediating the development of POCD, and its extensive cross-reaction with other elements in 5 hippocampal neurons remain elusive and need to be further explored. Dysfunction of microglia is involved in the amplification of the immune response in the pathophysiology of neurological disorders, including ischemic stroke  and neurodegenerative diseases [9, 10]. Overexpression of inflammatory cytokines in the brain after surgery and anesthesia exposure [5, 6] can be caused by microglial release of cytokines . Microglia constitute 10%-15% of the glial cell population in the parenchyma in the adult brain . Hence, anti-inflammatory therapies can alleviate the progression of brain disorders. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, is also associated with an anti-inflammatory effect , but its role in surgeryinduced neuroinflammation in aged brains is unclear. Hence, our study explored whether TSA can reduce microglia-mediated neuroinflammation and improve surgery-induced cognitive dysfunction as well as the possible mechanism. In the present study, a surgery model was established in aged rats by isoflurane exposure. This study aimed to examine the dynamic changes in microglial activation, surgeryinduced hippocampal neuroinflammation and cognitive impairment in aged rats. Then, the isobaric tags for the relative and absolute quantitation (iTRAQ) method combined with nano liquid chromatography-mass spectrometry (NanoLC-MS/MS), developed for protein quantitation, was used to reveal the profile of differentially altered proteins in the hippocampus underlying the animal model of POCD, and bioinformatics analyses were performed to explore the correlated signaling pathway. The study aimed to reveal the pathogenesis of POCD and provide potential therapeutic targets for its prevention and treatment in the vulnerable older brain. Materials And Methods Animals Aged male Sprague-Dawley rats (20 months old; weight, 550-650 g) were used for all on surgery-induced proinflammatory cytokine expression. Beginning at 6 h after surgery, the expression levels of IL-1 and TNF-in the hippocampus were significantly increased until 72 h after surgery (Fig. 3 A and B , p < 0.05). In contrast, TSA treatment resulted in a significant reduction in IL-1 and TNF-expression levels within 72 h. TSA inhibits surgery-induced neurofilaments (NFs) upregulation Compared with the control, surgery + TSA and TSA groups, the results from the iTRAQ analyses of proteins in the surgery group, including the intermediate filament (IF) rod domain profile (ProSiteProfiles), IF protein (Pfam), and IF rod domain signature showed fold changes were greater than 1.4, and these proteins exhibited significant increases in IPR039008 (ProSiteProfiles), IPR039008 (Pfam) and PR018039 (ProSitePatterns) peptides (p = 0.01570) at 6 h after surgery (Fig. 4 A) . Based on the results of the MS analysis, the expression levels of three dysregulated proteins (NEFH, NEFM and NEFL) at 6 h after surgery were validated using immunofluorescence (Fig. 4 B) and western blotting (Fig. 4 C and D) . These three proteins were significantly upregulated in the surgery-treated group compared with the control group, and TSA pretreatment significantly prevented the surgery-induced increase in these proteins in the hippocampus. When given alone, TSA had no effect on the expression of NEFH, NEFM and NEFL at 6 h after surgery. Therefore, the altered expression levels of proteins were consistent with the results from the MS analysis. KEGG pathways analysis An analysis of significantly differentially expressed proteins in the two groups was performed using the KEGG Pathway database (https://www.genome.jp/kegg/pathway.html). A total of 32 pathways were enriched in the surgery + TSA vs. the surgery group, and 14 among them, nine pathways showed statistically significant enrichment (Fig. 5 A and B , p < 0.05), including focal adhesion (rno04510), ECM-receptor interaction (rno04512), protein digestion and absorption (rno04974), AGE-RAGE signaling pathway in diabetic complications (rno04933), amoebiasis (rno05146), platelet activation (rno04611), glutamatergic synapse (rno04724), primary bile acid biosynthesis (rno00120), and phospholipase D signaling pathway (rno04072). Of these, the "focal adhesion" (p = 0.005433) and "ECM-receptor interaction" (p = 0.007986) pathways were the top two significantly enriched pathways and were chosen for analysis. In Fig. 5C , red represents upregulated proteins, and blue represents downregulated proteins. Discussion The laparotomy model was used in our study, which is a classic animal model that is widely used by researchers for POCD studies [7, 16] . Accumulated evidence demonstrates that the interaction of microglia and inflammatory cytokines participates in the pathology of cognitive impairment  . However, the role that microglia play in surgery-induced neuroinflammation and how the precise progression contributes to cognitive dysfunction, as well as the related signaling pathways, remain unexplored. We have demonstrated for the first time that TSA pretreatment alleviates surgery-induced NFs damage in the hippocampus of aged rats by decreasing microglial activation and inflammatory factor (IL-1 and TNF-) release, as well as changing the cargo of the proteins associated with the extracellular matrix (ECM). These results support the notion that microglial activation and its derived neuroinflammation play a role in surgery-induced cognitive dysfunction. The results also provide some insight into the NFs damage triggered by neuroinflammation observed in the aged brain, which was suppressed by TSA through the "focal adhesion" and "ECM-receptor interaction" pathways, which are major pathways involved in this the role of significantly differentially expressed signaling pathway proteins in the improvement of cognitive dysfunction is not necessarily proportional to these differences, and further verification of which signaling pathways are more effective in improving surgery-induced POCD is still needed.