To the Editor: Post-operative cognitive dysfunction (POCD) is described as a series of changes in the neurocognitive conditions and behaviors of patients, which occur within several weeks or even months after anesthesia and surgery. POCD occurs in 20% to 50% of patients, resulting in burden on patients, and exerting a significant social and economic impact.[1] Although an increased understanding of the effects of neuroinflammation and oxidative stress after surgery and anesthesia have clarified the underlying pathophysiology of POCD, many questions remain to be answered. At present, the treatment of POCD mainly focuses on prevention strategies, early identification, and perioperative risk factor management; and an effective treatment is still lacking. Dexmedetomidine (Dex) is a novel and highly selective α2 adrenergic receptor agonist. Perioperative Dex treatment can significantly reduce the incidence of POCD and inflammation, and it can improve post-operative neurocognitive function. Dex may inhibit the massive release of high mobility group protein B1 (HMGB1) by acting on the α2 adrenergic receptor to activate the PI3K/Akt signaling pathway, thus reducing the binding of HMGB1 with Toll-like receptor 4 and other membrane proteins, thus playing an anti-inflammatory role.[2] Dex can also enhance HMGB1-induced cognitive decline in inflammatory mice via vagal nerve stimulation. HMGB1 is released extracellularly after cell activation, stress, injury, or death and can promote an inflammatory response. High HMGB1 expression is associated with the occurrence of POCD. The inflammatory immune response may play an important role in the pathogenesis of POCD. Regulatory T (Treg) cells are key regulatory cells involved in inflammation and play an important role in immune tolerance and homeostasis. HMGB1 can downregulate the immune function of Treg cells by decreasing the expression of marker molecules on their surface and inhibiting cytokine secretion. Extracellular HMGB1 also intensifies the autoimmune process by impairing the stability of Treg cells.[3] In this study, we aimed to explore the effects and possible mechanisms of action of Dex in POCD. Twenty-four male Sprague Dawley rats were randomly divided into three groups: sham, POCD, and Dex + POCD (n = 8 per group). This study was approved by the Ethics Committee of the Experimental Animal Center of Guangxi Medical University (No. 201909020). Rats in the POCD and Dex + POCD groups were intra-peritoneally injected with normal saline and 2% pentobarbital sodium (50 mg/kg) 30 min before undergoing splenectomy. The Dex + POCD group was simultaneously intraperitoneally injected with Dex (20 μg/kg). No splenectomy was performed in the sham group, but the rats were otherwise treated the same as the POCD group. The Morris water maze (MWM) test was performed to assess cognitive function. Blood was collected from the abdominal aorta of the rats, and the proportion of Treg cells in the peripheral blood was determined. Inflammation-related factors were also identified. The hippocampal and spleen tissues of rats were used for subsequent analysis. The cognitive ability of the rats was measured using the MWM. Compared with the sham group, the escape latency time of rats was increased in the POCD group, while the swimming distance ratio in the original platform quadrant decreased. After Dex administration, the escape time was reduced and the ratio of swimming distance in the original platform quadrant increased [Figure 1A]. Furthermore, the serum tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and HMGB1 levels were significantly increased in the POCD group compared to those in the sham group, while the levels of TNF-α, IL-1β, and HMGB1 decreased after Dex administration, with the effects becoming more obvious with time [Figure 1B].Figure 1: Dex ameliorated POCD, reduces HMGB1, and modulates the activity of HMGB1-induced Treg cells in the sham, POCD, and Dex + POCD groups. (A) MWM was used to assay the cognitive ability of rats. (B) Serum TNF-α, IL-1β, and HMGB1 levels were detected at 1, 2, 3, 4, and 5 days. (C) Hematoxylin-eosin staining was applied to detect the morphological changes in the hippocampal tissue. (D) Immunochemistry was performed to detect HMGB1 expression in the hippocampus. (E) TUNEL was applied to detect the apoptosis rate of hippocampal cells. (F) Western blotting was performed to measure the expression of TNF-α, IL-1β, and HMGB1 in the hippocampus. (G) Proportion of Treg cells in the peripheral blood. (H) Western blotting was performed to detect HMGB1 and Foxp3 expression in the spleen tissue. (I) Western blotting was performed to detect IL-10, TGF-β, and Foxp3 expression in the hippocampus. ∗ P < 0.05 vs. sham group; † P < 0.05 vs. POCD group. Dex: Dexmedetomidine; HMGB1: High mobility group protein B1; MWM: Morris water maze; POCD: Postoperative cognitive dysfunction; Treg: Regulatory T cell.Histological analysis of hippocampal tissue showed that the hippocampal neuron cells in the POCD group were severely damaged, with an irregular arrangement and unclear cell structure. HMGB1 significantly decreased cell density, which was significantly rescued by Dex treatment [Figure 1C]. HMGB1 levels were significantly increased in the POCD group compared to the sham group, while the level of HMGB1 was decreased after Dex administration [Figure 1D and Supplementary Figure 1A, https://links.lww.com/CM9/B241]. The POCD group had an increased rate of apoptosis in the hippocampus compared to the sham group, whereas the Dex + POCD group had a decreased rate of apoptosis [Figure 1E and Supplementary Figure 1B, https://links.lww.com/CM9/B241]. Moreover, TNF-α, IL-1β, and HMGB1 expression in the hippocampus in the POCD group were significantly increased compared with that in the sham group, while the expression of TNF-α, IL-1β, and HMGB1 was decreased after Dex administration [Figure 1F]. Analysis of the proportion of Treg cells in the peripheral blood showed that, compared with the sham group, the proportion was decreased in the POCD group, and was increased after Dex administration [Figure 1G]. Foxp3 is a key transcription factor controlling the development and function of Treg cells. Foxp3 expression was decreased and HMGB1 expression was increased in the spleen tissue of the POCD group compared to the sham group. After Dex treatment, HMGB1 expression decreased, and Foxp3 expression increased [Figure 1H]. The hippocampal expression of IL-10, TGF-β, and Foxp3 was lower in the POCD group than in the sham group. After Dex treatment, IL-10, TGF-β, and Foxp3 expression levels increased [Figure 1I]. The pathological process of POCD is related to both neuroinflammation and microglial proliferation. Immune inflammation plays an important role in POCD progression. In rodents, POCD is associated with inflammatory activation of hippocampal microglia. HMGB1 is an abundant nuclear and cytoplasmic protein in mammalian cells; it is released from activated innate immune cells or dead cells, and its role in inflammatory diseases has garnered significant interest. Interestingly, Dex exhibits protective properties under inflammatory conditions, and can thus play a protective role in HMGB1-induced cell damage. Dex may exert a protective effect against traumatic brain injury-induced acute lung injury by acting on the HMGB1-RAGE pathway.[4] Dex has also been shown to inhibit spinal cord microglial activation in mice with spinal cord ischemia-reperfusion injury via the LeT-7a-1/2-3P/HMGB1 pathway.[5] Therefore, in this study, we constructed in vivo POCD animal models and found that Dex improved POCD. Furthermore, the levels of TNF-α, IL-1β, and HMGB1 decreased after Dex treatment. Tregs play a crucial role in maintaining immune homeostasis and preventing autoimmunity. Treg cells are defined by the expression of the transcription factor Foxp3, which exerts a strong regulatory effect on the function and plasticity of Treg cells. Dex has been shown to effectively improve the function of Treg cells and establishes a new helper T (Th)1/Th2 balance in patients with Graves’ disease.[6] HMGB1 aggravates lipopolysaccharide-induced acute lung injury by inhibiting the activity and function of Tregs. However, the knockout of HMGB1 in tumor cells weakens their ability to induce Treg cells. The protective effect of Dex against cystic echinococcosis may be related to the upregulation of IL-10 and TGF-β1 levels by Treg cells, and the inhibition of Th cells. In the absence of Tregs, the ability to inhibit inflammation is completely lost after treatment with Dex.[7] In this study, we found that IL-10 and TGF-β levels increased after Dex treatment and that Dex affected the activity of Treg cells. Furthermore, Foxp3 expression increased and HMGB1 expression decreased after Dex administration. Overall, our study demonstrated the effects and possible mechanisms of action of Dex on POCD. Dex ameliorates POCD, reduces HMGB1 release, and modulates HMGB1-induced Treg cell activity. Our study provides a reference and basis for the clinical treatment and prognosis of POCD in the future and will help to enrich new treatment strategies for POCD. Acknowledgements We are grateful for the technical support provided by Guangxi Medical University and Ningbo Medical Center Lihuili Hospital. Funding The present study was supported by grants from the Ningbo Nature Science Fundation (No. 2019A610267). Conflicts of interest None.

Background Quadratus lumborum block (QLB) is a novel and effective postoperative analgesia method for abdominal surgeries. However, whether QLB can affect early postoperative cognitive function by inhibiting surgical traumatic stress and the inflammatory response remains unclear. This study aimed to explore the effect of QLB on postoperative cognitive function in elderly patients undergoing laparoscopic radical gastrectomy. Methods Sixty-four elderly patients who underwent laparoscopic radical gastrectomy were randomly divided into the QLB group (Q group, n = 32) and control group (C group, n = 32). The Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) were used to measure cognitive function 1 day before and 7 days after surgery. Postoperative cognitive dysfunction (POCD) was defined as a decline of ≥ 1 SD in both tests. The visual analog scale (VAS) scores 6 h (T1), 24 h (T2), and 48 h (T3) after surgery were measured. The serum levels of high mobility group box protein 1 (HMGB1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were evaluated 1 day before surgery (baseline), and 1 day (day 1) and 3 days after surgery (day 3). The intraoperative remifentanil dosage, sufentanil consumption 24 h after surgery, recovery time from anesthesia, and adverse effects were also compared. Results POCD was present in two patients in the QLB group and eight patients in the C group 7 days after surgery (6.7 % vs. 27.6 %, P = 0.032). The MMSE and MoCA scores were similar in both groups preoperatively, and the two scores were higher in the QLB group than in the C group 7 days after surgery ( P < 0.05). The VAS scores were significantly lower in the Q group at all times after surgery ( P < 0.05). Compared with the C group, the levels of HMGB1, TNF-α, and IL-6 were significantly decreased 1 and 3 days after surgery in the QLB group ( P < 0.05). The remifentanil consumption intraoperatively and sufentanil 24 h postoperatively were significantly lower in the QLB group ( P < 0.05). The recovery time from anesthesia was shorter in the QLB group ( P < 0.05). No severe adverse effects occurred in either group. Conclusions QLB could improve postoperative cognitive function in elderly patients undergoing laparoscopic radical gastrectomy. This may be related to the suppression of the inflammatory response after surgery. Trial registration Chictr.org.cn identifier ChiCTR1900027574 (Date of registry: 19/11/2019, prospectively registered).

Postoperative cognitive dysfunction (POCD) is a common complication after surgery in the elderly and is induced by multiple factors including dysregulation of the immune system 1. Toll-like receptor 4 (TLR4) is a key pattern recognition receptor (PRR) that mediates innate and adaptive immune processes both related to infection, via signaling with pathogen-associated molecular patterns (PAMPs), and endogenous danger molecules, or damage-associated molecular patterns (DAMPs) 2. Within the brain, TLR4 is mainly expressed on microglia and neurons, and changes in its expression have been correlated with several central nervous system (CNS) disease states, such as Alzheimer's disease (AD) and cerebral ischemia 3. Upon activation, TLR4 signaling pathways lead to neuroinflammation 4, which can further develop into neurodegeneration and permanent cognitive deficits. Yet, there is no clear understanding on whether TLR4 contributes to the neuroinflammatory process of POCD. In this study, we sought to investigate the expression of TLR4 on neurons in the hippocampus after surgery in aged rats. Here, we reveal that high expression of TLR4 on neuron contributes to neuroinflammation after peripheral surgery and general anesthesia. Female Sprague-Dawley rats (22–23 month old, total n = 105) were used in accordance with the guidelines of experimental animal use established by the ethics committee of the Central South University. Animals underwent splenectomy under isoflurane anesthesia for 2 h (group S) or served as anesthesia (group A) and untreated controls (group C), respectively. Brains were harvested at 1, 3 and 7 days. Neurons (NeuN) and TLR-4 expression in the hippocampus was measured using double immunofluorescence combined with measurements of key pro-inflammatory cytokines including TNF-α and IL-1β in hippocampal homogenate using Western blot. Data are expressed as mean ± SD and analyzed by two-way ANOVA (SPSS 17.0, Shanghai, China). The expression of TLR4 on neurons was significantly up-regulated after surgery, especially on postoperative day 1 compared with control rats (Figure 1C and S1). TLR4 expression was decreased thereafter, with levels returning almost to baseline by postoperative day 7 (Figure 1S7). Remarkably, the expression of TLR4 was also increased after exposure to anesthesia only (Figure 1A), although levels were significantly lower than that after surgery. To corroborate the immunofluorescence findings, we measured TLR4 protein levels with Western blot (Figure 2). We also assessed levels of pro-inflammatory cytokines, IL-1β and TNF-α, in the hippocampus to further illustrate neuroinflammation after surgery and anesthesia. After splenectomy (Group S), we found a peak in hippocampal protein levels of IL-1β and TNF-α, returning to baseline by day 7. The increase in IL-1β and TNF-α after anesthesia exposure (Group A) was not as remarkable as after surgery. Peripheral surgery triggers both humoral and neuronal changes that cause CNS dysfunction and neuroinflammation 5. Systemic pro-inflammatory cytokines can enter the brain, disrupt the homeostasis of CNS microenvironment and initiate a local immune response by affecting the blood–brain barrier (BBB) function and permeability 6. TLR4 is a well-known pro-inflammatory receptor involved in the regulation of the immune response 7. In the CNS, it has been reported that TLR4 on microglia contributes to abundant production of various pro-inflammatory cytokines via MyD88- and TRIF-dependent signaling pathway. Furthermore, TLR4 is also associated with neurotoxicty and neuroapoptosis, for example, after ischemic brain injury 8. Few studies have focused on the role of TLR4 in POCD, and the molecular mechanism of TLR4 on neuron in neuroinflammation remains unclear. In this study, we found that the expression of TLR4 on hippocampal neurons was remarkably increased after surgery. Higher expression of TLR4 on microglia has been previously reported in models of AD, stroke and other CNS disorders. Neuron and microglia closely interact with each other to overall regulate CNS homeostasis and brain function. When the CNS is injured, for example, after surgical trauma, microglia becomes activated ultimately contributing to neuroinflammation and cognitive decline 9. It is possible that the high expression of TLR4 induced by surgery may also cause neuroapoptosis, and this may trigger microglia activation. Notably, microglia in the aged brain are “primed” and possibly more susceptible to changes in CNS homeostasis 10. Once activated, microglia produce and release multiple pro-inflammatory cytokines, leading to extensive inflammation, which can finally lead to cognition decline. Gradually after surgery, the expression of TLR4 returned to baseline and also the neuroinflammation disappeared, suggesting that TLR4 activation may regulate microglia activity and brain homeostasis. Our findings indicated that TLR4 on neuron plays a crucial role in modulating surgery-induced neuroinflammation by controlling microglia activation. Of interest, isoflurane anesthesia may also partly contribute to higher TLR4 expression, thus contributing to POCD. In conclusion, the present study revealed a close relationship between TLR4 and neuroinflammation after surgery, indicating that TLR4 might be a key factor in the development of POCD. To deeply understand the role of TLR4 in POCD, further studies should focus on the relevant molecular mechanism and selective therapies to modulate its expression in the brain. This study is supported by the National Natural Science Foundation of China (81172200) and 125 Program of The Third-Xiangya Hospital, Central South University (20100233). The authors declare no conflict of interest.

Postoperative cognitive dysfunction (POCD), common in elderly patients, refers to a decline in cognitive function following surgery, which may persist or even evolve into Alzheimer's disease (AD). Despite great efforts, the mechanism of POCD remains unclear. In the present study, we tested the hypothesis that Toll-like receptor 4 (TLR4) on microglia contributes to POCD. Shortly after surgery, aged rats demonstrated significant deficits in memory and learning, accompanied by the activation of microglia, marked upregulation of TLR4 on microglia in the hippocampus, as well as an increased expression of two downstream factors [myeloid differentiation factor 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF)] and pro-inflammatory cytokines [including tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β)]. With an increase in time following surgery, the expression of TLR4 and the aforementioned factors and pro-inflammatory cytokines gradually returned to normal, as did the cognitive function of the aged rats. In conclusion, our study suggests that the activation of TLR4 signaling on microglia may act as an underlying mechanism of POCD.

SummaryAimPostoperative cognitive dysfunction (POCD) is a growing and largely underestimated problem without defined etiology. Herein, we sought to determine the relationship between cognitive decline, blood–brain barrier (BBB) permeability, and inflammation, namely high mobility group box‐1 (HMGB1), after surgery in aged rats.MethodsAged rats were randomly assigned as surgery group (n = 45, splenectomy under general anesthesia), anesthesia (n = 45, 2% isoflurane for 2 h), and naïve control (n = 15). Markers of inflammation were measured in plasma and brain. Blood–brain barrier ultrastructure and permeability were measured by transmission electron microscope (TEM) and IgG immunohistochemistry. Cognitive function was assessed in a reversal learning version of the Morris water maze (MWM).ResultsSurgical trauma under general anesthesia caused distinct changes in systemic and central proinflammatory cytokines. Levels of HMGB1 and the receptor for advanced glycation end products (RAGE) were significantly upregulated in the hippocampus of operated animals. Immunohistochemistry and TEM showed BBB disruption induced by surgery and anesthesia. These molecular changes were associated with cognitive impairment in latency with the MWM up to postoperative day 3.ConclusionsHMGB1 and RAGE signaling appear pivotal mediators of surgery‐induced cognitive decline and may contribute to the changes in BBB permeability after peripheral surgical trauma.

Objective To investigate the effects of isoflurane anesthesia on hippocampus synaptosomes proteome in aged rats.Methods Twenty-seven 22- month-old SD rats weighing 480-550 g were randomly divided into 2 groups: control group (group C,n =6) and isoflurane group (group Ⅰ,n =21 ).In group C inhaled mixed gas containing 80％ oxygen for 2 h.In group Ⅰ the animals were endotracheal intubated after induction by 3％ isoflurane and inhaled 2％ isoflurane and 80％ oxygen for 2 h.Cognition function was evaluated by Y-maze at 24 h after anesthesia and the total training times were recorded.The total training times ＞ 75 was defined as cognitive dysfuction.In group Ⅰ the animals were divided into cognitive dysfuction group (group ⅠA) and non-cognitive dysfuction group (group IB) according to the results of Y-maze test.The animals were sacrificed and their hippocampi were removed and synaptosomes were extracted for two-dimensional gel electrophoresis.The different protein spots were analyzed by mass chromatographic analysis.Results Six rats had cognitive dysfuction (group IA) and another thirteen rats had no cognitive dysfuction (group IB).The total training times were significantly higher in group IA than in groups C and IB( P ＜ 0.05).There was no significant difference in the total training times between groups C and IB (P ＞ 0.05).There were 21 (11/10) different protein spots between groups IB and IA,and 19 (12/7) different protein spots between groups C and IA.Thirty-one protein spots were identified by means of MALDI-TOF-MS.Conclusion The cognitive dysfuction after isoflurane anesthesia in aged rats may be related to the changes of energy metabolism protein,cytoskeletal structure and regulatory protein in synapse of hippocampus.