It is well documented that α2A-AR is expressed in monocytes and macrophages (Kupffer cells, Langerhans cells, etc.), and that it plays a pivotal role in the inflammatory response of sepsis [15, 16, 20, 21]. Blood monocytes have been proposed as a source of microglia , it was therefore speculated that like tissue macrophages, microglia might also express α2A-AR. Although microglia in the spinal cord had been reported to express α2A-AR , it has remained uncertain whether brain microglia would also express α2A-AR. The present results have demonstrated for the fist time that α2A-AR was constitutively expressed in resting microglia and that it was upregulated in activated microglia induced by LPS. In the latter, α2A-AR expression was enhanced in LPS treated microglia in a dose-dependent manner, indicating that it plays specific roles in microglia functions.
Further analysis by ELISA in BV-2 microglia treated with α2A-AR agonist BHT933 and antagonist BRL44408 followed by LPS was then carried out to detect TNF-α secretion. It was previously reported that the LPS concentration in animal blood under sepsis condition was about 10− 8 g/mL. In the present study, the final LPS concentration was 100 ng/mL to simulate the condition in vivo. As shown in Fig. 1C, BHT933 or BRL44408 did not elicit microglia activation; however, TNF-α secretion was further increased and substantially by about 50% in LPS group exposed to BHT933 when compared with LPS group. TNF-α secretion was decreased to a level comparable to that of LPS group when pretreated with BRL44408, suggesting that α2A-AR activation promotes TNF-α secretion in LPS-activated BV-2 microglia; in other words, α2A-AR is instrumental to secretion of inflammatory mediators in LPS-activated BV-2. This would be highly consistent with the role of α2A-AR in peripheral macrophages.
To further demonstrate the role of α2A-AR in inflammation, the signaling pathways involved in TNF-α production, namely, inhibitor of nuclear factor kappa B (IκB), mitogen-activated protein kinases (MAPKs) of p38, c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (ERK) and the phosphorylated forms were explored. We confirmed that nuclear factor-kappa B (NF-κB) and JNK, but not ERK and P38 MAPKs are involved in promoting inflammatory response in activated microglia and that it is mediated by α2A-AR.
A pertinent question arose from this would be how activated α2A-AR might transmit intracellular signals and ultimately promote the inflammation-related pathways. α2A-AR belongs to G-protein-coupled receptors family, the cell signaling pathways of which include cAMP and phosphatidylinositol signaling pathway . The former depends on adenylate cyclase to guide the expression of various substrates and genes through cAMP-dependent protein kinase A (PKA) (cAMP-PKA pathway). The latter relies on phospholipase C to hydrolyze 4,5-diphosphate phosphatidylinositol (PIP2) to 1,4,5-triphosphate inositol (IP3) and diacylglycerol (DG). Among them, DG-specific activated protein kinase C (PKC) causes phosphorylation of various proteins or enzymes (DG-PKC pathway), and then regulates the biological effects of cells. Renal tubular cells treated with BHT933 showed no effect on intracellular cAMP, while DG increased 46% which were blocked by BRL44408 treatment , suggesting that α2A-AR signal in renal tubular cells may involve PKC pathway. It is relevant to note that α2A-AR in the prefrontal cortex was reported to increase testosterone-treated impulsivity through the PKA pathway .
Against the above background, we have focused our study on the role of PKA and PKC when α2A-AR is activated. When activated PKA enters the nucleus which activated cAMP-response element binding protein (CREB) and whose phosphorylation is indicative of PKA activation. The present results showed that the PKA pathway did not participate in the signal transduction of microglia α2A-AR. PKC binding to the second messenger is through phosphorylation of Thr566, C-terminal hydrophobic sites Ser729 and Thr710 in its catalytic region [28–30]. There is compelling evidence indicating that PKCε was indispensable in macrophage for the phosphorylation of I-κB and activation of MAPKs . Macrophages knocked out of PKCε had serious defects, and the host could not tolerate bacterial infection and the mortality increased . In view of this, we also detected the phosphorylation of PKCε on Ser729 in this study. The results showed that p-PKCε was increased significantly in BV-2 cells treated with BHT933, suggesting that PKC was involved in the signal transduction following α2A-AR activation. Furthermore, bisindolylmaleimide-1 interference ELISA showed that specific inhibition of PKC significantly reduced TNF-α secretion, confirming that PKC pathway is one of the main signal transduction pathways of α2A-AR activation. This was further substantiated by JNK and I-κB pathways that were detected in microglia incubated with bisindolylmaleimide-1.
We next investigated the effect of α2A-AR on sepsis brain damage. For this, we have used α2A-AR knockout mice. It was argued that α2A-AR would be activated in WT-LPS mice because sepsis induced the release of large amounts of NE . On the other hand, in KO mice α2A-AR was considered blocked because its gene was knocked out. It has been reported that peripheral inflammation induced by intraperitoneal injection of LPS can cause the inflammation in periventricular brain tissues and also the brain parenchyma. It is conceivable that this would activate microglia, cause neuroinflammation, and lead to cognitive impairment [33, 34]. We have used the sepsis model by intraperitoneal injection of LPS in mice. In order to mimic the actual clinical situation and observe the cognitive and motor functions in the middle and late stages of sepsis, we had opted a lower dose of LPS (15 mg/kg) to minimize the mortality rate of mice. The results showed that the mortality rate of sepsis mice induced by LPS was 33% which we considered to be acceptable.
Immunofluorescence showed that microglia lacking TNF-α expression were widely distributed in WT and KO-sham groups. When challenged with LPS, however, robust microglia activation occurred in WT-LPS group as manifested by the vigorous expression of TNF-α compared with KO-LPS mice. This strongly argued that excess release of NE in WT-LPS mice had activated and increased TNF-α production in activated microglia. This would be consistent with the results in vitro.
Additionally, double immunofluorescence of NeuN and synaptophysin showed that NeuN immunofluorescence in the pyramidal layer of the hippocampal CA2-3 regions in septic mice with α2A-AR gene knockout was comparable to the WT-LPS mice. However, synaptophysin, a synaptic marker in the radiation layer (second synaptic connection area) was more obvious in KO-LPS mice than that in WT-LPS mice, indicating that α2A-AR activation in sepsis had caused synaptic damage or synaptic dysfunction. This may offer an explanation for the decline in clinical cognitive impairment of learning and memory. Synaptic connections in the hippocampus are key units of learning and memory in the brain and convert short-term memory into long-term memory [35–37]. It has been reported that inflammatory factors can affect the function of synaptic connections. In a mouse model of sleep deprivation, an upsurge in inflammatory factors such as TNF-α, IL-1β and IL-6 derived from activated microglia was found in the dentate gyrus and CA2-3 areas of the hippocampus, which was coupled by a significant decline in spatial memory function . IL-1β can reduce the expression of synaptophysin in presynaptic membrane and PSD-95 in postsynaptic of septic rats model, thereby reducing LTP [39, 40] through MAPK pathway. TNF-α can directly inhibit the production of LTP , change excitatory protrusion transmission and reduce the occurrence of LTP .
Finally, we evaluated the learning and memory function, motor function and emotional response of mice  in different experimental groups. The latency of mice in WT-LPS was significantly longer than that of WT-sham at all time points within 4 weeks, while the latency of KO group showed no difference between the sepsis group and the sham group at 3–4 weeks. It is noteworthy that the latency of KO-LPS mice was significantly lower than that of WT-LPS mice during 1–4 weeks. A similar phenomenon was observed in the experiment of the targeting quadrant detention time and the frequency of platform crossings. The present Morris water maze experiment confirmed that sepsis can significantly reduce the learning and memory ability of mice as reported by many studies [44, 45]. Of note, blocking α2A-AR can improve the learning and memory function of sepsis mice, especially in the later sepsis stage. In terms of motor function, rotating-stick test was used whereby mice were placed and keep balance from falling down . If the mice had dyskinesia or poor coordination, they would fall off the stick very quickly. We showed here that sepsis could significantly reduce the motor function of mice, suggesting that this may be partly attributed to activation of α2A-AR.
Behavior includes adaptation to the natural and social environment, and this involves psychology and emotion. Anxiety, depression or post-traumatic stress disorder often occur in sepsis patients . Anxiety-like behavior was reported 10 days after operation in sepsis rats, accompanied by increased levels of inflammatory factors TNF-α, IL-1β and IL-6 in serum and brain tissues, suggesting that CNS inflammatory mediators are involved in anxiety-like symptoms in sepsis-associated encephalopathy . In light of above, we have used the elevated plus maze and open-field test to evaluate the emotional and psychological state of sepsis mice. The results showed that there was no difference in both wild type mice and α2A-AR knock-out mice, and that this was independent of sepsis. It stands to reason therefore that sepsis did not increase the anxiety and depression behavior, autonomous behavior, exploratory behavior and tension of animals and of animals in new and different environments; more importantly, α2A-AR was not involved in these situations.
In conclusion, the present results have shown unequivocally that microglia express α2A-AR. Remarkably, α2A-AR activation can amplify LPS-induced microglia activation and enhance the expression and secretion of inflammatory factor TNF-α. It was further demonstrated that this was via PKC transmission which activates intracellular JNK and NF-κB pathways. In vivo experiments confirmed that α2A-AR activation promotes the early activation of microglia and the expression of TNF-α in the hippocampus of LPS-treated mice. It is suggested that excess production of TNF-α would inhibit synaptic function, causing dysfunction of learning, memory and motor as demonstrated in the present neurobehavioral score study. On the basis of the present results, it is concluded that blocking α2A-AR may be a potential therapeutic strategy for amelioration of brain function-related damage and neurological dysfunctions caused by sepsis.