Dexmedetomidine Restores Autophagic Flux, Modulates Associated microRNAs and the Cholinergic Anti-inflammatory Pathway upon LPS-Treatment in Rats.

Infections and perioperative stress can lead to neuroinflammation, which in turn is linked to cognitive impairments such as postoperative delirium or postoperative cognitive dysfunctions. The α2-adrenoceptor agonist dexmedetomidine (DEX) prevents cognitive impairments and has organo-protective and anti-inflammatory properties. Macroautophagy (autophagy) regulates many biological processes, but its role in DEX-mediated anti-inflammation and the underlying mechanism of DEX remains largely unclear. We were interested how a pretreatment with DEX protects against lipopolysaccharide (LPS)-induced inflammation in adult male Wistar rats. We used Western blot and activity assays to study how DEX modulated autophagy- and apoptosis-associated proteins as well as molecules of the cholinergic anti-inflammatory pathway, and qPCR to analyse the expression of autophagy and inflammation-associated microRNAs (miRNA) in the spleen, cortex and hippocampus at different time points (6 h, 24 h, 7 d). We showed that a DEX pretreatment prevents LPS-induced impairments in autophagic flux and attenuates the LPS-induced increase in the apoptosis-associated protein cleaved poly(ADP-ribose)-polymerase (PARP) in the spleen. Both, DEX and LPS altered miRNA expression and molecules of the cholinergic anti-inflammatory pathway in the spleen and brain. While only a certain set of miRNAs was up- and/or downregulated by LPS in each tissue, which was prevented or attenuated by a DEX pretreatment in the spleen and hippocampus, all miRNAs were up- and/or downregulated by DEX itself - independent of whether or not they were altered by LPS. Our results indicate that the organo-protective effect of DEX may be mediated by autophagy, possibly by acting on associated miRNAs, and the cholinergic anti-inflammatory pathway. Preventive effects of DEX on LPS-induced inflammation. DEX restores the LPS-induced impairments in autophagic flux, attenuates PARP cleavage and alters molecules of the cholinergic system in the spleen. Furthermore, DEX alters and prevents LPS-induced miRNA expression changes in the spleen and brain along with LPS.

Diagnostic biomarkers for adult haemophagocytic lymphohistiocytosis in critically ill patients (HEMICU): a prospective observational study protocol.

INTRODUCTION: Haemophagocytic lymphohistiocytosis (HLH) in adults is characterised by toxic immune activation and a sepsis-like syndrome, leading to high numbers of undiagnosed cases and mortality rates of up to 68%. Early diagnosis and specific immune suppressive treatment are mandatory to avoid fatal outcome, but the diagnostic criteria (HLH-2004) are adopted from paediatric HLH and have not been validated in adults. Experimental studies suggest biomarkers to sufficiently diagnose HLH. However, biomarkers for the diagnosis of adult HLH have not yet been investigated. METHODS AND ANALYSIS: The HEMICU (Diagnostic biomarkers for adult haemophagocytic lymphohistiocytosis in critically ill patients) study aims to estimate the incidence rate of adult HLH among suspected adult patients in intensive care units (ICUs). Screening for HLH will be performed in 16 ICUs of Charité - Universitätsmedizin Berlin. The inclusion criteria are bicytopaenia, hyperferritinaemia (≥500 µg/L), fever or when HLH is suspected by the clinician. Over a period of 2 years, we expect inclusion of about 100 patients with suspected HLH. HLH will be diagnosed if at least five of the HLH-2004 criteria are fulfilled, together with an expert review; all other included patients will serve as controls. Second, a panel of potential biomarker candidates will be explored. DNA, plasma and serum will be stored in a biobank. The primary endpoint of the study is the incidence rate of adult HLH among suspected adult patients during ICU stay. Out of a variety of measured biomarkers, this study furthermore aims to find highly potential biomarkers for the diagnosis of adult HLH in ICU. The results of this study will contribute to improved recognition and patient outcome of adult HLH in clinical routine. ETHICS AND DISSEMINATION: The institutional ethics committee approved this study on 1 August 2018 (Ethics Committee of Charité - Universitätsmedizin Berlin, EA4/006/18). The results of the study will be disseminated in an international peer-reviewed journal and presented at international conferences. TRIAL REGISTRATION NUMBER: NCT03510650.

Physostigmine Restores Impaired Autophagy in the Rat Hippocampus after Surgery Stress and LPS Treatment.

Tissue damage and pathogen invasion during surgical trauma have been identified as contributing factors leading to neuroinflammation in the hippocampus, which can be protected by stimulation of the cholinergic anti-inflammatory pathway using the acetylcholinesterase inhibitor physostigmine. Macroautophagy, an intracellular degradation pathway used to recycle and eliminate damaged proteins and organelles by lysosomal digestion, seems to be important for cell survival under stress conditions. This study aimed to examine the role of autophagy in physostigmine-mediated hippocampal cell protection in a rat model of surgery stress. In the presence or absence of physostigmine, adult Wistar rats underwent surgery in combination with lipopolysaccharide (LPS). Activated microglia, apoptosis-, autophagy-, and anti-inflammatory-related genes and -proteins in the hippocampus were determined by Real-Time PCR, Western blot and fluorescence microscopy after 1 h, 24 h and 3 d. Surgery combined with LPS-treatment led to microglia activation after 1 h and 24 h which was accompanied by apoptotic cell death after 24 h in the hippocampus. Furthermore, it led to a decreased expression of ATG-3 after 24 h and an increased expression of p62/ SQSTM1 after 1 h and 24 h. Administration of physostigmine significantly increased autophagy related markers and restored the autophagic flux after surgery stress, detected by increased degradation of p62/ SQSTM1 in the hippocampus after 1 h and 24 h. Furthermore, physostigmine reduced activated microglia and apoptosis relevant proteins and elevated the increased expression of TGF-beta1 and MFG-E8 after surgery stress. In conclusion, activation of autophagy may be essential in physostigmine-induced neuroprotection against surgery stress.

Dexmedetomidine Prevents Lipopolysaccharide-Induced MicroRNA Expression in the Adult Rat Brain.

During surgery or infection, peripheral inflammation can lead to neuroinflammation, which is associated with cognitive impairment, neurodegeneration, and several neurodegenerative diseases. Dexmedetomidine, an α-2-adrenoceptor agonist, is known to exert anti-inflammatory and neuroprotective properties and reduces the incidence of postoperative cognitive impairments. However, on the whole the molecular mechanisms are poorly understood. This study aims to explore whether dexmedetomidine influences microRNAs (miRNAs) in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation. Adult Wistar rats were injected with 1 mg/kg LPS intraperitoneal (i.p.) in the presence or absence of 5 µg/kg dexmedetomidine. After 6 h, 24 h, and 7 days, gene expressions of interleukin 1-ß (IL1-ß), tumor necrosis factor-α (TNF-α), and microRNA expressions of miR 124, 132, 134, and 155 were measured in the hippocampus, cortex, and plasma. Dexmedetomidine decreased the LPS-induced neuroinflammation in the hippocampus and cortex via significant reduction of the IL1-ß and TNF-α gene expressions after 24 h. Moreover, the LPS-mediated increased expressions of miR 124, 132, 134, and 155 were significantly decreased after dexmedetomidine treatment in both brain regions. In plasma, dexmedetomidine significantly reduced LPS-induced miR 155 after 6 h. Furthermore, there is evidence that miR 132 and 134 may be suitable as potential biomarkers for the detection of neuroinflammation.

Neuroprotective effect of dexmedetomidine on hyperoxia-induced toxicity in the neonatal rat brain.

Dexmedetomidine is a highly selective agonist of α2-receptors with sedative, anxiolytic, analgesic, and anesthetic properties. Neuroprotective effects of dexmedetomidine have been reported in various brain injury models. In the present study, we investigated the effects of dexmedetomidine on neurodegeneration, oxidative stress markers, and inflammation following the induction of hyperoxia in neonatal rats. Six-day-old Wistar rats received different concentrations of dexmedetomidine (1, 5, or 10 µg/kg bodyweight) and were exposed to 80% oxygen for 24 h. Sex-matched littermates kept in room air and injected with normal saline or dexmedetomidine served as controls. Dexmedetomidine pretreatment significantly reduced hyperoxia-induced neurodegeneration in different brain regions of the neonatal rat. In addition, dexmedetomidine restored the reduced/oxidized glutathione ratio and attenuated the levels of malondialdehyde, a marker of lipid peroxidation, after exposure to high oxygen concentration. Moreover, administration of dexmedetomidine induced downregulation of IL-1ß on mRNA and protein level in the developing rat brain. Dexmedetomidine provides protections against toxic oxygen induced neonatal brain injury which is likely associated with oxidative stress signaling and inflammatory cytokines. Our results suggest that dexmedetomidine may have a therapeutic potential since oxygen administration to neonates is sometimes inevitable.

Acetylcholinesterase inhibitors reduce neuroinflammation and -degeneration in the cortex and hippocampus of a surgery stress rat model.

Exogenous stress like tissue damage and pathogen invasion during surgical trauma could lead to a peripheral inflammatory response and induce neuroinflammation, which can result in postoperative cognitive dysfunction (POCD). The cholinergic anti-inflammatory pathway is a neurohumoral mechanism that plays a prominent role by suppressing the inflammatory response. Treatments with acetylcholinesterase inhibitors enhance cholinergic transmission and may therefore act as a potential approach to prevent neuroinflammation. In the presence or absence of acetylcholinesterase inhibitors, adult Wistar rats underwent surgery alone or were additionally treated with lipopolysaccharide (LPS). Physostigmine, which can overcome the blood-brain barrier or neostigmine acting only peripheral, served as acetylcholinesterase inhibitors. The expression of pro- and anti-inflammatory cytokines in the cortex, hippocampus, spleen and plasma was measured after 1 h, 24 h, 3 d and 7 d using Real-Time PCR, western blot analysis or cytometric bead array (CBA). Fluoro-Jade B staining of brain slices was employed to elucidate neurodegeneration. The activity of acetylcholinesterase was estimated using a spectrofluorometric method. Surgery accompanied by LPS-treatment led to increased IL-1beta gene and protein upregulation in the cortex and hippocampus but was significantly reduced by physostigmine and neostigmine. Furthermore, surgery in combination with LPS-treatment caused increased protein expression of IL-1, TNF-alpha and IL-10 in the spleen and plasma. Physostigmine and neostigmine significantly decreased the protein expression of IL-1 and TNF-alpha. Neuronal degeneration and the activity of acetylcholinesterase were elevated after surgery with LPS-treatment and reduced by physostigmine and neostigmine. Along with LPS-treatment, acetylcholinesterase inhibitors reduce the pro-inflammatory response as well as neurodegeneration after surgery in the cortex and hippocampus. This combination may represent a tool to break the pathogenesis of POCD.