Clinical And Basic Research On Sepsis Associated Encephalopathy

OBJECTIVE:Sepsis associated encephalopathy (SAE) is a diffuse cerebral dysfunction resulted by systemic inflammatory response, without clinical or laboratory evidence of other encephalopathy including direct brain infection, cerebral hemorrhage, cerebral embolism, hepatic encephalopathy and pulmonary encephalopathy. Its main clinical manifestation is an acute deterioration of mental status, such as cognitive impairment, confusion, disorientation, agitation, stupor or coma. SAE is the most common form of encephalopathy in critical ill patients. Recently, with further study on sepsis, it has been found that SAE is associated with worse prognosis. However, comprehensive factors, for example, without diagnostic standards for SAE, clinical usage of sedative drugs, common underlying diseases such as organic brain diseases and potential nervous system injuring diseases, result in controversies on the incidence, epidemiology and mechanisms of SAE. This study investigated the epidemiology of SAE under current clinical diagnostic condition, demonstrated the effect of its incidence on prognosis, and analyzed its risk factors. Based on these results, we then established SAE animal models, used neurobiology score, electroencephalography (EEG), somatosensory evoked potentials for its early diagnosis, thus provide evidence for clinical diagnosis of SAE. Further, we studied Neuroglobin (Ngb), which has been found to protect again hypoxia ischemia brain injury, detected the change of its expression, demonstrated its brain protective role in SAE, thus provided an new perspective for the prevention and treatment of SAE.METHODS:Part1:All323septic patients admitting to the ICU in a single University Hospital from2008-2011were screened and91patients were excluded. Then the232included patients were divided into two groups, based on whether the patients had SAE or not. Differences between baseline information (gender, age, underlying diseases, disease types), vital signs (temperature, heart rate, blood pressure, breath), severity of disease (APACHE II score), Glasgow coma score (GSC score), etiological information, the sites of infection, biochemical indices:white blood cell count (WBC), neutrophil percentage (N%), hemoglobin (HB), platelets (PLT), total protein (TP), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), blood urea nitrogen (BUN), serum creatinine (Scr), blood sugar (BS), triglyceride (TG), cholesterol (CHO), PH, partial pressure of oxygen (PaO2), carbon dioxide partial pressure (PaCO2), central venous oxygen saturation (SCVO2), C-reactive protein (CRP), procalcitonin (PCT) and mechanical ventilation time, ICU time and28-day mortality were retrospectively analyzed.Part2:30rats were weighted, numbered, and placed with EEG monitoring electrodes10days before establishing models.10days later, rats were weighted, numbered, and randomized. Rat models of sepsis were made by cecal ligation and puncture (CLP). Observed the changes of their neurological behaviors, monitored the EEG at4,6,8,12,24hours after CLP, and recorded the values of EEG waveform change analysis and somatosensory evoked potentials. Rat models of sepsis were divided into sepsis+non-SAE group and SAE group based on whether EEG or somatosensory evoked potentials changed within24hours. Rats were sacrificed24hours later, brain tissue pathological and electron microscopy ultrastructural changes were observed. Thus we evaluate the feasibility of establishing early SAE animal model by monitoring the changes of neurological behaviors, EEG and somatosensory evoked potentials.Part3:Rats were weighted, numbered, and placed with EEG monitoring electrodes10days before establishing models.1Expression of Ngb in SAE group:50rats were divided into3groups, sham operation group (n=8);42rats underwent CLP surgery, in which20died within24hours, and the22survived rats were divided into sepsis+non-SAE group (n=12) and SAE group (n=10), according to neurobiology score, EEG and somatosensory evoked potentials.2Hemin up-regulating Ngb expression and its effect on SAE:60rats were divided into3groups, sham operation group (n=8);52rats underwent CLP surgery, and were randomly divided into sepsis group (n=26) and sepsis+hemin group (n=26). Sepsis+hemin group were intraperitoneal injected hemin50mg/kg right after and12hours after modeling. The onset of SAE were diagnosed by neurobiology score, EEG and somatosensory evoked potentials within24hours. Finally, we include sham operation group (n=8), SAE group (n=6), SAE+hemin group (n=5) into our study.3Antisense oligonucleotide nucleotide (AS-ODNS) inhibiting Ngb gene expression in SAE rats and its effect on SAE:100rats were dividied into3groups, sham operation group (n=8);92rats underwent CLP surgery, and were randomly divided into sepsis group (n=26), sepsis+AS-ODNS group (n=26), sepsis+S-ODNS group (n=20), sepsis+artificial cerebrospinal fluid (n=20). Sepsis+AS-ODNS group were intracerebroventricular injected AS-ODNS8ul right after and12hours after modeling, and the dose were determined by our pre-experiment; sepsis+S-ODNS group and sepsis+artificial cerebrospinal fluid group were intracerebroventricular injected S-ODNS or artificial cerebrospinal fluid8ul respectively at the same time points. The onset of SAE were diagnosed by neurobiology score, EEG and somatosensory evoked potentials within24hours. Finally, we include sham operation group (n=8), SAE group (n=6), SAE+AS-ODNS group (n=6), SAE+S-ODNS group (n=5) and sepsis+artificial cerebrospinal fluid group (n=4) into our study.Brain tissue pathology and electron microscopy ultrastructure changes within24hours were examined. ELISA, immunohistochemistry, Western blotting, RT-PCR were used to detect Ngb expression in blood and brain tissue, and to examine the changes of SOD, MDA and TUNEL apoptosis in brain.RESULTS:1Compared with non-SAE group, heart rate, arterial blood lactate and serum sodium was significantly higher, and platelets, serum albumin and PH value was significantly lower in SAE group (P<0.05). Other indices were not significantly different between two groups (P>0.05). Pathogen detection rate was70.7%(29/41) in SAE group and44.5%in non-SAE group (85/191), and the difference was significant (P=0.003). The infection rates of Acinetobacter, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Staphylococcus aureus and Enterococcus faecium were significantly higher in SAE group (P<0.05), and there were no significant differences in other bacterial species. 2In232patients included,41had SAE (17.67%), mechanical ventilation time of SAE group (8.2±13.8days) was significantly higher than that of non-SAE group (2.9±5.7days), P=0.021. ICU time was significantly longer in SAE group (12.4±15.5days vs.7.1±7.6days, P=0.042), and28days mortality of SAE group (56.1%,23/41)was significantly higher than that of non-SAE group (35.1%,67/191), P=0.013.3SAE could be early diagnosed by neurobiology score, reduced a wave and markedly increased delta wave on EEG, reduced amplitudes of evoked potentials P1, and significantly prolonged latency of S-P1and N1-P1. In survived septic rats,6had changes on neurological behaviors, EEG and somatosensory evoked potentials, thus were diagnosed as SAE. The incidence of SAE was46%.4Ngb expression was high in blood and brain tissue of SAE group, mainly localized in the frontal cortex and hippocampus. Blood SOD, MDA were significant higher and apoptosis was increased, while light microscopy and electron microscopy show neuronal damage, mitochondrial swelling, nuclear condensation, and apoptotic bodies could be seen.5Pretreating SAE rats with Chloride hemin could up-regulate Ngb gene expression in blood and brain tissue, reduce blood SOD, MDA and decrease apoptosis. Light microscopy and electron microscopy also show reduced neuronal damage than SAE group.6Intervening SAE rats with AS-ODNS could inhibit Ngb gene expression in blood and brain tissue, markedly increase blood SOD, MDA level and apoptosis. Light microscopy and electron microscopy also show aggravated neuronal damage than SAE group.CONCLUSION:1The incidence of SAE is high. Mortality is significantly higher in patients with SAE. Thus, a correct understanding of the differences between SAE and non-SAE, and active clinical interventions on its risk factors are of great significance on reducing incidence and mortality.2SAE can be early diagnosed by neurobiology score, EEG and somatosensory evoked potentials. SAE rat models are successfully established.3The incidence of SAE is high in septic rats. Significant changes can be detected within24hours, which can be used as observing time point in further studies. The main pathological changes are neuronal damage, mitochondrial swelling and apoptosis, but no obvious abscess and necrosis.4Ngb gene expression is high in brain and blood of SAE rats, and mainly localized in the frontal cortex and hippocampus.5Chloride hemin could up-regulate Ngb gene expression in brain tissue, and reduce SAE related brain injury. 6AS-ODNS-Ngb can inhibit Ngb gene expression and deteriorate SAE related brain injury.7The mechanisms of high Ngb gene expression in SAE rats'brain tissue and its brain protective effect are still unclear and waiting for further study.