| Endotoxin-induced brain lesion (EIBL) is commonly encountered in clinic . However , the pathological mechanism of EIBL has not yet been fully clarified. Endotoxin ( gram-negative cell wall lipopolysaccharide , LPS) is a major factor resulting in brain lesion . The papers showed that the LPS-induced brain lesion will activate the endogenous protective mechanism in the Pathological process of ischemia and hypoxia. Neuroglobin (Ngb) , the most important Oxygen-carrying protein in the neurons which is able to remove the free Radical in neurons likely participates in endogenous protective mechanism . The aim of this study was to observe the change of Ngb expression , so as to verify the endogenous nuroprotective mechanisms . The models of endotoxin shock and LPS brain lesion were established . Changes of Ngb expression in the frontal cortex , hippocampus , cerebrospinal fluid and plasma were examined by ELISA , Western Blot and IHC .PART I: EXPERIMENTâ… THE EXPRESSION OF NEUROGLOBIN IN THE RAT MODEL OF ENDOTOXIC SHOCKMETHODS: Seventy adult Sprague-Dawley rats were randomly divided into seven groups (10 rats in each group): the control group (0.5 mL normal saline was injected via vena caudalis of animals) and the experimental groups (0.5 mL Endotoxin was injected via vena caudalis of animals , 0.016 g/kg) The specimens were properly taken at 3 h , 6 h , 12 h , 24 h , 48 h and 72 h after endotoxin injection ). The expression of Ngb in each time points was measured by ELISA, WesternBlot and IHC . The brain water content was measured by dry and wet method .RESULTS:â‘ ELISA showed that the Ngb concentrations in the plasma, CSF , hippocampus and cortex of frontal lobe were significantly higher than those of control group(P<0.01 vs control)after endotoxin injection , and peaked at 48 h .â‘¡Western Blot showed that Ngb with relative molecular mass 17 kD was observed in all groups . The Ngb expression in experimental groups was significantly higher than that in the the control group. The expression of Ngb was the highest in 48 h group and 72 h group , and the lowest in 3 h group .â‘¢IHC showed that Ngb-positive cells were distributed in neurons of pyramidal layer, and Ngb-immunoreactive product was located in the cytoplasm of the pyramidal neurons , which were yellow-brown in color.â‘£After endotoxin injection, the brain water content was significantly higher(P<0.01 vs control), and peaked at 48 h .CONCLUSION: Our results indicate that there was a positive correlation between the up-regulation of Ngb expression and the severity of brain lesion. And the expressions of Ngb were up-regulated with a time-dependent manner . The up-regulation of Ngb maybe one of the endogenous nuroprotective mechanisms under endotoxic shock condition. PART II:EXPERIMENTâ…¡THE RELATION OF NEUROGLOBIN EXPRESSION CHANGE TO THE ENDOTOXIN INJECTION DOSE AND ENDOTOXIN INJECTION TIME IN RAT MODEL OF ENDOTOXIN INJECTIONMETHODS: Ninety adult Sprague-Dawley rats were randomly divided into 9 experimental groups ( n=10 , in each group ) by factorial design . The dose factor ( lower-dose , middle-dose and higher-dose ) and the time factor ( 12h , 24h , 48h) were analysised in this experiment . The expression of Ngb over different periods was measured by ELISA and Western-Blot .RESULTS:â‘ ELISA showed that Ngb expression in the cortex of frontal lobe and hippocampus of rats was increased with the dose and the duration of time after endotoxin injection .â‘¡Western Blot showed that Ngb with relative molecular mass 17 kD was observed in all groups . The tendency of Ngb expression change in the cortex of frontal lobe and hippocampus was the same as ELISA .CONCLUSION: Our results indicate that the changes of Ngb expression were correlated with the endotoxin dose and the duration of time . |
