| BackgroundSepsis and its complications are the most frequent causes of morbidity and mortality in intensive care unit (ICU), contributing to 750,000 cases per year in the United States. Epidemiologic studies indicate that sepsis occurs in approximately 2% of all hospitalizations and may be present in up to 75% of ICU patients.Sepsis-associated encephalopathy (SAE) is defined by diffuse cerebral dysfunction that accompanies sepsis in the absence of direct CNS infection, structural abnormality or other types of encephalopathy (for example, hepatic or renal encephalopathy), as detected by clinical or standard laboratory tests. SAE manifests as a spectrum of disturbed cerebral function ranging from mild delirium to coma. As mortality is increasing with severity of SAE,early identification and management of patients with SAE are important to reduce associated mortality. The pathophysiology of SAE is not still completely understood.The pathogenesis of septic encephalopathy may include the breakdown of blood brain barrier (BBB), microcirculation dysfunction, neuroinflammation reaction, amino acid and neurotransmitters deregulation, mitochondrial dysfunction, oxidative stress and apoptosis. The main stay of management of SAE hinges on early detection of delirium, determination of the underlying cause, accurate and prompt treatment of the infection, and provision of supportive care.Current evidence suggests that many’classical’pharmacological agents, such as ligands of alpha-adrenergic, beta-adrenergic, dopamine and adenosine receptors, in addition to control blood pressure, heart rate, cardiac contractility force, regulating airway response, some study also found that some of its involvement in regulating metabolism and central nervous system function.And adrenergic receptor plays an important role in the regulation of immune response. The results for the intervention in β receptor generated remains controversial. Part of the reason with which the effects produced by different subtypes of β, and may be associated with dose, time and manner of the drug. Isoproterenol (isoproterenol, ISO) non-selective β-agonists. studies found that isoproterenol regulate immune cells in the immune function and mitochondrial enzymatic activity. While the application of large doses will lead to organ dysfunction.ObjectiveThis experiment is aiming to the effect of research different dose isoproterenol to the sepsis rats serum levels of inflammatory factors, oxidative stress factors of brain mitochondrial function,and to find the best dose of a protective effect on brain septic rats, and to explore the mechanism of brain protection, providing a theoretical basis for clinical application.Methods1. Grouping and Building animal modelsBuy 30 Specific pathogen Free level SD male rats(animals batch number 44008500003278) fromYat-sen university,whose weight approximately 250-300g.fasting 12 hours but drink water freely before experiment. Using the simple random method,30 Sprague Dawley-(SD) rats were randomly divided into five groups (n=6 per group):control group, endotoxin group and ISO Intervention (small-dose, medium-dose and large-dose) group. Endotoxin group received intravenous injection of LPS 10mg/kg followed by an continuous intravenous infusion of 0.9% saline lml/h; ISO Intervention group received LPS 10mg/kg followed by an continuous intravenous infusion of ISO 0.06μg/(kg·min),0.3μg/ (kg·min) and 0.6μ/(kg·min); the control group received intraperitoneal injection and continuous intravenous infusion same amount of 0.9% saline. The endpoint of the study was 24h after intraperitoneal injection of 0.9% saline or LPS.2. Measuring method of the indicators2.1 General conditions in ratsTo observe the mental state, fever, diarrhea, vertical hair, skin and mucous bleeding before or after infusion ISO or NS, as well as to measure of basic vital signs,such as rectal temperature, heart rate and respiratory rate.2.2 Serum inflammatory reaction indexes in ratsRat serum TNF-a、IL-6、IL-1β levels were determined by enzyme-linked immunosorbent assay(ELISA) at 2h,6h and 24h after continues isoproterenol infusion or NS.2.3 Brain mitochondria oxidative stress indicators in ratsActivity of total-SOD was determined by xanthine oxidase technique; content of bain mitochondria MDA was determined by thiobarbituric acid(TBAC) method;content of brain mitochondria NO was determined with nitric acid deoxidize enzyme method; content of brain mitochondria iNOS was determined with L-arginineoxidation method2.4 The indicators related to brain mitochondria damageExtract brain mitochondria with the Pulitzer mitochondrial/cytoplasm preparation kit. Part of mitochondria were produced into electron microscopy ultrathin slices,brain mitochondrial morphology were observed under electron microscope.2.5 The pathological tissue damage of brainRat brain paraffin section observed under optical microscope3. The statistical analysisData were analyzed by SPSS 19.0, all results were reported as mean±standard deviation. Differences between groups were determined by one-way ANOVA. If there was statistical significance and homogeneity of variance between groups, Least-significant difference (LSD) was used, while if there was statistical significance and heterogeneity of variance between groups, Dunnett T3 was used.Results1. General state of ratNormal group rats are generally in good condition. The basic vital signs such as heart rate,breathing rate and body temperature were steady. While endotoxin group of rats were in poor response after intraperitoneal LPS injection about one hour.They emerged sepsis state such as lethargy, standing hair even bleeding tendency. The general state of ISO group were better than the sepsis group rats.2. The changes of serum inflammatory factors levels in rats2.1 The changes of serum TNF-a level in rats in each group(1)The level of serum TNF-a at 2h after continues isoproterenol infusion or saline among groupsComparing with control group,Levels of serum TNF-a significantly increased in endotoxin group and ISO intervene group (small-, medium- and large-dose group) (P all<0.05); Comparing with endotoxin group, levels of serum TNF-α significantly decreased in medium- and large-dose group (P all<0.05),and Comparing with small-dose ISO group, levels of serum TNF-α significantly decreased in medium-and large-dose group (P all<0.05).(2)The level of serum TNF-a at 6h after continues isoproterenol infusion or saline among groupsLevels of serum TNF-α significantly increased in endotoxin group and ISO intervene group (small-, medium- and large-dose group) compared with control group (P all<0.05); levels of serum TNF-α significantly decreased in medium- and large-dose group compared with endotoxin group (P all<0.05); levels of serum TNF-a significantly decreased in large-dose group compared with small-dose group (P<0.05).(3)The level of serum TNF-a at 24h after continues isoproterenol infusion or saline among groupsLevels of serum TNF-α significantly increased in endotoxin group and ISO intervene group (small-,and medium-) compared with control group (P all<0.05); levels of serum TNF-α significantly decreased in medium-and large-dose group compared with endotoxin group (P all<0.05); levels of serum TNF-a significantly decreased in large-dose group compared with small-and medium-dose group (P <0.05).2.2 The changes of serum IL-1β level in rats in each group(1) The level of serum IL-1β at 2h after continues isoproterenol infusion or saline among groups.Levels of serum IL-1β significantly increased in endotoxin group and ISO intervene group (small-,and medium-) compared with control group (P all<0.05); while levels of serum IL-10 has no statistical differences in all Intervention group compared with endotoxin group (P>0.05).(2) The level of serum IL-1β at 6h after continues isoproterenol infusion or saline among groupsLevels of serum IL-1β significantly increased in endotoxin group and ISO intervene group (small-,and medium-) compared with control group (P all<0.05); while levels of serum IL-1βhas no Statistical differences in all Intervention group compared with endotoxin group (P>0.05).(3) The level of serum IL-1β at 24h after continues isoproterenol infusion or saline among groupsLevels of serum IL-1βsignificantly increased in endotoxin group and ISO intervene group (small-,and medium-) compared with control group (P all<0.05); while levels of serum IL-1βhas no Statistical differences in all Intervention group compared with sepsis group (P>0.05).2.3 The changes of serum IL-10 level in rats in each group(1)The level of serum IL-10 at 2h after continues isoproterenol infusion or saline among groupsLevels of serum IL-10 has no significant increased in all Intervention group compared with endotoxin group (P>0.05).(2)The level of serum IL-10 at 6h after continues isoproterenol infusion or saline among groupsLevels of serum IL-10 significantly decreased in small-dose group compared with endotoxin group P<0.05)(3)The level of serum IL-10 at 24h after continues isoproterenol infusion or saline among groupsLevels of serum IL-10 significantly decreased in medium-and large-dose group compared with endotoxin group (P<0.05)3. Ultrastructure changes of the brain mitochondria among groupsIn the normal control group, the neurons nuclear membrane is integrity, no shrinkage,and can see round or oval mitochondrias in the cell matrix. clear boundary matrix uniform, no rupture of mitochondrial inner and outer membranes, Mitochondrial crista are clearly visible.Compared with normal control rats,The neurons nucleus nuclear membrane of the endotoxin group is shrinkage, chromatin dense, inner mitochondrial membrane rupture within the matrix environment, crest reduced, dissolved, disappeared,and vacuoles within the matrix. and the mitochondria structure within the dendrites almost is normal. Compared to the endotoxin group, the intervention group isoproterenol alleviate neuronal nuclear membrane shrinkage, mitochondria reduce mitigation, reduction of vacuolar matrix, the most significant improvement especially in the middle dose group, within the dendritic morphology of normal mitochondria.4.brain mitochondrial oxidative stress indicators in rats4.1 The changes of brain mitochondrial iNOS level in ratsCompared with normal control rats, mitochondrial iNOS level of endotoxin groups and ISO intervension groups increased significantly (P<0.05); compared with endotoxin groups, mitochondrial iNOS level concentration increased in medium-dose group (P< 0.05).4.2 The changes of brain mitochondrial NO level in rats:Compared with normal control rats, mitochondrial NO level of endotoxin groups increased significantly (P<0.05); while compared with endotoxin groups, mitochondrial NO level concentration reduced in all interversion group (small-, medium-and large-dose group) (P< 0.05),especially in medium-dose group4.3 The changes of brain mitochondrial totall SOD level in rats:Compared with normal control rats, mitochondrial totall SOD level of endotoxin groups decreased significantly (P<0.05); while compared with endotoxin groups, mitochondrial SOD level concentration increased in small-,and medium-dose group) (P<0.05)4.4 The changes of brain mitochondrial MDA level in rats:Compared with normal control rats, mitochondrial MDA level of endotoxin groups increased significantly (P<0.05); while compared with endotoxin groups, mitochondrial MDA level concentration decreased in small-,and medium-dose group) (P< 0.05);and compared with small-dose group, mitochondrial MDA level concentration increased in large-dose group) (P< 0.05)5. Pathological changes of the brain (HE stainning) among groupsUnder light microscopy, normal control group showed the shape and size of cells are normal,and the cell structure is clear; while in the endotoxin group, the endotoxin group see a large number of neuronal cell body shrinkage, nuclear condensation, stained cell structure is unclear.The gap is increased between cells, the perivascular edema severely,Part edema vascular bureaucratic occlusion; Compared to endotoxin group,the number of the shrinked,karyopyknosis,and stained neurons is decreased in the ISO intervension group,especially in the medium-dose group.Conclusions1.The inflammation factors such an TNF-α、IL-β3 and IL-10 are increased in early sepsis.2.The structure and function of brain mitochondrial are affected in early sepsis3. the medium-dose ISO continuous infusion in improving the septic inflammation and the structure and function of the brain mitochondria is most favorable. |
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