The Study On The Expression Of Caspase-3 And NF-κB In The Hippocampus Of Rats And Distribution After Ketamine Administration

The study on the expression of Caspase-3 and NF-κB in the hippocampus of rats after ketamine intraperitoneal injectionObjective: To investigate the behavial performance of rats, the pathological changes of tissue and expressions of NF-κB and Caspase-3 in the hippocampus of rats and their correlation after ketamine intraperitoneal injection (ip.) and to study the possible mechanisms of NF-κB in the ketamine abuse.Methods: 104 Sprague-Dawley rats were randomly divided into high dose, low dose and blank control groups which were respectively administered intraperitoneally with 60 , 10 mg/kg (qd) Ket and equal volume of 0.9 % saline solution (qd) for a week. The behavial performances of rats were observed, HE staining was conducted to assess the changes of the neuron cell in hippocampus, the expressions of NF-κB and Caspase-3 were detected by immunohistochemistry and the NF-κB expressions were determined byWestern blot. Results: (1) behavial performances of rats: The rats behavior were normal in the blank control group, the rats in the low-dose group appeared irritation and they moved frequently at about 10min after ketamine ip. The rats in the high-dose group appeared tachypnea, standing unsteadiness and always falling down at about 5min after ketamine ip. Afer 1 min, the rats appeared limbs trembling, muscular spasms and at last paralysis on the floor which persisted with 20~45min. The behavior change of rats were correlated to the dose of ketamine ip. (2)change of pathology: The gross appearance of tissue was not special in hippocamp of rats after ketamine ip. Under light microscope: the nerve cell arranged regulation, tight and their shape were complete in hippocampus of the rats in the control group. The nerve cell shape were complete and cellular structure was normal in hippocampus of the rats in the low-dose group. But the rats in the high-dose group, the nerve cell shape were observably loose, disorded arrangement and nucleus was anachromasis or not. (3)result of immunohistochemistry: Increased expression of NF-κB and Caspase-3 were found in the hippocampus of the high-dose ketamine rats group compared with controls in early 1 hour after injection and reached peak at 6h~12h and at 7d there have no obviously difference as compared with controls(p﹥0.05); Up-rugulating of NF-κB P65 and Caspase-3 were found in the low-dose group at 6h and reached peak at 12h~24h, but at 3d and 7d there have no obviously difference as compared with controls(p﹥0.05), the positive cell populations of the high-dose group were obviously increased compared with that of low-dose group(p< 0.01). The expression of NF-κB and Caspase-3 were found to be positive correlated. (4) result of Western blot: Expression of NF-κB can not be found in the hippocampus of the rats in the control group. Increased expression quantities of NF-κB reached peak at 12h in the hippocampus of the low-dose ketamine rats group after ketamine ip. and which have obviously difference as compared with controls and there have no obviously difference at the rests of time; Increased expression of NF-κB were found in early 1 hour after injection,and reached peak at 6h ~12h and at 7d there have no obviously difference as compared with controls(p﹥0.05). The positive cell quantities of NF-κB in the high-dose group were obviously increased and the lasting time was longer compared with that of low-dose group.Conclusions: (1) ketamine induced abnormal behavior change is corralted with the damagement of nerve cell in rat hippocampus, this corralation showed an dose dependence manner.(2) Repeatly using ketamine induced increasing expression of NF-κB and Caspase-3 in the hippocampus and the expression profiles of two markers is positive correlated and had a time-dose dependent tendency. Distribution of Ketamine in Acute Poisoned RatsObjective:1.To study the in vivo distribution of ketamine in acute poisoned rats.2. To observe signs and pathological changes of main rats organs after a fatal intragastric administration of ketamine.Methods: 1. GC/MS and GC were used to analyze and detect the ketamine in the blood and tissues. The samples were extracted by aether method and alkalified lidocaine was used as internal standard. The extracts are qualitated by GC/MS method and quantitated by internal standard and working curve methods.2. Distribution study: The rats in control group and experimental group were given intragastric administration of 0.9% saline and ketamine with a dose of 2LD50(460mg/kg), respectively. One hours later, they were executed by decapitation and cardiac blood, cardiac muscle, liver, brain, lung, kidney and spleen were taken for detecting ketamine at room temperature.3. Pathology: The cardiac blood, cardiac muscle, liver, brain, lung, kidney and spleen of acute poisoned rats were fixed with 4% formaldehyde, cut into sections and performed a HE staining for a light microscopic examination.Results: 1.Poisoned symptoms of rats: Temperament of the rats in control group were mild, moving activities were normal and easy for catching during all the experimental procedures. While for the rats in experimental group, irritation and moving activity were increased, tachypnea was observed at 2~5min after the intragastric administration of ketamine. Standing unsteadiness, sustained falling down, exophthalmos, limbs trembling, weak respiration, sialorrhea, muscular spasms and paralysis were observed after about 10min after the intragastric administration of ketamine.2.Pathology: postmortem examination of main organs showed the common signs of non-specific sudden death.3. Ketamine distribution: the concentration of ketamine in the rats of experimental group was detectable.The concentration of ketamine was comparatively higher in tissues and lower in blood(P<0.01). The concentration of ketamine content in rats was as follow: kidney>liver>brain>spleen>cardiac muscle >lung >cardiac blood.Conclusion: 1.Pathology changes of postmortem organs were similar which showed the common signs of non-specific sudden death;2. Ketamine was generally distributed in blood and tissues of rats. The concentration of ketamine in tissues was higher than that in blood.