The Model Of Chronic Infection Of Toxoplasma Gondii And Its Application On The Study Of Learning Capacity On Rats

Toxoplasmosis is a causative agent for zoonotic disease responsible for a wide variety of infections in animal and humans. Toxoplasma gondii has the ability of eroding neurons in brain, which lead to a chronic, hidden or latent infection in brain. Susceptivity is varied in different animal, as well as the symptom and sequel of the infection. A suitable animal model is a key point in researches. It has great value to established chronic infected animal model is important in research about diagnosis, vaccine and therapy of toxoplasmosis. Like human beings, rats is not susceptive to Toxoplasma gondii, it is generally asymptomatic after infected with Toxoplasma gondii. So, rat’s infection is more similar to human’s toxoplasmosis. It has more value in researching diagnosis, pathogenic biology, vaccine of toxoplasmosis.Recent studies have shown that chronic infection by Toxoplasma gondii can change host behavior and especially damage discretion and learning of host. Hippocampus is the damage zone crucial for memory formation. Only around the hippocampus or hippocampal brain regions damaged, there will be the spatial location of memory impairment. At present, mechanisms of central nervous system damage caused by Toxoplasma infection is poorly understood, and therefore need further research. The purpose of this study is to construct rat model which chronic infected by toxoplasma gondii. The contents of the study are as follows:1. Construction of rat model with chronic infection of Toxoplasma gondii RH strainBy mouth or abdominal cavity infected with Toxoplasma gondii RH strain, with the mothod of immune suppression, we constructed, compared, researched the rat model. The results showed that when dissected the rat infected with Toxoplasma gondii RH strain by mouth and abdominal cavity 90 days later, the tachyzoite or cyst could not be checked in the smear of brain, liver or homogenate, with the result of PCR using gene B1 or double PCR using gene B1 and 529bp, showed negative, when innoculated with the homogenate of rat’s brain, the mouse appeared no clinical symptom or death. But when immune suppression firstly, then infected with Toxoplasma gondii RH strain, we can separate the tachyzoite. And then, after infected the rat with tachyzoite, with the result of immune suppression, a few tachyzoites could be checked in 4 rats, meanwhile,7 positive rats were checked by the method of double PCR. All of these showed that by infected with Toxoplasma gondii RH strain using intraperitoneal injection, the infected model can be constructed.2.The effects of chronic Toxoplasma gondii infection on capacity of learning and memory and cytokinese of hippocampus in ratsAfter 9 weeks of infection, passive avoidance tests and Morris water maze tests were carried out to observe behavior changes such as learning and memory ability. The level of IL-1β、IL-6 and TNF-αof hippocampus in rats were examined by radioimmunoassay, the activity of SOD with xanthine oxidase, the content of MDA with thiobarbituric acid. The results showed that there were no difference in memory acquirement in all rats, but memory retention of infected rats was earlier than control group’s. The escape latency of the rats in Toxoplasma gondii infection groups was significantly greater than the control group’s and the percent of distance between the quadrant was previously decreased (P< 0.05). Compared with control groups, the level of IL-1βand TNF-αin hippocampus increased significantly in the Toxoplasma gondii infection group (P< 0.05), but the level of IL-6 was opposite (P> 0.05). SOD activity in hippocampus in Toxoplasma gondii infection rats mgprot is lower than in the control group (P<0.05). MDA content in hippocampus of the infected group increased, compared with the control group (P<0.05). Toxoplasma gondii infection could lead to obvious effects in learning and memory capacity of rats. Regulating the level of IL-1β、TNF-αcytokines and decrease hippocampal scavenging and increase oxidative stress in rats hippocampus by chronic Toxoplasma gondii infection maybe one of the mechanisms for affecting learning and memory ability of Toxoplasma gondii infection rats.3. Expression effects of subunits NR2A, NR2B of neurotrophic factor (BDNF) and N-methyl-aspartate receptor (NMDA) derived from rat hippocampus which were infected by Toxoplasma gondii Four-week old rats were infected by Toxoplasma gondii tachyzoite. After 9 weeks, use the application of immunohistochemistry, insitu hybridization and image processing to analysis the expression of hippocampal BDNF, CA1, CA3 and the dentate gyrus NR2A and NR2B. The results showed that the positive immunohistochemical staining granules of Toxoplasma gondii infection in rats’hippocampal BDNF were significantly higher than control groups, situ hybridization showed that rats infected with Toxoplasma gondii brain BDNF mRNA expression were increased, positive hybridization signals higher than the hippocampus. The expression in the CA3 area gray value of immune response was significantly higher than the control groups, while the expression of immune response in CA1 and dentate gyrus gray value had no significant differences compared with the control groups. The expression of NR2B protein expression in the CA1 and CA3 areas of the immune response gray were significantly higher than control groups(P<0.05); while the expression in the dentate gyrus gray value of immune response was no significant difference(P>0.05), which suggested that chronic infection of Toxoplasma gondii prompted hippocampus BDNF and BDNF mRNA expression. Toxoplasma chronic infection can affect the expression of hippocampal NMDA receptors.4. Effects of Toxoplasma gondii chronic infection in rats’on object recognition and brain monoamine nerrotransmitters norepinephrine, dopamine (DA), 5-hydroxytryptamine(5-HT) contentThe object recognition test and morris water maze tests were carried out to observe such behavior change as the learning and memory ability. Meanwhile, the monoamine neurotransmitter NE, DA and 5-HT contents in Toxoplasma gondii infection rats brain were detected with high performance liquid chromatography-electrochemical detection (HPLCECD). For ORT, infection groups of three doses showed significantly less exploration time on new object (P<0.05); the discrimination index (DI) of infection groups showed very significant lower DI (P<0.01), compared with control group. The contents of NE,5-HT in infected rats significantly reduced, while the DA was significantly increased. Compared with the control groups, there were significant differences (P<0.05). Suggest that Toxoplasma chronic infection may influence the monoamine neurotransmitter synthesis and therefore reduce the learning and memory ability.5. Effect of Toxoplasma gondii infection on levels of neurofilameat mRNA and cellular immunity in ratsFour-week old male SD rats were injected with Toxoplasma. After 9 weeks, mRNA levels of light molecular NF (NF-L), medium molecular NF (NF-M) and high molecular NF (NF-H) in cerebrum was determined by RT-PCR. The percentages of CD4+,CD8+ T lymphocytes were examined by means of flow cytometery, and the peripheral blood serum levels of IFN-y, TNF-a, IL-4 were analyzed by ELISA. The results showed that the mRNA levels of NF-L in rat cerebrum decreased significantly to 64.56%, the NF-M decreased to 95.83%, and NF-H declined to 89.47% of the control. Compared with the control group, no significant changes were observed on the levels of CD4+ T lymphocytes and CD8+ T lymphocytes. The levels of IFN-y, TNF-a, IL-4 in experimental rats’ sera raised.6. Changes of apoptosis and its related protein expressions of hippocampal neurons in rats with Toxoplasma gondii chronic infectionDetect Cell cycle and the percentage of the hippocampal neuron apoptosis in the rats suffered from Toxoplasma infected rats was measured with flow cytometry (FCM) and their mitochondrial membrane potential was measured with FCM under the Rhodamine 123 dying. The protein expressions of Cyt c and caspase-3 were detected with immunohistochemical methods. The results show that apoptosis in hippocampal neurons was significantly increased after 10 weeks of toxoplasma gondii infection (P<0.05). The cell percentage of G0/G1 phase increased significantly (P<0.05), while the cell percentage of S phase decreased significantly (P<0.05); the mitochondrial membrane potential decreased significantly (P<0.05), and the expressions of caspase-3 and Cyt c proteins increased significantly (P<0.05). It shows that Toxoplasma gondii chronic infection can lead to the apoptosis of the hippocampal neuron, and then lead to a certain learning and memory capabilities.7. Comparative proteome analysis of rat hippocampus from Toxoplasma gondii chronic infected and healthy ratsEstablish animal model of the Toxoplasma inection rat. Isolated hippocampus and extract hippocampal protein sample, and then total protein were analyzed by 2-DE. We obtained proteins of the satisfactory 2-DE patterns of the spinal cord. Totally 268±13 and 301±15 protein spots were obtained in the Toxoplasma gondii infection and control rats spinal cord maps respectively, which 7 spots increased or decreased in quantity.3 protein spots were identified by MALDL-TOF-MS,and similar to glyceraldehyde-3-phosphate dehydrogenase、valosin-containing protein and Gamma-actin. Proteomics method of Toxoplasma chronic infection was established in spinal cord. The differentially displayed proteins in the spinal cord may provide further insight into molecular mechanisms and useful clues for developing new drugs for its treatment.8. Proteome research of Toxoplasma gondii infection spinal cord by two -dimensional gel electrophoresis analysis and mass spectrometry identificationTo establish animal model of the Toxoplasma infection rat, and spinal total protein were analysed and extracted by 2-DE, blue staining and analyzed with PDQuest 1.0 software. We obtained proteins of the satisfactory 2-DE patterns of the spinal cord. Totally 268±13 and 301±15 protein spots were obtained in the Toxoplasma gondii infection and control rats spinal cord maps respectively,of which 7 spots increased or decreased in quantity.3 protein spots were identified by MALDL-TOF-MS,and similar to glyceraldehyde-3-phosphate dehydrogenase、valosin-containing protein and Gamma-actin. The differentially displayed proteins in the spinal cord may provide further insight into molecular mechanisms and useful clues for developing new drugs for its treatment.In summary, Toxoplasma gondii chronic infection damage learning and memory abilities of rats. The mechanism of Toxoplasma gondii infection may be related to hippocampus of cytokines, monoamine neurotransmitter, hippocampal antioxidant levels and protein differential expression and so on. The results provided basis for discovering new drug targets.