| Backgroud:Toxoplasma gondii is a highly prevalent, intracellular protozoan parasite with an indirect life cycle. It infects a very broad spectrum of warm-blooded vertebrates, including humans, as intermediate hosts but can reproduce sexually only in the feline intestine. It is estimated that adults worldwide, at least1/3of the people are infected, some developed countries in Europe, the serum antibodies against T.gondii were detected in more than80%. The incidence of Toxoplasma gondii encephalopathy is increasing, there are more than90%of the reported systemic Toxoplasma,which is died by Toxoplasma encephalitis. T.gondii is an opportunistic pathogen that generally forms cysts in an immunocompetent host which is generally asymptomatic. In hosts who are severely immunocompromised, the tachyzoites of T.gondii may frequently cause a dissemination and subsequent complications, sometimes become fatal. Tachyzoites disseminated via blood or the lymphatic system to different organs during acute stage disease cause toxoplasmo sis,which is characterized by lymphadenopathy and reticular cell hyperplasia. Chronic infection, which is characterized by parasite encystment in the host muscle and brain cells, persists following the resolution of acute infection and continues with seropositivity throughout the host’s lifetime.Vertical transmission occurs in most cases when a mother has a primary infection during pregnancy and this can present itself as either subclinical infection or congenital toxoplasmosis with a wide spectrum of severe manifestation in newborns or relapse later in life, such as mental retardation, retinochoroiditis, and epilepsy. T.gondii is also a zoonosis and may give rise to great economic loss in the livestock industry. Due to its high prevalence in the human population, it is critical to better understand the effects of T. gondii infection in the brain.In recent years, the associations have been identified between T. gondii seroprevalence and schizophrenia. Acquired toxoplasmosis patients often showed diffuse encephalopathy, epilepsy, encephalitis, single or multiple of large mass and lesions, and can be found in the mental disorders.Schizophrenia is a serious hazard to human health of nerve mental disease (neuropsy chiatric disease), which etiology is not clear. Schizophrenia affects approximately1%of the adult population and in most cases is a lifelong disease with exacerbations. Toxoplasma infection is thought to be one of candidate for the cause of schizophrenia. However, some researchers considered that the increased infection rate in patients was the results of schizophrenia due to the abnormal behavior, poor living environment or special living style.During natural infections, T.gondii disseminates widely and crosses the placenta and the blood-brain barrier. There may cause severe acute pathology or establish chronic infections.Considering the lifelong presence of the parasite, hosts infected with T.gondii must develop a powerful immune response that has to be under tight control and persistently maintained in all infected tissues. It is conceivable that efficient transportation of parasites by infected leukocytes may locate parasites in target organs (e.g. the placenta and the CNS) and that passage across cellular barriers may be facilitated by the parasite motility driven transmigration, which are important propitious to understand the mechanism of passage to the fetus and to the CNS. Cell-mediated immune mechanisms play a major role in the control of T. gondii infection because the parasite is exclusively localized intracellularly.In chronic phase, toxoplasma can long-term latent in the central nervous system, whose a typical feature is a significant activated glial cells. Astrocytes are now recognized as specialized glial cells that exert many essential functions in the healthy central nervous system. These studies indicate that astrocytes are a prevalent host cell for the cyst stage in the brain. Astrocyte activation is a hallmark of Toxoplasmic encephalitis with activated astrocytes encircling the inflammatory infiltrates around T. gondii cysts.Until recently, several studies have suggested that T. gondii infection in humans can have serious neurological effects. During the chronic stage of infection, infected rodents which are a key intermediate host for T. gondii, exhibit a distinct repertoire of specific behavioral changes, including a loss of aversion to cat odors.T.gondii induces behavioral alterations in infected rodents that would facilitate the transmission of the parasite to its definitive feline host, however, the mechanism responsible for these changes remains unclear. Toxoplasma is neurotropic, which tend to invade the brain tissue. The predilection of T. gondii for the CNS places it in a privileged position to manipulate host behaviour. In addition to, some behavior studies have gathered abundant evidence that latent toxoplasmosis is associated with impaired motor performance, deficits in spatial learning and memory, reduced anxiety, sensory attention deficits, altered novelty seeking behavior, longer reaction times, and, the most importantly, reduced avoidance of feline predators. Moreover, recent associations have been made between parasite infection and neurological disorders. Although schizophrenia is a multifactorial disease, pharmacological and genetic evidence suggest that dysregulation of neurotransmitter metabolism is involved in schizophrenia. Thus, schizophrenia, multiple sclerosis, Alzheimer’s disease and Parkinson’s disease, is a chronic disease of the central nervous system; Toxoplasma gondii encephalitis may manifest with symptoms similar to those of schizophrenia and other psychiatric disorders. The neuronal degeneration induced by neuroinflammation is known to play a key role in the pathogenesis of chronic neurodegenerative diseases, and in particular, Alzheimer’s disease (AD) is the most common cause of dementia in the elderly causing progressive and permanent reductions in learning, memory, and cognitive abilities.A lot of studies have shown that toxoplasma infection can change some neurotransmitter in the brain. Schizophrenia usually result in abnormal changes of neurotransmitters, animal studies have shown that toxoplasma infection can affect the neurotransmitter metabolism. It is possible that the increased dopamine accumulation and release observed during T. gondii infection may contribute to T. gondii associated schizophrenia. These limbic brain regions are well known to contain dopamine that plays important functions in the control of movements(basal ganglia),reward to stimuli,pleasure, dependency(hippocampus), motivation and cognition, and species and stimuli specific fear (amygdala). Furthermore, the behavioral changes of Toxoplasma infection are not only reserved to rodents but also involve humans. In addition, some studies have reported some medications used to treat schizophrenia inhibit the replication of T.gondii in cell culture, which improve the behavior of toxoplasma infection hosts. Investigations on the possible link between T. gondii infection and central nervous system disease in man have been mainly limited in the serology testing of T.gondii-specificanti body.How neurotransmitters dysregulation plays a role in schizophrenia is still unknown.However, relatively few studies have addressed the influence of poly amines on animal behavior, learning and memory functions. Polyamine includes spermine, spermidine, putrescine. L-arginine is a critical metabolite for living organisms, which additionally plays key role in host pathogen interactions. Constitutive functions include its requirement for protein synthesis, and as an intermediate leading to essential polyamine synthesis.On the one hand, nitric oxide synthase(NOS)catalyzes the enzymatic of L-arginine to NO; On the other hand, Arginase catalyzes the enzymatic hydrolysis of L-arginine to L-ornithine and urea; Ornithine decarboxylase (ODC)catalyzes the enzymatic decarboxylation of L-ornithine to putrescine,putrescine can generate spermine and spermidine under the effection of spermine acetyl transferase(SSAT).Some studies reported that T.gondii acquire L-arginine from the host and synthesize arginase themselves to obtain L-ornithine.In this study, construct T.gondii infection mouse model with Prugniaud strain. At the same time, the bradyzoite antigen was analyzed by HE staining and immunohistochemical; To test the speed enzyme of polyamine metabolism, and single amine neurotransmitter in mouse brains was detected by the high performance liquid chromatography (HPLC) method. It is crucial to examine whether neurotransmitter metabolism is affected by T. gondii infection. In view of this, this study will establish a stable animal model of chronic toxoplasma infection, which is a experimental tool for further study of the behavior change of caused Toxoplasma infection.Objective:1. To construct the chronic toxoplasma infection mice model with Prugniaud strain;2. To observe time and space distribution characteristics of the cyst in the brain of experimental mice;3. To analyse the polyamine metabolism enzyme in the toxoplasma infection mice and normal mice;4. These monoamine neurotransmitters were detected in the toxoplasma infection mice and normal mice, which make for exploring the behavioral changes of toxoplasma infection.Methods:1. Kunming mice were oral inoculated with cysts of Prugniaud strains.Infected mice were sacrificed at1,2, and3months post infection; the brains were analyzed for cysts in the microscope;2. Infected mice were sacrificed at1and3months post infection, which were fixed brain tissue by heart perfusion. The brain were embedded by paraffin, the bradyzoite could be recognized by HE staining and immunohistochemical respectively, which is facilitate to observe the distribution of cyst in brain tissue of mice;3. The speed limit enzymes of polyamine metabolism ODC(ornithine decarboxylase)and SSAT(spermine acetyl transferase)were analyzed by PCR method in the brain of toxoplasma infection mice and normal mice; 4. The neurotransmitter (norepinephrine, dopamine and serotonin) of T. gondii infection mice and normal mice were detected by high performance liquid chromatography (HPLC) method.Results:1. T. gondii chronic infection mice model was successfully established; Typical symptoms of acute toxoplasmosis, i.e., lethargy, ruffled fur or hunched posture were apparent after peroral infection with20tissue cysts of the avirulent Prugniaud strain.2. Immunohistochemical shows that the density cyst of the prefrontal cortex, hippocampus, thalamencephalon, cerebellum, and amygdale in the30days of Toxoplasma infection had significant difference (F=17.949, P<0.001). In90th days, we also found that the density of cyst in the five regions had significant difference (F=36.634, P<0.001). Among them, the density of cyst in the thalamencephalon was significantly higher than the other four regions(P<0.01), but the density of cyst in the cerebellum was significantly less than the other four regions, in addition to,the density of cyst of the other three regions was no significant difference (P>0.05). In one month, the density of cyst in the amygdala of mice was significantly less than3months (F=18.574, P<0.001). but the density of cyst in the cerebellum was significantly higher than3months (F=18.314,P<0.001).3. The PCR results show that the content of ODC and SSAT in the brain of chronic toxoplasma infection mice was higher than normal mice.4. High performance liquid chromatography (HPLC) showed the concentration of serotonin and norepinephrine in toxoplasma infection mice was less than in the normal mice, and dopamine was more than in the normal mice. Conclusion1. Chronic T. gondii infection mice model was successfully established;2. The cysts were located throughout the brain of chronic infected mice, the distribution of cysts had significant differences in different brain regions of mice. We found that the most cysts were in the thalamencephalon, but also prevalent in the prefrontal cortex, hippocampus and amygdala, while the least intensively at cerebellum. The space-time distribution of cysts had significant differences in chronic infected mice, which may be the pathological basis of clinical manifestations of Toxoplasmosis.3. The PCR results showed that the content of ODC and SSAT in the brain of T. gondii infection mice was higher than in the normal mice.4. High performance liquid chromatography (HPLC) showed the concentration of norepinephrine, serotonin in the brain of toxoplasma infection mice was less than the normal mice, and the concentration of dopamine was more than in the normal mice. |
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