The Effect Of Rotenone On The Serotoninergic Neurons Of Brain In Rats

Objective: Parkinson's disease (PD) is a common neurodegenerative disease that afflicted the middle- and old-age patients. The main pathological features is the selective loss of dopaminergic neurons and Lewy body in the dopaminergic neurons. The symptoms are rest tremor, bradykinesia, hypermyotonia and posture balance disturbance.Many studies have confirmed that the morbility of PD is relative to genetic factors and environmental toxins; the latter plays an important role in the pathogenesis of PD. Epidemiological investigations revealed that the crowd of long-term exposure to certain pesticides, herbicides is more liable to PD than the others. Rotenone is a classical, high affinity inhibitor of complex I and is typically used to define the specific activity of the complex. It is also a commonly used, naturally occurring, organic pesticide, exerted in lakes and reservoirs to kill nuisance fish. Rotenone-infused animals developed symptoms of parkinsonism, including bradykinesia and rigidity. Rats affected severely also developed the flexed posture and motor'freezing'typical of advanced PD. Thus, chronic, systemic rotenone infusion recapitulated the anatomical, biochemical, pathological and behavioral features of PD. Recent studies have shown that in addition to movement disorders, PD sometimes accompanied by depression, anxiety, insomnia, paranoia and other symptoms of non-movement disorders. Depression is one of the most common psychiatric symptoms, the incidence can be as high as 70% and seriously affects the quality of life in the patients with PD. PD patients with depression often have serotoninergic dysfunction. However, the association of the depression in PD is not yet clear.Methods: Male Lewis rats and Wistar rats were randomly divided into experimental and control groups. Rotenone emulsified in sunflower oil was given subcutaneous three times a day for 50 days to experimental groups. Oil was injected as vehicle to the control rats (1ml/kg). Immunocytochemistry and flow cytometry were used to detect the changes and their relevance of tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), 5-HT and serotoninergic transporter (SERT) expression in the experimental rat brain. Hematoxylin and eosin staining and electron microscopy method were respectively used to observe the morphology of neurons in substantia nigra and dorsal raphe nucleus.Results: 1. The toxic effect of rotenone: Rotenone emulsified in sunflower oil at 1.5 mg/kg,2.0 mg/kg and 2.5 mg/kg was respectively given subcutaneously to the rats of three groups for 50 days. Oil was injected as a vehicle to the control rats. The death rate of the group of 2.0 mg/kg and 2.5 mg/kg is very high. Rotenone-infused animals showed symptoms of parkinsonism, including weight loss, bradykinesia and rigidity. Severely affected rats also developed the flexed posture and motor'freezing'typical of advanced PD. 2. The results of HE staining: The morphology of some survived neurons in substantia nigra was abnormal and Lewy body can be observed in dopaminergic neurons in rotenone-infused rats. 3. The experimental results of immunohistochemical staining: (1) The results of dopaminergic neurons immunohistochemical staining: When the rats were injected rotenone, dopaminergic neurons of the brain have different degrees of injury, which show that the expression of TH immunoreactivity in the striatum and substantia nigra have varying degrees of reduction. The count of substantia nigra TH-immunoreactive neurons shows the number of neurons in the experimental group was significantly decreased compared to the control(P <0.01). The gray scale mean value analysis of striatum showed the immunoreactivie expression of TH in the experimental group was significantly lower than the control. It is also a statistically significant difference (P <0.01). (2) The results of serotoninergic neurons immunohistochemical staining:â‘ 5-HT neurons in rat brain stem mainly concentrated in the segment of inferior colliculus. Dorsal raphe nucleus looks like fountain in cross section at low magnification. The 5-HT positive neurons in dorsal raphe nucleus have brown reaction product in their perikarya, are uniform-sized, and appeared oval-shaped where the cellular nucleus is clearly discernible. TPH immunoreactive pattern is similar to 5-HT.â‘¡Subcutaneous rotenone exposure can cause the varying degrees of damage of serotoninergic neurons in the rat brain. 5-HT and TPH immunoreactive neurons of the experimental group in dorsal raphe nucleus were significantly lower than that of control (P <0.01). The gray scale mean value analysis of 5-HT and SERT immunoreactive fibers in striatum increased significantly in the experimental which indicates the immunoreactive expression of 5-HT and SERT in striatum of the experimental are significently lower than that of control. 4. The Results of flow cytometric detection: The fluorescent intensity of TH, TPH, 5-HT were lower in midbrain of rotenone-infused animals than that of control. The expression of TH, TPH, 5-HT decreased 28.5%, 38.34% and 47.93% of that of control respectively. Despite no significant difference was found in statistical analysis (P> 0.05), there was a decerased trend. 5. The result of electron micrograph: Neurons in dorsal raphe nucleus of the experimental group can be seen swelling mitochondria.Conclusions: This study confirmed that the rotenone is obviously neurotoxic. The rotenone not only can damage dopaminergic neurons causing the rats PD-like symptoms, but also can damage the 5-HT neurons in the brain, which showed the decreased numbers of serotoninergic neurons and the lower expression of TPH, 5-HT and SERT that may trigger the depression of PD .