| Objective: Amyotrophic lateral sclerosis(ALS) was a central nervous system neurodegenerative disease, epidemiology researches demonstrated that men was higher than premenopausal women in morbidity rate, however, there was no obvious difference between men and postmenopausal women. A behavior study of ALS animal model proved that the life time of intact female mice was longer than that of ovariectomized female mice. These results indicated that female sex hormone was related with the production and progression of ALS. Aromatase, key enzyme for either circulated or localized estrogen synthesis, converts androstenedione, testosterone and 16-hydroxytestosterone to oestrone, oestrodiol, oestriol. Under normal physiological conditions, multiple tissues could express aromatase, ovaries, placenta, testis, prostate, adipose tissue, bone and brain were included. In the central nervous system, aromatase is not only confined to mediating sexual differentiation, regulation of reproductive behavior and neuroendocrine response, it can also play a neuroprotective role by increasing the synthesis of estrogen in injury sites. Many studies demonstrated that aromatase is expressed only in neurons but not in glial cells under physiological conditions. While, after various of injury, activated astrocytes started to express aromatase, accompanied by increased aromatase activity. It was also proved that localized estrogen played neuroprotective roles in animal model of Alzheimer’s disease, Parkinson’s disease and ischemic stroke. However, the expression characteristic of aromatase in lumbar spinal cord of wild type and ALS animal model is rarely reported. In consideration of the relationship between ALS and age gender, the purpose of this experiment is to observe expression characteristic of aromatase in apinal cord ventral horn of lumbar segments in familial ALS animal model, providing a basis for the study of estrogen’s neuroprotective role in ALS.Methods:1 Animal modelBreeding familial amyotrophic lateral sclerosis transgenic mice animal models(SOD1G93A). Experimental groups were SOD1G93 A transgenic positive mice of pre-symptomatic stage, diease onset stage and end stage, the SOD1G93 A transgenic negative mice of the same nest and same age with experimental ones were the control groups. Each group includes 5 mice.2 MaterialAfter 10% chloral hydrate 350mg/kg intraperitoneal injection of anesthesia, animal tissues were fixated by 4% paraformaldehyde via heart perfusion for 20 min, the spinal cord and brain were separated from animal and then fixed into 4% paraformaldehyde for 48 hour.3 Immunohistochemical stainingThe fixed lumbar spinal cord were cut into 20μm thick sections, using immunohistochemical staining method to observe the expression and distribution characteristic of aromatase in lumbar spinal cord of SOD1G93 A transgenic mice.4 Immunofluorescence stainingThe fised lumbar spinal cord were cut into 20μm thick sections, using immunofluorescence staining method to observe the expression characteristic of aromatase in different cell types.Results:In non-SOD1G93 A transgenic mice, aromatase was expressed in motor neurons of ventral horn of the lumbar spinal cord, these motor neurons were proved to be α-motor neurons through immunofluorescence staining methods. While in SOD1G93 A transgenic mice, motor neurons appeared to be aromatase immunoreactivity during presymptomatic, with deterioration of disease, the number of motor neurons decreased, we still can see a few of motor neurons, in the end stage of ALS, few motor neurons can be seen, increasing glial cells were activated and started to express aromatase during onset and end stages, which were proved to be microglial cells through immunofluorescence staining methods.Conclusion:Aromatase were expressed in α-motor neurons of lumbar spinal cord of non-SOD1G93 A transgenic mice and SOD1G93 A transgenic mice during pre-symptomatic and onset steges, microglial cells were activated and started to express aromatase during onset and end stages. |
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