The Expression Characteristic Of Estrogen Nuclear Receptors In Mice Lumbar Spinal Cord

Amyotrophic lateral sclerosis(ALS) is an adult-onset degenerative disorder characterized by the death of motor cortex, brain stem, and spinal cord motoneurons. Epidemiological studies have found that the incidence of ALS is four to three times in men as that in women. After menopause, however,the ratio decreased to 1:1.Researches found that the onset of disease is earlier in male SOD1G93 A transgenic mice than in female ones, ovariectomy reduced the lifespan, whereas treatment of ovariectomized mice with estrogen significantly delayed disease progression.Epidemiologic and animal studies suggest that estrogen may be responsible for neuroprotective role in ALS.Estrogen(E) is one kind of steroid hormone, The physiological role of estrogen is mainly to regulate reproductive function and sexual behavior, recent evidence suggest estrogen combines with estrogen receptor to plays an important role in anti-apoptosis, anti-inflammatory antioxidant and other neuroprotective effect in a variety of models of brain and spinal cord injury, neurodegenerative Diseases. Estrogenic biological effects is mediated by estrogen nuclear receptor ER α、ERβ and estrogen Membrane receptor GPR30. Protective mechanisms of estrogen in brain were studied in the term of AD, PD and stroke, the protective mechanism on spinal cord diseases are focused on the acute spinal cord injury, multiple sclerosis and in vitro single cell research.Protection mechanism of estrogen on amyotrophic lateral sclerosis has not been reported.As mentioned above, the estrogen plays its biological functions mainly by binding with receptors. Previous studies found that ER alpha and ER beta is mainly expressed in I-III layer of the ventral horn of spinal cord, involving in pain modulation, and no systematic description of distribution in the ventral horn of spinal cord has been made.In order to describe the protective mechanism of estrogen, we need to make a comprehensive understanding of the expressional characteristics of its receptor.Based on these studies, we observed the expression characteristic of estrogen nuclear receptors in lumbar segments of the spinal cord of SOD1G93 A transgenic mice by means of immunohistochemistry and confocal laser scanning, to provide theoretical basis and possible targets for further study on neuroprotection of estrogen in the pathogenesis of ALS. This study is divided into two parts, as follows:Part ⅠExpression of ER alpha in anterior horn of adult male mice spinalcordObjective: To observe the expression of ERα in anterior horn motor neurons of adult male mice spinal cord.Methods:1 AnimalWe chose adult male CD1 mice to do our study2 ImmunohistochemistryMice were anesthetized with 10% chloral hydrate and perfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer for 20 minutes.The lumbar enlargements were carefully dissected and fixed in 4% pareformaldehyde for 48 hours. The tissues were then cut into 25-um sections using a vibratome. we observed the distribution of ERαin l anterior horn motor neurons of adult male mice spinal cord by immunohistochemistry.3 The fixed lumber spinal cord were cut into 25 μm thick sections, using immunofluorescence staining method to observe the expression characteristic of ERαin anterior horn motor neurons of adult male mice spinal cord.Results: In the lumbar spinal cord of adult male mice, ER alpha expressed in the ventral horn, mainly located in the motor neurons, which immunofluorescence staining confirmed alpha motor neurons.Conclusion: ER alpha expressed in anterior horn motor neurons of adult male mice spinal cord. Part ⅡExpression characteristic of ER beta in lumbar spinal cord ofSOD1G93A transgenic miceObjective:To observe the expression characteristic of ER beta in lumbar spinal cord of SOD1G93 A transgenic miceMethods:1 Animal modelBreed male familial amyotrophic lateral sclerosis transgenic mice animal models(SOD1G93A) and divid them into 3 groups :pre-symptomatic stage(equivalent to 60 days), symptomatic stage(equivalent to 90 days) and end-stage(equivalent to 120 days), non transgenic littermates is as control group, each group had 3 mice.2 MaterialAfter 10% chloral hydrate 350mg/kg intraperitoneal injection of anesthesia, animal tissues were fixed via heart perfusion by 4% paraformaldehyde for 20 min, the lumbar spinal cord of the mice were cut,all the blocks were fixed in 4% paraformaldehyde for 48 hour.3 Immunohistochemical stainingAll the segments were then directly made into 25μm thick sections, then they were used to immunohistostaining for detection of ERβ expression and distribution. Count the number of the ERβ-ir Alpha motor neurons of two sides in the ventral horn in lumbar spinal cords.Count the motor neurons in the fifth section of every serial sections.4 ImmunofluorescenceAll the segment were cut into 25μm thick sections, Using a coordinate immunostaining approach to determine the location of ERβ in the lumbar spinal cord of the mice under a confocal laser scanning microscope.5 Statistical analysisStatistical analysis was performed using one-way ANOVA followed by Student’s t-test with SPSS 13.0 statistical software.Differences were considered significant at P < 0.05.Results:1 Animal modelB6SJL-BTg(SOD1G93A)1Gur/J hemizygous male and B6SJLF1/J+/+ female 1:1 hybrid,at a constant temperature, humidity, 12 hour light/ dark cycles, no special pathogens(the Specific pathogen free, SPF)environment. Sterilization of SPF particles rodent feed and sterile water, cut end of the tail to extract DNA, PCR, agarose gel electrophoresis results are shown(Fig. 1): at 200-300 bp of between the bands(236bp) for m SOD1 the PCR products, such mice for SOD1G93 A transgenic mice. This entry with a non-m SOD1 PCR products is non-SOD1G93 A transgenic mice.2 ABC immunohitochemicalstaining for ERβ 2.1 In the transverse section of spinal cord of the SOD1G93 A transgenic mice we showed the presence of ERβ-ir in both the gray and white matter, mainly expressed in nucleus,and more expressed in the gray matter. Dense accumulation of ERβ was observed in the I-III layer of the dorsal horn. ERβwas lightly expressed in spinal cord ventral horn.Similar ERβ expression patterns of were observed in pre-symptomatic stage group.2.2 With the progression of the disease, the number of the ERβ-ir Alpha motor neurons was decreased in the SOD1G93 A transgenic mice.The number in pre-symptomatic stage, symptomatic stage and end-stage was 24.30 ± 1.25、14.40 ± 1.50、3.1. ± 2.28 respectively. There was significant difference between each period( P<0.05).However,in the control group the number was 23.40 ± 3.17、24.00 ± 1.70、22.20 ± 1.32 respectively. There was no significant difference between each period(P>0.05). There were statistical difference between positive groups and control groups.3 Immunofluorescence stainingERβ was expressed densely in the neucleus of Alpha motor neurons,astrocytes and microglias in ventral horn of spinal cord,lightly in cytoplasm of Alpha motor neurons.With the progression of the disease,the number of the ERβ-ir Alpha motor neurons was decreased in SOD1G93 A transgenic mice,and the number of the ERβ-ir Alpha motor neurons was increased in astrocytes and microglias.Conclusion:1 ER beta expressed in motor neurons, astrocytes and microglia in mice anterior horn of spinal cord,mainly in nucleus,meanwhile light expression in motor neurons cytoplasm was observed.2 As the disease progresses, the number of ER beta-ir alpha motor neurons in ventral horn of spinal cord decreased gradually in SOD1G93 A transgenic mice, the reactived astrocytes and microglial cells also expressed ER beta.