The Androgen Receptor Expression Characteristics of Cells in Anterior Horn of the Lumbar Spinal Cord of Family Amyotrophic Lateral Sclerosis Animal Model
|School||Hebei Medical University|
|Keywords||anterior horn of spinal cord androgen receptor amyotrophiclateral sclerosis SOD1mutations immunohistochemistry immunofluoresce-nce|
Objective: Amyotrophic lateral sclerosis(ALS) is a neurodegenerativedisease with unknown etiology, which affects motor cortex neurons, brainstemmotor nuclei neurons, and spinal anterior horn a-motor neurons，causingprogressive muscular weakness, atrophy, finally died of respiratoryfailure.There are many epidemiological risk factors, such as exposure toheavy metals, electrical or mechanical trauma, vigorous athletic activity,which have influence on occurrence of ALS. But there are also two widelyaccepted risk factors: age and sex. The study found that the overallincidence was greater in men than in women across all age groups(ratio of3.98for onset at age <49years and1.63for onset at age>55years). The mean age at onset was younger in men than inwomen. The onset of disease was different for the two sexes: significantlyearlier in male than in female hSOD1mice, the survival time was greater infemale than in male hSOD1mice. Gender is probably the risk factor ofamyotrophic lateral sclerosis(ALS), sex hormone may play a role in theneuron degeneration and loss in the pathogenesis process of amyotrophiclateral sclerosis(ALS).Another study found that men have a greater likelihood of onset in thespinal regions, and women tend to have onset in the bulbar region.Kennedy’s disease is a kind of X-ray chain motor neuron disease caused byandrogen receptor gene mutations. The disease mainly influence the motorneurons of the segment of high cervical spinal cord and medulla oblongata,commonly onset in upper limb.For family amyotrophic lateral sclerosisanimal model (SOD1G93A transgenic mice),the clinical phenotype ischaracterized by an adult onset of motor symptoms in the hind limbs around 12weeks of age, which progresses to end stage by17-20week.Now theandrogen receptor (AR) expression in the spinal cord of ALS mice wasreported rarely.The purpose of the present study is to observe the expression anddistribution of androgen receptor of the lumbar spinal cord anterior horn cellsby using immunofluorescence and to count the number of motor neurons ofthe lumbar spinal cord anterior horn and to observe the expression of androgenreceptor of motor neurons by using immunohistochemical of family amyo-trophic lateral sclerosis animal model (SOD1G93A transgenic mice). Thisexperiment aims to explore the role of androgen receptor in the pathogenesisof amyotrophic lateral sclerosis(ALS) in order to providing certain theoreticalbasis for amyotrophic lateral sclerosis(ALS) pathogenesis research.Methods:1Animal modelB6SJL-BTg（SOD1G93A）1Gur/J hemizygous male and B6SJLF1/J+/+female1:1hybrid,at a constant temperature, humidity,12hour light/darkcycles, no special pathogens (the Specific pathogen free, SPF)environment.Sterilization of SPF particles rodent feed and sterile water, cut end of the tailto extract DNA, PCR, agarose gel electrophoresis results are shown (Fig.1): at200-300bp of between the bands (236bp) for mSOD1the PCR products, suchmice for SOD1G93A positive transgenic mice. This entry with a non-mSOD1PCR products is non-SOD1G93A transgenic mice.Reference Vercelli Adescription，4score as diease onset stage,1scoreas end stage. SOD1G93A mice of60days as pre-symptomatic stage.Controlgroups for the experiment were non-transgenic mice in the same nest and havesame duration with experimental one,3mice of male and female in eachgroup.2MaterialAfter10%chloral hydrate350mg/kg intraperitoneal injection of anesthe-sia, animal tissues were fixated via heart perfusion by4%paraformaldehydefor20min, the lumbar spinal cord of the mice were cut，all the blocks were fixated in4%paraformaldehyde for48hour.3ImmunofluorescenceAll the segment were directly made into25μm thick sections, using acoordinate immunostaining approach to determine the location of AR in theanterior horn cells of the lumbar spinal cord of the mice under a confocal laserscanning microscope.4Immunohistochemical stainingAll the segment were directly made into25μm thick sections,then theywere used to immunohistostain of SMI-32and AR applying ABC method tocount the number of motor neurons of spinal cord anterior horn and observethe expression of AR of motor neurons. Count the motor neurons in the fifthsection of every serial sections，observe10sections in each segment.Standards: AR-ir motor neurons located in the anterior horn, d＞25μm,the nucleus are clear.5Statistical analysisThe mean numbers of neurons and standard deviations were calculatedfor sum of two sides in the anterior horn in the lumbar spinal cords.Statistical analysis were performed using one-way ANOVA followed byStudent’s t-test with SPSS13.0statistical software. Differences wereconsidered significant at P <0.05.Results:1Animal modelResults are shown (Fig.1): at200-300bp of between the bands (236bp)for mSOD1the PCR products, such mice for SOD1G93A positive transgenicmice. This entry with a non-mSOD1PCR products is non-SOD1G93Atransgenic mice.2ImmunofluorescenceAndrogen receptor was expressed in the motor neurons of the lumbarspinal cord of SOD1G93A transgenic mice and was located in the cytoplasm.Androgen receptor was not expressed in the astrocytes and the microglial cellsof the lumbar spinal cord of SOD1G93A transgenic mice.The control group was the same.3Immunohistochemical staining（SMI-32）Cell counts of in the lumbar spinal cord of SOD1G93A transgenic malemice of different stage were20.672.92、14.732.84、8.472.72.P valueswere0.000,0.000,0.000comparing with each group and the differences wereall statistically significant.Cell counts of in the lumbar spinal cord ofSOD1G93A transgenic female mice of different stage were20.603.20、17.473.40、8.473.44.P values were0.015,0.000,0.000comparing witheach group and the differences were all statistically significant.Comparingbetween male and female mice, P values were0.953,0.024,1.000and onlythe difference in the onset stage was statistically significant.4Immunohistochemical staining（AR）4.1Agreeing with the results of immunofluorescence, androgen receptorwas expressed in the motor neurons of the lumbar spinal cord of SOD1G93Atransgenic mice and was located in the cytoplasm.The color of cytoplasm wasuniform, not presenting sample particle deposition along with the progress.The control group was the same.4.2Cell counts of in the lumbar spinal cord of SOD1G93A transgenic malemice of different stage were20.871.81、14.931.67、8.671.23.P valueswere0.000,0.000,0.000comparing with each group and the differences wereall statistically significant.Cell counts of in the lumbar spinal cord ofSOD1G93A transgenic female mice of different stage were20.801.97、17.672.13、8.671.59.P values were0.000,0.000,0.000comparing witheach group and the differences were all statistically significant.Comparingbetween male and female mice, P values were0.924,0.001,1.000and onlythe difference in the onset stage was statistically significant.5The comparison between immunohistochemical(SMI-32) and immunohisto-chemical (AR)For SOD1G93A transgenic male mice of different stage，comparingbetween SMI-32-positive neurons and AR-positive neurons，P values were0.823,0.816,0.798and the differences were all not statistically significant. For SOD1G93A transgenic female mice of different stage，comparing betweenSMI-32-positive neurons and AR-positive neurons，P values were0.839,0.848,0.840and the differences were all not statistically significant.Conclusion:1The lumbar spinal cord anterior horn cells of normal mice：Androgenreceptor was expressed in the motor neurons and was located in thecytoplasm and was not expressed in the astrocytes and the microglial cells.2The lumbar spinal cord anterior horn cells of SOD1G93A transgenicmice：Androgen receptor was expressed in the motor neurons and waslocated in the cytoplasm and was not expressed abnormally in the astrocytesand the microglial cells.3The androgen receptor of the lumbar spinal cord anterior horn cells ofSOD1G93A transgenic mice was reduced gradually，which was associatedwith the reducing of the number of motor neurons in the progression.