| Objective: Amyotrophic lateral sclerosis(ALS) is a devastating neurodegenerative disorder characterized by progressive muscular dystrophy, paralysis, and resulting from the progressive and selective loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem as well as spinal cord, finally died of respiratory failure. At present the pathogenesis of motor neuron degeneration remain unclear, while some epidemiological studies have indicated that there are gender differences in the incidence of ALS. This epidemiologic evidence raises the possibility that estrogen may be responsible for these differences by being neuroprotective in ALS.Moreover evidence supporting a role for estrogen in neuroprotection, estrogen has beneficial effects in a variety of models of neurological disease. Studies have shown that the brain is an additional extragondal site of estrogen synthesis, and it can synthesize estrogen from cholesterol. The expression level of the effective concentration of brain estrogen depends on regional brain aromatase.The report about the behaviours of the ovariectomied SOD1G93 A transgenic mice an animal model of ALS is really rare and the expression of aromatase in spinal cord is unclear. So the purpose of the present study was to observe the onset, behavior and survival of SOD1G93 A transgenic mice after ovariectomy and to determine the distribution of aromatase in the lumbar spinal cord of transgenic mice of different stages in order to study the effects of ovariectomy in a transgenic mouse model of familial amyotrophic lateral sclerosis.Methods1 Animal modelBreeding male familial amyotrophic lateral sclerosis transgenic mice animal models(SOD1G93A). The forty SOD1G93 A mice age of five weeks were randomly divided into three experimental groups(n≧12) :ovariectomized group(OVX), sham operation group(Sham), the positive control group(NO-OVX). The negative control group(CON) were non-transgenic mice have same duration with experimental one.2 Determination of Symptom Onset and Survival End Points of MiceSOD1G93A mice were assessed twice daily for morbidity and mortality. We used 1-5 Vercelli score of the reference standard [Vercelli A et al., 2008] 4 score, when as a time of onset, 1 score, when as a time of death.3 Evaluation of motor functionWe evaluate the motor function through assessment of general condition, evaluation of weight, the rotarod test, hanging wire test and the measurement of step length.4 Immunohistochemical stainingAll the segments were then directly made into 25μm thick sections, then they were used to immunohistostaining for detection of aromatase expression and distribution. Count the number of the aromatase-ir Alpha motor neurons of two sides in the ventral horn in lumbar spinal cords. Standards: aromatase-ir motor neurons located in the anterior horn, d > 25μm, the nucleus are clear. At the same time, using Im-age Pro Plus 6.0 automatic image analysis system, we measured microscope(10x10 times) the range of aromatase, central tube to the absorbance of the anterior horn of spinal cord.And we selected 5 sections of spinal cord in each mouse, recording the integrated optical density calculated value(IOD).5 Statistical AnalysisStatistical analysis was performed using one-way ANOVA or the Mann-Whitney U test followed by Student’s t-test with SPSS 13.0 statistical software. Differences were considered significant at P < 0.05.Results 1 Animal modelSterilization 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 positive transgenic mice. This entry with a non-m SOD1 PCR products is non-SOD1G93 A transgenic mice. 2 Disease onset time and lifespanThe onset time of OVX group was 78.07 + 3.091 days,and 92.50 + 3.907 days of the NO-OVX group, P=0.008. The onset time of Sham group and was 83.50 + 3.598 days compared to 92.50 + 3.907 days of NO-OVX group, P=0.107. Compared with OVX group and Sham group P=0.26(Table.1 and Fig.2a)Compared the survival of OVX group 137.71 + 2.352 and 137.93 + 2.215 days of group NO-OVX P=0.738, the survival of Sham group was 132.08 + 2.932 days comparing with the NO-OVX group P=0.08 showed no statistical difference(Table.1 and Fig.2b). 3 Praxiology 3.1 Evaluation of weightFrom ninth weeks to tenth weeks, there were no statistically significant differences between the four experimental groups. From eleventh weeks to 14 weeks, only OVX group and the CON group mice had significant difference. Compared with the CON group, three positive groups(OVX group and Sham group, NO-OVX group) were statistically significant difference from the fifteenth week( Fig.3 a). 3.2 Hanging wire testFrom ninth to tenth weeks, there were no statistical differences in evaluation of hanging wire test of four experiment groups. From the eleventh week to twentieth week, compared with the CON group, the hanging wire test of the three positive group were statistically significant difference( Fig.3 b). 3.3 Rotarod testFrom ninth to fourteenth weeks, there were no statistical differences in evaluation of rotarod test of four experiment groups. Fifteenth rotarod test time of OVX group and CON group were statistically significant difference. From sixteenth weeks to twentieth week, compared with the CON group, rotarod test time of the three positive groups had statistically significant difference. However, from the beginning to the end,there was no difference between the OVX group and Sham group and no different between the OVX group and NO-OVX group( Fig.3 c). 3.4 Step lengthFrom ninth to fifteenth weeks, there were no statistical differences in evaluation of rotarod test of four experiment groups. From sixteenth weeks to twentieth week, compared with the CON group, step length of the three positive groups had statistically significant difference. However, from the beginning to the end,there was no difference between the OVX group and Sham group and no different between the OVX group and NO-OVX group( Fig.3 d). 4 ABC immunohitochemicalstaining 4.1 Immunohistochemistry showed that: In pre-symptomatic stage, we found the expression of Aromatase-ir in anterior horn of spinal cord of every groups, and mainly expressed in αmotor neurons. While, there is no expression in glial cells. The number of motor neuron counts OVX 20.20 ± 6.603, Sham 20.07 ± 4.877, NO-OVX 17.07 ± 4.978, CON 21.53 ± 5.343, P>0.05(Analysis of variance). IOD showed that: OVX 4904±1190, Sham 6493±2106, NO-OVX 5775±1543, CON 4904±1190, P>0.05(Analysis of variance)(Fig.4). 4.2 In onset stage, the CON group had no obvious changes in aromatase expression. For OVX group, Sham group and NO-OVX group, we found the expression of aromatase both in the motor neurons and glial cells and the number of the Alpha motor neurons was tapered in the ventral horn of the spinal cord. The number of motor neuron counts were OVX 8.27 ± 4.543,Sham 6.87 ± 2.973, NO-OVX 9.33 ± 4.701, CON 18.73 ± 5.063, P>0.05(Analysis of variance). IOD showed that: OVX 4554±1797, Sham 5739±1382, NO-OVX 5933±2002,CON 4841±1965, P>0.05(Analysis of variance)(Fig.5). 4.3 In the end stage, Aromatase in group CON still expressed in alpha motor neuron in anterior horn of spinal cord. The number of the Alpha motor neurons of OVX group, Sham group and NO-OVX group was tapered in the ventral horn of the spinal cord, to the end few Alpha motor neurons were observerd. At the same time we found the increased expression of glial cells. The number of motor neuron counts were OVX 0.6 ± 0.737, Sham 3.13 ± 1.846,NO-OVX 2.20 ± 1.568,CON22.53 ± 3.642. Compared with the other three groups,the number of motor neuron of OVX group was different(P<0.05). IOD showed that: OVX 9502±2243, Sham 8072±2543,NO-OVX 8216±2002 CON 4740 ± 1511. There was statistically significant differences between the ovariectomized group and the sham operation group(P=0.007). The same as the ovariectomized group and the positive control group(P=0.02). There were no statistically significant differences between the sham group and the positive control group(Fig.6).Conclusion1 Ovariectomy could alter the onset age of the disease but there was no obvious influence to the survival and movement function of SOD1-G93 A transgenic mice.2 Mutation of SOD1G93 A gene can induce expression of glial and aromatase in the neurons of spinal cord.3 Ovariectomy may increase the synthesis of local estrogen in spinal cord of SOD1G93 A transgenic mice. |
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