| Stereological studies showed that there was a significant decline in thewhite matter volume of aged brain, and the age-related white matter changewas mainly due to the decline of the total length of the myelinated fibers inthe white matter. Previous studies showed that there was cognitive deficitsin aged brain. Postmenopausal women will accelerate the process ofage-related cognitive decline and increase the risk of developingAlzheimer’s disease (AD). The structural abnormalities of myelinated fibersand myelin sheaths might be one of important causes for the age-relatedcognitive decline. Central nervous system is a target organ of estrogen, and alarge number of studies have shown that estrogen could improve thecognitive ability and spatial reference memory of brain. Estrogenreplacement therapy could improve the brain structures including whitematter in postmenopausal women. Long-term estrogen replacement therapy could protect the white matter loss of aged brain. Therefore, there might beclose relationships among the estrogen, brain aging and myelin sheaths ofthe myelinated fibers in the white matter. In this experiment, middle-agedfemale Sprague-Dawley (SD) rats were underwent bilateral oophorectomy,which was designed to simulate the conditions of structural abnormalities ofthe myelinated fibers and myelin sheaths in the white matter within the stateof estrogen deficiency in postmenopausal women. Transmission electronmicroscope technique and the stereological techniques were used to studythe effects of estrogen replacement therapy on the white matter and themyelin sheaths ultrastructure of the myelinated fibers in the white matter, inorder to understand the effects of estrogen on the white matter of aged whitematter and demyelinating diseases. The results of this experiment willprovide a scientific basis for slowing the process of brain aging andpreventing and treating the demyelinating disease with estrogen replacementtherapy in the future.Part One1.Materials and methods1.110~12month middle-aged female Sprague-Dawley rats (40rats)were bilaterally ovariectomized, and one month after surgery, the serumestrogen levels were examined. The rats were injected intraperitoneally with 0.4ml/100g in a concentration of1%sodium pentobarbital for anesthesiabefore surgery, and then the rats were bilaterally ovariectomized. Aftersurgery, each rat was injected penicillin in a dose of units daily for threeconsecutive days in order to anti-infect.19rats whose serum estrogen levelswere around11pg/ml were screened out as successful ovariectomy model tosimulate menopausal conditions in women.1.2The female Sprague-Dawley rats (19rats) with successful castrationthose serum estrogen levels was about11pg/ml. These rats were randomlydivided into experimental group and control group, and were separatelygiven estrogen and placebo treatment. Four weeks after treatment, the serumestrogen levels of19rats were again detected with chemiluminescence.1.3The spatial reference and learning memory capacities of two grouprats were tested with Morris water maze after four weeks.1.4After the Morris water maze test, six rats were randomly selectedfrom the experimental group, and five rats were randomly selected from thecontrol group. These rats were successively anesthetized, perfused and fixed.Then these rat brains were removed.1.5Along the coronal line, the right and left hemispheres of brain werecut into the tissue sections with the thickness of1mm. One hemisphere wasrandomly selected out from each rat. To ensure that all parts of the whitematter had the same possibility to be sampled, the grid with points was randomly put on the tissue sections of brain, and the white matter tissueblocks were sampled for the places where the points hit white matter. Onaverage, five electron microscope specimens were sampled from each rat. Aultrathin section with the thickness of60nm was cut from each specimen.Under the magnification of20000,12fields of vision were randomlyselected, and the images were captured.60photos in total were obtainedfrom each rat.1.6The related stereological parameters were calculated with thestereological methods. These parameters included the total volume of whitematter, length density of myelinated fibers, total length of myelinated fibers,volume density of myelinated fibers, total volume of myelinated fibers, thevolume density of myelin sheaths, the total volume of myelin sheaths, innerperimeter of myelin sheaths, outer perimeter of myelin sheaths, thedifference between outer perimeter and inner perimeter of myelin sheaths,the ratio of the inner perimeter and outer perimeter of myelin sheaths,thickness of myelin sheaths, arithmetic mean diameter of myelinated nervefibers, arithmetic mean diameter of axons, the difference between arithmeticmean diameter of myelinated fibers and arithmetic mean diameter of axons,the ratio of arithmetic mean diameter of axons and arithmetic mean diameterof myelinated fibers in the white matter of rats in two groups. According tothe different diameter ranges of the myelinated fibers in the white matter, the stereological parameters of the myelinated fibers in the white matter werestatistically compared.2.Results2.1The results of estrogen levels: The estrogen levels detected in serumof two group rats after surgery were not significantly different (P>0.05).The estrogen levels detected in the serum of two group rats after estrogenreplacement therapy were significantly different (P <0.01).2.2The results of Morris water maze test: When compared to the controlrats, the spatial reference and learning memory capacities of theexperimental rats was significantly improved (P <0.01).2.3The stereological results: The related stereological parameters hadno significant difference (P>0.05). These parameters included the meanvolume of white matter, length density of myelinated nerve fibers, totallength of myelinated nerve fibers, volume density of myelinated nerve fibers,total volume of myelinated nerve fibers, arithmetic mean diameter ofmyelinated nerve fibers, arithmetic mean diameter of axons, the differencebetween arithmetic mean diameter of myelinated nerve fibers and arithmeticmean diameter of axons, the ratio of arithmetic mean diameter of axons andarithmetic mean diameter of myelinated nerve fibers in the white matter ofrats in two groups. The total length of myelinated nerve fibers in the mean diameter of1.2~1.4μm in experimental group was significantly increasedby46.1%(P <0.05) when compared to the control group. Compared withthe control group, the difference between the mean diameter of myelinatedfibers and the mean diameter of axons in the mean diameter of0~0.4μm inthe experimental group was significantly increased by21.6%(P <0.05).Part Two1.Materials and Methods1.1The related stereological parameters were calculated with thestereological methods. These parameters included the volume density ofmyelin sheaths, the total volume of myelin sheaths, inner perimeter ofmyelin sheaths, outer perimeter of myelin sheaths, the difference betweenouter perimeter and inner perimeter of myelin sheaths, the ratio of the innerperimeter and outer perimeter of myelin sheaths, thickness of myelin sheathsin the myelinated nerve fibers in the white matter in the experimental ratsand the control rats.1.2The correlation between the stereological parameters and thebehavioral parameters of the Morris water maze was statistically analyzedwith bivariate correlation analysis.2.Results2.1Compared with the control rats, the total volume of the myelin sheaths in the myelinated nerve fibers in the white matter in the experimentalrats was significantly increased by27%(P <0.05). The difference betweenouter perimeter and inner perimeter of myelin sheaths was significantlyincreased by12.6%(P <0.05).2.2The volume density of myelin sheaths, inner perimeter of myelinsheaths, outer perimeter of myelin sheaths, the ratio of the inner perimeterand outer perimeter of myelin sheaths, thickness of myelin sheaths ofmyelinated fibers in the white matter in two group rats were not significantlydifferent (P>0.05).2.3Compared with the control group, the difference between the meandiameter of myelinated fibers and the mean diameter of axons in the meandiameter of0~0.4μm in the experimental group was significantly increasedby21.6%(P <0.05). Compared with the control group, the differencebetween the inner perimeter and outer perimeter of myelin sheaths ofmyelinated fibers in the mean diameter of0.4~0.6μm in the experimentalgroup was significantly increased by20.7%(P <0.05). The differencebetween the inner perimeter and outer perimeter of myelin sheaths ofmyelinated fibers in the mean diameter of0.6~0.8μm in two groups hadsignificant difference in trend (P=0.058), and the difference in theexperimental group was non-significantly increased by14.3%, whencompared to the control group. 2.4The total myelin sheath volume of myelinated fibers in the whitematter was significantly and negatively correlated with the escape latency onthe first day of Morris water maze and the escape latency on the fourth day ofMorris water maze (P <0.05).General Conclusions1. Estrogen replacement therapy had positive protective effects on thespatial reference and learning memory capacities of the middle-agedovariectomized rats.2. The basic data on the white matter were obtained. These data includedthe mean volume of white matter, length density of myelinated fibers, totallength of myelinated fibers, volume density of myelinated fibers, totalvolume of myelinated fibers, the volume density of myelin sheaths, the totalvolume of myelin sheaths, inner perimeter of myelin sheaths, outerperimeter of myelin sheaths, the difference between the inner perimeter andthe outer perimeter of myelin sheaths, the ratio of the inner perimeter andouter perimeter of myelin sheaths, thickness of myelin sheaths, arithmeticmean diameter of myelinated fibers, arithmetic mean diameter of axons, thedifference between arithmetic mean diameter of myelinated fibers andarithmetic mean diameter of axons, the ratio of arithmetic mean diameter ofaxons and arithmetic mean diameter of myelinated fibers in the white matterof two group rats, which would provide a scientific basis for delaying the process of brain aging and combating demyelination diseases with estrogenreplacement therapy in the future.3. The present study indicated that the total volume of myelin sheathsand the difference between the inner perimeter and the outer perimeter ofmyelin sheaths in the experimental rats were significantly increased whencompared with the control rats.4. The present study indicated that the total length of myelinated fibers inthe mean diameter of1.2~1.4μm in the experimental group wassignificantly increased when compared to the control group. The differencebetween the mean diameter of myelinated fibers and the mean diameter ofaxons in the mean diameter of0~0.4μm in the experimental group wassignificantly increased compared to the control group. Compared to thecontrol group, the difference between the inner perimeter and the outerperimeter of myelin sheaths of myelinated fibers in the mean diameter of0.4~0.6μm in the experimental group was significantly increased. Thedifference between the inner perimeter and the outer perimeter of myelinsheaths of myelinated fibers in the mean diameter of0.6~0.8μm in twogroups had significant difference in trend. These findings demonstrated thesignificant increase of the total volume of myelin sheaths of myelinatedfibers in the white matter of experimental group.5. The present study found that the total myelin sheath volume of the myelinated fibers in the white matter was significantly correlated with theescape latency on the first day of Morris water maze and the escape latencyon the fourth day of Morris water maze. |
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