Studies Of The Effects Of Short-term Estrogen Replacement Therapy On Behaviors And White Matter Of Middle-aged Ovariectomized Rats

The stereological studies have demonstrated that age-related decline inhuman brain volume was mainly due to a loss of myelinated fibers with asmall diameter in the white matter. Numerous studies from postmenopausalwomen have revealed beneficial effects of estrogen replacement therapy（ERT） on cognition and brain structure, such as the increase of whitematter volume. Whether the ERT could protect the white matter and themyelinated fibers in the white matter to delay the ageing process of brainremains unknown. The middle-aged ovariectomized (OVX) rat was used asan animal model to mimic the low estrogen condition of the menopause inwomen. After one month ERT to middle-aged OVX rats, we used Morriswater maze test to investigate the behavioral difference between the ERTgroup rats and vehicle therapy group rats. Then, we applied thestereological methods and transmission electron microscope technique toinvestigate the white matter and the myelinated nerve fibers in the white matter and analyzed the relationship between changes of behaviors and thewhite matter morphology. The stereological methods provided in thecurrent study will provide useful tools for the future studies on the effectsof ERT on the demyelination/remyelination in normal aging and diseasemodels.Part One1. Materials and methods1.119female middle-aged (10-12months) rats were bilaterallyovariectomized. One month after ovariectomy, the rats were randomlydivided into estrogen replacement therapy (OVX+E) group (n=10) andvehicle replacement therapy (OVX+Veh) group (n=9). The OVX+E ratswere given a one-month long daily s.c.injection of17β-estradiol diluted insesame oil at nape of the neck. Meanwhile, OVX+Veh rats received aone-month long daily s.c.injection of sesame oil as control. To ensure thecomplete removal of all ovarian tissues and effectiveness of the ERT, theserum estrodial levels were detected after ovariectomized and afterreplacement, the uterus from each animal was examined at the time ofsacrifice.1.2One month after replacement, all the rats received the Morris watermaze test. 2. Results2.1Compared with the mean serum estradiol level of OVX rats and theOVX+Veh rats, the mean serum estradiol level of OVX+E rats wassignificantly higher (P<0.05). The mean serum estradiol levels of OVX ratsand the OVX+Veh rats were not significantly different.2.2The mean weight of the uteruses from OVX+Veh rats was significantlylower than the uteruses from OVX+E rats (P<0.05).2.3In the Morris water test, the mean escape latencies of OVX+E rats fromday1to5were significantly shortened when compared to those ofOVX+Veh rats (P<0.05). For visible-platform task on day6, there was nosignificant difference between the escape latency of OVX+Veh rats andOVX+E rats.Part two1.Materials and methods1.1After the behavioral test,5rats were randomly chosen from each group.Each hemisphere was then coronally cut into serial1-mm thick parallelslabs, starting randomly at the rostral pole. The cut slabs werephotographed under anatomical microscope with a magnification of10.The white matter volume was calculated according to the Cavalieri’sprinciple. 1.25tissue blocks were randomly sampled from white matter in asystematic random fashion. One section with the thickness of60nm wascut from each sampled block. From each cut section,8fields of view wererandomly photographed under transmission electron microscope with amagnification of6000.1.3The stereological methods were used to quantitatively investigate thetotal length and total volume of the myelinated fibers, the total volume ofthe myelin sheaths and the mean diameter of the myelinated fibers in thewhite matter of the OVX+Veh rats and OVX+E rats.2.Results2.1There was no significant difference in the white matter volume betweenOVX+Veh rats and OVX+E rats (P>0.05).2.2There were no significant differences in the total length and totalvolume of the myelinated nerve fibers in the white matter between twogroups (P＞0.05).2.3There was no significant difference in the total volume of the myelinsheaths in the white matter between two groups (P＞0.05).2.4There was no significant difference in the mean diameter of themyelinated nerve fibers in the white matter between two groups (P＞0.05).2.5There was no significant difference in the total length of the myelinatednerve fibers with the axon diameter of0~1.8μm in the white matter between two groups (P＞0.05).General conclusions1. The present study demonstrated that ERT could improve the spatialmemory of the middle-aged OVX rats.2. Combining transmission electron microscope technique withstereological methods, we quantitatively investigated the white matter andthe myelinated nerve fibers in the white matter of OVX+Veh rats andOVX+E rats. Our results would provide important reference values for thefuture related studies.3. There were no significant differences in the white matter volume, thetotal volume and the total length of the myelinated nerve fibers, the totalvolume of the myelin sheaths and the mean diameter of the myelinatedfibers in the white matter between OVX+Veh rats and OVX+E rats. Therewas no significant difference in the total length of the myelinated nervefibers with the axon diameter of0~1.8μm in the white matter between twogroups.