Stereological Study Of The Bilateral Difference In The Age-Related Changes Of The White Matter And The Myelinated Nerve Fibers Therein And The Age-Related Changes Of The Oligodendrocytes In The White Matter Of Rat Brain

It has been generally believed for several decades that there is significant death of the neurons in the cortex of aged brain. However, using the new stereological methods, Denmark scientists found that there was no significant death of the neurons in the cortex of aged human brain, but there was significant decline of the white matter volume in the aged human brain. Tang et al found that the total length of the myelinated nerve fibers in the white matter of aged human brain was significantly decreased when compared to that of young brain, maily due to the general loss of the the myelinated fibers with small diameter. To exclude the possibility that the cases of the early AD might be used in our human studies, our research team used the rats which could not develop AD to study the age-related changes of white matter and confirmed the findings in the human brains. Since the functions of the the left and right hemispheres are different, there might be bilateral difference of white matter and myelinated nerve fibers therein between the left and right hemispheres, and there might be bilateral differences in the age-related changes of white matter and myelinated nerve fibers therein between the left and right hemispheres. Until now, however, there has been no study of the bilateral differences in the age-related changes of the myelinated nerve fibers in white matter in the left and right hemispheres with the new stereological methods. In order to explore if there are significant bilateral differences in the white matter and the myelinated fibers between the left and right hemispheres of rats and if the age-related changes of the white matter and the myelinated fibers in the white matter are the same in both hemispheres, we used transmission electron microscope (TEM) and stereological methods to investigate the total length, total volume and mean dimeter of the myelinated nerve fibers in the white matter. Oligodendrocytes are the neuroglial cells responsible for myelin synthesis that are important for the condition of nervous impulse. However, there has been no study of the age-related changes of the olioligodendrocyte number in white matter. Therefore, we investiiged the age-related changes of the total number of the oligodendrocytes in rat white matter with immunohisochemical technique and stereological method.PARTⅠ1. Materials and methods 1.1 Five young male Long-Evans rats (6-month old) and four aged male Long-Evans rats (18-month old) were used.1.2 The rats were anaesthetized and perfusion-fixed. The cerebrum was taken out. The two hemispheres were embedded in agar. Each hemisphere was coronally cut into 2 mm-thick slabs. The white matter volume was calculated according to the Cavalieri's principle. One hemisphere was randomly sampled from each rat brain. From the sampled hemisphere, 4 tissue blocks were randomly sampled from the all tissue blocks. One ultra-thin section was cut from each sampled block. From each cut section, 4 fields of view were randomly photographed under TEM with a magnification of 6000.1.3 The white matter volume, the total length, total volume and mean diameter of the myelinated nerve fibers in the white matter were investigated with the new stereological methods.2. Results2.1 There were no significant differences of the white matter and the mylinated nerve fibers in the white matter between the left and right hemispheres in both young and aged groups (p>0.05).2.2 Although the white matter volume, the total length and the total volume of the myelinated nerve fibers in the white matter were reduced in both left hemisphere and right hemisphere, only the white matter volume and the total volume of the myelinated fibers in the white matter of right hemisphere and the total length of the myelinated fibers in the white matter of left hemisphere were significantly decreased by 32.9%, 28.6% and 49.3%, respectively (p<0.05). PARTⅡ1. Materials and methods1.1 Five young male Wistar rats (6-month old) and four aged male Wistar rats (27-month old) were used.1.2 The rats were deeply anesthetized with 4% chloralhydrate(10 ml/kg)by an introperitoned injection and perfused with 0.9% NaCl followed by fixation with 4% paraformaldehyde in 0.1M PBS (pH 7.4). The brain was taken out and divided into two hemispheres along the cerebral longitudinal fissure. The hemispheres were immerged in 4℃30% sucrose PBS solution and taken out until it sank. The brain were embedded in the Optimal Cutting Temperature (O.C.T Tissue-Tek) half an hour, quickly frozen in -196℃liquid nitrogen and stored at -80℃until being used.1.3 Consecutive 30μm coronary sections from rostral pole to caudal pole were cut on a cryostat. Sections were sampled in a systematic random fashion. One section was sampled systematically every 20th section, the first one was randomly sampled between 1 and 20. On average, 17 sections were sampled from the total sections of each rat hemisphere.1.4 The sampled sections were stained with anti-CNPase. Nucleuses were stained with haematoxylin. 1.5 The section area sampling fraction and section thickness sampling fraction were set in the stereological equipment. The number of anti-CNPase positive oligodendrocytes was counted .1.6 The total number of the anti-CNPase positive oligodendrocytes in the white matter of each hemisphere was calculated with the stereological methods.2. Results2.1 There was no significant difference of the total number of the anti-CNPase positive oligodendrocytes between the left and right hemispheres both in young and old groups (p>0.05).2.2 The total number of the anti-CNPase positive oligodendrocytes in the left white matter was significantly reduced from 7.13×106 in the young group to 4.53×106 in the old group by 36.5% (p<0.01). The total number of the anti-CNPase positive oligodendrocytes in the right white matter was significantly reduced from 7.14×106 in the young group to 4.55×106 in the old group by 36.0% (p<0.01).2.3 The total number of the CNPase positive oligodendrocytes in the white matter was 14.2×106 in the young group, and 9.68×106 in the old group. When compared to young rats, the total number of the anti-CNPase positive oligodendrocytes in the white matter of aged rats was significantly decreased by 32.1% (p<0.01).General conclusions 1.The present study combined the new stereological techniques and transmission electron microscope technique to quantitatively study the bilateral differences in the age-related changes of the white matter and the myelinated nerve fibers between the the left and right male rat hemispheres. The present study provided the normal data of the white matter volume, the total length, total volume and mean diameter of the myelinated nerve fibers in the both left and right white matter of male Long-Evans rats. These data will provide the important baseline values for future studies.2. The present results indicated that there were no significant bilateral differences in the white matter volume, the total length, total volume and mean diameter of the myelinated nerve fibers in the white matter of young and aged rats.3. The present results indicated that the age-related changes of the white matter in left hemisphere were different from those in right hemisphere. The white matter volume and the total volume of the myelinated nerve fibers in the right hemisphere were significantly decreased during aging, while the total length of the myelinated nerve fibers in the left hemisphere was significantly decreased.4. There was no significant bilateral difference in the number of the oligodendrocytes in the left and right white matter of both young and old groups. The present study suggested that the total number of the oligodendrocytes in the whole white matter, the total number of the oligodendrocytes in the white matter of the left and right hemispheres were significantly decreased during aging..