Stereological Study Of The White Matter And The Myelinated Nerve Fibers In The White Matter Of Rat Brain

Objective: It was generally believed for several decades that there was 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. We for the first time in the world 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 subjects. An effort was made to ensure the group of brains did not include those from people with Alzheimer's disease(AD). However, it is very difficult to exclude the brains from people with early stages of AD. The failure to address the issue of the possible presence of early stages of AD cases in the sample is a serious oversight that confounds the issue of"normal aging". Aged rats do not develop AD. In order to exclude the cases of early AD that confounds the issue of normal aging, the present study further investigated the age-related changes of the white matter and the myelinated nerve fibers in the white matter of Long-Evans rats by means of the new stereological techniques and the transmission electron microscope technique. PARTⅠ1. Materials and methods1.1 6～8 month Long-Evans rats, 5 females and 5 males, were used.1.2 One hemisphere was sampled from each rat brain. From the sampled hemisphere, 4 white matter blocks were sampled in a systematic random fashion. One ultra-thin section was cut from each sampled block. From each cut section, 4 fields of view were randomly photographed under TEM, with the magnification of 6000.1.3 In both female and male rats, the white matter volume, the length density and total length of the myelinated nerve fibers in the white matter, the mean diameter of the myelinated nerve fibers in the white matter, the volume density and total volume of the myelinated nerve fibers in the white matter, and volume density and total volume of the myelin sheathes in the white matter were investigated with the new stereological methods.2. Results2.1 The volume of the white matter of female rats was 119.57±7.52 mm3, and the volume of males was 173.22±21.03 mm3. The difference was 44.9% (P<0.05).2.2 The total length of the myelinated nerve fibers in the white matter of female rats was 129.26±28.78 km, and the total length of males was 190.79±73.49 km. The difference between the two groups was 47.7% (P>0.05), with no statistical significance.2.3 The mean diameter of the myelinated nerve fibers in the white matter of female rats was 0.56±0.03μm, and the mean diameter of males was 0.53±0.04μm. The difference was 4.3% (P>0.05), with no statistical significance.2.4 The total volume of the myelinated nerve fibers in the white matter of female rats was 58.13±9.34 mm3, and the total volume of males was 80.66±10.15 mm3. The difference was 38.7% (P< 0.01).2.5 The total volume of the myelin sheaths in the white matter of female rats was 26.83±3.62 mm3, and the volume of males was 36.92±6.62 mm3. The difference was 37.6% (P< 0.01). PARTⅡ1. Materials and methods1.1 6～8 month Long-Evans rats (5 females and 5 males) and 18month Long-Evans rats (5 females and 4 males) were used.1.2 One hemisphere was sampled from each rat brain. From the sampled hemisphere, 4 white matter blocks were sampled in a systematic random fashion. One ultra-thin section was cut from each sampled block. From each cut section, 4 fields of view were randomly photographed under TEM, with the magnification of 6000. 1.3 In both young and old rats, the white matter volume, the length density and total length of the myelinated nerve fibers in the white matter, the mean diameter of the myelinated nerve fibers in the white matter, the volume density and total volume of the myelinated nerve fibers in the white matter, and volume density and total volume of the myelin sheathes in the white matter were investigated with the new stereological methods.2. Results2.1 The volume of the white matter and the total length of the myelinated nerve fibers in the white matter were decreased from 133.34±20.79 mm3 and 160.02±61.81 km in young rats to 101.14±13.73 mm3 (24.1%, P<0.01) and 94.43±47.75 km (41%, P<0.05) in old rats, respectively. The volume density of the myelinated nerve fibers and the myelin sheathes, and the mean diameter in old rats were increased by 30% (P<0.01), 23.9% (P<0.05) and 31% (P<0.01), respectively, compared to young rats. The declines of the length density of the myelinated nerve fibers, the total volume of the myelinated nerve fibers and the total volume of the myelin sheathes were not statistically significant.2.2 The declines of the white matter volume, the length density and total length of the myelinated nerve fibers in the white matter of 18 month female rats were not statistically significant. The volume density and total volume of the myelinated nerve fibers in the white matter, the volume density and total volume of the myelin sheathes, and the mean diameter were significantly increased by 41.7% (P<0.01), 29.7% (P<0.01), 42.6% (P<0.05), 30.0% (P<0.05) and 21.6% (P<0.05), respectively, in old female rats compared to young female rats.2.3 The volume of the white matter, the total length of the myelinated nerve fibers, the total volume of the myelinated nerve fibers and the myelin sheathes, in the white matter, were significantly decreased by 38.8% (P<0.01), 66.8% (P<0.05), 38.1% (P<0.01) and 35.2% (P<0.05), respectively, while the mean diameter was significantly increased by 42.6% (P<0.01) in old male rats compared to young male rats. The length density and volume density of the myelinated nerve fibers, and the volume density of the myelin sheathes, in the white matter of aged male rats, were not significantly changed when compared to young male rats.General Conclusions1. The present study for the first time in the world combined the new stereological techniques and transmission electronic microscope technique to quantitatively study the sex differences and the age-related changes of the white matter and the myelinated nerve fibers in the white matter of rat brain.2. The results for the first time indicated that there was sexual dimorphism in the white matter volume, the total volume of the myelinated nerve fibers and the total volume of the myelin sheathes in the white matter of normal adult Long-Evans rats. 3. The present results confirmed our previous findings in human study, that is, there was age-related white matter atrophy, which was primarily caused by the decline of the total length of the myelinated nerve fibers with thinner diameters in the white matter.4. The present study showed that there was difference in the age-related white matter changes between male and female rats and that the age-related decline of the total length of the myelinated nerve fibers in the white matter of male rats was much early than female rats.5. 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 white matter of Long-Evans rats. These data will provide the important baseline value to compare with future studies of the effects of the neurodegenerative diseases in CNS on the white matter and the myelinated nerve fibers in the white matter of this animal model.