Age-Related Changes In The Myelinated Nerve Fibers And MBP In The Corpus Collusm Of Rat Brain

Aging is an inevitable physiological process, which causes many social problems. Aging of brain has been paid the most attention. 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, Pakkenberg and Gunderson found that there was no significant loss of cortical neurons but the white matter volume of the aged human brain was decreased by 28%. The results have been confirmed by a lot of other reseachers. Since the different areas in white matter are related to different functions of brain, it is necessary to investigate the relationship between the age-related changes in the special area of white matter and the function decline in aging brain. The corpus callosum ( CC ) is the commissural fibers linking the corresponding areas of the cerebral cortex in two hemispheres, and the anatomy and function of which are known. Therefore, the CC has got more and more attentions from researchers. Many previous studies with imaging techniques found that there was the atrophy of the CC in normal aging and abnormal aging. They speculated the atrophy of the CC was possibly due to the loss of fiber bundless. Those studies indicated that nerve fibers, especially the myelinated nerve fibers, in CC were important for the nerve impulse conduction between the corresponding lobes of cortex. Therefore, changes of nerve fibers in CC may be one of the structural foundations for the changes of brain cognitive functions. However, all previous studies did not precisely quantitate the nerve fibers in CC. One of the reasons for this is the lack of the methods to quantitate the nerve fibers in CC that were irregularly arranged in three dimensions. Tang et al. for the first time used new stereological methods to quantitatively investigate the white matte fibers and provided an useful methods for three-dimensional quantitation of nerve fibers. In this study, we used TEM and new stereological methods to study the age-related changes of CC and myelinated nerve fibers in CC of Long-Evans rats. Moreover, many studies have showed that there were certain myelin protein changes together with the damage and loss of myelinated nerve fibers, which were related to the decline of cognition. MBP is one kind of special myelin proteins in CNS, and it is of importance for the generation and formation of the myelin sheaths. In this study, we used Western-blot to study age-related change of MBP in CC and the relationship between the changes in the MBP and the myelinated nerve fibers of rat CC.PARTⅠ1. Materials and methods1.1 Five young (7 months), five middle-aged (18 months) and six old-aged (27 months) female Long-Evans rats were used. Body weight is 280±20 g.1.2 One hemisphere was randomly sampled from each rat brain. From the sampled hemisphere, 4 blocks were sampled from CC 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. There were 24 photographs from CC of each rat.1.3 In each female rat, the CC volume, the length density and total length of the myelinated nerve fibers in CC, the volume density and total volume of the myelinated nerve fibers in CC, the volume density and total volume of the myelin sheaths in CC and the mean diameter of the myelinated nerve fibers in CC were investigated with the new stereological methods.2. Results2.1 Demyelination changes, including double myelin sheathes, lamellar separation and vacuolization, were evidently observed in CC of old-aged group.2.2 The volume of CC was 70.7±5.6 mm3, 63.2±4.2 mm3 and 51.6±2.6 mm3 in young, middle-aged and old-aged groups, respectively. Compared to young group, the CC volume was significantly decreased by 10.6% (p<0.01) in middle-aged group and by 26.9% (p<0.01) in old-aged group. The CC volume in old-aged group was significantly decreased by 18.3% (p<0.01) when compared to middle-aged group.2.3 The total length of the myelinated nerve fibers in CC was 78.3±7.7 km, 75.7±11.0 km and 62.4±5.8 km in young, middle-aged and old-aged groups, respectively. Compared to young group, the total length of the fibers in middle-aged group was non-significantly changed (p>0.05), whereas that of old-aged group was significantly decreased by 20.3% (p<0.05). When compared to middle-aged group, the total length of the fibers in old-aged group was significantly decreased by 17.5% (p<0.05).2.4 The total volume of the myelinated nerve fibers in CC was 46.6±3.7 mm3, 43.4±3.0 mm3 and 39.4±2.5 mm3 in young, middle-aged and old-aged group, respectively. Compared to young group, the total volume of the fibers in middle-aged group was not changed (p>0.05), whereas that of old-aged group was significantly decreased by 15.5% (p<0.01). When compared to middle-aged group, there was no significant decrease of the total volume of the fibers in CC of old-aged group (p>0.05).2.5 The total volume of the myelin sheaths in CC was 21.3±2.5 mm3, 19.7 ±2.6 mm3, 18.4±1.4 mm3 in young, middle-aged and old-aged groups, respectively. There were no statistically significant changes of the total volume of the myelin sheaths in CC among three groups (p>0.05).2.6 The mean diameter of the myelinated nerve fibers in CC was 0.70±0.04μm, 0.66±0.53μm and 0.62±0.03μm in young, middle-aged and old-aged groups, respectively. Compared to young group, the increase of the mean diameter in the middle-aged group was non-significant (p>0.05), whereas the mean diameter of the fibers in old-aged group was significantly increased by 13.0% (p<0.01). There was no significant change of the mean diameter in old-aged group when compared to middle-aged group (p>0.05).2.7 When compared to young group, the total length of the myelinated nerve fibers in CC with diameter in the range of 0.2 - 0.4μm in old-aged group was significantly decreased by 47.3% (p<0.01), and the total length of fibers with diameter in the range of 0.4 - 0.6μm was significantly decreased by 35.6% (p<0.01). When compared to young group, the total length of the myelinated nerve fibers with the diameters of 0.2 - 0.4μm and 0.4 - 0.6μm in middle-aged group was not significantly changed (p>0.05). PARTⅡ1. Materials and methods1.1 Ten 7-month-old (young group) and ten 27-month-old (old group) female Long-Evans rats were randomly divided into two groups.1.2 One group of 7-month rats and one group of 27-month rats were used for electron microscopic observation and stereological analysis in partⅠ.1.3 The other group of 7-month rats and the other group of 27-month rats were used for the detection of the MBP expression level by Western-blot, repeated at least 5 times.1.4 The OD values of the MBP bands were analyzed by Quantity One image analysis, and the expression levels were demonstrated by the gray scale value rate of MBP bands andβ-actin bands.2. Results2.1 The total length of the myelinated nerve fibers in CC of old group was significantly decreased by 20.3% (p<0.01) and the total volume of the myelinated nerve fibers in CC of old group was significantly decreased by 15.5% (p<0.01) when compared to young group. The total volume of the myelin sheaths of CC was non-significantly different between young group and old group (p>0.05).2.2 The expression levels of all type of MBP were decreased in old group compared to young group. However, only the expression level of the Mr 21.5×104 MBP was decreased significantly (p<0.05). By analyzing the total length and total volume of the myelinated nerve fibers, the total volume of the myelin sheaths and the expression level of Mr 21.5×104 MBP with Spearman correlation analysis, we found that the decrease of Mr 21.5×104 MBP was positively correlated with the decrease of the total volume of myelinated nerve fibers (r = 0.770; p<0.01) and myelin sheathes (r = 0.636; p<0.05) in old rats, but the decrease of Mr 21.5×104 MBP was not correlated with the total length of myelinated nerve fibers (r = 503; p>0.05). Using multiplinear regression analysis, we found that the decrease of MBP was significantly correlated with the decrease of total volume of myelin sheath (β= -1.105;p<0.05), however, the decrease of MBP was not correlated with the decrease of the total length and total volume of myelinated nerve fibers (β= -0.438, p>0.05;β= -1.306, p>0.05).General Conclusions1. The present study for the first time combined the new stereological techniques and TEM to quantitatively study the age-related changes of the CC and the myelinated nerve fibers in CC of rat brain.2. Our study provided normal data of CC volume, the total length, total volume and mean diameter of the myelinated nerve fibers and the total volume of the myelin sheaths in CC of Long-Evans rats. It will provide important basic data for the future studies on the impact of the neurodegeneration diseases of CNS on the myelinated fibers in CC using this animal model.3. The present results comfirmed previous findings in human CC, that is, there was the age-related atrophy of CC.4. We for the first time found that there was significant decrease of the total length and the totel volume of the myelinated nerve fibers in CC of old female Long-Evans rats. The decrease of the total length of the myelinated fibers in old rats was primarily caused by the decrease of the myelinated fibers with small diameter in CC.5. The expression level of MBP in CC was decreased in old rats, which was positively correlated with the age-related changes of myelinated nerve fibers in CC.