| PARTâ… THE STEREOLOGICAL STUDIES OF THE AGE-RELATED CHANGES OF THE UNMYELINATED FIBERS IN THE SUBCORTICAL WHITE MATTER IN RAT BRAINObjectives To investigate the age-related changes of unmyelinated fibers in the white matter of Long-Evans rats using transmission electron microscopy (TEM) and modern stereological techniques in order to further understand the mechanism of the myelinated fiber length decline in aged white matter.Methods Five young (6-8 month), five middle-aged (18 month) and eight old-aged (28 month) female Long-Evans rats were used. Body weight was 280±20 g. One hemisphere was randomly sampled from each rat brain. From the sampled hemisphere, 4 white matter blocks were sampled in a systematic random fashion. One 60 nm-thick ultra-thin section was randomly cut from each sampled block. From each section, 8-12 fields of view were randomly photographed under TEM with the magnification of 20000. The white matter volume, the total length of the unmyelinated fibers in the white matter and the total volume of the unmyelinated fibers in the white matter were investigated with the new stereological methods.Results 1 The white matter volume of was 119±8.6 mm3 in young rats, 108±5.6 mm3 in middle-aged rats and 68.5±11.4 mm3 in old-aged rats, respectively. There was no significant difference in the white matter volume between middle-aged rats and young rats. When compared to young rats, the white matter volume in old-aged rats was significantly reduced by 42.5% (p<0.01). When compared to middle-aged rats, the white matter volume in old-aged rats was significantly reduced by 35.4% (p<0.01). 2 The total volume of the unmyelinated fibers in the white matter was 32.8±3.2 mm3 in young rats, 18.6±7.8 mm3 in middle-aged rats, 9.1±3.5 mm3 in old-aged rats, respectively. When compared to young rats, the unmyelinated fiber volume in the middle-aged rats was significantly decreased by 43.3% (p<0.05). However, the unmyelinated fiber volume was significantly decreased in old-aged rats by 72.1% (p<0.01) when compared to young rats. The unmyelinated fiber volume in old-aged rats was significantly decreased by 51.0% (p<0.05) when compared to middle-aged rats. 3 The total length of the unmyelinated fibers in the white matter was 409±130 km in young rats, 350±171 km in middle-aged rats and 128±48 km in old-aged rats, respectively。There was no significant difference in the unmyelinated fiber length between middle-aged rats and young rats. The unmyelinated fiber length was significantly decreased in old-aged rats by 68.6% (p<0.01) when compared to young rats. The unmyelinated fiber length in old-aged rats was significantly decreased by 63.4% (p<0.01) when compared to middle-aged rats.Conclusions 1 When compared to 6-8 month female rats and 18 month female rats, the total white matter volume, the total length and total volume of the unmyelinated fibers in the white matter were all significantly decreased in old-aged (28-month) female rats. The total volume of the unmylinated fibers in the white matter of 18 month female rats was significantly decreased when compared to that of 6-8 month female rats. However, the total length of the unmyelinated fibers in the white matter of 18 month female rats was non-significantly changed when compared to 6-8 month female rats. The present results indicated that the age-related loss of the total length of the unmyelinated fibers in white matter was mainly due to the degeneration of the unmyelinated fibers with larger diameters. 2 After analyzing the changes of the myelinated fibers and unmyelinated fibers in the aged white matter together, we concluded that there was a decrease of the unmyelianted fibers with small diameters in the white matter of middle-aged rats, but this decrease was masked by the demyelination of the myelinated fibers with small diameters in the white matter of middle-aged rats. Since the myelinated fibers with small diameters were demyelinated to become unmyelinated fibers, the total volume of the unmyelinated fibers formed from the demyelination of the myelinated fibers was small. Since, in the old-aged rat white matter, the amount of the unmyelinated fibers formed from the demyelination of the myelinated fibers with small diameter might not retrieve the loss of the unmyelinated fibers with small diameter with age, the total volume and the total length of the unmyelinated fibers were significantly decreased in the old-aged rat white matter.PARTâ…¡THE STEREOLOGICAL STUDIES OF THE AGE-RELATED CHANGES OF THE MYELIN SHEATS IN THE SUBCORTICAL WHITE MATTER IN RAT BRAINObjectives To investigate the age-related changes of the myelin sheaths in the white matter of Long-Evans rats using transmission electron microscopy (TEM) and modern stereological techniques in order to further understand the mechanism of the myelinated fiber length decline in aged white matter. Method Five young (6-8 month), five middle-aged (18 month) and five old-aged (28 month) female Long-Evans rats were used. Body weight was 280±20 g. One hemisphere was randomly sampled from each rat brain. From the sampled hemisphere, 4 white matter blocks were sampled in a systematic random fashion. One 60 nm-thick ultra-thin section was randomly cut from each sampled block. From each section, 8-12 fields of view were randomly photographed under TEM with the magnification of 20000. The white matter volume, the total length of the myelinated fibers, the total volume of the myelin sheaths, as well as the mean inner diameter and mean outer diameter of the myelinated fibers, the mean inner perimeter and mean outer perameter of the myelinated fibers and the mean thickness of the myelin sheaths in the white matter were investigated with the new stereological methods.Results 1 The myelin sheaths in the white matter of old-aged rats showed three types of age-related alterations: (1) The splitting of the major dense line: The most common alteration was the development of bulges in some parts of sheaths where pockets of electron-dense cytoplasm were located. The cytoplasm in the pockets contained dark amorphous bodies; (2) The splitting of the intraperiod line: Balloons appeared as large rounded areas. Structurally, the space usually appeared to be empty, but, occasionally, they contained membranous material or dense granules, some of which adhered to the inside wall of the balloon; (3) Some axons were surrounded by redundant myelin. Essentially, the sheath was too large for the enclosed axon. Consequently, a normal axon was located at one end of the profile of a sheath that looped off to the side. 2 The white matter volume was 115±8.6 mm3 in young rats, 110±5.6 mm3 in middle-aged rats and 69.0±12.7 mm3 in old-aged rats, respectively. There was no significant difference in the white matter volume between middle-aged rats and young rats. When compared to young rats, the white matter volume in old-aged rats was significantly reduced by 40.0% (p<0.01). When compared to middle-aged rats, the white matter volume in old-aged rats was significantly reduced by 37.2% (p<0.01). 3 The total length of the myelinated fibers in the white matter was 115±27 km in young rats, 135±47 km in middle-aged rats and 62±25 km in old-aged rats, respectively。There was no significant difference in the myelinated fiber length between middle-aged rats and young rats. The myelinated fiber length was significantly decreased in old-aged rats by 46.1% (p<0.05) when compared to young rats. The myelianted fiber length in old-aged rats was significantly decreased by 54.1% (p<0.05) when compared to middle-aged rats. 4 The absolute distributions of the myelinated fiber length against the myelin sheath thickness in three groups were shown. The thickness of the myelin sheaths for most of the myelinated fibers ranged from 0.08μm to 0.18μm. The comparisons of the absolute distributions of the myelinated fibers length against the myelin sheath thickness between young group and old-aged group and between middle-aged group and old-aged group clearly showed the age-related decline of the total length of the myelinated fibers in the white matter was mainly due to the loss of the myelinated fibers with myelin sheath thickness less than 0.15μm. 5 The diameters of the myelinated fibers and the thicknesses of the myelin sheaths were divided into six intervals. The total length of the myelinated fibers in each interval was calculated. Comparisons of the total length of the myelinated fibers in each interval between young female rats, middle-aged female rats and old-aged female rats clearly showed that in middle-aged white matter and old-aged white matter, the total length of the myelinated fibers with the diameter less than 1.0μm and myelin sheath thickness less than 0.1μm was decreased. The total length of the myelinated fibers with the diameter less than 1.4μm and the myelin sheath from 0.1μm to 0.14μm in old-aged female rats was significantly decreased when compared to young female rats and middle-aged female rats. The total length of the myelinated fibers with a myelin sheath thickness from 0.14μm to 0.18μm in the white matter of middle-aged female rats was significantly increased when compared to young female rats and old-aged female rats. The total length of the myelinated fibers with a diameter more than 0.4μm and the myelin sheath thickness from 0.18μm to 0.22μm in the white matter of middle-aged female rats was significantly increased when compared to that of young female rats and old-aged female rats. 6 The total volume of the myelin sheaths in the white matter was 34±3.6 mm3 in young rats, 42±5.1 mm3 in middle-aged rats, 30±5.3 mm3 in old-aged rats, respectively. The total volume of the myelin sheaths in the white matter of middle-aged female group was significantly increased by 19.0% when compared to the young female group (p<0.05). The total volume of the myelin sheaths in the white matter of the old-aged female group was decreased significantly by 28.6% when compared to middle-aged female group ( p<0.01). However, the total volume of the myelin sheaths in the white matter of old-aged group was not significantly different from that of young group (p >0.05). 7 On average, the volume of myelin sheaths per unit length of the myelinated fibers was 0.30±0.04 mm3/km at age of 6-8 month female rats, 0.35±0.16 mm3/km at age 18 month female rats and 0.53±0.16 mm3/km at age 28 month female rats. The volume of myelin sheaths per unit length of the myelinated fibers in the white matter of old-aged female rats was increased significantly by 43.4% when compared to young female rats (p<0.05). The volume of myelin sheaths per unit length of the myelinated fibers in the white matter of old-aged rats was not significant different from that of middle-aged rats, but the volume of myelin sheaths per unit length of the myelinated fibers in the white matter of old-aged rats was increased by 34.0% when compared to middle-aged rats (p>0.05). 8 The external diameter and internal diameter of the myelinated fibers in the white matter were not significantly different among the age groups (p >0.05). However, the external diameter of the myelinated fibers in the white matter of old-aged female rats was non-significantly increased by 17.9% when compared to young female rats. 9 The outer perimeter of myelin sheaths was 3.39±0.72μm in young rats, 3.5±0.95μm in middle-aged rats, 3.90±0.54μm in old-aged rats, respectively. The inner perimeter of myelin sheaths was 2.60±0.66μm in young rats, 2.55±0.81μm in middle-aged rats, 2.60±0.36μm in old-aged rats, respectively. There were no significant differences in the outer perimeter and inner perimeter of myelin sheaths between young female rats, middle-aged female rats and old-aged female rats (p>0.05). The difference between the outer perimeter and inner perimeter of myelin sheaths was 0.78±0.07μm in young rats, 0.94±0.14μm in middle-aged rats, 1.31±0.19μm in old-aged rats, respectively. There were no differences in the outer perimeter and inner perimeter of myelin sheaths in the white matter between young group and middle-aged group (p>0.05). However, the difference between outer perimeter and inner perimeter of myelin sheaths in the old-aged rats was significantly increased by 40.5% and 28.2%, respectively, when compared to young female rats and middle-aged female rats (p<0.01). 10 The mean thickness of myelin sheaths was 0.12±0.00μm in young rats, 0.15±0.02μm in middle-aged rats, 0.18±0.02μm in old-aged rats, respectively. There was no significant difference in the mean thickness of the myelin sheaths in the white matter between young female group and middle-aged female group (p>0.05). However, the mean thickness of the myelin sheaths in the white matter of old-aged rats was significantly increased by 33.3% and 16.7%, respectively, when compared to young female rats and middle-aged female rats (p<0.01).Conclusion 1 The comparisons of the total length of the myelinated fibers with different diameters and with different myelin sheath thicknesses in the three groups gave us a clear picture of the age-related changes of the total length of the myelinated fibers in white matter. The present study for the first time provided direct evidence that the earliest change of the myelinated fibers in aged white matter was the loss of the myelinated fibers with small diameter and thin myelin sheath. The demyelination of the myelinated fibers with small diameter and thin myelin sheath in white matter might be the main factor leading to the decrease of the total length of the myelinated fibers with age. The myelin defects might lead to a decrease in the conduction velocity along axons, which might underlie some of the age-related cognitive deficits. 2 Another major finding in the current study is the significant age-related increase of the myelinated fibers with bigger diameters and thicker myelin sheath thickness. Even though the outer perimeter and inner perimeter of myelin sheaths was not different among young, middle-aged female rats and old-aged female rats, the difference between the outer perimeter and inner perimeter of myelin sheaths and the mean thickness of myelin sheaths in the white matter of old-aged female rats was significantly increased when compared to that of young female rats and that of middle-aged female rats, respectively. The present study was the first quantitative study to provide evidence for the continued myelination of the myelinated fibers in aged white matter female rats.PARTâ…¢THE EFFECTS OF THE SHORT-TERM EXERCISE ON SPATIAL MEMORY OF AGED RATS AND THE MYELINATED FIBERS IN AGED RATS WHITE MATTERObjectives To investigate the effects of running exercise on the spatial learning ability of middle-aged rats and old-aged rats, and then use the new stereological methods to examine the effects of forced treadmill running on the white matter and the myelinated fibers in the white matter of middle-aged rats and old-aged rats.Methods Twenty 14 month-old (middle-aged) Sprague-Dawley rats (10 males and 10 females) and twenty 24 month-old (old-aged) Sprague-Dawley rats (10 males and 10 females) were used for the current study. The middle-aged rats and old-aged rats were randomly divided into running exercise (RE) group and standard environment (SE) group. Exercising rats run on a motorized treadmill for 20 min/day on 5 consecutive days per week for 4 months. To reduce the novelty stress, the rats were familiarized with the treadmill by running on a motorized treadmill at a speed of 10 m/min in the first week and 15 m/min in the second week. From the third week, the speed was kept constant at 20 m/min. Spatial memory was examined with Morris water maze tasks for consecutive five days with hidden platform trials in first four days and visible platform trials on the fifth day. Time latency was recorded. One hemisphere was randomly sampled from each rat brain. From the sampled hemisphere, 4 white matter blocks were sampled in a systematic random fashion. One 60 nm-thick ultra-thin section was randomly cut from each sampled block. From each section, 4 fields of view were randomly photographed under TEM with the magnification of 6000. The white matter volume, the total length of the myelinated fibers in the white matter, the total volume of the myelinated fibers in the white matter and the mean diameter of the myelinated fibers in the white matter were investigated with the new stereological methods.Results 1 In the hidden platform test, the latency of middle-aged female exercise group was significantly decreased when compared to the sedentary group (p<0.05). However, the spatial memory performances in the exercise group of the middle-aged male rats, old-aged male rats and old-aged female rats were not significantly different from those rats living in standard conditions (p>0.05). The spatial memory performances in the visible platform test was not different between exercise group and control group both in middle-aged rats and old-aged rats. 2 In the middle-aged exercise group, exercise increased the white matter volume of the female rats by 22.8% even though the difference was not statistically significant (p>0.05). The total length of the myelinated fibers in the white matter of exercise group was significantly increased by 54.5% in middle-aged female rats (p<0.05) when compared to controls. The absolute distributions of the myelinated fiber length between exercise groups and sedentary groups of middle-aged female rats indicated that the significant increase of the myelinated fiber length of exercise rats was mainly due to the marked increase of the myelinated fibers with smaller diameter, especially the fibers with diameters less than 0.8μm. There was no significant difference in the total volume of the myelinated fibers in the white matter between middle-aged female exercise group and middle-aged female sedentary group (p>0.05). In the middle-aged group, the mean diameter of the myelinated fibers in white matter was significantly smaller in female exercise group than in female sedentary group (p<0.05). 3 In the middle-aged group, the white matter volume, the total volume and mean diameter of the myelinated fibers in white matter were non-significantly different between male exercise group and male sedentary group (p>0.05). The total length of the myelinated fibers in the white matter of middle-aged male rats was significantly increased by 69.54% in exercise group (p<0.01) when compared to controls. The absolute distributions of the myelinated fiber length between exercise groups and sedentary group of middle-aged male rats indicated that the significant increase of the myelinated fiber length of exercise rats was mainly due to the marked increase of the myelinated fibers with smaller diameter, especially the fibers with diameters less than 0.8μm. 4 In the old-aged group, the white matter volume, the total length, total volume and mean diameter of the myelinated fibers in white matter was non-significantly different between female exercise group and female sedentary group (p>0.05). 5 In the old-aged male group, the white matter volume and the total volume of the myelinated fibers in the white matter of exercise group was significantly increased when compared to sedentary group (p<0.05). The total length of the myelinated fibers in the white matter of old-aged male rats was not significantly changed in exercise group (p = 0.056>0.05) when compared to old-aged sedentary rats. The absolute distributions of the myelinated fiber length between exercise group and sedentary group of old-aged male rats indicated that the significant increase of the myelinated fiber length of exercise rats was mainly due to the marked regeneration of the myelinated fibers with smaller diameter, especially the fibers with diameters less than 0.8μm.Conclusions 1 The spatial memory of middle-aged female rats was significantly improved by short-term running exercise. This result provided an important theoretical basis for the future application of similar strategy to delay the progress of brain function decline with aging. 2 The white matter volume, the myelinated fiber length and the myelinated fiber volume in the white matter of both middle-aged rats and old-aged rats were increased in the short-term running exercise group when compared to the control group. The present results might provide a new target and a new direction for the future studies that search for the strategies to delay the progress of brain aging.
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