| The hippocampal formation plays important role in learning ability and memory. Before, it was believed that the age-related decline of hippocampal formation function was related to the neuron death of aged hippocampal formation. However, there are some studies indicating that there is no significant neuron death in the aged hippocampal formation. Therefore, it is speculated that age-related decline of the spatial learning capacity might be related to the age-related changes of the nerve fibers in hippocampal formation. Quite a lot of studies indicated that enriched environment could enhance spatial learning capacity and increase dendritic arborization, gilogenesis, neurogenesis of dentate gyrus and neurotrophin mRNA expression. The spatial learning capacity starts to decline in middle -aged. Therefore, the enriched environment interference beginning of middle-aged might prevent or postpone the spatial learning capacity, The enriched environment is the simple and easily-used behavioral interference strategy. Until now, however, there is no study in china and abroad investigating the effects of the enriched environment on the spatial learning capacity and on the myelinated fibers in the hippocampus of middle-aged rats. The present study was designed to investigate the effects of enriched environment on the spatial learning capacity and the myelinated fibers in the hippocampal formation in the middle-aged male and female rats. The present study first used Morris water maze to test the effects of enriched environment on the spatial learning capacity of middle-aged male and female rats. Then, transmission electron microscope technique and unbiased stereological methods were used to quantitatively investigate the effects of the enriched environment on the hippocampal volume and on the total myelinated fiber length, total myelinated fiber volume and mean diameter of the myelinated fibers in the hippocampus of middle-aged male and female SD rats.1. Materials and methods1.1 22 female Sprague-Dawley rats (SD) (14 months old) and 22 male SD rats (14 months old) were randomly divided into two groups: enriched environment (EE) rats (12 rats) and standard environment (SE) rats (10 rats). EE rats were reared in enriched environment and SE rats were reared in standard environment for 4 months. Then, 10 female SD rats (18 months old) (5 EE rats and 5 SE rats) and ten male SD rats (18 months old) (5 EE rats and 5 SE rats) were randomly sampled for the behavioral test and morphological analysis. 1.2 Enriched environment rats were kept 12 per cage in large cages (120 cm×60 cm×50 cm) containing a variety of stimulating objects such as tunnels, platform, running wheels, ropes, boxes, toys, balls, figurines, building blocks, etc. Tunnels, toys and food location are changed frequently. Standard environment rats were kept 5 per cage in regular size cages(40 cm×30 cm×30 cm)as groups of 5 without any stimulating object. All rats were housed at a temperature of 21℃±2℃. They were kept under a constant 12 hr light-12 hr dark cycle. Food and water were available ad libitum. The rearing time for the two groups of rats were both 4 months.1.3 After 4 months, the spatial memory ability of rats was tested with Morris water maze for consecutive five days with hidden platform trials in first four days and visible platform trials on the fifth day. Time latency was recorded.1.4 One hemisphere was sampled from each rat brain. From the sampled hemisphere, 4~6 hippocampal blocks were sampled in a systematic random fashion. One ultra-thin section was cut from each sampled block. From each cut section, 15 fields of view were randomly photographed under TEM, with the magnification of 9000. 75 electron microscope images were obtained per rats.1.5 The total volume of the hippocampal formation, the length density and total length of the myelinated nerve fibers in the hippocampal formation, the volume density and total volume of the myelinated nerve fibers in the hippocampal formation, and the mean diameter of the myelinated nerve fibers in the hippocampal formation were investigated with the new stereological methods.2. Results2.1 In the hidden platform test, the escaped latency was decreased significantly in EE female rats when compared to SE female rats (P<0.05). However, there was no significantly differences in the escaped latency between EE male rats and SE male rats (P>0.05). In the visible platform test, there were no significantly differences between EE and SE female rats and between EE female rats and SE male rats in the escaped latency, respectively.2.2 The total volume of the hippocampal formation in EE female rats was increased by 11.7% when compared to SE rats, but the difference was not significant (P>0.05). The total volume of the hippocampal formation in EE male rats was non-significantly increased by 4.6% when compared to SE male rats (P>0.05).2.3 The total length of the myelinated fibers in the hippocampal formation of EE female rats was significantly increased by 47.4%, when compared to that in SE female rats (P<0.01). The total length of the myelinated fibers in EE male rats was significantly increased by 71.8% than those in SE male rats (P<0.05). The total volume of the myelinated fibers in the hippocampal formation of EE female rats was increased significantly by 43.4% compared to that in SE female rats (P<0.01). The total volume of the myelinated fibers in EE male rats was significantly increased by 102.9% than those in SE male rats (P<0.05). Then mean diameter of the myelinated fibers in the hippocampal formation of EE female rats was decreased by 5.2% when compared to SE female rats, but the difference was not significant (P>0.05). The mean diameter of the myelinated fibers in the hippocampal formation of EE male rats was significantly increased by 33.1% when compared to SE male rats (P<0.01). The absolute distributions of the myelinated fiber length in the hippocampal formation of male and female EE rats and male and female SE rats indicated that the enriched environment-induced increase of the myelinated fiber length in the hippocampal formation of male and female EE rats was mainly due to the marked increase of the myelinated fibers with smaller diameter.General Conclusions1. The present study for the first time used transmission electron microscope technique and unbiased stereological methods to quantitatively investigate the effects of enriched environment on the hippocampal volume, the total myelinated fiber volume and the mean diameter of the myelinated fibers in the hippocampal formation of middle-aged male and female SD rats. Meanwhile, we used Morris water maze to examine the effects of the short-term enriched environment on the behavioral tests of the middle-aged male and female rats. 2. The present study found that the short-term enriched environment significantly improved the spatial learning capacity of the middle-aged female rats. These results might provide an important theoretical basis for searching the behavioral strategy to delay the progress of brain ageing in the future.3. The present study found that the short-term enriched environment had a positive effect on the myelinated fibers in the hippocampal formation of middle-aged male and female rats. The total myelinated fiber volume in the hippocampal formation of middle-aged male and female EE rats were significantly increased when compared to SE rats. The mean diameter of the myelinated fibers in the hippocampal formation of middle-aged male EE rats was significantly increased when compared to SE male rats. The enriched environment-induced increase of the total myelinated fiber length in the hippocampal formation of middle-aged rats was mainly due to the marked increase of the myelinated fibers with smaller diameter. The present results might provide important morphological basis for further understanding of the mechanism for the fact that enriched environment improves brain functions. |
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