| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Introduction: The value of neurotrophic factors in the skeletal muscle and skin is very low compared with that in the peripheral nerve, so measurement of the neurotrophic factors in the denervated skeletal muscle and skin is very difficult. To clarify the changes of the NGFmRNA in the denervated skeletal muscle and skin, we developed RT-PCR method using deletion mutant RNA as internal standard (1995, Shimizu).
Materials and methods: Gastrocnemial muscle and calf skin of 30 female ICR mice with age of five weeks were used. Gastrocnemial muscles (50 mg) and calf skin (3 x 4 mm) were taken zero, one, two and four weeks after cutting off the sciatic nerve. The proximal end of the sciatic nerve was turned over to prevent the regeneration. The muscle from contralateral side was also examined as the control. The second control was set up by sham operation. After homogenation of the material, internal standard was added before RNA extraction. Then mRNA was amplified by RT-PCR and it was analyze
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d by HPLC. NGFmRNA was measured by comparison with peak height of the internal standard.
Results: With 26 cycles of PCR amplification, the amount of RT-PCR products was proportional to the added amounts of pNGA and dNGA in the range up to 15 fg/tube. When a fixed amount (5 fg/tube) of dNGA was added to various amounts (1-15 fg) of PNGA, a linear relationship was obtained between the ratios of the RT-PCR products and the added amounts of pNGA. This RT-PCR-HPLC method is suitable for measurement of mRNA, especially for small amount. In the control group, the NGFmRNA value was 8.86 fg/mg tissue in the gastrocnemial muscle and 24.56 fg/mg tissue in the calf skin. The NGFmRNA on the cutting side was increased to 24.45 fg/mg tissue in the gastrocnemial muscle and 85.65 fg/mg tissue in the calf skin at one week. On the other hand, NGFmRNA in the muscle and the skin of the contralateral side were same as that in the control group. NGFmRNA in the gastrocnemial muscle at four weeks after nerve cutting was approximately four times that of the control value. In contrast to this, NGFmRNA of the skin was returned to the control value within four weeks.
Conclusion: An extremely small quantity of the NGFmRNA involved in the skeletal muscle and the skin was analyzed by RT-PCR/HPLC method. From one to four weeks after nerve laceration, NGFmRNA in gastrocnemial muscle was significantly increased. NGFmRNA in the calf skin was significantly increased from one and two weeks, but it was rapidly decreased to control level at four weeks. Less
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