Jiang Y Q, Pickett J, Oblinger M M
Department of Cell Biology and Anatomy, Chicago Medical School, IL 60064.
J Neural Transplant Plast. 1994 Apr-Jun;5(2):103-14. doi: 10.1155/NP.1994.103.
To compare the long-term recovery of gene expression in dorsal root ganglion (DRG) neurons under conditions of regeneration vs. non-regeneration, Northern blotting and in situ hybridization were used to assess steady-state neurofilament (NF) and beta tubulin mRNA levels 12 weeks following axonal injury. Adult male rats sustained either a crush lesion of the mid-sciatic nerve (regeneration occurs), or a cut lesion of the sciatic nerve combined with ligation of the proximal nerve stump and removal of a large segment of the distal nerve (regeneration does not occur). In the latter case, neuroma formation physically prevented axonal regeneration. Results of Northern blotting of total RNA obtained from the DRG indicated that NF-L and NF-M mRNA levels had largely returned to control levels at 12 weeks following crush axotomy but were still substantially depressed following cut/ligation injury of the sciatic nerve at that time. In situ hybridization studies indicated that both crush and cut/ligation axotomy resulted in significantly lower NF-L mRNA levels in large-sized (> 1000 micron2) DRG neurons at 12 weeks post-axotomy. Discrepancies in the conclusions from Northern blotting and in situ hybridization experiments were also noted in the case of tubulin mRNA changes at long intervals after axotomy. In situ hybridization data derived from the large-sized DRG neurons using a coding region beta-tubulin cDNA (which recognizes both beta II and beta III mRNAs) showed complete recovery of beta-tubulin mRNA levels in surviving large-sized DRG neurons after crush axotomy, but significantly elevated tubulin mRNA levels in surviving large DRG cells at 12 weeks after cut/ligation axotomy. In contrast, Northern blotting results indicated that beta II-tubulin mRNA levels in the crush axotomy condition remained elevated relative to control while they were substantially lower than control in cut/ligation axotomy samples. Results from analysis of beta III-tubulin mRNA changes were not conclusive. The lack of complete correspondence in the results from the two different methods of analysis of mRNA changes (blotting vs. in situ) is likely to be due to selective loss of large-sized DRG neurons in the long-standing cut/ligation injury condition. This would influence results from blotting data, where RNA is derived from the DRG as a whole, more so than in situ hybridization experiments which specifically focus on the surviving large-sized neurons. Overall, data from these experiments indicate that altered patterns of gene expression remain in the DRG for long intervals after axonal injury, whether or not axonal regeneration has been successful.(ABSTRACT TRUNCATED AT 400 WORDS)
为比较背根神经节（DRG）神经元在再生与非再生条件下基因表达的长期恢复情况，采用Northern印迹法和原位杂交技术评估轴突损伤12周后稳态神经丝（NF）和β微管蛋白mRNA水平。成年雄性大鼠分别接受坐骨神经中段挤压损伤（可发生再生）或坐骨神经切断损伤并结扎近端神经残端及切除大部分远端神经（不可发生再生）。在后一种情况下，神经瘤形成从物理上阻止了轴突再生。从DRG获取的总RNA的Northern印迹结果表明，挤压性轴突切断术后12周，NF-L和NF-M mRNA水平已基本恢复至对照水平，但坐骨神经切断/结扎损伤后此时仍显著降低。原位杂交研究表明，挤压和切断/结扎轴突切断术后12周，大型（>1000平方微米）DRG神经元中NF-L mRNA水平均显著降低。在轴突切断术后较长时间微管蛋白mRNA变化的情况下，也注意到Northern印迹法和原位杂交实验结论存在差异。使用编码区β微管蛋白cDNA（可识别βII和βIII mRNA）从大型DRG神经元获得的原位杂交数据显示，挤压性轴突切断术后存活的大型DRG神经元中β微管蛋白mRNA水平完全恢复，但切断/结扎轴突切断术后12周存活的大型DRG细胞中微管蛋白mRNA水平显著升高。相比之下，Northern印迹结果表明，挤压性轴突切断条件下βII-微管蛋白mRNA水平相对于对照仍升高，而在切断/结扎轴突切断样本中则显著低于对照。βIII-微管蛋白mRNA变化的分析结果尚无定论。两种不同的mRNA变化分析方法（印迹法与原位法）结果缺乏完全对应，可能是由于在长期切断/结扎损伤条件下大型DRG神经元选择性丢失。这对印迹数据结果的影响更大，因为印迹数据中的RNA来自整个DRG，而原位杂交实验则专门关注存活的大型神经元。总体而言，这些实验数据表明，无论轴突再生是否成功，轴突损伤后DRG中基因表达模式的改变会持续很长时间。（摘要截短至400字）
