Long-Term Effects of Axotomy on β-Tubulin and NF Gene Expression in Rat DRG Neurons

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-Μ 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 μm(2)) 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 β-tubulin cDNA (which recognizes both β(II) and β(III) mRNAs) showed complete recovery of β-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 β(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 β(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 largesized 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. However, recovery of “normal8221; patterns of cytoskeletal gene expression in the DRG is considerably more complete after crush injury than after cut/ligation injury.