Preliminary Studies On Expression Of NNOS-associating Molecules NIDD, CAPON, And Dexras1 In Rat Models Of Sciatic Nerve Injury And CFA-induced Inflammatory Pain

Objective: To observe the gene expression patterns of nNOS-associating molecules NIDD, CAPON and Dexras1 in rat models of sciatic nerve injury and CFA-induced inflammatory pain as well as to approach their potential biological roles in peripheral nerve injury and regeneration, neuronal survival and death, regeneration of denevated skeletal muscle and pain process or modulation.Methods: First, TaqMan probes and cRNA probes for NIDD, CAPON, Dexras1, and nNOS were obtained using primers designing, RT-PCR, T-A cloning, in vitro transcription and dot bloting techniques. Then, Sprague-Dawley (SD) rats were subjected to unilateral sciatic nerve transection or crush injury. Gene expression patterns for NIDD, CAPON, Dexras1, and nNOS were detected in the lesion nerves, corresponding DRG and lumbar spinal cord by real-time fluorescence quantitative PCR as well as in situ hybridization. More measures such as Western blot, H-E staining, Masson staining, NADPH-d histochemistry, immunofluorescence and co-immunoprecipitation were taken besides real-time fluorescence quantitative PCR and in situ hybridization to study the expression patterns of CAPON and nitric oxide synthases (NOS) in ipsilateral gastrocnemius muscle after sciatic nerve crush to explore their roles in denervated-muscle regeneration. Finally, thermal hyperalgesia was induced by an injection of complete Freund's adjuvant (CFA) into one hindpaw of the SD rat, and the changes in mRNA for NIDD, CAPON, Dexras1, and nNOS were also examined in the corresponding DRG and lumbar spinal cord.Results: (1) The correlation coefficient of the standard curve for real-time fluorescence quantitative PCR was above 0.99, the reaction efficiency was around 1.0 and all the variation coefficients were below 10%. The efficiency of Digoxigenin (DIG) labeling was quantitated by dot blotting and was found around 50 (ng/μl).(2) In normal adult rat, NIDD, CAPON, Dexras1, and nNOS mRNAs were expressed in sciatic nerve, DRG and spinal cord (SC) with a higher abundance in the SC. All of them changed following unilateral sciatic nerve transection. They increased with a peak at 1 w in both the proximal and the distal stumps of the injured nerves and the upregulated mRNAs were mainly localized in the proliferated Schwann cells (SCs). Gene expressions for NIDD, CAPON, Dexras1, and nNOS peaked in the ipsilateral DRG (L4-L6) at 2 d, 2 d, 2 w, and 1 d respectively and a majority of their increased mRNAs was in neurons and a minority in satellite cells. While in the lumbar SC, all their expressions increased with a peak at 1 w after nerve injury, mainly in the neurons of the bilateral dorsal horns (DH) and some of the bilateral ventral horns (VH). However, glial cells sometimes showed positive mRNA signals.(3) Gene expressions for NIDD, CAPON, Dexras1, and nNOS also suffered alterations after sciatic nerve crush injury. In details, they increased with a peak at 2 w in the crushed nerves, especially in the proliferated SCs of the very proximal lesion segments. In the ipsilateral DRG, they all markedly increased at 2 w as well, mainly in the neurons and sometimes in the satellite cells. In the SC, significant increase in mRNA for NIDD, CAPON, and Dexras1 were found 1 w post injury, also mainly in the neurons of the bilateral DH and some of the bilateral VH. Still some glial cells expressed positive mRNA signals. In contrast, nNOS mRNA underwent a downregulation, lowest at 1 w, and restored the baseline at 4 w.(4) The ipsilateral gastrocnemius muscle after sciatic nerve crush suffered atrophy because of denervation. However, satellite cells were activated meanwhile, which contributed to muscle regeneration. The expressions for CAPON and three isoforms of nitric oxide synthase (NOS) thus neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), changed during such process. CAPON mRNA and protein increased early after nerve lesion with a peak around 1 d; its protein was kept at a high level while the mRNA markedly increased again at 4 w. Both mRNA and protein for CAPON were expressed largely in the activated satellite cells or new-formed fibers. In contrast, nNOS mRNA and protein were decreased early after nerve injury, lowest at 1 w, and then returned to normal gradually. Expression of mRNA or protein for nNOS was also detected in the activated satellite cells or new-formed fibers. However, eNOS protein was kept increasing while iNOS showed two significant increases, one at 6 h and another at 2 w. Both eNOS and iNOS protein were found to have some colocalization with the satellite cell marker Myf-5. Co-immunoprecipitation revealed the in vivo interactions between CAPON and nNOS, but not between CAPON and eNOS or iNOS.(5) Thermal hyperalgesia was induced by CFA injection in the ipsilateral hindpaw of the SD rat, which was predominant in 4–6 h and last to 72 h. However, this did not happen in the contralateral side or in the normal saline (NS) group. In this inflammatory pain model, nNOS mRNA significantly increased in the ipsilateral corresponding DRG as early as 1 h post injection, and maintained a high level from 12 h to 72 h with a peak at 24 h. While in the lumbar SC, nNOS mRNA markedly increased from 48 h to 72 h after a transient upregulation at 4 h. NIDD mRNA was significantly increased at 24 h both in ipsilateral DRG and lumbar SC; however, it last until 72 h in the SC. Histological studies showed that NIDD mRNA was localized in small- and medium-diameter neurons of DRG, several of which co-express IB4, a marker for subpopulations of nociceptive and thermoreceptive neurons. Abundant mRNA signals of NIDD were shown in superficial laminae of the spinal dorsal horn (ⅠandⅡ), which predominantly receive input from nociceptive and thermoreceptive primary afferents. CAPON mRNA turned on evident decrease since 1 h and last until 72 h in the corresponding DRG. While in the lumbar SC, it decreased at 1 h, transiently backed to normal at 4 h, reduced again from 6 h and persisted to 48 h, and recovered at 72 h. Dexras1 mRNA increased remarkably since 1 h and almost stayed at the high level except for a tiny transient return at 4 h in the ipsilateral DRG. In contrast, it decreased in the lumbar SC at 1 h, transiently recovered at 4 h, reduced significantly again from 6 h to 12 h, and returned to normal from 24 h.Conclusions: (1) NIDD, CAPON, Dexras1and nNOS may be involved in the peripheral nerve regeneration after injury and may play an important role in the neuronal survival or death in the DRG or SC after the peripheral nerve lesion. They may even have an effect on the pain process and modulation.(2) CAPON and three NOS isoforms may take great part in the satellite cell activation and muscle regeneration in the denervated skeletal muscle after peripheral nerve injury.(3) NIDD, CAPON, Dexras1or nNOS might be involved in the pain process or modulation and may be associated with the development and (or) maintenance of pain induced by CFA hindpaw injection.