Macrophages, Nitric oxide synthases and Heme oxygenases: possible roles during peripheral nerve regeneration

The purpose of this study was to investigate the suggestions that macrophages, and the systems generating nitric oxide (NO) and carbon monoxide (CO), i.e. nitric oxide synthases (NOS) and heme oxygenases (HO), could be important for peripheral nerve regeneration.

Immunocytochemistry was used to visualise invading and resident macrophages, inducible NOS (iNOS), two isoforms of HO (HO-1 and HO-2), biliverdin reductase, and cGMP in regenerating peripheral ganglia in vitro and in vivo. HO-1 was also localised by in situ hybridisation and neuronal NOS (nNOS) by histochemistry. Two types of sensory ganglia (dorsal root ganglia (DRG) and nodose ganglia) and three autonomic ganglia (superior cervical ganglia (SCG), otic ganglia and sphenopalatine ganglia) were studied. Regeneration changes in the ganglia were induced by crush lesions or axotomy in vivo, or by excision and culturing.

Principal findings were that following nerve injury there is a rapid activation of resident and a massive invasion of macrophages to the site of an injury but also in the ganglia. The time course of this activation and invasion precedes certain regeneration-induced changes in the neurones of the ganglia, including neuropeptide synthesis. The results are consistent with the suggestion that products released by macrophages could be important for the regenerative response. However, invading macrophages do not seem to be crucial for initiation of axonal outgrowth of sensory axons.

iNOS was found in neurones of nodose ganglia and increased after injury both in these ganglia and SCG. An increase in iNOS was also observed in macrophages of regenerating SCG. nNOS was expressed in neurones of both intact and cultured sensory ganglia, although there was a marked difference in the number of nNOS positive cells in the DRG along the spinal cord. In satellite cells of sensory ganglia an increase of cGMP immunoreactivity was found. In cultured sensory and autonomic ganglia the inducible form of HO (HO-1) increased dramatically in satellite cells. HO-2 immunoreactivity was observed in neurones of all types of ganglia, but did not change in response to an injury. These findings suggests that both NO and CO may be important for intercellular signalling during regeneration.

Biliverdin reductase, the enzyme generating the antioxidant bilirubin, was observed in axons, nerve cell nuclei and nerve cell bodies of regenerating sensory and autonomic ganglia, implying an increased demand for antioxidants following injury.

Together these results show that systems with the potential to generate NO and CO are upregulated following a peripheral nerve injury, although the increase occurs in various cell types in different ganglia. I suggest that signalling between neurones and satellite cells occurs through NO/CO-cGMP, and that this signalling is enhanced during regeneration.