Activating transcription factor 3 immunoreactivity identifies small populations of axotomized neurons in rat cervical sympathetic ganglia after transection of the preganglionic cervical sympathetic trunk.

Activating transcription factor 3 (ATF3) has been proposed as a marker for injured neurons. Thus, while undetectable normally in sensory, motor, or sympathetic neurons, ATF3-like immunoreactivity (ATF3-IR) is readily detectable in such cells after axotomy. Here we examined ATF3-IR in the superior cervical ganglion (SCG) and the middle and inferior cervical ganglia (MICG) after transection of the predominantly preganglionic cervical sympathetic trunk (CST). The purpose of the study was to determine whether neurons in the SCG would exhibit ATF3-IR after decentralization and, if they did not, whether the induction of ATF3-IR was sensitive enough to identify the small numbers of neurons in the SCG and MICG that project their axons into the CST. Following transection of the CST, the majority of deafferented neurons in the SCG showed no ATF3-IR; however, a small group of neurons in both the SCG and MICG were labeled, and the location of the labeled neurons within these ganglia corresponded to that of neurons axotomized by this procedure. Furthermore, the ATF3-positive neurons in the MICG could be retrogradely labeled from the transected CST. In addition, a large number of smaller cells were labeled in the SCG, though not in the MICG, and some of these cells were double labeled with an antiserum to the glial protein S-100. These data indicate that, after transection of the CST, neuronal labeling in the SCG and MICG is restricted to axotomized neurons but that in addition there is extensive labeling of glial cells associated with anterograde degeneration within the SCG.

A comparison of the changes in the non-neuronal cell populations of the superior cervical ganglia following decentralization and axotomy.

Transecting the axons of neurons in the adult superior cervical ganglion (SCG; axotomy) results in the survival of most postganglionic neurons, the influx of circulating monocytes, proliferation of satellite cells, and changes in neuronal gene expression. In contrast, transecting the afferent input to the SCG (decentralization) results in nerve terminal degeneration and elicits a different pattern of gene expression. We examined the effects of decentralization on macrophages in the SCG and compared the results to those previously obtained after axotomy. Monoclonal antibodies were used to identify infiltrating (ED1+) and resident (ED2+) macrophages, as well as macrophages expressing MHC class II molecules (OX6+). Normal ganglia contained ED2+ cells and OX6+ cells, but few infiltrating macrophages. After decentralization, the number of infiltrating ED1+ cells increased in the SCG to a density about twofold greater than that previously seen after axotomy. Both the densities of ED2+ and OX6+ cells were essentially unchanged after decentralization, though a large increase in OX6+ cells occurred after axotomy. Proliferation among the ganglion's total non-neuronal cell population was examined and found to increase about twofold after decentralization and about fourfold after axotomy. Double-labeling experiments indicated that some of these proliferating cells were macrophages. After both surgical procedures, the percentage of proliferating ED2+ macrophages increased, while neither procedure altered the proliferation of ED1+ macrophages. Axotomy, though not decentralization, increased the proliferation of OX6+ cells. Future studies must address what role(s) infiltrating and/or resident macrophages play in regions of decentralized and axotomized neurons and, if both are involved, whether they play distinct roles.

Novel changes in gene expression following axotomy of a sympathetic ganglion: a microarray analysis.

Neurons of the peripheral nervous system are capable of extensive regeneration following axonal injury. This regenerative response is accompanied by changes in gene expression in axotomized neurons and associated nonneuronal cells. In the sympathetic nervous system, a few of the genes affected by axonal injury have been identified; however, a broad sampling of genes that could reveal additional and unexpected changes in expression has been lacking. We have used DNA microarray technology to study changes in gene expression within 48 h of transecting the postganglionic trunks of the adult rat superior cervical ganglion (SCG). The expression of more than 200 known genes changed in the ganglion, most of these being genes not previously associated with the response to injury. In contrast, only 10 genes changed following transection of the preganglionic cervical sympathetic trunk. Real-time RT-PCR analysis verified the upregulation of a number of the axotomy-induced genes, including activating transcription factor-3 (ATF-3), arginase I (arg I), cardiac ankyrin repeat protein, galanin, osteopontin, pituitary adenylate cyclase-activating polypeptide (PACAP), parathyroid hormone-related peptide, and UDP-glucoronosyltransferase. Arg I mRNA and protein were shown to increase within neurons of the axotomized SCG. Furthermore, increases in the levels of putrescine and spermidine, a diamine and polyamine produced downstream of arg I activity, were also detected in the axotomized SCG. Our results identified many candidate genes to be studied in the context of peripheral nerve regeneration. In addition, the data suggest a potential role for putrescine and spermidine, acting downstream of arg I, in the regenerative process.