Reduction in nerve growth factor availability leads to a conditioning lesion-like effect in sympathetic neurons.

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target-derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF-treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy.

A conditioning lesion enhances sympathetic neurite outgrowth.

Axonal regeneration can be influenced by a conditioning lesion (an axonal injury made prior to a second test lesion). Previously, sympathetic neurons in vivo were shown to respond to a conditioning lesion with decreased neurite outgrowth, in contrast to the enhanced outgrowth observed in all other peripheral neurons examined. The present experiments tested the effects of a conditioning lesion on neurite outgrowth in vitro from the superior cervical ganglion (SCG) and the impact of several factors on that response. Ganglia axotomized 1 week earlier and then explanted in Matrigel or collagen gel responded with a significant increase in neurite extension compared to sham-operated ganglia. A distal axotomy produced by unilateral removal of the salivary glands (sialectomy) caused an increase in neurite outgrowth similar to that of a proximal axotomy. These conditioning lesions induced both an increase in the rate of elongation, and, in the case of the proximally axotomized SCG, a shorter initial delay of outgrowth. The enhanced outgrowth following sialectomy was specific to the nerve containing the majority of axons projecting to the salivary glands, suggesting that the conditioning lesion effect is restricted to previously injured neurons. Deletion of the gene for leukemia inhibitory factor (LIF), a gene induced by axotomy, did not abolish the conditioning lesion effect in SCG explants or dissociated cell cultures. In conclusion, sympathetic neurons are capable of responding to a conditioning lesion with increased neurite outgrowth. The hypothesis that the neuronal cell body response to axotomy plays an important role in the conditioning lesion response is discussed.

Monocyte chemoattractant protein (MCP)-1 is rapidly expressed by sympathetic ganglion neurons following axonal injury.

EDI-immunoreactive macrophages, absent from the superior cervical ganglia (SCG) of normal rats, appear in these ganglia within 48h after postganglionic axotomy. Further, resident macrophages show changes after axotomy. Since chemokines function as chemoattractants and activators of leukocytes, the effects of axotomy on chemokine expression in the SCG were examined. Within 6 h after nerve transection, increases were seen in mRNA levels for monocyte chemoattractant protein (MCP)-1. MCP-1 mRNA was concentrated in a population of neurons, while MCP-1 protein was localized to endothelial cells. This axotomy-induced neuronal MCP-1 expression may trigger the infiltration and/or activation of macrophages in SCG after injury.

The levels of leukemia inhibitory factor mRNA in a Schwann cell line are regulated by multiple second messenger pathways.

Axotomy of sympathetic and sensory neurons leads to changes in their neuropeptide phenotypes. These changes are mediated in part by the induction of leukemia inhibitory factor (LIF) by nonneuronal cells. In the present study, we identified satellite/Schwann cells as a possible source of the injury-induced LIF. Using a Schwann cell line, SC-1 cells, we examined mechanisms of LIF induction. LIF mRNA levels increased rapidly when the cells were treated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, phorbol 12-myristate 13-acetate (PMA), or A23187. Among these reagents, PMA was the most efficacious. Inhibition of protein kinase C (PKC) by GF-1 09203X significantly reduced the PMA-induced LIF mRNA levels. As PKC is known to activate the extracellular signal-regulated kinase (ERK) signaling pathway, the involvement of this pathway in the PMA-stimulated induction of LIF mRNA was examined. Phosphorylation of ERKs was increased following PMA treatment in SC-1 cells. Moreover, inhibition of ERK kinase activity by PD98059 dramatically reduced PMA-stimulated phosphorylation of ERKs and induction of LIF mRNA. These results indicate that LIF mRNA levels can be regulated by ERK activation via stimulation of PKC in Schwann cells.

Nerve growth factor inhibits sympathetic neurons' response to an injury cytokine.

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

Chemical sympathectomy and postganglionic nerve transection produce similar increases in galanin and VIP mRNA but differ in their effects on peptide content.

Large changes in neuronal gene expression occur in adult peripheral neurons after axonal transection. In the rat superior cervical ganglion, for example, neurons that do not normally express vasoactive intestinal peptide (VIP) or galanin do so after postganglionic nerve transection. These effects of axotomy could result from a number of aspects of the surgical procedure. To test the idea that the important variable might be the disconnection of axotomized neuronal cell bodies from their target tissues, we examined the effects of producing such a disconnection by means of the compound 6-hydroxydopamine (6-OHDA), a neurotoxin that causes degeneration of sympathetic varicosities and avoids many of the complications of surgery. Two days after 6-OHDA treatment, VIP and galanin immunoreactivities had increased two- and 40-fold, respectively. Nevertheless, these increases were substantially smaller than the 30- and 300-fold changes seen after surgical axotomy. When expression of VIP and galanin was examined at the mRNA level, however, comparable increases were found after either procedure. The results indicate that chemical destruction of sympathetic varicosities produces an equivalent signal for increasing VIP and galanin mRNA as does axonal transection. The differences in the neuropeptide levels achieved suggests that peptide expression after nerve transection is regulated both at the mRNA and protein levels.

Changes in neuropeptide phenotype after axotomy of adult peripheral neurons and the role of leukemia inhibitory factor.

Adult peripheral neurons undergo dramatic shifts in gene expression following axotomy that are collectively referred to as the cell body reaction. Changes in neuropeptide expression are a prominent feature of these axotomized neurons. For example, while sympathetic, sensory, and motor neurons do not normally express the neuropeptides galanin and vasoactive intestinal peptide, they begin to do so within days after axotomy. In contrast, the expression of other peptides, which these neurons normally express, such as neuropeptide Y in sympathetic neurons and substance P in sensory neurons, is decreased. Recent studies in sympathetic neurons have demonstrated that leukemia inhibitory factor plays an important role in triggering these changes in neuropeptide phenotype in adult neurons. Future studies will be directed at determining to what extent LIF triggers the many other changes in gene expression after sympathetic axotomy and whether this cytokine plays a similar role in sensory and motor neurons.

Galanin expression in sympathetic ganglia after partial axotomy is highly localized to those neurons that are axotomized.

The neuropeptide phenotype of adult sympathetic neurons changes dramatically after postganglionic nerve transection. Studies, thus far, have been done on the superior cervical ganglion; however, one limitation of this preparation is that it is necessary to transect the postganglionic axons quite close to the ganglion. In the present study, we examined the effects of axonal damage on galanin-like immunoreactivity in the middle and inferior cervical ganglion complex. With these ganglia, it is possible to transect postganglionic axons at a considerable distance from their cell bodies and, therefore, to examine the extent to which local tissue damage, rather than specific axonal transection, is required for these changes in neuropeptide phenotype to occur. The anatomy of this system also allowed us to determine the extent to which the changes in galanin expression are restricted to those neurons that have been axotomized. The axons of a small population of the neurons in the middle and inferior cervical ganglia complex project into the cervical sympathetic trunk. Within two days after this trunk was transected, there was an increase in the level of galanin-like immunoreactivity in the complex and in the number of immunostained principal neurons. These neurons were concentrated primarily in the most rostral part of the complex. An increase in galanin-like immunoreactivity also occurred in response to the systemic administration of the sympathetic neurotoxin 6-hydroxydopamine. In that case, many more neurons were affected than after transection of the cervical sympathetic trunk, and the neurons were distributed evenly throughout the ganglion complex.(ABSTRACT TRUNCATED AT 250 WORDS)