Noxious Colorectal Distention in Spinalized Rats Reduces Pseudorabies Virus Labeling of Sympathetic Neurons

Authors

Hanad Duale, Travis S. Lyttle, Bret N. Smith, Alexander G. Rabchevsky

Abstract

The retrograde transsynaptic tracer pseudorabies virus (PRV) has been widely used as a marker for synaptic connectivity in the spinal cord. Notably, the PRV-152 construct expresses enhanced green fluorescent protein (EGFP). We recently reported a significant attenuation of PRV-152 labeling of the intermediolateral cell column (IML) and celiac ganglia after complete T4 spinal cord transection versus sham injury in rats at 96 h after PRV-152 inoculation of the left kidney. Here we found a significant increase in noxious colorectal distention (CRD)-evoked c-Fos expression in spinal cords of injured versus sham rats without PRV infection. In order to assess whether enhancing neuronal activity in spinalized rats might increase PRV-152 labeling, we subjected awake spinalized rats to 1.5 h of intermittent noxious CRD either: (1) just prior to inoculation, or (2) 96 h after inoculation (n = 3/group). Equal numbers of spinalized rats in both groups received PRV-152 inoculations without CRD (non-stimulated; n = 3/group). At 96 h post-inoculation fixed spinal cords and left celiac ganglionic tissues were assessed for the distribution and quantification of EGFP-labeled cells. The injured cohort that received CRD just prior to PRV injection showed a significant reduction in EGFP-labeled cells in both the IML and left celiac ganglion compared to non-stimulated injured rats. In contrast, the injured cohort that received CRD 96 h after PRV-152 inoculation showed no differences in EGFP-labeled cell numbers in the IML or celiac ganglia versus non-stimulated injured rats. Interestingly, microglia near c-Fos-positive cells after acute CRD appeared more reactive compared to non-stimulated spinalized rats, and activated microglial cells markedly reduce viral transduction and progression following PRV inoculation of the CNS. Hence our results imply that increased CRD-induced c-Fos expression in the injured paradigm, prior to but not after PRV injection, further attenuates PRV-152 uptake, perhaps through changes in neuronal activity and/or innate neuro-immune responses.