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

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 ¼ 3/group). Equal numbers of spinalized rats in both groups received PRV-152 inoculations without CRD (non-stimulated; n ¼ 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.

Spinal Interneurons and Forelimb Plasticity after Incomplete Cervical Spinal Cord Injury in Adult Rats

“…PRV transneuronal labeling has not been shown definitively to be associated with function; however, it is noteworthy that we 1,70 and others 51,52 have observed delayed first-order labeling of motoneurons after SCI. Although this may be associated in part with deafferentation, it is more likely a reflection of neuromuscular junction dynamics post-injury 71 or changes in PRV receptors.…”

“…[47][48][49][50] Trans-synaptic tracing techniques 1,4,51,52 were used in the present study to anatomically characterize the distribution of interneurons within the ECRL motor circuit. The results show an extensive bilaterally distributed propriospinal network with pre-motor interneurons being prominent in the intermediate gray matter (laminae VII and X) of the midcervical spinal cord, ipsilateral to the motoneuron pool.…”

“…Although this may be associated in part with deafferentation, it is more likely a reflection of neuromuscular junction dynamics post-injury 71 or changes in PRV receptors. 51,52 Irrespective of the underlying mechanism, delayed first-order infection can influence quantitative analyses, most likely resulting in an underestimation of interneuronal labeling post-injury. We found no evidence of altered interneuronal distribution post-injury; therefore, conservative interpretations of these results would conclude that either there was no impact of injury and/or plasticity on the number of neurons within the ECRL circuitry or that changes in the circuitry were underrepresented.…”

Respiratory function following bilateral mid-cervical contusion injury in the adult rat

“…While there is a growing use of PRV to transynaptically trace neuronal connectivity following SCI (Bareyre, et al, 2004, Duale, et al, 2009, Duale, et al, 2010, Im, et al, 2008, Kim, et al, 2002, Lane, et al, 2009, Lane, et al, 2008b, Yu, et al, 2003), we (Lane, et al, 2009) and others (Duale, et al, 2009, Duale, et al, 2010) have reported a reduction in the extent of first-order PRV neuronal infection as a function of time after SCI. In other on-going studies (unpublished), we have determined that the rate of infection is delayed such that maximum first-order labeling of PhMNs occurs 24 hours later than normal.…”

Translational spinal cord injury research

“…Many physiological, neurophysiological, neurochemical, histological, imaging and behavioral outcome measures are presently available that can provide more comprehensive, quantitative assessments of motor, autonomic, and sensory functions at different spinal levels. There are also methods available for assessing neuroprotection relative to specific functions and their underlying motor neuron and intraspinal substrate circuits using conventional (McKenna et al, 2000) and, when feasible, transneuronal (Bareyre et al, 2004; Lane et al, 2008b, 2009; Duale et al, 2009, 2010) retrograde tracing in combination with anterograde tracing.…”