The role of c-AMP-dependent protein kinase in spinal cord and post synaptic dorsal column neurons in a rat model of visceral pain

Abstract

Visceral noxious stimulation induces central neuronal plasticity changes and suggests that the c-AMP-dependent protein kinase (PKA) signal transduction cascade contributes to long-term changes in nociceptive processing at the spinal cord level. Our previous studies reported the clinical neurosurgical interruption of post synaptic dorsal column neuron (PSDC) pathway by performing midline myelotomy effectively alleviating the intractable visceral pain in patients with severe pain. However, the intracellular cascade in PSDC neurons mediated by PKA nociceptive neurotransmission was not known. In this study, by using multiple experimental approaches, we investigated the role of PKA in nociceptive signaling in the spinal cord and PSDC neurons in a visceral pain model in rats with the intracolonic injection of mustard oil. We found that mustard oil injection elicited visceral pain that significantly changed exploratory behavior activity in rats in terms of decreased numbers of entries, traveled distance, active and rearing time, rearing activity and increased resting time when compared to that of rats receiving mineral oil injection. However, the intrathecal infusion of PKA inhibitor, H89 partially reversed the visceral pain-induced effects. Results from Western blot studies showed that mustard oil injection significantly induced the expression of PKA protein in the lumbosacral spinal cord. Immunofluorescent staining in pre-labeled PSDC neurons showed that mustard oil injection greatly induces the neuronal profile numbers. We also found that the intrathecal infusion of a PKA inhibitor, H89 significantly blocked the visceral pain-induced phosphorylation of c-AMP-responsive element binding (CREB) protein in spinal cord in rats. The results of our study suggest that the PKA signal transduction cascade may contribute to visceral nociceptive changes in spinal PSDC pathways.

Figure 1

The role of PKA inhibitor, H 89 in the control of rat exploratory behavioral activity was investigated by using a computerized photo-beam monitoring FlexField system. During a consecutive period of 45 minutes (nice intervals with five minutes per interval), six parameters were measured: entry activities (number of beam broken, in Panel A), traveled distance (Panel B), active time (Panel C), resting time (Panel D), rearing events(Panel E) and rearing time (Panel D). Left panels in each individual figure show the effect of the surgical procedure on the rat’s behavioral changes after placement of an intrathecal catheter (the groups shown as Surgery) when compared to naïve rats (the groups shown as Naive) following mineral oil and mustard oil treatments. There was no significant difference in the exploratory behavioral activity between naïve rats and animals receiving the surgical procedure within the same treatment group (NS: no significant, n=5 in each group with the same treatment). However, the intracolonic injection of mustard oil (groups shown as hatched bar) results significant changes in the exploratory behavioral activity when compared to that of mineral oil treatment (groups shown as open bar, *: p< 0.05, mustard oil vs mineral oil). Mustard oil injection decreases the entry activity, traveled distance, active time, rearing events and rearing time while increases resting time. In the groups of rats receiving H89 administration intrathecally, significantly reduced the effects of mustard oil-induced changes (#: p< 0.05 in each individual panels, H 89 treatment vs ACSF treatment) without affecting that of mineral oil-treated group (NS: no significant, n=8).

Figure 2

Detection of PKA protein in spinal cord tissue of rats at 30 and 60 minutes following intracolonic injection of mineral oil (as a control) and mustard oil (noxious stimulation). Upper panel: A representative picture of immunoblotted bands of PKA protein (42 KD) and β–actin (40 KD, as a loading control) from rats with different treatments. Lower panel: Bar graph summarizing the relative density of the immunoblot bands of PKA protein (as the ratio to the value of β–actin). *, p <0.05; the value from the group with mustard oil treatment vs. mineral oil treatment (n=5 in each group). Open bar, at 30 minutes post-injection; hatched bar, at 60 minutes post-injection.

Figure 3

Immunofluorescent staining of PKA protein expressed on a retrogradely pre-labeled PSDC neuron collected from a rat treated with mustard oil injection (60 minutes after injection). Retrogradely labeled profile of a PSDC neuron is clearly marked by the fluorescent dye (Red color, Panel A). Immunoreactivity for PKA protein could be found on the cytoplasma (Green color, Panel B). Panel C shows the merged result (Yellow color). Lower bar graph indicated the increased cellular profiles of PKA protein in PSDC neurons from rats treated with mustard oil when compared to the value of rats treated with mineral oil. (n=6 in each group). Open bar, mineral oil treatment (as a control) group; hatched bar: mustard oil treated group. Scale bar =5 μm.

Figure 4

Effect of intrathecal infusion with a PKA inhibitor, H89 (for 30 minutes) on the expression of phospho-CREB protein and β-actin in spinal cord in rats at 60 minutes after visceral painful stimulation. Upper Panel Representative immunoblot bands of phospho-CREB protein (45KD) andβ-actin (42 KD) expression. Lower Panel Bar graph showing the relative density of the immunoblotting bands of phospho-CREB protein 25 (ratio to the value of β-actin). (*, p <0.05; the value from mineral oil treatment vs. mustard oil treatment in both of ACSF-treated and H89-treated groups; #, p< 0.05, the value of rats with mustard oil treatment between ACSF treatment and H89 administration; n=6 in each group). Open bar, mineral oil-treated group (labeled as MI); hatched bar, mustard oil-treated group (labeled as MO).