PKCγ/P2X3 Pathway Mediates Visceral Nociception And Hyperalgesia Following Exposure To PTSD-like Stress

Background and objectivePost-traumatic stress disorder (PTSD) is classified as a mental and somatoform disorder, the patient with which has experienced unexpected extreme traumatic events that is life-threatening and emergent. PTSD will cause acute, chronic or delayed mental and somatoform disorder. The characteristic symptoms of PTSD include persistent re-experiencing of the traumatic event, persistent avoidance of stimuli associated with the trauma, emotional numbing and hyperarousal with a chronic processing. These symptoms have the significant impacts on the quality of life of patients and cause social dysfunction and maladjustment problems. At present, with the increasing emergence of accidental events, specifically in the challenges of high-tech warfare for the soldiers in the modern society, the incidence of PTSD shows significant rising tendency.There is an intimate connection between PTSD and visceral pain. Visceral sensitization/desensitization related diseases exposed to PTSD-like stress, such as irritable bowel syndrome (IBS), interstitial cystitis were very likely to suffer from PTSD. It was indicated that the PTSD-like stress is a determinative factor to trigger the first onset or exacerbation of visceral hyperalgesia. Previous studies on PTSD have focused predominantly on the hypothalamic, hippocampus, amygdala, medial prefrontal cortex, and anterior cingulate of the brain. However, the efficacy of the drug target on the hypothalamic-pituitary-adrenal axis poor response to the visceral hyperalgesia. However, there is a growing appreciation that the dorsal horn neurons of the spinal cord and the dorsal root ganglia (DRG) are also heavily affected by the stress, which can last for weeks. Thus, spinal and DRG sensitization is presently well-recognized as an important factor to maintain allodynia and hyperalgesia in various models of chronic pain and stress-induced visceral hypersensitivity. So the molecular mechanisms and prevention strategy has been an active research area in recent years. However, the underlying molecular mechanisms altered central and peripheral neurons plasticity about the visceral sensitization/desensitization following exposure to PTSD-like stress remain unclarified. As thus, exploring the underlying molecular mechanisms and potential prevention strategy for visceral sensitization/desensitization has attracted the most attention in recent years.Previous studies indicate that the dorsal horn neurons of the spinal cord and DRG are also heavily affected by the stress, which can last for weeks. Stress-induced spinal and DRG sensitization has been an active research area in recent years. However, the underlying molecular mechanisms of the visceral sensitization/desensitization following exposure to PTSD-like stress remain unclarified.The gamma isoform of protein kinase C (PKCy) is a well-dispersed distributed throughout the nervous system, particularly in the interneurons of the inner part of lamina II of the dorsal horn, and purinergic P2X3 (P2X3) predominantly expressed in a subset of small-to medium-sized nociceptive neurons. Numerous studies have demonstrated that PKCy/P2X3 mediated the nociceptive signaling process and the development and progression of hyperalgesia. The rest PKCy is in the inactive form and the external stimuli can activate PKCy by increasing Ca2+ of neurons. The active PKCy is involved in modulation of channel conductivity, releasing the excitatory neurotransmitters and increasing the trafficking of receptors of pain, and then spur a cascade of reactions. Previous studies implied that PKCy expression is upregulated under pain conditions, resulting in neuropathic pain, cancer pain and inflammatory pain in animal models. In addition, GF109203X9 (PKCy receptor antagonist) administered through the intrathecal catheter can reverse the hyperalgesia and PKCy gene knockout mice also show reduced the hyperalgesia. All these growing evidences indicated PKCy might play a pivotal role in the generation of nociception. P2X3 expression is significantly increased under inflammatory, chronic pain, cancer pain and in neuropathic pain sensation in animal models. P2X3 mediated the adenosine triphosphate (ATP)-activated currents were enhanced. This excitation was mimicked by application of alpha, beta-methylene adenosine triphosphate (α,β-me ATP) and attenuated by TNP-ATP, a potent antagonist of P2X3 receptors. P2X3 gene knockout mice also show reduced the pain sensation. Some studies have been an interaction between PKCγ and P2X3, and co-regulation the generation of pain sensitization/desensitization. In the intracellular N-terminus of the P2X3 receptor is a conserved consensus sequence for protein kinase C (PKC)-mediated phosphorylation. The α,β-me ATP activated currents were reduced by administration of PKC inhibitors or changing the binding site. The α,β-me ATP activated currents were also increased by action of PKC.So we hypothesize that PKCy/P2X3 mediated visceral nociception and neuronal sensitization following exposure to PTSD-like stress of rats. In this study, we use the behavioral approach to indicate that the SPS treatment increased both visceromotor response (VMR) and abdominal withdrawal reflex (AWR) score in response to phasic ascending colorectal distention (CRD) over time following exposure to PTSD-like stress; we use both immunostaining and western blotting approaches to demonstrate that the SPS-induced expression of PKCy increased in the spinal cord and P2X3 increased in the DRG of rats over time after SPS treatment; We administrate the specific P2X3 receptor antagonist TNP-ATP and PKCy inhibitors GF109203X to effect the behavioral changes; Whole-cell patch clamp were used to record the characterization of P2X3 receptor channels in DRG neurons following exposure to PTSD-like stress. The purpose of this study was to provide novel insights into visceral sensitization/desensitization and explore the good foundation of stress induced clinical disease.Methods1. Experiments were done on on adult female Sprague-Dawley rats. We use the single-prolonged stress (SPS) and combined with the electric foot shock to establish animal model of visceral hyperalgesia for PTSD.2. VMR and AWR were measured by grading the behavioral response of rats to phasic CRD at various time points (on days 0,1,6,7,9,14,21,28 and 29) in rats following exposed to PTSD-like stress.3. Immunofluorescence staining and western blotting were performed using to examine the PKCγ protein expression in the spinal cord and P2X3 receptor of the DRG over time following exposed to PTSD-like stress.4. The effects of intrathecal GF109023X (the specific PKCγ inhibitor) and TNP-ATP (the specific P2X3 inhibitor) on VMR and AWR responses to CRD following exposure to PTSD.5. Whole-cell patch clamp were used to record the changes of α,β-me ATP-induced inward currents in the DRG neurons and the effect of TNP-ATP on α,β-me ATP-induced current following exposure to PTSD-like stress.ResultsPart 1 The alteration of chronic visceral hyperalgesia over time following exposure to PTSD-like stress1. VMR in response to CRD over time following exposure to PTSD-like stressThe VMR to CRD was recorded at different time points (days 0,1,6,7,9,14,21,28 and 29) following exposure to PTSD-like stress. On day 1, the rats exposed to PTSD-like stress exhibited an analgesic response and its VMR to CRD at 40 and 60 mmHg was reduced compared with the control group. On day 6, the VMR returned to the baseline value. However, at 7 days exposure to SPS, VMR significantly increased compared with control group and this increase persisted for 14 days,21 days,28 days, with the peak on day 9. On day 29, the VMR decreased to a baseline level.2. AWR in response to CRD over time following exposure to PTSD-like stressThe AWR to CRD was recorded at different time points (days 0,1,6,7,9,14,21,28 and 29) following exposure to PTSD-like stress, exhibiting a consistent trend in the changes of VMR. On day 1, the rats exposed to PTSD-like stress exhibited an analgesic response and its AWR to CRD at 40 and 60 mmHg was reduced compared with the control group, respectively. On day 6, the AWR returned to the baseline value. However, at 7 days exposure to SPS, AWR significantly increased, when compared with control group and this increase persisted for 14 days,21 days,28 days, with the peak on day 9. On day 29, the AWR decreased to a baseline level.Part 2 PKCy mediates visceral nociception and hyperalgesia following exposure to PTSD-like stress of rats in the spinal cord1. PKCy expression in the dorsal horn of spinal cord over time following exposure to PTSD-like stressThe most striking PKCy immunostaining neurons were located in the lamina Ⅰ, Ⅱ and Ⅲ in the dorsal horn of the L4-6 spinal cord, especially in the inner plexus of lamina Ⅱ and ventral to the plexus in lamina Ⅲ. PKCγ protein exhibited a down-regulated expression in the dorsal horn of the spinal cord on day 1 exposed to PTSD-like stress compared with the control rats. However, PKCy expression was replenished on day 6 under PTSD-like stress. However,as early as 7 days after exposure to PTSD-like stress, PKCy expression significantly increased in the dorsal horn of the spinal cord compared with the control group. And this increase persisted on 28 days, with the maximal expression of PKCy on day 9. On day 29 under exposure to PTSD-like stress, PKCγ reduced afterward, but it is merely a subtle change, relative to control rats.Western blot analysis was a consistent trend with immunofluorescence assay. PKCy protein exhibited a down-regulated expression on day 1 exposed to PTSD-like stress compared with the control rats. However, PKCy expression was replenished on day 6 under PTSD-like stress. As early as 7 days, PKCy expression significantly increased compared with the control group. And this increase persisted on 14,21 and 28 days, with the maximal expression of PKCy on day 9. On day 29 under exposure to PTSD-like stress, PKCy reduced afterward, but it is merely a subtle change, relative to control rats.2. Effects of GF109023X on VMR and AWR to CRD and on PKCy expression following exposure to PTSD-like stressSince the peaking PKCy expression was on day 9 after exposure to SPS, we chose this time point to investigate the dose-response effect of GF109023X(0.05-0.50 nmol/10 μL) on VMR and AWR to CRD, and of PKCy expression, determined the effective dose that completely inhibits.GF109203X at low doses ranging from 0.05-0.15 nmol/10μL has no significant effect on VMR when gradually increasing intensity of phasic CRD. However, GF109023X at 0.30 nmol/10 μL significantly attenuated VMR to CRD at 40 mmHg on day 9 after exposure to SPS group compared with the vehicle group but no such effect was recorded at 60 mmHg. GF109023X at 0.50 nmol/10 μL completely inhibited the VMR to CRD at both 40 mmHg and 60 mmHg on day 9 after exposure to SPS group compared with the vehicle group.GF109203X at low doses (0.05-0.15 nmol/10 μL) did not significantly affect AWR to the graded intensities of phasic CRD. However, GF109023X at 0.30 nmol/10 μL significantly attenuated AWR to CRD at 40 mmHg on day 9 after exposure to SPS group compared with the vehicle group, but no such effect was recorded at 60 mmHg. GF109023X at 0.50 nmol/10 μL completely inhibited the AWR to CRD at both 40 mmHg and 60 mmHg on day 9 after exposure to SPS group compared with the vehicle group.In addition, the administration of GF109203X at 0.50 nmol/10 μL abolished visceral hyperalgesia and attenuated SPS-induced increase in VMR and AWR to CRD at 40 and 60 mmHg on days 7,14,21 and 28 after exposure to PTSD-like stress, when compared with vehicle injection. In control rats, GF109203X had no significant effects on the VMR and AWR to CRD.Part 3 P2X3 mediates visceral nociception and hyperalgesia following exposure to PTSD-like stress of rats in the DRG1. P2X3 expression in the DRG over time following exposure to PTSD-like stressThe P2X3 receptor is cloned and shown to be largely located on nociceptive sensory neurons in the DRG. P2X3 receptors are predominantly expressed in a subset of small-to medium-sized nociceptive neurons. P2X3 immunostaining was measured at time points 0, 1.6,7,14,21 days in rats exposed to SPS. In L4-6 DRG neurons, strong P2X3 immunostaining was located in the small and medium diameter neurons, consisting of strongly immunoreactive cell bodies. By immunofluorescence analysis, SPS-treated rats exhibited down-regulated P2X3 expression in DRG on day 1 after exposure to SPS compared with control rats. However, the neuronal stores were replenished on day 6 after exposure to SPS. P2X3 expression in DRG was significantly increased on day 7 compared with the control rats. The up-regulated expression was maintained from day 14 to day 21 in SPS-treated rats compared with the control rats. The peak P2X3 expression was reached on day 7.2. Effects of TNP-ATP on VMR and AWR to CRD following SPS exposureIntrathecal administration of 50 μg/5μL TNP-ATP 10 min before CRD abolished visceral hyperalgesia and attenuated SPS-induced increase in VMR to CRD at 40 and 60 mmHg at day 7 after SPS when compared with vehicle injection. In control rats TNP-ATP had no significant effects on the VMR to CRD and caused significant reduction of AWR to CRD at 40 and 60 mmHg in SPS-treated rats compared with vehicle injection. In control rats, TNP-ATP had no significant effects on the AWR to CRD.3. Characterization of purinergic P2X3 receptor channels in DRG neurons in a rat of chronic visceral hyperalgesia following SPS exposureThe α,β-me ATP, a selective agonist of P2X3 receptors) evoked a fast desensitizing inward current (Iα,β-me ATP) in the DRG neurons of control and SPS-treated rats. The average peak current density of the Iα,β-me ATP in SPS-treated group increased more than 3.6-fold. This value was significantly bigger than that of the control group. This suggests that chronic visceral hyperalgesia is associated with potentiation of α,β-me ATP-activated currents in DRG neurons following exposure to SPS. At 100 nM, the selective P2X3 antagonist TNP-ATP markedly blocked all fast α,β-me ATP-induced inward currents in the DRG neurons both in controls and SPS-treated rats, suggesting that P2X3 receptor mediated the current responses. After 5 minutes wash out (time necessary for recovery from α,β-me ATP-induced desensitization), the current amplitude was almost completely restored.Conclusion1. The SPS and combined with the electric foot shock can successfully establish animal model of visceral hyperalgesia for PTSD; PTSD-like conditions alters visceral sensitivity over time.2. PKCy mediates visceral nociception and hyperalgesia following exposure to PTSD-like stress of rats in the spinal cord; PKCy expression has been changed over time following exposure to PTSD-like stress; GF109023X attenuates SPS-induced visceral hyperalgesia.3. P2X3 mediates visceral nociception and hyperalgesia following exposure to PTSD-like stress of rats in the DRG; P2X3 expression have been changed over time following exposure to PTSD-like stress; TNP-ATP can abolish SPS-induced visceral pain.