Xuerong Miao

Functional up-regulation of P2X3 receptors in dorsal root ganglion in a rat model of bone cancer pain

Cancer‐induced bone pain remains a clinical challenge due to the poor understanding of the mechanisms. Recent study revealed extracellular adenosine triphosphate (ATP) and P2X receptors may be implicated in nociceptive signalling under cancer pain state. Therefore, here we investigated the potential role of P2X3 receptor in a rat model of bone cancer pain.

Bilateral downregulation of Nav1.8 in dorsal root ganglia of rats with bone cancer pain induced by inoculation with Walker 256 breast tumor cells

BackgroundRapid and effective treatment of cancer-induced bone pain remains a clinical challenge and patients with bone metastasis are more likely to experience severe pain. The voltage-gated sodium channel Nav1.8 plays a critical role in many aspects of nociceptor function. Therefore, we characterized a rat model of cancer pain and investigated the potential role of Nav1.8.MethodsAdult female Wistar rats were used for the study. Cancer pain was induced by inoculation of Walker 256 breast carcinosarcoma cells into the tibia. After surgery, mechanical and thermal hyperalgesia and ambulation scores were evaluated to identify pain-related behavior. We used real-time RT-PCR to determine Nav1.8 mRNA expression in bilateral L4/L5 dorsal root ganglia (DRG) at 16-19 days after surgery. Western blotting and immunofluorescence were used to compare the expression and distribution of Nav1.8 in L4/L5 DRG between tumor-bearing and sham rats. Antisense oligodeoxynucleotides (ODNs) against Nav1.8 were administered intrathecally at 14-16 days after surgery to knock down Nav1.8 protein expression and changes in pain-related behavior were observed.ResultsTumor-bearing rats exhibited mechanical hyperalgesia and ambulatory-evoked pain from day 7 after inoculation of Walker 256 cells. In the advanced stage of cancer pain (days 16-19 after surgery), normalized Nav1.8 mRNA levels assessed by real-time RT-PCR were significantly lower in ipsilateral L4/L5 DRG of tumor-bearing rats compared with the sham group. Western-blot showed that the total expression of Nav1.8 protein significantly decreased bilaterally in DRG of tumor-bearing rats. Furthermore, as revealed by immunofluorescence, only the expression of Nav1.8 protein in small neurons down regulated significantly in bilateral DRG of cancer pain rats. After administration of antisense ODNs against Nav1.8, Nav1.8 protein expression decreased significantly and tumor-bearing rats showed alleviated mechanical hyperalgesia and ambulatory-evoked pain.ConclusionsThese findings suggest that Nav1.8 plays a role in the development and maintenance of bone cancer pain.

Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA) targeting Toll-like receptor 4 (TLR4) gene in ameliorating lipopolysaccharide- (LPS-) induced acute lung injury (ALI). Methods. In vitro, alveolar macrophages (AMs) were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL) for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group), 300 μL of Ad-EGFP (Ad-EGFP group), or 300 μL of Ad-siTLR4 (Ad-siTLR4 group) and then were intravenously treated with LPS (50 mg/kg) to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

Acute PAR2 activation reduces GABAergic inhibition in the spinal dorsal horn.

We investigated the mechanism underlying inhibition of spinal dorsal horn GABAergic neurotransmission to elucidate the role of protease-activated receptor-2 (PAR2). Initially, we confirmed that PAR2 agonist SL-NH(2) applied intrathecally produced mechanical hyperalgesia. Then we performed patch-clamp experiments in substantia gelatinosa neurons of spinal cord slice, and found that spontaneous inhibitory post-synaptic currents (sIPSCs) were significantly decreased in both frequency and amplitude when neurons were incubated with PAR2 agonist SL-NH(2) for a brief time period (2 min). The GABA-mediated currents were significantly reduced, and there was no impact on glycine-mediated currents during this SL-NH(2) treatment. These results suggest that PAR2 activation enhanced the pain response, potentially via inhibition of dorsal horn GABAergic neurotransmission.

Acute PAR2 activation reduces α, β-MeATP sensitive currents in rat dorsal root ganglion neurons

It has been reported that proteinase-activated receptor 2 (PAR2) receptor activation enhances the animals pain response and PAR2 coexpresses with P2X3 in dorsal root ganglion neurons. However, whether PAR2 activation has a direct impact on P2X3 currents is still not clear. In this study, we performed the patch-clamp experiments in cultured dorsal root ganglion neurons and found that when incubated with trypsin or the PAR2 agonist SL-NH2 for a short time (3 min), instead of increasing, P2X3 currents amplitude decreased significantly. Meanwhile, the opening of P2X3 ion channel accelerated. Protein kinase A inhibitor H89 could not reverse above phenomenon, but played a synergistic effect on the contrary. These results suggest that the enhanced pain response caused by PAR2 activation is not through direct increase of the P2X3 current amplitude, and the acceleration of P2X3 opening may participate in the enhanced pain response in a long-time view. Moreover, protein kinase A does not participate in the inhibition of P2X3 currents caused by PAR2 activation.

Suppression of acute morphine withdrawal syndrome by adenovirus-mediated β-endorphin in rats.

BACKGROUND Endogenous β-endorphin (β-EP) in the central nervous system (CNS) is decreased upon opioid addiction. The current study examined whether exogenous β-EP, delivered using an adenoviral vector into the CNS could attenuate morphine withdrawal syndrome in rats. METHODS The model of opioid-dependent rats was set up by receiving subcutaneous injection of morphine using an escalating regimen for 6days (5, 10, 20, 40, 50, 60mg/kg, three times/day). The adenovirus mediated β-EP gene was constructed based on our previous work. The ilea of opioid-dependent rats were isolated and treated with the supernatant of Ad-NEP. The basic and naloxone-induced (4μm/l) contractions of dependent ilea were recorded. The Ad-NEP was injected into the left lateral ventricle of the addition rats. The expression of the β-EP gene was verified by radioimmunoassay of the cerebrospinal fluid (CSF) and immunocytochemistry for β-EP. Withdrawal syndrome was evaluated after intraperitoneal injection of naloxone. RESULTS The contractions of dependent ilea were attenuated with supernatant containing β-EP expressed by Ad-NEP. Injection of the Ad-NEP resulted in significant increases in β-EP level in the CSF and β-EP-positive neurons. Rats receiving adenovirus carrying the β-EP gene had significantly less severe withdrawal symptoms upon naloxone challenge. CONCLUSIONS Exogenous β-EP mediated by adenovirus could attenuate withdrawal syndrome in morphine-dependent rats.

Trypsin-protease activated receptor-2 signaling contributes to pancreatic cancer pain

Pain treatment is a critical aspect of pancreatic cancer patient clinical care. This study investigated the role of trypsin-protease activated receptor-2 (PAR-2) in pancreatic cancer pain. Pancreatic tissue samples were collected from pancreatic cancer (n=22) and control patients (n=22). Immunofluorescence analyses confirmed colocalization of PAR-2 and neuronal markers in pancreatic cancer tissues. Trypsin levels and protease activities were higher in pancreatic cancer tissue specimens than in the controls. Supernatants from cultured human pancreatic cancer tissues (PC supernatants) induced substance P and calcitonin gene-related peptide release in dorsal root ganglia (DRG) neurons, and FS-NH2, a selective PAR-2 antagonist, inhibited this effect. A BALB/c nude mouse orthotopic tumor model was used to confirm the role of PAR-2 signaling in pancreatic cancer visceral pain, and male Sprague-Dawley rats were used to assess ambulatory pain. FS-NH2 treatment decreased hunch scores, mechanical hyperalgesia, and visceromotor reflex responses in tumor-bearing mice. In rats, subcutaneous injection of PC supernatant induced pain behavior, which was alleviated by treatment with FS-NH2 or FUT-175, a broad-spectrum serine protease inhibitor. Our findings suggest that trypsin-PAR-2 signaling contributes to pancreatic cancer pain in vivo. Treatment strategies targeting PAR-2 or its downstream signaling molecules might effectively relieve pancreatic cancer pain.Pain treatment is a critical aspect of pancreatic cancer patient clinical care. This study investigated the role of trypsin-protease activated receptor-2 (PAR-2) in pancreatic cancer pain. Pancreatic tissue samples were collected from pancreatic cancer (n=22) and control patients (n=22). Immunofluorescence analyses confirmed colocalization of PAR-2 and neuronal markers in pancreatic cancer tissues. Trypsin levels and protease activities were higher in pancreatic cancer tissue specimens than in the controls. Supernatants from cultured human pancreatic cancer tissues (PC supernatants) induced substance P and calcitonin gene-related peptide release in dorsal root ganglia (DRG) neurons, and FS-NH2, a selective PAR-2 antagonist, inhibited this effect. A BALB/c nude mouse orthotopic tumor model was used to confirm the role of PAR-2 signaling in pancreatic cancer visceral pain, and male Sprague-Dawley rats were used to assess ambulatory pain. FS-NH2 treatment decreased hunch scores, mechanical hyperalgesia, and visceromotor reflex responses in tumor-bearing mice. In rats, subcutaneous injection of PC supernatant induced pain behavior, which was alleviated by treatment with FS-NH2 or FUT-175, a broad-spectrum serine protease inhibitor. Our findings suggest that trypsin-PAR-2 signaling contributes to pancreatic cancer pain in vivo. Treatment strategies targeting PAR-2 or its downstream signaling molecules might effectively relieve pancreatic cancer pain.

Posttraumatic stress disorder: from diagnosis to prevention

Posttraumatic stress disorder (PTSD) is a chronic impairment disorder that occurs after exposure to traumatic events. This disorder can result in a disturbance to individual and family functioning, causing significant medical, financial, and social problems. This study is a selective review of literature aiming to provide a general outlook of the current understanding of PTSD. There are several diagnostic guidelines for PTSD, with the most recent editions of the DSM-5 and ICD-11 being best accepted. Generally, PTSD is diagnosed according to several clusters of symptoms occurring after exposure to extreme stressors. Its pathogenesis is multifactorial, including the activation of the hypothalamic–pituitary–adrenal (HPA) axis, immune response, or even genetic discrepancy. The morphological alternation of subcortical brain structures may also correlate with PTSD symptoms. Prevention and treatment methods for PTSD vary from psychological interventions to pharmacological medications. Overall, the findings of pertinent studies are difficult to generalize because of heterogeneous patient groups, different traumatic events, diagnostic criteria, and study designs. Future investigations are needed to determine which guideline or inspection method is the best for early diagnosis and which strategies might prevent the development of PTSD.