Neural pathways from hypothalamic orexin neurons to the ventrolateral preoptic area mediate sleep impairments induced by conditioned fear.

Introduction: Fear and sleep impairments common co-exist, but the underlying mechanisms remain unclear. Hypothalamic orexinergic neurons are involved in the regulation of sleep-wake and fear expression. The ventrolateral preoptic area (VLPO) is an essential brain region to promote sleep, and orexinergic axonal fibers projecting to the VLPO are involved in the maintenance of sleep-wake. Neural pathways from hypothalamic orexin neurons to the VLPO might mediate sleep impairments induced by conditioned fear. Methods: To verify above hypothesis, electroencephalogram (EEG) and electromyogram (EMG) were recorded for analysis of sleep-wake states before and 24 h after conditioned fear training. The retrograde tracing technique and immunofluorescence staining was used to identify the projections from the hypothalamic orexin neurons to the VLPO and to observe their activation in mice with conditioned fear. Moreover, optogenetic activation or inhibition of hypothalamic orexin-VLPO pathways was performed to observe whether the sleep-wake can be regulated in mice with conditioned fear. Finally, orexin-A and orexin receptor antagonist was administered into the VLPO to certify the function of hypothalamic orexin-VLPO pathways on mediating sleep impairments induced by conditioned fear. Results: It was found that there was a significant decrease in the non-rapid eye movement (NREM) and rapid eye movement (REM) sleep time and a significant increase in the wakefulness time in mice with conditioned fear. The results of retrograde tracing technique and immunofluorescence staining showed that hypothalamic orexin neurons projected to the VLPO and observed the CTB labeled orexin neurons were significantly activated (c-Fos+) in the hypothalamus in mice with conditioned fear. Optogenetic activation of hypothalamic orexin to the VLPO neural pathways significantly decreased NREM and REM sleep time and increased wakefulness time in mice with conditioned fear. A significant decrease in NREM and REM sleep time and an increase in wakefulness time were observed after the injection of orexin-A into the VLPO, and the effects of orexin-A in the VLPO were blocked by a pre-administrated dual orexin antagonist (DORA). Conclusion: These findings suggest that the neural pathways from hypothalamic orexinergic neurons to the VLPO mediate sleep impairments induced by conditioned fear.

Synergistic interaction between DAMGO-NH2 and NOP01 in peripherally acting antinociception in two mouse models of formalin pain.

The most commonly used opioid analgesics are limited by their severe side-effects in the clinical treatment of pain. Preliminary reports indicate that the combination of classical opioids and N/OFQ receptor (NOP) ligands may be an effective strategy to reduce unwanted side-effects and improve antinociception. But the interaction of these two receptor ligands in pain regulation at the peripheral level remains unclear. In this study, the antinociception of a designed amide analogue of the mu opioid receptor (MOP) peptide agonist DAMGO, DAMGO-NH2, and its antinociceptive interaction with the peripherally limited NOP peptide agonist NOP01 was investigated in two mouse models of formalin pain. Our results showed that DAMGO-NH2 acted as a MOP agonist in in vitro functional assays. Moreover, local subcutaneous or intraplantar injection of DAMGO-NH2 exerted dose-related antinociception in both phases of the formalin orofacial and intraplantar pain, which could be mediated by the classical opioid receptor. Peripheral but not central pretreatment with the peripherally restricted opioid antagonist naloxone methiodide inhibited local DAMGO-NH2-induced antinociception, supporting the involvement of the peripheral opioid receptor in local DAMGO-NH2-induced antinociception. Furthermore, co-administration of the inactive doses of DAMGO-NH2 and NOP01 produced effective antinociception. More importantly, isobolographic analysis indicates that the combination of DAMGO-NH2 and NOP01 elicited supra-additive antinociception in these two models of formalin pain. In addition, the combination of DAMGO-NH2 and NOP01 did not change motor function of mice in rotarod test. In conclusion, these data suggest that peripheral DAMGO-NH2 and particularly its combination therapy with NOP01 may be effective for pain management.

OCP002, a Mixed Agonist of Opioid and Cannabinoid Receptors, Produces Potent Antinociception With Minimized Side Effects.

BACKGROUND: Increasing attention has been attracted to the development of bifunctional compounds to minimize the side effects of opioid analgesics. Pharmacological studies have verified the functional interaction between opioid and cannabinoid systems in pain management, suggesting that coactivation of the opioid and cannabinoid receptors may provide synergistic analgesia with fewer adverse reactions. Herein, we developed and characterized a novel bifunctional compound containing the pharmacophores of the mu-opioid receptor agonist DALDA and the cannabinoid peptide VD-Hpα-NH2, named OCP002. METHODS: The opioid and cannabinoid agonistic activities of OCP002 were investigated in calcium mobilization and western blotting assays, respectively. Moreover, the central and peripheral antinociceptive effects of OCP002 were evaluated in mouse preclinical models of tail-flick test, carrageenan-induced inflammatory pain, and acetic acid-induced visceral pain, respectively. Furthermore, the potential opioid and cannabinoid side effects of OCP002 were systematically investigated in mice after intracerebroventricular (ICV) and subcutaneous (SC) administrations. RESULTS: OCP002 functioned as a mixed agonist toward mu-opioid, kappa-opioid, and cannabinoid CB1 receptors in vitro. ICV and SC injections of OCP002 produced dose-dependent antinociception in mouse models of nociceptive (the median effective dose [ED50] values with 95% confidence interval [CI] are 0.14 [0.12-0.15] nmol and 0.32 [0.29-0.35] µmol/kg for ICV and SC injections, respectively), inflammatory (mechanical stimulation: ED50 values [95% CI] are 0.76 [0.64-0.90] nmol and 1.23 [1.10-1.38] µmol/kg for ICV and SC injections, respectively; thermal stimulation: ED50 values [95% CI] are 0.13 [0.10-0.17] nmol and 0.23 [0.08-0.40] µmol/kg for ICV and SC injections, respectively), and visceral pain (ED50 values [95% CI] are 0.0069 [0.0050-0.0092] nmol and 1.47 [1.13-1.86] µmol/kg for ICV and SC injections, respectively) via opioid and cannabinoid receptors. Encouragingly, OCP002 cannot cross the blood-brain barrier and exerted nontolerance-forming analgesia over 6-day treatment at both supraspinal and peripheral levels. Consistent with these behavioral results, repeated OCP002 administration did not elicit microglial hypertrophy and proliferation, the typical features of opioid-induced tolerance, in the spinal cord. Furthermore, at the effective analgesic doses, SC OCP002 exhibited minimized opioid and cannabinoid side effects on motor performance, body temperature, gastric motility, physical and psychological dependence, as well as sedation in mice. CONCLUSIONS: This study demonstrates that OCP002 produces potent and nontolerance-forming antinociception in mice with reduced opioid- and cannabinoid-related side effects, which strengthen the candidacy of bifunctional drugs targeting opioid/cannabinoid receptors for translational-medical development to replace or assist the traditional opioid analgesics.

Inhibitory effects of antibiotic-induced gut microbiota depletion on acute itch behavior in mice.

BACKGROUND: The gut microbiota is known to be associated with the regulation of many neurological diseases and behaviors, including chronic pain. However, it is unclear whether the gut microbiota is critical to the itch sensation. In this study, we investigated the effects of gut microbiota depletion on acute itch. METHODS: First, an antibiotic cocktail was orally administered to deplete the gut microbiota in male C57BL/6 mice. Then, pruritogens were intradermally injected to induce acute itch behavior. In addition, antibiotic-treated mice received transplantation of fecal microbiota from untreated mice, followed by tests for acute itch. The changes in c-Fos expression in trigeminal ganglia (TG) neurons were also investigated by immunofluorescence staining. RESULTS: Our results indicated that chronic antibiotic treatment significantly reduced the diversity and richness of the gut microbiota of mice. Compared to vehicle-treated mice, antibiotic-treated mice showed reductions in acute itch behavior induced by compound 48/80, chloroquine (CQ), and serotonin (5-HT), respectively. Moreover, repositioning of microbiota reversed the reductions in acute itch behavior in antibiotic-treated mice. In addition, immunofluorescence staining revealed that antibiotic-treated mice displayed decreased c-Fos expression in ipsilateral TG compared to controls. CONCLUSIONS: Our study, for the first time, discovered that antibiotic-induced gut microbiota depletion could reduce acute itch behavior, which may be connected with decreased TG neuronal activity.

A potential biomarker based on clinical-radiomics nomogram for predicting survival and adjuvant chemotherapy benefit in resected node-negative, early-stage lung adenocarcinoma.

BACKGROUND: We aimed to construct a clinical-radiomics nomogram to predict disease-free survival (DFS) and the added survival benefit of adjuvant chemotherapy (ACT) for node-negative, early-stage (I-II) lung adenocarcinoma (ADC). METHODS: In this retrospective study including 310 patients from two independent cohorts, the CT-derived radiomics features were selected by least absolute shrinkage and selection operator Cox regression to generate a radiomics signature associated with DFS. The radiomics signature was incorporated to construct a clinical-radiomics nomogram along with the independent clinical risk predictors. The model performance was evaluated with reference to discrimination quantified by Harrell concordance index (C-index), integrated discrimination improvement (IDI) and net reclassification index (NRI), calibration and clinical utility. The risk score (RS) for clinical-radiomics nomogram was calculated. The association between ACT and survival benefit was assessed in high and low RS subgroup. RESULTS: The clinical-radiomics nomogram achieved the highest C-index of 0.822 [95% confidence interval (CI): 0.769, 0.876] in training cohort and 0.802 (95% CI: 0.716, 0.888) in validation cohort. The incorporation of radiomics signature into clinical-radiomics nomogram showed an incremental benefit over clinical nomogram according to the improved NRI and IDI. The calibration curves and decision curve analysis further verified the clinical utility of clinical-radiomics nomogram. Further, patients with high RS based on clinical-radiomics nomogram were more prone to benefit from ACT. CONCLUSIONS: The clinical-radiomics nomogram approach can feasibly conduct risk prediction and have potential to identify the beneficiaries of ACT among patients with node-negative, early-stage ADC, which might serve as a helpful tool in informing therapeutic decision-making.

Spinal microglia-derived TNF promotes the astrocytic JNK/CXCL1 pathway activation in a mouse model of burn pain.

Burn injury-induced pain (BIP) is an extremely complicated condition usually resistant to analgesic drugs, while its pathogenesis remains unknown. Considerable attention has been attracted to elucidate the glial mechanisms in chronic pain. In this study, we initiatively used a mouse model of second-degree BIP to investigate the underlying non-neuronal mechanisms at the spinal cord level. Our behavioral results showed that hind-paw burn injury caused persistent allodynia and hyperalgesia for 2 weeks in mice. Further studies revealed that both microglia and astrocytes activated in a spatially- and temporally-dependent manner in spinal cord after burn injury. In addition, the phosphorylated p38 mitogen-activated protein kinase (MAPK)-mediated tumor necrosis factor (TNF) release in spinal microglia is essentially attributed to the early stage of BIP, while the c-Jun N-terminal kinase (JNK) MAPK-dependent chemokine CXCL1 expression is mainly involved in the maintenance of pain hypersensitivity. Most strikingly, burn injury-induced pain symptoms and the activation of astrocytes were significantly suppressed by TNF inhibitor Thalidomide. On the contrary, intrathecal injection of TNF caused apparent pain hypersensitivity, accompanied by the activation of astrocytes and the upregulation of CXCL1 via the JNK MAPK signaling pathway, indicating that TNF is the key cytokine in the interaction between microglia and astrocytes at the spinal level. Moreover, treatment with the CXCR2 receptor antagonist SB225002 to block the biological activities of CXCL1 significantly attenuated the mechanical allodynia and thermal hyperalgesia in this BIP model. Taken together, this study indicates that intervention of glial pathways provides a new perspective in the management of BIP.

NOP01, a NOP receptor agonist, produced potent and peripherally restricted antinociception in a formalin-induced mouse orofacial pain model.

Orofacial pain is one of the most common medical challenges. A preliminary report indicates that the NOP receptor may act as a therapeutic target in orofacial pain. Previous studies have shown that [(pF)Phe4, Aib7, Aib11, Arg14, Lys15]N/OFQ-NH2 (NOP01) functions as a potent NOP receptor peptide agonist. This work aims to investigate the antinociception of NOP01 and its possible action mechanisms in a formalin-induced mouse orofacial pain model at different levels. Our results demonstrated that local, intraperitoneal (i.p.) or intrathecal (i.t.) injection of NOP01 produced dose-related antinociception in both phases of the formalin pain, which could be inhibited by the NOP receptor antagonist but not the classical opioid receptor antagonist. Furthermore, the antinociception induced by systemic NOP01 was blocked by local but not spinal pretreatment with the NOP receptor antagonist, suggesting the involvement of the peripheral NOP receptor in NOP01-induced systemic antinociception. Moreover, local injection of NOP01 markedly suppressed the expression of c-Fos protein induced by formalin in ipsilateral trigeminal ganglion (TG) neurons. In conclusion, this work suggests that NOP01 exerts significant antinociception on orofacial pain at both peripheral and spinal levels via the NOP receptor. Notably, NOP01 cannot readily penetrate the blood-brain barrier. Thus, NOP01 may behave as a potential compound for developing peripherally restricted analgesics.

Development of Multifunctional and Orally Active Cyclic Peptide Agonists of Opioid/Neuropeptide FF Receptors that Produce Potent, Long-Lasting, and Peripherally Restricted Antinociception with Diminished Side Effects.

We previously reported that a multifunctional opioid/neuropeptide FF receptor agonist, DN-9, achieved peripherally restricted analgesia with reduced side effects. To develop stable and orally bioavailable analogues of DN-9, eight lactam-bridged cyclic analogues of DN-9 between positions 2 and 5 were designed, synthesized, and biologically evaluated. In vitro cAMP assays revealed that these analogues, except 7, were multifunctional ligands that activated opioid and neuropeptide FF receptors. Analogue 1 exhibited improved potency for κ-opioid and NPFF2 receptors. All analogues exhibited potent, long-lasting, and peripherally restricted antinociception in the tail-flick test without tolerance development after subcutaneous administration and produced oral analgesia. Oral administration of the optimized compound analogue 1 exhibited powerful, peripherally restricted antinociceptive effects in mouse models of acute, inflammatory, and neuropathic pain. Remarkably, orally administered analogue 1 had no significant side effects, such as tolerance, dependence, constipation, or respiratory depression, at effective analgesic doses.

Experimental Research on Viscosity Characteristics of Grouting Slurry in a High Ground Temperature Environment.

The grouting method is a technical means to prevent and control the thermal damage of the tunnel with high ground temperature in the underground hot water area, and the viscosity characteristic of the slurry is the key factor of grouting treatment. When grouting in high ground temperature geological conditions, the slurry inevitably has both time-varying and temperature-varying characteristics of viscosity in the process of filling high-temperature fissures and plugging geothermal water. At present, the research on the viscosity characteristics of slurry at high temperature is rarely reported in the literature. In this paper, laboratory tests were carried out to measure the time-varying viscosity of ordinary cement slurry; cement-sodium silicate slurry, widely used in engineering; and high ground temperature grouting slurry (HGTGS), independently developed by our research group, at different preheating temperatures (20, 40, 60, 80 °C). The viscosity function curves of the three kinds of slurry were obtained by function fitting method, and the viscosity variation law of slurry was analyzed. The study found that the time-varying process of viscosity of the cement-sodium silicate slurry and the HGTGS in specific temperature conditions can be divided into two stages: The slow rising period of viscosity and the rapid rising period of viscosity. Whereas, the time-varying process of viscosity of ordinary cement slurry in specific temperature conditions includes only one stage of the slow rising period of viscosity. The viscosity of ordinary cement slurry and cement-sodium silicate slurry increases with the increase of temperature, while the viscosity of the HGTGS decreases with the increase of temperature. There are corresponding viscosity time-varying equations for the three kinds of slurry in different temperature conditions. The viscosity time-varying equation of the ordinary cement slurry accords with the linear function form, and the viscosity time-varying equation of the cement-sodium silicate slurry accords with the power-law function form. The viscosity time-varying equation of the HGTGS conforms to the exponential function form. On this basis, the unified description equation of the viscosity characteristics of grouting slurry in high ground temperature and the applicability of different types of grouting materials are obtained. This study has a certain reference value and guidance for theoretical analysis, numerical simulation and engineering application of grouting in high ground temperature environment.

VF-13, a chimeric peptide of VD-hemopressin(α) and neuropeptide VF, produces potent antinociception with reduced cannabinoid-related side effects.

Pharmacological evidence indicated a functional interaction between neuropeptide FF (NPFF) and cannabinoid systems, and the cannabinoids combined with the NPFF receptor agonist neuropeptide VF (NPVF) produced antinociception without tolerance. In the present study, VF-13, a chimeric peptide containing the pharmacophores of the endogenous cannabinoid peptide VD-hemopressin(α) (VD-Hpα) and NPVF, was synthesized and pharmacologically evaluated. In vitro, VF-13 significantly upregulated the phosphorylated level of extracellular signal-regulated kinase 1/2 (ERK1/2) in CHO cells stably expressing CB1 receptors and inhibited forskolin-induced cAMP accumulation in HEK293 cells stably expressing NPFF1 or NPFF2 receptors. Moreover, VF-13 induced neurite outgrowth in Neuro 2A cells via CB1 and NPFF receptors. These results suggest that VF-13 exhibits multifunctional agonism at CB1, NPFF1 and NPFF2 receptors in vitro. Interestingly, intracerebroventricular VF-13 produced dose-dependent antinociception in mouse models of tail-flick and carrageenan-induced inflammatory pain via the TRPV1 receptor. In contrast, the reference compound (m)VD-Hpα-NH2 induced CB1 receptor-mediated supraspinal antinociception. Additionally, subcutaneous injection of (m)VD-Hpα-NH2 and VF-13 produced significant antinociception in carrageenan-induced inflammatory pain model. In the tetrad assay, our data demonstrated that VF-13 elicited hypothermia, but not catalepsy and hypoactivity after intracerebroventricular injection. Notably, VF-13 produced non-tolerance forming antinociception over 6 days treatment in both acute and inflammatory pain models. Furthermore, VF-13 had no apparent effects on gastrointestinal transit, pentobarbitone-induced sedation, food intake, and motor coordination at the supraspinal level. In summary, VF-13, a novel chimeric peptide of VD-Hpα and NPVF, produced non-tolerance forming antinociception in preclinical pain models with reduced cannabinoid-related side effects.

Experimental Study of Split Grouting Reinforcement Mechanism in Filling Medium and Effect Evaluation.

In view of the deficiency of the split grouting theory for the filling area, a 3D simulated grouting test system was designed to explore the slurry diffusion law, reinforcement mechanism of split grouting in a filling soil, and effect of grouting reinforcement. The test system included an experiment bench system, grouting system, and information monitoring system, using which experimental research on split grouting in a filling soil was conducted. The grouting model experiment procedure was introduced first, following which the diffusion rule of slurry in the filling medium and the reinforcement mechanism of split grouting were analyzed according to the properties and distribution characteristics of grouting veins after grouting reinforcement. Finally, a uniaxial compression test, light dynamic contact test, permeability test, and laboratory geotechnical test were conducted on the soil before and after grouting. The relationship between the zoning characteristics of different properties of veins and the mechanical properties of filling soil were discussed. The results showed that there were three types of grouting veins: trunk grouting, branch grouting, and permeable grouting. The injected soil body was strengthened by the three-stage grouting vein network of the mentioned vein types and the compaction between soils. After the grouting, the uniaxial compressive strength of the filling soil increased by an average of 186%, and the permeability coefficient decreased by an average of 47 times. The cohesion and internal friction angle increased by 45.3% and 44.9%, respectively. Additionally, density, water content, and other indicators of filling were improved. The bearing characteristics reflected by a dynamic contact test were consistent with the distribution of grouting veins. The research results offer significant guidance for the reinforcement mechanism of split grouting and the evaluation of the grouting effect.

Spinal administration of the multi-functional opioid/neuropeptide FF agonist BN-9 produced potent antinociception without development of tolerance and opioid-induced hyperalgesia.

Chronic opioids treatment is impeded by the development of analgesic tolerance and opioid-induced hyperalgesia. Recent studies have shown that multi-functional opioid compounds produce analgesic activities with limited side effects. We developed a novel multi-functional peptide targeting opioid and neuropeptide FF receptors named BN-9, which produced potent and non-tolerance forming antinociceptive effect after supraspinal and systemic administrations. In the present study, the analgesic properties and potential side effects of intrathecal BN-9 were investigated in a range of preclinical rodent models. In complete Freund's adjuvant-induced inflammatory pain model, intrathecal BN-9 dose-dependently produced analgesic effect via opioid receptors, and the spinal antinociceptive effect was augmented by the neuropeptide FF receptor antagonist RF9. In contrast, in plantar incision-induced postoperative pain model, BN-9 exhibited potent anti-allodynic effect via opioid receptors and, at least partially, neuropeptide FF receptors. In mouse models of acetic acid-induced visceral pain and formalin pain, BN-9-induced spinal antinociception was mainly mediated by opioid receptors, independent of neuropeptide FF receptors. Furthermore, at the spinal level, chronic treatments with BN-9 did not lead to analgesic tolerance and cross-tolerance to morphine. Moreover, opioid-induced hyperalgesia was observed after repeated administration of morphine, but not BN-9. Taken together, our present study suggests that intrathecal BN-9 produces potent and non-tolerance forming antinociception, and does not cause opioid-induced hyperalgesia. Thus, BN-9 might serve as a promising lead compound in the development of multi-functional opioid analgesics with minimized side effects.

Central and peripheral modulation of gastrointestinal transit in mice by DN-9, a multifunctional opioid/NPFF receptor agonist.

BACKGROUND: The nonapeptide DN-9 functions as a multifunctional agonist to opioid and neuropeptide FF (NPFF) receptors and exhibits antinociceptive effects at the central and peripheral levels. METHODS: The effects of DN-9 on small and colonic intestinal transit were evaluated using the upper gastrointestinal (GI) transit test and colonic bead expulsion assay, respectively. Opioid and NPFF receptor antagonists were used to investigate the mechanisms of DN-9-induced GI inhibition. Furthermore, the agonism of the DN-9 analog [Phg9 ]-DN-9 to opioid and NPFF receptors was tested by the cAMP assay. KEY RESULTS: Intracerebroventricular administration of DN-9 dose-dependently slowed upper GI transit and colonic expulsion via mu- and kappa-opioid receptors in the brain, independent of the delta-opioid receptor. Similarly, intraperitoneal injection of DN-9 dose-dependently inhibited GI propulsion via the peripheral opioid receptors. DN-9-induced GI transit inhibitions were significantly aggravated by the NPFF receptor antagonist RF9. Moreover, the DN-9 analog [Phg9 ]-DN-9, an agonist at mu-, delta-, and kappa-opioid receptors but not NPFF receptors, inhibited GI more potently than DN-9. In addition, intracerebroventricular NPFF significantly attenuated the central inhibitory effects induced by [Phg9 ]-DN-9 and morphine. However, central and peripheral injections of NPFF or RF9 almost had no significant effects on GI transit by itself. CONCLUSION AND INFERENCES: Intracerebroventricular and intraperitoneal administrations of DN-9 inhibit GI transit via opioid receptors in mice by central and peripheral mechanisms, respectively. In addition, the NPFF agonism of DN-9 possesses antiopioid effects on GI transit, which might explain the reduced constipation at the antinociceptive doses.