[Determination of catecholamines in urine by disperse solid-phase extraction-liquid chromatography based on Ti3C2Tx/polyimide composites].

Neurotransmitters (NTs) are basic signaling chemicals used for communication between cells. The most well-known catecholamines (CAs) are epinephrine, norepinephrine, and dopamine. CAs are an important class of monoamine NTs that contain catechins and amine groups. The accurate determination of CAs in biological samples can provide essential information on potential pathogenic mechanisms. However, biological samples generally contain only trace levels of CAs. Therefore, sample pretreatment is necessary to separate and enrich CAs before instrument analysis. Dispersive solid-phase extraction (DSPE) technology combines the principles of liquid-liquid extraction and solid-phase extraction and is a useful method for purifying and enriching the target analytes in complex matrices. This method has the advantages of low solvent consumption, environmental safety, and high sensitivity and efficiency. In addition, the adsorbents used in DSPE do not need to be packed into a column and can simply be completely dispersed in the sample solution; this excellent feature greatly improves the extraction efficiency and simplifies the extraction process. Therefore, the development of new DSPE materials with high efficiency and adsorption capacity using simple preparation procedures has received wide attention from the research community. Carbon nitrides (MXenes) are a class of two-dimensional layered materials that possess good hydrophilicity, a large number of functional groups (-O, -OH, and -F), large layer spacing, different elemental compositions, excellent biocompatibility, and environmental friendliness. However, these materials have a small specific surface area and poor adsorption selectivity, which limits their applications in SPE. The separation selectivity of MXenes can be significantly improved by functional modification. Polyimide (PI) is a crosslinking product that is mainly formed by the condensation polymerization of binary anhydride and diamine. It has a unique crosslinked network structure, as well as a large number of carboxyl groups, and shows excellent characteristics. Therefore, the synthesis of new PI-functionalized Ti3C2Tx (Ti3C2Tx/PI) composites by growing a PI layer on the surface of two-dimensional MXene nanosheets in situ may not only overcome the adsorptive limitations of MXenes but also effectively improve their specific surface area and porous structure, thereby enhancing their mass transfer capacity, adsorption capacity, and selectivity. In this study, a Ti3C2Tx/PI nanocomposite was fabricated and successfully applied as a DSPE sorbent to enrich and concentrate trace CAs in urine samples. The prepared nanocomposite was examined using various characterization methods, including scanning electron microscopy, Fourier transform-infrared spectroscopy, X-ray diffraction, and zeta potential analysis. The effects of the extraction parameters on the extraction efficiency of Ti3C2Tx/PI were also investigated in detail. The adsorption performance of Ti3C2Tx/PI can be described by pseudo-second-order kinetics and the Freundlich isotherm model. The adsorption process appeared to occur on the outer surface, as well as surface voids, of the nanocomposite. The adsorption mechanism of Ti3C2Tx/PI indicated a chemical adsorption process based on multiple electrostatic, π-π, and hydrogen-bonding interactions. The optimal adsorption conditions included an adsorbent dosage of 20 mg, sample pH of 8, adsorption and elution times of 10 and 15 min, respectively, and eluent composed of acetic acid-acetonitrile-water (5∶47.5∶47.5, v/v/v). A sensitive method for detecting CAs in urine was subsequently developed by coupling Ti3C2Tx/PI as a DSPE sorbent with HPLC-FLD analysis. The CAs were separated on an Agilent ZORBAX ODS analytical column (250 mm×4.6 mm, 5 µm). Methanol and an aqueous solution of 20 mmol/L acetic acid were used as the mobile phases for isocratic elution. Under optimal conditions, the proposed DSPE-HPLC-FLD method exhibited good linearity in the range of 1-250 ng/mL with correlation coefficients >0.99. The limits of detection (LODs) and limits of quantification (LOQs) were calculated based on signal-to-noise ratios of 3 and 10 and found to be in the range of 0.20-0.32 and 0.7-1.0 ng/mL, respectively. The recoveries of the method were in the range of 82.50%-96.85% with RSDs≤9.96%. Finally, the proposed method was successfully applied to the quantification of CAs in urine samples from smokers and nonsmokers, thereby indicating its applicability for determining trace CAs.

Distinctive response of CNS glial cells in oro-facial pain associated with injury, infection and inflammation.

Oro-facial pain following injury and infection is frequently observed in dental clinics. While neuropathic pain evoked by injury associated with nerve lesion has an involvement of glia/immune cells, inflammatory hyperalgesia has an exaggerated sensitization mediated by local and circulating immune mediators. To better understand the contribution of central nervous system (CNS) glial cells in these different pathological conditions, in this study we sought to characterize functional phenotypes of glial cells in response to trigeminal nerve injury (loose ligation of the mental branch), infection (subcutaneous injection of lipopolysaccharide--LPS) and to sterile inflammation (subcutaneous injection of complete Freund's adjuvant--CFA) on the lower lip. Each of the three insults triggered a specific pattern of mechanical allodynia. In parallel with changes in sensory response, CNS glial cells reacted distinctively to the challenges. Following ligation of the mental nerve, both microglia and astrocytes in the trigeminal nuclear complex were highly activated, more prominent in the principal sensory nucleus (Pr5) and subnucleus caudalis (Sp5C) area. Microglial response was initiated early (days 3-14), followed by delayed astrocytes activation (days 7-28). Although the temporal profile of microglial and astrocyte reaction corresponded respectively to the initiation and chronic stage of neuropathic pain, these activated glial cells exhibited a low profile of cytokine expression. Local injection of LPS in the lower lip skin also triggered a microglial reaction in the brain, which started in the circumventricular organs (CVOs) at 5 hours post-injection and diffused progressively into the brain parenchyma at 48 hours. This LPS-induced microglial reaction was accompanied by a robust induction of IκB-α mRNA and pro-inflammatory cytokines within the CVOs. However, LPS induced microglial activation did not specifically occur along the pain signaling pathway. In contrast, CFA injection led to minor microglial morphological changes and an induction of IκB-α mRNA in the CVO regions; a significant increase in IL-1ß and IL-6 mRNA started only at 48 hours post-injection, when the induced pain-related behavior started to resolve. Our detailed analysis of CNS glial response clearly revealed that both nerve injury and oro-facial infection/inflammation induced CNS glial activation, but in a completely different pattern, which suggests a remarkable plasticity of glial cells in response to dynamic changes in their microenvironment and different potential involvement of this non-neuronal cell population in pathological pain development.

Statins alleviate experimental nerve injury-induced neuropathic pain.

The statins are a well-established class of drugs that lower plasma cholesterol levels by inhibiting HMG-CoA (3-hydroxy-3-methyl-glutaryl-coenzyme A) reductase. They are widely used for the treatment of hypercholesterolemia and for the prevention of coronary heart disease. Recent studies suggest that statins have anti-inflammatory effects beyond their lipid-lowering properties. We sought to investigate whether statins could affect neuropathic pain by mediating nerve injury-associated inflammatory responses. The effects of hydrophilic rosuvastatin and lipophilic simvastatin were examined in the mouse partial sciatic nerve ligation model. Systemic daily administration of either statin from days 0 to 14 completely prevented the development of mechanical allodynia and thermal hyperalgesia. When administered from days 8 to 14 after injury, both statins dose-dependently reduced established hypersensitivity. After treatment, the effects of the statins were washed out within 2 to 7 days, depending on dose. Effects of both statins in alleviating mechanical allodynia were further confirmed in a different injury-associated neuropathic pain model, mental nerve chronic constriction, in rats. Both statins were able to abolish interleukin-1ß expression in sciatic nerve triggered by nerve ligation. Additionally, quantitative analysis with Iba-1 and glial fibrillary acid protein immunoreactivity demonstrated that rosuvastatin and simvastatin significantly reduced the spinal microglial and astrocyte activation produced by sciatic nerve injury. The increase of interleukin-1ß mRNA in the ipsilateral side of spinal cords was also reduced by the treatment of either statin. We identified a potential new application of statins in the treatment of neuropathic pain. The pain-alleviating effects of statins are likely attributable to their immunomodulatory effects.