Projections from anteromedial thalamus nucleus to the midcingulate cortex mediate pain and anxiety-like behaviors in mice.

Prior research has demonstrated the involvement of the midcingulate cortex (MCC) and its downstream pathway in pain regulation. However, the mechanism via which pain information is conveyed to the MCC remains unclear. The present study utilized immunohistochemistry, chemogenetics, optogenetics, and behavior detection methods to explore the involvement of MCC, anteromedial thalamus nucleus (AM), and AM-MCC pathway in pain and emotional regulation. Chemogenetics or optogenetics methods were employed to activate/inhibit MCCCaMKIIα, AMCaMKIIα, AMCaMKIIα-MCC pathway. This manipulation evokes/relieves mechanical and partial heat hyperalgesia, as well as anxiety-like behaviors. In the complete Freund,s adjuvant (CFA) inflammatory pain model, chemogenetic inhibition of the AMCaMKIIα-MCCCaMKIIα pathway contributed to pain relief. Notably, this study presented the first evidence implicating the AM in the regulation of nociception and negative emotions. Additionally, it was observed that the MCC primarily receives projections from the AM, highlighting the crucial role of this pathway in the transmission of pain and emotional information.

The analgesic effects of ß-elemene in rats with neuropathic pain by inhibition of spinal astrocytic ERK activation.

Neuropathic pain takes a heavy toll on individual well-being, while current therapy is far from desirable. Herein, we assessed the analgesic effect of ß-elemene, a chief component in the traditional Chinese medicine Curcuma wenyujin, and explored the underlying mechanisms at the level of spinal dorsal horn (SDH) under neuropathic pain. A spared nerve injury (SNI)-induced neuropathic pain model was established in rats. Intraperitoneal injection (i.p.) of ß-elemene was administered for 21 consecutive days. Mechanical allodynia was explored by von Frey filaments. The activation of the mitogen-activated protein kinase (MAPK) family (including ERK, p38, and JNK) in spinal neurons, astrocytes, and microglia was evaluated using immunostaining 29 days after SNI surgery. The expression of GFAP, Iba-1, p-ERK, p-JNK, and p-p38 within the SDH was measured using immunoblotting. The levels of proinflammatory cytokines (including TNF-α, IL-1ß, and IL-6) were measured with ELISA. The levels of oxidative stress indicators (including MDA, SOD, and GSH-PX) were detected using biochemical tests. Consecutive i.p. administration of ß-elemene relieved SNI-induced mechanical allodynia (with an EC50 of 16.40 mg/kg). SNI significantly increased the expression of p-ERK in spinal astrocytes but not microglia on day 29. ß-elemene reversed spinal astrocytic ERK activation and subsequent upregulation of proinflammatory cytokines in SNI rats, with no effect on the expression of p38 and JNK in spinal glia. ß-elemene also exerted antioxidative effects by increasing the levels of SOD and GSH-PX and decreasing the level of MDA. Our results suggest that SNI induces robust astrocytic ERK activation within the SDH in the late phase of neuropathic pain. ß-elemene exerts remarkable analgesic effects on neuropathic pain, possibly by inhibiting spinal astrocytic ERK activation and subsequent neuroinflammatory processes. Our findings suggest that ß-elemene might be a promising analgesic for the treatment of chronic pain.

The perceptions of anatomy teachers for different majors during the COVID-19 pandemic: a national Chinese survey.

During the spring semester of 2020, medical school anatomists in China were forced by the COVID-19 pandemic to transition from face-to-face educators or part-time online educators to full-time online educators. This nationwide survey was conducted to assess online anatomy education during the pandemic for medical students from nonclinical medicine and clinical medicine majors at medical schools in China via WeChat. The total of 356 responders included 293 responders from clinical medicine and 63 respondents from nonclinical medicine majors (i.e., 21 from preventive medicine, 13 from stomatology, and 29 from traditional Chinese medicine). The survey results showed that several aspects of online anatomy education were quite similar in clinical and nonclinical majors' classes, including theoretical and practical sessions, active learning, assessments and evaluations. However, there were statistically significant differences in class size, implementation of active learning activities prior to the pandemic, and the evaluation of the effectiveness of online learning during the pandemic, between clinical and nonclinical medicine majors. These results indicated that, compared with teachers of anatomy courses in clinical medicine, teachers of nonclinical medicine majors using online learning in medical schools in China had relatively poor preparation for online learning in response to the unforeseen pandemic.

Projecting neurons in spinal dorsal horn send collateral projections to dorsal midline/intralaminar thalamic complex and parabrachial nucleus.

Itch is an annoying sensation that always triggers scratching behavior, yet little is known about its transmission pathway in the central nervous system. Parabrachial nucleus (PBN), an essential transmission nucleus in the brainstem, has been proved to be the first relay station in itch sensation. Meanwhile, dorsal midline/intralaminar thalamic complex (dMITC) is proved to be activated with nociceptive stimuli. However, whether the PBN-projecting neurons in spinal dorsal horn (SDH) send collateral projections to dMITC, and whether these projections involve in itch remain unknown. In the present study, a double retrograde tracing method was applied when the tetramethylrhodamine-dextran (TMR) was injected into the dMITC and Fluoro-gold (FG) was injected into the PBN, respectively. Immunofluorescent staining for NeuN, substance P receptor (SPR), substance P (SP), or FOS induced by itch or pain stimulations with TMR and FG were conducted to provide morphological evidence. The results revealed that TMR/FG double-labeled neurons could be predominately observed in superficial laminae and lateral spinal nucleus (LSN) of SDH; Meanwhile, most of the collateral projection neurons expressed SPR and some of them expressed FOS in acute itch model induced by histamine. The present results implicated that some of the SPR-expressing neurons in SDH send collateral projections to the dMITC and PBN in itch transmission, which might be involved in itch related complex affective/emotional processing to the higher brain centers.

VGLUT1 or VGLUT2 mRNA-positive neurons in spinal trigeminal nucleus provide collateral projections to both the thalamus and the parabrachial nucleus in rats.

The trigemino-thalamic (T-T) and trigemino-parabrachial (T-P) pathways are strongly implicated in the sensory-discriminative and affective/emotional aspects of orofacial pain, respectively. These T-T and T-P projection fibers originate from the spinal trigeminal nucleus (Vsp). We previously determined that many vesicular glutamate transporter (VGLUT1 and/or VGLUT2) mRNA-positive neurons were distributed in the Vsp of the adult rat, and most of these neurons sent their axons to the thalamus or cerebellum. However, whether VGLUT1 or VGLUT2 mRNA-positive projection neurons exist that send their axons to both the thalamus and the parabrachial nucleus (PBN) has not been reported. Thus, in the present study, dual retrograde tract tracing was used in combination with fluorescence in situ hybridization (FISH) for VGLUT1 or VGLUT2 mRNA to identify the existence of VGLUT1 or VGLUT2 mRNA neurons that send collateral projections to both the thalamus and the PBN. Neurons in the Vsp that send collateral projections to both the thalamus and the PBN were mainly VGLUT2 mRNA-positive, with a proportion of 90.3%, 93.0% and 85.4% in the oral (Vo), interpolar (Vi) and caudal (Vc) subnucleus of the Vsp, respectively. Moreover, approximately 34.0% of the collateral projection neurons in the Vc showed Fos immunopositivity after injection of formalin into the lip, and parts of calcitonin gene-related peptide (CGRP)-immunopositive axonal varicosities were in direct contact with the Vc collateral projection neurons. These results indicate that most collateral projection neurons in the Vsp, particularly in the Vc, which express mainly VGLUT2, may relay orofacial nociceptive information directly to the thalamus and PBN via axon collaterals.

A UPLC-MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study.

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra-high liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid-liquid extraction with ethyl acetate. The UPLC-MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra- and inter-day precision were both <10.2%, and the accuracy ranged from -11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.

Antioxidant therapy for pain relief in patients with chronic pancreatitis: systematic review and meta-analysis.

BACKGROUND: Currently, there is no specific therapy for chronic pancreatitis (CP). The treatment of micronutrient antioxidant therapy for painful CP has been sporadically used for more than 30 years, however, its efficacy are still poorly understood. OBJECTIVE: The purpose of this meta-analysis is to investigate the safety and efficacy of antioxidant therapy for pain relief in patients with CP. SETTING: University Hospital in China STUDY DESIGN: Systematic review and meta-analysis METHODS: Two authors independently reviewed the search results and extracted data and disagreements were resolved by discussion. Effects were summarized using standardized mean differences (SMDs), weighted mean differences, or odds ratio (OR) according to the suitable effect model. MEDLINE, PsycINFO, Scopus, EMBASE, and the Cochrane Central Register of Controlled Trials  were searched from 1980 through December 2012. Randomized controlled trials (RCTs) that studied antioxidant supplementation for pain relief in patients with CP were analyzed. RESULTS: Nine randomized controlled trials (RCTs) involving 390 patients were included. Overall, there was no association of antioxidant therapy with pain reduction in CP patients (SMD, -0.55; 95% CI, -1.22 to 0.12; P = 0.67). However, antioxidant therapy significantly increased blood levels of antioxidants in CP patients versus the placebo group (SMD, 1.08; 95% CI, 0.74 to 1.43; P < 0.00001). Interestingly, combined antioxidant (selenium, ß-carotene, vitamin C, vitamin E, methionine) therapy was found to be associated with pain relief (SMD, -0.93; 95% CI, -1.72 to -0.14; P = 0.02), while the trials in which a single antioxidant was used revealed no significant pain relief (SMD, -0.12; 95% CI, -1.23 to 0.99; P = 0.83) in CP patients. Strong evidence was obtained that the antioxidants increased adverse effects (OR, 6.09; 95% CI, 2.29 to 16.17, P < 0.01); nevertheless, none was serious. LIMITATIONS: Because of the small sample, a consolidated conclusion cannot be reached based on current RCTs. Large-sample RCTs are needed to clarify the analgesic effect of antioxidants in CP patients. CONCLUSIONS: Combined antioxidant therapy seems to be a safe and effective therapy for pain relief in CP patients. Measures of total antioxidant status may not help to monitor the efficacy of antioxidant therapy for patients with CP.

Triptolide prevents and attenuates neuropathic pain via inhibiting central immune response.

BACKGROUND: Current treatments for neuropathic pain are far from satisfactory. Considering the essential contribution of central immune factors to the pathogenesis of neuropathic pain, targeting inflammatory response is well accepted as an effective strategy for treating neuropathic pain. Triptolide has a long history in traditional Chinese medicine for treating inflammatory diseases and has been proven to inhibit cytokines released from glial cells. OBJECTIVE: In the present study, we tested whether systemic treatment with triptolide could prevent or attenuate nocifensive behaviors associated with neuropathic pain. We further tried to explore the underlying mechanism of the potential anti-allodynia effect of triptolide. STUDY DESIGN: A randomized, double blind, controlled animal trial. METHODS: Triptolide was administered systemically in a rat model of neuropathic pain induced by spinal nerve ligation (SNL) in the single bolus and repeated treatment manners. In the single bolus treatment experiment, triptolide (30 ug/kg, 100 ug/kg, 300 ug/kg) or vehicle was given to SNL and sham-operated rats once on day 1 or on day 10 after surgery (n = 6 each). In the repeated treatment study, prophylactic treatment with triptolide (30 ug/kg, 100 ug/kg, 300 ug/kg) was given to rats during the period of day -3 (3 days prior to SNL) to day 7 (7 days post-SNL) inclusively (n = 6 each). Another set of SNL and sham rats on postoperative day 10 received treatment with triptolide (30 ug/kg, 100 ug/kg, 300 ug/kg) or vehicle during the period of days 11-20 inclusively (n = 6 each), to assess potential reversal of established pain behavior. Mechanical allodynia of the rats was tested with von Frey filaments. Astrocytic and microglial activation in the spinal dorsal horn was evaluated with immunofluorescent histochemistry. Phosphorylation of mitogen-activated protein kinases (MAPKs), and expression of inflammatory cytokines (interleukin-6, interleukin-1beta, monocyte chemotactic protein-1, and tumor necrosis factor-alpha) were examined with Western blot analysis and real-time reverse transcription polymerase chain reaction study. RESULTS: A single bolus treatment with triptolide could neither prevent the induction nor reverse the maintenance of SNL-induced mechanical allodynia. However, repeated administration of triptolide dose-dependently inhibited neuropathic pain behavior in both preventative and interventional paradigms. Triptolide hampered SNL-induced activation of glial cells (astrocytes and microglia) in the spinal dorsal horn without influencing neurons. In addition, SNL-induced phosphorylation of MAPKs could be inhibited by triptolide. Furthermore, up-regulated expression of inflammatory cytokines in neuropathic pain states could be remarkably blocked by triptolide. LIMITATIONS: The direct target site (such as a specific receptor) of triptolide is still to be determined. In addition, triptolide could not completely block the SNL-induced mechanical allodynia. CONCLUSIONS: Our data suggest that triptolide may be a potential novel treatment for neuropathic pain through modulating immune response in the spinal dorsal horn.

Origins of endomorphin-2 immunopositive fibers and terminals in the rat medullary dorsal horn.

Endomorphin-2-immunoreactive (EM2-IR) fibers and terminals are densely present in the medullary dorsal horn (MDH) and are key factors in regulating central nociceptive processing. However, the origins of these EM2-IR fibers and terminals remain elusive. It was hypothesized that there were at least three possible origins of the EM2-IR fibers and terminals in the MDH: intrinsic dorsal horn neurons, primary afferent fibers, and projection fibers from higher parts of the brain. Different kinds of measures were employed in the current study to elucidate this hypothesis. After intracerebral ventricle administration of colchicine, no EM2-IR neuronal cell bodies were detected in the MDH, suggesting that there was no intrinsic EM2-IR dorsal horn neuron. Disruption of bilateral primary afferents (exposed to the primary afferent neurotoxin, capsaicin) decreased bilateral EM2 expression but did not eliminate it. Transection of the trigeminal nerve sensory root significantly decreased EM2 expression on the ipsilateral but not on the contralateral MDH. After injecting FluoroGold (FG) into the MDH, FG retrogradely labeled some EM2-IR neurons in the bilateral hypothalamus and nucleus tractus solitarii (NTS), and some of the FG retrogradely labeled neurons in the ipsilateral trigeminal ganglion also showed EM2-immunoreactivities. These results indicate that EM2-IR fibers and terminals in the MDH come not only from ipsilateral primary trigeminal afferents but also from bilateral fibers from the hypothalamus and NTS.

Synaptic connections between GABAergic elements and serotonergic terminals or projecting neurons in the ventrolateral orbital cortex.

The ventrolateral orbital cortex (VLO) is part of an endogenous analgesic system, consisting of the spinal cord-thalamic nucleus submedius-VLO periaqueductal gray (PAG)-spinal cord loop. The present study examined morphological connections of GABAergic (gamma-aminobutyric acidergic) neurons and serotonergic projection terminals from the dorsal raphe nucleus (DR), as well as the relationship between GABAergic terminals and VLO neurons projecting to the PAG, by using anterograde and retrograde tracing combined with immunofluorescence, immunohistochemistry, and electron microscopy methods. Results indicate that the majority (93%) of GABAergic neurons in the VLO also express the 5-HT(1A) (5-hydroxytryptamine 1A) receptor, and serotonergic terminals originating from the DR nucleus made symmetrical synapses with GABAergic neuronal cell bodies and dendrites within the VLO. GABAergic terminals also made symmetrical synapses with neurons expressing GABA(A) receptors and projecting to the PAG. These results suggest that a local neuronal circuit, consisting of 5-HTergic terminals, GABAergic interneurons, and projection neurons, exists in the VLO, and provides morphological evidence for the hypothesis that GABAergic modulation is involved in 5-HT(1A) receptor activation-evoked antinociception.

GABAergic modulation is involved in the ventrolateral orbital cortex 5-HT 1A receptor activation-induced antinociception in the rat.

The ventrolateral orbital cortex (VLO) is a component of an endogenous analgesic system consisting of an ascending pathway from the spinal cord to VLO via the thalamic nucleus submedius (Sm) and a descending pathway relaying in the periaqueductal gray matter (PAG). This study examines whether the activation of 5-HT 1A receptors in VLO produces antinociception and whether GABAergic modulation is involved in the VLO 5-HT 1A receptor activation-evoked antinociception. The radiant heat-evoked tail flick (TF) reflex was used as an index of nociceptive response in lightly anesthetized rats. Microinjection of the 5-HT 1A receptor agonist 8-OH-DPAT (1.0, 2.0, 5.0 microg) into VLO produced dose-dependent antinociception, which was reversed by the 5-HT 1A receptor antagonist (NAN-190, 20 mug). We also found that VLO application of the GABA A receptor antagonist bicuculline or picrotoxin (100 ng) enhanced the 8-OH-DPAT-induced inhibition of the TF reflex, whereas the GABA A receptor agonist muscimol (250 ng) or THIP (1.0 microg) significantly attenuated the 8-OH-DPAT-induced inhibition. These results suggest that 5-HT 1A receptors are involved in VLO-induced antinociception and that GABAergic disinhibitory mechanisms participate in the 5-HT 1A receptor-mediated effect. These findings provide support for the hypothesis that 5-HT 1A receptor activation may inhibit the inhibitory action of the GABAergic interneurons on the output neurons projecting to PAG leading to activation of the brainstem descending inhibitory system and depression of nociceptive inputs at the spinal cord level.

Origins of endomorphin-immunoreactive fibers and terminals in different columns of the periaqueductal gray in the rat.

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are endogenous ligands for mu-opioid receptors (MOR). In the central nervous system, EM-immunoreactive (IR) neuronal cell bodies are located mainly in the hypothalamus and the nucleus tractus solitarius (NTS). EM-IR fibers and terminals are found widely distributed in many brain areas, including the different columns of the periaqueductal gray (PAG). The hypothalamus, NTS, and PAG are closely involved in modulation of vocalization, autonomic and neuroendocrine functions, pain, and defensive behavior through endogenous opioid peptides that bind to the MOR in these regions. Projections exist from both the hypothalamus and the NTS to the PAG. In order to examine whether there are EM1- and/or EM2-ergic projections from the hypothalamus and NTS to the PAG, immunofluorescence histochemistry for EM1 and/or EM2 was combined with fluorescent retrograde tracing. In rats that had Fluoro-Gold (FG) injected into different columns of the PAG, some of the EM1- or EM2-IR neurons in the hypothalamus, but none in the NTS, were labeled retrogradely with FG. The majority of the EM1/FG and EM2/FG double-labeled neurons in the hypothalamus were distributed in the dorsomedial nucleus, areas between the dorsomedial and ventromedial nucleus, and arcuate nucleus; a few were also seen in the ventromedial, periventricular, and posterior nucleus. The present results indicate that the EM-IR fibers and terminals in the PAG originate principally from the hypothalamus. They also suggest that EMs released from hypothalamus-PAG projecting neurons might mediate or modulate various functions of the PAG through binding to the MOR.

Endomorphin 1- and endomorphin 2-like immunoreactive neurons in the hypothalamus send axons to the parabrachial nucleus in the rat.

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are the endogenous peptides with high affinity and selectivity for the mu-opioid receptor (MOR). We examined whether or not EM1- and EM2-expressing hypothalamic neurons might send their axons to the parabrachial nucleus (PBN), where many MOR-expressing neurons have been observed. Immunofluorescence histochemistry was combined with fluorescent retrograde tract-tracing method. In the rats injected with Fluoro-Gold (FG) into the PBN, some of EM1- and EM2-immunoreactive hypothalamic neurons were labeled retrogradely with FG. The majority of the EM1/FG and EM2/FG double-labeled neurons were distributed in the dorsomedial hypothalamus nucleus, centromedial hypothalamic region, and arcuate nucleus; a few of them were also seen in the periventricular hypothalamic nucleus and posterior hypothalamic nucleus. Endomorphins released from PBN-projecting hypothalamic neurons may modulate the gustatory, autonomic and nociceptive functions through MOR-expressing PBN neurons.