Prefrontal engrams of long-term fear memory perpetuate pain perception.

A painful episode can lead to a life-long increase in an individual's experience of pain. Fearful anticipation of imminent pain could play a role in this phenomenon, but the neurobiological underpinnings are unclear because fear can both suppress and enhance pain. Here, we show in mice that long-term associative fear memory stored in neuronal engrams in the prefrontal cortex determines whether a painful episode shapes pain experience later in life. Furthermore, under conditions of inflammatory and neuropathic pain, prefrontal fear engrams expand to encompass neurons representing nociception and tactile sensation, leading to pronounced changes in prefrontal connectivity to fear-relevant brain areas. Conversely, silencing prefrontal fear engrams reverses chronically established hyperalgesia and allodynia. These results reveal that a discrete subset of prefrontal cortex neurons can account for the debilitating comorbidity of fear and chronic pain and show that attenuating the fear memory of pain can alleviate chronic pain itself.

Quanty-cFOS, a Novel ImageJ/Fiji Algorithm for Automated Counting of Immunoreactive Cells in Tissue Sections.

Analysis of neural encoding and plasticity processes frequently relies on studying spatial patterns of activity-induced immediate early genes' expression, such as c-fos. Quantitatively analyzing the numbers of cells expressing the Fos protein or c-fos mRNA is a major challenge owing to large human bias, subjectivity and variability in baseline and activity-induced expression. Here, we describe a novel open-source ImageJ/Fiji tool, called 'Quanty-cFOS', with an easy-to-use, streamlined pipeline for the automated or semi-automated counting of cells positive for the Fos protein and/or c-fos mRNA on images derived from tissue sections. The algorithms compute the intensity cutoff for positive cells on a user-specified number of images and apply this on all the images to process. This allows for the overcoming of variations in the data and the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. We validated the tool using data from brain sections in response to somatosensory stimuli in a user-interactive manner. Here, we demonstrate the application of the tool in a step-by-step manner, with video tutorials, making it easy for novice users to implement. Quanty-cFOS facilitates a rapid, accurate and unbiased spatial mapping of neural activity and can also be easily extended to count other types of labelled cells.

Layer-specific pain relief pathways originating from primary motor cortex.

The primary motor cortex (M1) is involved in the control of voluntary movements and is extensively mapped in this capacity. Although the M1 is implicated in modulation of pain, the underlying circuitry and causal underpinnings remain elusive. We unexpectedly unraveled a connection from the M1 to the nucleus accumbens reward circuitry through a M1 layer 6-mediodorsal thalamus pathway, which specifically suppresses negative emotional valence and associated coping behaviors in neuropathic pain. By contrast, layer 5 M1 neurons connect with specific cell populations in zona incerta and periaqueductal gray to suppress sensory hypersensitivity without altering pain affect. Thus, the M1 employs distinct, layer-specific pathways to attune sensory and aversive-emotional components of neuropathic pain, which can be exploited for purposes of pain relief.

Prolonged Suppression of Neuropathic Hypersensitivity upon Neurostimulation of the Posterior Insula in Mice.

Neurostimulation-based therapeutic approaches are emerging as alternatives to pharmacological drugs, but need further development to optimize efficacy and reduce variability. Despite its key relevance to pain, the insular cortex has not been explored in cortical neurostimulation approaches. Here, we developed an approach to perform repetitive transcranial direct current stimulation of the posterior insula (PI tDCS) and studied its impact on sensory and aversive components of neuropathic pain and pain-related anxiety and the underlying neural circuitry in mice using behavioral methods, pharmacological interventions and the expression of the activity-induced gene product, Fos. We observed that repetitive PI tDCS strongly attenuates the development of neuropathic mechanical allodynia and also reverses chronically established mechanical and cold allodynia for several weeks post-treatment by employing descending opioidergic antinociceptive pathways. Pain-related anxiety, but not pain-related aversion, were inhibited by PI tDCS. These effects were associated with a long-term suppression in the activity of key areas involved in pain modulation, such as the cingulate, prefrontal and motor cortices. These data uncover the significant potential of targeting the insular cortex with the objective of pain relief and open the way for more detailed mechanistic analyses that will contribute to improving cortical neurostimulation therapies for use in the clinical management of pain.

Nerve Growth Factor (NGF) Encourages the Neuroinvasive Potential of Pancreatic Cancer Cells by Activating the Warburg Effect and Promoting Tumor Derived Exosomal miRNA-21 Expression.

Background: It has been reported that signaling from the nerve growth factor (NGF) pathway associated with peripheral nerves is able to contribute to perineural invasion (PNI) of pancreatic cancer (PC). Nevertheless, the underlying mechanism by which NGF leads to PNI remained poorly understood. Methods: Western blotting was employed to determine NGF level in PC and paracarcinoma tissues and in PC cell lines as well as pancreatic ductal epithelial cells. MiaPaCa-2 and CFPAC-1 cells were treated with 100 ng/ml of NGF or the NGF inhibitor Tanezumab for 24 h, CCK-8 and Transwell assays were employed to test cell proliferation, invasion, and migration, respectively. TrkA expression was knocked down in MiaPaCa-2 and dorsal root ganglion (DRG) cells treated with NGF to determine its effect on the Warburg effect. To reveal that the NGF-TrkA signaling pathway was closely associated with PC PNI, in vitro neuroinvasion model was established by using MiaPaCa-2 cells via coculturing DRG cells in Matrigel. Further, exosomes were extracted from PC cells and identified by examining the levels of specific markers for exosomes. Then RT-qPCR was applied to test miR-21-5p level in tumor derived exosomal (TDE-miR-21-5p). RIP assay was performed to validate NGF and miR-21 binding ability in MiaPaCa-2 cells. Rescue experiments were performed by using coprocessing of Tanezumab and miR-21-5p mimic on MiaPaCa-2 cells, followed by coculture with DRG cells. Subsequently, we used a model of neuroinvasion in nude mice to assess the effect of NGF in vivo on tumor nerve invasion as well as on nociceptive transmission. Results: NGF level was preeminently higher in PC tissues and cell lines than in paracarcinoma tissues and normal pancreatic epithelial cell lines. NGF promoted MiaPaCa-2 and CFPAC-1 cell invasion and migration, while Tanezumab treatment showed the opposite results. Besides, NGF binding to TrkA receptors encouraged the intracellular Warburg effect in PC and DRG cells. TrkA blocking-up could restrain NGF induced PC cell migration and neural invasion. Mechanistically, NGF could upregulate TDE-miR-21-5p levels, and DRG cells took up TDE to activate the Warburg effect and stimulate nociceptor gene expression. miR-21-5p inhibitor could abolish the facilitative effect of NGF on PNI in MiaPaCa-2 cells. In vivo tumorigenesis experiments, Tanezumab markedly alleviated nerve invasion of PC cells as well as relieved nociceptive conduction in animal models. Conclusions: These findings displayed that NGF/TrkA encouraged the neuroinvasive potential of PC cells by activating the Warburg effect in DRG cells through upregulation of TDE-miR-21-5p expression.

Clinical Observation of Alternative Wave Electroacupuncture Combined with Lee's Naprapathy in Treating Knee Osteoarthritis (Blood Stasis due to Qi Stagnation).

Objective: We aim to explore the clinical therapeutic effect of alternative wave electroacupuncture combined with Lee's naprapathy therapy on knee osteoarthritis (KOA) (blood stasis due to qi stagnation). Method: 122 patients with KOA treated in our hospital from January 2018 to October 2021 were randomly grouped into a combined group (n = 61) and a control group (n = 61). The combined group was treated with alternative wave electroacupuncture combined with Lee's naprapathy, while the control group was treated with alternative wave electroacupuncture alone. Clinical efficacy of the two groups was observed. The Visual Analogue Scale (VAS), Lysholm Scale, Indexes of Severity for Osteoarthritis (ISOA), and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were compared before and after treatment, followed up for 3 months and 6 months. The adverse reactions of the two groups were observed. Result: The overall response rate of the combined group (96.72%) was higher than that of the control group (81.97%), and the difference was statistically significant (P < 0.05). After treatment and follow-up for 3 months and 6 months, the Lysholm score of the combined group was higher than that of the control group, while the VAS, ISOA, and WOMAC scores were lower than those of the control group, and the difference between the two was statistically significant (P < 0.05). There were no serious adverse reactions in both groups (P > 0.05). Conclusion: The alternative wave electroacupuncture combined with Lee's naprapathy is effective and safe in treating KOA (blood stasis due to qi stagnation).

Cholinergic basal forebrain nucleus of Meynert regulates chronic pain-like behavior via modulation of the prelimbic cortex.

The basal nucleus of Meynert (NBM) subserves critically important functions in attention, arousal and cognition via its profound modulation of neocortical activity and is emerging as a key target in Alzheimer's and Parkinson's dementias. Despite the crucial role of neocortical domains in pain perception, however, the NBM has not been studied in models of chronic pain. Here, using in vivo tetrode recordings in behaving mice, we report that beta and gamma oscillatory activity is evoked in the NBM by noxious stimuli and is facilitated at peak inflammatory pain-like behavior. Optogenetic and chemogenetic cell-specific, reversible manipulations of NBM cholinergic-GABAergic neurons reveal their role in endogenous control of nociceptive hypersensitivity, which are manifest via projections to the prelimbic cortex, resulting in layer 5-mediated antinociception. Our data unravel the importance of the NBM in top-down control of neocortical processing of pain-like behavior.

Suppression of neuropathic pain and comorbidities by recurrent cycles of repetitive transcranial direct current motor cortex stimulation in mice.

Transcranial, minimally-invasive stimulation of the primary motor cortex (M1) has recently emerged to show promise in treating clinically refractory neuropathic pain. However, there is a major need for improving efficacy, reducing variability and understanding mechanisms. Rodent models hold promise in helping to overcome these obstacles. However, there still remains a major divide between clinical and preclinical studies with respect to stimulation programs, analysis of pain as a multidimensional sensory-affective-motivational state and lack of focus on chronic phases of established pain. Here, we employed direct transcranial M1 stimulation (M1 tDCS) either as a single 5-day block or recurring blocks of repetitive stimulation over early or chronic phases of peripherally-induced neuropathic pain in mice. We report that repeated blocks of stimulation reverse established neuropathic mechanical allodynia more strongly than a single 5-day regime and also suppress cold allodynia, aversive behavior and anxiety without adversely affecting motor function over a long period. Activity mapping revealed highly selective alterations in the posterior insula, periaqueductal gray subdivisions and superficial spinal laminae in reversal of mechanical allodynia. Our preclinical data reveal multimodal analgesia and improvement in quality of life by multiple blocks of M1 tDCS and uncover underlying brain networks, thus helping promote clinical translation.

Repetitive non-invasive prefrontal stimulation reverses neuropathic pain via neural remodelling in mice.

Chronic neuropathic pain presents a major challenge to pharmacological therapy and neurostimulation-based alternatives are gaining interest. Although invasive and non-invasive motor cortex stimulation has been the focus of several studies, very little is known about the potential of targeting the prefrontal cortex. This study was designed to elucidate the analgesic potential of prefrontal stimulation in a translational context and to uncover the neural underpinnings thereof. Here, we report that non-invasive, repetitive direct anodal current transcranial stimulation (tDCS) of the prefrontal cortex exerted analgesia in mice with neuropathic pain for longer than a week. When applied at chronic stages of neuropathic pain, prefrontal tDCS reversed established allodynia and suppressed aversion and anxiety-related behaviours. Activity mapping as well as in vivo electrophysiological analyses revealed that although the cortex responds to acute tDCS with major excitation, repetitive prefrontal tDCS brings about large-scale silencing of cortical activity. Different classes of different classes of GABAergic interneurons and classes of excitatory neurons differs dramatically between single, acute vs and repetitive tDCS. Repetitive prefrontal tDCS alters basal activity as well as responsivity of a discrete set of distant cortical and sub-cortical areas to tactile stimuli, namely the rostral anterior cingulate cortex, the insular cortex, the ventrolateral periaqueductal grey and the spinal dorsal horn. This study thus makes a strong case for harnessing prefrontal cortical modulation for non-invasive transcranial stimulation paradigms to achieve long-lasting pain relief in established neuropathic pain states and provides valuable insights gained on neural mechanistic underpinnings of prefrontal tDCS in neuropathic pain.

CXCL14 Overexpression Attenuates Sepsis-Associated Acute Kidney Injury by Inhibiting Proinflammatory Cytokine Production.

CXCL14 is a relatively novel chemokine with a wide spectrum of biological activities. The present study was designed to investigate whether CXCL14 overexpression attenuates sepsis-associated acute kidney injury (AKI) in mice. Sepsis model has been established by cecal ligation and puncture (CLP). CLP induced AKI in mice as assessed by increased renal neutrophil gelatinase-associated lipocalin (NGAL) expression and serum creatinine levels. We found that renal CXCL14 expression in the kidney was significantly decreased at 12 hours after CLP. Correlation analysis demonstrated a negative association between renal CXCL14 expression and AKI markers including serum creatinine and renal NGAL. Moreover, CXCL14 overexpression reduced cytokine (TNF-α, IL-6, and IL-1ß) production and NGAL expression in the kidney and decreased serum creatinine levels. In vivo and in vitro experiments found that CXCL14 overexpression inhibited M1 macrophage polarization but increased M2 polarization. Together, these results suggest that CXCL14 overexpression attenuates sepsis-associated AKI probably through the downregulation of macrophages-derived cytokine production. However, further studies are required to elucidate the underlying mechanism.

Electrochemistry-Regulated Recyclable SERS Sensor for Sensitive and Selective Detection of Tyrosinase Activity.

Tyrosinase (TYR) which can catalyze the oxidation of catechol is recognized as a significant biomarker of melanocytic lesions, thus developing powerful methods for the determination of TYR activity is highly desirable for the early diagnosis of melanin-related diseases, including melanoma. Herein, we develop a novel portable and recyclable surface-enhanced Raman scattering (SERS) sensor, prepared by assembling gold nanoparticles and p-thiol catechol ( p-TC) on an ITO electrode, for detecting TYR activity via the SERS spectral variation caused by the conversion of p-TC into its corresponding quinone under TYR catalysis. The developed SERS sensor has a rapid response to TYR within 1 min under the optimized conditions and shows high selectivity for TYR with the detection limit at 0.07 U/mL. Importantly, this SERS sensor can be easily regulated by applying negative voltage to achieve circular utilization, favoring the automation of SERS detection. Furthermore, the presented recyclable SERS sensor can perform well on both the determination of TYR activity in serum and the assessment of TYR inhibitor, demonstrating huge potential in the sensitive, selective, and facile detection of TYR activity for disease diagnosis and drug screening related with TYR.

Lower vitamin D levels are associated with depression in patients with gout.

BACKGROUND: Depression is commonly observed among patients with gout. Low levels of vitamin D have been associated with depression in non-gout subjects. We examined the association of vitamin D levels with depression in patients with gout. METHODS: We conducted a cross-sectional study of 186 gout patients at the Endocrinology Department of First Affiliated Hospital of Jiaxing University. Levels of serum 25-hydroxyvi-tamin D (25(OH)D) were determined using a competitive protein-binding assay. The 17-item Hamilton Depression Scale was used for screening for depressive symptoms. Diagnosis of depression in gout patients was made in accordance with Diagnostic and Statistical Manual of Mental Disorders, fifth edition criteria for depression. Multivariate analysis was performed using logistic regression models. RESULTS: Thirty-two gout patients (17.2%) were diagnosed as having depression. Patients with depression showed significantly lower 25(OH)D levels as compared to patients without depression (46.4±19.0 vs 57.0±17.3 nmol/L, P<0.001). Significant differences in 25(OH)D quartiles of gout patients were observed between the patients with depression and the patients without depression (P=0.003). In multivariate analyses, serum 25(OH)D levels (≤40.0 nmol/L) were independently associated with depression in patients with gout (OR 3.833, 95% CI 1.406-10.453, P=0.009). CONCLUSION: Our study demonstrates an important association between serum vitamin D levels and depression in patients with gout.

Discrepant Views of Oncologists and Cancer Patients on Complementary and Alternative Medicine in a Chinese General Hospital.

PURPOSE: Complementary and alternative medicine (CAM) has been widely used by cancer patients but rarely discussed by oncologists. This study was designed to evaluate the communication gap between China's oncologists and cancer patients on CAM. METHODS: Two parallel cross-sectional studies assessed 83 oncologists and 402 cancer patients on CAM communication between patients and oncologists, and attitudes toward CAM use and clinical decisions about CAM. RESULTS: A majority (75.1%) of the cancer patients (302/402) were identified as CAM users within the most recent three months while 77.6% of the cancer patients (312/402) were identified as CAM users since diagnosis of cancer. Oncologists and patients responded differently ( P < .001) on CAM communications. Both oncologists and patients expected that CAM could improve the immune system. They both agreed that oncologists usually discouraged their patients from using CAM. Regarding the effectiveness of CAM, cancer patients were more likely to believe that CAM was effective while oncologists had more concerns about adverse effects of CAM use. CAM use by patients was predicted by disease duration (≥9 months) in the multivariable logistic regression model. CONCLUSION: China's oncologists and cancer patients may hold discrepant views on CAM. China's oncologists are encouraged to improve their knowledge on CAM and to initiate more discussions with their patients regarding effective and the safe use of CAM.

Knockout of microRNA-155 ameliorates the Th1/Th17 immune response and tissue injury in chronic rejection.

BACKGROUND: MicroRNAs (miRNAs) are integral for maintaining immune homeostasis and self-tolerance. The influence of miRNAs on T-cell differentiation and plasticity are critical in the development of chronic rejection of transplanted hearts. In this study, we sought to determine whether the knockout of miR-155 affects the development of cardiac allograft vasculopathy (CAV) in a murine model. METHODS: miRNA microarray and quantitative polymerase chain reaction (qPCR) analyses were performed for allograft neointimal lesion samples in chronic rejection. A model of heterotopic murine heart transplantation (bm12 to miR-155+/+ or miR-155-/- mice) was then used to analyze allograft survival, histology, mRNA expression and T-cell sub-populations in spleens. The accelerated experiments were performed by intraperitoneal injection of either recombinant interleukin-17A or phosphate-buffered saline (PBS) after heart transplantation. For the competitive transfer experiments, CD4+ splenocytes from wild-type (WT) or miR-155-/- mice were mixed and injected into Rag1-/- mice, and cardiac transplantation was performed after 24 hours. The differentiation of T-helper subsets (Th1/Th17/iTreg) was investigated in vitro. RESULTS: miR-155-/- mice showed resistance to cardiac rejection along with weakened T-cell-mediated inflammation, especially for Th17 cells. Recombinant IL-17A could restore this relieved injury. The competitive experiments implied that miR-155 plays a vital role in the stability of the Th17 phenotype. In vitro, we also demonstrated that miR-155-/- mice exhibit a defect in Th17 differentiation. CONCLUSIONS: miR-155 regulates Th1/Th17-related inflammation in chronic cardiac rejection and may be a potential therapeutic target to attenuate cardiac allograft rejection. Despite advancements in immunosuppressive therapy, the immunologic mechanisms responsible for allograft rejection remain an important issue for both clinicians and researchers. Allograft rejection is a T-cell-dependent phenomenon and is critically dependent on inflammation mediated by CD4+ Th subsets, including Th1, Th2, Th17, Th9 and regulatory T (Treg) cells.

China's growing contribution to military Chinese medicine (2005-2014): a ten-year literature survey.

OBJECTIVE: In China, people have relied on traditional Chinese medicine (TCM) for thousands of years to keep healthy and treat diseases. TCM also plays an important role in military health services and now forms a new discipline called military Chinese medicine (MCM). However, the type, quality and focus of research articles about MCM have not been reported. The present study was performed to analyze the growing trends of MCM and investigate China's contribution to military health services. METHODS: China's MCM publications were retrieved from the PubMed database, as well as China National Knowledge Infrastructure, Wanfang Data and Chongqing VIP database from 2005 to 2014. RESULTS: The study found that the number of published articles increased markedly from 2005 to 2014. Basic research studies comprised a small percentage of the literature. Among these studies, military training injury and special military environmental medicine were the most common research subjects in MCM. Military hospitals were the main institutions generating MCM literature. CONCLUSION: The quality of MCM research is generally low, as indicated by the proportion of publications in core journals. Studies on MCM still lack high-quality publications and international cooperation.

Disease burden of enterovirus 71 in rural central China: A community-based survey.

In recent years, the epidemics of hand, foot, and mouth disease (HFMD) centered in the Asian-Pacific region have been characterized by high morbidity and mortality. Enterovirus 71 (EV71) infections were responsible for the majority of the infections leading to severe cases of HFMD and death. This is a community-based survey aimed to estimate the disease burden of EV71 in rural central China, especially for HFMD. From 2011 to 2013, demographic and socio-economic data were gathered from 343 ill children and their parents using a structured questionnaire. We quantified the health burden of disease resulting from EV71 infection in disability-adjusted life years (DALYs). Among 343 cases, 303 had confirmed HFMD, 6 presented with herpangina, 25 presented with respiratory symptoms, and 9 presented with non-specific symptoms. The number of severe cases was 47 (including 1 death) and all of these presented with HFMD. The total cost per patient for severe HFMD, mild HFMD, herpangina, respiratory disease, and non-specific disease was $2149.47, $513.22, $53.28, $31.95, and $39.25, respectively. The overall cost of EV71-related diseases as a proportion of local farmers' per capita net income ranged from 0.18% for those with non-specific disease to 187.12% for those with severe HFMD. The loss of DALYs for the 5 forms of disease were 3.47, 1.76, 1.07, 1.44, 1.22 person-years per 1000 persons, respectively. This study provides data on cost of treatment and health burden for diseases caused by EV71, which can be used in the evaluation of EV71 vaccine cost-effectiveness.

[Protective effect of melatonin in rats with phosgene-induced lung injury].

OBJECTIVE: To investigate the antioxidant effect of melatonin (MT) in the rats with phosgene-induced lung injury and its possible mechanism. METHODS: Fifty male SD rats were equally randomized into phosgene exposure group, air control group, MT treatment group, dexamethasone (DX) treatment group, and negative control group. All groups except the air control group were exposed to 8.33 mg/L phosgene for 5 min, and the MT treatment group, DX treatment group, and negative control group were injected with MT (10 mg/kg), DX (2.5 mg/kg), and 1% ethanol saline (1 ml/kg), respectively, via the caudal vein 1 hour after exposure. The rats were sacrificed 6h later. Then, the wet/dry ratio of the lung, the total protein content and neutrophil count in bronchoalveolar lavage fluid (BALF), and the malonaldehyde (MDA) content and superoxide dismutase (SOD) and myeloperoxidase (MPO) activities in lung homogenate were measured; pathological observation was made on the lung tissue under an optical microscope; the protein expression of inducible nitric oxide synthase (iNOS) and NF-κB in the lung tissue was measured by Western blot. RESULTS: Compared with the air control group, the phosgene exposure group showed significantly increased wet/dry ratio of the lung and total protein content and neutrophil count in BALF (P < 0.01) as well as significantly increased MDA content and MPO activity in the lung tissue (P < 0.05). Compared with the phosgene exposure group, the MT treatment group showed significantly decreased MDA content and MPO activity and significantly increased SOD activity (P < 0.01), and the MT treatment group and DX treatment group showed significantly decreased protein expression of iNOS and NF-κB (P < 0.01). CONCLUSION: MT has protective effect in phosgene-induced lung injury, and its protective mechanism may be associated with scavenging free radicals and inhibiting expression of iNOS and NF-κB.