Cell cycle associated protein 1 associates with immune infiltration and ferroptosis in gastrointestinal cancer.

Background: Cell Cycle-Associated Protein 1 (CAPRIN1) play an important role in cell proliferation, oxidative stress, and inflammatory response. Nonetheless, its role in tumor immunity and ferroptosis is largely unknown in gastrointestinal cancer patients. Methods: Through comprehensive bioinformatics, we investigate CAPRIN1 expression patterns and its role in diagnosis, functional signaling pathways, tumor immune infiltration and ferroptosis of different gastrointestinal cancer subtypes. Besides, immunohistochemistry (IHC) and immune blot were used to validate our esophagus cancer clinical data. The ferroptotic features of CAPRIN1 in vitro were assessed through knockdown assays in esophagus cancer cells. Results: CAPRIN1 expression was significantly upregulated, correlated with poor prognosis, and served as an independent risk factor for most gastrointestinal cancer. Moreover, CAPRIN1 overexpression positively correlated with gene markers of most infiltrating immune cells, and immune checkpoints. CAPRIN1 knockdown significantly decreased the protein level of major histocompatibility complex class I molecules. We also identified a link between CAPRIN1 and ferroptosis-related genes in gastrointestinal cancer. Knockdown of CAPRIN1 significantly increased the production of lipid reactive oxygen species and malondialdehyde. Inhibition of CAPRIN1 expression promoted ferroptotic cell death induced by RAS-selective lethal 3 and erastin in human esophagus cancer cells. Conclusion: Collectively, our results demonstrate that CAPRIN1 is aberrantly expressed in gastrointestinal cancer, is associated with poor prognosis, and could potentially influence immune infiltration and ferroptosis.

Thrombospondin 4, a mediator and candidate indicator of pain.

Pain is the most common symptom for which patients seek medical attention. Existing treatments for pain control are largely ineffective due to the lack of an accurate way to objectively measure pain intensity and a poor understanding of the etiology of pain. Thrombospondin 4(TSP4), a member of the thrombospondin gene family, is expressed in neurons and astrocytes and induces pain by interacting with the calcium channel alpha-2-delta-1 subunit (Cavα2δ1). In the present study we show that TSP4 expression level correlates positively with pain intensity, suggesting that TSP4 could be a novel candidate of pain indicator. Using RNAi-lentivirus (RNAi-LV) to knock down TSP4 both in vivo and in vitro, together with electrophysiological experiments involving paired patch-clamp recordings of evoked action potentials and post-synaptic currents in cultured neurons, we found that TSP4 contributes to the development of bone cancer pain, neuropathic pain, and inflammatory pain. This effect is mediated by regulation of neuron excitability via inhibition of synapsin I (Syn I) and modulation of excitatory and inhibitory presynaptic transmission via regulation of vesicular glutamate transporter 2(Vglut2), vesicular GABA transporter (VGAT), and glutamate decarboxylase (GAD) expression. The present study provides a replicable, predictive, valid indicator of pain and demonstrated the underlying molecular and electrophysiological mechanisms by which TSP4 contributes to pain.

MHC-I in the hippocampus promotes comorbid depressive symptoms in bone cancer pain via the upregulation of microglial TREM2/DAP12 signaling.

Pain and depression comorbidity affects patients' physical and mental health, as well as quality of life. Comorbid depressive symptoms in cancer pain have a severe impact on the recognition and treatment of pain. Similarly, cancer pain patients with depression are inclined towards more despair and greater impairment. The mechanisms responsible for the comorbid depressive symptoms in bone cancer pain (BCP) have not been fully delineated. Here, it was reported that the implantation of carcinoma cells into the femoral cavity of mice led to the upregulation of major histocompatibility complex class I (MHC-I) in the hippocampus. This was associated with the activation of microglial signaling pathway mediated by the triggering receptor expressed on myeloid cells 2 protein (TREM2) and DNAX-activating protein of 12 kDa (DAP12). Pain and depression-like behaviors were reversed by the knockdown of hippocampal MHC-I via a lentiviral vector harboring ribonucleic acid interference (RNAi) sequence. Moreover, MHC-I knockdown exhibited a marked reduction in the expression of TREM2 and DAP12. These results suggested that hippocampal MHC-I was involved in BCP and depression comorbidity via upregulating the signals mediated by TREM2/DAP12 in microglia. The suppression of MHC-I could be a potential therapeutic target for BCP.

SFXN1 as a potential diagnostic and prognostic biomarker of LUAD is associated with 18F-FDG metabolic parameters.

BACKGROUND: Sideroflexin 1 (SFXN1) has been discovered as a novel tumor marker for lung adenocarcinoma, but data on its importance in the development of lung adenocarcinoma is still limited. This study evaluated the correlation between SFXN1 and parameters related to 18F-flurodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT), and further explored the role of SFXN1 in the value-added and glycolytic processes of LUAD. METHOD: The expression and prognostic value of SFXN1 mRNA in LUAD were analyzed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data base. Retrospective analysis of 18F-FDG PET imaging and metabolic parameters in 42 patients to explore the relationship between the expression of SFXN1 and glucose metabolism levels in lung adenocarcinoma and its clinical significance. H1975 cells were selected as the in vitro research object, and the biological effects of SFXN1 on LUAD were further elucidated through Edu proliferation assay, CCK8 activity assay, wound healing experiment, and cell flow cytometry. RESULT: SFXN1 is highly expressed in various tumors, including LUAD, and its high expression can serve as an independent predictor of overall survival in lung adenocarcinoma. In addition, the expression of SFXN1 in LUAD was significantly correlated with 18F-FDG PET/CT parameters: maximum and average standardized uptake values (SUVmax and SUVmean), as well as total lesion glycolysis (TLG) (rho = 0.574, 0.589, and 0.338, p < 0.05), which can predict the expression of SFXN1 with an accuracy of 0.934. In vitro functional experiments have shown that knocking down SFXN1 inhibits the proliferation and migration of LUAD cells, promotes cell apoptosis, and may inhibit tumor activity by regulating the expression of glycolytic related genes SLC2A1, HK2, GPI, ALDOA, GAPDH, ENO1, PKM, and LDHA. CONCLUSION: The overexpression of SFXN1 is closely related to FDG uptake, and SFXN1, as a promising prognostic biomarker, may mediate the development of LUAD through the glycolytic pathway.

Upregulation of Calhm2 in the anterior cingulate cortex contributes to the maintenance of bilateral mechanical allodynia and comorbid anxiety symptoms in inflammatory pain conditions.

Peripheral inflammation-induced chronic pain tends to evoke concomitant anxiety disorders. It's common knowledge that the anterior cingulate cortex (ACC) plays a vital role in maintaining pain modulation and negative emotions. However, the potential mechanisms of chronic inflammation pain and pain-related anxiety remain elusive. Here, it was reported that injecting complete Freund's adjuvant (CFA) unilaterally resulted in bilateral mechanical allodynia and anxiety-like symptoms in mice via behavioral tests. In addition, CFA induced the bilateral upregulation and activation of calcium homeostasis modulator 2 (Calhm2) in ACC pyramidal neurons by quantitative analysis and double immunofluorescence staining. The knockdown of Calhm2 in the bilateral ACC by a lentiviral vector harboring ribonucleic acid (RNA) interference sequence reversed CFA-induced pain behaviors and neuronal sensitization. Furthermore, the modulating of ACC pyramidal neuronal activities via a designer receptor exclusively activated by designer drugs (DREADD)-hM4D(Gi) greatly changed Calhm2 expression, mechanical paw withdrawal thresholds (PWTs) and comorbid anxiety symptoms. Moreover, it was found that Calhm2 regulates inflammation pain promoting the upregulation of N-methyl-D-aspartic acid (NMDA) receptor 2B (NR2B) subunits. Calhm2 knockdown in ACC exhibited a significant decrease in NR2B expression. These results demonstrated that Calhm2 in ACC pyramidal neurons modulates chronic inflammation pain and pain-related anxiety symptoms, which provides a novel underlying mechanism for the development of inflammation pain.

SLC17A9 as a prognostic biomarker correlated with immune infiltrates in human non-small cell lung cancer.

The vesicular nucleotide transporter (SLC17A9) has been overexpressed in various cancers. Nonetheless, little is known about its influence on non-small cell lung cancer (NSCLC), including human lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Integrative bioinformatics analysis was performed to investigate the prognostic significance and underlying mechanisms of SLC17A9 in patients with NSCLC. Here, we found that SLC17A9 up-regulation was significantly correlated with overall survival in LUAD and LUSC (P < 0.05). Gene set enrichment analysis and protein-protein interaction results revealed that SLC17A9 up-regulation was linked to metabolic process, the hallmark of MYC targets, DNA repair, coagulation and complement. SLC17A9 expression was negatively associated with overall survival and positively related to most LUSC immune cells and immunoinhibitor (20/23), particularly immuno A2aR, PD-1, and CTLA-4 (P < 0.001). High SLC17A9 was associated with infiltrating levels of B cells, CD4+ T cells, M1 macrophages, and T cell exhaustion checkpoints such as PD-1, CTLA4, and LAG3 in LUAD. Moreover, Real-time PCR, MTS assay, EdU assay, ATP production assays and cell cycle analysis were performed to validate SLC17A9 knockdown in LUAD cells. SLC17A9 knockdown significantly inhibited cell proliferation and ATP levels by affecting P2X1, Cytochrome C, and STAT3 expression in lung cancer cells. In conclusion, the present study suggested that SLC17A9 could potentially serve as a prognostic biomarker and correlated with immune infiltrates in LUAD and LUSC.

Caprin-1 plays a role in cell proliferation and Warburg metabolism of esophageal carcinoma by regulating METTL3 and WTAP.

BACKGROUND: Cytoplasmic activation/proliferation-associated protein-1 (Caprin-1) is implicated in cancer cell proliferation and tumorigenesis; however, its role in the development of esophageal carcinoma (ESCA) has not been examined. METHODS: Biological methods and data analysis were used to investigate the expression of Caprin-1 in ESCA tissue and cell lines. We comprehensively analyzed the mRNA expression and prognostic values, signalling pathways of CAPRIN1 in ESCA using public databases online. Biological functions of CAPRIN1 were performed by clorimetric growth assay, EdU staining, colony formation, flow cytometry, apoptosis analysis, Western blot, lactate detection assay, extracellular acidification rates. The underlying mechanism was determined via flow cytometric analysis, Western blot and rescue experiments. In addition, xenograft tumor model was constructed to verify the phenotypes upon CAPRIN1 silencing. RESULTS: Caprin-1 expression was significantly elevated in both ESCA tumor tissues and cell lines compared with that in normal adjacent tissues and fibroblasts. Increased CAPRIN1 mRNA expression was significantly associated with clinical prognosis and diagnostic accuracy. The GO enrichment and KEGG pathway analysis CAPRIN1 might be related to immune-related terms, protein binding processes, and metabolic pathways. A significant positive correlation was observed between high Caprin-1 protein levels and lymph node metastasis (P = 0.031), ki-67 (P = 0.023), and 18F- FDG PET/CT parameters (SUVmax (P = 0.002) and SUV mean (P = 0.005)) in 55 ESCA patients. At cut-off values of SUVmax 17.71 and SUVmean 10.14, 18F- FDG PET/CT imaging predicted Caprin-1 expression in ESCA samples with 70.8% sensitivity and 77.4% specificity. In vitro and in vivo assays showed that Caprin-1 knockdown affected ESCA tumor growth. Silencing Caprin-1 inhibited ESCA cell proliferation and glycolysis, and decreased the expression of methyltransferase-like 3 (METTL3) and Wilms' tumor 1-associating protein (WTAP). However, this effect could be partially reversed by the restoration of METTL3 and WTAP expression. CONCLUSIONS: Our data suggest that Caprin-1 could serve as a prognostic biomarker and has an oncogenic role in ESCA.

Neuroligins facilitate the development of bone cancer pain via regulating synaptic transmission: an experimental study.

BACKGROUND: The underlying mechanism of chronic pain involves the plasticity in synaptic receptors and neurotransmitters. This study aimed to investigate potential roles of Neuroligins (NLs) within the spinal dorsal horn of rats in a newly established Bone Cancer Pain (BCP) model. The objective was to explore the mechanism of neuroligin involved in the occurrence and development of bone cancer pain. METHODS: Using our rat BCP model, we assessed pain hypersensitivity over time. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to investigate NL expression, and NLs were overexpressed in the rat spinal cord using lentiviral vectors. Immunofluorescence staining and whole-cell patch-clamp recordings were deployed to investigate the role of NLs in the development of BCP. RESULTS: We observed reduced expression levels of NL1 and NL2, but not of NL3, within the rat spinal cord, which were found to be associated with and essential for the development of BCP in our model. Accordingly, NL1 or NL2 overexpression in the spinal cord alleviated mechanical hypersensitivity of rats. Electrophysiological experiments indicated that NL1 and NL2 are involved in BCP via regulating γ-aminobutyric acid-ergic interneuronal synapses and the activity of glutamatergic interneuronal synapses, respectively. CONCLUSIONS: Our observations unravel the role of NLs in cancer-related chronic pain and further suggest that inhibitory mechanisms are central features of BCP in the spinal dorsal horn. These results provide a new perspective and basis for subsequent studies elucidating the onset and progression of BCP.

Differential epitranscriptome and proteome modulation in the brain of neonatal mice exposed to isoflurane or sevoflurane.

BACKGROUND: Repeated neonatal exposure to anesthetics may disturb neurodevelopment and cause neuropsychological disorders. The m6A modification participates in the gene regulation of neurodevelopment in mouse fetuses exposed to anesthetics. This study aims to explore the underlying molecular mechanisms of neurotoxicity after early-life anesthesia exposure. METHODS: Mice were exposed to isoflurane (1.5%) or sevoflurane (2.3%) for 2 h daily during postnatal days (PND) 7-9. Sociability, spatial working memory, and anxiety-like behavior were assessed on PND 30-35. Synaptogenesis, epitranscriptome m6A, and the proteome of brain regions were evaluated on PND 21. RESULTS: Both isoflurane and sevoflurane produced abnormal social behaviors at the juvenile age, with different sociality patterns in each group. Synaptogenesis in the hippocampal area CA3 was increased in the sevoflurane-exposed mice. Both anesthetics led to numerous persistent m6A-induced alterations in the brain, associated with critical metabolic, developmental, and immune functions. The proteins altered by isoflurane exposure were mainly associated with epilepsy, ataxia, and brain development. As for sevoflurane, the altered proteins were involved in social behavior. CONCLUSIONS: Social interaction, the modulation patterns of the m6A modification, and protein expression were altered in an isoflurane or sevoflurane-specific way. Possible molecular pathways involved in brain impairment were revealed, as well as the mechanism underlying behavioral deficits following repeated exposure to anesthetics in newborns.

High expression of HNRNPR in ESCA combined with 18F-FDG PET/CT metabolic parameters are novel biomarkers for preoperative diagnosis of ESCA.

BACKGROUND: The aim of this study was to determine the expression and function of heterogeneous nuclear ribonucleoprotein R (HNRNPR) in esophageal carcinoma (ESCA), the correlation between its expression and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computerized tomography scan (PET/CT)-related parameters. We also investigated whether 18F-FDG PET/CT can be used to predict the expression of HNRNPR in ESCA. METHODS: We analyzed patients with ESCA who underwent 18F-FDG PET/CT before surgery, and their tissues were stained with HNRNPR IHC. The associated parameters were derived using the 18F-FDG PET imaging data, and the correlation with the IHC score was evaluated. The Oncomine, TCGA, and GEO datasets were used to investigate HNRNPR expression in the pan- and esophageal cancers, as well as its relationship with N6-methyladenosine (m6A) modification and glycolysis. The R software, LinkedOmics, GeneMANIA, and StringOnline tools were used to perform GO/KEGG, GGI, and PPI analyses on the HNRNPR. RESULTS: HNRNPR is highly expressed in the majority of pan-cancers, including ESCA, and is associated with BMI, weight, and history of reflux in patients with ESCA. HNRNPR is somewhat accurate in predicting the clinical prognosis of ESCA. HNRNPR expression was positively correlated with SUVmax, SUVmean, and TLG in ESCA (p < 0.05). The combination of these three variables provides a strong predictive value for HNRNPR expression in ESCA. GO/KEGG analysis showed that HNRNPR played a role in the regulation of cell cycle, DNA replication, and the Fannie anemia pathway. The analysis of the TCGA and GEO data sets revealed a significant correlation between HNRNPR expression and m6A and glycolysis-related genes. GSEA analysis revealed that HNRNPR was involved in various m6A and glycolysis related-pathways. CONCLUSION: HNRNPR overexpression correlates with 18F-FDG uptake in ESCA and may be involved in the regulation of the cell cycle, m6A modification, and cell glycolysis. 18F-FDG PET/CT-related parameters can predict the diagnostic accuracy of HNRNPR expression in ESCA.

eIF6 is potential diagnostic and prognostic biomarker that associated with 18F-FDG PET/CT features and immune signatures in esophageal carcinoma.

BACKGROUND: Although eukaryotic initiation factor 6 (eIF6) is a novel therapeutic target, data on its importance in the development of esophageal carcinoma (ESCA) remains limited. This study evaluated the correlation between eIF6 expression and metabolic analysis using fluorine-18 fluorodeoxyglucose (18F-FDG) -Positron emission tomography (PET) and immune gene signatures in ESCA. METHODS: This study employed The Cancer Genome Atlas (TCGA) to analyze the expression and prognostic value of eIF6, as well as its relationship with the immune gene signatures in ESCA patients. The qRT-PCR and Western blot analyses were used to profile the expression of eIF6 in ESCA tissues and different ESCA cell lines. The expression of tumor eIF6 and glucose transporter 1 (GLUT1) was examined using immunohistochemical tools in fifty-two ESCA patients undergoing routine 18F-FDG PET/CT before surgery. In addition, the cellular responses to eIF6 knockdown in human ESCA cells were assessed via the MTS, EdU, flow cytometry and wound healing assays. RESULTS: Our data demonstrated that compared with the normal esophageal tissues, eIF6 expression was upregulated in ESCA tumor tissues and showed a high diagnostic value with an area under curve of 0.825 for predicting ESCA. High eIF6 expression was significantly correlated with shorter overall survival of patients with esophagus adenocarcinoma (p = 0.038), but not in squamous cell carcinoma of the esophagus (p = 0.078). In addition, tumor eIF6 was significantly associated with 18F-FDG PET/CT parameters: maximal and mean standardized uptake values (SUVmax and SUVmean) and total lesion glycolysis (TLG) (rho = 0.458, 0.460, and 0.300, respectively, p < 0.01) as well as GLUT1 expression (rho = 0.453, p < 0.001). A SUVmax cutoff of 18.2 led to prediction of tumor eIF6 expression with an accuracy of 0.755. Functional analysis studies demonstrated that knockdown of eIF6 inhibited ESCA cell growth and migration, and fueled cell apoptosis. Moreover, the Bulk RNA gene analysis revealed a significant inverse association between eIF6 and the tumor-infiltrating immune cells (macrophages, T cells, or Th1 cells) and immunomodulators in the ESCA microenvironment. CONCLUSION: Our study suggested that eIF6 might serve as a potential prognostic biomarker associated with metabolic variability and immune gene signatures in ESCA tumor microenvironment.

Schnurri-2 Promotes the Expression of Excitatory Glutamate Receptors and Contributes to Neuropathic Pain.

Neuropathic pain is a type of chronic pain with complex mechanisms, and current treatments have shown limited success in treating patients suffering from chronic pain. Accumulating evidence has shown that the pathogenesis of neuropathic pain is mediated by the plasticity of excitatory neurons in the dorsal horn of the spinal cord, which provides insights into the treatment of hyperalgesia. In this study, we found that Schnurri-2 (Shn2) was significantly upregulated in the L4-L6 segments of the spinal cord of C57 mice with spared nerve injury, which was accompanied by an increase in GluN2D subunit and glutamate receptor subunit 1 (GluR1) levels. Knocking down the expression of Shn2 using a lentivirus in the spinal cord decreased the GluN2D subunit and GluR1 levels in spared nerve injury mice and eventually alleviated mechanical allodynia. In summary, Shn2 regulates neuropathic pain, promotes the upregulation of GluN2D in glutamatergic neurons and increases the accumulation of GluR1 in excitatory neurons. Taken together, our study provides a new underlying mechanism for the development of neuropathic pain.

NWD1 facilitates synaptic transmission and contributes to neuropathic pain.

Neuropathic pain is the most common symptom for which patients seek medical attention. Existing treatments to control pain are largely ineffective because of poor understanding the underlying mechanisms. Synaptic plasticity is fundamental to the spinal sensitivity of neuropathic pain. In the present study, we showed that SNL induced significant allodynia and hyperalgesia as well as upregulation of Nwd1 and GluN2B, which were reversed by knockdown of NWD1. Electrophysiological experiments demonstrated that SNL enhanced synaptic transmission, which was prevented by knockdown of NWD1. In vitro experiments showed that knockdown of NWD1 inhibited dendritic growth and synaptogenesis. Taken together, our results suggest that NWD1 enhances synaptic transmission and contributes to the development of neuropathic pain by enhancing GluN2B synaptic expression and anchor and promoting excitatory synaptogenesis.

18F-FDG PET/CT metabolic parameters correlate with EIF2S2 expression status in colorectal cancer.

Background: We sought to investigate whether the expression of the gene EIF2S2 is related to 18F-FDG PET/CT metabolic parameters in patients with colorectal cancer (CRC). Materials and methods: The expression of EIF2S2 in CRC and its relationship with clinicopathological features were obtained through the ONCOMINE, UALCAN and GEPIA databases. EIF2S2 and GLUT1 expression were examined by immunohistochemistry in 42 CRC patients undergoing preoperative PET-CT examination. Spearman correlation analysis was used to assess the relationship between EIF2S2 and GLUT1 levels and clinical parameters. Correlation analysis between EIF2S2 and Reactome-Glycolysis signatures was performed using GEPIA2. We describe the effect of EIF2S2 knockdown on lactate production and the mRNA levels of glycolysis-related genes in human colon cancer SW480 cells. Results: Immunohistochemistry revealed an upregulation of EIF2S2 protein expression in tumor tissues of colorectal cancer patients, which is consistent with the significant upregulation of EIF2S2 transcript levels in the database. These colorectal cancer patients included 24 cases of colon cancer and 18 cases of rectal cancer, ranging in age from 31 to 78 years. The transcription was significantly related to histological subtypes and TP53 mutations (P <0.05). The value of SUVmax in CRC significantly correlated with the expression of EIF2S2 (rho = 0.462, P <0.01). Although SUVmax and SUVmean was not correlate with the expression of GLUT1 (P <0.05), a significant correlation was observed between the expression of GLUT1 and the volumetric PET parameters, such as MTV and TLG (P < 0.01). GLUT1 expression in CRC was positively correlated with EIF2S2 status (rho = 0.470, P <0.01). In SW480 cells, RNAi-mediated depletion of EIF2S2 inhibited lactic acid production (P <0.05) and SLC2A1, SLC2A3, SLC2A10, HK2, PKM2, LDHA mRNA level (P <0.01). Conclusions: Primary CRC FDG uptake is strongly associated with the overexpression of EIF2S2, and EIF2S2 may promote glycolysis in CRC by mediating GLUT1.

Electrophysiological Properties of Substantia Gelatinosa Neurons in the Preparation of a Slice of Middle-Aged Rat Spinal Cord.

A patch-clamp recording in slices generated from the brain or the spinal cord has facilitated the exploration of neuronal circuits and the molecular mechanisms underlying neurological disorders. However, the rodents that are used to generate the spinal cord slices in previous studies involving a patch-clamp recording have been limited to those in the juvenile or adolescent stage. Here, we applied an N-methyl-D-glucamine HCl (NMDG-HCl) solution that enabled the patch-clamp recordings to be performed on the superficial dorsal horn neurons in the slices derived from middle-aged rats. The success rate of stable recordings from substantia gelatinosa (SG) neurons was 34.6% (90/260). When stimulated with long current pulses, 43.3% (39/90) of the neurons presented a tonic-firing pattern, which was considered to represent γ-aminobutyric acid-ergic (GABAergic) signals. Presumptive glutamatergic neurons presented 38.9% (35/90) delayed and 8.3% (7/90) single-spike patterns. The intrinsic membrane properties of both the neuron types were similar but delayed (glutamatergic) neurons appeared to be more excitable as indicated by the decreased latency and rheobase values of the action potential compared with those of tonic (GABAergic) neurons. Furthermore, the glutamatergic neurons were integrated, which receive more excitatory synaptic transmission. We demonstrated that the NMDG-HCl cutting solution could be used to prepare the spinal cord slices of middle-aged rodents for the patch-clamp recording. In combination with other techniques, this preparation method might permit the further study of the functions of the spinal cord in the pathological processes that occur in aging-associated diseases.

Neuroligin1 Contributes to Neuropathic Pain by Promoting Phosphorylation of Cofilin in Excitatory Neurons.

Neuropathic pain is a kind of chronic pain that remains difficult to treat due to its complicated underlying mechanisms. Accumulating evidence has indicated that enhanced synaptic plasticity of nociceptive interneurons in the superficial spinal dorsal horn contributes to the development of neuropathic pain. Neuroligin1 (NL1) is a type of excitatory postsynaptic adhesion molecule, which can mediate excitatory synaptic activity, hence promoting neuronal activation. Vglut2 is the most common marker of excitatory glutamatergic neurons. To explore the role of NL1 in excitatory neurons in nociceptive regulation, we used transgenic mice with cre recombinase expression driven by the Vglut2 promoter combined with viral vectors to knockdown the expression of NL1 in excitatory neurons in the spinal dorsal horn. We found that NL1 was upregulated in the L4-L6 spinal dorsal horn in Vglut2-cre+/- mouse subjected to spared nerve injury (SNI). Meanwhile, the expression of phosphorylated cofilin (p-cofilin) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 1 (GluR1) was also increased. Spinal microinjection of a cre-dependent NL1-targeting RNAi in Vglut2-cre+/- mouse alleviated the neuropathic pain-induced mechanical hypersensitivity and reduced the increase in p-cofilin and GluR1 caused by SNI. Taken together, NL1 in excitatory neurons regulates neuropathic pain by promoting the SNI-dependent increase in p-cofilin and GluR1 in the spinal dorsal horn. Our study provides a better understanding of the role of NL1 in excitatory neurons, which might represent a possible therapeutic target for alleviating neuropathic pain.

Shank3 contributes to neuropathic pain by facilitating the SNI-dependent increase of HCN2 and the expression of PSD95.

Neuropathic pain is a very complex chronic pain state, the detailed molecular mechanisms of which remain unclear. In the present study, Shank3 was found to play an important role in neuropathic pain in rats following spared nerve injury (SNI). Shank3 was upregulated in the spinal dorsal horn of rats subjected to SNI, and mechanical hypersensitivity to noxious stimuli in these rats could be alleviated by knock down of Shank3. Shank3 also interacted with hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) and promoted the expression of HCN2 in central neurons of the spinal dorsal. Together with the SNI-dependent increase of HCN2, we also found that the postsynaptic protein of excitatory synapse (PSD95) was increased in rats following SNI. Taken together, our results showed that Shank3 modulated neuropathic pain by facilitating the SNI-dependent increase of HCN2 and the expression of PSD95 in spinal dorsal horn neurons. Our findings revealed new synaptic remodeling mechanisms linking Shank3 with neuropathic pain.

Virus Injection to the Pituitary via Transsphenoidal Approach and the Innervation of Anterior and Posterior Pituitary of Rat.

The theory holds that the anterior pituitary in mammals receives humoral regulation. Previous studies have reported that the pars distalis of the anterior pituitary of several mammalian species contains substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive nerve fibers, but the origins of these nerve fibers are unclear. Removal of the pituitary gland, also called hypophysectomy, involves methods that access the pituitary gland via the transauricular or parapharyngeal pathways. However, these methods are not applicable for viral tracer injection to investigate the innervation of the anterior pituitary. The transauricular technique leads to inaccuracies in locating the pituitary gland, while the parapharyngeal approach causes high mortality in animals. Here, we introduce a protocol that accesses the pituitary gland in the rat via the transsphenoidal pathway. This method imitates surgical manipulations such as endotracheal intubation and sphenoid bone drilling, which involve the use of custom-made devices. Using the transsphenoidal pathway greatly improves the survival rate of rats because no additional dissection of blood vessels and nerves is required. Moreover, the pituitary gland can be viewed clearly and directly during the operation, making it possible to accurately inject pseudorabies virus (PRV) 152-expressing enhanced green fluorescent protein (EGFP) into the anterior or posterior pituitary, respectively. After injecting PRV 152 into the anterior pituitary, we found no evidence of direct innervation of the anterior pituitary in the rat brain. However, PRV 152 injection into the posterior pituitary revealed retrograde transneuronal cell bodies in many brain areas, including the CA1 field of the hippocampus, the basolateral amygdaloid nucleus, posterior part (BLP), the arcuate hypothalamic nucleus (Arc), the dorsal portion of the dorsomedial hypothalamic nucleus (DMD), the suprachiasmatic nucleus (SCh), and the subfornical organ (SFO). In the present study, we provide a description of a possible model of hypophysectomy or pituitary injection, and identify brain regions involved in regulating the rat pituitary gland using transneuronal retrograde cell body labeling with PRV.

Visualization of gene therapy with a liver cancer-targeted adeno-associated virus 3 vector.

Background: To evaluate the feasibility of a self-complementing recombinant adeno-associated virus 3 (scrAAV3) vector targeting liver cancer and non-invasively monitor gene therapy of liver cancer. Materials and methods: An scrAAV3-HSV1-TK-kallistatin (ATK) gene drug was constructed, which contained the herpes virus thymidine kinase (HSV1-TK) reporter gene and human endogenous angiogenesis inhibitor (kallistatin) gene for non-invasive imaging of gene expression. Subcutaneous xenografted tumors of hepatoma in nude mice were generated for positron emission tomography/computed tomography (PET/CT) imaging. The ATK group was injected with the ATK gene through the tail vein, and an imaging agent was injected 2 weeks later. PET/CT imaging was performed at 1 hour after injection of the imaging agent. The control group was injected with phosphate-buffered saline at the same volume as the ATK gene drug. HE staining is used for pathological observation of tumor sections. HSV1-TK and kallistatin expression was identified by immunofluorescence, real-time quantitative PCR, and western blotting. Results: Radioactivity on PET/CT images was significantly higher in the ATK group compared with the control group. 18F-FHBG uptake values of left forelegs in ATK and control groups were 0.591±0.151% and 0.017 ± 0.011% ID/g (n=5), respectively (P<0.05). After injection of the ATK gene drug, mRNA and protein expression of HSV1-TK and kallistatin in subcutaneous xenograft tumors was detected successfully. In vitro analysis demonstrated significant differences in the expression of HSV1-TK and kallistatin between ATK and control groups (P<0.05). Conclusions: The scrAAV3 vector has a strong liver cancer-targeting ability, and the ATK gene drug can be used for targeted and non-invasive monitoring of liver cancer gene therapy.