Astrocyte activation in the periaqueductal gray promotes descending facilitation to cancer-induced bone pain through the JNK MAPK signaling pathway.

Descending nociceptive modulation from the supraspinal structures has an important role in cancer-induced bone pain (CIBP). Midbrain ventrolateral periaqueductal gray (vlPAG) is a critical component of descending nociceptive circuits; nevertheless, its precise cellular and molecular mechanisms involved in descending facilitation remain elusive. Our previous study has shown that the activation of p38 MAPK in vlPAG microglia is essential for the neuropathic pain sensitization. However, the existence of potential connection between astrocytes and c-Jun N-terminal kinase (JNK) pathway in CIBP has not yet been elucidated. The following study examines the involvement of astrocyte activation and upregulation of p-JNK in vlPAG, using a CIBP rat model. Briefly, CIBP was mimicked by an intramedullary injection of Walker 256 mammary gland carcinoma cells into the animal tibia. A significant increase in expression levels of astrocytes in the vlPAG of CIBP rats was observed. Furthermore, stereotaxic microinjection of the astrocytic cytotoxin L-α-aminoadipic acid decreased the mechanical allodynia as well as established and reversed the astrocyte activation in CIBP rats. A significant increase in expression levels of p-JNK in astrocytes in vlPAG of CIBP rats was also observed. Moreover, the intrathecal administration of JNK inhibitors SP600125 reduced the expression of glial fibrillary acidic protein, while microinjection of the SP600125 decreased the mechanical allodynia of CIBP rats. These results suggested that CIBP is associated with astrocyte activation in the vlPAG that probably participates in driving descending pain facilitation through the JNK MAPK signaling pathway. To sum up, these findings reveal a novel site of astrocytes modulation of CIBP.

Crosstalk between NFκB-dependent astrocytic CXCL1 and neuron CXCR2 plays a role in descending pain facilitation.

BACKGROUND: Despite accumulating evidence on the role of glial cells and their associated chemicals in mechanisms of pain, few studies have addressed the potential role of chemokines in the descending facilitation of chronic pain. We aimed to study the hypothesis that CXCL1/CXCR2 axis in the periaqueductal gray (PAG), a co-restructure of the descending nociceptive system, is involved in descending pain facilitation. METHODS: Intramedullary injection of Walker 256 mammary gland carcinoma cells of adult female Sprague Dawley rats was used to establish a bone cancer pain (BCP) model. RT-PCR, Western blot, and immunohistochemistry were performed to detect pNfkb, Cxcl1, and Cxcr2 and their protein expression in the ventrolateral PAG (vlPAG). Immunohistochemical co-staining with NeuN, GFAP, and CD11 were used to examine the cellular location of pNFκB, CXCL1, and CXCR2. The effects of NFκB and CXCR2 antagonists and CXCL1 neutralizing antibody on pain hypersensitivity were evaluated by behavioral testing. RESULTS: BCP induced cortical bone damage and persistent mechanical allodynia and increased the expression of pNFκB, CXCL1, and CXCR2 in vlPAG. The induced phosphorylation of NFκB was co-localized with GFAP and NeuN, but not with CD11. Micro-injection of BAY11-7082 attenuated BCP and reduced CXCL1 increase in the spinal cord. The expression level of CXCL1 in vlPAG showed co-localization with GFAP, but not with CD11 and NeuN. Micro-administration of CXCL1 neutralizing antibody from 6 to 9 days after inoculation attenuated mechanical allodynia. Furthermore, vlPAG application of CXCL1 elicited pain hypersensitivity in normal rats. Interestingly, CXCR2 was upregulated in vlPAG neurons (not with CD11 and GFAP) after BCP. CXCR2 antagonist SB225002 completely blocked the CXCL1-induced mechanical allodynia and attenuated BCP-induced pain hypersensitivity. CONCLUSION: The NFκB-dependent CXCL1-CXCR2 signaling cascade played a role in glial-neuron interactions and in descending facilitation of BCP.

Nuclear factor kappa B regulated monocyte chemoattractant protein-1/chemokine CC motif receptor-2 expressing in spinal cord contributes to the maintenance of cancer-induced bone pain in rats.

BACKGROUND: Chemokine, monocyte chemoattractant protein-1 (MCP-1), is a potential factor to cause cancer-induced bone pain (CIBP). NF-κB signaling is very important in mediating the expression of chemokines and may have a role in CIBP. However, the mechanism is still unclear. This study investigates the role of NF-κB in CIBP by regulating MCP-1/chemokine CC motif receptor-2 (CCR2) signaling pathway. METHODS: A rat CIBP model was established by injecting Walker-256 cells into the tibia medullary cavity. Nine days later, animals were intrathecally administrated with MCP-1 neutralizing antibody, CCR2 antagonist (RS504393), or NF-кB inhibitor (BAY11-7081). Mechanical paw withdrawal threshold was used to assess pain behavior and sciatic functional index, and radiographic images were adopted to evaluate the damage of nerve and bone. The spinal cords were harvested for Western blot and quantitative reverse transcription polymerase chain reaction. The distribution of MCP-1, CCR2, and NF-кB was detected by double immunofluorescent staining. RESULTS: CIBP caused remarkable bone destruction, injury of sciatic and femoral nerve, and persistent (>15 days) mechanical allodynia in rats. Tumor cell inoculation upregulate MCP-1 and NF-кB in activated neurons as well as CCR2 in neurons and microglia of the spinal cord. MCP-1 antibody, RS504393, and BAY11-7081 partially reversed CIBP-induced mechanical allodynia, and CIBP regulated the expression levels of pro-inflammatory cytokines, tumor necrosis factor-α and interferon-γ, and anti-inflammatory cytokine, interleukin 4, and BAY11-7081 lowered CIBP-induced MCP-1 and CCR2 expressions in a dose-dependent manner. CONCLUSION: In conclusion, NF-кB signaling pathway regulates the expressions of MCP-1/CCR2-induced inflammatory factors in the spinal cord of CIBP rats.

Growth restriction and congenital heart disease caused by a novel TAB2 mutation: A case report.

Congenital heart disease (CHD) is a malformation present from birth caused by the abnormal development of the heart and large blood vessels during the prenatal development. The TGF-ß activated kinase 1 (MAP3K7) binding protein 2 (TAB2) gene plays an important role in the embryonic development of heart tissue. When haploid dosage is insufficient, it can lead to CHD or cardiomyopathy. The present study reported a case study of a Chinese child with growth restriction and CHD. The results of whole exome sequencing suggested that a novel frameshift mutation (c.1056delC/p.Ser353fsTer8) occurred in TAB2. The parents of this patient are wild-type at this locus; therefore, it may be a de novo mutation. The mutant plasmid was constructed in vitro, and the western blotting results showed that the mutation may cease protein expression. This indicated the pathogenic harmfulness of this mutation. In conclusion, the present study emphasizes that TAB2 defects should be investigated in patients with unexplained short stature and CHD, irrespective of family history regarding CHD or cardiomyopathy. The current study provided new data on the mutation spectrum and provided information for second pregnancies and genetic counseling of the parents of patients.

Development of Henoch-Schoenlein purpura in a child with idiopathic hypereosinophilia syndrome with multiple thrombotic onset: A case report.

BACKGROUND: The incidence of pulmonary embolism (PE) in children is low, but its mortality is high. Hypereosinophilic syndrome (HES) is a group of diseases caused by an abnormal increase in eosinophilic granulocytes resulting in multiple-organ dysfunction. The urgent event of thromboembolism in the pulmonary region provoked by eosinophils in idiopathic HES (IHES) is relatively unusual. This article reports a case of IHES with multiple PEs and left leg venous thrombosis as the first manifestation. One month later, the patient developed Henoch-Schonlein purpura (HSP), which is very rare. CASE SUMMARY: We report the case of a 12-year-old boy who was admitted to the hospital with dyspnea, left leg pain, and aggravation. He had bilateral PE and left leg venous embolism with mild eosinophilia. Low-molecular-weight heparin and urokinase were given. At the same time, the interventional department was contacted about filter implantation, followed by urokinase thrombolysis. The left leg thrombus was aspirated under ultrasound guidance. He was discharged from the hospital on rivaroxaban. One month later, he developed a rash on both legs and ankle pain consistent with HSP, with severe eosinophilia and motor and sensory disturbances. The patient was diagnosed with IHES with multiple embolisms complicated by HSP after excluding other causes of the eosinophil elevation. After glucocorticoid treatment, the symptoms were relieved, but the patient later developed purpura nephritis. CONCLUSION: We report a rare and life-threatening case of IHES with multiple embolisms associated with HSP. A mild elevation of eosinophils early in the disease leads to difficulties in diagnosis and delayed treatment.

Development and Validation of a Diagnostic Nomogram to Predict the Anthracycline-Induced Early Cardiotoxicity in Children with Hematological Tumors.

This study aimed to establish and validate an effective nomogram to predict the risk of cardiotoxicity in children after each anthracycline treatment. According to the inclusion and exclusion criteria, the eligible children were randomly divided into the training cohort (75%) and the validation cohort (25%). Least absolute shrinkage and selection operator (LASSO) regression was used to select the predictors and a nomogram was developed. Then, concordance index (C-index), the area under the curve (AUC), Hosmer-Lemeshow (H-L) test, and decision curve analysis (DCA) were employed to evaluate the performance and clinical utility of nomogram. Internal validation was processed to inspect the stability of the model. A total of 796 eligible children were included in this study and divided into a training set (n = 597) and a validation set (n = 199). LASSO regression analysis revealed that cumulative anthracycline dose, ejection fractions, NT-proBNP, and diastolic dysfunction were effective predictors of cardiotoxicity. The nomogram was established based on these variables. The C-index and the AUC of the predicting nomogram were 0.818 in the training cohort and 0.773 in the validation cohort, suggesting that the nomogram had good discrimination. The calibration curve of the nomogram presented no significant deviation from the reference line, and the P-value of the H-L test was 0.283, implying a preferable degree of calibration. The threshold of DCA also reflects that the nomogram is clinically useful. A nomogram was developed to predict anthracycline chemotherapy-induced cardiotoxicity in children with hematological tumors. The nomogram has a good prediction effect and can provide a reference for clinicians' diagnosis and treatment.

SAP102 contributes to hyperalgesia formation in the cancer induced bone pain rat model by anchoring NMDA receptors.

The pathogenesis of cancer induced bone pain (CIBP) is extremely complex, and glutamate receptor dysfunction plays an important role in the formation of CIBP. Synapse-associated protein 102 (SAP102) anchors glutamate receptors in the postsynaptic membrane. However, its effect on hyperalgesia formation in CIBP has not been clarified. This study investigated the role of SAP102 in the formation of hyperalgesia in rats with CIBP SAP102 is present in spinal dorsal horn neurons, but not in astrocytes or microglia. NMDAR-NR2B is localized with neurons. In addition, SAP102 and NMDAR-NR2B expression levels in spinal dorsal horn tissues were detected by Western blot and co-immunoprecipitation. Intrathecal injection of lentiviral vector of RNAi to knockdown SAP102 expression in the spinal dorsal horn significantly attenuated abnormal mechanic pain when compared to non-coding lentiviral vector. These findings indicate that SAP102 can anchor NMDA receptors to affect hyperalgesia formation in bone cancer pain.

Phosphoproteomic profiling of oxycodone­treated spinal cord of rats with cancer­induced bone pain.

Treatment of cancer­induced bone pain (CIBP) is challenging in clinical settings. Oxycodone (OXY) is used to treat CIBP; however, a lack of understanding of the mechanisms underlying CIBP limits the application of OXY. In the present study, all rats were randomly divided into three groups: The sham group, the CIBP group, and the OXY group. Then, a rat model of CIBP was established by inoculation of Walker 256 tumor cells from rat tibia. Phosphoproteomic profiling of the OXY­treated spinal dorsal cords of rats with CIBP was performed, and 1,679 phosphorylated proteins were identified, of which 160 proteins were significantly different between the CIBP and sham groups, and 113 proteins were significantly different between the CIBP and OXY groups. Gene Ontology analysis revealed that these proteins mainly clustered as synaptic­associated cellular components; among these, disks large homolog 3 expression was markedly increased in rats with CIBP and was reversed by OXY treatment. Subsequent domain analysis of the differential proteins revealed several significant synaptic­associated domains. In conclusion, synaptic­associated cellular components may be critical in OXY­induced analgesia in rats with CIBP.

All-trans-retinoid acid (ATRA) suppresses chondrogenesis of rat primary hind limb bud mesenchymal cells by downregulating p63 and cartilage-specific molecules.

P63 null mice have no or truncated limbs and mutations in human p63 cause several skeletal syndromes that also show limb and digit abnormalities, suggesting its essential role in bone development. In the current study, we investigated the effect of ATRA on chondrogenesis using mesenchymal cells from rat hind limb bud and further examined the mRNA and protein expression of Sox9 and Col2a1 and p63 in rat hind limb bud cells. Limb buds were isolated from embryos from euthanized female rats. Growth of hind limb bud mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays. Formation of cartilage nodules was examined by Alcian blue-nuclear fast red staining. The expression of Sox9, Col2al and p63 was determined by Real-time RT-PCR and immunoblotting assays, respectively. Our MTT assays revealed that ATRA at 1 and 10µM significantly suppressed the growth of mesenchymal cells from rat hind limb bud at 24 and 48h (P<0.01 vs. controls). Alcian blue staining further showed that ATRA caused a significant dose-dependent reduction in the area of cartilage nodules (P<0.05 in all vs. controls). At 1µM ATRA, the area of cartilage nodules from hind limb bud cells was reduced to 0.05±0.03mm from 0.15±0.01mm in controls. Real-time RT-PCR assays further indicated that 1 and 10µM ATRA markedly reduced the mRNA expression of Sox9, Col2al and p63 in hind limb bud cells (P<0.05 in all vs. controls). In addition, ATRA time-dependently inhibits the mRNA expression of p63, Sox9 and Col2al. Western blotting assays additionally showed that ATRA dose-dependently reduced the expression of Sox9, Col2al and p63 (P<0.05 in all vs. controls). Together, our results suggest that ATRA suppresses chondrogenesis by modulating the expression of Sox9, Col2al and p63 in primary hind limb bud mesenchymal cells.