Enhanced focal cortical dysplasia detection in pediatric frontal lobe epilepsy with asymmetric radiomic and morphological features.

Objective: Focal cortical dysplasia (FCD) is the most common pathological cause for pediatric epilepsy, with frontal lobe epilepsy (FLE) being the most prevalent in the pediatric population. We attempted to utilize radiomic and morphological methods on MRI and PET to detect FCD in children with FLE. Methods: Thirty-seven children with FLE and 20 controls were included in the primary cohort, and a five-fold cross-validation was performed. In addition, we validated the performance in an independent site of 12 FLE children. A two-stage experiments including frontal lobe and subregions were employed to detect the lesion area of FCD, incorporating the asymmetric feature between the left and right hemispheres. Specifically, for the radiomics approach, we used gray matter (GM), white matter (WM), GM and WM, and the gray-white matter boundary regions of interest to extract features. Then, we employed a Multi-Layer Perceptron classifier to achieve FCD lesion localization based on both radiomic and morphological methods. Results: The Multi-Layer Perceptron model based on the asymmetric feature exhibited excellent performance both in the frontal lobe and subregions. In the primary cohort and independent site, the radiomics analysis with GM and WM asymmetric features had the highest sensitivity (89.2 and 91.7%) and AUC (98.9 and 99.3%) in frontal lobe. While in the subregions, the GM asymmetric features had the highest sensitivity (85.6 and 79.7%). Furthermore, relying on the highest sensitivity of GM and WM asymmetric features in frontal lobe, when integrated with the subregions results, our approach exhibited overlaps with GM asymmetric features (55.4 and 52.4%), as well as morphological asymmetric features (54.4 and 53.8%), both in the primary cohort and at the independent site. Significance: This study demonstrates that a two-stage design based on the asymmetry of radiomic and morphological features can improve FCD detection. Specifically, incorporating regions of interest for GM, WM, GM, and WM, and the gray-white matter boundary significantly enhances the localization capabilities for lesion detection within the radiomics approach.

Glucocorticoid-loaded pH/ROS dual-responsive nanoparticles alleviate joint destruction by downregulating the NF-κB signaling pathway.

Rheumatoid arthritis (RA) is an autoimmune disease causing severe symptoms that are difficult to treat. Nano-drug delivery system is recognized as a promising strategy for management of RA. However, how to thoroughly release payloads from nanoformulations and synergistic therapy of RA needs to be further investigated. To address this issue, a pH and reactive oxygen species (ROS) dual-responsive, methylprednisolone (MPS)-loaded and arginine-glycine-aspartic acid (RGD)-modified nanoparticles (NPs) was fabricated using phytochemical and ROS-responsive moiety co-modified α-cyclodextrin (α-CD) as a carrier. In vitro and in vivo experiments verified that the pH/ROS dual-responsive nanomedicine could be efficiently internalized by activated macrophages and synovial cells, and the released MPS could promote transformation of M1-type macrophages into M2 phenotype, thereby down-regulating pro-inflammatory cytokines. In vivo experiments demonstrated that the pH/ROS dual-responsive nanomedicine was remarkably accumulated in the inflamed joints of mice with collagen-induced arthritis (CIA). The accumulated nanomedicine could obviously relieve joint swelling and cartilage destruction without obvious adverse effects. Importantly, the expression of interleukin-6 and tumor necrosis factor-α in the joints of CIA mice were significantly inhibited by the pH/ROS dual-responsive nanomedicine in comparison with free drug and non-targeted counterparts. In addition, the expression of the NF-κB signaling pathway molecule P65 was also significantly decreased by nanomedicine-treatment. Our results reveal that MPS-loaded pH/ROS dual-responsive NPs can effectively alleviate joint destruction via down-regulation of the NF-κB signaling pathway. STATEMENT OF SIGNIFICANCE: Nanomedicine is recognized as an attractive method for the targeting treatment of rheumatoid arthritis (RA). To thorough release of payloads from nanoformulations and synergistic therapy of RA, herein, a phytochemical and ROS-responsive moiety co-modified α-cyclodextrin was used as a pH/ROS dual-responsive carrier to encapsulate methylprednisolone to manage RA. The fabricated nanomedicine can effectively release its payloads under pH and/or ROS microenvironment, and the released drugs dramatically promote transformation of M1-type macrophages into M2 phenotype to reduce the release of pro-inflammatory cytokines. The prepared nanomedicine also obviously decreased the NF-κB signaling pathway molecule P65 expression in the joints, thereby down-regulating pro-inflammatory cytokines expression to alleviate joint swelling and cartilage destruction. We provided a candidate for the targeting treatment of RA.

Microglia activation and temporal changes in rat model of trigeminal neuralgia.

OBJECTIVES: This study aimed to investigate whether the microglia in the spinal trigeminal nucleus caudal part (Sp5C) were activated in a rat model of trigeminal neuralgia and to explore whether the activation level of microglia is consistent with maxillofacial pain level. METHODS: Chronic constriction injury of trigeminal nerve (CCI) was induced by partial ligation of infraorbital nerve (IoN) in rats. The behavioral change of rats observed at D1, D5, D10, D15, and D30 days post-surgery and the change of pain threshold were detected with electronic Von Frey filaments served as an evaluation index of maxillofacial pain. Weight change was measured by weighing. Ionized calcium binding adaptor molecule-1 (Iba-1) expression level of Sp5C at each time point was detected, and three microglia morphological categories were analyzed by immunohistochemical staining. RESULTS: The changes of behavioral and pain threshold suggested the maxillofacial pain level first increased and then decreased post-surgery in the IoN-CCI group. Both the expressions of Iba-1 and proportion of ameboid morphology in ipsilateral Sp5C increased from D1 and reached their peaks in D10 and D5, respectively. Then, they recovered nearly to the same level with contralateral Sp5C on D30. This trend was consistent with the maxillofacial change. CONCLUSIONS: The model of trigeminal neuralgia in rats constructed by partial ligation of infraorbital nerve can induce the activation of microglia in Sp5C, and the activation level is consistent with maxillofacial pain, which reached its peak at around D10 post-surgery.

Klotho upregulates the interaction between RANK and TRAF6 to facilitate RANKL-induced osteoclastogenesis via the NF-κB signaling pathway.

BACKGROUND: α-Klotho (Klotho) plays a wide range of roles in pathophysiological processes, such as low-turnover osteoporosis observed in klotho mutant mice (kl/kl mice). However, the precise function and underlying mechanism of klotho during osteoclastogenesis are not fully understood. Here, we investigated the effects of klotho on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL). METHODS: The effects of klotho deficiency on osteoclastogenesis were explored using kl/kl mice both in vivo and in vitro. In in vitro experiments, lentivirus transfection, real-time quantitative PCR (RT-qPCR) analysis, western blot analysis, immunostaining, RNA-seq analysis, differential pathway analysis, Energy-based protein docking analysis and co-immunoprecipitation were used for deeply investigating the effects of klotho on RANKL-induced Osteoclastogenesis and the underlying mechanism. RESULTS: We found that klotho deficiency impaired osteoclastogenesis. Furthermore, in vitro studies revealed that klotho facilitated osteoclastogenesis and upregulated the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) during osteoclastogenesis. Mechanistically, we confirmed that klotho co-localized with nuclear factor kappa B (RANK) and facilitated the interaction between activated RANK and TNFR-associated factor 6 (TRAF6), thus klotho exerts its function in osteoclastogenesis through the activation of the NF-κB signaling pathway. CONCLUSIONS: Klotho promotes RANKL-induced osteoclastogenesis through upregulating the interaction between RANK and TARF6, Targeting on klotho may be an attractive therapeutic method for osteopenic diseases.

Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3.

Orthodontic tooth movement elicits alveolar bone remodeling and orofacial pain that is manifested by tooth mechanical hyperalgesia. Nerve growth factor (NGF) is upregulated in periodontium and may modulate tooth mechanical hyperalgesia. The objectives were to examine the role of NGF in tooth mechanical hyperalgesia and to elucidate the underlying mechanisms. Tooth mechanical hyperalgesia was induced by ligating closed coil springs between incisors and molars in Sprague-Dawley rats. Retrograde labeling was performed by periodontal administration of fluor-conjugated NGF and the detection of fluorescence in trigeminal ganglia (TG). Lentivirus vectors carrying NGF shRNA were employed to knockdown the expression of NGF in TG. The administration of agonists, antagonists, and virus vectors into TG and periodontium was conducted. Tooth mechanical hyperalgesia was examined through the threshold of biting withdrawal. Our results revealed that tooth movement elicited tooth mechanical hyperalgesia that could be alleviated by NGF neutralizing antibody and that NGF was upregulated in periodontium (mainly in periodontal fibroblasts) and TG. Retrograde labeling revealed that periodontal NGF was retrogradely transported to TG after day 1. Acid-sensing ion channel 3 (ASIC3) and NGF were co-expressed in trigeminal neurons and the percentage of co-expression was significantly higher following tooth movement. The administration of NGF and NGF neutralizing antibody into TG could upregulate and downregulate the expression of ASIC3 in TG, respectively. NGF aggravated tooth mechanical hyperalgesia that could be alleviated by ASIC3 antagonist (APETx2). Moreover, NGF neutralizing antibody mitigated tooth mechanical hyperalgesia that could be recapitulated by ASIC3 agonist (GMQ). NGF-based gene therapy abolished tooth mechanical hyperalgesia and downregulated ASIC3 expression. Taken together, in response to force stimuli, periodontal fibroblasts upregulated the expressions of NGF that was retrogradely transported to TG, where NGF elicited tooth mechanical hyperalgesia through upregulating ASIC3. NGF-based gene therapy is a viable method in alleviating tooth-movement-induced mechanical hyperalgesia.

N/OFQ modulates orofacial pain induced by tooth movement through CGRP-dependent pathways.

BACKGROUND: Nociceptin/orphanin FQ (N/OFQ) has been revealed to play bidirectional roles in orofacial pain modulation. Calcitonin gene-related peptide (CGRP) is a well-known pro-nociceptive molecule that participates in the modulation of orofacial pain. We aimed to determine the effects of N/OFQ on the modulation of orofacial pain and on the release of CGRP. METHODS: Orofacial pain model was established by ligating springs between incisors and molars in rats for the simulation of tooth movement. The expression level of N/OFQ was determined and pain level was scored in response to orofacial pain. Both agonist and antagonist of N/OFQ receptor were administered to examine their effects on pain and the expression of CGRP in trigeminal ganglia (TG). Moreover, gene therapy based on the overexpression of N/OFQ was delivered to validate the modulatory role of N/OFQ on pain and CGRP expression. RESULTS: Tooth movement elicited orofacial pain and an elevation in N/OFQ expression. N/OFQ exacerbated orofacial pain and upregulated CGRP expression in TG, while UFP-101 alleviated pain and downregulated CGRP expression. N/OFQ-based gene therapy was successful in overexpressing N/OFQ in TG, which resulted in pain exacerbation and elevation of CGRP expression in TG. CONCLUSIONS: N/OFQ exacerbated orofacial pain possibly through upregulating CGRP.

Non-coenzyme role of vitamin B1 in RANKL-induced osteoclastogenesis and ovariectomy induced osteoporosis.

Vitamins B are co-enzymes participating in energy metabolic pathways. While some vitamins B are known affecting bone homeostasis, the effects of vitamin B1 (thiamine) on bone health remains unclear. In our study, we used cell counting kit-8, tartrate-resistant acid phosphatase stain, actin cytoskeleton stain, and pit formation assay to evaluate the effect of thiamine on osteoclast differentiation, formation, and function, respectively. Then we used dichloro-dihydro-fluorescein diacetate assay to investigate reactive oxygen species (ROS) generation and removal. Osteoporosis model by ovariectomy was established for animal experiments. We found that thiamine had inhibitory effect on osteoclast differentiation. And its inhibitory role on osteoclast differentiation is in a dose-dependent way. Mechanistically, ThDP suppresses intracellular ROS accumulation and unfolded protein response signaling during osteoclastogenesis via inhibiting Rac-Nox1/2/4 and intracellular inositol-requiring protein-1α/X-box-binding protein pathways, respectively. Osteoporotic mice treated with thiamine rich dietary showed better bone strength relative to thiamine deficient dietary. Our study explored the non-coenzyme inhibitory functions of B1 vitamin in receptor activator of nuclear factor κB ligand induced osteoclastogenesis and uncovered the significance of B1 vitamin in bone health.

Physalin D inhibits RANKL-induced osteoclastogenesis and bone loss via regulating calcium signaling.

We investigated the effects of physalin A, B, D, and F on osteoclastogenesis induced by receptor activator of nuclear factor κB ligand (RANKL). The biological functions of different physalins were first predicted using an in silico bioinformatic tool (BATMAN-TCM). Afterwards, we tested cell viability and cell apoptosis rate to analyze the cytotoxicity of different physalins. We analyzed the inhibitory effects of physalins on RANKL-induced osteoclastogenesis from mouse bone-marrow macrophages (BMMs) using a tartrate-resistant acid phosphatase (TRAP) stain. We found that physalin D has the best selectivity index (SI) among all analyzed physalins. We then confirmed the inhibitory effects of physalin D on osteoclast maturation and function by immunostaining of F-actin and a pit-formation assay. On the molecular level, physalin D attenuated RANKLevoked intracellular calcium ([Ca(2＋)](i)) oscillation by inhibiting phosphorylation of phospholipase Cγ2 (PLCγ2) and thus blocked the downstream activation of Ca2＋/calmodulindependent protein kinases (CaMK)IV and cAMP-responsive element-binding protein (CREB). An animal study showed that physalin D treatment rescues bone microarchitecture, prevents bone loss, and restores bone strength in a model of rapid bone loss induced by soluble RANKL. Taken together, these results suggest that physalin D inhibits RANKL-induced osteoclastogenesis and bone loss via suppressing the PLCγ2-CaMK-CREB pathway. [BMB Reports 2020; 53(3): 154-159].

Bone Marrow-Derived CD44+ Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair.

BACKGROUND AND AIMS: Host-derived cells play crucial roles in the regeneration process of tissue-engineered constructs (TECs) during the treatment of large segmental bone defects (LSBDs). However, their identity, source, and cell recruitment mechanisms remain elusive. METHODS: A complex model was created using mice by combining methods of GFP+ bone marrow transplantation (GFP-BMT), parabiosis (GFP+-BMT and wild-type mice), and femoral LSBD, followed by implantation of TECs or DBM scaffolds. Postoperatively, the migration of host BM cells was detected by animal imaging and immunofluorescent staining. Bone repair was evaluated by micro-CT. Signaling pathway repressors including AMD3100 and SP600125 associated with the migration of BM CD44+ cells were further investigated. In vitro, transwell migration and western-blotting assays were performed to verify the related signaling pathway. In vivo, the importance of the SDF-1/CXCR4-JNK pathway was validated by ELISA, fluorescence-activated cell sorting (FACS), immunofluorescent staining, and RT-PCR. RESULTS: First, we found that host cells recruited to facilitate TEC-mediated bone repair were derived from bone marrow and most of them express CD44, indicating the significance of CD44 in the migration of bone marrow cells towards donor MSCs. Then, the predominant roles of SDF-1/CXCR4 and downstream JNK in the migration of BM CD44+ cells towards TECs were demonstrated. CONCLUSION: Together, we demonstrated that during bone repair promoted by TECs, BM-derived CD44+ cells were essential and their migration towards TECs could be regulated by the SDF-1/CXCR4-JNK signaling pathway.

A Standardized and Quality-Controllable Protocol of Constructing Individual Tissue-Engineered Grafts Applicable to Treating Large Bone Defects.

Patient-specific individual tissue-engineered bones (iTEBs) have been recognized as a promising strategy for treating large bone defects. However, current construction protocols of iTEBs vary between lots and lack standardization and quality control, hampering further research and application. This study was aimed to detail a standardized constructing protocol for iTEBs, which can be used for both clinical and experimental purposes. The procedure was designed and described as follows: scaffold preparation, cell isolation and culture, and fabrication of iTEBs. Manipulation and caution points in each section were detailed. A series of scales on the quality control and safety monitoring was developed. The effectiveness and safety of iTEBs were evaluated. Eventually, the preparing portion, from cell culture to scaffold treatment, usually required 21 days. Generally, the fabrication section took 5 days. The main advantage of this protocol was that each step was standardized and quality controlling and safety monitoring were performed throughout the process to ensure the homogeneity, reliability, and safety. The resulting iTEBs were effective and applicable to both clinical and experimental purposes. Thus, we have established a refined and standardized protocol detailing the construction process of patient-specific iTEBs that comply with strict quality control and safety criteria. This protocol is relatively easy for graduate students or staff working in the field of bone tissue engineering to implement.

IL-8 Enhances Therapeutic Effects of BMSCs on Bone Regeneration via CXCR2-Mediated PI3k/Akt Signaling Pathway.

BACKGROUND/AIMS: Tissue engineering bone transplantation with bone marrow mesenchymal stem cells (BMSCs) is an effective technology to treat massive bone loss, while molecular regulation of the bone regeneration processes remains poorly understood. Here, we aimed to assess the role of interleukin-8 (IL-8) in the recruitment of host cells by seeded BMSCs and in the bone regeneration. METHODS: A transwell assay was performed to examine the role of IL-8/CXCR1/CXCR2/PI3k/Akt on the migration potential of hBMSCs. The in vitro chondrogenic differentiation of hBMSCs was assessed by examination of 2 chondrogenic markers, Sox9 and type 2 collagen (COL2). mBMSCs were used in tissue engineered bone (TEB) with/without IL-8 implanted into bone defect area with CXCR2 or Akt inhibitors. Density and Masson staining of the regenerated bone were assessed. The chondrogenesis was assessed by expression levels of associated proteins, Sox9 and COL2, by RT-qPCR and by immunohistochemistry. RESULTS: IL-8 may trigger in vitro migration of hBMSCs via CXCR2-mediated PI3k/Akt signaling pathway. IL-8 enhances osteogenesis in the TEB-implanted bone defect in mice. IL-8 induces chondrogenic differentiation of hBMSCs via CXCR2-mediated PI3k/Akt signaling pathway in vitro and in vivo. CONCLUSIONS: IL-8 enhances therapeutic effects of MSCs on bone regeneration via CXCR2-mediated PI3k/Akt signaling pathway.