Neuroprotective effects of Chrysanthemum morifolium on cerebral ischemia- reperfusion injury contributes to the oxidative stress suppression and related Keap1/Nrf2 pathway.

BACKGROUND: Ischemic stroke, the cause of death and disability worldwide, is closely related to oxidative stress damage. Chrysanthemum has profound antiantioxidant activity. We aimed to verify whether Chrysanthemum morifolium extract (CME) influences brain injury in cerebral ischemia-reperfusion injury (CR/RI) model. METHODS: In vitro, rat hippocampal H19-7 neurons were pretreated with CME, CR/RI was simulated with oxygen glucose deprivation/reoxygenation (OGD/R). The cell viability, apoptosis, lactate dehydrogenase release, reactive oxygen species (ROS) generation, malonaldehyde (MDA) content and superoxide dismutase（SOD） activity were detected. In vivo, middle cerebral artery occlusion (MCAO) model rats were pre-administered with CME, and then behavioral test, triphenyltetrazolium chloride (TTC), hematoxylin-eosin staining (HE), terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), ROS immunofluorescence, MDA and SOD activity were tested. Furthermore, Keap1/Nrf2 signaling of CME in CI/RI was investigated. RESULTS: In OGD/R induced in H19-7 cells, CME increased OGD/R-induced cell viability and reduced cell apoptosis, which was reversed by siNrf2 transfection . In MCAO rats, CME improved the neurological deficits and alleviated brain injury. However, co-treatment with MLK385 counteracted these neuroprotective effects of CME on MCAO rats. CONCLUSION: CME could significantly reduce oxidative stress and nerve injury in vitro and in vivo models of CI/RI by regulating the Keap1/Nrf2 pathway.

Clinical independent prognostic factors and overall survival prognostic nomogram for intracranial subependymoma: A SEER population-based analysis 2004-2016.

Purpose: This study was launched to ascertain the independent prognostic factors influencing the overall survival (OS) prognosis of intracranial subependymoma and construct a prognostic model to predict OS time. Materials and methods: We collected data from patients with intracranial subependymoma, including treatment data, follow-up data, and clinical and pathological characteristics from the SEER database within 2004 to 2016, and patients were randomly classified into training and validation cohorts. Univariate and multivariate analyses were applied to the training group through building a Cox proportional hazards model. According to the results of multivariate analysis, we established a nomogram to forecast the OS rate of the per-case patient graphically, then calculated the accuracy of verification in both training and validation cohorts by concordance index (C-index). Univariate and multivariate analyses were used for different subgroups of unoperated versus operated, gross total resection (GTR), subtotal resection (STR), and biopsy after using the propensity score matching (PSM) analyses. Results: A total of 667 patients were enrolled, and we randomly assigned 535 patients (80.21%) into the training cohort and 132 patients (19.79%) into the validation cohort. Age [hazard ratio (HR) = 6.355; 95% confidence interval (CI), 2.240-18.029; p = 0.001] and sex (HR = 0.475; 95% CI, 0.232-0.974; p = 0.042) were the independent prognostic factors in the training cohort. On the basis of age and sex, the nomogram was established to predict the OS for every patient (C-index = 0.733 ± 0.065 in the training cohort and 0.850 ± 0.065 in the validation cohort), and calibration plots reflected the reliability of the nomogram. Age, gender, or laterality was the independent prognostic factor for OS in the different matched subgroups of unoperated versus operated, GTR, STR, and biopsy. Surgical treatment, race, year of diagnosis, insurance, tumor location, tumor size, pathology, tumor grade, and radiation were not statistically significantly different in OS for subependymoma in our research. Conclusion: Age and sex were the independent prognostic variables for OS in intracranial subependymoma. According to our research, we should not be more inclined to choose conservative or surgical treatment. Nonetheless, the information that we present might be useful to suggest potential hypotheses to be tested in the clinical research setting.

Clinical prognostic factors for central neurocytoma and subgroup analysis of different treatment measures: A SEER database-based retrospective analysis from 2003 to 2019.

Purpose: The study aimed to identify clinical prognostic factors affecting overall survival (OS) in patients with central neurocytoma (CN) and to determine independent prognostic factors in the subgroups of different treatment modalities using a retrospective analysis based on the SEER database from 2003 to 2019. Materials and methods: Data regarding patients with CN, including basic clinical characteristics, treatment measures, and prognosis follow-up, were extracted from the SEER database. The prognostic variables for all patients were assessed using log-rank test as well as univariate and multivariate analyses based on the Cox proportional hazards model. The same statistical methods were used for analysis in different subgroups of gross total resection (GTR), subtotal resection (STR), no surgery, radiotherapy (RT), and no RT. Results: In total, 413 patients were enrolled in this study. Tumor size, primary site surgery, and RT were independent prognostic factors in all patients with CN. In subgroup analyses, RT was not an independent prognostic factor in patients with GTR. However, sex and race were independent prognostic factors in patients with STR. Additionally, tumor size was an independent prognostic factor in patients who did not undergo surgery. Furthermore, sex and primary site were independent prognostic factors in patients who received RT. Size and primary site surgery were independent prognostic factors in patients without RT. Conclusion: In our study, patients with small tumors or GTR or those who did not receive RT showed a better prognosis. GTR was the preferred treatment for CN. RT was not recommended for patients after GTR. Men and African American showed certain advantages after STR surgery. Tumors with a size of >4 cm were recommended for active treatment. In the RT subgroup, patients with tumors outside the ventricle or women had a poorer prognosis than those with tumors within the ventricle or men, respectively. These findings will help clinicians and patients understand the treatment and prognosis of CN visually and intuitively.

Evaluation of the TRPM protein family as potential biomarkers for various types of human cancer using public database analyses.

The Transient Receptor Potential Melastatin (TRPM) protein family members have been demonstrated to be involved in a variety of different types of human cancer. However, to the best of our knowledge, there has not yet been a systematic study regarding the mRNA expression of the TRPM protein family or its prognostic value in human cancer. The present study investigated TRPM expression and its prognostic value in various human cancer types via the Oncomine database, Kaplan-Meier plotter, and the PrognoScan and Gene Expression Profiling Interactive Analysis databases. It was revealed that the transcriptional levels of TRPM1, TRPM3 and TRPM6 were decreased in the majority of cancer tissues, while TRPM2 was increased in most cancer types. In addition, the high or low transcriptional levels of the TRPM protein family members were associated with survival outcomes of different types of solid tumors. The present study suggested that certain TRPM protein family members may serve as useful biomarkers for cancer prognosis and anticancer targets for cancer treatment.

Hypertonic Saline Alleviates Brain Edema After Traumatic Brain Injury via Downregulation of Aquaporin 4 in Rats.

BACKGROUND Hypertonic saline (HS) has been successfully used for treatment of various forms of brain edema. Decreased expression of aquaporin (AQP)4 and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1b have been linked to edema pathogenesis. This study examined the effect of 3% HS on brain edema in a rat model of traumatic brain injury (TBI). MATERIAL AND METHODS Sprague-Dawley rats were subjected to TBI induced by a controlled cortical impactor. The HS group was injected with 3% NaCl until the end of the study period. AQP4, TNF-α, IL-1ß, and caspase-3 levels were measured by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay, and quantitative real-time PCR. Brain water content was also measured. Apoptotic cells in brain tissue were detected with terminal deoxynucleotidyl transferase dUTP nick-end labeling. Brain water content decreased following treatment with 3% HS relative to the TBI group. RESULTS This was accompanied by decreases in AQP4, TNF-α, and IL-1ß mRNA and protein levels. TBI resulted in increases in caspase-3 mRNA expression and the number of apoptotic cells; treatment with 3% HS suppressed apoptosis as compared to the TBI group. CONCLUSIONS Treatment with 3% HS ameliorated TBI-induced brain edema, possibly by suppressing brain edema, pro-inflammatory cytokine expression, and apoptosis.

In Vivo MRI Tracking of Polyethylenimine-Wrapped Superparamagnetic Iron Oxide Nanoparticle-Labeled BMSCs for Cartilage Repair: A Minipig Model.

OBJECTIVE: To evaluate the feasibility of tracking polyethylenimine (PEI)-wrapped superparamagnetic iron oxide (SPIO) nanoparticle-labeled, bone marrow-derived mesenchymal stem cells (BMSCs) by in vivo magnetic resonance imaging (MRI) in articular cartilage repair in a minipig model. METHODS: Eighteen Guizhou minipigs were randomly divided into three groups (groups A, B, and C). In group A, PEI-wrapped SPIO nanoparticle (PEI/SPIO) and green fluorescent protein (GFP) colabeled, autologous BMSCs seeded in type II collagen gel were transplanted into the articular cartilage defects of the minipig model. In group B, GFP-labeled, autologous BMSCs seeded in type II collagen gel were transplanted. In group C, no treatment was applied for cartilage defects. All minipigs underwent clinical 3.0-T MR imaging at 4, 8, 12, and 24 weeks postsurgery. The findings were compared histologically. RESULTS: Prussian staining and transmission electron microscope showed that BMSCs were efficiently labeled by PEI/SPIO. Cell viability, proliferation, and differentiation were comparable between labeled and unlabeled cells. MRI SET2WI sequence revealed that marked hypointense signal void areas representing the transplanted labeled BMSCs could be observed for at least 24 weeks. Histochemical staining confirmed the presence of Prussian blue-positive cells and GFP-positive cells at the hypointense signal void areas. At 24 weeks postsurgery, both MR signals and histologic staining of minipigs in groups A and B at the cartilage defect were close to the normal cartilage. CONCLUSIONS: 3.0-T MRI in vivo tracking of PEI/SPIO-labeled BMSCs seeded in type II collagen gel on cartilage repair following transplantation is feasible in minipigs.

In vivo tracking of superparamagnetic iron oxide nanoparticle labeled chondrocytes in large animal model.

Chondrocytes have been widely used as tissue engineered seed cells for repair of focal cartilage lesions in clinic. However, in vivo behaviors of delivered chondrocytes are still poorly understood. In this study, the feasibility of in vivo tracking of superparamagnetic iron oxide nanoparticle (SPIO)-labeled chondrocytes by magnetic resonance imaging (MRI) for articular cartilage repair in minipig model was investigated. Results showed that chondrocytes were efficiently labeled by SPIO at optimal low dosages while maintaining essential cell properties. MRI SET2WI sequence revealed that marked hypointense signal void areas representing the transplanted labeled chondrocytes could be observed for at least 12 weeks. Histochemical staining confirmed the presence of Prussian blue-positive cells and GFP-positive cells at the hypointense signal void areas. These findings provide knowledge on the in vivo tracking of SPIO labeled chondrocytes on cartilage repair following transplantation in minipigs.

[Effects of superparamagnetic iron-oxide particles-labeling on the multi-diffentiation of rabbit marrow mesenchymal stem cell in vitro].

The aim of this study was to label rabbit bone derived mesenchymal stem cells (BMSCs) with superparamagnetic iron oxide particles (SPIO) and to study the effects of magnetic labeling on the multi-differentiation of BMSCs. Rabbit BMSCs were isolated, purified, expanded, then coincubated with SPIO(25 microg/ml) complexed to protamine sulfate (Pro) transfection agents overnight. Prussian blue staining and transmission electron microscopy were performed to show intracellular iron. Cell differentiation was evaluated. Both labeled and unlabeled BMSCs were subjected to osteogenic, adipogenic and chondrogenic differentiation to assess their differentiation capacity for 21 d. Osteogenic cells were stained with alizarin red to reveal calcium deposition, adipogenic cells were stained with oil redO' respectively. Chondrogenic cells stained with Safranin-O, glycosamino glycans, and type II collagen production was assessed by standard immunohistochemistry. Cell with immunohistochemistry staining were detected by polarized light microscopy and analysed by Image-Pro Plus software. The results showed that intracytoplasmic nanoparticles were stained with Prussian blue and observed by transmission electron microscopy clearly except the unlabeled control. As compared with the nonlabeled cells, it showed no statistically significant difference on the differentiation of the labeled BMSCs. And the differentiation of the labeled cells were unaffected by the endosomal incorporation of SPIO. In summary, BMSCs can be labeled with SPIO without significant change in cell multi-differentiation capacity.

In vitro targeted magnetic delivery and tracking of superparamagnetic iron oxide particles labeled stem cells for articular cartilage defect repair.

To assess a novel cell manipulation technique of tissue engineering with respect to its ability to augment superparamagnetic iron oxide particles (SPIO) labeled mesenchymal stem cells (MSCs) density at a localized cartilage defect site in an in vitro phantom by applying magnetic force. Meanwhile, non-invasive imaging techniques were use to track SPIO-labeled MSCs by magnetic resonance imaging (MRI). Human bone marrow MSCs were cultured and labeled with SPIO. Fresh degenerated human osteochondral fragments were obtained during total knee arthroplasty and a cartilage defect was created at the center. Then, the osteochondral fragments were attached to the sidewalls of culture flasks filled with phosphate-buffered saline (PBS) to mimic the human joint cavity. The SPIO-labeled MSCs were injected into the culture flasks in the presence of a 0.57 Tesla (T) magnetic force. Before and 90 min after cell targeting, the specimens underwent T2-weighted turbo spin-echo (SET2WI) sequence of 3.0 T MRI. MRI results were compared with histological findings. Macroscopic observation showed that SPIO-labeled MSCs were steered to the target region of cartilage defect. MRI revealed significant changes in signal intensity (P<0.01). HE staining exibited that a great number of MSCs formed a three-dimensional (3D) cell "sheet" structure at the chondral defect site. It was concluded that 0.57 T magnetic force permits spatial delivery of magnetically labeled MSCs to the target region in vitro. High-field MRI can serve as an very sensitive non-invasive technique for the visualization of SPIO-labeled MSCs.

In Vitro Targeted Magnetic Delivery and Tracking of Superparamagnetic Iron Oxide Particles Labeled Stem Cells for Articular Cartilage Defect Repair / 华中科技大学学报（医学）（英德文版）

To assess a novel cell manipulation technique of tissue engineering with respect to its ability to augment superparamagnetic iron oxide particles (SPIO) labeled mesenchymal stem cells (MSCs) density at a localized cartilage defect site in an in vitro phantom by applying magnetic force.Meanwhile,non-invasive imaging techniques were use to track SPIO-labeled MSCs by magnetic resonance imaging (MRI).Human bone marrow MSCs were cultured and labeled with SPIO.Fresh degenerated human osteochondral fragments were obtained during total knee arthroplasty and a cartilage defect was created at the center.Then,the osteochondral fragments were attached to the sidewalls of culture flasks filled with phosphate-buffered saline (PBS) to mimic the human joint cavity.The SPIO-labeled MSCs were injected into the culture flasks in the presence of a 0.57 Tesla (T) magnetic force.Before and 90 min after cell targeting,the specimens underwent T2-weighted turbo spin-echo (SET2WI) sequence of 3.0 T MRI.MRI results were compared with histological findings.Macroscopic observation showed that SPIO-labeled MSCs were steered to the target region of cartilage defect.MRI revealed significantchanges in signal intensity (P＜0.01).HE staining exibited that a great number of MSCs formed a three-dimensional (3D) cell "sheet" structure at the chondral defect site.It was concluded that 0.57 T magnetic force permits spatial delivery of magnetically labeled MSCs to the target region in vitro.High-field MRI can serve as an very sensitive non-invasive technique for the visualization of SPIO-labeled MSCs.

The promotion of the vascularization of decalcified bone matrix in vivo by rabbit bone marrow mononuclear cell-derived endothelial cells.

The neovascularization of bone grafting represents an important challenge in bone regeneration. Prevascularization of tissue-engineered bone using endothelial cells (ECs) in vitro sheds light on accelerating the vascularization of bone replacements. In the present study, decalcified bone matrix (DBM) was prevascularized by seeding fibrin gels with ECs that are derived from rabbit bone marrow mononuclear cells (BMMNCs). The compound was then transplanted autologously into bone defects of rabbits to observe the vascularization in vivo. At 2, 4 and 8 weeks after grafting, the microvessel density of new bone tissues was significantly higher in the experimental group than in the control group (P<0.05), which suggests that prevascularization of BMMNC-derived cells may be suitable for improving vascularization in tissue-engineered bone.

[In vivo MR imaging tracking of supermagnetic iron-oxide nanoparticle-labeled bone marrow mesenchymal stem cells injected into intra-articular space of knee joints: experiment with rabbit].

OBJECTIVE: To evaluate the feasibility of in vivo magnetic resonance imaging (MRI) with 1.5T system tracking of the survival, migration and differentiation of magnetically labeled seed cells-bone marrow-derived mesenchymal stem cells (MSCs) injected into the articular cavity. METHODS: Rabbit MSCs were isolated, purified, expanded, and then coincubated in vitro with supermagnetic iron oxide particles (SPIO) and 5-bromo-2-deoxyuridine (BrdU). Prussian blue staining and transmission electron microscopy were performed to observe the intracellular iron. Some labeled MSCs were subjected to chondrogenic differentiation and the phenotype was examined to assess their chondrogenic differentiation capacity. MSCs colabeled with SPIO nanoparticles and (BrdU were suspended in chitosan and glycerophosphate (C-GP) gel. Eighteen rabbits underwent damage to the femoral trochlea to create cartilage defect models, and randomly divided into 3 groups 1 week later: Group A (n=6) undergoing injection of the MSC suspension in C-GP gel into the intra-articular space of knee joints, Group B (n=6), injected with un-labeled MSC suspension in C-GP gel, and Group C (n=6), without injection. MRI of the knee was performed 1, 4, 8, and 12 weeks after the injection respectively on a certain numbers of rabbits. and then the rabbits were killed with their knee joints taken out to undergo immunohistochemistry. The MR imaging findings were compared with the histological findings. RESULTS: Prussian blue staining and transmission electron microscopy showed intracytoplasmic nanoparticles in the SPIO-labeled cells. Safranin-O staining showed deposition of proteoglycan and type II collagen outside both the labeled and unlabeled MSCs, showing chondrogenesis. GRE T2-weighted MR image showed marked hypointense signal void areas, representing the implanted MSCs, in the intra-articular space after the MSC injection in Group A that persisted for 12 weeks at least; 2 week after the MSC injection hypointense signal could be seen in the defect, which peaked in the signal intensity about 4 weeks later, and then gradually decreased in the signal intensity; and 12 weeks after the injection no recognizable hypointense signal in the defect was detected. Immunohistochemical staining demonstrated the presence of Prussian blue-positive cells and BrdU-positive cells in the tissue sections in the areas corresponding well to the signal intensity loss regions in the MRI images. Group B and Group C showed no signal intensity loss in the intra-articular spaces by GRE T2-weighted MR imaging. Histological observation showed that the defects were repaired with fibrocartilage in Groups A and B, and with fiber tissue in Group C. CONCLUSION: Labeled with SPIO, the MSCs remains their ability of chondrogenic differentiation. It is feasible to track the fate and dynamic redistribution of magnetically labeled MSCs, the seed cells, injected into the articular cavity by 1.5T MRI, an efficient noninvasive technique.

[Cell apoptosis in atrophic skeletal muscle induced by immoblization in rabbits--an experimental study using TUNEL].

This experiment was designed to explore the correlation between the mechanism of immobilization-induced skeletal muscle atrophy and the apoptosis of muscular cells. The models of skeletal muscle atrophy induced by immobilization for different length of time were established according to Sievanen II methods. 24 rabbits, each of them having one hind leg fixed by the tubal plaster and the other one free as control, were randomly divided into four groups depending on time of fixation (3, 7, 14, and 28 days respectively). The animals were sacrificed by the end of fixation. TdT-mediated d-UTP nick end labeling (TUNEL) was used to investigate the apoptotic muscle cells in the animal's bone. By comparing the apoptotic muscle cells with the morphology of the skeletal muscle, the correlation between cell apoptosis and skeletal muscle atrophy were analyzed. Apoptotic muscle cells did appear after immobilization in the atrophied skeletal muscle. In various groups, some cells with false positive stained TUNEL were found in the atrophic muscle, which could be distinguished from apoptotic cells by their characteristics. In conclusion, cell apoptosis participates in the process of skeletal muscle atrophy induced by immobilization; the amount of apoptotic cells is strongly associated with the time of immobilization, its peak appears on the 14th day of immobilization; the distribution of apoptotic skeletal muscle cell varies with the time of fixation. The severity of skeletal muscle atrophy is associated with the degree of the muscle cell apoptosis.