Objective To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice. Methods BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2(Runx2) , collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1mg/kg) treatment in the Rspo2-intervention group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expression levels of ALP, OCN, osteogenic type Ⅰ procollagen amino-terminal peptide (PINP), and bone resorption β-collagen degradation product (β-CTX) by ELISA. Micro-CT was used to detect the bone mineral content and bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the thickness of trabeculae (Tb.Th), the number of trabeculae (Tb.N), the separation of trabeculae (Tb.Sp), and the bone volume fraction (BV/TV). Results CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic (P>0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group. Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group (P<0.05), with the highest showed in the 20 nm/L Rspo2 group. The mRNA expression levels of the osteogenesis-related genes (Runx2, Col1, and OCN) significantly increased, and the differences were significant between Rspo2grooups and control group (P<0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body weight and uterine weight after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant (P<0.05). ELISA showed that compared with the control group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP (P<0.05). After Rspo2 intervention, PINP expression was significantly reversed and increased, with significant differences compared to the control group and OVX group (P<0.05). The bone resorption marker β-CTX was higher in the OVX group than in the sham group (P<0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group (P<0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased (P<0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group (P<0.05) except Tb.Th. Conclusion Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.
Objective To study the impact of chronic hypoxia on white matter (WM) injury and brain development delay using a neonatal rat model, and to explore its value in simulating chronic hypoxic brain damage in cyanotic congenital heart disease (CHD). Methods Three-day-old Sprague-Dawley (SD) rats were randomly distributed to an experiment group (n=36, FiO2 10.5%±1.0%) and a control group (n=36, FiO2 21.0%±0.0%) and were raised for 12 days. (1) Body weight of SD rats was recorded every day and fresh brain weight was measured on P14. (2) H&E staining was performed on sections of brain tissue to observe pathological changes and ventricular size. (3) Immunohistochemistry (IHC) was applied to reveal alterations of oligodendroglial progenitor cells (OPC), preoligodendrocytes (PreOL) and myelin basic protein (MBP) in brain WM area. (4) Protein was extracted from 50 mg of brain tissue in WM area and expression of MBP was determined using Western blotting. (5) Motor function and coordination of rats (P30) were assessed via rotation experiment. Results (1) Body weight and brain weight were significantly less in the experiment group compared with the control group on P14 (body weight 14.92±1.26 gvs. 30.26±1.81 g, t=7.51, P<0.01; brain weight 0.68± 0.05 gvs.0.97±0.04 g, t=13.26, P<0.01); (2) HE staining: Sections of brain tissue from the experiment group showed ventricular size enlargement with a statistical difference (P<0.01), disordered cell organization, local neuronal death and leukomalacia. (3) The number of OPC and PreOL in the experiment group were significantly less than those in the control group (64.8±6.3vs. 126.2±8.4, t=11.19, P<0.01; 19.1±7.6vs. 46.7±9.5, t=7.28, P<0.01, respectively). MBP distribution was sparse and disorganized in the experiment group. (4) Western blotting: Expression of MBP was less in the experiment group (P<0.01). (5) Behavioral test: Time on rotarod was less in the experiment group with a statistical difference (P<0.01). Conclusion Chronic hypoxia can result in WM injury and brain development delay in neonatal rats, with features comparable to those seen in infants with cyanotic CHD.
ObjectivePulmonary vein banding was used to establish a piglet model of pulmonary vein stenosis. We investigated the pathomorphological alterations of pulmonary veins in the model and compared it with the vascular tissue of recurrent stenosis after total anomalous pulmonary venous connection (TAPVC).MethodsTen pigs of 6 weeks old were selected and randomly divided into 2 groups: 5 in a sham operation group and 5 in a pulmonary vein banding group. The operation had two stages, in which thoracotomies through intercostal space were done respectively on both sides. Biocompatible materials were applied around the pulmonary veins in the experimental group. The same method was used in the sham group. But the pulmonary veins were not banded. Six weeks after the operation, the pulmonary veins of the animals were harvested for hematoxylin-eosin staining and immunofluorescence staining to observe the pathological alterations of pulmonary veins. The proliferative tissues of patients with recurrent stenosis after TAPVC repair were collected and observed by hematoxylin-eosin staining and immunofluorescence staining.ResultsBoth the sham operation group and the pulmonary vein banding group survived. But the pulmonary vein banding group had obvious clinical manifestations of pulmonary venous stenosis. Compared with the sham group, the pulmonary vein banding group showed intimal hyperplasia, decreased expression of endothelial marker and increased expression of mesenchymal markers, and co-expression of endothelial and mesenchymal markers in intimal cells. Human pathology also showed intimal hyperplasia and co-expression of endothelial and mesenchymal markers in intimal cells.ConclusionThe surgical pulmonary vein stenosis in piglets shows intimal hyperplasia and myofibroblasts, which was consistent with clinical pathology.