The Profiles of Non-stationarity and Non-linearity in the Time Series of Resting-State Brain Networks.

The linearity and stationarity of fMRI time series need to be understood due to their important roles in the choice of approach for brain network analysis. In this paper, we investigated the stationarity and linearity of resting-state fMRI (rs-fMRI) time-series data from the Midnight Scan Club datasets. The degree of stationarity (DS) and the degree of non-linearity (DN) were, respectively, estimated for the time series of all gray matter voxels. The similarity and difference between the DS and DN were assessed in terms of voxels and intrinsic brain networks, including the visual network, somatomotor network, dorsal attention network, ventral attention network, limbic network, frontoparietal network, and default-mode network. The test-retest scans were utilized to quantify the reliability of DS and DN. We found that DS and DN maps had overlapping spatial distribution. Meanwhile, the probability density estimate function of DS had a long tail, and that of DN had a more normal distribution. Specifically, stronger DS was present in the somatomotor, limbic, and ventral attention networks compared to other networks, and stronger DN was found in the somatomotor, visual, limbic, ventral attention, and default-mode networks. The percentage of overlapping voxels between DS and DN in different networks demonstrated a decreasing trend in the order default mode, ventral attention, somatomotor, frontoparietal, dorsal attention, visual, and limbic. Furthermore, the ICC values of DS were higher than those of DN. Our results suggest that different functional networks have distinct properties of non-stationarity and non-linearity owing to the complexity of rs-fMRI time series. Thus, caution should be taken when analyzing fMRI data (both resting-state and task-activation) using simplified models.

A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.

Effects of disruption of epiphyseal vasculature on the proximal femoral growth plate.

BACKGROUND: Proximal femoral growth disturbance is a major complication associated with ischemic osteonecrotic conditions, such as Legg-Calvè-Perthes disease. The extent of ischemic damage and the mechanisms by which ischemic injury to the growing femoral head produces growth disturbance of the proximal femoral growth plate remain unclear. The purpose of this study was to investigate the effects of disruption of the epiphyseal vasculature on the morphology and function of the proximal femoral growth plate in a porcine model. METHODS: Ischemic osteonecrosis of the femoral head was surgically induced in sixty-five piglets by placing a ligature tightly around the femoral neck. Radiographic, histological, micro-computed tomographic, cellular viability, hypoxia marker, and cellular proliferation studies were performed. RESULTS: Disruption of the epiphyseal vasculature did not lead to diffuse growth plate damage in the majority of the ischemic femoral heads. One of the twelve femoral heads analyzed at four weeks and six of the twenty-six femoral heads analyzed at eight weeks had severe disruption of the growth plate that precluded histological assessment of the growth plate zones. In the remaining animals, the proximal part of the femur continued to elongate following induction of ischemia, albeit at a slower rate than on the normal side. Histologically, normal developmental thinning of the growth plate was seen to be absent on the ischemic side. Severe hypoxia and cellular death were limited to the area of the growth plate bordering on the infarcted osseous epiphysis. Normal chondrocytic organization and continued proliferation were observed in the proliferative zone of the growth plate. CONCLUSIONS: In our porcine model, the proximal femoral growth plate was not diffusely damaged following disruption of the epiphyseal vasculature in the majority of the ischemic femoral heads. The majority of the growth plates remained viable and were able to function despite total disruption of the epiphyseal vasculature. These findings suggest that the source of nutrition for the proximal femoral growth plate is not solely the epiphyseal vasculature as has been traditionally believed.

Hypoxia and HIF-1alpha expression in the epiphyseal cartilage following ischemic injury to the immature femoral head.

UNLABELLED: HIF-1alpha has been shown to be a central mediator of cellular response to hypoxia. The role it plays after ischemic injury to the immature femoral head is unknown. The purpose of this study was to determine the region of the femoral head affected by hypoxia following ischemic injury to the immature femoral head and to determine the site of HIF-1alpha activation and revascularization. We hypothesize that the epiphyseal cartilage, rather than the bony epiphysis, is the site of HIF-1alpha activation following ischemic osteonecrosis and that the epiphyseal cartilage plays an important role in the revascularization process. MATERIALS AND METHODS: Femoral head osteonecrosis was surgically induced in 56 immature pigs. Hypoxyprobe staining, cell viability assay, HIF-1alpha western blot, RT-qPCR of HIF-1alpha, VEGF, VEGFR2, and PECAM, and micro-CT assessments of microfil-infused femoral heads were performed. RESULTS: Severe hypoxia was present in the bony epiphysis and the lower part of the epiphyseal cartilage following ischemia. In the bony epiphysis, extensive cell death and tissue necrosis was observed with degradation of proteins and RNAs which precluded further analysis. In the epiphyseal cartilage, the loss of cell viability was limited to its deep layer with the remainder of the cartilage remaining viable. Furthermore, the cartilage from the ischemic side showed a significant increase in HIF-1alpha protein level and HIF-1alpha expression. VEGF expression in the cartilage was dramatically and significantly increased at 24 h, 2 and 4 weeks (p<0.05 for all) with 5 to 10 fold increase being observed on the ischemic side compared to the normal side. PECAM and VEGFR2 expressions in the cartilage were both significantly decreased at 24 h but returned to the normal levels by 2 and 4 weeks, respectively. Micro-CT showed revascularization of the cartilage on the ischemic side with the vessel volume/total volume equaling the normal side by 4 weeks. CONCLUSIONS: Acute ischemic injury to the immature femoral head induced severe hypoxia and cell death in the bony epiphysis and the deep layer of the epiphyseal cartilage. Viable chondrocytes in the superficial layer of the epiphyseal cartilage showed HIF-1alpha activation and VEGF upregulation with subsequent revascularization occurring in the cartilage.

Ischaemic injury to femoral head induces apoptotic and oncotic cell death.

AIMS: The mechanism of cell death in ischaemic osteonecrosis of the femoral head is not clear. Therefore, this study was designed to clarify the mode of cell death following ischaemic osteonecrosis of the femoral head in an established pig model. METHODS: Morphological assessment, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, detection of DNA laddering and transmission electron microscopy studies were performed to determine whether apoptosis is one of the pathways of cell death following ischaemic osteonecrosis of the femoral head. RESULTS: Mode of cell death was investigated from 2 to 14 days following the surgical induction of ischaemia. Ischaemic femoral heads showed morphological evidence of cell death by oncotic and apoptotic pathways in earlier stages of osteonecrosis. TUNEL positive cells were seen from 2 to 14 days following the induction of ischaemia. DNA samples obtained from ischaemic femoral heads following the induction of ischaemia showed nucleosomal ladders indicating apoptotic cell death. Electron micrographs also showed morphological changes associated with apoptosis. CONCLUSIONS: This study demonstrates that oncosis is not the sole mechanism of cell death following ischaemic injury of the femoral head. Both apoptosis and oncosis are involved as a result of ischaemic injury to the femoral head.

Retention, distribution, and effects of intraosseously administered ibandronate in the infarcted femoral head.

UNLABELLED: The local distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted femoral heads were studied. Intraosseous administration effectively delivered and distributed ibandronate in the infarcted femoral heads and decreased the femoral head deformity in a large animal model of Legg-Calve-Perthes disease. INTRODUCTION: Bisphosphonate therapy has gained significant attention for the treatment of ischemic osteonecrosis of the femoral head (IOFH) because of its ability to inhibit osteoclastic bone resorption, which has been shown to contribute to the pathogenesis of femoral head deformity. Because IOFH is a localized condition, there is a need to explore the therapeutic potential of local, intraosseous administration of bisphosphonate to prevent the femoral head deformity. The purpose of this study was to investigate the distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted head. MATERIALS AND METHODS: IOFH was surgically induced in the right femoral head of 27 piglets. One week later, a second operation was performed to inject (14)C-labeled or unlabeled ibandronate directly into the infarcted head. (14)C-ibandronate injected heads were assessed after 48 h, 3 weeks, or 7 weeks later to determine the distribution and retention of the drug using autoradiography and liquid scintillation analysis. Femoral heads injected with unlabeled ibandronate were assessed at 7 weeks to determine the degree of deformity using radiography and histomorphometry. RESULTS: Autoradiography showed that (14)C-Ibandronate was widely distributed in three of the four heads examined at 48 h after the injection. Liquid scintillation analysis showed that most of the drug was retained in the injected head, and almost negligible amount of radioactivity was present in the bone and organs elsewhere at 48 h. At 3 and 7 weeks, 50% and 30% of the (14)C-drug were found to be retained in the infarcted heads, respectively. Radiographic and histomorphometric assessments showed significantly better preservation of the infarcted heads treated with intraosseous administration of ibandronate compared with saline (p < 0.001). CONCLUSIONS: This study provides for the first time the evidence that local intraosseous administration is an effective route to deliver and distribute ibandronate in the infarcted femoral head to preserve the femoral head structure after ischemic osteonecrosis. In a localized ischemic condition such as IOFH, local administration of bisphosphonate may be preferable to oral or systemic administration because it minimizes the distribution of the drug to the rest of the skeleton and bypasses the need for having a restored blood flow to the infarcted head for the delivery of the drug.

Local bioavailability and distribution of systemically (parenterally) administered ibandronate in the infarcted femoral head.

Recent studies show that bisphosphonates can decrease the development of femoral head deformity following ischemic osteonecrosis by inhibiting osteoclast-mediated bone resorption. Given the potential new indication, improved understanding of pharmacokinetics of bisphosphonates as it applies to the infarcted head would be beneficial. The purpose of this study was to investigate the local bioavailability and the distribution of ibandronate in the infarcted head at the avascular and vascular phases of the disease process. Ischemic osteonecrosis of the femoral head was surgically induced in 15 piglets. One, 3, and 6 weeks following the induction of ischemia, which represent various stages of revascularization and repair, 14C-labeled ibandronate was administered intravenously. Twenty-four hours following 14C-drug administration, the level of radioactivity and its distribution in the infarcted heads were determined using liquid scintillation analysis and autoradiography. A significant correlation was found between the extent of revascularization and the level of radioactivity measured in the infarcted heads (r=0.80, P<0.05). The radioactivity level in the infarcted heads measured by liquid scintillation was similar to the negative controls at 1 week when revascularization was absent, but it increased significantly at 6 weeks when extensive revascularization was present (P

Ibandronate for prevention of femoral head deformity after ischemic necrosis of the capital femoral epiphysis in immature pigs.

BACKGROUND: Femoral head deformity is the most serious sequela of ischemic necrosis of the immature femoral head. The purpose of this study was to determine if a highly potent antiresorptive agent, ibandronate, can inhibit bone resorption during the repair of the infarcted femoral head and thus alter the repair process. We hypothesized that preservation of the trabecular framework by inhibiting osteoclastic bone resorption would minimize the development of deformity in a piglet model of ischemic necrosis. The effect of ibandronate on long-bone growth was also assessed. METHODS: Ischemic necrosis of the right femoral head was produced in twenty-four piglets by placing a ligature tightly around the femoral neck. The animals were divided into three groups according to whether they received saline solution, prophylactic treatment, or post-ischemia treatment. The contralateral, untreated femoral heads from the animals that had received saline solution served as the normal control group. At eight weeks, the femoral heads were assessed for deformity with radiography and for trabecular bone indices with histomorphometry. Also, the length of femur from the untreated side was measured on the radiographs and compared among the groups. RESULTS: Radiographic assessment showed that the epiphyseal quotient, determined by dividing the maximum height of the osseous epiphysis by the maximum diameter, was better preserved in the prophylactic (p < 0.001) and post-ischemia (p = 0.02) treatment groups than in the group treated with saline solution. Histomorphometric assessment also showed that the trabecular bone indices were better preserved in the prophylactic and the post-ischemia treatment groups than in the group treated with saline solution (p < 0.01). The mean femoral length on the untreated side of the animals treated with ibandronate was reduced compared with the length on the untreated side of the animals that had received saline solution (p

Increased VEGF expression in the epiphyseal cartilage after ischemic necrosis of the capital femoral epiphysis.

UNLABELLED: Ischemic injury to the immature femoral head produces epiphyseal cartilage damage and cessation of endochondral ossification. This study suggests that VEGF facilitates the repair of the necrotic epiphyseal cartilage, which is essential for restoration of endochondral ossification and re-establishment of the growth of the immature femoral head after ischemic necrosis. INTRODUCTION: Legg-Calve-Perthes disease (LCPD) is a childhood form of osteonecrosis that produces growth arrest of the secondary center of ossification. The cessation of growth is caused by ischemic damage to the hypertrophic zone of the epiphyseal cartilage where endochondral ossification normally occurs. The role of vascular endothelial growth factor (VEGF) in restoring endochondral ossification in the epiphyseal cartilage after ischemic necrosis was investigated in a piglet model of LCPD because the resumption of normal growth is important for maintaining the spherical shape of the femoral head. MATERIALS AND METHODS: Piglet femoral heads were assessed 24 h to 8 weeks after the surgical induction of ischemia. Western blot analysis, ribonuclease protection assay (RPA), immunohistochemistry, and in situ hybridization were performed. RESULTS: Western blot analysis and RPA showed increased VEGF protein and mRNA expression, respectively, in the epiphyseal cartilage of the infarcted heads compared with the contralateral normal heads. In the normal femoral heads, VEGF-immunoreactivity (VEGF-IR) and transcripts were observed in the hypertrophic zone of the epiphyseal cartilage. In the infarcted heads, VEGF-IR and transcripts were no longer observed in the hypertrophic zone because of diffuse cell death in that zone from ischemia. However, VEGF-IR and transcripts were observed in the proliferative zone above the necrotic hypertrophic zone. At 8 weeks, vascular granulation tissue invasion of the necrotic hypertrophic zone was observed with active resorption of the necrotic cartilage. In some areas where the necrotic cartilage was completely resorbed, restoration of endochondral ossification was observed. In these areas, VEGF transcripts were observed in the newly formed hypertrophic zone. CONCLUSIONS: VEGF expression was increased, and its spatial expression was altered in the epiphyseal cartilage after ischemic necrosis of the immature femoral head. VEGF upregulation in the proliferative zone after ischemic damage may play a role in stimulating vascular invasion and granulation tissue formation in the necrotic hypertrophic zone of the epiphyseal cartilage. This may be an important step toward facilitating the resorption of the necrotic cartilage and restoration of endochondral ossification leading to further growth and development of the femoral head.