The authors report the 6-year results of the Barrow Ruptured Aneurysm Trial (BRAT). This ongoing randomized trial, with the final goal of a 10-year follow-up, compares the safety and efficacy of ...surgical clip occlusion and endovascular coil embolization in patients presenting with subarachnoid hemorrhage (SAH) from a ruptured aneurysm. The 1- and 3-year results of this trial have been previously reported.
In total, 500 patients with an SAH met the entry criteria and were enrolled in the study. Of these patients, 471 were randomly assigned to the treatments: 238 to surgical clipping and 233 to endovascular coiling. Six patients who died before treatment and 57 patients with nonaneurysmal SAHs were excluded, leaving a total of 408 patients who underwent clipping (209 assigned) or coiling (199 assigned). Whether to treat patients within the assigned group or to cross over patients to the other group was at the discretion of the treating physician; 38% (75/199) of the patients assigned to coiling were crossed over to clipping and 1.9% (4/209) assigned to clipping were crossed over to coiling. The outcome data were collected by a dedicated nurse practitioner. The primary outcome analysis was based on the assigned treatment group; poor outcome was defined as a modified Rankin Scale (mRS) score > 2 and was independently adjudicated. Six years after randomization, 336 (82%) of 408 patients who had been treated were available for examination.
On the basis of an mRS score of > 2, and similar to the results at the 3-year follow-up, no significant difference in outcomes (p = 0.24) was detected between the 2 treatment groups. Complete aneurysm obliteration at 6 years was achieved in 96% (111/116) of the clipping group and in 48% (23/48) of the coiling group (p < 0.0001). In the period between the 3- and 6-year follow-ups, 3 additional patients assigned to coiling and none assigned to clipping received retreatment, for overall retreatment rates of 4.6% (13/280) for clipping and 16.4% (21/128) for coiling (p < 0.0001). When aneurysm location was considered, the 6-year results continued to match the previously reported results, with no difference in outcome for anterior circulation aneurysms at most time points. Of the anterior circulation aneurysms assigned to coiling treatment, 42% (70/168) were crossed over to clipping treatment. The outcomes for posterior circulation aneurysms continued to favor coiling. The randomization process was unexpectedly skewed, with 18 of 21 treated aneurysms of the posterior inferior cerebellar artery (PICA) being assigned to clipping, but even when PICA aneurysms were removed from the analysis, outcomes for the posterior circulation aneurysms still favored coiling.
Although BRAT was statistically underpowered to detect small differences, these results suggest little difference in outcome between the 2 treatments for anterior circulation aneurysms. This was not the case for the posterior circulation aneurysms, where coil embolization appeared to provide a sustained advantage over clipping. Aneurysm obliteration rates in BRAT were significantly lower and retreatment rates significantly higher in the patients undergoing coiling than in those undergoing clipping. However, despite the fact that retreatment rates were higher after coiling, no recurrent hemorrhages were known to have occurred in patients undergoing coiling in BRAT who were followed up for 6 years. Sufficient questions remain about the relative benefits of the 2 treatment modalities to warrant further well-designed randomized trials.
Signal Transducer and Activator of Transcription 3 (STAT3) has recently been shown to be involved in bone development and has been implicated in bone diseases, such as Job’s Syndrome. Bone growth and ...changes have been known for many years to differ between sexes with male bones tending to have higher bone mass than female bones and older females tending to lose bone mass at faster rates than older males. Previous studies using conditional knock mice with Stat3 specifically deleted from the osteoblasts showed both sexes exhibited decreased bone mineral density (BMD) and strength. Using the Cre-Lox system with Cathepsin K promotor driving Cre to target the deletion of the Stat3 gene in mature osteoclasts (STAT3-cKO mice), we observed that 8-week old STAT3-cKO female femurs exhibited significantly lower BMD and bone mineral content (BMC) compared to littermate control (CN) females. There were no differences in BMD and BMC observed between male knock-out and male CN femurs. However, micro-computed tomography (μCT) analysis showed that both male and female STAT3-cKO mice had significant decreases in bone volume/tissue volume (BV/TV). Bone histomorphometry analysis of the distal femur, further revealed a decrease in bone formation rate and mineralizing surface/bone surface (MS/BS) with a significant decrease in osteoclast surface in female, but not male, STAT3-cKO mice. Profiling gene expression in an osteoclastic cell line with a knockdown of STAT3 showed an upregulation of a number of genes that are directly regulated by estrogen receptors. These data collectively suggest that regulation of STAT3 differs in male and female osteoclasts and that inactivation of STAT3 in osteoclasts affects bone turnover more in females than males, demonstrating the complicated nature of STAT3 signaling pathways in osteoclastogenesis. Drugs targeting the STAT3 pathway may be used for treatment of diseases such as Job’s Syndrome and osteoporosis.
Abstract Scanning probe microscopy (SPM) has been in use for 30 years, and the form of SPM known as atomic force microscopy (AFM) has been around for 25 of those years. AFM has been used to produce ...high resolution images of a variety of samples ranging from DNA to carbon nanotubes. Type I collagen and many collagen-based tissues (including dentin, tendon, cartilage, skin, fascia, vocal cords, and cornea) have been studied with AFM, but comparatively few studies of bone have been undertaken. The purpose of this review is to introduce the general principles of AFM operation, demonstrate what AFM has been used for in bone research, and discuss the new directions that this technique can take the study of bone at the nanoscale.
Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide ...fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many “bioorthogonal” reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo6.1.0non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3–7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN–tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli, and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.
Bone is a hierarchical material formed by an organic extracellular matrix and mineral where each component and their physical relationship with each other contribute to fracture resistance. Bone ...quality can be affected by nutrition, and dietary supplements that are marketed to improve overall health may improve the fracture resistance of bone. To test this, 11 week old female C57BL/6 mice were fed either collagen, chondroitin sulfate, glucosamine sulfate, or fish oil 5 times a week for 8 weeks. Femurs, tibiae, and vertebrae were scanned with micro-computed tomography and then mechanically tested. Glucosamine and fish oil lowered elastic modulus, but did not alter the overall strength of the femur. There were no differences in bone mechanics of the tibiae or vertebrae. Overall, the data suggest that dietary supplements did little to improve bone quality in young, healthy mice. These supplements may be more effective in diseased or aged mice.
Osteogenesis imperfecta (OI) increases fracture risk due to changes in bone quantity and quality caused by mutations in collagen and its processing proteins. Current therapeutics improve bone ...quantity, but do not treat the underlying quality deficiencies. Male and female G610C+/− mice, a murine model of OI, were treated with a combination of raloxifene and in vivo axial tibial compressive loading starting at 10 weeks of age and continuing for 6 weeks to improve bone quantity and quality. Bone geometry and mechanical properties were measured to determine whole bone and tissue-level material properties. A colocalized Raman/nanoindentation system was used to measure chemical composition and nanomechanical properties in newly formed bone compared to old bone to determine if bone formed during the treatment regimen differed in quality compared to bone formed prior to treatment. Lastly, lacunar geometry and osteocyte apoptosis were assessed. OI mice were able to build bone in response to the loading, but this response was less robust than in control mice. Raloxifene improved some bone material properties in female but not male OI mice. Raloxifene did not alter nanomechanical properties, but loading did. Lacunar geometry was largely unchanged with raloxifene and loading. However, osteocyte apoptosis was increased with loading in raloxifene treated female mice. Overall, combination treatment with raloxifene and loading resulted in positive but subtle changes to bone quality.
•Combination treatment of anabolic loading and raloxifene improves bone quantity and quality in female OI mice.•Improvements were mostly with the strength of the bone, not the post-yield properties.•Osteocyte apoptosis was higher with loading in female untreated OI mice, but not in raloxifene-treated OI mice.
Osteogenesis imperfecta (OI) is a hereditary bone disease where gene mutations affect Type I collagen formation resulting in osteopenia and increased fracture risk. There are several established ...mouse models of OI, but some are severe and result in spontaneous fractures or early animal death. The Amish Col1a2.sup.G610C/+ (G610C) mouse model is a newer, moderate OI model that is currently being used in a variety of intervention studies, with differing background strains, sexes, ages, and bone endpoints. This study is a comprehensive mechanical and architectural characterization of bone in G610C mice bred on a C57BL/6 inbred strain and will provide a baseline for future treatment studies. Male and female wild-type (WT) and G610C mice were euthanized at 10 and 16 weeks (n = 13-16). Harvested tibiae, femora, and L4 vertebrae were scanned via micro-computed tomography and analyzed for cortical and trabecular architectural properties. Femora and tibiae were then mechanically tested to failure. G610C mice had less bone but more highly mineralized cortical and trabecular tissue than their sex- and age-matched WT counterparts, with cortical cross-sectional area, thickness, and mineral density, and trabecular bone volume, mineral density, spacing, and number all differing significantly as a function of genotype (2 Way ANOVA with main effects of sex and genotype at each age). In addition, mechanical yield force, ultimate force, displacement, strain, and toughness were all significantly lower in G610C vs. WT, highlighting a brittle phenotype. This characterization demonstrates that despite being a moderate OI model, the Amish G610C mouse model maintains a distinctly brittle phenotype and is well-suited for use in future intervention studies.
Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment ...epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.
Down syndrome (DS), affecting ∼1 in 800 live births, is caused by the triplication of human chromosome 21 (Hsa21). Individuals with DS have skeletal features including craniofacial abnormalities and ...decreased bone mineral density (BMD). Lowered BMD can lead to increased fracture risk, with common fracture points at the femoral neck and lumbar spine. While the femur has been studied in DS mouse models, there is little research done on the vertebrae despite evidence that humans with DS have affected vertebrae. Additionally, it is important to establish when skeletal deficits occur to find times of potential intervention. The Dp(16)1Yey DS mouse model has all genes triplicated on mouse chromosome 16 orthologous to Hsa21 and displayed deficits in long bone, including trabecular and cortical deficits in male but not female mice, at 12 weeks. We hypothesized that the long bone and lumbovertebral microarchitecture would exhibit sexually dimorphic deficits in Dp(16)1Yey mice compared to control mice and long bone strength would be diminished in Dp(16)1Yey mice at 6 weeks. The trabecular region of the 4th lumbar (L4) vertebra and the trabecular and cortical regions of the femur were analyzed via micro-computed tomography and 3-point bending in 6-week-old male and female Dp(16)1Yey and control mice. Trabecular and cortical deficits were observed in femurs from male Dp(16)1Yey mice, and cortical deficits were seen in femurs of male and female Dp(16)1Yey mice. Male Dp(16)1Yey femurs had more deficits in bone strength at whole bone and tissue-estimate level properties, but female Dp(16)1Yey mice were also affected. Additionally, the L4 of male and female Dp(16)1Yey mice show trabecular deficits, which have not been previously reported in a DS mouse model. Our results indicate that skeletal deficits associated with DS occur early in skeletal development, are dependent on skeletal compartment and site, are sex dependent, and potential interventions should likely begin early in skeletal development of DS mouse models.
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•Dp(16)1Yey mice model lumbar spine traits found in with Down syndrome.•Down syndrome mouse model skeleton is affected early in bone development.•Both the axial (lumbar vertebrae) and appendicular (femur) are affected.•There is a sexual dimorphism depending on skeletal site and bone compartment.•Mechanics of femur are affected in both sexes at the whole bone and tissue level.
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