X-ray Micro-Computed Tomography: An Emerging Technology to Analyze Vascular Calcification in Animal Models.

Vascular calcification describes the formation of mineralized tissue within the blood vessel wall, and it is highly associated with increased cardiovascular morbidity and mortality in patients with chronic kidney disease, diabetes, and atherosclerosis. In this article, we briefly review different rodent models used to study vascular calcification in vivo, and critically assess the strengths and weaknesses of the current techniques used to analyze and quantify calcification in these models, namely 2-D histology and the o-cresolphthalein assay. In light of this, we examine X-ray micro-computed tomography (µCT) as an emerging complementary tool for the analysis of vascular calcification in animal models. We demonstrate that this non-destructive technique allows us to simultaneously quantify and localize calcification in an intact vessel in 3-D, and we consider recent advances in µCT sample preparation techniques. This review also discusses the potential to combine 3-D µCT analyses with subsequent 2-D histological, immunohistochemical, and proteomic approaches in correlative microscopy workflows to obtain rich, multifaceted information on calcification volume, calcification load, and signaling mechanisms from within the same arterial segment. In conclusion we briefly discuss the potential use of µCT to visualize and measure vascular calcification in vivo in real-time.

A multi-factorial analysis of bone morphology and fracture strength of rat femur in response to ovariectomy.

BACKGROUND: Postmenopausal osteoporosis develops due to a deficiency of estrogen that causes a decrease in bone mass and changes in the macro- and micro-architectural structure of the bone, leading to the loss of mechanical strength and an increased risk of fracture. Although the assessment of bone mineral density (BMD) has been widely used as a gold standard for diagnostic screening of bone fracture risks, it accounts for only a part of the variation in bone fragility; thus, it is necessary to consider other determinants of bone strength. Therefore, we aimed to comprehensively evaluate the architectural changes of the bone that influence bone fracture strength, together with the different sensitivities of cortical and trabecular bone in response to ovariectomy (OVX). METHODS: Bone morphology parameters were separately analyzed both in cortical and in trabecular bones, at distal-metaphysis, and mid-diaphysis of OVX rat femurs. Three-point bending test was performed at mid-diaphysis of the femurs. Correlation of OVX-induced changes of morphological parameters with breaking force was analyzed using Pearson's correlation coefficient. RESULTS: OVX resulted in a decline in the bone volume of distal-metaphysis trabecular bone, but an increase in distal-metaphysis and mid-diaphysis cortical bone volume. Tissue mineral density (TMD) remained unchanged in both the trabecular and cortical bone of the distal metaphysis but decreased in cortical bone of the mid-diaphysis. The OVX significantly increased the breaking force at mid-diaphysis of the femurs. CONCLUSIONS: OVX decreased the trabecular bone volume of the distal-metaphysis and increased the cortical bone volume of the distal-metaphysis and mid-diaphysis. Despite the reduction in TMD and increased cortical porosity, bone fracture strength increased in the mid-diaphysis after OVX. These results indicate that analyzing a single factor, i.e., BMD, is not sufficient to predict the absolute fracture risk of the bone, as OVX-induced bone response vary, depending on the bone type and location. Our results strongly support the necessity of analyzing bone micro-architecture and site specificity to clarify the true etiology of osteoporosis in a clinical setting.

The involvement of C5a in the progression of experimental arthritis with Porphyromonas gingivalis infection in SKG mice.

BACKGROUND: Epidemiological evidence to suggest that periodontal disease (PD) is involved in the progression of rheumatoid arthritis (RA) is increasing. The complement system plays a critical role in immune responses. C5a has been implicated in chronic inflammatory diseases, including PD and RA. Porphyromonas gingivalis is the major causative bacteria of PD and can produce C5a. Therefore, it is hypothesized that P. gingivalis infection is involved in the progression of RA by elevating C5a levels. In the present study, P. gingivalis-infected RA model mice were established to investigate the involvement of C5a. METHODS: SKG mice orally infected with P. gingivalis were immunized with intraperitoneal injection of laminarin (LA) to induce arthritis. Arthritis development was assessed by arthritis score (AS), bone destruction on the talus, histology, and serum markers of RA. In order to investigate the effects of serum C5a on bone destruction, osteoclast differentiation of bone marrow mononuclear cells was examined by using serum samples from each group of mice. The relationship between C5a levels and antibody titers to periodontal pathogens in patients with RA was investigated by enzyme-linked immunosorbent assay. RESULTS: P. gingivalis oral infection increased AS, infiltration of inflammatory cells, bone destruction on the talus, and serum markers of RA in mice immunized with LA. The addition of serum from LA-injected mice with the P. gingivalis oral infection promoted osteoclast differentiation, and the addition of a neutralization antibody against C5a suppressed osteoclast differentiation. C5a levels of serum in RA patients with positive P. gingivalis antibody were elevated compared with those in RA patients with negative P. gingivalis antibody. CONCLUSIONS: These results suggest that P. gingivalis infection enhances the progression of RA via C5a.

A role for telomere length and chromosomal damage in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis is a fatal lung disease characterized by a progressive formation of fibroblastic foci in the interstitium. This disease is strongly associated with telomere dysfunction but the extent of telomere shortening and consequent chromosomal damage within IPF lungs and with regional disease severity remains unknown. METHODS: Explanted IPF lungs (n = 10) were collected from transplant surgeries with six samples per lung analysed to capture the regional heterogeneity ranging from mild to severe disease. Non-used donor lungs (n = 6) were collected as "healthy" controls. Structural changes related to disease severity (microCT surface density), relative telomere length (real-time qPCR), and quantitative histology of chromosomal damage (γ-H2A.X) and extracellular matrix (elastin, total collagen, collagen 1, and collagen 3) were measured. A multivariate linear mixed-effects model controlling for subject was used to identify association of disease severity or fibrotic markers with telomere length and chromosomal damage. RESULTS: We observed shorter telomere length (p = 0.001) and increased chromosomal damage (p = 0.018) in IPF lungs compared to controls. In IPF lungs, telomere length was associated with total collagen (p < 0.001) but not with structural changes of disease severity. Chromosomal damage was positively associated with increased elastin (p = 0.006) and negatively with structural disease severity (p = 0.046). Extensive γ-H2A.X staining was also present in airway epithelial cells. CONCLUSIONS: Telomere length and chromosomal damage are involved in IPF with regional variation in telomere length and chromosomal damage associated with pathological changes in tissue structure and the extracellular matrix.

3D printing from microfocus computed tomography (micro-CT) in human specimens: education and future implications.

Microfocus CT (micro-CT) is an imaging method that provides three-dimensional digital data sets with comparable resolution to light microscopy. Although it has traditionally been used for non-destructive testing in engineering, aerospace industries and in preclinical animal studies, new applications are rapidly becoming available in the clinical setting including post-mortem fetal imaging and pathological specimen analysis. Printing three-dimensional models from imaging data sets for educational purposes is well established in the medical literature, but typically using low resolution (0.7 mm voxel size) data acquired from CT or MR examinations. With higher resolution imaging (voxel sizes below 1 micron, <0.001 mm) at micro-CT, smaller structures can be better characterised, and data sets post-processed to create accurate anatomical models for review and handling. In this review, we provide examples of how three-dimensional printing of micro-CT imaged specimens can provide insight into craniofacial surgical applications, developmental cardiac anatomy, placental imaging, archaeological remains and high-resolution bone imaging. We conclude with other potential future usages of this emerging technique.

Propionibacterium acnes Incubation in the Discs Can Result in Time-Dependent Modic Changes: A Long-Term Rabbit Model.

STUDY DESIGN: A case-control study of animal model of Modic changes (MCs) on rabbits. OBJECTIVE: To evaluate the feasibility of inducing of MCs by injection of Propionibacterium acne (P. acnes) into the lumbar intervertebral discs of rabbits. SUMMARY OF BACKGROUND DATA: MCs have been widely observed, and assume to be closely associated with low back pain and P. acnes, but there are few animal models showing the progression of MCs. METHODS: Ten rabbits were used for the study. The L3-4 and L4-5 discs of all rabbits were injected with 100 µL P. acnes (1.6 × 10 CFU/mL) as P. acnes group, L2-3 disc were injected with 100 µL normal saline as vehicle, and L5-6 disc was untreated (blank). MCs were investigated by magnetic resonance imaging before operation and at 2 weeks, 1, 3, 4.5, 6, and 9 months postoperatively. Following sacrifice, histological analysis, blood test and micro-computed tomography were performed. Cytokine expression in nucleus and endplate tissues was quantified using real-time polymerase chain reaction. RESULTS: From 3 months postoperatively, the P. acnes group showed significantly decreased T1-weighted signal intensity, whereas the T2-weighted signal was significantly higher at 3 and 4.5 months, and then decreased remarkably at 6 and 9 months. Eleven of 20 inferior endplates were identified as type I MCs at 4.5 months, and 9 of 20 were identified as type II MCs at 9 months. Real-time polymerase chain reaction showed that expression of interleukin-1ß, tumor necrosis factor α, interferon-γ, matrix metalloproteinase-9, and thrombospondin motifs-5 in the nucleus pulposus, and interleukin-1ß, tumor necrosis factor α, and thrombospondin motifs-5 in the endplates, were significantly upregulated after injection of P. acnes. Histological slices of discs injected with P. acnes showed disc degeneration, endplate abnormalities, and inflammatory response, with micro-computed tomography confirming bone resorption. CONCLUSION: P. acnes infection of the disc can induce degeneration of the disc and an inflammatory response in the endplate region, presenting as MCs type I and II time dependently. LEVEL OF EVIDENCE: N/A.

Long-term wheel-running can prevent deterioration of bone properties in diabetes mellitus model rats.

OBJECTIVES: The purpose of this study was to examine the effects of long-term wheel-running on tibia bone properties in T2DM Otsuka Long-Evans Tokushima Fatty (OLETF) rats. METHODS: Ten five-week-old male OLETF rats were used as experimental animals and 5 Long-Evans Tokushima Otsuka (LETO) rats as controls. Half of OLETF rats performed daily voluntary wheel-running for 17 months (OLETF-EXE), while neither the remainder of OLETF nor LETO rats had exercise. At the end of experiment, in addition to serum biochemical and bone formation/resorption marker analyses, bone mass, trabecular bone microarchitecture and cortical bone geometry were analyzed in left tibia, and bone mechanical strength of right tibia was measured. RESULTS: Tibia bone mass, trabecular bone microarchitecture, cortical bone geometry and bone mechanical strength deteriorated in diabetic OLETF rats. However, such deterioration was obviously attenuated in OLETF-EXE rats, which maintained normal levels of blood glucose, HbA1c and blood urea nitrogen. CONCLUSIONS: Daily wheel-running could prevent the deterioration of bone properties in OLETF rats. This would be induced mainly by suppressing the development of T2DM. Regular physical exercise may be a potent strategy for preventing not only the development of diabetes but also the deterioration of bone properties in patients with chronic T2DM.

Variation in Brain Morphology of Intertidal Gobies: A Comparison of Methodologies Used to Quantitatively Assess Brain Volumes in Fish.

When correlating brain size and structure with behavioural and environmental characteristics, a range of techniques can be utilised. This study used gobiid fishes to quantitatively compare brain volumes obtained via three different methods; these included the commonly used techniques of histology and approximating brain volume to an idealised ellipsoid, and the recently established technique of X-ray micro-computed tomography (micro-CT). It was found that all three methods differed significantly from one another in their volume estimates for most brain lobes. The ellipsoid method was prone to over- or under-estimation of lobe size, histology caused shrinkage in the telencephalon, and although micro-CT methods generated the most reliable results, they were also the most expensive. Despite these differences, all methods depicted quantitatively similar relationships among the four different species for each brain lobe. Thus, all methods support the same conclusions that fishes inhabiting rock pool and sandy habitats have different patterns of brain organisation. In particular, fishes from spatially complex rock pool habitats were found to have larger telencephalons, while those from simple homogenous sandy shores had a larger optic tectum. Where possible we recommend that micro-CT be used in brain volume analyses, as it allows for measurements without destruction of the brain and fast identification and quantification of individual brain lobes, and minimises many of the biases resulting from the histology and ellipsoid methods.