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Background: The apoE-/-/LDL-/- double knockout mice are bearing considerable structural homology to human atherosclerosis. We hypothesized, that advanced lesion formation in the renal artery is associated with kidney alterations in these mice.Methods: Kidneys from apoE-/-/LDL-/- double knockout mice at the age of 80 weeks (n = 6) and C57/BL control mice (n = 5) were infused with Microfil, harvested and scanned with micro-CT (12 mum cubic voxels) and Nano-CT (900 nm cubic voxels). We quantitated the total vascular volume using micro-CT. Number and cross-sectional area (microm2) of glomeruli were measured using histology.Results: At the age of 80 weeks, the renal total vascular volume fraction decreased significantly (p < 0.001) compared to controls. Moreover, the renal artery showed advanced atherosclerotic lesions with adventitial Vasa vasorum neovascularization. Perivascular inflammation was present in kidneys of apoE-/-/LDL-/- double knockout mice, predominantly involved are plasma cells and leucocytes. Glomeruli cross-sectional area (9959 +/- 1083 microm2) and number (24.8 +/- 4.5) increased in apoE-/-/LDL-/- double knockout mice compared to controls (3533 +/- 398 microm2; 17.6 +/- 3, respectively), whereas 41% of the total number of glomeruli showed evidence for lipoprotein associated glomerulopathy (LPG). Moreover, immunohistochemistry demonstrated capillary aneurysms of the glomeruli filled with factor 8 containing emboli.Conclusion: The reduced intra-renal total vascular volume is associated with systemic atherosclerosis and glomeruli alterations in the apoE-/-/LDL-/- double knockout mouse model. [ABSTRACT FROM AUTHOR]

The present study deals with the characterization of bone quality in a sheep model of postmenopausal osteoporosis. Sheep were sham operated ( n = 7), ovariectomized ( n = 6), ovariectomized and treated with deficient diet ( n = 8) or ovariectomized, treated with deficient diet and glucocorticoid injections ( n = 7). The focus of the study is on the microscopic properties at tissue level. Microscopic mechanical properties of osteoporotic bone were evaluated by a combination of biomechanical testing and mathematical modelling. Sample stiffness and strength were determined by compression tests and finite-element analysis of stress states was conducted. From this, an averaged microscopic Young's modulus at tissue level was determined. Trabecular structure as well as mineral and collagen distribution in samples of sheep vertebrae were analysed by micro-computed tomography and time-of-flight secondary ion mass spectrometry. In the osteoporotic sheep model, a disturbed fibril structure in the triple treated group was observed, but bone loss only occurred in form of reduced trabecular number and thickness and cortical decline, while quality of the residual bone was preserved. The preserved bone tissue properties in the osteoporotic sheep model allowed for an estimation of bone strength which behaves similar to the human case.

Fractures of the first thumb ray are common and need accurate differential treatment to restore gripping hand functions. Displaced fractures of the distal and proximal phalanx of the thumb are often treated with screws or Kirschner wires. Stable fractures can also be treated non-operatively. Fractures of the base of the first metacarpal should be differentiated into extra-articular Winterstein fractures and intra-articular Bennett or Rolando fractures. Traction forces by the abductor pollicis longus tendon regularly lead to displacement of the shaft of the first metacarpal; therefore, these fractures usually require reduction and fixation. Good functional results can be achieved by operative treatment. Fractures of the trapezium are rare. If they are displaced, operative treatment is recommended to prevent osteoarthritis of the first carpometacarpal joint.

Purpose: Neovascularization is essential for bone regeneration in fractures. This study aimed to investigate the microvascular morphology and distribution in the non-injured femur and the neovascularization of the metaphyseal critical size defect in a small animal model of osteoporosis., Materials and Methods: Female rats (n=7) were ovariectomized (OVX) and received a multideficiency diet. Three months after OVX, a 5mm wedge shaped critical size defect was cut at the distal femoral metaphysis and stabilized with a T-shaped mini-plate. After six weeks, the animals were euthanized, and femora were removed and decalcified for micro-CT measurement of fracture neovascularization., Results: No fracture healing was observed along the critical size defects. In the non-injured bone, micro-vessel distribution showed a specific pattern, thereby enabling a differentiation between epi-, meta- and diaphysis. Micro-CT based morphometry revealed a significant reduction of the vascular volume fraction as well as the vascular thickness (p<0.001) in the critical size defect compared to the intact contralateral femur. Blood volume related vascular surface (vascular surface/volume) increased significantly (p<0.001). Connectivity density and tissue volume related vascular surface (vascular surface density) did not change significantly., Conclusions: Micro-CT based vascular morphometry demonstrated differences between epi-, meta- and diaphysis in the non-injured bone as well as differences between the critical size defect and the non-injured metaphysis. As angiogenesis is a crucial prerequisite that precedes osteogenesis, our results may influence further evaluation of osteoconductive or osteogenic biomaterials in this small animal model of osteoporosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female α7KO compared to their corresponding wild-type mice (α7WT)., Methods: Vertebrae and long bones of female 16-week-old α7KO (n = 10) and α7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM)., Results: Bone of female α7KO revealed a significant increase in bending stiffness (p < 0.05) and cortical thickness (p < 0.05) compared to α7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N(+) (p < 0.05) and C4H8N(+) (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM., Conclusions: Our results on female α7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that α7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit α2 the α7-nAChR favours reduction of bone strength thereby showing similar effects as α7β2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing α- and β-subunits as well as the subunits α7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric α7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis.

Purpose: The prevalence of systemic atherosclerosis and overactive bladder/detrusor overactivity increases almost simultaneously with age but an association between these diseases has not yet been proved. We evaluated changes in bladder function and morphology, including vascularization, in apoE(-/-)LDLR(-/-) double knockout mice with systemic atherosclerosis but without central nervous system involvement., Materials and Methods: Cystometry was performed in awake, freely moving 60-week-old apoE(-/-)LDLR(-/-) mice and C57BL/6N controls. The mice were sacrificed and perfused with Microfil® contrast medium. The bladder was excised, dissected and scanned by nano-computerized tomography, including 3-dimensional reconstruction. Samples then underwent histomorphological analysis., Results: In apoE(-/-)LDLR(-/-) mice cystometry revealed a significant decrease in the peak-peak interval, micturition interval, functional bladder capacity and micturition volume. However, maximum bladder pressure increased. Nano-computerized tomography revealed a significant reduction in bladder wall thickness, segment volume, vascular volume and the vascular volume fraction. Histomorphologically bladder specimens showed a thickened media of intramural vessels, activated endothelial cells and intramural inflammatory cells., Conclusions: To our knowledge this study presents a new in vivo mouse model of nonneurogenic detrusor overactivity caused by systemic atherosclerosis. Decreased bladder wall vascularization seems to be a major factor for detrusor overactivity onset. Capillaries are rarified with reduced lumina due to thickened media. Activated endothelial cells and the infiltration of inflammatory cells in apoE(-/-)LDLR(-/-) mice underlines once more that atherosclerosis is an inflammatory process that may also be relevant to the onset of detrusor overactivity., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Repetitive freeze/thaw cycles lead to a progressive loss of structural and molecular integrity in deep frozen specimens. The aim of this study was to evaluate a micro-CT stage, which maintains the cryoconservation of large specimens throughout micro-CT imaging. Deep frozen ovine vertebral segments (-20 °C) were fixed in a micro-CT stage made of expanded polystyrene and cooled with dry ice (0 g, 60 g and 120 g). The temperature inside the stage was measured half-hourly over a time span of three hours with subsequent measurement of surface temperature. The method was validated in a series of 30 deep frozen vertebral specimens and in liver tissue after repetitive micro-CT scanning. Isolation without cooling resulted in defrosting. Cooling with 60 g of dry ice led to a temperature rise inside the stage (max. 5.1 °C) and on the specimen surfaces (max. -3 °C). Cooling with 120 g of dry ice resulted in a significant (p < 0.001) and sufficient lowering of the temperature inside the stage (max. -14 °C) and on the surface of the specimens (max. -13.9 °C). The surface temperature during the subsequent micro-CT validation study did not exceed -16 °C (processing time 1 h 45 min). The resolution was 33 μm isotropic voxel side length, enabling a binarization of bone microstructures. Temperature can reliably be maintained below -10 °C during a micro-CT scan by applying the described technique. The resulting spatial resolution and image quality permits a binarization of bone microstructure., (© Wiley Periodicals, Inc.)

Nonalcoholic fatty liver disease has been linked to cardiovascular diseases and atherosclerosis. The aim of the current study was to characterize the hepatic pathology leading to fibrosis and tumors in a murine model of atherosclerosis. Male apolipoprotein E/low-density lipoprotein receptor double-knockout mice (AL) mice were fed with a high fat and high cholesterol western diet for 35 weeks (AL mice on WD). Protein and mRNA analysis as well as micro-computed tomography (micro-CT) were performed to assess oxidative stress, liver damage, inflammation, fibrosis, signaling pathways, vascularization, and tumorigenesis. Controls were chosen to distinguish between genetically and dietary effects in steatohepatitis and associated tumorigenesis. Hepatic inflammation and dyslipidemia were increased in AL mice on WD compared with wild-type mice on WD. Uniquely, AL mice on WD showed a spontaneous development of tumors (30% of cases) and thickening of intrahepatic vessel walls. Functionally relevant underlying signaling pathways such as NF-κB, Stat3, JNK, and AKT were differentially regulated between AL and wild-type mice on WD. Micro-CT was capable of visualizing and quantitatively distinguishing tumor neovascularization from vascularization in non-neoplastic liver tissue. AL mice on WD diet represent a novel model combining atherosclerosis and nonalcoholic fatty liver disease. Signaling pathways of liver cell damage and compensatory liver regeneration in combination with enhanced inflammation appear to be crucial for the spontaneous development of tumors in AL mice on WD. Micro-CT represents a new and powerful technique for the ultrastructural and three-dimensional assessment of the vascular architecture of liver tumors.

Purpose: In humans, glucocorticoid-induced osteoporosis is the most common cause of medication-induced osteoporosis. Recent clinical data suggest that glucocorticoid therapy increases the risk of vertebral fractures within a short treatment period. Therefore, this study aimed at investigating vertebral bone in a rat model of glucocorticoid-induced postmenopausal osteoporosis., Methods: Fifty Sprague-Dawley rats were randomly assigned into three groups: 1) untreated controls, 2) Sham-operated group, and 3) ovariectomized rats treated with glucocorticoid (dexamethasone) for 3 months (3M) after recovery from bilateral ovariectomy. Osteoporotic bone status was determined by means of the gold standard dual energy X-ray absorptiometry (DEXA) scan. Vertebral bodies were examined using µCT, histological analysis, mRNA expression analysis, and biomechanical compression testing. Further systemic effects were studied biochemically using serum marker analysis., Results: Dexamethasone treatment showed at 3M a significantly lower bone mineral density in ovariectomized rats compared to Sham-operated control (p < 0.0001) as analyzed in vivo by DEXA. Furthermore, Z scores reached levels of -5.7 in the spine indicating sever osteoporotic bone status. Biomechanical testing of compression stability indicated a lower functional competence (p < 0.0001) in the spine of treated rats. µCT analysis showed significant reduction of bone volume density (BV/TV%; p < 0.0001), significantly enhanced trabecular spacing (Tb.Sp; p < 0.0001) with less trabecular number (Tb.N; p < 0.001) and complete loss of trabecular structures in glucocorticoid-treated ovariectomized rats. Histological analysis by osteoblast and osteoclast activities reflected a higher bone catabolism reflected by osteoclast counts by TRAP (p < 0.019) and lower bone catabolism indicated by ALP-stained area (p < 0.035).Serum analysis showed a significant increase in osteocalcin (p < 0.0001), osteopontin (p < 0.01) and insulin (p < 0.001) at 3M. Expression analysis of molecular markers in the vertebral body revealed lower expression in tenascin C in the OVX-steroid animals at 3M., Conclusions: Short-term glucocorticoid treatment of ovariectomized rats indicates according to DEXA standards a severe osteoporotic bone status in vertebral bone. Nonetheless, dysfunctional bone anabolism and enhanced bone catabolism are observed. Alterations of bone extracellular matrix proteins that correlate to inferior mechanical stability and affected microstructure were noticed and suggest further investigation. Treatment with dexamethasone was also seen to affect insulin and osteopontin levels and thus osteoblast function and maturation. This described animal model presents a recapitulation of clinically obtained data from early phase glucocorticoid-induced osteoporosis observed in patients.

Plaque progression in atherosclerosis is closely connected to angiogenesis due to vasa vasorum (VV) growth. Objective of this study was to determine the unknown long-term effect of thalidomide on adventitial VV neovascularization and plaque progression using nano-focussed computed tomography (nano-CT). Proliferation and migration assays in human coronary artery endothelial cells (HCAEC) measured number of viable cells after incubation with thalidomide. Male ApoE(-/-)/LDLR(-/-) (AL) mice (n = 5) received a thalidomide containing western diet (WD) over 29 weeks. Another five male AL mice (WD without thalidomide) served as control group. Descending aortas were scanned with nano-CT at (1.5 μm)(3) isotropic voxel size. Number and area of adventitial VV as well as plaque cross sectional area were measured. Results were complemented by histology. Thalidomide inhibited proliferation and migration of HCAEC dose-dependently. VV neovascularization decreased in number per cross section (7.66 ± 0.301 vs. 8.62 ± 0.164, p < 0.001) and in cross sectional area (0.0183 ± 0.0011 vs. 0.0238 ± 0.0008 mm(2), p < 0.001). Cross sectional area of plaque decreased significantly when treated with thalidomide (0.57 ± 0.0187 vs. 0.803 ± 0.0148 mm(2), p < 0.001). Nano-CT imaging revealed a reduced plaque growth and VV neovascularization after long-term application of thalidomide. Therefore, nano-CT can be considered as a new method to detect therapeutic effects in experimental models of atherosclerosis.

For assessing mechanical properties of osteoporotic bone, biomechanical testing combined with in silico modeling plays a key role. The present study focuses on microscopic mechanical bone properties in a rat model of postmenopausal osteoporosis. Female Sprague-Dawley rats were (1) euthanized without prior interventions, (2) sham-operated, and (3) subjected to ovariectomy combined with a multi-deficiencies diet. Rat vertebrae (corpora vertebrae) were imaged by micro-CT, their stiffness was determined by compression tests, and load-induced stress states as well as property changes due to the treatment were analyzed by finite-element modeling. By comparing vertebra stiffness measurements with finite-element calculations of stiffness, an overall microscopic Young's modulus of the bone was determined. Macroscopic vertebra stiffness as well as the microscopic modulus diminish with progression of osteoporosis by about 70 %. After strong initial changes of bone morphology, further decrease in macroscopic stiffness is largely due to decreasing microscopic Young's modulus. The micromechanical stress calculations reveal particularly loaded vertebra regions prone to failure. Osteoporosis-induced changes of the microscopic Young's modulus alter the fracture behavior of bone, may influence bone remodeling, and should be considered in the design of implant materials.

Discrepancies in bone healing between osteoporotic and non-osteoporotic bone remain uncertain. The focus of the current work is to evaluate potential healing discrepancies in a metaphyseal defect model in rat femora. Female Sprague-Dawley rats were either ovariectomized (OVX, n=14) and combined with a calcium-, phosphorus- and vitamin D3-, soy- and phytoestrogen-free diet or received SHAM operation with standard diet rat (SHAM, n=14). Three months post-ovariectomy, DEXA measurement showed a reduction of bone mineral density reflecting an osteoporotic bone status in OVX rats. Rats then underwent a 3 mm wedge-shaped osteotomy at the distal metaphyseal area of the left femur stabilized with a T-shaped mini-plate and allowed to heal for 6 weeks. Biomechanical competence by means of a non-destructive three-point bending test showed significant lower flexural rigidity in the OVX rats at 3 mm lever span compared to SHAM animals (p=0.048) but no differences at 10 mm lever span. Microcomputer tomography (μCT) showed bridging cortices and consolidation of the defect in both groups, however, no measurable differences were found in either total ossified tissue or vascular volume fraction. Furthermore, histology showed healing discrepancies that were characterized by cartilaginous remnant and more unmineralized tissue presence in the OVX rats compared to more mature consolidation appearance in the SHAM group. In summary, bone defect healing in metaphyseal bone slightly differs between osteoporotic and non-osteoporotic bone in the current 3 mm defect model in both 3mm lever span biomechanical testing and histology., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Remodelling of trabecular bone is essentially affected by the mechanical load of the trabeculae. Mathematical modelling and simulation of the remodelling process have to include time-consuming calculations of the displacement field within the complex trabecular structure under loading. We present an adaptive diffuse domain approach for calculating the elastic bone deformation based on micro computer tomogram data of real trabecular bone structures and compared it with a conventional voxel-based finite element method. In addition to allowing for higher computational efficiency, the adaptive approach is characterised by a very smooth representation of the bone surface, which suggests that this approach would be suitable as a basis for future simulations of bone resorption and formation processes within the trabecular structure.

Purpose: To assess the feasibility of micro-CT for obtaining quantitative volumetric and morphologic information of changes in soft tissue, respiratory tracts and vascularization in fibrotic, emphysematous and non-diseased human lung specimens., Materials and Methods: Specimens from autopsy or lung explantation with lung fibrosis of UIP pattern (n = 22) or centrilobular emphysema (n = 10) were scanned by micro-CT and compared to controls (n = 22). Imaging was performed subsequent to intravascular contrast enhancement for the assessment of the vascular volume fraction. The soft tissue and air fraction were quantified after the fixation of ventilated lungs followed by tissue contrast enhancement using osmium. Aiming an artifact-free 3 D reconstruction of lung acini, synchrotron-based micro-CT scans of specimens with emphysema (n = 5) and non-diseased tissue (n = 6) was performed. Micro-CT imaging was complemented by histology for the demonstration of comparable findings., Results: Quantitative analysis showed a significant increase of the soft tissue fraction, equivalent to a decrease of the air fraction in fibrotic lungs compared to controls (p < 0.001) and a significant reduction of the vascular volume fraction compared to controls (p < 0.02). Specimens with emphysema demonstrated a significant increase of the air fraction with a decrease in soft tissue compared to controls (p < 0.001). 3 D reconstructions of lung acini worked successfully in non-diseased tissue but failed in fibrotic and emphysematous lungs., Conclusion: Our findings indicate micro-CT's technical feasibility to assess quantitative and morphological data from diseased and non-diseased human lung specimens., (© Georg Thieme Verlag KG Stuttgart · New York.)

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.

Background: Osteoporosis is a multi-factorial, chronic, skeletal disease highly prevalent in post-menopausal women and is influenced by hormonal and dietary factors. Because animal models are imperative for disease diagnostics, the present study establishes and evaluates enhanced osteoporosis obtained through combined ovariectomy and deficient diet by DEXA (dual-energy X-ray absorptiometry) for a prolonged time period., Material/methods: Sprague-Dawley rats were randomly divided into sham (laparotomized) and OVX-diet (ovariectomized and fed with deficient diet) groups. Different skeletal sites were scanned by DEXA at the following time points: M0 (baseline), M12 (12 months post-surgery), and M14 (14 months post-surgery). Parameters analyzed included BMD (bone mineral density), BMC (bone mineral content), bone area, and fat (%). Regression analysis was performed to determine the interrelationships between BMC, BMD, and bone area from M0 to M14., Results: BMD and BMC were significantly lower in OVX-diet rats at M12 and M14 compared to sham rats. The Z-scores were below -5 in OVX-diet rats at M12, but still decreased at M14 in OVX-diet rats. Bone area and percent fat were significantly lower in OVX-diet rats at M14 compared to sham rats. The regression coefficients for BMD vs. bone area, BMC vs. bone area, and BMC vs. BMD of OVX-diet rats increased with time. This is explained by differential percent change in BMD, BMC, and bone area with respect to time and disease progression., Conclusions: Combined ovariectomy and deficient diet in rats caused significant reduction of BMD, BMC, and bone area, with nearly 40% bone loss after 14 months, indicating the development of severe osteoporosis. An increasing regression coefficient of BMD vs. bone area with disease progression emphasizes bone area as an important parameter, along with BMD and BMC, for prediction of fracture risk.

Nanomedicine comprises a significant potential to approach the therapy of severe diseases. Knowledge of nanoparticle behavior at the target site would contribute to the development of specialized tools for respiratory medicine. Here, we were interested in the potential of micro-computed tomography (μCT) imaging to monitor the pulmonary distribution of polymeric nanoparticles. Composite nanoparticles were analyzed for physicochemical properties, morphology and composition. μCT was employed to visualize the pulmonary distribution of composite nanoparticles in an ex vivo lung model. Employed composite nanoparticles were composed of poly(styrene) cores coated by a thin shell of colloidal iron oxide. Particles were mainly located in the interstitial space and associated with pulmonary cells, as observed by light microscopy. μCT detected enhanced X-ray opacities in the conducting (linear pattern) and respiratory airways (aciniform X-ray attenuations). In conclusion, multifunctional nanoparticles will prompt the development of novel therapeutic and diagnostic tools in respiratory medicine., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aims: Increasing collagen synthesis was observed in lung after stimulation of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) on fibroblasts. Since collagen synthesis is an important process during fracture healing and bone remodelling, we asked whether cholinergic receptors are involved in bone collagen production., Main Methods: In the present study we analysed 16 week old male knockout mice for nAChRα7 (α7-KO) and mAChR M3R (M3R-KO) in correlation to their corresponding wild types (WT). Microarchitecture of right femora, vertebrae Th13 and L1 were analysed by 3D Micro-CT, left femora by a three-point bending test and humeri by real-time RT-PCR., Key Findings: A significant decrease in relative bone volume, trabecular thickness, trabecular number, bone surface density, and a significant increase in trabecular separation and structure model index were measured for the M3R-KO using Micro-CT analysis. Bending stiffness of M3R-KO was significantly reduced in comparison to WT as well as the collagen 1α1 and 1α2 mRNA expression was down-regulated. No changes were detected for α7-KO using Micro-CT, biomechanical testing, and collagen mRNA expression., Significance: Our results indicate that nAChRα7 are not involved in the regulation of bone collagen synthesis whereas M3R exert stimulatory effects on cancellous bone microarchitecture, flexural rigidity, and bone matrix synthesis. Since the M3R-KO exhibit bone structures similar to systemically diseased bone it might be valuable to establish new therapeutic strategies using administration of agonists for the M3R to improve bone qualities., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Age-related testicular changes are associated with declining spermatogenesis and testosterone levels. A relationship to atherosclerosis has never been investigated systematically. The ApoE(-/-)/LDL receptor(-/-) double knockout mouse model, providing a remarkable homology to human atherosclerosis, is an ideal tool to investigate spermatogenetic alterations in this context. Testes (n = 10) from ApoE(-/-)/LDL receptor(-/-) double knockout mice at the age of 80 weeks were perfused in vivo with contrast agent, harvested and scanned with micro-CT at (4.9 μm³) voxel size. Testes (n = 8) of C57/BL mice at the same age served as controls. Testis volume (mm³) and total vascular volume fraction (mm³) were quantified using micro-CT. Serum testosterone levels were determined. Testicular histology and epididymal sections were analysed for tubular structure, spermatogenetic scores and sperm count. The expression of protamine 2 as a marker for elongated spermatids, inflammation markers (CD4, F4/80) and hypoxia inducible factor 1 alpha (HIF1 alpha) were investigated using immunohistochemistry. ApoE(-/-)/LDL receptor(-/-) double knockout mice exhibit diminished testis and vascular volume fraction with respect to that of controls (p < 0.001). These findings were associated with a reduction of testosterone levels (p < 0.001). Mixed atrophy was present in 41% of the seminiferous tubuli in ApoE(-/-)/LDL receptor(-/-) double knockout mice at the age of 80 weeks. Sperm counts from the epididymis demonstrated a significant decrease in ApoE(-/-)/LDL receptor(-/-) double knockout mice (p < 0.001). In addition, sperm specific protamine 2 expression was decreased in testicular tissue and epididymis of ApoE(-/-)/LDL receptor(-/-) double knockout mice compared with that of control mice. Peritubular inflammatory infiltration and the expression of the hypoxia related marker was observed. Mixed testicular atrophy in ApoE(-/-)/LDL receptor(-/-) double knockout mice is linked to reduced testis volume, vascular volume fraction and low testosterone serum levels, suggesting a direct relation between atherosclerosis and disturbed spermatogenesis., (© 2011 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.)

Background: As women are the population most affected by multifactorial osteoporosis, research is focused on unraveling the underlying mechanism of osteoporosis induction in rats by combining ovariectomy (OVX) either with calcium, phosphorus, vitamin C and vitamin D2/D3 deficiency, or by administration of glucocorticoid (dexamethasone)., Material/methods: Different skeletal sites of sham, OVX-Diet and OVX-Steroid rats were analyzed by Dual Energy X-ray Absorptiometry (DEXA) at varied time points of 0, 4 and 12 weeks to determine and compare the osteoporotic factors such as bone mineral density (BMD), bone mineral content (BMC), area, body weight and percent fat among different groups and time points. Comparative analysis and interrelationships among osteoporotic determinants by regression analysis were also determined., Results: T scores were below-2.5 in OVX-Diet rats at 4 and 12 weeks post-OVX. OVX-diet rats revealed pronounced osteoporotic status with reduced BMD and BMC than the steroid counterparts, with the spine and pelvis as the most affected skeletal sites. Increase in percent fat was observed irrespective of the osteoporosis inducers applied. Comparative analysis and interrelationships between osteoporotic determinants that are rarely studied in animals indicate the necessity to analyze BMC and area along with BMD in obtaining meaningful information leading to proper prediction of probability of osteoporotic fractures., Conclusions: Enhanced osteoporotic effect observed in OVX-Diet rats indicates that estrogen dysregulation combined with diet treatment induces and enhances osteoporosis with time when compared to the steroid group. Comparative and regression analysis indicates the need to determine BMC along with BMD and area in osteoporotic determination.

The luminal composition of the auditory tube influences its function. The mechanisms involved in the monitoring are currently not known. For the lower respiratory epithelium, such a sentinel role is carried out by cholinergic brush cells. Here, using two different mouse strains expressing eGFP under the control of the promoter of choline acetyltransferase (ChAT), we show the presence of solitary cholinergic villin-positive brush cells also in the mouse auditory tube epithelium. They express the vesicular acetylcholine (ACh) transporter and proteins of the taste transduction pathway such as α-gustducin, phospholipase C beta 2 (PLC(β2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCβ2 was found regularly, whereas α-gustducin was absent in approximately 15% of the brush cells. Messenger RNA for the umami taste receptors (TasR), Tas1R1 and 3, and for the bitter receptors, Tas2R105 and Tas2R108, involved in perception of cycloheximide and denatonium were detected in the auditory tube. Using a transgenic mouse that expresses eGFP under the promotor of the nicotinic ACh receptor α3-subunit, we identified cholinoceptive nerve fibers that establish direct contacts to brush cells in the auditory tube. A subpopulation of these fibers displayed also CGRP immunoreactivity. Collectively, we show for the first time the presence of brush cells in the auditory tube. These cells are equipped with all proteins essential for sensing the composition of the luminal microenvironment and for communication of the changes to the CNS via attached sensory nerve fibers.

Bony integration of biomaterials is a complex process in which angiogenesis plays a crucial role. We evaluated micro- and nano-CT imaging to demonstrate and quantify neovascularization in bony integration of a biomaterial and to give an image based estimation for the needed resolution for imaging angiogenesis in an animal model of femora defect healing. In 8 rats 5mm full-size defects were created at the left femur that was filled with silica-collagen bone substitute material and internally fixed with plate osteosynthesis. After 6 weeks the femora were infused in situ with Microfil, harvested and scanned for micro-CT (9 μm)(3) and nano-CT (3 μm)(3) imaging. Using those 3D images, the newly formed blood vessels in the area of the biomaterial were assessed and the total vascular volume fraction, the volume of the bone substitute material and the volume of the bone defect were quantitatively characterized. Results were complemented by histology. Differences were statistically assessed using (ANOVA). High-resolution nano-CT demonstrated new blood vessel formation surrounding the biomaterial in all animals at capillary level. Immunohistochemistry confirmed the newly formed blood vessels surrounding the bone substitute material. The mean vascular volume fraction (VVF) around the implant was calculated to be 3.01 ± 0.4%. The VVF was inversely correlated with the volume of the bone substitute material (r=0.8) but not with the dimension of the fracture zone (r=0.3). Nano-CT imaging is feasible for quantitative analysis of angiogenesis during bony integration of biomaterials and a promising tool in this context for the future., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

It is well known that recanalization of thrombosed cerebral sinuses occurs early but without marked influence on the long-term outcome and on final venous infarct volume on magnetic resonance imaging. To better understand the possible microvascular mechanisms behind these clinical observations, we evaluated the sequels of subacute superior sagittal sinus (SSS) thrombosis in rats using micro- and nano-CT imaging of the same specimen to provide large volume and high resolution CT image data respectively. SSS thrombosis was induced in 11 animals which were euthanized after 6h (n=4) or 6 weeks (n=7). Eight sham-operated rats served as controls. After infusion of contrast into the vasculature of the brains, these were isolated and scanned using micro-, nano-, and synchrotron-based micro-CT ((8 μm³), (900 nm)³, and (1.9 μm³) voxel sizes). The cross-sectional area of the superior sagittal sinus, microvessels and cortical veins were quantified. Tissue sections were stained against VEGF antigen. Immunohistochemistry was confirmed using quantitative rtPCR. SSS thrombosis led to a congestion of the bridging veins after 6h. After 6 weeks, a network of small vessels surrounding the occluded SSS was present with concurrent return towards the diameter of the draining bridging veins of controls. This microvascular network connected to cortical veins as demonstrated by nano- and synchrotron-based micro-CT. Also the volume fraction and number of cortical veins increased significantly. Immunohistochemistry in the region of the microsvascular network demonstrated a strong immunoreactivity against VEGF, confirmed by rtPCR. The sequel of subacute SSS thrombosis induced a network of microvessels ("venogenesis") draining the bridging veins. Also the volume fraction of cortical veins increased significantly., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: To investigate the distribution of vasa vasorum (VV) relative to advanced atherosclerotic lesions (calcified, fibrotic or hemorrhaged) along the aortic wall of apoE-/-/LDL-/- mice at the age of 25 and 80 weeks using high-resolution nano-CT., Methods: Aortas from male apoE-/-/LDL-/- mice at the age of 25 weeks (n=4) and 80 weeks (n=7) were infused in situ with contrast agent and harvested for scanning with nano-CT. The spatial distribution of vasa vasorum [number and area/cross-section (mm2)] was compared to aortic luminal cross-sectional area and plaque cross-sectional area in the ascending aorta, aortic arch and descending aorta. Results were complemented with co-localized histology., Results: The number and total luminal cross-sectional area of VV showed a significant decrease in the ascending aorta and aortic arch from 25 to 80 weeks but not in the descending aorta. The number and cross-sectional area of VV showed significant local differences depending on whether it was near a fibrotic, and hemorrhaged or calcified plaque in animals at the age of 80 weeks. Area of VV progressively increased along the aorta from least in the ascending aorta