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Item Assessing the inter- and intra-animal variability of in vivo OsteoProbe skeletal measures in untreated dogs(Elsevier, 2016-12) McNerny, Erin M.B.; Organ, Jason M.; Wallace, Joseph M.; Newman, Christopher L.; Brown, Drew M.; Allen, Matthew R.; Department of Anatomy and Cell Biology, School of MedicineThe OsteoProbe is a second-generation reference point indentation (RPI) device without a reference probe that is designed to simplify RPI testing for clinical use. Successful clinical implementation of the OsteoProbe would benefit from a better understanding of how its output, bone material strength index (BMSi), relates to the material properties of bone and under what conditions it reliably correlates with fracture risk. Large animal models have the potential to help fill this knowledge gap, as cadaveric studies are retrospective and limited by incomplete patient histories (including the potential use of bone matrix altering drugs such as bisphosphonates). The goal of this study was to assess the intra and inter-animal variability of OsteoProbe measures in untreated beagle dogs (n = 12), and to evaluate this variability in comparison to traditional mechanical testing. OsteoProbe measurements were performed in vivo on the left tibia of each dog and repeated 6 months later on the day of sacrifice. Within-animal variation of BMSi (CV of 5–10 indents) averaged 8.9 and 9.0% at the first and second timepoints, respectively. In contrast, inter-animal variation of BMSi increased from 5.3% to 9.1%. The group variation of BMSi was on par with that of traditional 3-point mechanical testing; inter-animal variation was 10% for ultimate force, 13% for stiffness, and 12% for total work as measured on the femur. There was no significant change in mean BMSi after 6 months, but the individual change with time across the 12 dogs was highly variable, ranging from − 12.4% to + 21.7% (mean 1.6%, SD 10.6%). No significant correlations were found between in vivo tibia BMSi and femur mechanical properties measured by ex vivo 3-pt bending, but this may be a limitation of sample size or the tests being performed on different bones. No relationship was found between BMSi and tissue mineral density, but a strong positive correlation was found between BMSi and tibia cortical thickness (ρ = 0.706, p = 0.010). This report shows that while the OsteoProbe device has inter-individual variability quite similar to that of traditional mechanical testing, the longitudinal changes show high levels of heterogeneity across subjects. We further highlight the need for standardization in post-testing data processing and further study of the relationships between OsteoProbe and traditional mechanical testing.Item Crosstalk between MLO-Y4 osteocytes and C2C12 muscle cells is mediated by the Wnt/β-catenin pathway(Wiley, 2017) Huang, Jian; Romero-Suarez, Sandra; Lara, Nuria; Mo, Chenglin; Kaja, Simon; Brotto, Leticia; Dallas, Sarah L.; Johnson, Mark L.; Jähn, Katharina; Bonewald, Lynda F.; Brotto, Marco; Department of Anatomy and Cell Biology, School of MedicineWe examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ∼25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1. We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt/ β-Catenin pathway.Item Detection of different kidney stone types: an ex vivo comparison of ultrashort echo time MRI to reference standard CT(Elsevier, 2016-01) Ibrahim, El-Sayed H.; Cernigliaro, Joseph G.; Pooley, Robert A.; Bridges, Mellena D.; Giesbrandt, Jamie G.; Williams, James C.; Haley, William E.; Department of Anatomy and Cell Biology, School of MedicineBACKGROUND AND PURPOSE: With the development of ultrashort echo time (UTE) sequences, it may now be possible to detect kidney stones by using magnetic resonance imaging (MRI). In this study, kidney stones of varying composition and sizes were imaged using both UTE MRI as well as the reference standard of computed tomography (CT), with different surrounding materials and scan setups. METHODS: One hundred and fourteen kidney stones were inserted into agarose and urine phantoms and imaged both on a dual-energy CT (DECT) scanner using a standard renal stone imaging protocol and on an MRI scanner using the UTE sequence with both head and body surface coils. A subset of the stones representing all composition types and sizes was then inserted into the collecting system of porcine kidneys and imaged in vitro with both CT and MRI. RESULTS: All of the stones were visible on both CT and MRI imaging. DECT was capable of differentiating between uric acid and nonuric acid stones. In MRI imaging, the choice of coil and large field of view (FOV) did not affect stone detection or image quality. The MRI images showed good visualization of the stones' shapes, and the stones' dimensions measured from MRI were in good agreement with the actual values (R(2)=0.886, 0.895, and 0.81 in the agarose phantom, urine phantom, and pig kidneys, respectively). The measured T2 relaxation times ranged from 4.2 to 7.5ms, but did not show significant differences among different stone composition types. CONCLUSIONS: UTE MRI compared favorably with the reference standard CT for imaging stones of different composition types and sizes using body surface coil and large FOV, which suggests potential usefulness of UTE MRI in imaging kidney stones in vivo.Item Different brain responses to electro-acupuncture and moxibustion treatment in patients with Crohn's disease(Nature Publishing Group, 2016-11-18) Bao, Chunhui; Liu, Peng; Liu, Huirong; Jin, Xiaoming; Calhoun, Vince D.; Wu, Luyi; Shi, Yin; Zhang, Jianye; Zeng, Xiaoqing; Ma, Lili; Qin, Wei; Zhang, Jingzhi; Liu, Xiaoming; Tian, Jie; Wu, Huangan; Department of Anatomy and Cell Biology, School of MedicineThis study aimed to investigate changes in resting state brain activity in remissive Crohn's Disease (CD) patients after electro-acupuncture or moxibustion treatment. Fifty-two CD patients and 36 healthy subjects were enrolled, and 36 patients were equally and randomly assigned to receive either electro-acupuncture or moxibustion treatment for twelve weeks. We used resting state functional magnetic resonance imaging to assess Regional Homogeneity (ReHo) levels, and Crohn's Disease Activity Index (CDAI) and Inflammatory Bowel Disease Questionnaire (IBDQ) scores to evaluate disease severity and quality of life. The results show that (i) The ReHo levels in CD patients were significantly increased in cortical but decreased in subcortical areas, and the coupling between them was declined. (ii) Both treatments decreased CDAI, increased IBDQ scores, and normalized the ReHo values of the cortical and subcortical regions. (iii) ReHo changes in multiple cortical regions were significantly correlated with CDAI score decreases. ReHo changes in several subcortical regions in the electro-acupuncture group, and those of several cortical regions in the moxibustion group, were correlated with reduced CDAI. These findings suggest that both treatments improved cortex-subcortical coupling in remissive CD patients, but electro-acupuncture regulated homeostatic afferent processing network, while moxibustion mainly regulated the default mode network of the brain.Item The fracture toughness of small animal cortical bone measured using arc-shaped tension specimens: Effects of bisphosphonate and deproteinization treatments(Elsevier, 2017-12) Hunckler, Michael D.; Chu, Ethan D.; Baumann, Andrew P.; Curtis, Tyler E.; Ravosa, Matthew J.; Allen, Matthew R.; Roeder, Ryan K.; Department of Anatomy and Cell Biology, School of MedicineSmall animal models, and especially transgenic models, have become widespread in the study of bone mechanobiology and metabolic bone disease, but test methods for measuring fracture toughness on multiple replicates or at multiple locations within a single small animal bone are lacking. Therefore, the objective of this study was to develop a method to measure cortical bone fracture toughness in multiple specimens and locations along the diaphysis of small animal bones. Arc-shaped tension specimens were prepared from the mid-diaphysis of rabbit ulnae and loaded to failure to measure the radial fracture toughness in multiple replicates per bone. The test specimen dimensions, crack length, and maximum load met requirements for measuring the plane strain fracture toughness. Experimental groups included a control group, bisphosphonate treatment group, and an ex vivo deproteinization treatment following bisphosphonate treatment (5 rabbits/group and 15 specimens/group). The fracture toughness of ulnar cortical bone from rabbits treated with zoledronic acid for six months exhibited no difference compared with the control group. Partially deproteinized specimens exhibited significantly lower fracture toughness compared with both the control and bisphosphonate treatment groups. The deproteinization treatment increased tissue mineral density (TMD) and resulted in a negative linear correlation between the measured fracture toughness and TMD. Fracture toughness measurements were repeatable with a coefficient of variation of 12–16% within experimental groups. Retrospective power analysis of the control and deproteinization treatment groups indicated a minimum detectable difference of 0.1 MPa·m1/2. Therefore, the overall results of this study suggest that arc-shaped tension specimens offer an advantageous new method for measuring the fracture toughness in small animal bones.Item Intraluminal measurement of papillary duct urine pH, in vivo: a pilot study in the swine kidney(Springer, 2016-06) Handa, Rajash K.; Lingeman, James E.; Bledsoe, Sharon B.; Evan, Andrew P.; Connors, Bret A.; Johnson, Cynthia D.; Department of Anatomy and Cell Biology, School of MedicineWe describe the in vivo use of an optic-chemo microsensor to measure intraluminal papillary duct urine pH in a large mammal. Fiber-optic pH microsensors have a tip diameter of 140-µm that allows insertion into papillary Bellini ducts to measure tubule urine proton concentration. Anesthetized adult pigs underwent percutaneous nephrolithotomy to access the lower pole of the urinary collecting system. A flexible nephroscope was advanced towards an upper pole papilla with the fiber-optic microsensor contained within the working channel. The microsensor was then carefully inserted into Bellini ducts to measure tubule urine pH in real time. We successfully recorded tubule urine pH values in five papillary ducts from three pigs (1 farm pig and 2 metabolic syndrome Ossabaw pigs). Our results demonstrate that optical microsensor technology can be used to measure intraluminal urine pH in real time in a living large mammal. This opens the possibility for application of this optical pH sensing technology in nephrolithiasis.Item Motion artifacts in kidney stone imaging using single-source and dual-source dual-energy CT scanners: a phantom study(Springer, 2015-10) Ibrahim, El-Sayed H.; Cernigliaro, Joseph G.; Pooley, Robert A.; Williams, James C.; Haley, William E.; Department of Anatomy and Cell Biology, School of MedicinePURPOSE: Dual-energy computed tomography (DECT) has shown the capability of differentiating uric acid (UA) from non-UA stones with 90-100% accuracy. With the invention of dual-source (DS) scanners, both low- and high-energy images are acquired simultaneously. However, DECT can also be performed by sequential acquisition of both images on single-source (SS) scanners. The objective of this study is to investigate the effects of motion artifacts on stone classification using both SS-DECT and DS-DECT. METHODS: 114 kidney stones of different types and sizes were imaged on both DS-DECT and SS-DECT scanners with tube voltages of 80 and 140 kVp with and without induced motion. Postprocessing was conducted to create material-specific images from corresponding low- and high-energy images. The dual-energy ratio (DER) and stone material were determined and compared among different scans. RESULTS: For the motionless scans, all stones were correctly classified with SS-DECT, while two cystine stones were misclassified with DS-DECT. When motion was induced, 94% of the stones were misclassified with SS-DECT versus 11% with DS-DECT (P < 0.0001). Stone size was not a factor in stone misclassification under motion. Stone type was not a factor in stone misclassification under motion with SS-DECT, although with DS-DECT, cystine showed higher number of stone misclassification. CONCLUSIONS: Motion artifacts could result in stone misclassification in DECT. This effect is more pronounced in SS-DECT versus DS-DECT, especially if stones of different types lie in close proximity to each other. Further, possible misinterpretation of the number of stones (i.e., missing one, or thinking that there are two) in DS-DECT could be a potentially significant problem.Item Osteocitos y la regulación de la formación ósea(2016) Bellido, Teresita; Pelegrini, Gretel; Department of Anatomy and Cell Biology, School of MedicineFor many years, osteocytes have been the forgotten bone cells and considered as inactive spectators buried in the bone matrix. We now know that osteocytes detect and respond to mechanical and hormonal stimuli to coordinate bone resorption and bone formation. Osteocytes are currently considered a major source of molecules that regulate the activity of osteoclasts and osteoblasts, such as RANKL and sclerostin; and genetic and pharmacological manipulations of either molecule markedly affect bone homeostasis. This article summarizes recent findings demonstrating the mechanisms by which osteocytes regulate the number and activity of osteoblasts and thus affect bone formation.Item Pharmacological depletion of serotonin in the basolateral amygdala complex reduces anxiety and disrupts fear conditioning(Elsevier, 2015-11) Johnson, Philip L.; Molosh, Andrei; Fitz, Stephanie D.; Arendt, Dave; Deehan, Gerald A.; Federici, Lauren M.; Bernabe, Cristian; Engleman, Eric A.; Rodd, Zachary A.; Lowry, Christopher A.; Shekhar, Anantha; Department of Anatomy and Cell Biology, School of MedicineThe basolateral and lateral amygdala nuclei complex (BLC) is implicated in a number of emotional responses including conditioned fear and social anxiety. Based on previous studies demonstrating that enhanced serotonin release in the BLC leads to increased anxiety and fear responses, we hypothesized that pharmacologically depleting serotonin in the BLC using 5,7-dihydroxytryptamine (5,7-DHT) injections would lead to diminished anxiety and disrupted fear conditioning. To test this hypothesis, 5,7-DHT(a serotonin-depleting agent) was bilaterally injected into the BLC. Desipramine (a norepinephrine reuptake inhibitor) was systemically administered to prevent non-selective effects on norepinephrine. After 5days, 5-7-DHT-treated rats showed increases in the duration of social interaction (SI) time, suggestive of reduced anxiety-like behavior. We then used a cue-induced fear conditioning protocol with shock as the unconditioned stimulus and tone as the conditioned stimulus for rats pretreated with bilateral 5,7-DHT, or vehicle, injections into the BLC. Compared to vehicle-treated rats, 5,7-DHT rats had reduced acquisition of fear during conditioning (measured by freezing time during tone), also had reduced fear retrieval/recall on subsequent testing days. Ex vivo analyses revealed that 5,7-DHT reduced local 5-HT concentrations in the BLC by ~40% without altering local norepinephrine or dopamine concentrations. These data provide additional support for 5-HT playing a critical role in modulating anxiety-like behavior and fear-associated memories through its actions within the BLC.Item PTHrP-Derived Peptides Restore Bone Mass and Strength in Diabetic Mice: Additive Effect of Mechanical Loading(Wiley, 2017-03) Maycas, Marta; McAndrews, Kevin A.; Sato, Amy Y.; Pellegrini, Gretel G.; Brown, Drew M.; Allen, Matthew R.; Plotkin, Lilian I.; Gortazar, Arancha R.; Esbrit, Pedro; Bellido, Teresita; Department of Anatomy and Cell Biology, School of MedicineThere is an unmet need to understand the mechanisms underlying skeletal deterioration in diabetes mellitus (DM) and to develop therapeutic approaches to treat bone fragility in diabetic patients. We demonstrate herein that mice with type 1 DM induced by streptozotocin exhibited low bone mass, inferior mechanical and material properties, increased bone resorption, decreased bone formation, increased apoptosis of osteocytes, and increased expression of the osteocyte-derived bone formation inhibitor Sost/sclerostin. Further, short treatment of diabetic mice with parathyroid hormone related protein (PTHrP)-derived peptides corrected these changes to levels undistinguishable from non-diabetic mice. In addition, diabetic mice exhibited reduced bone formation in response to mechanical stimulation, which was corrected by treatment with the PTHrP peptides, and higher prevalence of apoptotic osteocytes, which was reduced by loading or by the PTHrP peptides alone and reversed by a combination of loading and PTHrP peptide treatment. In vitro experiments demonstrated that the PTHrP peptides or mechanical stimulation by fluid flow activated the survival kinases ERKs and induced nuclear translocation of the canonical Wnt signaling mediator β-catenin, and prevented the increase in osteocytic cell apoptosis induced by high glucose. Thus, PTHrP-derived peptides cross-talk with mechanical signaling pathways to reverse skeletal deterioration induced by DM in mice. These findings suggest a crucial role of osteocytes in the harmful effects of diabetes on bone and raise the possibility of targeting these cells as a novel approach to treat skeletal deterioration in diabetes. Moreover, our study suggests the potential therapeutic efficacy of combined pharmacological and mechanical stimuli to promote bone accrual and maintenance in diabetic subjects.